Structure and Function

Structure and Function
VOLUME 1
Polymeric Biomaterials
Polymeric Biomaterials: Structure and Function, Volume 1
Polymeric Biomaterials: Medicinal and Pharmaceutical Applications, Volume 2

Structure and Function
VOLUME 1
Founding Editor Editor

Severian Dumitriu Valentin Popa
Boca Raton London New York
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Contents
Preface...............................................................................................................................................ix
Acknowledgments..............................................................................................................................xi
Editors............................................................................................................................................. xiii
Contributors...................................................................................................................................... xv
Chapter 1 Synthesis and Fabrication of Polyesters as Biomaterials.............................................. 1

Philippe Lecomte and Christine Jérôme
Chapter 2 Hydrogels Formed by Cross-Linked Poly(Vinyl Alcohol).......................................... 37

Gaio Paradossi
Chapter 3 Development and Evaluation of Poly(Vinyl Alcohol) Hydrogels as a

Component of Hybrid Artificial Tissues for Orthopedics Surgery Application......... 57
Masanori Kobayashi
Chapter 4 Polyphosphazenes as Biomaterials.............................................................................. 83

Meng Deng, Cato T. Laurencin, Harry R. Allcock, and Sangamesh G. Kumbar
Chapter 5 Biodegradable Polymers as Drug Carrier Systems................................................... 135

Abraham J. Domb and Wahid Khan
Chapter 6 Bioresorbable Hybrid Membranes for Bone Regeneration....................................... 177

Akiko Obata and Toshihiro Kasuga
Chapter 7 Mucoadhesive Polymers: Basics, Strategies, and Future Trends.............................. 193

Andreas Bernkop-Schnürch
Chapter 8 Biodegradable Polymeric/Ceramic Composite Scaffolds to Regenerate

Bone Tissue............................................................................................................... 221
Catherine Gkioni, Sander Leeuwenburgh, and John Jansen
Chapter 9 Amphiphilic Systems as Biomaterials Based on Chitin, Chitosan,

and Their Derivatives................................................................................................ 243
Jacques Desbrieres
Chapter 10 Biomaterials of Natural Origin in Regenerative Medicine....................................... 271

Vijay Kumar Nandagiri, Valeria Chiono, Piergiorgio Gentile,
Franco Maria Montevecchi, and Gianluca Ciardelli
v
vi Contents
Chapter 11 Natural Polymers as Components of Blends for Biomedical Applications...............309

Alina Sionkowska
Chapter 12 Metal–Polymer Composite Biomaterials.................................................................. 343

Takao Hanawa
Chapter 13 Evolution of Current and Future Concepts of Biocompatibility Testing.................. 377

Menno L.W. Knetsch
Chapter 14 Biocompatibility of Elastomers................................................................................. 415

Dominique Chauvel-Lebret, Pascal Auroy, and Martine Bonnaure-Mallet
Chapter 15 Preparation and Applications of Modulated Surface Energy Biomaterials.............. 495

Blanca Vázquez, Luis M. Rodríguez-Lorenzo, Gema Rodríguez-Crespo,
Juan Parra, Mar Fernández, and Julio San Román
Chapter 16 Electrospinning for Regenerative Medicine.............................................................. 539

Toby D. Brown, Cedryck Vaquette, Dietmar W. Hutmacher, and Paul D. Dalton
Chapter 17 Polymeric Nanoparticles for Targeted Delivery of Bioactive Agents and Drugs..... 593
Cesare Errico, Alberto Dessy, Anna Maria Piras, and Federica Chiellini
Chapter 18 Polymeric Materials Obtained through Biocatalysis................................................ 617

Florin Dan Irimie, Csaba Paizs, and Monica Ioana Tosa
Chapter 19 Polymer-Based Colloidal Aggregates as a New Class of Drug Delivery Systems.......659

Cesare Cametti
Chapter 20 Photoresponsive Polymers for Control of Cell Bioassay Systems............................. 683

Kimio Sumaru, Shinji Sugiura, Toshiyuki Takagi, and Toshiyuki Kanamori
Chapter 21 Lignin in Biological Systems.................................................................................... 709

Valentin I. Popa
Chapter 22 Carbohydrate-Derived Self-Crosslinkable In Situ Gelable Hydrogels

for Modulation of Wound Healing............................................................................ 739
Lihui Weng, Christine Falabella, and Weiliam Chen
Contents vii
Chapter 23 Dental and Maxillofacial Surgery Applications of Polymers................................... 783

E.C. Combe
Chapter 24 Biomaterials as Platforms for Topical Administration of Therapeutic Agents

in Cutaneous Wound Healing................................................................................... 837
Rhiannon Braund and Natalie J. Medlicott
Chapter 25 Polymers for Artificial Joints.................................................................................... 851

Masayuki Kyomoto, Toru Moro, and Kazuhiko Ishihara
Index............................................................................................................................................... 885
Preface
The field of biomaterials has developed rapidly because of the continuous and ever-expanding
practical needs of medicine and health-care practice. There are currently thousands of medical
devices, diagnostic products, and disposables on the market, and the range of applications contin-
ues to grow. In addition to traditional medical devices, diagnostic products, pharmaceutical prepa-
rations, and health-care disposables, the list of biomaterials applications includes smart delivery
systems for drugs, tissue cultures, engineered tissues, and hybrid organs.
Undoubtedly, biomaterials have had a major impact on the practice of contemporary medicine
and patient care, resulting in both saving and improving the quality of lives of humans and animals.
Modern biomaterials practice is continuing to develop into a major interdisciplinary effort involv-
ing chemists, biologists, engineers, and physicians. It also takes advantage of developments in the
traditional, nonmedical materials field, and much progress has been made since the beginning of the
research in biomaterials that made possible the creation of a high-quality and much improved vari-
ety of devices, implants (permanent or temporary), and drug carrier devices. All of these now dis-
play a greater than ever biocompatibility and biofunctionality. The variety of chemical substances
used in these materials is currently very broad, and most biomedical applications are associated
with various polymers and materials based on them.
The pace of research in the field of polymeric biomaterials is so fast that two editions of Polymeric
Biomaterials have already been edited by Severian Dumitriu. Due to the interest generated and the
success of these books, Severian was working on a third edition. Unfortunately, he passed away
before this could be finalized. Many of the scientists who accepted his invitation to cooperate for
this new edition agreed to contribute to the book in memory of the contribution that Severian made
to the field of polymeric biomaterials. Together with Daniela, his beloved daughter, and Barbara
Glunn and Jessika Vakili from Taylor & Francis Group, we decided to continue the work and final-
ize this book.
This book is organized in two volumes consisting of 53 chapters that systematically provide the
latest developments in different aspects of polymeric biomaterials. Thus, we can mention contribu-
tions to the field of synthesis and applications of polymers such as polyesters, poly(vinyl alcohol),
polyphosphazenes, elastomers, bioceramics, blends or composites, enzymatic synthesis, along with
natural ones such as mucoadhesives, chitin, chitosan, lignin, carbohydrates derivatives, heparin, etc.
Drugs carriers and delivery systems, gene and nucleic acids delivery represent other subjects
of some chapters, dealing with both supports (biodegradable and biocompatible) and techniques
(nanoparticles, electrospinning, photo- and pH responsive polymers, hydrogels, lipid-core micelles,
biomimetic systems, medical devices) aspects. In some cases, biomaterials can be synthesized,
modified, and processed by different methods to ensure biocompatibility and biodegradability to
be used as membranes, composites, scaffolds, and implants. Some examples of specific utilizations
of polymeric biomaterials are presented, such as orthopedic surgery, bone regeneration, wound
healing, dental and maxillofacial surgery applications, artificial joints, diabetes, anticancer agents
and cancer therapy, modification of living cells, myocardial tissue engineering—repair and recon-
struction, and bioartificial organs.
Publishing this book was accomplished with the contributions of renowned scientists from all
over the world. They are all experts in their particular field of biomaterials research and have made
high-level contributions to various fields of research. We are very grateful to these scientists for
their willingness to contribute to this reference work as well as for their engagement. Without their
commitment and enthusiasm, it would not have been possible to compile such a book.
ix
x Preface
I am also grateful to the publisher for recognizing the demand for such a book, for taking the risk
to bring out such a book, and for realizing the excellent quality of the publication.
I would like to thank Daniela for her inestimable help and assistance. I dedicate this book to
memory of Severian, one of my best friends.
Last but not least, I would like to thank my family for their patience. I sincerely apologize for the
many hours I spent in the preparation of this book, which kept me away from them.
This book is a very useful tool for many scientists, physicians, pharmacists, engineers, and other
experts in a variety of disciplines, both in academe and industry. It may not only be useful for
research and development but may also be suitable for teaching.
This book has a companion CD that contains color figures as noted at the applicable text figures.
Valentin I. Popa
Acknowledgments
My father was passionate about polymeric biomaterials. He was very happy when this project was
planned with Taylor & Francis Group. He had worked tirelessly toward this. He would have loved
to have seen this book published, but destiny willed otherwise.
I am extremely grateful to Professor Popa for accepting to serve as the editor, to all the authors
for their precious contributions, and to the staff at Taylor & Francis Group.
The positive response from the authors to pursue their contribution to this book was amazing
and is testimony of their appreciation for the scientific contribution that my father made to the field
of polymeric biomaterials.
I trust the book is of great quality and reflects the efforts and dedication that have been put into
it by my father and all the contributors.
My small contribution to this book is dedicated to the memory of my parents, Severian and
Maria, for their unconditional love and for being the best teachers ever. And to finish on a positive
note, I want to cite one quote of Dr. Seuss that I particularly like:
“Don’t cry because it’s over. Smile because it happened”.
Daniela Dumitriu
xi
Editors
Severian Dumitriu (deceased) was a research professor, Department of Chemical Engineering,
University of Sherbrooke, Quebec, Canada. He edited several books, including Polymeric
Biomaterials, second edition, Polysaccharides in Medicinal Applications, and Polysaccharides:
Structural Diversity and Functional Versatility (all three titles were published by Taylor & Francis
Group [previously Marcel Dekker]), and authored or coauthored over 190 professional papers
and book chapters in the fields of polymer and cellulose chemistry, polyfunctional initiators, and
bioactive polymers. He also held 15 international patents. Professor Dumitriu received his BSc
(1959) and MS (1961) in chemical engineering and his PhD (1971) in macromolecular chemistry
from the Polytechnic Institute of Jassy, Romania. Upon completing his doctorate, he worked with
Professor G. Smets at the Catholic University of Louvain, Belgium, and was a research associate
at the University of Pisa, Italy; the Hebrew University Medical School, Jerusalem, Israel; and the
University of Paris, South France.
Valentin I. Popa earned his BSc and MSc in chemical engineering (1969) and PhD in the field of
polysaccharide chemistry (1976) from Polytechnic Institute of Iasi, Romania. He was awarded the
Romanian Academy Prize for his contributions in the field of seaweed c hemistry (1976). He has
published more than 500 papers in the following fields: wood chemistry and biotechnology, bio-
mass complex processing, biosynthesis and biodegradation of natural compounds, allelochemicals,
bioadhesives, and bioremediation. He is also the author or coauthor of 37 books or book chapters.
Dr. Popa holds six patents and has been involved in many Romanian and European research proj-
ects as scientific manager. He was visiting scientist or visiting professor at Academy of Sciences
(Seoul, Korea, 1972), Technical University of Helsinki (Finland, 1978), Institute of Biotechnology
(Vienna, Austria, 1995), Research Institute for Pulp and Paper (Braila, Romania, 1976), “Petru Poni”
Institute of Macromolecular Chemistry (Iasi, Romania, 1985, 1986), Université de Sherbrooke
and University McGill (Canada, 2003), STFI–Packforsk (now known as Innventia, Stockholm,
Sweden, 2008), and Institute of Wood Chemistry (Riga, Latvia, 2009). Dr. Popa is a member of the
International Lignin Institute, International Association of Scientific Papermakers, International
Academy of Wood Science, Romanian Academy for Technical Sciences, and American Chemical
Society. He is also a professor of wood chemistry and biotechnology in “Gheorghe Asachi”
Technical University of Iasi, PhD supervisor (30 students defended their theses), and editor-in-
chief of Cellulose Chemistry and Technology.
xiii
Contributors
Harry R. Allcock Weiliam Chen
Department of Chemistry Department of Surgery
The Pennsylvania State University Division of Wound Healing and Regenerative
University Park, Pennsylvania Medicine
School of Medicine
Pascal Auroy New York University
Université d’Auvergne New York, New York
Clermont-Ferrand, France
Federica Chiellini
Andreas Bernkop-Schnürch Laboratory of Bioactive Polymeric Materials
Institute of Pharmacy for Biomedical and Environmental
University of Innsbruck Application (BIOLab)
Innsbruck, Austria Department of Chemistry and Industrial
Chemistry
Martine Bonnaure-Mallet University of Pisa
UFR Odontologie Equipe de Microbiologie Pisa, Italy
EA 1254
Université Européenne de Bretagne Valeria Chiono
Rennes, France Department of Mechanical and Aerospace
Engineering
Rhiannon Braund Politecnico di Torino
School of Pharmacy Torino, Italy
University of Otago
Gianluca Ciardelli
Dunedin, New Zealand
Department of Mechanical and Aerospace
Engineering
Toby D. Brown
Politecnico di Torino
Institute for Health and Biomedical
Torino, Italy
Innovation
Queensland University of Technology E.C. Combe
Brisbane, Queensland, Australia School of Dentistry
University of Minnesota
Cesare Cametti Minneapolis, Minnesota
Department of Physics
University of Rome “La Sapienza” Paul D. Dalton
and Institute for Health and Biomedical Innovation
CNR-INFM-SOFT Queensland University of Technology
Rome, Italy Brisbane, Queensland, Australia
Dominique Chauvel-Lebret Meng Deng

Université Européenne de Bretagne Institute for Regenerative Engineering
UFR d’Odontologie-UMR CNRS 6226 Raymond and Beverly Sackler Center for
Sciences Chimiques de Rennes-UR1, Biological, Physical and Engineering
CHU-pole d’Odontologie et de chirurgie Sciences
Buccale The University of Connecticut
Rennes, France Storrs, Connecticut
xv
xvi Contributors
Jacques Desbrieres Catherine Gkioni

Department of Physics and Chemistry Department of Periodontology and Biomaterials
of Polymers Radboud University Nijmegen Medical Center
Pau and Adour Countries University Nijmegen, the Netherlands
Pau, France
Takao Hanawa
Alberto Dessy Institute of Biomaterials and Bioengineering
Laboratory of Bioactive Polymeric Materials Tokyo Medical and Dental University
for Biomedical and Environmental Tokyo, Japan
Application (BIOLab)
Department of Chemistry and Industrial Dietmar W. Hutmacher
Chemistry Institute for Health and Biomedical Innovation
University of Pisa Queensland University of Technology
Pisa, Italy Brisbane, Queensland, Australia
Florin Dan Irimie

Abraham J. Domb
Department of Biochemistry and Biochemical
Faculty of Medicine
Engineering
School of Pharmacy
“Babeş-Bolyai” University
Institute of Drug Research
Cluj-Napoca, Romania
The Hebrew University of Jerusalem
Jerusalem, Israel
Kazuhiko Ishihara
Department of Materials Engineering
Cesare Errico and
Laboratory of Bioactive Polymeric Materials Department of Bioengineering
for Biomedical and Environmental School of Engineering
Application (BIOLab) The University of Tokyo
Department of Chemistry and Industrial Tokyo, Japan
Chemistry
University of Pisa
John Jansen
Pisa, Italy
Department of Periodontology and Biomaterials
Radboud University Nijmegen Medical Center
Christine Falabella Nijmegen, the Netherlands
Department of Biomedical
Engineering Christine Jérôme
Stony Brook University Center for Education and
State University of New York Research on Macromolecules
New York, New York University of Liège
Liège, Belgium
Mar Fernández
Institute for Health and Biomedical Toshiyuki Kanamori
Innovation National Institute of Advanced
Queensland University of Technology Industrial Science and
Kelvin Grove, Queensland, Australia Technology
Tsukuba, Japan
Piergiorgio Gentile
Department of Mechanical and Aerospace Toshihiro Kasuga
Engineering Graduate School of Engineering
Politecnico di Torino Nagoya Institute of Technology
Torino, Italy Nagoya, Japan
Contributors xvii
Wahid Khan Philippe Lecomte

Institute of Drug Research (IDR) Center for Education and Research on
School of Pharmacy-Faculty of Medicine Macromolecules
The Hebrew University University of Liège
of Jerusalem Liège, Belgium
Jerusalem, Israel
Sander Leeuwenburgh
Menno L.W. Knetsch Department of Periodontology
Department of Biomedical and Biomaterials
Engineering/Biomaterials Science Radboud University Nijmegen Medical Center
Maastricht University Nijmegen, the Netherlands
Maastricht, the Netherlands
Natalie J. Medlicott
Masanori Kobayashi School of Pharmacy
Department of Biomedical Engineering University of Otago
Daido University Dunedin, New Zealand
Nagoya, Japan
Franco Maria Montevecchi
Department of Mechanical and Aerospace
Sangamesh G. Kumbar Engineering
Department of Orthopaedic Surgery, Chemical, Politecnico di Torino
Materials and Biomolecular Engineering Torino, Italy
Institute for Regenerative Engineering
Raymond and Beverly Sackler Center for Toru Moro
Biological, Physical and Engineering Science for Joint Reconstruction
Sciences Graduate School of Medicine
The University of Connecticut The University of Tokyo
Storrs, Connecticut Tokyo, Japan
Masayuki Kyomoto Vijay Kumar Nandagiri

Department of Materials Engineering Department of Mechanical and Aerospace
School of Engineering Engineering
and Politecnico di Torino
Science for Joint Reconstruction Torino, Italy
Graduate School of Medicine
The University of Tokyo Akiko Obata
and Graduate School of Engineering
Research Department Nagoya Institute of Technology
Kyocera Medical Corporation Nagoya, Japan
Tokyo, Japan
Csaba Paizs
Cato T. Laurencin Department of Biochemistry and Biochemical
Connecticut Institute for Clinical and Engineering
Translational Science “Babeş-Bolyai” University
and Cluj-Napoca, Romania
Institute for Regenerative Engineering
Raymond and Beverly Sackler Center for Gaio Paradossi
Biological, Physical and Engineering Department of Chemical Sciences
Sciences and Technologies
The University of Connecticut University of Rome Tor Vergata
Storrs, Connecticut Rome, Italy
xviii Contributors
Juan Parra Alina Sionkowska

Institute for Health and Biomedical Faculty of Chemistry
Innovation Nicolaus Copernicus University
Queensland University of Technology Torun, Poland
Kelvin Grove, Queensland, Australia
Shinji Sugiura
Anna Maria Piras National Institute of Advanced Industrial
Laboratory of Bioactive Polymeric Materials Science and Technology
for Biomedical and Environmental Tsukuba, Japan
Application (BIOLab)
Kimio Sumaru
Department of Chemistry and Industrial
National Institute of Advanced Industrial
Chemistry
Science and Technology
University of Pisa
Tsukuba, Japan
Pisa, Italy
Toshiyuki Takagi
Valentin I. Popa National Institute of Advanced
Department of Natural and Synthetic Industrial Science and
Polymers Technology
Faculty of Chemical Engineering and Tsukuba, Japan
Environmental Protection
“Gheorghe Asachi” Technical University Monica Ioana Tosa
of Iasi Department of Biochemistry
Iasi, Romania and Biochemical Engineering
“Babeş-Bolyai” University
Gema Rodríguez-Crespo Cluj-Napoca, Romania
Institute for Health and Biomedical
Innovation Cedryck Vaquette
Queensland University of Technology Institute for Health and Biomedical
Kelvin Grove, Queensland, Australia Innovation
Queensland University of Technology
Brisbane, Queensland, Australia
Luis M. Rodríguez-Lorenzo
Institute for Health and Biomedical Blanca Vázquez
Innovation Institute for Health and Biomedical
Queensland University of Technology Innovation
Kelvin Grove, Queensland, Australia Queensland University of Technology
Kelvin Grove, Queensland, Australia
Julio San Román
Institute for Health and Biomedical Lihui Weng
Innovation Department of Radiology
Queensland University of Technology University of Minnesota
Kelvin Grove, Queensland, Australia Minneapolis, Minnesota
1 Synthesis and Fabrication
of Polyesters as Biomaterials
Philippe Lecomte and Christine Jérôme
CONTENTS
1.1 Introduction............................................................................................................................... 1
1.2 Step-Growth Polymerization..................................................................................................... 2
1.3 Ring-Opening Polymerization of Cyclic Esters........................................................................3
1.3.1 Anionic Polymerization................................................................................................. 4
1.3.2 Coordination Polymerization......................................................................................... 6
1.3.3 Metal-Free Ring-Opening Polymerization....................................................................8
1.3.4 Enzymatic Ring-Opening Polymerization.................................................................. 13
1.3.5 Polymerization of Substituted and Functionalized Cyclic Esters................................ 14
1.4 Radical Ring-Opening Polymerization of Cyclic Ketene Acetals..........................................25
1.5 Macromolecular Engineering of Aliphatic Polyesters.............................................................26
1.5.1 Copolymerization........................................................................................................26
1.5.2 Modification of the Architecture................................................................................. 27
1.6 Conclusions..............................................................................................................................28
Acknowledgments.............................................................................................................................28
References.........................................................................................................................................28
1.1 INTRODUCTION
Nowadays, biomaterials are produced from a wide range of polymers. Among them, biodegradable
and biocompatible aliphatic polyesters occupy a key position. Their chemical structure can be eas-
ily modified, which allows the tailoring of important properties such as bioadherence, mechanical
properties, and kinetics of biodegradation. Aliphatic polyesters are thus widely used in biomedical
applications as implants, as scaffolds in tissue engineering, and as carriers for drug delivery.
This chapter aims at reviewing the most important techniques used for the synthesis of aliphatic
polyesters in biomedical applications. The simplest approach relies on step-growth polymerization
(Figure 1.1, routes a and b). Nevertheless, the control imparted to the polymerization is limited by
this technique, and the synthesis of high molar mass polyesters is difficult. These drawbacks can
be tackled by implementing another technique, the ring-opening polymerization of cyclic esters
(Figure 1.1, route c). Under appropriate conditions, this polymerization is living and enables the
synthesis of controlled high molar mass aliphatic polyesters. The chemical structure of the chain-
ends can be controlled and it is thus possible to functionalize them on demand. Besides, aliphatic
polyesters are produced by the ring-opening polymerization of cyclic esters at the industrial scale.
Moreover, the livingness of the ring-opening polymerization of cyclic esters opens up the possibility
to prepare various architectures such as star-shaped, comb-shaped, hyperbranched polymers and net-
works. The versatility of the ring-opening polymerization of cyclic esters thus allows fine tailoring
of the properties of aliphatic polyesters in view of biomedical applications. A very important section
of this chapter will thus be dedicated to this technique. It must be noted that other polymerization
1
2 Polymeric Biomaterials: Structure and Function, Volume 1
O O
a
HO R OH + HO R OH
O
b
HO R OH
O O
O c
R O n
R
O O d
FIGURE 1.1 Main techniques for the synthesis of aliphatic polyesters.
techniques were investigated even though they were not so popular. In this regard, special attention
will be paid to the ring-opening polymerization of cyclic ketene acetals (Figure 1.1, route d).
For biomedical applications, it is mandatory that aliphatic polyesters exhibit a very high purity
and are not contaminated by potentially toxic impurities. Unfortunately, usual polymerization tech-
niques are based on catalysts and initiators made up of toxic metals such as tin and aluminum,
especially as far as the ring-opening polymerization of cyclic esters is concerned. Special attention
will thus be paid to the recent advances in the implementation of polymerization processes based
on less toxic metals or, even better, on metal-free processes.
1.2 STEP-GROWTH POLYMERIZATION

A straightforward approach for the synthesis of aliphatic polyesters is based on the step-growth
polymerization of a mixture of diacids and diols or, more directly, of hydroxy acids, by imple-
menting an esterification reaction. This technique allows the synthesis of a very wide range of
aliphatic polyesters because of the easy synthesis of various diols, diacids, and hydroxy acids. Some
of these can even be obtained from renewable resources, and bio-based aliphatic polyesters can then
be produced. Step-growth polymerization has been in use for a long time, and hence we will not
develop further the theory of polycondensation. It is just worth noting that, recently, Kricheldorf
revised the concept of kinetically controlled (Kricheldorf and Schwarz 2003) and thermodynami-
cally controlled (Kricheldorf 2003) step-growth polymerization.
The main limitation of all step-growth polymerizations remains the difficult synthesis of high
molar masses. Indeed, the synthesis of high molar mass polyesters by step-growth polymerization
requires to reach high conversions very close to 100% and to stay close to the ideal 1/1 stoichiometry
between alcohol and acid functions.
The synthesis of polyhydroxyalkanoates (PHAs) by step-growth polymerization is a naturally
occurring process (Lu et al. 2009). These aliphatic polyesters are produced by microorganisms
as an intracellular reserve of carbon storage compounds and energy (Lu et al. 2009). Poly(3-(R)-
hydroxybutyrate) (PHB) is a typical example, but PHAs with a huge variety of chemical structures
can be synthesized from a very wide range of hydroxyalkanoates. The potential of PHAs has been
assessed for several biomedical applications such as controlled release, tissue engineering, surgical
sutures, and wound dressings (Williams et al. 1999).
The limitations of step-growth polymerization urged chemists to search for more efficient tech-
niques for the synthesis of high molar mass aliphatic polyesters with a fine control of the chemical
structure and the molar mass. This goal was achieved by implementing the ring-opening polymer-
ization of cyclic esters, as will be shown in the next section.
Synthesis and Fabrication of Polyesters as Biomaterials 3
1.3 RING-OPENING POLYMERIZATION OF CYCLIC ESTERS

Ring-opening polymerization is a very popular technique to synthesize aliphatic polyesters in view
of biomedical applications. The main reason for its success lies in the very easy synthesis of aliphatic
polyesters with high and controlled molar masses (Stridsberg et al. 2002, Penczek et al. 2007). The
main limitation remains, for the time being, the scarcity of cyclic esters, despite the recent pro-
gresses being made in the last few years, especially compared to diacids, diols, and hydroxyacids
used in step-growth polymerization.
The ring-opening polymerization of cyclic esters has been in use for a long time. van Natta
et al. (1934) already reported in 1934 the ring-opening polymerization of ɛ-caprolactone (ɛCL).
Nowadays, polylactide (PLA) and poly(ɛ-caprolactone) (PCL) are produced at the industrial scale
(Figure 1.2).
PCL is a semicrystalline biodegradable and biocompatible polyester (Tm = 60°C; Tg = −60°C)
(Woodruff and Hutmacher 2010). Nevertheless, the degradation of PCL is slow, making it suitable
in the field of drug delivery applications for long-term applications (Sinha et al. 2004). PLA and
copolymers of lactide and glycolide (PLGA) have the advantage to be more hydrophilic than PCL
and thus to degrade faster. Polylactide contains a chiral center (R or S), and its properties depend
on tacticity. It is indeed well known that isotactic PLA is semicrystalline, whereas atactic PLA is
amorphous. Semicrystalline PLA exhibits interesting mechanical properties but at the expense of
biodegradability. Isotactic PLLA is synthesized by the polymerization of enantiomerically pure
l-lactide, whereas atactic PLLA is obtained from a racemic mixture of l- and d-lactides.
The ring-opening polymerization of ɛCL and LA is carried out at the industrial level. Interestingly
enough, PLA is a bio-based polyester produced from agricultural renewable resources such as
starch, whereas biodegradable PCL is produced from oil.
Although ɛCL, GA, and LA are very important monomers, other cyclic esters can be polymer-
ized by ring-opening depending on their size. First, there is an important question to determine
whether a cyclic ester can be polymerized or not. The answer to this question can be found by
considering thermodynamic data. According to the microreversibility rule, there is a competition
between polymerization and depolymerization. Table 1.1 shows the values of the enthalpy (ΔHp) and
entropy (ΔSp) of polymerization, the monomer concentration at equilibrium ([M]eq), and the ceiling
temperature (Tc) (Duda et al. 2005).
Table 1.1 shows that the polymerization of low-membered cyclic esters is an enthalpy-driven
process (Penczek et al. 2000, Duda et al. 2005). The release of the ring strain is obviously a key
parameter in favor of polymerization. Nevertheless, the behavior of five-membered lactones is
completely different, as shown by the high-ceiling temperature, because depolymerization is faster
than polymerization. Although the ring-opening polymerization of γBL is difficult, it is possible to
O
O
O
O
n
ε-Caprolactone Poly(ε-caprolactone) (PCL)

O
O
R
O O
O
R n
R
O
R = H: glycolide R = H: polyglycolide (PGA)
R = Me: lactide R = Me: polylactide (PLA)
FIGURE 1.2 Polymerization of ɛ-caprolactone (ɛCL) and lactide (LA) and glycolide (GA).
TABLE 1.1
Thermodynamics for the Polymerization of Cyclic Esters
Ring Monomer
Monomer Size Polymer States ΔHp (kJ/mol) ΔSp (J/mol K)p [M]eq (mol/L) Tc (K)
βPL 4 1c −82.3 −74 3.9 × 10−10 1112
γBL 5 1c 5.1 −29.9 3.3 × 10−3 −171
LA 6 88 −22.9 −41 5.5 × 10−2 914
GA 6 11 −13.8 −45 2.5 520
VL 6 Lc −27.4 −65 3.9 × 10−1 —
εCL 7 1c −28.8 −53.9 5.3 × 10−2 534
TABLE 1.2
Nomenclature of Cyclic Esters
Ring Size IUPAC Name Usual Name
4 βPL oxetan-2-one β-propionolactone
5 γBL dihydrofuran-2(3H)-one γ-butyrolactone
6 δVL tetrahydro-2H-pyran-2-one δ-valerolactone
6 LA lactide 3,6-dimethyl-1,4-dioxane-2,5-dione
6 GA glycolide 1,4-dioxane-2,5-dione
7 εCL oxepan-2-one ε-caprolactone
obtain low molar mass oligomers under suitable conditions and also to copolymerize γBL with other
cyclic esters (Penczek et al. 2000).
It is worth noting that lactide, caprolactone, and glycolide are not the names recommended by
IUPAC. Table 1.2 shows the usual and official names for several cyclic esters. For the sake of sim-
plicity, the usual names will be mentioned in this review to avoid confusion.
So many initiators and catalysts have been used for the ring-opening polymerization of cyclic
esters for the last few years that it is impossible to list all. This review will thus focus on the most
popular processes with special attention being paid to the livingness and kinetics of polymeriza-
tion, and the contamination of aliphatic polyesters by toxic residues not tolerated in the frame of
biomedical applications.
1.3.1 Anionic Polymerization

Metal alkoxides are nucleophilic species prone to initiate the ring-opening polymerization of cyclic
esters. Polymerization takes place usually by the cleavage of the acyl-oxygen bond. Figure 1.3 shows
the initiation and propagation steps in the case of polymerization of ɛCL.
O O O
RO
O
–
O O O
– –
R O + RO 5 5 n O
Initiation Propagation
O
FIGURE 1.3 Anionic ring-opening polymerization of ɛCL.

The mechanism shown in Figure 1.3 assumes that polymerization is living and takes place
only by initiation and propagation reactions according to Szwarc (1956), which means that
no t ermination and transfer reactions are present. This view is unfortunately oversimplified because
termination and transfer reactions are often observed. Indeed, anionic species are deactivated by
traces of water or by other protic substances, which is nothing but a termination reaction. It is
thus necessary to perform these anionic polymerizations with carefully purified monomers under
strictly anhydrous conditions. Besides, although metal alkoxides react as nucleophiles, they react
also as bases. For instance, potassium tert-butoxide reacts with ɛCL to form the corresponding
potassium enolate.
Transfer reactions by transesterification reactions are commonly observed and are at the origin
of loss of control of polymerization (Figures 1.4 and 1.5). Indeed, alkoxides are often too reactive
to be selective, and they can react not only with the ester functions of monomers but also with the
ester functions present all along the polymer chains. Depending upon whether alkoxide and ester
are located on the same chain or not, transesterification reaction can be intramolecular or intermo-
lecular. Intramolecular transesterification reactions result in a decrease of the molar mass and in
the formation of cyclic oligomers (Figure 1.4). Conversely, intermolecular transesterification does
not result in a decrease of the number-average molar mass but rather results in the reshuffling of the
length of chains and thus in the modification of the polydispersity index (Figure 1.5). For the sake
of simplicity, the mechanism of ɛCL is shown in Figures 1.4 and 1.5, but it can be extended to other
cyclic esters as well.
Special attention has to be paid to the four-membered lactones because of their unusual behavior.
Indeed, polymerization of β-lactones by the mechanism based on the cleavage of the acyl-oxygen
bond is disfavored, which was accounted for by stereo-electronic effects (Coulembier et al. 2006a).
Another mechanism based on the scission of the alkyl-oxygen bond is then observed (Figure 1.6).
O O O
O O– Cyclic oligomer + O–
O 5 n 5 O 5 m O 5 n
FIGURE 1.4 Intramolecular transesterification reactions during the polymerization of ɛCL.
O O O
–
O + O
O 5 n O 5 m 5 O 5 p
O O
O –
+ O
O 5 n 5 O 5 p O 5 m
O O
FIGURE 1.5 Intermolecular transesterification reactions during the polymerization of ɛCL.
O O
R
O O O–
R O–
FIGURE 1.6 Ring-opening polymerization of β-lactones by cleavage of the alkyl-oxygen bond.

R O O
R O O–
+ +
O O– O R=H, Me O OH O
n R n R
Intramolecular
elimination reaction
O
RHC
O–
FIGURE 1.7 Transfer reaction for the polymerization of β-lactones.
Interestingly enough, ring-opening affords a carboxylate anion rather than an alkoxide. This polym-
erization can thus also be initiated by carboxylic salts even though they are less nucleophilic than
alkoxides (Penczek 2007).
It is worth noting that another transfer reaction mechanism shown in Figure 1.7 is reported as far
as β-lactones are concerned (Penczek 2007).
1.3.2 Coordination Polymerization
Anionic metal alkoxides are too reactive to be selective and transfer transesterification reactions are
very difficult to get rid of, at the expense of control of polymerization. In order to improve the con-
trol of polymerization, it is mandatory to use less reactive initiators. This can be achieved by playing
with steric and electronic effects (Lecomte and Jérôme 2004). The use of cumbersome ligands is the
first possible route to obtain more selective species. The most common approach relies on the use of
less electrophilic metals. The works of Teyssié and coworkers who used bimetallic μ-oxo-alkoxides
as initiators have to be mentioned (Hamitou et al. 1973, Ouhadi et al. 1976). Since then, a wide range
of transition metals have been investigated.
Aluminum alkoxides occupy a key position because of the very good control imparted to the
polymerization of cyclic esters. Commercially available aluminum triisopropoxide is a widely used
aluminum alkoxide. Firstly, the three alkoxide bonds initiate polymerization. Nevertheless, the
behavior of aluminum triisopropoxide is complicated due to its aggregation in solution. Indeed,
aluminum isopropoxide exists as a mixture of trimers (A3) and tetramers (A4; Ropson et al. 1995).
As long as polymerization of ɛCL is carried out by aluminum isopropoxide in toluene at 0°C,
only A3 is prone to initiate polymerization. The interconversion of A4 into A3 is slow compared
to the kinetics of polymerization, and A4 does not initiate polymerization under these conditions.
Conversely, polymerization of lactide is carried out at a higher temperature, typically at 70°C. The
interconversion of A4 into A3 is faster, and all species initiate polymerization. It is thus very impor-
tant to take into account the equilibrium between A3 and A4 species to determine the theoretical
molar mass on the basis of the monomer-to-initiator molar ratio. Last but not least, it is worth noting
that aluminum alkoxides can be prepared in the laboratory by the reaction of any alcohol with
triethylaluminum or aluminum isopropoxide (Dubois et al. 1989).
The mechanism of polymerization is quite similar to that of anionic polymerization. Nevertheless,
one has to take into account the coordination of the metal with oxygen atoms. The mechanism
shown in Figure 1.8 is referred in the state of the art as the coordination–insertion mechanism.
Firstly, the alkoxide (RO-M) coordinates with the carbonyl of the cyclic ester, followed by the
nucleophilic addition of the alkoxide onto the electrophilic C=O bond. Thereafter, ring-opening
takes place by an elimination reaction, resulting in the cleavage of the acyl-oxygen bond and in the
formation of a new alkoxide. The initiator can contain one functional group, provided it is tolerated
by aluminum alkoxide species (Dubois et al. 1989).
M
O R O O R O OM
O O O
RO M +
O O O O
n M M
O OR OR
M O O
O 5 5 OR
O n
FIGURE 1.8 Coordination–insertion mechanism for the ring-opening polymerization of cyclic esters.
During the propagation of the ring-opening polymerization of cyclic esters initiated by alu-
minum alkoxides, aggregation can again take place depending upon stereoelectronic factors, as
shown by NMR spectroscopy and kinetic studies (Duda and Penczek 1991). For instance, during
the polymerization of ɛCL, the three-arm growing species is a unimer when initiated by A3 and is a
trimer when initiated by Et2AlOR (Ropson et al. 1995).
Although aluminum alkoxides exerted an excellent control to the polymerization of cyclic esters,
they are suspected to be involved in Alzheimer’s disease. Moreover, catalytical remnants are very
difficult to withdraw and these toxicity issues are thus a huge limitation for biomedical applications.
Many researchers used tin(II) bis-(2-ethylhexanoate), also commonly referred to as tin octoate,
instead of aluminum alkoxides because of its recognition by the American Food and Drug
Administration (FDA). Another reason for the popularity of tin(II) bis-(2-ethylhexanoate) is its
lower sensitivity to water and other impurities. It is thus easier to achieve polymerization in labora-
tories or in industry. Nevertheless, it was found that tin(II) bis-(2-ethylhexanoate) is also cytotoxic
and should ideally be avoided for the synthesis of biomaterials.
Tin(II) bis-(2-ethylhexanoate) does not contain any alkoxide bond, and the mechanism remained
unclear for a long time and several proposals were reported. In 1998, Penczek and coworkers
reported that tin alkoxides are formed in the polymerization medium by the reaction of tin(II) bis-
(2-ethylhexanoate) with traces of water or with any other protic impurities. Polymerization thus
takes place by the usual coordination–insertion mechanism at least as long as it is carried out in
THF at 80°C (Kowalski et al. 1998). This proposal was substantiated by kinetic measurements
(Kowalski et al. 1998, 2000a), analysis of the chain-ends by MALDI-TOF (Kowalski et al. 2000b),
and proton-trapping agent experiments (Majerska et al. 2000).
It is worth noting that other tin-based initiators are also used. Hedrick and coworkers proposed
to use tin trifluoromethanesulfonate to improve the kinetics of polymerization (Möller et al. 2000).
Another possibility relies on the use of Sn(II) alkoxides (Duda et al. 2000, Kowalski et al. 2000c)
and Sn(IV) alkoxides (Kricheldorf and Eggerstedt 1998, Kricheldorf et al. 2001, Kricheldorf 2004).
Penczek and coworkers reported that the polymerization of lactide, initiated by Sn(OBu)2, is under con-
trol in a range of molar masses up to 106 g/mol (Duda et al. 2000, Kowalski et al. 2000c). Nevertheless,
whatever the tin derivative used to initiate polymerization, toxicity remains an issue. Albertsson and
coworkers proposed a smart approach to extract tin derivatives (Stjerndahl et al. 2007). They initiated
the polymerization of cyclic esters by tin(IV) alkoxides before adding 1,2-ethanedithiol to the polym-
erization medium to afford a sulfur-containing dibutyltin derivative, which can be extracted because
of its high solubility in organic solvents. For example, this process allowed synthesizing a sample of
PCL contaminated by only 23 ppm of tin residues (Stjerndahl et al. 2007).
If ring-opening polymerization can be carried out in bulk and in organic solvents, supercritical
carbon dioxide is also a valuable medium. The technology based on supercritical carbon dioxide is
particularly interesting in the field of biomaterials because of the remarkable extraction properties
of this medium with the prospect to purify aliphatic polyesters. Ring-opening polymerization of
ɛCL in supercritical carbon dioxide was first reported by Mingotaud and coworkers (Mingotaud
et al. 1999, 2000). Then, Jérôme and coworkers observed that tin(IV) alkoxides initiate the con-
trolled ring-opening polymerization of ɛCL in supercritical CO2 (Stassin et al. 2001). Slow kinetics
was accounted for by the reversible reaction of tin alkoxides and carbon dioxide (Stassin and Jérôme
2002). PCL is not soluble in supercritical carbon dioxide, and the polymerization of ɛCL is thus
heterogeneous. Nevertheless, in the presence of a suitable surfactant, nanoparticles can be obtained
(Stassin and Jérôme 2004). Last but not least, supercritical CO2 was used to withdraw quantitatively
unconverted monomer and metallic remnants.
The issues related to the toxicity of tin and aluminum derivatives urged researchers to initiate
the polymerization of cyclic esters by alkoxides based on less toxic metals. In this regard, bismuth
(Kim et al. 2004), magnesium (Shueh et al. 2004, Yu et al. 2005b), and calcium (Zhong et al. 2001,
Westerhausen et al. 2003) alkoxides are reported. The mechanism remains the usual coordination–
insertion mechanism. The kinetics and control of polymerization depend on the nature of the metal
and on its ligands.
So many metallic derivatives have been used to initiate or catalyze the ring-opening polymer-
ization of cyclic esters that it is almost impossible to list all. Among these derivatives, zinc octoate
(Libiszowki et al. 2002, Kowalski et al. 2007), aluminum acetyl acetonate (Kowalski et al. 2007),
scandium trifluoromethanesulfonate (Möller et al. 2000, Nomura et al. 2000), and scandium trifluo-
romethanesulfonimide [Sc(NTf2)3] (Oshimura and Takasu 2010) can be mentioned. Special atten-
tion has to be paid to lanthanides alkoxides (Metal = Er, Sm, Dy, La) as initiators because of the
very fast kinetics of polymerization (McLain and Drysdale 1992). Shen et al. (1996) showed that the
increase of steric hindrance of the ligand disfavors transesterification reactions. Yasuda and cowork-
ers polymerized ɛCL by SmOEt(C5Me5)2(OEt2), [YOMe(C5H5)2]2, and YOMe(C5Me5)2(THF)
(Yamashita et al. 1996). In 1996, yttrium isopropoxide was obtained “in situ” by the reaction of
isopropanol and yttrium tris(2,6-di-tert-butylphenolate (Stevels et al. 1996a,b). Then, a similar
approach was implemented by Jérôme and Spitz who synthesized Y(OiPr)3 (Martin et al. 2000,
2003a) and Nd(OiPr)3 (Tortosa et al. 2001) by the reaction of isopropanol with Y[N(SiMe3)2]3 and
Nd[N(SiMe3)2]3, respectively. In 2003, the polymerization of ɛ-caprolactone was carried out by
La(OiPr)3 (Save et al. 2002) and M(BH4)3(THF)3 (M = Nd, La, Sm) (Guillaume et al. 2003, Palard
et al. 2005). Cyclic esters other than ɛCL can be polymerized by the same family of lanthanide
alkoxides (Yamashita et al. 1996). Last but not least, Jérôme and coworkers grafted yttrium isop-
ropoxide onto a porous silica surface (Martin et al. 2003b,c). Two methods of immobilization were
reported. Firstly, the hydroxyl groups located on the surface of silica were allowed to react with an
excess of Y[N(SiMe3)2]3 into silylamido groups, which were finally allowed to react with 2-propanol
to obtain yttrium alkoxides. Secondly, Y[N(SiMe3)2]3 was made to react with less than three equiva-
lents of 2-propanol into an yttrium alkoxide, which was then grafted onto the surface. Hamaide and
coworkers supported alkoxides based on other metals (Al, Zr, Sm, and Nd) onto silica and alumina
(Miola-Delaite et al. 2000).
1.3.3 Metal-Free Ring-Opening Polymerization

Aluminum and tin alkoxides being too toxic for biomedical applications, chemists investigated
original metal-free processes for the ring-opening polymerization of cyclic esters. Their strategy
relies on the initiation of the polymerization by nucleophilic species such as alcohols and
amines. Nevertheless, these species are in general not nucleophilic enough to react with cyclic
esters. Nevertheless, there are exceptions to this general rule. For instance, highly reactive β-lactones
are polymerized by nucleophilic amines in the absence of any catalyst. Polymerization initiated by
tertiary amines is known as zwitterionic polymerization in the literature (Löfgren et al. 1995).
The mechanism shown in Figure 1.9 is based on the reaction of tertiary amines and cyclic esters
O
O
O R΄ R΄
O R΄ O–
O– N+ O N+
+ N R΄ R΄ R΄
R΄ R΄
R΄ O O O
n
Zwitterion
FIGURE 1.9 Zwitterionic polymerization of pivalolactone.
O H
RO
R O O
O H+
O +
O _H+
n H n OH
n
FIGURE 1.10 Ring-opening polymerization of cyclic esters catalyzed by acids and initiated by alcohols.
with a zwitterionic species made up of an ammonium cation and a carboxylate anion. Interestingly
enough, Kricheldorf et al. (2005) mentioned the possibility that chain extension takes place by step-
growth polycondensation at least at some stage of polymerization.
As a rule, alcohols and amines are in general not nucleophilic enough to react with cyclic esters
and the reaction has thus to be catalyzed. In order to do so, two main strategies might be imple-
mented. They rely on the activation of either the monomer or the initiator. Last but not least, some
processes combine both mechanisms of activation.
Cyclic esters can be activated by either acids or nucleophiles to allow their reaction with alcohols
and amines in the frame of a polymerization process. As far as acids are concerned as catalysts, the
mechanism of ring-opening polymerization relies on the activation of cyclic esters by protonation
of the exocyclic oxygen of cyclic diesters, which facilitates the reaction with the nucleophilic spe-
cies, that can be the initiator during initiation or the hydroxyl-end capped chain during propaga-
tion (Figure 1.10). Polymerization takes place by the scission of the oxygen-acyl bond. In 2000, an
example of ring-opening polymerization of ɛCL and δVL was reported by Endo and coworkers
by using alcohol as an initiator and HCl.Et2O as a catalyst (Shibasaki et al. 2000). Polymerization
was under control but the molar mass did not exceed 15,000 g/mol. It is worth noting that, later on,
Jérôme and coworkers reported that molar masses up to 50,000 g/mol were obtained as far as PVL
was polymerized (Lou et al. 2002a). Recently, the process was extended to a wider range of acids.
Trifluoromethanesulfonic is an efficient catalyst for the controlled ring-opening polymerization of
lactide (Bourissou et al. 2005) and ɛ-caprolactone (Basko and Kubisa 2006). Later, Bourrissou
showed that trifluoromethanesulfonic acid can be substituted for less acidic methanesulfonic acid
for the polymerization of ɛCL (Gazeau-Bureau et al. 2008). Polymerization of δVL was catalyzed
by trifluoromethanesulfonimide according to Kakuchi et al. (2010). Interestingly enough, polymer-
ization was under control and various functionalized alcohols were used as initiators. Very recently,
Takasu and coworkers extended this strategy to nonafluorobutanesulfonimide for the polymeriza-
tion of ɛCL (Oshimura et al. 2011). Interestingly enough, organic catalysts such as lactic acid (Casas
et al. 2004, Persson et al. 2006), citric acid (Casas et al. 2004), fumaric acid (Sanda et al. 2002, Zeng
et al. 2005), and amino acids (Casas et al. 2004) were also used. In addition, the acid catalyst can
be supported on silica (Wilson and Jones 2004). Amino acids exhibit a particular behavior because
they both catalyze and initiate polymerization (Liu and Liu 2004).
In the absence of any nucleophilic species such as an alcohol, the only nucleophilic species
remaining in the system is the cyclic ester. This is the typical case of cationic ring-opening polym-
erization of cyclic esters, which has been in use for a long time. The cationic polymerization can be
catalyzed not only by Bronsted acids but also by alkylating agents, acylating agents, and Lewis acids.
O O O
O + O
+
C C
O R O O R
O 5
+ +
R n
FIGURE 1.11 Mechanism for cationic ring-opening polymerization based on the reaction of the endocyclic
oxygen.
For instance, in 1984, Penczek reported the cationic polymerization of ɛCL and βPL by acylating
agents (Hofman et al. 1984). For a long time it was accepted that cationic ring-opening polymeriza-
tion mediated by alkylating agents takes place by the mechanism shown in Figure 1.11, which is
based on the reaction of the cation with the endocyclic oxygen followed by the cleavage of the acyl-
oxygen bond. In 1984, Penczek (Hofman et al. 1984) and Kricheldorf et al. (1986) proposed that the
cation reacts with the exocyclic oxygen rather than the endocyclic oxygen to afford a dialkoxycarbo-
cationic species, which finally reacts by cleavage of the alkyl-oxygen bond (Figure 1.12). It is worth
noting that both mechanisms shown in Figures 1.12 and 1.13 are observed when acylating agents are
used (Slomkowski et al. 1985).
In recent years, the ring-opening polymerization of cyclic esters by nucleophilic catalysts has
emerged as a very promising process under the impulse given by the group of Hedrick (Kamber
et al. 2007). Table 1.3 shows several nucleophiles known to activate cyclic esters and thus prone
to catalyze the ring-opening polymerization of cyclic esters initiated by alcohols by the mecha-
nism shown in Figure 1.13. Among nucleophilic species, N-heterocyclic carbenes (Table 1.3, entries
1–7), amines (Table 1.3, entries 8–11), and phosphines (Table 1.3, entries 12–17) can be mentioned.
Depending upon the cyclic ester that has to be polymerized, the nucleophilic catalyst must be care-
fully selected because its nucleophilicity and thus the kinetics of this ring-opening is influenced by
steric and electronic effects. Interestingly enough, polymerization is under control. Although this
approach is very promising, more work is needed to assess the impact of these catalysts on the bio-
compatibility of aliphatic polyesters.
Another mechanism relies on the activation of the initiator. Bases that are able to activate nucleo-
philic alcohols and catalyze the ring-opening polymerization of cyclic esters are shown in Table 1.4.
TBD and DBU catalyze the ring-opening polymerization of cyclic esters (Table 1.4, entries 1, 2).
R R
O O O
O
C+
O O O O
R+ +
O C+
O
FIGURE 1.12 Mechanism for cationic ring-opening polymerization based on the reaction of the exocyclic
oxygen.
O
O O
O R OH
Nu + + O– OH
Nu 5 RO 5
FIGURE 1.13 Activation of cyclic esters by nucleophiles.

TABLE 1.3
Nucleophilic Catalysts for the Ring-Opening Polymerization of Lactones
Entry Organocatalyst Monomer Initiator Reference
1 l-lactide EtOH, pyrenebutanol Connor et al. (2002)
N N εCL EtOH, pyrenebutanol
C
βBL EtOH, pyrenebutanol
rac-lactide — Dove et al. (2006)
meso-lactide — Dove et al. (2006)
2 εCL PhCH2OH Nyce et al. (2003)
N N δVL PhCH2OH
C
3 βBL PhCH2OH Nyce et al. (2003)

N N
C δVL PhCH2OH
εCL PhCH2OH
4 εCL PhCH2OH Nyce et al. (2003)
N N
C
5 Ph l-lactide MeOH, pyrenebutanol, Coulembier et al. (2005)
PEO-OH
N
N N βBL pyrenebutanol Coulembier et al. (2006b)
Ph C Ph
6 l-lactide — Connor et al. (2002)
N N
OR
7 Ph Ph rac-lactide — Dove et al. (2006)
meso-lactide —
N N
C
7 Ph Ph rac-lactide — Dove et al. (2006)

meso-lactide —
H N N H
Me Ph
C
Ph Me
8 Lactide EtOH, PhCH2OH Nederberg et al. (2001)
N N
DMAP
9 Lactide EtOH, PhCH2OH Nederberg et al. (2001)
N N
10 N Lactide Pyrenebutanol Pratt et al. (2006) and

Lohmeijer et al. (2006)
N N
MTBD
11 N Lactide Pyrenebutanol Lohmeijer et al. (2006)
N
DBU
(continued)
TABLE 1.3 (continued)

Nucleophilic Catalysts for the Ring-Opening Polymerization of Lactones
12 PBu3 Lactide PhEtOH Myers et al. (2002)
13 PPhMe2 Lactide PhEtOH Myers et al. (2002)
14 PPh3 Lactide PhEtOH Myers et al. (2002)
15 Lactide PhEtOH Myers et al. (2002)
16 Et Lactide PhEtOH Myers et al. (2002)
P
Et
Fe
Et
P
Et
17 Lactide PhEtOH Myers et al. (2002)
Pipr2
Fe
Pipr2
18 PCy2 Lactide PhEtOH Myers et al. (2002)
PCy2
Fe
The mechanism is pseudoanionic and is based on the activation of the alcohol by attracting the
proton. Phosphazene bases are also prone to catalyzing the ring-opening polymerization of cyclic
esters (Zhang et al. 2007a). In particular, 2-tert-butylimino-2-diethylamino-1,3-dimethylper-
hydrdro-1,3,2-diazaphosphorine (BEMP) is efficient (Table 1.4, entry 3). It is worth noting that
BEMP exhibits a higher basicity (pK BH+ = 27.6) compared to DBU (pK BH+ = 24.3) and MTBD
(pK BH+ = 25.4). As long as a dimeric phosphazene is used to catalyze the polymerization of racemic
lactide (Table 1.4, entry 4), isotactic polylactide is obtained (Zhang et al. 2007b).
Several catalysts have the ability to activate both the monomer and the initiator at the same
time (Table 1.5). 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) is an efficient organocatalyst for the
ring-opening polymerization of cyclic esters initiated by alcohols (Table 1.5, entry 1), and the
polymerization takes place by dual activation of both the monomer and the initiator. The activa-
tion of the monomer can take place either by an acyl transfer mechanism or by a mechanism
based on hydrogen bonding. This last mechanism was preferred as far as the polymerization
of l-lactide is concerned (Chuma et al. 2008). As long as 1,8-diaza[5.4.0]bicycloundec-7-ene
(DBU) and N-methylated TBD (MTBD) were used as catalysts instead of TBD, the polymer-
ization of lactide was slower. Polymerization of CL and VL did not take place. Indeed, these
experimental data can be easily understood if one takes into account that these catalysts can
only activate the alcohol but not cyclic esters (Lohmeijer et al. 2006). However, a possible trick
to overcome this issue relies on the addition of a thiourea activating cyclic esters by hydrogen
TABLE 1.4
Basic Catalysts for the Ring-Opening Polymerization of Lactones
1 Lactide 1-Pyrenebutanol Pratt et al. (2006) and
N Lohmeijer et al. (2006)
N N
MTBD
2 Lactide 1-Pyrenebutanol Lohmeijer et al. (2006)
N
N
DBU
3 l-lactide 1-Pyrenebutanol Zhang et al. (2007a)
δVL 1-Pyrenebutanol Zhang et al. (2007a)
N N εCL 1-Pyrenebutanol Zhang et al. (2007a)
P
Et2N
N
tBu
4 l-lactide 1-Pyrenebutanol Zhang et al. (2007a)
N N
N P N P N tBu
N N
bonding (Table 1.5, entry 2). Interestingly enough, both catalytical species can be combined in
a single molecule. Indeed, Hedrick and coworkers reported that the polymerization of cyclic
esters is efficiently catalyzed by 1-[3,5-bis(trifluoromethyl)phenyl]-3-[2-(dimethylamino)cyclo-
hexyl]thiourea with a very good control of the molecular parameters (Table 1.5, entry 3; Dove
et al. 2005). Polymerization takes place also in the presence of both 1-[3,5-bis(trifluoromethyl)
phenyl]-3-cyclohexylthiourea and N,N-dimethylcyclohexanamine (Table 1.5, entry 4; Dove et al.
2005). Whenever only one of these is present, no polymer was obtained, in agreement with a
dual activation mechanism.
1.3.4 Enzymatic Ring-Opening Polymerization

Enzymes are very promising nontoxic catalysts for the preparation of biomaterials. They are
green catalysts obtained from renewable resources and are easily separated from polyesters.
Among enzymes, lipases are known to catalyze the hydrolysis reaction of esters. Chemists used
these natural enzymes to catalyze the reverse reaction, for example, the esterification reaction.
The ring-opening polymerization of cyclic esters can be catalyzed by lipases as independently
discovered in 1993 by the groups of Kobayashi (Uyama and Kobayashi 1993) and Knani et al.
(1993). Since then, a wide variety of cyclic esters of different size were polymerized. Several
reviews have been published on this topic (Gross et al. 2001, Varma et al. 2005, Albertsson and
Srivastava 2008, Kobayashi and Makino 2009). Among the different lipases, Candida antarctica
(lipase CA, CALB or Novozym 435) is widely used. An alcohol can purposely be added in the
reaction medium to initiate polymerization. The course of polymerization is influenced by water.
TABLE 1.5
Dual Catalysts for the Ring-Opening Polymerization of Lactones
1 N Lactide Pyrenebutanol Pratt et al. (2006)
δVL Pyrenebutanol Lohmeijer et al. (2006)
N N εCL Pyrenebutanol Lohmeijer et al. (2006)
TBD
2 CF3 δVL Pyrenebutanol Lohmeijer et al. (2006)
εCL Pyrenebutanol Lohmeijer et al. (2006)
N S
+
N F3C N N
DBU H H
3 CF3 Lactide Pyrenebutanol Dove et al. (2005)
S
rac
F3C N N
H H
NMe2
4 CF3 Lactide Pyrenebutanol Dove et al. (2005)
S
+
F3C N N
H H
NMe2
Indeed, a minimum amount of water needs to be bound to the surface of the enzyme to maintain
its conformational flexibility and catalytic activity (Gross et al. 2001). Nevertheless, water also
initiates this polymerization. The synthesis of high molar mass aliphatic polyesters by enzymatic
ring-opening polymerization is thus difficult because it is not possible to carry out polymerization
in strictly anhydrous conditions. In terms of the control of polymerization, enzymatic polym-
erization cannot compete with coordination or organocatalytic polymerization. Nevertheless,
enzymatic polymerization has some practical interests. Polymerization proceeds under mild con-
ditions in terms of pH, temperature, and pressure and can be carried out in bulk, in organic media,
and even in supercritical carbon dioxide (Takamoto et al. 2001, Loeker et al. 2004). Moreover,
enzymes are able to catalyze the ring-opening polymerization of large-membered cyclic esters,
which are very difficult to polymerize by chemical catalysts and initiators such as metal alkoxides
and organocatalysts (Duda et al. 2002).
The mechanism of enzymatic polymerization is based on the activation of the monomer and is
very similar to the one already shown for nucleophilic organocatalysts. Briefly, a complex is formed
between the enzyme and cyclic esters. The hydroxyl group of a serine residue of the active site of the
enzyme reacts with cyclic esters affording an activated open form of the monomer. This activated
intermediate then reacts with an alcohol, which can be the initiator or a hydroxyl-end capped chain.
This mechanism is summarized in Figure 1.14.
1.3.5 Polymerization of Substituted and Functionalized Cyclic Esters

Although PCL, PLA, and PGA are widely used for biomedical applications, it is highly desirable
to synthesize a wider range of aliphatic polyesters in view of the development of novel biomedical
applications. Two main routes were investigated for this application: the first one relies on the direct
O
Lipase
O Lipase-OH O
Lipase/OH complex
n O n OH
R OH OR
– Lipase-OH O n OH
FIGURE 1.14 Enzymatic polymerization of cyclic esters.
chemical modification of aliphatic polyesters, and the second one is based on the synthesis and
polymerization of substituted and/or functionalized cyclic esters.
The direct functionalization of aliphatic polyesters is the most straightforward route. Vert and
coworkers functionalized PCL by a two-step process. Firstly, the metallation of PCL was carried out
by lithium diisopropylamide in order to obtain a poly(enolate), which was then reacted with any elec-
trophile such as naphthoyl chloride (Ponsart et al. 2000), benzylchloroformate (Ponsart et al. 2000),
acetophenone (Ponsart et al. 2000), benzaldehyde (Ponsart et al. 2000), carbon dioxide (Gimenez
et al. 2001), tritiated water (Ponsart et al. 2001), α-bromoacetoxy-ω-methoxy-poly(ethylene oxide)
(Ponsart et al. 2002), and iodine (Nottelet et al. 2006; Figure 1.15).
This approach is very simple, and many functionalized PCL can be synthesized just by
changing the nature of the electrophile. Moreover, the absence of any potentially toxic cata-
lyst is a huge advantage. Unfortunately, this strategy is very touchy because degradation of
aliphatic polyesters by nucleophilic enolates always takes place at a significant level. Under
optimized conditions, degradation can only be limited. Although the process was extended to
the functionalization of PLA, degradation is even more difficult to limit, this polymer being
more sensitive than PCL. Moreover, the content of functionalization is quite low (<30%) even
under optimized conditions.
4 O x
LDA
OLi
4 O x
Cl
H Cl O
MeI
O Ph O OBn
O O
O O
O
O 4 x
4 O 4 O x
4 x
x
O OBn O
HO Ph
FIGURE 1.15 Functionalization of PCL according to Vert and coworkers.

The important limitations of the process shown in Figure 1.15 prompted chemists to investigate a
less direct approach based on the synthesis and polymerization of functionalized cyclic esters (Lou
et al. 2003). For that sake, two strategies can be applied. The first one relies on the insertion of the
functional group inside the ring, and the second one is based on the substitution of the cyclic ester
by a pendent functionalized group.
Table 1.6 shows the conditions used for the polymerization of cyclic esters bearing, inside the
ring, functional groups such as an ether (Table 1.6, entries 1–3), a protected amine (Table 1.6,
entries 4,5), an unsaturation (Table 1.6, entries 6,7), a ketone (Table 1.6, entry 8), and an amide
(Table 1.6, entries 9–12) are present.
Table 1.7 shows cyclic esters bearing a substituent, functionalized or not. As functional groups,
aromatics (Table 1.7, entry 1), chloride (Table 1.7, entries 2,3), bromide (Table 1.7, entries 4–6),
iodide (Table 1.7, entry 7), alkyne (Table 1.7, entries 8, 9), alkene (Table 1.7, entries 10–15), and
epoxide (Table 1.7, entry 16) can be mentioned.
Cyclic esters reported in Tables 1.6 and 1.7 are usually polymerized by tin and aluminum
alkoxides. It is worth noting that 6,7-dihydro-2(3H)-oxepinone is an unusual case because this
monomer can be polymerized by another mechanism, the ring-opening metathesis polymerization
(ROMP) process by using the Schrock’s catalyst (Figure 1.16; Lou et al. 2002c).
In terms of chemoselectivity, enzymes are appealing catalysts because they tolerate a wide range
of functional groups and even epoxides (Table 1.7, entry 16). Unfortunately, tin and aluminum alk-
oxides are less tolerant and do not tolerate the presence of alcohols, carboxylic acids, and epoxides.
Noteworthily, ketones are tolerated by tin (IV) alkoxides but not by aluminum alkoxides for reasons
which still remain unclear (Latere et al. 2002). This issue was overcome by protecting these func-
tional groups prior to polymerization. Table 1.8 shows lactones substituted by protected ketones
(Table 1.8, entry 1), alcohols (Table 1.8, entries 2–6), diols (Table 1.8, entry 7), carboxylic acids
(Table 1.8, entries 8–15), and amines (Table 1.8, entries 16–19). The choice of the protection group
is essential for the success of this strategy. One the one hand, the protected functional groups have
to be stable enough to avoid any degradation prior to polymerization and to allow obtaining the
monomer in a very high purity, which is a prerequisite to achieve polymerization, at least as far
as sensitive initiators are concerned, such as aluminum alkoxides. On the other hand, the protec-
tion group has to be removed after polymerization under conditions that prevent degradation from
occurring. For this reason, benzylic alcohols and carboxylic esters are widely used because they can
be deprotected under neutral conditions rather than in acidic conditions. As a rule, these two condi-
tions are contradictory and it is not always that easy to find a satisfactory compromise between the
stability of the monomer and the easy deprotection.
Many of the cyclic esters shown in Tables 1.6 through 1.8 are chiral and possess at least one chi-
ral carbon (R or S). Although the stereoselective polymerization of lactide (Zhong et al. 2004) and
β-butyrolactone (Amgoune et al. 2006, Carpentier 2010) is reported in the literature, the stereoselectiv-
ity of their polymerization is in general not discussed and racemic mixtures are polymerized. Obviously,
more attention should be paid to these aspects in the future because stereoselective polymerization
allows the synthesis of aliphatic polyesters with different tacticities, and thus of different properties.
Last but not least, it must be noted that substituted cyclic esters can sometimes be expensive
because their synthesis can require several steps from commercially available compounds at the
expense of the global yield, which can be low. It is beyond the scope of this chapter to describe all
the syntheses of these functionalized cyclic esters, and the reader is invited to read the references
given in Tables 1.6 through 1.8. Another limitation relies on the necessity to synthesize a new cyclic
ester for any new functional aliphatic polyester.
The functionalization of aliphatic polyesters opens up new avenues in the field of biomaterials.
The presence of functional groups allows modifying the biodegradation rate by changing the crys-
tallinity and hydrophilicity or by adding functional groups such as carboxylic acids prone to catalyze
the degradation. Interestingly enough, pH-sensitive aliphatic polyesters can be obtained by grafting
carboxylic acids or amines along aliphatic polyesters. These groups can be under a neutral or an
TABLE 1.6
Ring-Opening of Cyclic Monoesters Functionalized inside the Ring
Entry Monomer Initiator Reference
1 O Bu2SnO, Sn(Oct)2 Mathisen and Albertsson (1989)
and Mathisen et al. (1989)
Al(OiPr)3 Löfgren et al. (1994)
O
O
2 O Et3Al, H2O Shirahama et al. (1993, 1996)
O
R
Me
O
3 O Zn(II) l-lactate Kricheldorf and Damrau (1998)
Al (OiPr)3 and Raquez et al. (2000)
4 O ROH/Sn(Oct)2 Trollsas et al. (2000)
N
O
CF3
5 O DBU Kudoh et al. (2009)
O
N
Ph
6 O Al(OiPr)3 Lou et al. (2001)
7 O Al(OiPr)3 Lou et al. (2002b)

Schrock’s catalyst Lou et al. (2002c)
(continued)

Ring-Opening of Cyclic Monoesters Functionalized inside the Ring
8 O l-Phenyl-2-propanol, Latere et al. (2002)
Sn(Oct)2
O
O
9 O O Porcine pancreatic lipase Feng et al. (1999a, 2000)
Lipase type XIII from Feng et al. (1999a,b)
Pseudomonas species
O N Lipase from Feng et al. (1999a,b)
Pseudomonas cepacia
Lipase type VII from Feng et al. (1999b)
Candida rugosa
10 O O Porcine pancreatic lipase Feng et al. (2000)
O N
O N
O N
ionic form depending upon the pH and their pKa, which affects their solubility in water. Another
application relies on the use of these functional groups for the covalent grafting of biologically active
molecules, drugs, targeting units, and other functional groups. For that sake, very efficient reactions
have to be used under nondegrading conditions. It is not very easy to find a reaction that meets these
criteria (Lecomte et al. 2006). Recently, it turned out that the click copper-catalyzed alkyne-azyde
Huisgen’s cycloaddition reaction (CuAAC) is particularly efficient (Parrish et al. 2005, Riva et al.
2007, Lecomte et al. 2008) in the presence of limited degradation (Lecomte et al. 2008). Moreover,
the reaction can take place in water and in organic solvents. Nevertheless, the use of copper salts
as catalysts is a severe limitation for biomedical applications. Although they are not as efficient as
the CuAAC reaction, several other metal-free reactions have been used such as the esterification
reaction between alcohols and carboxylic acids (Renard et al. 2003, Parrish and Emrick 2004), the
ring-opening of epoxides by thiols (Lou et al. 2002b), the thiol-ene reaction (Rieger et al. 2005), and
the coupling of ketones and oxyamines (Taniguchi et al. 2005, van Horn et al. 2008).
TABLE 1.7
Ring-Opening Polymerization of Functionalized Cyclic Esters
1 O 4-tBu-C6H4-CH2OH, Sn(Oct)2 Simmons and Baker
(2001)
O
O
2 Cl Sn(Oct)2 Liu et al. (1999)

Al(OiOr)3 Liu et al. (1999)
O
Pyridine, Et3N Liu et al. (1999)
CF3CO2H Liu et al. (1999)
O
3 O 2,2-Dibutyl-2-stanna-1,3- Lenoir et al. (2004)

Cl dioxepane
4 O Al(OiPr)3 Mecerreyes et al. (1999)
O
O
Br
5 O Al(OiPr)3 Detrembleur et al. (2000)
Br
6 O Al(OiPr)3 (copolymerization Wang et al. (2005)

Br with εCL)
(continued)

7 O MeOH, Sn(Oct)2 El Habnouni et al. (2009)
I
8 O EtOH, Sn(OTf)2 Parrish et al. (2005)
9 O 4-tBu-C6H4-CH2OH, Sn(Oct)2 Jiang et al. (2008)
10 O Al(OiPr)3 Mecerreyes et al. (2000a)
O
O
11 O PhCH2OH, Sn(Oct)2 Mecerreyes et al. (2000b)
12 O MeO-[Y] (copolymerization Ajellal et al. (2009)

O with βBL)

13 O PhCH2OH, Sn(Oct)2 Leemhuis et al. (2008)
14 O 2,2-Dibutyl-2-stanna-1,3- Li et al. (2006)

O dioxepane (block
O copolymerization with εCL)
O
15 O Novozym 435 Veld et al. (2007)

O
16 O Novozym 435 Veld et al. (2007)

O
N
CMe2Ph
O Mo
CH3(CF3)2OC O
OC(CF3)2CH3
O
ROMP O
n
FIGURE 1.16 Polymerization of 6,7-dihydro-2(3H)-oxepinone by ROMP.

TABLE 1.8
Ring-Opening Polymerization of Cyclic Esters Bearing Protected Functional Groups
1 O Al(OiPr)3 Tian et al. (1997)
2 O Sn(Oct)2 (copolymerization with δVL Pitt et al. (1987)

and a dilactone)
TBDMSO
3 O Al(OiPr)3 (copolymerization with εCL Stassin et al. (2000)
and TOSUO)
Et3SiO
4 O ROH, Sn(Oct)2 Trollsas et al. (2000)
PhCH2O
5 O Leemhuis et al. (2006)
N
O Zn
O
O iPrOH,
O
OBn
6 O Leemhuis et al. (2006)
N
Zn
O O
O iPrOH,
O
OBn

O O
O O
Ph
O
OBn
O
OtBn
10 O Al(OiPr)3 (copolymerization Lecomte et al. (2000)
with εCL)
O
OTBDMS
11 O Tetraethylammonium benzoate Bizzari et al. (2002)
O
OBn
O
(continued)

12 O Tetraethylammonium benzoate Barbaud et al. (2004)
O
OBn
O
13 O PEO-OH, Sn(Oct)2 Mahmud et al. (2006)
O
OBn
O
14 O PhCH2OH, Sn(Oct)2 Gerhardt et al. (2007)
O O
O
n
OBn
O n = 1,2
15 O Al(OiPr)3, Sn(Oct)2, Et2zn Kimura et al. (1988)
O
O O
16 O Tetrabutylammonium acetate Flétier et al. (1990)
O
TrN
17 O iPrOH, Sn(Oct)2 Blanquer et al. (2010)
HN OBn
O

18 O Sn(Oct)2 (copolymerization with εCL) Yan et al. (2010)
O
HN OBn
O
19 O PhCH2OH, Sn(Oct)2 Gerhardt et al. (2007)
O
H
O N OBn
4
O O
1.4 RADICAL RING-OPENING POLYMERIZATION OF CYCLIC KETENE ACETALS

Although the ring-opening polymerization of cyclic esters is a very widely used technique to
prepare aliphatic polyesters, special attention has to be paid to a less common process based on
the ring-opening polymerization of cyclic ketene acetals (Bailey 1985, Sanda and Endo 2001,
Agarwal 2010). For instance, the polymerization of ɛCL and 2-methylidene-1,3-dioxepane affords
an aliphatic polyester with the same chemical structure. Nevertheless, the polyester obtained from
ɛCL is semicrystalline and the polyester obtained from 2-methylidene-1,3-dioxepane is completely
amorphous due to a high degree of branching (Jin and Gonsalves 1997, Undin et al. 2010). The
degradation rate of amorphous polymers being faster compared to semicrystalline polymers, the
synthesis of amorphous aliphatic polyesters is promising in terms of biomedical applications.
The mechanism of the polymerization of 2-methylidene-1,3-dioxepane is shown in Figure 1.17.
Radicals add onto the double bond to afford a new radical. This radical rearrange by ring-opening
of the ketal into a new radical, which then propagate. The new C=O double bond is approximately
more stable than 50 kcal than the starting C=C double bond. This energy gain, combined with the
release of the ring-strain, is the driving force of polymerization (Bailey 1985).
Another possible mechanism is the direct radical polymerization without any ring-opening
(Figure 1.18). The polymerization selectivity (ring-opening versus direct polymerization) depends
on several parameters such as ring size, substituents, and temperature. As far as the seven-mem-
bered cyclic acetal 2-methylene-1,3-dioxepane is concerned, polymerization takes place with 100%
of ring-opening at room temperature (Bailey et al. 1982). Conversely, ring-opening is accompanied
by direct vinyl polymerization whenever five- and six-membered cyclic acetals are polymerized
(Bailey 1985). It is also worth noting that the presence of substituents can affect the selectivity of
polymerization. Indeed, substituents able to stabilize the radical formed after the ring-opening step
disfavor the direct vinyl polymerization. For instance, polymerization of 2-methylidene-4-phenyl-
1,3-dioxolane is fully selective with 100% ring-opening, whereas polymerization of unsubstituted
five-membered cycles is usually not selective (Bailey 1985).
R
R R O
O
O O C O O
O O O O
R + O
C
C
O
FIGURE 1.17 Polymerization of 2-methylidene-1,3-dioxepane.
R
R O O R
Ring-
opening
C O O
O O O O
R + O O
C O
C
O
Direct
polymerization
R O
C
O
O O
FIGURE 1.18 Polymerization of six-membered cyclic ketene acetals.
This technique of polymerization is not very popular because of its poor selectivity and the very
difficult synthesis and purification of cyclic ketene acetals with moderate or even low yields.
1.5 MACROMOLECULAR ENGINEERING OF ALIPHATIC POLYESTERS

The properties of aliphatic polyesters have to be tailored on demand to develop novel biomedical
applications. The previous sections of this review dealt with the main processes of polymeriza-
tion and the examples given were limited to homopolymerizations. It was already shown that the
functionalization of aliphatic polyesters is a first tool allowing the extension of the range of their
properties. A second possible approach is based on copolymerization or in the modification of the
architecture in the frame of macromolecular engineering.
1.5.1 Copolymerization
The simplest technique of copolymerization is the polymerization of a mixture of, at least, two
comonomers. The distribution of the two comonomers in the final polymer depends on their reac-
tivity ratios. As far as copolymerization is random, the so-obtained copolyesters exhibit averaged
properties of the corresponding homopolymers. The sequential polymerization of comonomers pro-
vides block copolymers, provided that the polymerization is living. In this review, it was shown that
a plethora of initiators and catalysts allows the ring-opening polymerization of cyclic esters to be
living. Accordingly, the literature reports a large number of examples dealing with the synthesis
of block copolymers by this technique. Interestingly enough, block copolymers exhibit brand new
properties compared to the corresponding homopolymers, and thus not just averaged properties as
is the case for random copolymers. In many examples, the ring-opening polymerization of cyclic
esters is just used to synthesize one block and the other block is obtained by another technique.
For example, PCL-b-PEO diblock copolymers are synthesized by successive polymerization of eth-
ylene oxide and ɛCL (Kim et al. 2004, Bednarek and Kubisa 2005). These polymers are amphi-
philic, PEO being soluble in water, which is not in the case of PCL. The self-association of these
amphiphilic copolymers in water affords micelles or hollow spheres, which are used as drug carriers
in drug delivery applications. It is worth recalling that PEO is not biodegradable but is biocompat-
ible and bioresorbable provided its molar mass is low enough. A plethora of other works describes
examples of fully degradable block copolyesters synthesized by ring-opening polymerization.
1.5.2 Modification of the Architecture

The synthesis of aliphatic polyesters with various architectures is achieved by using the ring-opening
polymerization of cyclic esters, this technique being living under appropriate conditions.
A first example is given by star-branched polyesters, which show particular properties such as
lower melt viscosities, lower crystallinity, and smaller hydrodynamic volume. Interestingly enough,
star-shaped copolyesters contain a higher number of chain-ends compared to linear polymers.
Whenever chemists desire to graft a drug, a targeting unit, or a probe, the use of star-shaped polyes-
ters rather than linear polyesters is thus an easy trick to increase their number. Two main techniques
allow synthesizing star-shaped copolyesters. In the frame of the arm-first technique, living chains
are coupled onto multifunctional (>3) electrophiles (Tian et al. 1994). Conversely, the initiation of
the ring-opening polymerization by multifunctional initiators is known as the core-first technique.
The most usual conditions are based on the initiation of the polymerization of cyclic esters by poly-
ols in the presence of tin octoate (Trollsas et al. 1998, Lang et al. 2002, Kricheldorf 2004, Choi
et al. 2005). Another approach less commonly used relies on the initiation of the polymerization by
a spirocyclic initiator (Kricheldorf and Lee 1996, Li et al. 2008). Finally, the arm-first and core-first
techniques can be combined to synthesize star-shaped copolymers in order to increase further the
range of properties of star-shaped copolymers (Van Butsele et al. 2006, Riva et al. 2011).
Graft polymers are other examples of polymers with a branched architecture (Dai et al. 2009).
These polymers can be synthesized by three main methods. The polymerization of chains end-capped
by a polymerizable unit, i.e., a macromonomer, is known as the “grafting through approach.” The
“grafting onto” process is based on the coupling of chains functionalized at one chain-end onto a
backbone bearing several complementary functions. The last process, the “grafting from” technique,
relies on the initiation of the polymerization by a macroinitiator bearing several initiating units.
Hyperbranched aliphatic polyesters are obtained by the polymerization of ABx inimers made up of an
initiator and a polymerizable group. Typical examples are lactones substituted by unprotected alcohols
(Liu et al. 1999a, Trollsas et al. 1999, Tasaka et al. 2001, Yu et al. 2005a, Parzuchowski et al. 2006).
The crystallinity, biodegradation rate, and the mechanical properties of biomaterials made up
of aliphatic polyesters can be modified by implementing cross-linking reactions. For instance,
Hedrick and coworkers reported the cross-linking of PCL bearing pendant acrylates (Mecerreyes
et al. 2001). An intramolecular cross-linking takes place under diluted conditions and nanoparticles
are then prepared. As far as the cross-linking is carried out at higher concentration, intermolecular
cross-linking affords three-dimensional networks. The cross-linking can be carried out in the pres-
ence of radicals (Mecerreyes et al. 2001) or photochemically (Riva et al. 2007, Vaida et al. 2008).
The cross-linking of linear or star-shaped aliphatic polyesters bearing an unsaturation at least at two
chain-ends is also possible (Turunen et al. 2001, Kweon et al. 2003). The cross-linking can be car-
ried out by processes based exclusively on ring-opening polymerization. In this respect, Albertsson
and coworkers reported on the ring-opening polymerization of tetrafunctional bis-(ɛ-caprolactones)
(Palmgren et al. 1997, Albertsson et al. 2000). Other cross-linking agents are made up of other
polymerizable heterocycles, such as bis-carbonates (Grijpma et al. 1993) or lactones substituted
by epoxides (Lowe et al. 2009). All these processes are based on the ring-opening polymerization
technique. Nevertheless, networks can also be obtained by the polycondensation of comonomers,
one of them being at least trifunctional (Kricheldorf and Fechner 2002, Theiler et al. 2010).
Finally, the cross-linking can be carried out by the coupling of telechelic polymers with polyes-
ters bearing along the chains the complementary functional groups. Several coupling reactions are
reported for that sake such as the Michael addition of amines onto acrylates (Theiler et al. 2010),
the coupling of ketones and oxyamines (van Horn and Wooley 2007), the click copper(II) catalyzed
azide-alkyne cycloaddition (Zednik et al. 2008), and the esterification reaction (Kricheldorf and
Fechner 2001, Kricheldorf 2004, Theiler et al. 2010).
Finally, it is worth noting that other examples of architectures such as macrocycles (Li et al.
2006, Jeong et al. 2007, Lang et al. 2002, Hiskins and Grayson 2009, Misaka et al. 2009, Xie et al.
2009) can be found in the literature. Nevertheless, their impact on the field of biomaterial is quite
limited and they will not be reported in this review. As far as macrocycles are concerned, the lack
of applications as biomaterials can be accounted for by their difficult synthesis even though some
recent progresses have been made in this field (Laurent and Grayson 2009).
1.6 CONCLUSIONS
The importance of biodegradable and biocompatible aliphatic polyesters as biomaterials and as environ-
mentally friendly thermoplastics prompted researchers to develop efficient processes for their synthesis,
mainly by step-growth polymerization and ring-opening polymerization of cyclic esters. Nowadays,
owing to the impressive progresses achieved in this field, it is possible to synthesize aliphatic polyesters
with high and controlled molar masses, to control the functionalities at the chain-ends and to graft
functional groups all along the chains. The architecture of aliphatic polyesters can also be modified on
demand in the frame of macromolecular engineering. Remarkably, ring-opening polymerization can
be carried out by a very wide range of polymerization techniques such as anionic, cationic, coordina-
tion, organocatalytic, enzymatic, and radical polymerizations. The synthesis of aliphatic polyesters can
thus be considered as a mature field. Currently, the challenge for a chemist remains to develop efficient
processes for the synthesis of ultrapure aliphatic polyesters, thus noncontaminated by toxic catalytical
residues. In this regard, tin(II) bis-(2-ethylhexanoate) is still widely used despite its known toxicity and
very difficult extraction. Obviously, more work needs to be done despite the very important progresses
being reported in the last few years. In the future, special attention will have to be paid to implement
green processes, for instance, by avoiding organic solvents and to the synthesis of a wider range of bio-
based aliphatic polyesters from new monomers produced from renewable resources.
ACKNOWLEDGMENTS
CERM is indebted to the “Belgian Science Policy” for general support in the frame of the
“Interuniversity Attraction Poles Programme (IAP 6/27)—Functional Supramolecular Systems.”
P.L. is research associate by the “Fonds National pour la Recherche Scientifique” (FRS-FNRS).
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2 Hydrogels Formed by
Cross-Linked
Poly(Vinyl Alcohol)
Gaio Paradossi
CONTENTS
2.1 Introduction............................................................................................................................. 37
2.2 Cross-Linking PVA with Di-Functional Molecules................................................................ 39
2.2.1 Low Molecular Weight Molecules as Di-Functional Cross-Linkers........................... 39
2.2.2 High Molecular Weight Molecules (PVA Macromers) as Di-Functional
Cross-Linkers..............................................................................................................40
2.3 PVA Macromers as Hydrogel Building Blocks....................................................................... 42
2.3.1 Photo-Cross-Linked PVA Hydrogels........................................................................... 43
2.3.2 Hydrogels Fabricated by Chemoselective Derivatization of PVA............................... 48
2.3.3 PVA “Click” Hydrogels............................................................................................... 49
2.4 Hybrid Hydrogels..................................................................................................................... 51
2.5 Concluding Remarks............................................................................................................... 54
List of Symbols................................................................................................................................. 54
References......................................................................................................................................... 55
2.1 INTRODUCTION
Poly(vinyl alcohol) (PVA) has a long-lasting history in polymer science [1]. It was first synthesized
in 1924 by Herrmann and Haechnel, and, during the time, the interest for this polymer has moved
from applications mainly as material for the fabrication of adhesives to the use as component for
the design of biomedical devices. The versatility of this synthetic polymer stems in its chemical
and physical features as well as in the low cytotoxic impact of derived materials/devices when they
interact with tissues and organs.
The hydrophilic nature of the polymer, due to the presence of a hydroxyl group in the repeating
unit, can be considered a key feature. The alcoholic functionality is also responsible for the unusual
chemical versatility of PVA as it allows the grafting of side chains with various chemical and
physical characteristics, providing the tuning of the hydrophilicity of the polymer backbone and the
possibility to formulate various cross-linking strategies. Backbone grafting is employed to intro-
duce side chains on PVA able to form cross-links directly with other PVA chains or able to react
selectively with complementary functional groups belonging to another PVA chain or to a different
polymer. The combination of these features opens a vast number of networking possibilities leading
to hydrogels based on new functionalities, degree of cross-linking, and 3D arrangements and pore
interconnections. Biomedical applications, addressing soft tissues or cartilage replacements, often
require an ad hoc design of hydrogels appropriate for not easily accessible physiological districts
37
and of procedures that should be as less invasive as possible. The approaches described here have
been developed to match such concepts with the final aim to provide new answers for the fast grow-
ing fields of tissue engineering and drug delivery.
Vinyl alcohol cannot be directly polymerized for the obtainment of PVA as the monomer exhib-
its cheto-enolic forms [1]. Bulk, solution, emulsion, and suspension radical polymerizations of vinyl
acetate monomers are standard methods used to obtain the parent poly(vinyl acetate) polymer. After
polymerization of vinyl acetate monomers, PVA is obtained by alkaline hydrolysis of the acetate
groups of the precursor polymer. The deacetylation degree is a key parameter influencing the PVA
behavior in solution. The already complex behavior of the starting material is further amplified
when substituted/grafted PVA chains are considered.
Without any presumption of completeness, this chapter highlights recent results on chemical
hydrogels based on PVA sorting out from the literature the most promising and attractive approaches
for the obtainment of PVA-based networks without reporting on PVA copolymers as most of the
considerations on PVA-based systems can be easily extended to chains having in the average repeat-
ing unit a vinyl alcohol residue.
The chapter is organized as follows: Section 2.2 reviews PVA-based chemical hydrogels formed
by di-functional cross-linkers (with small and high molecular weights), Section 2.3 considers
recent advancements in hydrogels synthesized by using functionalized PVA chains as precur-
sors (macromers) for photo- and chemo-selective cross-linking processes. In this section, “click
chemistry” as a method for the synthesis of chemical hydrogels is considered separately as one of the
newest and most promising approaches in this field. Section 2.4 addresses hybrid hydrogels combin-
ing the features of PVA with other polymer components into a multifunctional matrix.
The synthetic approaches to the chemical gelation of PVA-based networks consist mainly of
two synthetic routes: condensation, photo- or chemo-selective cross-linking of functionalized PVA
macromers. Within the chemo-selective approach, synthetic strategies involving “click chemistry”
[2–4] have been successfully used to design PVA chemical hydrogels. These different networking
methods offer the possibility to fabricate PVA-based chemical hydrogels with a wide range of struc-
tural, mechanical, and dynamic features addressing different types of applications. In this chapter,
we will confine the discussion to PVA hydrogels as materials for the design of polymer device for
biomedicine. Properties and potential applications of these hydrogels will be considered along with
their synthesis.
Characterization of PVA-based chemical hydrogels can be attempted using various physical
methods. Some of them will be reported in this chapter when considering specific examples of
PVA chemical networks. However, the analysis based on the concepts introduced by the Flory–
Rehner swelling theory and the rubber elasticity theory [5] developed for a cross-linking process
in the absence of solvent and further implemented by Peppas [6,7] for the case where the process
is carried out in solution still remains a rule of thumb in network characterization. According to
the earlier-mentioned approaches, swelling degree and elastic modulus are among the parameters
characterizing the hydrogel behavior and, within the assumptions of the method, their combined use
allows the determination of the effective degree of cross-linking, the value of the molecular weight
between cross-links and the determination of the polymer–solvent interaction parameter, χ, of the
Flory’s mean-field polymer solution theory.
In the Flory–Rehner theory, the swelling pressure, Πswell, is the sum of the uncoupled contribu-
tions πosm and G, the osmotic contribution and the elastic modulus, respectively:
Π swell = π osm − G (2.1)
The equilibrium condition is achieved by the hydrogel when these counter-acting contributions to
the swelling balance each other, zeroing the equilibrium swelling pressure. This approach assumes
that the network can be treated as a set of polymer chains in solution with the volume fractions in the
Hydrogels Formed by Cross-Linked Poly(Vinyl Alcohol) 39
relaxed and swollen states, φ0 and φ2, respectively. In terms of change of solvent chemical potential,
this is described by the following equation:
 ν   ϕ  2 ϕ2 
1/ 3
−  ln(1 − ϕ 2 ) + ϕ 2 + χϕ 22  = V1  e  ϕ 0  2  −  (2.2)
 V0   ϕ 0  f ϕ0 
 
where
(νe/V0) is the density of cross-links contributing to the elastic response of the hydrogel
χ is the polymer–solvent interaction parameter
According to this theory, it can be shown that the volumetric swelling ratio, Q, between the swollen
gel volume and the dry volume in the case of high hydration (Q > 10) is inversely proportional to the
cross-link density raised to the 3/5 power.
− 3/5
ν 
Q ∝ e (2.3)
 V0 
Also, the storage modulus, G′, is linked to the density of cross-links by the relation
1/ 3
νe G ′  ϕ 0  (2.4)
=
V0 RT  ϕ 2 
Equations 2.2 through 2.4, based on the swelling and rubber elasticity theories, bear several a ssumptions:
(1) uncoupling of the osmotic pressure from the elastic terms, (2) equivalence between the polymer–
solvent interaction parameter in the gel and in the corresponding polymeric solution, and (3) a strict
connection of the elastic force to individual cross-links (affine network model). Despite the criticisms
accumulated over decades [8,9], the outlined approach is still a valid investigation tool in the chemistry
of polymer networks as it gives direct evaluation of the network average degree of cross-links starting
from easily accessible experimental parameters.
2.2 CROSS-LINKING PVA WITH DI-FUNCTIONAL MOLECULES

PVA can be cross-linked by condensation of dialdehydes, diisocyanates, or any molecule containing
any two functionalities with hydroxyl groups of PVA chains. The cross-linker can be either low or high
molecular weight molecules. The cross-links, formed by condensation reactions, will form hydrogels
with different mechanical properties depending on the type of used di-functional molecules.
2.2.1 Low Molecular Weight Molecules as Di-Functional Cross-Linkers

Hydroxyl groups of PVA can be easily coupled with low molecular weight molecules bearing two
functionalities. The most used cross-linkers are glutaraldehyde, esa-methylene-diisocyanate, and
epichlorohydrin. The main drawback in the use of such cross-linkers is their toxicity, which can
jeopardize the compatibility of the polymer moiety. This is caused by the slow release of cross-
linker molecules after the gelation. For this reason, PVA hydrogels cross-linked with such mol-
ecules are usually not considered for biomedical applications. However, they can be regarded as
models for studying dynamic processes under controlled structural conditions as the diffusion in
confined environments and for probing the dependence of dynamic properties of polymer segments
and water from the hydrogel architecture.
Following this line, PVA has been cross-linked with glutaraldehyde by an acetalization reaction.
The diffusional properties of PVA chains have been studied during the hydrogel formation by pulse
field gradient 1H-NMR [10]. This approach allows the almost complete suppression of the diffusion
of water molecules, making possible the monitoring of NMR parameters regarding the polymer
as the spin–lattice and spin–spin relaxation times, T1 and T2, respectively, and the self-diffusion
coefficient, Dself, during the gel formation. The study of the time decay of the amplitude of the
echo signal revealed three self-diffusion coefficients as a consequence of the heterogeneity of the
system. Among these, the smaller Dself, accounting for the slowest diffusion process, depends on
the gelation time, whereas the other two Dself values were not influenced by the progressing of the
cross-linking reaction. The different dynamic behaviors found for the PVA/glutaraldehyde hydro-
gels can be explained by the local inhomogeneities of the matrix and with the presence of polymer
concentration gradients. Interestingly, the smaller Dself was characterized by a time dependence
similar to the trend displayed by the viscosity versus time, with an induction lag independent from
the gelation time, followed by a “secondary” gel regime characterized by a decrease of about two
orders of magnitude of the Dself value. Further time evaluation of the viscosity data was not possible
due to the abrupt increase of the viscosity close to the gel point.
Water dynamics in PVA-glutaraldehyde hydrogels was probed by quasi-elastic neutron scatter-
ing (QENS) showing a diffusionally quenched water due to the confinement present in the PVA
matrix [11].
Short chain cross-linkers were used recently in an investigation aimed to find a suitable
material among different polyalcohols to substitute natural lens [12]. The natural matrix con-
stituting the lens is a hydrogel containing a high molecular weight protein as polymer compo-
nent imparting a low elastic modulus and a high refractive index to the system. These features,
together with transparency, are crucial for adapting the lens when it is necessary to focus from
short to far distances and vice versa. In general, a low elastic modulus can be obtained with
hydrogels based on synthetic polymers with a small cross-linking degree; on the contrary, a
high refractive index is accomplished with a high polymer chain density in the hydrogel. PVA
was one of the polyalcohols considered as material for lens design. A butylated PVA was dis-
solved in N-methylpyrrolidone for the cross-linking reaction with 1,4-butane diisocyanate and
1,12-dodecyldiisocyanate. The reaction yielded opaque hydrogels due to macroscopic inhomo-
geneities caused by the difficulty to have an efficient mixing of the polymer solution with the
diisocyanates during the gelation process.
PVA networks were synthesized using epichlorohydrin as cross-linker. Mechanical and swelling
experiments confirmed the high cross-linking degree. Crystallization was promoted by drying the
PVA hydrogel at 90°C. The presence of chemical cross-links enhanced the crystalline zone of PVA
hydrogels [13].
2.2.2 High Molecular Weight Molecules (PVA Macromers)

as Di-Functional Cross-Linkers
In this section, we will discuss the exploitation of high molecular weight di-functional molecules
used as cross-linkers according to the same synthetic approach outlined in Section 2.1. In particular,
we will focus on the case of a PVA macromer with cross-linking functionalities at the ends of the
molecules, which allows the networking of PVA chains in order to obtain a hydrogel without the
use and the incorporation of external molecules in the polymer network as outlined in the previous
section.
As any other vinyl polymers, also PVA backbone bears head-to-head sequences, although in
much lower extent with respect to head-to-tail ones. Each head-to-head sequence contains a vicinal
diol that can be selectively and quantitatively splitted by metaperiodate, according to a well-known
reaction used in carbohydrate chemistry for the determination of the amount of equatorial hydroxyl
groups in a saccharide residue [14].
CH2 CH CH2 CH CH CH2 CH2 CH CH2 CH

OH OH OH OH OH
Head-to-head Head-to-tail
+ IO–4
CH2 CH CH2 C O
OH H
NT O C CH2 CH CH2 CH CH2 CH2 C O

H OH OH H
X
Telechelic PVA
+
H2O + IO–3 + O C CH2 CH CH2

H OH
SCHEME 2.1 Oxidation of head-to-head sequences in PVA.
The by-product of the splitting reaction of PVA chains are shorter PVA chains bearing aldehydes
as end groups, called telechelic PVA (see Scheme 2.1). The number of telechelic PVA macromers
originating from the parent PVA chains is determined by the amount of head-to-head sequences
present in the backbone, about 1%–2% (mol/mol) in commercially available samples. These PVA
macromers with a molecular weight of about 2000 g/mol can be used as cross-linkers of chains of
the same polymer via an acetalization in aqueous medium involving the aldehyde end groups of the
telechelic PVA and the hydroxyl moiety of the PVA backbone, both on telechelic and on intact PVA
chains. The main advantage of using reactive PVA macromers as cross-linkers is the absence of
toxic external molecules participating to the network formation which may impact the biocompat-
ibility of the resulting hydrogel.
The chain dynamics and the diffusive behavior of the confined water of this PVA-based hydrogel
were investigated by dynamic light scattering (DLS) around the sol ↔ gel transition and by QENS
to probe the dynamics of the water confined in the hydrogel [15,16]. The DLS autocorrelation func-
tion of the scattered light intensity, g2(t), displays a different phenomenology whether the state of
the investigated system is a polymer solution or a gel. In particular in a gel system, the g2(t) function
does not decay to zero, i.e., complete loss of correlation, even extending the delay times over several
decades. This is the evidence of the occurrence of an arrest of some of the diffusional processes
taking place in the system. In a polymer system undergoing a sol ↔ gel transition, a progressive
quenching of long-range dynamics will occur during gelation.
The autocorrelation functions of the intensity of light scattered by PVA systems collected around
the transition point clearly showed the passage from the solution to the gel state. The collected
autocorrelation functions were analyzed by means of a fractal model in order to assess a correlation
time, τ, accounting for the local collective mesh motions and a characteristic average length, δ, of
5 µm
FIGURE 2.1 (See companion CD for color figure.) Laser scanning confocal microscopy image after tag-
ging PVA shelled microballoons with fluorescein isothiocyanate (FITC).
the network meshes. These parameters changed during the network formation from about 300 to
100 μs and from 350 to 270 Å for τ and δ, respectively, when passing from the initial stage of the gel
formation to the final structured hydrogels. The results were corroborated by the comparison with
the equilibrium swelling and G′ measurements (Equations 2.1 and 2.2).
A particular application of this reaction involving PVA reactive macromers is represented by the
possibility to carry out the networking reaction at the water/air interface [17].
This is achieved by foaming the PVA aqueous solution by high shear stirring while the splitting
reaction described in Scheme 2.1 produces the telechelic PVA macromers. In this case, the network
formation is limited to the thin aqueous layer between the air contained inside the foam bubbles
and the outside. The buildup of hydrated cross-linked PVA shell generates well-dispersed and stable
microballoons with a gas (air)-filled core and with dimensions of about 2 μm with a standard devia-
tion of ±1 μm. Confocal laser scanning microscopy is a powerful technique for characterizing such
systems. As shown in Figure 2.1, fluorescein-labeled microballoons appear as fluorescent rings
when focused on their equatorial plane.
Colloidal dispersions of microbubbles are currently used in the ultrasound imaging, i.e., in
echography, as injectable ultrasound contrast agent (UCA). However, the marketed UCAs, based
on lipidic shells, present some drawbacks as their short time life in the circulation stream and a
poor control on the size distribution with shell diameters ranging from few to more than 10 μm.
The lipidic shells, stabilized by a core filled by hydrophobic gasses as perfluorocarbons or sulfur
hexafluoride, SF6, offer a time life of few minutes after parenteral administration. The new PVA
shelled microballoons have a shelf life of many months and a longer circulation life than the com-
mercial microbubbles can be expected as well. The biointerface of the PVA-based microballoons
has been investigated in terms of biocompatibility, localization of cancer cell lines, delivery of anti-
tumor drugs as doxorubicin, and impact on their viability. This is a step further toward the design
of a multifunctional device supporting diagnostic, i.e., ultrasound imaging, as well as therapeutic,
i.e., localized drug release, activity.
2.3 PVA MACROMERS AS HYDROGEL BUILDING BLOCKS

In this section, we will consider first the photo-activated cross-linking process occurring on PVA
macromers grafted with side chains with different length and chemical composition. In the sec-
ond part, we will highlight PVA macromers cross-linked via a chemoselective process. Hydrogels
obtained by click chemistry will be discussed in the third part.
2.3.1 Photo-Cross-Linked PVA Hydrogels

In situ formation of polymer networks offers a number of advantages as (1) reduced invasiveness of
surgical treatment as the polymeric precursors can be injected followed by a photo-curing process
and (2) complete compliance of the liquid (solutions) polymeric precursors to adapt to the walls
of the organ cavity and to its exact shape and dimensions for an efficient replacement. The recent
advent of photo-initiators sensible to near UV or visible light and the possibility to convoy and
confine light via thin fiber optic guides have contributed to finalize the application of photo-cross-
linkable polymer solution for tissue replacement in in vivo treatments.
Modified PVA has been considered as a key polymer component in this synthetic route for the
obtainment of chemically cross-linked hydrogels, based on the merging of features as hydrophilicity,
good biocompatibility, and tissue-like elasticity. The strategies for introducing vinyl groups ready
to photo-polymerize in the presence of a suitable initiator exploit the chemical versatility of PVA
secondary –OH groups present as pendants in every repeating unit. In particular, we will examine
two coupling reactions to graft PVA with photo-cross-linkable molecules:
1. Grafting acrylate and methacrylate functionalities on PVA by esterification of hydroxyl

groups with the vinyl acid.
2. Conjugation of macromers containing a carboxylic function and a vinyl moiety as end
groups to the hydroxyl moiety of PVA by activating the carboxylic group with the so-called
zero-length cross-linkers, consisting of different types of carbodiimide, as dicyclohexyl
carbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The
comb-like structure resulting from the coupling of the macromers to the PVA chains is then
cross-linked by photo-polymerization of the vinyl end groups contained in the side chains.
The networks based on both approaches have a common structural feature consisting in the forma-
tion of a photo-polymerized vinyl backbone zipping together two or more PVA chains. In the papers
describing the synthetic routes (1) and (2), the vinyl backbone obtained after photo-polymerization
is either a poly(acrylate) or a poly(methacrylate), according to the chemical composition of photo-
polymerized group grafted to PVA chain. A schematic description of this structure is given in
Scheme 2.2. The introduction of vinyl groups impacts with the hydrophilicity of PVA and limits
its solubility in water. For this reason, the degree of substitution of grafted PVA is low, typically
below 5%. However, this is not an obstacle to efficiently cross-link PVA chains due to the zipping
mechanism at the base of the networking.
Hydrogels obtained from photo-polymerization of PVA macromers grafted with acrylate accord-
ing to route (1) were comparatively studied with PVA chemically cross-linked with glutaraldehyde
(see Section 2.1) and physically cross-linked hydrogel domains obtained by freeze–thaw cycles [18].
This work is a systematic study on the factors influencing the network structure and evidences that
the macromer concentration and the type of networking process are key role factors influencing the
Spacing between chains changes as a

function of the side-chain length.
SCHEME 2.2 (See companion CD for color figure.) Representation of the zipping of the PVA chains (in
black) occurring during the photopolymerization of vinyl side chains.
network architecture. Equilibrium swelling ratio and compressive modulus of hydrogels based on
acrylated PVA macromers with a degree of substitution of 7% were photo-polymerized at different
concentrations. From Equation 2.2, the cross-linking density is readily obtained from the equilib-
rium degree of swelling, Q, with Q = 1/φ2. The structural parameter Mc, i.e., the average molecular
weight of the polymer chain between cross-linking points, is linked to the density of cross-links and
can be determined according to
ρp
Mc = (2.5)
( νe /V0 ) + (2ρ p /M n )
where
ρp is the density of the dry polymer
Mn is the number average molecular weight of the polymer chains before the cross-linking
The higher value of Mc measured in hydrogels derived from acrylated PVA macromers with respect
to the corresponding theoretical value, Mc,th, showed that the polymer network synthesized at lower
concentration contained a larger number of chain loops formed by the reaction of intramolecular
vinyl functionalities, not contributing to the elastic response to swelling of the hydrogels. Moreover,
a comparative study of the observed equilibrium swelling degrees of PVA hydrogels obtained (1) by
photo-polymerization of macromers with an acrylation degree of 7 mol%, (2) by freeze–thaw cycling
with a final crystallinity of 2% determined by differential scanning calorimetry, and (3) by acetaliza-
tion in the presence of 2 and 7 mol% glutaraldehyde, evidenced that for PVA hydrogels cross-linked in
the presence of 7% glutaraldehyde, a Q value of 4.5 was obtained, lower than the Q measured for the
network based on the acrylated PVA macromers. This finding was explained with the substantially
different cross-linking mechanisms occurring in the two types of processes. At the base of the photo-
polymerization, there is the chain polymerization propagating through the pendant vinyl groups
attached to PVA backbones, whereas the acetalization reaction is based on the random condensation
of fast diffusing glutaraldehyde molecules and of alcohol functionalities of the polymer. In the former
case, networking will contain a considerable amount of loops and inhomogeneities, whereas the latter
process will take place with a uniform cross-linker distribution in the reaction medium.
According to procedure (1), Mühlebach et al. [19] reported the modification of PVA with acrylic
and methacrylic acid and with 2-vinyl-4,4-dimethylazlactone. Photoinitiated cross-linking was
carried out by UV irradiation (280–320 nm) in the presence of Irgacure 2959, a photo-initiator
soluble in water at concentration lower than 0.1%. Biomedical applications generally require highly
hydrated polymer networks. In this respect, PVA is a suitable material for the design of polymer
scaffolds as hydrophilicity is one of the main features of this polymer.
Acrylamide-grafted PVA chains with different molecular weights and degree of acetylation were
prepared by reacting N-(2,2-dimethoxyethyl) acrylamide in water with PVA in the presence of HCl
[20]. The study was aimed to the assessment of the dependence of the swelling and of the mechani-
cal properties of the PVA-based network on structural parameters as degree of substitution (DS),
molecular weight, degree of acetylation (DA), and modality of hydrogel formulation as the percent
of macromer used in the photo-cross-linking reaction.
An interesting trans-esterification reaction proposed by Hennink et al. [21,22] between glycidyl
methacrylate and dextran, a natural water-soluble polysaccharide, was efficiently applied to PVA.
The trans-esterification of PVA with glycidyl methacrylate in dimethyl sulfoxide (DMSO) in the
presence of 4-dimethylaminopyridine (DMAP) allowed the obtainment of a wide range of DS [23].
As these chemical hydrogels are designed for biomedicine, the focus was on methacryloyl substi-
tuted PVA macromers with low DS values, suitable for the formation of hydrogel with high hydra-
tion degree. Once the methacrylate substitution was introduced on PVA, the photo-cross-linking
was accomplished by irradiation of UV light at 365 nm in the presence of Irgacure. The outlined
strategy is suitable for the formation of biocompatible hydrogel in the vitreal cavity, providing
an a lternative vitreous substitute for vitrectomy usually consisting in the replacement of the vit-
reous with silicone oil. With the method described earlier, many chemical PVA hydrogels were
designed as scaffolds for tissue regeneration. This application requires hydrogels in a monolithic or
“wall-to-wall” morphology. However, the possibility to fabricate nano- or micron-sized hydrogels,
i.e., microgels, opens other fields of application as drug delivery. In the context, it was reported that
a PVA-based chemical microgel can be obtained as a colloidal stable dispersion by carrying out the
photo-cross-linking reaction of methacryloyl derivative of PVA with different DS in the presence of
an aqueous solution of dextran with a molecular weight of about 40 kDa, Dextran T40. Due to their
incompatibility, PVA and dextran form in water a microemulsion under high shear stirring. This is
an application of the so-called water-in-water emulsion method. The absence of organic phases in the
synthesis of microgels makes this method particularly suitable for designing polymer-based micro-
devices for close contact with tissues or organs. The aqueous phase containing the methacryloyl PVA
derivative is sensible to photo-cross-linking in the presence of the photoinitiator Irgacure. In this
way, it was possible to obtain hydrogel beads based on cross-linked PVA with a diameter of about
2 μm and with a quite narrow distribution (σ = ± 1 μm), potentially usable as injectable drug delivery
system. The chemical versatility of PVA is also an asset for the surface coating of the microgel par-
ticles. Succinoylation of the microbeads surface imparts a negative ζ potential, the electric potential
at the microgel–liquid interface, allowing the loading of doxorubicin, a positively charged antitumor
drug, by favorable charge interaction with microgel particles. The cargo amount can be as much as
the 50% by weight of the dry microgel, providing a large dose range for treating tumor cells [24].
To enhance the biointerface features of these PVA-based microgels, other surface coatings were
also applied as hyaluronate. Hyaluronic acid (HA) is a glycosaminoglycan contained in synovial
fluids and cartilages of mammalians. It is a bioactive molecule as it participates to several cellular
processes, besides having an important dumping and lubrication function in the articular joints. In
1990, HA was assessed as a ligand for CD 44 transmembrane receptor [25], a protein regulating
bioadhesion and proliferation process and overexpressed by tumor cells. This has opened the pos-
sibility to target tumor cells and to enhance the bioavailability of the antitumor drug delivered by an
HA-functionalized device. The HA functionalization of PVA microgels surface is easily obtained
by partial oxidation of the polysaccharide by metaperiodate, followed by condensation with the
alcohol functionalities of the microgel particles [26].
The water-in-water emulsion polymerization is a suitable method to incorporate in the PVA
network other vinyl comonomers. The obtainment of thermoresponsive spherical-shaped micro-
gels using this method has been recently reported [27,28]. This was accomplished by carrying out
the “water-in-water” emulsion polymerization method in the presence of N-isopropylacrylamide
(NiPAAm) monomer. In this variant of the method, NiPAAm monomers distribute in both the
dispersing phase water/dextran T40 and the disperse phase containing water/methacrylated PVA.
The photo-polymerization incorporates NiPAAm in the PVA-rich phase, whereas oligomers of
NiPAAm are formed in the dextran-rich phase. After polymerization, NiPAAm oligomers are
washed out in the purification step. It is known that polymerized NiPAAm shows an LCST behav-
ior and, correspondingly, cross-linked NiPAAm displays a volume-phase transition. The behavior
of these PVA/poly(methacrylate-co-NiPAAm) microgel was studied with respect to their average
size and size distribution, swelling, and release properties. It was observed that the stirring speed
is a key parameter for controlling the amount of incorporated NiPAAm, the particle size and the
sharpness of the volume-phase transition. The volume-phase transition temperature (VPTT) of the
microgel was evaluated around 38°C and 34°C for microgels with a NiPAAm/methacrylate molar
ratio of 0.8 and 2.4, respectively. A volume reduction of about 50% of the microgel particles was
accompanied by a size decrease of the network meshes from 4 to 2 nm when temperature was
brought from 25°C to 40°C, as determined by size exclusion experiments with fluorescein-tagged
dextran, FITC-dextran, molecules. O-Succinoylated microgels were loaded with doxorubicin, a
well-known antitumor drug, by exploiting the favorable electrostatic interaction between negatively
charged microgel surface and positively charged doxorubicin. The drug release was enhanced at
temperatures higher than the VPTT, i.e., 40°C, due to the microgel shrinking and the consequent
increase of the surface/volume ratio.
Recently it has been shown [29] that structural and diffusion features of the PVA-based hydrogels
can be tuned by choosing the cross-linking strategy. The network structure and the macromolecular
drug release of hydrogels were discussed in terms of the cross-linking modality. Curing acryloyl-
derivative of PVA (DS = 2%, deacetylation degree 83%) aqueous solutions by photo- or by redox
reaction polymerizations yielded hydrogels with different behaviors in terms of swelling, release of
FITC-dextrans with 20 and 4 kDa, used as model high molecular weight drugs, and polymer mass
loss. The different curing methods gave indication of a higher extent of cross-linking in the case of
the photo-polymerized PVA network. The two curing strategies can be used for tuning the release
properties of PVA-based hydrogels based on radical cross-linking.
Synthetic routes based on approach (2) were reported by Anseth et al. [30] for obtaining bio-
degradable PVA-based chemical hydrogels. In this work, a degradable oligoester derived from
hydroxyethyl methacrylate, lactide and succinic anhydride, HEMA-(Lac)n-Suc, was prepared
(step 1) by ring-opening polymerization of molten lactide in the presence of stannous octoate at
110°C, followed by an esterification with succinic anhydride in DMSO using DMAP/pyridine as
catalyst (step 2). This macromer is then grafted by N,N′-dicyclohexylcarbodiimide (DCC) chemistry
to the PVA with DS ranging from about 1 to 15 mol% (see Scheme 2.3). Mass loss determinations
O
2 O O OH
O O
n
O O
HEMA-(Lac)n-Suc
OH OH OH OH OH
PVA
DCC, DMSO,
pyridine, DMAP
HO
O
O O O
O O
O n O
HO
HO
HEMA-(Lac)n-Suc grafted PVA
SCHEME 2.3 Synthesis of HEMA-(Lac)n -Suc grafted PVA.

as a function of time showed the dependence of network degradation in phosphate-buffered solu-

tion at pH 7.4 (PBS) and at 37°C from the number of lactide residues and from the degree of sub-
stitution. Hydrolysis of lactide residues in the side chains was effective in controlling the network
degradation. In addition, tuning of the hydrophobicity of the matrix was possible by controlling the
degree of substitution of PVA chains as demonstrated by the improvement of valve interstitial cells
bioadhesion to PVA grafted HEMA-(Lac)4-Suc surface, characterized by the higher hydrophobicity
among the investigated degradable scaffolds.
With a similar approach, degradable ester-acrylate chains terminating with a carboxylic acid
group were coupled by DCC chemistry to yield ester-acrylate anhydride molecules. These anhy-
drides were conjugated with PVA hydroxyl moiety in DMSO in the presence of triethylamine [31],
see Scheme 2.4.
The kinetic analysis of the hydrogel degradation caused by esters hydrolysis was treated as a pseudo-
first-order process:
nester
= exp( − k ′ t ) (2.6)
n0
where
nester and n0 are the number of ester blocks at time t and at t = 0, respectively
k′ is the apparent kinetic constant
O O O
2 O O
O O OH
O O
Acrylate-ester molecule
DCC
O O O O O O
O O O O
O O O O O
O O O O
Anhydride-acrylic-ester molecule
Triethylamine,
DMSO
n
OH
O O O
O O O
O O
O O
PVA acrylate-ester
SCHEME 2.4 Synthesis of PVA acrylate-ester for photopolymerization.

For the structural features of these photo-polymerized hydrogels, there is a direct connection of
the number of ester blocks with the cross-link density (νe/V0). It is then possible to write Equation 2.3
in the form
3 
Q ∝ exp  k ′ t  (2.7)
5 
2.3.2 Hydrogels Fabricated by Chemoselective Derivatization of PVA

A possible bias to photo-cross-linking is the production during the network formation of free radi-
cals triggered by the photoinitiation of the cross-linking reaction that may damage the vitreal cavity
tissues, although their presence should be expected in transient and low concentration. To circum-
vent the impact of cross-linking reaction based on radicals, Michael addition and more in general
nucleophilic additions offer the possibility to cross-link in situ and in aqueous medium several
polymers functionalized with suitable pairs of chemoselective coupling groups. In this way, the
outlined drawbacks of the photo-polymerization can be circumvented. Similarly to the approach
described in Section 2.3.1, (2), multifunctional PVA macromers were exploited for the obtainment
of hydrogels with high elasticity. However, in this approach, replacing the photo-polymerization of
vinyl moiety of the derivatized PVA, the cross-linking efficacy is based on the chemoselectivity of
the substituents and directly linked to the kinetic and thermodynamic driving forces of the reactions
involving the chemical functionalities born on the PVA chains.
Following this line, Ossipov et al. [32] prepared a set of PVA macromers bearing nucleophilic
and electrophilic complementary functional groups. In this study, cysteine-functionalized or thio-
lated PVA were coupled with maleimide functionalized PVA chains obtaining networks with 1:1
stoichiometry of the cross-linking reaction. Derivatization of the hydroxyl groups of the PVA main
chain with nucleophilic moieties was achieved by exploiting the chemical versatility of the alco-
hol functionality, coupled with known methods of peptide synthesis as 1,1′-carbonyldiimidazole
(CDI) chemistry. This synthetic route allowed the activation of PVA hydroxyl groups and the
coupling with molecules bearing appropriate groups, i.e., amines, thiols, and cysteine (see
Scheme 2.5). Protection is often required on the nucleophilic groups to avoid the conjugation with
the O-imidazolylcarbonyl PVA intermediate. The protection is removed in the presence of trifluo-
roacetic acid to allow coupling of the nucleophilic group with the complementary electrophilic
functions and chain cross-linking.
The approach used to introduce electrophilic moieties on PVA is based [32] on the coupling of
amino-derivatized PVA with N-hydroxysuccinimide ester of acids containing electrophilic groups
as maleimide, acrylate, α-iodoacetyl functionalities (see Scheme 2.6). The elastic properties and
the degree of swelling of the resulting hydrogels were, as expected, determined by the stability
and density of the cross-links contributing to the network elasticity. The same group [33] designed
hydrogel based on two PVA components, containing aldehydes and hydrazide side chains, respec-
tively. In this study, encapsulation of murine neuroblastoma cells in the hydrogel was carried out
during the cross-linking reaction occurring in aqueous medium at physiological condition of pH
and temperature.
Electrophilic–nucleophilic chemoselective coupling, based on Michael addition of cysteine-
modified PVA macromer on methacrylate-functionalized PVA, was also reported in the literature
[34]. Telechelic PVA macromers prepared according to Scheme 2.2 were coupled with the amine
group of cysteine, present in large excess, by reductive amination. The thiol end-capped PVA chains
were then conjugated with a methacrylic derivative of PVA with DS of 4 mol%, yielding a hydrogel
with thiol cross-links. The kinetics of the cross-links formation depended on the reactivity of the
functional groups involved in the reaction. A possible drawback of this thiol containing cross-links
could be the sensitivity of the hydrogel to oxidative conditions.
+ R
H2N
OH OH Amine with a Boc protected nucleophilic group,

PVA R: NuBoc
O
N N
N N
CDI
O OH
HN
R΄
Functionalized PVA via carbamate (red) coupling after deprotection
bearing a nucleofilic group R΄
R΄: NH2 , SH
SCHEME 2.5 (See companion CD for color figure.) CDI chemistry on PVA.
2.3.3 PVA “Click” Hydrogels

Polymer chemistry used “click chemistry” since early stages of this highly selective synthetic path-
way as an efficient cross-linking method for the formation of structurally controlled networks. The
general features of a “click” reaction are (1) mild reaction conditions, (2) high yields, (3) stereo-
specificity, (4) absence of by-products, (5) no side reactions, and (6) a high thermodynamic driv-
ing force (usually more than 20 kcal/mol). Moreover, the possibility to carry out click reactions in
water is fundamental for hydrogel fabrication. One of the most used click reactions, among the
copper(I)-catalyzed Huisgen reactions, is the chemoselective 1,3-cycloaddition between alkynyl-
functionalized and azido-functionalized macromers with the multiple formation of triazole cross-
links. The general features of this reaction are reported in Scheme 2.7.
The first application of click chemistry to PVA appeared recently in the literature [35]. In this
paper, click chemistry was exploited to construct hydrogels based on clicks between PVA chains
bearing acetylenic and azido functionalities on the side chains grafted by means of CDI chemistry.
The reported hydrogels are formed by activation of PVA chains with O-(imidazol-1-ylcarbonyl)-
functionality. In a second step, the reaction with primary or secondary amines containing alkyne or
azide functionalities allows the grafting of the PVA chains. In this way, the macromolecular precur-
sors are ready to form PVA-based hydrogels by clicking the complementary functionalities grafted on
the PVA chains. The design of this class of hydrogel should be bioorthogonal, i.e., the functionalities
+ O
N
O OH O R
O
O
NH
N-hydroxysuccinimide acid ester with R
containing an electrophilic group
H2N
I
Amine derivative of PVA
O
R:
O OH
O
NH
HN
O
R
SCHEME 2.6 Electrophilic functionalization of PVA chains.
Cu(I)
+ N N N
N
N N
: PVA chain
SCHEME 2.7 (See companion CD for color figure.) 1,3 Cycloaddition click reaction scheme for the forma-
tion of a triazole ring.
of PVA necessary to the “clicks” should be stable with respect to oxidation and hydrolysis pathways
in order to avoid any release of molecular species toxic to cells, and specificity should be enhanced
as much as possible with no side reactions involving metabolic or cellular materials. Moreover, the
kinetics of the hydrogel formation should be compatible with the time necessary to mix the starting
polymer components. The PVA “click” hydrogels described in the literature [35] do not exhaust the
possibilities provided by this enhanced chemoselective approach and many novelties concerning
hydrogel architectures and properties are to be expected in short time. Another “click chemistry”
approach [35] leads to a PVA-poly(ethylene glycol) hybrid hydrogel. Poly(ethylene glycol) (PEG)
was end-capped with azide functionalities, obtaining a telechelic diazide PEG spacer ready to click
the alkyne-functionalized PVA by exploiting the high thermodynamic driving force of the click
event. The hydrogels described in the paper provide a clear example of how “click chemistry” can
be used to link structure and properties of the hydrogels. The outlined azide end-capped PEG is
a two functional macromer, having two functional groups per polymer chain, f = 2, with a ratio
M/f = 1050, where M is the polymer molecular weight. The same M/f value was obtained with an
azide-functionalized PVA macromer with a DS of about 4.5%. However, “clicking” these types
of macromers with the same alkyne-grafted PVA produced hydrogels with very different behav-
iors. Comparison of gelation time, gel fraction, storage, and loss moduli for mixtures of polymers
indicates that sol ↔ gel transition occurred in much less time when azide-functionalized PVA is
clicked with the alkyne-derivative of PVA, hereafter called PVA-PVA. In a solution containing an
equimolar amount of azide and alkyne with a fixed total polymer and catalyst concentration, the
gel fraction, i.e., the dry polymer mass weighted after the hydrogel formation and swelling in water
with respect to the initial polymer mass, for the PVA-PVA was twice the gel fraction found for PVA-
PEG hydrogels, indicating that in the latter the occurrence of single chain links and intramolecular
loops is more probable than in the former hydrogel in agreement with a drop of the elastic modulus,
G′, of the PVA-PEG hydrogel. According to Equations 2.5 and 2.6, the observed G′ was converted
into the cross-linking density, νe/V0, and linked to the molecular weight of the chain between cross-
links, Mc. The molecular weight between cross-links found for the PVA-PVA hydrogel is lower than
the Mc of PVA-PEG, indicating a more frequent interchain cross-linking with a larger number of
chains participating to the elasticity of the network.
2.4 HYBRID HYDROGELS

The term hybrid hydrogels is used to indicate hydrated networks where two or more polymers partic-
ipate to the network. Together with hydrogels entirely based on PVA, in the last decade many exam-
ples of hybrid hydrogels have been reported in the literature. In principle, a network based on more
than one polymer component enables a larger number of functions with respect to one-polymer-based
network. Hybrid hydrogels can be formed by chemically cross-linked polymer chains or by the
interpenetration of networks, each one based on a single polymer component (IPN). Here, we will
consider only those formed by the chemical cross-linking between the polymer components.
Chemo-selective and “click chemistry” approaches are commonly used to generate hybrid
hydrogels, see, e.g., PEG-PVA hydrogel described in Section 2.3.3. Some examples of hybrid hydro-
gels have been already reviewed in the earlier sections. Coupling of nucleophilic with electrophilic
polymer partners in which one is a derivatized PVA chain and the other is a modified or intact
biopolymer, allows a number of hybrid hydrogels having different features as charge density, chain
stiffness, biodegradability, and bioelimination properties.
In the design of hybrid hydrogels, polymer compatibility is an often overlooked issue, although
being a key factor to avoid polymer phase segregation, breakdown of mechanical properties, low
and/or uneven cross-link distribution. Once the compatibility of the polymers at a given mass or
molar ratio is assessed, the fabrication of a multicomponent network is based on the functional
groups to link. Hybrid hydrogels have been used for mimicking complex natural networks and in
particular the extracellular matrix (ECM) with artificial scaffolds matching the manifold features
required for the controlled growth of a cell line. ECM is a complex multicomponent system, con-
taining low and high molecular weight constituents, produced by the cells themselves as suitable
medium for metabolic processes involving bioadhesion and proliferation. Many biological key fac-
tors are involved in different steps of cell culturing, from the RGD tripeptide sequences contained
in the fibronectin and enabling the bioadhesion process of the cells on the ECM matrix to cytokines
and growth factors triggering and supporting the growth, respectively. The proliferation step of the
cells requires a controlled hydrolysis of the polymer matrix promoted by the metallo-proteinases
expressed by the cells in order to have available a larger, destructured portion of the ECM. The
hydrolysis of the proteic part of the matrix is also paired by the progressive degradation of HA, a
polysaccharide component of the ECM, driven by the hydrolytic activity of the hyaluronidases, a
family of degradative enzymes also expressed by the cells hosted in the ECM. Only if this sche-
matically described interplay between the host ECM and the guest cells is properly established, the
growth of a cell colony leading to the final aim of the (re)generation of a tissue can be achieved.
An artificial scaffold for tissue regeneration should enable such complex interaction pattern,
functions, and processes in order to be a sound substitute of ECM. A possible approach in view of
the design of scaffolds for tissue regeneration is to favor cell growth incorporating some of the natu-
ral components in the artificially reproduced systems. HA, a key component in the ECM, is a can-
didate to be a polymeric partner in a PVA-based hybrid hydrogel. Ossipov et al. [32] focused on the
coupling between a nucleophilic derivatized PVA with a partially oxidized HA. PVA backbone was
modified with side chains containing nucleophilic functionalities as thiol, cysteine, 1,2-aminothiol,
and aminoxy side chains prepared according to the strategy outlined in Scheme 2.5, or, alterna-
tively, by introducing electrophilic functionalities in the PVA chains according to Scheme 2.6.
Nucleophilic or electrophilic functionalized PVA chains were coupled with HA after partial oxi-
dation with periodate. The action of periodate on HA consists in the specific oxidation of C2 and
C3 carbons of the d-guluronic acid residue of the hyaluronic repeating unit (Scheme 2.8) into
aldehyde groups.
The hybrid hydrogels obtained by the conjugation of oxidized HA with aminooxy-(PVAAO),
semicarbazide-(PVASemi) and hydrazide-(PVAH) modified PVAs yielded strong hydrogels ranging
H OH H
COO– Na+
H O H
O
O O
HO O NHCOCH3
H OH HO H
H H H H
+ NaIO4
H OH H
COO– Na+
O O H
O O
C
O O NHCOCH3
C H
O HO H
H H H
SCHEME 2.8 Oxidation of hyaluronate.

Cytotoxicity of medium from gels

0.6
Control HA/PVAH HA/PVAAO
0.5 HA/PVASemi HA/PVACys
Abs 570 nm
0.4
0.3
0.2
0.1
0.0
0h 24 h 48 h
Time
FIGURE 2.2 Cytotoxicity tests of different PVA-based/HA hybrid hydrogels on human dermal fibroblasts.
from compliable materials to more elastic networks. The chemoselective reaction responsible for the
cross-link formation takes place with the incorporation of the cells allowing the intimate contact
between the living material and the hydrogel constituents. Figure 2.2 shows the cytotoxic effect
of these hybrid hydrogels tested on human dermal fibroblasts. The impact of the hydrogels on cell
viability, monitored by MTT assay, was absent and the enzymatic degradation of HA by hyaluroni-
dase was accomplished within 3 days for all of the studied hydrogels. The degradation of HA due
to the hyaluronidase expressed by the cells incorporated in the PVA/HA co-hydrogels was not
investigated in the paper.
Another hybrid hydrogel based on the conjugation of PVA with heparin, a natural polysaccha-
ride belonging to the family of glycosaminoglycans, was described recently in the literature [36].
Methacrylated macromers of PVA and heparin, conjugated via photopolymerization of the vinyl
moiety, yielded a co-hydrogel with mechanical properties similar to the network generated by
the photo-cross-linking of methacrylated PVA and retained the biological activity character-
istic of heparin as its growth factor activity. Tests, performed with BaF3 fibroblasts seeded on
PVA/heparin co-hydrogels in the presence of 0.3 nM fibroblast growth factor FGF-2, showed
increased cells proliferation when compared with methacrylated PVA hydrogels (Figure 2.3).
1.00
Hydrogels
0.80 Controls
Proliferation index
0.60
0.40
0.20
0.00
PVA PVA/hep PVA/hep PVA/hep 0 nM 3 nM 30 nM
19.5/0.5 19/1 18/2 Heparin Heparin Heparin
(–ve) (+ve) (+ve)
FIGURE 2.3 BaF3 cell proliferation on PVA and PVA/heparin hybrid hydrogels, and in response to negative
(no heparin) and positive (3 and 30 nM heparin) controls, all in the presence of 0.3 nM FGF-2.
The effect was independent from the amount of heparin contained in the co-hydrogels, and this
was interpreted as evidence that the optimal signaling activity was already obtained with the co-
hydrogel incorporating the lowest amount of heparin.
The low molecular weight cross-linker, glutaraldehyde, as outlined in Section 2.2, was
exploited for the networking of PVA with chitosan, a polysaccharide derived from the deacety-
lation of chitin, the second most abundant polysaccharide after cellulose. The bioactivity of
this co-hydrogel was tested with respect to cell viability and cytotoxicity [37]. The use of
glutaraldehyde, a highly reactive and toxic cross-linker, did not impact with the viability of
the cells.
2.5 CONCLUDING REMARKS

The versatility of PVA is paramount in the fabrication of several chemical hydrogels enabling the
obtainment of a variety of internal architectures and overall morphologies, including monolithic
hydrogels, microgels, and microbubbles. This is combined with a favorable bio-interface provided
by several PVA-based hydrogels, validated by in vitro and in vivo studies with various cell lines.
Modulation of the properties and introduction of responsiveness to external stimuli are other assets
of recently formulated chemical hydrogels based on PVA.
The picture emerging from a literature survey shows a constantly ongoing research activity on
the synthesis of new hydrogels as well as on their characterization, including more advanced aspects
as the study of the network internal dynamics and the diffusive behavior of confined water ranging
from the molecular detail to the macroscopic level. Several, not less negligible, features are still
to be explored and new PVA-based hydrogels soon will enrich the panorama of the potential tools
available for biomedicine.
LIST OF SYMBOLS
Dself self-diffusion coefficient
f functionality of the cross-link
g2(t) autocorrelation function of the scattered intensity of light
G elastic modulus
G′ storage modulus
k′ apparent kinetic constant
Mc number average molecular weight between cross-links
Mn number average molecular weight
nest number of ester blocks at time t
n0 initial number of ester blocks
Q swelling ratio
V1 molar volume of the solvent (water)
VPTT volume-phase transition temperature
χ polymer–solvent interaction parameter
πosm osmotic pressure
Πswell swelling pressure
φ0 polymer volume fraction in the relaxed state
φ2 polymer volume fraction in the swollen state
νe/V0 density of cross-links
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3 Development and Evaluation
of Poly(Vinyl Alcohol)
Hydrogels as a Component
of Hybrid Artificial
Tissues for Orthopedics
Surgery Application
Masanori Kobayashi
CONTENTS
3.1 Introduction............................................................................................................................. 57
3.2 Development of PVA-H........................................................................................................... 58
3.3 Biocompatibility of PVA-H..................................................................................................... 59
3.4 Mechanical Properties of PVA-H............................................................................................60
3.4.1 Mechanical Properties and Fabrication Methods of PVA-H.......................................60
3.4.2 Tribology of PVA-H..................................................................................................... 63
3.5 Application for Arthroplasty Implant...................................................................................... 67
3.5.1 Development of Artificial Articular Cartilage............................................................ 67
3.5.2 Development of Artificial Articular Cartilage as a Hemi-Arthroplasty..................... 68
3.6 Development of Artificial Meniscus........................................................................................ 70
3.7 Development of Implant for Spine Surgery............................................................................. 74
3.7.1 Development of Artificial Intervertebral Disk............................................................ 74
3.7.2 Application of Artificial Intervertebral Disk Nucleus Implant................................... 76
3.8 Future and Potential of PVA-H................................................................................................ 77
3.8.1 Problems with PVA-H Fixation Techniques................................................................ 77
3.8.2 Future Possibilities of PVA-H...................................................................................... 78
Acknowledgments............................................................................................................................. 79
References......................................................................................................................................... 79
3.1 INTRODUCTION
The area of research of polymeric biomaterials covers a wide range of fields from basic research to
applied research. Due to the development of nanotechnology, biotechnology, and tissue engineer-
ing and the technical fusion of polymer sciences in recent years, the field of polymeric biomaterial
science is expanding rapidly. In vivo compartmentalized organic macromolecules (extracellular
matrix [ECM], proteins, polysaccharides, DNA, etc.) play an important role among cellular com-
ponents, and it is not an exaggeration to say that the living body and functional expression as such
57
depend on biological macromolecules. The essence of biomaterial research is to imitate the living
body, to substitute vital functions, and to actively influence the living body. One can say that it is
inevitable that research on polymer materials, the key elements that constitute the living body, is
wide-ranging. Among the research and development of such biomaterials, particularly regarding
the fields of application, eventually the most important point is a safe and reliable technical poten-
tial in order to meet the characteristics demanded in the desired medical field, as well as the specific
performance of clinically applicable material and devices and their evaluation. Due to its excel-
lent biocompatibility and mechanical properties, the biomedical application of poly(vinyl alcohol)
hydrogels (PVA-H) as artificial artery, artificial cartilage, and artificial muscle has been made
possible. Regarding the points mentioned earlier, PVA-H can be identified as a typical biomaterial
for which no specific clinical application has not been found yet in spite of a lot of research and
development being done in various fields for a long time. Since 1985, Oka et al. have been engaged
in research on the clinical application of PVA-H in the orthopedic surgery field and have also
performed many basic experiments on PVA-H, improved PVA-H, and developed PVA-H implants.
The author also joined this research group around 1995 and has been engaged in various researches
on the application of PVA-H. In this chapter, limiting this topic to the application of PVA-H in the
field of orthopedic surgery, and focusing on the implant study examples that have been developed
so far, the author will explain a series of findings with regard to the clinical application of PVA-H
and its possibilities.
3.2 DEVELOPMENT OF PVA-H

PVA was developed in 1924 by Hermann et al. and is a synthetic fiber with various excellent
mechanical properties. It is also the raw material of “vinylon,” the first high-strength, high-modulus
synthetic fiber that was developed in Japan. PVA-H is prepared from PVA. It is not only used in
the manufacturing of high-strength, high-modulus fiber but also serves as raw material for films
and acetal resin, as textile processing agent, adhesive agent, polyvinyl chloride polymerization
stabilizer, and inorganic binder. Especially, since the excellent biocompatibility of PVA-H has been
widely known, this has raised great expectations for its use as a biomaterial. Initially PVA-H was
not found to be a strong gel, and it was also turbid. Then, Hyon, who was a collaboration researcher,
and colleagues developed the freeze-thaw method [1,2]. Generally, hydrogels are defined as gels
that contain water but are not soluble in water. Later on, Hyon et al. made a complete homogenous
PVA solution by heating the mixture of PVA and water/dimethyl-sulfoxide (DMSO), after agitating
under nitrogen current, leaving the solution in low temperature (−20°C) for 10 h, and promoted the
crystallization and cross-linking of PVA molecules. This frozen gel was brought into contact with
water to exchange DMSO in the PVA gel with water. Repeating this cryogels process by freeze-
thaw cycling, a PVA-H with high mechanical strength, high water content, and excellent transpar-
ency was successfully produced. By using this technique, we have succeeded in the reinforcement
of the mechanical properties of gels by only the arrangement of water content and polymeriza-
tion of PVA-H without the chemical agent or other blend polymers. Adding the cross-linking by
γ-radiation, we have characterized further improved mechanical properties and biocompatibility
of PVA-H.
However, to develop PVA-H as an arthroplasty material, as shown in later Section 3.5, it is
necessary for PVA-H to infiltrate into the titanium fiber mesh as a composite device so that it
can be firmly fixed to the underlying bone. However, because of poor infiltration of PVA solution
into the pores of titanium mesh using the low-temperature crystallization method, the filtration
technique was improved by adopting high-pressure injection molding. The bonding strength of
PVA-H and titanium fiber mesh interface was increased remarkably with this improved fabrica-
tion technique. Figure 3.1 shows the flow chart of the fabrication process of two types of PVA-H.
Then, two types of PVA-H were selected and examined with the use of a medical device we tried
to investigate.
Development and Evaluation of Poly(Vinyl Alcohol) Hydrogels 59
20% Wt PVA-H
solution (DMSO)
<0°C, 12 h
Gelatine
PVA gel
(DMSO)
Solution with ethanol
PVA gel (Et-OH)
Dry in vacuum
Heating 100°C–120°C
Swelling in water
PVA hydrogel
PVA-H
(a)
Concentration of swelling
PVA PVA 30%
Mix Melting Injection molding
At 150°C, 160°C, 180°C, 200°C Injection pressure 120 MPa

Solvent by Kneading machine Injection velocity 10 cm3/s
Injection at 150°C, 160°C, 180°C, 200°C
H2O/DMSO =
20/80 (w/w)
Hydrogel
(b)
FIGURE 3.1 (See companion CD for color figure.) (a) Flow chart of the production process of poly(vinyl
alcohol)-hydrogel (PVA-H) by freeze-thaw method. (b) Flow chart of the production process of poly(vinyl
alcohol)-hydrogel (PVA-H) by injection molding method.
3.3 BIOCOMPATIBILITY OF PVA-H

It has already been reported that PVA-H has excellent biocompatibility; however, as the newly
improved PVA-H was not tested, de novo basic biocompatibility experiments using animals were
performed [3,4]. PVA-H was then implanted into various sites in rabbits and dogs, subcutaneously,
intramuscular, etc., and follow-up after surgery confirmed superior biocompatibility. In order to
make certain in vivo foreign-body immune reactions against PVA-H, fine particles of PVA-H
and ultra-high molecular weight polyethylene (UHMWPE) were injected into the right and left
knee joints, respectively, in the same rabbit. The PVA-H used were fine particles with a diam-
eter of 100 μm, with UHMWPE particles as control. Figure 3.2 is a histological photograph of
tissue observed 3 months after injection. In the periphery of the UHMWPE particles, accumulated
macrophages and foreign-body giant cells, and remarkable foreign-body reactions were observed,
whereas almost no reactions were seen around PVA gel particles on the opposite side. This bio-inert
characteristic of this PVA-H is speculated to be due to the strong hydrophilic effects of this gel,
which inhibits water circulation to the cells and makes it difficult for cells to adhere to its surface.
(a) (b)
FIGURE 3.2 (See companion CD for color figure.) Histological appearance of the implant material particles
and the surrounding tissue in rabbits knee joint (×100). (a) UHMWPE particle: many macrophage and giant cells are
surrounding particle due to intense immunoreactions. (b) PVA-H particle: Immunoreactions were hardly observed.
The results of these basic experiments confirmed that PVA-H has favorable biocompatibility. Even
in many animal experiments subsequently performed for the development of implants, no immune
reactions were observed for PVA-H.
3.4 MECHANICAL PROPERTIES OF PVA-H

PVA-H has been chosen for investigation as it possesses several useful properties, including perme-
ability, hydrophilicity, and low frictional function. It has been widely commercialized and studied
in the medical industries for the production of membrane, gel, and films for an artificial pancreas,
drug delivery system, and adhesion protection sheet [5–7].
However, the use of PVA-H as an application in the orthopedics surgery field has been thought
to be limited because of its low mechanical strength and durability. Actually, PVA-H is not such a
strong gel mechanically. In this chapter, a series of the development and evaluation concerning the
mechanical properties of PVA-H is mentioned.
3.4.1 Mechanical Properties and Fabrication Methods of PVA-H

Traditional cross-linking methods have been used to synthesize PVA-H materials with improved
mechanical properties; however, the chemical agents introduced in their preparation are often toxic,
inevitably effecting the biocompatibility of PVA-H. On the other hand, polymer blending is a use-
ful method of improving or modifying the physiochemical properties of polymer materials; blends
between synthetic polymers and biopolymers are of particular interest to current hydrogel research.
A polymer blend can be defined as a combination of two polymers without any chemical bond-
ing between them. Some chemical interactions can occur between components, but it sometimes
induces the deterioration of mechanical properties of gels.
In this academic background, Hyon et al. succeeded in gelling PVA by cryogenic crystalliza-
tion using a freeze-thawing method at low temperatures with the organic solvent DMSO in the
synthesis process, making it possible to prepare PVA of improved transparency and high strength.
Subsequently, the research and development of all kinds of implants was performed using an
improved version of this PVA-H, and it underwent improvements such as molecule cross-linking by
γ-irradiation in order to achieve higher strength.
However, as shown earlier, in order to develop PVA-H as an artificial articular cartilage [4,8],
PVA-H is required to infiltrate into titanium fiber mesh as a composite device to fix PVA-H firmly
to the underlying bone. Hence, the high-pressure injection molding technique was used instead
of the low-temperature crystallization method. Although the bonding strength of PVA-H and
titanium fiber mesh interface increased remarkably with this improved technique, the mechanical
properties of the PVA-H prepared by the new fabrication technique were found to be uncertain.
Accordingly, we have investigated the mechanical properties of novel PVA-Hs consecutively [9].
Figure 3.3 shows the tensile strength test results of PVA-H (degree of polymerization: 8800; water
content: 30%) developed in our laboratory. The mechanical strength and Young’s modulus of gels
by injection molding were all close to that of natural articular cartilage and PVA-H by freeze-
thawing, the difference of heating temperature and water content also showed no significant influ-
ence. The rheological behavior is also very important in investigating the mechanical properties of
hydrogels. Although its variability in measurement technique has been reported, we can evaluate
Err bar: ± SD
40
Tensile strength (MPa)
30
20
10
0
150°C 160°C 180°C 200°C Natural Freeze-thaw
articular method
Injection-molding method cartilage
(a) (heating temperature (°C))
25 Err bar: ± SD
Young’s modulus (MPa)
20 Natural
articular
15 cartilage
10
0
150°C 160°C 180°C 200°C Freeze-thaw
method
Injection-molding method
(b) (heating temperature (°C))
Err bar: ± SD
40 PVA-H
30
20
10
0
Water content Water content Natural articular
(c) 60% 45% cartilage
FIGURE 3.3 (See companion CD for color figure.) Mechanical properties of PVA-H prepared by injection
molding method. (a) Influence of heating temperature on ultimate tensile strength (MPa). (b) Influence of heat-
ing temperature on Young’s modulus (MPa). (c) Influence of water content on ultimate tensile strength (MPa).
PVA-H
10.0 Water content: 20%
Articular cartilage
PVA-H
Stress (MPa)
W.C. 45%
6.0
PVA-H
W.C. 90%
2.0
Meniscus
0 1.0 2.0 3.0 Strain
FIGURE 3.4 (See companion CD for color figure.) Representative stress–strain curves of various PVA-H
samples and natural tissue specimens in the compression test.
the potentiality of its viscoelastic characteristics by comparing with that of natural human tissue
[10]. Figure 3.4 shows the stress–strain curves in compression tests of various PVA-Hs in some
mechanical tests. As for the stress–strain curves of PVA-H, the appearance of viscoelastic curve
in high water content samples resembled that of natural tissue such as an articular cartilage, while
the elastic response (or solid-like response) means high Young’s modulus value dominated in low
water content. For yield stress, since PVA-H has high viscosity and does not reveal the fracture, a
definite yield point as observed in metals cannot be determined. Thus, with a gradually increas-
ing load on the PVA-H sample with a loading speed of 5 mm/min, a compression test consisting
of loading and unloading was repeatedly performed, and the load at which PVA-H deformation
remained even without loading after releasing was defined as the yield point. Figure 3.5 shows the
comparison of these yield stresses of PVA-H.
Figure 3.6 shows the results of stress–relaxation tests. PVA-H with a high water content tends to show
more marked stress relaxation. The human menisci exhibited the most acute stress relaxation. The results
PVA-H
Water content 12.6 ± 1.7
25%
PVA-H
45%
PVA-H
60%
PVA-H
90%
Meniscus
0.3 ± 0.03
specimen
0 2 4 6 8 10 12 14
(MPa)
FIGURE 3.5 (See companion CD for color figure.) Comparison of yield stress value of various PVA-H and
natural tissue specimens.
1.0
PVA-H
Stress (MPa)
Water content: 20%
0.5
Articular cartilage
PVA-H W.C. 45%
PVA-H W.C. 90%

Meniscus
0 1 2 3 4 5
Time (min)
FIGURE 3.6 (See companion CD for color figure.) Comparison of the stress–relaxation curves of PVA-H
and natural tissue specimens.
from mechanical testing indicated that changing the degree of cross-linking and water content could be
utilized as a means of achieving the desired mechanical properties, including the viscoelastic character-
istics of the PVA-H prepared by the freeze-thawing method or injection molding method.
3.4.2 Tribology of PVA-H
One of the important reasons that PVA-H has been widely studied as a biomedical application is its
excellent lubrication function. Numerous authors have already reported the lubricative mechanism of
PVA-H, which is said to be able to preserve the fluid film between articulating surface by elasto-hydro-
dynamic lubrication mode under a certain loading condition. However, boundary lubrication or solid
lubrication mode might also operate on the PVA-H as an articular cartilage substitute under the more
severe high loading such as athletic dynamic motion. In this chapter, the tribological property of PVA-H
obtained from our studies is described. The basic frictional function of PVA-H has been examined
using an end face–type friction test machine for the purpose of developing artificial cartilage [10,11].
Figures 3.7 through 3.9 explain these friction tests. Figure 3.8 shows the frictional coefficient of various
Load
Cotton
Leaf spring thread
Upper specimen
Lubricant Lower specimen
FIGURE 3.7 Cross-sectional view of friction testing machine.

Err bar: ± SD
Normal articular cartilage (sample number: n = 3)
Normal articular cartilage vs.

PVA-H (injection-molding; 150°C) (n = 3)
PVA-H (freeze-thaw method) (n = 3)
OA cartilage (1) (n = 5)
Alumina (n = 3)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

(a) Coefficient of friction
Normal articular cartilage Err bar: ± SD

OA cartilage (1) (n = 3 for each combination)
Injection-molding OA cartilage (2)
PVA-H vs.
(150°C) OA cartilage (3)
Injection-molding
PVA-H (150°C)
Injection-molding
PVA-H Injection-molding
(200°C) vs. PVA-H (200°C)
Freeze-thaw
vs. Freeze-thaw PVA-H
PVA-H
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

(b) Coefficient of friction
FIGURE 3.8 (See companion CD for color figure.) Histogram showing coefficients of friction for various
combination. (a) Friction coefficients of various materials specimens against normal articular cartilage. (b)
Friction coefficients of various materials specimens against PVA-H.
PVA-H vs. PVA-H vs.

PVA-H articular cartilage
0.08
0.07
Coefficient of friction
0.06
0.05
0.04
0.03
0.02
Saline HA 80 HA 200 Synovial fluid
(molecular 80 × 104
weight: 200 × 104)
FIGURE 3.9 (See companion CD for color figure.) Frictional coefficient of PVA-H versus articular cartilage
or PVA-H, and the influence of hyaluronic acid.
biomaterials against natural articular cartilage or PVA-H in synovial fluid simulated lubricants (saline
with 0.375% hyaluronic acid, 3.0% albumin, and 0.5% γ-globulin). All friction coefficients of PVA-H
against normal articular cartilage showed low values compared to that of articular cartilage versus artic-
ular cartilage, whereas the friction coefficients of PVA-H versus PVA-H was remarkably high. Figure
3.9 compares the friction coefficients of PVA-H versus articular cartilage, or PVA-H versus PVA-H with
each different lubricant. This result demonstrates that the coefficient of PVA-H versus PVA-H shows
high value against the dose of hyaluronic acid, while the lubrication of PVA-H versus articular cartilage
improve according to a hyaluronic acid dose. These frictional experimental data show that the adhesion
function of the lubricants such as hyaluronic acid, albumin, and γ-globulin on the PVA-H surface is
inferior to the articular cartilage surface. Therefore, as another experimental approach for tribological
function of PVA-H, the behavior of lubricating surfaces on two kinds of materials, natural articular
cartilage and PVA-H, was observed by confocal laser scanning microscopy (CLSM) [12,13].
Each specimen was placed on the table, the synovial fluid added to its surface, pressed with
a glass plate (0.15 mm thickness), and a load (12 N) applied for CLSM observation (Figure 3.10).
The compression of natural articular cartilage by a glass plate, which was equivalent to physi-
ological loading, caused the cartilage surface to exhibit two distinct areas: one in direct con-
tact area with a glass plate and another with a fluid pool between the cartilage and the plate
(Figure 3.11). Between these two areas, a third morphological area was observed, as shown
in Figure 3.12. In the area in direct contact with the glass plate, the detailed morphology in the
Laser
Weight
Grass plate
Cartilage Lubricant
Stage
FIGURE 3.10 (See companion CD for color figure.) Photograph of real-time 1LM21 confocal laser scan-
ning microscopy (CLSM) and schematic cross-sectional view of CLSM observation.
(a) (b)
FIGURE 3.11 (See companion CD for color figure.) (a) CLSM image of normal articular cartilage surface
without loading (×1400). (b) CLSM image of normal articular cartilage surface under the loading (×250). The solid
contact area, fluid pooling area with synovial fluid and intermediary area with fringe-like pattern were observed.
Intermediary area
Fluid area
Contact area 29.8 µm
(a)
Glass plate
Contact area
Fringe-like pattern
Articular cartilage
(b)
FIGURE 3.12 (a) CLSM image of normal articular cartilage surface under the loading (×1000). (b) Cross-
sectional view of CLSM image (×1000).
articular cartilage surface disappeared due to compression. In the area of the fluid pool, the
presence of a fluid membrane between the articular cartilage and the glass plate was confirmed,
but the state of the cartilage surface was unclear. In the third area, a fringe-like pattern was
observed around the contact area at higher magnification. In addition, the observation of this
area by a particular optical-isolation method revealed a reflected image that corresponded to
the third area, as shown in Figure 3.13. This finding indicates that the third area is composed
of a liquid crystal structure. In contrast, the CLSM images of the PVA-H surface under loading
were uniform, and distinguishing between the area of contact and the fluid pool was impossible,
as shown in Figure 3.14. In the cross-sectional view, the grass plate and PVA-H surface were
clear, but no images corresponding to the third area, which was seen in the articular cartilage
Fluid area
Intermediary area
Contact area
(a) (b)
FIGURE 3.13 (See companion CD for color figure.) CLSM images with optical-isolator technique about
the intermediary area. The reflection image (a) was obtained corresponding to the third area in the original
CLSM image (b).
(a)
Glass plate
Liquid layer
Polymer surface
50 µm
(b)
FIGURE 3.14 (a) CLSM image of PVA-H surface under the loading (×700). Third area and fluid pooling
area were unclear. (b) Cross-sectional view of CLSM image (×700).
specimens, were observed. The optical-isolation technique also did not reveal any image. To dis-
cuss the excellent lubricative mechanism of natural synovial joints under the high loading con-
ditions such as boundary lubrication, we must address the molecular nature of the protective
film layer that covers the articular cartilage surface. Many investigators have already proposed
the presence of this lubricin [14–16]. Our CLSM image on articular cartilage may indicate that
liquid crystal region reflected the protective film layer on cartilage surface. While PVA-H was
developed to have the same lubricative mechanism of natural joints, however, CLSM of PVA-H
did not reveal the presence of a film layer. Even though further analysis and discussion may be
demanded regarding these results, the finding means that PVA-H does not form a fluid film nor
liquid crystal, as does natural articular cartilage, and PVA-H has the limitation as an artificial
cartilage to imitate the lubricative mechanism of synovial joint cartilage.
3.5 APPLICATION FOR ARTHROPLASTY IMPLANT

3.5.1 Development of Artificial Articular Cartilage
Arthroplasty using implants as used in total hip joint arthroplasty (THA) and total knee joint
arthroplasty (TKA) is an established treatment for patients with severe arthrosis caused by diseases
such as osteoarthritis (OA) and rheumatoid arthritis (RA). However, the tolerance of artificial joints
to physiological loading is inferior to that of natural joints, and the number of revision operations
performed to adjust for loosening or wear of these implants has been increasing. The long-term
survival of THA and TKA is a significant problem in orthopedic surgery [17,18].
In contrast, it is known that natural joints have excellent lubrication. Though the mechanism
involved is not still clear, tribological studies, biomechanical studies, and morphological studies
have proposed various theories to clarify the mechanism behind the superior lubricative function of
natural joints. If the mechanism of natural joint lubrication could be imitated by artificial materials,
the properties of artificial joints can be much improved. In this respect, Oka et al. have attempted
to develop an artificial articular cartilage based on PVA-H. Initially, we thought that the medical
application as an artificial articular cartilage by PVA-H had a high potential as we were able to get
favorable in vitro data [4,19] and many studies had already reported the superior lubricative proper-
ties of PVA-H [20,21]. However, as shown in previous section, the frictional test results of PVA-H
exhibited high frictional coefficients and much wear, enough to predict the failure of arthroplasty
by PVA-H. Although the mechanical property of PVA-H was improved for medical application, the
use of lubrication remains a problem.
3.5.2 Development of Artificial Articular Cartilage as a Hemi-Arthroplasty

The development of total arthroplasty by PVA-H has some key issues. However, as mentioned
earlier, the friction coefficients of PVA-H against normal articular cartilage have shown low
values in frictional tests (4.2: tribology of PVA-H). This finding encourages us to attempt to use
PVA-H as a medical implant for partial hemi-arthroplasty replacement. Osteonecrosis of the
femoral head is a disabling disease that can lead to destruction of the hip joint. The appropriate
treatment, depending on the stage of the disease, remains controversial, yet progression to col-
lapse of the femoral head often necessitates total hip replacement (THR). Considering the age
of patients and the poor prognosis associated with THR, it is desirable to preserve as much of
the joint as possible during treatment as same as the concept of an artificial cartilage for osteo-
arthritis [22–24].
We have developed an articular cartilage composite device with PVA-H, which have character-
ized the mechanical properties and lubricating functions, and a titanium fiber mesh, which is used
to attach PVA-H firmly to underlying bone as shown in Figure 3.15 [25]. The PVA-H used was
polymerization degree 8800, water content 30%, which corresponds to the mechanical properties of
natural articular cartilage in vitro.
This composite has been implanted into the surface of canine femoral head for the purpose of
simulating partial surface replacement hemi-arthroplasty, as shown in Figure 3.16. In addition to
this PVA-H device, prostheses made from UHMWPE and alumina ceramics were also implanted
as a control group to compare with the present implant material. The dogs with implant were
allowed unrestrained weight-bearing and normal activity in their living area, and killed at time
intervals of 1, 3, 6, and 12 months. Dogs with all three types of devices showed good locomo-
tive function of the hip joint without dislocation, deformity, or limping during their survival
periods. Concerning the endurance of the PVA-H prosthesis, the transected specimens showed
good congruity of the PVA-H with the adjacent natural articular cartilage. Figure 3.17 shows the
histological and radiographic findings at 12 months after operation. It shows that the reduction
PVA-H
Titanium-fiber
FIGURE 3.15 (See companion CD for color figure.) Photograph of a synthetic composite osteochondral
device for partial replacement of the canine femoral head. The transparent portion is made of PVA-H.
Artificial PVA-H
Natural articular (artificial articular cartilage)
articular cartilage
cartilage
Articular (PVA-H) Titanium fiber mesh
cartilage
(a) (b)
FIGURE 3.16 (See companion CD for color figure.) Photograph of a specimen of the femoral head with
composite osteochondral device at 12 months after implant operation. (a) Macroscopic appearance; (b) cross-
sectional appearance.
(a) (b)
FIGURE 3.17 (See companion CD for color figure.) Cross-sectional view of a specimen of the femo-
ral head with composite osteochondral device at 12 months after implant operation (×2.5). (a) Histological
appearance (Giemusa stain). (b) Contact microradiograph appearance.
and position of femoral head were well maintained, and there was an increase in bone density
around the stem portion of the titanium fiber mesh and good fixation to the surrounding bone. To
evaluate the lubrication function of the PVA-H prosthesis, opposite canine acetabular articular
cartilages were also reviewed macroscopically and histologically. No adverse responses, such as
erosion or hemorrhage, were observed in the macroscopic appearance of the articular cartilage
of acetabulum of the PVA-H group, though a slight synovitis and discoloration in the alumina
group was observed. In the UHMWPE group, there was severe synovitis and erosion of the
acetabular cartilage 6 months after the operation. Histological evaluation of acetabular articular
cartilage was graded using the Mankin’s score [26]. These results are summarized in Figure 3.18.
The PVA-H group acetabular cartilage showed that structural integrity, surface regularity, and
thickness were well maintained throughout the experimental periods, whereas the histological
appearance of the other material groups deteriorated depending on the experimental periods.
Although this study has limitations as an evaluation, we think these results demonstrate the
superior potential of the clinical partial surface replacement hemi-arthroplasty using PVA-H
articular cartilage.
15
Alumina ceramics
10 UHMWPE
Score
PVA-H
0
1 3 6 12
Months
FIGURE 3.18 (See companion CD for color figure.) Mankin’s score for the changes in acetabular cartilage
in each group. Each point represents a single score for a joint.
3.6 DEVELOPMENT OF ARTIFICIAL MENISCUS

As well as the articular cartilage substitute for hemi-arthroplasty, the favorable lubricative function
of PVA-H against articular cartilage seems promising for the development of artificial meniscus.
Basic and biomechanical studies on the meniscus function of knee joint have suggested the impor-
tant role of meniscus, such as stabilization of knee joint movements, shock absorption, and stress
dispersion of loading, and there is an increasing understanding of the significance of meniscus
[27–29]. Furthermore, many clinical reports have also indicated the articular degeneration cases in
meniscetimized patients. Nowadays, it is well known that meniscectomy results in osteoarthritis
changes in articular cartilage [30–32].
Thus, the treatment of meniscus injury has been changed from meniscectomy to repair. However,
depending on the type of meniscus injury, meniscectomy cannot be avoidable. However, meniscus
implant replacement is the most favorable treatment for the reconstruction of meniscus function.
Thus, we have developed artificial meniscus using PVA-H [33,34]. The PVA-H used as an artificial
meniscus should have two major characteristics: (1) mechanical properties and (2) tribological
properties. The mechanical property of meniscus tissue is different from that of articular carti-
lage. The microstructural characteristics of the meniscus determine the mechanical properties.
Meniscus are composed of 70% water and 30% organic matter. Type-I collagen fibers constitute
75% of the organic matter, which provides the primary meniscal structural scaffolding. This type-I
collagen is one of the major differences between the menisci and hyaline or articular cartilage,
which is composed of predominantly type-II collagen. The remaining organic matter is composed
of proteoglycans. The proteoglycan aggregates make up only 1% of the wet weight of meniscus
but contribute most to mechanical properties such as compressive stiffness, hydration, and visco-
elasticity. The stress–strain curve and the stress–relaxation curve of human meniscus express the
typical viscoelasticity. Although PVA-H cannot fully substitute all the mechanical functions of
meniscus, the viscoelastic characteristics similar to human’s meniscus could be provided by adjust-
ing the water content of PVA-H in the gel production process. As for the lubricative function of
PVA-H meniscus, previous studies have proved favorable tribology of the PVA-H versus articular
cartilage. Based on these biomechanical data, we have selected high water content PVA-H (water
content: 90%, degree of polymerization: 17,500) as an implant sample and performed the in vivo
implantation experiment. The bilateral knees of rabbits were used, and PVA-H artificial meniscus
replacement was performed in the lateral compartment of one knee, with the lateral meniscectomy
in the other knee as a control (Figure 3.19). After operation, the rabbits were reared in cages, and
time-dependently sacrificed after a certain period of time. The status of the knee joint cartilage
and the degree of injury in the artificial meniscus were observed. The postoperative observation
period was 6 months, 1 year, 18 months, and 2 years. Figure 3.20 shows the macroscopic state
Natural meniscus
PVA-H meniscus
Artificial
meniscus
FIGURE 3.19 (See companion CD for color figure.) Photograph of PVA-H artificial meniscus and the
schema of artificial meniscus replacement.
Control
Time P.O. 6 month P.O. 1 year P.O. 18 month P.O. 2 year
Artificial meniscus
FIGURE 3.20 (See companion CD for color figure.) Macroscopic appearance of articular cartilage of rab-
bit’s knee joint up to 2 years after operation. In both groups, lateral side of knee joint was operated. In the con-
trol group, OA change appears in post operative 6 months, remarkable change becomes obvious in P.O. 1 year.
of the articular cartilage surface of the femoral condyles and tibia in rabbits knees replaced by
PVA-H artificial meniscus and the control knees. Six months after the operation, the erosion and
degeneration in the articular cartilage were noted in both group knees, and the degree of degen-
erative change was more marked in the control group. After 1 year, no more progressive osteoar-
thritis (OA) changes were observed in the artificial meniscus group, whereas more severe OA-like
changes such as partial disappearances of the cartilage surface and bone changes such as elevated
osteophyte were observed in the control knee group. Two years after the operation, articular carti-
lage in the control group showed progressive OA changes. On the other hand, all PVA-H artificial
menisci were attached to tibia side, and no breakage, displacement, or deformation of PVA-H was
observed. In addition, the color and texture of PVA-H were not seriously different from intact
PVA-H. Most of the articular cartilage surfaces also exhibit normal condition or only slight erosion
in the artificial meniscus group. These macroscopic changes in articular cartilage were assessed
using the scoring system concerning the degree of OA progress as shown in Figure 3.21. In the
control group, OA progressed with time; however, slight cartilage damage remained without pro-
gression of degenerative change in the artificial meniscus group between the follow-up period. On
the other hand, all PVA-H artificial menisci were attached to peripheral tissue, and no breakage,
dislocation, or deformation of PVA-H was observed. The color and texture of gels were also not
markedly different from intact PVA-H (Figure 3.22).
Control Artificial meniscus
10
8
Score
0
6 Month 1 Year 18 Month 2 Year
Time
FIGURE 3.21 (See companion CD for color figure.) Macroscopic evaluation of articular cartilage accord-
ing to classification by Chang.
(a) (b)
FIGURE 3.22 (See companion CD for color figure.) Macroscopic appearance of articular cartilage of
rabbit’s knee joint in 2 years after operation in PVA-H meniscus replacement group. (a) Neither breakage nor
dislocation of PVA-H is observed. (b) Articular cartilages of knee joint are also intact.
Figure 3.23 shows the microscopic appearance of articular cartilage of both groups 2 years after
the operation. In the control group, the cartilage layer was almost absent. In even survival cartilage
layer, the stainability was poor and the presence of chondrocytes was not clear. In the PVA-H menis-
cus group, abrasion and regressive changes in the cartilage layer were observed in some parts, but
the cartilage layer itself was almost similar to normal cartilage. These histological findings depen-
dent on postoperative time were scored numerically as shown in Figure 3.24 by Mankin’s scoring
system. With the observation time, a significant difference appeared in the degree of progression of
OA change in articular cartilage between the PVA-H artificial meniscus group and the control group.
Tibial side Femoral side

Control

Artificial meniscus
(a)

Control

Artificial meniscus
(b)
FIGURE 3.23 (See companion CD for color figure.) Histological appearance of articular cartilage of knee
joint 2 years after operation (×20, Safranin-O stain). (a) P.O. 1 year; (b) P.O. 1.5 year;
(continued)

250 µm
Control
250 µm
Tibial side 250 µm Femoral side

Artificial meniscus
250 µm
(c)
FIGURE 3.23 (continued) (See companion CD for color figure.) (c) P.O. 2 years.
Number Number
P.O. 24 month
P.O. 12 month
P.O. 6 month
15
10
Score
5
1
Control group Artificial meniscus group
(by Mankin’s grading system)
FIGURE 3.24 (See companion CD for color figure.) Histological evaluation of articular cartilage accord-
ing to Mankin’s grading system.
These results indicate that PVA-H with high water content is a promising material as an artificial
meniscus implant that reconstruct meniscus function and prevent OA changes in articular cartilage.
Further studies are needed to validate the PVA-H implant fixation method for clinical application.
3.7 DEVELOPMENT OF IMPLANT FOR SPINE SURGERY

3.7.1 Development of Artificial Intervertebral Disk
As regards the advantage of PVA-H that changing the degree of cross-linking and water content
could be utilized as a means of achieving the desired mechanical properties of gel, the application
as an intervertebral disk substitute that does not have lubrication issues would be favorable. Lesion
of the intervertebral disk is one of the most common causes of spinal disorders. The function of the
intervertebral disk is not only to stabilize the spine but also to give the spine its flexibility.
Spinal fusion is one of the most frequently performed procedures in spinal surgery. Spinal fusion,
although it restores stability, does not preserve flexibility, and biomechanical and clinical studies
have reported that it increases stress on the adjacent segments, leading to accelerated degeneration
in the long term. Thus, development of an artificial intervertebral disk (AID) to restore both the
stability and flexibility of the disordered spine is desirable [35,36].
PVA-H
TB
(a) (b)
FIGURE 3.25 (See companion CD for color figure.) (a) Diagram of artificial intervertebral disk for beagle
dog. (b) Macro appearance of the canine spine implanted with artificial intervertebral disk.
We have developed the AID made of PVA-H and titanium fiber mesh, and studied the mechani-
cal and biological behavior in vitro and in vivo [37,38]. Figure 3.25 shows the diagram and macro
appearance of AID for a beagle dog in animal experiment. A specially designed AID composed of
titanium fiber mesh and PVA-H was manufactured for canine lumbar vertebrae. To attach PVA-H
and titanium fiber mesh, PVA-H in fluid phase was permeated into the pores of titanium fiber mesh
under low pressure conditions, and these two materials were mechanically bonded together by
gelling the PVA. The degree of polymerization of PVA was 8800 and the water content of hydro-
gels was 30% and 50% according to the mechanical strength of canine lumbar intervertebral disk.
Adult beagle dogs were operated, and the lumbar spine L5/6 intervertebral disk was replaced by
AID. These dogs were sacrificed at 1, 3, 6, and 12 months after the operation, and the samples
were evaluated by histological method and radiographic examination. All the dogs survived with-
out any postoperative complication such as neuroparalysis. Figure 3.26 shows the radiographs of
Vertebral body
Bone ingrowth
into TFM
PVA-H
FIGURE 3.26 (See companion CD for color figure.) Contact microradiography findings of the artificial
intervertebral disk implanted into dog 1 month after operation. Massive bone ingrowth into the pores of
titanium fiber mesh was observed.
Failure
FIGURE 3.27 (See companion CD for color figure.) Micro-appearance of the interface between PVA-H
and titanium fiber mesh of artificial intervertebral disk at 6 months after operation into canine spine.
the lumbar spine implanted with this AID prosthesis in 3 months after the operation. Massive
bone ingrowth into the pores of the titanium fiber mesh was observed, with the implant attaching
firmly to the vertebral body. However, prosthesis failure occurred in some samples at 6 and 12
months after the operation (Figure 3.27), which were observed at the interface between PVA-H and
titanium fiber mesh. Furthermore, excessive osteophytes also were formed in the vertebral body
implanted with AID.
These phenomena have been observed at postoperative more than 6 months. We presume that
osteophyte was caused by the instability of spine with prosthesis due to the insufficient bonding
strength of PVA-H with titanium fiber mesh and low mechanical strength of PVA-H. Interface fail-
ure between PVA-H and titanium fiber mesh occurred.
In order to improve the mechanical properties and strength of PVA-H and the bonding strength
of the interface between titanium fiber mesh and PVA-H, we tried to use high-pressure injection
molding in PVA-H synthesis process. Using an improved method, a higher concentration of a stron-
ger PVA-H resulted. As a result, the AID with new PVA-H can solve these problems until postopera-
tive 6 months in animal experiment. Further studies are needed to assess the durability of implant
for this clinical application.
3.7.2 Application of Artificial Intervertebral Disk Nucleus Implant

Although the AID using PVA-H and titanium fiber mesh is useful, its treatment is invasive and
tasks on the patient in the view of concrete orthopedic surgical technique. It is more desirable if an
equivalent effective and the minimum invasive implant was achieved.
For the purpose of developing minimum-invasive treatment for spine disorders, Sou et al.
studied the intervertebral disk nucleus replacement using PVA-H implant [39]. The mechanical
properties and strength of PVA-H were controlled by changing the degree of polymerization and
water content of hydrogel. An AID nucleus similar to the mechanical properties of natural disk
nucleus has been developed. Figure 3.28 shows the histological appearance of a canine spine spec-
imen 3 months after the intervertebral disk nucleus replacement. In vivo data displayed neither
inflammation reaction nor displacement or breakage of PVA-H prosthesis. The minimum-invasive
replacement using this PVA-H seems to be a promising technique in the treatment of interverte-
bral disk hernia. Further evaluations regarding the in vivo results in the long term are needed for
clinical application.
Intervertebral
disk
PVA-H disk
nucleus
Vertebral body
FIGURE 3.28 (See companion CD for color figure.) Histological appearance of the partial disk replace-
ment by PVA-H artificial disk nucleus in an animal surgery.
3.8 FUTURE AND POTENTIAL OF PVA-H

PVA-H research and its development for clinical application as an orthopedics surgery implant
have been outlined in this chapter so far. The biocompatibility and in vivo safety of PVA-H can be
confirmed when taking into consideration previous studies. Furthermore, an advantage of PVA-H
is that a tissue substitute with appropriate mechanical properties for the desired tissues can be pre-
pared by adjusting the water content and the degree of polymerization.
Considering the other characteristic functions of PVA-H, such as permeability, hydrophilicity,
and transparency, depending on ideas and ingenuity, PVA-H is an extremely promising material in
not only orthopedic surgery but also other medical fields; however, in view of the new developments
in current biomaterial research, the future development of PVA-H will highlight various problems
in its application.
3.8.1 Problems with PVA-H Fixation Techniques

The greatest challenge regarding the future clinical application of PVA-H, whereby a method that
enables its fixation to living tissues is shown, has already been discussed earlier. PVA-H has strong
bio-inert properties, that is, it hardly adheres or binds to the living body. As mentioned earlier, in
order to couple PVA-H implants with a living bone, we tried to conjugate them with titanium fiber
mesh (Figure 3.29) [40–43]. Other reports have already confirmed the growth of the newly formed
bone into titanium mesh. If this method proves to be favorable to fix PVA-H and titanium mesh, then
PVA-H
Titanium
fiber mesh
Bone
ingrowth
(a) (b)
FIGURE 3.29 (See companion CD for color figure.) Histological appearance of composite device at
12 weeks after implantation of canine femoral condyle. Mature bone has grown into the pores of titanium
fiber mesh. (a) Giemusa stain (×10); (b) contact microradiograph (×10).
we can expect this method to be in practical use in the future. In order to achieve strong binding
between PVA-H and titanium mesh, a PVA solution injection molding method has been developed;
however, the conjugation of PVA-H with titanium mesh is first and foremost similar to joining
machine parts, and an uncertainty remains regarding its long-term fixation in vivo. However, as
research on the bioactivation of biomaterial surfaces and the interaction between artificial mate-
rial surfaces and the living body has made great progress in recent years, for this fixation method
between the living body and PVA-H, the development of other adhesion techniques with the liv-
ing body is greatly anticipated. As a result of a large number of researches, adhesion mechanisms
between artificial material surfaces and the cells of living tissues are already being gradually elu-
cidated and, using special surface treatment protein, adhesion has become possible even on bio-
material that was believed to be inert (stable) to living tissues; as a result, techniques enabling the
adhesion of living cells to biomaterial are being reported [44,45]. The greatest advantage of this
adhesion technique is that as long as the material surface has not lost the inducing functions of liv-
ing tissue, restoration and recombination by biological reactions can be expected even in the case of
rupture and damage to a portion of the adhesion site, which is a major stepping stone in solving the
problem of long-term fixation. As regards PVA-H, the report that adhesion to soft tissues is possible
using surface treatment has been addressed [46]. Other methods for bioactivation of the PVA-H gel
surface are also considered.
3.8.2 Future Possibilities of PVA-H

Currently, not only in the field of orthopedic surgery, but in many fields for the in vivo restoration
of soft tissues, tissue regeneration by regenerated medicine is attracting attention, and research is
thriving [47–49]. If tissue regeneration should advance successfully, it would be an ideal treatment
method as it assimilates with auto-tissue and has promising potential. However, specific control
techniques by which prepared regenerated tissues up to the micro-cell level are identical to the
desired tissues have not been established, and there is a long-term constant risk of carcinogenicity
and teratoma. Furthermore, in addition to such overall problems of regenerated medicine, there
are the unique problems regarding its application in the field of orthopedic surgery. The most
important problems that are needed to be assessed are how to handle a mechanical environment,
taking into consideration the biomechanics and the problem of a restoration period, considering
the time required for recovery with reduced mobility. The mechanical properties of orthopedic
surgery implants, which place a great deal of importance on biomechanical functions, present
major problems. Even in cartilage regeneration, in which current research is more advanced, the
fine viscoelastic properties of regenerating cartilage still often differ from the original cartilage.
Furthermore, in the actual treatment of arthropathy, which replaces THA and TKA, not only the
mechanical properties of the cartilage itself but also the tribology of joints and the shape of the
sliding surface must be taken into consideration. It is widely known that pathological joint defor-
mities can develop even in healthy normal cartilage as a result of a slight mechanical centralized
load and distortion of the alignment of the small articular surface. Whether it is possible to prepare
joints with an alignment that can maintain for a long period the same lubricating functions as
healthy natural joints, similar to the problem with THA and TKA, greatly depends on the fixation
of the regenerated cartilage over the whole articular surface. Another problem is the time needed
for tissue regeneration and the recovery of motor functions. Orthopedic surgery aims at the recov-
ery of motor functions, but this is difficult to achieve merely by surgical procedures using implants
and scaffolding material alone. Exercise from an early stage that considers rehabilitation is neces-
sary, and, from this aspect, the time required for tissue self-regeneration is an important factor in
functional recovery. Elderly people often suffer from femoral neck fractures, and implant replace-
ments, such as femoral head replacement, are performed early because of the reduced recovery
functions of the elderly and the problem of timing constraints for the early recovery of motor func-
tions. If regenerated medicine techniques advance in the future, such clinical issues will likely be
overcome, but in the short term, the advantages of artificial materials, which can recover motor
functions, can hardly be abandoned. On the other hand, it is expected that regenerated tissues
derived from artificial materials, such as PVA-H, and tissue engineering will not only be per-
ceived from the viewpoints of comparison and competition but will be expected to be developed
as treatment methods using new implants that combine and compensate for the advantages and
shortcomings of regenerated tissues derived from artificial implants and regenerative medicine.
Research is being conducted to use PVA-H with high mechanical durability as scaffolding material
with mechanical stability by using its properties as a gel in greater proportions, and hybridization
with other polymer molecules [50,51]. In scaffolding material, the material is ultimately degraded
and absorbed and substituted by regenerated tissue, and it is possible that by replacing a portion
of the implant with regenerated tissue and leaving a portion of the implant as artificial material in
the body, engineered tissues with additional new functions can be developed. For example, when
using regenerated cartilage instead of TKA, it is difficult to construct and retain the shape of the
knee joint surface directly, but it is possible to develop surface-substituted engineered artificial
cartilage, which combines the mechanical properties and shape by constructing the shape of the
original knee joint and covering the surface with regenerated cartilage using PVA-H, etc., which
is bioactivated on the PVA-H surface mentioned earlier, as a matrix. In the future, together with
developments in medical technology, not only in the field of orthopedic surgery, new methods for
implant treatments with combined characteristics will be developed for each clinical department
from the perspective of biomaterial and tissue engineering. As far as PVA-H is concerned, it will
be necessary to include the required new functions using our accumulated knowledge and tech-
niques, considering clinically specific treatment methods.
ACKNOWLEDGMENTS
This work was supported by the Department of Medical Engineering, Institute for Frontier Medical
Sciences, Kyoto University. The author would like to express special thanks to all staff including
Dr. S.-H. Hyon and Dr. Matsumura for their kind help and useful advice.
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4 Polyphosphazenes
as Biomaterials
Meng Deng, Cato T. Laurencin,
Harry R. Allcock, and Sangamesh G. Kumbar
CONTENTS
4.1 Introduction.............................................................................................................................84
4.2 Synthesis of Polyphosphazenes............................................................................................... 85
4.2.1 Ring-Opening Polymerization..................................................................................... 85
4.2.1.1 Mechanism of Ring-Opening Polymerization.............................................. 86
4.2.1.2 Thermodynamics of Ring-Opening Polymerization.................................... 86
4.2.2 Living Cationic Polymerization................................................................................... 87
4.2.2.1 Reaction Mechanism of Living Cationic Polymerization............................. 87
4.2.3 Macromolecular Substitution....................................................................................... 88
4.2.4 Other Synthesis Methods............................................................................................. 89
4.2.5 Polyphosphazene Architecture.................................................................................... 89
4.2.6 Structure–Property Relationships............................................................................... 89
4.3 Polyphosphazenes as a Versatile Class of Biomedical Materials............................................92
4.3.1 Biodegradable Polyphosphazenes................................................................................92
4.3.1.1 Aminated Polyphosphazenes........................................................................ 93
4.3.1.2 Alkoxy-Substituted Polyphosphazenes.........................................................94
4.3.1.3 Polyphosphazene Blends/Composites...........................................................94
4.3.2 Degradation of Biodegradable Polyphosphazenes......................................................99
4.3.2.1 Polymer Degradation Mechanisms...............................................................99
4.3.2.2 Degradation of Aminated Polyphosphazenes............................................. 101
4.3.2.3 Degradation of Alkoxy-Substituted Polyphosphazenes.............................. 105
4.3.2.4 Degradation of Polyphosphazene Blends/Composites................................ 105
4.3.3 Biocompatibility of Biodegradable Polyphosphazenes............................................. 108
4.3.3.1 Biocompatibility of Aminated Polyphosphazenes...................................... 108
4.3.3.2 Biocompatibility of Alkoxy-Substituted Polyphosphazenes....................... 109
4.3.3.3 Biocompatibility of Polyphosphazene Blends/Composites........................ 109
4.3.4 Biodegradable Polyphosphazenes for Biomedical Applications............................... 110
4.3.4.1 Drug Delivery............................................................................................. 110
4.3.4.2 Tissue Engineering..................................................................................... 118
4.4 Conclusions and Future Trends............................................................................................. 125
Acknowledgments........................................................................................................................... 126
References....................................................................................................................................... 126
83
4.1 INTRODUCTION
Biomaterials are generally “substances other than food or drugs contained in therapeutic or
diagnostic systems that are in contact with tissue or biological fluids” (Langer and Peppas 2003).
Synthetic or natural materials are thus designed to interface with biology and have been widely
used for a number of biomedical applications including drug delivery devices, temporary prosthe-
ses, and tissue engineering scaffolds (Langer and Peppas 2003; Ratner and Bryant 2004). In con-
trolled drug delivery, a biodegradable matrix that can be loaded with different biologically active
molecules is preferred to release various therapeutic agents in an erosion- or diffusion-controlled
fashion or a combination of both. For tissue engineering applications, a three-dimensional (3D)
biodegradable scaffold that mimics the properties of various tissues is required to support cellular
activities and promote neo-tissue formation. Even though biomaterials have already contributed
greatly to the improvement of human health, the need for better biomaterial systems is still increas-
ing (Ratner and Bryant 2004).
Among the existing biomaterials, biodegradable polymeric materials have played a paramount
role in the development of drug delivery systems and tissue engineering scaffolds due to their
biodegradability and inherent biocompatibility (Peppas et al. 2003; Nair and Laurencin 2007).
Biodegradable polymers including both natural and synthetic polymers degrade into low molecu-
lar weight fragments via hydrolysis or enzymolysis under physiological conditions (Nair and
Laurencin 2007). Synthetic biodegradable polymers are attractive, since they offer unique advan-
tages such as predictable properties and ease of tailoring for specific applications. Currently,
some major classes of synthetic biodegradable polymers include polyesters, poly(α-amino
acids), poly(ortho esters), and polyanhydrides (Nair and Laurencin 2007). Among them, ali-
phatic polyesters such as polymers of lactic (PLA) and glycolic (PGA) acid or their co-polymers
poly(lactide-co-glycolide) (PLAGA) are the most commonly investigated biodegradable poly-
mers. However, these polymers are associated with several distinct drawbacks. For example, they
degrade via unstable backbone ester hydrolysis into the corresponding acids, which can com-
promise the structural integrity and potentially affect biocompatibility both in vitro and in vivo
(Taylor et al. 1994; Agrawal and Athanasiou 1997; Böstman 1998; Fu et al. 2000). One example
is that in anatomical sites such as articular cartilage, a significant accumulation of these acidic
degradation products could occur and affect the cells and the tissues surrounding the implants
(Taylor et al. 1994; Böstman 1998). Furthermore, it is possible that these acids can inactivate the
sensitive molecules such as proteins used for drug delivery applications (Yang and Alexandridis
2000; Zhu et al. 2000). In order to overcome these limitations, PLA or PLAGA with high lac-
tide content has been investigated for various medical applications. However, several clinical
complications have been reported (Böstman 1998; Böstman and Pihlajamäki 2000; Landes et al.
2006). Additionally, the bulk erosion of polyesters often yields uncontrollable release kinetics
and catastrophic implant failure, which is not desirable for drug delivery and tissue engineer-
ing applications. These studies have propelled researchers to search for and develop alternative
biodegradable polymers with controlled degradation, nontoxic degradation products, appropriate
physicochemical and biological properties that match the requirements of each specific medical
application. Thus, polyphosphazene polymers that offer the combination of synthetic flexibility
and modulated properties (i.e., controlled degradability and biocompatibility) are highly pre-
ferred for these biomedical applications.
This chapter provides the fundamental knowledge of polyphosphazene synthesis, and dis-
cusses the types of biodegradable polyphosphazenes, the mode of degradation mechanisms,
and the inherent characteristics of biocompatibility. It further covers the medical applications
of biodegradable polyphosphazenes with a specific focus on the use of polyphosphazenes as
attractive candidate materials for developing drug delivery matrices and tissue engineering
scaffolds.
Polyphosphazenes as Biomaterials 85
4.2 SYNTHESIS OF POLYPHOSPHAZENES

Polyphosphazenes, a relatively new synthetic polymer class, are inorganic–organic hybrid polymers with
a backbone of alternating phosphorus and nitrogen atoms and with each phosphorus atom bearing two
organic or organometallic side groups (Allcock 1972b; Allcock et al. 1972; Allcock and Pucher 1991).
The general structure for polyphosphazenes is shown as Structure 4.1 (Allcock et al. 1977,
1994b; Nair et al. 2003). Nitrogen and phosphorus atoms on the backbone are linked by alternating
single and double bonds. Each phosphorus atom is substituted with two side groups R (organic or
organometallic or a combination of different functional groups). Generally polyphosphazenes are
high molecular weight compounds, and their physicochemical and biological properties are greatly
dependent on the nature and composition of the substituted side groups (Allcock 1967, 1972a;
Allcock 2003). So far, more than 700 polyphosphazenes with diverse properties have been reported
for a variety of applications including drug delivery and tissue engineering applications.
4.2.1 Ring-Opening Polymerization

The synthesis of polyphosphazenes was attempted by H.N. Stokes in 1895 via thermal ring-opening
polymerization of hexachlorocyclotriphosphazene ([NPCl2]3) (2; Scheme 4.1). But the product obtained
was an insoluble cross-linked rubbery material which was also hydrolytically labile and decomposed
into phosphates, ammonia, and hydrochloric acid upon the contact with moisture. The polymer would
N P
n
R
1
STRUCTURE 4.1 General structure of polyphosphazenes. R can be organic or organometallic or a combina-

tion of different functional groups.
Cl Cl
P
N N 250°C Cl
Cl Cl
P P Vacuum
N P
Cl N Cl
n
2
Cl
3
25°C
CH3 Cl PCl5
H3C Si N P Cl
CH3 Cl
4
SCHEME 4.1 Synthesis of the prepolymer poly(dichlorophosphazene) (3) via thermal ring-opening polym-
erization of trimer hexachlorocyclotriphosphazene (2) or living cationic polymerization of a phosphoranamine
monomer (4).
not find technological application for another 70 years because of its insolubility and hydrolytic insta-
bility (Allcock 1985). In 1965, Allcock and Kugel reported the first successful synthesis of linear
poly(dichlorophospazene) (3; Scheme 4.1) by carefully controlling the time and temperature for the
thermal ring-opening polymerization of trimer 2 (Allcock and Kugel 1965). Due to the highly reactive
and polar phosphorus-chlorine bonds (P-Cl), polymer 3 is hydrolytically unstable. Allcock’s group
synthesized the first hydrolytically stable, soluble, and high molecular weight polyphosphazene by
replacing the chlorine atoms with organic or organometallic nucleophiles in 1966. Different classes
of polyphosphazenes were then prepared by replacing chlorine atoms of the polydichlorophosphazene
intermediate by alkoxide or aryloxide (Allcock et al. 1966), primary or secondary amines (Allcock
and Kugel 1966), or organometallic reagents (Allcock and Chu 1979). In addition to this versatile
synthetic flexibility, another great advantage of polyphosphazene chemistry lies in the existence of
small molecular analogs of the high polymer for use as reaction and structural models (Allcock 1979).
Through a melt polymerization process, the thermal ring-opening polymerization produces high
molecular weight polymer 3 with an average of ∼15,000 repeating units (Allcock 2003). Briefly,
the trimer 2 is purified by recrystallization from heptane followed by sublimation. The sublimated
trimer 2 is then sealed in an evacuated glass tube and heated to 250°C for polymerization. The
polymerization is completed when the melt has become highly viscous. The sealed reaction tube
is then cooled to room temperature and broken carefully to harvest its contents. Pure polymer 3 is
isolated from the trimers and oligomers by sublimation. The polymerization step must be monitored
carefully because cross-linking can occur if the melt becomes too viscous. A high conversion from
cyclic trimer to polymer could also result in cross-linking. It is very important to limit the exposure
of the polymer synthesis to moisture because hydrolysis and cross-linking would occur. The melt
polymerization route generally produces polymer 3 with molecular weights over 1000 kDa.
4.2.1.1 Mechanism of Ring-Opening Polymerization

Ring-opening polymerizations are widely used to convert cyclic small molecules into linear polymers
(Allcock et al. 2003b; Odian 2004). The ring-opening polymerization for polyphosphazenes is a two-
step process, which involves the initial activation of trimer ring 2 and subsequent chain propagation
(Allcock 2003). At high temperature, ionization of a chlorine atom from the phosphorus atom occurs
and results in the formation a phosphazenium cation. In the second step, the formed phosphazenium cat-
ion attacks another trimer ring, which leads to chain cleavage of the attacked trimer and the consequent
transfer of positive charge to another phosphorus atom. The charge transfer process propagates to form
a linear chain. Since a ring structure is broken, the propagation rates are slow and often result in slower
molecular weight increases (Odian 2004). The chain continues to grow until all the trimers are used. If
the P-Cl unit in the middle portion of the polymer chain undergoes ionization, branching and growth
occur with the side chains. In addition, the presence of moisture results in cross-linking of the polymer.
4.2.1.2 Thermodynamics of Ring-Opening Polymerization
The ring-opening polymerization is governed by the fundamental thermodynamics. The change in
the Gibbs free energy (ΔG) for the polymerization reaction can be expressed as follows:
∆G = ∆H − T∆S
where
ΔH represents the enthalpy change
ΔS represents the entropy change
T is the reaction temperature
In general, the enthalpy change (ΔH) for most ring-opening polymerization is relatively small, since
the smaller rings, larger rings, and polymer chains are composed of the same repeating units as tri-
mer ring. Specifically in the ring-opening polymerization for polyphosphazenes, the bond angle of the
cyclic trimers should be around 109.5°. However, in a six-membered ring, the –N═P−N═bond angle is
increased to 120°. As the trimer is converted to linear polymer, the strain is released and hence ΔH would
be negative (Allcock 2003). Furthermore, the conversion of rigid trimer molecules to a flexible linear
polymer contributes to a slight increase in the entropy. However, there is a decrease in the translational
entropy when small molecules are converted into large macromolecules. Therefore, the temperature T is
a critical factor in determining the reaction process. At lower or moderate temperatures, ΔH is dominant
and ΔG is negative, which favors the polymerization. However, at higher temperatures, the contribution
of TΔS becomes dominant and ΔG is positive, which results in depolymerization. The transition temper-
ature at which a polymerization reaction shifts to depolymerization is called the “ceiling temperature.”
4.2.2 Living Cationic Polymerization

Although ring-opening polymerization is the most fully developed and commonly used method to syn-
thesize high molecular weight polymers (Allcock et al. 1977, 1994b; Nair et al. 2003), this process is
often associated with little or no control over molecular weight and with high polydispersities. Thus,
several attempts were made to synthesize polymer 3 with controlled molecular weight and small poly-
dispersities (Neilson and Wisian-Neilson 1988; Potin and De Jaeger 1991). For example, De Jaeger et al.
synthesized polymer 3 from dichlorophosphynoyliminotrichlorophosphorane Cl3P═N−P(O)Cl2 via
condensation polymerization at 240°–290° with POCl3 as by-products (Helioui et al. 1982; De Jaeger
et al. 1983). A certain degree of molecular weight control was achieved, but the limitations included the
necessity for high temperatures, the production of corrosive side products, and high polydispersities.
It was not until the early 1990s that Allcock, Manners, and co-workers reported a new method that
allows the synthesis of the macromolecular intermediate with molecular weight control and small poly-
dispersities by living cationic polymerization of phosphoranimines at ambient temperature (Scheme
4.1) (Honeyman et al. 1995; Allcock et al. 1996, 1997a,b; Nelson and Allcock 1997). It involves a cata-
lyzed condensation of the monomer trichloro(trimethylsilyl)phosphoranimine, (CH3)3 Si−N ═ PCl3 (4)
with loss of (CH3)3 SiCl. Briefly, purified trichloro(trimethylsilyl)phosphoranimineeither in solid state
or in solution (anhydrous dichloromethane or toluene) is treated with PCl5 in a well-defined monomer/
initiator ratio at 25°C for a variable period of time. In general, lower initiator concentration results in
a smaller number of initiated chains and higher molecular weight of polymer 3. Furthermore, increase
of reaction temperature reduces the reaction time. Additionally, the molecular weight of the poly-
mer 3 prepared in solution is higher with a smaller polydispersity than those prepared in solid state.
Branching of polymer could also occur if the cationic charge is delocalized down the chain.
This living cationic polymerization method has also been used for the direct synthesis of poly-
phosphazenes by the PCl5-induced polymerization of mono- and di-substituted organophosphora-
nimines, such as PhCl2P═NSiMe3, at ambient temperature (Allcock et al. 1997a). The chain length
of the polymer 3 can be varied by changing the ratio of the monomer to PCl5 catalyst. For example,
block copolymers with other phosphazene monomers or with organic monomers have been formed
through the living sites at the polymer chain ends in living cationic polymerization (Allcock et al.
1997b). In general, this route has several advantages over the thermal ring-opening route including
lower polymerization temperature, better control over polymer molecular weight with a smaller
polydispersity, and an active chain end that enables the synthesis of block copolymers and com-
plex macromolecular architectures such as star and dendrimer geometries (Allcock 1967, 1972a;
Honeyman et al. 1995; Allcock et al. 1996, 1997a,b; Nelson and Allcock 1997; Allcock 2003).
4.2.2.1 Reaction Mechanism of Living Cationic Polymerization

The reaction mechanism of living cationic polymerization has been reported (Honeyman et al. 1995;
Allcock et al. 1996, 1997a,b; Nelson and Allcock 1997; Allcock 2003). This polymerization involves
the addition of a cationic initiator, such as phosphorus pentachloride (PCl5), to the monomer 4. The
initiator causes the displacement of the trimethylsilyl group. This creates an active site where poly-
mer chain growth occurs with the addition of more monomers. The chain keeps growing until all the
monomers are used and the reaction is terminated with the addition of a terminator.
4.2.3 Macromolecular Substitution
As discussed earlier, the first step of polyphosphazene synthesis involves the synthesis of linear
polymer 3, which can be achieved via the thermal ring-opening polymerization of the commercially
available cyclic trimer [NPCl2]3 or living cationic polymerization (Scheme 4.1). In a second step, the
reactive chlorine atoms on the polymer 3 can be replaced via macromolecular substitution reactions
by various organic nucleophiles including amino acid esters, alkoxy, aryloxy, or a combination of
different functional groups as shown in Scheme 4.2.
The macromolecular substitution of polymer 3 makes polyphosphazene polymers a unique
platform for biomaterial development. For example, in contrast to direct synthesis from monomer
to polymer for many organic or inorganic macromolecules, polyphosphazene synthesis involves
the replacement of chlorine atoms in a macromolecular intermediate polymer 3. The two chlorine
atoms in polymer 3 are susceptible to nucleophilic attack by many nucleophiles such as alkoxides,
aryloxides, and amines (Scheme 4.2; Allcock 2003). Typically, about 30,000 chlorine atoms per
molecule are replaced in this step. Macromolecular substitution reactions allow introducing multi-
ple side groups that determine the polymer properties in a controlled manner. This unique synthetic
versatility has led to the development of an interesting class of polymers with different physico-
chemical and biological properties (Allcock 2003). In addition to single side group substitution,
multiple substituents can be covalently linked to the polymer backbone. For example, substitution
of two different types of side groups on the reactive intermediate polymer 3 can be achieved by
sequential substitution, allowing the bulkier group to react first followed by smaller reactive group
in solution phase during substitution (Allcock 2003). Secondary reactions can be further used to add
different functionality to the side groups. Thus, compared to the conventional monomer-dependent
Cl OR
RONa
N P N P
–NaCl
n n
Cl OR Single-substituent polymers
3 RNH2
NHR
–HCl
N P
n
RM
NHR
–MCl
RONa –NaCl R
N P
n
OR OR
Sequential substitution
RNH2
N P N P
–HCl
n n
Cl NHR
Mixed-substituent polymers
SCHEME 4.2 An illustration of the macromolecular substitution reactions that allow for a wide variety of
organic side groups to be linked to the phosphorus atoms to form single-substituent polymers (simultaneous
replacement of chlorine atoms) or mixed-substituent polymers (sequential substitution of chlorine atoms).
polymerizations, the macromolecular substitution is highly advantageous due to the systematic con-
trol of the material properties via side group chemistry rather than monomer polymerizability.
Another unique advantage of the macromolecular substitution reaction is the ability to introduce
two or more different side groups in different compositions, which allows fine-tuning the polymer
properties such as the degradation rate to suit a specific application. For example, in the case of a mixed-
substituted polymer having two side groups, one of which is resistant to hydrolysis and the other being
hydrolytically labile, the polymer erosion rate depends on the ratios of these two groups. Since many
combinations of different types of side groups are accessible to be linked to the polymer backbone, it
is possible to modulate the hydrolysis rate in a precisely controlled fashion. Allcock et al. have shown
that side groups such as amino acid ester units (Allcock et al. 1994b), imidazolyl (Allcock et al. 1982b),
glucosyl (Allcock and Scopelianos 1983), glyceryl (Allcock and Kwon 1988), and lactate or glycolate
(Allcock et al. 1994c) groups sensitize the polymer to break down over periods of hours, days, months,
or years at the physiological temperature depending on the specific molecular structure of the polymer.
4.2.4 Other Synthesis Methods

Several other polyphosphazene synthesis methods have also been reported in the literature for the gener-
ation of polymers with direct P–C bonds between the backbone and the sidegroups: (1) the ring-opening
polymerization of phosphazene cyclic trimers that have alkyl or aryl units linked to the ring (Allcock
1992); (2) condensation reactions of organophosphoranimines (Neilson and Wisian-Neilson 1988;
Montague and Matyjaszewski 1990); and (3) denitrogenation reactions of organophosphorus azides
(Franz et al. 1994). For example, Neilson and Wisian-Neilson et al. reported the direct synthesis of
poly(alkyl/aryl)phosphazenes with moderate molecular weights and polydispersities of 1.5–3.0 via the
condensation polymerization of N-silylphosphoranimines at 200°C (Neilson and Wisian-Neilson 1988).
4.2.5 Polyphosphazene Architecture
So far, the macromolecular substitution of polymer 3 has allowed for over 250 different side groups
to be covalently linked to the backbone via replacement of the labile chlorine atoms (Allcock 2003,
2010). This makes polyphosphazenes more versatile than many other polymer systems and has led to
the formation of various different phosphazene polymer architectures such as linear polymers, branched
or graft polymers, star polymers, and dendrimers (Scheme 4.3; Allcock 2003, 2010). Using the liv-
ing cationic route shown in Scheme 4.1, block polyphosphazene copolymers have also been synthe-
sized with various organic polymers including polystyrene (Allcock et al. 2004a; Chang et al. 2005),
poly(methyl methacrylate) (Allcock et al. 2004b), poly(ethylene oxide) (Chang et al. 2002, 2003), and
poly(dimethylsiloxane) (Prange and Allcock 1999; Allcock and Prange 2001). Furthermore, complex
structures that involve hexachlorocyclotriphosphazene, such as cyclo-linear polymers, have also been
synthesized with phosphazene rings linked between linear polymer backbones with polyethylene
(Scheme 4.3) (Allcock and Kellam 2001; Allcock et al. 2001c). The cyclic phosphazenes can also be used
as side chains for other polymers such as polystyrene (Allcock et al. 2001b), polynorbornene (Allcock
et al. 2001d, 2003a), and polyoctenamer (Allcock et al. 2005). Finally, 3D cross-linked networks “cyclo-
matrix” can be fabricated to form tough materials with high melting point and hardness using the cyclic
phosphazene ring through the multiple functional groups in the phosphazene system (Devadoss and Nair
1985; Zhang et al. 2005). In addition to phosphorus and nitrogen in the skeleton, a third element such as
sulfur or carbon has also been incorporated in the main chain for further functionalizations (Scheme 4.3).
4.2.6 Structure–Property Relationships

The torsional and angular freedom within phosphorus-nitrogen backbone offers high molecu-
lar weight and high flexibility to the polyphosphazenes. The bond torsional mobility allows the
polyphosphazene backbone to be stretched from a random coil. It is dependent on the electronic
Organic polymer chain

R
N P = R R R
Linear polyphosphazene N P N N P N N P N
R R R R R R R
n P P P P P P
R N R R N R R N R
Organic polymer chain
Star
Dendrimer Polyphosphazene pendent units
R R R
N P N P N S
n
Phosphazene block Organic block R R
Diblock copolymers Poly(thiophosphazenes)
R R R
R R R R
P P N P N P N C
N N N N
P P P P R R n
R N R R N R
Cyclolinear Poly(carbophosphazenes)
SCHEME 4.3 Different polymer architectures accessible through polyphosphazene platform.
character of the bond, bond angle and length, and the number, nature, and ratio of the attached
side groups (Allcock 2003). In a repeating unit of −P═N−, each phosphorus atom provides five
valence electrons while each nitrogen contributes another five. If the electron pairs are assigned
in a sigma bond framework, there are four electrons that are unaccounted for. Three of these elec-
trons are from the nitrogen atom and one is from the phosphorus atom. The electron from the
phosphorus atom resides in a 3d orbital forming a “stabilized excited state.” For the three electrons
on the nitrogen atom, two of them are confined to a lone-pair orbital, which gives the polymer its
basicity. The remaining electron from the nitrogen atom is in a 2p orbital. The pairing of these two
electrons from the 2p and 3d orbitals results in an “out of plane” dπ − p π bond. Due to the orbital
mismatch and nodes occurring at every phosphorus atom, delocalization is limited only over three
skeletal atoms and long range delocalization does not occur. Furthermore, the 2p orbital can be
brought into an overlapping position with a d-orbital at any torsion angle. Hence, the torsional
barrier in the backbone bonds is much smaller when compared to p π − p π double bond in organic
molecules (Allcock 2003). The bond angle at the nitrogen atom in the backbone ranges from 130°
to 160° while the bond angle at the phosphorus atom is 114° (Allcock 2003). The presence of the
side groups in alternating atoms in the backbone contributes to the less density of the side groups in
polyphosphazenes as compared to many other organic polymers and thus increases the backbone
flexibility. Bond length of typical −C═C− is ∼1.54 Å whereas the bond length between nitrogen
and phosphorus atoms ranges from 1.55 to 1.60 Å (Allcock 2003). The torsional barrier is further
reduced due to the increased distance between the side groups and the alternating bond lengths in
the backbone. Chatani et al. found a difference of 0.22 Å between the bonds in the polymer skeleton
(Chatani and Yatsuyanagi 1987). However, other studies reported the values of bond differences to
be 0.05 ∼ 0.07 Å (Allcock 2003).
The overall physicochemical and biological properties of polyphosphazenes such as solubility,

hydrophilicity/hydrophobicity, glass transition temperatures (Tg’s), mechanical properties, hydrolytic
sensitivity, and biocompatibility depend on the nature and composition of the side groups attached to
the backbone. Bulky and inflexible side groups or polar or hydrogen bonding units increase the torsional
barrier of the polymer backbone and result in a rigid polymer with high Tg’s. Furthermore, the incorpo-
ration of side groups that impart intermolecular hydrogen bonding such as glycylglycine dipeptide units
also leads to an increase in the torsional barrier of the motility. A large number of reagents such as alco-
hols, phenols, and amines have been used alone or in combination to synthesize polyphosphazenes with
a wide range of properties (Allcock et al. 1977, 1994b; Allcock and Fuller 1980; Allcock and Pucher
1991; Crommen et al. 1992a,b, 1993; Laurencin et al. 1993, 1996; Qiu and Zhu 2000a; Gümüsdereliolu
and Gür 2002; Nair et al. 2003, 2004; Conconi et al. 2004; Cui et al. 2004; Barrett et al. 2005; Yuan et al.
2005; Andrianov and Marin 2006; Sethuraman et al. 2006; Singh et al. 2006). Table 4.1 summarizes the
TABLE 4.1
Summary of Glass Transition Temperatures (Tg’s)
of Single-Substituent Polyphosphazenes Illustrating
the Great Tunability of Physico-Chemical Properties
by Modulating Side Group Chemistry
Polymer Side Group Tg (°C)
PNEG O −40
H2N
O
PNEA O −10
O
NH2
PNEL O 15
O
NH2
PNEPhA O 42
O
NH2
PNGEG H O 56
N
H2N O
O
PNMEEP HO O −84
O
PNmPh 2
OH
The selected polymers are (PNEG), poly[bis(ethyl glycinato)phosphazene];

(PNEA), poly[bis(ethyl alanato)phosphazene]; (PNEL), poly[bis(ethyl
leucinato)phosphazene]; (PNEPhA), poly[bis(ethyl phenylalaninato)
phosphazene]; (PNGEG), poly[bis(glycyl-ethylglycinato)phosphazene];
(PNMEEP), poly[bis(methoxyethoxyethoxy)phosphazene]; (PNmPh),
poly[bis(p-methylphenoxy)phosphazene].
physicochemical properties such as Tg’s of some of the representative polyphosphazenes for biomedical
applications (Allcock et al. 1977, 1994b; Allcock and Fuller 1980; Allcock and Pucher 1991; Crommen
et al. 1992a,b, 1993; Cohen et al. 1993; Laurencin et al. 1993, 1996; Schacht et al. 1996; Qiu and Zhu
2000a; Lee and Mooney 2001; Gümüsdereliolu and Gür 2002; Chaubal et al. 2003; Gunatillake and
Adhikari 2003; Katti and Laurencin 2003; Conconi et al. 2004; Cui et al. 2004; Gleria and De Jaeger
2004; Kricheldorf et al. 2004; Nair et al. 2004; Barrett et al. 2005; Yuan et al. 2005; Andrianov and
Marin 2006; Sethuraman et al. 2006; Singh et al. 2006). As seen from the table, a wide range of the glass
transition temperature from −84°C to 56°C can be obtained by changing the side groups. For example,
the polymer Tg is increased in the order of PNEG < PNEA < PNEL < PNEPhA, which is indicative of
the bulkier side group resulting in a higher Tg.
4.3 POLYPHOSPHAZENES AS A VERSATILE CLASS OF BIOMEDICAL MATERIALS

As discussed previously, the side groups largely determine the material properties of polyphos-
phazenes. Polyphosphazenes have an inherently flexible backbone due to the bonding nature of
the phosphorus-nitrogen atoms in the backbone. This flexibility offers great tunability of material
properties. This great synthetic flexibility of polyphosphazenes enables the design and development
of prospective biomedical materials with diverse properties. Most of the polyphosphazenes synthe-
sized so far are hydrolytically stable. These polymers exhibit unique properties including unusual
thermal properties (Sulkowski et al. 1997), optical properties (Allcock et al. 1998), electrolytic
properties (Allcock et al. 2001a), and electroactivity (Li et al. 2001). Early reports also attempted
to characterize tissue compatibility of several nondegradable fluorinated poly(organophosphazenes)
(Wade et al. 1978; Welle et al. 2000).
Hydrolytically labile polymers that can degrade into nontoxic products are highly preferable as
short-term medical implants for drug delivery and tissue engineering applications (Nair and Laurencin
2007). Due to their transient nature, biodegradable polyphosphazenes have attracted a great deal of
attention as a unique class of biomedical materials. The phosphorus–nitrogen backbone can be ren-
dered hydrolytical sensitivity by substituting with appropriate sidegroups (Allcock 2003). The polymers
undergo bulk erosion or surface erosion or a combination of both generating nontoxic degradation
products, such as phosphoric acid, ammonia, and the corresponding side groups. The degradation rate
is greatly influenced by the type and composition of the substituted side groups (Andrianov and Marin
2006; Kumbar et al. 2006; Singh et al. 2006). It is possible to design polyphosphazenes that can degrade
over a period of hours to days, months and years at body temperature depending on the structure and
composition of the polymer (Allcock et al. 1977, 1994b; Allcock and Pucher 1991). In addition, the
mode of side group attachment also plays an important role in determining the polymer degradability
(Allcock 2003; Allcock et al. 2003c). Thus, a versatile array of biodegradable polyphosphazenes can
be synthesized by modulating the side group chemistry. Specifically, this section is confined to vari-
ous biodegradable polyphosphazenes (aminated polyphosphazenes and alkoxy-substituted polyphopha-
zenes) and polyphosphazene blends/composites as well as their applications as drug delivery matrices
and tissue engineering scaffolds.
4.3.1 Biodegradable Polyphosphazenes
Biodegradable polyphosphazenes can be synthesized by substituting chlorine atoms of the polymer 3
with hydrolytically labile groups such as amino acid esters (5) (Allcock et al. 1994b), imidazolyl
(6) (Allcock et al. 1982b), glucosyl (7) (Allcock and Scopelianos 1983), glyceryl (8) (Allcock and
Kwon 1988), and glycolate or lactate ester (9) (Allcock et al. 1994c), to name a few (Figure 4.1). In
general, biodegradable polyphosphazenes alone can be classified into two categories based on the
type of side group substituents: aminated polyphosphazenes (those substituted with amines of low
acid dissociation constant pKa) and alkoxy substituted polyphosphazenes (those substituted with
activated alcohols). In addition, polyphosphazene blends/composites constitute another attractive
class of biodegradable polymer systems for drug delivery and tissue engineering applications.
OH
H O H
H
H O
N
HO HO
H HO
N N P
HNCHRCOOR´ OH H n
N P
n OH OH
N P
N H
n H O
HNCHRCOOR´ H O H
N OH
5 6 7
OH
OH
O C OCHRCOOR´ HNCHRCOOCHCH3COOCH2CH3
H2
N P N P N P
n n n
H2
O C OCHRCOOR´ HNCH2COOCH2CH3
OH 9 10
OH
8
N
HNCH2CONHCOOC6H5
N
N P N P
n n
O CH3
HNCH2COOCH2CH3
11 12
FIGURE 4.1 Structures of various biodegradable polyphosphazenes by substituting the chlorine atoms of
poly(dichlorophospazene) (3) with hydrolytically labile groups such as amino acid esters (5), imidazolyl (6),
glucosyl (7), glyceryl (8), and glycolate or lactate esters (9), to name a few. Polymers 10–12 are representative
aminated polyphosphazenes.
4.3.1.1 Aminated Polyphosphazenes

Aminated polyphosphazenes constitute the largest and most extensively investigated class of bio-
degradable polyphosphazenes. In 1966 Allcock and Kugel reported the first successful synthesis of
hydrolytically sensitive aminated polyphosphazenes (Allcock and Kugel 1966). Since then, com-
bined efforts of Allcock and coworkers have led to the synthesis of a series of aminated polyphos-
phazenes. Among them, polyphosphazenes substituted with amino acid ester (5) and imidazole (6)
side groups attracted much interest as candidate biomaterials due to their hydrolytic degradability
and nontoxic degradation products.
The amino acid ester polyphosphazenes comprise the most extensively investigated biodegrad-
able polyphosphazenes for biomedical applications due to their inherent biocompatibility and
bioactivity. Allcock et al. synthesized a wide spectrum of amino acid ester–substituted polyphos-
phazenes as new biomaterials (Allcock et al. 1977, 1994b). Efforts were also made to increase the
degradation rate of poly[(amino acid ester)phosphazenes] by incorporating side groups with hydro-
lytically sensitive ester functions (10, Figure 4.1; Crommen et al. 1992b). Furthermore, pH-sensitive
polyphosphazenes were synthesized by incorporating aminoacethydroxamic acid as cosubstituents
to poly[(amino acid ester)phosphazenes] (11, Figure 4.1; Qiu and Zhu 2000a). More recently, poly-
phosphazenes substituted with glycylglycine dipeptides have been designed and synthesized to ren-
der excellent degradability and hydrogen bond–forming ability (Krogman et al. 2007). Additionally,
polyphosphazenes cosubstituted with the amino acid ester/purine or pyrimidine polyphosphazenes
were reported to undergo hydrolytic degradation (Krogman et al. 2008a).
Specifically, the imidazole-substituted polymer (6) was found to be the most hydrolytically
sensitive among the aminated phosphazenes synthesized. Furthermore, the hydrolysis rate of
imidazole-substituted polyphosphazenes could be modulated by cosubstituting it with less hydro-
lytic sensitive groups such as p-methylphenoxy on the polymer backbone (12, Figure 4.1) (Allcock
and Kugel 1966).
4.3.1.2 Alkoxy-Substituted Polyphosphazenes

In addition to the class of aminated phosphazenes, some alkoxy-substituted polyphosphazenes were
also found to be hydrolytically labile (Allcock and Scopelianos 1983; Allcock and Kwon 1988;
Allcock and Pucher 1991; Allcock et al. 1994c). For example, Allcock and Kwon synthesized the
first glyceryl-substituted polyphosphazene (8) (Allcock and Kwon 1988). Hydrogels of this polymer
were further synthesized by cross-linking with various cross-linking agents such as adipoyl chloride
and hexamethylenediisocyanate. Moreover, polyphosphazenes substituted with glycosyl and methyl
amino side groups were also found to be hydrolytically labile (Allcock and Pucher 1991). Additionally,
Allcock et al. synthesized another alkoxy-substituted polyphosphazene bearing glycolic acid ester and
lactic acid ester side groups (9) (Allcock et al. 1994c). These polymers are not crystalline and hydro-
lyze much faster than the commonly used biodegradable polyesters such as PLA and PGA.
4.3.1.3 Polyphosphazene Blends/Composites

The synthetic flexibility of polyphosphazenes offers great tunability in polymer properties. Although
considerable advancement has been made in the development of polyphosphazenes with desirable
properties, some challenges still remain especially in the development of ideal candidate materi-
als with the right combination of desirable properties for a specific application. For example, the
incorporation of bulkier side groups such as phenylalanine ethyl ester provides higher mechanical
strength than other amino acid esters, but also results in a significant increase in polymer hydropho-
bicity, which substantially slows down the polymer degradation. The difficulties to attribute all the
required physical, chemical, and biological properties to a single polymer necessitate the continued
search for an ideal system (Deng et al. 2009).
Blending of two or more different polymers is a practical approach to develop novel biomateri-
als owing to the ease of blend preparation and the efficient control of blend properties via composi-
tional changes (Utracki 1989). It synergistically combines the advantages of the polymer components.
Moreover, the synthetic challenges of producing a copolymer are not encountered. Polymer blends/
composites are of great commercial interest and occupy over 30% of the polymer market (Utracki 1989).
For example, efforts have been made to improve the properties of polyesters by blending them with a
variety of other polymers such as 2-methacryoloxyethyl phosphoroylcholine (PMPC) (Iwasaki et al.
2002), poly(vinyl alcohol) (Pitt et al. 1992), chitin (Mi et al. 2003), polycaprolactone (PCL) (Chen
et al. 2003), and triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO),
PEO – PPO – PEO (Park et al. 1992). In addition, the effect of enantiomeric polymer blending on deg-
radation rate of polyesters has also been extensively investigated (Ikada and Tsuji 2000; Tsuji 2003).
In recent years, different blend systems of polyphosphazenes with a variety of classical synthetic
biodegradable polymers including polyesters such as PLA, PLAGA, and polyanhydrides have been
reported (Ibim et al. 1997; Qiu and Zhu 2000b; Ambrosio et al. 2002; Qiu 2002a,b; Nair et al. 2005;
Krogman et al. 2007). Among them, polyphosphazene–polyester blends have several unique advan-
tages compared to other polymer blend systems. In specific, the blend systems composed of biode-
gradable polyphosphazenes and polyesters are inherited with unique self-neutralizing ability where
the polyphosphazene degradation products such as ammonium phosphate act as a buffer system to
neutralize the acidic degradation products of polyesters. Furthermore, various functional groups
can also be easily introduced to the blends through the versatile side group chemistry in polyphos-
phazenes, and thus the blend properties can be easily tailored for different biomedical applications.
For example, carboxyl-substituted polyphosphazenes poly[bis(carboxylatephenoxy)phosphazene]
(PCPP) have been shown to interact with simulated body fluid (SBF) and form a bone-like apatite
layer on the surface (Brown et al. 2007). Such bioactivity would greatly improve the osteointegra-
tivity of scaffolds/implants for bone tissue engineering (Kokubo and Takadama 2006). However,
in order to achieve uniform and predictable properties of polyphosphazene–polyester blends, there
are several design criteria on the molecular structure of polyphosphazenes: (1) the polymer should
have appropriate functional groups that promote hydrogen bonding interactions with polyesters.
This would allow the formation of miscible blends with improved physical, chemical, and biologi-
cal properties (Deng et al. 2009); (2) the polymer should be biocompatible and should hydrolyze
into nontoxic small molecules that can be metabolized or excreted. The long-term collaborative
efforts between Laurencin and Allcock’s groups have led to the development of a series of promis-
ing polyphosphazene–polyester blends for various biomedical applications based on the polymers
substituted with amino acid esters, alkoxy, and aryloxy side groups (Figure 4.2). In addition, blends
of polyphosphazenes alone have also been investigated for a number of biomedical applications.
4.3.1.3.1 Theory of Hydrogen-Bonded Polymer Blends

For biomedical applications, blend miscibility is a critical factor that affects the material per-
formance. Uniform and predictable properties can only be achieved when blends are miscible.
Partially miscible or immiscible blends are often characterized by polymer phase segregation,
which adversely affects the material properties such as mechanical properties.
CH3
O
O HN
HN 1
O
O x N P
n
N P O
n
O
13: x = 0.5, y = 1.5

14: x = 1.0, y = 1.0
15: x = 1.5, y = 0.5
CH3 1
y
16
H O
O N
HN HN O
x
O l
N P O N P
n O n
O O
HN
N
H O y
17: x = 0, y = 2.0
18: x = 0.5, y = 1.5
19: x = 1.0, y = 1.0 1
20: x = 1.5, y = 0.5
21
FIGURE 4.2 Structures of several representative polyphosphazenes that are substituted with various amino
acid esters, alkoxy, and aryloxy side groups for the development of polyphosphazene-polyester blends.
Blend miscibility is obtained when the Gibbs free energy change of mixing is negative or
ΔGmix = ΔHmix − TΔSmix < 0. The miscibility of two polymers in a blend is generally a result of
intermolecular interactions between the polymers such as through hydrogen bonding, dispersion
forces, dipole–dipole interactions and/or van der Waals forces (Utracki 1989; Bates 1991; Coleman
and Painter 1995; He et al. 2004).
For hydrogen-bonded polymer blends, ΔGmix can be expressed in the following form:
∆G mix ΦA Φ ∆G H
= lnΦA + B lnΦB + χΦA ΦB +
RT MA MB RT
Vr
χ= [δ A − δ B ]2
RT
where Φi represents the volume fraction of polymer i in the blend, Mi is the degree of polymeriza-
tion for polymer i (i = A or B). There are three components that contribute to ΔGmix. The component
(ΦA/MA) ln ΦA + (ΦB/MB) ln ΦB represents a favorable contribution of combinatorial entropy; the
term χΦAΦB represents an unfavorable contribution of physical forces such as dispersion forces or van
der Waals forces; the ΔGH/RT represents a favorable contribution from hydrogen bonding. Since the
value of combinatorial entropy is usually very small, blend miscibility is largely determined by the
overall contributions from the χΦAΦB and ΔGH/RT components. When hydrogen bonding is absent,
miscibility can only be achieved when the critical value of χ determined by the solubility parameters
of the two polymers is extremely small. For example, χ must be less than 0.002 where only disper-
sive forces are present in Flory–Huggins model. When hydrogen bonding plays a dominant role, the
contribution of ΔGH/RT is greater than that of χΦAΦB and completely miscible blends are obtained.
In the polyphosphazene–polyester blend systems, the synthetic flexibility of polyphosphazenes
allows the incorporation of hydrogen bonding side groups on polymer backbone that interact with
polyesters to favor blend miscibility.
4.3.1.3.2 Miscibility of Polyphosphazene Blends

The concept and feasibility of fabricating various blends of polyphosphazenes with polyesters was
first demonstrated by Ibim et al. (1997). In the study, three different polyphosphazenes contain-
ing different molar ratios of ethyl glycinato to p-methylphenoxy (13, 14, and 15, Figure 4.2) were
synthesized and blended with PLAGA (50:50) at a weight ratio of 50:50. All blends showed homog-
enous surface morphologies as evidenced by scanning electron microscopy (SEM) and their misci-
bility was confirmed by differential scanning calorimetry (DSC).
Qiu et al. have investigated the miscibility of a series of degradable blends of PNEG (5) (where
R = H and R′ = CH2CH3) and polyesters or polyanhydrides (Qiu and Zhu 2000b; Qiu 2002a,b). In
the study, different blends of PNEG with PLA, PLAGA (80:20), poly(sebacic anhydride) (PSA) and
poly(sebacic anhydride-co-trimellitylimidoglycine)-block-poly(ethylene glycol) (30:50:20 by mole)
(PSTP) were fabricated in various ratios using a solvent-mixing technique (Qiu and Zhu 2000b).
The PNEG-PLA blends were completely immiscible, whereas the PNEG-PLAGA and PNEG-PSTP
blends were found to be partially miscible as evaluated by DSC, Fourier transform infrared spec-
troscopy (FTIR), and phase contrast microscopy.
In order to develop high-strength candidate materials for load-bearing regenerative applications,
several studies have indicated the feasibility of significantly increasing polyphosphazene hydro-
phobicity and mechanical properties by incorporating phenylphenoxy side groups (Singh et al.
2006; Sethuraman et al. 2010). Thus, Deng et al. (2008) fabricated blends of poly[(ethyl alanato)1
(p-phenylphenoxy)1phosphazene] (PNEAPhPh) (16, Figure 4.2) and PLAGA (85:15). Solvent effect on
the miscibility of polyphosphazene-polyester blends was investigated for the first time and tetrahydro-
furan (THF) was used to produce different blends at three weight ratios of PNEAPhPh:PLAGA namely
10 μm 10 μm 10 μm
(a) (b) (c)
FIGURE 4.3 SEM micrographs showing the surface morphologies of different blends. (a) BLEND25;
(b) BLEND50; and (c) BLEND75. BLEND25 showed uniform morphology indicating blend miscibil-
ity whereas the other two blends showed visible phase separation. (From Biomaterials, 29(3), Deng, M.,
Nair, L.S., Nukavarapu, S.P. et al., Miscibility and in vitro osteocompatibility of biodegradable blends
of poly[(ethyl alanato) (p-phenyl phenoxy) phosphazene] and poly(lactic acid-glycolic acid), 337–349.
Copyright 2008, with permission from Elsevier.)
25:75 (BLEND25), 50:50 (BLEND50) and 75:25 (BLEND75). Miscible blends were formed at lower
polyphosphazene content as characterized by the single Tg value with smooth surface morphology.
As shown in Figure 4.3, an increase in the polyphosphazene content of the blend caused phase separa-
tion due to the steric hindrance from the α-CH3 groups in the alanine units and bulky phenylphenoxy
side groups, as well as insufficient hydrogen bonding sites available to form miscible blends. This
further indicated that the solvent has a limited effect on blend miscibility (Deng et al. 2008).
Blend miscibility is one of the prerequisites in applications that require controllable and predict-
able degradation pattern. Efforts have also been made to improve blend miscibility by introducing
different side groups containing multiple hydrogen bonding sites in polyphosphazenes. In that direc-
tion, Krogman et al. (2009) developed polyphosphazenes having tris(hydroxymethyl)amino methane
(Trisma or THAM) side groups and with cosubstitutents glycine ethyl ester and alanine ethyl ester.
The THAM side group was linked to the polyphosphazene backbone via the amino function. The
polymer has three pendent hydroxyl groups. The polymers form completely miscible blends with
both PLAGA (50:50) and PLAGA (85:15) (Krogman et al. 2009). In another study, Krogman et al.
have synthesized different glycylglycine dipeptide–based polyphosphazenes (17–20, Figure 4.2) and
investigated the miscibility of their blends with both PLAGA (50:50) and PLAGA (85:15) (Krogman
et al. 2007). Among them, polymer 18 was found to form completely miscible blends with both
PLAGA, which was characterized by SEM, DSC, and attenuated total reflectance infrared spectros-
copy (ATR-IR). It was found that the Tg for each blend was lower than that for each parent polymer,
implying both polyphosphazenes and PLAGA in the blend acted as plasticizers for each other.
The aforementioned studies have highlighted the significant challenge in the development of
an ideal polyphosphazene blend system with the desirable balanced properties such as miscibility
and mechanical properties. More recently, Deng et al. (2010b,d) designed and fabricated blends of
poly[(glycine ethyl glycinato)1(p – phenylphenoxy)1phosphazene] (PNGEGPhPh) (21, Figure 4.2)
and PLAGA (50:50) in two different weight ratios (25:75 and 50:50, BLEND25 and BLEND50)
(Deng et al. 2010b,d). The two side groups for the PNGEGPhPh were selected based on the fol-
lowing rationale. The glycylglycine dipeptide was shown to provide two proton donors per mono-
mer for hydrogen bonding with PLAGA while the phenylphenoxy was beneficial for maintaining
mechanical function and hydrophobicity of the polymer (Sethuraman et al. 2006, 2010; Singh
et al. 2006; Krogman et al. 2007). Laurencin et al. demonstrated excellent osteocompatibility of
phenylphenoxy cosubstituted polyphosphazenes both in vitro and in vivo (Sethuraman et al. 2004,
2006). In addition, a mixed-substituent polymer with both hydrolysis sensitive groups and hydro-
phobic groups provided efficient tunability to achieve a suitable degradation rate. Specifically
the combination of the two side groups in a 50:50 ratio was expected to result in a complete
degradation time in the range of 12–24 weeks. Furthermore, the glycylglycine dipeptide was rec-
ognized to be biocompatible, with degradation into glycine units, one of the common amino acids
in the body (Adibi 1989). The miscibility of the blends was confirmed by a variety of analytical
techniques including DSC, ATR-IR, SEM, and high-resolution solid-state nuclear magnetic reso-
nance spectroscopy (NMR).
This mixed-substituent polymer resulted in a completely miscible blend system with the polyes-
ters through the hydrogen bonding between the amide and amine protons in the dipeptide units of
PNGEGPhPh and the carbonyl groups in PLAGA. For example, BLEND25 and BLEND50 showed
a single Tg of ∼36°C and ∼40°C, whereas PLAGA and PNGEGPhPh showed a Tg of ∼28°C and
∼53°C, respectively. The single Tg value for both blends was intermediate between those of the two
parent polymers indicating blend miscibility. Furthermore, for a miscible blend system, the Tg of
the blend can be predicted by the Wood equation (Wood 1958): Tg = w1Tg1 + w2Tg2 or by the Fox
equation (Fox 1956): Tg = Tg1Tg2/(w1Tg2 + w2Tg1) where wi and Tgi are the weight fraction and the
Tg of polymer i (i = 1, 2 designating polyphosphazene and PLAGA, respectively). The Tg’s esti-
mated using the Wood and Fox equations for Matrix1 and Matrix2 were 34°C and 40°C, and 32°C
and 37°C, respectively, which are very close to the experimental values from DSC thermograms
(Figure 4.4).
Recently, the synthesis of PLA grafted polyphosphazenes has been reported by Krogman et al.
(2008c). In addition, novel polyphosphazene block copolymers such as polyphosphazene-block-
polyester or -polycarbonate macromolecules have been recently synthesized (Krogman et al.
2008b; Weikel et al. 2010a). The lactic and glycolic acid blocks in the block copolymers could
act as compatibilizers in the blend, thereby leading to complete miscibility. Efforts are currently
underway to develop advanced polyphosphazene miscible blend systems using these polymeric
architectures as prototypes as well as polymers with bioactive side groups for further functional-
ization modifications.
60
50
40 y = 0.2439x + 28.562
R2 = 0.9929
30
Tg
20
Experiment
Wood
10
Fox
Linear (experiment)
0
0 20 40 60 80 100
PPHOS composition (%)
FIGURE 4.4 (See companion CD for color figure.) Miscibility of the polyphosphazene–polyester blends.
Linear relationship between blend Tg and polyphosphazene composition was based on the experimental values
from DSC thermograms, which was in line with Wood equation for miscible blends. Furthermore, the glass
transition temperatures estimated using the Fox equation for the blends were very close to the experimental
values from DSC thermograms. (From Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: In situ porous struc-
tures: A unique polymer erosion mechanism in biodegradable dipeptide-based polyphosphazene and poly-
ester blends producing matrices for regenerative engineering. Adv. Funct. Mater. 2010d. 20(17). 2794–2806.
Copyright Wiley-VCH Verlag GmbH & Co. KGaA. With permission.)
In the case of blends composed of polyphosphazenes alone, Lemmouchi et al. prepared

a blend (50/50, w/w) of PNEG with poly[(ethyl alanate)-co-(ethyl 2-(O-glycyl lactate))phosp-
hazene] (3% gly-lact-OEt) (Lemmouchi et al. 1998). A single Tg was observed for the blend
materials.
4.3.2 Degradation of Biodegradable Polyphosphazenes

4.3.2.1 Polymer Degradation Mechanisms
Allcock and coworkers first demonstrated that the incorporation of certain hydrolytically labile
side groups on polymer backbone could confer biodegradability to polyphosphazenes (Allcock
et al. 1982b). Since then, biodegradable polyphosphazenes have attracted a great deal of atten-
tion in biomedical applications. Biodegradation of hydrolytically sensitive polymers involves cleav-
age of hydrolytically labile bonds followed by polymer erosion. Although the polymer degradation
mechanism varies with the chemical composition, the mechanism of hydrolytic degradation can be
broadly classified into two types: bulk erosion and surface erosion. For example, synthetic biode-
gradable polyesters such as PLAGA are known to undergo bulk erosion whereas polyanhydrides
such as poly(carboxyphenoxy propane) degrade through surface erosion. In bulk erosion, the poly-
mer undergoes degradation with significant decrease in molecular weight and the corresponding
material properties (such as mechanical properties) as a function of degradation time. As illustrated
in Figure 4.5a, the matrix dimension remains constant until the structure fails catastrophically
during hydrolytic degradation. In the case of surface erosion, degradation occurs at the implant sur-
face with insignificant decrease in the molecular weight of the bulk material. The matrix becomes
smaller but maintains its original geometric shape as a function of degradation time until the struc-
ture is completely eroded as indicated in Figure 4.5b. In comparison, biodegradable polyphospha-
zenes can degrade by a combination of both bulk and surface erosion (Laurencin et al. 1992). More
recently, on the platform of biodegradable polyphosphazene blends, Deng et al. (2010d) demon-
strated for the first time a unique polymer erosion process through which polymer matrices evolve
Time
(a)
(b)
(c)
FIGURE 4.5 (See companion CD for color figure.) Schematic illustration of different types of polymer
erosion. (a) Bulk erosion; (b) Surface erosion; and (c) A unique polymer erosion through which the polymer
changes from a solid coherent film to an assemblage of microspheres with an interconnected porous structure.
(From Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: In situ porous structures: A unique polymer erosion
mechanism in biodegradable dipeptide-based polyphosphazene and polyester blends producing matrices for
regenerative engineering. Adv. Funct. Mater. 2010d. 20(17). 2794–2806. Copyright Wiley-VCH Verlag GmbH
& Co. KGaA. With permission.)
from a solid coherent film to an assemblage of microspheres with an interconnected 3D porous

structure (Figure 4.5c).
Polymer erosion results from polymer degradation. The process involved in the erosion of
a degradable polymer generally include several phases: (1) hydration with or without swelling,
(2) oligomers and monomers generation by water intrusion, (3) progressive degradation, and (4)
oligomers and monomers release leading to mass loss. There are several important factors that
affect the polymer degradation rate such as lability of the chemical bond, molecular weight,
hydrophilicity/hydrophobicity, polymer matrix water permeability, solubility of the degradation
products, environmental conditions (pH, temperature, in vitro, and in vivo, etc.), and copoly-
mer composition. For example, PLAGA is known to degrade by simple hydrolysis of the ester
bonds into lactic and glycolic acids. The degradation rate of polyphosphazenes mainly depends
on several factors such as molecular weight of the polymer backbone, nature of the substituted
side groups, composition and extent of side group substitution, or blend composition in the case
of polyphosphazene blends (Andrianov and Marin 2006; Kumbar et al. 2006; Singh et al. 2006).
A general degradation pathway for biodegradable polyphosphazenes is shown in Scheme 4.4.
During the polymer degradation, water molecules attack the organic side groups. The forma-
tion of P-OH units after the removal of side groups followed by the migration of the proton
from oxygen to nitrogen sensitizes the polymer skeleton to hydrolysis. The polymer ultimately
degrades into non-toxic degradation products comprised mainly of ammonia, phosphoric acid,
NHR (or OR΄) OH

H2O
N P N P
n –RNH2 (or R΄OH) n
NHR (or OR΄) NHR (or OR΄)
H O H O
H2O
N P N P
n –RNH2 (or R΄OH) n
OH NHR (or OR΄)
H2O H 2O
O
H2O
H3PO4 + NH3 NH2 + HO P
NHR (or OR΄)
SCHEME 4.4 A general degradation pathway for biodegradable polyphosphazenes. The degradation is initi-
ated by the water molecule attack on the organic side groups. The formation of P-OH units after the removal
of side groups followed by the migration of the proton from oxygen to nitrogen sensitizes the polymer skel-
eton to hydrolysis. The polymer ultimately degrades into nontoxic degradation products comprised mainly of
ammonium phosphate buffer and the corresponding side groups. The polymer degradation rate can also be
altered by modulating the side group chemistry. For example, polyphosphazenes substituted with hydrolyti-
cally sensitive groups such as amino acid esters or imidazolyl degraded faster than the bulkier hydrophobic
groups substituted polymers.
and the corresponding side groups (Andrianov and Marin 2006; Kumbar et al. 2006; Singh et al.
2006). In the case of a mixed-substituent polymer, the most hydrolytically sensitive group leaves
the polyphosphazene backbone first followed by bulkier side groups such as p-methylphenoxy,
p-phenylphenoxy, tyrosine, or pyrrolidone (Allcock et al. 1977, 1994b; Allcock and Fuller 1980;
Allcock and Pucher 1991; Crommen et al. 1992a,b, 1993; Laurencin et al. 1993, 1996; Qiu and
Zhu 2000a; Gümüsdereliolu and Gür 2002; Nair et al. 2003, 2004; Conconi et al. 2004; Cui et al.
2004; Barrett et al. 2005; Yuan et al. 2005; Andrianov and Marin 2006; Sethuraman et al. 2006;
Singh et al. 2006). Degradation products such as ammonium phosphate constitute a natural buffer
system for the microenvironment both in vitro and in vivo (Andrianov and Marin 2006; Kumbar
et al. 2006; Singh et al. 2006).
4.3.2.2 Degradation of Aminated Polyphosphazenes

Allcock et al. extensively investigated the effect of different amino side groups on the hydrolytic
behavior of polyphosphazenes as well as small molecular cyclic analogs (Allcock et al. 1982b).
Among a series of synthesized aminated cyclophosphazenes, hexaaminocyclophosphazenes
with amino, methyl amino, ethyl glycinato, methyl glycinamido, and imidazole groups were
found to be soluble in water. However, a detectable rate of hydrolysis at 25°C was only observed
for cyclophosphazene substituted with the imidazole group. Furthermore, all the aforementioned
aminated phosphazenes experienced hydrolysis at 100°C as evidenced by 31P NMR indicating
that an increase in temperature increased the degradation rate of polyphosphazenes. Hydrolysis
products of these compounds were found to be ammonia, phosphate, and the corresponding
side group amine or amino acid. Under the same reaction condition, the sensitivity to hydrolysis
for these compounds increased in the order of methyl amino-substituted cyclophosphazenes <
amino-substituted < methyl glycinamido-substituted < ethyl glycinato-substituted < imidazole-
substituted, as evidenced from changes in 31P NMR spectra and recovered starting materials.
Thus, highly hydrophilic imidazole groups can significantly increase the water permeability
and the resulting polymer degradationrate. Two mechanistic hydrolysis pathways have been pro-
posed for these compounds (Allcock et al. 1982b). The degradation of these compounds can be
triggered through the protonation of atoms in the skeleton or in the side groups. Protonation
of the skeletal nitrogen atom would directly result in ring cleavage. Ultimately this could lead
to the conversion of the compound to ammonium ion, phosphoric acid, and the free amine
salt. On the other hand, protonation of the side group nitrogen atom could take place, followed
by nucleophilic attack by water at phosphorus, yielding a monohydroxycyclophosphazene. The
monohydroxycyclophosphazene, by further hydrolysis, would undergo ring cleavage and even-
tual degradation. The detection of a monohydroxycyclophosphazene can be used as a key factor
to identify this pathway. However, both mechanisms are blocked in strongly basic media. It
has been further consolidated by experimental results where the rates of hydrolysis of all these
phosphazenes were very slow in basic media containing 1 N or stronger sodium hydroxide. The
overall hydrolysis rate of these phosphazenes in 1 N or stronger acid was higher than in water.
Thus, acidic media accelerate the hydrolytic breakdown of these aminated phosphazenes com-
pared to neutral or basic media.
Aminated polyphosphazenes followed the same general trend in hydrolytic sensitivity as the
aforementioned small molecular analogs. The imidazole-substituted polymer (poly[bis(imidazolyl)
phosphazene]) (6) was found to be the most hydrolytically labile among several synthesized ami-
nated polyphosphazenes. The high hydrolytic sensitivity of imidazole-substituted polyphospha-
zenes could be further modulated by co-substituting it with less hydrolytically sensitive groups on
the polyphosphazene backbone. This has been verified by the experiments where imidazolyl mixed-
substituent polymer (poly[(imidazolyl)(methylamine)phosphazene]) underwent a slow hydrolysis
over several months at 25°C in water. Similarly, Laurencin et al. evaluated the hydrolytic degradation
of imidazole cosubstituted polyphosphazenes (poly[(imidazolyl)(p-methylphenoxy)phosphazene])
(12) (Laurencin et al. 1987). It was found that the relative amount of hydrolytically labile imidazolyl
groups determined the degradation rate of the polymer. Degradation of 20% imidazole-substituted
polyphosphazene was quite slow, with only 4% of the polymer degraded in 600 h. The contact angle
measurement confirmed the hydrophobic characteristic of the polymer (Laurencin et al. 1993),
which in turn decreased the water permeability of polymer matrix. In the case of 45% imidazole-
substituted polymer, approximately 30% of the polymer degraded in approximately 300 h, whereas
80% imidazole-substituted polyphosphazene degraded in a few hours under ambient conditions.
Among the aminated polyphosphazenes, those substituted with amino acid ester groups have
been extensively studied. In general, these macromolecules degrade through either bulk or surface
erosion or a combination of both to body-friendly products such as amino acids and ammonium
phosphate buffer, which ensures excellent biocompatibility for the degradation micro-environment.
The selection of an amino acid ester as the substituent group rather than an amino acid is based on
the following rationales: (1) a free carboxylic group in an amino acid would participate in chlorine
substitution steps to form cross-links; (2) the free carboxylic groups in an amino acid could promote
polymer degradation. Therefore, Allcock et al. synthesized a series of amino acid ester polyphos-
phazenes as potential biomaterials (Allcock et al. 1977). The polymers were synthesized by intro-
ducing different amino acid ester groups (5) (where R = H, CH3, CH2CH(CH3)2 or CH2C6H5 and
R′ = CH2CH3) or an amino acid ester group first followed by treatment with methyl amino groups
sequentially to tune the polymer degradation rate. All the polymers underwent a slow decrease in
molecular weight both in solution and in the solid state at 25°C. In specific, PNEG underwent a
decrease in chain length from 8,000 to 20,000 repeating units to less than 1000 repeating units in
15 days in the solid state at 25°C. Additionally the mixed-substituent polymer with methylamine
side group underwent a chain shortening from about 8000 to 1000 repeating units in 25–30 days.
This study clearly demonstrated that incorporation of suitable cosubstituents along with appropriate
amino acid ester groups could efficiently modulate the polymer degradation pattern.
Allcock et al. (1994b) studied the influence of ester groups and α-substituents on the hydrolytic
sensitivity of amino acid ester-substituted polyphosphazenes. Methyl, ethyl, tert-butyl and benzyl
esters of glycine-, alanine-, valine- and phenylalanine-substituted polyphosphazenes were used for
the study. The degradation studies were performed in deionized water and the molecular weight
changes of these polymers were followed by gel permeation chromatography (GPC) analysis. The
hydrolytic sensitivity of the polymers was found to increase as the size of the ester groups decreased.
Thus, for the same amino acids, the sensitive to hydrolysis was found to increase in the order of ben-
zyl esters < tert-butyl esters < ethyl esters < methyl esters. Similarly, the smaller the group linked to
the α-carbon atom of the amino acid residue, the more sensitive was the polymer to hydrolysis. Thus,
phenylalanato units were less sensitive than the valinato units, which were less sensitive than the
alaninato groups, with the glycinato side groups being the most sensitive to hydrolysis. The hydro-
lysis products of these compounds were found to be phosphates, amino acid, the respective alcohol
and ammonia. The physical properties of these polymers allows for facile processing and retainment
of dimensional stability at physiological temperatures, making them prospective biomaterials for
short-term medical applications as drug delivery matrices and tissue engineering scaffolds.
Allcock et al. (1994) investigated the hydrolytic degradation of three polyphosphazenes as
potential matrices for controlled drug release. Those macromolecules were substituted with either
the same amino acid residue or the same ester group, i.e., PNEG, PNEA (5, where R = CH3
and R′ = CH2CH3) and poly[bis(benzyl alanato)phosphazene] (PNBA) (5, where R = CH3 and
R′ = CH2C6H5). The polymer degradation rate at physiological pH was found to decrease in
the order of ethyl glycinato-substituted > ethyl alanato-substituted > benzyl alanato-substituted
polyphosphazenes. For the ethyl glycinato-substituted polymer, the small hydrogen atom at the
α-position provides the least steric repulsion against hydrolytic attack on the phosphorus atom
in the skeleton or on the side group P–N bonds. The decreased degradability of ethyl alanato and
benzyl alanato compared to ethyl glycinato can be attributed to the relatively large methyl group
in ethyl alanato-substituted polyphosphazenesand the bulky hydrophobic benzyl group in benzyl
alanato-substituted polyphosphazenes. Thus, the substituents at the α-carbon of the amino acid
80 PPHOS-EG10
PPHOS-EG25
PPHOS-EG50
PPHOS-EG75
60
Percent mass loss (%)

40
20
0
0 200 400 600 800 1000 1200
Time (h)
FIGURE 4.6 Percentage mass loss versus degradation time (h) of ethyl glycinato-substituted polyphospha-
zenes of different compositions in 0.1 M sodium phosphate buffer at 37°C and pH 7.4. The standard deviation
(SD) of the mean is less than 3%. (From Laurencin, C.T., Norman, M.E., Elgendy, H.M. et al.: Use of polyphos-
phazenes for skeletal tissue regeneration. J. Biomed. Mater. Res. 1993. 27(7). 963–973. Copyright Wiley-VCH
Verlag GmbH & Co. KGaA. With permission.)
ester side group appear to have more bearing on the hydrolysis rate of amino acid ester polymers
than the types of ester groups. On the other hand, the ester groups at the chain end exhibited a
significant effect on the polymer surface properties, such as hydrophilicity or hydrophobicity, as
evidenced from contact angle measurements. Hydrophobic ester groups significantly decrease
water penetration into the matrix and thereby retard the hydrolytic breakdown of these polymers.
The hydrolysis products of these polymers were found to be phosphates, alanine or glycine, ben-
zyl alcohol or ethanol, and ammonia.
Laurencin et al. demonstrated that the degradation rate of ethyl glycinato-substituted poly-
phosphazene could be efficiently modulated by incorporating a hydrophobic substituent, such as
a methylphenoxy group, as co-substituent (Laurencin et al. 1993). Figure 4.6 shows the percent-
age mass loss versus degradation time of poly[(ethyl glycinato)(p-methylphenoxy)phosphazene]
of different compositions. It can be seen that the degradation rate of the polymer increases with
an increase in the percentage of the ethyl glycinato group. Furthermore, the degradation rate of
aminated-polyphosphazene can also be tuned by varying the bulkiness and composition of the sub-
stituted side groups. For instance, among the four different l-alanine-substituted polyphosphazenes
namely PNEA, poly[(ethyl alanato)1(ethyl glycinato)1phosphazene] (PNEAEG), poly[(ethyl alanato)1
(p-methylphenoxy)1phosphazene] (PNEAmPh) and PNEAPhPh (16), the water contact angle
increased in the order of PNEAEG < PNEA < PNEAmPh < PNEAPhPh. Water contact angles
are the measure of surface hydrophilicity indicating the reduced polymer hydrophilicity with
the increased side group bulkiness. Degradation studies carried out in phosphate buffer saline at
37°C showed a molecular weight loss pattern in the order of PNEAEG > PNEA > PNEAmPh >
PNEAPhPh due to the differences in the bulkiness of the side groups and initial molecular weight
of the polymer (Singh et al. 2006). SEM micrographs of polymer surface morphologies follow-
ing hydrolytic degradation suggested that PNEA, PNEAmPh, and PNEAPhPh degraded via sur-
face erosion mechanism, whereas the degradation of PNEAEG proceeded through a combination
of bulk and surface erosion. In addition, the degradation kinetics of these polymers can also be
modulated by adjusting the ratios of the two cosubstituents (Laurencin et al. 1993). These studies
systematically demonstrated the versatility of polyphosphazene synthesis and the efficient control
over polymer degradation by changing side group chemistry to suit the requirements of a specific
biomedical application.
Ibim et al. (1996) studied the effect of pH on the degradation rate of poly[(ethyl glycinato)1
(p-methylphenoxy)1phosphazene] (PNEGmPh) (14). Degradation studies were performed in dis-
tilled water with the pH adjusted continuously to 2.0, 7.4, and 10.0. The polymer degradation was
found to be very slow in neutral and basic solution (pH 7.4 and 10.0). In contrast, more than 80% of
the polymer degraded in 35 days in acidic solution (pH 2.0).
So far, several hydrolysis routes have been proposed for poly[(amino acid ester)phosphazenes]
(Allcock et al. 1982b, 1994b). The ester functionality could be involved in the breakdown of the
−P═N− skeleton via three different mechanisms. In the first, hydrolysis of the ester unit by water
molecules would lead to the formation of the corresponding polymer-bound amino acid. The car-
boxylic acid unit could then attack a nearby phosphorus atom in the polymer chain. This species
would further react with water molecules to release the amino acid and to form a hydrolytically
unstable phosphazane, which would ultimately break down to phosphates and ammonia. In a second
mechanism, the presence of water could facilitate an attack on the polymer backbone by the ester
functionality itself. Water molecules could then react with the unstable phosphorus–ester bond. As a
result of this reaction, the amino acid would be released and a hydrolytically sensitive phosphazane
could be formed. In a third mechanism, water molecules would displace the amino acid esters from
the phosphorus atoms to form a hydroxyphosphazene. This species could rearrange in the presence
of water to a phosphazane. The phosphazane would then react with water molecules to yield phos-
phates and ammonia. Since the evidence for all three mechanisms was found, it was suggested that
all three could occur simultaneously, although it is still not clear which mechanism predominates
(Allcock et al. 1982b, 1994b).
Since the biodegradation of poly[(amino acid ester)phosphazenes] could occur through hydroly-
sis of the amino acid ester function, leading to a carboxylic acid which may catalyze the polymer
backbone breakdown, the polymer degradation rate could be increased by incorporating side groups
with a hydrolytically sensitive ester functionality. Crommen et al. synthesized a series of polyphos-
phazenes bearing a number of hydrolytically labile substituents, such as ethyl 2-[O-glycyl] lactate
(10) (where R = H) or ethyl 2-[O-alanyl] lactate (10) (where R = CH3), and the remaining P–Cl
groups of polymer 3 substituted by ethyl glycinate or ethyl phenylalanate moieties (Crommen et al.
1992b). The degradation of these polymers was carried out in phosphate buffer solution at pH 7.4.
The in vitro degradation rate of these polymers was found to be directly dependent on the content
of the depsipeptide side groups. The activated ester compounds were found to be more sensitive to
hydrolysis than the normal alkyl esters. Thus, the incorporation of controlled amounts of a hydro-
lysis-sensitive amino acid ester is an elegant approach for controlling the degradation rate of amino
acid ester–substituted polyphosphazenes.
Nair et al. (2006) investigated degradation behavior of two ethyl alanato based polymers namely
poly[(ethyl alanato)1(ethyl oxybenzoate)1phosphazene] (PNEAEOB) and poly[(ethyl alanato)1(propyl
oxybenzoate)1phosphazene] (PNEAPOB) in phosphate buffer (pH 7.4) at 37°C. PNEAPOB showed
a faster degradation rate and higher water absorption compared to PNEAEOB. Both polymers were
found to be insoluble in common organic solvents following hydrolysis presumably due to cross-
linking reactions accompanying the degradation process.
Most aminated polyphosphazenes undergo faster degradation in acidic environment compared
to physiological or basic conditions. Qiu and Zhu (2000a) prepared polymers (11) that degrade
at a higher rate under physiological conditions, but are relatively stable at low pH. The polymer
degradation was studied at different pH values at 37°C. It took only 12 h for the sample to dissolve
completely in a pH 8.0 buffer, 1.5 days in pH 7.4, but more than 20 days in weakly acidic media
(pH 5.0 and 6.0). A two-stage degradation mechanism was proposed for these polymers. In the first
phase, the breakage of side groups played a prominent role, in that the benzyl ester of amino acethy-
droxamic acid was hydrolyzed to produce amino groups, probably accompanied by the hydrolysis of
glycine ethyl ester to glycine. As a consequence, the polymer degraded to water-soluble polymeric
products. In the second phase, the soluble polymeric products were further degraded by scission of
the backbone into phosphates and ammonia.
4.3.2.3 Degradation of Alkoxy-Substituted Polyphosphazenes

Glyceryl-substituted polyphosphazenes undergo hydrolysis generating glycerol and phosphate in water
at 100°C (Allcock and Kwon 1988). The hydrolysis was detectable after 12 h and was complete after
150 h. At 37°C the hydrolysis was essentially complete after 720 h. The polymer with glycosyl and
methyl amino substituents decomposed completely within 24–96 h at 100°C in aqueous buffer solutions
(pH 6.0, 7.0, and 8.0) (Allcock and Pucher 1991). Under physiological conditions the hydrolysis was
much slower, with half-lives in the range of 165–175 h. The degradation products of these polymers
are presumed to be phosphate, glucose, ammonia, and methylamine. Another type of biodegradable
alkoxy-substituted polyphosphazene consists of those with glycolic or lactic acid as side groups (Allcock
et al. 1994c). These polymers (9) (where R = H or CH3 and R′ = CH2CH3 or CH2C6H5) undergo
hydrolysis when exposed to water in the solid state as evidenced from 31P NMR and GPC. Polymer 9
showed evidence of initial backbone hydrolysis at 320 h (where R = H and R′ = CH2CH3), at 525 h
(where R = CH3 and R′ = CH2CH3), at 656 h (where R = H and R′ = CH2C6H5), and at 11:07 h (where
R = CH3 and R′ = CH2C6H5), respectively. The fastest decomposition of polymer 9 (where R = H and
R′ = CH2CH3) was attributed to the least sterically hindered groups present in it. Water molecules could
displace ethyl glycolato side units or hydrolyze the ester function to the acid, and the pendent carboxylic
acid could then induce skeletal degradation. Polymer 9 (where R = CH3 and R′ = CH2CH3) and poly-
mer 9 (where R = H and R′ = CH2C6H5) showed almost similar degradation profiles, much slower than
polymer 9 (where R = H and R′ = CH2CH3). Both had either a methyl group or a bulky benzyl ester
group at the α-position, which could hinder hydrolytic attack at the phosphorus atoms. Polymer 9 (where
R = CH3 and R′ = CH2C6H5) has both the hydrolysis-limiting factors of polymers 9 (where R = CH3
and R′ = CH2CH3) and polymer 9 (where R = H and R′ = CH2C6H5), specifically the methyl group
on the α-position as well as the benzyl ester group. The hydrolysis products of these compounds were
identified as benzyl alcohol or ethanol, glycolic or lactic acid, phosphates, and ammonia.
4.3.2.4 Degradation of Polyphosphazene Blends/Composites

As discussed in the earlier sections, it is possible to design polyphosphazenes with modulated degra-
dation rate through appropriate selection of side groups and their compositions. Polymeric blending
is an alternative approach to develop novel biomaterials with controllable properties and degrada-
tion kinetics (Utracki 1989; Deng et al. 2009). A wide variety of biodegradable blend/composite
materials can be developed by blending a biodegradable polyphosphazene with either another bio-
degradable polymer or with another biodegradable polyphosphazene having a different hydrolytic
sensitivity. The advantage of the blending process is that a series of materials with variable degrees
of degradability can be obtained from the two parent polymers (Utracki 1989; Deng et al. 2009).
The degradation studies performed by Ibim et al. demonstrated that blends degraded at d ifferent
rates, implying that the blend properties can be easily tuned for a variety of different biomedical appli-
cations (Ibim et al. 1997).The follow-up study by Ambrosio et al. investigated the self-neutralizing
ability of such blend systems and the possibility of maximizing the neutralization effect by opti-
mizing the blend composition (Ambrosio et al. 2002). Three types of samples including PLAGA
(50:50), PNEGmPh, and their blends were subjected to degradation in distilled water at pH 7.4
over a period of 40 weeks. The blend showed an intermediate degradation rate between those of
the parent polymers, and the hydrolysis of polyphosphazenes could have been catalyzed by the
acidic degradation products of PLAGA (Figure 4.7a). In addition, the amounts of neutralizing base
(0.01 N solution of sodium hydroxide) for titrating the medium back to pH 7.4 were measured for
each type of sample throughout degradation. Results showed that the blend required significantly
lower amounts of neutralizing base than PLAGA, which indicated that the blend system released
less acidic degradation products due to the buffering effect of polyphosphazene degradation prod-
ucts (Figure 4.7b). Hence, these two earlier studies demonstrated the advantages of self-neutralizing
blends and established the ground for developing polyphosphazene–polyester blends as promising
candidate materials for controlled drug delivery and tissue engineering applications.
120 16
Amount of NaOH used (meq/g)

14
100
12
80 PPHOS-EG50
% Mass loss
10 BLEND
PPHOS-EG50 PLAGA
60 8
BLEND
40 PLAGA 6
4
20
2
0 0
0 10 20 30 40 50 0 10 20 30 40 50
(a) Time in weeks (b) Time in weeks
FIGURE 4.7 Degradation of PNEGmPh-PLAGA blend and parent polymers in pH 7.4 distilled water at
37°C over 12 weeks. (a) Percent mass loss; (b) Amount of NaOH (meq/g) used to neutralize the acid released
during the degradation of the blend and its parent polymers. (From Biomaterials, 23(7), Ambrosio, A.M.A.,
Allcock, H.R., Katti, D.S., and Laurencin, C.T., Degradable polyphosphazene/poly([alpha]-hydroxyester)
blends: Degradation studies, 1667–1672. Copyright 2002, with permission from Elsevier.)
Meanwhile, Qiu et al. performed degradation studies on slabs of PNEG-PLAGA and PNEG-
PSTP blends in distilled water with or without Rhizopus delemer lipase at 37°C (Qiu 2002b). The
blend of PNEG-PLAGA (70/30, w/w) took 120 days to disappear completely, while PNEG-PSTP
(70/30, w/w) slabs needed only 20 days. The degradation rate of PNEG-PLAGA blends was strongly
accelerated by the enzyme and the degree of enzymatic degradation depended on the weight per-
centage of PLAGA in the blend. In vivo degradation of the polymer blends was carried out by
subcutaneously implanting them in the back of mice and some differences in degradation were
observed compared to in vitro degradation. Such variation was attributed to more complex physi-
ological environment, which indicated that the degradation of PNEG-PLAGA occurred through
a combination of hydrolysis and enzymolysis, while the degradation of PNEG-PSTP proceeded
through only hydrolysis. In addition, both in vitro and in vivo studies showed that the degradation
rate of the blends could be easily controlled by adjusting the blend composition.
Krogman et al. (2009) blended PLAGA with polyphosphazenes containing THAM side groups
and cosubstitutents such as glycine ethyl ester and alanine ethyl ester. However, the degradation
rates of the aforementioned polymers were found to be very low. The dipeptide side groups impart
hydrolytic sensitivity and significant hydrogen bonding capability to the polymers. Krogman et al.
(2007) investigated the degradation behavior of blends of PLAGA and different glycylglycine ethyl
ester–substituted polyphosphazenes (17–20). Hydrolysis studies showed the blends degraded at a
slower rate than both parent polymers. The pH of the degradation medium containing the blends
increased from 2.5 to 4.0, which was attributed to the buffering capacity of polyphosphazenes
degradation products. This study has demonstrated the feasibility of fine-tuning the blend degrada-
tion profile by adjusting either the side group ratios of polyphosphazenes or blend composition. In
another study, a class of biodegradable polyphosphazenes containing dipeptides alanylglycine ethyl
ester, valinylglycine ethyl ester, and phenylalanylglycine ethyl ester were synthesized by Weikel
et al. (2009). The polymers were found to be less sensitive to hydrolysis under neutral and basic
conditions than acidic conditions (pH 4.0) (Weikel et al. 2009).
More recently, Deng et al. (2010d) demonstrated for the first time a unique degradation
process for biodegradable biomaterials based on PNGEGPhPh-PLAGA blends both in vitro
(phosphate-buffered saline and distilled water) and in vivo (physiological conditions using a rat
subcutaneous implantation model). As shown in Figure 4.8, a unique polymer erosion process
was evident in all the blend compositions in which a solid blend film degraded to form a self-
assembled p olyphosphazene sphere-based 3D porous structure with interconnected porosity.
T=0 T = 4 weeks T = 7 weeks T = 12 weeks
Time
(a) (c) (e) (g)
BLEND25
(25% PPHOS)
(b) (d) (f ) (h)

BLEND50
(50% PPHOS)
Percentage
PPHOS
FIGURE 4.8 (See companion CD for color figure.) Surface morphologies of the blends during in vitro
degradation at 37°C over 12 weeks as a function of time. (a, c, e, g): SEM images showing surface morpholo-
gies of BLEND25 following 0, 4, 7, and 12 weeks of in vitro degradation. The insets of (e) and (g) show the
detailed 3D spherical structures. (b, d, f, h): SEM images showing surface morphologies of BLEND50 follow-
ing 0, 4, 7, and 12 weeks of in vitro degradation. The unique polymer erosion of the blend system resulted in
the change of matrix morphology from a solid coherent film to an assemblage of microspheres with intercon-
nected porous structures characterized by macropores (10–100 μm) between polyphosphazene spheres as well
as micro/nanopores on the sphere surface. (From Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: Biomimetic,
bioactive etheric polyphosphazene-poly(lactide-co-glycolide) blends for bone tissue engineering. J. Biomed.
Mater. Res. A 2010c. 92A(1). 114–125. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. With permission;
Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: In situ porous structures: A unique polymer erosion mechanism
in biodegradable dipeptide-based polyphosphazene and polyester blends producing matrices for regenera-
tive engineering. Adv. Funct. Mater. 2010d. 20(17). 2794–2806. Copyright Wiley-VCH Verlag GmbH & Co.
KGaA. With permission.)
Specifically, the polyphosphazene cosubstituted with hydrophilic glycylglycine dipeptide ester

and hydrophobic phenylphenoxy groups showed an exceptional ability to form inter- and intra-
molecular hydrogen bonds with a tunable degradation pattern. This copolymer resulted in a
completely miscible blend system with the polyester through intermolecular hydrogen bonding.
By altering the blend composition, it was possible to control blend physicochemical properties
and degradation pattern. The blends showed a degradation rate in the order of BLEND50 <
BLEND25 < PLAGA in vitro at 37°C over 12 weeks. Significantly higher pH values of degra-
dation media were observed for blends compared to PLAGA confirming the neutralization of
PLAGA acidic degradation by polyphosphazene hydrolysis products. In all the blend compo-
sitions both PLAGA and polyphosphazene components exhibited a similar molecular weight
loss degradation pattern. For example, the number and weight average molecular weights (M n
and Mw) of both the PLAGA and polyphosphazene components decreased exponentially with
degradation time throughout the subcutaneous implantation period. Furthermore, the appar-
ent degradation rate, K, can be obtained according to the following exponential relationship
between molecular weight and degradation time: lgM = lgM0 − Kt. For example, the apparent
degradation rates based on Mw were calculated to be 0.1014 week−1 and 0.0859 week−1 for the
polyphosphazene component in BLEND25 and BLEND50, respectively. This indicates that the
polyphosphazene degraded faster in BLEND25 due to higher PLAGA composition. A similar
trend in the apparent degradation rates was found for the PLAGA component in pristine PLAGA,
BLEND25, and BLEND50. This suggests that the PLAGA degraded the slowest in BLEND50
due to the higher polyphosphazene composition. These results agree well with in vitro findings
that the PLAGA hydrolysis accelerated the polyphosphazene degradation, whereas the degrada-
tion of polyphosphazene retarded the hydrolysis of PLAGA. Characterization of in vivo degra-
dation samples at 12 weeks revealed the formation of 3D interconnected porous structures with
82%–87% porosity. It was determined that the in situ porous structure resulted from inter- and
intra-molecular hydrogen bonding interactions and variations in the degradation rates of both
blend components promoting a three-stage degradation mechanism including intermolecular
hydrogen bonding dominant state (Stage I, 0 week), intermediate state (Stage II, 0–4 weeks), and
intramolecular hydrogen bonding dominant state (Stage III, 4–12 weeks).
Meanwhile, Weikel et al. synthesized polyphosphazenes cosubstituted with bioactive mol-
ecule such as choline chloride and with glycine ethyl ester, alanine ethyl ester, valine ethyl
ester, or phenyl alanine ethyl ester (Weikel et al. 2010b). These choline-based polyphosphazenes
were blended with PLAGA (50:50) and PLAGA (85:15) via solution casting techniques. It was
found that the increased choline content allowed these polymers to hydrolyze into water-soluble
products. Polymers were able to form miscible blends with both types of PLAGA. Hydrolysis
experiments in aqueous media revealed that the polymer blends hydrolyzed to near-neutral pH
media (∼5.8 to 6.8).
For the blends of polyphosphazenes alone, Crommen et al. prepared blends of a hydrolytically
sensitive depsipeptide-containing polymer with PNEG. It was found that the degradation rate of the
blend increases as the composition of the depsipeptide-containing polymer increases (Crommen
et al. 1993). Similarly, the degradation rate can also be adjusted by blending (50/50, w/w) poly[(ethyl
glycinate)-co-(ethyl 2-(O-glycyl lactate) )phosphazene] (3% gly-lact-OEt) with PNEA. However, it
was found that the presence of the depsipeptide-substituted polymer only promoted the release of
glycine and glycine ethyl ester but did not affect the release of the alanine side groups. Lemmouchi
et al. observed a similar phenomenon for a blend (50/50, w/w) of PNEG with poly[(ethyl alanate)-
co-(ethyl 2-(O-glycyl lactate) )phosphazene] (3% gly-lact-OEt) (Lemmouchi et al. 1998). Only the
release of alanine and alanine ethyl ester was significantly affected. Such observed degradation
behavior of depsipeptide-containing polyphosphazene blends might be attributed to the intramo-
lecular catalysis of the polymer by the pendant carboxylic acid.
4.3.3 Biocompatibility of Biodegradable Polyphosphazenes

Another important requirement for biodegradable materials in biomedical applications is biocom-
patibility. The efficacy of the drug delivery matrix to achieve controlled release kinetics as well as
the tissue engineering scaffold to support cell growth and achieve functional tissue repair largely
depends on the material biocompatibility.
4.3.3.1 Biocompatibility of Aminated Polyphosphazenes

Laurencin et al. first characterized the biocompatibility of the amino acid ester–substituted poly-
phosphazenes for tissue engineering applications (Laurencin et al. 1992). It was demonstrated that
biodegradable PNEG supported the adhesion of primary rat osteoblasts (PRO) to the same extent
as PLAGA and polyanhydrides. Since then, several amino acid ester polyphosphazenes have been
investigated for their in vitro cytocompatibility. For example, both PNEAEOB and PNEAPOB sup-
ported adhesion and proliferation of PRO cells, and were found to be suitable as reinforcing materi-
als for developing self-setting bone cements (Nair et al. 2006). Cytotoxicity study of the degradation
media of poly[bis(ethyl 4-aminobutyro)phosphazene] was performed using Swiss 3T3 and HepG2
cell lines (Gümüsdereliolu and Gür 2002). It was found that the presence of degradation media
did not affect the proliferation of both cell lines. These in vitro results confirmed the cytocom-
patibility of the polyphosphazene degradation products, and further supported them as potential
biomaterials for in vivo applications (Gümüsdereliolu and Gür 2002). Early studies focused on
characterizing these amino acid ester–substituted polyphosphazenes for their in vivo tissue compat-
ibility. For instance, alanine ethyl ester–substituted polyphosphazenes namely PNEA, PNEAmPh,
and PNEAPhPh were characterized for their in vivo tissue compatibility using a rat subcutaneous
implantation model (Sethuraman et al. 2006). All these polymers initially provoked a mild to mod-
erate immune response that subsided over time. After 12 weeks, a minimal immune response was
found for PNEAmPh and PNEAPhPh which was characterized by the presence of few neutrophils,
erythrocytes, and lymphocytes. Further in vitro osteocompatibility evaluation using PRO cells dem-
onstrated that cells adhered, proliferated, and maintained their phenotype on polymers of PNEA,
PNEAmPh, and PNEAPhPh (Sethuraman et al. 2010). Laurencin et al. (1998) also evaluated the
biocompatibility of PNEGmPh-based bone grafts in New Zealand white rabbits. This polymer sup-
ported the bone growth with a mild tissue response when compared to PLAGA grafts. Histological
evaluations revealed the presence of a thin layer of fibrous tissue at the implant interface and the
adjacent bone at 1 week. After 12 weeks of implantation, the interface contained lamellar bone and
showed the presence of fat and bone marrow cells adjacent to the implant, whereas PLAGA implants
were surrounded by a thin fibrous membrane that consisted of a few giant cells, occasional lym-
phocytes, and plasma cells. The superior biocompatibility of these novel polymers was further sup-
ported by the absence of implant fragmentation, fat necrosis, or granulomas formation (Laurencin
et al. 1998). These in vitro and in vivo characterizations demonstrated the excellent biocompatibility
of polyphosphazenes. Therefore, the aminated polymers are actively pursued as biomaterials for
4.3.3.2 Biocompatibility of Alkoxy-Substituted Polyphosphazenes

Alkoxy-substituted polyphosphazenes have not yet been extensively investigated as biocompatible
materials, although they possess great potential due to the presence of reactive hydroxyl groups in
many of them. For example, incorporation of alkoxy side groups to polymer backbone can modulate
polymer surface hydrophobicity/hydrophilicity, which in turn affects the biocompatibility of the
polyphosphazenes.
4.3.3.3 Biocompatibility of Polyphosphazene Blends/Composites

Since the first report on the biodegradable blends of poly[(amino acid ester)phosphazenes]
and polyesters, a series of polyphosphazene blends have been developed and characterized for
biocompatibility as prospective biomaterials. Most amino acid ester–based polyphosphazene
blends were found to be biocompatible. Nair et al. blended PNEA with PLAGA (85:15) at two
weight ratios of 1:3 and 1:1. Significantly higher PRO adhesion and proliferation were observed
for both the blends compared to the parent polymers (Nair et al. 2005). Meanwhile, Qiu et al.
have characterized a series of degradable blends comprising PNEG and polyesters or polyan-
hydrides for tissue biocompatibility using a rat subcutaneous implantation model (Qiu 2002a).
The tissue biocompatibility of PNEG-PLAGA was found to be better than that of PNEG-PSTP.
Moreover, the tissue biocompatibility of PNEG-PSTP blends was improved by increasing the
weight percentage of PNEG in blends. These findings suggested the blends of PNEG-PLAGA
or -PSTP may be used as drug delivery matrices or for other potential biomedical applications.
Deng et al. (2008) developed high-strength materials for tissue engineering applications from
the blends of PNEAPhPh and PLAGA at three weight ratios of 1:3, 1:1, and 3:1. In an in vitro
osteocompatibility study using PRO culture, these blends not only supported the PRO prolifera-
tion to the same extent as PLAGA but also showed enhanced differentiation characterized by
increased levels of alkaline phosphatase activity and mineralized matrix synthesis compared
to parent polymers.
In addition, in an effort to develop biomimetic and bioactive materials for achieving bet-
ter osteointegration, Deng et al. (2010c) also demonstrated that the novel etheric poly[(ethyl
glycinato)1(methoxyethoxyethoxy)1phosphazene]-PLAGA blends were able to nucleate bone-like
apatite via a biomimetic process. Enhanced phenotypic expression of the PRO was found on the
blends compared to parent polymers. Krogman et al. evaluated the in vitro osteocompatibility
of blends based on PLAGA and polyphosphazenes cosubstituted with both THAM and glycine
or alanine ethyl esters (Krogman et al. 2009). The polymer blends showed good adhesion and
proliferation of PRO during 14 days of culture. However, the dramatic decrease in cell number
from 14 to 21 days was attributed to the different degradation kinetics of the parent polymers.
This underlines the need to develop polymers with balanced properties in degradation rates and
biocompatibility. In that direction, Deng et al. (2010a,b) demonstrated that blends of PLAGA
and PNGEGPhPh resulted in improved osteoblast activity and biocompatibility compared to the
polyester materials due to their appropriate degradation pattern and self-neutralizing ability. The
blends demonstrated significantly higher osteoblast growth rates compared to PLAGA while
maintaining osteoblast phenotype over a 21 day culture. In a 12 week rat subcutaneous implan-
tation study, the dipeptide-based blends elicited minimal inflammatory response and exhib-
ited superior biocompatibility as compared to PLAGA as shown in Figure 4.9. More recently,
it was reported by Weikel et al. that the choline-based blends supported PRO cell growth with
an elevated osteoblast phenotype expression compared to the polyester (Weikel et al. 2010b).
Specifically, during the 21 day culture period, the elevated ALP expressions of osteoblast cells on
choline-based blends suggested the progression of cell maturity, whereas osteoblasts on PLAGA
were proliferating throughout the later time points. Choline-based blend system offers an advan-
tage in the development of a scaffold material with balanced properties such as the elevated cel-
lular activity from the release of choline and the increased buffering capacity from the hydrolysis
of the polyphosphazene backbone.
Thus, polyphosphazene–polyester blend systems are promising candidate materials for biomedi-
cal applications. Current research efforts are focused on the design and development of polyphos-
phazene blends with other synthetic or natural biodegradable polymers. Attempts also focus on the
incorporation of polyphosphazene block copolymers as compatibilizers to improve blend miscibil-
ity and other physicochemical and biological properties.
4.3.4 Biodegradable Polyphosphazenes for Biomedical Applications

Biodegradable polymer systems are widely investigated as controlled drug delivery matrices for
enhancing the therapeutic performance of drugs or 3D engineered scaffolds for tissue repair and
regeneration. The significant advantage of using biodegradable polymers is the ability to circumvent
the long-term biocompatibility concerns associated with many of the nondegradable polymers. In
addition to traditional polyesters, polyanhydrides, and poly(ortho esters), polyphosphazene poly-
mers have recently shown exponentially growing interest as a biodegradable material platform for
controlled drug release and tissue engineering applications as evidenced by some of the studies
discussed next.
4.3.4.1 Drug Delivery

4.3.4.1.1 Matrix Fabrication
Most studies have used biodegradable polyphosphazenes as pellets or films for drug delivery appli-
cations. These matrices are usually fabricated via compression molding (Crommen et al. 1992b)
or solvent casting due to great polymer solubility in a wide range of solvents (Laurencin et al.
1987, 1993; Allcock et al. 1994a; Ibim et al. 1996). In addition, Veronese et al. prepared naproxen-
loaded microspheres of poly[(phenylalanine ethyl ester)(imidazolyl)phosphazene] with side group
ratios of 71:29 and 80:20 by spray drying, emulsion/solvent evaporation, and emulsion/solvent
evaporation-extraction (polymer dissolved in 50:50 ethanol:dichloromethane mixture and injected
(a) (b)
(c) (d)
(e) (f )
FIGURE 4.9 (See companion CD for color figure.) Representative photomicrographs of hematoxylin
and eosin (H&E) stained section illustrating the chronic inflammatory response to PLAGA and blend matri-
ces during the post-implantation period. After 2 weeks, (a) PLAGA, (b) BLEND25, (c) BLEND50; After
4 weeks, (d) PLAGA, (e) BLEND25, (f) BLEND50. Both blend matrices elicited less inflammatory responses
than PLAGA. (From Biomaterials. 31(18), Deng, M., Nair, L.S., Nukavarapu, S.P. et al., Dipeptide-based
polyphosphazene and polyester blends for bone tissue engineering, 4898–4908. Copyright 2010b, with per-
mission from Elsevier.)
into phosphate buffer containing 5% Tween 80 as surfactant at pH 4.2) from a dichloromethane

or acetone solution of the polymer (Veronese et al. 1998). Microspheres prepared by spray drying
from a dichloromethane solution of the polymer yielded microcapsules with a hollow core, most
of which were found to collapse when freeze-dried. Microspheres prepared by spray drying using
acetone as the solvent maintained their spherical geometry with a narrow size distribution with a
mean diameter of 2–5 μm. The solvent evaporation and emulsion/solvent evaporation-extraction
technique yielded almost similar type of microspheres, with a larger size distribution of 25–80 μm
with more than 70% of the particles in the size range of 40–70 μm. Similarly, Calceti et al. used sus-
pension solvent evaporation, double emulsion-solvent evaporation, and suspension/double emulsion-
solvent evaporation for the preparation of insulin-loaded polyphosphazene microspheres (Caliceti
et al. 2000). SEM analysis demonstrated that all the preparation procedures produced spherical
microparticles with a porous surface and a honeycomb internal structure. The emulsion techniques
yielded microspheres with high porosity due to the elimination of the water microdrops of the
double emulsion inner phase during hardening of the microspheres. However, the microspheres pre-
pared by solvent evaporation also showed high porosity, indicating that microdrops of the continu-
ous phase can be entrapped in the polymeric matrix during microparticle formation. All the three
techniques yielded particles with a narrow size distribution, with the suspension solvent evaporation
method yielding 90% of the particles in the range of 30–90 μm, double emulsion-solvent evapora-
tion having 90% of the particles in the range of 10–60 μm and suspension/double emulsion-solvent
evaporation having 90% of the particles in the range of 0–60 μm.
4.3.4.1.2 Drug Release Kinetics

Degradation studies of biodegradable polyphosphazenes clearly demonstrate that the polymer
degradation rate can be efficiently tuned by incorporating different hydrophilic, hydrophobic, or
bulky substituent groups. This, combined with the nontoxic degradation products of the biode-
gradable polyphosphazenes, distinguished them from other biodegradable polymers as attractive
candidates for the controlled release of small and macromolecular drugs. The tunability in the
degradation characteristics of these polymers is so versatile that it could offer the ability to for-
mulate drug release systems based primarily on diffusion, erosion, or a combination of erosion
and diffusion.
The first attempt in that direction was carried out by Allcock and co-workers by attaching various
bioactive agents to a polyphosphazene backbone for subsequent hydrolytic release. Accordingly,
steroidal residues such as desoxoestrone, estrone, 17β-estradiol, 17α-ethynylestradiol, estradiol
3-methyl ether, and 1,4-dihydroestradiol 3-methyl ether were attached to the polyphospha-
zene backbone by Allcock and Fuller via the sodium salt of the steroidal hydroxy function
(Allcock and Fuller 1980). The degree of replacement of P–Cl bonds by P–OR bonds varied
in the range of 0.5%–40% and the remaining chlorine atoms were replaced by methylamine,
ethyl glycinate, or n-butylamine. Hydrolytically stable polymers were obtained when the ste-
roidal units were linked to phosphorus through an aryloxy residue, whereas linkage through
an alkoxy r esidue led to hydrolytically labile polymers. The use of ethyl glycinato residues as
cosubstituents yielded polymers that underwent chain cleavage in aqueous media. However,
detailed experiments on the degradation kinetics and release profiles of the attached steroids
have not been carried out. Similarly, local anesthetics such as procaine, benzocaine, chloropro-
caine, butyl-p-a minobenzoate, and 2-amino-4-picoline were attached to the polyphosphazene
backbone by direct a minolysis in order to modify the duration of biological activity of these
compounds (Allcock et al. 1982a). Preliminary studies indicated that the procaino-substituted
high polymers underwent a slow hydrolysis in buffered aqueous media at pH 7.0. In another
effort, Grolleman et al. developed a system consisting of 65%–90% of glycine ethyl ester and
10%–35% of a spacer group such as L-lysine ethyl ester linked to an anti-inflammatory agent,
naproxen (Grolleman et al. 1986). The degradation kinetics and naproxen release studies exhib-
ited a nearly zero-order release of the drug from the system. However, the amount of drug
released was found to be very low.
One of the earlier studies reported by Laurencin et al. investigated the feasibility of imidazole
co-substituted polyphosphazenes as a monolithic controlled drug delivery system (Laurencin
et al. 1987). Imidazole and methylphenoxy co-substituted polyphosphazene matrices, polymer
12 with 20%, 45%, and 80% imidazole content, were used for the in vitro release of small mole-
cules such as progesterone, p-nitroaniline, and macromolecule such as 14C-labeled bovine serum
albumin (BSA). The in vivo release of progesterone was studied via the subcutaneous implanta-
tion of matrices in Sprague–Dawley rats. The degradation rate of these matrices was discussed
previously in Section 4.3.2.2. It was found that release of progesterone from 20% imidazole-
substituted polyphosphazene matrices occurred over 10:00 h in vitro, whereas the in vivo study
demonstrated an initial lag period of approximately 80 h followed by a period of almost constant
release for 900 h. Furthermore, the release kinetics of 1% and 5% loaded p-nitroaniline from
20% imidazole-substituted polyphosphazene was determined as a function of square root of

time. A diffusion-controlled release of the small molecules occurred over approximately 250 h
(square root 15–16 h) and the release rate was found not to be very dependent on drug loading.
In contrast to the low molecular weight release, the release profile for BSA-loaded 20% imid-
azole-substituted polyphosphazene was characterized by an initial burst release of the protein
diffusing out of the matrix followed by a sustained release up to 500 h. The 25% burst release
within the first 4 h was successfully eliminated by coating the matrix with a thin layer of the
20% imidazole-substituted polymer alone. It was further determined that the coated matrices
released only 5% of protein during the first 4 h. Thus, prolonged release of low molecular weight
molecules as well as macromolecules in combination with the inherent biodegradability and
biocompatibility makes imidazole-substituted polymers (12) promising candidate matrices for
developing drug delivery systems.
Allcock et al. investigated the potential of using biodegradable poly[(amino acid ester)phos-
phazenes] for the controlled release of small molecules in a monolithic system (Allcock et al.
1994a). Three different poly[(amino acid ester)phosphazenes], PNEG, PNEA, and PNBA, were
examined as potential drug delivery matrices. The degradation rate of these polymers was
d iscussed previously in Section 4.3.2.2. The diffusion rate of small molecules such as ethac-
rynic acid and Biebrich Scarlet was found to be dependent on the hydrolysis rate of the poly-
mers. Bimodal release was observed for all three polymers: an initial slow release phase due
to diffusion of the drug through the polymer matrix, and a second phase with an increased
release rate owing to both diffusion and erosion, dominated by release through polymer erosion.
Thus, fine-tuning the polyphosphazene structure with a specific combination of amino acid ester
side groups could allow elegant control over the rate of polymer hydrolysis and the subsequent
drug release.
It has been extensively demonstrated that the amino acid ester side groups confer biodegradabil-
ity and biocompatibility to polyphosphazenes while the imidazole group can significantly increase
the polymer degradation rate. Thus, mixed-substituent polymers with both amino acid ester and
imidazole groups have been used for the controlled release of drugs to achieve desirable pharmaceu-
tical dosages. Conforti et al. (1996) prepared a discoid matrix based on this mixed-substituent poly-
mer with phenylalanine ethyl ester, imidazolyl group, and unsubstituted chlorine atoms in the side
group ratio of 75:18:7 for the prolonged release of naproxen. The feasibility of modulating the drug
release rate was demonstrated in vitro by either varying the drug loading or the matrix thickness.
In general, thinner disk matrices showed a faster release rate than thicker matrices. The drug release
from the thicker disks showed a release rate approaching zero-order kinetics. In vivo studies in a
rat model further showed the maintenance of naproxen concentration in plasma within the thera-
peutic range for several weeks. The pharmacological efficacy of the systems was demonstrated in
the inhibition of acute and chronic inflammatory diseases. In the acute inflammation model, the
implantation of thin matrices, which showed fast release in vitro, resulted in significant inhibition
of edema development, both 1 h and 1 week after matrix implantation. Interestingly, the implanta-
tion of thicker matrices resulted in a slow and steady drug release for inhibiting chronic adjuvant
arthritis up to day 28. Although these discoidal polyphosphazene matrices showed promise as a
controlled release system, the complexity associated with the need for implantation often limited
their applications. Thus, polymer microspheres with the ability to be delivered by a syringe were
developed for naproxen release. As mentioned earlier in Section 4.3.4.1.1, microspheres prepared by
emulsion/solvent evaporation or emulsion/solvent evaporation-extraction showed a size distribution
of 10–100 μm. About 80% loading of naproxen could be obtained in these microspheres (Veronese
et al. 1998). The in vitro release of naproxen from microspheres produced by spray drying was
very rapid and resulted in complete release of entrapped drug within 2 h irrespective of polymer
composition and drug loading (Veronese et al. 1998). On the other hand, naproxen release from
microspheres fabricated by solvent evaporation was slow and dependent on drug loading and poly-
mer composition. The increase of the imidazole content of the polymer led to greater drug release
due to the ease of polymer degradation and ease of water penetration into the matrix as a result of
increased hydrophilicity. The rate of drug release also increased as the drug loading in the matrix
increased. Thus, release from these microspheres was predominantly diffusion-controlled. In vivo
release of the drug, through the implantation in rats, demonstrated a constant plasma concentra-
tion (suitable for anti-inflammatory activity) of naproxen maintained up to 400 h. Therefore, the
drug release pattern from polyphosphazene matrices can be modulated by varying the polymer side
group composition, method of microsphere fabrication, and drug loading.
Ibim et al. (1998) developed a musculoskeletal delivery system for the controlled release of the
anti-inflammatory agent colchicines to joints based on two types of polyphosphazenes namely
poly[(imidazolyl)0.4(p-methylphenoxy)1.6phosphazene] (12, 20% imidazole-substituted) and
PNEGmPh (Ibim et al.). Polymer degradation and drug release studies were carried out using colchi-
cine-loaded matrices. The degradation rate of PNEGmPh was much higher than that of polymer 12 at
physiological pH. Colchicine release was found to be 20% and 60% from polymer 12 and PNEGmPh
over the 21 day period of study, respectively. The release profile of colchicine from these polymers
was found to be consistent with the degradation pattern of the respective polymers (i.e., the faster
polymer degradation yielded the faster colchicine release), although the release appeared to proceed
through a combination of erosion and diffusion mechanisms during the initial stages.
In another study, membranes and microspheres of polyphosphazenes PNEA and poly[(ethyl
phenylalanato)1.6(imidazolyl)0.4phosphazene] were adopted for the treatment of periodontal diseases
(Veronese et al. 1999). Drug release from polyphosphazene membranes was characterized both
in vitro and in vivo. It was found that drug released at a rate that ensured therapeutic concentrations
in the surrounding tissue. In vivo studies suggested that polyphosphazene membranes were much
more effective in promoting the healing of rabbit tibia defects than polytetrafluoroethylene mem-
branes. The drug release from polyphosphazene microspheres yielded local drug concentrations
that are useful per se or when mixed with hydroxyapatite (HAp) for better bone formation.
Due to their desirable properties such as biodegradability and matrix permeability, biodegrad-
able polyphosphazenes have also been extensively investigated as matrices for protein delivery.
Andrianov et al. reviewed the advantages of using polyphosphazene matrices for the protein
release (Andrianov and Payne 1998). For example, microspheres of poly[(ethyl phenylalanato)1.6
(imidazolyl)0.4phosphazene] were used for the controlled release of insulin (Caliceti et al. 2000).
The microsphere preparation was discussed in Section 4.3.4.1.1. The in vitro release of insulin
from these microspheres exhibited a bimodal profile, which was characterized by a burst release
during the initial 2 h and a subsequent slow release in the range of 2–70 h. Subcutaneous adminis-
tration of these microspheres prepared by suspension solvent evaporation and suspension/double
emulsion-solvent evaporation to diabetic mice rapidly reduced glucose levels to 80%, but they lost
most of their activity in 100 h. However, those obtained by double emulsion-solvent evaporation
induced a remarkable decrease in glucose level and the activity was maintained for 10:00 h. The
anti-insulin antibody production stimulated by all these preparations was found to increase con-
stantly over a period of 8 weeks. This attests to the potential of these materials in the development
of one-shot vaccines.
A polyphosphazene polymer (PNEGmPh) was also investigated by Ibim et al. as a substrate for
the controlled release of macromolecules (Ibim et al. 1996). 14C-labelled inulin, a polysaccharide
with a molecular weight of 5000, was the macromolecule used for the study. Inulin was incorpo-
rated into the matrices by a solvent casting technique at 1%, 10%, and 40% loading. The in vitro
degradation studies of these polymers were discussed in Section 4.3.2.2. The polymer degradation
was accelerated with higher hydrophilic macromolecule inulin content due to increased water per-
meability. It was found that rate of inulin release from PNEGmPh matrices was highest at pH 2.0
followed by pH 10.0 and 7.4, which was in line with the trend of polymer degradation. Here, drug
release also proceeded through a combination of erosion and diffusion mechanisms. Higher macro-
molecule loading increased the levels of initial drug burst release and resulted in increased amounts
of inulin released due to the increased matrix hydrophilicity.
Qiu and Zhu (2000a) synthesized biodegradable polyphosphazenes containing the glycine
ethyl ester and benzyl ester of aminoacethydroxamic acid as co-substituents by further modi-
fying polymer PNEG. The polymers were found to undergo hydrolysis at a greater rate under
physiological conditions, but were relatively stable at low pH. The potential of these polymers
for macromolecular release was evaluated using polysaccharide fluorescein isothiocyanate dex-
tran (FITC-dextran) and the protein myoglobin. The cumulative release of FITC-dextran from
the polymer matrix was found to be greater at pH 7.4 and 8.0 than at pH 6.0 and 5.0. However,
in the case of myoglobin, the matrix was found to retain the protein completely over 25 h at
pH 5.0 and 6.0, whereas there was an appreciable protein release from the matrix at pH 7.4
and 8.0. This complete retention of the protein below pH 6.0 may be attributed to the formation
of water-insoluble polyelectrolyte complexes between the protein and polyphosphazene below
the isoelectric point (6.9) of the protein.
Oredein-McCoy et al. (2009) recently investigated the feasibility of utilizing polyphospha-
zene poly[(ethyl phenylalanato)1.5(ethyl glycinato)0.5phosphazene] microsphere sintered scaffold
(Figure 4.10a) alone or incorporated with HAp (Figure 4.10b) to regulate the release of BSA. In the
study, polyphosphazene and its composite microspheres with nanocrystalline HAp were fabricated
via the emulsion-solvent evaporation technique. BSA-loaded microsphere matrices were created
using a solvent/nonsolvent sintering method. Confocal microscopy verified the presence of FITC-
BSA both in the adjoining regions between microspheres and within the surface of microspheres.
(a) (b)
Cumulative protein release

110 PPHOS
PPHOS-HA
90
Cumulative release (%)
70
50
30
10
0 5 10 15 20
–10
(c) Time point (days)
FIGURE 4.10 (See companion CD for color figure.) Polyphosphazene-based matrices for growth factor deliv-
ery. (a) SEM micrograph showing the morphology of polyphosphazene matrices via solvent/nonsolvent sinter-
ing; (b) SEM micrograph showing the morphology of polyphosphazene-HAp matrices via solvent/nonsolvent
sintering; and (c) Cumulative BSA release. Zero order release of BSA was demonstrated over the course of 21
days from both the BSA-loaded polyphosphazene and BSA-loaded polyphosphazene-HAp composite scaf-
folds. Electrostatic interactions between HAp and BSA contributed to the controlled release curve shown here.
(From Oredein-McCoy, O. et al., J. Microencapsul., 26(6), 544, 2009, with permission from Informa Healthcare.)
It was found that for both polyphosphazene and its composite microsphere scaffolds, BSA was
released over a 21 day period following a zero order release profile (Figure 4.10c). Furthermore, the
presence of nanocrystalline HAp particles in the composite scaffolds improved the release profile
pattern, which demonstrated the potential of HAp for controlled delivery of growth factors such as
angiogenic factors. The results of this work have established the platform for future development of
angiogenic factor-loaded polyphosphazene-based matrices to invoke neovascularization.
Thermosensitive polymer hydrogels have also attracted much attention as injected delivery sys-
tems of drugs, factors or cells for a variety of biomedical applications. Such polymer hydrogels show
sol-gel transitions under certain stimuli such as temperature and pH. Most of them are formed via
miscelle packing or hydrophobic interactions between polymer chains, and contain both hydro-
philic and hydrophobic regions. Earlier literatures have reported several synthetic polymer hydrogels
including N-isopropylacrylamide (NiPAAM), copolymers poly(lactide-co-glycolide)-poly(ethylene
glycol)-poly(lactide-co-glycolide) (PLAGA-PEG-PLAGA), and poly(ethylene oxide)-poly(propylene
oxide)-poly(ethylene oxide) (PEO-PPO-PEO) (Glatter et al. 1994; Chen and Hoffman 1995; Jeong
et al. 1999; Zentner et al. 2001). Polyphosphazenes that respond to external stimuli such as tempera-
ture and pH can be designed and applied for controlled drug release (Allcock et al. 1988; Allcock and
Ambrosio 1996; Lee et al. 2002; Zhang et al. 2006; Ghattas and Leroux 2009; Ilia 2009; Qiu and
Zheng 2009; Bi et al. 2010). For example, amphiphilic graft polyphosphazene with oligopoly(N-iso-
propylacrylamide) and ethyl 4-aminobenzoate as side groups were synthesized by Qiu and coworkers
with different hydrophobic/hydrophilic balances (Zhang et al. 2006). These polymers possess a lower
critical solution temperature (LCST) below which they remain water-soluble and above which they
precipitate out. Furthermore, pH-responsive hydrogels obtained by gamma irradiation of polyphos-
phazenes substituted with sodium oxybenzoate and methoxyethoxyethoxy side groups showed pH-
dependent swelling behavior (Allcock and Ambrosio 1996). Lee et al. synthesized thermosensitive
polyphosphosphazene gels bearing α-amino-ω-methoxy PEG (AMPEG) and hydrophobic amino
acid ethyl esters such as isoleucine ethyl ester, leucine ethyl ester and valine ethyl ester (Lee et al.
2002). By adjusting the nature and compositional ratios of the hydrophilic and hydrophobic moieties,
the gelation properties of the polymers can be finely tuned (Lee and Song 2005). For example, a
polyphosphazene gel that contains a greater hydrophobic component such as isoleucine ethyl ester
(IleOEt) generally shows a higher gel viscosity and lower gelation temperature. On the other hand, a
polyphosphazene polymer containing less hydrophobic or hydrolysis-sensitive groups such as ethyl-2-
(O-glycyl) lactate (GlyLacOEt) tends to exhibit lower gel viscosities and higher gelation temperatures.
On the polyphosphazene platform, a variety of prodrugs or bioactive molecule conjugates
have also been developed for controlled delivery. For example, drug conjugates of biodegradable
polyphosphazene–platinum (II) with a wide range of molecular weights showed high tumor selectivity
and excellent in vivo antitumor activity against both murine and human cancer cell lines than their
drug alone counterparts (Jun et al. 2005). Such an observed high antitumor activity was achieved
through the controlled release of the antitumor active platinum (II) moiety from the polyphosphazene
backbone in the degradation process. Anticancer drugs such as paclitaxel and doxorubicin have also
been successfully conjugated with polyphosphazenes for inhibiting local tumor growth (Chun et al.
2009a,b). The aqueous solutions of these conjugates showed a solgel transition behavior correspond-
ing to temperature changes. The tumor-active drugs from the polymer-drug conjugate hydrogels were
slowly released and effectively accumulated locally in the tumor sites. More recently, in an effort to
induce osteogenic differentiation of mesenchymal stem cells (MSCs) for enhancing ectopic bone for-
mation, an injectable and thermosensitive poly(organophosphazene)–RGD conjugate was successfully
synthesized through a covalent amide linkage between a cell adhesion peptide, GRGDS, and carboxylic
acid-terminated poly(organophosphazene) (Chun et al. 2009c). The polymer-GRGDS conjugates were
in fluid state at room temperature and immediately gelled at body temperature. The rabbit mesenchy-
mal stem cells (rMSCs) were cultivated in the polymer–GRGDS conjugate hydrogel constructs using
an injection method into a nude mouse. rMSCs were found to express markers at mRNA level for all
stages toward osteogenesis and showed a sharp increase of osteocalcin levels at 4 week post-induction.
Histological and immunohistochemical evaluations illustrated a significantly high level of mineral-

ization and collagen type I expression by the rMSCs cultivated on the polymer–GRGDS conjugate
hydrogels after 4 weeks. Furthermore, during the fabrication process of drug delivery matrices, bioac-
tive agents are exposed to conditions like elevated temperature or harsh organic solvents which might
compromise their bioactivity. It is possible to design hydrogels by the ionic cross-linking of a water-
soluble polyphosphazenes that can eliminate the exposure of bioactive agents to the aforementioned
harsh conditions. For instance, aqueous PCPP hydrogel microspheres can be obtained by cross-linking
with Ca2+ ions (Andrianov et al. 1998). Such formulations allow a sustained protein release (Payne
et al. 1995; Andrianov and Payne 1998; Andrianov et al. 2009). These biodegradable polyphosphazene
matrices have shown tremendous potential as carriers for controlled release of bioactive agents.
Compared to the aforementioned polyphosphazenes, alkoxy-substituted polyphosphazenes have
not been extensively investigated as drug delivery matrices. In fact, the presence of reactive hydroxyl
groups in many of alkoxy-substituted polymers makes them great candidates for controlled drug
release applications. Particularly, the glyceryl-substituted polyphosphazenes are very interesting,
since the presence of hydroxyl groups in the side units can generate water solubility and can also
provide potential sites either for cross-linking or for the attachment of bioactive agents (Allcock and
Kwon 1988). The capacity of this polymer to cross-link and form hydrogels offers the possibility of
using this system for the diffusion-controlled release of bioactive agents. Preliminary experiments
with the diffusion release of tripelennamine hydrochloride from this polymer hydrogel suggested
diffusion-controlled release followed by polymer hydrolysis. Furthermore, pharmaceutical agents
or peptides can be linked to the glucosyl-substituted polyphosphazenes (Allcock and Pucher 1991).
In vivo hydrolysis would release the glucose-bonded bioactive agents. In the case of polyphospha-
zenes with glycolic acid ester and lactic acid ester side groups, the lack of crystallinity of these poly-
mers combined with the faster degradation rate compared to PGA or PLA makes them appropriate
candidates for controlled drug release applications (Allcock et al. 1994c).
In addition, polyphosphazene blends/composites have also been explored as controlled drug deliv-
ery matrices due to the efficient control over the polymer properties and release profiles. Schacht
et al. evaluated the potential of the blends of two polyphosphazenes with different degradation rates
as a matrix for controlled drug release (Schacht et al. 1996, 1998). In vitro release of the antitumor
agent mitomycin C (MMC) was studied using polyphosphazenes containing ethyl glycinate and
phenylalanate side groups as well as blends of these two polymers. It was found that the MMC
release was much faster from PNEG than from PNEPhA. For the blends, the drug release decreased
with the increase of phenylalanato content, which may be attributed to the increase in hydropho-
bicity of the matrix and the possible decrease in the water permeability of the matrix. The release
profile was found to be diffusion-controlled. In the case of thermosensitive polymer hydrogels, the
precise control over the balance between hydrophilicity and hydrophobicity is required in order to
have the desired gelation properties. However, complexity and many unexpected factors involved in
polymer synthesis make such control unpredictable. Kang et al. (2005) demonstrated the efficacy
of controlling the gelation behavior of hydrogels via the compositional regulation over the hydro-
phobic and hydrophilic components in polymer blends. In the study, both hard and soft polyphosp-
hazenes were synthesized. A hard polymer such as [NP(IleOEt)1.16(AMPEG550)0.84]n exhibits a low
gelation temperature and a high viscosity, whereas a soft one like [NP(IleOEt)1.07(GlyLacOEt)0.02
(AMPEG550)0.91]n shows a high gelation temperature and a low viscosity. By blending these two
types of polyphosphazenes at an appropriate ratio, the blended aqueous solution was able to change
into a transparent hydrogel that showed great strength at 37°C. According to DSC and IR, the two
polymers blended homogenously and formed a thermosensitive injectable hydrogel with a Tmax of
37°C–38°C. Recently, Potta et al. synthesized a class of injectable, self-cross-linkable, and thermo-
sensitive polyphosphazene-based blends of functional thiolated and acrylated polymers to develop
an ideal injectable carrier (Potta et al. 2010). The gel properties such as mechanical strength, inner
3D network, and degradation rate can be modulated by varying the degree of cross-linking between
the thermosensitive and functional blended polymers.
In summary, the polyphosphazene polymers (aminated polyphosphazenes, alkoxy-substituted

polyphosphazenes, and polyphosphazene blends/composites) constitute a very promising matrix
platform capable of controlled delivery of both small molecules and macromolecules through the
mechanisms of diffusion, erosion, alone or in combination.
4.3.4.2 Tissue Engineering
4.3.4.2.1 Scaffold Fabrication
Tissue loss or organ failure is one of the most devastating and costly problems in health care. A variety
of musculoskeletal diseases including back pain, arthritis, osteoporosis, and bodily injuries are often
reported among the U.S. population. Each year, more than 3 million musculoskeletal procedures are
performed in the United States alone (Vacanti 1999). To overcome the limitations associated with the
current graft options, tissue engineering has emerged as a promising strategy that provides de novo
regenerated viable tissue substitutes. Tissue engineering has been defined as “the application of bio-
logical, chemical and engineering principles toward the repair, restoration or regeneration of tissues
using biomaterials, cells, and factors alone or in combination” (Laurencin et al. 1999).
All approaches in tissue engineering involve one or more of the three key components: (1) 3D
porous matrices with optimum properties to support and promote tissue regeneration, (2) a healthy
population of harvested cells, and (3) biological growth factors that can induce cellular migration,
differentiation, and tissue growth in vivo (Langer et al. 1993). Biodegradable scaffold plays crucial
roles in scaffold-based tissue engineering approach. During the regeneration process, the biode-
gradable scaffold provides structural and mechanical restoration of the damaged tissues, gradually
degrades into biocompatible products, presents an interconnected porous structure to accommodate
cell infiltration and vascularization, and promote matrix synthesis (Laurencin et al. 1999; Langer
2000). The success of a 3D scaffold in tissue regeneration is largely dependent on its nature, com-
position, and associated properties. Biomaterials including synthetic biodegradable polymers and
composites have shown great promise in a tissue engineering approach (Nair and Laurencin 2007).
Biodegradable polyphosphazenes have been identified as one of the important classes of biomaterials
in tissue engineering applications (Ibim et al. 1997; Ambrosio et al. 2002; Nair et al. 2005; Krogman
et al. 2007; Deng et al. 2010a). The synthetic flexibility of polyphosphazenes allows for the design and
development of 3D scaffolds with the right combination of desirable physical, chemical, and biological
properties for a specific tissue engineering application. A number of techniques including particulate
leaching, microsphere sintering, and electrospinning have been developed to fabricate polyphosphazene-
based porous scaffolds for a variety of tissue engineering applications based on aminated polyphospha-
zene, alkoxy-substituted polyphosphazenes, and polyphosphazene blends/composites (Laurencin et al.
1993, 1996; Ambrosio et al. 2003; Nair et al. 2004; Bhattacharyya et al. 2006, 2009; Brown et al. 2008;
Nukavarapu et al. 2008, 2009). In addition, several in situ scaffold systems have also been developed on
the polyphosphazene platform for tissue regeneration (Greish et al. 2005b; Deng et al. 2010d).
One of the early reports from Laurencin et al. detailed the fabrication of 3D porous scaffolds
from PNEGmPh with interconnected porosity and an average pore diameter of 165 μm using a salt
leaching technique for skeletal tissue regeneration (Laurencin et al. 1996). However, salt-leached
scaffolds are often not applicable to load-bearing applications and associated with several limita-
tions in generating the interconnected porous structures and eliminating the entrapped prorogens.
Thus, mechanically competent 3D porous microsphere scaffolds have been developed by Laurencin
et al. and showed great promise for regenerative applications (Borden et al. 2002a,b, 2003, 2004).
In brief, these 3D microsphere scaffolds are fabricated from polymers such as PLAGA by heating
the individual polymer microspheres above the polymer Tg in a steel mold of desired size and shape.
Polymer becomes rubbery above the Tg and holds the microspheres together to form a 3D scaffold
according to the mode shape with an interconnected porous structure at room temperature. It has
been shown that the pore size and porosity of the scaffolds can be varied by changing the duration/
temperature of sintering as well as the microsphere diameter. However, the applicability of heat
sintering technique to polyphosphazene microspheres is limited due to its dependence on specific
physicochemical properties such as Tg, crystallinity, viscosity, and surface tension of the polymer.
Thus, a novel route based on a solvent/nonsolvent approach was adopted to create porous polymeric
microsphere scaffolds suitable for tissue engineering purposes (Brown et al. 2008; Nukavarapu
et al. 2008, 2009). As shown in Figure 4.11, sintering the microspheres was based on the partial
dissolution of the microsphere surface in a solvent/nonsolvent composition at ambient conditions
that establishes a balance between polymer dissolution and precipitation. At this intermediate state,
the microsphere surfaces were susceptible to bonding with adjacent microspheres prior to precipi-
tation of the intertwined surface chains and sintering of the microspheres. The resultant 3D scaf-
fold properties such as the overall porosity, average pore diameter, and mechanical properties can
be fine-tuned by changing the nature and composition of solvent/nonsolvent. Brown et al. (2008)
used five different biodegradable polyphosphazenes exhibiting glass transition temperatures from
−8°C to 41°C to study the versatility of the process (Figure 4.11). For example, to achieve effective
Microsphere Sintered
Microspheres
slurry microspheres
Add sintering Solvent

solution evaporation
(a) (b)
(c) (d)
(e) (f )
FIGURE 4.11 Schematic illustration of the solvent/nonsolvent sintering approach and SEM micrographs
illustrating the flexibility of this dynamic solvent approach for creating 3D porous sintered microsphere scaf-
folds from a wide range of different polyphosphazene polymers where (a) PLAGA; (b) PNEA; (c) PNEPhA;
(d) PNMV; (e) PNEAPhPh; and (f) PNEAmPh. (From Brown, J.L., Nair, L.S., and Laurencin, C.T.: Solvent/
non-solvent sintering: A novel route to create porous microsphere scaffolds for tissue regeneration. J. Biomed.
Mater. Res. B. Appl. Biomater., 2008. 86B(2). 396–406. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.
With permission.)
sintering of PNEA, acetone was chosen as a solvent while hexane was a non-solvent. Similarly for
poly[bis(methyl valinato)phosphazene] (PNMV) and PNEPhA, the combination of THF/hexane
was used as the solvent/nonsolvent system. Thus fabricated polyphosphazene microsphere scaffolds
exhibited a maximum interconnected porosity of 37.6% and a maximum compressive modulus of
94.3 MPa. Nukavarapu et al. developed composite microspheres from PNEPhA and 100 nm sized
HAp with varying compositions in the range of 10%–30% (w/w) (Nukavarapu et al. 2008, 2009).
These composite microspheres were sintered into 3D architecture using a similar solvent/non-
solvent approach. These porous scaffolds exhibited mean pore diameters in the range of 86–145 μm
and compressive moduli of 46–81 MPa.
Polymeric nanofibers due to their similarity to natural extracellular matrix (ECM) are attrac-
tive candidates as tissue engineering scaffolds. They can be fabricated using various processing
techniques such as drawing (Ondarçuhu and Joachim 1998; Huang et al. 2003), self-assembly
(Whitesides and Grzybowski 2002; Huang et al. 2003), template synthesis (Martin 1996; Huang
et al. 2003), phase separation (Ma and Zhang 1999; Huang et al. 2003), and electrospinning (Huang
et al. 2003). Among these, electrospinning is the most general and simplest technique that can be
used to fabricate nanofibers from biodegradable polymers or polymer blends/composites (Huang
et al. 2003; Kumbar et al. 2008; Nair and Laurencin 2008). Figure 4.12a shows a general represen-
tation of an electrospinning apparatus used to create polymeric nanofibers (Kumbar et al. 2008).
In brief, a polymer jet is ejected from the surface of a charged polymer solution when the applied
electric potential overcomes the surface tension. The ejected jet under the influence of applied elec-
trical field travels rapidly to the collector and collects in the form of nonwoven web as the jet dries.
Before reaching the collector the jet undergoes a series of electrically driven bending instabilities
that results in a series of looping and spiraling motions. In an effort to minimize the instability due
to the repulsive electrostatic forces, the jet elongates to undergo large amounts of plastic stretch-
ing that consequently leads to a significant reduction in its diameter and results in ultrathin fibers.
Glass syringe
Needle
Grounded target
Polymer solution Polymer

solution
HV power supply jet
(a)
(b) (c)
100 um
FIGURE 4.12 Electrospinning of polyphosphazene polymers for tissue engineering. (a) Schematic repre-
sentation of an electrospinning apparatus to produce nonwoven nanofiber scaffolds, which consists of a high
voltage supply (5–40 kV), a source electrode, a grounded collector electrode, and a capillary tube with a needle
of small diameter; (b) SEM micrograph of electrospun PNmPh nanofibers (With permission from Nair, L. S.,
Bhattacharyya, S., Bender, J. D. et al., Fabrication and optimization of methylphenoxy substituted polyphospha-
zene nanofibers for biomedical applications, Biomacromolecules, 5(6), 2212–2220. Copyright 2004 American
Chemical Society); and (c) SEM micrograph of electrospun PNGEGPhPh-PLAGA blend nanofibers.
With low-viscosity polymer solutions, the ejected jet may break down into droplets and result in
electrospraying (Kumbar et al. 2007). Therefore, a suitable polymer solution viscosity is essential to
fabricate nanofibers without any beads or beads-on-a-string appearance. For example, increase in
viscosity or polymer concentration results in fiber diameter increase. By changing polymer concen-
tration alone it is possible to fabricate the fiber diameters in the range of few nanometers to several
micrometers while keeping other electrospinning parameters constant. In addition, the electrical
potential applied also plays an important role in the fiber diameter obtained by electrospinning. So
far, several poly[(amino acid ester)phosphazenes] have been successfully electrospun to produce
ECM-mimicking nanofiber scaffolds for a variety of tissue engineering applications (Conconi et al.
2004, 2006; Nair et al. 2004; Bhattacharyya et al. 2006, 2009; Krogman et al. 2010; Lin et al. 2010).
The morphologies of the PNmPh and PNGEGPhPh-PLAGA blend nanofibers under optimized elec-
trospinning conditions are presented in Figure 4.12b and c, respectively.
In addition to the aforementioned preformed matrices, several composite matrices including self-
setting polyphosphazene bone cements and in situ pore forming polyphosphazene blends have been
developed to offer a great advantage in a clinical setup by forming the scaffolds in situ (Greish et al.
2005b; Deng et al. 2010d).
Bone is a natural composite principally composed of collagen fibrils and HAp (Rho et al. 1998).
Collagen component provides bone with a structural framework, high tensile strength, and flexibility
while ceramic HAp component offers stiffness and high compressive strength. Fabrication of a self-
setting composite comprised of a polymer component and HAp closely mimics the bone composi-
tion while providing an easy way to form bone implants at the injury sites. Thus, a polyphosphazene
composite cement system has been developed by exploiting the favorable interactions between poly-
phosphazene side groups and calcium phosphate ceramics. For example, the formation of composites
has been achieved via an acid–base reaction of tetracalcium phosphate and anhydrous dicalcium
phosphate in the presence of polyphosphazenes bearing alkyl ester containing side groups at physi-
ological temperature (Greish et al. 2005b). Alkaline conditions resulted in the partial hydrolysis of
alkyl ester side groups of PNEOB and PNPOB producing carboxylate groups on the polymer chain.
Calcium ions in solution cross-linked these carboxylate groups resulting in the formation of a cal-
cium cross-linked polyphosphazene surface layer, which in turn nucleated the depositing HAp on the
surface (Greish et al. 2005b). Polymers such as PCPP and its water-soluble salts such as Na-PCPP or
K-PCPP also favored the composite formation at low polymer concentrations under physiologic con-
ditions (Greish et al. 2005a, 2006). Composites produced similarly using PNEAPhPh showed higher
compressive strength than PNEA composites due to bulkier side groups (Greish et al. 2008). Such bio-
mimetic deposition of HAp on the polymers closely resembles the mineralization of collagen, which
enables the potential of developing composite implants for tissue repair and regeneration in vivo.
Recently, a dipeptide-based polyphosphazene blend system has demonstrated a unique erosion
mechanism through which the polymer evolves from a solid coherent film to an assemblage of
microspheres with interconnected porous structures (Deng et al. 2010d). This allows to develop
and control 3D scaffolds for regenerative purposes with initial higher mechanical properties that
reduces over time as polymer degrades while generating interconnected porous structures to allow
tissue in-growth and matrix integration. Studies are currently underway to optimize dipeptide-
based polyphosphazene side group composition to achieve higher mechanical properties suitable
for load-bearing applications. We are also evaluating this blend system as a factor delivery vehicle
and tissue engineering scaffold for regenerative applications.
4.3.4.2.2 Scaffold-Based Tissue Regeneration

So far, biodegradable polyphosphazene scaffolds have demonstrated great potential for the regen-
eration of different tissue types such as bone, tendon, blood vessel, and nerve, to name a few.
For example, more than 6.5 million fractures occur in the United States each year (Braddock
et al. 2001). Reconstruction of large bone defects presents a significant medical challenge.
The porous 3D scaffolds of PNEGmPh developed by Laurencin et al. using a salt leaching
(a) (b)
FIGURE 4.13 (See companion CD for color figure.) Cytoskeletal actin distribution of PRO grown on
polyphosphazene-HAp composite microsphere scaffold for (a) 6 days; and (b) 12 days. Scale bar: 250 μm.
After 12 days, cells covered the entire scaffold including the microsphere surface as well as the microsphere
adjoining areas. (With permission from Nukavarapu, S.P., Kumbar, S.G., Brown, J.L. et al., Polyphosphazene/
nano-hydroxyapatite composite microsphere scaffolds for bone tissue engineering, Biomacromolecules, 9(7),
1818–1825. Copyright 2008 American Chemical Society.)
technique as mentioned earlier in Section 4.3.4.2.1 supported cell growth within the pores as
well as on surface of the polymer as early as day 1 (Laurencin et al. 1996). On these porous con-
structs, PRO cells proliferated in a steady state over the 21 day culture time whereas a decline in
cell number was found for 2D films after 7 days. Thus, 3D porous constructs provide increased
surface area and promote cell growth throughout the scaffold. In an effort to develop sintered
polyphosphazene microsphere scaffolds for bone tissue engineering, Nukavarapu et al. prepared
composite microspheres of PNEPhA with 100 nm sized HAp with varying HAp compositions
between 10%–30% (w/w) (Nukavarapu et al. 2008). These composite microspheres were sin-
tered into 3D architecture using a solvent/nonsolvent approach as discussed earlier in Section
4.3.4.2.1. The increased presence of cytoskeletal actin on the composite microsphere scaffolds
suggested the progressive growth of PRO (Figure 4.13). Quantitative analysis confirmed that the
composite scaffolds were able to support good osteoblast adhesion, proliferation, and alkaline
phosphatase expression throughout the 21 day culture. In another study, El-Amin et al. evalu-
ated the in vivo biocompatibility and degradation of 5 mm diameter plugs from PNEGmPh and
PNEG in a rabbit metaphyseal distal femur defect model (El-Amin et al. 2007). Histological
evaluations revealed that both polymers appeared to support bone growth comparable to the
PLAGA control. Femurs with polyphosphazene implants showed evidence of bone in-growth
and a mild fibrous response at 12 weeks.
Ambrosio et al. fabricated composites of PNEGmPh-HAp and evaluated their potential as scaf-
fold materials for bone tissue engineering (Ambrosio et al. 2003). These novel composites retained
their mechanical properties during degradation and supported osteoblast-like MC3T3-E1 cell pro-
liferation. Such in vitro studies strongly support the use of polyphosphazene-based composites as
promising new materials for developing biodegradable bone implants.
Recently, Deng et al. (2010d) fabricated the dipeptide-based polyphosphazene–polyester blends
and investigated their potential to accommodate cell infiltration using a rat subcutaneous implanta-
tion model. It was found that the in situ formed 3D interconnected porous structures enabled cell infil-
tration and collagen tissue in-growth throughout the void space between spheres. The robust tissue
in-growth of the dynamic pore forming scaffold attests to the utility of this system as a new strategy
in regenerative medicine for developing solid matrices that balance degradation with tissue formation.
Nair et al. evaluated the potential of the PNmPh nanofiber matrices for tissue engineering appli-
cations. Preliminary results showed that these polyphosphazene fiber matrices supported the adhe-
sion and proliferation of osteoblast-like MC3T3-E1 cells (Nair et al. 2004). PNmPh polymer can
be used to modulate the physico-chemical properties (hydrophobicity/hydrophilicity, mechanical
properties, and degradation pattern) and consequent regenerative efficacy of the mixed-substituent
polyphosphazenes. Bhattacharyya et al. fabricated PNEA as well as PNEA-HAp composite nano-

fiber matrices as scaffolds for bone tissue engineering applications (Bhattacharyya et al. 2006,
2009). Such polyphosphazene nanofiber structures closely mimic the ECM architecture, and have
shown improved cell performance over the conventional scaffold architectures (Nair et al. 2004;
Bhattacharyya et al. 2006, 2009; Conconi et al. 2006). Conconi et al. fabricated electrospun scaf-
folds from poly[(ethyl phenylalanato)1.4(ethyl glycinato)0.6phosphazene] and its blends with PLA and
PCL (Conconi et al. 2009). Osteoblast adhesion and growth were found to be less on electrospun
polyphosphazene scaffolds than on electrospun PLA scaffolds. However, a synergic effect on cell
proliferation was observed when cells were cultured on polyphosphazene-PLA blends. Although
the poor mechanical properties limit their applications to repair load-bearing defects, the inherited
buffering capacity of polyphosphazenes to neutralize the acidic degradation of PLA makes this
system interesting materials to repair cranial and maxillofacial defects.
As discussed earlier, nanofiber matrices have shown great promise for regeneration purposes
(Huang et al. 2003; Li and Xia 2004; Nair and Laurencin 2008). However, there are some significant
challenges that need to be addressed in order to enable their load-bearing applications in tissue engi-
neering. For example, nanofibers lack stability under compression, and hence may not be suitable to
support load-bearing bone defects. (Li et al. 2002; Whitesides and Boncheva 2002; Ma 2008). With
a more in-depth investigation into the constituents of bone and their unique properties, several scaf-
fold platforms have been developed to closely mimic the inherent characteristics of the native bone.
Brown et al. (2010) recently developed a novel structure that combines the robust mechanical
aspects of the sintered microsphere scaffold with a highly bioactive nanofiber structure to pro-
duce a composite scaffold that demonstrates an ability to mimic the mechanical environment of
trabecular bone while also promoting the osteoinduction of osteoblast progenitor cells. Exploiting
the chemistry of two biodegradable polymers, a 3D PLA nanofiber mesh was successfully incor-
porated within the void spaces between sintered PNEPhA microspheres (Figure 4.14a and b). The
non-load-bearing fiber portion of these scaffolds is sufficiently porous to allow cell migration and
ECM matrix production throughout the fibrous portion of the scaffold (Figure 4.14c and d). These
composite nanofiber/microsphere scaffolds promote osteoinduction through focal adhesion kinase
activity. Ultimately the focal adhesion kinase activity on the composite nanofiber/microsphere scaf-
folds demonstrated causality over the production of the mature osteoblast marker, osteocalcin, and
the development of a calcified matrix (Figure 4.14e and f).The phenotype progression of osteoblast
progenitor cells on the composite nanofiber/microsphere scaffolds illustrated a stronger and more
rapid progression leading to fully matured osteoblasts by 21 days.
Inspired by the hierarchical structures that enable bone function, Deng (2010) recently devel-
oped a mechanically competent 3D scaffold mimicking the bone marrow cavity, as well as, the
lamellar structure of bone by orienting electrospun polyphosphazene-polyester blend nanofibers in
a concentric manner with an open central cavity. The 3D biomimetic scaffold exhibited a similar
characteristic mechanical behavior to that of native bone. Compressive modulus of the scaffold was
found to be within the range of human trabecular bone. To our knowledge this is the first mechani-
cally competent 3D electrospun nanofiber scaffold with mechanical properties in the middle range
of human trabecular bone. The potential of this scaffold for bone repair was further investigated by
monitoring the cellular activity and mechanical performance over time using in vitro culture. These
blend nanofiber matrices supported PRO adhesion, proliferation, and showed an elevated pheno-
type expression compared to PLAGA nanofibers. This biomimetic scaffold supported the robust
PRO growth throughout the scaffold architecture and maintained osteoblast phenotype expression
in vitro, which resulted in a similar cell-matrix organization to that of native bone and maintenance
of structure integrity. When combined with the desirable polymer blend properties, the concentric
open macrostructures of nanofibers that structurally and mechanically mimic the native bone can
be a potential scaffold design for accelerated bone healing.
Besides bone repair, polyphosphazenes have also shown tremendous potential for regenerating
other types of tissue such as nerve (Langone et al. 1995; Aldini et al. 1997; Zhang et al. 2009;
(a) (b)
(c) (d)
PLAGA PPHOS Composite PLAGA PPHOS Composite

20 1.2
Alizarin red ODU/scaffold
1
15
pg OCN/ng DNA
0.8
10 0.6
5 0.4
0.2
0
3 7 14 21 0
–5 3 7 14 21
(e) Time (days) (f ) Time (days)
FIGURE 4.14 (See companion CD for color figure.) Polyphosphazene composite scaffolds bridging nano-
fiber and microsphere architectures to improve bioactivity of mechanically competent constructs for bone
regeneration. (a, b) SEM micrographs of the cross-sections of the composite nanofiber/microsphere scaffold
demonstrating incorporation of nanofibers of less than 1 μm within the void space generated by sintering
microspheres were (a) low magnification image and (b) high magnification image; (c, d) SEM micrographs of
preosteoblasts in the interior of composite scaffolds after 3 days (c) and 14 days (d) of culture demonstrating
the presence of preosteoblasts and the accumulation of matrix proteins in the interior of the composite scaf-
fold during cell culture. The arrows point to representative cells. Notably, the fibrous portion of the scaffold
has been extensively modified through the accumulation of ECM proteins after 14 days, to the extent that
identifying the cells is difficult. (e, f) Phenotype evaluation of preosteoblasts seeded on composite scaffolds,
(n = 4). Mature osteoblast marker such as osteocalcin (e) evaluated and normalized to total DNA demonstrates
a peak at 14 days for the preosteoblasts seeded on the composite scaffold; and (f) Total scaffold calcification
normalized to an unseeded construct demonstrates that the preosteoblasts seeded on the composite scaffold
produced a substantially more mineralized matrix than did the preosteoblasts on control PLAGA and poly-
phosphazene scaffold. (*) represents statistical significance, p < 0.05, from all other samples at a given time
point. All error bars represent standard deviation. (From Brown, J.L., Peach, M.S., Nair, L.S., Kumbar, S.G.,
and Laurencin, C.T.: Composite scaffolds: Bridging nanofiber and microsphere architectures to improve bio-
activity of mechanically competent constructs. J. Biomed. Mater. Res. A. 2010. 95(4). 1150–1158. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA. With permission.)
Deng et al. 2010a), vessel (Carampin et al. 2007; Deng et al. 2010a), and tendon (Huang et al. 2006;
Deng et al. 2010a). For example, in vivo nerve repair studies were carried out by using tubes of
poly[(ethylalanato)1.4(imidazolyl)0.6phosphazene] (Langone et al. 1995). Rat sciatic nerve stumps
were transectioned and immediately sutured into the ends of 10 mm long polymer tubes. After
implantation for 45 days, a tissue cable was found bridging the nerve stumps. Histological anal-
ysis illustrated that the tissue cable was mainly composed of a regenerated nerve fiber bundle.
Another study using the conduits of alanine ester–substituted polyphosphazenes demonstrated a
similar nerve regenerative capacity compared to autologous grafts in Wistar rats (Aldini et al. 1997).
Recently, a biodegradable polyphosphazene cosubstituted with both aniline pentamer and glycine
ethyl ester side groups showed an electrical conductivity of ∼2 × 10 −5 S/cm in the semiconduct-
ing region (Zhang et al. 2009). The polymer supported the attachment and proliferation of RSC96
Schwann cells, suggesting that it is a potential scaffold material for peripheral nerve regeneration.
As a first step to investigate the feasibility to construct human vessels or cardiac valve, Carampin
et al. demonstrated that electrospun nanofibers of biodegradable poly[(ethyl phenylalanato)1.4(ethyl
glycinato)0.6phosphazene] supported adhesion and growth of rat neuromicrovascular endothelial
cells (Carampin et al. 2007). Nair et al. (2004) also showed that the PNmPh nanofiber matrices sup-
ported the adhesion of bovine coronary artery endothelial cells (BCAECs).
In summary, the synthetic versatility of polyphosphazene materials combined with advanced
scaffold fabrication techniques hold great promise in developing optimal 3D structures with the
right combination of physical, chemical, and biological properties to suit the requirements of a spe-
cific tissue engineering application.
4.4 CONCLUSIONS AND FUTURE TRENDS

The development of biodegradable materials has led to the synthesis of new polymers with diverse
properties. The success of synthetic polymers as biomaterials for biomedical applications largely
depends on their inherent physicochemical properties, biodegradability, and biocompatibility.
Biodegradable polyphosphazenes are a unique class of polymers due to their synthetic flexibility and
versatile adaptability for biomedical applications. They are high molecular weight polymers hav-
ing an inorganic backbone of alternating phosphorus and nitrogen atoms bearing two side groups
attached to each phosphorus atom. The synthetic flexibility of polymer backbone allows controlled
tuning of physico-chemical properties, biodegradability, and biocompatibility via macromolecular
substitutions of more than 250 organic nucleophiles. Specifically, the substitution of appropriate
side groups such as amino acid esters or alkoxy substituents imparts biodegradability to polyphos-
phazenes. Biodegradable polyphosphazenes undergo degradation through surface erosion or bulk
erosion or a combination of both into nontoxic and neutral pH degradation products. Furthermore,
polyphosphazene blends/composites synergistically combine the advantages of the parent polymers.
The blend properties can be fine-tuned to satisfy the requirements of a specific application via
the blend compositional change. Additionally, the synthesis of novel polyphosphazene polyester
or polycarbonate block copolymers enables the development of advanced polymeric blend systems
using these polymeric architectures as prototypes. Thus, the polyphosphazene polymers based on
polyphosphazenes alone and polyphosphazene blends/composites are an excellent material platform
for developing controlled drug release matrices and tissue engineering scaffolds.
In controlled drug delivery, biodegradable polyphosphazenes are attractive materials due to
their hydrolytic lability, nontoxic degradation products, excellent biocompatibility, ease of matrix
fabrication, and matrix permeability. The controlled erosion rates and drug release profiles can
also be tuned through macromolecular substitution. Studies have demonstrated that the release of
small molecules and macromolecules from biodegradable polyphosphazene matrices occurs either
by diffusion, erosion, or a combination of these mechanisms. Their success in both the in vitro and
in vivo environments makes them potentially ideal candidates for the delivery of bioactive mol-
ecules. Future work will combine the versatile synthetic chemistry in polyphosphazenes with the
advancement in matrix fabrication (i.e., nanoparticles preparation) to develop targeted drug delivery
systems with controlled release kinetics.
In the field of tissue engineering, biodegradable polyphosphazenes have shown great promise
in the regeneration of different tissue types via a scaffold-based tissue regeneration approach
due to their excellent combination of physical, chemical, and biological properties. Recent stud-
ies have provided significant insights into the influence of topographic features in regulating
cell behavior, including preferential adhesion, migration, proliferation, and expression of cell-
specific phenotypes. With the advancement in micro- and nano-fabrication technology, bioma-
terials can be fabricated into various 3D structures that mimic the native tissue hierarchical
structures. Reflecting the biological processes during native tissue development, it is critical
to study and exploit the molecular mechanisms governing cell–cell and cell–material interac-
tion at the micro- and nano-scale to generate tissue-inducing scaffolds and achieve facilitated
tissue regeneration. Polyphosphazenes as a unique class of biomaterials, when appropriately
integrated with the essential physical and chemical cues present in natural ECM, will ultimately
enable successful regeneration of complex tissue systems using integrated graft systems in the
near future.
ACKNOWLEDGMENTS
This work was supported by NIH RO1 EB004051, RO1 AR052536, and NSF EFRI-0736002.
Dr. Laurencin was the recipient of Presidential Faculty Fellow Award from the National Science
Foundation.
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5 Biodegradable Polymers
as Drug Carrier Systems
Abraham J. Domb and Wahid Khan
CONTENTS
5.1 Introduction........................................................................................................................... 136
5.2 Biodegradable Polymers........................................................................................................ 136
5.2.1 Polyesters................................................................................................................... 136
5.2.1.1 Lactide/Glycolide and Their Copolymers................................................... 136
5.2.1.2 Polycaprolactones........................................................................................ 141
5.2.1.3 Poly(β-Hydroxybutyrate)............................................................................ 143
5.2.1.4 Polycarbonates............................................................................................ 144
5.2.1.5 Other Polyesters.......................................................................................... 145
5.2.2 Poly(Amides)............................................................................................................. 146
5.2.3 Poly(Phosphate Esters).............................................................................................. 147
5.2.4 Polyphosphazenes...................................................................................................... 148
5.2.5 Poly(Orthoesters)....................................................................................................... 149
5.2.5.1 Synthesis..................................................................................................... 150
5.2.5.2 Polymer Properties...................................................................................... 151
5.2.5.3 Cross-Linked Poly(Orthoesters)................................................................. 151
5.2.5.4 Polymer Hydrolysis..................................................................................... 152
5.2.5.5 Polymer Processing..................................................................................... 153
5.2.6 Polyanhydrides........................................................................................................... 153
5.2.6.1 Synthesis..................................................................................................... 153
5.2.6.2 Polymer Properties...................................................................................... 155
5.2.6.3 Polymer Hydrolysis..................................................................................... 155
5.2.6.4 Polymer Processing..................................................................................... 156
5.2.6.5 Other Polyanhydrides.................................................................................. 156
5.3 Biocompatibility and Toxicity............................................................................................... 158
5.3.1 Polyesters................................................................................................................... 159
5.3.1.1 Lactide/Glycolide Copolymers................................................................... 159
5.3.1.2 Poly(Caprolactone)...................................................................................... 160
5.3.1.3 Polycarbonates............................................................................................ 161
5.3.2 Poly(Amides)............................................................................................................. 161
5.3.2.1 Natural Polymers........................................................................................ 161
5.3.2.2 Pseudopoly(Amino Acids).......................................................................... 162
5.3.3 Polyphosphazenes...................................................................................................... 162
5.3.4 Poly(Orthoesters)....................................................................................................... 163
5.3.5 Polyanhydrides........................................................................................................... 163
References....................................................................................................................................... 165
135
5.1 INTRODUCTION
In recent years, there has been a rapid growth in the area of drug discovery, facilitated by novel
technologies such as combinatorial chemistry and high-throughput screening. These novel
approaches have led to drugs, which are generally more potent and have poorer solubility than
the drugs developed from traditional approaches of medicinal chemistry. The development of
these complex drugs has triggered a more urgent focus on developing novel techniques to deliver
these drugs more effectively and efficiently (Molinari 2009; Shanmugasundaram and Rigby 2009;
Yi Mok et al. 2009). Secondly, there is a continuous advance in biotechnology and drug devel-
opment which produces more pharmaceutically active agents that will be difficult to administer
by conventional means (Shire 2009). Thus, there is an increased demand for controlled or site-
specific delivery system, which thereby needs a material, i.e., biomaterial, to deliver the drug and
to perform its intended work.
Over the past two decades, the use of polymeric materials has increased dramatically for bio-
medical applications. The most important biomedical applications of biodegradable polymers are in
the areas of controlled drug delivery systems (Zheng et al. 2009; Bobo and Shelton 2010) and in the
form of implants and devices for bone and cartilage repair (Jung et al. 2008; Puppi et al. 2010), liga-
ment reconstruction (Ge et al. 2005; Sahoo et al. 2006, 2007; Frosch et al. 2009), surgical dressings
(Izumi et al. 2007; Xie et al. 2008), dental repairs (El-Backly et al. 2008), artificial heart valves,
contact lenses, cardiac pacemakers, vascular and bone grafts (Iwai et al. 2005; Shen et al. 2006;
Ngiam et al. 2009), tracheal replacements (Villegas-Cabello et al. 1994; Kaschke et al. 1996), and
organ regeneration (Seckel et al. 1984; Heng et al. 2005).
The purpose of this chapter is to review the chemistry, properties, and formulation procedures
of the different biodegradable polymers available, and to describe some of their release kinetics,
safety, and biocompatibility considerations related to these polymers.
5.2 BIODEGRADABLE POLYMERS

5.2.1 Polyesters
Polyesters represent a class of polymers with wide applications owing to their immense diver-
sity and synthetic versatility. This class includes poly(α-hydroxy acids) and other ester polymers
with or without oxygen atom adjacent to the α-carbon of the acid moiety (Farng and Sherman
2004). Among polyesters, the most extensively investigated polymers are the poly(α-hydroxy
acids), which include poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and their copolymer
poly(lactide-co-glycolide) (PLGA; Puppi et al. 2010). Several other aliphatic polyesters are now
attracting significant attention as biomaterials due to their good biocompatibility and controllable
degradation profiles.
5.2.1.1 Lactide/Glycolide and Their Copolymers

These are thermoplastic, aliphatic polyesters having hydrolytically labile aliphatic ester linkages
in their backbone and hence are biodegradable. Linear polyesters of lactide and glycolide have
been used for more than four decades for a variety of medical applications. Extensive research has
been devoted to the use of these polymers as carriers for controlled drug delivery of a wide range
of bioactive agents for human and animal use. They have been used for the delivery of steroids
(Ju et al. 2009), anticancer agents (Danhier et al. 2009; Mukerjee and Vishwanatha 2009; Park
et al. 2009), peptides and proteins (Santander-Ortega et al. 2009), antimicrobial agents (Kim et al.
2004; Xue et al. 2006; Nahar and Jain 2009), anesthetics, growth factors (Wenk et al. 2009), vac-
cines (Quintilio et al. 2009), and in tissue engineering (Zhang et al. 2009). Injectable formulations
containing microspheres of lactide/glycolide polymers have received the most attention and many
products (Table 5.1) are there in market.
Biodegradable Polymers as Drug Carrier Systems 137
TABLE 5.1
Commercially Available Biodegradable Polymeric Systems
Name of Active
Type of Polymer the Product Dosage Form Ingredients Reference
1. Polyesters
Lactide and glycolide Lupron Microspheres Leoprolide Hild et al. (2001), Wheeler et al. (1993),
copolymers depot Dlugi et al. (1990), Kappy et al. (1989)
Nutropin Microspheres Somatropin Kemp et al. (2004), Silverman et al.
depot (2002), Cook et al. (2002)
Risperdal Microspheres Triptorelin Gefvert et al. (2005), Bobo and Shelton
consta (2010)
Polycaprolactones Capronor Subdermal Levonorgestrel Anonymous (1991), Darney et al.
capsule (1989), Anonymous (1985), Ory et al.
(1983)
SynBiosys™ Drug delivery Peptides, Steendam et al. (2006), Gillissen et al.
system antibiotics (2006)
Polycarbonates Maxon™, Absorbable — Sonmez et al. (2009), Rodeheaver et al.
BioSyn™ sutures (1996)
2. Poly(amides)
3. Poly(phosphate esters) Paclimer® Microspheres Paclitaxel Pradilla et al. (2006), Armstrong et al.
(2006), Lapidus et al. (2004),
Harper et al. (1999)
4. Polyphosphazenes — Micelles Indomethacin Zhang et al. (2007)
5. Poly(orthoesters) Alzamer® Implant Pinholt et al. (1991), Heller (1990)
6. Polyanhydrides Gliadel® Wafer implant Carmustine Kleinberg et al. (2004), Whittle et al.
(2003)
5.2.1.1.1 Synthesis
Aliphatic polyesters can be synthesized by two mechanisms: (1) the step-growth polymeriza-
tion (polycondensation) and (2) the ring-opening polymerization(ROP) (polyaddition). When
polyesters are synthesized by the polycondensation, polymerization technique relies on the con-
densation of hydroxyl acids or of the mixtures of diacids and diols. But the major drawbacks of
polycondensation mechanism are the high temperatures and long reaction time, and it is difficult
to achieve high molecular weight polymers by this route (Edlund and Albertsson 2003; Jerome
and Lecomte 2008).
ROP of cyclic lactones has been developed as the most effective polymerization route to develop
high molecular weight homo- and co-polyesters. This process is free of polycondensation limitations
and is thus preferred. Linear lactide and glycolide-based polymers are most commonly synthesized
by ROP (Scheme 5.1) of lactide and glycolide by heating at 140°C–180°C for 2–10 h using catalyst/
initiators. A broad range of anionic, cationic, and coordinative initiators or catalysts have been
reported for the ROP (Dittrich and Schultz 1971; Nijenhuis et al. 1992; Urakami and Guan 2008).
Tin octoate (Sn(OCT)2) and alkoxides were the most widely used organometallic media-
tors for the ROP of lactones even if novel powerful metal-free catalytic systems are emerging.
O R O R
R
O O OH
HO O
O O O
R R n
O
SCHEME 5.1 Scheme for ROP of lactide/glycolide.

O O
CH3 CH3
O
O H.
O
O O
CH3 R'
O CH3
O
O O
O O C7H15 O– Sn+2 O C7H15
C7H15 O– Sn+2 O C7H15 (a)
O
CH3
O
O CH3
O H+
R O OH CH3
O -O O R
CH3 O O O
C7H15 O– Sn+2 O C7H15
(b)
SCHEME 5.2 Hypothetical mechanism of ROP using tin octoate as catalyst. (a) Activated ester-carbonyl
group in the dilactone, and (b) unstable intermediate which opens to the ester alcohol. (From Kissel, T. et al.,
J. Control Release, 16(1–2), 27, 1991.)
Yet, the common polymerization catalysts are tin derivatives such as tin octoate or tin hexanoate.
A hypothetical mechanism of the ROP of lactide using a tin catalyst was suggested by Kissel
et al. (1991) (Scheme 5.2).
In this mechanism, the Lewis acid character of the tin catalyst activates the ester-carbonyl group
in the dilactone (a, in Scheme 5.2). The activated species reacts with the alcohol initiator to form
an unstable intermediate which opens to the ester alcohol (b, in Scheme 5.2). The propagation reac-
tion proceeds by tin catalyst activation of another dilactone carbonyl group and reaction with the
hydroxyl end group. Tin catalyst activation of a carbonyl ester in the polymer chain results is an unde-
sirable depolymerization. The hydroxyl initiator is incorporated in the polymer at its carbonyl end
group. Lauryl alcohol is generally added to control the molecular weight (Dittrich and Schultz 1971;
Nijenhuis et al. 1992). Polymers with a molecular weight as high as 500,000 can be obtained by the
melt process when high-purity monomers (>99.9%) are used. Since Sn(OCT)2 promoted polymeriza-
tion are hardly controlled, a variety of other organometallic derivatives, particularly metal alkoxides,
are continuously tested as initiators in the polymerization of lactides. The aluminum alkoxide initia-
tors are the most versatile and readily available. The high selectivity of aluminum alkoxides is the
major reason for their choice to produce well-defined polyesters and molecular weight is very well
controlled. The very good control imparted to ROP by aluminum alkoxides is a unique platform for
the macromolecular engineering of aliphatic polyesters, for instance making comb-like, star-shaped,
graft, and hyper-branched polyesters. Both monoalkoxides (R2-AlOR) and trialkoxides (Al(OR)3)
were used. By using only one form of the trialkoxide isopropyloxode (Al(O-iPr)3), namely, its trimer,
a perfect control of polymerization can be achieved (Ma et al. 2005).
This type of initiator can also polymerize other cyclic monomers, such as adipic anhydride
and trimethylene carbonate (TMC) (Mecerreyes et al. 1999). For safety reasons, residual alu-
minum should be removed from the material before use in food or biomedical applications.
Aluminum can be efficiently extracted by EDTA complexation, tin (IV) alkoxides is rather
used as substitute for aluminum alkoxides, although the polydispersity of the chains is then
higher (∼1.5).
5.2.1.1.2 Polymer Properties

The polymer characteristics are affected by the comonomer composition, the polymer architecture,
and molecular weight. The crystallinity of the polymer, an important factor in polymer biodegrada-
tion, varies with the stereo regularity of the polymer. Polyglycolide is a highly crystalline polymer
(45%–55% crystallinity) and therefore exhibits a high tensile modulus and very low solubility in
organic solvents. The melting point of the polymer is greater than 200°C. Unlike glycolide, lactide
is a chiral molecule and exists in two optically active forms: l-lactide and d-lactide. The polym-
erization of these monomers leads to the formation of semi-crystalline polymers. The racemic dl
polymer is less crystalline than the d- or l-lactide homopolymers (Nair and Laurencin 2007).
A comprehensive review on the mechanical properties of several biodegradable materials used in
orthopedic devices has been published (Daniels et al. 1990). Polyglycolide shows excellent mechani-
cal properties due to its high crystallinity. Due to its good initial mechanical properties, polyglycolides
have been investigated as bone internal fixation devices, Biofix® (Steinmann et al. 1990). The thermal
properties, tensile properties, and the flextural storage modulus as a function of temperature were deter-
mined. The following polymers were compared: poly(l-lactic acid), poly(d,l-lactic acid), poly(glycolic
acid), poly(caprolactone), poly(hydroxybutyrate), and copolymers with hydroxyvaleric acid, and
poly(trimethylene carbonate) (PTMC). The thermal and mechanical properties of several of the poly-
mers tested are summarized in Table 5.2 (Engelberg and Kohn 1991). Different ratios of poly(lactide-co-
glycolides) have been commercially developed and are being investigated for a wide range of biomedical
applications according to mechanical properties and rate of degradation (Nair and Laurencin 2007).
The irradiated polymers continue to decrease in molecular weight during storage at room tem-
perature. This decline in molecular weight affects the mechanical properties and the release rate
from the polymers. Sterilization using γ-irradiation decreases the polymer molecular weight by
30%–40% (Montanari et al. 1998). PLA and its copolymers with less than 50% glycolic acid content
TABLE 5.2
Thermal and Mechanical Properties of Biodegradable Polyesters
Tensile Tensile Elongation
Strength Modulus
Polymer Mw Tg (°C) Tm (°C) (MPa) (MPa) Yield (%) Break (%)
Poly(lactic acid)
L-PLA 50,000 54 170 28 1200 3.7 6.0
L-PLA 100,000 58 159 50 2700 2.6 3.3
L-PLA 300,000 59 178 48 3000 1.8 2.0
DL-PLA 107,000 51 — 29 1900 4.0 5.0
Poly(glycolic acid)
PGA 50,000 35 210 NA NA NA NA
Poly(β-hydroxybutirate)
PHB 370,000 1 171 36 2500 2.2 2.5
P(HB-11%HV) 529,000 2 145 20 1100 5.5 17
Poly(e-caprolactone)
PCL 44,000 −62 57 16 400 7.0 80
Poly(trimethylene
carbonate)
PTC 48,000 −15 — 0.5 3 20 160
Poly(ortho esters)
t-CDM:1,6-HD 35:65 99,000 55 — 20 820 4.1 220
t-CDM:1,6-HD 70:30 101,000 84 — 19 800 4.1 180
Source: Engelberg, I. and Kohn, J., Biomaterials, 12(3), 292, 1991.

are soluble in common solvents such as chlorinated hydrocarbons, tetrahydrofuran, and ethyl acetate.
For poly(d,l-lactide-co-glycolide), study was reported aiming to determine solubility parameters for
different proportions of lactide to glycolide (Schenderlein et al. 2004). The solubility parameters
were in the range of 16.2–16.8, which is comparable to those of polystyrene and polyisoprene.
Frank et al. studied the effect of the chemical nature of the drug on matrix degradation and drug
release behavior of degradable polymers, using lidocaine as a model drug in base and salt forms.
The study showed that the drug in the base form has a substantial effect on the release characteris-
tics, through an accelerating effect on matrix degradation. Drug release study shows that lidocaine
salt follows a three-phase release pattern, in contrast to the biphasic release of the lidocaine base
(Frank et al. 2005). Blends of low and high molecular weight poly(d,l-lactide) were studied as car-
riers for drugs. As expected, the addition of low molecular weight polymer accelerated the release
of drugs from the blend formulations (Bodmeier et al. 1989).
Various other applications of polyester were reported in literature according to polymer proper-
ties. Intranasal administration of polylactide nanoparticles containing thyrotropin-releasing hormone
as a viable means to suppress seizures and perhaps epileptogenesis and become the lead compound
for intranasal anticonvulsant nanoparticle therapeutics has been reported. Results provide proof of
principle that intranasal delivery of sustained-release nanoparticles which may be neuroprotective
and suppress seizures and epileptogenesis (Kubek et al. 2009; Veronesi et al. 2009). Not only this,
diastereoisomeric complexes of insulin with d-poly(lactic acid) or stereocomplexes of d- and l-PLA
entrapping insulin were reported (Slager and Domb 2002). The complexes were spontaneously
formed when insulin and d-PLA were mixed together in acetonitrile solution. These macromolecu-
lar stereocomplexes may form the onset of the development of a new generation of controlled release
systems for peptides and proteins, by molecular complexation with enantiomeric polymers.
5.2.1.1.3 Biodegradation
The biodegradation of polyesters was reviewed by Lewis (1990), Gopferich (1997), and Holland et al.
(1986). The molecular weight and polydispersity as well as the crystallinity and morphology of the
polymers are important factors in polymer biodegradation. Polyesters mainly undergo bulk erosion,
i.e., the polymeric matrices degrade all over their cross-section and have erosion kinetics that are non-
linear and usually characterized by a discontinuity (Vert 2005). The factors that may affect the poly-
lactide degradation include chemical and configurational structure, molecular weight and distribution,
fabrication conditions, site of implantation, physical factors, and degradation conditions (Nakamura
et al. 1989). The degradation of semi-crystalline polymers proceeds in two phases; in the first phase,
the amorphous regions are hydrolyzed and then the crystalline regions. The polymers degraded by
bulk hydrolysis of the ester bonds, which resulted in a decrease in molecular with no weight loss.
A comprehensive investigation on the hydrolysis of lactide polymers was described by Vert et al.
(1991). In these studies, a standardized set of experiments was designed. All specimens were prepared
in a similar way and allowed to hydrolyze at 37°C in distilled water or isotonic phosphate buffer. The
changes in the polymer during hydrolysis were monitored by weighing for water uptake and weight
loss, gel permeation chromatography (GPC) for molecular weight change, and differential scanning
calorimetry (DSC) and X-ray scattering for thermal properties and crystallinity change, potentiom-
etry and enzymatic assays for pH change and lactic acid release, and dynamic mechanical tests for
changes in mechanical properties. The polymer, semi-crystalline PLA, lost about 50% of its mechani-
cal strength after 18 weeks in buffer, with no weight loss until about 30 weeks of hydrolysis. The degra-
dation of branched PLA was characterized as bulk erosion, like the linear polymers (Kissel et al. 1991).
The biodegradation of branched PLA with glucose or macromolecular polyol in rats was determined
by weight loss (Kissel et al. 1991). In vitro degradation was essentially the same, indicating minimal
involvement of enzymatic degradation. The branched materials degraded much faster than the reference
linear PLA. On the contrary, the linear PLA had a higher water uptake than the branched polymers.
For example, after 36 weeks, the linear PLA contained about 21 wt% of water while the corresponding
branched PLA contained only 2%. No adequate explanation was given for this phenomenon.
The degradation of several aliphatic polyesters in the form of microspheres in phosphate buffer
solution at 37°C and 85°C was reported. Lower molecular weight polymers degraded faster than
higher molecular weight polymers. Degradation at 85°C resulted in a similar degradation profile but
faster. The biodegradation of low molecular weight PLA used in tablets for oral delivery of drugs
was also studied (Moll and Koller 1990).
5.2.1.2 Polycaprolactones
The successful use of lactide and glycolide polymers in absorbable drug delivery systems and
medical devices and absorbable sutures, encouraged the evaluation of other polyesters for this
purpose. The most studied polymers in this category are the polycaprolactones, polyhydroxy-
butyrates, and polymers of other α-hydroxy acids. Among them, polycaprolactones is semi-
crystalline polyester; due to its slow degradation and biocompatibility, it offers an attractive
material for long-term biomedical and drug delivery applications, e.g., fibers of copolymer of PGA
and poly(ɛ-caprolactone) (PCL) are commercially available as monofilament suture (Monoacryl®)
(Sinha et al. 2004).
5.2.1.2.1 Synthesis
PCL is a semi-crystalline polyester and is of great interest as it can be obtained by the ROP of a
relatively cheap monomeric unit “ɛ-caprolactone.” PCL has been synthesized from the anionic,
cationic, or coordination polymerization of ɛ-caprolactone. The synthesis of polycaprolactones has
been reviewed extensively (Pitt 1990; Perrin and English 1997; Zhang and Guo 2003). A sche-
matic description of caprolactone polymerization using these three types of initiators is shown in
Scheme 5.3 (Pitt 1990).
Various initiators and polymerization conditions were reported for each type of catalyst. Effective
anionic reaction systems are tertiary amines, alkali metal alkoxides, and carboxylates in tetrahy-
drofuran, toluene, and benzene. The anionic method of polymerization is most useful for the syn-
thesis of low molecular weight hydroxy-terminated oligomers and polymers. Kinetics of the anionic
polymerization of ɛ-caprolactone initiated with (CH3)3SiO −K+ and carried out in THF solution has
been reported in the temperature range from 0°C to 20°C (Sosnowski et al. 1991).
O
O O
O :N
O– O
Initiation N Propagation N n
(a)
O
O O
O R+X–
+X– X
Initiation RO Propagation R- O
n
(b)
O R
O
δ+ H O O
O
OH OH
Initiation RO Propagation R- O
n
(c)
SCHEME 5.3 Polymerization of ɛ-caprolactone using (a) anionic, (b) cationic, and (c) coordination
catalysts.
Cationic polymerization (Ludvig and Belenkaya 1974) is affected by cationic catalysts, which
include protic acids, Lewis acids, acylating agents, and alkylating agents. The agents FeCl3, BF 3,
Et2O, alkyl sulfonate, and trimethylsilyl triflate have been used in 1,2-d ichloroethane at 50°C
to yield polymers with a molecular weight range of 15,000–50,000. High molecular weight
homopolymers and random copolymers with lactides and other lactones were obtained using
coordination catalysts such as di-n-butyl zinc, stannous octoate, and alkoxides and halides
of Al, Sn, Mg, and Ti. Polymerization occurs at 120°C under argon to yield polymers with
a narrow molecular weight distribution (Mw/Mn = 1.1) and molecular weights above 50,000
(Hamitou et al. 1977).
In another study, kinetic and PMR spectroscopic methods have been used to investigate the cat-
ionic polymerization of ɛ-caprolactone initiated by (C2H5)3O+SbF− in the presence of ethylene glycol
at 20°C–60°C. Alcohols markedly increase the rate of polymerization of ɛ-caprolactone. The dif-
ference in the basicity of the hydroxyl groups of the compounds formed in the initial linking of the
monomer causes preferential growth of the chain at one of the ends. In such systems, the molecular
mass of the polymer is determined by the monomer:addition ratio (Belen’Kaya et al. 1982).
In contrast to random copolymers, block and graft multicomponent systems are most often mul-
tiphase materials with properties that are different from the homopolymers or random copolymers.
They are useful in improving the phase morphology, the interfacial adhesion and, accordingly, the
ultimate mechanical properties of immiscible polymer blends. As an example, block copolymeriza-
tion of ɛ-caprolactone (CL) and lactides (LA) allows the permeability of the PCL to be combined
with the rapid biodegradation of PLA. Formation of large amount of homo PLA is observed and has
been attributed to the increase in the mean degree in association with aluminum alkoxide in toluene
from 1 to 3 in the presence of CL and LA, respectively. The homopolymer formation can be pre-
vented by the addition of a small amount of an alcohol, like 2-propanol, or the use of Al derivative
that bears only one alkoxide group (Endo et al. 1987).
The ROP of ɛ-caprolactone initiated by novel single lanthanide tris(4-tert-butylphenolate)s
[Ln(OTBP)3] is reported. Single-component La(OTBP)3 can effectively prepare polycaprolactone
with over 90% yield under quite mild conditions: molar ratio of ɛ-caprolactone to initiator is 1000,
60°C, 2 h in toluene (Susperregui et al. 2010). In another work, phosphotungstic acid (PTA) as a
novel initiator was reported for the ROP of ɛ-caprolactone. It was found that PTA was an efficient
initiator and ROP can be readily initiated by PTA at room temperature to form PCL with narrow
molecular weight distribution. Polymerization mechanism study indicates that the polymerization
proceeds via acyl-oxygen bond cleavage (Cheng et al. 2010).
5.2.1.2.2 Polymer Properties

PCL is a semi-crystalline polymer having glass transition temperature of −62°C and melting point
ranging between 57°C and 64°C, depending upon the crystalline nature of PCL. PCL is of great
research interest owing to its ease of processability and solubility in a wide range of organic sol-
vents. PCL is soluble in chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclo-
hexanone, and 2-nitropropane at room temperature. It has a low solubility in acetone, 2-butanone,
ethyl acetate, dimethylformamide, and acetonitrile and is insoluble in alcohol, petroleum ether, and
diethyl ether. PCL can be blended with other polymers to improve stress crack resistance, dyeability,
and adhesion. Copolymerization with lactide increases the Tg with the increase in the lactide content
in the polymer (Shindler et al. 1977; Sinha et al. 2004). The crystallinity of the polymer decreases
with the increase in polymer molecular weight; polymer of 5,000 is 80% crystalline, whereas the
60,000 polymer is 45% crystalline (Pitt et al. 1981a).
The biodegradation of PCL has been extensively studied in the past 40 years and several reviews are
available (Pitt 1990; Sinha et al. 2004). Like the lactide polymers, PCL and its copolymers degrade
both in vitro and in vivo by bulk hydrolysis of hydrolytically labile aliphatic ester linkages, with the
degradation rate affected by the size and shape of the device and additives (Pitt et al. 1981a). The
polymers degrade in two phases. In the first phase, a random hydrolytic chain scission occurs, which
results in a reduction of the polymer molecular weight. In the second phase, the low molecular
fragments and the small polymer particles are carried away from the site of implantation by solubi-
lization in the body fluids or by phagocytosis, which results in a weight loss. Complete degradation
and elimination of PCL homopolymers may last for 2–4 years. The degradation rate is significantly
increased by copolymerization or blending with lactide and glycolide. The rate of degradation can
also be increased by the addition of oleic acid or tertiary amines to the polymer which catalyzes the
chain hydrolysis (Pitt et al. 1981b; Woodward et al. 1985). Due to the slow degradation, high perme-
ability (to many drugs), and nontoxicity, PCL was initially investigated as a long-term drug/vaccine
delivery vehicle, one, e.g., is long-term contraceptive device Capronors for the long-term zero-order
release of levonorgestrel (Darney et al. 1989).
5.2.1.3 Poly(β-Hydroxybutyrate)
These are naturally occurring biodegradable polyesters produced by many bacteria as their energy
source. Poly(β-hydroxybutyrate) (PHB) was discovered in 1920 as produced by the bacteria Bacillus
megaterium (Nair and Laurencin 2007).
5.2.1.3.1 Synthesis
PHB is made by a controlled bacterial fermentation. The producing organism occurs naturally
(Arun et al. 2009). An optically active copolymer of 3-hydroxybutirate (3HB) and 3-hydroxyvalerate
(3HV) has been produced from propanonic acid or pentanoic acid by Alcaligenes eutrophus
(Holmes 1985):
O CH2CH3 O
O CH CH2 C O CH CH2 C
x x
CH3 n
3HB 3HV
The copolymer compositions (0–95 mole% 3HV content) can be controlled by the composition of
the carbon sources. Random copolymers of 3HB and 4HV were produced from 4-hydroxybutyric
acid and butyric acids by Alcaligenes eutrophus (Doi et al. 1988; Kunioka et al. 1988):
O O
O CH CH2 C O CH2 CH2 CH2 C
CH3 x y n
3HB 4HB
Studies aimed at screening and identifying a potential PHB accumulating Bacillus sp. and optimization
of media parameters for increased PHB production by the strain have been reported (Pal et al. 2009).
PHB production was found to be comparable to most of the Bacillus sp. reported to date. Whereas
some studies aim to study PHB formation, and degradation in sequencing batch biofilm reactor has
been reported. These reactors were operated in cycles comprising three individual phases: mixed fill,
aeration, and draw. A synthetic substrate solution with acetate and ammonium was used. PHB was
formed during the aeration phase immediately after acetate depletion and was subsequently consumed
for biomass growth, owing to the high oxygen concentration in the reactor (Nogueira et al. 2009).
5.2.1.3.2 Properties and Biodegradation

The polymers are characterized as having a high molecular weight (>100,000, [n] > 3 dL/g) with a
narrow polydispersity and a crystallinity of around 50%. The melting point depends on the polymer
composition; P(3HB) homopolymer melts at 177°C with a Tg at 9°C, the 91:9 copolymer with 4HB
melts at 159°C, and the 1:1 copolymer with 3HV melts at 91°C. The PHB properties in the living
cells of Alcaligenes eutrophus were determined using X-ray and variable-temperature 13C NMR
relaxation studies (Amor et al. 1991). PHB is an amorphous elastomer with a Tg around −40°C in its
“native” state within the granules. The biodegradation of these polymers in soil and activated sludge
show the rate of degradation to be in the following order:
P(3HB-co-9% 4HB) > P(3HB) = P(3HB-co-50% 3HV)
The hydrolytic degradation of HB polymers was studied (Brandl et al. 1988). Microspheres degraded
slowly in phosphate buffer at 85°C and after 5 months 20%–40% of the polymer eroded under these
conditions. Copolymers having a higher fraction of 3-HV and low molecular weight polymers were
more susceptible to hydrolysis.
The mass loss of this polymer follows zero-order release kinetics, and this property along with its
hydrophobic nature indicates that this polymer primarily undergoes surface erosion. This property
makes it an ideal candidate for developing drug delivery vehicles that can achieve zero-order drug
release (Nair and Laurencin 2007). Attempts are currently underway to increase the rate of degra-
dation of these polymers by blending them with more hydrophilic polymers or other low molecular
weight additives to increase water penetration and facilitate degradation (Chen et al. 2006). In another
biomedical application, the effects of biodegradable tubular conduit made from PHB scaffold with
predominantly unidirectional fiber orientation and supplemented with cultured adult Schwann cells
on axonal regeneration after cervical spinal cord injury in adult rats were studied by Novikova et al.
The results demonstrated that a PHB scaffold promotes attachment, proliferation, and survival of
adult Schwann cells and supports marked axonal regeneration within the graft (Novikova et al. 2008).
5.2.1.4 Polycarbonates
Poly(ethylene carbonate), poly(propylene carbonate), and PTMC have been tested as biodegradable
carriers for the drug delivery (Kawaguchi et al. 1982). They are linear thermoplastic polyesters of
carbonic acid with aliphatic dihydroxy compounds.
5.2.1.4.1 Synthesis
The polymers are synthesized from the reaction of dihydroxy compounds with phosgene or with
bischloroformates of aliphatic dihydroxy compounds by transesterification, and by polymerization of
cyclic carbonates. These polymers have been synthesized from carbon dioxide and the corresponding
epoxides in the presence of organometallic compounds as initiators (Kawaguchi et al. 1983).
R R O
HO CH2 CH OH + COCl2 CH2 CH O C O
n
R = H, CH3
High molecular weight flexible PTMC can be obtained by the ROP of TMC. Being elastomeric
aliphatic polyester with excellent flexibility and poor mechanical strength, PTMC has been inves-
tigated as a candidate implant material for soft tissue regeneration and as a suitable material for
developing drug delivery vehicles (Nair and Laurencin 2007).
Since the carbonate linkage may be labile to hydrolysis, the biodegradability of polycarbonates has
been studied (Kawaguchi et al. 1982, 1983). Poly(ethylene carbonate) is biodegraded by surface
erosion without significant change in the molecular weight of the residual polymer mass. The main
degradation product of poly(ethylene carbonate) in aqueous systems is ethylene glycol, formed pre-
sumably by hydrolysis of ethylene carbonate (Stoll et al. 2001).
Pellets of poly(ethylene carbonate) and poly(propylene carbonate) were implanted into the perito-
neal cavity of rats and the toxicity and weight loss of polymer pellets were determined. Poly(ethylene
carbonate) was completely eliminated 15 days post-implantation, whereas poly(propylene carbonate)
remained intact after 60 days. When pellets of the polymers were incubated in phosphate buffer pH 7.4
at 37°C, both polymers did not degrade even after 40 days. These data indicate that poly(ethylene car-
bonate) was degraded by enzymes. No visible inflammatory reaction was noted at the implantation sites.
PTMC undergoes surface degradation with the rate of in vivo degradation found to be much
higher than in vitro degradation. This is presumably due to the contribution of in vivo enzymatic
degradation process (Zhang et al. 2006). Copolymers of aliphatic carbonates and lactide showed
excellent biocompatibility and mechanical properties. Block copolymers of TMC and lactide were
synthesized from the reaction of the monomers with stannous octoate as catalyst at 160°C for 16 h:
O O
CH C O CH2 CH2 CH2 O C O
CH3 n m
PLA TMC
The polymers were soluble in common organic solvents and had a weight average molecular weight
of 90,000. They completely degraded in vivo in 1 year (Katz et al. 1985).
5.2.1.5 Other Polyesters

Poly(p-dioxanone) is clinically used as an alternative to poly(lactide) in absorbable sutures with
similar properties to poly(lactide) with the advantage of better irradiation stability during steriliza-
tion (Bezwada et al. 1990).
O
O CH2 CH2 O CH2 C
n
Poly(p-dioxanone)
5.2.1.5.1 Poly(p-Dioxanone)
The preparation of mono- and multifilament sutures incorporating ibuprofen as an anti-inflammatory
agent was reported using p-dioxanone (Zurita et al. 2006). The kinetics of both the loading p rocess and
the release in a Sorensen’s medium at 37°C has been investigated. Besides this, to produce an estro-
genic and bactericidal biomaterial for the treatment of infected nonunions or bone defects, a synthetic
degradable block copolymer of poly-d,l-lactic acid segments with randomly inserted p-dioxanone
and polyethylene glycol segments was mixed with recombinant human BMP-2 (rhBMP-2) and antibi-
otics at high concentration. The in vitro elution profile of an antibiotic (teicoplanin) from the polymer,
the effects of antibiotics on the bone-inducing capacity of rhBMP-2 or on ectopic new bone formation
induced by the rhBMP, and the ability of the polymer to repair bone in a rat cranial defect model were
determined. It was observed that the in vivo performance of pellets with antibiotics and rhBMP-2
revealed no significant change in bone yield within the ossicles after 3 weeks. Additionally, the bio-
logical activity of rhBMP-2 was retained irrespective of the presence of antibiotics. The data provide
important insights into the fabrication of implants that provide efficacious delivery of rhBMP-2 using
the lowest possible dose of this expensive osteoinductive protein. This information will be of value
for the clinical use of BMPs (Kato et al. 2006a,b; Suzuki et al. 2006).
Biodegradable polymers derived from naturally occurring, multifunctional hydroxy acids and
amino acid have been investigated by Lenz and Guerin (1983). The monomers, malic acid, and
aspartic acid were polymerized into a polyester or polyamide using a ROP process as follows:
COOH COOR COOR
COOR O
X CH X CH CH X
X CH CH2 C
CH2 CH2 CH2 C n
COOH COOH O
X = O (ester), X = NH (amide)
The molecular weights of the polymers were highly dependent on the purity of the cyclic monomers.
Polymers of 50,000 were obtained in 93% polymerization yield when very pure monomers were
used. Both polymers are water soluble; however, crystallinity and controlled number of acid
groups in the polymer chains can be used to alter water swellability and solubility of the poly-
mers. Biocompatibility evaluation in mice indicated that poly (β-malic acid) is nontoxic (Lenz and
Guerin 1983).
A series of polyesters, poly(propylene fumarate) (PPF), based on the reaction product of fumaric
acid (Domb et al. 1990, 1996), and tartaric acid with different aliphatic diols have also been evalu-
ated as drug carriers. The polymers were synthesized by a direct melt condensation of the acids with
the diols with acid catalysis.
5.2.2 Poly(Amides)
A poly(amide) is a polymer-containing monomer of amide joined by peptide bonds. Poly(amides)
are commonly used in textiles, automotives, carpet, and sportswear due to their extreme durability
and strength. The utilization of amide-based polymers, especially natural proteins, in the prepa-
ration of biodegradable matrices has been extensively investigated (Kemnitzer and Kohn 1997).
Microcapsules and microspheres of cross-linked collagen (Sehgal and Srinivasan 2009), gelatin
(Cao et al. 2009), and albumin (Mathew et al. 2009; Khan and Kumar 2011) have been used for
drug delivery.
Poly(amides) occur naturally (proteins, such as wool and silk) or can be made artificially (nylons,
and sodium poly(aspartate)) through step-growth polymerization or solid-phase synthesis. Synthetic
routes for new classes of biodegradable polymers derived from amino acids having alternating
anhydride or ester and amide bonds in the polymer backbone were described by Domb (1990).
This approach is useful in the synthesis of degradable polymeric drugs, when bioactive amino
acids are used, and possibly in the design of polymers with improved mechanical strength and
biocompatibility.
Various poly(amides) were reported, e.g., poly(l-glutamic acid) is a poly(amide) composed of
naturally occurring l-glutamic acid residues linked together through amide bonds. It is degraded
into monomeric l-glutamic acid by lysosomal enzymes, thus making it an ideal candidate as biode-
gradable biomaterial. The polymer is highly charged at physiological pH and has been identified as
a unique gene/plasmid delivery vehicle (Nicol et al. 2002). Poly(aspartic acid) is synthesized from
aspartic acid by thermal polymerization. It is a highly water-soluble ionic polymer with a carboxylate
content much higher than poly(glutamic acid) (Nair and Laurencin 2007). Poly(l-lysine) is a small
polypeptide of the essential amino acid l-lysine. Recently, the poly-l-lysine-coated PLGA micro-
spheres containing retinoic acid have been explored for nerve tissue engineering. Embryonic carci-
noma cells were seeded on them and found to exhibit differentiation into neural cells (Nojehdehian
et al. 2009).
The natural amino acid l-tyrosine is a major nutrient having a phenolic hydroxyl group. This
feature makes it possible to use derivatives of tyrosine dipeptide as a motif to generate diphenolic
monomers, which are important building blocks for the design of biodegradable polymers. Using
this approach, a wide variety of polymers have been synthesized (Bourke and Kohn 2003).
Bailey et al. reported two-dimensional thin films consisting of homopolymer and discrete
compositional blends of tyrosine-derived polycarbonates to elucidate the nature of different cell
responses that were measured in vitro. The data suggest strongly that optimal composition and pro-
cessing conditions can significantly affect the acute inflammatory and extracellular matrix produc-
tion responses (Bailey et al. 2006).
Poly(amides) are ubiquitous, naturally occurring biodegradable polymers; however, their appli-
cation as a biomaterial has been limited due to immunogenicity (Nair and Laurencin 2007). To
overcome these limitations, attempts have been made to develop pseudopoly(amino acids) com-
posed of amino acids linked by non-amide bonds such as esters, imino carbonates, and carbonates.
O
O
C
N
C O
R n
(a)
O NH
CH2 CH C NH CH CH2 O C
NH C O
n
Cbz O-(CH2)x CH3
(b)
SCHEME 5.4 Molecular structure of pseudo(poly amino acids). (a) Poly(N-acylhydroxyproline esters);
(b) Tyrosine-derived polyiminocarbonate, poly(CTTE), x = 1; poly(CTH), x = 4; poly(CTTP), x = 15.
Pseudopoly(amino acids) as an approach for biomaterials based on amino acids have been first
suggested by Kohn and Langer (1987) who prepared a polyester from N-protected trans-4-hydroxy-
l-proline and poly(iminocarbonate) from tyrosine dipeptide as monomeric starting material. The
structures of poly(N-acylhydroxyproline esters) and a homologous series of tyrosine derived poly-
mers are described in Scheme 5.4 (Kemnitzer and Kohn 1997).
The properties, biodegradability, drug release, and biocompatibility of this class of polymers
have been reviewed (Kemnitzer and Kohn 1997). Random copolymers of the α-amino acids
N-(3-hydroxypropyl)-l-glutamine and l-leucine were synthesized and used as carriers for naltrex-
one (Bennett et al. 1991). Naltrexone was covalently bound through the 3-phenolic or the 14-tertiary
hydroxyls to the polymer hydroxyl side chains via a carbonate bond. Naltrexone was released from
the polymer in a relatively constant way for 30 days, both in vitro and in vivo experiments in rats
(Bennett et al. 1991).
5.2.3 Poly(Phosphate Esters)

Poly(phosphate ester), polyphosphonates, and polyphosphazenes are three classes of phosphorus-
containing polymers that have received wide attention over the past decade for their utility in
biomedicine. Significant research in this area has led to niche polymers with morphologies
ranging from viscous gels to amorphous microparticles for utility in drug delivery (Chaubal
et al. 2003).
Poly(phosphate esters) were synthesized from the reaction of ethyl or phenyl phosphorodichlori-
dates and various dialcohols including bisphenol A and poly(ethylene glycol) of various molecular
weights.
O O
Cl P Cl + HO R´ OH P O–R´–O– + 2HCl
n
O–R O–R
Interfacial condensation using a phase transfer catalyst and bisphenol A as comonomer yielded
polymers with a weight average molecular weight around 36,000.
Poly(phosphate ester) polymers are among the attractive candidates, in the view of their high
biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under
physiological conditions, and a wide variety of physicochemical properties (Wang et al. 2001).
As an important class of eminent biomaterials, polyphosphates have good biocompatibility,
biodegradability probably due to the structural similarity to naturally occurring nucleic and
teichoic acids. They can be degraded naturally into harmless low molecular weight products
through hydrolysis or enzymatic digestion of phosphate linkages under physiological conditions.
In recent years, some linear random and block copolymers with phosphate units have been syn-
thesized and applied to drug and gene delivery. Some biocompatible and biodegradable hydro-
gels based on polyphosphates have also been produced for cell encapsulations (Liu et al. 2010a).
Poly(phosphate ester) has been studied as a potential biodegradable matrix for drug delivery
(Wen et al. 2004). The pentavalency of phosphorus offers the potential for covalent linking of
the drug (Chaubal et al. 2003).
The polymers based on bisphenol A release drugs for a long period of time; 8%–20% corti-
sone was released after 75 days in buffer solution. The degradation rate depends on the nature
of the polymer side chain; polymers with phenyl side chains degrade much slower than those
containing ethyl or ethoxyethyl side chains (Leong 1995). The in vivo degradation in rabbits was
faster than in vitro.
A biodegradable polyphosphoester, poly[[(cholesteryl oxocarbonylamido ethyl) methyl
bis(ethylene) ammonium iodide] ethyl phosphate] (PCEP), was synthesized and investigated for
gene delivery (Wen et al. 2004). Results obtained suggest the potential of this polyphosphoester for
naked DNA-based gene therapy (Huang et al. 2004). Cellular-specific micellar systems from func-
tional amphiphilic block copolymers are attractive for targeted intracellular drug delivery. Micelles
based on diblock copolymer of poly(ethyl ethylene phosphate) and poly(ɛ-caprolactone) were devel-
oped by Wang et al. The surface functionalized micellar system is promising for specific anticancer
drug transportation and intracellular drug release (Wang et al. 2008).
5.2.4 Polyphosphazenes
The uniqueness of the polyphosphazenes stems from its inorganic backbone (N=P) with certain
organic side groups which can be hydrolyzed to phosphate and ammonia. Several polymer struc-
tures have been used as matrix carriers for drugs or as a hydrolyzable polymeric drug, where the
drug is covalently bound to the polymer backbone and released from the polymer by hydrolysis
(Allcock and Kwon 1986). Various comprehensive reports on the synthesis, characterization, and
medical applications of polyphosphazenes were published by Allcock (Allcock 1976; Allcock and
Ambrosio 1996; Allcock et al. 2003, 2007).
The polymers are most commonly synthesized by a substitution reaction of the reactive
poly(dichlorophosphazene) with a wide range of reactive nucleophils such as amines, alkox-
ides, and organometallic molecules (Scheme 5.5). The reaction is carried out in general at room
temperature in tetrahydrofuran, or aromatic hydrocarbon solutions. Polymers containing mixed
substituent can be obtained from the sequential or simultaneous reaction with several nucleo-
phils (Scheme 5.5):
RX = RONa, RNH 2 , C6H 5ONa
Cl Cl
P Cl R
N RX
Cl N N P N P
P P Cl
N Cl Cl R n
Cl n
H2O
RH + H3PO4 + NH3
SCHEME 5.5 Synthesis and hydrolysis of polyphosphazenes. (From Allcock, H.R. and Kugel, R.L., Inorg.
Chem., 5, 1716, 1966.)
The properties of the polymers depend on the nature of the side groups. Hydrolytically degradable
polyphosphazene were obtained when amino acid and imidazole derivatives were used as sub-
stituent. The first bioerodible polymer was the ethylglycinato derivative (R=NHCH2COOEt) which
hydrolytically degrade to ethanol, glycine, phosphate, and ammonia (Allcock et al. 1977).
Imidazolyl substituted polyphosphazene are hydrolytically unstable and hydrolyze in room mois-
ture. The rate of hydrolysis can be slowed by the incorporation of hydrophobic side groups such
as phenoxy or methylphenoxy groups. The in vivo and in vitro release of progesterone and bovine
serum albumin (BSA) from imidazole, 4-methylphenoxy substituted polyphosphazene matrices
were reported (Laurencin et al. 1993). Almost 90% of the loaded progesterone was released in
30 days with about 60% released in 8 days when placed in phosphate buffer pH 7.4 at 37°C.
Tyrosine-functionalized polyphosphazenes were synthesized by Allcock et al. (2003) and their
hydrolytic stability, pH-sensitive behavior, and hydrogel-forming capabilities were investigated.
The physical and chemical properties of the polymers varied with the type of linkage between
the tyrosine unit and phosphazene backbone. The rate of hydrolysis was dependent on the ratio
of the two side groups, the slowest rate being associated with the highest concentration of tyrosine.
The hydrolysis products were identified as phosphates, tyrosine, glycine, ammonia, and ethanol
derived from the ester group.
Amino acid ester substituted polyphosphazene have been used for controlled release of the cova-
lently bounded anti-inflammatory agent, naproxen. Steroids having hydroxyl group were bound
to the polymer chain through the hydroxyl group (Allcock and Fuller 1980). These amino acid
ester substituted polyphosphazenes are attractive candidates because of their biocompatibility, con-
trollable hydrolytic degradation rates, and nontoxic degradation products. These polymers demon-
strated excellent tissue compatibility and in vivo biodegradability and can be potential candidates
for various biomedical applications (Sethuraman et al. 2006), e.g., in vivo orthopedic applications
(Nukavarapu et al. 2008).
Zheng et al. have reported a new class of amphiphilic methoxy-poly(ethylene glycol) grafted
polyphosphazene. Doxorubicin was encapsulated into polymeric micelles derived from these copo-
lymers. Findings suggest that these copolymers can encapsulate water-insoluble anti-cancer agents
and contribute to improve drug sensitivity of adriamycin-resistant cell line (Zheng et al. 2009). In
another study, Zheng et al. have reported the construction of self-assembled nanoparticles from
novel pH-sensitive amphiphilic polyphosphazenes. These nanoparticles provide fast pH-responsive
drug release and have the capability to disturb endosomal membranes. The pH-responsive nanopar-
ticles designed in this study have achieved their potential as a drug delivery system for tumor ther-
apy applications (Zheng et al. 2011).
5.2.5 Poly(Orthoesters)
Poly(orthoesters) (POEs) are amorphous hydrophobic polymers containing hydrolytically labile,
acid-sensitive, backbone linkages. These were developed by ALIZA Corporation (Alzamer®) as
hydrophobic surface eroding polymers particularly for drug delivery applications. POEs were
invented during the pursuit of developing a bioerodible polymer, subdermally implantable that
would release contraceptive steroids by close to zero-order kinetics for at least 6 months (Benagiano
and Gabelnick 1979). An additional objective was that the polymer erosion and drug release should
be concomitant so that no polymer remnants are present in the tissue after the entire drug has been
released.
The objectives mentioned earlier could only be met if the polymer was truly surface eroding. For
a surface eroding polymer, the erosion process at the surface of the polymer should be much faster
than in the interior of the device. To exhibit such a phenomenon, the polymer has to be extremely
hydrophobic with very labile linkages. Hence, it was envisioned that polymeric devices with an
orthoester linkage in the backbone, which is an acid-sensitive linkage, could provide a surface-
eroding polymer if the interior of the matrix is buffered with basic salts.
By using diols with varying levels of chain flexibility, the rate of degradation for these polymers,
pH sensitivity, and glass transition temperatures can be controlled. To date four different classes
of POEs have been developed (POE I, II, III, and IV). Few orthopedic applications of this class
of polymers have been explored, but major use of POEs has been limited to drug delivery systems
(Sokolsky-Papkov et al. 2007).
5.2.5.1 Synthesis
The first POEs were reported by Choi and Heller (1979), assigned to Alza Corporation. These pro-
prietary polymers were first designated as Chronomer and later as Alzamer. They were prepared
by a transesterification reaction (Choi and Heller 1979). The general synthesis involved the heating
of the reaction mixture to 110°C–115°C for about 1.5–2 h and then further heated at 180°C and
0.01 T for 24 h. The synthesis of such polymers, with minimal amount of cross-linkage, requires an
orthoester starting material in which one alkoxy group has a greatly reduced reactivity. This can be
achieved by using a cyclic structure as shown earlier.
EtO OEt
O O R
O O
+ HO-R-OH + EtOH
Hydrolysis of these polymers regenerates the diol and γ-butyrolactone. The γ-butyrolactone rap-
idly hydrolyses to γ-hydroxybutyric acid. The production of γ-hydroxybutyric acid would further
catalyze the breakdown of orthoester linkages leading to bulk erosion of the matrix. Thus, it was
decided to incorporate basic salts into the polymer matrix to neutralize the generated acid and keep
the hydrolysis process under control (Heller 1990). This polymer system was originally designated
as Chronomer@ but is now marketed as Alzamere. The polymer has been used in the treatment of
burns, in the delivery of the narcotic antagonist naltrexone, and in the delivery of the contraceptive
steroids norethindrone and levonorgestrel (Heller 1990).
Subsequently, another family of POEs were developed that were not related to Alzamer. These
polymers are prepared by the addition of polyols to diketene acetals. The general reaction can be
schematically represented as follows:
OR OR OR OR
H2C = C–O–R΄–O–C = CH2 + HO–R˝–OH O–C–O–R΄–O–C–O–R˝
n
CH3 CH3
Initial work was conducted with the monomer, diketene acetal, derived from pentaerythritol, and
1,6 hexanediol. The reaction is exothermic and proceeds to completion virtually instantaneously.
The reaction is as follows:
O-CH2 CH2-O
RCH = C C C=CHR + HO-R’-OH
O-CH2 CH2-O
RCH O-CH2 CH2-O CHR

C C C
O O-CH2 CH2-O O-R

n
where R = H, for 3,9-bis(methylene 2,4,8, 10-tetra oxaspiro [5,5] undecane). The diols investigated
were 1,6 hexandediol trans-1,4 cyclohexane dimethanol, 1,6-cyclohexanediol, ethylene glycol, and
biophenol A.
5.2.5.2 Polymer Properties

The molecular weight of POEs was significantly dependent on the type of diol and catalyst used
for synthesis. A linear, flexible diol like 1,6 hexane diol gave molecular weights greater than 200 K,
whereas bisphenol A in the presence of catalyst gave molecular weight around only 10,000 (Heller
et al. 1983).
The mechanical properties of the linear POEs can be varied over a large range by selecting
various compositions of diols. It was shown that the glass transition temperature of the polymer
prepared from DETOSU can be varied from 25°C to 110°C by simply changing the amount of
1,6 hexanediol in trans-1,4 cyclohexane dimethanol from 100% to 0% (Heller et al. 1985). There
seems to be a linearly decreasing relationship between the Tg and percentage of 1,6 hexanediol. One
could take advantage of the aforementioned relationship in selecting the polymer for in vivo applica-
tions because in vivo the Tg of the polymer would drop due to imbibitions of water. This can result
in the loss of stiffness and rigidity of the polymer.
5.2.5.3 Cross-Linked Poly(Orthoesters)

To prepare a cross-linked polymer, there should be at least one monomer that has functionality
greater than 2. In the case of POEs, it is possible for a ketene acetal or an alcohol to have a function-
ality greater than 2. Due to the difficulty in preparing trifunctional ketene acetals, triols were used
to prepare the cross-linked polymer.
In general, the synthesis of cross-linked polymers was synthesized by reacting prepolymer with
the triols or a mixture of diols and triols (Scheme 5.6; Heller et al. 1987).
The prepolymer is an acetal with a diol and is a viscous liquid at room temperature. Thus, the
compound of interest could be incorporated into the prepolymer along with the triol and the mixture
can be cross-linked at temperatures as low as 40°C. This can be a good method for incorporat-
ing thermo labile drugs. However, one should be cautious with using compounds with hydroxyl
functionality.
Another family of POEs can be prepared by reacting a triol with two vicinal hydroxyl groups
and one removed by at least three methylene groups with an alkyl orthoacetate (Heller et al.
1990). The intermediate does not have to be isolated and continuous reaction produces a polymer.
The use of flexible triols such as 1,2,6-hexanetriol produces highly flexible polymers that have
ointment-like properties even at relatively high molecular weights. However, properties such as
H3C O-CH2 CH2-O CH3 CH3

C=C C C=C + HO-CH2-CH-CH2-CH2-OH
H H
O-CH2 CH2-O
H3C O-CH2 CH2-O C2H5 H5C2 O-CH2 CH2-O CH3

C=C C=C C C C=C
C
H O-CH2 CH2-O O-CH-CH-CH-CH-O O-CH2 CH2-O
(Prepolymer)
Prepolymer + 1,2,6 Hexanetriol Cross linked poly(orthoester)
SCHEME 5.6 Synthesis of cross-linked poly(orthoesters).

viscosity and hydrophobicity can be readily varied by controlling molecular weight and the size
of the alkyl group R′.
CH2 CH R OH
OCH2CH3
H O O
H2C C R OH + R’ C OCH2CH3 C + CH3CH2OH
R’ OCH2CH3
OH OH OCH2CH3
R’ O CH2
C
CH3CH2OH +
O O CH R
In another experiment, Heller et al. (1992) replaced the flexible triol to a rigid one such as
1,1,4-cyclohexanetrimethanol to obtain a solid polymer, as shown next:
R OCH3
OH OH O O
R O H2
RC(OCH3)3
C
O O
HO HO
n
5.2.5.4 Polymer Hydrolysis

The primary mechanism for the degradation of POEs is via hydrolysis. Depending on the reactants
used during the synthesis of the polymer, the hydrolysis products are diol or mixture of diols, and
pentaerythritol dipropionate or diacetate if 3,9-bis (methylene-2,4,8,10-tetraoxaspiro [5,5] undec-
ane) was used (Sparer et al. 1984). The pentaerythritol esters hydrolyze at a slower rate to pentae-
rythritol and the corresponding acetic or propionic acid. The sequence of reaction is as follows:
O O
CH3CH2COCH2 CH2OCCH2CH3
C + HO-R-OH
HOCH2 CH2OH
HOCH2 CH2OH
CH3CH2COOH + + CH3CH2COOH
C
HOCH2
CH2OH
The difference in the sensitivity of the hydrolysis of orthoester linkages in acid versus alka-
line medium has been used to the advantage in designing the orthoester-based delivery systems.
This preferential sensitivity is used by incorporating acid anhydrides into the matrix to accelerate
the rate of hydrolysis. While, on the other hand, a base is used to stabilize the interior of the matrix.
5.2.5.5 Polymer Processing

The orthoester linkage is inherently unstable in the presence of water. However, because of the
polymer’s highly hydrophobic nature, they can be stored without careful exclusion of moisture.
Even though the polymer is relatively stable in trace amounts of moisture, it is unstable to heat and
undergoes disproportionation to an alcohol and ketene acetal. The combination of moisture and heat
can be fatal for the processing of POEs, which are designed to erode within days. Thus, if injection
molding is necessary to fabricate the device, then moisture must be rigorously excluded during fab-
rication. One should also consider the interaction between the incorporated anhydride as catalysts,
the polymer, and drug during the thermal processing.
Deng et al. reported the use of laboratory-scale spray-congealing equipment to fabricate injectable
microparticles consisting of poly(orthoester) and bupivacaine. Characterizations were performed to
determine the chemico-physical properties of poly(orthoester) before and after microparticle fabri-
cation. The microparticles have demonstrated the potential to be used for long-acting post-surgery
pain management by local injection (Deng et al. 2003).
The effect of local delivery of indomethacin by a bioerodible poly(orthoester) on the reossifica-
tion of segmental defects of the radius in rats was studied. The results of this study suggest that the
poly(orthoester) may be used as a bioerodible system for local delivery of indomethacin to inhibit
reossification of skeletal defects without tissue reaction, unabsorbed carrier, or systemic effects
(Solheim et al. 1995).
5.2.6 Polyanhydrides
The invention and development of polyanhydrides dates as far back as 1900s. In 1909, Bucher and
Slade reported the development of aromatic polyanhydrides composed of isophthallic acid and
terephthallic acid (Bucher and Slade 1909). Subsequently, Hill and Carothers (1932) reported a
series of aliphatic polyanhydrides. Systematic development of polyanhydrides, as substitutes for
polyesters in textile applications, was undertaken initially by Conix et al. (1958). They prepared
and studied a number of aromatic and heterocyclic polyanhydrides. It has been recognized that the
matrix should undergo heterogeneous degradation to maximize the control over release process.
These polymers were not suitable for textile applications because of the extreme reactivity of anhy-
dride linkage toward water. In early 1980s, Rosen et al. (1983) envisioned the use of hydrophobic
polyanhydrides in designing the surface eroding matrix for applications in controlled drug deliv-
ery. The fact that they are extremely hydrophobic and hydrolytically unstable renders them useful
in drug delivery applications (Leong et al. 1985, 1986). Therefore, although originally developed
for the textile industry, polyanhydrides found use in biomedical applications due to their biode-
gradability and excellent biocompatibility (Katti et al. 2002).
Efficient control over various physicochemical properties, such as biodegradability and biocom-
patibility, can be achieved for polyanhydrides due to the availability of a wide variety of diacid
monomers as well as by copolymerization of these monomers. Biodegradation of these polymers
takes place by the hydrolysis of the anhydride bonds and the polymer undergoes predominantly
surface erosion, a desired property to attain near zero-order drug release profile (Katti et al. 2002).
The properties of major classes of anhydrides and the impact of geometry on degradation and ero-
sion are discussed by Gopferich and Tessmar (2002).
5.2.6.1 Synthesis
5.2.6.1.1 Melt Polycondensation
The majority of the polyanhydrides are prepared by melt polycondensation. The sequence of reac-
tion involves first the conversion of a dicarboxylic acid monomer into a prepolymer consisting of a
mixed anhydride of the diacid with acetic anhydride. This is achieved by simply refluxing the diacid
monomer with acetic anhydride for a specified length of time. The polymer is obtained subsequently
by heating the prepolymer under vacuum to eliminate the acetic anhydride (Yoda 1963):
HOOC-(CH2)8-COOH + (CH3CO)2O
Reflux
O O O O
CH3-C-O-C-(CH2)8-C-O-C-CH3
10–4 mm Hg 180°C, 90 min
O O
-O-C-(CH2)8-C-O-
n
The polyanhydride thus obtained was of low molecular weight. For most of the practical applica-
tions, high molecular weight polyanhydrides are desirable. Hence, a systematic study was under-
taken to determine the factors that affected the polymer molecular weight (Domb and Langer 1987).
It was found that the critical factors were monomer purity, reaction time and temperature, and
an efficient system to remove the byproduct, acetic anhydride is required. The highest molecular
weight polymers were obtained using pure isolated prepolymers and heating them at 180°C for
90 min with a vacuum of 10 −4 mmHg, using a dry ice/acetone trap. Significantly higher molecular
weights were obtained in shorter times by using coordination catalysts such as cadmium acetate,
earth metal oxides, and ZnEt2.H2O (Domb and Langer 1987). The weight average molecular weight
varied from 90,000 to 240,000 when the concentration of cadmium acetate was changed from
0.5 to 3 mole%, with the reaction time of less than 1 h. Other catalysts, such as titanium and iron,
inhibited the polymerization reaction and the polymers were dark brown. Acidic catalyst, such as
p-toluene sulfonic acid, did not show any effect on polymer molecular weight while the basic cata-
lyst, 4-dimethyl amino pyridine, caused a decrease in molecular weight.
5.2.6.1.2 Solution Polymerization

Synthesis of polyanhydrides, using melt polycondensation, is useful to obtain high molecular weight
polymers but is not useful if the monomers are thermolabile. Hence, methods were developed to
synthesize polyanhydrides under ambient conditions for heat-sensitive monomers such as dipeptides
and therapeutically active diacids.
The solution polymerization is carried out by Schotten–Baumann technique. In this method,
the solution of diacid chloride is added dropwise into an ice-cooled solution of a dicarboxylic
acid. The reaction is facilitated by using an acid acceptor such as triethylamine. Polymerization
takes place instantly on contact of the monomers and is essentially complete within 1 h. The
solvents employed can be a single solvent or a mixture of solvents such as dichloromethane,
chloroform, benzene, and ethyl ether. It was found that the order of addition is very impor-
tant in obtaining relatively high molecular weight polyanhydrides. Addition of a diacid solution
dropwise to the diacid chloride solution consistently produced high molecular weight polymers
(Subramanyam and Pinkus 1985).
Base
HOOC-R-COOH + ClOC-R'-COCl -(R-C(O)-O-C(O)-R'-C(O)-O-C(O))- + Base - HCl
The drawback of this homogeneous Schotten–Baumann condensation reaction in solution is that

the diacid chloride monomer should be of very high purity. An alternate approach was the con-
version of dicarboxylic acid monomer into the polyanhydride using a dehydrative coupling agent
under ambient conditions. The dehydrative coupling agent, N′N bis[2-oxo-3-oxazolidinyl]phos-

phonic chloride, was the most effective in forming polyanhydrides with the degree of polymer-
ization around 20 (Leong et al. 1987). It is essential that the catalyst be ground into fine particles
before use and should be freshly prepared. A disadvantage of this method is that the final product
contains polymerization byproducts, which have to be removed by washing with protic solvents
such as methanol or cold dilute hydrochloric acid. The washing by protic solvents may evoke some
hydrolysis of the polymer.
Coupling agents such as phosgene and diphosgene could also be used for the polyanhydride for-
mation. Polymerization of sebacic acid using either phosgene or diphosgene as coupling agents with
the amine-based heterogeneous acid acceptor, poly(4-vinyl pyridine), produced higher molecular
weights in comparison to non-amine heterogeneous base K2CO3 (Domb et al. 1988).
5.2.6.1.3 Ring-Opening Polymerization

ROP offers an alternate approach to the synthesis of polyanhydrides used for medical applica-
tions. Albertsson and co-workers prepared adipic acid polyanhydride from cyclic adipic anhy-
dride (oxepane-2,7-dione) using cationic (e.g., AlCl3 and BF3.(C2H5)2O), anionic (e.g., CH3COO −K+
and NaH), and coordination-type inhibitors such as stannous-2-ethylhexanoate and dibutyltin oxide
(Albertsson and Lundmark 1988; Lundmark et al. 1991). ROP takes place in two steps: (1) prepara-
tion of the cyclic monomer and (2) polymerization of the cyclic monomers (Kumar et al. 2005).
5.2.6.2 Polymer Properties

Almost all polyanhydrides show some degree of crystallinity as manifested by their crystalline
melting points. An in-depth X-ray diffraction analysis was conducted with the homopolymers of
sebacic acid (SA), bis(carboxyphenoxy)propane (CPP), bis(carboxyphenoxy)hexane (CPH) and
fumaric acid, and the copolymers of SA with CPP, CPH, and fumaric acid. The results indicated
that the homopolymers were highly crystalline and the crystallinity of the copolymers was deter-
mined, in most cases, by the monomer of highest concentration. Copolymers with a composition
close to 1:1 were essentially amorphous (Mathiowitz et al. 1990).
The melting point, as determined by differential scanning calorimetry, of these aromatic poly-
anhydrides is much higher than the aliphatic polyanhydrides. The melting point of the aliphatic-
aromatic copolyanhydrides is proportional to the aromatic content. For this type of copolymers
there is characteristically a minimum Tm between 5 and 20 mole% of the lower melting component
(Domb and Langer 1987).
The majority of polyanhydrides dissolve in solvents such as dichloromethane and chloroform.
However, the aromatic polyanhydrides display much lower solubility than the aliphatic polyanhy-
drides. In an attempt to improve the solubility and decrease the Tm, copolymers of two different
aromatic monomers were prepared. These copolymers displayed a substantial decrease in Tm and
an increase in solubility than the corresponding homopolymers of aromatic diacids (Domb 1992).
The data on mechanical properties of polyanhydrides are very limited. The fibers of poly[1,2-
bis(p-carboxyphenoxy) ethane anhydride] showed a tensile strength of 40 kg/mm2 with an elonga-
tion of 17.2% and a Young’s modulus of 505 kg/mm2. A systematic study on the tensile strength of
the copolymers of CPP and SA showed that increasing the CPP content in the copolymer or the
molecular weight of the copolymer increased the tensile strength. Unsaturated polyanhydrides of
the structure [–(OOC–CH=CH–CO)x–(OOC–R–CO)y]n were developed to improve the mechanical
properties of the polymers. The advantage of the unsaturated polyanhydrides is that it can undergo
secondary polymerization of the double bonds to create a cross-linked matrix (Domb et al. 1991b).
5.2.6.3 Polymer Hydrolysis

Anhydride linkage is extremely susceptible to hydrolysis in the presence of moisture to generate
the dicarboxylic acids (Domb and Nudelman 1995). Hydrolysis of monomeric anhydrides is cata-
lyzed by both acid and base, and the hydrolytic degradation rate of polyanhydrides increases with
an increase in pH. It is believed that the poor solubility of the oligomeric products, under low pH
conditions, formed at the surface of the matrix impede the degradation of the core. In general, the
hydrophobic polymers such as P(CPP) and P(CPH) display constant erosion kinetics. The degrada-
tion rates of the polyanhydrides can be altered in a number of ways. The degradation rates can be
enhanced by incorporating the aliphatic monomer, such as sebacic acid, into the polymer. The deg-
radation can be slowed by increasing the methylene groups into the backbone of the polymer. For
example, in the case of poly[bis(p-carboxyphenoxy)alkane] series, increasing the methylene groups
from 1 to 6 increased the hydrophobicity of the polymer and the erosion rates underwent a decrease
of 3 orders of magnitude.
To achieve a variety of degradation rates, aliphatic-aromatic homopolyanhydrides of the structure
–(OOC–C6H4 –O(CH2)x–CO–)n were prepared with “x” varying from 1 to 10. Increasing the value
of “x” decreases the erosion rates (Domb et al. 1989). Increased erosion rates were also observed
when poly(sebacic acid) was branched with either 1,3,5 tricarboxylic acid or low molecular weight
poly(acrylic acid) (Maniar et al. 1990).
Apart from the reactivity of anhydride linkage toward water, aliphatic polyanhydrides and their
copolymers are found to undergo self-depolymerization under anhydrous conditions in the solid
state and in solution (Domb and Langer 1989). The depolymerization reaction mainly affects the
high molecular weight fraction of the polymer. Aromatic homopolymers show no sign of depoly-
merization when stored under anhydrous conditions. The depolymerization rate is found to follow
a first-order kinetics, accelerate with temperature, and increase with polarity of the solvent (Domb
and Langer 1989). The depolymerized polymer can be repolymerized to yield the original polymer,
suggesting that inter or intramolecular anhydride interchange takes place during depolymerization.
In order to tailor the erosion rate of polyanhydrides while retaining their surface erosion charac-
teristics, new three-component polyanhydrides of sebacic acid, 1,3-bis(p-carboxyphenoxy)propane
and poly(ethylene glycol), were synthesized by Hou et al. The incorporation of poly(ethylene glycol)
into traditional two-component polyanhydrides retains their surface erosion properties while mak-
ing the erosion rate tuneable. The new polyanhydrides hold potential for drug delivery applications
(Hou et al. 2007).
5.2.6.4 Polymer Processing

Drug incorporated matrix can be formulated either by compression or injection molding. The poly-
mer and drug can be ground in a Micro Mill grinder, sieved in to a particle size range of 90–120 μm,
and can be pressed into circular discs using Carver press. Alternatively, the drug can be mixed into
the molten polymer to form small chips of drug–polymer conjugate. These chips are fed into the
injection molder to mold the drug/polymer matrix into the desired shaped device. One must con-
sider the thermal stability of the polymer and the potential chemical interaction between drug and
polymer at high temperatures of injection molding.
The preferred method of drug delivery, in many instances, is by injection. This requires the develop-
ment of microcapsule or microspheres of the drug. Several different techniques have been developed
for the preparation of microspheres from polyanhydrides, which includes “Hot-melt’ microencapsula-
tion (Mathiowitz and Langer 1987) and solvent removal technique (Mathiowitz et al. 1988).
5.2.6.5 Other Polyanhydrides

In addition to the previously discussed aliphatic, aromatic, and the copolyanhydrides of the respec-
tive diacids, several other modifications of the backbone of the polyanhydrides have been reported.
These new polyanhydrides were developed to improve their physicochemical, mechanical, thermal,
and hydrolytic properties.
One of these new polyanhydrides is polyanhydride-imides, also referred to as copolyimides (Domb
1990). They showed good thermal resistance but were essentially insoluble in most organic solvents.
In an attempt to improve the solubility in more polar solvents, polyanhydrides were synthesized
using an imide-diacids containing aliphatic aromatic characteristics. A systemic study was reported
in which the starting monomers, imide-diacids, were prepared from aromatic acid anhydrides and
α-amino acids. Varying the number of methylenic units in the α-amino acids provided the variability
in the aliphatic character of the aromatic-aliphatic monomer. A typical example of such an aromatic-
aliphatic monomer is the one obtained by the reaction of trimellitic anhydride with glycine:
O
C
O + H2N-CH2-COOH
HOOC C
O
O
C
N-CH2-COOH
HOOC C
O
For the synthesis of polyanhydride, the aliphatic-aromatic diacid is first converted to the diacetyl
derivative by refluxing the diacid in the presence of excess acetic anhydride. The diacetyl deriva-
tive is polymerized either by melt polycondensation or in solution. The polyanhydride-imides thus
obtained are very soluble in polar organic solvents. However, they showed melt transitions at tem-
peratures of 245°C and above. Along with that, insufficient data are available on the hydrolytic sta-
bility of these materials, rendering them questionable materials as a carrier in drug delivery systems.
Another class of polyanhydrides is based on natural fatty acids. The dimers of oleic acid and
eurucic acid are liquid oils containing two carboxylic acids available for anhydride polymerization.
The homopolymers are viscous liquids. Copolymerization with increasing amounts of sebacic acid
forms solid polymers with increasing melting points as a function of SA content. The polymers are
soluble in chlorinated hydrocarbons, tetrahydrofuran, 2-butanone, and acetone (Domb and Maniar
1993).
Polyanhydrides synthesized from nonlinear hydrophobic fatty acid esters, based on ricinoleic,
maleic acid and sebacic acid, possessed the desired physicochemical properties such as low melting
point, hydrophobicity and flexibility to the polymer formed in addition to biocompatibility and bio-
degradability. The polymers were synthesized by melt condensation to yield film-forming polymers
with molecular weights exceeding 100,000 (Teomim et al. 1999).
O
OH
OH Ricinoleic acid
or Toluene
O O O O 80°C–90°C
O O
Succinic anhydride Maleic anhydride
O
OH
O
HO Ricinoleic acid succinate (1 double bond),
C O
Ricinoleic acid maleate (2 double bonds)
O
The properties of polyanhydrides were modified by the incorporation on long chain fatty
acid terminals such as stearic acid in the polymer composition, which alters its hydrophobicity
and decreases its degradation rate (Teomim and Domb 1999). Since natural fatty acids are mono-
functional, they would act as polymerization chain terminators and control the molecular weight.
A detailed analysis of the polymerization reaction shows that up to about 10 mole% content of stea-
ric acid, the final product is essentially a stearic acid terminated polymer. Whereas higher amounts
of acetyl stearate in the reaction mixture resulted in the formation of increasing amounts of stearic
anhydride byproduct with minimal effect on the polymer molecular weight, which remains in the
range of 5000. Physical mixtures of polyanhydrides with triglycerides and fatty acids or alcohols
did not form uniform blends.
The potential of poly 1,3-bis-(p-carboxyphenoxy)propane-co-sebacic acid (p(CPP:SA)) micro-
spheres was investigated for controlled delivery of basal insulin. The results indicate that CPP:SA
microspheres controlled insulin release in vitro and in vivo over a month and the released insulin
was conformationally and chemically stable and bioactive (Manoharan and Singh 2009).
Poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacic anhydride) (p(CPP-SA)) was used to
prepare microspheres, with human serum albumin (HSA) as the model protein. The structural
integrity of HSA extracted from microspheres was detected by gel permeation chromatography,
compared with native HSA. The results showed HSA remained its molecule weight after encapsu-
lated (Sun et al. 2009).
Slow drug release in osteomyelitis treatment is an important biomedical problem. Therefore, a
sustained-release bead system consisting of gentamicin sulfate in biodegradable poly(dimer acid-
tetradecandioic acid) copolymer [P(DA-TA), WDA: WTA = 50: 50] is prepared by melt casting for
osteomyelitis treatment. In vitro bacteriostatic activity studies demonstrated that the beads pos-
sessed desired bacteriostatic activity for Staphylococcus aureus and Escherichia coli, which are
common bacteria for infections in bone (Guo et al. 2007).
In another application, novel amphiphilic biodegradable systems based on polyanhydrides for the
stabilization and sustained release of peptides and proteins were reported (Torres et al. 2007). These
results indicated polyanhydride carriers for the stabilization and sustained release of therapeutic
peptides and proteins.
A series of biodegradable pasty poly(ester anhydride)s were prepared from alkanedicarboxylic
acids and ricinoleic acid and its oligomers by transesterification-repolymerization method by Krasko
and Domb. Pasty polymers can be mixed with drugs at room temperature and injected to tissue for
delivery of drugs, particularly for heat-sensitive drugs. The polymers release model drugs for a few
weeks while being degraded to their fatty acid counterparts. Copolymerization of alkanedicarbox-
ylic acids with ricinoleic acid resulted in pasty biodegradable polymers useful as injectable carriers
for drugs (Krasko and Domb 2007).
Recently as another application of polyanhydrides, Agueros et al. have reported the effect of the
combination between 2-hydroxypropyl-beta-cyclodextrin and bioadhesive polyanhydride nanopar-
ticles on the encapsulation and intestinal permeability of paclitaxel. A solid inclusion complex
between drug and cyclodextrin was prepared by an evaporation method. The complex was incor-
porated in polyanhydride nanoparticles. The association between these three components would
induce a positive effect over the intestinal permeability of paclitaxel (Agueros et al. 2009).
5.3 BIOCOMPATIBILITY AND TOXICITY

In all the potential uses of polymeric material, a direct contact between the polymer and biological
tissues is evident. Therefore, for the eventual human application of these biomedical implants and
devices, an adequate testing for safety and biocompatibility of the specific matrix polymer used in
each case is essential.
Whenever a synthetic polymer material is to be utilized in vivo, the possible tissue–implant
interactions must be taken into consideration. In the case of biodegradable matrices, not only the
possible toxicity of the polymer has to be evaluated, but also the potential toxicity of its degradation
products. The last section of this chapter reviews existing data about the biocompatibility and toxic-
ity of the different polymers actually available for biomedical applications.
5.3.1 Polyesters
5.3.1.1 Lactide/Glycolide Copolymers
Many biodegradable polymers have been developed for controlled drug delivery. The plethora
of drug therapies and types of drugs demand different formulations, fabrications conditions, and
release kinetics. Wen et al. have reported that no one single polymer can satisfy all the requirements
(Wen et al. 2003).
The feasibility of lactide/glycolide polymers as excipients for the controlled release of bioactive
agents is well proven, and they are the most widely investigated biodegradable polymers for drug
delivery. Most of the research work on the use of lactide/glycolide polymers as matrices for delivery
systems has been focused on the development of injectable microspheres formulations, although
implantable rod and pellet devices are also being investigated.
The lactide/glycolide copolymers have been subjected to extensive animal and human trials
without any significant harmful side-effects (Lewis 1990). No evidence of inflammatory response,
irritation, or other adverse effects has been reported upon implantation of lactide/glycolide polymer
devices.
Many conventional pharmaceutical agents formulated in lactide/glycolide polymer matri-
ces have been widely studied since almost three decades. One of the most successful lactide/
glycolide drug delivery formulations, in terms of clinical results obtained, is the steroid-loaded
injectable microspheres for the controlled release of contraceptives (Beck et al. 1981, 1983a,b).
Many animal and clinical trials with these systems were performed showing very good biocom-
patibility. The success of the steroid microsphere system based on lactide/glycolide matrices is
probably due to the combination of several factors: the reproducibility of the microencapsulation
process, the in vivo drug-release performance, reliability in the treatment procedure, and the
safety of the polymer.
Lactide/glycolide implants containing Naltrexone and other narcotic antagonist agents have also
been extensively studied (Schwope et al. 1975; Chiang et al. 1984). In one of these studies describing
the clinical evaluation of a bead preparation containing 70% naltrexone and 30% of a 90:10 lactide/
glycolide copolymer, a local inflammatory reaction at the site of implantation was reported in two of
three subjects after subcutaneous implantation of the beads containing the drug (Chiang et al. 1984).
This finding prevented further clinical testing of that particular formulation. No similar problems
were reported with other lactide/glycolide polymer preparations, and that incident was related to
some unique aspect of that product (Chiang et al. 1984).
Good biocompatibility data were also reported with lactide/glycolide copolymer matrices con-
taining antineoplastic drugs, antibiotics, and anti-inflammatory compounds (Lewis 1990). The
delivery of therapeutic molecules to the brain has been limited in part due to the presence of the
blood–brain barrier. The biocompatibility of lactide/glycolide copolymer in the brain was examined
regarding the gliotic response following implants of lactide/glycolide copolymer into the brains of
rats. It was found that lactide/glycolide copolymer is well tolerated following implantation into the
CNS and that the astrocytic response to lactide/glycolide copolymer is largely a consequence of the
mechanical trauma that occurs during surgery (Emerich et al. 1999).
Studies carried out for the encapsulation of bioactive macromolecules as proteins, peptides, and
antigens showed mixed biocompatibility results. Serious problems in achieving long-term release
have been reported in several cases where the macromolecules lost bioactivity in vivo after a few
days, as in the case of lactide/glycolide copolymer containing growth hormone. A complex interac-
tion apparently occurs in vivo between the acidic polymer and the hormone. In vitro release studies
have shown that growth hormone can become insoluble when incorporated in poly(lactide) films.
Similar problems in maintaining biological activity for longer than 5–10 days were also observed
with interferon-lactide/glycolide polymer formulation (Lewis 1990). On the other hand, promising
results were obtained with luteinizing hormone releasing hormone (LHRH) incorporated in lactide/
glycolide polymer showing long-term delivery of the macromolecule (Aschet al. 1985a,b). In general,
hydrophilic polypeptides of low molecular weight (<5000) are considered quite stable in the presence
of lactide/glycolide excipients and their acidic bioerosion byproducts.
Lactide/glycolide polymer implants, in the form of microbeads and pellets, containing insulin
were reported to be effective in lowering blood glucose levels in diabetic rats for about 2 weeks,
and no adverse effects, inflammation at the implant site, or deactivation of the macromolecule were
observed (Kwong et al. 1986).
Chen and Singh have designed injectable controlled release polymer formulations for growth
hormone using triblock copolymer PLGA-PEG-PLGA. The triblock copolymer used in this study
was able to control the release of incorporated growth hormone in vitro and in vivo for longer dura-
tion. Both in vitro and in vivo results support the biocompatible nature of these polymer delivery
systems (Chen and Singh 2008).
Bunger et al. have determined the feasibility and biocompatibility of a sirolimus eluting biode-
gradable poly(l-lactide) stent for peripheral vascular application. Histological analysis showed that
these stents were without any signs of excessive recoiling or collapse i.e., they showed sufficient
mechanical stability. The vascular inflammation against these stents was reduced by incorporation
sirolimus in these stents. No systemic toxicity or thrombotic complications was observed with these
stents (Bunger et al. 2007).
Aliphatic polyesters were tested for proliferation of human fibroblasts. Results obtained provide
the non-in vitro toxicity of the materials and of the processing method for the scaffolds preparation
in tissue engineering (Vaquette et al. 2006). Since biocompatibility of polylactic acid and polygly-
colic acid copolymers is affected by chemical composition, molecular weight, cell environment, and
by the methods of polymerization and processing, Di Toro et al. (2004) have determined all these
factors and reported that these materials do not show any negative influence on cell proliferation
and differentiation.
5.3.1.2 Poly(Caprolactone)
The biocompatibility and toxicity of poly(caprolactone) have mostly been tested in conjunction with
evaluations of Capronor™, which is an implantable 1 year contraceptive delivery system composed
of a levonorgestrel-ethyl oleate slurry within a poly(caprolactone) capsule. In a preliminary 90-day
toxicology study of Capronor in female rats and guinea pigs, except for a bland response at the
implant site and a minimal tissue encapsulating reaction, no toxic effects were observed (Pitt and
Schindler 1984).
The Capronor contraceptive delivery system was also tested implanted in rats and monkeys in
2 year period (Pitt 1990). The results of this study based on animal clinical and physical data as
blood and urine analysis, ophthalmoscopic tests, and histopathology after necropsy showed no sig-
nificant differences between the test and control groups.
Biocompatibility and toxicology of poly(caprolactone), which was synthesized using Ti(OBu)4 as
an initiator, were determined (Chen et al. 2000). The results obtained showed that poly(caprolactone)
films have a mild inflammatory reaction in the early days of planting and after 3 months the inflam-
mation disappeared. They conclude that poly(caprolactone) material possesses good biocompatibil-
ity and has a good developmental prospect.
Byrro et al. reported microspheres of poly(caprolactone) containing prednisolone acetate. There
microspheres were developed and evaluated as an injectable extended-release formulation. The
local biocompatibility of the system was verified by histopathological analysis of the deployment
region. Preliminary in vivo study showed the absence of local toxicity and confirmed the prolonged
release profile of drug, suggesting the viability of the developed system for the treatment of orbital
inflammatory diseases (Byrro et al. 2009).
Poly(caprolactone)-poly(ethylene glycol) copolymers are synthetic biomedical materials with

amphiphilicity, controlled biodegradability, and great biocompatibility according to Wei et al.
(2009). They have great potential application in the fields of nanotechnology, tissue engineering,
pharmaceutics, and medicinal chemistry.
5.3.1.3 Polycarbonates
Biodegradation and biocompatibility of poly(ethylene carbonate) were examined using an in vivo
cage implant system by Dadsetan et al. Exudate analysis showed the degradation products were bio-
compatible and induced minimal inflammatory and wound healing responses(Dadsetan et al. 2003).
To evaluate the technical feasibility of poly(ethylene carbonate), for injectable in situ forming
drug delivery systems, the physical properties of poly(ethylene carbonate) solutions were character-
ized. This study demonstrated that in situ depot forming systems can be obtained from poly(ethylene
carbonate) solutions (Liu et al. 2010b).
A study exploring the utility of poly(ethylene carbonate) as coating material for drug-eluting
stents under in vitro conditions was reported. Poly(ethylene carbonate) (Mw 242 kDa) was found to
be an amorphous polymer with thermoelastic properties. Tensile testing revealed a stress to strain
failure of more than 600%. These properties are thought to be advantageous for expanding coated
stents. In vitro cytotoxicity tests showed excellent cytocompatibility of poly(ethylene carbonate)
(Unger et al. 2007).
5.3.2 Poly(Amides)
5.3.2.1 Natural Polymers
The use of natural biodegradable polymers to deliver drugs continues to be an area of active research
despite the advent of synthetic biodegradable polymers. Natural polymers remain attractive primar-
ily because they are natural products of living organisms, readily available, relatively inexpensive,
and capable of a multitude of chemical modifications (Bogdansky 1990).
Most of the investigations of natural polymers as matrices in drug delivery systems have focused
on the use of proteins (polypeptides or polyamides) as gelatin, collagen, and albumin. Collagens are
the structural building blocks of the body. They are the most abundant group of organic macromol-
ecules in an organism and serve important. Collagen is a major structural protein found in animal
tissues where it is normally present in the form aligned fibers. The main applications of collagen
are collagen shields in ophthalmology, sponges for burns/wounds, minipellets for protein delivery,
and as basic matrices for cell culture systems in bone, tendon, and peripheral nerve repair (Sano
et al. 1998). Collagen can be processed into a number of forms such as sheets, films, tubes, rods,
sponges, powders, injectable solutions, and dispersions. In most drug delivery systems made of
collagen, in vivo absorption of collagen is controlled by the use of cross-linking agents such as glu-
taraldehyde. In order to render collagen suitable for tissue engineering applications, the mechanical
strength of collagen must be enhanced (Friess 1998; Sano et al. 1998; Lee et al. 2001).
Albumin in microspheres form was approved by the U.S. Food and Drug Administration in 1972
for clinical use (Schafer et al. 1994) and has subsequently found many applications in diagnostics
and therapeutics. As a result of this, more than 100 diagnostic agents and drugs have been incor-
porated into albumin microspheres (Dandagi et al. 2006). Noncollagenous proteins, particularly
albumin and to a lesser extent gelatin, continue to be developed as drug delivery vehicles (Adhirajan
et al. 2009; Ohta et al. 2009; Samad et al. 2009). The exploitable features of albumin include its
reported biodegradation into natural products and lack of toxicity. Although many examples on the
use of albumin microspheres were reported in the literature, there are only a few studies describ-
ing gelatin systems. Compared with albumin, gelatin offers the advantages of a good history in
parenteral formulations and lower antigenicity. A biocompatibility study with gelatin microspheres
reported no untoward effects when injected intravenously into mice over a 12 week period.
5.3.2.2 Pseudopoly(Amino Acids)

Pseudopoly(amino acids) belonging to the poly(amides) group represents one of the new and, there-
fore, less advanced biodegradable polymers for medical use. Only a few of them have been synthe-
sized and characterized. Preliminary results showed no gross toxicity or tissue incompatibility upon
subcutaneous implantation for present available pseudopoly(amino acids), and they are actively
investigated in several laboratories for medical applications ranging from biodegradable bone nails
to implantable adjuvant (Kemnitzer and Kohn 1997).
In a biocompatibility study for the cornea bioassay, implantation of four small pieces of the
polymer into four rabbit corneas elicited on pathological response in three of them and a very mild
inflammatory response in one cornea (Yu 1988). Histological examination of the corneas 4 weeks
after implantation showed no invading blood vessels or migrating inflammatory cells in the area
around the implants (Langer et al. 1981). In another biocompatibility study, 10 mg implants of
poly(N-palmitoylhydroxyproline ester) were inserted subcutaneously in mice in the dorsal area of
the animals between the dermis and the adipose tissue layer. The mice responses were examined
up to 1 year post-implantation (Yu 1988). No inflammatory reaction at the implantation site was
observed in any of the mice during the first 7 weeks. A thin but distinct layer of fibrous connective
tissue appeared around the implant by week 14 with a few multinucleated giant cells associated with
the fibrous connective tissue. Animal necropsy performed at 16 and 56 weeks post-implantation
showed no histopathological abnormalities (Yu 1988).
These preliminary biocompatibility results from two animal models, rabbit and mouse, indicate
that the poly(N-palmitoylhydroxyproline ester) elicits a very mild, local tissue reaction typical of
a foreign body response, resulting in the encapsulation of the foreign biomaterial without evidence
for any significant pathological abnormalities. However, additional safety and toxicity tests need
to be performed to evaluate possible systemic toxic effects, allergic reactions, or any mutagenic,
teratogenic, and carcinogenic activities.
Pseudopoly(amino acids) containing aromatic side chains of tyrosine in their backbone struc-
ture were also developed to investigate whether biodegradable polymers that incorporate aromatic
components would combine a high degree of biocompatibility with a high degree of mechanical
strength (Kemnitzer and Kohn 1997). Ertel et al. (1995) prepared a new pseudopoly(amino acid) and
determined the performance of this orthopedic implant material.
Two different types of polyphosphazenes are of interest as bioinert materials: those with strongly
hydrophobic surfaces characteristics and those with hydrophilic surfaces. Polyphosphazenes bear-
ing fluoroalkoxy side groups are some of the most hydrophobic synthetic polymers known. Such
polymers are as hydrophobic as poly(tetrafluoroethylene) (Teflon), but unlike Teflon, polyphospha-
zenes of this type are flexible or elastomeric, easy to prepare, and they can be used as coatings for
other materials.
Biocompatibility and safety testing of these polymers by subcutaneous implantation in animals
have shown minimal tissue response, similar in fact to the response reported with Teflon (Wade
et al. 1978). The connection between hydrophobicity and tissue compatibility has been noted for
classical organic polymers (Lyman and Knutson 1980). Thus, these hydrophobic polyphosphazenes
have been mentioned as good candidates for use in heart valves, heart pumps, blood vessel pros-
theses, or as coating materials for pacemakers or other implantable devices; however, more in vivo
testing and clinical trials are needed.
In their bioerosion reactions, polyphosphazenes display a uniqueness that stems from the pres-
ence of the inorganic backbone, which in the presence of appropriate side groups is capable of
undergoing facile hydrolysis to phosphate and ammonia. The phosphate can be metabolized and the
ammonia excreted. Theoretically, if side groups attached to the polymer are released by the same
process being excretable or metabolizable, then the polymer can be eroded under hydrolytic condi-
tions without the danger of a toxic response. Polyphosphazenes of this type are potential candidates
as erodible biostructural materials for sutures, or as matrices for controlled delivery of drugs (Kohn
and Langer 1987).
Polyphosphazenes containing amino acid ester side groups were the first bioerodible polyphos-
phazenes synthesized (Allcock et al. 1977). They are solid materials which erode hydrolytically
to ethanol, glycine, phosphate, and ammonia. These polymers were tested in subcutaneous tissue
response experiments showing no evidences of irritation, cell toxicity, giant cell formation, or tissue
inflammation (Allcock and Kwon 1986). Imidazoyl groups linked to polyphosphazene chains are
also hydrolyzed very easily showing good biocompatibility (Laurencin et al. 1993).
By changing the molar ratio of hydrophilic and hydrophobic segments, a series of novel amphi-
philic graft polyphosphazenes was synthesized (Qiu and Yan 2009). Doxorubicin was physically
loaded into micelles prepared by an O/W emulsion method. The results of cytotoxicity study showed
that amphiphilic graft polyphosphazenes were biocompatible while doxorubicin-loaded micelles
achieved comparable cytotoxicity with that of free doxorubicin.
5.3.4 Poly(Orthoesters)
Polymers with ester linkages in their main chain comprise a family of polymers with immense diver-
sity and versatility. Biodegradability is sought after in a wide range of applications, above all in the
preparation of environmentally friendly polymers and biomedical materials for temporary surgical
use and in drug delivery (Edlund and Albertsson 2003). As mentioned previously, the Chronomer®
poly(orthoester) material from Alza Corp. or Alzamer has been investigated as bioerodible inserts for
the delivery of the narcotic antagonist naltrexone and for the delivery of the contraceptive steroid nor-
ethisterone. The steroidal implant was tested in two separate human clinical trials causing local tissue
irritation, and therefore further work with this formulation was discontinued (Solheim et al. 1995).
The reasons for the local irritation were never properly elucidated. New types of POEswere devel-
oped but no data on the biocompatibility and safety of these materials were reported.
5.3.5 Polyanhydrides
Polyanhydrides are a novel class of biodegradable polymers under development as vehicles for the
release of bioactive molecules including drug peptides and proteins. A series of biocompatibility
studies reported on several polyanhydrides have shown them to be nonmutagenic and nontoxic
(Leong et al. 1986). In vitro tests measuring teratogenic potential were also negative. Growth of two
types of mammalian cells in tissue culture was also not affected by the polyanhydride polymers
(Leong et al. 1986); both the cellular doubling time and cellular morphology were unchanged when
either bovine aorta endothelial cells or smooth muscle cells were grown directly on the polymeric
substrate.
Subcutaneous implantation in rats of high doses of the 20:80 copolymer of bis-(p-carboxy-
phenoxy)propane (CPP) and sebacic acid (SA) for up to 8 weeks indicated relatively minimal
tissue irritation with no evidence of local or systemic toxicity (Laurencin et al. 1990). Since
this polymer was designed to be used clinically to deliver an anticancer agent directly into the
brain for the treatment of brain neoplasms, its biocompatibility in rat brain was also studied
(Tamargo et al. 1989). The tissue reaction of the polymer was compared to the reaction observed
with two control materials used in surgery, oxidized cellulose absorbable hemostat (Surgicel®,
Johnson and Johnson) and with absorbable gelatin sponge (Gelfoam®, Upjohn). The inflam-
matory reaction of the polymer was intermediate between the controls (Tamargo et al. 1989).
A closely related polyanhydride copolymer poly(CPP)-SA 50:50 was also implanted in rabbit
brains and was found to be essentially equivalent to Gelfoam in terms of biocompatibility evalu-
ations (Brem et al. 1989). In a similar study conducted in monkey brains, no abnormalities were
noted in the CT scans and magnetic resonance image, nor in the blood chemistry or hematology
evaluations (Brem et al. 1988a). No systemic effects of the implants were observed on histologi-
cal examinations of any of the tissues tested (Brem et al. 1988b).
Different classes of polyanhydrides have been synthesized and are undergoing extensive pre-
clinical testing, including a wide range of biocompatibility studies. Examples of these new materi-
als are polymers of sebacic acid poly(SA), and copolymers 1:1 of SA with fatty acid dimer (FAD),
fumaric acid (FA) and isophthalic acid poly(FAD:SA), poly(FA:SA), poly(ISO:SA). These materials
were implanted in rabbits intramuscularly, subcutaneously and in the cornea, and ocular and muscle
irritation studies were performed compared to the material controls Gelfoam, Surgicel, and Vycryl®
a synthetic absorbable suture (Ethicon) (Rock et al. 1991). Detailed observations of toxicity, bleed-
ing, selling, or infection of the implantation site were conducted daily. At the end of the study, the
animals were sacrificed and a gross necropsy examination of the tissues surrounding the implant
site was performed. No significant clinical signs or abnormalities of the incision sites were observed
during the study period (4 weeks). No meaningful differences could be seen in reaction between the
various polymer implants tested and the control materials (Rock et al. 1991).
In the rabbit cornea bioassay, no evidence of inflammatory response was observed with any of
the implants at any time. On an average, the bulk of the polymers disappeared completely between
7 and 14 days after the implantation (Rock et al. 1991). The cornea is a very sensitive indicator of
inflammatory reactions (Aronson and Horton 1971; Henkind 1978). The rabbit cornea possesses
clear advantages over other implant sites for studying implant–host interactions, due to the easy
accessibility for frequent observations without having to gain surgical access to the implantation
site; the transparency and avascularity of the cornea also enable the observer to distinguish among
the different inflammatory characteristics such as edema, cellular infiltration, and ingrowths of
blood vessels from the periphery of the cornea or neovascularization, which are strong indications
that the biomaterial under testing is unsuitable for implantation (Langer et al. 1981).
In similar animal experiments in which polyanhydride matrices containing tumor angiogenic
factor (TAF) were implanted in rabbit cornea, a significant vascularization response was observed
without edema or white cells. Moreover, and most importantly from the biocompatibility stand-
point, polymer matrices without incorporated TAF showed no adverse vascular response (Langer
et al. 1981, 1985).
The biocompatibility of polyanhydrides based on ricinoleic acid as compared to Vicryl™ surgi-
cal suture and sham surgery was tested in rats (Teomim et al. 1999). No evidence for tissue necrosis
was detected in any of the treated animals upon evaluation of the tissues 21 days post-implantation.
No indication for post-implantation test site contamination was noted. Generally, in all tested
groups there was a clear indication of time-related healing process (i.e., time-related reduction in
the incidence and severity of necrosis and acute to subacute inflammatory reaction, associated with
increased fibroplasia). The tissue reaction in the different treatment groups showed a clear trend of
healing with time. In particular, comparison of the tissue reactivity, along the three different time
periods (3, 7, and 21 days), indicated that the only noted remnants of tissue reaction at the 21 day
time period were minimal subacute inflammation and mild fibrosis. In comparison, under the same
conditions, Vicryl™ implant induced minimal fibrosis associated with the presence of minimal
quantity of giant cells and encapsulated foreign material.
Based on the biocompatibility and safety preclinical studies carried out in rats (Tamargo
et al. 1989; Laurencin et al. 1990), rabbits (Brem et al. 1989), and monkeys (Brem et al. 1988a,b)
reviewed here showing quite acceptability of the polyanhydrides for human use, a Phase I/II
clinical protocol was instituted (Domb et al. 1991a). In these clinical trials, a polyanhydride dos-
age form (Gliadel®) consisting of wafer polymer implants of poly(CPP-SA) 20:80 and containing
the chemotherapeutic agent Carmustine (BCNU) were used for the treatment of glioblastoma
multiforme, a universally fatal form of brain cancer. In these studies, up to eight of these wafer
implants were placed to line the surgical cavity created during the surgical debulking of the brain
tumor in patients undergoing a second operation for surgical debulking of either a Grade III or
IV anaplastic astrocytoma. In keeping with the results of the earlier preclinical studies suggest-
ing a lack of toxicity, no central or systemic toxicity of the treatment was observed during the
course of treating 21 patients under this protocol. Phase III human clinical trials have demon-
strated that site-specific delivery of BCNU from a P(CPP:SA)20:80 wafer (Gliadel®) in patients
with recurring brain cancer (glioblastoma multiforme) significantly prolongs patients survival
(Brem et al. 1995). Gliadel® finally won approval from the FDA adjust therapy for the treatment
of brain tumors.
Biocompatibility of an injectable gelling polymeric device for the controlled release of gentami-
cin sulfate in the treatment of invasive bacterial infections in bone of male Wister rats. The biode-
gradable delivery carrier, poly(sebacic-co-ricinoleic-ester-anhydride), designated as p(SA:RA), was
injected, with and without gentamicin, into the tibial canal. Rats were killed 3 weeks later and the
tibiae were processed histologically. The local tissue reaction to the polymer with or without anti-
biotic consisted mainly of mild reactive fibroplasia/fibrosis and mild to moderate increased reactive
bone formation. No evidence for any active inflammatory response to the polymer were observed;
thus, the injection of p(SA:RA) was well tolerated (Brin et al. 2009).
Low molecular weight hydroxy fatty acid based polyanhydrides were synthesized by Jain et al.
Control over drug release was accessed with drugs featuring different aqueous solubility, i.e., meth-
otrexate (hydrophobic) and 5-fluorouracil (hydrophilic). Due to their low melting temperatures, they
can be injected locally (SC or intratumorally) to from regional in situ depot and have a great poten-
tial as a drug carrier for localized delivery of anticancer drugs. These polymers were found good
to control the release of the entrapped drug and were found biocompatible in a preliminary in vivo
study (Jain et al. 2008).
Cell–polymer interactions in subcutaneous and bony tissue were examined for a novel class of
in situ forming and surface eroding polyanhydride networks. Specifically, photopolymerized disks
of several polyanhydride compositions were implanted subcutaneously in rats, and the tissue was
analyzed for an inflammatory response (Poshusta et al. 2003).
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6 Bioresorbable Hybrid
Membranes for Bone
Regeneration
Akiko Obata and Toshihiro Kasuga
CONTENTS
6.1 Introduction........................................................................................................................... 177
6.2 Siloxane-Doped Poly(l-Lactic Acid)/Calcium Carbonate (Vaterite) Composites (SiPVC)......178
6.2.1 Preparation of the Composites and bHA-Coating..................................................... 178
6.2.2 Cellular Compatibility............................................................................................... 179
6.3 Siloxane-Doped Vaterite Particle and Its Composites with Poly(l-Lactic Acid).................. 182
6.3.1 Preparation of Siloxane-Doped Vaterite Particles..................................................... 182
6.3.2 Preparation of Siloxane-Doped Vaterite/Poly(l-Lactic Acid) Composite Films........ 184
6.3.3 Cellular Compatibility of SiV-PLLA Composites..................................................... 185
6.4 Siloxane-Doped Vaterite/Poly(l-Lactic Acid) Composite Microfiber Membranes............... 186
6.4.1 Preparation of SiV-PLLA Microfiber Membranes.................................................... 187
6.4.2 Tissue Compatibility and Bone-Forming Ability of the Membrane......................... 190
6.5 Summary............................................................................................................................... 190
References....................................................................................................................................... 191
6.1 INTRODUCTION
Bioresorbable materials have been used as implants for bone reconstruction in clinical field. The
ideal implant materials should fulfill several criteria. The materials are basically required to have
biocompatibility (nontoxicity) and bioactivity. Bioactivity is regarded as an ability of bonding to the
host bone directly in body in the research field of implant materials. Various types of bioceramics
have been developed as the implant materials, particularly calcium phosphates have been reported
to show an excellent bioactivity. In fact, bioresorbable polymer and calcium phosphate compos-
ite materials also have been investigated to be applied to the implant materials enhancing bone
formation.
Poly(l-lactic acid) (PLLA) is one of the biodegradable polymers and has been applied clinically.
Bos et al. reported tissue reactions to PLLA implanted subcutaneously in the backs of rats (Bos et al.
1991). In the report, PLLA showed the degradation pattern of the biodegradable polymers during
26 weeks and no acute or chronic inflammatory reaction to PLLA was observed during 143 weeks
of implantation, except for the early part implant period. PLLA has been investigated for application
to scaffold materials for bone reconstruction (Mainil-Varlet et al. 1997). PLLA–ceramics compos-
ites were developed for bone reconstruction and showed good mechanical and bone-forming prop-
erties (Kasuga et al. 2003, Zhang et al. 2004, Kim et al. 2006). Calcium carbonate, for example, is
well known as a bioresorbable material, shows excellent bone-forming ability, and has been used
clinically as a bone filler (Liao et al. 2000, Vago et al. 2002). PLLA/calcium carbonate (vaterite)
177
composites showed excellent hydroxyapatite (HA)-forming ability, cellular compatibility, and bio-
compatibility by in vitro and in vivo tests (Maeda et al. 2004, 2006).
Silicon species is one of the essential, trace elements for healthy bone and has been recently
reported to stimulate functions of osteogenic cells (Carlisle 1986, Xynos et al. 2000). This has
been supported by the reports about bioactive glasses, such as Bioglass® (45% SiO2, 24.5% CaO,
24.5% Na2O, 6% P2O5). Ionic dissolution products released from Bioglass stimulated osteogenic
cells at the genetic level. Xynos et al. reported that some gene expressions in human osteoblasts
were stimulated by silicon species in a culture medium, derived from Bioglass. They also reported
that the expression of insulin-like growth factor II in human osteoblasts was enhanced by silicon
species in the culture medium, which stimulated the cells to proliferate. Reffitt et al. reported that
orthosilicic acid in a culture medium was effective to stimulate collagen type I synthesis and osteo-
blastic differentiation in human osteoblast-like cells (Reffitt et al. 2003).
Human mesenchymal stem cells (MSCs) and osteoblasts (HOBs) are one of the important and
useful cells in the field of bone tissue engineering. MSCs are capable of differentiating in vitro,
at the minimum, into the bone, cartilage, fat, and mature stromal cell lineages. When MSCs are
cultured on the surfaces of a ceramic block and a diffusion chamber, they can exhibit osteogenic
potential resulting in bone formation in vivo. MSCs have been reported to differentiate into osteo-
blasts and undergo mineralization when they are cultured in the presence of ascorbic acid (AsA),
β-glycerophosphate (βGP), and dexamethasone (Dex). These supplements stimulate HOBs to min-
eralize as well. AsA promotes synthesis and accumulation of type I collagen. βGP is used as an
exogenous phosphate source for the cells to synthesize mineralized material. Dex is used to induce
proliferation, osteoblast maturation, and extracellular matrix mineralization. Jaiswal et al., how-
ever, reported that MSCs showed osteogenic differentiation in Dulbecco’s modified Eagle’s medium
(DMEM) containing AsA, Dex, and βGP; moreover, the addition of AsA to MSCs in the absence
of Dex and βGP did not cause osteogenic differentiation of the cells (Jaiswal et al. 1997). We used
DMEM containing serum, antibiotics (penicillin/streptomycin), and AsA as the culture medium for
in vitro tests using MSCs or HOBs to establish whether the addition of commonly used osteogenic
factors is necessary for osteogenesis on our composite materials. Gough et al. reported that HOBs
in medium unsupplemented Dex and βGP was stimulated to mineralize in response to culture on a
bioactive glass in the SiO2-CaO-P2O5 system and also in response to dissolution products, silicon
and calcium species, from the glass (Gough et al. 2004). Jones et al. reported the mineralization
of HOBs was stimulated by culturing an SiO2-CaO bioactive glass in a medium unsupplemented
osteogenic factor (Jones et al. 2007). This chapter shows various types of PLLA/vaterite compos-
ites releasing silicon and calcium species that we have developed so far. The composites have been
found to have good HA-forming ability and cellular and tissue compatibilities.
6.2 SILOXANE-DOPED POLY(l-LACTIC ACID)/CALCIUM

CARBONATE (VATERITE) COMPOSITES (SiPVC)
Biodegradable composite materials releasing silicon and calcium species were prepared by mix-
ing silane coupling agent, aminopropyltrietoxysilane (APTES), into PLLA. We hypothesized it is
possible to get the materials silicon species are homogeneously mixed in by using APTES as it has
active sites for making hybrids with polymers. Silicon species were expected to be released from the
composite materials by the PLLA degradation. A composite containing no silicon species, PLLA/
vaterite (PVC), was prepared to clarify effects of silicon species on cell functions by comparing cell
behaviors on between the two types of composite materials.
6.2.1 Preparation of the Composites and bHA-Coating

SiPVC was prepared using PLLA (molecular weights, Mw; = 160 ± 20 kDa) dissolved in methy-
lene chloride and aminopropyltrietoxysilane (APTES; Si(OC2H2)3(CH2)3NH2) hydrolyzed through
Bioresorbable Hybrid Membranes for Bone Regeneration 179
(a) (b)
1 μm 1 μm
FIGURE 6.1 Surface morphologies of (a) SiPVC and (b) SiPVC+. SiPVC+ means SiPVC after soaking in
SBF for 3 days to coat its surface with bone-like hydroxyapatite. Arrows indicate vaterite particles with about
500 μm in diameter.
addition of carbodiimide and ammonium solution. The PLLA solution and the APTES solution
were mixed in weight ratio of PLLA:APTES = 1:0.67, and then added calcium carbonate (vaterite)
in weight ratio of PLLA:vaterite = 1:0.5. SiPVC and PVC coated with bone-like HA (bHA) (denoted
by SiPVC+ and PVC+, respectively) were prepared through immersion of the film in simulated body
fluid (SBF) for 3 days. The bHA is deposited on some bioactive materials after immersing in SBF.
SBF is a tris-buffer solution (pH 7.4) containing inorganic ions almost equal to those of human
plasma (Kokubo et al. 1990). The bHA formed in SBF has a leaf-like crystalline shape, and there-
fore can ensure a large surface area for cellular spreading.
Figure 6.1 shows the surface morphologies of SiPVC and SiPVC+. Many micropores of 0.1–1 μm
in diameter and vaterite particles of 0.5 μm in diameter (pointed by arrows in Figure 6.1a) were
observed on SiPVC surface. On the other hand, leaf-like bHA crystals were deposited on SiPVC;
the surface was completely covered with bHA. It has been observed that the thickness of SiPVC film
and the bHA coating was about 10 and ∼3 μm, respectively, and the bHA coating closely adhered
to SiPVC surface (Maeda et al. 2006). The bHA on SiPVC has been confirmed to contain silicon
species. The amount of silicon species in the bHA has been estimated to be 2.1 at% with an energy
dispersive spectrometer (EDS).
6.2.2 Cellular Compatibility

Osteoblast-like cells (MC3T3-E1 cells) from rat calvaria were seeded onto SiPVC+ or PVC+
sterilized using ethylene oxide gas in a 24-well plate at a density of 60,000 cells per well. Alpha-
modified minimum essential medium containing 10% fetal bovine serum (FBS) was used as the
culture medium and changed every 2 days. Cells were cultured at 37°C in a humidified incubator
with 5% CO2, harvested after 1, 3, and 5 days using 0.1% actinase in PBS, and counted by a hemo-
cytometer. Figure 6.2 shows the cellular numbers of MC3T3-E1 cells on SiPVC+ and PVC+ in a
24-well plate after incubation. The cells satisfactorily proliferated on both sample surfaces, and the
cellular numbers on SiPVC+ were significantly higher than that on PVC+.
HOBs were seeded on SiPVC and PVC at a density of 10,000 cells per well, and then cultured
for 21 days using medium, which is a Dulbecco’s modified Eagles medium (DMEM) containing
10% FBS with 0.05 mM l-ascorbic acid-2-phosphate (AaAP) and 2% penicillin/streptomycin.
The surfaces of dried samples were analyzed by laser Raman spectroscopy (LRS) and observed
by field emission scanning electron microscopy (SEM) after coated with osmium. Figure 6.3
shows SEM micrographs of HOBs cultured on SiPVC+ and PVC+ for 21 days. Figure 6.3a shows
an image of HOBs cultured on SiPVC+. The cells proliferated prosperously and appeared to
elongate and formed rough shape and some agglomerates. Figure 6.3b shows that the cells cul-
tured on PVC+. The cells appeared to elongate and aligned on the surface. No agglomerate was
40
35 PVC+
SiPVC+
30
Cell number (×104)

25
20
15
10
0
0 1 2 3 4 5 6
Culture time (/day)
FIGURE 6.2 Numbers of MC3T3-E1 cells on SiPVC+ and PVC+ after 1, 3, and 5 day culturing. Cellular
proliferation on SiPVC+ was better (*p < 0.05).
(a) (b)
1 μm 1 μm
PO4
SiPVC+ C–C
CO3 C–C
Intensity (a.u.)
C–O C–O
C–C–O
PVC+
1200 1100 1000 900

(c) Raman shift (/cm–1)
FIGURE 6.3 Results of culture tests using HOBs on SiPVC+ and PVC+. SEM micrographs of HOBs cul-
tured on (a) SiPVC+ and (b) PVC+ for 21 days. (c) LRS spectra of (a) and (b).
observed in the cells. Figure 6.3c shows the LRS spectra of SiPVC+ and PVC+ surfaces after
21 days of HOBs culturing. A peak corresponding to orthophosphate appeared at approximately
965 cm−1 on each spectrum. The peak on the spectrum of SiPVC+ was higher in intensity and
sharper in form than that of PVC+.
Bone nodule containing minerals forms after the maturation of extracellular matrix and the
agglomeration of HOBs. Although no agglomerate was observed on the PVC+ surface after HOBs
culturing, some agglomerates were observed on the SiPVC+ surface as shown in Figure 6.3a. In
the result of LRS, the intensity of PO4 peak on the spectrum of SiPVC+ after HOBs culturing was
higher and sharper than that on the spectrum of PVC+. The strong and sharp PO4 peak on the LRS
spectrum of SiPVC after culturing is expected to originate from calcium phosphate precipitation
in HOBs. According to the results of LRS, the agglomerates observed on the SiPVC+ surface are
regarded to be bone nodules. The bone nodule formation in HOBs was enhanced on the SiPVC+
surface. It was reported that the bone nodule formation was enhanced on some bioactive glasses
containing silica (Gough et al. 2004, Jones et al. 2007). It was also reported that calcium phosphate
significantly precipitated in HOBs cultured on silicon-substituted HA (Thian et al. 2006). The effect
of a trace amount of silicon species released from SiPVC or contained in bHA layer formed on
SiPVC was expected to enhance the mineralization of HOBs.
MSCs are capable of differentiating in vitro into various tissues when placed in an appropriate
environment. ALP activity in MSCs is known to increase during osteogenic differentiation. Jaiswal
et al. reported that MSCs showed osteogenic differentiation in DMEM containing AsAP, Dex, and
βGP, and that the addition of AsAP to MSCs in the absence of Dex and βGP did not cause osteo-
genic differentiation of the cells (Jaiswal et al. 1997). MSCs were seeded onto SiPVC+ and PVC+
in 24-well plates, at a density of 10,000 cells per well. The seeded cells were incubated in DMEM
supplemented with only AsAP, not Dex nor βGP. Table 6.1 shows ALP activity and cell number of
MSCs cultured on SiPVC+ and PVC+ for 21 days. Although the level of ALP activity in MSCs cul-
tured on PVC+ remained unchanged, that on SiPVC+ significantly increased for 21 day culturing.
The cells cultured on SiPVC+ showed larger number than that on PVC+ at all time points. The cell
proliferation was enhanced on SiPVC+.
There are few reports showing the effects of silicon species on the activity of osteogenic
differentiation of MSCs. Reffitt et al. reported that the collagen type 1 synthesis in MSCs was
enhanced in the medium containing 10–50 μM of orthosilicic acid (Reffitt et al. 2003). The
collagen type 1 synthesis by preosteoblasts or early undifferentiated osteoblast-like cells is
regarded to represent bone formation (Boskey et al. 1999). The ALP activity in MSCs increased
on SiPVC+; the osteogenic differentiation of MSCs was induced by the silicon species included
in/released from SiPVC+.
The effects of silicon species on the cellular activities have been mainly reported to enhance
the HOBs mineralization and bone formation. Silicon species also stimulate the MSC activity;
especially they induce the osteogenic differentiation of MSCs without supplementing the
TABLE 6.1
ALP Activity and Cell Number of MSCs Cultured
on SiPVC+ and PVC+ for 14 and 21 Days
14 Days 21 Days
ALP Number ALP Number
PVC+ 2.89 ± 0.0202 2.07 ± 0.0822 2.98 ± 0.115 3.61 ± 0.975
SiPVC+ 3.38 ± 0.250 6.59 ± 2.95 4.13 ± 0.125* 11.1 ± 1.35*
Unit ALP: p-nitrophenol nmol/min, number: ×104.

*p < 0.05, comparing the value between PVC+ and SiPVC+ on each stage.
osteogenic factors (Dex and βGP) in a culture medium. The mechanism for the effects of silicon
species on the MSCs activity has not been clarified yet. Some gene-expressions in HOBs were
reported to change by the addition of a trace amount of silicon species in the culture medium
(Xynos et al. 2001). Therefore, a change in gene-expression is believed to occur in MSCs cul-
tured on SiPVC+, which originates the stimulation to the proliferation and the osteogenic dif-
ferentiation of MSCs.
6.3 SILOXANE-DOPED VATERITE PARTICLE AND ITS

COMPOSITES WITH POLY(l-LACTIC ACID)
We have developed siloxane and PLLA hybrid materials and also successfully prepared the materi-
als with film or hollow sphere shape, as aforementioned. The matrix of the material consists of a
hybrid between amino groups in APTES and carboxyl groups in PLLA. The structure of the matrix
may be seriously changed by changing the amount of APTES; therefore, it is difficult to drastically
increase the silicon releasability of the material. It was assumed that vaterite particles containing
siloxane should be very useful to prepare composite materials releasing silicon species continuously
and controllably using various types of biodegradable materials.
A novel composite consisting of PLLA and calcium carbonate particles (vaterite) doped with sili-
con species was developed. The materials were successfully prepared using silane coupling agents
by a carbonation process. It was expected that silicon species were gradually released from the
composites through the degradation of PLLA matrix and vaterite and that the composites were able
to show the chronic effects of silicon species on cellular activities.
6.3.1 Preparation of Siloxane-Doped Vaterite Particles

Silicon-doped vaterite (SiV) powders were prepared by a carbonation process with methanol.
A sample of 150 g of Ca(OH)2 was mixed with 30 or 60 mL of aminopropyltriethoxysilane (APTES)
and 2000 mL of methanol with adding CO2 gas for 75 min at a rate of 2000 mL/min. The resulting
slurry was dried at 110°C, resulting in the SiV powders. The amount of silicon in SiV prepared using
30 and 60 mL of APTES was estimated to be approximately 1 and 3 wt%. SiV containing 1 and
3 wt% of silicon are hereafter denoted by SiV1 and SiV3, respectively.
Figure 6.4 shows SEM photographs of the SiV3 particles. The SiV3 particles have diameters
of ∼1 μm. These two types of particles consist of aggregates of nano-sized primary particles. The
sizes of SiV particles are larger than that of vaterite. The results of the nitrogen gas adsorption
method reveal that the specific surfaces of SiV3 and vaterite are 67.2 and 30.7 m 2/g, respectively.
1 μm
FIGURE 6.4 SEM photographs of SiV3 particles.

Intensity (a.u.)
20 25 30 35
(a) Diffraction angle, 2θ/ ° (CuKα)
T3
T2
Intensity (a.u.)
50 0 –50 –100 –150

(b) Chemical shift/ppm
FIGURE 6.5 Characterization of SiV particles: (a) XRD pattern and (b) 29Si MAS-NMR spectrum. SiV
consists predominantly of vaterite phase. No APTES exists as its monomer and siloxane was formed in SiV.
These results suggest that the conditions for the aggregation of the primary particles in the sus-
pension are influenced by adding APTES. Figure 6.5a shows the x-ray diffraction (XRD) pat-
tern of SiV. SiV consists predominantly of vaterite. The structure of APTES in SiV was further
characterized by 29Si magic angle spinning nuclear magnetic resonance (29Si MAS-NMR) spec-
troscopy. Figure 6.5b shows the 29Si MAS-NMR spectrum of SiV. A peak at −50 to −80 ppm is
seen. The peak may be superimposed by those corresponding to T 2 [NH 2(CH 2)3Si(OSi)2 (OH,
OC2H 5)] and T3 [NH 2(CH 2)3Si(OSi)3] (Yabuta et al. 2000). This indicates that no APTES in SiV
exists as its monomer, and siloxane was formed by the condensation of APTES through the
preparation of SiV.
6.3.2 Preparation of Siloxane-Doped Vaterite/Poly(l-Lactic Acid) Composite Films

A solution of PLLA (molecular weights, Mw = 160 ± 20 kDa) prepared by dissolving 1.5 g of PLLA
in 20 mL of methylene chloride was used to disperse portions of 0.75 g of the vaterite or SiV powders
under stirring for 30 min, resulting in the as-prepared composite. The composites prepared using
vaterite, SiV1 and SiV3 were denoted by V-PLLA, SiV1-PLLA, and SiV3-PLLA, respectively. The
composite films showed high flexibility. The surface observation of the film by SEM revealed that
the vaterite or SiV particles dispersively existed in PLLA.
For estimation of the silicon releasabilities of SiV1-PLLA and SiV3-PLLA, silicon concentra-
tions in α-minimum essential medium (α-MEM) containing 10% FBS after soaking SiV1-PLLA
and SiV3-PLLA were evaluated by inductively coupled plasma atomic emission spectroscopy
(ICP-AES). The medium was changed at days 1, 3, and 5 after soaking the sample and the silicon
concentration in the harvested medium was evaluated. Figure 6.6 shows the silicon concentra-
tion in the culture medium after soaking SiV1-PLLA and SiV3-PLLA. Both the samples showed
silicon releasability for 7 days of soaking in the culture medium. SiV1-PLLA and SiV3-PLLA
release approximately 2.2 and 9.2 mg/L of silicon ions, respectively, within 1 day of soaking.
The average value of the silicon released from SiV1-PLLA for 2–7 days of the incubation was
estimated to be 0.21 mg/L/day, while that of SiV3-PLLA was 0.51 mg/L/day. The SiV particles
existing on the composite sample surfaces are expected to be exposed to the culture medium
and then immediately release ionic silicon species to the medium during the first day of the
incubation. From day 2, the amounts of the released silicon species in both the samples sig-
nificantly decreased. The amount of silicon species released from SiV1-PLLA and SiV3-PLLA
decreased from 2.2 and 9.2 to 0.74 and 1.3 mg/L, respectively, at day 3. The releasing mechanism
of the silicon species from day 2 may be different from that within day 1. From day 2, the SiV
particles embedded in the PLLA matrix are gradually exposed to the culture medium through
the PLLA degradation and then release a trace amount of silicon species to the culture medium.
The silicon species release depends on the PLLA degradation from day 2 of incubation. The
molecular weight of PLLA was reported to decrease after implanted in body and the degrada-
tion speed of PLLA depends on its molecular weight (Kenawy et al. 2002). There was no sig-
nificant difference in Mw of the matrix PLLA among all of the composites. The Mw of V-PLLA,
14
SiV1-PLLA
12 SiV3-PLLA
10
Silicon amount (ppm)
0
0 1 2 3 4 5 6 7 8
Time (day)
FIGURE 6.6 Silicon concentration in the culture medium after soaking SiV1-PLLA or SiV3-PLLA. The
medium was changed at days 1, 3, and 5 after soaking of the sample and the total concentration in the medium
harvested so far was evaluated.
SiV1-PLLA, and SiV3-PLLA were 189, 179, and 175 kDa, respectively. Therefore, the difference
in the released amount of the silicon species between SiV1-PLLA and SiV3-PLLA is suggested
to originate from the amount of APTES in SiV, not the speed of the degradation of the PLLA
matrix. This indicates that the amount of silicon species released from SiV-PLLA composites is
controllable by changing the amount of APTES in SiV. That is, to control the APTES content
in SiV particles is regarded to be one of the most effective methods for controlling the silicon
releasability of SiV-PLLA. In fact, the amount of the silicon species released from SiV3-PLLA
per day was approximately 2.4 times as large as that released from SiV1-PLLA during day 2–7
of incubation. The value is evidently related to the difference in the amount of silicon species
between SiV1 and SiV3.
6.3.3 Cellular Compatibility of SiV-PLLA Composites

The cellular proliferations on two types of the siloxane-containing samples were examined to
investigate the effects on the cells of the silicon species amount in SiV-PLLA. Mouse osteo-
blast-like cells (MC3T3-E1 cells) were seeded onto the prepared samples in 24-well plates,
at a density of 30,000 cells per well. The α-MEM containing 10% FBS was used as a culture
medium. The medium was changed after 1 day of culturing and then changed every other day.
Figure 6.7 shows the number of cells cultured on SiV1-PLLA and SiV3-PLLA for 7 days. The
number of cells cultured on SiV3-PLLA is significantly higher than that on SiV1-PLLA at days
3, 5, and 7. The proliferation of MC3T3-E1 cells cultured on SiV3-PLLA, which released a
larger amount of silicon species, was higher than that on SiV1-PLLA. On the other hand, the
effects of the silicon species on the cellular functions may relate to their ionic states. The ionic
state of the silicon species released from SiV-PLLA has been not found yet. Further works to
clarify the ionic state and the correlation between the state and its effect on the cellular func-
tions are needed.
For estimating alkaline phosphatase (ALP) activity in MC3T3-E1 cells, they were seeded
onto the samples in 24-well plates, at a density of 10,000 cells per well. The culture medium
contained no differentiation agent. A Thermanox® plastic plate was used as a control sample.
40
SiV1-PLLA
35
SiV3-PLLA
30
Cell number (×104)
25
20
15
10
0
0 1 2 3 4 5 6 7 8
Culture time (day)
FIGURE 6.7 Numbers of MC3T3-E1 cells cultured on SiV1-PLLA or SiV3-PLLA for 7 days. The prolifera-
tion of MC3T3-E1 cells cultured on SiV3-PLLA, which released a larger amount of silicon species, was higher
than that on SiV1-PLLA (*p < 0.05).
8
V-PLLA
7
SiV3-PLLA
(p-nitrophenol μmol/min/104 cell)

Thermanox
6
ALP activity
4
0
6 8 10 12 14 16 18 20 22
Culture time (day)
FIGURE 6.8 ALP activities in MC3T3-E1 cells cultured on V-PLLA, SiV3-PLLA, and Thermanox®. The
levels of the activities of V-PLLA and Thermanox are almost same at all time points although those of SiV3-
PLLA increased with prolongation of the culture time (*p < 0.05).
Figure 6.8 shows the ALP activities in MC3T3-E1 cells cultured on V-PLLA, SiV3-PLLA, and
Thermanox. The activity levels of these samples increase during 21 days of culturing. In the
case of V-PLLA and Thermanox, the levels of the activities are approximately 0.1–0.6 at all
time points. On the other hand, the levels of SiV3-PLLA are approximately 1.2 at day 14 and
approximately 5.3 at day 21. These values of SiV3-PLLA are significantly higher than those of
V-PLLA at days 14 and 21. This indicates that the silicon species released from SiV3-PLLA
induced the differentiation of MC3T3-E1 cells.
6.4 SILOXANE-DOPED VATERITE/POLY(l-LACTIC ACID)

COMPOSITE MICROFIBER MEMBRANES
Three-dimensional scaffolds are common in biomaterials. Electrospinning is a process that can
generate polymer fiber meshes with high porosity and flexibility, both essential components of tis-
sue engineering (Still and von Recum 2008). In general, the process of electrospinning is mainly
affected by both system parameters and process parameters. System parameters include polymer
molecular weight (Mw) and polymer solution properties such as viscosity and conductivity. Process
parameters, on the other hand, involve flow rate of the polymer solution, electric potential, and the
distance between capillary and collector, among others (Still and von Recum 2008). It is possible to
control the fiber diameter and the thickness of the fiber mesh scaffolds by optimizing these param-
eters. To use electrospun fiber meshes in bone tissue engineering applications, their various proper-
ties, including material, fiber orientation, porosity, and surface modification, must be considered. To
optimize the mechanical and biomimetic properties of the fiber mesh, it is necessary to choose the
appropriate materials, since the orientation and diameter of the fibers in electrospun scaffolds can
influence cellular compatibility (Li et al. 2002, Zeng et al. 2003, Yang et al. 2005, Bandami et al.
2006, Xin et al. 2007). The fiber diameter in electrospun scaffolds, for example, has been reported
to influence osteoblast proliferation and mineralization.
There has been increasing interest in the use of electrospun fibrous meshes as scaffolds in
medicine. In particular, biocompatible polymers, such as PLGA and poly(caprolactone), have been
applied as materials for the meshes, incorporated with various growth factors or DNA (Still and von
Recum 2008). On the other hand, very few studies have been performed on fibrous meshes consist-
ing of polymer/ceramics composites. Previously, PLGA and HA composite scaffolds, which were
fabricated by electrospinning, contained only 5–10 wt% of HA nanocrystals (Nie and Wang 2007).
A larger amount of calcium is needed to enhance the bone-forming ability of the electrospun fibrous
mesh, and vaterite powders are effective materials for providing calcium.
The electrospun fiber mesh would contain a large volume of pores and would show flexibility, both
essential components of biomaterials, such as bone fillers. The fibrous mesh containing a large amount
of vaterite (up to 60 wt%) has been successfully prepared by kneading and subsequent electrospinning.
6.4.1 Preparation of SiV-PLLA Microfiber Membranes

The microfiber meshes were prepared by an electrospinning method. SiV powders were mixed
with PLLA (PURASORB®; Mw; 260 kDa, PURAC, the Netherlands) under 200°C for 10 min with
a kneader, resulting in the formation of the PLLA composites containing 60 wt% of the powders.
The composites were dissolved in chloroform at 10 wt% to prepare the solution for electrospinning.
Figure 6.9a and b shows morphologies of the SiV-PLLA-mesh. Microfibers approximately 10 μm
in diameter were observed at the SiV-PLLA-mesh surface and were found to twine one another. The
size of gaps between the microfibers varied between 10 μm and several hundred micrometers, as
shown in Figure 6.9a. The SiV particles observed at the microfiber surface were coated with a thin
film of the PLLA matrix, as shown in Figure 6.9b. The particles were found to be packed closely in
the microfiber from the fracture face of the microfiber. On the other hand, the V-PLLA mesh con-
sisted of short microfibers approximately 7 μm in diameter. The prepared SiV-PLLA mesh showed
flexibility without breaking, whereas the V-PLLA mesh was brittle and was easily broken by hand.
The prepared microfiber mesh was immersed in 10 mL of an aqueous solution containing ion
concentrations 1.5 times those of SBF (1.5 SBF) at 37°C for 1 day. Figure 6.9c and d shows mor-
phologies of the mesh after soaking. The particles were embedded in the PLLA matrix and after
the immersion in 1.5 SBF for 1 day, the surfaces of the microfibers were coated with leaf-shaped
deposits as shown in Figure 6.9d. No significant change was observed in the gap sizes between the
(a) (b)
200 μm 5 μm
(c) (d)
200 μm 5 μm
FIGURE 6.9 Morphologies of the SiV-PLLA-mesh (a and b) before and (c and d) after soaking in 1.5SBF for
1 day. The fibers have about 10 μm of diameter. The SiV particles are observed at the microfiber surfaces. After
soaking, the surfaces of the microfibers were coated with leaf-shaped deposits, bone-like hydroxyapatite (bHA).
microfibers before and after the immersion. After the bHA coating, further testing demonstrated
several cracks, but the coating did not peel off. The deposits were confirmed by XRD to be HA of
approximately 1 μm in thickness. The release of silicon ion from SiV-PLLA mesh coated with bHA
into the culture medium was evaluated over 5 days. The amount of the silicon species released from
it was 0.68 mg/L after 1 day of soaking in the culture medium. The sample kept releasing the spe-
cies; the amount in the culture medium was 0.39 mg/L at day 3 and 0.41 mg/L at day 5.
The flexibility of the mesh was felt to be a reflection of its structural properties such as the molecular
weight of the PLLA matrix and structural changes in the interface between the matrix and the SiV
powders. The structural analyses of SiV-PLLA were performed using 13C cross polarization magic
angle spinning nuclear magnetic resonance (13C CP/MAS-NMR) and Fourier transform infrared reflec-
tion (FT-IR) spectroscopy. In the 13C CP/MAS-NMR spectra of the prepared composites, the band
of the carboxy peak shifted slightly and a new peak appeared at the low magnetic field area of the
band, where carboxy groups form coordinate bonds with bivalent ions (Asada et al. 1991), as shown
in Figure 6.10a. The peaks further separated into two peaks, Peaks A and B. Peak A was defined at
one magnetic field in the band and Peak B as one magnetic field lower in the band. The intensity of
PLLA V-PLLA SiV3-PLLA

Intensity (a.u.)
Intensity (a.u.)
Intensity (a.u.)
A A
B B
180 175 170 165 160 180 175 170 165 160 180 175 170 165 160
(a) Chemical shift (ppm) Chemical shift (ppm) Chemical shift (ppm)
: PLA : CO32– : Amide I

Absorbance (a.u.)
Absorbance (a.u.)
V-PLLA
SiV3-PLLA
1800 1700 1600 1500 2000 1800 1600 1400 1200 1000 800 600
(b) Wavenumber (cm–1) Wavenumber (cm–1)
FIGURE 6.10 (See companion CD for color figure.) Results of characterization of SiV3-PLLA and V-PLLA.
(a) 13C CP/MAS-NMR spectra of the two samples and PLLA. The peaks further separated into two peaks, Peaks
A and B. The intensity of Peak B in the spectrum of V-PLLA is larger than that of SiV3-PLLA. This indicates that
more carboxy groups formed coordinate bonds with calcium ions in V-PLLA than SiV3-PLLA. (b) FT-IR spectra
of the two samples. A weak peak appears at around 1650 cm−1 in the spectrum of SiV3-PLLA. This peak appears
to originate from the amide bond between carboxy groups in PLLA and amino groups in APTES structure.
TABLE 6.2
Weight-Average Molecular Weight (Mw),
Number-Average Molecular Weight (Mn),
and Polydispersity (Mw/Mn) of PLLA and
PLLA Matrices in SiV3-PLLA or V-PLLA
Mw (kDa) 259 17 185
Mn (kDa) 168 7 86
Mw/Mn 1.5 2.4 2.1
Peak B in the spectrum of V-PLLA is larger than that of SiV-PLLA. This indicates that more carboxy
groups formed coordinate bonds with calcium ions in V-PLLA than SiV-PLLA and the formation of
the coordinate bonds caused the decrease in the molecular weight. In the FT-IR spectrum of SiV-PLLA,
a new weak peak was found at around 1650 cm−1 except for peaks corresponding to PLLA and CO32− ,
as shown in Figure 6.10b. This peak appeared to originate from the amide bond, which indicates that
carboxy groups in PLLA structure form chemical bonds with amino groups in APTES structure.
The molecular weights of PLLA matrix in the prepared meshes were measured by gel permeation
chromatography (GPC). Table 6.2 shows the weight-average molecular weight (Mw), number-average
molecular weight (Mn), and polydispersity (Mw/Mn) of PLLA and PLLA matrix in SiV-PLLA or
V-PLLA. Although the molecular weight of PLLA slightly decreased after kneading with SiV pow-
ders, it significantly decreased after kneading with vaterite powders. We suppose that the coordinate
bonds between calcium ions and carboxy groups caused breaks in polymer chains. This may be the
reason why the molecular weight of V-PLLA mesh was significantly smaller than that of SiV-PLLA
one. The SiV particles were partially coated with siloxane derived from APTES; the amount of cal-
cium ions explored to PLLA matrix was smaller comparing to that of vaterite. From these results of
structural analyses, the SiV-PLLA mesh structure is suggested, as shown in Figure 6.11.
SiV-PLLA fiber
Siloxane derived from APTES
Vaterite PLA
Siloxane Vaterite
C O
O Si
Si
O
Ca2+
O
O
C N (CH2)3 Si
C
O
O
O H
Si
PLA PLA
Estimated from Estimated from

13C CP/MAS-NMR spectra
FT-IR spectra
FIGURE 6.11 Schematic image of SiV-PLLA structure, based on the results of GPC, FT-IR, and 13C
CP/MAS-NMR. Carboxy groups in PLLA form coordinate bonds with calcium ions and also amide bonds
with amino groups in siloxane derived from APTES on the SiV surface.
Membrane
FIGURE 6.12 (See companion CD for color figure.) Histology of in vivo response to the bilayered meshes.
Villanueva Goldner stains show new bone formed in the meshes at the center of holes 8 mm in diameter
formed at frontal midline site of the rabbit calvaria. Asterisks indicate mineralized tissues. New bone forma-
tion was observed over almost the entire area of implanted Si-PVH/HA-mesh after 12 weeks.
6.4.2 Tissue Compatibility and Bone-Forming Ability of the Membrane

The tissue compatibility and bone-forming ability of the fiber meshes was evaluated by in vivo tests
using rabbits. An 8 mm-diameter hole was drilled into the front midline of the animal’s calvaria using a
bone cutter and then covered with the bilayered meshes consisting of SiV-PLLA mesh and PLLA one.
An 8 mm-diameter hole in bone is generally not regenerated if remained to be done. In our preliminary
experiment, a PLGA mesh was implanted in the same position for 12 weeks and a new bone was not
to be formed in/on the mesh at the center of an 8 mm hole. The bilayered mesh was fixed by putting
between skin and bone. The meshes were implanted with SiV-PLLA mesh on the side in contact with
the hole and dense PLLA mesh on the side in contact with the skin. Figure 6.12 shows the histology
of in vivo response to the bilayered meshes and the new bone formation at the center of the holes.
Villanueva Goldner stain showed that mineralized tissue was formed over almost the entire area of
implanted SiV-PLLA mesh after 12 weeks. There was no tissue inflammation observed by h istology.
The bone formation started from the inside of SiV-PLLA mesh and not from the edge of the hole.
6.5 SUMMARY
This chapter described various types of PLLA–vaterite composite materials releasing silicon species
that we have developed so far. These materials with various shapes have been prepared, such as film,
bulk, hollow sphere, and fibrous membrane. We can choose the shape of the materials to adapt to the
applications. Also, the shapability and mechanical elasticity of the materials enable us to allow good
technical flexibility in the application. The effects of silicon species on the osteogenic cell functions
have got much attention in the biomaterials for bone reconstruction and been gradually clarified in
their mechanisms. We believe applying the silicon species is useful to develop materials static and
chronic releasability of factors stimulating osteogenic cells and having good bone-forming ability.
ACKNOWLEDGMENTS
This chapter was based mainly on our projects: these works were supported in part by Grant-in-
Aids for Scientific Research (B) (No. 14380398 and 20390497) from Japan Society for Promotion
of Science, a Grant-in-Aid for Young Scientists (B) (No. 21700487) from The Ministry of
Education, Culture, Sports, Science and Technology (MEXT), New Regional Consortium R&D
Program from Ministry of Economy, Trade and Industry (METI), and a grant from Institute
of Ceramics Research and Education (ICRE), NIT. We thank Dr. Yoshio Ota and Dr. Takashi
Wakita for supporting our projects.
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7 Basics, Strategies, and
Mucoadhesive Polymers
Future Trends
Andreas Bernkop-Schnürch
CONTENTS
7.1 Introduction........................................................................................................................... 194
7.2 Mucus Gel Composition........................................................................................................ 195
7.3 Principles of Mucoadhesion.................................................................................................. 196
7.3.1 Chemical Principles................................................................................................... 197
7.3.1.1 Formation of Non-Covalent Bonds............................................................. 197
7.3.1.2 Formation of Covalent Bonds..................................................................... 197
7.3.2 Physical Principles..................................................................................................... 197
7.3.2.1 Interpenetration........................................................................................... 197
7.3.2.2 Mucus Dehydration..................................................................................... 198
7.3.2.3 Entanglements of Polymer Chains.............................................................. 199
7.4 Methods to Evaluate Mucoadhesive Properties..................................................................... 199
7.4.1 In Vitro Methods....................................................................................................... 199
7.4.1.1 Visual Tests................................................................................................. 199
7.4.1.2 Tensile Tests................................................................................................200
7.4.1.3 Rheological Techniques.............................................................................. 201
7.4.1.4 Spectroscopic Techniques........................................................................... 201
7.4.2 In Vivo Methods........................................................................................................ 201
7.5 Mucoadhesive Polymers........................................................................................................202
7.5.1 Non-Covalent Binding Polymers............................................................................... 203
7.5.1.1 Anionic Polymers....................................................................................... 203
7.5.1.2 Cationic Polymers.......................................................................................204
7.5.1.3 Non-Ionic Polymers....................................................................................204
7.5.1.4 Ambiphilic Polymers..................................................................................205
7.5.2 Covalent Binding Polymers.......................................................................................206
7.6 Multifunctional Mucoadhesive Polymers..............................................................................208
7.6.1 Mucoadhesion: Enzyme Inhibition............................................................................ 210
7.6.2 Mucoadhesion: Permeation Enhancement................................................................. 212
7.6.3 Mucoadhesion: Efflux Pump Inhibition.................................................................... 214
7.6.4 Mucoadhesion: Buffer System................................................................................... 215
7.6.5 Mucoadhesion: Controlled Release........................................................................... 215
7.7 Concluding Remarks............................................................................................................. 216
References....................................................................................................................................... 216
193
7.1 INTRODUCTION
In the early 1980s, academic research groups pioneered the concept of mucoadhesion as a new
strategy in order to improve the therapeutic effect of various drugs. Mucoadhesive polymers are
able to adhere to the mucus gel layer which covers various tissues of the body. These mucoadhesive
properties are in many cases advantageous, rendering such polymers interesting tools for various
pharmaceutical reasons:
1. Mediated by mucoadhesive polymers, the residence time of dosage forms on the mucosa
can be prolonged, which allows a sustained drug release at a given target site in order to
maximize the therapeutic effect. Robinson and Bologna, for instance, have reported that a
polycarbophil gel is capable of remaining on the vaginal tissue for 3–4 days and thus pro-
vides an excellent vehicle for the delivery of drugs such as progesterone and nonoxynol-9 [1].
2. Furthermore, drug delivery systems can be localized on a certain surface area for purpose
of local therapy or of drug liberation at the “absorption window.” The absorption of ribo-
flavin, for instance, which has its “absorption window” in the stomach as well as the small
intestine, could be strongly improved in human volunteers by oral administration of muco-
adhesive microspheres versus non-adhesive microspheres as illustrated in Figure 7.1 [2].
3. In addition, mucoadhesive polymers can guarantee an intimate contact with the absorption
membrane providing the basis for a high-concentration gradient as driving force of drug
absorption (IIIa), for the exclusion of a presystemic metabolism such as the degradation of
orally given (poly)peptide drugs by luminally secreted intestinal enzymes (IIIb) [3,4], and
for interactions of the polymer with the epithelium such as a permeation enhancing effect
[e.g., 5,6] or the inhibition of brush border membrane-bound enzymes (IIIc) [7,8].
Because of all these benefits, research work in the field of mucoadhesive polymers has strongly
increased within the last two decades, resulting in numerous promising ideas, strategies, systems,
0.7
0.6
Rate of urinary excretion of riboflavin (mg/h)
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12
Time (h)
FIGURE 7.1 Improvement in the oral uptake of riboflavin by utilizing a mucoadhesive delivery system (◽) in
comparison to the same delivery system without mucoadhesive properties (•) in human volunteers. (Adapted
from Akiyama, Y. et al., J. Pharm. Pharmacol., 50, 159, 1998.)
Mucoadhesive Polymers 195
and techniques based on a more and more profound basic knowledge. An overview reflecting the
status quo as well as future trends concerning mucoadhesive polymers should provide a good plat-
form for ongoing research and development in this field.
7.2 MUCUS GEL COMPOSITION

As mucoadhesive macromolecules adhere to the mucus gel layer, it is important to characterize
first of all this polymeric network representing the target structure for mucoadhesive polymers.
The most important component building up the mucus structure are glycoproteins with a relative
molecular mass range of 1–40 × 106 Da. These so-called mucins possess a linear protein core,
typically of high serine and threonine content that is glycosylated by oligosaccharide side chains
that contain blood group structures. The protein core of many mucins exhibits furthermore N-
and/or C-terminally located cysteine-rich subdomains, which are connected with each other via
intra- and/or intermolecular disulfide bonds. This presumptive structure of the mucus is illustrated
in Figure 7.2. The water content of the mucus gel has been determined to be 83% [9]. Generally,
mucins may be classified into two classes, secretory (I) and membrane-bound (II) forms. Secretory
mucins are secreted from mucosal absorptive epithelial cells as well as specialized goblet cells.
They constitute the major component of mucus gels of the gastrointestinal, respiratory, ocular, and
urogenital surface. The mucus layer based on secretory mucins represents not only a physical bar-
rier but also a protective diffusion barrier [9,10]. Membrane-bound mucins possess a hydrophilic
membrane-spanning domain and they are attached to cell surfaces. Up to now, eight different types
of human mucins have been discovered and characterized, which are listed in Table 7.1.
Secreted mucins are continuously released from cells as well as glands undergoing immediately
thereafter a polymerization process which is mainly based on an intermolecular disulfide bond for-
mation. This so-formed mucus layer, on the other hand, is continuously eroded by enzymatic and
mechanical challenges on the luminal surface. Although the turnover time of the mucus gel layer
seems to be crucial in order to estimate for how long a mucoadhesive delivery system can remain
on the mucosa in maximum, there is no accurate information on this time scale available. A clue
was given by Lehr et al., who determined the turnover time of the mucus gel layer of chronically
isolated intestinal loops in rats to be in the order of approximately 1–4 h [21]. Both mucus secre-
tion and mucus erosion, however, are influenced by so many factors such as mucus secretagogues,
mechanical stimuli, stress, calcium concentration, or the enzymatic activity of luminal proteases
leading to a highly variable turnover time, which is therefore quite complex and difficult to evaluate.
SS
SS
SH
SH
S S
FIGURE 7.2 Schematic presentation of the three-dimensional network of the mucus gel layer; protein core:
glycosidic side-chains: .
TABLE 7.1
Synopsis of Human Mucins
Cysteine-Rich
Mucin Characteristics High-Level Expression Subdomains References
MUC1 Membrane-bound epithelial mucin Breast, pancreas [12]
MUC2 Secreted intestinal mucin Intestine, tracheobronchus Yes [13]
MUC3 Secreted intestinal mucin Intestine, gallbladder, pancreas Yes [14]
MUC4 Tracheobronchial secretory mucin Tracheobronchus, colon, uterine [15]
endocervix
MUC5A/C Secretory mucin Tracheobronchus, stomach, ocular, Yes [16]
uterine endocervix
MUC5B Secretory mucin Tracheobronchus, salivary Yes [17]
MUC6 Major secretory mucin of the Stomach, gallbladder Yes [18]
stomach
MUC7 Salivary [19]
MUC8 Tracheobronchus, reproductive [20]
tract
Source: Campbell, B.J., Biochemical and functional aspects of mucus and mucin-type glycoproteins, bioadhesive drug
delivery systems, E. Mathiowitz, Chickering, D.E. III, and Lehr, C.-M., eds., Marcel Dekker, New York,
pp. 85–130, 1999.
7.3 PRINCIPLES OF MUCOADHESION

Since the concept of mucoadhesion has been introduced into the scientific literature, considerably
many attempts have been undertaken in order to explain this phenomenon. So far, however, no
generally accepted theory has been found. A reason for this situation can be seen in the fact that
many parameters seem to have an impact on mucoadhesion (Figure 7.3), which makes a unique
explanation impossible. Although there are many controversies in case of which theories should
be favored, at least two basic steps are generally accepted. In step I, the contact stage, an intimate
contact between the mucoadhesive and the mucus gel layer is formed. In step II, the consolidation
stage, the adhesive joint is strengthened and consolidated, providing a prolonged adhesion.
Swelling
behavior
Cohesiveness Mechanism
Mucoadhesion of binding
Characteristics
of the mucosa
FIGURE 7.3 Schematic presentation of effects influencing mucoadhesion. (From Bernkop-Schnürch, A. and
Steininger, S., Int. J. Pharm., 194, 239, 2000.)
7.3.1 Chemical Principles

7.3.1.1 Formation of Non-Covalent Bonds
Non-covalent chemical bonds include hydrogen bonding (I), which is based on hydrophilic func-
tional groups such as hydroxylic groups, carboxylic groups, amino groups, and sulfate groups,
ionic interactions (II) such as the interaction of the cationic polymer chitosan with anionic
sialic acid moieties of the mucus, and van der Waals forces (III) based on various dipole–dipole
interactions.
7.3.1.2 Formation of Covalent Bonds

In contrast to secondary bonds, covalent bonds are much stronger and are not any more influenced
by parameters such as ionic strength and pH value. Functional groups that are able to form covalent
bonds to the mucus layer are overall thiol groups. The way how such functional groups can form
covalent bonds with mucus glycoproteins is illustrated in Figure 7.4. Thiolated polymers are able to
mimic the natural occurring mechanism how mucus glycoproteins are immobilized in the mucus.
7.3.2 Physical Principles

7.3.2.1 Interpenetration
One theory in order to explain the phenomenon of mucoadhesion is based on a macromolecular
interpenetration effect. The mucoadhesive macromolecules interpenetrate with mucus glycopro-
teins as illustrated in Figure 7.5. The resulting consolidation provides the formation of a strong
stable mucoadhesive joint. The theory can be substantiated by the observation that chain flexibility
favoring a polymeric interpenetration is a crucial parameter for mucoadhesion. The cross-linking
of various polymers or the covalent attachment of large sized ligands [23] leading to a reduction in
chain flexibility results in a strong decrease in mucoadhesive properties.
Rheological approaches—as discussed in detail later on—demonstrating a synergistic increase in
the resistance to elastic deformation by mixing mucus with mucoadhesive polymer, i.e., the consoli-
dation of the adhesive joint and attenuated total reflection Fourier transform infrared (ATR-FTIR)
studies showing changes in the spectrum of a poly(acrylic acid) film because of interpenetrating
mucin molecules within 6 min, provide further evidence for this theory [24].
Polymer SH + Mucin S-S Mucin
Polymer S-S Mucin + Mucin SH
Polymer SH + HS Mucin
Ox.
Polymer S-S Mucin
FIGURE 7.4 Thiolated polymers forming covalent bonds with the mucus layer.
FIGURE 7.5 Interpenetration of a mucoadhesive matrix tablet ( ) and the mucus gel layer ( ).
4
Interpenetrated polymer (%)
0
PAA 2 PAA 45 PAA 450 PAA 3000
FIGURE 7.6 Size-dependent interpenetration of polyacrylates (PAA) of indicated molecular mass

(2–3000 kDa) in a mucus layer. (Adapted from Imam, M.E. et al., STP Pharma Sci., 13, 171, 2003.)
Recently, Imam et al. evaluated the degree of interpenetration of fluorescence labeled polyacry-
lates of increasing molecular mass with various mucus gel layers utilizing confocal laser microscopy
and fluorescence quantification techniques [25]. Results of these studies are illustrated in Figure 7.6,
demonstrating a clear correlation between the molecular mass of the mucoadhesive polymer and the
degree of interpenetration.
7.3.2.2 Mucus Dehydration

Dehydration of a mucus gel layer increases its cohesive nature, which was shown by Mortazavi
and Smart [26]. Dehydration essentially alters the physicochemical properties of a mucus gel layer,
making it locally more cohesive and promoting the retention of a delivery system. The theory can
be substantiated by studies with dialysis tubings. Bringing dry mucoadhesive polymers wrapped in
dialysis tubings into contact with a mucus layer leads to its dehydration rapidly [26]. Dehydrating
a mucus gel increases its cohesive nature and subsequently its adhesive behavior, which could be
shown by tensile studies [26]. An objection to the theory that no interpenetration but exclusively
mucus dehydration occurs is given by various rheological studies and tensile studies carried out
by Caramella et al. [27], who observed a significant increase in the total work of adhesion (TWA)
by mixing the polymer directly with mucin before tensile measurements. It is therefore likely that
glycoproteins of the mucus are carried with the flow of water into the mucoadhesive polymer which
leads to the already described interpenetration; an explanation which allows the combination of
both the interpenetration and mucus dehydration theory.
7.3.2.3 Entanglements of Polymer Chains

The mucoadhesive as well as cohesive properties of polymers can also be explained by physical
entanglements of polymer chains. The difference in cohesive as well as mucoadhesive proper-
ties of lyophilized and precipitated mucoadhesive polymers gives strong evidence for this theory.
Precipitated polymers display high cohesive as well as mucoadhesive properties with a likely high
number of polymer chain entanglements, whereas these properties are comparatively lower for the
corresponding lyophilized polymers, which do not exhibit such a high extent of entanglements [28].
7.4 METHODS TO EVALUATE MUCOADHESIVE PROPERTIES

7.4.1 In Vitro Methods
The selection of the mucoadhesive material is the first step in developing a mucoadhesive drug
delivery system. A screening of the adhesive properties of polymeric materials can be done by vari-
ous in vitro methods such as visual tests, tensile studies, and rheological methods, which are often
accompanied by additional spectroscopic techniques. Apart from these well-established methods
which are described in detail later, some novel methods such as magnetic [29] and direct force mea-
surement techniques [30] have been introduced into the literature as well.
7.4.1.1 Visual Tests

7.4.1.1.1 Rotating Cylinder
In order to evaluate the duration of binding to the mucosa as well as the cohesiveness of muco-
adhesive polymers, the rotating cylinder seems to be an appropriate method. In particular, tab-
lets consisting of the test polymer can be brought into contact with freshly excised intestinal
mucosa (e.g., porcine), which has been spanned on a stainless steel cylinder (diameter: 4.4 cm;
height: 5.1 cm; apparatus 4—cylinder, USP XXII). Thereafter, the cylinder is placed in the dis-
solution apparatus according to the USP containing an artificial gastric or intestinal fluid at 37°C.
The experimental setup is illustrated in Figure 7.7. The fully immersed cylinder is agitated with
250 rpm. The time needed for detachment, disintegration, and/or erosion of test tablets can be
determined visually [22].
7.4.1.1.2 Rinsed Channel

At this method, freshly excised mucosa is spread out on a lop-sided channel with the mucus gel layer
facing upward and placed in a thermostatic chamber, which is kept at 37°C. After applying the test
material on the mucosa, the rinse is flushed with an artificial gastric or intestinal fluid at a constant
flow rate and the residence time of the mucoadhesive polymer is determined visually or quantified
utilizing insoluble markers such as fluorescein diacetate [31]. The experimental setup is illustrated
in Figure 7.8 [32,33].
if
t c
m
FIGURE 7.7 Schematic presentation of the test system used to evaluate the mucoadhesive properties of tab-
lets based on various polymers. c, cylinder; if, intestinal fluid; m, porcine mucosa; t, tablet.
Test formulation
Mucosa
Buffer solution
FIGURE 7.8 Experimental setup in order to evaluate the mucoadhesive properties of test formulations on a
freshly excised mucosa spread out on a lop-sided channel.
7.4.1.2 Tensile Tests

7.4.1.2.1 Tensile Studies with Dry Polymer Compacts
Test polymers are thereby compressed to flat-faced discs. Tensiometer studies with these test discs
are carried out on freshly excised mucosa. Test discs are therefore attached to the mucosa. After a
certain contact time between test disc and mucosa, the mucosa is pulled at a certain rate (mm s−1)
from the disc. The TWA representing the area under the force/distance curve and the maximum
detachment force (MDF) are determined. The experimental setup is illustrated in Figure 7.9. It
represents one of the best established in vitro test systems which are used by numerous research
groups [e.g., 23,34,35].
7.4.1.2.2 Tensile Studies with Hydrated Polymers

In order to minimize the influence of an “adhesion by hydration,” tensile studies can also be car-
ried out with hydrated polymers as described by Robinson et al. [36]. Hydrated test polymers are
thereby spread in a uniform monolayer over excised mucosa which has been fixed on a flat surface.
In an artificial gastric or intestinal fluid at 37°C, the hydrated polymer is brought in contact with the
mucus layer of a second mucosa, which is fixed on a flat surface of a weight hanging on a balance.
The TWA and MDF are then determined as described earlier.
TWA/MDF
Tablet
Mucosa
FIGURE 7.9 Experimental setup for tensile studies with dry polymer compacts.
7.4.1.2.3 Tensile Studies with Microspheres

Tensile studies as described earlier are not designed for measuring microscopic interactions such
as those that may occur between microparticles and the mucus gel layer. Hence, a method was
developed for measuring mucoadhesive properties of microspheres. In vivo interactions are thereby
mimicked utilizing a miniature tissue chamber, which is heated by a water jacket. Thermoplastic
microspheres are mounted to the tips of fine iron wires using a melting technique. The unloaded
ends of the wires are then attached to a sample clip and suspended in the microbalance enclosure.
The freshly excised section of mucosa is clamped in the buffer-filled chamber at 37°C and the
microsphere is brought into contact with the tissue. To fracture the adhesive interactions, the tis-
sue is pulled off the microsphere and certain mucoadhesive parameters are calculated and graphs
of force versus position and time are plotted using appropriate software for the microbalance. The
method provides valuable information concerning the adhesive properties of microspheres. So far,
however, it is limited to microspheres not smaller than 300 μm [37].
7.4.1.3 Rheological Techniques

During the chain interpenetration of mucoadhesive polymers with mucin macromolecules, physical
entanglements, conformational changes, and chemical interactions occur. Thereby, changes in the
rheological behavior of the two macromolecular species are produced. An evaluation of the result-
ing synergistic increase in viscosity, which is supposed to be in many cases directly proportional to
results obtained with tensile studies [e.g., 38], can be achieved by mixing the mucoadhesive polymer
with mucus and measuring viscosity. The rheological behavior can be determined either by a classi-
cal rotational viscometry test at a certain shear rate or by dynamic oscillatory measurements, which
give useful information about the structure of the polymer–mucin network [39].
7.4.1.4 Spectroscopic Techniques

Since mucoadhesive properties of polymers have been investigated, these tests were accompanied
by additional spectroscopic analyses [40]. Kerr et al. [41], for instance, using 13C nuclear magnetic
resonance spectroscopy have provided evidence of hydrogen bonding between mucus glycoproteins
and the carboxylic acid groups of polyacrylic acid. Moreover, Tobyn et al., using Fourier transform
infrared spectroscopy, reported also interactions between the pig gastric mucus glycoproteins and
the test mucoadhesive [42]. Jabbari et al. could confirm the chain interpenetration theory by inves-
tigating a mucin and polyacrylic acid interface via attenuated total reflection Fourier transform
infrared spectroscopy (ATR-FTIR) [24]. Mortazavi et al., using infrared and 13C-NMR spectros-
copy, suggested the formation of hydrogen bonds between the mucoadhesive polyacrylic acid and
the terminal sugar residues on the mucus glycoprotein [43]. In another study, the nature of interac-
tions between the mucus gel and polyacrylic acid was investigated by tensile studies (I), dynamic
oscillatory rheology (II), and 13C-NMR spectroscopy (III) as well. The addition of hydrogen bond
breaking agents resulted thereby in a decrease in mucoadhesive strength, a reduction in viscoelastic
properties of polymer/mucin mixtures, and a positional change in the chemical shift of the poly-
acrylic acid signal [44].
7.4.2 In Vivo Methods

To date, the mucoadhesion of dosage forms on mucosal membranes has been evaluated in vivo by
direct observation (I), by gamma scintigraphy (II), and by using insoluble markers (III). Thereby
either the time period of mucoadhesion is determined or in case of the GI-tract to which extent the
transit time of the mucoadhesive dosage form can be prolonged.
The direct observation offers the advantage that neither radionuclides nor insoluble markers are
needed and that a pretreatment of the test formulation is in most cases not necessary. The tech-
nique can be used to evaluate mucoadhesion on various tissues in animal studies. Akiyama et al.,
for instance, administered mucoadhesive microspheres orally to rats. After 2.5 h, the extent of the
adhesion of these microspheres to the gastric mucosa was evaluated visually, demonstrating a high
amount of mucoadhesive microspheres being present in the stomach compared to non-adhesive
microspheres [45]. For studies in humans, however, the direct observation is limited only to a few
mucosal tissues such as the oral cavity. Bouckaert et al., for instance, determined the adhesion
of tablets in the region of the upper canine. Test tablets were thereby fixed for 1 min with a slight
manual pressure on the lip followed by moistening with the tongue to prevent sticking of the tablet
to the lip. The adhesion time was defined as the time after which the mucoadhesive tablet was no
longer visible under the lip upon control at 30 min interval [46]. In another study, which was car-
ried out in the same way, volunteers participating in this study were asked to record the time and
circumstances of the end of adhesion (erosion or detachment of the tablet) [47].
In contrast, no tissue limitations seem to exist for gamma scintigraphic methods. Using these
techniques makes it even possible to evaluate the increase in the GI-transit time of mucoadhe-
sive formulations. Radionuclides used for imaging studies include 99Tcm, 111Inm, 113Inm, and 81Krm.
Among them technetium-99m represents the most commonly used radionuclide, as it displays no
beta or alpha radiation and a comparatively short half-life of 6.03 h. Indium-113m, which has an
even shorter half-life of 1.7 h, has a different energy to technetium-99m and can therefore be used
in double-labeling studies. For many applications, the longer lived isotope indium-111m (half-life
2.8 days) seems to be more appropriate. Whereas a strongly prolonged GI-transit time of mucoadhe-
sive polymers was demonstrated in various animal studies [36], the same effect cannot in all studies
be shown in human volunteers (effect: [2]; no effect: [48,49]).
7.5 MUCOADHESIVE POLYMERS

A systematic for mucoadhesive polymers can be based on their origin (e.g., natural–synthetic), the
type of mucosa on which they are mainly applied (e.g., ocular–buccal), or their chemical struc-
ture (e.g., cellulose derivatives–polyacrylates). Apart from these approaches, mucoadhesive poly-
mers can also be classified according to their mechanism of binding as shown within this chapter.
An overview in the mucoadhesive properties of these different types of mucoadhesive polymers
was provided by Grabovac et al. A rank order of the 10 most mucoadhesive polymers as listed in
Table 7.2 was established by testing more than 50 polymers under standardized conditions [50].
TABLE 7.2
Overview on the Mucoadhesive Properties of the 10 Most
Mucoadhesive Polymers
Total Work of Adhesion (μJ);
Polymer pH Means ± SD (n = 5–8)
Chitosan-thiobutylamidine pH 3 lyophilized 740.0 ± 146.7
Polyacrylic acid (450 kDa)-cysteine pH 3 lyophilized 412.3 ± 27.3
Chitosan-thiobutylamidine pH 6.5 precipitated 408.0 ± 67.9
Polycarbophil pH 7 precipitated 342.0 ± 11.2
Carbopol 980 pH 7 precipitated 311.8 ± 42.0
Sodium carboxymethylcellulose-cysteine pH 3 precipitated 261.7 ± 32.8
Polycarbophil-cysteine pH 3 lyophilized 212.9 ± 14.4
Source: Grabovac, V. et al., Adv. Drug Deliv. Rev., 57, 1713, 2005.
7.5.1 Non-Covalent Binding Polymers

According to their surface charge which is important for the mechanism of adhesion, they can be
divided into anionic, cationic, non-ionic, and ambiphilic polymers.
7.5.1.1 Anionic Polymers

For this group of polymers, mainly –COOH groups are responsible for their adhesion to the mucus
gel layer. Carbonic acid moieties are supposed to form hydrogen bonds with hydroxyl groups of the
oligosaccharide side chains on mucus proteins. Further anionic groups are sulfate as well as sulfo-
nate moieties, which seem to be more of theoretical than of practical relevance. Important represen-
tatives of this group of mucoadhesive polymers are listed in Table 7.3. Among them one can find
the most adhesive non-covalent binding polymers such as polyacrylates and NaCMC [e.g., 34,62].
TABLE 7.3
Anionic Mucoadhesive Polymers
Polymer Chemical Structure Additional Information References
Alginate H [51–53]
COO– H
O
O COO–
H OH OH
H O
H
O OH OH
H H H
H H
Carbomer COOH COOH COOH Cross-linked with sucrose [34,53,54]
Chitosan–EDTA CH2OH Optionally cross-linked with [55,56]

O EDTA
H
H
O
O OH H –
H O
N
H NH N
O O–
O–
O
O
Hyaluronic acid COOH CH2OH [57–59]

O O O
H H H O
H
OH H H
O H HO H
H OH H HN
CH3
O
NaCMC H OH CH2OCH2COONa 0.3–1.0 carboxymethyl groups [34,52,60]

O per glucose unit
OH H H H H
H O OH H
H H
O
CH2OCH2COONa H OH
Pectins COOH H OH R = OH or methyl [61,62]

O
H H
O H OH H
OH H H
O H
H O
H OH COOR
Polycarbophil COOH COOH COOH Cross-linked with [34,60,63,64]

divinylglycol
(a)
(b)
FIGURE 7.10 (See companion CD for color figure.) Adhesive bond failure in case of insufficient cohesive
properties of the mucoadhesive polymer: (a) matrix tablets and (b) microspheres.
Because of their high charge density, these polymers display a high buffer capacity which might be
beneficial for various reasons as discussed in Section 7.6.4. In contrast to non-ionic polymers, their
swelling behavior, which is also crucial for mucoadhesion (Figure 7.3), strongly depends on the
pH value. The lower the pH value, the lower is the swelling behavior leading to a quite insufficient
adhesion in many cases. On the contrary, a too rapid swelling of such polymers at higher pH val-
ues can lead to an over-swelling, which causes a strong decrease in the cohesive properties of such
polymers. Even if the polymer sticks to the mucus layer, in this case, the drug delivery system will
not be any more mucoadhesive, as the adhesive bond fails within the mucoadhesive polymer itself.
This effect is illustrated in Figure 7.10. A further drawback of anionic mucoadhesive polymers,
however, is their incompatibility with multivalent cations such as Ca2+, Mg2+, and Fe3+. In the pres-
ence of such cations, these polymers precipitate and/or coagulate [65], leading to a strong reduction
in their adhesive properties.
7.5.1.2 Cationic Polymers

The strong mucoadhesion of cationic polymers can be explained by ionic interactions between these
polymers and anionic substructures such as sialic acid moieties of the mucus gel layer. In particular,
chitosan, which can be produced in high amounts for a reasonable price, seems to be a promis-
ing mucoadhesive excipient. Apart from its mucoadhesive properties, chitosan is also reported to
display permeation enhancing properties [66,67]. The most important cationic mucoadhesive poly-
mers are listed in Table 7.4. Their swelling behavior is strongly pH-dependent as well. In contrast
to anionic polymers, however, their swelling behavior is improved at higher proton concentrations.
Chitosan, for instance, is hydrated rapidly in the gastric fluid, leading to a strong reduction of its
cohesive properties, whereas it does not swell at all at pH values above 6.5 causing a complete loss
in its mucoadhesive properties.
7.5.1.3 Non-Ionic Polymers

The adhesion of anionic as well as cationic polymers strongly depends on the pH value of the
surrounding fluid. On the contrary, non-ionic mucoadhesive polymers are mostly independent
from this parameter. Whereas the formation of secondary chemical bonds due to ionic interac-
tions can be completely excluded for this group of polymers, some of them such as poly(ethylene
oxide) are capable of forming hydrogen bonds. Apart from these interactions, their adhesion to
the mucosa seems to be rather based on an interpenetration followed by polymer chain entan-
glements. These theoretical considerations are in good accordance with mucoadhesion studies,
demonstrating almost no adhesion of non-ionic polymers, if they are applied to the mucosa
already in the completely hydrated form, whereas they are adhesive if applied in dry form [69].
Hence, non-ionic polymers are in most cases less adhesive than anionic as well as cationic
TABLE 7.4
Cationic Mucoadhesive Polymers
Chitosan CH2OH Primary amino groups can [3]
CH2OH H
O be acetylated to some
H
H
O extent
H O OH H
H
O OH H
H H NH2
H NH2
Trimethylated chitosan H CH3NH

+ [68]
H
OH H
H O
H
O
CH2OH
Polylysine NH2 [61]

NH2
NH2
NH
NH
NH
O
O
O
mucoadhesive polymers. Well-known representatives of this group of mucoadhesive polymers

are listed in Table 7.5. In contrast to ionic polymers, non-ionic polymers are not influenced by
electrolytes of the surrounding milieu. The addition of 0.9% NaCl, for instance, to carbomer
leads to a tremendous decrease in its cohesiveness and subsequently to a strong reduction of its
mucoadhesive properties, whereas these electrolytes have no influence on non-ionic mucoadhe-
sive polymers.
7.5.1.4 Ambiphilic Polymers

Ambiphilic mucoadhesive polymers display cationic as well as anionic substructures on their
polymer chains. On the one hand, mucoadhesion of the cationic polymers is referred to be
caused by electrostatic interactions with negatively charged mucosal surfaces and for anionic
polymers, on the other hand, mucoadhesion can be explained by the formation of hydrogen
bonds of carboxylic acid groups with the mucus gel layer. The combination of positive as well
as negative charges on the same polymer, however, seems to compensate both effects lead-
ing to strongly reduced adhesive properties of ambiphilic polymers. Mucoadhesion studies of
chitosan–EDTA conjugates with increasing amounts of covalently attached EDTA can clearly
show this effect. Whereas the exclusively anionic chitosan–EDTA conjugate (Table 7.3) exhib-
iting no remaining cationic moieties and the exclusively cationic polymer chitosan displayed
the highest mucoadhesive properties, the mucoadhesion of chitosan–EDTA conjugates show-
ing both cationic moieties of remaining primary amino groups and anionic moieties of already
covalently attached EDTA was much lower. In addition, Lueßen et al. could show a strongly
increased intestinal buserelin bioavailability in rats using chitosan HCl as mucoadhesive
excipient. A mixture of this cationic polymer with the anionic polymer carbomer, however, led
to a significantly reduced bioavailability of the therapeutic peptide [71]. Representatives of this
type of mucoadhesive polymers are mainly proteins such as gelatin, which is reported as muco-
adhesive in various studies [e.g., 62,72].
TABLE 7.5
Non-Ionic Mucoadhesive Polymers
Hydroxypropyl-cellulose CH2OR H OR R = H or hydroxypropyl [35,70]
O H
H
H O OR H
OR H H
O
H H O
H OR CH2OR
Hydroxypropyl- R = H or methoxy [53,62]

methylcellulose or hydroxypropyl
Poly(ethylene oxide) OH [35,53]
O
HO
Poly(vinyl alcohol) [54,62]
OH OH OH
Poly(vinyl pyrrolidone) [62]
N N
O O
Due to the combination of cationic as well as anionic mucoadhesive polymers leading to ionic
interactions, however, the cohesiveness of delivery systems can be strongly improved [57,61]. If
the adhesive bond of a delivery system fails rather within the mucoadhesive polymer itself, than
between the polymer and the mucus layer as illustrated in Figure 7.10, this effect is more important
than the mucoadhesive properties of the polymer in order to improve the adhesiveness of the whole
dosage form.
7.5.2 Covalent Binding Polymers

Recently, a presumptive new generation of mucoadhesive polymers has been introduced into the
pharmaceutical literature [73]. Whereas the attachment of mucoadhesive polymers to the mucus
layer has to date been achieved by non-covalent bonds, these novel polymers are capable of forming
covalent bonds [39,74]. The bridging structure most commonly encountered in biological systems—
the disulfide bond—has thereby been discovered for the covalent adhesion of polymers to the mucus
layer of the mucosa. Thiolated polymers or so-called thiomers are mucoadhesive basis polymers,
which display thiol-bearing side chains (Figure 7.11). Based on thiol/disulfide exchange reactions
[75] as illustrated in Figure 7.12 and/or a simple oxidation process, disulfide bonds are formed
between such polymers and cysteine-rich subdomains of mucus glycoproteins (see Table 7.1). Hence,
thiomers mimic the natural mechanism of secreted mucus glycoproteins, which are also covalently
anchored in the mucus layer by the formation of disulfide bonds. Due to the covalent attachment of
SH SH SH
SH SH SH SH
FIGURE 7.11 Thiomer (thiolated polymer); mucoadhesive polymers, which display thiol moieties bearing
side chains.
S S S SH
S SH
COOH COOH
NH R NH R
SH SH
COOH
NH R SH SH
S COOH
S SH NH R
S
S SH
FIGURE 7.12 Schematic presentation of disulfide exchange reactions between a poly(peptide) and a cysteine
derivative according to G. H. Snyder [76]. The poly(peptide) stands here for a mucin glycoprotein of the mucus
and the cysteine derivative is a polymer–cysteine conjugate (R, mucoadhesive basis polymer). (Adapted from
Bernkop-Schnürch, A. et al., Pharm. Res., 16, 876, 1999.)
250
200
150
TWA (µJ)
100
50
0
0.06% 0.09% 0.25% 0.36% Contr. Chitosan HCl
FIGURE 7.13 Comparison of the adhesive properties of chitosan-thiobutylamidine displaying increasing

amounts of covalently attached thiol groups (%) and controls of unmodified chitosan. Represented values are
means of the TWA determined in tensile studies with dry compacts of indicated test material. (From Roldo, M.
et al., Eur. J. Pharm. Biopharm., 57, 115, 2004.)
thioglycolic acid to chitosan, for instance, the adhesive properties of this polymer could be strongly
increased [76]. Results of this study are shown in Figure 7.13. Apart from these improved muco-
adhesive properties, which could meanwhile also be shown for various other thiolated polymers,
thiomers exhibit strongly improved cohesive properties as well. Whereas, for example, tablets con-
sisting of polycarbophil disintegrate within 2 h, tablets based on the corresponding thiolated poly-
mer remain stable even for days in the disintegration apparatus according to the Pharmacopoeia
Europea [77]. The result as shown in Figure 7.14 can be explained by the continuous oxidation of
thiol moieties on thiomers which takes place in aqueous solutions at pH values above 5. Due to the
10
Disintegration time (h)

6
0
PCP
PCP-Cys
CMC
CMC-Cys
FIGURE 7.14 Comparison of the disintegration behavior of matrix-tablets (30 mg; 5 mm i.d.) containing indi-
cated lyophilized polymers (PCP, polycarbophil; CMC, carboxymethylcellulose; PCP-Cys, polycarbophil–
cysteine conjugate; CMC-Cys, carboxymethylcellulose–cysteine conjugate). Studies were carried out with a
disintegration test apparatus (Pharm. Eur.) in 50 mM TBS pH 6.8 at 37°C. Indicated values are means ±SD
of at least three experiments. Polycarbophil-cysteine tablets did not disintegrate even after 48 h of incubation.
(From Bernkop-Schnürch, A. et al., J. Control. Release, 66, 39, 2000.)
high density of negative charges within anionic mucoadhesive polymers, they can also function
as ion exchange resins displaying a high buffer capacity (see Section 7.6.4). According to this, the
formation of disulfide bonds within polymer–cysteine conjugates can be controlled by adjusting the
pH value of the system a priori. Adhesion of many quick swelling polymers, as already mentioned,
is limited by an insufficient cohesion of the polymer resulting in a break within the polymer network
rather than between the polymer and mucus layer (Figure 7.10). Although thiolated polymers are
rapidly hydrated, they are able to form highly cohesive and viscoelastic gels due to the formation
of additional disulfide bonds. The formation of an over-hydrated slippery mucilage can thereby be
excluded completely. Using the rotating cylinder (Section 7.4.1.1) in order to evaluate the mucoad-
hesive properties of various formulations, for instance, revealed a comparatively much longer adhe-
sion of tablets consisting of thiolated polymers [22]. Meanwhile, various anionic as well as cationic
thiolated polymers have been synthesized as listed in Table 7.6. They all display strongly improved
mucoadhesive properties compared to the corresponding unmodified polymers. The potential of
thiolated polyacrylates could be demonstrated even in clinical trials. As illustrated in Figure 7.15, a
sustained release of sodium fluorescein over several hours was achieved with thiolated polyacrylate
minitablets adhering to the eye of volunteers [86].
7.6 MULTIFUNCTIONAL MUCOADHESIVE POLYMERS

In the 1980s, the adhesive properties of polymers played a central role in the field of mucoadhesion,
whereas numerous scientists began in the 1990s to focus their interest also on additional features
of mucoadhesive polymers. These properties include enzyme inhibition, permeation enhancement,
high buffer capacity, and controlled drug release. For some of these properties, mucoadhesion is
substantial, and for others various synergistic effects can be expected due to adhesion.
TABLE 7.6
Thiolated Mucoadhesive Polymers
Chitosan-cysteine CH2OH 21 up to 100 μmol [78]
O thiol-groups per gram
H
H polymer
O OH H
H
H NH
O NH2
SH
Chitosan-glutathione SH [79]
Glutathione
H NH
H
OH
H
H
H O O
CH2OH
Chitosan-thiobutylamidine SH [80]
NH2 Cl–
+
H NH
H
OH
H
H
H O O
CH2OH
Chitosan-thioethylamidine HS [81]
NH2+Cl–
H NH
H
OH
H
H
H O O
CH2OH
Chitosan-thioglycolic acid CH2OH 11 up to 25 μmol [82]

O thiol-groups per gram
H
H
polymer
O OH H H
H NH
O
SH
(continued)

Thiolated Mucoadhesive Polymers
NaCMC-cysteine CH2OH H OH 22 up to 1280 μmol [22,77]
H
O H thiol-groups per gram
H O OH H
H
polymer
O OH H
H H O
H O CH2OH
COOH
NH
SH
O
Polyacrylate-cystamine 1 up to 20 μmol [83]

thiol-groups per gram
CONH COOH COOH polymer
SH
Polyacrylate-cysteine 1 up to 142 μmol [22,39,74,84,85]

thiol-groups per gram
CONH COOH COOH polymer
HS
COOH
300
250
Fluorescein (ng/mL)
200
150
100
50
0
0 1 2 3 4 5 6 7 8
Time (h)
FIGURE 7.15 Prolonged ocular residence time of fluorescein being embedded in thiolated polyacrylate ocu-
lar inserts (◽) in comparison to the same formulation but containing the unmodified polyacrylate (•). (Adapted
from Hornof, M.D. et al., J. Control. Release, 89, 419, 2003.)
7.6.1 Mucoadhesion: Enzyme Inhibition

Due to the great progress in biotechnology as well as gentechnology, the industry is capable of pro-
ducing a large number of potential therapeutic peptides and proteins in commercial quantities. The
majority of such drugs is most commonly administered by the parenteral routes that are often com-
plex, difficult, and occasionally dangerous. Besides so-called alternative routes of application such
as the nasal or transdermal route, there is no doubt that the peroral route is one of the most favored,
as it offers the greatest ease of application. A presystemic metabolism of therapeutic peptides and
proteins caused by proteolytic enzymes of the GI-tract, however, leads to a very poor bioavailability
after oral dosing. Attempts to reduce this barrier include the use of analogs, prodrugs, formulations
such as nanoparticles, microparticles, and liposomes that shield therapeutic peptides and proteins
from luminal enzymatic attack, and the design of delivery systems targeting the colon where the
proteolytic activity is relatively low. Moreover, considerable interest is shown in the use of muco-
adhesive polymers, since due to such excipients various in vivo studies could demonstrate a sig-
nificantly improved bioavailability of peptide and protein drugs after oral dosing. As formulations
containing mucoadhesive polymers provide an intimate contact with the mucosa, a presystemic
degradation of these drugs on the way between the delivery system and the absorbing membrane
can be excluded. Takeuchi et al., for instance, demonstrated a significantly stronger reduction in the
plasma calcium level after oral administration of calcitonin-loaded liposomes which were coated
with a mucoadhesive polymer in comparison to the same formulation without the mucoadhesive
coating [4].
In recent years, it could be demonstrated by various studies that certain mucoadhesive poly-
mers display also an enzyme inhibitory effect. In particular, poly(acrylic acid) was shown to
exhibit a pronounced inhibitory effect toward trypsin [e.g., 87,88]. Additionally, the immobi-
lization of enzyme inhibitors to mucoadhesive polymers acting only in a very restricted area
of the intestine seems to be a promising approach in order to improve their enzyme inhibitory
properties. Due to the immobilization of the inhibitor, it remains concentrated on the polymer,
which should certainly make a reduced share of this auxiliary agent in the dosage form sufficient.
Side effects of the inhibitor such as systemic toxicity, a disturbed digestion of nutritive proteins
and pancreatic hypersecretion caused by a luminal feedback regulation can be avoided. Hence,
the covalent attachment of enzyme inhibitors to mucoadhesive polymers such as those shown in
Figure 7.16 represents the combination of two favorable strategies for the oral administration of
poly(peptide) drugs, offering additional advantages compared to a simple combination of both
excipients without the covalent linkage. So far, various polymer–inhibitor conjugates have been
generated as listed in Table 7.7. Their efficacy could be verified by in vivo studies in diabetic mice
showing a significantly reduced glucose level after the oral administration of (PEGylated-)insulin
tablets containing a polymer–inhibitor conjugate [96,97]. The results of this study are shown in
Figure 7.17 [97]. In another study, the potential of the combination of mucoadhesive polymers and
polymer–enzyme inhibitor conjugates was demonstrated by the improved oral uptake of salmon
calcitonin in rats utilizing thiolated chitosan in combination with a chitosan–pepstatin conjugate
as illustrated in Figure 7.18 [98].
HN H O
N
HN H
O N C H
S
C S
H O H
C C N C H
H R H S
N S N N
H H O
O
H 2N O
HN
Polymer
O
FIGURE 7.16 Example for a mucoadhesive polymer–inhibitor conjugate. The elastase inhibitor elastatinal
is thereby covalently attached via a C8-spacer to polymers like polycarbophil or NaCMC. (From Bernkop-
Schnürch, A. et al., J. Control. Release, 47, 113, 1997.)
TABLE 7.7
Comparison of Various Mucoadhesive Polymer–Inhibitor Conjugates
Inhibited Enzymes
Based on Based on
Complexing Competitive Mucoadhesive
Polymer–Inhibitor Conjugate Properties Inhibition Properties References
Carboxymethylcellulose–elastatinal Elastase + [89]
conjugate
Carboxymethylcellulose–pepstatin Pepsin n.d. [90]
conjugate
Chitosan–antipain conjugate Trypsin ++ [91,92]
Chitosan–chymostatin conjugate Chymotrypsin n.d. [92]
Chitosan–elastatinal conjugate Elastase n.d. [92]
Chitosan–ACE conjugate Trypsin, chymotrypsin, n.d. [92]
elastase
Chitosan–EDTA Aminopeptidase N, +++ [8,55,56]
carboxypeptidase A/B
Chitosan–EDTA–antipain conjugate Aminopeptidase N, Trypsin n.d. [92]
carboxypeptidase A/B
Chitosan–EDTA–chymostatin Aminopeptidase N, Chymotrypsin n.d. [92]
conjugate carboxypeptidase A/B
Chitosan–EDTA–elastatinal Aminopeptidase N, Elastase n.d. [92]
conjugate carboxypeptidase A/B
Chitosan–EDTA–ACE conjugate Aminopeptidase N, Trypsin, + [92]
carboxypeptidase A/B Chymotrypsin,
Elastase
Chitosan–EDTA–BBI conjugate Aminopeptidase N, Trypsin, + [93]
carboxypeptidase A Chymotrypsin,
Elastase
Poly(acrylic acid)-Bowman–Birk Chymotrypsin ++ [94]
inhibitor conjugate
Poly(acrylic acid)–chymostatin Chymotrypsin +++ [23]
conjugate
Poly(acrylic acid)–elastatinal Elastase +++ [89]
conjugate
Polycarbophil–elastatinal conjugate Elastase +++ [89]
Poly(acrylic acid)–bacitracin Aminopeptidase N n.d. [95]
conjugate
Polycarbophil–cysteine Carboxypeptidase A/B ++++ [85]
Mucoadhesive properties are classified in poor (+), good (++), very good (+++), and excellent (++++).
ACE, antipain/chymostatin/elastatinal; BBI, Bowman–Birk inhibitor.
7.6.2 Mucoadhesion: Permeation Enhancement

A number of mucoadhesive polymers have also promising effects on the modulation of the absorp-
tion barrier by opening of the intestinal intercellular junctions [64,99]. In contrast to permeation
enhancers of low molecular size such as sodium salicylate and medium-chain glycerides [100,101],
these polymers might not be absorbed from the intestine, which should exclude systemic side
effects of these auxiliary agents. Permeation studies, for instance, carried out in Ussing cham-
bers on Caco-2 monolayers demonstrated a strong permeation enhancing effect of chitosan and
140
120
Glucose level (%)

100
80
60
40
0 10 20 30 40 50
Time (h)
FIGURE 7.17 Decrease in blood glucose level in diabetic mice after oral administration of PEGylated insu-
lin being incorporated in a thiolated polyacrylate. (Adapted from Caliceti, P. et al., Eur. J. Pharm. Sci., 22,
315, 2004.)
100
97.5
Blood calcium level (%)
95
92.5
90
87.5
85
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)
FIGURE 7.18 Comparison in the oral uptake of salmon calcitonin in rats determined via the decrease in blood
calcium level after oral administration of the therapeutic peptide with chitosan (×), with chitosan/chitosan–
inhibitor conjugate (◾) and with thiolated chitosan/chitosan–inhibitor conjugate (⚬). (Adapted from Guggi, D.
et al., J. Control. Release, 92, 125, 2003.)
carbomer [99,102]. This permeation enhancing effect on these mucoadhesive polymers can even
be significantly improved due to the immobilization of cysteine on these polymers [5,74]. This
improved permeation across the mucosa was accompanied by a decrease in the TEER, indicating a
loosening of the tightness of intercellular junctions, i.e., the opening of the paracellular route across
the epithelium for otherwise non-absorbable hydrophilic compounds such as peptides. Mechanisms
that are responsible for the permeation enhancing effect of mucoadhesive polymers, however, are
still unclear.
The permeation enhancing effect of cationic polymers such as chitosan might be based on the
positive charges of these polymers which interact with the cell membrane, resulting in a structural
reorganization of tight junction-associated proteins [6].
In case of thiolated polymers, protein tyrosine phosphatase might be involved in the underlying
mechanism. This thiol-dependent enzyme mediates the closing process of tight junctions by dephos-
phorylation of tyrosine groups from the extracellular region [103]. The inhibition of PTP by specific
inhibitors such as vanadate or pervanadate causes an enhanced opening of the tight junctions. As it
is also inhibited by sulfhydryl compounds such as glutathione forming a mixed disulfide with Cys
215 [104], it is likely that thiolated polymers might also lead to an inhibition of this enzyme.
7.6.3 Mucoadhesion: Efflux Pump Inhibition

Transmembrane efflux pump proteins are one of the factors influencing and restricting the absorp-
tion of non-invasively administered drugs. These proteins can effectively alter the pharmacokinetics
of various drugs such as anticancer drugs, immune suppressive drugs, and antibiotics. Efflux pump
inhibitors have therefore gained a considerable attention as tool to improve clinical efficacy of drugs
that act as substrates for efflux pumps. Among efflux pump inhibitors, polymeric excipients such as
polyethyleneglycols, pluronics, and certain polysaccharides have gained considerable attention, as
due to their comparatively high molecular mass these compounds are not absorbed from mucosal
membranes. Consequently, systemic toxic side effects—as it is the case for low molecular mass
efflux pump inhibitors—can be excluded for such mucoadhesive polymers. More recently, Werle
et al. revealed efflux pump inhibitory capability of thiolated polymers [105]. The transmucosal
transport of the P-gp substrate rhodamine 123, for instance, was strongly improved in the presence
of thiolated chitosan. In the following, these in vitro results were confirmed by in vivo studies in
rats. Föger et al. showed that the oral bioavailability of rhodamine 123 is even 3.0-fold improved
when this model P-gp substrate is embedded in thiolated chitosan minitablets administered orally to
rats (Figure 7.19) [106]. Furthermore, poly(acrylic acid)-cysteine showed to inhibit effectively Mrp2
efflux pump transporter, improving the permeation of sulforhodamine 101 4.67-fold [107]. In another
study, tumor growth in rats was significantly reduced when paclitaxel was given orally in combina-
tion with a thiolated polymer [108]. In contrast to PEGs showing a P-gp inhibitory effect, which
is completely independent from the molecular mass of the applied PEG [109], the inhibitory effect
of thiolated polymers is dependent on their molecular mass. Thiolated polyacrylates of 250 kDa
exhibit the highest P-pg inhibitory effect, for instance, whereas thiolated poly(acrylic acid) of
higher and lower molecular mass displays significantly lower inhibitory activity. Studies performed
in vivo in rats confirmed the efficacy of thiolated polyacrylates of 250 kDa as Mrp2 inhibitor [110].
50
40
Rho-123 (ng/mL)
30
20
10
0
0 2 4 6 8 10 12
Time (h)
FIGURE 7.19 Efflux pump inhibitory effect of thiolated chitosan (×) in comparison to unmodified chitosan (◾)
in rats using rhodamine 123 as model Pgp-substrate. (Adapted from Föger, F. et al., Biomaterials, 27, 4250,
2006.)
The postulated mechanism of efflux pump inhibition is based on an interaction of thiolated poly-
mers with the channel forming transmembrane domain of efflux pumps. Thiolated polymers seem
to enter in the channel and likely form subsequently one or two disulfide bonds with cysteine sub-
units located within the channel. Due to this covalent interaction, the allosteric change of the trans-
porter being essential to move drugs outside of the cell seems to be blocked [111].
7.6.4 Mucoadhesion: Buffer System

A further advantage of mucoadhesive polymers can be seen in the high buffer capacity of ionic
polymers. As these polymers can act as ion exchange resins, they are able to maintain their pH
value inside the polymeric network over a considerable period of time. Matrix tablets based on
neutralized carbomer, for instance, can buffer the pH value inside the swollen carrier system even
for hours in an artificial gastric fluid of pH 2 [112]. This high buffer capacity seems to be highly
beneficial for various reasons. For example, the epidermal growth factor (EGF) is recognized as an
important agent for acceleration of ulcer healing and has a peculiar biological property to repair
tissue damage by an enhanced proliferation and differentiation of epithelial tissues [113]. Itoh and
Matsuo demonstrated in a double-blind controlled clinical study the enhanced healing of rat gastric
ulcers after oral administration of EGF. This effect could even be drastically increased by using the
mucoadhesive polymer hydroxypropyl cellulose as drug carrier matrix [114]. As EGF is strongly
degraded by pepsin [115], the use of mucoadhesive polymers providing an additional protective
effect toward pepsinic degradation might therefore be helpful. It can be achieved by a comparatively
higher pH value inside the drug carrier matrix based on its high buffer capacity at which penetrating
pepsin is already inactive.
Apart from this likely advantage for the poly(peptide) administration, the high buffer capacity
of neutralized anionic polymer might also be highly beneficial in treatment of Helicobacter pylori
infection in peptic ulcer disease, as common antibiotics such as amoxicillin or metronidazole display
poor stability in the acidic pH of the stomach. The incorporation of these therapeutic agents in muco-
adhesive polymers displaying a high buffer capacity might improve their stability in the acidic milieu.
7.6.5 Mucoadhesion: Controlled Release

If the therapeutic agent is incorporated in the mucoadhesive polymer, the excipient can act both as
mucoadhesive and as a matrix system providing a controlled drug release. The release behavior of
drugs embedded in mucoadhesive polymers depends thereby mainly on their molecular size and
charge. According to the equation determining the diffusion coefficient, in which the radius of a
molecule indirectly correlates with the diffusion coefficient, small-sized drugs will be released
faster than greater ones. Apart from their size, the release of therapeutic agents from mucoadhesive
polymers can be simply controlled by raising or lowering the share of the polymer in the delivery
system. Whereas a low amount of the mucoadhesive polymer in the carrier matrix can guarantee
a rapid drug release, a sustained drug release can be provided by raising the share of the polymer
in the delivery system [77]. In addition, the drug release from mucoadhesive polymers can also be
controlled by the extent of cross-linking. The higher the polymer is cross-linked, the lower is the
release rate of the drug. Such a cross-linking can be achieved by the formation of covalent bonds, for
example, the cross-linking of gelatin with glutaraldehyde [72] or on the basis of ionic interactions.
The release rate of insulin from matrix tablets consisting of the anionic mucoadhesive polymer car-
bomer (C934P), for instance, can be strongly reduced by the addition of the divalent cationic amino
acid lysine [28]. In case of ionic drugs, a sustained release can also be guaranteed by the use of an
ionic mucoadhesive polymer displaying the opposite charge of the therapeutic agent. On the basis
of an ion exchange resin, for instance, a sustained release of the therapeutic agent α-lipoic acid over
a period of more than 12 h can be provided by the incorporation of this anionic drug in the cationic
mucoadhesive polymer chitosan [116].

Motivated by the great benefits that can be provided by mucoadhesive polymers such as a prolonged
residence time and an intimate contact of the dosage form on the mucosa, considerably intensive
research and development has been performed in this field since the concept of mucoadhesion has
been pioneered in the early 1980s. Merits of these efforts are the establishment of various useful
techniques in order to evaluate the mucoadhesive properties of different polymers as well as the
design of novel polymers displaying improved mucoadhesive properties. In addition, the develop-
ment of multifunctional mucoadhesive polymers exhibiting also features such as enzyme inhibitory
properties, permeation enhancing effects, high buffer capacity, and the possibility to control the
drug release made them even more promising auxiliary agents. Although there are already numer-
ous formulations based on mucoadhesive polymers on the market, the number of delivery systems
making use of these advantages will certainly increase in the near future.
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8 Biodegradable
Polymeric/Ceramic
Composite Scaffolds
to Regenerate Bone Tissue
Catherine Gkioni, Sander Leeuwenburgh,
and John Jansen
CONTENTS
8.1 Introduction........................................................................................................................... 222
8.2 Bone Structure....................................................................................................................... 222
8.3 Bone-Substituting Implants................................................................................................... 223
8.3.1 Implant Requirements for Bone Substitution............................................................ 223
8.3.2 Candidate Materials................................................................................................... 223
8.3.3 Composite Approach toward Novel Bone-Substituting Biomaterials.......................224
8.4 Polymers................................................................................................................................224
8.4.1 Degradation of Polymers........................................................................................... 225
8.4.2 Degradable Polymers of Natural Origin.................................................................... 225
8.4.3 Synthetic Degradable Polymers................................................................................. 226
8.4.3.1 Polyesters.................................................................................................... 226
8.4.3.2 Polyethylene Glycol/Polyethylene Oxide.................................................... 227
8.5 Ceramics................................................................................................................................ 227
8.5.1 Bioactive Ceramics.................................................................................................... 227
8.5.1.1 Calcium Phosphates.................................................................................... 227
8.5.1.2 Bioactive Glass............................................................................................ 228
8.5.2 Bioinert Ceramics...................................................................................................... 228
8.6 Composite Materials.............................................................................................................. 228
8.6.1 Preparation Techniques............................................................................................. 228
8.6.2 Polymer/Ceramic Composites Derived from Natural Polymers............................... 229
8.6.2.1 Collagen-Based Composite Materials........................................................ 229
8.6.2.2 Miscellaneous Polymer/Ceramic Composites Derived
from Natural Polymers............................................................................... 231
8.6.3 Synthetic Polyester-Based Polymer/Ceramic Composites......................................... 232
8.6.4 Miscellaneous Polymer/Ceramic Biodegradable Composites................................... 233
8.7 Composite Ceramic/Polymer Degradable Biomaterials........................................................ 233
8.8 Conclusions............................................................................................................................ 237
References....................................................................................................................................... 237
221
8.1 INTRODUCTION
According to the U.S. Healthcare Cost and Utilization Project, nationwide inpatient statistics show
that in 2004 more than 1,100,000 surgical procedures were conducted involving the partial excision
of bone, bone grafting, and inpatient fracture repair [1]. The total estimated cost of those procedures
was more than $5 billion [1]. The majority of those procedures are due to congenital defects, bone
tumors, or traumatic events in young patients and also from osteoarthritis in older patients [2].
The “gold standard” for bone grafting therapies is the use of autogenous bone, which is a part
of healthy bone tissue as harvested from the same patient. It is the only solution that is effectively
fulfilling all the requirements needed for the replacement of bone tissue [3]. Even though autog-
enous bone is osteogenic, osteoinductive, osteoconductive, and biocompatible, its use has a lot of
drawbacks. As an extra surgery is required in order to harvest the bone, great risk exists for septic
complications, viral transmission and morbidity at the site of the harvesting. The surgery can some-
times be painful, and the amount of the bone removed is always limited [2]. An alternative to the use
of autogenous bone is the use of allografts (bone from another human donor) or xenografts (bone
from animals). Allografts, however, are also of limited amount and there is always the risk of a viral
or bacterial infection from the donor or disease transmission [2,3], while the disadvantages of using
xenografts are similar to those of using allografts.
In this chapter, the major reasons that inspired scientists to develop polymer/ceramic composite
biomaterials for bone substitution are discussed. To this end, a short introduction to the biology of
bone including its mechanical and biological properties will provide the proper background to be
able to understand the requirements for bone-substituting materials. Basic polymers and ceram-
ics used for the manufacturing of bone-substituting composite biomaterials and their properties
that are relevant to tissue engineering applications are being discussed, since the properties of the
final composite greatly depend on the physicochemical characteristics of the single components.
Biodegradable polymer/ceramic composites are considered to be a promising solution for bone
regeneration, since the mechanical properties of these materials can be tailored to match the host
tissue. Moreover, these temporary biomaterials can be replaced by newly formed bone upon gradual
degradation of the original biomaterial. Therefore, this chapter will provide an overview of cur-
rently investigated biodegradable, polymeric/ceramic composite scaffolds that have been synthe-
sized to replace bone tissue.
8.2 BONE STRUCTURE

Nature is the ultimate example of how material properties can be tailored to desire by combining
single components. Wood, for example, is a composite material consisting of long cellulose fibers
in a lignin matrix. Other examples of the composite structure of natural materials include corals as
well as teeth and bone.
Bone is a very complex composite material with various functions. Among others it provides
structural support for all the muscles, protects very sensitive organs (e.g., the brain and the eyes),
and provides the body with minerals for metabolic reactions. The overall mechanical properties
of different types of bone are not only a result of the synthesis of bones (a combination of two
materials, i.e., an organic matrix and inorganic mineral crystals dispersed in this matrix) but also
the way those materials are combined together from the order of some angstroms until the macro-
level. Even though the structure remains always the same, the composition of bone is constantly
changing through the life span of humans, affecting the mechanical properties. As mentioned
before, bone mainly consists out of an organic collagen matrix and a dispersion of inorganic min-
eral crystals. The latter are plate-shaped carbonated apatite crystals of hexagonal crystallographic
symmetry (Dahlite) [4]. The average dimensions of the crystals are 50 nm length and 25 nm wide
with a thickness of 3 nm. Often a lot of impurities are present in the bone apatite such as HPO4, Na,
Mg, K, citrate, carbonate, and more [5].
Biodegradable Polymeric/Ceramic Composite Scaffolds to Regenerate Bone Tissue 223
The other major component of bone (collagen type I) has a fibrous structure that is created by the
self-assembly of three collagen molecules into a triple helix [5]. The fibrils are ordered to sublayers
that are parallel and have a gap of approximately 40 nm between the ends of the molecules. The
crystals of the mineral actually grow in that gap and are in alignment from one fibril to the next.
Five of these sublayers rotate one from each other approximately 30° and comprise a lamellar creat-
ing a plywood-like motif [6]. Lamellae (3–7 μm wide) are further arranged to osteons or Haversian
systems and to woven bone. Finally woven bone and osteons combine together to give two types of
bone, i.e., cortical and trabecular bone. These two types of bone are distinguished by their degree of
porosity, density, and histological appearance of the tissue’s microstructure [5].
Bone is mainly an anisotropic material [7,8] and it has a range of values for its mechanical prop-
erties rather than specific values. The Young’s modulus is between 7 and 30 GPa; it has a tensile
strength ranging between 50 and 150 GPa and elongation at fracture of about 1%–3%. From every-
thing described earlier, it is quite clear that the mechanical properties of bone are the result of many
factors, including the structure and the components of this fascinating composite material. The col-
lagen in bone (an elastic hydrogel network) in combination with a hard inorganic material such as
hydroxyapatite (HA) as well as the way they structurally combine together endow bone with unique
mechanical properties such as low stiffness, resistance to tensile and compressive forces, and high
fracture toughness [9].
8.3 BONE-SUBSTITUTING IMPLANTS

8.3.1 Implant Requirements for Bone Substitution
When the biology of the natural bone is taken into respect, it is becoming quite clear that the implant
meant to replace such a tissue should meet some standards. Mechanical properties close to that of
healthy tissue and also the maintenance of these properties throughout the implantation period are
one of the basic requirements that a bone replacing material or scaffold must possess in order to be
inserted into the body. Another important requirement is the biocompatibility of the scaffold, which
means that the scaffold should not elicit an inflammatory response when introduced in the body.
This also implies that the material itself but also any degradation products should not be immuno-
genic and cytotoxic. A very important issue is the sterilization ability of the scaffold. The material
should be easily sterilized not only on the surface but in the whole interior of the material in order
to prevent infections. Another preferable characteristic of the scaffold, especially when intended for
bone regeneration, is the porosity. The scaffold should have large interconnected pores to allow the
migration of cells into the implant and promote vascularization of the newly formed tissue. Finally,
all of the aforementioned characteristics should be combined in a way that leads to materials easily
fabricated with validated production methods at low production costs [10].
8.3.2 Candidate Materials

A very wide range of materials has been used as implants through the ages. Archeologists have men-
tioned findings that people were using ox teeth, ivory, shells, coral, wood, human teeth from corpses,
gold, and silver to replace missing human teeth or body parts [11]. It is quite obvious that as the living
standards of society are growing higher and higher, the demand for better solutions is becoming more
of a necessity. For bone replacement (such as hip-joint prostheses), metallic and ceramic implants
are used most frequently, even though these materials are associated with a variety of problems. The
most common problem, resulting from the interaction of the metal with bone and with the fluids of
the body, is corrosion of the metal surface and the release of metal ions that can be detrimental for
the organism at high concentrations [12]. Many metal alloys used for fracture repair contain Ni or Co
ions, which cause allergic, toxic, and carcinogenic reactions [13]. Another drawback is that metallic
materials are not resorbable, while ceramic scaffolds show very poor degradability [14,15].
Even though these materials are still widely used in clinics, there is a strong need for novel biomateri-
als with improved properties. Generally, metallic materials possess high moduli of elasticity, whereas
ceramic materials have different values for tensile strength and brittleness compared to bone [16,17].
These differences in mechanical properties between implant materials and surrounding tissue are
addressed by Wolff’s law [16]. According to “Wolff’s law,” bone will remodel so that stress or strain is
retained within specific levels, which means that that part of the mechanical load will be carried by the
implant when a stiff metal implant is placed in bone, resulting into bone resorption and implant loosen-
ing [16]. In some cases, revision of the surgery is necessary for the scaffold to be fixated in position [17].
So, the mechanical properties of the material as implanted should not differ from the mechanical prop-
erties of the host tissue because this could lead to changes in the host tissue after implantation. The
need for materials that combine mechanical properties similar to those of bone but also elicit biological
reactions that can lead to a better bonding between the implant and the surrounding tissue is becoming
more obvious. In order to fabricate materials that mimic bone’s functions and mechanical properties, the
understanding of the properties of the host tissue is very important.
8.3.3 Composite Approach toward Novel Bone-Substituting Biomaterials

During the past decades, much effort has been put in research on composite materials consisting
of a polymeric matrix and a ceramic dispersed phase. By the combination of the two materials,
scientists managed to eliminate many of the drawbacks that these materials might have when they
stand on their own, thereby tailoring and enhancing the final properties of the composite material
by controlling many of their properties such as the mechanical characteristics, bioactivity, and of
course their degradation time.
Polymers, in general, lack the ability to bond with the surrounding tissue when implanted in the
body. The basic concept for preparing polymer/ceramic composites is that the dispersed mineral
phase will provide nucleation sites for HA formation in order to connect with the surrounding
tissue by providing sites for the cells to attach [18]. While the polymer degrades gradually, the
newly formed bone will replace the temporary implant and will provide the necessary mechanical
stability. Among the many advantages of these composite materials is also the fact that ceramics
in the polymer matrix can act as a buffer to the acidic microenvironment of the implant during
polymer degradation [19]. This acidic environment is known to induce inflammation around the
implantation site. Titration experiments [20] showed that carbonated calcium phosphates are able
to buffer the acidic residues of polymer degradation and preserve the pH at a physiological value
(pH 7.4) [20]. By a careful selection of the polymer and the ceramic phases, degradation times and
mechanical properties of the scaffold can be controlled to a large extent [1]. Another characteris-
tic is the improvement of the processing properties of pure ceramics since with the addition of a
polymer the final material can be easily moldable and in some cases even injectable because of the
elasticity caused by polymeric components [10,21].
In what follows, the biodegradable polymers and ceramics that are mostly used for the fabri-
cation of biomaterials are discussed. Polymers that exist already in nature and are derived from
natural sources are called natural polymers, whereas polymers synthesized in the lab are called syn-
thetic polymers. Ceramics are categorized as bioactive (calcium phosphates and bioactive glasses)
or bioinert ceramics. Subsequently, composite materials made of polymers (matrix) and ceramics
(dispersed phase) mentioned earlier are discussed in the next section. The review ends with a short
discussion about composite materials having ceramic matrices reinforced with polymers.
8.4 POLYMERS
The first polymeric biomaterials were developed under the great need for materials that could be
used for repair, reconstruction, or regeneration of tissue during the Second World War in the decade
1940–1950 [22]. Biomaterials were first used for corneal substitution, hip-joint replacements,
intraocular lenses, and blood contact devices [22,23]. There are a wide range of polymers used for
replacement of diseased or damaged tissues. Owing to the tremendous progress in organic chemis-
try of the last century, it has become possible to synthesize copolymers, which can combine proper-
ties from the different polymers, such as degradability, biological response, and good mechanical
properties. This widens the horizons even more and makes polymers a perfect candidate for compos-
ite scaffolds material. Polymers are mainly categorized as non-degradable or degradable polymers.
An additional advantage of these polymers is that the fractions of the degradation can enter into the
physiological cycles of the human metabolism and can be excreted without having any malicious
effects for the body.
8.4.1 Degradation of Polymers
Polymers can degrade either by hydrolysis (passively) or by enzymatic reaction (actively). Enzymatic
degradation only occurs to natural polymers [24], whereas passive degradation occurs by hydrolysis
in the backbone of the polymer at a rate determined by the type of bonds that are affected by
hydrolysis. Other factors can also affect the degradation rates (pH, catalytic phenomena, and the
presence of functional groups in the polymer), but the most important ones are the composition and
amount of water uptake. For example, the change in composition upon introduction of a second
monomer in the polymer chain may lead to changes in the crystallinity or the glass transition of
the material affecting the degradation rates. Hydrolysis is a reaction of water with the bonds from
the polymer. Greater water concentrations will lead to faster degradation times; thus, hydrophilic
polymers will tend to degrade much faster than hydrophobic ones [24].
8.4.2 Degradable Polymers of Natural Origin

Since collagen is the main polymer phase of bone, collagen has been the most popular natural
polymer under investigation for the fabrication of bone substitutes. Even though at least 27 dif-
ferent types of collagen have been identified, bone and cartilage consist mainly of type I and
II collagen. One molecule of collagen is structured from the arrangement of three polypeptide
chains into a triple helix [25,26]. Collagen is highly biocompatible and degrades enzymatically
and it can be processed also very easily into different forms such as sheets, tubes, sponges, and
fibers [23]. The main disadvantage of collagen usage for scaffolds is that collagen can cause
allergic reactions. There is also concern about the transmission of diseases, since the main
collagen source is bovine or porcine. On the other hand, recombinant collagen often lacks the
biological activity of native tissue since this type of collagen does not undergo posttranslational
modifications [27].
Another common natural polymer used in tissue engineering is gelatin. Gelatin is produced
from collagen and it can be derived by hydrolysis of collagen under acidic or basic conditions. It is
considered to be a very good candidate for tissue engineering and it can replace collagen because it
has a composition similar to collagen, while gelatin is not likely to transmit pathogens since it is a
denatured protein [28].
Hyaluronic acid (HAc) is a polysaccharide that is also present in human organism, i.e.,
mainly found in the extracellular matrix. It is a glycosaminoglycan, which chemically consists
of d-glucuronic acid and 2-acetamido-2-deoxy-d-glucose monosaccharides. The main disad-
vantages of this polysaccharide are the high solubility and the fast degradation times. Those
two characteristics, though, can be tailored by cross-linking hyaluronic acid or by chemical
modification leading to materials with controllable properties more suitable for scaffold fabri-
cation [29,30].
Alginic acid is another natural polymer that is widely used for tissue engineering. Alginic
acid is a linear polysaccharide and is composed of repeating units of d-mannuronic acid and
l-guluronic acid. Its main source is brown algae and it is commercially available as sodium salt.
Alginate is widely used in the food industry as thickener or as emulsifying agents [31]. Due
to the presence of carboxylate groups in its structure, it can form gels by cross-linking using
divalent cations such as calcium [32]. Even though alginate is a natural polymer, it does not
exist in the human body. Thus, its presence in the human body can always result in an immune
response.
8.4.3 Synthetic Degradable Polymers

8.4.3.1 Polyesters
A very common polymer group used for fabricating scaffolds for tissue engineering or used as drug
delivery system is that of aliphatic polyesters. Polyesters can be synthesized by the polycondensation
of diacids and diols; self-polycondensation of hydroxyacids; or the ring-opening polymerization of
cyclic diesters, lactones, glycolides, and lactides [10,23]. Since esterification is a reversible process,
hydrolytic degradation of these polymers happens through a random scission of the ester bond yield-
ing products that can be easily metabolized via natural procedures [33]. Some of the factors that can
affect the degradation kinetics are the chemical composition and configurational structure, process-
ing history, molar mass, polydispersity, crystallinity, morphology, presence of the original mono-
mers, and the overall hydrophilicity [34].
8.4.3.1.1 Polyglycolic Acid

One of the polymers belonging to aliphatic polyesters is polyglycolide. Polyglycolide or poly
(glycolic acid) (PGA) is a highly crystalline polymer with a high tensile modulus and a poor solubil-
ity in organic solvents. Right after implantation the polymer comes in contact with the body fluids
and immediately degradation begins, resulting to a loss of strength in 1–2 months and a loss of mass
after 6–12 months [35]. It is widely used for surgical sutures since the 1970s [36], but it also has been
used as a material for devices for internal bone fixation [23].
8.4.3.1.2 Polylactic Acid

Another widely used aliphatic polyester is poly(lactic acid) (PLA). Due to the fact that lactide, the
monomer from which PLA is synthesized, is a chiral molecule, the polymer exists in four different
isotopes:
• Poly(l-lactic acid)
• Poly(d-lactic acid)
• Poly(dl-lactic acid)
• Meso-poly(lactic acid)
The l-isomer (PLLA) is also a crystalline polymer with almost 37% crystallinity. It is among the
most used polymers for biomaterials because of the good mechanical properties, good tensile
strength, low elongation, and high tensile modulus, and therefore, it has been used for load-bearing
applications [37]. In poly(dl-lactic acid), the l- and d-units are randomly distributed in the poly-
meric chain resulting in a completely amorphous polymer with lower mechanical properties than to
the l-isomer. Even though the polymer loses its strength in 1–2 months like PLLA, the loss in mass
can take from 12 to 16 months [35].
8.4.3.1.3 Poly(Caprolactone)
Poly(caprolactone) (PCL) is also part of the polyester group. It is obtained by the polymerization
of caprolactone, which is a very cheap starting material. PCL is semi-crystalline, with low tensile
strength, high elongation at breakage, and it is soluble in most organic solvents [38]. It attracts the
interest of most researchers because of its ability to blend perfectly with other polymers and it has
a long degradation time (over 2 years), a property that makes PCL a perfect candidate for long-term
drug delivery vehicles [35,39]. Copolymers of PCL with PLA have been investigated in order to
achieve faster degradation times, whereas copolymers of PCL with PGA have been developed for
the improvement of its mechanical properties [39].
As mentioned earlier, the overall goal is to find an optimal polymer with the best combina-
tion of properties. To this end, many of the aforementioned polymers are polymerized together to
make copolymers with tailored properties. Poly(lactic-co-glycolic) acid is one of these copolymers.
Degradation and mechanical properties change depending on the percentage of lactic and glycolic
acid in the copolymer. Since lactic acid is more hydrophobic, copolymers with low lactic acid con-
tent tend to degrade more rapidly [40].
8.4.3.2 Polyethylene Glycol/Polyethylene Oxide

A very important polymer used widely in tissue engineering for drug delivery systems and scaf-
folds is polyethylene glycol (PEG) or polyethylene oxide (PEO) when it is of higher molecular
weight. This polymer is extremely hydrophilic, it can degrade hydrolytically, and the products of
the hydrolysis can be cleared by the kidneys [41]. A very important property of PEG is that it is not
recognized by the molecules that provoke immune response, making it highly biocompatible [42].
It can be combined with a lot of polymers to form copolymers that adopt many of PEG’s proper-
ties such as biocompatibility and biodegradability. Such a synthetic polymer is oligo(poly(ethylene
glycol)fumarate) (OPF). This relatively new copolymer was synthesized by Jo et al. [43], by the
copolymerization of PEG with the presence of fumaric acid, a naturally derived dicarboxylic acid.
The main benefits of OPF hydrogels for tissue engineering applications include the ability to tailor
the macromolecular structure in order to elicit different degradation rates as well as the good cell
viability in vitro and tissue response in vivo [44,45].
8.5 CERAMICS
Ceramics are non-metallic inorganic materials that can have different degrees of crystallinity. In
general, ceramic materials have a high compressive strength, but their resistance to deformation is
limited resulting into a high brittleness [14].
8.5.1 Bioactive Ceramics

8.5.1.1 Calcium Phosphates
The class of materials that can create a firm bond with bone at the site of implantation, by the
formation of a layer of HA, are called bioactive materials [46]. Calcium phosphates are a group
of ceramics that are widely used as biomaterials because they resemble the ceramic part of bone,
or they appear in human organisms at places of pathological calcifications such as urinary stones,
dental calculi, and crystalluria [47].
Monocalcium phosphate monohydrate (MCPM, Ca(H 2PO4)2 · H 2O) is the most water soluble
compound of the calcium phosphate family. It is precipitated from highly acidic solutions, and
when it is heated above 100°C it is transformed to anhydrous monocalcium phosphate. In gen-
eral, MCPM is not found in biological calcifications and it is mostly used in self-hardening cal-
cium phosphate cements (CPC) [48,49]. Dicalcium phosphate dihydrate (DCPD, CaHPO 4 · 2H 2O)
(also known as brushite) is a mineral often found in pathological calcifications. It can be easily
precipitated from aqueous solutions, and when it is heated at temperatures above 80°C, it is
transformed to its anhydrous component. Both DCPD and the anhydrous component are also
used for the synthesis of CPC [50,51]. Octacalcium phosphate (OCP, Ca8(HPO4)2(PO4)4 · 5H 2O)
is also found in humans as the stable phase of dental and urinary calculi. Its structure consists of
apatitic layers separated by hydrated layers. It is supposed that OCP acts as a precursor in vivo
for biological apatite formation. It is used in medicine as implant for bone substitution in vari-
ous forms such as coatings or granules [52–54]. β-Tricalcium phosphate (β-TCP, β-Ca 3(PO4)2)
cannot be precipitated from aqueous solutions but it can be obtained by thermal decomposition
of other calcium phosphates (such as calcium-deficient HA) and by calcining of bones. When it
is heated above 1125°C, it transforms in α-TCP. Both β-TCP and α-TCP have the same chemi-
cal composition, but different crystal structure and thus solubility. β-TCP is more stable at room
temperature and less soluble than α-TCP and it is usually mixed with HA to form biphasic
calcium phosphate (BCP). Both phases of TCP and BCP are used widely for the preparation of
bone-substituting products [47]. Amorphous calcium phosphate (ACP, Ca xHy(PO4)z · nH 2O) is
considered to be the first phase that precipitates in solutions containing calcium and phosphate
ions. In humans it is often found in pathological calcifications of soft tissues such as heart
valves [55]. It is widely used as dental and bone implant material [56].
Hydroxyapatite (HA, Ca5(PO4)3) is a very stable component with low solubility. It can be pre-
cipitated in aqueous solutions by mixing exactly stoichiometric quantities of calcium and phosphate
containing solutions at alkaline pH, followed by boiling in CO2 free atmosphere [47]. Even though
pure HA is never met in pathological calcifications, its similarity with the mineral found in bone and
teeth makes it a perfect candidate for the use as bone-replacing material. It has been used in a vari-
ety of different forms such as coatings, granules, blocks, and as a component for bone cement [57].
8.5.1.2 Bioactive Glass

Another group of ceramics that is widely used in research for biomedical applications is bioac-
tive glass. The first bioactive glass Bioglass®, a quaternary (SiO2-CaO-P2O5-Na2O) system, was
introduced by L.L Hench [58] and was prepared by the quenching method. Extensive research has
been performed since then and there are many glasses with various components that are used in the
preparations of biomedical devices [59]. The problems generated by the high-temperature process
and of the limited composition of these glasses can be overcome with the preparation of glasses
using the sol–gel method [60,61]. Glasses made by the sol–gel method are more porous than the
melt-derived ones. Due to their mesoporous structure, they tend to be more bioactive and more
bioresorbable compared to the traditional glasses [62].
8.5.2 Bioinert Ceramics

Ceramic materials that are encapsulated by a fibrous tissue layer between the implant surface and
surrounding bone tissue are called bioinert. Alumina (Al2O3) and zirconia (ZrO2) are bioinert
ceramic materials that are most frequently used for the manufacturing of biomedical devices. High-
density and highly pure alumina (α-Al2O3) has been used for femoral heads of hip prostheses and
acetabular cups. Zirconia is being used clinically as a material in the hip prosthesis due to its good
mechanical properties such as high strength, low wear and high hardness [63].
8.6 COMPOSITE MATERIALS

8.6.1 Preparation Techniques
All of the aforementioned materials have been combined extensively by biomaterials scientists in
search of the optimal combination of material properties in terms of mechanical and biological
characteristics.
In case of a dispersed ceramic phase, this ceramic phase can be introduced into the organic
matrix using either biomimetic, wet-chemical or physical mixing methods.
Biomimetic methods: Using this method, ceramic phases are formed in organic matrices according
to synthesis strategies that resemble the way in which calcium phosphates are synthesized in nature.
By soaking the matrix into saturated solutions (like simulated body fluids, SBF), the organic matrix
is mineralized by precipitation of nano-crystallized minerals. Alternatively, these matrices can be
calcified by alternate soaking in solutions rich in calcium or phosphate ions.
Wet-chemical methods: This method involves mixing of the polymer matrix in the solution in
which the ceramic precursors are present either as dissolved salts or as precipitated particles.
The advantage of this method is that it uses very small particles, which remain suspended in the
solution, resulting in composite materials with a very good dispersion avoiding the formation of
agglomerates.
Physical mixing: This method is the most common one and is used not only for particles but
also for the preparation of fiber composites. The size and the shape of the dispersed phase of
the composite material play a key role to the final mechanical properties of the composite. For
example, fine particles tend to agglomerate by forming strong aggregates. It is very important
to break down the agglomerates during processing and distribute the particles homogeneously
in the matrix.
The packing behavior of a material is strongly dependent on particle size, shape, and surface char-
acteristics. In general, bi-modal size distribution of the ceramic particles can be beneficial for com-
posite preparation resulting into composites with high ceramic densities since the smaller particles
occupy the space between the larger particles [64].
The first effort to create a material with a structure and mechanical properties similar to human
bone was performed during the 1980s. This so-called Bone Analogue was introduced by Bonfield
et al. [65,66], and it is a composite material consisting of a polymeric matrix of polyethylene (PE)
mixed with HA crystals. This biomaterial is commercially available under the name HAPEX and it
has been successfully used as middle-ear implant.
8.6.2 Polymer/Ceramic Composites Derived from Natural Polymers

8.6.2.1 Collagen-Based Composite Materials
Since collagen is the polymer found in human tissues, it is a logical consequence that a lot of
work has been performed on composite materials consisting of a collagen matrix with a min-
eral dispersed phase. Collagen-HA composites show remarkable properties. They have been
shown to be biocompatible both in humans and in animals; they accelerate osteogenesis and
they revealed better osteoconductive properties in comparison to monolithic HA implants [67].
Yamauchi et al. [68], reported the fabrication of multilayer collagen/HA sheets, which present
better stability, minimum swelling compared to the collagen reference, and better mechanical
properties.
There are several collagen-HA composites commercially available. The commercialization of
these materials means that they have been subjected to pre-clinical and clinical studies in animals
and in humans and have been proved safe for use. Collapat II® (BioMet, Inc) is made from calf skin
collagen of type I and synthetic HA granules. Another material derived from bovine collagen, HA,
and bone marrow aspirate is Healos® (Depuy Orthopedics, Inc). The collagen consists of cross-
linked fibers coated with the HA. Biostite® (Vebas) is a composite material made from equinine col-
lagen of type I, synthetic HA and chondroitin-6-sulphate. Collagraft® (Neucoll, Inc and Zimmer, Inc)
(a bovine collagen type I matrix mixed with HA and TCP granules and autologous bone marrow) is
another commercially available material.
In a comparative study performed by Leupold et al. [69], three commercially available bone
graft substitutes (including a ceramic one (ProOsteon®), demineralized bone matrix (DBX®)
and Collagraft) were implanted in New Zealand white rabbits. After 3 months of implantation,
Collagraft showed the most pronounced bony ingrowth compared to the other two and to empty
controls.
Another interesting material comprised of recombinant human-like collagen with HA assembled
into fibrils was dispersed into a PLA matrix and implanted into defects made in the radius of
rabbits [70]. After 24 weeks of implantation, bone density increased, while cell ingrowth, new bone
formation, scaffold degradation, and complete bone union were observed.
Serre et al. [71], studied the in vitro behavior of Biostite and compared it with the in vitro behav-
ior of collagen sponges and HA crystals. The materials were analyzed with transmission electron
microscopy and X-ray microanalysis to investigate the synthesis and calcification of organic matrix
when cultured with human osteoblast-like cells. The composite biomaterial showed an earlier and
more intense activity, while mineral deposits were observed on their surface.
Kikuchi et al. [72] developed a nanocomposite consisting of collagen and HA by the method of
co-precipitation. Using Ca(OH)2, H3PO4, and collagen solutions under alkaline conditions (pH 9)
and at a temperature of 40°C, a bone-like nanostructured material was formed with HA crystals of
up to 50 nm aligned along the collagen fibers similar to the nanostructure of bone. The collagen/HA
blocks were compacted by removing water under cold isostatic pressure. After implantation of
12 weeks in the tibiae of Beagle dogs, the composite material was shown to be biodegradable and
osteoconductive, but revealed a low resorption rate without significant tissue invasion in the implant
due to its dense body. The same group [73] used glutaraldehyde cross-linking to the nanocompos-
ites in order to improve the mechanical properties and the biodegradation rate. The results showed
that biodegradability was affected by the addition of the glutaraldehyde as the resorption rate of the
material was reduced with increasing concentration of the cross-linker, but the mechanical proper-
ties were improved.
In order to overcome the problems that were caused by the dense structure of the collagen/HA
composite described earlier, Sotome et al. [74], tried to incorporate the collagen/HA nanocomposite
into an alginate matrix. After implantation of the new composite in rats, the new material showed
extensive bone formation and a satisfactory tissue invasion in the collagen-alginate/HA composite
mainly due to swelling. Because of the different swelling ratios between the collagen/HA and the
alginate, fragmentation was observed to occur in the implants. The tissue invaded the implant from
the fragmentation sites and the collagen/HA composite was released from the implant inducing
more bone formation around the material.
Song et al. [75] manufactured a porous composite HA/collagen membrane using a co-precipitation
technique and a dynamic filtration technique. The membranes, designed for use as guided bone
regeneration, were compared with a membrane made only out of collagen. The mechanical proper-
ties of the composite membrane (stiffness and tensile strength) and the enzymatic stability were
significantly improved compared to the membranes made only from pure collagen.
Wu et al. [76] prepared collagen/HA microspheres with a size of 50–60 nm using a water-in-
oil emulsion. After the preparation of the emulsion at 4°C, glutaraldehyde was used to cross-link
collagen. The spheres were then cultured with rat osteoblasts. It was concluded that the composite
microspheres can be an excellent carrier for osteoblast cells.
Joseph et al. [77] studied a bovine fibril type I collagen matrix reinforced with a BCP phase
consisting of HA and TCP to a 60:40 ratio combined with a small amount of autogenous bone.
The material was used instead of autogenous bone graft substitute into a spinal fusion model in
canines for 12 months. The overall behavior of the composite was tested using biomechanical and
biochemical methods. No differences were observed in the behavior of the composite and the con-
trol groups upon biomechanical testing, and they propose that the material is a good alternative to
autologous bone.
Zou et al. [78] prepared a β-TCP/collagen scaffold by mechanically mixing the TCP powder
with an acidic suspension of collagen. The TCP powder was prepared after a precipitation reac-
tion and calcination at 900°C. After mixing the β-TCP powder with the suspension of the col-
lagen, the material was cross-linked with glutaraldehyde and it was put into molds. β-TCP has
a good biodegradability, making it a good candidate for use in composite materials. As a result,
many research groups have also studied β-TCP/collagen materials [79–81], but the particle size
was big (5–50 μm) and the mechanical mixing that was used for the preparation of the compos-
ite resulted in inhomogeneous distribution of the particles and resulted in a weak interaction
between the particles and the matrix.
Another scaffold, as described by Shinji et al. [82], was made from porcine collagen sponge com-
bined with OCP. The authors implanted collagen/β-TCP, collagen/HA, and their scaffold in critical
sized defects in rat crania and compared the bone regeneration induced by the different scaffolds.
Even though after week 4 of implantation there was no significant difference between the three
types of implants, the authors report that at week 12 bone regeneration of collagen/OCP implant was
significantly higher than those of the collagen/β-TCP, and collagen/HA implants. They also report
that the collagen/OCP implant was more degradable.
Du et al. [83,84] developed a series of calcium phosphate/collagen composites by immersing
commercially available type I collagen sheets into solutions containing calcium and phosphate ions.
By controlling the reaction conditions, various materials were obtained including ACP/collagen
and poorly crystalline carbonate apatite/collagen. The later ceramic is the one that resembles the
mineral of bone. The nano-HA-sized composite was also implanted in a marrow cavity. The implant
was shown to be bioactive and biodegradable and it had mechanical properties that could reach the
lower limits of bone.
A different approach was followed by Pederson et al. [85], who developed a novel injectable bio-
material that consisted of a collagen matrix mixed with calcium and phosphate loaded liposomes.
Phospholipids and phospholipids concentration was carefully chosen such as the liposomes would
melt at body temperatures. In addition, the acidic collagen monomers self-assemble to a gel near the
same temperatures. The combination of both the thermally gelling collagen and thermally triggered
mineralization induced by the melting of the phospholipids resulted into a novel injectable scaffold
that gels and mineralizes in situ triggered by an increase in temperature from room temperature to
body temperature.
In a study performed by Eglin et al. [86], two composite materials were prepared consisting of
the same polymer, i.e., type I collagen, and different reinforcing agents (CaO-P2O5-SiO2 bioactive
glass and silica particles derived from the sol–gel method). The materials were tested for their in
vitro ability to induce HA mineralization and the results were compared with pure collagen, bioac-
tive glass, silica and alumina/collagen scaffolds. It was also proven that both manufactured scaf-
folds induced the formation of HA after immersion in SBF. Surprisingly, collagen/silica particles
induced mineralization even though the single components did not, which implies a synergistic role
of collagen and silica that needs further investigation.
8.6.2.2 Miscellaneous Polymer/Ceramic Composites Derived from Natural Polymers

A three-dimensional porous alginate/HA scaffold with interconnected pores was manufactured
by Lin et al. [87] using a three-step process. The final scaffolds were sponges with weight ratios
of alginate:HA equal to 75/25 and 50/50 and were cross-linked with different divalent cations.
The porous structure in the scaffolds was well defined and the average pore size was 150 μm
with a porosity of 85%. The mechanical properties of the scaffolds were tested and a rat osteo-
sarcoma cell line was used to evaluate the in vitro response. The results showed that the 50/50
scaffold had better mechanical properties than the 75/25 and the pure alginate that was used as
a control. Both the composite scaffolds showed a better capacity for cell attachment than pure
alginate.
Panzavolta et al. [88] prepared new porous composite scaffolds of gelatin containing α-TCP.
The α-TCP particles partially hydrolyze during the foaming procedure to yield OCP. After immer-
sion in phosphate buffer solution, the mineral undergoes further hydrolysis to give poorly crystal-
line carbonated apatite. To stabilize and prevent dissolution of gelatin, the authors cross-linked
the scaffolds with genipin, a naturally occurring cross-linking agent. Another porous scaffold was
prepared by Kim et al. [89]. The scaffold consisted of gelatin and up to 30 wt% of HA particles.
The suspensions of gelatin and HA particles were first freeze-dried and then immersed in a solu-
tion containing a cross-linking agent, yielding cross-linked, highly porous composite scaffolds.
The mechanical properties of the reinforced gelatin foam and the pure foam were tested and the
results showed that the HA particles improved the compressive stress and the elastic modulus of
the composites in both dry and wet states. The authors also made coatings on titanium disks with
the composite gelatin/HA and measured the ALP activity of both the coatings and the composite
foams. Both materials revealed enhanced ALP activity in comparison with scaffolds and coatings
made out of pure gelatin.
8.6.3 Synthetic Polyester-Based Polymer/Ceramic Composites

Nazhat et al. [90] prepared a hot-pressed three-phase composite material combining a matrix of
PDLA reinforced with HA particulates and semi-crystalline unidirectional fibers of PLLA. The
authors studied the effect of the reinforcing phases on some of the mechanical properties and the
glass transition temperature of the PDLA matrix. Following a similar pathway, Nehad et al. [91]
prepared a HA/PLLA composite and evaluated how the hot-pressing method affects the structural
characteristics of the material. They reported that there might be a change in bioresorption due to
change in crystallinity of the PLLA during hot-pressing.
A composite porous material composed of HA/PDLLA was studied in terms of biocompat-
ibility, osteoconductivity, and biodegradability by Hasegawa et al. [92]. Both the composite
and the porous HA were implanted in rabbit femoral condyles. The composite scaffold was
surrounded by bone in direct contact with the implant surface after 6 weeks of implantation.
The amount of newly formed bone in the composite scaffold increased after 26 weeks and by
that time newly formed bone was formed inside the pores and bone marrow was present. The
bone in the HA scaffold did not show any significant increase after 12 weeks. The HA/PDLLA
scaffold was resorbed much faster than pure HA, which was hardly resorbed throughout the
implantation period.
Ignatius et al. [93] tested in vivo two composite materials consisting of α-TCP or GB14N glass
and PLA polymer matrix and compared the results with an autogenous implant. The group tested the
implants for up to 24 months implantation period in sheep. Both composite materials showed a good
biocompatibility, osseous integration, and compression strengths in the range of autogenous grafts
after 6 and 12 months of implantation. After 24 months of implantation, both implants presented an
inflammation around the tissue and the mechanical properties were significantly decreased result-
ing from severe degradation of the polymer component.
PLA was also combined with heat-treated and non-heat-treated glass fibers of the system 50P2O5-
40CaO-5Na2O-5Fe2O3 by Ahmed et al. [94]. The material had a flexural strength close to that of
cortical bone, but the modulus values were much lower and after 6 weeks of degradation in distilled
water it maintained 50% of the initial values. The authors concluded that the composite with the
heat-treated fibers maintained its mechanical properties and showed higher cell viabilities mainly
due to the slower degradation of the heat-treated fibers.
Kim et al. [95] prepared a porous HA scaffold coated with a composite solution of PCL
and HA. The antibiotic tetracycline hydrochloride was entrapped in the coating layer resulting in
a scaffold with drug release capability. They reported that the thickness of the coating played a
more significant role than the HA:PCL ratio to the mechanical properties of the scaffold. Higher
amounts of HA in the coating increased the biodegradation, while the antibiotic was released in
an uncontrolled manner during the first 2 h, but after that period it was released with more sus-
tained manner.
Causa et al. [96] prepared a composite material using PCL and HA. The mechanical perfor-
mance of the composite material was improved compared to pure PCL. After seeding osteoblasts
from human trabecular bone in the scaffolds, they showed that the composite materials had better
osteoconductivity than the PCL alone. Guarino et al. [97], prepared a PCL matrix reinforced with
two different phases. The biodegradable PLA fibers helped in maintaining the spaces required for
cellular ingrowth, while the calcium phosphate particles reacted in the matrix and produced needle-
like calcium-deficient HA that improved the mechanical properties.
A guided bone regeneration membrane comprising β-TCP and poly(l-lactide-co-glycolide-co-

ɛ-caprolactone) (PLGC) or poly(l-lactide-co-ɛ-caprolactone) (PLCL) made by the heat-kneading
method was described by Kikuchi et al. [98]. The scaffolds were first soaked in physiological and
buffer saline and seemed to maintain their mechanical properties after 12 weeks. The composites
were also implanted in the mandible of Beagle dogs for up to 6 weeks and showed good bone
regeneration.
8.6.4 Miscellaneous Polymer/Ceramic Biodegradable Composites

Song et al. [17] fabricated a pHEMA/carbonated apatite scaffold using a biomimetic process. The
hydrogel was soaked in an acidic solution of HA in the presence of urea. As the solution was heated,
urea started to decompose and during the change to a more basic pH partial hydrolysis of the hydro-
gel was achieved. The hydrolysis on the surface of the hydrogel promoted not only the nucleation
and growth of a several microns thick surface layer of apatite but also into the whole interior.
Khan et al. [99] prepared a novel material by chemically binding nano-composite HA with
polyurethane polymer. The HA nanoparticles were prepared by the sol–gel method and they were
chemically bonded on the backbone of the polyurethane through solvent polymerization. This new
material was then dissolved in DMF and the solution was electrospun to make nano-fibers. The
material seemed to be very promising for the use in dental applications even though more in vivo
and in vitro tests should be conducted. Bil et al. [100] created polyurethane/bioglass and polyure-
thane/PDLLA/bioglass composites by the slurry-dipping method. The bioglass coatings were very
homogeneous on the surface of the polymer foams and after immersion in SBF there was a forma-
tion of a calcium phosphate layer within only 7 days, while the uncoated polyurethane foams did not
show any calcium phosphate formation even after 21 days of immersion. The degradation rate of the
coated polyurethane in SBF was slightly higher than the non-coated one.
An in vitro and in vivo study of a composite scaffold consisting of two PVA polymers and
gelatin combined with HA ceramic in a weight ratio of 6:1:3 was conducted by Wang et al. [101].
The scaffold was synthesized by emulsification resulting in a highly interconnected porous net-
work. The pores were about 100–500 μm allowing the ingrowth of fibrous tissue after implanta-
tion in rats.
A study by Leeuwenburgh et al. [102] involved the synthesis of a functionalized OPF hydrogel
with finely dispersed calcium phosphates. The composite scaffolds were fabricated by synthesizing
calcium phosphate nanocrystals directly in the OPF hydrogel by a wet-chemistry reaction. Even
though the composite material had finely dispersed nano-apatite crystals throughout the polymer
matrix and reduced swelling behavior, no mineralization was observed after 28 days in SBF.
The synthesis of a composite hydrogel/calcium phosphate material was reported by Demitreas
et al. [103]. The authors fabricated a highly porous hydrogel scaffold based on poly-acrylamide
and reinforced it with HA beads with size between 25 and 45 μm. The HA beads were added in
the monomer and the polymerization occurred in the presence of the HA. The mechanical prop-
erties were improved and as reported the interconnectivity of the pores was maintained. Upon
testing in vitro using cell culture, the material proved to be cytocompatible and did not release
any cytotoxic compounds in the cell culture media. In Table 8.1, all the aforementioned materials
are listed.
8.7 COMPOSITE CERAMIC/POLYMER DEGRADABLE BIOMATERIALS

All the aforementioned examples of composite scaffolds are mainly examples of polymeric matri-
ces reinforced with a ceramic phase in the form of granules, particles fibers, or coatings. The oppo-
site approach toward enhanced mechanical and biological properties involves functionalization of
ceramic matrices with a polymer phase. The dispersed polymeric phase can be either in the form of
fibers or in the form of particles to provide the final material with certain porosity.
TABLE 8.1
Composite Polymer-Ceramic Biomaterials for Bone Substitution
Polymer Ceramic Author Reference Description
PE HA Bonfield et al. [65,66] “The Bone Analogue”
The first composite material
manufactured for use as bone
substitute
Collagen HA Yamauchi et al. [68] Multilayer collagen/HA sheets (2–10
layers)
Collagen (calf skin type I) HA Collapat II® (BioMet Inc)
Commercially available
Collagen (bovine type I) HA Healos® (DePuy Orthopedics, Inc)
Commercially available, contains also
bone marrow aspirate
Collagen (equinine) HA Serre et al. [71] Biostite® (Vebas), commercially
available material, contains also
chondroitin-6-sulfate
Collagen (bovine type I) HA+TCP Leupold et al. [69] Collagraft® (Neucoll, Inc and Zimmer,
Inc), commercially available material,
contains also autologous bone marrow
Collagen (recombinant HA Wang et al. [70] Fibrils of mineralized collagen in a
human like) PLA matrix
Collagen HA Kikuchi et al. [72,73] Bone-like nanocomposite material
made by co-precipitation of HA on
collagen fibrils
Sotome et al. [74] The nanocomposite of Ref. [85] was
introduced in an alginate matrix
Collagen HA Song et al. [75] Porous membranes
Collagen HA Wu et al. [76] Microspheres with the size of 50–60 nm
cross-linked with glutaraldehyde
Collagen (bovine type I) HA+TCP Joseph et al. [77] Collagen fibrils combined with biphasic
calcium phosphate and autogenous
bone
Collagen β-TCP Zou et al. [78] Mechanically mixed β-TCP powder
with a collagen suspension cross-
linked with glutaraldehyde
Collagen (porcine) OCP Shinji at al. [82] Collagen sponges combined with OCP
Collage (type I) ACP, poorly Du et al. [83,84] Collagen sheets were immersed in
crystalline solutions containing calcium and
carbonated phosphate ions. By controlling the
apatite reaction conditions, different CaP
phases were obtained
Collagen Calcium Pederson et al. [85] Liposomes loaded with calcium and
phosphate phosphate ions that melt at body
temperatures were introduced in a
collagen matrix
Collagen (type I) SiO2 particles Eglin et al. [86] Sol–gel derived reinforcing phase,
CaO-P2O5-SiO2 physically mixed with collagen
bioactive glass suspensions
Alginate HA Lin et al. [87] Porous sponges with interconnected
porosity of 85%

Composite Polymer-Ceramic Biomaterials for Bone Substitution
Polymer Ceramic Author Reference Description
Gelatin α-TCP Panzavolta [88] Porous scaffolds. The ceramic phase
et al. partially hydrolyzes yielding OCP and
poorly crystalline carbonated apatite
Gelatin HA Kim et al. [89] Freeze-dried sponges
PDLA HA Nazhat et al. [90] Hot-presses three phase material
PLLA HA Nehad et al. [91] Hot-pressed material
PDLLA HA Hasegawa et al. [92] Porous scaffold
PLA α-TCP Ignatius et al. [93] Particles of the reinforced phase
GB14N dispersed in the matrix
bioactive glass
PLA 50P2O5-40CaO- Ahmed et al. [94] Reinforcing phase consisting of
5Na2O-5Fe2O3 heat-treated and untreated fibers
bioactive glass
PCL HA Kim et al. [95] Composite including antibiotic factors,
used as coating on porous HA
scaffolds
PCL HA Causa et al. [96] Particles dispersed in polymer scaffold
PCL HA Guarino et al. [97] Polymer matrix reinforced with HA
particles and PLA fibers
PLGC β-TCP Kikuchi et al. [98] Membrane made by heat-kneading
method
pHEMA HA Song et al. [17] Hydrogel mineralized through a
biomimetic process in the presence
of urea
PU HA Khan et al. [99] HA nanoparticles prepared by the
sol–gel method and chemically
bonded on the polymer
PU Bioglass Bil et al. [100] PU foams coated with bioglass
PVA HA Wang et al. [101] Three-phase composite material.
PVA/gelatin/HA ratio of 6:1:3
OPF Poorly Leeuwenburgh [102] A ceramic phase was synthesized
crystalline et al. directly in the hydrogel using a wet
carbonated chemistry method
apatite
Poly-acrylamide HA Demitreas [103] HA beads added in the monomer
et al. solution and polymerization in the
presence of HA
CPC are mineral powders that are mixed with an aqueous solution to produce a paste that sets
at room temperatures due to the precipitation and subsequent entanglement of calcium phosphate
crystals [104]. These materials are biocompatible and osteoconductive but have slow degradation
times. Ruhe et al. [105] incorporated rhBMP-2 into and on the surface of PLGA microspheres
and added them in CPC. The release of the growth factor was slowed down by an interaction of
rhBMP-2 with the cement. Fei et al. [106] also incorporated loaded PLGA particles with rhBMP-2.
The growth factor was released in a controlled manner even after 28 days while the ALP and osteo-
calcin tests showed a dose response to the rhBMP-2, indicating that the activity of the growth factor
was maintained.
TABLE 8.2
Composite Ceramic-Polymer Biomaterials for Bone Substitution
Ceramic Polymer Author Reference Description
HA PCL Kim et al. [95] Porous HA scaffold coated with a composite film of PCL
with HA. The coating also included antibiotic agents
CPC PLGA Ruhe et al. [105] Commercially available cement (Calcibone®) reinforced
with rhBMP-2 loaded microparticles
CPC PLGA Fei et al. [106] Loaded microspheres with rhBMP-2 were used as
reinforcement in the cement
CPC Collagen Tamimi et al. [107] Acidic collagen solution was used as the liquid phase for
the preparation of brushite CPC
CPC Collagen Mai et al. [108] Mineralized collagen fibrils in suspensions used as liquid
phase for the preparation of the ceramic matrix
CPC Alginate, PLGA Qi et al. [109] Alginate hydrogel used as liquid phase for the preparation
of CPC to make porous scaffolds. The pores of the
scaffolds were filled with PLGA
CPC Aramid Xu et al. [110] CPC reinforced with fibers of variable length
Polyglactin
HA+TCP Chitosan Zhang et al. [111] The ceramic porous substrate was prepared by using an
organic mould that was burned out. After the immersion
of the scaffold in a chitosan solution, chitosan sponges
grew in the pores of the ceramic scaffold
Tamimi et al. [107] prepared a composite material consisting of a brushite CPC and collagen.
In their study, they reported about the utilization of the solubility of collagen and the setting of
the brushite cements at low pH. The collagen was dissolved in a solution containing citric acid
and was used as the liquid phase for the preparation of the cement. The composite cements were
tested in terms of mechanical properties and the combination of citric acid and collagen not only
accelerated the precipitation of brushite crystals but also improved the cell adhesion capacity of
the cement.
CPC intermixed with collagen was fabricated by Mai et al. [108]. The collagen fibers were min-
eralized before the addition into the cement using a wet-chemical precipitation method. The com-
posite material was implanted in minipigs for a total of 18 months. After 12 months of implantation,
the cement was completely replaced by spongeous bone and no left composite cement could be
detected. The authors concluded that the degradation of the scaffold occurred simultaneously with
the formation of new bone. As such, this material was considered to be a desirable material for
clinical applications.
The group of Qi et al. [109] fabricated composite CPC with the addition of alginate. The alginate
powder was dissolved in water in order to make a hydrogel that was used as the liquid phase for
the cement synthesis. In order to control the pore size and the interconnectivity of the scaffolds,
different liquid-to-powder ratios were used. After manufacturing the scaffold, its pores were filled
with PLGA polymer by immersing the scaffolds in a PLGA polymer solution and imposing vacuum
infiltration for 30 min. The addition of the PLGA polymer into the pores improved the mechanical
properties of the alginate/CPC scaffolds significantly.
The effect of fiber reinforcement on CPC was studied by Xu et al. [110]. Two polymer-derived
fibers (aramid and polyglactin) were tested among others, the latter being a copolymer of glycolide
and lactide. The composite materials with the aramid fibers showed the highest tensile strength
and the polyglactin/CPC showed the lowest. The fiber length in the composite seems to play an
important role since an increase in ultimate strength was observed when longer fibers of the same
polymer were used.
A very interesting approach is followed by Zhang et al. [111]. The group prepared a three-
dimensional macroporous calcium phosphate scaffold with chitosan sponges. The CPC scaffold
was prepared by dipping PU in a solution containing HA and TCP by burning the organic compo-
nent (PU) out at 250°C. The result was a pure ceramic scaffold with interconnected pores of a mean
size of 300 μm. The prepared scaffolds were then immersed in a chitosan solution under vacuum in
order to achieve the penetration of the solution into the pores. The chitosan solution containing the
ceramic scaffold was then transferred to −20°C to induce a solid–liquid phase separation and it was
finally transferred to the freeze-dryer to result in a ceramic scaffold with chitosan sponges grown
in its pores. The mechanical properties of the reinforced scaffold were improved in comparison
with the porous scaffold without the polymer. Table 8.2 summarizes the aforementioned composite
materials based on a continuous ceramic matrix.
8.8 CONCLUSIONS
In this chapter, several composite polymer/ceramic materials used for bone implants have been
reviewed. Most of these materials have mechanical properties close to those of bone. The mechani-
cal properties of these composites can be modified by a careful choice of the matrix and the dis-
persed phase. The mineral phase can provide nucleation sites for the crystallization of HA and
subsequent bone formation.
Also, several classes of biodegradable composite materials for bone substitution were discussed.
Compared to conventional single-component materials based on either ceramics or polymers, biode-
gradable polymer/ceramic composite materials display several advantages that are mainly related to
the possibility to fine-tune the mechanical properties, degradation rate, and biological response by
a careful selection of suitable composite components and appropriate composite preparation tech-
niques. The ultimate goal is to tailor the functional properties of the composites in such way that
their mechanical properties become similar to native bone tissue, while adjusting the degradation
profile of the composites in order to match the rate of new bone formation. In that way, novel, tem-
porary scaffolds can be created that instruct the physiological environment toward regeneration of
diseased or damaged bone tissue.
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9 Amphiphilic Systems as
Biomaterials Based on Chitin,
Chitosan, and Their Derivatives
Jacques Desbrieres
CONTENTS
9.1 Introduction........................................................................................................................... 243
9.2 Amphiphilic Polymeric Systems........................................................................................... 245
9.3 Amphiphilic Systems Based on Chitin and Chitosan Derivatives........................................246
9.3.1 Elaboration.................................................................................................................246
9.3.1.1 Amphiphilic Covalent Derivatives............................................................. 247
9.3.1.2 Amphiphilic Block Copolymers.................................................................248
9.3.1.3 Surfactant-Polyelectrolyte Complex or Polyelectrolyte Complex............... 249
9.3.1.4 Hydrogels.................................................................................................... 250
9.3.2 Properties................................................................................................................... 251
9.3.2.1 “Self-Assembly” Properties........................................................................ 251
9.3.2.2 Interfacial Properties.................................................................................. 253
9.3.2.3 Interactions.................................................................................................. 255
9.3.2.4 Swelling...................................................................................................... 255
9.4 Biomedical Applications of Amphiphilic Systems................................................................ 256
9.4.1 Drug Delivery............................................................................................................ 256
9.4.2 Gene Transfection...................................................................................................... 258
9.4.3 Tissue Engineering....................................................................................................260
9.4.4 Cells and Proteins Adhesion and Other Applications............................................... 261
References....................................................................................................................................... 262
9.1 INTRODUCTION
Chitin is the second most abundant polysaccharide in nature after cellulose. It is widely distributed
in the animal and vegetal kingdom, constituting an important renewable resource. It was first iso-
lated from fungi by Braconnot in 1811,1 but the name chitin, from the Greek language, which means
tunic or cover, was given by Odier, who in 1923 isolated it from the elytrum of the cock-chafer
beetle by treatment with hot alkaline solutions.2 Because of its insolubility in the vast majority of
common solvents, chitin was considered an intractable polymer and for many years it remained
mainly a laboratory curiosity. However, at present, chitin and its derivatives have become poly-
mers of great interest in a large variety of areas of human activity. Chitin is generally represented
as a linear polysaccharide composed of β (1 → 4) linked units of N-acetyl-2-amino-2-deoxy-d-
glucose. A great structural similarity exists between chitin and cellulose; the difference between
243
them consists in that the hydroxyl group of carbon C2 in cellulose is substituted by an acetamide
group in chitin. Both biopolymers play similar roles, since they both act as structural support and
defense materials in living organisms.
Chitin is the most abundant organic component of the skeletal structure of many classes compris-
ing the group of invertebrates, such as arthropods, mollusks, and annelids. In animals, chitin occurs
associated with other constituents, such as lipids, calcium carbonate, proteins, and pigments. It has
been estimated that the crustacean chitin present in the sea amounts to 1560 million tons.3 Chitin is
also found as a major polymeric constituent of the cell wall of fungi and algae. Fungal chitin exhib-
its some advantages as compared with animal chitin, such as a greater uniformity in composition, a
continuous availability in time, and the absence of inorganic salts in its matrix. However, in fungi,
chitin is associated with other polysaccharides, such as cellulose, glucan, mannan, and polygalac-
tosamine, which makes its isolation difficult.4
Chitosan is a linear polysaccharide obtained by extensive deacetylation of chitin. It is mainly
composed of two kinds of β (1 → 4) linked structural units: 2-amino-2-deoxy-d-glucose and
N-acetyl-2-amino-2-deoxy-d-glucose. However, since it is virtually impossible to completely
deacetylate chitin, what is usually known as chitosan is a family of chitins with different degrees
of acetylation (defined as the molar fraction of acetylated units) lower than 50%. The capacity
of chitosan to dissolve in dilute aqueous solutions is the commonly accepted criterion to differ-
entiate it from chitin. Chitosan is also present in significant quantities in some fungi, such as
Mucor rouxii (30%) and Choanephora cucurbitarum (28%), although again associated with other
polysaccharides.
Chitin and chitosan are structurally similar to heparin, chondroitin sulfate, or hyaluronic acid,
which are biologically important mucopolysaccharides. By themselves they have a wide range of
applications. Among them, they are remarkable biomaterials because of their numerous biological
and immunological properties.5 They show excellent biological properties such as biodegradation
in the human body,6,7 and immunological,8,9 antibacterial,10,11 and wound healing activity.12–14 But
the restricted solubilization restrained their use and it is necessary to consider their chemical or
enzymatic modification to enlarge their domain of use. These polymers have two reactive groups
suitable for this purpose, namely, primary (C6) and secondary (C3) hydroxyl groups in the case of
chitin, whereas chitosan has additionally the amino (C2) group on each deacetylated unit. All these
functions are susceptible to a variety of classical reactions that can be applied in a controlled fashion
to obtain a vast array of novel materials based on the two polysaccharides which can also be modi-
fied by either cross-linking or graft copolymerization.
The presence of hydroxyl and highly reactive-free amino groups offers a great potential for
further derivatization.15,16 In a wider consideration, the application of chitin and chitin deriva-
tives in many areas including food,17–19 cosmetics,20 biotechnology, and medicine is due to their
promising colloidal properties such as the formation of complexes with surfactants (includ-
ing fatty and amino acids, phospholipids), interaction with biomembranes, self-organization
in ordered nano-structures at the interfaces or in solution, stabilization of emulsions or foams,
formation of pH-, electrolyte-, or thermo-responsive physical gels. Moreover, the great inter-
est in medical applications of chitosan and some of its derivatives is readily understood. The
cationic character of chitosan is unique: it is the only artificial (derivative of a natural polymer)
cationic polymer.
Several chitosan-based molecules have been shown to be non-toxic, biodegradable, and biocom-
patible with low immunogenicity.17,21 These cationic polyelectrolytes are potential candidates for
gene delivery22–25 since they bind efficiently negatively charged DNA and protect it against nuclease
degradation.26–30 DNA-chitosan complexes have been applied for in vitro transfection assays and
have been found useful for gene delivery.31–33
Numerous modifications of chitin and chitosan were described for biomedical applications34
but we focused our review on the amphiphilic systems based on chitin, chitosan, and their
derivatives.
Amphiphilic Systems as Biomaterials Based on Chitin, Chitosan, and Their Derivatives 245
9.2 AMPHIPHILIC POLYMERIC SYSTEMS

Amphiphilic polymeric systems combine in the macromolecular backbone or in grafted compo-
nents a range of hydrophilic and hydrophobic groups. The former are typically based on hydroxyl,
urethane, and carboxylic moieties, to name but a few, and the latter on alkyl or long fatty acid
chains to cite the more prevalent examples. Amphiphilic polymers are, on one hand, able to self-
assemble and as a consequence form mono- and polymolecular micelles or aggregates in aqueous
dispersions35–43 and, on the other hand, exhibit enhanced adsorption characteristics at interfaces.
These results lead to sharp reductions in surface and interfacial tensions. In addition, aqueous dis-
persions tend to display enhanced viscosities due to intermolecular interactions between hydropho-
bic groups, thus leading to the formation of polymolecular associations and long-range structures.
According to their structure, the aggregation mechanism will be different as the properties. But in
any case the basic phenomenon will be the hydrophobic interactions between hydrophobic parts of
the polymeric chains leading to junction zones. According to medium conditions, a physical gel,
which is a loose tridimensional network, may be obtained. Hence, a large viscosity increase was
observed. Self-assembly of such polymers allows also the elaboration of specific materials such as
nanoparticles and gel-like aggregates. Among the many uses for polymers of this type are included
environmentally friendly formulations or viscosity modifiers in tertiary oil recovery systems.44–47
Moreover, such amphiphilic polymers may present hydrophobic interactions with other chemicals
and especially coming from living systems. Hence although polysaccharide based systems have
been considered for a range of applications48–52 of particular interest, those allied to their use in
biomedicine will be focused.
Polysaccharides are natural polymers that exhibit good biocompatibility and have been targeted
for use in site-specific drug delivery, encapsulation, and rheological modifications due to their inher-
ent stiffness. The amphiphilic characteristics of polysaccharides may be altered through modifying
either hydrophilic or hydrophobic groups. As most polysaccharides are in fact polyelectrolytes,
the hydrophilic character may be controlled by varying the charge density (or the number of ionic
charges per saccharide unit), or when the ionic sites are weak acids or bases by changing the pH of
the medium, which modifies the degree of ionization. The hydrophobic character may be adjusted,
however, either by the chemical modification of the macromolecular chain structure through
covalently bonding hydrophobic groups onto the hydrophilic backbone of the p olysaccharide to
give, for example, an alkylated polyelectrolyte, a so-called polysoap (see Figure 9.1a) or graft
copolymerization,53 or by the formation of non-stoichiometric dynamic associations between the
polysaccharide and an oppositely charged molecule as a surfactant to give a surfactant-polyelectrolyte
complex (SPEC), as exemplified by the modification of an O-carboxymethylchitin (Figure 9.1b) to
the SPEC shown in Figure 9.1c or an amphiphilic polyelectrolyte leading to a polyelectrolyte com-
plex (PEC) as shown in Figure 9.1d.
Thus, this allows adjusting the chemical structure and the architecture of polymeric chain to
required properties. In these systems, hydrophobic associations through intra- or interpolymer
interactions compete with electrostatic repulsions, which operate within the same polymer chain or
between different chains. The preference of intra- or interpolymer hydrophobe association depends
on the polymer concentration and the structural characteristics of the macromolecule, notably the
content and the nature of the hydrophobic groups and the sequence distribution of electrolyte and
hydrophobic monomer units along the polymer chain. These effects and the ionic strength of the
system are factors that also are important54,55 for the association efficiency and subsequently the
dynamical behavior of solutions of hydrophobically modified polyelectrolytes.
Among the more discussed biomedical applications, encapsulation and controlled release of
active compounds, on one hand, and stimuli-responsive polymeric gels,56 on the other hand, are
predominant. To be used in such domains polymeric systems need solubility control, specific con-
trolled interactions, mechanical properties, fast response time in physiological environment, etc.
The limitation of chitosan for the preparation of sustained release systems arises from its rapid
– +
H OH H OCH2COO , Na
H OH H OH
H O H
H O O
H O
HO O
H NHCOCH3 HO O HO O HO O
H H NHR H NHCOCH3
NH2
H H
DS
DA H 1-DA-DS H H H H
H n
(a) R: Alkyl chain (b)
Me CH2
Me
Me N+ H2C CH
+ Cl–
+
–
CH2OCH2COO–Na+ CH2OCH2COO Na+ CH2OCH2COO–Na+
CH2OCH2COO–Na+
O O
O O
OH O OH O
OH O OH O
NHCOCH3 NHCOCH3
NH2 NH2
(c) (d)
FIGURE 9.1 Chemical structures of chitin derivatives: (a) alkylchitosan, DA being the degree of acetylation
and DS the degree of alkyl substitution, (b) O-carboxymethylchitin (CMCh), (c) SPEC formed from CMCh
and cationic surfactant, and (d) PEC formed from CMCh and an amphiphilic cationic polyelectrolyte.
water absorption and high swelling degree in aqueous environments leading to fast drug release.
The amphiphilicity is an important characteristic for the formation of self-assembled structures such
as nanoparticles, potentially suited for drug delivery applications. The hydrophobic core of these
nanoparticles could act as reservoirs or microcontainers for various active substances. Moreover,
the small size of these particles leads them to be administrated via the intravenous injection for
targeted drug delivery. For other applications such as DNA transfection, it was observed a small
efficiency of chitosan assumed to be due to the low hydrophobicity of the complexes resulting from
the condensation of DNAs by the chitosan chains. As a consequence it is important to be able to
bring amphiphilic character to chitosan macromolecules. In order to overcome these problems, chi-
tosan was modified with alkyl halides bearing hydrophobic alkyl chains. The utility of modifying
chitosan with hydrophobic branches for controlling solubility properties has been demonstrated.57,58
More recently, it was reported that chitosan derivatives with both hydrophobic groups (as long acyl
groups) and hydrophilic groups (sulfate groups) could form micelles and solubilize hydrophobic
compounds.59 The possibility to modify water-soluble chitosan derivative (N-methylene phosphonic
chitosan) allows to obtain an amphiphilic system in which the hydrophobic moiety counterbalances
the electrostatic repulsion but it also gets more tensioactive properties.58
9.3 AMPHIPHILIC SYSTEMS BASED ON CHITIN AND CHITOSAN DERIVATIVES

Amphiphilic materials may exist under different types discussed from the chemical point of view.
Moreover, they can be elaborated under several forms and among those specially devoted to bio-
medical applications is the hydrogel one.
9.3.1 Elaboration
Chitin possesses one functional group, the hydroxyl one, whereas chitosan has two, the hydroxyl
and the amino ones. This allows various chemical reactions on each of them and the capacity
to perform specific reactions on the amino group, by contrast to cellulose or starch polymers.60
Moreover, amphiphilic systems may be obtained from other strategies considering interactions that
chitin or chitosan (and its derivatives) may build with other chemicals such as surfactants and other
polyelectrolytes.
9.3.1.1 Amphiphilic Covalent Derivatives

The alkylated derivatives of chitosan and chitin were generally obtained by reductive amination
adapting the procedure described by Yalpani.61 This is a versatile and specific method for creating a
covalent bond between a substrate and the amine function of the glucosamine unit. For considered
chitosan it involves the reaction between the amine function of the unit and an aldehyde function.
Generally the reaction of substitution of polymers is not homogeneous. Indeed, many reactions
(e.g., on cellulose or starch) are performed in heterogeneous and the chemical modification can be
observed only on the accessible sites of the macromolecular chain. Due to lack of accessibility, a
block-wise substitution distribution is obtained and the compound is structurally inhomogeneous.
A procedure was developed for performing homogeneous chemical reactions allowing a uniform
distribution of substituants.62 The reaction mechanism is as follows:
CH2OH CH2OH CH2OH
O H O O
O Slow O BH3CN– O
OH + O OH OH
Fast
R
NH2 n HN+ n NH n
C R
R H H H
The reduction of imminium ion was performed using NaCNBH3, which is more reactive and selec-
tive than usual reducing agents such as NaBH4, selephenol (PhSeH) or pentacarbonyl ion in alco-
holic potassium hydroxide. The advantage of such reducing agent is its stability in the acidic media.63
Moreover, the reduction of imminium ion by BH3CN− anion is rapid at pH values of 6–7 and the
reduction of aldehydes or ketones is negligible in this pH range (it becomes quick at pH values smaller
than 3.5). Using the same process cyclodextrin-chitosan was synthesized using β-cyclodextrin
monoaldehyde.64 Cyclodextrins are cyclic oligosaccharides built from 6 to 8 d-glucose units and
are formed during the enzymatic degradation of starch and related compounds.65 The d-glucose
units are covalently linked together by 1,4 linkages to form torus-like structures. The formed cavity,
hydrophobic in nature, is capable of binding aromatic and other small organic molecules and there-
fore provides ideal binding sites.66,67
New techniques were developed to elaborate such amphiphilic derivatives. Graft copolymer-
ization is a versatile method for providing functionality and regulating polymer properties for the
resulting polymers.68 Graft copolymerization may be initiated either by chemicals such as ammo-
nium or potassium persulfate (APS or KPS) and ceric ammonium nitrate (CAN) or by γ-irradiation
and enzymes. The grafting parameters such as grafting efficiency are greatly influenced by several
parameters such as the type and concentration of initiator, monomer concentration, reaction time
and time. Combining chitosan with poly(1,4-dioxan-2-one) (PPDO) would provide an amphiphilic
copolymer. Chitosan-g-PPDO copolymer was synthesized in bulk by ring-opening polymerization
of p-dioxanone initiated by the hydroxyl group or amino group of chitosan in the presence of stan-
nous octoate (SnOct2)68 (Scheme 9.1).
The incorporation of PPDO into chitosan would lead to the controllability of physical properties
by controlling the chemical structure of the graft copolymer. Similar architecture was obtained by
grafting ɛ-caprolactone (ɛ-CL) oligomers onto the hydroxyl groups of chitosan via ring-opening
polymerization.69 In this reaction, methanesulfonic acid plays a dual solvent and catalyst role in
the graft polymerization process. Due to protective protonation of amine group of chitosan in
OH OH NH OR OR
O O SnOct2 NHR
2 * O
* O
O * + O *
HO O RO O
O O 100°C–110°C O
H3C C NH OH H3C C NH OR
m n m n
O O
O O
H2 H2 H2 H2 H2
R = H or R = * C C O C O C C O C C OH
p
SCHEME 9.1 Grafting of chitosan with p-dioxanone.
acidic medium, grafting of ɛ-CL takes place mainly on hydroxyl groups of chitosan. The resul-
tant chitosan-g-PCL showed improved solubility in a variety of organic solvents, and easy to be
processed into nanofibers via electrospinning. Owing to the negative membrane potential of cells,
it was promising that these amino-reserved chitosan-g-PCL copolymer fibers could generate a cat-
ionic surface to promote cell adhesion when used as tissue engineering scaffolds. Radiochemical
techniques have recently caught the attention of researchers as an alternate process. Yu et al.70 used
γ-irradiation for grafting butylacrylate. They observed that grafting efficiency increases when the
monomer concentration and total dose increase or when chitosan concentration and reaction tem-
perature decrease. Compared with chitosan films, chitosans grafted with butylacrylate show larger
hydrophobicity and best mechanical strength. Singh et al.71 have grafted poly(acrylonitrile) onto
chitosan using a microwave irradiation technique under homogeneous conditions. They obtained
70% efficiency within 90 s.
Recently, hydrophobically modified glycolchitosan was prepared by partially reacting the
amino groups of glycolchitosan with 5β-cholanic acid by in situ activation using 1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS;
Scheme 9.2).72 EDC catalyzes the formation of amide bonds between the carboxylic acid groups of
organic acids and the amine groups of chitosan.
The enzymatic approach to the modification of chitin and chitosan is interesting owing to its
specificity and environmental advantages compared with chemical modification. With respect to
health and safety, enzymes offer the potential of eliminating the hazards associated with reactive
agents. Chen73 grafted hexyloxyphenol to chitosan using tyrosinase. On the basis of contact angle
measurements, the heterogeneous modification of chitosan film was found to produce a hydrophobic
surface due to the substituent. From the biochemically relevant quinines studied, it would be pos-
sible to prepare materials of medical interest; for instance, menadione, a synthetic naphtoquinone
derivative having the physiological properties of vitamin K, is particularly prone to rapid reaction
with chitosans, greatly modifying its spectral characteristics and increasing the surface hydropho-
bicity of treated chitosan films.74
Finally, another type of reaction was carried out to reduce the hydrophilic character of chitosan:
Bordenave75 reported an oxidation of the primary alcohol to carboxylic group of the chitosan units
to produce a reactive site to further chemical modification such as hydrophobic chain grafting. The
work focuses on the selective oxidation of the primary alcohols mediated by TEMPO. The oxida-
tion was first conducted on the raw chitosan and degradation was observed. This phenomenon
confirmed the necessity of a protection on the amino group.
9.3.1.2 Amphiphilic Block Copolymers

Poly(ethyleneglycol) (PEG) is amphiphilic and non-toxic. As chitosan is hydrophilic, mPEG-PEI-
chitosan block copolymers were synthesized. Poly(ethylenimine) (PEI), a highly branched poly-
amine, is one of the most commonly used polycations for gene delivery. Methoxy PEG (mPEG) was
used to increase the slow-releasing property and water-solubility when PEI was used to improve
OCH2CH2OH COOH
OCH2CH2OH
* O
O +
HO O
NH *
HO O
O C NH2
x y+z
EDC, NHS
3:1 MeOH/H2O
OCH2CH2OH
OCH2CH2OH
* O
OCH2CH2OH
O
HO O
NH * O
HO O
O C NH2 HO O *
x y NH
z
C
O
SCHEME 9.2 Reaction of glycolchitosan with 5β-cholanic acid.
transfection efficiency. To perform this synthesis, mPEG iodide was first prepared,76 then synthesis
of mPEG-PEI was carried out via the mPEG iodide attacking the amino groups of PEI.77 Finally, the
reaction of mPEG-PEI with N-phtaloyl chitosan iodide was performed in DMF.
9.3.1.3 Surfactant-Polyelectrolyte Complex or Polyelectrolyte Complex

PECs are formed by the reaction of a polyelectrolyte with an oppositely charged polyelectrolyte in
an aqueous solution when SPECs are formed by the interaction of a polyelectrolyte with an oppo-
sitely charged surfactant. Essentially this is the result of electrostatic interactions between both
ions (or polyions). They were applied in wide applications.56 Yoshiwaza78,79 prepared PEC films
composed of a cationic chitosan and an anionic polyalkylenoxide/maleic acid (PAOMA) copolymer
to be applied as drug carriers. PAOMA has an excellent biocompatibility and unique amphiphilic
properties and PEC films were demonstrated to have pH- and temperature-sensitivity for equilib-
rium swelling.
The complexes between DNA and chitosan are now under investigation for elaboration of
nanoparticles as gene carriers. It is necessary to have a minimal DP around 6–9 for stability of
such complexes.80 Their stability is reduced above a pH value of 7.4 (close to the physiological pH)
and this is interpreted as one of the reasons for the observed high transfection activity of oligomer-
based complex. The nanoparticles were prepared using a complex coacervation process81 and the
important parameters for this synthesis are concentrations of DNA and chitosan, molecular weights
of polymers, temperature, and pH of solutions. Efficiency was improved by modifying the chitosan
through hydrophobic modification.82
9.3.1.4 Hydrogels
Materials that can be elaborated from these polymeric systems are of two types according to the
cross-linking process. Polymeric self-assembly materials (often called SA) are one of them and they
were extensively studied concerning their autoassociative rheological or their micellar behavior.
They form “hydrogels” using non-covalent cross-linking by exploiting either hydrogen bonding
or hydrophobic interactions. These are biocompatible as gel formation does not require the use of
organic solvents or chemical cross-linking reactions, which may be potentially deleterious to the
active matter load. They are also called “physical gels.”
The “chemical gels” (cross-linked by covalent bonds), the second type of hydrogels, can be
obtained either by employing cross-linking agents such as epichlorohydrin or by grafting/cross-
linking with acrylic monomers. Epichlorohydrin, as many other cross-linking agents, is known
to be cytotoxic.83 Moreover, short cross-linking bridges introduced by such a cross-linking agent
between polymeric backbones would lead to a final product having a high rigidity in dry state. The
increase of cross-linking bridge length can be accomplished by grafting/cross-linking of polysac-
charide with monomers, among which acrylics occupy a significant position.84,85 For this purpose,
the polysaccharide should be at first modified with unsaturated binding functionalities. There are
several advantages of this method, including introduction of longer and/or exhibiting temperature or
pH-sensitive behavior bridges between polysaccharide chains. A less rigid structure, and an easier
control of network density, as a function of number of double bonds introduced on the polysaccha-
ride backbone, may thus be attained.
Among different kinds of hydrogels, “smart hydrogels” are of great interest because they can
undergo a reversible discontinuous phase change in response to various external physicochemical
factors. Among them, temperature- and pH-responsive hydrogels have been the most likely consid-
ered because these two factors can be applicable in vivo. Thermosensitive hydrogels are of great
interest in therapeutic delivery systems and in tissue engineering as injectable depot systems. In
a positive thermo-reversible system, polymer solutions having an upper critical solution tempera-
ture (UCST) shrink by cooling below this temperature. Polymer solutions having a lower critical
solution temperature (LCST) contract by heating above LCST. These negative thermo-reversible
hydrogels can be tuned to be liquid at room temperature (20°C–25°C) and to undergo gelation in
contact with body fluids (36°C–37°C) due to an increase in temperature. Therefore, polymers hav-
ing LCST below human body temperature have a potential for injectable applications. Different
thermal setting gels have been described in the literature including, for example, the acrylic acid
copolymers and the N-isopropylacrylamide (NIPAm).86,87 Poly(N-isopropylacrylamide) (PNIPAm)
is well known to have a thermally reversible property, exhibiting a LCST around 32°C in aqueous
solution.88 For biomedical applications, attention was paid to the development of stimuli-responsive
polymeric gels with unique properties such as biodegradability and biocompatibility. They may be
prepared by combining thermoresponsive polymers with natural-based polymeric components to
form smart hydrogels.89,90 When we have an assembly of two cross-linked polymers and at least one
of which is synthesized and cross-linked in the presence of the other in the linear form, the system
is termed as semi-Inter-Penetrating Network (semi-IPN). A number of polysaccharides were con-
sidered, including chitosan and alginate. Moreover due to the pH-sensitive character of chitosan, a
combination of this polymer with a thermoresponsive material will produce dual-stimuli-responsive
polymeric systems that can be used as delivery vehicles which respond to localized conditions of pH
and temperature in the human body.
Graft copolymerization of NIPAm onto chitosan was carried out using usual initiator such as
AIBN and CAN. Then, chitosan-g-PNIPAm particles were synthesized by a soapless emulsion copo-
lymerization process91 using APS or 2,2′-azobis-(2-methylpropionamidine) dihydrochloride (AIBA)
as initiator. In this case, the swelling ratio of the copolymer decreased with increasing cross-linking
density and pH value. Chemically cross-linked polymers are good candidates to improve wet
strength. PNIPAm-containing chitosan/glutaraldehyde gels were elaborated.92,93 Another example
is glutaraldehyde cross-linked semi-IPN of chitosan and poly(acrylonitrile) (PAN).94 Finally, full-

IPN hydrogel was synthesized by the chemical combination of methylenebis(acrylamide) cross-
linked PNIPAm network with a formaldehyde cross-linked chitosan network.95 Hydrogels based on
PNIPAm-grafted chitosan may be also obtained by applying γ-irradiation.96 Kim et al. synthesized
hydrogels based on grafting chitosan with epoxy-terminated poly(dimethylsiloxane) (PDMS) by
using UV irradiation.97
9.3.2 Properties
9.3.2.1 “Self-Assembly” Properties
Macromolecules may aggregate due to secondary binding forces, solvation, steric factors, and
interpenetration. As reflected by the reaction mechanisms in vivo, specific interactions between
macromolecules are necessary for the display of a functionality regardless of the polymer chain
structure. Amphiphilic polymers generate specific interactions called “hydrophobic” ones. They
are not caused by direct cohesive forces between molecules but by the specific structure of water
molecules. In order to minimize the contact surface area of hydrophobic groups with water, result-
ing in a decrease of the entropy, hydrophobic groups aggregate with each other, leading to a “self-
assembly” phenomenon. The increase in the hydrophobicity of an amphiphilic polymer induces
stronger intermolecular interaction and aggregation. In the chitosan-based aqueous system, there
are electrostatic interaction, hydrophobic interaction, and hydrogen bonding, which will influence
the solubility or the assembly behaviors of chitosan derivatives.98 Hydrophobic modified chitosans
prefer to form self-assembled aggregates in aqueous media. The main microstructural characteristic
of such polymeric systems is their ability to give rise to weak intra- and intermolecular hydropho-
bic interactions in water solutions. Indeed the hydrophobic groups distributed along the macromo-
lecular chain associate to minimize their exposure to water. As an example, hydrophilic polymers
containing a few hydrophobic units can form temporary hydrophobic association networks in aque-
ous media at relatively low concentrations.99 More generally different types of materials may be
elaborated from amphiphilic polymers: micelles, gel-like or fiber-like aggregates, nanoparticles, etc.
In the semi-dilute or moderately concentrated regime, these intermolecular interactions are pre-
dominant and they form a temporary associated network which serves to reinforce the entangled
network. Hence, a large viscosity increase was observed. The tendency for such polymeric systems
to undergo association leads to various interesting and unusual linear and non-linear rheological
behavior, which make them candidates as rheology modifiers in many industrial applications.100 The
preference of intra- or interpolymer hydrophobe association depends on the polymer concentration
and the structural characteristics of the macromolecule, notably the content and the nature of the
hydrophobic groups and the sequence distribution of electrolyte and hydrophobic monomer units
along the polymer chain. These effects and the ionic strength of the system are factors that are also
important55 for the association efficiency and subsequently the dynamical behavior of solutions of
hydrophobically modified polyelectrolytes. It is possible to adjust the chemical structure (content of
hydrophobic and hydrophilic moieties) to reach desired properties.
Rheological curves (variation of the viscosity as a function of the shear rate) are determined for
different polymer concentrations at different temperatures. At constant temperature and polymer
concentration, the chitosan solution has a Newtonian behavior when the alkylchitosan solutions are
more and more viscoelastic when the hydrophobicity (related with the length of the alkyl chain)
was increased.101 For the longest alkyl chain, a yield stress fluid was observed related to the forma-
tion of a gel as will be shown later and no Newtonian plateau was described in the used shear rate
range. The higher the hydrophobic properties of the macromolecular chain, the larger the gap to
the Newtonian behavior. Considering the viscosity at a low shear rate as a function of the polymer
concentration (Figure 9.2), the viscosity rises with increasing polymer concentration. This increase
is stronger as the hydrophobicity of the polymer increases (as the length of the alkyl chain or the
104
Chitosan
CC8 5%
CC10 5%
1000 CC10 12%
CC12 5%
Viscosity (mPa s)
100
10
1
10–2 10–1 100 101 102
Concentration (g/L)
FIGURE 9.2 Role of polymer concentration on the zero-shear rate viscosity of chitosan and hydrophobic
derivatives solutions (solvent AcOH 0.3M/AcONa 0.05 M, T = 298 K).
degree of substitution). Moreover, the concentration at which this increase is significant compared
with the chitosan curve decreases when the hydrophobicity increases. This is due to the presence
of interactions between polymer chains. The association effect on the viscosity becomes gradually
more marked as the polymer concentration increases. This threshold concentration was sometimes
called as the critical aggregation concentration (cac) and this viscosity increase was observed for
polymer concentration lower than the overlap concentration. The viscosity increase is concomitant
with the apparition of hydrophobic domains in the solution and it is shown that the junction points
for the 3D-network formation are constituted of alkyl aggregates looking like a micellization.101
As the hydrophobic interactions are favored by temperature, it may be expected to obtain formu-
lations presenting either thermal stability or a viscosity increase with temperature as already dem-
onstrated for methylcellulose102 or polyacrylate polymers grafted with poly(ethylene oxide) chains103
as examples, these polymers belonging to the class of thermoassociative polymers. According to
the structure of the polymer and the polymer concentration, temperature stable formulations or
formulations presenting a viscosity increase with temperature may also be obtained.104 Hence, it
is possible to adjust the structure of the amphiphilic chitosan to the required rheological behavior.
Application to cosmetic formulations was proved and solutions of alkylated carboxymethylchitin
were used to stabilize such formulations. These are stable even when the temperature is increased
and sensorial evaluation was found to be improved.105
In more concentrated media, self-assembly leads to aggregation. The morphologies of aggre-
gates produced from amphiphilic polymer self-assembly can be diverse, such as liposomes, micro-
sphere, vesicles, rods, lamellas, tubes, and large compound micelles.106–110 They can be tailored by
changing the hydrophobic/hydrophilic ratio or preparation conditions, since the self-assembly pro-
cess has proven to be dominated by both thermodynamic and kinetic controls. The self-assembled
polymer aggregates with unique structures and surface properties lead to a wide range of industrial
applications, especially in pharmaceutical such as drug targeting devices, large quantity guest mol-
ecule encapsulation and gene therapy.111–114 The effect of the descendible groups of chitosan deriva-
tives on the aggregate morphology is far from well-understood. Chitosan-based self-aggregates
were difficult widely apply to drug delivery systems because chitosan aggregates are insoluble
in biological solution (pH ∼ 7.4) and they are readily precipitated within a few days. To increase
their stability in biological solution and decrease the cytotoxicity by acidic solution, where chito-
san is soluble, water-soluble chitosan derivatives such as glycol chitosan were synthesized. It was
then hydrophobically modified with deoxycholic acid115 and nanoparticles were obtained from
self-aggregation. The mean diameter of self-aggregates was controllable by changing the degree
of substitution of deoxycholic acid in the range of 245–450 nm. They are stable for 10 days in
physiological conditions and they provide the availability of nanoscale containers for hydrophobic
drugs and genes. N-succinyl-chitosan can self-assemble regular nanosphere morphology in distilled
water.116 Within these nanospheres, there are hydrophobic domains formed and as they have non-
toxic and cell-compatible properties, they present a great potential as a drug matrix in the controlled
release delivery. Similarly hydrophobically modified glycolchitosan may form nanoparticles.72,117
They have a globular shape with an average diameter of 350 nm. They present an enhanced distri-
bution in the whole cells compared to the parent hydrophilic glycol chitosans. N-octyl-O-sulfate
chitosan can therefore be used as a potential drug carrier.118 To develop chitosan-based efficient
gene carriers, highly purified chitosan oligosaccharides were chemically modified with deoxycho-
lic acid. Owing to the amphiphilic character, the modified oligochitosans formed self-aggregated
nanoparticles in aqueous medium.119 Their physicochemical characterization revealed that the par-
ticle size of the nanoparticles was in the range of 200–240 nm and the cac was 0.012–0.046 g/L,
depending on the degree of substitution.
N-maleoylchitosan was able to self-assemble, at concentrations higher than the cac, as fiber-like
aggregates with an averaged diameter of around 2.5 μm and length of more than 100 μm.120 These
fiber-like aggregates have potential application in tissue engineering scaffold.
Amphiphilic copolymers consisting of hydrophilic and hydrophobic segments can finally form
micelle structures with the hydrophobic inner core and the hydrophilic outer shell in aqueous media.
The hydrophobic inner core is surrounded by a hydrophilic outer shell, which provides a stabiliz-
ing interface between the micelle core and the aqueous environment. Adjusting the structure of
the amphiphilic copolymers, the size and morphology of the polymeric micelles can be easily con-
trolled. In addition, polymeric micelles were also considerably more stable than surfactant micelles.
Thus, these core-shell-type micelles may be used as drug delivery vehicles for poorly water-soluble
drugs, especially when the micelles are made with suitable biodegradable polymers. By reacting
dl-lactide (DLLA) and water-soluble chitosan in dimethylsulfoxide solution, an amphiphilic graft
copolymer was synthesized121 and micelles in water were obtained. According to the composition of
the copolymer, the critical micelle concentration (cmc) was between 10 −2 and 10 −1 g/L. These values
are lower than the critical micelle concentration of low molecular weight surfactants, indicating the
stability of micelles from graft copolymers at dilute conditions. The increasing hydrophobicity by
introduction of a large amount of hydrophobic groups further reduces the cmc values. The size of
polymeric micelles was measured by dynamic light scattering and it varies between 150 and 200 nm.
Finally, modifying sulfated chitosan by adding long alkyl chains to the amino groups at C-2 induces
the creation of an amphiphilic molecule that forms micelles in water and therefore offers the poten-
tial to increase the solubility of poorly soluble drugs. With N-alkyl-O-sulfate chitosan (the length
of the alkyl chain being between 8 and 12 C atoms) polymeric micelles of size around 100–400 nm
were formed in water.118
9.3.2.2 Interfacial Properties

Interfacial or surface properties of such materials depend on the interactions that these may build
with other media or molecules. As many of interesting media may be hydrophobic (or contain
hydrophobic moieties), the amphiphilic character of used derivatives is of great importance. These
interactions allow adsorption of hydrophobic active matter on surfaces, the formation of stable com-
plexes, the ability of particles to migrate through membranes, etc.
Cationic polyelectrolyte chitosan (Ch) as well as anionic carboxymethylchitin (CMCh) has no
tensioactive properties. This corresponds to the known property of weakly hydrophobic polyelectro-
lytes to manifest poor adsorption activity at both oil–water and air–water interfaces at relatively high
degree of ionization.122,123 But the surface activity of polyelectrolytes may be modified by preparing
an amphiphilic chemical (as surfactants). As an example the alkyl carboxymethylchitin presents
80
70
Surface tension (mN/m)
60
CMCh
50 CMCh-C14
Z = 0.001
Z = 0.03
TTAB
40
30
–6 –5 –4 –3 –2 –1
Polymer or surfactant concentration, log C (monomol/L)
FIGURE 9.3 Surface tension isotherms of solution as a function of concentration of TTAB, chitin deriva-
tives and SPECs (solvent: water, T = 298 K).
some activity when the polymer concentration is larger than 10 −3 monomol/L for C14 alkyl chain and
a degree of substitution of 0.04 (CMCh-C14, Figure 9.3). This critical concentration depends upon
the length of the alkyl chain and the degree of alkyl substitution and this effect is larger with the
hydrophobicity (e.g., longer alkyl chain for the same degree of alkylation).124 This is not surprising
taking into account that by increasing the hydrophobicity of the polysoaps their adsorption at the
air–water interface is expected to be increased. Similar observations were done on (2-hydroxyl-3-
dodecanoxyl)propyl-carboxymethylchitosans (HDP-CMCHS).125 The introduction of hydrophobic
substituents made the resulting derivatives become amphiphilic polymers, which decrease the sur-
face tension. But different from the low molecule surfactants, the decrease of surface tension is slow
and no clear transition points on the surface tension–concentration curve. Moreover, the addition
of NaCl to the DHP-CMCHs solution can make the surface tension lower. It is because that the
electrolyte can compress the electric double layer of the anionic carboxymethyl group on the hydro-
philic backbone of the DHP-CMCh. So it is easy for the polymers to concentrate on the surface of
the solution to reduce surface tension and to form aggregates by hydrophobic group association in
solution as already observed from other amphiphilic derivatives.102
But the addition of a cationic surfactant (tetradecyltrimethylammonium bromide, TTAB, with a
C14 alkyl chain as CMCh-C14) able to interact with the highly charged anionic polymer causes a sig-
nificant effect. With carboxymethylchitin, ion pairs are formed in the presence of TTAB by electro-
static interactions and tensioactive properties are affected (Figure 9.3). The effect is directly related
to the stoichiometric ratio Z (defined as Cs/Cp, Cs being the concentration of surfactant molecules
in mol/L and Cp, expressed in base-mol/L, the polymer concentration): the higher Z gives the lower
surface tension at a given monomole concentration; in addition, it goes to a characteristic limit over
a critical monomole concentration depending directly on Z. This quantity corresponds to a cac much
lower than the critical micellar concentration (cmc) of TTAB (cmc ∼ 4.10 −3 mol/L). It was concluded
that strong interactions were due to a mixed micelle formation including polyelectrolyte and surfac-
tant molecules.126–128 Comparing the isotherms σ(Cp) for the alkylated CMCh sample with those of
SPECs formed between nonalkylated CMCh and TTAB at different compositions Z of mixed solu-
tions (Figure 9.3), one concludes that from the point of view of their effect on the surface activity
the SPECs reveal to be formally much more effective than the alkylated CMCh. The SPECs formed
in a mixed solution with the composition Z ≅ 0.001 produce the same effect on the surface tension
as the alkylated samples (polysoaps) with degree of alkylation τ = 0.04 (for C14 chain), meaning that
40 times less of hydrophobic C14 alkyl chains is necessary to reach similar tensioactive properties.
With the approximate same content of hydrophobic groups (Z = 0.03), the tensioactive properties
are much more improved. This is probably related to the impossibility for the adsorbed alkyl side
chains of the alkylated CMCh to form a dense packing at the interface because of the steric hin-
drance originating from the local stiffness of macromolecule backbone. The nonalkylated pieces
of the polymeric backbone localized between the neighboring alkyl side chains “anchored” at the
interface cannot form the “loops” if their average length l is not large enough. In contrast, the non-
alkylated CMCh macromolecules form electrostatic dynamic associations with alkyl chains of the
surfactant macromolecules, which are considered as more labile. As an expected result, more alkyl
chains of bound surfactants could be anchored at the interface. This explains why the surface activ-
ity of the alkylated samples of CMCh reveals to be relatively low with regard to the nonalkylated
CMCh which forms the dynamic complexes SPECs of the same expected content of alkyl chains of
oppositely charged TTAB molecules. The very small value of the cac, compared to the cmc of the
surfactant in pure solution, leads to the use of smaller quantities of surfactants to observe surface
activity (Figure 9.3). This regime of very small addition of surfactants is the important domain
for all the interfacial mechanisms in relation with the stabilization of emulsions and foams. With
chitosan-SDS complexes, similar results were obtained.
Moreover, using the drop tensiometry technique,129 the kinetics of formation of the film and the
viscoelastic properties of the interfacial film may be studied. According to the hydrophobicity and
the steric hindrance, the kinetics is not the same. Different observations were interpreted in terms
of the formation of a gel-like structure in the adsorption layers of polysoaps and SPECs.127,130 It
has been found that the elastic modulus of the adsorbed layer increases with increasing alkyl chain
content in the case of both polysoaps and SPECs, but unexpectedly, the kinetics of the adsorp-
tion and the elasticity of the SPEC adsorption layer are remarkably higher with regard to that of
polysoaps. This is explained by higher diffusion mobility of alkyl groups of surfactants inside the
SPEC with regard to that of polysoaps allowing the formation of more compact gel-like structure
in the adsorption layers. It has been found that the conformation of the polysoap macroion and of
SPEC in the bulk of solution has an impact on the kinetics of the structure formation in the adsorp-
tion layers.
Finally, wettability of surfaces was determined from static water contact angles on the films.
9.3.2.3 Interactions
Specific interactions of amphiphilic chitosan derivatives may lead to particular behavior and
properties. As an example interactions between proteins (bovine serum albumin, BSA) and
N-succinyl chitosan (NSCS) were characterized and it was demonstrated that chain entangle-
ments are easily formed between BSA and NSCS and mainly driven by H-bond and hydrophobic
interactions.131 The NSCS entrapped BSA shows nanosphere morphology with a 100–200 nm
diameter at high concentration of NSCS. The conformation of BSA entrapped in the matrix of
NSCS does not change significantly, suggesting that NSCS is a potential matrix to load proteins
or other bioactive drugs.
It may be mentioned that the association of chitosan with hydrophobic (or less hydrophilic) com-
pounds such as fatty acid allows increase film resistance to water transmission.132
9.3.2.4 Swelling
Swelling of hydrogels in water and aqueous solutions is of great importance in relation with bio-
medical applications. It allows inclusion of active drugs and then their release considering diffu-
sion process. The swelling behavior of a material refers to the case when water molecules enter
the material and combine with the hydrophilic groups in the material molecules. Consequently,
the content of hydrophilic groups and the intermolecular force are the two main factors that influ-
ence the material’s swelling property. On one hand, the increase of the substitution degree lessens
the intermolecular hydrogen bonds and reduces the compactness of the network. This will raise
the swelling ration. On the other hand, the hydrophobicity of the network is enhanced upon increas-
ing the amount of the hydrophobic groups. The increased hydrophobicity will result in a decrease
of the swelling ratio.
When chitosan-g-PNIPAm hydrogels are more specially considered, below the LCST, the increase
of either the chitosan/NIPAm weight ratio or the cross-linking agent decreased their swelling ratio.
Indeed, both PNIPAm and chitosan have a hydrophilic behavior in this temperature range when for
a temperature above LCST, PNIPAm was hydrophobic and difficult to swell in aqueous medium.
So the hydrophilic behavior of chitosan dominated the swelling ratio of chitosan-g-PNIPAm. The
increase of the chitosan/NIPAAm weight ratio increased the swelling ratio of the copolymer.56
9.4 BIOMEDICAL APPLICATIONS OF AMPHIPHILIC SYSTEMS

9.4.1 Drug Delivery
Chitosan has interesting biopharmaceutical characteristics such as pH sensitivity, biocompatibility,
and low toxicity.133,134 Moreover, chitosan is metabolized by certain human enzymes, especially
lysozyme, and is biodegradable.133 As a consequence, the use of chitosan and its derivatives in drug
delivery applications was extended. Moreover, in such applications it is also extremely important
that chitosan be water-soluble and positively charged. This character enables it to interact with nega-
tively charged polymers in an aqueous medium. But the limitation of chitosan for the preparation of
sustained release systems arises for its rapid water absorption and high swelling degree in aqueous
environments leading to fast drug release. In order to overcome these problems, chitosan was modi-
fied to increase its hydrophobicity. As mentioned earlier, self-assembly of hydrophobic polymers
allows elaboration of materials with different morphologies.
Park et al.135 elaborated glycol chitosan nanoparticles modified with hydrophobic bile acid ana-
logs from self-assembly. They comprise the hydrophilic shell of glycol chitosan and hydropho-
bic multicores of bile acid analogs and they are promising therapeutic agents in the anticancer
drug delivery system.136,137 Varying the molecular weight of the polymer, the mean diameters of
the nanoparticles (ranging from 230 to 310 nm when the molecular weight is between 20,000 and
250,000 g/mol) did not differ significantly. Furthermore, their in vitro colloidal stability and sur-
faces charges were similar. However, the higher molecular weight glycol chitosan particles circulate
in the blood for longer periods of time and display higher tumor selectivity compared to those with
lower molecular weight. This pharmacokinetic alteration in blood circulation may be associated
with increased in vivo stability on nanoparticles, leading to high tumor selectivity. Particles elabo-
rated from hydrophobically modified glycol chitosan with 5β-cholanic acid had a globular shape
with an average diameter of 360 nm.72 The internalization efficiency of Cy5.5-labeled nanoparticles
(Cy5.5, a monofunctional cyanine as a carboxylic derivative being a fluorophore) in the HeLa cell
cultures was higher than that of the glycolchitosan polymer analog or the control Cy5.5. The higher
binding affinity of the hydrophobically modified glycol chitosan nanoparticles at the cellular mem-
brane may account for their facile internalization. These nanoparticles possessing tunable physico-
chemical properties, low toxicity, and biocompatibility and exhibiting a fast cellular uptake through
various routes are promising macromolecular carriers. N-succinyl chitosan was demonstrated to
have potential applications for encapsulation of protein and other hydrophilic bioactive drugs.131
Chitosan-g-PPDO (poly(p-dioxanone) ) copolymer was synthesized by ring-opening polymeriza-
tion of p-dioxanone.68 The incorporation of PPDO into chitosan would lead to the controllability of
physical properties by controlling the chemical structure of the graft copolymer. This copolymer
was used as Ibuprofen carrier. The higher the degree of polymerization (DP) of PPDO grafts, the
lower the cumulative release percentage, and it was observed that its release rate decreased com-
pared with that of pure chitosan carrier. Those phenomena indicated that the release of ibuprofen
was controlled by the diffusion and that the hydrophobicity of the carrier was crucial. The higher
DP value made the copolymer more hydrophobic. The strong hydrophobicity of the carrier made
water penetration difficult, and the drug release slowed. N-lauryl carboxymethylchitosan with both
hydrophobic and hydrophilic groups was studied in connection with the delivery of taxol to cancer-
ous tissues.59 Chitosan-based systems bearing β-cyclodextrin cavities were considered as a matrix
for controlled release.138,139 Due to the presence of the hydrophobic cyclodextrin rings, these systems
provide a slower release of the entrapped hydrophobic drugs.
Chitosan was modified with alkyl halides bearing different hydrophobic alkyl side chain140 and
delivery of vitamin B2 was considered.141 Liu et al.142 examined the use of N-(aminoalkyl)chitosan
microcapsules as a carrier for gene delivery. It was reported that N-(aminoalkyl)chitosan-modified
poly(lactic acid) films (PDLLA) improved the biocompatibility of PDLLA.143 Li et al.144 prepared
N-butylchitosan and it was demonstrated that the mechanical properties may be strengthened and
the biocompatibility of materials with osteoblasts improved, leading this material to be a promising
candidate for bone regeneration. Using the same chemical reaction (reductive amination), Taboada
et al.145 prepared new arylamine chitosan derivatives for trapping proteins.
Other systems were considered as non-covalent gels (or physical gels) based on chitosan; for
example, the amphiphilic vesicle formed from palmitoyl glycol chitosan.146 Varying the hydropho-
bicity of the gel material leads to variations of gel hydration, gel erosion, bioadhesion, and the
release of entrapped macromolecules. Chitosan derivatives are ideal candidates materials for use
in the fabrication of buccal drug delivery dosage forms. Indeed, polycations (such as chitosan) are
reported to increase epithelial permeability by enhancing paracellular transport.147,148 Systems com-
bining chitosan and PNIPAm have drug release profiles which can be controlled by both pH and
temperature,149,150 constituting very promising materials.
Polyelectrolyte complexes (PEC) films prepared from chitosan and polyalkyleneoxide-maleic
acid copolymer (PAOMA) were shown to change drug release rate in response to change in envi-
ronmental pH.78 The drug release was higher in pH 7.2 media compared to pH 3.8 because of the
increase of repulsive force between carboxyl groups in PAOMA and anionic groups in model drugs
(salicylic acid) and the increase of effective diffusion area. Temperature-sensitive drug release stud-
ies showed that the increase in temperature from 25°C to 50°C yielded the increase in the rate
of drug release when using PAOMA with molecular weight of 20,000 and a cloud point equal
to 35°C. This is attributed to the increase of release area due to the phase transition of PAOMA
and the increase of repulsive forces between carboxyl groups in PAOMA and anionic groups in
model drugs.
N-alkyl-O-sulfate chitosan derivatives carrying long alkyl chains (n = 8, 10, or 12) as hydropho-
bic moieties and sulfated groups as hydrophilic ones were used to elaborate polymeric micelles.118,151
Taxol, a water-insoluble anticancer drug, was solubilized into the polymeric micelles by physical
entrapment. The taxol concentration in the docyl derivative micellar solution was around 2 mg/mL,
which is much higher than in water (<0.001 mg/mL). Another water-insoluble drug, Paclitaxel, was
successfully tested.151 As a consequence, N-octyl-O-sulfate chitosan may be used as a potential drug
carrier. According to the nature of the drug to be released, it is obvious that the nature of the chito-
san derivative can alter its profile release. By tailoring the properties of the polymer (hydrophilic/
hydrophobic in our context), the desired drug release rate can be obtained. As an example, for bone
implant and calcein release, a higher percentage of drug was released when the hydrophilic polymer
N-octyl-sulfated chitosan was present in tablets compared with the tablets containing the hydropho-
bic polymer N-octyl chitosan.152 Through grafting dl-lactide onto chitosan, leading to amphiphilic
chitosan-polylactide graft copolymers, polymeric micelles were prepared.121 As these micelles have
a hydrophobic core, they are used as delivery carrier for the entrapment and controlled release of
hydrophobic drugs.
Chitosan was hydrophobically modified by cholesterol to obtain amphiphilic cholesterol suc-
cinyl chitosan conjugates, which are used as the anchoring materials to coat on the liposome
surface.153 Indeed, chitosan coated liposomes (CCL) were hoped to be used as the carrier of anti-
cancer drugs, but the chitosan layer formed by the electrostatic attractive interaction between
the positively charged amino groups in chitosan molecules and the negatively charged surface
of liposomes was not effective enough to significantly stabilize liposomes in vitro. To improve
this stability hydrophobically modified chitosan derivatives (such as cholesterol succinyl or pal-
mitoyl conjugates) were used as anchoring materials. Chitosan anchored liposomes (CAL) demon-
strate larger sizes, higher zeta potential, better physical stability, and more sustained drug release
behavior. Epoxypseudoisoeugenol-2-methyl butyrate (EPB) was chosen as a model anticancer drug
to assess the potential of chitosan derivative anchored liposome as a novel carrier of anticancer
drugs. Current results showed that cholesterol succinyl chitosan anchoring on the surface of EPB-
loaded liposomes could significantly improve their physical stability and sustain the release of EPB
in vitro.
Hydroxypropylchitosan and dihydroxypropylchitin154 were considered for release of oxytetracy-
cline and it may be adjusted according to the nature of the chitin derivative.
Sui et al. have synthesized amphiphilic derivatives of succinyl-chitosan ( (2-hydroxypropyl-3-
butoxy) propyl-succinyl chitosan).155 It can concentrate on surface to decrease the surface tension
and can associate with hydrophobic chains to form aggregates in solution. These derivatives may be
used in controlled release of hydrophobic drug.
As already discussed SPECs may be obtained from ionic derivatives of chitin or chitosan and
oppositely charged surfactant. The physical gels based on SPECs may be produced in the form of
microbeads and capsules by the diffusion enhanced gelation.156,157 When a droplet of an aqueous
chitosan solution containing bioactive substance (as enzymes) falls into a receiving aqueous solu-
tion of oppositely charged surfactants (or polyelectrolytes), the gel-like wall of SPECs forms at the
interface, whose thickness grows with time and finally the droplet transformed into a microcapsule.
The gel-like wall of these capsules contains micelle-like aggregates with highly ordered nanostruc-
ture evidenced by small-angle X-ray scattering.158 These capsules and microcapsules made by this
method are polyfunctional: they may be loaded by enzymes in their aqueous core and contain in
their gel-like walls the micelle-like aggregates able to be solubilized by the oil-soluble drugs. A pre-
liminary study on the immobilization of enzymes (α-chymotrypsin, urease, etc.) in capsules made
of chitosan-SDS complexes159 has shown that these enzymes conserve their activity after encapsula-
tion in spite of the possible contact with detergent molecules inside the gel.
9.4.2 Gene Transfection
Gene therapy is to introduce therapeutic genes into specific cells for the purpose of correcting the
dysfunction of specific genes or over-expressing therapeutically useful proteins to take curative
effects for human diseases or to provide immunologic protection. Potential clinical application of
gene therapy depends greatly on the development of safe and efficient delivery systems, to which
the most distinct drawback is the lack of effective gene ferrying vectors. Non-viral vectors have
been attracting increasing research efforts owing to the consideration of safety and feasibility for
manufacturing. Cationic polymers are among the most attractive non-viral gene carriers, which
include PEI, polyallylamine, poly-l-lysine (PLL), and chitosan.160 The advantage of chitosan-based
vectors lies not only in avoiding cytotoxicity problems that are inherent in most synthetic polymeric
vehicles but also in their unique capability for transcellular transport. The oligochitosans are water
soluble in a wide range of pH and are still able to condense DNA. Nevertheless, recent reported
data161,162 have shown a rather limited transfection efficiency of oligochitosans in vitro as compared
to cationic surfactants. These observations have been made also with quaternized forms of these
oligomers where quaternary amino groups have been added in order to increase both their solubility
and their electrostatic interaction with oppositely charged DNAs.163 This rather low transfection effi-
ciency is assumed to be due to low hydrophobicity of condensing agents, the oligochitosans, which
produce the electrostatic condensation of DNA into the relatively bulky coil-like complexes. The
higher transfection efficiency is attributed to increased entry into cells facilitated by hydrophobic
interactions and easier unpacking of DNA from alkylated chitosans due to the weakening of elec-
trostatic attractions between DNA and alkylated derivatives.
Indeed, the factors that are supposed to play an important role in the transfection efficiency of
cationic surfactants are, primarily, the formation by these surfactants of micelle-like aggregates via
hydrophobic interaction between the alkyl chains, and, secondly, the interaction of alkyl chains with
the bilayer membranes of living cells by insertion of alkyl chains into the membrane hydrophobic
core. The first factor, the hydrophobic attraction between the alkyl chains of cationic surfactants
electrostatically bound to the oppositely charged DNA macroions, induces a transition (a collapse)
from a coil-like conformation of DNA-surfactant complexes into smaller size and stable globule-
like aggregates.164 Smaller is the size of the complexes, higher is their transfection efficiency. Unlike
cationic surfactants, cationic oligochitosans are giving only bulky coil-like electrostatically sta-
bilized complexes with DNA macroions, with low transfection efficiency. The second factor, the
hydrophobic interaction with the membrane bilayer, allows the absorption of the DNA-surfactant
complexes onto the living cell surface by insertion of the alkyl chains of surfactants into the mem-
brane core. This hydrophobic attachment is assumed to increase the ability of the DNA to pen-
etrate into the cytoplasm. Unlike the surfactants, the oligochitosans whose electric charge is already
compensated within the DNA-oligochitosan complexes are not able to interact with the negatively
charged membranes of living cells even by electrostatic attraction.
Thus, covalent grafting of alkyl chains to oligochitosans is believed to increase, on one hand, their
ability to form stable and small-size complexes with DNA and, on the other hand, the adhesion of DNA-
oligochitosan complexes to the living cell surface. At the same time, the alkylation of oligochitosans is
expected to decrease their solubility and their instability toward the hydrophobic aggregation in water.
For example, as it has been reported earlier,165,166 the alkylation of high molar mass chitosans decreases
their solubility in aqueous media and consequently leads to the formation of complexes of larger size
including ∼50–100 DNA molecules per complex. Assuming that the solubility of the alkylated chito-
sans can be improved by the decrease of their molecular weight, a series of hydrophobically modified
oligochitosans with different degree of covalent substitution of C12-chains in the form of N-/2(3)-
(dodec-2-enyl)succinoyl groups were prepared167 using a process already described by Hirano et al.168
Chitosan oligosaccharides were chemically modified with deoxycholic acid (DOCA).119 They are
able to form core-shell-type nanoparticles in aqueous medium. Compared to unmodified chitosan
oligosaccharides, the hydrophobic derivative based nanoparticles showed elevated potential as gene
carriers by efficient DNA condensation, and protection of the condensed DNA from DNase I attack.
Based on a chitosan oligosaccharide of molecular weight in the range 1000–3000, more than 20 and
100 times enhanced gene transfection was observed by the particle mediated gene delivery than that
of PLL in the absence and presence of FBS, respectively. Although the slightly lower pH value than
physiological value for the efficient transfection may prevent systemic applications, hydrophobically
modified chitosan oligosaccharides-mediated gene transfections can be a potential methodology
for local gene deliveries such as mucosal and intestinal gene delivery. The self-aggregate DNA
complex from deoxycholic acid-modified chitosan was shown to enhance the transfection efficiency
over monkey kidney cells.169 Self-aggregates can form charge complexes when mixed with plasmid
DNA. These self-aggregate DNA complexes are considered to be useful for transfer of genes into
mammalian cells in vitro and served as good delivery system composed of biodegradable polymeric
systems. PEGylation of chitosan in order to increase its solubility, elongate the plasma circulation
time, and prolong the gene transfer has been another proposed technique for the sustained DNA
release. Microspheres physically combining PEG-grafted chitosan (PEG-g-Chi) with poly(lactide-
co-glycolide) (PLGA) were formulated by Yun et al.170 They reported that these microspheres were
capable of sustained release of PEG-g-Chi/DNA for at least 9 weeks, and the rate of DNA release
was not modulated by varying the amount of PEG-g-Chi.
The interaction of LMW-DDC-chitosans with model lipid membranes has been tested.
The decrease of membrane fluidity (increase of membrane microviscosity) in the liquid crystalline
phase induced by the interaction with LMW-DDC(18%)-chitosan molecules may be explained by
the insertion of the dodecyl chains covalently bound to the glucosamine backbone into the mem-
brane bilayer along to the fatty acid chains. Unlike the low molecular weight cationic detergents
which exhibit some toxicity because of their high lateral mobility at the interface and their flip-
flop ability between the two membrane leaflets, the LMW-DDC-chitosans are obviously much less
prone to move laterally and across the bilayer, which probably explains their low toxicity.82
Cationic methoxy poly(ethyleneglycol) (mPEG)-polyethylenimine (PE)-chitosan was synthe-
sized77 and it showed a good DNA condensation capability, exhibiting negligible toxicity. The trans-
fection of human embryonic kidney 293 (HEK 293) cells proved that mPEG-PEI-chitosan/VRaft-1
plasmid has little toxicity on the growth and gene expression of cells. Using this copolymer as
transfection agent, the conversion of ω-6 to ω-3 fatty acids was successfully realized. Based on
these superior properties, mPEG-PEI-chitosan should be a promising non-viral gene carrier for
biomedical and biological applications.
Self-assembled nanoparticles were prepared using a hydrophobically modified glycol chitosan
obtained by the reaction of the amino group with 5β-cholanic acid.117 The modified chitosan sponta-
neously formed DNA nanoparticles by a hydrophobic interaction between hydrophobically modified
glycolchitosan (HGC) and hydrophobized DNA. As the HGC content increased, the encapsulation
efficiencies of DNA increased while the size of HGC nanoparticles decreased. Upon increasing
HGC contents nanoparticles became less cytotoxic. The increased HGC content also facilitated
endocytic uptakes of HGC nanoparticles by COS-1 cells. Moreover, the HGC nanoparticles showed
increasing in vitro transfection efficiencies in the presence of serum. In vivo results also showed that
the HGC particles have superior transfection efficiencies to naked DNA. Animal studies confirmed
that HGC nanoparticles could be used as a potent gene delivery vehicle in vivo.
Low molecular weight chitosans grafted with N-/2(3)-(dodec-2-enyl)succinoyl groups with an
average molecular weight of 5000 were considered as a new class of non-viral vectors for gene
therapy.82 These molecules were found to form micelles through hydrophobic interactions involving
their tetradecenoyl chains and non-protonated glucosamine units. Interaction with large unilamel-
lar vesicles taken as model membranes indicated that these chitosan derivatives interact mainly
with vesicles mimicking the inner leaflet of biomembranes both through electrostatic and hydro-
phobic interactions. This preferential interaction may destabilize endosomal membranes and favor
the DNA release into the cytoplasm in gene delivery applications. Moreover, since this interaction
significantly decreased the membrane fluidity of these vesicles, the hydrophobically modified chito-
sans are thought to exhibit limited lateral mobility and flip-flop ability, and thus limited cytotoxicity.
Other derivatives such as N-methylenephosphonic chitosan (NMPCS) were synthesized and
investigated as non-viral vector.171 NMPCS/DNA or chitosan (CS)/DNA complexes were prepared
using a complex coacervation method. MTT assay was employed to evaluate the cytotoxicity of the
polymers and pGL3-control luciferase plasmid was utilized as a reporter gene to assess the trans-
genic efficacy of the polymers.172 It was demonstrated that NMPCS was able to fully entrap the
DNA at N/P ratio of 2:1, whereas chitosan entrapped the DNA completely at N/P ratio of 1:1. The
expression of transgene mediated by NMPCS was much higher (more than 100-fold) than that medi-
ated by chitosan, indicating that NMPCS was more efficacious gene ferrying vector than chitosan.
9.4.3 Tissue Engineering
Tissue engineering research is nowadays based on the seeding of cells onto porous biodegrad-
able polymer matrixes. Chitosan and its derivatives have been reported as attractive candidates
for scaffolding materials because they degrade as the new tissues are formed, eventually without
inflammatory reactions or toxic degradation.169,173 In tissue engineering applications, the cationic
nature of chitosan is responsible for electrostatic interactions with anionic glycosylaminoglycans,
proteoglycans, and other negatively charged molecules. Mixing biological properties of chitosan
and mechanical properties of l-poly(lactic acid) (PLLA) may be of promising interest. Surface
functionalization of biodegradable PLLA was performed by plasma coupling reaction of chitosan
with PLLA.174 The proliferation and morphology studies of ell lines (mouse fibroblasts and human
hepatocytes) cultured on this surface showed that these cells hardly spread and tended to become
round, but could proliferate at almost the same speed as cells cultured on glass surface. More
recently, PLLA-chitosan hybrid scaffolds were proposed as tissue engineering scaffolds and simul-
taneously drug release carriers.175 In this system, a chitosan porous structure, in which cells and
tissues would mostly interact, is created within the pore structure of a stiffer PLLA scaffold.
N-maleoylchitosan was also considered for such applications.121 The sample with a maleoyl sub-
stitution degree of 70% was synthesized from the reaction of maleic anhydride and the amino group
of chitosan. With increasing concentration to around 0.5 mg/mL, which is 10 times the cac, these
molecules self-assemble to fiber-like aggregates. The nanofibrous structure facilitates the attach-
ment of human osteoblasts and chondrocytes. Therefore, these fiber-like aggregates might have
potential applications in tissue engineering scaffold.
By grafting ɛ-caprolactone oligomers onto the hydroxyl groups of chitosan, chitosan-graft-
polycaprolactone (PCL) copolymers were achieved.69 The specimen synthesized from the feed
molar ratio of glucosamine units in chitosan versus ɛ-caprolactone as 1:12 showed equivalent cyto-
toxicity to the neat chitosan and PCL against KB (human carcinoma) cell line, and the cell viability
was almost close to 100%. In addition, chitosan-g-PCL exhibited good solubility in organic sol-
vents, facilitating the formation of PCL/chitosan-g-PCL blend nanofibers via electrospinning with
the use of DMF/CHCl3 as solvent. Owing to the enhanced cellular attachment from cationic amino
groups, it is promising that these copolymers are interesting substances for developing drug carriers
and tissue engineering scaffolds.
9.4.4 Cells and Proteins Adhesion and Other Applications

Surface modification of biomaterials was significantly studied since it is the surface of such materi-
als that first comes into contact with the biological surrounding. The change of surface property was
found to affect adsorption of platelets, cells, and biomacromolecules such as proteins on the poly-
mer surface. Numerous techniques were used to alter the chemical composition and thus the sur-
face property of the materials (plasma treatment…). Grafting of polymer or oligomer species was
considered and especially of monomethoxyethylene glycol oligomers on chitosan surface.176 The
hydrophobicity of the modified surface, determined by air–water contact angle, decreased when
the ethyleneglycol derivatives were grafted on the film. The presence of such oligomers reduced
the adsorption of proteins (albumin and lysozyme) on the films. PEG was recognized to suppress
non-specific protein adsorption. Grafting was obtained from similar reaction than the elaboration
of alkylated derivative (reductive amination). The choice of the chemical compound attached to the
chitosan can affect its response to protein adsorption: hydrophobic (N-stearoyl chitosan) surface
enhances protein adsorption when ethyleneglycol oligomer could reduce the protein adsorption.
A series of temperature-sensitive poly(NIPAm-co-CSA) hydrogels were synthesized by the
copolymerization of acrylic acid derivatized chitosan (CSA) and N-isopropylacrylamide (NIPAm)
in aqueous solution.177 The investigation of L929 cell adhesion and detachment of poly(NIPAm-
co-CSA) hydrogels indicated the cell adhesion and spreading was higher on the surface of these
hydrogels than that of PNIPAm hydrogel at 37°C due to the incorporation of chitosan, which have
excellent cell affinity. They showed more rapid detachment of cell sheet compared to PNIPAm
hydrogel because of the highly hydrophilic and hygroscopic nature of chitosan chains when reduc-
ing the culture temperature from 37°C to 20°C.
The permeability of chitosan membranes grafted with hydroxymethylmethacrylate may be con-
trolled by means of a plasma treatment for applications in dialysis domain.178

Due to wide availability and their specific properties (biocompatibility, biodegradability, etc.) chitin,
chitosan, and their amphiphilic-based systems have found applications in a large variety of areas
of biomaterials. They are elaborated using various processes either from the chemical modification
of the macromolecular chain or from specific interactions with surfactants or oppositely charged
polyelectrolytes. Physicochemical properties (rheological, interfacial, and swelling) allow a great
use of such systems as biomaterials. As a consequence, the domains of applications are wider and
wider considering among them drug delivery, gene transfection, tissue engineering, and cell or
protein adhesion. Further studies and development of such amphiphilic systems for new biomedical
applications can be expected in the next century.
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10 Biomaterials of Natural Origin
in Regenerative Medicine
Vijay Kumar Nandagiri, Valeria Chiono, Piergiorgio Gentile,
Franco Maria Montevecchi, and Gianluca Ciardelli
CONTENTS
10.1 Introduction........................................................................................................................... 271
10.1.1 Historical Background............................................................................................... 272
10.2 Natural Polymers as Biomaterials......................................................................................... 272
10.2.1 Polysaccharides as Biomaterials................................................................................ 273
10.2.1.1 Cellulose..................................................................................................... 273
10.2.1.2 Starch.......................................................................................................... 276
10.2.1.3 Agar-Agarose.............................................................................................. 278
10.2.1.4 Alginate.......................................................................................................280
10.2.1.5 Chitin and Chitosan.................................................................................... 281
10.2.1.6 Hyaluronan/Hyaluronic Acid/Hyaluronate................................................. 282
10.2.2 Proteins as Biomaterials............................................................................................ 286
10.2.2.1 Collagen...................................................................................................... 286
10.2.2.2 Gelatin......................................................................................................... 288
10.2.2.3 Fibrin-Fibrinogen-Fibronectin.................................................................... 291
Abbreviations.................................................................................................................................. 297
References....................................................................................................................................... 298
10.1 INTRODUCTION
Biomaterials are nonviable materials used as medical devices intended to interact with biological
systems. They display a combination of properties including chemical, mechanical, physical, and
biological properties that render them suitable for safe, effective, and reliable use within a physi-
ological environment. Biomaterials are developed to support damaged tissues, through suitable
scaffolds for promoting cell differentiation and proliferation toward the formation of a new tissue
(Williams 1987). The basic aim of scaffolds is to provide mechanical, morphological, and bio-
logical support for the damaged tissues and to recover them. To be successful, a scaffold should
effectively repair the defect it covers without eliciting an adverse tissue reaction while maintain-
ing mechanical and biological integrity for a desired amount of time from a few weeks to several
years (Puppi et al. 2010). Natural-based polymers are or resemble the biological macromolecules,
which the biological environment is prepared to recognize and deal with metabolically. The ECM
is the optimized environment that nature has developed to maintain homeostasis and to direct tissue
development (Mano et al. 2007). Owing to their similarity to the ECM macromolecules, natural
polymers may serve as potential biomaterials. Natural polymers may also bypass the stimulation of
chronic inflammation or immunological reactions and toxicities encountered by implants. The deg-
radation of natural polymers is directed by naturally occurring enzymes and/or simple hydrolysis or
271
oxidative processes which ensures that the implant degrades completely during normal metabolic
processes. Furthermore, the rate of degradation of natural polymers can be modified by chemical
cross-linking or other modifications to attain a predetermined biodegradation rate. So, with natural
polymers, biomaterials can be designed such that they can function biologically at molecular level.
10.1.1 Historical Background

The utilization of natural polymers as biomaterials is dated back to centuries. Ancient Egyptians
used animal tendons as sutures. In the first century AD, in both Greece and India, physicians used
natural biomaterials for surgery to repair mutilations from war. Susrutha, a medical encyclopedia of
ancient India, describes a procedure similar to “autograft” which involved the transplantation of a
tissue from one part of the body to another in the same individual. The method was used to obtain
a prosthetic nose for patients whose nose had been cut off, by using a skin graft from the patient’s
cheek, and molded into the new prosthetic nose. Physicians succeeded to keep an open wound at
the nose clean and viable, to detach a portion of patient’s skin while maintaining the blood supply
as to avoid necrosis and finally to reattach it to the patient so that it revascularized and the new nose
sustained itself (Chari 2003).The Romans were able to perform simple surgical techniques such as
repairing damaged ears around the first century BC. Due to religious reasons, they did not approve
the dissection of both human beings and animals, thus their knowledge was based on the texts of
their Greek predecessors (Santoni-Rugiu 2007). At around 1460, physicians performed rhinoplasty
in which the skin from arm was used as prosthetic nose (Zimmerman and Veith 1993).
After two centuries, the first two cases of “xenograft” were performed in France and the
Netherlands. In July 1667, Jean-Baptiste Denis, a French physician, transfused the blood of a sheep
into a 15-year-old boy, who recovered. He also performed transfusion into a laborer, who also sur-
vived. But, in winter 1667, Denis performed several transfusions on Antoine Mauroy with calf’s
blood who on the third account died and the procedure was banned in France (Maluf 1954). A Dutch
surgeon, Job van Meekeren, was the first to document the use of bone graft in 1668. He used a piece
of canine skull to replace a skull defect in a Russian nobleman. The operation was successful but
resulted in the excommunication of the patient by the church (Pacaccio 2005).
Whilst these attempts to advance medicine were opposed for political and religious reasons of
that period, they set the stage for the introduction of prosthetics in regenerative medicine. Though
early physicians lacked the sophistication and technology of modern surgeons, some of them were
able to intuitively grasp concepts of biomimicry, necessary to successfully plan these operations. Of
course, without knowledge of the immunogenicity of the implants they were using, many patients
experienced complications or died soon after surgery. Implant technology progressed slowly until
aseptic technique, anesthesia, and a basic understanding of cellular-level mechanisms matured.
Understanding the history of the development of biomaterials can give a better perspective for the
development of biomaterials (Coburn and Pandit 2007).
10.2 NATURAL POLYMERS AS BIOMATERIALS

Living organisms produce a wide variety of polymers such as polysaccharides, proteins, and poly-
nucleotides which can function as biomaterials. These natural-based polymers offer the advantage
of being similar to ECM allowing the design of biomimetic biomaterials, i.e., materials that func-
tion biologically at the molecular level. Natural ECM consists of nanofibers containing different
chemical ligands that interact with cell surface receptors. The ECM is a complex mixture of matrix
molecules, including the glycoproteins fibronectin, collagens, laminins, proteoglycans, and nonma-
trix proteins including growth factors. Therefore, natural ECM provides cell signaling molecules
to cells, including cell adhesion molecules and growth factors. A biomimetic substrate is able to
emulate the functions of native ECM by mimicking its three-dimensionality, nanofibrous topog-
raphy and plethora of chemical motifs. The use of natural polymers, which are component of the
Biomaterials of Natural Origin in Regenerative Medicine 273
ECM or similar to ECM macromolecules, allows the obtainment of substrates able to interact spe-
cifically with cell surface receptors (Albertsson and Karlsson 1996). Another interesting property
of natural polymers is their ability to be degraded by naturally occurring enzymes guaranteeing
that the implant will be finally metabolized by physiological mechanisms. Hence, it is possible to
implant a natural-based scaffold which can deliver a specific function for a pre-determined period
of time, following which it is expected to be degraded completely by normal metabolic processes.
Moreover, the degradation rate of naturally occurring materials can be manipulated by introduc-
ing functionalities or cross-links which enable the regulation of the in vivo life-time of the implant
(Elsie et al. 2009).
10.2.1 Polysaccharides as Biomaterials

Polysaccharides are crucial components of life matter. They display perfect biocompatibility
and biodegradability, which are the basic characteristics for polymers used as biomaterials.
Polysaccharides (called also glycans) consist of monosaccharides linked together by O-glycosidic
linkages. Polysaccharides containing only one kind of monosaccharides are homopolysaccharides,
or homoglycans, whereas those containing more than one kind of monosaccharides are hetero-
polysaccharides or heteroglycans. Polysaccharides differ not only in the nature of their compo-
nent monosaccharides but also in the length of their chains and in the amount of chain branching.
Although a given sugar residue has only one anomeric carbon and thus can form only one glycosidic
linkage with hydroxyl groups on other molecules, each sugar residue carries several hydroxyls,
one or more of which may be an acceptor of glycosyl substituents. This ability to form branched
structures distinguishes polysaccharides from proteins and nucleic acids, which are linear polymers
(Kobayashi and Uyama 2003).
Polysaccharides have several characteristics not found in other natural polymers such as water
solubility, flow behavior, gelling potential, and/or surface and interfacial properties depending
on the differences in the monosaccharide composition and linkage types (Barbosa et al. 2005).
Polysaccharides perform different physiological functions and may offer a variety of potential
applications as biomaterials. Three factors generate increased interest toward polysaccharide-based
biomaterials: (1) the large and growing body of information pointing to the critical role of sac-
charide moieties in cell signaling schemes and particularly in the area of immune recognition;
(2) the recent development of new powerful techniques with the potential for automated synthesis
of biologically active oligosaccharides, able to allow decoding and exploiting the language of oli-
gosaccharide signaling; (3) the explosion in tissue engineering research and the associated need
for new materials with specific, controllable biological activity and biodegradability (Lakshmi and
Cato 2007).
The presence of reactive functional groups along the polymer chains of polysaccharides facilitates
their modification and allows the formation of hydrogels. Depending on the source of availability,
polysaccharides can be further classified into plant polysaccharides (including algae), animal poly-
saccharides, and microbial polysaccharides. Different polysaccharides will be described in detail in
the following with particular attention to their relevant applications in regenerative medicine.
10.2.1.1 Cellulose
Cellulose is a linear polysaccharide of repeated d-glucose units with β-1,4 linkages; it is an abun-
dant, biodegradable, and biocompatible polymer and the principle constituent of plants and natural
fibers, such as cotton and linen (Klemm et al. 2005). Plant cellulose (PC) forms the structural
framework of plants and it is isolated in the form of microfibrils and possesses repeated d-glucose
units linked by β-(1–4) glycosidic bonds which account for the high crystallinity of cellulose
(usually in the range of 40%–60% for PC). In nature, the cellulose molecules assemble to form
cellulose I-type crystal structures, with regularly packed, parallel-chain alignments along the
longitudinal axis (Hieta et al. 1984). The regular intra- and intermolecular hydrogen bonds yield
self-assembly and hierarchical organization of cellulose which corresponds to the crystal structure
and polymorphism resulting in its insolubility in common solvents (Atalla and VanderHart 1984,
Nishiyama et al. 2002, 2003). The biodegradation of cellulose leads to progressive decrease in
molecular weight and mechanical strength and increase in water solubility. The presence of amor-
phous regions in cellulose can increase its biodegradability in tissue. In vivo biodegradability can
be enhanced by lowering the crystallinity and intensifying the hydrophilicity of cellulose-based
materials (Miyamoto et al. 1989). In order to enhance the water solubility and decrease the crystal-
linity of cellulose, various substitutions have been attempted along the anhydroglucose backbone.
Introduction of hydrophobic groups such as methyl or hydroxypropyl moieties to the cellulose back-
bone yields the derivatives with hydrophilic-lipophilic balance values ranging from 10.0 to 11.25.
Furthermore, introduction of these hydrophobic groups gives the polymer surface activity (as in
polysoap) and unique hydration-dehydration characteristics and improves the water affinity of the
cellulose (Sarkar and Walker 1995).
Cellulose can be derivatized in the form of ethers, esters, and acetals with varying solubility
properties like methyl cellulose (MC), hydroxyl propyl cellulose (HPC), hydroxyl propyl methyl cel-
lulose (HPMC), and carboxy methyl cellulose (CMC) which can be termed as cellulosics. Cellulose-
based materials have been used in a number of biomedical applications. These include construction
of cellulose membranes for hemodialysis (Wu 2002), construction of enzyme carriers for biosen-
sors (Utpal et al. 2006) drug delivery (Malafaya et al. 2007), and preparation of scaffolds for the
regeneration of various tissue types, such as bone (Barbosa et al. 2005), cartilage (Svensson et al.
2005, Muller et al. 2006), liver, skin, and blood vessels (Miyamoto et al. 1989, Seal et al. 2001).
Precisely, due to their high diffusional permeability to most of the toxic metabolic solutes, cellulose
membranes have been extensively investigated as hemodialysis membranes. Sevillano et al. (1990)
designed hemodialysis membranes based on cellulose acetate (CA) and they observed that the mem-
branes based on CA improved red blood cell function in hemodialysis patients. Artanareeswaran
et al. (2004) demonstrated the influence of poly(ethylene glycol) (PEG) addition to CA membrane
on pore size, permeate flux, and the protein rejection rates. Furthermore, Ani Idris et al. investigated
the effect of incorporating PEGs with different molecular weights, such as PEG 200 kDa, PEG
400 kDa, and PEG 600 kDa on the dialysis membrane performance in terms of urea clearance and
also on the morphology of membrane. They reported that the membrane performance in terms of
solute clearance was significantly influenced by the amount of additives and their molecular weights
(PEG molecular weight). For example, PEG with a low molecular weight produced superior dialysis
membranes that can clear urea (Idris and Yet 2006).
Then, Entcheva et al. synthesized diffusion-controlling membranes and membrane carriers for
enzyme immobilization in biosensors. They constructed biosensor membranes with glucose oxi-
dase, covalently bound to acetylcellulose carriers activated with urea, and tested them to evalu-
ate their application in the measurement of the glucose concentration in blood serum and plasma
(Entcheva and Yotova 1994). Successively, Liang et al. prepared biodegradable cellulose/chitin
blend porous-like membranes from blend solution of cellulose and chitin in 9.5 wt% NaOH/4.5 wt%
thio urea aqueous solution coagulating with 5.0 wt% (NH4)2SO4 and water as solvent. They studied
the permeability, the partition coefficients, and the diffusion coefficients of three different drugs in
these membranes. They concluded that the introduction of chitin influenced the morphology and
enhanced the pore size and porosity of the membranes and thus the crystal structure of cellulose
in the blend membranes was partly destroyed, resulting in a significantly different permeability.
According to them, the diffusion of drugs through these membranes resulted by dual transport
mechanism (pore mechanism and partition mechanism) with some hindrance of molecular diffu-
sion via polymer obstruction (Liang et al. 2007).
Apart from these, cellulose derivatives have been extensively investigated for their applications
such as dressings in the treatment of surgical incisions, burns, wounds, and various dermatologi-
cal disorders (Peppas 1986). The dressings based on CMC fibers form a strong cohesive gel upon
hydration and have been marketed under the trade name AQUACEL®. Cellulose derivatives can
also undergo sol-gel transition in water at an optimum balance of substituting hydrophilic and
hydrophobic moieties and can be employed for hydrogel fabrication (Walker et al. 2003).
Oxidized cellulose has been used for wound dressing in the form of a gauze which becomes gelati-
nous after contact with blood, thus adhering to the surrounding tissue and for osseous regeneration
since decades under the trade name of Surgicel® (Johnson and Johnson) (Degenshein et al. 1963, Dias
et al. 2003). Skoog demonstrated that Surgicel promotes osseous and soft tissue regeneration in maxil-
lary reconstructive surgery, when used as superiosteal graft before flap closure (Skoog 1967). Owing to
its biocompatibility, ability to integrate into surrounding tissue, and mechanical properties, cellulose is
considered as a promising candidate for scaffolds in bone tissue engineering. To improve the osteocon-
duction of the membranes, Rhee et al. deposited a surface apatite layer on cellulose cloth with the aid
of citric acid. Hydroxyapatite (HAp) formation was induced by soaking cellulose cloths in simulated
body fluid (SBF) solutions (1.5× SBF) with ion concentrations 1.5 times that of citric acid (1.0 M citric
acid) and they concluded that the formation of HAp could be due to the hydrogen bonding of citric acid
to the cellulose cloth and its chelating ability toward calcium ions (Rhee and Tanaka 2000).
Moreover, regenerated cellulose (RC; cellulose regenerated by the viscose process, CRV®)
sponges were demonstrated to promote bone regeneration and to have slow degradability (Martson
et al. 1998). Regenerated cellulose hydrogels have also been implanted as an attachment material
for the femoral component in hip prostheses, in place of the acrylic cement (Poustis et al. 1994).
Hofmann et al. attempted to coat nonwoven cellulose (regenerated, oxidized) fabrics with
hydroxy carbonated apatite (HCA) using a procedure based on biomimetic method. They employed
a high degree of supersaturation (5× SBF) to accelerate the biomimetic formation of bone-like apa-
tite on the cellulose fabrics. Furthermore, the crystal growth was accelerated by successive soaking
of the coated fabric into a second 5× SBF with reduced Mg2+ and HCO3− concentrations. They found
that the HCA layer thickness increased by increasing the soaking time in the second solution and
concluded that the amount of CO32− substituting PO3− 4 in the HAp lattice of the precipitates can be
varied by changing the soaking time (Hofmann et al. 2006).
In a biomimetic approach (with the aim to mimic ECM functionalities), de Taillac et al. proposed
the immobilization of bioactive molecules, like peptides bearing the RGD (Arg- Gly-Asp) sequence,
onto cellulose. The ability of this peptide sequence to bind a variety of cells facilitates bone cell
attachment and proliferation. In this approach, silane-derivatized spacer arms were first attached
to the surface of cellulose as an intermediate for the covalent linkage of RGD-containing peptide
(de Taillac et al. 2004). They reported that the biomimetic modification of cellulose surface enhanced
the adhesion of human bone marrow stroma cells and proliferation of osteoprogenitor cells.
In other applications in tissue engineering, Entcheva et al. prepared scaffolds for growing func-
tional cardiac cell constructs based on CA and RC to demonstrate the properties of CA and RC
surfaces. They observed that CA and RC helped in promoting cardiac cell growth, enhancing cell
connectivity (gap junctions) and electrical functionality (Entcheva et al. 2004). Luo et al. reported
the preparation of cellulose/soya protein isolate (SPI) sponges by freeze drying process for neural
tissue regeneration. They observed that the sponges were porous in structure and that the size of
the pores increased and the thickness of the pore walls decreased as the SPI content of the sponges
increased. The authors hypothesized that the incorporation of SPI into cellulose followed by freeze-
drying process resulted in the formation of pores and thin pore walls in the composite sponges,
which facilitated the migration of cells and tissue into the sponges, leading to gradual fusing with
the implants. Thus, the new cellulose/SPI sponges exhibited good biocompatibility using L929
fibroblast cells and biodegradability (Luo et al. 2010).
Recently, Zaborowska et al. attempted to refine the method for preparing microporus bacte-
rial cellulose scaffolds in order to attain a porosity of 300–500 μm and evaluated its suitabil-
ity as a 3D scaffold for bone tissue engineering. They observed that the refined microporus
cellulose-based scaffolds improved the osteoprogenitor cell seeding onto the scaffolding mate-
rial resulting in the formation of cell clusters within the scaffolds and ultimately improved the
bone regeneration (Zaborowska et al. 2010).
To summarize, cellulose has been attempted either directly or in modified form for various tis-
sue engineering applications such as fabrication of dialysis membranes, surgical dressings, hydro-
gels, and as scaffolding material for bone, cartilage, and other tissue regeneration applications.
Researchers also studied composite scaffolding materials based on cellulose either with addition of
other polymeric materials or inorganic components.
10.2.1.2 Starch
Starch is a mixture of glycans that plants synthesize as their principal food reservoir. It is deposited
in the chloroplasts of plant cells as insoluble granules composed of α-amylose (normally 20%–30%)
and amylopectin (normally 70%–80%) (Morrison and Karkalas 1990). α-Amylose is a linear poly-
mer of several thousands of glucose residues linked by α-(1–4) bonds. Amylopectin, consisting
mainly of α-(1/4)-linked glucose residues, is a branched molecule with (1/6) branch points at every
24–30 glucose residues in average. Amylopectin molecules contain up to 106 glucose residues,
making them some of the largest molecules in nature (Voet et al. 1999).
The physico-chemical and functional characteristics of starch and its uniqueness in various prod-
ucts vary with the biological origin of starch (Svegmark and Hermansson 1993). Starch is insoluble
in cold water, but it is very hygroscopic and binds water reversibly. Heating starch solution results in
rupturing of interior hydrogen bonding in the starch granule leading to the gelatinization of starch
(Appelqvist and Debet 1997). Furthermore, the linear amylose molecules leach out of the granules
into the solution which forms a suspension that contains a mixture of linear amylose molecules,
swollen granules, and granule fragments which in turn result in the formation of a thick paste or
gel, depending on the amount of water present (Whistler and Daniel 2005). Starch is degraded
by amylase (EC number 3.2.1.1) and glucosidase (EC number 3.2.1.20) enzymes. The α (1 → 4)
linkages are broken down by the enzyme amylase and the α (1 → 6) linkages are attacked by glu-
cosidases. The degradation products of starch are low molecular weight starch chains, fructose,
and maltose. Starch-based polymers and their derivatives exhibit a wide range of properties like
ease in shaping them into thin films (Rindlava et al. 1997), fibers (Pavlov et al. 2004), or porous
matrices (Gomes et al. 2001) which make them suitable for uses in several biomedical applications
such as bone plates, screws, drug delivery carriers, and tissue engineering scaffolds (Marques et al.
2002). Starch-based materials were firstly proposed for bone-related applications in 1995 by Reis
and Cunha (1995). These starch-based materials consist of blends of corn starch with ethylene
vinyl alcohol (SEVA-C), cellulose acetate (SCA), poly (caprolactone) (SPCL), and poly (lactic acid)
(SPLA). Up to now, several studies have shown the potential of these materials for several bio-
medical applications, namely, as bone substitutes (Sousa et al. 2000, 2003), drug delivery carriers
(Malafaya et al. 2001, Elvira et al. 2002), partially degradable bone cements (Espigares et al. 2002,
Boesel et al. 2004), and hydrogels (Pereira et al. 1998, Elvira et al. 2002).
Marques et al. (2002) demonstrated the in vitro cytocompatibility of two different blends of
corn-starch SEVA-C and SCA and their respective composites with HAp, by cytotoxicity and
cell adhesion tests using L929 mouse fibroblast cell line, and they reported that both blends are
cytocompatible and can be adopted as promising material for bone replacement and/or tissue engi-
neering applications. Then, Gomes et al. also attempted to fabricate three-dimensional porous
architectures made of SEVA-C and SCA blends to obtain adequate mechanical properties. They
also used hydroxyapatite as a reinforcement material for the polymeric scaffold. The scaffolds
were fabricated by standard conventional injection molding process, in which a solid blowing agent
based on carboxylic acids was used to generate the foaming of the bulk of the molded part in order
to produce scaffolds with a compact skin and a porous core. They observed that scaffolds based
on SCA have displayed better tensile properties than that of SEVA-C scaffolds and the stiffness
of the materials was slightly increased (a maximum increase of 12% for SEVA-C and around 25%
for SCA) until a certain value of added carboxylic acids as a blowing agent. They concluded that
such blends are noncytotoxic and can be employed for various biomedical applications (Gomes
et al. 2001).
Similarly, Salgado et al. have developed porous starch scaffolds based on a 50/50 (wt%) blend
of corn starch/ethylene-vinyl alcohol by melt-based technology for bone tissue-engineering appli-
cations. The scaffolds showed 60% porosity degree with pore sizes between 200 and 900 μm and
a reasonable degree of pore interconnectivity; furthermore, scaffolds were nontoxic and did not
inhibit cell growth. These scaffolds showed a high compressive modulus of 117.50 ± 3.7 MPa and a
compressive strength of 20.8 ± 2.4 MPa (Salgado et al. 2004).
Tuzlakoglu et al. designed nano- and microfiber combined scaffolds for bone tissue engineering
based on starch and polycaprolactone (SPCL) by electrospinning method to mimic the biophysi-
cal structure of natural ECM. They tested the developed structures with two different cell types,
a human osteoblast-like osteosarcoma SaOs-2 cell line and rat bone marrow stromal cells. They
demonstrated that the nanofibers affected cell shape and cytoskeletal organization of the cells on
the nano-/microfiber combined scaffolds. They concluded that the developed structures exhibited a
great potential on the 3D organization and guidance of cells that is provided for engineering of 3D
bone tissues (Tuzlakoglu et al. 2005).
Furthermore, Gomes et al. performed studies in dynamic culture conditions (by the use of a
bioreactor) to demonstrate the expression of an array of bone growth factors by marrow stromal
cells cultured on SPCL fiber-mesh scaffolds. Bone marrow cells cultured on starch-based scaf-
folds responded to flow perfusion conditions by augmenting the production of several bone-related
growth factors, namely, TGF-β1, FGF-2, vascular endothelial growth factor (VEGF), and BMP-2
(Gomes et al. 2006), and Santosa et al. illustrated that SPCL fiber meshes supported the mainte-
nance of extra cellular morphological structure and endothelial integrity and augmented the vascu-
larization process. They suggested that SPCL fiber meshes are potential scaffolds for bone tissue
engineering (Santosa et al. 2007).
Recently, Martins et al. developed a novel hierarchical multilayered starch-based scaffold by
a combination of starch-polycaprolactone micro- and polycaprolactone nano-motifs, respectively
produced by rapid prototyping and electrospinning techniques. Such multilayered scaffold dis-
played a combination of parallel aligned microfibers in a grid-like arrangement, inserted by a mesh-
like structure with randomly distributed nanofibers. They concluded that such an integration of
nanoscale fibers into 3D rapid prototyped scaffolds enhanced scaffold biological performance in
bone tissue–engineering strategies (Martins et al. 2009). Azevedo and Reis (2009) reported com-
pression molding method to tailor the degradation rates of starch by encapsulating α-amylase
(a thermostable enzyme).
Starch can also be applied as a slow-release drug delivery system. Tuovinen et al. attempted
to control the hydrophobicity and rate of enzymatic degradation of starch by acetyl substitution.
They emphasized that the hydrophobicity of starch increases as a function of degree of substitution
(Tuovinen et al. 2003). Furthermore, they prepared microparticles based on starch acetate having
various DS from 2 to 6 and loaded with calcein to target retinal pigment epithelium (RPE) for ocular
treatment. They observed that starch acetate microparticles are taken up by the RPE cells and the
polymer can be degraded by the enzymes in these cells (Tuovinen et al. 2004).
Malafaya et al. developed starch microspheres as drug delivery carriers in tissue engineer-
ing applications. They loaded microparticles with a model nonsteroidal anti-inflammatory drug
(Meclofenamic sodium salt) which is widely used in the treatment of arthritic inflammations
and immobilized them into scaffolds or administrated separately with scaffolds. Furthermore,
they speculated that by altering processing conditions such as pH and ionic strength, such a sys-
tem can be potentially incorporated with different growth factors or even living cells and can
be utilized for improving bone regeneration in tissue engineering (Malafaya et al. 2006). Silva
et al. have synthesized composite microparticles composed of a starch-based material and a bio-
active glass (BG 45S5®). These particles displayed a bioactive behavior by the formation of a
calcium-phosphate layer resembling biological apatite at their surface upon immersion in SBF.
The combination of bioactive behavior and the ability to encapsulate bioactive agents conferred
these microparticles a great potential for controlled growth factor release in bone applications
(Silva et al. 2005). Starch-based biomaterials (SBB) surface properties can be changed by using
oxygen-based radio frequency glow discharge (rfGD) treatment to modulate bone cells response
to SBB. This Oxygen rfGD treatment uniformly functionalizes/activates the surface of SBB with-
out affecting the bulk properties. RfGD-treated surfaces showed an increase in hydrophilicity and
surface energy when compared to nonmodified SBB. The synergic effect of surface plasma treat-
ment on the adhesion, growth, and morphology of bone-like cells was also reported (Alves et al.
2007). Duarte et al. described a method to prepare starch-based porous matrixes by using super
critical fluid technology. They used supercritical immersion precipitation technique to prepare
scaffolds of a polymeric blend of starch and poly (l-lactic acid) for tissue engineering purposes
by using two organic solvents, dichloromethane and chloroform, and concluded chloroform as the
most favorable solvent for the process as it dissolved the raw materials better and resulted in the
formation of thicker and more porous matrix. They also revealed that pressure mostly affected the
porosity, interconnectivity, and pore size of the matrix, whereas temperature had negligible affect
(Duarte et al. 2009).
To conclude, starch has been used as a promising biomaterial for tissue regeneration applications
either directly or in blended form with thermoplastic polymers to improve thermo-mechanical deg-
radation, and make them less brittle and more easily processed. Blends of starch with ethylene vinyl
alcohol, cellulose acetate, polycaprolactone, and poly(lactic acid) have been proposed as potential
alternative biodegradable materials for a wide range of biomedical applications, including bone
cements, hydrogels for controlled drug delivery, and bone substitutes. The structure and functional
properties of these materials depend on blend components, material processing technique, incorpo-
ration and nature of additives, and reinforcement fillers. By incorporating α-amylases and cellulose
enzymes into starch-based scaffolding systems, the degradation rates can be tailored.
10.2.1.3 Agar-Agarose
Agar is made of two components: agarose (a non-sulfated fraction) and agaropectin (a mixture of var-
ious sulfated molecules). The structure of agarose consists of a linear chain of β-d-galactopyranose
residues linked through positions 1 and 3 (A units) and α-d/l-galactopyranose residues linked
through positions 1 and 4 (B units) arranged in an alternating sequence (AB)n. A unit may carry
methyl ether groups on position 6, sulfate hemiester groups on positions 2, 4, or 6; some of A units
may carry pyruvic acid, linked as a cyclic ketal bridging O-4 and O-6 (1-carboxyethylidene
groups). B units can occur in either d or l form which carry methyl groups on position 2 or
4-oxymethyl-α-l-galactopyranosyl groups on position 6 or/and sulfate hemiester groups on posi-
tion 2 or 6 or both (Painter 1983). This arrangement allows the chains to join together and adopt
a double helix. The two chains are wrapped together so tightly that any gaps are closed, trapping
any water inside the helix. Agarose forms thermally reversible gels (cold setting gels at approx.
38°C); however, the melting temperature is much higher, approximately 85°C; this gives agar gel
a very large gelling/melting hysteresis (Izydorczyk et al. 2005). Degree of gelation depends on the
methoxyl and sulfate content and it involves the formation of double helices followed by a phase
separation to form a gel (Rinaudo 2008). Agarose gels have been attempted to prepare scaffolds
for neural and cartilage tissue engineering (Kong and Mooney 2004); in particular, agarose-based
hydrogels have been reported to be potential scaffolds for cartilage tissue engineering owing to
their swelling nature and high water content (Elisseeff et al. 2005). For nerve tissue regeneration,
agarose hydrogels have been reported to stimulate and maintain three-dimensional neurite exten-
sion from primary sensory ganglia in vitro (Luo and Shoichet 2004). However, a successful scaffold
for neural tissue engineering is required to have suitable mechanical properties for neurite extension
(Nisbet et al. 2008). Balgude et al. attempted to demonstrate the structure–function relationship
between the mechanical stiffness of the agarose hydrogel and its ability to initiate and maintain
maximal neurite extension. They explored agarose gels in the range of 0.75%–2.00% (wt/vol, incre-
ments of 0.25%), by dissolving powdered agarose in sterile saline and showed that pore size of
agarose hydrogels increased with the increase in agarose concentration. Furthermore they explored
the role of mechanical stiffness as a potential parameter that can be perturbed to enhance neurite
extension in agarose hydrogel (Dillon et al. 1998, Balgude et al. 2001).
Moreover, agarose hydrogels have been used to provide a three-dimensional environment to
investigate in vitro chondrocyte mechano-transduction pathways which involve (1) the encapsula-
tion of cells within agarose hydrogel, (2) the application of cell-seeded hydrogel of defined levels
of mechanical deformation or loading, and (3) the observation of the resulting changes in cellular
structure and organization (Buckley et al. 2009).
Suspending chondrocytes in a three-dimensional matrix similar to their natural environment has
been reported to permit the cells to retain their native phenotype and to produce their extracellular
components. Benya et al. demonstrated that the culture of rabbit cartilage in agarose permitted
restoration of the differentiated phenotype (Benya and Shaffer 1982). Chondrocytes cultured in
agarose scaffolds have been reported to maintain their phenotype and synthesize normal levels of
collagen II. Furthermore, chondrocyte-seeded agarose hydrogels have been shown to produce a
functional extracellular matrix in free swelling culture (Mauck et al. 2002). Agarose hydrogels have
been applied for cartilage tissue engineering applications to assess the elaboration of pericellular
and extracellular matrix. Quinn et al. have demonstrated the matrix deposition around individual
chondrocytes during de novo matrix synthesis in agarose suspension culture for a period of 1 month
(long enough for cartilage-like material properties to begin to emerge). Cartilage regeneration was
promoted by a differential recruitment, pericellular distributions of proteoglycan, and matrix pro-
tein deposition around the hydrogel (Quinn et al. 2002).
Mouw et al. (2005) compared the similarities and differences among scaffolding materials in
proteoglycan accumulation and GAG composition. Maximum cell proliferation and extracellular
matrix accumulation have been observed in agarose constructs in comparison with PGA, algi-
nate, fibrin, and collagen. Furthermore, they reported that agarose constructs had the highest sGAG
(sulfated GAG) to DNA ratio, while alginate and collagen I had the lowest levels (Mouw et al. 2005).
Even though agar is compatible with most proteins and other polysaccharides at near neutral pH,
it has limited applications owing to its brittle mechanical properties (Freile-Pelegrin et al. 2007).
In tissue engineering, the hierarchical organization of cells to promote the in vitro development of
functional tissue has been benefited from the spatially controlled placement of cells in specific loca-
tions on porous, often heterogeneous, substrates. The use of printing technologies may facilitate the
invasion of cells into the scaffold spatially and the generation of organized tissues. Agarose replica
printing have been successfully used for generating patterns of cells with circular shapes and diam-
eters of 200, 700, or 1000 μm on the surface of HAp scaffolds and glass slides (Stevens et al. 2005).
Agarose has been recently combined with ceramics as natural biodegradable binder to increase
the biomaterial flexibility facilitating its placement into the bone defect. Roman et al. optimized the
fabrication of biodegradable scaffolds composed of β-tricalcium phosphate (β-TCP) and agarose
based on the thermal gelation technique. They succeeded in internalizing vancomycin during the
scaffold preparation stage, without disturbing the scaffold consolidation process in order to improve
the graft performance. They proposed these scaffolds as easily injectable scaffolds with reinforced
hydrogel-like behavior (Roman et al. 2008).
Furthermore, Alcaide et al. studied the behavior of hydroxyapatite- β-TCP/agarose disks cul-
tured with L929 fibroblasts and Saos-2 osteoblasts. They observed that both cell types adhered and
proliferated on the biomaterial surface maintaining their characteristic morphology and that the
biomaterial induced apoptosis in Saos-2 osteoblasts and a transitory stimulation of fibroblast mito-
chondrial activity. They concluded that the obtained scaffold can be potentially applied for bone
substitution and repair (Alcaide et al. 2008).
Agar has also been used to encapsulate cells (such as islets of Langerhans, chondrocytes,
Adherent Crandall-Reese feline kidney cells). Shinji et al. (2007) prepared spherical capsules of
around 140 μm diameter by extruding aqueous agarose-gelatin conjugate and then encapsulated
adherent crandall-reese feline kidney cells, reporting the enhancement of degree of proliferation
and mitochondrial activity.
These studies demonstrate that agarose gels promote cell retention, proliferation, and chondro-
genesis in vivo and in vitro. The intrinsic mechanical and cellular mechano-transduction properties
of agarose hydrogels have been reported to be dependent on the agarose concentration. However,
there is significant variation in the reported values for the compressive properties of agarose gel at
any given concentration. Complete and concise characterization of the mechanical properties of
these hydrogels at different concentrations is necessary to facilitate their use as a scaffold material
in cartilage tissue engineering and to properly interpret the results of cellular mechano-transduction
studies that utilize these gels.
10.2.1.4 Alginate
Alginate is a high-molecular mass polysaccharide extracted from various species of kelp. Alginates are
also produced extracellularly by Pseudomonas aeruginosa and Azotobacter vinilandii (Johnson et al.
1997, Ertesvag and Valla 1998). The function of alginates in algae is primarily skeletal with the gel
located in the cell wall and intercellular matrix conferring the strength and flexibility necessary to with-
stand the force of water in which the seaweed grows (Ertesvag et al. 1996). Alginates are composed of
linear anionic block copolymers of heteropolysaccharides of β-d-mannuronic acid and α-l-guluronic
acid residues, which can be arranged in different proportions and sequences along the polymer chain.
Another important property of alginate gels is the occurrence of self sol-gel transition without any
alteration of temperature. This sol-gel transition results in the formation of channel-like pores. The
dimensions of these pores depend on concentration, nature, and conformation of the alginate.
The derivatives of alginates with monovalent metals (like sodium) are soluble in water and may
form viscous solutions. Sodium alginate forms relatively stable hydrogels through ionotropic gela-
tion in the presence of multivalent ions like Ca2+. During calcium cross-linking, hydrogels do not
undergo excessive swelling and subsequent shrinking; thus, they can maintain their form. The
cross-linking process can be carried out under very mild conditions, at low temperature and in the
absence of organic solvents and hydrogels of different shapes can be prepared. Alginate gels with
uniform concentration can be obtained by cross-linking via internal setting method as described
by Rassis et al. (2002) and Onsoyen (1992). By this method, a very regular gel network of alginate
is formed through a slow release of calcium ions by using an inactive form of the cross-linking ion,
either bound by a sequestering agent such as phosphate, citrate, or Ethylenediaminetetraacetic acid
(EDTA) or as a very low solubility salt, as CaSO4, or as a salt insoluble at neutral pH like CaCO3,
in association with a slowly hydrolyzing lactone, usually d-glucono-δ-lactone (GDL). Since GDL
generates an acidic pH, the calcium ions are gradually released and captured by guluronic residues
of alginate (Smidsrod and Draget 1996, Kuo and Ma 2001).
Owing to an outward flux of cross-linking calcium ions into the surrounding medium, alginate
gels can gradually lose their mechanical stability in biological fluids. The instability is also favored
due to the presence of high concentration of nongelling ions such as sodium and magnesium. To
address this problem, Yeom and Lee (1998) have attempted to introduce stable covalent cross-links
using bi-functional cross-linkers, such as glutaraldehyde. Stabilization can also be achieved by add-
ing free calcium ions to the medium while maintaining Na:Ca ratio at 25:1 for high-guluronate
alginates and 3:1 for low-guluronate alginates.
Alginate scaffolds with tube-like pores can be fabricated by using alginate/calcium phosphate
composites without heating them to higher temperatures. Additionally, nanocrystalline hydroxyapa-
tite could be synchronously precipitated during the sol-gel transition by adding phosphate ions into
the alginate solution (Dittrich et al. 2006).
Gelinsky et al. have produced biphasic monolithic alginate scaffolds for the repair of joint osteo-
chondral defects. The scaffolds contained both HAp phase and an alginate/hyaluronic acid compos-
ite hydrogel phase. Even if the scaffold consisted of two layers, the channel-like pores were running
through the whole scaffold crossing the interface (Gelinsky et al. 2007).
Several therapeutic agents, including antibiotics, enzymes, growth factors, and DNA, have
already been successfully incorporated in alginate gels, retaining a high percentage of biological
activity (Rowley et al. 1999, Albarghouthi et al. 2000). Moreover, alginate hydrogels have been
widely studied for cartilage and bone regeneration applications as scaffolds and vehicles for biologi-
cally active molecules or cells (Tortelli and Cancedda 2009).
10.2.1.5 Chitin and Chitosan

Chitin is the principal structural component of the exo-skeletons of invertebrates, such as crusta-
ceans and insects, and it is also present in the cell walls of most fungi and many algae. It is a semi-
crystalline homopolymer of β-(1/4)-linked N-acetyl-d-glucosamine residues (GlcNAc) (Lezica and
Quesada-Allue 1990). Depending on its source of availability, chitin occurs in two allomorphic
forms, namely, α and β forms, α-Chitin being the most abundant. The allomorphic forms can
be distinguished by IR spectroscopy and solid-state NMR and XRD (Rinaudo 2006). Owing to
its biodegradability, nontoxicity, antimicrobial, and gel-forming properties, chitin has been widely
employed in biomedical field (Kifune 1992, Shigemasa and Minami 1995, Hirota et al. 1996, Freier
et al. 2005b). Chitin can be converted into soluble derivatives such as chitosan, carboxy methyl chi-
tin, and glycol chitin (Kurita 2001). Among all the products of hydrophilic modifications of chitin,
carboxymethylated-chitin (CMCH) attempted as a promising material for biomedical applications
(Sashiwa and Aiba 2004). CMCH is an amphiprotic ether derivative, containing –COOH groups
and –NH2 groups in the molecule. CMCH is prepared by using monochloracetic acid in the presence
of concentrated sodium hydroxide. CMCH exhibits high viscosity, large hydrodynamic volume, and
film and gel-forming capacity which makes it an attractive option for biomedical applications. It was
also observed that CMCH possesses wound healing and inhibition of keloid formation properties
(Wongpanit et al. 2005).
Chitosan, a de-acetylated derivative of chitin, is a fiber-like substance and it is soluble in dilute
acids. Chitosan is a high molecular, nontoxic, mainly biodegradable polymer similar to cellulose
except in C-2 position. C-2 position of chitosan is occupied by chemically reactive free amino
groups (–NH2), whereas C-2 position of cellulose is occupied by hydroxyl (–OH) groups. Chitosan
possesses positive ionic charges, which facilitates its chemical binding with negatively charged
fats, lipids, cholesterol, metal ions, proteins, and macromolecules (Li et al. 1992). Cationic nature
of chitosan is primarily responsible for electrostatic interactions with anionic glycosaminoglycans,
proteoglycans, and other negatively charged molecules. Chitosan has free amine groups which are
potentially reactive sites for many chemical reactions (Knaul et al. 1999).
The degree of deacetylation (DD) of chitosan varies between 56% and 99% depending on the
crustacean species and the preparation methods. The number fraction (%) of N-acetyl-d-glucosamine
GlcNAc residues in the polymer chain is designated by degree of acetylation and it influences chi-
tosan physico-chemical properties such as solubility, reactivity, biodegradability, and cell response
(Khor and Lim 2003 Freier et al. 2005a). Deacetylation also influences the biodegradability and
immunological activity of chitosan (Tolaimate et al. 2000).
The degree of deacetylation can be determined by ninhydrin test (Curotto and Aros 1993), linear
potentiometric titration, nuclear magnetic resonance spectroscopy, and infrared spectroscopy (Tan
et al. 1998). Among these, the IR spectroscopy method is commonly used for the estimation of
chitosan DD values (Rinaudo 2006). Chitosan can be chemically modified by attaching functional
groups in order to favor its solubility in neutral or alkaline solutions and also to control some of
its physical properties. The utilization of chitosan as a biomaterial can be drastically broadened by
modifying it with hydrophilic and hydrophobic moieties. The hydrophilic modification includes
PEG grafting, sulfonation, and carboxymethylation. Hydrophobic modifications have been attained
by using palmitic acid, linolenic acid, linoleic acid, and deoxycholic acid (Kim et al. 2007). The
chemical modification of chitosan also controls the interaction of chitosan with drugs/bioactive
components, enhances the load capability, and tailors the release profiles (Prabaharan 2008).
Grafting of chitin and chitosan to synthetic polymers has also been performed as an interesting
way to extend their use as biomaterials for tissue engineering application. The physico-chemical
and biological properties of grafted co-polymers mainly depend on molecular structure, length, and
number of side chains attached to it. Various grafting techniques have been exploited for chitosan
such as radical or radiation-induced method, microwave irradiation, and dendronization. Graft copo-
lymerization results in the formation of tailored hybrid materials of chitosan and synthetic polymers
which are widely used as potential drug carriers (Carreira et al. 2010). For instance, grafting PEG
onto chitosan has led to water-soluble chitosan derivatives, which have been proposed as carriers
for anticancer drugs. The PEG-g-chitosans, prepared by using carbodiimide as initiator, have been
found to aggregate in aqueous medium due to hydrogen bonding. When such derivatives have been
prepared via reductive alkylation using PEG monoaldehydes, followed by further cross-linking,
their water solubility has been found restricted and they have been found to gradually re-aggregate
further, then becoming insoluble (Dal Pozzo 2000). Such a property is highly useful in wound
dressings to prevent tissue adhesion in internal surgery. Ideally, such materials are insoluble when
positioned in the surgical wound, but undergo progressive bioerosion leading to complete resorption.
Based on choice of PEG substitution and cross-linking, derivatives having high swelling capac-
ity and enhanced hydrophilicity can be obtained. Moreover, chitosan was largely used to prepare
composite scaffolds for tissue regeneration. In particular, Zhang and Zhang (2001) have produced a
strong macroporous, bioactive, and biodegradable composite based on chitosan and calcium phos-
phate. Two types of calcium phosphates, β-tricalcium phosphate and calcium phosphate inverted
glass, have been incorporated into the composite. They have demonstrated that chitosan provides a
structural skeleton fixing the scaffold form whereas the calcium phosphate bioactivity enhanced the
attachment of osteoblasts. The mechanical strength of the scaffold depended on the weight ratio of
chitosan with respect to the two types of calcium phosphates (Zhang and Zhang 2001).
On the other hand, several injectable materials based on chitosan and its derivatives have been
attempted for bone regeneration (Kim et al. 2008). The mechanical characteristics of chitosan can
be augmented by HAp and resulting materials can be applied as bone substitutes for bone repair
and reconstruction (Huang et al. 2008). Another interesting application of hydroxyapatite–chitin–
chitosan composites is the formation of self-hardening paste which can be employed as bone-filling
material for guided tissue regeneration for the treatment of periodontal bony defects (Xu et al. 2008).
In a recent study, Madhumathi et al. have reported chitin–HAp composite membranes by using an
alternate soaking method. They employed CaCl2 and Na2HPO4 solutions to prepare Chitin–Hap
composite membranes within very short time based on the wet process of Hap. They observed
the formation of apatite crystals on the surface of chitin membranes and revealed that the size
and deposition of the crystals are directly proportional to the number of immersion cycles. They
hypothesized that the presence of apatite layers on the surface of chitin membranes increased the
biocompatibility and provided a suitable substratum to facilitate cell attachment, adhesion, and
spreading (Madhumathi et al. 2009).
To conclude, many researchers have been attempting chitin and its derivatives, particularly chito-
san, as promising materials for tissue regeneration applications. The advantage of chitin derivatives
over other polysaccharides such as cellulose, starch, agar, etc., attributed to its chemical structure,
which allows specific modifications without too many difficulties at the C-2 position, as described
earlier. Furthermore, chitosan can be easily molded to various forms and its derivatives are biode-
gradable by lysozomal enzymes which make chitosan as an exciting and promising material for tis-
sue regeneration applications. Table 10.1 represents some of the recent research publications (from
2009) which are based on chitin and/or chitosan and their modified forms.
10.2.1.6 Hyaluronan/Hyaluronic Acid/Hyaluronate

Hyaluronic acid (HA) is a GAG component of connective tissue, synovial fluid of vertebrates, and
the vitreous humor of the eye (Drury and Mooney 2003). It also occurs as an extracellular poly-
saccharide in a variety of bacteria and it can be produced from Streptococcus zooepidemicus and
Streptococcus equi. HA is composed of alternating disaccharide units (250–25,000) of d-glucuronic
acid and N-acetyl-d-glucosamine with β(1 → 4) interglycosidic linkage. HA is an important compo-
nent of ECM and displays a vital role in lubrication; water sorption; water-retention; and a number
TABLE 10.1
Scaffolds Based on Chitosan
Tissue
Active Bio Engineering
Scaffold Type Method of Preparation Molecule/Cell Types Application References
Collagen/hyaluronan/ EDCI crosslinking — Potential Lin et al. (2009)
chitosan composite followed by freeze biomedical
sponges drying applications
Hybrid chitosan-silica Sol–gel process followed TMOS, CaCl2 and Bone regeneration Lee et al. (2009)
xerogels by freeze drying TEP as calcium
and phosphorous
oxide precursors
Chitosan–nanoHAp Ultrasonic dispersion of Nano HAp Bone tissue Han and Misra
composite scaffolds Hap in chitosan solution engineering (2009)
followed by freeze
drying
Chitosan–gelatin Cross linking with GTA Hepatocytes Hepatic tissue Jiankang et al.
hybrid scaffolds solution followed by engineering (2009)
molding in SL apparatus
and freeze drying
CMCH/poly(vinyl Electrospinning followed HAp Bone tissue Shalumon et al.
alcohol) nanofibrous - by cross linking with engineering (2009)
HAp composite GTA followed by
scaffolds soaking in Hap
suspension
Alginate dialdehyde Freeze drying CPP Meniscus tissue Wang et al. (2010)
cross-linked engineering
chitosan/calcium
polyphosphate
composite scaffold
Chitosan-graft-β- Grafting of CD on Ketoprofen Tissue Prabaharan and
cyclodextrin chitosan in presence of engineering Jayakumar (2009)
scaffolds EDCI and NHS applications
followed by freeze
drying. Drug loaded
scaffolds prepared by
immersing in drug
solutions in presence of
GTA
Chitosan–gelatin/ Mixing and GTA nBGC Alveolar bone Mathew et al.
nano-bioactive glass crosslinking followed tissue (2010)
ceramic composite by freeze drying engineering
scaffolds
Combined brushite- Immersing chitosan β-TCP VEGF and Bone regeneration De La Riva et al.
chitosan system sponges in TCP cement PDGF (2010)
mixture
Temperature- Mixing CH-GP solution Sheep chondrocytes Articular cartilage Hao et al. (2010)
responsive chitosan with cross-linking repair
hydrogels solution consisting
filter-sterilized HEC
Chitosan collagen Mixing of appropriate hBMSC- Bone tissue Wang and
composite hydrogels volumes engineering Stegemann (2010)
of cellular functions such as attachment, migration, and proliferation. In water solutions, HA

occupies a volume approximately 1000 times higher than that in its dry state (Mano et al. 2007).
Furthermore, its abundant repelling anionic groups bind cations and water molecules leading to a
peculiar visco-elastic behavior of solutions (Gatj et al. 2005). Hyaluronate can be used as excellent
biological absorber and lubricant. Moreover, HA enhances tissue formation and repairs, provides a
protective matrix for reproductive cells, and serves as a regulator in the lymphatic system and acts
as a lubricating fluid in joints.
Various composite materials based on HA with proteins like gelatin and collagen have been
fabricated and successfully used for biomedical applications. Composites based on HA and gela-
tin have been obtained in the form of porous sponges from gelatin-hyaluronan solutions into 90%
(vol/vol) acetone/water mixtures containing a small amount of a carbodiimide (EDCI) as cross-
linking agent (Choi et al. 1999). This sponge-type biomaterial was constructed for wound dressings
or scaffolds for tissue engineering. When impregnated with antibacterial silver sulfadiazine, this
hyaluronan-gelatin sponge has been found to facilitate epidermal healing (Choi et al. 2001).
Composite materials of hyaluronan and collagen have been prepared by dissolution of the two
components in aqueous acetic acid followed by cross-linking with glyoxal or a periodate-oxidized
starch dialdehyde (Rehakova et al. 1996). These composites possess resistance to collagenase and
stimulate fibroblast growth (Rehakova et al. 1996). Alternatively, composite materials consisting
of hyaluronan and collagen have also been prepared using polyethylene oxide and hexamethylene
diisocyanate by cross-linking the dried hyaluronan/collagen coagulates. In addition, composite
materials of hydroxyapatite-collagen-hyaluronan have been prepared by adding hydroxyapatite
particles to a hyaluronan solution followed by blending with an aqueous dispersion of collagen
fibers. The final material, which consisted of 90% hydroxyapatite, 9.2% collagen, and 0.8% hyal-
uronan (wt/wt), was biocompatible and mechanically robust and was used as bone defect filler
(Bakos et al. 1997). Alternatively, a porous collagen-hyaluronan matrix was prepared by cross-
linking collagen with periodate-oxidized hyaluronan; the resulting material supported new bone
formation and has potential uses for the delivery of growth factors or as an implantable cell-seeded
matrix (Alini et al. 2003).
HA can be chemically modified to produce mechanically and chemically sturdy m aterials.
It can be modified at three positions per repeat unit of its structure: (1) –OH in C-6 position of
the N-acetyl glucosamine; (2) –COOH of the d-glucuronic acid, and (3) –NH position of the
N-acetyl-d-glucosamine. Among these, the main reactions were performed on –OH groups by
sulfation, esterification, or periodate oxidation. The sulfation of hydroxyl group in HA with a
sulfur trioxide–pyridine complex in dimethyl formamide (DMF) produced HyalSx, where x is the
degree of sulfation per disaccharide (x = 1–4). The Hyalsx compounds are promising components
as blood-compatible materials for medical device coating. For instance, immobilized HyalS3.5 onto
plasma-processed polyethylene has been reported to produce blood-compatible, antithrombotic PE
surfaces. Furthermore, Chen et al. (1997) have converted HyalSx to a photolabile azidophenylamino
derivative and photoimmobilized onto a poly (ethylene terephthalate) film. They have described that
HyalSx -coated surfaces display significant reduction of cellular attachment, fouling, and bacterial
growths compared with uncoated surfaces and the coating prevents the degradation by chondroitin-
ase and hyaluronidase.
Many chemical modifications have been performed also on the carboxylic acid position of HA
to increase its hydrophobicity. For example, Benedetti et al. have attempted to esterify HA by using
its alkyl salt, tetra (n-butyl) ammonium salt, by alkylation with an alkyl halide in DMF solution
(Benedetti et al. 1993). As the percentage of esterification increased, the hydrophobicity increased
resulting in a water insoluble material, which has been named as HYAFF® (Fidia Advanced Bio-
polymers). These HA esters have been used in the production of membranes and fibers, lyophilized
sponges, and spray-dried to microspheres. These polymers have shown good mechanical strength
in a dry state and lower robustness in a wet state. The degree of esterification has been found to
influence the size of hydrophobic patches leading to a polymer chain network which is more rigid
and stable, and less susceptible to enzymatic degradation. Finally, hyaluronan benzyl ester mate-
rials have been used as meshes and sponges for growth of human fibroblasts and for culture of
chondrocytes and bone marrow–derived mesenchymal cells for repair of cartilage and bone defects
(Benedetti et al. 1993). Recently, Hirakura et al. reported a novel hybrid hydrogel composite mate-
rial by using cross-linked HA as the biocompatible controlled release matrix and partially modi-
fied pullulan with cholesteryl group nanogels as the host molecule with molecular chaperone as a
model protein to support neurite growth. They demonstrated that low substituted HA hydrogels
degraded more quickly owing to higher recognition for enzymatic cleavage and bioactivity and they
concluded that upon addition of bioactive components, HA hydrogels can serve as potential compo-
nents to support neurite growth (Hirakura et al. 2010).
In another approach, Segura et al. have shown the construction of hydrogels based on cross-
linked HA with poly (ethylene glycol) diglycidyl ether. They also have incorporated collagen into
the cross-linked network in order to support cellular adhesion in vitro. This construct possessed low
water content and low degradation rate and it could be topographically patterned during gelation.
The authors have also functionalized HA and HA/collagen scaffolds by introducing biotin into the
HA backbone before cross-linking reaction in order to introduce avidin-streptavidin and neutra-
vidin molecules on the surface of hydrogel which can be widely employed for tissue engineering
applications (Segura et al. 2005).
Pouyani et al. have hypothesized that hydrazides, which have pKa values for the corresponding
conjugate acids range between 2 and 4, would have retained their nucleophilicity at pH 4.75 and
would have coupled efficiently to carbodiimide-activated glucuronic acid residues of hyaluronan.
They have referred the utilization of dihydrazide compounds, such as adipic dihydrazide (ADH),
to modify the chemical structure of HA. This chemical modification has provided multiple pen-
dant hydrazide groups which may be further derivatized with drugs, biochemical probes, and for
cross-linking agents. These abundant surface hydrazide groups of ADH-modified hyaluronan can
be loaded with drug molecules and then cross-linked into a hydrogel which facilitates confine-
ment of the drug in the bulk of the hydrogel (Pouyani et al. 1994). Alternatively, a limited number
of the hydrazide groups could be cross-linked and the remaining hydrazide groups on the result-
ing hydrogel could be loaded with a therapeutic agent. For example, Luo et al. have synthesized
HA-Taxol® bioconjugates by the conjugation of HA-ADH and ester-activated Taxol. This conjugate
can be labeled with a fluorescent dye allowing the monitoring of uptake by human breast, ovarian,
and colon cancer cells and measurement of the corresponding selective toxicity of the polymeric
prodrug (Luo et al. 2000).
Hahn et al. have reported different hydrogel preparation techniques based on HA for three differ-
ent applications, such as the management of tissue adhesion formation and soft tissue augmentation.
They have investigated the influence of functional groups involved and type of cross-linking cova-
lent bonds and density on degradation of HA hydrogels. Initially they have grafted HA with adipic
acid dihydrazide by cross-linking with bis(sulfosuccinimidyl) suberate (BS3) and then modified
with methacrylic anhydride in the presence of dithiothreitol (DTT; Michael addition), Traut’s reagent
(imminothiolane) in order to produce methacrylated HA (HA-MA) and thiolated HA (HA-SH),
respectively. The degradation rates were reported as HA-SH > HA-ADH > HA-MA. The degrada-
tion rate of HA hydrogels can be controlled by changing the functional group of HA derivatives and
the cross-linking reagents (Hahn et al. 2007).
In addition, the gelling state of HA hydrogel was another factor in controlling the degradation
rate. HA and HA-modified surfaces show potential medical applications. Important new products
are already available in market. For example, Synvisc is a specific device for visco supplementation
therapy in osteoarthritis which increases the viscoelastic properties of the synovial fluid and the
intercellular matrix of the synovial tissue and capsule. Similarly, Hylans are hydrogels marketed
by Biomatrix obtained by cross-linking hyaluronan-containing residual protein with formaldehyde
in a basic solution. Hylan gels have greater elasticity and viscosity than soluble hylan materials,
while still retaining the high biocompatibility of native hyaluronan. Hylans have been investigated
in a number of medical applications. In the treatment of degenerative joint disease and rheuma-
toid arthritis, hylans have been found to protect cartilage and prevent further chondrocyte injury,
although the effect is reversible and viscosity dependent.
10.2.2 Proteins as Biomaterials

Natural proteins are a class of materials with enormous different properties which perform very
specific biochemical, mechanical, and structural properties. Amino acids are the building blocks of
proteins and consist of a central carbon linked to an amine group, a carboxyl group, a hydrogen atom,
and a side chain (R groups). R groups can be classified as nonpolar groups, uncharged polar groups,
or charged polar groups, and their distribution along the protein backbone gives the proteins distinct
characteristics. The amino acid sequences of polypeptides and proteins possess multiple functional
groups, exact molecular weights, defined branch points, and predisposed three-dimensional struc-
tures conferring them unique and complex bioactive properties. Despite the tremendous research
in synthesizing synthetic materials able to mimic the properties of natural proteins, these attempts
were not successful. Protein-based polymers mimic many features of the extracellular matrix and
potentially direct the physiological functions, such as cell migration, growth, and organization of
cells during tissue regeneration and wound healing and the stabilization of encapsulated and trans-
planted cells. The major drawback of the natural–origin proteins is their possible batch variation.
One interesting strategy to overcome this issue is the recombinant protein technology where the
mono dispersity and precisely defined properties of proteins as well as the predictable placement
of cross-linking groups (binding moieties at specific sites along the polypeptide chain) or their
programmable degradation rates make them very attractive and useful for drug delivery and tissue
engineering (Rodriguez-Cabello et al. 2005). In the following, the most important natural polymers
and their application in biomedical field are discussed.
10.2.2.1 Collagen
Collagen is a major natural constituent of connective tissue, staging approximately 30% of all
vertebrate body protein and a major structural protein of any organ. Collagen is synthesized by
fibroblasts originated from pluripotential adventitial cells or reticulum cells. Collagen is the major
protein component of the ECM and provides support to connective tissues such as skin, tendons,
bones, cartilage, blood vessels, and ligaments. In its native environment, collagen interacts with
cells in connective tissues and transduces essential signals for the regulation of cell anchorage,
migration, proliferation, differentiation, and survival (Malafaya et al. 2007).
The basic unit of collagen is a polypeptide consisting of the repeating sequence of glycine, pro-
line, and hydroxyproline. These three polypeptide chains twine around one another as in a three-
stranded rope and combines with 12 others to form the left-handed triple helix structure in collagen.
Each chain has an individual twist in the opposite directions. The principal feature that affects a
helix formation is a high content of glycine and amino acid residues (Piez 1985).
The strands are held together primarily by hydrogen bonds between adjacent -CO and -NH
groups, and also by covalent bonds. The basic collagen molecule is rod shaped with a length and a
width of about 3000 and 15 Å, respectively, and has an approximate molecular weight of 300 kDa.
The mechanical stability and other physical characteristics of collagen result from intermolecular
cross-linking forming macromolecular fibers (Shoulders and Raines 2009).
The applications of collagen as a biomaterial was first reported by Ehrmann and Gey in 1956,
who compared the growth of cells and tissue explants on collagen and on glass. They found that
collagen gels augmented cellular growth. Since then, collagen substrates have been known to affect
the morphology, the migration, the adhesion and, in certain cases, the differentiation of cells. Till
date, more than 28 types of collagens have been identified, among which type I collagen is the
most abundant. However, the most extensively investigated types are collagen type I, II, III, and IV.
Type I collagen is present in tendons, ligaments, and bones and consists of striated fibers between
80 and 160 nm in diameter. Type II collagen consists of fibers that are less than 80 nm in diameter
and it is present in cartilage and intervertebral disks. Type III forms reticular fibers in tissues and
strengthens the walls of hollow structures such as arteries, intestine, and uterus. Type IV is a highly
specialized form of collagen present as a loose fibrillar network in the basement membrane (Friess
1998). Type VI is microfibrillar collagen and type VII is anchoring fibril collagen (Samuel 1998).
Type IX, XI, XII, and XIV collagens are fibril-associated collagens and possess small chains, which
contain some nonhelical domains.
Biomaterials made of collagen offer several advantages as they are biocompatible and non-
toxic and have well-documented structural, physical, chemical, and biological suitable properties.
Additionally, collagen can be cross-linked to modulate its mechanical, degradation, and water
uptake properties. Furthermore, it promotes blood coagulation, it can be formulated in a num-
ber of different forms, and it is compatible with many synthetic polymers. The disadvantages for
the utilization of collagen as a biomaterial includes the high cost for purification (particularly for
type I collagen) and the variability of isolated collagen in terms of cross-linking density, fiber size,
and presence of impurities. Apart from this, the hydrophilicity of collagen may lead to swelling
and more rapid release of incorporated bioactive components. Other drawbacks are variability in
enzymatic degradation rate as compared with hydrolytic degradation. The degradation of collagen
is influenced by many enzymes such as collagenase and gelatinase and many other nonspecific
proteinases (Friess 1998). Moreover, handling of collagen degradation is difficult because several
factors have an impact on degradability of collagen, for instance, the penetration of cells into the
structure may cause contraction. Side effects of collagen include the risk of bovine sponge form
encephalopathy (BSF) and the occurrence of undesired mineralization (Chvapil 1979, Todhunter
et al. 1994, Brown and Timple 1995, Liu et al. 1995, Ramshaw et al. 1995, Rao 1995, Friess 1998,
Exposito et al. 2002).
As collagen can form fibers with extra strength and stability through its self-aggregation and
in vivo cross-linking, it is widely used for tissue engineering applications. But, once collagen is
extracted and reprocessed from its original source, it has weaker mechanical properties, thermal
instability, and it may undergo rapid proteolytic breakdown. To overcome these problems, colla-
gen has been cross-linked by a variety of cross-linking agents (Lee et al. 2001). The modified
cross-linked collagen degrades at a much slower rate than native collagen further enhancing its in
vivo efficacy as a biomaterial. The degree of cross-linking of collagen can be estimated via free
amino group analysis and resistance to enzymatic digestion. Glutaraldehyde (GTA) has been fre-
quently used as the cross-linking reagent as it is easily available and inexpensive and can effectively
cross-link collagenous tissues quite rapidly (Khor 1997). However, GTA is toxic if released into
the host. As a consequence, natural, milder, efficient agents were attempted to strengthen collagen.
Transglutaminases (EC 2.3.2.13) are a group of enzymes that can catalyze several post-translational
reactions. The most important of these reactions results in the cross-linking of peptides or pro-
teins to form multimers via ɛ- (γ-glutamyl)lysine linkage using the side chains of lysine and gluta-
mine residues. Hence, transglutaminases can be adopted to cross-link collagen matrices to further
increase their strength and also to incorporate cell adhesion factors within the gel matrix, resulting
in an enhancement of cell proliferation. Collagen modified with transglutaminases demonstrated
greater resistance to the total complement of cell-secreted proteases and improved resistance to
cell-mediated degradation. Transglutaminase-cross-linked collagen may be more robust than native
collagen, either through the presence of cross-links which may disturb the native conformation, or
via disruption of the native fibrillar form during fibrillogenesis. Alternatively, due to the increased
resistance of the cross-linked collagen to Matrix metallo proteinase (MMP) degradation, fibroblasts
may elicit an enhanced MMP response in a futile attempt to increase the rate of collagen break-
down. The cross-linked collagen elicits different cellular response compared to untreated collagen.
TG-treated collagen matrix is in contact with the cells longer due to its increased proteolytic resis-
tance, thus providing the required integrin-mediated signal to the Human osteoblastic cells (HOB
cells) necessary for differentiation (Yamane et al. 2004).
The other cross-linking methods include carbodiimide treatment, acylazide treatment, and phys-
ical methods like dry heat treatment and exposure to ultraviolet (Avery and Bailey 2008). Cross-
linking with carbodiimides, especially 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI),
involves the formation of amide bonds between carboxylic and amino groups on the collagen mol-
ecules without becoming part of the actual linkage. At pH 5, carbodiimides first couple to carbox-
ylic groups to form o-isoacylurea structures. The resulting activated intermediate is attacked by a
nucleophilic primary amino group to form an amide cross-link and the iso-ureaderivative of the
applied carbodiimide is then eliminated and can be washed out (Grabarek and Gergely 1990). The
susceptibility of carbodiimide-cross-linked collagen to enzymatic degradation can be controlled by
varying the degree of cross-linking via the reaction conditions (Olde et al. 1996). Alternatively, acyl
azide method involves the formation of an intermediate deriving from induction of amide bonds
(Petite et al. 1990). The azide functionalities couple with amino groups to give amide cross-links
with superior cytocompatibility (Petite et al. 1995). Physical methods in cross-linking of collagen
include dehydrothermal treatment (DHT) and/or exposure to ultraviolet light at 254 nm. In case of
DHT treatment, the degradation of the triple helices can be minimized through removal of water
content via vacuum prior to heating. Even small amounts of residual moisture can cause breakdown
of the helical structures and proteolysis (Yannas and Tobolksy 1967). The dehydration itself already
induces amide formation and esterification between carboxy and free amino and hydroxyl groups,
respectively. Cross-linking of collagen with UV irradiation is initiated by the formation of free
radicals on aromatic amino acid residues (Weadock et al. 1995) which indicates a rather limited
maximum degree of cross-linking due to the small number of tyrosine and phenylalanine residues
in collagen (Weadock et al. 1996).
Owing to its physico-chemical properties, flexibility in physico-chemical modifications and
versatile cross-linking properties, collagen has been applied for various tissue engineering appli-
cations including dermal, bone, and cartilage regeneration (Glowacki and Mizuno 2008). Collagen-
based scaffolds containing other biopolymers or bio-ceramics have also been widely investigated.
Particularly, in case of bone tissue engineering applications, the composites of osteoconductive
hydroxyapatite and collagen have been extensively investigated as promising scaffolding materials
where the addition of hydroxyapatite induces mechanical strength (Murphy et al. 2010). Table 10.2
represents some of the recent research activities which involve the use of collagen either in native
form or in modified form as a scaffolding material for tissue regeneration applications.
10.2.2.2 Gelatin
Gelatin is a protein derived from collagen by thermal denaturation either with very dilute acid or
alkaline water solution treatment (Zhou and Regenstein 2005). Depending on the pretreatment of
collagen, gelatin is classified into two types: type A and type B. Type A gelatin is synthesized from
pig skin by acid denaturation whereas type B gelatin is produced by alkaline denaturation of cattle
hides and bones. Such treatments result in partial cleavage of collagen cross-links, resulting in the
formation of warm water-soluble collagen, that is, gelatin (Schrieber and Gareis 2007, Seal et al.
2001). Collagen breakdown into gelatin is dependent on three factors: temperature, time, and pH.
High temperatures and long periods of exposure to heat accelerate the process. Gelatin exhibits
similar amino acid composition and to some extent similar backbone structure of collagen, but it
also exhibits its own properties (Olsen et al. 2003). Gelatin is a heterogeneous mixture of single or
multi-stranded polypeptides, each with extended left-handed proline helix conformations and con-
taining between 50 and 1000 amino acids. Gelatin contains many glycine (almost one in three resi-
dues, arranged every third residue), proline, and 4-hydroxyproline residues. A typical structure of
gelatin is -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-Gly-Pro- (Gomez et al. 2002). Recent studies revealed
that the helical structure of gelatin is composed of a 7/1 helix (the three strands forming a 7/2 helix)
with a 60 Å axial repeat, with tripeptides forming each unit (Okuyama et al. 2006). Each of the
three strands in the triple helix requires about 21 residues to complete one turn and typically there
would be between one and two turns per junction zone (Oakenfull and Scott 2003).
TABLE 10.2
Collagen Based Matrices/Scaffolds for Tissue Regeneration
Active Bio Tissue
Molecule/Cell Regeneration
Scaffold Type Method of Preparation Types Application References
Tailor-made porous Covalent attachment of Immobilized Regeneration of Pieper et al. (2000)
collagen–GAG GAGs to collagen matrices GAGs CS tissues like
matrices performed using EDCI and and HS tendon and skin.
NHS followed by
lyophilization
Cross-linked type I Crosslinking by using EDCI Chondroitin Regeneration of Buma et al. (2003)
and type II collagen followed by lyophilization sulfate articular
matrices cartilage tissues
Two-layered DHT crosslinking followed Growth-factor Treatment of deep Gotterbarm et al.
collagen–tricalcium by lyophilization mixture (GFM) osteochondral (2006)
phosphate cell-free defects
construct
Thin collagen Cross-linking under a Cultured RPE Age-related Lu et al. (2007)
scaffolds 254 nm UV lamp for 3.5 h cells for in vivo macular
and then vacuum- characteristics degeneration
desiccated for 48 h with
fresh Drierite
pH-responsive alkali Crosslinking with MAD — Tissue Saito et al. (2007)
treated collagen engineering and
hydrogels drug delivery
systems
Porous collagen- Chemical crosslinking by Nano- HAp Bone regeneration Pek et al. (2008)
apatite nano- using EDCI-NHS followed crystals
composite foams by freeze drying
Collagen biomimetic Obtained a film through Calcium acetate Skin regeneration Chirita (2008)
films forced evaporation of the and sodium
solvent in air at 25°C by silicate
using glycerine as plasticizer
Biomimetic collagen/ Compositional and structural Nano-HAp Bone regeneration Liu (2008)
nano-hydroxyapatite gradient composite scaffold crystals
scaffold was formed by
precipitation of nano-HAp
crystallites within the
collagen fibril matrix
mPCL–TCP/collagen Fused deposition modeling rhBMP-2 Skull regeneration Sawyer et al. (2009)
composites in a rat calvarial
defect
Collagen membranes By grafting Basic fibroblast Bladder Chen et al. (2010)
growth factor regeneration
Porous collagen Homogenization followed Hyaluronic acid Improved Perng et al. (2009)
scaffolds with by freezing for 24 h and oligosaccharides revascularization
hyaluronic acid freeze drying for 24 h process for rapid
oligosaccharides followed by EDCI wound healing
crosslinking.
Three-dimensional Crosslinking with mTGase Hydroxyapatite Bone tissue Ciardelli et al.
porous HAp/collagen followed by freeze drying regeneration (2009)
composite scaffolds for 36 h
Gelatin is a natural derived polymeric product and it has been widely utilized for the fabrication
of scaffolds owing to its peculiarities: (1) the presence of both acidic and basic functional groups
in the gelatin macromolecules, which render superior chemical modification properties to gelatin;
(2) its capacity to form a specific triple-stranded helical structure not observed in synthetic poly-
mers (this structure is formed in solutions at low temperatures) (particularly, the rate of formation
of a helical structure depends on many factors such as the presence of covalent cross-bonds, gelatin
molecular weight, the presence of amino acids, and the gelatin concentration in the solution); and
(3) its specific interaction with water which is different than that observed with synthetic hydro-
philic polymers. This peculiarity governs the structural and physico-mechanical properties of gela-
tin in the solid state (de Wolf 2003, Okuyama et al. 2006).
At optimal concentrations, gelatin solutions are in the sol state at around 40°C and change into
gels when they are cooled down to room temperature. This gelation normally occurs by physical
cross-linking, which results in the formation of “junction zones” and finally a three-dimensional
branched network. The sol-gel transformation is due to a conformational disorder-order transition of
the gelatin chains which form thermo-reversible networks by associating helices in junction zones
which are stabilized by hydrogen bonds (Bigi et al. 2000).
Throughout this dynamic process, bonds are continually breaking and reforming within the gel,
due to weak polymer–polymer interactions. Thus, at elevated temperatures, gelatin macromolecules
assume the conformation of a statistical coil. Under specific conditions (temperature, solvent, pH),
gelatin macromolecules can display a flexibility sufficient to realize a wide variety of conforma-
tions. This makes it possible to vary gelatin characteristics depending on its molecular structure
(Djabourov et al. 1988). Upon aging, the gel gradually assumes a more stable conformation. The
detailed structure of the final product is highly dependent on the temperature at which the gel is set.
Although the structure continues to change and the helix content of the gels continues to increase,
the physical restrictions on the polymers prevent the gel from ever reaching equilibrium. Hence,
the formation of gelatin gels is often described as a “frustrated renaturation process” (Djabourov
et al. 1988).
Gelatin, similar to synthetic high polymers, displays a wide molecular weight distribution. Gelatin
can form a large variety of super molecular structures; from the simplest globular structure, typical
amorphous polymeric structure, to a well-developed fibrillar structure with various intermediate
states (Young et al. 2005). Furthermore, gelatin displays various physico-mechanical properties.
(1) First, gelatin behaves very differently depending on macromolecular conformations. In case
of collagen-like helical conformation of gelatin macromolecules, gelatin samples exhibit proper-
ties suitable for service at ordinary temperatures and humidities, while in the coiled conformation
conditions, gelatin behaves as a brittle impractical material. (2) In the dry state, gelatin behaves as a
brittle material regardless of the molecular conformations. (3) Gelatin features a wide temperature
range of the glassy state with the upper limit of 205°C–210°C. This characterizes gelatin as a mate-
rial with a fairly wide range of heat resistance. Moreover (4) gelatin, as a hydrophilic biopolymer,
specifically interacts with water and undergoes drastic changes of its physico-mechanical proper-
ties, depending on the moisture content (Lakshmi and Cato 2007). With respect to collagen, gelatin
does not express antigenicity in physiological conditions, and it is much cheaper and easier to obtain
in concentrated solutions. On the other hand, gelatin exhibits poor mechanical properties which
limit its possible applications as a biomaterial. Moreover, gelatin is soluble in aqueous solution,
so gelatin materials for long-term biomedical applications have to be cross-linked to improve both
their thermal and the mechanical stability (Michon et al. 1997).
Several attempts have been made to modify the physico-mechanical properties of gelatin by
chemical cross-linking. In chemical cross-linking methods, cross-linkers are used to bond functional
groups of amino acids. Commonly used chemical cross-linkers include formaldehyde, glutaralde-
hyde, polyepoxy compounds, dimethyl suberimidate, carbodiimides, and acyl azide (Khor 1997),
similar cross-linking agent described earlier (Section 10.2.2.1). Such cross-linking methods
have been widely used in the manufacture of various gelatin-based biomaterials. However these
chemical cross-linking reagents are generally cytotoxic, impairing the biocompatibility of biopros-
theses, resulting in an increasing demand for a cross-linking reagent that can form stable and bio-
compatible cross-linked products, without causing problems of cytotoxicity. Frequently, a low-toxic
and naturally occurring reagent, genipin, has been used as a cross-linking agent for fixing biological
tissue. Genipin and its related iridoid glucosides, extracted from the fruits of Gardenia jasminoides
Ellis, can react with amino acid or proteins to form stable cross-linked products (Sung et al. 1999).
The mechanism of the reaction of amino acids or proteins with genipin is still not well understood
at present. The mechanism proposed by Touyama’s research group (Touyama et al. 1994) for the
formation of the genipin-methylamine monomer is through a nucleophilic attack by methylamine
on the olefinic carbon at C-3 in genipin, followed by the opening of the dihydropyran ring and an
attack by the secondary amino group on the resulting aldehyde group. The blue-pigment polymers
are presumably formed through the oxygen radical-induced polymerization and dehydrogenation of
several intermediary pigments.
Gelatin has been widely used in the pharmaceutical industry as well as in the biomedical fields to
make hard and soft capsules, microparticles, sealants for vascular prostheses, wound dressing, and
adsorbent pad for surgical use and for scaffold preparation for regenerative applications (Lakshmi
and Cato 2007). Table 10.3 depicts some of the published articles on gelatin-based scaffolding mate-
rial either in native form or in cross-linked form. Apart from this, researchers have also prepared
hybrid scaffolding materials based on gelatin and other polymers. Table 10.4 represents some of the
recent publications which involve scaffolding materials based on blends between gelatin and other
natural polymers. Many researchers also fabricated scaffolds based on the combination of gelatin
and synthetic polymers such as PLGA, PLA, PCL, etc., to improve the biocompatibility of the sub-
strates through bioartificial blending. Bioartificial polymeric materials were initially conceived to
produce new systems combining the characteristics of synthetic polymers, such as good mechanical
properties, good processability, low production and transformation costs, with the specific prop-
erties of biodegradability and compatibility with cells and tissues associated to biopolymers. In
addition to these studies, scientists also synthesized composite scaffolds based on native gelatin or
hybrid polymers with inorganic components such as hydroxyapatite and bioactive glass to attain
synergic bioactivity and biomechanical properties (Table 10.5).
10.2.2.3 Fibrin-Fibrinogen-Fibronectin
Fibrin is a nonglobular, fibrous, insoluble protein matrix which forms immediately at the site of skin
injury in order to prevent blood loss. Fibrin formation involves the enzymatic cleavage of fibrino-
peptides A and B followed by noncovalent polymerization of thrombin (Mosesson et al. 2001).
Fibrinogen is a monomer of fibrin and is composed of three pairs of nonidentical polypep-
tide chains, which are arranged as one central domain (E domain) and two terminal domains
(D domains) linked together by disulfide bridging within the N-terminal E domain. The central
domain contains fibrinopeptide E and fibrinopeptides A and B whereas the two terminal domains
are based on fibrinopeptide D linked to the central domain by a coiled coil segment and sites that
participate in cross-linking. These domains contain constitutive binding sites that participate in
fibrinogen conversion to fibrin, fibrin assembly, cross-linking, and platelet interactions. Fibrinogen
and fibrin play an important, interdependent role in clot formation, fibrinolysis, cellular and matrix
interactions, inflammation, wound healing and neoplasia by fibrin formation and the cascade of its
complementary interactions between specific binding sites on fibrinogen and extrinsic molecules
including proenzymes, clotting factors, enzyme inhibitors, and cell receptors.
Fibronectin is a large extracellular glycoprotein (a disulfide-bonded dimer of 220–250 kDa sub-
units) and is a multifunctional component of the ECM. It induces cell attachment and serves as
a substrate for cell adhesion and migration events in important physiological processes such as
embryogenesis, wound healing, hemostasis, and thrombosis (Hynes 1990, Pankov and Yamada
2002). It is also involved in many cellular processes like tissue repair, embryogenesis, and
blood clotting.
TABLE 10.3
Scaffolds Based on Crosslinked Gelatin
Active Bio Tissue
Molecule/Cell Engineering
Gelatin-resorcinol Cross linking with Resorcinol, fibrin Adhesive glue Kodama et al.
formaldehyde- formaldehyde (37%) and GTA glue and even in the (1997)
glutaraldehyde glue (50%) (9:1 weight ratio) antifibrinolytic presence of
agent (aprotinin) degenerative
tissue
Gelatin as a sealant Gelatin impregnation process Rifampin and Prevention of Javerliat et al.
for vascular graft by using partially succinylated tobramycin bacterial (2007)
mixture of USP ossein gelatin (antibiotics) infection in
with polyester, 20% aqueous vascular graft
formaldehyde as cross linker
Gelatin films as Film formation by freezing at EGF Wound healing Akane and
wound dressing −4°C for 3 days and Toshikati (2005)
sterilization by Gamma
irradiation 25 kGy
Gelatin sponges for GTA crosslinking and freeze Human recombinant Wound dressing Ulubayram et al.
wound dressing drying for 24 h EGF (2001)
Gelatin gels A citric acid derivative as a Divalent anions as Tissue adhesive Aoki et al. (2004)
novel cross-linker cross linkers
Aligned porous GTA crosslinking, — Aligned porous Wu et al. (2010)
gelatin scaffolds unidirectional freezing by scaffolds for
with micro tubules using liquid nitrogen and tissue
orientation freeze drying for 48 h engineering
structure applications
Gelatin scaffolds Salt leaching method by using Artificial skin Porous gelatin Lee et al. (2005)
containing artificial NaCl followed by freeze scaffolds for
skin drying. Crosslinking by skin defects
immersing dried samples in
acetone:water mixture (9:1 by
volume) containing 30% EDCI
by gelatin weight for 24 h
Porous gelatin Cryogenic treatment and a Human umbilical Cell delivery Van Vlierberghe
cryogels subsequent freeze-drying vein endothelial tool in tissue et al. (2006)
cells (HUVEC), engineering
osteoblasts (MG-63
and CAL 72),
human foreskin
fibroblasts, glial
cells (U373-MG)
and epithelial cells
(HELA)
Nanofibrous gelatin/ Combination of thermally Paraffin spheres Bone tissue Liu et al. (2009b)
apatite composite induced phase separation and were used as a engineering
scaffolds porogen leaching by using template to create
freeze-drying in a salt-ice bath macropores and
for 4 days and then vacuum MC3T3-E1
drying at room temperature osteoblast cells
for another 3 days
TABLE 10.4
Hybrid Polymeric Scaffolds Based on Gelatin and Other Natural Polymers
Active Bio
Scaffold Type Method of Preparation Molecule/Cell Types Application References
Chitosan–gelatin Freeze-drying technique — Tissue engineering Mao et al.
hybrid polymer (2003)
network
Genipin cross linked Genipin cross linking — Tissue engineering Chiono et al.
chitosan-gelatin blends followed by freeze drying (2008)
Chitosan-gelatin Combination of rapid Hepatocytes isolated Hepatic tissue Jiankang et al.
hybrid scaffolds with prototyping (RP), from male Sprague– engineering (2009)
well-organized micro-replication and Dawley rats
microstructures freeze–drying techniques
Hydroxypropyl Thermo-crosslinking by — Corneal stroma Wang et al.
chitosan-gelatin using 1,4-butanediol tissue engineering (2009)
scaffolds diglycidyl ether for 12 h
followed by freezing at
−50°C and drying for 12 h
Glycidyl methacrylated BMP-free or BMP-loaded BMPs and human Periodontal Fa-Ming et al.
dextran (Dex-GMA)/ Dex-GMA/PEG PDLCs regeneration (2007)
gelatin scaffolds with microspheres were
microspheres loaded prepared by
with BMPs polymerization of
Dex-GMA in the
discontinuous phase of an
aqueous PEG solution and
scaffolds prepared by
radical crosslinking and
low dose γ-irradiation
Silk sericin gelatin 3-D Sericin/ gelatin 3-D — Tissue engineering Mandal et al.
scaffolds scaffolds were fabricated and biomedical (2009)
by pre-freezing sericin/ applications
gelatin mixture solution
(s) at −20°C, followed by
lyophilization
Porous chitosan- Freezing at −20°C for 1 h, Plasmid DNA as Cartilage defects Guo et al.
gelatin scaffold −80°C for 2h, and finally expression vector regeneration (2006)
containing plasmid lyophilizing for 48 h consisting of the
DNA encoding coding sequence of
transforming growth TGF-β1 and the
factor-β1 cytomegalovirus
enhancer inserted at the
upstream (pCMV-
TGF-β1, 6896 bp)
Agarose-gelatin Coupling using — A promising Shinji et al.
conjugate scaffolds 1,1-carbonyldiimidazole matrix for tissue (2007)
(CDI) at 25°C engineering
applications
Silk fibroin/gelatin Layer-by-layer (LbL) Trypan blue, FITC- Controlled release Mandal et al.
multilayered films assembly of various inulin and FITC-BSA of a wide spectrum (2009)
blends as model components of bioactive
for controlled release molecules
TABLE 10.5
Composite Polymeric Scaffolds Based on Gelatin and Bioceramics
Active Bio Tissue
Molecule/Cell Engineering
Porous gelatin–starch Microwave vacuum Nano hydroxy Hard tissue (bone) Sundaram et al. (2008)
composite scaffold drying apatite crystals engineering
with
nanohydroxyapatite
Composite chitosan- GTA crosslinking and Nano-HAp crystals Bone tissue Peter et al. (2010a)
gelatin/ freeze drying for 48 h engineering
nanohydroxyapatite
scaffolds
Composite gelatin- Step 1: Cross-linking of
nano hydroxyl gelatin with GTA in
apatite/fibrin the presence of
scaffold loaded with medical-grade nHAp
rh BMP-2 granules to form
hydrogel.
Step 2: Infusion of Nano HAP crystals Bone regeneration Liu et al. (2009a)
above hydrogel into a and rhBMP-2
solution containing
100 μg rhBMP-2 and
50 mg fibronectin +
400 IU thrombin in
1 mL of CaCl2 and
freeze drying
Chitosan-gelatin/ GTA cross-linking and Bioactive glass Alveolar bone Peter et al. (2010b)
nano-bioactive glass freeze drying for 48 h ceramic tissue
ceramic composite nanoparticles engineering
scaffold
Osteochondral Composite ceramic part Osteochondral cells Articular cartilage Lien et al. (2009)
scaffold of was prepared by repair
ceramic-gelatin pressure compression
assembly (CGA) of TCP powder in the
presence of amorphous
calcium polyphosphate
(CPP) powders.
Genipin crosslinking
and freeze drying for
48 h
3-dimensional gelatin/ Glutaraldehyde The rat stromal stem Tissue Zheng et al. (2007)
montmorillonite– crosslinking and freeze cells cells and engineering
chitosan scaffolds drying for 48 h montmorillonite applications
Composite gelatin Genipin crosslinking Hydroxyapatite/bio Bone tissue Gentile et al. (2010)
films with followed by drying active glass engineering
hydroxyapatite and at room temperature
or bioactive glass for 48 h
Fibronectin exists in two main forms: (1) as an insoluble glycoprotein dimer that serves as a
linker in the ECM (extracellular matrix), which is made by fibroblasts, chondrocytes, endothelial
cells, macrophages, as well as certain epithelial cells and (2) as a soluble disulfide-linked dimer
found in the plasma (plasma FN) which is synthesized by hepatocytes. Each monomer is composed
of many repeats of three types (I, II, and III) of small domains (Baron et al. 1991).
Fibronectin can be incorporated onto the surface of numerous biomaterials to enhance the avail-
ability of its cell-binding site. The repeat domains of fibronectin reversibly unfold in a distinctive
sequence under applied tension. Under certain mechanical conditions, cellular activity changes the
tension applied to fibronectin molecules resulting in the formation of cryptic sites. These cryptic
sites include additional sequences which bind integrin receptors on cells, other matrix molecules,
and other fibronectin molecules, as well as antiadhesive sites. Thus, exposure of cryptic sites by
mechanical unfolding may act as a trigger for a variety of cell-matrix interactions (Watanabe et al.
2000). On a scaffold with the surface having oriented fibronectin layer, human umbilical vein endo-
thelial cells can spread significantly faster in a more spherical way (Calonder et al. 2005).
Fibrin-based materials are biocompatible and biodegradable, can be produced from human
serum, and have been widely used in short-term conditions such as adhesion and sealing as they
are soluble and unstable due to degradation by fibrinolysis. Furthermore, as they are autologous, the
risks of toxic degradation or inflammatory reactions are negligible. The key aspect for the versatile
utilization of fibrin as biomaterial relays on characteristic in vitro structural fiber network formation
which exactly mimics that of in vivo plasma clots (Ye et al. 2000).
In the field of tissue engineering, fibrin has been attempted for a variety of tissue engineer-
ing applications such as to fabricate sealants in surgery and porous scaffold for bone regeneration
(Eyrich et al. 2007). Biomaterials produced from fibrin display varying mechanical and structural
properties depending on the initial fibrinogen concentrations, and hence, they are applied for various
tissue regeneration applications. For instance, biomaterials produced by using higher concentrations
of fibrinogen have been used as tissue sealants (Traver and Assimos 2006), whereas fibrin hydro-
gels, which have been produced at lower fibrinogen concentrations, have been studied as cardiovas-
cular grafts (Tiwari et al. 2002, del Mel et al. 2008) and bioengineered cartilages. For use in tissue
engineering applications, fibrin gels have been prepared by combining fibrinogen and thrombin
solutions containing calcium ions. These preparations are not suitable in conditions where the scaf-
folds are needed to be intact for longer durations. One of the major challenges in using fibrin as a
scaffold relays on its rapid degradation nature. However, the rate and extent of its degradation varies
depending on cell type and culture conditions; in some cases, it is too rapid as compared to the rate
at which ECM is produced by the cells. For instance, this happens in case of smooth muscle cells
which rapidly degrade the fibrin, leading to the complete degradation of scaffold before significant
ECM is produced to replace it (Ahmed et al. 2008). Rapid degradation of the fibrin matrix can be
controlled by using serine protease inhibitor aprotinin. Tuan et al. have reported the successful inhi-
bition of fibrin degradation by using aprotinin in concentrations of 500 KIU/mL (Tuan et al. 1996).
Herbert et al. have studied lysine analog, 1-aminocaproic acid (ACA), as an inhibitor to slow
down fibrin degradation which competitively inhibits attachment of plasmin and plasminogen to
fibrin (Herbert et al. 1996). Bach et al. utilized fibrin glue as a suspending vehicle for the delivery
of urothelial cells in urethral reconstruction. They applied such suspension to a connective tissue
capsule tube formed in vivo. The urothelial cells spread and formed an adherent and confluent cell
layer within 2 weeks of implantation, at which time the fibrin clot had already been replaced with
other connective tissue. Similar technique has also been applied to construct skin grafts and a com-
posite neotrachea (Bach et al. 2001).
Han et al. have constructed bioengineered ocular surface by using fibrin as a matrix for corneal
epithelial stem cells. They hypothesized that fibrin provides a favorable matrix environment for epi-
thelial cell growth and differentiation during wound healing. In their work, fibrinogen and thrombin
were isolated from blood plasma in the lab and contained a number of other plasma proteins. This
demonstrated the possibility of using autologous sources for the fibrin gel. Human corneal stem cells
suspended in the fibrin proliferated and exhibited markers of differentiation, expressing keratin 3,
as well as keratin 19 in some cases. Keratin 3 is a marker of corneal-type epithelial differentiation,
while keratin 19 is proposed as a marker for corneal stem cells. No functional tests were performed,
but the tissue was found to be soft, pliable, and elastic (Han et al. 2002).
Fibrin has also been attempted for cardiac tissue engineering applications such as construction of
bio-artificial arteries and tissue-engineered heart or venous valves based on the tendency of cells to
remodel it and replace it with new ECM. Ye et al. attempted to construct heart valves by combining
myofibroblasts with commercially available fibrinogen and thrombin to form a fibrin gel. However,
the mechanical properties of the myofibroblast-seeded fibrin gel were not examined (Ye et al. 2000).
Neidert et al. enhanced the mechanical properties of fibrin by using growth factors such as
TGF-β (transforming growth factor-β), plasmin, and insulin to increase the amount of ECM pro-
duced and observed that the fibrin constructs contained nearly 8 times the amount of collagen as
those without the additives after 51 days of incubation. In addition, the ultimate tensile strength and
modulus have also been improved by the addition of these growth factors. The results suggested
that the tensile strength of fibrin with growth factors was improved 10-fold than that of the samples
without growth factors (Neidert et al. 2002). Osathanon et al. have developed micro/nanoporous
scaffolds based on fibrin and calcium phosphate with well-controlled porous architecture for bone
regeneration. The scaffolds were fabricated by using poly (methyl methacrylate) (PMMA) sphere-
templating and leaching technique (Osathanon et al. 2008).
Apart from this, significant efforts have also been made to develop fibrin as a drug delivery
vehicle through both modification of the molecule and engagement of the native polymer system
biology. For instance, functionalized polyethylene glycol was explored as a means to covalently
bind the therapeutic proteins to fibrinogen backbone thus facilitating their delivery through fibrin
polymer (Barker et al. 2001). Recently, Soon et al. (2010) introduced tetra peptide knob sequences
of fibrin to other proteins to empower the binding capacities to them via knob-pocket interactions,
thus improving their properties. They adopted integrin-binding fibronectin 9th and 10th type III
repeats (FnIII9–10), which do not bind to fibrinogen as a model protein domain to demonstrate that
knob–protein binds stably to fibrinogen via specific knob-pocket interactions.
To summarize, fibrin has been attempted to fabricate variety of scaffolds for tissue regenera-
tion applications based on its inherent characteristics such as biocompatibility, biodegradability,
and capability of entrapping cells directly. Furthermore, it has adequate mechanical properties and
promotes cell growth and remodeling. Fibrin gels with large mesh size, low elastic modulus, and
adequate binding sites for integrins and other cellular targets have been demonstrated to be suit-
able for tissue engineering applications. While there are some challenges to be addressed, fibrin
has shown promise as a biopolymer scaffold in several tissue engineering applications including
ocular implants, skin, blood vessels, and heart valves. The in vivo efficacy of fibrin gels cannot
be assessed from its in vitro efficacy without considering the possible instability of gels at desired
sites and the host immunologic response. Hence, researchers have attempted to derive fibrin from
nonmammalian source such as farmed salmon to minimize the host immunological responses
(Tavares-Dias and Oliveira 2009). More work is necessary to further develop these applications
and examine new ones.

Regenerative medicine is an alternative tool for reconstructive surgery and organ transplantation
which relies on the induction of tissue regeneration through suitable scaffolding materials. At the
current stage of research, tissue regeneration is still in its early development, and it will take a long
time to become well established, although some approaches are nearly ready for clinical applica-
tion. The basic mechanism of normal tissue regeneration involves the action of a variety of growth
factors and bioactive components on cells so as to form their mutual networks. Time of release,
site of release, and concentration of growth factors action are delicately regulated in the body.
Nonetheless, it will be impossible to replicate living systems only by the scientific knowledge and
currently available technologies. If a key growth factor is supplied to the target site at the right
time, for the appropriate period of time, and at the right concentration, the living body system will
be naturally directed toward the process of tissue regeneration. Once the right direction is taken,
it is highly possible that the intact biological system of the body will start to function, resulting
in automatic achievement of tissue regeneration. Hence, successful tissue regeneration demands a
collaborative research between material, pharmaceutical, biological, and clinical scientists. A great
deal of research work is needed in order to obtain an increased number of commercially avail-
able and clinically successful natural-based systems for tissue regeneration. At the current sce-
nario of research, several scaffolds based on natural origin polymers have been widely emerged as
suitable tools for tissue regeneration applications. They display advantageous features in terms of
intrinsic cellular interaction, degradability, biocompatibility, low cost and availability, and simi-
larity with the extracellular matrix. However, these materials do also exhibit some disadvantages
such as poor mechanical properties, difficulties in controlling the variability from batch to batch,
mechanical properties or limited processability, which are the major obstacles for their widespread
use. Therefore, it is necessary to increase the awareness about these natural polymers in order to
enable the development of new approaches such as utilization of recombinant DNA technologies for
producing more biocampatible polymers and better purification techniques to render them suitable
material properties. To conclude, natural-based polymers or nature-inspired materials are attractive
materials of choice for tissue regeneration applications. The purpose of this chapter can be accom-
plished if it provokes the reader’s enthusiasm toward research field of tissue regeneration based on
natural polymers and by using recent emerging suitable techniques to warranty complete functional
tissue regeneration.
ABBREVIATIONS
ACA 1-aminocaproic acid
ADH adipic dihydrazide
BMP-2 bone morphogenic protein-2
BSA bovine serum albumin
BSF bovine sponge form encephalopathy
CA cellulose acetate
CD cyclodextrin
CGA ceramic-gelatin assembly
CH-GP chitosan-β-sodium glycerophosphate
CMC carboxy methyl cellulose
CMCH carboxymethylated-chitin
CPP calcium polyphosphate
DD degree of deacetylation
Dex-GMA glycidyl methacrylated-dextran
DHT dehydrothermal treatment
DMF dimethyl formamide
DNA deoxyribonucleic acid
ECM extracellular matrix
EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
EGF epidermal growth factor
FGF-2 fibroblast growth factor
FITC fluorescein isothiocyanate
FN fibronectin
GAGs glycosaminoglycans
GFM growth-factor mixture
GlcNAc N-acetyl-d-glucosamine
GTA glutaraldehyde
HA hyaluronic acid
Hap hydroxyapatite
hBMSC human bone marrow-derived stem cells
HEC hydroxy ethyl cellulose
HELA human epithelial cells
HOB human osteoblastic cells
HPC hydroxyl propyl cellulose
HPMC hydroxyl propyl methyl cellulose
HUVEC human umbilical vein endothelial cells
MAD malic acid derivative
MC methyl cellulose
MMP matrix metallo proteinase
mPCL methylated poly caprolactone
nBGC bioactive glass ceramic nanoparticles
NHS N-hydroxysuccinimide
PC plant cellulose
pCMV plasmid cyto megalo virus vector
PDGF platelet derived growth factor
PDLC periodontal ligament cells
PEG poly(ethylene glycol)
PLA poly(lactic acid)
PLGA poly(lactic-co-glycolic acid)
RC regenerated cellulose
rfGD radio frequency glow discharge
RGD (Arg-Gly-Asp)
RPE retinal pigment epithelium
SBB starch-based biomaterials
SBF simulated body fluid
SCA starch with cellulose acetate
SEVA-C starch with ethylene vinyl alcohol
SL stereo lithography
SPCL starch with polycaprolactone
SPI soya protein isolate
SPLA starch with poly(lactic acid)
TCP tri-calcium phosphate
TEP triethyl phosphate
TG transglutaminase
TGF transforming growth factor
TMOS tetramethylorthosilane
UV ultra violet region
VEGF vascular endothelial growth factor
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11 Natural Polymers as
Components of Blends
for Biomedical Applications
Alina Sionkowska
CONTENTS
11.1 Introduction...........................................................................................................................309
11.2 Miscibility and Compatibility of Blends............................................................................... 311
11.3 New Materials Based on Blends of Collagen with Synthetic Polymers................................ 313
11.4 New Materials Based on Blends of Chitosan with Other Polymers...................................... 318
11.5 Materials Based on Blends of Elastin and Other Polymers................................................... 324
11.6 Silk and Keratin Blends with Other Polymers....................................................................... 327
11.7 New Materials and Composites Based on Natural Polymers, Synthetic Polymers,
and Their Blends Containing Inorganic Particles................................................................. 331
11.8 Conclusions............................................................................................................................ 332
Acknowledgment............................................................................................................................ 332
References....................................................................................................................................... 333
11.1 INTRODUCTION
The biomedical sector requests more and more complex structures in attempts to fulfill the
requirements of a multitude of different applications. For this reason, polymer scientists exploit
existing compounds and also design new compounds with properties that can be considered as new
materials for biomedical applications [1,2]. The applications of polymers are essential in surgery,
for prosthetic systems, and in pharmacology, for drug formulation and controlled drug delivery.
Polymeric biomaterials are usually used under complex, demanding conditions, and must fulfill
stringent requirements relating to their chemical, biological, and physicomechanical properties.
Therefore, there is an urgent need for new polymeric biomaterials that can satisfy the demand-
ing performance requirements and standards required for these applications [3]. The development
of innovative polymeric biomaterials can be an engine of innovation for new medical treatments
and therapies. The existing knowledge clearly shows that the careful optimization of the chemical
structure of polymers used in medical implants can result in better clinical outcomes for the implant
recipients. Biodegradable polymers play a significant role in the preparation of polymeric materials
for biomedical applications. They are preferred candidates for developing therapeutic devices such
as temporary prostheses, three-dimensional porous structures as scaffolds for tissue engineering,
and as controlled/sustained release drug delivery vehicles [4].
At the end of the twentieth century, a new trend in the development of polymeric materials was
observed [5]. Many scientific laboratories started to work with polymer blends as potential new mate-
rials for medicine (biomaterials), the food industry (biodegradable and photodegradable food pack-
aging materials), and for the electronic industry (conducting polymers). Especially, new materials for
biomedical applications based on the blends of two polymers were studied. Cell-based transplantation,
309
tissue engineering, and gene therapy are important therapeutic strategies for current and future regen-
erative medicine. One challenge is to present the target cells in a suitable matrix to allow the cells
to survive wound contraction, tissue repair, and remodeling in certain tissues. Recently, functional
biomaterial research has been directed toward the development of improved scaffolds and new drug
delivery systems for regenerative medicine. During the last three decades, the increasing interest in
new materials based on the blends of two or more natural polymers and synthetic polymers has been
observed. Blends of two (or more) natural polymers and/or blends of synthetic and natural polymers
can form a new class of materials with improved mechanical properties and biocompatibility com-
pared with those of single components. In scientific literature, new words had been designed for the
blends of natural polymers with synthetic ones. Materials based on such blends have been called
bioartificial or biosynthetic polymeric materials [5–9]. In many scientific articles, one can find that
this new vocabulary has been commonly used. Natural biomacromolecules, so-called biopolymers,
serve as intrinsic templates for cell attachment and growth. In general, natural nonspecific polymers
can offer a range of advantages when compared with synthetic polymer-based materials. Natural
polymers are usually biocompatible, whereas synthetic polymers can contain a residue of initiators
and other compounds/impurities that do not allow cell growth. Synthetic polymers possess good
mechanical properties and thermal stability, much better than several naturally occurring polymers.
There is also a limitation in the performance of several natural polymers in comparison to synthetic
polymers. New polymeric materials in many cases should be biocompatible, and at the same time
they should possess good thermal and mechanical properties. For this reason, blends of natural poly-
mers and synthetic polymers have become promising materials for biomedical applications. That is
why the new materials designed should contain both natural polymers and synthetic ones.*
New materials based on blends of natural and synthetic polymers are a rapidly growing research
area [5–44]. Intensive research is currently underway in areas in which biopolymeric–polymeric
materials can be used in the engineering of bone, tissue, and organs [5].
New materials based on the blends of natural polymers and man-made polymers can also be
applied as packaging materials for drugs and medical devices. They can also be applied in the
food packaging industry. The biodegradability of natural polymers is very useful in the packaging
industry. Blends of synthetic and natural polymers are required in this field as bacteria start to
degrade the natural polymer and therefore decrease the natural component making it easier
to degrade the synthetic component. The blends of natural polymers and synthetic polymers can
more readily undergo photodegradation than synthetic polymers. Natural polymers possess many
chemical groups (chromophores) absorbing UV light from the sun. Free radicals produced in the
natural polymers may initiate photochemical degradation in synthetic polymers [16–18]. Bacterial
degradation of the blend is more efficient after partial photodegradation of synthetic polymers. On
the other hand, UV light is used for sterilization of biomedical materials. Natural polymers are
usually sensitive to the action of UV radiation, and they might be photochemically more stable
in the blend with synthetic polymer [18]. Moreover, the thermal stability of the blend is usually
different from that of the single component of the blend. Thermal and photochemical stability are
very important during processing and sterilization by high temperature and UV irradiation [37].
Two components of the blend need to be combined into one versatile material. The compo-
nents can be combined in solid state and/or dissolved in the same solvent. The problem is that
many naturally occurring polymers are insoluble in common solvents. One has to find a soluble
derivative of natural polymers or one has to hydrolyze the macromolecule to shorter chains.
When a soluble biopolymer is achieved, then it is necessary to decide what kind of synthetic
polymer to combine with the natural one. After preparing the mixture of two components in the
same solvent, one has to study the interaction between the natural and synthetic polymers. One
has to remember that the third component, solvent, is also present in the blend. Miscibility of the
* Reprinted from Prog. Polym. Sci., 36, Sionkowska, A., Current research on the blends of natural and synthetic polymers
as new biomaterials: review, 1254–1276, 2011. Copyright 2011, with permission from Elsevier.
Natural Polymers as Components of Blends for Biomedical Applications 311
components is an important aspect, and it determines the properties of the blend. The miscibil-
ity in polymer blends is assigned to specific interactions between polymeric components, which
usually give rise to a negative free energy of mixing in spite of the high molecular weight of
polymers. Several interactions can appear between the components of a polymeric blend. The
most common interactions in the blends are hydrogen bonding (when polymers contain chemical
groups capable of forming hydrogen bonds), ionic and dipole, pi-electrons, and charge-transfer
complexes. Most polymers in the blend are immiscible with each other due to the absence of
specific interactions. Sometimes, two polymers are partially miscible, only in solution, but they
are immiscible in the solid state. Miscibility of two or more synthetic polymers has been studied
by many research groups by viscometry, Fourier Transform Infrared spectroscopy (FTIR), and
differential scanning calorimetry (DSC). The miscibility of two natural polymers as well as
natural polymers with synthetic polymers is poorly understood. In the assessment of miscibility
in solution, the main problem is to find a solvent suitable for both natural and synthetic polymers.
The next problem is the hierarchical native structure of many natural polymers. It may happen
that during mixing of biopolymers with synthetic polymers the natural one can change its native
structure.
The most common natural polymers that can be used for the preparation of biomaterials based
on the blends of synthetic polymers and natural ones are collagen, cellulose, chitosan, elastin,
starch, and silk. Natural polymers, such as collagen and elastin, are usually insoluble in water and
in organic solvents. Collagen extracted from young animals’ tissues is soluble in dilute acetic acid.
Chitosan is soluble in dilute acetic acid solution, but the concentration that can be reached is low
and strongly depends on the molecular weight of this biopolymer. The solubility of collagen and
chitosan in acetic acid may allow them to be blended with other water-soluble polymers. Silk, elas-
tin, and keratin are highly insoluble natural polymers, and hence processing of these biopolymers
is a problem. In many laboratories, elastin, keratin, and silk have been hydrolyzed to aid solubility.
The blends of collagen with other polymers have been studied widely by several research groups
[5–7,12–14,16–62]. Collagen possesses several interesting properties that make it a good material
for biomedical applications [63–83]. Chitosan also possesses several interesting properties to be
applied as a potential biomedical material [82,84–102]. Chitosan can be blended with synthetic
and/or other natural polymers [10,13–15,19,20,46,48,54,62,102–157]. Elastin as a natural polymer
has also been considered for biomaterial preparation [69,158–166]. A very promising biopolymer to
prepare biomaterials is silk [167–195].
11.2 MISCIBILITY AND COMPATIBILITY OF BLENDS

Natural polymers and synthetic polymers do not exist together as blends in nature, but the specific
properties of each may be used to produce man-made blends that confer unique structural and
mechanical properties. Two natural polymers sometimes exist together as a blend in nature, and
in fact such blends possess unique structural and mechanical properties. To mimic nature and to
produce new materials for biomedical applications, it is necessary to prepare polymeric blends in
the laboratory under controlled conditions. If we use a relatively low-cost, low-pollution biopolymer
with specific properties and an appropriate synthetic polymer, we could increase the potential for
the development of a new generation of prosthetic implants.
An important aspect of the properties of a blend is the miscibility of its components. Miscibility
in polymer blends is assigned to specific interactions between polymeric components, which usually
give rise to a negative free energy of mixing in spite of the high molecular weight of polymers. The
most common interactions in the blends are hydrogen bonding, ionic and dipole, π-electrons, and
charge-transfer complexes. Most polymer blends are immiscible with each other due to the absence
of specific interactions.
Several methods have been employed to assess the interactions between two components in the
blend and then miscibility of two polymers [5–8,12,13,39,103,107].
In several laboratories, viscosity measurements have been employed to study the miscibility of
two components. The main problem is that it is necessary to dissolve two polymers in the same
solvent. In our laboratory, viscosity measurements have been used to assess the miscibility between
components in the blend. For calculation, the following equations have been used [196]:
ηsp
= η + bc, (11.1)
c
where
[η] is the intrinsic viscosity
b is the polymer–polymer interactions term at finite concentration related to the Huggins coef-
ficient kH by
2
b = kH η . (11.2)
Equation 11.1 can be adapted to a ternary system containing a solvent (component 1) and two poly-
mers (components 2 and 3). For the ternary system, the total polymer–polymer interactions (bmc2)
are given by c2(c2 b22 + c3b23) for component 2 and c3(c2 b23 + c3b33) for component 3.
bm c 2 = b22 c22 + b33c32 + 2b23c2 c3 , (11.3)
where
c2 and c3 are concentrations of components 2 and 3, respectively, in a mixed polymer solution
c is the concentration of mixed polymer solution
b22 and b33 are specific interaction coefficients of components 2 and 3 in a single polymer solution
b23 represents the interaction between different types of polymer molecules in a mixed polymer
solution
bm =
(b c + b33c32 + 2b23c2 c3
2
22 2 ). (11.4)
c2
The intrinsic viscosity of mixed polymer solution

 ηspm  c  c  (11.5)
η m =   = η 2  2  + η 3  3  .
 c c→0  c c→0  c c→0
The intrinsic viscosity of a mixture of two polymers is the weight average of the intrinsic viscosity
of the two polymers taken separately. For a ternary system the equation is
ηspm c
=  η 2  2
  c3
(
  b22 c2 + b33c3 + 2b23c2 c3
2 2
)  c (11.6)
 +  η 3  c  +
c  c     c2 
 
ηspm
c
(
= η 2 w2 + η 3 w3 + b22 w22 + b33 w32 + 2b23 w2 w3 c, ) (11.7)
where w designates the normalized weight fraction of polymer (w2 = c2/c, w3 = c3/c).
The equation for ideal mixed polymer solution
ηspm = η 2 c2 + η 3 c3 + b22 c22 + b33c32 + 2 b22 b33 c2 c3 . (11.8)

Polymer mixtures might exhibit positive or negative deviations from the defined ideal behavior
because of the existence or absence of interactions. The polymer–polymer miscibility can be deter-
mined by a parameter Δb.
∆b = b23 − b22 b33 , (11.9)
where b23 is obtained experimentally by substituting all the terms in Equation 11.4.
∆b > 0 − miscibility. (11.10)
In our research group, we used the viscosimetry method to investigate polymer–polymer interac-
tions and for assessment of the miscibility between collagen and synthetic polymers as well as
between chitosan and synthetic polymers.
11.3 NEW MATERIALS BASED ON BLENDS OF COLLAGEN

WITH SYNTHETIC POLYMERS
Collagen is the main protein in an animal’s body. This structural protein forms molecular lines that
strengthen the tendons and vast, resilient sheets that support the skin and internal organs. Hard tissues
such as bones and teeth are made of collagen with the addition of mineral crystals, mainly hydroxy-
apatites. There are 20 genetically distinct collagens in the collagen family. Collagen type I, type II, and
type III are fibril-forming ones: type I (skin, tendon, and bone), type II (cartilage), and type III (skin
and vasculature) [63–69]. In each collagen chain, there are 1000 amino acids, which form the sturdy
structure by a repeated sequence of three amino acids. Every third amino acid is glycine, a small
amino acid that fits perfectly inside the helix. In other positions in the chain, the following are located:
proline and a modified version of proline, hydroxyproline. It is believed that hydroxyproline is respon-
sible for collagen stability because of additional hydrogen bonds formed by the OH group. Hydrogen
bonds play the main role in the stabilization of the collagen triple helix [64]. Each collagen molecule
undergoes a strong molecular connection with neighboring collagen molecules by hydrogen bonding
and other crosslinks [63–65,67–69]. Highly crosslinked collagen is usually insoluble in water [70].
Additional crosslinking of collagen can be made by exposure of collagen to UV irradiation [71].
Like most proteins, collagen loses all of its structure during heating. In the collagen molecule,
atoms in the individual chains are held together with strong covalent bonds. However, the three chains
are held in the triple-helical structure by weaker bonds (mainly hydrogen bonds). During heating,
these bonds are broken and the three chains separate from one another and collapse into random coils
[67,72,73]. In solution, the unfolding temperatures of collagen are within only a few degrees of the
animal’s body temperature, but when the molecules are aggregated to form fibers there is an increase
in the transition temperature [74,75]. The temperature of thermal denaturation of collagen depends on
water content, pH of environmental medium, and degree of crosslinking [76–80].
The next material that is widely used in the biomedical field is gelatin. Gelatin is usually obtained
from collagen by exposing animal skins and bones to a controlled extraction process. Commercially,
gelatin is most typically obtained from cattle hides and bones and pigskins. Based on their struc-
tural roles and compatibility within the body, collagen and gelatin are commonly used biomaterials
in the medical, pharmaceutical, and cosmetic industries. Gelatin can also be obtained from the
processing of tannery waste, but this requires further processing to serve as a commercially useful
material. Gelatin is widely used in food preparation and also in many households in the form of
cosmetics and toiletries, which utilize gelatin for its hypoallergenic and hydrating properties.
Collagen-based materials and gelatin-based materials are widely used in biomedical field and the cos-
metics industry [5,10,19,21,27,41,43,49,51,54,56, 62,63,81,82]. The properties of collagen-based materials
depend on the source of collagen and the method of preparation involving purification, fibril formation or
casting, and subsequent crosslinking. Collagen has been identified as a mildly antigenic fibrous protein,
and for this reason it has been considered suitable for employment as a biomaterial. Moreover, as a
biological macromolecule, collagen has been found to have low immunogenicity, absorbability, biodeg-
radation rate, and cell interaction. For these reasons, collagen has been identified as a candidate material
to combine with water-soluble synthetic polymers to produce hybrid systems with various properties,
compositions, and forms. The blending of collagen with synthetic polymers can be modulated by many
factors. From the blends of collagen with other hydrophilic polymers, it is possible to get scaffolds for tis-
sue engineering and good quality hydrogels. Hydrogels are three-dimensional polymeric networks held
together by crosslinked covalent bonds and weak cohesive forces in the form of either hydrogen bonds
or ionic bonds. Hydrogels are a broad class of hydrophilic polymeric materials that have the inherent
ability to swell in water and other suitable solvents, capable of imbibing and retaining more than 10% of
their weight in water within the gel structure. Attributes such as permeability to small molecules (such
as tissue metabolites), a soft consistency, and a low interfacial tension between the gel and an aqueous
solution are some of the important properties that have helped to generate interest in hydrogels as use-
ful biomaterials. New materials based on the blends of collagen and synthetic polymer can be used as
biodegradable polymeric scaffolds, which are widely used in medical applications. A basic advantage is
the avoidance of surgery to remove the polymer from the body, when the implanted device is no longer
needed. Highly porous structures of scaffolds maintain large areas for the cells to attach and proliferate.
The blends of collagen and synthetic polymer can be used for preparation of drug delivery devices.
Biodegradable polymers provide flexibility in the design of delivery systems of large molecular weight
drugs such as peptides and proteins, which are not suitable for diffusion controlled release through non-
biodegradable polymeric matrices. These systems release the bioactive substances as they degrade to
the desired area with an effective dose, for a predetermined rate and duration. Drugs formulated in
polymeric devices are released either by diffusion through the polymer barrier or by erosion of the
polymer material, or by a combination of both. The polymers selected as delivery devices must meet
several requirements like biocompatibility, drug compatibility, the possibility of their processing, and
suitable biodegradation kinetics and mechanical properties. A wide variety of natural and synthetic
biodegradable polymers have been investigated for drug targeting or prolonged drug release. However,
only a few of them are biocompatible. Natural biodegradable polymers like bovine serum albumin
(BSA), human serum albumin (HSA), collagen, gelatin, and hemoglobin have been studied for drug
delivery [9,61,62]. Biodegradable polymeric systems can be designed in various shapes such as mem-
branes, pellets, sponges, micro- or nanospheres, depending on the purpose of application. However, the
use of natural polymers in the biomedical field is limited due to their high cost and questionable purity.
To summarize, the applications of collagen in tissue regeneration/engineering may include arti-
ficial skin, bone graft substitutes, dental implants, implants for incontinence, artificial tendons and
blood vessels, corneal implants, and nerve regeneration [81,82].
Nevertheless, it should be emphasized that the use of animal-derived materials in the preparation
of tissue-engineered products is generally viewed as a problem in the area of biomaterials. Although
the extensive processing used provides a wide margin of safety, the preference for products that do
not contain animal-derived materials is growing. For these reasons, we can expect that polymers
and biomaterials derived from recombinant sources will become increasingly important in future
products, potentially decreasing the importance of bovine collagen. Current tissue engineering
approaches have employed the use of hybrid polymer systems composed of natural and synthetic
macromolecules for various applications in biomaterials science. These hybrid polymeric materials
are often used as hydrogels and other implantable specimens. These materials are often biocompat-
ible and possess good physicochemical and mechanical properties.
New materials based on the blends of collagen and other polymers could result in a new genera-
tion of biodegradable polymeric materials that could be utilized as packaging material. However,
packaging materials should be as cheap as possible. For this reason, mainly collagen wastes are con-
sidered and studied. To get soluble material, collagen wastes are usually hydrolyzed to short peptide.
After enzymatic hydrolysis of collagen waste, it is possible to use collagen hydrolysate for different
applications (fertilizer, adhesives, concrete mixtures, etc.) and also as additive to plastics increasing
their sustainability. Synthetic polymers modified with collagen hydrolysate can form a sustainable
plastic material workable to films for use in agriculture, forestry, packaging, etc. Collagen hydroly-
sate can be obtained using leather industry wastes, which are abundant [47].
The properties of new polymeric blends that have often been evaluated are the mechanical prop-
erties, like ultimate tensile strength (UTS), and percentage of elongation at break (EL). The barrier
properties, like water vapor and oxygen permeability, are also important. Although most of the devel-
oped films show desirable oxygen permeability, their moisture barrier and mechanical properties are
poor in comparison to the synthetic polymeric films. Inclusion of lipids in the film in emulsified form
or following the technique of formation of a composite bilayer has been found to improve the mois-
ture barrier property of the films. The next important factor for biopolymeric blends is their surface.
Surface modifications that directly affect the biological function can be performed by producing
specific surface functional groups, adsorption-adhesion of specific biomolecules and atoms, and by
deposition of microparticles such as functional inorganic species, catalysts, and microcapsules to
deliver specific reagents depending on environmental conditions. The surface can be also modified
by UV irradiation [83]. The medical device and its packaging can be degraded irreversibly by the
sterilization process. Most often, the earliest evidence of material degradation will appear in the sur-
face layers of the product or its package. Surface plays a very important role in biology and medicine
because most biological reactions occur at surfaces and interfaces. It is also important for most tissue
engineering approaches aiming at the repair or regeneration of living tissues and the interactions of
cells and polymeric biomaterial. UV irradiation is a good method of sterilization of polymeric bio-
medical materials, but it may also degrade them. The energy of UV light has been extensively applied
for surface graft polymerization of polymers. The surface of a synthetic polymer can be grafted by
natural polymers, and this procedure improves the biocompatibility of polymeric materials.
In our laboratory, water-soluble synthetic polymers have been blended with natural poly-
mers like collagen, chitosan, and gelatin. We have used the following synthetic polymers:
poly(vinylpyrrolidone) PVP, poly(vinylalcohol) PVA, poly(ethylene glycol) PEG, and poly(ethylene
oxide) PEO. From the blends, we attempted to form films, hydrogels, and sponges. Collagen films
can be obtained by solvent evaporation from collagen solution and by treating collagen gel solu-
tions with different concentrations of glutaraldehyde. In our laboratory, the polymeric films were
obtained by solvent evaporation from collagen solution in acetic acid [5,12,14,16–18,39].
Miscibility between two components has been studied by viscometry methods, Fourier trans-
form infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) methods [39].
According to the classical Huggins equation (11.1), from the intercept and slope of the plots of ηsp/c
versus c, [η] and bm (for solutions of individual polymers) are obtained. The value of b23 is obtained
by substituting all the terms in Equation 11.4. Viscometric data for binary blends containing collagen
and PVP show that collagen and PVP are miscible. Similar results have been obtained for collagen/
PVA blends. The parameter Δb > 0 signifies the miscibility and is shown in Table 11.1. The intrinsic
TABLE 11.1
Δb Parameter for Binary Blends Containing Collagen and Synthetic Polymer
X2 (Collagen) X3 (Synthetic) Δb Coll/PVP Δb Coll/PVA Δb Coll/PEO Δb Coll/PEG
0.00 1.00
0.20 0.80 0.0867 1.9811 −0.2189 −0.2901
0.50 0.50 0.3942 0.0839 0.0765 −0.3811
0.80 0.20 1.2901 1.2691 1.4131 3.7500
1.00 0.00
Source: Reprinted from J. Mol. Liq., 145, Sionkowska, A., Skopińska, J., and Wiśniewski, M., Collagen synthetic poly-
mer interactions in solution and thin films, 135–138, 2009. Copyright 2009, with permission from Elsevier.
TABLE 11.2
Amide Band Position of Collagen before and after
Blending with Synthetic Polymers
Amide Band Position [cm−1]
Sample A B I II
Collagen 3326 3094 1654 1557
Collagen/PVP blend 3330 3088 1658 1557
Collagen/PEO blend 3328 3082 1656 1557
Collagen/PVA blend 3325 — 1654 1559
Collagen/PEG blend 3228 3090 1656 1557
Source: Reprinted from J. Mol. Liq., 145, Sionkowska, A., Skopińska, J.,
and Wiśniewski, M., Collagen synthetic polymer interactions
in solution and thin films, 135–138, 2009. Copyright 2009,
with permission from Elsevier.
viscosity measurements for the blends are different from the values from theoretical calculations
(Table 11.1). The miscibility of these polymers in solid state has been confirmed by FTIR spectra of
collagen and collagen/synthetic polymer blend. FTIR is a powerful technique to detect the intermo-
lecular interactions between two polymers. The intermolecular interaction through hydrogen bonding
can be characterized by FTIR, because the specific interaction affects the local electron density and
the corresponding frequency shift can be observed. The position of amide band A in a blend with
PVP is shifted to higher frequencies, while amide B to lower frequencies (Table 11.2). It suggests
interaction between collagen and PVP by hydrogen bonds. Collagen, which is a hydrogen donor,
forms a hydrogen bond with the carbonyl group from PVP. The pyrrolidone rings in PVP contain a
proton-accepting carbonyl moiety, while collagen presents hydroxyl and amino groups as side groups.
Therefore, a hydrogen-bonding interaction may take place between these two chemical moieties in a
blend of collagen and PVP. The formation of hydrogen bonds between two different macromolecules
competes with the formation of hydrogen bonds between molecules of the same polymer.
Viscometric data for binary blends containing collagen/PEO and collagen/PEG show that collagen
and those polymers are immiscible, parameter Δb < 0 (Table 11.1) [39]. However, from the mixture it
was possible to prepare films that can have a potential for future applications. FTIR spectra revealed
some minor interactions between collagen and PEO and/or PEG in a solid state (thin film). The position
of amide bands for collagen in blends was slightly different from that for pure collagen (Table 11.2).
FTIR spectra of collagen and collagen/PVA blends show that the positions of amide bands are
at the same wavenumbers for all the blends and only slightly different than for pure collagen film
(the example of FTIR spectra is shown in Figure 11.1, data are listed in Table 11.2). It suggests
only a minor interaction between collagen and PVA in the solid state. It seems that hydrogen bond
formation between collagen molecules competes with the formation of hydrogen bonds between
molecules of PVA and collagen in the solid state.
In Figure 11.2, the biocompatibility of new polymeric materials based on the blends of col-
lagen and synthetic polymers is compared with that of pure collagen film. The biocompatibility
test of collagen–polymer blend sheets was carried out as follows: polymeric films were soaked in
ethyl alcohol for 15 min to sterilize, then an appropriate medium was added and incubated until
cells proliferate. When cells had spread adequately, the Resazurin assay test was performed [5,39].
After the Resazurin test, the fluorescence of each sample was measured with a spectrofluorometer.
Figure 11.2 shows that fibroblasts grow with the fastest rate on collagen films. However, the biocom-
patibility of new polymeric materials based on the blends of collagen, PEG, and PVA seems to be
sufficient for biomedical applications.
0.7
0.6
0.5
Absorbance 0.4
0.3
0.2
0.1
0
3500 3000 2500 2000 1500 1000
Wavenumber (cm–1)
FIGURE 11.1 FTIR spectra of collagen before blending with synthetic polymers. (Reprinted from J. Mol.
Liq., 145, Sionkowska, A., Skopińska, J., and Wiśniewski, M., Collagen synthetic polymer interactions in solu-
tion and thin films, 135–138, 2009. Copyright 2009, with permission from Elsevier.)
1000
900
800
700
Fluorescence
600
500
400
300
200
100
0
Control Collagen Collagen- Collagen- Collagen- Collagen-
surface PEG PEO PVP PVA
(petri dish)
Film type
FIGURE 11.2 Fluorescence of Resazurine after 3.25 h reaction–fibroblast growth time of 5 days (gain = 60).
(Reprinted from J. Mol. Liq., 145, Sionkowska, A., Skopińska, J., and Wiśniewski, M., Collagen synthetic poly-
mer interactions in solution and thin films, 135–138, 2009. Copyright 2009, with permission from Elsevier.)
The FTIR and viscosimetry results show that collagen macromolecules could form different
types of hydrogen bonds with the macromolecule of synthetic polymer. The following hydrogen
bonds can be formed:
• Between two hydroxyl groups (OH……OH)

• Between the hydrogen of the amide group and oxygen of the hydroxyl group (NH….OH)
• Between the hydrogen of heteroatoms and the carbonyl group of the amide (C=O….HO,
C=O….HN)
These specific interactions between two polymers can modify their physicochemical properties.
New polymeric materials based on the blends of collagen and synthetic polymer possess lower bio-
compatibility than pure collagen, but the biocompatibility can be further modified by UV irradiation.
It is commonly known that living cells like to attach to polar chemical groups at the material sur-
face [163]. During UV irradiation, photooxidation process takes place and new polar groups can
be formed. The interaction of UV light with the blends is important, as the behavior of the blends
needs to be understood under potentially harsh conditions. UV light may be used to refine the blend
preparation process to produce blends of specific structural and chemical characteristics. After dif-
ferent doses of UV irradiation, the mechanical and surface properties of the film were investigated.
TABLE 11.3
Thermal Parameters of Collagen, Collagen/PVP, and Collagen/PVA Blends before
and after UV Irradiation
Time of Peak 1 ΔH1 Peak 2 ΔH2 Glass Transition
Specimen Irradiation [h] T1 [°C] [J/g] T2 [°C] [J/g] Tg [°C]
Collagen 0 80.3 222.3 212.8 3.6 —
Collagen 8 91.9 249.4 212.4 4.4 —
PVP 0 82.5 306.3 — — 178.3

PVP 8 76.1 450.3 — — 177.7
Collagen/PVP 0 87.1 119.5 206.8 2.4 —

Collagen/PVP 8 87.9 156.6 207.4 2.0 —
PVA 0 92.5 366.3 227.3 132.5 —

PVA 8 86.1 350.3 225.8 136.5 —
Collagen/PVA 0 85.1 129.5 216.8 122.4 —

Collagen/PVA 8 82.9 146.6 217.4 143.0 —
Source: Reprinted from Sionkowska, A., in: R.K. Bregg, ed., Current Topics in Polymer Research, Nova Science
Publisher, New York, 2005. With permission.
During UV irradiation of polymers, excited molecules are formed in the first step and then, the sec-
ondary processes such as chain scission, crosslinking, and oxidation take place. For UV-irradiated
blends, thermal properties were investigated. In the presence of thermally stable synthetic poly-
mers, collagen is usually more stable and undergoes thermal degradation in higher temperature than
collagen alone (Table 11.3).
Interesting materials for potential biomedical applications can also be obtained using the blends
of collagen with chitosan. Such blends are considered in the next chapter.
11.4 NEW MATERIALS BASED ON BLENDS OF CHITOSAN

WITH OTHER POLYMERS
Chitosan is a natural polymer derived from chitin. Chitin is a polysaccharide, a homopolymer com-
posed of 2-acetamido-2-deoxy-β-D-glucopyranose units. Chitosan can be obtained from chitin by the
deacetylation process. Some units in chitosan chains exist in the deacetylated form as 2-amino-2-deoxy-
β-D-glucopyranose. Chitosan is a biodegradable natural polymer with great potential for pharmaceuti-
cal applications and cosmetic industry due to its biocompatibility, high charge density, nontoxicity, and
mucoadhesion. It has been shown that it not only improves the dissolution of poorly soluble drugs but
also exerts a significant effect on fat metabolism in the body [82,84–88]. Chitosan is widely applied
in the biomedical field and also in waste water treatment [86]. It has been reported that chitosan is a
potentially useful pharmaceutical material owing to its biocompatibility and low toxicity [85]. For this
reason, it can be used in many applications in the formulations employed in drug delivery. Furthermore,
the macromolecular chain of chitosan is stiff enough to stabilize a liquid crystalline phase in acetic acid
solution where the persistence length is reported to be between 190 and 300 Å depending on the degree
of acetylation [87]. Chitosan and its derivatives can be used to prepare nonviral gene delivery [88].
Chitosan itself is a very good material for biomedical applications, but after modification of
properties of chitosan one can obtain versatile biomaterial for cell therapy, tissue engineering, and
gene therapy [89–91].
Many research groups have studied chitosan-based materials and their applications in the field
of orthopedic tissue engineering. Interesting characteristics that render chitosan suitable for this
purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be
molded in various geometries and forms such as porous structures, suitable for cell ingrowth and
osteoconduction. The cationic nature of chitosan allows it to complex DNA molecules making it
an ideal candidate for gene delivery strategies [92–96]. Covalent and ionic crosslinking of chitosan
leads to the formation of hydrogels, which can work as a drug delivery system under pH-controlled
conditions [97]. Chitosan derivatives can also be applied in various tissue engineering applications,
namely, skin, bone, cartilage, liver, nerve, and blood vessel [98–102].
In the scientific literature, one can find information about blending of chitosan with other natural
polymers and synthetic polymers. There are several methods of preparing polymer blends of cellu-
lose, chitin, and chitosan with natural and synthetic polymers [103]. It is possible to use the method
of solid-phase blending of these polymers under conditions of high pressure and shear deformation.
Several synthetic polymers can be blended with chitosan. Water-soluble synthetic polymers,
such as polyvinylalcohol (PVA), polyethyleneoxide (PEO), and polyvinylpyrrolidone (PVP), have
been blended with chitosan to design new materials. Several research groups showed miscibility of
chitosan and PVP and the possibility of using the blend material. Other studies showed that chito-
san and PVA form an immiscible system in which interaction between macromolecules of PVA is
stronger than that between PVA and chitosan [104,105]. Chitosan blends with hydrophilic polymers
including polyvinylalcohol (PVA), polyethyleneoxide (PEO), and polyvinylpyrrolidone (PVP) were
investigated as candidates for oral gingival delivery systems [15,104–106]. Moreover, chitosan/PVP
blends can be used as a drug release system and to control the release profile of a poorly water-
soluble drug. The mechanical and surface properties of chitosan, polyvinylpyrrolidone (PVP), and
chitosan/PVP blends have been studied, and the influence of UV irradiation on these properties has
been compared [107]. The results showed that the mechanical properties of the blends were greatly
affected by UV irradiation with 254 nm wavelength, but the level of the changes of these properties
was smaller in the blend than in pure chitosan and strongly dependent on the dose of irradiation
and the composition of the blends (Figures 11.3 and 11.4). The surface alterations of chitosan-based
blends were studied by contact angle measurements. The contact angle and the surface free energy
of PVP/chitosan blends were altered by UV irradiation. The polarity of the surface of the blend was
altered by UV irradiation due to the photooxidation process (Table 11.4) [107].
120
100
Ultimate tensile strength (MPa)
1
80 2
3
60
4
40 5
20
0
0 2 4 6 8 10
Irradiation time (h)
FIGURE 11.3 The influence of UV irradiation on ultimate tensile strength of chitosan (1) and chitosan/
PVP films (2—80:20, 3—60:40, 4—40:60, 5—20:80). Each value is reported with its standard deviation.
(Reprinted from Polym. Deg. Stab., 88, Sionkowska, A., Wisniewski, M., Skopinska, J., Vicini, S., and
Marsano, E., The influence of UV irradiation on the mechanical properties of chitosan/poly(vinyl pyrrolidone)
blends, 261–267, 2005. Copyright 2005, with permission from Elsevier.)
12
Ultimate percentage elongation (%)

10 1 1
2
8 3
4
6 5
4
4 5
2 3
2
0
0 2 4 6 8 10
Irradiation time (h)
FIGURE 11.4 The influence of UV irradiation on ultimate percentage elongation of chitosan (1) and
chitosan/PVP films (2—80:20, 3—60:40, 4—40:60, 5—20:80). Each value is reported with its standard
deviation. (Reprinted from Polym. Deg. Stab., 88, Sionkowska, A., Wisniewski, M., Skopinska, J., Vicini, S.,
and Marsano, E., The influence of UV irradiation on the mechanical properties of chitosan/poly(vinyl pyr-
rolidone) blends, 261–267, 2005. Copyright 2005, with permission from Elsevier.)
TABLE 11.4
Surface Energy Calculated by Owens-Wendt Method of the
Chitosan, PVP, and Chitosan/PVP Blend after UV Irradiation
Irradiation Surface Energy
Sample Time [h] (Total) [mN/m] Disperse Part Polar Part
Chitosan 0 27.2 21.8 5.4
4 39.3 22.8 16.5
8 50.0 20.0 30.0
PVP 0 38.7 31.7 7.0
4 42.4 39.8 12.6
8 49.4 26.5 22.9
Chitosan/PVP 0 27.7 22.4 5.3
80:20 4 38.7 21.4 17.3
8 47.4 21.7 25.7
Chitosan/PVP 0 28.4 25.3 3.1
60:40 4 34.7 26.8 7.9
8 43.1 23.4 19.7
Chitosan/PVP 0 28.6 26.1 2.5
50:50 4 34.3 26.6 7.7
8 39.9 24.5 15.4
Chitosan/PVP 0 29.0 25.1 3.9
40:60 4 34.2 26.6 7.6
8 43.0 22.3 20.7
Chitosan/PVP 0 31.8 24.2 7.6
20:80 4 35.3 30.6 4.6
8 49.5 18.8 30.7
Source: Reprinted from Polym. Deg. Stab., 88, Sionkowska, A., Wisniewski, M.,
Skopinska, J., Vicini, S., and Marsano, E., The influence of UV irradiation
on the mechanical properties of chitosan/poly(vinyl pyrrolidone) blends,
261–267, 2005. Copyright 2005, with permission from Elsevier.
The interaction between chitosan and synthetic polymers is mainly due to hydrogen b onding.
Another kind of highly crosslinked material can be obtained by chemical crosslinking using
crosslinking agents. The most common crosslinking agent is glutaraldehyde. By crosslinking of
chitosan/polyvinyl pyrrolidone blend by glutaraldehyde, a semi-interpenetrating polymer network
can be obtained as potential controlled drug release system for antibiotic delivery [108]. Chitosan/
PVP blends can be turned to hydrogels with porous structures using the freeze-drying method and/
or electron beam irradiation at room temperature [109,110]. The blends of poly(vinyl alcohol) and
chitosan can also be used for hydrogel preparation [111–113]. From the blends of PVA and chitosan
in solution, nanofibers can be obtained using the electrospinning method. Nanofibrous microporous
PVA/chitosan scaffolds can be fabricated using a cryogenic grinding method with subsequent com-
paction. Such multiscale porous structures may offer ideal matrices for tissue engineering applica-
tions [114]. The blend of poly(ethylene oxide) PEO and chitosan was used to develop novel drug
delivery systems with pH-sensitive swelling and drug release properties for localized antibiotic
delivery in the stomach [105,115,116]. The blend could be useful for localized delivery of antibiotics
in the acidic environment of the gastric fluid.
Chitosan can be blended with nylon [117,118], polyacrylamide [119], and poly(lactic acid) [120].
Poly(lactic acid) is a biodegradable and resorbable polymer widely used in biomedical applications.
Biodegradable film blends can be prepared (mainly by mixing the components in solution and
film casting) [120]. Interesting membranes can also be obtained from the blend of chitosan and
polyhedral oligomeric silsesquioxane derivatives [121], as well as poly(caprolactone) [122,123].
Poly(caprolactone) PCL, a synthetic polymer, can be used together with chitosan for preparation of
several materials for medical applications. The blends of chitosan with PCL have been studied not only
as a membrane but also as particles [122,123]. Such particles can form stable complexes with DNA.
New materials based on chitosan can be made by mixing of three components. By mixing of chito-
san and poly(caprolactone) with a third component (a whey-protein isolate), an increase in the water
vapor resistivity of chitosan was observed [124].
New materials based on the blends of chitosan with synthetic polymers show interest-
ing properties not only for biomedical applications [125–129]. Chitosan can also be blended
with polymers that possess electrical properties, like conductivity [130–132]. The conduc-
tive materials based on the blends of chitosan with other polymers can be used in biomedical
devices. Thermal properties of chitosan can be modified by blending it with thermally stable
polymers [133].
Chitosan can be blended with several other naturally occurring polymers. The interaction of chi-
tosan and collagen leads to the opportunity for preparation of new materials based on their blends
[5,13,14,20,44,62,134–137]. Several research groups have shown that collagen and chitosan are mis-
cible in both solution and solid state [13,134,135]. The blending of collagen with chitosan gives the
possibility of producing new bespoke materials for potential biomedical applications [44,135]. The
addition of chitosan does not denature collagen fibers [20]. Thermal properties of collagen/chitosan
blends depend on the composition of the blend and are not significantly altered by UV irradiation
(Table 11.5) [136]. However, the mechanical properties of the blends were greatly affected by the
duration of UV irradiation (Figure 11.5) [136].
From collagen/chitosan blends, nanofibers have been prepared by electrospinning [137].
Intermolecular interactions varied with the chitosan content in electrospun fibers. New fibers were also
produced by blending chitin with tropocollagen. The blended fiber was chemically modified at the fiber
state by treatment with a series of carboxylic anhydrides and aldehydes to afford the corresponding
N-modified fiber. Such fibers have improved blood compatibility [62]. Collagen and gelatin have been
used as components of a binary blend with chitosan for biomedical applications [138–141]. Gelatin itself
is a mixture of water-soluble proteins derived primarily from collagen. Usually, gelatin is obtained
from collagen by exposing animal skins and bones to a controlled extraction process. Gelatin comes in
various types. Based on their structural roles and compatibility within the body, collagen and gelatin
TABLE 11.5
Values of Thermal Parameters of Collagen and Its Blends
with Chitosan Determined by Thermal Analysis in Nitrogen
Stage I: Weight Loss%
Collagen/Chitosan
Time of Irradiation [h] Collagen 70/30 50/50 30/70 Chitosan
0 13 14 11.3 12.4 10.9
8 11.5 14.5 11.9 15.4 14.2
Stage II: Weight Loss%
Collagen/Chitosan
0 67.9 67.5 54.8 59.6 57.7
8 55.1 57.6 50.1 63.9 62.9
Stage II: Tmax
Collagen/Chitosan
0 313 308 289 289 289
8 312 311 290 290 287
Source: Reprinted from Polym. Deg. Stab., 91, Sionkowska, A., Wisniewski, M.,
Skopinska, J., Poggi, G.F., Marsano, E., Maxwell, C.A., and Wess, T.J.,
Thermal and mechanical properties of UV irradiated collagen/chitosan thin
films, 3026–3032, 2006. Copyright 2006, with permission from Elsevier.
100
90
80 1
70 2 3
60
50
40 4
30
20
10
0
0 2 4 6 8 10
Time of irradiation (h)
FIGURE 11.5 (See companion CD for color figure.) The influence of UV irradiation on ultimate tensile
strength of collagen and collagen/chitosan films: (1) collagen; (2) 90/10 collagen/chitosan; (3) 70/30 collagen/
chitosan; (4) 60/40 collagen/chitosan. Each value is the means of 20 determinations and is reported with its
standard deviation. (Reprinted from Polym. Deg. Stab., 91, Sionkowska, A., Wisniewski, M., Skopinska, J.,
Poggi, G.F., Marsano, E., Maxwell, C.A., and Wess, T.J., Thermal and mechanical properties of UV irradiated
collagen/chitosan thin films, 3026–3032, 2006. Copyright 2006, with permission from Elsevier.)
are commonly used biomaterials in the medical, pharmaceutical, and cosmetic industries. It was found
that several enzymes can catalyze the gel formation from gelatin and chitosan blends [138]. A novel
chitosan-based membrane was made of hydroxypropyl chitosan, gelatin, and chondroitin sulfate [139].
It can potentially be used as a carrier for corneal endothelial cell transplantation. Glucose permeation
results demonstrated that the blend membrane had higher glucose permeability than natural human cor-
nea. The optical transparency of the membrane was as good as the natural human cornea [139]. Blends
between chitosan and gelatin with various compositions were produced using genipin as crosslinking
agent. Different amounts of genipin were used to crosslink the components of the blend, promoting the
formation of amide and tertiary amine bonds between the macromolecules and the crosslinker. The
mouse fibroblasts adhesion and proliferation on substrates depend on the blend composition and on the
amount of crosslinker. Such materials are promising candidates for use in the field of nerve regeneration
[140]. Blends based on chitosan and gelatin have gained much attention as scaffolds in various tissue
engineering applications [141]. The results obtained by several research groups show significant influ-
ence of blending gelatin with chitosan on scaffold properties and cellular behavior.
Chitosan was blended with other proteins such as soy protein and silk fibroin [142–147]. From such
blends, mainly biomaterials for applications in wound healing and skin tissue engineering scaffolding
are considered. The formation of scaffolds from chitosan/fibroin blends from aqueous solution is possi-
ble but it depends on the pH value [144,145]. By electrospinning of the silk fibroin/chitosan blends with
a chitosan content of up to 30%, the continuous fibrous structure of material can be obtained [147].
In scientific literature, one can also find reports that chitosan can be blended with starch [148,149],
cellulose and its derivatives [150,151], alginates [102,152–154], pullulan [155], and dextran [156].
Cellulose can be used together with chitosan for biomedical applications. However, cellulose is a
highly insoluble biopolymer, so it is not easy to get a chitosan/cellulose blend. The main problem is
which solvent to choose to prepare a blend. Polysaccharide-based membranes of chitosan and cellulose
blends were prepared using trifluoroacetic acid as a co-solvent [150]. The mechanical and dynamic
mechanical thermal properties of the cellulose/chitosan blends appear to be dominated by cellulose and
suggest that cellulose/chitosan blends are immiscible. It is believed that the intermolecular hydrogen
bonding of cellulose breaks down to form cellulose–chitosan hydrogen bonding [150]. The chitosan/
cellulose blend membranes may be suitable as a wound dressing with antibacterial properties. Chitosan
can be blended with cellulose derivatives like hydroxypropyl cellulose, and the membranes can be
prepared by the solution casting method followed by crosslinking with urea-formaldehyde–sulfuric
acid mixture [151]. Material prepared in such a way possesses a high selectivity of moisture uptake.
Alginates are another class of biopolymers from marine sources and have been studied and uti-
lized in the pharmaceutical and biotechnological fields as well as in product development for many
years [152–154]. Alginates are usually extracted from brown algae. Alginates and chitosan have
the common characteristic of forming gels, in addition to possessing viscosity-related properties.
Alginates and chitosan can be utilized as hydrophilic drug carriers as coating material (they form film)
and as matrix materials. Recently, these polysaccharides have received attention as potential rate-
controlling excipients in drug release systems. Both chitosan and alginates can be used separately as
materials for biomedical application as well as blends of these two biopolymers [152–154]. In addi-
tion to drug delivery systems, these polysaccharides have been used in encapsulation and immobi-
lization of living cells. They can also be used as artificial organs consisting of hormone-producing
cells. Several research groups have published articles regarding chitosan and alginate blends. The
blend of alginate and carboxymethyl chitosan has been used for fiber preparation by spinning the
mixture solution through a viscose-type spinneret into a coagulating bath containing aqueous
CaCl2 [152]. Improved miscibility between alginate and carboxymethyl chitosan due to the strong
interaction from the intermolecular hydrogen bonds has been shown. Introduction of carboxymethyl
chitosan in the blend fiber improved water-retention properties of the blend fiber compared with
pure alginate fibers. From these alginate-chitosan blends, gel beads could be prepared based on Ca
ions or dual crosslinking with various proportions of alginate and chitosan [153]. The homogeneous
solution of alginate and chitosan was dripped into the solution of calcium chloride; the resultant Ca
ion single crosslinked beads were dipped in the solution of sodium sulfate sequentially to prepare
dual crosslinked beads. The dual crosslinkage effectively promoted the stability of beads under
gastrointestinal tract conditions. The dual crosslinked beads have potential small intestine or colon
site-specific drug delivery properties [153]. Based on the blends of chitosan and synthetic polymers,
cationic and anionic microparticles can be prepared [102]. Such modified microparticles showed an
enhanced model protein delivery into phagocytic cells.
Chitosan blended with pullulan is a potential moisture adsorbent and can be intended for wound
dressing application [155]. The design and development of chitosan as a wound dressing can be
improved by the addition of cornstarch or dextran and propylene glycol to obtain the films with
optimal properties for wound management [156].
When chitosan is blended with egg phosphatidylcholine, advanced injectable drug delivery sys-
tems for pharmaceutical applications can be obtained [157]. The stability of such blends is due to
the specific interactions between the molecules. Microdomains can be formed within the blends,
which increase the stability.
11.5 MATERIALS BASED ON BLENDS OF ELASTIN AND OTHER POLYMERS

The next biopolymer that can be used for biomaterial preparation is elastin [69,158–160]. Elastin
is composed of several amino acids, where the main amino acids are glycine, proline, and alanine.
Elastin is a structural protein in mammals, where it is the main component of skin, blood vessels,
such as the aorta, and tissues of the lung [69]. Elastin is a highly crosslinked material, and it is
insoluble in water [158,159]. In biomedical applications, elastin can be used as raw material for
preparation of several biomaterials, such as arterial prosthesis, dermal substitute, and hydrogels
[161–163]. However, elastin is not a good material for blend preparation as it is highly insoluble.
The only way to obtain soluble derivatives of elastin is hydrolysis of this biopolymer to smaller pep-
tide and polypeptide [164–166]. In the scientific literature, there is a lack of publications regarding
blends of elastin and elastin hydrolysates with synthetic polymers.
In our laboratory, we prepared new material based on the blend containing elastin hydrolysates
and soluble collagen [163,166]. Elastin for hydrolysis was obtained from pig aorta. The molecular
weight of the elastin hydrolysates was characterized by gel filtration using a superose gel column.
The distribution of molecular weight of elastin hydrolysates was broad (Figure 11.6). As can be
seen in Figure 11.6 (curve b), elastin hydrolysate was eluted as a broad peak, covering a molecular
weight range from 180 to 10 kDa: about 70% of total elastin-derived peptides are in the range of
65–12 kDa. High molecular weight heterogeneity is observed compared with marker globular pro-
teins (ovalbumin and myoglobin), which are eluted as well-separated peaks (Figure 11.6, curve a),
superimposed. We tried to modify elastin hydrolysates with UV irradiation (λ = 254 nm). The short
duration of UV irradiation (0.5 h) did not cause any larger differences in molecular weight distribu-
tion of elastin hydrolysates (Figure 11.6, curve c). After 2 h of UV irradiation, elastin hydrolysate was
eluted as a broader peak than elastin before irradiation (Figure 11.6, curve d). These results showed
high molecular weight heterogeneity of UV-irradiated elastin hydrolysates in comparison to nonirra-
diated elastin hydrolysates. The clear fraction of higher molecular weight than that for elastin before
irradiation suggests that elastin hydrolysates undergo crosslinking reactions during UV irradiation.
The mixtures of elastin/collagen were prepared by mixing of appropriate volumes of elastin hydroly-
sates and collagen in acetic acid solution. From elastin/collagen mixture, thin films were obtained by sol-
vent evaporation from solutions poured onto glass plates covered by polypropylene sheets. The obtained
films were 0.015–0.030 mm thick. An example of elastin/collagen film is presented in Figure 11.7.
For elastin/collagen films, the surface properties were studied. Our results showed that the sur-
face of a collagen/elastin film is enriched in the less polar component–collagen. Moreover, the
surface of elastin/collagen films was modified by the exposure to UV light. To assess polarity of
the surface, the contact angle can be measured and the surface free energy can be calculated. The
surface free energy (γs) is one of the parameters that determine the quality of the material’s surface
0.7
0.6
0.5 Myoglobin
Ovalbumin
Protein (abs 280 nm)
0.4
0.3
0.2
c b
0.1
a d
0
40 50 60 70 80 90 100 110
Elution volume (Ve, mL)
FIGURE 11.6 Gel filtration chromatograph of the standard protein mixture (a) (ovalbumin, 45 kDa and
horse myoglobin, 17.8 kDa)—for comparative purposes and elastin hydrolysates before (b) and after 0.5 h (c),
2 h (d) irradiation. (Reprinted from J. Photochem. Photobiol. B Biol., 86, Sionkowska, A., Skopińska, J.,
Wiśniewski, M., and Leżnicki, A., Spectroscopic studies into the influence of UV radiation on elastin hydro-
lysates in the presence of collagen, 186–191, 2007. Copyright 2007, with permission from Elsevier.)
FIGURE 11.7 (See companion CD for color figure.) Image of thin film based on collagen and elastin
hydrolysates. (Reprinted from Appl. Surf. Sci., 225, Skopinska-Wisniewska, J., Sionkowska, A., Kaminska,
A., Kaznica, A., Jachimiak, R., and Drewa T., Surface properties of collagen/elastin based biomaterials for
tissue regeneration, 8286–8292, 2009. Copyright 2009, with permission from Elsevier.)
and its possible applications. It characterizes the disruption of intermolecular bonds that occur dur-
ing the formation. The surface free energy can be calculated on the basis of the contact angle (Θ),
which measures the noncovalent forces between a liquid and the first monolayer of material. Owens-
Wendt method is one of the most commonly used calculating ways for polymeric materials. The
method allows to estimate the dispersive ( γ s ) and polar (γs) components of surface free energy,
d
which provides more detailed information on the studied surfaces. In order to calculate the surface
free energy of collagen, elastin, and its blends, two measuring liquids were used—glycerol (G) and
TABLE 11.6
Surface Free Energy (γs) of Elastin, Collagen, and Elastin/
Collagen Films before and after UV Irradiation (Calculated
by Owens-Wendt Method)
Surface Free Energy
Time of Irradiation [h] Elastin Elastin-1:3 Collagen 5:95 Collagen
0 32.31 37.52 36.61 36.76
2 37.11 37.00 37.88 38.90
4 37.35 37.45 39.86 38.64
12 37.08 41.12 42.69 43.77
24 37.76 46.90 47.24 48.52
Source: Reprinted from Appl. Surf. Sci., 225, Skopinska-Wisniewska, J.,

Sionkowska, A., Kaminska, A., Kaznica, A., Jachimiak, R., and Drewa T.,
Surface properties of collagen/elastin based biomaterials for tissue regen-
eration, 8286–8292, 2009. Copyright 2009, with permission from Elsevier.
TABLE 11.7
Polar Component of the Surface Free Energy gs of Elastin,
p
Collagen, and Elastin/Collagen Films before and after UV

Irradiation (Calculated by Owens-Wendt Method)
Polar Component of Surface Free Energy
Time of Irradiation [h] Elastin Elastin-1:3 Collagen 5:95 Collagen
0 8.45 5.81 4.93 3.77
2 11.87 6.42 7.67 6.27
4 15.59 7.18 8.87 8.09
12 11.77 11.83 12.83 13.60
24 13.27 18.00 18.00 18.93
Source: Reprinted from Appl. Surf. Sci., 225, Skopinska-Wisniewska, J.,

Sionkowska, A., Kaminska, A., Kaznica, A., Jachimiak, R., and Drewa T.,
Surface properties of collagen/elastin based biomaterials for tissue regen-
eration, 8286–8292, 2009. Copyright 2009, with permission from Elsevier.
diiodomethane (D). The results of the surface free energy for films made of elastin, collagen, and
the blend of these two biopolymers are presented in Tables 11.6 and 11.7.
The value of surface free energy is lower for nonirradiated elastin films than that for collagen film
before irradiation. In addition, the surface free energy for the elastin/collagen blends is higher than
that for elastin films. However, the differences between surface free energy for collagen film and the
blend made of elastin and collagen are not significant. After UV treatment, we observed an increase
in surface free energy of elastin/collagen blends. This change in the surface free energy may lead to
the improvement of adhesion ability of the material. The values of polar component of surface free
energy have been presented in Table 11.7. The results prove that elastin is more polar than collagen
in spite of the presence of hydrophobic domains in the structure of the first one. It could be caused
by relatively short length elastin hydrolysate chains and, as an effect, large content of polar terminal
groups. The presence of collagen in the blended films causes the drop of the value γ s . The polar
p
component of the surface free energy for all kinds of studied samples increases after UV irradiation.
800.00 nm
2 2
4 4
6 6
8 8
(a) μm (b) μm
FIGURE 11.8 (See companion CD for color figure.) AFM images of: (a) nonirradiated film based on the
blend of elastin/collagen (25/75) (b) the same film after 24 h of irradiation. (Reprinted from Appl. Surf. Sci.,
225, Skopinska-Wisniewska, J., Sionkowska, A., Kaminska, A., Kaznica, A., Jachimiak, R., and Drewa T.,
Surface properties of collagen/elastin based biomaterials for tissue regeneration, 8286–8292, 2009. Copyright
2009, with permission from Elsevier.)
Such results indicate that efficient photochemical reactions take place on the surface of biopolymeric
films. The increase in polarity leads to improved interaction between the material’s surface and
cells. The higher content of elastin in biomaterial promotes cell adhesion and their viability on the
surface. A suitable dose of UV light may improve the biocompatibility of the materials.
Atomic force microscopy (AFM) images have showed that the topography and roughness of the
materials based on elastin/collagen blends were affected by UV irradiation (Figure 11.8).
11.6 SILK AND KERATIN BLENDS WITH OTHER POLYMERS

Silk fibroin produced by the domesticated Bombyx mori silkworm has been used as a raw material
for decades. Natural fibroin is comprised of repeat sequences of alanine and glycine that readily
form the β-sheet crystals responsible for the mechanical properties of silk. Natural (and also syn-
thetic) silks’ elasticity and strength make them important candidates for application in synthetic
bone, ligament, and cartilage. Pure fibroin is biocompatible, can degrade slowly in vivo, supports
attachment and proliferation of many cells, and supports osteoblastic differentiation. Silk fibroin
can be processed into films and scaffolds to improve tissue regeneration in skin, nerve, bone, and
cartilage. Blending the natural fibroin with the less expensive synthetic polymers is one approach
to reduce the cost of materials. The surface of silk fibroin is usually improved when silk-based
materials are used as blood-contacting materials, such as introducing heparin into them. In tissue
engineering recently a big amount of silk is used.
Polymeric biomaterials based on the blends of silk with synthetic polymers and/or another natu-
ral polymer are widely studied [167–186]. From such blends, thin films can be produced, and also
nanofiber can be prepared by the electrospinning method [170,172].
Several materials made from a chitin/silk fibroin blend were considered as a potential candidate
for tissue engineering scaffolds with excellent cell attachment properties [178–180]. Silk fibroin
was blended with chitosan [180], poly(vinyl alcohol) (PVA) [181–184], poly(ethylene glycol), and
nylon 66 [182]. Silk fibroin after blending with poloxamer 407 macromer was considered as a
matrix for wound dressing [185]. Using chitosan/silk fibroin blend films containing nanoparticles
several nanocomposites can be obtained [186].
In our laboratory, we prepared several biomaterials based on the blends of chitosan and silk
fibroin. Thin films were obtained from solution by solvent evaporation. For films made of the blend
of chitosan and silk fibroin, several properties were measured. To assess the surface properties of
TABLE 11.8
The Surface Free Energy (γs) of Silk Fibroin, Chitosan, and Their Blends (Films)
before and after UV Irradiation (Calculated by Owens-Wendt Method)
γs [mN/m]
Time of UV
Irradiation [h] SF 90/10 80/20 60/40 50/50 40/60 20/60 10/90 CTS
0 27.90 23.24 20.01 23.12 21.17 22.72 23.05 20.23 10.18
1 33.86 41.75 40.50 34.10 36.18 36.54 36.89 35.22 30.68
2 43.64 50.09 52.69 49.28 48.93 44.44 49.57 40.50 40.35
4 37.69 51.61 46.52 51.34 48.76 49.97 50.17 50.09 46.06
8 41.72 48.09 51.04 50.68 52.23 52.04 42.23 42.04 49.63
12 20.85 26.65 34.70 28.83 27.10 24.32 28.72 30.78 40.15
24 48.43 54.81 57.17 57.64 57.58 57.86 57.33 57.26 54.38
TABLE 11.9
The Polar Component of the Surface Free Energy gs of Surface Free Energy
p
(γs) of Silk Fibroin, Chitosan, and Their Blends (Films) before and after UV
Irradiation (Calculated by Owens-Wendt Method)
gsp [mN/m]
Time of UV [h] SF 90/10 80/20 60/40 50/50 40/60 20/60 10/90 CTS
0 2.87 1.97 1.83 2.28 3.07 3.80 4.15 4.12 0.26
1 7.52 11.08 10.14 4.89 4.07 3.34 5.66 4.20 0.50
2 17.35 19.50 22.04 20.78 18.95 17.32 21.08 9.93 39.72
4 10.68 19.89 11.71 14.36 11.59 12.27 12.38 11.49 18.59
8 22.32 13.48 17.02 16.49 17.06 17.08 7.01 8.14 21.15
12 10.50 8.91 7.09 21.98 22.14 13.77 16.73 9.00 16.74
24 16.55 20.53 22.77 25.27 28.15 26.48 24.88 25.11 18.69
thin films before and after UV treatment, the contact angle measurements were done and surface
free energy was calculated. The results of the surface free energy for films made of chitosan, silk
fibroin, and the blend of these two biopolymers are presented in Tables 11.8 and 11.9.
The value of surface free energy is lower for nonirradiated chitosan films than for silk fibroin
film before irradiation. The surface free energy for the chitosan/silk fibroin blends is higher
than for chitosan films. However, the differences between surface free energy for silk fibroin
film and the blend made of silk fibroin and chitosan are not significant. After UV treatment, the
increase in surface free energy of chitosan/silk fibroin blends was observed. The values of polar
component of surface free energy have been presented in Table 11.9. The results prove that silk
fibroin film is more polar than chitosan film. It could be caused by several hydrophilic functional
groups in silk chain. The presence of chitosan in the blended films causes the drop in the value
of the polar component of surface free energy ( γ s ). The polar component of the surface free
p
energy for chitosan/silk fibroin blends increased after UV irradiation. One can say that efficient
photochemical reactions take place on the surface of biopolymeric films based on the blends of
chitosan/silk fibroin. The increase in polarity can lead to improved interaction between the mate-
rial’s surface and cells. The polar component of surface free energy had the highest value for the
blend with a composition 50% of chitosan and 50% of silk fibroin after 24 h of UV irradiation.
Structural changes on the surface of chitosan/silk fibroin films induced by UV irradiation were
observed using AFM microscope. The microphotographs of nonirradiated and irradiated chitosan/silk
5.00 150.0 nm
75.0 nm
0.0 nm
2.50
Digital instruments nanoscope

Scan size 5.000 μm
Scan rate 1.973 Hz
Number of samples 512
Image data Height
Data scale 150.0 nm
0
0 2.50 5.00
(a) μm
5.00 150.0 nm
75.0 nm
0.0 nm
2.50

Scan size 5.000 μm
Scan rate 1.973 Hz
Image data Height
Data scale 150.0 nm
0
0 2.50 5.00
(b) μm
FIGURE 11.9 (See companion CD for color figure.) AFM images of (a) nonirradiated film based on the
blend of chitosan/silkfibroin (50/50) (b) the same film after 2 h of irradiation.
TABLE 11.10
Surface Roughness of Chitosan
Films, Silk Fibroin Films, and Films
Based on the Blends of SF/CTS
Rms [nm]
Time of UV [h] SF 50/50 CTS
0 22.9 13.4 4.8
2 19.2 9.1 3.4
8 3.9 4.2 2.8
TABLE 11.11
Mechanical Properties of the Blends of Chitosan/
Silk Fibroin
Young’s Breaking
Specimen % Elongation Modulus [GPa] Load [N]
Chitosan 1.15 0.54 5.88
CTS/SF (80/20) 0.97 1.11 15.48
CTS/SF (60/40) 1.14 2.68 15.69
CTS/SF (50/50) 0.48 2.01 5.04
CTS/SF (40/60) 0.75 2.14 13.52
CTS/SF (20/80) 0.07 0.58 0.11
fibroin films are shown in Figure 11.9. AFM images show differences in surface properties of the films
before and after UV irradiation. The surface roughness is decreasing with increasing UV irradiation
time. The atomic force microscope is often used to obtain a reasonable measure of surface roughness
on the nanometer scale. Typically, AFM users rely on root mean square (Rms) roughness. The values
of root-mean-square (Rms) surface roughness of films made of silk fibroin, chitosan, and their blends
before and after UV irradiation are shown in Table 11.10. Rms value for silk fibroin film is much bigger
than for pure chitosan film and also bigger than for chitosan/silk fibroin film. UV irradiation caused
the decrease in surface roughness of silk fibroin films, chitosan films, and films made of chitosan/silk
fibroin blend.
Mechanical properties of chitosan films and films based on the blends of chitosan and silk fibroin
are shown in Table 11.11. Several films made of the blend of chitosan and silk fibroin with different
composition were obtained after solvent evaporation from chitosan/silk fibroin solution. As one can
see in Table 11.11, for chitosan/silk fibroin film with a composition of 60% of chitosan and 40% of
silk fibroin, the mechanical properties, such as elongation at break, ultimate tensile strength, and
Young’s Modulus have the highest value. Thin silk fibroin films were very fragile, and it was not
possible to measure mechanical properties for such films.
Keratin is a biopolymer, the major component of hair, feathers, nails, and horns of mammals,
reptiles, and birds. This fibrous protein is composed of several repeating sequences of amino acids
along its chain. In the biopolymer chain amino acid containing sulfur, cystine is present. Cystine
in the keratin chain is able to form characteristic inter and intramolecular disulphide bonds, which
determine the properties of keratin [187,188]. Keratin, similarly to elastin is a highly insoluble protein
[189]. Keratin can be modified by UV radiation and chemical crosslinking [190–192]. In scientific
literature, there are only a few papers regarding the blends of keratin with other polymers. There are
reports regarding blends of keratin with poly(ethylene oxide) [193,194] and with c hitosan [170,195].
11.7 NEW MATERIALS AND COMPOSITES BASED

ON NATURAL POLYMERS, SYNTHETIC POLYMERS,
AND THEIR BLENDS CONTAINING INORGANIC PARTICLES
Most natural polymers have a complex structure with interesting mechanical properties. These com-
plex structures, which have risen from hundreds of million years of evolution, are inspiring scientists
in the design of novel materials. Their defining characteristics, hierarchy, multifunctionality, and
self-healing capability are often investigated [197]. Self-organization is also a fundamental fea-
ture of many biological materials and the manner by which the structures are assembled from the
molecular level up. The basic building blocks are described, starting with the 20 amino acids and
proceeding to polypeptides, polysaccharides, and polypeptide–saccharides. Biological materials
very often contain inorganic nanoparticles. The “hard” phases are primarily strengthened by min-
erals, which nucleate and grow in a biomediated environment that determines the size, shape, and
distribution of individual crystals. The most important mineral phases are hydroxyapatite, silica,
and aragonite [197].
New materials based on the blends of two polymers or biopolymers that contain inorganic
nanoparticles can be used as an implant in the context of both hard and soft tissues. Natural
polymers can be used for preparation of such biomaterials for biomedical applications without
any additives. Pure and blended collagen were widely studied [63–83], together with pure chito-
san and its blends [15,20,44,62,82–156]. Nanostructured materials can be created by combining
organic and inorganic compounds. It can be achieved by the intercalation of inorganic nanopar-
ticles in a polymeric matrix. In order to develop novel bone substitutes, hydroxyapatite has been
intensively investigated from the viewpoints of biocompatibility, bioactivity, and biodegradation.
It is extremely difficult to connect nanoparticles with natural polymers, as many of them are
insoluble. In many projects, insoluble natural polymers such as collagen, elastin, chitosan, keratin,
and silk have been converted to soluble derivatives by hydrolysis (acidic, base, and enzymatic).
These hydrolysates, after characterization, could be used to design and make new biomaterials for
medical applications.
Regenerative medicine is currently using three strategies: transplantation of cells to form new tis-
sue in the transplant site, implantation of bioartificial tissues constructed in vitro, and induction of
regeneration in vivo from healthy tissues adjacent to an injury. New materials based on the blends of
natural and synthetic polymers containing nanoparticles can be used as bioartificial implants in the
context of both hard and soft tissues. The next several years will be critical for the maturation of nano-
material science and its role in clinical medicine. Elucidation of the fundamental structure–function
relationships in normal and diseased or damaged tissue through sound basic research will continue to
be a necessary prerequisite. In addition, consideration of the different elements in the product devel-
opment continuum from inspiration, product design, and bench studies, to the clinic is vital. Although
natural polymers have separately been proposed as in vitro extracellular matrix materials, the influ-
ence of biopolymer–polymer composite matrices on cell morphology, differentiation, and function is
not well studied. Recently, attention has been given to the applications of nanoparticles in combina-
tion with polymers and biopolymers. Biopolymer-based materials can be combined with a bonelike
filler material (as bioactive glass, calcium phosphates, and other calcium salts). The properties of
biomaterials that will be used in tissue regeneration as bone, scaffolds, and guiding structures are
of key importance. In addition to gross properties, such as mechanical strength, biodegradability,
biocompatibility, and lack of toxic or immune reactions, the biomaterial should exhibit the capac-
ity to interact specifically with cell surfaces, in particular with cell-membrane receptors that are
active in recognition, adhesion of cells to the extracellular matrix, and in mediation of signals to the
cell. Not only should the biomaterial surfaces contain the respective recognizable structures, but
they should also present these structures on a nanoscale, which would compare to the dimensions
of adhesive plaques and clusters of receptor molecules at the surfaces of adhering cells. Properties
of the biomaterial surface (such as topography and chemistry) control the type and magnitude of
cellular and molecular events at the tissue–implant interface. Therefore, nanotechnologies repre-
sent a new approach to materials science. Several papers have been published so far in the area of
biomaterials based on the blends of natural polymers and synthetic polymers containing inorganic
nanoparticles [147,181,198–206]. We can expect that in the near future several new materials will be
designed based on the blends of two or more natural polymers as well as with synthetic polymers
containing nanoparticles. An economic impact can be expected, as materials based on the blends
of natural polymers and synthetic polymers should be less expensive than materials made strictly
from biopolymers.
11.8 CONCLUSIONS
Most natural materials are complex composites with outstanding mechanical properties. These
complex structures, which have risen from many years of evolution, are inspiring materials scien-
tists in the design of novel materials. Their defining characteristics, hierarchy, multifunctionality,
and self-healing capability, are illustrated by several researchers. Self-organization is also a funda-
mental feature of many biological materials and the manner by which the structures are assembled
from the molecular level up. The main biopolymers used in preparation of materials for biomedi-
cal applications are collagen, chitin, chitosan, keratin, silk, and elastin. These biopolymers can be
blended with several synthetic polymers. Moreover, the blends can be strengthened by minerals,
which nucleate and grow in a polymeric blend to produce the appropriate size, shape, and distribu-
tion of individual crystals, similar to hard tissue. The main goal of several research groups is the
search for bioinspired materials and structures. Traditional approaches focus on design methodolo-
gies of biological materials using conventional synthetic materials. The new approach focuses on
design methodologies of biological materials using blends of synthetic and natural polymers. The
new frontiers reside in the synthesis of bioinspired materials through processes that are charac-
teristic of biological systems; these involve nanoscale self-assembly of the components and the
development of hierarchical structures. The development of new biomaterials for medical applica-
tions is one of the challenging tasks for many scientists today. Due to the mechanical, chemical,
and biological properties of the natural-based composites, multifunctional biomaterials based on
the blends of natural polymers with synthetic ones can be designed to be similar to those of normal
human tissue. It is believed that it will present significant advantages over current materials such
as human and animal grafts or artificial materials. In the near future, we can expect the develop-
ment of adequate three-dimensional scaffolds from both natural and synthetic materials and also
their blends. Such materials can subsequently be used as highways for cells in order to induce
cell activity, being physiologically suitable for cell activity (adhesion, proliferation, differentiation)
and having mechanical properties similar to those of the natural tissue. We can expect also the
development of 3D culture technology by which functional tissue can be grown ex vivo in the
three-dimensional scaffolds. The application of the blends can be very wide, including membranes
for dialysis, wound dressings, artificial skin, and implantable devices for the delivery of biologi-
cally active substances and materials. New materials based on the blends have the potential to
develop new material for packaging of medical devices and drugs. Our research and that of several
research groups has shown that it is not easy to study the interaction between natural and synthetic
polymers [207–218]. Very often, the properties of the blends do not meet the scientific and practical
expectations. Nevertheless, substantial gains are expected both from a medical and an economic
standpoint. We hope that in the near future there will be a huge increase in new products based on
the blends of natural and synthetic polymers.
ACKNOWLEDGMENT
Financial support from the Ministry of Science (MNII, Poland) Grant No. N N507 3495325 is grate-
fully acknowledged.
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12 Metal–Polymer Composite
Biomaterials
Takao Hanawa
CONTENTS
12.1 Introduction...........................................................................................................................344
12.2 Properties of Metals against Polymers..................................................................................344
12.3 Surface of Metallic Biomaterials........................................................................................... 349
12.3.1 Importance of Metal Surface..................................................................................... 349
12.3.2 Surface of Metals....................................................................................................... 349
12.3.3 Surface Oxide Film................................................................................................... 350
12.3.3.1 Titanium...................................................................................................... 350
12.3.3.2 Titanium Alloy............................................................................................ 352
12.3.3.3 Stainless Steel............................................................................................. 353
12.3.3.4 Cobalt–Chromium Alloy............................................................................ 353
12.3.3.5 Dental Precious Alloys............................................................................... 353
12.3.4 Surface Active Hydroxyl Group................................................................................ 353
12.3.4.1 Formation.................................................................................................... 353
12.3.4.2 Acidic and Basic Properties........................................................................ 353
12.4 Adhesion of Polymers to Metals in Dentistry....................................................................... 354
12.4.1 Adhesive Reagents..................................................................................................... 354
12.4.2 Bonding to Base Metal Alloys................................................................................... 355
12.4.3 Durability in Water.................................................................................................... 356
12.4.4 Bonding to Precious Alloys....................................................................................... 356
12.5 Stents and Stent Grafts.......................................................................................................... 357
12.5.1 Stents.......................................................................................................................... 357
12.5.2 Drug Eluting Stents................................................................................................... 358
12.5.3 Stent Grafts................................................................................................................ 359
12.6 Immobilization of Poly(Ethylene Glycol)..............................................................................360
12.6.1 Poly(Ethylene Glycol)................................................................................................360
12.6.2 Chemical Immobilization..........................................................................................360
12.6.3 Electrodeposition....................................................................................................... 362
12.7 Immobilization of Biomolecules...........................................................................................364
12.7.1 Metal–Biomolecule Interface....................................................................................364
12.7.2 Peptides......................................................................................................................364
12.7.3 Protein and Collagen................................................................................................. 366
12.7.4 Hydrogel and Gelatin................................................................................................. 366
12.8 Other Metal–Polymer Composites........................................................................................ 367
12.8.1 Bonding of Polymers with Metal through Silane Coupling Agent............................ 367
12.8.2 Polymers Condensed in Porous Titanium.................................................................. 367
12.8.3 Metallization of Polymers......................................................................................... 368
12.9 Conclusions............................................................................................................................ 370
References....................................................................................................................................... 370
343
12.1 INTRODUCTION
The fast technological evolution of polymers has made it possible to apply these materials to medi-
cal devices over the last three decades. In particular, because of their excellent biocompatibility
and biofunctions, polymers are expected to show excellent properties for use as biomaterials; in
fact, many devices made from metals have been replaced by others made from polymers. In spite
of this fact, about 80% of implant devices are still made from metals, and this percentage remains
unchanged because of their high strength, toughness, and durability. Medical devices consisting of
metals cannot be replaced with polymers at present.
The use of metals as raw materials has a long history, and it can be said that present materials
science and engineering have been based on research into metals. However, metals are sometimes
thought of as “unfavorite materials” for biomedical implants because of memories of the environ-
mental and human damages caused by heavy metals. Since an improvement in the safety of met-
als for medical use is vital, strenuous efforts have been made to improve corrosion resistance and
mechanical durability. In addition, metals are typically artificial materials and have no biofunctions,
which makes them fairly unattractive as biomaterials.
On the other hand, polymers are widely used as biomaterials because of their high degree of
flexibility, biocompatibility, and technologic properties.1 Also, it is relatively easy to design biofunc-
tional polymers based on biomimic technique, because biofunctional polymers exist in the human
body as parts of biomolecules, cells, tissues, and organs. Therefore, some polymeric materials are
widely known as biocompatible and biofunctional materials, as shown in other chapters in this book.
If these advantages of metals and polymers are mixed and disadvantages are eliminated by
manufacturing metal–polymer composite materials, then humankind will obtain ideal materials
with excellent mechanical properties and biofunctions. Two metal–polymer composite materials
are feasible to design: one of them is a combination of bulk polymeric materials and bulk metallic
materials, the other is immobilization of polymers to metal surfaces. In this chapter, these metal–
polymer composite materials for biomedical use are explained and the corresponding researches are
reviewed. To understand metallic materials against polymeric materials, an outline of metals is first
presented. Then, surface properties of metals are described to know the reaction of metal surface
with polymers that govern the manufacture of metal–polymer composites, followed by the main
subject, metal–polymer composites.
12.2 PROPERTIES OF METALS AGAINST POLYMERS

Materials are categorized to metals, ceramics, and polymers (Figure 12.1). Metallic materials are
generally multicrystal bodies consisting of metal bonds. For example, metal oxide, metal salt, metal
complex, etc., contain metal elements; however, these are compounds consisting of ionic bonds
or covalent bonds; these properties are completely different from those of metals consisting of
metal bonds. Therefore, in the field of materials engineering, ceramics and metals are clearly distin-
guished, although they are categorized together in inorganic compounds. Each material has its own
advantages and disadvantages, and the applications are determined according to their properties.
Metals have been utilized for dental restoration and bone fixation since 2500 years ago; they have
a long history as biomaterials. Advantages of metals as biomaterials are listed as follows. These
properties are caused by metal bonds:
1. Great strength
2. Great ductility, easy working
3. Fracture toughness
4. Elasticity and stiffness
5. Electroconductivity
Metal–Polymer Composite Biomaterials 345
Metallic Metal; alloy Metallic bond

Inorganic
materials
compounds
Inorganic Ceramics; glass Ionic bond; covalent bond
materials
Organic
Organic Natural polymer Covalent bond; ionic bond
compounds
materials synthesized polymer
Composites
FIGURE 12.1 Category of materials.
1500
Co-Cr-Mo ally
(ASTM F799 wrought)
×
1200
900
Stress/MPa
Zirconia
× Ti-6Al-4V alloy
× (ASTM F136 wrought)
β-type Ti alloy
600 (TNTZ alloy)
× Alumina SUS316L
(ASTM F138 wrought)
CP Ti Grade 2
300 (ASTM F67) ×
×
× Bone
UHMWPE
0
50 100
Strain/%
FIGURE 12.2 Stress–strain curves of various materials.
Metals and alloys are widely used as biomedical materials and are indispensable in the medical
field. Advantages of metals compared with ceramics and polymers are great strength and difficulty
to fracture. In particular, toughness, elasticity, rigidity, and electrical conductivity are essential
properties for metals used in medical devices. Figure 12.2 shows stress–strain curves of typical
metals, ceramics, and ultra high molecular weight polyethylene (UHMWPE). The strength and
elongation of metals against ceramics and polymers are clearly understood.
Conventionally, metals are essential for orthopedic implants: Bone fixators, artificial joints,
external fixators, etc., can substitute for the mechanical function of hard tissues in orthope-
dics. Stents and stent grafts are placed at angusty in blood vessels for dilatation. Therefore,
elasticity or plasticity for expansion and rigidity for maintaining dilatation are required for the
devices. In dentistry, metals are used for restorations, orthodontic wires, and dental implants.
TABLE 12.1
Comparison of Properties of Metals for Implant
Mechanical Corrosion
Property Workability Resistance
Tensile Wear
Materials Strength Resistance Plasticity Machinability Pitting
Stainless steel Type316L Good Excellent Excellent Excellent Excellent
 Poor  Poor
Co-Cr alloy Cast Good Good Poor Poor Good
 Good
Annealed Excellent Good Good Good Fair
 Good
Ti; Ti alloy CP Ti Excellent Excellent Fair Fair Excellent
 Poor  Poor
Ti-6Al-4V Excellent Excellent Fair Excellent Excellent
 Poor  Poor
For mechanical reliability, metals must be used and cannot be replaced with ceramics or
polymers. In noble metals and alloys, gold (Au) marker for the imaging of stent, platinum (Pt)
for embolization wire, and Au alloys and silver (Ag) alloys for dental restoratives are utilized.
In base metals and alloys, austenitic stainless steels, cobalt–chromium (Co-Cr) alloys, titanium
(Ti), and Ti alloys, whose corrosion resistance is maintained with surface oxide film as pas-
sive film, are utilized for implant materials. On the other hand, wear resistance is required
to decrease the generation of wear debris. Co-Cr-molybdenum (Mo) alloys have good wear
resistance and are used for sliding parts of artificial joints. Comparison of various properties
of metals used for implant is summarized in Table 12.1. The metals used for such devices are
listed in Table 12.2. Safety to the human body is essential in biomaterials; no toxic material is
used for biomaterials. Metals implanted in tissues do not show any toxicity without metal ion
dissolution by corrosion and generation of wear debris by wear. Therefore, corrosion resistance
is absolutely necessary for metals in biomedical use, resulting in the uses of noble or corrosion-
resistant metals and alloys for medicine and dentistry.
A disadvantage of using metals as biomaterials is that they are typically artificial materials and
have no biofunction. Therefore, additional properties are required for metals. Requests for met-
als for biomedical use are summarized in Table 12.3. To respond to these requests, new design of
alloys and many techniques for surface modification of metals are attempted on a research stage
and some of them are commercialized. Surface modification is necessary because biofunction can-
not be added during manufacturing processes of metals such as melting, casting, forging, and heat
treatment. Surface modification is a process that changes a material’s surface composition, struc-
ture, and morphology, leaving the bulk mechanical properties intact. Reviews on surface modi-
fication of Ti have already been published on plasma spraying2 and electrochemical treatments.3
The most important difference between a metal and polymer is cohesive and noncohesive b odies.
In other words, the interface of those is a boundary between a rigid and condensed solid and a
flexible and mobile molecule. Of course, metals mainly consist of metal bonds; polymers mainly
consist of covalent bonds. Therefore, design and fabrication of metal–polymer composites are to
decrease the mismatch of their properties at the interface. In addition, the mechanical and chemical
properties of metallic materials are not identified, even though the compositions are determined.
The phase of metallic materials is easily changed by working and heat treatment, and various fac-
tors influence the resultant structure. Factors governing the mechanical and chemical properties of
metallic materials are summarized in Figure 12.3.
TABLE 12.2
Metals Used for Medical Devices
Clinical Division Medical Device Material
Orthopedic surgery Spina fixation 316L stainless steel; Ti; Ti-6Al-4V;
Ti-6Al-7Nb
Bone fixation (bone plate, screw, wire, 316L stainless steel; Co-Cr-W-Ni; Ti;
bone nail, mini-plate, etc.) Ti-6Al-4V; Ti-6Al-7Nb
Artificial joint; bone head 316L stainless steel; Fe-Cr-Ni-Co;
Co-Cr-Mo; Ti-6Al-4V; Ti-6Al-7Nb;
Ti-15Mo-5Zr-3Al:
Ti-6Al-2Nb-1Ta-0.8Mo
Spina spacer 316L stainless steel; Ti-6Al-4; Ti-6Al-7Nb
Cardiovascular medicine and Implant-type artificial heart (housing) Ti
surgery
Pace maker (case) Ti; Ti-6Al-4V
(electric wire) Ni-Co
(electrode) Ti; Pt-Ir
(terminal) Ti; 316L stainless steel; Pt
Artificial valve (frame) Ti-6Al-4V
Stent 316L stainless steel; Co-Cr-Fe-Ni;
Co-Ni-Cr-Mo; Co-Cr-Ni-W-Fe; Ti-Ni; Ta
Guide wire 316L stainless steel; Ti-Ni; Co-Cr
Embolization wire Pt
Clip Ti-6Al-4V; 630 stainless steel;
631 stainless steel; Co-Cr-Ta-Ni;
Co-Cr-Ni-Mo-Fe; Ti; Ti-6Al-4V
Otorhinology Artificial inner ear (electrode) Pt
Artificial eardrum 316L stainless steel
Dentistry Filling Au foil; Ag-Sn(-Cu) amalgam
Inlay, crown; bridge; clasp; denture Au-Cu-Ag; Au-Cu-Ag-Pt-Pd; Ag-Pd-Cu-Au;
base Ag-(Sn-In-Zn); Co-Cr-Mo; Co-Cr-Ni;
Co-Cr-Ni-Cu; Ti; Ti-6Al-7Nb; 304 stainless
steel; 316L stainless steel
Thermosetting resin facing crown; Au-Pt-Pd; Ni-Cr
porcelain-fused-to-metal
Solder Au-Cu-Ag; Au-Pt-Pd; Au-Cu;
Ag-Pd-Cu-Zn
Dental implant Ti; Ti-6Al-4V; Ti-6Al-7Nb; Au
Orthodontic wire 316L stainless steel; Co-Cr-Fe-Ni; Ti-Ni;
Ti-Mo
Magnetic attachment Sm-Co; Nd-Fe-B; Pt-Fe-Nb; 444 stainless
steel; 447J1 stainless steel; 316L stainless
steel
Treatment device (bar, scaler, 304 stainless steel
periodontal probe, dental tweezers,
raspatory, etc.)
General surgery Needle of syringe 304 stainless steel
Scalpel 304 stainless steel; 316L stainless steel; Ti
Catheter 304 stainless steel; 316L stainless steel;
Co-Cr; Ti-Ni; Au; Pt-In
Staple 630 stainless steel
TABLE 12.3
Requests of Metals for Medical Devices
Required Property Target Medical Devices Effect
Elastic modulus Bone fixation; spinal fixation Prevention of bone absorption by stress
shielding
Superelasticity shape memory effect Multipurpose Improvement of mechanical
compatibility
Wear resistance Artificial joint Prevention of generation of wear
debris; improvement of durability
Biodegradability Stent; artificial bone; bone fixation Elimination of materials after healing;
unnecessity of retrieval
Bone formation bone bonding Stem and cup of artificial hip joint; Fixation of devices in bone
dental implant
Prevention of bone formation Bone screw; bone nail Prevention of assimilation
Adhesion of soft tissue Dental implant; trans skin device; Fixation in soft tissue; prevention of
external fixation; pacemaker inflectional disease
housing
Inhibition of platelet adhesion Devices contacting blood Prevention of thrombus
Inhibition of biofilm formation All implant devices; treatment tools Prevention of infectious disease
and apparatus
Low magnetic susceptibility All implant devices; treatment tools No artifact in MRI
and apparatus
Composition
Additional element
Manufacturing process
Melting
Casting
Forging
Rolling
Heat treatment
Structure
Matrix phase
Uniformity of solute atom in solid solution
Impurity
Segregation
Cavity
Dislocation
Stacking fault
Grain boundary
Surface oxide
Mechanical property
Chemical property
FIGURE 12.3 Factors governing the mechanical and chemical properties of metallic materials through the
manufacturing process.
12.3 SURFACE OF METALLIC BIOMATERIALS

12.3.1 Importance of Metal Surface
When a metallic material is implanted into a human body, immediate reaction occurs between its
surface and the living tissues. In other words, immediate reaction at this initial stage straightaway
determines and defines a metallic material’s tissue compatibility. Since conventional metallic bio-
materials are usually covered with metal oxides, surface oxide films on metallic materials play an
important role not only against corrosion but also in tissue compatibility. To fabricate metal–polymer
composites, knowledge of the material’s surface composition is absolutely necessary. Adhesion of
polymers and immobilization of molecules to metals are governed by the surface property of the
substrate metals. Immobilization of molecules is a kind of surface modification of metals, because
surface modification is a process that improves surface property by changing the composition and
structure, while leaving the mechanical properties of the material intact. Surface properties of a
metallic material may be controlled with surface modification techniques.
12.3.2 Surface of Metals

Atoms in metallic materials located at the surface are considered partly reactive to the environment
because atomic configuration terminates at the surface. The surface represents a property different
from the inside of the material. Due to high surface energy, a single molecular layer forms easily
on solid surface, where gas molecules are adsorbed at 1 Pa in 10 −4 s. For example, in the presence of
oxygen atoms, oxygen atoms and metal atoms chemically bond together to form an oxide layer. This
phenomenon occurs even at the surface of Au—which is the most noble metal. Unlike polymers and
ceramics, enrichment of component elements occurs easily at metal surfaces. This means that the
surface composition of a metal is different from its inside composition in the order of nanometers.
Therefore, the variant surface composition of a metallic material contributes significantly to defin-
ing the overall properties of the material.
A metal surface is usually covered with a surface oxide film. The surface oxide layer, on the
other hand, is always covered with surface hydroxyl groups that are adsorbed by water, as shown in
Figure 12.4. In particular, the surface oxide film and surface active hydroxyl groups are important
to understand the surface reaction of metals.
Adsorbed water
Hydroxyl groups
M M M M M M
O O O O O O O O O O O
M M M M M M
Metal oxide O O O
O O O
M M M M M M
O O O O O O O O O O O
M M M M M M
M M M M M M
M M M M M M
Metal substrate
M M M M M M
M M M M M M
FIGURE 12.4 Schematic model of the structure of surface layer consisting of oxide layer, hydroxyl group
layer, and adsorbed water layer on metals and alloys.
12.3.3 Surface Oxide Film

Except in reduction environments, the corrosion process always causes a reaction film to form on
metallic materials. Passive film is one such reaction film, and it is particularly significant for corro-
sion protection. When solubility is extremely low and pores are absent, adhesion of film—which is
formed in an aqueous solution—to the substrate will be strong. The film then becomes a corrosion-
resistant or passive film. Passive film is about 1–5 nm thick and is transparent. Due to the tremen-
dously fast rate at which it is formed, passive film readily becomes amorphous. For example, film on
a Ti metal substrate was generated in 30 ms. This was estimated from the time transient of current of
the Ti at 1 V versus a saturated calomel electrode (SCE) after exposing the metal surface, as shown
in Figure 12.5. Since amorphous films hardly contain grain boundary and structural defects, they
are corrosion resistant. However, corrosion resistance decreases with crystallization. Fortunately,
passive films contain water molecules that promote and maintain amorphousness.
Metallic materials such as stainless steels, Co-Cr alloys, commercially pure Ti, and Ti alloys
used for biomedical devices are covered by their characteristic passive films. These films self-repair
when they are disrupted by some causes. Noble metals and alloys such as dental alloys are also
covered with an oxide layer. While the oxide layer protects against corrosion, it is not chemically
strong like the passive film.
12.3.3.1 Titanium
When Ti is polished in deionized water and analyzed using X-ray photoelectron spectroscopy (XPS),
the Ti 2p spectrum obtained from the Ti gives four doublets according to valence: Ti0, Ti2+, Ti3+, and
Ti4+. Published data4 are used to determine the binding energy of each valence. Figure 12.6 shows an
example of the decomposition of Ti 2p spectrum. A distinct Ti0 peak at metallic state is observed, which
accounts for a very thin surface oxide film at less than a few nanometers only. Besides, Ti4+ (TiO2), Ti3+
(Ti2O3), and Ti2+ (TiO) are detected. Though Ti2+ oxide exists in the surface oxide film, Ti2+ formation is
always thermodynamically less favorable than Ti3+ formation at the surface. On the other hand, Figure
12.7 shows O 1s spectrum obtained from polished Ti, which is decomposed to three peaks originating
from oxide (O2−), hydroxide or hydroxyl group (OH−), and water (H2O). The surface oxide film on Ti
4
Current density, J/A cm–2
2
Mostly repassivated
0
0 20 40 60 80
Time, t/ms
Exposure of metal substrate
FIGURE 12.5 Time transient of anodic current of titanium in Hanks’ solution under the 1 V charge versus
SCE. Anodic current is generated with the dissolution and repassivation of titanium.
Ti 4+
2p1/2 2p3/2
Ti 3+
Intensity (a.u.) Ti 2+ Ti 0
470 460 450

Binding energy, Eb/eV
FIGURE 12.6 Decomposition of Ti 2p XPS spectrum obtained from titanium abraded and immersed for
300 s in water into eight peaks (2p3/2 and 2p1/2 electron peaks in four valences). Numbers with arrows are
valence numbers.
O2–
OH–
Intensity (a.u.)
H2O
542 538 534 530 526 522

Binding energy, Eb/eV
FIGURE 12.7 Typical O 1s spectrum obtained from polished Ti and its deconvolution into O2−, OH−, and
H2O components.
contains these chemical states. Therefore, the surface oxide film on Ti consists mainly of nonstoichio-
metric TiO2 containing the hydroxyl group and water.
Since a considerable portion of oxidized titanium stays as Ti2+ and Ti3+ in the surface film, the
oxidation process may proceed to the end just at the uppermost part of the surface film. As shown in
Figure 12.8, the proportion of Ti4+ among titanium cations (Ti2+, Ti3+, and Ti4+) in the film decreases
with an increase in photoelectron take-off angle,5 indicating that more Ti4+ exists near the outer
layer in the film. Deducing from the take-off angle dependence of [OH−]/[O2−] in Figure 12.8, oxy-
gen atoms in the hydroxyl group are mainly located in the outer part of the surface film. This means
that dehydration proceeds inside the surface film and only partly for Ti4+ oxide.
Thickness of the film is about 2 nm just after polishing and about 5 nm at 1 week after polishing
(as shown in Figure 12.9). Note too that the thickness of the surface oxide film increases according
to the logarithmic rule, which is common in the initial growth of oxide films of metallic materials.
Take-off angle of photoelectron (degree)

0 10 20 30 40 50 60 90
1.0
[OH–]/[O2–] and proportion of Ti4+

0.8
0.6
0.4
[OH–]/[O2–]
Ti4+
0.2
0.0
0.0λ 0.2λ 0.4λ 0.6λ 0.8λ 1.0λ
Effective escape depth of photoelectron
Outer Inner
layer layer
FIGURE 12.8 The ratio of the proportion of the concentration of OH− to that of O2−, [OH−]/[O2−], and
proportion of cationic fraction of Ti4+ among titanium species, in surface oxide film on Ti polished in water
plotted against the average effective escape depth of photoelectrons for angle-resolved XPS measurements.
Lambda (λ) is the average escape depth of O 1s and Ti 2p3/2 photoelectrons, and the effective escape depth is
the escape depth times sin (take-off-angle). The values at small take-off angle of photoelectron or effective
escape depth of photoelectron represent outer region information, and the larger ones represent inner region
information.
6 6d
Thickness of surface oxide film, l/s
4 10 min
3 1d
2
102 103 104 105 106
Time after polished, t/s
FIGURE 12.9 Thickness of the surface oxide film on Ti as a function of time after polishing.
12.3.3.2 Titanium Alloy

The film on Ti-6 aluminum (Al)-4 vanadium (V) alloy is almost the same as that on Ti containing
a small amount of aluminum oxide.6,7 In other words, the surface oxide film on Ti-6Al-4V is a TiO2
containing small amounts of Al2O3, hydroxyl groups, and bound water. V contained in the alloy
is not detected in the oxide film after the alloy is polished. The Ti-56 nickel (Ni) shape-memory
alloy is covered by TiO2-based oxide, with minimal amounts of Ni in both the oxide and metal-
lic states.6,7 The film on Ti-56Ni is a TiO2 containing Ni(OH)2, hydroxyl groups, and bound water.
In Ti-zirconium (Zr) alloy, the surface oxide film consists of titanium and zirconium oxides.8 The
relative concentration ratio of Ti to Zr in the film is almost the same as that in the alloy. The thick-
ness of the film increased with increase in Zr content. The chemical state of Zr is more stable than
that of Ti in the film.
12.3.3.3 Stainless Steel

Compositions of surface oxide films on stainless steels are well understood in the field of engi-
neering. In an austenitic stainless steel, the surface oxide film consists of iron (Fe) and chromium
containing a small amount of molybdenum. However, it does not contain nickel while in the air and
in chloride solutions.9,10
On the other hand, surface oxide film on 316L steel polished mechanically in deionized water
consists of oxide species of Fe, Cr, Ni, Mo, and manganese (Mn), and its thickness is about
3.6 nm.11 The surface film contains a large amount of OH− —the oxide which is hydrated or oxy-
hydroxidized. The surface oxide film is also enriched with Fe, while the alloy substrate just under
the film is enriched with Ni, Mo, and Mn.
12.3.3.4 Cobalt–Chromium Alloy

Surface oxide film of a Co-Cr-Mo alloy is characterized as containing oxides of cobalt and chro-
mium without molybdenum.12 On the other hand, the surface oxide film on another Co-Cr-Mo
alloy polished mechanically in deionized water consists of oxide species of cobalt, chromium, and
molybdenum, and its thickness is about 2.5 nm.13 This surface film contains a large amount of
OH− —the oxide which is hydrated or oxyhydroxidized. There are also more traces of Cr and Mo
distributed at the inner layer of the film. In the surface oxide on Co-Cr-Ni Mo alloy for metallic
stent known as MP35N, Cr is enriched and Co and Ni are depleted.14
12.3.3.5 Dental Precious Alloys

Au-copper (Cu)-Ag alloys and Ag-palladium (Pd)-Cu-Au alloys for dental restoration are covered
by copper oxide and silver oxide.15 An Ag-indium (In) alloy is covered by zinc oxide and indium
oxide, and an Ag-Sn-zinc (Zn) alloy is covered by tin oxide and zinc oxide. While these oxides serve
as a protection film against corrosion, they are not as chemically strong as the passive film.
12.3.4 Surface Active Hydroxyl Group

12.3.4.1 Formation
The surface of oxide reacts with moisture in air, and hydroxyl groups are rapidly formed. In the
case of Ti, the surface oxide immediately reacts not only with water molecules in aqueous solutions
but also with moisture in air and is covered by hydroxyl groups.16,17 The surface oxide is always
formed on conventional metallic biomaterials, and the surface of the surface oxide is active because
of the same reason described earlier. Therefore, the oxide surface immediately reacts with water
molecules and hydroxyl groups are formed, as shown in Figure 12.10a. The surface hydroxyl groups
contain both terminal OH and bridge OH in equal amounts. Concentration of hydroxyl groups on
the unit area of the surface is determined with various techniques.
12.3.4.2 Acidic and Basic Properties

Active surface hydroxyl groups dissociate in aqueous solutions and form electric charges, as shown
in Figure 12.10b.16–19 Positive or negative charge due to the dissociation is governed by the pH of
the surrounding aqueous solution: Positive and negative charges are balanced and apparent charge
is zero at a certain pH. This pH is the point of zero charge (pzc). The pzc is the unique value for
an oxide and an indicator that the oxide shows acidic or basic property. For example, in the case of
TiO2, the pzc of rutile is 5.3 and that of anatase is 6.2.16 In other words, anatase surface is acidic
H H Terminal OH
O H H
Bridge OH
+ O H δ+
O H
δ– δ+ δ– δ–
O Ti O O Ti O O Ti O
(a)
H+
OH–
OH O–
M+ M M
Smaller pH Point of zero Larger pH
charge (pzc)
(b)
FIGURE 12.10 Formation process of hydroxyl group on titanium oxide (a) and dissociation of the hydroxyl
group in aqueous solution and pzc (b).
at smaller pH and basic at larger pH than 6.2. Active surface hydroxyl groups and electric charges
formed by the dissociation of the groups play important roles for the bonding with polymers and
immobilization of molecules. Therefore, the concentration of surface hydroxyl group and pH are
important factors for the bonding with polymeric materials and immobilization of molecules.
12.4 ADHESION OF POLYMERS TO METALS IN DENTISTRY

12.4.1 Adhesive Reagents
Metal restorations and prostheses such as inlay, crown, and bridge must be retained in a fixed
position in a mouth. For this purpose, dental cements are used: glass ionomer cements, zinc phos-
phate cements, polyacrylate cements, resin-based cements, etc. In particular, resin-based cements
generate chemical adhesion between tooth and metal. Cements based on resin composites are now
used for cementation of crown and bridges and direct bonding of orthodontic brackets to enamel.
Polymer-based filling, restorative, and luting materials are specified in ISO 4049.
Resin cements based on methyl methacrylate (MMA) have been available since 1952 for the use
of cementation of inlays, crowns, etc. The resin composite for crown and resin was invented in the
early 1970s. Cementation of alloy restorations is performed with self-cured composite cements.
Two-paste systems are adopted for self-cured composite cements. One paste mainly consists of a
diacrylate oligomer diluted with lower molecular weight dimethacrylate monomers. The other con-
sists of silanated silica or glass. Peroxide amine is used for the initiator accelerator.
Although many efforts have been made to use industrial adhesives such as epoxy resins and
cyanoacrylate as dental adhesives, adequate adhesion is not achieved under the extreme condi-
tions of the oral cavity. Much effort has been expended to achieve adhesion between artificial
compounds and tooth substances. In 1978, Takeyama et al.20 synthesized a new dental adhesive
that meets the requirements stated here. The dental adhesives contained 4-methacryloxyethyl-
trimellitic anhydride (4-META) as an adhesive monomer with MMA. In 1983, Omura21 synthe-
sized an adhesive containing a dimethacrylate monomer bis-glycidyldimethacrylate (Bis-GMA)
and phosphate monomers. 4-META is an ethyl anhydride of trimellitic acid and hydroxyethyl
methacrylate and has both hydrophilic and hydrophobic groups, as shown in Figure 12.11. The
4-META cement is formulated with MMA monomer and acrylic resin filler and is catalyzed
by tributylborane (TBB). Another adhesive resin cement is phosphonate cement supplied with a
two-paste system, containing Bis-GMA resin and silanated quartz filler. Phosphonate molecule
is very sensitive to oxygen, so a gel is provided to coat the margins of a restoration until setting
has occurred.
CH3
CH2 C COOCH2 CH2 OOC CO

O
CO
(C4H9)3B(O)x
FIGURE 12.11 4-Methacryloxyethyl trimellitate anhydride (4-META) (top) and tri-butyl Borane (TBB)
(bottom).
One of the main problems in dental adhesion techniques is that bonds between adhesives and metals
are strong in a dry environment and weaken in a wet environment. For example, there are clinical reports
of dental adhesion bridges breaking off from teeth after extended use, and this has led to a loss of confi-
dence in dental adhesion techniques. Hence, a major problem awaiting solution in dentistry is the dura-
bility of bonding joints exposed to water. Researches on dental adhesives are well reviewed elsewhere.22
12.4.2 Bonding to Base Metal Alloys

The phosphate end of the phosphonate reacts with calcium of the tooth or with a metal oxide.
The double-bonded ends of both 4-META and phosphonate cements react with other double bonds
when available. Setting of resin cements results from self- or light-cured polymerization of carbon–
carbon double bonds.
There are numerous studies that have been made on the adhesion to dental alloys; pioneering
work by Tanaka on Ni-Cr alloys23 and gold alloys;24 studies on Ni-Cr alloys, Co-Cr alloys, and
precious metal alloys.25 There are several surface pretreatment methods for Ni-Cr alloys: immer-
sion in concentrated HNO3 solution after etching with HCl,23 immersion in solutions containing
an oxidizing agent after sandblasting, etching and passivating by an electrochemical method, and
spraying of molten metal on the alloy surface. These methods increase bond strength by increas-
ing the mechanical retention and chemical affinity between the adhesive and alloys. However, it is
necessary to distinguish the contribution of these two effects, mechanical and chemical, to evaluate
how the alloy surface structure improves the resin adhesion.
The adhesion of 4-META resin to Co-Cr and Ni-Cr alloys is examined by tensile test with and
without thermally induced stress.26 The resin bond to the as-polished surface of Co-Cr and Ni-Cr
alloys is stronger than that to the oxidized surfaces. The thermal cycles cause clear differences in
the surface states that affected the adhesion. The adhesive ability of 4-META resin to Ni-Cr alloy
improves remarkably when the alloy surface is treated by concentrated HNO3 solution. Adhesion to
the alloy surfaces treated by concentrated HNO3 is excellent, comparable with as-polished Co-Cr
alloy, and is resistant to and protects against thermal cycling using liquid nitrogen.
The effect of a thick water layer adsorbed on the top of oxide surfaces on the bonding ability
of 4-META resin is examined with a Co-Cr alloy and 18–8 stainless steel.27 The alloys are heated
500°C in air and dehydrated by heating to 700°C at 1 × 10 −4 Pa in silica glass tube. Dental adhesive
resin containing 4-META is bonded to these alloy surfaces, and after thermal cycle treatment the
bonding strengths and failure types for the dehydrated surfaces are compared with surfaces heated
at 500°C. In both alloys, the specimens oxidized at 500°C in air show partial interface (alloy/resin)
failure at the periphery. However, except for a few cases, dehydrated specimens display cohesive
failure and the bonding strength is similar to that of the as polished Co-Cr alloy, showing excellent
bonding ability. The bonding ability of the 4-META resin to the dehydrated oxide layer surface is
excellent when adhesion procedures are performed in an atmosphere excluding water vapor. The
cleaned metal surface obtained by hydrogen gas reaction shows excellent adhesive ability on Cr, Co,
and 304 type stainless steel, showing a passive oxide film on the metal substrates.28 Figure 12.12
shows the possible adhesion mechanism models of 4-META: Models (a), (b), and (c) are for the as-
polished surface and models (d), (e), and (f) are for the oxidized surface.
1
M MM 4-META PMMA
Alloy matrix
(a) 2
O M OH 4-META PMMA
Passive film 4
(b) 3
H 4-META PMMA
O M O
H
(c) Bond water 2
(M O M) OH 4-META PMMA
(d) Oxide layer 6
5
H 4-META PMMA
(M O M) OH O
(e) H 6
7 H
5 4-META PMMA
H O
(M O M) OH O H
(f ) H
FIGURE 12.12 Suggested adhesion model of PMMA resin containing 4-META on as polished (a, b, and c)
and oxidized (d, e, and f) surface: M is metal atom—is hydrogen bond. The chemical bonds that are similar in
bonding strength are labeled by the same number at the bonding position.
12.4.3 Durability in Water
Generally, joints of dental adhesive resin bonded to dental alloys weaken in a wet environment, although
adhesion is strong in a dry environment. Durability of the adhesion in a wet environment, such as
the oral cavity, is predominantly important in the bonding of dental adhesive materials to teeth and
dental alloys.
With aluminum/epoxy resin,29 aluminum/Li-polysulfone,30 and mild-steel/4-META resin31 in wet
environments, water molecules enter the adhesion interface by diffusion through the adhesive resin
rather than by passage along exposed bond lines. Polymethyl methacrylate (PMMA) film bonded by
4-META dental adhesive resin to mild steel is used as a specimen to study the mechanism of water
permeation into the adhesive interface. Water enters the interface by diffusion through the resin rather
than by passage along the interface. The water content penetrated to the interface is calculated from the
solution to Fick’s second equation.31 After 3 days of immersion, no change is observed on the mild steel
surface through the clear resin layer, but XPS analysis reveals the same chemical state as in the 2 week
immersion specimen.32 The hydrogen bonds appear to be destroyed with water penetrating through the
resin layer. This was followed by corrosion, resulting in a complete destruction of the adhesion interface.
The intensity of thermal stress due to thermal shock, calculated by the three-dimensional finite
element method, increases with increasing resin film thickness, indicating that the resin film sepa-
rates without degradation due to water when the resin film is thick. Total interface failure occurs
on specimens with resin thicker than 0.5 mm. The critical thermal shearing stress was calculated
as 22.5 MPa. When the adhesion interface degrades, the adhesion is broken by lower shear stresses
than the value for 0.25 mm layers, 16 MPa. Immersion time to reach equilibrium water content is
0.5 days for a 0.25 mm resin film.33
12.4.4 Bonding to Precious Alloys

4-META adheres to hydroxyl groups on metal surface as explained already, and noble metals and
precious alloys have a smaller number of hydroxyl groups on their surfaces. Therefore, the bond-
ing strength of 4-META with noble metals and precious alloys is weaker than base metal alloys.
The resins bond independently to dental precious metal alloys because they have low chemical
affinity for the precious metals. Several surface modification methods have been developed for
improvement of adhesion to dental alloys: high-temperature oxidation, immersion in oxidizing
agent, immersion in concentrated nitric acid,23 anodizing, electropolishing with tin, SiOx coating,34
and ion coating.35 The principle of these techniques is to form oxide layer on precious metal alloys.
However, these methods have drawbacks, such as complicated procedures, expensive equipment,
and degradation of chemical agent.
The other method to obtain bonding of 4-META to precious metal alloy is to design new alloys
containing In, Zn, and tin (Sn).36 The water durability and bonding strength of 4-META resin to
binary alloys of Au, Ag, Cu, or Pd containing In, Zn, or Sn are studied. With In in Au-based alloys,
the In2O3 on the alloy surface plays an important role in the adhesion with 4-META. To obtain
excellent adhesion, the element in an oxide with chemical affinity for 4-META must cover at least
50% of the alloy surface. The poor adhesive ability of 4-META resin to pure gold is considered to
be caused by chemisorbed H2O molecules on the surface.37 The adhesion ability of binary alloy was
improved by adding In, Zn, or Sn.
12.5 STENTS AND STENT GRAFTS

12.5.1 Stents
Stents and stent grafts are tubes used for dilatation to counteract decreases in vessel or duct
diameter and to maintain localized flow in stenotic blood vessels. Stents are also applied to ves-
sels of the bile duct, esophagus, and other passages for dilation. Therefore, elasticity or plastic-
ity for expansion and rigidity for the maintenance of dilatation and resistance to elastic recoil
are required. The expandability and plasticity of a balloon-expandable stent and the elasticity of
a self-expandable stent, as well as rigidity and resistance to the elastic recoil of blood vessels,
are required for stent materials. Conventional metals cover these properties when proper metals
are selected.
Stents are expandable tubes of metallic mesh that were developed to address the negative
sequelae of balloon angioplasty. At the beginning of the twentieth century, glass tubes that became
a prototype of stents were implanted into blood vessels of animals. In the next stage, the ori-
gin of percutaneous transluminal angioplasty (PTA), the expansion of blood vessels obtained by
increasing the diameter of catheter tube, was attempted.38 This trial failed because of migration
and the development of thrombus. In 1985, Palmaz and his colleagues developed the first balloon-
expanded stent,39 and just 1 year later, Gianturco developed a balloon-expanded coil stent.40 These
stents were made of stainless steel. The first self-expandable stent, Wallstent®, made of a Co-Cr
alloy (Elgiloy), was clinically applied in 1986.41 Another popular self-expandable stent, SMART®,
consists of a superelastic Ni-Ti alloy.42 Since the 1990s, stents have been used in coronary arteries.
Typical popular stents for this purpose are the Palmaz-Schatz® stent and the Gianturco-Roubin®
stent. New designs and functions of stents have also been developed. Today, for example, the
majority of patients undergoing percutaneous transluminal coronary angioplasty (PTCA) receive
a stent. Since the mid-1990s, stents have also been applied to the treatment of cerebrovascular
disease, and carotid stenting to prevent stroke, recoil, and restenosis has been attempted.43–45 In
addition, the first treatment for abdominal aortic aneurysms with stent grafts started in the 1990s.46
Stent grafts are generally constructed of stainless steel or a Ni-Ti alloy and coated with compounds
such as expanded polytetrafluoroethylene (ePTFE) that is a metal–polymer composite. Typical
bare stent and stent graft are shown in Figure 12.13.
Metals are the main materials utilized for stents because of their mechanical properties and
visibility by X-ray imaging. Metallic stents and stent grafts are now important devices in noninva-
sive medicine. New techniques for treatments using metallic stents are continuously being devel-
oped, and such development will continue into the foreseeable future. While pioneering ideas for
FIGURE 12.13 (See companion CD for color figure.) Examples of bare metallic stent and stent graft.
treatment are constantly being announced, the development of proper materials for stents is back-
ward from the viewpoint of materials science.
12.5.2 Drug Eluting Stents

Initial complications associated with stenting generally involve subacute stent thrombosis, which
occurs in 1%–3% of patients within 7–10 days. The major long-term complication is in-stent reste-
nosis, which occurs within 6 months in 50% of patients. Although it is unknown whether the origi-
nal cause of these problems is the metal from which the stents are made, the use of polymer-coated
drug-eluting stents (DESs) is increasing.47–49
The most promising results have been attained with polymer-coated DESs1 and DES is one
of the metal–polymer composites. Two of the drugs currently in clinical trials are rapamycin
and paclitaxel. These drugs are embedded in a polymer matrix (such as a copolymer of poly-n-
butyl methacrylate and polyethylene-vinyl acetate or a gelatin–chondroitin sulfate coacervate
film) that is coated onto the stent. The drug is related by diffusion and/or polymer degrada-
tion over varying periods of time that can be engineered by the specifics of the polymer-drug
system. These coated stents have had excellent initial success, virtually eliminating restenosis
over time periods of 2 years and longer and are felt to represent a major breakthrough in the
treatment of coronary artery disease. Commercial DESs and their structures are summarized
in Table 12.4.
In DES, chemicals such as immunosuppressants are coated on metal frames. Because direct
immobilization of drugs to metals or coating of the polymers and ceramics impregnating the
drugs is necessary to control their release rate, attempts have been made to coat the photoset-
ting gelatin impregnating the drug.50 To improve the biofunction of stents, a stent coated with
a phospholipid-like 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer to inhibit the
adsorption of proteins and the adhesion of platelets has been developed.51 For the same purpose,
the immobilization of poly(ethylene glycol) (PEG) and compounding with medical polymers
are effective.52
In DESs, all fractures occurred around areas of increased rigidity due to the overlapping of
metals, which may have formed a fulcrum for metal deformation due to vessel movement.53
TABLE 12.4
Various Drug Eluting Stents
Coating Stent
Manufacturer Brand Name Drug Material Name Material
Johnson & Cypher™ Sirolimus Nonabsorbable polymer Bx Velocity 316L stainless steel
Johnson
Boston Scientific Taxus™ Paclitaxel Nonabsorbable polymer Express 2 316L stainless steel
Medtronics Endeavor™ Zotarolimus PC coating Driver Co-Cr alloy
Abbot/Boston
Scientific Xience™ V Everolimus Acryl/fluorine polymer ML-Vision Co-Cr alloy
Biosensors/Termo Nobori™ Biolimus Absorbable polymer (PLA) S-Stent 316L stainless steel
12.5.3 Stent Grafts

The other metal–polymer composite is stent graft. Stents for treatment of peripheral vascular
disease are generally constructed of stainless steel or nitinol and may be coated with com-
pounds such as ePTFE. Stent grafts are composed of a metallic frame covered by a fabric
tube and combine the features of stents and vascular grafts; they can be deployed endovascu-
larly. Stent grafts are used to treat aortic aneurysms, where the aortic wall has been weakened
and threatens to rupture, as well as stenosis of other arterial sites. The graft portion, usually
composed of polyester or ePTFE, can sit on either the luminal or abluminal (outside) aspect of
the metallic stent and is intended to provide a mechanical barrier to prevent intravascular pres-
sure from being transmitted to the weakened wall of the aneurysm, thus excluding the aneurysm
from the flow of blood. These stents and stent grafts are developed in a similar manner to those
in the coronary circulation, either as self-expanding units or covering an inflatable balloon.
The stent is used for a given application unit or over an inflatable balloon. The stent used for a
given application is selected by diameter, length, and geometry of the lesion and location of side
braches or branch points.
Current synthetic vascular grafts are typically fabricated from poly(ethylene terephthalate)
(Dacron) or ePTFE, with the Dacron graft being used for larger vessel applications and the ePTFE
to bypass smaller vessels. These grafts can be made porous to enhance healing, but they are then
impregnated with connective tissue proteins to aid clotting, reduce the blood loss through the pores
of the graft upon implantation, and stimulate tissue ingrowth, and with antibiotics to reduce the risk
of infection of the grafts that are not impregnated, which need to be preclotted with the patient’s own
blood for this same purpose.
In light of the complications associated with vascular grafts, current research has focused on
improvement of synthetic vascular grafts and on alternatives such as tissue-engineered blood
vessels. The purpose of obtaining the luminal surface is as follows: (1) prevent coagulation, (2)
prevent platelet adhesion/aggregation, (3) promote fibrinolysis, (4) inhibit smooth muscle cell adhe-
sion/proliferation, and (5) promote endothelial cell adhesion and proliferation. Endotherialization
of the entire graft may also help prevent bacterial attachment to the graft and subsequent infection.
Engineering an artery muscle and endotherial cells is a promising approach to solving the problem
of developing an adequate small-diameter vascular graft.54,55
Severe pitting and crevice corrosion originating from crevice corrosion are observed in Ni-Ti
alloys in stent grafts, as shown in Figure 12.14.56 These problems may occur in Ni-Ti alloys and
stainless steel at crevices between an artificial blood vessel and a metallic stent because of the elec-
trochemical properties of the alloys. Corroded sites may then become the initiation site of a fracture.
Alloys that are resistant to pitting and corrosion must be developed and used.
FIGURE 12.14 Severe crevice corrosion observed in Ni-Ti alloy used as stent graft for 6 months. (From
Heintz, C. et al. 2001. Corroded Nitinol wires in explanted aortic endografts: An important mechanism of
failure? J Endovasc Ther 8: 248–253.)
12.6 IMMOBILIZATION OF POLY(ETHYLENE GLYCOL)

12.6.1 Poly(Ethylene Glycol)
PEG is an oligomer or polymer of ethylene oxide, but historically PEG has tended to refer to oligomers
and polymers with a molecular weight below 20,000. PEG has the structure shown in Figure 12.15.
PEGylation is the act of covalently coupling a PEG structure to another larger molecule, for example,
a therapeutic protein (which is then referred to as PEGylated). PEG is soluble in water, methanol,
benzene, and dichloromethane and is insoluble in diethyl ether and hexane. It is coupled to hydro-
phobic molecules to produce nonionic surfactants. This property, combined with the availability of
PEGs with a wide range of end functions, contributes to the wide use of PEGs in biomedical research:
drug delivery, tissue engineering scaffolds, surface functionalization, and many other applications.57
12.6.2 Chemical Immobilization
The immobilization of biofunctional polymers on noble metals such as Au is usually conducted by
using the bonding –SH or –SS– group; however, this technique can only be used for noble metals.
The adhesion of platelets and adsorption of proteins, peptides, antibodies, and DNA are controlled
by modifications of this technique. On the other hand, PEG is a biofunctional molecule on which
adsorption of proteins is inhibited. Therefore, immobilization of PEG to metal surface is an impor-
tant event to biofunctionalize the metal surface. Examples of immobilization of PEG to oxide surface
are shown in Figure 12.16. A class of copolymers based on poly(L-lysine)-g-poly (ethylene glycol),
PLL-g-PEG, has been found to spontaneously adsorb from aqueous solutions onto TiO2, Si0.4Ti0.6O2,
and Nb2O5 to develop blood-contacting materials and biosensors.52,58 In another case, TiO2 and Au
surfaces are functionalized by the attachment of PEG-poly(DL-lactic acid), PEG-PLA, and copoly-
meric micelles. The micelle layer can enhance the resistance to protein adsorption to the surfaces up
H H
H O C C O H
H H n
FIGURE 12.15 Chemical structure of PEG.

Ψ
O N
HN S
Ψ O O
Heterofunctional O N
cross linker
Ψ S
O
S
N O
Heterofunctional NH
cross linker O O O Ψ Grafted PEG
O N
NH
S
O
O
Si O O
HN O N O
Interpenetrating
Si H polymer network
H2N O O N
Aminosilane
Si Si O
Si O O O
O
Silane
TiO2 TiO2
(a) (b)
O O O O
O O
O O
O Ψ
O O
O O O
O O O
O x x x
O O O O
O
O O
O O
O O O O O O
PEG O O O
O O O
O O O O O O O O
O
O O O
O O O
O O O C
O C
NH NH NH
Poly-L-lysine O O
N CH C N CH C N CH C N CH C N CH C O Functionalized
H H H H H alkane
O O O
O O O O O O O O
phosphate P P P P
+O + +O
NH3 NH3 O O+ +
O O+ +O
O
+
+ +
TiO2 TiO2
(c) (d)
FIGURE 12.16 Various techniques for immobilization of PEG to titanium oxide. (From Xiao, S.J. et al.,
Biochemical modification of titanium surfaces, in: D.M. Brunrtte, P. Tenvall, M. Textor, and P. Thomsen,
eds., Titanium in Medicine, Springer, Amsterdam, the Netherlands, p. 421, 2001.) (a) Silanization using
APTES followed by covalent attachment of maleimide; (b) silanized surface followed by formation of a
non-adhesive P(AAm*co*EG/AA); (c) polycationic poly(amino acid) grafted with poly(ethylene grycol) side
chain; (d) self-assembled monolayers of long-chain alkanephosphate.
to 70%.59 A surface of stainless steel was first modified by a silane coupling agent, SCA, (3-mercap-
topropyl)trimethoxysilane. The silanized stainless steel, SCA-SS, surface was subsequently activated
by argon plasma and then subjected to UV-induced graft polymerization of poly(ethylene glycol)
methacrylate, PEGMA. The PEGMA graft-polymerized stainless-steel coupon, PEGMA-g-SCA-SS,
with a high graft concentration and, thus, a high PEG content was found to be very effective to pre-
vent the absorption of bovine serum albumin and γ-globulin.60 These processes require several steps
but are effective for immobilization; however, no promising technique for the immobilization of PEG
to a metal surface has been so far developed. Photoreactive PEG is photoimmobilized on Ti.61
12.6.3 Electrodeposition
Both terminals of PEG (MW: 1000) are terminated with –NH2 (NH2–PEG–NH2), but only one ter-
minal is terminated with –NH2 (NH2–PEG). The cathodic potential is charged to Ti from the open
circuit potential to −0.5 V versus an SCE and maintained at this potential for 300 s. During charging,
the terminated PEGs electrically migrate to and are deposited on the Ti cathode, as shown in Figure
12.17. Not only electrodeposition but also immersion leads to the immobilization of PEG onto a Ti
surface. However, more terminated amines combine with Ti oxide as an NH-O bond by electrode-
position, while more amines randomly exist as NH 3+ in the PEG molecule by immersion (Figure
12.18).62,63 A scanning probe microscopic image is shown in Figure 12.19. The amounts of the PEG
layer immobilized onto the metals are governed by the concentrations of the active hydroxyl groups
on each surface oxide in the case of electrodeposition, which is governed by the relative permittivity
of the surface oxide in the case of immersion.64 The PEG-immobilized surface inhibits the adsorption
of proteins and cells, as well as the adhesion of platelets65 and bacteria66 (Figure 12.20), indicating
O–
NH+3
O–
O– NH+
3
Ti O–
O– NH+
3
O–
O– PEG
FIGURE 12.17 Attraction of PEG with positively charged terminal to cathodic Ti surface by electrodeposition.
–NH oriented to
titanium
Random direction Electro-
Immersion NH+3 . . . O– deposition
Unstable bonding NH–O
Stable bonding
NH2-PEG-NH2 NH2-PEG-NH2
immersion NH2-PEG
electrodeposition
electrodeposition
NH+3 NH+3
NH+3 NH+3 NH+3
NH+3 NH+3
NH+3
NH+3 NH+3 NH+3 NH NH NH NH NH NH NH NH NH NH NH NH NH
NH+3 NH+3 NH+3 NH+3
O–
O– O– O–
O –
O– O– O O O O O O O O O O O O O O
Ti Ti Ti
Random Brudh-shape U-shape
FIGURE 12.18 Schematic model of the deposition manner and chemical bonding state of PEG by immer-
sion and electrodeposition.
60.00
(nm)
0.00
0.20 0.80
0.40 0.60
0.60 0.40 60.00

(nm)
0.80 0.20
0.00
1000.00 × 1000.00 (nm) Z 0.00–60.00 (nm) 0.00
kshakeB-PEG
FIGURE 12.19 (See companion CD for color figure.) Scanning probe microscopic image of electrodepos-
ited PEG to Ti surface.
(a1) (b1)
15.0 kV 9.9 mm × 2.00 k SE 20.0 μm 15.0 kV 10.0 mm × 2.00 k SE 20.0 μm
(a2) (b2)
20 μm
15.0 kV 10.1 mm × 2.00 k SE 20.0 μm 15.0 kV 10.1 mm × 2.00 k SE 20.0 μm
FIGURE 12.20 Platelet adhesion and fibrin network formation (a1) and bacterial adhesion (a2) are active on
Ti, while they are inhibited on PEG-electrodeposited Ti surfaces (b1 and b2).
that this electrodeposition technique is useful for the biofunctionalization of metal surfaces. It is also
useful for all electroconductive materials and materials with complex surface topography.
12.7 IMMOBILIZATION OF BIOMOLECULES

12.7.1 Metal–Biomolecule Interface
Organic coatings have been scarcely used on metallic implants. Nevertheless, it is expected
that these materials will significantly improve their market share as improved biocompatibility
becomes the decisive criterion for patients and surgeons in the future. This approach, which is
based on the latest medical and cellular biological results, employs biopolymers (proteins) that
have been immobilized on the surface of metallic implants. The intention is to reduce the region
character of the implant for the body. This is accomplished by coating with substances that are
normally found on the surface or in the vicinity of the tissue that has to be substituted by the
implant. It has been found that these coatings act as local mediators of cell adhesion and, in conse-
quence, as a stimulating factor for the growth and proliferation of the cells normally found around
the substituted tissue. The tight attachment at the oxide-coated surface of the metallic implant and
the conservation of the biological function of the proteins involved are prerequisites for obtaining
these highly desirable properties.
Since the natural environment around the implant is aqueous while the surface of the implant
is either bare or oxidized metal, specific demands are imposed on the coating to mediate success-
fully between these different structural entities. The purpose of these demands is to obtain the
native conformation of all proteins and cells that are in contact with the coating and to avoid all
forms of aggregation and other conformational changes that might lead to protein denaturaliza-
tion or cell death.
One approach is the immobilization of biological molecules (growth factors, adhesive pro-
teins) onto the implant surface in order to induce a specific cellular response and promote osseo-
integration. The application of large extracellular matrix proteins, however, can be unpractical
due to their low chemical stability, solubility in biological fluids, and high cost. In addition,
entire ECM molecules are usually of allogenetic or xenogenetic origin and thus associated with
the risk of immune reaction and pathogen transfer. To immobilize biomolecules to metal sur-
faces, the following techniques are used: modification through silanized titania (thiol-directed
immobilization, amino- and carboxyl-directed immobilization), modification through photo-
chemistry, electrochemical techniques, and chemical modifications based on self-assembled
monolayers.
The immobilization of biomolecules to a metallic surface can be achieved using self-assembled
monolayers as cross-linkers. Self-assembled monolayers provide chemically and structurally
well-defined surfaces that can often be manipulated using standard synthetic methodologies.67 Thiol
on self-assembled monolayers68,69 and siloxane-anchored self-assembled monolayers70 has been
particularly well studied. A problem related to the application of immobilized biomolecules via
silanization techniques is the hydrolysis of siloxane films when exposed to aqueous (physiological)
conditions.71 More recently, alkyl phosphate films that remain robust under physiological condi-
tions72 have been used to provide an ordered monolayer on tantalum oxide surfaces,73,74 and
alkylphosphonic acids have been used to coat the native oxide surfaces of metals and their alloys
inducing iron,75 steel,76 and Ti.77
12.7.2 Peptides
In a living tissue, the most important role played by the extracellular matrix has been highlighted
to favor cell adhesion.78 Studies have shown that interactions occur between cell membrane
Electrodeposition Ti
of PEG zwitterion
Potentiostat
– +
Ti
Pt
Ti SCE PEG/Ti
–3 V, 15 min, 310 K
24 h, 310 K Ti Ti
Immobilization of RGD/Ti RGD/PEG/Ti
RGD with immersion
FIGURE 12.21 PEG twitter ion is electrodeposited to Ti firstly and RGD is immobilized on the PEG.
receptors and adhesion proteins (or synthetic peptides) derived from the bone matrix, such as
type I collagen or fibronectin.79 These proteins are characterized by an RGD (Arg-Gly-Asp)
motif with special transmembrane connections between the actin cytoskeleton and the RGD
motif, and the whole system can activate several intracellular signaling pathways modulating
cell behavior (e.g., proliferation, apoptosis, shape, mobility, gene expression, and differentia-
tion).80 Due to the main role of the RGD sequence in cell adhesion, several research groups
have developed b iofunctionalized surfaces by immobilization of RGD peptides. Grafting RGD
peptides has been performed on different biomaterials, such as Ti,81–83 and has been shown to
improve osteoconduction in vitro. Methodologies differ by the conformation of RGD (cyclic or
linear) and by the technique used for peptide immobilization.78,79,82–84 Since the graft of an RGD
peptide is known to be efficient in bone reconstruction,85 the challenge is to develop simple and
cheap methods to favor cell anchorage on biomaterial surfaces.83,84
Self-assembled molecular monolayers bearing RGD moieties have been grafted to numer-
ous surfaces, using either silanes,86 phosphonates on oxidized surfaces,83 or thiols on Au84 but
have analyzed some application problems for large-scale production. Phosphonates are known
to adsorb on Ti. To be mechanically and physiologically stable, phosphonate layers have to be
covalently bound to the material surface by using drastic conditions77,87 (anhydrous organic sol-
vents, high temperature), which are not compatible with biomolecule stability. Monolayers of
RGD phosphonates have been achieved using a complex multistep process, which necessitates
tethering a primer onto Ti surface, then a linker, and finally the peptide.88 To immobilize RGD
to the electrodeposited PEG on Ti, PEG with an −NH2 group and a −COOH group (NH2−PEG−
COOH) must be employed. One terminal group, −NH2, is required to bind stably with a sur-
face oxide on a metal. On the other hand, the other terminal group, −COOH, is useful to bond
biofunctional molecules such as RGD, as shown in Figure 12.21.89 This RGD/PEG/Ti surface
accelerates calcification by MC3T3-E1 cell.90 The calcification is the largest on the RGD/PEG/Ti
surface (Figure 12.22).
Glycine (G)-arginine (R)-glycine (G)-asparaginic acid (D)-serine (S) sequence peptide, GRGDS
peptide, is coated with chloride activation technique to enhance adhesion and migration of osteo-
blastic cells.91 The expression levels of many genes in MC3T3-E1 cells are altered.
PEG
Ti Ti Ti
RGD/PEG/Ti RGD/Ti Ti
FIGURE 12.22 Calcification (dark regions) by MC3T3-E1 cells is more active on RGD/PEG/Ti specimen
than on RGD/Ti and Ti. Scale bar represents 5 mm.
12.7.3 Protein and Collagen

Among the relevant molecules involved in biochemical modification of bone-contacting surfaces,
growth factor, such as bone morphogenetic protein-2 (BMP-2), is of primary interest. BMP-2 has
been known to play an important role in bone healing processes and to enhance therapeutic effi-
ciency. Ectopic bone formation by BMP-2 in animals has been well established following the first
reports of BMP-2 by the Urist research group.92–94 Synthetic receptor binding motif mimicking
BMP-2 is covalently linked to Ti surfaces through a chemical conjunction process.95 A complete
and homogeneous peptide overlayers on the Ti surfaces; the content is further measured by gamma
counting. Biological evaluations show that the biochemically modified Ti were active in terms of cell
attachment behavior. Ti surfaces can enhance the rate of bone healing as compared with untreated
Ti surface. Bone morphogenetic protein-4 (BMP-4) is immobilized on a Ti-6Al-4V alloy through
lysozyme to improve the hard tissue response.96 Proteins are silane coupled to the oxidized surfaces
of the Co-Cr-Mo alloy, the Ti-6Al-4V alloy, Ti, and the Ni-Ti alloy to improve tissue compatibility.97
Biomolecules are also used to accelerate bone formation and soft tissue adhesion on a material.
Type I collagen is immobilized by immersion in the collagen solution.98 Type I collagen production
increases with modification by ethane-1,1,2-triphosphonic acid and methylenediphosphonic acid
grafted onto Ti.99 Type I collagen is grafted through glutaraldehyde as a crosslinking agent.100 For
electrodeposition, it is found that an alternating current between −1 V and +1V versus SCE with 1 Hz
is effective to immobilize type I collagen to Ti and durability in water is high.101
Fibronectin is immobilized directly on Ti using the tresyl chloride activation technique.102
L-threonine and O-phospho-L-threonine are immobilized on acid-etched Ti surface.103
12.7.4 Hydrogel and Gelatin

Immobilization or coating of hydrogel to metal surface is currently attempting to add a drug
delivery ability to orthopedic implant and stents or fluorescent sensing ability to microchips.
Currently, synthetic polymeric hydrogels like poly(hydroxyethylmethacrylate) (pHEMA) and
poly(hydroxyethylacrylate) (pHEA) are widely used as compliant materials particularly in the case
of contact with blood or other biological fluids.104 Despite hydrogel’s good flexibility in the swollen
state, hydrogels usually lack suitable mechanical properties, and this could greatly impair their use
as coating materials for surgical procedure. Moreover, in case of inadequate adhesion between the
hydrogel coating and the metal surface, a breakage at the coating–steel interface might occur.105
A spray-coated method has been set up with the aim to control the coating of pHEMA onto the
complex surface of a 316L steel stent for percutaneous coronary intervention (PCI).106 The pHEMA
coating evaluation of roughness wettablity together with its morphological and chemical stability
after three cycles of expansion-crimping along with preliminary results after 6 months demon-
strates the suitability of the coating for surgical implantation of stent.
An alternative promising synthetic route is represented by electrochemical polymerization,
which leads to thin film coatings directly on the metal substrates with interesting applications either
for corrosion protection or for the development of bioactive films.107–110 As far as the orthopedic
field is concerned, in recent years, many procedures based on surface modification have been sug-
gested to improve the biocompatibility and biofunctionality of Ti-based implant.111 2-Hydroxy-
ethyl-methacrylate (HEMA), a macromer poly(ethylene-glycol diacrylate)(PEGDE), and PEGDE
copolymerized with acrylic acid were used to obtain hydrogels. A model protein and a model drug
were entrapped in the hydrogel and released according to pH change.112
12.8 OTHER METAL–POLYMER COMPOSITES

12.8.1 Bonding of Polymers with Metal through Silane Coupling Agent
The interfacial chemical structure governing the bonding strength, especially at the nanometer
level, is one of the most challenging aspects of the development of composite materials. The
combination of a Ti alloy with a resin for crown facings has been attempted.113 In particular,
SCAs containing S-H groups and Si-O-CH3 groups are comprehensively used to combine den-
tal alloys with resins.114 The S-H groups work as a bonding agent with polymers; the Si-O-CH3
works as a bonding agent with metals. The mechanical properties and durability of composite
resin increase with silanized filler.115–118 However, in most studies about materials using SCAs
in the field of dentistry, only the bonding strength is evaluated and discussed, and there are few
reports that examine and discuss the chemical structures at the bonding interface and how they
influence the bonding strength. Other studies on SCAs to combine polymers with metals have
been performed in other fields. An aluminum–vegetable oil composite using a SCA has been
developed.119,120 Rubber-to-metal bonding by a SCA was investigated.121 In addition, the surface
modification of stainless steel by grafting of PEG using a SCA has been reported.60 However,
only the chemical structure is investigated in these studies. In other words, the relationship
between the bonding strength and the interfacial chemical structure containing a SCA layer has
not been studied.
The unequivocal relationship between the shear bonding strength and the chemical struc-
ture at the bonding interface of a Ti-segmentated polyurethane (SPU) composite through a SCA
(γ-mercaptopropyl trimethoxysilane [γ-MPS]) is investigated.122 Schematic models of the fractured
region before and after the shear bonding test in the case of a thin γ-MPS layer and a thick γ-MPS
layer are shown in Figure 12.23. On the other hand, the shear bond strength of the Ti/SPU interface
increased with ultraviolet (UV) irradiation according to the increase in the cross-linkage in SPU.
Platelet adhesion to Ti is inhibited by SPU, as shown in Figure 12.24. This technique is used for
the creation of a new meta-based material with high strength, high toughness, and biofunction. UV
irradiation to a Ti-SPU composite is clearly a factor governing the shear bond strength of the Ti/
SPU interface.123 In addition, active hydroxyl groups on the surface oxide film are clearly factors
governing the shear bond strength.124 After a good bonding between metal and polymer is pro-
duced, biofunctionalization techniques developed in the field of polymers could be applied to the
composite materials.
12.8.2 Polymers Condensed in Porous Titanium

The Young’s modulus of metallic materials is relatively larger than that of cortical bone: about
200 GPa in stainless steels and Co-Cr-Mo alloys, about 100 GPa in Ti and Ti alloys, and 15–20 GPa
in cortical bone. When fractured bone is fixed with metallic bone fixator such as bone plate and
Hard
Polyurethane group segment Soft
segment
γ-MPS
SPU SH SH
Ti O Si O Si O Si O
Thin γ-MPS O O O
(a)
γ-MPS
SPU
SH SH SH SH
Ti
Thick γ-MPS
Si O Si O Si O Si O
O O O O
(b)
FIGURE 12.23 Schematic model of the fractured region before and after the shear bonding test in the case
of a thin ã-MPS layer (a) and a thick γ-MPS layer (b).
screws and bone nail, during healing a load to fixation part is mainly received by metallic fixators
because of the difference in their Young’s modulus. This phenomenon is well known as the so-
called “stress shielding” in orthopedics. This large Young’s modulus generates other problems.
When a metal is used as a metallic spacer in spinal fixation, the spacer is mounted in matrix bone. In
the case of a dental implant, occlusal pressure is not absorbed by the implant and directly conducts
to jaw bone.
To solve these problems, metals with low Young’s modulus are required. Two approaches are
feasible: the decrease in the Young’s modulus of a metallic material itself and decrease in the apparent
Young’s modulus by forming a porous body. In the latter case, the pores are sometimes filled by poly-
mers to control the apparent Young’s modulus. UHMWPE125 and PMMA126 are attempted to fill the
pores in porous Ti. Figure 12.25 shows porous Ti whose pores are filled by UHMWPE.
12.8.3 Metallization of Polymers
The properties of polymer surfaces with regard to their chemical, electrical, mechanical, and other
properties are modified by metallization, and this technique could be applied to biomaterials.127 In
the metallization process, the metal atoms arrive as mobile individuals at a polymer surface. The
metal layer formation on polymer will be influenced by the type and strength of metal–polymer
interaction and the structure of the polymer. Metallization techniques are categorized by evapora-
tion, sputtering, chemical vapor deposition, and electrochemical deposition.
Ti SPU
10 μm 10 μm
10.0 kV 12.6 mm × 5.00 k SE 10 μm 10.0 kV 14.9 mm × 5.00 k SE 10.0 μm
Fibrin network No platelet adhesion
Ti-SPU composite
10 μm
10.0 kV 10.3 mm × 5.00 k SE 10.0 μm
No platelet adhesion
FIGURE 12.24 Platelet adhesion on Ti is inhibited by SPU layer.
FIGURE 12.25 Porous Ti whose pores are filled by UHMWPE after bending test.
A number of conditions must be determined to accomplish metal penetration into polymers. The
metal deposition rate must be low enough to prevent metal cluster formation at the polymer surface
in an initial stage of the metallization process. The metal bonds in such clusters immobilize the
atoms, and a strong interaction between the metal atom and polymer chain reduces the diffusion.
The metal atoms must not react with the polymer. The polymer substrate temperature must be well
above the glass transition of the polymer surface region. The polymer segment motions assist the
metal atom transport deep into the material.
The diffusion front of a metal is not smooth, but it is rough and creates mechanical interlocking,
which in turn enhances the mechanical strength between metal overlayer and polymer. The mechani-
cal properties increase in the composite region compared with the pure polymer. Electric conductivity
may be affected and the thermal expansion coefficients are matched to some extent by interphase.
12.9 CONCLUSIONS
Metallic materials are widely used in medicine for not only orthopedic and dental implants but also
cardiovascular devices and other purposes. The metal surface may be biofunctionalized by various
techniques such as dry and wet processes, the immobilization of biofunctional molecules, and the
creation of metal–polymer composites. These techniques make it possible to apply metals to a scaf-
fold in tissue engineering. Artificial materials such as metal–polymer composites will continue to
be used as biomaterials in the future, because of their excellent biocompatibilities and biofunctions.
Some metal–polymer composites are reviewed in other textbooks.127–129
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13 Evolution of Current
and Future Concepts
of Biocompatibility Testing
Menno L.W. Knetsch
CONTENTS
13.1 Introduction: Biocompatibility Testing of Medical Devices................................................. 378
13.1.1 History of Medical Devices and Biocompatibility Testing....................................... 379
13.1.2 Biocompatibility Regulations.................................................................................... 380
13.1.3 Definition of Biocompatibility................................................................................... 383
13.2 Biocompatibility Testing........................................................................................................ 384
13.3 In Vitro Biocompatibility Testing.......................................................................................... 385
13.3.1 Control Materials....................................................................................................... 386
13.3.2 Cytotoxicity............................................................................................................... 386
13.3.2.1 Indirect Testing........................................................................................... 387
13.3.2.2 Direct Contact Testing................................................................................ 389
13.3.2.3 Cell Viability Assays.................................................................................. 390
13.3.2.4 Evaluation Cell Viability or Cytotoxicity................................................... 393
13.3.3 Cell Proliferation In Vitro.......................................................................................... 393
13.3.4 Cell Function In Vitro................................................................................................ 394
13.3.4.1 Evaluation of In Vitro Functional Biocompatibility Tests.......................... 396
13.3.5 Genotoxicity.............................................................................................................. 396
13.4 Ex Vivo Biocompatibility Testing......................................................................................... 399
13.5 In Vivo Biocompatibility Testing: Introduction....................................................................400
13.5.1 Short History of In Vivo Biocompatibility Testing................................................... 401
13.5.2 In Vivo Biocompatibility Testing.............................................................................. 401
13.5.2.1 Local and Systemic Toxicity.......................................................................402
13.5.2.2 Sensitization................................................................................................402
13.5.2.3 Irritation......................................................................................................402
13.5.2.4 Systemic Toxicity........................................................................................403
13.5.2.5 Acute Systemic Toxicity.............................................................................404
13.5.2.6 Subchronic and Chronic Systemic Toxicity................................................405
13.5.2.7 Local Implant Effects.................................................................................405
13.5.3 Functionality of Medical Devices In Vivo: Choice of Model...................................406
13.5.4 Future of In Vivo Testing...........................................................................................407
13.6 In Silico Testing.....................................................................................................................408
13.7 Example: Biocompatibility Testing of a Central Venous Catheter........................................ 410
References....................................................................................................................................... 411
377
13.1 INTRODUCTION: BIOCOMPATIBILITY TESTING OF MEDICAL DEVICES

The clinical success of medical devices is ultimately determined by their biocompatibility.
Medical devices have to possess all the required characteristics to ensure clinically acceptable
biocompatibility. The definition of biocompatibility that is most commonly used these days
was formulated by D. Williams some years ago as “The ability of a material to perform with
an appropriate response in a specific application” [1,2]. This definition takes into account that
a biomaterial does not only have a passive, supportive, or mechanical function but also has an
important role in guiding the host response toward the biomedical implant. This means that
the biofunctionality of the implant is an intricate characteristic of the device. It is unavoidable
that the body reacts to a newly implanted material with a wide range of defense mechanisms.
This response is known as the foreign body response [3]. Over the past decades, different strat-
egies to obtain acceptable host response to medical implants have been applied. These range
from trying to achieve perfect inertness to implants with highly bioactive or biofunctionalized
surfaces to actively induce a desired host-tissue response. There has been success with many
biomedical implants, but most of these do not carry bioactivity in or on the device [4]. Clinical
practice demonstrates that design of a truly bioactive implant is difficult since the exact host-
response cannot be predicted. The way in which the tissue will respond to the invading foreign
material is dependent on many factors and differs between patients significantly. After a while,
a fragile dynamic equilibrium is reached between the materials and the surrounding tissue. The
implanted materials will perform well until a slight change in one of the interaction parameters,
causing a series of events that will lead to the disturbance of the delicate equilibrium and may
result in failure of the device. This also means that the variation between the treated patients
makes prediction of the performance of a biomaterial virtually impossible. For instance when
considering hip replacement, the quality of the bone in which the stem and cup are fixed are of
critical importance for the clinical outcome of the device. One can easily imagine that patients
suffering from osteoporosis may have a poorer clinical outcome than patients who do not suf-
fer from this bone disease [5]. Another example that demonstrates the effect of, on first sight,
minor details is the performance of mechanical heart valves. The thrombotic complications of
metallic and polymeric parts of this medical device are suppressed by anticoagulants and/or
antiplatelet drugs. A simple visit to the dentist, e.g., to remove a tooth, will require the reduc-
tion of this anticoagulant therapy for a short while. The risk of thrombosis will increase in this
short period, although the materials do not change. There is a significant change in one of the
parameters that determines the delicate equilibrium between the blood and the blood contacting
surfaces of the mechanical valve. Also for patients that have received a coronary stent, patient
compliance concerning the anticoagulation therapy has been shown to be the cause of late-stent
thrombosis, leading to failure of the device and in worst cases death [6,7]. These examples show
that reduction in the number of parameters determining the tissue-implant equilibrium can be
beneficial for the clinical success of the medical device. In some cases this means that biofunc-
tionalization of biomaterial surfaces is a viable strategy to increase the long-term performance.
In the cases of the stents and heart valves, a coating that would inhibit/suppress surface-induced
blood coagulation will not only decrease side effects, like prolonged bleeding, but also would
prevent failure of the device because of poor patient compliance concerning the anticoagulation
therapy. Therefore, the introduction of bioactivity in theory is a useful characteristic of any bio-
material. We should however not forget that next to the practical problems concerning bioactive
biomaterials (sterilization, costs, limited shelf life, storage conditions, etc.), the intended bio-
logical response to the implant may differ considerably between patients. Even after thorough
testing, the intended functionality of the implant may vary because of the subtle differences of
the tissue environment that are encountered in different patients.
Of course, also the intended site of the implants determines the nature of the biomaterials
used as well as the biofunctionalization. A hip implant will require totally different materials
Evolution of Current and Future Concepts of Biocompatibility Testing 379
from a synthetic vascular prosthesis. The first will have to initiate bone formation close to and
in direct contact with the surface. This may be achieved by the application of hydroxyapatite
coatings [8,9], while a more sophisticated, but not more successful, method is the immobi-
lization of specific growth factors that can stimulate bone growth locally [10,11]. For blood
vessel prostheses, the biomaterials have to possess a certain compliance to avoid blood flow
disturbances and the surface will have to accommodate vascular endothelial cells to obtain a
biofunctionalized surface at the lumen of these devices. One can easily imagine that the nature
of the bioactive compounds used for both applications is very different and also the need for the
use of bioactive compounds differs greatly. Additionally, the expectation of patients and society
in general determines the need for complicated and costly modifications of biomaterials. When
patients are satisfied with a hip prosthesis that will function without noticeable problems for
15 years, a well-constructed inert metal implant will be the implant of choice. However, when
younger patients require a much longer lifetime of the implant, the choice of implant may be
very different, relying on the manipulation of natural processes improving the functionality of
the hip prosthesis. For synthetic blood vessels and stents, the problem of biomaterial-induced
thrombosis can be quite easily handled pharmacologically, by administering the right amounts
of anticoagulant drugs. There is however a downside to this that the long-term use of such drugs
can have some undesired side effects, like increased bleeding tendencies or interference of the
drugs with other physiological processes, like the effect of coumarins on osteoporosis and vas-
cular calcification [12].
All this means that for a (bio)material to be tested, the intended site of implantation strongly
determines the characteristics of the materials used as well as the production parameters. This
has to be taken into account when testing the biomaterials and medical devices before they can be
applied clinically. The choice of the right method of biocompatibility and biofunctionality testing is
therefore critical to obtain any useful information concerning the chance the implant will function
satisfactory in patients.
Biocompatibility testing procedures have been developed to predict the performance of a bio-
medical implant. Most and for all, the biosafety of implants is thoroughly tested. The functional-
ity of the biomedical implants is very hard to predict, if possible at all. The use of animal model
systems is nowadays required before any implantation in human patients can be considered. In this
chapter, these methods of testing are described extensively and also the value of the different bio-
compatibility testing methods is discussed.
13.1.1 History of Medical Devices and Biocompatibility Testing

Biomaterials have been used for thousands of years, and one might think that some form of testing
has been in place during the same period of time. The discovery of a prosthetic tooth in an Egyptian
mummy, dated at 5500 BC, provokes the question if the surgeons of that time would have put an experi-
mental implant directly in a high-society person, or that they first would have tested this implant in
slaves or humans belonging to a low caste [13]. This would be the ultimate way of testing (in humans),
and one can easily imagine that ethical considerations as we know today would not have been an
obstacle for the surgeons in the antiquities. The situation might be a little different in the case of the
cranioplasty found in the Inca culture, approximately 1500 years ago [14]. It is still not clear why and
under which circumstances the Inca surgeons would have decided to make holes in skulls. Because the
Inca had a strong tradition in warfare, skull injuries would be frequently encountered, and closing the
wounds the only option to save the warrior. Already then, and still today, wartime situations acceler-
ate the application of new and untested treatments of severely wounded patients, who have nothing
to lose and preferable need to return to the battlefield as soon as possible. The Inca surgeons closed
skull holes with gold plates, and natural materials like shells, and in many cases the patients survived
this treatment. Skulls were found in which the “biomaterial” was well incorporated in the bone of the
patient. There is however no information about the quality of life of these patients, so the question if
the biomaterial functioned satisfactory cannot be answered, but the lack of signs of severe infection
or inflammation near the site of implantation does suggest successful treatment. The Inca were smart
enough, or just discovered by chance, to use inert metals to close the skull holes, or to use materials that
are close to the structure and composition of bone, like calcium-carbonate shells.
Some of the first detailed reports on the use of biomaterials concern cranioplasty, the repair
of large cranial defects. At the start of the sixteenth century, Fallopius described the use of gold
plates to replace fractured bone [14]. The first described bone graft for cranioplasty was reported
by J.J. van Meekeren, a surgeon in Amsterdam, in 1668 [14]. He described the use of a canine bone
graft in the treatment of a Russian nobleman, repairing his cranium successfully with a piece of
dead dog’s cranium, which was formed to fit the cranial defect exactly. The nobleman recovered but
was excommunicated by the church because “the Christian head was too pure to be graced with the
bone of a dog.” When the nobleman asked for the removal of the piece of dog cranium, the surgeon
was helpless because the bone was firmly fused to the patient’s cranium. The lack of knowledge
prompted the surgeons of those times to perform implantations of foreign body materials into their
patients. Although extensive literature is not available, it is highly likely that many failures occurred
during these implantations of such foreign materials in patients. Of course, some of the materials
and methods were tested on animal models, but since there was no regulation and standardization
concerning such experiments, the results were of limited use. Of course, severely toxic materials
or just bad surgical methodology were eliminated before they could be used for human treatment.
Several developments in the nineteenth and twentieth centuries, like the discovery of microor-
ganisms, sterilization procedures, and better hygiene conditions in general, improved the success
rate of implantation procedures. However, it became more and more clear that some sort of regula-
tion was required to ensure efficient and safe treatment of all patients. In the mean time, the fast
increase in animal experimentation, which formed the basis of many of the developed implantation
treatments, prompted the British government in 1876 to pass a law on animal cruelty (the Cruelty
to Animals Act), fixing, among others, rules for the (more humane) treatment and experimental
use of animals [15]. One could see this as the first regulatory action for biomedical experiments
on animals. Up to that time, virtually all testing of materials to be applied in humans was done
in live animals, under sometimes horrendous conditions. This is the so-called in vivo testing, in a
living animal so that also functionality of the implant can be observed and studied. Later in this
chapter, we extensively discuss the current in vivo testing procedures.
In the mean time, a number of scientists had been working for a long time with isolated organs,
which were kept “alive” and functioning for some time outside of the body. This so-called ex vivo
strategy bypassed all legislation concerning animal experimentation and was seen as an alterna-
tive for testing procedures [16]. Later in the twentieth century, it became possible to isolate cells of
animal, and also human, origin and culture these for some time in the controlled environment of
the laboratory. Cells from fast growing tumors were found to be able to grow indefinitely and soon
became an easy tool for experimentation in vitro. The construction of immortalized cell lines by
molecular genetic techniques, developed largely in the 1970s, gave rise to huge libraries of different
cell lines [17]. It soon became clear that it was necessary to organize all these new cell lines into
a central facility like the ATCC (American Type Culture Collection) and the ECACC (European
Collection of Cell Cultures; now run by the British Health Protection Agency). The testing of bio-
compatibility soon embraced all new developments and was done in vivo, ex vivo, and in vitro. In
the rest of this chapter, the three different forms of testing are extensively discussed, also in the
context of different sorts of implants.
13.1.2 Biocompatibility Regulations

During this time of great advance in biology and medicine, the first strict rules on the use of bioma-
terials in humans or in contact with human tissue came from the field of pharmacology in the form
of a U.S. pharmacopeia regulation on the use of plastic cups for tissue, like e.g., blood, collection.
Without going into too much detail, both the FDA and European Union decided that a specific
norm for the testing of biocompatibility of medical devices was necessary to tightly regulate
and control the quality of safety of implanted medical devices and devices that contact human
tissue [18]. First in the early 1980s, the American Association for Testing and Materials (ASTM)
published the F748 standard and subsequent refinements over the years. In 1989, the International
Standards Organization (ISO) installed Technical Committee (TC) 194 concerning the biological
evaluation of medical devices. The resulting regulations and standards were finally published in
the ISO-10993 norm that currently contains 20 different parts/chapters, all describing a different
facet of biocompatibility testing. A summary of the ISO norm is given in Table 13.1, and the matrix
on which the biological evaluation is based is in Table 13.2. The FDA Office of Device Evaluation
(ODE) issued a slightly adapted guidance to the biomaterials reviews process, described in the
ISO-10993 norm. This means that in general the ISO-10993 norm, and the therein described test-
ing matrix, is the best guideline to decide which biocompatibility tests are required for testing of
medical devices and implants. Also a flowchart, adapted from ISO-10993, describing the informa-
tion and test required to make funded decisions about what steps to take in medical device testing
is depicted in Figure 13.1.
It has become clear in the past, that the regulations or biocompatibility testing are ruled by the
political situation and the public’s request for safety and humane treatment of test animals. The risks
associated with the implantation or use of medical devices have to be minimized as much as pos-
sible by well-planned testing procedures that utilize the most modern and reliable techniques avail-
able. Over the centuries, the increase in scientific knowledge, development of different attitudes
toward the treatment of living beings, and changing reluctance to accept safety risks associated with
medical procedures have led to a steady evolution of biocompatibility testing concepts.
TABLE 13.1
Different Chapters of the ISO-10993 Norm
ISO-10993-Part Description
1 Guidance to biological evaluation and testing of medical products
2 Animal welfare requirements
3 Tests for genotoxicity, carcinogenicity, and reproductive toxicity
4 Selection for tests for interactions with blood
5 Tests for in vitro cytotoxicity
6 Tests for local effects after implantation
7 Ethylene oxide sterilization residuals
8 Selection of reference materials
9 Framework for identification and quantification of potential degradation products
10 Tests for irritation and delayed-type hypersensitivity
11 Test for systemic toxicity
12 Sample preparation and reference materials
13 Identification and quantification of degradation products from polymeric medical devices
14 Identification and quantification of degradation products from ceramics
15 Identification and quantification of degradation products from metals and alloys
16 Toxicokinetic study design for degradation products and leachables
17 Establishment of allowable limits for leachable substances
18 Chemical characterization of materials
19 Physicochemical, morphological, and topographical characterization of materials
20 Principles and methods for immunotoxicology testing of medical devices
The different parts describe separate aspects of biocompatibility testing of medical devices and products.
TABLE 13.2
Biocompatibility Testing Matrix Adapted from the ISO-10993 Norm
Biological Effect
Device Category Initial Evaluation Tests Supplementary Tests
Intracutaneous Reactivity
Systemic Toxicity (Acute)

Contact
Subchronic Toxicity
Hemocompatibility
(Subacute Toxicity)
Duration
Chronic Toxicity
Carcinogenicity
Developmental
Biodegradation
Reproductive/
Genotoxicity
Implantation
Sensitization
Cytotoxicity
Irritation or
S = Limited
(≤24 h)
M = Prolonged
(24 h–30 Days)
L = Permanent
Body Contact (>30 Days)
Surface devices Skin S X X X
M X X X
L X X X
Mucosal S X X X
membrane M X X X • • •
L X X X • X X • •
Breached or S X X X •
compromised M X X X • • •
surfaces L X X X • X X • •
External Bloodstream S X X X X X
communicating indirect M X X X X • X
devices L X X • X X X • X X X
Tissue/bone/ S X X X •
dentine M X X X X X X X
communicating L X X X X X X X X X
Circulating S X X X X ▲ X
blood M X X X X X X X X
L X X X X X X X X X X
Implant devices Tissue/bone S X X X •
M X X X X X X X
L X X X X X X X X X
Blood S X X X X X X X
M X X X X X X X X
L X X X X X X X X X X
The X checked boxes indicate required tests for the medical device. The • indicates optional tests according to the ISO
norm, while the ▲ indicates a potential test as described for devices used in extracorporal circulation. Boxes that are
not checked indicate these tests are not required or recommended but can be performed voluntarily. For the contact
duration S = short-, M = medium-, and L = long-period of contact.
For biocompatibility testing the material(s) or the medical device of interest and some form of liv-
ing tissue or organism are required. The choice of the materials and isolated cells, tissue, or animal
model that should be used is of critical importance for the significance of the results. Some of the
experimentation procedures are standardized and are described, e.g., in the ISO-10993 norm. The
norms and the value of standardization are obvious when considering extrapolation of results from
in vitro, ex vivo, or in vivo testing of biomaterials and medical implants for their biocompatibility.
Material Yes Direct or No No biological

identification and indirect contact evaluation
characterization with patient required
New material in device

Material identical Yes Sufficient test data or
identical in: Yes
to approved existing justification based Yes
-manufacturing
medical device on similarity
-medical application
-sterilization and packaging No
No No
Yes
Sufficient toxicology Material characterization

data for justification No sufficient for safety data?
No Yes
Toxicity tests (biosafety) Selection biological Qualitative characterization

mimicking clinical exposure evaluation tests -residues, leachables, surface
biocompatibility
Yes No
Yes
Leachables present: Sufficient for
stabilizer, lubricant, antioxidant, No justification
monomer, plasticizer, contaminant
No
Yes No
No
Toxicology data Yes Quantitative characterization
available for -residues, leachables, surface
risk assessment
Standard requirements Final risk

met for ISO 10993 assessment
FIGURE 13.1 Flowchart describing decision making in biocompatibility testing. (Adapted from ISO-10993
Part 1; ISO norms can be purchased from www.iso.org)
Of course, the exact definition of biocompatibility declares if the results of such testing make the
material or device acceptable for clinical application.
13.1.3 Definition of Biocompatibility
Biocompatibility is one of those words of which the exact meaning has constantly changed over
time. The word biocompatibility is obviously composed of the words bio and compatibility. When
analyzing these words, one could come to the definition for biocompatibility as “the capacity of a
foreign, synthetic material to be introduced into the body of a (specified) individual without exciting
a destructive reaction.” This definition is deducted from the biological meaning of compatibility,
which was derived from early blood-transfusion experiments in which it was noted that the recipient
often demonstrated a violent reaction to the received blood, but this did not occur in monozygotic
twins. This observation induced the development of “compatibility test” which should be performed
before a transfusion to determine if the recipient had antibodies against the blood of the donor
[18,19]. The other definite meaning of compatibility is mutual tolerance. This means that in case
of a biological situation both the patient and the implant tolerate each other without any negative
responses. After this look at the classical dictionaries, a Google search with the keywords “biocom-
patibility” and “definition” results in thousands of hits. The definition that comes up with the highest
frequency is the definition that appears in the Williams Dictionary of Biomaterials [2]:
The ability of a material to perform with an appropriate host response in a specific application.
Biocompatibility is a term that is still frequently used to describe the biosafety of a synthetic mate-
rial that is destined to function in contact with tissues, organs, and cells of a living patient. Many
scientists and students still feel that biocompatibility comprises the somewhat older definition:
The capacity of blood, tissue, or an organ to be introduced into the body of a (specified) individual
without exciting a destructive reaction.
Therefore, most of biocompatibility testing over the last 50 years has concentrated on testing for
toxicity of the materials. Often this was done in vitro using immortalized cell lines, and still today
many new biomaterials are presented, accompanied by data from in vitro cytotoxicity assays. This
is of course invalid concerning the biocompatibility of the material or medical device, since func-
tionality of materials or medical device is not tested. Therefore, the misconception that toxicity, at
least the absence of toxicity, is what determines biocompatibility is still present in the biomaterials
field. Especially David Williams has been instrumental in educating the biomaterial scientists in
proper biocompatibility thinking. Recently, he also fine-tuned the definitions of biocompatibility,
taking into account the upcoming field of tissue engineering. Additionally, the effects of long-term
implants were not clearly defined in previous definitions, and so the slow and systemic effects were
incorporated in the current biocompatibility definition, which now reads:
Biocompatibility refers to the ability of a biomaterial to perform its desired function with respect to a
medical therapy, without eliciting and undesirable local or systemic effects in the recipient of benefi-
ciary of that therapy, but generating the most appropriate beneficial cellular or tissue response in that
specific situation, and optimizing the clinically relevant performance of that therapy.
The above definition implies that a range of biological and physiological tests have to be performed
before a biomaterial or medical device can be labeled as biocompatible. Not only in vitro toxicity
measurements but also in vivo functionality and systemic toxicity determination have to be com-
bined to make any funded statement about biocompatibility. In addition, the definition indicates that
the current testing for biocompatibility will always be flawed by the use of some model system or
model organism used for the initial testing. The simplification in testing and the proposed extrapo-
lation from these simplified systems to the human situation make the decision on biocompatibility
scientifically unreliable. It means that the definitive answer on the question if a biomaterial or medi-
cal device is biocompatible can only be gained from the clinical use in the patient.
13.2 BIOCOMPATIBILITY TESTING

Before a medical implant can be applied clinically, a series of tests have to be performed to ensure safe
and efficient treatment of the patient with the medical device. The exact tests that have to be performed
are laid down in a number of regulations of which the ISO-10993 norm is the most referred to norm
(Table 13.2). However, the exact regulations for testing of biocompatibility differ somewhat between dif-
ferent countries, but we will not go into these minor differences here. It is good and useful to know that
biocompatibility testing regulations differ slightly between for instance the United States and Europe.
The tests that will have to be performed for a new biomaterial or medical device can be deduced from a
flowchart that is given in Figure 13.1. This flowchart is an adaptation of the chart that has been published
earlier in ISO-10993 part 1. One of the shortcomings of the chart is the absence of tissue-engineered
products, but this class of bioengineered products is so different from the “classical” medical devices that
a completely new set of tests is required for the biocompatibility testing of these products. One should not
forget the fact that almost all tissue-engineered products contain some form of synthetic material, which
is necessary as a scaffold to shape the engineered tissue as well as giving it the mechanical strength for
the period of constructing the tissue in a biocontainer. Consequently, in the end also the scaffold or the
remains of the scaffold are transferred into the patient’s body. This means that, in principle, the scaf-
fold materials should undergo the whole range of biocompatibility tests. It is however not completely
clear what regulation will be applied in case of tissue-engineered products with scaffold still in. Next,
ex vivo testing is performed for a range of biomaterials. One could think here about perfused organs
or working with freshly obtained tissue, like cornea or pieces of skin. Another interesting option is to
attach blood-contacting devices, in a flow setup, to extracorporal circulatory machines. In this way, a
longer testing time can be achieved than with freshly obtained blood, which will deteriorate within hours
after collection, thus limiting the window of testing blood-contacting devices ex vivo. The final step in
testing for biocompatibility is of course implantation in an animal model, so-called in vivo testing. The
way in which animal testing has been performed over the centuries strongly depended on the political
situation. After the acceptance of the animal cruelty act (Great Britain in 1876), the use of test animals
became more “humane.” This is also one of the key mistakes that has been made in the early years of
controlled animal testing, the fact that animals would make a reliable source of information about the
performance, the biocompatibility, of a medical device or implant in human patients. The choice of the
animal model as well as other parameters chosen in the experiment strongly influence the outcome of the
test. The goal of implantation can range from relatively straightforward biosafety testing to functionality
testing of the implant or device. Recently, there has been some discussion about the value of this animal
testing since the extrapolation to the human situation has been shown to be very difficult and unreliable
in many cases [20]. A well-known example is synthetic small-diameter vascular prostheses [21]. These
will perform satisfactorily in animal models like dogs or pigs, but they perform with an extremely high
failure rate in humans. Some of the considerations for in vivo testing are discussed later in this chapter.
13.3 IN VITRO BIOCOMPATIBILITY TESTING

In vitro biocompatibility tests are mainly concerned with safety of materials as well as adverse reac-
tions of body fluids like blood. Immunological tests like irritations and long-term hypersensitization
cannot be performed in vitro because no reliable alternatives for the complete immune system are
available. In addition, experiments concerning the long-term systemic toxicity in a three-dimensional
(3D) environment of tissue can currently not be reproduced in in vitro tests. Although there are now
various bioreactor systems, in which it is possible to grow cells in a 3D environment on a carrier
material, this is only useful for the testing of potential scaffold materials. But the further develop-
ment of these 3D systems might in the near future lead to the “implantation” of a material in a small
piece of 3D cell mass, followed by incubation for a chosen period of time. At the moment, only very
small pieces of tissue can be produced in vitro, up to a couple of cubic centimeters. This of course is
not of much use for material testing in vitro, but it makes for an outlook in which in vivo biosafety
testing might be minimized, saving a large number of animals and also reducing the total costs of
biocompatibility testing. Changes to the genetic information in the DNA can be studied relatively
easily and reliably in simple laboratory tests. The underlying message is that in vitro testing will
never suffice for medical devices. The real answer on the biocompatibility of a medical device is opti-
mally studied in animal models and ultimately in human patients. Of course, in vitro tests are useful
to filter out really “bad” materials that have very strong effects as toxins, mutagens, or carcinogens.
However, one should remember that materials are tested in an environment that does not resemble the
conditions they encounter after implantation. The presence of proteins, extracellular matrix proteins,
enzymes, and cells of the immune system makes the environment of medical devices highly complex
and the in vitro reaction of the materials or substances that leach from the materials unpredictable.
Therefore, the in vitro biocompatibility testing should be seen as the attempt to minimize the risk
for the patient and increase biosafety. One can identify several levels of in vitro biocompatibility
testing, namely:
1. Cytotoxicity—the effect of a synthetic material or the substances that leach from this
material on stable cell lines in liquid culture media.
2. Cell growth—do cells attach and proliferate on the test material?
3. Functionality—does the material influence the “normal” cellular functions of differenti-
ated cells?
These three main levels of in vitro testing are discussed in detail.
13.3.1 Control Materials
For biocompatibility testing, in general, the use of controls is vital for critical assessment of results.
In biocompatibility testing, a comparison to positive (toxic) and negative (nontoxic) controls is nec-
essary. The positive control demonstrates the sensitivity of the used system, and can also be used
as a background measurement. The fact that cells, tissues, or animals will show a noncompatible
response demonstrates that an appropriate model was chosen. Also for quantitative measurements,
especially in vitro, the signal obtained with the positive control can be considered the background
level of the assay used. The negative control can be compared to mock operations when assessing
the efficiency and functionality of an implanted medical device or to the placebo in pharmaco-
logical clinical testing. A good negative control should have the same shape and physical proper-
ties as the tested material. Therefore, a negative control for a ceramic powder should also be a
powder variant of standard material. A hydrophilic polymeric material should not be compared to
poly(tetrafluoroethylene), which is highly hydrophobic. Finally, a degradable polymer should prefer-
ably be compared to a degradable polymer, or two different control materials should be used: one
for the bulk, nondegraded state and one for the soluble degradation products.
A variety of materials is being used as control materials in (cyto)toxicity testing, but the ISO-10993
norm describes organo-tin stabilized poly(vinylchloride) as the preferred positive control, although
many scientists use phenol or ethanol dilutions as such. The use of liquid materials is not preferred,
because the prescribed extraction conditions are not mimicked in the positive control. As negative
control materials, high- and low-molecular weight polyethylene are recommended [22]. These control
materials represent the far ends of the biocompatibility scale. There is no reliable scale of materials
that represent intermediate biocompatibility or toxicity. This leaves the field open to interpretation
and subjective assessment of the results of biocompatibility tests. Several attempts on producing stan-
dardized materials with intermediate toxicity have been made, but the use of such a scale is unverified
and remains to be determined. It is however clear that for in vitro biocompatibility testing a series of
standard materials ranging from highly cytotoxic to noncytotoxic would improve objective evaluation
of biocompatibility testing. Additionally, the interlaboratory variation between results and interpreta-
tion of results could be decreased. The biggest problem is how this range of standard materials cor-
relates to in vivo biocompatibility performance. The choice of animal model and surgical procedures
may significantly influence the biocompatibility and the performance of the materials in vivo. So the
search for a measuring stick to create an objective biocompatibility score is still ongoing.
13.3.2 Cytotoxicity
The ISO-10993 part 5 norm describes the correct way to test the effect of biomaterials and medical
devices on the viability of cells in vitro. The determination of cytotoxicity is one of the first tests
that are performed after obtaining a new material that can be incorporated into a medical device. In
addition, when a material is manufactured using a different procedure, when the manufacturing or
sterilization protocol is changed, a simple start is to test the in vitro cytotoxicity. First, one should
make a clear division in direct and indirect testing.
In direct testing, the living cells make direct physical contact with the test material, while in
indirect testing the cells are merely used as a sensor to detect possible toxic substances released from
the material. Also, indirect testing can be used to determine the toxicity of degradation products of
(bio)-degradable biomaterials. The committee recognized that the preparation of the materials to be
tested is of critical importance for the results of in vitro toxicity and compatibility testing. Therefore,
separate parts of the ISO-10993 norm were created to specify conditions by which material extracts
and degradation products should be produced. The ISO-10993 part 12 contains protocols describ-
ing the extraction conditions for obtaining leachables from biomaterials and medical implants. The
extraction media, incubations times and temperatures, storage conditions, etc. are all specified. The
ISO-10993 part 9 was included to specify identification and quantification of degradation products
and describes how degradation products should be obtained. The correctly obtained material extracts
or degradation products can then be used in standard cytotoxicity testing. This emphasis on carefully
chosen conditions for sample preparation before in vitro biocompatibility testing is of critical impor-
tance to get to an objective and transparent testing procedure that mimics clinical conditions as good
as possible. This should ensure results with optimal information concerning in vitro biocompatibility.
13.3.2.1 Indirect Testing
ISO-10993 is very clear in how indirect cytotoxicity testing should be performed and how the
leachables from the test materials should be extracted (Figure 13.2). In the first possible cyto-
toxicity assay, a piece of material is extracted in for instance cell growth medium. This extract
is then added to a semiconfluent cell layer and incubated. After the desired incubation period
(>24 h), cells are analyzed for cell viability. Another possibility is that the material is placed on
top of a filter (pores approximately 1–5 μm), which fits perfectly in the cell culture dish and keeps
Test material Test material

Test material
Agar
Monolayer cells Extraction in Monolayer cells Insert with filter-bottom Monolayer cells Agar + cell-growth-medium
cell-growth-medium
Incubate cells with material extract Place well-insert above cells Incubate cells under agar
incubate with cell-growth-medium place test material on agar
Determine cell viability with Determine cell viability with Observe cell-death/cell viability
- MTT assay MTT assay using microscope
- LDH assay LDH assay
FIGURE 13.2 (See companion CD for color figure.) Overview of indirect-contact cytotoxicity testing. On
the left is depicted cytotoxicity testing of material extracts that are separately isolated by incubating a sample
of the material in an appropriate extraction medium. The middle part shows indirect incubation with the test
material, the leachables can diffuse through the filter, and the cells will detect cytotoxic components. On the
right, the material and cells are separated by a layer of soft agar through which the extractable compounds
from the material can diffuse. A zone of cytotoxicity around the material may appear since diffusion is limited.
the test material a little distance above the cells. After incubation, the insert and test material
are removed, and cell viability is determined. The last method shown in Figure 13.2 is an agar
overlay assay. Cells are overlaid with a layer of (soft) agar, and the test material is placed on top
of this agar. The leachables then diffuse though the agar and reach the cells. Here, the viability
of cells is examined microscopically, and a zone of nonviable cells just beneath the test material
might be observed. The extracts should be obtained in such a way that the extraction conditions
exaggerate the conditions of clinical use. One should be careful as not to change the physical
properties of the material during the testing, so fusion, melting, and alteration of the chemical
structure should be avoided. The choice of the extraction medium also limits the extraction
conditions. When choosing a cell culture medium with serum, the use of elevated temperatures
above 50°C is not recommended to avoid denaturation and/or deactivation of important com-
pounds from this serum. In such a case, a prolonged extraction at 37°C is most appropriate, for
instance for more than 24 h and up to 120 h. When an accelerated extraction is required, higher
extraction temperatures, 50°C or 70°C, can be used, but then only culture medium without
serum or a buffered physiological salt solution are useful.
Although these conditions are described in the ISO norm, one has to be cautious about such
extraction conditions since the presence of proteins in the extraction medium can greatly influ-
ence the extraction kinetics and the amount of substances leaching from the material. The next
parameter important for the extraction of leachables is the material/medium ratio. How much
of the material should be incubated in which volume of medium? There is no straightforward
answer to this, since the shape and the physical structure of the test material are vital for the
extraction efficiency. A solid piece of pMMA will have much slower extraction kinetics than a
porous polyurethane piece. A rule of thumb describes the use of 6 cm 2/mL of extraction medium.
In practice, this is sometimes impossible to calculate, as for porous materials, or materials that
are powdered or broken up into nonuniform particles. Then for sheets of material, one could
easily calculate the total surface area, but the thickness or bulk of the material will also greatly
influence the total concentration of extractable substance in the extract. It is therefore clear that
great care has to be taken during the extraction procedure. A guideline for the amount of mate-
rial that has to be used for extraction of potential toxic chemicals, adapted from ISO-10993-12,
is given in Table 13.3.
For the testing of cytotoxicity of extracts, the choice of cell line is not that critical. The cell
in this case is only used as a sensor, albeit a living sensor, for toxins that influence the metabo-
lism and integrity of the cell. The most commonly used cells are mouse fibroblasts, largely
because these are easy to maintain in the laboratory at low cost. In addition, these fibroblasts
are sensitive to a large array of chemical compounds, which is essential for the cell to be a use-
ful toxin sensor.
TABLE 13.3
Amount or Surface Area to Volume Ratio Required for Proper Extraction
of Test Materials according to the ISO-10993 Norm Part 12
Thickness of Extraction Ratio ± 10% (Surface
Material (mm) Area or Mass per Volume) Examples of Test Materials
≤0.5 6 cm2/mL Synthetic polymers, ceramics, metals, sheets,
composite films, tubings
>0.5 3 cm2/mL Synthetic polymers, ceramics, metals, composite
films or tubings, preformed implants
≤1.0 3 cm2/mL Natural elastomers, latex, rubber
>1.0 1.25 cm2/mL Natural elastomers, latex, rubber
Nonregular 0.1–0.2 g/mL or 6 cm2/mL Microspheres, nanospheres, pellets, powders
13.3.2.2 Direct Contact Testing

For direct contact testing of biomaterials and medical devices or implants, a somewhat different
approach must be taken. First of all, the fact that most used materials are not opaque makes them
unfit for use in standard microscopic analysis. Also in direct contact testing, it is vital that the cells
attach well to the material. This is important to realize because the lack of cells on a particular
material can be caused by toxicity of the surface, inhibition of proliferation, or by lack of cell
attachment. Direct contact testing should therefore be combined with testing of material extracts as
described earlier. Also, the test material should first be extracted, so that no more toxic compounds
leach from the material. Then the “clean” surface of the material can be tested, and the cells will
demonstrate the effect of the surface on cell viability [23].
If this is not done properly, no real additional information is gained from performing such a
direct-contact biocompatibility assay. The topography, charge, free energy, and the physicochemi-
cal composition of the surface all play a role in the interaction with cells and tissues. In addition, a
pretreatment of the test material with proteins, i.e., medium including serum, can be of importance
since protein adsorption may dramatically alter the interaction of cells with surfaces. In the in vivo
situation, this also occurs, and thus it is advisable to mimic the in vivo situation as closely as pos-
sible. For instance, for a synthetic blood vessel the luminal surface of the device optimally will get
covered with functional endothelial cells to prevent thrombotic complications. The “naked” surface
of the device will never be encountered by the endothelial cells since plasma proteins will rapidly and
irreversible adsorb to this surface. For in vitro testing of material endothelialization, plasma proteins
should be allowed to adsorb to the surface prior to endothelial cell interaction. Only then will the situ-
ation as encountered after implantation be mimicked appropriately. It has been shown in several stud-
ies that the adsorption of proteins can dramatically change the behavior of cells on synthetic surfaces.
Direct contact testing can be performed in several ways, of which two are most popular
(Figure 13.3). First, the test material can be placed on top of a (semi-)confluent cell layer, and
Negative (nontoxic) Test material with

control unknown toxicity
F-actin+nuclei
Test material
Layer of cells Test material Isolated cells

Live
Dead
Incubation of material Incubate cells in direct

in direct contact with cells contact with material
Remove
test material
Fluorescence microscopy
Objective (microscope)
Cover slip
Sample (material with cells)
Microscopical evaluation Glass slide
vital staining
viability assay
FIGURE 13.3 (See companion CD for color figure.) Direct-contact testing in vitro of test materials of medical
devices. Materials are either placed on top of or act as a support for cells. After the appropriate incubation time,
cells can be analyzed for cell viability, morphology, or cell function. Fluorescent techniques are often used because
of the opaque nature of most synthetic materials. The cytoskeleton of attached cells can be visualized by staining
with fluorescently labeled phalloidin (right top panel). Cells can also be analyzed for viability or cell death (right
middle and bottom panels, respectively).
after incubation the viability and functionality of the cells directly underneath the material can
be analyzed. Another, more elegant method is to place the cells directly on the test material. In
this way, the cells will not obtain mechanical damage or suffer from nutrient or oxygen depletion
due to limited diffusion underneath the piece of test material. Then, the analysis of the attached
cells is problematic. In most cases, standard microscopic observation of the cells will not be
possible due to the bad optical qualities of most medical devices and materials. However, the
development of fluorescent probes and immuno-histological techniques overcomes this prob-
lem (Figure 13.3). Also, the cells can be isolated from the material and the differentiation and
functionality can be tested by studying protein expression or cell-specific enzymatic activities.
Overall, a combination of direct and indirect contact testing will deliver the most complete and
reliable information concerning cytotoxicity and support of cell-type specific function. This is the
first step in biocompatibility testing, and these in vitro techniques will be developed further to
improve their predictive power and extrapolation to the in vivo situation. One should however always
be cautious with the extrapolation since the medical device is only one part of the patient’s treat-
ment, and the actual surgical procedure is another vital part. Often this is disregarded in biocom-
patibility testing, and devices are produced that are impossible to use properly in a clinical setting.
13.3.2.3 Cell Viability Assays

Cell viability is tested as a general marker for toxicity of a material or substance. The most
widely used cell viability tests are based on conservation of mitochondrial integrity and activ-
ity. Loss of mitochondrial activity is one of the crucial steps during necrosis and apoptosis of
cells. Therefore, chemicals like MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide) and XTT (Sodium 3,3,-(Phenylamino)carbonyl]-3,4-Tetrazolium-Bis(4-methoxy-6-nitro)
benzenesulfonic acid hydrate) are used as viability agents. These compounds are converted by the
succinate dehydrogenase in the mitochondrion in an insoluble colored or fluorescent dye, respec-
tively (Figure 13.4) [24].
Another variation on cell viability test is based on cell membrane integrity. The leaching of the
enzyme lactate dehydrogenase (LDH) from treated cells can be used as a marker for cytotoxicity.
The amount of LDH in the culture medium is then a measure for the (non-)viability of the cells.
NH
N N N N N
N N N N N N N N N
S S S
MTT MTT-formazan MTT

(yellow soluble) (purple crystals) (yellow soluble)
(a) (b)
FIGURE 13.4 MTT is converted by viable cells (a) into a purple, water-insoluble compound that can easily
be quantified by microscopy or spectrophotometry. Nonviable cells (b) will not convert MTT because mito-
chondrial activity has been lost.
Trypan blue
NH2 OH OH NH2
N N N N
–O +
O– –O O–
S S 4Na+ S S
O O O O O O O O
Neutral red
N N NH2
FIGURE 13.5 Chemical structure of the viability stains trypan blue and neutral red. Trypan blue is excluded
from viable cells and only stains necrotic cells, with compromised plasma membrane, blue. Neutral red is
exclusively incorporated in viable cells.
A known disadvantage of this method is that many sera used for supplementing culture medium
have a high basal level of LDH. The basal level of LDH activity will therefore be high and the sen-
sitivity of the LDH-viability assay low.
The advantage of the MTT type dyes is that it is relatively easy to quantify cell viability of a large
number of samples in a short period of time. Of course, the method critically depends on the use of
suitable positive (toxic) and negative (nontoxic) controls. Also, MTT staining can be used for stain-
ing of tissue-engineered constructs and cells grown on top of medical devices in a 3D environment.
Simple bright-field microscopy can then reveal viable cells, or by lysing the cells and dissolving the
formed purple formazan, a measure for the number of viable cells can be obtained.
Other frequently used compounds to determine cell viability are the so-called viability stains
or vital stains. The most widely used of these dyes are Neutral Red and Trypan Blue (Figure 13.5).
Trypan Blue, which is negatively charged, cannot permeate through an intact plasma membrane
of viable cells. When cell viability is lost and the plasma membrane gets compromised, the dye
can enter the cell and the cell will take on the color of this blue dye. Trypan blue is often used in
standard cell culture to determine the viability of cells. Cells that take up a blue color have a com-
promised cell membrane and should be considered as nonviable. Neutral red however is only taken
up by viable cells and stored in lysosomes. Standard microscopic examination can then be used to
determine the level of cell viability in a population of cells.
In the last few decades, a large number of more sophisticated fluorescent dyes were developed to
assess cell viability [25]. These dyes can freely diffuse into the cell and are converted by intracel-
lular enzymes into a fluorescent and membrane-impermeable compound inside the cell. Therefore,
when cells are challenged with such a dye, only the fluorescent cells are viable in the population.
The fluorescent dye Calcein AM is a compound that becomes fluorescent when hydrolyzed by
intracellular esterases in intact, viable cells (Figure 13.6). The amount of observed fluorescence is
a direct measure for the number of viable cells. Cells that have a compromised plasma membrane
will not be able to retain the intracellular esterases as well as the formed polyanionic product of
Calcein-AM hydrolysis.
Thus, nonviable cells will not be fluorescently labeled. The quantification of cell viability using
these types of viability dyes is a bit more laborious since the fraction number of fluorescent cells has
to be determined using flow cytometry. For this, the cells have to be released from the tissue cul-
ture plate and each sample has to be analyzed separately, which results in a rather time-consuming
analysis. Also, a number of cells may get damaged during the procedure, influencing the measure-
ment in this way, increasing the number of nonviable cells.
Calcein AM
ROOC COOR
ROOC N N COOR
O O O
O O
O
R: CH2OCOCH3
O
494 nm
–
OOC CH2OO–
517 nm
–OOC N N COO–
HO O O
COO–
FIGURE 13.6 Calcein AM staining of viable cells results in uptake and hydrolysis of the dye. The result
is an anionic fluorescent compound now trapped inside the cell. The excitation wavelength for detection is
494 nm and the emission 517 nm.
Another variation of these assays that are based on membrane integrity makes use of fluorescent
dyes that are membrane impermeable. Only nonviable cells with a compromised plasma membrane
will incorporate the dye and be visible under the appropriate wavelength of light. Since exclusively
nonviable cells are stained by these dyes, they are often called “dead-dyes.” Many of these dyes are
nucleotide-binding dyes, like propidium-iodide or ethidium bromide analogues, that dramatically
increase their fluorescence upon binding of DNA, which is normally unreachable inside the nucleus
(Figure 13.3). With FACS analysis, the proportion of fluorescent cells is a direct measurement for
the percentage of dead cells in a population.
Mostly the cell viability dyes are used as a rapid qualitative method to observe large differences in
cell viability. Using two separate dyes, one viability dye like Calcein-AM, combined with a nonperme-
able dead dye (e.g., propidium iodide or ethidium-homodimer 1), a rapid evaluation on the cell viability
in a population of cells can be made using a standard fluorescence microscope setup (Figure 13.3).
All these viability dyes have in common the fact that they are most suited for qualitative analysis
of a cell population. Quantitative analysis using these dyes is possible but can be time-consuming
and can suffer from a high background level of dead cells due to the procedure. Therefore, for
quantitative analysis, the metabolic dyes are more practical since an accurate measurement of cell
viability for a large number of samples can be determined easily and rapidly.
13.3.2.4 Evaluation Cell Viability or Cytotoxicity

Determining the level of cell viability or cytotoxicity in a population of cells is rather straightforward,
but the interpretation of the results is far from that. Where the ISO-10993 norm lacks detail is in the
way in which the results of cytotoxicity assays should be interpreted. For qualitative assays, like micro-
scopic examination of cells that have been in contact with toxic materials or solutes, a scale describ-
ing exact toxicity levels is lacking. The interpretation is now completely dependent on the subjective
interpretation of the researcher analyzing the cells. The interpretation should be classified on the cyto-
toxicity scale as 0 = noncytotoxic, 1 = mildly cytotoxic, 2 = moderately cytotoxic, and 3 = severely
cytotoxic. The problem of this qualitative evaluation is that there are no strict rules describing when a
material is mildly cytotoxic or moderately cytotoxic. What percent of the cell population needs to be
dead or nonviable for the material to be considered nonsafe and cytotoxic? This problem also arises
for the quantitative assays. The results can be determined objectively, a percentage of cell viability of
a standard control material. The question that remains is when does one consider a material toxic? Is a
material that displays a cell viability of 75% of the negative control cytotoxic? What is missing here is
a series of materials that have intermediate cytotoxicity (i.e., biocompatibility), from which is known
how they behave both in vitro as well as in vivo concerning biosafety.
Obviously, the functionality of a material, which is an intricate part of biocompatibility, can-
not be determined with these relatively simple toxicity tests. To conclude, in vitro cytotoxicity and
viability testing is very important in determining the biosafety of a material or medical device, but
the interpretation of the results is not fully standardized. This makes it very hard to compare the
in vitro biocompatibility data between labs and even in the same lab between different investigators.
There is a clear need for a series of standard materials that cover a range from severely cytotoxic to
noncytotoxic, to standardize interpretation of in vitro biocompatibility and cytotoxicity tests.
13.3.3 Cell Proliferation In Vitro

Next to viability, the proliferation rate of cells can be of importance for the performance of implanted syn-
thetic materials. Especially for materials that should allow regeneration of cells and tissues, cell growth
is an important parameter. There are numerous ways to determine cell proliferation on top of synthetic
materials. Most are based on the increase in DNA of biomass. The simplest method to determine cell
proliferation is counting cells in a well-defined area or volume and compare the cell counts at different
time intervals. The advantage is that except for a microscope, no equipment is required. When cells are
cultured on a translucent material, the cell number can be directly determined in a defined field of vision
and recorded over time. In case the cells are attached to an opaque material, the cells have to be released
from this material and then counted. The disadvantage is that the efficiency of harvesting the cells prior
to cell counting is crucial for the accuracy of the measurement. Therefore, this method is implemented
as a crude indication of cell proliferation in general. Other classic cell proliferation assays were per-
formed by addition of nucleotide analogues, both radioactive and nonradioactive. The incorporation of
for example 3H-thymidine over time is a direct measure for the amount of DNA synthesis and directly
proportional to cell proliferation. The radioactive compound (3H-thymidine) was slowly replaced by the
nonradioactive 5-bromo-2' deoxyuridine (BrdU) because of practical considerations. The comparison of
cells in contact with a standard material (toxic or nontoxic) demonstrates the rate of cell proliferation on
the studied materials or implant. The setup of the experiment is the same as for cell viability experiments
as shown in Figure 13.3. Another method to determine the increase in genetic material inside a cell
population is based on the use of DNA-dependent fluorescent dyes. For these, cells must be lysed so that
the dye can bind tightly to DNA and become fluorescent. The amount of fluorescence can be compared
to a standard curve, linking fluorescence to cell number. One should of course beware of the difference
between cell types and the standard curve should always be of the identical cell type (or mixture of cells)
that is being studied. Another method to determine the number of cells undergoing cell division and thus
proliferation is to study cell-cycle markers. One of these markers is the Ki-67 protein, which is only pro-
duced by cells undergoing cell division. The number of cells positive for this marker is a direct measure
for cell proliferation in the population of cells. The more Ki-67 can be observed, the more cells are going
through the cell cycle and cell division, so the higher the proliferation rate of this population of cells.
Another method uses the activity of intracellular enzymes measured over a period of time to determine
an increase in cell number. The assumption is that the number of, for instance, mitochondria per cell does
not change over time and does not change by contact with a synthetic material or its soluble compounds.
The most used substance here is the MTT dye that is frequently used for cell viability measurements.
The disadvantage of the use of these metabolic dyes is that one does not actually determine the number
of cells, but the relative number of living cells compared with a control or standard. For the answer if a
synthetic material or a medical device influences the proliferation of a certain cell type, the results of all
the described assays is satisfactory. One has to keep in mind that loss of viability and loss of proliferative
potential are two different things. Cells may lose their ability to proliferate, to multiply by cell division,
without losing their viability. Therefore, a combined approach determining the effect of biomaterials or
their leachables on both viability and proliferation is preferred.
When screening a large library of different synthetic compounds, the cells used are just a sensor for
the ability of the material to allow for cell proliferation. In that case, one may choose a cell line that is
intrinsically fluorescent. This fluorescence can be achieved by incorporation of a fluorescent protein,
i.e., green-fluorescent-protein (GFP). The gene encoding this protein is put under control of a constitu-
tive promoter so that all the cells are brightly fluorescent. These cells are then incubated with the bio-
materials to be tested, and the change in fluorescence over time is a direct measure for the proliferation
rate. One can imagine that this method is easily applicable for large-scale and high-throughput screen-
ing of libraries of materials. Of course, one remark that has to be made is that the lack of fluorescent
cells on a material can be caused by loss of viability, lack of proliferation, or the inability to adhere to
the material. The problem with all the described proliferation assays is that the recorded increase in
cell number is not only dependent on the rate of cell division but also on the attachment of the cells
to their solid support. Cell growth is the addition of proliferation and attachment. The attachment of
cells to a test surface can be easily determined. A high density of cells is incubated with the material
for a short time so that no significant proliferation takes place. The number of attached cells can be
determined as described here. In some studies, the force needed to detach cells from the surface is
determined as a measure for the strength of attachment. The force needed to remove the cells is pro-
portional to the attachment force of the cells for that specific material surface.
13.3.4 Cell Function In Vitro

The next level of in vitro biocompatibility testing is functionality of cells in contact with biomaterials
(Figure 13.7). One can imagine that a material that is intended as a filler of gaps in bones should be
nontoxic, support the growth and attachment of the bone-forming cells (osteoblasts), and should not
interfere with the bone-forming characteristics of these cells. The state of differentiation of the cells
should not be influenced by the materials, so no re- or de-differentiation should occur. This means that
the production of functional proteins is a vital part of biocompatibility of materials in vitro. In the case
of osteoblasts, the formation of calcified extracellular matrix is the important functional parameter.
There are several different methods to determine whether the right set of genes gets expressed. The
steady developments in the field of molecular genetics and biology are exploited to facilitate the bio-
compatibility studies. In the early days of gene-expression determinations, mRNA was isolated from
cells in contact with biomaterials, and the expression of the gene of interest was determined using
the northern blotting technique [26]. This method is rather time-consuming because there are a large
number of steps involved before a signal is obtained. The development of polymerase chain reaction
(PCR) in combination with the use of reverse transcriptase (RT) resulted in the RT-PCR technique. In
the earlier version of this technique, a low amount of RNA was converted to DNA using RT and then
used as a template in the subsequent PCR technique. By choosing the right primers for the reactions, a
specific set of genes can be amplified. The produced DNA was analyzed on agarose gels followed by
DNA staining. The quantification of this technique was rather inaccurate, and this problem was tackled
Northern blotting
qRT-PCR
RNA isolation
Test material Isolated cells

Micro-array/DNA chip
Western blotting
Incubate cells in direct ELISA

contact with material
Protein isolation
Enzyme activity
Substrate
Product
FIGURE 13.7 (See companion CD for color figure.) Scheme depicting possible test for cell function in vitro.
Cells in direct contact with the test material can be analyzed for the presence of cell-type specific gene expres-
sion and protein production.
by the development of quantitative PCR (qRT-PCR). The incubation of fluorescent nucleotides during
the PCR reaction, coupled to a sensitive fluorescent measurement of the produced DNA, is a real-time
measure for the amount of mRNA present in the original sample. So comparing the cells to the appro-
priate controls delivers detailed information to the relative gene expression of cell-type specific genes. A
more recent method to determine the level of gene expressions of cells is to make use of DNA microar-
rays. These libraries of DNA sequences, printed or synthesized on glass chips, are ideal for comparing
mRNA profiles in a large variety of cell-types. In one reaction, a large number of different genes can be
studied, so there is a massive amount of information available in one experiment. At this moment, the
commercially available DNA arrays are rather expensive, but they can be reused. Also only one sample
can be incubated with one array at the same time. The bioinformatics attached to DNA array analysis
is improving all the time, and also prices for arrays are coming down. Therefore, it is reasonable to
assume that most of the analyses of gene expression in biocompatibility testing will be performed by
microarray in the near future. Different microarrays will become available for analyzing the transcrip-
tional activity of a certain cell types, enabling a rapid estimation of the differentiation state of the cell.
The advantage of these molecular genetic methods is that they are fast and, in most cases, a
large number of samples can be analyzed simultaneously. The information is also rather direct,
and relatively small differences in gene-expression can be detected. The disadvantage is that the
produced mRNA is not the functional component in the cell. The protein that is produced from the
RNA is what determines the functionality of the cell. The change in mRNA is not always directly
and proportionally reflected in the production of the encoded protein. From this point of view, the
study of proteins to determine the differentiation state of the cells is much more direct and sensible.
The proteins produced by a cell can be analyzed by western blotting or ELISA (enzyme linked
immunosorbent assay) methods that are based on the use of specific antibodies. For western blotting
isolated proteins are separated by molecular weight and the protein of choice is visualized by the
specific interaction with an antibody [27]. For ELISA, the protein of interest is caught out of a crude
protein sample by a surface-attached antibody [28]. The immobilized protein can now be quantified
with a second specific antibody and a standard curve. This analysis of the cell’s protein make-up gives
a direct representation of the differentiation and functionality of the cell. The development of pro-
teomic sciences has greatly improved the determination of protein identities over the last decade. The
production of reliable antibody-, protein- or peptide arrays is still not possible but is expected to be
available over the next years. Proteins can then be identified on the basis of specific interaction with
an antibody or peptide sequence on a glass chip, very similar to that described for the DNA array. For
the moment, however, this technique is not fully developed, and the analysis of protein production by
the classical techniques is preferred. In addition, the composition of deposited extracellular matrix is
only possible by these methods. The production of the right proteins and enzymes is a direct measure
of the functionality of the cells and therefore also for the biocompatibility of the tested material.
These described assays can be performed in an array setup, so that a large number of materials
can be tested simultaneously. High-throughput screening will become more and more available
when some remaining technical problems have been solved.
13.3.4.1 Evaluation of In Vitro Functional Biocompatibility Tests

One of the drawbacks of in vitro biocompatibility testing is to assess the relevance of the obtained
results to the in vivo, the in patiento situation. It is of course important to show that a cell is remain-
ing functional despite interaction with a foreign material. Also, the production of cell-type specific
proteins and enzymes is a clear indicator for conserved functionality of cells. In the case of tissue
engineering, often precursor or stem cells are used, which are stimulated by signaling molecules
to adapt a certain phenotype. The analysis of the gene-expression profile can then confirm that the
desired differentiation has indeed taken place and that a functional tissue is being formed. Because
only a specific set of active genes defines the differentiation of a specific cell type, the number
of genes that has to be studied is large. The set of studied genes must be large enough to exclude
some intermediate differentiation state, in which the cell-type specific genes are expressed, along
with genes specific for another, sometimes closely related, cell type. One can imagine that with the
appropriate DNA micro arrays, this analysis will be possible. The increase in the expression of cer-
tain genes and decreased expression of others are then clear indicators of cellular function.
As stated before, the produced mRNA molecules are not the active compounds of the cell, but the
proteins that are encoded by these messenger molecules. The analysis of large numbers of different
proteins is a focus of the proteomic field, and the techniques are steadily developing. Therefore, it is fea-
sible that in the near future a protein profile of a specific cell type can be analyzed in a chip format with
similar speed as the DNA microarray. Until that time the somewhat slower protein analysis and identi-
fication techniques will have to do. A last possibility is histological analysis of cells and cell constructs
that were grown in vitro. The overall morphology and the interaction with the produced extracellular
matrix can all be assessed via microscopic evaluation of proper samples. The primary bottleneck how-
ever remains the lack of a 3D network of cells, a tissue that demonstrates proper homeostasis in vitro.
13.3.5 Genotoxicity
This form of testing is relatively modern, since 100 years ago nobody even considered toxic effects
on the genetic material, which had only just been identified as being DNA. It was already known that
some chemicals could cause phenotypic changes in Drosophila or plants, but the mechanism remained
completely unknown for a long time. The developments in bacterial and eukaryote genetics, combined
with cell culturing techniques, led to the development of several different genotoxicity assays. The pub-
lication of the structure of DNA stimulated the discovery of many chemical mutation mechanisms over
the following decades. Subsequently, regulation was put in place to avoid human exposure to chemical
mutagens [29]. The effect of biomaterials and their degradation products or leached compounds on
the stability of the DNA is of great concern when determining biocompatibility. This specific form
of toxicity is mostly observed after long-term incubations in vivo, but the consequences can be disas-
trous. Consider the use of asbestos in building projects during long periods of the twentieth century as
insulating and heat-resistant material. Only decades later it turned out that inhaled fibers of asbestos
remained present in the lungs, got encapsulated, and caused cancer long after the inhalation. The study
of so-called genotoxicity has therefore gained importance over the past decades. Very important prog-
ress was made by the development of the Ames test in the 1970s by Bruce Ames and coworkers [30].
The test was designed for analysis of mutagenicity of chemical compounds and is still widely used
today. Genotoxicity can be subdivided in different forms of damage to the DNA. Mutagenicity are
changes to DNA that change the sequence in functional genes, resulting in an altered function of the
gene product. These mutations mostly only affect small portions of the DNA and include frame shifts,
base-pair mutations, and small insertions or altered splice sites. The most frequently used test to detect
such DNA changes is the Ames test, which is described in Figure 13.8. The test is based on the use of
bacterial auxotrophic markers. A histidine-dependent Salmonella typhimurium strain is treated with
the test material, and the number of revertants is then assessed. A mutagenic substance will randomly
evoke changes in the DNA of the bacteria. Some mutations will restore the defective gene in the histi-
dine operon, and the resulting bacterium will now be autotrophic for histidine, so be able to grow on a
Rat liver Control plate

extract
Low
dose
Test-
material Medium
extract dose
High
dose
Culture
His- Medium
Salmonella lacking Experimental
typhimurium histidine Incubation plates
FIGURE 13.8 Scheme showing the Ames test. An extract of the test material is mixed with a rat liver extract
and incubated with Salmonella typhimurium His− strain. The number of revertants, histidine-independent
growing bacterial colonies, is a measure for the mutagenic potential of the test material. Low, medium, and
high doses of the extract are tested to improve reliability of the test and minimize false positives.
medium without added histidine. The number of bacteria that become histidine independent after treat-
ment is a direct measure for the mutagenicity of a material.
One of the main criticisms on this assay is that it involves a prokaryote, and the DNA structure
and DNA repair mechanisms are different from those found in eukaryotes. There is an alternative
assay using mouse lymphoma cells, L5178Y. This method of determining genotoxicity is also based
on the dependence of cells on certain vital nutrients in their medium. The mutations generated
by incubation with a test material, in both metabolically active and inactive cells, result in clonal
growth of cells under restricted medium conditions. The analysis of these mutant colonies can be
directly linked to the level of mutagenicity of the test material.
Next to rather subtle changes to the DNA sequence, also larger DNA damage can occur, which
destroys the integrity of chromosomes. The breaks in chromosomes, gaps, deletions, and exchanges
can be relatively easily demonstrated in Chinese Hamster Ovary (CHO) cells after incubation with
the test material. Staining of the CHO cells with a DNA stain and subsequent microscopic evalua-
tion can reveal a large number of different defects caused by a mutagen.
Another assay, popular for the detection of chromosomal aberrations, is the mouse bone-marrow
micronucleus test. From the bone marrow of mice, erythroblasts are isolated and these cells are treated
with the test material. When the cells subsequently divide, only unaffected cells will give rise to two
daughter cells with the entire chromosomal DNA in the nucleus. When however either the chromo-
somes or the mitotic apparatus gets damaged, small parts of chromosomes will be localized outside of
the main nucleus in a so-called secondary nucleus, or the micronucleus. This micronucleus will remain
present in the cells that will develop to form erythrocytes. These red blood cells normally do not con-
tain a nucleus, but formed micronuclei will remain present and can be easily identified by staining for
DNA. The number of erythrocytes with micronucleus is a direct measure for the mutagenicity of the
test material or chemical.
A rather new method to determine genotoxicity is the alkaline comet assay, also known under the
name single cell gel electrophoresis (SCGE). This is an ex vivo/in vivo assay that makes use of isolated
single cells from tissue or peripheral blood. The assay is depicted in Figure 13.9. Isolated cells are
Blood cell Tissue

isolation homogenization Cell culture
Isolated cells in agarose
Cell lysis
alkaline unwinding
Electrophoresis
wash and stain
FIGURE 13.9 (See companion CD for color figure.) Alkaline comet assay for determining genotoxicity of
materials. Isolated cells are incorporated in an agarose gel, lysed, and the DNA denatured. Damaged DNA will
migrate away from the original site of the cell in an electric field, resulting in comet-like structures after staining
of the DNA.
challenged with a potential genotoxic material and incorporated in an agarose layer. After alkaline
(pH > 13) treatment, the DNA remains in the agarose, localized where the nucleus was. DNA-fragments
will now migrate away from the site of the nucleus to the cathode in an electric field. The shape of the
DNA, like a comet, determines if and how much of the DNA was damaged. The nondamaged DNA
remains in the head of the comet, with the damaged DNA pieces forming the tail. The exact shape of the
comet can also indicate the type of damage that was induced. The method is still under scrutiny, and the
predictive value is under investigation [31]. Because large cohort studies in multilaboratory validation
trials are still lacking, the reliability is still not clear. The assay is increasingly popular, but small differ-
ences in the protocols between different labs lead to different results [32]. Therefore, a definitive stan-
dardized protocol will be necessary for this assay to be useful in general for biocompatibility studies.
13.4 EX VIVO BIOCOMPATIBILITY TESTING

Because the direct extrapolation of in vitro data to the human, clinical situation is not feasible and cer-
tainly not accepted, further testing of biomaterials and medical devices is required. The in vitro testing
lacks the 3D aspect in which cells in tissues normally exist. Furthermore, the interaction and communi-
cation between these cells is not present or very much inhibited in 2D cell cultures. Also in tissue-engi-
neered constructs, the small piece of tissue lacks the communication between the cells since a constant
flow of medium will dilute signaling molecules rapidly, so that no intercellular communication beyond
direct cell-cell contact is unlikely. One option to circumvent this shortcoming is to use isolated tissues
and/or organs for short-term biocompatibility testing. The problem, with this form of ex vivo testing, is
the limited time the isolated tissue can be maintained outside of the body. An incubation of more than
2–3 days is not possible since the cells in the tissue will start deteriorating, leading to nonreliable results.
Perfusion of for instance kidneys or hearts can be used to test surgical patches or implants that
deliver drugs to these organs. Another ex vivo approach is used in hemocompatibility testing, so
the contact between blood and a biomaterial. For this, the material can be formed into a tube, or
a patch of the material can be incorporated in a tube. This tube can then be attached to a vein of
a test animal or subject, and the blood can be allowed to flow through the conduit for a speci-
fied time (Figure 13.10). The blood is then monitored during the experiment to detect changes in
Shunt tubing
Artery Vein
Blood flow
Test surface
direction
FIGURE 13.10 Ex vivo testing of a blood contacting material. The material is shaped or incorporated in a
tube and attached to the circulation of an animal or human volunteer. The blood is analyzed for the generation
of thrombin or other coagulation indicators. After the incubation period, the material is studied separately for
thrombus formation on this surface.
blood composition. The appearance of for instance D-dimer indicates that a thrombus has formed
inside the extracorporal conduit, and thus the materials could be thrombogenic, so have poor hemo-
compatibility. After the incubation period, the tube can be analyzed for different parameters, among
which the formation of thrombi on the surface is the most important. Later in this chapter, the
determination of hemocompatibility is discussed separately as an example for a strategy of biocom-
patibility testing.
Ex vivo testing with perfused organs is not frequently used because of the limited incuba-
tions that are possible. In addition, it is not easy to test a series of biomaterials simultaneously,
since the number of organs of good quality is limited. Furthermore, perfusion of tissues and
organs is relatively expensive, making it an unpopular method. Finally, although the cells are
now organized in a 3D matrix, extrapolation of the results to an in vivo situation, as encoun-
tered in a patient, is not trivial. A careful prediction on the behavior of the biomaterial inside an
organ is possible. However, the lack of blood circulation and cells of the immune system being
transported to the site of implantation or contact makes this prediction limited. Therefore, the
question is if ex vivo testing has a significant role to play in biocompatibility testing. Maybe
in the future when tissue-engineered organs and tissues become readily available, this form of
testing outside the human body may pose a relevant method of testing of medical devices in
a 3D cellular environment. But up to that time ex vivo test results should be interpreted with
extreme care.
13.5 IN VIVO BIOCOMPATIBILITY TESTING: INTRODUCTION

The use of animal model systems to test surgical procedures or pharmaceuticals dates back to the
start of medical science. Most animal testing was based on vivisection, the anatomical study of
animal physiology. In addition, surgeons might have practiced operating techniques on live ani-
mals or human cadavers. However, this form of animal experimentation cannot be seen as in vivo
testing of medical implants. Up to the end of the nineteenth century, there was no significant use of
synthetic medical implanted devices. The Cruelty to Animals Act, which was introduced in Great
Britain in the early nineteenth century, regulated the treatment of animals in general as well as
the use of animals for scientific purposes. The Act of 1876 (fully called An Act to amend the Law
relating to Cruelty to Animals) stipulated that researchers would be prosecuted for cruelty, unless
they conformed to its provisions, which required that an experiment involving the infliction of pain
upon animals to only be conducted when “the proposed experiments are absolutely necessary for
the due instruction of the persons to save or prolong human life.” Additionally, the act prescribed
that the animal should only get anesthetics once and should be killed after the experiment was
finished. These regulations appear obvious to us, but up to that time several scientists denied the
perception of pain by animals. The steady evolution of medical science and synthetic chemistry
resulted in the synthesis of medical implants in the middle of the twentieth century. It also became
clear that the testing of such medical implants should include testing in a living organism, before
clinical application could be warranted. However, for a long time the in vivo testing of medical
devices was completely arbitrary and dependent on the choices made by the researchers. In addi-
tion, the patient rights improved steadily, urging the companies that were marketing the medical
devices to pay more attention to patient safety and functionality of the medical device or implant.
The safety of medical devices as well as longevity and near perfect functionality became important
for the marketing strategy of the devices. As a result, the researchers started to take more care in
the strategy of in vivo testing. The choice of animal model system was long guided by convenience
but is nowadays often directed by the suitability of the model to assess biocompatibility and func-
tionality in vivo. Still, the extrapolation of in vivo test results to the clinical situation, i.e., to use in
patients, remains a point of discussion. The limited predictive value remains a matter of concern,
and therefore recently some researchers have openly discussed the value of and need for biocom-
patibility testing in animal models.
13.5.1 Short History of In Vivo Biocompatibility Testing

The evaluation of synthetic materials and implants with in vivo test methods is as old as the use of
medical devices and implants. There is little evidence that the ancient Egyptian and Inca surgeons
used to test their procedures on animals. From current understanding, it seems almost impossible
to comprehend that also these surgeons did not test their implants and procedures in an animal
model before treating patients. One should also not forget that in the past and also in some so-called
“primitive” societies, the use of implants is widely distributed. The insertion of materials to pierce
tissue or subcutaneous implants to create a desired look (cosmetic surgery) obeys the same rules as
the implantation of a medical device today. There should be a desired response of the tissue, and
the “patient” should not be harmed by the implant. This means that by simple trial and error, some
materials were used and others were rejected because of unwanted effects or toxicity in the recipient.
In principle, this is similar to the in vivo testing that is required today before clinical application of
a medical device is considered. Of course, biocompatibility testing evolved together with improving
biological and chemical techniques. Therefore, in the nineteenth century, strict rules were adopted for
“humane” treatment of animals, and consequently more care and planning went into animal testing.
Also the risks accepted for medical treatments of patients are under constant change. Electroshock
therapy was widely used 50 years ago, and today it will be considered cruel by a vast majority of
people. Heroine and cocaine were recognized drugs for treating a variety of disorders and were ini-
tially hailed as the compounds to combat morphine addiction. Changing opinions in society influence
decision on the use of medical devices and how these are supposed to be tested prior to clinical use.
Often in the past, discovery of biocompatible materials was more or less coincidental and driven
by current technical possibilities. The use of ceramic or glass for eye prostheses was driven by the
possibility of producing these materials in the shape of eyes and coloring these to mimic the lost eye.
The materials turned out to be inert enough not to cause damaging effects in continuous contact with
the eye socket. The use of ceramic or glass eyes was thus the result of a more or less trial and error
concept. Also the clinical use of poly(methylmethacrylate) (pMMA) was initiated after it had become
clear that shrapnel from Second World War fighter planes (the windows of the cockpit) in the eyes
of pilots remained intact without adverse effects for years. So the scientists concluded that pMMA
was a biocompatible material although, in hindsight, only the biosafety of pMMA was established.
In vivo biocompatibility testing comprises the use of animal models for testing biocompatibility
as well as implantation studies in human patients. Implantation studies are the final step in testing
of a new medical device. The local and systemic response of cells and tissues is of critical impor-
tance for the efficiency of the treatment and the success of the medical device. The lack of toxicity
can be predicted from the various in vitro cytotoxicity experiments. Genotoxicity can be studied by
in vitro mutagenicity studies although a mouse-based assay has been developed, namely the comet
assay (see Figure 13.9). So for a material to turn out toxic only in the latter stage of in vivo testing is
rare. Of course, the completely assembled medical device, sterilized and packed, is generally tested
only in vivo, because in vitro testing of a complete medical device is often not feasible. The disas-
sembly of the device should be done and the parts then tested in vitro for toxicity, but this is seldom
done properly. Wear within a medical device can reveal new particles or compounds that can only
be studied realistically in vivo. Also the effect of different sterilization procedures on the medical
device is often not tested properly in vitro. Part 7 of the ISO-10993 norm describes how the residues
of ethylene oxide sterilization should be investigated, because these residues are highly toxic. Since
the sterilization procedures for clinically used medical devices is different from those in laboratory
testing, analyzing the effect of these procedures on the biosafety of the medical device is important.
13.5.2 In Vivo Biocompatibility Testing

There are some biocompatibility aspects that can only be observed in vivo, namely: (1) local toxicity,
sensitization, and irritation; (2) systemic toxicity; (3) local effects of solid implants in a 3-D tissue;
(4) degradation or wear in living organism and effects of the degradation and wear particles; (5)
functionality of the medical device in a living organism. Most of these biocompatibility aspects are
discussed in the following sections.
13.5.2.1 Local and Systemic Toxicity

The most obvious test that should be performed in vivo is local and systemic toxicity, basically a
biosafety determination. This is prompted by the requirement that the medical device should not
harm the patient. This is the logical next step after in vitro cytotoxicity, which is a good predictor
of toxicity of the material and medical device and chemicals released by these. Several levels of
toxicity can be identified, namely sensitization, irritation, and direct toxic effects on neighboring
and distant cells and tissues, called systemic toxicity. The exact tests that have to be performed are
of course guided by the intended use of the medical device as depicted in Table 13.2. However, for
all medical devices intended for clinical use, sensitization and irritation tests have to be performed.
13.5.2.2 Sensitization
Sensitization testing is rather straightforward, although in practice the test requires experience and
is time-consuming. A film of the biomaterial, or extracts of the medical implant in saline solution
or oil, is applied to the shaven skin on the back of a guinea pig. Guinea pigs are chosen because
the skin resembles the human skin in sensitization reactions. In addition, the relatively low cost of
working with these small rodents is of importance. The film of material or an extract-soaked patch
is allowed to have contact with the skin for up to several weeks. The skin can be repeatedly chal-
lenged after recovery periods of approximately 2 weeks, to observe additive sensitization reactions.
The skin reactions are analyzed, by determining the reddening and swelling of the skin, and are
compared to those in negative controls.
In another experimental setup, the material extracts are first injected into the test animal along
with an adjuvant, intended to enhance the immune response. Then after a couple of weeks, the skin
of these animals is challenged with a topical patch soaked with the extract. Again after the incuba-
tion periods, the reddening and swelling of the skin are analyzed and compared to those of a nega-
tive control (e.g., the extraction medium).
In the past decades, the use of mice for a local lymph node assay (LLNA) has been introduced
(Figure 13.11). LLNA is based on the proliferation of lymphocytes in the lymph nodes after contact
with a test material. The increased incorporation of 3H-thymidine is a direct measure for lympho-
cyte proliferation and immune reaction [33,34]. The method has a number of advantages above the
earlier described guinea pig assay in that less animals are needed for the assay and these animals
suffer much less trauma. Also the assay has a quantitative outcome, which can be objectively deter-
mined, this in contrast to the subjective evaluation by a skilled technician for the guinea pig assay.
Extensive testing of this method and comparison with the standard guinea pig assay also revealed
much less interlaboratory variation. Therefore, the LLNA is the method of choice for large-scale
sensitization and irritation testing as for instance under the new REACH (registration, evaluation,
authorization and restriction of chemicals) program from the European Union [35].
13.5.2.3 Irritation
According to the ISO definition, irritation is a “localized inflammatory response to single,
repeated, or continuous application of a test substance, without involvement of an immunologi-
cal mechanism.” The effect of chemicals on patients can be studied in different ways. The ISO-
10993-10 norm proposes first a thorough search of the literature to find data on the test materials.
Second, an in vitro cytotoxicity test as described earlier can also identify the presence of extract-
able chemicals that have unacceptable effects on cells in vitro. When no information on irritation
can be obtained by these steps, in vivo experiments should follow. Finally, noninvasive clinical
tests in human subjects are the definitive test, but they should only be performed after some initial
form of irritation testing.
Test
Test
or material
material
extract
Day 0
Day 1,2
3H-thymidine
Day 5
Isolate lymph nodes from ear
Determine 3H-thymidine incorporation
Measure for lymphocyte proliferation Day 5 + 5 h
FIGURE 13.11 Local lymph node assay (LLNA). Mice are treated with a material or material extract for a
defined period. After the incubation, 3H-thymicine is injected vial the tail vein, and after several hours lymph
nodes from the ear are isolated and analyzed for lymphocyte proliferation.
The in vivo tests are very similar to standard allergy tests. The test material fluid extracts are
injected into the skin of shaven albino rabbits. For medical devices, the use of saline and vegetable
oil is sufficient to ensure the extraction of hydrophilic and hydrophobic compounds. A set volume
of the extracts is injected and the injection sites are examined after 1, 2, or 3 days of incubation. The
resulting reaction is scored and compared to adequate controls. Depending on the intended use of
the medical device, additional tests are performed at the site of use. For this the ISO-10993-10 norm
describes a number of tests for oral, penile, vaginal, rectal, and ocular irritation tests. The ocular
irritation tests for instance are only performed for eye-contacting devices. The extract or a powder
of the test material is directly put under the lower eyelid of rabbits. This irritation testing has been
considered particularly cruel, also because it was mainly used in the cosmetic industry to test the
safety of their products. Additionally, this method was rather laborious and not very reliable for pre-
dicting human skin and eye reactions. Therefore, a number of alternatives have been developed over
the last 10–20 years. Most of these assays are however based on some form of cytotoxicity testing.
The only advance in alternatives for ocular irritation testing is that not only cultured cells are used
but also corneal tissues, isolated eyes, and pieces of epidermis. The common denominator is that
all the assays evaluate adverse responses of the tissue or cells. The readout therefore is often loss of
cell viability (as measured by MTT or viability dyes), loss of cell integrity (hemolysis, the disruption
of red blood cells), or swelling of the tissue (isolated eyes and corneas). When the in vitro tests are
performed carefully, the predictive power is as good as the tests on rabbits. At the end, none of the
in vitro assays can fully replace the in vivo or clinical testing, but the large majority of the animal
testing may be omitted in the near future [36].
13.5.2.4 Systemic Toxicity

The study of systemic toxicity is based on a rather simple idea, that when an exaggerated extract
of a test material is injected into a test animal, observation of behavior and physiology can indicate
acute or long-term toxicity. Acute effects are observed in a matter of days, while subchronic effects
take 14–28 days (less than 10% of the expected life span of the animal). Chronic effects may take
more than 10% of the animal’s life span and are therefore only required for permanent implants. At
the end of a systemic toxicity experiment, the organs can be examined and possible systemic toxic
effects can be observed by an animal pathologist. Of course, the exact way in which the experiment
is performed is vital for the reliability and significance of the obtained results. Is the extract of the
material administered once or multiple times, at what doses, what intervals are chosen, how long
are the animals allowed to recover before final examination, etc. These are all decisions that can
seriously affect the outcome of systemic toxicity testing. It will therefore not come as a surprise
that before extraction and test conditions were standardized, the variability of test results between
different labs was enormous. Additionally, the result of the tests is dependent on the researchers’
ability to observe aberrant behavior and or changes in the animals’ physiology. In addition, the
method of application of the extract or chemical is important, and several different ways have been
suggested. Subcutaneous or intramuscular injection is the most obvious but also intraperitoneal (for
oils) and intravenous (for saline extracts) are frequently used. Also application via inhalation has
become more popular especially in the toxicity study of airborne nano- and microparticles.
In general, large numbers of rats or mice are used to perform these tests. In some cases, it might
be preferable to use the implanted device in systemic toxicity tests, for instance for resorbable and
degradable implants. Then the systemic toxicity of the degradation products is a vital part of bio-
compatibility testing.
13.5.2.5 Acute Systemic Toxicity

For acute systemic toxicity, an exaggerated extract of the medical device is prepared and applied
to the animal. When toxic effects are observed with in 1 or 2 days, the material can be rejected
because of acute systemic toxicity. This acute effect is not always caused directly by the materials
comprising the medical device but can originate from contaminants, called pyrogens, that have
attached to the device during its production. Rest of ethylene oxide, which is used for steriliza-
tion of the device, can cause rapid and dramatic toxicity. Also residues of bacteria that attach to
the surface of the device, also known as endotoxins, can cause acute systemic toxicity. Therefore,
methods were designed to determine the possible contamination of materials with such pyrogens.
Often the pyrogens or endotoxins are introduced during manufacturing by the used machinery or
the people working in the production line. The endotoxins are organic molecules of bacterial origin,
like membrane components or proteoglycans that stick to the medical device. The classical assay to
detect pyrogens makes use of rabbits that are injected with an extract of the medical device or test
material. The reaction in body temperature of the rabbit is a measure for the presence of unaccept-
able levels of pyrogens. If most of the test rabbits demonstrate a rise in temperature of more than
0.5°C and the total rise in temperature over all affected rabbits in a group of 10 is more than 3.3°C,
the test materials contained unacceptable levels of pyrogens. The assay detects both chemical and
material-mediated pyrogenicity [37].
An alternative assay was developed that makes use of the lysate of Limulus Amoebocyte (the
so-called LAL test) [37]. This test replaced the rabbit testing for materials and devices. An a queous
extract of the blood cells (amoebocytes) of the horseshoe crab Limulus polyphemus is used to detect
the presence of membrane components of bacterial origin (endotoxins). The LAL test is the pre-
ferred way to detect bacterial endotoxins. The clotting of the limulus extract is strongly induced
by endotoxins, and this clotting reaction can be detected. Some of the newer available LAL assays
make use of recombinant forms of enzymes in the clotting cascade of the extract, and fluorescent
substrates replace the formation of a solid clot as readout. The commercially available LAL test
kits show a high sensitivity for endotoxins and demonstrated that it is very difficult to produce
endotoxin-free medical devices.
Another alternative in vitro pyrogens test relies on the interaction of human blood with the pyro-
gens present on the test material or medical device. Upon interaction of the leukocytes in the blood
with the pyrogens, interleukin-1 will be produced, and this can be detected using a specific ELISA.
The advantages of this assay are obvious since the blood of the targeted species (human) is used.
Additionally, the assay is cheap and sensitive to all pyrogens, not exclusively for gram-negative
endotoxins as is the case for the LAL test [38].
13.5.2.6 Subchronic and Chronic Systemic Toxicity

For long-term toxic effects, the methodology does not necessarily change, but the time span over
which the animals are allowed to be exposed to the test material is longer. The rule of thumb is
that for subchronic testing the incubation period does not exceed 10% of the animal’s lifespan.
Chronic systemic toxicity measurements take longer, in general > 10% of the animal’s lifespan, and
are therefore more expensive and only required for permanent implants. The testing for long-term
systemic toxicity can be replaced by good chemical characterization of the test material. The identi-
fication and quantification of extractable chemicals from the test material should precede biological
evaluation. If no detectable extractable chemicals can be identified, the necessity for systemic toxic-
ity testing can be irrelevant. In addition, when there is extensive literature available on the toxicity of
the exact chemicals that are identified in the extract, toxicity testing can be precluded (Figure 13.1).
The most used organism for systemic toxicity tests is the rat. This is because enough biological
materials like blood and urine can be obtained from these animals to perform a range of testing.
If not enough blood can be obtained, only limited analysis of the blood is possible and insufficient
toxicity data will be the result. Therefore, rats and in earlier times also dogs, are the animal model
of choice for systemic toxicity. After the desired time of exposure to the test material, the animal
is sacrificed and a standard pathological examination is performed. This includes the examination
of the organs, both macroscopically and microscopically. The organs are weighed and microscope
samples are prepared according to standardized protocols. Furthermore, the clinical chemistry
examination will yield valuable information concerning toxicity. The blood analysis can reveal
changes in electrolyte balance, liver and kidney functions, lipid and carbohydrate metabolism, and
many specific parameters that vary with the nature of the investigated medical device.
Of course, also systemic carcinogenicity and mutagenicity of the test material can be studied
using the comet assay that was described earlier (Figure 13.9). For this, parts of organs are homog-
enized and the isolated cells are subsequently subjected to the alkaline comet assay [31]. The ani-
mals should also be carefully examined for the appearance of unexplained tumors. The long-term
effects on stability of the DNA are of critical importance to assure safety of the material and medi-
cal implant.
13.5.2.7 Local Implant Effects

Part 6 of the ISO-10993 norm gives guidelines for examining the effects of implants on their direct
environment, although no real border, where the local environment stops, for this is given. Mostly
a test strip of a material (dimensions 1–3 × 1 × 10 mm [h × w × l]) is implanted in the paralumbar
muscle of a rat, mouse, guinea pig, or rabbit. Simultaneously, a number of nontoxic control mate-
rials are implanted in the same animal. For short-term implants, the implants are assessed after
1, 4, and 12 weeks, while for long-term implants 12, 26, 52, and 78 weeks are specified by the ISO
norm. After each incubation period, the implant is examined for the presence of a capsule and the
implant is removed together with the surrounding tissue. The negative control materials will not
have induced formation of a capsule around the implant. For further processing, often the implant
is removed, although this is not preferred since the interface between the material and the tissue is
actually the most interesting. The removal of the implant from the tissue will destroy this delicate
interface layer of tissue and diminish the quality of the data. Of course, the embedding of the tissue
with implant is critical, since a discrepancy between the mechanical properties of the embedding
polymer and the test material can lead to problems during microscope sample preparation. The
lack of necrotic cells close to the implant and the absence of inflammatory cells indicate a minimal
response of the tissue toward the implant. High numbers of inflammatory cells can be accompa-
nied by necrotic cells around an implant with inherent toxicity. The assay is rather straightforward,
although there are some pitfalls for this method of testing. The choice of model organism can be of
importance for the observed tissue effects [39]. In addition, the site of implantation should be con-
sidered carefully, since the subcutaneous implantation of a bone support device is not very logical.
For simple biosafety experiments of medical implants, subcutaneous (on the back), or intramuscular
(in the hind leg musculature) is sufficient. This is also because the animal will suffer the least dis-
comfort and pain at these sites. In addition, because the animal has two virtually identical sides,
there is always place for a control implant in the same animal.
In short, the implantation of a medical device can serve the purpose of examining the direct
response of the local, contacting tissue. The preparation of the tissue sample after the specified time
intervals is of critical importance to preserve the structures formed at the interface of the implant
and tissue. In addition, the formation of a capsule that can be observed with a low-power microscope
should be recorded, as it is a clear sign for the activation of a defense mechanism of the animal. The
embedding and staining procedures should be carefully chosen so that no structures are damaged
during the processing of the samples. The readout from the experiments is very direct, the presence
of dead cells or large numbers of inflammatory cells indicates toxicity of the implant.
13.5.3 Functionality of Medical Devices In Vivo: Choice of Model

A very important part of biocompatibility testing is the evaluation of the function of the medical
device. For instance, a heart valve needs to perform perfectly for the patient to benefit fully. Hip
replacements have to perform for a long period of time without any complications as loosening
and wear to prevent premature revision. Of course, most of the functions of the medical device can
only be fully assessed in human patients. The use of model animal systems is complicated since
the animals react often very differently to the implanted materials. Also the choice of animals and
implantation procedure can be of great influence on the results.
Let us consider the following example. The replacement of occluded blood vessels is of great
importance for a large group of human patients. Not only stenosis of coronary arteries but also of
peripheral vessel (e.g., carotid arteries and femoral-popliteal artery) can often give rise to serious
medical complications. With the sharp increase in diabetes mellitus type 2, the number of patients
with peripheral artery disease is increasing rapidly. The diabetes foot is a well-known example of
this. Therefore, the researchers of the biomedical engineering field have been looking for decades
for synthetic solutions to this problem. The Holy Grail would be a fully synthetic vascular prosthesis
that performs well over a long period of time. The prosthesis should regulate hemostasis as well as
inhibit inflammation that can lead to failure of the device. The patients that would normally receive
such a prosthesis are not healthy and mostly of higher age. It is therefore puzzling that the potential
synthetic vascular prostheses are almost exclusively tested in young, healthy animals that have no
underlying disease causing peripheral artery complications. Therefore, most of the synthetic vas-
cular prostheses perform very well in the animal models. However, when applied in humans the
efficiency of these implanted vascular grafts is disappointing [21]. The underlying problem is that
in animals the inside of the prosthesis gets rapidly covered by a layer of vascular endothelial cells.
This is possible because these cells are healthy and motile and proliferate well. In patients however
the endothelial cells do not proliferate well and do not cover the luminal side of the prosthesis.
Therefore, the regulation of hemostasis and inflammation is flawed and the implant will eventually
fail. To really get a good testing procedure, older, preferably diseased animals should be used in
biocompatibility testing, especially concerning functionality of the medical device. This is mostly
not allowed by the medical and ethical commissions overseeing the animal testing procedures.
Another example is the testing of bulking agents used for treatment of stress urinary inconti-
nence (SUI). These materials are injected next to the muscles of the pelvic floor to enhance stiffness
of the soft tissue. This stiffening then allows for better closure of the urethra, and a decrease in SUI.
The results of the SUI treatment are largely subjective since it is virtually impossible to get an objec-
tive readout (e.g., lower frequency of incontinence events and less urine leakage during an event) of
the treatment. It is how the patients perceive their situation that indicates whether the treatment was
successful and whether the patients feel that there is a decrease in urine leakage and an increase
in quality of life. It is almost impossible to test such materials for their function in animal models.
First of all, there are no real stress incontinent animal models available. Then it is very difficult to
get an objective readout of the animals concerning the treatment. The animals cannot tell how they
are doing or feeling. In such cases one feels that after well-performed biosafety experiments, there
is no other option than to apply the medical implants in the clinic and carefully assess the outcome
for each patient.
The choice of the right animal model is therefore of critical importance. Preferably, an animal as
close to humans as possible should be used. The use of primates however is highly controversial and
in most cases impossible, not only because of the extremely high costs but also because institutions
that are allowed to work with primates are rare. The political pressure to avoid the use of primates
in biomedical research is high, and it is virtually impossible to get approval for testing of medical
devices in primates. The use of large mammals, pigs, horses, goats, dogs, etc., is limited because of
the relatively high cost and limited numbers of animals. The time it takes to breed a new generation
of these animals is high, and therefore a constant flow of test animals is not available, at least not
in large numbers.
Additionally, the implantation procedure should be as similar as possible to the intended clini-
cal application. The collaboration with surgeons that work in the clinic daily is of great value to
improve the medical implant at this stage, since they can indicate exactly what can be improved to
obtain the best and most efficient medical device. Only when the surgeon is happy with the implant
and the most appropriate test model is chosen will the results of the experiments have any predic-
tive value.
A last consideration is whether the use of clonal animals that are genetically all identical is
appropriate [40,41]. In practice, the patients that await treatment are a genetically heterogeneous
pool, which will demonstrate a wide range of reactions to the device. The problem arises when
the identical genetic makeup of test animals (for instance a mouse strain or rat strain) has a direct
influence on the tissue- or systemic response to the implant or device. The results are then biased
and only valid for animals with that genetic makeup. In this way, good materials and devices could
be eliminated and missed, or some devices are considered safe and good. The increase in variation
between the test animals may improve the reliability of the obtained results. Recent studies have
shown that this increase in heterogeneity does not significantly affect the reproducibility of the
results [42]. Therefore, choosing a representative population of animals in preclinical studies would
be an improvement over the current testing procedure. It seems logical to increase this heterogene-
ity, because in this way the animal group used resembles a patient population much more closely.
To conclude, the results of animal testing in general should be treated with extreme care since
the animals are just different from the patients for whom the medical device was designed. The
final, conclusive answer whether a medical device is biocompatible can only come from the clinical
application of the device in real patients.
13.5.4 Future of In Vivo Testing

The rate of failure for medical implants in the clinic is relatively high and prompted a discussion
during the World Biomaterials Conference 2008 in Amsterdam with the provocative thesis that pre-
clinical testing in animal models is a waste of time [20]. The predictive value of many animal models
systems is limited. In addition, the in vitro use of cell lines and many ill-characterized primary cells
is in most cases imperfect and inadequate to get sensible information concerning the biocompat-
ibility of the medical device. At the end of the 2008 biomaterials conference, the consensus was that
preclinical testing is not a complete waste of time, and the severely toxic and completely nonfunc-
tional materials and devices can be filtered out quite well. However, biofunctionality testing remains
a matter of concern, and researchers should take great care in the design of their animal experiments.
Another, maybe unexpected, reason for many unnecessary biocompatibility experiments is the
reluctance to publish failed experiments or experiments describing toxic and noncompatible newly
synthesized materials, composites, implants, and devices. One can imagine that by carefully study-
ing the literature on identical materials or combination of materials, and how these performed in
vitro and in vivo, we could eliminate many animal experiments, since the fact that these materials
do not perform satisfactory has already been shown. When researchers and journals do not publish
these data, other groups will have to perform the same, unnecessary experiments, coming to the
same conclusion again.
Then there is the continuous pressure from the “animal rights and alternative experiments”
lobby, practicing political pressure to minimize the use of animals for biomedical experimentation.
Although the majority of the population in western countries is convinced of the necessity of animal
testing of biomedical devices and procedures, the pressure to invent alternatives for animal testing
may be useful to improve current in vivo experimentation or accelerate the development of proper
alternatives. Already some alternatives have been published, which could replace some forms of
animal testing.
One example we have already discussed is the use of LAL and human blood test for determina-
tion of pyrogen and endotoxin levels in medical devices (systemic toxicity). The assay has for a large
part replaced the existing rabbit assay, lowering the number of required animals dramatically.
Another example is the development of in vitro cultured skin, which resembles natural skin ever
more close. The recently published method for producing skin in the lab is a critical step in provid-
ing material for chemical and biomaterial testing in vitro on a living tissue that is identical to the
“real” tissue [42]. In this way, the number of animals needed for testing can be reduced in the near
future.
A last example is based on the use of cellular matrices, harvested from animals that are used as a
scaffold for specialized cells. The vascular network and extracellular matrix are used for supporting
and feeding specialized cells during a prolonged time [43]. This pseudo-tissue can then be used to
test the effect of chemicals or material extract on for instance liver or kidney cells. This is impor-
tant because especially in the liver, chemicals can be converted and subsequently show completely
different toxicity profiles. So by performing testing with the help of such engineered tissue-like
structures, unexpected toxicities can be minimized and thereby the number of unnecessary animal
experiments can be decreased.
With the speed of evolution of biocompatibility testing increasing steadily, it is disappointing
how few new biomaterials have reached clinical practice over the last few decades. The medical
device companies seem to stick to the old and trusted materials that have been in use for a long
time. The risk of introducing a new material is too large, and the requirements for risk assessment
and compatibility testing sometimes work paralyzing for innovation. The constant improvement
in biocompatibility testing, reducing the risk for the patient, will eventually break the deadlock
and induce a greater use of more sophisticated, better performing, and safer materials for medical
devices and implants.
13.6 IN SILICO TESTING

The development of increasingly larger databases, also in biomedical sciences, may present
a good opportunity to develop in silico biocompatibility testing procedures. In silico design
of novel drugs has been common practice for more than a decade now. However, in the field
of biomedical engineering and biomaterials science, computer-aided design is underexplored.
A potential biocompatibility database would consist of all experimental data obtained worldwide
concerning in vitro, ex vivo, and in vivo biocompatibility testing on all biomaterials that are
interesting for biomedical scientists. Of course, the completion of such a database can be con-
sidered a utopia, but the perceived advantages are enormous. First, and maybe most important,
many unnecessary biocompatibility tests can be avoided, because these have been performed
Biomaterial micro-array
Material
spots
Marrow
Mesenchymal
stem cells
Blood
interaction
Protein
Cultured
Material spot adsorption
cells
Cell adhesion Protein adsorption

Cell viability Protein activity/function
Cell proliferation Platelet adhesion/activation
Cell function Leukocyte adhesion/activation
Differentiation Blood coagulation
FIGURE 13.12 (See companion CD for color figure.) Scheme showing one possibility for high-throughput
screening of biomaterials. Small spots of the materials are deposited on a glass slide. Cells are cultured on
these spots and can be analyzed for viability, proliferation, or functionality. Alternatively, the materials can be
challenged with proteins or body fluids like blood, and the subsequent interaction analyzed.
before, at some time in another laboratory. This would save not only time, effort, and funds but
also would reduce the use of animals for in vivo testing.
An important development for such a biocompatibility database could be high-throughput
screening, which is starting to gain ground in the biomaterials field [44,45]. In Figure 13.12, a
possible scenario for high-throughput screening is depicted. A glass chip is covered with spots of:
(1) chemically different biomaterials, (2) identical materials with different surface characteristics
(roughness, topography, etc.), (3) different composites and blends of materials, (4) identical materi-
als with varying biofunctionalization (e.g., protein adsorption of attachment).
The chip can then be submitted to a number of tests to assess the different aspects of biocom-
patibility. Interaction with a cell line that is genetically engineered to indicate different levels of
cytotoxicity would be of great interest. Differentiation of stem cells into a desired differentiated cell
lineage (for instance bone forming osteoblasts from mesenchymal stem cells) is another possibility.
Cell adhesion and proliferation studies are also easy experiments that can be adapted for such high-
throughput conditions. Interaction of the materials with blood, analyzing coagulation or immune
response could prove a great tool in the design of blood-contacting materials. In fact, one can think
of many different in vitro experiments to test some aspects of biocompatibility. These data should
then be collected in an open-access database so that no unnecessary duplication of experiments is
required. In the flowchart in Figure 13.1, one of the first steps that are taken by researchers working
on a new medical device is to investigate whether biocompatibility and/or toxicity data are available
on the materials they intend to use. So it is logical that the collection of all biocompatibility tests
will help the development of medical devices and implants.
For in vivo biocompatibility experiments, it is a bit harder. There are so many different param-
eters influencing the final results, but a (central) database would be of use. If in vivo experiments
on a biomaterial, composite, ceramic, or metal alloy have already been performed, the additional
number of in vivo experiments may be reduced. The largest gain would be in saving funds and valu-
able time. The funds that are saved could then be used for new, valuable experiments. The gain in
time might eventually even save the lives of patients, because an improved treatment comes to the
clinic faster.
Of course, there are huge problems with such a database, and the chance of it ever seeing the
light of day is slim. How are the results in such a database verified? What data are entered and what
data are considered unwanted? The decision to include experimental data into a database means that
other researchers have to be able to rely on these data. Because all data concerning biocompatibility
are scattered over the scientific literature, the need for some structuring in these data would be of
great use. Anyhow, the biocompatibility database is far, far away; however, some effort should be
made to achieve this goal.
13.7 EXAMPLE: BIOCOMPATIBILITY TESTING

OF A CENTRAL VENOUS CATHETER
Central venous catheters (CVCs) are percutaneous (through the skin) catheters that are frequently
used in critical care patients. The catheters are mostly polymeric tubes that allow for monitoring
of the patients as well as therapeutic treatment, i.e., the application of medication directly in the
circulation. These catheters suffer from different problems among which the infection rate and
material-induced thrombosis are most prominent. The development of new coatings for the central
venous catheters has therefore attracted much attention of biomedical engineers and medical doc-
tors. Here we take an existing polymeric catheter with a newly developed coating as an example to
enlighten the process of biocompatibility testing. Because the coating is completely new, there are
no existing biocompatibility or toxicity data available. Therefore, the new coating material needs to
be carefully analyzed and biocompatibility tests according to the ISO10993 matrix have to be per-
formed. From Table 13.2 we can deduce that the device is an external communicating device that is
in contact with the circulating blood for a medium-long period of time (up to 30 days). The required
tests therefore are cytotoxicity (in vitro), sensitization, irritation, acute and subchronic systemic
toxicity, genotoxicity, implantation (local effects on tissue), and hemocompatibility. For the design
of the experiments, careful planning and consideration of the intended use of the device determine
how the experiments will be performed.
For the in vitro cytotoxicity, direct contact and indirect contact testing are warranted since the
new material will be in direct contact with cells and tissue. The extract used for indirect testing
is best made in medium containing serum, since this mimics the intended blood contact best. The
choice of cell strains for the in vitro toxicity tests is relatively straightforward. Fibroblasts and
possibly keratinocytes are the most obvious choices, although the use of endothelial cells could
be considered since these cells cover the inside of blood vessels. Sensitization and irritation tests
are very much standardized, and the only variation can be the way the material is presented to the
animals. In this case, it would be best to produce strips of the tube-polymer with the new coating.
These strips could then be applied to the skin of the animals. Extracts of the final CVCs can be
produced in saline and vegetable oil and applied subcutaneously or intravenous to study the acute
and subchronic systematic toxicity. Genotoxicity of the CVC can be easily analyzed with a standard
AMES- or Comet test as described in Figures 13.8 and 13.9. Systemic genotoxicity assay is not
required, since the amount of chemicals leaching from the thin coating of the CVC is extremely
low. For the study of the local effect on tissue upon implantation, a clear choice can be made.
Small parts of the catheter can be implanted subcutaneously or intramuscularly in small rodents.
Another option would be to produce a smaller version of the catheter and do the implantation study
according to clinical use, so through the skin and into a vein. This is of course more work; one has
to produce the miniature version of the catheter, and only relatively large animals (e.g., rabbits or
dogs) are suited for such a study. This approach is closer to the clinical situation but the ISO norm
does not prescribe such testing, so in this case subcutaneous implantation is for many scientists the
preferred option. The number of hemocompatibility tests is large (ISO-10993-4). Many scientists
study hemocompatibility by analyzing the interaction between the material and platelets from the
blood. Also, often the adsorption of proteins from the blood-plasma is used as an indicator for blood
compatibility. But when one carefully thinks about the function of CVCs, these tests are relatively
meaningless. In the case of CVC, the actual problem encountered is formation of thrombus on the
surface, which could lead to thromboemboli being released and vessel occlusion in other tissues.
Therefore, the most relevant experiments that address this process are the generation of thrombin
and subsequent thrombus formation at the material’s surface. Which can be done in vitro. Small
pieces of catheter are brought in contact with blood or platelet-rich plasma, and the thrombin gen-
eration is determined. Alternatively, the thrombogenicity of the CVC can be analyzed in an ex vivo
approach (Figure 13.10), attaching a tube of the CVC material to the extracorporal circulation
device of a patient. The readout will be the formation of thrombus at the surface and the generation
of thrombin and clotting indicators (e.g., D-dimer) in the contacting blood. Of course, the interac-
tion of the CVC surface with erythrocyte and leukocytes should be considered. Hemolysis of red
blood cells should be determined because the device should not harm the patient. The interaction
with white blood cells, however, is more difficult. The release of inflammatory markers in the blood
can be determined, but the question remains what the actual consequences of this proinflammatory
response for CVCs are. Activation of monocytes can result in faster blood coagulation. Because
most CVCs are only used for several days, study of long-term immunological responses is not
required for these devices.
The use of a good control material is of vital importance for the evaluation of the CVC. The
basic CVC without the new coating can be used as a control, since this device is already approved
for clinical use. The new coating should improve the performance of the CVC in the functional tests
(hemocompatiblity and implantation) and should not perform worse in the biosafety tests (sensitiza-
tion, irritation, cytotoxicity, genotoxicity).
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14 Biocompatibility
of Elastomers
Dominique Chauvel-Lebret, Pascal Auroy,
and Martine Bonnaure-Mallet
CONTENTS
14.1 Overview................................................................................................................................ 416
14.1.1 Introduction............................................................................................................. 416
14.1.2 Miscellaneous.......................................................................................................... 417
14.1.2.1 Definition.................................................................................................. 417
14.1.2.2 Classification of Elastomers...................................................................... 417
14.1.2.3 Elastomer Chemistry and Synthesis......................................................... 419
14.1.3 Silicone Elastomers................................................................................................. 426
14.1.3.1 Introduction............................................................................................... 426
14.1.3.2 Manufacture.............................................................................................. 427
14.1.3.3 Homopolymerization of Silicone Elastomers........................................... 428
14.1.3.4 Copolymerization of Silicone Elastomers................................................ 429
14.1.3.5 Fillers........................................................................................................ 431
14.1.3.6 Surface Treatments................................................................................... 431
14.1.4 Polyurethane Elastomers......................................................................................... 433
14.1.4.1 Introduction............................................................................................... 433
14.1.4.2 Manufacture.............................................................................................. 434
14.1.4.3 Mechanical Behavior of Polyurethane Elastomers................................... 435
14.1.4.4 Biodegradation of Polyurethane Elastomers............................................. 438
14.1.4.5 Biostable Polyurethane Elastomers........................................................... 441
14.1.4.6 Shape Memory Polyurethane Elastomers................................................. 442
14.1.4.7 Grafted Polyurethane Elastomers............................................................. 442
14.1.4.8 Modified Polyurethane Elastomers........................................................... 443
14.1.4.9 Biodegradable Polyurethane Elastomers..................................................444
14.1.5 Saturated Polyesters and Biopolyesters...................................................................444
14.1.6 Polyvinyl Chlorides and Phthalate.......................................................................... 445
14.1.7 Polyolefins................................................................................................................ 447
14.1.8 Styrene Copolymers................................................................................................448
14.1.9 Natural Rubbers.......................................................................................................448
14.1.10 Hydrogels.................................................................................................................449
14.1.11 Polypeptide, Collagen, Poly-Glycerol-Sebacate, Elastomers..................................449
14.1.12 Polyphosphazenes.................................................................................................... 450
14.1.13 Polyamides............................................................................................................... 450
14.1.14 Polyacrylics.............................................................................................................. 451
415
14.1.15 Fluorinated Elastomers............................................................................................ 452

14.1.16 Elastomeric Medical Devices.................................................................................. 452
14.2 Methods to Evaluate the Biocompatibility of Elastomers..................................................... 455
14.2.1 Definitions............................................................................................................... 455
14.2.2 General.................................................................................................................... 457
14.2.3 Testing...................................................................................................................... 457
14.2.3.1 Cytotoxicity............................................................................................... 457
14.2.3.2 Sensitization Assays—Irritation or Intercutaneous
Reactivity—Implantation..........................................................................460
14.2.3.3 Hemocompatibility and Blood Interaction Assays................................... 462
14.2.3.4 Biodegradation Assays.............................................................................. 463
14.2.3.5 Reproduction and Embryo Development—Assays...................................464
14.2.3.6 Genotoxicity..............................................................................................464
14.2.3.7 Biocompatibility Testing in Humans........................................................464
14.2.3.8 Biocompatibility Testing—Microbial Colonization.................................464
14.3 Biocompatibility of Different Classes of Elastomers............................................................465
14.3.1 Elastomers in General..............................................................................................465
14.3.2 Local Reactions.......................................................................................................465
14.3.2.1 Protein Adsorption....................................................................................465
14.3.2.2 Adherence and Activation of Inflammatory Cells....................................466
14.3.2.3 Local Complications.................................................................................466
14.3.3 General Reactions....................................................................................................468
14.3.3.1 Degradation...............................................................................................468
14.3.3.2 Carcinogenic Reactions.............................................................................468
14.3.4 Silicones...................................................................................................................469
14.3.5 Polyurethanes........................................................................................................... 470
14.3.6 Biodegradable Polyurethanes.................................................................................. 474
14.3.7 Polyesters................................................................................................................. 475
14.3.8 Polyvinylchloride: PVC........................................................................................... 475
14.3.9 Polytetrafluoroethylene: PTFE................................................................................ 476
14.3.10 Hydrogels................................................................................................................. 476
14.3.11 Polyethylenes........................................................................................................... 476
14.3.12 Natural Rubbers....................................................................................................... 477
14.3.13 Styrene Copolymers................................................................................................ 477
14.3.14 Poly(Glycerol-CO-Sebacate).................................................................................... 477
References....................................................................................................................................... 478
14.1 OVERVIEW
14.1.1 Introduction
Elastomers, like plastics, are polymers. The term elastomer is currently used to designate all
rubbers, i.e., natural or synthetic macromolecular materials with rubber-like elasticity. Medical
applications for elastomers appeared soon after they began being produced on an industrial scale
(Yoda 1998) following the discovery of vulcanization (crosslinking) in 1839 by the American
Charles Goodyear. Over the past 15 years, the literature on biomedical applications for elasto-
mers has grown considerably and reports have appeared regularly (Leeper and Wright 1983,
McMillin 1994).
Biocompatibility of Elastomers 417
14.1.2 Miscellaneous
14.1.2.1 Definition
While they are members of the broad family of polymers, elastomers behave differently than plastic
materials or plastomers. Generally speaking, for materials to be considered elastomers they must be
• Flexible, i.e., have low rigidity (several megapascals)

• Highly deformable, i.e., are able to withstand strong deforming forces without ruptur-
ing and have an elongation at rupture over 200% while possessing relatively high tensile
strength at ultimate elongation
• Elastic or resilient, i.e., are able to return to their original shape and size after the deform-
ing force is removed and quantitatively release the energy used to deform them
(Table 14.1) (Pinchuk 1994).

Many so-called biomedical elastomers do not meet the second criterion. While it is difficult to set
an elastic limit beyond which a polymer stops being a plastomer and becomes an elastomer, we
will use the broadest medical definition and refer to all polymers with sufficient elasticity to return
to their original shape after a substantial deformation as elastomers (Mardis et al. 1993). However,
it will occasionally be more appropriate to talk about elastic polymers than true elastomers.
14.1.2.2 Classification of Elastomers
14.1.2.2.1 The Various Families of Elastomers
There are some 15 families of elastomers, some of which include 10–20 different grades. Modern
polymerization processes increasingly allow the manufacture of customized products that address
specific application problems and provide specific elastomeric properties.
The current classification system separates elastomers into four categories (the acronyms are
based on the ISO 1629:1995 standard):
1. General-use elastomers, which include natural rubber (NR), synthetic polyisoprene (IR),
styrene butadiene copolymers (SBR), and polybutadienes (BR)
2. Special elastomers, which include ethylene propylene and diene copolymers (EPM and
EPDM), isobutylene and chloro- and bromo-isobutylene isoprene copolymers (IIR, BIIR,
CIIR), nitrile butadiene copolymers (NBR), and polychloroprenes (CR)
3. Very special elastomers with high thermal and/or chemical resistance, including silicone
elastomers (VMQ), fluoro-elastomers (FPM), chloro-polyethylenes and chloro-sulfonyl
polyethylenes (CM, CSM), polyacrylates (ACM), ethylene vinyl acetate elastomers (EVA),
ethylene methyl acrylate (EAM), hydrogenated nitrile elastomers (HNBR), and epichlor-
hydrin elastomers (CO, ECO, GECO)
4. Thermoplastic elastomers, which are in a separate category in that they do not need to be
vulcanized since they can be used like any thermoplastic material
The first two families of elastomers account for 97% of total world consumption.
14.1.2.2.2 Very Special Elastomers
Polysiloxanes or silicone elastomers are the most abundantly produced very special elastomers.
The backbone of these elastomers is not a chain of carbon atoms but an arrangement of silicon and
oxygen atoms. Their most remarkable quality is their outstanding thermal performance. They retain
their service properties from −85°C to 250°C. However, their mechanical properties are relatively
weak. They are used for a broad range of applications in the electrical, electronic, and automotive
industries. Their physiological inertness makes them well suited for use in the biomedical field.
TABLE 14.1
Some Commercially Available Elastomers Arranged by Tensile Strength
Tensile Tensile Ultimate
Trade Strength Strength Elongation
Elastomer Name Supplier (PSI) (MPa) (%)
Butyl rubber Butyl Polysar Inc., Akron, OH 700–1,500 4.8–10.3 200–250
(polyisobutylene)
Polyisoprene Santoprene Monsanto Co., Akron, OH 1,640 11.3 520
Silicone rubber Silastic Dow Corning, Midland, MI 500–1,500 3.4–10.3 200–1000
Med McGhan NuSil Corp., 750–1,750 5.2–12.1 850
Carpenteria, CA
SM Sil-Med Corp., Taunton, MA 500–1,500 3.4–10.3 250–900
Silicone rubber C-Flex Concept polymer, Inc., 1,000–2,000 6.9–13.8 200–600
copolymers Clearwater, FL
Polyvinylchloride Goodtouch B.F. Goodrich, Cleveland, OH 1,700–1,850 11.7–12.7 430–460
(PVC) Tygon Norton Perform. Plastic, 1,200–2,300 8.3–15.9 375–410
Wayne, NJ
Ethylene vinyl acetate Ultrathene U.S. Industrial Chem. Co., 2,000 13.8 700
(EVA) New York, NY
Acrylonitrile-butadiene Krynac Polysar Inc., Akron, OH 500–2,250 3.4–15.5 150–650
Polyhexene Hexsyn® Goodyear Tire CO., Akron, OH 1,500–2,400 10.3–16.5 250–350
Polychloroprene Neoprene E.I. DuPont, Wilmington, DE 1,000–2,500 6.9–17.2 200–600
Fluoro-elastomers Fluorel 3M, St. Paul, MN 1,500–2,500 10.3–17.2 100–400
Styrene butadiene Krayton Shell Chem. Co., Houston, TX 750–2,650 5.2–18.3 550–850
rubber (SBR) Plioflex Goodyear Tire Co., Akron, OH 200–2,825 1.4–19.5 290–900
Ethylene vinyl acetate EVA E.I. DuPont, Wilmington, DE 3,200 22.1 600–750
(EVA)
Natural rubber Natsyn Goodyear Tire CO., Akron, OH 2,200–3,450 15.2–23.8 500–600
(cis-polyisoprene)
Polyester terephthalate Hytrel® E.I. DuPont, Wilmington, DE 4,000–6,800 27.6–46.9 350–700
glycolated
Nylon (amorphous) Pebax Atochem Polymers, 4,200–7,400 29.0–51.0 350–680
Philadelphia, PA
Thermoplastic Tecoflex Thermedics, Inc., Woburn, MA 3,000–6,000 20.7–41.4 250–600
polyether urethanes
Thermoplastic Estane B.F. Goodrich, Cleveland, OH 4,000–8,000 27.6–55.2 400–600
polyester urethanes
Thermoplastic Corethane® Corvita Corp., Miami, FL 4,500–9,000 31.0–62.1 250–500
polycarbonate ureth
Chlorotrifluoroethylene Aclar Allied-Signal, Morristown, NJ 7,500–11,000 51.7–75.8 200–300
Saran Dow Chemical, Midland, MI 8,000–16,800 55.1–115.8 70–90
Source: Pinchuk, L., J. Biomater. Sci. Polym. Ed., 6, 225, 1994.
Fluorocarbons are another family of high-performance elastomers containing 70% fluoride,

which gives them outstanding resistance to heat and chemicals. They can be used at temperatures
up to 250°C. Their cold resistance, however, is less than that of silicones (−30°C). Because of their
passivity and insolubility, they are useful in the biomedical field.
Polyacrylates are specially designed to resist oils containing sulfur-based additives. They have
high heat (180°C) and oil swell resistance. They are used as components of grafted and linear bio-
medical elastomers (Polyurethane (PU), Styrene Ethylbutylene Styrene (SEBS)).
Thermoplastic Thermosetting
(a) B : Branch (b) : Crosslinking point
FIGURE 14.1 (See companion CD for color figure.) Schematic drawing of (a) thermoplastic and (b) thermo-
setting elastomer structure.
Chloro-polyethylenes and chloro-sulfonyl polyethylenes compete with polychloroprenes

because of very similar chemical compositions. The high chemical saturation of the polymer chain
gives them better heat resistance (120°C–130°C), and they are often used for wires and cables.
Hydrogenated nitrile, one of the most recent elastomers to arrive on the market, has hydroge-
nated double bonds, which provide it with better heat resistance (150°C) than conventional nitrile
rubber as well as with excellent resistance to oils and ozone.
14.1.2.2.3 Thermoplastic Elastomers

Polymers are commonly defined by their thermal properties, i.e., thermoplastics and thermosetting
materials. Thermoplastics are composed of independent, linear molecules (which may have side
branches). They can be melted (e.g., polyethylene) and are processed by heating. Thermosetting
materials are composed of networks of covalently linked chains. They are initially polymerized at
room temperature or by heating and cannot be remelted once they have solidified (e.g., silicone elas-
tomers). The reality is a little more complex since certain elastomers have intermediate structures
and properties (Figure 14.1).
Many types of thermoplastic elastomers (TPEs) are being developed. They have certain advan-
tages with respect to classic elastomers and are replacing them in some applications. Thermoplastic
elastomers can be defined as materials that combine the flexibility or elasticity of rubber com-
pounds with the thermoreversible versatility of thermoplastic polymers. Most require little or no
formulation. If formulation is required, it can be performed by the producer or by an intermediary
between the producer and the processor—the compounder. Thermoplastic elastomers are rela-
tively simple to use and require fewer processing steps than thermosetting elastomers (no mixing
or crosslinking), thus reducing costs. Processing time is also much shorter, reducing energy con-
sumption. Processing methods are nonspecific and more numerous, being the same as those used
for plastomers. Unlike classic elastomers, thermoplastics can be recycled. Manufacturing scraps
and clippings can be reused with little or no change in their properties. Despite these numerous
advantages, most of these elastomers do have drawbacks, including their temperature sensitivity.
According to the Freedonia Group at the 11th International Thermoplastic Elastomers Conference
in 2008, global TPE demand is expected to grow more than 6% per year and reach 3.7 million met-
ric tons by 2011 for a market value of approximately U.S$12 billion. The use of TEPs in medical
applications has risen from 3% to 5% in 10 years.
14.1.2.3 Elastomer Chemistry and Synthesis

14.1.2.3.1 Structure
Elastomers are composed of macromolecular chains that occasionally exceed 100,000 atoms in length,
fold back on themselves when at rest, and have a very high degree of self-cohesion (bond energies of
several hundred kJ/mole). On the other hand, cohesion between different chains is weak because of the
small number of bonds and their relatively low energy (covalent, Van der Waals, polar bonds). Because
of their ability to rotate around their carbon-carbon or silicon-silicon catenary backbones, the chains
can twist into various shapes and are thus very flexible at their service temperature. When subjected
to a deforming stress, these entangled macromolecular chains begin to unfold, sliding over each other,
and separating along the direction of the stress, giving the material its elastomeric property. The
greater the number of bonds between chains and the more chemically stable the bonds (crosslinking),
the less likely the material is to deform under stress. What limits rotation of the catenary backbone and
sliding is the formation of chemical bonds (generally covalent) between the chains, thereby leading to
the formation of a stable, three-dimensional network. The greater the crosslinking, the more rigid the
polymer. The formation of chemical bonds produces various active sites on the macromolecular chains
(double bonds, hydrogen atoms, labile halogen atoms, etc.). From a thermodynamic perspective, when
an elastomer is subjected to a tensile stress, it may stretch to several times its free length when the
macromolecular chains unfold. The energy transferred to the elastomer makes it thermodynamically
unstable. When the stress is removed, the macromolecules return to their initial, energetically stable,
intertwined state, releasing most of the input energy.
14.1.2.3.2 Polymerization
True polymerization only involves the formation of macromolecules by the creation of multiple
carbon–carbon bonds. However, in a larger sense, it simply means the formation of macromole-
cules. Monomers with accessible double bonds, i.e., bonds that are not obstructed by large substitu-
ents may form giant molecules. The double bonds can be rearranged by catalysts such as radiation,
oxygen, and free radicals (like those produced by the dissociation of peroxides), giving a new radi-
cal that attacks another monomer, in a chain reaction. This is called free radical polymerization.
H H H H Cl
R +C C R –C C …… — CH2 CH CH2 CH CH2 CH —
H CCl H CCl Cl Cl
The catalyst may attack the main chain, producing side chains (Figure 14.2). However, in principle, bridges
never form between two chains (see Crosslinking). Certain molecules with different functional groups
can combine into a single molecule, e.g., acid and alcohol or aldehyde and amine, etc. Molecules with dif-
ferent functional groups can combine into macromolecules. Molecules with identical functional groups
can also react with other molecules with antagonistic functional groups to produce m acromolecules.
Siloxanes are an example of this. They condense to form polysiloxanes (silicon elastomer).
H H H H
Si OH + HO Si Si O Si + H2O
H+
H H H H
CH2
CH2
CH2
CH2 CH3
CH2 CH2 CH CH2 (CH2)p CH
CH CH2 CH2 (CH2)q CH
CH2
CH3
CH2
CH3
FIGURE 14.2 An example of branched homopolymer; Polyethylene.

Another example is carbonyl chloride (phosgene) and bisphenol A, which combine to produce
polycarbonate.
CH3
n Cl CO Cl + n HO C OH
CH3
CH3
CO O C O + n HCl
CH3
n
In the two examples given earlier, for each bond created, a small molecule (H2O, HCl) is lost in a
reaction called polycondensation. In other cases, as with the formation of urethanes
CH3 CH3
a O—
— C—
—N N— — O + n HO — R — OH
—C— — CO — NH NH— CO — OR — O —
and other forms of polysiloxanes, no molecules are lost. This is referred to as polyaddition.
H H H H
Heat
Si CH3 + CH2 —
— CH Si Si CH2 CH2 CH2 Si
Peroxide
H H H H
A wide variety of catalysts (specific to each reaction) are involved in free radical polymerization
reactions (polyaddition or polycondensation), all of which lead to long polymers that concatenate
either the same monomer (homopolymers) or different monomers (copolymers).
14.1.2.3.2.1 Homopolymerization The linear bonding of structurally identical small molecules

(monomers) leads to the formation of homogenous polymers. Most commonly used biomedical
elastomers are homopolymers (Figure 14.2). However, the possibility of mixing different monomers
and/or small polymers is leading to the synthesis of new polymers with infinitely varied proper-
ties, which, because of their improved performance, are increasingly replacing homopolymers for
specific applications.
14.1.2.3.2.2 Copolymerization Different types of monomers can be copolymerized or mixed

with small reactive polymers to obtain alternating (-A-B-A-B-A-B-), sequential (-A-A-A-B-B-B-A-
A-A-B-B-B-), or random (A-B-B-A-B-A-A-B-) copolymers with different block structures contain-
ing two, or occasionally more, identical monomers (Figure 14.3).
Polyether
(–CH2)4 OCNH CH2 NHCO (–CH2CH2CH2O)n CNH CH2 NHCO–

O O O O
Urethane Diisocyanate
FIGURE 14.3 Different blocks of a terpolymer segmented polyetherurethane; Pellethane® (A-(Bn)-C).

COOH COOH
CH2 CH2
CH CH2 CH CH2 CH CH CH2 CH2 CH2 CH3
CH2 CH2
CH2 CH2
CH2 CH2
CH2 CH2
COOH COOH
CH2 CH2
CH CH2 CH CH2 CH CH2
CH2 CH2
CH2 CH CH2 CH2 CH2 CH3
CH2 CH2
Acrylic polyacid
Ethylene-hexene grafted copolymer
grafted polyethylene
FIGURE 14.4 (See companion CD for color figure.) Monomers attached to existing chains to create
branched or grafted copolymers.
Other types of monomers or small polymers can also be attached to existing chains to create
branched or grafted copolymers (Figure 14.4). Alterations to the catenary or terminal structures of
copolymers change their properties, creating new types of elastomers, some of which have poten-
tially interesting biomedical applications. It would be more accurate to talk about bi and terpoly-
mers when a reaction involves two or three types of monomers, but the term copolymer is used to
describe all polymers containing two or more different types of monomers.
14.1.2.3.3 Crosslinking
When polymers have reactive sites within the chain (e.g., the double bonds of unsaturated polyesters) or at
the terminal (e.g., the hydroxyl group of polyols), they can be reacted with a polyfunctional agent to cre-
ate a network in a process referred to as crosslinking or vulcanization. Such agents are generally called
catalysts, hardeners, or coupling agents, and the initial polymer is called a prepolymer (Figure 14.5).
Crosslinking can be performed by free radical polymerization using organic peroxides, which
greatly increases the thermal resistance of the network by creating very strong carbon-carbon
bridges. Other types of crosslinking agents can be used, especially for elastomers with specific
reactive groups (carboxyls, halogens, etc.).
14.1.2.3.4 Formulation
Apart from a few rare exceptions, the properties of “green” elastomers do not allow them to be used
as is. Various additives, all with well-defined functions, must be incorporated. This is what is called
formulation. Despite a certain empirical approach, formulation requires a great deal of know-how to
compensate for the occasionally antagonistic properties required by certain applications.
A standard formulation contains
• Elastomer(s): Specific properties, especially chemical properties, must address application

specifications. A mixture of elastomers may be used.
• Filler(s): Fillers are used to provide superior mechanical properties, and also to improve
handling and to lower production costs. Precipitated silica is the most common additive in
biomedical elastomers. Chalks and kaolins are also occasionally used.
• Plasticizers: Plasticizers are used to make processing easier and improve low temperature
flexibility. Heavy solvents, oils, and esters are generally used.
• Protective agents: These are used to protect the macromolecular chains from oxygen,
ozone, and ultraviolet radiation. Amine and phenol derivatives are generally used.
(a) (b)
FIGURE 14.5 Crosslinking of a polymer. (a) Crosslinking of a polymer with reactive side groups.
(b) Crosslinking of a polymer with reactive terminal groups.
• A crosslinker: These allow the formation of a stable, three-dimensional network without

which the elastomer would be useless.
• Various other ingredients: Numerous other ingredients with very specific functions such as
colorants, conditioning agents, swelling agents for manufacturing cellular elastomers, etc.
It is clear that the release of additives like plasticizers, antioxidants, initiators, catalysts, fillers, etc.
by biomedical elastomers may have a significant impact on host tissues.
14.1.2.3.5 Elastomer Reinforcement

Reinforcement fillers are quite often added because elastomers have fairly low breaking strengths
at ambient temperature. Adding more precipitated silica enhances breaking strength, but decreases
flexibility. The amount of filler can be changed to generate variable stress-strain curves and pro-
duce elastomers with specific properties. Fillers like semi-reinforcing kaolins can be used to gen-
erate economical, clear mixtures. The same is true of chalks, which are considered as diluents.
Elastomers are also occasionally reinforced by the addition of bulk fibers or tissues (glass, carbon,
aramide) for very specialized aeronautics and medical applications for example.
Exogenous reinforcing materials can affect the biocompatibility of finished materials. However,
natural networks composed of two or three interpenetrating polymers can have major advantages.
Interpenetrating networks are based on at least two polymers chemically linked to form at least one
network and are synthesized in the presence of or at the same time as the other constitutive poly-
mers (Sperling and Hu 2002, Sperling and Misra 1997). The properties of interpenetrating networks
depend on the kinetics of formation, network crosslinking, and polymer miscibility during synthesis.
However, numerous other factors play a major role, including the synthesis method itself, the com-
position of the polymers and their respective quantities, the density of potential crosslinking sites of
each polymer, their glass transition temperature, and their crystallinity (Lipatov 1990, Xu et al. 2000).
Many classes of polymers can be used to synthesize interpenetrating networks, notably polyure-
thanes, which readily lend themselves to such a use. The mechanical properties of the synthesized mate-
rials are improved while there is little or no effect on their biocompatibility (Karabanova et al. 2006).
14.1.2.3.6 Surface Modification of Elastomeric Biomaterials

In recent years, progress in understanding the interactions between biomaterials and cells has
revealed that the predominant factors in play are the physical and chemical properties of the surface
of the biomaterials. The development of tissue-engineered biomaterials, that is, biomaterials capa-
ble of eliciting cell behavior (adhesion, migration, proliferation, differentiation, etc.) is testimony to
this progress. The adhesion of cells to a biomaterial surface is a sine qua non for eliciting other cell
behaviors by the biomaterial. The fact that cell adhesion is influenced by the surface properties of
biomaterials (wettability, surface energy, roughness, topography, ionic interactions, electric charge,
etc.) explains why so much effort is expended to modify surface properties to improve biological
interactions. Of course, these modifications do not alter the bulk properties of the materials.
Surface modification techniques make it possible to change the morphology and physicochemi-
cal properties of the surface and to deposit biologically active molecules such as peptides and pro-
teins on it. (Table 14.2) (Jiao and Cui 2007). Ma et al. recently published a complete review on this
topic (Ma et al. 2007).
14.1.2.3.7 Elastomeric Biomaterials and Micro- and Nanotechnology

The emergence and rapid development of nano- and microtechnologies—i.e., the development and
exploitation of structures and devices at nano- and micrometer levels—has opened vast new vistas
in the life, health, biomaterials, and biomedical elastomer sciences. Nanotechnologies and nano-
manufacturing can be used to optimize the polymer structure, formulations, strength, and surface
properties of elastomeric biomaterials. Nanomanufacturing relies on the mastery of surface science,
especially in biology. The nano- and microtopographies of surfaces are as important as physico-
chemical properties for biological interfaces (Kasemo 1998).
The creation of surfaces that copy the molecular organization of natural tissues at the nanometer
level makes it possible for biomaterials to present the desired biological signal with much greater
precision. The biomolecules used in what are called “precision immobilization strategies” to create
biomimetic surfaces include proteins, polypeptides, nucleotides, lipids, and polysaccharides. The
biomolecules immobilized on the surface of the material in one position, one conformation, and
one specific density provide the surrounding tissue with one surface organized to better control
cell interactions. (Castner and Ratner 2002). For example, the deposition of nanostructure films
of amphiphilic macromolecules, that are macromolecules with both hydrophilic and hydrophobic
domains, has been used to control cell adhesion (Hung et al. 2009b, Jiao and Cui 2007).
Nanoencapsulation of elastomeric biomaterials is another approach for optimizing surface prop-
erties through the deposition of nanofilms. Based on its physicochemical properties and thickness,
a nanofilm deposited on the surface of a biomaterial will behave as a selective barrier regulating
exchanges between the underlying biomaterial and the surrounding tissue. For example, encapsulat-
ing the silicone-hydrogel of contact lenses with a plasma-deposited nanofilm prevents the absorption
of proteins by the hydrogel and the diffusion of constitutive macromolecules from the material while
remaining permeable to small molecules such as water, carbon dioxide, and oxygen (Yasuda 2006).
Nanofibers are found naturally in living tissues. The fabrication of biomaterials with synthetic
biomimetic nanofibers either incorporated in the biomaterial, exposed on their surfaces, or formed
into networks has given rise to numerous biomedical applications, tissue engineering scaffolds, and
drug delivery systems. Most of the polymer fibers, which have diameters ranging from a few nano-
meters to a few microns, are produced by electrospinning.
TABLE 14.2
Surface Modifications of Elastomeric Biomaterials
A. Surface modification techniques used to produce various surface properties of biomaterials
Type of Modification Properties of Modified Surface
Morphological 1. Coating containing pores to encourage tissue ingrowth (porosity
modifications and roughness)
2. Grooved surface to prevent epithelial down-growth on dental
implants and direct bone formation along the particular region
of an implant
Chendeal 1. Glow discharge to increase surface free energy and tissue adhesion
modifications (composition and charge)
2. Crosslinked polymeric surface to decrease surface permeability
and increase surface hardness
3. Plasma treatment with reactive gases to create new functional groups
on polymer surface
4. Ion implantation to cause chemical and structural alterations
to increase corrosion/wear resistance
5. Grafting macromolecules such as PEO to reduce protein adsorption
and cell adhesion. Functional groups used to produce positively
(amino) or negatively (acidic and sulfonate) charged surface
Biological 1. Immobilizing molecules (ROD, heparin/heparin sulfate-binding
modifications peptides, proteins like fibronectin and grotl1 factors) on biomaterials
to enhance cell adhesion and cell growth
2. Immobilizing phosphorylcholine on biomaterials to resist protein and
cell adhesion for applications where the material comes into contact
with blood
B. Surface modifications with biomacromolecules

Modification Mechanism Method
Chemical graft Immobilizing bioactive peptides, Chemical graft, photochemical
proteins, and polysaccharides to graft, plasma graft
improve cell adhesion and
biocompatibility
Surface coating Immobilizing bioactive Physical adsorption
macromolecules and growth
factors to promote cell affinity
Entrapment Immobilizing bioactive or Swelling in a mixture solution and
water-soluble biomacromolecules entrapment in a nonsolvent to
to improve hydrophilicity and produce a stably modified surface
blood compatibility
Electrostatic Immobilizing charged, hydrophilic Depositing polyanions and
and bioactive macromolecules polycations alternately
Source: Jiao, Y.P. and Cui, F.Z., Biomed. Mater., 2, R24, 2007.
This process is being used in protective clothing, air filtration, template nanofiber, and poly-
mer nanotube applications. It uses an electrical charge to draw very fine fibers from a liquid.
The process is noninvasive and does not require the use of coagulation chemistry or high tem-
peratures to produce solid threads from solution. This makes the process particularly suited to
the production of fibers using large and complex molecules of nano- and microfiber biomateri-
als. These fibers can be used, among other things, to create nanofiber tubes from biodegradable
polyester to guide the regeneration of peripheral nerves, as well as bone, muscles, and endothe-
lial tissues (Berkland et al. 2004, Bini et al. 2004, Gu and Ren 2005, Kim et al. 2003, Kwon et al.
2005, Luu et al. 2003, Mo et al. 2004, Wutticharoenmongkol et al. 2006a,b, Yang et al. 2005).
The addition of metallic nanoparticles makes it possible to produce polymer–metal composites
with better properties than elastomeric biomaterials. For example, adding gold nanoparticles
to polyurethane induces a morphological transformation of the surface of the nanocomposite,
providing it with long-lasting resistance to biodegradation (Chou et al. 2008, Hsu et al. 2006).
Many biomaterials never come in direct contact with living tissues as such, just with molecules,
proteins, fluids, and tissue extracts of living organisms. This is the case for materials specifically
developed for biological laboratory applications. Micro- and nanobiotechnologies have also given
rise to new applications in this area. The infinitesimal machines that Richard Feynmann dreamed
of (Feynmann 1992, 1993) are now a reality in a broad range of major applications in cellular
biology, immunological testing, DNA sequencing, and protein crystallization (Balagadde et al.
2005, Fu et al. 2002, Hansen et al. 2002, Hong et al. 2004, Jiang et al. 2003, Kartalov and Quake
2004, Liu et al. 2002, 2003, Murakami et al. 2004, Sohn et al. 2000, Wu et al. 2004).
Many of these nanomachines are composed of polydimethylsiloxane elastomer, which is used
in various nanolithography techniques (Kuncova-Kallio and Kallio 2006, Rogers and Nuzzo 2005,
Wacaser et al. 2003, Whitesides et al. 2001). The moldability of silicone elastomers makes it possible
to reproduce details less than 10 nm in size (Kartalov et al. 2006). In addition, they have no effect
on molecules, proteins, cells, or tissues because of their chemical inertness and biocompatibility.
They are thus often used to construct microfluidic channels that consist of a surface micromachined
labyrinth with several inlets and outlets.
Soft polysiloxane elastomers have a promising future in micro- and nanotechnology biomedical
applications and research (Kartalov et al. 2006, Quake and Scherer 2000).
14.1.3 Silicone Elastomers
14.1.3.1 Introduction
Silicones (a contraction of silicon ketone) make up a vast family of polymers with remarkable
properties due to the presence of both silicon–oxygen and silicon–carbon bonds. Depending
on the nature of the functional groups attached to the silicon atoms, silicone polymers may be
resins, oils, or elastomers, making them well-suited for diverse applications in a wide variety
of areas. Silicon elastomers are composed of very long polymers containing several thousand
silicon atoms per molecule (Bischoff 1972). The first biomedical applications for silicone elasto-
mers can be traced back to the 1950s, while industrial-scale manufacturing dates to 1965 (Jones
1988, Toub 1987). Today, silicone elastomers are the most widely used polymers in medical
applications (Yoda 1998) because of the strong, very mobile bonds of their Si-O-Si (siloxane)
catenary backbone, which provide elevated chemical inertness and exceptional flexibility. They
are also very stable over time and at body temperature, show little tissue reactivity, and are
highly resistant to chemical attack and heat, which allows them to be autoclaved. They also
have exceptional mechanical properties such as high tear strength, outstanding elasticity, and
high gas permeability, which make them suitable for many medical applications (contact lenses,
special dressings, air/blood filter membranes, tissue expanders, artificial dermises, artificial
esophaguses, intraocular lenses, artificial cornea, etc.) (Boone and Braley 1966, Ren et al. 2008,
Vondracek 1981) (Table 14.3).
TABLE 14.3
Properties of Silicones for Use in Biomedical Applications
Good elastomeric and relatively uniform properties over a wide temperature range
Physiological indifference
Good low-temperature resistance and stable at high temperatures
Excellent resistance to biodegradation
Excellent resistance to oxidation and ultraviolet light
Moderate biocompatibility
Outstanding resistance to aging
Excellent dielectric behavior over a wide range of temperatures
14.1.3.2 Manufacture
Chlorosilanes, especially dichlorosilanes and trichlorosilanes (where R is an organic radical), are
the basic building blocks of silicone elastomers.
R R
Cl Si Cl Cl Si Cl
R Cl
The main organic radicals used are methyl (−CH3) and phenyl (−C6H5) groups. Others (ethyl, vinyl,
etc.) are used for special application silicones. Chlorosilanes are complicated and costly to produce.
They react with water to give silanols, which, being unstable, lose water molecules and condense
into polymers—polysiloxanes.
R R R
n Cl Si Cl + 2n H2O n HO Si HO + 2n HCL HO Si O H
R R R n
Depending on the dichlorosilane-trichlorosilane ratio, different polymers are obtained. The greater
the percentage of dichlorosilane, the more linear the polymer, and the greater the percent of tri-
chlorosilane, the more branched the polymer. Depending on the reaction conditions, polymers with
varying molecular masses can be produced. The terminal hydroxyl groups can react with other
components or with each other in the presence of catalysts. The termination reaction produces the
final silicone product.
CH2 CH3 CH3 CH3 CH3 CH3
HO Si O Si O Si O OH + H2O CH3 Si O Si O Si CH3
CH3 CH3 n CH3 CH3 CH3 n+2 CH3
CH3 CH3
CH3 Si O Si CH3
CH3 CH3
The properties of the finished product depend on the nature of the organic radicals grafted
onto the catenary backbone, meaning that an infinite number of chemical structures—and
properties—are possible.
The end products of this industrial process are polycondensates of basically linear or weakly
branched polysiloxane molecules of varying molecular masses and consistencies with terminal
reactive groups such as –Cl or –OH. These reactive polycondensates or prepolymers serve as the
raw materials to produce the final silicone products by room temperature vulcanization (RTV) or
high temperature vulcanization (HTV).
14.1.3.3 Homopolymerization of Silicone Elastomers

Most commonly used biomedical silicone elastomers are homopolymers. Increasingly, however,
various polysiloxane prepolymers as well as other types of prepolymers with varying properties
are being combined to synthesize copolymers for the biomedical market. Different polymerization
processes can be used to produce a variety of silicone elastomers.
One-component RTV silicones result from a reaction between atmospheric moisture and a mixture
of polycondensates and catalyst. Crosslinking thus occurs from the outside in, limiting the useful
thickness.
H H H H
Si OH + HO Si Si O Si + H 2O
H+
H H H H
Such silicones are only used for adhesives, coatings, and sealants. Their biomedical utility
arises from the absence of fillers and additives, which gives them excellent biocompatibility
(Ashar et al. 1981).
Two-component RTV silicones result from a reaction between a polycondensate and a crosslinker
added to initiate the reaction. They are used in the medical field for limited production runs of
molded products.
• Crosslinking can occur by polycondensation of a relatively low molecular weight polydimethyl

siloxane with a reactive hydroxyl group in the presence of tin octoate (i.e., condensation cure)
H H H H
Si H + HO Si Si O Si + H2
Metal
H H salt H H
• By polyaddition of a relatively low molecular weight vinylsiloxane, a polysiloxane with

a terminal hydrogen, and an organometallic catalyst like chloroplatinum acid (e.g., RTV
71556 Silbione from Rhodia, formerly Aventis or Rhône-Poulenc).
H H H H
Si H + CH3 CH Si Si CH3 CH2 Si

Organometallic catalyst
H H H H
HTV elastomers are produced by heat vulcanizing similar but higher molecular weight polyconden-
sates premixed with crosslinkers (peroxides).
H H H H
Heat
Si CH3 + CH2—
— CH Si Si CH2 CH2 CH2 Si
Peroxide
H H H H
They are used in small and large-scale industrial production runs by injection molding, extrusion,
calendaring, etc. (e.g., MDX 4 4210 Silastic from Dow Corning).
14.1.3.4 Copolymerization of Silicone Elastomers

Silicone elastomer copolymers can be produced by addition or condensation. Condensation gener-
ally involves low to medium molecular weight prepolymers. For example, vinyl polymethylsiloxane
is condensed with hydroxy polymethylsiloxane to create a new prepolymer—siloxyethanol ether—
which can in turn be condensed with diisocyanate to produce silicone urethane copolymers whose
blood compatibility and stiffness have been improved to the point where they can be used as blood
catheters or blood pump coatings.
CH3 CH3 CH3
*HO Si O Si O n
Si OH*
CH3 CH3 CH3
Silanol terminated
CH3 CH3 CH3

*CH Si O Si O n
Si CH*
CH3 CH3 CH3
Vinyl terminated
*CH2
CH3 CH CH3 CH3
CH3 Si O Si O Si O Si CH3
m n
CH3 CH3 CH3 CH3

Vinyl terminated
CH3 *H CH3
CH3 Si O Si O n
Si CH3
CH3 CH3 CH3
Polymethyl hydrosiloxane
Examples of different reactive silicone prepolymers (Whitford 1984)

Me Me Me
HO (Si O)n Si OH CH2 CH2(Si O)n CH2 CH2
Me Me Me
O
+ +H2O
CH2 CH2
Me
HOCH2CH2 O (Si O)n CH2CH2OH
Me
Siloxyethanol ether
Silicone urethone OCN R΄ NCO

Di-isocyanate
Me
CH2CH2 (Si O)n CH2CH2 OCONHR΄ NHCOO
Me
Polysiloxane copolymers — condensation
Copolymerization (Whitford 1984)

Condensation has also been used to produce silicone polycarbonate copolymers designed to trans-
port ions. Their good mechanical resistance and high stability in aqueous environments make them
useful for microelectrodes.
CH3 CH3 CH3 O O
NH(CH2)3 (Si O)x (Si O) y Si(CH2)3NH C ( O BPA O C) z
CH3 CH3 CH3
CH2CN
n
m
BPA = Bisphenol A
CH3
HO C OH
CH3
Silicone polycarbonate
(Whitford 1984)
Certain linear silicone elastomer copolymers are also prepared by addition curing. These are gener-
ally higher molecular weight polymers that can be processed or molded using industrial processes.
Siloxane and methacrylate copolymers were developed to improve the properties of elastomer con-
tact lenses (increasing their hardness, stiffness, transparency, wettability, and gas permeability) by
combining the properties of the various monomers used to produce the copolymer.
CH2 = CCOCH2)3Si(OSiMe3)3
CH3
Methacrylaxypropyltris (trimethylsiloxy) silane
O Me Me
CH2 = CCOCH2)3SiOSiMe
CH3 Me Me
Methacryloxypropylpentamethyldisiloxane
O Me Me
CH2 = CCOCH2)3Si(OSiMeSiMe)3
CH3 Me Me
Methacryloxypropyltris (pentamethyldisiloxanyl)-silane
O OH OSiMe3
CH2 = CCOCH2)2OCH2CHCH2O(CH2)3SiMe
CH3 OSiMe3
Various siloxane methacrylate copolymers (Whitford 1984)

Graft silicone elastomer copolymers can have two types of grafted side chains, one type on all the
molecules in the elastomer and a second added by graft polymerization at the surface (see Surface
Treatments). Silicone elastomers can be mixed relatively easily with numerous copolymers
(Styrene butadiene styrene [SBS], SEBS, Polypropylene [PP], PU). Silicone elastomer copolymers
are all very biocompatible because the addition of silicone gives them the elasticity that only the
incorporation of plasticizers (phthalates) would otherwise provide. They are suitable for large-
scale production runs by molding or injection because of the thermoplasticity provided by the
SBS, SEBS, and PP copolymers. They also combine the surface properties of silicones with the
intrinsic mechanical performance of the added copolymers. Polyurethanes are good examples of
copolymers with greatly enhanced blood compatibility and mechanical strength. Polyurethane
products are produced by mixing a vinylsiloxane polycondensate with a curing agent (crosslinker)
followed by injection into an appropriate mold. The components crosslink to form a thermoplastic
silicone–polyurethane network with remarkable physical and biomedical properties. These bio-
medical silicone elastomers can be further modified by the addition of other components before
the final cure. These components may or may not be involved in the crosslinking, and they can
have an impact on the physical and chemical properties and thus on the biocompatibility of the
final elastomer products (Kossovsky 1995, Kossovsky and Freiman 1995).
14.1.3.5 Fillers
Biomedical silicone elastomers may be considered composite materials in the sense that they are
composed of organomineral polymer matrixes containing fillers that are more or less linked to
the network. Fillers are used to improve the mechanical performance of elastomers, notably to
increase tear and tensile strength. They are generally mineral in nature, but may also be organic,
like high molecular weight polyacrylic acid (Maturri et al. 1991). Amorphous silica is the most
widely used filler and can be modified using a coupling agent that improves adherence to the poly-
mer matrix (sizing). This is generally done by silanization because the most common coupling
agents are silanes like Y X3Si(CH2)nY, where n = 0 to 3, X is the hydrolysable group, and Y is the
organic group selected on the basis of the polymer matrix. Silica with reactive Si-H covalent bonds
can be obtained by a reaction with methyldichlorosilane for example (Yoda 1998). Other more spe-
cific treatments have been reported like the attachment of antithrombogenic heparinoid substances
to amorphous silica or nanohydroxyapatite fibers (Khan et al. 2008, Von Recum and LaBerge 1995,
Von Recum and Van Kooten 1995). Other fibers (glass, aramide, carbon, etc.) can also be used to
reinforce the structure of biomedical silicone elastomers. Sizing the fibers ensures good adherence
between the reinforcement and the matrix (Auroy et al. 1996).
The fillers must never reduce material biocompatibility, either because they are not biocompat-
ible themselves or are released in situ due to insufficient bonding to the polymer.
14.1.3.6 Surface Treatments

Changes to the basic silicone elastomer may be insufficient for special applications. The desire to
improve the materials has led to a number of surface treatments to optimize the biocompatibility
of surfaces in contact with living tissue, seal in undesirable residues or additives using a coating,
and regulate excretion and/or absorption by the sealed elastomer using a selectively permeable sur-
face (Colter et al. 1977). Silicone elastomers are hydrophobic and have high coefficients of fric-
tion, which in combination with their low surface energy (10–18 dynes/cm) ensures low wettability
(Brown 1995, Mardis et al. 1993). While these biophysical characteristics can be advantageous
(no tissue adherence to mammary implants, no bacterial adherence (Rodrigues et al. 2007), no
sliding of urethral stents or blood catheters (Bambauer et al. 2004) due to surface friction caused by
contact with living tissue), they can cause numerous biological and technical problems that need to
be solved if they are to be used for other applications.
Various surface treatments can be performed using a variety of techniques:
• Plasma deposition of simple substances or composites. Heating a gas to a very high

temperature using an electric arc (electric glow discharge) causes extreme atomic exci-
tation resulting in partially or almost totally ionized gases, or plasmas. When the ions
come into contact with a “cold” surface, they are deposited and become chemically
bonded to the surface. Exposure of elastomers to such plasmas (He, Ar, etc.) allows the
deposition and chemical bonding of monomers or polymers to the exposed surface in a
process called plasma polymerization, or glow discharge polymerization to avoid con-
fusion with blood plasma. The atomic, molecular, or macromolecular deposits obtained
using this technique create a thin, smooth film several Å to several hundred Å thick.
Most polymers or molecules with suitable biological or technical properties (polyeth-
ylene, polyvinylchloride, polytetrafluoroethylene, polycarbonate, polymethylmethacry-
lates, polysiloxanes, acrylic acid, allylamine, etc.) (Ren et al. 2008) can be applied to
silicone elastomers.
The plasma deposition of H2 +, N2+, O2+, and Ne+ ions breaks existing chemical bonds
on the surface and creates new radicals such as =C=O using O+ plasma for example. The
affinity of certain proteins (albumin, fibrin) for the surface of silicone elastomers modified
in this way creates a layer of adsorbed protein that prevents the adherence of other blood
constituents by steric repulsion, a well-known effect in colloidal chemistry (Park and Park
1996). The result is a powerful antithrombogenic effect.
The deposition of ionic oxygen, argon, carbon dioxide, or ammonia makes the silicone
elastomer surface more hydrophilic. However, the migration of free polymer to the elasto-
mer surface affects the durability of such treatments (Everaert et al. 1995, Hsiue et al. 1996).
The deposition of these ions also allows for polymer grafting on the elastomer surface.
• Surface polymer grafting is a more long-lasting solution, but more technically compli-
cated. After argon plasma or solvent activation, the silicone elastomer is placed in a reac-
tor containing a monomer solution (acrylic acid, hydroxyethyl methacrylate, bis amino
polyethylene oxide, etc.). Heating promotes chemical bonding of the monomers to the
elastomer and crosslinking among the grafted monomers, producing a smooth, continu-
ous coating of grafted copolymer (polyacrylic or polyethylene for example) covering the
elastomer surface. By alternating the chemical treatment of the various surfaces of an
elastomer, a bifunctional, homobifunctional, or heterobifunctional material can be created
(Figure 14.6). Such treatments have been used to improve the adherence and growth of
Polymer type A
Bifunctional Silicon rubber Polymer type B
SR-grafted-polymer type A
Homobifunctional Silicon rubber
Polymer type A-grafted-SR-grafted-polymer type A
Eterobifunctional Silicon rubber
Polymer type A-grafted-SR-grafted-polymer type B
FIGURE 14.6 Schematic diagrams for grafted silicon rubber surfaces.

corneal epithelial cells by grafting 2-hydroxyethylmethacrylate to the outside surfaces of

silicone elastomer corneas and to prevent adherence by grafting bis amino polyethylene
oxide to the inside surfaces (Lee et al. 1996a,b).
• The denucleation of silicone elastomers helps remove the micro air bubbles in the sur-
face irregularities of the elastomer. The goal is to remove air/blood interfaces to prevent
complement activation and platelet aggregation. The procedure is relatively long but tech-
nically simple. It involves rinsing the elastomer in double distilled water for 12 h, soaking
in 99% ethanol for 24 h, soaking in a buffer solution with vacuum degassing for 6 h, then
gradually replacing the buffer solution with ethanol over a 6 h period. The elastomer is then
stored in the buffer solution until used (Kalman et al. 1991). This treatment helps increase
hemocompatibility without modifying the structure or composition of the silicone elasto-
mer surface.
• Acylation or hydroxylation of the silicone elastomer surface increases its affinity for serum
proteins (Tsai et al. 1991). Hydroxylation is performed by an oxymercuration/demercura-
tion reaction or hydroboration, which transforms the polydimethylsiloxanes on the surface
into hydroxymethylvinylsiloxane. Acylation of -OH groups is achieved through simple
esterification of exposed carbons on the siloxane side chains (Tsai et al. 1991).
14.1.4 Polyurethane Elastomers

Otto Bayer developed the first polyurethanes in the late 1930s. They now make up the largest fam-
ily of thermoplastic and thermosetting plastics and range from very rigid to very flexible and from
compact to cellular. They are used to produce varnishes, fibers, textiles, plastics, and elastomers.
Polyurethane elastomers were studied in great detail in the 1950s and 1960s (Bonart 1968, Cooper
and Toblosky 1966, Frisch and Saunders 1962). Pangman (Pangman 1958), Boretos (Boretos and
Pierce 1967), and Yoda (Yoda 1998) proposed their use in biomedical applications (Pangman 1958).
They have been widely used in the medical field for over 35 years now (Autian 1974, Boretos
and Pierce 1967, Coury et al. 1984, Lelah and Cooper 1986, Stokes and Cobian 1982, Wilkes and
Samuels 1973) to manufacture biomaterials that come into contact with blood, including catheters,
valve protheses, artificial blood vessels, etc. They have also recently been used to fabricate micro-
sphere vectors for drugs and bioactive substances, tissue-engineered scaffolds (membranes, biode-
gradable porous polymer structures, etc.) (Bouchemal et al. 2004, Haugen et al. 2006, Shukla et al.
2002). Cf lower 1 4 7 modified polyurethane elastomers (Table 14.4).
They are produced by polyaddition reactions between polyisocyanates and polyalcohols (polyols).
Broadly speaking, all elastomers are isocyanate derivatives.
TABLE 14.4
Current Biomedical Applications of Polyurethanes
Blood bags, closures, fittings Leaflet heart valves
Blood oxygenation tubing Mechanical heart valve coatings
Breast prostheses Orthopedic splints, bone adhesives
Cardiac assist pump bladders, tubing, coatings Percutaneous shunts
Catheters Reconstructive surgery materials
Dental cavity liners Skin dressing and tapes
Endotracheal tubes Surgical drapes
Heart pacemaker connectors, coatings Suture materials
Hemodialysis tubing, membranes, connectors Synthetic bile ducts
Lead insulators, fixation devices Vascular grafts and patches
14.1.4.2 Manufacture
Polyurethane elastomers are formed by reactions between isocyanate and substances with mobile
hydrogen ions, generally polyols, amines, and water.
R–N=C=O+H—A R — NH — C = O
—
A
Alcohols give urethanes:
R − N = CO + H − OR ′ → R − NH − CO − OR ′
Nontertiary amines give ureins or substituted ureas.

Water also give ureins together with carbon dioxide.
In most cases, the components, i.e., the raw materials, the catalysts, and the adjuvants, are mixed as
liquids and then injected into molds or cavities. Aromatic polyisocyanates are the most frequently
used raw materials. Toluenediisocyanate (TDI) is used as is or is dimerized to synthesize elastomers
with high tear resistance. Diphenylmethanediisocyante (MDI) and naphthylenediisocyante (NDAI)
are used to produce polyester elastomers.
CH3 CH3
NCO OCN NCO
N–CO
2-4 2-6
Toluene diisocyanate (TDI)
O
OCN
C
CH3 N N CH3
C NCO
O
Dimerized toluene diisocyanate (TDI)
OCN CH2 NCO
Diphenylmethane 4-4′diisocyanate (MDI)
NCO
OCN
Polyether
soft segment
(–CH2)4 OCNH CH2 NHCO (–CH2CH2CH2O)n CNH CH2 NHCO–

O O O O
Urethane Diisocyanate
hard segment
FIGURE 14.7 Polyetherurethane-segmented Pellethane®.
Naphthylene 1-5-diisocyanate (NDI)

Polyethers are the most frequently used polyols and result in polyetherurethanes. Polyesters are used
less often, but the resulting polyesterurethanes are much more chemically stable.
R΄ R΄
R — OH + n CH2 — CH –––––> R — O — (CH2 — CH — O —)n— H
Formation of polyether-polyols
(n + 1) HO – (CH2)2 – OH + n HOOC – (CH2)4 – COOH
Butanediol Adipic acid
HO – (CH2)2 O – C – (CH2)4 – C – O – (CH2)2 – OH + nH2O
O O n
Formation of polyester-polyols
Pellethane® is a poly(ether urethane) that has been used for many years in the medical field, and it is
the result of a reaction between MDI and polytetramethyleneglycol (PTMG), which produces a soft
segment ending in an isocyanate that reacts with the diol moiety of butanediol to extend the chain
(Yoda 1998) (Figure 14.7).
The greater the number of reactive groups on the polyols used to synthesize the polyurethane,
the more the end product is crosslinked and thus rigid. The polyols with the highest molecular mass
and the least crosslinking give the most flexible elastomers. To improve the mechanical resistance
of certain polyurethane elastomers, all or part of the polyol may be replaced by a reactive diamine
(methylene dianailine or MDA, methylene bisorthochloroaniline or MOCA, ethylene dianiline or
EDA), giving rise to segmented polyetherurethaneurea elastomers such as Biomer® and Mitrathane®,
which are used in cardiovascular applications. Prepolymer processing (foaming) using expansion
agents produces cellular elastomers: either water is reacted with isocyanate to produce a urein and
carbon dioxide (chemical expansion) or low boiling point inert liquids are added and subsequently
expelled by degassing during high temperature polymerization (physical expansion). Adjuvants are
also required for crosslinking: catalysts (tertiary amines, organic tin salts), surfactants (silicones),
fillers, reinforcements, pigments, etc. During manufacture (whatever the process) and storage, poly-
urethanes must be kept in a dry environment because their affinity to and reactivity with water can
result in changes to their composition and/or create defects in the end product.
14.1.4.3 Mechanical Behavior of Polyurethane Elastomers

The hard segments crosslink among themselves within the mass of the elastomer to form
agglomerations that act as fillers, improving the mechanical resistance of the material. The soft
segments remain free and randomly arranged (Figures 14.8 and 14.9). Under a tensile stress
O O
Hard segment: urethane O (CH2)4 O CNH CH2 NHC
n
Soft segment: urethane
O (CH2)4
O O
Diisocyanate
O CNH CH2 NHC
FIGURE 14.8 Linear polyetherurethane chain. (From Coury, A.J. et al., J. Biomater. Appl., 3, 130, 1988.)
FIGURE 14.9 Distribution of hard and soft segments in a polyurethane elastomer. (From Coury, A.J. et al.,
J. Biomater. Appl., 3, 130, 1988.)
FIGURE 14.10 Crystallization of soft segments under a weak vertical stress. (From Coury, A.J. et al.,
J. Biomater. Appl., 3, 130, 1988.)
of 150% (Bonart 1968), the polyether soft segments line up along the elongation axis, dis-
placing the urethane hard segments so that they are more or less perpendicular to the vertical
axis. Soft segments are brought into close proximity with each other by stress crystallization,
a process that is completed when the elongation reaches approximately 250% (Figure 14.10).
When the elongation increases even more (∼500%), the chemical bonds crosslinking the hard
segments break down and the now separate hard segments align along the vertical axis. At this
point, the soft segments relax because of the stretching due to the release of the hard segments
(Figure 14.11).
Segmented polyurethane elastomers thus have elastic behavior under low stress (deformation)
conditions, which becomes plastic when the hard segment network breaks down. Deformed
samples do not immediately return to their original shape following removal of the stress.
However, rearrangement of the hard segments eventually results in a total or partial return to the
initial shape, depending on the elastomer structure. The greater the concentration of hard seg-
ments, the more plastic the deformation. On the other hand, the lower the concentration of hard
segments, the more elastic the behavior.
Chemical properties also depend on the soft/hard segment ratio. A large proportion of hard
segments makes the material harder and provides better stress resistance but decreases elasticity
and resistance to abrasion (Szycher and Reed 1992). The presence of long polyether or polyester
soft segments provides superior break elongation but makes the material more sensitive to oxida-
tion and degradation by biological fluids and thus to failure under repeated stress (Table 14.5).
Other strategies for synthesizing segmented polyurethanes result in the formation of biodegradable
FIGURE 14.11 Reorientation of hard segments and relaxation of soft segments under a strong vertical stress.
(From Coury, A.J. et al., J. Biomater. Appl., 3, 130, 1988.)
p olyurethanes that are extremely elastic (elongation at break 4700%) because they contain very few
hard segments (Asplund et al. 2007).
14.1.4.4 Biodegradation of Polyurethane Elastomers

In vivo degradation of polyurethane elastomers occurs via four main mechanisms: calcifi-
cation, macromolecular chain breakdown (Yoda 1998), hydrolysis, and environmental stress
cracking. Calcification involves the deposition of calcium hydroxyapatite on the surfaces of
implanted elastomers (Schoen et al. 1988). Macromolecular chain breakdown of polyurethane
elastomers occurs via oxidation reactions with the soft segments and at the junctions between
hard and soft segments (Takahara et al. 1991). Corrodable metallic residues favor oxidation by
acting as catalysts in vivo (Pinchuk 1994, Stokes 1988). Hydrolysis depends on the suscepti-
bility of the urea and urethane bonds and the concentration of hydrolytic enzymes at the cell/
polymer interface. The extreme sensitivity of polyesterurethanes to hydrolysis limits their use
in biomedical applications as opposed to polyetherurethanes, which are more resistant (Yoda
1998). However, polyurethane elastomers in general cannot be autoclaved because of the risk
of hydrolysis.
Environmental stress cracking (ESC) occurs when mechanical stresses are combined with inter-
actions with living tissue (Phillips et al. 1988) and can lead to the complete destruction of the
implanted material. Three phenomena must coincide for ESC to occur: the presence of enzymes as a
result of an inflammatory response at the interface (Anderson et al. 1992, Anderson and Miller 1984,
Kao et al. 1994, Zhao et al. 1992), a susceptible poly(ether urethane) elastomer, and a mechanical
stress on the elastomer (Phillips et al. 1988, Rambour 1973). ESC, which has never been observed
Biocompatibility of Elastomers
TABLE 14.5
Commercially Available Polyurethanes Used in Biomedical Applications
Type and Molecular Architecture Processing Trade Name Supplier
Segmented polyetherurethane Extrusion Pellethane™ Dow Chem. Co. USA

Injection molding
(–CH2)4 OCNH CH2 NHCO (–CH2CH2CH2CH2O) n CNH CH2 NHCO Solution casting
O O O O
Extrusion Tecoflex™ Thermedics Inc. USA

(–CH2)4 OCNH H CH2 H NHCO (–CH2CH2CH2CH2O) n CNH H CH2 H NHCO
O O Injection molding
O O
Solution casting
Segmented polyetherurethaneurea
NH C NH CH2 NHCO (–CH2CH2CH2CH2O)n CNH CH2 NHCNHCH2CH2 Solution casting Biomer™ Ethicon Co. USA
O O O O
NH C NH CH2 NHCO (–CH2CH2CH2CH2O)n CNH CH2 NHCNHCH2CH2 Solution casting TM3™, TM5™ Toyobo Co. Japan
O O O O CH3
Copolymer of segmented polyetherurethane

and polydimethlysiloxane
(–CH2)4 OC N CH2 NHCO (–CH2CH2CH2CH2O)n CNH CH2 NHCO Solution casting Cardiothene51™ Kontron Inc. USA Japan
O O O O Nippon Zeon Co.
CH3
( Si O ) n C CH3
CH3 O
Source: Yoda, R., J. Biomater. Sci. Polym. Ed., 9, 561, 1998.
439
Activated cell O2– + H+ HOO
- O - CH2 - CH2- CH2- CH2 - O- CH2- CH2 -

HOO
- O - CH2 - CH2 - CH2 - CH2- O - CH2 - CH2 - + H2O2
HOO
- O - CH2- CH2 - CH2 - CH2 - O - CH2 - CH2-

OOH
–H2O
- O - CH2- CH2 - CH2 - C - O - CH2 - CH2 -

O
H2O H+ Esterases
- O - CH2- CH2 - CH2 - C - OH + HO - CH2 - CH2 -
O
FIGURE 14.12 The degradation mechanism of a polyurethane at the carbon alpha to the ether group.
(As proposed by Anderson, J.M. et al., Cell/polymer interactions in the biodegradation of polyurethanes, in
Biodegradable Polymers and Plastics, eds. Vert, M., Feijen, J., Albertson, A., Scott, G., Chiellini, E., Royal
Society of Chemistry, Cambridge, U.K., pp. 122–136, 1992.)
O
RO CH2 CH2 CH2 CH2 OR΄
+H
Sn1
H
+
O
RO CH2 CH2 CH2 CH2 OR΄
Slow
RO CH2 CH2+ + HO CH2 CH2 OR΄
–X
(Fast)
RO CH2 CH2 X + HO CH2 CH2 OR΄
FIGURE 14.13 Cleavage of a polyether urethane by Sn1 type acid hydrolysis. (From Pinchuk, L., J. Biomater.
Sci. Polym. Ed., 6, 225, 1994.)
with polyurethanes in vitro, does not occur if a single factor is missing (Szycher and Reed 1992).
ESC seems to be due to the oxidation of the methyl group on the soft segment polyether chains
(Szycher and Reed 1992) (Figures 14.12 through 14.14).
Numerous studies have shown that soft segments are prone to oxidation after varying peri-
ods of exposure in vivo (Gogolewski 1991, Schubert et al. 1997, Zhao et al. 1990, 1991). It is
now generally accepted that reactive oxygen species released by macrophages and foreign body
giant cells are responsible for the biodegradation because they capture one proton on the alpha
methylene of the polyether leading to either the rupture of the polymer or the creation of a new
bond between two chains of neighboring polymer (Schubert et al. 1995, Zhao et al. 1991). The
splitting of the polymer chains inexorably breaks down the biomaterial while, at the same time,
O
H C
R OCH2 CH2 CH2 C O N R2
H H
O O
H C
R OCH2 CH2 CH2 C O N R2
H H
R
O
H C
R OCH2 CH2 CH2 C O N R2 + RH
H
O
C
R OCH2 CH2 CH2 + H C N R2
O H
FIGURE 14.14 The degradation mechanism of a polyurethaneurea at the carbon alpha to the urethane group
as proposed by Tyler and Ratner. (From Pinchuk, L., J. Biomater. Sci. Polym. Ed., 6, 225, 1994.)
the increase in the number of bonds between the chains makes it more rigid, making it less and
less elastomeric. At last, the biomaterial breaks down more and more and each piece becomes
increasingly rigid.
Chemical properties are not the only factors at play. The hardness, mechanical resistance, and
morphology or microstructure as well as the affinity for proteins also influence sensitivity to bio-
degradation (Zhang et al. 1994). The microporous surfaces of polyetherurethane vascular devices
promote tissue colonization, neointima formation, and fibrotic capsule formation that in turn favor
biodegradation by increasing the size of the biological interface with respect to the volume of the
device (Marois et al. 1989, White 1988).
14.1.4.5 Biostable Polyurethane Elastomers

The impact of ESC on polyetherurethanes has stimulated research on polyurethane elastomers
that do not contain ether groups and that remain stable when subjected to environmental stresses
in vivo, i.e., biostable polyurethanes (Stokes 1983, Stokes et al. 1987, Szycher and Reed 1992).
Coury et al. (Coury and Hobot 1991) have filed a patent for a polyurethane that contains no ether
groups. An aliphatic complex replaces the soft segment polyethers with a very rigid biostable
skeleton that makes the polymer much too hard for many applications. Numerous attempts have
been made to modify or replace the soft segments in order to improve biostability. Soft segment
polyethers have been replaced by polybutadienes and polymethylsiloxanes (Hergenrother et al.
1994, Lim et al. 1994, Takahara et al. 1991), polycarbonates (Capone 1992, Christenson et al.
2004b, Stokes et al. 1995), aliphatic hydrocarbons (Capone 1992), and sulfonated polyethylene
oxide (Han et al. 2006). Polysiloxanes are attractive substitutes for polyethers because of their
low toxicity, good thermal and oxidative stability, low coefficient of friction, and good hemo-
compatibility, and the fact that they do not affect the mechanical properties of the polyurethanes
because their modulus of elasticity is similar to that of the soft segment they are replacing
(Briganti et al. 2006, Martin et al. 2000, Simmons et al. 2004). Polycarbonate soft segments are
also less sensitive to oxidation, although the hydrolysis of carbonate linkages in vivo is always
Polycarbonate
soft segment
O O O O H O
O
OH-CH2CH2 CH2 CH2O C N CH2 N-C O CH2 O C O CH2 CH2 O C O CH O C O CH2 N -C
2
H H 6 6
N H O
Diisocyanate CH2
CH2
CH2
O O O O CH2
O
OH-CH2CH2 CH2 CH2O-C N CH2 N-C O CH2 O C O CH2 CH2 O C O CH O C O O
2 CH2 N-C
H H 6 H O
m 6
N H
Urethane
hard segment
FIGURE 14.15 An example of a biostable polyurethane: Corethane® (polycarbonate urethane) produced by

reacting poly(1,6-hexyl 1,2-ethyl carbonate)diol with MDI. (From Pinchuk, L., J. Biomater. Sci. Polym. Ed.,
6, 225, 1994.)
possible (Capone 1992, Coury et al. 1990, Pinchuk 1994, Pinchuk et al. 1993, Reed et al. 1994,
Stokes et al. 1995, Ward et al. 1996) (Figure 14.15).
Polycarbonate urethanes are gradually supplanting polyurethanes because they are more resistant
to oxidation. They were first commercialized by Corvita Corporation in 1992 and more recently, by
the Polymer Technology Group as corethane, coremer, vascugraft, carbothane, chronoflex, bionate,
etc. Like polyether urethane, they can be used in applications ranging from vascular to orthopedic.
The addition of a triglyceride to 12-hydroxyoleic acid (the main component of castor oil) during
the synthesis of polycarbonate urethanes produces significant improvements, including an affinity
for albumin, antithrombogenic properties, and enhanced physical properties (Szelest-Lewandowska
et al. 2003, 2007). The addition of carbon fibers or hydroxyapatite improves the physical and bio-
logical properties of polycarbonate urethane elastomers destined for orthopedic applications (Geary
et al. 2008).
The complementary qualities of polyurethane and polyvinylsiloxane elastomers led to the idea of
producing “alloys” in the hope of creating new polymers with enhanced resistance to biodegradation
(Iwamoto et al. 1986, Nyilas and Ward 1977, Yoda 1998). This complex mixture of polyurethane
and silicone (Cardiotane 51®) is difficult to process because it is neither soluble nor thermoplastic
(Coury et al. 1988).
14.1.4.6 Shape Memory Polyurethane Elastomers

Shape memory elastomers are a class of shape memory polymers (SMPs) used in various medi-
cal applications. Light, easy to manufacture, and adaptable to a wide range of applications,
most are shape memory polyurethanes. Most are rigid polymers, but some with the consistency
of elastomers are used for specific applications such as stretchable stents, prosthetic valves,
and self-tensioning sutures. They are generally biocompatible, nontoxic, and nonmutagenic.
Patented in 1996 by Mitsubishi Heavy Industries, they are now distributed by its Diaplex
subsidiary in the form of thermoset resin or thermoplastic (Cabanlit et al. 2007, Sokolowski
et al. 2007).
14.1.4.7 Grafted Polyurethane Elastomers

The biomedical properties of polyurethane elastomers can be improved by encapsulation to isolate
them from living tissue. Grafting polysiloxane molecules on the surface makes the material hydro-
phobic and protects the soft segments from oxidation and hydrolysis in vivo (Macocinschi et al.
2009, Mathur et al. 1997, Ward 1995, Ward et al. 1995). Grafting alkyl or polyethylene (oligoethylene
monoalkyl(aryl)alcohol ether) groups to the sodium atoms of urethane hard segments or incorporat-
ing sulfate groups by nucleophilic molecular displacement of N-H urethane groups creates sulfonate
polyurethane anionomers with chemical properties that limit biodegradation and thrombogenicity
(Baumgartner et al. 1997, Grasel and Cooper 1989, Grasel et al. 1987, Han et al. 1998, Keogh et al.
1996, Lelah et al. 1985, Lim et al. 1993, Okkema et al. 1987). Researchers have also grafted pro-
teins and other molecules on polyurethane elastomers in an attempt to improve hemocompatibility
(albumin [Keogh and Eaton 1994]; heparin [Albanese et al. 1994]; adenosine diphosphate [Bakker
et al. 1991]; fibronectin and gelatin [Hess et al. 1992]; amino acids [Wang et al. 2003]). The graft-
ing processes, or impregnation in the case of gelatin, are complex and require numerous steps.
Collagen grafted to carboxyl group-enriched polyurethanes via 1,2-bis(2,3-epoxypropoxy)ethane
links allows better epithelial cell growth than raw polyurethanes (Lee et al. 1996a,b).
Seeding the lumen of microvessel vascular prostheses prepared by glow discharge allows the
in vivo proliferation of a multicell layer close to but distinct from the elastomer wall. The cell layer
is histologically similar to a native artery with antithrombogenic endothelial cells on the surface
(Williams et al. 1992).
Plasma processing can be used to modify surfaces by depositing metallic silver (Boswald et al.
1995, Greil et al. 1999, Jansen et al. 1994, Li and Zhao 1995, Oloffs et al. 1994), fluorinated films
(Pizzoferrato et al. 1995), nitrogen, and hexamethyldisiloxan polymers (Li and Zhao 1995) to, among
other things, improve the antithrombogenic and antibacterial properties of cardiovascular implants.
14.1.4.8 Modified Polyurethane Elastomers

Solvent extraction (toluene, acetone, methanol, etc.) of small molecular weight surface poly-
mers (which are different in composition from the bulk polyurethane) increases the molecular
weight of the surface fraction and enriches it in hard segments, making it more polar and
improving hemocompatibility (Grasel et al. 1988, Lelah and Cooper 1986, Lelah et al. 1986,
Ratner 1983).
Since biogradation mainly affects the ether bonds of soft segments, which are very sensitive
to oxidation, certain biomedical polyetherurethanes contain high concentrations of antioxidants
(Schubert et al. 1996). Unfortunately, these elastomers (or their degradation products) may be toxic
to host tissues (Szycher 1988). Researchers have recently begun examining natural antioxidants like
vitamin E as possible alternatives to industrial additives.
Attempts to inhibit tissue responses have also been made. Adding hydroepiandrosterone
(DHEA) to poly(etherurethane urea) reduces biodegradation by limiting macrophage activation.
However, the effect is dose-dependent and since DHEA is very hydrosoluble, it rapidly dif-
fuses away, making it useless for long-term protection (Collier et al. 1998, Szelest-Lewandowska
et al. 2007).
Toyo Cloth Co. produces high molecular weight poly(ether/urethane/amide) from polytetra-
methylene oxide, MDI, ethylene glycol, and nylon 66. The addition of calcium chloride to this
polyurethane-nylon polymer modifies its physical and biomedical properties (Kawashima and Sato
1997) by increasing water permeability, hydrophilicity, biocompatibility, thrombogenicity, and
elasticity, and by decreasing breaking strength (tensile modulus). An affinity for calcium ions points
to potential thrombogenic or antithrombogenic applications for heparinized versions of the elasto-
mer (Albanese et al. 1994).
The addition of chitin during the synthesis of polyurethane improves its hydrophobicity and
enhances its resistance to biodegradation in vivo (Zia et al. 2009a,b).
While polyurethanes can be encapsulated, they can also be used to coat medical devices
like pacemakers and endovascular stents (De Scheerder et al. 1995a,b, Holmes et al. 1994,
Schellhammer et al. 1999). Such coatings have various degrees of hemocompatibility (Rechavia
et al. 1998) and occasionally shift because of a lack of adherence to vessel walls (Schellhammer
et al. 1999).
R O O O H H
O O O O O N
O O ; O O N
x
O R o p O O
y O O
n
FIGURE 14.16 Example of a biodegradable polyurethane. (From Saad, B. et al., J. Biomed. Mater. Res., 36,
65, 1997.)
14.1.4.9 Biodegradable Polyurethane Elastomers

The development of biodegradable polymers is a major advance in the research on biomaterials.
Designed to gradually degrade in vivo over the implantation period, these polymers are used
in numerous medical and surgical applications Biodegradable polyesterurethanes have been
developed in recent years (Hirt et al. 1996, Saad et al. 1996, 1997). DegraPol/btc® is a polyes-
terurethane copolymer with polyhydroxybutyric acid hard segments and polycaprolactone soft
segments (Figure 14.16).
The hard and soft segments can be replaced by a variety of substitutes that make the end
product more or less biodegradable in vivo. By selecting the right substitute, it is possible to
design cellular polyesterurethanes with varying degrees of rigidity and with degradation rates
suitable for osteoblast colonization of bone implants (Saad et al. 1996). Polyesterurethane foams
can also be used to guide soft tissue regeneration (van Minnen et al. 2005). They can lose half
their mass within several months of implantation. They are not strictly speaking elastomers, but
lengthening their soft segments may provide new mechanical properties and open the door to
soft tissue applications.
The controlled degradation of biodegradable polyurethanes in vivo facilitates the growth of new
tissue. Tissue engineering scaffolds are fabricated from biodegradable polyurethanes using various
processes such as carbon dioxide injection foaming, electrospinning, wet spinning, thermal phase
separation, and salt leaching.
These scaffolds support the growth of cells and tissues both in vitro and in vivo, and their pro-
gressive degradation can be controlled through their formulation and composition. In addition, the
degradation products are not cytotoxic (Guelcher 2008).
14.1.5 Saturated Polyesters and Biopolyesters

These thermoplastic polymers have long been used in textiles (Dacron®, Tergal®) and plastic films.
Polyethylene terephthalate (PET) holds the lion’s share of the market.
O [CH2]2 O C C O [CH2]4 O C C
O O O O n
n
Polyethylene terephthalate (PET) Polybutylene terephthalate (PBT)
PET was first used as a medical elastomer when DuPont introduced Hytrel® copolymer in 1972.
PET, together with polybutylene terephthalate (PBT), which is used in injection molding, poly-
cyclohexylenedimethylene terephthalate (PCT), and polyester-glycol copolymers (ethylenes and
cyclohexane dimethanols) are excellent elastomers (polyesters and copolyesters) for biomedical
applications. They are used in surgical field, bag, flask, bottle, and bandage-backing materials. PET
and PBT are especially suitable for making copolymers with polycarbonate, and are also available
with fiberglass reinforcements (∼30%). PET and PBT, which theoretically can be esterified on their
diacid or dialcohol groups, are in practice obtained by transesterification of dimethylterephthalate

by ethylene glycol and butylene glycol, with the release of methanol.
Diméthyltéréphtalate Ethyléneglycol
CH3 O C C O CH3 + OH [CH2]2 OH

O O
O [CH2]2 O C C + 2 CH3OH
O O Méthanol
n
PET
The reaction is controlled by gradually increasing the temperature until the desired molecular mass
is obtained. They are easy and cheap to produce on an industrial scale. They are sensitive to hydro-
lysis but are chemically resistant to a wide range of chemicals and solvents, are relatively nontoxic,
contain no plasticizers, can be easily washed and sterilized (autoclave, gamma radiation), are very
rigid, have the lowest coefficient of friction of any thermoplastic, and are very esthetic (smooth
surface, very translucid). Thin films are permeable to water vapor and are very flexible and tear
resistant (White 1991). When copolymerized with polycarbonate, they are very easily colonized by
endothelial cells and may see use as vascular prostheses.
The first natural biopolyester—poly(hydroxyalkonoate) or PHA—was isolated by Lemoigne in
1925 (Chen et al. 2008). These water-insoluble polyesters are synthesized and used as storage mate-
rials by many bacterial species.
R1 O R2 O
CH C CH C
O (CH2)x O (CH2)x O
n
The general structure of PHAs (x = 1,2,3,4;

n =100–30000; R1, R2 = alkyl groups, CI–C13)
An infinite variety of PHAs can be synthesized industrially using a variety of processes and
substrates (Huijberts et al. 1992, Poirier et al. 1995). Biopolyesters produced by living organisms
are interesting because they are easily degraded by living cells, tissues, and organisms and are
a renewable biodegradable source of plastic polymers and elastomers (Barak et al. 1991, Imam
et al. 1998).
Most PHAs are somewhat rigid but, since the 1990s, biodegradable elastomers have been fabri-
cated using chemical reactions in the presence of sulfates or peroxides (Gagnon et al. 1994a,b) or
by photopolymerization (Ashby et al. 1998, de Koning et al. 1991). These industrial polymers can
be enzymatically hydrolyzed just like natural PHAs. They can be produced using various processes
(injection, extrusion) and with different structures (granules, pasts) and show promise in biomedical
applications (Duvernoy et al. 1995, Hocking and Marchessault 1994, Lafferty et al. 1988, Williams
et al. 1999).
14.1.6 Polyvinyl Chlorides and Phthalate

Polyvinylchloride is a widely used polymer and PVC products can be produced with a broad range
of properties (flexible, rigid, cellular). PVC has been widely used in the medical field for some
40 years (gloves, tubes, tubing, catheters, bags, etc.) because of its low cost and ease of processing.
The (CH2-CHC1) monomer is obtained from ethylene and chloride.
H Cl
C C
H H
n
The high pressure polymerization of vinyl chloride monomer, which is a gas at room tempera-
ture and pressure, is activated by the addition of an initiator (peroxide) and controlled using
temperature, mixing, and surfactants. The result is a white pulverulent polymer whose struc-
ture varies depending on the process (bulk, suspension, or emulsion polymerization). Vinyl
chloride monomer lends itself to copolymerization, especially with vinyl chloroacetate (VCA).
PVC/VCA copolymers generally contain 5%–15% vinyl chloroacetate. They can be processed
at lower temperatures and pressures than PVC alone, more filler can be added, and they can
be used to produce very transparent products. PVC is almost never used on its own. Small
amounts of specific adjuvants such as heat stabilizers (to protect the resin during processing)
and lubricants (to facilitate or make the processing possible) are often added. Fillers, pigments,
reinforcing agents, light stabilizers, antioxidants, etc. may also be added. In addition to the
adjuvants mentioned before, PVC elastomers also contain plasticizers (5–70 parts per 100 parts
of resin). Since conventional plasticizers (phthalates) are incompatible with biomedical applica-
tions, PVC elastomers intended for biomedical applications are specially designed not to release
toxic products (Fabre et al. 1998, Lindner et al. 1994). Phthalate-free PVC elastomers either
contain plasticizers that are not released or are nontoxic (azelate, phosphate ester and polyester,
citrate), or they are made without plasticizer by combining PVC with other elastomers to pro-
duce copolymers (polyurethanes, vinyl chloroacetate) or simple phase mixtures. The elastomer
phase plasticizes the rigid PVC (polyadipates, polyesters, polyacrylonitrile-butadiene) (Branger
et al. 1990).
PVCs contain plasticizers that have been known to be potentially toxic since the late 1970s
(Fishbein 1984). Prolonged exposure to phthalates has been suspected since the early 1980s to
have a profound, cumulative effect on the structure and function of living organisms (Ganning
et al. 1984).
The most commonly used plasticizer, di(2-ethylhexyl)phthalate (DEHP), is toxic in animals,
but its effects in man were in doubt until 2005 (Blass 1992, Hill et al. 2001, Hoenich et al. 2005,
de Lemos et al. 2005).
According to the international organization Health Care Without Harm (2004), dioxins, poly-
vinylchloride, and di(2-ethylhexyl) phthalate are the three main toxins interfering with the goal
to maintain a healthy environment. Exposure to these chemical products has been correlated with
the appearance of cancers, cardiac and liver diseases, and reproduction and development disorders
in animals and is strongly suspected to cause the same problems in man (Loff et al. 2008, Satoh
et al. 2008, Tickner et al. 2001). Epidemiological studies have also shown an association between
exposure to phthalates and the development of asthma and allergies in children (Jaakkola and
Knight 2008).
Phthalates have been found in the saliva of children who place plastified PVC objects in
their mouths. While the phthalate concentrations are low and the amounts ingested probably
even lower than acceptable limits (Corea-Tellez et al. 2008), biomedical devices made of PVC
elastomers such as mouth protectors, oral ortheses worn by patients with algo dysfunction of the
manducator apparatus, oral respiratory devices worn by patients with sleep apnea syndrome, etc.
are placed in the mouth much more frequently by adults than PVC toys by children. As such,
other elastomers that do not contain plasticizers or DEHP-free elastomers should be used instead
of plastified PVCs.
It is technically possible to replace the toxic plasticizers in PVCs with other substances that are
reputed to be nontoxic and that do not leach from the PVC (Branger et al. 1990). This is crucial and
urgent for medical and food packing materials and toys (Hansen 2008) and, a fortiori, elastomeric
biomaterials. BASF began developing a new plasticizer, diisononyl phthalate or Hexamoll DINCH,
in the late 1990s with an excellent toxicological profile. However, BASF only began using it in their
PVC biomedical products in 2008 (Hansen 2008).
The surface properties of PVC elastomers can be altered by plasma treatments and hydrophilic
monomer grafts (carboxylation, acrylic and metacrylic acid, vinyl pyrrolidone) (Singh and Agrawal
1992). Single-use antithrombogenic endovascular materials can be produced by grafting PU/SI
copolymers on the surface of PVC tubing.
14.1.7 Polyolefins
Polyolefins are produced by the polymerization of ethylene molecules where the hydrogen
atoms are replaced by various hydrocarbon groups. Ethylene (CH 2 =CH 2) can be used to pro-
duce polyethylene, polypropylene, etc. by simple substitution of the side group (H, CH 3, C2H 5,
C4H9, etc.).
CH2 CH2 Polyethylene

n
CH2 CH Polypropylene
n
CH3
CH3
CH2 C Polyisobutylene
CH3
CH2 CH Polymethylpentene
n
CH2
CH3 CH CH3
Polyethylenes are produced by polymerizing gaseous ethylene under different conditions.

Polyisobutylenes (PIB), for example, are polymerized in the presence of catalysts. Propylene/
ethylene copolymers (often called polypropylene copolymers) are elastomeric in nature and have
improved shock resistance properties (block copolymers). Random copolymers are especially
well suited for manufacturing plastic films, hollow objects, plates, etc. Polymethylpentenes
(PMPs) are obtained by high pressure polymerization in the presence of a catalyst. PMPs have
good water and gas permeability and superior physicochemical properties, making them resistant
to autoclaving and inert to most pharmaceuticals. Numerous compounds may be added to facili-
tate processing. PMPs must, however, be washed to eliminate potentially toxic residues. They
are used for all sorts of packaging materials, as well as for flexible tubing, stoppers, implants,
catheters, etc. The excellent resistance to flexural fatigue of polypropylene copolymers makes
them more suitable than other elastomers for certain applications (extracorporeal pump dia-
phragms, synthetic finger joints made of Hexsyn®, for example). Grafting various monomers
(vinyl pyrrolidone, ethyl methacrylate, acrylamide) on the surfaces of polypropylenes can provide
excellent wettability while conserving their superior mechanical performance (Katbab et al.
1992, Mirzadeh et al. 1993). The recent production of polypropylene homopolymers containing
alternating isotactic and atactic segments (relative configurations of successive asymmetric ele-
ments in the polymer chain) by a new mettalocene polymerization process opens the way to new
medical applications for polyolefins (Dopico-Garcia et al. 2007, Hotta et al. 2006, Yoda 1998).
14.1.8 Styrene Copolymers
The styrene family contains a wide variety of polymers. Polystyrene is obtained by the polymeriza-
tion of styrene produced from the alkylation of benzene by ethylene and the dehydrogenation of
ethylbenzene. Liquid styrene can be polymerized in two ways:
1. Bulk polymerization: delicate method

2. Suspension polymerization: fine droplets (0.1–10 μm) of styrene monomer dispersed in an
aqueous phase
CH2 CH CH2 CH
n
C C
H C C H H C C H
H C C H H C C H
C C
H H
Polystyrene
Styrene can be copolymerized with polybutadiene or ethylene/butylene to produce graft styrene-
butadiene-styrene (SBS) and styrene-ethylene/butylene-styrene (SEBS) or Styrene-block-IsoButylene-
block-Styrene) (“SIBS”) which are widely used in medical applications. These elastomers not only
have improved shock resistance but also do not require additives. They are very flexible and pure and
do not release any residues in situ (Freij-Larsson et al. 1993). However, they are not very histocompat-
ible and numerous attempts have been made to improve them. Polyhydroxyethylmethacrylate grafts
improve blood compatibility, polysiloxane grafts impart the properties of silicone elastomers (Deisler
1987), while N+, F+, and Ar+ ions improve cell adhesion (Bacakova et al. 1996, El Fray et al. 2006).
Poly(styrene-block-isoButylene-block-styrene) (SIBS) is a biostable thermoplastic elastomer
with physical properties that overlap silicone rubber and polyurethane. Research so far has shown
that (1) SIBS does not activate thrombogenesis in the vascular system, (2) inflammatory reactions
are not triggered around implants in the in the eye, skin, or vascular system, (3) it does not cause
fibrosis or calcification in vivo, and (4) no degradation of the polymer has been observed to date in
vivo. It appears that the inertness of SIBS is responsible for its remarkable stability (Pinchuk et al.
2008). SBS, SEBS, and SIBS elastomers can be sterilized by heat, vapor, gamma radiation, or ethyl-
ene oxide without losing their physical properties. They are used to manufacture catheters, stoppers,
nonrigid containers, surgical fields, condoms, gloves, etc.
14.1.9 Natural Rubbers
Natural rubbers are the most elastic and resistant of all biomedical elastomers, but are also the least
hemocompatible due to the release of accelerator (dithiocarbamate) residues (Nakamura et al. 1990).
Many attempts have been made to improve their blood compatibility. Methylmethacrylate grafts have
shown great promise because they make natural rubber more hemocompatible than many silicone
elastomers (Razzak et al. 1988). Another approach has been to vulcanize natural rubber by gamma
radiation without additives. Natural rubbers polymerized in this way are very pure and demonstrate
remarkably good histocompatibility (Balabanian et al. 2006). Untreated natural rubbers are mainly
used to manufacture latex gloves while treated natural rubbers are used to produce catheters and tubing.
Latex gloves have been shown to prevent cross-infections (Tucci et al. 1996). The well-balanced physi-
cal properties of natural rubbers (elasticity/tear resistance) make them ideal for this use. Hydrosoluble
proteins are responsible for allergies associated with latex products. Simple washing is sufficient to
completely remove the proteins and eliminate the risk of allergic reactions (Cormio et al. 1993).
14.1.10 Hydrogels
Biomedical uses for hydrogels first appeared over 30 years ago (National Heart and Lung Institute
1971, Ratner et al. 1975). A number of polymers can be designed to act as hydrogels and retain
water. Polyvinyl alcohol is the most widely used such polymer in ophthalmological applications
(Yoda 1998). Polyvinylpyrrolidone is a relatively nontoxic, hydrosoluble polymer that can be
impregnated with water or organic solvents once it has been crosslinked (Vijayasekaran et al.
1996). It can be grafted on the surfaces of silicone polymers (Ratner et al. 1975) and polyure-
thanes to improve thromboresistance (Chapiro 1983). Thermogelling polyurethane copolymers
and newly synthesized tri-component multiblock poly(ether ester urethane)s have a release rate
that can be controlled by the adjusting the composition of the poly(ether ester urethane)s or the
concentration of the copolymer (Loh et al. 2006, 2007), which will make it possible to better
control the quantities and concentration of bioactive molecules released by this type of hydrogel
(Xinming et al. 2008). It is also suitable for use in soft contact lenses (Yoda 1998).
14.1.11 Polypeptide, Collagen, Poly-Glycerol-Sebacate, Elastomers

Polypeptide elastomers of varying composition that mimic natural biological elastic polymers with
different physical properties have been synthesized to and can be designed to be either biostable or
biodegradable (Yoda 1998). They contain polypeptides (synthetic and/or natural) that can be copo-
lymerized or not with artificial polymers (Ifkovits and Burdick 2007). Collagen is the most widely
used substance for producing bulk or graft polypeptide elastomers. Elastin is also used to synthe-
size elastomeric biological tissues for numerous promising biomedical applications (Table 14.6).
TABLE 14.6
Polypeptide Elastomers for Medical Applications
Elastomer Composition
Polypentapeptide (Val-Pro-Gly-Val-Gly)n
Polypeptide Poly(ethylene-graft-γ-benzyl l-glutamate)
A-B-A type block A: Poly(γ-benzyl l-glutamate)
copolymers B: Polybutadiene
A-B-A type block A: Poly(γ-ethyl l-glutamate)
A-B-A type block A: Poly(γ-methyl l- or d, l-glutamate)
A-B-A type block A: Poly (ε-N-benzyloxycarbonyl l-lysine)
A-B-A type block A: Poly(γ-benzyl l-glutamate)
copolymers B: Polyisoprene
Collagen Collagen-polyacrylates graft copolymer
Gelatin/chitin Gelatin/carboxymethylchitin complex
Pseudo-poly(amino acids) Poly(desaminotyrosyltyrosine hexylester carbonate)

Poly(glycerol sebacate) (PGS) is a biodegradable elastomer synthesized from biocompatible

monomers. It forms a covalently crosslinked three-dimensional network with hydroxyl groups
attached to the backbone. The hydrogen bonding interactions between the hydroxyl groups con-
tribute to the unique properties of this elastomer. In vitro and in vivo studies have shown that it has
very good biocompatibility and is completely absorbed within several weeks of implantation (Wang
et al. 2002). It can also be used to fabricate macroporous elastomer scaffold sponges with numer-
ous micropores. The macropores are generated by glycerol vapor formed during curing. When the
elastomer sponges are implanted, the micropores facilitate the colonization of the biomaterial by
host cells.
The physicochemical properties and biodegradability of this biomaterial make it potentially very
useful for nerve tissue engineering (Chen et al. 2008, Fidkowski et al. 2005, Sundback et al. 2005).
It can also be modified to adhere strongly to tissues (Mahdavi et al. 2008). Since PGS can be pre-
pared by photocuring, it can be used to encapsulate bioactive molecules that are destroyed by high
temperature polymerization (Nijst et al. 2007).
Polyphosphazenes have long flexible chains with alternating phosphorus and nitrogen atoms.
2 NaOR OR
Cl Cl N P n
P Cl OR
Cl N N Cl N P n
P P Cl NaOR OR
Cl N Cl N P n
NaOR΄ OR
Synthesis of poly (dichlorophosphazene) and poly (organophosphazenes)
(Yoda 1998)
Several hundred different polyphosphazene polymers have been produced simply by changing the
radical on the phosphorus atom. The flexibility of the chain provides good elasticity and superior
flexibility. Fluorinated polyphosphazenes are very hydrophobic, which means they are theoreti-
cally weakly thrombogenic. Given their mechanical properties, they are used in external devices
that come into contact with blood (Heyde et al. 2007). Their good biocompatibility means that
microporous polyphosphazene polymer matrices can be seeded with osteoblasts to serve as bone
fillers or to induce bone regeneration. Blending etheric biodegradable polyphosphazene, poly[(50%
ethyl glycinato)(50% methoxyethoxyethoxy)phosphazene] [PNEG(50)MEEP(50)] with poly(lactide-
co-glycolide) [PLAGA] enhances the adherence of osteoblasts, pointing to a number of interesting
orthopedic applications for this biodegradable polymer (Conconi et al. 2006, Deng et al. 2010,
Nukavarapu et al. 2008). While they are also used to coat metallic vascular stents to improve bio-
compatibility, they can occasionally provoke strong tissue reactions (De Scheerder et al. 1995a,b,
Deng et al. 2010) Certain polyphosphazenes with aminoester acid radicals are biodegradable
(Crommen et al. 1993).
14.1.13 Polyamides
Polyamides have an amide (–CO–NH–) bond that is the result of the condensation of an organic
acid group on an amine. They make up a large family of textile fibers and widely used techni-
cal polymers. The first polyamide (Nylon 66), which was invented by Carothers, is a member
of the group of polymers obtained by reactions between diacids (HO–CO–(CH 2)b–CO–OH)

and diamines (NH 2 –(CH 2)a–NH 2). Two other types, derived from polycondensation reac-
tions between neighboring caprolactam and amino acid molecules (–CH 2 –COOH + H 2N–CH
and –CH 2 –CO–NH–CH 2 –+H 2O), appeared a little later. Gradual heating to 250°C results in
the elimination of the water and an increase in molecular mass. Diacid/diamine polyamides
(e.g., Nylon 66) are produced by isolating and purifying a crystalline intermediate (nylon salt),
which then undergoes polycondensation. A wide variety of copolymers can be produced in
this way. Polyether block amides (PEBA) are a very large family of copolymers that have
alternating sequences of crystalline polyamides and amorphous polyethers. These thermoplas-
tic copolymers are intermediates between plastics and elastomers. Ferruti and Ranucci (1991)
have produced numerous functional polyethylene glycol and polyamidoamine polymers that
are used as grafts on a number of elastomeric biomaterials. Polyamide elastomers are used in
a wide variety of applications: sterile packing material, cardiovascular surgery, and perfusion
and transfusion materials. Nerve chambers composed of collagen and synthetic biodegrad-
able polymers filled with polyamide filaments oriented along the tube axis or highly porous,
insoluble analogs of the ECM are very effective at orienting the growth and regeneration of
peripheral nerves in vitro (Yannas et al. 2007). When covalently linked, hairpin polyamides
made up of N-methylpyrrole and N-methylimidazole can bind to DNA in a sequence-specific
manner (Murty and Sugiyama 2004).
14.1.14 Polyacrylics
Polyacrylics are the result of the esterification of methacrylic acid by methanol. Methylmethacrylate
is a clear liquid at 20°C. Polymerization can be achieved in two ways:
1. Emulsion polymerization is catalyzed by adding peroxide to an aqueous monomer

emulsion. The result is a low molecular weight white power ready for molding.
2. Molding polymerization involves heating a monomer and catalyst in an oven between
two glass plates, which act as the mold. Thermoplastic objects are rarely obtained
directly by polymerization. The higher molecular weight (compared to emulsion polym-
erization) facilitates thermomolding by increasing the temperature range of the rubber
platform.
H CO–OCH3
C C
n
H CH3
Polymethylmethacrylate
PMMA is mainly known for its exceptional optical properties. This amorphous polymer is remark-
ably transparent (92% light transmission). A 3 m thick block of PMMA only absorbs 50% of incident
light. This makes it ideal for use in ophthalmological devices (contact lenses, crystalline lenses).
PMMA is also very biocompatible (Hollick et al. 1998, Jongebloed et al. 1994). The addition of
plasticizers (phthalates) provides PMMAs with elastomeric properties, enabling them to be used for
odontological applications (maxillofacial prostheses, mouthguards) (Chauvel-Lebret et al. 1999).
They are also used in combination with other biomedical elastomers (polyurethanes, SEBS, etc.)
to produce linear and graft copolymers. In addition, they are widely used to fabricate intraocular
lenses with the capacity to release bioactive molecules (Kaur and Smitha 2002, McCulley 2003,
Xiong et al. 2006).
They can only be sterilized using ethylene oxide or gamma radiation (Henry 1985) since they
melt at low temperatures (80°C–100°C).
14.1.15 Fluorinated Elastomers
Fluorinated elastomers are a group of technical polymers with exceptional properties. Replacing
the hydrogen with fluorine greatly improves heat and chemical resistance. Industrial fluorinated
monomers are gases that liquefy at very low temperatures. They are polymerized at very high pres-
sures using radical catalysts, generally by suspension or aqueous emulsion polymerization in order
to control the strong exothermic reaction.
F F
F2C = CF2 C C
Tétrafluoroéthyléne F F
n
Polytétrafluoroéthyléne (PTFE)
The very strong electronegativity of the PTFE fluoride ions protects the carbon chain and gives
these elastomers their extreme chemical stability. They are inert and insoluble (Bischoff 1972,
Wortman et al. 1983). They are naphthalene-plasticized resins and are extruded and made micro-
porous for vascular graft prostheses (Teflon®, Impra®, Goretex®) (Huang et al. 1992, Sigot-Luizard
et al. 1993). Their vascular biocompatibility is adequate but less than that of polyurethanes (Jeschke
et al. 1999). They are used in orthopedic surgery to reduce friction between metallic and/or compos-
ite joint prostheses (Defrere and Franckart 1992) both alone and in combination with polyurethanes
(Jahangir et al. 2002, Massa et al. 2005, Xie et al. 2008).
14.1.16 Elastomeric Medical Devices

See Tables 14.7 through 14.12 and Figure 14.17.
TABLE 14.7
Grouping of Medical Devices
Type Environment Duration Application
I Internal devices Less than 30 days Intravenous catheters
Drainage tubes
More than 30 days Hip implants
Pacemakers
Artificial heart valves
II External devices Less than 30 days Devices that contact the skin such as gloves, tapes
dressings and orthopedic casts
More than 30 days Devices that contact the mucous membranes such
as urinary catheters and intravaginal devices
III Indirect devices (do not Hypodermic syringes
contact the body) Transfusion assemblies
Dialysis components
IV Non-patient contact devices Dressing trays
(do not touch the body) Packaging materials

TABLE 14.8
Typical Research Concerning Small Diameter (Less than 6 mm I.D.) Vascular Grafts
Material Important Design Feature
Polyetherurethane Tube walls are porous and microfibrous three-dimensional mesh composed
of polyurethane fibers of l μm diameter. Penetration depth of living tissue cell
is limited to about 20–30 μm
Polyetherurethane Graft with pore size 30–100 μm
Polyetherurethaneurea It is of sufficient porosity to permit tissue ingrowth
Its stress-strain curve is similar to that of the thoracic aorta
Polyetherurethaneurea/silicones Microporous replamineform graft (20–30 μm pore size) composed of silicones
was coated with commercial Biomer™
Polyetherurethaneurea Fibrillar microporous hydrophobic graft made from commercial Mitrathane™
Polyetherurethane Grafted with albumin and adenosine-cyclic monophosphate
Surface has better blood compatibility
Modified the surface further with urokinase
Degradable, aliphatic It has well defined chemical and physical properties and controlled rates of
Polyesterurethane degradation
It induces the growth of functional neoartery which has cellular structure similar
to that of the native artery
Expanded Teflon coated with silicones Platelet accumulation was reduced by coating the graft surfaces with a smooth
layer of silicones
Polyesterurethane (Vascugraft™) Made from commercial polyesterurethane free from migrating additives such
as catalysts and stabilizers
Microfibrous, open pore structure
Polyurethane/poly (l-Lactide) Microporous, compliant and biodegradable
Function as a temporary scaffold for the generation of small-caliber arteries
Polyurethane/poly (l-Lactide) Microporous and biodegradable graft consisted of two layers: the inner layer
made from aliphatic polyetherurethane, the outer ply constructed by
precipitating a physical mixture of polyesterurethane and poly (l-Lactide)
Polyetherurethane Microporous graft prepared by an excimer laser ablation technique
The pore size (100 μm) and the pore-to-pore distance (200 μm) are constant
Polyetherurethane Made by a filament-winding technique comprising Lycra™ elastomeric fibers
embedded in an elastomeric matrix of Pellethane™
Poly (carbonate urethane)/ Glycine or fibronectin are covalently bound to polyurethane surface by succinyl
pentapeptide dichloride coupling
Biodurable poly-(carbonate urethane) Microporous grafts with excellent physical and mechanical characteristics.
(ChronoFlex™) Self-sealing and maintenance of compliance
Polyurethane/poly Polyurethane modified by coating with poly (HEMA- block-styrene)
(HEMA-block-styrene) from solution
Copolymers of MPC Copolymers of 2-methacryloyloxyethyl phosphorylcholine (MPC)
with hydrophobic alkyl methacrylates
Polyurethane/ Endothelial cell The hybrid artificial graft made of an endothelialized microporous polyurethane
Polyester/human vein Vascular graft made of human vein and a highly porous polyester fabric vascular
prosthesis

TABLE 14.9
Elastomers Used for Making Artificial Hearts
Supplier or Investigator Type of Blood Pump Elastomer
Novacor’s LVAD Pusher-plate Biomer™
Symbion Diaphragm Biomer
Aviomed Tuberous Angioflex™
Nimbus Pusher-plate Hexsyn™/gelatine
Thermedics LVAD Tuberous Tecoflex™
Nippon Zeon VAD Sac Cardiothane™
Totobo VAD Diaphragm TM-3™, TM-5™
Berlin University Diaphragm Pellethane™
TABLE 14.10
Implant Applications for Medical Grade Silicone Elastomers
Treatment Application
Plastic and reconstructive surgery Reconstruction of nose, chin, ear armature, etc.
Breast reconstruction
Ophthalmology Correction of detached retina
Prosthetric eye
Repairing fracture of the floor of the orbit
Orthopedic surgery Reconstruction of fingers, thumbs, wrists, elbows feet, tendons,
temporomandibular joint, etc.
Maxillofacial prosthesis
Penile prosthesis.
Cardiovascular surgery Ball in the ball-and-cage heart valve
Coatings on pacemakers and leads-wires
Construction in artificial hearts and heart assist devices
TABLE 14.11
Elastomers Used in Transdermal
Therapeutic Systems
Classification Elastomer
Hydrophobic polymers Poly(ethylene-co-vinyl acetate)
Silicone rubber
Polyurethane
Poly(vinyl chloride)
Collagen-based materials
Ethylcellulose
Cellulose acetate
Hydrogels Poly(hydroxyethyl methacrylate)
Crosslinked poly(vinyl alcohol)
Crosslinked poly(vinylpyrrolidone)
Polyacrylamide

TABLE 14.12
Typical Materials for Contact Lenses
Classification Polymer and Characteristics
Hydrogel soft lenses Poly (hydroxyethyl methacrylate) (PHEMA)
Generally crosslinked with ethylene glycol dimethacrylate, at a level of 26%–42%
sufficient to give water content in equilibrium
It may be copolymerized with other hydrophilic monomers, such as N-vinyl
pyrrolidone, acrylamide and methacrylic acid
Elastic soft lenses Polydimethylsiloxane and its copolymers
They have good flexibility and very high oxygen permeability, but strong
hydrophobicity results in considerable discomfort for the patient
Surface treatment may improve wettability
Perfluoropolyethers and its copolymers
They have attractive combinations of flexibility and gas permeability
Rigid lenses Poly(methyl methacrylate) (PMMA)
It has good properties: very clear optically, good biocompatibility and good
handling characteristics
The main problem relates to poor oxygen permeability. There have been attempts
to introduce arrays of very small holes (15 μm) to facilitate oxygen transport
Gas-permeable rigid lenses Cellulose acetate butyrate and others
The butyrate is a highly oxygen-permeable and relatively rigid material
The copolymerization of siloxany alkyl methacrylates of fluorosilicone
methacrylates with methyl methacrylate provides a rigid methacrylate backbone
with rubbery, highly permeable alkylsiloxane side groups
14.2 METHODS TO EVALUATE THE BIOCOMPATIBILITY OF ELASTOMERS

14.2.1 Definitions
Most medical devices use synthetic biomaterials as their principal component. A biomaterial refers
to any nonvital material intended to interact with biological systems within or on the human body.
In general, elastomers, like other biomaterials, must be compatible and inert, must be nontoxic
to living tissues, and must not destroy the cellular constituents of the body fluid with which they
interface. A battle takes place between the organism and the implanted elastomer. Possible out-
comes include acceptance, slow digestion of the implant, or rapid ejection accompanied by unde-
sired secondary effects (Dumitriu and Dumitriu 1990). Medical devices must thus be designed to
prevent rejection. This has led to the development of a new class of materials called biomaterials,
which includes elastomers used in the medical field. The ability of biomaterials to fulfill their role
in medical devices depends on their degree of biofunctionality and biocompatibility.
Biocompatibility is the ability of a material to perform it function in a host in a given situation
(Williams 2003) when applied as intended. However, a “biocompatible” material may not be com-
pletely inert. Because of this, the host response is decisive (Schmalz and Arenholt-Bindslev 2009).
It is difficult to talk about the biocompatibility of elastomers since they are only in contact with
physiological systems in the form of medical devices. In addition, the biocompatibility of elastomers
depends on the type of device and its function. In theory, an evaluation of the biocompatibility of
an elastomer should include
• The behavior of host tissue and the host itself in the presence of the biomaterial
• The behavior over time of the biomaterial in the host environment
Forehead prosthesis
Auricle prosthesis Macular plombe
External auditory canal Eyelid bag prosthesis

dilating prosthesis
Contact lens
Synthetic eyeball
Tracheal cannula Orbital floor implant
Expanding prosthesis
for the contracted
esophagus
Nasal cartilage
Mammary prosthesis Nasal cartilage block
Suction prosthesis
for nipple
Mandibular prosthesis
Artificial skin Wrist joint
Finger joint
Elbow-joint cap
Artificial nail Hip-joint cap
Colostomy kit
Synthetic testicle
Penis implant
Retainer for
elytroplasty
Toe joints
FIGURE 14.17 Medical device in the human body.

However, in practice, in vivo studies of all these parameters are difficult if not impossible to con-
duct, which is why the evaluation of biocompatibility begins with in vitro testing. Briefly, biomateri-
als have evolved through three generations:
1. First generation: technical performance (noncytotoxic = tolerance)

2. Second generation: biological performance (cytocompatibility)
3. Third generation: biological performance (biofunctionality)
Four trends currently dominate the evolution of biomaterials:
1. Giving materials a surface that mimics the biological properties required for the intended
application, which requires fundamental knowledge of molecular/cellular reactions at the
material/tissue interface
2. Taking implantation in young subjects into account, which implies long-term use and the
need for immunological, mutagenic, and carcinogenic evaluations
3. Decreasing animal testing, which requires the development of alternative methods
4. Setting up marketing regulations, which includes the development of standard tests
14.2.2 General
Elastomers are very important in the manufacture of medical devices that may come into contact
with host tissues for varying periods of time. Exposure times have been arbitrarily divided into
three categories: limited exposure (<24 h), prolonged exposure (24 h to 30 days), and permanent
exposure (>30 days). The tests used to evaluate biocompatibility depend on exposure time. It is thus
easy to understand why elastomers used to make implants have to meet stricter standards than those
used for dental impressions. This classification system is only a guide.
Various standards (mainly ISO 10993-(ISO 2003a) ) apply to medical devices that are
1. In contact with surfaces like the skin or mucous membranes other than the skin
2. In direct or indirect contact with other tissues, including blood
3. Implantable devices
14.2.3 Testing
14.2.3.1 Cytotoxicity
In vitro cytotoxicity testing evaluates lysis, growth inhibition, and other impacts on cells by
medical devices, component materials, and leachates using morphological, biochemical, and
metabolic criteria. These techniques have well-defined standards for material preparation,
sterilization, cell lines, and the type of testing required. Whatever methods are used, con-
trols must be included. Cell culture studies are usually the first step of the evaluation of the
biocompatibility.
14.2.3.1.1 Material Preparation

The shape of the material used for biocompatibility testing varies greatly from author to author.
Generally speaking, however, elastomers are tested as disks the size of multiwell plates used for cell
cultures. Lehle et al. (2009) used 6 mm diameter disks, Bal et al. (2009) used 14-mm-diameter disks,
while Briganti et al. (2006) used 10 × 15 mm pieces to evaluate polyurethanes. Other techniques involve
producing blocks, which are then cut into thick slices using a microtome. Special attention must be paid
to the cut surface, which must not only be identical in test products and controls but must also resemble
the surface of the finished product. It is often preferable in the industrial research phase to test materials
that correspond as closely as possible to the finished product. In the development and marketing phases,
evaluations can be performed on randomly selected “parts” to avoid testing bias.
Many studies have been conducted on the cytotoxicity of finished elastomers. Extracts are pre-
pared on the basis of recommended normative standards. A range of extraction protocols is avail-
able, including dynamic extraction, static extraction, and heat extraction.
The chemical substances extracted from elastomers are usually residues from accelerators
(e.g., mercaptobenzothiazole and tetramethylthiuram disulfite), activators (e.g., zinc oxide), inert
fillers (e.g., wax), plasticizers (e.g., oil), lubricants (e.g., stearic acid), and vulcanization products
(e.g., zinc stearates and dithiocarbonate zinc salts). To minimize the amount of residue extracted
from elastomers, they must be either cleaned or coated, or both.
14.2.3.1.2 Sterilization
The sterilization protocols for the various items used for cell cultures vary depending on the
elastomer being evaluated. Most authors report using ethylene oxide (500–540 mg/L EO in 80%
CO2 at a relative humidity of over 35% for 3 h at 35°C–41°C). Others have used gamma radiation
(2.5 Mrad for 5 h) (Pizzoferrato et al. 1995, Saad et al. 1996), dry heat or autoclaving (Bal et al.
2009), antiseptic solutions (Hunt et al. 1996, Menconi et al. 1992), antibiotics occasionally combined
with UV radiation for 48 h (Menconi et al. 1992) or 30 min (Ozdemir et al. 2009), and immersion in
70% ethanol for 30 min followed by immersion in phosphate-buffered saline containing antibiotics
(Morrison et al. 1995). In recent years, sterilization by exposure to gamma irradiation has increased
in popularity with device manufacturers because it does not produce toxic residues. Most elastomers
resist gamma radiation sterilization with minimal changes to their properties. All polymers are
affected to some degree by irradiation, with crosslinking and molecular scission reported. While
there have been reports that sterilization of polymers influences their performance in cell cultures
with changes in cell attachment and morphology, certain authors do not mention the sterilization
method used. There is an international standard regulating the management of residues following
sterilization by ethylene oxide, glutaraldehyde, and formaldehyde (ISO 1995, 2002a), but there are
no standards regulating sterilization procedures per se.
14.2.3.1.3 Cell Lines

Fibroblast cell lines (3T3, L929) are frequently used in cytotoxicity tests (Chen et al. 2008, Ozdemir
et al. 2009, Zia et al. 2009a,b). Tests based on the medical end uses of materials are conducted
with cell lines that are as similar as possible to the cells or tissues that will be in contact with the
elastomers in vivo. For example, MG 63 osteoblast cell lines and RAW 264.7 macrophages (Yim
et al. 2009) are used for testing materials to replace cartilage, and SV-HUC1 cells are used to test
materials that will be in contact with the urinary tract (Pariente et al. 1998).
Primary cultures are more suitable for cytotoxicity testing. Various cells from different origins
have been successfully used to evaluate the toxicity of elastomers. While primary cultures, espe-
cially those derived from human tissues, provide superior biocompatibility testing results because
they are only one step removed from in vivo conditions, they do not allow interlaboratory com-
parisons to be made. Ideally, researchers should include both an established reference line and a
primary culture in their studies, as was the case with Lehle et al. (Lehle et al. 2008).
Cells can be also grown in vitro in three dimensions, which allows better in vivo stimulation.
A three-dimensional full-thickness engineered human oral mucosal model has recently been opti-
mized (Moharamzadeh et al. 2008).
In dentistry, an ISO 7405 standard applies specifically to dental materials (semi-horizontal
standard) (ISO 2008).
14.2.3.1.4 Tests
The crystal violet, neutral red, and trypan blue cell viability screening tool developed over 30 years
ago for evaluating the toxicity of plastics is the most common test (Rosenbluth et al. 1965). Whatever
the cell type, two methods can be used: direct tests where the cells are placed in direct contact with
the material, and indirect tests where extracts of the medium used to culture the cells are placed
in contact with a cell culture either by diffusion across a membrane or in an agar overlay (Johnson
et al. 1983, Kirkpatrick and Mittermayer 1990). While the incubation period can vary from 24 h to
6 days, depending on the protocol, it also depends on the type of cells used. Generally speaking, the
incubation period can be longer for established cell lines than for primary cultures. A tetrazolium-
based colorimetric assay (MTT) is often used to evaluate polymer cytotoxicity or, more precisely,
the metabolic activity of cells in contact with the material. The MTT assay is simple, reproducible,
and does not use radioisotopes. Tests can be carried out using a fully automated microtiter plate sys-
tem, making it possible to economically test polymers of limited availability. It has also been used
to evaluate polyurethanes (Haugen et al. 2006, van Minnen et al. 2005), polyesterurethanes (Saad
et al. 1997), elastomers (Chauvel-Lebret et al. 1999), and urinary stents (Pariente et al. 1998) The
in vitro WST-1 cytotoxicity test based on the European ISO 10993-5 standard (ISO 1999a) is also
used and, like the MTT test, involves the release of water soluble products from a tetrazolium salt
by metabolically active cells. However, the results obtained using this test are different from those
of the MTT test (Haugen et al. 2006).
In addition to cell viability and metabolic activity assays, cell morphology is also verified to
complete the first-phase screening of elastomers. The impact on cell morphology is generally deter-
mined by scanning electron microscopy (Guidoin et al. 1992, Haugen et al. 2006, Lee et al. 1996a,
Menconi et al. 1992). Samples are generally dehydrated by critical point drying and then metal-
licized with colloidal gold. Elastomers that have not been in contact with the cells are used as con-
trols. While cell viability and morphology tests provide an initial screening for cytotoxicity, many
researchers have been working to examine the biofunctionality of cells in contact with elastomers
and elastomer extracts. Various tests have been used, including the evaluation of protein synthesis
and mRNA expression. Fluorescence-activated cell sorting (FACS) and Western blotting are used
to detect the impact of biomaterials on cell metabolism, especially on signaling pathways. These
methods are used to evaluate radical oxygen species concentrations, apoptosis, DNA damage and
repair, and synthesis of specific inflammation mediators (Hsu et al. 2008, Hung et al. 2009b, Lorenz
et al. 2006).
The concept of biocompatibility is based on the interaction between a material and a biological
environment, most often expressed as an inflammatory response. Implanted elastomers provoke
an inflammatory response, which initiates the process of tissue repair and regeneration. Various
in vitro tests have been developed to evaluate the inflammatory response and repair phases. The
tests most frequently reported in the literature to determine the biocompatibility of polyurethanes
involve measuring the concentrations of NO and TNFα, which are produced when macrophages
and osteoblasts interact with polyesterurethanes (Saad et al. 1996, 1997), the concentrations of vari-
ous types of fibronectin and collagen (Lee et al. 1996b, Saad et al. 1996, 1997, Sprague and Palmaz
2005), and the adsorption of proteins.
Techniques based on molecular approaches are being developed to characterize the interactions
between cells, biological fluids, and biomaterials. Most of these techniques have become more
accessible with the arrival of commercially available molecular biology kits. Gene expression using
microarray test systems reveals information on the genes involved in the cellular stress response
(Schweikl et al. 2008, Tare et al. 2009) and has been proposed for biocompatibility tests (Pernagallo
et al. 2009). These tests obviously depend on the end use of the elastomer. With increasing informa-
tion available on interactions between cells and biomaterials, new guiding principles for the in vitro
evaluation of biocompatibility, including the use of relevant cells types and biological parameters
and the need to use dynamic conditions will be necessary in the future (Owen et al. 2005).
14.2.3.1.5 Controls
Tests cannot be valid or objective unless internal controls are included. Tissue culture plastic is a
commonly used negative control, although silicone rubber has also been used. Asbestos, phenol, and
latex, which cause major disruptions to metabolic activity, are often used as positive or cytotoxic
controls (Pariente et al. 1998). It should be noted that latex has been used as both a negative and
positive control. Its cytotoxicity arises from the presence of accelerators and oxidants used during
the vulcanization process (van Minnen et al. 2005, Oshima and Nakamura 1994). As indicated by
Park and Park (Park and Park 1996), in the absence of clear criteria for evaluating biocompatibility,
many materials have been mistakenly thought to be biocompatible and have been used as controls.
Silicone rubber is a case in point. For a long time, silicone rubber was believed to be totally bio-
compatible. It is clearly necessary to reevaluate the biocompatibility of existing biomaterials and a
fortiori, the negative controls. Positive controls are not reported in many studies, which undermines
the validity of the results and proposed evaluation technique.
14.2.3.2 Sensitization Assays—Irritation or Intercutaneous Reactivity—Implantation

General Considerations: Even today, there are no totally satisfactory in vitro elastomer evalua-
tion systems. While such assays provide an initial screening, in vivo testing on animals is required
before implanting, injecting, or using elastomers in humans. A nonexhaustive search of the litera-
ture between 1990 and 2010 reveals that too many animals have been sacrificed just to study the
implantation response of elastomers. It is vitally important that researchers meticulously plan their
studies to extract the maximum amount of information from each animal.
14.2.3.2.1 Material Preparation

For solid implants, the physical properties (shape, hardness, surface finish) must be identical for each
implant, except if one of the parameters is being singled out for study. The chemical and morphological
aspects of elastomeric surfaces must be controlled (Chesmel and Black 1995). All contaminants must
be removed from the implants, which must then be sterilized using the method that will be applied
to the finished device. Careful attention must be paid to comparisons between in vivo responses and
in vitro assays. For example, Rosengren et al. (2005) compared the in vivo tissue responses to in vitro
cytotoxicity before and after implantation to evaluate the predictive value of cytotoxicity testing. They
observed that when zinc diethyldithiocarbamate was added to polyurethane, fewer cytotoxins were
released in vitro than in vivo conditions. They concluded that many clinically useful implant materials
may be unnecessarily rejected due to the results of in vitro tests.
Tests on nonsolid materials such as powders, liquids, and particles (including mixtures) can
be conducted in PTFE tubes. Some authors dispute the suitability of PTFE as a negative control,
especially as regards surface properties. Cylindrical cages can be fabricated using stainless-steel
wire mesh (Christenson et al. 2004a). At the very least, control tubes or cages must have the same
physical properties as test tubes.
14.2.3.2.2 Animals
The choice of animal depends on the size of the implant, the test period, and known differences in
biological responses of soft and hard tissues. Rats are the animals of choice for many studies. Implants
are placed bilaterally in the back muscles. Two animals per material per implant period are used with
a minimum of two implants per animal (Fabre et al. 1998, Hunt et al. 1996, van Minnen et al. 2005,
Pomerantseva et al. 2009). When rabbits are used, 8–12 implants are placed in the back muscles
(Briganti et al. 2006, Oloffs et al. 1994). Swine are also used to test biocompatibility and hemocom-
patibility (Richard et al. 2009). Dogs are used to study new materials for vasectomies and arterio-
venous fistulas (Carroll et al. 1993, Schellhammer et al. 1999) and to test materials for temporary
replacement organs. The number of animals used varies greatly depending on the type of research
and the complexity of the procedure. Because of the differences, few studies can be compared.
14.2.3.2.3 Implantation Period

The implantation period varies greatly depending on the animals used and the material being evalu-
ated. In the few chronic inflammation studies that have been reported, positive controls that could lead
to a major inflammatory response within 12 weeks are missing. Generally speaking, observations are
made over a 30 day (Fabre et al. 1998) or 12 week (Carroll et al. 1993, Huang et al. 1992, Hunt et al.
1996) period. The implantation period must be chosen to obtain a stable biological reaction, which
depends on the nature and properties of the material and the damage caused by the surgery. A recent
study published results of implantation after a 2 year period in dogs (Welsing et al. 2008).
14.2.3.2.4 Evaluation of Biological Reactivity

Evaluations of biological reactivity begin with an analysis of health of the animals (vigilance, nutri-
tion, coat, weight, temperature, etc.). Macroscopic and histopathological reactions are evaluated
over time. Histopathological examinations include the degree of inflammation, the number and type
of inflammatory cells (leukocytes, PMNs, lymphocytes, plasmocytes, macrophages, polynuclear
cells, etc.), necrosis, and the presence of material debris, granulomas, fat, and calcified tissue.
14.2.3.2.5 Irritation and Sensitization Assays

Irritation is a nonimmunological inflammatory reaction. Sensitization assays (contact allergies)
and delayed hypersensitivity reactions involve the immune system. These assays are vital for
understanding acute toxicity. Rabbits are often used for skin irritation assays with observations at
1, 24, 48, and 72 h. The test period may be prolonged in the event of persistent lesions to determine
whether the lesions are reversible or permanent, but must in no case exceed 14 days. Less than
0.2 mL of extract is injected intradermally. Irritation is scored based on the level of edema and
erythema. Generally speaking, 10 injections are made on the anterior and posterior portions of
the trunk (including controls). Ocular and oral mucosal assays may also be conducted. To evaluate
immune responses, tests developed for other purposes such as research on autoimmune disease ani-
mal models and conjunctive tissue pathologies are used. These tests are based on delayed hypersen-
sitivity reactions induced by a given autoantibody (thyroglobulin), anti-collagen, etc. In the research
reported by Naim et al. (1995), silicone gels were tested for toxicology, antigenic, and adjuvancy
properties. In vitro skin irritation tests are being as alternatives to animal models.
14.2.3.2.6 Implantation
All elastomers act as foreign bodies when they are implanted, leading to an acute inflammatory
response and the accumulation of phagocytes (Anderson et al. 2008). Elastomers are relatively inert,
unreactive, and nontoxic. It is thus difficult to understand how they are detected by the immune
system and how they provoke an inflammatory response. However, considering the mechanisms
involved in such responses, a good starting point would be the initial interaction between the bio-
material and proteins in the surrounding host tissue. Immediately after implantation, hydropho-
bic polymers like polyurethanes, polyethylenes, polydimethylsiloxanes, and dacron become coated
with host protein. Plasma and denaturated interstitial fluid proteins rapidly colonize the implant,
binding together to form a disorganized matrix on the implant surface. Since proteins rapidly adsorb
to the surface of the biomaterial, host inflammatory cells and fibroblasts cannot come into direct
contact with the implant. This protein coat determines future cell reactions to the implant and it
is undoubtedly this initial phase that is key in determining biocompatibility. Albumin, immuno-
globulin G, and fibrinogen are the main proteins coating the surfaces of implants. Conventional
histological techniques involving hematoxylin, eosin, Von Gieson, and Schiff periodic acid stains
are often used to study the inflammatory response. The inflammatory cells, which include macro-
phages, PMNs, and lymphocytes, are identified based on morphological criteria. The distribution
of these cells around the implant provides a qualitative description of the response. A number of
assays using computer assisted image analysis have been developed. Discriminating between differ-
ent cells types has also greatly evolved with the arrival of specific monoclonal antibodies. Specific
enzymatic assays and signal amplification kits (avidin, biotin, etc.) have considerably enhanced
the ability to gauge implant inflammatory responses. In addition to these histochemical staining,
immunocytochemical, and counting techniques, certain authors have published the results of in situ
hybridization techniques for evaluating inflammatory protein production (TNFα) and detect the
expression of TNFα mRNA. These techniques provide a clear picture of the intensity of the inflam-
matory response. The reaction of TNFα and other growth factors or enzymes must reflect a balance
between the tendency to promote biocompatibility and the tendency to cause inflammatory damage
and implant rejection (Hunt et al. 1996, Lobler et al. 1999).
A number of researchers have analyzed the quality and quantity of the inflammatory exudate
using the in vivo cage implant system originally developed by Marchant et al. (1983). A number
of biochemical tests (protein analyses, albumin concentration, extracellular enzymes) are possible.
The exudate may also be analyzed by cytofluorometry for quantitative results (Fabre et al. 1998,
Huang et al. 1992). Flow cytometry appears to be particularly well suited for studying exudate cell
composition. The literature on exudate cell responses is relatively meager compared to that on tissue
reactions. To our knowledge, there have been no reports correlating tissue inflammation (as mea-
sured by histomorphometric image analysis) and cellular response (as measured by flow cytometry).
14.2.3.3 Hemocompatibility and Blood Interaction Assays

14.2.3.3.1 General
Only materials in prolonged or permanent contact with blood pathways must be subjected to this
type of testing, which can be divided into five categories depending on the primary process (throm-
bus, coagulation, platelet aggregation) or biological system involved (blood, immune). Various stan-
dards determine which tests will be used to evaluate hemocompatibility:
• Devices whose surfaces are not in direct contact with the blood
• Devices whose surfaces are in direct contact with the blood
• Implantable devices
In certain cases, it is difficult to determine whether biocompatibility and/or hemocompatibility tests

should be performed. In general, experience has shown that materials that are hemocompatible will
also be biocompatible. However, not all biocompatible materials are also hemocompatible. The
situation is even more complex because there has been a tendency to designate materials as being
biocompatible on the basis of toxicological tests while others have been designated as being hemo-
compatible on the basis of a single whole blood clotting test (Bruck 1980).
14.2.3.3.2 Tests
A given material may be adequate for one type of application, but not for others. Differences between
arterial and venous blood, varying blood flow patterns, and the design and mechanical operation of a
medical device are among the parameters that influence the performance of materials when they are
in contact with blood. Most biomaterial researchers would like to be able to predict biological perfor-
mance using simplified methodologies. However, no single procedure is adequate for this purpose.
Tests that merely measure the amount of thrombus give only the final result of the blood coagulation
sequence without any insight into the initial events. A rheology-based test allows the sequence of events
to be measured over time. ISO 10993 guidelines for the biological evaluation of implantable medical
devices (ISO 2002a,b) list a number of recommended in vitro and in vivo tests that can be used to study
hemocompatibility. ISO recommends the hemolysis test as the standard screening method.
Many researchers use the acute ex vivo canine femoral anteriovenous series shunt technique,
which allows the testing of a number of materials under similar physiological and hematological
conditions for in vivo hemocompatibility studies (Chen et al. 1998, Peek et al. 2002). High levels
of platelet and fibrinogen deposition are generally associated with a more thrombogenic polymeric
surface. The morphology of the adherent platelets examined by SEM also provides valuable infor-
mation. A measure of thrombogenicity is the tendency of a surface to cause adherent platelets to
change shape and activate.
Hemocompatibility tests include evaluations of platelet adhesion, aggregation, activation, and

release reactions under dynamic blood flow conditions together with measurements of protein
adsorption and coagulation factor activation. The most common test for evaluating platelet adhesion
uses fresh blood from healthy donors. The platelets are isolated and a standard microscopic or flow
cytometric analysis is performed (Chen et al. 1998, Rechavia et al. 1998, Tarnok et al. 1999). Platelet
counts can also be determined using the Petri disk model with flat sheet membranes. Complement
activation and release reactions have also been explored. To evaluate the extent of in vitro comple-
ment activation by elastomers, the concentration of C3a des Arg has been measured according to
the method of Wagner and Hugli (1984) using radioimmunoassay kits available from a number of
manufacturers (Branger et al. 1990).
Tests evaluating contact activation, the intrinsic coagulation system, the Hageman factor (f XII)
and dependent kinin formation and fibrinolysis pathways have been developed. A quantitative mea-
surement of the activation of the contact system is required in studies examining the effect of arti-
ficial surfaces on blood. Van der Kamp and Van Oeveren (1994) proposed analyzing these cascades
by using the kallikrein inhibitor aprotinin to calculate the activity of factor f XII and kallikrein.
One approach to improving the hemocompatibility of biomaterials has been to immobilize anti-
coagulants like heparin at the interface (Albanese et al. 1994, Hong et al. 1999). Tests including
clotting assays (TT, reptilase time, and anti-XA activity), coagulation time, platelet counts, and
Resonance Thrombography (RTG) have been used.
Previous studies have shown that blood-contacting materials activate blood monocytes. A highly
sensitive technique has been proposed to detect changes at the mRNA level in circulating mono-
cytes and to find suitable “gene markers” for assessing the hemocompatibility of biomaterials.
Human blood can be recirculated in a modified Chandler Loop formed of test tubes. The recircula-
tion of human blood in an in vitro model in combination with the immunomagnetic separation of
monocytes and the Duplex RT-PCR method is a powerful tool for getting reliable results (Weber
et al. 2001).
Also, with the development of nanomaterials, a new test has been developed to validate an
in vitro method designed to analyze the potential of a variety of nanoparticles to damage red blood
cells. Specifically, the study describes approaches to identify nanoparticle/erythrocyte interfer-
ences, when they occur and how to resolve them to get accurate results, i.e., to avoid false-positive
or false-negative results (Dobrovolskaia et al. 2008).
14.2.3.4 Biodegradation Assays

These assays are used to evaluate both biodegradation and biostability. Degradation products are
generated by the decomposition or chemical degradation of a material. Biodegradation is the deg-
radation of a biological material involving a loss of integrity and/or performance during exposure
to a physiological or simulated environment. Elastomer degradation is studied using toxicokinetic
modeling. Many types of surgically implantable devices and drug delivery systems that only func-
tion for a relatively short time in vivo can be made from polymers that are eliminated from the
body by hydrolytic degradation and subsequent metabolism after serving their intended purpose.
Biomaterials made of biodegradable polymers are designed to degrade in vivo in a controlled man-
ner over a predetermined period. The main in vivo degradation mechanism of polymers is hydro-
lytic degradation in which enzymes may play a role. In vitro cell culture and animal models may
also be used (Piskin 1994, Singh et al. 1979).
So-called nonbiodegradable biomaterials may be slowly degraded over time by the organism.
One example is the failure of PU medical devices like pacemaker leads, which are manufactured
from poly(ether) polymers. PU failure consistently occurs 5–10 years after implantation. The mech-
anism of this process is still not well understood (Santerre et al. 2005). Although in vitro studies
have been performed with single enzyme systems, which showed the release of products, the in vivo
situation involves complex biosystems that act synergistically. Other assays using sophisticated tech-
nology such as attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR)
(Mathur et al. 1997), transmission Fourier transform infrared analysis (T–FTIR), and scanning elec-
tron microscopy (SEM) (de Brito Alves et al. 2003, Collier et al. 1998) have also been developed.
These techniques can be used to evaluate inflammatory responses and throw light on the entire
degradation process of a biomaterial.
No progress on the evaluation of degradation has appeared in the literature since 2000. An ISO
framework (ISO 1999b) and standard (ISO 1998) are available.
14.2.3.5 Reproduction and Embryo Development—Assays

It is not known whether implant materials can have an impact on reproduction and embryo develop-
ment. The few tests that have been conducted in this area involve the functional testing of biomate-
rials used for preparing sperm prior to artificial insemination, in vitro fertilization procedures, and
embryo transfers. These assays can be conducted in vitro (e.g., spermatozoid cultures) or in vivo
(e.g., sperm penetration tests using hamster ova and mouse embryo compatibility studies) (Hunter
et al. 1988). To our knowledge, no standards have been established for these assays.
14.2.3.6 Genotoxicity
ISO 10993-3 (ISO 2003b) is a set of criteria based on the intended use of a product or device and
establishes testing guidelines depending on the implantation period and the nature of the contact
with the biomaterial. The criteria stipulate that any material or implantable device placed in con-
tact with mucosal, bone, or oral tissue, when the contact exceeds 30 days, must be subjected to
genotoxicity testing prior to commercialization. The tests are intended to detect genetic anomalies
(mutations, chromosomal alterations) in cells (prokaryote, yeast, mammalian) placed in contact
with the material. A review of the literature has revealed that four main techniques are used to
evaluate genotoxicity: the sister chromatid exchange method, the micronucleus test, the Ames test,
and the chromosomal aberration test (Nadeenko et al. 1997, Schendel et al. 1995). Other tests appear
to evaluate genotoxicity using a micronucleus assay (Elespuru et al. 2009, Kleinsasser et al. 2006)
and a high-throughput comet assay (Stang and Witte 2009). The high-throughput comet assay was
developed to reduce processing time and increase sample-throughput of the assay (Tice et al. 2000).
The number of published studies addressing the mutagenicity of elastomers is comparatively low.
14.2.3.7 Biocompatibility Testing in Humans

Clinical tests are better indicators of biocompatibility and also help improve biomaterials. While
implantable devices are supposed to be biocompatible based on in vitro testing, in vivo clinical test-
ing is the only way to be sure. Human tests are conducted using ethical research practices. These
tests have been used in randomized clinical research protocols (Baumann et al. 2003, Hollick et al.
1998, 1999), medium to long-term epidemiological studies (Bambauer et al. 2004, Boldin et al.
2008, Johnell et al. 2005, Rubin and Yaremchuk 1997), and in one study with a 20 year follow-up
(Donawa 2006a,b,c, Pritchett 2008).
14.2.3.8 Biocompatibility Testing—Microbial Colonization

There are no bacteriological standards or compulsory tests in the field of reproduction and
embryo development. Elastomers used in implantable medical devices are by definition sterile
(see Sterilization). Nevertheless, certain authors have included microbial (including yeast) adher-
ence and colonization in their battery of tests (Gottenbos et al. 1999, Trevisani et al. 2005). Such
testing is recommended for certain elastomers for which implantation failure may be caused by bac-
terial infections. The plasma proteins deposited on implant surfaces may mediate bacterial adher-
ence, especially that of Staphylococcus aureus, a pathogen associated with recurrent infections.
Bacterial adherence testing is conducted in radial flow chambers mounted on the motorized stage of
a video microscopy system. Image processing software is used to perform automated data collection
and image analysis (Baumgartner et al. 1997, Santos et al. 2008). Bacteria-induced infections in the
presence of polymers have been studied in animals. It would be of interest to determine how bacteria
adhere to polymer substrates and, in certain cases, to evaluate the efficacy of antibiotic/antiseptic
treatments (Kockro et al. 2000). A better understanding of bacterial genomes should also lead to a
better understanding of virulence factors produced by bacteria. These virulence factors may vary
depending on the biomaterial as suggested by changes in bacterial phenotypes (unpublished data).
14.3 BIOCOMPATIBILITY OF DIFFERENT CLASSES OF ELASTOMERS

14.3.1 Elastomers in General
Biomaterials are materials designed for use with living tissues and/or biological fluids in order
to evaluate, treat, modify, or replace a tissue, organ, or bodily function (Kalman et al. 1991). For
materials to be biocompatible, they must not provoke allergic, inflammatory, or immunological
reactions, they must be nonthrombogenic, nontoxic, and noncarcinogenic, and they must not dam-
age surrounding tissues, plasma proteins, or enzymes (Kalman et al. 1991, Piskin 1994). However,
in most cases, it is not a lack of response that is important but rather an appropriate host response to
the specific application for which the biomaterial was designed. Biocompatibility is thus defined by
the response of the biological system to the biomaterial, which is seen as a foreign body and which
provokes a cascade of interrelated reactions both systematically as well as locally at the interface
with the biomaterial (Piskin 1994).
Biocompatibility is directly dependent upon the magnitude and duration of the host inflamma-
tory response (Kao et al. 1999, Roach et al. 2007). The body initiates a cascade of reactions against
the foreign material (Brodbeck et al. 2005).
Biomaterials may thus lead to numerous undesirable side effects (clotting, infections, fibrosis,
thrombosis, tissue necrosis, carcinogenesis, etc.). These effects can be caused by the degradation or
mineralization of the biomaterial. In many cases, these adverse effects are associated with the rapid
accumulation of large numbers of phagocytes (Anderson et al. 2008, Hu et al. 2001).
Over the long-term, the service life of the biomaterials depends on the mechanical and physico-
chemical properties of the biomaterial on the micro and nano-scales (Roach et al. 2007).
14.3.2 Local Reactions
When a biomaterial comes in contact with an intra or extravascular system, a complex sequence
of events involving protein adsorption, monocyte/macrophage adhesion, and macrophage fusion to
form foreign body giant cells (FBGC) takes place (Anderson et al. 2008, Schmidt and Kao 2007).
The initial responses are acute inflammation within a few days, followed by chronic inflamma-
tion (Anderson et al. 2008, Brodbeck et al. 2005, Schmidt and Kao 2007).
14.3.2.1 Protein Adsorption

Implanted biomaterials very quickly acquire a layer of host proteins well before the arrival of
inflammatory cells (Hu et al. 2001, Roach et al. 2007). The first to adsorb are the more abundant
small proteins, which are then replaced by larger proteins with a greater affinity for the surface of
the biomaterial. The initial adsorption prevents direct interactions between cells and the implant
material (Roach et al. 2007).
Protein cascades may be closely involved in the dynamic phenomenon of protein adsorption and
desorption know as the Vroman effect (Anderson et al. 2008, Roach et al. 2007).
Protein adsorption is affected by surface chemistry and nanoscale topography. Protein deforma-
tion is induced by interactions with a surface and is affected by electrostatic forces, entropic effects,
hydrophobic interactions, and conformational changes (Roach et al. 2007).
The presence of adsorbed proteins such as albumin, IgG, complement, vitronectin, gamma glob-
ulin, fibrinogen, and fibronectin modulate the host inflammatory response, cell activation, adhesion,
and wound healing (Anderson et al. 2008, Lloyd 2002, Tang and Eaton 1995).
The primary interactions between cells and proteins occur via integrins. Integrins bind specifi-
cally to an arginine-glycine-aspartic acid (RGD) in adhesins such as fibronectin, vitronectin, lam-
inin (Roach et al. 2007, Vasita et al. 2008).
The differences in integrin expression observed on different materials may account for the varia-
tions in cell attachment. Integrins are a large family of cell surface receptors that mediate binding to
the extracellular matrix (ECM) and are also involved in intracellular signaling and a diverse range
of cell functions (Anderson et al. 2008, Wilson et al. 2005).
14.3.2.2 Adherence and Activation of Inflammatory Cells

The type, concentration, and conformation of adsorbed proteins and thus the adhesion and sur-
vival of cells bound to the proteins depend on the surface properties of the biomaterial (Anderson
et al. 2008).
Polymorphonuclear leukocytes, monocytes, and lymphocytes are the predominant cell involved
in the inflammatory response and the development of foreign body reactions and fibrous capsule
formation (Brodbeck et al. 2005).
Macrophages bound to the surface of a biomaterial fuse to form FBGC. IL4 induces macrophage
fusion on biomaterial surfaces while IL3 induces the fusion of monocyte-derived macrophages
(Anderson et al. 2008). All phagocytes contain lysosomes, intracellular organites that contain a
great variety of proteolytic enzymes. When liberated in situ, these enzymes can destroy the ECM
that supports connective and epithelial tissues. Lysosomes also release vasodilatating substances
and activate the complement and kallicrein/kinin systems, coagulation, and fibrinolysis (Diebold
et al. 1977, Kalman et al. 1991, Rakhorst et al. 1999, Van der Kamp and Van Oeveren 1994).
Platelets, which are very important in homeostasis, can act as inflammation mediators via sub-
stances in their intracytoplasmic granules (Trowbridge and Emling 1997).
14.3.2.3 Local Complications

Host inflammatory responses to contact with a biomaterial are normal reactions. In certain cases,
local complications may occur, the most frequent being the formation of thromboses, fibrotic hyper-
plasia around the implant, and bacterial infections (Park and Park 1996, Sigler et al. 2005, Tang and
Eaton 1995, Werner and Jacobasch 1999).
In humans and animal studies, protracted enhanced inflammation is associated with increased
susceptibility to infections (Boelens et al. 2000).
While platelet aggregation may be a normal reaction when a biomaterial comes in contact with
blood, it may become iatrogenic, with the formation of a thrombus and the risk of emboli (Park and
Park 1996, Piskin 1994).
Thrombus formation is not only due to endothelial damage but also to a foreign body surface
directly exposed to the blood stream. The consequences of a thrombus can be vessel occlusion or
embolization (Sigler et al. 2005).
Factor XII activates the complement system. Activated fractions C3a and C5a cause platelet
aggregation. C3b promotes cell adherence, increasing the risk of thrombosis (Kalman et al. 1991,
Keogh et al. 1996).
Certain authors have reported that hemocompatibility is influenced by the type of proteins
adsorbed to the surface of the implant. Albumin seems to prevent the adhesion of platelets, pro-
viding acceptable hemocompatibility by reducing the early proinflammatory and thrombogenic
responses of inflammatory cells, while fibrinogen and gamma-globulin increase platelet adhe-
sion, leading to bio-incompatibility (Asberg and Videm 2005, Casenave 1986, Dumitriu and
Dumitriu 1990).
Platelets and neutrophils can bind to IgG adsorbed to a biomaterial surface. IgG-bound platelets
induce neutrophils to release toxic reactive oxygen species (Asberg and Videm 2005).
Thrombogenicity is regulated by surface texture, electrostatic potential, and composition
(Kannan et al. 2006).
Various techniques have been developed to diminish thrombogenicity, including coating the
external surface of catheters with methacryloyloxyethyl phosphorylcholine-based copolymer
(MPC) or silicone. These techniques improve hemocompatibility because the coatings have a low
degree of thrombogenicity (Bambauer et al. 2004, Lloyd 2002, Nakabayashi and Williams 2003,
Rose et al. 2005).
When vascular grafts are implanted, the response of endothelial cells is crucial because they play
a role in maintaining the antithrombogenicity of the prosthesis. Endothelial cells (EC) have both
anticoagulant and thrombostatic functions. Under physiological conditions, negatively charged EC
prevent blood from clotting by releasing vascular relaxing factors, antiplatelet adhesion/aggregation
factors, antithrombogenic factors, and fibrinolysis promoters. Under pathological or traumatic con-
ditions, EC secrete repair proteins to maintain the integrity and continuity of blood vessels (Wang
et al. 2003).
To achieve optimal integration of a vascular implant, the lumen of the prosthesis must be quickly
and completely endothelialized, which prevents the formation of blot clots and vascular occlusion.
Various techniques have been developed to enhance EC adhesion and proliferation and optimize
cell-polymer interfaces by giving the surface a positive charge and by immobilizing cell growth-
promoting factors on it (fibronectin, laminin, Poly-d-lysine) (Wang et al. 2003).
The endothelial damage caused by the implantation activates the inflammatory and coagulation
systems, which in turn activates the smooth muscle cells of the media. These cells then migrate into
the intima leading to the formation of intimal hyperplasias (Sigler et al. 2005).
The formation of a fibrotic capsule is a normal reaction to the implantation of a foreign body. The
process may become pathological if the fibroblasts proliferate too extensively and lead to fibrotic
hyperplasia. The thickness of a fibrous capsule around an implant is part of the biological response
to the degree of biocompatibility of the implant (Kaluzny et al. 2007). An intense host inflammatory
response to a biomaterial stimulates phagocytes, especially macrophages and neutrophils, which are
attracted in large number and produce cytokines, chemokines, and other mediators (Cabanlit et al.
2007, Miller and Anderson 1989, Park and Park 1996, Tang and Eaton 1995).
A lack of implant/tissue adherence and the space between the two surfaces causes a certain
amount of friction that results in the accumulation of serous fluid and inflammatory cells. An
intense inflammatory response at this interface can result in fibrotic hyperplasia (Von Recum and
Van Kooten 1995). The contraction of the fibrotic capsule around the implant, which is more fre-
quent with silicone elastomers than PUs, is a common complication with implanted biomaterials
(Szycher and Siciliano 1991, Tang and Eaton 1995).
Bacterial infections are frequently associated with implanted biomaterials. Fibrinogen is known
to mediate the adhesion of Staphylococcus aureus, the main pathogen involved in implant-associated
infections. Fibrinogen can act as an opsonin and promote the aggregation of bacterial cells, espe-
cially staphylococci and streptococci (Trowbridge and Emling 1997). The access route for catheter-
related infections remains a point of contention. Certain authors (Cooper and Hopkins 1985, Maki
et al. 1977, 1991) have suggested that infections involving percutaneously inserted catheters are
due to contamination of the external surfaces. Others have suggested that the contamination of the
catheter hub can lead to intralumenal colonization and catheter-related septicemia (Boswald et al.
1995). Bacteria and fungi colonize the polymer surface, forming an adherent biofilm. The micro-
organisms embedded in these highly adherent films may, over time, secrete products that degrade
certain biomaterials (Bakker et al. 1990a). In addition, biofilms make antibiotic treatments and host
defense mechanisms less effective (Baumgartner et al. 1997, Jansen et al. 1994, Kossovsky 1995,
Kossovsky and Freiman 1995).
The development of microdomain-structured surfaces (PUr SMA-coated catheters) is a promis-
ing technology for reducing bacterial colonization (Bambauer et al. 2004).
A novel silicone elastomer catheter grafted with a poly(vinylpyrrolidone) hydrogel (SEpvp) has
been used to show that the adherence of Staphylococcus epidermidis on SEpvp in vitro is signifi-
cantly lower than on conventional silicone elastomer catheters (Boelens et al. 2000).
14.3.3 General Reactions
14.3.3.1 Degradation
Degradation is a complex mechanism that affects all elastomeric biomaterials and depends on the
chemical, physical, and structural properties of the implant and the inflammatory response of the
host (Pinchuk 1994). Degradation occurs at the molecular, macromolecular, microscopic, and mac-
roscopic levels (Maki et al. 1991). All implanted materials provoke an inflammatory response from
the host, which attempts to attack, destroy, and reject the foreign body. Leukocytes, especially
macrophages and FBGCs, are involved in this mechanism, which leads to the degradation of the
elastomer (Szycher and Reed 1992, Tang and Eaton 1995).
Macrophages appear to play a predominant role in the degradation of elastomers. They are
involved in the pinocytosis and phagocytosis of cell and tissue debris as well as polymer contami-
nants on the surfaces of biomaterials. Phagolysosomes digest the particles they have ingested and
the degradation products are then released into the extracellular environment together with lyso-
somal enzymes, peroxides, and hydrogen ions (Collier et al. 1998, Marchant et al. 1984b, Szycher
and Reed 1992, Tang and Eaton 1995). Lysosomal enzymes like enzymes—cholesterol esterase
(CE) and phospholipase A2 (PLA) have often been implicated in the degradation process despite
the difficulties in analyzing this phenomenon in vivo (Labow et al. 1997, Marchant et al. 1984a).
The release of lysosomal enzymes, peroxides, and hydrogen ions, which are very concentrated at the
cell/polymer surface interface, leads to significant degradation at this site. The degradation is faster
for porous elastomers than dense elastomers. Stress and tension combined with the chemical proper-
ties of the elastomer are also involved in the degradation process (Collier et al. 1998). Degradation
leads to surface cracking and a loss of molecular weight, which in turn results in a loss of tensile
strength (Pinchuk 1994).
14.3.3.2 Carcinogenic Reactions

The carcinogenic potential of materials implanted in rodents has been recognized since 1950. This
process is called the Oppenheimer effect. Many substances have been shown to induce foreign body
carcinogenesis at the implantation site (Pinchuk 1994). In 1975, Brand et al. (Brand et al. 1975)
described the various steps in foreign body tumorigenesis:
• Proliferation and tissue infiltration

• Progressive formation of a fibrotic tissue capsule
• Quiescence of the tissue reaction
• Direct contact of clonal preneoplastic cells on the foreign body surface
• Maturation of preneoplastic cells
• Sarcomatous proliferation (Brand et al. 1975)
Tumorigenesis is controlled by a number of factors. Foreign body carcinogenesis is influenced by

physical features such as shape, size, hardness, and porosity (Nakamura et al. 1992, Pinchuk 1994,
Stokes and Cobian 1982). Implant size is critical. Tumorigenesis does not occur unless the implant
is of sufficient size. The bigger the implant, the greater the probability that preneoplastic clones will
attach to the foreign body surface (Brand et al. 1975). Other researchers have reported that chemi-
cal properties play an important role in tumorigenesis (Nakamura et al. 1992). Numerous studies
have examined the incidence of tumor formation as a function of substrate and, in particular, PUs
and silicones. PUs appear more carcinogenic than silicones. Differences between the two elasto-
mers in terms of cell adhesion and thus cell proliferation are important factors in the carcinogenic
process (Nakamura et al. 1992, Tsuchiya et al. 1995a,b). The higher thermal sensitivity and water
absorption of PUs with respect to silicones also plays an important role in this process (Stokes and
Cobian 1982). In addition, a degradation product of PU (MDA or 4,4′ methylene dianiline) has been
implicated in the development of cancer in rats (Brinton and Brown 1997, Nakamura et al. 1992,
Pinchuk 1994, Tang and Eaton 1995). Heat (autoclave) and gamma radiation sterilization have also
been implicated in the formation of this toxic, carcinogenic compound (Yoda 1998). The mecha-
nism remains very controversial. On the other hand, a 1995 study produced no evidence that steril-
ization leads to the formation of MDA (Shintani 1995). The tumorigenesis mechanism is not clear
despite numerous studies on the carcinogenic potential of biomaterials. Nakamura (Nakamura et al.
1992) has tried to provide an explanation by suggesting a two-step mechanism: initiation followed
by promotion. The initiation step depends on active oxygen species produced by cells like macro-
phages and foreign body giant cells or on initiators in the biomaterial (Tsuchiya et al. 1995a). The
role of surface properties in the promotion of tumors has been examined by gap junction intercel-
lular communication inhibition studies using collagen immobilized on polyethylene film. Studies
have shown that the inhibition of gap junction intercellular communication reduces the tumorigenic
potential of polyethylene by decreasing tumor promotion activity (Nakaoka et al. 1997).
14.3.4 Silicones
Silicone elastomer is one of the most commonly used biomaterials in medical devices such as voice
protheses, artificial dermises, artificial phages, tissue expanders, intraocular lens, and artificial cor-
nea (Ai et al. 2003, Ren et al. 2008).
While it is accepted that silicone elastomers have good hemocompatibility, low toxicity, and
long-term stability, the biocompatibility of these elastomers remains an open question. Undesirable
effects from silicone implants have been reported (Spiller et al. 2007). Iatrogenic effects are caused
by the formation of a hypertrophic fibrotic capsule around the implant, which later leads to the con-
traction of the capsule, causing severe pain, local tissue damage, and implant function impairment
(Backovic et al. 2007). A comparison of silicone elastomer with methacrylate hydrogel, both of
which are nonabsorbable, and with absorbable crosslinked Na-hyaluronate, showed that crosslinked
Na-hyaluronate induces the least inflammation and a thicker fibrous capsule around the implant
than silicone (Kaluzny et al. 2007). Silicone has occasionally been associated with severe foreign
body reactions and with autoimmune disorders (Caballero et al. 2003). An analysis of epithelial
cell adhesion to various elastomers gave similar results for both silicones and PUs. However, cell
growth was poorer on silicone elastomers than on PUs. These results tend to minimize the role of
cell adhesion to the substrate.
Cytotoxicity is likely related to the release of toxic substances (Bakker et al. 1988). Scanning
electron microscopic analyses of the behavior of cells grown in contact with silicone elastomers
have shown that the cells grow in aggregates with little spreading and produce numerous adher-
ence structures linking them to one another, but few binding them to the substrate (Chauvel-Lebret
et al. 1999). The properties of silicone elastomers described before affect cell adherence to the
substrate and thus cell growth but have no effect on viability. Various techniques have been used to
enhance cell behavior and growth on silicone. Microwave plasma surface modification of silicone
with allylamine causes an increase in cell growth and viability with respect to unmodified silicone
(Ren and Mahon 2007). Modifying silicone by physical entrapment of a synthetic hydrophilic lipid
increases cells growth and enhances biocompatibility (Hodgins et al. 2007).
In vivo studies on the biocompatibility of elastomer implants have shown that a moderate inflam-
matory response occurs with the appearance of low numbers of FGBC (Kalicharan et al. 1991,
Wolfaardt et al. 1992, Wortman et al. 1983).
Hemocompatibility studies on silicones have shown that albumin and fibrinogen strongly
adsorb to these elastomers and that platelets also adhere in large quantities. Wettability can be
calculated using contact angle measurements. There is a very large decrease in the contact angle
of silicone elastomers over time, changing them from hydrophobic to hydrophilic biomaterials.
According to the authors of one study (Rakhorst et al. 1999), variations in wettability have a
greater impact on hemocompatibility than initial wettability (Van der Kamp and Van Oeveren
1994, Vignon 1995). A study on these systems has shown that silicones in contact with blood
preferentially activate the kallicrein/kinin system. This system is more active on hydrophobic
surfaces, which leads to v asodilatation, platelet aggregation, and an increase in permeability
(Van der Kamp and Van Oeveren 1994). Various treatments have been attempted to improve the
hemocompatibility of silicones. The elimination of micro air bubbles on the surface of the elasto-
mer by denucleation reduces complement activation and platelet aggregation (Kalman et al. 1991).
The affinity of albumin, which acts as an activation inhibitor of various biological reactions,
can be increased by a number of different processes. This reduces inflammatory cell migration,
greatly improves hemocompatibility, decreases complement activation, and prolongs contact time
in vivo (Tsai et al. 1990, 1991). The presence of fibers in polymethylvinylsiloxane does not modify
hemocompatibility (Fountain et al. 1979).
Chronic inflammation and myofibroblast proliferation often results in dense collagenous fibrosis
shortly after implantation (Backovic et al. 2007). Another study showed that the FBGC have greater
difficulty phagocytosing silicone particles than Teflon particles. When FBGC do not phagocytose
silicone particles, they develop into large multinucleated cells surrounding the silicone particle,
and a fibrous layer covers both the granulomatous lesion and the implant (Caballero et al. 2003).
Monocytes and macrophages secrete more functional IL-1ß when grown on a film of fibrinogen
and fibronectin than on a surface without a protein film. It has also been shown that the substrate
itself plays a role in the production of IL-1ß. In the presence of an identical protein film on differ-
ent substrates, monocytes/macrophages produce more functional IL-1ß when grown on silicone
than on Teflon, Dacron, polyethylene, or Biomer (Bonfield and Anderson 1993). The nature of the
substrate and the adsorbed protein film also plays a role in molecular conformation (Kossovsky
and Freiman 1995). Backovic et al. (2007) showed that silicone promotes the adhesion of altered
self proteins, which may trigger an autoimmune response. The cells of the ECM are particularly
susceptible to modification when they adsorb to silicone. The proteins in the hydrophobic domain
act like crystallization foci for other ECM proteins and are recognized as foreign by the immune
system. Still other proteins adsorbed to the silicone surface are involved in the innate (complement
component, C reactive protein) and immune (IgG, alpha 1 microglobulin) response. The process
of protein modification is slow end allows metabolic and immune reactions but prevents the acute
foreign body reaction (Backovic et al. 2007).
14.3.5 Polyurethanes
PUs have a number of medical uses because of their acceptable level of biocompatibility, their
high mechanical resistance, and their elastomeric properties (Spiller et al. 2007, Yoda 1998). PUs,
together with silicones, are among the few elastomers than can be implanted for long periods in
man (Pinchuk 1994). They are materials of choice for soft tissue and cardiovascular applications,
especially for catheters (Brown 1995, Jeschke et al. 1999, Szycher and Reed 1992). PUs have also
been cited as the best biomaterial for applications requiring a combination of hardness, durability,
biocompatibility, and biostability (Coury et al. 1987). They are divided into two main families: poly-
etherurethanes and polyesterurethanes. Since the ester bonds of the polyesterurethanes are subject
to hydrolysis (vascugraft), these elastomers are used less frequently in medical applications. Another
class of PUs includes poly(ether urethane-urea) in which the diol group is replaced by a diamine.
Numerous studies have compared the biocompatibilities of various PUs. In vitro biocompatibility
analyses comparing PU elastomers (polyether and polyester) and copolymers (polyether-polyester)
have shown that epithelial cells have the same growth patterns and explant morphologies as cells
grown on control substrates. Comparable results have obtained with the artificial aging technique
(Bakker et al. 1988). Endothelial cells proliferative more strongly on PUs within 1 week despite
slower spontaneous endothelialization than on other polymers like PTFE (Bschorer et al. 1994). The
biocompatibilities of a polyetherurethane and a porous copolymer (polyether-polyester) have been
studied following implantation in rats. The degradation of these PUs, which is less rapid than other
elastomers, does not result in the release of toxic products. The proliferation of fibroblasts and the
growth of fibrous and bone tissue are signs of acceptable implant fixation despite the presence of
macrophages and foreign body giant cells (Bakker et al. 1990b).
A number of biocompatibility studies have been conducted to evaluate the role of wettability
and implant structure (porosity) on cell adhesion, endothelial cell proliferation, inflammation, and
cicatrization. The studies evaluated a polyesterurethane (Vascugraft) with interconnected pores and
two polyetherurethaneureas (Mitrathane), one hydrophilic with closed pores and the other hydro-
phobic with open pores. Following implantation in rats, the histological analysis revealed a slight
cell reaction to the polyether and a moderate reaction to the polyester. The fibrotic capsule sur-
rounding the polyester implant pointed to good cicatrization (Huang et al. 1992). Cell adhesion to
the polyester was weak, slightly stronger to the hydrophobic polyether, and equivalent to the control
substrate for the hydrophilic polyether. Cell proliferation followed the same pattern, although a
uniform monolayer of endothelial cells formed on the polyester while multiple layers formed on the
hydrophilic polyether (Guidoin et al. 1992, Huang et al. 1992). Similar results were reported in a
study on hydrophilic and hydrophobic polyetherurethaneureas. The formation of cell monolayers on
a hydrophobic substrate is facilitated more by the presence of interconnecting pores than wettability
(Sigot-Luizard et al. 1993). A porous structure promotes fibroblast proliferation and the production
of new collagen on polyester. Tissue growth is a function of material porosity (Huang et al. 1992,
Pollock et al. 1981). If there are no interconnecting pores, a thin but linear fibrotic capsule is depos-
ited around the implant, but fibrotic tissue does not form within the implant (Ives et al. 1984, Martz
et al. 1987). Open pores should allow the development of fibrotic tissue within the implant. A porous
structure thus prevents deformation and reduces the risk of infection and contraction of the capsule
around the implant (Huang et al. 1992, Szycher and Siciliano 1991, Tang and Eaton 1995). These
reports show the importance of the physical properties of substrates such as porosity and surface
wettability in elastomer biocompatibility. A recent study reported a novel processing method for
injection-molded polyether urethane scaffolds. NaCl was used as a porogen to generate an open cell
culture. This study showed an increase in fibroblast viability and uniform proliferation on the scaf-
fold surface (Haugen et al. 2006).
Polyetherurethanes (PUs) can be aromatic or aliphatic. When aliphatic PUs are produced using
HMDI (hydrogenated methylene diisocyanate) rather than MDI (methylene diisocyanate), they no
longer release 4.4′ methylene dianiline as a decomposition product, which has been shown to be
carcinogenic in rats. In addition, these aliphatic PUs are not affected by ultraviolet radiation (Yoda
1998). As for hemocompatibility, platelet adherence is similar for both aromatic and aliphatic PUs,
although more fibrinogen is deposited on aliphatic PUs (Lelah et al. 1986). Aliphatic PUs are more
difficult to synthesize and have slightly weaker mechanical properties than aromatic PUs (Lelah
et al. 1986, Yoda 1998).
Aliphatic polyether-coated stents (Tecoflex) and uncoated stents display the same level of endo-
thelialization and neointimal formation. On the other hand, the inflammatory tissue response asso-
ciated with aliphatic PU-coated stents may be an indication of low biocompatibility (Rechavia et al.
1998). A new type of aliphatic PU (UTA) has been shown to increase the speed of cell adhesion,
making it equal to that of hydrophilic polyetherurethaneurea. After 1 week, both the UTA and
hydrophilic PU surfaces were covered by multiple cell layers while the hydrophobic PU was only
covered by a monolayer (Sigot-Luizard et al. 1993). Extraction cytotoxicity analyses have revealed
that this family of elastomers does not release toxic contaminants and has no clear superiority
in terms of biocompatibility over the other elastomers tested (polyetherurethaneurea and PTFE:
Goretex, Impra) (Bischoff 1972). The author of this study also pointed to the role of biomaterial
hydrophobicity in cell adhesion.
The rate of degradation of a biomaterial is affected by morphological variations in the surface of
the material in direct contact with living tissue, indicating that there is a correlation between the sur-
face properties of the biomaterial, cell behavior, and degradation (Wang et al. 2000). The degrada-
tion of PUs has been studied in great detail and involves three main mechanisms—mineralization,
oxidation, and environmental stress cracking (ESC).
Mineralization or calcification (Marchant et al. 1984a) involves the incorporation of crystal-

line inorganic substances on the surface of the implanted material. This mechanism often results
in hardening, distortion, and mechanical degradation of the elastomer (Coury et al. 1987). This
mechanism most often comes into play when elastomers are in contact with the bloodstream or are
in zones subject to dynamic flexing. Microdefects in the material can catch small particles of cel-
lular debris, which in turn can act as nuclei for the formation and growth of calcium-phosphate-rich
crystals (Coury et al. 1987, Szycher and Reed 1992, Yoda 1998).
Oxidation is induced by free radicals that break bonds, opening the elastomer up to attack by
oxygen molecules. This can be influenced by factors such as the flexibility of the medical device,
the type of soft segment, and the ability to interact with ions (Pinchuk 1994).
Environmental stress cracking is mediated by surface oxidation, stress, polyether soft segment
chemistry, molecular morphology, and the presence of monocyte-derived macrophages, FBGC, and
unknown biological elements (UBE) (Santerre et al. 2005). The degradation process may be better
described by the term environmental biodegradation (EB), which designates a unique environment
with multiple parameters, stress, and degradation of the polymer in a biological medium (Santerre
et al. 2005). Szycher and Reed (1992) have mentioned the notion of BI-ESC in vitro. Cell and
humoral-mediated inflammatory responses may also be involved in the surface degradation of PUs.
ESC first occurs in the direction of the stress and then takes on a three-dimensional aspect that may
extend to the very heart of the material, leading to total failure (Szycher and Reed 1992). Oxidative
degradation in vivo is implicated in the degradation of PU, but the role of enzymatic hydrolysis
remains controversial. Several authors think that hydrolytic enzymes, especially cholesterol ester-
ase, are involved in the biodegradation of PU (Santerre et al. 2005). Others think that hydrolytic
enzymatic degradation in vivo is negligible (Christenson et al. 2004b, 2006b). The problem with
degradation has been minimized with the introduction of biostable PUs. Coury (Coury and Hobot
1991) have synthesized PUs with no ether functions. In vitro, the absence of an ether bond removes
a point of attack for enzymes and chemical oxidants, thus lowering the risk of degradation (Pinchuk
1994, Szycher and Reed 1992). Szycher and Reed (1992) detected no degradation, cracking, or fail-
ure in biostable PUs after 12 weeks of implantation.
Modified PU elastomers (polycarbonate urethanes (PCU) have also been produced using poly-
carbonate glycol as a soft segment. While polyether urethanes undergo significant degradation after
6 months of implantation, the surface of PCUs remains smooth and crack-free (Pinchuk 1994).
PCUs are more biostable than polyether urethane (PEU). PCUs display better cell attachment and
proliferation in vitro, cause less platelet activation, and induce a lower inflammatory response (Hsu
et al. 2004). Christenson compared the biodegradation in vitro of PCUs and PEUs, and showed
that monocytes adhere to, differentiate on, and fuse to form FBGCs on these two PUs. The reac-
tive oxygen released by macrophages and FBGCs initiates the degradation of PEUs. PCUs are also
susceptible to biodegradation by products released by adherent cells (Christenson et al. 2004b).
PCU degradation is caused by oxidation. The biodegradation rate of PCUs is significantly less than
for PEUs, with PCUs being resistant to surface cracking. The degradation appears to be confined
to a very thin surface layer (Christenson et al. 2004a, 2006a). The even greater biostability of poly-
carbonate PUs has been attributed to their carbonate bonds (Mathur et al. 1997). When used as
vascular prostheses, polycarbonate PUs become endothelialized much more quickly than PTFEs.
In addition, chronic proliferation is lower than with PTFEs, decreasing the risk of hyperplasia and
occlusion (Jeschke et al. 1999).
New elastomers such as polycarbonate PUs and PUs endcapped with polydimethylsiloxane
have made their appearance. Studies have recently been published comparing the biocompatibility
and biostability of these new PUs with classic polyetherurethanes and PTFEs. They show that all
PUs share the same biological reactions (acute and chronic inflammation). The hydrophobicity of
polydimethylsiloxane leads to a lower accumulation of FBGCs on the surface. Polydimethylsiloxane
endcapped PUs are less subject to biodegradation because the endcap provides protection against
the oxygenated free radicals released by macrophages and FBGCs. Few studies have examined the
biocompatibility of poly(ether)urethane modified by the addition of polydimethylsiloxane to pro-

duce PCU-PDMS. Briganti showed that these news elastomers have no cytotoxic effects on human
endothelial cells and mouse fibroblasts (Briganti et al. 2006). Another study showed that PEU-
PDMS are more hemocompatible (Spiller et al. 2007). PU elastomers are synthesized with varying
proportions of poly(hexamethylene)oxide and polydimethylsiloxane(PDMS) macrodiols. The for-
mulation with 80% PDMS produced the best result in terms of flexibility, strength, and biostability
(Martin et al. 2000). A study with silicone-modified PEU and PCU showed that silicone modifica-
tion did not fully inhibit the oxidative degradation of PEU and PCU soft segments but the rate of
chain scission seemed to be slower than for the control PU soft segments (Christenson et al. 2005).
The biocompatibility of elastomers can be improved by modifying the surface or internal proper-
ties of polymers. However, these physical, chemical, and biological modifications must not affect
their bulk properties. To quote Han et al., “practically every physical and chemical material prop-
erty has been suggested as being important in biocompatibility for areas such as thrombus, calcifi-
cation and infection.” (Han et al. 1998).
Other techniques have been developed to improve the biocompatibility of PU elastomers. The
incorporation of glycerophosphocholine as a chain extender in poly(tetramethylene oxide)-based PU
significantly decreases bacterial adhesion and protein adsorption. A new MDI-based poly(ether)ure-
thane with glycerophosphorylcholine (GPC) and glycerophosphorylserine(GPS) were synthesized
and their biocompatibilities compared. This study showed that the elastomers were not cytotoxic
while the GPC elastomer displayed a low level of interaction with blood (D’Arrigo et al. 2007). The
incorporation of dehydroepiandrosterone (DHEA) in polyetherurethaneurea decreases macrophage
adhesion and FBGC formation for up to 7 days. After 7 days, the biocompatibility of all the PUs
(with and without DHEA) was similar (Collier et al. 1998). The incorporation of vitamin E (tocoph-
erol) generates a very stable PU, something that is not observed with DHEA (Collier et al. 1998).
Calcium chloride added to poly(ether-urethane-amide) improves biocompatibility, hydrophilicity,
and plasticity. However, it also increases thrombogenicity (Kawashima and Sato 1997). Grafting
collagen onto PU increases endothelial cell growth, which is dependent on the morphology and
diameter of the collagen fibers. However, the collagen may also activate and aggregate platelets,
making the material thrombogenic (Lee et al. 1996a). Coating prostheses (PU + collagen) with a
confluent monolayer of endothelial cells should resolve this problem. All these studies clearly dem-
onstrate the complexity of the biocompatibility of elastomers in general. PU has been modified with
incorporation of chitin and modified chitin. These materials are nontoxic, biodegradable, antibacte-
rial, and have good biocompatibility (Zia et al. 2009a,b).
Manufacturing processes, composition, and the surface properties of PU all play a role in
PU/blood interactions (Lelah et al. 1986). The roles of the various components of PU have been
studied. The nature of the soft segment makes PU more or less thrombogenic. Polyethylene oxide is
more thrombogenic than polypropylene oxide or polytetramethylene oxide, and retains more plate-
lets (Lelah et al. 1986). The nature of the hard segments is also important in the blood response.
Aliphatic PUs do not have the same properties as aromatic PUs. They both attract platelets to an
equal degree, but aliphatic PUs evoke an increased fibrinogen response (Bschorer et al. 1994). The
quantity of soft and hard segments influences hemocompatibility. PUs with large proportions of
hard segments are subject to rapid cell adhesion and sustained cell proliferation (Lelah et al. 1986).
The lower the proportion of hard segments, the weaker the blood interactions (Chen et al. 1998). The
molecular mass of the hard segments is important when elastomer devices must be in contact with
blood. PTMO 1000 is more hemocompatible than PTMO 2000 (Chen et al. 1998). This shows that
both the concentration and the conformation of the hard segments play a role in hemocompatibility
(Lelah et al. 1986). Two main types of chain extender can be used—diols (polyether urethanes) and
diamines (polyetherurethane urea). Diamines provide enhanced thromboresistance and hydrophi-
licity (Lelah et al. 1986).
Solvent casting affects the blood response by changing the orientation and/or conformation of the
diisocyanate and the components of the chain extender (Bschorer et al. 1994). The biocompatibility,
durability, and adhesive properties of aliphatic PUs increase in line with the decrease in the amount
of plasticizer (Lindner et al. 1994).
To get around the problem of thromboresistance, researchers have tried adding and/or replac-
ing components. Sulfonated PUs (PEO SO3) produced by various techniques exhibit a lower level
of platelet adhesion and platelet factor IV release from the time they first come in contact with
blood to up to 12–24 h later (Han et al. 1998, McCoy et al. 1990). Hemocompatibility parallels
bacterial repellence—the more hemocompatible a polymer, the more resistant it is to infections
(Han et al. 1998).
The hemocompatibility of sulfonated PUs modified by a surface derivation technique using
2-acrylamido-2-methyl-propane-sulfonic-acid (AMPS) varies depending on the species, with in
vivo and in vitro studies providing very contradictory results (Keogh et al. 1996). While very hemo-
compatible in vitro, sulfonated PUs attract large numbers of neutrophils when implanted in mice,
increase macrophage recruitment to unexpected levels, and lead to the formation of thromboses
when implanted in dogs (Keogh et al. 1996).
In a recent study, a sulfonated-polyethylene oxide-grafted PU (PU-PEO-SO3) was used to
coated heart valves and vascular grafts. Less calcium was deposited on the PU-PEO-SO3 while
platelet adhesion and thrombus formation appeared to be significantly lower on PU-PEO-SO3
(Han et al. 2006).
Applying a layer of heparin to PUPA [PU + poly(amido-amine) + HMDI] provides excellent
antithrombogenic properties, increasing hemocompatibility (Albanese et al. 1994).
PUs that incorporate gold or silver nanoparticles have been developed. Nanocomposites have
been prepared from water-borne polyether–type PU and water-borne polyester-type PU containing
different amounts of gold nanoparticles. PU-Au enhances cell proliferation, and reduces platelet
and monocyte activation, bacterial adhesion, and oxidative degradation (Hsu et al. 2006, 2008).
The gold or silver particles improve the biostability of water-borne PU by inhibiting oxidation and
crosslinking polyether soft segments (Chou et al. 2008).
To reduce the risk of infections associated with medical devices, a number of metallic ions
known for their bactericidal effect, especially silver, have been investigated. Numerous studies
have examined the biocompatibility, infection rates, and thrombogenicity of silver-impregnated and
silver-coated PUs and silicones (Boswald et al. 1995, 1999, Greil et al. 1999, Jansen et al. 1994,
Oloffs et al. 1994). Coating PU, silicone, and Dacron catheters with silver ions prevents microbial
colonization both in vitro and in vivo (Boswald et al. 1995, Boswald et al. 1999, Jansen et al. 1994,
Oloffs et al. 1994, Zhao and Stevens 1998).
14.3.6 Biodegradable Polyurethanes

The chemical properties of elastomers have also been modified to produce biodegradable products.
Studies on biodegradable polyesterurethanes have shown that these elastomers have good cell com-
patibility and that cell/substrate interactions do not lead to the release of toxic substances or the
activation of macrophages. Relatively strong adhesion and acceptable macrophage and osteoblast
growth occurs. Fibroblasts keep their phenotype for 12 days. The main problem seems to the actual
degradation of the elastomer, which results in the production of crystalline poly(r-3-hydroxybutyric
acid) or PHB-P particles. Macrophages, and to a lesser degree osteoblasts, may internalize the par-
ticles by phagocytosis. These particles may be toxic for macrophages and osteoblasts at concentra-
tions of 200 pg PHB-P/cell and 400 pg PHB-P/cell, respectively (Saad et al. 1996, 1997).
A few studies have analyzed the biocompatibility of biodegradable PU. Three types of biode-
gradable PU membranes were synthesized—smooth membrane, shrunken, and particulate. Cell
adhesion was the best and the cells were better spread out and flattened on the particulate mem-
brane. The smooth membrane was degraded the fastest, while the particulate membrane had not
changed significantly 8 weeks post-implantation (Wang et al. 2000). A few studies have shown
that biodegradable PUs display no signs of cytotoxicity in vitro and in vivo and do not provoke
a toxic tissue response (Asplund et al. 2007, van Minnen et al. 2005). These elastomers support
the ingrowth of cells and tissues in vivo and are degraded in a controlled fashion to noncytotoxic
decomposition products (Guelcher 2008).
14.3.7 Polyesters
Polyethylene Terephthalate: Dacron
Dacron, which has been used since 1940 as a bone and cartilage substitute, is considered to be
biocompatible (Schellhammer et al. 1999, Yoda 1998). It is used in numerous medical applica-
tions, especially in vascular surgery and for stent coatings (Schellhammer et al. 1999). However,
chronic, systemic inflammation has been reported following the implantation of a Dacron device.
Lymphocytes appeared after 30 days and the size of giant cells increased over time (Blum et al.
1996, Cavallini et al. 1987, Feldman et al. 1983, Granstroem et al. 1986, Link et al. 1996, Lodi
et al. 1988, Maturri et al. 1991, Sato et al. 1986). Their tendency to provoke intimal hyperplasia
and platelet stimulation—and thus their thrombogenicity—limits their clinical use to blood ves-
sels greater than 6 mm in diameter (Yoda 1998). Polytetraphthalate-coated stents are preferred to
PU stents, which are much more subject to dislocation (Schellhammer et al. 1999) The polyesters,
such as poly(l-Lactide) PLLA have been reported to have an excellent biodegradability, biocom-
patibility and non toxicity (Jiao and Cui 2007). When a bioadsorbable (polylactide)acid PLA was
compared with silicone sponge, the PLA displayed good biocompatibility, but the fibrous tissue
encapsulation was thicker and there were more inflammatory cells than on the silicone sponge
(Lansman et al. 2005).
Few polyesters have been modified to enhance the biocompatibility such as surface modifi-
cation (Jiao and Cui 2007). A film of poly(3 hydroxybutyrate) modified by microwave ammonia
treatment resulted in a hydrophilic surface with good long-term stability (Nitschke et al. 2002).
A new class of biodegradable elastomeric APS polymers has been developed by copolymeriza-
tion of poly(esteramide) elastomer and a polyol and a diacid. The cells interacted favorably with
the elastomer, and displayed good morphology and metabolic activity. The reduced fibrous cap-
sule thickness and the limited macrophage recruitment suggest that this elastomer would be well
accepted in vivo (Bettinger et al. 2008).
14.3.8 Polyvinylchloride: PVC

PVC is increasingly being used for medical applications, with an annual increase of 5.4%. It has
been used in the healthcare field since 1940 because of its broad biocompatibility, ease of manu-
facture, and low cost (Yoda 1998). A study in 1991 reported chronic inflammation associated with
PVC (Jayabalan et al. 1991). Two days after implantation, the inflammatory exudate around the PVC
contained mainly neutrophils, while the exudate around the PU and silicone contained a mixture of
neutrophils and monocytes (Fabre et al. 1998).
Additives have often been implicated in harmful host responses. Additives include plasticizers,
antioxidants, pigments, and UV stabilizers. Plasticizers in particular can release substances that
are incompatible with biological applications (Yoda 1998). A study in 1994 (Lindner et al. 1994) on
the biocompatibility of and cell response to PVC showed that there is a clear correlation between
the amount of plasticizer and the host inflammatory response. Various processes have been used
in an attempt to make PVCs more biocompatible. The most commonly used plasticizer is phthalic
ester, but it can be replaced by trioctyl trimellitate, azelate, or phosphate ester, which are nontoxic
and are not released (Hansen 2007, Yoda 1998). It has been shown that the loss of adenoviral vector
infectivity by effect of DEHP can be prevented by preflushing PVC tubes with albumin or methanol
(Bilbao et al. 2004).
The effect of plasticizers on hemocompatibility has been studied using PVC blood tubing manu-
factured using various plasticizers (phthalate alone, trimellitate or TD 360, and phthalate coextruded
with PU). The concentration of C3a anaphylatoxin and protein morphology were examined by elec-
tron microscopy. Phthalate-free PVC is more biocompatible while PU-coated PVC releases less C3a
anaphylatoxin, although its hemocompatibility did not change (Branger et al. 1990).
PVC surfaces have been coated with three different substances—heparin, phosphatidylcholine
(DMPC), and phosphatidylethanolamine (DMPE). The most biocompatible material was heparin-
coated PVC, followed by DMPE and DMPC (Moreira Pda et al. 2004).
14.3.9 Polytetrafluoroethylene: PTFE

Polytetrafluoroethylene (PFTE) is used for many medical applications, especially for peripheral
vascular surgery (Bschorer et al. 1994). in vitro extraction and direct contact analyses have revealed
no cytotoxic contaminants (Sigot-Luizard et al. 1993). In vivo, the inflammatory response resorbs
within 9 weeks, indicating acceptable biocompatibility (Huang et al. 1992, Sigot-Luizard et al.
1993). Fibroblasts respond to vascular PFTE prostheses by forming a thick external fibrotic capsule
and a thin layer coating the pores (Guidoin et al. 1992, Sigot-Luizard et al. 1993). Despite an initial
quick, spontaneous endothelialization process, complete endothelialization of the lumen of a PTFE
prosthesis can take up to 6 months because of the rough surface, which, compared to PUs, makes
endothelial cell proliferation more difficult. Chronic proliferation is, however, significant. After
6 months, the cells are still dividing, increasing neointimal formation and thus the risk of hyperpla-
sia and thrombosis (Bschorer et al. 1994, Jeschke et al. 1999). Surface texture also influences cell
migration. Cell migration, which can prevent thrombosis and occlusion, is slow on PTFE (Jeschke
et al. 1999).
Following a 500 min contact between human plasma and a PTFE prosthesis, variations in contact
angle were minimal and there was little fibrinogen adsorption, while platelet adhesion was elevated
(Rakhorst et al. 1999). Blood contact with hydrophobic materials preferentially activates the kal-
licrein/kinin system, leading to vasodilatation, platelet aggregation, and an increase in vascular
permeability (Van der Kamp and Van Oeveren 1994).
14.3.10 Hydrogels
Hydrogels have been used in a wide variety of medical applications such as contact lenses, pros-
thetic devices, and drug delivery systems for over 30 years (Yoda 1998). General properties include
swelling in water, high mobility of surface chains, low interfacial tension, low surface friction,
and appreciable elasticity, making them reasonably biocompatible. Their hydrophilicity and low
surface tension decreases protein adsorption and cell adhesion, which makes them suitable for use
in contact lenses. The low surface friction reduces fibroblast stimulation and prevents the formation
of a fibrotic capsule (Park and Park 1996). The high elasticity and low mechanical resistance means
that they have to be combined with other more solid elastomers to produce hydrogels with excellent
mechanical and biological properties.
Aqueous solutions of poly(ethylene glycol) dimethacrylate (PEGDM) and poly(ethylene glycol)
urethane dimethacrylate (PEGUDM) have been photopolymerized to yield hydrogels. The biocom-
patibility studies showed that the cells remained completely viable in both types of hydrogels after
2 weeks (Lin-Gibson et al. 2004).
14.3.11 Polyethylenes
A 1983 study evaluated the cell response to polyethylene following implantation in the peritoneal
cavities of mice (Wortman et al. 1983). The rough surface of the elastomer becomes covered with
numerous macrophages and FBGCs. The tissue reaction to polypropylene oxide includes an acute
inflammatory response involving numerous macrophages and lymphocytes. Polyethylenes are
degraded within 5 months of implantation. The resulting host reactions indicate that the degradation
products are toxic. The study by Bakker et al. (1990a) showed that polypropylene oxide cannot be used
for alloplastic tympanic membranes. Hemocompatibility studies have shown strong albumin adsorp-
tion and weak platelet adhesion to high-density polyethylene. In addition, analyses of the kallicrein/
kinin system and factor XII have shown that both systems are activated to the same extent, indicating
acceptable hemocompatibility (Rakhorst et al. 1999, Van der Kamp and Van Oeveren 1994).
14.3.12 Natural Rubbers
While natural rubbers are used in surgical gloves, urinary catheters, and tubing, their low hemo-
compatibility means they cannot be used in many other applications. Methylmethacrylate grafts can
improve hemocompatibility (Razzak et al. 1988, 1989). Natural rubbers also cause cytotoxic and
allergic reactions of various etiologies (Cormio et al. 1993). The proteins in natural rubbers cause
Type I IgE-induced allergic reactions. It is possible to remove these proteins by simple washing.
Gamma radiation vulcanization can be used to produce nitrosamine-free, low toxicity elastomers
(Yoda 1998). The cytotoxicity of natural rubbers is due to dithiocarbamates (Nakamura et al. 1990).
Various improvements mean that natural rubbers could soon see more frequent use in medical
applications.
In a recent study, granules of natural latex were implanted in the dental alveoli of rats after
extraction. This study shows that the latex was biocompatible, with the granules integrating into the
alveolar bone, accelerating bone formation, and playing an important role in the healing process
(Balabanian et al. 2006).
14.3.13 Styrene Copolymers
Polystyrene block- isoButhylene-Block-styrene (SIBS) is a thermoplastic elastomer and is oxida-
tively, hydrolytically and enzymatically stable and is biostable with a relatively low foreign body
reaction. SIBS does not activate platelets in the vascular system. In this study, the authors have
observed a few of polymorphonuclearleukocytes around SIBS implants, no encapsulation, no calci-
fication and no degradation of SIBS implants. The excellent biocompatibility is due to the inertness
of SIBS and lack of cleavable moieties (Pinchuk et al. 2008).
ASIBS with 30% of polystyrene has been studied and compared with silicone. The results show
that SIBS 30 has similar biocompatibility and superior dynamic properties in comparison with
silicone (El Fray et al. 2006).
14.3.14 Poly(Glycerol-CO-Sebacate)
Poly(glycerol-co-sebacate) (PGS) is an elastomer with excellent biocompatibility, the inflammatory
and fibrotic responses to PGS implants are minimal in subcutaneous and intramuscular implanta-
tion sites. The mechanisms of PGS degradation are most likely controlled by an enzymatic surface
erosion process and the hydrolytic cleavage may have played a minor role in PGS degradation
(Pomerantseva et al. 2009).
Another study has synthesized three PGS at 110°C, 120°C, and 130°C and the degradation has
been studied. The PGS have a wide range of degradability from being degradable in 2 weeks to
being nearly inert (Chen et al. 2008).
A photocurable polymer has been developed: the poly(glycerol-co-sebacate) acrylate PGSA. In
this study, PGSA has a good in vitro biocompatibility by sufficient cell adherence and subsequent
proliferation into a confluent cell monolayer (Nijst et al. 2007).
A porous scaffold prepared from PGSA could be used to support the growth and differentiation
of encapsulated cells. The biocompatibility is similar between the porous PGSA and no porous
PGSA, but porous PGSA promotes tissue ingrowth as compared with nonporous PGSA (Gerecht
et al. 2007).
ACKNOWLEDGMENTS
The authors thank Céline Allaire (Université de Rennes 1) for editorial assistance and for the prepa-
ration of the manuscript. English revision was made by Gene Bourgeau (Canada).
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15 Preparation and Applications
of Modulated Surface
Energy Biomaterials
Blanca Vázquez, Luis M. Rodríguez-Lorenzo,
Gema Rodríguez-Crespo, Juan Parra,
Mar Fernández, and Julio San Román
CONTENTS
15.1 Introduction........................................................................................................................... 496
15.2 Surface Modification Methods.............................................................................................. 499
15.2.1 Physical Methods....................................................................................................... 499
15.2.1.1 UV/Ozone Ablation.....................................................................................500
15.2.1.2 Radio Frequency Plasma Treatment...........................................................500
15.2.1.3 Corona Discharges...................................................................................... 502
15.2.1.4 Ion Implantation.......................................................................................... 502
15.2.2 Chemical Methods..................................................................................................... 503
15.2.2.1 Wet Chemical Methods............................................................................... 503
15.2.2.2 Grafting Polymerization.............................................................................504
15.2.2.3 Polyelectrolyte Complex............................................................................. 505
15.2.2.4 Click Chemistry.......................................................................................... 505
15.2.2.5 Biochemical Modification...........................................................................506
15.3 Methods of Analysis of Polymer Surfaces............................................................................ 507
15.3.1 Spectroscopic Methods.............................................................................................. 507
15.3.1.1 X-Ray Photoelectron Spectroscopy............................................................ 507
15.3.1.2 Fourier Transform Infrared Spectroscopy.................................................. 508
15.3.1.3 Secondary Ion Mass Spectrometry.............................................................509
15.3.2 Nonspectroscopic Methods........................................................................................509
15.3.2.1 Contact Angle.............................................................................................509
15.3.2.2 Surface Plasmon Resonance....................................................................... 510
15.3.2.3 Zeta Potential.............................................................................................. 511
15.3.2.4 Quartz Crystal Microbalance..................................................................... 511
15.3.3 Microscopic Methods................................................................................................ 512
15.3.3.1 Atomic Force Microscopy.......................................................................... 512
15.3.3.2 Scanning Electron Microscopy and Transmission Electron Microscopy........513
15.4 Interactions with Biological Environment and Applications................................................. 514
15.4.1 Cell–Polymer Interaction Determines Biocompatibility........................................... 514
15.4.2 Passivating Polymer Surfaces for Protein Repelling................................................. 517
15.4.3 Bioactivity of Polymer Bioconjugate Surfaces.......................................................... 520
References....................................................................................................................................... 523
495
15.1 INTRODUCTION
Surface properties of materials are the key parameters in the interaction between an implanted
device and the host tissue (Chen et al. 2008; Xu and Siedlecki 2009). Hydrophobicity or hydrophi-
licity of a material is usually expressed as surface wettability and determines protein adsorption,
platelet adhesion, and cell and bacterial adhesion (Xu and Siedlecki 2007) and also their subse-
quent structural and biological activity (Hylton et al. 2005; Wu et al. 2005b). Hydrophobic surfaces,
namely poorly wettable, tend to absorb proteins in a greater proportion than hydrophilic surfaces,
namely highly wettable. In hydrophilic surfaces, repulsive solvation forces arising from strongly
bound water occur (Israelachvili and Wennerstrom 1996; Noh and Vogler 2006). A stark transi-
tion between protein adherent and protein nonadherent materials has been found to happen on
water contact angles 60°–65° (Xu and Siedlecki 2007), which is consistent with the appearance of
hydrophobic interactions (Yoon et al. 1997). However, surface wettability seems not to be the only
parameter affecting the adhesion of proteins. The phenomenum of protein adhesion is rather a com-
petition between the hydrophobic effect and the dehydration of the surface. The former one expels
proteins from solution but proteins will only be adsorbed on a surface when the energy necessary
for dehydrating the surface is lower, i.e., ΔG0 (Phobic effect) > ΔG0 (dehydration) (Noh and Vogler 2006). The
hydrophobic effect is the energy that expels protein from solution to recover hydrogen bonds among
water molecules otherwise separated by proteins.
The desirable biological response to a surface depends largely on the specific applications and
remains as an important target area of research in the preparation of biocompatible polymeric mate-
rials. It is necessary to remember at this point that biocompatibility is defined as the ability of a
material/device to perform in a specific environment (Williams 2008). And it is still an open issue,
whether highly energetic surfaces, i.e., highly wettable or low energetic surfaces, i.e., poorly wet-
table, are more or less adequate for each specific application (Navarro et al. 2006).
The design of blood contacting devices must involve the engineering of surfaces to avoid pro-
tein adhesion. Platelet adhesion is the cause of thrombus formation and severe associated vascular
problems (Rodrigues et al. 2006). Fibrinogen, which is one of the most abundant protein plasmas in
blood, is a ligand for the platelet integrin receptor RIIb_3 (GPIIb/IIIa), and the interaction between
this ligand and receptor is responsible for immobilization, activation, and aggregation of plate-
lets. As an example, several platelet adhesion and activation studies with and without preadsorbed
fibrinogen, albumin, and plasma have been performed based on the effect of surface wettability of
self-assembled monolayers (SAMs) containing different ratios of methyl- and hydroxyl-terminated
alkanethiols (Martins et al. 2003a; Rodrigues et al. 2006; Goncalves et al. 2009).
There are also numerous reports of adsorption on hydrophilic surfaces, either ionic or hydro-
gels (Vogler 1998; Navarro et al. 2006). On hydrogels, complications introduced by water-hydrated
polymers must be considered since it becomes very difficult to distinguish between absorption and
adsorption. Some adsorption phenomena on hydrophilic surfaces have been reported which cannot
be easily dismissed. Adsorption on hydrophilic surfaces must be explained assuming that adsor-
bents are not physicochemically bonded to the surface but rather associated through a hydration
layer or “bound” to the water layer.
Mammalian cell attachment has been described as a different process from protein adsorption
and it occurs efficiently on hydrophilic surfaces but inefficiently on hydrophobic ones. Thus, the
hydrophobic/hydrophilic balance in the biological response to materials, often disputed in bioma-
terials science, should be studied from the perspective of water structure and reactivity at surfaces
(Vogler 1998).
Attachment and adhesion are different phenomena. Adhesion is related to the force necessary to
separate adherents and is controlled by short-range forces that result from the formation of cova-
lent, ionic, hydrogen, and charge-transfer bonds. Thus, adhesion occurs only when cellular surfaces
(or some extension of the cellular surface) touch the substratum and contact at atomic level can be
made. Attachment can be a physical phenomenon where cells are held close to the surface by an
Preparation and Applications of Modulated Surface Energy Biomaterials 497
attractive potential where attractive dispersion forces overcome electrostatic repulsion. Attachment
happens before adhesion. Once cells are attached, they can proliferate and spread on a surface.
The results obtained from studies carried out with culture medium supplemented with 10% fetal
calf serum (FCS) suggested that the complex mixture of proteins present in FCS presented a higher
affinity for hydrophilic surfaces.
Cell cultures performed with MG63 osteoblast-like human cells on materials with different sur-
face wettability have shown that the initial attachment of the cells is better for the poly(lactic acid)
(PLA)/phosphate glass (G5) composite, the most hydrophilic of the tested materials, than for the
other two materials. PLA, the most hydrophobic of the tested materials, is the substrate with the
lowest amount of attached cells. Besides, proliferation and differentiation assays have suggested
that the most hydrophilic surface triggered the differentiation process earlier than the hydrophobic
surfaces (Navarro et al. 2006).
For biosensing applications, the surface energy must be tailored to bind targeted proteins while
rejecting all other proteins. Experiments have been carried out to monitor protein adsorption as
a function of the hydrophilic or hydrophobic nature of the terminal functional groups in ethylene
glycol (EG) (Clare et al. 2005). Because the hydroxyl group of EG can, in theory, be oxidized
to a charged carboxylate group, the hydroxyl-terminated EG could lead to an increased nonspe-
cific adsorption of positively charged proteins (Ostuni et al. 2001). Replacing the terminal hydroxyl
with a methoxy group to form Me-EG3-ene could therefore provide additional resistance to protein
adsorption, especially under oxidizing conditions. However, some reports have shown that methyl-
terminated EG3 (Me-EG3) and hydroxyl-terminated EG3 are comparably effective at reducing the
amount of nonspecifically adsorbed protein. Some proteins such as fibrinogen possess both hydro-
philicity and hydrophobic domains and they can be adsorbed onto both hydrophilic and hydrophobic
surfaces. The elongated structure of fibrinogen produces orientation-dependent changes, and these
physical packing forces may dominate the adsorption dynamics, thus weakening the effect of sur-
face termination (Figure 15.1).
The problem with many synthetic materials is that they absorb a complex nonspecific layer of
proteins (Castner and Ratner 2002). Nonspecific protein layers are not used in nature. Nature uses
specific proteins in fixed conformations and orientations so that they optimally deliver signals. The
host may react to this nonphysiological proteinaceous layer as to a foreign invader that must be
walled off. Thus, surfaces of biomaterials must be designed to control the conformation and orienta-
tion of the proteins they adsorb.
B B B B
HO HO
O O O O
HN NH2 NH2 HN HN O O HN
6 6
100% amino 90% EG6-ene,

S/NS = 1.21‡ 10% amino
S/NS = 22.8*
1.5 mm 1.5 mm
(a) (b)
FIGURE 15.1 Scheme of avidin nonspecifically adsorbed onto (a) 100% amino-terminated and (b) 90%
EG6/10% amino-terminated monolayer on silicon. (From Clare, T.L. et al., Langmuir, 21, 6344, 2005.)
TABLE 15.1
Summary of Strategies for Surface Modification
Surface strategy Comment
PEG a Effective but dependent on chain density at the surface; damaged by oxidants
PEG-like surfaces by plasma deposition Applicable for the treatment of many substrates and geometries; highly
effective
PEG oligomers in SAMs Highly effective; applicable for precision molecularly engineered surfaces;
durability to elevated temperature is low
PEG-containing surfactants adsorbed to A simple method for achieving nonfouling surfaces; durability may be low and
the surface high surface densities are hard to reach
PEG blocks in other polymers coated on May provide a relatively low density of surface PEG groups
the surface
Hydrogen bond acceptors Possibly, this principle imparts nonfouling properties to PEG surfaces; this is
leading to new discoveries of surface functional groups for nonfouling surfaces
Protein layer A preadsorbed protein layer resists further adsorption of proteins; this
approach, long used by biologists, is easy to implement but of low durability
Hydrogels PEG is in this class; many other hydrogelshave shown nonfouling behavior
a Also called poly(ethylene oxide) (PEO).
Many strategies to inhibit nonspecific protein adsorption, i.e., nonfouling surfaces, have been
developed as expressed in Table 15.1 taken from Castner and Ratner (2002).
Cells relate to surface through proteins, thus, cells require surfaces with specific adhesive prop-
erties (Falconnet et al. 2006). Cells require an adhesive surface to exert forces and consequently
spread. The ability to constrain the spreading to a specific cell–surface contact area has been shown
to dramatically affect cellular development. Mechanical compliance of cell-adhering substrates
can also substantially affect the cellular response and development. The ability to spatially and
temporally control the chemistry, the pattern geometry, and the local substrate stiffness will con-
tinue to provide new insights into the fundamental aspects of cell–surface interactions and ligands
(peptides, proteins, etc.) (Andersson et al. 2003; Barber et al. 2003; Falconnet et al. 2006). Thus, the
ability to suppress nonspecific interactions between the surface and the protein-containing media is
crucial in order to generate unbiased experimental outcomes.
Advances in surface chemistry have made possible the synthesis of numerous so-called nonfoul-
ing surfaces that significantly reduce or eliminate the nonspecific adsorption of proteins and other
biomolecules from biological fluids, e.g., through the incorporation of molecules such as carbohy-
drates, agarose, and mannitol as well as albumin (Luk et al. 2000). The most widely used system
is poly(ethylene glycol) or PEG with the monomeric repeat unit [–CH2–CH2–O–]– (also known
as poly(ethylene oxide) or PEO). The factors governing protein resistance of a PEG-graft copoly-
mer (PLL-g-PEG) has been investigated, with quantitative information provided on the interfacial
architecture of PEG chains and their influence on protein resistance (Daria et al. 2002; Dalsin et al.
2003). Many different PEG surface-immobilization strategies have been successfully applied. One
example is the use of triblock copolymers PEG–PPO–PEG that assemble spontaneously on hydro-
phobic surfaces through hydrophobic–hydrophobic interactions. This polymer class is also known as
poloxamers or Pluronics® and has been extensively studied (Daria et al. 2002; De Silva et al. 2004).
One major issue that biopolymers must deal with is the possibility of bacterial adhesion, which
causes biomaterial-centered infection, and, as a consequence, the lack of successful tissue integration
(Gristina 1987). Modifications of biomaterial surfaces at the atomic level will allow the program-
ming of cell-to-substratum events, thus diminishing infection. Bacterial adhesion is the consequence
of a competition between binding proteins of the host tissue and binding proteins of the bacteria.
Several approaches can be found on literature to overcome this shortcoming, the first one is to
modify the surface of the biomaterial to enhance tissue compatibility or integration (Gristina 1987)
and the second one is to modify the material to directly inhibit bacterial adhesion. This second
approach can distinguish between those materials with intrinsic antimicrobial properties, such as
those functionalized with amine or ammonium groups (Kenawy et al. 2005; Kenawy et al. 2006)
and those that immobilize water-soluble or emulsifiable disinfectants onto macromolecular surfaces
such as acrylic derivates of eugenol on hydrophilic 2-hydroxyethyl methacrylate (Rojo et al. 2008).
Finally, cellular adhesion and spreading, which is the ultimate purpose of surface modification
on biomaterials, must deal with differences in behavior from in vitro to in vivo experiments. RGD
peptide sequence is found in a wide variety of ECM proteins and implicated in a number of ligand–
receptor interactions (Reyes and Garcia 2003), making it exceptionally useful for promoting general
cell adhesion. However, the RGD tripeptide sequence exhibits limited specificity for specific integrins
and allows minimal control over cellular responses resulting in the loss of full biological activity. This
has been attributed to the absence of additional functional domains present in the native, three-dimen-
sional structure of the ECM molecule (Reyes and Garcia 2003). Modification of surfaces with peptide
sequences containing a three-dimensional structure has shown better effectiveness on in vivo experi-
ments. The glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) sequence
that can be found on the α1(I) chain of type I collagen is recognized by the integrin α2β1. Integrin rec-
ognition is entirely dependent on the triple-helical conformation of the ligand similar to that of native
collagen (Reyes and Garcia 2004). Modifications with the GFPGER sequence are lately being pro-
posed as a robust strategy for enhancing cell–material interactions and encouraging biospecific cell
adhesion that may provide with good results on in vivo experiments (Reyes and Garcia 2003, 2004).
Thus, there are several proposals on what surface properties and what surface modifications are
the most appropriate to tailor biological interaction of biomaterials to specific applications. The
purpose of this chapter is to review methods that can be found in the current literature to modify
biomaterial surfaces, surface characterization methods, and the interactions of host tissues with
different sorts of surfaces.
15.2 SURFACE MODIFICATION METHODS

Chemical, physical, and physicochemical modifications of biomaterial surfaces comprise a number
of ways to control cellular functions on a biomaterial surface. Surface characteristics such as hydro-
philicity, surface charge density, surface micro-morphology, free energy, and specific chemical
groups can affect and regulate a wide variety of biological functions. Several chemical modification
techniques have been developed to improve wetting, adhesion, and printing of polymer surfaces by
introducing a variety of polar groups and specific functional groups.
15.2.1 Physical Methods

Dry surface treatment techniques, such as corona discharges (Zhu et al. 2006c), plasma (Kwang
et al. 2009), or ultraviolet (UV) radiation, are among the most common methods used to modify the
physical–chemical exterior properties of biopolymers.
The principal effects of these treatment techniques are summarized in the following (Sham et al. 2009):
1. Cleaning: through conversion of contaminants into gaseous, volatile products

2. Ablation: etching out the weak layer on the microscopic scale or selective leaching of low–
molecular weight polymer chains
3. Surface strengthening: further cross-linking or branching the near surface polymer
molecules
4. Modification of chemical structures of surface through reactions of free radicals with sur-
rounding gases
15.2.1.1 UV/Ozone Ablation

UV light surface modification is an easy process that does not require vacuum, avoiding compli-
cated machinery, or chemical reagents that avoid the production of residual polluting by-products.
UV light can modify polymers through two different mechanisms. It can directly affect the
bonds inside the polymer by local excitations, which introduce photocrosslinking and/or photo-
degradation. Or it can form a strongly oxidant ozone layer, containing some atomic oxygen and
oxygen radicals as well, on the air layer outside the polymer (Øiseth et al. 2004). The ozone attacks
and chemically modifies the top layers of the polymer, which are oxidized and/or etched primarily
through the following reaction:
O3 + Polymer → O2 + Oxidized polymer
The extent of these photo-induced reactions depends on the mobility of the polymer chains, the
UV absorption coefficient, the photon penetration depth into the polymer, the glass transition tem-
perature, the diffusion coefficients of gases in the polymer core, and the wavelength (Andrady
et al. 2003). Polyethylene is mostly activated at wavelengths around 330 nm. However, a very strong
absorption below 160 nm that originates from the dissociative excitation of the C–C and C–H
bonds can be observed (Øiseth et al. 2004). Effects of UV light and ozone have been studied for
various polymer surfaces, including polyethylene (PE) (Øiseth et al. 2004; Poulsson et al. 2009),
polyethylenetherephthate (PET) (Teare et al. 2000), polypropylene (PP) (Macmanus et al. 1999),
polyetheretherketone (PEEK) (Romero-Sánchez et al. 2001), poly(vinylmethyl siloxane) (PVMS)
(Efimenko et al. 2005), polydimethylsiloxane (PDMS) (Ye et al. 2006), polystyrene (PS) (Mitchell
et al. 2005), as well as polyesters and mix of fabric surfaces (Michael et al. 2004).
Using PDMS as an example, the atomic oxygen originating from ozone splitting reacts with the
PDMS surface forming radicals that will oxidize and remove organic parts of the polymer in the
form of CO2, H2O, and other volatile compounds. UV-light of 355 nm may break Si–CH3 bonds
since the energy of that radiation is 3.5 eV, which is close to the bond energy in Si–CH3 (3.2 eV), but
it will not affect the backbone of the polymer since Si–O bond energy is 4.6 eV. The surface of the
PDMS will then have an SiOx structure that will be covered with –OH groups. Thus, it becomes
more hydrophilic than when it is covered with the usual –CH3 groups of a normal PDMS surface
(Sham et al. 2009).
15.2.1.2 Radio Frequency Plasma Treatment

Plasma technology is used to alter the surface properties of polymers without affecting bulk
properties. Physicochemical properties such as surface free energy, hydrophilicity, and surface mor-
phology can be modified to make the surface of the biomaterial adequate for cell–material interac-
tions. Plasma treatment can improve wettability, oxidize the surface, and enhance cell growth and
adhesion. The effects of plasma on a polymer surface can be classified as surface modification,
grafting, and film deposition. Plasma surface modification can be achieved using gases such as
oxygen, nitrogen, argon, and helium. The main effects are removal of chemical contaminants (Kim
et al. 2005) and/or weakly bound polymer layers but mainly, introduction of chemical groups such
as hydroxyl, carbonyl, carboxylic, amino, or peroxyl groups on the surface that permit covalent
bonding (Sanchis et al. 2006). Oxidation, nitration, hydrolization, and amination processes induced
by plasma are used to improve the surface energy and reactivity. In plasma-induced grafting, free
radical formation using inert gas plasma is followed by the introduction of unsaturated monomers
into the reaction chamber that react with the free radicals to yield a grafted polymer (Choi et al.
2004). Plasma polymerization process is used to produce thin films with unique physicochemical
properties (Cheng et al. 2004). Methane, ethylene, propylene, fluorocarbon monomers, and organo-
silicon compounds can be polymerized by this method. The plasma-polymerized thin films are
generally homogeneous, highly cross-linked, and strongly bound to the surface.
Radio frequency (RF) plasma treatment of polymeric biomaterials has been used to improve bio-
compatibility. The effects of nitrogen and oxygen plasma treatment on surface modification of polyeth-
yleneterephthalate (PET) have been studied (Vesel et al. 2008) and used to produce protein-resistant
properties (Vandencasteele et al. 2008). The surface of PET polymer was modified by this method
to achieve improved attachment of fucoidan, with antithrombogenic properties (Junkar et al. 2009).
Helium plasma treatment has been applied on polyurethane film coated on glass substrate to enhance
endothelial cell growth and adhesion (De et al. 2004). The critical surface tension (γC) of polyurethane
(PU) film increased by 2 dynes/cm due to helium plasma treatment (De et al. 2005). Griesser et al.
studied the attachment and growth of human endothelial cells and fibroblasts on polymer surfaces
fabricated by the polymerization of volatile amine and amide compounds in a low-pressure gas plasma
and by the treatment of various surfaces in ammonia plasmas, which served to increase the nitrogen
content of the surface layers (Griesser et al. 1994). In other studies, they demonstrated that the covalent
surface immobilization of polysaccharides, proteins, and synthetic oligopeptides can be achieved via
nanometres thick, interfacial bonding layers deposited by gas plasma methods (Griesser et al. 2002).
The adhesion of endothelial cells on plasma-treated PS varied with plasma treatment time, increas-
ing with longer periods of time (Van Kooten et al. 2004). Surface energy increased after oxygen
plasma treatment of fiber-reinforced PET composite and enhanced cell adhesion (Cioffi et al. 2003).
Poly(lactide-co-glycolide) (PLGA) films were treated by oxygen plasma. Under specific conditions,
cells stretched very well and the ability to endure the shear stress was improved greatly. Results showed
that improved cell adhesion was attributed to the combination of surface chemistry and surface mor-
phology of PLGA during plasma etching (Wan et al. 2004). Wan et al. (2003) modified poly(l-lactic
acid) (PLLA) films by plasma treatment using O2, N2, Ar, and NH3 gas atmospheres under different
conditions. Cell retention on the NH3 plasma-modified PLLA surface was much higher suggesting
that a high polar component and N-containing groups may play a role in enhancing cell resistance
to shear stress. Ultrathin antibacterial polyammonium coating given on plasma-treated polyethyl-
ene (PE) surface required much smaller quantities of the antibacterial agent than other conventional
methods to produce the same effects (Thome et al. 2003). PE coated with triclosan and bronopol
was treated with oxygen plasma followed by argon and hydrogen plasma, hydrogen plasma result-
ing more efficient to improve antibacterial properties (Zhang et al. 2006c). Carbon fiber-reinforced
polyether ether ketone (PEEK) for joint endoprostheses and fracture fixation plates was treated by
oxygen plasma and N2/O2 plasma to get better surface activation for joining and coating processes
(Ha et al. 1997). A poly(ε-caprolactone) (PCL) surface was modified by an O2 plasma surface treat-
ment to form oxygen-containing functional groups to promote the precipitation of a bone-like apatite
layer in vitro (Oyane et al. 2005). Film deposition was performed on porous PU matrix loaded with
ciprofloxacin. The antibiotic released at a controlled rate when the film was coated with poly(n-butyl
methacrylate) by RF plasma deposition (Kwok et al. 1999). To prevent biofilm formation in dental
implants, adsorption of histatin 5 was produced onto poly(methyl methacrylate) (PMMA) surfaces
after modification of the polymer surface using a cold plasma technique. Adsorption increased due
to the increased surface hydrophilicity and the formation of the carboxyl groups (Yoshinari et al.
2006). Improvement of corneal epithelial cell growth on different substrates for artificial corneas
such as poly(vinyl alcohol), silicone rubber, PS, and polycarbonate was analyzed and it was found that
plasma-induced graft copolymerized poly(hydroxyethyl methacrylate) (pHEMA) on silicone rubber
provided the best growth rate (George and Pitt 2002). Plasma processes have been used to obtain
immobilization of biomolecules on various substrates. Insulin and heparin were immobilized on PET
films grafted previously with acrylic acid using oxygen plasma to improve blood compatibility (Kim
et al. 2000) and to enhance growth of smooth muscle cells (Gupta et al. 2002). Chen et al. (2004c)
investigated the allylamine polymerization using pulsed RF plasma as an adhesion layer immobilizing
DNA probe for DNA hybridization. Patterning of amine groups was achieved by selectively depos-
iting plasma-polymerized ethylenediamine films on specific areas of a glass surface. These results
demonstrated that the formation of amine groups on glass slides by plasma-enhanced chemical vapor
deposition resulted an excellent tool for DNA array technology (Jung et al. 2006).
15.2.1.3 Corona Discharges

Corona discharge was first commercially used in the 1960s to modify the surface of different poly-
meric materials such as PP, PS, and polyethylene-terephthalate (Romero-Sánchez et al. 2001). A
corona is a process that is developed from an electrode with a high potential in a neutral fluid that
results in the ionization of the fluid. When the ionized fluid finds its way to an oppositely charged
object, a spark or continuous arc of plasma is produced. A setup for corona discharge is shown in
Figure 15.2.
When a corona discharge is applied to a polymeric surface, relatively stable free radicals are formed
that produce changes in surface properties. Some examples of corona discharge uses follow. It has
been used for changing the surface energy on rubber (Romero-Sánchez et al. 2001). The procedure
removes low-molecular weight moieties on the surface, increases the amount of silica, and incorporates
oxygen that is associated to Si as SiO2. This is attributed to the fact that the energy of part of photons
from corona discharge is larger than the binding energy of Si-O-Si (8.3 eV) and Si-CH3 (4.5 eV) bonds.
Hydrophilic OH groups can be formed as by-products instead of the hydrophobic CH3 on the surface
of rubber, which allows the hydrophobicity of polymeric materials to be reduced (Zhu et al. 2006c).
Corona discharges have been also applied to produce covalently linked hydroxyapatite (OHAp)
on silicona. Hydrophobic surfaces are unable to nucleate OHAp due to a low Lewis acid–base sur-
face tension (Wu et al. 1997). A corona-discharge treatment has been used for graft polymerization
of acrylic acid onto silicone sheets. A composite with an amino-modified OHAp was then possible
to prepare (Furuzono et al. 2001).
15.2.1.4 Ion Implantation

In ion implantation processes, ions are accelerated with energies in the range 20–200 keV and directed
to the surface of a polymer. Penetration of these ions is of several hundred nanometers and can mod-
ify the surface of the materials without affecting bulk properties (Cui et al. 2008). Properties that
can be tailored following this procedure include hardness, wear resistance, coagulability, wettabil-
ity, and biocompatibility of the material (Cui and Luo 1997). Two different mechanisms have been
found to explain the character of chemical processes in ion implantation processes. The first one is
electronic excitation, also named as inelastic collision and the second one is nuclear collisions, also
named as elastic collisions (Sviridov 2002). The electronic excitation is responsible for the formation of
mobile radical species, e.g., H+, whereas nuclear collisions produce massive ruptures of chemical bonds
as a consequence of atom displacements. High-energy light ions deposit their energy by electronic
Electrode
Resistor (50 kΩ)
OSC Sample
PC
OSC: Oscilloscope PC: Personal computer
FIGURE 15.2 (See companion CD for color figure.) A corona discharge setup. (Taken from Zhu, Y. et al.,
Polym. Degrad. Stab., 91, 1448, 2006c.)
excitations while the main mechanism for heavy ions is nuclear collision (Sviridov 2002). Thus, stop-
ping ions involves the deposition of high energy within the tracks followed by the ions and the for-
mation of radicals, secondary ions, and electrons. Ion implantation of partially halogenated aliphatic
polymers causes radical-induced dehydrogenation and carbonization on the surfaces (Rao et al. 1993).
The effects of ion implantation can be modulated by changing the energy of the ions used. An irradia-
tion of Sb + (150 keV) on polyamide-6 results in an important change of polarity, whereas N+ irradiation
on poly(ethylene therephthalate) (PETP) only changes polarity by 30% (Sviridov 2002). Concerning
applications in the medical field, a few works have reported the use of this procedure to improve the
blood biocompatibility and anti-thrombogenicity of PDMS surfaces (Dupas-Bruzek et al. 2009).
15.2.2 Chemical Methods
15.2.2.1 Wet Chemical Methods
This technique involves the treatment of the biomaterial surface with liquid chemical reagents to
generate reactive functional groups. It is adequate for porous three-dimensional substrates due to
the high penetration of the liquids inside the substrate (Liu and Ma 2004); however, this method
can produce a range of oxygen-containing functional groups, and the degree of surface function-
alization may not be repeatable depending on molecular weight, crystallinity, or tacticity of the
polymer. Chromic acid oxidation has been applied to polyethylene (PE) films to introduce carbox-
ylic acid groups (Goddard and Hotchkiss 2008). Partial oxidation of sodium alginate with potassium
permanganate (KMnO4) has been attempted to improve degradability of alginate for biomedical
applications (Lu et al. 2009). Acid-catalyzed hydrolysis and aminolysis have been used to yield
carboxyl and amine-terminated PMMA surfaces (Brown et al. 2006). George and co-workers
(Zhu et al. 2004b) introduced free amino groups onto polyurethane (PU) scaffolds which increased
surface energy and provided a convenient way to further immobilize bioactive species and to
enhance the cell–PU interaction in the endothelium regeneration process. Electrospun nanofibers
have been chemically functionalized after surface modification by wet chemical method and other
different routes including plasma treatment, surface graft polymerization, and co-electrospinning
of surface active agents and polymers (Yoo et al. 2009). Dalton et al. (1998) studied the effect of
hydrophilicity on colonization by human bone-derived (HBD) cells, comparing untreated PS and
a sulfuric acid-treated PS surface for mechanisms of cell migration. The chemically treated PS
surface contained various oxidative groups and a minor amount of sulfonate groups and HBD
cells migration was dependent on the presence of vitronectin (Vn) and it was higher on the hydro-
philic acid-treated surface. Alferiev et al. (2006) investigated a chemical approach consisting
of bulk carboxylation of polyurethanes via bromoalkylation to enable surface heparinization for
thromboresistance to improve hemocompatibility of vascular prostheses. Some investigators have
reported that surface modification of biodegradable polymers in alkaline solution could be used to
generate a hydrophilic and rough surface for cell attachment (Nam et al. 1999). The treatment of
poly(lactic-co-glycolic acid) (PLGA) with Na-OH solution enhanced vascular smooth muscle cell
adhesion and proliferation but decreased those of endothelial cells (Miller et al. 2004). Aminolysis
of PLLA membranes improved the cytocompatibility of PLLA using human umbilical endothe-
lial cells culture (Zhu et al. 2004). Two chemical methods, base hydrolysis and aminolysis, were
used to introduce carboxylic acid or primary and secondary amine groups onto PLGA surface
and provide satisfactory results, creating a truly biomimetic scaffold for tissue engineering (Croll
et al. 2004). Surface thiolation of poly(methacrylic acid)-based microparticles was carried out by
coupling l-cysteine with a water-soluble carbodiimide. This method is proposed as an interest-
ing strategy to improve paracellular permeability of hydrophilic macromolecules (Sajeesh et al.
2010). Biological ligands (biotin and peptides) were immobilized onto hydrolyzed poly(glycolic
acid) (PGA) sutures through an amide bond between the amine (ligand) and the carboxylic groups
of the modified surface. This strategy can be applied to other polyesters to tailor their properties
for biomedical applications (Lee et al. 2003). Modification of poly(ethylene terphathalate) fabric
with glycerol polyglycidyl ether has been achieved by means of supercritical carbon dioxide using
a series of immobilization processes to finish with the incorporation of natural agents such as
sericin, collagen, or chitosan (Ma et al. 2010).
15.2.2.2 Grafting Polymerization

Grafting polymerization is used to produce surface modification when the bioactive compound loses
activity if it is directly linked to the hydrophobic polymer surface. Acrylic acid has been UV graft polym-
erized to PCL films pretreated with Argon plasma to generate carboxylic acid groups for immobilization
of collagen (Cheng and Teoh 2004). Surface modification of a PLLA with collagen has been carried out
by covalent bonding to polymer via gamma irradiation grafting with poly(acrylic acid) as a coupling
agent (Yang et al. 2003). Vinyl acetate, acrylic acid, and acrylamide were grafted onto PLLA surface
films separately or in combinations, using a UV-induced photo-polymerization process. Combinations
of acrylamide with vinyl acetate or acrylic acid produced the hydrophilicity of the modified surface
(Janorkar et al. 2006). Kubies and co-workers studied the influence of different hydrophilic groups,
hydroxyl, carboxyl, or amide, introduced onto PLLA membrane surface via photocopolymerization of
the corresponding monomers, on the cytocompatibility for chondrocytes. Results showed that whereas
for hydroxyl and amide groups cytocompatibility was greatly improved, for carboxyl groups it was even
worse although all modified surfaces possessed a similar hydrophilicity (Kubies et al. 2003). Polysulfone
(PSF) membranes were treated with ozone to introduce peroxides, and then grafted with either acrylic
acid or chitosan, followed by the immobilization of heparin to improve the hydrophilicity and blood
compatibility of PSF membrane (Yang and Lin 2003). Surface of nylon membranes was modified by
graft copolymerization of glycidyl methacrylate using a redox initiation system to promote further anti-
body immobilization (Jackeray et al. 2010). PEG was covalently immobilized to a layer of branched
poly(ethylenimine) (PEI) to control bacteria adhesion (Kingshott et al. 2003). Silk fibroin surfaces were
PEGylated by reaction with cyanuric chloride-activated PEG and proposed as useful antiadhesion and
antothrombogenic biomaterials (Vepari et al. 2010). Different functional monomers, like hydroxyethyl
methacrylate, acrylic acid, N-vinyl pyrrolidone, and glycidyl methacrylate, have been grafted onto the
surface of films, using simultaneous photo-grafting and cold plasma-grafting techniques, to alter the
surface properties of elastomeric substrates, such as hydrophilicity and biocompatibility. Hydroxyethyl
methacrylate and acrylic acid showed exceptionally high cell compatibility in terms of cell adhesion
and proliferation (Desai et al. 2003). Synthetic phospholipid analogs have been photografted onto PP
hemodialysis membranes to reduce fouling and thrombus formation and improve biocompatibility (Xu
et al. 2004). Graft polymerization of a phospholipid polymer 2-methacryloyloxyethyl phosphorylcholine
(MPC) onto the polyethylene surface has been investigated by Moro et al. (2004) to reduce friction
factors frequently involved in periprosthetic osteolysis—bone loss in the vicinity of a prosthesis. PU
membranes were modified by grafting the zwitterion of sulfobetaine monomer using a novel three-step
procedure to enhance hemocompatibility with good results (Huang and Xu 2010; Figure 15.3).
+ + + +
+ + + +
+ + + +
+ + + +
+ + + ++ +
–– – – – –– – –– – – ––
Electrostatic interaction Affinity interaction Covalent attachment
Bioactive compound Avidin/streptavidin
Biotin
– + Charged species
Reactive functional groups
FIGURE 15.3 Mechanism of immobilization. (Taken from Goddard, J.M. and Hotchkiss, J.H., Prog. Polym.
Sci., 32, 698, 2007.)
15.2.2.3 Polyelectrolyte Complex

A polyelectrolyte complex (PEC) is a compound made from an electrically neutralized molecular
complex of polyanions and polycations. The effects of PEC films composed of polysaccharides on
cell behavior have been studied and it was reported that PEC can stimulate differentiation of osteo-
blasts and periodontal ligament fibroblasts (Hamano et al. 2002; Nagahata et al. 2005). Chitosan
covalently immobilized onto PLA film surface was made to react with heparin (Hp) solution to form
a PEC on the PLA surface. PLA surface modified by chitosan/heparin complex inhibited platelet
adhesion and activation and showed enhanced cell adhesion (Zhu et al. 2002). PECs were formed
through the mixing of aqueous solutions of naturally occurring sulfated fucoidans with hydroxy-
ethyl cellulose derivatives containing cationic and hydrophobic substituents. Formation of PECs
was determined by the type and arrangement of substituents and the degree of charging, as well
as the composition of polysaccharide molecules (Shchipunov et al. 2003). Based on this principle,
a way of surface modification has been focused on the PEC formation on the biomaterial surface
to attain different goals. Polyelectrolyte films built up by the alternated adsorption of cationic and
anionic polyelectrolyte layers constitute a novel and promising technique to modify surfaces in a
controlled way (Ladam et al. 2002). Poly-(dl-lactide) substrates were activated by PEI to obtain
stable positively charged surface and polyelectrolytes such as alginate and poly-(l-lysine) were
alternatively deposited onto the activated substrates. Results indicated that the multilayer-modified
substrates promoted the chondrocyte cytocompatibility (Zhu et al. 2004c). The surface modifica-
tion with PEC multilayer of hyaluronic acid (HA) and poly(l-lysine) (PLL) resulted in drastically
reduced peripheral blood mononuclear cell attachment according to the lactate dehydrogenase assay
for cell counting. This nano-scale control of material deposition may be successfully applied for
surface modification of various biomaterials (Hahn and Hoffman 2005). Poly(lysine)/poly(glutamic
acid) (PLL/PGA) multilayer films ending by several PLL/PGA-g-PEG bilayers, PGA-g-PEG cor-
responding to PGA grafted by PEG, can be used to drastically reduce both protein adsorption and
bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat
any type of biomaterial for protection against bacterial adhesion and therefore limiting its patho-
logical consequences (Boulmedais et al. 2004). Modification of intraocular lens of poly(methyl
methacrylate) using layer-by-layer deposition of poly(sodium 4-styrenesulfonate) and PEI has been
proposed to facilitate anti-bacterial drug adsorption (e.g., ampicillin) onto the surface as a way to
protect eye and the device from infections (Manju and Kunnatheeri 2010). The adhesion of L929
cells toPCL nanofibers was successfully improved via coating with polyelectrolyte multilayer thin
films of poly(diallyldimethylammonium chloride) and poly(sodium 4-styrene sulfonate), which
enhanced the potential of this material as a scaffold in tissue engineering applications (Dubas et al.
2009). A new type of polymeric hybrid coating consisting of poly(acrylic acid) and poly(acrylamide)
was created by layer-by-layer deposition of polyelectrolyte multilayers onto nano-patterned polymer
brushes derived from a surface-reactive rod-coil block copolymer, PS-block-poly[3-(triethoxysilyl)-
propylisocyanate], offering a new organic hybrid coating with novel surface properties (Yang
et al. 2008). Layer-by-layer polyelectrolyte films offer extensive potentials to enhance surface
properties of vascular biomaterials. Three films composed of poly(sodium 4-styrenesulfonate)/
poly(allylamine hydrochloride), PLL/hyaluronan, and PLL/poly(l-glutamic acid) were built onto
isolated PET filaments, thread, and vascular prostheses, demonstrating improved mechanical resis-
tance (Rinckenbach et al. 2008). The layer-by-layer technique applied to the coating of an oral pros-
theses gave successful results and it was proposed for the coating of other oral prosthesis devices
(Etienne et al. 2006).
15.2.2.4 Click Chemistry
The “click” concept introduced by Sharpless and co-workers in 2001 is undeniably one of the most
promising trends in synthesis and functionalization chemistry (Kolb et al. 2001). Among the different
chemical reactions that can be considered of the “click” type, the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) is generally regarded as the example of click chemistry (Rostovtsev et al. 2002).
CuAAC has been used for building, crosslinking, or functionalizing polymeric micelles, nanogels, or
liposomes (Lutz 2007). Wooley et al. first reported the advantages of click CuAAC for functionaliz-
ing block copolymer micelles of PS core and poly(acrylic acid) shell (Joralemon et al. 2005; O’Reilly
et al. 2006). The in situ shell-functionalization of micelles composed of a cholesterol-based hydro-
phobic core and a thermoresponsive PEG-based polymer shell has been reported (Lutz et al. 2007).
Functionalization of lipid vesicles have been made using different alkyne-functional surfactants, assur-
ing that, by this method, only the outer surface of the vesicles was modified (Cavalli et al. 2006; Hassane
et al. 2006). An artificial oxygen carrier was constructed by conjugating hemoglobin molecules to
biodegradable micelles using the click chemistry. First, a series of triblock copolymers (PEG-PMPC-
PLA) in which the middle block contains pendant propargyl groups were self-assembled into core-shell
micelles in aqueous solution, and then, micelles were conjugated to azidized hemoglobin molecules
via click reaction between the propargyl and azido groups (Shi et al. 2009). N-azidated chitosan was
prepared using click chemistry by four different methods (Kulbokaite et al. 2009). In other studies
thrombin inhibitors were fixed on poly(ethylene terephthalate) (PET) and poly(butylene terephthal-
ate) (PBT) by wet chemistry treatment (activation of hydroxyl chain-ends) and photochemistry (nitrene
insertion by photoactivation of aromatic azide) (Salvagnini and Marchand-Brynaert 2006). UV grafting
of a carbohydrate-bearing photoreactive azide group has been performed on poly(ethylene terphthalate)
film (Roger et al. 2010).
15.2.2.5 Biochemical Modification

The attachment of biomolecules to polymer surfaces could be an important tool for controlling
cell–biomaterial interaction. Covalent immobilization of various integrin-binding peptides as well
as entire proteins has been shown to mediate the adhesion of many types of cells. Polymeric
surfaces must have specific functionality to be covalently attached to a bioactive compound.
Common functional groups in bioconjugation chemistry include thiols, aldehydes, carboxylic
acids, hydroxyls, and primary amines. Crosslinking agents are commonly used to link the bioac-
tive compound directly to the functionalized surface or through a spacer of several angstroms
(Hermanson 1996). Electrically conductive polypyrrole (PPY) was surface functionalized with
HA and sulfated hyaluronic acid (SHA) by the use of a cross-linker having the appropriate func-
tional groups to improve its surface biocompatibility (Cen et al. 2004). Gelatin and chitosan have
been immobilized onto biodegradable poly(lactide-co-glycolide) acid (PLGA) producing a sig-
nificant increase in cell adhesion and spreading (Zhu et al. 2006b). Surface functionalization of
biodegradable PLLA was achieved by plasma coupling reaction of chitosan. The results suggest
that the new substrate can be used to control the morphology of cells and has potential applications
in tissue engineering (Ding et al. 2004). The conjugation of heparin with a biodegradable PLA
has been achieved to improve blood compatibility and related biocompatibility and suggested for
implantable medical devices and tissue engineering (Jee et al. 2004). Since Arg-Gly-Asp (RGD)
sequences have been found to promote cell adhesion in 1984, numerous polymers have been func-
tionalized with RGD peptides to achieve biomimetic materials (Niu et al. 2005). RGD has been
covalently attached to amine-functionalized PLA using a glutaraldehyde linkage and the results
indicated that modifying the surface of PLA scaffolds with RGD, bone cell attachment, and differ-
entiation were enhanced, suggesting that this strategy may improve bone tissue regeneration more
efficiently in wound models (Hu et al. 2003). BisGMA/TEGDMA thermosets and composites
have been surface modified to expose carboxylic groups on the surface and the activated material
was further modified by deposition of a lactose-modified chitosan decorated with RGD peptide
driven by strong electrostatic interactions. The modified thermoset was carried out in vitro very
similar to clinically used roughened titanium (Travan et al. 2010). Biochemical modification of
fibrous scaffolds by interfacial PEC was achieved by immobilization of RGD chemically conju-
gated to a thiol-active maleimidylated form of the scaffold (Tai et al. 2010). Surface immobiliza-
tion of fibronectin-derived PHSRN peptide has been attained by spontaneous adsorption on thin
polysiloxane thin films having different surface energy. The effect of the immobilization of this
sequence, which induces cell invasion and accelerates wound healing, on fibroblasts was different
depending on the hydrophilicity of the surface (Satriano et al. 2010).
On the other hand, PEG has been used for conjugation of proteins since 1970 (Davis 2002).
Nowadays, this technique is well established and it is called PEGylation (Milton Harris and Chess
2003). PEG possesses interesting properties for conjugation. It is a highly water-soluble polymer that
extends to give a hydrodynamic radius greater than that of a globular protein of equivalent molecu-
lar weight and it can act as a hydrophilic spacer between the polymeric surface and the bioactive
compound. Acrylated antibodies were photografted on polymer surfaces previously treated with PEG
to facilitate rapid and sensitive antigenic detection (Sebra et al. 2005). A dodecapeptide (H12) has
been conjugated to the end of PEG chains on the surface of a phospholipid vesicle with an average
diameter of 220 nm to prepare H12-PEG-vesicles (Okamura et al. 2005). Covalent immobilization of
heparin onto electrically conductive polypyrrole (PPY) film was attained after graft copolymeriza-
tion with poly(ethylene glycol) methacrylate (PEGMA) and activation with cyanuric chloride. After
surface modification, the film improved wettability while retaining significant electrical conductivity
and with immobilized heparin, platelet adhesion and platelet activation on PPY film were significantly
suppressed (Li et al. 2003). Peptides incorporating the RGD and PHSRN integrin-binding sequences
were immobilized onto a polyethyleneglycol-based polymer substrate to study the effects of fibro-
nectin in supporting macrophage fusion to form foreign body giant cells (Kao and Hubbell 1998).
Chemoselectivity of click chemistry is another route presently used to obtain polymer bioconjugates. It
has been demonstrated that sequence-defined oligopeptides can be efficiently linked to synthetic mac-
romolecules using CuAAC ligation (Dirks et al. 2005; Parrish et al. 2005). Click cycloaddition of short
peptides such as RGD with well-defined polymers synthesized by atom transfer radical polymerization
(ATRP) has been investigated (Lutz et al. 2006). Peptide bioconjugation via huisgen cycloadditions
were explored on linear or dendritic synthetic macromolecules with carbohydrates (Wu et al. 2005a;
Fernandez-Megia et al. 2006). Other more complex biological entities such as proteins (Reynhout
et al. 2007) or even viruses, bacteria, or cells (Deiters et al. 2003; Link et al. 2004) have also been
conjugated to polymers or low-molecular weight functional groups using azide-alkyne click chemistry.
15.3 METHODS OF ANALYSIS OF POLYMER SURFACES

Surface characterization of biomaterials is a central issue in biomaterials research since surface
properties can have an enormous effect on the success or failure of a biomaterial device. This char-
acterization can be carried out with different analytical tools whose selection should be based on the
nature of the surface, the specificity required, and the resources available. A general classification
of these techniques divides them into the following three categories:
• Spectroscopic methods
• Nonspectroscopic methods
• Microscopic techniques
While nonspectroscopic and microscopic techniques describe in a precise manner the changes in
surface chemistry, equipment is costly and analysis can be time-consuming. Therefore, spectro-
scopic analytical techniques are more useful for laboratory routine due to its simplicity and the
speed with which results can be obtained.
15.3.1 Spectroscopic Methods
15.3.1.1 X-Ray Photoelectron Spectroscopy
X-ray photoelectron spectroscopy (XPS), also called electron spectroscopy for chemical analysis
(ESCA), is a powerful technique to characterize the chemical composition of polymer surfaces,
where a depth of typically 7 nm is probed. Therefore, the information provides only affects to some
surface atomic layers of the material.
The principle of XPS is based on the emission of photoelectrons from the surface in response
to irradiation by a beam of monochromatic x-rays. The kinetic energy (K E) of these emitted photo-
electrons is unique for the different elements and is also sensitive to the chemical state of the atoms;
therefore, the comparison of K E obtained to known values allows the user to identify the elements
present on the surface and its oxidation state (McArthur 2006). The resulting spectrum is a plot of
intensity (arbitrary units) versus binding energy (eV), the intensity of the ejected photoelectrons is
directly related to the material surface atomic distribution and, therefore, can be used to quantify
atomic compositions and stoichiometric ratios.
XPS can be used to identify specific functional groups present in complex polymers by using
derivatization reactions that provide a more accurate identification and determination of functional
groups by selective labeling. For example, trifluoroacetic acid anhydride (TFAA) is often used to deter-
mine hydroxyl groups, while aldehydes like pentafluorobenzaldehyde or trifluoromethylbenzaldehyde
are reported as derivatization agents for quantification of amines (Pippig et al. 2009; Strola S 2010).
In biomaterials field, XPS has been widely used to determine elemental composition of solid sur-
faces. It has been applied to different surfaces and surface modifications in studies of adsorption and
retention of chemicals such as antibiotics, for the detection of immobilized proteins, understanding
the chemistry of the structure, formation and stability of plasma-treated surfaces, as well as for the
characterization of the steps formation of thin coatings (Sabbatini and Zambonin 1996; McArthur
2006; Hook et al. 2010).
15.3.1.2 Fourier Transform Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a spectroscopic technique based on the use of
infrared beam to analyze the chemical functionalities present in a molecule. When an infrared
beam (IR) hits a sample, chemical bonds stretch, contract, and bend causing it to absorb IR radiation
in a defined wavelength number (Goddard and Hotchkiss 2007). This technique provides informa-
tion about the molecular structure, inter- and intramolecular interactions, crystallinity, conforma-
tion, and orientation of molecules.
Infrared spectroscopy can also be used in attenuated total reflection mode (ATR-FTIR), which is
widely used in biomaterial surface analysis. In this mode, the incident IR beam first passes through
an internal reflection element made from inorganic crystals like germanium, ZnSe, or diamond.
Total reflection occurs at the interface between the sample and this internal reflection element, but
instead of reflecting totally, a fraction of the incident IR enters into the sample in a form of elec-
tromagnetic wave with an exponentially decreasing magnitude and with a penetration depth from
several hundred nanometers to more than 1 μm. This kind of electromagnetic wave is called the
evanescent wave, which has a sampling depth depending on the wavelength and the refraction index
of both the reflection element and the sample.
Advancement in FTIR characterization of materials has been achieved with the development of
FTIR microscopy (Bhargava and Levin 2001). This technique has significant advantages compared
to many other imaging methods for the characterization of biomedical materials, because it relies
on the characteristic absorbance of corresponding molecular vibrations in the sample. Therefore, it
does not require the use of added dyes or labeling methods for visualization of different chemical
components in the sample (Kazarian and Chan 2006).
An IR microscope is similar to an optical microscope, being the optical paths for both the visible
and the IR radiation identical through the sample plane. The user can focus on specific features of
the specimen, use the apertures to delineate the sample region, and collect the single beam IR spec-
trum for the sample. As a microscopic analysis, this technique has a resolution limit dictated by dif-
fraction effects. When two samples are less than 50 μm apart, they cannot be clearly resolved over
the mid-IR range; thus, pure spectra from each sample cannot be obtained. This means that when
the spectrum of one sample is obtained, a certain amount of the energy from the second sample is
being detected. Thus, pure spectra from each sample cannot be obtained (Sahlin and Peppas 1997).
This technique has been extensively used in analyzing polymer/polymer and polymer/solute dif-
fusion (Sahlin and Peppas 1997; Miller-Chou and Koenig 2002; Gupper et al. 2004; Gupper and
Kazarian 2005; González-Benito and Koenig 2006; Peter 2006), drug release and tablet dissolution
or water sorption in pharmaceutical formulations (Chan and Kazarian 2004a,b; Kazarian et al.
2005). Kazarian and Chand (2003, 2006) have used ATR-FTIR imaging to investigate the in situ
dissolution of polymer/drug formulations in water based on PEG and different model drugs.
15.3.1.3 Secondary Ion Mass Spectrometry

Secondary ion mass spectrometry (SIMS) is a mass spectroscopy-based technique for analyzing
elemental and chemical compositions of the outermost molecular or atomic layer of a solid surface
(Stamm 2008). This technique provides a high surface specificity, since only several atomic layers
on the sample surface are analyzed. The material surface is bombarded with a beam of energetic
ions, usually argon or gallium. When the ions hit the surface, atoms, clusters, and molecules will
be removed from the material surface in a process called sputtering. A fraction of the sputtered
particles will be ionized to produce secondary ions, which are separated as a function of the ratio of
mass per electric charge in a mass spectrometer.
SIMS can be developed in static or dynamic mode (Ma et al. 2007). Static SIMS uses a low-
energy primary ion beam to scan the sample surface in a static surface analysis. In this mode, only a
monolayer is removed from the surface, it allows, e.g., the determination of surface contaminants. In
dynamic SIMS, a high-energy primary ion beam is applied in short times eroding away the material
surface continuously. Real-time signal is recorded simultaneously. The signal is plotted against the
depth of the sample to obtain a high-resolution depth (several nanometers) profiling the chemical
compositions/structure from the surface into the bulk of the sample.
Time-of-flight SIMS (ToF-SIMS) is a very efficient method for characterizing the elemental
(including H and isotopes) and molecular composition of the top surface of biomaterials (Belu et al.
2003). In this case, the secondary ions are accelerated to a constant kinetic energy and then allowed
to pass in a field-free environment before they are collected on an intensity-sensitive detector. The
time of flight of the ions is correlated with individual masses; the positively charged secondary ion
fragments are characteristic of chemical structures on the material surface and can provide signifi-
cant information for the surface.
In the last 20 years, SIMS has progressed from a novel research methodology to an analytical
tool for determination of the surface chemistry of polymers in biomedical applications. SIMS spec-
tra can provide characteristic fingerprints for biomaterial surfaces. This technique has also been
used to obtain direct proof of covalent binding of molecules to a surface or to correlate surface
chemistry with cell growth, as well as to evaluate surface homogenicity by two-dimensional chemi-
cal maps (Chilkoti et al. 1991; Perez-Luna et al. 1997; Leonard et al. 1998; Kingshott et al. 2003;
Mouhib et al. 2010).
15.3.2 Nonspectroscopic Methods

15.3.2.1 Contact Angle
Measurement of the contact angle of a liquid test droplet on a solid surface is a useful technique that
reveals surface energetic information inaccessible by surface spectroscopy. Although this method
represents perhaps one of the earliest methods used to investigate surface structure, it still yields
very useful information. Basically, a drop of fluid is placed on the biomaterial surface of interest and
an equilibrium position is achieved. The contact angle is determined from the tangent associated
with the drop/polymer surface. For immobile surfaces, contact angles are believed to be sensitive to
the outermost 3–10 Å of a surface (Merrett et al. 2002).
Measurements can be carried out by several different methods such as the Wilhelmy plate method,
the sessile drop method or the captive bubble method. In biomaterials bibliography, contact angles are
typically measured using a goniometer by the sessile drop method using water as test fluid, as well as
other liquids with different polarities such as diiodomethane or glycerol. Water contact angle gives infor-
mation on the hydrophilic/hydrophobic balance of the surface by measuring how much a droplet of water
spreads on it. The lower the contact angle, the more hydrophilic the surface is. Hydrophilicity of materi-
als is an important parameter to predict the interaction of biomaterials with the surrounding tissues, and
therefore to evaluate their biocompatibility (Hernández et al. 2007; Servoli et al. 2009; Xiong 2010).
In addition, contact angle measurements can be used to study the influence of surface topography
on wettability (Öner and McCarthy 2000). For this purpose, dynamic wettability has to be addressed
by measuring the hysteresis, i.e., the difference between the increase (advancing contact angle) and
decrease (receding contact angle) in the test droplet volume. Contact angle hysteresis has also been
reported to occur with surfaces that are chemically heterogeneous or contaminated with surface-active
reagents, as well as for surfaces that reorient upon exposure to different fluids (Mathieu et al. 2003).
15.3.2.2 Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a surface-sensitive technique able to monitor any dynamic pro-
cess at the biomaterial surface, such as adsorption or degradation, in real time and without the need
to label the adsorbate or complex sample preparation. It is an optical technique that has been widely
used in biomaterials field to investigate biological interactions like protein adsorption process and
is capable of real-time in situ analysis to define protein adsorption and desorption rate (Green et al.
1999; Green et al. 2000; Vicente 2010).
SPR is based on the excitation of surface plasmons. A surface plasmon is a longitudinal charge den-
sity wave that propagates along the interface of two media, one is a metal and the other is a dielectric.
The metal used must exhibit free electron behavior, silver and gold being the most commonly used.
Most SPR instruments are designed based on the Kretchman configuration (Figure 15.4), which is
based on the phenomenon of total internal reflection. This occurs when light traveling through an
optically dense medium, e.g., glass, reaches an interface between this medium and reflects back into
the dense medium. Although the incident light is internally reflected, a component of this light, the
evanescent wave or field, penetrates the interface into the less dense medium to a distance of one
wavelength. In SPR, a monochromatic p-polarized light source is used and the interface between
the two optically dense media is coated with a thin metal film of thickness less than one wavelength
of light. The evanescent wave of the incoming light is able to couple with the free oscillating elec-
trons (plasmons) in the metal film at a specific angle of incidence, and thus the surface plasmon is
Flowcell
V = 0.3 µL
Flow rate = 10 µ Tmin
AuCr film
d = 41/2 nm Prism
Beamsplitter
Si photodetector 5 mm
Laser
λ = 670 nm
FIGURE 15.4 The Kretchmann configuration for SPR. (From Aguilar, M.R. et al., Macromol. Biosci., 4(7),
631, 2004.)
resonantly excited. This causes a loss of energy from the incident light to the metal film, resulting in
a reduction in the intensity of the reflected light which can be detected by a two-dimensional array
of photodiodes or charge-coupled detectors (CCD). Therefore, if the refractive index above the metal
film changes by the adsorption of a protein layer, a change in the angle of incidence required for
exciting a surface plasmon will occur. By monitoring the angle at which resonance occurs during the
adsorption process with respect to time, an SPR adsorption profile can be obtained.
SPR has been extensively used to follow interactions between polymers and biomolecules, espe-
cially proteins (Li et al. 2005; He et al. 2007; Servoli et al. 2008, 2009). Aguilar et al. (2004)
investigated the formation of PECs between chitosan and synthetic polymers derived from 2-acryl-
amido-2-methylpropane sulfonic acid (AMPS), as well as the absorption of albumin and fibrinogen
on these complex surfaces.
15.3.2.3 Zeta Potential

When a charged solid surface is in contact with a liquid phase, an electrical potential develops at the
interface. A double layer is established, one of them between surface ionizable groups and tightly
bound liquid phase ions of opposite charge forming the fixed layer and the other with loosely bound
liquid phase ions of opposite charge forming the mobile layer. The zeta potential is the change in
potential across this double layer (Goddard and Hotchkiss 2007). In general, zeta potential is mea-
sured indirectly using one of the three means: (1) by measuring electro-osmotic mobility, (2) by
measuring streaming current or streaming potential generated by pressure-driven flow through a
conduit, or (3) by measuring response of a small spherical particle in an applied E-field (Kirby and
Hasselbrink 2004).
Zeta potential can be used to determine surface isoelectric points and quantify a change in sur-
face ionisable groups (Richey et al. 2000). It is also a useful tool to predict the stability of colloidal
suspensions or emulsions, and therefore, in biomaterials field, it is widely used for the investigation
of physicochemical stability of micro- and nanoparticles for drug delivery (Mosqueira et al. 2000;
Heurtault et al. 2003; Hoffart et al. 2003; DeVolder 2010).
15.3.2.4 Quartz Crystal Microbalance

A quartz crystal microbalance (QCM) is a high-resolution mass-sensing technique, which measures
mass by measuring the change in frequency of a piezoelectric quartz crystal, when it is disturbed
by the addition of tiny masses such as proteins or any other tiny objects intended to be used. This
technique possesses a wide detector range. At the low mass end, it can detect a monolayer surface
covered by small molecules or polymer films. At the upper end, it is capable of detecting much
larger masses bound to the surface, such as complex arrays of biopolymers and biomacromolecules
or even whole cells (Marx 2003).
The instrument consists of a thin quartz disk with electrodes placed on it. The application of
an external electrical potential to a piezoelectric material produces internal mechanical stress. As
the QCM is piezoelectric, an oscillating electric field applied across the device induces an acoustic
wave. This wave propagates through the crystal and meets minimum impedance when the thick-
ness of the device is a multiple of a half wavelength of the acoustic wave. The deposition of a thin
film on the crystal surface decreases the frequency in proportion to the mass of the film. A resonant
oscillation is achieved by including the crystal into an oscillation circuit where the electric and the
mechanical oscillations are near to the fundamental frequency of the crystal. Changes in resonant
frequency are simply related to the mass accumulated on the crystal. Thus, for detection of analytes
in air, the frequency is simply related to the change in mass (O’Sullivan and Guilbault 1999).
QCM is becoming a good alternative analytical method in a great deal of applications such as
biosensors, analysis of biomolecular interactions, study of bacterial adhesion at specific interfaces,
study of polymer film-biomolecule or cell-substrate interactions, immuno-sensors, and an extensive
use in fluids and polymer characterization, and electrochemical applications between others (Hook
et al. 2002; Arnau 2008; Olsson et al. 2010).
A special variation of QCM technique is the so-called quartz crystal microbalance with dissipation
monitoring (QCM-D). The main advantage of QCM-D compared with conventional QCM is that the
former estimates changes in both the mass and the viscoelastic constant for the adsorbed layer through
measurements of frequency and dissipation (Dixon 2008; Huang 2010; Martins et al. 2010).
15.3.3 Microscopic Methods

15.3.3.1 Atomic Force Microscopy
With atomic force microscopy (AFM), three-dimensional images of a surface are created by scan-
ning a tip attached to a flexible cantilever across the sample surface and monitoring the minute
forces of interaction between the sample surface and probe. This technique allows the measure-
ment of surface morphology at atomic scale. The change in surface height is then measured by
the location of the reflected laser beam in the quadrant photo-detector, and a topographical map is
generated from which surface roughness can be calculated (Alessandrini and Facci 2005). Surface
roughness is an important parameter in biomedical materials since it can affect cell adhesion,
specially important for blood-contacting materials, in which an increase of surface roughness can
compromise hemocompatibility since turbulent flow can promote hemolysis (Aksoy et al. 2008).
The AFM can operate in different modes (Jandt 2001), as shown in Figure 15.5. In the constant
force mode, also called contact mode, the force between the tip and the sample surface is kept
(a) (b)
Out of phase High elastic

modulus
region
Low elastic
modulus
Scan direction Scan direction region
(c) (d)
(e)
FIGURE 15.5 Schematic representation of the most common operating modes in AFM. (a) Nondynamic mode:
contract mode, (b) dynamic mode: tapping mode, (c) phase imaging, (d) force modulation, (e) dynamic mode:
noncontract mode. (From Jandt, K.D. et al., Surf. Sci., 491(3), 303, 2001.)
constant via an electronic feedback loop. An image of the sample’s surface topography is created
from the cantilever deflection required to maintain a constant force over the surface. In this mode,
the tip is in permanent physical contact with the sample; therefore it may not always be appropriate
for biomaterials applications.
The tapping mode uses an oscillating tip (frequencies of 50–500 kHz in air and approxi-
mately 10 kHz in fluids) that lightly taps the sample surface with amplitude typically ranging
from 20 to 100 nm. A split photodiode detector monitors the deflection of the laser light reflected
from the tip of the cantilever. A feedback loop maintains constant the oscillation amplitude or
frequency as the scanner moves laterally across the surface. This mode maintains the high-
resolution capabilities of contact mode but it is not destructive since there are no lateral fric-
tional forces applied to the sample. For this reason, it has been proved to be very successful for
high-resolution studies of polymeric biomaterials allowing the characterization of nanometer-
scale features not visible by other microscopic techniques (Ortega-Vinuesa et al. 1998; Bowen
et al. 2000; Willemsen et al. 2000).
Phase imaging mode can be used to map the surface composition of a sample. In this mode, the can-
tilever vibrates at or near its resonance frequency and lightly taps the sample surface. A phase image
shows the phase difference between oscillation of the piezoelectric crystal that drives the cantilever
and oscillation of the cantilever itself as it interacts with the surface. AFM in phase imaging mode can
be performed at the same time as topographic imaging with tapping mode in a single scan. The main
advantage of this mode is that the tip–sample interactions depend not only on the sample’s topography
but also on different sample characteristics, e.g., sample hardness, elasticity, or adhesion.
In noncontact mode, attractive forces are measured. The scanning tip is oscillated perpendicular
to, and just above the sample surface with amplitude typically less than 10 nm. This mode may work
well with hydrophobic polymers but it is not recommended for hydrophilic polymers or samples in
a liquid environment due to the lower resolution.
AFM can be also developed using coated tips. In this mode, tips are modified with functional groups
or even biomolecules to achieve very high sensitivity in the detection of surface properties and molecu-
lar recognition. This technique has been applied to study specific interactions of biomaterials with
biological systems (Schneider et al. 1999; Flores et al. 2006; Weder et al. 2010). It can also be applied
for the production of chemical affinity maps. In this sense, the introduction of carbon nanotube tips,
which can be specifically functionalized even with single molecules, has remarkably increased the
performances of AFM, giving the potential for even greater affinity map resolution (Alessandrini and
Facci 2005).
15.3.3.2 Scanning Electron Microscopy and Transmission Electron Microscopy

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) make use
of a primary beam of electrons to bombard the sample in a vacuum environment, resulting in
a secondary electron emission and x-rays (Merrett et al. 2002). The intensity of the secondary
electrons is detected to generate a high-resolution three-dimensional surface image. X-rays can be
detected to develop elemental analysis.
SEM sample preparation involves fixation (if cells or tissue are present), followed by drying,
attachment to a metallic stub and coating with a metal prior to data collection. The metallic coating
(20–30 nm) is usually applied by sputter coating, gold, platinum, or gold/platinum alloy being the
most common conductive metals used. The drying and metal coating of the sample might alter sur-
face morphology in polymeric materials, particularly those that could undergo changes in hydrated
environment. In this sense, the introduction of environmental scanning electron microscopy
(ESEM) has represented a new perspective. ESEM does not operate at high vacuum and allows
imagery of the material sample in wet mode without further sample preparation (Muscariello et al.
2005). This instrument has a unique secondary electron detector capable of forming high-resolution
images at pressures in the range of 0.1–20 Torr. At these relatively high pressures, nonconductive
specimens are observed without metallic coating or any special preparation.
In addition to imaging the surface morphology of polymeric biomaterials, SEM can be combined
with other analysis methods such as energy dispersive x-ray analysis (EDX) to determine elemental
composition and IR and Raman spectroscopy to monitor surface modification procedures. EDX
results are typically obtained from a sampling depth on the order of micrometers; thus, they are
more representative of the bulk material rather than the surface.
TEM sample preparation involves fixation (if proteins, cells or tissue are present), processing,
embedding, and sectioning. Embedding media can include methacrylates, polyester, and acrylic
resins. Specimens are typically sectioned using a microtome and need to be very thin since elec-
trons with an accelerating voltage of 100 kV will not penetrate specimens more than 1 μm thick.
Good resolution and clarity of detail can normally be obtained with sample thickness in the order
of 50–90 nm. It should be noted that embedding and sectioning used in the preparation of some
polymeric materials may alter the polymeric material itself or the quality of the image obtained due
to factors such as drying, thickness variations, wrinkling, or compression.
SEM and ESEM have been extensively used for studying both surface morphology and cel-
lular response to biomaterials (Rodríguez-Lorenzo et al. 2006, 2009; Kirsebom et al. 2007;
López Donaire et al. 2009). Moreover, TEM techniques are more useful for the investigation of the
interface between tissue and biomaterials (Porter 2006; Lamers et al. 2010).
15.4 INTERACTIONS WITH BIOLOGICAL ENVIRONMENT AND APPLICATIONS

The biocompatibility study includes the analysis of the interaction between materials and biological
systems in a wide range of applications and molecular processes, taking into account both the host
response against the material as well as the evolution of the material properties after insertion into a
biological environment (Tirrell et al. 2002). Due to the fact that the interaction between the material
and the environment is developed through the interface by means of “bio-recognition processes”
(Elbert and Hubbell 1996), it will consequently depend heavily on the properties of the material
surface. When a material is in contact with a biological environment, its structure, chemical compo-
sition, and topography will determine the adsorption of proteins, the interaction with the cells and,
ultimately, the response of the host.
The material incorporation into a biological system triggers an immediate and nonspecific
adsorption of proteins on its surface, independent of its nature. This phenomenon will deter-
mine the subsequent biological reactions (platelet adhesion, activation of the foreign body
reaction, etc.) so that the design of biocompatible surfaces requires a deep knowledge of the
interaction between proteins and biomaterial surfaces (Ratner and Bryan 2004). Recently, the in
vitro and in vivo studies on the interaction between biomaterial surfaces and biological systems
have shown a remarkable progress which has led to the development of surfaces resistance to
nonspecific protein adsorption as well as surfaces with selectivity toward specific proteins by
immobilization of biomolecules or biomimetic ligands (Anderson et al. 2004; Levenberg et al.
2004; Goldberg et al. 2007; Vasita et al. 2008). Currently, the main lines of research in the field
of polymeric biomaterials are focused on studying in depth the biological mechanisms involved
in the interaction between the polymer surface and proteins that influence cellular response, on
developing polymer surfaces for the repulsion of proteins; and developing polymeric bioactive
surfaces by selective adsorption of biomolecules.
15.4.1 Cell–Polymer Interaction Determines Biocompatibility

Proteins are natural biopolymers that result from the polymerization of at least 20 l-α-amino
acids and have very complex physical and chemical characteristics. Proteins are involved in
most cellular functions leading to many forms of cell–substrate specific interactions that arise
from the presence of one or more active domains available to react with specific substrates.
When a polymer comes into contact with a biological system, cells in this system tend to identify
the polymer as a foreign body, a fact that triggers an immune response and inflammation
(Anderson 2001; Anderson et al. 2008). However, if the polymer includes any ligand capable of
being specifically recognized by the proteins of host cells, a specific and selective response can
be obtained.
There are two basic kinds of molecules involved in establishing such interactions, extracellular
proteins and cell membrane proteins. Extracellular proteins play an important role in cell adhesion,
propagation, and growth regulation and are divided into extracellular matrix (ECM) components
and plasma proteins (Schwartz and Ginsberg 2002). The ECM components are directly or indi-
rectly attached to the cells. In the organic component of the ECM, there are the two most important
types of extracellular macromolecules: fibrous proteins and proteoglycans. The plasma proteins
circulate in blood plasma and are primarily responsible for the regulation of cell proliferation, dif-
ferentiation, and immune response (Chen et al. 2008). The proteins embedded in cell membranes
are classified, according to their functionality, in transporters, receptors, adhesion molecules, and
enzymes. They are responsible for substrate transfer and signal transmission across the membrane,
connection and recognition among cells, interaction with the extracellular matrix, immune response
to foreign objects, and matrix formation for cell embedding (Barclay 2003; Wierzbicka-Patynowski
and Schwarzbauer 2003; Ehrhardt et al. 2004; Arnaout et al. 2005; Leckband and Prakasam 2006).
These proteins, usually modified with carbohydrates and lipids, directly interact with the matrix
substrates or signal like growth factors via its extracellular domain. Growth factors, together with
ECM proteins are responsible for the regulation of cell proliferation, differentiation, and adhesion
(Yamada and Even-Ram 2002). Adhesion between cells is accomplished by connections through
ECM proteins and specific receptors anchored on cell surface. The connections of ECM proteins
and these receptors trigger a specific focal adhesion formation and signal transduction. These focal
adhesion phenomena entrench the union of the cell with the surrounding microenvironment. The
signal transduction to adjacent cells is essential in regulating cell migration, proliferation, and dif-
ferentiation (Schwartz and Ginsberg 2002).
The main biomolecules involved in cell adhesion are proteins. This fact has motivated the
development of various research projects that aim to focus on the study of cell–surface inter-
actions by immobilizing adhesive proteins derived from the ECM onto polymer surfaces, as
fibronectin (FN), collagen, laminins, or vitronectin (Linnola et al. 2000; Vasita et al. 2008). The
key advantage of such modifications lies in that ECM proteins are natural ligands of integrins so
that their presence will not cause direct harmful effects. However, its application can also lead
to several problems: the orientation of immobilized ECM proteins is difficult to control, their
conformation can be changed, ECM proteins are easily degraded by enzymes so that the long-
term applications may be impeded, and the ECM proteins can induce immune responses in the
host (Chen et al. 2008).
The mechanisms employed in the FN immobilization on polymer surfaces have been devel-
oped by both physical adsorption and covalent conjugation. UV ozone has been used to activate
hydrophobic materials derived from poly(dimethylsiloxane), obtaining hydrophilic surfaces with
physically adsorbed FN that promote cell adhesion and spreading significantly (Toworfe et al.
2004). Koenig and co-workers analyzed the influence of the FN adsorption in different systems
of varied chemical composition, using human umbilical vein endothelial cells (HUVECs) cul-
tures, concluding that the adsorption of FN on the surface induced an increase in cell adhesion
and spreading dependent on the polarity of these systems (Koenig et al. 2003). With increasing
surface hydrophobicity, they observed a decrease in both cell adhesion and effective growth in
these cultures, probably because the surface of such formulations induced the denaturalization
of the FN. The latter result has been corroborated in other studies, such as that developed by Qu
et al., who obtained a higher growth of HUVECs and rabbit aorta smooth muscle cells (SMCs) on
the surface of a copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate treated with ammonia
plasma (hydrophilic variant), compared with the same copolymer untreated (hydrophobic vari-
ant), although the amount of adsorbed FN was greater in the latter. This result incises again on
the importance of the maintenance of the FN proper conformation to preserve unchanged its
bioactivity (Qu et al. 2005).
There are also several examples of successful immobilization of such proteins on the surface
of polymers by covalent conjugation, among them the functionalization of the poly(l-lactide-
co-caprolactone) surface with both FN and collagen, using the bridge of amino group linkages
introduced on the surface of these systems through aminolyzing with 1,6-hexanediamine and
glutaraldehyde as coupling agents (Zhu et al. 2006a) or the development of FN-modified Pluronic
conjugates, whose surfaces have shown a specific bioactivity to the dorsal root ganglia neurite out-
growth of postnatal rats (Zhang et al. 2005). Covalent conjugates have also been developed with
other proteins, such as growth factors. Different studies have produced systems with enhanced
bioactivity through the covalent binding of epidermal growth factor (EGF) (Gobin and West
2003; Klenkler et al. 2005), vascular endothelial growth factor (Zisch et al. 2001), transforming
growth factor-β (TGF-β) (Mann et al. 2001), or nerve growth factor (Chen et al. 2004b), using
PEG spacers or not.
With regard to the result obtained with the two methods described earlier, it is noteworthy that
several systems with proteins covalently conjugated showed a performance advantage over systems
with physically adsorbed proteins on its surface (Biran et al. 2001; Webb et al. 2001; Klee et al.
2003), although other researchers have suggested that the latter have a more favorable behavior for
certain cell lines (Zhang et al. 2007).
With the aim of contributing to the inactivation of cellular recognition processes arising from
conformational changes that affect proteins, specific polypeptides were covalently anchored to the
surface of different polymers with diverse functional domains involved in molecular adhesion.
Fibronectin has several cell membrane integrin-binding domains (RGD sequence, in repeat III10
module, PHSRN, in repeat III9 module, LDV y REDV from spliced V region, IDAPS y KLDAPT,
in repeats III14 y III5 modules, y EDGIHEL, in spliced Eda segment) and heparin-binding domains
(PRRARV sequence, in III13 module, and WQPPRARI, in III14 module) (Wierzbicka-Patynowski
and Schwarzbauer 2003; Chen et al. 2008). Other peptide sequences are involved in cell adhesion
processes through ligand–acceptor interaction, as are the sequences KKQRFRHRNRKG, from vit-
ronectin; CDPGYIGSR, YIGSR, IKVAV, RNIAEIIKDI, YFQRYLI and PDSGR, from laminin;
DGEA, from collagen I; VAPG, from elastin; and FHRRIKA, from bone sialoprotein (Tirrell et al.
2002; Chen et al. 2008).
Arg–Gly–Asp (RGD) sequence is present in several ECM proteins, a fact which has moti-
vated numerous studies to develop biomimetic surfaces using this sequence. There are other
advantageous characteristics in the use of this sequence, such as its low immunogenic activity,
the ability to synthesize it with a controlled conformation, its high stability against sterilization
methods, high temperatures or pH variations, or its low price compared with the ECM proteins.
Some of the disadvantages associated with their use are the risk of enzymatic degradation and
the lower binding activity obtained for specific subtypes of integrins compared with that shown
by ECM proteins.
The methodologies used to incorporate the RGD sequence are varied, the first one being sim-
ple adsorption (McConachie et al. 1999; Dettin et al. 2002). This method does not significantly
improve cell adhesion due to structural problems, such as the entrapment of peptides into depoly-
mers and undesirable peptide density. Other methods are spacing (Rezania and Healy 1999), blend-
ing (Quirk et al. 2001), copolymerization (Shin et al. 2002), and chemical or physical treatment
(Marchand-Brynaert et al. 1999; Carlisle et al. 2000). One of the techniques most commonly used
in biomedical applications is the chemical conjugation. By applying this type of technique, RGD
or RGD-containing peptides have been immobilized onto polymer surfaces using specific reactions
or chemical functional groups. Systems that improve the adhesion and differentiation of osteogenic
precursor cells were obtained using RGD peptide conjugated with PLA surface via the reaction
of –CHO and –NH2 (Hu et al. 2003), as well as conjugates with RGD-containing peptides that also
promote the cellular functions (Ebara et al. 2004; Smith et al. 2005). The RGD sequence can also
be immobilized by copolymerization techniques, obtaining copolymers such as poly[N-p-vinyl-

benzyl-d-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-GRGDS], in which the inclusion
of the GRGDS peptide improves growth and specific functions of hepatocytes and phechromocy-
toma cells (PC12), albumin secretion, and urea synthesis in hepatocytes (Park 2002), and dopamine
secretion by PC12 cells (Park et al. 2004). Another example is the RGD-grafted poly-d-lysine-graft-
(polyethylene glycol), adsorbed onto substrates by electrostatic interaction between positive charges
in poly-d-lysine and negative charges in the substrate, which inhibit nonspecific protein adsorp-
tion in an aqueous medium and allow the adhesion and proliferation of human dermal fibroblasts
(VandeVondele et al. 2003).
During the cell adhesion process, four overlapping steps occur: (1) cell attachment, when
a specific integrin–ligand binding between the cell and the surface is established, (2) cell
spreading, (3) actin–cytoskeleton organization, and (4) focal adhesion formation (Lebaron and
Athanasiou 2000). One of the key points to achieve a solid union between cells and polymeric
surface is the correct formation of focal adhesion complex. The main participants in this com-
plex formation are integrins along with several transmembrane, membrane, and cytosolic mol-
ecules, such as tetraspanins, growth factor receptors, syndecans, lipids, tensin, vinculin, talin,
paxillin, and focal adhesion kinase (Pande 2000; Petit and Thiery 2000; Geiger and Bershadsky
2001; Zamir and Geiger 2001). Integrins connect the extracellular components with intracellular
proteins and are mainly responsible for signal transduction that regulates important processes
such as cell proliferation, differentiation, migration, or apoptosis. Integrins consist of two trans-
membrane subunits (α and β subunits) associated by noncovalent bonds. The human genome
can code for different types of subunits with specificity for certain ligands, resulting in 24 dif-
ferent heterodimers with different specificities (van der Flier and Sonnenberg 2001). Some of
these integrins exhibit a high specificity, while others have the ability to bind to a large number
of ligands, such as αvβ3 integrin which binds to vitronectin, fibronectin, von Willebrand factor,
osteopontin, tenascin, bone sialoprotein, and thrombospondin. Moreover, certain ECM mol-
ecules such as fibronectin are ligands for several types of integrins (Plow et al. 2000; van der
Flier and Sonnenberg 2001). The transmembrane signaling by integrin mechanisms and binding
mechanisms to different ligands are very complex and involve numerous conformational change
(Gottschalk et al. 2002; Gallant et al. 2005). Integrins have a major role in the application of
RGD-modified polymers, since at least half of the heterodimers existing in humans recognize
this peptide as its ligand (van der Flier and Sonnenberg 2001). There are numerous examples
in which the development and application of RGD-modified polymers have improved adhesion,
proliferation, and/or differentiation mediated by integrins in different cell lines: osteogenic
active cells (Rezania and Healy 1999; Itoh et al. 2002; Barber et al. 2003; Behravesh and Mikos
2003; Hu et al. 2003; Huang et al. 2003; Zreiqat et al. 2003; Shin et al. 2004), fibroblast (Koo
et al. 2002; Gümüşderelioğlu and Karakeçili 2003; VandeVondele et al. 2003; Rajagopalan et al.
2004), hepatocytes (Bhadriraju and Hansen 2000; Park 2002), myoblast (Rowley and Mooney
2002; Smith et al. 2005), endothelial cells (Ebara et al. 2004; Sagnella et al. 2005; Chen et al.
2006; Sheardown et al. 2007), marrow stromal cells (Mapili et al. 2005), and neuron-like cells
(Park et al. 2004). Moreover, other studies have shown that a more “complete” cellular response
(e.g., cell attachment, spreading, focal contact formation and organized cytoskeletal assembly)
is obtained through application of systems with two types of peptide sequences, cell-binding
(RGD domain) and heparin-binding sequences of FN or bone sialoprotein (Tirrell et al. 2002).
15.4.2 Passivating Polymer Surfaces for Protein Repelling

One of the main objectives in the development of materials with biomedical applications, crucial for
those that will be in contact with blood vessels, is the reduction of foreign body immune response by
the inhibition of the nonspecific protein adsorption that occurs when the material comes in contact
with the biological environment (Hench and Polak 2002; Rodriguez et al. 2009) (Figure 15.6).
Platelets
Platelets
Proteins Adsorption
Thrombin
Proteins
Thrombus
formation
Platelets
Fibrin matrix
FIGURE 15.6 (See companion CD for color figure.) An example where the nonspecific adsorption of
proteins to a surface can lead to serious problems is the thrombus formation. (Taken from Tirrell, M. et al.,
Surf. Sci., 500, 61, 2002.)
Within the basic variables that regulate the adsorption of proteins, the host response is necessary
to emphasize the hidrophilic/hydrophobic balance of the material*** surface (Ma et al. 2007) that can
be defined using a specific parameter, pure water adhesion tension (τ°) (Vogler 1998). The surfaces
identified as hydrophilic have a value of τ° > 30 dyn/cm, while for hydrophobic, it is τ° > 30 dyn/cm.
Hydrophobic surfaces favor nonspecific adsorption from aqueous solution thermodynamically and
can contribute to denaturalize the native conformation of proteins involved in recognition and cell
adhesion processes, and hence may affect their bioactivity. Moreover, hydrophilic materials tend to
inhibit protein adsorption. Although the results obtained in this field do not always provide matching
conclusions, it seems clear that an improvement in the surface hydrophilicity of the hydrophobic mate-
rials is necessary to adsorb an appropriate amount of proteins while maintaining its natural conforma-
tion and, hence, to obtain a positive cellular response (Vogler 1998; Tziampazis et al. 2000).
Along with the physical techniques developed to modulate the hydrophilicity/hydrophobicity
ratio of synthetic polymers, the main research trends are focused on improving the resistance of
materials against nonspecific adsorption of proteins by introducing chemical modifications on their
surfaces (Shen et al. 2003; Roach et al. 2005, 2007; Ladd et al. 2008; Zhang et al. 2008). The num-
ber and type of functional groups resistant to protein adsorption has been determined by combining
SAMs and SPR spectroscopy. This knowledge has allowed establishing the basic principles to be
considered in the design of inert groups. These groups must be polar, hydrogen-bond acceptors, not
hydrogen-bond donors and electrically neutral groups (Chapman et al. 2000; Ostuni et al. 2001),
although not all inert surfaces possess all these features. One example is SAMs bearing mannitol
groups with a high number of hydrogen-bond donors that are inert to protein absorption (Luk et al.
2000). According to these principles and the great diversity of interactions between proteins and
material surfaces, different methodologies have been developed to obtain protein-repelling surfaces
(Higuchi et al. 2002; Zhao et al. 2003; Zhou et al. 2005). In order to obtain inert surfaces, several
polymeric layers grafted to a substrate have been developed (Gupta et al. 2002; Higuchi et al. 2002;
Martins et al. 2003b; Vermette and Meagher 2003; Combellas et al. 2004; Wan et al. 2005; Joshi
and Sinha 2006). The interaction between proteins and tethered polymer chains is very complex
and depends on the properties of both proteins and polymer surface. Despite the high complexity
of this interaction, three generic modes of protein adsorption in such systems have been described:
primary adsorption, when the particle size is less than the distance between grafted chains, a fact
which causes the diffusion of particles inside the brush, and their subsequent adsorption on the sub-
strate surface; secondary adsorption, when the particles cannot physically penetrate into the brush,
a fact which causes its adsorption to the brush-solvent interface; and ternary adsorption, when the
particles can diffuse into the brush, where they are retained (Halperin 1999; Currie et al. 2003).
Two of the most commonly used polymers in the preparation of modified surfaces are PEG
and PEO, which share the same repeating unit but are derived from different monomers via dif-
ferent polymerization methods (Chen et al. 2008). The modification of surfaces with these poly-
mers is very interesting not only because they are efficient inhibitors of protein adsorption but
also because they provide easy ether linkages with proteins. With regard to resistance to protein
adsorption on PEG-modified surfaces, it is necessary to emphasize the role of different thermo-
dynamic factors (Hoffman and Ratner 2004), the steric repulsion, and the hydration or water
structuring layer formation (Morra 2000; Vermette and Meagher 2003) along with structural fac-
tors, as the length of the PEG chain, the density of surface coverage or the chains organization
on the material surface (Currie et al. 2003). Ideally, the inhibition of protein adsorption requires
a high density and a long chain. In the event in which PEG chains of different lengths are incor-
porated on the same support, it is possible to obtain surfaces with high efficiency in specific
repulsion for certain proteins and not for others, depending on their size (Lazos et al. 2005). It is
also possible to control the hard charged proteins adsorption, alternating the charge of PEG-
modified surfaces (Pasche et al. 2005). Moreover, it is possible to modulate the bioactivity and
conformational changes of adsorbed proteins, which will allow monitoring of cellular interaction
and biocompatibility by varying the PEG concentration. For example, it was found that both the
amount of adsorbed FN and its specific activity decreased with increasing PEG concentration.
The decrease in the activity of this protein results from the globular conformation, more com-
pact, which appears when the PEG concentration on the material surface is high, and hinders
the participation of the RGD domain in the process of adhesion (Thom et al. 2000; Tziampazis
et al. 2000). There are numerous examples that illustrate the influence of polymer surfaces modi-
fied with PEO or PEG, by physical adsorption techniques, by establishing covalent bonds, or by
surface grafting of PEO/PEG-containing molecules. Among these examples, there are systems
based on polyurethane (Cornelius et al. 2002; Kim et al. 2003; Archambault and Brash 2004a,b)
and silicone (Chen et al. 2004a, 2005a,b) polymers with anticoagulant activity, that inhibit plate-
let adhesion or protein adsorption; PS systems (Byun et al. 2004; Bosker et al. 2005; Lazos et al.
2005), which inhibit specifically the adsorption of certain proteins depending on their size; or
poly(methylmetacrylate) (Liu et al. 2004; Bi et al. 2006), polylactide- (Otsuka et al. 2000) and
polyaniline- (Li and Ruckenstein 2004) based polymers that also significantly reduce platelet
adhesion and adsorption of certain proteins.
Another type of surface modification aimed at obtaining biomimetic systems with nonthrom-
bogenic characteristics is carried out using phosphorylcholine (PC)-based polymers. The develop-
ment of this approach arises after discovering that the lipids that form part of the outer membrane
of erythrocytes possess antithrombogenic character, unlike those found in the inner membrane,
resulting from the presence of PC moieties (Zwaal et al. 1977). This fact has motivated the syn-
thesis of different PC-containing monomers, such as MPC (Ishihara et al. 1990), which has a
zwitterionic PC head group and whose polymerization has given rise to polymers with different
properties, e.g., the ability to control the behavior of adsorbed proteins (Feng et al. 2004, 2005;
Iwata et al. 2004). Different mechanisms have been proposed to explain protein adsorption resis-
tance of these systems (Lewis 2000). One of them attributed this property to the formation of an
organized bilayer of plasma lipid, which would act as a protective shield for the material surface
(Heiden et al. 1998). Other works consider that the large hydration layer formed by interaction of
numerous free water molecules with PC groups on the material surface is responsible for the bio-
mimetic character of these systems by not allowing significant changes in protein conformation
(Sheng et al. 1995; Ishihara et al. 1998). It has also been shown that the flexibility and mobility of
the PC group plays an important role in the reduction of protein–surface interactions (Parker et al.
2005). Different MPC-modified polymers based on polyurethane were obtained through blending,
semi-IPN, coating, chemical or photochemical graft, photoinduced graft, ozone-induced graft, or
swelling-deswelling methods, systems (Korematsu et al. 2002; Morimoto et al. 2002, 2004). These
systems significantly reduce the adhesion of FN, reduce or suppress platelet adhesion, or inhibit the
fibroblasts adhesion. Other systems based on polyethylene (Iwasaki et al. 2002; Ishihara et al. 2004),
PSF (Hasegawa et al. 2002; Ishihara et al. 2002), and PDMS (Xu et al. 2003; Goda et al. 2006; Seo
et al. 2008) also reduce platelet adhesion and total adsorption of proteins, or modulate specifically
the adsorption of certain proteins.
Like the PC-containing polymers, sulfobetaine- and carboxybetaine-based polymers have
cationic and anionic groups on the same monomer residue; this, coupled with the fact that both
methacrylic derivatives of sulfobenaine (SBMA) and carboxybetaine (CBMA) are more stable
and easier to obtain than MPC makes them suitable candidates for the development of biomedi-
cal systems with reduced protein adsorption (Wang et al. 2003; Zhang et al. 2006a). The most
important factors affecting the effectiveness of sulfobetaine and carboxybetaine surfaces as
inhibitors of protein adsorption are the modification method, the elaboration of surface pack-
ing, and the surface density. Sulfobetaine polymers have been incorporated on polyurethane,
poly(ether urethane), and segmented poly(ether urethane) by graft polymerization and ozone-
induced polymerization (Yuan et al. 2003a, 2004a; Yuan et al. 2003b, 2004b), silicone (Yuan
2004c), and cellulose (Zhang et al. 2003), giving systems with a high biocompatibility with sur-
faces that inhibit adsorption protein and/or platelet adhesion. With regard to the carboxybetaine
methacrylic derivative, poly(carboxybetaine methacrylate) (PCBMA), it is noteworthy that this
polymer not only exhibits a high efficacy to inhibit protein adsorption but also has several func-
tional groups available for immobilizing biomolecules, such as fibrinogen, lysozyme, or human
chorionic gonadotropin (hCG) (Zhang et al. 2006b). This dual functionality makes such systems
of great interest for various biomedical applications (diagnostics, biomedical/tissue engineering,
drug delivery).
15.4.3 Bioactivity of Polymer Bioconjugate Surfaces

To achieve biomaterial–biological environment interactions, one of the most innovative strategies
is based on incorporating bioactive molecules on the biomaterial surfaces, to obtain hybrid mac-
romolecules with unprecedented properties (Lutz and Zarafshani 2008; Lutz and Börner 2008;
Börner 2009). The possibility of incorporating a “biological functionality” to biomaterials by com-
bining synthetic materials and biological structures at different hierarchical levels of organization
(monomeric, oligomeric, polymeric, supramolecular or highly organized structures) is a powerful
and flexible tool that multiplies the applications of the new systems developed, through the regula-
tion of protein–protein, protein–nucleic acid and protein–carbohydrates interactions. Although it is
possible to modify biologically the surface of most biomaterials, synthetic and natural polymers are
especially useful for this purpose because their physical or chemical combination with different bio-
logical moieties to obtain polymer bioconjugates is relatively simple (Goddard and Hotchkiss 2007;
Hermanson 2008). The polymer surface may contain numerous reactive groups or can be easily
changed by adding reactive groups that can be used to link biomolecules covalently. In addition, the
possibility of varying composition and molecular structure through the synthesis of homopolymers
and random, alternating, block and grafting copolymers and the versatility of polymers to be pro-
cessed in many forms (films, membranes, tubes, hollow fibers, fabrics, particles, capsules, porous
systems, etc.), further multiplies the possibilities. Biological molecules used in the preparation of
these bio-hybrids and bio-inspired systems can be found in living organisms (lipids, nucleic acids,
peptides or carbohydrates), but they can also be produced synthetically (de Graaf et al. 2009). There
are different techniques to obtain man-made copies of natural molecules such as oligonucleotides
or peptides with automated synthesizers. Although the combination of polymers with peptides and
proteins has been widely practiced in the field of pharmaceutical chemistry during the past decades
(Langer and Tirrell 2004), the recent and explosive development of biotechnology and nanotechnol-
ogy has enlarged the applications of polymer bioconjugates in materials science, e.g., as biosensors,
artificial enzymes, biometrics, light-harvesting systems or photonics, and nano-electronic devices
(Klok 2005; Whitesides 2005).
Most of the polymer bioconjugates are obtained by applying covalent chemistry techniques,
although it is notable that there are different alternatives to obtain bioconjugates by applying non-
covalent methods, among which are electrostatic interactions and ligand–receptor pairing (as in
biotin–avidin) (Niemeyer 2001). The biotin–(strept) avidin system is based on the recognition of the
low-molecular weight biotin by larger glycoproteins such as avidin or streptavidin. The fact that
the noncovalent bond established in these systems is the strongest reported (up to 250 pN) justifies
the application of this system in the biotechnology field (DNA and proteins microarrays, immu-
nological assays, or bio-separation devices), and the increased effort applied to the development
of new biofunctionalized systems by anchoring of avidin-modified biological entities (Goddard and
Hotchkiss 2007). There are other possibilities to prepare polymer bioconjugates such as cofac-
tor reconstitution routes for modifying enzymes and transport proteins (Boerakker et al. 2002;
Reynhout et al. 2007) and the supramolecular recognition of sugar moieties and corresponding
lectins (You et al. 2002). On the other hand, less specific noncovalent interactions can be used for
connecting synthetic polymers and biological objects. The ionic complexation of oppositely charged
entities is a powerful alternative to conjugate biological structures that exhibit charged surfaces.
For example, the charged surfaces of proteins, enzymes, or carbohydrates may be modified with
appropriate polyelectrolytes, in order to implement them for the development of bioactive polymer
bioconjugates by complexation (Dufresne and Leroux 2004; Jaturanpinyo et al. 2004). Moreover,
the negatively charged backbone of DNA can be complexed with various synthetic polycations or
block-copolymers containing polycationic segments, resulting compact biohybrid particles known
as polyplexes, which have been extensively studied in the field of nonviral gene delivery (Lutz and
Börner 2008).
Considering the nature of the biomolecules used in the preparation of polymer conjugates,
three major types of polymer conjugation can be distinguished: (1) peptide bioconjugation,
(2) bioconjugation with nucleobases, oligonucleotides, and nucleic acids, and (3) bioconjugation
with carbohydrates. Moreover, the possibility of modifying highly organized biosystems also
deserves attention.
Depending on the complexity of the peptide sequence incorporated into the polymer chain,
peptide bioconjugates can be classified into three categories: (1) amino acid building blocks,
(2) oligo and polymeric peptides, and (3) high-molecular weight proteins (Lutz and Börner 2008).
The incorporation of single amino acids in synthetic polymers is the lowest level of complexity.
This modification can modulate certain properties of polymer conjugates, such as the hydrophobic/
hydrophilic balance (Geng et al. 2006). The increase in the length of the sequence improves the
chemical diversity and the number and complexity of biological functions in which these sys-
tems may be involved. Thus, the control of more complex properties, such as selective catalytic
activity, highly specific recognition or directional functions, and the combination of multiple
functions can only be achieved through the preparation of polymer systems with native or high-
molecular weight proteins (Klok 2005; Maskarinec and Tirrell 2005). Although the development
of this latter type of polymers is significantly more expensive, and their interaction with the bio-
logical environment is less predictable, remarkable progress was attained in the field of protein
engineering and biomaterials design in recent years (Langer and Tirrell 2004; Maskarinec and
Tirrell 2005; Smeenk et al. 2005).
Another group of biomolecules used in polymer bioconjugates are the nucleobases, oligo-
nucleotides, and nucleic acids, due to both structural and functional reasons. These biomolecules
provide exceptional properties for organizing supramolecular synthetic nanomaterials and com-
prise a valuable tool for the development of biotechnology devices among which are biosensors,
diagnostic kits, or bioseparation devices (Sessler and Jayawickramarajah 2005; Sivakova and
Rowan 2005; He and He 2009). Polysaccharides and carbohydrates are biomolecules involved in
many biological functions such as storage and transport of energy (starch, glycogen), generation
of structure and support (cellulose, chitin, chitosan), control of osmotic pressure in connective
tissue (proteoglycans), control of lubricity and cushion in the extracellular matrix (glycosami-
noglycans), and control of blood coagulation (heparin) (Yoshida 2001). The cell membrane has
carbohydrates, glycoproteins, and glycolipids, involved in biological processes as cell growth
phenomena, inflammatory processes, or different specific molecular recognition processes that
modulate cell adhesion and recognition mechanisms for toxins and viruses (Dwek 1996; Yoshida
et al. 1999). Given the great diversity of biological functions in which carbohydrates are involved,
and the plasticity of its chemical structure, the development of carbohydrate-containing syn-
thetic polymers has grown rapidly in order to implement the biofunctionality of these natu-
ral macromolecules in biomedical, pharmaceutical, and cosmetic applications (Ladmiral et al.
2004). Glycoconjugates combining a synthetic polymer backbone and pendent mono- or oligo-
saccharide segment in the side chain of each monomer repeat unit have been developed with the
aim of incorporating the lubricating, cushioning, or anti-inflammatory properties of native car-
bohydrates (Yoshida 2001; Ladmiral et al. 2004). Maltoheptose polymeric systems containing
units such as side-chains based on poly(methyl methacrylate), which posses anti-HIV activity
and prevent blood coagulation (Yoshida et al. 1999), and polymers containing statistical distri-
butions of d-glucaric residues, which inhibit the activity of β-d-glucuronidase (Hashimoto et al.
2006), are also described. Likewise, systems that mimic the native properties of proteoglycans
have been developed by grafting sugar-containing methacrylates from each repeat unit of a
poly(methyl acrylate) backbone (Muthukrishnan et al. 2005). Among the polymer–carbohydrate
conjugates, the systems with heparin need to be highlighted. Heparin is a sulfated polysaccha-
ride that participates in various physiological and pathophysiological processes resulting from
its interaction with heparin-binding proteins. The result of the immobilization of heparin on the
material surface is characterized by an improvement in the in vitro and in vivo biocompatibility
(Tanzi 2005) derived from the catalytic effects of heparin on antithrombin III and the accompa-
nying forces of the thrombin complexes (Sánchez et al. 1997), the heparin-induced reduction in
the selective adsorption of other plasma proteins, and the contribution to the maintenance of the
native structural state of these proteins (Wendel et al. 1999; Wendel and Ziemer 1999).
Finally, it is noteworthy to mention that those biological entities with an extremely high morpho-
logical and functional complexity such as viruses, bacteria, and cells are difficult to be explored.
Their complexity hinders the design and characterization of bioconjugated systems, since it is dif-
ficult to predict how they will interact with biological systems. However, several research lines
are aimed at the functionalization of polymers using these types of highly organized biological
structures (Raja et al. 2003; Croyle et al. 2004; Prescher et al. 2004; Scott and Chen 2004; Lee et al.
2005; Lee et al. 2006) by applying in most cases PEO or PEG coatings. The main property that
this modification provides to the system is the reduction of the immunogenic response, because the
polymeric coating inhibits the adsorption of proteins that triggers the immune response cascade,
when viruses, bacteria, or exogenous cells penetrate into the host organism. Among the works
that illustrate this application, modified retroviruses or adenoviruses with a modified immunogenic
activity (Raja et al. 2003; Croyle et al. 2004; Lee et al. 2005) are used as delivery vectors in gene
therapy (Verma and Somia 1997) after replacing their genetic material by therapeutic genes. Living
cells have also been used with this type of coating, in order to reduce the immune response in blood
transfusion or tissue transplantation (Scott and Chen 2004; Lee et al. 2006).
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16 Electrospinning for
Regenerative Medicine
Toby D. Brown, Cedryck Vaquette,
Dietmar W. Hutmacher, and Paul D. Dalton
CONTENTS
16.1 Introduction...........................................................................................................................540
16.2 Fundamentals of Electrospinning.......................................................................................... 541
16.3 Polymers Used for Electrospinning in Regenerative Medicine............................................. 542
16.3.1 Biologically Derived Polymers.................................................................................. 542
16.3.2 Synthetic Polymers.................................................................................................... 543
16.4 Electrospinning Parameters...................................................................................................544
16.4.1 Polymeric Parameters................................................................................................544
16.4.1.1 Solution Viscosity.......................................................................................544
16.4.1.2 Surface Tension........................................................................................... 545
16.4.1.3 Solvent Conductivity................................................................................... 545
16.4.2 Instrument Parameters...............................................................................................546
16.4.2.1 Flow Rate....................................................................................................546
16.4.2.2 Applied Voltage..........................................................................................546
16.4.2.3 Spinneret Diameter..................................................................................... 547
16.4.2.4 Temperature................................................................................................ 547
16.4.2.5 Collection Distance..................................................................................... 547
16.4.2.6 Humidity..................................................................................................... 548
16.4.3 Instrument Configuration.......................................................................................... 548
16.4.3.1 Single Collection Systems........................................................................... 549
16.4.3.2 Dual Collector Systems.............................................................................. 550
16.4.3.3 Structured Electroconductive Collectors.................................................... 550
16.4.3.4 Rotating Collectors..................................................................................... 551
16.4.3.5 Combining Electric Field Manipulation with a Rotating Collector............ 553
16.4.3.6 Electrospinning Writing............................................................................. 553
16.5 Cell-Invasive Electrospun Materials...................................................................................... 554
16.5.1 Hydrospinning........................................................................................................... 554
16.5.2 Electrospinning onto Ice Crystals............................................................................. 555
16.5.3 Simultaneous Electrospinning and Electrospraying.................................................. 555
16.5.4 Bimodal Electrospun Scaffolds................................................................................. 555
16.5.5 Combining Conventional Technologies with Electrospinning.................................. 555
16.5.6 Electrospinning Small Diameter Microfibers............................................................ 557
16.6 Growth Factor Delivery from Electrospun Materials............................................................ 557
16.7 Surface Modification of Electrospun Fibers.......................................................................... 558
16.7.1 Postprocessing Approaches....................................................................................... 558
16.7.1.1 Adsorbing Proteins onto Electrospun Fibers.............................................. 558
16.7.1.2 Chemical Binding Bioactive Functionalities.............................................. 558
16.7.2 Protein Resistant Biofunctionalization...................................................................... 558
539
16.8 In Vitro Studies of Electrospun Materials........................................................................... 559

16.8.1 Fibroblasts............................................................................................................. 559
16.8.2 Epithelial Cells...................................................................................................... 561
16.8.3 Endothelial Cells...................................................................................................562
16.8.4 Smooth Muscle Cells............................................................................................. 563
16.8.5 Cardiomyocytes, Cardiac Stem Cells, and Cardiac Cell Lines............................. 565
16.8.6 Mesenchymal Stem Cells...................................................................................... 565
16.8.7 Osteoblasts............................................................................................................. 567
16.8.8 Chondrocytes......................................................................................................... 567
16.8.9 Neurons and Glia................................................................................................... 567
16.8.10 Ligament Cells...................................................................................................... 570
16.8.11 Keratinocytes........................................................................................................ 573
16.8.12 Other Cells............................................................................................................. 573
16.9 In Vivo Results of Electrospun Materials............................................................................ 574
16.9.1 Biocompatibility and In Vivo Cell Penetration..................................................... 574
16.9.2 Neural Tissue Engineering.................................................................................... 575
16.9.3 Bone Tissue Engineering....................................................................................... 576
16.9.4 Cartilage Tissue Engineering................................................................................ 576
16.9.5 Vascular Tissue Engineering�� 577
16.10 Summary.............................................................................................................................. 577
References....................................................................................................................................... 578
16.1 INTRODUCTION
Regenerative medicine is an interdisciplinary field combining knowledge of cell biology, materials sci-
ence, and medicine with the aim to create constructs capable of healing or replacing lost and damaged
tissue due to sickness or accidents, eliminating the need for donated tissue. For example, the effective
healing of critical size bone defects, as a consequence of injury or disease, is an increasing problem in an
aging population. The majority of current treatment strategies involve autografts, where bone is extracted
from another part of the patient’s body, usually from the iliac crest or the fibula. Although this approach
is often successful, there are issues associated with infections and hematomas due to the creation of
a secondary injury at the donor site, while the amount of bone available is relatively scarce. Another
approach involves using allografts; however, donated bone introduces the risk of transferring diseases
between the donor and host, as well as increases the risk of infections and donor site rejection. Tissue
engineering (TE) offers the possibility to avoid these problems by incorporating three-dimensional (3D)
constructs, or scaffolds, with osteoinductive and osteoconductive properties to replace the auto/allograft.
Scaffold-based TE concepts often involve the combination of viable cells, biomolecules, and a struc-
tural scaffold combined into a tissue-engineered construct (TEC) to promote repair or regeneration [1].
The TEC is intended to support cell migration, growth, and differentiation and guide tissue development
and organization into a mature and healthy state. Preferably, the scaffold should be absorbed by the sur-
rounding tissue at the same rate as new tissue is formed so that by the time it breaks down the newly
formed tissue will take over mechanical loads. Because this field is still in its infancy, the requirements
for an ideal scaffold are complex and still not clear and may vary depending on specific tissue types and
applications but involve consideration of factors including architecture, structural mechanics, surface
properties, degradation properties and by-products, and the changes of these factors with time. However,
it is widely accepted that scaffolds in TECs have minimum essential requirements. They should
1. Provide sufficient initial mechanical strength and stiffness to maintain a degree of mechan-
ical function of the diseased or damaged tissue
2. Maintain sufficient structural integrity during the tissue growth and remodeling process in
order to achieve stable biomechanical conditions and vascularization
Electrospinning for Regenerative Medicine 541
3. Have a scaffold architecture that allows for initial cell attachment and subsequent migra-
tion into and through the matrix, mass transfer of nutrients and metabolites, and provide
sufficient space for development and later remodeling of organized tissues
4. Have degradation and resorption kinetics based on the relationships between required
mechanical properties, mass loss, and tissue development
5. Possess optimized surface properties for the attachment and migration of cell types
depending on the desired tissue. Furthermore, the gross size and shape may be customized
for a particular application and individual patient [2]
In TE, cellular regeneration requires scaffold support systems to guide the growth of cells and stimulate
the development of native extracellular matrix (ECM). Cell proliferation and adhesion are dependent
on the specifications of the scaffolds, especially upon the porosity and interconnectivity. Scaffolds for
TE are obtained through a variety of fabrication techniques, described elsewhere, each with advantages
and disadvantages [3]. These include rapid prototyping, 3D printing; selective laser sintering; extrusion/
direct writing; inkjet or organ printing; solid free-form fabrication; and stereo-lithography. However,
these techniques have limits to the degree of miniaturization possible. The latest trend in TE has been
to use solution electrospun scaffolds, which offer fibers with diameters comparable to those of the
fibrils typically found in tissue ECM, rather than other methods for in vitro studies. Electrospinning is
a technique that fabricates continuous nano- and microscale filaments with potential for regenerative
medicine, in particular for growth factor delivery and TE. Even though the production of filamentous
material for such biomedical applications has been used for many decades, electrospinning was only
recently proposed for TE (in 2002) [4,5] and growth factor delivery (2005) [6,7]. Electrospun materi-
als (also described as meshes) have widely been proposed as TE scaffolds, despite the clear evidence
that many of these materials do not form a cell-invasive structure, which is a major criterion for a TE
scaffold. Exceptions to this include specific strategies that introduce porosity into the electrospun mesh,
such as co-electrospraying [8,9], electrospinning onto ice crystals [10,11], and the formation of bimodal
scaffolds, which deliberately contain differently scaled fibers [12,13]. In comparison, growth factor
delivery from these meshes does not necessarily require cell penetration and has been used in surgi-
cal paradigms. In this review, we focus on the approaches to make and modify electrospun scaffolds,
rendering the material suitable for applications in regenerative medicine.
16.2 FUNDAMENTALS OF ELECTROSPINNING

There are numerous configuration permutations for electrospinning, but a schematic of the most
common electrospinning design—using a single collector—is shown in Figure 16.1. In electros-
pinning, an electrical potential difference is generated between a liquid and a collector, which is
Syringe pump
High voltage (HV)
FIGURE 16.1 (See companion CD for color figure.) Schematic of a typical electrospinning setup.
achieved by applying high voltage to either the liquid [5,14], collector [13,15], or both [16]. A spin-
neret (small orifice or flat-tipped needle) may be used; however, nonspinneret “nozzle-less” systems
have also been adopted [17,18]. Typically, as shown in Figure 16.1, a (positive or negative) voltage
is applied to a polymer solution or melt that is pumped through a spinneret facing a collector at a
different electrical potential. As the polymer droplet emerging from the spinneret becomes charged,
electrostatic repulsion counteracts the surface tension and the droplet is stretched. Upon reaching
a critical voltage, the surface tension of the polymeric fluid is overcome by the charge buildup,
and the droplet elongates into a (Taylor) cone, where a continuous jet is ejected and drawn by the
electrostatic force toward the collector. The potential difference between the spinneret and collector
typically ranges from 10 to 20 kV, although voltages as low as 0.5 kV [19] and as high as 130 kV [20]
have been reported.
The ejected electrospinning polymeric jet, which normally consists of a stable region and an
unstable zone (usually called bending or whipping zone), has been subject to significant math-
ematical analysis. If there are sufficient molecular entanglements in the polymer, the jet does not
break up into droplets due to Raleigh instabilities. Initially, the electrified jet is stable and travels
directly toward the collector. The surface charge density of the liquid (which caused the ejection of
the jet from the droplet) again increases with decreasing distance to the collector and “twists” the
jet, resulting in bending instabilities. In this zone, the instabilities in the jet’s path cause it to spiral
or “whip,” where it rapidly accelerates laterally to the flight path in a conical envelope, leading to
further stretching. As the electrified jet passes through the air, the solvent evaporates or the molten
polymer cools, depending on whether electrospinning of a polymer solution or melt is occurring,
and the jet undergoes magnitudes (up to 5 orders) in diameter reduction by the time it reaches the
collector. Electrospinning can be performed with the spinneret(s) and collector(s) configured so
that the path of the jet is either vertical or horizontal, since the effects of aerodynamics and gravity
are minimal compared to those of the electrostatic forces involved [21]. The path of the polymer
solution jet, in particular, is extremely dynamic, and the fiber collection is rapid. Such high speeds,
plus long spiraled traveling distances, make accurately controlled deposition of the electrospun fiber
technically challenging. Electrospinning commonly results in a nonwoven mat of fibers, although
other collection techniques shown here are expanding the morphological nature of the scaffolds.
A variation of electrospinning is melt electrospinning [22], which avoids issues such as toxicity
and solvent removal associated with solution electrospinning. Melt electrospinning behaves differ-
ently to solution electrospinning, in that the initial stable region of the jet is relatively much longer
and thus there is less lateral movement of the jet [23] resulting in less reduction in the diameter of
the jet. However, the larger micron scale fibers that typify melt electrospinning may offer the ability
to fabricate thicker structures with greater pore size and porosity, promoting more cell invasiveness.
Advances in how melt electrospun fibers can be collected may provide patterned aligned fibrous sub-
strates useful for building complex 3D structures that are of interest in applications such as TE [22,24].
16.3 POLYMERS USED FOR ELECTROSPINNING IN REGENERATIVE MEDICINE

There are literally hundreds of different polymers that have been electrospun, and extensive lists
of these polymers can be found in other articles [25–28]. Tables 16.1 through 16.3 provide a selec-
tion of polymers chosen for electrospinning applications in regenerative medicine. Polymers can
be categorized into two distinct classes—those derived from biological sources and those that are
synthetically produced.
16.3.1 Biologically Derived Polymers

It is understandable that when trying to replace ECM fibrils within the body, the most obvi-
ous polymers to use are those that constitute the natural ECM. Such biologically derived
polymers (Table 16.1) provide biological signals to cells and are typically degradable materials.
TABLE 16.1
Selected Biologically Derived Polymers
Polymer Reference Polymer Reference
Collagen [5,29,32] Hyaluronic acid [33,34]
Gelatin [35,36] Cellulose [37]
Silk [31,38] Laminin [39,40]
Chitosan [41] Fibronectin [42,43]
Fibrinogen [44,45] Chitosan/agarose [46]
TABLE 16.2
Selected Synthetic Polymers
PLGA [13,48,49] PLLA [50,51]
PCL [52] PLLA-CL [53,54]
PS [55,56] PEG-PCL [57,58]
PU [8,59–61] PEO [62,63]
PNIPAM [64,65] sP(EO-stat-PO)-PLGA [66]
TABLE 16.3
Selected Hybrid Electrospun Systems
Collagen/PCL [67] Silk/PEO [68]
Gelatin/PLGA [69] HAp/PLLA [70]
Chitosan/PVA [71] Hap/Collagen/PVA [72]
However, the process of dissolving the polymer into a solvent can create problems, particularly
denaturation. For example, collagen can be denatured to gelatin during electrospinning, due to
both the solvent used (often 1,1,1,3,3,3-hexa-fluoro-2-propanol [HFIP]) and the high voltage
applied [29]. Silk fibroin is also used; however, it requires treatment in order to dissolve into a
solution capable of being electrospun. A second issue with silk is also a generic issue with the
use of aqueous solutions: a degree of post-treatment is needed after generating the electrospun
fibers so that they do not redissolve upon placement in aqueous media [27,30,31]. Biologically
derived polymers have also been blended with synthetic polymers to improve the processing
ability of the fiber.
Numerous studies that blend different biologically derived polymers, such as chitosan and
agarose [46], gelatin and hyaluronic acid, are reported [47]. In addition to the selected articles
listed in Table 16.1, in-depth reviews on the electrospinning of biologically derived polymers are
available [27,28].
16.3.2 Synthetic Polymers
The number of different synthetic polymers electrospun (Table 16.2) is definitely greater than that of
those derived from biological sources. Synthesized polymers have widely been electrospun due to the
benefits of reproducibility and the ability to tailor important material properties such as molecular
weight, which appears to facilitate improved electrospinning than when using biologically derived
polymers. There are many specialized reviews on various polymers, with poly(caprolactone) (PCL)
and polyethylene glycol (PEG) being two of the most studied polymers for regenerative medicine.
The lack of biological signals provided by synthetic polymers (to trigger and control cellular
responses) requires the adsorption of proteins onto the surface, or a surface-engineered strategy:
one common approach is to blend biologically derived polymers into a synthetic polymer solution
in order to incorporate protein into the fibers. However, denaturation of the protein is still an issue.
Furthermore, the two different polymers often phase separate into separate domains because of their
intrinsic properties. For example, collagen (hydrophilic) and PCL (hydrophobic) are immiscible and
will phase segregate. Table 16.3 provides a list of some commonly blended “hybrid” polymers.
16.4 ELECTROSPINNING PARAMETERS

It is widely appreciated that three broad factors influence the electrospinning process, and thus
the morphology of the resulting fibers: the properties of the polymeric fluid used; the instrument
conditions (e.g., voltage and flow rate); and the configuration of the instrument. The most widely
reported characteristic of electrospun material is the diameter of the fibers. The diameter is known
to increase with increasing polymer concentration and also decrease with voltage applied. While
the diameter is an important aspect and is discussed later, the morphology and structure of the
electrospun materials significantly have impacts on other important properties such as pore inter-
connectivity and cell invasiveness. Due to the large number of parameters that can be altered, and
the interdependency of most of these parameters, studying the effects of the processing conditions
on scaffold morphology can become excessively difficult. However, through careful experimental
design it is possible to deduce definitive relationships.
16.4.1 Polymeric Parameters

When electrospinning polymeric solutions, there are a number of parameters that can be altered to
generate different types of structures or different fiber diameters.
16.4.1.1 Solution Viscosity

One of the most important electrospinning parameters is the viscosity of the polymer solution:
i.e., the degree of macromolecular entanglements in the polymer that prevents breakup of the liquid
jet into droplets. One way by which viscosity can be controlled is by adjusting the concentration of
the polymer solution. Chain entanglement can be determined by the molecular weight used and also
depends on the polymer type (linear or branched). There is a strong relationship between polymer
concentration and fiber diameter, which is well described for a large number of polymers in a large
number of studies. Usually highly concentrated solutions produce relatively thick and bead-free
fibers. Mckee et al. [73] extensively studied the electrospinnability and resultant fiber morphology of
polymer solutions with respect to their rheological properties. The dependence of polymer viscosity
on solution concentration can be described by identifying four distinct concentration regimes: the
dilute, semidilute unentangled, semidilute entangled, and the concentrated. The boundary between
the semidilute un/entangled regimes is named entanglement concentration Ce and corresponds to
the point at which there is significant overlap of the polymer chain to constrain the chain motion
causing entanglement and couplings [73]. McKee observed that for a c oncentration below Ce only
polymer beads were obtained. Whereas for concentrations between Ce and 2–2.5 times Ce, beaded
fibers form, for concentrations above 2.5 times Ce, uniform bead-free fibers occur. This work showed
that there exists a minimum polymer concentration required for solution electrospinning. A fiber
diameter prediction model was proposed where the diameter universally scales with the normalized
concentration (C/Ce)2.6, where C is the c oncentration of the polymer solution and Ce the entangle-
ment concentration [73].
Typically, electrospinning of natural polymers in aqueous solutions is challenging due to a num-

ber of factors including polymer chain rigidity (which reduces the degree of polymer chain entan-
glements) and high surface tension in the solution. Increasing the entanglements has been shown to
be an efficient way to facilitate the aqueous electrospinning of alginate, a natural polymer [74]: in
this strategy better electrospinnability was achieved by using glycerol, a strong polar solvent, which
is believed to disrupt the strong intramolecular structure of the polymer. Glycerol also forms new
hydrogen bonds with the polymer chains and improves their flexibility. As a result, the viscosity of
the solution usually increases due to increased entanglements, whereas surface tension decreases,
enhancing the electrospinnability of these polymers. A similar effect was observed when electros-
pinning hyaluronic acid in a mixture of water, formic acid, and dimethylformamide [75]. Here again
the rigid inner structure of the natural polymer was disrupted by the formic acid, which changed the
chain conformation from alpha helix to a coil conformation improving chain flexibility, increasing
entanglements and therefore electrospinnability.
16.4.1.2 Surface Tension

During the electrospinning process, a jet forms once the electrostatic forces overcome the sur-
face tension in the droplet emerging from the spinneret. Therefore, the surface tension is a critical
parameter and in polymer solutions it can be controlled by blending solvents [76–78], adding a sur-
factant or salt [79], or varying the temperature [80]. It is generally assumed that high surface tension
solutions are difficult to electrospin and require higher voltage for the initiation of the jet. Because
it tends to minimize the surface area per mass of a fluid, resulting in a spherical shape, surface ten-
sion is also thought to participate in the formation of beads on fibers when electrospinning relatively
low or moderately viscous solutions. Generally, reduced surface tension produces bead-free smooth
fibers [79].
16.4.1.3 Solvent Conductivity

The forces that tend to elongate the electrospinning jet are created by a buildup of charge within
and on the surface of the polymer droplet. For polymer solutions, the net charge carried by the jet
is directly bound to the conductivity and the mobility of these charges in the solution. Generally,
greater stretching of the jet, and thus thinner fibers, is to be expected when the net charge of the jet
is increased, which is facilitated by an increased conductivity. Furthermore, in most cases increas-
ing the conductivity of the solution enhances the bending instability (because it enhances the charge
repulsion). Therefore, the jet lengthens and is subject to more whipping, causing more stretching and
leading to further reduction in fiber diameter [81].The significance of this electrospinning parameter
is demonstrated by the fact that changes in the conductivity of the same solvent can lead to discrep-
ancies in fiber diameter and scaffold morphology from one experiment to another [55]. For the same
conditions and using the same solvent but from different suppliers, Uyar et al. [55] obtained both
beaded and smooth fibers. They correlated these varying fiber morphologies to the differences in
the solution conductivities, which varied from 0.7 to 7 μS/cm. The highest conductivity produced
the smallest fibers.
The solvent or combination of solvents that are part of electrospun fiber preparation affects the
fiber structure, which in turn affects cell behavior [82]. Solvent mixtures have widely been used to
alter, generally to increase, the conductivity of the electrospun polymer solution. This approach is
valid as long as the viscosity of the solution does not vary too greatly; otherwise, conclusive obser-
vations on the effects of conductivity may be difficult. In some cases, adding a highly conductive
solvent without a significant variation in the viscosity of the solution is possible [76]. When dimeth-
ylformamide (DMF) is added to methylene chloride, the viscosity of a 13 wt% PCL solution does
not change, but the conductivity increases up to 10-fold, responsible for a sharp decrease in fiber
diameter [76]. The stability of the process may also benefit from the addition of a highly conductive
solvent. For example, chloroform or dichloromethane solutions can have an instable Taylor cone,
and consequent spinneret clogging can require the operator to clean it frequently. However, upon
addition of DMF to the solution the process is stabilized and can be run for several hours without
the need to clean the spinneret.
Another way to enhance the conductivity of a polymer solution is to add salt. A substantial
amount of literature on this subject has generally shown that thinner fibers are produced when the
salt concentration is increased [81,83,84]. It has also been observed that increasing the salt concen-
tration tends to reduce bead formation [85]. Interestingly, while comparing several types of salt,
Zong et al. [83] observed that ions with smaller atomic radii produce thinner fibers. This effect was
attributed to the higher charge density of smaller ions, providing higher mobility in the solution and
therefore producing stronger elongational forces.
Although an increase in polymer solution conductivity generally leads to the production of thin-
ner electrospun fibers, the opposite trend has also been reported [80]. This may be due to additional
mass flow created by higher elongational forces.
16.4.2 Instrument Parameters
16.4.2.1 Flow Rate
The effect of mass delivery, or flow rate, on the nature of electrospun scaffolds has been studied
extensively [24,83,86–88]. It is widely accepted that increasing the flow rate produces larger fibers:
as more material is extruded through the spinneret per unit time, the volume of the Taylor cone
increases for the same collection distance. This leads not only to larger diameter fibers but also to
the deposition of wet fibers, which may fuse together due to a greater amount of residual solvent.
Therefore, more fusion at the points of contact between solution electrospun fibers is generally
expected for higher feed rates. This can significantly affect the mechanical properties of the resul-
tant meshes (high fusion density results in tougher scaffolds) and affect the interconnectivity and
therefore the cell invasiveness of the construct. For given conditions, increasing the flow rate can
induce instability into the Taylor cone. This usually happens when the amount of polymer solution
exceeds the amount of polymer that can be drawn by the electrostatic forces. As a result, break-
ing up of the jet can be observed, and fibers with large beads can also be formed [89]. Therefore,
increasing the voltage may be necessary to maintain a stable jet when the flow rate is increased.
16.4.2.2 Applied Voltage

Since an electrical field is inherently required for electrospinning to be possible, the applied voltage
plays a major role in the electrospinning process. In most cases, a direct-current (DC) apparatus is
utilized to produce the high voltage. However, alternative-current (AC) setups are also possible. The
main advantage of using AC voltage resides in the ability to electrospin or electrospray onto non-
conductive collectors [90]. Using AC voltage, the alternating polarity induces a neutralizing effect,
leading to the reduction of net charge on the jet. Consequently, compared to using DC voltage, the
electrostatic forces that produce jet instability and thus the whipping instability are significantly
reduced. As a result, fibers obtained using AC voltage are generally larger than those fabricated
using DC voltage [90].
Several phenomenological observations can be made when the DC voltage is increased during
electrospinning. Firstly, the shape of the Taylor cone will change from a droplet to a spindle. Several
studies agree that there is a reduction in the volume of the Taylor cone with increasing voltage (for a
given flow rate) [83,91]. As the voltage is increased, the jet acceleration is greater and more volume
is drawn from the Taylor cone; therefore, the cone volume and also the jet diameter is reduced [83].
Secondly, increasing the voltage may cause the formation of multiple jets from the Taylor cone [92]:
higher voltage favors the formation of thin secondary jets, which may broaden the fiber diam-
eter distribution [81] or produce bimodal fiber distributions [93]. Usually, when secondary jets are
not formed the fiber diameter distribution appears to narrow [94–96]. When the voltage is further
increased, the Taylor cone can even recede into the spinneret. Thirdly, when the voltage becomes
too high corona discharge can be observed, perturbing the deposition [97].
It is generally accepted that an increase in electrospinning voltage produces a decrease in fiber

diameter [86,94,95]: as the jet is increasingly accelerated it is further stretched. Katti et al. observed
that after an initial sharp decrease in fiber diameter, a constant diameter is obtained when the volt-
age is further increased [95]. However, several other studies report no change or increase in the fiber
diameter at higher voltages. Therefore, there is no definitive consensus on the effect of the applied
voltage; which appears to be dependent on the solution used.
At each voltage extreme (too low or too high voltage), bead formation has frequently been
reported. However, once beads appear during fiber deposition, the higher the voltage the higher the
bead density [86,91,98]. Lee et al. observed that the morphology of beads from a low viscosity solu-
tion could also be controlled by the applied voltage, where they adopted a more pronounced spindle
shape at higher voltages [99].
16.4.2.3 Spinneret Diameter

The diameter of the spinneret through which the polymer is delivered during electrospinning plays
a significant role in controlling fiber diameter as it mostly determines the initial size of the Taylor
cone. Several studies have shown that smaller spinneret diameters produce smaller fibers [94,95].
A smaller diameter orifice also increases the surface tension of the emerging droplet and thus the
critical voltage required to initiate the Taylor cone. It is also believed to reduce bead formation and
clogging of the spinneret [94], while a smaller Taylor cone provides less surface area in contact with
the air for solvent evaporation. However, it can prove difficult to extrude highly viscous solutions
through small diameter orifices, and the buildup of back pressure in syringe-based systems may
affect the feed rate and therefore consistent fiber deposition.
16.4.2.4 Temperature
The control over the solution/melt temperature can be obtained by developing a jacket-type heat
exchanger in which water [80,100,101] or silicone oil [102] is circulated. Other technical solutions
can be utilized such as glass fiber heating tape or infrared emitter heating systems [103]. Increasing
the temperature generally decreases the fiber diameter since the viscosity of the solution is greatly
reduced [92]. Wang et al. systematically studied the effect of elevated temperature over the proper-
ties of polyacrylonitrile solution in DMF and on the morphology of the resulting fibers [102]. This
study found, as expected, that the viscosity of the solution decreased, but also that the entanglement
density was not significantly affected by the elevated temperature. In addition, the surface tension
of the solution was found to decrease with temperature, whereas the opposite trend was observed
for the conductivity, which increased with temperature. The later phenomenon was attributed to
the higher mobility of the molecules caused by the increased temperature. The reduction of the
viscosity, surface tension, and the increase in the conductivity lead to smaller fiber diameters.
Besides reducing the fiber diameter, elevated temperatures have other implications for the pro-
cess of electrospinning. Demir et al. observed improved uniformity in fiber diameter distribution
when electrospinning a polyurethane solution at 70°C compared with room temperature [92]. Wang
et al. also reported the increase in the Taylor cone volume, which resulted in an increase in the volt-
age required to electrospin the polymer solution [102]; In Wang’s and the work by Givens et al. [103],
a high electrospinning temperature was responsible for a slight decrease in polymer crystallinity.
Electrospinning at elevated temperatures can also be used to dissolve polymers that are insoluble
at room temperature: linear low density polyethylene (LLDPE) was successfully electrospun at
105°C–110°C using p-xylene as a solvent [103]. This strategy can also be used to improve the incor-
poration efficacy of poorly soluble drugs into fibrous polymer matrices, because the solubility of
most compounds is improved at high temperatures [104].
16.4.2.5 Collection Distance

Varying the collection distance, also known as “tip-target distance” or “working distance,”
strongly impacts the strength of the electrical field and the time of flight of the polymer jet.
Although it is generally accepted that a collection distance that is too short will create adverse
effects, such as excessive fiber fusion caused by the low solvent evaporation, there is no global
consensus on the effects of collection distance on fiber morphology. Several studies have shown
very small or insignificant changes in fiber diameter and morphology when increasing the col-
lection distance [81,105,106], while others have observed significant changes in the fiber diam-
eter [107]. However, the collection distance directly affects the volume of the Taylor cone and
plays a significant role in the initial diameter of the jet [105]: small distances increase the field
strength and tend to reduce the volume of the Taylor cone. Smaller distances may also lead to
greater acceleration of the jet, but not necessarily greater stretching, where induced bead for-
mation has been reported [86]. Longer distances reduce the field strength and have also been
associated with the formation of beads or increased bead diameter [108]. Collection distance
appears to have less of an effect on fiber diameter and morphology than deposition area. As the
collector is moved further away from the spinneret, the base of the cone formed by whipping
becomes larger, resulting in the deposition of the same quantity of material over a larger surface
area. Therefore, thinner scaffolds are obtained for the same deposition time. When the working
distance is increased, the time of flight is also increased. Consequently, this allows more sol-
vent to evaporate and reduces the fusion density between the fibers. This can have a significant
impact upon the mechanical properties of the scaffolds, as more fiber fusion increases the ten-
sile strength of the electrospun mat. Therefore, varying the collection distance could also be a
means to control the macroscopic mechanical properties of the resultant scaffolds. Although the
deposition of melt electrospun fibers is relatively more focused compared to solution electrospin-
ning, a similar effect is experienced where longer collection distances result in less focus of the
deposited fibers [24].
16.4.2.6 Humidity
Humidity is not always controlled during the electrospinning process; however, it seems to play
a significant role in the morphology of the fibers obtained. There are several studies that demon-
strated the possibility of tailoring the surface morphology of the fiber by controlling the ambient
humidity of the electrospinning chamber. It has been shown that increasing the relative humidity
leads to the formation of spherical “pores” on the fiber surface, and it is generally accepted that the
higher the relative humidity, the larger the pore diameter until the pores fuse together [109,110]. The
formation of such pores is attributed to the condensation of water molecules onto the fibers during
deposition. As a consequence, the specific surface area is greatly increased by the presence of these
pores on the fibers [110]. Relative humidity can have significant impact not only on the superficial
pattern of a single fiber but also on the gross architecture of an entire scaffold. When electrospin-
ning an aqueous-based solution with various humidity contents, it has been observed that larger
diameter fibers are formed at low humidity percentages, due to the higher evaporation rate of the
water in the “dry” environment, resulting in less drawing of the jet by the applied voltage. Whereas
the fiber diameter was observed to decrease when the relative humidity was increased, up to a point
where beads were formed [111].
16.4.3 Instrument Configuration
This section focuses on the utilization of different configurations to generate discrete structures
within an electrospun matrix. The variation of instruments described in the literature is far
greater than presented in this chapter. However, the focus here is on the ability to discretely
position and place fibers in specific locations and patterns. Central to many fibrous systems are
woven materials, and therefore creating woven structures or threads using electrospinning is an
interesting and relevant component of TE. Initially, static electrospinning instrument configura-
tions are discussed, followed by dynamic collection systems that result in structured electros-
pun substrates.
16.4.3.1 Single Collection Systems

Using a single, solid collector is the most commonly used electrospinning configuration and results
in a nonwoven mesh that is non-cell invasive. It is usually the arrangement that most polymeric
materials are initially electrospun onto, since the nonwoven mesh is readily handled and manipu-
lated. In this configuration, the electric field between the spinneret and the collector is uniform
or symmetrical. Although in most cases electrospun fibers are deposited onto a solid collector
(Figure 16.2a and b), common liquids such as water can be used for fiber collection [112,113].
A straightforward method of collecting a continuous yarn from electrospun fibers by first deposit-
ing them onto a liquid medium (as shown in Figure 16.2c) was not demonstrated until 2005 by Smit
et al. and Khil et al. [114,115], even though Formhals had patented various continuous fiber yarn set-
ups in the 1930s [116,117]. This was an important technical breakthrough, because of the potential
for continuous yarns of electrospun nanofibers to be woven into textiles, with applications including
protective clothing, high performance fabrics, composites, and TE [118]. The method proposed
by Smit and colleagues [114] is worthwhile for further discussion. It involved the deposition of
(a) (b)
(c) (d)
FIGURE 16.2 (See companion CD for color figure.) Various collection systems for electrospinning.
(a) Single collector; (b) Patterned collector; (c) Collection onto water with rotating mandrel; and (d) Collection
across two parallel collectors.
randomly oriented electrospun fibers onto the surface of a water bath, which were then drawn to the
edge and collected on a rotating mandrel. This caused elongation of the mesh and alignment of the
fibers as they were drawn over the water. Surface tension caused the fibrous mesh to collapse into a
yarn when it was lifted off the surface of the water onto the rotating collector. As the resultant yarn
was rolled onto the mandrel at a rate of 3 m/min, more fibers were deposited on the water surface to
feed the drawing process. Electrospun poly(vinyl acetate), poly(vinylidene fluoride), and polyacrylo-
nitrile nanofiber yarns were fabricated using this method. Khil et al. employed a similar concept but
rotated the mandrel at 30 m/min and used a mixture of water and methanol [115]. However, no com-
ment was made on the difference between yarn collected on water or water/methanol. According to
Teo and Ramakrishna [118], the high surface tension provided by water may be favorable to other
liquids with lower surface tension because the electrospun fibers may become submerged, creating a
higher drag force as they are drawn to the rotating collector. More studies are required to determine
the influence of the liquid bath properties on the yarn.
16.4.3.2 Dual Collector Systems

An example of a dual collector system is schematically illustrated in Figure 16.2d while a photo-
graph in Figure 16.3, shows highly aligned fibers across the separation between two rings placed in
parallel, equidistant from the spinneret were deposited [119]. An important advantage of this type
of approach, which is also termed the “gap method of alignment,” is that the perimeters of the elec-
trode rings where the fibers deposit allow precise and consistent controlled positioning of the fiber
bundle (Figure 16.4) [120]. Such a gap method of alignment has been used to manufacture oriented
fibers for in vitro use, particularly for applications in the nervous system [16,67,121–123].
16.4.3.3 Structured Electroconductive Collectors

Methods to create patterned 2D and 3D electrospun structures using static electroconductive pat-
terned collectors have also been developed. However, the random formation of honeycombed
Spinneret
Electrospun fibers
1 cm
FIGURE 16.3 (See companion CD for color figure.) Oriented electrospun PCL fibers collected using the
gap method of alignment. The collectors are spaced 10 cm apart, and the oriented fibers that collect in between
are generated in approximately 30 s. (Previously unpublished photograph.)
+ 12 kV
Insulating tape
Steel blade
– 3 kV
x
FIGURE 16.4 Experimental setup for precise deposition of electrospun fibers over two known points.
(Reproduced from Teo, W.E. and Ramakrishna, S., Nanotechnology, 16, 1878, 2005.)
[63,124] and dimpled structures [125] using this approach has been observed, due to the buildup
of electrostatic charges on the deposited fibers and/or the collector, which prevent new deposition
directly onto the collector. Furthermore, it is currently reported that the fibers forming 3D structures
are very loosely packed and can be easily compressed: rendering them unsuitable for applications
where structural integrity is required [125]. Thus, the conditions required to form 3D structures
using this method are still unclear.
Patterned electro-conductive collectors, either in the form of ridges and indentations or
grids with pores in between, have been used to form patterned 2D solution electrospun meshes.
Ramakrishna et al. reported the use of a collector with conducting ridges and nonconducting
indentations where the electrospun fibers were randomly deposited according to time; however,
the resultant mesh resembled the pattern of the collector [125]. Aligned fibers formed on the con-
ducting ridges and randomly deposited fibers on the nonconducting regions. Zhang and Chang
also demonstrated that protrusions/patterns in the collectors are important parameters that may
greatly affect the structures of electrospun meshes (Figure 16.5) [126,127]. The density of uniaxi-
ally aligned fibers deposited between the protrusions on a collector was shown to be dependent
on the distance between two protrusions, decreasing with increasing distance between the protru-
sions. Woven structures were then generated through time-dependent control of the arrangement
of a patterned series of electroconductive protrusions: one pair of thin parallel-aligned protru-
sions, insulated from one another, was mechanically raised higher than the other pairs of protru-
sions. Fibers were drawn to deposit on the higher pair due to stronger electric attraction forming
a parallel-aligned fiber bundle between the protrusions. After a certain time, the first pair was
lowered and another pair oriented at 90° was raised and allowed to collect for a similar period.
The newly deposited fibers were also parallel but oriented perpendicular to the initial fiber bundle.
The process was repeated with additionally located protrusions at 90° to create woven fibrous
architectures [127].
16.4.3.4 Rotating Collectors

Several researchers have shown that it is possible to obtain aligned fibers by using a rotating
collector/mandrel [5,128,129]. Matthews et al. demonstrated the effect of the rotating speed
of a mandrel on the degree of solution electrospun collagen fiber alignment [5]. At a speed of
SICCA SEI 20.0 kV ×50 100 µm WD 10 mm 100 µm

(a) (b)
FIGURE 16.5 (a) Patterned electro-conductive collector and (b) resulting patterned electrospun mesh.
(Reproduced from Zhang, D.M. and Chang, J., Adv. Mater., 19, 3664, 2007.)
less than 500 rpm, a random mix of collagen fibers was collected. However, when the rotating
speed of the mandrel was increased to 4500 rpm (approximately 1.4 m/s at the surface of the
mandrel), the collagen fibers showed significant alignment along the axis of rotation. At such
a high rotational speed, the velocity of the electrospinning jet may be slower than the linear
velocity of the rotating mandrel. The reported average velocity range of the electrospinning jet
is from 2 m/s [130,131] to 186 m/s [114]. A separate study by Zussman et al. using a rotating disk
collector demonstrated that at high enough rotational speeds, necking of the electrospun fibers
occurs [132]. Therefore, the mandrel rotational speed directly influences fiber orientation as
well as the material properties of the engineered fibrous structure. In cases where disoriented
fibers are collected using a rotating mandrel at sufficient speed, residual charge accumulation
on the deposited fibers may interfere with the alignment of incoming fibers. According to Teo
and Ramakrishna, reducing this residual charge accumulation on the rotating mandrel as well as
reducing the chaotic path of the jet are possible ways to achieve greater fiber alignment, in solu-
tion electrospinning applications [120]. Kessick et al. used an AC high-voltage supply instead
of the typically used DC supply when solution electrospinning polyethylene oxide (PEO) [90]
onto a rotating mandrel to improve fiber alignment. Using an AC potential to charge the solution
may have a dual function: the electrospinning jet may consist of short segments of alternating
polarity, which significantly reduce its chaotic path and allows the fibers to be wound onto the
rotating mandrel with greater ease and alignment. Then, given the presence of both positive and
negative charges on the surface of the rotating mandrel, there may be a neutralizing effect over
the area of the fibers, thus minimizing residual charge accumulation. Another simple approach
to reduce charge accumulation is to apply the opposite polarity to the collector than to the spin-
neret, instead of grounding the collector [16].
Although the methods incorporating a rotating device described so far have all formed 2D
aligned fiber meshes, 3D conduits can be fabricated by depositing fibers onto smaller diameter
(<5 mm) collectors. For the construction of vascular grafts, Stitzel et al. electrospun a conduit
120 mm long and 1 mm thick using a polymer solution containing a mixture of collagen type I,
elastin, and poly(d,l-lactide-co-glycolide) on a circular mandrel of diameter 4.75 mm [133].
However, in this case the rotational speed was only 500 rpm to facilitate homogeneous deposi-
tion of random fibers. Longitudinal and circumferential mechanical testing showed no signifi-
cant difference between the two directions. Another advantage offered by the electrospinning
process when forming small diameter conduits for vascular grafts is the ease of spinning dif-
ferent materials to form layered composites [134]. This way, materials such as collagen, which
encourages cell proliferation and attachment but is mechanically weak, can be electrospun as
an inner layer, while artificial materials such as polymers with superior mechanical properties
can form an outer layer.
16.4.3.5 Combining Electric Field Manipulation with a Rotating Collector

The combination of electric field manipulation and a dynamic collector has been used to achieve
greatly ordered fiber assemblies. Theron et al. used a knife-edged rotating disk collector to take
advantage of the fiber alignment created by rotary motion as well as the convergence of electric
field lines toward the edge of the disk [135]. To collect the fibers, glass coverslips or any noncon-
ducting substrate can be attached around the edge of the discs used in TE studies [136]. Crossed
fiber arrays were obtained by Bhattarai et al. using a similar concept to the rotating disk collector,
but instead winding copper wire as an electrode on an insulated cylinder and then rotating it at an
optimum speed of ∼2000 rpm [137]. The size of the fiber bundle was controllable by varying the
diameter of the collector. Instead of using static electrodes placed in parallel, Katta et al. used a
rotating wire drum [138], whereas Sundaray et al. used a sharp pin as an electrode on a rotating
collector to focus deposition of the electrospinning jet and introduced lateral movement to the col-
lector to obtain cross-bar patterned fibers [131]. Instead of situating an electrode directly below
the spinneret to focus electrospun fiber deposition, Teo et al. incorporated a negatively charged
knife-edged electrode placed at an oblique angle to the spinneret [120]. The electrospinning jet was
observed to travel at the same angle as that of the diagonal electric field lines from the tip of the
spinneret to the knife edge. Diagonally aligned fibers were then collected on a rotating tube placed
in the path of the electrospinning jet. This enabled the fabrication of laminar composites with
aligned fibers in different orientations. While the combination of dynamic mechanical devices and
electrical field manipulation leads to parallel arrays and grids of electrospun fibers with improved
alignment, these methods do not provide sufficient control over the pitch width or the ability to cre-
ate complex patterns such as circles [139].
16.4.3.6 Electrospinning Writing

Currently, a limited number of writing approaches using electrospinning are available.
Buckling phenomena have been studied where electrically charged solution jets depositing
onto collectors moving laterally at a constant velocity produced buckling patterns with limited
controllability [140]. While in a different study, the bending and buckling of a molten polymer
jet was shown to be close to the collector [141]. There are processes that take advantage of the
initially stable region of the solution electrospinning jet, by significantly reducing the spinneret-
to-collector distance to below that at which bending instabilities develop along the jet. This
results in relatively focused fiber deposition. Then, matching the speed of a translating collector
to that of the electrospinning jet enables the ability to write with a continuous filament. Scanning
tip electrospinning [128] and near-field electrospinning [19,139,142] processes have demonstrated
relatively predictable control over the deposition of submicron fibers using an automated x–y
stage as the translating collector. Alternatively, a polymer melt jet will remain stable over rela-
tively large distances and is well directed toward the collector. Dalton and colleagues utilized
this phenomenon in combination with a translating collector, demonstrating the accurate pat-
terning and drawing of a cell adhesive scaffold with a PEG-b-PCL/PCL blended melt across the
surface of a microscope slide [24]. Recently Brown et al. demonstrated a direct writing process
using melt electrospun pure PCL, where complex patterns could be drawn with a continuous
filament. Layers of melt electrospun fibers were then consistently located on top of each other,
to build complex 3D structures up to 1 mm in thickness (Figure 16.6). This process also enabled
the fabrication of porous TE scaffolds, where control over the location of fiber placement was
demonstrated with micron scale resolution [143].
(a) (b)
(c) (d)
FIGURE 16.6 Electrospinning writing of a polymer melt. Photograph of Queensland University of

Technology logo “QUT” written on a microscope slide (a), showing stacking of fibers upon each other.
SEM image of stacked and interwoven fibers until the stacking becomes affected by either the height of the
sample or the uneven z-direction height, the latter caused by differences due to the interweaving of the fibers
(b and magnified in c). The underneath of this box-like structure is shown in (d). Scale bars for b, c, and d are
1 mm, 100 μm, and 1 mm, respectively. Unpublished images from the same study as reported in Ref. [143].
16.5 CELL-INVASIVE ELECTROSPUN MATERIALS

Solution electrospinning offers relatively consistent control over fiber diameters, generally in the sub-
micron range. However, due to the nature of the process, it is characterized by disorderly fiber deposi-
tion, which has limited its full potential. Despite this, numerous techniques have been developed and
are still evolving, which have improved the orderly placement of fibers, including mechanical devices
such as rotating collectors, liquid deposition methods, manipulation of the electric field, combining
electric field manipulation with a rotating collector, static patterned collectors, and translating col-
lectors. These approaches are important as they progress electrospinning toward true TE scaffold
manufacture. Presented here is a selection of techniques developed to produce cell-invasive scaffolds.
16.5.1 Hydrospinning
In this approach, water is used as a collection medium, and a layer of electrospun fibers forms on the
surface. The film of fibers is transferred to a microscope slide and the process repeated, generating
multiple layers [113]. The water is then removed with a vacuum and the resulting scaffolds contain
considerable volume, are both highly porous, and cell invasive. In this same study, by Levenberg
and colleagues, myoblasts were introduced into the process and successfully cultured with an equal
density throughout the electrospun scaffold.
16.5.2 Electrospinning onto Ice Crystals

In another study aimed at increasing the porosity of electrospun scaffolds, Simonet et al. electrospun
fibers directly onto ice crystals, as they grew off the surface of a rotating drum. The rotating drum was
hollow and filled with solid CO2, causing water vapor in the surrounding air to crystallize on the col-
lector [10]. The ice/fiber mixture was lyophilized, and the remaining scaffold was impressively large,
with interconnected pores. Extending on this process, the porous material was mechanically pulled and
transformed into a cotton wool-like material, which was then used as a TE scaffold in this form [11].
16.5.3 Simultaneous Electrospinning and Electrospraying

A similar concept involving building a conductive substrate up from the surface of a collector was
devised by researchers trying to manufacture TE constructs. However, instead of using ice crys-
tals, a layer of hydrogel was “grown” from the collector surface: gelatin containing smooth muscle
cells was electrosprayed concurrently while electrospinning polyurethane [8]. Similarly, Ekaputra
et al. electrosprayed a thiolated hyaluronic acid solution while electrospinning PCL/collagen onto a
collector [9]. Human fetal osteoblasts were then observed to migrate up to 200 μm into the scaffold
in a mixture of electrospun fibers and gel. In both these instances, a rotating collector was used and
the two spinnerets were oriented at 180° to each other.
16.5.4 Bimodal Electrospun Scaffolds

It is recognized that ultrafine electrospun fibers do not readily create a cell-invasive scaffold, because
they are too tightly packed and thus do not provide adequate pore size and porosity. However, com-
bining these fibers with a larger structure may allow the benefits of both “nano” and “micro” struc-
tures. This approach leads to the generation of scaffolds with high surface area due to the smaller
fibers (promoting cell attachment and assisting in higher seeding of the scaffold), as well as suitable
volume and pore sizes due to the microfibers. Gentsch et al. showed that solution electrospinning at
very low flow rates induced both small and large diameter electrospun materials from a single spin-
neret. In this instance, PCL was used in conjunction with Chinese hamster ovary (CHO-K1) cells
for infiltration experiments with improvements using a bimodal approach. Alternatively, simultane-
ous melt electrospinning (which produced micron-diameter fibers of 28.0 ± 2.6 μm) and solution
electrospinning (which produced submicron diameter fibers of 530 ± 240 nm) of PLGA were per-
formed onto a rotating mandrel, to form a thick cell-invasive bimodal scaffold [13].
16.5.5 Combining Conventional Technologies with Electrospinning

Electrospun fibers can be mixed with conventionally fabricated fibers to create a scaffold similar in
principle to a bimodal electrospun scaffold, where the ultrafine electrospun fibers are mechanically
supported by the larger structures. In one case, this was achieved by melt spinning poly(l-lactide-co-
epsilon-caprolactone) (PLCL) into a cylindrical structure for vascular TE [144]. In another notable
case, Kim et al. reported 3D extrusion combined with electrospinning to significantly increase poros-
ity (Figure 16.7a). In a different study, an automated scaffold assembly system was used to construct the
scaffold: a fused deposition modeling (FDM) process was interrupted between each layer to deposit
3D plotter
556
Electrospinning
Repetitive
process
Nanofiber
Hybrid Microfiber
structure
Direct polymer
Electrospinning
molt deposition
Hybrid scaffold

No
(a) nanofiber matrix
Power supply
1 Hybrid
nanofiber matrix
Heating nozzle
Syringe 4 Layered 1 Layered
Collection woodpile shaped nanofiber matrices
5 mm
microfiber scaffolds
drum
Syringe pump
Oil circulator
(c) (b)
FIGURE 16.7 (See companion CD for color figure.) (a) Schematic of a hybrid process combining 3D extrusion and electrospinning. (Reproduced from Kim, G. et al.,
Macromol. Rapid Commun., 29, 1577, 2008.) (b) Combining Electrospinning with a melt extrusion technique to create an electrospun carrier. (Reproduced from Park, S.H. et al.,
Acta Biomater., 4, 1198, 2008.) (c) Combining melt electrospinning and solution electrospinning to create a bimodal scaffold. (From Kim, S.J. et al., Polymer 51, 1320, 2010.)
an electrospun PCL/collagen fiber mesh (Figure 16.7b) [145]. The resultant constructs contained large
pores, while the electrospun fibers provided suitable structures for cell adhesion, effectively increas-
ing the surface area available for penetrating cells to attach. Following this approach, a tubular con-
struct for vascular TE was manufactured, incorporating nanostructured electrospun substrates and
FDM fibers for sufficient mechanical strength [146]. Finally, a bimodal scaffold was successfully
formed by simultaneously solution electrospinning (low diameter fibers) and melt electrospinning
(large fibers) onto a rotating mandrel, using PLGA for both types of electrospinning (Figure 16.7c).
16.5.6 Electrospinning Small Diameter Microfibers

There is a reported correlation between fiber diameter and pore size in electrospun meshes: a mini-
mum fiber diameter (2–3 μm) is required for cell penetration (pore size 10–15 μm) [147]. Thus, the
lack of pore size and interconnectivity resulting from the chaotic deposition of electrospun nanofibers
creates a barrier to cell invasion. Interestingly, there are only a handful of studies on cellular inter-
actions on meshes with fiber diameters between 2 and 10 μm, meaning that fibrous scaffolds with
both high surface area and cell invasiveness are underinvestigated for TE. When such scaffolds are
seeded, cells penetrate 1200 μm into the scaffold under flow and 300 μm under static conditions [147].
16.6 GROWTH FACTOR DELIVERY FROM ELECTROSPUN MATERIALS

Electrospun fibers can be used to deliver growth factors and other proteins to their surrounding
environment. The growth factor can be included by simple addition to the polymer solution to be
electrospun or incorporated using a variety of other means [148,149]. It is important in this case
that the bioactivity of the growth factor is retained, since dissolution within a solvent can denature
proteins, thus altering their bioactivity. Table 16.4 provides a summary of different growth factors
incorporated within electrospun fibers.
As shown in Table 16.4, the delivery of growth factors can be achieved through the release of
soluble factors or by tethering the molecules to the surface of the electrospun fibers. Drug delivery in
the latter approach requires the sustained availability of the growth factor, in a bioactive and natural
form. Therefore, the surface conjugation of biomolecules to electrospun fibers is an important area of
research and includes single and multistep conjugation approaches for tethering bioactive molecules.
TABLE 16.4
Selected Examples of Growth Factors Delivered from Electrospun Fibers
Growth Factor Polymer Comments References
BMP-2 Silk fibroin [150]
BMP-2 PLGA/Hap [151,152]
BMP-2 PLGA/Hap BMP-2 plasmid delivered [153]
BDNF PCL Surface functionalized [154]
EGF PLLA GF both adsorbed and heparin bound [155]
FGF-2 PLGA Two approaches used—normal and coaxial [156]
FGF-2 Gelatin or collagen Introduced with perlecan binding domain [157]
FGF-2 PLLA Via surface bound heparin [155,158]
GDNF PCLEEP [159]
IGF-1 Polyurethane Delivered via PLGA microspheres in fibers [160]
NGF PLCL Emulsion electrospinning [161]
NGF PCLEEP NGF co-delivered with BSA [7]
PDGF Dextran/PLCL Coaxial [162]
VEGF Gelatin/PCL Coaxial—VEGF surface bound to heparin [163]
16.7 SURFACE MODIFICATION OF ELECTROSPUN FIBERS

Biofunctionalization of the interface between a polymer and a biological entity is an important
means to direct cell behavior and/or prevent bacterial infection [164]. The surface of the polymer
fiber also influences the properties of the electrospun fiber mesh, such as wettability. Broadly, there
are two methods possible to modify the electrospun fiber surface: postprocessing of the fibers, or
more recently biofunctionalization was performed in a single electrospinning step [122,165].
16.7.1 Postprocessing Approaches

The treatment of electrospun fibers after their manufacture is a common route to biofunctionalization.
It is simple, because traditional conjugation chemistry such as 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide hydrochloride (EDC) or N-hydroxysulfosuccinimide (sulfo-NHS) can be adopted. One prob-
lem with this approach, however, is that chemical removal and washing in different media is required,
and the repetitive transition to different liquids may damage delicate parts of an electrospun scaffold.
16.7.1.1 Adsorbing Proteins onto Electrospun Fibers

Proteins can also adsorb onto surfaces, particularly hydrophobic surfaces. This is a simple method
of introducing biological signals onto electrospun fibers but needs to be compared with includ-
ing the protein as part of the electrospinning solution. For example, Ramakrishna and colleagues
adsorbed laminin onto electrospun fibers. However, when laminin was included within the elec-
trospinning solution, neurite growth was significantly higher compared to electrospun fibers with
adsorbed laminin [40]. Some drugs adsorb onto electrospun fibers through simple physical adsorp-
tion using electrostatic interactions, hydrogen bonding, and hydrophobic interactions [166,167].
16.7.1.2 Chemical Binding Bioactive Functionalities

The direct conjugation of bioactive molecules onto electrospun fibers is an important approach to
functionalization. Through various chemistries, brain-derived neurotrophic factor (BDNF) [154],
heparin [158], Cibacron blue F3GA (CB), and bovine serum albumin (BSA) [168] have been cova-
lently attached onto electrospun fibers. By incorporating heparin onto the surface of the fibers,
growth factors such as FGF-2 and EGF-1 were bound [155,158]. This set the platform for many
different growth factors to be bound using heparin binding sites. Combining adsorption and chemi-
cal conjugation, DNA was electrostatically incorporated onto electrospun fibers that have matrix
metalloproteinase (MMP) cleavable functionalities [169].
16.7.2 Protein Resistant Biofunctionalization

There has been some focused work on generating protein resistant electrospun fibers with bioactive
surface functionalities. Using amphiphilic block copolymer polymers with the correct hydrophilicity
and chemistry, the resulting fibers were hydrophilic [58]. When these amphiphilic polymers were end
modified with arginine-glycine-aspartic acid (RGD), biotin, or were thiolated, then they were protein
resistant, hydrophilic, and functionalized [57,58]. Interestingly, only when mixed solvents are used
(chloroform/methanol instead of chloroform only) there is strong resistance to BSA or streptavidin
adsorption. Thiolated electrospun fibers in particular are able to then chemically bind with numerous
bioactive molecules [170]. A more simplified approach to that described here has been devised by
the same group that uses an amphiphilic and isocyanate star-shaped macromer, a reactive additive to
a PCL or PLGA solution, to create both biofunctionalized and hydrophilic fibers [122,165]. Protein
resistant fibers were produced using solutions containing only 7% star-shaped macromer relative
to the PLGA or PCL. Functionalization was achieved by adding a bioactive peptide to the mac-
romer/polymer solution prior to electrospinning with RGDS peptide and RDES modified electrospun
substrates. The functionalized and hydrophilic fibers exhibited distinct differences in cell adhesion
50 µm
FIGURE 16.8 (See companion CD for color figure.) RGD-modified electrospun fibers deposited onto reac-
tive substrates that prevent adhesion of cell—fibroblasts can only adhere onto the fibers. (Reproduced from
Grafahrend, D. et al., Nat. Mater., 10, 67, 2011.)
when investigated in vitro for neural applications [122]. When such electrospun fibers are placed
upon reactive substrates that prevent cell adhesion, cells can only adhere to the fibers (Figure 16.8).
16.8 IN VITRO STUDIES OF ELECTROSPUN MATERIALS

As previously described, typical electrospinning does not result in cell-invasive scaffolds. Despite
this, there are numerous excellent reviews on the interactions between electrospun fibers and cells
in vitro that address aspects of cell biology [26,171] as well as demonstrate the diversity of poly-
meric, morphological, and structural properties. In general, electrospun fibers are excellent sub-
strates for cells: except for a few examples, when the fibers are hydrophilic and protein resistant
[57,58,165,170], cells adhere readily onto electrospun material. The cell shape, however, changes
with electrospun fiber morphology: random meshes allow cell spreading similar to that seen on flat
substrates, whereas cells tend to self-orient onto aligned fibers, particularly if there is no other sub-
strate for them to adhere to Ref. [123]. In studies where “dilute” quantities of fibers were collected
onto reactive substrates, the cells were only permitted to adhere to the fibers, inducing a very ori-
ented structure. Interestingly, phenotypical differences have been observed between cells cultured
on oriented fibers rather than random meshes.
16.8.1 Fibroblasts
Fibroblasts are the most abundant cells in connective tissue, and for various reasons, they are by far
the most investigated cell type with electrospun material. Fibroblasts excrete ECM, including colla-
gen, and are an important part of the wound-healing process. In addition to collagen, they produce
glycosaminoglycans, reticular and elastic fibers, and glycoproteins. Electrospun fibers have often
been compared to collagen, which can be fibrillar in structure. Since collagen is widely present in
such connective tissue, it is appropriate that electrospun fibers and fibroblasts are so widely studied.
For example, there are studies where the integration of fibroblasts into an electrospun scaffold is
a crucial component of the final application such as in ligament of skin tissue engineering. Table
16.5 provides a list of selected references where fibroblasts were cultured with electrospun fibers.
TABLE 16.5
Selected In Vitro Studies with Fibroblasts
Polymer Comments References
PLGA Cells adhered and spread according to fiber direction [4]
PLGA Bimodal d = 530 nm and 28 μm. Higher cell attachment/spreading in the [13]
nano/microfiber than the microfibrous scaffolds without nanofibers
PCL/PEG-b-PCL blends Electrospun fibers were drawn onto substrates for discrete cell adhesion [24]
Fibrinogen Neonatal rat cardiac fibroblasts readily migrated into and remodel [44]
electrospun fibrinogen scaffolds with deposition of native collagen
PEG-b-PCL Fibers were both protein resistant and surface functionalized with RGDS [57,58]
PCL/PEG-b-PCL blends Direct in vitro electrospinning with melt electrospinning [100]
PCL/sP(EO-stat-PO)-RGDS Single step functionalization—fibers were both protein resistant and [165]
surface functionalized with RGDS
Thiolated PEG-PDLLA Adherence and proliferation on RGDC-functionalized fibers with reduced [170]
protein adsorption
Collagen The aligned scaffold exhibited lower rabbit conjunctiva fibroblast adhesion [179]
but higher cell proliferation
Polyurethane Rotating cylindrical collector aligned fibers. After mechanical strain, [180]
ligament fibroblasts had a spindle shape on aligned fibers and secreted
more ECM than random ones
Polyamide Cells rearranged their actin cytoskeleton to a more in vivo–like [181]
morphology
Collagen and PCL Supported attachment and proliferation [182]
Chitosan/silk fibroin Supported attachment and proliferation [183]
Ag-containing PVA The Ag ions and Ag nanoparticles are cytotoxic to cells [184]
PLLA/silk fibroin/gelatin After 12 DIV, the scaffold supported attachment, spreading, and growth [185]
Collagen and hyaluronate The ratio of foreskin fibroblasts expression of TIMP1 to MMP1 was lower [186]
than collagen fibers
PDLA/PLLA (50:50) After 5 DIV, the fibroblasts adhered, migrated, and proliferated [187]
PLGA and PLGA/MWCNTs MWCNT incorporation promoted attachment, spreading, and proliferation [188]
PLGA/collagen Effective as wound-healing accelerators in early stage wound healing [189]
PEG/PPG/PCL Supported excellent cell adhesion, comparable to pure PCL [190]
PCL and PCL/PEO Proliferated fastest with pores greater than 6 pm. Conformation changed as [191]
the pores grew from 12 to 23 pm; cells aligned along single fibers instead
of attaching to multiple fibers
PHB/chitosan Promoted attachment/proliferation [192]
pDNA PEI/HA in PCL/PEG Complexes of pDNA with PEI-HA released from fiber mesh scaffolds [178]
core shell could successfully transfect cells and induce expression of enhanced
green fluorescent protein (EGFP)
PLGA/chitosan and PLGA/chitosan and core-shell PLGA/chitosan showed better adhesion/ [193]
core-shell PLGA/chitosan viability than PLGA
PCL and ECM proteins Presence of collagens, tropoelastin, fibronectin, and glycosaminoglycans in [194]
scaffolds
PDLLA/PEG Dermal fibroblasts interacted and integrated with the fibers containing 20% [195]
and 30% PEG
Plasma silk fibroin O2-treated fibers showed higher cellular activities for fibroblasts [196]
Silk fibroin L929 fibroblasts showed good cell adhesion after 1, 3, and 7 DIV with [197]
confluence at 7 DIV
Polyurethane/MWNT MWNT incorporation exhibited highest cell adhesion/proliferation and [198]
migration/aggregation
PVA Cells grew significantly after 7 DIV but were round-shaped [199]

Selected In Vitro Studies with Fibroblasts
Collagen Type I Qualitative analysis of rabbit corneal fibroblasts morphology and protein [200]
expression suggests fibers suitable for engineering a corneal tissue
replacement
Bombyx mori silk fibroin Attachment/growth with no difference between fibers electrospun with [201]
different solvents
Polyurethane elastomer Human fibroblasts adhere and migrate, proliferate, and produce [202]
components of an ECM
PLGA Human skin fibroblasts spread and showed growth. Collagen type III gene [172]
expression was upregulated on matrices with fiber diameters in the range
of 350–1100 nm
Hexanoyl chitosan Fibrous scaffolds supported attachment/proliferation [174]
Polyethyleneimine Normal human fibroblasts attached/spread on fibers [203]
PCL PCL was electrospun using an auxiliary electrode and chemical blowing [204]
agent. Good adhesion with the blown web relative to a normal
electrospun mat
Because wound closure is an important part of fibroblast function, these cells have been
used in wound-healing applications. Experiments with electrospun materials have focused on
fibroblast adhesion, proliferation, and migration: the majority of electrospinning publications
involving fibroblasts are superficial, in that the cells help to determine adhesion and toxicity
toward the fiber, rather than as part of a focused wound-healing experiment. However, there are
some studies providing in depth analysis of fibroblast function, including upregulation of impor-
tant genes, such as collagen III production, which is optimal on fibers with diameters between
350 nm and 1.1 μm [172].
In many instances, fibroblasts are used as a proof of cytotoxicity [173] and biocompatibility [174].
These cells can also be used to ascertain whether a strategy of electrospinning produces feasible
fiber morphology [175], if surface modification of a polymer was successful [176], or if a drug deliv-
ery system is viable [177]. These issues are important, as cells may be the most sensitive measure of
changes in bioactivity of a surface, since instrumentation techniques have detection limitations. For
example, fibroblasts have successfully been transfected with a nonviral vector incorporated within
electrospun fibers so that they express green fluorescent protein [178]. In another study, fibroblasts
were used as part of the collection system, to demonstrate that melt electrospun fibers could be
deposited directly onto cells without a loss in vitality [100]. Similarly, recent work on making pro-
tein resistant and functionalized fibers involved fibroblasts to determine the success of the surface
modification approach [57,58,122,165,170]. Although many of the aforementioned electrospinning
investigations could be undertaken with almost any other cell type, fibroblasts are inexpensive,
readily accessible, and relatively simple to work with. Suspended fibers have also been used with
in vitro studies, with a minimum separation distance between fibers required for fibroblasts to
bridge the gap (Figure 16.9) [345].
16.8.2 Epithelial Cells
Simple (one cell thick) or stratified epithelium covers and lines connective tissue (containing
fibroblasts) surfaces in the body—from skin, cornea, to the inside of blood vessels, and intes-
tine—thus protecting the underlying tissues. Epithelium also functions to secrete enzymes,
hormones, as well as ECM and is separated from connective tissue by a basement membrane.
(a) (b) (c)
Bridged
Failed-to-bridge
0 50 100 150 200 250 300 350 400

(a) (b) Gap between fibers (μm)
FIGURE 16.9 (See companion CD for color figure.) (a) Human dermal fibroblasts cultured on suspended
aligned fibers at different distances apart. (b) The effect of a gap between suspended fibers and whether cells are
able to bridge the gap. (Reproduced from Sun, T. et al., Biotechnology and Bioengineering, 97, 1318–1328, 2007.)
TABLE 16.6
In Vitro Studies with Epithelial Cells
P(LLA-CL) 75:25 Adhered and proliferated well [94]
Polyamide T47D breast epithelial cells underwent morphogenesis to form [181]
multicellular spheroids
Chitosan and alginate Layer-by-layer scaffolds have biocompatibility with Beas-2B human [205]
bronchial epithelial cells
PDLA Both protein adsorption and porcine esophageal epithelial cells attachment [206]
PHBV; PHBV/collagen, and Co-culture of hair follicular cells with epithelial outer root sheath/dermal [207]
PHBV/gelatin sheath cells 3–5 DIV showed PHBV promoted wound closure and
reepithelization more than PHBV/collagen
Fibronectin-g-PLLCL Promotes epithelium regeneration, using esophageal epithelial cells [208]
PCL and PVA co-spinning Human prostate epithelial cells attached/proliferated more with PVA [209]
electrospun PCL mats
It is substituting the basement membrane that attracts researchers to electrospun fibers. These
specialized epithelial cells have been used with electrospun fibers to create layered cellular
structures. However, there has been less emphasis on creating cell-invasive substrates, since
epithelium should sit on top of electrospun meshes and form a layered construct. Where elec-
trospun fibers fit into epithelium regenerative medicine strategies is not as well defined as com-
pared to other cell types. Despite this, epithelia are crucial to wound-healing strategies, and
they will be increasingly used in electrospun fiber research. Table 16.6 lists a selection of epi-
thelial studies using electrospun fibers.
16.8.3 Endothelial Cells
Endothelial cells are specialized epithelial cells that line the interior surface of blood vessels to cre-
ate a barrier between circulating blood and the rest of the vessel wall. Endothelia are of wide inter-
est to electrospinning researchers, which may be due to the structure they comprise: although the
most widely used electrospinning collection configuration is a single flat collector, the second most
TABLE 16.7
In Vitro Studies with Endothelial Cells
PLLCL (70:30) Human coronary artery endothelial cells were evenly distributed and well [210]
spread from 1 to 10 DIV and maintained expression of PECAM-1
(P(LLA-CL)) Both random and aligned fibers preserved human coronary artery endothelial [211]
cell phenotype; expression of PECAM-1, fibronectin, and collagen type IV
Polyurethane Small-diameter grafts with microfibers and microgrooves endothelial cells [212]
formed confluent monolayers. The cells expressed cadherin
PCL and CNTs Good cerebro-microvascular endothelial cell viability [213]
Collagen/chitosan Endothelial cells proliferated well [214]
PCL/collagen Endothelial cells showed enhanced cellular orientation and focal adhesion [215]
Coaxial cellulose acetate As mechanical stiffness increased, endothelial cell growth/adhesion and [216]
and chitosan migration promoted. Fibers did not induce platelet aggregation or activation
Silk fibroin Iliac endothelial cells attached and proliferated in comparison with cast SF films [217]
Silk fibroin/PLLCL Silk fibroin/PLLCL blended fibers increased Iliac endothelial cell growth [218]
compared with PLLCL
Polyurethane/collagen Iliac endothelial cells migrated inside the scaffold within 24 h of culture [219]
Span-80/PLLACL coaxial No differences in iliac endothelium cell growth on the fiber types [220]
core/shell
HMA:MMA:MAA Cytocompatible allowing human blood outgrowth, endothelial growth, [221]
terpolymer maintained phenotype
Polyphosphazene After 16 DIV, neuromicrovascular endothelial cells formed a monolayer on [222]
the whole surface
P(LLA-CL 70:30) Collagen-coated fibers enhanced spreading, viability, and attachment of [223]
human coronary artery endothelial cells and preserved phenotype
Gelatin-grafted-PET Gelatin grafting improved the spreading/proliferation of the ECs and [224]
preserved phenotype
PLLA Endothelial cell function was enhanced on smooth solvent cast rather than on [225]
the rough electrospun fibers. Electrospun substrates favored vascular smooth
muscle cell behavior
commonly used is a single, rotating collector to generate tubular structures. Since blood vessels are
such an important tissue, numerous researchers have looked at incorporating such cells and fibers
together to create a functional epithelium on electrospun tubes, as shown in Table 16.7. Desirable
mechanical properties of the electrospun tubular mesh are a critical requirement. Electrospun tubes
of poly(urethane) and PLLCL in particular have been used, due to their elastic nature and strength
[210–212]. In addition, nano and microfibers have been combined to obtain nanoscale structures
with sufficient mechanical strength.
Endothelial cells derived from umbilical cords (human umbilical vascular endothelium
cells; HUVECs), with the potential to function in regenerative medicine, are an important and
increasingly researched cell type, as illustrated in Table 16.8. HUVECS are popular because
they are derived from an unrequired tissue available at birth, and there are few ethical issues
with using this form of human tissue [226].
16.8.4 Smooth Muscle Cells

Blood vessels also consist of nonstriated muscle, and smooth muscle cells (SMCs) are an impor-
tant cell type in this tissue. SMCs are also found in other tissues, including the aorta, gas-
trointestinal tract, lymphatic vessels, uterus, urinary bladder, the iris, and the ciliary muscle.
TABLE 16.8
In Vitro Studies with Human Umbilical Vascular Endothelium Cells
HMA:MMA:MAA terpolymer Cytocompatible fibers allowed growth, maintained phenotype [221]
Methacrylic terpolymer Fiber morphology controlled proliferation, metabolic activity, and [227]
morphology of cells
MWCNT/PU Fibers activated Rac and Cdc42, while CNT regulated the activation of Rho [228]
PCL D = 0.8 or 3.6 μm. The larger fibers had higher vitality and improved [229]
interaction
Silk/gelatin Gelatin improved the mechanical and biological properties [230]
PLLCL VE-cadherin expression not detected, but a high degree of tissue integrity [231]
was achieved
PLCL (50:50) Adhered well and proliferated on small diameter fiber fabrics (0.3 and [232]
1.2 μm; reduced adhesion, cell spreading and no signs of proliferation
on the 7.0 μm diameter fibers
Table 16.9 shows a list of electrospun material cultured with SMCs, where emphasis is placed on
seeding cells on tubular constructs mostly for vascular tissue engineering [233,234]. Cell seed-
ing onto 3D constructs creates many challenges and the more anisotropic the scaffold, the more
complex and difficult cell seeding becomes. This step is crucial in the in vitro culture of cellular
implants since homogenous cell distribution appears necessary for successful TE strategies,
TABLE 16.9
In Vitro Studies with Smooth Muscle Cells
[P(LLA-CL)] (75:25) Rotating disk collector aligned fibers. SMCs attached and migrated along [136]
aligned fibers; the distribution of cytoskeleton proteins were parallel to
the fibers
Type I and II collagen Mandrel rotated at approximately 500 rpm. Fibers possessed the typical [5]
67 nm banding pattern observed in native collagen
PU Microintegrated cells into a biodegradable elastomer fiber matrix [8]
[P(LLA-CL)] (75:25) Adhered and proliferated well [94]
PCL Larger fiber diameter improved SMC infiltration into the bilayered [215]
vascular scaffolds
PLGA/COL Co-culturing of SMCs with ECs, under a pulsatile perfusion system, [233]
leads to the enhancement of vascular EC development, as well as the
retention of the differentiated cell phenotype
PU Cells integrated into a strong, compliant biodegradable tubular matrix [234]
Collagen-coated PCL Proliferation on collagen-coated PCL fibers was not different than that [236]
on TCP
PCL Contains heparin [237]
Collagen I, II, and elastin A three-layered vascular construct, integrating fibroblasts and endothelial [235]
cells
PLLA + PLLA/PVP Morphology/proliferation improved with decreasing of the weight ratio [238]
of PLLA/PVP. In platelet adhesion decreased with increment of PVP
content in the films
COL-coated PCL Same proliferation rate on polycaprolactone (PCL) nanofibrous matrices [239]
coated with collagen or tissue culture plates. PCL coated with collagen
showed more migration
which explains why there is such an emphasis placed on developing cell seeding techniques in
this area. Multilayered vascular constructs have been prepared, demonstrating how complex tis-
sue can be generated using the electrospinning technique [235]. Boland et al. fabricated a three-
layered collagen/elastin vascular construct, which was built with separate media and adventitia
seeded with SMCs and fibroblasts for the structural component, while the lumen was seeded
with endothelial cells. Stankus et al. also addressed the cell-invasive issue associated with using
submicron fibers by simultaneously electrospinning fibers and electrospraying cells in gelatin, to
form tubular constructs, which were then included into a bioreactor [8]. The fibers and cells were
laid down simultaneously, rather than constructing a scaffold and seeding them in a second step.
16.8.5 Cardiomyocytes, Cardiac Stem Cells, and Cardiac Cell Lines

An excellent article on contractile cells was provided in 2004 by the Vacanti laboratory, where
cardiomyocytes were grown on a mesh of PCL, which had been collected and suspended on a
metallic ring [240]. This thin sheet began contractile movements, and the metallic wire ring acted
as a passive mechanical support for the cells. Important cardiac tissue markers such as myosin,
connexin 43, and cardiac troponin I were expressed after 14 DIV (days in vitro) [241]. Interestingly,
electrospun fibers were also embedded into collagen sponges, with an increase in cardiac stem cell
attachment on the collagen scaffolds with fibers embedded. Table 16.10 provides a list of in vitro
studies with cardiomyocytes, cardiac stem cells, and cardiac cell lines.
16.8.6 Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent stem cells that can differentiate into different cell
types, including chondrocytes, osteoblasts, and adipocytes. They are found in bone marrow and
can be extracted using a biopsy, with MSCs often but not only sourced from humans. Table 16.11
provides a list of different publications where MCSs were cultured on electrospun materials and
demonstrates that this is one of the most investigated cell types with electrospun materials due to
their ability to differentiate. With MSCs, the lineage of the cultured cell is of particular interest.
Some researchers use differentiation media with electrospun fibers to induce a particular type of
cell line with success [246,247]. Therefore, electrospun fibers do not interfere with conditions that
are designed to differentiate MSCs into particular lineages. Alternatively, electrospun scaffolds can
be used as a drug delivery platform to induce stem cell differentiation into the targeted cell lineage.
TABLE 16.10
In Vitro Studies with Cardiomyocytes, Cardiac Stem Cells, and Cardiac Cell Lines
Polymer Cells Comments References
PCL Cardiomyocytes A 10 μm thick mesh is suspended across a wire ring. Myosin, [240,241]
connexin 43, and cardiac troponin I expressed after 14 DIV
PLLA Cardiomyocytes Cells with fiber-guided filipodialike protrusions and developed into [242]
sarcomeres
PGA Cardiac stem cells Fibers embedded in collagen sponge and more cells attached to the [243]
collagen sponge incorporating fibers compared to without. The
attachment and proliferation were enhanced by incubation in a
bioreactor perfusion system
Polyurethane Cardiomyocytes Cells were elongated, but morphological changes induced by [244]
material macrostructure did not directly correlate to functional
differences
PPDL H9c2 Fibers were not cytotoxic and supported cell proliferation [245]
TABLE 16.11
In Vitro Studies with Mesenchymal Stem Cells
Polymer Comments Reference
PLGA/TCP Cotton wool-like fibers from collection on ice. Osteocalcin showed osteogenic [11]
differentiation
HAp/TCP/PCL The solvent or combination for fiber preparation affects properties and cell [82]
behavior
PLLA/PCL Combined electrospinning with FDM to make a tubular construct [146]
Silk/PEO/HAp HAp particles improved bone formation. BMP-2 and HAp resulted in the highest [150]
calcium deposition and upregulation of BMP-2 transcript levels
PLGA/HAp Encapsulated DNA/chitosan nanoparticles have higher cell attachment, viability, [153]
and transfection
PLGA/HAp Encapsulation of HAp could enhance cell attachment to scaffolds and lower [152]
cytotoxicity
PLGA Under hypoxia/nutrient starvation conditions, the IGF-1 loaded scaffolds improved [160]
MSC survival
PLCL and PLCL/coll Neural differentiation was induced with soluble factors [246]
PCL Chondrogenic differentiation was enhanced after incubation in chrondrogenic [247]
media
PCL Delayed initial attachment and proliferation on meshes, but enhanced [248]
mineralization at a later time point
PLGA and PCL Supported attachment, proliferation, and differentiation [249]
PCL Differentiated to adipogenic, chondrogenic, or osteogenic lineages by specific [250]
differentiation media
PCL Differentiated to chondrocytic phenotype—a zonal morphology is described [251]
PCL Matrix mineralization and collagen type I throughout after 4 weeks [252]
Collagen Combined electrospinning with SFF; Enhanced initial cell attachment and cell [253]
compactness between pores of the scaffold relative to SFF 3D collagen scaffold only
HAp/PLGA Increased ALP activity and expression of osteogenic genes w calcium [254]
mineralization of MSCs
Gelatin/siloxane Resulted in apatite formation and stimulated proliferation and differentiation [255]
PCL Increased osteocalcin and osteopontin 3 weeks of culture [256]
PCL Multimodal distribution of fiber size allowed increased cell seeding [257]
Collagen or PLLA Early adhesive behavior is affected by texture of surface. Collagen and collagen [258]
coated PLLA fiber had similar outcomes
PCL Thicker scaffolds provide a better substrate for cell proliferation [259]
PLCL MSCs expressed CD44 and CD105 but did not express CD34 or CD14 [260]
P(3HB-co-4HB) Porcine aortic heart valves were decellularized, coated with electrospun fibers [261]
Silk fibroin Fiber alignment affected morphology and orientation [262]
Chitin/PVA Fibers supports cell adhesion/attachment and proliferation [263]
PLA or PLA/DBP Mineralization with osteogenic supplements was greater PLA after 14 DIV but [264]
same level 21 DIV
PCL or PCL/COL Multilayer construct, supported cell attachment similar tissue culture polysterene plate [265]
From a practical viewpoint, it was shown that insulin growth factor (IGF-1) loaded electrospun
scaffolds improved MSC survival under hypoxia or nutrient reduced conditions [160]. With such
a complex and heterogeneous environment generated during stem cell culture, all conditions and
parameters need to be reported in order to allow comparison between studies and to draw definitive
conclusions.
16.8.7 Osteoblasts
Ideally, bone TE should provide a substitute for the autologous bone graft, and there is a lot of
research to develop a load-bearing material that can integrate with bone defects. Since electrospun
fibers do not generate sufficient compressive strength, their use is more to support bone formation
in other surgical paradigms, including an artificial periosteum and as a structure to contain hemato-
mas between bone fractures [266]. Mineralization is a crucial process in bone formation, and many
of the in vitro studies with osteoblasts and electrospun fibers measure the alkaline phosphatase
activity (ALP), which indicates deposition of mineralized matrix (early bone formation). A distinc-
tive feature of electrospun fibers within bone TE is the inclusion of inorganic substances within
the fibers to improve the biological outcome when using osteoblasts by rendering scaffolds more
osteoinductive. Such materials include hydroxyapatite (HAp), silica, titanium dioxide (TiO2), and
bioactive glass. The cells used for bone TE can also be MSCs, as they are the stem cell precursor
for osteoblasts. Another issue in this type of TE strategy is the degree of cell invasiveness of the
electrospun scaffolds, which has been addressed by Ekaputra et al. [9]. These authors simultane-
ously electrospun and electrosprayed different materials, which made the fibrous mesh less dense.
For example, when a hyaluronic acid solution is electrosprayed with electrospun PCL onto the same
collector, there was increased osteoblast penetration into the scaffold.
A further step in the development of osteoinductive scaffolds is the deposition of a biomimetic
calcium phosphate coating on the fibers. This appears to enhance the ALP activity and the osteo-
pontin expression of a MC3T3-E1 mouse osteoblastic cell line under osteoblastic induction [267]. In
a different study, the same cell line displayed similar behavior onto biomimetically coated electro-
spun PCL. The expressions of ALP, RunX2, collagen I, and osteocalcin were upregulated confirm-
ing the beneficial effect of a calcium phosphate coating on osteoblast differentiation [268].
As seen in Table 16.12, there is significant interest in the interactions between osteoblasts and
electrospun fibers. This partial list contains only selected journal articles, and the actual publication
list is much greater. Jang et al.’s work is an excellent review on electrospinning for bone regenera-
tion [284].
16.8.8 Chondrocytes
There is a clear medical need for transplantable cartilage tissue, as cartilage has a limited capacity
to repair itself. Cartilage TE research emphasizes regeneration, rather than repair, and an ideal car-
tilage scaffold should possess similar mechanical properties to the natural tissue. The ECM, which
contributes the most to the mechanical properties, is comprised of collagen fibrils (mainly Type II)
and proteoglycans and is distributed into zones with distinct mechanical properties [285,286].
A promising approach to cartilage repair is 3D printing of hydrogels [287,288]. As previously
described, this approach can be combined with electrospinning to form a different class of “bimodal
scaffold,” where the scaffold consists of structures with distinctly different scales. In this instance,
the extruded hydrogels form struts approximately 100 to 200 μm in diameter, while electrospun
fibers are incorporated within the larger struts (Figure 16.7b) [289]. The smaller diameter fibers
aid in increased cell seeding efficiency and are included in the printing process by electrospinning
separate layers during the printing process. Table 16.13 summarizes in vitro studies with chondro-
cytes and electrospun fibers.
16.8.9 Neurons and Glia

Electrospun fibers have been investigated for the guidance of neurons and glia, which naturally exist
in highly oriented and structured tissues, particularly in the peripheral nerve and the white matter
of the central nervous system (CNS). The research with such cells can be divided into two main
areas of interest—in vitro guidance systems and tubular structures for containing damaged nerves.
TABLE 16.12
In Vitro Studies Performed with Osteoblasts
Polymer Cell Type Comments References
PCL/collagen and Fetal osteoblasts All three scaffold types supported attachment/proliferation; [9]
PEO; PCL/collagen/ better penetration was with mPCL/Collagen fibers
Heprasil co-electrosprayed with Heprasil
PCL/b-TCP MC3T3-E1 cells Viscoelastic and biomechanical properties increased with [269]
DIV
Gelatin and HAp Fetal osteoblasts Combining of electrospinning of gelatin and electrospraying [270]
of HA increased ALP activity and enhanced mineralization
PCL-PEG-PCL and Osteoblasts Scaffolds could provide a suitable environment for [271]
HAp attachment, and MTT revealed the scaffolds had good
biocompatibility and nontoxicity
HA/TiO2 MC3T3-E1 cells Collagen treated HA/TiO2 fibers possess better cell [272]
adhesion and significantly higher proliferation and
differentiation than untreated fibrous mats
PCL-silica Pre-osteoblast Good proliferation and differentiation of cells [273]
cells
PLA/bioactive glass Osteoblasts Bioactive glass phase enhanced the in vitro apatite [274]
formation and cells adhered
PLLA Osteoblasts After mechanical expansion of the microfibrous mats, the [275]
scaffolds proliferated osteoblasts that actively penetrated
the inside of the 3D scaffold
p(TMC-co-CL)-block- MC3T3-E1 Cells proliferated 1.2 times faster at 4 DIV, 1.5 times faster [276]
p(p-dioxanone) at 7 DIV after seeding; At 28 DIV alkaline phosphatase
was four times more than control
RGD-mod PLCL Osteoblasts Adhesion and proliferation was greater on RGD modified [277]
fibers meshes up to 7 DIV. ALP activity and calcium
content on the RGD-AAc-PLCL meshes were
approximately 7.5 and 6.7 times higher than those on the
other meshes, respectively. Expression of Cbfa1, ALP, and
Osteocalcin (OCN), was upregulated (at least 5–9.7 times
greater) on the RGD-AAc-PLCL meshes
PHBV and cHAp SaOS-2 Both PHBV and CHA/PHBV supported the proliferation; [278]
The alkaline phosphatase was higher than the CHA/PHBV
after 14 DIV
PCL/collagen Pig bone marrow Osteogenic differentiation markers were more pronounced [279]
mesenchymal on PCL/Col fibrous meshes
cells
PLCL/gelatin/HAp Fetal osteoblasts Electrospinning PCL/gelatin and electrospraying of HA [280]
nanoparticles made a rough surface with better proliferation,
mineralization, and alkaline phosphatase activity
Silica MC3T3-E1 cells ALP activity and expressions of type I collagen and [281]
osteocalcin were higher for cells cultured on nonwoven
silica gel fabrics than on TCP
PLLA and PLLA/HAp Osteoblasts n-HA deposition improved seeding efficacy at 20 min time [282]
point
PLA/bioactive glass As bioglass concentration increased (from 5% to 25%), [283]
the bioactivity improved. Cells attached, grew well,
and secreted collagen. ALP expression was higher than
pure PLA
TABLE 16.13
In Vitro Studies with Chondrocytes
PCL/collagen Favorable conditions for cell adhesion/proliferation, attributed to [145]
nanotopography, chemical composition, and an enlarged surface for cell
attachment and growth
Chitosan Fibers allowed chondrocytes to proliferate and produce glycosaminoglycan [290]
PCL and 3D prototyping This approach is feasible for fabricating 3D scaffolds for chondrocytes [289]
PCL Fetal bovine chondrocytes upregulated collagen type IIB expression after [291]
3 weeks
HA/collagen Chondrocyte adhesion/proliferation were enhanced maintained chondroblastic [292]
morphology
3D printed PEOT or BPT Combined scaffolds enhanced articular chondrocyte entrapment compared to [293]
and electrospun PCL 3DF scaffolds and higher GAG/DNA ratio after 28 DIV. Spread morphology
was observed on 3DF scaffolds, suggesting a direct influence of fiber
dimensions on cell differentiation
PCL on PLA microfibers Increased cellular infiltration with higher porosity [294]
PHBV Rabbit auricular chondrocytes attached more efficiently to electrospun fibers [295]
(30% after 2 h) than cast films (19%)
Collagen type II Scaffolds produce a suitable environment for chondrocyte growth [296]
Fibers have an inherent guiding property on neuron processes when their diameter is below approxi-
mately 250 μm [297,298]. Further reducing the fiber diameter down to submicron levels, as seen in elec-
trospinning, still maintains this guidance effect [67,121,123]. Since nervous tissue is often bundled into
organized arrays, the orientation of electrospun fibers is a particularly relevant aspect of the technique.
As mentioned previously, electrospun fibers can be aligned by collecting them onto a rapidly rotating
mandrel. The oriented electrospun sheet can then be flattened with minimal curvature if the mandrel
is of sufficiently large diameter [299]. Alternatively, it is possible to collect electrospun fibers across
the gap between two collectors, previously outlined in Section 16.4.3. This has been performed using
parallel bars, sharp blades, and metallic rings. When short collection times are used in this approach,
the fibers that are suspended across the gap remain “individual,” in the sense that they do not adhere to
other fibers. However, performing cell culture on such individual fibers can prove difficult, as changes
in culture media are difficult to perform.
Recent work on chemically reactive substrates has permitted the study of a single cell on a single
oriented fiber, quantifying the effects of changes in surface chemistry by measuring cell elongation
and process extension [67,121,123]. Fibers are transferred to a substrate by passing a prepared cover
slip through an oriented suspension of fibers. The reactive substrate binds the fibers, but then con-
tinues to react with water, so that it is ultimately resistant to both protein and cell adhesion. In this
configuration, neurons, oligodendrocytes, Schwann cells, astrocytes, and dorsal root ganglia have
all been cultured, and changes in the fiber composition (and reactive substrate chemistry) provide
distinct differences in migration and adhesion of the cell. Table 16.14 summarizes these experi-
ments and other investigations where oriented electrospun fibers are cultured with neural derived
cells. Co-culture of DRGs on astrocytes/electrospun fibers induced directional growth along the
fibers with increased neurites in the presence of astrocytes (Figure 16.10).
The other main regenerative medicine approach involving neurons and glia is to manufacture
tubular structures to enclose nervous tissue, either the spinal cord or more commonly, the peripheral
nerve. Here, tubular electrospun materials have been implanted within animal models, described
further in Section 16.9. Such tubular materials must resist compressive forces and be flexible enough
to avoid significant mechanical mismatch with the nerve [310].
TABLE 16.14
In Vitro Studies with Neurons and Glia
Laminin/PLLA PC-12 Functionalized fibers increased axonal extensions; [40]
blended laminin and PLLA is a simple method to
modify fibers
PCL/collagen Schwann Cells; OECs; Cells aligned on oriented fibers; collagen/PCL [67]
DRG explants blends provided better process growth
PCL/collagen Dissociated Schwann Schwann cell processes were longer on collagen [121]
Cells; DRGs containing PCL fibers. When substrate is made
adhesive, fibers reduced their guidance
PCL/sP(EO-stat-PO)-RGDS DRG Resistance to protein adsorption with [122]
functionalization
PCL/collagen U373; hNP-ACs; Glia aligned on the oriented electrospun substrates [123]
SH-SY5Y
PLGA C17.2 nerve stem cells Cells attached and differentiated along [300]
the direction of the fibers
PCL-gelatin Schwann Cells Aligned and random PCL/gelatin are better [301]
substrates compared with PCL
Polysialic acid Schwann Cells Good viability and directed cell proliferation along [302]
the fibers
Matrigel™ DRG Supported attachment, elongation of neurites, and [303]
migration of Schwann cells similar to electrospun
collagen type I fibers
PLLA DRG; Schwann Cells Guided growth along the aligned fibers. Schwann [304]
cells had bipolar phenotype, average neurite
length was not different from fiber densities
PPy coated PLLA/PLGA DRG Electrical stimulation increased maximum neurite [305]
length by 83% and 47%, respectively, for random
and aligned samples
PCL PC-12 Aligned fibers had similar growth irrespective of [306]
diameter. Neurites on aligned fibers were longer
than on randomly oriented fibers
Plasma PCL and PCL/COL Schwann Cells 17% increased proliferation on plasma modified [307]
PCL compared with PCL/collagen. Schwann cells
had bipolar elongations
PCL/chitosan and PCL Schwann Cells 48% more cell proliferation on PCL/chitosan after [308]
8 DIV
Polydioxanone Astrocytes; DRG Astrocytes had directional growth along the fibers. [309]
Increased neurites when co-cultured with DRGs
16.8.10 Ligament Cells
Ligaments and tendons are soft connective tissues that respectively attach bones to bones or bones to
muscles. Their major function is usually to transmit movement and to ensure joint stability. The most
predominant proteins in these tissues are collagen type I and III [311]. Ligaments and tendons display
a specific structure made of crimped aligned collagen fibers in which cells are embedded, providing
high tensile strength and elasticity. Electrospun nano- or microfibers have been mainly used to develop
scaffolds for the rotator cuff [312], periodontal ligament [313–315], and also for orthopedic applications
[316–319] (such as regeneration of anterior cruciate ligament or tendon). As the structure of the rotator
cuff and the anterior cruciate ligament are similar, the scaffolds developed for their regeneration are
(a) (b)
(c) (d)
3.5
3
Extent of growth (mm)
2.5
1.5
0.5
0
DRGs alone On astrocytes
(e) (f )
FIGURE 16.10 (See companion CD for color figure.) Images showing DRGs grown on astrocytes and how
the directionality of growth is affected by the astrocytes. (a) and (b) are random electrospun meshes while
the cells in (c) and (d) are grown on oriented fibers. (e) shows how neurite processes change direction when
encountering an astrocyte. (f) is a graph showing that astrocytes increase neurite growth. Scale bars: a–d,
100 μm; e, 30 μm. (Reproduced from Chow, W.N. et al., Neuron Glia Biol., 3, 119, 2007.)
also alike. In these regenerative strategies, the electrospun fibers were utilized as a cell culture support
providing a topographic guide to the cells. There is evidence showing that cell orientation occurs onto
aligned electrospun fibers mimicking the morphology of the native tissue. Even though the guidance
provided by the aligned fibers appears to be cell specific, it is generally assumed that collagen type I
and type III are upregulated as a result of the cell alignment [180,320]. It also seems that aligned fibers
enhance the deposition of orientated ECM [319,321].
As discussed previously, there are a number of means to fabricate scaffolds with aligned fibers.
It is also possible to obtain self-crimped aligned electrospun fibers (as long as the polymer used is
not too rigid), in order to further mimic the morphology of the native tissue [322]. When aligning
fibers onto a rotating mandrel, residual stress is created in the electrospun fibers. Once the fibers
are removed from their collection support, spontaneous fiber relaxation occurs, resulting in fiber
shrinking producing crimped structures. The crimping process can be controlled by adjusting the
temperature at which the fibers are left to relax. This crimped structure has a significant effect on
the mechanical properties at low strain (<6% strain) as it results in a J-shape stress–strain curve sim-
ilar to that observed for native tissue. The particular shape of the tensile curve is explained by the
recruitment of the collagen fibers during stretching; as the fibers are being aligned no stress increase
is observed (corresponding to the initial toe-region in the J-curve); once the fibers are aligned they
take over the load applied resulting in a rapid stress increase (known as the linear part of the curve).
Although it is possible to provide topographic cell guidance and to obtain scaffolds with similar
stretching mechanisms to ligaments or tendons, one major drawback of the use of aligned electros-
pun scaffolds comes from the weak mechanical performance of these structures. There is a striking
difference between the ultimate tensile force of a human anterior cruciate ligament (around 2500 N
[323]) and an electrospun membrane (several tenths of N). Therefore, several groups have developed
composite scaffolds in an attempt to overcome this limitation [316–319]. These structures are made
of a mechanically resistant knitted construct onto which nano- or microfibers are electrospun. Here
again it is also possible to fully mimic the morphology of the ligament and to provide a topographic
cell guide by aligning the electrospun fibers onto the knitted structure [319]. These structures dis-
played a higher ultimate strength of up to 250 N (compared to several tenths of Newtons for electro-
spun scaffolds only), but this is still far from the ultimate strength of a human ligament. Therefore,
research must be pursued in this direction, that is, to increase the mechanical properties of the scaf-
folds in order to develop functional tissue-engineered constructs.
There are two different strategies for periodontal applications. The first strategy concerns the so-
called tissue-guided regeneration in which a biodegradable membrane is used to isolate the damaged
region from the surrounding connective tissue. In this strategy, the membrane has to be biocompatible
but cell-occlusive in order to prevent gingival cell infiltration and to allow the remaining periodontal
cell to proliferate onto the root surface. Therefore, electrospun membranes can be considered good
candidates due to their intrinsic small pore size that prevents cell infiltration. Gelatin with several levels
of crosslinking [314] as well as synthetic biodegradable materials [324] have been used in the past, and
they showed promising outcomes in terms of barrier property and stability. Another strategy involved
the design of a graded electrospun composite in which hydroxyapatite nanoparticles were incorporated
in the bulk of the fibers to enhance osteo-integration while maintaining cell occlusiveness [315]. The
opposite side of the graded membrane in contact with the root surface can also be loaded with drugs
to combat periodontal pathogens and/or favor periodontal fibroblast proliferation. The second strat-
egy using the principles of TE utilizes a cellular scaffold to regenerate the damaged regions [313].
Here, electrospun scaffolds can be used as cell culture supports enabling the fabrication of a multilay-
ered cellular construct upregulating the expression of osteopontin and sialoprotein. Other structures
for periodontal tissue regeneration consisted of a complex arrangement of parallel and cross-aligned
electrospun fibers, which were drawn onto a rotating mandrel [325]. These structures displayed higher
tensile strength than randomly orientated fibers and also enhanced the proliferation and migration of rat
periodontal ligament fibroblasts. Cell elongation onto the aligned fibers was also observed to promote a
higher level of tissue organization in these scaffolds (Table 16.15).
TABLE 16.15
In Vitro Studies with Ligament Cells
Polyurethane Human ligament Aligned nanofibers had spindle-shaped, oriented cells; [180]
fibroblast more collagen was synthesized on aligned fibers
PLGA (85/15) Human rotator cuff Aligned nanofibers upregulated collagen type I from 3 DIV [312]
fibroblast and type III after 1 DIV, integrin α2 expression was
higher for the aligned fibers
PLGA Periodontal ligament Osteogenic differentiation and osteopontin, osteocalcin, [313]
cells and bone sialoprotein marker expression. At 3, 6, 9, and
12 DIV cell membrane layers deposited and multilayered
structures established
Gelatin Periodontal ligament Cells cultured on the membrane in vitro exhibited good [314]
cells attachment, growth, and proliferation
PLGA Bone marrow Electrospun onto PLGA knitted scaffold. Higher [316]
stromal cells expression of collagen I, decorin, and biglycan genes
PLGA (65:35) Porcine bone Nanofiber coated knitted scaffolds could sustain cell [317]
marrow stromal growth and proliferation better than woven scaffolds and
cells promoted easy cell seeding
PLGA(85:15); Rabbit bone marrow More resistant scaffolds when cell seeded after 21 DIV [318]
stromal cells
PLCL Rat bone MSCs Collagen type III started to form a fibrous network 14 DIV [319]
Poly(ester urethane) Bone marrow Spindle-shaped morphology increased with fiber diameter [320]
stromal cells and degree of alignment. Expression of collagen 1 alpha 1,
decorin, and tenomodulin was greatest on the smallest fibers
PLCL (80/20) Bovine ligament Immunohistochemistry staining revealed ligament ECM [321]
fibroblasts markers collagen type I, collagen type III, and decorin
organizing and accumulating along the fibers
PLCL (80/20) Bovine ligament Deposition of ECM onto the fibers [322]
fibroblasts
PLLA/MWNTs/HA Periodontal ligament PLLA/MWNTs/HA enhanced adhesion/proliferation and [324]
cells inhibited the adhesion and proliferation of gingival
epithelial cells
PLGA (50:50) Rat periodontal Cells orientation along the fibers, higher level of tissue [325]
ligament cells organization than random scaffold
16.8.11 Keratinocytes
For effective skin reconstruction, both the dermis and the epidermis need to be reconstructed.
This requires the successful culture of keratinocytes. Such culture on a Petri dish presents chal-
lenges including the transfer of the cells. Therefore, thin membranes acting as mechanical supports
and reinforcements are used during cell culture to aid in handling the cells, thus facilitating their
transplantation. Transfer materials such as Laserskin, for example, assist in growing and expanding
keratinocytes ready for implantation [326]. In this way, electrospun fibers can be seen as a potential
transfer material when fabricated as a sheet and used for in vitro culture. Table 16.16 provides a list
of studies where electrospun materials are used as a substrate for keratinocyte culture.
16.8.12 Other Cells
Due to the number of specialized cells, tissue, and organs in the body, it is not possible to list all of
the different areas where electrospun fibers are used in regenerative medicine approaches. However,
Table 16.17 provides a selection of these different cell types and electrospun fibers.
TABLE 16.16
In Vitro Studies with Keratinocytes
PLGA Bimodal d = 530 nm and 28 μm. Higher cell attachment/spreading in the [13]
nano/microfiber than the microfibrous scaffolds without nanofibers
Silk fibroin Electrospun material was more preferable than SF film and SF microfibers [30]
Ag-containing PVA The Ag ions and Ag nanoparticles are cytotoxic to cells [184]
Silk fibroin O2-treated fibers showed higher cellular activities for normal human [196]
epidermal keratinocytes
TABLE 16.17
In Vitro Studies with Other Cell Types
PCL CHO-K1 Bimodal d = 400 nm and 6 μm; or d = 4 μm. Increased [12]
penetration of cells into scaffolds
Polyamide Rat kidney cells Cells rearranged their actin cytoskeleton to a more [181]
in vivo–like morphology
PCL Human amniotic Kinetics of osteogenic differentiation were observed between [248]
fluid stem cell hMSCs and hAFS cells, with the hAFS cells displaying a
delayed alkaline phosphatase peak, but elevated mineral
deposition, compared with hMSCs
PCL CHO Proliferation was influenced by the thickness of the scaffold [259]
(0.1 and 0.6 mm); scaffolds with 0.6 mm thickness were a
better substrate for proliferation
PCL and PCL/gelatin Adipose-derived Good attachment, although migration was more with [327]
stem cells PCL/gelatin
PCL and PCL/HAp Embryonic stem Cells proliferated at the same rate as cells growing on TCP [328]
cells and maintained pluripotency markers
16.9 IN VIVO RESULTS OF ELECTROSPUN MATERIALS

There are understandably a greater number of in vitro experiments using electrospun polymers than
there are in vivo studies, due to the increased time and effort to perform animal studies. To date,
in vivo experiments with electrospun fibers are all generally positive, with the electrospun material
demonstrating potential for the particular application.
16.9.1 Biocompatibility and In Vivo Cell Penetration

Electrospun scaffolds do not usually produce any major adverse reaction once implanted. Several stud-
ies have demonstrated relatively good biocompatibility with nano- and microfibers [329–331]. Cao et al.
compared the foreign body response of PCL randomly orientated nanofibers, aligned nanofibers, and
films in a subcutaneous rat model. They found that films and the two types of fibrous scaffold displayed
good biocompatibility as shown by a very early resolution of acute and chronic inflammation. Two weeks
post implantation no, or minimal, inflammation was observed. It was also found that aligned nanofibers
induced a less severe healing response when compared to random nanofibers. Although no definitive
conclusion could be made, it was hypothesized that the aligned nanofibers induced a more moderate
inflammation due to better tissue integration as higher cell infiltration was found in this type of scaffold.
As opposed to most in vitro studies, implanting electrospun materials generally leads to full [265] or
nearly full colonization of the scaffolds [332]. However, increasing the pore size, using for example the
sacrificial fiber removal technique, produced better cell infiltration and tissue integration [332].
16.9.2 Neural Tissue Engineering

In neural TE, most approaches involve attempts at mimicking the natural ECM by fabricating pores,
ledges, and fibers so as to present the growing axons with appropriate cues and a permissive environ-
ment. Perhaps the most numerous studies involve nerve guides, where cylinders of electrospun mate-
rial are generated on a rotating mandrel and used to bridge a gap defect in the peripheral nerve. Nerve
guidance channels (NGCs) are one of the most frequently implanted electrospun material constructs;
however, the in vivo results are not particularly compelling so far, when compared with previous nerve
guides. For peripheral nerve injuries, a critical gap length for different species dictates what maximum
distance regenerates 50% of the nerves [333]. For NGCs from electrospun fibers, regeneration does not
appear to extend beyond what would be expected for traditional biomaterials. However, electrospin-
ning is still developing and there are approaches to manufacture electrospun tubes that may provide
an advantage over traditional techniques. For example, the Bellamkonda laboratory has been filling
NGCs with electrospun meshes, showing encouraging outcomes for repair (Figure 16.11) [334].
1-film 1-film, NF160 (axons)
Axon core
Thin-film
Tube wall
(a) (b)
3-film 3-film, NF160
400 μm
(c) (d)
FIGURE 16.11 (See companion CD for color figure.) Nerve guides implanted within a peripheral nerve
regeneration model, containing single (a&b) or three (c&d) sheets of electrospun meshes. (Reproduced from
Clements, I.P. et al., Biomaterials, 30, 3834, 2009.)
(a) (b) (c) (d)
(e)
rhBMP-2 release from alginate

80
Cumulative release (ng)
60
(f )
40
20
0
12 4 7 10 14 21
(g) Day
FIGURE 16.12 (See companion CD for color figure.) (a) SEM image of electrospun PCL. (b) Hollow elec-
trospun tubular implants without perforations and (c) with perforations. (d) Scheme of implant in segmental
bone defect. (e) Defect after placement of a perforated mesh tube with alginate seen inside the tube through
the perforations. (f) One week postsurgery and alginate was present inside the defect, with hematoma present
at the bone ends. (g) Alginate release kinetics of the rhBMP-2 during the first 4 days.
16.9.3 Bone Tissue Engineering

Electrospun scaffolds were also used in bone TE although for non-load bearing implantation due
to the poor intrinsic mechanical properties of the fibrous mats. Generally, electrospun scaffolds
do not induce spontaneous and significant bone formation unless surface treated with osteo-
inductive materials [335,336] or seeded with osteo-inducted cells [337], or loaded with growth
factors such as BMP-2 [338]. However, when one or several of these conditions are used bone
formation can occur in vivo even in a nonbony environment such as subcutaneous implantation
[335,336]. A recent study on perforated electrospun tubes between bone defects shows some
fascinating outcomes, in that the perforated holes were crucial for attaining good bone forma-
tion (Figure 16.12).
16.9.4 Cartilage Tissue Engineering

TE using electrospun scaffolds offers the potential to develop treatments for defective articular
cartilage, where isolated articular chondrocytes or MSCs are seeded onto a scaffold before
implantation into a damaged joint. Here, the scaffold architecture is critical, as it should simu-
late the ECM of cartilage in order to promote cellular adhesion, proliferation, differentiation,
and migration, whilst also providing resistance to tensile, compressive, and shear stresses. Such
a strategy was used to regenerate a cartilage defect in a mini-pig model. Li et al. observed that
MSCs performed better than chondrocytes seeded onto PCL nanofiber constructs. Fibrous scaf-
folds seeded with MSCs resulted in the formation of hyaline cartilage–like tissue at 6 months
TABLE 16.18
In Vivo Studies with Electrospun Materials
Polymer Organ Comments Reference
PCL Peripheral nerve Preliminary data on scaffold made using gap method of [16]
alignment
Poly(urethane) Wound dressing Rate of epithelialization is increased and dermis is well [59]
organized if wounds are covered with electrospun
membrane
P(LLA-CL) (70:30) Inferior superficial Tubular scaffolds kept structural integrity for 7 weeks [210]
epigastric vein
PCL/COL Subcutaneous Good integration/neovascularization [265]
PCL-silica Bone Biocompatible and bioresorbable [273]
PLLA/MWNTs/HA Periodontal Cell/fibers were implanted into the leg muscle pouch [324]
ligament cells
P(AN-co-MA) Peripheral nerve Sheet-like structures in a conventional nerve guide [334]
PCL Bone Woven bone-like appearance, with mineralized matrix [337]
Chitosan Peripheral nerve Similar to autograft [341]
PLGA/PCL Peripheral nerve Modest regeneration over 10 mm gap after 16 weeks [342]
Bilayered chitosan tube Peripheral nerve YIGSR modified tubes regenerated similar to isograft after [343]
5 and 10 weeks
PCL Abdominal aortic Good patency, endothelialization, and cell ingrowth [344]
substitute
postimplantation, whereas the chondrocytes produced a fibrocartilage tissue [339]. It is also pos-
sible to surface treat the nanofibers with cationized gelatin to enhance their tissue integration
while maintaining or even promoting the secretion of cartilage ECM such as collagen II [340]
(Table 16.18).
16.9.5 Vascular Tissue Engineering

Alternatively, suitable vascular graft substitutes must be able to withstand pulsation and the high
pressure and flow rate of the blood stream. Electrospinning offers the potential to control the com-
position, mechanical properties, and structure of a graft while making it theoretically possible to
match the compliance of the synthetic scaffold to the native artery. Topographically aligned sub-
micron fibers have similar circumferential orientations to the cells and fibrils found in the medial
layer of the native artery. Because of these similarities between electrospun scaffolds and natural
ECM, and because of the large variety of materials that can be used, cell viability is often superior
to other scaffold designs. One crucial point in developing vascular grafts is the stability of the
implants. Any blockage of the inner part of the cylinder due to thrombosis and/or collapse of the
electrospun materials should be avoided. PCL-collagen tubular scaffolds have been demonstrated
to be very stable after 4 weeks postimplantation under normal hemodynamic conditions in a rabbit
aortoiliac by-pass model. This type of scaffold maintained structural integrity and could resist the
development of aneurisms [330].
16.10 SUMMARY
With the sudden interest in electrospinning and the desire to control the process to fabricate
different assemblies, various setups have been reported. The use of both rotating and translat-
ing devices, the manipulation of the electric field profile, and liquid and patterned stationary
collectors have proven to be successful in the fabrication of various fibrous assemblies. However,
most of the work has focused on solution electrospinning. Here, since increased deposition of
fibers will often result in an accumulation of charges, it remains to be seen whether these methods
are able to obtain thick, patterned fibrous meshes. The conditions required to form 3D structures
are still unclear: currently, the fibers forming reported 3D structures are very loosely packed
and easily compressed, rendering them unsuitable for applications where structural integrity is
required. Further advances in how scaffolds can be collected using melt electrospinning, coupled
with improvements in yield, may provide methods to fabricate aligned fibrous substrates with
increased porosity: useful for building complex structures that are of interest in TE applications.
By far the most studied of the two types of electrospinning, solution electrospun substrates are
interesting in many applications for regenerative medicine. More importantly, the diversity of
materials employed is excellent, and polymers from those both biologically derived and syntheti-
cally generated (and blends of both) can be readily used. The ability to generate electrospun
scaffolds with a variety of morphologies is also important, with highly oriented, random, and pat-
terned meshes already demonstrated. In this approach, surface segregation of polymeric additives
is interesting, since it provides a simple route to both biofunctionalize a fiber and render it protein
resistant. Undoubtedly, electrospinning will remain dominated by the use of solutions for the
foreseeable future, and advances in controlling the morphology and utility of polymeric scaffold
fabrication can advance electrospinning even further. At this point, it is pertinent to consider that
electrospinning for TE was proposed less than a decade ago and that the next decade will provide
further advances and breakthroughs in the process. We anticipate that electrospinning will remain
an important aspect of regenerative medicine research and become clinically important as the
field builds on the foundation of the last 10 years.
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17 Polymeric Nanoparticles
for Targeted Delivery
of Bioactive Agents and Drugs
Cesare Errico, Alberto Dessy, Anna Maria Piras,
and Federica Chiellini
CONTENTS
17.1 Introduction........................................................................................................................... 593
17.2 Techniques for Nanoparticle Preparations............................................................................. 596
17.2.1 Emulsion–Solvent Evaporation/Extraction Methods................................................ 596
17.2.1.1 Single Emulsion Method............................................................................. 596
17.2.1.2 Double Emulsion Method........................................................................... 597
17.2.2 Miniemulsion Methods.............................................................................................. 597
17.2.3 Phase Separation........................................................................................................ 598
17.2.4 Solvent Dispacement.................................................................................................. 598
17.2.4.1 Nanoprecipitation Method.......................................................................... 598
17.2.4.2 Coprecipitation Method.............................................................................. 599
17.2.5 Dialysis Method......................................................................................................... 601
17.2.6 Self-Assembling.........................................................................................................602
17.2.7 Rapid Expansion of Supercritical Fluid Solution......................................................603
17.2.8 Spray Drying..............................................................................................................603
17.2.9 Colloidal Coating.......................................................................................................604
17.3 Targeting Approaches of Polymeric Nanoparticles...............................................................605
17.3.1 Passive Targeting.......................................................................................................606
17.3.2 Direct Application of Drugs......................................................................................607
17.3.3 Physical Targeting......................................................................................................608
17.3.4 Active Targeting........................................................................................................608
17.4 Conclusions............................................................................................................................ 610
References....................................................................................................................................... 611
17.1 INTRODUCTION
The interdisciplinary field of nanobiotechnology, which combines chemistry, biology, engineer-
ing, and medicine, is revolutionizing the development of drug-delivery systems (DDS) and devices.
Novel materials and formulations enable the site-specific targeting and controlled release of tradi-
tional pharmaceuticals, recombinant proteins, vaccines, and nucleic acids (Goldberg et al. 2007);
safeguard drugs from spoilage as caused by attacks of enzymes, and enhance the penetration of the
active agent in the diseased tissues improving its bioavailability, with increase in efficacy and toxic-
ity reduction (Veronese and Caliceti 2002).
The field of drug delivery has attracted the attention of not only academic researchers but also phar-
maceutical industries because it offers a strategic tool to expand current drug markets; new delivery
593
technologies could repackage classic drugs, thus offering a competitive edge after theexpiration of
patents and preclude competition from generics (Parveen et al. 2011). According to a recent estimate,
the development of a new drug for human use involves around $800 million and 10–12 years of
research inputs. However, 9 out of 10 drugs fail in their clinical study phase causing huge loss to the
investigating organization. For DDS development, an U.S. estimate shows the development cost to
be around $40 million and a period between 3 months and 3 years. Thus the burden on company’s
exchequer is quite less with good chances of ensured returns (Mandal and Mandal 2010).
Similarly, the development of nanotechnology products may play an important role in adding a
new armamentarium of therapeutics to the pipelines of pharmaceutical companies (Farokhzad and
Langer 2009) and is likely to have a significant impact on the drug-delivery sector (Parveen et al.
2011). Depending on the final role of the developed DDS, its dimension and shape play a fundamen-
tal role, but an engineered nanoscale system is preferred for a defined interaction with the biological
environment at the molecular level (Wagner et al. 2006).
Nanoparticles can be correctly envisioned as the future of drug-delivery technology as they have
the potential to become useful therapeutic and diagnostic tools in the near future, with many poten-
tial applications in clinical medicine and research (Parveen et al. 2011).
Nanoparticles are frequently defined as solid, colloidal particles with size in the range of
10–1000 nm. The term “nanoparticles” is a collective term given for any type of polymer nanopar-
ticles, but specifically, it can be distinguished in nanospheres and nanocapsules. Nanospheres are
matrix particles, i.e., particles whose entire mass is solid, and bioactive molecules may be adsorbed
at the sphere surface or encapsulated within the particle bulk. In general, they are spherical, but
“nanospheres” with a nonspherical shape and those that are uniformly distributed are also described
in the literature. Nanocapsules are vesicular systems, acting as a kind of reservoir, in which the
entrapped substances are confined to a cavity consisting of a liquid core (either oil or water) sur-
rounded by a solid material shell (Rao and Geckeler 2011).
The relative interest of using nanoparticulate rather than microparticulate systems in pharma-
ceutical applications is in their potential to increase the absorption rate, improve bioavailability, and
enable target drug delivery and intravenous delivery systems.
Nanoparticles are taken up by cells more efficiently than larger micromolecules and, therefore,
could be used as effective transport and delivery systems. Drugs can either be integrated in the
matrix of the particle or attached to the particle surface (Suri et al. 2007) and their release kinetics
(Goldberg et al. 2007) can be tuned by surface or bulk erosion, diffusion through the matrix, swell-
ing followed by diffusion, or in response to the local environment inputs (Peer et al. 2007).
For a rapid and effective clinical translation, the developed nanocarriers should be made from a
material that is biocompatible, well characterized, and easily functionalized; exhibit high differen-
tial uptake efficiency in the target cells over normal cells (or tissue), either soluble or colloidal under
aqueous conditions for increased effectiveness; and have an extended circulating half-life, a low rate
of aggregation, and a long shelf life (Peer et al. 2007). Currently, both polymers and lipids are typi-
cally used as drug delivery vectors. However, polymers are the most commonly explored materials
for constructing nanoparticle-based drug carriers (Figure 17.1).
Polymeric nanoparticles can be made from synthetic polymers, including among the other bio-
compatible poly(lactic acid) (PLA) and poly(lactic co-glycolic acid), or from natural polymers such
as chitosan and collagen and may be used to encapsulate drugs without major chemical modifica-
tion (Peer et al. 2007). The role of polymers in DDS covers multiple aspects, from the enhancement
of the physical–chemical stability of the drug to the regulation of drug release profile and target-
ing (Vasir et al. 2005). They have highly tunable physical–chemical characteristics, and in some
cases, polymeric materials can be further processed or functionalized to more coherent systems.
Accordingly, they very likely represent the best-suited class of materials for the modern drug deliv-
ery technology (Kashyap et al. 2004).
Several different types of nanoparticles have successfully made their way into preclinical stud-
ies in animals, clinic trials in patients, or even successful commercial products used in routine
Polymeric Nanoparticles for Targeted Delivery of Bioactive Agents and Drugs 595
* *
x y
n
O O O O O O
OH O (CH2)3CH3 OH O (CH2)3CH3
PEG–OCH3 (CH2)2OCH3
VAM41-PEG
O H
HO O
CH3 O y
x
PLGA
O H
H O
n
CH3
PLA
HO O
O H
y
x
PLA-PEG
H OH
n
PCL
FIGURE 17.1 List of commonly used polymer as matrices for drug-loaded nanoparticle preparation.
clinical practice (Wang et al. 2009). Nanoscale albumin-based drug carriers have recently reached
clinic exploitation. Examples include Abraxane®, a nanoparticle of albumin and paclitaxel, and
Albuferon-α®, a conjugate of albumin and interferon-α (Hoffman 2008). A few drugs have been
marketed as nanoparticulate systems such as Amphotericin B, some antineoplastics, and others
including proteins, peptides, and macromolecules have been investigated in animal and clinical
models. The promises of nanoparticles as DDS are only limited by the choice of suitable biocom-
patible polymers (Mandal and Mandal 2010). Compared with the total pharmaceutical and medical
device market, nanostructured medical formulations currently constitute a tiny niche. However,
great strength lies in the versatility of nanomedicine: Nanotechnology has the potential to add inno-
vative functionality and bioefficacy to many pharmaceutical products and medical devices (Wagner
et al. 2006).
17.2 TECHNIQUES FOR NANOPARTICLE PREPARATIONS

For the preparation of nanoparticles using biodegradable or biostable, biocompatible polymers, it is
important to choose an appropriate encapsulation process which meets the following requirements:
chemical stability and biological activity of the incorporated drug, high encapsulation efficiency,
reasonable size range, and reproducible release profile.
There are a number of techniques available for the preparation of drug-loaded nanoparticulate
systems such as the emulsion solvent evaporation/extraction method, spray drying, phase separa-
tion–coacervation, interfacial deposition, and in situ polymerization. Each method has its own
advantages and disadvantages. The choice of a particular technique depends on polymer and drug
features, site of action, and therapy regimes (Errico et al. 2009b).
The methods currently used to produce nanoparticles can be divided into three main groups:
1. Physicochemical methods, e.g., the creation of nanoparticles using preformed polymers

and inducing their precipitation by emulsification–solvent evaporation, diffusion, or
reverse salting-out
2. In situ chemical synthesis methods of macromolecules, giving rise for instance to bulk
polymerization or interfacial polycondensation reactions
3. Mechanical methods, e.g., use of high-energy devices like high-pressure homogenizers,
sonifiers, or high-energy wet milling apparatuses [Li 2010]
The techniques that follow concise descriptions concerning the preparation of nanoparticles from
preformed polymers are reported.
17.2.1 Emulsion–Solvent Evaporation/Extraction Methods

17.2.1.1 Single Emulsion Method
The solvent evaporation technique is generally used for the preparation of microspheres based on
polyesters. However, microspheres prepared by the typical technique have a relatively large particle
size of 5–100 μm, do not show a very narrow size distribution, and the solvent used may not be
completely removed. It is a very popular method because it is easy and it mainly allows efficient
encapsulation of numerous compounds of lipophilic nature (Desgouilles et al. 2003).
This method has been primarily used to encapsulate hydrophobic drugs through oil-in-water
(o/w) emulsification process (Park et al. 2005). The hydrophobic drug is dissolved or dispersed in
an organic solvent into the polymer solution, and the resulting mixture, after emulsification by high-
speed homogenization or sonication, is added into an aqueous solution to make an o/w emulsion with
the aid of amphiphilic macromolecules, which are known as emulsifier/stabilizer/additives (Si-Feng
2004). The solvent in the emulsion is removed by either evaporation at elevated temperatures or
extraction in a large amount of water, resulting in the formation of compact particles (Figure 17.2).
The solvent evaporation method has been used extensively to prepare PLA and PLGA micro-
and nanoparticles containing many different drugs (Fonseca et al. 2002; Kim and Martin 2006).
Several variables have been identified that can influence the properties of the nanoparticles, includ-
ing drug solubility, internal morphology, solvent type, diffusion rate, temperature, polymer com-
position, medium viscosity, and drug loading (Panyam et al. 2004). This method, however, is only
available for the hydrophobic drugs because the hydrophilic drugs may diffuse out or may partition
out from the dispersed oil phase into the aqueous phase, leading to poor encapsulation efficiencies
Water Nanoparticles
Emulsion
Polymer
drug
Organic solvent
FIGURE 17.2 Nanoparticles preparation: oil in water emulsion technique.
(Park et al. 2005). Many types of drugs with different physical and chemical properties have been
formulated into polymeric systems, including anticancer drugs, narcotic agents, local anesthetics,
steroids, and fertility control agents (O’Donnell and McGinity 1997).
17.2.1.2 Double Emulsion Method

In double emulsion formulations, microparticles with different morphologies are generated due to
the presence of the aqueous-phase microdroplets inside the emulsion droplet. During the solvent
elimination, these microdroplets generally coalesce under the pressure of the precipitating polymer.
Depending mainly on the polymer concentration and emulsification energies, the final micropar-
ticles will be a mixture of honeycomb, capsule, or plain structures. During the shrinkage due to the
incompressibility of the inner microdroplets, the precipitating polymer wall around them may break
forming holes through which the encapsulated substance partly leaks. Through these holes, the encap-
sulated substance is further partitioned with the external aqueous phase during solvent evaporation
and contributes to the initial burst release during the therapeutical administration (Rosca et al. 2004).
Several water-soluble drugs have been encapsulated by water-in-oil-in-water (w/o/w) methods.
The aqueous solution of the water-soluble drug is emulsified with polymer-dissolved organic solu-
tion to form the water-in-oil (w/o) emulsion. The emulsification is carried out using either high-
speed homogenizers or sonicators. This primary emulsion is then transferred into an excess amount
of water containing an emulsifier under vigorous stirring, thus forming a final w/o/w emulsion. In
the subsequent procedure, the solvent is removed by either evaporation or extraction process.
One advantage of this method is bound to the possibility of encapsulating hydrophilic drugs in
an aqueous phase with a high encapsulation efficiency. For this reason, the w/o/w emulsion system
has been used widely for the development of protein delivery systems. The characteristics of the
particles prepared by the double emulsion method are dependent upon the properties of the poly-
mer (such as composition and molecular weight), the ratio of polymer to drug, the concentration
and nature of the emulsifier, temperature, and the stirring/agitation speed during the emulsifica-
tion process (Park et al. 2005). Polymers such as poly(lactic acid), poly(lactic acid)/poly(ethylene
glycol), poly(d,l-lactic acid-co-glycolic acid), and poly(ethylene oxide)/poly(lactic acid) are the
most utilized in the described technical methodology (Rao and Geckeler 2011).
17.2.2 Miniemulsion Methods
In recent years, miniemulsions have been studied and successfully applied in cosmetics and thera-
peutics for skin treatments. They can be defined as heterophase systems consisting of thermo-
dynamically stable nanodroplets (possessing an average diameter between 50 and 500 nm) in
a continuous phase. Miniemulsions can be basically divided into o/w and w/o systems (W/O);
the aforementioned systems are commonly studied both in the development of miniemulsion
polymerization processes and for the production of emulsion-based DDS for the time-controlled
administration of a variety of drug and bioactive agents. These comprise antitumor agents, peptide
drugs, sympatholytics, local anaesthetics, steroids, anxiolytics, anti-infective drugs, vitamins, anti-
inflammatory drugs, and dermatological products. The use of miniemulsions in the development of
skin care products presents many advantages related to the system stability against sedimentation
and to the use of skin-friendly ingredients. Dead Sea Minerals (DSM)-loaded polymeric nanopar-
ticles can be prepared by means of a w/o combined miniemulsion/solvent evaporation process. In
the treatment of skin diseases, the beneficial effects of Dead Sea Water (DSW) are well known.
Therapeutic baths in the Dead Sea are commonly used as a treatment both for psoriasis, atopic
dermatitis, and UV-damaged skin. DSW salt composition is represented by a high concentration of
different minerals such as magnesium, calcium, bromide, sodium, potassium, zinc, and strontium.
Ahava, an Israeli cosmetics company manufactures skin care products made of mud and mineral-
based compounds from the DSW.
The first step consists in the development of a stable nanoemulsion. Nanoparticles were prepared
by dissolving an amphiphilic ionomeric polymer such as 2-methoxyethyl hemiester of poly(maleic
anhydryde-alt-butylvinylether) grafted with 5% of methoxy-PEG2000 (VAM41-PEG) and DSM in
a water/ethanol mixture while nonionic surfactants are dissolved in paraffin oil. The two phases are
mixed by means of ultrasounds to obtain a miniemulsion. An oily nanoparticles suspension can then
be obtained by removing ethanol and water under vacuum (Dessy et al. 2011a).
17.2.3 Phase Separation
This method involves phase separation of a polymer solution by adding an organic nonsolvent.
Drugs are first dispersed or dissolved in a polymer solution. To this solution an organic nonsolvent
(e.g., silicon oil) is added under continuous stirring, by which the polymer solvent is gradually
extracted and soft coacervate droplets containing the drug are generated. The rate of nonsolvent
addition affects the extraction rate of the solvent, the size of the particles, and encapsulation
efficiency of the drug. The commonly used nonsolvents include silicone oil, such as dimethyl veg-
etable oil, light liquid paraffin, and low-molecular-weight polybutadiene. The coacervate phase
is then hardened by exposing it into an excess amount of another nonsolvent such as hexane,
heptane, and diethyl ether. The final characteristics of the colloidal product are affected by the
molecular weight of the selected polymer, viscosity of the nonsolvent, and polymer concentration.
The main disadvantage of this method is represented by the high possibility of ending with large
aggregates. Infact extremely sticky coacervate droplets frequently adhere to each other before
complete phase separation.
This technique is promising for preparation of protein-loaded microcapsules. For example,
conventional methods of preparing microparticles involve extensive exposure of proteins to the
interface between aqueous and organic phases, to hydrophobic polymer matrix, and to acidic/basic
microenviroments resulting from degradation of the polymer. These unfavorable interactions are
reported to induce conformational changes of proteins. On the contrary, the interfacial phase sepa-
ration technique is shown to minimize these sources of protein inactivation (Yeo et al. 2004) thus
enabling a satisfactorily maximum therapeutical efficacy of the prepared systems.
17.2.4 Solvent Dispacement
17.2.4.1 Nanoprecipitation Method
The nanoprecipitation method, also called solvent displacement, was developed by Fessi et al. It
is one of the easiest preparation procedures of nanospheres. Additionally due to its simplicity, this
procedure is reproducible, fast, and economic and it uses preformed polymers as starting materials
rather than monomers (Vauthier and Bouchemal 2008).
The polymers commonly used are biodegradable polyesters, especially poly(ε-caprolactone)

(PCL), polylactide (PLA), and poly(lactide-co-glycolide) (PLGA). Eudragit, poly(butyl methacylate-
co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate), can also be used as many other
polymers such as polyalkylcyanoacrylate (PACA). Natural polymers such as allylic starch and dex-
tran ester were also used, though synthetic polymers have higher purity and better reproducibility
than chemically modified natural polymers. Some of the used polymers are characterized by the
presence in the macromolecular structure of poly(ethylene glycol) (PEG) fragments as grafts or as
blocks inherent in the chain backbone. The presence of PEG in the polymer matrix prevents the
opsonization of the nanoderived formulations with consequent abatement of the decrease nanopar-
ticle recognition by the reticular endothelial system (Rao and Geckeler 2011).
The nanoparticle formation is instantaneous and the entire procedure is carried out in only one
step. The polymer and the drug are dissolved together and precipitated into a nonsolvent, miscible
with the former one. Nanoprecipitation occurs by a rapid desolvation of the polymer when the poly-
mer solution is added to the nonsolvent. Indeed, as soon as the polymer-containing solvent has dif-
fused into the dispersing medium, the polymer precipitates, involving immediate drug entrapment.
The rapid nanoparticle formation is governed by the so-called Marangoni effect (Scriven and
Sternling 1960), which is due to interfacial turbulence taking place at the interface of the solvent
and the nonsolvent and results from complex and cumulated phenomena such as flow, diffusion, and
surface tension variations. Nanoprecipitation often enables the production of small nanoparticles
(100–300 nm) with narrow unimodal distribution and a wide range of preformed polymers can be
used, such as PLGA, cellulose derivatives, or poly caprolactones.
This method does not require extended shearing/stirring rates, sonication, or very high tempera-
tures, and is characterized by the absence of oily-aqueous interfaces, all conditions that might dam-
age the structure of very sensitive bioactive agents and drug as proteins. Moreover, surfactants are not
always needed and unacceptable toxic organic solvents are generally excluded from this procedure.
However, the original nanoprecipitation method suffers because of some limitations correlated
to the nature of the drug to be loaded. This technique is mostly suitable for compounds having a
hydrophobic nature such as indomethacin, which is soluble in ethanol or acetone, but displays very
limited solubility in water. Recently, formulation and process modifications were investigated to
improve the versatility of the nanoprecipitation technique, particularly with respect to the encapsu-
lation of hydrophilic drugs (e.g., proteins and poly saccharides) (Bilati et al. 2005).
The nanoprecipitation method has been used, however, extensively to prepare nanoparticles con-
taining many different drugs (Fonseca et al. 2002; Dong and Feng 2004).
17.2.4.2 Coprecipitation Method

The coprecipitation method is an original and straightforward procedure recently developed
(Cowdall et al. 1999a,b) that appears advantageous for the loading of protein drugs into a poly-
mer matrix. In this case, the polymer is dissolved in a water-miscible organic solvent and added
dropwise to an aqueous solution containing the selected loading water soluble drug (proteins or
oligonucleotides) and appropriate stabilizers (protective colloids). During the coprecipitation
process, the polymeric material gives rise to microphase separation because of its low water
solubility and the concurrent interaction with macromolecular drug leads to nanoparticle forma-
tion (Figure 17.3).
This methodology does not entail the use of chlorinated solvents and it does not require vigorous
shear mixing, preventing appreciable denaturation of the entrapped bioactive macromolecular drug
that typically occurs with other techniques. It is well established, in fact, that exposure of bioac-
tive drug such as proteins to denaturation factors including organic solvent and sonication during
the encapsulation process can reduce the biological activity of proteic active agents (Park et al. 1995;
Zambaux et al. 1999; Pawar et al. 2004). Albumin, λ-interferon, trypsin, urokinas, and hemoglobin
(Hb) have been loaded into bioerodible polymeric nanoparticles by following the coprecipitation tech-
nique (Chiellini et al. 2006, 2007; Piras et al. 2006, 2007; Dinucci et al. 2007).
FIGURE 17.3 Nanoparticles preparation: coprecipitation technique.
In particular the feasibility of loading Hb into polymeric nanoparticles by means of a nonag-

gressive technique, like the coprecipitation, can lead to potential applications in the biomedical
field. The encapsulation of Hb into nanostructures has in fact attracted the attention of the scien-
tific community over the past decades, due to the perspective of developing artificial systems that
can substitute blood transfusions in restoring oxygen homeostatic concentration. At present, blood
transfusions are in fact mostly applied for this purpose, but the need of the right type of blood and
its short shelf life are still serious problems to be overcome. In this case, the use of autologous trans-
fusions prevents the need of cross-matching and, although autologous transfusions are considered
the safest, they are not always feasible because they may cause perioperative anemia and are more
expensive than allogenic transfusions (Pape 2007). In the meanwhile, the direct administration of
Hb is not feasible due to its short half life, abnormal high oxygen affinity, and the incoming of seri-
ous sides effects, namely malaise, abdominal pain, hemoglobinuria, and renal toxicity (Ness and
Cushing 2007). The potential benefits of developing artificial blood substitutes can thus be related
to the universal compatibility, the on-need availability, freedom from disease transmission, and
long-term storage potential. In the past decades, many strategies have been investigated in order
to obtain artificial oxygen carriers that could solve the aforementioned donor transfusion associ-
ated drawbacks. Many studies regarding the employment of fluorocarbons (Clark and Gollan 1966;
Riess 2001; Castro and Briceno 2010) or chemically modified Hb are reported in the literature (Li
et al. 2006; Asanuma et al. 2007; Hu et al. 2007); anyway both approaches have revealed the pres-
ence of severe side effects and altered oxygen carrier ability once injected in vivo (Sharma et al.
2011). The encapsulation of Hb into structures able of mimicking red blood cell membranes (such as
polymeric nanoparticles, liposomes, and solid lipid nanoparticles) represents an alternative strategy
that has shown favorable results in terms of biocompatibility and maintenance of protein physico-
chemical properties. Different polymeric matrices have been tested for the production of Hb-loaded
polymeric nanoparticles by means of the coprecipitation technique; among these the employment
of VAM41-PEG polymer has shown favorable results. As reported in literature (Dessy et al. 2011b),
stealth nanoparticles possessing suitable features to be used as injectable systems were obtained by
applying the coprecipitation technique under controlled atmosphere and temperature (Figure 17.4).
Moreover, the introduction of different reducing agents in the VAM41-PEG Hb-loaded nanoparticles
formulation system, aimed at minimizing protein oxidative phenomena, did not alter nanoparticles fea-
tures in terms of dimension, shape, and protein loading, highlighting the extreme versatility of the
coprecipitation technique (Dessy et al. 2011b).
(a)
(b)
FIGURE 17.4 VAM41-PEG Hb-loaded polymeric nanoparticles: (a) SEM micrograph and (b) water
suspension.
17.2.5 Dialysis Method
Some disadvantages of the conventional methods include the difficulties and necessities of removal
of solvent and surfactant residues, low particle yields, excessive time-consuming steps for prepara-
tion, and the necessity to use a high concentration of surfactant for the preparation of small spherical
particles and polymeric micelles. The dialysis method is a simple and effective preparation method
for small and narrow-sized distributed nanoparticles mostly using either block or graft copolymers
and other amphiphilic materials. Polymer, drug, and surfactants are dissolved in the same organic
solvent and placed inside a dialysis tube with proper molecular weight cut-off. Dialysis is performed
against a nonsolvent, miscible with the former one. The displacement of the solvent inside the mem-
brane is followed by the progressive aggregation of polymer, drug, and surfactants due to a loss of
solubility and by the formation of homogeneous suspensions of micro- nanoparticles (Errico et al.
2009) (Figure 17.5).
Trials of preparing nanoparticles in the absence of surfactants were performed. The dialysis
method was applied to PLGA starting from different solvent solutions and avoiding the use of
surfactants. The solubility of the particle components in the initial solvents may affect the physi-
cochemical properties such as particle size and drug contents, whereas the absence of surfactant
led to some drawbacks such as instability of the re-dispersed freeze-dried PLGA nanoparticles
and low drug-loading efficiency (Jeong et al. 2001). PLA/Tween 80 copolymers were synthe-
sized, aiming at incorporating a widely used emulsifier for biomedical nanoparticles preparation
directly in the polymer backbone. Nanoparticles loaded with anticancer drugs were obtained by
using the dialysis method and without the use of additional surfactants/emulsifiers (Zhang and
Feng 2006).
Dialysis method was applied in the preparation of PLGA and PHB nanoparticles encapsulat-
ing retinoic acid (RA) and some processing conditions varied in order to observe their effect on
nanoparticle characteristics. The use of a binary solvent solution composed of a mix of polymer and
drug solvent and nonsolvent proved to be effective for preparing nanoparticles with small size and a
narrow distribution of diameters. The small diameter of PHB nanoparticles prepared by this method
was quite exceptional when compared with those of PHB microsphere prepared by other techniques
commonly used such as solvent evaporation (Errico et al. 2009).
Dialysis tube
Receiving Organic solvent

compartment
Water
FIGURE 17.5 Nanoparticles preparation: dialysis technique.
17.2.6 Self-Assembling
The concept of self-assembly has been extensively used to prepare organic nanoparticles. For exam-
ple, liposomes and nanoscaled vesicles that were prepared using self-assembly of phospholipids can
serve as powerful nanocarriers for drug and gene delivery. Self-assembled amphiphilic copolymer
building blocks spontaneously form nanoparticles, which can be used for drug delivery and molecu-
lar imaging (Wang et al. 2009).
Such association depends on many factors including coulombic interactions, hydrophobicity of
the polymer–molecule pair, and the conformational features of the polymer. One special class of
such systems is represented by the complexes formed from macromolecular monomers of oppo-
site charges. The solution behavior of these complexes strongly depends on their composition.
Electroneutral complexes that contain equivalent amounts of polyion units and monomers are
water insoluble.
Nonstoichiometric complexes containing an excess of one of the components are generally soluble
in water. Since these complexes are capable of forming aggregates of nanometer size, they have been
termed as polyion complex (PIC) micelles or block ionomer complexes (BICs) (Gupta et al. 2006).
Micellar DDS enjoy several advantages over other particulate configurations. For example,
they can significantly enhance the water solubility of hydrophobic drugs for improved bioavail-
ability. They usually exhibit low critical micelle concentration (CMC), rendering the drug-loaded
micelles stable in the bloodstream to achieve fairly long circulation time (Liu et al. 2003).
Typical studies were focused on polystyrene-b-poly(ethylene oxide), polystyrene-b-poly(acrylic
acid), polystyrene-b-poly(methacrylic acid), etc. The traditional micelles are unable to sense a sig-
nal and respond by changing their structures. Incorporating external control over structure and
physical properties offers the possibility of constructing systems capable of both tunable transfor-
mation and controlled transmission of energy or information. For this purpose, thermosensitive
(Liu et al. 2003; Wei et al. 2006), pH-sensitive (Hruby et al. 2005; Hsiue et al. 2006), and pH- and
temperature-sensitive micelles (Determan et al. 2005) had been engineered.
17.2.7 Rapid Expansion of Supercritical Fluid Solution

Supercritical fluids and dense gases have unique properties that make them suitable for various
applications such as extractions, reactions, and particle formation processes. In recent years, consid-
erable interest has been expressed in the field of supercritical fluid and dense gas technologies in an
attempt to find alternative and improved methods of pharmaceutical processing where biodegrad-
able compounds are used frequently (Mishima 2008).
A supercritical fluid is loosely defined as a solvent at a temperature above the critical tempera-
ture, at which the fluid remains a single phase regardless of pressure. However, for practical pur-
poses, such as high density for solubility considerations, fluids of interest in materials processing are
maintained typically at near-critical temperatures.
Among the most important properties of a supercritical fluid are the low and tunable densities,
which can be easily varied from gaslike to liquidlike through a simple change in pressure at constant
temperature, and the unusual solvation effects at densities near the critical density (often discussed
in terms of solute–solvent and solute–solute clusterings). The production of polymeric nanoparticles
through “Rapid Expansion of a Supercritical Solution” into either air (RESS) or Liquid Solvent
(RESOLV) may conceptually be divided into two somewhat related processes. One is the initial
formation of nanoparticles in the rapid expansion, and the other is the stabilization of the suspended
nanoparticles. Evidently, the protection of initially formed polymeric nanoparticles represents a
different set of technical challenges, which are largely independent of the rapid expansion process
itself, especially if the protection agent is added immediately after the expansion. The good point is
that many methods aimed at stabilizing nanoparticle suspensions are already available in the litera-
ture, some of which have shown promise in use with RESOLV.
Supercritical fluid processing techniques can play a significant role in the particle formation and
production. In particular, the RESOLV technique offers a unique way to prepare clean and narrowly
distributed polymeric nanoparticles. Since the nanoparticles obtained in RESOLV are suspended,
they may be protected from agglomeration by using existing stabilization methods and agents. These
stable nanoparticle suspensions may find many interesting and important applications, as already
discussed in the literature (Sun et al. 2005; Meziani et al. 2006). Furthermore, a new method called
Supercritical Fluid Extraction of Emulsions (SFEE) has been investigated. The method combines
the advantages of traditional emulsion-based techniques, namely control of particle size and surface
properties, with the advantages of continuous supercritical fluid extraction process, such as efficient
scale-up, higher product purity, and shorter processing times (Chattopadhyay et al. 2006).
17.2.8 Spray Drying
Spray drying technology is widely known and used to transform liquids (solutions, emulsions, sus-
pension, slurries, pastes, or even melts) into solid powders. A traditional spray dryer is generally
used to transform liquid substances into powders rapidly and efficiently. The speed of the process
and the consequently short drying time enable the drying of even temperature-sensitive products
without degradation. This spray drying process is particularly used to improve product conservation
in dried solid form (Li et al. 2010).
Compared to other conventional methods, spray drying offers several advantages. It shows good
reproducibility, involves relatively mild conditions, allows for control of the particle size, and is
less dependent on the solubility of the drug and the polymer. Generally, the polymer is dissolved in
volatile solvents and the drug is dispersed or dissolved in the polymer solution.
Solutions or dispersions are sprayed against a stream of cold air (−60°C; top-spraying) using
a two-fluid pneumatic nozzle with heating facilities. The frozen droplets formed by this spray-
freezing step are dried during the following atmospheric freezedrying in the cold desiccated air
stream by sublimation. A filter holds the fine product back in the drying chamber, while the water
vapor is removed by the circulating air in the cooling systems, where the humidity condenses on
the refrigerated surfaces (Leuenberger 2002). Recently this method has been used to prepare dry
powder aerosol particles (Azarmi et al. 2006), a powder formulation for controlled delivery of
Paclitaxel (Mu et al. 2005), and powders for aerosol delivery to the lung (Sham et al. 2004).
In an attempt to minimize aggregation of the microparticles, a double-nozzle spray-drying
technique was developed. While the polymer/drug solution is sprayed from one nozzle, aqueous
mannitol solution is simultaneously sprayed in order to coat the particulate with an anti-adherent
agent. The results indicate that the coating of the microspheres with mannitol reduces the extent
of aggregation and augments the yield of the product (Takada et al. 1995). When a protein drug is
encapsulated by means of spray drying, a loss of its biological activity may occur especially when
aqueous phase systems are involved (Elversson and Millqvist-Fureby 2005). The atomization of a
w/o emulsion or a cryogenic nonaqueous processes can be used as an alternative to spray drying
(De Rosa et al. 2005). In the latter technique, the liquid droplets of the polymer/drug solution are
produced through the spraying nozzle, collected in liquid nitrogen containing frozen ethanol, and
hardened by placing them at −80°C where the solvent extraction occurs (Johnson et al. 1996).
In a recent study, Li et al. presented the Nano Spray Dryer B-90, a revolutionary new sprayer
developed by Büchi, use of which can lower the size of the produced dried particles by an order
of magnitude attaining submicron sizes. The strength of the BüchiNanoSprayDryerB-90 lies in
its vibration mesh spray technology, creating tiny droplets (before evaporation) in a size range
of a smaller order of magnitude than in classical spray dryers. This is a revolution in spray dry-
ing technology, making it possible to produce powders in the submicron-size range with very
narrow distributions and high formulation yields. The Büchi Nano Spray Dryer B-90 appeared
to provide very satisfactory results for the formulation of submicron particles, with relatively
high yields (70%–90%) for small sample amounts. Five representative polymeric wall materials
(Arabic gum, whey protein, polyvinyl alcohol, modified starch, and maltodextrin) were spray
dried and the resulting size distributions were shown to be mainly below the 1-μm scale, attain-
ing sizes as low as ∼350 nm, which is a very noteworthy result for spray drying technology
(Li et al. 2010).
17.2.9 Colloidal Coating
Although a number of different methods exist for the preparation of nanoparticles, it is highly desir-
able to provide alternative methods, aimed at producing polymeric particles that do not entail the
utilization of organic solvents, which are undesirable for their potential toxicity. Such methods are
expected to receive a particular attention in biotechnological field. An organic solvent-free method
has been developed for the preparation of nanoparticles based on a copolymer of maleic anhydride
and butyl vinyl ether (VAM41) nanoparticles loaded with RA. This method is easy to perform,
effective for the preparation of relatively small nanoparticles with narrow size distribution, and
operator friendly since toxic organic solvents need not be handled.
The novel nanoparticle preparation method was conceived by taking into account the charac-
teristics of the drug, polymer, and stabilizing agent (HSA) and the behavior of their different mix-
ture in response to variation of water pHs. At pHs below 7, RA self-associates into a micellar-like
structure while it is soluble at pHs above 8 in its deprotonated form. Alike, VAM41, due to the
presence of the carboxylic ionizable group, is soluble in water at pHs above 8 and is insoluble at
pHs below 7. A basic solution of the drug, polymer, and stabilizing agent was prepared and the
subsequent acidification of the solution provoked the polymer to absorb and coat uniformly the sur-
face of the forming micellar suspension of RA, leading to the formation of small and narrow-size
distribution nanoparticles.
Somehow this process resembles the practice of tablet coating in which tablets are loaded in
large rotating pans and coated with synthetic polymers or polysaccharides. In the case of tablet
coating, it is the evaporation of the solvent that triggers the coating process, while in this case, it is
the decrease of pH (Errico et al. 2009).
17.3 TARGETING APPROACHES OF POLYMERIC NANOPARTICLES

The biophysical-chemical properties of the vehicle, such as size, charge, surface hydrophilicity, and
the nature and density of the ligands on their surface, can all impact the circulating half-life of the
particles as well as their biodistribution (Farokhzad and Langer 2009).
The introduction of three key technologies stimulated the immense activity and clinical success
of nanotherapeutics from the late 1980s to the present. The first was the concept of “PEGylation,”
which refers to polyethylene glycol-conjugated drugs or drug carriers (Veronese and Pasut 2005).
The second is the concept of “active targeting” of the drug conjugate by conjugating cell membrane
receptor antibodies, peptides, or small molecule cell ligands to the polymer carrier (Timko et al.
2011) (Figure 17.6).
The third was the discovery of the “enhanced permeation and retention (EPR) effect”, wherein
nanoscale carriers are entrapped within solid tumors due to leaky vasculature of the fast-growing
tumor. This is called “passive” targeting as contrasted with active targeting (Hoffman 2008),
whereas the former two were considered a kind of active targeting (Figure 17.7).
More recently, surface properties of nontargeted drug-delivery vehicles such as ordered stria-
tions of functional groups as well as their shape and size have also been shown to enhance particle
uptake (Farokhzad and Langer 2009).
Cell membrane
receptor antibodies
Drug
PEGylation
FIGURE 17.6 Representation of a PEGylated and targeted nanoparticle.
(a) (b)
FIGURE 17.7 Schematic representation of passive (a) and active (b) targeting.
To reach the site of action, the carrier has to cross many biological barriers such as other organs,
cells, and intracellular compartments, where it can be inactivated or can express undesirable effects.
The striking advantages of drug targeting can be summarized as (1) simplification of drug admin-
istration protocols, (2) reduction of the drug quantity required to achieve a therapeutic effect with
abatement of therapy costs, and (3) sharp increase of the drug concentration in the required sites
without negative effects on nontarget compartments.
17.3.1 Passive Targeting
Passively targeting nanocarriers first reached clinical trials in the mid-1980s, and the first products,
based on liposomes and polymer–protein conjugates, were marketed in the mid-1990s (Peer et al.
2007). Passive targeting is generally based on the relation between the size of the drug carrier and
the tissue characteristics, such as their permeability.
A typical example of passive targeting based on particle density, shape, and size is the targeted
delivery of drugs to specific lung tissues. Before releasing the pharmacological agent, inhalated par-
ticles need to reach and deposit in the site of action. The deposit of particles in the respiratory tract
is governed by impaction (inertial deposition), sedimentation (gravitational deposition), brownian
diffusion, interception, and electrostatic precipitation. In mammals, the upper airways (nose, mouth,
larynx, and pharynx) and the branching anatomy of the tracheobronchial tree act as a series of filters
for inhaled particles. Thus, aerosol particles bigger than 100 μm generally do not enter the respira-
tory tract and are trapped in the naso/oropharynx. Particles bigger than 10 μm will not penetrate the
tracheo-bronchial tree; they must be less than 5 μm to reach the alveolar space and bigger than 0.5 μm
to not be exhaled without deposition (Crowder et al. 2002). The carrier particles should meet special
requirements such as an appropriate mass median aerodynamic diameter (MMAD) and a suitable
fine particle fraction (FPF). Direct delivery of drug-loaded nanoparticles to the lungs combines the
concepts of localized delivery with the advantages of using nanoparticles in lung cancer therapy. The
recent developments of inhalable biodegradable nanoparticles, large porous particles, and liposomal
dry powders make inhalation a feasible alternative approach to deliver macromolecules such as insu-
lin and for the treatment of lung-specific diseases such as tuberculosis. One issue with nanoparticle
pulmonary delivery is that their size is not suitable for deep lung deposition. In fact, a carrier system
such as lactose microparticles is required for deep lung delivery (Roa et al. 2011).
Concerning the intravenous administration of nanoparticles, passive targeting to body compart-
ments may be achieved by extravasation in the diseased tissue or organ (Palmer et al. 1984). The
enhanced therapeutic potencies are mainly due to the EPR effect that is attributed to high vascular-
ization and enhanced permeability of tumor blood vessels combined with limited lymphatic clear-
ance of macromolecules from the tumor environment (Min et al. 2008).
The ability of vascular endothelium to increase its permeability was first noticed in tumors and
in hypoxic areas of infarcted myocardium (Palmer et al. 1984).
In tumors, the defective vascular architecture is due to the rapid vascularization necessary to serve
fast-growing cancers, coupled with poor lymphatic drainage (Brannon-Peppas and Blanchette 2004).
In such areas with increased vascular permeability, even relatively large particles ranging from
10 to 500 nm in size can extravasate, accumulate inside the interstitial space, and release the loaded
active agent. Because the “cut-off” size of the permeabilized vasculature can vary from case to case,
the size of the DDS may be used to control the efficacy of such spontaneous or “passive” drug delivery.
However, to exploit this targeting method, drug carriers should circulate in blood long enough to
provide acceptable accumulation of the bioactive molecule in the area of interest.
Particles with 0.3- to 7-μm diameter have short circulation half-lives due to their fast removal
by the natural defense mechanisms of the body. After opsonization, these particles are seized by
the Mononuclear Phagocytic System (MPS), also known as RES. Therefore, in order to take advan-
tage of the EPR effect, the particle surfaces need to be modified to be “invisible” to opsonization
(Maeda et al. 2009).
In a recent study, Min et al. (2008) used a hydrophobically modified glycol chitosan-based (HGC)
nanoparticle as a Camptothecin (CPT) delivery carrier because it has been previously confirmed
that such nanoparticles are promising therapeutic DDS for hydrophobic drugs such as doxorubicin,
Paclitaxel, and therapeutic peptide drugs. CPT, a natural plant alkaloid extracted from Camptotheca
acuminate, has been shown to be a potent anticancer drug targeting intracellular topoisomerase I.
CPT and CPT analogs are increasingly explored in clinical treatment and show great utility in the
treatment of various cancer forms including primary and metastatic colon carcinoma, small-cell
lung carcinoma, and ovarian, breast, pancreatic, and stomach cancers. The achieved results showed
that compared to free CPT, CPT-HGC nanoparticles showed a longer circulation time in the blood,
a better targeting of drug to tumor tissue, and a substantial enhancement of antitumor activity (Min
et al. 2008).
In a site of inflammation, one of the cardinal features of the inflammatory reaction is the increase
in vascular permeability to solutes and macromolecules (Rossler et al. 1995). This phenomenon
determines the extravasation of the nanoparticles at the diseased site (Coester et al. 2000). As an
example, during multiple scleroses (MS), the permeability of the blood brain barrier (BBB) is
increased and the passage of nanoparticulate systems could be facilitated. PACA nanoparticles have
been investigated in experimental allergic encephalomyelitis (EAE), which serves as an animal
model for MS, in order to determine their capacity to reach the central nervous system (CNS). Their
concentration has been found to increase in the CNS, particularly in the white substance. Long-term
circulating PACA nanoparticles with PEG moieties were found in the CNS in greater amounts than
non-PEGylated PACA nanoparticles (Gupta et al. 2004).
Seki et al. developed and studied the biodistribution of lipoprotein-like particles, Lipid Nano-
Sphere (LNS®), incorporating dexamethasone palmitate (DMP). LNS® easily and selectively passed
through the leaky capillary wall at the inflamed sites by passive diffusion. Because of the lower
uptake of these particles by the liver, LNS® showed good recovery from the liver and prolonged
plasma half-life of DMP after intravenous injection. High efficiency in the targeting of DMP into
inflammation sites and high anti-inflammatory efficacy were observed (Seki et al. 2004).
17.3.2 Direct Application of Drugs

The simplest method of driving a drug to a specific region within the body consists of locally apply-
ing the drug to the selected area. Even if this strategy cannot properly be assimilated to targeted
delivery, it is at the borderline between systemic delivery and targeted delivery. Indeed, typical
applications reach the goal to limit the area within which the drug can diffuse, thus avoiding most
systemic side effects. When a drug is dispensed systemically, most of the product is wasted because
of its diffusion through healthy organs, which can suffer damage, and only a small amount of
the drug can reach the target tissue or organ. Direct application allows for better control over the
amount of administered drug that reaches the area of interest; increasing the local concentration
while reducing the drug diffusion within the rest of the body.
The successful examples of this approach include the intra-articular administration of hormonal
drugs in the therapy of arthritis (Williams et al. 1996), intracoronary infusion of thrombolytic
enzymes in the therapy of thrombus-induced myocardial infarction (Khaw et al. 2007) and ocular
formulations (Vandervoort and Ludwig 2007). Nanoparticles also can be delivered to distant tar-
get sites by localized delivery using a catheter-based approach with a minimal invasive procedure
(Panyam and Labhasetwar 2003), which allows the drug to be retained at the site action for a long
period of time. However, the applicability of such straightforward approaches is rather limited.
BBB, which is formed by the tight junctions within the capillary endothelium of the brain, con-
stitutes a formidable barrier to the CNS delivery of therapeutic agents. Although selective transport
mechanisms are present in the BBB such as diffusion, carrier-mediated transport, receptor-mediated,
adsorptive, and fluid-phase endocytosis, the transport of therapeutic agents via systemic administra-
tion to the brain is limited. To overcome these challenges, the direct injection of drugs into the brain
has been investigated by means of microinjection techniques. This approach could be effective in
targeted delivery to a certain area of the brain; however, it would not be as effective if the whole
brain is addressed, due to the poor diffusion of the therapeutic agents through the brain tissue. To
improve diffusion of the injected drug, some of the strategies include conjugation of the therapeutic
proteins with nontoxic neuronal binding domain of tetanus toxin (Tetanus Toxin fragment C-TTC).
Proteins linked to TTC show not only a better retention, but also a superior distribution in the extra-
cellular space on direct injection into brain (Francis et al. 2004).
In recent years, a novel approach to direct application of drugs has been studied. The active
agent or the nanocarrier can be loaded in implants, such as cardiac valves and vascular stents, and
be released from there. The loading of a nanoparticulate carrier instead of the direct loading of the
drug would provide a fine control over drug release kinetics (Finkelstein et al. 2003).
Concerning growth factors (GF) delivery, it has been shown that their direct injection into the site
to be regenerated is generally not effective because they rapidly diffuse from the injected site and
are enzymatically digested or deactivated (Muzzarelli and Muzzarelli 2005). Therefore, there are
demands for either extremely higher doses and/or frequent injections. For clinically beneficial out-
come, these GF require a delivery system to guide tissue regeneration and prevent the rapid dispersal
of the factors from the site (Douglas and Tabrizian 2005). First, it is essential to identify the key GF
or factors for a particular tissue application. The mode of factor delivery must target the desired cell
population and minimize signal propagation to nontarget tissues and cells. Moreover, the controlled
delivery of GF requires a relatively long-term maintenance of biologic activity within the time.
Finally, the release profile of the GF from the system should be controlled temporally and spatially,
making these systems similar to an artificial Extracellular Matrix (ECM) (Agnihotri et al. 2004).
17.3.3 Physical Targeting
The concept behind physical targeting consists of the targeted delivery of drugs mediated by physi-
cal alterations of the area of interest. This technique can be applied following one of the two main
approaches. The first one relies on the intrinsic properties of the injured area; indeed, inflamed or
neoplastic areas differ from normal tissues, often showing higher temperature or lower pH. In such
conditions, it is possible to employ drug carriers that are able to survive in normal tissues, but sub-
jected to degradation at lower pH or higher temperatures; in this case, drugs are only released in the
injured area, without undesired systemic effects. The second approach consists of the application of
an external stimulus, like heat or a magnetic field, to degrade the carrier and release the drug. When
the application is strictly localized, the drug accumulates only inside the area of interest.
The latter is already employed in medicine for contrast agents in magnetic resonance-based diag-
nostics and it is now widely studied for magnetic drug targeting processes (Chithrani et al. 2009;
Hoeffel et al. 2009). In fact, by surrounding iron oxide nanoparticles in supermagnetic conditions
with a biocompatible polymer able to absorb a drug, it is possible to direct them onto a specific
body target, by means of an external localized magnetic field (Zhang et al. 2009). DDS based on
nanoparticles mainly endowed with magnetic properties resulted to be also very interesting for the
gene therapy (Pan et al. 2007; McBain et al. 2008). Furthermore, the presence of magnetic par-
ticles is useful for the treatment of tumors, by creating a synergistic effect of drug release and local
hypertermia therapies. Local heating of the tumoral cells between 37°C and 45°C typically 42.5°C
induces cellular apoptosis. Over 45°C, thermoablation is meant to occur (Müller 2009).
17.3.4 Active Targeting
The use of targeting moieties is an active strategy that relies on specific interactions at target sites
and such interactions include antibody–antigen and ligand–receptor interactions. To maximize
specificity, a surface marker (antigen or receptor) should be overexpressed on target cells relative to
normal cells (Peer et al. 2007).
The “magic bullet” initially theorized by Paul Ehrlich (1906) has evolved in a three-parts system
composed of a therapeutic agent a carrier for the drug, and a targeting moiety combined together.
The targeting moiety has to be specific for the area of interest, thus making the distribution of the
drug independent from the EPR effect. This targeting strategy provides evident advantages such as
high specificity for the injured area and reduced side effects, but it can show its best performances
in cancer therapy, especially for the cure of delocalized tumors or tumors in their early stages of
development, when the vasculature is still immature (Solaro et al. 2010). The identification and
characteristics of the targeting moiety are important for circulation time, cellular uptake, affinity,
and extravasation (Byrne et al. 2008).
Several cellular receptors or integral membrane proteins have been investigated as target for
tumor-specific drug delivery because they are overexpressed in tumor tissues, compared to normal
tissues. Folate receptor (FR) is a well-known tumor marker that binds vitamin folate and folate–
drug conjugates with a high affinity, and transferrin receptor is a carrier protein for Transferrin that
imports iron by internalizing the transferrin–iron complex through receptor-mediated endocytosis.
Glycans, which are expressed on the extracellular side of the plasma membrane, also play an impor-
tant role as targeting moieties. Cancer cells often express different glycans compared with their
normal counterparts (Cho et al. 2008).
Despite many of these still standing challenges, targeted nanocarriers represent a promising
strategy for future development of targeted delivery therapeutics.
Today, over 200 delivery systems based on antibodies or their fragments are in preclinical and
clinical trials (Peer et al. 2007).
Monoclonal antibodies (mAb) can be attached to particles by physical adsorption (Kubiak et al.
1988) or by covalent linkages (Cirstoiu-Hapca et al. 2007). Antibody fragments containing only the
variable region of the antibody are now more commonly used for active targeting of therapeutics
because they retain the specificity of their target, while lacking the constant “Fc effector region” that
could result in complement activation or undesirable interaction with other cells (Chapman 2002).
Furthermore, animal-derived antibodies are easily recognized as foreign elements and may cause
strong immune responses. Chimeric antibodies, combining human constant regions and mouse
variable regions, have shown to reduce but not completely prevent immune reactions (Stacy 2005).
Humanized antibodies, which contain only the binding regions of the mouse antibodies fused with a
human antibody, have shown reduction in immunogenicity but sometimes at the expense of affinity
for the target (Stacy 2005). Antibodies which are 100% human have been developed in transgenic
animals (Van Dijk and van de Winkel 2001) and through phage display techniques (Goletz et al.
2002). The advantages of using small-molecular weight fragments of mAb are the lower steric hin-
drance and the possibility of grafting a larger number of targeting moieties on carrier surfaces, thus
leading to improved pharmacokinetics profiles.
The final concern in the incorporation of antibodies or antibody fragments (Fab) to DDS is
the method of conjugation. Abs or Fabs fragments are normally conjugated either directly to
nanoparticle surfaces or through linker molecules such as PEG. Conjugation of the antibody can
be random or site specific. Random conjugation is commonly carried out by carbodiimide-medi-
ated chemistry, which creates stable amide bonds between carboxylic acid groups in the nano-
carrier and primary amine groups, including lysines and the N-terminus amine, of the antibody
(Chapman 2002). Due to the lack of specificity of this conjugation route, antibodies can be bound
to the nanocarrier in a number of ways, some of which can even block access to the binding site
of the antibody (Lee et al. 1999).
Lectin-carbohydrate is another classical example of active drug targeting. Lectins are proteins
of nonimmunological origin capable of recognizing and binding to glycoproteins expressed on
cell surface or capable of circulating. The interaction of lectins with certain carbohydrates is very
specific. This interaction is as specific as the enzyme–substrate or antigen–antibody interactions.
Lectins may bind free sugar or sugar residues of polysaccharides, glycoproteins, or glycolipids.
One major property of lectins is their specific saccharide-binding sites. Some lectins are composed
of subunits with different binding sites. This ligand–carbohydrate interaction can be made use of
in the development of nanoparticles containing carbohydrate moieties that are directed to certain
lectins (direct lectin targeting) as well as incorporating lectins into nanoparticles that are directed
to cell surface carbohydrates (reverse lectin targeting).
Presently, a powerful targeting strategy is represented by the use of aptamers. Aptamers are
RNA or DNA molecules that fold by intramolecular interaction into unique three-dimensional
conformations for target recognition. They can be selected by a process referred to as SELEX
(Systematic Evolution of Ligands by Exponential Enrichment) from a pool of DNA or RNA by
repetitive binding of the target molecules (Ellington and Szostak 1990; Turek and Gold 1990).
Aptamers possess numerous advantageous characteristics, including small size, lack of immunoge-
nicity, and ease of synthesis, all of which rival those of other molecular probes such as antibodies
(Brody and Gold 2000). While most aptamers reported so far have been selected for single targets,
such as protein, drug, or amino acid, whole living cells have been used as targets for the selection of
a panel of aptamers (cell-SELEX) for specific cell recognition (Shangguan et al. 2006; Tang et al.
2007). Aptamers are also amenable to a wide variety of chemical modifications to insert radioscopic
or fluorescent reporters and affinity tags for molecular recognition or to create nuclease-resistant
aptamers. It is also possible to chemically modify aptamers to facilitate covalent conjugation to
nanomaterials, e.g., with 5′ or 3′ amino or thiol groups.
Aptamers that were conjugated to nanoparticles resulted in increased targeting and more effi-
cient therapeutics, as well as more selective diagnostics. For example, nanoparticle–aptamer con-
jugates could target the prostate-specific membrane antigen (Farokhzad 2004), a transmembrane
protein that is upregulated in prostate cancer (Lupold 2002). This was the first report of an aptamer
that enabled the targeted delivery of drug-encapsulated nanoparticles. In addition, aptamers have
been used as vehicles to deliver anthracycline chemotherapeutics by their own, through intercala-
tion within the aptamer double-stranded regions (Bagalkot et al. 2006). This concept has enabled
the engineering of polymeric nanoparticle systems to co-deliver two drugs in a temporally distinct
manner (Zhang 2007).
Localized diseases such as inflammation not only have leaky vasculature but are also character-
ized by an overexpression of some epitopes or receptors (Coester et al. 2000) and an accumulation
of macrophages (Gupta et al. 2004) that can be used as target moieties. Therefore, nanomedicines
can also be actively targeted to these sites. In this view, macrophage-targeted therapies can be
applied for atherosclerosis (Gupta et al. 2004) and rheumatoid arthritis (RA) (Furusawa et al. 2004).
Paclitaxel (PCX) is an antitumor agent with a unique mechanism of action promoting the assem-
bly of microtubules from tubule dimers and prevents them from depolarizing. This leads to the loss
of normal microtubule dynamics necessary for cell division and other vital processes and conse-
quently causes cell death. The ability of this drug to stabilize microtubules makes it significantly
effective against various types of solid tumors including breast cancer, advanced ovarian carci-
noma, lung cancer, head and neck carcinomas, and acute leukemias. However, its clinical applica-
tion is mainly limited by its narrow therapeutic index and very poor solubility in water or in other
pharmaceutically acceptable solvents (Bilensoy et al. 2008). Zhang et al. formulated PCX-loaded
nanoparticles that exposed folate moieties on the surface for targeting the cancer cells rich of the
FRs. They showed that the nanoparticle formulations significantly promoted targeted delivery of
the drug to the corresponding cancer cells, substantially enhancing its therapeutic effects (Zhang
and Feng 2006).
17.4 CONCLUSIONS
Polymeric nanoparticles are rapidly progressing and are being implemented to improve the thera-
peutic value of various water-soluble/insoluble medicinal drugs and bioactive molecules, by solving
several limitations of conventional DDS such as nonspecific biodistribution and targeting, poor oral
bioavailability, and low therapeutic indices.
The present chapter is focused on the technical aspects of nanoparticles preparation and provides an
overview of the exploitable targeting approaches that make them suitable for the treatment of a multitude
of diseases. The flexibility to modify and adapt nanoparticles to meet the needs of pathological condi-
tions or as a diagnostic tool (targeted molecular imaging) is just one of the important characteristics of
this technology that appears to be promising for far-reaching consequences in drug-delivery practice.
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18 Polymeric Materials Obtained
through Biocatalysis
Florin Dan Irimie, Csaba Paizs, and Monica Ioana Tosa
CONTENTS
18.1 Introduction........................................................................................................................... 617
18.2 Enzymatic Synthesis of Oligo- and Polysaccharides............................................................ 618
18.2.1 Glycosyltransferases-Assisted Synthesis of Oligosaccharides.................................. 618
18.2.1.1 Leloir Glycosyltransferases......................................................................... 620
18.2.1.2 Non-Leloir Glycosyltransferases................................................................. 621
18.2.1.3 Phosphorylases............................................................................................ 621
18.2.2 Glycosidases (Glycoside Hydrolases)-Assisted Synthesis of Oligo- and
Polysaccharides.......................................................................................................... 624
18.2.2.1 Polymerization of the Activated Substrates with Glycosidases.................. 625
18.2.2.2 Transglycosidases....................................................................................... 628
18.2.2.3 Glycosynthases............................................................................................ 628
18.3 Enzymatic Synthesis of Polyesters........................................................................................ 631
18.3.1 Stereoselectivity of Lipases....................................................................................... 632
18.3.2 Polycondensation Synthesis of Polyesters................................................................. 634
18.3.2.1 Polycondensation of Hydroxyacids............................................................. 634
18.3.2.2 Polycondensation of Carboxylic Acids/Esters with Diols.......................... 635
18.3.3 Ring-Opening Polymerization of Cyclic Monomers................................................. 636
18.3.3.1 Enantioselectivity in Ring-Opening Polymerization of Lactones..............640
18.3.3.2 End-Functionalized Polyesters.................................................................... 641
18.3.3.3 Polycarbonates from Cyclic Carbonates..................................................... 642
18.4 Enzymatic Synthesis of Phenolic Polymers..........................................................................644
18.4.1 Phenolic Polymers by Peroxidase-Assisted Polymerization......................................644
18.4.2 Phenolic Polymers by Laccase-Assisted Polymerization..........................................648
18.5 Enzymatic Polymerization of Vinylic Monomers................................................................. 650
18.6 Conclusions............................................................................................................................ 652
References....................................................................................................................................... 653
18.1 INTRODUCTION
Modern life would be inconceivable without synthetic or artificial polymers. By catalytically
assisted processes, polymers of a large diversity are produced in impressive amounts; their fabrica-
tion consumes both energy and material resources. Therefore, any measure that leads to lowering
the cost and increasing the quality of the polymer product, or reduces environmental impact and
the costs related to it, can make the difference between the producing companies. During the last
two decades we assist to an explosion of papers that deal with the topic of enzyme utilization in
polymerization reactions.
The use of enzymes is legitimated by the advantages they possess. Enzymes are proteins that
catalytically assist in an integrated manner, all the biochemical reactions in a living organism
617
(metabolic reactions). Being produced and constantly improved by nature over millions of years of
evolution, they possess in relation to natural substrates performances that are unattainable in the
world of chemocatalysts.
Enzymes have all the properties of catalysts: they assist the transformation of substrate(s) by
providing alternative mechanistic pathways with more reduced activation energies; their quantities
are not modified during reactions, and therefore, they can participate in several catalytic cycles.
Moreover, as compared to chemocatalysts, enzymes have in dedicated instances the advantages
of outstanding selectivity and activity, of mild reaction conditions regarding temperature, pressure,
ionic strength and, last but not least, an almost perfect compatibility with the environment.
A high enzymatic activity can be achieved when the protein provides alternative reaction routes;
an optimum combination of proximity effect, general and specific acid-base catalysis with covalent
and ionic catalysis will lower the activation energy of the reaction. Thus more molecules would have
access to transformation in the same period; in other words the transformation rate would increase.
The high, but not absolute, selectivity enables enzymes to assist transformation of non-natural
substrates. They can be used in pure state or as enzyme preparations with different degrees of
purity. To increase their usability enzymes can be immobilized on various supports, entrapped, or
used in reticulated form.
Enzymes can assist polymer production: (i) in vivo, when biopolymers are obtained by trans-
forming a precursor or an environmental component, in a metabolic process, with the participation
of several different enzymes; (ii) in vitro, when a polymerization (polycondensation) reaction is
assisted by a single enzyme, present in a more or less purified state, and (iii) enzymatic modification
of already existing polymers.
At industrial scale all these procedures are applicable. Industrial in vivo obtaining of polymers
is achieved by fermentative processes conducted with intact cells. Application of such a process
requires a complex metabolic approach that exceeds the topic we discuss here.
A modified definition of that found in literature [1] can be enzymatic polymerization is an in vitro
polymerization process of a non-natural substrate, catalyzed by a more or less purified enzymatic
preparation, via a nonmetabolic process.
18.2 ENZYMATIC SYNTHESIS OF OLIGO- AND POLYSACCHARIDES

There are serious difficulties in providing stereo- and regioselectivity to successive glycosy
lation for polymer synthesis by manipulating the leaving groups and the activators, or the selec-
tive protection and de-protection. These difficulties, together with the challenging aspects of
chemical achievement of a sugar oligomer or polymer, involve a great work expense with mostly
modest outcomes.
Therefore, the need for improvement focused the attention of researchers on enzymatic assis-
tance of polymerization process. The target enzymes studied for polymerization (polyglycosylation)
process were glycosidases (glycosid hydrolases) and glycosyl transferases. The so-called glycosyn-
thases, more adapted for in vitro synthesis of multimeric structures, have been created by genetic
manipulations during these investigations.
18.2.1 Glycosyltransferases-Assisted Synthesis of Oligosaccharides

The idea of using enzymes for the synthesis of polysaccharides led to the question: Which are the
most suitable enzymes that could assist the formation of certain polysaccharides, in conditions of
regio- and stereoselectivity, with acceptable rate and molecular mass dispersion?
Seemingly, the most useful type of enzymes for the synthesis of oligo- and polysaccharides are
glycosyltransferases. In biosynthetic pathways they can ensure the gradual generation of glycosidic
bonds between a growing, accepting oligo/polysaccharide chain and a carbohydrate component
originating from an activated donor. In vivo, one glycosyltransferase can transfer processively the
Polymeric Materials Obtained through Biocatalysis 619
sugar to form homopolysaccharides, or in combination with other glycosyltransferases, to form

heteropolysaccharides. In vivo, glycosyltransferases are responsible for the synthesis of most
cell glycoconjugates. An exhaustive classification of glycosyltransferases can be found in CAZy
(Carbohydrate-Active enZymes) database. Recent syntheses, of high quality, on in vivo utilization
of transferases, including glycosyltransferases, to obtain oligo- and polysaccharides are those of
Visser [2] and van de Vilst [3].
Glycosyltransferases are type II membrane-bound glycoproteins consisting of a short N-terminal
cytoplasmic domain, a transmembrane domain with a “stem” or “neck” region which is easily cleav-
able by proteases, and the C-terminal catalytic domain [4].
The canonical model at hand is cellulose synthase (UDP forming), E.C. 2.4.1.12 [5] (Scheme
18.1) [1] responsible for cellulose synthesis. A similar enzyme is cellulose synthase (GDP forming),
E.C. 2.4.1.29 [6].
Glycogen and starch are similarly produced by glycogen (starch) synthase (E.C. 2.4.1.11.)
Attempts to use these enzymes at a reasonable scale to produce artificial/synthetic cellulose or
amylose have been abandoned due to the unavailability of a stable enzyme and the prohibitive price
of the substrate.
Glycosyltransferases are a subclass of transferases (E.C. 2.4.x.x.) which transfer carbohydrates
from their conjugates with nucleoside monophosphates (CMP NeuAc), diphosphates (UDP-Glc,
UDP-Man, UDP-GlcNAc), lipids (dolichol phosphate oligosaccharides), or unsubstituted phos-
phates. Glycosyltransferases that use sugar nucleotides as carbohydrate donors are known as Leloir
glycosyltransferases, whereas all the others are non-Leloir glycosyltransferases. Most of the gly-
cosyltransferases responsible for the biosynthesis of mammalian glycoproteins and glycolipids are
Leloir glycosyltransferases. The products of glycosyltransferase reactions are conjugates of the
transferred carbohydrates with (oligo-, poly-)saccharides, lipids, proteins, or nucleic acids.
The glycosyltransferases transfer the sugar with either retention or inversion of configuration at
the anomeric center (Scheme 18.2).
OH
O O
OH
HO
HO O O NH Cellulose O
OH O + nUDP
O P O P O synthase
N O HO
O O O OH
n
OH OH Cellulose
SCHEME 18.1 Synthesis of cellulose chain assisted by cellulose synthase. The monomer is a very expensive
UDP-α-d-glucose.
O O O
D D A
HO HO O
ion
Invers OR1
O(PO3 )–R 2– R
–(PO 4)
Reten O D,A: donor, acceptor
O tion D
A HO
HO –(PO 2 O
4 ) –R
OR1 O A
OR1
SCHEME 18.2 Formation of glycosidic bond between two adjacent sugars can be done with retention or
inversion of anomeric carbon of donor sugar.
18.2.1.1 Leloir Glycosyltransferases

The use of Leloir glycosyltransferases encountered two major drawbacks: limited enzyme availabi
lity, the high costs of donors, and, related to this, the necessity of their regeneration.
Nevertheless, for the preparation of complex and highly pure oligosaccharides, methods based
on the application of glycosyltransferases are currently recognized as being the most effective [2].
The methods are extremely useful particularly for monoglycosylation of a sugar or oligosaccharide.
By performing the reactions with various donors and enzymes, oligosaccharide structures of high
complexity can be obtained.
For example, a crude β-1,3–N-Acetylglucosaminyltransferase (β3GnT) prepared from bovine
serum by precipitation with 25%–30% saturated ammonium sulfate (Scheme 18.3) was used to
obtain certain oligosaccharide sequences which are present in human milk [7]. The synthesis of
these compounds includes the transfer of N-acetylglucosamine (GlcNac). These sequences contain-
ing N-acetylglucosamine are useful for investigation of their immunomodulating, probiotic, and
inhibitor in human infections properties.
Another modality of attaching a galactose residue by an α(1–3) bond was the use of a bifunc-
tional fusion enzyme, obtained by cloning, which contains both uridine-5′-diphospho-galactose
4-epimerase and galactosyltransferase (GalT). GalE allows in situ generation of the expensive UDP-
Gal, from the cheaper UDP-Glc and galactose (Scheme 18.4) [8].
Many experimental investigations have been patented [9–12].
UDP formed in the transglycosylation reaction is a powerful inhibitor for transglycosidase and
therefore it should be removed. By introducing in the reaction environment a calf intestinal phospha-
tase, an enzyme that degrades UDP, a significant increase of the conversion rate has been noticed [13].
So, if enzyme availability, including its high price, could be resolved by recombinant DNA tech-
niques, the price contribution of nucleotidic conjugates to the product value could be reduced by
using in situ regeneration systems [14].
OH UDP
UDP-GlcNAc OH
OH O NHAc
HO O OH O
HO HO O O
O β3GnT HO HO
OH O O
OH OH from bovine OH
Lactose OH OH OH
serum
GlcNacβ1,3Galβ1,4Glc
SCHEME 18.3 The transfer of GlcNAc from UDP-GlcNAc assisted by β1,3–N-acetylglucosaminyl-

transferase, (β3GnT).
OH OH
O HO O
HO
HO HO
OH OH
OH O-UDP OH
OH
O
HO OH OH
OH O OH
OH OH
HO
OH OH
NH2 OH
O OH OH NH2
OH O-UDP O OH
O HO
O OH O
HO O H O O H
OH N OH HO N
HO GalE-GalT
OH OH
O O
SCHEME 18.4 Use a bifunctional fusion enzyme for Gal transfer and “in situ” formation of UDP-Gal from
inexpensive UDP-Glc.
Moreover, to obtain the desired structures with financially advantageous conditions, a multi
enzymatic procedure should be used and also substrate engineering is often necessary. An approach
to these techniques can be found in an excellent review by Homann and Seibel [15].
18.2.1.2 Non-Leloir Glycosyltransferases

Besides Leloir glycosyltransferases which ensure the biosynthesis in vivo of the oligo- and polyglu-
cidic structures, as stated earlier, the researcher and the engineer now have at their hands glycosyl-
transferases functioning in vivo as catabolic, depolymerizing enzymes (phosphorylases, sucrases).
The enzyme that normally catalyzes the direct path can be used for the assistance of reverse
reaction by handling the factors that influence the equilibrium: raising the substrate concentration or
reducing the concentrations of products by eliminating them from the system. In this way, enzymes
that are commonly depolymerizing can be used to obtain large amounts of polymers.
18.2.1.3 Phosphorylases
Among non-Leloir glycosyltransferases, α-glucan phosphorylases are most used in the attempts to
obtain oligo- or glycopolymers. These enzymes that catalyze phosphorolysis at the nonreducing ends of
α-1,4-glucans with retention of configuration at the anomeric carbon constitute a large family of very
similar enzymes, of which glycogen phosphorylase, or simply phosphorylase, is best investigated [16].
The overall reaction catalyzed by phosphorylase is
(1,4-α-D-glucosyl)n + inorganic phosphate (Pi) ∆ (1,4-α-D-glucosyl)n +1 + glucose-1-phosphate
The enzyme functions only in the presence of pyridoxal phosphate. The role of this one in ensur-
ing the stereoselectivity of the phosphorolytic attack is still questionable [16]. The mechanism
proposed by Helmreich remains likely: the breaking of the glycosidic bond and the forming of
the new one take place on the same side of the plan of the oxocarbenium intermediary, the reten-
tion of configuration being achieved at C1 [17]. Scheme 18.5 illustrates the reversibility of the
O O
H
O
Phosphorolyse
+
–O
O–
P
O O O H
O O
O O– O
O P H
O LP O O–
H –O
O
–
Inorganic O O P
P O O–
phosphate O
O– Condensation H
H –O
O α-Glucopyranose-1-
–O
O O P phosphoric acid
P + LP O O–
LP O O–
O
H
–O
O–
P
O O H
O O–
P
LP O O–
SCHEME 18.5 The phosphorylase-assisted phosphorolysis and condensation.

catalyzed reaction: it shows both the direct sense—phosphorolysis and the inverse one, of utility
in polymerization—the condensation.
The problem concerning the production of synthetic amylose by condensation of glucose-1-
phosphate is that the process cannot flow directly from glucose-1-phosphate and needs the presence
of a pre-formed chain or primer (a poly- or oligomaltosaccharide).
The smallest primer able to trigger the polymerization was maltotriose (Weibull and Tisselius,
cited by Vilst [3]).
In situ generation of glucose-1-phosphate was investigated and solved by the team of Kurichi, using
an auxiliary enzymatic equipment, consisting of sucrose phosphorylase, or cellobiose phosphorylase
[18–20], and also using auxiliary substrates: sucrose or cellobiose, respectively, and inorganic phosphate.
The dependence on primers of the glycogen phosphorylase-assisted polycondensation opens the
way for making hybrid structures, starting from modified primers, recognized by the enzymatic
equipment that performs the polycondensation.
A synthesis of the most recent results can be found in the paper of Vlist and Loos [3].
By the enzymatic polycondensation of alkylated maltose primers, structures with modified
hydrophilicity and interesting properties were produced. Attaching of primers to appropriate sur-
faces allows the growing of polymers on these surfaces—a technique named “grafting from.” The
initial surface takes a “brush” appearance with hydrophilic hairs and can acquire new properties,
such as biocompatibility, wettability, and corrosion resistance.
The stands initially used were silica or silicone surfaces. After an activation of the surfaces with
(γ-aminopropyl)trimetoxysylane or chlorodimethylsilane, the primers were attached chemically.
Using glucose-1-phosphate as monomer, the growing of primers was assisted by potato phosphorylase.
The consumption of monomer was monitored by spectrometric determination of inorganic phos-
phate, and it was correlated with the average length of the amylose polymers. Due to the chiral
nature of glucose units, the modified surfaces have a good chiral discrimination, when employed as
stationary phases in the chiral chromatography.
The investigations done, since 1954 (Bailey cited in Vlist and Loos [3]), have shown that the mecha-
nism of the reaction is of a “multichain” type, which means that after each step of monomer binding,
enzyme dissociates from the elongating chain, whereas a “single-chain”-type mechanism ensures the
continuous growth of the primer length without dissociation. Moreover, it has been noticed that the
length of the primer influences the distribution of amylose chain dimension. Starting from four mono-
saccharide units, the lengths of amylose chains show a Poisson-type distribution, compatible with a
“multichain”-type mechanism: the attachment of monomers is random, but the chains grow with a
constant rate. When maltotriose was used as a primer, a bimodal distribution was noticed, which can be
explained by a more difficult, and consequently slower attachment of the first monomers on the initial,
short chain, followed by the attachment of the next monomers at a constant, 400 times higher rate [3].
The known helicoidal structure of amylose allows the formation of inclusion compounds,
even during the polymerization reaction. Through phosphorylase assisted by polycondensa-
tion, a α-d-glucose 1-phosphate (Glc-1-P), initiated by maltoheptose, in the presence of polyTHF
[poly(tetrahydrofuran]—a hydrophobic polymer, the polycondensation proceeded with the formation
of the amylose-polyTHF inclusion complex (Scheme 18.6). By degree of polymerization and length
measurements, it was shown that one unit of polyTHF is included by two amylose units, with a degree
of polymerization of 75–90 for each. This inclusion complex could not be formed by mixing amylose
and polyTHF having the same length [1]. The helicoidal structure wrapped around the hydrophobic
polymer resembles vines of a plant growing around a rod. Therefore, this polymerization system was
named “vine twining polymerization [21].” Besides polyTHF, other hydrophobic polymers have been
used: poly(oxetane), poly(ε-caprolactame), and poly(γ-valerolactone). During polycondensation, the
growing polymer chooses the hydrophobic polymer from a mixture of two resembling polymers. For
example, if amylose synthesis is done in the presence of polyTHF and poly(oxetane), the polymer that
will participate in the inclusion complex will be polyTHF and not poly(oxetane). Similarly, from a
mixture of poly(ε-caprolactame) and poly(γ-valerolactone), the last one will be chosen [22].
O
HO H
PolyTHF n
HO HO
O O
HO HO
HO HO HO
OH O
OPO2– O
3
HO
O
Maltoheptose O
primer HO
5
OH OH
Amylose
PolyTHF
SCHEME 18.6 Poly(THF) template polycondensation of glucose-6-phosphate using maltoheptose primer.
Moreover, when the synthesis of amylose is performed in the presence of polyTHF with diffe
rent molecular weights, the inclusion complex partner is selected from a specific range of degree of
polymerization [23].
Besides phosphorylase, branching enzymes (E.C. 2.4.1.18) could be successfully used in vitro.
The glycogen branching enzyme belongs to the transferase family and is able to transfer short,
α-(1–4) linked, oligosaccharides from the nonreducing end of starch to an α-(1–6) position in intra-
or intermolecular manner.
The group of Katja Loos reported the use of an enzymatic tandem reaction in which the potato
phosphorylase and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) catalyze the
synthesis of branched polyglucans from glucose-1-phosphate (G-1-P) (Scheme 18.7). The resulting
branched polyglucans have a high degree of branching of 11%. The reaction was primer dependent.
O O O O O O O O
O O O O O O O OH
O O O O O O O
O O O O O O OH
Glycogen branching enzyme
O O O O O
O O O O OH
O
O
O O
O
O
O O O O O O O
O O O O O O OH
SCHEME 18.7 Using branching enzymes for introducing 1-6-α-glycosidic bonds in amylose chain.
The fact is important since it allows synthesizing by modification of the primer at the reducing
side, a class of amylose-based materials, including star-shaped structures, block copolymers, like
amylose block-polystyrene, PEG-block-amylose, l-glutamic acid-block-amylose, comb-type copo-
lymers like polysiloxane-graft-amylose, and amylose-functionalized silica particles [24].
18.2.2 Glycosidases (Glycoside Hydrolases)-Assisted

Synthesis of Oligo- and Polysaccharides
In normal conditions, these enzymes hydrolytically split the glycosidic bonds of oligo- and poly-
saccharides, thus producing lower weight sugars. Through their characteristic degradation mecha-
nism exercised over polysaccharides, glycosidases contribute to degradation of cellulose biomass,
and participate in body defense through lysis of the bacterial peptidoglycan wall (lysozyme), to
expression of pathogenicity (neuraminidase), etc. In suitable conditions, glycosidases can catalyze
the opposite reaction, i.e., condensation reaction, in which water is no more a reactant, but a prod-
uct, i.e., condensation reaction. These conditions can be achieved either through thermodynamic
control, which forces the equilibrium toward the condensation reaction (increasing the monomer
concentration, using an organic co-solvent, to reduce water activity) or by using activated donors in
a kinetic control. More than that, the condensation reaction is facilitated because the solubility of
the obtained polymer is lower than that of substrates and it will leave the system by precipitation.
The benefits of using glycosidases include their stability, availability, the large number of sources,
with a diversity of substrate selectivity, and last but not least, the fact that glycosyl donors are much
cheaper compounds as compared to those of glycosyltransferases [25].
As glycosyltransferases, some glycosidases can function with configurational retention at the
anomeric carbon atom involved in the hydrolyzed/formed glycosidic bond, or with configura-
tional inversion.
Most glycosidases used for synthetic purposes are retaining glycosidases [26]. The mechanism
of this reaction, as established by Koshland in 1953, is based on the existence of two reaction centers
[27] against which the anomeric carbon and oxygen atom of the glycosidic bond that will be cleaved
or formed are positioned. This mechanism is confirmed today by numerous experimental data [28]
(Scheme 18.8).
The two centers are represented by carboxyl group (the acid-base center) and carboxylate (the
nucleophile center), respectively. The reaction undergoes with the retention of configuration and
involves two steps: (1) the carboxylate attacks the anomeric carbon, which weakens the C–O bond,
and produces a partial shift of the pyranose oxygen electron pair. In addition, the glycosidic oxygen
comes near carboxyl group proton. The first in sequence transition state, with oxocarbenium ion-like
structure, evolves to the covalent intermediate glycosyl–enzyme. The anomeric carbon of the newly
formed glycosyl bond has a changed configuration as compared to the substrate, as an effect of the
nucleophile attack on the opposite side of the leaving group. (2) This intermediate can be further
transformed by a nucleophilic attack of oxygen from a water molecule or from a glycosidic bond of
an acceptor oligosaccharide structure. Similar to the first step, the nucleophilic attack will be done
on the opposite side of the leaving group (anomeric carbon–carboxylic oxygen bond). The substitu-
tion reaction is facilitated by the attack of the conjugated base (the carboxylate ion) which will take
the proton attached to the nucleophile. Transition state 2 has a similar structure to the first one and
it will evolve toward the reaction product. The latest one depends on the nucleophile that attacks the
glycosyl–enzyme intermediate. When the nucleophile is water, a hydrolysis reaction takes place,
and when the nucleophile is a sugar compound, the reaction is a transglycosylation. A relative reduc-
tion of the water content is done by using a co-solvent. Water cannot be totally removed, because a
minimal amount is needed to ensure the conformational mobility of the enzyme.
Thermodynamic forcing of the direction of hydrolysis/transglycosylation reaction through
water concentration is limited to the synthesis of some simple disaccharides and alkyl glycosides.
O O δ– O O
H δ+
O O H δ+
δ–
O O
R R
δ+
δ– O O R1OH ROH
–
O O O O
–
Oxacarbenium ion-like
H
transition state 1
O O
R1
‡
O O O O
δ– O O
H
δ+
O O δ+ H Covalent intermediate
δ+ glycosyl-enzyme
O O 1
R1 δ– R
O O
δ–
– O
O O Glycosylation
R 1: O
Oxacarbenium ion-like Hydrolyze
R 1: H
transition state 2
SCHEME 18.8 Biocatalytic mechanism of retaining glycosidases.
Therefore, the strategies of polysaccharide synthesis are based either on kinetic control of polym-
erization reaction or on the use of engineered glycosidases. In the first case, activated donor sugar
(fluorosugars or nitrophenyl esters) is used and an additional thermodynamic control is provided
by a reduced water concentration. In the second case, by dissolution of the nucleophilic site, the
enzymatic action is reduced to the control of a single nucleophilic step (the attack of the acceptor
sugar), which, as will be shown, will lead to polymers with a higher molecular mass, and obtained
with a superior yield.
18.2.2.1 Polymerization of the Activated Substrates with Glycosidases

There are commonly two types of activated monomers that can be used to obtain multimeric struc-
tures: sugar fluoride–type monomer used for synthesis of cellulose and its derivatives via “polycon-
densation” and sugar oxazoline–type monomer used in synthesis of chitin and related polymers, all
of these polymers having a 2-N-acetyl group in the core used as donor.
When sugar fluoride–type monomer is used, the classic example is the polycondensation of di- or
trisaccharide glycosyl fluoride donor units, as substrates for self-polycondensation by known glyco-
sidases such as cellulases, xylanases, amylases, and β-glucanases.
The use of fluorine, attached to the anomeric carbon of the di- or triglucidic monomer (cello-
biose, cellotriose, or derivatives), in the same configuration as of the oxygen of the newly formed
glycosidic bond, is motivated by the following: (1) cellobiose is the smallest monomer unit recog-
nized as acceptor by cellulase, (2) the dimension of fluorine (covalent radius 0.64 Å) is very close to
that of oxygen (covalent radius 0.68 Å), which enables it to accommodate in the catalytic site of the
enzyme, (3) the fluoride anion is a better leaving group than oxygen because of both its electronega-
tivity and its capability to form hydrogen bonds in the presence of suitable partners, and (4) among
sugar haloids, fluoro derivatives are the most stable [1].
A particular mention must be done for synthetic cellulose. This was the first polymer obtained through
enzymatic synthesis by Kobayashi [29] starting from cellobiosyl-fluoride in acetonitrile/acetate buffer,
R3 R3
HO O R1 O R1
Cellulase HO O
HO O O HO O R3
R2 HO F R2 HO O O R1
OH HO O O
OH
R1 R2 R3 R2 HO OH
a CH2HO OH CH2OH n OH
b CH2OCH3 OH CH2OH
c CH2OH NHAc CH2OH
d H OH H
SCHEME 18.9 Cellulose derivatives obtained by cellulose or cellulose like enzymes from fluorodiglucides.
with a cellulase from Trichoderma viride. The degree of polymerization was 22, superior to the
limit of 10 which is the demarcation between oligo- and polysaccharides.
Further investigations of Kobayashi team demonstrated that the structure of cellulose obtained
by polymerization with this cellulase depends on enzyme source and its purity [25].
Besides cellulose, some of its unnatural derivatives have been synthesized, starting from ade-
quately derivatized monomers (Scheme 18.9).
A related example, an alternating 6-O-methylated cellulose derivative has been produced
(Scheme 18.9b). A hybrid cellulose–chitin polysaccharide was also synthesized in the presence of
a cellulase from T. Viride as well as with (Scheme 18.9c) a chitinase from Bacillus sp. (see the fol-
lowing). The polymerization degree of the hybrid is only 8. Starting from a β-d-xylobiosyl fluoride
as self-polycondensation monomer, a synthetic xylan has been obtained (Scheme 18.9d). The main
degree of polymerization grade was 23, with a perfect stereo- and regioselectivity [30].
Besides enzymatic polycondensation of cellobiosyl fluoride and its derivatives, the polycondensation
of α-maltosyl fluoride in the presence of α-amylases of different sources has also been investigated.
A preference of the enzyme for the self-condensation reaction was noticed in which the fluoromaltose
donor attached to the elongating chain, as compared to the normal hydrolysis reaction (Scheme 18.10).
Unfortunately, the maximum polymerization degree was only 6 (12 glucosyl units) [31].
The activation of the monomer by introducing an oxazolinic structure at C1 can be a valuable
method used to obtain chitin, chondroitin, chondroitin sulfate, and keratan-type carbohydrate poly-
mers. These polymers contain a N-acetyl group in position C2, adjacent to the anomeric carbon
involved in the formation or breakdown of glycosidic bond. Therefore, the utilization of oxazolinyl-
monomer provides it a structural similarity with the intermediate of hydrolysis reaction. This
intermediate possesses a strongly positivated C1 atom, induced by the topology of the two carboxyl-
carboxylate residues of the catalytic site and by the susceptibility to protonation of the oxazolinic
cycle. Therefore, the intermediate will be attacked from the opposite side of the C1-oxazolinic oxy-
gen bond by the nucleophilic water. (Reaction I-B in Scheme 18.11, which shows the mechanisms of
hydrolysis/polymerization reactions catalyzed by chitinase).
If the oxazolinic monomer is in excess and the water amount is reduced, the nucleophile which
will open the oxazolinic ring will be the hydroxylic oxygen attached to C4 of the nonreducing end
of the acceptor (Reaction II-B in Scheme 18.11). In the condition of an activation similar to that of
water molecule, the nucleophilic attack takes place with the same enantiopreference. The result is the
formation of a new glycosidic bond. The repetitive process is, in fact, a ring-opening polymerization.
Certain data were published concerning the polymerization of oxazoline-activated monomers
with chitinase from Bacillus sp [32]. (Scheme 18.12).
OH OH OH OH OH OH
O O α-Amylase O O O O
OH F OH O OH
HO OH O HO OH O OH O OH
HO HO HO HO HO HO
n
SCHEME 18.10 Polycondensation of maltose units assisted by α-amylase.

Polymeric Materials Obtained through Biocatalysis
I-A Hydrolysis of chitin
OH OH
NHAc NHAc
HO O O O O
O O HO O O O
N NHAc
OH H O OH
H
O O O O
H Polymerization to chitin
I-B Chitinase II-B Donor Acceptor
OH OH OH OH
NHAc NHAc NHAc NHAc
HO O O H HO O O HO O O HO O O
O HO O HO O HO O O HO O HO O
O O H O
NHAc NHAc
OH O H OH OH O OH
N N
H O H
O O O
O O H O O H
I-C Chitinase II-C Chitinase

OH OH OH
NHAc NHAc OH
O HO O O NHAc NHAc
HO O HO O O HO O O
O O HO OH HO O HO O
O O HO O O HO O
N NHAc
OH N NHAc
OH H O OH H O OH
H H
O O O O O O O O
H H
SCHEME 18.11 Mechanisms of hydrolysis/polymerization reactions catalyzed by chitinase.
627
OH OH OH OH R1 R2
O O Chitinase O O H H
HO O O O O CH3 H
R2O NHAc R1O R2O NHAc R1O
O NHAc H CH3
N CH3 CH3
n
CH3
SCHEME 18.12 Polymerization of oxazoline-activated monomers with chitinase from Bacillus sp.
R1 OH OH
O OH R1 R1 R2
+Na–O Hyaluronidase O
2C O R2 O R2 Artificial
HO O O O O H OH
hyaluronan
HO O R2O NHR
OH NHAc
N OH H Artificial
chondroitin
R n
SCHEME 18.13 Hyaluronidase-assisted polymerization of oxazoline activated diglucides monomers.
Besides chitinase, hyaluronidase, an extremely promising catalyst for the synthesis of glycos-
aminoglycans, was another enzyme used in obtaining polysaccharides starting from oxazoline-
activated monomers.
Ovine testicular hyaluronidase was found to be more efficient than bovine testicular hyaluronidase
and allowed the formation with 40%–53% yield of hyaluronan-type polysaccharides with (8–13.3) × 104
molecular weight values. The same enzyme was used for the synthesis of chondroitin-type polysac-
charides, giving a 19%–50% yield with molecular weight values of (2.1–4.6) × 103 (Scheme 18.13) [33].
18.2.2.2 Transglycosidases
These enzymes of bacterial origin are very helpful in the synthesis of polysaccharides because they
can directly act on unactivated substrates, using the energy of the broken bonds to re-form new glyco-
sidic bonds [34]. All these enzymes work by the same retaining mechanism as retaining glycosidases.
The name of transglycosidases is questionable, as far as glycosyltransferases make the same
sugar transfer; however, the latest enzyme prefers the substrates that are activated by phosphoryla-
tion. Glucansucrases are a special class of transglycosidases which use sucrose as glucose donors in
oligomerization reaction, releasing d-fructose.
Glucan sucrases are transglycosidases classified in the glycoside-hydrolase (GH) family 70
according to the CAZy system. These enzymes can produce homopolyglucans as dextran, con-
taining above 50% α-1,6 linkages by dextransucrase (E.C. 2.4.1.5), mutan, containing above 50%
α-1,3 linkages by mutansucrase (E.C. 2.4.1.5), reuteran, containing above 50% α-1,4 linkages by
reuteransucrase (E.C. 2.4.1.5), and alternan, containing alternating α-1,3 and α-1,6 linkages by
alternansucrase (E.C. 2.4.1.140).
Amylosucrase (E.C. 2.4.1.4), another transglycosidase classified in GH family 13, assists the
transfer of glucose moiety to a growing chain of amylase [35]:
Sucrose + (1,4-α-D-glucosyl)n ∆ D-fructose + (1,4-α-D-glucosyl)n +1
18.2.2.3 Glycosynthases
The difficulties encountered when glycosyltransferases and glycosidases were used for the synthe-
sis of oligo- and polysaccharides have been partly overcome with the occurrence of glycosynthases.
Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides [36].
These enzymes derived from glycosidases have been engineered to catalyze the formation of gly-
cosidic bond without catalyzing the hydrolysis reaction. This modification has been done by replacing
the amino acid that bears the nucleophilic carboxylate in the catalytic site (Glu, Asp) with a less volu-
minous amino acid that bears a non-nucleophilic residue (Gly, Ala) (Scheme 18.14). By removing the
–O
O O O
H
H
O O O
O
HO OH O O
HO OH
F F–
CH3 CH3
SCHEME 18.14 Condensation of two sugars assisted by a synthase.
nucleophilic center, the hydrolytic activity of the mutated enzyme decreases with five orders of mag-
nitude, being virtually abolished. In this case the covalent intermediate glycosyl–enzyme intermediate
cannot form. It can be mimed by a substrate having attached, at the anomeric carbon, a substitute with
good fugacity, and also less voluminous, that could accommodate in the free space remained in the
catalytic site by the replacement of a methylene carboxylate or carboxylate with a methyl or hydrogen.
It is obvious that the position of this substitute must be opposite to the forming glycosidic bond. The
most used substitute is fluorine, reasons for its suitability being discussed in a reference paper [37]. The
intact acid-base site in its acid form will facilitate the discharge of the donor leaving group, or in its con-
jugated base form (carboxylate) will generate the nucleophile that forms the glycosidic bond [38]. The
functioning of the mutant as a condensation enzyme (synthase) will be more efficient than using glyco-
sidases for transglycosylation, because this reaction will no more compete with the hydrolysis reaction.
Glycosynthases are obtained from both exoglycosidases and endoglycosidases. Similar to wild-
type enzyme, exoglycosynthases derived from exoglycosidases show moderate substrate- and regi-
oselectivity, similar to those of the wild-type enzymes. They can catalyze the forming of a large
variety of glycosidic bonds only in short-chain oligosaccharides (di-, tri- and tetrasaccharides) as
major products. On the other hand, endoglycosynthases derived from endoglycosidases have high
regioselectivity; they can catalyze the forming of a single type of glycosidic bond. Unlike exoglyco-
synthases, they can use longer glycosyl donors which can form polysaccharides by self-condensation
or transfer to suitable acceptors [39].
The first types of used glycosynthases were derived from retaining glycosidases. These enzymes
keep the acceptor-related regioselectivity of original glycosidases, but use donors with a configura-
tion of glycosidic carbon opposed to the one of the condensation product. The mutant E358A of
β-glycosidase from Agrobacterium sp. (Abg) is inactive in hydrolysis, but performs, with a high
yield, glycosylation of acceptors bearing carbohydrate units, including self-glycosylation. It forms
β-(1,4) and β-(1,3) bonds, respectively, in the case of acceptors bearing xylose units, using as donors
both α-glucosyl fluoride and α-galactosyl fluoride. However, these donors cannot glycosylate accep-
tors bearing a hydroxyl in the position 4 axial [40].
Beside sugar polycondensation, glycosynthases are able to mediate not only the glycosylation of
sugars, but also glycosylation of sugars with noncarbohydrate acceptors (aglicons). For example, Abg
glycosynthase catalyzed the formation of 4-methylumbelliferil cello-trioside and cello-tetraoside,
used as chromogenic substrates for cellulases. The same enzyme catalyzed the synthesis of
2,4-dinitrophenyl 2-deoxy-2-fluoro-β-cellobioside and -cellotrioside, which are mechanism-based
inactivators for cellulases [41].
Some papers report glycosynthases derived from Thermus thermophilus β-glycosidase, which
are able to catalyze with a yield higher than 90% the formation of β-1,3 glycosidic bonds [42].
Besides mutant enzymes from Agrobacterium with xylosynthase activity, starting from β-xylanases
from Cellulomonas fimi and Geobacillus stearothermophilus, other engineered enzymes have also
been created. Mutant β-glucuronidases produced by Thermotoga maritima [43] and Escherichia
coli [44] are to transfer both glucuronyl and glucuronyl moieties.
The synthesis of polysaccharides with a high polymerization degree became possible through
endoglycosynthases, which possess a wider binding site than that of exoglycosynthases.
During the last decade a great progress was achieved concerning the endoglycosynthases-medi-
ated preparation of oligo- and polymers. Usually self-condensation has been used, namely glycosyl
fluoride serving as donor and acceptor. Numerous mutants have been employed, such as E197 of
cellulase Cel7B from Humicola insolens, E231 of β(1→3) glucanase from barley, and E134A of
(1→3,1→4)-β-d-glucanase from Bacillus licheniformis.
Each of these mutants accepts several types of substituted or nonsubstituted glycosyl fluorides
as donors.
18.2.2.3.1 β-(1→4)-Glucans (Synthetic Cellulose–Type Glycans)

The mutant E197 of cellulase Cel7B from H. insolens through β-(1→4) bonds can catalyze the
attachment of α-cellobiosyl- or lactosyl fluoride to different donors. Moreover, the mutant accepts
donors functionalized at C6′, with amino, bromo, azido groups, or even glycosyl substitution
(R1 substituent in Scheme 18.15), which allows it to be used in a large variety of applications to
produce cellodextrins or diversely functionalized cellodextrinic structures [45]. Although the trans-
fer yields of lactobiosyl fluoride on different mono- or diglucidic acceptors were relatively high
(51%–100%), only trimers and tetramers were obtained, according to the acceptors used, because
galactose at the nonreducing end is not an acceptor.
Using the same glycosynthase, but with substituted α-cellobiosyl fluorides as donors, polymers
with a molecular weight of 5000 ± 1000 have been obtained; this is the case of polymer with R1:NH2
(Scheme 18.15) which means a polymerization degree of about 16 ± 3 [45].
18.2.2.3.2 β-(1→3)-Glucans (Synthetic Laminarin–Type Glucans)

The mutant E231 of β(1→3) glucanase from barley is capable of catalyzing the self-condensation of
fluorine-activated monomer glucidic units, which will be linked through β(1→3) bonds (Scheme 18.16).
The resulting polymer is similar to recrystallized curdlan, and has a degree of polymerization in the
range of 28–44, with an average of 30 [46]. The mutant can assist the self-condensation of 3-thio-
α-laminaribiosyl fluoride, producing oligosaccharides in which monomers are linked through alter-
nating O- and S-glycosidic bonds, respectively.
18.2.2.3.3 β-(1→3)-β-(1→4) Alternating Mixed Glucans

An interesting use of glycosynthases in the synthesis of sugar polymers refers to the self-condensa-
tion of α-laminaribiosyl fluoride catalyzed by the mutant E134A of (1→3,1→4)-β-d-glucanase from
R1 R1
OH OH
HO O O
O E197A R1
HO O O O OH
OH HO cellulase HO O O
OH OH HO O O
F
OH HO
n
OH OH
SCHEME 18.15 Polycondensation reaction catalyzed by the E197A cellulase (glycosynthase) from H. insolens.
OH OH OH OH OH OH
HO O HO O E231G HO O HO O HO O HO O
HO x 1 3-β-glucanase HO x O x
OH OH OH OH
F
n
SCHEME 18.16 Polycondensation reaction catalyzed by the E231Gβ (1 → 3)-β-d-glucanase (glycosynthase)

from barley.
OH E134A OH
OH OH OH OH
(1 3,1 4)-β-
HO O HO O D-glucanase HO O HO O O O
O O HO
HO O O
glycosynthase HO
OH OH
F
OH OH
OH OH OH
n
SCHEME 18.17 Polycondensation reaction catalyzed by the E134A (1 → 3,1 → 4)-β-d-glucanase (glycosyn-
thase) from B. licheniformis.
B. licheniformis, which leads to the generation of a mixture of oligosaccharides, the most abundant
and having a degree of polymerization of 12 [47] (Scheme 18.17).
The H. insolens GH7 glycosynthase, HiCel7B E197S, is capable of synthesizing nongalactosyl-
ated, XXXG-based homoxyloglucan up to Mw 60,000 [G = Glcβ(1→4); X = Xylα(1→6)Glcβ(1→4);
L = Galβ(1→2)Xylα(1→6)Glcβ(1→4)], which is among the largest products so far obtained with
glycosynthase technology [48].
18.3 ENZYMATIC SYNTHESIS OF POLYESTERS

In the living world polyesters are the fourth class of biopolymers after nucleic acids (DNA and
RNA), proteins (polypeptides), and polysaccharides. They can be found as structures of omega
hydroxy acids and their derivatives, which are interlinked via ester bonds, forming an indeterminate
size polyester-type polymer. They are main components of the plant cuticle which covers all aerial
surfaces of plants. Polyhydroxyalkanoates are another class of natural polyesters found in both
plants and bacteria. It seems that they serve as energy and carbon stores. The interest for polyhy-
droxyalkanoates is rising, because they possess the general properties of synthetic polyesters, and in
addition have the advantages of biodegradability and biocompatibility. These qualities make them
useful in a wide range of applications, including the medical ones [49].
Another example of a natural polyester is shellack produced by female lac bug, Laccifer
(Tachardia) lacca, and stored on tree barks, in India and Thailand, from where it is scraped.
Generally, polyesters are high tonnage synthetic products with broad utilization, starting with
textile industry and finishing with the well-known PETs (PolyEthyleneTerephthalate). Today, when
more and more environmental restrictions are imposed on chemical synthesis, the enzymatic alter-
native becomes increasingly attractive, not only due to the general advantages of enzyme utilization
but also due to the high diversity of existing lipases, the most used enzymes, in both the formation
and the breaking of esteric bonds [50].
However, utilization of enzymes in polymerization processes has certain peculiarities, as compared
to organic synthesis biotransformations. Temperature introduces a number of restrictions which need
to be optimized. In chemocatalysis, polycondensation temperatures can reach values of 180°C–280°C.
These high values are required by the catalyst, and also by the need to maintain a reduced viscosity of the
environment in which a polymer with high melting point is formed. The latter condition is necessary for
the efficient mass, impulse, and heat transfer. On the other hand, the high temperature can induce secon
dary reactions in thermolabile polymers, as water release in the case of diols, opening of oxiranic cycles,
uncontrolled branching in unsaturated polymers, etc. It is evident that polymer thermolability increases
with their complexity. Besides temperature, the metals existing in the structure of chemocatalysts bring
additional problems concerning the environmental impact. Moreover, these metals affect biocompa
tibility, as it is difficult to totally eliminate them from the product destinated to be used in the organism.
Not least, as compared to chemocatalysts, enzymes have the advantage of chemo-, regio-, and
stereoselectivity. A summary of the advantages offered by enzymes in obtaining polyesters through
polycondensation is presented in a recent, excellent review [51].
Lipases are present in all organisms, but the majority of commercial ones come from microor-
ganisms. In vivo, lipases (E.C. 3.1.1.3.) split esteric bonds in triacylglycerols, converting them to
diacylglycerols, monoacylglycerols, glycerol, and fatty acids.
1 2
3
O O O
H N N H O C H N N H O
C H N N O
O O O O– O
R1 X C R1 X C O
R1 XH C
R2 R2 R2
Enzyme–substrate complex Tetrahedric intermediate Acyl–enzyme
6 5 4
O O O
C N H O C H N N H O C H N N O
H N
O O– O H O
O O C
Y C Y C Y
R2 R2 R2
Enzyme–product complex Tetrahedric intermediate Acyl–enzyme
SCHEME 18.18 Catalytic mechanism of lipases.
The mechanism by which lipases assist the breaking of the esteric or amidic bond is shown in
Scheme 18.18 and is based on the existence in the catalytic site of the catalytic triad consisting
of a carboxylate (derived from Asp/Glu), an imidazole residue derived from His, and a hydroxy-
methylene residue derived from a Ser unit. The topological sequence of these residues ensures an
increased nucleophilicity to the serine oxygen.
The reaction occurs in two main steps. In the first one, a covalent bond is formed between the car-
bonylic carbon atom of esteric/amidic bond to be hydrolyzed and the serine from the catalytic site.
The acyl–enzyme intermediate is formed through a negatively charged transition state at the car-
bonylic oxygen atom. The role of general base played by the catalytic His facilitates the generation
of this transition state. This role is potentiated by the Asp/Glu residue which stabilizes the partial
positive charge that occurs at the imidazolic nucleus of His. The serinic oxygen becomes strongly
nucleophilic and therefore can generate a tetrahedric transition state at the carbonylic carbon. This
transition state is stabilized by hydrogen bonds which attach the oxyanion of the carbonylic carbon
(intermediate 2—Scheme 18.18). The hydrogen bonds form in the so-called oxyanionic pocket.
Breaking of the esteric/amidic bond leads to the formation of acyl–enzyme (intermediate 3).
In the second step, under the influence of the same general base but in the presence of water or
of another nucleophile Y–H (Y=HO, RO, HOO, RNH), a nucleophilic attack will occur at the same
C1 (state 4). In the second transition state, the oxyanion is also stabilized by hydrogen bonds pres-
ent in the same oxyanion pocket (state 5). The enzyme is regenerated by breaking the esteric bond
between serine and the fragment R2CO– (state 6).
18.3.1 Stereoselectivity of Lipases
The chiral center may belong to the acyldonor (R2) or can be attached to the nucleophile or to the
acylated constituent (X or Y).
Lipase enantioselectivity toward the acyldonor is low to moderate [52], while enantioselectivity
toward secondary alcohols, acylated amines, or amines in train to be acylated is high.
Lipases show an enantiopreference for the chiral center of the substrate to be transformed,
located in the component that is acylated. The enantiopreference is preserved, whatever the
nature of the substituent at the chiral center. Moreover, the so-called rule of Kazlauskas has been
formulated on a statistical basis, applicable to secondary alcohols and to their O-Acyl derivatives.
According to this rule, in most cases the enantiomer R will be transformed faster than enantiomer S
(Scheme 18.19).
Acylation Hydrolysis
Acyl Acyl
H OH H OH H O H O
S L S L S L S L
S S S S
Acyl
HO H Acyl O HO H O H
H
S L S L S L S L
R R R R
SCHEME 18.19 Enantiopreference R of lipases, illustrated on a chiral secondary alcohol.
Structurally, this enantiopreference is reflected in the topology of lipases’ active site. The attach-
ment of the acylated, or in train to be acylated, component to the enzyme is done at the stereo-
complementary chiral center with two pockets: a small pocket that can easily accommodate a small
(S) substituent, usually a methyl or at most ethyl group, and a large pocket that can accommodate
a large substituent (L). In this way, the attachment of the other enantiomer is less firm, and a diffe
rence in the binding energy between the two stereoisomers occurs. This energy difference will be
expressed as a preference for the complementary enantiomer.
It should be noted that enantiopreference in both steps is a manifest of acyl–enzyme intermediate
formation and the transacylation step, when the enzyme is deacylated.
Activity, selectivity, availability, and lack of cofactor have made the lipases to be some of the
most used enzymes in the biotransformation of organic substrates [53–55]. In polymer chemistry,
the valorization of the enzymes’ enantioselective potential leads to the obtaining of stereoregulated
polyesters, with the most exquisite applications.
Lipases can be used in (poly)condensation by providing conditions in which the system can
evolve in the opposite direction (high alcohol and acid/acyl derivative concentration, as low as pos-
sible concentration of water).
Practically, the lipase-assisted obtaining of polyesters can be achieved in two different ways:
1. Polycondensation
a. Polycondensation of hydroxyacids
b. Polycondensation of diacids/their derivatives with diols/their acylated derivatives
2. Polymerization (with opening of the lactone ring)
The most common ways of lipase-assisted forming of the esteric bond in polycondensation
reaction are
1. The direct reaction of the acid and alcohol:
RCO2H + R1OH ∆ RCO2 R1 + H 2O
2. Transesterification, which implies a direct interaction of the ester with the alcohol (alco-
holysis), with the acid (acidolysis), or with another ester (interesterification):
RCO2R1 + R2OH Δ RCO2R2 + R1OH (alcoholysis)

RCO2R1 + R2CO2H Δ R2CO2R1 + RCO2H (acidolysis)
RCO2R1 + R2CO2R3 Δ RCO2R3 + R2CO2R1 (interesterification)
18.3.2 Polycondensation Synthesis of Polyesters

18.3.2.1 Polycondensation of Hydroxyacids
Because it is a dehydration reaction, the polycondensation of hydroxyacids must be conducted in
conditions which do not favor the hydrolysis of the formed product, implying a reduced water con-
centration. Hydrophobic organic solvents, such as benzene, toluene, and diphenylether, are pre-
ferred. These solvents remove water from the reaction, solubilize the polymeric product, and force
the shift of equilibrium toward the product. However, in hydrophobic solvents, the enzyme may
suffer a partial denaturation. This is produced by transforming the intermolecular hydrogen bonds
in intramolecular ones. Intermolecular hydrogen bonds are established between hydrophilic compo-
nents of the enzyme and the existing water molecules in the natural environment of the enzyme. In
a hydrophobic solvent, the hydrophilic groups on the surface of the enzyme will interact, reducing
the conformational mobility of the enzyme, and consequently reducing its activity. A solution to
maintain the enzyme activity in a reasonable range is the attachment of polyethylene glycol (PEG)
chains on its proteic structure. These chains interact with the hydrophobic solvent and provide to
enzyme the necessary mobility to interact with the substrate. In case of monomers and polymeriza-
tion products that are in a liquid state at the working temperature (60°C–80°C), the reaction can be
done in a solvent-free environment [56,57].
Moreover, water can be removed from the system by using low-pressure, molecular sieves or
temperatures as high as enzymes can tolerate.
The polymerization of a hydroxyacid was first mentioned in 1985. 10-hydroxydecanoic acid was
polymerized in benzene using a PEGylated lipase [58], with a degree of polymerization higher than 5.
An oligomer of glycolic acid was also obtained with a PEGylated esterase [58].
The degree of polymerization of polyesters obtained from hydroxyacids is generally low and
depends on carbon chain length. It was noticed that for the lipase from Candida antarctica,
10-hydroxydecanoic acid was close to optimum length, as monomers with shorter and longer carbon
chains (4-hydroxybutyric acid, dl-2-hydroxybutyric acid, glycolic acid, and 16-hydroxyhexadeca-
noic acid) failed to polymerize.
The degree of polymerization can be increased by raising the enzyme/monomer mass ratio.
An interesting result is the polycondensation of ricinoleic acid ( (R,Z)-12-hydroxyoctadec-9-
enoic acid) in presence of an immobilized lipase (from C. antarctica or Burkholderia cepacia) and
a synthetic zeolite (Scheme 18.20). The working temperature ranges from 60°C to 80°C, the time
was 168 h, and MS 4A was used as zeolite [59]. In the conditions described in the patent, the average
molecular mass was not less than 20,000.
In the same license the alternate method of polycondensation by transesterification, starting from
the methyl- or ethyl ester, is described. The authors demonstrated that the polymer obtained by
crosslinking has elastomer properties comparable with those of synthetic rubbers.
Another interesting example of polycondensation, which emphasized the regioselective character
of the enzyme, was the obtaining of cholic acid oligomers (Scheme 18.21). Their average mass, of
only 920, was modest, but the esteric bound was achieved only with the hydroxy in position 3 [60].
The chemocatalysis of the polycondensation reaction can be made more performant using car-
boxyl-activated substrates through electron-withdrawing groups (acid chlorides, trifluoroethyloxy).
The enzymatic catalysis does not need such an expensive activation.
Immobilized lipase
HO O
Synthetic zeolite
OH O
n
O O
SCHEME 18.20 Polycondensation of ricinoleic acid.

O
OH OH
CH3 CO2H CH3
Candida antarctica
CH3 lipase CH3
H H
HO OH O OH
H H
n
SCHEME 18.21 Polycondensation of cholic acid.
Polycondensation of diacids and of their diol derivatives offer more diverse possibilities. Instead
of free dicarboxylic acids, the use of dicarboxylic acid esters, is largely employed, because they are
liquid at the working temperature. Besides, in case of vinyl esters, during transesterification, usu-
ally alcoholysis, the released vinyl alcohol tautomerizes to acetaldehyde which, due to its volatility,
leaves the system and forces the shift of the equilibrium toward the condensation reaction. The
average molecular mass and the reaction time depend on the working temperature, on the presence
or absence of the solvent and on the solvent’s nature.
The structure of monomers is an important influence factor. Lipases usually accept monomers
with a relatively long carbon chain, because the substrate is attached through the hydrophobic por-
tion, and the reaction occurs in the hydrophilic area of the substrate.
The formation of acyl–enzyme intermediate is a direct condensation reaction between the car-
boxyl group of the free acid and the serine hydroxyl group, or a transesterification (alcoholysis)
between the substrate ester and the serine hydroxyl. The second step is almost always an alcoholysis
of acyl–enzyme bond and the alcohol substrate (monomer).
All the earlier-mentioned parameters deeply depend on the nature of the enzyme. There is no
such thing as an universal enzyme suitable for all reactions. Therefore, trials remain at hand; if the
enzyme’s structure is known, docking calculations can be made, or evolutionary means for adapting
its structure and functionality to the reaction’s needs can be ultimately used.
In the enzyme-assisted synthesis of polyesters, starting from diesters of dicarboxylic acids and
diols, linear polyesters are obtained together with oligoesteric macrocycles. The concentration of
the latter ones in the product mixture depends on the structure of monomers and the initial concen-
tration of the reactive functions [61].
18.3.2.2 Polycondensation of Carboxylic Acids/Esters with Diols

The easiest reaction is simple dehydration. The first paper on polymerization by condensation (poly-
condensation) in enzymatic catalysis of α,ω-dicarboxylic acids with α,ω-glicols reports the iden-
tification, among esterification products, of α,ω-dicarboxylic acids (a = 4,5,6,8,10,11,12) with 1,2
ethanediol b = 2 and 1,3 propanediol b = 3 (Scheme 18.22) [62].
Oligomers: a trimer (m = 1), a pentamer (m = 2), and a heptamer (m = 3), respectively, have been
identified only for the couple 1,13-tridecanoic acid (a = 11) and 1,3-propanediol (b = 3). The reac-
tions have been conducted at 30°C for 16 h. The reaction mixture has not been based on equimolar-
ity of the two types of components.
Usually (poly)condensation reactions are run in a nonaqueous environment, on the reason
of shifting the equilibrium toward water and also (poly)ester generation. However, it has been
noticed that certain polycondensations can run in water with good yields. Moreover, it has been
O O
Aspergillus niger lipase
+ H
HO2C aCO2H HO bOH
O bO O bOH
a
m
SCHEME 18.22 General polycondensation between of a α,ω-dicarboxylic acid and 1,2-ethanediol or

1,3-propane diol.
Consumption Accumulation Consumption Accumulation

k1 k1
MR PR MR PR
k1 > k2
krac krac > k1, k2
k2 k2
MS PS MS PS
Accumulation Consumption
Kinetic resolution Dynamic kinetic resolution
SCHEME 18.23 Kinetic resolution versus dynamic kinetic resolution.
stated that in the polycondensation of α,ω-dicarboxylic acids with α,ω-glicols, the length of the
carbon chain influences the yield; the best results were obtained with compounds having similar
hydrophobicity [63].
18.3.2.2.1 Enantioselective Polycondensation

As it has been shown, one advantage of enzymes in general, and of lipases in particular, is their
stereoselectivity. This property can be valorized in obtaining polymers with high enantiopurity.
Enantioselective polymerization is based on the higher incorporation rate of a certain stereoisomer,
say (M R), as compared to its optic antipode (MS). The process is developed in organic chemistry
biotransformations and is known as (enzymatic) kinetic resolution (Scheme 18.23, Kinetic resolution);
its application in obtaining chiral polyesters is similar. Despite the advantage of obtaining a highly
enantiopure polymer, the major disadvantage of the process is a maximum 50% conversion of the
racemic mixture; the untransformed enantiomer (MS in our example) may have a use difficult to
identify. More than that, its separation greatly complicates the whole process.
The solution is to find a system that could racemize the monomer, at a rate higher than its incor-
poration into the polymer. This system is known as (enzymatic) dynamic kinetic resolution and it
is applied in enzymatic biotransformations of organic substrates. In the absence of racemization
system, the preferred enantiomer (M R) is consumed, and the relative concentration of the enantio-
mer (MS) increases. When the racemization system is present, the enantiomer (MS), which is in a
higher concentration, is racemized to (M R) and (MS); the (M R) enantiomer is further incorporated
into the product, while (MS) enantiomer is transformed to (M R) and so on (Scheme 18.23, dynamic
kinetic resolution).
The most suitable example is the obtaining of a high enantiopurity polyester, starting from a
racemic mixture of 1,1′-(1,4-phenylene)diethanol and dimethyl adipate (Scheme 18.24) [64].
Racemization was achieved with the catalyst [RuCl2Cymene]2. The reaction lasted for 4 days, the
molecular mass reached 3000–4000, and the optical rotation of the mixture increased from −0.6°
to +128°.
18.3.3 Ring-Opening Polymerization of Cyclic Monomers

Aliphatic polyesters prepared by ring-opening polymerization of lactones are now used worldwide
as bioresorbable devices in surgery (orthopedic devices, sutures, stents, tissue engineering, and
adhesion barriers) and in pharmacology (controlled drug delivery) [65].
Lipase
(Novozym 435) O CH3
HO OH
Ru catalyst O
+ MeO2C CO2Me
4 H 3C O O
H3C CH3 –MeOH
4
n
SCHEME 18.24 Dynamic kinetic resolution used for synthesis of a high enantiopure polymer.
Enz-OH
O
Monomer activation O Enz-O (CH2)n O H

CH2
n
O Acyl–enzyme covalent intermediate
enzyme-activated monomer (EAM)
Enz-OH
O O
EAM
Initiation Enz-O (CH2)n O H R-O (CH2)n O H
Oxyacid/oxyester
R=H, alkyl
O
H R
O
EAM
Enz-OH
Enz-O (CH2)n O H
O O
Elongation
R–O (CH2)n O H R–O (CH2)n O H
m m+1
SCHEME 18.25 The mechanism of lactone polymerization by ring opening.
The first references on the use of cyclic monomers in enzymatic polymerization by ring open-
ing issued in 1993. Knani et al. have described the polymerization of ε-caprolactone in n-hexane,
in the presence of crude porcine pancreatic lipase (PPL) with a degree of polymerization (DP)
of 35 [66]; practically in the same time, Uyama and Kobayashi reported the polymerization of
δ-valerolactone and ε-caprolactone using lipases from Pseudomonas fluorescens, Candida cil-
indracea, and porcine pancreatic lipase. They worked in the absence of solvent (bulk polymeriza-
tion), obtaining after 10 days, at 60°C a polymer having a Mn = 7000, and a conversion of the
monomer of 92% [67,68].
The mechanism of the polymerization reaction, as described by Kobayashi [69], is essentially
depicted in Scheme 18.25.
The main step of the process is the reaction of the enzyme with the lactone, which leads to mono-
mer activation, by attaching to the enzyme. The enzyme is positioned with the serine hydroxyl close
to the lactone carbonyl. This configuration actually represents the complex enzyme–substrate, in
which the two components are not attached by covalent bonds. In this enzyme–substrate complex,
the attack of serine hydroxyl on the carbonylic carbon opens the lactone ring, with the formation of
the covalent intermediate acyl–enzyme (monomer-enzyme or enzyme-activated monomer—EAM).
Once formed, the acyl–enzyme intermediate will release the oxyacid, through the nucleophilic
attack of water or of an aliphatic alcohol present in the reaction environment, or the oxyester respec-
tively, which resulted from the initial lactone. This is the initiation step that generates the structure
from which elongation begins. For the monomer 12-dodecanolide (DDL) it has been shown, from
the similarity of its conversion-time curves, in the presence and absence of the alcohol that releases
the enzyme in the initiation step that this nucleophile does not influence the consumption rate of the
monomer. Consequently, the release step is much faster than the formation of EAM intermediate,
this latter one being the rate limiting step.
In the elongation step, the oxyacid or oxyester will interact with the activated monomer (EAM)
and attach it releasing the enzyme, which will be able to activate another monomer molecule.
This “monomer-activated mechanism” found in case of lipases is different from the conventional
anionic mechanism of ring-opening polymerization, which is an “active-chain mechanism.”
In chemocatalysis, lactone reactivity increases from macrolides to medium ring-sized mono-

mers and finally to small-size compounds. The reactivity of lactones in the polymerization reaction
directly correlates with the ring-strain.
In enzymatic catalysis of ring-opening polymerization, the minimum reactivity is found for five-,
six-, and seven-membered lactones, which do not have a sufficiently high ring-strain to energetically
facilitate the ring opening, nor an extended hydrophobic area, to provide an efficient contact with
the enzyme, for the interaction of serine in the active site with the ester group. Lactones with smaller
rings are more reactive because of the tension accumulated in the ring, and those with higher dimen-
sions are more reactive than the five-, six-, and seven-membered ones, because of the increased
hydrophobic contact with the lipase. So the polymerization of ε-caprolactones is a challenge.
When monomer concentration lowers and oxoesteric oligomer concentration rises, the enzyme
will attach such an oligomer, obviously at the free or esterified carboxyl group. Its detachment can
be performed with the participation of an OH group of an oligomer—when another, linear oligomer
results, or with the participation of the OH group of the molecule attached to the enzyme, when a
cyclic oligomer will be formed (Scheme 18.26).
The use of organic solvents leads to the lowering of monomer concentration and consequently to
an increased probability of obtaining cyclic oligomers [70].
Ring-opening polymerization was investigated on lactones with a different number of members,
unsubstituted and substituted. Several very good quality reviews synthesize this area of research.
The general features of ring-opening polymerization show that enzymatic polymerization, simi-
lar to chemical polymerization, conducts to polymers with higher molecular weights and higher
conversions, as compared to polymerization through condensation of oxyacids [71].
Investigations showed that lipases acted as efficient catalysts for the ring-opening polymerization
of simple 4–17-membered unsubstituted lactones, which demonstrates that enzymes are an increas-
ingly competitive alternative to chemocatalysts [72].
Enzymatic ring-opening polymerization can be performed in bulk or in organic solvents. When a
low molecular mass alcohol is not present in the environment, the obtained polymer will always have
at its ends a carboxyl group and a hydroxyl group, respectively, which means that the nucleophile
necessary for monomer detachment from the enzyme is water (R=H in Scheme 18.26). Therefore,
the polymers obtained by ring-opening polymerization belong to the class of telechelics.
The most studied lactone was ε-caprolactone. Although it has a lower reactivity, as compared to
four-membered lactones and to macrolides, using of lipases conducted to good results. When using
Porcine Pancreas Lipase in the polymerization of ε-caprolactone to heptanes, a polymer with an
average molecular mass Mn = 2300 was obtained in 4 days at 65°C. Lipases from other sources have
been also tested. Among them, C. antarctica lipase B (CALB) used as a commercial product immo-
bilized on acrylic resin (Novozym 435) proved to be the most efficient. The performance parameters
were clearly superior to other tested lipases. Thus, in a bulk polymerization, the percentage ratio
R-O (CH2)n O H
m–k
Oxyacid/oxyester
O
O O (CH2)n
O
R-O (CH2)n O H Enz-O (CH2)n O O
O
m k–1 (CH2)n–OH k–1
Oxyacid/oxyester n(H2C)
O
Enz-OH
Cyclic oligomer
Enzyme-activated
oxyacid/oxyester
SCHEME 18.26 Formation of cyclic oligomer in polycondensation of oxyacids/oxyesters.

enzyme/monomer lowered from 20%–50% to 1%, and the reaction time was reduced to 10 h, at a
working temperature of 60°C [72].
Polymerization of methyl-substituted ε-caprolactones has also been studied. If α-Me-ε-
caprolactone and γ-Me-ε-caprolactone had approximately the same polymerizability as unsub-
stituted ε-caprolactone, ω-Me-ε-caprolactone showed a significantly lower polymerizability than
unsubstituted ε-caprolactone [71]. The reason seems to be the crowding of the nucleophilic center
(the terminal OH, with a crucial role in the propagation step).
Four-membered lactones unsubstituted or substituted have been easily polymerized due to the
ring tensions (Scheme 18.27).
A description of the polymerization level of these lactones is done by Matsumura [71].
The significance of β-butyrolactone polymerization must be noticed, which conducts to poly(3-
hydroxybutirates), polymers similar to those existing in bacteria [73]. The polymerization products
contain, besides linear polyesters with OH and COOH ends, cyclic oligo-/polyesters and crotonate-
type polyesters, which underwent elimination at the level of the last hydroxyl (Scheme 18.28).
Polymalate is also a natural product. It has been first isolated from cells of Penicillium
cyclopium [74]. Polymalate is an attractive polymer for the biomedical field because of its water
soluble, biodegradable structure [75]. Its degradation in vivo leads to the nontoxic malic acid.
O O
O Lipase
H
HO O
n
β-Propiolactone
O
O CH3
O Lipase
H
HO O
n
CH3
β-Butyrolactone
O O
O Lipase H
HO O
n
H3C CH3
α-Methyl–β-propiolactone
O O OH
O O
O Lipase O O
H2
O H H
HO O Pd/C HO O
n n
O
Benzyl–β-malolactonate
SCHEME 18.27 Polymerization of four-membered lactones unsubstituted or substituted.
O O CH3 O CH3 O CH3

Lipase O CH3
O
H + +
HO O O HO O H
CH3 n p k
–Butyrolactone Linear Cyclic Crotonate type
SCHEME 18.28 β-Butyrolactone polymerization products.

The presence of carboxyl side-chain groups attached to a stereogenic center allows, through conju-
gation with other structures, the fine tuning of physical and physiological properties of the polymer.
It is obtained by ring-opening polymerization of benzyl–β-malolactonate. The product,
poly(benzyl malate) with a Mw higher than 7000, is submitted to a quantitative debenzylation by
catalytic reduction, to generate poly(β-malic acid). The sodium salt of the polyacid is formed by
neutralization [76].
From the five-membered lactones series, γ-butyrolactone has been polymerized. Chemical
polymerization of this compound is not possible; the reaction was conducted with lipase from pork
pancreas or from Pseudomonas sp. [73].
From the six-membered lactones series several structures have been polymerized, such as
δ-valerolactone, α-methyl valerolactone [77], and derivatives as 1,4-dioxan-2-one [78]. The latter
is of interest due to its biocompatibility, good flexibility, and tensile strength. Another type of six-
membered lactone is lactide (the di-ester of lactic acid), which conducted by polymerization with
lipase to a polymer with high molecular weight, but with a low yield [71].
Higher-membered lactone lipase polymerizations are detailed in literature [1,72].
18.3.3.1 Enantioselectivity in Ring-Opening Polymerization of Lactones

Unsubstituted lactones are nonchiral. The introduction of a substituent generates an asymmetric car-
bon atom. Enzymes in general, and lipases in particular, are enantioselective catalysts, which show
preference for one of the two enantiomers in an equimolar or nonequimolar mixture. Enzymatic
polymerization with lipases is a selective process, but can be influenced by numerous factors,
and their interaction may produce hardly predictable effects. The essential characteristic of ring-
opening polymerization of substituted lactones is that a monomer structure bearing an asymmetric
center contacts an enzyme at least twice. The first contact takes place at the ring opening of lactone
and formation of enzymea-activated monomer—EAM (Scheme 18.25). Then the opened structure,
released by the enzyme, will contact the enzyme once again, in order to retrieve the activated
monomer from the enzyme through the nucleophilic attack with the OH group. Each contact will
represent an enantiomeric selection; therefore the overall selectivity of the lipase should be high.
The first systematic studies on ring-opening polymerization of substituted lactones have been
done by Kobayashi and coworkers, using P. fluorescens and C. antarctica lipases. They have clearly
revealed the enantiopreference of the enzyme [77,79]. The type of enantiopreference, (R) or (S), and
its magnitude calculated as E (enantiomeric ratio), defined as the ratio of the specific constants,
E = (kcat/K M)R /(kcat/K M)S, depend on the position of the substituents, the size of the substituents, and
the size of the ring.
The effect of substituent position was evaluated in an experiment on methyl-substituted
ε-caprolactone [80]. Methyl-substituted substrates in positions 3–6 were polymerized in
1,2,3,4-trimethylbenzene at 45°C. Substituents in positions 3, 4, and 6 induced an (S) selectivity,
while the substituent in position 5 was transformed with (R) selectivity. The recorded E values
ranged from 93 ± 27 for 4-methyl ε-caprolactone to 13 ± 4 for 3-methyl ε-caprolactone; due to an
extremely low conversion, E for 6-methyl ε-caprolactone was not recorded, only its (S) selectivity
was established.
The size of substituent also influences the polymerization rate and selectivity. Thus, Novozym
435-catalyzed ring-opening polymerizations of 4-substituted ε-caprolactones showed that the poly
merization rate decreases by a factor of 2 upon the introduction of a Me substituent at the 4-position.
Moreover, 4-EtCL polymerizes five times slower than 4-MeCl and 4-PrCL is even 70 times slower. The
decrease in polymerization rate is accompanied by a strong decrease in enantioselectivity: while the
E-ratio of 4-MeCL polymerization is 16.9, the E-ratios of 4-EtCL and 4-PrCL are 7.1 and 2.0, respec-
tively. Interestingly, Novozym 435 displays S-selectivity for 4-MeCL and 4-EtCL in the polymerization
reaction, but the enantioselectivity is changed to the (R)-enantiomer in the case of the 4-PrCL [80].
The influence of lactone ring size on its lipase-catalyzed ring opening by transesterification
showed that enzymes have a pronounced tendency for transforming (S) stereoisomers of rings up
O O
O O
Cisoid Transoid
(small lactones) (larges lactones)
FIGURE 18.1 Cisoid and transoid lactone rings.
to seven members; for rings with more than seven members, the tendency is directed toward (R)
stereoisomers [81]. (S)-enantiopreference is unexpected, in relation with statistical data that allowed
validation of Kazlauskas’s rule. This switch was explained by conformational change adopted by
large, less tense rings, from transoid structures to cisoid conformation specific for small rings [82]
(Figure 18.1). The enantiopreference switch is accompanied by an increase of the transformation
rate from small (cisoid) rings to large (transoid) rings. The rate increase is about one order of
magnitude. The rate of alkaline hydrolysis changes in the opposite direction, when compared to
ring-opening polymerization rate. Small rings will be hydrolyzed more rapidly than large rings.
The decrease of hydrolysis rate with the increase of ring size is more than four orders of magnitude.
18.3.3.2 End-Functionalized Polyesters

Ring-opening polymerization can be used for the synthesis of polyester macromonomers using
functionalized initiators and/or terminators.
Initiation of the polymerization reaction (release of the structure from which elongation begins)
can be done, besides water, with an alcohol (R=alkyl, Scheme 18.25), thiol, or amine [83]. In this
case, one end of the polymer will be a hydroxyl and the other end will be an ester, thiolester, or
amine, as appropriate. The alcoholic end group can be modified by introducing in the environment
of a “terminator” that contains a carboxyl group capable to acylate, in the presence of lipase, the
terminal OH group of the polyester. By modifying the end groups of the oligomer/polymer, end-
functionalized polyesters can be obtained, useful in block-copolymerization processes or in further
derivatization.
18.3.3.2.1 Initiator Method

This method allows the introduction of desired groups using alcohols that contain them. For exam-
ple, the introduction of vinyl group can be done by using hydroxy (met)acrylates or ω-alkenyl/
alynyl-1-alcohol as initiators [84] (Scheme 18.29).
When using alcohols that contain esteric bonds as initiators (see the HEMA example), a problem
occurs with the transesterification reaction. This reaction takes place because lipases interact, with
O
O
+ R-OH
R-OH
(CH2)n
R1
Lipase
O (CH2)n–OH
OH
O O
(CH2)n–OH R O
R1= CH3: 2-hydroxyethyl methacrylate (HEMA) O (CH2)n H
m
R1= H : 2-hydroxyethyl acrylate
End-functionalized
polymer
SCHEME 18.29 Obtaining end-functionalized polymers.

O
O
O
O
OH O (CH2)5 H
n
O HO O
HO
HO O PPL, 96 h HO O
OH 70˚C OH
SCHEME 18.30 Using ethyl glucoside as initiator in ring-opening polymerization of ε-caprolactone.
a higher or smaller selectivity, with esters leading to acidolysis, alcoholysis, or interesterification.

Therefore, 1,2-ethanediol or methacrylated hydroxyl end-groups [85] can be found, besides HEMA,
among terminal structures attached to the carboxylic end of the polyester.
An interesting example initiated by Gross was the use of ethyl glucoside [86] as an alcoholic
component. Ethyl glucoside in the presence of porcine pancreatic lipase (PPL), in the reaction with
ε-caprolactone and in the absence of solvent, initiated a polymerization process that, after 96 h at
70°C, resulted in a polymer with Mn = 2200, in which the carboxylic end was esterified with ethyl
glucoside in position 6 (Scheme 18.30).
18.3.3.2.2 Terminator Method

The OH end of the obtained polyesters can be acylated, using the acylation potential of lipases,
with structures that bear reactive groups. Thus, the quantitative acylation of the terminal OH was
achieved by using lipases derived from Pseudomonas family. Methacryl- and ω-alkenyl-type poly-
ester macromonomers were synthesized by the polymerization of 12-dodecanolide in the presence
of vinyl methacrylate or vinyl 10-undecenoate. Vinyl methacrylate is an useful acylating agent, as
through the alcoholysis produced during the formation of acyl–enzyme intermediate, the acetalde-
hyde that ensures the irreversibility of the process is released. This process could be extended to
single-step synthesis of polyester telechelics. The polymerization in the presence of divinyl sebacate
produced a telechelic polymer having a carboxylic acid group at each end. By using a fatty acid ethyl
ester or an acetic acid alkyl ester as additive, on the other hand, the quantitative introduction of the
corresponding acyl group was not achieved [87].
18.3.3.3 Polycarbonates from Cyclic Carbonates

Cyclic carbonates are actually carbonic acid esters. By their ring-opening polymerization, poly-
carbonates are generated. These macromolecular structures are biodegradable, because of their
biocompatibility properties, and have a high applicative potential, especially in the biomedical area,
as a result of their reduced toxicity.
The first cyclic carbonate submitted to ring-opening enzymatic polymerization was trimethy
lene carbonate [88]. Enzymatic polymerization was first approached, almost simultaneously, by the
groups of Kobayashi et al., Matsumura et al., [89] and Gross et al., [90] which communicated the
results on lipase-catalyzed polymerization of trimethylenecarbonate (Scheme 18.31).
The enzymes used were Porcine Pancreatic Lipase and Novozym 435 (Lipase B from
C. antarctica). The highest molecular weight of poly(trimethylenecarbonate) has been obtained
by Matsumura et al. (Mw = 156,000, Mw/Mn = 3.8). In the paper of Gross, it has been found that
O
O
O O Lipase
O O
Bulk n
Trimethylenecarbonate Poly(trimethylcarbonate)
SCHEME 18.31 Polymerization of trimethylene carbonate.

Enz-OH
O
O (CH2)3
Monomer activation Enz-O O OH
O O
Acyl enzyme covalent intermediate
enzyme-activated monomer (EAM)
Trimethylenecarbonate
EAM Enz-OH CO2

O
(CH2)3
Initiation Enz-O O OH HO (CH2)3 OH
Propane-1,3-diol
O
H H
Elongation (a) Formation of dimer

EAM Enz-OH
O
O
(CH2)3 HO (CH2)3 O O (CH2)3 OH
Enz-O O OH
Bis(3-hydroxypropyl) carbonate
HO (CH2)3 OH
(b) Polymerization Enz-OH
EAM O
O O
(CH2)3 HO (CH2)3 O O (CH2)3 O O (CH2)3 OH
Enz-O O OH
k
O O Poly(trimethylenecarbonate)
HO (CH2)3 O O (CH2)3 O O (CH2)3 OH
k–1
SCHEME 18.32 Mechanism of polymerization of trimethylenecarbonate in enzyme catalysis.
the polymer obtained by Novozym 435 [90] catalysis has both ends terminated in –CH2–OH. The
author explains this by a mechanism that involves a decarboxylation step (Scheme 18.32).
Although molecular masses did not exceed 24,400, monomer conversions were almost com-
plete. Trimethylcarbonate polymerization was resumed in 2008 with Porcine Pancreatic Lipase
immobilized on silica particles; good conversions were obtained and also high molecular
weight [91].
An interesting example is the synthesis of an enantiomerically pure functional polycarbon-
ate from a novel seven-membered cyclic carbonate monomer derived from naturally occur-
ring l-tartaric acid. The monomer was synthesized in three steps and was polymerized at 80°C.
An optically active polycarbonate with Mn = 15,500 g/mol was obtained by catalysis with lipase
Novozyme-435. Deprotection of the ketal group was achieved with minimal polymer chain cleavage
(Mn = 10,000 g/mol) [92] (Scheme 18.33).
Other examples of cyclic carbonates which were polymerized by ring-opening enzymatic cataly-
sis can be found in the literature (Scheme 18.34) [93].
O
OH O O O OH
1. lipase
CO2H 80˚C bulk O
HO2C O
2. CF3CO2H/aq n
OH OH
O O
L-tartaric acid
SCHEME 18.33 Polymerization of cyclic carbonate derived from a l-tartaric in lipase catalysis.
O
O
O O O
O O O
O O O
O O
O O
5-Methyl-5-benzoyloxycarbonyl- 5-(Benzyloxy)-1,3- 4-Methyl-1,3- 5,5-Dimethyl-1,3-

1,3-dioxane-2-one dioxan-2-one dioxan-2-one dioxan-2-one
SCHEME 18.34 Cyclic carbonates submitted to ring-opening polymerization in hydrolases assistance.
18.4 ENZYMATIC SYNTHESIS OF PHENOLIC POLYMERS

Phenolic compounds are widely spread in the living world, as, for example, the ubiquitary tyrosine.
In plants, phenolic structures are well expressed. They are present in tree bark, in the content of
vacuole, as well as in intercellular spaces. Flavonoids, anthocyans, and resveratrol are several poly-
phenolic structures with known antioxidant properties, used in cancer and circulatory disorders
prevention, as antimicrobial and anti-inflammatory agents. As a general rule, the term polyphenol
refers to the existence of more than two OH groups attached to an aromatic structure. Polymers
produced starting from phenolic monomers can also be called polyphenols. To avoid confusion,
we stick to the convention proposed by Ritter [94] to use the term “polyphenol” for nonpolymer
compounds, and the term “phenolic polymer” for macromolecular phenolic compounds. Phenolic
polymers can be produced in the desired amounts by combining radicals resulted from phenolic
structures. Radicals are generated with the help of enzymes able to mediate a monoelectronic trans-
fer from a phenol to an oxidizing agent that can be a peroxide (even hydrogen peroxide) or oxygen.
The most significant results have been obtained with peroxidases and laccases.
18.4.1 Phenolic Polymers by Peroxidase-Assisted Polymerization

Peroxidases (E.C. 1.11.1.7.) are a large family of heme-containing enzymes widely spread in living
organisms. Most of the peroxidases studied so far are heme enzymes with ferric protoporphyrin IX
(protoheme) as a prosthetic group (Figure 18.2). However, the active centers of some peroxidases
CH3
H3C
N N
Fe
N N
H 3C CH3
HO2C CO2H
FIGURE 18.2 Ferric protoporphyrin IX as prosthetic group of peroxidases.

also contain selenium (glutathione peroxidase), vanadium (bromoperoxidase) most commonly

found in marine environments, manganese (manganese peroxidase), and flavin as a nonmetal cofac-
tor (flavoperoxidase) in case of some bacterial peroxidases [95].
These enzymes perform monoelectronic oxidation of a substrate using as an oxidative cosub-
strate, usually hydrogen peroxide. But in some cases an organic peroxide (alkyl or benzyl). They
have a variety of functions in the organism. Most of them are involved in detoxification of hydrogen
peroxide in antimicrobial activity, in biosynthesis of plant hormones, in cytochrome c metabolism,
and in degradation of lignin [94].
Peroxidases transform electron-rich substrates in radicals, allowing their further transformation
by recombinations, or by radical transfer with nonradical molecules, in particular cases, to polymers.
The need to replace certain chemical transformations with enzymatic ones, the biodegradation of
phenols, as easily oxidizing compounds, from industrial wastes became a consistent object of study.
One of the first systematic studies on phenol polymerization dates back to 1987 [96]. Its authors
showed that in an appropriate environment (solvent, buffer) these compounds, in the presence
of peroxidase, can be transformed into polymers. On the one hand, phenol polymerization with
peroxidase has found applications in replacing the production of phenol-formaldehyde-type resins
like resitol, resole, and novolak, avoiding in the manufacturing process the use of the highly toxic
formaldehyde, and on the other hand provides a reliable alternative for the treatment of wastewa-
ters. In the first case, the obtained polymers have similar properties to those of commercial phenol-
formaldehyde polymers. The tentative to impose the oxidative polymerization with peroxidase in the
treatment of wastewaters with high phenol content is based on the insolubility of the obtained polymer.
The mechanism of electron transfer put to work by the peroxidase engine is still in debate. Based
on spectral data, a model of the process is described in detail by Montellano [97].
A simplified model, which captures the essence of electron and atom transfer, is presented in
Scheme 18.35. According to this model, the oxidation of the phenolic substrate takes place in three
steps and leads to the formation of a phenoxy radical. Through recombinations or radical transfer
steps, this radical will be able to form polymers.
In the first stage, the resting state, the central ion having the state Fe(III) will react with H2O2 (or
with another peroxide) generating the iron-oxo derivative known as compound I and releasing water.
This compound has two additional oxidizing equivalents as compared to the resting state (Fe(III) ),
also having a ferilic structure Fe(IV)=O and a structure of π cation radical. Compound I is able to
R
+ H2O
O FeIII
H2O2
Resting
R state
H2O
OH
Catalytic cycle of
O HRP with phenol as O
reducing substrate
+
FeIV FeIV
Compound II Compound I
O R
OH
SCHEME 18.35 A simplified catalytic cycle of peroxidase.

H2O
OH O O O OH OH
0.5H2O2
HRP
SCHEME 18.36 Radical species generated by peroxidase from phenol.
OH OH OH OH
+… OH +…
HO
SCHEME 18.37 Radical coupling reaction(s).
HO HO
OH OH OH OH
OH + OH +…
SCHEME 18.38 Radical transfer reaction(s).
extract an electron from the phenolic substrate, which will be transformed into a phenoxy radical.
The extraction is assisted by a base which captures the proton from the same phenolic hydroxyl
(Ar–OH=Ar–O• +H+ + 1e−). By this transfer compound I is reduced to compound II.
The resting state is regenerated by a new monoelectronic reduction of compound II, while a new
phenoxy radical is generated. The ferryl oxygen, together with two protons derived eventually from
phenol, will generate a new water molecule.
The resting state is regenerated by a new monoelectronic reduction of compound II, with the
generation of a new phenoxy radical. The ferryl oxygen, together with two protons derived eventu-
ally from phenol, will generate a new water molecule.
Due to the conjugation of the unpaired electron with the π-electrons of the aromatic ring, pheno-
lic radicals will possess to reaction centers (Scheme 18.36).
The formed radicals, which initially are in high amounts, will further undergo coupling reactions
(Scheme 18.37). In these reactions, the concentration of radical species lowers rapidly because through
reaction of two radical species will result a nonradical species. Monoelectronic transfer reactions take
place in parallel, but also sequentially, with a dynamics depending on the environment, but especially
on the structure of phenolic components (Scheme 18.38). In these reactions, from small radicals which
are continuously generated, oligomeric radicals are formed, which recombine with other radical spe-
cies, thus increasing the chain. The process continues until radical species are depleted.
When a radical species cannot participate in a recombination or an electron transfer reaction, it
will be oxidized to a ketone structure.
Phenol oxidation at room temperature, in an aqueous environment, precipitates polymers composed
of phenylene and oxyphenylene units (Scheme 18.39). From the point of view of oxidative polymer-
ization, phenol is a multifunctional monomer. The radicalic mechanism, which involves highly reac-
tive species with similar affinity to their reaction partners, explains the difficulty in controlling the
regio- and chemoselectivity of the process, for the obtaining of a stereoregulated polymer. In the case
of phenol, a certain control of regioselectivity can be obtained by changing the solvent. Obviously,
regioselectivity is measured (expressed) as phenylene/oxyphenylene unit ratio (Scheme 18.39). Thus,
H2O
OH OH O
0.5nH2O2
HRP
SCHEME 18.39 Phenolic monomer units in the polymer obtained using peroxidase.
H3C O H3C O
Peroxidase
HO CO2H O CO2H
H2O2
H 3C O H3C O
H2O, CO2
n
SCHEME 18.40 Peroxidase-assisted polymerization of syringic acid.
a polymer with a molecular mass of 2100–6000, soluble in DMF, was obtained by polymerization in
aqueous methanol. A modification of the share of phenylene unit between the limits of 32% and 66%
has succeeded, by changing the solvent composition [98].
The presence of the substituents on the phenol ring increases the solvent-mediated control on the
regioselectivity of enzymatic polymerization [99]. This control was investigated for the 4-substituted
phenols. Besides phenol, p-phenylphenol [95], meta-cresol [100], 4,4′-Dihydroxydiphenylether [101],
A and F bisphenols, have also been polymerized.
An interesting example is offered by syringic acid polymerization, which undergoes by decar-
boxylation and is catalyzed by HRP or by soja beans peroxidase. The molecular weight of the
obtained polymer was of approximately 1.3 × 104 (Scheme 18.40) [102].
The working environment for enzymatic oxidative polymerization must provide, on the one
hand, solubilization of phenolic monomers and oligomers, and an efficient buffering on the other
hand, taking into consideration the existence of a proton transfer, associated to electron traffic
between the substrate and the enzyme. Polar solvents such as methanol, ethanol, acetone, N,N′-
dimethylformamide, 1,4-dioxane, and methyl formate have been proved to be the most efficient
organic solvents.
Utilization of modified cyclodextrins, such as 2,6-di-O-methyl-β-cyclodextrin, eliminates the
need for organic solvents because these structures, when are used in catalytic amounts, include the
substrate in a hydrophilic coat, allowing the enzyme to interact with the exposed substrate area.
Thus, by phenol polymerization in the presence of cyclodextrin in buffer only, a polymer with the
weight in the range 1000–1400 was obtained with a high yield [103]. The polymer was soluble in
acetone and DMF.
Phosphate buffer and the organic buffer N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic
acid) (HEPES) were most often used as they maintain the pH in the neutral domain.
The reaction kinetics depends on the structure of phenolic monomers. Phenols and aromatic
amines with electron-withdrawing groups could hardly be polymerized by HRP catalysis, but phe-
nols and aromatic amines with electron-donating groups could easily be polymerized. The reaction
rate of either the para-substituted or meta-substituted substrate was higher than that of ortho-
substituted substrate. When ortho- and para position related to the hydroxylic group of phenols are
occupied by an electron-repulsive group, the reaction rate increased [104].
One solution to avoid the introduction of hydrogen peroxide or other peroxides is to use peroxi-
dase together with glucose oxidase. In this way, hydrogen peroxide is generated in situ using glucose
as a co-substrate. This one will be oxidized to gluconolactone (Scheme 18.41) simultaneously with
the generation of hydrogen peroxide that will be used by peroxidase [105].
OH OH
O HO O
HO
HO HO
OH OH OH O
Glucose
oxidase
H2O H2O2
HRP
Polymerization
O OH
SCHEME 18.41 The combined action of two enzymes for in situ generation of hydrogen peroxide.
H O H3C CH3 H O
H3C CH3
Coprinus cinereus
+ H3C CH3 peroxidase (+ H2O2) HO OH H3C CH3
HO OH O O O O
OH O
Bisphenol A Syringaldehyde n
SCHEME 18.42 Copolymerization of syringaldehyde and bisphenol A.
An interesting case is that of syringaldehyde. This compound is abundant in plants, especially as

glycosides. In contrast to the earlier-presented syringic acid, which can be polymerized by decarbo
xylation with the participation of peroxidase, syringaldehyde seems unable to polymerize, because
positions 2, 4, and 6 related to the hydroxylic group are firmly blocked. In a recent paper it has been
demonstrated that although the products of radicalic enzymatic polymerization of syringaldehyde
with peroxidase from Coprinus cinereus (CiP) have a low molecular mass, this monomer may be
included, by a radicalic mechanism, into a co-polymer with bisphenol A [106].
The incorporation into the copolymer can be explained by the attachment of syringaldehyde to
the ends of the polymer, its grafting on bisphenol A units, and also by transformation of syringal-
dehyde, through an oxidative decarboxylation process, in 2,6-dimethoxybenzoquinone, followed by
a reduction in the reaction environment, to 1,4-dihydroxy-2,6-dimethoxy-benzene. This diphenolic
product can be incorporated into the main chain.
The yield of its incorporation into the polymer, the number average molecular weight (Mn),
weight average molecular weight (Mw), and also its polydispersity increase with the lowering of
syringaldehyde content in the initial mixture. The presence of syringaldehyde in the copolymer has
been proven by FT-IR spectrometry and also by 13C NMR. Incorporation of syringaldehyde with
bisphenol A leads to a copolymer with an increased thermal stability (Scheme 18.42) [102].
18.4.2 Phenolic Polymers by Laccase-Assisted Polymerization

Laccases (E.C. 1.10.3.2.) are monophenoloxidases that oxidize a phenol group to a radical and trans-
fers hydrogen via an enzyme-bound cupric ion to a molecular oxygen. Hence, in contrast to peroxi-
dases, laccase uses Cu instead of Fe and the electron acceptor is oxygen instead of hydrogen peroxide.
Laccases are widely distributed in plants, fungi, and bacteria. If in plants their main biological role
is achieved together with peroxidases and consists in participation to lignification process, in patho-
genic fungi laccase participates inter alia to inactivation of certain phytoalexinic and taninic toxins.
Laccase or, if we refer to the multitude of sources from which they come, laccases have a his-
torical determination as being the first applicative example of enzyme use in polymerization [107].
This refers to the fact that, because of a high laccase content, the latex from Japanese/Chinese
lacquer tree (Toxicodendron vernicifluum or formerly Rhus vernicifera) hardened in presence of air
[108]. Through this report, laccase also became one of the first enzymes described in the literature.
The common substrates for laccases are phenols and aliphatic or aromatic amines. The electron
transfer process takes place through a cluster of four cupric ions which form the catalytic core
of the enzyme. Two of these ions directly interact. This interaction confers to the enzymes an
intense blue color [109]. By completing a catalytic cycle which involves the four cupric ions, from
one molecule of oxygen are obtained four water molecules, respectively four radicals, which can
interact according to the model shown in peroxidases, through combination and electronic transfer.
Laccase-mediated electron transfer from the substrate is not always possible, either because of the
large dimensions of the substrate which does not have access to the active site of the enzyme, or
because of the elevated redox potential of the substrate [110]. Using the model of nature, human
factor can introduce mediators. These are substances able to participate in redox reactions. They
will be inserted into the original electron chain. The role of mediators is not only to increase the
efficiency of substrate use, but also to widen the range of substrates (Scheme 18.43) [111].
Laccases normally oxidize substrates with a redox potential inferior to the source potential (E° in
the range +475 to +790 mV according to the source of laccase). Using mediators, laccases can oxi-
dize molecules with a superior redox potential [111]. A couple of mediators are shown in Figure 18.3.
Although more than 100 types of mediators are known, the most commonly used are still 1-hydroxy-
benzotriazole (HBT) and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).
There are relatively few examples of polymers obtained by action of laccases:
Polymerization of syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) produces a poly-
mer (poly(oxy-2,6-dimethoxy-1,4-phenylene) similar to the one formed by polymerization of
2,6-dimethyphenol. It is important to note that laccase-assisted polymerization, just as the HRP-
assisted one, occurs through decarboxylation. Polymers with masses up to 1.8 × 104, slightly higher
than in peroxidase-assisted polymerization have been obtained in the presence of laccases from
Pycnoporus coccineus and Myceliophthore [102].
Substratered Substrateox
Substratered Substrateox Mediatorox Mediatorred
Laccaseox Laccasered Laccasered Laccaseox
H2O O2 O2 H2O
SCHEME 18.43 Insertion of mediators in the redox chain of substrates oxidation by laccase.
N N
N N N
N +H N–O S S N
4 3
OH S SO3–NH4+
1H-benzo[d][1,2,3]triazol-1-ol (HBT) 2,2΄-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
FIGURE 18.3 Two laccase mediators.

Polymerization of 4-hydroxy-benzoic acid assisted by peroxidases from horse radish, soybean,

and Coprinus cinerius, or by laccases from P. coccineus, Myceliophtore, and Pyricularia oryzae did
not succeed, as mentioned by Ikeda [102] in the earlier study. On the contrary, it was found that in
the presence of laccase from Trametes villosa, expressed in an Aspergillus host, 4-hydroxy-benzoic
acid can polymerize. A polymer with weight-average molecular mass (Mw) of 17,550 was obtained
after 4 h, and of 17,800 after 24 h, at 45°C. The ability of laccase to bind 4-hydroxy-benzoic acid
on lignin structures has been also highlighted [112]. Because of a more reduced steric hindrance,
4-hydroxy-benzoic acid is grafted easily on lignin components in the presence of laccase, as com-
pared to vanillic or syringic acids.
18.5 ENZYMATIC POLYMERIZATION OF VINYLIC MONOMERS

The first mention on the possibility of enzymatic polymerization of acrylic monomers was issued
in 1951 and is due to Parravano [113]. He describes the possibility to initiate the polymerization of
aqueous methyl methacrylate with biocatalytic systems such as E. coli cells and xanthine oxidase,
in the absence of oxygen. Until the 1990s, this topic was not significantly approached. In 1992, a
paper of Derango and co-workers reported the polymerization of certain acrylic monomers (acryl-
amide, hydroxyethyl metacrylate, acrylic acid, and methyl acrylate in the presence of xanthine
oxidase, horseradish peroxidase, chloroperoxidase, and alcohol oxidase) [114]. They motivated the
use of these enzymatic equipments by the fact known at that time that acrylic monomers do not
polymerize through an ionic mechanism, but through a radicalic one. Therefore, the used enzymes
were selected on the basis of their ability to produce radicals. Although this paper does not specify
the working conditions and the characteristics of the obtained polymers, we can state that it is
the first modern presentation of enzymatically assisted polymerization of vinylic monomers. Since
then, there was a constant increase of interest for enzymatic polymerization of vinylic monomers.
The most investigated were acrylic and styrenic monomers [1].
Oxidative polymerization of aromatic compounds by peroxidase or laccase systems was
described earlier. These enzymes develop, by specific mechanisms, in presence of hydrogen perox-
ide or molecular oxygen, respectively, radical species which can conduct to macromolecular prod-
ucts by recombination with a reduced specificity.
Further studies showed that in the polymerization of acrylic substrates, peroxidases are the most
useful, followed by laccases, even if the first ones have the disadvantage of using hydrogen peroxide
as an oxidizing agent to obtain the initiator in free radical state.
Several factors can influence the parameters of polymerization reaction. Among these are the
nature, the presence, and the concentration of initiator.
It has been emphasized that utilization of a species that can generate free radicals (the initiator,
or the initiator’s analogue in chemical polymerization) in the enzyme–substrate polymerization
system greatly improves the yield of the process. Thus, enzymatic polymerization differs from the
chemical one only by the way radicals are generated (formation of the active initiator). Propagation
and termination steps are similar.
In principle, polymerization of a vinylic substrate can also take place without an initiator; in
this case the source of radicals would be one of the nondedicated components in the environment.
This statement is valid for laccases, but not for peroxidases, which need the presence of the ternary
system: substrate, initiator source (usually a β-diketone), and the oxidant. However, for laccases,
the yield of reaction increases in the presence of a dedicated initiator. Thus, polymerization of
acrylamide without an initiator was carried out in water, at relatively low temperature (50°C), with
laccase from P. coccineus. The result was a polymer of high molecular weight. In the presence of
2,4-pentanedione, laccase efficiently mediated the vinyl polymerization at room temperature [115].
β-diketones can be used as radicalic initiators because of the liability of the proton (hydrogen
atom) located between the two keto groups. The taking over of this atom (proton + electron) pro-
duces a mesomeric stabilized radical (Scheme 18.44).
H2O
H H2O2/O2
O O O O O O O O
ln
R1 R2 R1 R2 R1 R2 R1 R2
H
R R R R
ln ln
SCHEME 18.44 Mechanism of generation, stabilization, and initiation of polymerization by radicals derived
from β-diketonic initiators, in polymerization with peroxidase.
Acrylamide polymerization in presence of horse radish peroxidase at room temperature, con-

ducted to polymers with Mn in the range 0.15–0.46 × 106 g/mol, and a dispersion Mw/Mm = 2.0–2.4.
The yield was in the range of 72%–95%; these values lowered, with a decrease in hydrogen peroxide
concentration under 1.0/0.66 (mol × L−1/mol × L−1) [acrylamide]0/[H2O2]0. The absence from the
reaction environment of hydrogen peroxide, of enzyme or of 2,4-pentanedione, was independently
tested. In all cases when one of the three components was missing, the reaction did not occur, prov-
ing that polymerization does not take place in absence of the oxidant, the reducer (initiator source),
and the enzyme [116].
Again in case of acrylamide polymerization, but in the presence of laccase from P. coccineus,
and without diketone, at 50°C–80°C, a polymer was obtained with a degree of polymerization simi-
lar to the one in the previous example: Mn ≈ 1 × 106 g/mol and Mw/Mm ≈ 2.0 [116].
In a study dedicated to styrene polymerization catalyzed by horseradish peroxidase at ambient
temperature, molecular weight and yield of polystyrene were influenced by solvent, concentration
of hydrogen peroxide, and initiator (β-diketones, coumarin). THF:H2O (v/v) and hydrogen per-
oxide (0.082 mol/L) provided maximum yield of polymer (21.2% weight conversion of styrene to
polystyrene) with 2,4-pentanedione as initiator. 1,3-Cyclopentanedione and dibenzoylmethane as
initiators resulted in higher yield of polymer (60%) and a higher molecular weight (Mn = 96,504,
polydispersity = 2.16), respectively. This enzymatic strategy was also used for the synthesis of poly-
mers from styrene derivatives, 4-methylstyrene and 2-vinylnaphthalene, the latter resulting in a >90%
yield of polymer [117]. The presence of the initiators in the polymer chains was reported, which is
in complete agreement with the mechanism shown in Scheme 18.44. Several types of β-diketones as
well as cumarin have been used as initiators in enzymatic polymerization experiments (Figure 18.4).
The molecular weight of polymer depends on enzyme concentration. When the enzyme concen-
tration is higher, the average molar weight of the obtained polymer is smaller [118]. It is obvious that
O O O O O O
O O O O O O
O O
FIGURE 18.4 Types of initiators used in styrene polymerization.

H2C H 2C CH3 CH3 CH3

H2C CH3 CH3
C C C C C
C C C
O O O O O O O O O O
HRP Δ/hν
OH OH O OH O
n n
SCHEME 18.45 Chemoselective polymerization of a phenol derivative having a methacryl group by per-
oxidase catalyst.
high enzyme concentrations simultaneously initiate more polymerization chains; thus the average
molar mass will be smaller, even if monomer conversion is greater.
The influence of hydrogen peroxide concentration on polymerization reaction parameters is
insignificant, within the limits of a small inferior concentration, necessary for initiator formation by
the existing (small) amount of enzyme, and a high concentration, from which oxidation processes
of the enzyme may take place [118].
Methyl metacrylate polymerization can be carried out at ambient temperatures in the presence of
low concentrations of hydrogen peroxide and 2,4-pentanedione in a mixture of water and a water-
miscible solvent [119]. Polymers of MMA formed were highly stereoregular with predominantly
syndiotactic sequences (syn-dyad fractions from 0.82 to 0.87). Analyses of the chloroform-solu-
ble fraction of syndio-PMMA products by GPC showed that they have number-average molecular
weights, Mn, that ranged from 7,500 to 75,000. By using 25% v/v cosolvents: dioxane tetrahydrofu-
ran, acetone, and dimethylformamide, 85%, 45%, 7%, and 2% product yields, respectively, resulted
after 24 h. Increasing the proportion of dioxane to water from 1:3 to 1:1 and 3:1 resulted in a decrease
in polymer yield from 45% to 38% and 7%, respectively. An increase in the enzyme concentration
from 70 to 80 and 90 mg/mL resulted in increased reaction rate. By adjustment of the molar ratio
of 2,4-pentanedione to hydrogen peroxide between 1.30:1.0 and 1.45:1.0, the product yields and Mn
values were increased. On the basis of the catalytic properties of HRP and studies herein, we believe
that the keto—enoxy radicals from 2,4-pentanedione are the first radical species generated. Then,
initiation may take place through this radical or by the radical transfer to another molecule.
Based on the aforementioned experiments, it can be concluded that peroxidases can initiate oxida-
tive polymerization of aromatic rings and also polymerization of (meth)acrylic motifs. The polym-
erization reaction of acrylic monomers is much slower than that of phenol-type substituted aromatic
structures. When both moieties coexist in the same molecule, due to the difference in reaction rate,
peroxidases will selectively polymerize the aromatic structures (chemoselectivity). Completion of
the reaction, in the sense of also achieving a polymerization of the vinylic structure, can be done
chemically by thermic or photochemical classical initiation; in this case structures with two chains
will be obtained, as for monomers of 4-hydroxyphenethyl methacrylate type (Scheme 18.45) [120].
18.6 CONCLUSIONS
This review, with no claim of completeness, presents the state of the art in obtaining polymers by
enzymatic biotransformation. Enzymatic obtaining of polymers is certainly an area with great pros-
pects. The research orientations required in the future are optimization of the reaction conditions, to
produce polymers with desired properties; identification of new enzymes or biocatalysts for specific
structural purposes, and improvement, by evolutionary techniques, of existing enzymes performances.
All these can be achieved by joint efforts whose motivation is highlighted by the utility of poly-
mer product.
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19 Polymer-Based Colloidal
Aggregates as a New Class
of Drug Delivery Systems
Cesare Cametti
CONTENTS
19.1 Introduction........................................................................................................................... 659
19.2 Polymers in Controlled Drug Delivery.................................................................................. 661
19.2.1 Basic Mechanisms of the Controlled Release........................................................... 661
19.2.2 Polymer Aggregates................................................................................................... 661
19.2.3 Polymeric Micelles.................................................................................................... 662
19.3 Polymerosomes...................................................................................................................... 667
19.3.1 Polymer Vesicles........................................................................................................ 669
19.3.1.1 Microcapsules............................................................................................. 670
19.3.1.2 Microspheres............................................................................................... 670
19.4 Stimuli-Responsive Polymers................................................................................................ 673
19.4.1 Thermo-Responsive Polymers................................................................................... 673
19.4.2 Electro-Responsive and Magnetically Responsive Polymers.................................... 674
19.4.3 Light-Responsive Polymers....................................................................................... 675
19.5 Internal Stimuli-Responsive Systems.................................................................................... 675
19.5.1 pH-Responsive Polymers........................................................................................... 675
References....................................................................................................................................... 677
19.1 INTRODUCTION
Drug delivery occurs when an appropriate carrier, whether natural or synthetic, is combined in a
suitable manner with a drug or other active bioagents in such a way that the drug itself is transferred
to the target (biological structures, both tissues or biological cells) and then released in a prede-
signed and controlled manner.
The idea of pairing up a bioactive agent (the drug) with a transporter vehicle was borne in the
early 1970s and forcefully advanced by Ringsdorf [1] who attracted attention on how drug con-
jugation technique requires two equally contributing elements, i.e., the polymer which acts as a
carrier and transport vehicle and the drug that is inert during the transport process and exerts its
bioactivity, under environmental conditions, only in the target tissue. In practical terms, the tech-
nique comprises the bioreversible binding of a bioactive agent, for example, an antitumor drug, to
a water-soluble sub-micrometer-molecular carrier designed and synthesized in conformance with
pharmaceutical requirements. This strategy has been proved to be particularly advantageous in the
treatment of solid tumors, because polymer conjugates tend to accumulate thanks to the enhanced
intra-tumoral vascular permeability, allowing for substantial leakage of the polymeric molecules
into the tumor tissue [2].
659
This methodology presents several advantages compared with more traditional methods, namely
the possibility to concentrate the drug in the region of interest, leaving practically unaffected the
surrounding regions, the maintenance of the drug level within the desired range, the optimization of
the drug treatment to which it follows an increase patient compliance. Moreover, the great flexibility
of this technique, due to the variety of the structures the polymers are able to produce, allows modu-
lating the drug release in a large extent in a controlled way, eliminating or reducing the potential
risk of both the under- and overdosing.
So far, viral vectors are the most efficient and widely used gene delivery systems. However, their
use in clinical trials is limited by safety problems. For this reason, large effort has been devoted in
developing nonviral vectors employing nucleic acid-based drugs, nanoscopic or mesoscopic struc-
tures based on lipid self-assembly and, more recently, on polymeric aggregates of different struc-
tural complexity.
Until recently, structures based on lipid self-assembling have assumed a primary importance.
When phospholipids are placed in an aqueous medium, the hydrophilic interaction of the lipid
head groups with water results in the formation of multilamellar and/or unilamellar vesicles. These
structures (lipoplexes), which resemble a biological membrane, form spontaneously as a result of
electrostatic interactions between positively charged groups of the polycation and the negatively
charged phosphate groups of DNA. Because of their entrapping ability, liposomes are considered
as drug carrier structures. Lipoplexes introduce anionic polymers (DNA) inside cells in vitro and
in vivo by means of adsorptive endocytosis and pore formation and/or fusion [3]. These structures
promise to be a viable alternative to the viral vectors, because of absence of risk of infections, the
low immunogenicity, as well as the ease of manufacturing and their broad applicability.
Many factors are known to affect the efficiency of the gene transfer by means of lipoplex carriers.
Among these parameters, there are the composition of the liposomes, the cationic lipid-to-DNA
ratio, and even more there is the transfection protocol, and various efforts have been made in the
direction of a rationalization of this very complex phenomenology. However, there are also some
drawbacks, such as the lack of colloidal stability, the relatively low transfection efficiency, the rela-
tively low duration of expression and, most importantly, the nonspecific interactions with many cells
and tissues.
The past few years have witnessed important advances in polymer-based controlled release drug
delivery systems and significant efforts have been made [4–6]. Up to earlier years, these methods
are based on natural polymers such as carbohydrates or proteic compounds, giving rise to adverse
features including immunogenicity and backbone toxicity, moderate biodegradation and poor sta-
bility. More recently, synthetic polymers imposed themselves in drug conjugation for different rea-
sons, which encompass synthetic versatility, easy control of physical and chemical behavior and,
above all, minimization in synthetic toxicity and immunogenic properties.
The ideal drug delivery system should be biocompatible, capable of achieving high drug loading,
and biodegradable. Most of these advantages are based on the nano- or meso-scale size of these
delivery systems.
This approach is based on the premise that polymers, whose physicochemical characteristics
must be assigned, can be used to formulate biodegradable polymer particle delivery compositions,
for time release of bioactive agents in a consistent and reliable manner. The trend has been toward
utilizing biodegradable polymer excipientes which allow sustained release of therapeutic agents
while eliminating the necessity of removing the polymer after the drug is spent [7].
Polymer-based approaches to the delivery of DNA have advanced notably over the past few years.
The incorporation of multiple functional components within a single carrier, for example, combin-
ing tumor targeting, stimulated release of drugs and delivery of imaging agents has been proved
essential to address the challenges of tumor heterogeneity in order to obtain treatment of cancer,
realizing a nanomedicine platform that can diagnose, deliver, and monitor cancer pathologies [8].
Future directions in controlled drug delivery are oriented toward responsive systems where
drugs, from implantable devices, are released in response to changes in the biological environment,
Polymer-Based Colloidal Aggregates as a New Class of Drug Delivery Systems 661
i.e., the system is able to deliver, or cease to deliver, drugs according to these changes. Successful
developments of these novel formulations are based on responsive polymers with specifically
designed microscopic structural and chemical features.
In the following, we will review most of the recent progresses obtained in polymer-based drug
delivery systems, focusing on preparation, characterization, and potential applications of the most
important types of polymeric aggregates as drug carriers, showing how polymers can be used in
a smart fashion leading to multiple responses at the desired point of action. Over recent years, the
synthesis of new polymeric materials has become more sophisticated and is presently in continuing
evolution. There are a vast number of papers available on this topic and therefore only a selection of
examples will be reviewed and discussed.
19.2 POLYMERS IN CONTROLLED DRUG DELIVERY

19.2.1 Basic Mechanisms of the Controlled Release
The basic mechanisms according to which the active agent can be released from the delivery system
can be classified into three different classes, i.e., diffusion, degradation, and swelling, followed by
diffusion. Here, we will briefly comment about each one of them.
Diffusion, which is the most common mechanism that occurs, takes place when the drug passes
from the polymer matrix to the external environment. The rate of the drug release normally decreases
upon time since the drug has a progressively longer distance to travel and therefore requires a longer
diffusion time to release.
The swelling increases the aqueous solvent content within the formulation, accompanied by an
increase of the polymer mesh size. This effect enables the drug to diffuse through the swollen network
into the external environment. Most of the materials employed in swelling-controlled release systems
are based on hydrogels, which can adsorb a great deal of fluid (on the order of 60%–90% of the
total mass). As stated earlier, in these systems, the drug release is a consequence of the polymer swelling.
Because in the most cases the swelling is controlled by pH and pH-sensitive polymers swell at high pH
and collapse at low pH, the drug delivery occurs only upon an increase of pH in the environment. This
peculiar feature is exploited, for example, in systems such as oral delivery, in which the drug is not
released at low pH values, as in the stomach, but rather in the upper small intestine, at higher pH values.
Finally, biodegradable systems are based on materials that degrade within the body as a result of
natural biological processes. This class of drug release has the advantage that it is unnecessary to
remove the drug delivery system after the release of the active agent.
Most biodegradable polymers degrade as a result of hydrolysis of the polymer chains into pro-
gressively smaller compounds. For example, in the case of polylactides, polyglycolides and their
copolymers, the polymers break down to lactic acid and glycolic acid, which, entering into the
Kreb’s cycle, will be further broken into carbon dioxide and water and finally excreted through
normal processes. The degradation may take place through bulk hydrolysis or may occur only at the
surface of the polymer matrix, as, for example, in the case of polyanhydrides and polyorthoesters,
resulting in a release that is proportional to the surface area of the drug delivery system.
The most commonly used colloidal particle-based delivery systems have been reviewed by
Kostarelos [9]. They are summarized in Figure 19.1, together with their typical average dimensions,
ranging from 1 nm, in the case of polymer-drug complexes, to some micrometers, in the case of
hydrogels. This wide size interval can be conveniently exploited in the different formulations.
19.2.2 Polymer Aggregates

Various types of particles, such as polymer vesicles, microcapsules, microspheres, micelles, or col-
loidal dispersions, have been designed, according to the particular purpose to be attained. The
possible structures, resulting from the polymer aggregation, have a different hierarchy and act in
Polymer drug Micelles Liposomes and Nanoparticles Microparticles Macrospores and

conjugates polymerosomes hydrogels
1 nm 10 nm 100 nm 1000 nm 0.2–5 µm 0.5–20 µm
Typical size
FIGURE 19.1 A sketch of the different polymeric structures involved in drug delivery systems, ordered by
increasing size. They vary from polymer-drug conjugates employing water-soluble polymers, micelles based
on amphiphilic di- (and tri-) block copolymers, liposomes, and polymerosomes based on phospholipid vesicles
up to micro- and macro-particles and hydrogels. The typical size of the aggregates ranges from 1 nm, in the
case of the smallest structure, up to 10–20 μm, in the case of hydrogels.
a different way. Selected examples will be described and we will review key advances of some of
these polymeric structures and discuss their potential in drug delivery.
19.2.3 Polymeric Micelles

Polymeric micelles are characterized by a supramolecular core-shell structure and were first pro-
posed as drug carriers by Bader et al. [10] in 1984. These structures have been the object of a grow-
ing interest as carriers for water-insoluble drugs and for their ability of avoiding scavenging by the
phagocyte system and offering considerable advantage for cancer diagnosis and therapy [11].
These kinds of micelles are generally formed in an aqueous environment by self-assembling of
biocompatible amphiphilic block copolymers having an A-B diblock structure, where the hydro-
phobic chains form the exterior shell (a coronal layer) and the hydrophilic chains form a semisolid
core. A sketch is shown in Figure 19.2. The term “block” denotes a linear polymer in which the end
of one segment is covalently linked to the end of the other adjacent segment to give the diblock A-B
or multiblock (A-B)n copolymers.
This special arrangement makes polymeric micelles suitable as long-circulating drug nanocon-
tainers. Being loaded in the hydrophobic core, the drug is well protected from inactivation under the
effect of biological surrounding.
Recently, block copolymer micelles have been drawing significant attention as promising car-
riers in drug delivery systems [12]. Micelles entrapping drugs exhibited several important positive
features such as high colloidal stability, reduced interaction with biological components, prolonged
circulation in the blood, and a relatively low toxicity, which are all particularly advantageous for
in vivo applications. One of the unique features that have made polymeric micelles an attractive
(a) (b)
FIGURE 19.2 Schematic representation of a polymeric micelle formed by amphiphilic diblock copolymers
(a) and the drug-encapsulated structure drug carrier, (b).
method for the tumor-targeted delivery of chemotherapeutic agents is the flexibility of the polymer
organization, which permits the simultaneous chemical modification of the micellar core and the
shell structure without one affecting the other.
Insoluble drugs can be incorporated into the micelles by chemical conjugation or by physi-
cal entrapment through, for example, dialysis or emulsion techniques. In the first case, a covalent
bond between a specific group of the drug molecules and the hydrophobic chain of the polymer is
required. In the second case, the physical entrapment may occur in two different ways. After the
solubilization of the drug and the polymer (its hydrophobic part) in a solvent in which they are both
soluble, an extensive dialysis provokes a continuous replacement of the solvent with one that is suit-
able for the hydrophilic part of the polymer, in most cases, water. As a consequence, the hydropho-
bic portions of the polymers associate into a micellar core, incorporating the drug too.
On the other hand, the emulsion method consists in preparing an aqueous solution of the copoly-
mer to which drug in a water-insoluble solvent is added. The mixture forms an oil-in-water emulsion
and the following solvent evaporation favors the micelle-drug complexes.
The hydrophobic core generally consists of biodegradable polymers such as poly(β-benzyl-L-
aspartate) [PBLA], poly(LD-lactic acid) [PLA], poly(ε-caprolactone) [PCL], and many others.
The shell, which is responsible for the micelle stabilization in an aqueous solvent, consists of
hydrophilic, biocompatible, but not biodegradable polymers such as poly(ethylene oxide) [PEO] and
poly(ethylene glycol) [PEG], just to name a few.
The size of the micelles is determined in a large extent by the balance between opposite forces,
i.e., the interfacial energy and the conformational entropy of the polymer strands. Moreover, an
important factor that influences the size of the polymeric micelles is the length of the charged
block copolymer. For example, in the case of poly(ethylene glycol)-b-poly(L-lysine) [PEG-b-PLL]
diblock copolymer, micelles have a hydrodynamic radius of 24 and 37 nm as the degree of polym-
erization of poly(L-lysine) [PPL] increases from 18 to 78, the molecular weight of poly(ethylene
glycol) [PEG] being equal (12 kD) [13]. These aggregates generally present a considerably small
size distribution characterized by a low polydispersity index (in the most case less than 0.1) and by
a unimodal size distribution.
The final geometrical arrangement these micelles assume is, in some ways, conditioned by the
nature of the specific compound entrapped. While micelles entrapping oligonucleotides, oligopep-
tides, or proteins have a spherical or spheroidal shape, cationic micelles entrapping plasmid DNA
present a large variety of topologies, the most frequent of them being the toroidal shape. These dif-
ferent topologies can affect the transfection efficiency and these aspects are the subject of intense
investigation, especially as far as the DNA condensation is concerned.
The physicochemical parameters that characterize the behavior of a polymeric micelle are those
typical of a charged colloidal particle in an aqueous medium. Among these, the ζ-potential controls in a
large extent the colloidal stability of the aggregate and modulates the interaction of the micelle with the
biological entities. Most of the properties of colloidal systems are determined by the electrical charge
on the particle surface and hence by the surface potential. Moreover, ζ-potential and the size R of the
aggregates are generally strongly correlated. A typical example of this connection, taken from Ref. [14],
is shown in Figure 19.3, where the dependence of ζ-potential and the size R on the concentration of the
agent that induces aggregation is reported. Even if these data refer to a system composed by polymeric
particles stuck together by an oppositely charged polyion, and therefore a system that differs from the one
consisting of polymeric micelles, as those we are dealing with, nevertheless, the basic behavior holds.
Polymeric micelles have found practical applications in a variety of pharmacological fields,
even if, up to today, the most promising applications have been made as anticancer drug delivery.
Compared to conventional small-molecule-based therapy, polymeric micellar systems have several
potential advantages including higher payload capacity, higher circulation time, and a generally
reduced toxicity to healthy tissues.
Typical examples are polymeric micelles formed by Adriamycin [ADR, an anthracycline anticancer
drug] bound to the poly(aspartic acid) chain of a diblock copolymer formed by poly(ethylene glycol)
20
1500
Hydrodynamic size (nm)

ζ-Potential (mV)
0
1000
–20
500
–40
0.1 0.2 0.3 0.4 0.5 0.6
Polylysine concentration C (mg/mL)
FIGURE 19.3 ζ-potential (on the left) and hydrodynamic size (on the right) of poly(lactic acid) particles
aggregated together to form a cluster, as a function of ε-polylysine concentration in the aqueous solution. The
maximum in the aggregate size corresponds to a ζ-potential close to zero. The behavior of the two parameters
evidences two typical phenomena in the aggregation of colloidal particles governed exclusively by electro-
static interactions, i.e., the charge inversion (documented by the change of sign in the ζ-potential close to the
isoelectric point) and the reentrant condensation (documented by the nonmonotonous increase of the aggre-
gate size). (Data taken and redrawn from Zuzzi, S. et al., Langmuir, 25, 5910, 2009.)
and poly(aspartic acid) [PEG-P[Asp(ADR)]] [15]. These complexes form a micellar structure of size
on the order of 50 nm, with a narrow size distribution and a very good stability in a phosphate-buffered
saline solution. The system works, for example, against mouse leukemia, demonstrating a reduced
toxicity as compared to an administration of free Adriamycin.
This polymeric micelle has been proved to be efficient also against solid tumors [16] and more-
over it was observed in blood at much higher concentrations with a longer half-life than ADR, after
the intravenous injection. An example of the efficiency of these systems in reducing the tumor
growth after the inoculation is shown in Figure 19.4. The stabilization of ADR in blood, by binding
Reduced tumor volume
10
0.1
0 20 40 60
Day after first injection
FIGURE 19.4 Tumor growth after the drug injection. Tumor volumes are plotted in ratios to the volume
at the first drug injection. Control: (⚬, ▫, Δ, ∇) 0.9% NaCl solution in a volume of 0.1 mL/10 g body weight.
(▪): C26 tumor, 150 mg/kg PEG-P[Asp(ADR)]; (•): M5076 tumor, 150 mg/kg PEG-P[Asp(ADR)]; (▴): C38
tumor, 150 mg/kg PEG-P[Asp(ADR)]; (▾): MX-1 tumor, 150 mg/kg PEG-P[Asp(ADR)]. (Data redrawn from
Yokoyama, M. et al., Cancer Res., 51, 3229, 1991.)
to the block copolymer, was attributed to the micellar structure of the carrier which possesses the
hydrated outer layer composed by poly(ethylene glycol) chains.
Polymeric micellar systems allow the use of a large variety of existing drugs otherwise deemed
too hydrophobic to be inherently water insoluble. For example, the low water solubility of paclitaxel
(PTX, an anticancer agent which inhibits the microtubule growth) can be increased from 0.0015
to 2 mg/mL (more that three order of magnitude) when encapsulated within the hydrophobic core
of a polymeric micelle [17]. Several polymeric systems are currently in phase I/II clinical trials for
delivery of doxorubicin [DOX] and paclitaxel [PTX].
As mentioned earlier, the hydrophilic shell with its brush-like coating inhibits or reduces the
phagocytic clearance by the reticuloendothelial system [18].
While in other nanotherapeutic systems, such as polymer-drug conjugates or dendrimers, it has
proved to be necessary the covalent conjugation of the drug molecules to the polymer (the carrier), poly-
meric micelles offer the advantage to need a noncovalent encapsulation (compatible with hydrophobic
drugs). This fact extends the use of this approach to a large class of drugs since the presence of function-
alizable chemical groups on the drug molecules necessary to the covalent conjugation is not necessary.
An aspect that makes polymeric micelles an emerging and powerful platform for therapeutic
applications is the possibility to incorporate multiple drug components within a single micelle.
Furthermore, polymeric micelles can be employed in cancer diagnosis, for example, by incorpora-
tion of gadolinium-based contrast agents at their surface for use in NMR imaging. The presence of
gadolinium complexes on the surface of polymeric micelles modifies the relaxation time T1, increas-
ing the sensitivity of detection [19,20].
A typical example that summarizes in a clear way the multifunctional use of a polymeric micelle
is reported by Nasongka et al. [21]. These authors employ a micelle that incorporates different func-
tions due to the presence of targeting ligand and of an MRI-visible agent and of an anticancer drug,
realizing micelles 45 nm in size containing doxorubicin [DOX] and a cluster of superparamagnetic
iron oxide [SPIO] particles into the core of a poly(ethylene glycol)-poly(lactic acid) [PEG-PLA]
micelle with a cyclic(Arg-Gly-Asp-D-Phe-Lys) [cRGD] ligand on the micelle surface. The peptide
targets the ανβ3 integrin overexpressed on the surface of angiogenic tumor vessels.
Figure 19.5 shows the antitumor efficacy of cRGD-DOX-SPIO micelles against subcutaneous
A549 tumor xenografts in nude mice [data redrawn from Ref. [21]]. As can be seen, a notable tumor
volume reduction is observed.
2.5
2.0
Relative tumor volume
1.5
1.0
0.5
0 5 10 15 20
Days
FIGURE 19.5 Antitumor efficacy data for PBS control (▪), DOX-SPIO micelles with 0 (⚬) and 16% (▫) surface
density of cRGD in subcutaneous A549 tumor xenografts in nude mice. (Data redrawn from Nasongka, N.
et al., Nano Lett., 6, 2427, 2006.)
A similar approach was developed by Xiong et al. [22], who investigated multi-functional poly-
meric micelles based on RGD decorated poly(ethylene oxide)-b-poly(ester) diblock copolymer with
potential for selective delivery of DOX to metastatic tumor cells. In order to provide a selective
intracellular delivery of DOX in tumor over healthy cells, these authors decorated the exterior
micellar shell with internalizing antagonist peptide for ανβ3 integrin. DOX loading was carried out
through physical encapsulation or chemical conjugation. Mechanism of DOX release is different.
In the case of physical encapsulation, DOX release is dependent on the rate of drug diffusion from
the micellar core and the core degradation. In the case of DOX conjugation, the drug release is con-
trolled essentially by the core degradation.
Polymeric micelles can be considered as stimulus-responsive drug release systems and several
strategies have been explored including pH-, temperature-, and ultrasound-stimulated release. We
will go into details of these aspects in the next section which is handling general aspects of stimuli-
responsive polymers.
While polymeric micelles characterized by a hydrophilic shell have been extensively studied,
fewer studies have been reported on micelles in organic solvents (reverse micelles), where the
role of the core and the shell is exchanged (a polar hydrophilic core and a hydrophobic shell).
In the last decade, reverse micelles have been prepared from dendrimers [23] or hyperbranched
polymers [24]. The amphiphilic polymers were obtained from the hydrophobic modification of
poly(glycerol) of poly(amidoamine), and the hydrophobic shell-forming segments consisted of
covalently linked hydrocarbon chains resulting in micelles soluble in organic solvents. To these
structures, a potential as drug delivery systems has been attributed and, because of these properties,
they are mentioned here.
Recently, the water-soluble poly(glycerol methacrylate) backbones were modified through the
esterification of pendant hydroxyl functions with acyl chlorides and the resulting compounds were
shown to self-assemble into reverse micelles (20–60 nm in size) in organic solvents [25]. These
structures, besides having applications in organic chemistry (extraction of anionic dyes from water),
have a large use in pharmacological technology. For example, Jones et al. [25] reported that the
subcutaneous administration of loaded reverse micelles to rats prolonged the effect of vasopressin
for more than a factor of four.
Most of the properties suitable for drug delivery depend on the architecture of the hydrophobic
tail of the amphiphilic copolymer employed in the polymeric micelle formation. For applications
in drug delivery, the shape of the micelle, that is ultimately governed by the self-assembly of the
hydrophobic core, influences in a rather strong way its area of application. Micelles with different
sizes would locate at different tissues and organs with a consequent different ability to stabilize their
circulation time in blood.
Recently, the effect of four different tail architecture on the self-assembly of amphiphilic copo-
lymers in dilute solutions was investigated through Brownian dynamics simulations by Cheng and
Cao [26], who considered linear, dendritic, starlike, and branched copolymers. Their main results
reveal that linear tail copolymers begin to associate at a very low critical micellar concentration
[cmc] forming spheroidal polymeric micelles with a large size and a narrow size distribution. In the
case of branched polymers, the cmc is inversely proportional to the branching parameter and the
size of the aggregates increases exponentially with the branching parameter, presenting a higher
thermal stability. This study offers the possibility of providing important information to design the
self-assembling systems to prepare polymeric micelles for applications in drug delivery.
Up to today, there are several poly(amino acid) micellar carriers under clinical evaluation.
Following the recent review by Matsumura [27], the first example at the stage of phase I clinical
trial we are dealing with, concerns a polymeric micelle incorporating paclitaxel [PTX], one of the
most useful anticancer agent helpful in various cancers, including ovarian, breast, and lung can-
cers. Micelles were self-assembled from diblock copolymers consisting of poly(ethylene glycol) and
poly(aspartate), the PTX being incorporated by physical entrapment owing to hydrophobic interac-
tions with poly(aspartate) chains. The resulting formulation, named NK105, had a weight average
diameter of about 85 nm. These micelles present a marked antitumor activity against colon cancers
as compared to administration of free PTX. This is a clear example of how the drug, in nanostruc-
ture, increases its efficiency. Moreover, in this specific case, tumors disappeared after the first dos-
ing to mice treated with NK105 (with an equivalent dose of 100 mg/kg) up to 40 days after the drug
injection. In his review, Matsumura [27], besides antitumor activity, considers the neurotoxicity and
the radiosensitizing effect of NK105 in comparison to free PTX effect. As far as the last property
is concerned, the radiosensitization is considered to be important in combined treatments based on
PTX-based chemotherapy and radiotherapy, as demonstrated by clinical studies [28].
The second example deals with cisplatin-incorporating polymeric micelles [29], prepared starting
from poly(ethylene glycol) [PEG], which constitutes the outer shell of the micelle, and the coordinate
complex of poly(glutamic acid) [P(Glu)] and cisplatin [CDDP], which constitutes the inner core of the
micelle. The resulting aggregate [NC-6004] has an average size of 30 nm, with a narrow size distri-
bution. Cisplatin [CDDP] is a chemotherapy drug for cancer including lung and gastrointestinal can-
cers. The driving force for the complex formation is the ligand substitution of platinum(II) atom from
chloride to carboxylate in the side chain of P(Glu). In this case too, even if the CDDP and NC-6004
administration at the same dose level showed no significant difference in the tumor growth rate, the
nephrotoxicity and the neurotoxicity are clearly in favor of the nanostructured delivery system.
The third and last example taken from the review by Matsumura [27] and which is worth men-
tioning here is the use of a camptothecin [CPT] analog, irinotecan hydrochloride [CPT-11], which
is converted to 7-ethyl-10-hydroxy-CPT [SN-38] by carboxylesterases. SN-38 is a potent cytotoxic
agent against various cancer cells. The polymeric micelles were built up by self-assembling of copo-
lymers PEG-P(Glu)(SN-38) [16]. The resulting aggregate [NK012] is about 20 nm in size, within a
relatively narrow range.
Recently, a micellar-based carrier for honokiol delivery in cancer chemotherapy has been pro-
posed by Wei et al. [30]. Honokiol is an active component derived from Chinese traditional herb
magnolia able to inhibit growth and induce apoptosis in different cancer cell lines (human ovarian
tumor SKOV3 and COC1 cells [31], lung cancer CH27 cells [32], and leukemia HL-60 cells [33]). In
this case, honokiol-loaded polymeric micelles were prepared by self-assembling of triblock copoly-
mer from biodegradable poly(ε-caprolactone)-poly(ethylene glycol) polymers. This system, result-
ing in particles of 60 nm in size with a relatively low surface charge density (ζ-potential of about
−0.5 mV), offers the advantage of a slow honokiol release over a prolonged time up to several weeks,
maintaining a comparable cytoxicity with free honokiol.
Micelles formed by self-assembly of copolymers specifically designed for protein drug delivery
have been proposed by Yiang et al. [32], who presented the synthesis of a series of hyper-branched
poly(amine ester)-co-poly(DL-lactide) copolymers. As we have stated earlier, poly(DL-lactic acid)
is widely used in drug delivery and tissue engineering because of its nontoxic biocompatible and
bioabsorbable properties. However, poly(DL-lactide) deactivates proteins when it forms protein-
loaded nanoparticles. The combining of poly(DL-lactide) with hyper-branched poly(amine ester)s
overcomes this difficulty. The system has been proved to be effective in the interaction with bovine
serum albumin [BSA]. BSA-loaded micelles showed a marked encapsulation efficiency and, overall,
the structural stability of BSA was maintained over the whole release process.
19.3 POLYMEROSOMES
Polymerosomes (also referred to as polymeric vesicles) represent a class of vesicles ranging in size
from 50 nm to 1–2 μm made up of amphiphilic synthetic block copolymers which form the vesicle
membrane. The term polymerosomes was coined in 1999 by Discher et al. [34] to indicate a new
structure for encapsulation with an increased stability and reduced permeability when compared
with more traditional liposomes and with more efficiency in manipulating the physicochemical
characteristic of the membrane. Polymerosomes are similar to liposomes formed from naturally
occurring lipids, but, compared with these, they exhibit increased stability and reduced permeability.
Polymerosomes are generally composed of diblock copolymeric amphiphiles, where one block is
hydrophilic and the other block is hydrophobic. In aqueous solution, the diblocks form an arrange-
ment where the hydrophobic blocks tend to associate with each other in order to minimize the direct
exposure to water, whereas the more hydrophilic blocks form the two interfaces of a typical bilayer
membrane.
These structures have rapidly attracted growing interest thanks to their intriguing aggregation
phenomena, their virus-mimicking size and, above all, to their potential applications in medicine
and biotechnology [35].
Polymerosomes have a fluid-filled core with walls that consist of entangled polymer chains sepa-
rating the core from the external medium. Compared to liposomes, polymerosomes have many
different properties that render these structures particularly attractive. They mainly concern with
the elasticity, permeability, and mechanical stability associated with the membrane thickness which
can be controlled by the molecular weight of the hydrophobic block of the polymers. Moreover, due
to the high molecular weight of the polymers as compared to lipids, the membrane of polymero-
somes are generally thicker, stronger and are more stable than conventional liposomes. Finally,
polymerosomes can encapsulate hydrophilic molecules within the aqueous interior and can inte-
grate hydrophobic molecules within the membrane.
The use of diblock copolymers to generate, in general, polymer particles and, in particular,
polymerosomes is widely recognized as something very frequently done. Polymerosomes can be
spontaneously formed by precipitating the block copolymers by adding a poor solvent for one of
the diblocks [36]. Analogous to the standard method to form lipid vesicles from dried phospho-
lipid films, polymerosomes can also be formed by rehydration of a dried film of the copolymers
[37] followed by sonication or extrusion, generating unilamellar micrometer-sized vesicles, as well
as monodisperse sub-micro-meter-sized vesicles [38]. More recently, Lorenceau et al. [39] have
described a new method to create highly uniform polymerosomes using a one-step process where
the inner and the outer fluids are maintained as completely separated streams. In this way, a highly
efficient encapsulation is assured.
There are a number of polymers available for in vivo applications, as controlled release drug
delivery systems. They include both hydrophilic and hydrophobic block copolymers. To the ones of
the former class belong poly(ethylene glycol) [PEG/PEO] [40], poly(2-methyleoxazoline) [41], while
to the second class belong poly(dimethyl siloxane) [PDMS] [41], poly(caprolactone) [PCL] [42],
poly(lactide) [40], poly(methyl methacrylate) [PMMA] [43].
Recently, Rameez et al. [42] describe the preparation of polymerosomes made by biodegradable
and biocompatible amphiphilic diblock copolymers composed of poly(ethylene oxide) [PEO] and
poly(caprolactone) [PCL] or poly(ethylene oxide) [PEO] and poly(lactide) [PLA]. In the diblock
copolymers, PEO acts as the hydrophilic block and either PLC or PLA can function as the hydropho-
bic blocks. The important feature of this formulation is that PEO, PCL, and PLA are biocompatible
polymers and the last two polymers are biodegradable. Polymerosomes based on amphiphilic block
copolymers are interesting vesicles for drug delivery (and also for diagnostic purpose), because, by
selecting different block copolymers, the membrane properties, the stability, and the rate of deg-
radation can be conveniently tuned. For example, PEO-PCL and PEO-PLA polymerosomes have
been demonstrated to be able to encapsulate hemoglobin and they are promising candidates for
hemoglobin-based oxygen carriers.
Figure 19.6 shows a comparison of the hemoglobin encapsulation efficiency of the two types
of polymerosomes (PEO-PCL and PEO-PLA, respectively) (data redrawn from Ref. [42]). As the
molecular weight of the PEO block of the copolymer increases, the hemoglobin encapsulation effi-
ciency decreases. This is a simple consequence of the fact that, with the increase of the molecular
weight, the length of the outer leaflet of the polymerosomes becomes larger and less volume in the
core will be available for hemoglobin encapsulation. Moreover, the hemoglobin-polymer weight
ratio can be appropriately modified in order to make the oxygen carrying capacity of these poly-
merosomes comparable to the one of blood.
25
Low molecular weight
Hb encapsulation efficiency (%)

20
15
10 High molecular weight
0
PEO-PCL PEO-PCL PEO-PLA PEO-PLA
FIGURE 19.6 Comparison of hemoglobin encapsulation efficiency in polymersosomes composed of

poly(ethylene oxide-b-caprolactone) [PEO-PCL] and of poly(ethylene oxide-b-lactic acid) [PEO-PLA]. The
polymers have different molecular weights, in the range 10–15 kDa (referred to as high molecular weight) and
in the range 1–3 kDa (referred to as low molecular weight). Darker histograms refer to bovine hemoglobin and
lighter histograms refer to human hemoglobin. (Redrawn and adapted from Rameez, S. et al., Bioconj. Chem.,
19, 1025, 2008.)
Together with polymerosomes, we mention a new multicomponent structure, known as veso-

some, consisting of drug-loaded liposomes encapsulated with another bilayer. Even if vesosomes
are currently too large for optimal intravenous use, they represent a promising and interesting drug
carrier, as far as the retention and stability are concerned, compared with traditional liposomes.
19.3.1 Polymer Vesicles

One of the major problems with the aggregation of lipidic molecules into closed lipid bilayers (lipo-
somes) is a reduced stability that, as far as the drug delivery context is concerned, results in a rapid
clearance of the encapsulated drug, for example, after the intravascular administration. This disad-
vantage can be avoided, and the stability increased, by surface grafting of hydrophilic polymers [44]
or by polymerization of reactive lipid molecules in the vesicular aggregates [45].
Another approach involves the layer-by-layer deposition of polyelectrolytes on the surface of
charged nanoparticles followed by the subsequent dissolution of the templating particles [46].
Finally, a different approach consists in the self-assembly of amphiphilic diblock copolymers into
micelles, in a selective cross-linking of their hydrophilic shell and the subsequent degradation of
the hydrophobic core [47].
As stated earlier, block copolymers have been studied extensively because of their interesting
properties and their self-assembling behavior in aqueous solution. Recently, attention has been
focused on a specific class of hybrid block copolymers, employing a well-defined peptide sequence
combined with synthetic polymers, resulting in novel aggregates for drug delivery. Just to mention
some of them, we remember here copolymers of β-sheet folding peptides prepared by Sogah and
coworkers [48], consisting of alanine (or alanyl-glycine) repeats and poly(ethylene glycol) or the
diblock copolymers of polybutadiene and poly(glutamic acid) prepared by Klok et al. [49] that form
vesicles whose size could be altered by variation of the pH.
More recently, a well-defined ABA triblock copolymer based on the atom transfer radical polym-
erization of methyl methacrylate was mentioned by Ayres et al. [43], resulting into aggregates that
were a mixture of polymerosomes and large compound micelles, ranging from 400 nm to 10 μm
in size. As noted by these authors, their method for the preparation of peptide-containing triblock
copolymers leads to a wide range of fascinating structures in the nanometer size range whose char-
acteristics can be appropriately tuned by varying the peptide, the length of the polymer, or even the
kind of the monomer that is polymerized.
Finally, the synthesis of a triblock copolymer composed by poly(2-methyleoxazoline) [PMOXA],
poly(dimethylsiloxane) [PDMS], PMOXA-PDMS-PMOXA, carrying polymerizable methacrylate
groups at both chain ends has been reported by Nardin et al. [41]. This polymer forms vesicular
structures in dilute aqueous solution which can be polymerized to hallow nanospheres.
19.3.1.1 Microcapsules
A microcapsule can be considered as a microsphere containing many small cavities inside. When a
drug is included in one of these cavities, before being released, it should permeate through several
regions of polymer, sustaining a release over a long period of time. For example, in the case of poly-
peptide microcapsules [50] built up by poly[L-Lys(Z)]-block-poly(Sar) [PKZ-b-PS] and poly[L-
Glu(OMe)-block-poly(Sar) [PMG-b-PS], the release of fluorescein-labeled dextran [FITC-dextran]
entrapped within the microcapsule during the stage of preparation occurs over period of time on the
order of ten days (Figure 19.7).
One of the more intriguing properties of polypeptide microcapsules is that these structures are
capable of modulating the drug release in response to external signals due to the easiness of a chem-
ical modification at the site of the functional groups of the amino acid components. If we stick to
the examples described by Kimura et al. [50], a pH-responsive microcapsule can be prepared by the
connection of poly(L-Glu) to the side chain amino groups in partially deprotected poly[L-Lys(Z)]-
b-poly(Sar), [PKZ-b-PS]. In this case, the carboxyl groups are located in the graft chains connected
to the positively charged main chain. The influence of pH in the release of fluorescein-labeled dex-
tran [FITC-dextran] encapsulated in the microcapsule is shown in Figure 19.8.
Figure 19.9 shows the release of FITC-dextran from microcapsules when the temperature goes
up and down between 25°C and 40°C (data taken and redrawn from ref. [51]). When the temperature
was raised to 40°C, the pNIPAAM gel in the small cavities shrank, producing a pathway where
dextran could diffuse more or less freely.
19.3.1.2 Microspheres
Some of the most widely studied materials for drug delivery are aliphatic polyester-based poly-
mers which include poly(lactic acid) and its copolymers. Poly(lactic acid) is a biodegradable and
70
60
FITC-dextran released (%)
50
40
30
20
10
–2 0 2 4 6 8 10 12 14 16 18
Time (day)
FIGURE 19.7 Amount of FITC-dextran released from microcapsules built up by PZK-b-PS (⚬) and by
PGM-b-PS (▪). (Redrawn and adapted from Kimura, S. et al., Polym. Adv. Technol., 12, 85, 2001.)
3.0
pH = 7
FICT-dextran released
2.0
pH = 3
1.0
0.0
1 2 3 4
Time (days)
FIGURE 19.8 Release of FITC-dextran from poly(L-Glu)-grafted-PKZ-b-PS microspheres for different val-
ues of pH, as a function of time. (▪): pH = 7; (⚬): pH has been changed from 7.0 to 3.0 and then after brought
back to the value of pH = 7.0. (Redrawn and adapted from Kimura, S. et al., Polym. Adv. Technol., 12, 85, 2001.)
12
10
FITC-dextran release (a.u.)
T = 40°C
2
0 T = 25°C
0 100 200 300 400 500 600

Time (min)
FIGURE 19.9 Release of FITC-dextran from pNIPAAM-loaded microcapsule in response to temperature

jump between 25°C and 40°C. (Redrawn and adapted from Kidchob, T. et al., J. Chem. Soc. Perkin Trans.,
2, 2195, 1997.)
thermoplastic polymer (that has been approved by the Food and Drug Administration [USA]) for
use in human subjects. As biocompatible polymer, it has been employed as suture material, inter-
body cages, scaffolds for tissue engineering and for biodegradable drug delivery systems [52–54].
This poly(lactic acid) has been one of the most widely used biodegradable polymers for drug deliv-
ery systems and porous scaffolds [55,56].
Another classic biodegradable polymer able to form nanospheres and microspheres is poly(DL-
Lactide-co-Glycolide) [PLGA] polymer. Together with poly(DL lactic acid), PLGA has been one
of the most widely used polymers because of its excellent tissue compatibility and safety for use in
humans [57].
Nanoparticles, because of their surface properties, have been proved to guarantee a prolonged
in vivo circulation and a sustained drug release.
Hyaluronic acid-coated poly(butyl cyanoacrylate) nanoparticles (of about 300 nm in size) were
synthesized by He et al. [58]. These particles represent an effective and safe vehicle for hydrophobic
anticancer drugs such as paclitaxel [PTX], a model anticancer drug. In this system, whereas the
PTX encapsulation was obtained with an efficiency up to 90%, the in vitro drug release profile
clearly shows that the presence of hyaluronic acid slows down the drug release and reduces the ini-
tial burst in the first 10 h. Moreover, the particles have a very good biocompatibility, the hemolysis
ratio being well less than 2%.
Recently, an increasing interest as drug carrier in clinical setting has been turned to albumin
[59]. Albumin is an acidic protein stable in the pH range of 4–9, that preferentially uptakes in tumor
and inflamed tissues. These properties, besides ready availability, biodegradability, and lack of
toxicity, make it a very interesting candidate for drug delivery. A recent review that gives account of
albumin in different drug delivery systems has been made by Kratz [59]. Here, we will report some
results dealing with micro- and nanoalbumin particles. Albumin microspheres, which are usually in
the range 1–100 μm, can carry therapeutic or diagnostic agents. However, the size is the main fac-
tor for the biodistribution of the microspheres. Whereas small (1–3 μm) microspheres are taken up
by the reticuloendothelial system, accumulating in the liver and spleen, as well as in solid tumors,
larger microspheres (>15 μm) accumulate in capillary bed of the lungs.
Recently, albumin nanoparticles ideal for the encapsulation of lipophilic drugs in the size range
comparable with the size of small liposomes (100–200 nm) have been developed by American
Bioscience, Inc. As an example, albumin nanoparticles with paclitaxel [nab-paclitaxel], approxi-
mate diameter of 130 nm, have been extensively investigated preclinically and clinically, showing
superior antitumor efficacy over paclitaxel in a number of human tumor models [60].
Figure 19.10 shows the antitumor activity of nab-paclitaxel (30 mg/kg/d) and paclitaxel (13.4 mg/
kg/d) in the MX-1 xenograft model.
Among polymeric particles, we mention hydrogel nanoparticles that have gained considerable
attention as a promising drug delivery system, combining the characteristics of a hydrogel (hydro-
philicity and large water content) with the one of the nanoparticles (size on the order of 50–100 nm).
In the recent years, several polymeric hydrogel nanoparticles have been prepared and characterized,
based on both natural and synthetic polymers [61].
The most common monomers employed in hydrogels for pharmaceutical applications include
Hydroxyethyl methacrylate [HEMA], Ethylene glycol dimethacrylate [EGDMA], N-isopropyl Aam
2000
1500
Tumor volume (mm3)
1000
500
10 20 30 40 50 60 70
Time (h)
FIGURE 19.10 Antitumor activity of nab-paclitaxel (30 mg/kg/d) (▪) and paclitaxel (13.4 mg/kg/d) (⚬) in the
mamma carcinoma MX-1 model (at equitoxic dose). (Data redrawn and adapted from Desai, N. et al., Clin.
Cancer Res., 12, 1317, 2006.)
[NIPAAm], Ethylene glycol [EG], and many others. Hydrogels must have an acceptable biodegrad-
ability and biocompatibility that can be assured by the different improved crosslinking methods.
Hydrogel-based systems can be divided into two categories, according to the manner they pro-
vide the drugs, i.e., time-controlled systems and stimuli-responsive systems. These latter systems
will be considered in detail in the next section. As far as the former one is concerned, the drug
release is controlled by diffusion, according to Fick’s first law of diffusion (with constant or variable
diffusion constants), depending on the mesh size within the matrix of the gel. Typical mesh size
in biomedical hydrogels range from 5 to 100 nm [62,63], the drug diffusion being not considerably
inhibited in the swollen state.
Hydrogel particles of nanoscopic size (nanogel), merging the features and the characteristics
that hydrogel and nanoparticles separately possess, have been the subject of considerable amount
of effort in drug delivery approaches. They summarize the hydrophilicity, flexibility, high water
content, biocompatibility typical of the hydrogels with the advantages of nanoparticles which allow
long life in the circulation system and the possibility of targeting the desired site. Detailed classifi-
cation and applications of different hydrogel polymeric materials have been reviewed by Coviello
et al. [64], Lin et al. [65] and Peppas et al. [66].
19.4 STIMULI-RESPONSIVE POLYMERS

An ideal drug delivery system should respond to physiological requirements and modify the drug
release profile accordingly. Much research has been directed toward single- or dual-stimuli-responsive
hydrogel for drug delivery.
Stimuli-responsive polymeric drug delivery is a method of increased importance in which drugs
can be delivered to the body that is able to respond to physiological requirements and modify the
drug-release profile accordingly [67]. The main characteristic of these systems is their ability to
undergo changes from a hydrophilic to a hydrophobic state triggered by small changes in the envi-
ronment. Moreover, these changes are reversible in the sense that the system is able to return to its
initial state as the external stimulus is removed. Compared to the more conventional delivery
methods, where the drug concentration results in a peak followed by a rapid fall, these systems
allow maintaining the desired drug concentration within the single administration.
The external stimuli able to induce a response in the polymer structure are extremely various,
ranging from electrical, magnetic, physicochemical, thermal, or mechanical stimuli. A summary of
them, together with the associated release mechanisms, has been reviewed by Soppimath et al. [68].
Here, we will consider only the most important of them and will report and discuss some of the
more typical examples.
19.4.1 Thermo-Responsive Polymers
The characteristic of this class of polymers, which are the most commonly studied class of stimuli-
responsive polymers, is the presence of a critical solution temperature at which a phase transition
occurs. In other words, if the polymer becomes insoluble upon heating, hydrophobic interactions pre-
vail and therefore hydrogels made of these polymers shrink as the temperature increases. These hydro-
gels are made of polymer chains that possess a mixture of hydrophilic and hydrophobic segments.
The most widely synthetic thermo-responsive polymer is poly(N-isopropyl acrylamide)
[pNIPAAM] which exhibits a coil-globule transition at its lower critical solution temperature
[LCST]. Its biocompatibility and the occurring of the LCST at 32.3°C makes pNIPAM a very inter-
esting material for controlled release applications. Moreover, the LCST is largely independent of the
molecular weight and concentration and its phase transition temperature can be easily tuned around
an appropriate temperature (≃ 40°C) by shifting the hydrophilic-hydrophobic balance by means of
copolymerization achieved by the introduction of a hydrophilic co-monomer [69]. Hydrophilic copo-
lymers decrease the LCST whereas hydrophobic copolymers have the opposite effect.
80 100
70
60 80
Cell viability (%)

50 60
40
40
30
20 20
10
0 0
0 20 40 60 80 100 120 140 160 180 0.1 1 10 100
(a) Time (h) (b) Doxorubicin (µg/mL)
FIGURE 19.11 (a) DOX release behavior from DOX-mixed micelles under acidic (pH = 5.0, (⚬)) and neutral
(pH = 7.4, (▪)) conditions. (b) Cell viability of HeLa cells treated with various concentrations of free DOX for
1 day (•) and 3 days (⚬) incubation. (Data redrawn from Lo, C.-L. et al., Biomaterials, 30, 3961, 2009.)
Polymers that exhibit a temperature phase transition have been the subject of many investigations
[70], leading to many intriguing applications such as artificial muscle, nanoactuators, and motor
systems [71,72].
Recently, two other polymers attracted attention. They are poly(N,N′-diethyl acrylamide)
[pDEAAM] that has lower critical solution temperature in the range of 25°C–35°C [73] and poly(N-
(1)-1-hydroxymethyl propyl methacrylamide) [HMpMAAM], which is a novel thermo-responsive
polymer designed for optical activity [74].
Mixed micelles formed from critical micelle concentration and temperature-sensitive di-block
copolymers for biomedical applications have been widely investigated. However, different impedi-
ments prevent their use. Among these, there is the lack of biocompatibility of the most part of tem-
perature sensitive copolymers, the uncontrolled mechanism of drug release, because of their phase
transition temperature, and, over all, the poor micellar stability.
A new class of mixed micelles composed of temperature-sensitive diblock copolymers and Cmc
diblock copolymers has been recently proposed by Lo et al. [75], which exhibit an improved sta-
bility under various physiological conditions and can be applied to intravenous drug delivery of
different anticancer drugs. In this particular case, these authors employed methoxy poly(ethylene
glycol)-block-poly(DL-Lactide) [mPEG-b-PLA], a biocompatible Cmc diblock polymer, and
methoxy poly(ethylene glycol)-block-poly(N,n-propylacrylamide-co-vinylimidazole) [mPEG-b-
P(NnPAAm-co-Vim)], a temperature-sensitive copolymer.
The drug release from mixed micelles incorporating doxorubicin [DOX] is shown, as an exam-
ple, in Figure 19.11, together with the cell viability of human cervical epithelioid carcinoma cells
treated with DOX-mixed micelles for one (or three) day incubation.
This new micellar system has various remarkable characteristics important for practical applica-
tions such as smaller and uniform particle size, marked stability, and a rapid response to stimuli,
besides an easy and economic method of preparation.
19.4.2 Electro-Responsive and Magnetically Responsive Polymers

Polyelectrolytes, which are polymers with a high concentration of ionizable groups along the
backbone chain, are sensitive to an external electric field. Under this influence, electro-responsive
hydrogels generally shrink or swell, allowing the release of a pre-loaded drug under controlled
conditions.
Magnetic targeting is based on the attraction of magnetic particles to an external magnetic field
and a translational force will be exerted on the drug-encapsulated particle complex (magnetic drug
targeting [76]).
19.4.3 Light-Responsive Polymers
A typical example of a light-response polymer is a polymer network where a leuco derivative mol-
ecule has been inserted in order to produce a UV light–responsive hydrogel [77]. Leuco derivatives
are normally neutral but dissociate into ion pairs under an ultraviolet irradiation. This causes a swell
due to an increase in osmotic pressure due to the increased ion concentration. Notably, hydrogels
shrink when the light is removed.
An interesting and versatile system investigated by Sumaru et al. [78] is based on pNIPAM and
acrylamide-functionalized spirobenzopyran that is able to inter-convert between neutral and zwit-
terionic form upon irradiation. This photochemically switchable polymer undergoes four distinct
states consisting of a colorless spiro form, a colored merocyanine, and of the corresponding proton-
ated merocyanine and spiropyran, which exhibit different solubilities with temperature. This behav-
ior, which is extremely photosensitive during UV irradiation, is caused by the hydrophilic character
of merocyanin and the hydrophobic character of spiropyran.
19.5 INTERNAL STIMULI-RESPONSIVE SYSTEMS

19.5.1 pH-Responsive Polymers
The physiological pH varies according to the various tissues and cellular compartment. Its value
is strongly acid in the stomach (pH = 1.0–3.0), shifting to basic values (pH = 5–8) in the intes-
tine or close to neutral in the blood (pH = 7.35–7.45). This property can be favorably addressed in
pH-responsive polymers. Basically, weak acids and bases linked to a polymer chain may change
their ionization upon variation of pH, with a consequent conformational change of the whole poly-
mer chain or a swelling behavior in the case of a hydrogel. The pH-responsive swelling and the col-
lapsing behavior has been used to induce release of caffein [79], indomethacin [80], or lysozyme [81].
A change in pH is used in drug release in cancer targeting, because tumor tissue has generally an
extracellular pH slightly lower than normal tissue (pH = 6.5–7.2 compared with pH = 7.4 in normal
condition) [82].
The pH-sensitive micelles appear to be a promising candidate for photosensitizer delivery for
photodynamic therapy. As a matter of fact, it is advantageous in therapy of this kind if a drug deliv-
ery system is used that releases the drug (in this case the photosensitizer) selectively at the target
site. In this context, pH-triggered photosensitizer release using polymeric micelle formulation, has
been suggested by Leroux el al. [83] and by the group of Kataoka [84]. Their main results have been
recently summarized by van Nostrum [85]. For example, the latter author employed a polycationic
porphyrin dendrimer mixed with a polyanionic poly(ethylene glycol)-poly(aspartic acid) diblock
copolymer forming high-stable micelles [PIC micelles [86]], with an average diameter of 56 nm.
These micelles are internalized in Lewis lung carcinoma cells with no marked toxicity exhibiting
a high photodynamic efficiency. These systems offer many opportunities to be further optimized,
for example, using degradable systems in order to prevent accumulation and long-term toxic effects.
Stimuli-responsive polymers may respond as well to applied stimuli originated from internal phys-
icochemical changes occurring within various body tissues. A typical example is a pH-responsive
polymer designed for oral delivery which experiences marked pH changes along different gastrointes-
tinal tracts, passing from pH ≃ 1.0−3.0 in the stomach to pH ≃ 7.0−7.5 in the colon [87].
This class of polymers present acidic or basic groups that allow the accepting or the release of protons
in response to a change in pH. Polymers undergo a conformational change with a consequent change
in the swelling behavior of the hydrogel when ionizable groups are linked to the polymer structure.
To this class of polymers, poly(acrylamide) [PAAM], poly(acrylic acid) [PAA], poly(methacrylic acid)
[PMMA], and many others belong. On the exposure to aqueous media of appropriate pH, pen-
dant groups ionize and produce a charge distribution on the polymer network causing swelling
or deswelling of the hydrogel originated by electrostatic repulsive forces. This process ultimately
controls the drug delivery [88].
pNIPAM has been extensively studied for oral delivery of calcitonin and insulin. These sub-
stances are immobilized in the polymeric beads which are stable passing through the stomach.
Conversely, in the alkaline intestin, the beads disintegrate with the consequent drug release. This
approach was followed by Serres et al. [89] for the intestinal delivery of calcitonin and by Kim et al.
[90] for the delivery of insulin. In these cases, the thermal stimulus from the pNIPAM is not needed
for the delivery, but it is appropriately exploited for the preparation of the loaded beads.
For drug delivery, a response to a local environmental stimulus at the disease site is, in principle,
an ideal way to direct a therapeutic drug toward the target. At least from a theoretical point of view,
factors able to control a polymer response are extremely wide, including biological factors such as
over-expressed intracellular enzymes, cell surfaces receptors, inflammation signaling molecules, or
physicochemical factors such as hyperthermia. This is the reason that has led to increased interest
in polymers in biomedical and biological research, giving rise to a branch known as bioresponsive
chemistry in drug delivery.
As an example of this approach, we can mention the method proposed by Shabat and co-workers
[91], who used a “self-immolating” system, i.e., macromolecules able to break down by a cascade
process following biochemical reactions and triggered by biochemical stimuli. In the earlier-stated
example, the first process in realizing the target molecule employs a nucleophilic intramolecular
cyclization to generate a stable cyclic species and the second employs a quinine methide rearrange-
ment followed by a self-elimination to release the therapeutic agent.
When a controlled drug release profile is required, for example, in diseases such as diabetes,
reversible responsive polymers have been implemented. Diabetes is one disease that received great
attention because of the potential for therapies using controlled drug release.
Polymers that respond to changes in local glucose concentration by releasing insulin have been
studied extensively investigated by Miyata et al. [92]. Acidic copolymer hydrogels in which glucose
oxide has been immobilized represent a significative example. In the initial state, because of the
extended polycarboxylates, the system is impermeable to macromolecular transport (e.g., to insulin)
but permeable to sugar, resulting in the production of gluconic acid. Because of the protonation of
the gel, caused by pendant glucose oxidase, following the ingress of glucose, a contraction of the
polymer matrix occurs, allowing insulin through.
The “molecular gates” systems, shown in Figure 19.12, consist of an insulin-containing reservoir
with a polymeric membrane in which glucose-oxidase has been immobilized. The gel expands at
H+
H+
Glucose oxidase
Insulin
FIGURE 19.12 Sketch of a glucose-responsive polymer for “gating” a flow of insulin. Left-hand side: chain-
extended polycarboxylates are impermeable to macromolecular transport (insulin), but permeable to glucose.
Right-hand side: Pendant glucose oxidase generates protons following ingress of glucose, which in turn causes
polymer protonation and chain collapse. The membrane opens and allows insulin through. (Redrawn and
adapted from Alexander, C. and Shakesheff, K.M., Adv. Mater., 18, 3321, 2006.)
high pH values, closing the gate, and shrinks at low pH values (due to the interaction of glucose
with immobilized glucose-oxidase), opening the gates. Control of the insulin delivery depends on
the size of the gates and on the rate of the gates’ opening and closing [93].
There is more. Since conversion of glucose to gluconic acid needs the presence of oxygen, the effec-
tiveness of the system could be limited by the oxygen availability in the hydrogel matrix. This limita-
tion can be at least partially overcome by incorporation of a further enzyme, catalase, into the polymer
matrix, with the charge of reducing hydrogen peroxide generated during the glucose oxidation.

Polymers, that represent an exceptional versatile class of materials, have led to the development of
several novel drug delivery systems. The new strategy, derived from the amalgamation of polymer sci-
ence and pharmaceutical science, is based on the symbiotic union between two partners, the polymer,
that acts as a carrier to move the drug to the target, and the drug itself, that, in virtue of the polymer
interaction, is preserved during the transport and exerts its bioactivity only in the target tissue.
Within this context, polymeric aggregates are promising candidates for a wide range of applica-
tions in the field of drug delivery systems. This strategy, based on polymer aggregates of different
physicochemical characteristics and started more than a quarter of a century ago, with the advent
of polymer-drug conjugates, is destined to get on in the delivery of multiple agents to target several
pathways to overcome the challenge of tumor heterogeneity.
Incorporation of a drug in a polymeric structure provides a lot of opportunities for optimizing
polymer-based drug delivery systems, such as enhancing drug loading efficiency, biocompatibility,
a controlled release of the drug from the polymeric matrix, and the minimization of the side-effects.
Future advances in polymer science will be based on modifying the physical and chemical prop-
erties of the polymer in order to reach a wide acceptability and distinct advantages such as the
possibility of tuning the polymer aggregate size, high solubility, simple sterilization and a wide
biocompatibility and, finally, a controlled release of drugs. It is expected that continuous investiga-
tions on polymer aggregates with various structures will lead to an optimal formulation for drug
delivery systems, opening a new stage in the chemotherapy development to achieve a personalized
therapy of cancer.
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20 Photoresponsive Polymers
for Control of Cell
Bioassay Systems
Kimio Sumaru, Shinji Sugiura, Toshiyuki Takagi,
and Toshiyuki Kanamori
CONTENTS
20.1 Introduction........................................................................................................................... 683
20.2 Wet and Soft System Manipulated by Light Irradiation........................................................684
20.3 Photoresponsive Polymers..................................................................................................... 685
20.4 Development of Photoresponsive Hydrogels......................................................................... 689
20.5 Micromanipulation of Photoresponsive Hydrogels............................................................... 691
20.6 Photo-Control of Cell Adhesion............................................................................................ 695
20.7 Elaborate Cell Patterning on Photoresponsive Culture Substrate.........................................700
20.8 Summary and Future Outlook............................................................................................... 703
References....................................................................................................................................... 705
20.1 INTRODUCTION
Corresponding to recent increase in need for innovative technologies in terms of tools for biological
research and medical diagnosis, cell chips have attracted lots of attention of many researchers and
pharmaceutical industry as new biochips following DNA chips, protein chips, and glyco chips
(Singhvi et al. 1994; Ito et al. 1997; Ziauddin and Sabatini 2001; Anderson and van den Berg 2004;
Suh et al. 2004; Hui and Bhatia 2007). A number of critical problems arising in the integration of
bioassays on the basis of conventional cell-screening methods have been expected to be cleared
feasibly by using the cells cultured in array on a chip in a cost-effective manner. However, unlike
existing biochips to array molecules such as DNA, proteins, and sugar chains (Fodor et al. 1991;
Chee et al. 1996; Kiessling and Cairo 2003), cell chips are made of living cells and need certain
conditions to keep cells alive until the end of bioassays, imposing severe restriction in preparation,
storage, and distribution of the cell chips. Therefore, compatibility to the on-site preparation is also
considered in designing the construction and the operation of cell chips.
In order to implement high-throughput screening, integrated microfluidic systems need to be
designed to generate myriad bioassay conditions; mediums or drug solutions should be dispensed to
each culture chamber according to each different schedule.
Further, it is required to induce the cultured cells to express and maintain their high-order func-
tions by bringing the environment of the cell chips close to that in vivo, since the cell bioassay is
based on functions of cells in internal organs or body tissues (Fukuda and Nakazawa 2005; Nakazawa
et al. 2006; Sakai and Nakazawa 2007; Tamura et al. 2008). Therefore, the methods to construct
elaborated co-culture systems using multiple cell types are a key to prepare biomimetic cell chips
that mimic in vivo conditions in addition to the development of microfluidic portions of cell chips.
683
Thus, the development of practically applicable cell chips needs to break down many obstacles,
which are hard to get over with existing technologies. In these circumstances, many research-
ers are developing a variety of new technology components, which could make a breakthrough.
Among them, we proposed a scheme to manipulate cell adhesion on culture substrate and fluid
flow in microfluidic systems freely by means of computer-controlled patterned light irradiation.
This scheme is to manipulate living cells and fluid flows individually in a parallel manner taking
full advantage of light irradiation as a means of signaling: the applicability to the object locally
and instantaneously in a noncontact manner. In this chapter, we will briefly review the research
on photoresponsive biopolymers as technology components to construct and control integrated
cell bioassay system.
20.2 WET AND SOFT SYSTEM MANIPULATED BY LIGHT IRRADIATION

Not to point out, light has been playing quite an important role in biological research. Optical micro-
scopes have been widely used as the most general means to observe cells and tissues since the
dawn of biology. Confocal scanning laser microscopes which enable us to observe the 3-dimensional
structure of cells and tissues, as well as fluorescent microscopes, have spread rapidly since 1990 in
the research field of biology, and have become common tools for most researchers nowadays. Further,
accompanied with the rapid progress in optoelectronics, the single molecule analysis (Kitamura et al.
1999) and super-resolving microscopy (Fujita et al. 2007; Kawata et al. 2008) have been developed
and applied in mainly biological research.
As seen earlier, the use of light has been common especially in a passive scheme such as a
detection of the light from the objects in the research field of biology. On the other hand, a few
technologies utilizing light as a means to control the objects in an active manner have been
developed. Above all, “optical tweezer,” which is a method to capture a small object by applying
light focused with an objective lens of high power (Ashkin 1970, 1992), has been applied to
manipulate suspended cells (Ashkin and Dziedzic 1989a,b). More recently, pulsed laser irradia-
tion with high energy was introduced as a tool to kill living cells or to make a hole in cellular
membrane (Hosokawa et al. 2004; Iwanaga et al. 2006; Smith et al. 2006), and several products
based on this method have already been released (Szaniszlo et al. 2006).
In addition to these existing technologies to let the light affect the objects directly, other strate-
gies to manipulate small objects such as cultured cells and fluid flows in microfluidic system via the
photoresponsive materials have been investigated (Figure 20.1), and new class of photoresponsive
materials, which are wet and soft, have been developed so far to construct the systems implementing
such strategies. In practice, several research groups including us have developed culture substrates,
whose cell adhesion is controlled by light irradiation, and the hydrogels, which exhibit volume
change in response to light irradiation. Mediated by these materials, the light irradiation applied as
an input signal is transduced to the change in cell adhesion of culture substrate, or to change in the
volume and the shape of a hydrogel.
On the other hand, an apparatus to irradiate light in arbitrary patterned area in the microscope
field of view at arbitrary timing is necessary in order to control the properties of photorespon-
sive materials locally and instantaneously in a noncontact manner. To meet the requirement, a
computer-controlled micropattern irradiation system (Kikuchi et al. 2009) was developed by the
cooperation of our research group and Engineering System Co. (Matsumoto, Japan). A schematic
illustration is shown in Figure 20.2. A projection unit and an image sensor unit, built in the opti-
cal system of an inverted biological microscope, are both controlled by a computer software, and
the users can specify the area, where the light should be irradiated, just by dragging the pointer
on the monitored microscope field of view. Also, light irradiation and microscopic observation,
which are associated with each other, can be easily automated due to their high compatibility to
information technology.
Photoresponsive Polymers for Control of Cell Bioassay Systems 685
Microscopic
observation
Detection
Cell adhesivity Volume/ Magnification

change profile change
Photo- Photo-
responsive responsive
substrate hydrogel
Light Light Reduction
Projection
“On”
On-demand
patterned
irradiation
“Off ”
FIGURE 20.1 Scheme to manipulate small objects based on the conversion of light to material property.
20.3 PHOTORESPONSIVE POLYMERS

Organic photochromic materials whose chemical structures and properties are varied by light
are promising as a means to transduce an input signal of light irradiation to the surface property
or the morphology of material. Among them, spiropyrans, azobenzenes, and diarylethenes are
known to exhibit reversible changes (Figure 20.3). Research on their application has been carried
out very actively, and some of them have already been used practically in, for example, optical
storage media.
In terms of the attempts to provide polymer materials with photoresponsive property by introduc-
ing photochromic groups, Goodman and Falxa reported in 1967 that the conformational transition
was induced by light irradiation for an azobenzene-functionalized polypeptide in trifluoroacetic
acid (Goodman and Falxa 1967), and many related researches were carried out subsequently
(Pieroni et al. 1980, 1985; Fissi et al. 1996). Then, Ishihara et al. reported that the surface property
such as water wettability and dye adsorption of azobenzene-functionalized poly(2-hydroxyethyl
methacrylate) was changed by light irradiation (Ishihara et al. 1982). Later, in the first report
on the photoresponsive solution property observed in aqueous system, Kungwatchakun and Irie
described that the lower critical solution temperature (LCST, transition temperature between mis-
cible and immiscible conditions) was largely shifted by light irradiation for an aqueous solution
of poly(N-isopropylacrylamide)(pNIPAAm) functionalized with azobenzene (Kungwatchakun
and Irie 1988). Later, on pNIPAAm functionalized with azobenzene in aqueous system, several
researches have improved the photoresponsive properties (Desponds and Freitag 2003; Akiyama
and Tamaoki 2004).
Microscopic image Irradiation pattern
Computer
FIGURE 20.2 Schematic illustration showing construction of computer-controlled micropatterned irradia-

tion system.
On the other hand, Kröger et al. found that the LCST of aqueous solution of poly(N,N-dimethyl-
acrylamide) functionalized with azobenzene was shifted by at most 20°C in response to light irradi-
ation (Kröger et al. 1994). Afterward, Shimboji et al. modified an enzyme with this photoresponsive
copolymer and demonstrated that the enzymatic activity could be controlled by light irradiation
(Shimoboji et al. 2002). As aqueous systems of photoresponsive polymers other than azobenzene-
functionalized ones, there were several reports on aqueous solutions of polymer functionalized with
leuco chromophores (Irie and Hosoda 1985).
Also, there were some reports on photoresponsive polymers functionalized with spiropyrans
which were among the most studied organic photochromic materials along with azobenzenes and
leuco dyes since 1970s (Smets et al. 1978; Irie et al. 1979, 1985; Menju et al. 1981; Pieroni et al.
1992). However, all these reports were on the polymer property in organic solvents, and research on
aqueous system had hardly been carried out until 2000s.
Later, we found that pNIPAAm functionalized with spiropyran chromophore (pSpNIPAAm)
exhibited drastic photoresponsive dehydration in acidic aqueous solution of pH 4 or less (Sumaru
et al. 2004a,b). The polymer solution which had been transparent and yellowish in the dark turned
colorless in quick response to blue light irradiation due to the structural change (isomerization) of the
hv
O– N+ O N
hv ’, ∆
Spiropyran
N=N
hv
N=N
hv ’, ∆
Azobenzene
NC CN NC CN
hv
hv ’
S S S S
Diarylethene
FIGURE 20.3 Structural change of organic photochromic materials through photoisomerization.
chromophore. In addition to the change in color, the change in the physical properties of the chromo-
phore accompanied by the photoisomerization is so substantial as to influence much on the hydration
of pNIPAAm main chain; drastic dehydration was triggered by the isomerization of the chromophore
introduced at a small functionalization ratio of only one monomer mol%. As a result, drastic turbidity
increase followed by the precipitation was observed in the polymer solution after light irradiation at
temperature from 30°C to 37°C (Figure 20.4). After stopping the irradiation, however, the solution
returned to its original transparent state in one or a few hours, and this reversible change was repeat-
able for at least 10 times without any detectable degradation.
Thereafter, our detailed analysis revealed that the spiropyran chromophores introduced at side
chain of pNIPAAm were in a protonated open-ring form, which exhibited yellow color due to strong
absorbance at around 420 nm. These positively charged chromophores contributed to stabilize the
0.8
0.7
0.6
Turbidity at 700 nm
0.5
0.4
0.3
0.2
0.1
0
24 26 28 30 32 34 36 38
Temperature (°C)
FIGURE 20.4 Influence of the light irradiation and temperature change on the turbidity of 0.10 wt%
pSpNIPAAm aqueous solution containing 0.20 mM HCl. Open squares: under irradiation, closed circles: in
the dark.
x : y = 1 : 99
O O O NH
x y
O ∆
N
OH +
+ +N
H+ Light
λmax : 422 nm
pNIPAAm
main chain
Light
2.0
1.5
Absorbance + turbidity
After irradiation
1.0
In the dark
0.5
0.0
300 350 400 450 500 550 600 650 700
Wavelength (nm)
FIGURE 20.5 Schematic illustration showing the photo-induced structural change and the following dehy-
dration of pSpNIPAAm.
hydration of pNIPAAm main chain even at temperatures higher than LCST of pNIPAAm aque-
ous solution. Irradiated with blue light, on the other hand, most of the chromophores isomerized
effectively to a nonionic and hydrophobic form through the photo-induced ring closure and proton
dissociation, destabilizing the hydration of main chain (Figure 20.5).
As a spiropyran-functionalized water-soluble polymer other than the pSpNIPAAm described ear-
lier, we examined dextran functionalized with nitrospiropyran (NSp-Dex) (Edahiro et al. 2006). An
aqueous mixture solution of dextran and poly(ethylene glycol) (PEG) at concentrations in a certain
range was known to separate into two aqueous phases at temperatures higher than a certain critical
temperature, while the mixture solution was uniform at the lower temperature (Tjerneld et al. 1985;
Andersson and Hahn-Hagerdal 1990). By functionalizing dextran with nitrospiropyran composing
O
O O OH
OH
O
y
OH
OH x OH
O
O
+ H ∆
N O NO2 N O NO2
+ H+
Light
Unstable region Unstable region

NSp-Dex fraction
Light
Stable region Stable region

PEG fraction PEG fraction
FIGURE 20.6 Schematic illustration showing the photo-induced structural change of NSp-Dex and the
mechanism of photo-induced phase separation of aqueous mixture solution of NSp-Dex and PEG.
such a mixture solution, phase separation was induced by light irradiation under the condition of
constant temperature (Figure 20.6). Since the physicochemical stability of such a solution is quite
sensitive to the balance between the hydrophilicities of the two polymer components, drastic phase
separation was triggered by light even though the functionalization ratio was only 0.3 mol% of the
total number of hydroxyl groups in dextran. Phase separation of aqueous mixture solutions, which
had been examined for the application to the less-invasive separation of proteins and living cells
(Albertsson 1958; Guan et al. 1996; Edahiro et al. 2005a), was expected to gain wider application
due to the property controllable by light as well as temperature change.
20.4 DEVELOPMENT OF PHOTORESPONSIVE HYDROGELS

Many researchers have studied so far not only for aqueous polymer solutions, but also for photores-
ponsive hydrogels. In the pioneering research on photoresponsive hydrogels, Irie et al. reported that the
volume of the hydrogel functionalized with leuco chromophore increased largely by UV irradiation
(Irie and Kunwatchakun 1986; Mamada et al. 1990). Although the photoresponsive swelling was so
slow as to take more than an hour, it was shown experimentally for the first time that the signal of light
irradiation could be transduced to the change in the volume of material. Later, several research groups
reported independently the photoresponsive ion permeability of leuco-functionalized hydrogel mem-
branes (Kimura et al. 1995; Kono et al. 1995; Kodzwa et al. 1999). The leuco chromophore was advanta-
geous in modifying the hydration of hydorgel since it is ionized in response to light irradiation. However,
its photoreaction requires high-energy UV light of the wavelength less than 300 nm, and therefore its
degradation was critical due to the side reaction.
Without using photochromic materials, Suzuki and Tanaka demonstrated that the pNIPAAm
hydrogel colored with chlorophyll shrank in response to the intensive irradiation with visible light
(Suzuki and Tanaka 1990). This photoresponsive shrinking was due to the temperature rise induced
through the conversion of light absorbed by chlorophyll to heat (“heat-mode”). Because of inevitable
influence of the thermal diffusion, however, such a thermal-mode scheme is not valid in application
to the manipulation in micrometer scale, where the use of light seemed mostly suitable. Although
Garcia et al. reported later that the hydrodynamic radius of the polyarylamine microgel containing
spiropyran in water decreased by light irradiation, its variation was at most 20% and the tempera-
ture range to obtain the photoresponse was limited (Garcia et al. 2007).
In these circumstances, we prepared the hydrogel composed of the pSpNIPAAm described ear-
lier, and observed that the hydrogel, which had been swollen in the acidic aqueous system, shrank
by 70% in quick response to blue light irradiation, and returned gradually to the former swollen
state after stopping the irradiation (Figure 20.7) (Sumaru et al. 2006; Szilagyi et al. 2007). This
drastic and quick photoresponsive shrinking was observed in the wide temperature range between
20°C and 30°C, and was repeatable for many times.
In order to examine the applicability of the pSpNIPAAm hydrogel to mass transfer control, we
functionalized a porous membrane with the hydrogel, and evaluated the influence of light irradia-
tion on its permeability for acidic aqueous solution (Sumaru et al. 2006). As a result, the permeabil-
ity of the membrane was doubled by light at 30°C, maintained the increased value under irradiation,
and returned gradually to the former low value over a few hours. On the other hand, we prepared
a rod composed of the photoresponsive hydrogel and observed that the rod in an aqueous solution,
when irradiated with blue light from one side, bent toward the light source.
O NH O NH
O O
x y z
HN O
OH
Light
+N Isomerization O
N + H+
+ Light
+ Shrinking
Swelling
+
FIGURE 20.7 Photo-induced shrinking of pSpNIPAAm hydrogel.

20.5 MICROMANIPULATION OF PHOTORESPONSIVE HYDROGELS

As described at the beginning, light is an advantageous means of signaling due to its local and
instantaneous applicability to small object. In order to demonstrate this feature, we composed a
microfluidic system having photoresponsive microvalves using the pSPNIPAAm hydrogel (Sugiura
et al. 2007). Three photoresponsive microvalves were fabricated by incorporating pieces of the
hydrogels at prescribed positions in microfluidic channel with 500 μm width and 100 μm depth,
through in situ photo-polymerization. In order to visualize the fluid flow through the microvalves,
dilute acidic aqueous solution of indigocalmin was introduced into a microchannel with constant
applied pressure. Before irradiation, all the hydrogels were in swollen state, stopping the fluid flow
in these microvalves stably. In response to the local irradiation of blue light with 436 nm wavelength
at 20 mW/cm2 intensity, a hydrogel in the right-side microvalve shrank to allow the fluid flow toward
downstream. The openings of the center and the left-side microvalves were also achieved indepen-
dently by the following local irradiation of blue light in series (Figure 20.8). Further, simultaneous
opening of multiple microvalves by temperature rise was demonstrated utilizing the temperature-
dependence of pSpNIPAAm hydrogel.
In clear contrast to the methods based on the conversion of light to heat (Sershen et al. 2005;
Chen et al. 2008), the photoresponsive valve openings working in pure “photon mode” would be
insusceptible to thermal interference between adjacent microvalves even in such a case that many
of the highly integrated microvalves were manipulated at the same time. And unlike the methods
based on the thermal melting (Moriguchi et al. 2002; Hattori et al. 2004; Park et al. 2007; Hua
et al. 2008) and wettability change (Caprioli et al. 2007) of materials caused by light irradiation,
fluid flow could be controlled reversibly.
By using a computer-controlled micropattern irradiation system described previously, we can
control the photoresponsive hydrogel with resolutions down to a micrometer scale. So we tried
on-demand control of the microrelief profile on the surface of photoresponsive hydrogel sheet by
modulating its local thickness by micropatterned light irradiation (Szilagyi et al. 2007). First, we
prepared a pSpNIPAAm hydrogel sheet of initial thickness of ∼250 μm on an acrylated glass surface
so that the hydrogel sheet was attached covalently to the surface of the flat glass plate. In order to
characterize the photoresponse of the hydrogel sheet prior to the experiment of microrelief forma-
tion, we measured the change in its thickness induced by blue light irradiation. As a result, the
thickness in the irradiated area decreased by half shortly after irradiation for a few seconds, and
the shrunk state was retained for about 10 min. Then the thickness returned gradually to the former
swollen state over 2.5 h (Figure 20.9).
Then, we applied the micropatterned irradiation onto the pSpNIPAAm hydrogel sheet by using
a computer-controlled micropattern irradiation system. As a result, a very sharp microrelief was
etched on the surface of hydrogel sheet right after micropatterned irradiation for a second. The
resultant fine microrelief was retained stably after stopping irradiation, indicating determinately
that the shrinking of the hydrogel was not due to the thermally induced process through light-heat
conversion (“heat mode” process) but due to pure “photon mode” process through the photoisomer-
ization of chromophores. The microrelief was erased by leaving the sheet in the dark for a while,
and new microrelief was formed by other micropatterned irradiation. Further, as a demonstration of
3-dimensional control of the microrelief profile, a microrelief with four-step height was formed by
the stepwise micropatterned irradiation (Figure 20.10).
In order to apply such a controllability of surface profile to a new microfluidic system with high
flexibility of design, we developed a universal microfluidic system composed of the photoresponsive
hydrogel in which we could instantly form a microchannel along arbitrary path just by micropat-
terned light irradiation (Sugiura et al. 2009). The working principle is shown in Figure 20.11. The
system was constructed by mounting another glass plate with multiple inlet and outlet ports, on the
photoresponsive hydrogel sheet attached to a glass plate. As a result of light irradiation along an
pSpNIPAAm hydrogels
1 mm
Irradiated area
FIGURE 20.8 Independent control of multiple photoresponsive microvalves by means of local light
irradatiation.
300
250
200
Thickness (µm) 150
100
50
0
0 20 40 60 80 100 120 140 160
Time (min)
FIGURE 20.9 Change in the thickness of a pSpNIPAAm hydrogel sheet after blue light irradiation.
arbitrary path from one of the inlet ports to one of the outlet ports, the hydrogel in the irradiated area
shrank, and a microchannel was formed to lead a fluid supplied from the inlet port to the outlet port.
Figure 20.12 shows the experimental result of the practical demonstration using the constructed
universal microfluidic system. After micropatterned irradiation, a microchannel was formed in the
hydrogel sheet along the irradiated pattern, and the fluorescent latex suspension supplied from an
inlet port started to flow through the newly formed microchannels within a few minutes. A straight
microchannel, a bent microchannel, a serpentine microchannel, and branched microchannels were
repeatedly formed by micropatterned light irradiation. Note that it took several minutes to 10 min
to fill up the long microchannels. In this scheme of microfluidic control based on the conversion of
light to the volume change of the photoresponsive hydrogel, microchannels with an arbitrary width
and depth along arbitrary path could be formed at an arbitrary timing. This result demonstrated that
the universal microfluidic system based on this scheme would provide microfluidics with a powerful
method of mass transfer control.
Further, combining the universal microfluidic system described earlier and conventional fixed
microchannel, we attempted the construction of new integrated photoresponsive microvalve array
(Sugiura et al. 2009). The system was constructed by stacking a polydimethylsiloxane microchan-
nel chip and a glass plate with mechanically fabricated through-holes, on a photoresponsive hydro-
gel sheet attached to a glass plate (Figure 20.13). As a result of micropatterned light irradiation,
the hydrogel in the irradiated area adjacent to a pair of through-holes shrank, inducing the opening
of the microvalve. Figure 20.14 shows the construction of microvalve array and the experimen-
tal result of the practical demonstration. In the microvalve array, 10 photoresponsive microvalves
were integrated within 1 cm2. Each microvalve connected a 1.2 mm-wide inlet microchannel to
400 μm-wide outlet microchannel via two 400 μm through holes and the photoresponsive hydrogel
sheet. The individual microvalves were successfully controlled through the opening of a single
microvalve by local light irradiation while they had stopped stably the flow of fluid applied at the
pressure of 5 kPa before the irradiation. Parallel control of multiple microvalves was also suc-
cessfully demonstrated as two microvalves were simultaneously opened within several minutes by
micropatterned irradiation.
In the photoresponsive microvalves fabricated by incorporating pieces of the hydrogels at pre-
scribed positions in microfluidic channel as shown in Figure 20.8, reproducible polymerization with
high precision was required to achieve both the stable stopping under unirradiated condition and the
prompt opening upon irradiation (Sugiura et al. 2007). On the other hand, such a requirement was
easily cleared in the construction stacking a flat glass plate on a photoreponsive hydrogel sheet with
uniform thickness attached to another flat glass plate.
200 μm
FIGURE 20.10 The process of a microrelief with 4-step light by stepwise micropatterned irradiation on
pSpNIPAAm hydrogel sheet.
Fluid Outlet ports

Inlet port
Photoresponsive
hydrogel sheet
Glass substrate
Fluid
Light irradiation
Fluid
FIGURE 20.11 Schematic illustration showing a working principle of a universal microfluidic system com-
posed of photoresponsive hydrogel sheet.
20.6 PHOTO-CONTROL OF CELL ADHESION

To implement the integrated cell bioassay system, which was mentioned at the beginning, a tech-
nology to control the cell culture system precisely is required in addition to the photo-controlled
manipulation of microfluidics. Considering the advantage of light control as mentioned earlier, it is
important to develop the cell culture substrate that transduces light irradiation to the change in its
cell adhesion property. As attempts to control cell adhesion property of culture substrates by light
irradiation, patterned culture substrates were developed by applying conventional photo-resist pro-
cess from 1980s to 1990s (Pierschbacher and Ruoslahti 1984). Later, Nakayama et al. developed a
culture surface whose cell adhesion property was increased by UV irradiation with the wavelength
of 290–400 nm (Nakayama et al. 2003). Since these methods require photo-resist process or the
irradiation of high-energy UV, light irradiation must be done in prior to cell seeding on the substrate.
696
Patterned irradiation <Dark field image>
Irradiated area Latex flow
Inlet Outlets Inlet Fluorescent

latex
dispersion
2 mm
Patterned irradiation <Dark field image> Patterned irradiation

Irradiated area
Latex flow
Irradiated area
<Dark field image> Patterned irradiation <Dark field image>
FIGURE 20.12 On-demand formation of microchannels with arbitrary pathways in the universal microfluidic system by micropatterned light irradiation. (From Ref. [77].
With permission.)
PDMS
Glass
Shrinking
Glass Photo-
Light responsive
hydrogel
FIGURE 20.13 Schematic illustration showing the structure and working principle of microvalve array
composed of photoresponsive hydrogel sheet.
In such situations, we proposed a new scheme of cell manipulation through in situ control of cell
adhesion property of the culture substrate by light irradiation after cell seeding (Figure 20.15). First,
living cells are seeded and cultured on a culture substrate whose surface property can be changed by
mild light irradiation. These cells are observed and classified, for example, by the shape and/or fluo-
rescence markers, then the light is irradiated in the area where the classified cells are. As a result,
cell adhesion property of the culture substrate changes (increases in the case shown in Figure 20.15)
only in the irradiated area, and subsequent washing of the substrate surface selectively removes the
cells whose adhesion to the substrate surface is relatively weak. Implementation of such a technol-
ogy will enable us to separate or purify the cultured cells having a certain specific property in the
adherent state without exfoliating from the substrate.
We synthesized a number of photoresponsive polymers functionalized with spiropyrans and
azobenzenes, prepared cell culture substrates, and examined the influence of light irradiation on
their cell adhesion property. After a number of experiments which turned negatively, we observed
that the cell adhesion on the surface of nitrospiropyran-functionalized pNIPAAm (pNSpNIPAAm)
increased significantly in response to the light irradiation in the presence of living cells under a
specific condition (Edahiro et al. 2005b, 2008). CHO-K1 cells were seeded and cultured for 24 h
on a photoresponsive culture substrate functionalized with pNSpNIPAAm, and confluent cells were
irradiated with the patterned UV light of 365 nm wavelength at 30 mW/cm2 for 5 min. After subse-
quent cooling at 10°C for 20 min, the surface was washed uniformly with a phosphate buffer solu-
tion cooled at 10°C. As a result, it was observed that the cells were removed from the unirradiated
area effectively, while most cells remained in the irradiated area. After cell pattern was successfully
formed, cells were stained with CMTPX, a fluorescence dye staining only living cells. Fluorescence
was observed from most of the cells, indicating that a series of the process including light irradiation
and cooling did not cause critical damages on the cell viability (Figure 20.16).
698
Patterned irradiation <Dark field image> Patterned irradiation
Fluorescent
latex
dispersion
Inlet
Latex flow

2 mm
Irradiation area
<Dark field image> Patterned irradiation <Dark field image>
FIGURE 20.14 Independent and parallel flow control in photoresponsive microvalve array composed of pSpNIPAAm hydrogel sheet. (From Ref. [77]. With permission.)
1
Photoresponsive
substrate Cultured cells
Local light
irradiation
Light Light
Uniform
flushing
FIGURE 20.15 Scheme of cell manipulation through in situ control of cell adhesion property of the culture
substrate by light irradiation after cell seeding.
500 µm
FIGURE 20.16 Fluorescence microscopic image of CHO-K1 cells patterned by in situ photo-control of cell
adhesion and stained with CMTPX.
This photoinduced increase of cell adhesion was applicable only to the cells in a certain adherent
condition, and this unique anchoring effect could not be produced with suspended cells and firmly
adhering cells cultured for a long time. In spite of this practical problem, the result was important as
the first report on in situ control of cell adhesion by light irradiation. Further, we also demonstrated
that the different kinds of cells were immobilized in certain areas in a closed microchannel by using
this method (Tada et al. 2006b).
20.7 ELABORATE CELL PATTERNING ON PHOTORESPONSIVE

CULTURE SUBSTRATE
As seen in Figure 20.16, cell patterning could be easily achieved even in the culture environment
by using this photoinduced increase of cell adhesion mentioned earlier. However, the photoin-
duced anchoring was effective only in temporary cell adhesion increase, and this method did not
restrict the growth or movement of the cells. So, the patterned cells continued to grow and spread
beyond the irradiated area in the further incubation. To keep cells within fine patterns, making
confined cell adhesive areas in the nonadhering surface by light irradiation seemed reasonable
to explore.
Nakanishi et al. developed a new class of photoresponsive culture substrate based on “caged”
(protected by a photocleavable protecting group) technology (Nakanishi et al. 2004, 2007, 2008).
They prepared a substrate by coating adhesion inhibitors such as bovine serum albumin (BSA) or
block copolymer of ethylene glycol and propylene glycol (Pluronic) on a caged glass surface to pro-
vide the property of cell adhesion inhibition. By irradiation with UV light of 365 nm wavelength,
which is compatible with the optical systems of widely used biological microscopes, the adhe-
sion inhibitors were released via the “uncaging” mechanism, allowing cell adhesive moieties such
as fibronectin to be adsorbed on. In clear contrast to the conventional photo-patterned substrates,
which require the photo-resist process or the irradiation of high-energy UV as mentioned earlier,
the cell adhesion property of the substrate can be changed by light irradiation, even being in cell
culture environment. By using the substrate, they prepared various cellular patterns at single-cell
resolution. Further, they demonstrated that the single cells, which had localized within independent
confined areas respectively, were able to migrate along the paths appended afterward by the subse-
quent irradiation.
On the other hand, we developed a substrate, whose surface attribute could be switched from
being cell-inhibitory to being cell-adhesive just by light irradiation without any treatment before cell
introduction (Tada et al. 2006a; Kikuchi et al. 2009). Figure 20.17 shows the mechanism of the pho-
toresponsive change in cell adhesion property and a scheme to construct an elaborately patterned
co-culture system using the substrate. We prepared the substrate by immobilizing PEG on the sur-
face as the cell adhesion inhibitor via poly(methyl methacrylate) functionalized with nitrospiro-
pyran (pNSpMMA). In response to irradiation with UV light of 365 nm wavelength, pNSpMMA
became hydrophilic through photoisomerization of nitrospiropyran and released the PEG from the
surface, and then cell adhesive area appeared instantly. When the suspension of adherent cells
was introduced onto the surface, the cells adhered only to the previously irradiated area and grew
within the area in the subsequent incubation. After removing the floating cells above unirradiated
area, highly contrasted cellular pattern appeared, and the constructed cell pattern was maintained
stably. The cell adhesive area was surrounded by the barrier of PEG, which prevented the adhering
cells effectively from further spreading. Since the cell adhesive area could be appended just by light
irradiation even in the culture environment, we could construct the elaborately co-culture system,
which was composed of multiple kinds of living cells arranged in arbitrary patterns by repeating
patterned irradiation, introducing and removing cells on the photoresponsive culture substrate in a
stepwise manner.
In the earlier-mentioned method based on caged technology, application of cell adhesive moi-
eties such as fibronectin was necessary after light irradiation to obtain sufficient cell adhesion
Light
irradiation
FIGURE 20.17 Mechanism of the photoresponsive change in cell adhesion property of photoresponsive
culture substrate and a scheme to construct an elaborately patterned co-culture system via stepwise cell pat-
terning on the substrate.
in the irradiated area (Nakanishi et al. 2004, 2007). In contrast, the bare surface of pNSpMMA
which appears after photoinduced removal of PEG from our photoresponsive substrate was suf-
ficiently cell adhesive, and most anchorage-dependent cells could adhere. As a result, highly
contrasted cellular pattern was available just by light irradiation without further treatment. This
feature is advantageous especially in stepwise fabrication of elaborate cellular pattern. PEG
used here as a cell adhesion inhibitor is known to be basically harmless and is used in a large
amount in cell fusion (Wojcieszyn et al. 1983; Khetani and Bhatia 2008). Additionally, the
amount of PEG immobilized on the pNSpMMA layer was quite small, and the PEG released into
medium by light irradiation was removed from the culture environment by subsequent medium
replacement.
Here we show several examples of practical cell patterning implemented by the photore-
sponsive culture substrates and a computer-controlled micropatterned irradiation system.
Figure 20.18 shows the appearance of a simple cellular pattern composed of MDCK cells. The
MDCK cells were seeded on a surface of a photoresponsive culture substrate which had been
irradiated with a stripe pattern of 80 μm line width. After incubation for 3 h, the cells floating
FIGURE 20.18 The microscopic image of MDCK cells patterned on a photoresponsive culture substrate.
above unirradiated area were removed subsequently. Figure 20.18 shows the appearance of the
cells after additional incubation for half a day, and all the irradiated area was filled with the
grown MDCK cells. The constructed cellular pattern was maintained stably even after 3 days.
As a result, it was demonstrated that sufficient cell adhesion was obtained on the substrate
surface just by light irradiation in the culture environment while potent inhibition was stably
maintained in unirradiated area.
Meanwhile, the regulation of the culture environment by this cell-patterning method has much
influence on the bioactivity of cultured cells. The HepG2 cells were seeded on a surface of a pho-
toresponsive culture substrate which had been irradiated with a polka-dot pattern. HepG2 is a cell
line derived from human hepatoma which is widely used in the research concerning the function
of human liver. After incubation for 9 h, the cells floating above unirradiated area were removed.
The patterned cells continued to grow and propagated within the irradiated area confined with a
wall of PEG, forming evenly spaced uniform organoids in 5 days (Figure 20.19). Then balb/3T3
fibroblasts were introduced into the space among HepG2 organoids to form patterned co-culture
system. Analysis on liver-specific gene expressions proved that the hepatic function of HepG2 such
as cytochrome P450 activities and albumin production improved remarkably when cultured in such
a patterned co-culture system compared with the conventional monoculture systems (Kikuchi et al.
2009). This experimental result suggested strongly that the patterned co-culture would increase the
reliability of bioassay using liver-derived cells culture in vitro, which had been used as a means of
high-throughput and low-cost drug screening (Norde and Gage 2004; Otsuka et al. 2004; Fukuda
et al. 2006; O’Brien et al. 2006).
500 µm
FIGURE 20.19 Microscopic image of HepG2 organoid array fabricated by using photoresponsive culture
substrate.
500 µm
FIGURE 20.20 Microscopic image of balb/3T3 fibroblasts oriented by using photoresponsive culture
substrate.
Figure 20.20 shows the appearance of balb/3T3 fibroblast cultured on a photoresponsive culture
substrate which had been irradiated with a fine stripe pattern of 4 μm-wide lines and 12 μm-wide
spaces. Adhering cells extended along the direction of stripe pattern, showing that the photorespon-
sive culture substrate enables to control through patterned light irradiation not only the distribution
of adhering cells, but also the cell orientation.
Although the cell adhesion ability varies widely with the type of cells, the cell adhesion property
of the photoresponsive culture substrate is easily tunable by varying the amount and molecular
weight of PEG immobilized on the pNSpMMA layer (Tada et al. 2006a). By using properly tuned
substrate, most of anchorage-dependent cells can be patterned in this way. We have confirmed that
this cell-patterning method is applicable to all the cell types we examined such as CHO-K1, HeLa,
MEF, Hek293, HOS, HUVEC, and NIH/3T3, as well as MDCK, HepG2, and balb/3T3 mentioned
earlier. In addition, we can irradiate light in arbitrary patterns according to monitored microscope
field of view with very high reproducibility by using a computer-controlled micropatterned irra-
diation system we developed. Taking these advantages, we demonstrated to fabricate more elabo-
rate culture system than the previously mentioned system composed of HepG2 and balb/3T3, by
repeating cell patterning process in a stepwise manner as shown schematically in Figure 20.17.
Figure 20.21 shows the result of stepwise fabrication of an elaborate cellular pattern composed of
MDCK cells. As patterned irradiation and cell seeding were repeated, cellular patterns were added
on the substrate sequentially. The cell patterning process was repeated three times taking a day in
total. The cellular pattern fabricated in former patterning process was maintained stably during the
latter process including the removal of floating cells. The MDCK cells introduced in each step were
stained differently in advance, and we confirmed that the cells in the resultant pattern are intro-
duced successfully into the prescribed areas as we intended.
20.8 SUMMARY AND FUTURE OUTLOOK

In this chapter, we briefly reviewed the researches on photoresponsive biomaterials as technology
components to construct and control integrated cell bioassay system by means of light irradiation.
In particular, we focused on two technology components: the method to fabricate elaborated
co-culture systems by light and photo-controlled manipulation of microfluidics. Combination of
these technologies is expected to realize an integrated cell bioassay system with high reliability, in
which all the process including cell culture and cell bioassay is thoroughly automated via computer-
controlled micropatterned light irradiation. The schematic illustration of such system is shown in
Figure 20.22. The use of light is advantageous to control high-throughput cell bioassay since non-
contact regulation of closed and sterile systems and parallel control of massive objects are feasible.
Since the photoresponsive hydrogels we have developed so far can be driven only under acidic
500 µm
FIGURE 20.21 Stepwise fabrication of an elaborate cellular pattern composed of MDCK cells on photore-
sponsive culture substrate.
Drug A Drug B Drug C
Cell culture chamber
Photo-
responsive
Light hydrogel
Patterned culture system Drug supply at
fabricated on photoresponsive substrate arbitrary amount and timing
FIGURE 20.22 Construction of integrated cell bioassay system fully controlled by light irradiation.
conditions, we cannot apply these hydrogels to the components of cell bioassay system as they are
now. To solve this problem, we are now studying the properties of various copolymers function-
alized with structurally modified photochromic materials to achieve precise photo-control in the
culture environment.
In recent years, there is a tendency to create new technologies by gathering and combining
technology components developed in various different research fields. Also the photo-manipulation
methods for integrated cell bioassay system introduced in this chapter have been accomplished
only by gathering and combining the various technology components such as synthesis and char-
acterization of photoresponsive polymer materials, culture and analysis of cells, and construction
and control of optoelectronic systems. The cell bioassay, which is biologically accurate and fully
controllable by computer, will provide the drug screening, which has already been highly auto-
mated, with higher reliability and throughput. In these days, the rapid progress in optoelectronics is
launching various new devices, and we expect that the photo-control technology for cell culture and
bioassay will contribute to the progress in the research field of biology.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Dr. Jun-ichi Edahiro, Dr. Andras Szilagyi, Dr. Koji Hattori,
Ms. Kyoko Kikuchi, Mr. Yuichi Tada, and Ms. Yuki Ooshima for their cooperation in our research on
the development and the application of photoresponsive polymer materials. The authors also acknowl-
edge financial supports by Industrial Technology Research Grant Program in 2002 and 2005 from the
New Energy Development Organization (NEDO), the Creation and Support Program for Start-ups from
Universities in 2005 from Japan Science and Technology Agency (JST), and KAKENHI (20350110).
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21 Lignin in Biological Systems
Valentin I. Popa
CONTENTS
21.1 Introduction........................................................................................................................... 709
21.2 Lignins as Antibacterials....................................................................................................... 711
21.2.1 In Vitro Studies.......................................................................................................... 713
21.2.2 In Vivo Studies.......................................................................................................... 714
21.2.3 Prebiotic Effects of Purified Lignin.......................................................................... 715
21.3 Lignins as Antioxidants and Photoprotectors........................................................................ 715
21.4 Lignins in Reduction of Carcinogenesis................................................................................ 723
21.5 Anti-HIV Properties of Lignins............................................................................................ 728
21.6 Lignin as Spermicide............................................................................................................. 730
21.7 Mechanism............................................................................................................................. 730
21.8 Conclusions............................................................................................................................ 735
References....................................................................................................................................... 735
21.1 INTRODUCTION
In the polymer industry, utilization of plant and animal compounds is the key to sustainable
development. Carbohydrates have already been used significantly in the food, medical, and cosmetic
industries. Plant materials containing cellulose, hemicelluloses, and lignin are the largest organic
resources but with the exception of cellulose, they are not very well utilized. Hemicelluloses are
significantly underutilized. Lignin, production of which is over 20 million tons per year worldwide,
is mostly burnt as fuel, although it is one of the most useful natural resources (Ranby 2001).
Lignins are three-dimensional polymers consisting of phenylpropane or C9 units. There are three
types of structural units that are available in a wide range of proportions in the woody tissue of dif-
ferent plant species: p-hydroxyphenylpropane, guiacyclopropane, and syringylpropane units. The
proportion of these three C9 units in lignin available in the woody tissues of plants depends on the
nature of plant species. In general, gymnosperm (softwood) lignins consist mostly of guiacyclo-
propane units and angiosperm (hardwood) lignins are mainly composed of guiacyl- and syringyl-
propane units with varying ratios depending on the wood species. By contrast, grass (Gramineae)
lignins are composed of p-hydroxyphenyl-, guiacyl-, and syringylpropane units and consist of lignin
cores and peripheral units (Le Digabel and Averous 2006). The lignin cores are essentially lignins
of the guiacyl-syringyl type, while the peripheral units consist of p-hydroxycinnamic acid and feru-
lic acid groups that are bonded by ester linkages mostly to hydroxyl groups at C-γ in the C9 units of
lignin cores (Figure 21.1).
It has been shown that lignin is a versatile molecule that possesses multiple properties such as
antioxidant (i.e., radical scavenger), UV-absorption antifungal, and antibiotic activity. It has been
suggested that lignin can be applied for stabilization of food and feed, due to their antioxidant,
antifungal, and antiparasitic properties. Also, anticarcinogenic, apoptosis-inducing antibiotic, and
anti-HIV activities of lignin have been reported.
However, very little attention has been paid to the biological activities and mechanism of action
of lignins or lignin-containing/derived substances. To enhance the industrial use of lignins, there is
709
H2C OH
HC O
HC OH
H2C OH
H2C OH
HC O
H3C O HC OH
HC OH CH3
O CH O
H2C OH O
CH
CH
CH HC OH
H3C O H 3C O
H3C O OH O
H2C OH O
H2C HC HC O H 2C CH
O H2C OH
O CH H3C O HC CH
CH3 O CH H2C OH
HC OH HC CH2
HC OH HO CH O
OH
H3C O HC OH
CH2
O CH3
O CH H3C O O CH3
O O
HC O HO CH2 H3C
HO CH2 HC O
HC CH2
O CH3
HC O
H3C O
H2C OH H2C OH
H3C O
O CH HC O
HC OH C O
H3C O
OH OH
FIGURE 21.1 Schematic representation of lignin molecules from wheat straw.
a need for a continuous supply of lignin products with constant quality as related to purity, chemical
composition, functional properties, and their reproducibility (Popa 2007).
Sometimes, the use of lignin has not yet been successfully achieved, because lignin molecules
lack stereoregularity and also the repeating units in the molecule are heterogeneous and complex.
In addition, lignocellulose components cannot be separated by simple extraction processes such as
the pulping process, which make lignin molecules even more heterogeneous. That is why, lignophe-
nols, as lignin derivatives are characterized by high content of phenolic function, high stability, and
less heterogeneity compared with conventional lignin preparation. It was assumed that lignophenols
derived from native lignin have different phenolic functionalities to exhibit various biological activ-
ities (Akao et al. 2004). Moreover, the functionality of lignophenols is able to be easily modulated
by various modifications (Figure 21.2).
Lignophenols, such as lignocatechol, lignoresorcinol, and lignopyrogallol, which have many
phenolic hydroxyl groups, are highly hydrophilic. On the other hand, lignophenols combined with
monohydric phenols such as cresol are water insoluble. When water-insoluble lignophenols were
carboxymethylated (CM) and/or alkaline treated (AT), they became highly hydrophilic and water
soluble. Especially, CM-lignophenols showed a high binding affinity for proteins because of the
carboxyl groups that were introduced.
Lignin in Biological Systems 711
H3C
CH3
HO O OH
OH
OH OH
HO O
Phase-separation system
HO direct conversion of native H3C O HO
O
lignin to lignophenol
O O
CH3 H3C O
H3C
O CH3 Selective phenolation at C
O CH3
OH α-position
OH
Partial structure of native lignin Molecular-weight control
by the cleavage of benzyl
aryl ethers
Partial structure of ligno-p-cresol
O H 3C
OH
H3C OH
+ OH
Alkaline treatment
O CH3
HO HO
OH OO
H3C
Modifications of lignophenol
CH3
Additionally functionality
Fragmentation to lower molecular weights of control of lignophenol
ligniphenol by the cleavage of original aryl-O-Cβ
linkages, resulting in
O
Lignophenol –OH Lignophenol – O – CH2 – C – OH
Substitution of hydroxyl groups in lignophenol by

carboxymethyl ether groups, resulting in greater Carboxymethylation
hydrophilicity
FIGURE 21.2 Conversion of native lignin into lignophenol derivatives and control of their functionality.
Today, lignin or lignin-derived compounds are not used in the neutraceutical/para-pharmaceuti-

cal and cosmetic industries. However, it can be imagined that lignin could represent an alternative/
substitute for a number of plant polyphenol-containing extracts currently used in such industries,
suggesting that lignin could have a more direct protective effect on health, thereby opening up pos-
sibilities of using this molecule as a source of bioactive molecules for use in medicine. That is why in
this review some examples of the possibilities to use lignins in the biological systems are discussed.
However, important biochemical and epidemiological research efforts are necessary before lignin
can be used in a medical (as opposed to para-pharmaceutical) context. Additionally, it would also
be necessary to demonstrate the advantages of using such medicaments/food additives as compared
to simply changing to a more fiber-rich diet, containing lignin-rich fiber. Nevertheless, a number of
potentially important niches exist for the use of lignin-derived products in the healthcare sector and
these aspects will be discussed in the following sections.
21.2 LIGNINS AS ANTIBACTERIALS

The role of lignin in the human and animal metabolism has been mentioned many years ago (Csonka
et al. 1929). A loss of lignin in human beings as well as in dogs has been reported. The experiments
carried out with dogs and cows point to degradation of lignin. The conclusions are based on the fact
that an increase in the hippuric acid eliminated was evidenced after lignin was investigated, as well
as a decrease of the methoxyl group content was observed. Thus, experiments performed with a dog
showed a loss of 15% of the methoxyl groups initially present in lignin, and in an experiment with a
cow, a 37% loss of the methoxyl groups was observed. It was supposed that in these experiments there
was direct evidence that lignin is metabolized in the animal body. The results on the benzoic acid
eliminated in the urine when lignin was fed support this claim. Experiments were carried out in vitro
in which known quantities of lignin mixed with fresh material taken from the four compartments of
a cow stomach were incubated. A loss of methoxyl groups was observed, indicating that their partial
disappearance of this takes place in vitro in the stomach of the animal and it is not brought about by
bacteria but rather by some other agents, possibly an enzyme in the gastric juice of the animal.
As is known, the lignin is taken up as a “dietary fiber” when vegetables and fruits are consumed.
It is thought that dietary fiber is useful for the prevention of some diseases (such as cancer), although
most is excreted without being digested. To clarify the influence of dietary fiber on an organism, it is
important to examine the relationship between the dietary fiber and enterobacteria. In respect to the
influence of lignin on enterobacteria, it was reported that woody lignin shows antibacterial activity
against Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus
aureus (Nelson et al. 1994).
The influence of lignin extracted from rice bran on the growth of enterobacteria and their effect
on lignin was also studied. E. coli, Enterococcus faecium, Lactobacillus minutus, Staphylococcus
epidermis, Bacteroides vulgatus, and Streptococcus intermedius isolated from human feces were
identified and used along with Bifidobacterium bifidum (NBRC 13952), Bifidobacterium adoles-
centis (JCM 7044), and Bifidobacterium longum (JCM1217). The turbidities (OD600) in the medium
including enterobacteria with or without rice bran lignin were determined (Toh et al. 2007). Thus,
the OD600 values of medium cultivated with L. acidophilus and S. intermedius in the presence of
lignin were significantly greater than those without lignin. The OD600 values of E. coli, S. epidermis,
L. minutus, and B. bifidum in the presence of lignin were significantly lower than those without
lignin. Small values of OD600 were observed when inhibition of growth of bacteria or bacteriolysis
occurred. In the gel permeation chromatography (GPC) of the rice bran lignin in the case of E. coli,
it was found that molecular mass was reduced from about 5,000 to 10,000 in 24 h.
The change in molecular mass of lignin that was observed in this study means that E. coli metab-
olized lignin and the number of microorganisms was reduced by lignin. Whether the products of the
metabolized lignin contributed to the growth of the bacterium is unclear. However, the degradation
of rice bran lignin occurred within 24 h after the incubation of E. coli, and lignin taken as a meal
may be degraded in the intestine by this bacteria. Both herb and wood lignins showed antimicrobial
activity for E. coli, in spite of the differences in structures and molecular mass between them. As
concerning the mechanisms in the case of E. coli it is thought that metabolism of lignin by bacteria
contributes to its growth. In other cases, there is some possibility that lignin physically functions
as the structure of medium, which might contribute to the growth of enterobacteria because lignin
is a biopolymer.
The link between uses of growth-promoting antibiotics and antibiotic resistance of pathogenic
bacteria in humans has led member countries of the European Union to ban use of these antibiotics
in animal agriculture. At the same time, recent findings demonstrate that purified lignin encom-
passes production and health benefits in animals in the absence of antibiotics. For this reason,
purified lignin is one of several natural compounds that have received new interest and scientific
consideration as a natural feed additive. Purified lignin may potentially mitigate production and
economic losses after the use of antibiotics in livestock production is discontinued. In its purified
form, lignin contains several low-molecular mass phenolic monomers that possess biological effects
not characteristic of native lignin. The possibility exists that monophenolic fragments of purified
lignin may have beneficial effects on productivity of farm animals, safety of animal products, and
the environment. Lignin is not digested by monogastric animals, but the rumen bacteria flora facili-
tates degradation of benzyl-ether linkages of lignin polymers.
On the other hand, the sorption capacity of lignin has been the base in the development of drugs
for the therapy of gastroduodenal disorders. Those functional groups allow lignin to adsorb and
retain various types of microorganisms and cholesterol, bilirubin, lipid bile acids, and other carci-
nogenic substances.
21.2.1 In Vitro Studies

The phenolic components of lignin have been reported to inhibit growth of some microorganisms.
The comparison of bioactivity toward E. coli of nonwood soda-lignin applied in soluble form has
revealed that the antimicrobial properties strongly depend on the origin of lignins and, consequently,
their structure. The side chain structure and nature of the functional groups of the phenolic com-
pounds are major determinants of the antimicrobial effects of lignin. In general, phenolic fragments
with functional groups containing oxygen (–OH,–CO, COOH) in the side chain are less inhibitory,
whereas the most inhibitory phenolic fragments are those containing a double bound in α, β posi-
tions of the side chain, and a methyl group in the γ position.
The clearest effects were observed for lignins obtained from sisal, abaca, and flax (Telysheva
et al. 2005). The same lignins are characterized with the high radical-scavenging activity in tests
with DPPH-free radical and superoxide anion radical. This could indicate some relationship between
these lignin properties (Figure 21.3).
The antimicrobial effect of lignin products under study was also detected against Bacillus subti-
lis and a range of phytopathogenic bacteria (Erwinia carotovora, Bacillus polymixa, Pseudomonas
syringae, X. vesicatoria) and fungi (Fusarium oxysporium, Cladosporium herbarum, Botrytis
cinerea, Rhizoctonia solani, Micriodochium nivale). The results obtained confirmed that the level
of microorganism growth inhibition depends on a lignin sample prehistory, its concentration in
cultivation medium, and type of microorganisms. Kraft lignin provided a bactericide effect toward
E. carotovora and X. vesicatoria at lower concentration under study (0.25% on nutrition medium).
However, in the case of P. syringe and B. polymyxa, the kraft lignin did not show a bactericide
effect even at the 2% concentration. Spruce hydrolysis lignin (HL) showed the bactericide effect
toward P. syringe and Xanthomonas vesicatoria in 2% concentration, so at lower concentration only
slight bacteriostatic activity was observed. The modification with quaternary ammonium compound
increases bactericidal properties of lignins; e.g., for HL a stable bactericide effect was achieved at
the both concentrations used. Fungal species revealed different resistance to lignin products, and in
total, the investigated products can be arranged according to their inhibitory effect in the following
range: kraft lignin < quaternized HL lignin < quaternized kraft lignin. The inactivation of pathogenic
4
3
1,000,000,000 CFU/mL
0
Control Flax Straw Hemp Sisal Jute Abaca Bagasse
FIGURE 21.3 Influence of different lignin samples on bacteria Escherichia coli development.
Sorbtion efficacy, %
100
80
60
40
20
0 E. coli
HL
Quatern.HL B. subtilis
Cu-HL
Curan
Cu-Curan
FIGURE 21.4 Influence of different lignin samples on pathogenic bacteria sorption. (Curan is commercial
kraft lignin produced by Borregaard Ltd, Norway.)
microorganisms activity may be explained also by their sorption properties onto lignin products:
nonmodified HL and kraft lignin are capable of adsorbing 45%–60% of the gram-negative and gram-
positive bacteria E. coli and B. subtilis, respectively, in 1 h (Figure 21.4).
The lignins modified with the quaternary ammonium compounds, well-known antimicrobial
agents, revealed the higher sorption capacity (70%–80% in 1 h) toward the same microorganisms.
In contrast to the nonmodified lignin samples, quaternized lignin limited the retained microorganisms’
vital activity. The Cu-containing modified lignins are characterized with the highest activity toward
both the bacteria and besides that totally suppress the vital activity of the bacteria adsorbed.
The polyphenolic compounds of lignin cause cell membrane damage and lysis of bacteria with
subsequent release of cell contents. Monophenolic compounds, such as carvacrol and cinnamal-
dehyde, also possess antibacterial properties. Carvacrol and thymol exhibit antimicrobial effects
determining bacterial cell membrane disintegration and releases of cell contents. In contrast, cinnam-
aldehyde penetrates the bacterial cell membrane to reduce intracellular pH and cause ATP depletion.
The antibacterial mechanistic actions seem to vary among phenolic compounds. To date there is no
report establishing the mode of action of a product consisting of multiple phenolic compounds.
Some lignin products owing to their microstructure and diversity of sorption centers can realize
a possibility of simultaneous sorption of organic pollutants and microorganisms and thus activate
the process of pollutant biodegradation on interfaces. This was confirmed by sorption experiments
carried out with 2,4-D and its bacteria-degrader Burkholderia cepacia, which was used along with
HL and lignin modified with Si-containing compounds.
21.2.2 In Vivo Studies

Indulin (40 and 100 g/kg of dry matter [DM]) reduced volatile fatty acids (VFA) concentrations in
the ceca and large intestine of chickens, suggesting that purified lignin altered the fermentation pat-
tern of the chicken intestinal tract by inhibiting growth of certain bacteria. In an attempt to provide
a better evaluation of the antibacterial effects of lignin, the broiler chickens were orally challenged
with pathogenic strains of E. coli and fed antibiotic-free diets or one containing Alcell lignin or
antibiotics. Alcell lignin reduced cecal concentrations of total E. coli after 3 and 9 days. E. coli inhi-
bition was more pronounced at higher concentrations (25 versus 12.5 g/kg of DM), suggesting that
the antibacterial effects of Alcell lignin mostly occur at higher doses. Further research d emonstrated
that dietary Alcell lignin reduced concentrations of E. coli in poultry litter when compared to
antibiotic-free diets or one containing antibiotics. Intestinal E. coli contaminate poultry carcasses
during slaughter, thereby representing an important cause of food-borne illnesses in humans. At the
same time, poultry litter may contribute to the spread of antibiotic-resistant genes into the food
chain. Moreover, litter E. coli is the major causative pathogen implicated in cellulitis, a major cause
of carcass condemnation at processing plants. These findings suggest that purified Alcell lignin
may represent a dietary strategy to reduce E. coli load in the chicken intestine and litter that could
offer an opportunity to improve the safety of poultry products and the control of cellulitis. Effects
of purified lignin on these variables remain to be studied.
In a mouse model, Alcell lignin did not alter aerobic bacterial growth in the cecum, but reduced
translocation of these bacteria in the lymph nodes and liver after burn injury. In mice, a pine wood
lignin was reported to possess antitumor and antibacterial properties against the pathogens E. coli
and Pseudomonas.
Ruminant responses to dietary lignin are variable. Alcell lignin had no effect on rumen fermen-
tation in sheep and did not alter fecal concentrations of anaerobic, aerobic, and coliform microor-
ganisms in calves. This could be the result of degradation of the phenolic compounds of lignin by
rumen microbes. Para-coumaric and ferulic acids are extensively converted into reduced phenolics
by ruminal microorganisms, as evidenced by their ruminal disappearances. The infusion of phenolic
compounds, such as quinic acid, phenolic benzoic, and phenyl acetic acids, into sheep rumen caused
large increments in urinary outputs of phenolic acids and phenols. These observations suggest that
phenolic monomers are extensively metabolized by the rumen microbial flora to more chemically
reduced forms, which are subsequently absorbed and metabolized. It seems, therefore, that the anti-
microbial action of lignin may be species dependent and occurs mostly in nonruminants.
21.2.3 Prebiotic Effects of Purified Lignin

Prebiotics are indigestible feed ingredients that selectively stimulate growth or metabolic activ-
ity of a limited number of intestinal microorganisms in birds and mammals. Lactobacilli and
Bifidobacteria are beneficial bacteria that limit intestinal colonization of pathogens by competing
for nutrients and binding sites and by secreting antibacterial substances. However, at higher level
(i.e., 25 g/kg of DM), lignin inhibited growth of these bacteria. Prebiotics may also play important
roles in improving the intestinal morphology of animal species. Longer villi are generally cor-
related with better intestinal health and improved efficiency of digestion and absorption. Goblet
cells are responsible for the production of mucins, which destroy intestinal pathogens. In broiler
chickens, Alcell lignin (12.5 g/kg of DM) increased villi height and goblet cell number. However,
there was no benefit at a higher lignin level (i.e., 25 g/kg of DM) suggesting that prebiotic effect of
Alcell lignin only occurs within a limited inclusion range (Baurhoo et al. 2008).
21.3 LIGNINS AS ANTIOXIDANTS AND PHOTOPROTECTORS

An antioxidant is defined as any substance that in low concentrations diminishes or inhibits the
oxidative damage in a target molecule.
The researches on naturally occurring polyphenols have received more attention in human medi-
cine, versus animal agriculture, as polyphenols exert several health benefits in humans. First, they
inhibit oxidation of low-density lipoproteins, thereby decreasing risks of heart diseases. Second,
polyphenols possess anti-inflammatory and anticarcinogenic properties and third, they are effective
antioxidants for food lipids. Third, the neuronal toxicity is mediated and enhanced by reactive oxygen
species (ROS) or reactive nitrogen species (RNS) and thus that the oxidative stress accelerates the
cell death of neuronal cells in neurodegenerative disorders. In fact, such neuronal cell death has been
reported to be attenuated by antioxidants and free radical scavengers (Catignani and Carter 1982).
120
100
80
Inhibition, %
60
40 LG; IC50: 134.7 μg/mL

BG; IC50: 45.3 μg/mL
SE; IC50: 76.6 μg/mL
20 CU; IC50: 81.9 μg/mL
0
0 50 100 150 200
Concentration, μg/L
FIGURE 21.5 Inhibitory effects of different lignin solutions on hemolysis induced by AAPH. LG, ligno-
sulfonates; BG, lignin from bagasse; SE, lignin from steam explosion; and CU, Curan, a commercial lignin.
Thus, the phenolic fragments of kraft lignin, obtained as by-products during pulp manufacture
process are as effective as vitamin E as an antioxidant in humans.
The antioxidant effect of different types of lignin has been evaluated by their capacity to inhibit
human erythrocyte hemolysis induced by AAPH [2,2′-azobis (2-amidopropane) dihydrochloride], a
peroxyl radical initiator (Vinardell et al. 2005).
The antioxidant activity is expressed as IC50 (concentration determining 50% inhibition on hemo-
lysis induced by AAPH). The values presented in Figure 21.5 are obtained for 45.3–134 μg/mL,
bagasse lignin having the highest antioxidant activity, because of its low IC50 value.
The photoprotective activity has been determined after incubating the erythrocytes suspension
for 30 min in the presence of different lignins and chlorpromazine (CPZ), a photohemolytic com-
pound (Figure 21.6).
A product could be considered as photoprotector if it increases the concentration of chlorproma-
zine necessary to induce hemolysis. The values of chlorpromazine (CPZ) concentrations that deter-
mine hemolysis and photohemolysis range from 60 to 189 mg/mL in the presence of different lignins
at a single concentration of 100 μg/mL. The three lignins studied showed no protective activity at
this concentration. Nevertheless, the lignins showed a protective effect on the hemolysis induced by
CPZ when red blood cells are not irradiated. These results confirm the antioxidant activity of the
lignins, since CPZ also induced hemolysis by oxidative stress (Vinardell et al. 2005).
The hemolysis of erythrocytes induced by AAPH was also studied in the presence of Ligmed-A.
This product which is composed of about 90% lignin was prepared from sugarcane and is commer-
cially available as antidiarrheal drug (Mitjans et al. 2001). The antioxidant effect of Ligmed-A was
expressed as the percentage of hemolysis inhibition at several concentrations. The inhibitory effect
was dependent on concentration of lignin, the greatest effect (the IC50 was 106.63 μg/mL) being
obtained at the highest concentration studied (Garcia et al. 2006).
The antioxidant efficiency of different lignin samples was also studied by Boeriu et al. using
two models (Boeriu et al. 2004). The first approach was to measure the ability of lignin samples
to react with free radicals in aqueous solution. The ABTS+⋅ [ABTS—2,2′-azino-bis(3-ethylbenzo-
thiazoline-6-sulphonate)] cation radicals were generated by an enzymatic system consisting of per-
oxidase and hydrogen peroxide. The antioxidant activities of lignins were determined by measuring
the rate of disappearance of the ABTS radical in the presence of lignin. All lignin samples tested
reacted with ABTS radical (Figure 21.7), but their efficiency was about 20%–30% (on mass) of that
measured for the commercial antioxidant BHT (butylated hydroxytoluene). Lignin samples from
Nonirradiated
120
CPZ
CU
LG
100 BG
80
% Hemolysis
60
40
20
0
60 90 105 120 135 180
(a) CPZ (mg/mL)
Irradiated
120 CPZ
CU
LG
100 BG
80
% Hemolysis
60
40
20
0
60 90 105 120 135 180
(b) CPZ (mg/mL)
FIGURE 21.6 Hemolysis and photohemolysis of CPZ in the presence and absence of different lignins (for the
significance of lignin samples see Figure 21.5). (a) nonirradiated (b) irradiated results are expressed as mean ± SE.
abaca, sisal and jute, and the softwood lignins SW-Kr-1 and SW-Kr-3 show the highest scavenging
activity against the ABTS radical.
Further, the effect of lignin on the oxidation of unsaturated compounds by molecular oxygen has
been evaluated. The oxidation of linoleic acid in micellar solution initiated with azo-compounds
[2,2′-azobis-(2-amidopropane)-dihydrochloride-ABAP] has been studied in the absence and pres-
ence of lignins and tocopherol (Vitamin E), which is a commonly used as antioxidant. Antioxidant
activities of lignin compounds were determined by measurement of the rate of generation of linoleic
acid peroxide during inhibited peroxidation of linoleic acid initiated by ABAP with known rate of
initiation. All lignins show some ability to act as chain-breaking antioxidants during lipid peroxi-
dation, but only lignins from abaca, sisal, hardwood, and jute and the sulfur-free softwood lignin
SW-SF-2 have significant antioxidant effects (Figure 21.8).
The most effective chain-breaking antioxidant lignin showed about 30% (on mass basis) of the
activity of tocopherol in the same system.
The results suggest that the radical scavenging activity of lignins, although induced by hydroxyl
phenolic groups, is not linearly related to the concentration of the phenolic groups, but is influenced
also by other structure-related factors such as the steric availability of aromatic hydroxyl group and the
1.4
1.2
RSAABTS, (mmolABTS/min.mg) 1
0.8
0.6
0.4
0.2
0
He
Si
Ab
St
Ju
FI-1
SW-Ls-1
SW-Kr-1
SW-LS-2
FI-2
FI-ox
SW-Kr-2
SW-SF-1
SW-Kr-3
HW
SW-SF-2
SW-Kr-4
.
FIGURE 21.7 Relative ABTS-radical scavenging activity of lignin samples. He, hemp; Si, sisal; Ab, abaca;
Ju, jute; Fl-1, flax; SW-Ls-1, lignosulfonate from softwood (Boresperse 3A); SW-Kr-1, kraft from softwood
(Indulin AT); SW-Ls-2, lignosulfonate from softwood (Wafex P); Fl-2, soda flax (Bioplast); Fl-ox-soda, flax
oxidized; SW-Kr-2, kraft from softwood (Curan 100); SW-SF-1, soda from softwood (precipitated at high pH);
SW-Kr-3, kraft from softwood; HW, organosolv (Alcell) from mixed hardwoods; SW-SF-2, soda softwood
(precipitated at low pH); SW-Kr-4-kraft (Curan 2711P).
25
RSAABTS, (mmolABTS/min.mg)
20
15
10
0
SW-LS-2
SW-Kr-2
SW-SF-1
SW-Kr-3
SW-SF-2
SW-Kr-4
SW-Ls-1
SW-Kr-1
FI-ox
FI-1
FI-2
HW
He
Ab
Si
St
Ju
FIGURE 21.8 Relative chain-breaking antioxidant effect of lignin in lipid peroxidation. Significance of the
samples is the same as in Figure 21.7.
stabilization of phenolic radicals. Adsorption phenomena at the oil–water interface may be of impor-
tance for reactions in micellar systems (e.g., inhibition of oxidation of polyunsaturated fatty acids).
The antioxidant capacity of black liquor lignin was evaluated by the inhibition of formation
of free radicals such as superoxide anion, hydroxyl radical, and radicals generated by UV-light
(Perez-Perez et al. 2005). In addition, isolated lignin antioxidant activity (AOA) and total antioxi-
dant activity (TAA) values were determined and the effect of isolated lignin on lipid peroxidation
process was examined using TBA and FOX methods. In all cases, the antioxidant capacity of lignin
was compared to natural antioxidant (melatonin, quercetin, and commercial lignin). In relation to
superoxide anion, all the analyzed samples could decrease superoxide anion formation (Table 21.1).
TABLE 21.1
Inhibition Percentages of Superoxide Anion
and Hydroxyl Radical Generation
Sample Superoxide Anion Hydroxyl Radical
Precipitated lignin 51.44 ± 1.29 33.68 ± 0.91
Commercial lignin 47.15 ± 2.04 27.81 ± 1.30
Melatonin 79.06 ± 0.32 53.89 ± 1.07
Quercetin 71.46 ± 0.85 53.07± 1.13
Precipitated lignin 3 mg/mL; melatonin 1 μM; quercetin 1 μM; com-

mercial lignin 3 mg/mL. Mean ± SE (n = 9). Means within a column
sharing the same letter were not significantly different by Newman–
Keuls multiple comparison test (p < 0.05).
TABLE 21.2
AOA and TAA Values of Precipitated
Lignin and Compared Antioxidants
Sample AOA (mM) TAA (mM)
Precipitated lignin 0.91 ± 0.03 56.71 ± 0.27
Commercial lignin 0.82 ± 0.01 4.24 ± 0.24
Melatonin 0.83 ± 0.02 6.99 ± 0.17
Quercetin 0.86 ± 0.03 10.55 ± 0.23
Precipitated lignin 3 mg/mL; melatonin 1 μM; quer-

cetin 1 μM; commercial lignin 3 mg/mL. Mean ± SE
(n = 9). Means within a column sharing the same
letter were not significantly different by Newman–
Keuls multiple comparison test (p < 0.05).
Precipitated lignin had an inhibition percentage for superoxide anion similar to commercial lignin
but lower than melatonin and quercetin. In the case of hydroxyl radical, the same behavior was observed,
precipitated lignin being better able to reduce hydroxyl radical formation than commercial lignin, but
less effective than melatonin and quercetin. This can be due to the fact that melatonin is a natural anti-
oxidant widely studied and which lost in aging prevents the kidnapping of free radicals and organism
protection, and quercetin which has antitumoral, anti-inflammatory, and antioxidant potential.
AOA values of precipitated lignin were determined and the results are summarized in Table 21.2.
Precipitated lignin had the highest AOA values, even higher than natural and commercial anti-
oxidants used in these experiments. AOA values of precipitated lignin were high as compared
with other samples analyzed by this method (Koracevic et al. 2001), like urine (0.17 mM), saliva
(0.84 mM), and brain–spinal fluid (0.095). TAA values of precipitated lignin were determined by
ABTS+⋅ TAA values are considered higher according to the results presented by Re et al. (2003), in
which a value of TAA superior to 3.0 mM is considered high, and values below 3.0 mM are consid-
ered low. Precipitated lignin sample displays TAA values that were comparable and even greater
than commercial lignin and lower than melatonin and quercetin. In addition, these values of TAA
are high when compared with other samples analyzed by this method, like 1.16 ± 0.18 mM for blood,
0.33 ± 0.02 mM for white onion, and 4.00 ± 0.20 mM for feluric acid. It is important to emphasize
that quercetin displayed values of TAA superior to that of the Trolox, which is used as a standard
in this method. This behavior has been reported for quercetin, which was approximately 3.3 times
TABLE 21.3
Inhibition Percentages of Formation of MDA and LHPs Determined by FOX
and TBA methods
Sample/Methods Enzymatic TBA Nonenzymatic TBA FOX UV-Light TBA UV-Light FOX
Precipitated lignin 82.1 ± 0.4 55.4 ± 0.5 40.0 ± 0.8 52.29 ± 0.4 55.3 ± 0.6
Commercial lignin 69.9 ± 0.5 57.4 ± 0.4 61.4 ± 0.5 39.93 ± 0.6 48.5 ± 0.5
Melatonin 82.3 ± 0.4 55.4 ± 0.1 62.4 ± 0.4 51.95 ± 0.8 57.5 ± 0.3
Quercetin 73.6 ± 0.5 58.7 ± 0.6 56.0 ± 0.1 55.69 ± 0.2 58.2 ± 0.5
Precipitated lignin 3 mg/mL; melatonin 1 μM; quercetin 1μM; commercial lignin 3 mg/mL. Mean ± SE (n = 9).
Means within a column sharing the same letter were not significantly different by Newman–Keuls multiple com-
parison test (p < 0.05).
more powerful than Trolox to scavenge radical cation ABTS, whereas the mono- and dichloride
derivatives of quercetin were 5.8 and 6.1 times more efficient than the Trolox, respectively.
To verify the antioxidant capacity of precipitated lignin, two methods were used to measure the
products of lipid peroxidation. These products were lipid hydroperoxides (LHPs), one of first prod-
ucts of this process, and malondialdehyde (MDA), a final product. The results of these experiments
are shown in Table 21.3, in which the inhibition percentages of the formation of each one in different
methods are reported. All the studied samples can inhibit formation of MDA and LHPs, in a similar
way than melatonin, quercetin, and commercial lignin. The effect of the precipitated lignin was also
studied on the radicals generated by UV-light. It was observed clearly that all the studied compounds
had the capacity to decrease the formation of MDA and LHPs in a statistically significant way.
For the moment, the mechanism by which lignin acts like antioxidant is not known, but it can be
presumed that it can be a scavenger of free radicals thanks to its phenolic structure. Several mechanisms
can be proposed: (1) lignin acts like a chelating agent scavenging metals of Fenton’s reagent (Fe II and
Mn II); (2) lignin acts as a “suicide” antioxidant, receiving the attack of the hydroxyl radical avoiding
the action of this radical on target molecules; or (3) lignin inhibits enzymes involved in the metabolic
pathways able to generate free radicals. Phenols are efficient in scavenging of peroxyl radicals because
of their molecular structures, which include hydroxyl groups that contain mobile hydrogens. In addition,
the action of phenolic compounds can be related to their capacity to reduce or to chelate necessary diva-
lent ions for several reactions. On the other hand, phenolic antioxidants can interrupt the oxidation reac-
tions by hydrogen atom transfer or by electron transfer with the formation of the radical cation phenoxy,
which is fast and reversible deprotonated forming the phenoxy radical. These two mechanisms always
happen in parallel but with different rates. Although these mechanisms have the same net results, the
electron transfer is preferable because the radical cation formed by electron transfer can be mutagenic.
To assess the radical scavenging activity of a potential antioxidant, the stable free radical
1,1-diphenyl-2-picryl hydrazyl (DPPH⋅) is used as active radical (Telysheva et al. 2000). The reac-
tivity of DPPH⋅ is far lower than that of oxygen-containing free radicals (HO⋅, RO⋅, ROO⋅, and
O2−⋅), and unlike them, the interaction rate is not diffusion controlled, but is ruled by the electronic
structure of the antioxidants. To evaluate the radical scavenging activity, potential antioxidants are
allowed to react with DPPH, usually in mixtures of water with organic solvent. Due to the presence
of an unpaired electron, DPPH⋅ radical gives a strong absorption band having a maximum at 517 nm
in the visible region. In the presence of a free radical scavenger, the absorption decreases, and
the resulting discoloring is stoichiometric with respect to the number of electrons taken up. Thus,
DPPH⋅ test provides information on the capacity of compounds to scavenge free radicals indepen-
dent from such factors as the presence of initiators or enzymes in a system. The methanol-soluble
fractions from commercial softwood kraft and hydrolysis lignin as well as laboratory acid-soluble,
alkaline, and ethanol lignin from softwood were studied.
2.1
1/DPPH* relative concentration

2
DPPH* relative concentration

1 1
1.9
0.9 3
1.7
0.8
1.5
0.7 4 4
1.3
0.6 3 1.1
1
0.5 0.9
0 500 1000 1500 2000 0 500 1000
(a) Time (s) (b) Time (s)
FIGURE 21.9 The kinetics of DPPH decay (a) and the dependence of the reciprocal DPPH⋅ concentration
on the interaction time (b) in the presence of (1) aspen alkaline lignin; (2) methylated aspen alkaline lignin;
(3) birch alkaline lignin; (4) spruce alkaline lignin.
For lignin fractions soluble in methanol, it has been confirmed that the OHphen group content plays a
crucial role in radical scavenger activity of lignin in the DPPH⋅ assay (Dizhbite et al. 2004). This was
proved by the methylation of lignin with diazomethane (Figure 21.9, curve 2), and dimethoxy units
(syringyl derivatives) exhibited higher antioxidant efficacy (Figure 21.9 curves 1,3 versus curve 4).
Methanol-soluble fraction of kraft lignin is characterized by a higher antiradical activity than
hydrolysis lignin. According to the data obtained, lignin fractions have stoichiometries expressed as
a number of reduced DPPH, of 2 and 1.3, respectively, for kraft and hydrolysis lignin, which could
be correlated with 240 mg of gallic acid equivalent (GAE) per 1 g for fraction from kraft lignin
against 126 mg GAE for that from hydrolysis lignin. These results are in good agreement with the
bioactivity of these fractions against phytopathogenic microorganisms, which could be connected,
to some extent, with the inhibition of radical processes by mobile lignin fractions (Figure 21.10).
Regarding the antioxidant activity of isolated lignins, the presence of π-conjugation system, for
example, stilbenes in kraft lignin, should be taken into account besides the aforementioned factors.
These systems are characterized with high ability to form self-donor-acceptor complexes, thus
decreasing the energy of polymer molecules excitement from singlet into triplet state and increasing
100
80
Bacteria numbers,
% of control
60
40
20
Co
n
Ins trol
olu
So
0 lub ble
a Kr le
tori vor
a
ae aft
ica ato ing yxa lig
ve s car syr lim
nin
X. E. P s . po
B .
FIGURE 21.10 (See companion CD for color figure.) The influence of lignin on phytopathogenic
microorganisms.
the reactivity of polymer. Π-conjugation systems of lignin could act as catalyst or activators of the
lignin phenolic structures interaction with DPPH⋅, but do not react with DPPH⋅ directly. Therefore,
rate constant for DPPH⋅ disappearance in the case of different lignins depends on both the number
of phenolic hydroxyls and degree of π-conjugation systems development. It has been established
that the extended π-conjugation systems of polymeric lignins significantly increase rate constants
of the second-order one-side process of DPPH⋅ decay, whereas the high molecular mass, enhanced
heterogeneity and polydispersity are among the factors decreasing the radical scavenging activity.
The ability of various lignophenols derivatives to protect SH-Y5Y cells from hydrogen peroxide
(H2O2)-induced cell death has been studied (Akao et al. 2004). It was observed that lignocresol
derivatives were more potent in the neuroprotective activity compared with other lignophenols.
Especially, the carboxymethylated lignocresol (lig-8) from bamboo displayed the most potent apop-
tosis preventing activity at 20 and 30 μM. This process was explained by blocking the caspase-3
activation, which is responsible by apoptosis (Figure 21.11). Results from in vivo and in vitro experi-
ments suggested that lig-8 possibly blocked the activation of caspase-3.
It is known that mitochondria play a major limiting role in the apoptosis of neuronal cells. H2O2-
induced apoptosis is considered to be mediated via the mitochondrial pathway, and lignin derivative
was shown to ameliorate the apoptosis by preventing depolarization of mitochondrial membrane PT.
The results have proved that lignophenol derivatives protected human dopaminergic neuroblastoma
SH-SY5Y cells against cell death due to apoptosis induced by oxidative stress.
The properties of lignins, especially the network structures, differ greatly depending on the lig-
nocellulosics, because the precursors building up lignin differ in their species. Generally, softwood
lignins consist mainly of guaiacyl aromatic units, and hardwood lignins contain evenly guaiacyl
and syringyl units. On the other hand, herb lignins including bamboo have para-hydroxy phenyl
units rather than these units. Certainly, the structure containing the para-hydroxy phenyl units in
bamboo lignin and additional phenolic groups may contribute to the neuroprotective activity.
2h 4h 6h
Control – + – + – + lig-8 (30 μM)
35 (kDa)
Caspase-3
17
(a)
100
ZDEVD-FMK
87.5
Z-VAD-FMK
75
Apoptotic cells (%)
62.5
50
37.5
25
12.5
0
10 20 30 50 100
(b) Concentrations of inhibitors (µM)
FIGURE 21.11 Involvement of caspase-3 activation in the H2O2-induced apoptosis and inhibition of caspase-3
activation by lig-8. (a) Western blot analysis of caspase-3. The arrow indicates the active form of caspase-3.
(See companion CD for color figure.) (b) Apoptosis inhibition assay using caspase-3 inhibitor ZDEVD-FMK or
pan-caspase inhibitor Z-VAD-FMK. The apoptotic cell death was evaluated by Hoechst 33342 staining. Results are
the mean ± SD of three independent experiments. The percentage of apoptotic cells without the inhibitor is as 100%.
21.4 LIGNINS IN REDUCTION OF CARCINOGENESIS

Damage of deoxyribonucleic acid (DNA) by oxygen radicals and various chemicals is thought to be
an important etiologic factor in the development of chronic diseases such as cancer. Carcinogenicity
and mutagenicity of chemicals may be modulated by other chemicals mostly prepared by organic
synthesis. As to the several drawbacks of synthesis, compounds for human organism examination of
preparations of plant origin for this purpose received more attention in the last few years. Lignin com-
ponent of biomass was derived from kraft pulping and prehydrolysis of wood was investigated with
regard to its ability to bind N-nitrosamines, well-known carcinogens and to reduce DNA strand breaks
in H2O2 and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-damaged mammalian cells as well as to
induce SOS response 4-nitroquinoline-N-oxide (4NQO) in E. coli PQ37 (Kosikova et al. 2002).
The most effective lignin sorbents of N-nitrosodiethylamine were modified spruce and beech
kraft lignins and these were examined with respect to their influence on the ability of mutagenic
compound 4NQO to induce SOS response in the microbial system E. coli (Figures 21.12 through
21.14). The SOS response is a postreplication DNA repair system that allows DNA replication to
bypass lesions or errors in the DNA. The SOS uses the RecA protein. The RecA protein, stimulated
15.00
10.00
IF
5.00
0.00
0.00 1.04 2.08 4.16 8.30
Concentration of lignin (µg/test)
FIGURE 21.12 Antimutagenic activity of modified kraft spruce lignin against 4-nitroquinoline-N-oxide.
15
10
IF
0
0 1.04 2.08 4.16 8.3
Concentration of lignin (μg/test)
FIGURE 21.13 Antimutagenic activity of prehydrolysis lignin against 4-nitroquinoline-N-oxide.

15
IF 10
0
0 1.04 2.08 4.16 8.3
Concentration of lignin (µg/test)
FIGURE 21.14 Antimutagenic activity of modified kraft beech lignin against 4-nitroquinoline-N-oxide.
by single-stranded DNA, is involved in the inactivation of the LexA repressor thereby inducing the
response. It is an error-prone repair system. SOS box is the region in the promoter of various genes
to which the LexA repressor binds to repress the transcription in the absence of DNA damage.
The observed antimutagenic activity of lignin samples is probably caused by their low degree of
cross-linking density resulting in highly absorptive capacity toward 4NQO. The mutual differences
of both modified lignins could be explained in terms of different ratios of guaiacyl and syringyl
structures in beech and spruce lignin. The obtained results indicate that antimutagenic potential of
lignin is based on its ability to absorb xenobiotics and thus to protect DNA damage.
The antioxidative effect of lignin sample 1 (prehydrolysis lignin) on DNA in human cells VH 10
exposed to oxidative treatment with H2O2 was investigated. The protective antioxidative effect of
lignin sample 1 results from the distribution of H2O2-induced DNA breakage in pretreated (+lignin)
and nonpretreated human cells (−lignin) Figure 21.15.
It is clear that in cells preincubated with lignin before oxidative treatment, there are significantly
fewer heavily damaged cells (percent tail DNA > 40) than in those treated with H2O2 alone. It is
known that lignin, which has unique hindered phenolic hydroxyl groups, acts as a stabilizer of reac-
tions induced by oxygen and its radical reduction products. Based on this, it can be suggested that
VH 10
100
80
Mean of % tail DNA
Control
60 Lignin 1
*** ***
40
20
0
Control H2O2 MNNG
(100 µmol/L) (13.6 µmol/L)
FIGURE 21.15 Influence of lignin 1 on level of DNA strand breaks induced by H2O2 and MNNG. Values are
means of three independent experiments. Significant difference (***p = 0.001).
phenolic groups of lignin matrix with active dissociable hydrogen are responsible for scavenging of
reactive oxygen species and reduction of oxidative DNA damage in lignin-pretreated cells.
In the case of the experiments with human Caco-2 colon carcinoma cells exposed to oxidative
damage, sample 1 (prehydrolysis lignin) and sample 2 (condensed kraft lignin) were used as anti-
oxidants. Both lignin samples tested were found to be effective in the reduction of strand breaks.
Further experiments with H2O2-treated Chinese hamster cells V79 confirmed the reduction of
DNA strand breaks by both lignin samples. Occurrence of oxidative DNA lesions induced by H2O2
was reduced more significantly in the case of lignin 1 probably due to its lower molecular mass,
which allows a better transfer of lignin macromolecules into cells.
Others experiments were performed using N-methyl-N′-nitro-N-nitrosoguanidine (MNNG).
In contrast to H2O2, MNNG does not induce any significant level of oxidative damage to DNA but
it alkylates several positions on DNA. The level of single strand breaks of DNA induced by MNNG
was also reduced by both lignin samples. The protective effect of lignin tested on MNNG-treated
cells may be associated with their low cross-linking density resulting in high binding affinity for car-
cinogenic and mutagenic compounds. The obtained results could be correlated with the suggestion
that the reduction of single-strand breaks of DNA by lignin has a different character in MNNG and
H2O2-treated cells. Reduction of H2O2-induced DNA may be caused by scavenging of OH radicals.
On the other hand, reduction of alkylating activity of MNNG to cause instability of N-glycosyl bond
on DNA lesions may correlate with the adsorption affinity of lignin tested for N-nitrosodiethylamine.
The effect of lignin on cytotoxicity, mutagenicity, and SOS response induced by nitroquino-
line-N-oxide (4NQO), 3-(5-nitro-2-furyl) acrylic acid (5NFAA), 2-nitrofluorene (2NF) as well as
hydrogen peroxide was investigated in bacterial assay system, i.e., the Ames test with Salmonella
typhimurium TA 98, TA 100, TA 102, and the SOS chromo test with E. coli PQ37 (Mikulasova and
Kosikova 2003).
The experiments were performed in order to establish to which extent antimutagenic properties
of lignin are influenced by its genetic origin and methods of isolation. As shown in Figure 21.16, all
modified lignins were effective in reducing IF by 4NQO as well by H2O2 (Figure 21.16).
Effect of wood-derived lignins on 4NQO (13.15 nmol) and H2O2 (1.1 mmol) induced SOS
response. The data are the mean from at least three independent experiments.
Kraft lignin derivatives 1 and 2 isolated from spruce and beech wood exhibited a similar effi-
ciency (Figure 21.16a and b). Sample 3 derived from beech wood prehydrolysate had an effect
comparable with both kraft lignins (Figure 21.16c). The modification of sample 3 yielded lignin
100
4NQO
H2O2
80
60
IF (%)
40
20
0
0 1.04 2.08 4.16 8.32
(a) Lignin (µg/assay)
FIGURE 21.16 Effect of wood-derived lignins on 4NQO (13.15 nmol) and H2O2 (1.1 mmol) induced SOS
response. The data are the mean from at least three independent experiments: (a) Spruce kraft lignin.
(continued)
100
4NQO
H2O2
80
60
IF (%)
40
20
0
0 1.04 2.08 4.16 8.32
(b) Lignin (µg/assay)
100
4NQO
80 H2O2
60
IF (%)
40
20
0
0 1.04 2.08 4.16 8.32
(c) Lignin (µg/assay)
100
4NQO
80 H2O2
60
IF (%)
40
20
0
0 1.04 2.08 4.16 8.32
(d) Lignin (µg/assay)
FIGURE 21.16 (continued) Effect of wood-derived lignins on 4NQO (13.15 nmol) and H2O2 (1.1 mmol)
induced SOS response. The data are the mean from at least three independent experiments: (b) beech kraft lig-
nin, (c) beech prehydrolysis lignin, and (d) beech condensed prehydrolysis lignin.
preparation 4 which exhibited the strongest ability to decrease activity of 4NQO (Figure 21.16d).
The determination of molecular parameters of lignins 3 and 4 shows that the modification increased
the average molecular mass about 62.5% and decreased cross-links about 10.5%. The increased
efficiency of lignin 4 could be explained by degradation of cross-links and linearization of the lig-
nin macromolecule during modification. The samples prepared from steam-treated annual plants
showed a relatively lower efficiency than those isolated from wood.
Therefore, the results obtained indicate that tested lignin derivatives decrease mutagenic
activity of 4NQO, 2NF, and H2O2, and the differences could be correlated with their genetic origin
and methods of modification (Mikulasova and Kosikova 2003).
The DNA-protective effects of lignin were examined in rat testicular cells and rat peripheral
blood lymphocytes using in vitro and ex vivo experiments. H2O2 and visible light-excited methylene
blue (MB) were used as DNA-damaging agents. Testicular cells were chosen because the germinal
epithelium of tests is one of the most proliferative active tissues potentially susceptible to DNA-
damaging effects. As a second target, peripheral blood lymphocytes were chosen because dietary
lignin or its metabolites circulate in the animal organism probably through the blood system. For
the in vitro experiments, isolated cells were preincubated with lignin for 2 h before treatment with
one of the oxidative agents. In ex vivo experiments, the cells were exposed to H2O2 or visible light-
excited MB after isolation from rats fed either a common diet or a lignin-supplemented diet. The
water-soluble, sulfur-free lignin used in experiments was obtained by fractionation of hardwood
hydrolysate. The level of direct single-strand DNA breaks in H2O2-treated cells was measured by
the classical Comet assay, and the level of oxidative DNA lesions in visible light-treated cells was
measured by a modified Comet assay. It was found that lignin reduced DNA lesions induced by
H2O2 or visible light-excited MB both in vitro and ex vivo. Therefore, the lignin polymer obtained
by fractionation of hardwood hydrolysate manifested a specific type of antimutagenic effect (Labaj
et al. 2004).
Antitumor substances were separated from pinecones of Pinus parviflora. Spectral analysis
of one isolated fraction revealed that this fraction included a lignin-related structure as its main
constituent. This fraction along with various commercially lignins (alkali lignin, dealkali lignin,
and lignosulfonates) significantly stimulated iodination of human peripheral blood polymorpho-
nuclear (PMN) cells. The PMN iodination increased with incubation time under the influence of
alkali lignin and reached a transient plateau at 150 min after start of incubation with Na125I. The
PMN iodination again rapidly increased after 180 min. This might be due to some damage of PMN,
since PMN aggregation occurred at this time. Optimal concentration of alkali lignin was 50 μg/mL
(Sakagami et al. 1991).
But it was observed that the PMN-stimulating activity of alkali lignin was significantly inhibited
by the presence of serum. This suggests the importance of structurally modifying lignins to reduce
their nonspecific binding to various proteins.
The capacity for stimulation of PMN iodination is substantially related to the lignified or tannin-
like polyphenolic structure, although the details of structural requirement need further investigation.
It was found that the activity of mentioned products depended more on molecular mass than on the
number of hydroxyl groups on each benzene ring in the molecule or on the presence of sugars
or hexahydroxyldiphenyl groups. The stimulation of PMN iodination by lignin-related phenolic
substances or a tannin-related compound was observed in only myeloperoxidase-containing cells
and was significantly reduced by myeloperoxidase inhibitors. These results suggest that the mecha-
nism involved might specifically potentate the active oxygen-mediated host defense capacity against
microbial and viral infections and possibly tumor growth and metastasis. The mechanism might
have played a role in the antimicrobial, antitumor, antiparasite, and antiviral activities displayed by
the pinecone extract and some lignified materials (Sakagami et al 2005).
Lignin derivatives demonstrated differential responses between cells derived from tumor tissues
and from normal tissues and strongly inhibited the growth of mouse sarcoma cells (FRUKTO)
and rat fetal cells (Ad 12-3Y1-Z-19) transformed with adenovirus type 12 (Sorimachi 1992). The
production of tumor necrosis factor (TNF-α), which induced multinucleated giant cell formation,
is regulated by polyanions such as lignin derivatives. ELISA for TNF-α showed that polyanion
induced TNF-α production by macrophages. The secretion of TNF-α from the cells reached a max-
imum at 3–6 h, and then showed a slight decline. Northern blotting of TNF-α mRNA evidenced
that the amount of TNF-α reached a maximum within 1 h of macrophage culture in the presence of
a lignin derivative. On the other hand, TNF-α mRNA was undetectable in the control cells. It was
concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF-α
mRNA, which is then followed by translation of TNF-α (Sorimachi et al 1995).
It was also observed that the soluble lignin derivatives obtained from an extract of the culture
medium of Lentinus edodes mycelia induced the secretion of tumor necrosis factor TNF-α. At the
same time, lignin derivative markedly induced the secretion interleukin-8, nitric oxide, along with
TNF-α in macrophages. The level of these compounds could be negatively and positively modu-
lated by interferon-γ (IFN-γ). It seems that IFN-γ is one of the important cytokines that regulate
macrophage functions, particularly in the presence of foreign bodies. In the coculture of macro-
phages and human urinary bladder carcinoma cells, lignin derivative induced the cytotoxic effect of
macrophages on the tumor cells. Therefore, IFN-γ could regulate macrophage–tumor cell interac-
tion (Sorimachi et al. 2003).
21.5 ANTI-HIV PROPERTIES OF LIGNINS

Natural compounds can be used to reduce the signals that tell macrophages they are in the sec-
ond and third stages, thereby easing entry into the fourth stage. Macrophages play an essential
role in the immune response. Natural compounds could be used to stimulate macrophage (and
other immune cell) activity and to reduce immune evasion. Natural compounds could promote the
antiangiogenic mode of macrophages by reducing the signals that tell macrophages angiogenesis
is necessary. Compounds that support the immune system do so by providing proper nutrition to
immune cells or by acting as antioxidants or both. (Immune cells such as macrophages require
large amounts of antioxidants for protection from the free radicals they generate.) Some natural
compounds also help the immune system by providing a regulatory influence, thereby normaliz-
ing the extremes in activity found in diseases like cancer. Compounds that stimulate the immune
system often do so by increasing the production of cytokines, including IL-2 and interferons, but
may act in other ways as well. These natural compounds tend to increase production of a broad
range of cytokines.
Macrophages play an important role in the immune system and their function is regulated by
substances (lymphokines) produced by sensitized lymphocytes. It is known that human immuno-
deficiency virus (HIV) induces the acquired immune deficiency syndrome (AIDS), which results
in life-threatening opportunistic infection and malignancies. HIV infects not only human T cells
but also transformed B cells and macrophages. Indeed HIV particles were detected in macrophages
obtained from AIDS patients. In addition, it was shown that receptor protein (CD4) for HIV exists
on cell surface in the HIV-infected cells.
It has been reported that several samples of lignin extracted from natural products in land
plants and fungi had anti-HIV activities toward cultured cells. Thus, a water-solubilized lignin
derivative (EP3), chromatographically purified from water extract of culture medium of Lentinus
edodes mycelia (LEM), inhibited the cytopathic effect of HIV in AH8 cells derived from human T
cells in vitro (Suzuki et al. 1990). The culture medium of Lentinus edodes mycelia was extracted
with hot water and this extract was called LEM. LEM was further fractionated with ethanol
followed by hydrophobic column chromatography. The most active fraction was called EP3.
Lignosulfonate (LS) was prepared by ethanol precipitation from waste liquor of the acid sulfite
pulping process of wood.
When macrophages were cultured for 24 h in the presence of EP3, the number of macrophages
increased. In addition, EP3 accelerated the glucose consumption of macrophages. EP3 increased
thymidine uptake and glucose consumption in murine macrophages. Giant cell formation was not
observed during the first 24 h of incubation of macrophages with EP 3 at 37°C, but increased mark-
edly during the next 24 h. Multinucleation further proceed between 2 and 3 days of culture. When
EP3 was removed from culture medium after macrophages were incubated with the drug for 24 h,
giant cell formation still took place over the next 24 h. Thus, macrophages retain the memory of
contact with EP3. The giant cells were active in carrying out phagocytosis of India ink.
Lignosulfonate also induced giant cell formation of macrophages. This substance is also known
to increase glucose consumption and thymidine uptake in murine macrophages and to inhibit the
cytopathic effect of HIV. These suggest that lignin derivatives in general may induce the multi-
nucleation of macrophages in vitro. The plasma membrane of macrophages is presumably altered
by EP3 and LS to induce the cell fusion, and this effect seems to be specific for macrophages
(Sorimachi et al. 1990, 1997)
However, examining the antiviral activity of EP3 and LS, it was found that these drugs prevented
the cytopathic effects of DNA viruses (herpes simplex viruses) and RNA viruses (Western equine
encephalitis virus, polio virus, and measles virus) in VERO-317 cells derived from African green
monkey kidney, although giant cell formation did not occur. In addition, the oral administration of
lignin derivative (LEM) improved hepatic functions in hepatitis-B patients. These results suggest
that lignin derivatives can affect cellular metabolism in cells other than macrophages.
In other experiments, a purified fraction EPS4 also obtained from LEM by ethanol precipitation fol-
lowed by hydrophobic chromatography and gel filtration chromatography completely inhibited the HIV-
1-induced cytopathic effect in vitro at concentrations of greater than or equal to 10 μg/mL. Chemical and
spectral analysis revealed that EPS4 is composed of water-soluble lignins containing minor amounts of
protein (3.2%) and sugars (12.2%). Taken together with the previously reported observation that EPS 4
promotes the activation of macrophages and the proliferation of bone marrow cells, the fraction appears
to possess both an immunostimulating activity and anti-HIV effect in vitro (Suzuki et al. 1998).
A water-soluble lignin derivative of high molecular mass was found in boiling water extracts of
an edible mushroom, Fuscoporia oblique. This product inhibited protease of human immunodefi-
ciency virus type 1 (Ichimura et al. 1990).
HIV-1 protease was inhibited by lignin extracted from wheat bran and by synthetic lignin-like
compounds, and at the same time, these induced cytopathicity in cultured cells. To examine the
molecular mass dependency of the protease inhibition by these materials, synthetic dehydrogenated
polymers (DHP) start in from p-coumaric acid and ferulic acid were fractionated into four ranges
of molecular mass by passing through ultra-filtration membranes. All the DHP fractions exhib-
ited HIV-1 protease inhibitory activity, although there was a tendency for the inhibitory activity to
increase as the molecular mass increased.
The anti-HIV-1 activities of oligomers of p-coumaric and ferulic acids and wheat bran lignin were
examined by using MT-4 cells. It was observed, while wheat bran lignin inhibited the cytopathicity
induced by HIV-1 replication in MT-4 cells like several other lignins, it showed cytotoxicity at twice
the effective dose (Ichimura et al 1999).
When rat bone marrow macrophages were incubated with acetyl lignin in the presence of fetal
bovine serum, the macrophages secreted tumor necrosis factor (TNF-α) in a dose-dependent manner.
This was followed by macrophage multinucleation. Lignin derivative was found to have a signifi-
cant effect on TNF-α secretion at a minimum dose of 2 μg/mL and produced no significant further
increase at levels above 50 μg/mL, while multinucleation was most active at 10 μg/mL. However,
multinucleation did not occur at higher concentration of acetyl lignin (50 and 100 μg/mL). Secretion
of TNF-α was significantly reduced in the absence of fetal bovine serum, whereas multinucleation
was very active, starting after 6 h of incubation. At a concentration of 100 μg/mL, lignosulfonate
only induced low levels of TNF-α secretion from macrophages, but induced active multinucleation.
The multinucleation induced by addition of LS was inhibited by further addition of acetyl lignin.
Thus, macrophages multinucleation was most active when a low level of TNF-α was secreted from
macrophages (Okazaki et al. 1996).
21.6 LIGNIN AS SPERMICIDE

Lignin-derived macromolecules (LDMs) that are highly sulfonated (LSA) are negatively charged
compounds that exhibit a number of biological activities. LSA and other LDMs are potent inhibi-
tors of HIV in vitro, possibly through interference with the CD-4 receptor/HIV interaction.
In vitro microbicidal activity of LDMs has been demonstrated with several other viruses as well.
This antiviral and antipathogen activity is cell surface mediated and is likely related to similar
inhibitory effects of other sulfonated and sulfated macromolecules. Selective effects of LDMs on
various cell types have also been observed. For example, an LDM has been shown to dramati-
cally inhibit the growth of fibroblast and sarcoma cells, but yet activate murine macrophages and
induce proliferation of bone marrow cells. LDMs have also been shown to inhibit fertilization in
echinoderms without showing cytotoxic effects through a mechanism that involves inhibition of
the sperm acrosome reaction. LSA inhibits the sea urchin sperm acrosome reaction and it com-
petes with the natural sulfated ligand, egg jelly, on the sperm surface. LSA binds to the head of
capacitated macaque sperm, a location consistent with its biological activity. LSA significantly
inhibited the binding of macaque sperm to macaque zonae pellucidae both when the compound
was added to sperm after capacitation and when it was added to sperm before Percoll separation
and capacitation (Tollner et al. 2002).
LSA has a complex highly branched structure that may promote “binding” to a large number of
sperm surface receptors that recognize anionic domains. LSA may provide a useful tool for eluci-
dating the relationship between capacitation and the sperm functions involved in binding to the zona
pellucida. Due to its lack of cytotoxicity and its antifertility effect on noncapacitated sperm, LSA is
a strong candidate for development as a vaginal contraceptive. In addition, formulations containing
LSA have been shown to prevent infection with sexually transmitted herpes simplex virus in the
mouse without causing vaginal irritation.
21.7 MECHANISM
The information concerning the biological properties of lignin could be correlated with the
similar well-known properties of lignans. This subject is very interesting having in mind the
data existing on the part of lignin and lignans as antibacterial, antioxidants, anti-HIV and in the
prevention of breast and prostate cancers, osteoporosis, or cardiovascular diseases. Thus, it was
supposed that lignins are major dietary precursors of mammalian lignan. Lignins are structur-
ally related to lignans but differ from them by their polymeric nature and distribution in plants
(Begum et al. 2004).
The most common monomers in lignins are guiacyclopropane (G). Therefore, lignins are struc-
turally related to secoisolariciresinol (SECO) and matairesinol (MAT) (Figure 21.17).
The rats were fed with cereal brans that had and had not had lignans removed by solvent extrac-
tion to examine the respective contributions of plant lignans and lignins to the formation of mam-
malian lignans. The rats were also fed with a deuterated synthetic lignin (DHP) and the excretion of
deuterated enterolactone (ENL) was measured in urine. Thus, it was demonstrated for the first time
that lignins are precursors of lignan phytoestrogens (Nicolle et al. 2002).
Therefore, lignin can be metabolized into ENL in rats. The yield of D2-ENL was 655 nmol/g
DHP (195 μg/g). This yield is ≈10 times higher than that calculated for bran lignins. These differ-
ences could be explained either by a limited accessibility to the substrate for bran lignins embed-
ded in the cell wall or to structural differences between native bran lignins and synthetic lignins.
Lignins, because of their polymeric nature and of their embedding in the cell wall, are usually
considered inert in the digestive tract. However, the results obtained show that they are metabolized
by the gut microflora to form part of the ENL excreted in urine. But more data on lignan and lignin
contents in foods will be required to determine their respective contributions to the mammalian
lignan formation.
HO CR2 HO CH2OH
O
CH2OH
O
OH OH
Enterolactone R = H ne-etichetat Enlerodiol
R = D deuterat
H3C O H3C O CH2OH H3C O CH2OH

O
HO CH2OH HO CH2OH
HO
O
O CH3 O CH3 O CH3

OH OH OH
Matairesinol Secoisolariciresinol Isolariciresinol
O CH3 O CH3
O CH3
OH OH
OH
O OH
O
O CH3 H2C
O H3C O O
O
HO HO
HO
O CH3 O CH3
O CH3
Pinoresinol Syringaresinol Lariciresinol
FIGURE 21.17 Chemical structures of mammalian lignans and of the plant lignans. (Adapted from Begun
et al. 2004.)
A significantly higher formation of enterolactone was found in pigs fed with the rye diet (high in
lignans) and higher fecal and urinary excretion and circulating levels of mammalian lignans than
pigs fed with wheat diet (low in lignans). The conversion of mammalian lignan precursor to entero-
lactone was 48% with the wheat diet and 60% with the rye diet. Mammalian lignans are absorbed
by passive diffusion from the large intestine and the resulting fraction of the absorbed mammalian
lignans undergoes enterohepatic circulation, resulting in low diurnal variation in plasma levels of
enterolactone (Bach Knudsen et al. 2003).
One the other hand, the experimental results could suggest a mechanism whereby the free
radical-scavenging activity of lignin in dietary fiber may be involved in the fiber–colon cancer interac-
tion. Thus, the ability of dietary fiber to protect against colon cancer may be partly determined by the
amount of lignin in dietary fiber as well as the free radical-scavenging ability of lignin. Data showing
that lignin is a free radical scavenger were obtained when the NADH-phenazine methosulfate-nitro
blue tetrazolium free radical-producing system was used. An alkali lignin solution with a concentration
of 46.29 μg/mL causes 50% (IC50) inhibition of uric acid production by xanthine oxidase. Alkali lignin
with an IC50 of 59.08 μg/mL inhibits the activity of xanthine oxidase, one of the enzymes related to
the production of superoxide anion radicals, and presents a mixed-type noncompetitive inhibition pat-
tern. Using deoxyribose method, it was found that alkali lignin is a hydroxyl radical scavenger with an
IC50 of 250 μg/mL, and using the thiobarbituric acid method, it was observed that alkali lignin inhibits
nonenzymatic and enzymatic lipid peroxidation with an IC50 of 72 and 100 μg/mL, respectively. Alkali-
lignin also hinders the activity of g lucose-6-phosphate dehydrogenase, another enzyme related to the
generation of superoxide anion radicals with IC50 of 123.6 μg/mL and obstructs the growth and viabil-
ity of cancer interaction (HeLa) cells in a dose-dependent manner (Lu et al. 1998).
Various natural and synthetic compounds including alkaloids, terpenoids, and phenolics were
tested for inhibition of the cell surface expression of intercellular adhesion molecule-1 (ICAM-1)
and vascular cell adhesion molecule-1 (VCAM-1), both of which are crucial in the regulation of
immune response and inflammation (Figure 21.18).
Preliminary studies on the structure–activity relationships suggested that chalcone and isofla-
vone seem to be superior to dehydrochalcone, flavone, and flavanone in down regulating the cell
surface expression of VCAM-1 and ICAM-1:
3 3 3´
2 2 4 2´ 4´
4
5´ A 5´ 8 1´
6´
1 5
4´ 6´
1 5 O 5´
4´ 7
B 6 6 6´
3´ 3´ 6
1´ 1´
2´ 2´ 5
O O O
(a) (b) (c)
3´
2´ 4´ 8
7
O
8 1´
O 5´
2´
7 6
6´ 1´ 3´
6 5
5 O 6´ 4´
O 5´
(d) (e)
Flavonoids affecting cell expression of VCAM-1 and ICAM-1 are: a-chalcone; b-dehydrochal-
cone; c-flavone; d-flavanone; e-isoflavone (genistein).
Studies on the relationship between the substitution pattern of the hydroxy groups and the
VCAM-1 inhibitory activity in 24 chalcone derivatives have suggested that chalcone bearing one or
two hydroxy group at C-2′ of the ring B and C-4 of the ring A are potentially active in decreasing
the cell surface level of VCAM-1 expression. However, the inhibitory activities of these com-
pounds were restored to the control level by the chemical conversion (acetylation, benzylation, or
methylation) of hydroxy groups. These results seem to indicate that the coplanarity formed by the
hydrogen bonding between the 2′-hydroxy group of the ring B and the adjacent conjugated ketone
may be important in inhibiting the cell surface expression of VCAM-1 (Tanaka et al. 2001).
The relationship between macromolecular and mutagenic properties of lignin-containing com-
pounds (LCC) was studied. LCC in water medium and in swollen state (at a moisture ≥ 60%) behave
as polyanions and/or neutral molecules according to a value of pH, whose action on biological systems
is connected with competition mechanisms. They disturb the structural–functional systems of cells:
at different levels (gene, membrane, enzymes). The influence of molecular mass value both of natu-
ral and technical LCC prepared by different methods (fractionation by solvents, neutron irradiation
plasma treatment) on their mutagenic properties was proved by cytogenetical testing. The biological
properties of LCC were studied for molecular mass values of 4000–7000. The oligomers with these
molecular masses induce the chromosomal aberration in sexual cells of male Benedictia baicalensis
(Baikal lake mollusc) and root cells of plants at a concentration more than 0.1 and 0.016 g/L, respec-
tively, regardless of the LCC nature and mode of preparation. Relationship between logarithm of
molecular mass and mutagenic effect on mollusks is approximately linear and may be used both for
estimating and prediction of LCC influence on hydrobionts. LCC with an average molecular mass
O HO
O
Cl–
N N CH3
O + CH3 HO
H H C O
O CH3
O
H
CH3 OGlc
Berberic chloride (1) Ipecoside (2)
CH3
O
O CH3 H3C O NH
HO
N N
H3C O CH3 CH3 OH
H H
O
O O
O CH3 CH3
Isotetrandrine (3) (±)-N-norreticuline (4)
O
H CH3
H3C CH3 H3C
O H3C H3C
N
H3C + O H
Cl– H2C H3C
O
Sanguinarine chloride (5) β-Caryophyllene (6) α-Humulene (7)
HO CH2 O
O O OH
O OH OH OH
HO
HO HO OH O
O
HO
O O CH2
O OH OH O
OH O
11,13-Dihydro-taraxinic acid Aloe-emodin (9) Amentoflavone (10)
14-O- β-D-glucoside (8)
H3C O
O
H3C O
O HO C C C C C O O CH3
H H H2
(t)
O CH3 Asparenyol (12)

OH
( ±)-Arctigenin (11)
H3C O
O
OH
HO
HO O
O CH3
OH O OH
Isoliquiritigenin (13)
(±)-Matairesinol (14)
FIGURE 21.18 Chemical structure of various compounds affecting the expression of cell adhesion molecules.
below 4000 and above 1000 had no cytogenetical effect on mollusks and plants. The increase of
hydrophobicity and toxicity was, however, observed at the relationship Calk:Car = 3–5:1 (content of
carbon atoms in aliphatic chain is more than 10). The increasing oxidation value of aromatic rings and
increasing content of quinone and methylquinone structures as result of LCC transformation by the
action of biogenic and abiogenic factors causes enhancement of toxic and mutagenic properties. The
frequency of chromosomal aberration in sexual cells of mollusks was increased in response to more
oxidized high-molecular mass fractions of LCC, but the reduction of LCC (by neutron irradiation)
decreased their mutagenic activity. A comparison between microheterogeneity calculated based on
potentiometric titration data and biological activity showed its influence on the mutagenic and growth
regulating activity. The fractions of LCC with molecular masses of 4000–7000 and narrow distri-
bution factor revealed the high growth-regulating activity depended on content of carboxylic and
hydroxylic groups, as well as density of negative charge of macromolecules.
Thus, the biological properties of LCC are determined by the content of labile fractions and
active functional groups, density of charges on macromolecules and depend both on value of molec-
ular mass and its distribution and conditions of environment that favored the transformation of
macromolecules and inactivation of reactive groups (Novikova et al. 1998).
Plant lignans from sources such as flaxseeds, whole grain cereals, berries, vegetable, and fruits are
metabolized in the colon by microflora into enterodiol and enterolactone. Previous research has focused
on plant lignans as reducing risk of prostate cancer and improving menopause health, gene health,
breast health, heart health, hair loss, acne, and inflammation. The metabolites of lignans may also be
beneficial for people with colorectal adenomas growth in the colon and rectum that are considered to
be precursors for colorectal cancer. The benefits of lignans appeared to be dose dependent meaning
that a higher plasma level of the metabolites enterodiol and enterolactone were associated with a greater
benefit. A plasma level of enterolactone of more than 26.3 nmol/L was associated with a reduction in
the risk of colorectal adenomas of 37%, compared with people with intakes of less than 4.6 nmol/L.
Enterodiol was similarly associated with colorectal adenomas as enterolactone, although con-
centrations of enterodiol were 5- to 10-fold lower. This suggests that in the human body, enterodiol
might be more active than enterolactone.
The hydroxymatairesinol (HMR lignan) and to a greater extent its human metabolite enterolac-
tone may suppress the growth of prostate cancer cells. The results are reported to show that both
enterolactone and hydroxymatairesinol increased the programmed cell death (apoptosis) in a dose-
dependent manner. Enterolactone is said to be two times as effective as hydroxymatairesinol but less
than half that of cycloheximide (cytotoxic agent) and estradiol.
A high intake of plant lignans could reduce the risk of breast cancer for premenopausal women.
In women with high plasma levels of enterolactone (above 12.96 nmol/L), high lignan intake was
associated with a 58% reduction of breast cancer risk, while average plasma enterolactone levels of
24.96 nmol/g were associated with a reduction of 62%.
A high intake of enterolignans (enterodiol and enterolactone) on the basis of dietary intake
(846 μg/day) combined with a high plasma enterolactone level was associated with a reduction in the
risk of breast cancer of 64%. Research has shown enterolactone to stimulate the synthesis and circu-
lating levels of a biochemical called sex hormone-binding globulin (SHBG). Through this activity,
enterolactone may reduce the free bioavailable pool of circulating estrogen, thereby reducing estro-
gen penetration in tissues and the risks of an adverse estrogen balance. There is also evidence that
enterolactone may inhibit biosynthesis of estrogen by blocking aromatase, a key enzyme in biosyn-
thesis of estradiol. Collectively through these multiple mechanisms of action, lignans appear to have
a positive influence on the estrogen balance in the body.
Flaxseed, the richest known source of plant lignans and omega-3 fatty acid, has been shown to
have chemoprotective effects in animal and cell studies. Omega-3 fatty acid suppresses the produc-
tion of interleukin-1 (IL-1), tumor necrosis factor (TNF) and leukotriene B4 (LTB4), and of oxygen
free radicals (OFRs) by polymorphonuclear leukocytes (PMNLs) and monocytes. Lignans possess
antiplatelet activating factor (PAF) activity and are antioxidant. PAF, IL-1, TNF, and LTB4 are
known to stimulate PMNLs to produce OFRs. Modest dietary flaxseed supplementation is effective
in reducing hypocholesterolemic atherosclerosis markedly without lowering serum cholesterol. Its
effectiveness against hypercholesterolemic atherosclerosis could be due to suppression of enhanced
production of OFRs by PMNL in hypercholesterolemia. Dietary flaxseed supplementation could,
therefore, prevent hypercholesterolemia-related heart attack and strokes (Parasad 1997).
Flaxseed and its lignan secoisolariciresinol diglucoside (SDG) inhibit mammary tumor develop-
ment in rats. At the same time, the increased plasma insulin-like growth factor I (IGF-1) concentra-
tions are associated with increased breast cancer risk. The anticancer effect of flaxseed and SDG
may be related, in part, to reductions in plasma IGF-I (Rickard et al. 2000).
SDG is an antioxidant. It scavenges for certain free radicals like hydroxyl (HO⋅). Our bodies
produce free radicals continually as we use (oxidize) fats, proteins, alcohol, and some carbohydrates
for energy. Free radicals can damage tissues and have been implicated in the pathology of many
diseases like atherosclerosis, cancer, and Alzheimer’s disease. The flax lignan secoisolariciresinol
(SECO) and the mammalian lignins enterodiol, and enterolactone also act as antioxidants. Indeed
the antioxidant action of SECO and enterodiol is greater than that of vitamin E.
The flaxseeds’ effects may be mediated through its influence on endogenous hormone produc-
tion and metabolism. Two competing pathways in estrogen metabolism involve production of the
2-hydroxylated and 16-α-hydroxylated metabolites. Because of the proposed differences in biologi-
cal activities of these metabolites, the balance of the two pathways has been used as a biomarker
for breast cancer risk. The effects of flaxseeds consumption on urinary estrogen metabolite excre-
tion were examined. The results obtained suggest that flaxseed may have chemoprotective effects
in postmenopausal women. Lignans bind to estrogen receptors on sex hormone-binding globulin
(SHBG), thus blocking the binding of estrogen and testosterone. As SHBG is found in breast cancer
cells, the binding of mammalian lignans to SHBG may interfere with cancer processes that are
controlled by estrogen (Haggans et al. 1999).
It has been shown that secoisolariciresinol diglycoside (SDG) decreases some early markers of
colon cancer risk over the short term. Further studies followed to establish whether over the long term
flaxseed still exerts a colon cancer-protective effect, whether its effect may, in part, be due to its high
content of SDG and whether any change in β-glucuronidase activity plays a role in the protective
effect. Urinary lignan excretion, which is an indicator of mammalian lignan production, was signifi-
cantly increased in the case of rats that were fed with flaxseed and defated flaxseed. It was concluded
that flaxseed has a colon cancer-protective effect, which is due, in part, to SD and that the protective
effect of flaxseed is associated with increased β-glucuronidase activity (Jenab and Thompson 1996).
21.8 CONCLUSIONS
Little attention has so far been paid to the utility in biological science of lignins, which have long
been regarded as waste from the pulp industry. However, the existing information evidences that
lignin and its derivatives have a multifunctional role in biological systems (antibacterials, antioxi-
dants and photoprotectors, anticarcinogen, anti-HIV, and spermicide) influencing cell metabolism.
Having in mind the polyphenolic character of lignins, one may suppose that the action of lignins
is based on their interactions with cell surface at the level of membrane lipids. Thus, the action of
lignins can be correlated with their molecular mass and hydroxyl group content by hydrophilic–
hydrophobic interactions. At the same time, lignins can be transformed by different metabolic path-
ways into products (e.g., lignans) that can be useful for human and animal health.
Since many molecular species of lignin are available, depending on the natural sources and
methods of extraction and modification, further investigations are definitely worthwhile for isolat-
ing and identifying the principle responsible for their notable biological activity. In this context, we
need to have information emanating from studies of the molecular biology of genomics and pro-
teomics in order to enhance our comprehension of intricate roles/functions at the molecular level of
the lignin and its derivatives.
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22 Carbohydrate-Derived
Self-Crosslinkable In Situ Gelable
Hydrogels for Modulation
of Wound Healing
Lihui Weng, Christine Falabella, and Weiliam Chen
CONTENTS
22.1 Introduction........................................................................................................................... 740
22.1.1 Hyaluronic Acid (HA)............................................................................................... 740
22.1.2 Dextran...................................................................................................................... 741
22.1.3 Chitosan..................................................................................................................... 742
22.1.4 Gelatin....................................................................................................................... 742
22.2 Acrylated Chitosan/Oxidized Dextran for Acceleration of Wound Healing......................... 743
22.2.1 Modification of Dextran and Chitosan...................................................................... 743
22.2.2 Rheological Analysis of Hydrogel Formation........................................................... 744
22.2.3 Morphology of the Hydrogels.................................................................................... 745
22.2.4 Swelling Properties of the Hydrogels........................................................................ 746
22.2.5 In Vitro Hydrolytic Degradation of the Hydrogels.................................................... 746
22.2.6 Cytotoxicity of the Hydrogels and Cell Viability Analysis....................................... 747
22.2.7 Cell Encapsulation in Hydrogels............................................................................... 748
22.2.8 Acceleration of Wound Healing in Murine Transcutaneous Full-Thickness
Wounds...................................................................................................................... 749
22.3 Gelatin/Oxidized Hyaluronan (Amenable to Cell Migration)............................................... 750
22.3.1 Preparation of oHA and Formulation of oHA/Gelatin Hydrogels............................ 750
22.3.2 Hydrogel Morphological Analyses............................................................................ 754
22.3.3 Swelling Properties of Hydrogels.............................................................................. 755
22.3.4 Rheological Analyses of Hydrogels........................................................................... 755
22.3.5 Three-Dimensional Infiltration and Distribution of Cells in Hydrogels................... 756
22.3.6 Long-Term Cell Viability in the Presence of Hydrogels and In Vivo
Biocompatibility........................................................................................................ 758
22.4 Carboxyethyl Chitosan/Oxidized Dextran Hydrogel for Prevention of Post-Surgical
Abdominal Adhesion............................................................................................................. 762
22.5 Suppression of Wound Healing by Hydrogel-Mediated Delivery of an iNOS Inhibitor....... 765
22.5.1 Microspheres for GED Delivery................................................................................ 766
22.5.2 Characterization of Microspheres............................................................................. 766
22.5.3 In Vitro Release of GED............................................................................................ 768
22.5.4 Macrophage Inhibition by GED Released from Microspheres................................. 768
22.5.5 Suppression of Cell Infiltration into Hydrogel in Mice Full-Thickness
Transcutaneous Wound Models................................................................................. 772
739
22.6 Conclusions............................................................................................................................ 773

Abbreviations.................................................................................................................................. 774
References....................................................................................................................................... 774
22.1 INTRODUCTION
Repair of wounds after trauma or surgery is of significant clinical and research importance. Wound
healing is a dynamic interactive process; it typically evolves from an initial inflammatory response,
and progresses through various stages to finally achieve complete resolution [1]. The capacity of
an implantable to maintain a moist environment is recognized as an important factor in normal
wound repair. It is recognized that moisture retention is very important in burn injury as heavy
water loss from the wound bed by exudation and evaporation may lead to a fall in body temperature
and increase in the metabolic rate. Hydrogels, with their high water retention capacity, appear to
be optimal media to assist wound healing [2]; thus, much interest has been focused on developing
hydrogel-based wound dressings from biomaterials [3–5].
Hydrogels are three-dimensional, hydrophilic, polymeric networks capable of maintaining large
amounts of water or biological fluids [6]. Injectable, flowable, and in situ gelable systems have
received much attention in tissue engineering because of several advantages. First, this type of
in situ gel-forming matrix, initially a fluid and readily conforms to the surrounding, could be directly
introduced through a needle/catheter and thereby obviates surgical implantation [7]. Second, thera-
peutic agents, such as cells, drugs and growth factors, could be incorporated into the matrix by
simple pre-mixing. Third, the aqueous in situ gel-forming matrix is completely devoid of residual
solvents as compared with other systems formed by various processing methods [8].
Among all the materials proposed for preparation of hydrogels, natural polysaccharides col-
lectively represent a class of abundant renewable resources; they are advantageous over synthetic
polymers in biocompatibility and biodegradability. Moreover, many of these carbohydrates are clas-
sified by the U.S. FDA as GRAS (Generally Regarded As Safe). Another widely used GRAS polymer
for the preparation of hydrogel is gelatin, which is also a renewable resource. In recent years, many
of these natural polymers, such as hyaluronan, dextran, and chitosan, have been utilized to formu-
late hydrogels, and their applications in drug delivery, tissue engineering, and gene therapy have
been extensively investigated.
22.1.1 Hyaluronic Acid (HA)

Hyaluronan (HA) is a linear non-sulfated polysaccharide consisting of alternating β-1, 4-linked
units of β-1, 3-linked glucuronic acid and N-acetyl-d-glucosamine (Figure 22.1a). It presents mainly
in human connective tissues such as cartilages and in the vitreous, where its primary role is to
lubricate body tissue and block the spread of invading microorganisms [9]. HA is also a main con-
stituent of the extracellular matrix (ECM), which is known to play important roles in both cell dif-
ferentiation/growth and wound repair [10,11]. Due to the remarkable viscoelastic properties of HA
and its lack of immunogenicity, it is widely used in ophthalmologic surgery and in orthopedics for
the treatment of joint diseases. In addition, HA inhibits both platelet adhesion and aggregation, and
stimulates angiogenesis, making it attractive for vascular applications [12].
Native HA is very soluble and can be degraded rapidly in vivo by free radicals or enzymes
such as hyaluronidase [13]. Various crosslinking techniques aimed at enhancing HA’s longevity
and physicochemical properties without compromising its biocompatibility have been developed to
formulate hydrogels, films and microspheres [14–18]. The crosslinkers generally used are diepoxide
[14], disulfide [16], glycidyl ether [19], glutaraldehyde [20], and water-soluble carbodiimides [15].
HA hydrogels and their variants have attracted great interest as new biocompatible and biodegrad-
able materials with applications in drug delivery and tissue engineering. Much effort has also been
made to develop in situ gelable HA hydrogels; a system was produced by chemical crosslinking of
Carbohydrate-Derived Self-Crosslinkable In Situ Gelable Hydrogels 741
O O– OH
C
O O
HO O
HO O
OH NH
O C CH3
n
(a)
O
HO
HO
OH
O
n
(b)
OH OH
O
O
O O
HO HO
NHCOCH3
NH2 x y
(c)
FIGURE 22.1 The structures of (a) hyaluronan, (b) dextran, and (c) chitosan.
adipic dihydrazide-derivatized HA and periodate oxidized HA [21]. Simple mixing of the polymer
solutions with pre-dissolved bupivacaine resulted in producing a prolonged local anesthetic effect
of the drug [21].
Extracellular matrix HA is prominent in both fetal development and the early phases of wound
healing, especially in fetuses where the wound repair is scar-free [22]. In contrast, accumulation of
HA is reduced in adult tissues, where wound healing is characterized by scarring, or in keloids char-
acterized by exuberant collagen deposition [23]. The suggestion that extracellular HA might prevent
fibrosis have prompted the investigations on the effect of HA derivatives and hydrogels on promot-
ing wound healing [24–26]. Moreover, thiol-modified hyaluronan hydrogels have been developed to
slowly release bFGF, and the results showed that the bFGF released from the hydrogel dramatically
stimulated blood vessel formation [25].
22.1.2 Dextran
Dextran is a bacterial derived polysaccharide comprising α-1,6 linked d-glucopyranose as well
as some α-1,2-, α-1,3- or α-1,4-linked side chains [27,28] (Figure 22.1b). Other than exceptional
biocompatibility, dextran is also biodegradable; it has been used as a blood volume expander and
for purification of biologics [29]. Dextran has been widely used in the biomedical field to formulate
hydrogels for sustained protein (lysozyme, albumin, immunoglobulin G) and drug delivery [30–33].
A methacrylate moiety is the key feature of these dextran derivatives and they are photocrosslinked;
the hydrogels formed swell in aqueous media and are partially hydrolyzable by dextranase. Drugs
can be incorporated into dextran hydrogels and the versatility of this type of matrices renders it pos-
sible to modulate the rate of drug release.
Heparin-like dextran derivatives can interact with heparin-binding growth factors FGF-1, FGF-2
and TGF-b, protecting them from proteolytic and enzymatic degradations; this maneuver also
favors the binding to their high-affinity tyrosine kinase receptors [34–36]. They also exhibit both
in vitro and in vivo anti-complement properties [37] and anticoagulant activities [38]. Regenerating
agent (RGTA) dextran polymers have been shown to promote several tissue-repair processes by
stimulating cell growth, differentiation or migration in various in vitro and in vivo models, includ-
ing muscles [39–41], skin [42], gingival tissue [43,44], and bone [45]. Moreover, part of the biologi-
cal effects of the RGTA family is attributable to their influence on extracellular matrix remodeling
either by modulating collagen biosyntheses [43,46] or by regulating the enzyme activities of matrix
metalloproteases [42,43,47], plasmin and cathepsin G [48,49]. For example, film containing glycidyl
methacrylate-derivatized dextran/acrylic acid nanoparticles loaded with an antibacterial agent has
been prepared and used as an investigational wound dressing material [50]. This film not only main-
tains a moist environment over a wound bed but also provides a continuous and sustained release
of the antibacterial agent.
22.1.3 Chitosan
Chitosan is a partially de-acetylated derivative of chitin found in arthropod exoskeletons. It com-
prises α-1, 4-linked d-glucosamine residues with a variable number of randomly located N-acetyl-
glucosamine groups [51] (Figure 22.1c). Chitosan is approved by the FDA as a biocompatible,
biodegradable, non-toxic, and bioresorbable biomaterial [52–55], and it has been widely utilized in
many fields including pharmaceutics, tissue engineering, food processing, textiles, and so on [56–59].
Chitosan is a semi-crystalline polymer and its crystallinity is dependent upon the degree of deacet-
ylation; the crystallinity is the maximum for both chitin (i.e., 0% deacetylated) and fully deacety-
lated (i.e., 100%) chitosan, with the minimum crystallinity at intermediate degrees of deacetylation.
Due to its stable crystalline structure, chitosan is insoluble in aqueous solutions above pH 7 [51].
Various chemical modifications have been employed to enhance the aqueous solubility of chitosan;
some examples are reductive amination with phosphorycholine-glyceraldehyde [60], sulfation [61],
carboxymethylation [62], carboxyethylation [63], and N- or O-acylation [64,65]. Amongst these,
introducing the highly hydrophilic acrylic acid into the chitosan chains through Michael’s reaction
is both facile and effective to render it soluble in water [63].
Chitosan has been reported as a wound-healing accelerator [66]. At the inflammatory stage,
infiltrating neutrophils cleanse the wound of foreign agents, chitosan accelerates the infiltration of
inflammatory cells, consequently accelerating wound cleansing; various chitosan hydrogels have
been developed for wound dressing. A photocrosslinked chitosan hydrogel with hemostasis prop-
erty was formulated and used as a dressing for wound occlusion and tissue adhesive [67]. Moreover,
chitosan hydrogel with epidermal growth factor (EGF) incorporated was developed and used in
second-degree burn wounds in rat models aiming to accelerate epithelialization [68].
22.1.4 Gelatin
Gelatin is a collagen-derived protein with unique gelation properties; due to a partial recovering of
the triple-helix conformation of native collagen [69], gelatin is a liquid above its gelation tempera-
ture (tgel) while a gel below it.
Gelatin exhibits several advantages; it is amphipathic and bioresorbable and its physicochemi-
cal properties can be easily modified [70–72]. However, gelatin hydrogels melt within the body
temperature range, and it is susceptible to in vivo degradation by gelatinases. The rapid degradation
of gelatin does not afford sufficient functional longevity to its structural properties in vivo, thereby
limiting its utility in biomedical applications. Much effort has been devoted to modulate the physical
properties of gelatin hydrogels via chemical crosslinking [73–77]; both glutaraldehyde and formal-
dehyde have been employed as crosslinking agents to reinforce this protein. This type of treatment
increases gelatin’s resistance to enzymatic degradation [78–80] by masking the specific enzymatic
recognition sites; however, the disadvantage for this approach is a reduction in biocompatibility [81].
Recently, non-cytotoxic crosslinking methods mediated by transglutaminases have been developed.

Transglutaminases are a class of natural enzymes capable of catalyzing the acyl-transfer reaction
between the amino group of lysine and the carboxyamide group of glutamine on proteins [70,82,83].
In particular, calcium-independent microbial transglutaminase is able to catalyze the formation of
covalent crosslinking between glutamine and lysine residues [84,85].
Gelatin is typically used in wound dressing products instead of collagen as the immunogenic
potential of the former is generally lower than that of the latter [86–88]. The high absorptive capac-
ity of gelatin hydrogel prevents fluid accumulation; excess wound fluids and cell debris would be
absorbed and retained inside the hydrogel applied. Tissue ingrowth could also take place in the bio-
degradable gelatin matrix with the regenerated tissue and implant fully integrated. Furthermore, EGF
has also been incorporated into the gelatin hydrogel wound dressings for efficacy enhancement [89].
In this chapter, we summarize the investigations conducted on several different in situ hydrogel
systems composed of the above four biomacromolecules. The physicochemical properties of these
hydrogels and their variants are described in detail; the efficacies of these hydrogels in various
in vivo experimental models are also revealed.
22.2 ACRYLATED CHITOSAN/OXIDIZED DEXTRAN

FOR ACCELERATION OF WOUND HEALING [90]
Native chitosan is insoluble in organic solvents but can be dissolved in aqueous dilute acid; this
aspect renders chitosan relatively difficult to process by conventional industrial methods, thereby
limiting its versatility in biomedical applications. Introducing carboxyethyl groups into the polymer
chain enables chitosan to dissolve in water. Polysaccharides generally degrade relatively rapidly
and do not have the endurances of synthetic polymers; crosslinking of polysaccharide chains can
enhance their stability and thus durability. Various methods on utilizing small molecule cross-
linkers for formulating chitosan hydrogels have been reported [27,91–94]; however, cytotoxicity
potential are known to associate with many small molecule crosslinkers. Furthermore, the slow
gelation times of some of these hydrogel formulations limits their applicability when rapid in situ
gelation is required.
Oxidizing dextran with periodate is a classic method to functionalize dextran, and this maneuver
produces a macromolecular crosslinker with multiple functional aldehyde groups capable of reacting
with those polymers bearing free amino groups to form hydrogels [95]. Water-soluble carboxyethyl
chitosan (CEC) was crosslinked with oxidized dextran (Odex), and the structure, cytotoxicity, bio-
degradability, and gelation mechanism of the Odex/CEC system were investigated; the gelation time
of the hydrogel produced could be modulated and it was both non-cytotoxic and biodegradable. The
hydrogel’s efficacy in enhancing the healing of murine full-thickness dermal wounds was validated.
22.2.1 Modification of Dextran and Chitosan

The vicinal hydroxyl groups of dextran were modified with periodate to form aldehyde groups. As
summarized in Table 22.1, the actual oxidation degrees of periodate-treated dextrans as determined
by 1H NMR with the theoretical oxidation degrees of 5%, 15%, and 25% were in actuality 4.6%,
7.4%, and 15.8%, respectively, and the corresponding molecular weights determined by HPLC were
48.1, 38.5, and 36.8 kDa, respectively. The substitution degree of CEC was determined as approxi-
mately 45% by 1H NMR. Introducing carboxyl groups into the structure of chitosan partially dis-
rupted the strong hydrogen bonds of native chitosan, rendering it water-soluble. Mixing Odex and
CEC readily formed transparent hydrogels that gelled rapidly, and the gelation time could be modu-
lated by varying the formulation parameters, including polymer concentration and temperature.
The mechanism was proposed in our previous work [96]; essentially, the aldehyde groups on Odex
formed Schiff base with the free amino groups on the CEC, leading to the formation of a 3-D cross-
linked network structure (Figure 22.2).
TABLE 22.1
Oxidation Degree of Odex Determined from 1H NMR and Number
Average of Molecular Weight (Mn) Estimated by GPC
Theoretical Oxidation Degree
Oxidized Dextran Oxidation (%) 1H NMR (%)a Mn (kDa) Gelling Time (s)b
Odex-I 5 4.6 48.1 359 ± 5
Odex-II 15 7.4 38.5 103 ± 4
Odex-III 25 15.8 36.8 79 ± 4
Source: Modified from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-
cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dex-
tran, 3905–3913. Copyright 2008, with permission from Elsevier.
a Calculated by integrating the anomeric proton peak at δ 4.8 ppm and the tert-butyl proton peak at δ
1.3 ppm.
b Gelling time for 2.5% solutions of Odex and 2.5% CEC solutions in 0.01 M PBS at 37°C.
OH OH OH
O O O O O
O O
O + HO NH
O NH2 HO HO NH
O n x y z
O
Oxidized dextran N-Carboxyethyl chitosan O
OH
OH OH OH
O O O O O
O
HO N x HO NH NH
y HO z
O
O
Schiff base
O O
n O
N
O H
H
OH O
O
O HN O Hydrogen bonding
HO x O
OH
O
HN
HO Oy
O OH
O
HO z
Oxidized dextran crosslinked N-carboxyethyl chitosan
FIGURE 22.2 Schematic presentation of the reaction between Odex and CEC. (Reprinted with permission
from Weng, L., Chen, X., and Chen, W., Rheological properties of in situ crosslinkable hydrogels from dextran
and chitosan derivatives, Biomacromolecules, 8(4), 1109–1115. Copyright 2007 American Chemical Society.)
22.2.2 Rheological Analysis of Hydrogel Formation

Figure 22.3 showed the variation of the elastic (G′) and viscous moduli (G″) as well as the complex
viscosity (η*) versus time for a 2.5% Odex/CEC (ratio 5:5) preparation. With G′ lower than G″, the
mix initially exhibited the behavior of a viscous fluid corresponding to a sol. Both moduli elevated
rapidly, and the buildup rate of G′ was considerably higher than that of G″ attributable to the rapid
103 105
G΄
G˝
104
η*
102
103
G΄ and G˝ (Pa)
η* (Pa.s)
101 102
101
100
tgel
100
0 1000 2000 3000

t (s)
FIGURE 22.3 Time evolution of the storage modulus (G′) and loss modulus (G″) for 2.5% (w/v) Odex-III/CEC
(ratio 5:5) at 37°C. The time where G′ and G″ crossover is denoted as tgel (the gelation point). (Reprinted from
Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels com-
posed of N-carboxyethyl chitosan and oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
occurrence of crosslinking. G′ and G″ eventually intersected (t = tgel, where tgel is the gel point),
indicative of the transition of the Odex/CEC system from a viscous behavior-dominated liquid-
phase to an elastic behavior-dominated solid-phase [97]; eventually, both moduli leveled off, imply-
ing the formation of a well-developed three-dimensional network. The η* also underwent a similar
transformation. The tgel for this particular formulation was approximately 78 s and the gelation
speed/rate could be modulated [96]. The relationship between the gelation time and the oxidation
degree of Odex is shown in Table 22.1. Using Odex solutions and CEC solutions at 2.5% concentra-
tion, the gelation time rapidly decreased with increase in the oxidation degree of the Odex (from
5% to 25%), indicating that gelation was strongly correlated with the abundance of aldehyde groups.
22.2.3 Morphology of the Hydrogels

The Odex/CEC hydrogel was transparent and yellowish (Figure 22.4a) with its color intensity increasing
gradually with time. Figure 22.4b depicts the SEM image of a fractured lyophilized hydrogel prepared
50 μm
(a) (b)
FIGURE 22.4 (See companion CD for color figure.) 2.5% (w/v) Odex-III/CEC (ratio 5:5) hydrogels: (a) the
photographic image and (b) the SEM image (b); scale bar: 50 μm. (Reprinted from Biomaterials, 29, Weng, L.,
Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl
chitosan and oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
from a 2.5% Odex/CEC (ratio 5:5) mixture, revealing a highly porous and interconnected interior struc-
ture; the average pore size was approximately 50 μm. It could be inferred that the hydrogel had a high
water retention capacity, and both small and macromolecules could diffuse into and out of the structure.
22.2.4 Swelling Properties of the Hydrogels

Figure 22.5 showed the relationship between the swelling ratio (q) of the Odex/CEC hydrogels prepared
from a 2.5% Odex/CEC (ratio 5:5) mixture. q initially decreased relatively rapidly with an increase in
the Odex content from 30% to 50%; this was followed by a slight decline when the Odex content was
increased to 60%; and finally, q increased when the Odex content was increased to 70%. This observa-
tion was attributed to the variation of the crosslinking density in the hydrogels, with an elevation of the
Odex content. As depicted in Table 22.1, when the Odex content was in the range of 50%–60%, the
theoretical ratio of the –CHO on the Odex to the –NH2 on the CEC was approximately 1 (i.e., mixing
the 2.5% Odex and 2.5% CEC solutions in equal volume); it could be inferred that the crosslinking
density increased as the Odex content of the hydrogel increased from 30% to 50% (with –NH2 groups in
excess), leading to a decrease in the swelling ratio. When the crosslinking density reached its maximum
(50%–60%), q was at the minimum. An excess of –CHO from an increase in the Odex content resulted
in the lowering of the degree of crosslinking, assuring a higher swelling ratio.
22.2.5 In Vitro Hydrolytic Degradation of the Hydrogels

Degradation of the Odex/CEC hydrogels was dependent upon the relative amount of Odex and CEC used
for the formulation. In vitro degradation of various 2.5% Odex/CEC hydrogels (ratio 3:7, 5:5, and 7:3)
was conducted by monitoring their weight losses. The results shown in Figure 22.6 indicates that all
hydrogels underwent relatively fast weight loss initially with the rates of degradation moderated thereaf-
ter, implying that they were degradable hydrolytically. The 7:3 formulation degraded considerably faster
than the 5:5 formulation, which had the lowest degradation rate. The rate of hydrogel degradation was
40
35
Swelling ratio q
30
25
20
30 40 50 60 70
Odex content (%)
FIGURE 22.5 Dependence of swelling ratio of Odex-III/CEC hydrogels (ratio 5:5) on the Odex content
of the hydrogel. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-
cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dextran, 3905–3913.
60
Weight loss (%)

40
20
3:7
5:5
0
7:3
0 1 2 3 4 5
Time (week)
FIGURE 22.6 Hydrolytic degradation of three Odex-III/CEC hydrogels. (Reprinted from Biomaterials, 29,
Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of
N-carboxyethyl chitosan and oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
dependent upon the crosslinking density [98]; greater crosslinking typically resulted in forming more
stable hydrogels. The 5:5 formulation had the highest crosslinking density and degraded the slowest; the
7:3 formulation degraded slower than the 3:7 formulation despite their comparable theoretical crosslink-
ing densities. The direct implication was hydrogel degradation was dependent upon both the breakdown
of the crosslinking as well as the stability of both Odex and CEC.
22.2.6 Cytotoxicity of the Hydrogels and Cell Viability Analysis

The cytotoxicity and cell viability of the Odex/CEC (3:7, 5:5, and 7:3) hydrogels were evaluated by seed-
ing cells in either the presence or absence of the hydrogel. Cell viabilities were determined by MTS assay
on days 3, 7, 12, and 30, respectively, and the results were depicted in Figure 22.7. The hydrogel did not
appear to affect cell growth as there was no detectable difference in the viabilities between the cells incu-
bated with the hydrogels and the control within 1 week. However, on days 3 and 7, the medium of the 3:7
1.2
3:7 Odex/CEC
5:5 Odex/CEC
1.0 7:3 Odex/CEC
Absorbance at 490 nm
Control
0.8
0.6
0.4
0.2
0.0
Day 0 Day 3 Day 7 Day 12 Day 30
Time (day)
FIGURE 22.7 MTS assay of the cell viabilities in the presence of 3:7, 5:5, and 7:3 Odex-III/CEC hydro-
gels compared with monolayer cells control by the indirect contact method. (Reprinted from Biomaterials, 29,
Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of
N-carboxyethyl chitosan and oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
Odex/CEC hydrogel showed a slightly lower absorbance than those of the other two hydrogels (5:5 and
7:3) as well as the control; this could be attributed to the excess carboxyl functionalities in the presence
of excess CEC. After 30 days of culture the obtained results showed that the extent of cell proliferation
was comparable to that of the controls, further confirming the hydrogel’s non-cytotoxicity; it could also
be inferred that the byproducts from hydrogel degradation were nontoxic. Hydrogels were broken down
by the enzymes produced by the cells, evidently due to degradation of the polymer network [99].
22.2.7 Cell Encapsulation in Hydrogels

Dermal fibroblasts were encapsulated by the Odex/CEC hydrogel. Cells entrapped by the hydro-
gel initially assumed round shapes (Figure 22.8a), and they were different from the cells residing
H
H
100 μm 50 μm
(a) (b)
H
H
100 μm 50 μm
(c) (d)
100 μm 200 μm
(e) (f )
FIGURE 22.8 Morphologies of cells encapsulated in the Odex-III/CEC (ratio 5:5) hydrogel. (a) Day 3,
(c) 1 week, (e) 2 week, (b and d) 1 week stained with crystal violet, and (f) 2-D control. : cell assuming
round shape, : spread out cells, and H: hydrogel. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A.,
Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and
oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
200 μm 200 μm
(a) (b)
FIGURE 22.9 (See companion CD for color figure.) Live/Dead™ staining of the cells encapsulated in the
Odex-III/CEC hydrogel. (a) Day 3; (b) 1 week. : live cells; : dead cells; *: spread out cells, H: hydrogel.
(Reprinted from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic, in situ gel-
able hydrogels composed of N-carboxyethyl chitosan and oxidized dextran, 3905–3913. Copyright 2008, with
permission from Elsevier.)
on the 2D culture with their typical spiny shapes (Figure 22.8f). Cells did not initially adhere to
the hydrogel, some of the cells started to adhere after 1 week as indicated by them assuming oval/
or rod shape (Figure 22.8b through d). Figure 22.7c showed the living cells; they were spreading
out to form clusters indicating proliferation. Cell-laden hydrogels were fixed with ethanol (70%)
and stained by crystal violet (Figure 22.8b and d), the cells and their nuclei could readily be iden-
tified (“ ”); they were evidently adapting to the hydrogel surrounding them several days after
incubation. Fibroblasts secrete and deposit extracellular matrix (ECM) on polymers [100,101].
The ECM rendered the hydrogel more amenable to cell growth, functioning and cell–cell interac-
tions [102]. The cell density steadily increased with time (Figure 22.8e), and the cells clearly were
proliferating in the hydrogel network. Eventually, cells broke down the hydrogel via the degrada-
tive enzymes they produced, further confirming the biodegradability of the hydrogel network.
Live/Dead™ staining was performed to differentiate the living cells (green) from the dead cells
(red). Figure 22.9 depicted the fluorescent microscopy of stained cells residing in the 2.5% Odex/
CEC (ratio 5:5) hydrogels. Cells maintained their characteristic round shape on day 3 (Figure 22.9a)
with nearly 95% cells alive (green color); they started to spread out (marked with “*”) by day 7 and
this was in good agreement with the results shown in Figure 22.8. The viability of cells was not
affected by their entrapment inside the hydrogel.
22.2.8 Acceleration of Wound Healing in Murine

Transcutaneous Full-Thickness Wounds
In situ forming hydrogels capable of fully conforming to wound beds are advantageous over preformed
scaffolds. Chitosan is known to be both biocompatible and bacteriostatic [103] and it has been shown to
promote wound healing [104,105]. The precursor of the 2.5% Odex/CEC mix (5:5) was applied to mouse
full-thickness transcutaneous wound models (n = 7 per group) to form hydrogel in situ. Figure 22.10
showed the typical wound beds shortly after the procedure and closure with Tegaderm™. The histologi-
cal sections of two typical wound beds after 7 days were depicted in Figure 22.11. Figure 22.11a showed
the histology of a typical wound bed implanted with the Odex/CEC hydrogel, its counterpart receiv-
ing PBS was depicted in Figure 22.11b. The implant showed evidence of partial degradation, and the
granulation tissues were present in the dermis of both groups; the infiltrated inflammatory cells were
more abundant in the group receiving the Odex/CEC hydrogel. The degrees of healing were quanti-
fied as% granulation tissue formation and% re-epithelialization. The extent of granulation tissue
(a) (b)
FIGURE 22.10 (See companion CD for color figure.) Wound beds of mice covered by Tegaderm™. (a)
PBS treated, and (b) Odex/CEC hydrogel treated. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A.,
Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and
oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
formation in the wound bed with the hydrogel was 84.9% ± 9.0% (Figure 22.11a) as compared with
the 46.3% ± 8.3% for the control (Figure 22.11b) (p < 0.001). Granulation tissue fills the wound bed
and set the stage for re-epithelialization [2]. Application of the Odex/CEC hydrogel resulted in 100%
re-epithelialization, as compared with the counterpart treated with PBS (54.3% ± 8.4%, p < 0.001).
22.3 GELATIN/OXIDIZED HYALURONAN (AMENABLE TO CELL MIGRATION)

Both collagen and HA are the major components of the extracellular matrix (ECM); impairment
of ECM is a fundamental cause of many soft tissue dysfunctions. Hydrogels composed of HA and
gelatin could serve as transient substitutes for impaired soft tissues. HA is inherently rigid due to the
presence of β-1,4 backbone linkages and repeated pyranoid rings; therefore, HA hydrogels are brittle.
Partial oxidation of HA introduces open rings into its structure which is a strategy to circumvent the
chain rigidity issue, thereby improves the elastic properties of the HA hydrogels formed [18]. Gelatin
melts at body temperature and this feature limits the potential biomedical applications of pure gelatin
hydrogels. Chemical crosslinking has been extensively utilized as a strategy to modulate the physical
properties of gelatin hydrogels [69,75,76]. Hydrogels composed of HA and gelatin have been formu-
lated by small molecule crosslinker-mediated reactions [77,106]. Here we describe an approach that
does not require any extraneous crosslinker and the hydrogel formulated is very amenable to deep
cell infiltration; this is in contrast to other comparable systems where cell attachment occurs mostly
on the surface of the hydrogels [77]. These temperature-insensitive self-crosslinkable hydrogels were
formulated from partially oxidized HA (oHA) and gelatin in the presence of borax and their physi-
cal properties were characterized; dermal fibroblast and mice subdermal models were also used to
characterize their in vitro and in vivo interactions, respectively.
22.3.1 Preparation of oHA and Formulation of oHA/Gelatin Hydrogels

Figure 22.12a depicted the modifications of HA’s hydroxyl groups to aldehydes and the opening of
the sugar ring to form a linear chain [18]. The degree of oxidation could be controlled by adjusting
the feed ratio and it was estimated by periodate consumption [107]; 1H NMR on tert-butyl carbazate–
treated oHA was performed for validation [18]. The NMR spectra of both HA and oHA were depicted
Re-epithelialization
Hydrogel residual
Hydrogel
residual
Granulation tissue
(a)
Granulation tissue
(b)
FIGURE 22.11 (See companion CD for color figure.) Acceleration of wound healing by Odex/CEC hydro-
gels in mouse full-thickness dermal wounds. (a) A representative wound bed implanted with hydrogel with
both robust epidermal hyperplasia and granulation tissue formation. Part of the implanted hydrogel was frac-
tured and neo-epidermal tissue apparently developed transversely along the side of the hydrogel fragment. (b)
A representative wound bed treated with PBS (no hydrogel). Moderate epidermal hyperplasia and granulation
tissue formation were observed. The images were captured under 20 × magnification, whereas the insets were
captured under 200 × magnification. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J.,
and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized
dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
in Figure 22.13, the peaks at 1.87 and 1.32 ppm were from the methyl groups on HA (3H) and tert-
butyl groups from oHA (9H, denoted by “*” in Figure 22.13), respectively [18]. The oxidation degree
(Table 22.2) increased from 16.7% to 44.4% when the periodate:HA ratio was changed from 20% to
70%; the corresponding average molecular weight of oHA decreased from 183.8 to 35.5 kDa. The
FTIR spectra of native HA (Figure 22.14a) and oHA (Figure 22.14b) confirmed oxidation; the peak at
1735 cm−1, emerged as a shoulder on the oHA spectrum, was attributed to the symmetric vibration of
aldehyde groups on the oHA [108].
Figure 22.12b depicted the reaction between oHA and gelatin, the presence of borax (a FDA
GRAS material) favored Schiff base formation [75,109,110]. Various formulations composed of
OH OH
COOH COOH
O HO NaIO4 O HO
O O O O O
O
HO
OH O
NH O NH
O O
CH3 CH3
(a) HA oHA
OH OH
COONa COONa
O HO
O O HO
O O O O
O
O NH O
O N NH
O
O
CH3
H3C CH3
oHA
0.1 M borax O N
+
HN N
NH2 NH2
O O O
NH2 OH O
COOH
OH
(b) Gelatin oHA/Gelatin gel
FIGURE 22.12 (a) Oxidation of HA by sodium periodate and (b) crosslinking reaction between oxidized
HA and gelatin. (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed of partially
oxidized hyaluronan and gelatin: In vitro and in vivo responses. J. Biomed. Mater. Res. 2008. 85A. 352–365.
Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
(b)
*
(a)
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm
FIGURE 22.13 1H NMR spectra of (a) HA and (b) oHA treated with tert-butyl carbazate. * denotes the peak
of tert-butyl groups (9H). (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed of
partially oxidized hyaluronan and gelatin: In vitro and in vivo responses. J. Biomed. Mater. Res. 2008. 85A.
352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
TABLE 22.2
Oxidation Degree of oHA Prepared
Oxidation Degree Oxidation Degree
Preparation (Theoretical) (%) (Experimental) (%) Mn (kDa)
1 20 16.7 183.8
2 30 20.3 71.1
3 40 23.4 57.8
4 50 27.8 41.2
5 70 44.4 35.5
Source: Modified from Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydro-
gels composed of partially oxidized hyaluronan and gelatin: In vitro and in
vivo responses. J. Biomed. Mater. Res. 2008. 85A. 352–365. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA Reproduced with permission.
Note: Calculated by NMR and number average of molecular weight (Mn) mea-
sured by HPLC.
(d)
(c)
1646 cm–1
1544 cm–1
(b)
1735 cm–1
(a)
4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm–1)
FIGURE 22.14 FT-IR spectra of (a) HA, (b) oHA, (c) oHA/gelatin hydrogel, and (d) gelatin. (From Weng, L.,
Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed of partially oxidized hyaluronan and gelatin:
In vitro and in vivo responses. J. Biomed. Mater. Res. 2008. 85A. 352–365. Copyright Wiley-VCH Verlag
GmbH & Co. KGaA. Reprinted with permission.)
oHA of different oxidation degrees and different gelatin contents were prepared (see Table 22.3)
and evaluated. The FTIR spectra of a lyophilized oHA/Gelatin hydrogel and native gelatin were
depicted in Figure 22.14c and d, respectively. The two absorption peaks (3300 and 1650 cm−1) in
Figure 22.14d were the N–H and the C=O stretching, respectively [18]. The peaks at 1646 and
1544 cm−1 were stretching of the C=N bonds [111], indicating crosslinking.
TABLE 22.3
Characteristics of the oHA/Gelatin
Hydrogels Used
Oxidation Degree
Hydrogel of oHA (%) oHA:Gelatina
oHG-1 16.7 5:5
oHG-2 20.3 5:5
oHG-3 23.4 5:5
oHG-4 27.8 5:5
oHG-5 44.4 5:5
oHG-6 27.8 4:6
oHG-7 27.8 6:4
Source: Modified from Weng, L., Pan, H., and Chen,

W.: Self-crosslinkable hydrogels composed of par-
tially oxidized hyaluronan and gelatin: In vitro and
in vivo responses. J. Biomed. Mater. Res. 2008. 85A.
352–365. Copyright Wiley-VCH Verlag GmbH &
Co. KGaA. Reproduced with permission.
a The concentrations of oHA and gelatin solutions used are
20% (w/v) each.
22.3.2 Hydrogel Morphological Analyses

The typical cross-sectional SEM images of three typical lyophilized hydrogel formulations (oHG-4,
oHG-7 and oHG-8) (oHA of 27.8% degree of oxidation) were depicted in Figure 22.15. The struc-
tures were highly porous (average pore size: ∼60 μm), implicating the materials’ potential to accom-
modate cell migration. The hydrogels formulated from oHA:gelatin at the ratios of 4:6 (oHG-7) and
(a) (b)
10μm 10μm
(c) (d)
10μm 10μm
FIGURE 22.15 SEM images of lyophilized hydrogels: (a) oHG-8, (b) oHG-4, (c) oHG-7, and (d) 20% (w/v)
pure gelatin hydrogel. Scale bar: 10 μm. (From Weng, L., Pan, H., Chen, W: Self-crosslinkable hydrogels
composed of partially oxidized hyaluronan and gelatin: In vitro and in vivo responses. J. Biomed. Mater. Res.
2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.)
6:4 (oHG-8) resulted in less compact structures (Figure 22.15a and c), as compared with oHG-4 (5:5)
(Figure 22.15b), suggested the highest crosslinking density at a ratio 5:5. The structure of pure gela-
tin hydrogel was shown in Figure 22.15d for comparison with its highly porous network attributable
to its triple helical physical crosslinks [69].
22.3.3 Swelling Properties of Hydrogels

Figure 22.16 depicted the correlation between hydrogel swelling and the oxidation degrees of oHA
(Figure 22.16a) and the gelatin contents (Figure 22.16b). The swelling ratio (q) sharply declined with
the increase of the oxidation degrees of oHA from 16.7% to 23.4%; this was followed by a rapid mod-
eration when the oxidation degree of oHA was increased to 44.4%. Initial increase in free aldehyde
residues resulted in extensive Schiff base formation leading to the rapid drop in water uptake. As a
large amount of free amino residues on the gelatin was occupied, subsequent increase in the oxidation
degree of oHA resulted in substantially less Schiff base formation and thus the swift decrease of water
uptake. Lastly, with the bulk of the amino residues consumed, further increase in the oxidation degree
of oHA only resulted in a very modest additional decrease of water uptake. q reached its minimum at
a gelatin content of 50%, suggesting the greatest degree of crosslinking.
22.3.4 Rheological Analyses of Hydrogels

Figure 22.17a depicted the typical frequency sweep profiles of three representative hydrogel for-
mulations (oHG-3, oHG-4, and oHG-5) showing their viscoelasticities under periodic strain. Loss
moduli (G″) were weakly dependent on the frequencies in the range of 0.5–100 Hz; storage moduli
(G′) appeared to be constant. G″ was considerably larger than G′, indicating the dominance of the
elastic properties over the viscous properties, thereby, the presence of well-developed networks
[69,112]. G′ elevated from 3,600 to 10,000 Pa with the corresponding increase in the oxidation
degree of oHA from 23.4% to 44.4% (oHG-3, oHG-5), this was due to the increase in aldehyde
residues and therefore greater Schiff base linkages. The magnitude of elevation in the oxidation
degree of oHG-3/oHG-4 was 4.4% as compared with that of oHG-4/oHG-5 at 16.6%; however, the
extent of G′ increase for the former was considerably greater than that from the latter, suggesting the
30
30
25
Swelling ratio q
Swelling ratio q
20 20
15
10
10
0 5
10 20 30 40 50 30 40 50 60 70
(a) Oxidation degree (%) (b) Gelatin content (%)
FIGURE 22.16 Correlation of swelling ratio (q) of hydrogels on the oxidation degree of oHA (a) and gela-
tin content (b). (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed of partially
10,000
G' G''oHG-5
G' G''oHG-4
G' G''oHG-3 oHG-7
oHG-8
Storage and loss modulus
Storage modulus G' (Pa)

10,000 oHG-4
G', G'' (Pa)
1,000
1 10 100 100 1,000

(a) Frequency ω (Hz)
(b) Shear stress τ (Pa)
FIGURE 22.17 Rheological spectra of hydrogels: (a) Correlation of the storage modulus G′ and the loss
modulus G″ to the oscillating frequency of hydrogels. (b) Correlation of the storage modulus G′ on the oscilla-
tory shear stress of hydrogels. (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed
of partially oxidized hyaluronan and gelatin: In vitro and in vivo responses. J. Biomed. Mater. Res. 2008. 85A.
352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
consumption of most of the amino residues on gelatin when the oxidation degree of oHA reached
27.8% (i.e., oHG-4). These results were in strong agreement with those depicted in Figure 22.16.
The oscillation stress sweep profiles of three other hydrogel formulations (oHG-4, oHG-7, and
oHG-8) at 1 Hz were depicted in Figure 22.17b. The linear viscoelastic region (LVR) is the range
where G′ is independent of the applied stress [113]. Due to a slight slippage at high shear stress, The
G′s of oHG-4 and oHG-7 showed a moderate decrease with shearing stress increases; the critical shear
stress (i.e., breakdown, at the end of LVR) was different from the slippage-initiated decrease in G′. The
G′ was the highest for the oHG-4 hydrogel formulation composed of oHA (at 27.8% oxidation): Gelatin
at a 5:5 ratio; the breakdown shear stress was in the order of 5:5(oHG-4) > 4:6(oHG-7) > 6:4 (oHG-8),
suggesting that the key contributory role of the oHA:Gelatin ratio to the mechanical strengths of the
hydrogels. oHG-4 appeared to have the most optimal ratio for maximizing the interaction of aldehyde
and amino groups and thus resulting in a hydrogel with the greatest mechanical strength.
22.3.5 Three-Dimensional Infiltration and Distribution of Cells in Hydrogels

Sections of cell-laden hydrogels (∼400 μm thick) were stained with crystal violet. Cells attached to
all hydrogel formulations 1 day after seeding (Figure 22.18a) with cell numbers increased with time
(a) (b)
FIGURE 22.18 (See companion CD for color figure.) Cell attached to hydrogels (top view). (a) Day 1, and
(b) day 3 after cell seeding. Cells were stained with crystal violet. Scale bar: 20 μm. (From Weng, L., Pan, H.,
and Chen, W.: Self-crosslinkable hydrogels composed of partially oxidized hyaluronan and gelatin: In vitro
and in vivo responses. J. Biomed. Mater. Res. 2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH &
Co. KGaA. Reprinted with permission.)
(a) (b)
(c) (d)
(e) (f )
FIGURE 22.19 (See companion CD for color figure.) Cross sections of cell-laden hydrogel (oHG-7):
(a, b) 3 days, (c, d) 5 days, and (e, f) 5 days after cell seeding. Samples are stained with crystal violet (a, c, e)
and Live/Dead dye (b, d, f), respectively. The demarcation line marked as: S: Hydrogel surface, I: Hydrogel
interior, F: Fibroblasts, : dead cells (red), *: live cells (green). Scale bar: 20 μm. (From Weng, L., Pan, H.,
and Chen, W.: Self-crosslinkable hydrogels composed of partially oxidized hyaluronan and gelatin: In vitro
and in vivo responses. J. Biomed. Mater. Res. 2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH &
Co. KGaA. Reprinted with permission.)
(Figure 22.18b), eventually forming multiple cell layers. The cell attachment rate, the distribution pattern
and the numbers of cells attached were comparable in all hydrogel formulations. The hydrogels that were
more densely crosslinked had smaller pores and thus, slower cell infiltration. Cell infiltrated the fastest
into the hydrogels composed of a 4:6 oHA to Gelatin ratio (oHG-7), deep infiltration was self-evident
at 3 and 5 days after seeding (Figure 22.19a, c, and e). The highly elongated cell morphology observed
was distinctively different from the typical shapes of cells seeded on culture dishes. The arrays formed
were highly organized with the leading cells assuming spherical conformations while the trailing cells
were elongated. Both cell infiltration and their corresponding penetration depth increased with time
(compare Figure 22.19a and c); there was a corresponding decrease in material cohesiveness signifying
cell-mediated degradation and mechanical deterioration.
Both the structure and components of oHA/Gelatin hydrogels bear resemblance to the in vivo
environment. Cells attaching to hydrogels were described [114–116], but the robust cell infiltration
into the oHA/Gelatin hydrogel had not previously been unambiguously demonstrated. Methods
devised to induce cell ingrowth into hydrogels included pre-mixing cells in hydrogel precursor
solutions, integrating chemo-attractants into hydrogels, stamping and centrifugation were less than
optimal [117–121]. The majority of these studies involved embedding of cell-laden particles into
hydrogels and the out-migration of cells was inferred as cell infiltration [122,123].
Despite HA being a major component of ECM, cells do not infiltrate into the structures formed
by native HA [124]. Blending oHA with gelatin to form hydrogel formed a structure very ame-
nable to cell infiltration; the morphological/organizational patterns of cells resembled the in vivo
counterparts. The implication is that the extent of cell infiltration and the physicomechanical proper-
ties of the oHA/Gelatin could be modulated by adjusting the formulation parameters. These hydro-
gels have the potential to be applied as soft tissue substitutes, as scaffolds for tissue reconstruction,
and they could be modified as drug delivery vehicles, particularly for bioactive agents.
22.3.6 Long-Term Cell Viability in the Presence

of Hydrogels and In Vivo Biocompatibility
The viabilities of the cells infiltrated into the hydrogels were depicted in Figure 22.20. In general, all
samples reached full confluence in 1 week and both the hydrogels and their degradation byproducts
did not have any apparent effect on cell viability. The results obtained for all oHA/Gelatin hydrogels
were virtually identical and they further corroborated with those shown in Figure 22.19b, d, and f.
Fibroblasts produce and maintain ECM [125], and dysfunctional ECM protein distribution/deposition
are associated with many pathological conditions [126]. Deposition of ECM on the hydrogel 5 days after
cell seeding was demonstrated (Figure 22.21a and b); evidently, the pores inside the hydrogels where
cells attached were mostly covered by ECM and the structures where cells had not yet reached were
comparable to those of the cell-free controls (Figure 22.21c). The morphological changes of partially
degraded hydrogels were also examined (Figure 22.21c and d). The orderly porous structure of the
hydrogel surface had turned into fibrous structure suggesting surface erosion (Figure 22.21d), whereas
the interior of the hydrogel where cells did not reach remained intact (Figure 22.21c and d). HA and gela-
tin in their native states are rapidly degraded both in vitro and in vivo [127]. The cell-mediated degrada-
tion was a more accurate reflection of in vivo degradation as compared with in vitro degradation studies
conducted in either PBS or enzyme solutions [75,119]. Fibroblasts produce many enzymes including
0.9
oHG-1
0.8
oHG-2
0.7 oHG-3
oHG-4
0.6 oHG-5
Absorbance at 490 nm
oHG-7
0.5
Control
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4
Time (week)
FIGURE 22.20 MTS assay for testing cell viability on various hydrogel formulations compared with mono-
layer fibroblast control (absence of hydrogel). (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydro-
gels composed of partially oxidized hyaluronan and gelatin: In vitro and in vivo responses. J. Biomed. Mater.
Res. 2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
(a) (b)
20 µm 10 µm
(c) (d)
20 µm 10 µm
(e) (f )
100 µm
20 µm
FIGURE 22.21 In vitro (a, b, c) deposition of ECM, and in vitro cell-mediated degradation of oHG-6 hydro-
gel (d). (a, b) Pores of hydrogel are masked and filled with ECM at day 5 after cell seeding; : deposited ECM.
(a) hydrogel surface (scale bar: 20 μm), and (b) cross section of fractured hydrogel (scale bar: 10 μm); right
side: pores are masked by ECM; left side: unmasked pores; (c) interior of pristine hydrogel remained intact and
are not masked by cell deposited ECM (scale bar: 20 μm), and (d) morphology of hydrogel showing degrada-
tion (scale bar: 10 μm). Pore sizes on the surface are larger than those in the interior, indicating cell-mediated
degradation. S: Hydrogel surface; I: Hydrogel interior. In vivo (e, f) deposition of ECM. (e) The hydrogel
surface is completely covered by ECM (scale bar: 10 μm), and (f) the explanted hydrogel cross-sectional 1
week post-implantation (scale: 100 μm), *: unmasked pores are the tunnels created by cell infiltration, and :
deposited ECM. (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed of partially
40
35
30
Cells + medium
25 Medium
Time (day)
20
15
10
0
oHG-1 oHG-2 oHG-3 oHG-6 oHG-7 oHG-8 oHG-9
Gel sample
FIGURE 22.22 Cell mediated degradation time of various hydrogel formulations. Hydrogels in cell culture
medium are used as controls.
hydrolases such as hyaluronidase and collagenases, capable of degrading oHA/Gelatin. Cell infiltrated
into the hydrogel broke down the crosslinks and hastened the degradation leading to disintegration.
The degradation results of the hydrogels after 5 weeks of incubation with fibroblasts were shown
in Figure 22.22. Hydrogel degradation was defined as the time span from cell seeding to material
disintegration, signified by a loss of cohesiveness. The degradation rates were controlled by the
degree of oxidation of oHA and thus the crosslinking densities of the hydrogel. The disintegra-
tion time of the hydrogel formulations composed of 5:5 (oHA:gelatin, oHG-1, oHG-2 and oHG-3)
increased from 11 to 32 days when the oxidation degree of oHA was elevated from 16.7% (oHG-1)
to 23.4% (oHG-3). In contrast, the hydrogels formulated from oHA of 27.8% oxidation degree in dif-
ferent ratios (oHG-6(3:7), oHG-7(4:6), oHG-8(6:4), oHG-9(7:3)) had disintegrated in 7 days. These
results suggested that a greater degree of crosslinking was achieved at a ratio of 5:5, which appeared
to be the most optimal for material stability and hence resistance to degradation.
Hydrogels were implanted subcutaneously in mice. The gross appearance of the explanted mate-
rial and the adjoining tissues indicated that the hydrogels and their degradation byproducts were
non-cytotoxic; the inflammatory response typically induced by implantation was mild with no sign
of tissue necrosis. The implanted hydrogels were evidently degrading (reduced to approximately
half the pre-implant size) and appeared to be integrating with the adjoining tissues 3 days post-
implant. The hydrogels maintained their cohesiveness after 7 days; their sizes were further reduced
to approximately one-fourth of their pre-implant sizes and they were surrounded by very thin
fibrous capsules. After 3 weeks, the implants were almost fully resorbed, as indicated by a reduc-
tion of their sizes to approximately one-tenth of their initial sizes.
An explanted hydrogel was depicted in Figure 22.23. The hydrogel was surrounded by both neu-
trophils and macrophages 3 days after implantation (a, b); it was encapsulated in a thin fibrous layer of
connective tissue, typically observed in material implanted subdermally [128], 7 days after implanta-
tion. There were similarities between the extents of cell infiltration observed in both the in vivo and
in vitro studies (depicted in Figure 22.20). One week after implantation (c, d), both the hydrogel and
its surrounding capsule were populated by fibroblasts, with inflammatory cells (macrophages, neutro-
phils and mast cells) scattered throughout. Fibroblasts secrete hydrolases and macrophages secrete both
hydrolytic and oxidative enzymes in conjunction with its phagocytic activities were all contributory to
material degradation [129]. Three week after implantation (e, f), most of the hydrogel had apparently
(a) (b)
(c) (d)
(e) (f )
(g) (h)
FIGURE 22.23 (See companion CD for color figure.) Explanted hydrogels stained with H&E. (a, b) day 3,
(c, d) day 7, (e, f) day 21 post-implantation of hydrogels, (g) intact mouse skin, and (h) day 21 post-implantation
of Vicryl™ suture. C: Collagenous capsule, H: Hydrogel, D: Dermis, L: Loose connective tissue, : Macrophage,
: Fibroblast, and *: Suture. Scale: 20 μm. (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels
2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
been replaced by loose connective tissues (g), indicating full resolution of the implant site; the notice-
able absence of macrophages implied the inflammation had subsided. A thin collagenous capsule in
concert with a lack of foreign body giant cells suggested that the inflammatory response induced by the
implanted hydrogel was both mild and transient. For comparison, the tissue adjoining the implanted
poly-lactide-co-glycolide Vicryl™ sutures (as the benchmark) showed intense inflammation with con-
spicuous cell infiltration, they were enclosed by thick fibrous tissues (h).
Consistent with the histology depicted earlier, the SEM images of ECM deposition on the explanted
hydrogel (Figure 22.21e and f) suggested a more metabolically active in vivo environment resulted
in extensive new ECM formation on hydrogels and the original porous hydrogel structure could not
be easily distinguished 1 week after implantation (Figure 22.21e). For comparison, Figure 22.21f
depicted the cross section of the hydrogel, some of the “tunnels” (“*”) created by cells during their
infiltration could be identified. An orderly replacement of the h ydrogel by ECM suggested restora-
tion of the damaged implant site. A combination of robust host cell infiltration and ECM deposition
resulted in the hydrogels’ maintenance of their cohesiveness after implantation.
22.4 CARBOXYETHYL CHITOSAN/OXIDIZED DEXTRAN HYDROGEL

FOR PREVENTION OF POST-SURGICAL ABDOMINAL ADHESION
A great majority (>90%) of the patients undergoing abdominal surgery experience some form of
adhesions. Adhesions are the results of prolonged inflammation of injured tissues, fibroblast in-
growth and neovascularization leading to the formation of fibrous tissue [130], triggering symp-
toms including severe pain, bowel obstruction, and infertility [131]. Clinical products targeting
reduction of abdominal adhesions generally fall into two categories: non-absorbable and absorb-
able. Non-biodegradable polytetrafluoroethylene membranes are highly effective in preventing
adhesions; however, these membranes have to be surgically removed, thus, biodegradable barriers
are appealing alternatives. One of more prominent biodegradable products is a dry uncrosslinked,
carboxymethylcellulose/hyaluronan film [132], which forms a gel via absorbing moisture from the
surrounding tissues after deployment. The occlusive barrier formed between the site of injury is to
reduce adhesion formation without interfering with the repair of tissues under the barrier [133,134].
However, these films are brittle and also relatively difficult to apply as they easily adhere to any
moist surface, these drawbacks limit the acceptance of this type of film among surgeons [135].
A resorbable rapid in situ gelable hydrogel barrier that can be applied as a flowable fluid precur-
sor and capable of fully conforming to any complex geometries will be an effective transient bar-
rier, moreover, it is also compatible with endoscopic/laparoscopic procedures. We investigated the
feasibility of using the in situ gelable hydrogel formulation composed of partially oxidized dextran
(Odex) and N-carboxyethyl chitosan (CEC) [96] as a potential anti-adhesion barrier. There are two
main advantages for this system: (1) both chitosan and dextran have long safety records; (2) Odex
and CEC readily crosslink to form hydrogels at physiological pH/temperature by electrostatic inter-
action, hydrogen bonding, and eventually stabilize by Schiff base formation [136]. Odex serves as
a macromolecular crosslinker to react with CEC to form a macromolecular network obviating the
need of small molecule crosslinking agents, thereby, decreasing the cytotoxicity concerns [136]
(see Figure 22.2 for illustration of the hydrogel formation). The efficacy of this hydrogel system
was validated in a 21 day, rat cecum abrasion model and commercially available uncrosslinked,
carboxymethylcellulose/hyaluronan film was used as a benchmark for comparison.
The results of the rat cecum abrasion study were summarized in Table 22.4. The mean adhesion score
for the control animals was 2.1 ± 1.2. Animals treated with the carboxymethylcellulose/hyaluronan
TABLE 22.4
Anti-Adhesion Scores
Control Odex/CEC Carboxymethylcellulose/
Score (N = 11) Hydrogel (N = 9) Hyaluronan (N = 8)
0 2 3 0
1 1 4 7
2 2 1 0
3 6 1 1
Source: Modified from J. Surg. Res. Copyright (2009) (in press), with per-
mission from Elsevier.
film and the Odex/CEC hydrogel had adhesion scores of 1.3 ± 0.7 and 1.0 ± 1.0, respectively. The
adhesion score of the Odex/CEC hydrogel treated group was significantly lower than that of the
control (p = 0.029). Animals treated with the carboxymethylcellulose/hyaluronan film also showed
a lower adhesion score (p = 0.056). There was no statistical difference between the two treatments,
although the Odex/CEC hydrogel group showed a slight trend toward a lower score than that of the
group treated with the carboxymethylcellulose/hyaluronan film (p = 0.214). Twenty one days after
intervention, modest amounts of hydrogel remnants (as small fragments), localized along the areas
adjoining the cecum and abdominal wall, were identified in 6 out of the 9 animals treated with the
Odex/CEC hydrogel (Figure 22.24a and b) [137]. Amber-colored remnants of carboxymethylcel-
lulose/hyaluronan film were also identified in 4 animals (score = 1).
Histological analysis of tissues either received no treatment (control) or Odex/CEC hydro-
gel were performed. The control specimen was consistent with a severe adhesion score of 3
(Figure 22.25a), a robust and fibrous tissue layer formed between the cecum and abdominal wall.
The Odex/CEC treated specimen (adhesion score 1, with visible remnant fragments of hydrogel)
showed completely healed abdominal wall (Figure 22.25b) and cecum surface (Figure 22.25c);
the ongoing hydrogel degradation was also captured, the presence of multi-nucleated macrophages
ingesting the residual hydrogel pieces within the re-epithelialized tissue were self-evident (circled
area, Figure 22.25b).
(a) (b)
(c)
FIGURE 22.24 (See companion CD for color figure.) Examination of cecal and abdominal wall tissues
from rats 21 days after surgery: (a) An example of a severe adhesion (score = 3) case: a control rat with adhesion
of the cecum (CE) to the abdominal wall (AW). The arrow indicates the area where the adhesion is p resent.
Odex/CEC hydrogel remnants found on both (b) the cecum, and (c) the abdominal wall of one animal subject.
This animal received an adhesion score of 1 due to some light, filmy adhesions of fat/omentum to the abdomi-
nal wall. The circled regions of (b) and (c) highlight the presence of the hydrogel pieces while the arrows in (c)
point out the light adhesions that formed inside the rat abdomen after 21 days. (Reprinted from J. Surg. Res. 159,
Falabella, C.A., Melendez, M.M., Weng, L., and Chen, W., Novel macromolecular crosslinking hydrogel to
reduce intra-abdominal adhesions, 772–778. Copyright 2009, with permission from Elsevier.)
(a) (b)
(c)
FIGURE 22.25 Photomicrographs of abdominal and cecum tissue excised 21 days after injury. (a) Cross
section of rat tissue from untreated, control animals with severe adhesion (*) between the abdominal
wall (AW) and the cecum (CE) surface (score = 3). (b) Site of abdominal wall defect in a rat treated with
the Odex/CEC hydrogel. Remnants of the hydrogel are highlighted with arrows and located inside the
re-epithelialized (RE) abdominal wall defect site. The circled area highlights a fused, giant cell that
has ingested a portion of the hydrogel remnant. (c) Site of cecum serosal abrasion from the same rat in
(b). Fragments of residual hydrogel are located inside the re-epithelialized tissue on the serosal surface.
Magnification 4X for all photomicrographs. (Reprinted from J. Surg. Res. Copyright 2009 (in press), with
permission from Elsevier.)
The window for efficacy of an adhesion barrier is recognized as between 12 and 36 h after surgery
[138,139]. A longer-term endpoint (21 day after surgery) was chosen to observe the adhesion sever-
ity in conjunction with validating the biodegradability of the hydrogel. The typical post-operative
sacrifice time in comparable studies performed on rat models range from 7 to 14 days [140,141].
Adhesions are normally manifested within the first few days after surgery and more organized,
rigid and vascular tissues typically form between 5 and 7 days [132,135]. Any adhesions present
after 7 days could persist for several months due to the remodeling occurring inside the adhesion
tissue [132].
Analyses of the histological results of the Odex/CEC hydrogel remnants in conjunction with the
adjoining tissue at the injured site did not show cellular infiltration into the hydrogel bulk, suggesting
a degradation mechanism involving surface erosion [90]. This feature suggested that the hydrogel
served as an effective, occlusive barrier to prevent inflammatory cells and fibroblasts from forming
aggressive tissue adhesions between the injured cecum and the abdominal wall. Granulation tissue,
an indicator of wound healing, was observed at the site of injury with re-epithelialization occur-
ring underneath the hydrogel suggesting tissue repair/restoration. Some residual hydrogel fragments
inside the granulation tissue were detected and they were surrounded by macrophages with a notice-
able presence of foreign body giant cells, the presence of internalized hydrogel fragments suggest-
ing finalizing of the biodegradation process. This was comparable to the ingestion of other dextran
containing biodegradable hydrogel particulates by foreign body giant cells [142], it should be noted
that the presence of the multi-nucleated giant cells surrounding any foreign material introduced
into the abdominal cavity during surgery at 21 days is not unusual [143–145]. The hydrogel did
not appear to interfere with the normal immune response inside the abdomen; therefore, the main
mechanism of moderating tissue adhesion was likely through the maintenance of a physical barrier
between the injured surfaces.
There was no statistical significance between the severity of adhesion scores between the
groups treated by carboxymethylcellulose/hyaluronan film and the Odex/CEC hydrogel; how-
ever, the capacity of the hydrogel to conform to complex geometries is very advantageous.
The former is typically applied to damaged tissues as a rigid but brittle sheet thus creating a
dimensional limitation on open surgeries. It has been reportedly demonstrated that the carboxy-
methylcellulose/hyaluronan film could be utilized in conjunction with laparoscopic surgeries;
nonetheless, the brittle nature of the film could cause difficulties in deployment in addition to
the precaution needed to avoid any moisture on the application tools [146]. Conversely, the
Odex/CEC hydrogel is a conformal tissue sealant; it could easily be adapted in both conven-
tional surgeries and laparoscopic procedures as either a syringe-borne configuration or through
a laparoscopic port. Upon mixing, a steady but rapid increase in both the viscosity and adhe-
siveness of the precursor mix ensures its localization. The Odex/CEC hydrogel also circum-
vents the need to dry the bowel surface which could cause further irritation and this is generally
regarded as undesirable.
In this study, we have applied an in situ gelable Odex/CEC hydrogel formulation to reduce the
incidence of abdominal adhesions in a rat cecum abrasion model. The hydrogel is easy to apply,
fully conformal to any geometries, and has achieved comparable, overall efficacy in adhesion pre-
vention as the commercially available carboxymethylcellulose/hyaluronan film.
22.5 SUPPRESSION OF WOUND HEALING BY HYDROGEL-MEDIATED

DELIVERY OF AN iNOS INHIBITOR [147]
Macrophage plays significant roles in the post-implant inflammatory response mounted against
various biomaterials; it also directs the progress of the wound healing process [148,149]. Activated
macrophages impede the growth of microorganisms and malignant cells; they are also involved
in ECM production, cell proliferation and angiogenesis [150], moreover, macrophages have been
implicated in the pathophysiologies of various inflammatory diseases [151]. Nitric oxide (NO) is
one of the major mechanisms for macrophages to mediate inflammatory response and biomate-
rial degradation [152–154]. NO produced by inducible nitric oxide synthase (iNOS) plays impor-
tant roles in many physiological and pathophysiological functions [155]; it has also been strongly
implicated in dermal wound healing [153–156]. Application of a selective iNOS inhibitor has been
shown to reduce inflammatory extravasations [157], a condition known to closely associate with
scar or formation of polyposis in wound healing. Therefore, scar formation could theoretically be
reduced by timely suppression of iNOS. Guanidinoethyl disulfide (GED, Figure 22.26) is a potent
iNOS inhibitor [158] and it has been shown to suppress experimental gingivitis linked to focal
increase in iNOS level [159].
H2N + NH
2
N S S N . 2HCO3–
H2N + H H NH2
FIGURE 22.26 The structure of GED. (Reprinted from Biomaterials, 29, Weng, L., Ivanova, N.D.,
Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of cellular activity by guanidinoethyl disulfide
released from hydrogel microspheres composed of partially oxidized hyaluronan and gelatin, 4149–4156.
The self-crosslinkable hydrogel prepared by blending oxidized oHA with gelatin we shown
previously was used as the basic experimental vehicle for the delivery of GED. It was also previ-
ously shown that biodegradable hydrogel microspheres were capable of enhancing the efficacy of
various bioactive agents [160–162]; we therefore developed microspheres using the oHA/Gelatin
blend as the carrier for GED. The structural and physical properties of the microspheres were
characterized and the formulation parameters affecting the characteristics of microspheres and
their GED release profiles were investigated. Finally, the in vivo efficacy of the GED micro-
spheres in suppressing macrophages infiltration was validated in mice full-thickness dermal
wound models.
22.5.1 Microspheres for GED Delivery

The GED-carrying microsphere was formulated using a water-in-oil emulsion process. oHA
(oxidation degree: 44.7%) and gelatin solutions were blended at approximately 40°C and it was
emulsified with soya oil using Span 80 as a surfactant, the emulsion was maintained at 50°C over-
night. Microspheres were crosslinked/stabilized by the formation of Schiff base between alde-
hyde groups and amino groups in the mixture via a similar mechanism revealed previously [99].
GED-loaded oHA/gelatin microspheres were prepared by incorporating GED solutions of various
concentrations (0.2%, 1%, and 2%). The encapsulation efficiencies of these microspheres were
between 37% and 76%; the amount of GED captured could be adjusted (see Table 22.5).
22.5.2 Characterization of Microspheres
The SEM images of plain oHA/gelatin microspheres and GED containing microspheres
(1% loading) in both dried and swollen state were shown in Figure 22.27. The microspheres were
distorted and it was likely the results of the drying process. The surfaces of these microspheres
in their dried state were moderately coarse (Figure 22.27a and c) with scattered small pores.
Moreover, the diameters of the GED containing microspheres were considerably smaller than their
counterparts with no GED. In contrast, the microsphere surfaces in their swollen state exhibited
highly porous morphologies.
TABLE 22.5
Particle Size Distribution of Blank and GED Incorporated
oHA/Gelatin Microspheres
GED Encapsulation
Microsphere Composition Mean Diameter (μm) ± SD Yield (%)
oHA/gelatin 91.56 ± 18.76 —
oHA/gelatin/0.2% GED 86.28 ± 19.69 76.64
oHA/gelatin/1% GED 56.39 ± 9.43 70.98
oHA/gelatin/2% GED 31.39 ± 8.67 37.4
Source: Modified from Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and
Chen, W., In vitro and in vivo suppression of cellular activity by guanidino-
ethyl disulfide released from hydrogel microspheres composed of partially
oxidized hyaluronan and gelatin, 4149–4156. Copyright 2008, with permis-
sion from Elsevier.
Note: Their GED encapsulation efficiencies were also presented.
20 μm 20 μm
(a) (b)
80 μm 80 μm
(c) (d)
FIGURE 22.27 SEM photomicrographs of: (a, c) plain oHA/gelatin microspheres, and (b, d) 1% GED-loaded
oHA/gelatin microspheres. (a, b): original dry state, and (c, d): lyophilized after fully swollen. (Reprinted from
Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of cel-
lular activity by guanidinoethyl disulfide released from hydrogel microspheres composed of partially oxidized
hyaluronan and gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
The size distributions of four microspheres formulations were depicted in Figure 22.28;
in general, their spreads broadened considerably with decrease in their GED contents. The
mean sizes of the plain microspheres, and the GED containing microspheres with 0.2%, 1%,
and 2% GED incorporated, were 91.6 ± 18.8 μm (a), 86.3 ± 19.7 μm (b), 56.4 ± 9.4 μm (c), and
31.4 ± 8.7 (d) μm, respectively (as summarized in Table 22.5). The mean size of the former
was generally larger than that of the latter; moreover, there was an inverse correlation between
the microsphere size and the GED loading. GED-loaded microspheres were generally smaller
when compared with its counterpart containing no GED; and it decreased from 91 to 31 μm,
with the concentration of GED solution incorporated during the preparation increased from
0% to 2%. Decrease in solution viscosity caused by the increase of GED concentration dur-
ing emulsification resulted in the formation of smaller emulsion droplets and thus the smaller
microspheres formed [162].
Fully swollen oHA/gelatin microspheres (containing 1% GED loading) and the swelling
kinetics were depicted in Figure 22.29. All microspheres formulations reached their full swol-
len state very quickly (< 500 s); the microspheres containing GED had higher equilibrium swell-
ing ratios compared with their counterpart without GED, and this ratio appeared to be linearly
related to their GED contents. The presence of GED interfered with the interactions between
amino groups and aldehyde groups on the gelatin and oHA, respectively; increase in the micro-
spheres’ GED content led to a corresponding reduction of their crosslinking density and thus
greater swelling.
8 8
6 6
Frequency
Frequency
4 4
2 2
0 0
60 80 100 120 140 40 60 80 100 120 140
(a) Microsphere size (μm) (b) Microsphere size (μm)
14
10
12
10 8
Frequency
Frequency
8 6
6
4
4
2
2
0 0
40 50 60 70 80 90 10 20 30 40 50 60
(c) Microsphere size (μm) (d) Microsphere size (μm)
FIGURE 22.28 Frequency and size distribution plots of oHA/gelatin microspheres. (Reprinted from
Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of
cellular activity by guanidinoethyl disulfide released from hydrogel microspheres composed of partially oxi-
dized hyaluronan and gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
22.5.3 In Vitro Release of GED

GED release was performed in 0.01 M PBS at 37°C and the GED HPLC eluent-time profiles were
depicted in Figure 22.30. Typical eluent profiles of GED solutions (3 h time-point) were shown in
Figure 22.30a and b. The retention time for GED was approximately 13 min and multiple peaks
also appeared on the eluent profile of the release samples in the vicinity of 13 min suggesting
the release of GED was accompanied by the release of matrix materials from the microspheres.
Figure 22.31 showed the release kinetics of three oHA/gelatin microspheres formulated using
0.2%, 1%, and 2% GED solutions. The initial release kinetics (first 7 h) hi-lighted in the inset indi-
cated that GED release was related to the microspheres’ initial GED content; this was consistent with
other comparable microspheres prepared by physically entrapping the active principles [163,164].
Typical burst releases signifying the discharge of free residual GED bound to microspheres’ surface
were observed in all formulations [165,166]. After the initial burst, the release of GED from the
microspheres continued at a modest pace.
22.5.4 Macrophage Inhibition by GED Released from Microspheres

Macrophage play important roles in mediating post-implant tissue/biomaterial inflammatory responses
and wound healing [167–169]. In the presence of inflammatory stimuli, the production of NO by
100 μm
(a)
25
20
No GED
Swelling ratio (q)
0.2 % GED
15
1% GED
2 % GED
10
0 500 1000 1500 2000

(b) Time (s)
FIGURE 22.29 The effect of GED contents on the swelling dynamics of the oHA/gelatin microspheres.
(Reprinted from Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo
suppression of cellular activity by guanidinoethyl disulfide released from hydrogel microspheres composed
of partially oxidized hyaluronan and gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
macrophages rises sharply [170,171]; therefore, suppression of the iNOS activity and thus the NO secre-
tion is a potential strategy to modulate the inflammatory responses evoked by the implant. Macrophage
was used as the model cell type for validating the effect of the GED-loaded oHA/Gelatin microspheres.
GED containing microspheres were incorporated into the oHA/Gelatin hydrogels (abbrevi-
ated as GOG hydrogel film) and macrophages were cultured directly with them. As controls,
macrophages were seeded in either cell culture medium (i.e., background, control) or in the
presence of the oHA/Gelatin hydrogel films (abbreviated as OG, as the negative blank control).
The effects of GED on macrophages growth and their distributions were observed at 3–6 days
after seeding and the results were depicted in Figure 22.32. GED-loaded microspheres evi-
dently suppressed the proliferation of the macrophages; the effect was particularly prominent
on day 3, and cells attached to both the culture well (bottom) and the hydrogels. Macrophages
residing on the culture wells (Figure 22.32a) and on the OG hydrogel (Figure 22.32c) assumed
their characteristic round morphology with a considerable number of them also spread out,
13.68
10.45
13.88
11.0
11.0
(a) (b)
FIGURE 22.30 Typical HPLC elution profiles: (a) A standard GED solution and (b) a sample from the
release test of 1% GED-loaded oHA/gelatin microspheres. (Reprinted from Biomaterials, 29, Weng, L.,
Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of cellular activity by guanidino-
ethyl disulfide released from hydrogel microspheres composed of partially oxidized hyaluronan and gelatin,
4149–4156. Copyright 2008, with permission from Elsevier.)
Cumulative GED released (μg/mg microspheres)
30 35
Cumulative GED released
(μg/mg microspheres)
30
25
0.2% 20
1% 15
10
20 2%
5
0
0 1 2 3 4 5 6 7 8
Time (h)
10
0 50 100 150 200

Time (h)
FIGURE 22.31 GED release profiles of oHA/gelatin microspheres formulated with 0.2%, 1%, and 2% GED
solutions. (Reprinted from Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and
in vivo suppression of cellular activity by guanidinoethyl disulfide released from hydrogel microspheres com-
posed of partially oxidized hyaluronan and gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
50 μm 50 μm
(a) (b)
50 μm 50 μm
(c) (d)
50 μm 50 μm
(e) (f )
FIGURE 22.32 Morphology of macrophages in the presence hydrogel. Control: (a) 3 days and (b) 6 days.
OG hydrogel: (c) 3 days and (d) 6 days. GOG hydrogel: (e) 3 days and (f) 6 days. The GED-loaded micro-
spheres in the GOG hydrogel was formulated with a 1% GED solution. (Reprinted from Biomaterials, 29,
Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of cellular activity by
guanidinoethyl disulfide released from hydrogel microspheres composed of partially oxidized hyaluronan and
gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
indicating their activation (i.e., inflammatory response) [168]. Conversely, virtually all the
cells co-cultured with the GOG hydrogel maintained their typical non-activated round mor-
phology (Figure 22.32e); GED evidently fully suppressed their activation. Cells also prolifer-
ated rapidly in the control group (Figure 22.32b) with a more moderate cell growth in the OG
group (Figure 22.32d); macrophage proliferation was suppressed in the presence of the GOG
hydrogel (Figure 22.32f). These results were quantitatively summarized in Figure 22.33. The
control group had higher cell numbers as compared with both OG and GOG groups, strongly
suggesting the release of GED resulted in the suppression of macrophage proliferation. By day
250
Control
Number of macrophages in 100 μm2

OG hydrogel
200
GOG hydrogel
150
100
50
0
3 6
Time (day)
FIGURE 22.33 The densities of macrophages after cultured for 3 days and 6 days, respectively. The
GED-loaded microspheres in the GOG hydrogel was formulated with a 1% GED solution. (Reprinted from
Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of cel-
lular activity by guanidinoethyl disulfide released from hydrogel microspheres composed of partially oxidized
hyaluronan and gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
6, cells in the control group have reached full confluence and they were growing over each
other (see Figure 22.32b), whereas the cells in the GOG groups reached 80% confluence
(Figure 22.32d); in contrast, the increase in cell numbers in the GOG group was very mod-
est (Figure 22.32f). Continual cell suppression was self-evident despite daily culture medium
changes, implicating sustained release of GED from the microspheres entrapped inside the
oHA/gelatin hydrogels.
22.5.5 Suppression of Cell Infiltration into Hydrogel in Mice

Full-Thickness Transcutaneous Wound Models
The efficacy of the GOG hydrogel in suppressing cell infiltration was validated by mouse full-
thickness incisional dermal wound models. The predominant cell types interacting strongly with
implants are macrophages and fibroblasts during wound healing; they also interact and modulate
the functions of each other [136,172]. As a typical response to injury, the number of macrophages in
the wound bed surges almost immediately as the initial inflammatory reaction, their number usually
peaks between days 3 and 4, and begins to subside by days 6–7; the number of fibroblasts surges at
day 1, peaks by days 5–6, and begins to subside by days 7–8 [173,174]. Days 3 and 7 appeared to be
the most optimal time-points for showing GED suppression of cell activities. On day 3, tissue forma-
tion, typically manifested as an emerging white ring around the edge of the wound bed, during the
early stage of healing was observed in both the unimplanted control and the OG hydrogels. No tissue
formation was observed in the wound beds of the mice implanted with the GOG hydrogels, indicat-
ing the suppression of wound healing. The histology sections of the hydrogels and the tissues adja-
cent to them after 3 days of implantation were depicted in Figure 22.34. The capacity of the oHA/
gelatin hydrogel to accommodate cell infiltration/proliferation in subdermal implantation was shown
earlier [99] (Figure 22.23); the cell infiltration depicted in Figure 22.34a (OG hydrogel) was in good
agreement with those shown previously [99] (Figure 22.23). However, the numbers of cells infiltrated
into the GOG hydrogel (Figure 22.34b) were substantially lower than their OG counterparts shown
in Figure 22.34a; moreover, the maximum penetration depth was approximately half the thickness
of the hydrogel. Under high magnifications, the great majority of these infiltrated cells were identi-
fied as macrophages (“ ”) with some neutrophils and occasionally fibroblasts, which resided pre-
dominantly along the fibrous tissues adjacent to the hydrogel. Cell infiltration was suppressed by
(a)
(b)
FIGURE 22.34 (See companion CD for color figure.) H&E staining of typical wounds after 3 days. The
wound beds were (a) implanted with OG hydrogel, and (b) implanted with GOG hydrogel containing GED-
loaded microspheres formulated from 1% GED solution. C: Fibrous tissue formed, I: Implant, L: Loose con-
nective tissue, : Macrophage, : Fibroblast. The images were captured under 20 × magnification. (Reprinted
from Biomaterials, 29, Weng, L., Ivanova, N.D., Zakhaleva, J., and Chen, W., In vitro and in vivo suppression
of cellular activity by guanidinoethyl disulfide released from hydrogel microspheres composed of partially
oxidized hyaluronan and gelatin, 4149–4156. Copyright 2008, with permission from Elsevier.)
GED, and by manual cell counting, the ratio of cells in the GOG and OG hydrogels was determined
as 4.8:1. There were also clear difference between both the thicknesses and densities (as reflected
by the staining intensity) of the two types of hydrogels. Macrophages produce enzymes capable of
degrading implanted materials [171], and both hyaluronan and gelatin are very susceptible to these
enzymes. Partial digestion of the OG hydrogel caused breakdown of crosslinks, leading to a partial
loss of the hydrogel’s structural integrity, rendering it less resistant to water uptake and, thus, swell-
ing. Collectively, the net effect is an increased thickness of the OG hydrogel on the histology section
and less intense staining. Conversely, GED in the GOG hydrogel suppressed the macrophage activi-
ties leading to a greater degree of preservation of the structural integrity of the hydrogel.
22.6 CONCLUSIONS
This chapter summarizes our investigations on natural material derived and crosslinker-free in situ
gelable formable/conformable hydrogels; this type of hydrogels is versatile and advantageous
over their counterparts requiring extraneous crosslinking reagents or photo-induction. They can
be formulated to either prevent or facilitate cell adhesion/migration and their physicomechanical
properties could be tailored and optimized for different purposes. The potential biomedical appli-
cations (enhance wound healing and prevent post-surgical adhesion) have also been demonstrated.
The hydrogel can also be formulated into drug delivery vehicles.
ABBREVIATIONS
CEC Carboxyethyl chitosan
ECM Extracellular matrix
EGF Epidermal growth factor
FGF Fibroblast growth factor
GED Guanidinoethyl disulfide
GRAS Generally regarded as safe
iNOS Inducible nitric oxide synthase
HA Hyaluronan
Odex Oxidized dextran
oHA Oxidized hyaluronan
TGF Transforming growth factor
LVR Linear viscoelastic region
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23 Dental and Maxillofacial
Surgery Applications
of Polymers
E.C. Combe
CONTENTS
23.1 Introduction........................................................................................................................... 785
23.1.1 Polymers in the Oral Environment............................................................................ 785
23.1.2 Overview of Applications.......................................................................................... 787
23.2 Overview of Mechanisms and Materials............................................................................... 787
23.2.1 Free Radical Addition Polymerization...................................................................... 788
23.2.1.1 Activation/Initiation Systems..................................................................... 788
23.2.1.2 Methacrylates and Dimethacrylates........................................................... 789
23.2.1.3 Factors Associated with Polymerization..................................................... 789
23.2.1.4 Inhibition of Polymerization....................................................................... 791
23.2.2 Reaction of a Basic Powder with an Acidic Polyelectrolyte in Aqueous Solution.......793
23.2.3 Material Types........................................................................................................... 795
23.2.3.1 Cements and Luting Agents........................................................................ 795
23.2.3.2 “Continuum”............................................................................................... 795
23.2.3.3 Resin Composites........................................................................................ 796
23.2.3.4 Glass-Ionomer (Polyalkenoate) Cements.................................................... 797
23.2.3.5 Resin-Modified Glass-Ionomer Cements.................................................... 797
23.2.3.6 Polyacid-Modified Resin Composites (“Compomers”).............................. 797
23.2.3.7 General Comparison of Material Properties.............................................. 797
23.2.3.8 Critique of the Concept of a “Continuum”................................................. 797
23.3 Interfaces and Adhesion........................................................................................................ 798
23.3.1 Applications and Dental Significance....................................................................... 798
23.3.2 Mechanisms and Materials........................................................................................ 799
23.3.2.1 Chemical Adhesion to Tooth Tissue........................................................... 799
23.3.2.2 Micromechanical Adhesion to Tooth Enamel............................................ 799
23.3.2.3 Hybridization Bonding to Tooth Dentin.....................................................800
23.3.2.4 Bonding to Ceramics..................................................................................804
23.3.2.5 Bonding to Metallic Materials....................................................................804
23.4 Prevention of Disease and the Effects of Trauma.................................................................804
23.4.1 Pit and Fissure Sealants.............................................................................................804
23.4.1.1 Purpose.......................................................................................................804
23.4.1.2 Classification of Dimethacrylate-Based Sealants.......................................804
23.4.1.3 Biocompatibility.........................................................................................805
23.4.1.4 Bonding to Enamel and Retention Rates.................................................... 805
23.4.1.5 Effect on Bacteria Levels in Caries Lesions............................................... 805
23.4.1.6 Application of Self-Etch Adhesives............................................................ 805
783
23.4.1.7 Application of Compomers.................................................................... 805

23.4.1.8 Application of Polyelectrolyte Materials............................................... 805
23.4.1.9 Summary of Current Clinical Recommendations.................................806
23.4.1.10 Research Needs......................................................................................806
23.4.2 Mouth Protectors....................................................................................................806
23.4.2.1 Purpose and Requirements....................................................................806
23.4.2.2 Materials................................................................................................806
23.4.2.3 Classification..........................................................................................807
23.4.2.4 Properties...............................................................................................807
23.4.2.5 Conclusion..............................................................................................807
23.5 Orthodontics.........................................................................................................................809
23.5.1 Orthodontic Adhesives...........................................................................................809
23.5.2 Orthodontic Elastics............................................................................................... 810
23.6 Esthetic Dentistry: Veneers.................................................................................................. 810
23.7 Tooth Restoration................................................................................................................. 811
23.7.1 Tooth Filling Materials........................................................................................... 811
23.7.1.1 Resin Composites................................................................................... 811
23.7.1.2 Siloranes................................................................................................. 815
23.7.1.3 Ormocers................................................................................................ 816
23.7.1.4 Compomers............................................................................................ 816
23.7.1.5 Glass-Ionomers and Resin-Modified Glass-Ionomers........................... 817
23.7.1.6 Summary of Data on Clinical Longevity of Tooth-Colored Restorations..... 817
23.7.2 Endodontic Materials............................................................................................. 817
23.8 Impression Materials............................................................................................................ 818
23.8.1 Introduction and Classification............................................................................... 818
23.8.2 Hydrocolloids......................................................................................................... 818
23.8.2.1 Agar: Reversible Hydrocolloids............................................................. 818
23.8.2.2 Alginates: Irreversible Hydrocolloids.................................................... 819
23.8.3 Non-Aqueous Elastomers....................................................................................... 819
23.8.3.1 Introduction............................................................................................ 819
23.8.3.2 Polysulfides............................................................................................ 820
23.8.3.3 Condensation Silicones.......................................................................... 820
23.8.3.4 Addition Silicones (Vinyl Polysiloxane)................................................ 820
23.8.3.5 Polyether Materials................................................................................ 821
23.8.3.6 General Comparison of Elastomers....................................................... 821
23.8.3.7 Disinfection of Impression Materials..................................................... 821
23.9 Provisional (Interim) Restorations....................................................................................... 822
23.9.1 Purpose and Requirements..................................................................................... 822
23.9.2 Critique of Materials.............................................................................................. 822
23.10 Prosthodontic Materials....................................................................................................... 822
23.10.1 Complete Dentures................................................................................................. 822
23.10.1.1 Poly(Methyl Methacrylate): Techniques of Polymerization................... 822
23.10.1.2 Limitations of Acrylic Denture Materials............................................. 822
23.10.1.3 Hypoallergenic Denture Polymers......................................................... 823
23.10.2 Denture Teeth......................................................................................................... 823
23.10.3 Resilient Polymers as Denture Linings..................................................................824
23.10.3.1 Temporary Lining Materials..................................................................824
23.10.3.2 Permanent Resilient Lining Materials...................................................824
23.10.4 Denture Adhesives..................................................................................................824
Dental and Maxillofacial Surgery Applications of Polymers 785
23.11 Polymer Applications in Periodontology............................................................................. 825

23.11.1 Membranes for Guided Tissue Regeneration......................................................... 825
23.11.2 Other Constituents.................................................................................................. 825
23.12 Dental Implants.................................................................................................................... 825
23.13 Soft Tissue Augmentation.................................................................................................... 825
23.13.1 Objectives and Requirements................................................................................. 825
23.13.2 Materials................................................................................................................. 825
23.14 Maxillofacial Prostheses..................................................................................................... 826
23.14.1 Requirements.......................................................................................................... 826
23.14.2 Materials................................................................................................................. 826
23.14.2.1 Silicone Polymers................................................................................... 826
23.14.2.2 Methacrylates......................................................................................... 826
23.14.2.3 Other Polymers...................................................................................... 826
23.15 Wound Closure Materials.................................................................................................... 826
23.16 Conclusion........................................................................................................................... 827
References....................................................................................................................................... 827
23.1 INTRODUCTION
23.1.1 Polymers in the Oral Environment
There is a long history of the application of polymers in dentistry. In the 1800s, before the advent of
synthetic polymers, India-rubber and gutta-percha were employed; the latter material is still in use
in endodontic therapy.
Given the current vast range of available synthetic polymers, and the ability to modify polymer
properties—for example, by co-polymerization, cross-linking, incorporation of plasticizers and the
inclusion of reinforcing constituents—it is not surprising that polymers are playing an essential
major role in oral and maxillofacial treatments. To demonstrate the broad scope of the subject,
Table 23.1 lists a wide range of types of polymer-containing materials, giving their principal dental
and maxillofacial surgery application(s).
The fundamental question to be asked is this—how will a synthetic material behave in the oral
and maxillofacial environment? In contrast to other parts of the body, materials in the mouth are
exposed to saliva, bacteria, and components of drinks and foodstuffs. Extremes of temperature
(0°C–90°C) and pH (gastric secretions have pH values from 1.0 to 3.5) can occur [1].
In addition to, or as well as biological effects, materials may suffer chemical or mechanical
breakdown under the aggressive conditions of the oral environment. This includes corrosion,
fatigue, fracture, and surface wear. Thus, there must be consideration for the safety, durability and
efficacy of biomedical polymers. Figure 23.1 is a general illustration of the principal concerns about
the placement of a synthetic material in a living environment.
Thus, there are stringent requirements that apply to polymers designed to be placed in the oral
cavity. In certain cases there may be poor biocompatibility. Biocompatibility may be defined as
“the ability of a material to perform with an appropriate host response in a specific situation” [2,3].
Problems of biocompatibility of polymers may occur from a number of sources, including the pos-
sibility of elution of unreacted monomers or co-monomers, polymerization initiators, stabilizers,
contaminants, and products of chemical decomposition [4]. In addition to chemical considerations,
surface features—for example, roughness, surface charge, porosity—may influence host response.
Potentially, a material may be irritant, toxic, allergenic, teratogenic, mutagenic, or carcinogenic.
Biocompatibility is a recurring theme of this review.
Before detailed consideration of each polymer application, an overview of polymers and their
principal applications is given. This is desirable because classification of the subject is made difficult
TABLE 23.1
Classification and Principal Dental Applications of Polymers
Hardening
Type of Polymer Polymer Name Application Mechanism
Synthetic 1. Condensation polymers Polyamides—e.g., nylon Prosthodontics Polymerization
polymers Polycarbonates
2. Free-radical addition, Poly(methyl methacrylate) Prosthodontics Polymerization
linear polymers — acrylic resins
— PMMA
3. Free-radical addition, Dimethacrylates Resin composites, Polymerization
cross-linked sealants and cements
4. Flexible polymers Plasticized acrylics Denture soft linings Polymerization
Elastomers: Impressions Polymerization
Polysulfides Soft linings
Siloxanes Mouth protectors
Polyether Maxillofacial polymers
5. Water soluble polymers Aqueous poly(acrylic acid) Adhesive dental cements Acid-base
and related co-polymers reaction
Naturally 6. Polyisoprenes Latex Orthodontic elastics
occurring Gutta percha Endodontics
polymers 7. Polysaccharides Alginates Impressions Gel formation
8. Polysaccharide Hyaluronic acid Soft tissue augmentation
9. Protein Collagen Soft tissue augmentation
Chemical or enzymatic degradation?

Electrochemical corrosion?
Mechanical effects:
Mechanical shock?
Fatigue?
Abrasion of surfaces?
Efficacy and durability
Interface
The oral Polymer

environment
Biocompatibility: safety
aspects
Is the material or any substance
eluted from the material, or
any breakdown product:
Irritant?
Estrogenic?
Toxic?
Allergenic?
Teratogenic?
Mutagenic?
Carcinogenic?
FIGURE 23.1 Considerations in the placement of a synthetic material in a living environment.

by the fact that a given polymer type may have multiple biomedical applications, and a given appli-
cation may have the choice of a number of materials. In particular, there are some dental procedures
(e.g., placement of sealants, orthodontic bonding, restoring teeth materials and adhesives), where
there is “competition” between two fundamentally different types of polymer-containing material—
that is, those based on the free radical polymerization of dimethacrylates and those containing aque-
ous solutions of acidic synthetic polyelectrolytes that react with basic powders.
In addition to being structural materials (e.g., components of tooth restoration and prosthetic
materials), polymers also make a significant contribution to cements and adhesives. A review of
three adhesive mechanisms is given, all of which utilize polymers, and are widely applicable in
contemporary dental practice.
23.1.2 Overview of Applications
The extensive range of applications of dental and maxillofacial applications of polymers is listed in
a logical sequence, in general working from the least invasive procedures to the most invasive. Thus,
the prevention of disease and trauma is considered, followed by applications such as orthodontics
and esthetic treatments, then with the repair of diseased teeth, followed by the replacement of teeth
and then maxillofacial surgery.
Dental and maxillofacial applications of polymers and polymer-containing materials include
• The prevention of decay—pit and fissure sealants for teeth.

• The prevention of the effects of trauma—resilient polymers can be used for mouth guards
for sports players.
• Orthodontic treatment—polymer-containing cements for bonding brackets and bands to
teeth can be employed. In some cases, orthodontic appliances will include polymeric elastics.
• Esthetic treatment—the cementation of translucent veneers on to tooth enamel.
• The direct restoration of teeth.
• Endodontic therapy.
• The recording of impressions, for the preparation of inlays, crowns and fixed partial den-
tures and removable complete dentures.
• Indirect restorative materials, that is, those that are fabricated outside of the mouth and
cemented into place.
• Provisional (temporary) crown and bridge materials.
• Complete dentures.
• Denture teeth.
• Resilient polymers as linings for dentures.
• Periodontology—guided tissue regeneration.
• Soft tissue augmentation.
• Maxillofacial prosthetic materials.
• Obturators.
• Wound closure materials (sutures and tissue adhesives).
One critically important factor to consider is that in many cases bonded interfaces are required,
thus adhesion to tooth enamel and dentin, and to ceramics and alloys is essential for success. There
exists a wide range of bonding agents, many of which are polymer-based.
23.2 OVERVIEW OF MECHANISMS AND MATERIALS

In classifying the synthetic polymers, it is important to distinguish between two categories of
polymer-containing material, which are tooth-colored and which harden directly in the mouth. Some
materials harden by a polymerization reaction (e.g., number 3 in Table 23.1), and in others an acidic
polyelectrolyte in aqueous solution reacts with a basic or amphoteric powder to form a set material
(number 5 in Table 23.1). As stated earlier, in a number of applications there is a choice between
examples of these general material types; where relevant the different types of material will be com-
pared and contrasted.
23.2.1 Free Radical Addition Polymerization*

Many dental biomaterials set by a mechanism of free radical addition polymerization (though some
contemporary materials employ a cationic mechanism). Three different mechanisms of activation/
initiation are employed—namely, thermal, photochemical and redox methods.
23.2.1.1 Activation/Initiation Systems

23.2.1.1.1 Thermal Activation
Some materials, such as denture materials based on poly(methyl methacrylate)—see Section
23.10—contain benzoyl peroxide. On heating, this compound forms free radicals by the homolytic
decomposition of the weak –O–O– bond.
23.2.1.1.2 Photochemical Activation

Photoactivated initiators can be employed. Frequently the diketone camphorquinone and an amine
are used [5], and free radicals are generated in the presence of light of wavelength approximately
470 nm (Figure 23.2). A more recent alternative diketone that is sometimes employed is 1-phenyl-
1,2-propanedione [6].
CH3
H3C
CH3
O O O
H3C C C
O
Camphor quinone 1-Phenyl-1,2-propanedione
Tertiary
amine
•N Exciplex
•
CH2
hv • _ +
O *O O •N
~470 nm Activated CH3
triplet
state
• N
OH
Two free radicals • CH2
FIGURE 23.2 Photochemical activation.
* Condensation polymerization reactions are also used (Section 23.8.3), but not for tooth-colored materials that harden in
the mouth.
CH3
H3C CH3
FIGURE 23.3 N,N-dimethyl-p-toluidine.
23.2.1.1.3 Redox Activation

Alternatively, a redox system be employed—for example, when free radicals to initiate the
polymerization are formed by the reaction of benzoyl peroxide with a tertiary amine such as
N,N-dimethyl-p-toluidine (Figure 23.3). These systems are sometimes referred to as “self-curing,”
“autopolymerizing,” or “chemical curing.”
23.2.1.1.4 Dual Cure

In some instances, dual cure materials are available—that is, they have both a photochemical reac-
tion and redox activation.
23.2.1.2 Methacrylates and Dimethacrylates

An extensive list of dentally used methacrylates and dimethacrylates monomers has been given by
Van Landuyt et al. [7]. These monomers are classified as follows:
• Monofunctional acrylates and methacrylates—used in adhesive and prosthodontic appli-

cations (Section 23.10).
• Cross-linking monomers based on dimethacrylates (Figure 23.4)—widely used as the
basis of sealants (Section 23.4.1), orthodontic adhesives (Section 23.5.1), restorative mate-
rials (Section 23.7.1) and temporary inlay, crown and bridge materials (Section 23.9). Of
these, the most widely used are Bis-GMA, UDMA, and TEGDMA.
• Methacrylate monomers with functional group(s) to promote adhesion. The functional
groups have been classified as (i) sulfonic acid (e.g., in Figure 23.5), (ii) phosphate
(Figure 23.6), phosphonate (Figure 23.7), carboxyl (Figure 23.8), and alcohol
(Figure 23.9) [7]. These are discussed in more detail in Section 23.3.2.
23.2.1.3 Factors Associated with Polymerization

23.2.1.3.1 Reaction Exotherm
The fact that free radical addition polymerization reactions are exothermic has a number of dental
ramifications. First, a polymer-based restorative material should not generate enough hear to dam-
age the sensitive pulp of the tooth [8] (Section 23.7.1). Second, the substantial temperature during
the polymerization of methyl methacrylate has to be taken into account into the processing of acrylic
dentures (Section 23.10.1) to avoid volatilization of unpolymerized monomer [9].
23.2.1.3.2 Polymerization Shrinkage

Polymerization reactions are usually accompanied by shrinkage. For example, the volumetric shrink-
age during the polymerization of methyl methacrylate is 21%. This is an important factor in the fab-
rication of complete acrylic dentures (Section 23.10). Contraction is also critical in polymer-based
O O
O O O O
OH OH
Bis-GMA (Bowen's resin): Bisphenol A diglycidyl methacrylate
O
O O
O
TEGDMA: triethylene glycol dimethacrylate
O O
O O
O N N O
O H H O
UDM: urethane dimethacrylate
O O
O O
O O
Bis EMA: ethoxylated bisphenol A glycol dimethacrylate
FIGURE 23.4 Dimethacrylate monomers.
O
NH
S O
O OH
AMPS: 2-acrylamido-2-methyl-1
propanesulfonic acid
FIGURE 23.5 Methacrylate monomer with sulfonic acid functional group.
tooth restorative materials—the stress resulting from shrinkage during polymerization may compro-
mise bonding of the material to tooth substance [10] (Section 23.7.1.1.4). As will be discussed later,
there is intensive research to overcome this concern.
23.2.1.3.3 Degree of Conversion of Monomer to Polymer

It needs to be recognized that not all double bonds react in free radical addition polymerization. In
the case of the polymerization of methyl methacrylate, there is always a certain amount of residual
O O
O O
CH2 H2C
O O O
OH CH2 CH2
O O O O
O
O P OH O
O P OH
O O
OH
HEMA-phosphate:
2-hydroxyethyl methacryl PENTA: dipentaerythritol pentaacrylate
dihydrogenphosphate monophosphate
OH
O OH
P O
O O
O P OH
O O
O O
Phenyl-P:
2-(methacryloyloxymethyl)phenyl 10-MDP: 10-methacryloyloxydecyl dihydrogenphosphate
hydrogenphosphate
FIGURE 23.6 Monomers with phosphate groups.
OH OH
OH P OH
P
O O
O O
O
O
O O
EAEPA: MAEPA: 2,4,6 trimethylphenyl

ethyl 2-[4-(dihydroxyphosphoryl) 2-[4-dihydroxyphosphoryl
-2-oxabutyl]acrylate -2-oxabutyl]acrylate
FIGURE 23.7 Monomers with phosphonate groups.
monomer. This may have deleterious biological consequences. In the case of dimethacrylates, the
degree of conversion of –C=C– bonds may influence biocompatibility, mechanical properties and
durability of the material [11].
23.2.1.4 Inhibition of Polymerization

23.2.1.4.1 Oxygen
Free radical addition polymerization is inhibited in the presence of oxygen [12]. As shown in
Figure 23.10, oxygen can form an adduct with a free radical; this will slow the rate of polymerization.
This may have implications for the clinical placement of fissure sealants (Section 23.4.1) and polymer-
containing tooth restorative materials (Section 23.7.1).
OH
O N COOH
O
O
O
O
O O NPG-GMA: N-phenylglycine
4-META: 4-methacryloyloxyethyl glycidyl methacrylate
trimellitate anhydride O
O
O OH
H2O
O O N COOH
O
O O
COOH
4-MET: 4-methacryloyloxyethyl
trimellitic acid COOH
CH3
NTG-GMA: N-tolylglycine glycidyl methacrylate
COOH
O NH OH
O
O
O O COOH
5-NMSA:
MMEP:
N-methacryloyl--
mono-2-methacryloyloxyethyl phthalate
5-aminosalicylic acid
HOOC COOH
O O
O O
O O
O O
BPDM: biphenyl dimethacrylate
FIGURE 23.8 Monomers with carboxyl groups.
O
OH
O
HEMA:
2-hydroxyethyl methacrylate
FIGURE 23.9 Monomer with an –OH group.
CH3 CH3 CH3
R CH2C • + O2 R CH2COO CH2COO •

n
COOR' COOR' COOR'
FIGURE 23.10 Oxygen inhibition of polymerization.

CH3
H3C CH3
C
C H3C O OH
CH3
H3C OH CH3
CH3
BHT: Butylated MEHQ: Monomethyl
hydroxytoluene ether hydroquinone
FIGURE 23.11 Inhibitors of polymerization.
23.2.1.4.2 Eugenol
3-(4-hydroxy-3-methoxyphenyl)-1-propene (eugenol) is used as a constituent of some dental cements.
Since this compound is a scavenger for free radicals, it is imperative that it should not come into
contact with a material that is polymerizing by a free radical mechanism [13].
23.2.1.4.3 Butylated Hydroxyl Toluene (BHT) and Monomethyl

Ether Hydroquinone (MEHQ)
Because of the tendency of monomers to polymerize under unfavorable storage conditions, minute
quantities of free radical scavengers such as BHT or MEHQ (Figure 23.11) are added to dental
materials to confer stability on storage [4]. When the polymerization is activated, there is an induc-
tion period during which the scavenger reacts with free radicals. When the scavenger is consumed,
the polymerization will commence [14]. Given that these compounds can be eluted from resins, the
biocompatibility implications need to be considered (Section 23.7.1) [4,15,16].
23.2.2 Reaction of a Basic Powder with an Acidic

Polyelectrolyte in Aqueous Solution
Some dental biomaterials contain an aqueous solution of a synthetic polyelectrolyte acid, which is
designed to undergo an acid–base reaction with an inorganic solid. For example, zinc oxide reacts
with aqueous poly(acrylic acid)—PAA—to give zinc polycarboxylate. This was the first chemically
adhesive dental adhesive cement [17].
Subsequently, glass-ionomer cements were developed using similar polyelectrolyte solutions, but
with a powdered fluoro-aluminosilicate (FAS) glass [18]. Early materials used an acrylic acid homo-
polymer, but today, copolymers with itaconic or maleic acid are often employed (Figure 23.12). The
general term for these polymers and copolymers is polyalkenoic acids, and glass-ionomers materials
are sometimes referred to as polyalkenoates. Material properties are dependent on molecular weight
(MW) of the polymer, higher MWs giving stronger material for a given concentration of the acid.
H H H CH2COOH H H
C C C C C C
H COOH H COOH HOOC COOH

Acrylic acid Itaconic acid Maleic aicd
FIGURE 23.12 Component monomers of water-soluble polyelectrolytes used in glass-ionomer cements.

Aqueous
poly (acrylic acid) FAS
glass Al3+ and Ca2+ ions
The polymeric are generated,
acid chemically causing the
attacks surface material to set
of glass particles
Fluoride ions can Fluoride ions are

migrate out of and into also released, but
cement without they are not part
affecting its strength of the structure of
or structure the set material
FIGURE 23.13 Setting and fluoride release of glass-ionomer cements.
However, there are limitations—viscosity of the solution increases with molecular weight for a given
concentration. In practice, typical figures are a 47.5% solution of MW 23,000, given that if the viscos-
ity of the cement liquid was too great, it would be impossible to mix with the FAS glass powder [19].
After mixing the polyalkenoate acid with FAS glass, a number of events occur, as depicted in Figure
23.13. The acid attacks the surfaces of the glass particles, resulting in extraction of ions from the glass.
Three of these are of great significance. Ca2+ and Al3+ can crosslink the polyalkenoate anions, contrib-
uting to the development of strength and rigidity of the material. F− ions are also released, but remain
in the aqueous phase. They are not part of the structure of the material, and they can diffuse through
the aqueous medium, without alteration in mechanical properties of the material. They can migrate
both out of and into the cement; the clinical significance of this will be discussed later. It should be
noted that the structure of the set material is composite (although dental terminology usually restricts
the term “composite” to refer to resin composite materials—see the following text), since only the outer
portion of the FAS glass particles react. The set material is composed of unreacted glass surrounded
by a siliceous hydrogel, and held together by the cross-linked polyalkenoate chains (Figure 23.14) [20].
(a) (b) (c)
0.5 μm
FIGURE 23.14 Structure of set polyalkenoate cement. a, FAS glass particle; b, siliceous hydrogel; c, matrix
of reaction products. (Courtesy of J. Perdigão.)
23.2.3 Material Types
23.2.3.1 Cements and Luting Agents
A luting cement is defined as a material used to attach indirect restorations to prepared teeth [21].
There is a wide range of such materials, both for temporary and permanent luting (Table 23.2)
[22,23], many of which are polymer-containing.
23.2.3.2 “Continuum”
Based on the earlier discussion, we can now give a preliminary discussion of four types of dental
biomaterial, which are all capable of hardening in the mouth, and all of which are polymer-
containing and tooth-colored. They all harden by free radical polymerization and/or an acid-base
reaction. They are used for a host of dental applications, including orthodontics, pedodontics,
restorative dentistry and prosthodontics, often with several materials being advocated for any one
application.
The four types of material have been described as a “continuum” [24]. Because there is often
a choice of material for a given application, it is essential that both the similarities and differ-
ences between these various materials be understood. Figure 23.15 shows the main types of
material, and describes their principal characteristics. The general principles of these materi-
als are discussed here, and in later sections of the review (e.g., tooth restorations) more details
will be given, including detailed chemical composition, current concerns, and potential research
developments.
The two original materials of the “continuum” are the resin composites and the glass-ionomers.
These will be considered first. The other two materials were derived from these, as shown by the
arrows on Figure 23.15.
TABLE 23.2
Dental Luting Cements
Material Main Constituents as Supplied Setting Mechanism(s) Comments
Zinc oxide-eugenol Zinc oxide + eugenol Acid-base Not polymer-containing;
for temporary cementation
Zinc phosphate Zinc oxide + buffered aq. H3PO4 Acid-base Not polymer-containing
Zinc polycarboxylate Zinc oxide + aq. poly(acrylic acid) Acid-base Chemically adhesive to tooth
substance – see
Section 23.2.2
Glass-ionomer FAS glass + aq. polyalkenoic acid Acid-base Chemically adhesive – see
Section 23.3.2
Resin-modified FAS glass + aq. polyalkenoic acid with, Acid-base + Improved mechanical
glass-ionomer e.g., pendant methacrylate groups polymerization properties compared to
(Figure 23.17) glass-ionomer
Polyacid-modified Resin composite + unreacted Free radical addition See Section 23.2.3.6
resin composite glass-ionomer type constituents polymerization
(“compomer”)
Polymer-based Monomers (dimethacrylate) and Free radical addition Similar to tooth restorative
adhesives inorganic fillers polymerization materials, but with lower
filler content (Section 23.7.1)
Adhesive polymers Contains bifunctional adhesive Free radical addition Adhesion to metals as well
monomer such as 4-META polymerization as tooth substance
(Figure 23.8) or 10-MDP (Figure 23.6) (Section 23.3.2.5)
Source: Hill, E.E., Dent. Clin. N. Am., 51, 643, 2007.

Polyacid-
Resin-
Resin modified Glass-
modified
composites resin ionomer
glass-
composites cements
ionomers
(compomers)
Setting Polymerization Acid-Base

Polymerization Polymerization
Mechanism(s) and Acid-Base Reaction
Involved in
Inorganic Chemically inert Chemically inert Involved in acid-
acid-base
constituent during setting during setting base reaction
reaction
Structure of set
Composite Composite Composite Composite
material
Aqueous No aqueous
No Yes Yes
phase? phase as placed
Low potential for Very limited
Fluoride release release of scope for fluoride Fluoride release Fluoride release
fluoridea release
Mechanical Quite tough Not as tough as Not as brittle as Quite brittle
properties materials resin composites glass-ionomers materials
Tooth
Luting, some
restorations, Some tooth Luting, some
Applications tooth
fissure sealants, restorations tooth restorations
restorations
adhesive resins
a Note that some resin composites have been developed with special additives (e.g., YbF3), claimed to
release fluoride in vivo.
FIGURE 23.15 The “continuum” of tooth-colored restorative materials.
23.2.3.3 Resin Composites

In many applications, such as sealants, restorative materials, orthodontic bonding and adhesive
cements, a dimethacrylate polymer is employed, following the pioneering work of Bowen [25]. The
main difunctional monomers are BisGMA (or derivatives thereof) or UDMA (Figure 23.4). These
monomers are very viscous, so are diluted with TEGDMA (Figure 23.4) to facilitate the incorpora-
tion of particulate inorganic filler, which may comprise up to 86 mass% of the material. The types
of filler particles and their functions are classified in Section 23.7.1. To aid bonding between the
two phases of the composite, the inorganic fillers are treated with a silane coupling agent (typically
3-methacryloxypropyl trimethoxysilane—Figure 23.16). Other constituents include polymerization
initiators and stabilizers, as discussed earlier. Materials for restoring teeth usually contain pigments
to enhance the esthetic appeal of the material.
OCH3
O Si OCH3
OCH3
O
3-Methacryloxypropyl
trimethoxy silane
FIGURE 23.16 Silane coupling agent.

COOH COOH COOH COOH
FIGURE 23.17 Modified poly(alkenoic acid) with pendant methacrylate group.
23.2.3.4 Glass-Ionomer (Polyalkenoate) Cements

These materials have two potentially useful properties for dentistry. First, the ability to leach fluo-
ride to the surrounding tooth tissue can have great potential benefit for the prevention or reduction
of tooth decay (caries), although the relevance of in vitro studies to long-term clinical benefits is not
well established [26]. Second, they are capable of chemical adhesion to many materials, including
tooth material, as discussed later (Section 23.3.2).
23.2.3.5 Resin-Modified Glass-Ionomer Cements
Subsequently, materials have been developed in which both an acid-base reaction and a polymerization
reaction occur—these are termed resin-modified glass-ionomers. They can contain 2-hydroxyethyl
methacrylate (HEMA, Figure 23.9), which can be polymerized because of the incorporation of a
photo-sensitive initiator. Additionally, some materials contain poly(acrylic acid) on to which are
grafted branches with vinyl groups (Figure 23.17). In such cases, co-polymerizaton with HEMA
can occur, in addition to the acid-base reaction (Figure 23.18). This modification has the effect of
producing tough materials, which retain the merits of fluoride leaching and chemical adhesion [27].
23.2.3.6 Polyacid-Modified Resin Composites (“Compomers”)
Compomers are essentially a subset of resin composites, which have been modified by the incor-
poration of additional monomers with acidic functional groups [28]—for example, the di-ester
of 2-hydroxyethyl methacrylate with 1,2,3,4-butanetetracarboxylic acid or a cycloaliphatic dicar-
boxylic dimethacrylate [29]. These additional monomers comprise less than 10 wt% of the total
material. In addition, some reactive glass powder, as used in glass-ionomers, is included. When
compomers were introduced they were sometimes wrongly confused with “glass-ionomers” [30].
The materials harden by free radical addition polymerization only, usually with visible light
activation (as for composites, but contrast with resin-modified glass-ionomers). One product uses
a redox system [31]. Once the material has polymerized, it can absorb water, and in the long term,
some glass-ionomer type reaction can occur, giving some potential for the release of fluoride.
23.2.3.7 General Comparison of Material Properties
Given that the aforementioned four classes of material are often recommended for multiple applica-
tions, it is helpful to compare and contrast their properties, before considering their dental applica-
tions. Some very general comparisons can be given:
• In terms of adhesion to tooth substance, the materials containing polyelectrolytes (glass-

ionomer and resin-modified glass-ionomer) are chemically adhesive (Section 23.3.2.1).
• Materials with an aqueous phase (glass-ionomer and resin-modified glass-ionomer) have
the potential for fluoride release.
• Mechanical properties such as fracture toughness are usually superior for the resin com-
posites compared with the polyelectrolyte materials [32].
23.2.3.8 Critique of the Concept of a “Continuum”
Although the concept of a continuum is helpful in understanding the origin of each type of material, and
in comparing and contrasting properties, it needs to be noted that there is a discontinuity in the diagram,
represented by a vertical line in Figure 23.15. To the right of this line the materials contain an aqueous
phase, and to the left they do not; this distinction is significant in terms of potential for fluoride release.
COOH COOH COOH COOH
Acid-base
reaction + FAS glass
COO COO COOH COO

Al Al
HOOC COO OOC COO
Cross-linking by
visible light cure + HEMA
and/or autopolymerization
COO COO COOH COO
Al Al
HOOC COO OOC COO
FIGURE 23.18 Mechanisms of setting of a resin-modified glass-ionomer.
23.3 INTERFACES AND ADHESION

23.3.1 Applications and Dental Significance
In the last 50 years, there has been considerable emphasis on the development of adhesive materials for
dentistry [33]. In relation to bonding to tooth tissue it was realized, for example, that non-adhesive
dental restorations could not seal the tooth cavity, resulting in “microleakage” of saliva, bacteria and
bacterial toxins to the pulp of the tooth. Thus postoperative sensitivity, recurrent decay and staining
at the margins of the restorations can occur.
In addition to bonding to teeth, the following applications of adhesion technology involving
polymerizing materials are important:
• Bonding to surfaces of ceramic and alloy restorations and prostheses that are fabricated in
a laboratory before being cemented in place.
• Adhesion between the phases of a composite material—for example, by the use of a silane
compound as already discussed.
TABLE 23.3
Principal Dental Applications of Adhesion
1. Chemical adhesion
a. Glass-ionomer cements to tooth substance
b. Adhesive resins to alloys
c. Silane agents to ceramics
d. Soft tissue adhesives
2. Micromechanical adhesion
a. Adhesion to etched enamel
b. Contributes to dentin bonding
c. Abraded alloy surfaces
d. Etched ceramics (HF)
3. Hybridization bonding
a. Bonding to dentin
There are essentially three mechanisms of adhesion, and each is applied to the bonding to tooth
tissue:
• Chemical adhesion, where is chemical attraction between adhesive and adherend.

• Micromechanical adhesion, where a material is applied to a roughened or porous surface;
on setting a micromechanical bond is formed.
• Hybridization bonding, in which the adhesive is absorbed by the adherend; the adhesive
sets to form a hybrid of adhesive and adherend.
It should be noted that (1) surface preparation is critical to good bonding and (2) in some instances
more than one of the mechanisms may be responsible for the observed bonding between materials.
Table 23.3 lists the dental applications of adhesion in terms of mechanism of action; in very many
instances a polymeric material has been developed for this purpose.
23.3.2 Mechanisms and Materials

23.3.2.1 Chemical Adhesion to Tooth Tissue
The mechanism of the chemical bonding of the polyelectrolyte cements has been explored by Wilson
et al. [34]. They studied the interaction between polyacrylate ions and hydroxyapatite (the main con-
stituent of tooth enamel and a significant component of dentin), and postulated that polyelectrolyte
chains are embedded in the enamel or dentin surface with the release of calcium and phosphate ions
(Figure 23.19). The pretreatment of dentin with a dilute poly(acrylic acid) conditioning solution did
not improve clinical bonding [35].
23.3.2.2 Micromechanical Adhesion to Tooth Enamel

This depends on roughening of the surface of enamel, followed by the application of a mono-
meric material that can flow into pores on the rough surface, and polymerize in situ, resulting in
a micromechanical lock between the materials. This is widely used in sealants, orthodontics and
restorative dentistry. It is based on the discovery by Buonocore in 1955 [36] that enamel can be
roughened by the application of phosphoric acid (Figure 23.20). Resins applied to such a surface
form “prism-like” tags both in vitro [37] and in vivo [38]. Though this technique was not devel-
oped or applied for quite a number of years, it is now very widely applied (see Sections 23.4.1,
23.5.1, and 23.6).
Ca2+ Ca2+ Ca2+

O O O O O O
Hydroxyapatite
P P P
surface
O O O O O O
H
CH2 C
COO n
COO
CH2 CH2
CH CH
O O + Ca2+ and PO43–
Hydroxyapatite
C P C
surface
O O O O O O
FIGURE 23.19 Suggested mechanism for the adhesion of polyelectrolyte cements. (After Wilson, A.D.
et al., J. Dent. Res., 62, 590, 1983.)
36% HP 5.0 kV × 7.00 k SE(M) 5.00 μm
FIGURE 23.20 Enamel etched by 36% phosphoric acid gel. (Courtesy of J. Perdigão.)
23.3.2.3 Hybridization Bonding to Tooth Dentin

23.3.2.3.1 Nature of Dentin
A number of approaches have been used to bond to dentin, which has been the subject of intensive
research for many years [33]. The first factor to be considered is how to treat the dentin surface for
bonding. Dentin consists of a composite of hydroxyapatite particles in a matrix of collagen. It has
fluid-filled tubules that converge toward the pulp. It is imperative that any adhesive system should
ensure that the tubules are sealed to prevent fluid movement in either direction. Following dental
instrumentation of dentin, a smear layer is formed, composed of denatured collagen and mineral.
This penetrates into the dentinal tubules to form smear plugs [39]—see Figure 23.21. An important
fundamental question is whether the smear layer should be removed or retained before bonding.
Another factor to be considered for a bonding system is that it should be hydrophilic, given
that the dentin contains water, but also hydrophobic to be compatible with a polymer-containing
(a)
(b)
(c)
Smear 4.5 kv 12.5 mm × 11.0 k SE(M) 5.00 µm
FIGURE 23.21 Smear layer and smear plugs. a, smear layer; b, smear plug; c, dentinal tubule. (Courtesy of
J. Perdigão.)
restorative material. Thus, many monomers are used in the different products, which contain a
polymerizable group (e.g., acrylate or methacrylate) and also hydrophilic functional groups—which
are shown and classified later in the chapter in Figures 23.5 through 23.9. However, if a material is
too hydrophilic, there may be increased degradation of the resin–dentin bond [40].
23.3.2.3.2 Historical Developments

Early work in dentin bonding proposed glycerophosphoric acid dimethacrylate as a dental adhesive
[41,42] studied the interaction of this compound with dentin. The first commercial adhesive mate-
rials contained phosphorylated methacrylate monomers [43]. However, the resultant shear bond
strengths were low (typically 5–7 MPa, about one-third of the strength of the bond to etched enamel)
[43] because the hydrophobic monomer did not penetrate the water-containing smear layer [44].
Fusayama [45] introduced the idea of preparing dentin by etching it with phosphoric acid (the
enamel of the cavity can be simultaneously etched), and this led to adhesive systems that are clas-
sified as “etch and rinse”; that is etching (or conditioning) is followed by copious rinsing to remove
any reaction products. This procedure has a significant effect on the smear layer; it removes the
smear layer and demineralizes the most superficial hydroxyapatite crystals [46]. As a result, the con-
ditioned dentin can be readily infiltrated by monomers. Nakabayashi et al. etched dentin with a 10%
citric acid and 3% ferric chloride mixture and applied 4-META (see Figure 23.8), a monomer with
both hydrophobic and hydrophilic groups [47]. These authors demonstrated the infiltration of mono-
mers into the treated dentin; these monomers polymerized in situ to form a hybrid layer composed
of resin, collagen, residual hydroxyapatite and a small quantity of water. The resultant tensile bond
strength was 18 MPa [47]. This hybrid layer (Figure 23.22) was reported to be about 10 μm in thick-
ness, following etching with 20% H3PO4 for 30 s [48]. In contrast, Hashimoto et al. reported a hybrid
layer of <5 μm after 30 s etching with 35% H3PO4 [49]. Subsequently “self-etching primers” were
developed—for example, it was shown that an aqueous solution of 20% phenyl-P (Figure 23.6) and
30% 2-hydroxyehtyl methacrylate (Figure 23.9) creates diffusion channels in the dentin, which can
be infiltrated by monomers [50]. However, durability of bonding was a concern—if the dentinal col-
lagen is not completely protected by resin it can be hydrolyzed, causing defects in bonding.
23.3.2.3.3 Current Materials: Classification and Constituents

Today, there is a wide range of available dentin bonding systems and the details of their composi-
tion have been reported [7,46]. They may be classified into four types, which are illustrated in
Figure 23.23 together with the practical stages involved in the application of each. The first two
(a)
(b)
(c)
XP-WET-3 5.0 kv 12.3 mm × 2.50 k SE(M) 20.0 µm
FIGURE 23.22 Bonding to dentin. a, resin composite restorative material; b, adhesive layer; c, hybrid layer.
(Courtesy of J. Perdigão.)
Total etch
3-step
Apply Apply Apply
etch and Wash
conditioner primer adhesive
rinse
2-step Apply combined

etch and Apply Wash
conditioner primer and adhesive
rinse
Self–etch
2-step Apply
Apply self-etching primer
self etch adhesive
1-step Apply combined self-etching primer

self etch and adhesive
FIGURE 23.23 Classification of dentin bonding systems.
types involve removal of the smear layer (“etch and rinse” systems), and the second two do not (“self
etch”). One of the objectives of these developments has been to reduce the number of treatment
steps, with the potential to reduce treatment errors (but see the following text). Using the classifica-
tion of Van Landuyt et al. [7] the materials are
• Three-step etch and rinse adhesives; a conditioner (typically a 35% phosphoric acid gel),
similar to the etchant for enamel. After washing off the debris, a primer is applied, fol-
lowed by an adhesive.
• Two-step etch and rinse adhesives; the primer and adhesive are combined, thus eliminating
one step of the practical procedure.
• Two-step self-etch adhesives, in which the conditioner and primer are combined (“self-
etching primer”).
• One-step self-etch adhesives—a single material is applied to the dentin.
It should be noted that self-etch systems are also being recommended for application to enamel.
Dentin bonding systems contain the following types of constituents, though not all products
utilize all these components types [7]:
• Dimethacrylate monomers(s) such as Bis-GMA, UDMA, and TEGDMA (Figure 23.4)—

these are the same monomer types as used in resin composites (see the following text).
These monomers enable the bonding material to covalently bond to the composite restor-
ative material.
• A hydrophilic monomer that is polymerizable, particularly HEMA (Figure 23.9).
• A few products contain polyalkenoic acids, capable of chemical bonding to the calcium of
hydroxyapatite.
• Methacrylate monomers with functional groups—including those detailed in Figures 23.5
through 23.8. Note that these monomers contain both hydrophilic and hydrophobic groups.
• Inorganic fillers (also used in resin composite restorative materials).
• Solvating agent (alcohol/water, acetone).
• Polymerization initiators, for example, for visible light curing.
23.3.2.3.4 Durability
Reporting in 1988, Van Meerbeek et al. claimed that there had been important improvements in
materials, particular in terms of retention [51]. However, no material could prevent microleakage
over a long time period. More recently, it was claimed that the materials with fewer practical steps
were not as effective [52]. To quote, “Simplification so far appears to induce loss of effectiveness” [53].
De Munck et al. [54] regard three-step etch and rinse systems to be the “gold standard.”
23.3.2.3.5 Current Concerns and Developments

A major problem is that when polymerizing, a resin composite undergoes shrinkage, and this places
stress on the bond to dentin. In the later discussion on composites (Section 23.7.1.1.4), the attempts
to minimize this problem are discussed.
The long-term stability of bonding is also an issue, particularly the hydrolytic stability of chemi-
cal bonding to hydroxyapatite. Inoue et al. [55] and Van Landuyt et al. [56] showed that durability
of bonding depends on the chemical bonding of the functional monomer. Yoshida et al. [57] deter-
mined that 10-MDP formed very stable bonds with hydroxyapatite, superior to the performance of
4-MET and phenyl-P.
There are some issues of concern in relation to one-step self etch adhesives. Those that do not
contain HEMA are prone to display droplets within the material, attributed to phase separation
between monomer and solvent; this can compromise bond durability [58]. In adhesives that were
HEMA-rich, droplets were also observed due to water absorption from dentin, caused by osmosis [59].
The term nanoleakage has been introduced to describe nanometer-sized spaces filled with water [60]—
distinguish from microleakage described earlier.
Although the presence of HEMA can prevent phase separation, its allergenic potential is a concern.
Due to low molecular weight, it can penetrate through gloves to cause contact dermatitis [61].
Currently, the problem of degradation of the hybrid layer is being investigated. When the dentin
bonding system used acetone as solvent, extensive degradation is observed; this can be reduced
by the application of chlorhexidine gluconate [62]. It is believed that this compound increases the
stability of the collagen fibrils in the hybrid layer against endogeneous metaloproteinases [63].
In the etch-and-rinse adhesives, primer or combined primer-adhesive is applied to moist dentin.
Replacing water with ethanol has been reported to increase the durability of bond strength [64].
23.3.2.4 Bonding to Ceramics

Ceramics based on feldspathic porcelain can be etched with hydrofluoric acid, then treated with a
silane agent (Figure 23.16), which bonds both to the ceramic surface and to the resin-based com-
posite cement adhesive [65].
However, many modern tougher dental ceramics are based on alumina or zirconia, which are
not capable of being etched by HF. For glass-infiltrated alumina restorations, currently there is a
choice of bonding technique—either coat the surface with silica, apply a silane compound, and use
an adhesive based on bis-GMA resin or roughen the surface with airborne Al2O3 particle abrasion,
followed by application of a phosphate-containing polymer cement. For zirconia-based materials,
this latter procedure appears to be the best available at this time [66].
23.3.2.5 Bonding to Metallic Materials

Silica coating, as described earlier, can also be applied to cast dental alloys, followed by silane treat-
ment. Alternatively, tin plating has been applied. An adhesive resin cement, containing 4-META or
10-MDP may be employed [67].
23.4 PREVENTION OF DISEASE AND THE EFFECTS OF TRAUMA

23.4.1 Pit and Fissure Sealants
23.4.1.1 Purpose
Dental decay (caries) has been described as the most common disease of school children [68].
Rough pit and fissure surfaces of teeth are difficult to clean, and are therefore more susceptible to
caries. In addition, fluoride protection is less beneficial in protecting pits and fissures, compared
with smooth enamel surfaces. Epidemiological research has emphasized the need for some means
of protection of pits and fissures [69].
This problem has been tackled by the application of sealants. A fissure sealant has been defined as “a
material that is placed in pits and fissures of teeth in order to prevent or arrest the development of dental
caries” [70]. These polymeric materials are applied to the pits and fissures that are prone to decay. They
provide a physical barrier that protects pits and fissures from microorganisms and particles of food [71].
However, although sealants have been available for over 30 years, they were described in 2005 as being
“perhaps the most tested yet most underused technique in clinical preventive dentistry” [72].
Early attempts at pit and fissure sealing included the use of materials containing methyl-2-cyano-
acrylate. However, clinical tests showed “almost total failure of the fissure sealant” [73].
Representatives of all the groups of material within the “continuum” of tooth-colored materials
(Section 23.2.3) have been advocated as sealants. However, materials based on dimethacrylates are
the most widely used and thus will be considered first, followed by a critique of alternative materials.
23.4.1.2 Classification of Dimethacrylate-Based Sealants

There is a plethora of different materials, often confusingly designated as “first generation,” “sec-
ond generation” etc. Differences have occurred in activation system. Some early materials were
activated by UV radiation. However, there were two grounds for their criticism. In the first place,
the safety of using 365-nm radiation was called into question because of potential biological dam-
age such as skin cancer, eye damage and mutagenic affects [74]. Furthermore, the efficiency of
the curing lights was sometimes poor because of insufficient intensity of radiation [75].
In today’s materials, there are many different choices—for example,
• There is a choice of “self-cure” vs. visible light cure (VLC) materials.

• Filled and unfilled polymers are available; the filled materials resemble resin composite
restorative materials (Section 23.7.1), but usually with lower filler concentration. In fact, one
type of composite restorative material (flowable composites) is being suggested for sealant use.
• Sealants may be clear or opaque or tinted. Some change color—for example, from clear
to a green color, or from pink to opaque white, after polymerization, in order to ensure
adequate placement of sealant.
• Some sealants contain fluoride.
23.4.1.3 Biocompatibility
Free-radical addition polymerized sealants often have an oxygen-inhibited layer, estimated to be
from 7–84 μm in depth [76]. Is this harmful? A major concern has been the claim that sealants
may leach compounds that are estrogenic, such as bisphenol-A (2,2-bis[4-hydroxyphenyl]propane
(BPA), a component in the synthesis of Bis-GMA. Olea et al. [77] claimed that “the use of bis-
GMA resins in dentistry, and in particular the use of sealants in children, appears to contribute
to human exposure to xenoestrogens.” Joskow [78] reported that one brand of sealant leached
BPA. However, more recently, a systematic review has shown that there is no risk of exposure
to BPA from sealants, though removal of the soft layer of un- or under-polymerized material is
recommended [79]. It has also been shown that the formation of an oxygen-inhibited layer can be
prevented by placing a thin layer of oil to form an air barrier, and then shining the polymerizing
light through this barrier [80].
23.4.1.4 Bonding to Enamel and Retention Rates

Having looked at the safety of sealants, it is now necessary to consider efficacy. We consider how well
they are retained on enamel and whether there has been success in caries prevention. A Cochrane
evidence-based review showed that caries reduction from 87% at 1 year to 60% at 4–4½ years can
be achieved. Retention of the sealant on the enamel was from 79% to 92% after 1 year, 71% to 85%
at 2 years, and 61% to 80% at 3 years [81].
23.4.1.5 Effect on Bacteria Levels in Caries Lesions

The efficacy of sealants has assumed their application to sound enamel. What if inadvertent seal-
ing over dental caries is carried out? Consistent with the finding that bacteria levels are reduced if
covered by sealants [82], a review by Griffin et al. supported the technique of placing sealants over
caries lesions that were not cavitated [83].
23.4.1.6 Application of Self-Etch Adhesives

Since self-etch adhesives have been introduced for bonding to dentin (Section 23.3.2), it was sug-
gested that they could be employed for bonding sealants to enamel [84–87].
23.4.1.7 Application of Compomers

In his review on compomers, Nicholson [28] claimed that these materials performed well in a num-
ber of applications, including fissure sealing, despite a decrease of strength in vivo associated with
water uptake. Two short-term clinical trials appear to confirm this [88,89], but the outcomes of
longer term clinical data are awaited.
23.4.1.8 Application of Polyelectrolyte Materials

Glass-ionomer and resin-modified glass-ionomer cements have two features that render them poten-
tially attractive as sealants—namely, their adhesion to enamel and their ability to release fluoride.
Thus, they have been tested as fissure sealants. A systematic review showed that neither resin-based
or glass-ionomer was superior to the other in the prevention of dentin lesion development [90].
However, the retention of resin-based sealants (by micromechanical adhesion) has been shown to be
greater than that of chemically adhered glass-ionomers [91].
23.4.1.9 Summary of Current Clinical Recommendations

Evidence-based clinical recommendations have been published by the American Dental Association
Council on Scientific Affairs [92].
• Sealants are recommended for pits and fissures of both primary and permanent teeth if
there is risk of caries development. They can also be applied to early carious lesions, to
reduce the probability of progression of the lesion [83,92].
• Although glass-ionomer cement can be used as interim preventive agent, particularly if
moisture control during placement is difficult, resin-based materials should be the first
choice of material [93].
• In relation to bonding to enamel, if self-etching bonding agents are used without the sepa-
rate step of enamel etching, there may be less retention that the standard acid-etch proce-
dure. If such bonding agents are used on already etched enamel, sealant retention would
be enhanced [92].
• For maximum effectiveness, it is important that sealant be monitored regularly, and reap-
plied as necessary [92].
23.4.1.10 Research Needs

Beauchamp et al. [92] have also listed numerous research needs, including studies of preventive
effectiveness and cost-effectiveness, timing of sealant application, and exploration and evaluation
of new materials.
23.4.2 Mouth Protectors
23.4.2.1 Purpose and Requirements
Mouth protectors (sometimes referred to as mouth guards or gum shields) are recommended for
participants in many types of sport to prevent orofacial injuries and concussion. A mouth protec-
tor have been defined by the American Society for Testing and Materials as a “resilient device or
appliance placed inside the mouth (or inside and outside), to reduce mouth injuries, particularly
to teeth and surrounding structures” [94]. The American Dental Association has listed some
29 sporting activities that pose hazards resulting from a blow to the head or mouth [95].
The ideal requirements for a mouth protector include its function, its fit and its durability.
In terms of function, the material must have the ability to absorb impact. In terms of fit, there
should be accurate adaptation to the individual’s mouth structures and should have smooth well-
contoured edges. The protector should be comfortable to wear and not interfere with speech or
breathing. The material should resist tearing and should be easily cleanable to avoid unpleasant
odors or tastes.
23.4.2.2 Materials
Over the years, five types of polymer have been advocated for mouth protector construction [96,97].
These include
• Ethylene-vinyl acetate copolymer (EVA)—this is currently the most widely used material,
owing to its ease of fabrication.
• Poly(vinyl chloride)—noting that there are concerns relating to the biocompatibility of
phthalate esters used as plasticizers—these materials have been banned in the European
Union [98].
• Natural rubber.
• Soft acrylic resin.
• Polyurethane.
23.4.2.3 Classification
Mouth protectors can be classified into three types [94]:
1. Type I—Thermoplastic type. These may be either mouth-formed or vacuum-formed.

2. Type II—Thermosetting type.
3. Type III—Stock type.
It should be noted that alternative classifications have been given by the American National
Standards Institute and Standards Australia International [99].
Of these three types, Type III is generally considered to be the least satisfactory. Such materials
do not provide good fit and comfort, may not stay in place comfortably, as they have no retention and
they can interfere with mouth breathing and speech [97]. Currently, the choice is between mouth-
formed custom-made protectors.
23.4.2.3.1 Mouth-Formed Protectors
There are two varieties of mouth-formed protectors. Some are thermoplastic “boil and bite” materi-
als, usually an EVA copolymer. The material is softened in hot water, briefly cooled and then shaped
in the mouth [100]. A problem with this approach to fabrication is that the material may become
over-thinned in some regions, thus giving insufficient protection to the wearer [97].
Alternative mouth-formed protectors are the shell-liner materials, in which a PVC shell contains
an inner lining of a resilient material such as a plasticized acrylic or a silicone rubber [100].
23.4.2.3.2 Custom-Made Protectors
These are frequently prepared on a gypsum model or cast, using low heat and vacuum, using EVA.
Sometimes, there may be insufficient thickness of material in the formed protector. This problem
can be minimized by using a pressure lamination technique [100].
23.4.2.4 Properties
EVA is a random co-polymer produced by high-pressure free radical polymerization. Properties
are dependent on the vinyl acetate content—as this is increased it disrupts the polymer crystallin-
ity, which has important consequences for the properties of the co-polymer. Increase vinyl acetate
content (up to 28%) linearly decreases the hardness (Figure 23.24), flexural modulus (Figure 23.25),
and softening temperature (Figure 23.26), and increases the impact strength (Figure 23.27) [101].
23.4.2.5 Conclusion
A recent extensive meta-analysis by Knapik et al. [102] has concluded that the “overall risk of an
orofacial injury is 1.6–1.9 times higher when a mouthguard is not worn, relative to wearing a mouth-
guard.” It is as yet unknown if mouthguards afford protection from concussion.
100
95
Hardness (shore A)
90
y = –0.5298 x + 99.034
85 R2 = 0.9582
80
0 10 20 30
% PVA
FIGURE 23.24 Effect of quantity of PVA in EVA copolymer on hardness.

200
Flexural modulus (MPa)

150 y = –4.9318x + 143.56
R2 = 0.9397
100
50
0
0 10 20 30
% PVA
FIGURE 23.25 Effect of quantity of PVA in EVA copolymer on flexural modulus.
100
Softening temperature (°C)
y = –1.7071x + 93.723
R2 = 0.968
80
60
40
0 10 20 30
% PVA
FIGURE 23.26 Effect of quantity of PVA in EVA copolymer on softening temperature.
50
y = 0.7585x + 26.49
R2 = 0.9847
Tensile impact (J/cm3)
40
30
20
0 10 20 30
% PVA
FIGURE 23.27 Effect of quantity of PVA in EVA copolymer on impact energy.

23.5 ORTHODONTICS
23.5.1 Orthodontic Adhesives
The role of polymers in orthodontic treatment is now considered. Orthodontics deals with the treat-
ment of malocclusion and other irregularities of the teeth and jaws. Polymer-containing materials
are used in orthodontics as agents to bond brackets to tooth enamel (Figure 23.28), and also as
cements to retain bands around teeth (Figure 23.29). The requirements for orthodontic adhesives are
quite stringent, and include good biocompatibility and durable adhesion to enamel.
As with pit and fissure sealants (see the earlier text), cyanoacrylates were tested and found to
lack bonding durability [103]. Today, all four classes of material in the “continuum” (see the earlier
text) are employed.
For cementing orthodontic bands, a recent systematic review has found insufficient evidence
to establish which adhesive is the most effective adhesive for bands, though polyelectrolyte
FIGURE 23.28 Orthodontic brackets cemented to tooth enamel. (Courtesy of J. Beyer.)
FIGURE 23.29 Cemented orthodontic band. (Courtesy of J. Beyer.)

FIGURE 23.30 Orthodontic elastics. (Courtesy of J. Beyer.)
materials can adhere to both enamel and stainless steel, can release and take up fluoride, and have
antibacterial properties [104].
By way of contrast, in relation to the bonding of brackets, there is widespread use of polymer-
izing materials, using a range of bonding techniques (acid etch, self etch, etc.). However it has been
predicted that in the future use of compomer and glass-ionomer based materials will increase [105].
The potential iatrogenic effects of direct orthodontic bonding need to be considered.
• Is there danger from BPA, as was once alleged for fissure sealants? Eliades et al. [106] have
measured BPA release from orthodontic adhesives and demonstrated the absence of any
estrogenicity [107].
• One problem is that decalcification of enamel (white spot lesions) can occur during orth-
odontic treatment [108]. Reportedly this can be reduced by the application of a fluoride-
containing varnish [109] or a fissure sealant [110].
• Is there permanent damage to enamel after debonding? Studies have shown that enamel
cracking [111] and facture [112] can occur.
23.5.2 Orthodontic Elastics
Orthodontic elastics are used in some treatments to aid tooth movement (Figure 23.30). Traditionally
these have been made of cis-polyisoprene (latex). However there is concern about hypersensitiv-
ity [113,114]. As a result synthetic non-latex elastics are available, reportedly containing styrene-
butadiene with mineral oil and small amounts of stearic acid, magnesium carbonate, silicic acid, and an
antioxidative stabilizer [115]. As a result of dynamic mechanical testing it was shown that the synthetic
materials need more frequent replacement than the latex products because of force loss [115,116].
23.6 ESTHETIC DENTISTRY: VENEERS

A veneer is a thin layer of an esthetic material, which is placed over a tooth surface, to mask tooth
defects or discoloration (e.g., tetracycline staining), to correct the appearance of uneven or crooked
teeth, to cover chips in teeth and to close diastemas. They play a very significant role in elective
dental esthetics [117]. A merit of this treatment approach is that there is little or no removal of tooth
structure—up to about 0.5 or 0.75 mm [117,118].
Veneers may be made from translucent ceramics such as feldspathic porcelain, cemented into
place with a visible light cured polymeric material, in conjunction with enamel etching and silane
treatment of the ceramic. In terms of clinical durability, long-term clinical success has been claimed
[119]. Survival rates of veneers up to 16 years have been determined [120].
Alternatively, tooth bleaching followed by application of a resin composite (see Section 23.7.1.)
may be used in certain circumstances, as a more conservative approach to esthetic dentistry [121].
23.7 TOOTH RESTORATION

23.7.1 Tooth Filling Materials
For many years, the main options for the restoration of teeth were silver amalgam for posterior teeth
and silicate cements for anterior teeth. Polymer-containing materials have now completely replaced
silicates for anterior restorations, and have also partially displaced amalgam. The early history of
usage of polymers for tooth restoration has been recorded by Puckett et al. [122].
The requirements for materials for restoring teeth are stringent. Such materials are in an aggres-
sive environment, encountering dietary acids, plaque-forming bacteria, and enzymes; in addition,
masticatory forces have the potential to cause degradation. In addition to biocompatibility, and the
ability to adhere durably to tooth tissue, chemical stability, mechanical toughness, surface wear
resistance, esthetic properties, and ease of handling are also important.
In this section, each of the four classes in the “continuum” (Section 23.2.3.2) is considered and
critiqued, with the greatest emphasis on resin composites. Emphasis is placed on the potential limi-
tations of these materials, and the research efforts to bring about improvements. Also some new
types of material that have become recently available are then reviewed.
23.7.1.1 Resin Composites

The basic principles of these materials have already been presented (Section 23.2.3.3). As supplied
they consist of dimethacrylate monomers mixed with silane-coated inorganic particles, initiators,
stabilizers and pigments. Early materials were supplied as two pastes with a peroxide/tertiary amine
redox system. Most current products are visible light cured. It should also be noted that indirect
composite resins are available for laboratory fabrication [123].
23.7.1.1.1 Classification by Inorganic Filler Content

Fillers have included silica, barium glass, strontium glass, and zirconia/silica. Composites are
often classified according to their filler content [124]. Table 23.4 classifies current materials.
Different particle size and size distribution have been used, including macrofilled materials
(Figure 23.31), microfillers (Figure 23.32), a combination of different filler sizes (Figure 23.33)
and nanofillers (Figure 23.34). The functions of these fillers include: improvement in mechanical
properties such as compressive strength and hardness, reduction in shrinkage associated with
polymerization and conferment of radiopacity on the material if a heavy metal (Ba, Sr, Zr) is
TABLE 23.4
Classification of Resin Composite Tooth Restorative Materials
according to Their Filler Content
Class of Composite Particle Size of Filler
Macrofilled 1–50 μm (e.g., Ba glass, SiO2)
Large particle hybrid 1–20 μm glass + 0.04 μm SiO2
Midifilled hybrid 0.1–10 μm glass + 0.04 μm SiO2
Minifilled hybrid 0.1–2 μm glass + 0.04 μm SiO2
Continuum filled 0.01–3.5 μm zirconia/silica (mean 0.6 μm)
Microfilled 0.04 μm SiO2
Nanofilled (translucent) 75 nm SiO2 + clusters of agglomerated 75 nm particles;
cluster size range = 0.6–1.4 μm
Nanofilled (radiopaque) Non-agglomerated 20 nm SiO2 + 0.6–1.4 μm clusters
of zirconia/silica particles of size 5–20 nm
Concise 5.0 kV 14.1 mm × 5.00 k SE(M) 10.0 µm
FIGURE 23.31 Macrofilled composite—see Table 23.4. (Courtesy of J. Perdigão.)
A110 5.0 kV 14.0 mm × 20.0 k SE(M) 2.00 µm
FIGURE 23.32 Microfilled composite—see Table 23.4. (Courtesy of J. Perdigão.)
present. Good esthetics depends on translucency of the material, but a mismatch of refractive
index between polymer and filler can be detrimental [125]. Developments in filler technology
have included the incorporation of fluoride, for example, ytterbium trifluoride.
23.7.1.1.2 Classification by Rheology

Composites have been produced with a range of consistencies. Flowable materials, with 52–68 wt%
filler are capable of being injected through a syringe. They may contain colloidal silica which con-
fers thixotrpy on the material [126] so that it is easy to syringe but will stay in place after syringing,
until it is adapted to the tooth cavity. There is considerable variation in available products in terms
of composition and properties [127]. Some of the lower viscosity products are being used as fissure
sealants. Others are being recommended for Class III and Class V restorations, as liners under other
restorations and for repairing small defects in indirect restorations.
In contrast, materials with higher viscosity have been produced; they are termed “packable com-
posites.” They were designed to mimic the handling properties of dental amalgam. Placement and
Grandio 5.0 kV 14.2 mm × 5.00 k SE(M) 3/1/09 10.0 µm
FIGURE 23.33 Hybrid filled composite—see Table 23.4. (Courtesy of J. Perdigão.)
SupremeA3D 5.0 kV 14.2 mm ×20.0 k SE(M) 2.00 µm
FIGURE 23.34 Nanofilled composite, showing clusters of nanoparticles—see Table 23.4. (Courtesy of
J. Perdigão.)
handling properties may be better than that of other composites for posterior restorations [128] but
they are not necessarily superior to them in mechanical properties [129].
23.7.1.1.3 Issues Relating to Biostability, Biocompatibility,

and Chemical and Mechanical Durability
It needs to be realized that the conversion of monomer to polymer is not a chemically efficient reac-
tion since 25%–60% of methacrylate groups remain unreacted [130]. Thus, there is concern about
the elution of monomers; lower molecular weight monomers are more likely to be extracted than
higher molecular weight ones [131]. TEGDMA has been shown to be extracted from composites
[132,133], with potential toxic effects on gingival fibroblasts [134]. TEGDMA and HEMA have
been shown to express cyclooxygenase-2 (COX-2) which is involved in inflammatory reactions [135].
Another issue is degradation of polymers in the oral environment—that is—biostability. Santerre
et al. [136] have listed the potential types of polymer degradation processes. Of particular interest
is the possibility for breakdown of the silane coupling agent and also enzymatic degradation of
polymer components.
OCH3
O Si
OCH3
OCH3
3-(3-methoxy-4-methacryloylphenyl)propyl-
trimethoxysilane
FIGURE 23.35 Alternative silane coupling agent (cf. Figure 23.16).
To counteract the possibility of hydrolytic breakdown of 3-methacryloxypropyl trimethoxysilane

(Figure 23.16), Nihei et al. [137] have synthesized a silane with a hydrophobic phenyl group—
3-(3-methoxy-4-methacryloyloxyphenyl)propyltrimethoxysilane (Figure 23.35). They demonstrated
that composites with this silane had better wear resistance, which was attributed to the formation of
a hydrophobic layer on the filler surface.
Enzymatic degradation of the resin components of composites has been extensively studied, given
that saliva contains esterases [138] which can catalyze the hydrolysis of resin composites [139]. The
biochemical stability of a composite is dependent on the material’s composition [140]. Following
incubation in cholesterol esterase, degradation products such as methacrylic acid, TEGMA and
bishydroxypropoxyphenylpropane can be produced [140].
Chemical and/or biochemical breakdown can cause physical breakdown of the material. This has
been extensively reviewed by Drummond [141], including the effects of aging in different chemical
media, and cyclic loading.
23.7.1.1.4 Polymerization Shrinkage

It is well documented that composites contract during polymerization. Smaller monomer molecules
show the greatest shrinkage on polymerization. For example, the volumetric shrinkage of pure bis-
GMA and TEGDMA is 6.1% and 14.3%, respectively [142], or 5.2% and 12.5%, respectively [143].
For dental resin composites, the volumetric shrinkage is typically around 2% [144]. On polymeriza-
tion, a shrinkage stress results. Of particular importance is the stress developed after the material
has become solid—the so-called “post-gel” shrinkage [145] The stress depends not only on the
magnitude of the shrinkage but also on the viscoelastic properties of the material and the configura-
tion of the tooth cavity [143]. Deleterious outcomes can result from this phenomenon. These include
tooth deformation, debonding of the restoration, propagation of cracks in enamel and post-operative
sensitivity [146].
Substantial research has been, and is being, carried out to solve this problem. Approaches
include:
• The development of polyfluorinated prepolymer multifunctional urethane methacrylate—

reported to give low shrinkage and a high degree of conversion of monomer [147].
• Cyclopolymerizable monomers (oxy bis-methacrylates) have been synthesized
(Figure 23.36) and found to be capable of producing cross-linked polymers from bulk
polymerization. These have been tested in combination with Bis-GMA; less contraction
and good mechanical properties were claimed [148].
• The ring-opening polymerization of spiro orthocarbonates has been suggested as a means
of providing expansion to counteract the shrinkage associated with polymerizing dimeth-
acrylates [149–151]. By using bis-GMA derivatives (in place of bis-GMA) with spiro
orthocarbonates, Moon et al. [152] claimed to achieve reduction in shrinkage without com-
promise of mechanical properties.
X
CO2R
X
RO O OR
RO2C
O
O O
RO2C CO2R
O
n
FIGURE 23.36 Cyclopolymerization of an oxy bis-methacrylate monomer. (After Stansbury, J.W., J. Dent.
Res., 69, 844, 1990.)
• New high molecular weight dimethacrylate monomers, based on hydrogenated dimer

acid have been developed. Although these materials have low modulus of elasticity
and mechanical strength, they are claimed to low water absorption and polymerization
shrinkage [153].
• Urethane derivatives of bis-GMA have been shown to have favorable properties com-
pared with bis-GMA—namely, higher degree of polymerization and lower polymerization
shrinkage. However, lower flexural strength was reported [154].
Fluorinated monomers have been developed in attempts to improve the hydrophobicity

of the composite, hence potentially reducing its liability to degrade under oral conditions.
Early work led to hydrophobic polymers with poor mechanical properties [155]. Later research
[156] claims the development of fluorinated polymers with a high degree of polymerization
conversion.
The chemistry of the monomers used in both conventional and experimental materials has been
extensively reviewed by Peutzfeldt [130] and Moszner et al. [157].
Two chemical developments in composites have led to the production of clinically used dental
materials—namely, the so-called siloranes and ormocers. These are considered separately later.
23.7.1.2 Siloranes
The word silorane is derived from a combination of siloxane and oxirane. The silorane monomer
is shown in Figure 23.37. The siloxane component was chosen for its well known hydrophobicity,
and oxiranes are known to give stable polymers with low shrinkage on polymerization. Siloranes
can undergo cationic addition polymerization, in contrast to (di)methacrylate polymerization
which is usually a free radical reaction. The cationic initiator is a combination of camphorqui-
none (as used in visible light cure free radical systems), an iodonium salt and an electron donor.
In the presence of visible light, cations are generated. The reaction proceeds as shown in Figure
23.38 [158]. Volumetric shrinkage on polymerization is 1%—about half of that for typical com-
posites. This is attributed to the effect of the ring opening reaction. Fillers include quartz and
radiopaque yttrium fluoride [159]. Mechanical properties are similar to those of conventional
composites [160].
An adhesive system has been developed for use with the silorane composite. This includes a self-
etch primer containing phosphorylated methacrylates and a hydrophobic adhesive [161] aimed at
bonding the hydrophobic restorative material to the more hydrophilic tooth material.
O Si
Si O
O Si
Si O
FIGURE 23.37 Silorane monomer.
O-cation O-cation
O
O C
Cation+ C
+
O
+ etc.
FIGURE 23.38 Cationic polymerization of siloranes.
23.7.1.3 Ormocers*
An ormocer is an organically modified ceramic. These materials were developed by Wolter, Storch
and Ott [162], who report the synthesis of thioether- and urethane-(meth)acrylate silanes. The gen-
eral features of the chemistry of the latter compounds are outlined in Figure 23.39. In addition to
an ormocer, the commercially available dental composite includes a quantity of traditional diethac-
rylates (e.g., bis-GMA) and inorganic filers [159]. The material demonstrated good wear resistance
in in vitro testing [163].
23.7.1.4 Compomers
The chemistry of these has been discussed earlier (Section 23.2.3.6) [28]. Given that they are not
as tough as resin composites, their application is mainly in areas of the mouth where they will be
subjected to little or no stress—for example, in deciduous teeth and cervical and proximal restora-
tions [164].
* Ormocer® is a trademark of the Fraunhofer Gesellschaft, Germany.

R
Si
O
O
Si O O O
Si Si
O
O
Si
O
FIGURE 23.39 Ormocer structure.
23.7.1.5 Glass-Ionomers and Resin-Modified Glass-Ionomers

As recorded earlier (Section 23.2.3.7), these polyelectrolyte-based materials are more brittle than
resin composites and thus cannot withstand high forces [32]. However, different formulations of the
material type are very versatile, being used for provisional restorations, tooth cavity lining and base
materials, luting restorations, fissure sealants (Section 23.4.1) and bonding agents [165].
23.7.1.6 Summary of Data on Clinical Longevity of Tooth-Colored Restorations

The data for clinical longevity of polymer-containing tooth restorative materials present a challenge to
both clinician and research worker. It has been shown in clinical studies that resin composite is not as
durable as dental amalgam, and that “routine clinical practice may be producing suboptimal results” [166].
23.7.2 Endodontic Materials
When trauma or oral disease affects the pulp of the tooth, endodontic treatment needs to be carried out.
A root filling is placed with the aim of obturating the root canal system. Sealing of the root canal
is important to prevent micro-organisms and/or toxins from tracking from the coronal aspect of the
root to the root apex.
Traditionally, gutta percha [trans-poly(isoprene)] has been employed. Often this core material is
used in conjunction with a “sealer” composed of zinc oxide-eugenol cement. However, there is no
adhesion, either between the sealer and the dentin or between the sealer and the gutta percha. Thus,
there has been extensive work in attempts to develop adhesive systems to eliminate undesirable
leakage.
Alternative sealers include an epoxy-resin-based material and glass-ionomer cement [167]. The
former material shows some toxicity in the unset state [168,169]. The latter sealer is hard and adhe-
sive, but makes retreatment difficult if this should be necessary [170].
More recent developments include the development of endodontic restorations where the compo-
nents act as a single unit—this is termed a “monoblock.” One example is a composite core material
consisting of thermoplastic polycaprolactone with bioactive glass and bismuth and barium salts to
confer radiopacity. This is used in conjunction with a sealer which is a composite resin, based on filled
dimethacrylates—not dissimilar from the restorative materials discussed earlier (Section 23.7.1). This
system comes with a self-etch primer for dentin, composed of a sulfonic acid terminated monomer,
HEMA, water and initiator for polymerization. It is claimed that adhesion, both to the dentin and
between sealer and core, is obtained. However, in vitro fluid filtration tests on this system have shown
that it does not eliminate leakage [171]. Moreover, it has been shown this system, in common with
others poses “significant cytotoxic risks and that cytotoxicity generally increased with time” [172].
An alternative self-etch sealing material, based on 4-META technology, has been designed for
use with either gutta percha or polycaprolactone cores. However, it has been shown to have limited
etching potential, which may reduce the ability to seal [173].
A recent development is gutta percha coated with methacrylate resin, designed to be used with a
urethane dimethacrylate-based sealer [174].
Polyelectrolyte-containing materials have also been proposed. A new commercial system uses
gutta percha coated with glass-ionomer particles to be used in conjunction with a glass-ionomer
sealer [174]. An experimental system incorporated high-molecular-weight poly(acrylic acid) par-
ticles into both gutta percha and a sealer and was shown to have low leakage [175].
23.8 IMPRESSION MATERIALS

23.8.1 Introduction and Classification
Discussion now turns to dental situations where a restoration, prosthesis or appliance is fabricated out-
side the mouth. A clinical impression of the tissues is required. There are stringent demands for such
materials, including appropriate rheological properties, a high degree of dimensional accuracy and sta-
bility, elastic recovery on displacement from the tissues, and hydrophilicity. Currently used impression
materials, which set to form an elastic solid, are polymer-containing. They may be classified as follows:
1. Hydrocolloid materials:
a. Agar (reversible)
b. Alginate (irreversible)
2. Synthetic (non-aqueous) elastomers:
a. Polysulfides (condensation polymerization)
b. Condensation silicones (condensation polymerization)
c. Addition silicones (addition polymerization)
d. Polyethers (addition polymerization)
23.8.2 Hydrocolloids
23.8.2.1 Agar: Reversible Hydrocolloids
Agar is a reversible hydrocolloid. The material is heated to form a sol, and it gels in the mouth (with
an appropriate cooling technique) to form the impression. The principal constituent is agar, which is a
sulfuric ester of a linear polymer of galactose. The composition of this material is given in Table 23.5.
This material is seldom used, as it involves use of an elaborate procedure and equipment for control-
ling the heating and cooling.
TABLE 23.5
Constituents of Agar Hydrocolloid Impression Materials
Constituent wt. % Function
Agar 14 Colloid
Borax 0.2 Strengthens the gel but retards the setting
of dental stone model and die materials
Potassium sulfate 2 Accelerates the setting of dental stone
Water 83.8 Dispersion medium
TABLE 23.6
Constituents of Alginate Impression Materials
Constituent wt. % Function
Soluble salt of alginic acid (sodium, potassium 12 Reacts with calcium ions to give calcium alginate gel
or triethanol amine salt)
Slowly soluble calcium salt (for example, 12 Releases calcium ions to reacts with salt of alginic acid
calcium sulfate)
Trisodium phosphate 2 Reacts with calcium ions go give calcium phosphate
and delay gel formation
Filler (for example, diatomaceous earth) 70 Increases cohesion of mix and strengthens gel
Silicofluorides or fluorides Trace Improves surface of stone model
Flavoring agents Trace Improves patient acceptability
Chemical indicator (present in some materials) Trace Changes color with pH change during setting to
indicate correct timings for the impression procedure
23.8.2.2 Alginates: Irreversible Hydrocolloids

Alginates are irreversible hydrocolloids, that is, they form a gel irreversibly. They have been in use
for over 60 years, and their chemistry is well known [176,177]. They are supplied as powders to be
mixed with water. A typical composition is given in Table 23.6. Three of the components take part
in two chemical reactions that occur sequentially. The gel is formed by the reaction of the alginic
salt with calcium sulfate:
Na n Alg + n/2 CaSO 4 

→ n/2 Na 2SO 4 + Ca n / 2 Alg
However, this reaction would lead to very rapid gel formation, and not permit sufficient time
for recording the impression before gelation. Thus, trisodium phoshate is included as a retarder. It
reacts preferentially with calcium sulfate until it has all reacted:
→ Ca 3 ( PO 4 )2 + 3Na 2SO 4
2Na 3PO 4 + 3CaSO 4 
There are some important limitations to this material. The gel is not tough, and is liable to tear,
although a constituent such as diatomaceous earth is included to strengthen the material. A more
major limitation is the fact that the gel is dimensionally unstable and is susceptible to changes in
fluid content. Three things can occur depending on the conditions: (1) syneresis, which is the con-
traction of the gel which causes a separation of liquid, (2) loss of moisture—a diffusion controlled
process [178]—which causes shrinkage, and (3) imbibition or uptake of moisture, which can cause
swelling.
Alginates are most commonly used for impressions for complete dentures, and in orthodontics
for the preparation of study casts. However, they are not usually considered to have adequate dimen-
sional accuracy and stability for impressions for cast alloy restorations or prostheses.
23.8.3 Non-Aqueous Elastomers
As noted earlier, two of the elastomers set by a condensation reaction and two by an addition reac-
tion. Loss of volatile reaction products from a set impression can result in shrinkage, hence loss of
dimensional stability. As a result, addition polymers are now the more widely used materials.
C2H5
HS C2H4 O CH2 O C2H4 S S C S S C 2H 4 O CH2 O C2H4 SH

m n
SH
FIGURE 23.40 Polysulfide molecule with one pendant and two terminal –SH groups.
23.8.3.2 Polysulfides
These materials are supplied as two pastes. The “base” paste contains a polysulfide, the formula
of which is given in Figure 23.40. This molecule contains three –SH groups; two of these groups
are terminal, one is pendant. The base paste also contains a filler, between 11% and 54% for dif-
ferent materials, for example, TiO2. This paste is usually colored white, because of the color of the
filler. The reactor paste (sometimes called “accelerator” or “catalyst” paste) contains lead dioxide
(PbO2) that causes polymerization and cross-linking, by oxidation of –SH groups (Figure 23.40).
Sulfur is also present. An oil (an ester or chlorinated paraffin) is included to form a paste of the
correct consistency. This paste is brown colored due to the lead dioxide. On mixing the pastes,
the –SH groups can be oxidized by PbO2, giving S-S linkages, resulting in both chain lengthening
and cross-linking.
23.8.3.3 Condensation Silicones

These are also two paste materials. The base paste contains a silicone polymer with terminal
hydroxyl groups and a filler. This is mixed with a reactor paste containing a cross-linking agent
such as an alkoxy ortho-silicate or a polymer thereof. In the reactor paste there is also an activator,
usually an organo-tin compound, such as dibutyl-tin dilaurate. On mixing, cross-linking occurs
with the elimination of alcohol (Figure 23.41).
23.8.3.4 Addition Silicones (Vinyl Polysiloxane)

Silicones that set by an addition reaction are now widely used, and are often termed vinyl polysilox-
anes. These materials contain an organo-hydrogen siloxane, which can undergo an addition reaction
with a siloxane compound containing a terminal vinyl group in the presence of a precious metal
catalyst such as H2PtCl6. This type of reaction can lead to the formation of a cross-linked silicone
rubber (Figure 23.42) [179].
OH RO OR
+ Si + HO
OH RO OR
(One chain of
(Two chains of (Alkoxy ortho- silicone polymer)
silicone polymer) silicate; cross-
linking agent)
O O
Si + 3ROH
O OR (Alcohol)
(Three polymer chains
linked together)
FIGURE 23.41 Condensation polymerization of silicones.

R O
O Si CH CH2 + H Si R
R O
R Si H + H2C CH Si O
R O
O Si CH CH2 + H Si R R O
R O O Si CH2 CH2 Si R
R O R
H2PtCl6
R Si CH2 CH2 Si O
R O R
O Si CH2 CH2 Si R
R O
FIGURE 23.42 Addition polymerization of silicones.
O R R O
H3C CH CH2 C O C CH2 O C CH2 C O CH2 CH CH3

n n
m
N H H N
H2C CH2 H2C CH2
FIGURE 23.43 A polyether with imine end groups.
23.8.3.5 Polyether Materials

The base paste contains a polyether with imine end groups (Figure 23.43), a plasticizer and a filler.
The reactor paste contains an aromatic sulfonate, a plasticizer and a filler. Setting is by cross-link-
ing reaction of the imine groups by cationic addition polymerization [180].
23.8.3.6 General Comparison of Elastomers

In comparing and contrasting vinyl polysiloxane and polyether materials, some general points can
be made:
• Both yield impressions of high accuracy and dimensional stability

• Vinyl polysiloxanes are not hydrophilic, which can be problematic if the material does not
adequately wet moist tissues during impression taking. Some products have added surfac-
tants in attempts to minimize this problem [181].
• Polyether materials are intrinsically hydrophilic, in contrast to the silicone-based materials.
23.8.3.7 Disinfection of Impression Materials

It is now becoming mandatory for impressions to be disinfected before transmission to the dental
laboratory. Numerous experiments have been carried out on chemical disinfection by spraying or
immersing impression materials. Usually disinfection does not affect the dimensional stability of
an impression, though there are occasional exceptions to this [182]. In general non-aqueous elas-
tomeric impressions are less likely to be adversely affected by immersion than the hydrocolloid-
based materials.
23.9 PROVISIONAL (INTERIM) RESTORATIONS

23.9.1 Purpose and Requirements
Interim restorations are essential in fixed prosthodontic treatment. During the interval between
tooth preparation and delivery of the definitive restoration, protection from thermal changes, mar-
gin breakage, caries and pulpal irritation is essential. An interim restoration also provides tooth
stability and aids the patient in terms of oral function.
The requirements for such materials are essentially similar to those for definitive materials,
although strength and esthetics may not be so important. One factor to consider is reaction exo-
therm—if this is too high, pulpal damage could ensue.
23.9.2 Critique of Materials
The available polymer-based materials include
• Poly(methyl methacrylate), supplied as a polymer powder to be mixed with monomer, and

initiated by a redox reaction
• Materials based on higher methacrylates such as poly(ethyl methacrylate)
• Filled dimethacrylate polymers, similar to resin composites, but with less inorganic filler
Of these materials, the latter are probably the most widely used, given that the acrylic materials
have the highest exotherm and the lowest flexural strength. The materials have been reviewed by
Gratton et al. [183].
23.10 PROSTHODONTIC MATERIALS

23.10.1 Complete Dentures
23.10.1.1 Poly(Methyl Methacrylate): Techniques of Polymerization
Complete dentures are usually fabricated from acrylic polymers-poly(methyl methacrylate) or
PMMA. Traditionally the procedure involves preparing a dough from monomer and beads of
polymer, with benzoyl peroxide. This dough is packed under pressure in a two part mold and
heated to activate the polymerization of the monomer. Temperature control is important. Too
little heating will result in a denture with excessive residual monomer (see the following text).
However very rapid initial heating can cause the dough temperature to exceed the boiling point
of the monomer (100.9°C), because of the exothermic heat. This can result in a polymer with
gaseous porosity.
Alternative polymerization methods have been explored, including autopolymerizing resins used
in a “fluid resin” technique, for example, for emergency treatment [184], visible light curing [185],
including a methyl methacrylate-free urethane dimethacrylate [186] and microwave heating [187].
23.10.1.2 Limitations of Acrylic Denture Materials

There are several areas of concern about PMMA as a denture material.
23.10.1.2.1 Biocompatibility and Residual Monomer Content

The residual monomer (RM) content of dental PMMA is related to the method of polymerization.
For example,
RM of fluid resin > RM of heat cured resin ≥ RM of microwave cures resin [188].
The RM of a heat-cured material has been reported to be 0.045%–0.106%, compared with a
figure of 0.185% for an autopolymerizing material [189].
The reasons for concern about RM are twofold. Firstly it may act as a plasticizer and compromise
the mechanical properties of the material. Secondly, RM can be a problem in relation to biocom-
patibility. RM can cause irritation, inflammation and an allergic response [190]. Autopolymerizing
acrylics have been found to be the most cytotoxic [190].
23.10.1.2.2 Mechanical Properties

PMMA dentures can fracture from flexural fatigue and impact forces [191]. As a result alternative
polymers have been tested, including epoxy resins, polystyrene, and polycarbonate [192].
Modifications to PMMA have been advocated, including rubber-acrylic graft co-polymers, and
the incorporation of up to 30% by weight low molecular weight butadiene styrene rubber [193].
Fiber reinforcement studies have included the use of carbon fibers [194], high modulus poly(p-
phenylene terephthalamide) fibers (Kevlar®) [195], glass fibers [196–198], ultra-high molecular
weight polyethylene fibers [199–201], and PMMA fibers [202].
Metallic reinforcement of PMMA has met with problems, including stress concentration around
the metallic material which can cause weakening [191]. Also, bonding between phases has been
difficult to ensure. In attempts to overcome this, the following have been tested:
• Surface roughening treatment of the metal [203]

• Silanization techniques [204]
• An adhesive resin containing 4-META [205]
23.10.1.2.3 Radiopacity
A problem with PMMA dentures is that if fracture occurs, there may be ingestion or aspiration of
broken fragments. Radiological detection of such fragments is impossible, and deaths have resulted
[206]. Attempts have been made to solve this problem by including constituents with elements such
as iodine, barium or bismuth, but as yet there is no radiopaque denture material that can match
PMMA in terms of esthetics and mechanical properties [206].
23.10.1.3 Hypoallergenic Denture Polymers

For patients who are allergic to methacrylates there are two possible alternative chemistries available.
An injection molded nylon denture base material is available [207]. Also, a number of urethane-
based materials are being produced [208].
23.10.2 Denture Teeth
Although porcelain is sometimes used as a synthetic tooth material, most dentures are fabricated
with polymeric teeth. Options include conventional PMMA, cross-linked PMMA with conventional
PMMA coating, or an interpenetrating polymer network (IPN) of cross-linked and uncrosslinked
PMMA [209]. Composite teeth (nano-filled and micro-filled) are also produced [210]. It appears
that the conventional PMMA teeth have the lowest fracture toughness [209] and least resistance to
abrasive wear [211].
Good bonding between PMMA denture and teeth is essential. It appears that heat-cured poly-
mers bond better to teeth than visible light cured resins [212]. Further, when heat curing is used,
higher polymerization temperature results in greater diffusion of monomer from the denture base
into the tooth, resulting in better bond strength [213].
23.10.3 Resilient Polymers as Denture Linings

23.10.3.1 Temporary Lining Materials
Temporary resilient lining materials for dentures may be used as tissue conditioners in order to
condition inflamed and distorted soft tissues underlying poorly fitting dentures. They may also be
used for recording functional impressions [214].
These materials contain a polymer powder which is mixed with a liquid plasticizer. Traditional
materials have been based on poly(ethyl methacrylate) with an ethanol/ester mixture. On mixing,
the ethanol increases the rate of gel formation. However, it is lost from the material within 24 h.
Thus a product has been developed without ethanol; it is based on a n-butyl methacrylate/i-butyl
methacrylate copolymer [215]. It should be noted that some materials whose composition has been
reported by Murata et al. [215] contain phthalate esters—compared to mouth protectors (Section
23.4.2), where these compounds have been banned [98].
23.10.3.2 Permanent Resilient Lining Materials

These can be used when the tissues of the denture beading area are atrophied and pressure from the
denture during mastication would cause pain. They can also be used for utilizing undercut areas to
achieve maximum retention of the denture.
Requirements for resilient materials are stringent, and at this time there is no ideal material.
Obviously the materials should not be toxic or irritant, and should ideally be free from odor and
taste. Secure permanent bonding to the PMMA denture base is essential. There should be no
adverse effect from the oral environment; thus loss of plasticizers (if present) is undesirable, as also
is absorption of water causing swelling. The material should be permanently resilient. The composi-
tion and properties of these materials have been extensively reviewed [216].
23.10.3.2.1 Methacrylate-Based Materials

Heat-activated higher methacrylate polymers are used, in which poly(ethyl methacrylate) or co-
polymers are mixed with ethyl, butyl or 2-ethoxyethyl methacrylate. Butyl phthalyl butyl glycolate
or a phthalate ester plasticizer is incorporated. In general, these materials adhere well to PMMA
dentures, but may leach plasticizer components, causing hardening and dimensional change.
23.10.3.2.2 Siloxane-Based Materials

These are usually chemically similar to the condensation silicone impression materials discussed
earlier (Section 23.8.3). One addition polymerizable material, requiring heat at 100°C has been
produced. In general, it is difficult to secure durable bonding of a silicone to PMMA. In attempts to
overcome this, a silicone product with pendant methacrylate groups is available. A major concern
with silicone soft linings is that they support the growth of Candida albicans [217].
23.10.3.2.3 Suggested Modified and Alternative Materials

In the development of modified or new materials, the following experimental approaches, among
others, have been tested:
• The use of polymerizable plasticizers [218,219]

• The application of a fluoroalkyl methacrylate polymer [220]
• The use of a poly(fluoroalkoxy) phosphazine elastomeric system [216]
23.10.4 Denture Adhesives
These products are usually a blend of polymer salts such as sodium carboxymethyl cellulose and
zinc and calcium salts of poly(methyl vinyl ether-co-maleic anhydride), as well as oils, flavors, bind-
ing materials and preservatives [221,222].
23.11 POLYMER APPLICATIONS IN PERIODONTOLOGY

23.11.1 Membranes for Guided Tissue Regeneration
Guided tissue regeneration (GTR) is a surgical procedure that utilizes barrier membranes to direct
the growth of new bone and soft tissue. It is necessary to exclude unwanted cell lines from healing
sites to allow growth of desired tissues. A barrier membrane provides primary closure and stability
of the wound that promotes undisturbed and uninterrupted healing, creates and maintains space to
facilitate bone in-growth and migration of undifferentiated mesenchymal cells [223–225]. Nyman
et al. [226] demonstrated the potential of this technique in dentistry.
Two membrane types are currently used in GTR. These are
1. Synthetic non-resorbable PTFE membranes such as Gore-tex®

2. Synthetic resorbable membranes formed from gylcolide and lacide copolymers, collagen, etc.
23.11.2 Other Constituents
A membrane may be used alone or with a bone graft (an autograft or allograft) or bone substitute.
Growth factors like bone morphogenic proteins (BMP) and human recombinant platelet derived
growth factors (rhPDGF) can also be used in conjunction with a barrier membrane. BMPs can be
produced using recombinant DNA technology. BMP-2 and BMP-7 have FDA approval for human use.
The biological basis of bone morphogenesis was shown by Urist [227]. A recent review has been
presented by Bashutski et al. [228].
23.12 DENTAL IMPLANTS

Titanium-based implants are finding increasing use in dentistry, for the functional replacement
of missing teeth. They have achieved a considerable measure of success, related to the process of
osseointegration, in which newly formed bone develops and integrates with the implant surface [229].
Extensive research is being carried out on surface modification to enhance the interaction with
bone. Such surface treatments include (but are not limited to) the use of polymers, which may be
naturally occurring or synthetic materials. Research has included collagen and biomimetic poly-
mers similar to the extracellular matrix of bone [230].
23.13 SOFT TISSUE AUGMENTATION

23.13.1 Objectives and Requirements
Numerous materials have been employed for facial rejuvenation, in attempts to correct the effects of
aging. Such procedures are increasing in popularity. Material requirements include: non-allergenic,
minimal side effects, longevity, yet with the ability to reverse the treatment and ease of use [231].
23.13.2 Materials
Materials may be classified according to (a) chemical composition—naturally occurring vs. syn-
thetic polymers [232], (b) temporary vs. permanent materials [233], or (c) reversible vs. nonreversible
materials [234]. Cross-linked hyaluronic acid (HA) is the most popular of the polymeric materials
[234]; this may be avian-derived or bacterial-cultured [232]. HA materials are reversible because
treatment can be reversed by the injection of hyaluronidase [234]. Collagen-containing materials
(from bovine, cadaveric or cell-cultured sources) are available [232]. Other polymers include poly-l-
lactic acid microparticles, PMMA microspheres in 3.5% bovine collagen, silicone liquids, poly(alkyl
imide), and polyacrylamide hydrogel. Not all of these materials have received FDA approval [232].
23.14 MAXILLOFACIAL PROSTHESES

23.14.1 Requirements
Facial defects resulting from surgery, trauma or malformation require prosthetic rehabilitation
when reconstructive surgery is not feasible or advisable [235]. The polymers employed for such
treatment include silicone and polyurethane elastomers and methacrylates [236,237] though no type
of material shows all the ideal properties. Applications include the use of rigid materials (e.g., for
orbital prostheses) and materials that can mimic the flexibility of living tissue. Some prostheses are
fabricated from a silicone bonded to a rigid acrylic base.
The requirements for such materials are stringent, including the obvious ones of biocompatibility and
durability. From the patient’s viewpoint, color, texture and hygiene considerations are paramount. The
material must also have suitable mechanical properties including flexibility and resistance to tearing.
23.14.2 Materials
23.14.2.1 Silicone Polymers
Medical grade silicones—both heat vulcanizing and autopolymerizing—are used, but the latter
are preferred. Their chemistry is somewhat similar to the impression materials discussed earlier
(Section 23.8.3), consisting of filled siloxanes. They may be polymerized by a condensation reaction
or an addition reaction, as for the impression materials. Fillers may include silica or kieselguhr;
though these increase strength, they may reduce the polymer’s translucency [237].
Aziz et al. [238] have compared the mechanical properties, such as hardness, tensile strength, tear
strength and water absorption of a range of silicone products. In a separate study, Hulterström et al.
[239] claimed that addition-type silicones showed little or no water absorption, in contrast to two con-
densation materials. However, this may be related to the silica filler; –OH groups on the surface of the
silica may facilitate water absorption, whereas surface-treated hydrophobic fillers may inhibit this [238].
In attempts to achieve good esthetics, many pigments and coloring techniques have been suggested
[240], including intrinsic and extrinsic coloration [237]. However, color stability is difficult to achieve,
given that factors such as ultraviolet light exposure, application of cosmetics and cleaning agents may
operate. Inorganic pigments appear to be more stable over time compared with organic colorants [241].
A methacryloxypropyl-terminated silicone has been developed to facilitate adhesion to non-
silicone-based adhesives [242]. Improvements in physical properties of maxillofacial silicones have
been achieved by the incorporation of nanosized oxides (TiO2, ZnO, CeO2) at concentrations of
2%–2.5% [243].
23.14.2.2 Methacrylates
PMMA can be used for restoration of defects with tissue beds that are relatively non-mobile [237],
for ocular prostheses [244] and for hollow obturator prostheses. For this latter application, visible
light curing denture materials are being advocated [245].
23.14.2.3 Other Polymers
Other polymeric maxillofacial materials include polyurethanes [246] and chlorinated polyethylene [247].
Glass fiber-reinforced composite have been proposed for a reinforcing substructure to reinforce a silicone
elastomer [248].
23.15 WOUND CLOSURE MATERIALS

The requirements for suturing in dentistry are exacting, given the presence of saliva, and functions
such as speech, mastication and swallowing [249]. Non-resorbable sutures such as silk resist the
effects of oral fluids, but require a clinical appointment for removal. Resorbable sutures include
gut, chromic gut, polyglactin, and poly(glycolic acid) [250]. Gassner [251] has extensively reviewed
wound closure materials.
23.16 CONCLUSION
It has been shown that there is extensive ongoing research in biomedical polymers for dental and
maxillofacial applications. The limitations of current materials present great challenges to the
researcher. Providers of treatment are challenged to make wise decisions on the selection and uti-
lization of materials. The future is exciting as the possibilities of new technologies are demanding
radically new approaches to the subject [252].
ACKNOWLEDGMENTS
The author is indebted to colleagues for helpful discussion, particularly, Dr. C. Aparicio, Dr. J. Beyer,
Dr. Yasser F. Gomaa, Dr. V. Thumbigere Math and Dr. J. Perdigão. Also Drs. Perdigão and Beyer
kindly supplied some of the illustrations.
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24 Biomaterials as Platforms
for Topical Administration
of Therapeutic Agents in
Cutaneous Wound Healing
Rhiannon Braund and Natalie J. Medlicott
CONTENTS
24.1 Introduction........................................................................................................................... 837
24.2 Cutaneous Wounds................................................................................................................ 837
24.2.1 Acute Wounds and the Normal Wound Healing Response....................................... 839
24.2.2 Chronic Wounds: Impaired Wound Healing.............................................................840
24.3 Biomaterials Used in Wound Healing Applications..............................................................840
24.4 Topical Delivery of Drugs to Wounds................................................................................... 843
24.4.1 Anti-Infective Agents Delivery from Biomaterials...................................................844
24.4.2 Growth Factors Delivery from Biomaterials.............................................................844
24.5 Pharmacokinetic Models of Drug Delivery to Wounds........................................................ 845
24.6 Concluding Comments..........................................................................................................846
References.......................................................................................................................................846
24.1 INTRODUCTION
Biomaterials have found application in cutaneous wound healing either as the basis of wound
dressings or as platforms for the delivery of therapeutic agents into wounds. Increased wound epi-
thelialization was first reported in pigs following application of occlusive dressings made from
polyethylene films by Winter in 1962 and has led to the current practice of maintaining a moist
wound environment to optimize wound healing. Since this time, much has been discovered on the
mechanisms of wound healing to support this practice. This together with advances in biomaterial
science and increased availability of a range of biocompatible materials has led to the development
of modern products used for treatment of wounds today (Yudanova and Reshetov 2006, Singer and
Dagum 2008). Still remaining, however, is the fundamental challenge of improving the healing of
chronic wounds such as foot ulcers in diabetic patients (Greenhalgh 2003, Mekkes et al. 2003).
The aim of this chapter is to review the current use of biomaterials in wound healing and, from
this, to gain an appreciation of the potential of biomaterials to enhance wound healing outcomes
through their promotion of an optimal environment for healing and use as drug delivery platforms.
24.2 CUTANEOUS WOUNDS

The skin is a vital organ in all animal species, it covers the body and provides protection to the
underlying tissues from the environment in which we live. However, periodic damage to the skin
can and does occur leaving the affected individual vulnerable. Many reviews are available on the
837
1. Inflammation
2. Tissue formation
(epithelialization and granulation
tissue formation)
3. Tissue remodeling
(change to collageneous scar)
Approximate time line for wound healing stages (days)
0 3 20 100
FIGURE 24.1 Stages of normal wound healing. Approximate timeline is from Clark. (From Clark, R.A.F.,
Wound repair: Overview and general considerations, in R.A.F. Clark (ed.), The Molecular and Cellular
Biology of Wound Repair, 2nd edn., Plenum Press, New York, 1995.)
physiological, cellular, and biochemical response to cutaneous wounds and readers are directed to
these reviews for a greater depth of discussion of the wound healing process while a brief overview
follows (Kiritsy et al. 1993, Scaffer and Nanney 1996, Singer and Clark 1999, Yamaguchi and
Yoshikawa 2001, Monaco and Lawrence 2003, Bao et al. 2009).
The normal physiological response to wounds is termed the “wound healing response.” It aims to
reduce loss of blood, by initiation of the clotting cascade, then to trigger an inflammation response
with the formation of granulation tissue and ultimately results in re-epithelialization with tissue
maturation and remodeling (Singer and Clark 1999, Monaco and Lawrence 2003). These stages of
wound healing are generally considered as sequential, but overlap in time as shown in Figure 24.1.
The cellular and biochemical responses associated with each stage are becoming increasingly under-
stood (Clark 1995) and this information is being used to propose therapeutic targets to enhance
wound healing outcomes.
Wounds in general do not represent a single well-defined entity, but rather cover a range of pre-
sentations classified by both cause and severity from minor cuts and abrasions to extensive loss of
large areas of skin in, for example, burns (Singer and Dagum 2008). The body’s ability to recover
unaided appears highly dependent on the type and severity of the wound and on the existence of
underlying diseases and/or infection. The importance of these factors is reflected in the often high
mortality associated with burn injury, where patient age >60 years, burn area >40% of the body
surface, and coexisting inhalation injury have been identified as risk factors for mortality (Ryan
et al. 1998). At the time of any cutaneous wounding, there is a dramatic increase in skin perme-
ability with potential for entry of toxic substances and infective organisms. From transdermal drug
delivery literature, the importance of the stratum corneum (outmost layer of the skin) in preventing
entry of compounds applied to the skin is well understood (Walker and Smith 1996, Menon 2002,
Magnusson et al. 2004). Microorganisms also cannot easily penetrate intact skin but see no such
barrier once wounding has occurred. Colonization of wounds by microorganisms can both delay
healing and increase the risk of systemic infection (Thomsen 1994, Heggers 2003). A committee of
the Wound Healing Society in 1994 developed definitions of wounds, wound healing, and wound
attributes to facilitate comparison between wound healing studies. In this they defined an acute
wound as “one that proceeds through an orderly and timely reparative process to establish sustained
anatomic and functional integrity” and a chronic wound as “one that has failed to proceed through
an orderly and timely reparative process to produce anatomic and functional integrity or has pro-
ceeded through the repair process without establishing a sustained anatomic and functional result”
(Lazarus et al. 1994).
Biomaterials as Platforms for Topical Administration 839
Modern biomaterials and tissue-engineered skin substitutes form an integral part of today’s
wound treatments. They are used firstly to assist in the reduction of blood loss through hemostatic
mechanisms and action as a replacement physical barrier to prevent entry of microorganisms.
Then if the mechanisms of wound repair are well understood, these same materials may be
developed into treatments that actively promote wound healing. As part of this advance, bioma-
terial dressings are becoming increasingly recognized for their potential to be used as platforms
for the local delivery of therapeutic agents (Lawrence and Diegelmann 1994, Sakiyama-Elbert
and Hubbell 2000, Wong et al. 2003, Braund et al. 2007a, 2009, Bader and Kao 2009, Mulder
et al. 2009).
24.2.1 Acute Wounds and the Normal Wound Healing Response

In response to clean, acute, cutaneous wounds, there is a reasonably orderly interplay between
the vascular and cellular responses and the chemical mediators such as growth factors and cyto-
kines to control the progression of wound healing (Kiritsy et al. 1993, Scaffer and Nanney 1996,
Monaco and Lawrence 2003, Frokjaer and Otzen 2005). Risk factors and underlying diseases that
impede successful wound healing are recognized, with diabetes mellitus and peripheral vascular
disease being two of the most well recognized (Greenhalgh 2003, Brem and Tomic-Canic 2007,
Milic et al. 2009). The goals of acute wound management include ensuring rapid and complete
wound closure, optimal return of skin functions with a low level of tissue scarring (Singer and
Dagum 2008). In order to achieve this, risk factors recognized to delay healing are identified
and reduced, thereby creating a wound environment that has an increased chance of optimal
healing. A number of references give guidelines for optimizing wound healing, and some com-
mon approaches that are summarized in Figure 24.2 include removal of dead tissue, prevention
of wound infection, pressure off-loading of the wounded area, and correction of any underlying
vascular deficits (Franz et al. 2008). Efforts are also directed at reducing the extent of scarring
through maintenance of a moist wound environment during healing. To achieve this, occlusive
and moisture controlling dressings are commonly used (Field and Kerstein 1994, Singer and
Dagum 2008). Burns constitute a significant group of acute wounds, with the body area affected
being highly variable. Outcomes are dependent on surface area, depth of injury, and whether or
not the lungs are involved in the burn injury (Ryan et al. 1998). For burn wounds of moderate or
lower severity, treatment may focus on the local area of the burn and includes skin grafting and
use of biomaterial dressings.
Wound
debridement
Pressure Wound Infection

off-loading healing control
Correction of
underlying
vascular
deficiencies
FIGURE 24.2 Factors contributing to resolution of acute wounds.

TABLE 24.1
Some Systemic and Local Factors That Contribute to
Delayed Wound Healing
Systemic Factors Local Factors
Aging Arterial occlusion/insufficiency
Alcoholism Foreign bodies
Diabetes mellitus Ischemia
Drugs, e.g., steroids Pressure (e.g., bed-ridden patients)
Immunodeficiency Tissue necrosis
Liver disease Ulcerating tumors
Nutritional deficiencies Ulcerating skin diseases
Peripheral vascular disease Wound infection
Vasculitis
Venous insufficiency
Source: Burns, J.L. et al., Clin. Plast. Surg., 30, 47, 2003.
24.2.2 Chronic Wounds: Impaired Wound Healing

The mechanisms by which acute wounds turn into chronic wounds are not completely under-
stood, but as research on the biochemistry and cellular biology of chronic wounds continues,
new understandings are gained on the differences between acute and chronic wound environ-
ments so that new therapeutic targets may be identified in the future. In the meantime, the high
financial and patient morbidity costs associated with treatments for chronic wounds make this
an important health-care area especially as the population ages and diseases such as diabetes
mellitus increase in prevalance (Sen et al. 2009). Treatments described in many published guide-
lines aim to minimize the risk factors identified as impeding wound healing (Robson and Barbul
2006, Franz et al. 2007, Hinchliffe et al. 2008). The conditions and coexisting diseases known
to make a significant contribution to delayed wound healing are listed in Table 24.1 (Burns et al.
2003) and although treatment of the underlying cause of impaired wound healing would seem
the logical course of action to improve wound healing outcomes in patients, complete resolu-
tion is often difficult if these underlying diseases cannot be well controlled. Newer treatments
for chronic wounds have included the use of biomaterial dressings to maintain the moist wound
environment together with active treatments with antiseptics and antibiotic agents to control
wound infection. Topical delivery of growth factors has also been investigated with mixed suc-
cess. These areas are discussed further in the next sections.
24.3 BIOMATERIALS USED IN WOUND HEALING APPLICATIONS

Modern wound dressings are usually presented in the form of gels, thin films, or foam sheets
(Boateng et al. 2008). Properties of an ideal wound dressing have been suggested by many including
Zhang et al. (2005). These include provision of hemostatic action, sufficient bacterial barrier, adsor-
bant to exudates, ability to maintain a moist wound environment, flexibility to cover wound surface,
adherent to health tissue while nonadherent to wound tissue, easy and painless to remove, and low
cost. Along with these properties, materials used should exhibit tissue biocompatibility so they do
not promote negative inflammatory changes in the wound tissue (Anderson et al. 2008) including
development of foreign body reactions (Rosenberg 2006). There is also an optimal size and shape
requirement for individual wounds so that biomaterials whose size and shape can be e asily manipu-
lated to fill and/or cover non-uniform shaped wounds have potential advantage.
The most appropriate dressing to use is often determined by the type and extent of the wound
and some general guidelines are available in the literature (Singer and Dagum 2008). Numerous
polymeric materials have been reported as the basis of modern dressings in wound healing literature
and many of these have successfully reached the market (Yudanova and Reshetov 2006, Boateng
et al. 2008). Yudanova and Reshetov (2006) suggested a need for systematic classification of wound
dressings according to their characteristic features. Boateng et al. (2008) gave three classifications:
firstly, according to the dressing function; secondly, according to the type of material used; and
thirdly, according to the physical form of product. Jeffcoate also gave a similar classification of
wound dressings into films, foams, hydrogels, hydrocolloids, alginates, hydrofibers, and medicated
dressings (Jeffcoate et al. 2004).
Synthetic polymeric materials have a variety of physical properties and those used in wound
dressings are generally processed into gels, thin sheets or films, or foams. The resulting dressings
have different fluid absorbencies, shapes, and water and vapor permeabilities to allow selection of
appropriate dressings to maintain moist, but not too wet, wound environments (Table 24.2).
The biological dressings are prepared from materials that have been reported to play an active part
in the wound healing process and include alginates, collagen, hyaluronic acid, and fibrin. Alginate
polymers have formed the basis of wound dressing products for many years. Both sodium and cal-
cium alginates are used and the degree of alginate cross-linking and hence physical properties of
the material are dependent on the calcium concentration within the alginate (Ichioka et al. 1998).
Following application to wounds, sodium and calcium ions can exchange between the wound fluid
and the applied alginate so that physical properties can be influenced by the biological environment.
Comparative studies have shown that alginates typically remain on the wound for longer than hydro-
colloid materials (Ichioka et al. 1998). Calcium alginate also appears to increase fibroblast prolifera-
tion in the wound (Doyle et al. 1996) and calcium ions may help with clotting through a hemostatic
effect (Blair et al. 1988).
Collagen is a major constituent of connective tissue and forms a large portion of the wound
granulation tissue. Its use in wound dressings aims to provide a ready supply of collagen for incor-
poration into granulation tissue. Sources of collagen with low immunogenicity and good biodegrad-
ability are available and have been used in wound dressings and for dermal reconstruction (Macleod
et al. 2005, 2008, Benito-Ruiz et al. 2006, Shevchenko et al. 2008). Arul et al. (2007) showed
that collagen alone gave an increased intra-wound collagen (compared with saline-treated con-
trols) 21 days after experimental wounds in diabetic rats. Then, if collagen was used as a platform
for delivery of a therapeutic agent, biotinylated tripeptide Gly-His-Lys, wounds showed complete
re-epithelialization and densely packed collagen tissue at day 21. Shevchenko et al. (2008) reported
that a paste form of collagen allowed better cellular infiltration over sheet collagen in full-thickness
wounds. Hence, the biomaterial physical form as well as chemical composition can influence the
wound healing outcome.
Chitosan is a versatile polysaccharide material; it is made up of linear chains of d-glucosamine and
N-acetyl-d-glucosamine units. Hydrogels can be prepared using a variety of cross-linking agents to
produce materials with varying physical properties and geometries, for example, gels, powders, beads,
films, tablets, capsules, microspheres, microparticles, sponges, and nano and microfibers (Bhattarai et
al. 2010). Dressings composed of chitosan (Rossi et al. 2007, Liu et al. 2008, Meng et al. 2010) or chito-
san with alginates (Khor and Lim 2003, Liu et al. 2008, Ho et al. 2009, Meng et al. 2010) or hyaluronic
acid (Choi et al. 2001) have been reported widely for their beneficial effects on wound healing.
Tissue-engineered artificial skin can be acellular where the biomaterial provides a scaffold
architecture into which surrounding cells migrate and proliferate. Alternatively, cells (usually
fibroblasts or keratinocytes) can be seeded into the biomaterial scaffold to initiate tissue regenera-
tion. Nondegrading and biodegradable polymers have been used as the scaffold forming materi-
als and they are especially useful for replacing lost tissue in full-thickness wounds (Miller and
Patrick 2003, Kamolz et al. 2008, Priya et al. 2008, Dieckmann et al. 2010). Bota et al. (2010)
showed that for expanded polytetrafluoroethylene (ePTFE) implants, a porous structure with an
TABLE 24.2
Biomaterial Dressings Used in Cutaneous Wound Healing
Polymers Properties
Hydrocolloids Carboxymethyl cellulose Colloidal materials
Prepared as films or thin sheets
Gelatin Adhere to dry and moist areas
Pectin Initially impermeable to water vapor
Absorb fluid to form a gel, which becomes increasingly
permeable to air and water as water content increases
Useful for moderately exudating wounds
Alginates Sodium or calcium alginate Prepared as porous sheets or flexible fibers. Fibers can be
(polysaccharide monomers: packed into wounds
mannuronate and guluronic acid) Form gel structure on contact with water. High mannuronic
content gives softer gels. Higher guluronic content gives
firmer gels
Can be cross-linked with calcium ions to give a material
with a stronger barrier function
Useful for moderate to heavily exudating wounds due to
high absorbency
May dehydrate drier wounds
Often require frequent changing
Hydrogels Poly(methacrylates) Insoluble, swellable hydrophilic materials
polyvinylpyrrolidine Prepared as amorphous gels or elastic solid sheets or films
Cross-linking produces matrices that can entrap water
Application of amorphous gels usually requires covering
with a secondary dressing
Sheets may be polymer backed with adhesive edges to
minimize water loss through the hydrogel
Due to preparation with high water content, they do not
adsorb much exudate
Fluid accumulation under dressing can cause tissue maceration
Useful for deep abrasions and wounds with crusted surface
exudate
Semipermeable films Nylon derivatives Permeable to water vapor but not liquid exudate
Polyurethane Thin, often transparent films
Acrylic derivatives Highly flexible and elastic so that they can conform easily to
the skin contours
Useful for relatively shallow wounds
Foam dressings Polyurethane Prepared with an open porous polyurethane structure
High absorbency
Provide a protective padding
May be cut into shaped to fit within wounds
Useful for partial or full-thickness wound as well as heavily
exudating wounds
Biological dressings Collagen Made from biomaterials that play an active part in the wound
Hyaluronic acid healing process (also called bioactive dressings)
Chitosan See text for further properties
Alginates
Elastin
Fibrin

Biomaterial Dressings Used in Cutaneous Wound Healing
Polymers Properties
Tissue-engineered Synthetic or biological polymers, May be acellular or cell-containing matrices. The cellular
artificial skin e.g., polyesters, collagen, component is usually fibroblasts or keratinocytes
silicone, hyaluronic acid Polymeric material forms a scaffold that replicates dermal
derivatives, fibrin architecture through which the regenerating tissue can form
The scaffolds should have optimal mechanical properties
initially to support regenerating tissue, and then may
degrade once tissue is established
Sources: Boateng, J.S., J. Pharm. Sci., 97, 2892, 2008; Singer, A.J. and Dagum, A.B., N. Engl. J. Med., 359, 1037, 2008.
internodal distance of 4.4 μm allowed cellular invasion and resulted in the formation of a thinner
fibrous capsule surrounding the implant. Smaller pore sizes (internodal distances of 1.2 and 3.0 μm)
and nonporous PTFE in contrast were associated with little cellular infiltration and thicker fibrous
capsules surrounding the implanted material. In vitro studies were also reported and showed an
increased secretion of early proinflammatory cytokines for the larger pore sized ePTFE incubated
with monocyte/macrophage cells (Bota et al. 2010). Results were suggestive of an increased mac-
rophage activation in response to ePTFE topography and an ability to modify the tissue foreign
body response through scaffold topography (Bota et al. 2010). Chang et al. (2008) showed that
surface hydrophobicity affected interactions with lymphocytes and macrophages. They measured
cytokines, chemokines, and matrix proteins produced on culture of monocytes and lymphocytes
with polyethylene terephthalate–based surfaces. Hydrophilic anionic or neutral surfaces produced
greater pro-inflammatory responses with less breakdown of extracellular matrix than hydrophilic
cationic materials, where a more anti-proinflammatory response was generated with increased ratio
of m
etalloproteinases (MMP-9) to tissue inhibitors of MMP (TIMP) observed. Since MMPs nor-
mally show a 1:1 stoichiometry with their inhibitors (TIMP), an increase in the ratio of MMP/TIMP
could be expected to result in increased extracellular matrix breakdown in the wound.
24.4 TOPICAL DELIVERY OF DRUGS TO WOUNDS

A further consideration in the development of an improved wound dressing relies on integration
of biomaterial and drug delivery sciences. The goal is to use the biomaterial as a platform for drug
release, and then future dressings may enhance wound healing both through moist wound environ-
ment and pharmacological mechanisms. Two classes of therapeutic agents that have received con-
siderable attention for topical administration are the anti-infective agents and growth factors. While
the topical application of anti-infective agents has been part of wound management for many years,
growth factors have more recently been investigated (Kiritsy et al. 1993, Lawrence and Diegelmann
1994, Werner and Grose 2003, Wong et al. 2003, Robson et al. 2004, Amery 2005, Braund et al.
2007a,b, Davidson 2007). Becaplermin, a recombinant platelet-derived growth factor (PDGF-BB),
is available for topical applications under the name Regranex® in many countries (Papanas and
Maltezos 2010). Clinical trials have shown becaplermin to be beneficial in the treatment of diabetic
foot ulcers with adequate blood supply (Embil et al. 2000, Papanas and Maltezos 2010). Akopian et
al. (2006) reported benefit in the treatment of larger, higher grade wounds, following topical applica-
tion of growth factors (platelet-derived wound healing factor or recombinant platelet-derived growth
factor-BB) at the early stage of treatment. Routine use of becaplermin in wounds has, however, been
somewhat limited. Proposed reasons for this have been the product’s relatively high cost and its
perceived reduced efficacy outside the well-controlled clinical trial environment. More recently, an
increased cancer risk has been reported for patients treated with three or more tubes (i.e., 45 g total)
of Regranex® (Papanas and Maltezos 2010).
24.4.1 Anti-Infective Agents Delivery from Biomaterials

Topical application of anti-infective agents aims to reduce wound colonization by microorganisms
and subsequent infection, thereby exerting a positive effect on wound healing.
Silver preparations have been used as semi-solid creams and ointments in patients with burns
(Boateng et al. 2008, Singer and Dagum 2008). Incorporation into wound dressing materials has
also occurred with products such as Aquacoat®, which produces a slow release of silver into the
wound. The silver in this product is deposited onto the surface of the material as a 1 μm thick
layer using a physical vapor deposition method. The potential advantage suggested for this mode
of delivery of silver to wounds was that the total silver dose could be reduced thereby lowering
systemic exposure (Tredget et al. 1998). A further advantage of application of silver in the wound
dressing is that it is easier to remove than a cream formulation at the dressing change. Leaper
reviewed the use of silver-containing dressings in wounds and recognized the emergence of an
increasing number of silver-containing dressings (Leaper and Durani 2008). The incorporation of
nanocrystallized silver seems to be associated with good sustained-release properties and lower
risk of systemic toxicity.
Other antiseptic agents incorporated into wound dressings include povidone iodine (Reimer et al.
2000) and chlorhexidine (Rossi et al. 2007, Sulea et al. 2011). Both these agents have be applied
to wounds as solutions in conventional treatment protocols, but incorporation into biomaterials
offers the advantage of sustained release and reduced frequency of application. Antibiotic agents,
for example, gentamicin (Zilberman et al. 2009) and minocycline (Aoyagi et al. 2007), have also
been incorporated into biomaterials for direct application to wounds to reduce bacterial infection.
While the topical application of antiseptic agent has not usually been associated with the occur-
rence of resistant microorganism (Leaper and Durani 2008, Milstone et al. 2008), release of topical
antibiotics needs to be carefully controlled so that treatments prevent wound infection but do not
increase the emergence of antibiotic-resistant strains of microorganisms.
24.4.2 Growth Factors Delivery from Biomaterials

Topically delivered growth factors to promote wound healing have long been proposed in the scien-
tific literature (Kiritsy et al. 1993, Werner and Grose 2003, Robson et al. 2004, Fu et al. 2005, Braund
et al. 2007a, Davidson 2007, Papanas and Maltezos 2007). Whether topically applied growth fac-
tors can be used for the treatment of poorly resolving chronic wounds remains to be seen. Because
these compounds are protein molecules, processing into biomaterials will need consideration of the
effects of processing variables on protein physical stability, that is, secondary and tertiary structures
(Arakawa et al. 2001, Frokjaer and Otzen 2005). Protein instability with generation of aggregated
protein can result in both decreased biological activity and increased risk of adverse immunological
reactions (Rosenberg 2006).
It is known that chemokines, cytokines, and growth factors, when produced in an orderly
sequence in response to wounds, mediate and control the biochemical and cellular events of
the healing response (Kiritsy et al. 1993, Frokjaer and Otzen 2005). How exogenously applied
growth factors modify this process is unknown so that further research is needed to improve our
understanding in this area. Benefits have been shown in wound closure rates in animal models
following application of growth factors (Chan et al. 2006). Chan et al. showed in diabetic mice
that although application of recombinant platelet-derived growth factor (Regranex®) did not
increase the rate of wound closure of 1.5 cm2 wounds, it did increase the amount of g ranulation
tissue in the wound. Positive reports have also been described in human clinical trials (Smiell
1998, Rees et al. 1999, Smiell et al. 1999, Embil et al. 2000, Papanas and Maltezos 2010). A
recent review has examined the potential of growth factor delivery to wounds (Braund et al.
2007a). From this, it appears that mixed clinical outcomes have been observed. Still little
research has been done on the optimal time-course of exogenous growth factor application.
If this is important, then research into biomaterials that can precisely control release of incor-
porated growth factors may provide a key to future development of pharmacologically active
wound dressings.
24.5 PHARMACOKINETIC MODELS OF DRUG DELIVERY TO WOUNDS

A simple model is shown in Figure 24.3 where the concentration of free drug in a wound envi-
ronment is determined by its release from the applied biomaterial and loss through systemic
absorption, binding to local tissues and degradation within the wound. As the wound heals, the
barrier function of the outermost layer will change. Cross and Roberts (1999) reported the change
in permeability of full-thickness excisional wounds to solutes of different molecular sizes. In
this work, they showed that large molecules (FGF-2 and epidermal growth factor) could only
penetrate to superficial layers of the developing granulation tissue. Braund et al. have shown that
fluorescent-labeled FGF-2 applied as single dose (0.3 μg) in a hypromellose gel or film could be
detected in wound tissue sections by confocal microscopy at least 8 h after application. They also
measured intra-wound FGF-2 tissue concentration as a function of depth into the wound (Braund
et al. 2009).
Fibroblast growth factors belong to the heparin-binding family of growth factors (Burgess
and Maciag 1989). The interaction between FGF and heparin is reported to improve physical
stability of the growth factor (Gospodarowicz and Cheng 1986, Zakrewska et al. 2009), and
heparin has been used to stabilize this protein in the development of some FGF-releasing bio-
materials (Tanihara et al. 2001). There is some question as to whether heparin is needed in the
interaction between FGF and its receptor (FGFR) in vivo. Zakrewska et al. (2009) suggested
that heparin was not necessary if the physical stability of FGF was maintained in some other
way. Hence, it may be possible that interaction with polymeric biomaterials may fulfill this
function.
Topically applied compound
Local
X* X
degradation
Systemic absorption
FIGURE 24.3 Schematic illustrating the potential fate of topically administered drugs in a model wound.
X = amount of drug in the wound, X* = tissue-bound drug. (Modified from Braund, R. et al., Curr. Drug
Deliv., 4, 195, 2007a.)
24.6 CONCLUDING COMMENTS

Both synthetic and biological polymeric materials have shown application in wound healing
treatments. The greatest advances have been in the development of materials with ranges of
physical forms, fluid absorbencies, and water vapor transmission rates. These modern wound
dressings address the clinical needs of maintaining a moist, but not wet, wound environment
and acting as an artificial replacement for lost dermal tissue. Release of biologically active
agents from wound dressing is receiving increased attention and may offer future enhancements
in the area of difficult to treat chronic wounds. Extended release of antimicrobial agents from
wound dressings can help to control wound infection, whereas the topical application of growth
factors may have the potential to directly influence the biochemical course of the wound h ealing
response.
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25 Polymers for Artificial Joints
Masayuki Kyomoto, Toru Moro, and Kazuhiko Ishihara
CONTENTS
25.1 Artificial Joint Replacement.................................................................................................. 851
25.1.1 Hip Joint Replacement and Its Clinical Performance............................................... 851
25.1.2 Knee Joint Replacement and Its Clinical Performance............................................. 853
25.1.3 Increase in Hip Joint Arthroplasty Procedures......................................................... 854
25.1.4 Problems of Joint Replacement: Osteolysis............................................................... 855
25.2 Bearing Materials for Joint Replacements............................................................................. 857
25.2.1 Polyethylene Bearing Material.................................................................................. 857
25.2.1.1 History of Polyethylene in the Orthopedic Field........................................ 859
25.2.1.2 Cross-Linked Polyethylene.........................................................................860
25.2.1.3 Antioxidants for Polyethylene..................................................................... 862
25.2.2 Phospholipid Polymers for Mimicking Articular Cartilage......................................864
25.2.2.1 Hydration Lubrication.................................................................................864
25.2.2.2 Articular Cartilage and Material Design....................................................864
25.2.2.3 Photo-Induced Surface “Grafting from” Polymerization........................... 865
25.2.2.4 Poly(MPC)-Grafted Polyethylene............................................................... 867
25.2.3 Poly(Ether-Ether Ketone) Bearing Materials............................................................ 872
25.2.3.1 Structure and Properties............................................................................. 872
25.2.3.2 Tribological Properties............................................................................... 873
25.2.3.3 Surface Modification.................................................................................. 873
25.3 Fixation Materials for Joint Replacements............................................................................ 875
25.3.1 Poly(Methyl Methacrylate) Bone Cement................................................................. 875
25.3.2 Bone Cement History................................................................................................ 876
25.3.3 Problems with PMMA Bone Cement........................................................................ 876
25.3.4 Solutions for the Problems of PMMA Bone Cement................................................ 876
25.3.4.1 Antibiotic-Loaded Acrylic Cement............................................................ 876
25.3.4.2 Bone Bioactive Organic–Inorganic Hybrid Bone Cement......................... 877
25.4 Future Perspectives................................................................................................................ 877
References....................................................................................................................................... 878
25.1 ARTIFICIAL JOINT REPLACEMENT

25.1.1 Hip Joint Replacement and Its Clinical Performance
A normal joint in our body is made up of bones that are lined by surface cartilage. The joint is sur-
rounded by a capsule with a thin lining of synovial cells that produce a thin layer of lubrication film.
The lubrication film (synovial fluid) together with the surface cartilage (articular cartilage) acts as a
shock absorber and allows the joint to move smoothly; this protective action endures for many years
(such as 50–60 years). If the surface cartilage is badly damaged or if the joint surfaces are mis-
aligned (e.g., hip dysplasia), then the cartilage will wear out much quicker than in normal wear and
tear, and as a result, the bone under the cartilage layer becomes exposed. The exposed bone starts to
rub against the other and the process of osteoarthritis (wear and tear) is established. Osteoarthritis
851
is, therefore, the result of mechanical wear and tear on a joint. Its main feature is a loss of surface
cartilage with bone rubbing on bone and this may include joint pain, tenderness, stiffness, creaking,
and locking of joints. This process produces pain and local inflammation. In osteoarthritis, a variety
of potential forces—hereditary, developmental, metabolic, and the mechanical—may initiate pro-
cesses leading to loss of cartilage. As the body struggles to contain ongoing damage, immune and
regrowth processes can accelerate damage. Sometimes the body tries to relieve this pain by increas-
ing the amount of fluid in the joint. This is why joints are swollen. The formation of bone spurs and
cysts around the joint is another hallmark of osteoarthritis.
The most common type of arthritis leading to total hip replacement is degenerative arthritis
(e.g., osteoarthritis) of the hip joint. This type of arthritis is generally seen with aging, trauma, or
congenital abnormality (dysplasia) of the hip joint. Other conditions leading to total hip replace-
ment include bone fractures, rheumatoid arthritis, and bone death (aseptic necrosis) of the femoral
head. Bone necrosis can be caused by fracture of the hip, alcohol and drugs (such as prednisone
and prednisolone), diseases (such as systemic lupus erythematosus), and conditions (such as kidney
transplantation).
Total hip arthroplasty (THA) or hemi-arthroplasty is a surgical procedure whereby the diseased
cartilage and the bone of the hip joint are surgically replaced with an artificial joint to restore joint
movement (Figure 25.1). In general, total hip joint replacement consists of three (cement fixation) or
four (cementless fixation) parts as follows:
• A plastic cup and metal shell that replaces the hip socket (acetabulum)
• A metal or ceramic ball that replaces the fractured femoral head
• A femoral metal stem that is attached to the shaft of the bone to add stability to the
prosthesis
If a hemi-arthroplasty is performed, either the femoral head or the hip socket (acetabulum) will be
replaced with a prosthetic device.
Upon inserting the prosthesis into the central core of the femur, it is fixed with a bone cement of
poly(methyl methacrylate). Alternatively, a “cementless” prosthesis is used, which has microscopic
Pelvis
Plastic (polyethylene) cup
Acetabulum
Acetabular metal shell

(cementless fixation)
Metal or ceramic ball

Total hip joint replacement
Femoral metal stem
Femur
FIGURE 25.1 Schematic model of total hip joint replacement and typical product.
Polymers for Artificial Joints 853
pores that allow bone ingrowth from the normal femur into the prosthesis stem. This “cementless”
hip is considered to have a longer duration and will be chosen especially for younger patients.
THA is one of the most successful joint surgeries performed today. The operation relieves pain
and stiffness symptoms, and most patients (over 80%) need no help in walking. In well-selected
patients who are appropriate candidates for total hip replacements, the effects of the procedure
last for at least 10 years in nearly 95% of patients [1,2]. However, with time, many problems have
been observed due to the limited long-term fixation of the replacement. Hence, improvements with
new devices and techniques are necessary. The future will provide newer devices that will fur-
ther improve patient outcomes and lessen the potential for complications. Moreover, with improved
devices and techniques, the operation could be recommended for younger individuals.
25.1.2 Knee Joint Replacement and Its Clinical Performance

The most common type of arthritis leading to knee replacement is also degenerative arthritis
(i.e., osteoarthritis) of the knee joint. For patients with mild arthritis, which is confined to one of the
condyles of the knee, the surgeon might decide to perform unicondylar knee arthroplasty (UKA).
If the arthritis is more serious and both the condyles of the knee are diseased, the surgeon may
perform a bicondylar total knee arthroplasty (TKA) (Figure 25.2). Finally, in the case of extreme
circumstances, such as a revision operation or in the event of tumor resection, semi-constrained
hinged knee design with metal or ceramic materials might be employed.
TKA includes a prosthesis consisting of three or four parts as follows:
1. A metal or ceramic femoral component

2. A plastic insert or all-plastic tibial component
3. A metal or ceramic tibial tray
4. A plastic patellar component
The plastic ultra high molecular weight polyethylene (UHMWPE) insert or all-plastic tibial com-
ponent plays a primary role: articulating either against a metallic or ceramic femoral component; in
several cases in patellar resurfacing, the UHMWPE may articulate against cartilage.
TKA continues to be a remarkably successful operation for pain relief. Cementless fixation of the
artificial knee joint is much less common than cement fixation. Today, TKA yields beneficial and
predictable results with a survival rate of over 90% at 10 years after surgery [3].
On the other hand, the number of primary TKA has continuously increased. Although the fre-
quency of revision surgery has not changed, the failure mechanism that necessitates revision TKA
appears to be increasingly related to UHMWPE wear and tear along with osteolysis. Rand et al.
Femoral ceramic or
Femur
metal component
Polyethylene insert
Tibial metal
component
Tibia Total knee joint replacement
FIGURE 25.2 Schematic model of total knee joint replacement and typical product.
reported that implant loosening was the major mode of failure [4]. Aseptic loosening was the lead-
ing reason for revision arthroplasty, followed by osteolysis and polyethylene wear. However, the
true wear and tear rate of polyethylene in TKA is not exactly known for a number of reasons as
follows: no radiographic methods for measuring it have been established, the complex geometry of
knee implants makes this task far more difficult than in THA, and the activity levels of the patient
are largely unreported. In clinical outcome studies, the wear and tear of the tibial UHMWPE insert
should be correlated with not only the period of clinical use but also the condition and type of use.
The orthopedic community has also recognized that oxidative degradation can adversely affect
the wear properties of UHMWPE. Oxidative degradation occurs due to prolonged shelf life and
clinical use. An oxidatively degraded UHMWPE insert generally shows bad results with failure by
wear and tear or osteolysis.
Wear and tear in TKA is far more dependent on alignment and ligament balancing techniques
than wear and tear in THA is. The understanding of alterations in knee kinematics in TKA has
markedly improved, but the surgical technique for TKA remains largely unchanged. Improvements
in surgical technique with experience and teaching should reduce the frequency of component posi-
tioning that tends to accelerate wear and tear.
25.1.3 Increase in Hip Joint Arthroplasty Procedures

THA is widely recognized as a successful and effective treatment for degenerative hip joint dis-
ease. Worldwide, the number and rate of artificial hip joints used for primary and revised THA are
substantially increasing every year. For example, the rate of primary THA per 100,000 persons
increased by 46%, and the rates of revision THA increased by 60%, respectively, during 1990–2002
in the United States [1]. The number of primary THA increased from 119,000 in 1990 to 193,000 in
2002. Taking into account the population changes according to the United States Census Bureau,
the overall rate of primary THA is 15 procedures per 100,000 persons per decade. On the other
hand, in Japan, the number of primary THA and hemi-arthroplasties increased from 15,040 and
27,916 in 1994 to 35,793 and 49,315 in 2006, by 138% and 77%, respectively (Figure 25.3) [5].
Hip joint arthroplasties have important implications for health costs in Japan. For example, if the
85,108 hip joint arthroplasties performed in 2006 were to increase by 1%, the potential increase in
cost could be 581 million (≈$5.81 million) based on recent procedural cost estimates of 0.7 million
(≈$0.007 million) for each hip joint arthroplasty.
A general trend pointing to an increase in both the number and the rate of revision arthroplasties
has also been observed. The number of revision THA increased from approximately 24,000 in 1990
to 43,000 in 2002 in the United States [1]. Further, the mean revision burden for THA was noted to
be 17.5% (15.2%–20.5%). Taking into account the population changes according to the U.S. Census
Bureau, the overall rate of revision THA was 3.7 procedures per 100,000 persons per decade.
5 Hemi-arthroplasty
Procedures (×104)
2
THA
1
0
1992 1997 2002 2007
Year
FIGURE 25.3 Procedures of primary THA and hemi-arthroplasty in Japan. *Forecast value.
TABLE 25.1
Revision Burdens for Hip Arthroplasty
in Various Countries
Revision
Country Period Burden (%) Comments
Australia 1999–2002 18.2 —
Canada 2002–2003 13.1 —
Finland 1980–2001 15.7 —
Finland 1990–2001 18.3 —
Norway 1987–1998 15.0 —
Norway 1994–1998 16.4 —
Sweden 1979–2000 7.7 —
Sweden 1992–2000 11.0 —
Sweden 1992–2000 6.4 ≥65 years old
United States 1990–2002 17.5 —
United States 1990–2002 16.9 ≥65 years old
With the exception of THA performed in Sweden, the revision burden in the United States com-
pared favorably with that in several countries with established total joint registries (Table 25.1) [1,2].
Overall, the THA revision burden of 17.5% in the United States from 1990 through 2002 fell within
the range of revision burdens of 15.0%–18.3% observed in Norway, Finland, and Australia. In Canada,
the revision burden for THA was lower (13.1% for 2002–2003). The overall revision burden for THA
in the United States was substantially greater than the revision burden reported for Sweden (7%–11%).
25.1.4 Problems of Joint Replacement: Osteolysis

Table 25.2 illustrates the reasons for revision in the 14,081 first revisions for THA performed in
the previous study [2]. The majority (75.3%) of the revision surgeries were performed because of
aseptic loosening with or without focal osteolysis, 7.6% were performed to treat primary or second-
ary infection, and 8.8% were performed for technical reasons and dislocation that could have been
mainly related to misalignment of the implants. Periprosthetic fractures (5.1%), implant fractures
(1.5%), and a number of less prevalent reasons constituted the balance of the reasons.
TABLE 25.2
Reasons for Revision THA
Reason Number Share (%)
Aseptic loosening 10610 75.3
Primary deep infection 948 6.7
Dislocation 810 5.8
Fracture only 716 5.1
Technical error 425 3.0
Implant fracture 215 1.5
Secondary infection 128 0.9
Polyethylene wear 126 0.9
Pain 46 0.3
Miscellaneous 56 0.4
Missing 1 <0.1
Total 14081 100
Wear debris
from polyethylene cup
Phagocytosis of debris
by macrophage
Post-operation Release of cytokines
and prostaglandin
Loosening
Osteolysis
Osteoclast
formation
9 years after operation
FIGURE 25.4 Schematic model of the mechanisms by which wear debris leads to osteolysis.
As shown in Table 25.2, a consensus statement on total hip joint replacement concluded that the
major remaining issues of concern included long-term fixation of the acetabular component, oste-
olysis due to wear debris, the biological response to debris, and problems related to revision surgery.
Although acetabular fixation is no longer a problem, wear and related complications continue to be
the major issue affecting the longevity of total hip joint replacements. The bone loss associated with
osteolysis can result in pelvic dissociation and instability and major segmental cortical defects in the
femur. Young active patients are most at risk for wear and osteolysis.
The precise mechanisms by which wear debris leads to osteolysis will ultimately be deter-
mined by defining how specific types of particles combine with environmental factors to permit
interactions with specific types of cells that then communicate with each other through the release
of soluble mediators (Figure 25.4). Generation of wear debris occurs immediately after implant
insertion and ultimately results in a profile of particles that includes all total hip joint replacement
materials [6,7]. The extent of bone resorption at the implant–bone interface varies with the severity
of the granulomatous tissue response to wear debris and determines the time lapsed before implant
loosening occurs. Wear particle-induced macrophage activation plays a role in periprosthetic oste-
olysis. Essentially, this occurs by two biological mechanisms. First, wear particle-associated mac-
rophages release proinflammatory factors (e.g., cytokines, growth factors, prostaglandins) that
enhance the activity of osteoclasts, the cells that carry out bone resorption. Second, osteoclasts are
formed from mononuclear precursors that are present in the wear particle-induced macrophage
infiltrate. These processes are not mutually exclusive; other stromal and inflammatory cell ele-
ments found at the bone–implant interface likely influence both the extent of osteoclast formation
and bone resorption.
The contribution of the cells present within the macrophage-rich inflammatory tissue to the
induction of bone resorption and implant loosening involves multiple cellular mechanisms. The
macrophages are activated by the particles and subsequently release proinflammatory cyto-
kines and other agents that induce bone resorption. Macrophage products capable of inducing
bone resorption include interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, ara-
chidonic acid metabolites, and degradative enzymes. The existence of multiple factors at one
site is likely to accelerate bone destruction. IL-1α, IL-1β, and TNF-α may also induce sec-
ondary effects on other cell types (such as osteoblasts) in the interfacial membrane, resulting
in the release of matrix-degrading enzymes, including collagenase, stromelysin, gelatinases,
and plasminogen activators. Granulocyte/macrophage colony-stimulating factor (GM-CSF) has
also been implicated in cellular proliferation in the interfacial membrane around implants.
Other cytokines that may exhibit immunomodulatory roles include IL-12, which is increased
in the pseudosynovial fluid in patients with aseptic loosening of hip joint replacement. A pri-
mary response of macrophages to particulate debris is the increased release of TNF-α. TNF-α
release results in part from the exposure of macrophages to particles, which activates the tran-
scription factor NF-κB; this reaction is related to membrane receptor events. Alteration of
the bone surface by these proteases may stimulate osteoclast bone-resorbing activity and may
influence the recruitment and adhesion of mononuclear phagocyte osteoclast precursors at the
bone–implant interface.
A second important mechanism relevant to the role of macrophages in implant loosening is
revealed by data demonstrating that wear particle-associated macrophages are capable of differen-
tiating into multinucleated cells that exhibit all the phenotypic features of osteoclasts. Osteoclasts
are highly specialized multinucleated cells that are uniquely capable of carrying out lacunar
resorption. Osteoclasts are formed by fusion of bone marrow-derived mononuclear precursors that
circulate in the monocyte fraction. A number of cellular and humoral factors are known to influ-
ence RANKL and osteoprotegerin (OPG) expression. Osteoclast formation in periprosthetic tis-
sues can effectively be viewed as a balance between the productions of these two factors. Various
cytokines and growth factors (apart from macrophage CSF) abundant in periprosthetic tissues in
aseptic loosening, such as IL-1 and TNF-α, increase the OPG mRNA expression by osteoblasts,
suggesting that these factors that stimulate osteoclastic bone-resorbing activity appear to act con-
versely to downregulate osteoclast formation. Prostaglandins such as PGE2 have also been shown
to increase RANKL production and to decrease OPG release, thus stimulating osteoclast formation
and bone resorption. Inflammatory cells, such as T-cells, are present in the arthroplasty membrane
and may influence osteoclast differentiation and periprosthetic osteolysis by modulating RANKL
expression and OPG production. Recent studies have also highlighted the role of certain cytokines
(e.g., TNF-α, IL-1β, and IL-1) in inducing osteoclast formation both in the presence and absence
of RANKL.
25.2 BEARING MATERIALS FOR JOINT REPLACEMENTS

25.2.1 Polyethylene Bearing Material
Polyethylene is a polymer formed from ethylene (C2H4), which is a gas with a molecular weight
of 28. The generic chemical formula for polyethylene is –(C2H4)n−, where n is the degree of polym-
erization. For UHMWPE, the molecular chain can consist of as many as 0.2 × 106 ethylene repeat
units, i.e., the molecular chain of UHMWPE contains up to 0.4 × 106 carbon atoms.
There are several kinds of polyethylene, which are synthesized with different molecular
weights and chain architectures. Low-density polyethylene (LDPE) and linear low-density poly-
ethylene (LLDPE) generally have branched and linear chain architectures, respectively, each with
a molecular weight of typically less than 5 × 104 g/mol. High-density polyethylene (HDPE) is a
linear polymer with a molecular weight of up to 0.2 × 106 g/mol. In comparison, UHMWPE has
a molecular weight of up to 6 × 106 g/mol. In fact, the molecular weight is so ultra-high that it
cannot be measured directly by conventional methods and must instead be inferred by its intrinsic
viscosity. Table 25.3 summarizes the physical and chemical properties of LDPE, LLDPE, HDPE,
and UHMWPE.
TABLE 25.3
Typical Physical and Chemical Properties of LDPE, LLDPE, HDPE,
and UHMWPE
Property LDPE LLDPE HDPE UHMWPE
Molecular weight (10 g/mol)
6 — — 0.05–0.25 2–6
Melting temperature (°C) 110–115 110–125 130–137 125–138
Poisson’s ratio — — 0.40 0.46
Specific gravity 0.910–0.930 0.910–0.925 0.952–0.965 0.932–0.945
Tensile modulus of elasticity (GPa) 0.1–0.4 0.1–1.6 0.4–4.0 0.8–1.6
Tensile yield strength (MPa) 7–14 7–42 26–33 21–28
Tensile ultimate strength (MPa) 3–57 8–46 22–31 39–48
Elongation (%) 145–1000 460–1100 10–1200 350–525
Crystallinity (%) <50 — 60–80 39–75
Implant products
Molding;
compression,
ram extruding, etc. Machining
PE resin powder
Sheet and bar stocks
FIGURE 25.5 Typical processing steps in the manufacture of UHMWPE implants.
Three industrial steps are needed to manufacture orthopedic implants. First, UHMWPE must be
polymerized from ethylene gas. Second, the polymerized UHMWPE, in the form of resin powder,
needs to be consolidated into a sheet (i.e., compression molding), rod (i.e., ram extrusion), or near-
net shaped implant (i.e., direct compression molding). Finally, in most instances, the UHMWPE
implant needs to be machined into its final shape (Figure 25.5).
Since the 1950s, UHMWPE powders have been produced by Ruhrchemie (currently known as
Ticona GmbH, Oberhausen, Germany) using the Ziegler process. The main ingredients for process-
ing UHMWPE are reactive ethylene gas, hydrogen, and titanium tetrachloride catalyst. The polym-
erization takes place in a solvent used for mass and heat transfer. The requirements for medical-grade
UHMWPE powder are specified in the American Society for Testing and Materials (ASTM) stan-
dard F648 and the International Organization for Standardization (ISO) standard 5834-1 [8,9].
Historically, the UHMWPE powder has been converted by compression molding since the 1950s,
because the industries in the area around Ruhrchemie already were experienced in this processing
technique. Today, compression-molded sheets of the UHMWPE are produced commercially by two
companies (Orthoplastics, Ltd., Lancashire, United Kingdom, and Meditech Poly Hi Solidur, Ltd.,
Fort Wayne, IN). One UHMWPE sheet is pressed between the upper and middle plates, and the
second is produced between the middle and lower plates. The plates are oil heated and hydraulically
actuated from below. The heating and loading systems are all computer controlled. Finally, the entire
press is contained in a clean room, to reduce the contamination of extraneous matter into the sheet.
In contrast, ram extrusion of UHMWPE was developed by converters in the United States during
the 1970s. Today, only few converters supply medical-grade ram extrusion UHMWPE to the ortho-
pedic industry. Medical-grade extrusion facilities are owned by Orthoplastics, Ltd., Meditech Poly
Hi Solidur, Ltd., and Westlake Plastics Co. Ltd. (Lenni, PA). The process is as follows. UHMWPE
powder is fed continuously into an extruder. The extruder itself consists essentially of a hopper that
allows powder to enter a heated receiving chamber, a horizontal reciprocating ram, a heated die, and
an outlet. Within the extruder, the UHMWPE is maintained under pressure by the ram as well as by
the backpressure of the molten UHMWPE. The backpressure is caused by frictional forces of the
molten resin against the heated die wall surface as it is forced horizontally though the outlet. Beyond
the outlet, the UHMWPE rod is slowly cooled in a series of electric heating mantles.
25.2.1.1 History of Polyethylene in the Orthopedic Field
The load-bearing articulating surface materials used in total joint arthroplasty comprise metal-
lic alloys, ceramics, and polymers. The articulating couples of primary concern—those that gen-
erate considerable amounts of wear leading to osteolysis—include UHMWPE cups and inserts.
Accordingly, in the past several decades, most research and development have been focused on
improving the wear resistance of UHMWPE (Table 25.4).
Introduced clinically in November 1962 by Charnley, UHMWPE articulating against a metal-
lic femoral ball remains the gold standard bearing surface combination in total hip joint replace-
ment [11]. Considering how rapidly technology can change in the field of orthopedics, the long-term
role that UHMWPE has played in hip joint replacement since the 1960s is fairly remarkable.
In the 1970s, the properties of UHMWPE were modified by including carbon fibers within the
matrix of polyethylene, thereby creating a carbon fiber-reinforced UHMWPE, known as Poly II
(Zimmer, Inc., Warsaw, IN) [13]. However, this UHMWPE composite was not found to exhibit con-
sistent and improved clinical results relative to the conventional UHMWPE introduced by Charnley.
The material was designed with orthopedic bearing applications in mind, under the assumption that
increasing the modulus and ultimate tensile strength of the bearing as well as decreasing its creep
properties would increase its longevity. This assumption was reasonable, since bearing surfaces are
subject to high contact stresses, conditions under which conventional UHMWPE had often been
observed to be pitted or delaminated. The inclusion of short chopped carbon fibers in a UHMWPE
matrix resulted in a composite material with improved mechanical properties in vitro. Thus, the
expectation was that Poly II would be more resistant to the pitting and delamination often seen in
TABLE 25.4
History of UHMWPE Development for Joint Replacement
Year Comments
1958 Clinical use of polytetrafluoroethylene as bearing material of implants in hip
arthroplasty by Charnley et al. [10]
1962 Charnley et al. adopts UHMWPE for use in hip arthroplasty [11]
1969 UHMWPE was gamma-ray sterilized in air with a minimum dose of 25 kGy [12]
1970 Commercial release of the Poly II-carbon fiber-reinforced UHMWPE for hip
arthroplasty by Zimmer, Inc. [13]
1971 Clinical introduction of the 100 Mrad PE—extremely highly CLPE by more
than 1000 kGy of gamma-ray irradiation in air by Oonishi et al. [16]
1982 Commercial release of alumina ceramic balls articulating against UHMWPE by
Kyocera, Corp. [19]
1986 Clinical introduction of silane cross-linked HDPE by Wroblewski et al. [20]
1991 Commercial release of the Hylamer—highly crystalline UHMWPE for hip
arthroplasty by DePuy Orthopedics, Inc. [21]
1997 Commercial release of highly CLPE with an energy-ray irradiation of
50–105 kGy by several orthopedic product manufacturers [13,24]
2006 Clinical use of the vitamin E-blended UHMWPE in knee arthroplasty produced
by Nakashima Medical, Co. Ltd., as a trial [44]
2007 Clinical use of the PMPC-grafted CLPE in hip arthroplasty produced by Japan
Medical Materials Corp. as a trial [42]
joint replacements. Further, wear testing of Poly II conducted by the manufacturer revealed it to
have lower wear than conventional UHMWPE, suggesting that the strength benefits would result in
longer lasting hip arthroplasties. However, unfortunately, the promise shown by Poly II in vitro was
not borne out in the clinical setting, and within a short time after implantation, many patients pre-
sented with osteolysis and complete mechanical failure of their bearing surfaces [14]. One possible
explanation for the mechanical failure was that the poor crack propagation resistance of Poly II was
due to the carbon fibers not bonding with the UHMWPE matrix, instead serving as stress concen-
trators and crack nucleation sites [15].
In Japan, during the 1970s, an important technological advancement occurred: the clinical
introduction of an extremely highly cross-linked polyethylene (CLPE) with more than 1000 kGy
of gamma-ray irradiation in air by Oonishi et al., the so-called 100 Mrad PE [16,17]. A similar
advancement in extremely highly CLPE also occurred in South Africa during the 1970s, where
researchers in Pretoria clinically introduced a UHMWPE that was gamma-ray irradiated with up
to 700 kGy in the presence of acetylene [18]. During the 1980s, two other noteworthy developments
occurred relative to polyethylene in joint replacements. Chas F. Thackray-DePuy International Ltd.
(Leeds, United Kingdom) began the development of an injection-molded HDPE that could be cross-
linked by silane coupling. Only 22 of these implants were produced and implanted by Wroblewski
et al. starting in 1986 [20]. After an initial wear period (initial bedding-in period), these cross-linked
HDPE components have been found to exhibit very low clinical wear rates.
In 1991, a highly crystalline UHMWPE known as Hylamer was patented by Li et al. from
E. I. Du Pont de Nemours and Company (Wilmington, DE) and marketed by the DePuy-DuPont
Orthopedics joint venture (Newark, DE) [21]. Hylamer is a hot isostatically pressed UHMWPE,
leading to the formation of an extended-chain crystallite morphology with thick (200–500 nm)
lamellae and higher crystallinity (65%–71%) [13]. In contrast, conventional low-pressure sintered
UHMWPE displays a folded-chain crystalline morphology with much thinner lamellae (10–50 nm
in thickness) and a crystallinity of 50%–55%. By varying the postconversion heating, pressure, and
cooling sequence, a family of materials was developed with varying crystalline morphologies and
sizes. Hylamer has a higher density and crystallinity than conventional UHMWPE. Although the
yield and ultimate strength of Hylamer are slightly higher, the most noticeable change occurs in the
elastic modulus, which is nearly double for Hylamer as compared to conventional UHMWPE. The
clinical results for the highly crystalline UHMWPE, which were clarified in the 1990s, have been
mixed and are therefore controversial. Although several studies reported worse clinical perfor-
mance using the Hylamer compared with conventional UHMWPE, other studies reported several
satisfactory or even improved performances [13].
25.2.1.2 Cross-Linked Polyethylene

High-energy ray irradiation cross-linking and thermal treatment of UHMWPE has aroused intense
scientific and commercial interest within the orthopedic field since the late 1990s (Figure 25.6).
For decades, the cross-linking of polyethylene has been known to improve the abrasion resistance
of the polymer for industrial applications. However, only a few applications of this technology have
been reported in orthopedics literature [13,22,23]. All high-energy ray irradiation, including the
standard 25- to 40-kGy dose of gamma-ray irradiation used for sterilization, leads to the formation
of free radicals in polymeric materials through homolytic chain cleavage. In UHMWPE, some of
these free radicals recombine with each other to form cross-links or trans-vinylene bonds, while
others remain as highly reactive species in the structure for extended periods of time. Although
the gel content of UHMWPE may be increased to 80% by an average gamma-ray radiation dose of
25 kGy, the polymer becomes highly cross-linked (corresponding to a gel content of 90%–100%)
after an absorbed dose of 50 kGy [13,24]. Despite the plateau in gel content, the cross-linking
density in UHMWPE did not reach an asymptotic value until a dose of 100–150 kGy had been
absorbed. Therefore, several CLPEs, irradiated with 50–105 kGy, have been launched since 1998
and used extensively.
Amorphous
region Cross-link
Gamma-ray
irradiation
Crystalline
region
Tie molecule
FIGURE 25.6 Schematic illustration of cross-linking induced by gamma-ray irradiation.
TABLE 25.5
Wear Reductions in Early- and Mid-Term Clinical Studies of
CLPE Cups Compared with Conventional UHMWPE Cups
Manufacturing Process Mean Follow-Up Wear
for CLPE Period (Years) Reduction (%) Reference
Cold-irradiated and annealed 2.0 85 [27]
2.3 42 [28]
2.3 94 [23]
4.0 58 [29]
4.9 60 [30]
Cold-irradiated and remelted 2.8 72 [31]
3.2 45 [32]
5.3 73 [24]
5.5 95 [33]
Warm-irradiated and 2.0 54 [34]
remelted 2.6 94 [35]
2.9 44 [36]
3.0 23 [37]
3.8 83 [38]
5.0 55 [39]
In several independent reviews of the literature, it was found that osteolysis is rare in patients in
whom the UHMWPE cup is wearing at a rate of less than about 0.1 mm/year, but osteolysis becomes
much more frequent and extensive as the wear rate increases substantially above this “threshold”
value [25,26]. In several studies with a mean duration of follow-up of ∼5 years or longer, the mean
rates of wear of CLPE cups (Table 25.5) were all well below 0.1 mm/year [23,24,27–39].
On the other hand, the osteolysis threshold of 0.1 mm/year was established for hip joints with
conventional UHMWPE cups—i.e., those that either were not cross-linked or were moderately
cross-linked during gamma-ray sterilization. Some investigators have reported that the mean par-
ticle size is smaller with CLPE and that, in equivalent volumes, smaller particles tend to be more
likely to cause osteolysis [40]. If that is correct, these factors could lead to the osteolysis threshold
being somewhat lower for CLPE. We are aware of only one published case report of clinically rel-
evant osteolysis in a hip with a CLPE cup [41]. However, the hip in question also had a forged-steel
surface-grit-blasted femoral component that, at revision, was found to be loose at the stem–cement
interface. Because the osteolysis in this hip joint occurred endosteally around the loosened stem,
with no acetabular osteolysis, it is highly possible that the lesions were primarily due to debris pro-
duced at the stem–cement interface rather than from the CLPE cups. Continued close monitoring
of patients with CLPE cups is essential to determine if the improved wear resistance that has been
observed in the mid-term, as summarized here, will translate into a substantial reduction in the
prevalence and severity of osteolysis at long-term follow-up.
25.2.1.3 Antioxidants for Polyethylene

Recently, there has been an explosion of interest in the research and development of vitamin E as an
antioxidant for UHMWPE in the orthopedic field. The primary role of vitamin E (α-tocopherol) is
to stabilize the active free radicals resulting from oxidation. The antioxidant activity of vitamin E
is due to hydrogen abstraction from the –OH group on the chroman ring by a peroxy free radical,
which can combine with another free radical (Figure 25.7). In a gamma-ray irradiated UHMWPE
with vitamin E, peroxy free radicals abstract a hydrogen from vitamin E, forming hydroperoxides.
The oxidative degradation cascade in the gamma-ray irradiated UHMWPE is hindered in the pres-
ence of vitamin E.
The idea of vitamin E-blended UHMWPE is popular in the industrial field: the first widespread
applications of the vitamin E-blending technology actually appeared in food packaging since the
1980s. In the orthopedic field, Tomita et al. demonstrated the use of vitamin E-blended UHMWPE
in order to prevent delamination by reducing crack formation at the grain boundaries of UHMWPE
in 1998 [43]. Then, they demonstrated that the vitamin E-blended UHMWPE with gamma-ray ster-
ilization exhibited a higher resistance to oxidation and fatigue wear compared with conventional
UHMWPE. In light of its acceptance as an effective antioxidant, the vitamin E-blended UHMWPE
ROOH + R*
Gamma-ray RH
C H
O2 ROO*
or R* ROO* ROO OOR R1 C R2 + O2 + HOR
C C O
UHMWPE α-tocH
ROOH + α-toc*
α-Tocopherol
(α-tocH)
CH3
HO CH3 CH3 CH3
CH3
H3C O CH3
CH3
α-Tocopherol free radical

(α-toc*) CH 3
*O
CH3 CH3 CH3
CH3
H3C O CH3
CH3
CH3 Dimer
α-toc*
O * CH3 CH3 CH3
CH3
H 3C O CH3
CH3 ROO* α-toc OOR
FIGURE 25.7 Schematic illustration of reaction of vitamin E (α-tocopherol).

insert in TKR was produced by Nakashima Medical Co. Ltd. (Okayama, Japan) and is being used in
a clinical trial in Japan since 2006 [44]. Although this trial has taken place, the clinical results have
not been published yet.
Subsequently, many orthopedic manufacturers have developed CLPE with vitamin E for joint
replacements. However, several new problems have arisen, in particular for the procedures of intro-
duction of vitamin E into the polyethylene as follows: (1) blending during compression molding
or extrusion before the cross-linking and (2) diffusion after the cross-linking and machining [45].
The disadvantages of the former are that the cross-link density is suppressed to a low value during
the cross-linking procedure with (e.g., gamma-ray) irradiation (Figure 25.8). On the other hand,
those of the latter are that it is difficult to control the concentration and distribution of diffused
vitamin E.
In both the cases, the hypothesized advantage of the vitamin E-blended/diffused CLPE is that
the vitamin E protects the CLPE against oxidative degradation (Figure 25.9).
1.8
1.5
Cross-link density (mol%)
1.2
0.9
0.6
0.3 : CLPE
: Vitamin E-blended CLPE
0.0
0 25 50 75 100 125 150
Irradiation dose (kGy)
FIGURE 25.8 Cross-link density of vitamin E-blended CLPE as a function of the gamma-ray irradiation.
Bar: Standard deviations.
12
: Conventional PE (EOG sterilization)
: Conventional PE (γ-ray sterilization)
10 : Vitamin E-blended CLPE (γ-ray sterilization)
8
Oxidation index
0
0 3 6 9 12 15
Accelerated aging duration (w)
FIGURE 25.9 Oxidative degradation (oxidation index) of vitamin E-blended CLPE as a function of the
accelerated aging duration in air at 80°C. Bar: Standard deviations.
25.2.2 Phospholipid Polymers for Mimicking Articular Cartilage

25.2.2.1 Hydration Lubrication
Water attracted by hydrophilic macromolecules in the surface layer plays an important role in lubrication.
As macromolecules are flexible, they cannot support a load by themselves. The water in the surface
layer would support most of the load because the water is attracted by the macromolecules. Frictional
forces arise due to the adhesion of macromolecules to the counter surface. The time-dependent prop-
erties of friction forces can be interpreted as follows (Figure 25.10) [46]. Under a load, water exudes
slowly from the surface layer with or without sliding. As the result of water loss, the thickness of the
surface layer reduces and the water content of the surface layer decreases. Consequently, the degree of
adhesion to the opposite bearing surface increases and the frictional force also increases. Therefore,
it may be concluded that friction depends essentially on the water content of the surface layer. This
hydration would lead to low friction and wear, by acting as “hydration lubrication.”
25.2.2.2 Articular Cartilage and Material Design

Although the lubrication mechanism of human joints has been studied since the 1930s, it has not yet
been understood clearly. However, it is well known that the composition elements of the articular
cartilage surface consist of the collagen network, hyaluronic acid, and proteoglycan subunits. The
proteoglycan subunits form a gel-like surface layer due to hydration along with the joint synovia.
Although the binding between the proteoglycan subunits and hyaluronic acid can be visible [47], the
binding between hyaluronic acid and the collagen network has not yet been confirmed. It was reported
by Obara et al. [48] that the friction coefficient of joints increases when the gelled material on the car-
tilage surface is removed by gauze. After this, the joint surface is lubricated only by joint synovia or
hyaluronic acid, i.e., following the loss of the gel comprising proteoglycan subunits, the friction coef-
ficient of the joint cannot be lowered again. This fact indicates that the proteoglycan aggregates are not
combined with the collagen network by physical adsorption and that the hydrophilic macromolecules
on the joint surface play an important role in keeping the friction at low levels. A previous study
reported that the hydrophilic macromolecules of the cartilage surface are assumed to have a brush-like
structure: a part of the proteoglycan aggregate brush is bonded with the collagen network on the car-
tilage surface (Figure 25.11) [49]. The rest of the proteoglycan aggregate floats freely in joint synovia.
Load Bearing surface Water film

Water film
recovery
Water flow
Surface layer
FIGURE 25.10 Schematic model of hydration lubrication.
Proteoglycan subunit
Hyaluronic acid
Collagen network
FIGURE 25.11 Schematic model of the brush-like structure of the cartilage surface.
Longfield et al. [50] and Ikeuchi et al. [51] reported that the lubrication mechanism of joints
mainly comprises hydration lubrication. Hydrophilic macromolecules induce low friction by pro-
moting the formation of a fluid film that is retained by the water molecules’ attraction forces,
indicating that the water molecules’ attraction forces are important for realizing low friction. The
lubrication of the human joint appears to occur by hydration lubrication because the surface layer
of the joint resembles the structure of the gelled material in the human joint. Sasada et al. proposed
a new idea for joint lubrication, named “Surface gel hydration lubrication” [49] for a lubrication
mechanism peculiar to such hydrophilic macromolecules. A bearing surface with a brush-like struc-
ture comprising hydrophilic macromolecules in artificial hip joints was therefore assumed to be
similar to that of articular cartilage. The hydration lubrication interface can also be regarded to
mimic the natural joint cartilage in vivo. The novel material design with this hydration lubrication
should be necessary.
25.2.2.3 Photo-Induced Surface “Grafting from” Polymerization

The grafting of polymers onto various surfaces has been studied for over 50 years and has played
an important role in many areas of biomaterial science and technology, e.g., colloidal stabiliza-
tion, adhesion, lubrication, tribology, and rheology. Recent work has focused on the synthesis of
so-called polymer brushes whereby the polymer chains stretch out away from the surface or sub-
strate [52,53]. There are three primary methods for modifying a planar substrate with an organic
polymer: (a) physical coating, (b) chemical coating and/or “grafting to,” and (c) “grafting from”
(Figure 25.12). This includes physical coating such as spin or dip coating; however, the polymer
is merely adsorbed onto the substrate and may diffuse away when the substrate is immersed into
a solvent in which it is soluble. Chemical coating utilizes the functional group of the polymer to
chemically attach onto the substrate via several coating techniques. Robust layers may be created
by utilizing a self-assembled monolayer (SAM) in order to immobilize a reactive functionality.
Thus, the polymer can be attached to the surface, provided the preformed polymer possesses a
functional group that is capable of bonding with the surface (e.g., a polymer containing a pri-
mary amine could form an amide bond with a carboxylic acid-terminated SAM). This approach
Polymer
(a)
Functional group
(b)
Monomer
Initiator
hν or Δ
(c)
FIGURE 25.12 Approaches for modifying a substrate with a polymer: (a) physical coating (adsorption),
(b) chemical coating (immobilization) and/or “grafting to,” and (c) “grafting from.”
is known as the “grafting to” technique and has been considerably successful at synthesizing
robust layers of 1–50 nm in thickness. However, the “grafting to” technique is limited by diffusion
barriers that prevent the preformed polymer from intercalating through the tethered polymer to
the reactive substrate. Therefore, the “grafting to” method yields a low-density brush. In contrast,
the “grafting from” approach has been utilized to synthesize high-density polymer brushes [54].
The conformation of these polymer brushes in a solvent can dramatically change with the graft
density. At low-graft density, they will assume a “mushroom” conformation with a coil dimen-
sion similar to that of free chains. With increasing graft density, the graft chains will be obliged
to stretch away from the substrate, forming a “polymer brush.” These high-density brushes can
be much thicker and range in size from a nanometer-scale to greater than a micrometer-scale.
The great increase in thickness for “grafting from” layers is due to much higher grafting densities
compared to those of “grafting to” layers.
Photochemical initiation has several advantages over thermal initiation. First, certain func-
tional groups are not thermally stable; therefore, it is desirable to activate polymerization at
room temperature. This also simplifies manufacturing processes. Furthermore, most alkylthio-
late SAMs are not stable above 70°C and may begin to degrade at the temperatures required
for most thermal initiations. Second, photoinitiation is generally faster than thermal initia-
tion. Third, the initiation process may be activated at almost every temperature; this yields
great flexibility when controlling the reactivity and processability of a layer. Surface-initiated
polymerization has been carried out with a variety of initiators, and Figure 25.13 describes
some of the most common photoinitiators [55]. For surface-initiated polymerization applica-
tions, these initiators are typically modified and covalently bonded to the substrate to yield a
“grafting from” polymerization. Alternatively, photo-sensitizers can be added to bulk solu-
tions in order to abstract hydrogen atoms from the substrate. For example, benzophenone (BP,
Figure 25.13d) is converted to a reactive triplet state after ultraviolet (UV) irradiation; this trip-
let is capable of abstracting hydrogen atoms from various moieties. Tertiary amines or thiol-
ene systems have been activated with photo-sensitizers, but until recently have not been used
for surface-initiated polymerization. Other photoinitiators include peroxides (Figure 25.13a)
and benzoin derivatives (Figure 25.13c); of the two, only peroxides have been used for sur-
face-initiated polymerization. The most common free radical photoinitiators are derivatives of
2,2′-azobisisobutyronitrile (AIBN, Figure 25.13b), and these have been used by several research
groups for “grafting from” polymerizations from various substrates. Recently, controlled free
radical polymerizations have gained much recognition owing to their low polydispersities and
“living”-like properties [55]. Indeed, “living” polymerizations have a tremendous advantage
for surface-initiated polymerization since it is possible to grow block copolymers and to ter-
minate the polymerization with specific end-groups. However, most living free radical polym-
erizations utilize thermal initiation; for example, atom transfer radical polymerization may be
O
O
NC
O O N
N
O CN H3CO
(a) (b) (c)
+
C OC10H21 S N(CH2CH3)2
O SbF6– S
(d) (e) (f )
FIGURE 25.13 Various types of photoinitiators: (a) benzoylperoxides, (b) 2,2′-azobisisobutyronitrile com-
pounds based on AIBN, (c) benzoin methylethers, (d) triplet photo-sensitizers, benzophenone (e) onium salts
for cationic polymerization, and (f) controlled free radical polymerization with photoiniferters.
used for surface-initiated polymerization, but the rate of initiation and propagation is relatively
slow as compared to that of photoinitiation and typically leads to layers that are less than 50 nm
thick. Thus, there is a rich variety of photopolymerization strategies that may be utilized in
the future, although very few examples of photosurface-initiated polymerization have been
reported to date.
25.2.2.4 Poly(MPC)-Grafted Polyethylene

Surface modification is important for improvements in bearing materials. Moro et al. have
demonstrated the creation of an artificial hip joint based on the novel concept of “hydration
lubrication” by using poly(MPC) (PMPC)-grafted onto the surface of CLPE (PMPC-grafted
CLPE); this device is designed to reduce wear and suppress bone resorption [56–58]. A pre-
vious study has reported that the hydrogel cartilage surface is assumed to have a brush-like
structure: a part of the proteoglycan aggregate brush is bonded with the collagen network on
the cartilage surface [49]. Therefore, the bearing surface with PMPC in artificial hip joints is
assumed to have a brush-like structure similar to that of articular cartilage (Figure 25.14). The
hydration lubrication interface can therefore be regarded to mimic the natural joint cartilage
in vivo.
MPC, a methacrylate monomer with a phospholipid polar group in the side chain, is a novel
biomaterial designed and developed by Ishihara et al. that mimics the neutral phospholipids of
cell membranes [59]. MPC polymers are one of the most common biocompatible and hydrophilic
polymers studied thus far, which have potential applications in a variety of fields, such as biology,
biomedical science, and surface chemistry, because they possess the unique properties of good bio-
compatibility, high lubricity and low friction, anti-protein adsorption, and cell membrane-like sur-
face [59–62]. Hence, MPC is hydrophilic and can form a thin film of free water under physiological
conditions. Several medical devices have already been developed by utilizing the MPC polymers.
These devices have been subjected to clinical use with the approvals of the Ministry of Health,
Labour and Welfare (MHLW) of Japan and the Food and Drug Administration (FDA) of the United
States; therefore, the efficacy and safety of the MPC polymer as a biomaterial are well established
(Table 25.6) [63–82].
Water thin film
Proteoglycan subunit
Micrometer
scale Hyaluronic acid
Collagen network
(a)
Water thin film
Phosphorylcholine subunit
Nanometer
scale
Methacrylate
Cell membrane
(b) CLPE substrate
FIGURE 25.14 Schematic model of PMPC-grafted CLPE surface mimicking cartilage. (a) Cartilage. (b)
PMPC-grafted CLPE.
TABLE 25.6
Medical Devices with MPC Polymer
Medical Device Product Name Manufacturer Clinical Introduction Reference
Artificial heart Evaheart Sun Medical Current trial [63]
Artificial joint Aquala Japan Medical Current trial [42]
Materials
Artificial lung Mimesys Sorin Biomedica 2002 [64]
Synthesis Sorin Biomedica 2003 [65]
Physio Sorin Italia 2005 —
Catheter Eliminate Clinical Supply — MHLW approval
Contact lens Proclear Cooper Vision 1998 FDA approval
Guide wire Aqua diver Clinical Supply — MHLW approval
Inter through Clinical Supply — —
Hunter Biocompatible 1997 FDA approval
Micro catheter Londis Clinical Supply 2005 MHLW approval
Stent Endeavor Medtronic Current trial [66]
Endeavor I Medtronic Current trial [67,68]
Endeavor II Medtronic Current trial [67–69]
Endeavor II CA Medtronic Current trial [67,68]
Endeavor III Medtronic Current trial [67,68,70]
TriMaxx Abbott Laboratories 2005 [71]
ZoMaxx Abbott Laboratories — [72]
Biodiv Ysio Biocompatible 2000 [73–82]
Tympanostomy tube — Gyrus, Grace Medical 2000 FDA approval
C [BP]T
hν
O
BP
CH2 CH2 CH* CH2 + C*

l l
OH
CLPE (or UHMWPE)
O
O O– OH
O P N+
MPC O C
O +
m C
CH CH2 OH
n
PMPC-grafted CLPE Benzopinacol
FIGURE 25.15 Schematic illustration of MPC graft polymerization by using the BP system.
The grafting of biocompatible and hydrophilic PMPC with CLPE has been accomplished by
using a photo-initiated “grafting-from” polymerization. The photo-initiated “grafting-from” polym-
erization reaction by using a typical BP photoinitiator is shown in Figure 25.15. First, the physi-
cally coated BP on CLPE is excited by UV irradiation. The BP excited to the triplet state extracts a
hydrogen atom from the –CH2− group and then generates a radical that is capable of initiating the
graft polymerization of MPC.
PMPC layer PMPC layer PMPC layer

PMPC layer
CLPE CLPE CLPE CLPE CLPE
(a) (b) (c) (d) (e)
FIGURE 25.16 Cross-sectional TEM images of PMPC-grafted CLPE obtained with a 0.5 mol/L MPC con-
centration and various photo-irradiation times. Bar: 200 nm. (a) 11 min. (b) 23 min. (c) 45 min. (d) 90 min.
(e) 180 min.
This technique has several important benefits as follows: direct grafting of PMPC to CLPE—
thereby forming C–C covalent bonding between the PMPC and CLPE substrate, high mobility of
the chains of the PMPC, a high density, and controlling the length of the introduced PMPC.
Figure 25.16 shows cross-sectional transmission electron microscope (TEM) images of PMPC-
grafted CLPE produced with various photo-irradiation times during polymerization [83]. With
photo-irradiation times longer than 45 min, a 100- to 200-nm-thick PMPC-grafted layer was clearly
observed on the surface of the CLPE substrate. The MPC-covered region was coexistent with
uncovered regions after a photo-irradiation time of 23 min, although the thickness of the covered
region on the PMPC layer remained the same (100–200 nm). With photo-irradiation for 11 min, no
PMPC layer was observed on the surface of the CLPE. These results indicate that the density of
the grafted PMPC can be controlled by the polymerization time. This is attributable to the fact that
the number of polymer chains produced in a radical polymerization reaction is generally correlated
with the photo-irradiation time.
Figure 25.17 shows the static water-contact angle of PMPC-grafted CLPE as a function of the
photo-irradiation time used for polymerization (0.50 mol/L MPC concentration) [83]. The static
water-contact angle of untreated CLPE was 90° and decreased markedly with a decrease in
the photo-irradiation time. The static water-contact angle decreased as the irradiation time was
increased.
100
: CLPE (untreated)
: PMPC-grafted CLPE
80
Contact angle (°)
60
40
20
0
0 50 100 150 200
Photo-irradiation time (min)
FIGURE 25.17 Static water-contact angle of PMPC-grafted CLPE as a function of the photo-irradiation
time with a 0.5 mol/L MPC concentration. Bar: Standard deviations.
0.12
0.10
Dynamic coefficient of friction

0.08
y = 0.001x – 0.0016
0.06 R2 = 0.920
0.04
0.02
0
0 20 40 60 80 100
Contact angle (°)
FIGURE 25.18 Relationship between dynamic coefficient of friction and contact angle in the PMPC-grafted
CLPE surface. Bar: Standard deviations.
10
0
Weight change (mg)
–5
–10
–15
: 0 min (untreated CLPE, 0.00)*
–20 : 23 min (0.11)*
: 45 min (0.32)*
–25 : 90 min (0.46)*
: 180 min (0.48)*
–30
0 1 2 3 4 5
Test duration (×106 cycles)
FIGURE 25.19 Weight change (gravimetric wear) of the PMPC-grafted CLPE cups obtained with a
0.5 mol/L MPC concentration and various photo-irradiation times in the hip joint simulator wear test. Bar:
Standard deviations. *P–O group indexes are in parentheses.
Figure 25.18 shows the relationship between the dynamic coefficient of friction and the con-
tact angle [84]. The dynamic coefficient of friction tended to increase with the contact angle. This
increase was linear to a degree of accuracy, and the correlation coefficient was 0.920.
Figure 25.19 shows the gravimetric wear of PMPC-grafted CLPE with various photo-
irradiation times during the hip joint simulation test. The PMPC-grafted CLPE cups were found
to wear significantly less than the untreated CLPE cups. The wear of the PMPC-grafted CLPE
cups subjected to 23-min photo-irradiation time started to increase after 2.5 × 10 6 cycles. The
PMPC-grafted CLPE cups exhibited a slight increase in weight. This was partially attributable
to enhanced fluid absorption in the tested cups than in the load-soak controls. When using the
gravimetric method, the weight loss in the tested cups is corrected by subtracting the weight
gain in the load-soak controls; however, this correction cannot be perfectly achieved because
only the tested cups are continuously subjected to motion and load. Fluid absorption in the
tested cups is generally slightly higher than that in the load-soak controls. Consequently, the
(untreated CLPE)
(a) (b) (c)
(d) (e)
FIGURE 25.20 FE-SEM images of wear particles of the untreated CLPE and PMPC-grafted CLPE with
various photo-irradiation times during the 4.5 × 106 –5.0 × 106 cycles of the hip joint simulation test. Arrows:
wear particles. Bar: 5 μm. (a) 0 min. (b) 23 min. (c) 45 min. (d) 90 min. (e) 180 min.
correction for fluid absorption by using the load-soak data as the correction factor leads to a
slight underestimation of the actual weight loss. The initial wear rate is defined as that from
the start to 0.5 × 10 6 cycles, and the steady wear rate is considered as that from 4.0 × 106 to
5.0 × 10 6 cycles. All the untreated CLPE and PMPC-grafted CLPE cups showed low initial wear
rates of −1.42 to −3.74 mg/10 6 cycles. The steady wear rate of the untreated CLPE cups and the
PMPC-grafted CLPE cups with a low P–O group index of 0.11 (23-min photo-irradiation time)
increased to 5.11 and 5.48 mg/10 6 cycles, respectively. In contrast, the wear rates of the PMPC-
grafted CLPE cups with high P–O group indexes, i.e., 0.46 (90-min photo-irradiation time) and
0.48 (180-min photo-irradiation time), were markedly lower at 0.32 and −0.02 mg/10 6 cycles,
respectively.
Figure 25.20 shows field emission scanning electron microscope (FE-SEM) images of wear
particles of the untreated CLPE and PMPC-grafted CLPE with various photo-irradiation times
during the 4.5 × 10 6 –5.0 × 10 6 cycles of the hip joint simulation test. The wear particles of
the untreated CLPE and PMPC-grafted CLPE cups, as characterized by FE-SEM, were pre-
dominantly submicrometer-sized granules. The wear particles of the PMPC-grafted CLPE
cups with 45-, 90-, and 180-min photo-irradiation times were found to be significantly lesser
than those for the untreated CLPE cups and the PMPC-grafted CLPE cups with 23-min photo-
irradiation time.
In summary, an artificial hip joint based on the novel concept of “hydration lubrication” was cre-
ated by using PMPC grafted onto the surface of CLPE for reducing the wear debris of UHMWPE.
The approach using “hydration lubrication” is surely novel in the field of orthopedic biomaterials
science, and joint replacement with hydration lubrication can pioneer the “next generation” artificial
joint. Furthermore, these joint replacements have the potential to be applied in the orthopedic field
in the near future [85]. The clinical trial for such joint replacements with hydration lubrication
(i.e., PMPC-grafted CLPE acetabular cup) has been started at the University of Tokyo and other
hospitals in Japan since 2007. For this novel PMPC-grafted CLPE material, close monitoring of
clinical performance and accurate quantification of wear rates would be essential for the early rec-
ognition of unforeseen problems.
25.2.3 Poly(Ether-Ether Ketone) Bearing Materials

25.2.3.1 Structure and Properties
Poly(aryl-ether-ketone) (PAEK), including poly(ether-ether-ketone) (PEEK), is a new family of
high-performance thermoplastic polymers, consisting of an aromatic backbone molecular chain
interconnected by ketone and ether functional groups, i.e., a BP unit is included in its molecular
structure. Polyaromatic ketones exhibit enhanced mechanical properties, and their chemical struc-
ture is stable, resistant to chemical and radiation damage, and compatible with several reinforcing
agents (such as glass and carbon fibers; carbon fiber-reinforced PEEK [CFR-PEEK], Figure 25.21).
Therefore, they are considered to be promising materials for not only industrial applications but also
In the 1980s, the in vivo stability of various PAEK materials and the tissue response to the
same were investigated [86]. Recently, PEEK has emerged as the leading high-performance super-
engineering plastic candidate for replacing metal implant components, especially in the field of
orthopedics and spinal surgery (Table 25.7) [87]. In recent studies, the tribological and bioactive
properties of PEEK, which is used as a bearing material and flexible implant in orthopedic and
spinal surgeries, has been investigated [88–90]. However, conventional single-component PEEK
cannot satisfy these requirements (e.g., antibiofouling, wear resistance, and fixation to a bone) for
use as an artificial joint or intervertebral body fusion cage [87]. For further improving the capabili-
ties of PEEK as an implant biomaterial, various studies have focused upon the lubricity and antibi-
ofouling of the polymer, either via reinforcing agents or surface modifications [91,92]. Therefore,
multicomponent polymer systems have been designed in order to synthesize new multifunctional
Carbone fiber
PEEK substrate
100 um
FIGURE 25.21 Fluorescence microscopic images of CFR-PEEK. Bar: 100 μm.
TABLE 25.7
Typical Physical and Chemical Properties of PEEK and CFR-PEEK
Property PEEK 30% CFR-PEEK 60% CFR-PEEK
Molecular weight (106 g/mol) 0.08–0.12 0.08–0.12 0.08–0.12
Melting temperature (°C) 343 343 —
Poisson’s ratio 0.36–0.40 0.40–0.44 0.38–0.44
Specific gravity 1.3 1.4 1.6
Flexural modulus (GPa) 4 20 135
Tensile ultimate strength (MPa) 93–97 170–228 >2000
Elongation (%) 30–40 1–2 1
Crystallinity (%) 30–35 30–35 30–35
biomaterials. In order to use PEEK and related composites in the implant applications, they can be
engineered to have a wide range of physical, mechanical, and surface properties.
25.2.3.2 Tribological Properties

In the 1990s, CFR-PEEK was evaluated as a bearing material for hip and knee joint replacement [88].
Wang et al. carried out a more comprehensive tribological investigation of PEEK composites for both
hip and knee joint replacements. CFR-PEEK formulations were blended with 20%–30% mass dis-
continuous polyacrylonitrile (PAN) or pitch fibers. Under higher stress, cylinder-on-flat loading con-
ditions, the PEEK composites exhibited higher wear rates than conventional UHMWPE. In contrast,
under the lower stress hip simulator test conditions, all the PEEK composites had substantially lower
wear rates than conventional UHMWPE, with the lowest wear observed between 30% pitch CFR-
PEEK against ceramics. In contrast, unreinforced PEEK wore at six times the rate of UHMWPE.
The results of this study underscored the importance of fiber reinforcement in lowering stress and
conforming contact applications and provided a further basis for exploring PEEK composites for hip
joint replacements, especially in combination with ceramics as opposed to Co-Cr heads. Therefore,
alumina became the femoral head material of choice for THA applications with CFR-PEEK. Co-Cr
heads, when used in conjunction with CFR-PEEK liners, exhibited substantially higher wear, with
observations of scratching of the metallic surface by the carbon fibers. On the other hand, this study
also suggested that PEEK composites were unsuitable for knee applications, regardless of the fiber
content of the composite or the type of the counter surface. The authors recommended that the com-
posite materials should not be used as a tibial insert for knee joint replacement.
To validate the in vivo wear behavior and compatibility of CFR-PEEK wear debris, a clini-
cal study was initiated in Italy starting in 2001 using the ABG II total hip system (Stryker SA,
Montreux, Switzerland). The CFR-PEEK liners were fabricated from injection-molded PEEK
blended with 30% pitch fibers, and the bearing surfaces were machined to achieve the desired final
tolerance. After a mean follow-up period of 3 years, none of the liners needed to be revised due
to aseptic loosening. This clinical trial is still ongoing, and the detailed results have not yet been
published. Overall, the available preliminary clinical data support the short-term effectiveness of
CFR-PEEK as a bearing material for hip joint replacement. However, in a conventional hip joint
replacement design, the current data do not yet demonstrate a long-term clinical advantage of CFR-
PEEK over other well-established bearing alternatives, such as CLPE.
25.2.3.3 Surface Modification

On the other hand, surface modification is one of the most important technologies for the prepara-
tion of new multifunctional biomaterials for satisfying several requirements. Surface modifications
used today include coating, blending, and grafting.
It is well known that when BP is exposed to photo-irradiation such as ultraviolet-ray (UV)-
irradiation, a pinacolization reaction is induced; this results in the formation of semi-benzopinacol
radicals (i.e., ketyl radicals) that act as photo-initiators. Therefore, in this study, we have focused
upon a BP unit in PEEK and formulated a novel self-initiated surface-graft polymerization method
that utilizes the BP unit in “graft from” polymerization (Figure 25.22) [93,94]. This polymeriza-
tion reaction involving free radicals is photoinduced by UV-irradiation. Under UV-irradiation, a
BP unit in PEEK can undergo the following reactions in monomeric aqueous solutions [95–101] as
follows: the pinacolization reaction (photoreduction by H-abstraction of a BP unit in PEEK) results
in the formation of a semi-benzopinacol radical, which can initiate the graft-from polymerization of
the feed monomer as the main reaction, and the graft-to polymerization (the radical chain end of the
active-polymer couples with the semi-benzopinacol radical of the PEEK surface) as a subreaction.
In addition, a photoscission reaction occurs as a subreaction, which may not need a hydrogen (H)
donor. The cleavage reaction induces recombination and graft-from polymerization. When water
polymerization is performed in the presence of an H-donor, a phenol unit may be subsequentially
formed due to H-abstraction. This technique enables the direct grafting of the functional polymer
hν MPC
O O C [PEEK]T
O n
PEEK *
O O C* + M* O O C
OH n OH n
O
PMPC-grafted PEEK O–
O
O P N+
O
O PMPC layer
m
O O C PEEK
OH n : 100nm
FIGURE 25.22 Schematic illustration for the preparation and cross-sectional TEM image of PMPC-grafted
PEEK.
onto the PEEK surface in the absence of a photoinitiator, thereby resulting in the formation of a C–C
covalent bond between the functional polymer and PEEK substrate.
Kyomoto et al. demonstrated the fabrication of a biocompatible and highly hydrophilic
nanometer-scale-modified surface by PMPC-grafting onto the self-initiated PEEK surface using a
photo-induced pinacolization reaction (Figure 25.22) [93,94].
This novel and simple self-initiated surface-graft polymerization on the PEEK surface induces
unique properties such as lubricity and anti-protein adsorption by PMPC grafting, which are novel phe-
nomena in the field of orthopedic and spinal surgery (Figure 25.23). Moreover, the fabrications of the
PMPC-grafted PEEK and CFR-PEEK can result in next-generation orthopedic and spinal applications.
120 0.25 0.7

Hydrophilic Lubricious Anti-fouling
0.6
Amount of adsorbed BSA (μg/cm2)
100
Coefficient of dynamics friction
0.20
0.5
80
Contact angle (°)
*
0.15
0.4
60
0.3
0.10
40 *
0.2 *
*
0.05
20 0.1
* *
0 0.00 0.0
PMPC- PMPC- PMPC- PMPC- PMPC- PMPC-
Untreated grafted Untreated grafted Untreated grafted Untreated grafted Untreated grafted Untreated grafted
(a) PEEK CFR-PEEK (b) PEEK CFR-PEEK (c) PEEK CFR-PEEK
FIGURE 25.23 (a) Static water-contact angle, (b) coefficient of dynamic friction, and (c) amount of adsorbed
BSA of PMPC-grafted PEEK and CFR-PEEK. *t-test, significant difference (p < 0.05) as compared to the
untreated PEEK and CFR-PEEK, respectively.
25.3 FIXATION MATERIALS FOR JOINT REPLACEMENTS

25.3.1 Poly(Methyl Methacrylate) Bone Cement
Bone cements based on poly(methyl methacrylate) (PMMA) are important polymer materials in
joint replacement surgery: PMMA bone cements are primarily used for the fixation of joint replace-
ment. In the fixation of joint replacement, the self-curing bone cement fills the free space between
the joint replacement and bone and constitutes a very important interface.
The PMMA bone cements are two-component systems, comprising a polymer powder and a
liquid methyl methacrylate (MMA) monomer. The polymer powder component is composed of
PMMA and/or methacrylate copolymer. The polymer powder contains benzoyl peroxide (BPO),
which acts as an initiator for radical polymerization. The polymer powder also contains an X-ray
contrast agent. In the liquid monomer, MMA is the main constituent; however, at times, other meth-
acrylates—such as butyl methacrylate—are used. In order for the MMA to be used in bone cements,
it must be polymerizable, i.e., it must contain a carbon double bond that can be broken. Furthermore,
the liquid monomer contains an aromatic amine, such as N,N-dimethyl-p-toluidine (DMT), as an
activator of radical formation. Additionally, it contains an inhibitor (e.g., hydroquinone) to avoid
premature polymerization in storage aging.
PMMA bone cements are polymeric materials produced by the radical polymerization of MMA
(Figure 25.24). The polymerization process starts when the polymer powder and liquid monomer
are mixed, resulting in a reaction between the BPO initiator and DMT activator, forming radicals.
Consequently, the DMT causes a breakdown of the BPO in the reaction process by electron
transfer, resulting in the formation of benzoyl radicals. The C=C of the MMA monomer has a pair
of electrons that is attacked by the free radical to form a new chemical bond between the initiator
fragment and one of the C=C bond of the monomer molecule. The other electron of the C=C bond
stays on the C atom that is not bonded to the initiator fragment, creating a new free radical. This
unpaired electron is capable of attacking the C=C bond of a new monomer unit. This process, with
the breakdown of the initiator molecule to form radicals, is called the “initiation reaction” of the
radicals’ polymerization. The new radical reacts with another MMA molecule in the same way as
the initiator fragment reaction. Another radical is always formed when this reaction takes place,
over and over again. This process of the growing polymer chain is called a “propagation reaction.”
As the polymerization continues, the rate of termination (termination reaction) is decreased,
O
O
O O or *
O*
BPO
O
+ +
CH3 CH3 CH3
H3C N H3C N* + H3C N + H*
CH3 CH3 CH2*
DMT
MMA
* *
n
O O
O O
PMMA
FIGURE 25.24 Schematic illustration of formation of radical polymerization chains in PMMA bone cement.
because the diffusion of chain growth and the combination of chain ends is reduced. The system
becomes depleted of free radicals by recombination of the two radical chains and the polymeriza-
tion ceases.
25.3.2 Bone Cement History

In 1936, Heraeus Kulzer GmbH & Co. KG (Wehrheim, Germany) found that dough material could
be produced by mixing PMMA powder and a liquid MMA monomer, which was cured when BPO
was added and the blend was heated to 100°C [102]. In 1958, Charnley et al. successfully fixed a
femoral stem prosthesis by using PMMA as bone cement [103]. Since the introduction of PMMA
bone cement by Charnley et al., many people have had their lives dramatically and remarkably
improved by this innovation. At the beginning of the 1970s, in an effort to alleviate periprosthetic
infection—the most feared complication after joint replacement—Buchholz et al. advocated the
addition of antibiotics to bone cement [104]. Their idea was to add antibiotics to the bone cement in
order to reduce the incidence of infection. The PMMA bone cement with a gentamicin powder was
demonstrated to be stable and offered suitable antibiotic activity.
25.3.3 Problems with PMMA Bone Cement

Since the introduction of PMMA bone cement in the orthopedic field, bone cement is considered
to have become one of the effective polymer materials for fixation in joint replacement. However,
aseptic loosening remains a serious issue on a multifactorial basis as follows. Aseptic loosening
is suggested to be a result of monomer-mediated bone damage; during end-polymerization, there
is volumetric shrinkage of the cement, potentially compromising the bone–cement interface.
Using a fluid displacement model, Charnley observed that the volume of cement increases to a
maximum during polymerization, before shrinking slightly, although not to its initial volume.
This may be a largely theoretical concern, but there is a conflict between the stiffness of cement
and the adjacent bone. The Young’s modulus is 0.5–1.0 GPa for cancellous bone; 15–20 GPa,
for cortical bone; 2 GPa, for bone cement; 1 GPa, for titanium alloy; and 220 GPa, for cobalt–
chromium–molybdenum alloy. The bone cement may provide a shock-absorbing layer between
elastic bone and a stiff implant. The conflict between degrees of stiffness is therefore much
greater for cementless implants, and in some instances, the cement mantle and its interfaces may
be the weak link in the construct. The bone–cement interface is the key to the survival of a THR.
The combination of matte-surfaced collared femoral stems and poor cementing technique are
intrinsic to the failure of some implants. Polished collarless tapered stems generally give better
fixation, while cement particles were once considered a biological cause of aseptic loosening.
However, wear particles of polyethylene are now seen as primary initiators of the biological reac-
tions in aseptic osteolysis.
25.3.4 Solutions for the Problems of PMMA Bone Cement

25.3.4.1 Antibiotic-Loaded Acrylic Cement
In 1969, Buchholz et al. incorporated gentamicin in PMMA bone cement for the treatment of
infection in prosthetic joints [104]. Initially, the antibiotic was added during the operation, and
subsequently during manufacture, making antibiotic-loaded PMMA bone cement widely available
as part of antimicrobial prophylaxis in primary arthroplasty. There is valid evidence to support
the prophylactic use of antibiotic-loaded PMMA bone cement, which remains a standard practice
in Europe, and is in transition in the United States. In 2003, the Food and Drug Administration
accepted the use of three commercial antibiotic-loaded PMMA bone cements in the second stage
of revision surgery for prosthetic joint infection. Their use in primary THA has not been autho-
rized. The use of antibiotic-loaded PMMA bone cement in joint replacement provides short- to
medium-term protection against prosthetic infection. It aims to overlap with, and then replace, the
prophylaxis provided by perioperative intravenous antibiotics. To achieve this, the antibiotic must
be released from cement in adequately high concentrations that exceed the minimum inhibitory
concentration of potential colonizing bacteria. Gentamicin is the most common additive because
it has, among other features, a good spectrum of concentration-dependent bactericidal activity,
thermal stability, and high water solubility. In 1980, Wahlig et al. gave robust evidence of gen-
tamicin release from PMMA bone cement for up to 5.5 years in patients who had undergone
hip joint replacement [105]. Others have also confirmed the reliable release of gentamicin from
PMMA bone cement. However, concerns regarding antibiotics in cement still persist, including
concerns regarding the induction of antibiotic resistance. In 1989, Hope et al. found that 90% of
Staphylococcal strains isolated from infected hip joint replacements were resistant to gentamicin,
but if plain cement had been used at the initial operation, the resistance rate was only 16% [106].
Other studies have confirmed that antibiotic-loaded PMMA bone cement reduces infection in
total joint replacement at the price of increasing bacterial resistance. These problems have not
been demonstrated clinically, although they have been postulated. Despite the aim of achieving
early and total release, all in vitro studies show that only 5%–8% of the added antibiotic is ever
freed. Clinical studies have shown a low concentration of the release of gentamicin in failing
hip joint replacements up to 25 years after the primary operation, a potent stimulus for antibi-
otic resistance. In summary, we need to design guidelines regarding the use of antibiotic-loaded
PMMA bone cement and advise its use only in cases where the patient has significant risk factors
for infection.
25.3.4.2 Bone Bioactive Organic–Inorganic Hybrid Bone Cement

Recently, Ishihara et al. reported that PMMA bone cement containing hydroxyapatite as a bone
bioactive-filler was developed using 4-methacryloyloxyethyl trimellitate anhydride (4-META) to
promote adhesion to both bone and hydroxyapatite [107]. The mechanical properties of this PMMA
cement with 4-META and hydroxyapatite did not decrease significantly with increasing hydroxy-
apatite filler content in the cement. In contrast, the mechanical properties decreased with increasing
hydroxyapatite filler content in the absence of 4-META. The fracture surface of the cement clearly
showed that there are no gaps between hydroxyapatite filler and PMMA matrix resin; this means
that the hydroxyapatite filler adhered to the PMMA matrix resin by addition of 4-META [108]. The
hydroxyapatite filler along the surface increased with increased hydroxyapatite filler content in the
cement. The PMMA cement with 4-META and hydroxyapatite adhered also to bone with a tensile
bond strength of higher than 10 MPa [109–111].
The sol–gel process is popular for the preparation of nanometer-scaled composites of organic–
inorganic components. It has been reported that organically modified silicates can be synthesized
through hydrolysis and polycondensation of tetraethoxysilane and poly(dimethylsiloxane). The
nanometer-scaled organic–inorganic composite has the potential to show the properties of both
organic and inorganic components. Based on the finding that a CaO–SiO2 glass is effective in pro-
ducing bone bioactivity, Ohtsuki et al. applied organic modification with Si–OH and Ca2+ to PMMA
bone cement in order to induce the bone bioactivity in the bone cement (Figure 25.25) [112,113].
This bone bioactive PMMA cement has the potential to demonstrate bone-bonding properties in the
bone–cement interface (Figure 25.25d).
25.4 FUTURE PERSPECTIVES

Every year, the number and prevalence of primary and revision hip, knee, and other joint replace-
ments are increasing substantially worldwide. As a result, the quality of all artificial joints is becom-
ing increasingly important. Therefore, we can see that functional, durable, and natural joint-like
artificial joint replacements will be necessary for all artificial joints. We consider that the important
research goal for the future is the creation of the ultimate artificial joint interface that mimics the
n m
Body fluid O O
Ca2+ O O
PO43–
Ca2+ OH– Apatite
OH OH OH OH
HO Si Si Si Si
–O O O O
O–
2+ Si
O Ca
O O O H3CO OCH3
Bone cement OCH3
(a) (b)
0d 7d
Bone
Bone cement
10 μm 10 μm 5 mm
Bone cement surface Apatite layer

(c) (d)
FIGURE 25.25 Schematic illustration of bone bioactive PMMA bone cement: (a) concept of bone bioactive
cement, (b) chemical structure, (c) apatite formation in simulated body soaking fluid by SEM observation, and
(d) pQCT image after 9 weeks implantation, Animal experiment.
natural joint cartilage. Additionally, a functional, durable, and natural surface not only would be of
great service to applications as medical devices such as artificial joints but also would be important
to biomaterial and bioengineering sciences. For example, it is well known that the composition
elements of the articular cartilage surface consist of the collagen network, hyaluronic acid, and
proteoglycan subunits. However, the functions of the articular cartilage surface have not been well
explained. We consider that a bioengineering surface with new polymeric biomaterial would help
in clarifying these phenomena; further, researches in biotribological science can elucidate the func-
tions of articular cartilage surface. We believe that the designs of polymeric biomaterials and bioen-
gineering surfaces will act as key technologies for further evolving biomaterial and bioengineering
sciences, and we hope that this issue will be addressed by future scientists in polymeric biomaterial
and bioengineering sciences.
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Index
A natural compounds, 728
TNF-α, 729
Acrylated chitosan/oxidized dextrans Antioxidants and photoprotectors, lignin
cell encapsulation, see Cell encapsulation ABTS radical, 716, 718
conventional industrial methods, 743 AOA and TAA values, 719
hydrogels cytotoxicity and cell viability analysis, apoptosis, 722
747–748 CPZ, 716–717
in vitro hydrolytic degradation, 746–747 DPPH, 720–721
macromolecular crosslinker, 743 inhibitory effects, 716
morphology, 745–746 MDA and LHPs, 720
murine transcutaneous full-thickness wounds, 749–751 phytopathogenic microorganisms, 721
Odex and CEC reaction, 743, 744 polyphenols, 715
oxidation degree, Odex determination, 743, 744 relative chain-breaking, 717, 718
rheological analysis, 744–745 scavenging activity, 717
swelling properties, 746 superoxide anion and hydroxyl radical generation,
vicinal hydroxyl groups, 743 718–719
water-soluble CEC, 743 APTES, see Aminopropyltrietoxysilane (APTES)
Activation/initiation systems Arg-Gly-Asp (RGD)
dual cure, 789 application, 517
photochemical, 788 ECM proteins, 516
redox, 789 Artificial intervertebral disk (AID)
thermal, 788 application, 76–77
Acute and normal wound healing response development
occlusive and moisture controlling dressings, 839 beagle dog, 75
optimal healing, 839 massive bone ingrowth, 75–76
resolution factors, 839 PVA-H and titanium fiber mesh, 76
vascular and cellular, 839 spinal fusion, 74
AFM, see Atomic force microscopy (AFM) Artificial joint replacement
Agar-agarose hip, 851–853
chondrocytes, 279 knee, 853–854
encapsulate cells, 279 osteolysis, see Osteolysis
hydrogels, 279, 280 THA, see Total hip arthroplasty (THA)
printing technologies, 279 Atomic force microscopy (AFM)
structure, 278 coated tips, 513
AID, see Artificial intervertebral disk (AID) operating modes, 512
Alginate phase imaging mode, 513
calcium phosphate composites, 280–281 surface height, 512
derivatives, 280 tapping mode, 513
Aminopropyltrietoxysilane (APTES), 178
Amphiphilic systems B
amino groups, 244
biomedical applications BBB, see Blood brain barrier (BBB)
cells and proteins adhesion, 261 BHT, see Butylated hydroxyl toluene (BHT)
drug delivery, 256–258 Bimodal electrospun scaffolds, 555
gene transfection, 258–260 Biocompatibility, elastomers
tissue engineering, 260–261 biodegradable polyurethanes, 474–475
chitin, 243–244 biodegradation assays, 463–464
chitin and chitosan derivatives, see Chitin and chitosan biomaterials, 465
derivatives carcinogenic reactions, 468–469
chitosan, 244 categories, 457
polymers, 245 complications, 466–467
polysaccharides, 245 cytotoxicity, 457–460
Antibiotic-loaded acrylic cement, 876–877 degradation, 468
Anti-HIV properties, lignin evaluation, 455, 457
dehydrogenated polymers, 729 FBGC, 465
macrophages, 728 genotoxicity, 464
885
886 Index
hemocompatibility and blood interaction assays, Biomaterials

462–463 description, 271–272
hydrogels, 476 historical background, 272
inflammatory cells, 466 natural polymers
medical devices, 455–456 ECM, 272–273
microbial colonization, 464–465 polysaccharides, see Polysaccharides
PFTE, 476 proteins, see Proteins
PGS, 477 Blends
polyesters, 475 biodegradable polymers, 309
polyethylenes, 476–477 chitosan, 318–324
polyurethanes, 470–474 collagen, see Collagen
protein adsorption, 465–466 components, 310–311
PVC, 475–476 elastin, see Elastin
reproduction and embryo miscibility and compatibility
development-assays, 464 natural and synthetic polymers, 311
rubbers, 477 polymer–polymer interactions, 312–313
sensitization assays, 460–462 viscosity measurements, 312
silicones, 469–470 natural and synthetic polymers
styrene copolymers, 477 nanoparticles, 332
test, humans, 464 regenerative medicine, 331
Biocompatibility testing natural polymers, 311
blood-contacting devices, 385 silk and keratin, 327–330
cellular matrices, 408 Blood brain barrier (BBB), 607–608
CVCs, see Central venous catheters (CVCs) BMPs, see Bone morphogenic proteins (BMPs)
ex vivo, 399–400 Bone bioactive organic–inorganic hybrid bone
in silico testing cement, 877
chip, 408–409 Bone morphogenic proteins (BMPs), 825
drugs, 408 Bone tissue
high-throughput screening, 409 candidate materials, 223–224
in vitro, see In vitro biocompatibility testing ceramics, see Ceramics
in vivo, see In vivo biocompatibility testing composite materials
medical devices alginate/HA scaffold, 231
cranioplasty, 380 biodegradable, polymer/ceramic, 233–235
decision making, 381, 383 collagen, 229–231
definition, 383–384 CPC, 235–237
human tissue, 380–381 gelatin, 231–232
in vivo functionality, 406–407 polymer/ceramic, 232–233
implants, 378–379 polymer phase, 233
ISO norm, 381–382 preparation techniques, 228–229
metallic and polymeric parts, 378 grafting therapies, 222
skull injuries, 379–380 implant requirements, substitution, 223
skin, 408 polymers
Biodegradable polymers alginic acid, 225–226
application, 136 collagen and gelatin, 225
biocompatibility and toxicity degradation, 225
description, 159 description, 224–225
lactide/glycolide, 159–160 HAc, 225
poly (amides ), 161–162 PEG, see Polyethylene glycol (PEG)
poly(caprolactone), 160–161 polyesters, see Polyesters
poly (orthoesters ), 163 structure, 222–223
polyanhydrides, 163–165 substituting biomaterials, composite approach, 224
polycarbonates, 161 Bovine serum albumin (BSA)
polyphosphazenes, 162–163 HAp and, 115
drug discovery, 136 microsphere matrices, 115–116
POEs, see Poly(orthoesters) (POEs) BSA, see Bovine serum albumin (BSA)
poly(amide), 146–147 Butylated hydroxyl toluene (BHT), 793
poly(phosphate ester), 147–148
polyanhydrides, see Polyanhydrides C
polyesters, see Polyesters
polyphosphazenes, 148–149 Calcium phosphate cements (CPC)
Biologically derived polymers alginate, 236
collagen denaturing, 543 and collagen, 236
ECM fibrils, 542 synthesis, 227
types, 543 Camptothecin (CPT), 667
Index 887
Carbohydrate-derived self-crosslinkable in situ gelable Ceramics

hydrogels, see Wound healing bioactive glass, 228
Carboxylic acids/esters with diols polycondensation bioinert, 228
dehydration, 635 calcium phosphates, 227–228
α,ω-dicarboxylic acid and 1,2-ethanediol/1,3-propane description, 227
diol, 635 Chinese Hamster Ovary (CHO) cells, 398
enantioselective, 636 Chitin and chitosan derivatives
oligomers, 635 amphiphilic covalent
water equilibrium, 635 glycolchitosan reaction, 248, 249
Carboxymethylated-chitin (CMCH), 281 grafting, 247–248
Carcinogenesis reaction mechanism, 247
DNA, 723 CMCH, 281
DNA breakage, 724–725 grafting, 281–282
kraft beech lignin, 723, 724 hydrogels, 250–251
kraft spruce lignin, 723 hydrophilic modification, 281
MNNG, 725 hydroxyl, 246–247
prehydrolysis lignin, 723 injectable materials, 282
TNF-α, 728 PEC, 249
wood-derived lignins, 725–726 PEG, 248, 249
Cartilage tissue engineering, 577–578 properties
CDDP, see Cisplatin (CDDP) hydrophobic groups, 251
Cell adhesion interactions, 255
CHO-K1 cells pattern, 697, 699 interfacial/surface, 253–255
manipulation, 697, 699 N-maleoylchitosan, 253
photoinduced increase, 700 rheological curves, 251
UV irradiation, 695, 697 swelling, 255–256
Cell encapsulation viscosity, 251–252
Live/Dead™ staining, 749 scaffolds, 282, 283
morphologies, 748–749 Chitosan
Cell function in vitro alginates, 323–324
biocompatibility testing, 394–395 collagen and gelatin, 321, 323
evaluation, 396 crosslinking agents, 321
molecular genetic methods, 395–396 macromolecular chain, 318
PCR technique, 394, 395 mechanical properties, 321, 322
western blotting/ELISA, 396 Owens-Wendt Method, 319, 320
Cell-invasive electrospun materials synthetic polymers, 319
bimodal electrospun scaffolds, 555 thermal properties, 321–322
conventional technologies, 555–557 tissue engineering applications, 319
description, 554 UV irradiation
electrospinning and electrospraying, 555 elongation, 319, 320
electrospinning onto ice crystals, 555 tensile strength, 319
hydrospinning, 554–555 Chlorpromazine (CPZ)
small diameter microfibers, 557 hemolysis and photohemolysis, 716–717
Cell–polymer interactions lignins and, 716
cell adhesion process, 517 Chondrocytes, 567, 569
ECM proteins, 516 Chronic wounds, 840
extracellular and cell membrane Cisplatin (CDDP), 667
proteins, 515 CLPE, see Cross-linked polyethylene (CLPE)
FN immobilization, 515 CMCH, see Carboxymethylated-chitin (CMCH)
RGD, 516–517 Collagen
Cellulose amide band position, 316
biodegradation, 273–274 amino acids, 313
CA, 274 animal-derived materials, 314
derivatives, 274–275 applications, 286–287, 314
membranes, 274 broblasts grow, 316, 317
microporus cellulose-based scaffolds, cross-linking methods, 287–288
275–276 Δb parameter, 315, 316
regenerated cellulose, 275 degradation, 287
Central venous catheters (CVCs) FTIR, see Fourier transform infrared spectroscopy
cytotoxicity, 410 (FTIR)
evaluation, 411 gelatin, 313
function, 411 hydrophilic polymers, 314
genotoxicity, 410–411 polypeptide chains, 286
thrombogenicity, 411 properties, 313–314
888 Index
thermal parameters, 318 polyphosphazene blends/composites

tissue regeneration, 288, 289 choline chloride, 108
UV irradiation, 315 depsipeptide-substituted polymer, 108
wastes, 314–315 PNEGmPh-PLAGA blend, 105, 106
water-soluble synthetic polymers, 315 surface morphologies, 106–107
Complete dentures Dental and maxillofacial surgery, polymers
biocompatibility and residual monomer adhesives, see Orthodontic adhesives
content, 823 cements and luting agents, 795
hypoallergenic denture polymers, 823 comparison, material properties, 797
mechanical properties, 823 “continuum”, 795–797
PMMA, 822 elastics, see Orthodontic elastics
radiopacity, 823 esthetic dentistry, veneers, 810
Compomers, 817 free radical addition polymerization, see Free radical
“Continuum” of tooth-colored restorative materials, addition polymerization
795–797 glass-ionomer (polyalkenoate) cements, 797
Coprecipitation method hydrocolloids, see Hydrocolloids
nanoparticles preparation, 599–600 interfaces and adhesion, see Interfaces and adhesion
VAM41-PEG Hb-loaded polymeric nanoparticles, interim restorations, 822
600–601 mouth protectors, see Mouth protectors
versatility, 600 MWs of polymers, 793
CPC, see Calcium phosphate cements (CPC) non-aqueous elastomers, see Non-aqueous elastomers
CPT, see Camptothecin (CPT) in oral environment, 785–787
CPZ, see Chlorpromazine (CPZ) in periodontology, 825
Cross-linked polyethylene (CLPE) pit and fissure sealants, see Pit and fissure sealants
continued close monitoring, 861–862 polyacid-modified resin composites, 797
gamma-ray irradiation, 860, 861 polymer-based materials, interim restorations, 822
high-energy ray irradiation, 860 prostheses, see Maxillofacial prostheses
osteolysis threshold, 861 prosthodontic materials, see Prosthodontic materials
UHMWPE, 860 resin composites, 796
wear reductions, early-and mid-term clinical resin-modified glass-ionomer cements, 797, 798
studies, 861 setting and fluoride release, glass-ionomer
CVCs, see Central venous catheters (CVCs) cements, 794
Cytotoxicity soft tissue augmentation, see Soft tissue
Calcein-AM hydrolysis, 391–392 augmentation
cell lines, 458 structure, set polyalkenoate cement, 794
cell morphology, 459 synthetic polyelectrolyte acid solutions, 793
controls, 459–460 titanium-based implants, 825
description, 457 tooth restoration, see Tooth restoration
determination, 386–387 water-soluble polyelectrolytes, glass-ionomer
direct contact testing, 389–390 cements, 793
direct tests, 458–459 wound closure materials, 826
dyes, 392 DESs, see Drug-eluting stents (DESs)
evaluation, 393 Dextrans
indirect testing, 387–389 antibacterial agent, 742
LDH, 390–391 definition, 741
living cells, 387 heparin-like derivatives, 741
material preparation, 457–458 in vitro and in vivo models, 742
MTT, 390 structure, 741
sterilization, 458 1,1-Diphenyl-2-picryl hydrazyl (DPPH)
kinetics, 720–721
D lignin activity, 721
Direct contact testing, cytotoxicity
DDS, see Drug-delivery systems (DDS) and indirect, 390
Degradation test materials, 389–390
alkoxy-substituted polyphosphazenes, 105 DLS, see Dynamic light scattering (DLS)
aminated DPPH, see 1,1-Diphenyl-2-picryl hydrazyl (DPPH)
amino acid ester, 102, 104 Drug delivery, amphiphilic systems
ethyl glycinato group, 102–103 alkyl halides, 257
hydrolysis, 104 chitosan coated liposomes, 257–258
hydrolytic behavior, 101 glycol chitosan nanoparticles, 256
imidazole-substituted polymer, 101–102 N-alkyl-O-sulfate chitosan, 257
polymer mechanisms PEC, 257
erosion, 98–99 poly(p-dioxanone), 256
pathway, 100 SPECs, 258
Index 889
Drug delivery, biodegradable polyphosphazenes natural rubbers, 448–449

matrix fabrication PGS, 450
microparticles, 111–112 polyacrylics, 451–452
microspheres, 110–111 polyamides, 450–451
release kinetics polyolefins, 447–448
BSA, 115–116 polypeptide, 449
glyceryl-substituted, 117 polyphosphazenes, 450
imidazole and methylphenoxy, 112 polyurethane, see Polyurethane elastomers
imidazole-substituted polymer, 113 PVC and phthalate, 445–447
matrices, growth factor delivery, 115 saturated polyesters and biopolyesters
matrix permeability, 114 PET and PBT, 444–445
microspheres, 113–114 water-insoluble, 445
PNEGmPh, 114 silicone, see Silicone elastomers
poly(amino acid ester), 113 styrene copolymers, 448
polymer hydrogels, 116 Electron spectroscopy for chemical analysis (ESCA),
steroidal residues, 112 508–509
Drug-delivery systems (DDS), 594 Electro-responsive and magnetically responsive
Drug-eluting stents (DESs) polymers, 674
immobilization, 358 Electrospinning, regenerative medicine
structures, 358–359 biologically derived polymers, see Biologically derived
Dual collection systems polymers
experimental setup, electrospun fibers deposition, cell-invasive electrospun materials, see cell-invasive
550, 551 electrospun materials
oriented electrospun PCL fibers, 550 cell proliferation and adhesion, 541
Dynamic light scattering (DLS), 41 characteristics, electrospun materials, 544
description, 540
E growth factor delivery, electrospun materials, 557
in vitro studies, see In vitro studies, electrospun
ECM, see Extracellular matrix (ECM) materials
Elastin in vivo studies, see In vivo studies, electrospun
amino acids, 324 materials
collagen and, 324, 325 instrument configuration, see Instrument
protein mixture, 324–325 configuration, electrospinning
surface free energy, 326 instrument parameters, see Instrument parameters,
UV irradiation, 327 electrospinning
Elastomers nonwoven mat of fibers, 542
biocompatibility, see Biocompatibility, elastomers polymeric parameters, see Polymeric parameters,
chemistry and synthesis electrospinning
biomaterials, 424, 425 Raleigh instabilities, 542
crosslinking, 422 scaffold-based TE concepts, 540
formulation, 422–423 selected hybrid electrospun systems, 542, 543
nano-and microtechnologies, 424, 426 selected synthetic polymers, 542, 543
polymerization, 420–421 setup, 541–542
reinforcement, 423–424 surface modification, see Surface modification,
structure, 419–420 electrospun fibers
classification synthetic polymers, 543–544
chloro-polyethylenes and sulfonyl TECs, requirements, 540–541
polyethylenes, 419 Electrospinning writing, 553, 554
current system, 417 ELISA, see Enzyme linked immunosorbent assay (ELISA)
fluorocarbons, 418 Enantioselective polycondensation, 636
hydrogenated nitrile, 419 End-functionalized polyesters
polyacrylates, 418 description, 641
polysiloxanes/silicone, 417 initiator method, 641–642
TPEs, 419 terminator method, 642
description, 416–417 Endodontic materials
fluorinated, 452 gutta percha, 817
hydrogels, 449 in vitro fluid filtration tests, 817–818
medical devices 4-meta technology, 818
grouping, 452 “monoblock”, 817
implant applications, silicone, 454 polyelectrolyte-containing, 818
making artificial hearts, 454 root canal sealing, 817
materials, contact lenses, 455 Endothelial cells
transdermal therapeutic-systems, 454 description, 562
vascular grafts, 453 electrospun tubes and PLLCL, 563
890 Index
human umbilical vascular, 563, 564 gene upregulation, 561

with in vitro studies, 563 ligament, skin tissue engineering, 559
Enhanced permeation and retention (EPR) effect, 605 with selected in vitro studies, 559–561
ENL, see Excretion of deuterated enterolactone (ENL) suspended fibers, 561, 562
Environmental stress cracking (ESC) wound closure, 561
impact, 441 FITC, see Fluorescein isothiocyanate (FITC)
polyurethanes, 438, 440 Flory-Rehner theory, 38–39
Enzymatic synthesis, polymeric materials Fluorescein isothiocyanate (FITC), 42
advantages, 617–618 Foreign body giant cells (FBGC), 472
catalystic properties, 618 Fourier transform infrared spectroscopy (FTIR)
description, 617 attenuated total reflection mode, 508
in vivo, in vitro and enzymatic modification, 618 characterization, 508
oligo-and polysaccharides, see Oligo-and collagen, 316
polysaccharides synthesis DSC, 315
phenolic polymers, see Phenolic polymers IR microscope, 508–509
polyesters, see Polyesters Free radical addition polymerization
transformation rate, 618 activation/initiation systems, 788–789
vinylic monomers, see Vinylic monomers degree of conversion, monomer to polymer, 790–791
Enzyme linked immunosorbent assay (ELISA), 396 description, 788
Epithelial cells inhibition, see Inhibitions of polymerization
cell-invasive substrates, 562 methacrylates and dimethacrylates, see Methacrylates
definition, 561 and dimethacrylates monomers
enzymes and harmones, 561 reaction exotherm, 789
with in vitro studies, 562 shrinkage, 789–790
EPR, see Enhanced permeation and retention (EPR) FTIR, see Fourier transform infrared spectroscopy
ePTFE, see Expanded polytetrafluoroethylene (ePTFE) (FTIR)
ESC, see Environmental stress cracking (ESC)
ESCA, see Electron spectroscopy for chemical analysis G
(ESCA)
Excretion of deuterated enterolactone (ENL), 730 GED, see Guanidinoethyl disulfide (GED)
Expanded polytetrafluoroethylene (ePTFE), 842–843 Gelatin
extracellular and cell membrane proteins, 515 advantages and disadvantages, 742
Extracellular matrix (ECM) composite polymeric scaffolds, 291, 294
components, 515 cross-linking methods, 290–291
molecules, 517 definition, 742
polysaccharide component, 51–52 description, 288
proteins, 515 hybrid polymeric scaffolds, 291, 293
structure, 499 in situ hydrogel systems, 743
Ex vivo biocompatibility testing immunogenic, 743
blood contacting material, 399–400 macromolecules, 290
tissues and organs, 400 non-cytotoxic crosslinking methods, 743
scaffolds, 291, 292
F Gelatin/oxidized hyaluronans
description, 750
Fabrication methods, PVA-H hydrogel morphological analysis, 754–755
freeze-thawing method, 60 long-term cell viability, see Long-term cell viability,
mechanical properties, 61 hydrogels
polymer blend, 60 oHA/gelatin hydrogels, see oHA/gelatin hydrogels
stress-relaxation curves, 62–63 rheological analysis, 755–756
stress-strain curves, 62 swelling properties, 755
yield stress value, 62 temperature-insensitive self-crosslinkable
FCS, see Fetal calf serum (FCS) hydrogels, 750
Fetal calf serum (FCS), 497 three-dimensional infiltration and distribution,
Fibrin-fibrinogen-fibronectin cells, 756–758
description, 291 Gene transfection
forms, 295 alkyl chains, oligochitosans, 259
gels, 296 clinical application, 258
keratin, 295–296 factors, 259
tissue engineering, 295 LMW-DDC-chitosans, 259–260
Fibroblasts NMPCS, 260
aforementioned electrospinninginvestigations, 561 self-assembled nanoparticles, 260
cytotoxicity and biocompatibility, 561 Genotoxicity
definition, 559 alkaline comet assay, 398–399
electrospun fibers, 559 Ames test, 397–398
Index 891
CHO cells, 398 Human recombinant platelet derived growth factors

description, 396 (rhPDGFs), 825
mutagenicity, 397 Hyaluronic acid (HA)
Glass-ionomer (polyalkenoate) cements, 797 and collagen, 284
Glycolide (GA), 3 definition, 740
Glycosidases (glycoside hydrolases)-assisted synthesis description, 282
activated substrates polymerization, 625–628 extracellular matrix, 741
biocatalytic mechanism, 624, 625 hyaluronan structure, 740, 741
carboxyl and carboxylate groups, 624 hydrogels, 285
characteristic degradation mechanism, 624 hylans, 285–286
configurational retention, anomeric carbon atom, 624 longevity and physicochemical properties, 740
glycosynthases, see Glycosynthases materials, 284
polysaccharide synthesis, 625 viscoelastic properties, 740
thermodynamic forcing, 624 Hybridization bonding
transglycosidases, 628 classification and constituents, 801–803
Glycosyltransferases-assisted synthesis, oligosaccharides degradation, hybrid layer, 803
CAZy database, 619 durability, 803
cellulose chain, cellulose synthase, 619 etch-and-rinse adhesives, 803
description, 618–619 HEMA, 803
glycosidic bond formation, 619 historical developments, 801, 802
leloir, see Leloir glycosyltransferases long-term stability, 803
mammalian glycoproteins and glycolipids nature, dentin, 800–801
biosynthesis, 619 Hydrocolloids
non-leloir, see Non-leloir glycosyltransferases agar, 818
phosphorylases, see Phosphorylases alginates, 819
starch synthase, 619 Hydrogel morphological analysis,
type II membrane-bound glycoproteins, 619 754–755
Glycosynthases Hydrospinning, 554–555
acceptor-related regioselectivity, 629 Hydroxyacids polycondensation
β-(1→3)-β-(1→4) alternating mixed glucans, 630–631 acyl-enzyme formation, 635
condensation, sugars assisted by synthase, 628–629 in Candida antarctica, 634
endoglycosynthases-mediated preparation, 630 cholic acid, 634, 635
exoglycosidases and endoglycosidases, 629 enzyme-assisted synthesis, polyesters, 635
β-(1→3)-glucans, 630 hydrophobic organic solvents, 634
β-(1→4)-glucans, 630 PEGylated lipase and esterase, 634
mutant β-glucuronidases, 629 ricinoleic acid, 634
oligo-and polysaccharides synthesis, 628 structure of monomers, 635
sugar polycondensation, 629 vinyl esters, 635
Growth factor delivery
biomaterials, 844–845 I
electrospun materials, 557
GTR, see Guided tissue regeneration (GTR) Indirect testing, cytotoxicity
Guanidinoethyl disulfide (GED) ISO-10993, 387–388
cell infiltration suppression, 772–773 potential toxic chemicals, 388
in vitro release, 768, 770 Inhibitions of polymerization
macrophage inhibition, 768, 769, 771–772 BHT and MEHQ, 793
oHA/gelatin microspheres, 767, 768 eugenol, 793
particle size distribution, 766 oxygen, 791, 792
SEM photomicrographs, 766, 767 iNOS inhibitors
swelling dynamics, oHA/gelatin microspheres, 768, 769 GED, see Guanidinoethyl disulfide (GED)
water-in-oil emulsion process, 766 inflammatory extravasations, 765
Guided tissue regeneration (GTR), 825 macrophage, 765
self-crosslinkable hydrogel preparation, 766
H structural and physical properties, microspheres, 766
structure, GED, 765
HA, see Hyaluronic acid (HA) Instrument configuration, electrospinning
Hip joint replacement description, 548
bone necrosis, 852 dual collection systems, 550, 551
cementless fixation parts, 852 electric field manipulation, 553
“cementless” prosthesis, 852–853 electrospinning writing, 553, 554
lubrication film, 851 rotating collectors, 551–553
osteoarthritis, 851–852 single collection systems, see Single collection systems
pain and stiffness symptoms, 853 structured electroconductive collectors, see Structured
THA, 852 electroconductive collectors
892 Index
Instrument parameters, electrospinning irritation, 402–403

applied voltage, 546–547 local and systemic toxicity, 402
collection distance, 547–548 pMMA, 401
flow rate, 546 sensitization, 402
humidity, 548 subchronic and chronic systemic toxicity, 405
spinneret diameter, 547 synthetic materials and implants, 401
temperature, 547 systemic toxicity, 403–404
Interfaces and adhesion In vivo studies, electrospun materials
ceramics bonding, 804 biocompatibility and cell penetration, 574–575
chemical, tooth tissue, 799, 800 bone TE, 576
hybridization bonding, tooth dentin, see Hybridization cartilage TE, see Cartilage tissue engineering
bonding neural TE, see Neural tissue engineering
metallic materials bonding, 804 vascular TE, 577
micromechanical to tooth enamel, 799, 800
non-adhesive dental restorations, 798 J
polymerizing materials, 798
principal dental applications, 799 Joint replacements
Internal stimuli-responsive systems, pH-responsive artificial, see Artificial joint replacement
polymers bearing material, PEEK, 872–874
acidic copolymer hydrogels, 676 bone cements, PMMA, see Poly(methyl methacrylate)
biological factors, 676 (PMMA)
cancer targeting, 675 phospholipid polymers, see Phospholipid polymers
classification, 675 polyethylene bearing material, see Polyethylene
and collapsing behavior, 675 bearing material
in diabetes, 676
glucose-responsive polymer, “gating” flow of insulin, K
676–677
internal physicochemical changes, 675 Keratinocytes, 573, 574
limitations, 677 Knee joint replacement
pNIPAM, 676 description, 853
“self-immolating” system, 676 orthopedic community, 854
tissues and cellular compartment, 675 TKA, 853
In vitro biocompatibility testing UHMWPE, 853
cell function, see Cell function in vitro wear and tear, TKA, 854
cell proliferation, 393–394
control material, 386 L
cytotoxicity, see Cytotoxicity
3D environment, 385 Laccase-assisted polymerization
genotoxicity, 396–399 description, 648
levels, 386 historical determination, 648
In vitro hydrolytic degradation 4-hydroxy-benzoic acid, 650
description, 746 Japanese/Chinese lacquer tree, 649
Odex-III/CEC hydrogels, 747 mediators insertion, 649
theoretical crosslinking densities, 747 redox reactions, 649
In vitro studies, electrospun materials in syringic acid, 649
cardiomyocytes, cardiac stem cells and cardiac cell Lactate dehydrogenase (LDH), 390–391
lines, 565 Lactide (LA), 3
characteristic diversity, 559 LCC, see Lignin-containing compounds (LCC)
chondrocytes, 567, 569 LCST, see Lower critical solution temperature (LCST)
endothelial cells, see Endothelial cells LDH, see Lactate dehydrogenase (LDH)
epithelial cells, 561–562 Leloir glycosyltransferases
fibroblasts, see Fibroblasts bifunctional fusion enzyme, Gal transfer and “in situ”
keratinocytes, 573, 574 formation, 620
ligament cells, see Ligament cells drawbacks, 620
MSCs, see Mesenchymal stem cells (MSCs) β3GnT preparation, 620
neurons and glia, 567, 569–571 recombinant DNA techniques, 620
osteoblasts, 567, 568 substrate engineering, 621
with other cell types, 573, 574 sugar/oligosaccharide monoglycosylation, 620
phenotypical differences, cell culture, 559 Ligament cells
SMCs, see Smooth muscle cells (SMCs) aligned fibers, 572
In vivo biocompatibility testing collagen type I and type III upregulation, 572
acute systemic toxicity, 404–405 description, 570
animal model systems, 400 electrospun nano-/microfibers, 570
implant effects, 405–406 osteo-integration enhancement, 572
Index 893
tissue-guided regeneration, 572 vinyl chains, 43–44

topographic cell guidance and scaffolds, 572 VPTT, see Volume-phase transition temperature
with in vitro studies, 572, 573 (VPTT)
Lignin “water-in-water” emulsion polymerization
antibacterials method, 45
animal agriculture, 712 Malondialdehyde (MDA), 720
in vitro studies, 713–714 Materials, joint replacements
in vivo studies, 714–715 bone cements, PMMA, see Poly(methyl methacrylate)
influence, 712 (PMMA)
prebiotics, 715 PEEK, 872–874
role, 711–712 phospholipid polymers, 864–871
sorption capacity, 713 polyethylene bearing material, 857–863
anti-HIV properties, 728–729 Maxillofacial prostheses
antioxidants and photoprotectors, methacrylates, 826
715–722 polyurethanes and chlorinated polyethylene, 826
carcinogenesis, 723–728 requirements, 826
derived compounds, 711 silicone polymers, 826
lignophenols, 710, 711 MEHQ, see Monomethyl ether hydroquinone (MEHQ)
mechanism Meniscus
cell adhesion molecules, 732, 733 articular cartilage, 70–72
ENL, 730 function, 70
enterodiol, 734 gels, peripheral tissue, 72
LCC, 732 knee joint, articular cartilage, 73–74
mammalian and plant lignans, 730–731 Mankin’s grading system, 73, 74
SDG, 735 OA progress, 72
VCAM-1 and ICAM-1, 732 replacement, 70–71
molecules, wheat straw, 709, 710 treatment, 70
spermicide, 730 Mercaptopropyl trimethoxysilane (MPS), 367, 368
Lignin-containing compounds (LCC), 732 Mesenchymal stem cells (MSCs)
Lipase stereoselectivity definition, 565
acyldonor (R2), 632 HOBs, 178
enantiopreference, 632–633 IGF-1 loaded electrospun scaffolds, 566
poly condensation reactions, 633 with in vitro studies, 565, 566
Lipid hydroperoxides (LHPs), 720 4-META, see 4-Methacryloxyethyl trimellitate anhydride
Live/Dead™ staining, 749 (4-META)
Long-term cell viability, hydrogels Metalloproteinases (MMPs), 843
cell mediated degradation, 760 Metals
explanted hydrogels stained with H&E, biomaterials
760, 761 atoms, 349
in vitro deposition, 758, 759 cobalt–chromium alloy, 353
MTS assay, 758 dental precious alloys, 353
poly-lactide-co-glycolide Vicryl™ sutures, 761 hydroxyl group, 353–354
SEM images, ECM deposition, 761–762 passive film, 350
Lower critical solution temperature (LCST), 688 stainless steel, 353
surface importance, 349
M surface oxide layer, 349
titanium, 350–352
mAb, see Monoclonal antibodies (mAb) titanium alloy, 352–353
Macromers biomolecules
“click chemistry” hydrogel and gelatin, 366–367
application, 49 immobilization, 364
cycloaddition reaction, 49, 50 peptides, see Peptides
features, 49 protein and collagen, 366
oxidation and hydrolysis pathways, 51 description, 344
hydrogels fabrication PEG, see poly(ethylene glycol) (PEG)
CDI chemistry, 48–49 polymers
electrophilic functionalization, 48, 50 metallization, 368, 369
electrophilic moieties, 48 porous titanium, 367–369
photo-cross-linked hydrogels SCA, see Silane coupling agent (SCA)
acrylate-ester, photopolymerization, 47 SPU layer, 367, 369
HEMA-(Lac) n-Suc, 46–47 thick and thick γ-MPS layer, 367, 368
photo-polymerization, 43 polymers adhesion
polymer networks, 43 alloys, 355–356
trans-esterification reaction, 44 bonding, precious alloys, 356–357
894 Index
dental, 355 description, 194–195

durability, water, 356 efflux pump inhibition, 214–215
4-META, 354–355 enzyme inhibition
restorations and prostheses, 354 blood glucose level, 211, 213
stents and stent grafts, see Stents conjugates, 211–212
properties description, 211
category, 344, 345 in vitro methods
disadvantage, 346 rheological techniques, 201
factors, materials, 346, 348 spectroscopic techniques, 201
medical devices, 346, 347 tensile tests, 200–201
orthopedic implants, 345–346 visual tests, 199–200
requests, biomedical use, 346, 348 in vivo methods, 201–202
stress–strain curves, 345 mucus gel composition
Methacrylates and dimethacrylates monomers human mucins, 195–196
with an-OH group, 789, 792 structure, 195
with carboxyl groups, 789, 792 vs. non-adhesive microspheres, 194
description, monomers, 789, 790 non-ionic, 204, 205
with phosphate groups, 789, 791 permeation enhancement, 212–214
with phosphonate groups, 789, 791 principles
with sulfonic acid functional group, 789, 790 covalent bonds formation, 197
4-Methacryloxyethyl trimellitate anhydride dehydration, 198
(4-META) interpenetration, 197–198
adhesive ability, 355 non-covalent bonds formation, 197
and TBB, 354–355 parameters, 196
N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 725 polymer chains entanglements, 199
Microcapsules, 670, 671 properties, 208
Microscopic methods types, 202
AFM, see Atomic force microscopy (AFM) Murine transcutaneous full-thickness wounds
SEM, 513–514 mice wound beds, Tegaderm™, 749, 750
Microspheres Odex/CEC hydrogels acceleration, 749–751
albumin nanoparticles, 672
antitumor activity, 672 N
hydrogel-based systems, 673
nanoparticles, 671 Nerveguidance channels (NGCs), 575
pharmaceutical applications, 672–673 Neural tissue engineering
PLGA polymer, 671 description, 575
poly(lactic acid), 670–671 nerve guides implantation, 575
PTX encapsulation, 672 NGCs, 575
MMPs, see Metalloproteinases (MMPs) Neurons and glia, 567, 569–571
MNNG, see N-methyl-N-nitro-N-nitrosoguanidine NGCs, see Nerveguidance channels (NGCs)
(MNNG) NiPAAm, see N-isopropylacrylamide (NiPAAm)
Monoclonal antibodies (mAb), 609 N-isopropylacrylamide (NiPAAm), 45
Monomethyl ether hydroquinone (MEHQ), 793 Nitrospiropyran (NSp-Dex)
Mouth-formed protectors, 807 photoresponsive polymers, 688–689
Mouth protectors structural change, 689
classification, 807 N-methylenephosphonic chitosan (NMPCS), 260
description, 806 Non-aqueous elastomers
materials, 806 comparison, elastomers, 821
properties, 807–808 condensation and addition, silicones,
requirements, 806 820–821
MSCs, see Mesenchymal stem cells (MSCs) disinfection, impression materials,
Mucoadhesive polymers 821–822
ambiphilic, 205, 206 loss, dimensional stability, 819
anionic, 203–204 polyether materials, 821
buffer system, 215 polysulfides, 820
cationic, 204 Non-leloir glycosyltransferases, 621
controlled release, 215 Non-spectroscopic methods
covalent binding contact angle
chitosan-thiobutylamidine, 207 dynamic wettability, 510
matrix-tablets, 208 measurement, 509
sodium fluorescein, 208, 210 QCM, 511–512
thiolated, 208–210 SPR, 510–511
thiol/disulfide exchange reactions, 206, 207 zeta potential, 511
thiomer, 206 NSp-Dex, see Nitrospiropyran (NSp-Dex)
Index 895
O modified and alternative, 824

siloxane-based, 824
oHA/gelatin hydrogels Peroxidase-assisted polymerization
characteristics, 753, 754 catalytic cycle, 645
crosslinking reaction, 751, 752 cyclodextrins, 647
FT-IR spectra, 753 enzymatic oxidative, 647
1H NMR spectra, 751, 752
enzymes in in situ generation, 647, 648
oxidation degree, 751, 753 Fe(III) reaction, 645
sodium periodate oxidation, 750, 752 ferric protoporphyrin IX, 644
Oligo-and polysaccharides synthesis monoelectronic oxidation, 645
description, 618 phenol, 645
glycosidases (glycoside hydrolases)-assisted, see phenolic monomer units, 646–647
Glycosidases (glycoside hydrolases)-assisted phenols and aromatic amines, 647
synthesis phosphate and organic buffers, 647
glycosyltransferases-assisted, see Glycosyltransferases- radical coupling reactions, 646
assisted synthesis, oligosaccharides radical species, 646
stereo-and regioselectivity, 618 radical transfer reactions, 646
Ormocers syringaldehyde and bisphenol A copolymerization, 648
description, 816 of syringic acid, 647
structure, 816, 817 PGA, see Polyglycolic acid (PGA)
Orthodontic adhesives PGS, see Poly(glycerol-co-sebacate) (PGS)
brackets cemented to tooth Pharmacokinetic models
enamel, 809 FGF-2 tissue concentration, 845
cemented band, 809 potential fate, wound, 845
iatrogenic effects, 810 PHB, see Poly(β-hydroxybutyrate) (PHB)
malocclusion, teeth and jaws, 809 Phenolic polymers
pit and fissure sealants, 809 description, 644
polymerizing materials, 810 laccase-assisted polymerization, 648–650
Orthodontic elastics, 810 peroxidase-assisted polymerization, 644–648
Osteoblasts, 567, 568 Phospholipid polymers
Osteolysis articular cartilage and material design, 864–865
bone–implant interface, 856 hydration lubrication, 864
GM-CSF, 857 photo-induced surface “grafting from” polymerization,
macrophages, 856–857 865–867
mechanisms, wear debris, 856 poly(MPC)-grafted polyethylene, 867–871
osteoclasts, 857 Phosphorylases
periprosthetic fractures, 855 amylose-based materials, 624
prostaglandins, 857 branching enzymes, 1-6-a-glycosidic bonds, 623
reasons, revision THA, 855 description, 621
transcription factor NF-κB, 857 enzymatic polycondensation, 622
in young active patients, 856 helicoidal structure, amylose, 622
Owens-Wendt Method, 319, 320 in situ generation, glucose-1-phosphate, 622
phosphorylase-assisted phosphorolysis and
P condensation, 621
poly(THF) template polycondensation,
Paclitaxel (PTX), 665, 666, 672 glucose-6-phosphate, 622, 623
PAOMA, see Polyalkylenoxide/maleic acid (PAOMA) silica/silicone surfaces, 622
Passivating polymer surfaces, protein repelling single chain and multi chain mechanisms, 622
PEG and PEO, 519 stereoselectivity, 621
polymer chains, 518–519 synthetic amylose production, 622
proteins, 517–518 Photoresponsive hydrogels
PEC, see Polyelectrolyte complex (PEC) development
PEEK, see Poly(ether-ether-ketone) (PEEK) leuco chromophore, 689
PEG, see Poly(ethylene glycol) (PEG) pSpNIPAAm, 689, 690
PEO, see Poly(ethylene oxide) (PEO) micromanipulation
Peptides light irradiation, 691, 692
MC3T3-E1 cells, 365–366 microfluidic system, 691, 695
RGD, 364–365 microrelief, 691, 694
Periodontology microvalve array, 693, 697
BMP and rhPDGF, 825 pSpNIPAAm, 691, 693
GTR, 825 universal microfluidic system,
Permanent lining materials 693, 696
description, 824 Photoresponsive polymers
methacrylate-based, 824 cell adhesion, see Cell adhesion
896 Index
cell patterning Poly(methyl methacrylate) (PMMA)

balb/3T3 fibroblasts, 702–703 antibiotic-loaded acrylic cement, 876–877
caged technology, 700 biosafety, 401
fabrication, 703, 704 bone bioactive organic–inorganic hybrid bone
HepG2 organoid array, 702 cement, 877
MDCK cells, 701–702 description, 875–876
mechanism, 700–701 history, 876
description, 683–684 problems, 876
hydrogels, see Photoresponsive hydrogels Poly(orthoesters) (POEs)
integrated cell bioassay system, 703, 705 cross-linked
LCST, 685, 686 properties, 151, 152
light irradiation, 684–685 synthesis, 151–152
light irradiation and temperature, 687 description, 149–150
micropatterned irradiation system, 685–686 polymer hydrolysis, 152–153
NSp-Dex, 688–689 polymer process, 153
organic photochromic materials, 685 properties, 151
pSpNIPAAm, 688 synthesis, 150–151
spiropyrans, 686 Poly(vinyl alcohol) (PVA)
Pit and fissure sealants characterization, 38
bacteria levels, caries lesions, 805 description, 37–38
biocompatibility, 805 Flory-Rehner theory, 38–39
classification, dimethacrylate-based, 804–805 high molecular weight molecules, di-functional
compomers, 805 cross-linkers
“continuum” of tooth-colored materials, 804 autocorrelation functions, 41–42
defined, 804 DLS, 41
description, dental decay, 804 FITC, 42
enamel and retention rates bonding, 805 head-to-head sequences, 40–41
evidence-based clinical recommendations, 806 microbubbles, 42
polyelectrolyte materials, 805 hybrid hydrogels
research needs, 806 BaF3 cell proliferation, 53
self-etch adhesives, 805 cytotoxicity tests, 53
PLLA, see Poly(l-lactic acid) (PLLA) ECM, 51–52
PMMA, see Poly(methyl methacrylate) (PMMA) glutaraldehyde, 54
PNEGmPh, see Polyphosphazene polymer (PNEGmPh) hyaluronate oxidation, 52
POEs, see Poly(orthoesters) (POEs) low molecular weight molecules, di-functional
Poly(β-hydroxybutyrate) (PHB) cross-linkers
description, 143 hydroxyl groups, 39
properties and biodegradation, 143–144 properties, 40
synthesis, 143 short chain cross-linkers, 40
Poly(ε-caprolactone) (PCL) macromers, see Macromers
degradation, 3 Polyacid-modified resin composites, 797
functionalization, 15 Polyalkylenoxide/maleic acid (PAOMA)
metallation, 15 anionic groups, 257
polyesters, 226–227 PEC, 257
Poly(ether-ether-ketone) (PEEK) phase transition, 257
structure and properties, 872–873 Polyanhydrides
surface modification, 873–874 hydrolysis, 155–156
tribological properties, 873 imide-diacids, 156–157
Poly(ethylene glycol) (PEG) invention and development, 153
electrodeposition melt polycondensation, 153–154
cathodic Ti surface, 362 polymer processing, 156
immersion, 362 properties, 155
platelet adhesion and fibrin network formation, ROP, see Ring-opening polymerization
362, 363 (ROP)
scanning probe microscopic image, 362, 363 solution polymerization, 154–155
immobilization Polybutylene terephthalate (PBT), 444–445
PEGMA, 361 Polycaprolactones (PCL)
techniques, 360, 361 anionic reaction systems, 141
and PEO, 519 biodegradation, 142–143
structure, 360 cationic polymerization, 142
Poly(ethylene oxide) (PEO), 519, 663, 668, 669 description, 141
Poly(ethyleneglycol) (PEG), 249–250 kinetic and PMR spectroscopic methods, 142
Poly(glycerol-co-sebacate) (PGS), 450, 477 polymer properties, 142
Poly(l-lactic acid) (PLLA), 177–178 ROP, 142
Index 897
Polycarbonates dynamic coefficient relationship, 870

biodegradation, 144–145 FE-SEM images, wear particles, 871
synthesis, 144 “hydration lubrication”, 871
Polycondensation synthesis medical devices, 867, 868
carboxylic acids/esters with diols, see Carboxylic polymers, 867
acids/esters with diols polycondensation static water-contact angle, 869
hydroxyacids, see Hydroxyacids polycondensation surface modification, 867
Polyelectrolyte complex (PEC) weight change, 870–871
effects, 505 Poly(vinyl alcohol) hydrogels (PVA-H)
PAOMA, 249, 257 AID, see Artificial intervertebral disk (AID)
Polyesters artificial articular cartilage
aliphatic femoral head, 69
architectures, 27–28 Mankin’s score, 69–70
copolymerization, 26–27 osteochondral device, 68
properties, 26 THA and TKA, 67–68
carbonylic carbon atom, esteric/amidic bond, 632 biocompatibility, 59–60
catalytic mechanism, lipases, 631, 632 description, 57–58
chemo-, regio-and stereoselectivity, 631 development, 58–59
cyclic ketene acetals, 26 mechanical properties
description, 1–2, 137, 226 fabrication methods, 60–63
lactide/glycolide tribology, 63–67
aliphatic polyesters, 137 meniscus, see Meniscus
biodegradation, 140–141 potential
commercially available, 136–137 characteristic functions, 77
linear polyesters, 136 possibilities, 78–79
polymer properties, 139–140 titanium mesh, 77–78
ROP, 137–138 Polylactic acid (PLA), 226
lipases, 631 Polymerase chain reaction (PCR), 394
nucleophilic attack, 632 Polymer-based colloids, drug delivery
PCL, see Polycaprolactones (PCL) advantages, nano-or meso-scale size, 660
PETs, 631 aggregates, 661–662
PGA, 226 description, 659
PHB, see Poly(β-hydroxybutyrate) (PHB) drawbacks, 660
PLA, 226 drug conjugation technique, 659
poly(p-dioxanone), 145–146 future directions, 660–661
polycarbonates, see Polycarbonates internal stimuli-responsive, see Internal stimuli-
polyhydroxyalkanoates, 631 responsive systems, pH-responsive polymers
polymerization process, 631 lipid self-assembling, 660
ring-opening polymerization, see Ring-opening lipoplexes, 660
polymerization, cyclic monomers mechanisms, controlled release, 661, 662
ROP, cyclic esters, see Ring-opening polymerization micelles, see Polymeric micelles
(ROP) multiple functional components, 660
step-growth polymerization, 2 physicochemical characteristics, 660
stereoselectivity of lipases, see Lipase stereoselectivity polymeric aggregates, 661
techniques, aliphatic, 1, 2 polymerosomes, see Polymerosomes
Polyethylene bearing material stimuli-responsive, see Stimuli-responsive polymers
antioxidants, 862–863 synthetic toxicity and immunogenic properties, 660
CLPE, see Cross-linked polyethylene (CLPE) traditional methods, 660
ethylene (C2H4), 857 treatment, solid tumors, 659
manufacture, UHMWPE implants, 858 viral vectors, 660
mass and heat transfer, 858 Polymer bioconjugate surfaces
medical-grade extrusion facilities, 858 avidin system, 521
molten resin vs. heated die wall surface, 859 biomaterial–biological environment interactions,
in orthopedic field, 859–860 520–521
physical and chemical properties, 857, 858 heparin, 522
types, 857 maltoheptose polymeric systems, 522
Polyethylene glycol (PEG), 227, 544 types, 521
Polyethylene terephthalate (PET), 444–445 Polymeric micelles
Polyglycolic acid (PGA), 226 A-B diblock structure, 662
Poly(MPC)-grafted polyethylene amphiphilic diblock copolymers, 662
BP system, 868 antitumor efficacy data, PBS control, 665
clinical uses, 867 CDDP, 667
CLPE surface mimicking cartilage, 867 clinical evaluation, 666
cross-sectional TEM images, 869 CPT, 667
898 Index
definition, 662 monomers, 422

dendrimers/hyperbranched preparation, 666 polyaddition, 421
drug delivery applications, 666 polycondensation, 421
drug existance, 665 polyethylene, 420
emulsion method, 663 Polymerosomes
examples, 663–664 definition, 667
geometrical arrangement, 663 diblock copolymeric amphiphiles, 668
honokiol delivery, cancer chemotherapy, 667 fluid-filled core for separation, 668
insoluble drugs, 663 hemoglobin encapsulation efficiency comparison,
long-circulating drug nanocontainers, 662 668, 669
nanotherapeutic systems, 665 hydrophilic and hydrophobic block copolymers, 668
nontoxic biocompatible and bioabsorbable monodisperse sub-micro-meter-sized vesicles, 668
properties, 667 multicomponent structure, vesosome, 669
pharmacological fields, 663 PEO and PCL, 668
physicochemical parameters, 663 polymer vesicles, see Polymer vesicles
positive features, 662–663 stability and permeability, 667
ζ-potential and hydrodynamic size, poly(lactic acid) Polymer vesicles
particles, 663, 664 ABA triblock copolymer, 669–670
selective intracellular delivery, DOX, 666 description, 669
self-assembly of amphiphilic copolymers, 666 hybrid block copolymers, 669
size, 663 layer-by-layer deposition of polyelectrolytes, 669
stimulus-responsive drug release systems, 666 microcapsules, see Microcapsules
tumor growth after drug injection, 664 microspheres, see Microspheres
water-soluble poly(glycerol methacrylate) Poly methyl methacrylate (PMMA), 822
backbones, 666 Polyolefins, 447–448
Polymeric nanoparticles Polyphosphazene polymer (PNEGmPh)
colloidal coating, 605 biocompatibility, 109
DDS, 596–597 degradation, 105–106
dialysis method, 601–602 3D scaffolds, 121–122
drug delivery, 593–594 fabricated composites, 122
polymers, 594, 595 matrices, 114
preparation, techniques PNEG, 122
description, 596 Polyphosphazenes
double emulsion method, 597 biocompatibility
miniemulsion methods, 597–598 alkoxy-substituted, 109
phase separation, 598 aminated, 108–109
single emulsion method, 596–597 polyphosphazene blends/composites, 109–110
self-assembling, 602 biodegradable
solvent dispacement alkoxy-substituted, 94
coprecipitation method, 599–601 aminated, 93–94
nanoprecipitation method, 598–599 biocompatibility, 108–110
spray drying technology, 603–604 blends/composites, 94–99
supercritical fluid solution, 603 drug delivery, 110–118
targeting approaches structures, 92–93
antigen/receptor, 608 tissue engineering, 118–125
BBB, 607–608 degradation, see Degradation
drug, 606 description, 84
EPR, 605 synthesis
growth factors, 608 architecture, 89
inflammation, 610 glass transition temperatures, 91–92
lectin-carbohydrate, 609–610 living cationic polymerization, 87
mAb, 609 macromolecular substitution, 88–89
passive targeting, 606–607 phosphorus-nitrogen backbone, 89–90
PCX, 610 physicochemical and biological properties, 91
PEGylated, 605–606 ring-opening polymerization, see Ring-opening
physical targeting, 608 polymerization
Polymeric parameters, electrospinning structure, 85
solution viscosity, 544–545 synthetic flexibility, 92
solvent conductivity, 545–546 Polyphosphazenes blends/composites
surface tension, 545 blend systems, 94–95
Polymerization concept and feasibility, 96
copolymerization, 421 hydrogen-bonded polymer, 95, 96
free radical, 420 mixed-substituent polymer, 98
homopolymerization, 421 polymer, 94
Index 899
structures, 95 Proteins
surface morphologies, blends, 97 collagen, 286–288
Polysaccharides description, 286
agar-agarose, 278–280 fibrin-fibrinogen-fibronectin, see
alginate, 280–281 Fibrin-fibrinogen-fibronectin
cellulose, 273–276 gelatin, see Gelatin
chitin and chitosan, 281–282 PTX, see Paclitaxel (PTX)
description, 273 PVA-H, see Poly(vinyl alcohol) hydrogels (PVA-H)
factors, 273 PVC, see Polyvinylchloride (PVC)
HA, see Hyaluronic acid (HA)
starch, 276–278 Q
Polytetrafluoroethylene (PFTE), 476
Polyurethane elastomers QCM, see Quartz crystal microbalance (QCM)
biodegradable, 444 Quartz crystal microbalance (QCM)
biodegradation instrument, 511
chemical properties, 441 variation, 512
cleavage, 440
ESC, 438, 440 R
mechanism, 440
biomedical applications, 433 Regranex®, 844
biostable, 441–442 Resilient polymers
grafted, 442–443 permanent lining materials, 824
manufacture temporary lining materials, 824
Pellethane®, 435 Resin composites
TDI and MDI, 434 biostability and biocompatibility, 813
mechanical behavior chemical and mechanical durability, 813–814
biomedical applications, 437, 439 classification, inorganic filler content, 811–813
hard and soft segments, 435, 436 description, 796
hard segments reorientation, 437–438 laboratory fabrication, 811
linear polyetherurethane chain, 435–436 polymerization shrinkage, 814–815
stress crystallization, 437 rheology classification, 812–813
modification, 443–444 silane coupling agent, 796
shape memory, 442 Resin-modified glass-ionomer cements, 797, 798
Polyurethanes, 470–474 Ring-opening polymerization (ROP)
Polyvinylchloride (PVC) anionic
products, 445–446 alkyl-oxygen bond, 5
surface properties, 447 β-lactones, 6
Post-surgical abdominal adhesion εCL, 4–5
advantages, 762 intramolecular transesterification reactions, 5
anti-adhesion scores, 762 coordination
carboxy-methylcellulose/hyaluronan film, 765 anionic metal alkoxides, 6
cecal and abdominal wall tissues examination, 763 auminum alkoxides, 6
conventional surgeries and laparoscopic carbon dioxide, 7–8
procedures, 765 insertion mechanism, 6–7
granulation tissue, 764 metallic derivatives, 8
in situ gelable hydrogel barrier, 762 tin(II) bis-(2-ethylhexanoate), 7
multi-nucleated giant cells, 764–765 cyclic monomers
non-absorbable and absorbable, 762 “active-chain mechanism”, 637
non-biodegradable polytetra?uoroethylene aliphatic polyesters, 636
membranes, 762 β-butyrolactone products, 639
Odex/CEC hydrogel remnants, 764 ε-caprolactone, 638–639
photomicrographs, abdominal and cecum tissues, chemocatalysis, 638
763, 764 enantioselectivity, 640–641
post-operative sacrifice time, 764 end-functionalized polyesters, see
Primary rat osteoblasts (PRO) cells, 121–122 End- functionalized polyesters
Prosthodontic materials enzymatic, 638
complete dentures, see Complete dentures enzyme-substrate complex, 637
denture adhesives, 824 five-, six-, and seven-membered lactones, 638
denture teeth, 823 four-membered lactone polycondensation, 639
resilient polymers, see Resilient polymers lactone mechanism, 637
Protein resistant biofunctionalization monomer 12-dodecanolide (DDL), 637
amphiphilic block copolymer polymers, 558 oxyacids/oxyesters polycondensation, 638
cell-fibroblasts adhesion, 559 polycarbonates, cyclic carbonates, 642–644
macomer/polymer solution, electrospinning, 558 polymalate, 639
900 Index
six-membered lactones series, 640 polymeric biomaterials, 327

δ-valerolactone and ε-caprolactone, 637 surface free energy, 328
εCL, LA and GA, 3 surface roughness, 330
enzymes, 13–15 Siloranes
lactide/glycolide, 137 adhesive system, 815
mechanism, 86, 137–138 definition, 815
metal-free monomer, 815, 816
acids and alcohols, 9 volumetric shrinkage, 815
activation, nucleophiles, 10 Siloxane-doped poly(l-lactic acid)/calcium carbonate
catalysts, 12–13 (vaterite) composites (SiPVC)
dual catalysts, 13–14 cellular compatibility
mechanism, cationic, 10 bone nodules, 181
nucleophilic catalysts, lactones, 10–12 culture tests, 179, 180
Z-witterionic, pivalolactone, 8–9 MC3T3-E1 cells, 179–180
nomenclature, 4 MSCs, 181, 182
polyphosphazenes, 85 SiV3 particles, 185
substituted and functionalized characterization, 182–183
cyclic ketene acetals, 16, 25 description, 182
description, 14, 15 lactic acid/calcium carbonate, 178–179
6,7-dihydro-2(3H)-oxepinone, 16, 21 microfiber
2-methylidene-1,3-dioxepane, 16, 25 electrospinning, 186
monoesters, 16–18 electrospun, 186–187
PCL, 15 preparation, 187–188
thermodynamics, 3, 4, 86–87 tissue compatibility and bone-forming ability, 190
ROP, see Ring-opening polymerization (ROP) MSCs, see Mesenchymal stem cells (MSCs)
PLLA, see Poly(l-lactic acid) (PLLA)
S preparation, 178–179
silicon species, 178
SCA, see Silane coupling agent (SCA) SIMS, see Secondary ion mass spectrometry (SIMS)
Scanning electron microscopy (SEM) Single collection systems
ESEM, 513–514 collector on liquid medium, 549
TEM, 513 description, 549
SDG, see Secoisolariciresinol diglycoside (SDG) electrospun fibers, solid collector, 549
Secoisolariciresinol diglycoside (SDG), 734–735 surface tension, 550
Secondary ion mass spectrometry (SIMS), 509 Single emulsion method
Segmentated polyurethane (SPU), 367, 369 nanoparticles preparation, 596, 597
Self-etch adhesives, 805 solvent evaporation technique, 596
SEM, see Scanning electron microscopy (SEM) SMCs, see Smooth muscle cells (SMCs)
Sensitization assays Smooth muscle cells (SMCs)
animals, 460 description, 563
biological reactivity evaluation, 461 multilayered vascular construction, 565
considerations, 460 with in vitro studies, 564
implantation period, 460–462 Soft tissue augmentation
material preparation, 460 materials, 825
Silane coupling agent (SCA), 367 requirements, 825
Silicone elastomers Solution viscosity
copolymerization chain flexibility improvements, 545
condensation, 429 concentration regimes, 544
reactive prepolymers, 429 electrospinning parameter, 544
siloxane and methacrylate, 430 fiber diameter prediction model, 544
siloxane methacrylate, 431 glycerol, 545
fillers, 431 Solvent conductivity
homopolymerization, 428 addition of salt, 546
manufacture, 427–428 beaded and smooth fibers, 545
properties, 426–427 electrospinning jets, 545
surface treatments higher elongational forces, 546
acylation/hydroxylation, 433 mixtures, 545
denucleation, 433 Taylor cone and spinneret clogging, 545
description, 431 Spectroscopic methods
grafting, 432 FTIR, 508–509
techniques, 432 SIMS, 509
Silk and keratin XPS, 507–508
fibroin films, 328–330 Spray drying technology, 603–604
mechanical properties, 330 SPU, see Segmentated polyurethane (SPU)
Index 901
Starch postprocessing approach

characteristics, 276 chemical binding bioactive functionalities, 558
drug delivery system, 277 proteins adsorption, 558
SBB, 278 protein resistant biofunctionalization
scaffolds, 277 amphiphilic block copolymer polymers, 558
SEVA-C and SCA, 276 cell-fibroblasts adhesion, 559
Stents macomer/polymer solution, electrospinning, 558
bare, 357–358 Surface plasmon resonance (SPR)
designs and functions, 357 Kretchmann configuration, 510
DESs, see Drug-eluting stents (DESs) polymers and biomolecules, 511
and stent grafts, 359–360 Synthetic cellulose-type glycans (β-(1→4)-glucans), 630
Stimuli-responsive polymers Synthetic laminarin-type glucans (β-(1→3)-glucans), 630
drug delivery system, 673 Synthetic polymers
electro-responsive and magnetically responsive blended “hybrid” polymers, 543, 544
polymers, 674 lack of biological signals, 544
light-responsive polymers, 675 material properties, 543–544
physiological requirements, 673 PCL and PEG, 544
thermo-responsive polymers, see Thermo-responsive selected electrospuns, 543
polymers
Stress urinary incontinence (SUI), 406–407 T
Structured electroconductive collectors
3D structures, 551 TBB, see Tri-butyl Borane (TBB)
and patterned electrospun mesh, 551, 552 TE, see Tissue engineering (TE)
static electroconductive patterned, 550–551 TEM, see Transmission electron microscopy (TEM)
woven fibrous architectures, 551 Tensile tests
SUI, see Stress urinary incontinence (SUI) dry polymer compacts, 200
Surface energy biomaterials hydrated polymers, 200
adhesion, 496–497 microspheres, 201
approaches, 498–499 THA, see Total hip arthroplasty (THA)
biological environment and applications Thermoplastic elastomers (TPEs), 419
cell–polymer interaction, 514–517 Thermo-responsive polymers
material incorporation, 514 ability and physiological conditions, 674
passivating polymer surfaces, protein repelling, characteristics, 673
517–520 DOX release behavior, 674
polymer bioconjugate surfaces, 520–522 mixed micelles, 674
biosensing applications, 497 pNIPAAM, 673
blood contacting devices, 496 temperature phase transition, 674
cells, 498 Tissue engineering
chemical methods regeneration
biochemical modification, 506–507 alanine ester, 125
click chemistry, 505–506 dipeptide-based polyphosphazene,
grafting polymerization, 504 122–123
PEC, see Polyelectrolyte complex (PEC) microsphere architectures, 123–124
wet, 503–504 nanofiber matrices, 123
FCS, 497 PRO cells, 121–122
hydrophilic, 496 scaffold fabrication
physical methods approaches, 118
corona discharges, 502 biodegradable polyphosphazenes, 118
dry surface treatment techniques, 499 dipeptide-based, 120
ion implantation, 502–503 3D porous microsphere, 118–119
radio frequency (RF) plasma treatment, 500–501 polymeric nanofibers, 120
UV/ozone ablation, 500 polyphosphazene polymers, 120
polymer surfaces solvent/nonsolvent sintering approach, 119
characterization, 507 Tissue engineering (TE)
microscopic methods, 512–514 bone, 576
non-spectroscopic methods, 509–512 cartilage, 577–578
spectroscopic methods, 507–509 neural, 575
proteins, 497 vascular, 577
strategies, 498 Titanium
Surface hydroxyl group oxygen atoms, hydroxyl group, 351, 352
acidic and basic properties, 353–354 surface oxide film, 351–352
formation, 353, 354 Ti 2p XPS spectrum decomposition,
Surface modification, electrospun fibers 350–351
polymers biofunctionalization, 558 TNF-α, see Tumor necrosis factor (TNF-α)
902 Index
Tooth filling materials methyl metacrylate polymerization, 652

classes, “continuum”, 811 oxidative polymerization, aromatic compounds, 650
clinical longevity, tooth-colored restorations, 817 Visual tests
compomers, 816 rinsed channel, 199–200
glass-ionomers and resin-modified glass-ionomers, 817 rotating cylinder, 199
ormocers, 816, 817 Volume-phase transition temperature (VPTT),
requirements, 811 45–46
resin composites, see Resin composites VPTT, see Volume-phase transition temperature (VPTT)
siloranes, 815–816
usage, polymers, 811 W
Tooth restoration
endodontic materials, 817–818 “Water-in-water” emulsion polymerization method, 45
filling materials, see Tooth filling materials Wound closure materials, 826
Total hip arthroplasty (THA) Wound healing
description, 854 acrylated chitosan/oxidized dextran, see Acrylated
primary and hemi-arthroplasty, Japan, 854 chitosan/oxidized dextrans
revision burdens, countries, 854–855 acute and normal, see Acute and normal wound
Total knee joint arthroplasty (TKA), 67 healing response
TPEs, see Thermoplastic elastomers (TPEs) anti-infective agents delivery, 844
Transglycosidases, 628 biocompatible materials, 837
Transmission electron microscopy (TEM), 513 biomaterial dressings, 841, 842
Tribology, PVA-H cause and severity, minor cuts and abrasions, 838
articular cartilage, 63, 64 chitosan, 742
CLSM, 65 chronic, see Chronic wounds
frictional coefficient, 63, 64 clinical trials, 843
friction testing machine, 63 collagen and chitosan, 841
lubricative mechanism, 67 colonization, 838
optical-isolator technique, 66 comparative studies, 841
Tri-butyl Borane (TBB), 354–355 definition, 740
Tumor necrosis factor (TNF-α), 728 description, 837
dextran, see Dextrans
drug delivery sciences, 843
U ePTFE topography, 843
gelatin, see Gelatin
UBE, see Unknown biological elements (UBE)
gelatin/oxidized hyaluronan, see Gelatin/oxidized
UHMWPE, see Ultra high molecular weight polyethylene
hyaluronans
(UHMWPE)
growth factors delivery, 844–845
Ultra high molecular weight polyethylene (UHMWPE)
HA, see Hyaluronic acid (HA)
description, 853
hydrogels, 740
gamma-ray sterilization, 862
importance, stratum corneum, 838
high-energy ray irradiation cross-linking, 860
iNOS inhibitor, see iNOS inhibitors
history, joint replacement, 859
MMPs, 843
implants, 858
pharmacokinetic models, drug delivery, see
molecular weight, 857
Pharmacokinetic models
orthopedic community, 854
physiological response, 838
PEEK, 873
polymeric materials, 841
Unknown biological elements (UBE), 472
post-surgical abdominal adhesion, see Post-surgical
abdominal adhesion
V Regranex®, 844
skin, 837
Vascular cell adhesion molecule-1 (VCAM-1), 732 stages, 838
Vascular tissue engineering, 577 synthetic polymers, 740
VCAM-1, see Vascular cell adhesion molecule-1 (VCAM-1) therapeutic agents, 839
Veneers, 810 tissue biocompatibility, 840
Vinylic monomers tissue-engineered skin and biomaterials, 839, 841
acrylamide polymerization, 651
acrylic and styrenic, 650 X
chemoselective polymerization, 652
description, 650 XPS, see X-ray photoelectron spectroscopy (XPS)
β-diketones mechanism, 650–651 X-ray photoelectron spectroscopy (XPS)
free radicals, 650 ESCA, 508–509
initiators, styrene polymerization, 651 principle, 508
5 µm
FIGURE 2.1 Laser scanning confocal microscopy image after tagging PVA shelled microballoons with
fluorescein isothiocyanate (FITC).
Spacing between chains changes as a
function of the side-chain length.
SCHEME 2.2 Representation of the zipping of the PVA chains (in black) occurring during the photopoly-
merization of vinyl side chains.
+ R
H2N
OH OH Amine with a Boc protected nucleophilic group,

PVA R: NuBoc
N N
N N
CDI
O OH
HN
R'
Functionalized PVA via carbamate (red) coupling after deprotection
bearing a nucleofilic group R'
R': NH2 , SH
SCHEME 2.5 CDI chemistry on PVA.

Cu(I)
+ N N N
N
N N
: PVA chain
SCHEME 2.7 1,3 Cycloaddition click reaction scheme for the formation of a triazole ring.
20% Wt PVA-H
solution (DMSO)
<0°C 12 h
Gelatine
PVA gel
(DMSO)
Solution with ethanol
PVA gel (Et-OH)
Dry in vacuum
Heating 100°C–120°C
Swelling in water
PVA hydrogel
PVA-H
(a)
Concentration of swelling
PVA PVA 30%
Mix Melting Injection molding
At 150°C, 160°C, 180°C, 200°C Injection pressure 120 MPa

Solvent Injection velocity 10 cm3/s
by Kneading machine
Injection at 150°C, 160°C, 180°C, 200°C
H2O/DMSO =
20/80 (w/w)
Hydrogel
(b)
FIGURE 3.1 (a) Flow chart of the production process of poly(vinyl alcohol)-hydrogel (PVA-H) by freeze-
thaw method. (b) Flow chart of the production process of poly(vinyl alcohol)-hydrogel (PVA-H) by injection
molding method.
(a) (b)
FIGURE 3.2 Histological appearance of the implant material particles and the surrounding tissue in rabbits
knee joint (×100). (a) UHMWPE particle: many macrophage and giant cells are surrounding particle due to intense
immunoreactions. (b) PVA-H particle: Immunoreactions were hardly observed.
Err bar: ± SD
40
30
20
10
0
150°C 160°C 180°C 200°C Natural Freeze-thaw
articular method
Injection-molding method cartilage
(a) (heating temperature (°C))
25 Err bar: ± SD
Young’s modulus (MPa)
20 Natural
articular
15 cartilage
10
0
150°C 160°C 180°C 200°C Freeze-thaw
method
Injection-molding method
(b) (heating temperature (°C))
Err bar : ± SD
40 PVA-H
30
20
10
0
Water content Water content Natural articular
(c) 60% 45% cartilage
FIGURE 3.3 Mechanical properties of PVA-H prepared by injection molding method. (a) Influence of heat-
ing temperature on ultimate tensile strength (MPa). (b) Influence of heating temperature on Young’s modulus
(MPa). (c) Influence of water content on ultimate tensile strength (MPa).
PVA-H
10.0 Water content: 20%
Articular cartilage
PVA-H
Stress (MPa)
W.C. 45%
6.0
PVA-H
W.C. 90%
2.0
Meniscus
0 1.0 2.0 3.0 Strain
FIGURE 3.4 Representative stress–strain curves of various PVA-H samples and natural tissue specimens in
the compression test.
PVA-H
25%
PVA-H
45%
PVA-H
3.2 ± 0.08
Water content
60%
PVA-H
90%
Meniscus 0.3 ± 0.03

specimen
0 2 4 6 8 10 12 14
(MPa)
FIGURE 3.5 Comparison of yield stress value of various PVA-H and natural tissue specimens.
1.0
PVA-H
Stress (MPa)
Water content: 20%

0.5
Articular cartilage
PVA-H W.C. 45%
PVA-H W.C. 90%

Meniscus
0 1 2 3 4 5
Time (min)
FIGURE 3.6 Comparison of the stress–relaxation curves of PVA-H and natural tissue specimens.
Err bar: ± SD
Normal articular cartilage (sample number: n = 3)
Normal articular cartilage vs.
PVA-H (freeze-thaw method) (n = 3)
Alumina (n = 3)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

(a) Coefficient of friction
Normal articular cartilage Err bar: ± SD

OA cartilage (1) (n = 3 for each combination)
Injection-molding OA cartilage (2)

PVA-H vs.
(150°C) OA cartilage (3)
Injection-molding
PVA-H (150°C)
Injection-molding
PVA-H Injection-molding
(200°C) vs. PVA-H (200°C)
Freeze-thaw
vs. Freeze-thaw PVA-H
PVA-H
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

(b) Coefficient of friction
FIGURE 3.8 Histogram showing coefficients of friction for various combination. (a) Friction coefficients of
various materials specimens against normal articular cartilage. (b) Friction coefficients of various materials
specimens against PVA-H.
PVA-H vs. PVA-H vs.
PVA-H articular cartilage
0.08
0.07
Coefficient of friction
0.06
0.05
0.04
0.03
0.02
Saline HA 80 HA 200 Synovial fluid
(molecular 80 × 104
weight: 200 × 104)
FIGURE 3.9 Frictional coefficient of PVA-H versus articular cartilage or PVA-H, and the influence of hyal-
uronic acid.
Laser
Weight
Grass plate
Cartilage Lubricant
Stage
FIGURE 3.10 Photograph of real-time 1LM21 confocal laser scanning microscopy (CLSM) and schematic
cross-sectional view of CLSM observation.
(a) (b)
FIGURE 3.11 (a) CLSM image of normal articular cartilage surface without loading (×1400). (b) CLSM image
of normal articular cartilage surface under the loading (×250). The solid contact area, fluid pooling area with
synovial fluid and intermediary area with fringe-like pattern were observed.
Fluid area
Intermediary area
Contact area
(a) (b)
FIGURE 3.13 CLSM images with optical-isolator technique about the intermediary area. The reflection
image (a) was obtained corresponding to the third area in the original CLSM image (b).
PVA-H
Titanium-fiber
FIGURE 3.15 Photograph of a synthetic composite osteochondral device for partial replacement of the
canine femoral head. The transparent portion is made of PVA-H.
Artificial Natural articular PVA-H
articular cartilage (artificial articular cartilage)
cartilage
Articular (PVA-H) Titanium fiber mesh
cartilage
(a) (b)
FIGURE 3.16 Photograph of a specimen of the femoral head with composite osteochondral device at
12 months after implant operation. (a) Macroscopic appearance; (b) cross-sectional appearance.
(a) (b)
FIGURE 3.17 Cross-sectional view of a specimen of the femoral head with composite osteochondral device
at 12 months after implant operation (×2.5). (a) Histological appearance (Giemusa stain). (b) Contact micro-
radiograph appearance.
15
Alumina ceramics
10 UHMWPE
Score
PVA-H
0
1 3 6 12
Months
FIGURE 3.18 Mankin’s score for the changes in acetabular cartilage in each group. Each point represents a
single score for a joint.
Natural meniscus
PVA-H meniscus
Artificial
meniscus
FIGURE 3.19 Photograph of PVA-H artificial meniscus and the schema of artificial meniscus replacement.
Control
Time P.O. 6 month P.O. 1 year P.O. 18 month P.O. 2 year
Artificial meniscus
FIGURE 3.20 Macroscopic appearance of articular cartilage of rabbit’s knee joint up to 2 years after opera-
tion. In both groups, lateral side of knee joint was operated. In the control group, OA change appears in post
operative 6 months, remarkable change becomes obvious in P.O. 1 year.
Control Artificial meniscus
10
8
Score
0
6 Month 1 Year 18 Month 2 Year
Time
FIGURE 3.21 Macroscopic evaluation of articular cartilage according to classification by Chang.

(a) (b)
FIGURE 3.22 Macroscopic appearance of articular cartilage of rabbit’s knee joint in 2 years after oper-
ation in PVA-H meniscus replacement group. (a) Neither breakage nor dislocation of PVA-H is observed.
(b) Articular cartilages of knee joint are also intact.
Control

Artificial meniscus
(a)

Control

Artificial meniscus
(b)

250 µm
Control
250 µm
Tibial side 250 µm Femoral side

Aritificial meniscus
250 µm
(c)
FIGURE 3.23 Histological appearance of articular cartilage of knee joint 2 years after operation
(×20, Safranin-O stain). (a) P.O. 1 year; (b) P.O. 1.5 year; (c) P.O. 2 years.
Number Number
P.O. 24 month
P.O. 12 month
P.O. 6 month
15
10
Score
5
1
Control group Artificial meniscus group
(by Mankin’s grading system)
FIGURE 3.24 Histological evaluation of articular cartilage according to Mankin’s grading system.
PVA-H
TB
(a) (b)
FIGURE 3.25 (a) Diagram of artificial intervertebral disk for beagle dog. (b) Macro appearance of the
canine spine implanted with artificial intervertebral disk.
Vertebral body
Bone ingrowth
into TFM
PVA-H
FIGURE 3.26 Contact microradiography findings of the artificial intervertebral disk implanted into dog
1 month after operation. Massive bone ingrowth into the pores of titanium fiber mesh was observed.
Failure
FIGURE 3.27 Micro-appearance of the interface between PVA-H and titanium fiber mesh of artificial inter-
vertebral disk at 6 months after operation into canine spine.
Intervertebral
disk
PVA-H disk
nucleus
Vertebral body
FIGURE 3.28 Histological appearance of the partial disk replacement by PVA-H artificial disk nucleus in
an animal surgery.
PVA-H
Titanium
fiber mesh
Bone
ingrowth
(a) (b)
FIGURE 3.29 Histological appearance of composite device at 12 weeks after implantation of canine femoral
condyle. Mature bone has grown into the pores of titanium fiber mesh. (a) Giemusa stain (×10); (b) contact
microradiograph (×10).
60
50
40 y = 0.2439x + 28.562
R2 = 0.9929
30
Tg
20
Experiment
Wood
10 Fox
Linear (experiment)
0
0 20 40 60 80 100
PPHOS composition (%)
FIGURE 4.4 Miscibility of the polyphosphazene–polyester blends. Linear relationship between blend Tg and
polyphosphazene composition was based on the experimental values from DSC thermograms, which was in
line with Wood equation for miscible blends. Furthermore, the glass transition temperatures estimated using
the Fox equation for the blends were very close to the experimental values from DSC thermograms. (From
Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: In situ porous structures: A unique polymer erosion mechanism
in biodegradable dipeptide-based polyphosphazene and polyester blends producing matrices for regenera-
tive engineering. Adv. Funct. Mater. 2010d. 20(17). 2794–2806. Copyright Wiley-VCH Verlag GmbH & Co.
Time
(a)
(b)
(c)
FIGURE 4.5 Schematic illustration of different types of polymer erosion. (a) Bulk erosion; (b) Surface ero-
sion; and (c) A unique polymer erosion through which the polymer changes from a solid coherent film to an
assemblage of microspheres with an interconnected porous structure. (From Deng, M., Nair, L.S., Nukavarapu,
S.P. et al.: In situ porous structures: A unique polymer erosion mechanism in biodegradable dipeptide-based
polyphosphazene and polyester blends producing matrices for regenerative engineering. Adv. Funct. Mater.
2010d. 20(17). 2794–2806. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. With permission.)
T=0 T = 4 weeks T = 7 weeks T = 12 weeks
Time
(a) (c) (e) (g)
BLEND25
(25% PPHOS)
(b) (d) (f ) (h)

BLEND50
(50% PPHOS)
Percentage
PPHOS
FIGURE 4.8 Surface morphologies of the blends during in vitro degradation at 37°C over 12 weeks as a
function of time. (a, c, e, g): SEM images showing surface morphologies of BLEND25 following 0, 4, 7, and
12 weeks of in vitro degradation. The insets of (e) and (g) show the detailed 3D spherical structures. (b, d, f, h):
SEM images showing surface morphologies of BLEND50 following 0, 4, 7, and 12 weeks of in vitro degrada-
tion. The unique polymer erosion of the blend system resulted in the change of matrix morphology from a solid
coherent film to an assemblage of microspheres with interconnected porous structures characterized by macro-
pores (10–100 μm) between polyphosphazene spheres as well as micro/nanopores on the sphere surface. (From
Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: Biomimetic, bioactive etheric polyphosphazene-poly(lactide-co-
glycolide) blends for bone tissue engineering. J. Biomed. Mater. Res. A 2010c. 92A(1). 114–125. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA. With permission.; Deng, M., Nair, L.S., Nukavarapu, S.P. et al.: In
situ porous structures: A unique polymer erosion mechanism in biodegradable dipeptide-based polyphospha-
zene and polyester blends producing matrices for regenerative engineering. Adv. Funct. Mater. 2010d. 20(17).
2794–2806. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. With permission.)
(a) (b)
(c) (d)
(e) (f )
FIGURE 4.9 Representative photomicrographs of hematoxylin and eosin (H&E) stained section illustrat-
ing the chronic inflammatory response to PLAGA and blend matrices during the post-implantation period.
After 2 weeks, (a) PLAGA, (b) BLEND25, (c) BLEND50; After 4 weeks, (d) PLAGA, (e) BLEND25,
(f) BLEND50. Both blend matrices elicited less inflammatory responses than PLAGA. (From Biomaterials.
31(18), Deng, M., Nair, L.S., Nukavarapu, S.P. et al., Dipeptide-based polyphosphazene and polyester blends
for bone tissue engineering, 4898–4908. Copyright 2010b, with permission from Elsevier.)
(a) (b)
Cumulative protein release

110 PPHOS
PPHOS-HA
90
70
50
30
10
0 5 10 15 20
–10
(c) Time point (days)
FIGURE 4.10 Polyphosphazene-based matrices for growth factor delivery. (a) SEM micrograph showing the
morphology of polyphosphazene matrices via solvent/nonsolvent sintering; (b) SEM micrograph showing
the morphology of polyphosphazene-HAp matrices via solvent/nonsolvent sintering; and (c) Cumulative BSA
release. Zero order release of BSA was demonstrated over the course of 21 days from both the BSA-loaded poly-
phosphazene and BSA-loaded polyphosphazene-HAp composite scaffolds. Electrostatic interactions between
HAp and BSA contributed to the controlled release curve shown here. (From Oredein-McCoy, O. et al., J.
Microencapsul., 26(6), 544, 2009, with permission from Informa Healthcare.)
(a) (b)
FIGURE 4.13 Cytoskeletal actin distribution of PRO grown on polyphosphazene-HAp composite micro-
sphere scaffold for (a) 6 days; and (b) 12 days. Scale bar: 250 μm. After 12 days, cells covered the entire
scaffold including the microsphere surface as well as the microsphere adjoining areas. (With permission from
Nukavarapu, S.P., Kumbar, S.G., Brown, J.L. et al., Polyphosphazene/nano-hydroxyapatite composite micro-
sphere scaffolds for bone tissue engineering, Biomacromolecules, 9(7), 1818–1825. Copyright 2008 American
Chemical Society.)
(a) (b)
(c) (d)
PLAGA PPHOS Composite PLAGA PPHOS Composite

20 1.2
Alizarin red ODU/Scaffold
1
15
pg OCN/ng DNA
0.8
10 0.6
5 0.4
0.2
0
3 7 14 21 0
–5 3 7 14 21
(e) Time (days) (f ) Time (days)
FIGURE 4.14 Polyphosphazene composite scaffolds bridging nanofiber and microsphere architectures to
improve bioactivity of mechanically competent constructs for bone regeneration. (a, b) SEM micrographs of
the cross-sections of the composite nanofiber/microsphere scaffold demonstrating incorporation of nanofibers
of less than 1 μm within the void space generated by sintering microspheres were (a) low magnification image
and (b) high magnification image; (c, d) SEM micrographs of preosteoblasts in the interior of composite
scaffolds after 3 days (c) and 14 days (d) of culture demonstrating the presence of preosteoblasts and the
accumulation of matrix proteins in the interior of the composite scaffold during cell culture. The arrows point
to representative cells. Notably, the fibrous portion of the scaffold has been extensively modified through
the accumulation of ECM proteins after 14 days, to the extent that identifying the cells is difficult. (e, f)
Phenotype evaluation of preosteoblasts seeded on composite scaffolds, (n = 4). Mature osteoblast marker such
as osteocalcin (e) evaluated and normalized to total DNA demonstrates a peak at 14 days for the preosteoblasts
seeded on the composite scaffold; and (f) Total scaffold calcification normalized to an unseeded construct
demonstrates that the preosteoblasts seeded on the composite scaffold produced a substantially more min-
eralized matrix than did the preosteoblasts on control PLAGA and polyphosphazene scaffold. (*) represents
statistical significance, p < 0.05, from all other samples at a given time point. All error bars represent standard
deviation. (From Brown, J.L., Peach, M.S., Nair, L.S., Kumbar, S.G., and Laurencin, C.T.: Composite scaf-
folds: Bridging nanofiber and microsphere architectures to improve bioactivity of mechanically competent
constructs. J. Biomed. Mater. Res. A. 2010. 95(4). 1150–1158. Copyright Wiley-VCH Verlag GmbH & Co.
Intensity (a.u.)
Intensity (a.u.)
Intensity (a.u.)
A A
B B
180 175 170 165 160 180 175 170 165 160 180 175 170 165 160
(a) Chemical shift (ppm) Chemical shift (ppm) Chemical shift (ppm)
FIGURE 6.10 Results of characterization of SiV3-PLLA and V-PLLA. (a) 13C CP/MAS-NMR spectra of the
two samples and PLLA. The peaks further separated into two peaks, Peaks A and B. The intensity of Peak B
in the spectrum of V-PLLA is larger than that of SiV3-PLLA. This indicates that more carboxy groups formed
coordinate bonds with calcium ions in V-PLLA than SiV3-PLLA.
Membrane
FIGURE 6.12 Histology of in vivo response to the bilayered meshes. Villanueva Goldner stains show new
bone formed in the meshes at the center of holes 8 mm in diameter formed at frontal midline site of the rabbit
calvaria. Asterisks indicate mineralized tissues. New bone formation was observed over almost the entire area
of implanted Si-PVH/HA-mesh after 12 weeks.
(a)
(b)
FIGURE 7.10 Adhesive bond failure in case of insufficient cohesive properties of the mucoadhesive polymer:
(a) matrix tablets and (b) microspheres.
100
90
80 1
70 2 3
60
50
40 4
30
20
10
0
0 2 4 6 8 10
Time of irradiation (h)
FIGURE 11.5 The influence of UV irradiation on ultimate tensile strength of collagen and collagen/chitosan
films: (1) collagen; (2) 90/10 collagen/chitosan; (3) 70/30 collagen/chitosan; (4) 60/40 collagen/chitosan. Each
value is the means of 20 determinations and is reported with its standard deviation. (Reprinted from Polym.
Deg. Stab., 91, Sionkowska, A., Wisniewski, M., Skopinska, J., Poggi, G.F., Marsano, E., Maxwell, C.A., and
Wess, T.J., Thermal and mechanical properties of UV irradiated collagen/chitosan thin films, 3026–3032,
2006. Copyright 2006, with permission from Elsevier.)
FIGURE 11.7 Image of thin film based on collagen and elastin hydrolysates. (Reprinted from Appl. Surf. Sci.,
225, Skopinska-Wisniewska, J., Sionkowska, A., Kaminska, A., Kaznica, A., Jachimiak, R., and Drewa T.,
Surface properties of collagen/elastin based biomaterials for tissue regeneration, 8286–8292, 2009. Copyright
800.00 nm
2 2
4 4
6 6
8 8
(a) μm (b) μm
FIGURE 11.8 AFM images of: (a) nonirradiated film based on the blend of elastin/ collagen (25/75)
(b) the same film after 24 h of irradiation. (Reprinted from Appl. Surf. Sci., 225, Skopinska-Wisniewska, J.,
Sionkowska, A., Kaminska, A., Kaznica, A., Jachimiak, R., and Drewa T., Surface properties of collagen/elastin
based biomaterials for tissue regeneration, 8286–8292, 2009. Copyright 2009, with permission from Elsevier.)
5.00 150.0 nm
75.0 nm
0.0 nm
2.50

Scan size 5.000 μm
Scan rate 1.973 Hz
Image data Height
Data scale 150.0 nm
0
0 2.50 5.00
(a)
μm
5.00 150.0 nm
75.0 nm
0.0 nm
2.50

Scan size 5. 000 μm
Scan rate 1. 973 Hz
Image data Height
Data scale 150.0 nm
0
0 2.50 5.00
(b) μm
FIGURE 11.9 AFM images of (a) nonirradiated film based on the blend of chitosan/silkfibroin (50/50) (b)
the same film after 2 h of irradiation.
FIGURE 12.13 Examples of bare metallic stent and stent graft.
60.00
(nm)
0.00
0.20 0.80
0.40 0.60
0.60 0.40 60.00

(nm)
0.80 0.20
0.00
1000.00 × 1000.00 (nm) Z 0.00–60.00 (nm) 0.00
kshakeB-PEG
FIGURE 12.19 Scanning probe microscopic image of electrodeposited PEG to Ti surface.

Test material Test material
Test material
Agar
Extraction in Monolayer cells Insert with filter-bottom Monolayer cells Agar + cell-growth-medium
Monolayer cells
cell-growth-medium
Incubate cells with material extract Place well-insert above cells Incubate cells under agar
incubate with cell-growth-medium place test material on agar
Determine cell viability with Determine cell viability with Observe cell-death/cell viability
MTT assay MTT assay using microscope
LDH assay LDH assay
FIGURE 13.2 Overview of indirect-contact cytotoxicity testing. On the left is depicted cytotoxicity testing of
material extracts that are separately isolated by incubating a sample of the material in an appropriate extraction
medium. The middle part shows indirect incubation with the test material, the leachables can diffuse through
the filter, and the cells will detect cytotoxic components. On the right, the material and cells are separated by a
layer of soft agar through which the extractable compounds from the material can diffuse. A zone of cytotoxic-
ity around the material may appear since diffusion is limited.
Negative (nontoxic) Test material with
control unknown toxicity
F-actin+nuclei
Test material
Layer of cells Test material Isolated cells
Live
Dead
Incubation of material Incubate cells in direct
in direct contact with cells contact with material
Remove
test material
Fluorescence microscopy
Objective (microscope)
Cover slip
Sample (material with cells)
Microscopical evaluation Glass slide
vital staining
viability assay
FIGURE 13.3 Direct-contact testing in vitro of test materials of medical devices. Materials are either placed on
top of or act as a support for cells. After the appropriate incubation time, cells can be analyzed for cell viability,
morphology, or cell function. Fluorescent techniques are often used because of the opaque nature of most synthetic
materials. The cytoskeleton of attached cells can be visualized by staining with fluorescently labeled phalloidin
(right top panel). Cells can also be analyzed for viability or cell death (right middle and bottom panels, respectively).
Northern blotting
qRT-PCR
RNA isolation
Test material Isolated cells

Micro-array/DNA chip
Western blotting
Incubate cells in direct ELISA

contact with material
Protein isolation
Enzyme activity
Substrate
Product
FIGURE 13.7 Scheme depicting possible test for cell function in vitro. Cells in direct contact with the test
material can be analyzed for the presence of cell-type specific gene expression and protein production.
Blood cell Tissue
isolation homogenization Cell culture
Isolated cells in agarose
Cell lysis
alkaline unwinding
Electrophoresis
wash and stain
FIGURE 13.9 Alkaline comet assay for determining genotoxicity of materials. Isolated cells are incorporated
in an agarose gel, lysed, and the DNA denatured. Damaged DNA will migrate away from the original site of the
cell in an electric field, resulting in comet-like structures after staining of the DNA.
Biomaterial micro-array
Material
spots
Marrow
Mesenchymal
stem cells
Blood
interaction
Protein
Cultured
Material spot adsorption
cells
Cell adhesion Protein adsorption

Cell viability Protein activity/function
Cell proliferation Platelet adhesion/activation
Cell function Leukocyte adhesion/activation
Differentiation Blood coagulation
FIGURE 13.12 Scheme showing one possibility for high-throughput screening of biomaterials. Small spots
of the materials are deposited on a glass slide. Cells are cultured on these spots and can be analyzed for
viability, proliferation, or functionality. Alternatively, the materials can be challenged with proteins or body
fluids like blood, and the subsequent interaction analyzed.
B
Thermosetting
Thermoplastic
(a) B : Branch (b) : Crosslinking point
FIGURE 14.1 Schematic drawing of (a) thermoplastic and (b) thermosetting elastomer structure.
COOH COOH
CH2 CH2
CH CH2 CH CH2 CH CH CH2 CH2 CH2 CH3
CH2 CH2
CH2 CH2
CH2 CH2
CH2 CH2
COOH COOH
CH2 CH2
CH CH2 CH CH2 CH CH2
CH2 CH2
CH2 CH CH2 CH2 CH2 CH3
CH2 CH2
Acrylic polyacid
Ethylene-hexene grafted copolymer
grafted polyethylene
FIGURE 14.4 Monomers attached to existing chains to create branched or grafted copolymers.
Electrode
Resistor (50 kΩ)
OSC Sample
PC
OSC: Oscilloscope PC: Personal computer
FIGURE 15.2 A corona discharge setup. (Taken from Zhu, Y. et al., Polym. Degrad. Stab., 91, 1448, 2006c.)
Platelets
Platelets
Proteins Adsorption
Thrombin
Proteins
Thrombus
formation
Platelets
Fibrin matrix
FIGURE 15.6 An example where the nonspecific adsorption of proteins to a surface can lead to serious
problems is the thrombus formation. (Taken from Tirrell, M. et al., Surf. Sci. 500, 61, 2002.)
Syringe pump
High voltage (HV)
FIGURE 16.1 Schematic of a typical electrospinning setup.

(a) (b)
(c) (d)
FIGURE 16.2 Various collection systems for electrospinning. (a) Single collector; (b) Patterned Collector;
(c) Collection onto water with rotating mandrel; and (d) Collection across two parallel collectors.
Spinneret
Electrospun fibres
1 cm
FIGURE 16.3 Oriented electrospun PCL fibers collected using the gap method of alignment. The collectors
are spaced 10 cm apart, and the oriented fibers that collect in between are generated in approximately 30 s.
(Previously unpublished photograph.)
3D plotter
Electrospinning
Repetitive
process
Nanofiber
Hybrid Microfiber
structure
Electrospinning Direct polymer

molt deposition
Hybrid scaffold
No
(a) nanofiber matrix
Power supply
1 Hybrid
nanofiber matrix
Heating nozzle
Syringe 4 Layered 1 Layered
Collection woodpile shaped nanofiber matrices
microfiber scaffolds 5 mm
drum
Syringe pump
Oil circulator
(c) (b)
FIGURE 16.7 (a) Schematic of a hybrid process combining 3D extrusion and electrospinning. (Reproduced from Kim, G. et al., Macromol. Rapid Commun., 29, 1577, 2008.)
(b) Combining Electrospinning with a melt extrusion technique to create an electrospun carrier. (Reproduced from Park, S.H. et al., Acta Biomater., 4, 1198, 2008.)
(c) Combining melt electrospinning and solution electrospinning to create a bimodal scaffold. (From Kim, S.J. et al., Polymer 51, 1320, 2010.)
50 µm
FIGURE 16.8 RGD-modified electrospun fibers deposited onto reactive substrates that prevent adhesion of
cell—fibroblasts can only adhere onto the fibers. (Reproduced from Grafahrend, D. et al., Nat. Mater. 10, 67, 2011.)
(a) (b) (c)
Bridged
Failed-to-bridge
0 50 100 150 200 250 300 350 400

(a) (b) Gap between fibers (μm)
FIGURE 16.9 (a) Human dermal fibroblasts cultured on suspended aligned fibers at different distances apart.
(b) The effect of a gap between suspended fibers and whether cells are able to bridge the gap. (Reproduced from
Sun, T. et al., Biotechnology and Bioengineering, 97, 1318–1328, 2007.)
(a) (b)
(c) (d)
3.5
3
Extent of growth (mm)
2.5
1.5
0.5
0
DRGs alone On astrocytes
(e) (f )
FIGURE 16.10 Images showing DRGs grown on astrocytes and how the directionality of growth is affected
by the astrocytes. (a) and (b) are random electrospun meshes while the cells in (c) and (d) are grown on ori-
ented fibers. (e) shows how neurite processes change direction when encountering an astrocyte. (f) is a graph
showing that astrocytes increase neurite growth. Scale bars: a–d, 100 μm; e, 30 μm. (Reproduced from Chow,
W.N. et al., Neuron Glia Biol., 3, 119, 2007.)
1-film 1-film, NF160 (axons)
Axon core
Thin-film
Tube wall
(a) (b)
3-film 3-film, NF160
400 μm
(c) (d)
FIGURE 16.11 Nerve guides implanted within a peripheral nerve regeneration model, containing single (a&b)
or three (c&d) sheets of electrospun meshes. (Reproduced from Clements, I.P. et al., Biomaterials, 30, 3834, 2009.)
(a) (b) (c) (d)
(e)
rhBMP-2 release from alginate

80
Cumulative release (ng)
60
(f )
40
20
0
12 4 7 10 14 21
(g) Day
FIGURE 16.12 (a) SEM image of electrospun PCL. (b) Hollow electrospun tubular implants without perfo-
rations and (c) with perforations. (d) Scheme of implant in segmental bone defect. (e) Defect after placement of
a perforated mesh tube with alginate seen inside the tube through the perforations. (f) One week postsurgery
and alginate was present inside the defect, with hematoma present at the bone ends. (g) Alginate release kinet-
ics of the rhBMP-2 during the first 4 days.
100
80
Bacteria numbers,
% of control
60
40
20
Co
n
Ins trol
olu
So
0 lub ble
a Kr le
ori vor
a aft
sic
at gae
ato rin yxa lig
. ve car sy lim
nin
X E. Ps. . po
B
FIGURE 21.10 The influence of lignin on phytopathogenic microorganisms.

100
ZDEVD-FMK
87.5
Z-VAD-FMK
75
Apoptotic cells (%)
62.5
50
37.5
25
12.5
0
10 20 30 50 100
(b) Concentrations of inhibitors (µM)
FIGURE 21.11 (b) Apoptosis inhibition assay using caspase-3 inhibitor ZDEVD-FMK or pan-caspase inhibitor
Z-VAD-FMK. The apoptotic cell death was evaluated by Hoechst 33342 staining. Results are the mean ± SD of
three independent experiments. The percentage of apoptotic cells without the inhibitor is as 100%.
50 μm
(a) (b)
FIGURE 22.4 2.5% (w/v) Odex-III/CEC (ratio 5:5) hydrogels: (a) the photographic image and (b) the SEM
image (b); scale bar: 50 μm. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and
Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dex-
tran, 3905–3913. Copyright 2008, with permission from Elsevier.)
H
200 μm 200 μm
(a) (b)
FIGURE 22.9 Live/Dead™ staining of the cells encapsulated in the Odex-III/CEC hydrogel. (a) Day 3;
(b) 1 week. : live cells; : dead cells; *: spread out cells, H: hydrogel. (Reprinted from Biomaterials, 29, Weng, L.,
Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic, in situ gelable hydrogels composed of N-carboxyethyl
chitosan and oxidized dextran, 3905–3913. Copyright 2008, with permission from Elsevier.)
(a) (b)
FIGURE 22.10 Wound beds of mice covered by Tegaderm™. (a) PBS treated, and (b) Odex/CEC hydrogel
treated. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic,
in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dextran, 3905–3913. Copyright
Hydrogel residual
Hydrogel
residual
Granulation tissue
(a)
Re-
epithelialization
Granulation tissue
(b)
FIGURE 22.11 Acceleration of wound healing by Odex/CEC hydrogels in mouse full-thickness dermal
wounds. (a) A representative wound bed implanted with hydrogel with both robust epidermal hyperplasia and
granulation tissue formation. Part of the implanted hydrogel was fractured and neo-epidermal tissue appar-
ently developed transversely along the side of the hydrogel fragment. (b) A representative wound bed treated
with PBS (no hydrogel). Moderate epidermal hyperplasia and granulation tissue formation were observed.
The images were captured under 20 × magnification, whereas the insets were captured under 200 × magnifi-
cation. (Reprinted from Biomaterials, 29, Weng, L., Romanov, A., Rooney, J., and Chen, W., Non-cytotoxic,
in situ gelable hydrogels composed of N-carboxyethyl chitosan and oxidized dextran, 3905–3913. Copyright
(a) (b)
FIGURE 22.18 Cell attached to hydrogels (top view). (a) Day 1, and (b) day 3 after cell seeding. Cells were
stained with crystal violet. Scale bar: 20 μm. (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydro-
gels composed of partially oxidized hyaluronan and gelatin: In vitro and in vivo responses. J. Biomed. Mater.
Res. 2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
(a) (b)
(c) (d)
(e) (f )
FIGURE 22.19 Cross sections of cell-laden hydrogel (oHG-7): (a, b) 3 days, (c, d) 5 days, and (e, f) 5 days
after cell seeding. Samples are stained with crystal violet (a, c, e) and Live/Dead dye (b, d, f), respectively.
The demarcation line marked as: S: Hydrogel surface, I: Hydrogel interior, F: Fibroblasts, : dead cells (red),
*: live cells (green). Scale bar: 20 μm. (From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels
2008. 85A. 352–365. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission.)
(a) (b)
(c) (d)
(e) (f )
(g) (h)
FIGURE 22.23 Explanted hydrogels stained with H&E. (a, b) day 3, (c, d) day 7, (e, f) day 21 post-implantation
of hydrogels, (g) intact mouse skin, and (h) day 21 post-implantation of Vicryl™ suture. C: Collagenous capsule,
H: Hydrogel, D: Dermis, L: Loose connective tissue, : Macrophage, : Fibroblast, and *: Suture. Scale: 20 μm.
(From Weng, L., Pan, H., and Chen, W.: Self-crosslinkable hydrogels composed of partially oxidized hyaluronan
and gelatin: In vitro and in vivo responses. J. Biomed. Mater. Res. 2008. 85A. 352–365. Copyright Wiley-VCH
Verlag GmbH & Co. KGaA. Reprinted with permission.)
(a) (b)
(c)
FIGURE 22.24 Examination of cecal and abdominal wall tissues from rats 21 days after surgery: (a) An
example of a severe adhesion (score = 3) case: a control rat with adhesion of the cecum (CE) to the abdominal
wall (AW). The arrow indicates the area where the adhesion is p resent. Odex/CEC hydrogel remnants found on
both (b) the cecum, and (c) the abdominal wall of one animal subject. This animal received an adhesion score
of 1 due to some light, filmy adhesions of fat/omentum to the abdominal wall. The circled regions of (b) and
(c) highlight the presence of the hydrogel pieces while the arrows in (c) point out the light adhesions that formed
inside the rat abdomen after 21 days. (Reprinted from J. Surg. Res. 159, Falabella, C.A., Melendez, M.M.,
Weng, L., and Chen, W., Novel macromolecular crosslinking hydrogel to reduce intra-abdominal adhesions,
772–778. Copyright 2009, with permission from Elsevier.)
(a)
(b)
FIGURE 22.34 H&E staining of typical wounds after 3 days. The wound beds were (a) implanted with OG
hydrogel, and (b) implanted with GOG hydrogel containing GED-loaded microspheres formulated from 1%
GED solution. C: Fibrous tissue formed, I: Implant, L: Loose connective tissue, : Macrophage, : Fibroblast.
The images were captured under 20 × magnification. (Reprinted from Biomaterials, 29, Weng, L., Ivanova, N.D.,
Zakhaleva, J., and Chen, W., In vitro and in vivo suppression of cellular activity by guanidinoethyl disul-
fide released from hydrogel microspheres composed of partially oxidized hyaluronan and gelatin, 4149–4156.

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Structure and Function

Polymeric Biomaterials: Structure and Function, Volume 1

Polymeric Biomaterials: Medicinal and Pharmaceutical Applications, Volume 2

Founding Editor Editor

Boca Raton London New York

CRC Press is an imprint of the

© 2013 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4200-9471-8 (eBook - PDF)

and the CRC Press Web site at

Chapter 1 Synthesis and Fabrication of Polyesters as Biomaterials.............................................. 1

Chapter 2 Hydrogels Formed by Cross-Linked Poly(Vinyl Alcohol).......................................... 37

Chapter 3 Development and Evaluation of Poly(Vinyl Alcohol) Hydrogels as a

Chapter 4 Polyphosphazenes as Biomaterials.............................................................................. 83

Chapter 5 Biodegradable Polymers as Drug Carrier Systems................................................... 135

Chapter 6 Bioresorbable Hybrid Membranes for Bone Regeneration....................................... 177

Chapter 7 Mucoadhesive Polymers: Basics, Strategies, and Future Trends.............................. 193

Chapter 8 Biodegradable Polymeric/Ceramic Composite Scaffolds to Regenerate

Chapter 9 Amphiphilic Systems as Biomaterials Based on Chitin, Chitosan,

Chapter 10 Biomaterials of Natural Origin in Regenerative Medicine....................................... 271

Chapter 11 Natural Polymers as Components of Blends for Biomedical Applications...............309

Chapter 12 Metal–Polymer Composite Biomaterials.................................................................. 343

Chapter 13 Evolution of Current and Future Concepts of Biocompatibility Testing.................. 377

Chapter 14 Biocompatibility of Elastomers................................................................................. 415

Chapter 15 Preparation and Applications of Modulated Surface Energy Biomaterials.............. 495

Chapter 16 Electrospinning for Regenerative Medicine.............................................................. 539

Chapter 18 Polymeric Materials Obtained through Biocatalysis................................................ 617

Chapter 19 Polymer-Based Colloidal Aggregates as a New Class of Drug Delivery Systems.......659

Chapter 20 Photoresponsive Polymers for Control of Cell Bioassay Systems............................. 683

Chapter 21 Lignin in Biological Systems.................................................................................... 709

Chapter 22 Carbohydrate-Derived Self-Crosslinkable In Situ Gelable Hydrogels

Chapter 23 Dental and Maxillofacial Surgery Applications of Polymers................................... 783

Chapter 24 Biomaterials as Platforms for Topical Administration of Therapeutic Agents

Chapter 25 Polymers for Artificial Joints.................................................................................... 851

“Don’t cry because it’s over. Smile because it happened”.

Dominique Chauvel-Lebret Meng Deng

Jacques Desbrieres Catherine Gkioni

Florin Dan Irimie

Wahid Khan Philippe Lecomte

Masayuki Kyomoto Vijay Kumar Nandagiri

Juan Parra Alina Sionkowska

FIGURE 1.1 Main techniques for the synthesis of aliphatic polyesters.

1.2 STEP-GROWTH POLYMERIZATION

1.3 RING-OPENING POLYMERIZATION OF CYCLIC ESTERS

ε-Caprolactone Poly(ε-caprolactone) (PCL)

1.3.1 Anionic Polymerization

FIGURE 1.3 Anionic ring-opening polymerization of ɛCL.

FIGURE 1.4 Intramolecular transesterification reactions during the polymerization of ɛCL.

FIGURE 1.5 Intermolecular transesterification reactions during the polymerization of ɛCL.

FIGURE 1.6 Ring-opening polymerization of β-lactones by cleavage of the alkyl-oxygen bond.

FIGURE 1.7 Transfer reaction for the polymerization of β-lactones.

1.3.3 Metal-Free Ring-Opening Polymerization

FIGURE 1.9 Zwitterionic polymerization of pivalolactone.

FIGURE 1.13 Activation of cyclic esters by nucleophiles.

3 βBL PhCH2OH Nyce et al. (2003)

7 Ph Ph rac-lactide — Dove et al. (2006)

10 N Lactide Pyrenebutanol Pratt et al. (2006) and

TABLE 1.3 (continued)

16 Et Lactide PhEtOH Myers et al. (2002)

2.2.2 High Molecular Weight Molecules (PVA Macromers)