Review in Electrospinning 2

Progress in Polymer Science 35 (2010) 868892
Contents lists available at ScienceDirect
Progress in Polymer Science

journal homepage: www.elsevier.com/locate/ppolysci
Polymer nanobrous structures: Fabrication, biofunctionalization, and

cell interactions
Vince Beachley a , Xuejun Wen a,b,c,d,
a
b
c
d
Clemson-MUSC Bioengineering program; Department of Bioengineering, Clemson University, Charleston, SC 29425, USA
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425, USA
Department of Orthopedic Surgery, Medical university of South Carolina, Charleston, SC 29425, USA
The Institute for Advanced Materials and Nano Biomedicine (iNANO), Tongji University, Shanghai 200072, Peoples Republic of China
a r t i c l e
i n f o
Article history:
Received 23 July 2009
Received in revised form
26 November 2009
Accepted 11 March 2010
Available online 17 March 2010
Keywords:
Polymer
Nanober
Scaffold
Biomaterial
Tissue engineering
Regeneration
Electrospinning
Phase Separation
Self assembly
a b s t r a c t
Extracellular matrix bers (ECM) such as collagen, elastin, and keratin provide biological
and physical support for cell attachment, proliferation, migration, differentiation and ultimately cell fate. Therefore, ECM bers are an important component in tissue and organ
development and regeneration. Meanwhile, polymer nanobers could play the same critical role in tissue regeneration process. Fibrous structures can be fabricated from a variety
of materials and methods with diameters ranging throughout the size scale where cells can
sense individual bers (several nanometers to several microns). Polymer nanober scaffolds
can be designed in a way that predictably modulates a variety of important cell behaviors towards a desired overall function. The nanobrous topography itself, independent
of the ber material, has demonstrated the potential to modulate cell behaviors desirable
in tissue engineering such as: unidirectional alignment; increased viability, attachment,
and ECM production; guided migration; and controlled differentiation. The versatility of
polymer nanobers for functionalization with biomolecules opens the door to vast opportunities for the design of tissue engineering scaffolds with even greater control over cell
incorporation and function. Despite the promise of polymer nanobers as tissue engineering scaffolds there have been few clinically relevant successes because no single fabrication
technique currently combines control over structural arrangement, material composition,
and biofunctionalization, while maintaining reasonable cost and yield. Promising strategies
are currently being investigated to allow for the fabrication of optimal polymer nanober
tissue engineering scaffolds with the goal of treating damaged and degenerated tissues in
a clinical setting.
2009 Elsevier Ltd. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods of polymer nanober scaffold fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Electrospinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Self assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Phase separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author at: Clemson-MUSC Bioengineering Program, Department of Bioengineering, Clemson University, 173 Ashley Avenue,
CRI#605B, Charleston, SC 29425, USA.
E-mail address: xjwen@clemson.edu (X. Wen).
0079-6700/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2010.03.003
869
871
871
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3.
4.
5.
6.
7.
V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892
869
2.4.
Bacterial cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Templating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.
Drawing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.
Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.8.
Vapor-phase polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.9.
Kinetically controlled solution synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.10.
Conventional chemical oxidative polymerization of aniline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biofunctionalization of polymer nanobers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Natural ECM molecule nanobers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.4.
Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.5.
Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.6.
Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.7.
Laminin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.8.
Hyaluronic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Surface functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Physical absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2.
Covalent surface bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3.
Examples of biomolecule surface functionalized nanobers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Bulk biomolecule incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1.
Mixing biomolecules directly into a polymer solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2.
Co-axial incorporation (electrospinning) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3.
Incorporation of biofunctional peptide sequences (self assembly) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cell behavior on polymeric nanobers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Cell morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Cell alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Viability: survival/proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
ECM production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.
Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7.
Cell migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.8.
Potential mechanisms of cellnanostructure interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tissue engineering applications of polymer nanober scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cell incorporation into nanobrous electrospun scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Cell incorporation during fabrication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Cell population by migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1.
Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.2.
Scaffold permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.3.
Strategies to increase cell permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.4.
Layer by layer assembly of pre-seeded nanober sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
The brous component of the extracellular matrix
(ECM) in the tissue is made up of protein bers such as
collagens, elastin, keratin, laminins, bronectin and vitronectin. These protein bers provide structural support
to tissues and regulate many aspects of cell behavior. The
building blocks of protein nanobers are synthesized intracellularly by cells, and then secreted into the extracellular
space by exocytosis. Soluble precursors are enzymatically
modied within the ECM for formation into bers. ECM
bers provide structural support and mechanical integrity
to tissues as well as locations for cell adhesion and regulation of cell functions such as proliferation, shape, migration,
and differentiation [1]. Tissues requiring high levels of
mechanical strength such as tendons, ligaments, and bone,
have high levels of organized bers to impart mechanical

strength, while some soft tissues have much higher levels of unorganized bers. Composition and arrangement of
ECM bers in a tissue can impart ne tuned mechanical
properties. For example, arteries exhibit precise mechanical behavior characterized by highly elastic behavior at
low levels of distension and stiffer behaviors at high levels
of distention due to a highly organized microstructure of
elastin and collagen bers [2]. ECM bers also offer locations for cell adhesion and can regulate cell shape and
migration patterns based on composition and arrangement. Structural brous proteins can also act as storage
locations for the release of small bioactive peptides and
growth factors upon release by proteolytic cleavage.
The ECM in natural tissue is constantly being remodeled by proteolytic cleavage and cellular reassembly, and
870
Table 1
Comparison of methods for fabricating polymer nanober scaffolds.
Electrospinning
Advantages
Disadvantages
Easy to setup
Poor cell inltration into the core of

the scaffolds
2-Dimensional pore or
microstructure arrangement
Toxic solvents often used
Cost effective
High level of versatility allows
control over ber diameter,
microstructure and arrangement
Vast materials selection
Self Assembly
Easy incorporation of cells during

ber formation
3-Dimensional pore arrangement
Injectable for in vivo assembly
Complex procedure
Lack of control of ber orientation
and arrangement
Limited ber diameter 2-30 nm and
length 10 m
Phase Separation
3-Dimensional pore arrangement
Complex procedures
Lack of control of ber arrangement
Bacterial Cellulose
Low cost
High yield
Limited material selection

Lack of versatility for
functionalization
Templating

Control over ber diameter and
length
Sacricial materials
Limitation on ber dimensions and
arrangement
Drawing
Low productivity (One single ber at

a time)
Difcult to form bers with
consistent diameter
Simple procedure
Extraction
Natural materials

Limited control of ber diameter and
length (a few microns)
Vapor-Phase Polymerization
Polymer synthesized directly into

nanobers

length (hundreds of microns)
Complicated procedures
Kinetically controlled solution synthesis
Polymers synthesized directly into

nanobers

length (60 m)
Chemical Polymerization of Aniline
Polymers synthesized directly into

nanobers

length
accelerated ECM remodeling is critical during embryonic

development and during tissue regeneration. For example,
assembly of a brillar matrix from bronectin is crucial for
embryonic development and wound healing [3] and multiple ECM brous proteins, such as bronectin and laminin
are required for the process of branching morphogenesis
evident in the development of organs such as the kidney, lung, and prostate [4,5]. Remodeling in adult bone
tissue involves an initial assembly of collagen bers with
entrapped cells, followed by calcium salt precipitation on
the collagen bers to form bone. In the peripheral nervous
system after axonal degeneration following injury, regenerating axons grow back to their targets by following the
laminin and bronectin containing basal lamina tube that
surrounded the original nerve ber [6].
Tissue engineering strategies utilizing brous components attempt to ll the role of brous components in
natural tissue. Fibrous components of tissue engineering
scaffolds can impart mechanical strength, structure for cell

attachment, and act as reservoirs for biomolecule delivery
in much the same way as the natural brous components
of the ECM. For example, in many bone tissue engineering strategies a brous mesh is used as a template for
subsequent mineralization in a similar way to collagen
bers in natural remodeling. The fabricated matrix forms
a bonelike structure and provides an attractive microenvironment for osteogenic function in resident cells. Blood
vessel tissue engineering with nanobrous scaffolds benets from its properties of high tensile strength, good
cell attachment properties for endothelium formation, and
directional alignment for smooth muscle cells in the vessel wall. In vivo, injectable self-assembling nanobrous
networks introduced to injury sites have been used to provide a 3-dimensional structural environment that has been
shown to increase the inltration and retention of endogenous cells [7,8].
871
Fig. 1. (A) schematic (A) of a standard electrospinning setup (reproduced with permission from Year 2006 Dove Medical Press Ltd. [11]) and a scanning
electron microscope (SEM) image (B) of electrospun polyurethane nanobers.
2. Methods of polymer nanober scaffold

fabrication
Polymer nanobers have been fabricated using a number of different techniques. The methods of nanober
fabrication are varied and utilize physical, chemical, thermal, and electrostatic fabrication techniques. The methods
of polymer nanober fabrication most commonly associated with tissue engineering scaffolds in the literature
are electrospinning, self assembling peptide reactions, and
phase separation [911]. There are however several other
ways to fabricate polymer nanobers as well. Several of
these fabrication methods are briey described in the
following section and some of the advantages and disadvantages of each method are listed in Table 1.
2.1. Electrospinning
Electrospinning is an electrostatically driven method
of fabricating polymer nanobers. Nanobers are formed
from a liquid polymer solution or melt that is feed through
a capillary tube into a region of high electric eld. The electric eld is most commonly generated by connecting a high
voltage power source in the kilovolt range to the capillary
tip (Fig. 1). As electrostatic forces overcome the surface tension of the liquid, a Taylor cone is formed and a thin jet
is rapidly accelerated to a grounded or oppositely charged
collecting target. Instabilities in this jet cause violent whipping motions that elongate and thin the jet allowing the
evaporation of some of the solvent or cooling of melts to
form solid nanobers on the target site. Nanober size
and microstructure can be controlled by several processing parameters including: solution viscosity, voltage, feed
rate, solution conductivity, capillary-to-collector distance,
and orice size [12]. The electrospinning technique is very
versatile and a wide range of polymer and copolymer
materials with a wide range of ber diameters (several
nanometers to several microns) can be fabricated using
this technique. Many different types of molecules can be
easily incorporated during the electrospinning fabrication
process to produce functionalized nanobers. Electrospun
nanobers are usually collected from an electrospinning

jet as non-woven randomly or uniaxially aligned sheets or
arrays.
2.2. Self assembly
Self assembly is a process by which molecules organize and arrange themselves into patterns or structures
through non-covalent forces such as hydrogen bonding,
hydrophobic forces, and electrostatic reactions. Dialkyl
chain amphiphiles containing peptides were developed to
mimic the ECM. These peptide amphiphiles (PA), derived
from a collagen ligand, allow for a self assembling system
that consists of a hydrophobic tail group and a hydrophilic
head group (Fig. 2) [13]. The specic composition of amino
acid chains in peptide amphiphile systems determines the
assembly, chemical, and biological properties of the system, and therefore PA systems can be tailored to specic
applications [14,15]. Nanobers with diameters around
525 nm can be formed by the self assembly process, and
systems have been developed where nanober assembly can be induced by appropriate pH values [16,17].
Cells can be encapsulated in a nanobrous PA structure
if they are added during the self assembly process and
PAs can also be injected in vivo where they subsequently
self assemble into a nanobrous network. It has been
demonstrated that self assembled peptide nanobers can
spontaneously undergo reassembly back to a nanober
scaffold after destruction by sonication, and after multiple
cycles of destruction and reassembly, the peptide nanober
scaffolds were still indistiquishable from their original
structures [18]. While peptides are most commonly used
in self assembly of tissue engineering structures, synthetic polymer nanobers have also been fabricated by self
assembly [19,20].
2.3. Phase separation
Nanobrous foam materials have been fabricated by
a technique called thermally induced liquid-liquid phase
separation [22]. This fabrication procedure involves (a)
872
Fig. 2. Schematics of the (A) molecular structure and (B) nanostructure, and images of the (C) micro and macro structure of a self assembling peptideamphiphile nanober network (reproduced with permission from Year 2005 Wiley-VCH Verlag GmbH & Co. KGaA [21]).
Fig. 3. A schematic (A) of nanober formation by phase separation (reproduced with permission from Year 2005 World Scientic Publishing [23]), and an
SEM image (B) of nanobrous structure fabricated by this technique [22].
the dissolution of polymer in solvent (b) phase separation and polymer gelatination in low temperature (c)
solvent exchange by immersion in water and (d) freezing and freeze-drying (Fig. 3). The morphology of these
structures can be controlled by fabrication parameters
such as gelatination temperature and polymer concentration. Interconnected porous nanober networks have been
formed from polymers such as, poly-L-lactide acid (PLLA),
poly-lactic-co-glycolic acid (PLGA), and poly-DL-lactic acid
(PDLLA) with ber diameters from 50500 nm, and porosities up to 98.5%.
2.4. Bacterial cellulose

Cellulose nanobers produced by bacteria have been
long used in a variety of applications, including biomedical applications [24]. Cellulose synthesis by Acetobacter
involves the polymerization of glucose residues into chains,
followed by the extracellullar secretion, assembly and
crystallization of the chains into hierarchically composed
ribbons (Fig. 4). Networks of cellulose nanobers with
diameters less than 100 nm are readily produced, and bers
with different characteristics may be produced by different
Fig. 4. Schematic of Acetobacter cells depositing cellulose nanobers (A), and an SEM image of a cellulose nanober mesh produced by bacteria (B)
(reproduced with permission from Year 2007 American Chemical Society [24]).
873
Fig. 5. (A) Schematic of the fabrication of polymer nanobers using a nondestructive templating technique (grey: alumina template, green: resin, blue:
polymer nanobers, pink: silica replica template. (B) SEM images of 120 nm (B&C) and 1 m (D&E) polymer bers fabricated by the above technique
(reproduced with permission from Year 2008 American Chemical Society [27]).
strains of bacteria [24]. Copolymers have been produced

by adding polymers to the growth media of the cellulose
producing bacteria [25,26].
2.5. Templating
Polymer nanobers can be fabricated using templates
such as self-ordered porous alumina. Alumina network
templates with pore diameters from 25 to 400 nm, and
pore depths from around 100 nm to several 100 m have
been be fabricated. Polymer nanober arrays can be
released from these molds by destruction of the molds
or mechanical detachment (Fig. 5) [27,28]. The length of
polycaprolactone (PCL) nanobers fabricated from alumina
templates can be controlled as a function of parameters
such as melt time and temperature [29].
2.6. Drawing
Nanobers can be mechanically drawn from viscous
polymer liquids directly [30]. In one example, nanobers
were drawn directly when a rod was placed in a polymer
melt and moved up forming a thin lament that cooled to
form a nanober (Fig. 6). This method was used to fabricate
poly(trimethylene terephthalate) nanobers with diameters as low as 60 nm, and lengths up to 500 mm [31]. An
automated drawing technique utilized a pipette dispensing
liquid polymer solution while intermittently contacting a
substrate and moving the x-y direction across the substrate
[32]. This process allowed the formation of thin suspended
nanobers connecting droplet shaped dots on the substrate. This technique was used to fabricate polystyrene
nanobers with diameters ranging from tens nanometers
to several microns in highly ordered patterns.
2.7. Extraction
Nanobers can be extracted from natural materials
using chemical and mechanical treatments. Cellulose brils
can be disintegrated from plant cell walls. In one example, cellulose nanobers were extracted from wheat straw
and soy hull with diameters ranging from 10 to 120 nm
and lengths up to a few thousand nanometers (Fig. 7)
[33]. Invertebrates have also been used as a source for the
extraction of nanobers. Chitin nanobers 3-4 nm in diameter and a few micrometers in length were extracted from
squid pen and Poly-N-acetyl glucosamine nanobers isolated from a marine diatom demonstrated prothrombotic
interactions with red blood cells [34,35].
2.8. Vapor-phase polymerization
Polymer nanobers have also been fabricated from
vapor-phase polymerization. Plasma-induced polymerization of vapor phase vinyltrichlorosilane produced
organosiloxane bers with diameters around 25 nm and
typical lengths of 400600 nm and cyanoacrylate bers
with diameters from 100 to 400 nm and lengths of hundreds of microns (Fig. 8) [37,38].
2.9. Kinetically controlled solution synthesis
Nanobers and nanowires have been fabricated in
solution using linear aligned substrates as templating
agents such as iron-cation absorbed reverse cylindrical
micelles and silver nanoparticles [39]. Poly(vinyl alchohol)poly(methyl methacrylate) nanobers were fabricated
using silver nanoparticle that were linearly aligned in solution by vigorous magnetic stirring (Fig. 9) [40]. These
874
Fig. 6. (A) Schematic of nanober fabrication by the drawing technique. (B) Transmission electron microscope (TEM) image of a polymer nanober fabricated
using the drawing technique (reproduced with permission from Year 2008 The Optical Society [31]). .
nanoparticle chain assemblies acted as a template for further polymerization of nanobers with diameters from 10
to 30 nm and lengths up to 60 m.
can be utilized to fabricate tissue engineering scaffolds that

guide cells toward desired behavior and function.
3.1. Natural ECM molecule nanobers
2.10. Conventional chemical oxidative polymerization of

aniline
Chemical oxidative polymerization of aniline is a traditional method for synthesizing polyaniline and during the
early stages of this synthesis process polyaniline nanobers
are formed (Fig. 10). Optimization of polymerization conditions such as temperature, mixing speed, and mechanical
agitation allows the end stage formation of polyaniline
nanobers with diameters in the range of 30120 nm
[41,42].
Molecules that are naturally occurring in the extracellular matrix are ideal materials for cell attachment, survival,
proliferation, and differentiation. In addition, substrate
interactions between cells and ECM molecules may modulate certain cell functions. Biofunctional nanobers can be
directly fabricated from natural ECM materials or synthetic
polymers can be blended with natural ECM molecules to
form copolymer bers. Blended nanobers generally benet from improved physical properties due to the synthetic
polymer component and improved bioactivity due to the
natural ECM component [43,44].
3. Biofunctionalization of polymer nanobers

Polymer nanobers are excellent structures for the
design of tissue engineering scaffolds because of the wide
variety of biocompatible polymer materials that can be
formed into nanobrous structures. Different types of biocompatible polymers demonstrate a variety of mechanical
properties, degradation rates, and cell-material interactions, and new types of polymers are continuously being
synthesized. In addition to the vast material selection
available, one of the most favorable characteristics of polymer materials in tissue engineering scaffolds are their
suitability for biofunctionalization. Biofunctional polymer
nanobers can be fabricated entirely from materials found
in the ECM, and a variety of biomolecules and drugs
can be incorporated into polymer nanobers during the
fabrication process or by using post processing surface
modication techniques. Biofunctionalization techniques
3.1.1. Collagen
Pure collagen nanobers are commonly electrospun
from solutions of soluble type I collagen dissolved
in organic solvents such as 1,1,1,3,3,3-hexauoro-2propanol (HFIP) or aqueous acids. Bead free pure
collagen nanobers have been fabricated with diameters ranging from 100 to 500 nm [45,46]. Water soluble
collagen nanobers must be crosslinked before use in
cell culture with crosslinking agents, such as glutaraldehyde vapor, 1,6-diisocyanatohexane, genipin, 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC), and so on [45,47,48]. Nanober blends containing
collagen and synthetic polymers such as PCL are also easily
fabricated by electrospinning when both materials are dissolved in the same solvents [49]. In nanobers electrospun
from single solvent solutions containing collagen and PCL,
collagen was well dispersed as small spherical aggregates
Fig. 7. Images of natural wheat straw [36], wheat straw microbers [33] after chemical treatment and wheat straw nanobers [33] after chemical treatment.
875
Fig. 8. (A) Schematic describing a proposed mechanism for nanober formation by vapor-phase polymerization (B) Arial (1) and side views (2) of polymer
nanobers fabricated from vapor-phase polymerization at high (B), intermediate (C) and low (D) packing densities [37]. (Reproduced with permission from
Year 2007 American Chemical Society [37]).
at low concentrations (10 wt%) and as much larger irregular

shapes at higher concentrations (50 wt%) [50]. Cells cultured on synthetic nanobers blended with collagen have
shown better attachment, growth, and ECM production
than bers without collagen incorporation [51,52]. Blends
allow for the fabrication of collagen containing nanobers
with a greater range of mechanical properties and ber
diameters. It has been shown that electrospinning collagen
from organic solvents causes extensive denaturation and
it was suggested that similar bioactivity may be obtained
using gelatin in these cases [53]. However, several studies directly comparing electrospun collagen and gelatin
nanobers indicate that electrospun collagen nanobers
may retain some favorable bioactivity when compared to
gelatin nanobers despite solvent denaturation [51,54,55].
3.1.2. Gelatin
Gelatin nanobers have been electrospun by dissolution
in organic solvents such as HFIP and 2,2,2-triuoroethanol
(TFE) or aqueous acids. Pure gelatin nanobers have been
electrospun with ber diameters around 50 to 500 nm
[45,56]. Gelatin nanobers are crosslinked before use
in cell culture with crosslinking agents, such as glutaraldehyde vapor, 1,6-diisocyanatohexane, genipin, EDC,
and so on [45,48]. Gelatin nanober blends can also be
electrospun by combination of gelatin and other polymers
in one solution with a variety of ber diameters. Cells
attached and proliferated better on synthetic bers when
they were blended with gelatin [51,5759]. Increases in
cell attachment and proliferation have been showed to be
a function of the ratio of gelatin in the ber blends [44].
Fig. 9. (A) Schematic of silver nanoparticle embedded polymer nanobers fabrication (B) SEM and TEM images of silver nanoparticle embedded polymer
nanobers (reproduced with permission from Year 2006 Royal Society of Chemistry [40]).
876
Fig. 10. Schematic showing the nucleation of polyaniline nanobers. (I) Under non-ideal nucleation conditions aggregate formation is present. (II) When
ideal nucleation conditions are predominant, well-dispersed polyaniline nanobers are formed. Typical images of the reaction vials and microstructure are
displayed next to the schematic (reproduced with permission from Year 2009 American Chemical Society [42]).
PCL bers blended with gelatin also enhanced nerve differentiation as compared to plain PCL nanobrous scaffolds
[57].
3.1.3. Elastin
Alpha-elastin and tropoelastin bers have been electrospun from HFIP solutions and aqueous acids with diameters
ranging from 1 to several microns [45,46,48]. Electrospun
elastin bers have been observed to possess a ribbon like
morphology as opposed to the uniform cross-section lament shape most common in other electrospun bers.
Elastin nanobers must be crosslinked before use in cell
culture with various crosslinking agents such as glutaraldehyde vapor, 1,6-diisocyanatohexane, EDC, and so
on [45,46,48]. Elastin ber blends with collagen and synthetic materials have also been successfully electrospun
[45,60].
3.1.4. Chitosan
Pure chitosan nanobers are difcult to electrospin due to limited solubility, ionic character and
three-dimensional networks of strong hydrogen bonds
[61]. High molecular weight chitosan nanobers have
however been successfully electrospun using high concentration acid solutions (diameter = 130 nm) and organic
solvents (diameter = 60330 nm) [6163]. Chitosan can
be mixed with other polymers in organic solvent
solutions such as HFIP or acid solutions to more easily form blended nanobers [43]. Chitosan nanobers
have been electrospun as blends with very low concentrations of polyethylene oxide (PEO) (10%) with
diameters from 150 to 200 nm [64]. Chitosan/cellulose
blends have also been produced by bacteria fabrication with chitosan concentrations of 710% dry weight
[26]. Nanober scaffolds containing chitosan have demon-
strated favorable properties as tissue engineering constructs [64].

3.1.5. Dextran
Dextran bers with a wide range of ber diameters
have been electrospun from aqueous solutions and organic
solvents such as dimethylformamide (DMF) and dimethyl
sulfoxide (DMSO) mixtures [65]. Dextran is highly soluble in an aqueous environment, but methacrylated dextran
nanobers photocrosslinked after electrospinning formed
stable hydrogels in the aqueous environment [65]. Blended
PLGA/dextran nanobers have also been fabricated and
have demonstrated favorable tissue engineering properties
[66].
3.1.6. Fibrinogen
Fibrinogen nanobers with diameters from 80 to
700 nm have been electrospun from solutions of brinogen
dissolved in HFIP and 10 minimal essential medium (9:1)
[67]. Fibrinogen nanober scaffolds were able to maintain
their structure when hydrated without crosslinking and
demonstrated good interactions with cells in vitro culture
conditions [68,69]. The protease inhibitor, aprotinin can be
added to cell culture media to slow down the degradation
rate of brinogen nanobers [68,69].
3.1.7. Laminin
Laminin puried from EngelbrethHolmSwarm (EHS)
tumor was solubilized in HISP and electrospun into 90 to
300 nm bers with various amounts of bead composition
[70]. Bioactivity of laminin nanobers was conrmed by
the in vitro behavior of PC12 and human adipose stem cells.
Laminin nanobers were able retain their structure under
hydrated conditions without crosslinking.
3.1.8. Hyaluronic acid

Hyaluronic acid nanobers are very difcult to fabricate,
but a combination procedure involving electrospinning from an acidic solution with heated air blown
around the electrospining jet was developed to obtain
uniform bers [71]. Pure hyaluronic acid nanobers
(diameter = 110 nm) were also fabricated by electrospinning hyaluronic acid:PEO blended nanobers followed
by extraction of the PEO component of the bers
[72,73]. Scaffolds assembled from these hyaluronic acid
nanobers demonstrated good cell interaction properties
in vitro.
3.2. Surface functionalization
One of the major advantages of polymeric materials
in tissue engineering scaffold design is vast versatility for
surface modications. Biomolecules can be absorbed or
chemically bonded to the surface of polymer nanobers
in order to modulate specic cell functions in tissue
engineering. In this review only surface functionalization
techniques that have previously been carried out with polymer nanober structures are included, but in theory any
method of polymer surface modication may be applicable
to polymer nanobers as long as some part of the process
does not destroy the sometimes fragile nanober structures.
3.2.1. Physical absorption
The simplest method for surface biofunctionalization
of polymer nanobers with biomolecules is to incubate
nanober meshes with solutions containing solubilized
biomolecules to allow for physical absorption of the
biomolecules onto the nanober surfaces. Biomolecules
may adhere to the surface of the nanobers because of
interactions such as Van der Waals force, electrostatic
forces, hydrophobic interactions, and hydrogen bonding.
The efciency of physical adsorption onto hydrophobic
nanober scaffolds can be increased by treatment with
air plasma to make them more hydrophilic and allow
greater inltration of aqueous solutions containing water
soluble biomolecules. Plasma treatment can also be used
to improve the hydrophilicity of nanober meshes for
improved covalent grafting modications explained in the
following section [74]. The efciency of plasma treatment
may be improved by placing a sample at a distance downstream of the plasma source to allow surface modication
while minimizing damages due to etching and ablation
[75].
3.2.2. Covalent surface bonding
Covalent surface bonding can be used to chemically
bond biomolecules to exposed functional groups directly
on the nanober surface. Covalent surface bonding provides a much more efcient coating and greater long
term retention of biomolecules [76]. Covalent coating of
biomolecules on nanobers usually involves at least two
requirements: (1) exposed functional groups on the ber
surface, (2) covalent bonding of biomolecules to these functional groups.
877
3.2.2.1. Exposing functional groups. Depending on the

molecular conguration of the specic polymer requiring
modication, functional groups may already be present, or
can be added to the surface by various chemical treatments.
The functional groups most commonly used in polymer
nanober surface modication reactions in the literature
are carboxyl and amine groups. Carboxyl groups can be
exposed on the surface of nanobers made from materials,
such as PLLA and PCL, by treatment with chemicals such
as sodium hydroxide (NaOH) [77,78]. Amine groups were
grafted on the surface of degradable polyester nanobers
by treatment with 1,6-hexanediamine/propanol solution
or ethylenediamine (ED) [79,80]. When the desired functional groups cannot be easily grafted directly on the
polymer nanober surface then a linker molecule with the
desired exposed functional groups can be tethered to the
ber surface. Acrylic acid in the gas phase was grafted
to various polymer nanobers as a plasma treatment
to add carboxyl groups [81]. An additional di-aminopoly(ethylene glycol) (di-NH2 -PEG) was used as a linker
molecule to add functional amine groups to polyester
nanobers that had previously been soaked in NaOH
to expose carboxylic acid groups [82]. Exposed carboxyl
groups were added to polyethersulfone (PES) nanobers
by photo polymerization grafting of poly (acrylic acid) as
a linker molecule [83]. When adding linker molecules to
the surface of polymer nanobers it may be important to
consider the properties of the specic linker molecules
used because several research groups have shown that
linker properties such as length have an effect on the cell
response to functionalized polymer nanobers [8385].
Polymer blends and modied polymers can also be synthesized to include desired exposed functional groups on their
structure. A PCL-PEG block copolymer was synthesized
with functional amine groups on the surface via PEG linkers
and electrospun into nanobers that were further functionalized [86]. A copolymer of methyl methacrylate (MMA)
and acrylic acid (AA) was synthesized and electrospun
into nanobers with different carboxyl group contents by
varying the ratio of MMA to AA [87]. In another case PLGAb-PEG-NH2 di-block copolymer was directly blended with
PLGA in organic solvents and electrospun to form PLGA
nanobers with exposed functional amino groups [88].
3.2.2.2. Covalently attaching biomolecules to functional

groups. Covalently attaching biomolecules to polymer
nanober surfaces may require the activation of exposed
functional groups on the polymer surface, the biomolecules
or both. Carboxyl groups can be activated for reaction
with primary amines by treatment with 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC).
This method of activation is commonly referenced in the
literature for bonding biomolecules to polymer nanobers
[55,74,75]. The efciency of EDC initiated bonding can be
improved by adding N-hydroxysuccinimide (NHS), which
converts an unstable amine-reactive intermediate formed
by the EDC reaction into an amine-reactive NHS ester
[55,74]. Glutaraldehyde, and 1-hydroxybenzole (HOBt)
with EDC have been used to attach biomolecules to exposed
amine groups on polymer nanobers [79,86].
878
3.2.3. Examples of biomolecule surface functionalized

nanobers
A variety of biomolecules have been attached to
polymer nanobers to increase their bioactivities. Many
research groups have used the physical absorption method
to coat nanobers with nanocrystalline apatites by immersion in simulated body uid (SBF), which is an aqueous
solution that contains ions in concentrations similar to
those of human body plasma [89]. The degree of coating
can be controlled by incubation time and SBF solution composition [77,78]. Hydroxyapatite coatings stimulate the
expression of osteogenic genes by osteoblastic cells when
compared to uncoated scaffolds [78]. The physical absorption method has also been used to coat synthetic nanobers
with biomolecules such as collagen, laminin, E-selection,
and many others and successful biomolecule incorporation
is reected in enhanced cell spreading, viability, attachment, and phenotype preservation [76,90,91]. The physical
absorption method has the advantage of being a very simple and gentle procedure. Modications preformed by this
method limit damage to fragile nanober structures and
biomolecules, but binding can be relatively weak.
Many biomolecules have been covalently bonded to the
surface of polymer nanober scaffolds as well. Covalent
attachment of collagen and laminin to polymer nanobers
enhanced the suitability of the nanobers as neural tissue engineering scaffolds [76,87]. Collagen synthesis by
esophageal epithelial cells was increased by covalent bonding of bronectin to nanobers, and covalent attachment of
an RGD peptide enhanced the attachment and proliferation
of broblasts [79,88]. When compared to physical absorption, covalent bonding provides a more efcient and stable
surface attachment of biomolecules. The major disadvantages of covalent bonding are more complex procedures
and the potential use of harsh conditions that may limit the
types of biomolecules that can be incorporated. For example, the organic solvents used in some covalent binding
procedures may deactivate biomolecules such as growth
factors.
Combinational techniques to utilize both covalent
bonding and physical absorption can also be used to biofunctionalize polymer nanober surfaces. The physical
absorption of hydroxyapatite coatings with SBF can be
made more efcient if carboxyl groups are exposed by
NaOH prior to incubation [77,78]. Growth factors can be
bound to polymer nanobers using a combinational technique where molecules such as heparin are covalently
bonded to act as reservoirs that stabilize, store, and protect
growth factors introduced by physical absorption [55,82].
This technique allows for strong, stable covalent binding
of heparin to the nanober surface, without exposing the
growth factors to harsh conditions that could reduce their
biofunctionality.
3.3. Bulk biomolecule incorporation
In addition to surface functionalization, biomolecules
can also be incorporated into the bulk material of
nanobers during the fabrication process. The three most
common methods of bulk biomolecule incorporation into
polymer nanobers are direct mixing into a polymer
solution, co-axial electrospinning, and insertion of biofunctional peptide sequences into peptide amphiphile
molecules for self assembly. It has been shown that
the direct incorporation technique can allow for greater
amounts of biomolecule incorporation and improved
bioactivity when compared to surface modication techniques [76]. In addition, biomolecules incorporated by this
technique are embedded into the bulk material of the bers
and can facilitate extended release of biomolecules by diffusion through the nanobers or release by degradation
of the nanobers in the case of biodegradable materials.
The release kinetics of biomolecules contained in polymer
nanobers commonly involves an initial burst followed by
steady extended release.
3.3.1. Mixing biomolecules directly into a polymer
solution
Many biomolecules can be directly incorporated into
a polymer solution during fabrication. This method of
biomolecule incorporation can be employed for nanober
fabrication techniques that utilize a polymer solution such
as electrospinning, phase separation, templating, drawing,
and so on, most notably electrospinning. Biomolecules may
be directly dissolved in a polymer solution if a common
solvent is available [92], dissolved in a miscible solvent
and incorporated as a suspension [9294], or dissolved in
an immiscible solvent and incorporated as an emulsion
with agitation [9597]. When two miscible solvents for a
particular polymer and biomolecule solution are not available, a multi-component system may be used. For example
a three component solution containing dichloromethane
(DCM), methanol, and water was used to incorporate
water soluble heparin into DCM soluble PCL nanobers
[98]. Heparin was dissolved in water, which is miscible
in methanol, which is miscible in DCM. Electrospinning
of emulsions formed by stirring, vortexing, or sonication
resulted in beadlike pockets containing an aqueous phase
or even a core-shell structure [99101]. Inclusion of miscible solvents and emulsions may however disturb the
electrospinning process and make ber formation more
difcult due to inclusion of multiple phases in the jet
[95]. Distribution of a biomolecule in a single phase solution was found to be even thoughout the nanober, while
biomolecule distribution was aggregated for a two phase
system [92,97]. However, even biomolecule distribution
has also been observed in nanobers electrospun from miscible solvent suspensions [98]. Reported efcacy of direct
biomolecule incorporation in electrospun nanobers varies
greatly. This should be expected due to the high level of
variations in material selection and electrospinning parameters reported in the literature. Heparin electrospun as a
miscible suspension had an efciency of around 100% [98],
laminin directly blended in organic solvent with PLLA had
an efciency of around 75% [76], and nerve growth factor (NGF) electrospun into nanobers from an emulsion
had a reported efciency of just 2.5% [95]. Incorporation
of biomolecules can have an effect on the morphology
of electrospun nanobers because biomolecule incorporation can change the solution properties such as viscosity
and charge density [92,93,98]. For example, ber diameter decreased with increasing loading levels of retinoic
879
Fig. 11. Deagglomeration of nanoparticles during stages of continuous processing in twin screw extrusion electrospinning apparatus (scale bar is 20 m
[103].
acid, while ber diameter increased with increased loading

levels of bovine serum albumin (BSA) [92]. Incorporation
of biomolecules can also have an effect on the mechanical properties of nanobers because of the inclusion of
aggregates. Increased loading levels of BSA were observed
to decrease the ductility of PCL and ethyl ethylene phosphate nanobers, while the presence of retinoic acid
increased the strength of the nanobers independent of
the ber diameter [92]. A variety of biomolecules have
been directly incorporated into electrospun nanobers.
NGF was mixed with BSA as a carrier protein and electrospun from an emulsion with sustained release up to
3 months after a 20% burst effect and retention of at
least some degree of bioactivity [95]. Antibiotics have also
been incorporated into electrospun polymer nanobers
with sustained release and bioactivity retention [93]. DNA
has also been successfully incorporated into electrospun
nanobers with a sustained release of intact DNA capable
to cellular transfection and protein encoding [94]. Vitamins were also loaded into electropsun polymer nanobers
with expended release [102]. Nanoparticles have also been
loaded directly into polymer solutions and electrospun
as dispersions [103]. A special extruder (Fig. 11) used
to feed polymer solution/nanoparticle dispersions into
an electrospinning capillary helped to break up particle
aggregates and maintain a uniform solution composition

[103].
3.3.2. Co-axial incorporation (electrospinning)
An alternative approach to directly mixing biomolecules
in an electrospinning solution is to use a special co-axial
nozzle, which allows the formation of core-shell nanobers
where one material makes up a core lament that is surrounded by another material in a concentric geometry
[104]. Multiple solutions are feed though a special nozzle
in a concentric geometry to form a two phase liquid electrospun jet where a solution at the center is surrounded
by an outer solution, forming solid core shell nanobers
upon solvent evaporation. Hollow tube nanobers have
also been electrospun using the co-axial technique [105].
Core shell nanobers offer many attractive properties in
tissue engineering applications, especially in biomolecule
incorporation. Biomolecules incorporated inside of core
shell nanober are not affected by initial burst and they do
not require extended contact with any toxic solvents used
in the formation of the shell materials [106]. The ow rate
of each component of the nanober can be independently
controlled to vary the overall diameter of the bers and the
core to shell volume ratio. Coaxial electrospinning can also
be used to electrospin materials that cannot be electrospun
880
using conventional methods. For example, nanobers were

formed from poly(glycerol sebacate), a material that cannot
be electrospun directly, by encapsulating its precursors in
a constraining shell material that was removed after curing
[107].
3.3.3. Incorporation of biofunctional peptide sequences
(self assembly)
Biofunctional peptide sequences can be directly incorporated into peptide amphiphile nanober scaffolds. Biofunctional sequences are inserted into peptide amphiphile
molecules and these biofunctional sequences are presented
to cells on the nanober surface after self assembly. In one
example, a peptide sequence called RADA16 was mixed
with modied RADA16 sequences directly coupled with
various bioactive motifs to promote cell adhesion and
osteogenic functions [108]. Compared to pure RADA16
scaffolds, scaffolds containing bioactive motifs increased
cell proliferation and markers of osteogenic differentiation. Incorporation of a designed chemokine similar
to stromal cell-derived factor-1 improved cardiac function after myocardial infarction [109] and cell adhesion,
differentiation and bone marrow homing motifs added
to RADA16 sequences improved neural cell survival in
nanober scaffolds [110]. A peptide amphiphile nanober
scaffold with the incorporation of the pentapeptide epitope isolucinelysine-valine-alanine-valine (IKVAV), known
to promote neurite sprouting and to direct neurite growth
was designed as a tissue engineering scaffold specically
for neuronal cells [15]. The effect of IKVAV incorporation in
the peptide nanobers was demonstrated by the superior
bioactive properties of these scaffolds versus controls without the IKVAV sequence. The arrangement of biofunctional
peptide sequences can have an effect on their bioactivity
in nanobrous scaffolds. RGD peptide sequences incorporated into self assembling peptide scaffolds had better cell
attachment properties when the motif was displayed at
a branched site than with linear incorporations of RDG
because of increased motif availability at the nanober surface [111].
4. Cell behavior on polymeric nanobers
Cells in their natural in vivo surroundings are exposed
to a complex chemical and structural environment. The
natural extracellular matrix is made up of structural components that are of nanoscale dimensions. Major brous
extracellular molecules such as collagen bers, elastin
bers, keratin bers, etc. have nano-scale diameters. It is
highly important to mimic the natural environment when
culturing cells in vitro because cell behavior is determined
by both genetic make up and the surrounding environmental cues. Cellular behaviors such as proliferation,
differentiation, morphology, and migration are commonly
controlled in culture by modulation of the chemical environment. Cells also respond to different morphological cues
that can be determined by the growth substrate in vitro and
in vivo. Four components may be involved in the growth,
differentiation, functions and morphology of cells on biomaterial surfaces: (1) adsorption of serum components
(2) extracellular matrix components secreted by the cell
(3) cell adhesion molecules and (4) cytoskeleton mechanics [112]. It has been shown that the structural substrate
property of surface roughness can cause selective protein
absorption, and that higher surface roughness increases
total protein absorption [113]. In this case, increased protein adsorption could be attributed to an increase in surface
area for rough surfaces and thus could be important in relation to nanotopographical materials because these exhibit
extremely high surface area. In relation to nanostructure and cell interactions, the cytoskeletal mechanics is
of importance because cells cultured on substrates with
nanoscale features can take on different shapes in response
to the specic features that are encountered.
Nanotopography can affect cellular behavior through
known mechanisms such as the regulation of cell shape
and surface protein absorption properties, but it is possible
that there are unknown effects associated with nanotopographies as well. Cells can react to objects as small as
5 nm [114] and it is possible that nanostructures, especially those with similar dimensions to the natural ECM,
can inuence cell behavior through unknown mechanisms. It has been shown that cell behavior can be highly
dependent on the substrate that they are cultured on,
and the understanding of cell substrate interactions with
nanostructures could provide valuable information that
would allow for the design of better tissue engineering
scaffolds. Nanobrous scaffolds present nanostructured
features, mimic the brous components of natural tissue, allow three-dimensional congurations, and provide
a unique mode of presentation of chemical and biological
cues to cells. In order to gain a better understanding of the
effect that nanobrous architecture has on cell behavior,
experimental results have been summarized according cell
morphology, alignment, attachment, viability, ECM production, differentiation, and migration.
4.1. Cell morphology
Cell morphology can be inuenced by the substrate that
a cell is attached to. Cells many times adopt a different
morphology on nanobrous substrates compared to at
substrates, and cell morphology on nanobrous substrates
can be inuenced by the ber diameter. Cell morphology
is commonly described by the projected area/degree of
spreading or the aspect ratio (ratio of the long and short
axis). Cell morphology is an important characteristic in tissue engineering scaffold design because of its signicance
in controlling cell arrangement and the translational effects
that cell morphology have on other cell functions.
Cells may adopt a more rounded shape with a smaller
projected area when cultured on nanobers as opposed
to at surfaces. Osteoprogenitor cells cultured on electrospun polymer ber meshes with diameters of 140 and
2100 nm displayed signicantly smaller projected areas
than cells cultured on smooth surfaces [115], and fetal
bovine chondrocytes cultured on nanobrous scaffolds
maintained a round or spindle-like shape in contrast to
a at well spread morphology observed on tissue culture
plate [116]. Rounded morphologies may correspond with
lack of organized actin bers compared to cells with more
well spread morphologies [116]. Chondrocytes grown on
polymer nanobers (500900 nm) also adopted a rounded

shape with a disorganized actin cytoskeleton in contrast
to cells grown on 15 m microbers, which adopted a well
spread shape [117]. Because fteen microns approaches the
size of a cell, they may react to these bers in a manner
similar to a at substrate in the previous example.
Cell also may adopt a more elongated shape on
nanobers of certain diameters. Osteoprogenitor cells cultured on 140 nm bers adopted a similar aspect ratio to
those on smooth surfaces, but cells on 2100 nm bers had
a statistically higher aspect ratio [115]. In agreement with
this result, dermal broblasts had statistically signicant
increase in aspect ratio for ber diameters of 8640 and
970 nm versus at controls, but not for ber diameters
of 160 and 650 nm [118]. Human umbilical vein endothelial cells also showed enhanced elongated cell morphology
on larger bers (15 m) than smaller nanober scaffolds (2001000 nm and 10200 nm) [119]. This elongating
effect could be due to cells growing along single bers,
as it would be logical that cells would be more likely to
grow along single bers in nanober meshes with larger
diameter bers.
4.2. Cell alignment
One of the most important cell morphologies associated with tissue engineering is elongated unidirectional
cell alignment. Many tissues such as nerve, skeletal and
cardiac muscle, tendon, ligament, and blood vessels contain cells oriented in a highly aligned arrangement, thus it
is desirable that scaffolds designed for these tissue types
are able to induce aligned cell arrangements. It is well documented that cells adopt a linear orientation on aligned
substrates such as grooves and bers. Aligned nanobers
arrays can be easily fabricated using the electrospinning
method [120,121] and many studies have shown that cells
align with the direction of the bers in these scaffolds.
Primary cardiac ventricular cells and smooth muscle cells
aligned in the direction of 1.8 m and 550 nm aligned
nanober meshes respectively [122,123]. Neuron elongation and outgrowth was parallel to the direction of
aligned 113 and 431 nm nanobers and neuronal stem
cells seeded on random and aligned 300 nm and 1.5 m
nanobers turned through large angles in order to grow
parallel to the ber alignment independent of ber diameter [57,124]. Human skeletal muscle cells grown on 300 nm
nanober scaffolds aligned on aligned bers scaffolds in
contrast to a random orientation adopted on randomly oriented scaffolds [125]. The effect of nanober alignment
on neurite alignment of dorsal root ganglion (DRG) was
measured quantitatively for nanobrous scaffolds with different degrees of alignment using Fourier image analysis.
Neurite alignment corresponded to the degree of ber
alignment as demonstrated by full-width-half max of the
intensities of the fast Fourier transform images of 38, 69,
and 84% (p < 0.0001) for highly aligned, intermediate, and
randomly oriented samples respectively [126].
In addition to the inuence on ber arrangement, cell
alignment can have positive effects on cell growth within
tissue engineering scaffolds. Myotubes formed on aligned
nanober scaffolds were more than twice the length of
881
myotubes grown on randomly oriented bers (p < 0.05) and

neurites extending from DRG explants on highly aligned
scaffolds were 16 and 20% longer than those grown on
intermediate and randomly aligned scaffolds respectively
[125,126].
4.3. Attachment
When designing tissue engineering scaffolds it is critically important that cells readily attach to the constructs.
Many studies have conrmed that nanobrous architectures have cell attachment properties superior to at
surfaces. Two factors that may lead to improved cell
attachment on nanobers could be the physical entrapment of cells penetrating inside of nanober meshes or
improved focal adhesions. Many more lopia have been
observed projecting from the edges of cells cultured on
small diameter bers compared to at surfaces [118] and
uniform focal adhesion distribution has been observed in
cells cultured on nanobers as compared to a distribution
around the cell perimeter for cells cultured on at surfaces [118]. An extreme example of physical entrapment
was demonstrated when large hepatocyte spheroids completely enveloped nanobers and demonstrated improved
adhesion to at controls upon agitation [127].
Relatively consistent results have been reported on the
improved attachment of various cell types on nanobers
as opposed to at surfaces. Fibroblasts, adipose stem
cells, and smooth muscle cells that were allowed 15 min
to 8 h to attach, adhered to nanobers (100500 nm) at
rates 50150% higher than cells on at substrate controls
[70,123,128]. Bone marrow-derived hematopoietic stem
cells attached to nanobrous scaffolds fabricated from various materials at levels at least four times that of tissue
culture plate at time points from 10 to 60 min [91]. After
long term cell culture of 10 days, 40% of total hematopoietic stem cells grown on polymer nanober meshes were
adherent after washing, while only 25% of total cells stayed
on at substrates [129].
Fiber diameter can have an affect on the cell attachment
properties of nanobrous scaffolds, however optimum
ber size seems to vary for different types of cells and conditions. The attachment of human mesenchymal stem cells
was found to be around 40% greater on 1 m diameter
bers as compared to 500 nm bers [130], but in contrast, attachment of 3T3 broblasts on nanober scaffolds
was greatest on 425 nm diameter scaffolds and decreased
with increasing ber diameter for 641 and 900 nm scaffolds [131]. On a smaller size scale, astrocytes seeded on
60, 100, 120, and 200 nm carbon ber disks attached best
to the largest ber diameter disks [132].
4.4. Viability: survival/proliferation
Cell survival and proliferation must be well controlled in
tissue engineering scaffolds. In most cases, maximum survival and proliferation are desired, but in some applications
reductions in cell survival and proliferation are required.
For example, astrocyte proliferation at an implant/tissue
interface in the central nervous system causes the formation of an undesirable glial scar. It is well documented that
882
nanobrous architecture can have a signicant effect on

the survival and proliferation of cells, however the specic effects may vary for different cell types and conditions.
Nanobers have been shown to both increase and decrease
cell proliferation for various cell types and conditions when
compared to at control. Fiber diameter effects on cell proliferation also vary for different cell types and conditions.
Self assembling peptide nanober scaffolds were found
to improve the viability of transplanted islet cells after 7
days of standard culture and in hypoxia injury model [133].
Cell viability as conrmed by MTT assay was increased
from 58.6% to 81.0% in standard culture and from 29.2%
to 60.6% in injury model. Viable broblast cell numbers
after 813 days of culture were increased up to 3 times on
300500 nm bers vs. at control [128]. It was further conrmed by transwell culture that nanobrous architecture
caused cells to release soluble factors that could stimulate
increased cell numbers. In contrast, hematopoietic stem
cells cultured on three different types of 500 nm bers had
1050% fewer cells than those cultured on at lms after
10 days [129], and Schwann cells cultured on ve different polymer materials for 35 days had decreased MTT
viabilities on four types of polymer bers (14 m) compared to lms and similar viability on one type of nanober
(130 nm) when compared to lm [134].
Specic ber diameter also has a variable effect on the
proliferation of cultured cells. 3T3 broblasts cultured on
polymer nanobers with 6 diameters from 117 to 1051 nm
had similar cell counts on day 1, but on days 3-7 cell
number was greatest on 428 nm scaffolds and decreased
towards the ends of the range up to 40% for the smallest and
largest ber diameters [131]. In contrast, human umbilical vein endothelial cells demonstrated higher viability
and cell densities on 1000 to 1500 nm bers as compared to 10200 nm and 2001000 nm bers [119]. Carbon
nanobers with diameters of 60 and 100 nm resulted in
osteoblast numbers nearly 3 times that of 125 and 200 nm
bers after 7 days of culture, but astrocyte cell counts were
up to 66% higher on 125 and 200 nm bers as compared to
60 and 100 nm bers after 5 days of culture [132,135].
4.5. ECM production
The goal of many tissue engineering approaches is to
provide a framework for the assembly of natural ECM
secreted by resident cells in vitro or in vivo. For example,
biodegradable tissue engineering scaffolds are designed to
provide initial structural support that is intended to be
replaced by naturally produced ECM as the original scaffold materials degrade. Thus it is important that a well
designed tissue engineering scaffold promotes ECM production from resident cells. Several studies have shown
that nanobrous architecture can elicit increased ECM production from resident cells and that deposited ECM may be
more highly organized. Chondrocytes seeded on 700 nm
nanober scaffolds produced more sulfated proteoglycan
rich, cartilaginous matrix than those seeded on tissue culture plate [116] and chondrocytes grown on nanober
(500900 nm) meshes showed a nearly 2 fold increase in
glycosaminoglycan (GAG) production at 28 days as compared to microber (15 m) culture [117]. Cartilage ECM
markers, such as collagen II and IV, aggrecan, and cartilage

proteoglycan link protein were also greater in chondrocyte
cultures grown on nanobers than microbers as conrmed by immunostaining [117].
Fiber diameter and orientation can affect the ECM production of cells residing in nanobrous scaffolds. Calcium
production in osteoblasts from 7 to 21 days was signicantly higher in cells cultured on 60 and 100 nm carbon
bers compared to cells cultured on 125 and 200 nm bers
[135]. Lee et al. found that human ligament broblasts cultured on 650 nm nanobers synthesized signicantly more
collagen on aligned bers than on randomly oriented bers
as conrmed by collagen assay normalized by DNA content
at 3 and 7 days [136].
In addition to stimulating increased ECM production,
nanobrous structure can also result in organized deposition of ECM products. Immunohistochemistry at 7 and 14
days revealed that rotator cuff broblasts deposited collagen matrix in an aligned orientation on aligned nanober
scaffolds but not on randomly oriented scaffolds [137].
Baker et al. linked [138] an increase in scaffold strength
after in vitro culture of mesenchymal stem cells (MSCs) and
meniscal brochondrocytes on aligned ber scaffolds to
organized collagen arrangement independently of collagen
production. Collagen produced by both cell types oriented
parallel to aligned nanobers, and despite similar overall
collagen content, aligned ber scaffolds showed a >7 MPa
increase in modulus over 10 weeks in culture as compared
to an 1 MPa increase in modulus for randomly oriented
ber scaffolds.
4.6. Differentiation
Many tissue engineering strategies require the selective
differentiation of stem or progenitor cells into a specic lineage. It is well established that various soluble cues can be
applied to certain cell types to induce them to differentiate
to a specic fate. There is also evidence that the physical environment can affect cell differentiation. Nanobrous
architecture can have strong effects on cell fate versus at
culture surfaces and ber properties, such as diameter and
arrangement can affect cell differentiation. The effect of
nanobers on cell differentiation can be quite strong, but
varies for different cell types and conditions. Nanobrous
architecture has also been shown to promote, prevent, and
have no effect on differentiation when compared to at
surfaces.
Mouse embryonic broblasts cultured in self assembling peptide scaffolds were observed to undergo strong
osteogenic differentiation after osteogenic induction while
cells cultured on at culture plate did not differentiate
[139]. Furthermore, mouse embryonic stem cells cultured
in nanobers without osteogenic induction expressed
enhanced early stage markers of osteoblast differentiation compared to tissue plate culture [139]. Primary
cardiac ventricular cells cultured on 2 m diameter ber
scaffolds also shifted to a more mature phenotype than
those grown on tissue culture plate [122]. In another
study, differentiated chondrocyte phenotype was maintained on 700 nm nanober scaffolds for 21 days of culture
while chondrocyte cells seeded on at tissue culture plate
de-differentiated [116]. In contrast MSCs cultured on 501000 nm nanobers showed no differences in osteogenic
phenotype markers compared to tissue culture plate after
12 days of osteogenic differentiation induction [140].
Nanobrous scaffolds have also been observed to prevent differentiation and allow for the proliferation and
maintenance of a pluripotent niche, which is important in
vitro stem cell expansion. Hematopoietic stem cells cultured in 529 nm polymer nanober meshes for 10 days
mediated a greater percentage of primitive progenitor cells
when compared to the at surface [129] and proliferation
and self-renewal of pluripotent mouse embryonic stem
cells were greatly enhanced in nanobrillar meshes when
compared to smooth culture surface [141]. While proliferation with self-renewal was allowed to continue in
nanober topography, the cells were observed to maintain
their ability to differentiate when exposed to differentiation factors [141]. In another study, a small fraction of
mouse embryonic stem cells appeared to develop into
small embryo body like colonies [139]. It was found that the
frequency of these colonies was remarkably higher in self
assembling peptide nanober cultures than in at tissue
plate culture.
In addition to possessing the capability to support both
stem cell differentiation and self renewal, nanobrous
topography can selectively inuence differentiation based
on ber diameter and alignment. Differentiation of neural stem cells cultured on aligned and random nanober
meshes with ber diameters of 300 and 1500 nm was
observed to be highly dependent on ber diameter [124].
The quantitative differentiation rates evaluated on the
basis of shape change were approximately 80% and 40%
for 300 and 1500 nm bers respectively. Expression of ligament markers by bone marrow stromal cells was effected
by both ber diameter and alignment [142]. While expression of Col-11, decorin, and tenomodulin in cells cultured
on 270 nm bers was greater than on lms, expression of all
three markers was signicantly repressed in cells cultured
on 820 and 2300 nm bers. In contrast, expression of the
ligament marker Scleraxis increased with increasing ber
diameter and ber alignment. Primary cardiac ventricular cells cultured on aligned ber scaffolds also expressed
more markers of a mature phenotype than those grown on
randomly oriented bers [122].
4.7. Cell migration
Cell migration is a critical process in determining the
success of tissue regeneration. In many designs, tissue scaffolds are populated by cells due to cell migration, either
from cells seeded on the scaffold surface in vitro or endogenous cell migration in vivo. Tissue engineering scaffolds
designed with controlled conduction of cell migration are
desirable, and nanobrous architecture can have a signicant effect on cell migration properties in vitro and in vivo.
Traditional agarose droplet spreading experiment conrmed that nanobrous surface topography can have an
effect on the specic migration patterns of adult human
dermal broblasts [118]. Migration and aggregation of hepotocyte cells appeared to be restricted by nanobrous
architecture and this resulted in the formation of smaller
883
more uniform aggregates as compared to at lms [127].

MSCs also had reduced migration distances on three different ranges of collagen nanobers (50200, 200500,
and 5001000 nm) that were quantied and found to be
3756% of those observed on TCPS control [140].
Endogenous cell migration in vivo was increased with
the addition of nanobrous architecture. A 3D peptide
nanober mesh assembled in the in vivo myocardium
was able to recruit endothelial progenitor cells, smooth
muscle cells, and myocyte progentitor cells and promote
vascularization and tissue regeneration. Implantation of
matrigel without bers as control resulted in few numbers of endothelial cells and no myocyte progenitors [7]. In
this case nanobrous structure may facilitate cell migration
by providing structure in 3-dimensional space for them to
move along and attach to.
In addition to the structural roles of nanobers, they
may also elicit functional responses in cells to migratory and remodeling behaviors. Nanobrous structure
induced broblasts to increase production of collagenase
that degrades the adhesive collagen between the scaffold
surface and cell membrane, and this lead to the hypothesis
that the nanobrous architecture may have been encouraging migratory or remodeling behavior in comparison to
a smooth surface [128].
4.8. Potential mechanisms of cellnanostructure
interactions
It is clear that nanobrous architecture has major effects
on modulating a wide variety of cell behaviors. It is not
surprising that nanobrous architecture has such an effect
on cell behavior when considering the differences in local
environmental conditions experienced by a cell in a nanobrous substrate as compared to a at culture surface. It is
theorized that nutrient inltration, surface molecule presentation, and cell shape many be mechanisms by which
nanobrous architecture modulate cell behavior. It is intuitive that cells in a 3D nanobrous structure would be
able to exchange nutrients and utilize receptors throughout their surface, while cells in at culture conditions are
limited to nutrient exchange on only one side. By limiting
the useable surface area of cells, at culture conditions may
also limit their function.
The surface molecule presentation of nanobers may
be another mechanism by which nanobers modulate cell
behavior. Interest in nanobrous architecture in biology is
many times credited to mimicking the bers of the natural
ECM, but the more general property that has fuelled the
study of nanobers is their extremely high surface area to
volume ratio. It has been shown that this property results
in higher protein absorption and more efcient presentation of biomolecules to cells in nanobrous architectures
and that this translates to modulation of cell behavior.
Leong et al. [143] found that solid poly(D,L-lactide) bers
(850 nm) absorbed 16 times as much protein as lms,
and they fabricated porous nanobers that absorbed 80%
more protein than the solid bers. Cell attachment on the
three scaffolds increased in agreement with the observed
increases in protein absorption. Baker et al. [144] found that
polystyrene nanober scaffolds (200 nm) also absorbed
884
16 fold more serum than the equivalent ask area and

hypothesized that stromal cell differentiation into smooth
muscle cells was inhibited by non-specic serum protein
absorption. Observed increases in myoblastic mouse cell
attachment, proliferation and differentiation on brous
pressed carbon nanotubes as compared to at pressed
graphite were amplied by pre-culture incubation with
50% fetal bovine serum to increase the effect of surface
protein absorption on cell behavior [145]. It has also been
shown that biomolecules incorporated into nanobers are
much more efcient at modulating cell behavior than at
coated surfaces or soluble factors due to the high surface area to volume ratio of nanobers. Three dimensional
and two dimensional self-assembling peptide nanober
meshes incorporating the IKVAV motif of laminin induced
neural stem cell (NSC) differentiation into neurons at a rate
of 35% after 1 day, as compared to 15% after 7 days on
at laminin coated substrates [15]. Neuronal differentiation was not increased in non-bioactive ber meshes with
the addition of soluble IKVAV and it was hypothesized that
improved differentiation rates were caused by an increased
efciency in the presentation of the motif to the cells, which
was estimated to be amplied by1000 times compared to
at laminin coated surfaces.
Another pathway for modulation of cell behavior by
nanobrous architecture may be through cell shape. One
proposed mechanism for the transduction of cell shape
information into gene expression is by the transmission
of mechanic forces directly from the actin cytoskeleton to
the nucleus [146]. It has been shown that regulation of
cell shape can inuence the differentiation of multipotent
human mesenchymal stem cells (hMSCs) into adiogenic
or osteoblastic fate [147]. Human mesenchymal stem cells
allowed to atten and spread expressed osteoblastic markers, such as alkaline phosphatase, while constrained cells
that remained unspread and rounded expressed adiogenic lipid production. While cytoskeletal organization is
related to cell shape, the cytoskeleton can inuence gene
expression independently of cell shape. The inhibition of
myosin-generated cytoskeletal tension in hMSCs caused
decreased alkaline phosphatase activity and increased lipid
production without changing cell shape [147]. In a similar
study, Spiegelman and Ginty [148] found that differentiation of an adipogenic cell line could be inhibited when
it was allowed to attach and spread on bronectin coated
surfaces and that the inhibitory effect on cell differentiation could be reversed by keeping the cells rounded and by
chemically disrupting the actin cytoskeleton.
Nuclear shape has also been measured directly and correlated to gene expression and protein synthesis [149,150].
Intermediate values of nuclear distention promoted maximum collagen I synthesis in primary osteogenic cells
[149]. Two to nine fold increases in gene expression above
baseline accompanied signicant rounding of nuclei in
mesenchymal stem cells [150].
Nanobrous architecture is able to inuence a variety of cell behaviors through a variety of mechanisms,
therefore smart design of nanobrous components in tissue engineering scaffolds can allow for increased control
over resident cell behavior and thus improved overall function. It is however important to consider that the effect of
nanobers on cell behavior varies widely with specic cell

types, ber type and environmental conditions. For example, osteoprogenitor cells cultured on electrospun polymer
ber meshes exhibited a lower cell density than those on
smooth surfaces in the absence of osteogenic factors, but
when osteogenic factors were added the cell density of
ber surfaces was equal to or greater than that on smooth
surfaces [115].
5. Tissue engineering applications of polymer
nanober scaffolds
Many different applications for nanobers in tissue engineering have been explored. Because polymer
nanobers can provide three-dimensional architecture,
modulate cell behavior, and have the potential to deliver
biomolecules, they are a good candidate for a wide variety of tissue engineering applications. Some of the other
properties of polymer nanober scaffolds lead them to be
well suited for more specic applications. For example,
nanobers may offer good mechanical properties for load
bearing applications, directional alignment to tissues with
aligned structures, and nanober meshes with ne pores
act as membranes that allow transport of nutrients and
waste, but limit cellular inltration. Table 2 organizes several references to experiments where polymer nanobers
have been used as scaffolds for specic tissue regeneration applications. Each of these experiments is related to a
specic tissue or application based on (1) The incorporation of tissue specic biomolecues (2) eliciting a desirable
response from tissue specic cells in vitro (3) sharing a
similar microstucture or mechanical properties to a natural
tissue and (4) in vivo applications.
The mechanical integrity of polymer nanobers has lead
to a vast amount of research for bone tissue engineering
scaffolds. Many of these studies concentrated on the functionalization of nanobers with bone specic biomolecules
such as hydroxyapatite. It has been demonstrated that
nanobrous scaffolds can support osteogenic differentiation in vitro and promote in vivo bone growth into
nanobrous scaffolds in subcutaneous and bone defect
models. The mechanical properties of nanobrous structures also make them an attractive scaffold for use in
cartilage tissue engineering where chondrocytic function
and differentiation has been demonstrated in nanobrous
scaffolds. Polymer nanobers are a suitable material choice
for vascular grafts because of their tensile strength and
directional guidance properties. Polymer nanober vascular grafts have been tested in vitro under static and
pulsatile conditions and in vivo. In skeletal muscle, tendon and ligament tissue engineering, the tensile strength
and directional alignment of polymer nanobers are also
attractive properties. Nerve tissue engineering benets
from the directional guidance offered by aligned nanobers
as well. Polymer nanober scaffolds can improve the viability, growth, alignment, and differentiation of neural cells in
vitro and they have been applied to injuries in the peripheral and central nervous system to promote regeneration
in vivo. Many in vivo studies have been conducted using
nanober scaffolds in wound healing applications, including clinical trials [151]. Nanober scaffolds can provide
885
Table 2
Nanobrous scaffolds for tissue engineering applications.
Tissue Type
Functionalization with
tissue specic
biomolecules
Promotion of desired
behaviors from tissue
specic cells in vitro
Favorable Structural
properties for tissue
specic application
In vivo models
Bone
Secondary surface
mineralization
[14,77,78,89,152,153]
Bioactive peptide
incorporation [108]
Hydroxyapatite particle
incorporation [154158]
BMP-2 incorporation [157]
Osteoblastic and
pre-osteoblastic cells
[159161]
Mesenchymal stem cells
[28,162,163]
Selective pore size [164]
Subcutaneous
implantation
[165167]
Bone Defect-Skull
[167169]
Bone defect- Femur
[170]
Chondrocytes
[116,117,171,172]
Meniscal
brochondrocytes [138]
Mesenchymal stem cells
[138,173]
Endothelial cells [90,177]
Smooth muscle &
endothelial cells [178,179]
Physiological shape [174]

Suitable mechanical
properties [175]
Cartilage
Vascular
Elastin/collagen blend
[176]
Ligament/Tendon
Muscle
Neural
Bioactive peptide
incorporation [15,186]
Laminin surface
modication [82]
Wound Healing
Antibiotic incorporation
[193]
Ligament/tendon derived
broblasts[136,137,184]
Skeletal muscle derived
cells/myobasts [125,185]
Brain derived neural stem
cells [57,80,187]
Dorsal root ganglion (DRG)
[126]
DRG & perineural
membrane derived
primary neurons [188]
Hair follicular cells [51]
Dermal broblasts
[66,194,195]
Dermal broblasts &
keratinocytes [196,197]
Human skin equivalent
tissue model [198]
Intervertebral Disc
Islet/Hepotocyte
Transplantation
Shape & mechanical

properties [180,181]
Epigastric vein graft

[182]
Abdominal aortic graft
[183]
Shape [189]
Severed optic tract [8]

Spinal cord transaction
[190]
Tubular sciatic nerve
bridging device
[191,192]
Surgical wound model
[47,199,200]
Burn wound model
[86,201]
Subcutaneous
implantation [202]
Mechanical properties
[203]
Shape & Structure [204]
Islets [133]
Hepatocytes [205,206]
Stem cell derived hepatic
cells[207]
structure for the ingrowth of cells around a wound site and

provide a barrier to outside infection.
6. Cell incorporation into nanobrous electrospun
scaffolds
The success of a tissue engineering scaffold is determined by its ability to incorporate desired cell types and to
promote the desired functionality of the incorporated cells.
Despite great promise and widespread investigation there
have been few clinically relevant successes for nanobers
as tissue engineering scaffolds. One of the main reasons
for this limited success is the difculty of incorporating
cells into electrospun nanobrous scaffolds. Electrospinning is the most versatile and widely studied method of
nanobrous scaffold fabrication, but it still remains a major
challenge to fabricate electrospun scaffolds with antiquate
cell incorporation or permeability. Cells can be easily incor-
Islet transplantation
[208]
porated into self assembling peptide nanober scaffolds,

which is one of the major reasons for the success of this
technique. There are however some major limitations to
the self assembling peptide method such as, complex procedures, and limited control over ber material, size, and
arrangement. In contrast, the electrospinning technique is
a very simple and versatile process that allows the fabrication of nanobers from many types of materials and
offers control over ber size and arrangement. A feasible and practical method of quickly advancing the eld
of nanobrous tissue engineering scaffolds is through the
development of better methods to incorporate cells into
electrospun scaffolds.
Several creative approaches have been explored to
address this major challenge in polymer nanober scaffold
fabrication. Cell incorporation into nanobrous scaffolds
may be achieved in two ways: (1) Cell incorporation inside
of the scaffold during fabrication (2) Migration of cells into
886
the scaffold in vitro or by host tissue cells after in vivo

implantation. Some of the strategies for improving cell
incorporation into electrospun nanober scaffolds include
cell incorporation during fabrication, physically assisted
seeding methods and the design of cell permeable scaffolds
using composite techniques, layer by layer assembly, and
incorporation of sacricial materials.
6.1. Cell incorporation during fabrication
Several techniques for direct incorporation of cells into
electrospun nanober scaffolds have been developed. Cell
nanober constructs were fabricated by directly pipetting
cell suspension into an electrospun nanober scaffold during collection. Nanobers were electrospun onto a charged
ring on the surface of culture media and intermittent
pipetting of cells during the fabrication process resulted
in nanobrous constructs with uniform cell incorporation
[101]. No signicant toxicity was caused by residual solvent left on the electrspun nanobers. A similar approach
incorporated cells into an electrospun nanober scaffold by
electrospraying a cell containing solution onto a grounded
target while nanobers were simultaneously electrospun
onto the same target [209]. Cells integrated into the
nanober constructs and no signicant decrease in cell
viability from the fabrication process was apparent. Fibroblast and adipose-derived adult stem cells were directly
added into an aqueous PVA solution and electrospun to
form nanobers that encapsulated the cells [210]. Fibroblasts incorporated into PVA nanobers retained viability,
proliferation, and function.
6.2. Cell population by migration
When cells are not directly incorporated into a
nanober scaffold, the scaffold may be populated by cells
that migrate into the interior of the scaffold from the
outer surface. The extent of cell migration into nanober
scaffolds can be affected by both the architecture of the
scaffolds and the biological or chemical cues incorporated
into the scaffold.
6.2.1. Perfusion
Cells inltration into nanobrous scaffolds can be
enhanced by physical forces, such vacuum, ow perfusion, or centrifuge [172,211]. These types of techniques
can be utilized to increase the speed at which cells inltrate nanobrous scaffolds during in vitro seeding. Flow
perfusion speeds up cell inltration by moving cells with
hydrodynamic forces and increases nutrient transport
improving cell viability deeper inside the scaffolds [211].
6.2.2. Scaffold permeability
In order for cell migration into a nanobrous scaffold to
occur there must be adequate void space for cells to occupy
and move around. In the case of randomly aligned electrospun mats, pore size is generally related to ber diameter,
where scaffolds fabricated from larger bers have larger
pores and thus greater permeability to cell inltration
[212]. Pham et al. [211] fabricated electrospun microber
scaffolds with ber diameters ranging from 2 to 10 m. The
pore size of these scaffolds increased with increasing ber

diameter from a value of around 10 m for 2 m diameter bers, to around 40 m for 10 m diameter bers. In
contrast, overall porosity ranged from around 8589% with
the greatest porosity in smaller diameter ber scaffolds.
Cell inltration into 5 m diameter nanober scaffolds was
severely limited by the inclusion of a thin layer of electrospun 600 nm ber on its surface under both static and ow
perfusion culture conditions [211]. Migration of human
venous myobroblasts into nanober scaffolds with diameters ranging from 3.4 to 12.1 m increased with ber
diameter during 3 days of culture [213]. Even the relatively large 3.4 m diameter ber signicantly impeded
cell migration when compared to the 12.1 m ber scaffolds. Optimal pore size may be dependent on the specic
cell type or application, but the minimum pore size necessary for inltration must not be signicantly smaller than
that of the migrating cells. Small diameter nanobers can
be desirable because of the effects that ber diameter has
on cell behaviors as discussed in section 4, but pore size
requirements limit the use of small diameter nanobers in
tissue engineering scaffolds. The trade off between scaffold
porosity and ber diameter in electrospun scaffolds is one
of the current limiting factors of this technology in tissue
engineering.
6.2.3. Strategies to increase cell permeability
Several strategies have been developed to fabricate
nanober scaffolds that combine both small diameter
nanobers and adequate permeability for cell inltration.
Several groups have fabricated composite ber scaffolds
that combine microbers for large pore size and structural stability and nanobers for improved cell interactions
and increased area for attachment. A starch based scaffold made by ber bonding with 160 m microbers was
electrospun with 400 nm nanobers that allowed endothelial cells to span the spaces between microbers, migrate,
and organize into capillary-like structures while maintaining structural integrity and a porosity of 70% [214,215]. A
composite scaffold made from direct polymer melt deposition of microbers that included electrospun PCL/collagen
nanobers showed improved cell adhesion and proliferation [216]. Multilayered micro/nanober strucutures
have also been fabricated by modifying electrospinning
conditions to include layers of 5 m and 600 nm bers
[211]. Nanobers with diameters of 400500 nm were even
directly electrospun onto 30 m microbers, which were
subsequently formed into a highly porous scaffold containing nanobers [217].
Another method to produce nanober scaffolds with
improved cell inltration properties is by addition of large
pores within a nanobrous structure. One hundred micron
holes etched into 500 nm ber scaffolds by UV lithography allowed the migration of smooth muscle cells into the
scaffolds [218]. Porogen leaching has also been used as a
method of fabricating structures with large interconnected
pores within a nanobrous network. Silk broin electrospun nanober scaffolds were soaked in a salt containing
solution and lyophilized to create a porous structure. This
process formed 600900 m pores in a scaffolds with ber
diameters of 400 nm in contrast to an initial pore size of
12 m [219]. Salt particles have also been incorporated

into nanober meshes by mechanical addition of solid salt
particles directly to an electrospun jet during fabrication
[164]. Salt particles were deposited in intervals during electrospinning to form a layered structure containing regions
of salt particles. Cell inltration of depths up to 4 mm was
reported in these scaffolds after 3 weeks of in vitro culture.
Similar techniques have utilized other types of sacricial materials to create an evenly distributed enhanced
porous structure within electrospun nanobrous scaffolds.
Ice crystals were introduced to nanober scaffolds during fabrication by cooling a collecting mandrel to 30 C
while electrospinning at room temperature [220]. Ice
crystals simultaneously deposited on the mandrel during electrospinning and were distributed throughout the
mesh. Freeze drying resulted in a nanober mesh with
pores around 100 times larger than in control meshes
and pore size changed with the level of humidity. Larger
pore size resulted in nanober meshes that allowed cell
penetration up to 50 m, while control meshes allowed
only surface cell growth. A similar technique involves
simultaneously electrospinning water soluble sacricial
bers into a nanobrous scaffolds to create void space
after sacricial ber dissolution. In randomly oriented PCL
nanober (diameter 1 m) scaffolds, the depth of cell
penetration after 7 days was increased from 48 to 114 m
with the addition of soluble gelatin nanobers (diameter
1 m) [7]. Cell penetration into PCL scaffolds containing water soluble PEO bers increased with increasing
percentage of PEO bers [144]. In contrast, it has also
been reported that nanober meshes with sacricial PEO
and gelatin bers offered very limited improvement versus control meshes in an inltration experiment due to
the collapse of the structure after sacricial ber leaching
[221].
Cell permeable materials can be added as a ller material within a nanobrous scaffold to add space for cell
penetration. Ekaputra et al. fabricated a hybrid brous
PCL/Col mesh with regions of hydrogel matrix by simultaneously electrospinning nanobers and electrospraying
hydrogel onto a collection mandrel [221]. The depth of
cell inltration into nanober meshes containing electrosprayed hydrogel was increased from around 60 to
225 m when compared to control after 10 days of culture. Composite lms containing a protein matrix and
nanobers were fabricated by adding xed nanober
arrays to an aqueous gelatin solution [12]. After gelatination the gelatin matrix provided structural support to
fragile aligned nanober arrays with signicant void space
between individual bers.
Porous 3D nanobrous networks have been fabricated
by suspending electropun nanobers in liquids. A unique
method of dispersing hydrophobic nanobers in aqueous solutions was developed that utilized the attachment
of lipase onto the surface of electrospun polystyrene
nanobers [222]. Alcohol pretreatment of the surface
modied bers caused the tightly aggregated nanobers
to be dispersed into a loosely entangled structure with
greatly increased volume, and this dispersed geometry
remained when the bers were immersed DI water as
887
long as the bers remained hydrated throughout the washing step. Silk broin nanobers collected directly in a
methanol bath formed a three dimensional structure that
was retained after hydration and freeze drying [223]. Cells
were able to penetrate into the porous structure of these
scaffolds, while control scaffolds collected on a rotating
mandrel did not allow cell penetration. Three dimensional networks of PCL nanober yarns were fabricated
as suspensions in water by electrospinning into a novel
dynamic ow collecting system [224]. These nanober
yarn networks also retained their porous microstructure after freeze drying. It is hypothesized that these
loosely dispersed nanobers would allow improved cell
penetration.
6.2.4. Layer by layer assembly of pre-seeded nanober
sheets
Three-dimensional nanobrous constructs with uniform cell distributions have been fabricated with layer-bylayer stacking of thin cell containing nanobrous sheets. A
cylinder with an 8 mm diameter and a height of 3 mm was
constructed by stacking thirty 100 m thick cell seeded
nanober disks, and nourished with a perfusion bioreactor
[225]. Thin sheets of nanobers with a thickness of 10 m
were collected on a wire ring with a diameter of about
15 mm and seeded with cardiomyocytes [226]. Individual
layers adhered immediately when these thin cell-nanober
sheets were stacked, and constructs of up to 5 layers were
formed without incidence of core ischemia. Multilayer cellnanober structures have also been formed by stacking
additional layers of cell containing ber sheets every few
days during cell culture [184].
7. Concluding remarks
Many damaged or degenerated tissues cannot be
treated by conventional methods. Tissue engineering
presents a promising alternative to regenerate damaged
and degenerated tissues by guiding the formation of new
healthy tissue. An ideal matrix for promoting the formation of such tissue requires a supporting structure for
cell attachment within three dimensions as well as adequate void space for cell inltration and vascularization.
Polymer nanobers are an ideal material for assembling
structures that address both of these requirements. In
addition polymer nanobers posses many other advantages as tissue engineering scaffolds, such as versatility
for biofunctionalization, and the promotion of specic
desired cell behaviors that are elicited by the nanobrous
architecture. Despite a variety of techniques employed to
fabricate polymer nanobers and to assemble them into
structures, no optimal method has emerged that combines the potential advantages of polymer nanobers and a
truly 3-dimensional structure. The future clinical success of
polymer nanober tissue engineering scaffolds will depend
on whether new techniques are developed that allow for
the fabrication of polymer nanober scaffolds with control
over structural arrangement, material composition, and
biofunctionalization, while maintaining reasonable cost
and yield.
888
Acknowledgments
This work was made possible by the NIH/NINDS
USA (R01 NS050243) and American Heart Association
(09PRE2060154).
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Progress in Polymer Science 35 (2010) 868892

Contents lists available at ScienceDirect

Progress in Polymer Science

Polymer nanobrous structures: Fabrication, biofunctionalization, and

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

have high levels of organized bers to impart mechanical

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

Poor cell inltration into the core of

Easy incorporation of cells during

3-Dimensional pore arrangement

Limited material selection

Vast materials selection

Vast materials selection

Low productivity (One single ber at

Limited material selection

Polymer synthesized directly into

Limited control of ber diameter and

Kinetically controlled solution synthesis

Polymers synthesized directly into

Limited control of ber diameter and

Chemical Polymerization of Aniline

Polymers synthesized directly into

Limited control of ber diameter and

accelerated ECM remodeling is critical during embryonic

scaffolds can impart mechanical strength, structure for cell

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2. Methods of polymer nanober scaffold

nanobers are usually collected from an electrospinning

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

2.4. Bacterial cellulose

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strains of bacteria [24]. Copolymers have been produced

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

can be utilized to fabricate tissue engineering scaffolds that

2.10. Conventional chemical oxidative polymerization of

3. Biofunctionalization of polymer nanobers

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at low concentrations (10 wt%) and as much larger irregular

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

strated favorable properties as tissue engineering constructs [64].

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3.1.8. Hyaluronic acid

3.2.2.1. Exposing functional groups. Depending on the

3.2.2.2. Covalently attaching biomolecules to functional

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

3.2.3. Examples of biomolecule surface functionalized

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

acid, while ber diameter increased with increased loading

aggregates and maintain a uniform solution composition

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

using conventional methods. For example, nanobers were

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

polymer nanobers (500900 nm) also adopted a rounded

myotubes grown on randomly oriented bers (p < 0.05) and

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

nanobrous architecture can have a signicant effect on

markers, such as collagen II and IV, aggrecan, and cartilage

V. Beachley, X. Wen / Progress in Polymer Science 35 (2010) 868892

more uniform aggregates as compared to at lms [127].