Periodontology 2000, Vol.

24, 2000, 253–269 Copyright C Munksgaard 2000

Printed in Denmark ¡ All rights reserved
PERIODONTOLOGY 2000
ISSN 0906-6713

Tissue engineering:
a new paradigm for periodontal
regeneration based on molecular
and cell biology
P. M ARK B ARTOLD, C HRISTOPHER A . G . M C C ULLOCH,
A . S AMPATH N ARAYANAN & S ANDU P ITARU
Regenerative treatment of periodontal defects with an agent, or procedure, requires that each functional stage
of reconstruction be grounded in a biologically directed process.

With the above paradigm in mind, we have set about components of the periodontium to participate in
in this chapter to distill what is currently known and the regenerative process. This latter approach to re-
done and what is likely to be available in the near construction makes use of understanding of the de-
future in the context of periodontal regeneration. We velopment of the periodontium and the cellular pro-
contend that the way forward in the field of peri- cesses that are involved.
odontal regeneration is through the application of When considering periodontal regeneration, we
current knowledge in the fields of molecular and cell believe that at least four criteria must be met in
biology, developmental biology and tissue engineer- order for regeneration to have occurred. These in-
ing principles as applied to tissue regeneration. clude all the features of the normal dentogingival
Through a combination of transplanted biomaterials complex that would equate to restoration of these
containing appropriately selected and primed cells, tissues to their original form, function and consist-
together with an appropriate mix of regulatory fac- ency:
tors to allow growth and specialization of the cells
and matrix, we envision a new vista for periodontal O A functional epithelial seal must be re-established
regeneration becoming possible. at the most coronal portion of the tissues and be
The management of periodontal defects, includ- no more than 2 mm in length.
ing destruction of the periodontal ligament, ce- O New connective tissue fibers (Sharpey’s fibers)
mentum and the formation of intrabony defects, has must be inserted into the previously exposed root
always been a challenge in clinical periodontics. surface to reproduce both the periodontal liga-
During the 1950s and into the 1960s, resective surgi- ment and the dentogingival fiber complex.
cal therapy with or without osseous recontouring O New acellular, extrinsic fiber cementum must be
was considered the norm in the belief that attain- reformed on the previously exposed root surface.
ment of shallow pocket depths was a worthwhile O Alveolar bone height must be restored to within 2
goal. Since then attention has been focused more on mm of the cementoenamel junction.
regenerative and reconstructive therapies rather
than resective therapies. Today, attention appears to On the basis of current understanding of the mol-
focus on two main approaches for periodontal re- ecular and cell biology of periodontal development
generation. On the one hand there is the approach and regeneration, we hypothesize that the simplistic
that requires introduction of a ‘‘filler’’ material into approach of introducing a filler material into a peri-
the periodontal defect in the hope of inducing bone odontal bony defect is no longer tenable. Rather, ef-
regeneration. Alternatively, techniques are being de- forts must be made to recapitulate in wound healing
veloped to guide and instruct the specialized cellular the crucial events that were associated with the orig-

253
Bartold et al.

inal development and formation of the periodon- to previously diseased root surfaces (32, 69, 95, 109).
tium. Thus, we contend that serious efforts should For many years attempts have been made to ex-
be made to apply knowledge to the development of ploit emerging principles of dermal wound repair for
procedures based on sound biological principles periodontal therapy. The following is a brief overview
rather than the current and widely held view of ‘‘see of what has been tried in the past.
hole – must fill (with anything)’’.

Regenerative capacity of the An overview of regenerative

periodontal connective tissues procedures
Surgical approaches to regeneration
Regeneration of periodontal tissues so that their
original form, architecture and function are restored Over the years, a variety of surgical procedures have
has been an elusive but laudable goal. In order to been advocated to enhance periodontal tissue re-
fully appreciate what is involved, the ‘‘cross-talk’’ sponses and to ensure an outcome in which re-
among the components of the periodontium (cellu- generation can occur (49). However, regardless of the
lar and matrix), together with the inherent regenera- type of procedure used, the epithelial tissues always
tive capacity of this tissue, need to be considered. proliferate at a faster rate than the underlying mes-
Tissue damage associated with gingivitis is, for the enchymal tissues, with the resultant ‘‘long’’ junc-
most part, reversible provided that the causative tional epithelium forming and attaching to the root
agent(s) can be removed. Indeed, the gingival tissues surface. A complicating factor of this type of re-
have a remarkable capacity to regenerate to their sponse is that oral epithelium in adult mammals
original form and function following inflammatory does not appear to promote cementum or peri-
or traumatic injury, as evidenced by the lack of scar odontal ligament formation, as does odontogenic
tissue formation and ability to rapidly resume a epithelium does during the development of the peri-
functional anatomic relationship with the tooth sur- odontium. Although the formation of a long junc-
face and underlying structures following surgical or tional epithelium and formation of a new epithelial
nonsurgical therapies (67). However, in peri- attachment may be compatible with an acceptable
odontitis, once the destructive phase reaches the al- clinical outcome and clinical health, this form of
veolar bone and other deep periodontal structures, healing is classified as repair and not regeneration
regeneration does not usually occur on a clinically because the original form and architecture of the
predictable basis. The major challenges in contem- tissues have not been restored. This realization led
porary periodontal therapy are to re-establish soft to further modifications to surgical treatments in-
tissue attachment to newly formed cementum on cluding root surface conditioning and the use of
the root surface and to restore lost bone. This re- bone grafting materials in further attempts to attain
quires regeneration of gingival connective tissues de- the elusive goal of periodontal regeneration.
stroyed by inflammation, formation of new ce-
mentum and restoration of bone loss, and, most im-
Root surface conditioning for regeneration
portantly, new attachment of connective tissue fibers
In order to create an environment suitable to cell
repopulation it was considered that the root surface
needed to be cleaned and prepared in a manner
Table 1. Chemical treatment of root surfaces conducive to cell attachment and subsequent matrix
Acid etching synthesis. For these reasons root surfaces were ‘‘con-
Citric acid ditioned’’ in early attempts to prepare the root sur-
Tetracycline
Detergents face and allow for subsequent regeneration. A variety
Cetylpyridinium chloride of agents have been studied over the years (Table 1).
Sodium N-lauroyl sarcosine
Chelating agents Of these treatments, either demineralization of root
Ethylenediaminetetraacetic acid surfaces with acids or coating root surfaces with bio-
Egtazic acid
Enzymes logical attachment agents such as fibronectin, or
Attachment proteins both, have received the most attention. The demin-
Fibronectin
Growth factors eralization procedure was believed to reverse peri-
odontitis-induced hypermineralization and was

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 Periodontal tissue engineering

thought to expose old collagen fibers with which Table 2. Grafting materials used in periodontal
newly formed fibers could interdigitate (22). Exposed intrabony pockets
collagen fibers were also expected to discourage the Autografts
attachment of unwanted epithelial cells. However, Cortical bone
Cancellous bone
this procedure did not yield predictable regeneration Bone marrow
and often caused ankylosis and root resorption as Iliac crest
side effects instead (20, 60). The rationale for using Allografts
Demineralized freeze-dried bone allografts
fibronectin as a root surface coating (79) was also Freeze-dried bone allografts
unclear because serum contains high fibronectin Alloplastic materials
levels, and the added benefit of providing attach- Ceramics
Hydroxyapatite
ment proteins to facilitate reattachment is uncertain. Polymers
Although seemingly based on sound biological prin- Bioglass
ciples, these procedures did not produce the ex-
pected results; thus, regeneration remained an elus-
ive goal.
lation of the wound site with periodontal ligament
cells (36, 44, 76). This guided tissue regeneration
Implant materials for regeneration
procedure presumed that the periodontal ligament
Treatment of intrabony periodontal defects has often contained all of the progenitor cells required for the
focused only on the bony defect, and this has lead formation of bone, cementum and periodontal liga-
to the use of a number of grafting materials to stimu- ment and that selective repopulation of the wound
late bone repair. A wide variety of autografts, allo- site by the progenitor cells and their progeny would
grafts and alloplastic materials have been used and lead to improved clinical outcomes. This method
advocated (Table 2). Allografts and alloplastic ma- quickly gained wide clinical acceptance (44, 71).
terials, while convenient for the filling of defect vol- However, long-term studies and evaluations of this
ume, have little osteoinductive activity. Autogenous method have indicated that the clinical improve-
bone grafts are thought to be osteoinductive in vivo ments obtained by this procedure are of small mag-
but are still of limited value for inducing periodontal nitude and exhibit large variability (12, 84, 106). As
regeneration since their ability to induce new ce- with other procedures, regeneration of periodontal
mentum and periodontal ligament are limited. Al- ligament, cementum and bone can best be assessed
though utilization of various grafting materials may via histological means and, for guided tissue re-
result in some gain in clinical attachment levels and generation procedures, this has not always shown
radiographic evidence of bone fill, careful histologi- good results. For example, although the principle re-
cal assessment usually reveals that these materials lies on exclusion of epithelium and gingival connec-
have little osteoinductive capacity and generally be- tive tissue cells, histological assessments often show
come encased in a dense fibrous connective tissue. downgrowth of epithelial cells between the mem-
In addition, the problematic junctional epithelium brane and tooth surface (Fig. 1).
still seems to form between the graft and tooth sur- A further complicating factor in successful guided
face in spite of the placement of a graft (25, 70). tissue regeneration outcomes is the development of
Thus, these materials do not fulfill our criteria as infection following placement of the barrier mem-
useful periodontal regenerative materials. brane (24, 56, 100). This finding, together with the
inability to seal the healing tissue from the oral en-
vironment, is likely to be among the two most sig-
Guided tissue regeneration
nificant impediments to successful regenerative out-
In the 1980s, a novel procedure was proposed in comes with this method. If the wound site could be
which a physical barrier was introduced by surgically closed (as happens for guided bone regeneration)
placing a membrane between the connective tissue and infection by oral bacteria controlled, then one
of the periodontal flap and the curetted root surface. would expect that the regeneration process following
Based on biological theories of cellular domains and guided tissue regeneration would be far more pre-
the repopulation of wounds by specific cell popula- dictable.
tions, the membrane was expected to prevent apical In summary, guided tissue regeneration can be
migration of gingival epithelial and connective cells considered to have been a useful approach because
onto the root surface and to facilitate the repopu- it has stimulated thinking on the development of

255
Bartold et al.

therapeutic procedures based on some interesting formed under the influence of these factors. Further-
biological theories; however, its shortcomings are more, the continuing problem of inadequate means
manifest because of its limited clinical predictability. of isolating these agents from the oral environment
to allow their action within a ‘‘closed healing en-
vironment’’ remains unsolved.
Growth factors for regeneration

More recently, the application to root surfaces of a

variety of growth and differentiation factors to What has been learned from these
stimulate cell repopulation of periodontal defects studies?
and subsequently induce regeneration has been in-
vestigated. Growth factors are an attractive group of Paramount for successful periodontal regeneration is
agents to target for potential wound regeneration protection of the healing site from the oral environ-
studies because of their regulatory effects on im- ment. Initial sealing and isolation of the site to pre-
mune function and on the proliferation and differen- vent tissue damage induced by low-grade infections
tiation of cells from the epithelium, bone and soft may dramatically improve the regenerative process.
connective tissues. A list of various growth factors Whether this could be achieved by encouraging early
currently under in vitro and in vivo investigation is epithelial attachment to the root surface (yet pre-
shown in Table 3. Two of these growth factors, plate- venting its downgrowth) remains to be established.
let-derived growth factor and insulin-like growth fac- It would seem advantageous during the very early
tor-I, have been noted to enhance regeneration in stages of periodontal wound healing to form a
beagle dogs and monkeys with experimental peri- physiological epithelial seal and create a more favor-
odontitis (54, 89). Another promising group of poly- able closed environment for predetermined stages of
peptide growth factors is the bone morphogenetic regeneration to occur. Furthermore, it has become
proteins which offer good potential for stimulating clear that in order for periodontal regeneration to
bone and cementum regeneration (47, 87). At pres- occur, many events have to be regulated at the cellu-
ent there are not sufficient numbers of well-con- lar and molecular level (Table 4). While these mol-
trolled long-term studies to make any substantial ecules facilitate the recruitment of needed cells, at
comments about the clinical utility of growth factors the same time excluding unneeded cells, other mol-
in periodontal regeneration for humans. While they ecules that permit the expansion of needed cells and
certainly show some promise, it is possible that they their differentiation are also needed. Some of these
too will be less than satisfactory. For example, limi- molecules may be specific to the local environment
tations with growth factor use for periodontal re- in the cementum matrix. Additionally, the local en-
generation include restricted understanding of the vironment consisting of the extracellular matrix and
differentiation repertoire of the periodontal cells, the molecules that sequester in the extracellular matrix
exact target cells that are to be modulated by these is also needed for the differentiation of the cells. The
factors and the stability of the tissues that are to be most important requirement, of course, is the avail-
ability of the needed cells or their precursors (72, 80).
Previous regenerative technologies as well as
those in current use today show less than optimal
Table 3. Growth factors under investigation for outcomes for a number of reasons, including:
periodontal regeneration
Name In vitro In vivo O inability to control the formation of a long junc-
studies studies tional epithelium;
Basic fibroblast growth factor Yes Yes O inability to adequately seal the healing site from
Bone morphogenetic proteins Yes Yes the oral environment and prevent infection;
Epidermal growth factor Yes No O inability to maintain the wound as a closed rather
EmdogainA enamel matrix protein Yes Yes than open system;
Fibroblast growth factor Yes Yes O restriction of regeneration to the bone compart-
Insulin-like growth factor-1 Yes Yes ment while ignoring regenerative processes in the
Platelet-derived growth factor Yes Yes cementogenic and fibrous compartments;
Transforming growth factor-b Yes No O inability to define precisely the growth and differ-
a
Not strictly a growth factor but is believed to work in a similar fashion entiation factors needed for regeneration;
to growth factors, differentiation factors and the morphogenetic proteins.
O the possibility that growth factors may not be suf-

256
 Periodontal tissue engineering

cell populations, their proliferation and differen-

tiation and synthesis of matrix constituents (5). The
periodontal system is unique because, in order for
new connective tissue fibers to insert into ce-
mentum and bone, the healing components of both
the soft tissues and the hard tissues of the periodon-
tium need to be coordinated and integrated. Peri-
odontal regeneration presumably involves several
cell types: fibroblasts for soft connective tissues, ce-
mentoblasts for cementogenesis, osteoblasts for
bone and endothelial cells for angiogenesis (80).
During the regenerative process, these cells must in-
teract with a variety of soluble mediators such that
the course of regeneration is dictated by a combi-
nation of molecule-cell, cell-matrix and cell-cell in-
teractions. Very little is known about the signals that
initiate and regulate these interactions in vivo.
Healing of tissues damaged by any kind of injury re-
quires the participation of several regulatory mol-
ecules and cell types and involves a series of overlap-
ping stages that include inflammation, granulation
tissue formation and tissue remodeling (19). Whether
the damaged tissues heal by regeneration or are re-
paired by scar tissue depends upon at least three fac-
tors that are not mutually exclusive: (i) availability of
the appropriate cell type(s), (ii) soluble mediators of
cell function that activate these cells (66) and (iii) an
evolving (developing) extracellular matrix.
Fig. 1. Histology of guided tissue regeneration procedure.
Histological appearance of periodontal tissues 6 weeks
after placement of a guided tissue regeneration barrier
Cell selection, differentiation and maturation
membrane. Note significant downgrowth of junctional
epithelium (filled arrows) between ‘‘regenerating tissues’’ Adult mammal periodontal soft connective tissues
(NT) and the root surface. The Gore-TexA membrane is
contain heterogeneous populations of cells with di-
marked with open arrows. AB denotes alveolar bone.
verse lineages, metabolic properties, gene expression
and functions (29, 51, 62) (Fig. 2). Each periodontal
cellular compartment may contain the cells necess-
ficiently discriminative in their ability to induce ary for the regeneration of other periodontal struc-
regeneration, and thus the induction of particular tures, although in different concentrations, and their
transcription factors as an earlier event of cell
stimulation may be warranted; and
O infection of the implanted membrane or regene- Table 4. Events and processes required for
rative material postoperatively. periodontal regeneration
O Regeneration depends upon availability of
appropriate cell types and signals that activate these
cells.
Current understanding of the O ‘‘Wrong’’ cell types may have to be excluded.
regenerative process O Local environment plays a major role in the
recruitment of the right cells and preventing the
wrong cells. The local environment includes
The events associated with periodontal regeneration cementum matrix and cementodentinal junction.
are extraordinarily complex and require the partici- O Substances in the local environment affect cell
migration, adhesion, proliferation and
pation of many if not all cellular components of the differentiation.
periodontium. These include an initial inflammatory O The effect may be cell-specific and/or nonspecific.
cell component, recruitment of connective tissue

257
Bartold et al.

iety of molecules required for the appropriate re-

cruitment and functioning of the connective tissue
cells that participate in healing and regeneration.
In normal remodeling and repair processes of the
periodontium, the fibroblasts originate from pro-
genitors located within gingival and periodontal liga-
ment connective tissues. Cementoblasts are believed
to originate from ancestral cells in the periodontal
ligament and possibly the endosteal spaces of bone
(63–65). The origin and properties of cementoblasts
still remain enigmatic, in part because of their ap-
parent similarities to osteogenic cells, and in part be-
cause of the relative paucity of specific cementoblast
markers (2, 37, 52).
Irrespective of their genesis, for regeneration to
occur, sufficient numbers of cells responsible for
each process must be produced to participate at the
right location and in an appropriate temporal se-
quence. In addition, certain cells, especially the epi-
Fig. 2. Schematic representation of the sites of prolifer-
ation and migration of periodontal ligament cells in ro- thelial cells that can migrate from the adjacent oral
dent molar teeth. Cells adjacent to blood vessels are epithelium, should be excluded from the healing
thought to be enriched for progenitors for periodontal site. Overall, the cell selection process is presumably
ligament fibroblasts as well as some cementoblast and os- dictated by signaling systems in which soluble
teoblast populations within the vascular channels that
agonists, matrix proteins and cell surface receptors
connect with the periodontal ligament. Cell migration oc-
curs along the collagen fibers that traverse the width of participate and initiate responses in gene expression
the periodontal ligament. Source: adapted from Lekic & through defined intracellular signaling pathways.
McCulloch Anat Rec 1996: 245: 327.

Soluble mediators and regulators of cell function

participation may be regulated by other factors (57,
72, 80). Critical messages for cell activity are provided by
The cell types required for periodontal regenera- substances present in the local environment and
tion include epithelial cells to seal the wound area, mediate their effects through specific cell surface re-
fibroblastic cells for the soft connective tissues of the ceptors. Binding of soluble mediators to the cell sur-
gingiva and periodontal ligament, mineralized face receptors activates numerous intracellular sig-
tissue-forming cells (alveolar bone cells for bone for- naling molecules and mechanisms which, in turn,
mation and cementoblasts for cementogenesis), and lead to cell responses such as cell migration, changes
endothelial cells for forming blood vessels (Table 5). in cell shape and synthesis of extracellular matrix
During early stages of regeneration, inflammatory macromolecules. In addition, the expression of other
cells are also necessary since they secrete a wide var- cell surface molecules such as integrins is influenced

Table 5. Cell types and molecules participating in periodontal regeneration

Cells Epithelial Junctional epitheliuma
Fibroblasts Gingival fibroblasts, periodontal ligament fibroblasts
Osteoblastic cells Osteoblasts, alveolar bone cells, cementoblasts
Molecules Growth factors Fibroblast growth factor-1 and -2 (acidic and basic fibroblast growth factor),
insulin-like growth factor-I and II, bone morphogenetic proteins, epidermal growth
factor, platelet-derived growth factor, chlorella growth factor
Adhesion molecules Fibronectin, laminin, osteopontin, bone sialoprotein, collagens, cementum
attachment protein
Structural proteins Types I, III, V, XII and XIV collagens, proteoglycans, hyaluronan, osteocalcin,
non-collagenous proteins, tenascin, osteonectin, dentin/enamel matrix proteins
a
Although not classically considered to be involved in regeneration, the formation of an epithelial seal to protect the underlying regenerating tissues a functional
junctional epithelium is required. This should not be in the form of a ‘‘long’’ junctional epithelium.

258
 Table 6. Processes of gene expression
Event Description Controlling factors Function
Access of DNA for DNA in chromatin is not available for transcription.
transcription DNA must be made accessible for transcription by
RNA polymerases
Transcription RNA polymerases allows formation of RNA from RNA polymerases, Catalyze transcription reaction
open DNA template transcription factors Bind to cis-elements of DNA
– Basal factors – initiate transcription at correct site
– Upstream factors – facilitate and augment efficiency of transcription
– Inducible factors – respond to environmental agents to affect gene
expression
Formation of RNA Messenger RNA Template for polypeptides
Transfer RNA Carriers for amino acids
Ribosomal RNA Forms ribosome where transcription occurs
Small RNAs Accessory roles in transcription and translation
Modification of RNA Cleavage of pre-messenger RNA Endonuclease activity Cleaves messenger RNA to function size
Addition of bases Poly(A) polymerase Polyadenylation of messenger RNA
Splicing/removal of introns Transesterification Allows several isoforms of a protein to be made
Transport of messenger RNA Messenger RNA plus binding proteins 5ø cap acts as signal for transport.
to cytoplasm Messenger RNA binds poly(A)-
binding protein in cytoplasm
Commencement of Peptide chain formed by addition of amino acids Initiation factors Forms complex of guanosine triphosphate and
messenger RNA translation Elongation factors transfer RNA
and peptide synthesis
Postranslational Targeting or sorting Leader sequence of 25 amino acids Allows targetting to specific organelles
Modification Translocation Sequence in amino terminus Allows movement across membranes

259
Periodontal tissue engineering
Bartold et al.

by soluble mediators and these responses, in turn, ora of controlling and regulatory mechanisms
affect cell-matrix and cell-cell interactions. through which protein synthesis can be regulated:
It is well established that genes contain the for example, DNA methylation, synthesis or regula-
necessary coding information for the production of tion of transcription factors, RNA editing and spli-
proteins. In recent years significant inroads have cing as well as translational and posttranslational
been made in understanding how gene expression is modifications. At each stage of gene expression, RNA
regulated and, more importantly, how this may be synthesis and peptide synthesis, there is an oppor-
used to clinical advantage through ‘‘gene manipula- tunity for control or regulation – these areas offer
tion’’. Although beyond the scope of this discussion, exciting prospects for the future in terms of manipu-
some background knowledge of gene expression and lation of gene expression and regulation of cell func-
control is necessary for a general overview, see Bar- tion. However, before such external control can be
told & Narayanan (5). An overview of the events and effected, ongoing studies into the molecular nature
factors involved in gene expression and protein syn- of the control of cell function through the effects of
thesis is shown in Table 6 and Fig. 3. soluble mediators is needed.
Very simply, genes are specific portions of DNA Soluble mediators of importance in regeneration
that code for proteins. They are normally inactive yet include growth factors such as platelet-derived
can be activated by special proteins called transcrip- growth factor, transforming growth factor-b, and
tion factors (which themselves are gene products). fibroblast growth factor, cytokines including in-
Following activation, DNA partly unravels and allows terleukin-1, tumor necrosis factor-a and interleukin-
the binding of RNA polymerase, commencement of 4, lymphokines such as interferon-g as well as
transcription, or the reading of the DNA coding se- fibronectin and other adhesion molecules (Table 7).
quence, and the eventual assembly of the messenger Some of these have been used already in animal
RNA complex. The messenger RNA is eventually studies of periodontal regeneration (54, 89). During
transported to the cytoplasm, and within the rough inflammation and in wound healing, these sub-
endoplasmic reticulum, the assembly of amino acids stances are secreted by inflammatory cells, by resi-
occurs leading to the formation of a full-length pro- dent connective tissue cells and may also be derived
tein. Following the formation of the polypeptide from the tissue matrix. Many of these molecules
chain, substantial modification or posttranslational such as the bone morphogenetic proteins and the
modifications may occur before the protein reaches transforming growth factor family of proteins are
its final form ready for secretion from the cell. sequestered as components of the extracellular ma-
Throughout all of these processes, there are a pleth- trix of mineralized tissues (19, 40). The mechanism
of action of these soluble factors is considered to be
via cell surface receptors which, in turn, leads to an
effect on gene expression through the regulation of
inducible transcription factors (59, 105).
For convenience, the molecules necessary for
periodontal regeneration can be roughly grouped
into three families – polypeptide growth factors,
attachment or adhesion proteins and structural
components. The outcome of the action of each
group of these molecules may vary depending upon
the stage of healing and target cells available. Many
of these molecules have been identified in the ce-
mentum and alveolar bone matrix (57, 71, 72). These
include the adhesion proteins osteopontin, bone sia-
Fig. 3. Schematic representation of events involved in
loprotein and fibronectin that have been shown to
gene expression and protein synthesis. 1. Activation of be expressed early in development during cemento-
transcription via cell surface receptor. 2. Transcription of genesis, and growth factors platelet-derived growth
the DNA code into messenger RNA (mRNA). 3. Processing factor and fibroblast growth factor (fibroblast growth
of messenger RNA in preparation for transportation to factor-1 and -2), transforming growth factor-b, insu-
cytoplasm. 4. Transport of messenger RNA to cytoplasm.
5. RNA translation and peptide synthesis. 6. Polypeptide
lin-like growth factor-I and bone morphogenetic
elongation. 7. Postranslational modification. 8. Transport proteins. These growth factors have a broad range of
to and across cell membrane. biological activities on periodontal cells and affect

260
 Periodontal tissue engineering

Table 7. Some mediators affecting cell proliferation and matrix synthesis in periodontal fibroblastsa
Mediator Proliferation Collagen Collagenase Proteoglycan Hyaluronan
⇓ ⇓ ⇓
Transforming growth factor-b – ⇓
⇓ ⇓ ⇓ ⇓ ⇓
Platelet-derived growth factor
Interferon-g – ⇓ ⇓ ? ?
⇓ ⇓ ⇓ ⇓ ⇓
Interleukin-1a and b
⇓
Tumor necrosis factor-a – ⇓ ?? ??
⇓ ⇓
Prostaglandin E2 ⇓ ⇓ ⇓
⇓ ⇓
Interleukin-6 ? – ?
⇓ ⇓
Insulin-like growth factor-1 (weak) – ? nil
⇓
Basic fibroblast growth factor ⇓ ? ? ?
⇓ ⇓ ⇓
Epidermal growth factor (weak) – –
a
For references refer to the text.

their migration, proliferation and differentiation (57, through a similar sequence of synthesis and seques-
71, 72, 80). Interestingly, the presence of some of tration of soluble mediators, both of which result in
these components may be limited to some peri- homeostatic feedback control mechanisms (19). As
odontal tissues, and their activities may vary with detailed in the previous section, cells can respond
different periodontal cell types (2, 42, 68, 75, 90, 97). to their environment through the interaction of cell
Although the biological activation of many of surface receptors and the activation of intracellular
these substances has been studied in detail in sev- signalling events, which ultimately lead to a re-
eral in vitro and wound healing model systems, a sponse by the cell. Notwithstanding the importance
few general points can be made in the context of of soluble mediators in regulating cell function, cells
periodontal regeneration. First, availability and ac- also dynamically interact with components of the
tivity of polypeptide growth factors from inflamma- extracellular matrix via similar mechanisms (Fig. 4).
tory cells and from the matrix may appear redun- The specific mechanisms by which the matrix
dant and superfluous. Nevertheless, the same growth regulates gene expression are unclear but may differ
factors may play different roles depending upon the substantially from those noted for the soluble factors
healing stage, target cell type and availability of ma- and inducible transcription factors. Cells interact
trix components. For example, a growth factor may
have primarily a proliferative function during early
stages of healing and prior to extracellular matrix
formation. However, with the formation of an extra-
cellular matrix, the same molecule may promote cel-
lular differentiation. This concept that the presence
of a mature and functional extracellular matrix per-
mits the major action of growth factors to be di-
rected primarily towards the recruitment and differ-
entiation of cells is fundamental to our future pro-
gress in the field of periodontal regeneration.

Role of the evolving and developing extracellular

matrix
Fig. 4. Schematic representation of the interaction be-
Although the extracellular matrix does not directly tween extracellular matrix and gene expression. Compo-
promote regeneration, it plays a prominent role in nents located within the extracellular matrix (either ma-
regulating the events necessary for regeneration. In- trix molecules or bound growth factors) can interact with
specific cell surface receptors thereby activating a number
deed, the temporal and spatial sequence of extracel- of intracellular signalling mechanisms which ultimately
lular matrix macromolecule deposition during tissue lead to specific gene expression for matrix synthesis which
repair and regeneration is intimately coordinated ultimately can lead to a self-regulatory feedback loop.

261
Bartold et al.

with many matrix components via integrins on the the complexity of controlling cell proliferation
cell surface, which leads to reorganization of cyto- through the use of growth factors. Indeed, such
skeletal proteins and their formation of complexes complexity highlights the naivety of current ap-
with other intracellular proteins, which serve to proaches to using growth factors, and it may be
function as transduction molecules (1, 3, 21, 39, 88). that more precise means of targeting intracellular
In addition, reorganization of the cytoskeleton can controlling mechanisms are required should we
lead to restriction of cytokine receptor action as well wish to dictate cell behavior along the path of re-
as the sequestration or localizing of growth factors generation.
to the cell surface for subsequent receptor-mediated Through its component adhesion molecules, the
signal transduction and gene activation (31). extracellular matrix could participate in recruit-
Through the above mechanisms the extracellular ment of cell types. For example, fibronectin and
matrix, which consists of collagens, fibronectin, lam- type I collagen permit adhesion of most cell types,
inin and other non-collagenous proteins and proteo- whereas laminin and type IV collagen are selective
glycans, regulates many cellular activities (91, 110, to certain cell types, and tenascin is antiadhesive.
114). It affects the migration, division and differen- Therefore, the extracellular matrix (together with
tiation of cells, serves as a substratum for cell ad- any available growth factors) can determine what
hesion, and promotes cell spreading and cytoskeletal cells are enlisted and whether a wound heals by
organization (3, 4). The extracellular matrix also pro- regeneration or repair. Thus, it is possible that the
tects cells from apoptosis through mechanisms in- absence of a suitable extracellular matrix is one
volving integrins and downstream events involving reason why cementum does not form on planed
insulin signals, insulin receptor substrate-1, focal ad- tooth root surfaces.
hesion kinase and phosphatidylinositol-3 kinase (15,
28, 30, 50). In the context of periodontal regenera-
New cementum formation
tion, it will be important to determine which compo-
nents of the extracellular matrix regulate gene ex- Many studies indicate the important role of ce-
pression of growth factors, their receptors, how cells mentum in the formation and regeneration of
respond to various agonists and gene regulation of periodontal tissues (9, 94, 95, 112). Frequently new
cell movement, proliferation and differentiation (1, attachment requires new cementum formation to
4, 39, 53, 91). Clearly, the matrix elements of the vari- replace diseased root surfaces contaminated with
ous periodontal compartments contain the necess- bacterial endotoxins and which are removed dur-
ary instructive messages necessary for normal func- ing periodontal therapy. Although some infor-
tion. What is unclear at present is how to unravel mation is available on the biology of cementogen-
those messages of importance to induce and direct esis in mice during development (17, 96), how ce-
periodontal tissue regeneration. mentum formation is regulated in adult humans is
Adhesive interactions with the matrix play a cru- not clear. A major question in this context is what
cial role during development and whether cells di- is the source of the putative progenitor cemento-
vide or differentiate can also be determined by the blast populations and where is it located? The sec-
extracellular matrix (1, 39, 43, 91). As discussed ond key question is which molecules control the
above, cells interact with the extracellular matrix proliferation and concomitant migration of these
through specific integrin receptors, which initiate a populations towards the root surface, their attach-
cascade of signaling reactions that include tyrosine ment to this surface, and terminal differentiation?
phosphorylation of focal adhesion kinase, acti- The connective tissue matrix of cementum seques-
vation of mitogen-activated protein kinase cascade, ters growth factors such as the transforming
expression of c-fos and elevation of certain cyclin growth factor-b, fibroblast growth factors and ce-
levels (3, 4, 8, 27, 38, 43, 46, 92, 107, 111, 113, 115). mentum-derived growth factor as well as a battery
Many of these events are also induced by growth of other polypeptides, including osteopontin, bone
factors (16). The signalling pathways induced by sialoprotein-II and cementum-specific attachment
both extracellular matrix components and growth protein which mediate cell adhesion and spreading
factors cooperate in mediating their biological (57, 61, 73, 77, 81, 97). These molecules affect the
functions; for example, both integrin and growth migration, attachment and proliferation of peri-
factor mediated signals are necessary for express- odontal cells and their matrix synthesis and, more
ion of cyclins, which regulate cell cycle progression importantly, they manifest cell specificity, and
(3, 4). This information can help us to understand tissue specificity among the same cell type (73, 77,

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 Periodontal tissue engineering

82, 97). The extracellular matrix of cementum has cells or instructive messages incorporated into it, we
the potential to regulate the differentiation of pre- believe it will be possible to overcome many of the
cursor cells into cementoblasts (99). Thus, ce- limitations associated with conventional regenera-
mentum components are capable of providing in- tive technologies (listed earlier in this chapter). On
formational signals for the recruitment, prolifer- the basis of success in other anatomic sites, a tissue
ation and differentiation of periodontal cells and engineering approach for periodontal regeneration
regulate the regeneration of cementum as well as will better address our working paradigm that the
adjacent periodontal components. use of a material or procedure that takes into ac-
count the next functional stage of reconstruction will
be essential for regeneration (14, 33, 83, 85) (Fig. 5).
Periodontal tissue engineering – Before tissue engineering for periodontal defects
a new vista for periodontal becomes commonplace, a number of issues still need
regeneration to be addressed that relate to the success of tissue en-
gineering for any component of the musculoskeletal
Tissue engineering is the emerging field of science system. The requirements for successful tissue engin-
aimed at developing techniques for the fabrication eering have been divided into two main areas (14).
of new tissues to replace damaged tissues and is These include engineering issues related to main-
based on principles of cell biology, developmental taining an in vivo cell culture substratum such as bio-
biology and biomaterials science (74, 85, 102). This mechanical properties of the scaffold, architectural
contemporary area of applied biomedical research is geometry and space maintaining properties. The sec-
attracting considerable attention from both the pri- ond group of requirements relate to the biological
vate and government sectors because of its consider- functions of the engineered matrix, including cell re-
able economic potential (55). cruitment, permission of neovascularization and de-
The main requirements for producing an engine- livery of the requisite morphogenetic, regulatory and
ered tissue are the appropriate levels and sequencing growth factors for tissue regeneration. Many of these
of regulatory signals, the presence and numbers of specific features are listed in Table 8.
responsive progenitor cells and an appropriate
extracellular matrix or carrier construct. Recent ad-
Space maintenance within the defect site
vances in growth factor biology and biodegradable
polymer constructs have set the stage for successful For over 50 years, it has been recognized that bone will
tissue engineering of cartilage, bone and related grow into an adjacent tissue space provided that
tissues of which the periodontium could be con- space can be maintained and soft tissue ingrowth pre-
sidered a prime candidate for such procedures. Pre- vented (6, 41). Such natural phenomena should be
liminary studies have indicated that periodontal used to advantage when considering tissue engineer-
ligament and bone cells can be transplanted into ing and placement of bioengineered matrices for re-
periodontal sites with no adverse immunologic or generation. The engineered material should be of suf-
inflammatory consequences (48, 58, 103). ficient form to allow placement into a defect and pre-
It is clear that the regenerative events of peri- vent subsequent collapse of the repositioned tissues
odontal wound healing require recruitment of pro- into the defect site. Thus, the material should act in a
genitor cells, which have the potential to differen- manner consistent with the principles of guided
tiate into specialized regenerative cells, followed by tissue regeneration and have similar design features
proliferation of these cells, and synthesis of the spe- (93). As a result, a sufficient wound space and a suit-
cialized components of the connective tissues they able environment for regeneration will act synergist-
are attempting to repair. Thus a tissue engineering ically to permit the cascade of molecular and molecu-
strategy for periodontal regeneration that exploits lar events needed to drive the regenerative process.
the regenerative capacity of cells residing within the Design features to obtain satisfactory space main-
periodontium could involve the use of such cells tenance would include an ability to be easily cut or
grown within a three-dimensional construct and molded into a desired shape and be of a consistency
subsequent implanted into the defect. In doing so, compatible with easy handling (14). The scaffold
the need for recruitment of cells to the site is ne- should be of sufficient rigidity to withstand soft
gated and the predictability of the outcome may be tissue collapse into the defect (93). The internal
enhanced. Through the provision of a prefabricated architecture of the scaffold should be such as to
three-dimensional structure with the appropriate maximize rapid colonization by cells of the desired

263
Bartold et al.

Fig. 5. Schematic representation of a tissue engineering allow synthesis of an appropriate matrix suitable for im-
approach to periodontal regeneration. A. Synthetic scaf- plantation. D. Periodontal defect. E. The ‘‘engineered’’ ma-
folds can be prepared which can be cut and molded into trix is implanted into the periodontal defect site which,
desired shapes and forms. B. Cells from the periodontal with an adequate epithelial seal, will lead to a regenerative
tissues (either as primary cell cultures or even genetically response. F. Fully regenerated periodontal defect resulting
manipulated for the expression of specific matrix compo- from implanted engineered tissue. PDL: peridontal liga-
nents) can be seeded into the scaffold in vitro. C. The ment.
seeded cells are cultured in vitro for a specified time to

phenotype and ingrowth of tissue compatible with epithelium and connective tissue, yet also permit
those to be regenerated (108). selective ingrowth of regenerative tissues (18, 108).
To achieve this, design features will have to be incor-
porated whereby the external surface may be exclu-
Barrier or exclusionary functions
sionary yet the internal scaffold conducive to new
The engineered tissues should act as a barrier to the tissue ingrowth. Consistent with the principles of
ingrowth of unwanted tissues, for example, gingival guided tissue regeneration, an engineered matrix

264
 Periodontal tissue engineering

should exclude any unwanted tissue components yet Table 8. Requirements for successful tissue
promote growth, differentiation and maturation of engineering
desired tissues (cementum, periodontal ligament Biomechanical features
and bone). Perhaps of interest here is the important O Space maintenance
O Barrier or exclusionary features
sealing role the epithelium plays. Thus, a principle
Biological functions
aim of tissue engineering for the periodontium O Biocompatibility
should not be total exclusion of the epithelium from O Incorporation of cells
O Incorporation of instructive messages
the site but rather encouraging it to form a rapid
and successful biological seal to protect the fragile
underlying regenerating events (104).

edge of what constitutes cells with a ‘‘periodontal re-

Biocompatibility and design features
generative phenotype’’, it should be possible to cul-
The scaffold material should be either biocompat- ture and subsequently incorporate these cells into a
ible with the tissues to be regenerated or biodegrad- suitable biodegradable scaffold for immediate intro-
able, allowing for gradual replacement with regener- duction into a periodontal defect. The possibility of
ated tissue (34, 101). Furthermore, regeneration of genetically altering the ability of the cells to produce
tissues through the use of bioengineered products particular matrix components or instructive mess-
appears to be dependent upon the porosity and pore ages also exists (13, 26, 86). However, such procedures
size of the supporting three-dimensional structure introduce the problem of biosafety with regards to
(10, 108). Thus, design features need to include con- genetic manipulation.
siderations of pore size for both cell attachment and Likely sources of cells for periodontal tissue engin-
incorporation in vitro as well as subsequent tissue eering are to be from the host site (cementum, peri-
maturation during in situ regeneration. Materials odontal ligament and bone). Whether the so-called
tested to date include calcium phosphate, hydroxy- progenitor cells that reside in these tissues can be
apatite, extracellular matrix components (collagens, isolated and propagated in culture for future seeding
hyaluronan and fibronectin) polyglycolic acid and remains to be established (116).
other synthetic bioresorbable materials (14). The
issue of biosafety needs to be addressed as well. Al-
though no guidelines seem to have been established Incorporation and bioavailability of instructive
messages
for assessment of the safety and efficacy of cell-
based and tissue-based tissue-engineered products, The synthetic matrix should be bioresorbable but
clearly these materials should be free from transmit- constructed from a material with a suitable affinity
table disease, immunologically inert and not induce for the adsorption of appropriate growth/differen-
an overexuberant inflammatory response (78). tiation factors as well as integrins, cell receptors and
In the case of the periodontium, a biodegradable other instructive molecules normally found in re-
material that would degrade slowly over time and be generating tissues (7, 23, 85, 98). As more data be-
replaced with a soft connective tissue compatible come available, the precise signaling molecules re-
with periodontal ligament, together with new al- quired for growth, differentiation and subsequent
veolar bone and root surface cementum with appro- gene expression for particular extracellular matrix
priately orientated and inserted collagen fibers, proteins in an orderly manner may be incorporated
would be desirable. into engineered matrices for regenerative purposes.
Agents that promote cell adhesion, permit prolifer-
ation of progenitor cells and can be easily incorpor-
Incorporation of cells with appropriate phenotype ated into a scaffold or matrix for suitable slow re-
for ongoing periodontal regeneration
lease with adequate delivery kinetics will become the
Skin substitutes that contain cultured cells have been major focus of interest. One major obstacle to be
available for some time (11, 83). Although rather sim- overcome in this regard is the control of delivery. For
pler in their composition compared to the periodon- example, because the particular inductive agent is
tium, bioengineered skin substitutes with incorpor- released from the biomatrix, it may be capable of
ated cells can provide almost unlimited quantities of inducing ectopic tissue formation in neighboring re-
tissue for wound management and illustrate the po- gions (45). Thus, control and containment of the
tential of such an approach. With increasing knowl- agent is paramount for its effectiveness and safety.

265
Bartold et al.

Concluding comments – new oral health? At present, there is no good evidence

horizons for the management of the to suggest that current regenerative technologies will
inflammatory periodontal diseases enhance tooth retention or lead to improved oral
health. However, this does not preclude an ongoing
As the causative and pathogenetic processes of the search for regenerative technologies. Indeed, we be-
inflammatory periodontal diseases are better under- lieve the converse to be true. Continuing efforts
stood, new vistas in management of these problems should be made to explore means of attaining true
will evolve. These vistas may develop along three regeneration of all periodontal connective tissues
fronts and address issues relating to the causation of (cementum, ligament and bone) to a level consistent
the disease, the host response to the disease and the with health and thus restore original form and func-
anatomic changes occurring as a result of the dis- tion to the dentition with a resultant improved tooth
ease. With respect to the issues discussed in this retention rate.
chapter, future developments in the fields of mol-
ecular and cell biology, developmental biology and
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