TISSUE ENGINEERING: Part B

Volume 16, Number 2, 2010

ª Mary Ann Liebert, Inc.
DOI: 10.1089=ten.teb.2009.0562

Periodontal Tissue Engineering and Regeneration:

Current Approaches and Expanding Opportunities

Fa-Ming Chen, D.D.S., Ph.D.,1 and Yan Jin, D.D.S., Ph.D.2

The management of periodontal tissue defects that result from periodontitis represents a medical and socioeco-
nomic challenge. Concerted efforts have been and still are being made to accelerate and augment periodontal
tissue and bone regeneration, including a range of regenerative surgical procedures, the development of a variety
of grafting materials, and the use of recombinant growth factors. More recently, tissue-engineering strategies,
including new cell- and=or matrix-based dimensions, are also being developed, analyzed, and employed for
periodontal regenerative therapies. Tissue engineering in periodontology applies the principles of engineering and
life sciences toward the development of biological techniques that can restore lost alveolar bone, periodontal
ligament, and root cementum. It is based on an understanding of the role of periodontal formation and aims to
grow new functional tissues rather than to build new replacements of periodontium. Although tissue engineering
has merged to create more opportunities for predictable and optimal periodontal tissue regeneration, the tech-
nique and design for preclinical and clinical studies remain in their early stages. To date, the reconstruction of
small- to moderate-sized periodontal bone defects using engineered cell–scaffold constructs is technically feasible,
and some of the currently developed concepts may represent alternatives for certain ideal clinical scenarios.
However, the predictable reconstruction of the normal structure and functionality of a tooth-supporting apparatus
remains challenging. This review summarizes current regenerative procedures for periodontal healing and re-
generation and explores their progress and difficulties in clinical practice, with particular emphasis placed upon
current challenges and future possibilities associated with tissue-engineering strategies in periodontal regenera-
tive medicine.

Introduction lenge.7–11 Major scientific advances in periodontology over

the past 150 years have fundamentally changed how clini-

P eriodontitis is one of the most common inflammatory

diseases of humans and a leading cause of tooth loss in
adults. It is characterized by progressive destruction of the
cians detect and treat periodontal diseases; however, there is
still no ideal therapeutic approach today to cure periodontitis
or to achieve predictable and optimal periodontal tissue re-
tooth-supporting apparatus, including gingiva, alveolar bone, generation.6 Current treatment for periodontal defects there-
periodontal ligament (PDL), and root cementum; if left un- fore does not restore function to the multiple tissues in many
treated, it can lead to the loosening and subsequent loss of cases.2,5,6,12 Conventional mechanical or antiinfective peri-
teeth.1,2 Periodontitis has also been linked to many systemic odontal therapies have focused upon eliminating inflamma-
disorders, such as coronary artery disease, stroke, and diabe- tory processes and halting disease progression.12,13 To restore
tes, thus causing an increased risk of heart attack or other lost tooth-supporting structures, various regenerative proce-
serious health problems.2–4 Although the cornerstone of dures have been proposed, tested, and evaluated in the last
successful treatment starts with establishing excellent oral two decades. This has included the use of a range of surgical
hygiene, followed by the removal of bacterial plaque and procedures, the use of occlusive barrier membranes,14 the
calculus to control inflammation and stop progressive bone use of a variety of bone grafts and osteoconductive bioma-
loss, the complete repair and regeneration of functional hy- terials, and, more recently, the use of purified protein mix-
brid periodontal tissues remains an elusive but laudable goal tures from developing teeth such as Emdogain
(Biora AB,
in the treatment of periodontal disease.5,6 Periodontal regen- Malmö, Sweden) and growth factors from recombinant
erative medicine is a subject of clinical science, basic science, technology.7–9,15–19 Among them, the regeneration of dam-
and engineering concerns that represents a formidable chal- aged periodontal structures with various bone graft materials

1Department of Periodontology and Oral Medicine, School of Stomatology, and 2Research and Development Center for Tissue

Engineering, Fourth Military Medical University, Xi’an, Shaanxi, P.R. China.

219
220 CHEN AND JIN

and guided tissue regeneration (GTR) strategies has achieved amount of tissue can be highly beneficial to the patient. In
some success in certain ideal clinical scenarios, but outcomes implantology, there also appears to be an urgent need for the
are variable, depending upon multiple factors such as defect concept of tissue and bone regeneration around implants due
size and type, patient age and education, genetics, and, in- to the increasing need to utilize dental implants for growing
deed, operator skills.8,14 An analysis of the data published to population of patients and to enhance their function to sim-
date demonstrates that the outcomes of these therapies from ulate normal tooth physiology and proprioception.
both preclinical and clinical studies remain limited and un- Tissue-engineering strategies in regenerative medicine
predictable, especially in terms of cementum and functional have involved a combination of cells, engineering materials,
PDL regeneration.5,11,20 and suitable biochemical and physicochemical factors to im-
Notwithstanding such limitations, accepted periodontal prove or replace biological functions in an effort to effect the
therapies have, for some time, included implantable materi- advancement of medicine.21–25 The creative processes that
als, either bone substitutes or grafts aimed at replacing peri- define tissue engineering must be achieved by cells (host and=
odontal defects with some form of structure that can replicate or donor) by providing them with the appropriate spatial and
part, or all, of the function that has been lost.7,14,16,18 However, temporal cues to enable growth, differentiation, and synthesis
even with the current gold-standard material for bone re- of a functional extracellular matrix (ECM) of sufficient volume
generation, the failure rates have been reported to be as high and functional integrity.22,33–36 Although there are several
as 30% in maxillofacial and craniofacial surgery, in addition to general definitions of tissue engineering, the concept for
their drawbacks such as donor-site morbidity, high variability periodontal regeneration has been that periodontal tissue
in outcome, and limited availability.6 The failure rate can be engineering is a contemporary area of persuasion of the
easily estimated as much higher if the regeneration of multiple periodontal tissue to heal itself through the delivery of cells,
tissues in the periodontium is considered. Synthetic materials biomolecules, and=or supporting structures, including medi-
are primarily designed to be permanently implanted, where cal devices, matrix, cell=tissue scaffolds, or more complex
long-term complications include stress shielding, loosening, tissue-engineered constructs.21–24,29,30 The lack of clinically
and mechanical or chemical breakdown of the materials efficient modalities of treatment has prompted research into
themselves.15–19 In many cases, replacement or augmentation tissue engineering that exploits the potential of matrix-
of the periodontium using synthetic biomaterial devices ap- assisted cell therapy for the management of periodontal dis-
pears to lead to periodontal repair, but with restoration of eases. However, if we consider the strategies for protein
neither the original periodontal structure nor its physiologi- delivery and cell therapy that are currently being applied
cal function.5,6,11,18 It is clearly a highly challenging task to in periodontal tissue engineering, it is soon realized that
manage periodontal destructions using existing therapeutic much more can be done. In the near term, endeavors for this
modalities and regenerative techniques, because the ability end are likely to involve the identification of additional spe-
to completely and predictably regenerate lost periodontal cific growth factors and evaluation of the optimal combi-
structures has not been achieved in humans, and only limited nation of cells, growth factors, and scaffolds that is able to
data are available to recommend one form of clinical therapy mimic the critical aspects of the natural healing processes
over another.5,10 occurring in the periodontium. An enormous accumulation of
The logical conclusion arising from recent research in this scientific discoveries (theories, principles, concepts, and facts)
field is that periodontal regeneration rather than repair re- provides fuel for periodontal tissue engineering in modern
mains the desired optimal outcome.21–24 The desire to create dentistry.
more biological alternatives to the permanent implantation Use of the principles of tissue engineering in a rational
of static synthetic materials for the management of peri- manner offers promise for regenerating or developing de novo
odontal defects has inspired the field of periodontal tissue periodontal or other hybrid tissues; however, it is important
engineering and regenerative medicine. It is anticipated that to bear in mind that all tissue-engineering techniques at-
tissue-engineering methods could overcome some of the tempted to date are still in their early stages and have not yet
limitations associated with the current clinically available had a significant impact either clinically or commercially.21–27
strategies.21–23,25–30 Basic tissue-engineering approaches in- As will be discussed later in this review, the regeneration of
volve the utilization of in-vitro-expanded cells with regen- periodontal complex tissues requires a synthesis of biology,
erative capacity and their delivery to the appropriate sites medicine, and material science, where the underlying science
using biomaterial scaffolds. Along with genetic- and protein- thus far is largely related to cell, molecular, and develop-
based approaches, tissue engineering has become one of the mental biology, as well as embracing advances in biotech-
principal research areas that offer expanding opportuni- nology and bioengineering.22,27,28,37–41 This review considers
ties for the management of periodontal defects.23,30,31 Ideally, the possibilities for tissue-engineering research to contribute
in-vitro-expanded cells in sufficient quantities and possessing to this multidisciplinary endeavor and presents a broad over-
the potential to regenerate alveolar bone, cementum, and view of the collective knowledge of periodontal regenerative
PDL could be used with appropriate biomaterials to engineer medicine, utilizing a traditional rather than a systematic ap-
living tissues in vitro for subsequent transplantation into proach. Progress in our understanding of the principles of
defect sites.31,32 At the same time, the utilization of gene periodontal healing=regeneration and the structure–function
therapy to sustain the release and bioavailability of osteo- relationships within the periodontium will then be discussed
genic growth factors also offers potential for periodontal as this provides the foundation for developing strategies for
tissue-engineering and regeneration applications.21,23,26 The reparative and regenerative medicine. We emphasize that
use of tissue-engineering protocols for periodontal regener- tissue-engineering strategies that have been developed via
ation presents numerous advantages. One of the most re- cell- and=or matrix-based approaches have great potential for
markable advantages is that even the regeneration of a small future periodontal tissue regeneration strategies, highlighting
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 221

the current status and reported achievements as well as dif- gival collagen fibers to both the cementum and alveolar
ficulties and opportunities in the clinical therapeutic appli- bone, that is, to obtain total regeneration of the period-
cation of periodontal tissue engineering. ontium, deterioration of which during periodontal disease
can ultimately lead to the loss of teeth.11 Although not the
The Periodontium: Physiology emphasis of this article, a brief overview of the structure–
and Structure–Function Relationships function relationships of periodontal tissues is critical for
the development and optimization of any regenerative pro-
Given that periodontal tissue regeneration is based
cedures.
upon the principles of developmental, cellular, and molecu-
lar biology, then an understanding of the formation and
PDL fibers
structure of periodontal tissues, their physicochemical char-
acteristics, and the molecular events that define normal The PDL, the fiber-like tissue that joins the teeth to the
periodontal function is vital, especially for those scientists sockets (alveoli), is a group of specialized connective tissue
from material science who contribute their efforts and time in fibers that essentially connect the surface of the tooth root
the ambitious purpose of periodontal regeneration.11,20,27,42 with the bony tooth socket. These fibers help the tooth with-
The development of the periodontium is initiated after crown stand the naturally substantial compressive forces that occur
formation. Hertwig’s epithelial root sheath (HERS) ruptures during chewing and remain embedded in the bone. Com-
and allows mesenchymal cells to contact root dentin and pared with most other ligaments of the body, they are highly
form cementum. When the crown of the tooth erupts toward vascularized. Once destroyed, there are currently no artificial
the oral mucosa, fibroblasts become active and begin to biomaterials that can be employed to replace or repair the
produce collagen fibrils, which eventually form the PDL.43 highly regular and oriented principal fibers of the PDL. Thus,
Thus, it can be seen that the periodontium is an extremely regeneration offers the only approach to restore the PDL after
complex tissue comprised of at least two hard and two soft damage. The PDL fibers are composed primarily of type I
tissues, that is, root cementum, PDL, bone lining the tooth collagen, but type III fibers are also involved. These fibers
socket (alveolar bone), and the part of the gingival that faces make up of many bundles of connective tissue in six distinct
the tooth (dentogingival junction) (Fig. 1). The periodontium groups according to their different orientations, distributions,
functions to anchor the teeth to the jaws (mandible and and functions and are linked with blood vessels, lymph ves-
maxilla) as well as to provide nourishment to the teeth.1,2,43 sels, and nerves. The six groups are as follows:27,42
When considered the development of new regenerative strat-
the transseptal group,
egies to enhance periodontal therapy outcome, it is impor-
the alveolar group,
tant to understand that each of the periodontal components
the horizontal group,
has a very specialized structure and that these structural
the oblique group,
characteristics directly define function. Indeed, proper func-
the apical group, and
tioning of the periodontium is only achieved through struc-
the interradicular group.
tural integrity and interaction between its components.1
Therefore, a critical challenge for regenerative periodontal All fibers have physiological as well as supportive responsi-
treatment is to restore the attachment of the PDL and gin- bilities to the dentition and function in a cooperative manner.
Sharpey’s fibers are the ends of principal fibers of the PDL that
insert into the cementum. Fibroblasts, osteoblasts, and ce-
mentoblasts can all be identified in healthy adult PDL.42,43 It is
found that the healthy PDL harbors stem cell niches and ECM
microenvironments where multipotent stem cells can syn-
thesize and remodel the three tissues of mesenchymal origin
that form the periodontium; thus, the PDL retains regenera-
tion potential to a certain degree.27,42 The ligament functions
to provide a protective sheath for vessels and nerves to
transmit occlusal forces to bone, to attach teeth to bone, to
maintain the gingival tissues, and to resist the impact of oc-
clusal forces. The formation of optimal alveolar bone and ce-
mentum is crucial for the regeneration of a functionally
effective PDL, including the optimal orientation and insertion
of Sharpey’s fibers into exposed root surfaces and bone, that
can support a healthy tooth and has adaptive responses to
occlusal force loading.11

Root cementum
Root cementum, a specialized mineralized tissue lining
the tooth root surface, is another essential component of the
FIG. 1. Schematic illustration of the tooth-supporting ap- periodontium and is a calcified avascular mesenchymal tissue
paratus in normal periodontium, which is comprised of existing in acellular and cellular forms.1 Although cemen-
gingival, alveolar bone, cementum, and periodontal ligament tum is anatomically an integral part of the tooth, functionally,
(PDL). it is a component of the periodontium, and its major role is to
222 CHEN AND JIN

serve as the site of attachment for principal collagen fibers fective approaches for bone regeneration are needed to reverse
(Sharpey’s fibers).20 Cementum is excreted by cells called ce- bone loss caused by periodontitis. Unfortunately, the science
mentoblasts within the root of the tooth and is thickest at the of bone regeneration itself is still its infancy in the mineralized
root apex. Its coloration is yellowish, and it is softer than tissue field, with all current regenerative therapeutics hav-
enamel and dentin due to being less mineralized. Although ing serious limitations. The reconstruction of the bone–PDL–
cementum tissue composition resembles that of bone, there cementum apparatus is a highly challenging task, presenting a
are distinct structural and functional differences between more difficult and complex process as compared with simple
these two mineralized tissues.20 Acellular extrinsic fiber ce- bone regeneration due to the multiple tissues involved in the
mentum (AEFC) (primary cementum or acellular cementum) periodontium.10,20,29
contains extrinsic fibers (Sharpey’s fibers) and serves to an-
chor the root to the PDL. It develops very slowly and is con-
Physiology and wound healing in the periodontium
sidered to be acellular since the cells that form it remain on its
surface. It is formed first and covers approximately the cer- The periodontium’s main responsibility is to support teeth
vical third to half of the root.1,11,20 Cellular intrinsic fiber ce- during function, and it relies upon the stimulation it receives
mentum (secondary cementum or cellular cementum) is during normal functioning for the preservation of its struc-
formed after the tooth reaches the occlusal plane and is more ture.29 Due to external forces, the alveolar bone undergoes
irregular in composition when compared with AEFC. Ce- constant remodeling. The PDL can also accommodate in-
mentum is very permeable in young patients and becomes creased function by increasing in width. When forces are re-
less so with age. The normal thickness ranges from 16 to 30 mm duced, the PDL will effectively atrophy. Vascularization of
at the coronal half and reaches 150 to 200 mm at the apex. the supporting structures is derived from the inferior and
Pathological conditions associated with cementum include superior alveolar arteries. The PDL receives blood from the
resorption (caused by either local or systemic causes) or an- apical vessels, penetrating vessels from the bone, and anas-
kylosis (fusion of cementum and alveolar bone).1 tomosing vessels from the gingiva. Venous drainage accom-
It has been suggested that the regeneration of acellular panies the arterial supply, whereas lymphatics supplement
cementum is the gold standard in periodontal regeneration.20 the venous drainage system.45 The regeneration of periodon-
However, new AEFC formation and the restoration of a tal tissues such that their original form, architecture, and
connective tissue attachment to that cementum have yet to function are restored has been a major goal of periodontal
be predictably demonstrated.11 Although the composition of research. To fully appreciate what is involved, the cross-talk
the ECM of cementum closely resembles that of other min- among the components of the periodontium (cellular and
eralized tissues, it has a unique physiology and contains matrix), together with the inherent regenerative capacity of
molecules previously not described in other tissues.20 The this tissue, needs to be considered.22 Although the general
cementum matrix appears to play a key role in maintaining principles of healing and the cellular and molecular events
the homeostasis of cementum under healthy conditions.11,20 observed in nonoral sites also apply to the healing processes
Therefore, for new cementum regeneration to occur, the local that take place after periodontal surgery, a more difficult and
environment must be conducive to the recruitment and complex situation presents itself when a mucoperiosteal flap
function of cementum-forming cells, and the wound matrix is apposed to an instrumented root surface previously de-
should be favorable for the regeneration of these three quite prived of its periodontal attachment.46 The events associated
different tissues.27 with periodontal regeneration are extraordinarily complex
and require the participation of many, if not all, cellular
components of the periodontium, wherein the healing com-
Alveolar bone
ponents of both the soft tissues and the hard tissues of the
The alveolar process is the thickened ridge of bone (alveolar periodontium need to be coordinated and integrated by a
bone) that contains the tooth sockets on bones that bear teeth. signaling system for new connective tissue fibers to insert into
In humans, the tooth-bearing bones are the maxilla and the cementum and bone. Unfortunately, very little is known
mandible. The mineral content of alveolar bone is mostly hy- about the signals that initiate and regulate these interactions
droxyapatite (HA), which is also found in enamel as the main in vivo.22 Therefore, therapeutic failure appears to be more
inorganic substance.1 The alveolar bone is developed before frequent in periodontal surgery than in other traumas.46,47
mineralization by osteoblasts that produce matrix vesicles. However, advances in our understanding of the pathobi-
These vesicles contain enzymes that seed the nucleation of HA ology of periodontitis, its association with systemic diseases,
crystals. The crystals are aligned with their long axes parallel and advances in molecular biology, cell biology, genetics,
to the collagen fibers and appear to be deposited within the and biomaterials science have stimulated new innovations
fibers in mature lamellar bone.44 The alveolar bone consists of in regenerative biology based upon tissue-engineering ap-
an external plate of cortical bone, the inner socket of thick, proaches.11,20–24,47
compact bone, and cancellous trabeculae interposed.1 During
fetal growth, alveolar bone is formed by intramembranous
Periodontal Regeneration: Difficulties and Potential
ossification and constantly remodels by osteoblast and osteo-
clast activity. The outer bone surfaces are covered by perios- As mentioned above, the complexity of the tooth-
teum, and the inner surfaces are lined with endosteum. The supporting apparatus provides the periodontium with a very
contour of the bone follows the topography of the roots. De- limited capacity for regeneration. Although a number of
fects in the bone structure can be seen as isolated areas in procedures have been investigated in an attempt to regen-
which the root is denuded of bone (fenestrations) or as defects erate lost periodontal tissue, none have yet led to predictable
extended through the marginal bone (dehiscence). Safe, ef- new cementum formation, remodeling of the PDL, and new
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 223

bone formation.2,10 During the early phases of periodontal Tissue engineering offers a new option to supplement
disease, some minor regeneration of the periodontium may existing treatment regimens for periodontal disease. It is
be seen. However, once periodontitis becomes established, clear that the regenerative events of periodontal wound
only effective therapeutic intervention has the potential to healing require the recruitment of progenitor cells that can
induce periodontal tissue reconstruction.8 A number of stud- differentiate into specialized regenerative cells, followed by
ies have indicated that reduced probing depths, clinical at- the proliferation of these cells and synthesis of the special-
tachment gains, and radiographic bone fill are consistently ized components of the periodontium.22 While a major
achieved for intrabony and furcation defects after scaling tissue-engineering strategy for periodontal regeneration fo-
and root debridement alone, open flap debridement, auto- cuses on exploiting the regenerative capacity of cells residing
genous bone grafting, and implantation of biomaterials in- within the periodontium, a potential approach in the future
cluding bone derivatives and bone substitutes.7,18 Despite may involve the incorporation of progenitor cells and in-
obvious gains in clinical attachment levels and radiographic structive messages in a prefabricated 3D construct=scaffold,
evidence of bone fill obtained using some grafting materials, subsequently implanted into the defect after a sufficient
careful histological assessment usually reveals that these in vitro culture.22 The cells and their origins, the optimization
materials have little osteoinductive capacity and generally of engineering methods, and biomaterial modifications are
become encased in a dense fibrous, nonmineralized connec- all highly challenging hurdles that require novel solutions.
tive tissue.34 To date, various periodontal therapies can lead Although there is significant potential for advances in tissue
to different patterns as well as different degrees of peri- engineering to cause a paradigm shift in the way surgeons
odontal healing and=or regeneration (Table 1).11–14,18,29,48,49 reconstruct patients with periodontal defects, ultimately the
As a result of periodontal regenerative research, a series of most basic question of which general approach to use re-
clinical techniques has emerged for high degree and more mains unknown. Current data are far from sufficient for
efficient regeneration and repair of periodontal defects and tissue-engineering therapeutics within periodontology, es-
also for improved implant site development.21,23,25 With the pecially preclinical evaluation in well-characterized rodent
availability of purified protein mixtures and growth factors,27 screening models. Clinical evaluation is largely limited to
a new era of tissue engineering has emerged whereby bio- therapies showing promising results in these preclinical
logical mediators can be used for periodontal regeneration.23 models.46 Therefore, periodontal tissue engineering for
The course of regeneration therefore is largely dependent periodontal regeneration is still in its infancy, at least, in
upon the availability of appropriate cell sources, inductive discriminating between outcomes in large animals to confirm
and differentiation factors, and, indeed, ECM secreted by its clinical potential and efficacy. Clearly, it is not likely that
these cells.34,46 Although the exact events that occur during tissue-engineering technology will progress into widespread
the healing process are still unclear, appropriate progenitor clinical practice for periodontal regeneration in the very near
cells must migrate toward and attach to the denuded root future.
surface and then proliferate and mature into the components The recent development of biomedical engineering as well
that contribute to a functional tooth-supporting attachment. as stem cell biology has enabled us to induce cell-based re-
Moreover, current scientific evidence indicates that wound generation of body tissue to self-repair defective tissue or
stability after regenerative surgery, space provision by a substitute biological functions of damaged organs. Clearly,
three-dimensional (3D) framework with implantation into the the future application of regenerative and tissue-engineering
defect, and primary intention healing are fundamental bio- techniques to periodontal therapy is one of immense poten-
logical and clinical challenges that must be met to obtain true tial, capable of meeting a variety of patient needs. High-
periodontal regeneration.20,46 Therefore, a profound under- quality clinical research from current available therapeutics is
standing of the complex factors that may influence the clinical always paramount to ensuring that the development of novel
outcome will allow periodontal surgeons to effectively ma- clinical treatments is supported by robust data and that such
nipulate biological and clinical factors to optimize the clinical approaches are effective. A brief review of outcomes from
result and increase the predictability of periodontal regener- existing therapeutic modalities can guide successful innova-
ative therapies.11,46 tions to an enhanced periodontal regeneration, highlighting

Table 1. Different Patterns of Periodontal Healing=Regeneration

Patterns Characterizations

Long junctional epithelium Healing of periodontal defect by an new epithelial attachment along the instrumented
healing root surface, which is formed by keratinocytes that migrate into the pocket from the
crevicular epithelium
Connective tissue repair Healing of periodontal defect by collagen fibers oriented parallel or perpendicular to a
instrumented root surface previously exposed to periodontal disease or otherwise
deprived of its periodontal attachment
Bone and=or bone-like Healing of periodontal defect by bone or bone-like tissue formation without specific PDL
tissue repair and=or acellular extrinsic fiber cementum regeneration
Periodontal tissue Healing of the periodontal defect by regeneration of tooth cementum, a functionally
regeneration oriented PDL, alveolar bone, and gingiva in periodontal defect

PDL, periodontal ligament.

224 CHEN AND JIN

the need for close partnerships between basic research and preclinical and clinical trials over the past three decades have
clinical scientists. been comprehensively reviewed.8,54–58 Traditionally, peri-
odontal defects, if left empty after open flap debridement, fill
Candidate Therapeutics for Periodontal Defects with the first cells, that is, epithelial cells and fibroblasts, to
reach the area after cell proliferation, which generates a core of
Over the last five decades, much of the armamentarium
fibro-epithelial tissues that ultimately prevent the subsequent
available to the periodontist and general dentist has been fo-
orderly and sequential regeneration of true periodontal tissues.
cused upon disease prevention, slowing or arresting disease
The GTR technique therefore employs a barrier membrane
progression, regenerating lost periodontium, and maintain-
of variable porosity, to prevent epithelial down-growth and
ing achieved therapeutic outcomes.8,10,12,16 On the basis of the
fibroblast trans-growth into the wound space, thereby also
priority of establishing an excellent oral hygiene, nonsurgi-
facilitating the maintenance of the space site for target peri-
cal therapy such as scaling and root surface debridement
odontal tissue regeneration.54,55,57–59 Thus, it can be seen that
is performed before surgical open flap debridement for peri-
the goal of the membranes is to retard the migration of oral
odontitis, and surgery is only indicated when nonsurgical
epithelium into the space between the decontaminated root
methods fail.12,13 To date, a variety of different surgical
surface and adjacent alveolar bone, thereby avoiding the for-
techniques have been used, including subgingival curettage,
mation of a long junctional epithelial attachment.58 This is
gingivectomy, modified Widman flaps, and full- or split-
believed to afford time for selective repopulation of the root
thickness flap procedures with or without defect correction.50
surface by undifferentiated stem cells from within the PDL
Although the results of longitudinal preclinical and=or clinical
space. This approach may also permit putative progenitor cells
trials have highlighted the advantages and disadvantages of
within the periodontal defect to differentiate into the specific
each technique, the optimal approach remains controversial
cell types required for the regeneration of a functional peri-
because evidence has shown that all of these approaches
odontal attachment under the stimulus of as yet poorly defined
generally result in repair of the diseased site with a non-
signaling=growth factors.7,16,18,34 Normally, GTR supports
physiological epithelial attachment, rather than regeneration
substantial gains at the clinical attachment level, reductions in
of a true periodontal attachment between the root surface and
probing depth, and gains in defect fill as compared with open
neighboring alveolar bone.8,51–53 Periodontal therapy has
flap debridement alone in intrabony defects; however, the
evolved from the days of scaling and root planning and=or
outcome is associated with a relatively high degree of vari-
gingivectomy to currently include a wide array of sophisti-
ability and the overall amount or type of regeneration is un-
cated plastic and regenerative procedures such as GTR and a
predictable. Besides the resorbability of the GTR membrane,
variety of implantable substances (Fig. 2).51 When considering
the size of the defect, the morphology of the defect, and op-
which approach to use, the defect complexity and specific
erator preference, differences may be also related to variations
patient situation take priority for medical attention, and the
in the numbers of putative progenitor stem cells and the con-
one that is most simple, straightforward, reliably successful,
centrations of appropriate signaling factors at the periodontal
and safest should be chosen. The ultimate goal of these tech-
defect site between patients.10,53
niques is the predictable 3D reconstruction of the lost at-
The first reports of GTR as a clinical periodontal surgical
tachment including root cementum, alveolar bone, and
procedure appeared more than 20 years ago.56 The key aim of
intervening PDL in a coordinated fashion, although differ-
periodontal therapy is to improve periodontal health and, by
ent degrees of success have been reported with different
this means, to satisfy a patient’s aesthetic and functional de-
procedures.10,18,53
mands. Mostly, this is achieved through repair of the peri-
odontium. Human histology has demonstrated that such
Guided tissue regeneration
regeneration can occur to some extent following GTR proce-
GTR is an approach to regaining lost periodontal attachment dures. However, histological differences in outcomes are
that utilizes the surgical implantation of a cell-impermeable not sufficient to recommend which form of therapy is better
barrier between a detoxified root surface and the crevicular than another. To consider a change in clinical practice, a new
epithelium. The principle of GTR and its achievements in both therapy must offer superior outcomes and may include clin-

FIG. 2. Diagram of currently established

alternative techniques for periodontal de-
fects.
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 225

ical, patient-centered, or economic outcomes. In view of the
The science of bone regeneration is still in its infancy,
limited number of long-term clinical research reports inves- with all current or emerging strategies having serious
tigating GTR and the lack of split mouth investigations, limitations; thus, the replacement of bone itself is a
pooling of data is essential to understand the characteristics of complex and demanding undertaking.
the intervention and to investigate possible causes of differ-
The formation of alveolar bone, apart from ideal, is
ences in outcomes.51 not true periodontal regeneration, and the regeneration
Several clinical studies have demonstrated that GTR is a of a functional bone–PDL–cementum apparatus is even
successful remedy, and it has become an accepted procedure more challenging.
in most periodontal practices today, either by itself or in
Surgical challenges, infection problems, and difficulties
combination with other treatment modalities.5,8,57 Different associated with different bone grafting materials still
modified therapeutics based on GTR may offer additional exist.
benefits in terms of radiographic resolution of the intrabony A brief description of bone biology and constitutional ele-
defect and predictability of clinical outcomes. The clinical ments is helpful in understanding the challenges that must
measurements of attachment level and probing depths, along be met in its replacement by grafting. Bone formation occurs
with radiography, are good methods of evaluating tooth when osteoblasts secrete collagen molecules and ground
survival and prognosis, but they do not indicate true bio- substance. The collagen molecules polymerize to form col-
logical regeneration. In addition, there is marked variability lagen fibers. Calcium salts precipitate in the ground sub-
between studies, and the clinical relevance of these beneficial stance along the collagen fibers to form osteoid. Osteoblasts
changes remains unknown.11,46 Indeed, histological analysis become trapped in the osteoid and become osteocytes.
of GTR-mediated healing demonstrates that new connective Basically, bone formation after the implantation of different
tissue attachment to the root surface forms with minor con- bone grafts=substitutes may exhibit different biological per-
tributions from new cementum and bone formation, which, formance, where the terms osteogenesis, osteoinduction, and
by definition, is not true periodontal tissue regeneration. osteoconduction are frequently, but not always correctly,
Although histological analysis is a gold-standard tool for used in describing the ability of bone regrowth induced by
the evaluation of periodontal tissue regeneration in animal bone grafts (Table 2).16,18,60
models, there is no standard evaluation method for clinical In general, there are two types of bone grafts, autogenic and
parameters to date, and it is extremely difficult to know the allogenic grafts. A number of reviews have already summa-
exact amount and type of regenerated tissues that have been rized the advantages and disadvantages of different grafts as
achieved after clinical regenerative therapies. As a result, it is well as bone substitutes.7,8,10,16,18,62 Allogenic and xenogenic
still difficult to draw general conclusions about the clinical grafts are widely available and do not require a second sur-
benefits of GTR with the currently available evidence. gical site for the patient to harvest autogenous bone. How-
ever, allogenic bone must undergo processing techniques
Bone grafts
such as lyophilization, irradiation, or freeze-drying to remove
Bone grafts aim to restore the height of the alveolar bone all immunogenic proteins, to avoid any risk of immunological
around a previously diseased tooth; thus, the principles and reactions. In turn, these processing techniques have a negative
techniques from bone regeneration have been directly trans- effect on the osteoinductive and osteoconductive potential of
ferred to periodontal therapy with or without modifica- the allografts, which consequently decreases their biological
tions.5,7,8,18,38,46,60 During the past two decades, various types performance as compared with autografts.16,17,63–65 Accord-
of bone grafts have been investigated to determine their ingly, there is limited evidence from histological examinations
ability to stimulate new bone regeneration or to serve as bone of periodontal regeneration after the use of decalcified freeze-
replacements for defects in both preclinical and clinical tri- dried allogeneic bone grafts obtained from commercial tissue
als,7,18 and a number of reviews have focused on the biology banks. It is believed that alloplastic grafts can support, to a
characteristics and preclinical and clinical applications of certain degree, the formation of a new attachment apparatus
bone grafting materials with extensive discussions.30,40,61–64 in intrabony defects; however, the available data indicate that
The outcome and predictability of bone grafts, alone or as- they do not induce cementogenesis and therefore do not have
sociated with GTR, in periodontal regenerative therapy re- the potential to completely regenerate the architecture of the
main unclear because of the following:8,46 original periodontium.11 Drawbacks such as the possibility of

Table 2. Three Interrelated, But Not Identical, Biological Performances Induced by Bone Grafts

Performances Characterizations

Osteogenesis The formation of new bone by the primitive, undifferentiated, and pluripotent cells derived from
the graft material itself, which are somehow stimulated to develop into the bone-forming cell
lineage, for example, osteoblasts
Osteoinduction The ability of a material to recruit immature cells and to induce these cells to develop into
preosteoblasts and osteoblasts from the surrounding tissue at the graft-host site, which results
in bone growth
Osteoconduction The ability of a material to support the growth of bone over a surface, where an osteoconductive
surface is one that permits bone growth on its surface or down into pores, channels, or pipes.
This phenomenon is regularly seen in the case of bone implants
226 CHEN AND JIN

eliciting a host immune response, the risk of disease trans-
the existence of a small population of bone marrow stem
mission, and the apparent wide variability in the concentra- cells, and
tion of bone and periodontal-inductive agents (and hence
surgery-related issues.
biological activity) between different preparations have sig- Disadvantages with the use of fresh autogenous grafts in-
nificantly influenced their final applications.64 clude root resorption (iliac crest grafts) and the requirement
Autogenous grafting may include cortical, cancellous, or for an additional invasive surgical procedure that may result
mixed cortico-cancellous bone, which can appear in one piece, in donor-site morbidity, chronic postoperative pain, hyper-
en bloc, or in a particulate form. The grafted bone may be sensitivity, and infection. Another important drawback to
regarded as a partially necrotic tissue, which, in an undefined the use of autografts is their limited availability. Moreover,
time frame, goes through stages of resorption and revascu- histological evidence of true periodontal regeneration has
larization and subsequently acts as a scaffold for new bone been limited. In many instances, alveolar bone regeneration
formation.63,66–68 Alternatively, swift and gentle handling of is seen in association with the formation of a long junc-
the bone graft may permit substantial cell survival and revi- tional epithelium, representing periodontal repair and not
talization of the graft in situ. Because the survival of osteocytes true regeneration.8,15,64 Although autogenous bone grafts are
relies upon the presence of a vascular supply within a distance considered the gold standard in bone substitutes for bone
of 0.1 mm, cortical bone grafts lacking vascular and cellu- reconstruction, the regeneration of hybrid tissues and inter-
lar pools on endosteal and periosteal surfaces may not be able faces in the periodontium presents a challenge that exceeds
to sustain cellular viability.7,16,19,69 Cancellous bone grafts, that of engineering just a single tissue. However, the use of
however, have a greater likelihood of supporting cell survival autogenous bone grafts in clinical practice is still an alter-
due to the possibility for diffusion of nutrients and revascu- native and necessary choice before novel approaches are
larization from the recipient bed.17 Currently, autogenously successfully developed for the treatment of patients with
harvested cancellous bone grafts are most commonly used periodontal defects.
for the replacement of bone material in bone-repair-related
research, especially due to the absence of an immunogenic Biodegradable biomaterials
reaction postsurgery. Such grafts also demonstrate a very
good biological performance in terms of osteogenicity, os- Biomaterials offer surgeons a powerful set of clinical tools
teoinductivity, and osteoconductivity (the ability of three for patient treatment, usually in the form of ECMs, bone
types of bone formation as mentioned in Table 2). Evidence substitutes, and scaffolds, as well as delivery vehicles for cells
suggests that cancellous bone grafts, obtained from iliac crest, and growth factors.16,27,28 Implantable biomaterials for peri-
the maxillary tuberosity, or healing extraction sockets, are odontal regeneration are mostly a replacement technology,
capable of producing statistically significant bone fill.8,17,62–64 frequently modified through different strategies such as im-
The healing of autogenous bone grafts entails both osteo- proving biological properties in terms of biocompatibility,
conduction, where new bone is gradually formed around the biodegradability, and permeability, introducing intercon-
resorbing graft, and osteoinduction, where released proteins nected networks and incorporating growth factor(s) and=or
are capable of stimulating osteoblasts or preosteoblasts to cells,16,27,28,34,70–74 the latter will be discussed later in this re-
form new bone. In many aspects, the healing of bone grafts is view (Fig. 3). An understanding of the materials available and
similar to the healing of fractures, where factors of importance their basic properties contributes to better and more effective
for the successful incorporation of autogenous bone grafts are outcomes in preclinical and clinical practice.
summarized as follows:7,8,10,16,18,30 The biomaterials used for periodontal surgery are now
virtually all biodegradable because any remaining materials

the embryonic origin and source of the graft employed, often become physical obstacles to tissue regeneration.67,75,76

the rate and extent of revascularization, Over the past few decades, synthetic biodegradable polymers

structural and biomechanical features, used clinically are homopolymers of lactide and its copoly-

the rigid fixation of the graft to the recipient site, mers formed with glycolide, that is, biodegradable polyesters,

graft orientation, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-

the availability of specific growth factors within the co-glycolic acid). The biodegradable polyester family has been
bone matrix, regarded as one of the few synthetic biodegradable polymers

the ability of the bone to act as a biocompatible scaffold, with controllable biodegradability, excellent biocompatibil-

FIG. 3. Implantable biomaterials devel-

oped for the management of periodontal
defects: (A) bone substitutes fabricated
from glycidyl methacrylated dextran and
poly(ethylene glycol) (a general view of
the samples as shown in Ref.27, Fig. 2A);
(B) hybrid hydrogel scaffolds fabricated
from glycidyl methacrylated dextran and
gelatin with interconnected pore struc-
tures (image from the same batch of scaf-
folds as shown in Ref.105, Fig. 2B).
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 227

Table 3. Four Main Groups of Synthetic Bone Substitutes

Groups Examples

Metallic implants Titanium and its alloys, stainless steel, and cobalt–chromium alloys
Ceramics Calcium phosphate, alumina, carbon, and glass ceramics
Synthetic polymers Poly(methyl methacrylate), poly(urethane), ultra-high molecular weight polyethylene, silicon,
and polylactide
Composites Calcium phosphate–ceramic coatings on metallic implants and polymer–ceramic composites such
as biphasic synthetic ceramic (Ceraform
, Teknimed SA, Vic-en Bigorre, France) and bovine
collagen=hydroxyapatite mixtures

ity, and high safety. Their biodegradable properties can be therapy.5,7,8,10,15–19,60,64 On the basis of their chemical com-
widely and readily controlled by changing the molecular position, synthetic bone grafts=substitutes can be divided into
weight and composition of the copolymers.27,77–79 Of the four main groups (Table 3).15 Synthetic bone grafts have the
natural polymers, proteins (collagen, gelatin, fibrin, and al- advantage that they do not induce an immune response and
bumin) and polysaccharides (chitin, hyaluronic acid, cellu- can be easily tailored to the intended application. However,
lose, and dextran) have been medically and pharmaceutically the biological performances of synthetic bone grafts with re-
employed.48,49,80–82 Generally, the degradation of each natu- spect to the initiation and support of bone growth are inferior
ral polymer is driven by hydrolytic and enzymatic cleavage to those of natural bone grafts.17,19
of their principal chain.27,80–82 Collectively, for tissue regen- The real interest in osteoinductive biomaterials is derived
eration, it is necessary to increase the number of cells within from the hypothesis that a material with osteoinductivity that
the tissue as well as to reconstruct an ECM structure to sup- is able to ectopically induce bone will also perform better in an
port the proliferation and differentiation of cells for the in- orthotopic manner.15,91 Therefore, unraveling the mechanism
duction of regeneration.34,82,83 It is well known that the ECM of osteoinduction by biomaterials is an important scientific
not only supports cells physically but also has an important challenge. The story of the development of osteoinductive
influence on cell proliferation and differentiation or mor- therapy, from the discovery of bone morphogenetic proteins
phogenesis, which contributes to the resultant tissue regen- (BMPs) in 1965 to the publication of randomized clinical trials
eration and organogenesis.29,82–84 It is unlikely that large in 2001 for tibial nonunions and 2002 for spinal fusion, is truly
tissue defects will regenerate naturally by simply supplying fascinating.39 These advances unfortunately have not lead to
cells to the defect without ECM support. Therefore, one rapid application of using osteoinductive biomaterial therapy
promising method involves building an environment suit- for reconstructing periodontal defects, because the complex-
able for inducing tissue regeneration at the defect site by ity and unique characteristics of periodontal defects will likely
providing a biomaterial as an artificial ECM to temporarily require multiple robust osteoinductive signals for clinically
support cell attachment and subsequent proliferation and meaningful bone and periodontal regeneration.5,7,8,10,19,30,39,67
differentiation.85,86 In periodontal regenerative therapies, it Due to an incomplete understanding of the mechanisms by
can be assumed that self-derived cells residing around the which biomaterials induce bone formation, a significant num-
implanted biomaterial infiltrate into the biomaterial and pro- ber of studies still focus upon ectopic implantation tech-
liferate and differentiate therein, provided that the artificial niques, as this remains one of the few ways of demonstrating
ECM is biologically compatible with those cells.11,20 Once a osteoinduction.15 There are few studies to date that have
new tissue is regenerated, the tissue eventually produces an convincingly demonstrated that a material’s osteoinductive
intact ECM. To ensure that the remaining biomaterial is not a potential directly equates with its orthotopic performance.
physical antagonist to tissue regeneration, it is essential to The number of studies in which osteoinductive materials have
control the retention time profile of the remaining biomaterial been evaluated for clinically relevant orthotopic defects is
within the defect. Hence, biodegradable biomaterials serving even more limited.61,62,65,92
as an in vivo cell growth platform are preferred for peri- Despite this, those studies that have been published sup-
odontal implantation,69,87 thus guiding an appropriate peri- port the hypothesis that materials capable of ectopically in-
odontal and bone tissue regeneration.82,84,88,89 In cases where ducing bone formation are also able to increase the amount
the tissue around the defect lacks any inherent potential for of bone at clinically more relevant orthotopic sites.51,93 De-
regeneration, tissue regeneration obtained by supplying the spite the fact that we still do not completely understand ei-
biomaterials (scaffold) alone is unlikely to succeed. Therefore, ther the mechanisms of osteoinductive biomaterials or their
scaffolds are frequently used in combination with cells and=or exact effect on orthotopic bone formation, recent studies all
biomolecular signals that have the potential to accelerate and suggest that osteoinductive biomaterials possess a greater
facilitate the regeneration process; this essentially embraces ability to orchestrate their in vivo surroundings to form new
tissue engineering.80,90 bone than nonosteoinductive biomaterials in periodontal
therapy.5,10,16,93
Osteoinductive implants have already been widely em-
Osteoinductive substances
ployed in the clinical arena; however, substances that do not
Due to the previously discussed limitations of natural incite immunological reactions and have the same biological
bone grafts, a large number of synthetic grafts have been performance as autologous bone grafts are still a long way
developed for bone repair and, more recently, periodontal from clinical application in periodontal regeneration.63,94 As
228 CHEN AND JIN

FIG. 4. Guided tissue and

bone regeneration membranes
obtained from collagen-based
biomaterials containing mi-
croparticle vehicles (unpub-
lished data): (A) unloaded
microparticles immobilized
in the membranes; (B) protein-
loaded microparticles
immobilized into the
membranes.

discussed in the literature, recent research has demonstrated carriers.28,103 The carrier may also protect the growth factor
that the biological performance of osteoinductive biomaterials, against proteolysis, facilitating prolonged retention of growth
in terms of orthotopic new bone growth, is significantly better factor activity in vivo. Ideally, the carrier should be degraded
than that of nonosteoinductive biomaterials.15 Future research within the body, as there is more than required after the com-
needs to focus on completely unraveling the mechanisms of pletion of growth factor release.90 Other than the controlled
osteoinduction to enable the development of materials that release of the growth factor, the prolongation of its half-life,
may successfully replace the autologous bone graft.15 improvements in adsorption, and growth factor targeting are
Other osteoinductive technologies that have been in- also primary objectives of such delivery systems.27,104 For
vestigated recently involve the incorporation of growth fac- example, a promising approach involves the promotion of
tors into implantable biomaterials, including membranes and periodontal tissue regeneration by targeting growth factors
scaffolds.95 For example, an in vitro release study demon- with extended half-lives to sites of desired tissue regenera-
strated that the use of functionalized dextran-derived hy- tion.27,28,104 Moreover, it has been proposed that successful
drogels as BMP carriers allowed the retention of recombinant tissue engineering requires the combination of tissue=cell
human bone morphogenetic protein-2 (rhBMP-2) growth scaffolds and growth factor vehicles into a single device to
factor to varying degrees, depending upon the functionali- create the optimal environment for the induction of tissue
zation ratio.95 However, GTR membranes cannot adsorb regeneration.49,105–107
sufficient growth factor because they are thin films. Therefore, Particulate forms of delivery vehicles have also been em-
the process of encapsulating growth factor into microparticle ployed across diverse biomedical applications.48,108–110 In
delivery systems and then introducing those microparticles this scenario, the potential of particulate systems for growth
into prefabricated membranes represents a promising method factor delivery has been promptly perceived as a means to
to develop functionalized GTR membranes (Fig. 4). The eval- protect growth factors during tissue regrowth and to offer
uation of those functionalized substances for periodontal re- adequate control over release rate. This is derived from key
generation is an area in need of greater research effort. properties such as size, surface area, and physicochemical
properties, which stem from the heterogeneity of the mate-
Recombinant protein therapy rials and the methods combined during their production
(Fig. 5). Two recently published reviews have focused on
Wound healing is an evolutionarily conserved, complex,
different vehicles fabricated from different materials and
multicellular process that is executed and regulated by an
using different manufacturing strategies for periodontal tis-
equally complex signaling network involving numerous
sue engineering and regeneration.27,28 Collectively, con-
growth factors, cytokines, and chemokines. The application of
trolled delivery systems offer promise in overcoming the
morphogenetic or mitogenic growth factors to support bone
inherent limitations of conventional delivery methods.111–115
formation at localized alveolar ridge defects has become an
Indeed, periodontal tissue regeneration is one area of tissue
area of increasing interest.9 Growth factors have an important
regeneration that can obviously benefit from the specific
role in regulating the proliferation, migration, and=or ECM
properties of materials in particulate form or microparticle=
synthesis of a variety of cell types, including those derived
scaffold composites.27,28,48,49,104,108–110
from the periodontium.96–100Although there are some cases in
A variety of delivery vehicles have been employed to tar-
which growth factors are required to promote tissue regen-
get growth factors to wound sites to optimize bioavailability.
eration, the direct injection of growth factor solutions into the
Depending on the device and application of the growth
site to be regenerated appears to be generally ineffective. This
factor or cytokine, dosage, release pattern (pulsatile, con-
is because the injected growth factor rapidly diffuses away
stant, or time programmed), kinetics of release, and duration
from the site. To enable the growth factor to efficiently exert its
of delivery can all be optimized.27,28 As alluded to previ-
biological effects, drug delivery systems are available.101,102
ously, devices serving as drug delivery vehicles as well as
Topical delivery of growth factors to chronic wounds must
tissue=cell scaffolds offer the following properties:27,28,103
be resistant to rapid degradation form the wounds proteo-
lytic environment and have sustained release. One promising
controlled release of target growth factor(s);
technique involves the controlled release of growth factors,
highly porous, 3D interconnected pore networks for cell
both at the desired site of action and over an extended time in-growth and flow of transport nutrients and metabolic
period, by incorporating the growth factor into appropriate waste;
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 229

FIG. 5. Particulate vehicles developed

for protein delivery across diverse bio-
medical applications in periodontal
regenerative medicine and tissue
engineering: (A) microparticles with
smooth surface; (B) microparticles with
corrugated surface and pore structure; (C)
microparticles loaded with protein drugs;
(D) microparticles incorporated into the
pore structure of scaffolds (a magnified
local view of the sample scaffolds as
shown in Ref.136, Fig. 1E).

biocompatible and bioresorbable with a controllable growth factors (bFGF)128,133–135 have all been investigated for
rate of degradation=resorption to match the cell=tissue periodontal regeneration.16,28,96,98,99,104 Although significant
growth in vitro and=or in vivo; progress has been made in the delivery of signaling molecules

suitable surface chemistry for cell attachment, prolifer- to facilitate periodontal healing, the short half-life and insta-
ation, and differentiation; bility of those proteins requires the delivery of milligram

mechanical properties that match those of the site of quantities of the factor or multiple dosages in clinical prac-
implantation. tice.27,28 The delivery of the effective doses of factors with
The development of new delivery devices for periodontal short half-lives and the maintenance of an environment with
regeneration has largely improved the efficacy of growth an appropriate combination of molecular signals to induce
factor delivery in vivo in recent years. In fact, bioabsorbable proper cell function and regenerate clinically useful amounts
scaffolds can work as a delivery platform that sequesters of new tissue in vivo have been critical challenges. Current
growth factors and releases them at optimal doses in a timely delivery systems still suffer from several limitations for clin-
manner depending upon the biological demand of the target ical periodontal applications such as loss of bioactivity, lim-
tissue.27,28,49,105,116 A body of literature has demonstrated ited control over dose administration, nontargeted delivery,
that this approach is feasible and results in constructs with and=or lack of availability.
biological activity.50,70,72,86,105 It is well recognized that periodontal tissue regeneration is
It should be emphasized that although treatment is more a complex process in which responding cells are regulated by
straightforward and appears safe when utilizing osteo- the coordinated action of several environmental cues and
inductive protein therapy as compared with cell-based or signals, which suggests that the appropriate presentation of
gene therapy, there are still, unfortunately, many inherent multiple regulatory signals may be a prerequisite for effective
limitations. Even with optimal scaffolds=vehicles, the local periodontal tissue engineering.22,46,136 Thus far, difficulties
application of growth factors often requires a large amount associated with dual or multiple growth factor delivery have
of protein to stimulate significant effects in vivo, which in- not been overcome.
creases the risk of undesirable side effects.28,71,72 An alter-
Periodontal Tissue-Engineering Triad
native approach using gene transfer may have the advantage
of transferring specific promoters into specific cells, using Tissue engineering is a relatively new but rapidly emerg-
appropriate vectors, to attain a sustained gene expression ing field with great potential to profoundly improve patient
and a more efficient method of in vivo delivery. Both strat- care. Based upon principles of cell biology, developmental
egies have been discussed in detail in three separate reviews biology, and biomaterials science, it aims to develop proce-
from different groups at different stages.26,117,118 dures and biomaterials to facilitate the development of new
Rutherford et al. conducted some of the first studies to in- tissues to replace damaged tissues.23,25,28,29,78 The major goal
vestigate the feasibility of using growth factors for periodontal of periodontal tissue engineering, however, is to harness the
tissue regeneration applications.119,120 To date, growth factors body’s own capacity to regenerate functionally active peri-
such as BMPs (e.g., BMP-2,49,89,96,105,109,121–124 BMP-7,68,125 odontal tissue that physiologically responds to metabolic
and BMP-12126), transforming growth factor (TGF)-b1,127,128 cues rather than to replace tissue with autografts, allografts,
platelet-derived growth factors (PDGFs),119,120,129–132 insulin- bionics, or metallic devices that are currently available in the
like growth factor (IGF)-1,48,119,130,132 and basic fibroblast market.21–23,28,29,47,106,107,117 In this case, a new therapeutic
230 CHEN AND JIN

FIG. 6. The key determi-

nants of periodontal tissue
regeneration. The triad con-
sists of responding cells, the
extracellular matrix scaffolds,
and an inductive signaling
system. Color images avail-
able online at www
.liebertonline.com=ten.

trial, in which disease healing can be achieved based upon mentum components influence the activities of periodontal
the natural healing potential of patients, has been explored. cells, and they manifest selectivity toward some periodontal
This therapy is termed ‘‘periodontal regenerative medicine.’’ cell types relative to others.11,20,22 Recently, the biological as-
As noted previously in this review, periodontal tissue regen- pects of cementum and the molecular and cellular aspects of
eration occurs through the recapitulation of key events that cementogenesis relevant to periodontal healing have been
occur during tissue formation and growth, which include detailed elsewhere.11,20 If cementum ECM-induced regener-
cellular migration, proliferation=apoptosis, differentiation, ative therapy is realized, it will increase the therapeutic
inductive=inhibitory interactions, and periodontal tissue choices of clinicians and ultimately lead to therapeutic bene-
patterning=morphogenesis.24,25,28,107 Tissue engineering is fits for those patients who had not previously received clini-
one such biomedical engineering method for building up cally effective therapies.9,11,20,22
the environment for regeneration induction, where the inte- More recently, biomimetic design and fabrication of tissues
gration of three key elements, that is, signaling molecules, and organs have become a popular strategy for tissue and
responding cells, and the ECM scaffold, is necessary to re- organ replacements. Thus, the emerging paradigm of ‘‘bio-
produce this developmental sequence of events.23,107 Given logical solutions for biological problems’’ has been introduced
these requirements for periodontal regeneration based on to both clinical dentistry and medicine. As detailed above, an
tissue-engineering technology, the major therapeutic areas of understanding of the basic principles of embryogenesis and
investigation have concentrated on a crucial combination of wound healing will lead us to the principles of biologically
one or more of these three components (Fig. 6).22,74 Each based therapies;20,24,46 however, finding the right signaling
component of the tissue-engineering triad may be imple- molecules and then expressing and delivering them in the
mented in a variety of ways. Some of the challenges in tissue correct spatial and temporal sequence is a daunting task. For
engineering involve identifying the most appropriate form of example, the structural integrity and biochemical composi-
each constituent of the tissue-engineering triad for a specific tion of the cementum matrix is severely compromised in
application.74 periodontal disease, and the provisional matrix generated
Biomaterials are clearly central to the advancement of tis- during periodontal healing is different from that of mature
sue engineering. To realize the regeneration of lost tissue, it is cementum.11,20,22 Nevertheless, recent developments in the
necessary to provide cells with a local in vivo environment of management of periodontal diseases using biotechnology and
artificial ECM (scaffold) that is suitable to their proliferation bioengineering indicate that tissue-engineering approaches
and differentiation, where the natural scaffolding of the ECM are undoubtedly feasible and, given their promise, worthy of
is faithfully duplicated. In addition to matrix-based means of further exploration. Overall, a successful periodontal tissue
controlling cell fate, soluble or insoluble instructional mole- regeneration based on tissue-engineering strategies requires
cules (biofactors) may be used to provide guidance to cells.74 a delicate interplay between the following three key elements:
It has become more and more clear that although the ECM
of cementum resembles other mineralized tissues in compo-
the presence of appropriate numbers of responsive
sition, its physiology is unique and contains biomolecules, progenitor=stem cells that will create the new hybrid
cementum-derived growth factor, and specific cementum periodontal tissues,
protein, for example, that have not previously been detected
an appropriate ECM or carrier construct to act as a
in other tissues or in other periodontal structures.11,20,22 Ce- scaffold to hold the cells, and
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 231

appropriate levels and sequencing of regulatory signals itors are committed to several developmental lineages, that
that instruct the cells to form the multiple tissue types in is, osteoblastic, fibroblastic, and cementoblastic. This prop-
the periodontium (signaling molecules). erty determines the PDL cells as a desirable cell population
It should also be emphasized that successful PDL and ce- capable of regenerating a functional periodontal attachment
mentum formation only occurs when these components are apparatus. It has been shown that PDL cells (Fig. 7A) may
placed in an appropriate environment that is capable prevent ankylosis and root resorption and may possibly also
of supporting regeneration.10,11,20 Each of these will now be produce a new PDL inserted into the tooth and adjacent bone.
reviewed in sequence. Moreover, roots covered with alveolar bone cells induce a
cementum-like tissue formation, suggesting that cemento-
Candidate cells for periodontal tissue engineering blast and osteoblast precursors commonly originate from the
alveolar bone.141–143 Thus, PDL-derived cell sources are one
Cells, both tissue-specific cells and stem cells, have an of the most promising candidates for cell-based therapies and
enormous influence on the success of tissue-engineering tissue-engineering-associated periodontal regeneration, and
protocols. Candidate cell sources for tissue engineering may positive preclinical results have already been achieved both
be classified into three main types on the basis of differences in vitro and in vivo.144–151 Taken together, the results of these
in living species: investigations demonstrate that PDL cells are capable of

autologous (patient’s own), preventing epithelial down-growth and root resorption, with

allogenic (donor’s), and the periodontal healing pattern being characterized by con-

xenogenic (animal origin). nective tissue adaptation involving parallel bundles resting
on root dentin.142,143 The formation of new bone and cemen-
Different cell sources have their own advantages and disad- tum varies from a complete absence to coverage of parts of the
vantages.78,137,138 It seems unlikely that allogenic or xenogenic debrided root surfaces mainly at the borders of the defects, to
feeder cells could be used successfully for periodontal regen- a thin layer of newly formed cementum with complete new
eration in the near future, because many of the challenges bone formation covering the entire previously denuded root
and risks associated with them remain unsolved.78,138,139 surface.144–151 The heterogeneity of results obtained may be
Therefore, autologous cells appear the most appropriate for explained by the small number of specimens used, the dif-
tissue engineering in so far as their activity remains high, ferent types of periodontal defects observed, and the stability
whereas allogenic and xenogenic cells are immunogenic and of the carriers utilized to deliver the cells (i.e., hyaluronic
would likely require adjunctive immunosuppressive therapy acid and blood clots). In addition, one may speculate that PDL
(Table 4).83,140 cell differentiation is highly sensitive to differences in the
Due to the difficulties encountered in isolating specialized microenvironment, resulting in different types of periodontal
cells and the associated morbidity involved, stem cells serve healing. However, the clinical application of autologous PDL-
as a better alternative. On the basis of their potency to dif- derived cells can be challenging due to60,78,106,138–140
ferentiate, stem cells can be classified into embryonic stem

insufficient cells available (it is difficult to harvest suf-
cells and postnatal stem cells, that is, adult (tissue-specific)
stem cells.6 Although embryonic stem cells offer greater ficient cells directly from patients),

difficulties in harvesting (it is extremely difficult to
plasticity, their sourcing is controversial and is surrounded by
ethical and legal issues, which reduces the attractiveness of harvest PDL cells from a patient without extracting the
these cells for developing new therapies. This explains why tooth),

time-consuming cell culture (the amount of harvested
many researchers are now focusing attention on developing
stem cell therapies using postnatal stem cells donated by the cells is frequently insufficient for clinical treatment; thus,
patients themselves or their close relatives.140 Although the cells need to be expanded by cell culture for several
most valuable cells for regenerative medicine are stem cells, weeks),

high-quality laboratory conditions (the in vitro culture
periodontal tissue engineering was clearly began with several
specialized cell resources such as cells from PDL and bone procedure requires a clean cell-processing centre to avoid
marrow. From a careful examination of the literature, it is contamination), and

possible viral infection that may accompany the fetal
soon realized that both cell sources may also contain a small
subpopulation of stem cells with the innate ability to maintain calf serum, which is most commonly used during cell
a stem-cell pool by self-replication and generate more com- culture.
mitted progenitors through differentiation along multiple A number of studies have attempted to clarify the role and
lineages. It is difficult to say which cell population contributes potential of PDL cells in periodontal regeneration. Despite the
more to the outcomes obtained. Beginning with a brief sum- limited success and predictability of procedures developed to
mary of adult cells derived from PDL and bone marrow in this date, they have contributed substantially to our understand-
section, our translational emphasis is placed upon using stem ing of the mechanisms involved in the development of the
cells for periodontal regeneration. periodontium, showing great potential for designing pre-
dictable therapies in the near future.151
PDL-derived cells. During periodontal regenerative pro-
cedures, the remaining healthy PDL plays a key role in the Bone marrow cells and cementoblasts. Bone marrow
regeneration of new compartments. The regenerative capacity stromal cells (BMCs; Fig. 7B) have been shown to have the
of the PDL itself is attributed to a few progenitor cells main- potential to develop into mature cells that form liver, fat,
taining their proliferation and differentiation potential in the cartilage, bone, epithelium, neuronal, and muscle tissue,
periodontium.22 It has been demonstrated that such progen- according to the conditions under which they are cultured.
232 CHEN AND JIN

Table 4. Candidate Cells for Periodontal Tissue Engineering: Representative Examples

and Their Main Results in Animal Experiments

Cell types Animal model Main results References

PDL cells Dogs (extracted dental roots were All of the roots exhibited ankylosis and 142
& gingival cells covered with PDL cells, gingival resorption. Only the roots bearing cultured
cells, or no cells and then trans- PDL cells were associated with fragments
planted to holes drilled in the of new fibrous tissue, which was identified
edentulous premolar region) as PDL on the basis of the orientation of
its fibers, the presence of Sharpey’s fibers
in bone and tooth, and cells located in
positions normally occupied by osteoblasts
and cementoblasts.
PDL cells Mini-pigs (extracted dental roots No cell controls showed widespread resorp- 143
& alveolar bone were covered with PDL cells, tion and ankylosis; alveolar bone cells
cells alveolar bone cells, or no cells and synthesized a calcified cellular tissue
then implanted into palatal bone resembling cellular cementum; PDL cells
defects) exhibited no calcified tissue formation
in vivo, but cells synthesized a connective
tissue with orientated fiber bundles
attached to both host bone and root,
resembling PDL.
PDL fibroblast-like Dogs (class II furcation defects, cells Healing by new connective tissue attachment 145
cells in autologous blood coagulum) with cementum formation was found in
75% of the cell-seeding defects, whereas it
was found in 71% of controls. Bone forma-
tion was found to fill 51% of furcation
defects; however, it was only found in 35%
of the defects at control sites.
Dogs (artificial fenestration defects, New bone formation was greater in the cell- 146
cells in autologous blood coagu- seeding group (84%) compared with the
lum) control (39%). However, the connective
tissue located between newly formed bone
and the root surface was observed to adapt
to the dentin surface, with limited cemen-
tum formation.
PDL-derived cells Dogs (periodontal fenestration The seeded cells (collagen sponge scaffold) 150
defects, cells seeded onto a induced cementum regeneration on the
collagen sponge scaffold) root surface, indicating the potential of
in situ tissue engineering using autologous
cells for the regeneration of periodontal tissues.
BMCs Mice (BMCs were inoculated onto New cellular cementum-like tissue formed 152
the surface of root slices and then along EMP-treated root slices. This is the
transplanted subcutaneously) first time differentiation of BMCs into
cementoblasts using an EMP-based proto-
col had been demonstrated.
Dogs (horizontal alveolar bone The height of repaired alveolar bone and the 259
defects, cells were mixed with average level of buccal alveolar ridge in
calcium alginate to form a gel experimental groups was higher than both
form of cell–scaffold construct) control groups.
Cementoblasts & Athymic rat (rodent periodontal Cementoblasts have a marked ability to 157
dental follicle cells fenestration model, cells in PLGA induce mineralization in periodontal
polymer sponges) wounds when delivered via polymer
sponges, whereas implanted dental follicle
cells seem to inhibit periodontal healing.
PDL stem cells Miniature swine (periodontal PDL stem cells were capable of regenerating 164
defects in a model periodontal tissues, leading to a favorable
of periodontitis) treatment for periodontitis.
Immunocompromised mice PDL stem cells generated cementum-like 176
(transplanted subcutaneously structures associated with PDL-like
with HA=TCP) connective tissue when transplanted into
immunocompromised mice.
BMSCs Dogs (cells were mixed with atelo- The defects were regenerated with cemen- 173,174
collagen and autotransplanted tum, PDL, and alveolar bone in the MSC-
into experimental class III defects) atelocollagen groups, and less periodontal
tissue regeneration was observed in the
control group.
(continued)
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 233

Table 4. (Continued)

Cell types Animal model Main results References

Dogs (cryopreserved cells were Both freshly isolated and cryopreserved 175
transplanted into the periodontal BMSC transplantations induced signifi-
fenestration defects using a colla- cantly better periodontal regeneration with
gen scaffold carrier) newly formed cementum, alveolar bone,
and PDL compared with the application of
collagen scaffold alone.
Goats (immediate implant The experimental side: periodontal-like tissue 176
placement post canine teeth with newly formed bone was demonstrated
extraction with a porous hollow both at 1 day and after 1 month, whereas
root-form poly[DL-lactide-co- the control specimens showed early signs
glycolide] scaffold seed with cells of connective tissue regeneration around
around the titanium fixture) the titanium fixture at 10 days, but
was not shown in the 1-month specimens.
ASCs Rats (periodontal tissue defects, cells A small amount of alveolar bone regeneration 179
were mixed with PRP obtained was observed 2 and 4 weeks after
from inbred rats) ASC=PRP implantation. Moreover, 8 weeks
after implantation, a PDL-like structure
was observed along with alveolar bone.

PDL, periodontal ligament; BMCs, bone marrow stromal cells; EMP, enamel matrix protein; PLGA, poly(lactic-co-glycolic acid); HA,
hydroxyapatite; TCP, tricalcium phosphate; BMSCs, bone mesenchymal stem cells; MSC, mesenchymal stem cell; ASCs, adipose-derived
stem cells; PRP, platelet-rich plasma.

Extensive renewing potential in combination with their de- bone.157 However, it is well recognized that cementoblasts can
velopmental plasticity has given rise to a tremendous interest play a key role in PDL-cementum apparatus formation during
in the prospective use of BMCs to regenerate damaged peri- the periodontal regenerative process.
odontal tissues.152–155 It has also been shown that cultured
porcine BMCs, after induction in appropriate media, can Tissue-specific stem cells. Stem cells have the unique
differentiate into cementoblasts using an enamel matrix capacity to regenerate functional tissue for the lifetime of an
protein-based protocol.152 Therefore, BMCs offer promise as organism, offering an attractive raw material for multiple
candidate cells for periodontal regeneration because they are biomedical and bioengineering applications.158,159 They are
easy to harvest and are present in greater numbers in the body also multipotent because they can differentiate into numer-
than PDL cells. Other cells such as cementoblasts have shown ous types of specialized functional cells, such as cells needed
a marked ability to induce mineralization in periodontal for periodontal reconstruction (Fig. 8).158 Recent findings
wounds when delivered via polymer sponges, while dental have also shown that stem cells exist in most, if not all, tis-
follicle cells (DFCs) seem to inhibit periodontal healing.156 The sues and that stem cell tissue specificity may be more flexi-
limitations of periodontal regeneration largely depend on the ble than originally believed.158,159 Recently, reports have
regenerative potential at the root surface. Cellular intrinsic begun to emerge demonstrating that populations of adult
fiber cementum, the so-called bone-like tissue, may form in- stem cells reside in the PDL of humans and other animals.
stead of the desired AEFC, and interfacial tissue bonding may This opens the way for new cell-based therapies for peri-
be weak. The PDL harbors progenitor cells that can differen- odontal regeneration using stem cells. The PDL contains
tiate into PDL fibroblasts, osteoblasts, and cementoblasts, but fibroblasts, macrophages, undifferentiated ectomesenchymal
their precise location is unknown. It is also unclear whether cells, cementoblasts, osteoblasts, cell rests of Malassez, and
osteoblasts and cementoblasts arise from a common precursor vascular and neural elements that are capable of generating
cell line, or whether distinct precursor cell lines exist. Thus, and maintaining periodontal tissues. However, the existence
there is limited knowledge about how cell diversity evolves in of paravascular progenitor stem cells within the PDL has
the space between the developing root and the alveolar long been speculated, and recently, their isolation and

FIG. 7. Optical micrographs

of candidate cells for peri-
odontal tissue engineering:
(A) periodontal ligament cells;
(B) bone marrow stromal
cells. Color images available
online at www.liebertonline
.com=ten.
234 CHEN AND JIN

FIG. 8. Role of stem cells in periodontal tissue regeneration. Multipotent stem cells residing in the paravascular spaces of
the mature pulp, periodontal ligament (PDL), or bone marrow have the potential to differentiate into mature PDL fibroblasts,
cementoblasts, and osteoblasts for functional regeneration of hybrid periodontal tissues, that is, PDL fibrils, cementum, and
alveolar bone. Progress in the techniques of expansion of stem cells and the control of their lineage into multiple cell types
will ultimately lead to the development of immunocompatible off-the-shelf stem cells for periodontal tissue regeneration.
Color images available online at www.liebertonline.com=ten.

propagation in culture have been investigated using different such characteristics.163 Techawattanawisal et al. recently re-
methodologies.33,161–169 In an impressive study, Seo et al. ported the isolation of multipotent stem cells from rat PDL
(2004)166 isolated a population of multipotent stem cells in using a neurosphere-forming culture system. Enzymatically
human PDL (PDL stem cells), providing a unique reservoir of dissociated PDL cells were cultured in serum-free basal me-
stem cells from an accessible tissue resource. PDL stem cells dium containing epidermal growth factor (EGF), bFGF, and
exhibit mesenchymal stem cell (MSC) characteristics such as leukemia inhibitory factor (LIF). Free-floating spheres ex-
clonogenicity and high proliferation and express the putative pressing nestin, glial fibrillary acidic protein (GFAP), and vi-
stem cell marker STRO-1 and the perivascular cell marker mentin were formed after 7 days of culture. In addition, the
CD146. In addition, in-vitro-expanded PDL stem cells can spheres expressed mRNA of the neural-crest-associated tran-
form collagen fibers and generate cementum=PDL-like scription factors Twist, Slug, Sox2, and Sox9. The PDL-
structures. The transplantation of ex-vivo-expanded cells may derived spheres differentiated into multinucleated myotubes,
hold promise as a therapeutic approach for the reconstruction neurofilament M (NFM)-positive neuron-like cells, GFAP-
of tissues destroyed by periodontal diseases.166 The same positive astrocyte-like cells, and 20 ,30 -cyclic nucleotide 30 -
group also demonstrated that human postnatal stem cells can phosphodiesterase (CNPase)-positive oligodendrocyte-like
be recovered from cryo-preserved human PDL, thereby pro- cells. A methylcellulose colony-forming assay revealed that a
viding a practical clinical approach for the utilization of fro- single PDL cell could form a sphere at a frequency of ap-
zen tissues for stem cell isolation.167 Findings from Nagatomo proximately 0.01% of total cells. These data indicate that the
et al. (2006) indicate that PDL cells possess crucial stem cell PDL-derived spheres contained multipotent adult stem cells
properties, such as self-renewal and multipotency, and ex- capable of differentiating into both neural and mesodermal
press the MSC markers CD105, CD166, and STRO-1 on their progeny.168 This is the first report of the isolation of PDL-
cell surface, although there is some variability. This provides derived stem cells with primitive neural crest stem cell fea-
an underlying explanation as to why PDL cells appear ef- tures. To date, the use of autologous PDL stem cells to treat
fective in periodontal regenerative procedures.165 Recently, it periodontal defects in animal models of periodontitis has
has been claimed that stem cells have a potent ability to ex- demonstrated the feasibility of using stem-cell-mediated tis-
clude Hoechst dye (a cell nucleus stain); cells possessing this sue engineering to treat periodontal diseases.164
phenotype are termed ‘‘side population.’’ In addition, it is The generation of a cell line from PDL progenitor=stem cells
suggested that side population cells are ubiquitously present would facilitate the development of tissue-engineering-based
in adult tissues. Kawanabe et al. (2006) additionally demon- periodontal regenerative therapies. However, little is known
strated that PDL stem cells exhibit a stem cell function profile about the characteristics of PDL progenitor=stem cells because
and express ABCG-2 (a general stem cell marker) and Oct-4 PDL tissue consists of a heterogeneous cell population and
(a critical transcriptional factor for maintaining pluripotenti- there are no pure PDL cell lines, and until recently, no highly
ality); however, only 3.9% of all cultured PDL cells exhibit purified periodontal stem cell clone has yet been established
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 235

from adult human PDL tissue.170 Fujii et al. (2008) succeeded cells have been transplanted, although the bone marrow
in immortalizing primary human PDL fibroblasts by trans- must involve different kinds of stem cells), and
fecting them with SV40 T-antigen and human telomere re-
relatively easy to harvest and expand (compared with
verse transcriptase (hTERT) and isolated three clonal cell lines PDL-derived cells).
from these immortalized cells (lines 1–4, 1–11, and 1–24) that
The acquisition of core knowledge surrounding growth fac-
express RUNX-2, Col I, ALP, OPN, OCN, RANKL, OPG,
tors and cytokines to promote BMSC differentiation and
scleraxis, periostin, Col XII, and alpha-SMA mRNA. Im-
suitable scaffolds or carriers for their delivery is key to real-
munocytochemical analysis demonstrated that CD146 was
izing the potential of BMSCs in periodontal tissue engineer-
expressed in cell lines 1–4 and 1–11 and that STRO-1 was
ing.173–177 In recent years, adipose-derived stem cells,178,179
expressed in lines 1–11 and 1–24. Lines 1–4 and 1–11 differ-
human embryonic stem cells,180,181 and human dental stem
entiated into osteoblastic cells and adipocytes when cultured
cells182–184 have all been considered for periodontal applica-
in lineage-specific differentiation media. Four weeks after
tions. However, irrespective of the cell source, the number of
transplanting cell line 1–11 into immune-deficient mice with
harvested stem cells remains insufficient for clinical applica-
beta-tricalcium phosphate (b-TCP), the transplanted cells
tion, and cell expansion by culture is invariably necessary.
produced cementum=bone-like tissues around the (b-TCP).
This is a challenge because their proliferative ability is gen-
Eight weeks after transplantation, the 1–11 cell transplant
erally low, and dedifferentiation with repeated passages
formed PDL-like structures on the surface of the (b-TCP).
eventually takes place. When stem cells are cultivated on a
These data suggest that cell line 1–11 was derived from a
two-dimensional (2D) cell culture dish, cell proliferation pro-
progenitor=stem cell present in the PDL and could be very
ceeds at a reasonable rate but is accompanied by dedifferen-
useful for studying the biology and regeneration of the human
tiation. In contrast, dedifferentiation does not readily arise
periodontium.161,171 From the studies reviewed, it appears
when stem cells are cultured on 3D substrates, but the cell
that PDL stem cells possess all of the necessary MSC abilities,
proliferation rates are severely compromised.78,185 Since the
such as self-renewal and the potential for differentiation into
discovery and characterization of multipotent MSCs from
several phenotypes, and are responsible for the partial peri-
bone marrow, MSC-like populations from other tissues,
odontal regeneration obtained by previous periodontal re-
dental pulp, and PDL tissues, for example, have now been
generative approaches. Once isolated, it is possible to enhance
characterized based on the gold-standard criteria established
the number of PDL stem cells sufficiently and in the right lo-
for MSCs from bone marrow. The search for MSC-like cells in
cation to optimize the complex differentiation processes in-
specific tissues has led to the discovery of a variety of stem
volved in periodontal regeneration. Recently, Singhatanadgit
cells in every organ and tissue in the body in the past decades.
et al. (2009) has reported, for the first time, the clonal isolation
Dental-tissue-derived MSC-like populations are among many
and characterization of highly proliferative subpopulations
other stem cells residing in specialized tissues that have been
derived from primary nontransformed human PDL cells, in-
isolated and characterized, representing an alternative stem
cluding putative PDL stem cells with multilineage differentia-
cell sources for periodontal regeneration.169,186 A greater un-
tion potential, that appears to exhibit, at least in vitro, the
derstanding of the biology of these dental stem cell popula-
definitive features of an adult human stem cell.170 Further
tions is a prerequisite to understanding the extent of their
studies are needed to elucidate the real potential of PDL stem
efficacy for future regenerative medicine. Characterization of
cells to form periodontal tissues and develop novel stem-cell-
these cells, and determination of their potentialities in terms of
based therapies for periodontal regeneration.
specificity of regenerative response may help direct new
MSCs are of great interest to both clinicians and researchers
clinical treatment modalities. The use of stem cells in peri-
for their great potential to enhance tissue engineering. The
odontal regeneration is reviewed in greater detail in another
diverse in vivo distribution of MSCs includes bone mar-
two contributions.33,185
row, adipose, periosteum, synovial membrane, skeletal
muscle, dermis, pericytes, blood, trabecular bone, human
Candidate scaffolds for periodontal tissue engineering
umbilical cord, lung, dental pulp, and PDL.169 The bone
marrow contains MSCs (BMSCs) that are capable of differ- The development of biomaterials capable of supporting
entiating into several connective tissue cell types, including regrowth of the individual mineralized tissues of the period-
osteocytes, chondrocytes, adipocytes, tenocytes, myocytes, ontium and functional graded interfaces between these tis-
and BMCs.33,35 These cells contribute to the homeostasis of sues is an active area of research. As noted in this article, most
musculoskeletal tissue while also supporting the growth and current studies have used known osteo-conductive materials
differentiation of primitive hemopoietic cells. Recent ad- to guide periodontal engineering efforts, and these materials
vancements in tissue engineering and regenerative medicine parallel those used in engineering other mineralized tissues.74
have highlighted BMSCs as a potential source of cells that It is well established that cells reside, proliferate, and differ-
may differentiate into a variety of tissues tailored to individ- entiate inside the body with a complex 3D environment, in-
ual needs. The ability of BMSCs to give rise to multiple spe- dicating that an ECM is a pivotal factor with a significant role
cialized cell types along with their extensive distribution in in supporting or restoring periodontal regeneration. ECM is
many adult tissues (including those of dental origin) has made normally assembled from components synthesized and de-
them an attractive target for use in periodontal regenera- posited outside the cell surface that provide structural and
tion.171,172 The advantages of BMSCs are as follows:33,35,171–173 functional integrity to connective tissues and organs. The
synthesis and deposition of ECM largely occur in response to

high safety (no reported tumorigenesis), growth factors, cytokines, and mechanical signals mediated

localized tissue regeneration (bone marrow regenerates via cell surface receptors. These cell surface receptors provide
only the tissue specific to the site where the bone marrow points of attachment that cells can use to sense mechanical
236 CHEN AND JIN

disruptions and to remodel the deposited matrix to render and hence are mixed with soft polymers to yield tough hy-
it structurally and functionally viable. An artificial ECM, brid scaffolds, so that they still maintain high levels of bio-
carried out by scaffolding materials, therefore is a prerequi- activity.88,190,193,194 Biodegradable polymers and bioactive
site of most tissue regeneration strategies. Scaffolds are ceramics are being combined in a variety of composite ma-
porous, degradable structures fabricated from either natural terials as tissue-engineering scaffolds.46 Further challenges
materials (collagen,120,124,133,135,155 fibrin,80,86 or synthetic such as incorporating biomolecules, surface functionaliza-
polymers [polyglycolide, polylactide, and polylactide cogly- tion, and 3D scaffold characterization all offer potential
colide]).71,72,84,86,111,116 They can be sponge-like sheets, gels, or strategic solutions.192 CaP cement has also been confirmed to
highly complex structures with intricate networks of pores provide stable wound healing and enhanced periodontal
and channels fabricated using new material-processing tech- regeneration within periodontal defects in dogs with exper-
nologies. Virtually, all scaffolds used in tissue engineering are imental periodontitis.195 Although there were no statistically
intended to degrade slowly after implantation in the patient, significant differences between the two treatment groups in
being replaced by new tissue.160 Collagen and hyaluronan one study, histological observation indicated that CaP ce-
occur naturally and are involved in numerous physiological ment seemed to act as a scaffold for bone formation and
processes, thereby offering a safe scaffold material. Although provided histocompatible healing of periodontal tissues. This
collagen can be constituted into various forms such as cement might be applicable to periodontal therapy; however,
films, fibers, sheets, gels, and sponges, these forms frequently further investigations are required.196
lack the space provision needed to form scaffolds for tissue Fibrin is a critical blood component responsible for hemo-
engineering.90,187 However, collagen scaffolds can be formed stasis, which has been used extensively as a biopolymer scaffold
with greater mechanical strength by cross-linking with mol- in tissue engineering and represents exciting possibilities in
ecules such as formaldehyde, glutaraldehyde, polyepoxy periodontal bioengineering, where commercially available fi-
compounds, or carbodiimides 90,188 or by combining collagen brinogen and thrombin are combined to form a fibrin hydrogel
with inorganic materials.189 for diverse applications.80 Hydrogels are a new class of bio-
As discussed under the ‘‘Biodegradable Biomaterials’’ sec- materials that could potentially be injected into the period-
tion, synthetic polymers can be designed specifically in terms of ontium. These biomaterials are composed of a viscous polymer
material, mechanical properties, porosity, and degradation made of synthetic or natural hydrophilic macromolecules that
properties.189 The major function of scaffolds is similar to that are able to form a hydrogel after physical, ionic, or covalent
of the natural ECM that assists proliferation, differentiation, cross-linking.81,83,86 Future advances in the development of 3D
and biosynthesis of cells. In addition, a scaffold placed at the site scaffolds, such as hydrogels that might be able to support in vivo
of regeneration will prevent undesirable cells from infiltrating synthesis of hybrid tissues, should be the subject of further re-
the site of action. Basically, regenerative medicine involves two search efforts. Scaffolds for periodontal tissue regeneration can
concepts, cell therapy without any use of scaffolds and tissue also be designed to release growth factors that induce cellular
engineering that requires a scaffold to support the tissue re- differentiation and tissue growth in vitro or cell migration into
generation process. In the latter case, a scaffold serves as a bed the wound site in vivo (Table 5). For example, surgical im-
for cell attachment, penetration, migration, and in-growth. To plantation of rhBMP-2 in the resorbable collagen sponge ap-
fulfill the functions of a scaffold in tissue engineering, the pears to significantly enhance bone regeneration in intrabony
scaffold should meet a number of requirements:80,81,84,86 periodontal defects in baboons,197 and collagen scaffolds loa-


ded with BMP-2 and=or 7 have also been successfully used in
interconnected micropores for cell migration and in-
applications to induce bone formation, which has led to the
growth,


development of Food and Drug Administration–approved
optimal porosity with adequate surface area and me-
collagen-BMP products for the treatment of such bone de-
chanical strength, and


fects.198 The combination of rhBMP-2 delivered in an absorb-
controlled absorption kinetics or degradation.
able type I collagen sponge was approved by the Food and
Inorganic scaffolds have been used in addition to polymeric Drug Administration in 2004 as INFUSE
Bone Graft (Wyeth
scaffolds, specifically for alveolar bone defects. Biomaterials Pharmaceuticals, Philadelphia, PA) for anterior lumbar inter-
used for this purpose include HA and calcium phosphates body spinal fusion and open tibial shaft fractures and by the
(CaP).88,190–194 CaP biomaterials have outstanding properties, European Union in 2002 as InductOs
(Wyeth Pharmaceuticals,
which include the following:88,190–194 Maidenhead, Berkshire, United Kingdom) for open tibial shaft


fractures. At present, osteogenic protein-1 (OP-1) delivered in a
a similarity in composition to bone mineral,


particulate bone-derived type I collagen matrix is available in
bioactivity (ability to form bone apatite-like material or
the United States and the European Union as OP-1
Implant
carbonate HA on their surfaces),


(Stryker Biotech, Hopkinton, MA) through a Humanitarian
ability to promote cellular function and expression
Device Exemption for recalcitrant nonunion fractures.27
leading to the formation of a uniquely strong bone-CaP
Recently, an interconnected macroporous glycidyl metha-
biomaterial interface, and


crylated dextran=gelatin hydrogel scaffold has been success-
osteoconductivity (ability to provide the appropriate
fully developed in our laboratory.49,105 Scaffolds containing
scaffold or template for bone formation).
small, degradable beads that release BMP-2 can significantly
In addition, CaP biomaterials with appropriate 3D geometry enhance periodontal tissue regeneration. The fragile nature
that are able to bind and concentrate endogenous circulating of proteins has motivated the design of scaffolds that release
BMPs may become osteoinductive (capable of osteogenesis) naked plasmid DNA containing genes that encode growth
and can be effective carriers of bone cell seeds. These inor- factors.70,199,200 When a chitosan=coral composite scaffold
ganic scaffolds with interconnected pore structures are brittle fabricated to release the gene for PDGF was implanted sub-
 Table 5. Candidate Scaffolds (Growth Factors) Reported for Periodontal Tissue Engineering
and Regeneration: Selective Data and Their Principal Findings

Author, published year

(Ref.) Scaffolding (growth factors) Study design Principal findings

Kim et al., 2005 (67) A particulated autogenous bone Animal experiment (one-wall No significant healing aberrations such as root
and a coral-derived biomaterial intrabony periodontal defects resorption and ankylosis
(without growth factors) in dogs)
Chen et al., 2005 Dex-GMA=gelatin hydrogel In vitro studies and animal Promoted cell proliferation and osteoblastic
(109, 110); 2006 scaffolds (rhBMP-2); or experiment (class II furcation differentiation of PDL cells, enhanced significant new
(48, 108); 2007 (49) microparticles (BMP-2 or IGF-1) defects in dogs) bone, PDL, and cementum regeneration in artificial
in calcium phosphate cements periodontal defects
Blumenthal et al., An ACS or a calcium phosphate Animal experiment (intrabony Both scaffolds achieved significant tissue regeneration;
2002 (124) putty (aBSM, ETEX periodontal defects in baboons) rhBMP-2=ACS supported significantly greater new

237
Corporation, Cambridge, MA) cementum formation.
(rhBMP-2)
Sorensen et al., A bioresorbable calcium phosphate Animal experiment (bilateral, Sites receiving rhBMP-2=Ceredex (ETEX Corporation,
2004 (121) cement (Ceredex) (rhBMP-2) critical-size periodontal defects in Cambridge, MA) exhibited increased cementum
Hound Labrador mongrels) formation, but without a functionally oriented PDL,
and increased ankylosis and root resorption.
Takahashi et al., A combination of polylactic Animal experiment (class III Ankylosis was observed in some of the rhBMP-2=PGS
2007 (89) acid–polygricolic copolymer furcation defects in cats) group at 3 and 6 weeks, but not at 12 weeks. Residual
and gelatin sponge (rhBMP-2) PGS may play an important role in preventing
ankylosis.
Ripamonti et al., A bovine insoluble collagenous Animal experiment (furcation Induced cementogenesis on surgically denuded root
1996 (125) matrix (hOP-1) defects in nonhuman primates) surfaces, enhanced periodontal tissue regeneration
Wikesjö and An ACS (rhBMP-2 or rhBMP-12), Animal experiment (alveolar defects rhBMP-2=ACS has a limited effect alone in this
colleagues, 2000 or rhBMP-2=ACS combined with or critical-size periimplant defects augmentation model of class III alveolar ridge defects;
(187); 2002 (124); HA; or calcium phosphate putty in mongrels dogs, or large size, rhBMP-2=ACS elicited the promotion of bone
2003 (188); 2004 (aBSM, ETEX Corporation, three-wall intrabony defects in regeneration, whereas rhBMP-12=ACS exhibited a
(126) Cambridge, MA) carrier baboons) functionally oriented PDL bridging the gap between
(rhBMP-2) newly formed bone and cementum; a space-providing
macroporous ePTFE device defined rhBMP-2=
ACS-induced alveolar augmentation in critical-size
periimplant defects; rhBMP-2=ACS supported
significantly greater new cementum formation than
rhBMP-2=aBSM.

(continued)
 Table 5. (Continued)

Author, published year

(Ref.) Scaffolding (growth factors) Study design Principal findings

Mohammed et al., A 25% pluronic F-127 (TGF-b1) Animal experiment (standardized TGF-b1 encouraged bone regeneration in class II furcation
1998 (127) class II furcation in sheep) defects in sheep, an effect enhanced by the presence of
a barrier membrane.
Tatakis et al., 2000 A calcium carbonate carrier (CaCO3) Animal experiment (critical-size Surgical implantation of rhTGF-b1 resulted in minimal
(128) (TGF-b1) periodontal defects in dogs) stimulation of alveolar bone or cementum regeneration.
Lynch and A methylcellulose gel vehicle Animal experiment (ligature- PDGF-BB=IGF-1 combination resulted in significant
colleagues, 1996 (rhPDGF-BB and=or rhIGF-1) induced periodontitis in mon- increases in new attachment and osseous defect fill.
(212); 1997 (130); keys)
2003 (131)
A methylcellulose gel vehicle Clinical practice (bilateral osseous 150 mg=mL each of rhPDGF-BB and rhIGF-1 resulted in
(rhPDGF-BB & rhIGF-1) periodontal lesions in patients) a significant promotion in bone regeneration.
A bone allograft (rhPDGF-BB) Clinical practice (interproximal in- Use of purified rhPDGF-BB mixed with bone allograft
trabony defects and molar class II resulted in robust periodontal regeneration in both
furcation lesions in patients) class II furcations and interproximal intrabony defects.
Giannobile and col- A b-TCP product (rhPDGF-BB) Clinical practice (advanced period- Low-dose rhPDGF-BB application elicited the promotion
leagues, 2006 (129) ontitis in patients) of bone turnover at early stages of the repair process.
Takayama et al., 2001 A gelatinous carrier (FGF-2) Animal experiment (furcation class Local application of FGF-2 can enhance considerable
(219) II defects in primates) periodontal regeneration.
Nakahara et al., 2003 A sandwich membrane (b-FGF) Animal experiment (three-walled al- Induced successful regeneration (cementum and PDL)
(133) veolar bone defects in dogs) of the periodontal tissues in a short period of time

238
Sato et al., 2004 (135) A collagen gel (b-FGF) Animal experiment (experimentally Induced formation of dense fibers bound to alveolar bone
induced partial defects in dogs) and newly synthesized cementum in teeth treated
with 1 mg of b-FGF
Shirakata et al., 2002 An injectable calcium phosphate ce- Animal experiment (experimental Calcium phosphate cement provided stable wound
(196); Hayashi ment (without growth factors) periodontitis in dogs) healing and enhanced periodontal regeneration in
et al., 2006 (195) periodontal defects in dogs with experimental
periodontitis.
Zhang et al., 2007 Porous chitosan=coral composites In vitro and in vivo studies (im- PDGF-B gene-activated scaffolds promoted PDLCs
(201, 202) (PDGF-B gene or adenoviral vec- planted subcutaneously into proliferation, up-regulated their expression of PDGF-B
tor encoding BMP-7) athymic mice or implanted into and type-I collagen, and increased expression
defects on both sides of the man- of PDGF-B in vivo; scaffolds containing Ad-BMP-7
dible) exhibited the higher alkaline phosphatase activity,
and enhanced bone formation at dental implant defects.

Dex-GMA, glycidyl methacrylated dextran; rhBMP-2, recombinant human bone morphogenetic protein-2; BMP-2, bone morphogenetic protein-2; IGF-1, insulin-like growth factor-1; rhIGF-1,
recombinant human insulin-like growth factor-1; PDL, periodontal ligament; BSM, bone substitute material; PGS, poly (D, L-lactic-co-glycolic acid) copolymer/gelatin sponge; ACS, absorbable collagen
sponge; hOP-1, human osteogenic protein-1; ePTFE, expanded polytetrafluoroethylene; TGF-b1, transforming growth factor-b1; rhTGF-b1, recombinant human transforming growth factor-b1; PDGF-B,
platelet-derived growth factor-B; rhPDGF-BB, recombinant human platelet-derived growth factor-BB; FGF-2, fibroblast growth factor-2; b-FGF, basic fibroblast growth factor; BMP-7, bone
morphogenetic protein-7; PDLCs, periodontal ligament cells; Ad, adenoviral.
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 239

cutaneously into athymic mice, combined with human PDL On the basis of this concept, new biomaterial platforms
cells, results indicated that PDL cells showed enhanced pro- could be developed by combining instructive injectable scaf-
liferation properties on the gene-activated scaffolds compared folds (functionalized self-assembling peptide nanofibers)
with pure coral scaffolds, and expression of PDGF and type-I with a polymer matching the biomechanical properties of
collagen was up-regulated in the gene-activated scaffold. After the bone and periodontal tissue. This platform concept could
in vivo implantation, PDL cells not only proliferate but also be developed with a variable spectrum of self-assembling
increase their expression of PDGF.201 In another study from the materials, cell types, and mechanically flexible micropo-
same group, porous chitosan=collagen scaffolds were prepared rous materials, depending on the specific needs.207,208 Finally,
through a freeze-drying process and loaded with an adenoviral drug delivery devices (external dispensers or nanoparticles)
(Ad) vector encoding BMP-7. Results indicated that the scaffold may also offer highly desirable features in the complex process
containing Ad-BMP-7 exhibited higher alkaline phosphatase of regeneration: for example, independent time release of
activity and that expression of osteopontin and bone sialopro- multiple factors, each acting at a different time point, to pro-
tein were up-regulated. After implantation in defects around mote the development of functional periodontal tissue.209,210
implants, bone formation in Ad-BMP-7 scaffolds was greater
than that in other scaffolds at 4 or 8 weeks, demonstrating the
Candidate growth factors
potential of chitosan=collagen scaffolds combined with Ad-
for periodontal tissue engineering
BMP-7 in bone tissue engineering.202 Controlling the diffusion
rates of genes and proteins from scaffolds so that they are within Growth factors are biologically active polypeptide hor-
the physiological range is the next challenge.70,200 New bioac- mones that affect immune function as well as differentiation
tive materials, such as those that covalently incorporate growth of cells from the epithelium, bone, and connective tissue.96
factors and other molecules that regulate cell behavior, offer These proteins are endogenously secreted in the cell bodies
alternatives for enhancing scaffold performance.203,204 themselves (autocrine) or as a result of communication with
It has been recognized, for many years, that the microen- surrounding cells (paracrine). A major focus of periodontal
vironment in which a cell resides dictates many of its func- research has evaluated the impact of growth factor applica-
tions. Thus, it seems logical that the construction and design tions on periodontal tissue regeneration.27,28 Advances in
of a cell-seeding scaffold must take into account microenvi- molecular cloning have made available unlimited quantities
ronmental design features to induce the appropriate gene of recombinant growth factors for applications in tissue en-
expression in cells forming new tissues.205 By physical ad- gineering, where the healing and regeneration process is
sorption of biomolecules onto scaffold surfaces, physical controlled by spatio-temporal action of growth factors, cyto-
entrapment of biomolecules in polymer microspheres or kines, and chemokines leading through progression of heal-
hydrogels, and chemical immobilization of oligopeptides or ing and resulting in the reestablishment of tissue function. In
proteins on biomaterials, biologically active biomaterials and periodontal tissue engineering, of particular importance are
scaffolds can be derived. These bioactive systems show great PDGF, IGFs, FGF, TGF-b, and BMPs, and those that regulate
potential in tissue engineering by rendering bioactivity and= the epithelial–mesenchymal interactions involved in initial
or specificity to scaffolds. Apart from this, the concept of tooth formation (e.g., Embdogain, Biodenix Technologies,
molecular self-assembly is the driving force for the devel- Richmond, Canada) have been used in preclinical and clinical
opment of new biomaterials that support the growth and trials for the treatment of large periodontal or intrabony de-
functional differentiation of cells and tissues in a controlled fects and around dental implants.96,104 Proper periodontal
manner.206,207 Finally, the use of self-assembling materials in healing is guided by stringent regulation of these agents as
combination with a bioengineering platform has been pro- well as a local microenvironment that favors their activity.
posed to assist functional bone and periodontal regeneration Two of these growth factors, PDGF and IGF-1, enhance re-
in cases of larger defects. The fact that self-assembling pep- generation in beagle dogs and monkeys with periodontal
tides may promote matrix biomineralization in vitro as well disease,119,120 especially when combined.211–215 Bone-derived
as in vivo indicates a potential application for their use in BMPs, with a collagenous matrix as a carrier, induced ce-
bone-related therapies as biomaterials themselves, or in more mentum and alveolar bone regeneration in surgically created
complex tissue-engineering platforms. Semino (2008) recently furcation defects in primates.216 It is noteworthy that there
discussed in detail the features of this class of material:207 was morphogenesis of PDL and a true insertion of Sharpey’s
fibers into cementum. In the same furcation model, recombi-

they are fabricated by rational design and are easily nant human OP-1 (rhOP-1, also known as BMP-7), in con-
synthesized, producing chemically defined biomaterials; junction with the collagenous carrier, induced extensive

the peptides are injectable, gelling after interaction with cementogenesis with insertion of Sharpey’s fibers into the
body fluids, and adopting the geometry of the tissue newly formed cementum.124 The observation that BMPs in-
defect; duce cementogenesis and PDL formation indicates that

chemical functionality can be controlled, as can biome- these proteins may have multiple functions in vivo not limited
chanics; to cartilage and bone induction. The rapid advances in the

as far as it is known, they induce no apparent immune molecular biology of BMPs and their receptors bode well for
response; the emergence of novel strategies to engineer the regenera-

they are biodegradable, breaking down into constituent tion of periodontal tissue.100,124,125,217,218 Other growth factors
amino acids; as mentioned in the ‘‘Recombinant Protein Therapy’’ section,

they share many properties with natural ECMs, such as such as TGF-b1,127,128 PDGFs,119,120,129–132 IGF-1,48,119,130,132
truly 3D nanofiber matrices to support cell maintenance, and bFGF,133–135,219 are all candidates for periodontal regen-
proliferation, and differentiation. eration.16,27,28,96,98,99
240 CHEN AND JIN

The enamel matrix derivative (EMD) contains proteins cleavage of the bond that binds the protein to the
belonging to the amelogenin family, which is the hydrophobic polymer.48,49,67,90,105,109,110,119,120,124,133,211–216
constituent of the enamel matrix proteins. Enamel matrix
Use of a plasmids (DNA)=vector that include the gene
proteins, produced by HERS, are known to play an important encoding the desired growth factor and the vector (hav-
role in cementogenesis, as well as in the development of the ing the gene of interest) is directly injected to the wound
periodontal attachment apparatus. There is some evidence or sometimes, delivered using a polymer carrier—the
that these proteins also play a role in the regeneration of plasmid will biosynthesize the growth factor and secrete
periodontal tissues after periodontal therapy. Emdogain, a it from the cells where the plasmid resides, so long as the
formulation of EMD, is used clinically for periodontal re- plasmid remains active.201,202
generation, where it stimulates cementum formation and
A gene encoding the growth factor is transferred to a
promotes gingival healing, although long-term benefits specific type of cell (stem cells, fibroblasts, or the cells of
and histological data after treatment with EMD are still lim- interest) using a vector and the processed cells are al-
ited.220–222 The complex sequences of carefully orchestrated lowed to grow in vitro and increase in number, and then
natural molecular and cellular events triggered by EMD transplanted into the body where the desired tissue is
during the periodontal healing process are difficult to emu- to be engineered—the growth factor encoded by the
late. It is postulated that EMD gel contains both TGF-b- and transferred gene will be released into the body as long as
BMP-like growth factors, and maybe others that contribute to the gene stays active in the cells.36,39,233,234
the induction of biomineralization during periodontal re-
Aiming at periodontal tissue regeneration, we have devel-
generation. The synergistic interactions between multiple
oped a microsphere system using gelatin and dextrans (as
biomolecules may play an important regulatory role in peri-
well as their derivatives) for direct growth-factor delivery.
odontal tissue regeneration.223
There are two kinds of gelatin commercially available, acidic
Platelet-rich plasma (PRP) presents a new approach in tis-
and basic. Acidic gelatin can form ionic complexes with basic
sue regeneration and a developing area for clinicians and re-
proteins, while basic gelatin can form ionic complexes with
searchers. It is a component of autologous whole blood
acidic proteins. It is therefore expected that IGF-1 will be re-
isolated after the centrifugation of the plasma. PRP acts as a
leased from the ionic complex formed upon mixing acidic
source of growth factors, including PDGF and TGF-b, both of
gelatin with ionically basic IGF-1, when implanted in the
which appear to be critical growth factors involved in peri-
body, due to the enzymatic degradation of gelatin molecules.
odontal regeneration.129,131,211,223 The availability of several
Such polyion complexation between different charged species
commercial kits to isolate PRP at the chair-side has contrib-
is very effective in preventing drug burst release from gelatin
uted to its increasing popularity among clinicians. The in-
hydrogels, suggesting that complexed gelatin=biomolecule
corporation of PRP into the sinus graft has been proposed as a
fragments are mainly released by enzymatic degradation of
method to shorten periodontal healing times, enhance peri-
the carrier in vivo.48 However, it should be noted that this
odontal wound healing, and improve bone quality. However,
relationship cannot be applied to all proteins. Escherichia coli–
large-scale human studies are required before this technique
expressed BMP-2, for example, has an isoelectric potential
can be recommended for routine use.224–231 Although the
value close to 8.2, but may exhibit weakened electrostatic in-
growth factors and mechanisms involved are still poorly un-
teractions with acidic gelatin due to sugar residues covering
derstood, the easy application of PRP in the clinic and its
the BMP-2 molecule. That is to say, the BMP adsorption is
possible beneficial outcome, including reduction of bleeding,
higher in a basic gelatin than in an acidic one, regardless of
rapid soft tissue healing, and bone regeneration, hold promise
which kind of BMPs is delivered,235 which is why basic gelatin
for new treatment approaches.230,231
was used for BMP-2 delivery in our previous work.105,136,209
Critical in the successful application of growth factors
The use of both ex vivo and in vivo gene delivery strategies for
to tissue engineering are the methods of growth factor de-
periodontal tissue regeneration has also presented exciting
livery to the site of action.27 The principal drawback with
results, which were discussed in 2006 in two reviews.117,118
these techniques is that these growth factors, which generally
Currently, significant efforts have been made to induce neo-
have a short in vivo half-life, are delivered as a single
vascularization using various growth factors to supply suffi-
nonphysiological bolus. The development of controlled-
cient amounts of nutrients to the cells engaged in tissue
release delivery approaches that delivery their cargo in a
regeneration. The application of growth factors in periodontal
space- and time-dependent manner has the potential to sig-
tissue engineering will be accelerated when growth factors
nificantly increase growth factor clinical effectiveness. In
become more readily available and less expensive. The clinical
addition to autocrine and paracrine cell signaling, they pro-
application of these protein- and gene-based strategies for
vide specific extracellular information necessary to conduct
periodontal regeneration will eventually provide additional
tissue homeostasis and (re)generation.232 Three methods
treatment options for periodontal surgeons, while lowering
have been attempted for growth factor delivery in tissue
the protein or gene load required for an adequate healing
engineering:27,28,117,118
response.

Direct delivery of growth factors to the wound using
Tissue-Engineering Approaches
carriers—the growth factor is physically immobilized
in the carrier matrix and released in a sustained Limitations of current periodontal regeneration methods,
and=or controlled manner by degradation or dissolu- in terms of both predictability and extent of healing, un-
tion of the carrier or system in the body; in case that derscore the importance of developing novel therapeutics
the protein is chemically bound to the polymer back- that are capable of regenerating complex tooth-supporting
bone, it is released by the hydrolytic or enzymatic structures. Recently, a paradigm shift has taken place by
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 241

moving toward the utilization of tissue engineering and local in developing strategic and technological platforms. A par-
drug=gene delivery systems in periodontal tissue regenera- adigm shift is taking place in orthopedic and reconstructive
tion. By reviewing the literature and imposing a hierarchical surgery away from using medical devices and tissue grafts to
framework, tissue-engineering approaches for periodon- a tissue-engineering approach that employ biodegradable
tal regeneration are nondefinitively subdivided into three scaffolds combined with cells or biological molecules to re-
principal therapeutic strategies for introduction in this sec- pair and=or regenerate tissues.22,38,205,236
tion:21–23,29,47
Cell-based therapy

cell-based therapy—implantation of freshly isolated or
cultured cells, which can be autologous or allogenic cell Cell-based therapy involves the utilization of in-vitro-
suspensions or cell-sheets that are injected and=or expanded cells with regenerative capacity and their delivery
transplanted to periodontal defect sites; to the appropriate sites via vehicles or biomaterial scaffolds

in vitro approaches—implantation of tissues assembled or by direct injection of single cell suspensions into the target
in vitro from cells, scaffolds, and biomolecules (for ex- tissues. This approach is advantageous because the cells may
ample, growth factors, growth factor encoding genes, directly participate in the regenerative process, can be ge-
completely lyophilized cell fractions, peptides, and netically modified by ex vivo techniques to serve as vehicles
polysaccharides); for gene therapy, and can potentially develop into a variety

in vivo approaches—in situ tissue regeneration by im- of tissue types.34,82,84,139,237 The growth factors expressed by
plantation of different types of matrices (such as hy- the transplanted cells have both autocrine and paracrine ef-
drogels and microspheres=beads) in combination with fects, which should make the regenerative process more ro-
cells and=or biomolecules, or a scaffold containing bio- bust.232–234 The source and type of cells used are important
molecules implanted directly into the defects that stim- considerations when planning a cell-based therapeutic ap-
ulates the body’s own cells to promote local tissue proach for periodontal tissue regeneration. Although the
regeneration. exact source of periodontal precursor cells has yet to be de-
termined, it is believed that they are most likely located
These strategies are not mutually exclusive (Fig. 9). Tissue within the PDL tissues. For example, a population of multi-
engineering is thus not only a complex and interdisciplinary potent postnatal stem cells can be isolated from human
area but one in which research teams have multiple choices PDL that are capable of generating cementum=PDL-like

FIG. 9. A schematic representation of three major approaches based on tissue-engineering strategy for periodontal
healing=regeneration. Cell-based therapy is to harvest biopsies from patients, culture, and expand cell populations with select
inductive factors, directly injected into the periodontal defects or simply mixed with injectable cell carriers before injection.
Alternatively, cells were seeded into a three-dimensional scaffold containing growth factors, and immediately implant into
periodontal defects (in vivo approaches), or to create tissue-like constructs in culture room by combine three elements (scaffolds,
growth factors, and cells) before transplanting tissue-engineered tissue into periodontal defects, where cells grow and organize
on a three-dimensional support (scaffolds) (ex vivo approaches). Color images available online at www.liebertonline.com=ten.
242 CHEN AND JIN

structures when transplanted into immunodeficient rats.166 gineering, which seems to have great potential in the future
Therefore, PDL cells are believed to play a crucial role in the tissue engineering of periodontal tissues and has recently been
regeneration of periodontal tissues, and an undifferentiated discussed in more detail in another review.247
mesenchymal cell subset is also thought to exist within this
population. In vitro approaches
As discussed previously, it seems that many cell sources
In-vitro-expanded cells in sufficient quantities and possessing
are available for periodontal regeneration. However, PDL-
the potential to regenerate alveolar bone, cementum, and PDL
derived cells are the most frequently investigated in current
may be used with appropriate biomaterials to engineer living
studies because human PDL fibroblasts are known to play a
tissues in vitro for subsequent transplantation into defect
key role in the regeneration of the periodontal compart-
sites.137 In vitro tissue engineering involves tissue reconstruction
ment during GTR procedures.42 Recent results indicate
by cell culture methods and organ substitution with functional
that human PDL contains a subpopulation of cells capable
cells, termed ‘‘bioartificial hybrid organs.’’ If tissue can be re-
of undergoing osteogenic differentiation under 2D and 3D
constructed in vitro in factories or laboratories on a large scale, it
culture conditions and presumably contributes to the re-
can be supplied to patients when required.78,248 However, at
generation of bone defects in the adjacent area. Three oste-
present, it is difficult to complete in vitro tissue engineering
ogenic culture conditions (dexamethasone, ascorbic acid, and
because it is not possible to artificially arrange a biological en-
beta-glycerophosphate) have been established:238
vironment for cell-based tissue reconstruction. In addition, ox-
1. 2D culture as a single-cell monolayer, ygen limitations often create difficulties in the in vitro growth of
2. 3D static culture on mineralized poly-DL-lactic-co- large tissues.78,106 One of the simplest examples in tissue engi-
glycolic acid scaffolds, and neering involves the sourcing and expansion of cells and their
3. 3D culture on mineralized poly-DL-lactic-co-glycolic subsequent signaling by growth factors in a bioreactor. Besides
acid scaffolds inside a NASA-approved bioreactor stim- the cost, some of the drawbacks of this include the instability of
ulating microgravity conditions. many of these molecules and the fact that their optimal effects
are usually observed in vivo rather than ex vivo.
Results have demonstrated that human PDL-seeded mineral-
ized poly-DL-lactic-co-glycolic acid scaffolds in the micrograv-
In vivo approaches
ity bioreactor may be used to support osteogenic differentiation
in vitro.238 The osteogenic differentiation potential of human Distinct from in vitro tissue engineering, in vivo tissue en-
PDL fibroblasts was further assessed in a 3D osteogenic culture gineering has the advantage of facilitating cell-induced tissue
environment after encapsulation in chitosan-HA microspheres regeneration. Clearly, most biological components that are
with a size range of 350–450 mm.239 Thus, PDL fibroblasts are essential for tissue regeneration, such as growth factors and
proven to be a good cell resource for periodontal cell-based cytokines to accelerate the proliferation and differentiation of
therapies.71,240 cells, as well as stem and progenitor cells of high proliferative
Tissue engineering encompassing the general paradigm of and differentiation potential, are supplied by the host.47,137
seeding cells into biodegradable scaffolds has also evolved as a Therefore, almost all of the current approaches to tissue en-
method for the reconstruction of various tissues and organs.137 gineering for periodontal regeneration have been performed
However, regenerative therapies using cell sheet technology in vivo with biodegradable scaffolds or artificial ECM.21,23
have garnered significant interest and attention in periodon- There are a number of examples whereby in vivo tissue re-
tal regenerative therapies.241 For example, Hasegawa et al. generation has been achieved to a certain degree using cell
(2005) have developed a new method of cell transplantation scaffolds alone or in combination with cells.43,87,249–261 If the
using cell sheet engineering. The characteristics of human tissue to be repaired has a high propensity to regenerate,
PDL cell sheets retrieved from culture on unique temperature- a new tissue will be formed within the biodegradable scaf-
responsive culture dishes were investigated to address whe- fold matrix by active, immature cells that infiltrate from the
ther these cell sheets can regenerate periodontal tissues. The surrounding healthy tissues. However, additional means
results demonstrated PDL-like tissues that included an acel- may be required if the regeneration potential of the tissue is
lular cementum-like layer, and fibrils anchoring into this layer low, due to, for example, low concentrations of relevant cells
were identified in all of the athymic rats transplanted with and=or growth factors. The scientific basis for these novel
human PDL cell sheets. This fibril anchoring closely resembled approaches to periodontal regeneration lies, in part, with the
native PDL fibers, while such regeneration was not observed existence of putative precursor cells within the vicinity of the
in nontransplanted controls.242 The histological findings with PDL.33,162–167,169,170,252 These cells are believed to be capable
Azan stain showed that the outermost layers of curetted root of differentiating into the more specialized cell types required
dentin surfaces were positively stained blue only in the for the reconstruction of a functioning periodontal attachment
transplanted cases. In the regenerated PDL tissues, immature apparatus (osteoblasts, cementoblasts, and fibroblasts), under
fibers were obliquely anchored onto this layer. This orientation the influence of specific growth factors.
resembled that of native PDL fibers.242 The PDL cell sheets On the basis of current evidence from protein-based ther-
were also employed in beagle dog dehiscence models with apies, protein=gene delivery scaffolds suitable for implanta-
new cementum formation, including collagen fibers inserted tion at load-bearing sites offer a greater degree of versatility
perpendicularly into the newly formed bone and cemen- for clinical applications.27,49,70–72,86,105,116–117,190,199–202
tum.243 Further, it is also suggested that the human PDL cell The control of gene expression by cells within a scaffold can
sheet technique may be applicable for regeneration of the be regulated via interactions with the adhesive surface, with
clinical PDL–cementum complex.244–246 Cell sheet engineering other cells in the vicinity or, as described above, with growth
without scaffolds is a new, alternative approach to tissue en- and differentiation factors incorporated into the scaffold.
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 243

Accordingly, cell-seeding scaffolds must provide the correct rived from the periosteum.266 Developments in scaffolding
combination of these factors, depending upon the nature of matrices for cell, protein, and gene delivery have demonstrated
the tissues to be regenerated.27,117,118 To date, little work has significant potential to provide smart biomaterials that can in-
been carried out in this complex area, although early studies teract with the matrices, cells, and bioactive factors. The tar-
have begun to utilize specific cell-attachment peptide se- geting of signaling molecules or growth factors (via proteins or
quences, pore sizes, and surface textures in an attempt to genes) to periodontia has led to significant new knowledge,
improve tissue integration and regeneration. using factors that promote cell replication, differentiation, ma-
One of the primary principles of in vivo periodontal tissue trix biosynthesis, and angiogenesis.
engineering involves the utilization of an ideal supporting
scaffold to control cell behavior, thus reconstructing the pre- Challenges and Future Perspectives
viously lost tooth-supporting apparatus as closely as possi-
ble.262–264 Unfortunately, current scaffolds poorly mimic the Clinical evaluation
ECM that cells expect.79,111,199,200,234,253,262,264 Many would Proper evaluation of the clinical success rates of tissue-
therefore argue that a far more realistic strategy encompasses engineering techniques and products has been hampered by a
the development of hydrogel scaffolds with interconnecting lack of consistency in the experimental techniques used to
pore structures that more closely mimic the gel morphology of induce periodontal defects among different animal groups for
the ECM of natural cementum. A variety of protein, polysac- preclinical trials, as well as disparities in the methods used to
charide, and synthetic hydrogel structures have been devel- analyze outcomes obtained by different technologies. The
oped for this purpose.49,80–82,86,89,105,135,155,198 The cells within most popular animal models used for the assessment of peri-
this 3D matrix will experience the same type of stimulation odontal regenerative protocols today involve ligature-induced
received in the normal periodontal ECM, rather than the more periodontal defects in nonhuman primates (especially the cy-
random nature of interactions between a cell suspended in nomolgus and rhesus monkeys, which share marked similar-
culture medium and the porous polymeric network into which ity with the human periodontium in terms of structure, plaque
it is seeded.81,82,86,198 There is also the added advantage that the flora, and inflammatory infiltrate),89,119,120,197 and beagle
growth factors and cytokines required for periodontal regen- dogs48,49,67,79,121,126,128,134,142,145,146,187,195,196,243,246,256 or mini-
eration may be incorporated into these gels.86,198 Of consider- ature pigs164 (which have a different microflora and much
able importance in this research arena are the attempts to use faster bone turnover rate as compared with humans), although
matrix-immobilized growth factors to mimic the release of much more can be done to continuously improve and optimize
growth factors from natural ECM, in vivo, through cell- those models. A major challenge that has been overlooked has
controlled proteolysis.27,80–82 However, as discussed in a been the wound differences between induced periodontal
number of recently published reviews, these developments defects and human periodontitis; in the latter, the modulation
have been largely unsystematic in nature, and little information of the exuberant host response to microbial contamination that
is available about procedures for orchestrating release profiles, plagues the periodontal wound environment is much more
particularly the sequential release of multiple factors, to opti- complex and different as compared with animal models.
mize their effectiveness. Most therapies suggested until now Obvious ethical issues preclude the en bloc harvesting of
rely upon the supply of a single factor, whereas the natural tooth, PDL attachment, and supporting alveolar bone that
process is multifactorial, such that there is the need for a better would be required for micro-CT and histologic evaluation in
orchestration of growth factor profiles.27,70,198–200 human clinical trials. Therefore, by necessity, in the assess-
The study of scaffold materials for use in periodontal tissue ment of efficacy in clinical trials today, only a combination
engineering should lead to improved predictability of this new of intraoral radiographic evaluation and clinical assessment
technology based upon cell and molecular biology. In the fu- of attachment gain can be applied. However, the estimation of
ture, it will become increasingly important to consider the pocket depth or attachment level by the probing technique is
concept of scaffolds that are not only space making and exclu- questionable due to its lack of accuracy. In addition, it is very
sionary, but also biocompatible and able to elicit appropriate difficult to control and standardize the data obtained from
gene expression by the cells for which they provide the carrier radiographic evaluation and clinical assessment. Attempts to
capacity.78,60,247 Gene-activated matrices blend these two strat- statistically analyze the effectiveness of these techniques have
egies, serving as local bioreactors with therapeutic gene ex- been hampered by the observation that some subpopulations
pression and providing a structural template to fill the lesion appear to respond better to treatment than others and the fact
defects for cell adhesion, proliferation, and ECM synthesis.265 that the factors affecting clinical outcomes are unclear from
An understanding of the complex design features necessary for the literature and these might include study conduct issues
successful tissue engineering will help this technique to become such as bias. The priority of future research should be to
an accepted biomedical procedure. Gene transfer strategies in identify factors associated with improved outcomes as well as
combination with tissue engineering are under intense investi- investigating outcomes relevant to patients, where types of
gation due to their significant therapeutic potential and may research might include large observational studies to generate
achieve greater bioavailability of growth factors within peri- hypotheses for testing in clinical trials, qualitative studies on
odontal wounds, which may, in turn, provide greater regener- patient-centered outcomes, and trials exploring innovative
ative potential.111,112,199,203 Major advances have been made analytic methods such as multilevel modeling.267
over the past decade in the reconstruction of complex peri-
odontal and alveolar bone wounds that have resulted from
Gene therapy and periodontal tissue engineering
disease or injury, including several unestablished novel ap-
proaches such as the attempt to regenerate canine periodontal The cellular and molecular activities of periodontal healing
tissue defects by grafting autologous cultured membrane de- are coordinated and regulated by an elaborate system of
244 CHEN AND JIN

growth factors. Although significant work has been dedicated fore, research related to the management of growth factors
to developing controlled release systems that deliver growth through matrix- and gene-based strategies is of pivotal im-
factors directly for periodontal regeneration, a simpler ap- portance to the future of periodontal tissue engineering.
proach bypassing this dilemma involves converting cells into Cell-based therapies are unique in that the active compo-
protein-producing factories. This is done by a so-called gene nent consists of living cells, which are difficult to define in
delivery method. Gene delivery for periodontal regeneration their pharmacological characteristics and which produce
is a relatively new strategy, as nonviral vectors are continually variable and largely unknown amounts of bioactive mole-
optimized for clinical therapeutic purposes, and significant cules, similar to that found in the natural ECM.277 There is a
reservations about viral vectors have increasingly dampened pressing need for the development of quality controls in cell-
their appeal.26 It is anticipated that, in the future, gene- based therapies, including clinically relevant potency assays,
enhanced tissue-engineering approaches will afford great with prospective validation in human clinical trials.138,247
potential for both dentin-pulp and periodontal regeneration; Protagonists of this new scientific era promote stem cells as
nevertheless, this approach would currently face significant promising tools on which regenerative medicine can rely for
regulatory hurdles before government approval.117,119,268,269 the treatment of human pathologies. Stem cells can be ob-
With the continued development of improved methods of tained from various sources, exfoliated deciduous teeth,
gene delivery to cells, as well as advances in our knowledge of dental follicle, and apical papilla tissues. Once forced to ex-
the molecular basis of tooth formation and periodontal ho- pand and differentiate into functional progenies, stem cells
meostasis, it is reasonable to anticipate that a simple chair-side may become suitable for cell replacement and tissue engi-
protocol could be developed in the future.270 This might in- neering.158,171–173,179,185 Understanding the mechanisms of
volve either direct delivery of the DNA of interest to the self-renewal and the regulation of stem cells during differ-
periodontal tissues or the isolation of a small amount of gin- entiation and specific tissue production is of vital importance
gival tissue from the patient, transduction=transfection of the before development of effective cell-based therapies for peri-
DNA at the chair-side, and reimplantation of the gene- odontal regeneration.186 The manipulation and=or stimula-
enhanced cells into the PDL space.39,118,232,233 tion of adult stem cells seems to be particularly promising, as
We are now nearing the 20-year mark since the first gene it may improve endogenous regenerative potential without
therapy trial. Although success has been limited, the future the risk of rejection and it could overcome the ethical and
still seems overwhelmingly promising, and research is stea- political issues that surround embryonic stem cell research.
dily approaching an acceptable safety record.271 The increas- Stem cells are already leaving the bench and reaching the
ing progress of drug and gene delivery research in the bedside, despite an incomplete knowledge of the genetic
future will help progress tissue engineering.26–28,271–273 Gene control program driving their fate and plasticity.278 On the
delivery combined with tissue engineering has shown early basis of information obtained from basic and translational
progress in achieving an enhanced tissue regeneration in the research, several clinical trials have recently commenced to
periodontium, and some research projects have already come evaluate the safety and efficacy of autologous stem cells for
close to clinical application.274 Ongoing investigations in periodontal regeneration.158–172 The evaluation of both the
ex vivo and in vivo gene transfer to periodontia seek to examine benefits and the potential risks of these new tools for peri-
the extent of the potential effects in stimulating periodontal odontal regeneration is an immediate challenge for scientists
tissue engineering.118 Since gene therapy for periodontal tis- and clinicians alike.
sue engineering is still in its infancy, it can be only envisioned For successful periodontal regenerative therapy utilizing
as to how this new field of science might constantly evolve to cells, it is important to develop the technologies and meth-
redefine periodontal therapy in due time. odologies of tissue engineering using biomaterials that enable
molecular design and the creation of a local environment
(artificial ECM) conducive to cell proliferation and differen-
Optimization of cell sources, scaffolds,
tiation.279–281 Various biomaterial scaffolds have been inves-
and best growth factor combination
tigated to demonstrate the feasibility of such basic research in
It is essential to understand the cellular and molecular cell biology and the applications of their results to regenera-
mechanisms implicated in periodontal wound healing, spe- tive therapy.282,283 Collectively, recent advances in stem cell
cifically in relation to the need to clarify which molecular isolation, protein engineering, growth factor biology, gene
signals are the key players in the complex cascade of tissue therapy, and biodegradable polymer constructs have set the
events in periodontal regeneration. If key growth factors are stage for the successful engineering of many tissues, of which
supplied to the target site at the right time, for the appropri- the periodontium could be considered a prime candidate for
ate period of time and at the right concentration, it is hoped such procedures.26,27,185,271,284–289 Although stem cells from
that the defect sites can be naturally directed toward tissue PDL can be obtained (and certain other stem cells also have
regeneration.26–28,74,275,276 There is no doubt that when the potential for periodontal application), their direct appli-
growth factors are used in vivo, a controlled release system is cation necessitates the development of an environment suit-
essential. However, the present technology does not allow for able for cell proliferation and differentiation. Notably, dentin
accurate regulation of the quantum and temporality of matrix was recently demonstrated to be a suitable scaffold
growth factor release.27,28 Therefore, currently, one practical and inductive microenvironment for dentin regeneration,
approach is to release multiple growth factors necessary to implying its potential application in future periodontal bio-
increase the number of precursor or stem cells in vivo by gene engineering.290 One of the main problems today is a shortage
delivery methods.26 As clearly discussed in the literature,26–28 of active biomaterial scientists within the sphere of tissue
it is not presently possible to control cell differentiation using engineering, who focus on combining traditional drug de-
currently available methods of growth factor release. There- livery technologies and biomaterials with a goal of develop-
PERIODONTAL TISSUE ENGINEERING AND REGENERATION 245

ing regenerative technologies that mimic accurate natural eration and remodeling of periodontal tissues,294 but the exact
bone–PDL–cementum functions. Such researchers must have effects of dentin or dentin matrix on DFCs remain an open
knowledge in medicine, dentistry, biology, and pharmacol- question, although dentin noncollagenous proteins (DNCPs)
ogy, in addition to the material sciences. extracted from dentin was recently found to stimulate DFCs
to differentiate into cementoblast lineages.293 Despite of
DFCs, current evidence also showed that PDL stem cells
De novo regeneration of a biotooth
treated by DNCPs may present several features of cemento-
In light of first principle and roles of periodontal tissue blast differentiation.295 In addition, in vivo incubation of PDL
engineering as a technology, much effort has been paid to stem cells treated by DNCPs revealed that cementum-like
in situ regeneration of lost tissues that serves individual tissues formed along the chemical-conditioned root dentin
patient. In vitro approaches with expectation to create new surface with fibrous tissue adjacent to or inserted into those
periodontal tissues, or a tissue-engineering construct, for rou- mineralized layers.295 In 2006, a new population of stem cells
tine clinical applications can only be presumed to be achieved isolated from the root apical papilla of human teeth (SCAP,
associated with the development of a bioengineered tooth.280 stem cells from apical papilla) has demonstrated generation of
Progress in understanding the role of biofactors in tooth de- a root=periodontal complex capable of supporting a porce-
velopment, the demonstration of dental stem cells, and ac- lain crown, resulting in normal tooth function. It is confirmed
cumulating knowledge on biomedical scaffolds provided a that the root apical papilla contained MSCs that appear to
number of unique opportunities to de novo regeneration of a have a greater capacity for dentin regeneration than den-
biotooth and its associated tissues.280 Tooth development tal pulp stem cells. This hybridized tissue-engineering ap-
contains a series of events, including the instruction of HERS, proach, as a model system for engineering multiple tissues,
formation of PDL, tooth eruption, dentinogenesis, cemento- leads to a new concept over current strategies for periodontal
genesis, and osteogenesis, during which several kinds of cell tissue regeneration.296 Recently, data from our group suggest
types are involved such as HERS cells, dental papilla cells, that the combination of apical tooth germ cell-conditioned
DFCs, odontoblasts, cementoblasts, PDL cells, and osteo- medium and endogenous ECM could maximally mimic the
blasts.280 De novo synthesis of hybrid periodontal tissues that microenvironment of root=periodontal tissue development
functionally formed around the root is one of the key con- and enhance the reconstruction of physiological architecture
siderations for making a biotooth, where cementum regen- of a cementum=PDL-like complex in a tissue-mimicking
eration on the surface of root dentin followed by the insertion way.297 At the same time, Huang et al. (2009) provide the first
of oriented PDL fibers is truly challenging.28,280 Devel- evidence showing that pulp-like tissue can be regenerated
opmentally, the formation of root dentin and cementum may de novo in emptied root canal space by stem=progenitor cells
be stimulated by signals from the HERS, an apical extension from apical papilla and dental pulp stem cells that give rise to
of the enamel organ that disintegrates around the time of root odontoblast-like cells producing dentin-like tissue on existing
formation, leaving a few select cells to become cell rests of dentinal walls.298 It is the first step toward reaching the goal of
Malassez that lie within the PDL space.291 At about the same finally making a biotooth. It is also important to note that the
time as the disintegration of HERS and the initiation of possibility of utilizing dermal multipotent cells from skin
cementum formation, fibroblasts and collagen bundles reor- tissue has been proven to be a more practical strategy of stem-
ganize to orient perpendicular to the root surface. The first cell-based tooth regeneration research because tooth tissue
cementum laid down in the cervical portion of the root is engineering is somewhat challenged in the circumstances
acellular. More apically, toward the root tip, cementum be- where tooth-derived stem cells are not readily accessible.299
comes both thicker and more cellular.292 Cementoblasts, the In the near term, these advances are likely to be applied
cells responsible for synthesizing the ECM and promoting to endodontics and periodontal surgery; ultimately, they
mineralization of the cementum, become entrapped within may facilitate approaches regenerating whole teeth and their
the matrix of this thicker layer and are then termed ‘‘ce- hybrid periodontal tissues for use in clinical tooth replace-
mentocytes.’’ Although the mechanism of cementogenesis ment.298
is an area full of debate, the DFCs are thought to be the Implementation of tooth tissue-engineering-based thera-
precursors of cementoblasts.293 When the dental follicle pies is underway and results obtained so far suggest that stem
region disappears as tooth formation proceeds, cells retain- cell biology will be more and more a key fundamental.300–304
ing pluripotential properties reside in the PDL, where they We are still some distance from fully understanding the po-
may be available to differentiate and repair damage to the tentiality and behavior of tooth-derived progenitor cells, and
periodontium in adult tissues.292 A competing hypothesis subsequent clinical treatment modalities. Nonetheless, the
maintains that cementoblasts are the result of an epithelial– opportunities for their exploitation in tooth engineering are
mesenchymal transformation event that HERS cells undergo; becoming clearer and will lead to significant benefits in
thus, the processes of epithelial–mesenchymal interaction and the management of tooth defect or loss.304 Future efforts will
the role played by HERS cells are of critical importance in no doubt focus on140,186,280,300–310 (i) the in vitro enrichment
establishing cellular differentiation and tissue pattern forma- of dentin-, PDL-, cementum-, and bone-forming progenitors
tion during cementum development.292,294 At the onset of (multipotent stem cells); (ii) the continued optimization of
cementogenesis, DFCs come into contact with the root dentin biodegradable scaffolds that can induce dentinogenesis,
surface and undergo subsequent differentiation. Although cementogenesis, and osteogenesis; and (iii) the identification
concrete evidence is lacking, the inductive role played by the of artificial matrix mimicking dentin or cementum matrix
underlying dentin matrix has been proposed as a possibility. that is necessary for sequential cell differentiation. Although
TGF-b1 and perhaps other factors in dentin matrix might today there are many technical obstacles facing the biologi-
regulate cell behavior and, therefore, could influence regen- cal solution to de novo tooth reconstruction, attempt for
246 CHEN AND JIN

stem-cell-mediated root regeneration offers expanding op- coveries that will pave the way for the development of new
portunities to realize more and more perspective outcomes. tissue-engineering procedures needed for the predictable re-
generation of periodontal tissues.
Concluding Remarks
Acknowledgments
Regenerative treatment of periodontally damaged teeth is
Portions of the authors’ research discussed in this article
a demanding task. Advantages that come with the use of
were supported by a grant from the National Natural Science
biomaterials are mainly connected with the fact that tradi-
Foundation of China (30700173) as well as grants from the
tional, surgical treatment protocols often do not lead to bone
contributors’ own institution. The authors genuinely thank
regeneration. The purpose of research into periodontal tissue
Professor Iain L.C. Chapple (School of Dentistry, The Uni-
engineering is very clear: that is, to establish a new clinical
versity of Birmingham) for his critical reading and significant
technology that makes it possible to manage periodontal
modifications regarding the presentational nature of this pa-
diseases, beyond the traditional approaches that are based
per. The authors also wish to thank all contributors for their
solely upon infection control. Recent advances in this field
substantial information that was delivered in a body of pre-
have merged to create a promising new era in which it is
viously published reviews in the same or similar fields, as
possible that biological approaches may be routinely used to
cited in this article, which provided an important guideline
enhance the reconstruction of the tooth-supporting appara-
while preparing our work. The authors apologize to all sci-
tus.31,32 Periodontal clinicians and certainly periodontitis
entists whose work is not cited or included in this article due
patients have great expectations of regenerative medicine or
to knowledge limitations and space restrictions.
tissue engineering, although there is still a paucity of re-
ported human clinical trials in the contemporary literature. It
Disclosure Statement
seems that the clinical application of new tissue-engineering
strategies for periodontal defect management has been much No competing financial interests exist.
slower than our initial expectations. On one hand, the out-
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Address correspondence to:
of cementum=periodontal-ligament complex using a novel
Fa-Ming Chen, D.D.S., Ph.D.
three-dimensional pellet cultivation system for human peri-
Department of Periodontology and Oral Medicine
odontal ligament stem cells. Tissue Eng Part C Methods 15,
School of Stomatology
571, 2009.
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Tuan, R., and Shi, S. Stem=progenitor cell-mediated de Xi’an 710032
novo regeneration of dental pulp with newly deposited P.R. China
continuous layer of dentin in an in vivo model. Tissue Eng
Part A 2009. DOI:10.1089=ten.TEA.2009.0518. E-mail: cfmsunhh@fmmu.edu.cn
299. Huang, G.T., Sonoyama, W., Liu, Y., Liu, H., Wang, S., and
Shi, S. The hidden treasure in apical papilla: the potential Received: August 16, 2009
role in pulp=dentin regeneration and bioroot engineering. Accepted: October 23, 2009
J Endod 34, 645, 2008. Online Publication Date: January 11, 2010