Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2014; 59:(1 Suppl): 117–130

doi: 10.1111/adj.12100

Stem cells, tissue engineering and periodontal regeneration

J Han,* D Menicanin,* S Gronthos,† PM Bartold*
*Colgate Australian Clinical Dental Research Centre, School of Dentistry, The University of Adelaide, South Australia.
†School of Medical Sciences, The University of Adelaide, South Australia.

ABSTRACT
The aim of this review is to discuss the clinical utility of stem cells in periodontal regeneration by reviewing relevant lit-
erature that assesses the periodontal-regenerative potential of stem cells. We consider and describe the main stem cell
populations that have been utilized with regard to periodontal regeneration, including bone marrow-derived mesenchy-
mal stem cells and the main dental-derived mesenchymal stem cell populations: periodontal ligament stem cells, dental
pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla and dental follicle pre-
cursor cells. Research into the use of stem cells for tissue regeneration has the potential to significantly influence peri-
odontal treatment strategies in the future.
Keywords: Periodontium, repair, bone grafts, bioactive materials, scaffolds.
Abbreviations and acronyms: ADSC = adipose-derived stromal cell; BMP = bone morphogenetic protein; BMSC = bone marrow stro-
mal stem cell; CFU-F = colony-forming unit fibroblast; DPSC = dental pulp stem cell; EMD = enamel matrix derivative; GFP = green
fluorescent protein; IGF-I = insulin-like growth factor-I; iPS = induced pluripotent stem; ISCT = International Society for Cellular Ther-
apy; MSC = mesenchymal stem cell; PDGF = platelet-derived growth factor; PDL = periodontal ligament; PDLSC = periodontal liga-
ment stem cell; PRP = platelet-rich plasma; SCAP = stem cell from apical papilla; SHED = stem cell from exfoliated deciduous teeth.

economic cost. The ultimate goal of periodontal ther-

INTRODUCTION
Periodontal disease is a chronic inflammatory condi-
tion of the periodontium that is characterized by irre-
versible destruction of the tooth attachment and its
surrounding bone. The disease state, if left untreated,
can lead to progressive loss of gingival tissue, peri-
odontal ligament and supporting alveolar bone, ulti-
mately resulting in an aesthetically and functionally
compromised dentition, including premature tooth
loss.1 The pathogenesis of periodontal disease involves
a complex interaction between the host’s immune
response to microbial colonization of the periodontal
attachment, and modifying host factors, including
tobacco smoking2 and genetic susceptibility.3 Perio-
dontal disease has also been linked to many underly-
ing systemic disorders such as diabetes,4
cardiovascular disease and rheumatoid arthritis.6 Pro-
5
gressive periodontitis is seen in most adult human
populations, with a prevalence of either moderate or
severe periodontal disease in the Australian popula-
tion estimated at 22.9%.7 The consequences of
untreated periodontal disease have broad ranging
implications on an individual’s quality of life, and
thus impact upon the health system and carry a heavy
© 2013 Australian Dental Association
apy relies on the achievement of complete restoration
of all components of the periodontium to their origi-
nal architecture and function. This entails reconstruc-
tion of gingival connective tissue, cementum, alveolar
bone and periodontal ligament (PDL). In addition,
formation of Sharpey’s fibres, or the portion of the
PDL embedded in both newly formed cementum and
alveolar bone, is essential to restore appropriate con-
nections between the tooth and its supporting tissues.8
Current conventional techniques for the treatment of
periodontal disease show a limited potential for com-
plete periodontal regeneration. An improved under-
standing of periodontal biology coupled with current
advances in the development of scaffolding matrices
has introduced novel treatments that use cell and gene
therapy to enhance periodontal tissue reconstruction
and its biomechanical integration.
The periodontium
The periodontium is a complex organ consisting of
two soft connective tissues (gingival and periodontal
ligament) and two hard connective tissues (cementum
and alveolar bone).9
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J Han et al.

Formation of the periodontium with the dentine of the root.24 Approximately 45% to
50% of cementum is composed of inorganic hydroxy-
Periodontal regeneration aims to recapitulate the cru-
apatite whilst organic collagen and non-collagenous
cial stages of wound healing associated with periodon-
matrix proteins constitute the remaining 50%. Colla-
tal development in order to restore lost tissues to their
gen type III and XII are found alongside collagen type
original form and function.10 Hence, an understand-
I which constitutes up to 90% of the organic compo-
ing of the processes involved in the development of
nent in cementum.25 Additionally, non-collagenous,
the periodontium is essential in order to elucidate the
bone-associated proteins, identified in cementum,
cellular and molecular events that might occur during
include bone sialoprotein, osteopontin, osteocalcin
periodontal regeneration.
and osteonectin.26,27
The tooth is formed as a result of reciprocal inter-
Cementum can be broadly classified as cellular and
actions between the dental epithelium and neural crest
acellular but at least five types have been identified
cell-derived ectomesenchyme.11–15 Dental develop-
and described depending not only on their cellular
ment involves distinct morphological stages, namely
content but also the presence of extrinsic or intrinsic
bud, cap, bell and crown stage. At the bell stage, the
collagen fibres.28 The layer of cementum attached to
tooth germ consists of the enamel organ, dental
the root dentine and covering the cervical portion of
papilla and dental follicle, a fibrocellular layer envel-
the root is acellular and is termed primary cementum.
oping the dental papilla and enamel organ. The sup-
The apical portion of the root is covered by a cellular,
porting tissues of the tooth form from the dental
or secondary cementum, in which cementoblasts,
follicle.16 Development of the periodontal tissues
cementum forming cells, remain trapped in lacunae
begins when cells of the inner and outer dental epithe-
within their own matrix, characteristic of osteocytes
lium proliferate from the cervical loop of the enamel
in bone. These entrapped cells are termed cemento-
organ to form a double layer of cells known as Her-
cytes. Acellular cementum anchors the periodontal lig-
twig’s epithelial root sheath. The root sheath separates
ament fibre bundles to the tooth, while cellular
cells of the dental papilla from the dental follicle.
cementum has an adaptive role, allowing bodily tooth
Cells of the dental papilla differentiate into odonto-
movement within the jaw during orthodontic tooth
blasts to form root dentine, following which cells of
movement.25
the root sheath secrete a fine matrix of proteins onto
the dentine surface, known as the hyaline layer of
Hopewell Smith.17 Subsequent fragmentation of the Periodontal ligament
root sheath allows ectomesenchymal cells of the den-
The periodontal ligament is a highly specialized
tal follicle to attach to this protein matrix and differ-
fibrous connective tissue situated between the cemen-
entiate into cementoblasts.18,19 Apical development of
tum, covering the root of the tooth, and the bone
the root continues with continuing cementum deposi-
that forms the socket wall. It is a highly vascular-
tion. Collagen fibre bundles become embedded in
ized tissue, which reflects the high rate of turnover
newly-formed cementum, known as Sharpey’s fibres.
of its cellular and extracellular constituents. Similar
This process triggers the formation of the periodontal
to all other connective tissues, the periodontal liga-
ligament by fibroblasts also derived from the dental
ment consists of cells and an extracellular compo-
follicle.20,21 Likewise, bundle bone, the portion of the
nent of collagenous fibres and a non-collagenous
alveolar process that lines the tooth socket, is formed
extracellular matrix.16 The constituent cells include
by active osteoblasts originating in the dental follicle.
osteoblasts and osteoclasts (contained within the
Finally, insertion of Sharpey’s fibres into this newly-
ligament but functionally associated with bone),
forming bone completes the development of the
fibroblasts, epithelial cell rests of Malassez, macro-
attachment complex of the periodontium.22 The coor-
phages, undifferentiated mesenchymal cells,29 and
dinated formation of cementum, periodontal ligament
cementoblasts (also contained within the ligament
and alveolar bone, along with the transformation of
but functionally associated with cementum). The
reduced enamel epithelium to sulcular and junctional
extracellular compartment consists of well-defined
epithelium during tooth eruption, give rise to the com-
collagenous and non-collagenous proteins.30 The
plete periodontium.23 A summary of the biochemical
human periodontal ligament spans approximately
composition and function of each of the tissues is
0.2 mm in width, and provides mechanical resistance
presented below.
for the absorption of masticatory forces on the
tooth via the dense masses of collagen fibre bundles.
Cementum Furthermore, the periodontal ligament is mechano-
responsive and shows adaption to increased or
Cementum is an avascular, bone-like connective tissue
decreased mechanical loads by regulating its overall
which lines the tooth root and is firmly interlocked

118 © 2013 Australian Dental Association


width by as much as 50% without losing its original
architecture. The periodontal ligament also provides a
proprioceptive function for the neuromuscular control
of mastication.25,31
Alveolar bone
The alveolar bone constitutes the alveolar process,
which is firmly attached to the basal bone of the jaw.
The alveolar bone comprises inner and outer compo-
nents and is perforated by many foramina, which
transmit nerves and vessels. The bone directly lining
the socket, the inner aspect of alveolar bone, is specifi-
cally referred to as bundle bone. Embedded within
this bone are the extrinsic collagen fibre bundles of
periodontal ligament (Sharpey’s fibres). The bundle
bone generally contains less intrinsic collagen fibrils
than other types of bone. The alveolar bone is sub-
jected to remodelling during normal function and dur-
ing increased mechanical loading.25,32
Gingiva
The gingiva has the general pattern of stratified
squamous epithelium with an underlying connective
tissue lamina propria which bounds tightly to the
bone. The covering epithelium is keratinized to with-
stand masticatory and other minor traumatic forces
during normal oral function. Cells from the kerati-
nized surface layer are continually shed and replaced
from the underlying granular or intermediate layer,
prickle-cell layer and the basal layer. This represents
progressive epithelial maturation and is achieved by
proliferation of cells in the basal layer by mitosis,
cell differentiation at the supra-basal layers, and cell
death at the surface layer. The main cellular compo-
nent of the oral epithelium is the keratinocyte. Non-
keratinocytes also present in the oral epithelium
include Langerhans cells, Merkel’s cells, melanocytes
and inflammatory cells including lymphocytes. The
connective tissue layer, lamina propria, which under-
lies the oral epithelium contains fibroblasts, immune
cells, endothelial cells, blood and lymphatic vessels,
neural elements, as well as a complex extracellular
matrix composed of collagenous and non-collagenous
proteins.30
Periodontal wound healing
The unique anatomy and composition of the peri-
odontium, comprising hard and soft connective tissues,
as well as epithelium, make periodontal wound heal-
ing a complex process.33 The current conventional
techniques for periodontal repair focus on arresting
the progression of periodontal disease by reducing the
microbial levels in the periodontium, and modifying
© 2013 Australian Dental Association
Tissue engineered periodontal products
the local environment to reduce inflammation. Cur-
rent non-surgical techniques, such as subgingival
debridement, and surgical procedures, such as open
flap debridement, aim to remove diseased tissue and
clean the root surface to encourage reattachment of
tissues. These techniques typically result in a process
of repair (Table 1), leading to the healing of the
wound site by formation of a long junctional epithe-
lial attachment. This epithelial attachment represents
a non-physiologic epithelial attachment that does not
provide a strong connection between root surface and
the adjacent gingival connective tissue.34 This process
results in minimal repair of the damaged cementum or
alveolar bone and therefore, by definition, cannot be
considered true regeneration of the periodontium.8
While healing by repair may be effective in preventing
future progression of disease and therefore any ulti-
mate tooth loss, there are a number of limitations to
the therapeutic outcomes. Firstly, the treatment may
have limited effect in resolving the existing mobility
of the teeth due to lack of regeneration of lost tissues.
Secondly, such treatment frequently results in gingival
recession, which is both unsightly and may result in
predisposition of affected teeth to root caries. Finally,
it is postulated that, without restitution of the original
anatomy, the site may be more susceptible to recur-
rence of disease in the future.35
Current treatment regenerative approaches
A number of different procedures have been described
to promote true and predictable regeneration of the
periodontium since the 1980s. To describe the latest
trends, principles of these different treatment
approaches include the use of graft materials to com-
pensate for the bone loss incurred as a result of peri-
odontal disease,36 use of barrier membranes for
guided tissue regeneration,37,38 and use of bioactive
molecules.39–41
Table 1. Periodontal wound healing responses by
repair and regeneration
Repair Regeneration
Control of inflammation Formation of new functional
attachment, including formation
of cementum, periodontal ligament
and alveolar bone
Formation of long
junctional epithelium
Re-attachment of connective
tissue to adjacent root surface
New bone separation from
the root surface by long
functional epithelium,
accompanied by root
resorption, and/or ankylosis
Adapted from reference 8.
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Bone grafts formation. Among those characterized to date are the

platelet-derived growth factor (PDGF) and insulin-like
Various types of bone grafts have been investigated to
growth factor-I (IGF-I) which have shown to enhance
determine their ability to stimulate new bone forma-
regeneration in beagle dogs and monkeys with peri-
tion. These include introduction of alloplastic materi-
odontal disease.53,54 In addition, bone morphogenetic
als (generally synthetic fillers), autografts (grafted
proteins (BMPs) have also shown potential to stimu-
tissue from one site to another in the same individ-
late bone and cementum regeneration.55
ual), allografts (tissue between individuals of the same
The most widely clinically tested of these molecules
species but with different genetic composition) and
is the enamel matrix derivative (EMD), known to reg-
xenografts (grafted materials between different spe-
ulate the epithelial mesenchymal interactions involved
cies). The use of such grafting materials has resulted
in initial tooth formation. The mode of function of
in some gain in clinical attachment levels and radio-
these proteins remains unclear. However, it is known
graphic evidence of bone fill. However, histological
that they are produced by Hertwig’s epithelial sheath,
analyses of these treatments have shown minimal
and play an important role in cementogenesis and the
osteoinductive and cementogenic capacity as most
development of the periodontal attachment appara-
generally become encased in a dense fibrous connec-
tus.41 EmdogainTM (Straumann AG, Basel, Switzer-
tive tissue.42,43
land) is a mixture of enamel matrix proteins primarily
containing amelogenins derived from developing por-
Guided tissue regeneration cine teeth. Furthermore, EmdogainTM contains TGF-b
and BMP growth factors and, as such, the addition of
Based on the hypothesis of Melcher in 1976 that the
these proteins to intrabony defects may promote peri-
first cells to populate a wound will dictate the nature
odontal regeneration by recapitulating the environ-
and quality of tissue that forms there,33 Nyman et al.
ment during initial tooth attachment.39–41 A Cochrane
first demonstrated that by guiding the colonization of
review of 13 clinical trials revealed that while EMD
the periodontal wound healing site by progenitor cells
significantly improved probing attachment levels
from the periodontal ligament and alveolar bone, and
(1.1 mm) and pocket depth reduction (0.9 mm), the
eliminating cells of gingival epithelial and fibroblastic
data could not be interpreted fairly due to a high
lineages from this site, it is feasible to achieve peri-
degree of heterogeneity observed among the included
odontal regeneration in experimental animals and
trials.39 Overall, it was found that EmdogainTM pro-
humans.44,45 This technique, termed guided tissue
duces results similar to those seen with the use of con-
regeneration, has recently become a widely accepted
ventional guided tissue regeneration, and no evidence
clinical procedure, and is currently considered the
of clinically significant differences between the use of
‘gold standard’ upon which to base and compare
EMD and guided tissue regeneration could be
regenerative therapies.46,47 Guided tissue regeneration
found.39 In addition, clinical significance of these pro-
involves utilization of barrier membranes to prevent
cedures, and particularly the benefits that accrue to
unwanted epithelium and gingival connective tissue
patients from these outcomes, remain to be fully dem-
from entering the healing site while recruiting cells
onstrated as all of these procedures are only applica-
from the periodontium to re-populate the defect area.
ble in very specific indications, such as in infrabony
It has, however, been reported that the clinical
defects exhibiting vertical bone loss around single
improvements obtained through the use of guided tis-
teeth, or between furcation lesions. Consequently, in
sue regeneration are, at present, minor and highly var-
many cases where periodontal attachment loss is pres-
iable.48–50 The limited success of this approach has
ent in a regular horizontal pattern throughout the
been attributed to insufficient biocompatibility and
mouth, regenerative procedures currently have little or
poor mechanical properties of the common membrane
no utility.
materials used for their crucial role of wound stabil-
ization and space maintenance. Moreover, there is evi-
dence to suggest that the outcomes of treatment are Periodontal regeneration summary
technique sensitive.51,52
For regeneration to occur, healing events must pro-
gress in an ordered and programmed sequence both
Bioactive materials temporally and spatially, replicating the key events in
periodontal development.56 Currently, it is known
Another approach to induce periodontal regeneration
that appropriate progenitor cells must first migrate to
has involved the direct delivery of bioactive polypep-
the wound site and attach to the denuded root surface
tide growth factors to the root surface in order to
in order to proliferate and mature into the tissue com-
facilitate the cascade of wound healing events
ponents of a functional periodontal attachment
that lead to new cementum and connective tissue

120 © 2013 Australian Dental Association


apparatus. The success of proliferation, migration and
maturation of these progenitor cells is known to then
depend on the availability of appropriate growth fac-
tors and contact with extracellular matrix, controlling
gene expression.57,58 In the absence of appropriate
cellular, molecular and matrix signals and compo-
nents, healing may be compromised and occur by
repair rather than regeneration. Progenitor cells are of
particular interest in periodontal wound healing and
regeneration as they are most likely the parental cells
of synthetic cells such as osteoblasts, cementoblasts
and fibroblasts, responsible for the restoration of lost
periodontal tissues (Fig. 1). Recent evidence shows
that subset populations isolated from the periodontal
ligament have characteristics of stem cells. As a conse-
quence, current research trends have been directed
towards developing cellular-based techniques for peri-
odontal regeneration.
Cell-based tissue engineering
In light of the clinical unpredictability of currently
available treatment modalities to treat all types of
periodontal defects, tissue engineering has emerged as
an alternative approach to alleviate the shortcomings
of conventional therapeutic options, by regenerating
living and functional dental structures. Tissue engi-
neering draws on principles of cell biology, develop-
mental biology and biomaterials science to fabricate
new structures to replace lost or damaged tissues.59
The concept has been applied to recent applications
of reconstruction and regeneration of the periodon-
tium,56 the dentine-pulp complex60 and orofacial
tissues.61
In the context of periodontal therapy, a potential
tissue engineering approach to periodontal regenera-
tion involves incorporation of progenitor cells in a
prefabricated three-dimensional construct, which is
subsequently implanted into the defect site (Fig. 2).56
This strategy overcomes some of the limitations
Stem cell Progenitor cells Committed cells
Growth factors
Pre-
cementoblast
Pre-
fibroblast
Pre-
osteoblast
Extra cellular matrix
Cell adhesion molecules
Fig. 1 A schematic diagram of proposed differentiation of adult
mesenchymal stem cells and progenitor cells into periodontal cells.
(Reproduced with permission from reference 9.)
© 2013 Australian Dental Association
Tissue engineered periodontal products
Fig. 2 Schematic representation of periodontal tissue engineering. A
successful outcome of periodontal tissue engineering requires an ade-
quate supply of appropriate progenitor cells with the capacity to differen-
tiate into the required mature tissue-forming phenotypes, including
osteoblasts, cementoblasts and fibroblasts; the appropriate signalling mol-
ecules to modulate cellular differentiation; and a conductive three-dimen-
sional extracellular matrix scaffold to support and facilitate these
processes. Angiogenic signals, promoting new vascular networks, are
essential to provide the nutritional base for tissue growth and
homeostasis.
associated with conventional regenerative procedures
as it allows direct placement of growth factors and
progenitor cells into the defect, eliminating the
expected lag phase of progenitor cell recruitment to
the wound site.62 A successful outcome of periodontal
tissue engineering requires the following essential fac-
tors: (1) an adequate supply of appropriate progenitor
cells with the capacity to differentiate into the
required mature tissue-forming phenotypes, including
osteoblasts, cementoblasts and fibroblasts; (2) the
appropriate signals to modulate cellular differentiation
and tissue neogenesis; and (3) a conductive three-
dimensional extracellular matrix scaffold to support
and facilitate these processes.35 Additionally, angio-
genic signals, promoting new vascular networks, are
essential to provide the nutritional base for tissue
growth and homeostasis. Furthermore, appropriate
mechanical loading is required for the development of
highly organized, functional periodontal ligament
fibres. Finally, as periodontal defect sites accompany a
microbial load, strategies to control infection and host
response are required for optimum periodontal regen-
eration.63
Cementoblast One of the most critical factors in tissue engineering
is the choice of scaffold and optimal stem cell popula-
tion to employ.
Fibroblast
Scaffold-based tissue engineering
For successful tissue engineering to be achieved a
Osteoblast scaffold must be combined with living cells, and/or
biologically active molecules. This will create a ‘tissue
engineering construct’ to be used to promote regenera-
tion of tissues.64,65 These scaffolds need to support
various important cellular processes and functions,
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J Han et al.

including cell colonization, migration, growth and dif- Furthermore, tissue in-growth does not equate to tis-
ferentiation. Physico-chemical properties, morphology sue maturation and remodelling, in other words a
and degradation kinetics of the scaffold are also defect filled with immature tissue should not be con-
important design features which need to be taken into sidered ‘regenerated’. Hence, many scaffold-based
account. In order for constructs to be clinically suc- strategies have failed in the past as the scaffold degra-
cessful they need to be able to support and stimulate dation was more rapid than tissue remodelling and/or
both the onset and the continuance of tissue maturation. In this context it has been proposed that
in-growth and also allow subsequent remodelling and the onset of degradation should only occur after the
maturation. This can be achieved by providing opti- regenerated tissue within the scaffold has remodelled
mal stiffness together with predetermined external at least once in the natural remodelling cycle.67
and internal geometrical shapes. Tissue engineering
constructs must also provide sufficient initial mechani-
Stem cells
cal strength and stiffness to substitute for the loss of
mechanical function of the diseased, damaged or miss- By definition, a stem cell refers to a clonogenic, rela-
ing tissue. To achieve stable biomechanical conditions tively undifferentiated cell that is capable of self-
and vascularization at the host site, continuous cell renewal and multi-lineage differentiation.68 Therefore,
and tissue remodelling is important. In addition to a stem cell is considered to be capable of propagating
these requirements of mechanics and geometry, a use- and generating daughter stem cells, whilst its progeny
ful construct will: (1) possess a three-dimensional and holds the potential to differentiate and commit to
highly porous interconnected pore network with opti- maturation along multiple lineages, giving rise to a
mized surface properties to allow attachment, migra- range of specialized cell types. A stem cell may either
tion, proliferation and differentiation of cell types undergo prolonged self-renewal or differentiation
needed for tissue regeneration as well as enable satis- depending on intrinsic signals modulated by extrinsic
factory flow transport of nutrients and metabolic factors in the stem cell niche.69 Stem cells have now
waste; and (2) be biocompatible and biodegradable been isolated from a wide variety of tissues and upon
with a controllable rate to complement cell/tissue their characterization, these populations have been
growth and maturation.66 It is now recognized that categorized according to their respective developmen-
for successful tissue formation, remodelling, and mat- tal potential (Fig. 3).
uration at the defect site it is essential to understand Stem cells can be broadly divided into three catego-
and control the scaffold degradation process. In the ries: embryonic stem cells, adult, somatic or postnatal
early days of tissue engineering, it was believed that stem cells and more recently, through genetic manipu-
scaffolds should rapidly degrade and disappear as the lation, induced pluripotent stem cells. Embryonic stem
tissue in-growth was occurring. It is important to rec- cells are pluripotent cells derived from the early mam-
ognize that tissue in-growth, remodelling and matura- malian embryo with the capacity to proliferate exten-
tion differ temporally between various tissues. sively and differentiate into cells with features of all

Germ cells

Sperm Ovum
TOTIPOTENT STEM CELLS
Fertilisation Fertilised Egg (capable of differentiating into all possible cell types
including extraembryonic tissues)

Embryonic Stem Cells

Blastocyst
(derived from inner cell mass of pre-implantation embryo)

Embryonic Germ Cells PLURIPOTENT STEM CELLS

Foetus (derived from primordial germ cells isolated from embryonal (capable of differentiating into all possible cell types
gonad) including extraembryonic tissues)

Adult Embryonic Carcinoma Cells

(derived from primordial germ cells in embryonic gonad but
usually found as components of testicular tumours in adults)

Adult Stem Cells MULTIPOTENT STEM CELLS

(derived from many ectodermal and mesodermal organs in (capable of differentiating into multiple, but a limited
adults) number of cell lineages)

Adults cells undergone nuclear transformation (clonal) TOTIPOTENT

INDUCED PLURIPOTENT STEM CELLS

Adult cells induced to an embryonic stem cell (somatic cells genetically manipulated to a
phenotype pluripotent state, thought to possess comparable
cell potency to embryonic stem cells)

Fig. 3 Derivation of stem cell populations. Stem cells are classified according to their differentiation potency as defined by the site, stage of development
or cell culture induction environment in which the populations are maintained. Cells may be categorized as totipotent, pluripotent, multipotent or induced
pluripotent. (Adapted, with permission, from reference 62.)
122 © 2013 Australian Dental Association

three embryonic germ layers (mesoderm, endoderm
and ectoderm). These cells have the ability to develop
into almost all cell types; however, they lack the
capacity to contribute to extra embryonic tissue and
therefore cannot develop into a fetal or adult ani-
mal.70–72 Despite their developmental potential, the
use of embryos to obtain human embryonic stem cell
lines raises serious ethical concerns, which recently
prompted efforts to genetically reprogramme somatic
cells back to their pluripotent state. These efforts
resulted in the generation of induced pluripotent stem
(iPS) cells that are functionally similar to embryonic
stem cells. These cells are comparable to embryonic
stem cells in their morphology, gene expression pro-
files, proliferation and differentiation capacities.73–75
However, genetic manipulations fundamental to gen-
eration of iPS cells may alter their growth and devel-
opmental characteristics, which hinders the
predictability of their behaviour and, as such, limits
their use in tissue-regenerative purposes. Furthermore,
embryonic stem cells and iPSCs both carry tumouri-
genic properties, raising a significant safety challenge
in the use of these cells for regenerative therapies.76,77
Adult stem cells have been identified in numerous
tissue niches in the postnatal organism and are
thought to function in replenishing cell loss as a con-
sequence of tissue damage and death.78 Since their
discovery, it has been recognized that the developmen-
tal capabilities of adult stem cells are greater than ini-
tially thought. In addition to being responsible for the
maintenance of tissue homeostasis in their host tissue,
it has also been demonstrated that they have the
capacity to differentiate into other cellular lineages
beyond their tissue(s) of origin.79,80 Whilst these cells
exhibit a more restricted proliferation and differentia-
tion potential compared to embryonic stem cells, they
are easily accessible, immunocompatible and are not
associated with ethical concerns.
Mesenchymal stem cells
Mesenchymal stem cells (MSCs) are adult stem cells
able to give rise to multiple specialized cell types.79,81–85
Due to their extensive distribution in many adult
tissues, including those of dental origin, MSCs have
become an attractive target for use in periodontal
regeneration.
Defining properties of mesenchymal stem cells
MSCs were first described by Friedenstein in 1976 as
clonogenic, bone marrow-derived, plastic-adherent
cells which were termed colony-forming unit fibro-
blasts (CFU-F).86,87 The term now refers to a hetero-
geneous mix of progenitors, where subset populations
are capable of differentiating into cells of mesodermal
© 2013 Australian Dental Association
Tissue engineered periodontal products
(adipocytes, osteoblasts, chondrocytes, tenocytes, skel-
etal myocytes and visceral stromal cells),79,81–85 ecto-
dermal (neurons, astrocytes)88 and endodermal
(hepatocytes)89 origins.
Originally, MSCs were isolated from bone marrow
and stroma of spleen and thymus.86,87 Hence, these
cells have previously been referred to as bone marrow
stromal stem cells (BMSCs).90 Friedenstein and Owen
demonstrated that BMSCs can be identified by their
capacity to form clonal colonies when bone marrow
aspirates were plated at low density and cultured for
a period of two weeks.91 Clonal characterization indi-
cated that a proportion of cell clones displayed the
capacity to differentiate into multiple stromal lineages
including myelosuppurative stroma, bone, fat and car-
tilage, whilst other colonies displayed a restricted
capacity for differentiation and were more representa-
tive of committed progenitor cells, rather than multi-
potential BMSCs.89,92–94 The colony forming assay is
a widely accepted technique for establishing BMSC
cultures from bone marrow aspirates based on their
plastic-adherent properties, allowing for their isolation
from non-adherent haemopoietic stem cells. However,
this type of culture system is composed, not only of
BMSCs, but also other adherent accessory cells includ-
ing smooth muscle cells, endothelial cells, osteoblasts
and adherent macrophages.90 This makes character-
ization of the biological properties of primitive
BMSCs difficult as the contaminating accessory cells
could influence the growth rate and differentiation
potential of BMSC-derived colonies. Moreover, subse-
quent studies which characterized MSC-like popula-
tions from other tissues including trabecular bone,
periosteum, articular cartilage, synovium, synovial
fluid, skeletal muscle, adipose tissue,95 tendons,
blood,96 blood vessels, umbilical cord vasculature,97
placental tissue,98,99 fetal tissues100,101 and skin102
demonstrated that a cell’s ability to adhere to and
grow on plastic was not sufficient to classify them as
MSC. To account for the biologic properties of multi-
potential, clonogenic, plastic-adherent cells derived
from various stromal tissues, the International Society
for Cellular Therapy (ISCT) has proposed the use of
the term ‘mesenchymal stem cell’ and acronym ‘MSC’
only where characterized populations satisfy the mini-
mal criteria as outlined in Table 2.103
Use of mesenchymal stem cells in tissue engineering
MSCs are considered suitable candidates for cell-based
tissue engineering strategies owing to their extensive
expansion rate and potential to differentiate into cells
of multiple organs and systems. Although MSCs dem-
onstrate a lower developmental potential and a shorter
life span than pluripotent embryonic stem cells, MSCs
have not been reported to spontaneously form tumours
123
J Han et al.
Table 2. Minimal criteria for defining multipotent
mesenchymal stem cells (MSCs) as proposed by the
International Society for Cellular Therapy (ISCT)
Minimal criteria for defining multipotent
mesenchymal stem cells (MSCs)
Adherence to plastic Cells must be plastic-adherent when
cultured in standard culture conditions
in standard culture flasks.
A specific surface antigen At least 95% of the MSC population
(Ag) expression pattern must express immunophenotype
markers CD105, CD73 and CD90,
while less than 2% of the population
should express markers CD45, CD34,
CD14 or CD11b, CD79a or CD19
and HLA class II. These markers may
also be expressed by cultured
fibroblasts isolated from different
tissues that have little or no
multi-lineage potential.
Multipotent MSC populations must give rise to at
differentiation potential least three cell lineages: osteogenic,
chondrogenic and adipogenic under
standard in vitro differentiation
conditions.
Reproduced, with permission, from reference 99.
in vivo, and are not constrained by ethical consider-
ations.104 Additionally, MSCs seem to be not only
hypoimmunogenic and thus suitable for allogeneic
transplantation, but they further exhibit immunosup-
pressive properties upon transplantation.105
Considering their potential in regenerative applica-
tions, a variety of stem cells have been identified in
most organs and tissues in the body.106–108 Studies on
the developmental potential of MSCs have provided
the foundation for the proposed stromal hierarchy of
these cells (Fig. 4),109 a concept arising from a series
of landmark studies by Friedenstein and
Owen.87,91,93,94,110–112 Based on these findings, perio-
dontal ligament derived mesenchymal stem cell-like
cells are now being considered as a more appropriate
cell type for developing novel tissue periodontal tissue
engineering strategies. Nevertheless, aside from fulfill-
ing the essential factors of optimal cell populations,
signals and scaffolds, required for tissue engineering a
major obstacle that currently limits the clinical use of
MSCs for tissue regeneration is the heterogeneity of
the starting cell population, and the inability to estab-
lish optimal growth and differentiation conditions
in vitro which may result in poor reproducibility of
therapeutic outcomes.113
Cells for periodontal regeneration
Both extraoral and intraoral tissues have stem cell
populations that represent a viable and accessible
alternative source to harvest and expand multipotent
colonies for potential use in periodontal tissue
124
Embryogenesis Postnatal stem cells
Committed Mature
Progenitors Stromal Cells
Myelosupportive
Stroma
Muscle
Marrow Stromal Stem
Mesoderm Cell
Bone
Adipose
Cartilage
Dentin
Dental Pulp Stem Cell Fibrous Pulp
Adipose
Neural crest
Cementum
Periodontal Ligament
Ligament
Stem Cell
Adipose
Fig. 4 Proposed stromal hierarchy of cellular differentiation. Precursor
cells residing in the bone marrow stroma, dental pulp and periodontal
ligament tissues have the capacity to differentiate into lineage-specific
cells that give rise to various stromal tissues. (Reproduced, with permis-
sion, from reference 109.)
engineering. MSCs derived from multiple tissue
sources have been investigated in preclinical animal
studies for the treatment and regeneration of the peri-
odontium.
Extraoral mesenchymal stem cells in periodontal
tissue engineering bone marrow-derived
mesenchymal stem cells
Past research has demonstrated a potential for the
use of MSCs, derived from tissue sources outside the
oral cavity, for transplantation to the periodontal
complex.114 Auto-transplantation of bone marrow-
derived MSCs has been shown to promote up to
20% of cementum and alveolar bone regeneration in
experimental Class III defects in dogs.115 A cell-label-
ling technique in which bone marrow-derived MSCs
were engineered to express green fluorescent protein
(GFP) showed that, four weeks after transplantation,
the periodontal defects were almost completely
regenerated and contained GFP positive cemento-
blasts, osteoblasts, osteocytes and fibroblasts within
the regenerated periodontal tissue. These results sug-
gest that transplanted bone marrow-derived MSCs
differentiated into cementoblasts, periodontal liga-
ment fibroblasts, and alveolar bone osteoblasts
in vivo.116 In a subsequent small human clinical
trial, autologous, expanded bone marrow-derived
MSCs mixed with atelocollagen were transplanted
into periodontal osseous defects at the time of peri-
odontal surgery.117 Positive clinical results were
obtained, including a 4-mm reduction in probing
depths and respectively, a 4-mm attachment gain.
Furthermore, radiographic assessments showed that
the use of MSCs in conjunction with platelet-rich
plasma (PRP) demonstrated a reduction in intrabony
© 2013 Australian Dental Association

defect depth and resolution of bleeding. Interdental
papillae, supported by this tissue engineering technol-
ogy, had also regenerated.117
Thus, from the preclinical trials conducted to date,
bone marrow-derived MSCs have the capacity to pro-
mote periodontal regeneration through enhanced gen-
eration of alveolar bone and neovascularization.
Notably, transplanted bone marrow-derived MSCs
have also been reported to contribute to the formation
of new cementum and periodontal ligament. How-
ever, despite their proven capacity to regenerate peri-
odontal tissues, the difficulty associated with the
ascertainment of these cells for use in the clinical set-
ting has instigated exploration of dental-tissue-derived
MSC-like populations which can be simply obtained
chairside rather than via an invasive bone marrow
aspiration procedure in a secondary clinic.
Adipose-derived stromal cells
Adipose tissue is another extraoral and non-dental
derived source of MSCs potentially applicable in peri-
odontal tissue engineering. Adipose-derived stromal
cells (ADSCs) mixed with platelet rich plasma have
been shown to promote regeneration of periodontal
ligament-like structures along with alveolar bone in
rats.118 These observations suggest that ADSCs may
be useful in future clinical cell-based therapy for peri-
odontal tissue engineering and present a favourable
cell candidate due to ease of accessibility to human
lipoaspirates and low morbidity associated with their
procurement.
Intraoral (dental-derived) mesenchymal stem cells for
periodontal tissue engineering
Dental-tissue-derived mesenchymal stem cell-like pop-
ulations are among many other isolated and charac-
terized stem cells residing in specialized tissues. With
the exception of enamel, which lacks ameloblasts or
other cellular elements following tooth development,
the periodontium and dentine continue to retain some
regenerative or reparative capacities. This is thought
to be mediated by the presence of multipotent progen-
itor cells that are capable of differentiation into
functional, lineage-specific cells.31,119–121 However,
dental-tissue-derived progenitor cells are likely to be
more committed or restricted in their cellular potency
when compared to bone marrow-derived MSCs
because dental tissues do not undergo continuous
remodelling as do bone tissues. Additionally, dental
mesenchyme is termed ‘ectomesenchyme’ due to its
earlier interaction with the neural crest. Thus, it is
postulated that these ectomesenchyme-derived dental
stem cells possess characteristics similar to those of
neural crest cells (Fig. 4).122
© 2013 Australian Dental Association
Tissue engineered periodontal products
Initially, dental MSCs were isolated from human
pulp tissue and, upon their characterization, were
termed postnatal dental pulp stem cells (DPSCs).123
Subsequently, three more types of dental-MSC-like
populations were isolated and characterized: stem
cells from exfoliated deciduous teeth (SHED)124; peri-
odontal ligament stem cells (PDLSCs)29; and stem
cells from apical papilla (SCAP).125,126 Recent studies
have also identified more dental-tissue-derived progen-
itor cell populations, from the dental follicle and gin-
giva.127,128 The developmental relationship between
these different mesenchymal stem cell-like populations
has yet to be determined. The different cell popula-
tions differ in aspects of their growth rate in culture,
gene and protein expression profiles and cell differen-
tiation capacities, although the extent to which these
differences can be attributed to tissue of origin, func-
tion or culture conditions remains unclear. Nonethe-
less, the abovementioned intraoral progenitor cells
have recently been used for tissue engineering studies
in small and large animal models to assess their
potential in preclinical applications.
Of particular interest is the capacity of progenitor
cell populations derived from periodontal ligament as
the presence of multiple cell types within periodontal
ligament suggests that this tissue contains progenitor
cells that maintain tissue homeostasis and regenera-
tion of the periodontal tissues. Earlier evidence has
shown that periodontal ligament contains cell popula-
tions that can differentiate into either cementoblasts
or osteoblasts.129,130 These cells can form clonogenic
colonies with characteristics of postnatal stem cells,
are self-renewable and possess the ability to give rise
to a range of dental and non-dental tissues, including
cementoblast-like cells, adipocytes and collagen-form-
ing cells. When transplanted into immunocompro-
mised rodents, PDLSCs show the capacity to generate
a cementum/PDL-like structure and contribute to
periodontal tissue repair.29
In vivo differentiation capacity of periodontal
ligament stem cells
There has been a considerable amount of research
conducted to assess the regenerative capacity of
PDLSCs in a range of dental and craniofacial defects
in various animal models. It is evident from these
studies that implanted PDLSCs generate cementum
and periodontal ligament-like structures similar to
native periodontal complex.
In a very early study autologous re-implantation of
extracted dental roots lined with PDL cells in minipigs
resulted in the formation of connective tissue, resem-
bling PDL and mimicking the orientation of the fibre
bundles, within four weeks of implantation.131 Twelve
weeks post implantation, root surfaces were covered
125
J Han et al.
by organized connective tissue resembling periodontal
ligament and oriented fibre bundles were attached to
alveolar bone and root, penetrating the bone and root
surfaces in the same way as Sharpey’s fibres.131 In
subsequent studies reimplanted cultured cells from the
periodontium implanted into experimental furcation
and interdental defects in a variety of animal models
resulted in formation of new cementum and bone,
and new attachment.130,132–135
More recently periodontal ligament cell sheets have
emerged as a novel alternative approach for periodon-
tal tissue engineering as the technical handling of
implanted cell population bypasses disruption of criti-
cal cell surface proteins such as ion channels, growth
factor receptors and cell-to-cell junction proteins.132
Preclinical investigations have noted periodontal
regeneration involving formation of new bone, peri-
odontal ligament and cementum after eight weeks, in
three out of five test sites, when autologous periodon-
tal ligament cell sheets were implanted into dehiscence
and intrabony defects in dogs.136,137 Allogeneic trans-
plants of periodontal ligament cell sheets in miniature
swine have demonstrated periodontal tissue regenera-
tion in a manner no different to that observed in
autologous transplants with no evidence of rejection
of the allogeneic cells.138
In a more ambitious study, stem cells from root
apical papilla (SCAP) together with PDLSCs in a
HA/TCP and Gelfoamâ (absorbable gelatine) carrier
have been studied in an attempt to generate a func-
tional root/periodontal complex capable of supporting
a porcelain crown in miniature pigs.125 Following
extraction of the lower incisor, the site was cleaned to
ensure removal of all periodontal ligament tissues and
the implant constructs, coated with the cells/carrier,
were inserted into the extraction socket and sutured.
The surface of the constructed implant was surgically
re-opened at three months post implantation and a
porcelain crown resembling a miniature pig incisor
was inserted and cemented into a pre-formed site in
the implant. Four weeks post installation of the
crown, CT and histological analysis confirmed regen-
eration of the root/periodontal structure with signifi-
cantly better compression strength than defects which
did not receive stem cells. This proof-of-concept study
showed the efficacy of using a multi-type stem cell
approach to mimic a bio-physiological root/periodon-
tal reconstruction.125
Despite the general consensus observed in preclini-
cal animal studies assessing the potential of PDLSCs
in periodontal regenerative therapy, only two human
clinical studies have been conducted to date.139,140
The findings from these studies have shown some
encouraging but variable results which provide proof
of principle for the use of autologous periodontal liga-
ment cells for regenerative purposes. These studies
126
have also demonstrated potential efficacy and safety
of implanting autologous cells and displayed clinical
benefits from the treatment. However, important chal-
lenges remain to be addressed before such treatments
are applied as a standard in a clinical setting.
CONCLUSIONS
Complete and predictable regeneration of periodontal
tissues lost due to trauma or disease presents a major
challenge. Periodontal regeneration requires consider-
ation of many factors that parallel periodontal devel-
opment, including the use of optimal progenitor cell
population, signalling molecules and matrix scaffold
in an orderly temporal and spatial sequence. The peri-
odontal ligament is now known to be a rich source of
MSCs, and while this tissue appears to have high
regenerative potential, it is difficult to harness and uti-
lize this capacity for clinical utility.
To date, extraoral and dental-tissue-derived stem/
progenitor cells have been used for tissue engineering
studies in small and large animal models to assess
their potential in preclinical applications. While there
may be an overwhelming body of evidence to support
the notion that MSCs can be used for periodontal
regeneration, there are several main objectives that
need to be addressed before the development of effec-
tive cellular-based therapies for regenerative dentistry.
This requires better understanding of: (a) the mecha-
nisms of self-renewal in order to sufficiently regulate
adult stem cell growth in vitro to generate required
cell numbers needed for different applications; (b) the
regulation of stem cells during differentiation and
maturation into tissue-specific cell types, as well
as during wound healing; (c) the interactions between
stem cells and the immune system, in particular,
regarding use of allogeneic cell populations; and
(d) mechanisms needed to control and prevent ex
vivo-expanded mesenchymal stem cells from transfor-
mation.
DISCLOSURE
The authors have no conflicts of interest to declare.
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Address for correspondence:
Professor PM Bartold
Colgate Australian Clinical Dental Research Centre
School of Dentistry
The University of Adelaide
Frome Road
Adelaide SA 5005
Australia
Email: mark.bartold@adelaide.edu.au
© 2013 Australian Dental Association