Periodontology 2000, Vol.
51, 2009, 239–251
Printed in Singapore. All rights reserved
Stem cells and future
periodontal regeneration
N I -H U N G L I N , S T A N G R O N T H O S & P. M A R K B A R T O L D
Periodontitis is an inflammatory disease that causes
pathological alterations in the tooth-supporting tis-
sues, potentially leading to tooth loss. National sur-
veys have shown that the majority of adults in the
population suffer from moderate periodontitis, with
up to 15% of the population being affected by severe
generalized periodontitis at some stage in their lives
(2, 17). The significant burden of periodontal disease
and its impact on general health and patient quality
of life point to the need for more effective manage-
ment of this condition (113, 143). Several procedures
have been attempted to achieve periodontal regen-
eration, including root surface conditioning, bone
graft placement, guided tissue regeneration and
growth factor application. However, current regen-
erative procedures that are used either alone or in
combination have limitations in attaining complete
and predicable regeneration, especially in advanced
periodontal defects (108, 109, 123, 139, 146). Recent
advances in stem cell biology and regenerative
medicine have presented opportunities for tissue
engineering as well as gene-based approaches in
periodontal therapy (60, 63, 70, 73, 138). The follow-
ing discussion reviews our current understanding of
stem cells and their potential application in regen-
erative periodontal therapy. Major progress in stem
cell biology in the dental context is highlighted and
the challenges in translating stem cell research into
clinical practice are identified.
Definition of stem cells and their
types
Stem cells are the foundation cells for every organ
and tissue in the body, including the periodontium
(129, 130). A stem cell has two defining characteris-
tics: (i) the ability for indefinite self-renewal to give
rise to more stem cells; and (ii) the ability to differ-
 2009 John Wiley & Sons A/S
PERIODONTOLOGY 2000
entiate into a number of specialized daughter cells to
perform specific function(s) (95, 115). A stem cell can
divide asymmetrically, in which case one of the two
daughter cells retains the stem cell characteristics
while the other is destined for specialization under
specific conditions (83). A pluripotent stem cell can
differentiate into all cell types of the body, whereas a
multipotent stem cell can differentiate along multi-
lineages into many different cell types (95). The po-
tential for self-renewal vs. differentiation is governed
by extracellular signals coupled to intracellular
signaling cascades (140). Information that specifies
the fate of stem cells is encoded by the presence or
absence of signals, and also by the combinations,
localization, level and timing of these signals.
There are two main types of stem cells – embryonic
stem cells and adult stem cells – which are classified
according to their origin and differentiation potential
(Table 1). Human embryonic stem cells, derived from
the inner cell mass of blastocysts, are pluripotent
stem cells capable of differentiating into cells of all
three germ layers of the adult body (131). Human
embryonic stem cells have been derived primarily
from spare blastocysts created by in vitro fertiliza-
tion. Human embryonic stem cell lines are unique in
that they can be maintained in an undifferentiated
state in vitro for an indefinite period of time, while
retaining their ability to differentiate into all types of
specialized cells in the body (57, 107, 131). These cells
have been demonstrated to produce approximately
200 different types of cell within the adult body but,
because of ethical concerns, have not been tested for
their ability to participate in human embryonic
development or to contribute to germ lines in vivo. It
is estimated that at least 225 human embryonic stem
cells lines, including some with genetic disorders,
have been generated in different laboratories world-
wide (47, 51, 100). The use of human embryonic stem
cells for clinical therapy is a relatively new endeavor
239
Lin et al.

Table 1. Different stem cell populations and their differentiation potential

Development stage Type of stem cell Differentiation potential
Blastocyst Embryonic stem cells Pluripotent
Derived from the inner cell mass of the
pre-implantation embryo
Fetus Embryonic germ cells Pluripotent
Derived from primordial germ cells iso-
lated from the embryonal gonad
Adult Embryonal carcinoma cells Pluripotent
Derived from primordial germ cells in
embryonic gonad and usually found as
components of testicular tumors in adults
Adult stem cells Multipotent
Derived from ectodermal and mesoder-
mal organs of adults
Adult cells that have undergone nuclear Totipotent
transformation
Adult cells that can be induced to an Inducible pluripotent
embryonic stem cell phenotype

and, currently, this development has been hampered
by ethical concerns (145).
In 2006, cells with properties similar to those of
embryonic stem cells, termed induced pluripotent
stem cells, were created from specialized somatic
cells via the forced over-expression of four key factors
(126, 127). The authors reported that when plasmids
containing Oct3 ⁄ 4, Sox2, cMyc and Klf4 genes were
introduced into murine fetal fibroblasts, the cells
were reprogrammed to an embryonic-like state.
These murine-induced pluripotent stem cells exhibi-
ted the morphology and growth properties of murine
embryonic stem cells although they had different
gene-expression and DNA-methylation profiles
(Table 1). Follow-up reports with improved repro-
gramming techniques have yielded more embryonic
stem cell-like cells in mice, with the reprogrammed
cells contributing to every cell type in the body (77,
97, 141). Human counterparts of murine-induced
pluripotent stem cells have subsequently been gen-
erated using various combinations of Oct3 ⁄ 4, Sox2,
cMyc, Klf4, Nanog, LIN28, hTERT and SV40 large-T
factors (75, 99, 126, 155). Although the mechanism by
which each of these factors restores pluripotency is
not clearly understood, human-induced pluripotent
stem cells attract immense interest from stem cell
researchers because of the lack of ethical issues
involving the use of human oocytes or embryos.
Earlier techniques to restore pluripotency to human
somatic cells, involving somatic cell nuclear transfer
into enucleated oocytes (144) or fusion of somatic
cell nuclei with human embryonic stem cells (25,
125), have drawn intense debate regarding the source
of human oocytes ⁄ embryos and the use of oocyte
progenitors in the adult ovary (26). Furthermore,
somatic cell nuclear transfer has yet to be success-
fully demonstrated in humans, and the pluripotent
cells resulting from the fusion of somatic cells with
embryonic stem cells are often tetraploid with limited
practical application. By contrast, the technology
used to generate induced pluripotent stem cells can
create patient-specific and disease-specific cell lines
for research purposes with fewer ethical concerns,
but this method of reprogramming is still inefficient
and many fundamental questions remain unan-
swered. Thus, research on human pluripotent cells
derived using different methods should be continued
to identify the best method for regenerative therapy
(56).
Adult, or tissue-specific, stem cells are found in the
majority of fetal and adult tissues. They have been
derived from tissues that continually replenish
themselves [e.g. peripheral blood (3, 116), dermis
(134, 153) and gastrointestinal epithelium (12)] as
well as from tissues with lower regenerative capacity
[e.g. brain (117)]. Adult stem cells are thought to
function in long-term tissue maintenance and ⁄ or
repair by replacing cells that are either injured or lost
(71). They are generally multipotent stem cells that
can form a limited number of cell types corre-
sponding with their tissues of origin, although some
studies have suggested that they are more versatile
and can develop into many other cell types than
previously expected (15, 68, 80). The most common

240

source of adult stem cells is the bone marrow, which
contains hematopoietic stem cells (132) and bone
marrow stromal cells or mesenchymal stromal ⁄ stem
cells (29, 52). Hematopoietic stem cells were the
first stem cells to be successfully used in therapies,
particularly in the treatment of blood malignancies
and immunodeficiency syndromes, but they are
not capable of giving rise to supporting connective
tissues (65). Mesenchymal stem cells, by contrast, are
a cell type of interest with the therapeutic potential
to treat a range of musculoskeletal abnormalities,
cardiac diseases and immune abnormalities (65).
This review will focus on the properties and function
of mesenchymal stem cells as potential candidates
for periodontal regeneration (50, 63).
Human mesenchymal stem cells
Mesenchymal stem cells were initially identified in
rodent marrow as colony-forming unit fibroblasts
capable of forming bone, cartilage and fat, as well as
reconstituting the hematopoietic microenvironment
(37, 38). Colony-forming unit fibroblasts have sub-
sequently been detected in many species, including
humans (19, 20, 46, 101), and the formation of col-
ony-forming unit fibroblasts is considered to be a
distinguishing feature of mesenchymal stem cells.
Mesenchymal stem cells have also been characterized
both morphologically and immunophenotypically
using different cell-surface markers. Mesenchymal
stem cells occur as either large and flat cells or as
elongated and fibroblastic cells, but the functional
significance of this diverse morphology is unclear (9).
Phenotypically, mesenchymal stem cells can express
the characteristics of endothelial, perivascular, neu-
ral, bone or muscle cells, and also express a range of
surface markers (including CD49a ⁄ CD29, CD44,
STRO-1, CD90, CD105, CD106, CD146, CD140b,
CD166 and CD271). This suggests a common link
between different cell types because most of these
markers are expressed by all mesenchymal stem cells
(45, 46, 101, 104). However, clonal studies have
highlighted the heterogeneous nature of these cells,
given that different mesenchymal stem cell colonies
demonstrate different proliferative and developmen-
tal capacities both in vitro and in vivo (46, 67, 85,
101). Hence, characterization of candidate cells has
involved the combined evaluation of their surface
maker expression as well as of their proliferative
ability (to form colonies) and developmental poten-
tials (to differentiate into osteoblasts, adipocytes and
chondrocytes) (11, 101, 103).
Stem cells and future periodontal regeneration
Bone marrow mesenchymal stem cells are the most
widely studied mesenchymal stem cells because they
are easily accessible in quantities appropriate for
clinical applications (19, 20, 46). These cells are clo-
nogenic and have demonstrated the ability to form
bone and cartilage in vivo (101, 102). Because of their
differentiation potential, bone marrow mesenchymal
stem cells have been used in various phases of clinical
application, particularly in orthopedics (21, 53). Mes-
enchymal stem cells have also been shown to form
cementum, periodontal ligament and alveolar bone
in vivo after implantation into periodontal defects in
beagle dogs (50, 63), suggesting that bone marrow may
be a useful source of mesenchymal stem cells for
periodontal regeneration. Apart from the bone mar-
row, mesenchymal stem cell-like cells have also been
identified in many other tissues, including adipose
tissue, muscle, peripheral blood, fetal pancreas and
liver (6, 18, 54, 67, 157). Adipose tissue-derived mes-
enchymal stem cells exhibit stable growth kinetics
in vitro and display a multilineage differentiation
capacity (including osteogenesis, chondrogenesis and
adipogenesis) which is similar to that of bone marrow
mesenchymal stem cells (42, 59, 74, 159). As adipose
tissue can be obtained by using less-invasive methods
and in larger quantities than bone marrow cells, the
use of adipose-derived mesenchymal stem cells as an
alternative to bone marrow mesenchymal stem cells
is very appealing. Indeed, at least one study has
demonstrated that eight weeks after implantation
of adipose-derived stem cells into rat periodontal de-
fects, new periodontal ligament-like and alveolar
bone-like structures were formed, implying that
adipose-derived stem cells could promote periodontal
regeneration in vivo (133). The abundance of human
lipoaspirates, coupled with low host morbidity in their
procurement, makes them a unique stem cell reservoir
for regenerative periodontal therapy.
Stem cells in dental and
periodontal tissues
In addition to nondental stem cells, the potential of
dental mesenchymal stem cell-like cells for peri-
odontal regeneration and repair has been extensively
studied in recent years. The ability of mesenchymal
stem cells to differentiate into multiple specialized
cell types in many adult tissues (including dental
tissues) has made them a promising cell source for
use in periodontal regeneration.
The first human dental stem cells were isolated
from dental pulp tissue of extracted third-molar teeth
241
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and were characterized relative to bone marrow
mesenchymal stem cells (43). These dental pulp stem
cells were found to be highly proliferative, clonogenic
cells capable of differentiating into odontoblast-like
cells and forming dentin ⁄ pulp-like complex when
implanted into immunocompromised mice (41, 43).
Subsequently, human mesenchymal stem cells were
isolated from exfoliated deciduous teeth and were
noted to induce bone and dentin formation in vivo
(82). Periodontal ligament stem cells were first iso-
lated in 2004 and have been shown to give rise to
adherent clonogenic clusters resembling fibroblasts
(110). They are capable of developing into adipocytes,
osteoblast-like cells and cementoblast-like cells
in vitro as well as producing cementum-like and
periodontal ligament-like tissues in vivo (39, 110,
112). Recent studies have also shown their ability to
differentiate into neuronal precursors (24, 128).
Periodontal ligament stem cells also express an array
of cementoblast and osteoblast markers as well as the
STRO-1 and CD146 antigens, which are found on
dental pulp stem cells and bone marrow mesenchy-
mal stem cells (43, 135). These findings indicate that
periodontal ligament stem cells have properties
similar to those of dental pulp stem cells and bone
marrow mesenchymal stem cells, and this may rep-
resent another mesenchymal stem cell-like popula-
tion (Fig. 1).
In 2006, stem cells from the apical papilla of hu-
man teeth were isolated (1, 120). These cells form
adherent clonogenic clusters and, similar to other
mesenchymal stem cell populations, are capable of
differentiating into adipocytes and odontoblasts ⁄
osteoblasts in vitro. Stem cells from the apical papilla
Mesenchymal stem cell collection
Tooth
Pulp
Bone marrow Periodontal
ligament
Magnetically activated cell sorting
(MACS) or Dynal bead selection
using STRO-1 antibody
+ –
Fig. 1. Schematic illustration of the isolation of mesen-
chymal stem cells from bone marrow, dental pulp and
periodontal ligament.
242
have been shown to give rise to dentin tissue in
immunocompromised mice, and to generate a
root ⁄ periodontal complex capable of supporting a
porcelain crown after transplantation with perio-
dontal ligament stem cells in minipigs (119, 120).
These findings provide support for the use of com-
bined mesenchymal stem cell populations in root
and periodontal regeneration.
Mesenchymal progenitor cells have also been iso-
lated from the dental follicle of human third-molar
teeth (84). These fibroblast-like, colony-forming and
plastic-adherent cells express stem cell markers
(STRO-1 and nestin) and can be maintained in culture
for at least 15 passages. STRO-1-positive dental fol-
licular progenitors have been shown to differentiate
into cementoblasts in vitro (64, 148) and to form
cementum in vivo (48). The finding that immortalized
dental follicle cells can generate periodontal liga-
ment-like tissue after in vivo implantation (151) im-
plies that dental follicular progenitor cells may be a
useful research tool for studying periodontal ligament
formation and for regenerative periodontal therapies.
While the use of both dental and nondental mes-
enchymal stem cells for tooth formation and perio-
dontal regeneration are promising, mesenchymal cell
populations from different tissues display distinct
biological properties (27). Even if they carry common
genetic markers, they are likely to be conditioned by
their specific microenvironment and to be commit-
ted towards a specific differentiation pathway (87,
88). This notion is supported by the observation that
bone marrow mesenchymal stem cells display a
lower odontogenic potential than dental mesenchy-
mal stem cells (156). In this context, it remains to be
determined which source of dental mesenchymal
stem cells will be most suitable for regenerative
periodontal therapy. The use of dental follicular cells
is likely to be limited by the availability of tooth
germs, and nondental stem cells may require the
transfer of genes to enhance their odontogenic
potential. Although the best stem cell source is yet to
be identified, the prospect of using these cells for
regeneration represents a step forward in the devel-
opment of more predictable biologically based
therapies for the periodontium.
Potential uses of stem cells in
periodontal regeneration
Stem cells have been used extensively in many
medical disciplines for the repair and ⁄ or regenera-
tion of defective tissues and organs such as bone (53),

cartilage (86), heart (122) and spinal cord (81). In
dentistry, the identification of mesenchymal stem
cell-like populations from both dental and nondental
tissues has presented exciting possibilities for the
application of tissue engineering as well as gene-
based therapies. These techniques have the potential
to lead to the development of novel strategies for
regenerative periodontal therapy.
Periodontal tissue engineering
Tissue engineering is a contemporary area of science
based on the principles of cell biology, developmen-
tal biology and biomaterials science to develop new
procedures and biomaterials to replace lost or dam-
aged tissues (69, 93, 114). The main requirements for
producing an engineered tissue are appropriate pro-
genitor cells, signaling molecules, an extracellular
matrix or carrier construct and an adequate blood
supply (114, 121). Although tissue engineering
encompasses many different strategies, including cell
injection, tissue culturing, porous and injectable
scaffolds, and three-dimensional printing (7, 114),
this field is most frequently linked to the paradigm
of cell delivery within biocompatible scaffolds (70,
138). In this context, a potential tissue-engineering
approach to periodontal regeneration involves the
incorporation of progenitor cells and instructive
messages in a prefabricated three-dimensional con-
struct and subsequent implantation of the construct
into the defect site (7) (Fig. 2). This approach may
overcome a major shortcoming associated with
conventional regenerative procedures because the
recruitment of progenitor cells and growth factors is
negated and healing can be enhanced.
Tissue-engineering strategies have been applied to
reconstruct damaged periodontal apparatus, either
Fig. 2. Schematic representation of the concept of tissue
engineering for periodontal regeneration. A periodontal
defect may be filled with a biodegradable scaffold con-
taining specific molecular instructional messages and
stem cells to result in full regeneration of the defect. Note
Stem cells and future periodontal regeneration
by reiteration of tooth development based on epi-
thelial–mesenchymal tissue recombination or by
seeding of cells on biomaterial scaffolds (32, 63, 89–
92, 98, 120, 133). The tissue-recombination technique
aims to replicate key reciprocal interactions between
the dental epithelium and the ectomesenchyme
during odontogenesis to regenerate the periodon-
tium. For example, following the combination of
embryonic epithelial and mesenchymal cells derived
from mouse fetuses, the re-associated tooth germs
result in tooth-like structures including roots, peri-
odontal ligament and alveolar bone in vivo (55, 91).
Furthermore, the combination of oral epithelium
with nondental-derived mesenchyme (e.g. embryonic
stem cells, neural stem cells and adult bone-marrow-
derived cells), results in the formation of both tooth
crown and bone in vivo (96). Several studies have
reported that engineered tooth primordia give rise to
proper tooth development after being transferred
into the adult mandible (32, 91, 96), indicating that
cultured stem cells can replace lost or damaged
dental structures following transplantation into the
adult oral cavity. However, the periodontal structures
regenerated using tissue-recombination techniques
are not formed in isolation from other dental tissues
and this may pose problems for implantation into
periodontal defects. In addition, there is currently
no suitable substitute for the embryonic epithelial
compartment of the engineered tooth germ, and the
use of human embryonic tissues for periodontal
engineering may limit the practical application of this
approach.
The technique of cell transplantation, within
three-dimensional matrices, to regenerate the perio-
dontium has been extensively studied. Nondental
stem cells (such as bone marrow mesenchymal stem
cells and adipose-derived stem cells) have been
that this is an illustration only and the radiographs do not
represent an actual clinical outcome; they are used to
illustrate a potential outcome based on the concepts of
tissue engineering.
243
Lin et al.
investigated as cell sources for periodontal regener-
ation. Autologous bone marrow mesenchymal stem
cells and adipose-derived stem cells can regenerate
alveolar bone and periodontal ligament-like struc-
tures after transplantation in vivo, and this provides
support for the use of these cells in regenerative
periodontal therapy (63, 72, 133). Studies have also
used dental stem cells to achieve periodontal regen-
eration both in vitro (136, 137) and in vivo (32, 44, 66,
120, 152). In these studies, progenitor cells were
seeded directly onto biomaterial scaffolds (e.g. poly-
glycolic acid, calcium phosphate material, collagen
sponges) followed by transplantation into periodon-
tal defects in animal models (such as mice, rats, dogs,
pigs and sheep). Seeding of tooth bud cells (which
presumably include some progenitor cells) on bio-
degradable scaffolds resulted in the formation of new
periodontal ligament and bone tissues after implan-
tation into the omenta of adult rat hosts and at the
site of previously lost teeth (32, 152). A novel study
has reported the combination of swine stem cells
from the apical papilla with periodontal ligament
stem cells to regenerate root and periodontal struc-
ture in minipigs (117). The swine stem cells from the
apical papilla were seeded into a root-shaped scaffold
that was then wrapped with gelfoam containing
porcine periodontal ligament stem cells. After a
three-month healing period, periodontal ligament
tissue was noted to form around this bio-root struc-
ture and appeared to have a natural relationship with
the surrounding bone. Regenerated root structure
containing new cementum and periodontal ligament
has been reported to occur by culturing swine dental
bud cells on a cylinder scaffold and then grafting it
back into the alveolar socket (66). These promising
results reported in animal models using dental stem
cells are paving the way for human regenerative
periodontal therapy.
Overall, these findings demonstrate the feasibility
and potential of using dental and nondental stem
cells for functional periodontal and tooth regenera-
tion. An ideal source of stem cells for periodontal
regeneration should be nonimmunogenic, highly
proliferative, easy to harvest, resilient to prolonged
processing and have the ability to differentiate into
desired cell types (40). Recent findings confirming
that human adult dermal fibroblasts can be repro-
grammed to pluripotency (75, 99, 126, 155) may en-
able researchers to overcome immune rejection and
to produce large quantities of cells for regenerative
periodontal therapy. Human-induced pluripotent
stem cells can be derived from a dental source (e.g.
gingival fibroblasts of the patient) and then differ-
244
entiated into a variety of autologous cell types
(including dental stem cells and dental epithelium)
for in vivo administration.
Stem cell-mediated gene therapy
Gene therapy relies on genetic engineering involving
molecular techniques to introduce, suppress or
manipulate specific genes, thereby directing an
individualÕs own cells to produce a therapeutic agent
(4). In the context of periodontal regeneration, gene
therapy seeks to optimize the delivery of agents, such
as growth factors, to periodontal defects so that the
limitations associated with the efficacy of topical
application of these factors (e.g. a short duration of
action) can be overcome (106). Gene-delivery tech-
niques have been used in periodontal ligament and
alveolar bone regeneration in rats (60, 92), and
embryonic stem cells have been shown to express
tooth-initiation genes in mice (96, 149). Two poten-
tial strategies for delivering therapeutic transgenes
into human recipients are: (i) the direct infusion of
the gene of interest using viral or nonviral vectors
in vivo; and (ii) the introduction of the gene into
delivery cells (often a stem cell) outside the body
ex vivo followed by transfer of the delivery cells back
into the body (160). The inherent proliferative and
pluripotent capabilities of stem cells may offer life-
long opportunities for treatment by repairing,
replacing or regenerating the damaged tissues.
The use of adenoviral vectors to enable the over-
expression of growth-promoting molecules, such as
platelet-derived growth factor and bone morphoge-
netic protein-7, has been investigated for its potential
in periodontal regeneration (5, 60, 61). Sustained re-
lease of bone morphogenetic protein-7 and platelet-
derived growth factor by transformed cells implanted
into experimental periodontal defects results in en-
hanced regeneration of bone and cementum (33, 60).
The use of such technology with dental stem cells
may offer an alternative to conventional methods as a
result of their ability to provide a renewable source of
growth factor release for regeneration. STRO-1-
selected rat dental pulp stem cells transduced with
bone morphogenetic protein-2 demonstrate en-
hanced odontogenic differentiation compared with
nontransduced cells (147). However, much work is
still needed to optimize the number of cells that are
virally transduced and to maximize the duration and
extent of gene expression. Further research is also
needed to address the potential risks of viral recom-
bination and immune responses towards viral anti-
gens (36, 118), which may ultimately determine the

success of gene-transfer techniques in periodontal
regeneration. Many challenges remain in bringing
stem cell research to clinical practice in humans, and
it is important to overcome these challenges to fulfill
our goal of treating periodontal diseases with stem
cell-based therapy.
Challenges in bringing stem
cell-based research to practice
Biological challenges
Despite biological evidence showing that regenera-
tion can occur in humans, complete and predictable
regeneration still remains an elusive clinical goal
(especially in advanced periodontal defects). The
isolation and characterization of stem cells from
periodontal tissues have provided a good starting
point for understanding the role of progenitor cells in
periodontal healing. However, we have incomplete
understanding of the way that roots develop, and
little is known about the signaling mechanisms that
occur during this process (124, 150). The molecular
pathways that underlie stem cell self-renewal and
differentiation are also largely unknown (16). For
periodontal regeneration to occur, we need to repli-
cate the key cellular events that parallel periodontal
development and to understand the specific cell
types, the inductive factors and the cellular processes
involved in formation of the periodontium (7). Given
that the fate of stem cells is influenced by their
interaction with the microenvironment (including
soluble and immobilized factors, extracellular matrix
and signals from neighboring cells), understanding
the key components regulating the properties of stem
cells may elucidate ways to expand stem cells prop-
erly and control their differentiation precisely (34).
Numerous studies on stem cells to date have
emerged from work on cell culture or animal models.
While some species (e.g. primate) are more suited
than others (e.g. mouse) for understanding stem cell
behavior in humans, these findings may not always
be directly extrapolated to humans.
Technical challenges
The technical challenges in stem cell therapy are
associated with cell manipulations, scaffold materi-
als and delivery systems. First, culture conditions are
not sufficiently developed to mimic the cell micro-
environment in vivo and to ensure that both cell
proliferation and differentiation can be performed
Stem cells and future periodontal regeneration
safely and consistently. Furthermore, as cell-culture
medium often requires xenogenic products (such as
fetal bovine serum or mouse feeder layers), cell
cultures may not be completely free of pathogens
and infectious risks are a concern (79). The estab-
lishment of an optimal culture condition free of
potential cross-contaminations is relevant in pro-
ducing clinical-grade human stem cell lines and for
performing basic research involving the regulation of
their self-renewal and lineage determination. Sec-
ond, timing is an inherent constraint in cell therapy
and tissue engineering. Some autologous construct-
based approaches may involve weeks to months of
ex vivo processing (35). Although the use of stem
cells can often minimize the processing time com-
pared with somatic cells, possible karyotypic insta-
bility and gene mutations of the cells after prolonged
culture can also limit their usefulness (78). Third, the
search for the ideal biocompatible scaffolding
material(s) and delivery system is a significant
technical pursuit. The ideal matrix scaffold should
mimic native extracellular matrix, support cell
attachment, allow controlled release of bioactive
factors, be conducive to tissue ingrowth and facili-
tate laboratory handling (8, 10, 76, 142). There is
evidence to suggest that cultured human periodontal
ligament stem cells in a suitable scaffold implanted
into surgically created periodontal defects can result
in the formation of a periodontal ligament-like
structure (111). However, further refinement of the
methodology to propagate and incorporate these
cells into a carrier scaffold is needed, and this should
limit the ex vivo processing time and encourage the
integration and function of the implanted cells with
the host cells (62, 158).
Clinical challenges
Clinical challenges in stem cell-based periodontal
therapy relate to immune rejection after administra-
tion, oncogenic properties of stem cells and func-
tional integration of transplanted tissues into the host
(23). It is important to understand how the immune
system will respond to stem cells or their derivatives
upon transplantation. Generally, the immunogenicity
of a human cell depends on its expression of class I
and II major histocompatibility antigens, which allow
the body to distinguish its own cells from foreign cells
(14). Human embryonic stem cells express a low level
of class I major histocompatibility antigens, but this
expression is up-regulated with differentiation (30). A
potential solution to this problem lies in the use of
autologous stem cells (from cell ⁄ tissue banks) to
245
Lin et al.
overcome immune rejection (14, 58). Furthermore,
the production of patient-specific pluripotent stem
cells (or induced pluripotent stem cells) from adult
somatic cells is now feasible and the differentiation of
autologous induced pluripotent stem cells into cell
types desired for transplantation is being explored
(49). Recent findings relating to the immuno-
suppressive effects of mesenchymal stem cells both
in vitro and in vivo have also raised the possibility of
using allogenic stem cells without the need for donor
and recipient cross-matching (22, 94, 105).
The challenge relating to genomic stability and the
risk of tumorigenesis following stem cell transplan-
tation are major safety considerations because
reliable methods to eliminate undifferentiated
embryonic stem cells from culture are yet to be
established (13), and current studies lack long-term
follow-up to draw firm conclusions (154). It is likely
that the more specific and extensive the therapeutic
application, the longer the stem cells may have to
remain in vitro. During this extended period in cul-
ture, there is a greater likelihood for genetic or epi-
genetic changes in stem cells (31, 78). Improvement
in the understanding of such changes in vitro will
better enable researchers to address the risk of
tumorigenesis in stem cell therapy.
Finally, it is unclear whether human stem-cell
derivatives can integrate into the recipient tissue and
fulfill the specific functions of lost or injured tissues
(28). It will be necessary to demonstrate that stem
cells develop into stable cells and display the char-
acteristics and functions of normal host cells fol-
lowing their transplantation. It is hoped that, as our
knowledge on progenitor cells, growth factors and
delivery systems improves, we will make stem cell-
based therapy a safe and effective approach in
periodontal regeneration.
Conclusion
Restoration of tissues destroyed by periodontitis to
their original form and function has been a long-
standing goal of periodontal therapy. However, our
current available regenerative therapies are crude
and of poor clinical predictability. There is need for
novel regenerative technologies to be developed
based on contemporary understanding. In order for
this to become a reality it will be necessary for us to
obtain a complete understanding of periodontal
development and the progenitor cells involved in this
process. Subsequent tissue-engineering approaches
may then be developed using these progenitor cells
246
within a matrix scaffold, together with the introduc-
tion of various signaling molecules in an orderly
temporal and spatial sequence. To date, a number of
studies have reported that stem cells, in conjunction
with different physical matrices and growth factors,
have the capacity to regenerate periodontal tissues
in vivo. Notwithstanding these significant advances
there are still numerous biological, technical and
clinical hurdles to be overcome. In particular, a
thorough understanding of the underlying pro-
cesses in periodontal development, as well as the
mechanisms behind stem cell self-renewal and
differentiation, will be required. Refinement of cur-
rent laboratory techniques to facilitate handling of
stem cells and their translation to a clinical setting
will be equally critical in advancing the field. Studies
on both embryonic cells and adult stem cells should
continue as part of a collective effort to expand our
knowledge on how cells function and what fails in
the disease process. It is this combined and solid
knowledge base that will underpin future treatment
modalities and ultimately make stem cell-based
tissue engineering and gene therapy a realistic alter-
native in periodontal regeneration.
Acknowledgments
The authorsÕ work cited in this review has been fun-
ded variously by the National Health and Medical
Research Council of Australia (NHMRC), the Austra-
lian Dental Research Fund and the Australian Peri-
odontology Research Foundation.
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