601658

research-article2015
JDRXXX10.1177/0022034515601658Journal of Dental ResearchDental MSCs for Pulp-dentin Regeneration

Clinical Review
Journal of Dental Research
1­–8
Pulp-dentin Regeneration: Current State © International & American Associations
for Dental Research 2015

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DOI: 10.1177/0022034515601658
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Y. Cao1*, M. Song1*, E. Kim2, W. Shon1, N. Chugal1, G. Bogen1, L. Lin3,

R.H. Kim1,4, N.-H. Park1,4,5, and M.K. Kang1,4

Abstract
The goal of regenerative endodontics is to reinstate normal pulp function in necrotic and infected teeth that would result in
reestablishment of protective functions, including innate pulp immunity, pulp repair through mineralization, and pulp sensibility. In
the unique microenvironment of the dental pulp, the triad of tissue engineering would require infection control, biomaterials, and
stem cells. Although revascularization is successful in resolving apical periodontitis, multiple studies suggest that it alone does not
support pulp-dentin regeneration. More recently, cell-based approaches in endodontic regeneration based on pulpal mesenchymal stem
cells (MSCs) have demonstrated promising results in terms of pulp-dentin regeneration in vivo through autologous transplantation.
Although pulpal regeneration requires the cell-based approach, several challenges in clinical translation must be overcome—including
aging-associated phenotypic changes in pulpal MSCs, availability of tissue sources, and safety and regulation involved with expansion of
MSCs in laboratories. Allotransplantation of MSCs may alleviate some of these obstacles, although the long-term stability of MSCs and
efficacy in pulp-dentin regeneration demand further investigation. For an alternative source of MSCs, our laboratory developed induced
MSCs (iMSCs) from primary human keratinocytes through epithelial-mesenchymal transition by modulating the epithelial plasticity
genes. Initially, we showed that overexpression of ΔNp63α, a major isoform of the p63 gene, led to epithelial-mesenchymal transition
and acquisition of stem characteristics. More recently, iMSCs were generated by transient knockdown of all p63 isoforms through
siRNA, further simplifying the protocol and resolving the potential safety issues of viral vectors. These cells may be useful for patients
who lack tissue sources for endogenous MSCs. Further research will elucidate the level of potency of these iMSCs and assess their
transdifferentiation capacities into functional odontoblasts when transplanted into the root canal microenvironment.

Keywords: stem cells, biocompatible materials, endodontics, dental pulp calcification, epithelial-mesenchymal transition, cell- and
tissue-based therapy

Introduction Triad of Pulp Regeneration

Tissue regeneration in dental pulp entails resolution of chronic Control of inflammation. Elimination of root canal infection is
inflammation and restoration of damaged dentoalveolar tis- the primary objective in regenerative therapies to resolve api-
sues, including the organized pulp-dentin complex. The under- cal periodontitis. In addition, persistent pathologic inflamma-
lying rationale for endodontic regeneration is reinstitution of tory signals in the root canal space can interfere with
normal physiologic function in an otherwise necrotic pulp, differentiation and maturation of apical papilla cells, resulting
including the protective mechanisms—for example, innate in arrested root development. This is typically observed in
pulp immunity, pulp repair through tertiary dentin mineraliza- immature teeth with necrotic pulps and wide-open apices (Fig.
tion, and sensation of occlusal pressure and pain. Restoration 1). Boyle et al. (2014) demonstrated that long-term exposure of
of these pulpal functions will enhance long-term survival of dental pulp cells to tumor necrosis factor (TNF)-α, a potent
teeth and help patients to retain their natural dentition. Hence, inflammatory cytokine, led to loss of differentiation and
it is critical to develop a novel regenerative approach that will mineralization capacities, which could be rescued by inhibition
restore not only pulp vitality but organized pulp-dentin struc-
ture with the full spectrum of normal physiologic functions. In 1
general, tissue engineering encompasses 3 requirements— School of Dentistry, UCLA, Los Angeles, CA, USA
2
School of Dentistry, Yonsei University, Seoul, Korea
namely, scaffold, growth differentiation signals, and stem cells 3
New York University College of Dentistry, New York, NY, USA
(Alsberg et al. 2001). This triad is inevitably different for pulp 4
Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA
regeneration owing to the unique microenvironment of the root 5
David Geffen School of Medicine, UCLA, Los Angeles, CA, USA
canal space potentially harboring pathogenic bacteria and the *
Authors contributing equally to this article.
hard dentinal structure constantly under repeated masticatory
Corresponding Author:
pressure. Therefore, successful endodontic regeneration requires M.K. Kang, UCLA School of Dentistry, Center for the Health Sciences,
coordinated effects of infection control, biomaterials, and stem Room A3-077, 10833 Le Conte Ave, Los Angeles, CA, USA.
cells, which make up the triad of pulp tissue engineering. Email: mkang@dentistry.ucla.edu

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2 Journal of Dental Research 

antibiotic paste (TAP) composed of cip-

Figure 1. Revascularization leads to resolution of apical periodontitis with disorganized
radio-opaque changes within the root canal system. (A) Radiograph of a mandibular left second
premolar showing radiographic signs of apical periodontitis. (B) Postoperative radiograph of
completed revascularization procedure with final restoration and presence of continued apical
pathosis. (C) One-year radiographic recall showing resolution of apical periodontitis and apical
closure. Note presence of nonspecific radio-opaque calcific tissue within canal space (arrow).
Courtesy of Dr. Rashi Vohra. (D) Radiographic sequence of another revascularization case
performed on a maxillary right central incisor exhibiting immature root formation associated with
a wide-open apex. (E) Six-month radiographic review. Acute swelling and symptomology had
resolved after initial visit. After 6 mo (F) and 1 y (G) following the revascularization procedure,
recall radiographs show resolution of apical periodontitis and advanced root formation. However,
lumen of the root canal is filled with disorganized radio-opaque calcified hard tissue (arrows).
Courtesy of Dr. Fei-fei Wang.
of NF-κB signaling. These findings are in agreement with the
landmark study that prescribed the inhibitory role of NF-κB on
osteogenic differentiation of mesenchymal stem cells (MSCs)
through β-catenin degradation (Chang et al. 2013). It is appar-
ent that root formation depends on the odontogenic differentia-
tion of apical papilla cells in developing teeth (Sonoyama et al.
2008). Activation of the inflammatory cascade during active
root canal infections would interfere with the odontogenic dif-
ferentiation of these cells, resulting in apical periodontitis
associated with wide-open apices. Evidently, this process is
not a permanent arrest of root development, as root canal
debridement and disinfection lead to continued root formation
and apical closure (Jeeruphan et al. 2012).
In light of regenerative endodontic therapies, root canal dis-
infection is achieved primarily by chemomechanical debride-
ment and intracanal medicaments. A recent review of 60
relevant publications revealed that revascularization proce-
dures employed mechanical instrumentation (e.g., endodontic
files in only 32% of cases; Kontakiotis et al. 2015). This
emphasizes the importance and adequacy of root canal irriga-
tion and medication. In the interest of salvaging the apical
papilla cells, canal irrigation is currently recommended with
lower-strength sodium hypochlorite solutions at 1.5%, while
irrigation with 17% ethylenediaminetetraacetic acid (EDTA)
has shown to promote the release of growth factors (e.g., trans-
forming growth factor [TGF]-β, resulting in hard tissue forma-
tion; Becerra et al. 2014; Martin et al. 2014; Galler et al. 2015).
Intracanal disinfection is primarily achieved by use of calcium
hydroxide (CH) or a mixture of antibiotics known as triple
rofloxacin, metronidazole, and doxycy-
cline. Kontakiotis et al. (2015) reported
that the majority (80%) of revasculariza-
tion cases are performed with TAP as the
main medicament, although CH intraca-
nal dressing also leads to highly successful
treatment outcomes (Chen et al. 2012).
Currently, the American Association of
Endodontics recommends placement of
CH or low concentrations of TAP during
the first appointment of regenerative pro-
cedures, although this recommendation is
supported by limited case reports.
Importantly, CH appears to promote pro-
liferation of periapical MSCs, while
high concentration of antibiotics have
detrimental effects (Ruparel et al. 2012).
Failure of revascularization has also
been reported, primarily due to persis-
tent intracanal bacterial biofilms present
after canal debridement, indicating the
need for improved protocol/agents for
infection control to achieve predictable
outcomes (Lin et al. 2014).
Biomaterial. Inasmuch as pulp-dentin
regeneration requires direct contact
between the restorative material and the pulp, the dental mate-
rial suitable for pulp regeneration must satisfy the following 3
criteria: First, the material must be biocompatible, since it will
be in direct contact with the regenerated dental pulp; second,
the material must be hard enough to withstand repeated masti-
catory pressure; and third, the material should provide an excel-
lent seal against the dentinal wall to prevent leakage of oral
microorganisms into the pulp space. Mineral trioxide aggregate
(MTA), a calcium silicate cement–based material, has been the
material of choice for endodontic regeneration because of its
exceptional biocompatibility, hardness, and enhanced marginal
adaptation against dentin. One potential shortcoming of MTA,
in the context of pulp tissue engineering, is reduced washout
strength due to its extended setting period (Formosa et al. 2013).
Since pulp-dentin regeneration cases would require direct con-
tact between the restorative material and the intracanal medium
in forms of blood clot, hydrated scaffold, and/or cellular sus-
pensions, washout of the overlaying restorative material could
pose technical challenges.
In recent years, several fast-setting biomaterials with simi-
lar composition and physicobiochemical properties as MTA
have been introduced into the discipline, including BioDentin
(Septodont, Lancaster, PA, USA), EndoCem MTA (Maruchi,
Wonju, Korea), and OrthoMTA (BioMTA, Seoul, Korea).
EndoCem MTA, for instance, is an ultrafast-setting pozzolan-
containing cement, which demonstrates biocompatibility and
osteogenic capacities (Choi et al. 2013; Park et al. 2014).
Miniaturizing the particle size in EndoCem MTA increases the
surface area of the material, resulting in fast-setting reaction

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Dental MSCs for Pulp-dentin Regeneration
and increased washout strength (Byun et al. 2010; Choi et al.
2013). Consequently, fast-setting materials may be preferred
for regenerative endodontic cases because the material would
be in direct contact with the intracanal medium. However, the
comparative long-term efficacy of various silicate cements,
slow or fast setting, applied in regenerative procedures still
requires ongoing investigation.
Stem cells. MSCs are represented by stromal cell populations
demonstrating stem characteristics and multipotent differentia-
tion. The first characterized MSCs were identified in the bone
marrow (Friedenstein et al. 1966) and, since then, discovered
in other somatic tissues, including dentoalveolar tissues.
Although there are differences in MSCs derived from different
tissue origin, MSCs in general demonstrate common pheno-
types, including multipotent differentiation, as detailed else-
where (Huang et al. 2009). In addition, MSCs are generally
thought to be immunomodulatory and immunosuppressive,
supporting the concept of allotransplantation for therapeutic
purposes, although some reports indicate their potential immu-
nogenicity (Rasmusson et al. 2003; Chen et al. 2006; Kode
et al. 2009; Wang et al. 2012).
The first type of MSCs from dentoalveolar tissues was iso-
lated from the human dental pulp (Gronthos et al. 2000).
Subsequently, stem cells have been isolated from dental pulp in
the primary dentition, periodontal ligament (PDL), and apical
papillae of immature teeth with open apices (Miura et al. 2003;
Seo et al. 2004; Sonoyama et al. 2006). Pulpal MSCs are a
clonogenic and rapidly proliferative cell population associated
with adult human dental pulps (Gronthos et al. 2000).
Remarkably, pulpal MSCs are capable of regenerating the dentin-
pulp complex in vivo, as demonstrated in xenotransplantation
(Gronthos et al. 2000). When subcutaneously implanted in
immunocompromised mice, pulpal MSCs developed mineral-
ized matrix lined with odontoblastic cells and fibrous tissue
containing blood vessels similar to the pulp-dentin complex in
natural teeth (Gronthos et al. 2002). Pulpal MSCs from decidu-
ous teeth have a similar differentiation capacity as those from
adult teeth (Miura et al. 2003). These cells exhibited enhanced
proliferation capacity, suggesting that they originate from a
more immature population of multipotent stem cells when
compared with the adult counterpart (Miura et al. 2003; Sakai
et al. 2010). MSCs isolated from PDL expressed a higher level
of scleraxis, a tendon-specific transcription factor, than that of
bone marrow MSCs and pulpal MSCs (Seo et al. 2004), and
they can differentiate into cementoblast-like cells and colla-
gen-forming cells (Seo et al. 2004). They also show the poten-
tial for calcific tissue deposition in vitro similar to bone marrow
and pulpal MSCs. However, they form cementum-like miner-
alized nodules and surrounding fibrotic tissues, resembling the
cementum-PDL complex, which is distinct from dentin or bone
(Seo et al. 2004). MSCs from the apical papillae of immature
permanent teeth also show greater proliferation properties and
tissue regeneration capacities than those of other cell lineages
(Sonoyama et al. 2006). It is presumed that these MSCs at the
apical papillae in immature teeth contribute to root develop-
ment and apical closure during revascularization procedures,
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© International & American Associations for Dental Research 2015
3
although histologic evidence supports recruitment of other
MSCs to the pulp space during revascularization.
Cell-free approach for pulp regeneration: “regeneration” vs.
“repair.” Considering the triad of pulpal regeneration require-
ments, the initial attempt to regenerate dental pulp tissue was
made with a necrotic immature tooth exhibiting an open apex
and sinus tract (Iwaya et al. 2001). The procedure employed
in the landmark case report (Banchs and Trope 2004), which
included the use of TAP for root canal disinfection and intra-
canal bleeding, became the standard “model” for many similar
case reports that followed. Hence, this initial study brought
about a new approach in the endodontic treatment of immature
teeth with open apices, which were treated by apexification
before the introduction of this strategy. Subsequent to this, a
number of case reports and case collections with long-term fol-
low-up (up to 108 mo) demonstrated quite successful treatment
outcomes when measured by resolution of apical periodontitis
(Jung et al. 2008; Chueh et al. 2009). These studies incorpo-
rated revascularization protocols modified from the original
reported procedures—for example, use of CH as an intracanal
medicament in place of the antibiotic paste and use of growth
factors in the form of platelet-rich plasma rather than blood
clotting. Irrespective of the specific reagents and/or techniques
used, these case studies reported survival rates close to 100%
after revascularization (Jeeruphan et al. 2012). Some case
studies have actually reported pulp vitality reestablishment in
necrotic teeth after revascularization (Ding et al. 2009). These
reports clearly demonstrate the therapeutic efficacy of revascu-
larization in resolving apical periodontitis for immature teeth
with open apices and that procedural details have little influ-
ence on the treatment outcome.
Although revascularization seems successful in resolving
apical periodontitis and revitalization of pulpless teeth in lim-
ited cases, it had been largely unknown whether the revascular-
ized tissues truly represent pulp-dentin regeneration—that is,
formation of an organized odontoblastic layer. Several preclin-
ical and large animal studies have refuted the notion that revas-
cularization is bona fide tissue regeneration. Wang et al. (2010)
reported apposition of cementum, bone formation, and fibrous
tissues in the revascularized root canals in dogs lacking orga-
nized pulp-dentin complex. In our histologic report of a revas-
cularized human mandibular first molar, we also confirmed
that canals are primarily filled with ectopic bone, fibrous tis-
sues, and cementum apposition onto the inner wall of the radic-
ular dentin. The structures lacked an organized pulp-dentin
complex, irrespective of whether canals were filled with blood
clot of platelet-rich plasma (Fig. 2; Martin et al. 2013). These
studies strongly suggest that revascularization alone does not
support pulp-dentin regeneration but rather induces tissue
repair, resulting in the resolution of apical periodontitis.
With this information, we may gain further insight based on
radiographic evidence in revascularization cases and draw
some conclusions. As evinced in Figure 2, the distal root canal
of the mandibular molar readily visible preoperatively was
filled with disorganized radio-opaque materials 14 mo after
revascularization, presumably representing ectopic bone
4 Journal of Dental Research 

Figure 2. Revascularized pulp tissues lack organized pulp-dentin complex and are composed of ectopic bone, cementum, and fibrous tissues. (A)
Preoperative radiograph of mandibular right first molar displaying gross coronal caries and apical periodontitis. (B) Fourteen-month postoperative
radiograph after revascularization procedure showing root maturation and periradicular healing. Note disorganized radio-opaque calcified tissues in
the distal canal. (C) Histologic section showing ectopic bone formation within the lumen of the distal canal. Note absence of organized palisading
odontoblastic layer. (D) Boxed area from panel C enlarged. (E) Mesial canal space exhibiting ectopic bone formation (white arrow) and layered
cementum formation onto the existing dentinal surface (black arrows). Images modified from Martin et al. (2013).

formation in the root canal. In fact, a number of postoperative
radiographs of revascularization cases indicate resolution of
apical periodontitis characterized by a disorganized radio-
opacity inside the root canal space, indicative of ectopic calci-
fication. As shown in Figure 1, healing of apical periodontitis
and apical closure were evident at 1 y after revascularization.
However, the root canal space became partly obliterated with
disorganized radio-opaque material, presumably representing
ectopic bone formation. These cases further illustrate the point
that revascularization in the absence of exogenous cell
transplantation leads to resolution of inflammation without
pulp-dentin regeneration. Also, intracanal assessment of post-
operative radiographs can often confirm ectopic calcification
of the root canal space.
The discovery that revascularized tissues in the root canal
consist of ectopic bone, cementum, and fibrous tissues is not an
accident. Since revascularization is often performed with intra-
canal bleeding from the periapical tissues, which may include
cells from the PDL and alveolar bone, it is very plausible that
undifferentiated MSCs from these sources would migrate into
the root canal space and initiate tissue deposition. As illustrated in
the xenograft study by Seo et al. (2004), MSCs from pulp, PDL,
and bone marrow generate the same tissues as their origins when
transplanted in vivo. These data clearly demonstrate that MSCs
from different dentoalveolar tissues are unique and retain identi-
ties from their direct tissue sources. Hence, when MSCs are
recruited from periapical alveolar bone marrow and PDL during
revascularization, it is not surprising to find ectopic calcification
in the root canal space with no pulp-dentin regeneration. In con-
trast to these findings, it also indicates that pulp-dentin regenera-
tion requires the presence of pulpal MSCs in the root canal.
As an alternative to revascularization, the “cell homing”
approach has been developed to regenerate pulp-dentin com-
plex through a cell-free method that encourages recruitment of
endogenous resident MSCs to the site of tissue regeneration
(Mao et al. 2012). Therefore, cell-homing approaches would
not necessitate MSC transplantation and hence drastically
enhance the feasibility of pulp-dentin regeneration beyond
simple revascularization. Several preclinical studies have
shown promising results in terms of cellular acquisition in the
root canal space when the canal was filled with scaffold and
several homing factors—such as basic fibroblast growth fac-
tors, vascular endothelial growth factors, platelet-derived
growth factors, nerve growth factors, and bone morphogenetic
protein 7 (Kim et al. 2010). However, the efficacy of this
approach in pulp-dentin regeneration in vivo requires further
investigation. In summary, revascularization without MSC
transplantation is highly successful in resolving apical peri-
odontitis, while the revascularized pulpal tissues are evidently
composed of bone, cementum, and fibrous tissues, produced
by recruited MSCs derived from PDL and alveolar tissues.
Hence, pulp-dentin regeneration would require pulpal MSCs
present in the root canal.
Cell-based approaches in pulp-dentin regeneration: the cur-
rent state. Progress with the cell-based approach for pulp-
dentin regeneration has been hampered primarily due to safety
and regulatory issues regarding pulpal MSC production and
transplantation in patients. However, several notable preclini-
cal studies illustrate the efficacy of the cell transplantation
approach for pulp-dentin regeneration. As illustrated in the
study of Cordeiro et al. (2008), stem cells from human exfo-
liated deciduous teeth–seeded scaffolds had the potential to
form dental pulp–like tissue in vitro. The first in vivo evidence
was reported by Huang et al. (2010) in a murine model, which
showed pulp-dentin regeneration in human root fragments
only after MSC transplantation. On the contrary, those samples
with scaffold alone demonstrated ingrowth of fibrous tissues
with no evidence of organized pulp tissue. Similarly, ectopic
transplantation of human dentin disks filled with poly-L-lac-

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Dental MSCs for Pulp-dentin Regeneration
tic acid scaffold and dental MSCs led to regeneration of the
pulp-dentin complex, which was capable of forming nascent
mineralized dentin (Sakai et al. 2010). This study also illus-
trated the requirement of exogenous cell transplantation, as the
scaffold alone failed to form a functional odontoblast layer.
Large animal studies have also attempted to test the require-
ment of cell transplantation for pulp-dentin regeneration (Zhu
et al. 2013). These studies showed that pulp debridement and
disinfection protocols that involve intracanal medicaments
through TAP were successful in resolving apical periodonti-
tis but failed to induce genuine pulp-dentin regeneration, irre-
spective of the dental MSC transplantation. On the contrary,
Iohara et al. (2009) first demonstrated successful regeneration
of pulp-dentin complex in a pulpotomy model in dogs by trans-
plantation of fractionated side-population cells enriched with
the CD31–/CD146– immunophenotype. In subsequent studies,
the same group demonstrated regeneration of the entire pulp
when MSCs enriched with CD105+ dental pulp stem cells were
transplanted into root canals combined with stromal derived
factor–1 (SDF-1; Iohara et al. 2011). Although selection of
cells enriched with CD105+ immunophenotype enhanced pulp-
dentin regeneration, it is unknown whether cell fractionation is
required, since unfractionated pulp MSCs were also capable for
tissue regeneration, albeit to a much lesser extent when com-
pared with CD105+-enriched cells. Alternatively, heterologous
MSCs from adipose tissues showed a drastic reduction in pulp
tissue regeneration, even with CD105+ enrichment, indicating
inherent differences among MSCs of different tissue origin.
SDF-1 is a chemokine implicated in recruitment of MSCs to
an injury site requiring tissue repair (Kucia et al. 2005). It is
known to function directly by binding to its receptor CXCR4,
which facilitates MSC migration and tissue regeneration in
multiple tissue systems (Zwingenberger et al. 2014; Yang et
al. 2015). For this reason, SDF-1 is known as MSC “homing
factor,” which may, in the context of pulp regeneration, recruit
endogenous MSCs to the pulp-dentin complex to enhance tis-
sue regeneration. However, the study by Iohara et al. (2011)
demonstrated that pulp-dentin regeneration did not occur in
the absence of pulp MSCs even with the presence of SDF-1.
Therefore, it is highly likely that the homing factor alone is not
capable of endogenous MSC recruitment from surrounding tis-
sues to promote dental pulp cell differentiation. These studies
further underscore the importance of pulpal MSC presence in
the root canal space for pulp-dentin regeneration.
Although the cell-based therapies are somewhat futuristic in
endodontic regeneration, the strategy has shown promise for
other systemic conditions in patients who are refractory to con-
ventional therapies. Systemic MSC applications are primarily
regenerative or immunomodulatory in nature; the transplanted
MSCs would replace the damaged tissues/organs through tissue-
specific differentiation of the MSCs or suppress the inflamma-
tory and immunogenic conditions. Although there is controversy
regarding whether such MSC therapies represent bona fide tis-
sue regeneration, evidence supports the notion that the trans-
planted MSCs differentiate into the same terminal tissue as their
respective host. Hepatogenic differentiation of MSCs has been
documented after exposure to liver-specific factors such as
hepatocyte growth factor and nicotinamide (Chivu et al. 2009).
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© International & American Associations for Dental Research 2015
5
A more recent study showed differentiation of hepatic stellate
cells, representing liver-resident MSCs, into bile acid–synthesiz-
ing and bile acid–transporting hepatocytes (Kordes et al. 2014),
further confirming direct involvement of MSCs in tissue regen-
eration. However, cell-based therapies also harness the immuno-
modulatory effects of MSCs, as observed in immune cell
maturation and proliferation (Glennie et al. 2005; English et al.
2010). Systemic transplantation of allogeneic MSCs yielded
promising results for patients with systemic lupus erythemato-
sus who were refractory to conventional therapies (Sun et al.
2009). These ongoing observations support the concept of cell-
based therapy as a novel treatment option for patients in medi-
cine, and accumulating evidence indicates the need for this type
of approach in pulp-dentin regeneration.
Cell-based approaches: challenges and alternative
approaches. Certainly, one prominent question must be
addressed regarding the cell-based approach: “How is it pos-
sible to bring the technology to a dental office?” This question
is seemingly simple, but it considers a multitude of complex
issues in providing the practical logistics of MSC transplanta-
tion into root canals in the typical endodontic practice setting.
Primarily, the source and potency of MSCs are paramount and
could be limited by our available technology. Normal human
somatic cells such as pulpal MSCs undergo a finite number of
cell divisions during in vitro culture until the cells exhaustively
arrive at the terminal state called replicative senescence (Kang
et al. 2004). Our prior study showed a marked accumulation of
senescent pulpal MSCs using in vitro culture and loss of essen-
tial odonto-/osteogenic differentiation capacities in cells due to
induction of p16INK4A and loss of Bmi-1, a stem factor (Meh-
razarin et al. 2011). Moreover, in vitro expansion of pulpal
MSCs in preparation for cell transplantation procedures would
require an extensive number of cell doublings with congruent
loss of differentiation capacity. Furthermore, availability of tis-
sue sources for pulpal MSCs is age dependent due to the occur-
rence of age-related pulpal calcification that characterizes
older patients (Morse 1991). This physiologic process is not an
issue for immature teeth with open apices, which are primarily
found in pediatric patients. However, pulpal MSC transplanta-
tion in adult patients with closed apices and reduced cellularity
would be problematic due to lack of autologous pulpal tissues
from which MSCs could be harvested. Therefore, one major
limitation of the cell-based approach would be procurement of
donor pulp tissues in the aged population while being a viable
option for immature teeth.
Clinical translation of the cell-based approach must also over-
come the challenge of MSC expansion in vitro and the require-
ment of good manufacturing practice facilities to ensure reliable
cell production. This becomes a difficult requirement for autolo-
gous MSCs due to time constraints during a dental appointment
and the cost associated with individualized MSC cultivation at a
good manufacturing practice laboratory. As an alternative, allo-
geneic pulpal MSCs can be mass produced enabling a rapid turn-
around to generate potent MSCs ready for transplantation. For
systemic MSC therapies—for example, intravenous perfusion of
MSCs for systemic lupus erythematosus and placenta-derived
MSC infusion for diabetes—allogeneic MSCs have been used
6 Journal of Dental Research 

TGF-β. The resulting cells with EMT

phenotype had acquired stemness,
assessed by immunophenotype, marker
gene expression, and secondary struc-
ture formation in vitro. Likewise, EMT
in NHEKs induced by ΔNp63α demon-
strated the mesenchymal phenotype
and acquired stem characteristics,
including transdifferentiation with
osteo-/odontogenic differentiation
potential (Oh et al. 2011; Fig. 3).
Our recent investigations have also
shown that EMT can be induced by
transient transfection of small-interfer-
ing RNA targeting all isoforms of p63
in NHEKs (Yi et al. 2014). These cells,
named NHEK/Sip63, also demon-
Figure 3. Overexpression of ΔNp63α in normal human epidermal keratinocytes (NHEKs) led strated acquisition of stem characteris-
to epithelial-mesenchyme transition (EMT) and acquisition of stemness. (A) Western blotting tics, including altered
was performed with NHEKs expressing ΔNp63α (NHEK/ΔNp63α) and the control cells (NHEK/ immunophenotype and osteo-/odonto-
LXSN) for the markers of epithelial phenotypes, including E-cadherin (E-Cad) and cytokeratin 14
(K14). Note disappearance of E-Cad and K14 in NHEK/ΔNp63α at 40 population doublings (PDs), genic differentiation (Fig. 4). The
while the control cells infected with the empty vector (LXSN) showed no such change, indicating advantages of this new approach in
occurrence of EMT by ΔNp63α overexpression. To rule out the possibility of contaminating generating NHEK/Sip63 cells with
cells (e.g., sebocytes), we checked for the unique sebocyte marker K7, which was not expressed
in the NHEK/EMT cells. (B) The EMT cells demonstrated distinct morphology from sebocytes,
induced stemness is that it requires only
as well as transdifferentiation capacities into osteogenic cells producing mineralized matrix and transient transfection small-interfering
adipogenic cells. Original magnification, 100×. (C) Western blotting revealed induced expression RNA without the use of viral vectors,
of a reprogramming factor Nanog in NHEK/ΔNp63α but not in the control cells (NHEK/LXSN). thereby greatly mitigating safety con-
As controls, we included dental mesenchymal stem cells (MSCs) and mouse embryonic stem cells.
GAPDH was used as loading control. Images modified from Oh et al. (2011). cerns. Further experiments revealed
underlying mechanisms by which p63
and well tolerated by the patients, with acceptable safety in phase knockdown stimulates EMT in NHEKs and that p63 cooper-
1 clinical trials (Sun et al. 2009; Jiang et al. 2011). Allogeneic ates with other epithelial regulatory factors, including the
MSCs are known to possess immunomodulatory effects, which Grainyhead-like 2 and miRNA 200 family members, to define
in part provide some of their therapeutic efficacy when used for the epithelial identity of human keratinocytes (Mehrazarin et
inflammatory disorders. However, studies also indicate that allo- al. 2015). It appears that p63 is critical for establishing the epi-
geneic MSCs are short-lived in the host and can elicit immuno- thelial identity of NHEKs. Also, when cells lose their epithelial
genic responses in the recipient. In particular, an investigation by phenotype, they become their “default” phenotype, which
Eliopoulos et al. (2005) showed that allotransplantation of MSCs appears to be mesenchyme with induced stemness. Clinically,
in immunocompetent MHC-mismatched mice led to rapid loss of these unique cell populations may represent an alternative
MSC functionality and elicited adverse host immune responses. source of autologous MSCs for patients who lack adequate tis-
Although allotransplantation of MSCs may be more clinically sue availability for MSCs, since these iMSCs can be easily
feasible than that of autologous MSCs in pulp-dentin regenera- obtained from skin. Further investigation will elucidate the
tion, issues pertaining to host immunogenicity, long-term stabil- potency levels of these iMSCs and assess their transdifferentia-
ity of the stem cell engraftment, and therapeutic efficacy demand tion capacities into functional odontoblasts when transplanted
further examination. in the root canal microenvironment.
As an alternative, our laboratory developed a method to
generate MSCs from primary normal human epidermal kerati-
Author Contributions
nocytes (NHEKs) by inducing epithelial-mesenchymal transi-
tion (EMT). We coined the term induced MSCs (iMSCs) to Y. Cao, M. Song, M.K. Kang, contributed to conception, design,
describe these cells, which are distinct from induced pluripo- and data analysis, drafted and critically revised the manuscript; E.
tent stem cells generated by transduction of defined repro- Kim, W. Shon, N. Chugal, G. Bogen, L. Lin, R.H. Kim, N.-H.
gramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc; Park, contributed to data analysis, drafted and critically revised the
Takahashi and Yamanaka 2006). iMSCs were initially made manuscript. All authors gave final approval and agree to be
from NHEKs by overexpression of ΔNp63α (Oh et al. 2011). accountable for all aspects of the work.
The notion of “stemness” associated with EMT was first
described by Mani et al. (2008), who showed induction of stem Acknowledgments
characteristics in immortalized human mammary epithelial M.K.K. is supported by the Jack Weichman Endowment Fund.
cells by overexpression of Twist or Snail or exposure to We thank Dr. Domenico Ricucci for providing the histologic

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© International & American Associations for Dental Research 2015

Dental MSCs for Pulp-dentin Regeneration 7

Figure 4. Transient knockdown of p63 led to epithelial-mesenchyme transition (EMT) with acquired stem cell characteristics. (A) Western blotting
was performed with normal human epidermal keratinocyte (NHEK) transients transfected with siRNA targeting p63 (Si-p63) at 3, 6, and 10 d
posttransfection, along with those treated with nonspecific siRNA (Si-Cont). Whole cell lysate of NHEK/ΔNp63α (EMT) was used as comparison.
Note the loss of E-cadherin (E-Cad) and induction of fibronectin (FN) and vimentin in NHEK/Si-p63 after 10 d. GAPDH was used as loading control.
(B) NHEK/Si-Cont and NHEK/Si-p63 cells were maintained in culture and serially passaged in culture condition suitable for mesenchymal stem cells
(MSCs). NHEK/Si-Cont cells underwent terminal differentiation (arrows), while NHEK/Si-p63 cells exhibiting EMT phenotype continued to proliferate,
maintaining the spindle-shaped morphology. (C) NHEK/Si-p63 cells demonstrate alkaline phosphatase activity, along with the MSCs from pulp and
periodontal ligament (PDL), while the control cells did not. (D) NHEK/Si-p63 cells exhibiting the EMT phenotype were induced to differentiate into
cells producing mineralized matrix detected by alizarin S red staining. BM, bone marrow; NHOF, normal human oral keratinocyte. (E) NHEK/Si-p63
cells with the EMT phenotype demonstrate adipogenic differentiation capacity when assessed by oil red staining (red droplets).

slides of intracanal tissues. Studies in this review article were sup-
ported in part by grants from the American Association of
Endodontists Foundation. The authors declare no potential con-
flicts of interest with respect to the authorship and/or publication
of this article.
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