Pulp Regeneration—Translational Opportunities
Pulp Stem Cells: Implication in Reparative Dentin Formation
Sasha Dimitrova-Nakov, DDS, PhD, Anne Baudry, PhD, Yassine Harichane, DDS, PhD,
Odile Kellermann, Dr
es Sciences Naturelles, and
Michel Goldberg, DDS, PhD, Dr
es Sciences Naturelles
Abstract
Many dental pulp stem cells are neural crest derivatives
essential for lifelong maintenance of tooth functions and
homeostasis as well as tooth repair. These cells may be
directly implicated in the healing process or indirectly
involved in cell-to-cell diffusion of paracrine messages
to resident (pulpoblasts) or nonresident cells (migrating
mesenchymal cells). The identity of the pulp progenitors
and the mechanisms sustaining their regenerative ca-
pacity remain largely unknown. Taking advantage of
the A4 cell line, a multipotent stem cell derived from
the molar pulp of mouse embryo, we investigated the
capacity of these pulp-derived precursors to induce
in vivo the formation of a reparative dentin-like struc-
ture upon implantation within the pulp of a rodent
incisor or a first maxillary molar after surgical exposure.
One month after the pulp injury alone, a nonmineralized
fibrous matrix filled the mesial part of the coronal pulp
chamber. Upon A4 cell implantation, a mineralized os-
teodentin was formed in the implantation site without
affecting the structure and vitality of the residual pulp
in the central and distal parts of the pulp chamber. These
results show that dental pulp stem cells can induce
the formation of reparative dentin and therefore consti-
tute a useful tool for pulp therapies. Finally, reparative
dentin was also built up when A4 progenitors were per-
formed by alginate beads, suggesting that alginate is a
suitable carrier for cell implantation in teeth. (J Endod
2014;40:S13–S18)
Key Words
Cell niches, dentin repair, osteodentin, pulpoblasts, pulp
stem cells
From the INSERM UMR-S 747, Equipe 5: Cellules souches,
Signalisation et Prions, Paris, France; Universite Paris Descartes,
Sorbonne, Paris, France; and Biomedicale des Saint P
eres, Paris,
France.
This paper is based on a presentation from the International
Association for Dental Research (IADR) Pulp Biology and
Regeneration Group Satellite Meeting, which was held March
24–26, 2013 in San Francisco, California.
Address requests for reprints to Dr Michel Goldberg, Bio-
medicale des Saints P
eres and INSERM UMR-S 747, 45 rue
des Saints P
eres, 75006 Paris, France. E-mail address: michel.
goldberg@parisdescartes.fr
0099-2399/$ - see front matter
Copyright ª 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.01.011
JOE — Volume 40, Number 4S, April 2014
P ostnatal dental pulp contains heterogeneous cell populations responsible for its
maintenance, defense, and capacity of repair. The resident cells include stromal fi-
broblasts also named pulpoblasts by Baume (1), odonto-osteoprogenitors, neuronal
and vascular cells, and inflammatory and immune system cells (2). The pulp responds
to a variety of pathological injuries by a deposition of reparative dentin by pulp ‘‘pro-
genitors’’ (2, 3). However, the origin, localization, and precise identity of odontogenic
stem cells remain largely unknown. Identifying stem cells mobilized in response to pulp
injury is a prerequisite to design alternative strategies for pulp healing and regeneration
and/or for endodontic treatment (3, 4).
Mitotic divisions and apoptosis constantly renew the pulp cell population, in
contrast with odontoblasts and cells of the Hoehl’s layer, which are postmitotic cells.
Pulpoblasts are implicated in the synthesis and secretion of an extracellular matrix
(ECM). Enzymatic cleavages followed by the reuptake of ECM remnants are prerequisite
to promote dentin and/or pulp mineralization.
A few pulp cells are stem cells, probably less than 1% of the total cell population.
According to Kenmotsu et al (5), approximately 0.40% of the pulp cells may be stem
cells (also referred to as the side population) when they are found in young rats,
whereas only 0.11% is found in adult rats.
Variations in cell number and the respective distribution and functions have been
recorded between the central part of the pulp and the periphery and also between the
radicular and coronal pulp (2). This article highlights the pulp complexity and how it is
difficult to select genuine stem cell–based and cell-free approaches for pulp therapies.
Dental Pulp Stem Cells
Postnatal dental pulp stem cells (DPSCs) were firstly isolated from human teeth
(6). They are largely neural crest–derived cells expressing genes that are also present
in embryonic stem cells but lacking expression of mesodermal genes. It is difficult to
have a clear idea of the outcome of stem cells because of the fact that their origin
has not yet been elucidated although cell membrane markers and receptors specifically
identify them. Depending on the culture medium, they become odontoblasts, osteo-
blasts, chondrocytes, adipocytes, neurons, and smooth muscles. Implanted with
Hap/tricalcium phosphate, the clones differentiate into odontoblast-like cells produc-
ing a dentin-like structure, which are widely used to produce reparative dentin. In the
presence of bone morphogenetic protein 2 and 4 (BMP2 and BMP4), DPSCs differen-
tiate into dentin-forming odontoblasts. This hypothesis is strengthened by the fact that
the BMP antagonist Noggin inhibits the capacity of DPSCs to differentiate into odonto-
genic cells as suggested by the lack of expression of dentin sialophosphoproteins.
Populations of DPSCs exhibit generic mesenchymal stem cell (MSC)-like proper-
ties; display the ability to form colonies; and express in vitro osteoblastic, adipogenic,
chondrogenic, and even neuronal markers (6–9). DPSCs share many similarities with
MSCs of the bone marrow (BMSCs), which are the most studied stromal stem cell
populations. More than 4,000 human genes are expressed either by BMSCs or
DPSCs (9). Dental stem cell populations also express different panels of stem cell sur-
face markers used to characterize hematopoietic stem cells of the bone marrow
(BMSCs) (10). However, it is important to note that DPSCs and BMSCs do not have
the same embryonic origin. Cells derived from human or animal dental pulps have
not been able to support hematopoiesis in transplantation assays (10, 11). DPSCs
are thought to contribute to reparative dentin formation, and it appears that they
Pulp Stem Cells S13
Pulp Regeneration—Translational Opportunities
may correspond to heterogeneous populations of precursor cells or
represent distinct differentiation stages along the odontoblastic lineage.
Pulp stem cells are implicated in pulp repair (8). Nevertheless, the
formal demonstration that pulpal resident stem cells are actually the
reparative dentin-forming cells recruited in response to injury is still
lacking. The hypothesis that a subset of stem cells carried by the vascu-
lature replenishes the pulp after a lesion cannot be totally excluded.
Moreover, the responsiveness of the pulp provides a dynamic system
for tissue repair that may imply migration of stem cells from their resting
places to the site of injury. Undifferentiated mesenchymal/mesectoder-
mal cells present in the stroma and perivascular cells, such as Rouget’s
pericytes, have been proposed as potential progenitors mediating pulp
repair after destruction of the odontoblasts and the Hoehl’s subodonto-
blastic cell layer (12, 13). Advances in the identification of stem cell
markers are still needed to visualize stem/precursor cells in situ.

Different Dental Stem Cells

In addition to DPSCs, others stem cells have been obtained from
human dental tissues (deciduous teeth and the apical part of the papilla)
(6, 11, 14) and periodontal tissues (gingiva, cementum, alveolar bone,
and periodontal ligament) (15). Stem cells were also isolated from the
dental follicle. Other cellular candidates have been identified and impli-
cated as new tools in the formation of mineralized tissues. BMSCs gave
rise to osteoblast-like cells that generate a bone-like structure. DPSCs
are producing structures resembling dentin, whereas BMSCs create
bone condensation at some distance to capillaries (14).
Dental stem cells are considered as a population of MSC-like cells.
Therefore, the markers that have been used for identifying MSCs were
also instrumental for isolating dental stem cells. Subpopulations of DPSCs
or stem cells from human exfoliated deciduous teeth (SHED) expressing
c-kit/CD34/cell surface antigen expressed by stromal elements in human
bone marrow (STRO-1) are considered as multipotent stem cells
although c-kit and CD34 have also been shown to be markers for hemato-
poietic cell lineages (11–19). Therefore, dental stem cells and non–
dental stem cells sharing a high proliferation rate, multidifferentiation
ability, and easy accessibility are candidates for the development of
tooth regeneration and/or tooth engineering (6, 8, 10).
Can Nonresident Migrating Cells
Become Stem Cells?
In addition, 2 other groups of cells may potentially be stem cell
candidates. These possibilities have been experimentally addressed.
The Origin and Role(s) of Nonresident Progenitor
and Hematopoietic Cells in Pulp Repair
Investigating the potential roles of nonresident progenitors and
hematopoietic cells in the contribution of stem cells found in the dental
pulp, nonresident progenitors were suggested to contribute to repara-
tive dentinogenesis. Parabiosis was established between transgenic
(freen fluorescent protein + [GFP+]) and wild-type (GFP ) mice to
ensure cross-circulation between the 2 mice. Pulp exposure and
capping of the first maxillary molar were performed. GFP-positive cells
were detected in close association with reparative dentin formed at the
site of pulp exposure of GFP-negative mice (20).
This observation shows the participation of migrating nonresident
progenitor and hematopoietic cells in the formation of reparative
dentin. Used for the cell migration the blood circulation and moving
throughout the mesenchymal tissues, nonresident progenitor
GFP + cells were clearly implicated in the reparative process.
Figure 1. The multipotent pulp-derived A4 cell line cultured in monolayer for
15 days in an odonto-/osteogenic medium (bGP/AA/Dex) forms a mineralized
matrix as shown by the von Kossa staining. In absence of an inducer, the A4
cells never differentiate spontaneously.
MSCs and Cell Migration
MSCs are found in bone marrow, cord blood, and dental pulp.
They differentiate into osteoblasts, chondroblasts, adipoblasts, fibro-
blasts, myofibroblasts, and neuroblasts. The cells migrate from bone
marrow, transit through the vasculature, and arrive at the affected tis-
sues. Chemotaxis implicates enzymes, phosphorylases, and proteins
inducing cell polarization and directional movement. These cells take
origin in the bone marrow, and they are found in blood vessels. This
implies that they may penetrate through the open apex and colonize
the dental pulp.
Some years ago, a limited number of stem cells were identified, and
marked differences were detected between embryonic and adult stem
cells. Differences in gene expression, transcription factors, and growth
factors account for the dissimilarities noticed between embryonic and
adult lines. Adult stem cells alone are considered in this review within
the frame of a therapeutic armamentarium. The number, origin, and
stage of differentiation have increased, and selecting acceptable stem cells
among a heterogeneous cell population becomes quite a hard task.
Genomic Stability
Immortalized human stem cells have been obtained from dental
pulp. Genomic stability from transformed residing cells of ectomesen-
chymal origin has the potential to differentiate into odontoblast-like
cells. It is actually required that the cells and their derivatives maintain
their genomic stability. The presence of mosaicism and the accumula-
tion of karyotypic abnormalities have been reported within cultured
cell subpopulations. Gradually, many of the cultured cells (70%)
exhibit karyotypic abnormalities after some passages. The heteroge-
neous spectrum of abnormalities indicates a high frequency of chromo-
somal mutations that continuously arise upon extended

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 Pulp Regeneration—Translational Opportunities

Figure 2. The A4 clone behaves as a multipotent mesenchymal progenitor. Depending on the nature of the inducers, A4 cells are able to undergo odontogenic,
osteogenic, chondrogenic, or adipogenic differentiation in a mutually exclusive manner. Two other clonal pulpal cell lines, C5 and H8, appear as monopotent
progenitors, with a differentiation potential restricted to the odontoblastic program.

culture. Consequently, there is a need for careful analysis of the
cytogenetic stability before undertaking any clinical therapies (21).
To obtain stable cell lines, immortalized dental papilla derived
cell lines were used. The MO6-G3 mouse-derived cell line (22), the
cloned 3T6 cell (23), odontoblast-like cells immortalized by telome-
rase (24), and SV-40 immortalized cell lines (25–27) allowed us to
obtain the lines, which are required for further studies. This is the
only way to study the signaling cascade leading to the differentiation
of odontoblastic-like pulp cells. Even if it is not known yet to which
extent the immortalization procedure of cells or mice influences the
genes and protein expression, it seems impossible to circumvent this
strategic step.
Stem Cell Niches
Stem cell behavior is regulated by a local microenvironment
referred as to ‘‘the stem cell niche,’’ which is characterized by 3
essential properties (14, 28):
1. A niche provides an anatomic space where the stem cell number is
regulated.
2. It is the place where a stem cell is instructed to control the mainte-
nance, quiescence, self-renewal, and recruitment toward differenti-
ation, fate determination, and long-term regenerative capacity.
3. The niche will influence cell motility.
Hallmarks of a niche include the stem cell itself, stromal-
supporting cells that interact directly with the stem cells via secreted
factors, and cell surface molecules (29). The ECM provides a struc-
tural support to the niche and allows the diffusion of mechanical and
chemical signals. In teeth, the location and the elements of the pre-
sumptive niche providing the physiological environment for stem cell
self-renewal and odontogenic differentiation are unknown. It is
assumed that multiple niches exist in teeth, but specific markers al-
lowing the accurate identification of stem cells within the pulp are
lacking.
Many authors hypothesized a perivascular origin for stem cells.
MSCs are associated with perivascular niches and they coexpress
many markers in common with pericytes. Some pericytes differentiate
into specialized tooth mesenchyme-derived cells.
Some data suggest that pericytes could differentiate into osteo-
blast-like cells, and odontogenic stem cells may reside in a perivascular
niche (18). In this context, it is interesting to mention that many he-
matopoietic stem cells and neuronal stem cells are localized close to
the vascular network. Besides, alterations in ECM components and ma-
trix stiffness related to damage or aging may also provide mechanical
signals that could have a profound impact on stem cell activity (29).
Thus, it appears that the distribution of tissue stem cells is not random,
and, within the dental pulp, there are potentially several distinct niches
of stem/progenitor cells (30). This implies that signals ensure their

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Pulp Regeneration—Translational Opportunities
Figure 3. After 10 days, implantation of A4 cells in the surgically exposed dental pulp of the mouse incisor promotes the massive formation of reparative osteo-
dentin. The control group (nonimplanted with cells) exhibits no pulp repair.
survival and prevent their differentiation while maintaining their
responsiveness after pulp damage. Whether an endogenous pool of
stem cells associated with supportive stromal cells is mobilized at the
site of injury and/or whether attraction of migrating stem cell is neces-
sary to repopulate a niche and expand precursor cells at the appropriate
site for dentin repair is unknown. Summarizing the conclusions of some
reports, it seems obvious that some dental pulp side populations are
implicated in the stimulation of angiogenesis, neurogenesis, and pulp
regeneration (12) although PCNA immunostaining and the alpha-
actin labeling characteristic of microvascularization never overlap in
the dental pulp (7, 12).
The A4 Cell Line Induces the Formation
of Reparative Dentin In Vivo
Dental stem cell research is still confronted with the lack of
precise knowledge related to the location and identity of the cells
participating in reparative dentin formation upon tooth injury. In or-
der to characterize the dental pulp progenitors and study the molec-
ular and cellular mechanisms governing their differentiation, our
laboratory has obtained clonal cell lines from tooth germs of day
18 mouse embryos transgenic for an adenovirus-SV40 recombinant
plasmid (pK4) (25). Clonal cell lines derived from the dental pulp
provide valuable tools to answer several fundamental questions such
as the localization of the stem cells niches. Combining in vitro and
in vivo experimental approaches, the answers to these questions
lead to a better understanding of the potential of stem cells for tooth
repair (26, 27).
Whether stem cell phenotypes result from genuine multipotent
cells or from the coexistence of distinct progenitors is still an open
question. Three clonal pulp precursor cell lines (A4, C5, and H8)
were induced to differentiate toward the odonto-/osteogenic, chondro-
genic, or adipogenic program (Figs. 1 and 2). Implanted into mandib-
ular incisors or calvaria of adult mice, the A4 clone behaves as a
multipotent cell. In contrast, the C5 and H8 clones displayed a more
restricted potential. Altogether, isolation of these clonal lines shows
the coexistence of multipotential and restricted-lineage progenitors in
the mouse pulp, unraveling the specificities of the different types of
pulp progenitors (27). To explore for the first time the potential of
pulp-derived ‘‘stem’’ cells to improve dental repair upon injury, we
developed several in situ approaches.
S16 Dimitrova-Nakov et al.
Implantation into the Mouse Incisor
In the first in vivo pilot study, the A4 cells were implanted in the
mouse mandibular incisor, and this led to the formation of a mineral-
ized osteodentin within the dental pulp (Fig. 3). Because mouse incisor
is a continuously growing tooth, it was necessary to choose a model
closer to the human tooth physiology.
Implantation into the Rat Molar
The small size of the mouse molars and teeth of limited growth
make experiment procedures difficult. In contrast, the rat molar pro-
vides an easier experimental approach involving the preparation of
tooth lesions (Fig. 4).
We performed surgical pulp exposure with or without cell implan-
tation in the first maxillary molar of 9-week-old Sprague-Dawley rats old
following institutionally approved protocols for animal experimentation
research. After anesthesia, a cavity was drilled on the mesial aspect of
the tooth using a tungsten dental bur (ISO 006; Dentsply France SAS,
Montigny le Bretonneux, France) with a low-speed handpiece. Pulp
perforation was accomplished by pressure with the tip of a steel probe.
This method avoids rolling the pulp around the dental burr. About 105
A4 cells were collected in a tube and centrifuged to form a cell pellet
subsequently implanted into the pulp of 14 rat molars (31, 32).
Two, 14, and 30 days after surgery, the rats were euthanized. Block
sections including the hemimaxillaries were dissected, fixed in a 4%
paraformaldehyde solution, EDTA demineralized, and included in
paraffin. Seven-micrometer-thick sections were collected on glass
slides and observed after Masson trichrome staining.
The Sham Group
A surgical pulp exposure was performed in the first rat molars.
Two days after the injury, the pulp tissue appeared normal (Fig. 4A
and B). However, inflammatory cells were recruited near the implanta-
tion site (Fig. 4C). One month after the surgery, the mesial pulp cham-
ber was fibrotic but not mineralized (Fig. 4B).
The Experimental A4 Cell Implantation Group
After 1 month, the mesial part of the pulp chamber was massively
filled by mineralized osteodentin seen under a calciotraumatic line
(Fig. 4D). Thus, A4 cells have the capacity after implantation in a rat
molar and in the absence of any carrier or biomolecule to promote
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 Pulp Regeneration—Translational Opportunities

Figure 4. After a sham pulp exposure in the first maxillary rat molar, inflammatory processes are limited after (A) 2 days and (B) 28 days. (C) In the control
(nonimplanted with cells), a nonmineralized fibrous reaction is seen in the mesial part of the coronal pulp. (D) In contrast, A4 implantation promotes the massive
formation of reparative dentin in the mesial part of the coronal pulp beneath the calciotraumatic lines (arrows). Implantation of alginate beads loaded by A4 cells
induces the formation of a dentin barrier (db and asterisk) located between the mesial and central part of the pulp chamber. (E and F) A fibrous matrix (fm) is
seen in the mesial pulp chamber.

efficient dentin repair (26, 27). It is of note that the inflammatory
response normally observed upon pulp injury is not enhanced by the
implantation of mouse-derived pulpal cells in a rat molar.
Implantation of Alginate Beads Loaded
with or without A4 Cells
In order to improve the implantation protocol (ie, precisely visu-
alize the implantation site and control the number of cells implanted
within the pulp), we used as carrier alginate beads. Alginate, a linear
copolymer of anionic polysaccharide, was selected as a suitable and
potentially nontoxic biomaterial because its pH is neutral during and
after gelation. Moreover, this irreversible hydrocolloid is biocompat-
ible and biodegradable (33). Alginate beads alone or loaded with
cells were implanted in exposed pulp. After 1 month, the group im-
planted with the alginate beads alone was comparable with the sham
group. Conversely, 1 month after the implantation of alginate beads
loaded with 3.103 A4 cells, we observed the formation of reparative
osteodentin appearing as a dentin plug located in the isthmus located
between the mesial and central parts of the pulp chambers (Fig. 4E
and F). These preliminary data using alginate beads as a cell carrier
suggest that this biomaterial is suitable for cell implantation inducing
an efficient pulp repair. Hence, we get the first evidence that implan-
tation of pulpal precursor cells within the pulp promotes the forma-
tion of a robust dentin barrier separating the residual pulp from the
reparative area.
Conclusions and Perspectives
The stem cells appear as tools to get a better understanding of the
cellular mechanisms of pulp repair. They display innovating potential in
dental therapies. The present results indicate that the direct implanta-
tion of mouse progenitor cells in the dental pulp of a rat molar leads
to the formation of reparative osteodentin.
It is important to determine (1) whether precursor cells reintro-
duced in a pulpal ‘‘natural’’ environment differentiate into osteoodon-
togenic cells or whether the implanted cells recruit resident pulp stem
cells toward osteoodontogenic differentiation and indirectly promote
the formation of the dentinal bridge. Future prospects will determine
whether the implanted progenitor cells are directly involved in the for-
mation of the reparative dentin or whether they induce the recruitment
and differentiation of host progenitor cells.
In conclusion, our preclinical experimental approach paves the
way for the development of cellular therapies after pulp injury. The
long-term goal should provide new clinical strategies to restore
the functionality of an injured tooth by using pulp stem cells.
Acknowledgments
The author denies any conflicts of interest related to this study.
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