Pulp Regeneration—Translational Opportunities

Pulp Stem Cells: Implication in Reparative Dentin FormationSasha Dimitrova-Nakov, DDS, PhD, Anne Baudry, PhD, Yassine Harichane, DDS, PhD,Odile Kellermann, Dr �es Sciences Naturelles, andMichel Goldberg, DDS, PhD, Dr �es Sciences Naturelles

Abstract

Many dental pulp stem cells are neural crest derivativesessential for lifelong maintenance of tooth functions andhomeostasis as well as tooth repair. These cells may bedirectly implicated in the healing process or indirectlyinvolved in cell-to-cell diffusion of paracrine messagesto resident (pulpoblasts) or nonresident cells (migratingmesenchymal cells). The identity of the pulp progenitorsand the mechanisms sustaining their regenerative ca-pacity remain largely unknown. Taking advantage ofthe A4 cell line, a multipotent stem cell derived fromthe molar pulp of mouse embryo, we investigated thecapacity of these pulp-derived precursors to inducein vivo the formation of a reparative dentin-like struc-ture upon implantation within the pulp of a rodentincisor or a first maxillary molar after surgical exposure.One month after the pulp injury alone, a nonmineralizedfibrous matrix filled the mesial part of the coronal pulpchamber. Upon A4 cell implantation, a mineralized os-teodentin was formed in the implantation site withoutaffecting the structure and vitality of the residual pulpin the central and distal parts of the pulp chamber. Theseresults show that dental pulp stem cells can inducethe formation of reparative dentin and therefore consti-tute a useful tool for pulp therapies. Finally, reparativedentin was also built up when A4 progenitors were per-formed by alginate beads, suggesting that alginate is asuitable carrier for cell implantation in teeth. (J Endod2014;40:S13–S18)

Key WordsCell niches, dentin repair, osteodentin, pulpoblasts, pulpstem cells

From the INSERM UMR-S 747, Equipe 5: Cellules souches,Signalisation et Prions, Paris, France; Universit�e Paris Descartes,Sorbonne, Paris, France; and Biom�edicale des Saint P�eres, Paris,France.

This paper is based on a presentation from the InternationalAssociation for Dental Research (IADR) Pulp Biology andRegeneration Group Satellite Meeting, which was held March24–26, 2013 in San Francisco, California.

Address requests for reprints to Dr Michel Goldberg, Bio-m�edicale des Saints P�eres and INSERM UMR-S 747, 45 ruedes Saints P�eres, 75006 Paris, France. E-mail address: michel.goldberg@parisdescartes.fr0099-2399/$ - 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

Postnatal dental pulp contains heterogeneous cell populations responsible for itsmaintenance, defense, and capacity of repair. The resident cells include stromal fi-

broblasts also named pulpoblasts by Baume (1), odonto-osteoprogenitors, neuronaland vascular cells, and inflammatory and immune system cells (2). The pulp respondsto 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 odontogenicstem cells remain largely unknown. Identifying stem cells mobilized in response to pulpinjury is a prerequisite to design alternative strategies for pulp healing and regenerationand/or for endodontic treatment (3, 4).

Mitotic divisions and apoptosis constantly renew the pulp cell population, incontrast 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 prerequisiteto 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 stemcells (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 beenrecorded between the central part of the pulp and the periphery and also between theradicular and coronal pulp (2). This article highlights the pulp complexity and how it isdifficult to select genuine stem cell–based and cell-free approaches for pulp therapies.

Dental Pulp Stem CellsPostnatal dental pulp stem cells (DPSCs) were firstly isolated from human teeth

(6). They are largely neural crest–derived cells expressing genes that are also presentin embryonic stem cells but lacking expression of mesodermal genes. It is difficult tohave a clear idea of the outcome of stem cells because of the fact that their originhas not yet been elucidated although cell membrane markers and receptors specificallyidentify them. Depending on the culture medium, they become odontoblasts, osteo-blasts, chondrocytes, adipocytes, neurons, and smooth muscles. Implanted withHap/tricalcium phosphate, the clones differentiate into odontoblast-like cells produc-ing a dentin-like structure, which are widely used to produce reparative dentin. In thepresence of bone morphogenetic protein 2 and 4 (BMP2 and BMP4), DPSCs differen-tiate into dentin-forming odontoblasts. This hypothesis is strengthened by the fact thatthe 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 withMSCs of the bone marrow (BMSCs), which are the most studied stromal stem cellpopulations. More than 4,000 human genes are expressed either by BMSCs orDPSCs (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 havethe same embryonic origin. Cells derived from human or animal dental pulps havenot been able to support hematopoiesis in transplantation assays (10, 11). DPSCsare 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 orrepresent distinct differentiation stages along the odontoblastic lineage.

Pulp stem cells are implicated in pulp repair (8). Nevertheless, theformal demonstration that pulpal resident stem cells are actually thereparative dentin-forming cells recruited in response to injury is stilllacking. 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 systemfor tissue repair that may implymigration of stem cells from their restingplaces to the site of injury. Undifferentiated mesenchymal/mesectoder-mal cells present in the stroma and perivascular cells, such as Rouget’spericytes, have been proposed as potential progenitors mediating pulprepair after destruction of the odontoblasts and the Hoehl’s subodonto-blastic cell layer (12, 13). Advances in the identification of stem cellmarkers are still needed to visualize stem/precursor cells in situ.

Figure 1. The multipotent pulp-derived A4 cell line cultured in monolayer for15 days in an odonto-/osteogenic medium (bGP/AA/Dex) forms a mineralizedmatrix as shown by the von Kossa staining. In absence of an inducer, the A4cells never differentiate spontaneously.

Different Dental Stem CellsIn 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 thedental follicle. Other cellular candidates have been identified and impli-cated as new tools in the formation of mineralized tissues. BMSCs gaverise to osteoblast-like cells that generate a bone-like structure. DPSCsare producing structures resembling dentin, whereas BMSCs createbone 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 werealso instrumental for isolating dental stem cells. Subpopulations of DPSCsor stem cells from human exfoliated deciduous teeth (SHED) expressingc-kit/CD34/cell surface antigen expressed by stromal elements in humanbone marrow (STRO-1) are considered as multipotent stem cellsalthough c-kit and CD34 have also been shown to bemarkers for hemato-poietic cell lineages (11–19). Therefore, dental stem cells and non–dental stem cells sharing a high proliferation rate, multidifferentiationability, and easy accessibility are candidates for the development oftooth regeneration and/or tooth engineering (6, 8, 10).

Can Nonresident Migrating CellsBecome Stem Cells?

In addition, 2 other groups of cells may potentially be stem cellcandidates. These possibilities have been experimentally addressed.

The Origin and Role(s) of Nonresident Progenitorand Hematopoietic Cells in Pulp Repair

Investigating the potential roles of nonresident progenitors andhematopoietic cells in the contribution of stem cells found in the dentalpulp, nonresident progenitors were suggested to contribute to repara-tive dentinogenesis. Parabiosis was established between transgenic(freen fluorescent protein + [GFP+]) and wild-type (GFP�) mice toensure cross-circulation between the 2 mice. Pulp exposure andcapping of the first maxillary molar were performed. GFP-positive cellswere detected in close association with reparative dentin formed at thesite of pulp exposure of GFP-negative mice (20).

This observation shows the participation of migrating nonresidentprogenitor and hematopoietic cells in the formation of reparativedentin. Used for the cell migration the blood circulation and movingthroughout the mesenchymal tissues, nonresident progenitorGFP + cells were clearly implicated in the reparative process.

S14 Dimitrova-Nakov et al.

MSCs and Cell MigrationMSCs 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 bonemarrow, transit through the vasculature, and arrive at the affected tis-sues. Chemotaxis implicates enzymes, phosphorylases, and proteinsinducing cell polarization and directional movement. These cells takeorigin in the bone marrow, and they are found in blood vessels. Thisimplies that they may penetrate through the open apex and colonizethe dental pulp.

Some years ago, a limited number of stem cells were identified, andmarked differences were detected between embryonic and adult stemcells. Differences in gene expression, transcription factors, and growthfactors account for the dissimilarities noticed between embryonic andadult lines. Adult stem cells alone are considered in this review withinthe frame of a therapeutic armamentarium. The number, origin, andstage of differentiation have increased, and selecting acceptable stem cellsamong a heterogeneous cell population becomes quite a hard task.

Genomic StabilityImmortalized 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-likecells. It is actually required that the cells and their derivatives maintaintheir genomic stability. The presence of mosaicism and the accumula-tion of karyotypic abnormalities have been reported within culturedcell 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

JOE — Volume 40, Number 4S, April 2014

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 monopotentprogenitors, with a differentiation potential restricted to the odontoblastic program.

Pulp Regeneration—Translational Opportunities

culture. Consequently, there is a need for careful analysis of thecytogenetic stability before undertaking any clinical therapies (21).

To obtain stable cell lines, immortalized dental papilla�derivedcell lines were used. The MO6-G3 mouse-derived cell line (22), thecloned 3T6 cell (23), odontoblast-like cells immortalized by telome-rase (24), and SV-40 immortalized cell lines (25–27) allowed us toobtain the lines, which are required for further studies. This is theonly way to study the signaling cascade leading to the differentiationof odontoblastic-like pulp cells. Even if it is not known yet to whichextent the immortalization procedure of cells or mice influences thegenes and protein expression, it seems impossible to circumvent thisstrategic step.

Stem Cell NichesStem cell behavior is regulated by a local microenvironment

referred as to ‘‘the stem cell niche,’’ which is characterized by 3essential properties (14, 28):

1. A niche provides an anatomic space where the stem cell number isregulated.

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.

JOE — Volume 40, Number 4S, April 2014

Hallmarks of a niche include the stem cell itself, stromal-supporting cells that interact directly with the stem cells via secretedfactors, and cell surface molecules (29). The ECM provides a struc-tural support to the niche and allows the diffusion of mechanical andchemical signals. In teeth, the location and the elements of the pre-sumptive niche providing the physiological environment for stem cellself-renewal and odontogenic differentiation are unknown. It isassumed that multiple niches exist in teeth, but specific markers al-lowing the accurate identification of stem cells within the pulp arelacking.

Many authors hypothesized a perivascular origin for stem cells.MSCs are associated with perivascular niches and they coexpressmany markers in common with pericytes. Some pericytes differentiateinto 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 perivascularniche (18). In this context, it is interesting to mention that many he-matopoietic stem cells and neuronal stem cells are localized close tothe vascular network. Besides, alterations in ECM components and ma-trix stiffness related to damage or aging may also provide mechanicalsignals 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 nichesof stem/progenitor cells (30). This implies that signals ensure their

Pulp Stem Cells S15

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.

Pulp Regeneration—Translational Opportunities

survival and prevent their differentiation while maintaining theirresponsiveness after pulp damage. Whether an endogenous pool ofstem cells associated with supportive stromal cells is mobilized at thesite of injury and/or whether attraction of migrating stem cell is neces-sary to repopulate a niche and expand precursor cells at the appropriatesite for dentin repair is unknown. Summarizing the conclusions of somereports, it seems obvious that some dental pulp side populations areimplicated in the stimulation of angiogenesis, neurogenesis, and pulpregeneration (12) although PCNA immunostaining and the alpha-actin labeling characteristic of microvascularization never overlap inthe dental pulp (7, 12).

The A4 Cell Line Induces the Formationof Reparative Dentin In Vivo

Dental stem cell research is still confronted with the lack ofprecise knowledge related to the location and identity of the cellsparticipating 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, ourlaboratory has obtained clonal cell lines from tooth germs of day18 mouse embryos transgenic for an adenovirus-SV40 recombinantplasmid (pK4) (25). Clonal cell lines derived from the dental pulpprovide valuable tools to answer several fundamental questions suchas the localization of the stem cells niches. Combining in vitro andin vivo experimental approaches, the answers to these questionslead to a better understanding of the potential of stem cells for toothrepair (26, 27).

Whether stem cell phenotypes result from genuine multipotentcells or from the coexistence of distinct progenitors is still an openquestion. 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 amultipotent cell. In contrast, the C5 and H8 clones displayed a morerestricted potential. Altogether, isolation of these clonal lines showsthe coexistence of multipotential and restricted-lineage progenitors inthe mouse pulp, unraveling the specificities of the different types ofpulp progenitors (27). To explore for the first time the potential ofpulp-derived ‘‘stem’’ cells to improve dental repair upon injury, wedeveloped several in situ approaches.

S16 Dimitrova-Nakov et al.

Implantation into the Mouse IncisorIn 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 incisoris a continuously growing tooth, it was necessary to choose a modelcloser to the human tooth physiology.

Implantation into the Rat MolarThe 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 oftooth 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 oldfollowing institutionally approved protocols for animal experimentationresearch. After anesthesia, a cavity was drilled on the mesial aspect ofthe tooth using a tungsten dental bur (ISO 006; Dentsply France SAS,Montigny le Bretonneux, France) with a low-speed handpiece. Pulpperforation 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 pelletsubsequently implanted into the pulp of 14 rat molars (31, 32).

Two, 14, and 30 days after surgery, the rats were euthanized. Blocksections including the hemimaxillaries were dissected, fixed in a 4%paraformaldehyde solution, EDTA demineralized, and included inparaffin. Seven-micrometer-thick sections were collected on glassslides and observed after Masson trichrome staining.

The Sham GroupA surgical pulp exposure was performed in the first rat molars.

Two days after the injury, the pulp tissue appeared normal (Fig. 4Aand 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 GroupAfter 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 ratmolar and in the absence of any carrier or biomolecule to promote

JOE — Volume 40, Number 4S, April 2014

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 massiveformation of reparative dentin in the mesial part of the coronal pulp beneath the calciotraumatic lines (arrows). Implantation of alginate beads loaded by A4 cellsinduces 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) isseen in the mesial pulp chamber.

Pulp Regeneration—Translational Opportunities

efficient dentin repair (26, 27). It is of note that the inflammatoryresponse normally observed upon pulp injury is not enhanced by theimplantation of mouse-derived pulpal cells in a rat molar.

Implantation of Alginate Beads Loadedwith or without A4 Cells

In order to improve the implantation protocol (ie, precisely visu-alize the implantation site and control the number of cells implantedwithin the pulp), we used as carrier alginate beads. Alginate, a linearcopolymer of anionic polysaccharide, was selected as a suitable andpotentially nontoxic biomaterial because its pH is neutral during andafter gelation. Moreover, this irreversible hydrocolloid is biocompat-ible and biodegradable (33). Alginate beads alone or loaded withcells were implanted in exposed pulp. After 1 month, the group im-planted with the alginate beads alone was comparable with the shamgroup. Conversely, 1 month after the implantation of alginate beadsloaded with 3.103 A4 cells, we observed the formation of reparativeosteodentin appearing as a dentin plug located in the isthmus locatedbetween the mesial and central parts of the pulp chambers (Fig. 4Eand F). These preliminary data using alginate beads as a cell carriersuggest that this biomaterial is suitable for cell implantation inducingan 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 thereparative area.

Conclusions and PerspectivesThe stem cells appear as tools to get a better understanding of the

cellular mechanisms of pulp repair. They display innovating potential in

JOE — Volume 40, Number 4S, April 2014

dental therapies. The present results indicate that the direct implanta-tion of mouse progenitor cells in the dental pulp of a rat molar leadsto 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 stemcells toward osteoodontogenic differentiation and indirectly promotethe formation of the dentinal bridge. Future prospects will determinewhether the implanted progenitor cells are directly involved in the for-mation of the reparative dentin or whether they induce the recruitmentand differentiation of host progenitor cells.

In conclusion, our preclinical experimental approach paves theway for the development of cellular therapies after pulp injury. Thelong-term goal should provide new clinical strategies to restorethe functionality of an injured tooth by using pulp stem cells.

AcknowledgmentsThe author denies any conflicts of interest related to this study.

References1. Baume LJ. Biology of pulp and dentin: a historic, terminologicotaxonomic, histolog-

ic-biochemical, embryonic and clinical survey. In: Myers HM, ed. Monographs inOral Science. Basel: S Karger AG; 1980:1–220.

2. Goldberg M, Smith A. Cells and extracellular matrices of dentin and pulp. Abiological basis for repair and tissue engineering. Crit Rev Oral Biol Med 2004;15:1327.

3. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy.J Dent 2000;28:77–92.

4. Balic A, Mina M. Characterization of progenitor cells in pulps of murine incisors.J Dent Res 2010;89:1287–92.

Pulp Stem Cells S17

Pulp Regeneration—Translational Opportunities

5. Kenmotsu M, Matsuzaka K, Kokubu E, et al. Analysis of side population cells derived

from dental pulp tissue. Int Endod J 2010;43:1132–42.6. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells

(DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–30.7. Batouli S, Miura M, Brahim J, et al. Comparison of stem-cell mediated osteogenesis

and dentinogenesis. J Dent Res 2003;82:976–81.8. Huang GT. Dental pulp and dentin tissue engineering and regeneration—advance-

ment and challenge. Front Biosci (Elite Ed) 2012;3:788–800.9. Shi S, Robey PG, Gronthos S. Comparison of human dental pulp and bone marrow

stromal stem cells by cDNA microarray analysis. Bone 2001;29:532–9.10. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs.

those of other sources: their biology and role in regenerative medicine. J Dent Res2009;88:792–806.

11. Gronthos S, Brahim J, Li W, et al. Stem cell properties of human dental pulp stemcells. J Dent Res 2002;81:531–5.

12. Ishizaka R, Hayashi Y, Iohara K, et al. Stimulation of angiogenesis, neurogenesis andregeneration by side population cells from dental pulp. Biomaterials 2013;34:1888–97.

13. Fitzgerald M, Chiego DJ Jr, Heys DR. Autoradiographic analysis of odontoblast replace-ment following pulp exposure in primate teeth. Arch Oral Biol 1990;35:707–15.

14. Ema H, Suda T. Two anatomically distinct niches regulate stem cell activity. Blood2012;120:2174–81.

15. Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated decid-uous teeth. Proc Natl Acad Sci U S A 2003;100:5807–12.

16. Seo B, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cellsfrom human periodontal ligament. Lancet 2004;364:149–55.

17. Janebodin K, Horst OV, Ieronimakis N, et al. Isolation and characterization of neuralcrest-derived stem cells from dental Pulp of Neonatal Mice. PLoS One 2011;6:e27526.

18. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in humanbone marrow and dental pulp. J Bone Miner Res 2003;18:696–704.

19. Sonoyama W, Liu Y, Yamaza T, et al. Characterization of apical papilla and itsresiding stem cells from human immature permanent teeth—a pilot study.J Endod 2008;34:166–71.

S18 Dimitrova-Nakov et al.

20. Frozoni M, Zaia AA, Line SR, et al. Analysis of the contribution of nonresident pro-genitor cells and hematopoietic cells to reparative dentinogenesis using parabiosismodel in mice. J Endod 2012;38:1214–9.

21. Duailibi MT, Kulikowski LD, Duailibi SE, et al. Cytogenetic instability of dental pulpstem cell lines. J Mol Histol 2012;43:89–94.

22. MacDougall M, Thiemann F, Ta H, et al. Temperature-sensitive simian virus 40 largeT antigen immortalization of murine cell cultures: establishment of clonal odonto-blast cell line. Connect Tissue Res 1995;33:97–103.

23. Hanks CT, Sun ZL, Fang DN, et al. Cloned 3T6 cell line from CD-1 mouse fetal molardental papillae. Connect Tissue Res 1998;37:233–49.

24. Hao J, Narayanan K, Ramachandran A, et al. Odontoblast cells immortalized by telo-merase produce mineralized dentin-like tissue both in vitro and in vivo. J BiolChem 2002;277:19976–81.

25. Priam F, Ronco V, Locker M, et al. New cellular models for tracking the odontoblastphenotype. Arch Oral Biol 2005;50:271–7.

26. Harichane Y, Hirata A, Dimitrova-Nakov S, et al. Pulpal progenitors and dentinrepair. Adv Dent Res 2011;23:307–12.

27. Lacerda-Pinheiro S, Dimitrova-Nakov S, Harichane Y, et al. Concomitant multipotentand unipotent dental pulp progenitors and their respective contribution to miner-alised tissue formation. Europ Cells Materials 2012;23:371–86.

28. Huang GT, Sonoyama W, Liu Y, et al. The hidden treasure in apical papilla: the po-tential role in pulp/dentin regeneration and BioRoot engineering. J Endod 2008;34:645–51.

29. Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage spec-ification. Cell 2006;126:677–89.

30. Feng J, Mantesso A, De Bari C, et al. Dual origin of mesenchymal stem Cells contrib-uting to organ growth and repair. Proc Natl Acad Sci U S A 2011;108:6503–8.

31. Ohshima H. Ultrastructural changes in odontoblasts and pulp capillaries followingcavity preparation in rat molars. Arch Histol Cytol 1990;53:423–8.

32. Decup F, Six N, Palmier B, et al. Bone sialoprotein-induced reparative dentinogen-esis in the pulp of rat’s molar. Clin Oral Investig 2000;4:110–9.

33. Kl€ock G, Pfeffermann A, Ryser C, et al. Biocompatibility of mannuronic acid-rich al-ginates. Biomaterials 1997;18:707–13.

JOE — Volume 40, Number 4S, April 2014