Sasha Dimitrova-Nakov

Pulpal Progenitors and Dentin Repair

Mesenchymal stem cells are present in the dental pulp. They have been shown to contribute to dentin-like tissue formation in vitro and to participate in bone repair after a mandibular lesion. However, their capacity to contribute efficiently to reparative dentin formation after pulp lesion has never been explored. After pulp exposure, we have identified proliferative cells within 3 zones. In the crown, zone I is near the cavity, and zone II corresponds to the isthmus between the mesial and central pulp. In the root, zone III, near the apex, at a distance from the inflammatory site, contains mitotic stromal cells which may represent a source of progenitor cells. Stem-cell-based strategies are promising treatments for tissue injury in dentistry. Our experiments focused on (1) location of stem cells induced to leave their quiescent state early after pulp injury and (2) implantation of pulp progenitors, a substitute for classic endodontic treatments, paving the way for pulp stem-cell-based therapies.

Pulp stem cells: implication in reparative dentin formation.

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 capacity 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 structure 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 osteodentin 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 constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth.

In vitro bioactivity of Bioroot™ RCS, via A4 mouse pulpal stem cells.

OBJECTIVE To evaluate the biocompatibility and osteoinductive properties of Bioroot™ RCS (BR, Septodont, France) compared to Kerrs Pulp Canal Sealer™ (PCS, Kerr, Italy) using the mouse pulp-derived stem cell line A4, which have an osteo/odontogenic potential in vitro and contribute to efficient bone repair in vivo. METHODS A4 cells were cultured at the stem cell stage in the presence of solid disks of BR or PCS, whereas untreated A4 cells were used as control. After 3, 7, 10 days of direct contact with the sealers, cell viability was quantified using Trypan Blue exclusion assay. Immunolabelings were performed to assess the expression of odontoblast markers i.e. type 1 collagen, DMP1 or BSP. Finally, sealer-treated cells were induced toward osteo/odontogenic differentiation to assess the impact of the sealers on mineralization by Von Kossa staining. Statistical significance was evaluated by one-way analysis of variance and t-test (p<0.05). RESULTS BR did not alter the viability and morphology of A4 pulpal cells compared to control group (p>0.05); however, living cell percentage of PCS was significantly lower compared to control and BR groups (p<0.05). BR preserved the intrinsic ability of A4 cells to express type 1 collagen, DMP1 or BSP at the stem cell stage. It did not alter the integrity of collagen fibers surrounding the cells and promoted overexpression of BSP and DMP1 at the cell surface. In contrast to PCS, BR did not compromise the mineralization potential of pulpal A4 stem cells. SIGNIFICANCE Bioroot™ RCS was not as cytotoxic as PCS. It did not recruit the pulpal stem cells toward differentiation but preserve their osteo-odontogenic intrinsic properties. Bioroot™ RCS might provide more suitable environment to induce stem cells for hard tissue deposition.

Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets

Characterizing stem cell intrinsic functions is an ongoing challenge for cell therapies. Here, we report that two independent A4 and H8 stem cell lines isolated from mouse molar pulp display the overall functions of bioaminergic cells. Both clones produce neurotrophins and synthesize, catabolize, store, and transport serotonin (5‐hydroxytryptamine [5‐HT]) and dopamine (DA). They express 5‐HT1D,2B,7 and D1,3 autoreceptors, which render pulpal stem cells competent to respond to circulating 5‐HT and DA. We show that injury‐activated platelets are the source of systemic 5‐HT and DA necessary for dental repair since natural dentin reparation is impaired in two rat models with monoamine storage‐deficient blood platelets. Moreover, selective inhibition of either D1, D3, 5‐HT2B, or 5‐HT7 receptor within the pulp of wild‐type rat molars after lesion alters the reparative process. Altogether our data argue that 5‐HT and DA coreleased by pulp injury‐activated platelets are critical for stem cell‐mediated dental repair through 5‐HT and DA receptor signalings. Stem Cells 2015;33:2586—2595

Enamel alterations in serotonin 2B receptor knockout mice

The role of the serotonin 2B receptor (5-HT(2B) R) in enamel formation and mineralization was explored in adult 5HT(2B) R knockout (KO) mice compared with wild-type (WT) mice. In the molar, quantitative data obtained by micro-computed tomography imaging showed that the overall volume of the enamel layer was firmly reduced in KO mice. Defective mineralization was ascertained by energy-dispersive X-ray microanalysis. We also observed, using scanning electron microscopy, that parazones in the KO mice included two or three helicoidally twisted rods within Hunter-Schreger bands, instead of a single rod, as found in the WT mice. Minor disturbances were also detected in the incisors of KO mice. Structural modifications, thinner enamel crystallites, and porosities observed in KO mice indicate that the 5-HT(2B) R-mediated signaling pathways as part of the enamel formation process. These data provide a basis for evaluating the role of 5-HT(2B) R in ameloblast functions. Defects observed in the mineralization and structure of enamel in KO mice highlight that the 5-HT(2B) R interferes with the mechanisms directing amelogenesis.

Comparative studies between mice molars and incisors are required to draw an overview of enamel structural complexity

In the field of dentistry, the murine incisor has long been considered as an outstanding model to study amelogenesis. However, it clearly appears that enamel from wild type mouse incisors and molars presents several structural differences. In incisor, exclusively radial enamel is observed. In molars, enamel displays a high level of complexity since the inner part is lamellar whereas the outer enamel shows radial and tangential structures. Recently, the serotonin 2B receptor (5-HT2BR) was shown to be involved in ameloblast function and enamel mineralization. The incisors from 5HT2BR knockout (KO) mice exhibit mineralization defects mostly in the outer maturation zone and porous matrix network in the inner zone. In the molars, the mutation affects both secretory and maturation stages of amelogenesis since pronounced alterations concern overall enamel structures. Molars from 5HT2BR KO mice display reduction in enamel thickness, alterations of inner enamel architecture including defects in Hunter-Schreger Bands arrangements, and altered maturation of the outer radial enamel. Differences of enamel structure were also observed between incisor and molar from other KO mice depleted for genes encoding enamel extracellular matrix proteins. Thus, upon mutation, enamel analysis based exclusively on incisor defects would be biased. In view of the functional relationship between enamel structure and tooth morphogenesis, identification of molecular actors involved in amelogenesis requires comparative studies between mice molars and incisors.

Plithotaxis, a collective cell migration, regulates the sliding of proliferating pulp cells located in the apical niche.

Abstract Using the proliferating cell nuclear antigen (PCNA) immunostaining, we previously identified, after pulp exposure, three zones of proliferating cells in the rat molar pulp. Zones I and II were in the crown near the pulp. Zone III was near the apex revealing a recruitment of mitotic cells at distance from the lesion. To gain further insight into the spatio-temporal evolution of proliferating pulp cells of zone III, we performed a longitudinal study of PCNA staining in rat molar mesial root at 3, 8, and 15 d after pulp exposure associated to implantation of unloaded or amelogenin loaded agarose beads. At day 3 after implantation, PCNA-positive cells were located in the central part of the radicular pulp. At day 8, PCNA-labeled cells were aligned in the lateral part of the pulp beneath the odontoblast/sub-odontoblast layer. At day 15, PCNA labeling became undetectable in the root and was located in the coronal pulp. These results suggest that after pulp exposure, PCNA-positive cells may migrate from the central part of the radicular pulp to the sub-odontoblast cell layer and then from the apical root to the crown. Electron microscopy and immunostaining analysis showed that pulpal cells were linked by desmosome-like and gap-junctions. Extracellular matrix was composed of thin collagen fibrils associated with glycosaminoglycans favoring cell mobility. These data suggest that the syncytium-like structure formed by pulp radicular cells may be a pre-request for plithotaxis, a collective cell migration process. This emergent mechanism may govern pulp healing and regeneration after dental lesion.

BPA from dental resin material: where are we going with restorative and preventive dental biomaterials?

Since a number of years, the question of the impact of BPA released from dental resins and/or sealants (or other dental materials) is open. These materials contain different monomers and many additives that are used to induce or inhibit the polymerization reaction. The monomers are mainly urethane dimethacrylate, BPA glycidyl dimethacrylate (Bis-GMA), its ethoxylated version (Bis-EMA), and BPA dimethacrylate (Bis-DMA). Comonomers such as triethylene glycol dimethacrylate are used to dilute the thick consistency and provide a higher degree of cure because of their relatively lower molecular weight [1]. BPA is used in the manufacturing process of Bis-GMA, Bis-EMA, and Bis-DMA or other similar molecules, and thus, minute amounts of BPA residuesmay be present in the final material. Interestingly, it has been shown that some patients treated with Bis-DMA containing materials present salivary and urinary BPA concentrations two to tenfold higher than control patients [2], which, however, decreased to almost normal after 24 h. For Bis-GMA materials, very small amounts of BPA could be detected in saliva and only neglectable amounts in urine, which also decreased 24 h after placement. It has been published that BPA may act as an endocrine disruptor (ED), thus being able to alter functions of the endocrine system and cause adverse health effects to an intact organism or its progeny (World Health Organization (WHO) definition 2002). EDs with an estrogenic activity have been proposed to be associated with estrogenic effects such as testicular dysgenesis, inducing testicular, prostate, and mammary gland cancers, impairing semen quality, diabetes, and obesity [3]. Some authors [3–5] claim that a series of diseases including cancer risks, type 2 diabetes, obesity, and developmental impairments are related to exposure towards BPA [3–5]. In human cross-sectional studies, BPA plasma or urine concentrations were reported to be associated among others with prostate dysplasias and abnormal birth weight [4]. A main problem in this context is the correct chemical analysis of BPA, the sensitivity of the analytical methods, and the question of BPA conjugation/glucoronization. Whereas the measurements in urine may be considered reliable, unconjugated BPA is apparently difficult to be measured in plasma because the sensitivity of the methods may not allow the measure the concentrations in the picogram per milliliter range. It is accepted that reported results in plasma/serum may be artifacts due to external contamination of the samples. Experimental studies in animals could not confirm results from reported observational studies in humans and showed general toxicity of BPA of doses above 5 mg/kg bw/day. Proliferation of mammary gland at doses of 400 μg/kg bw/day in monkeys [6] and behavioral effects in rodents at the same dose level. Although there are effects to be noted in in vitro studies on cell and tissue cultures and in vivo animal experiments, which may indicate adverse endocrine related effects due to BPA, it is still difficult to extrapolate these findings to the human clinical situation. Its precise mechanism of action is still not clear, as the signaling pathways seem to be dependent on the BPA concentrations. However, its functional interaction with estrogen receptors has been recently demonstrated and could explain at least in part its endocrine disrupting effects [4]. In the EU, USA, and Canada, BPA was banned from feeding bottles. M. Goldberg (*) : S. Dimitrova-Nakov INSERM UMR-S U747 (Equipe 5) & Faculte des Sciences Fondamentales et Biomedicales, Universite Paris Descartes, 45 rue des Saints Peres, 75270 Paris Cedex 06, France e-mail: mgoldberg.goldberg004@gmail.com

Deletion of Serotonin 2B Receptor Provokes Structural Alterations of Mouse Dental Tissues

Rampant caries and periodontal diseases occur in patients treated with antidepressants such as serotonin reuptake inhibitors (SRIs; e.g., Prozac) which target the serotonin transporter (SERT). As the serotonin 2B receptor (5HT2BR) regulates SERT functionality and capacity to recognize SRIs, we investigated the potential role of 5HT2BR on dental tissues by exploiting 5HT2BR knockout (KO) mice. Compared to wild-type (WT) mice, several structural differences were identified in the teeth of KO mice. In the molar of KO mice, rod curvatures and twisting were altered compared to WT mice, suggesting involvement of 5HT2BR at early stages of enamel formation. The volume of the KO enamel layer was also reduced, and larger porosities were observed in the prismatic enamel, with smaller crystallite thickness. Crystallite pattern disorganization and occlusal abrasion were enhanced in female KO mice, indicating a sexual dimorphism. In the incisor, no difference was detected in the width of the enamel layer between KO and WT mice; however, enamel maturation differed in absence of 5HT2BR. Specifically, the outer aprismatic enamel border was 1.5- to 2-fold larger in KO compared to WT mice, together with a decreased etching pattern. Finally, although no noticeable difference was observed in dentin, the micro-CT three-dimensional pulp reconstruction evidenced a decrease in both length and width of dentin formation in the root canals of the KO versus WT mice. These data provide evidence that 5HT2BR-mediated signaling pathways are involved in enamel formation and dentinogenesis.