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 Kerr's 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.

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

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.

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.

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.

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.