Aim To investigate the involvement of Wnt signalling and cell cycle regulation in hard tissue formation after pulp capping with several materials in a rat molar pinpoint exposure model. Methodology Thirty‐two rat molar pulps were mechanically exposed and assigned to 4 groups according to the pulp capping materials used Ca(OH)2, mineral trioxide aggregate (MTA), Biodentine™ and an untreated control group. After 4 weeks, the teeth were collected for microcomputed tomography to quantify reparative dentine formation. Histological analysis was then performed to evaluate the quality of the reparative dentine and the dental pulp tissue inflammatory reaction. Cyclin D1 and β‐catenin expression was examined using immunofluorescence staining. The Kruskal–Wallis followed by Dunn’s multiple comparison test was performed to determine significant differences. Results The exposed dental pulps treated with Ca(OH)2, MTA and Biodentine™ exhibited reparative dentine formation near the exposure site. Fibrous tissues adjacent to the exposure site were observed in the untreated group. The microcomputed tomography evaluation of MTA and Biodentine™ groups revealed significantly greater reparative dentine formation compared with the control group (P = 0.0032 in the MTA group and P = 0.05 in the BiodentineTM group). From histological evaluations, the BiodentineTM group exhibited significantly greater reparative dentine formation grading compared with the control group (P = 0.0152). The pulp tissues treated with Ca(OH)2 and Biodentine™ exhibited a lower inflammatory score compared with those of the untreated control (P = 0.0291 in the Ca(OH)2 and P = 0.0110 in the BiodentineTM group). Ca(OH)2, MTA and Biodentine™ induced cyclin D1 expression in the dental pulp tissues adjacent to the reparative dentine. Moreover, the Biodentine™‐treated defects demonstrated β‐catenin expression in the pulp tissue adjacent to the newly formed reparative dentine, which was not observed with the other materials. Conclusion All test materials promoted dentine bridge formation and stimulated cyclin D1 expression. The favourable outcome after direct pulp capping with Biodentine™ involved Wnt/β‐catenin signalling. However, Wnt/β‐catenin signalling did not participate in the mechanism by which Ca(OH)2 and MTA promoted reparative dentine formation.

Aim:To investigate the effect of Wnt3a on odonto/osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs) and reparative dentine formation in a rat model. Methodology:Stem cells isolated from human exfoliated deciduous teeth were cultured in media with Wnt3a (50-200 ng/ml). Wnt activation was confirmed by βcatenin immunocytochemistry. Colony-forming unit assay (normalized percentage area), osteogenic gene expression analysis by real-time polymerase chain reaction and mineralization assays measured by the absorption at 540 nm were performed.Tertiary dentine formation in vivo was evaluated using 8-week-old, male Wistar rats.Cavities with pinpoint pulp exposure by a sharp instrument were prepared at the mesial surface of the first molars. Teeth were divided into (n = 6): (1) distilled water (negative control), (2) phosphate-buffered saline (PBS), (3) lithium chloride in DI (20 μM), and (4) Wnt3a in PBS (200 ng/ml). Collagen sponge was used as a scaffold.The cavity was sealed with glass ionomer restoration. Four weeks later, animals were euthanized by sodium pentobarbital (120 mg/kg body weight). Hard tissue formation was evaluated using micro-computerized tomography. Sixty consecutive slides from the initial plane were analysed and calculated as bone/dentine volume per total tissue volume. Paraffin sections (2 μm) were stained with haematoxylin and eosin and Masson's trichrome for morphological evaluation. Data are presented as the mean ± standard error. Mann-Whitney U test was used for two-group comparison.Kruskal Wallis followed by pairwise comparison was employed for three or more group comparisons. Statistical analysis was performed using GraphPad Prism 7.Differences were considered significant at p < .05.Results: Wnt3a decreased SHEDs colony formation and increased OSX, BMP2, and DMP1 expression, corresponding to an increase in mineralization. Additionally, a significant increase in dentine/bone volume per total tissue volume was observed in Wnt3a treated defects. Dentine bridge formation at the exposure sites treated with Wnt3a demonstrated, while fibrous tissues were observed in the control.