Analysis of hard tissue regeneration and Wnt signalling in dental pulp tissues after direct pulp capping with different materials.

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 ß-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 ß-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/ß-catenin signalling. However, Wnt/ß-catenin signalling did not participate in the mechanism by which Ca(OH)2 and MTA promoted reparative dentine formation.

Dental properties, ultrastructure, and pulp cells associated with a novel DSPP mutation.

OBJECTIVE: To investigate physical characteristics and behaviours of dental pulp cells of teeth isolated from a dentinogenesis imperfecta (DGI) patient with a novel dentin sialophosphoprotein (DSPP) mutation. SUBJECTS AND METHODS: Whole exome and Sanger sequencing were employed to identify mutations. Physical characteristics of the teeth were examined. Pulp cells' behaviours including cell proliferation, colony-forming unit, osteogenic differentiation, pluripotent markers, and mesenchymal stem cell markers were investigated. RESULTS: The proband had opalescent brown primary teeth with extensive loss of enamel. Mutation analysis revealed a novel heterozygous 4-bp deletion, c.1915_1918delAAGT (p.K639QfsX674), in exon 5 of the DSPP associated with DGI. Analysis of the extracted primary incisor demonstrated a decrease in brightness but an increase in yellow and red chroma. The dentin showed reduced mineral density. The dentinal tubules were present in the predentin, but progressively collapsed in the dentin. The pulp cells exhibited markedly reduced CD105 expression, decreased cell proliferation, and smaller colony-forming units. CONCLUSIONS: We identified a novel mutation in the DSPP gene which disturbed dentin characteristics and pulp cells' behaviours. Our study expands the mutation spectrum and understanding of pathologic dentin phenotypes related to the frameshift deletion in the dentin phosphoprotein (DPP) region of the DSPP gene.

Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. MATERIAL AND METHODS: hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. RESULTS: Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. CONCLUSION: These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.

Neurogenic differentiation of human dental pulp stem cells using different induction protocols.

OBJECTIVE: An investigation on neuronal differentiation capacity of human dental pulp stem cells (DPSCs) was still lacking. In this study, two different neuronal induction protocols were investigated and compared. METHODS: The neuronal differentiation was induced using chemical or growth factor induction protocol. The differentiation was confirmed by the neurogenic mRNA and protein expression using polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Chemical-induced neuronal differentiation protocol promoted morphological change and ß3-TUBULIN protein expression. Though, SOX2, SOX9, and ß3-TUBULIN mRNA levels were not different compared with the control, indicating a defective differentiation. For growth factor induction protocol, the cells were exhibited neurite-like cellular process and positively stained with ß3-TUBULIN. In addition, the increase in intracellular calcium was noted upon NMDA stimulation, implying the neuronal function. A dramatic increased mRNA expression of neurogenic markers [SOX2, SOX9, ß3-TUBULIN, and gamma-aminobutyric acid (GABA receptors)] was noted as compared to the control. In addition, a remarkable increased expression of Notch signaling target gene, HEY1, was observed in growth factor-induced DPSCs derived neuronal-like cells compared with the control. CONCLUSION: These data indicate that growth factor induction method is a preferable protocol for neuronal differentiation by DPSCs.

IL-6 regulated stress-induced Rex-1 expression in stem cells from human exfoliated deciduous teeth.

OBJECTIVE: To investigate the effect of mechanical stress on stem cells from human exfoliated deciduous teeth (SHED). METHODS: Cells were stimulated with mechanical stress ranging from 0 to 2.5 g cm⁻² for 2 h in serum-free condition. The expression of stem cell markers was examined by polymerase chain reaction and immunocytochemistry. The molecular mechanisms of these effects were investigated by means of inhibitors and siRNA. RESULTS: Mechanical stress induced Rex-1 expression in a force-dependent manner. The upregulation of interleukin-6 (IL-6) was also observed. Application of neutralizing antibody against IL-6 and IL-6 siRNA could attenuate the mechanical stress-induced Rex-1 expression. In addition, the increase in mRNA levels of Rex-1 was observed after treating SHED with an exogenous IL-6. This inductive effect was attenuated by JAK inhibitor, suggesting the involvement of JAK signaling pathway. CONCLUSION: These results suggest the role of mechanical stress in the regulation of stemness via IL-6-Rex-1 interaction.