Dental papilla cells (DPCs) belong to precursor cells differentiating to odontoblasts and play an important role in dentin formation and reproduction. This study aimed to explore the changes and and involvement of mitochondrial respiratory function during odontogenic differentiation. Primary DPCs were obtained from first molar dental papilla of neonatal rats and cultured in odontogenic medium for 7, 14, 21 days. DPCs, which expressed mesenchymal surface markers CD29, CD44 and CD90, had the capacity for self-renewal and multipotent differentiation. Odontoblastic induction increased mineralized matrix formation in a time-dependent manner, which was accompanied by elevated alkaline phosphatase (ALP), dentin sialophosphoprotein and dentin matrix protein 1 expression at mRNA and protein levels. Notably, odontogenic medium led to an increase in adenosine-5'-triphosphate content and mitochondrial membrane potential, whereas a decrease in intercellular reactive oxygen species production and NAD/NADH ratio. Furthermore, odontogenic differentiation was significantly suppressed by treatment with rotenone, an inhibitor of mitochondrial respiratory chain. These results demonstrate that enhanced mitochondrial function is crucial for odontogenic differentiation of DPCs.

N-Acetyl-5-methoxytryptamine (melatonin, MT) at pharmacological concentrations promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; however, its role at physiological concentrations (1 pM–10 nM) remains unclear. We explored the effects of 1 pM–1 µM MT on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mitochondrial dynamics-mediated mechanisms. T he PDLSC phenotype was detected by flow cytometry and evaluated for three-line differentiation. Alkaline phosphatase activity assay and Alizarin red staining were used to evaluate osteogenic differentiation. Osteogenesis-related gene and protein expression levels were measured by quantitative reverse transcription -polymerase chain reaction and western blotting. Mitochondrial function assays were performed using reactive oxygen species, ATP and NAD+/NADH kits and molecular mechanisms of mitochondrial dynamics-related proteins were assessed by western blotting. Our results have shown that physiological MT concentrations induced differentiation of hPDLSCs and down-regulated osteopontin (OPN) and osteocalcin (OCN) expression levels, which were restored or even up-regulated by 1 µM MT (lowest pharmacological concentration). Compared to the osteogenic induction alone, this treatment decreased the intracellular ATP content, whereas the intracellular reactive oxygen species level and NAD+/NADH ratio were increased. Mitochondrial function- and dynamics-related protein expression levels were consistent with those of osteogenic genes following osteogenic induction and MT treatment of hPDLSCs at various physiological concentrations. Physiological MT concentrations inhibited the osteogenic differentiation of hPDLSCs and simultaneously altered mitochondrial function. These findings provide insights into the stem cell tissue engineering and functions of MT.

Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation (p < 0.05). In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.