Effect Of Melatonin On The Osteogenic Differentiation Of Human Dental Pulp Mesenchymal Stromal Cells

Objective Human dental pulp mesenchymal stromal cells(h DPSCs)were isolated and cultured from healthy human dental pulp tissues by the tissue block method.The effect of melatonin on the osteogenic differentiation ability of h DPSCs was evaluated at first from the cellular and molecular level in vitro experiments.Then,the effect of melatonin on the osteogenic differentiation ability and inflammatory cytokine levels of h DPSCs in an inflammatory micro-environment stimulated by lipopolysaccharide and the potential mechanism of melatonin were further evaluated,providing theoretical basis for the application of melatonin in periodontal tissue engineering in the future.Methods 1 The primary h DPSCs were obtained by separating and culturing the pulp tissue of the premolars extracted for orthodontic treatment or the third molars extracted without reservation value by the tissue block method.And the cells were subcultured by enzyme digestion.After subculture,the cell morphology was observed under the microscope carefully,and the cell surface antigen markers were identified by flow cytometry.2 The effect of melatonin on the osteogenic differentiation ability of h DPSCs was evaluated at first.The cytotoxicity and the effect on cell proliferation ability of melatonin were detected by Cell Counting Kit-8(CCK-8).The effect of different concentrations of melatonin on alkaline phosphatase activity in cells were quantitatively analyzed by alkaline phosphatase(ALP)detection kit.Then qualitative evaluation of the effect of melatonin on the osteogenic mineralization ability of cells was analysed by alkaline phosphatase and alizarin red S staining.The m RNA and protein expression of osteogenic differentiation related genes were detected by q PCR and western blot.3 And then the effect of melatonin on the osteogenic differentiation ability and inflammatory cytokine levels of h DPSCs in an inflammatory micro-environment was investigated.MTT assay was used to detect the effect of different concentrations of lipopolysaccharide on cell viability and ELISA was used to detect the effect of different concentrations of lipopolysaccharide on the level of inflammatory factors in cell supernatant.CCK-8 was used to detect the protective effect of melatonin on cells stimulated by lipopolysaccharide.The m RNA and protein expressions of osteogenic differentiation related genes in cells stimulated by lipopolysaccharide were detected by q PCR and western blot while the m RNA and protein expressions of inflammatory cytokines in cells stimulated by lipopolysaccharide were detected by q PCR and ELISA.The effects of melatonin on the m RNA and protein expressions of key factors of TLR4/NF-?B signaling pathway in cells stimulated by lipopolysaccharide were detected by q PCR and western blot.Results 1 The primary and P3 generation h DPSCs were observed under the inverted phase contrast microscope,and they were long spindle and fusiform cells.The flow cytometry results showed that the cells were positive for the MSC markers CD73 and CD44 while negative for the hematopoietic markers CD45 and CD34,which was consistent with the characteristics of human mesenchymal cells.2 The results of CCK-8 showed that melatonin had no obvious toxic and side effects on cell proliferation.The quantitative analysis of ALP activity showed that melatonin could significantly increase the alkaline phosphatase activity of cells,and the most significant improvement was achieved when the concentration of melatonin was 1?M(P<0.05).The results of alkaline phosphatase and alizarin red S staining showed that the expression of alkaline phosphatase and the formation of calcium nodules were increased in the melatonin group.The results of q PCR showed that the expression level of OCN,OPN,BMP-2 and Runx2 were all increased significantly in the groups which cells were exposed to melatonin when compared to the control groups,and the increase was most significant when the melatonin concentration was 1?M(P<0.05)while the western blot results showed that the protein expression levels of OCN and OPN in the 1?M melatonin-treated group were significantly increased(P<0.05).3 The MTT assay showed that lipopolysaccharide inhibited cell viability in a dose-dependent manner and the ELISA results showed that lipopolysaccharide increased the level of inflammatory cytokines in cell supernatant in a dose-dependent manner as well.CCK-8 results showed that melatonin could enhance cell proliferation activity inhibited by lipopolysaccharide.The results of q PCR and western blot showed that melatonin could improve the osteogenic differentiation of cells inhibited by lipopolysaccharide,and the effect was the most significant when melatonin concentration was 10?M(P<0.05).While the results of q PCR and ELISA showed that melatonin could reduce the levels of IL-6,IL-1? and TNF-? in the microenvironment and the reduction effect was most significant when the concentration of melatonin was 10?M(P<0.05).And the q PCR and western blot results showed that melatonin played an anti-inflammatory role by inhibiting the TLR4/NF-?B signaling pathway activated by lipopolysaccharide.Conclusion In conventional culture,melatonin can promote the osteogenic differentiation of h DPSCs,while in the inflammatory micro-environment,melatonin not only promotes the osteogenic differentiation of h DPSCs,but also exerts an anti-inflammatory effect by inhibiting the TLR4/NF-?B signaling pathway,and melatonin has a potential therapeutic effect on periodontitis.