| Objective: Dental pulp stem cells(h DPSCs)is commonly used as bone tissue engineering seed cells,this experiment is intended to explore melatonin effects on the proliferation and osteogenesis differentiation h DPSCs and related regulatory mechanism.Materials and methods:(1)h DPSCs were extracted by tissue block adhesion method;Their surface antigens were detected by flow cytometry and combined with verification of osteogenic and lipogenic bidirectional differentiation ability to identify the cells.(2)The obtained h DPSCs were cultured in complete medium containing different concentrations of melatonin(0,1 μM,5 μM,10 μM,25 μM,50 μM,100 μM,250 μM,500 μM,1000 μM).CCK-8(Cellcountingkit-8)assay and scratch test were used to preliminatively analyze the effects of different concentrations of melatonin on the proliferation and migration of h DPSCs,and further clarify the appropriate concentration gradient of melatonin in subsequent osteogenesis induction experiments.(3)The cells were cultured with complete medium,osteogenesis induce medium(OIM),OIM+1 μM melatonin,OIM+10 μM melatonin,OIM+100 μM melatonin,and OIM+500 μM melatonin,respectively.The effect of melatonin on the osteogenic differentiation of h DPSCs was determined by alkaline phosphatase(ALP)staining and activity assay,alizarin red staining and calcium nodule assay,RT-q PCR analysis of osteogenic-related gene expression and Western Blot analysis of osteogenic-related protein expression levels.(4)RNA sequencing(RNA-Seq)was used to screen for possible signalling pathways involved in melatonin-promoted osteogenic differentiation of h DPSCs,and the addition of inhibitors of this pathway was further combined with Western Blot and early and late osteogenic phenotypes to elucidate the potential molecular mechanisms involved in melatonin-promoted osteogenic differentiation of h DPSCs.Results:(1)In this study,h DPSCs were successfully isolated from the pulp of orthodontic meiotic teeth or healthy third molar,and positive expressions of CD90 and CD44 and negative expressions of CD34 and CD45 on the surface of h DPSCs were detected by flow cytometry.14 days after osteogenesis induction,calcium nodules could be observed under the microscope by alizarin red staining.Lipid droplet formation was observed by oil red O staining microscope after 3 weeks of lipid induction.The results showed that h DPSCs with multidirectional differentiation ability could be successfully extracted from healthy pulp.(2)CCK-8 results showed that the proliferation ability of h DPSCs was significantly inhibited at three time points after co-culture when the melatonin concentration was above 500 μM.When melatonin concentrations were 100 μM and250 μM,the proliferation capacity of h DPSCs was significantly up-regulated.Scratch test further confirmed that 100 μM melatonin promotes the migration of h DPSCs best.(3)In the process of osteogenesis induction,the results of RT-q PCR showed that the h DPSCs induced by 100 μM melatonin produced the most significant up-regulation trend of osteogenesis related genes in different concentration groups.Further Western Blot results showed that the expression levels of several osteoblast-related proteins were up-regulated with the intervention of melatonin,and the expression levels of all proteins were the highest when the melatonin concentration was 100 μM.The ALP activity detection and alizarin red staining showed that the ALP activity and calcium salt deposition of h DPSCs were increased to varying degrees under the intervention of a certain concentration of melatonin,and melatonin showed the most ideal early and late osteoblast phenotypes under the concentration of 100 μM.(4)Bioinformatics analysis of RNA-Seq showed that multiple differential genes were enriched in osteogenic signaling pathways such as MAPK signaling pathway and PI3K/AKT signaling pathway.The addition of PI3 K pathway inhibitor LY294002 inhibited the expression levels of p-AKT protein.The results of alkaline phosphatase activity and alizarin red staining showed that the promotion effect of melatonin on early and late h DPSCs osteogenesis was also inhibited.Conclusions: At the concentration of 100 μM,melatonin can effectively enhance the proliferation and migration of h DPSCs,and significantly promote the osteogenic differentiation of h DPSCs.The promotion of melatonin in the osteogenic differentiation of h DPSCs is related to PI3K/AKT signaling pathway. |
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