| Objective:Previous studies have revealed that melatonin could promote osteogenesis and play an anti-osteoporosis role.However,how melatonin affects angiogenesis in the process of bone regeneration and what its effects are on osteoporotic bone defect are still unclear.This study was aimed to investigate the effect of melatonin on osteoporotic bone defect healing and its potential mechanism of action,with a view to finding an ideal approach for the increasing number of patients with osteoporotic bone defect who are difficult to treat in clinic.Methods:1.We evaluate the effects of melatonin on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)by CCK-8 cell proliferation assay,alkaline phosphatase(ALP)staining,and alizarin red S staining(ARS)staining.Subsequently,molecular and cell biological techniques such as qRT-PCR,Western blot,ELISA,and immunofluorescence staining were used to further investigate the pro-osteogenesis effect of melatonin and its potential influence on angiogenesis.2.The effect of melatonin on angiogenesis was further investigated by cell scratch wound assay,transwell migration assay,and matrigel tube formation assay.3.We first established the rat model of osteoporotic bone defect,and the rats were treated with melatonin or placebo.The effect of melatonin therapy on bone defect healing in osteoporosis rats were evaluated by micro-CT,HE staining,Masson staining,immunohistochemical staining,immunofluorescence staining,sequential fluorescence labeling and biomechanical test.Results:1.(1)The result of CCK-8 cell proliferation assay showed that the proliferation level of rat BMSCs in the melatonin-treated groups were obviously elevated compared with the control group.(2)The results of ALP staining and ARS staining showed that melatonin treatment enhanced the osteogenic differentiation ability of rat BMSCs.(3)The result of qRT-PCR showed that the mRNA expression levels of osteogenesis-related markers(ALP,OCN,and RUNX2)and angiogenesis-related marker(VEGFA)in the melatonin-treated groups were obviously elevated compared with the control group.(4)The results of Western blot,ELISA,and immunofluorescence staining showed that melatonin treatment up-regulated the protein expression levels of the above markers(ALP,OCN,RUNX2,and VEGFA).2.The results of cell scratch wound assay,transwell migration assay,and matrigel tube formation assay showed that melatonin treatment couldn’t stimulate angiogenesis in a direct manner,but melatonin treatment significantly enhanced BMSCs-mediated endothelial cell migration and angiogenesis in vitro.3.(1)The results of the micro-CT,HE staining,Masson staining,and sequential fluorescence labeling showed that the area of bone trabecula,the amount of new bone formation,and the bone mineral apposition rate in the bone defect area were significantly increased in the melatonin-treated groups compared with the control group.(2)The result of immunohistochemical staining showed that melatonin treatment up-regulated the protein expression levels of osteogenesis-related marker(OCN)and angiogenesis-related markers(VEGFA and CD31).(3)The result of immunofluorescence staining showed that the number of type H vessels in the melatonin-treated groups were significantly increased compared with the control group.(4)The result of biomechanical test showed that melatonin treatment increased bone strength of the defective tibia in osteoporosis rats.Conclusion:Melatonin could promote osteogenesis-angiogenesis coupling by up-regulating the expression of VEGFA,thus accelerating bone defect repair in osteoporosis rats.This finding is expected to provide a new perspective and idea for the study of bone regeneration,that is,the effective coupling of osteogenesis and angiogenesis should be attached great importance in the research of bone regeneration. |
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