Exosomes Derived From Melatonin-modified Bone Marrow Mesenchymal Stem Cells Promote Osteogenesis Of Bone Marrow Mesenchymal Stem Cells

Objective To explore the effects of exosomes derived from melatonin(MT)-modified bone marrow mesenchymal stem cells on the osteogenesis of bone marrow mesenchymal stem cells,and to analyze its feasibility as a cell-free method to promote osteogenic treatment of osteoporosis.Methods Primary BMSCs were isolated and characterized by whole bone marrow adherent method.Exosomes derived from BMSCs(NC-Exos)and melatonin(MT)pretreated BMSCs(MT-Exos)were extracted by ultracentrifuge,and identified.The uptaking of Exos into BMSCs was observed under confocal microscope.BMSCs were cultured in osteogenic induction medium.BMSCs were treated with MT-Exos and NC-Exos,separately.BMSCs were treated with PBS as a blank control group.Alkaline phosphatase activity and alizarin red S staining(ARS)were employed to evaluate the osteogenic differentiation of BMSCs in each group.The mRNA and protein expression of alkaline phosphatase(ALP)and Runx2 were measured through RT-PCR and western blot assay,respectively.Western blot was used to detect the protein expression of Wnt1 and β-catenin in BMSCs osteogenic differentiation.Results BMSCs had a typical spindle-like morphology and had the ability to differentiate into three lines.The majority of exosomes was smaller than 150 nm in diameter and expressed CD9,CD63,and CD81.Exosomes were taken up by BMSCs.At 7 days after osteoinduction,compared with the NC-Exos group,MT-Exos group presented higher alkaline phosphatase activity(P <0.01),mRNA and protein expression of alkaline phosphatase and Runx2(P <0.05),and Wnt1 and β-catenin protein expression(P < 0.001).At 14 days after osteoinduction,compared with the NC-Exos group,MT-Exos group showed more red calcium nodules.Conclusion MT-Exos can significantly increase the expression of Runx2,ALP protein and mRNA,and promote osteogenic differentiation of BMSCs by activating Wnt1/β-catenin signaling pathway.These results indicate that MT-Exos has great potential as a new cell-free method in the treatment of osteoporosis,and provides a new idea for the treatment of osteoporosis.