Effect Of MSC-derived Exosomes On Healing Of Diabetic Bone Defects

BackgroundWith the economic development and changes in people’s lifestyle,the incidence of diabetes has rapidly increased year by year.Concomitantly,diabetes and other types of diseases have brought severe challenges to the treatment of various specialties.Studies have found that 87%of diabetic patients will prolong the healing time after fracture,the risk of reunion will increase 3.4 times.These disorders bring physical and psychological trauma to patients,and additional economic burden to family and society.How to treat bone fractures in patients with diabetes and to shorten the fracture healing time of diabetic patients has become an urgent problem to be solved in clinical practice.Mesenchymal stem cell-derived exosomes(MSC-exosomes)have become new hotspots for tissue repair.It has been found that stem cell-derived exosomes have great potential in tissue regeneration.The use of MSC conditioned media in a diabetic rat bone defect model can promote tissue growth and angiogenesis in the tibial fracture model.However,it is still not clear which factors play a major effect.Therefore,we speculate that exosomes from MSC conditioned medium may be one of the main reasons that can promote the healing of diabetic fractures.We extracted MSC derived exosomes and explored whether exosomes can inhibit the negative effect of high glucose in vitro.A model of bone defect in diabetic rats was conduct to evaluate the ability of MSC-derived exosomes to accelerate bone healing in vivo.PurposeWe extract the exosomes derived from MSC in order to observe the therapeutic effects on bone-related cells with high-glucose environment in vitro.Then we perform diabetic bone defect model in rats to explore the mechanism of the promising healing ability in vivo.Methods1.MSC were extracted and cultivated by using whole bone marrow culture method.Morphological observation and flow cytometry were used to identify cell surface markers using microscope.2.Establishing a method for extracting exosomes of mesenchymal stem cells by using modified size exclusion chromatography.The characterization of exosomes were investigated by transmission electron microscope(TEM),nanosight and Western blot.3.Osteoblasts and mesenchymal stem cells are cultured under high glucose conditions in vitro.The extracted exosomes are co-cultured with the two types of cells.CCK-8 test and scratch test were used to examine the cell proliferation and migration ability.4.We construct a model of femur bone defect in type I diabetic rats and exosomes was given by in situ injection.We tested blood glucose changes,and used micro-CT to analyze the bone defect.Results1.The primary bone marrow mesenchymal stem cells were fibroblast-like spindle cells under the microscope.Flow cytometry showed that CD90,CD 105 were positive,and CD34,CD45 were negative.2.Transmission electron microscopy showed double capsule appearances.The size of exosomes derived from BMSCs ranged from 30 nm to 130 nm.Western blot results showed that the expression of GD9 and CD63 on the surface of exosomes was positive.3.MSC derived exosomes can promote stem cell self-proliferation(P<0.05)and migratory capacity(P<0.05)under high glucose conditions,but have no effect on the proliferation of osteoblasts(P>0.05)and migration ability.(P>0.05).4.MSC derived exosomes can accelerate bone defect healing in diabetic rats,promote bone formation,increase callus formation and bone density at the defect site.Conclusion1.We successfully extracted primaiy bone marrow mesenchymal stem cells and secreted exosomes from stem cells.2.MSC derived exosomes can enhance the proliferation and migration of mesenchymal stem cells under high glucose conditions,but have no significant effect on the proliferation and migration of osteoblasts.3.MSC derived exosomes can accelerate bone defect healing in diabetic rats.