Exosomes Derived From Human CD34~+ Stem Cells Transfected With MiR-26a Prevent Glucocorticoid-induced Osteonecrosis Of The Femoral Head By Promoting Angiogenesis And Osteogenesis

ObjectiveThe objective of this study was to explore whether exosomes(Exos)derived human CD34~+stem cells transfected with miR-26a could prevent the progression of glucocorticoid(GC)-induced osteonecrosis of femoral head(ONFH).MethodsWe constructed the human CD34~+stem cell line overexpressed miR-26a by the lentiviral transfection and extracted exosomes from serum-free medium.qPCR and fluorescence microscopy were used to measure transfection efficiency in human CD34~+stem cells and exosomes.Transmission electron microscopy(TEM),nanosight tracking analysis(NTA)and western blotting were used to identify exosomes.We further analyzed the effects of different exosomes on bone marrow stromal cells(BMSCs)and human umbilical vein endothelial cells(HUVECs)in vitro.Assays of Alizarin red staining,ALP staining and qPCR were used to evaluate osteogenesis-related activity.Transwell,scratching assay and tube formation experiment were used to analyze the angiogenic capacity of endothelial cells.Then,forty Sprague–Dawley rats of about 300 g were randomly divided into four groups:control group,model group,CD34~+-Exos group and miR-26a-CD34~+-Exos group.The rat model of GC-induced ONFH was established by intramuscular injection of methylprednisolone(MPS)at a dose of 20 mg/kg three times a week for three consecutive weeks.After each MPS injection,the intervention group was given 100μg exosomes(dissolved in 200μL PBS),and the control group and the model group were given the same dose of PBS.After 6 weeks,micro-CT scanning,HE staining and immunohistochemical assay were performed to observe results.ResultsIn vitro experiments demonstrated that CD34~+-Exos attenuated the inhibitory effects of GC on the migration and tube formation of HUVECs.In addition,miR-26a-CD34~+-Exos and CD34~+-Exos alone had almost the same effect on endothelial cells without statistical significance.Similarly,we have also demonstrated that miR-26a-CD34~+-Exos enhanced the depressed osteogenic differentiation capacity of BMSCs in vitro.Finally,in a GC-induced ONFH rat model,miR-26a-CD34~+-Exos increased the vascular density,maintained the trabecular bone integrity of femoral head,and inhibited the progression of ONFH.ConclusionsGC can inhibit osteogenic ability and damage local blood vessels in the femoral head area.miR-26a-CD34~+-Exos can promote the osteogenesis of BMSCs and angiogenesis of HUVECs,and prevent the occurrence and progression of GC-induced ONFH.