Co-transplantation Of Bone Marrow Mesenchymal Stem Cells And Endothelial Progenitor Cells For Treating Steroid-induced Osteonecrosis Of The Femoral Head

Background:Osteonecrosis of the femoral head(ONFH)is mainly caused by ischemia of the femoral head.In addition to the destruction of the blood supply of the femoral head,steroid-induced ONFH also involves a reduction in the number of mesenchymal stem cells(MSCs)and endothelial progenitor cells(EPCs),a decline in osteogenic/angiogenic cell function,and an enhancement of the adipogenic differentiation ability of MSCs.Therefore,achieving the ideal osteogenesis,adipogenesis and angiogenesis in the femoral head is the key to the treatment of steroid-induced ONFH.The existing clinical strategies play a very limited role in the treatment of steroid-induced ONFH.Tissue engineering and stem cell transplantation have become new directions for the treatment of steroid-induced ONFH.It is known that the combined transplantation of MSCs and EPCs is significantly better than single cell transplantation in the treatment of bone defects.This study assumes that combined transplantation of MSCs and EPCs can also effectively promote the repair of steroid-induced ONFH.Objective:BMSCs and EPCs were used to be seed cells and carboxymethyl chitosan/sodium alginate(CMC/ALG)were used to be the cell scaffold,then,the CMC/ALG/BMSC/EPC composite graft were constructed.The purpose of this study was to explore its therapeutic effect on steroid-induced ONFH.Methods:1.Isolation,culture and identification of BMSCs and EPCs Gradient density centrifugation was used to separate BMSCs and EPCs.The function of BMSCs was identified by alkaline phosphatase(ALP),Alizarin Red S and Oil Red O staining.The function of EPCs was identified by tubule formation and lipid droplet phagocytosis experiments.The phenotypes of BMSCs and EPCs were detected by flow cytometry.2.The effect of co-cultured BMSCs/EPCs on cell osteogenic,adipogenic and angiogenic differentiationDevelopment of BMSC/EPC co-culture system by direct contact means.Using ALP staining,ALP activity experiment and RT-PCR to detect the osteogenic differentiation of co-cultured system;Using Oil Red O staining,Oil Red O quantitative experiment and RT-PCR to detect the adipogenic differentiation of co-cultured system;Using tube formation experiment and RT-PCR to detect the angiogenic differentiation of co-cultured system.3.Construction of CMC/ALG scaffold material and cell compatibility testThe CMC/ALG scaffold material was constructed by freeze-drying method.The structure of the material was observed by scanning electron microscopy(SEM).CCK-8 was used to detect the cytotoxicity of the scaffold material.The cell adhesion and vascularization of the material were detected by cell fluorescent labeling.ELISA was used to detect the secretion of osteogenic and angiogenic factors of BMSCs/EPCs in a three-dimensional culture environment.4.Construction of steroid-induced ONFH model and implantation of composite graftsThe rabbit steroid-induced ONFH model was constructed by injection of methylprednisolone and lipopolysaccharide,and the necrotic femoral head was detected by MRI,Micro-CT and H&E staining.On the basis of core decompression,CMC/ALG/BMSCs/EPCs were transplanted into the necrotic femoral head.5.Radiographical and histological evaluation of bone repairMicro-CT was used to detect bone repair;H&E staining was used to observe the percentage of empty lacunae in the femoral head;OCN,Adiponectin,and CD31 immunohistochemistry were used to detect the osteogenesis,adipogenesis and angiogenesis in the femoral head.Results:1.The isolated and cultured BMSCs can differentiate towards osteogenic and adipogenic cells,and express MSCs phenotype;EPCs can differentiate towards endothelial cells and express endothelial-related phenotypic.2.In vitro co-culture of BMSCs and EPCs by direct contact way can promote cell osteogenic differentiation,angiogenic differentiation,and inhibit adipogenic differentiation.3.CMC/ALG scaffold material has no cytotoxicity,which is beneficial to the adhesion,survival,osteogenesis and angiogenesis of BMSCs/EPCs.4.CMC/ALG/BMSCs/EPCs composite graft can promote the repair of steroid-induced ONFH by promoting bone formation,vascularization and reducing fat formation.Conclusion:In vitro co-culture of BMSCs and EPCs in direct contact way can effectively promote osteogenic and angiogenic differentiation and inhibit adipogenic differentiation;combined transplantation of BMSCs and EPCs can promote the repair of steroid-induced ONFH by promoting bone and blood vessel regeneration and reducing lipid formation.