Cd41-deficient Exosomes From Non-traumatic Femoral Head Necrosis Tissues Impair Osteogenic Differentiation And Migration Of Mesenchymal Stem Cells

Background:Non-trauma osteonecrosis of the femoral head(ONFH)is a common and disabling orthopedic disease with a high incidence in young adults aged 20-50 years.Glucocorticoid use is a common and well-known cause for ONFH,however,the exact mechanism of Glucocorticoid-induced ONFH is still unknown.Thus,there is no effective treatment for this disease.In recent years,stem cell transplantation has been used to treat ONFH,and has achieved a certain effect.However,the osteogenic differentiation and migration ability of transplanted MSCs is reduced,which results in a high failure rate of the treatment.In addition,we found that the disease continued to progress when the causal factors were removed.Therefore,it is speculated that the necrotic bone tissue of the femoral head was releasing some signals to impair the restorative effect of normal MSCs in bone tissue and the transplanted MSCs.Exosome is a common extracellular vesicle with function of signaling delivery.Thesignal molecules included in the exosome are more stable and the range of signaling delivery is more extensive due to its double-layer membrane structure,and therefore,the influence of this delivery approach is stronger than other delivery approaches.However,there are reports about that ONFH-exos cause ONFH through damaging the osteogenic differentiation and migration of MSCs.So,we explored the role of ONFH exosomes from the levels of cells,human tissues and animal models in ONFH,and made an attempt to explore the mechanism involved in this process.Aims:Our study aimed to investigate the effect of ONFH exosomes(ONFH-exos)on osteogenic differentiation and migration of MSCs,and prove the pathogenic effect of ONFH-exos in vivo.Next,our study aimed to investigate the changed protein spectrum of ONFH-exos,find out the key molecule involved in the disease process and figured out the pathogenesis of ONFH bone exosomes.At last,this study aimed to verify the osteogenic differentiation and migration repair of normal bone tissue exosomes on dexamethasone-treated MSCs,as well as the therapeutic effect on ONFH.Methods:Exosomes were extracted from bone tissue via ultracentrifugation and identified by transmission electron microscopy,nanoparticle tracker analysis and exosome markers measured by Western Blotting.S-D ratswere treated with ONFH-exos,and the effects of this exosome were examined by micro-CT and histological sections.Primary human mesenchymal stem cells(HMSCs)and C3H10T1/2 cells were treated with ONFH-exos,and the osteogenic differentiation of the two cells was detected by Western blotting and alizarin red assay.Migration of the cells was detected by wound healing assay.Cell proliferation was detected by CCK-8 and flow cytometry and apoptosis was detected by flow cytometry.Then,the protein spectrum of ONFH-exos was analyzed by itraq-based TOF-LC-MS/MS to identify the differential proteins,and the key molecules were selected out through bioinformatics analysis.The role of the molecule and its related pathways were verified via Western blotting and wound healing assay.Finally,micro-CT and histological examination were used to verify the preventive effect of normal exosomes on ONFH in S-D rats.Results:ONFH-exos could cause femoral head necrosis in S-D rats through the tail vein injection.ONFH-exos were taken up by HMSCs and C3H10T1/2cells,and inhibited the expression of osteogenetic markers,such as alkaline phosphatase(ALP),bone gla protein(BGLAP),collagen Ⅰ(COL Ⅰ),osteopontin(OPN),and inhibited calcium deposits.It was found that ONFH-exos could inhibit the migration of HMSCs and C3H10T1/2 cells,through the wound healing assay.By proteomic analysis,64 significantlyaltered differential proteins were figured out,and finally,integrin α2b(CD41)was selected out as the key molecule.It was also demonstrated that the lack of CD41 in ONFH-exos resulted in a decrease in osteogenic differentiation and migration of HMSCs and C3H10T1/2 cells,where CD41/integrin β3-Fak-PI3k/Akt-Runx2 pathway played an important role.At last,our study also revealed that CD41-rich exosomes from normal bone tissue can repair the damaged osteogenic differentiation and migration of HMSCs and C3H10T1/2 cells against glucocorticoid,and injection of CD41-rich exosomes can prevent ONFH in S-D rats.Conclusion:Necrotic bone tissue of the femoral head can inhibit the differentiation and transplantation of mesenchymal stem cells by releasing exosomes,resulting in high failure rate of stem cell transplantation and irreversibility of the disease.CD41 in the exosomes might play a vital role in this process where CD41/integrin β3-Fak-PI3k/Akt-Runx2 pathway was involved.CD41-rich normal exosomes can effectively prevent ONFH.