Research On The Pathogenesis Mechanism Of Several Kinds Of Cells In Aseptic Loosening Of Artificail Joint

The aseptic loosening of artificial joint prosthesis is one of the main causes of the failure of joint replacement,which mainly caused by the effect of various kinds of cells and cytokines and the subsequently osteolysis.However,the pathogenesis mechanism is not yet fully understood.In a mouse calvarial osteolysis model,the level of LC3 puncta in the osteoblast was upregulated in wear particles-treated mice,which suggesting that the wear particles could induce the increase of autophagy in osteoblasts.To verify this finding,the osteoclast cells was treated with wear debris directly in vitro.The expression of autophagy marker protein LC3 was detected by western blot,the level of LC3 puncta was observed by immunofluorescence and the autophagosome and autolysosome were observed by transmission electron microscopy.All the results confirmed that wear particles markedly increased the autophagy levels in osteoblasts.As osteoblast apoptosis is one of the critical causes of osteolysis and various studies have shown that autophagy and apoptosis are closely related,we explored the effect of autophagy in the process of the stimulating of wear debris on osteoblasts.Flow cytometry assay showed that the wear particles could increase the apoptosis of osteoblasts in a dose-dependent manner.However,when cotreated cells with autophagy inhibitor 3-MA/siATG5 and wear particles,the apoptosis induced by particles was decreased.These results indicating that autophagy was involved in the upregulation of wear debris-induced osteoblast apoptosis.In the study of molecular mechanism,the data obtained by autophagy inhibitor and interfering RNA suggesting that the increase of autophagy by wear particles was through ERN1/MAPK8 signaling.Further,autophagy can promote osteoblast apoptosis by upregulating the expression of BAX.In animal experiments,inhibition of autophagy can reduce the wear debris-induced osteoblast apoptosis and the function inhibition in osteoblast,and ameliorate osteolysis.By regulating autophagy level in osteoblasts represents a new treatment strategy for the prosthesis aseptic loosening.Osteocytes are the most abundant cells in the skeletal system and can regulate bone resorption and bone formation independently.Previously,little attention was focused on the role of osteocytes in the pathogenesis of aseptic loosening.So in the present study,we have explored the role of osteocytes in the pathogenesis of the disease.We first confirmed that the osteocyte supernatant can inhibit the osteoclastogenesis from mouse bone marrow macrophages(BMMs),however,when the osteocyte cotreated with wear debris,the inhibition effect of supernatant on osteoclast differentiation was attenuated.Further studies showed that wear particles reduced the secretion of IFN-(3 from osteocytes.By functional simulation and suppression experiments,the downregulation of IFN-β played the role in the enhancement of osteoclastogenesis.In the study of the mechanism,it was found that autophagy was involved in the decrease of IFN-β in osteocytes.Downregulation of autophagy could partially restore the inhibition effect of osteocyte supernatant on osteoclastogenesis.This study suggests a new mechanism for the pathogenesis of aseptic loosening.Previously,we found that the expression of endoplasmic reticulum stress(ER stress)marker protein was upregulated in membranes from patients with aseptic loosening.Considering that the fibroblast was one of the major cell types in the interface membrane,we tested the effects of endoplasmic reticulum stress on the pathogenesis of aseptic loosening in vitro and in vivo.In the clinical interface membrane and animal models,endoplasmic reticulum stress marker protein were significantly increased.Wear particles upregulated the expression of ER stress marker protein and its downstream protein XBP1 s in fibroblasts.Further studies showed that ER stress/XBP1s were involved in the upregulation of RANKL expression in fibroblasts that induced by wear particles.In vivo,the inhibition of ER stress/XBP1s reduced wear particles-induced osteoclast formation and decreased osteolysis.In conclusion,the upregulation of RANKL mediated by ER stress/XBP1s pathway in wear debris treated fibroblasts promoted osteolysis,and the inhibition of ER stress/XBP1s pathway may be a new approach in treating aseptic loosening.