The MTOR/ULK1 Signaling Pathway Mediates The Autophagy-Promoting And Osteogenic Effects Of Dicalcium Silicate Nanoparticles

BackgroundDicalcium silicate nanoparticles is a kind of bioglass material with good biological activity and biocompatibility,which has been gradually applied in the field of biology,and has been used as bone substitute material and important implant coating material.However,there is still a lack of systematic studies on the osteogenic effect and potential osteogenic mechanism of dicalcium silicate nanoparticles in vitro and in vivo.ObjectiveTo determine whether dicalcium silicate nanoparticles has osteogenic effect in vivo and in vitro;To investigate the mechanism of dicalcium silicate nanoparticles osteogenesis effect in vivo and in vitro.Materials and MethodsDicalcium silicate nanoparticles was characterized by SEM and EDS.The SD rat unilateral cranial defects were used as animal model,and mice bone marrow mesenchymal stem cells(BMSCs)was used to establish a vitro osteoblast model.The animal and cell models were treated with dicalcium silicate nanoparticles respectively.The osteogenic effect and its underlying mechanism of dicalcium silicate nanoparticles in vivo and in vitro was determined by micro CT,immunohistochemistry,alizarin red staining and Western Blot.Results1.Dicalcium silicate nanoparticles appeared as irregular crystals with an average diameter of about 100 nm,and had some degree of agglomeration in culture medium,saline and pure water.2.The dicalcium silicate nanoparticles could be safely applied in vivo without significant effects on survival rate,body weight and local tissue inflammation in rats.3.In the rat in vivo cranial defect model,micro-CT results and bone fluorescence double-staining results showed promotion of new bone regeneration at the bone defect.4.Dicalcium silicate nanoparticles could be taken up by BMSCs into the cells without causing significant decrease in cell viability of BMSCs or causing significant apoptosis.It also caused cell cycle transition from G1 phase to S phase,and promoted cell proliferation of BMSCs,as well as promoted their mineralization.5.In vivo experiments,immunohistochemical results showed that the expression levels of autophagy-associated protein LC-3 and osteogenic protein BMP2 were increased when zinc oxide nanoparticles were applied to skull defects.Dicalcium silicate nanoparticles also promoted the expression of osteogenic proteins in vitro,and promoted the formation of autophagic vesicles and the expression of osteogenic proteins in BMSCs.6.Inhibition of cellular autophagy in BMSCs with 3-MA inhibited the level of mineralization,and promotion of autophagy in BMSCs with RAPA promoted the mineralization of BMSCs,while the use of 3-MA partially inhibited the level of mineralization and the expression of osteogenic-associated proteins.7.Dicalcium silicate nanoparticles promoted the expression of mTOR/ULK1 pathway proteins and Wnt/β-Catenin,and 3-MA inhibited the expression of mTOR/ULK1 pathway proteins and Wnt/β-Catenin.ConclusionDicalcium silicate nanoparticles had good biocompatibility both in vitro and in vivo,and did not cause significant cytotoxicity or apoptosis.In vitro experiments have shown that dicalcium silicate nanoparticles promoted the cell cycle transition from G1 to S phase in BMSCs and promoted cell proliferation.Dicalcium silicate nanoparticles promoted new bone formation and mineralization,as well as the expression of osteogenic-related proteins in both in vivo and in vitro cellular models of cranial defects.Dicalcium silicate nanoparticles induced the formation of autophagic vesicles in BMSCs,and both in vivo tissue sections and in vitro experiments showed that dicalcium silicate nanoparticles induced cellular autophagy,and inhibition of autophagy in vitro resulted in down-regulation of mineralization and osteogenesisrelated protein expression.This indicated that dicalcium silicate nanoparticles could induce cellular autophagy promote osteogenesis in BMSCs.BMSCs treated with dicalcium silicate nanoparticles could able to activate the activation of autophagyrelated pathway proteins mTOR/ULKl and osteogenic pathway Wnt/β-Catenin.The expression levels of mTOR/ULK1 and Wnt/β-Catenin were inhibited by the use of 3MA.Therefore,dicalcium silicate nanoparticles were able to induce cellular autophagy through the mTOR/ULK1 pathway to promote the osteogenesis of BMSCs.