Porous Silica Nanoparticles Loading BMP-2 Plasmid Promote Osteoblast Differentiation Through Autophagy

The defects of oral and maxillofacial bone tissue caused by trauma,infection,tumor,etc.,seriously affect the physical and mental health of patients.In the studies of bone formation promoted by nanomaterials,our group demonstrate that porous silica nanoparticles loading BMP-2 plasmid promote osteogenesis in vivo,indicating that these nanoparticles have intrinsic biological activity.However,the intracellular fate and the mechanism(s)involved remains to be elucidated.In this study,the effects of autophagy on MC3T3-E1 cells differentiation promoted by porous silica nanoparticles will be investigated by experiments in vitro,which provided an experimental basis for the study of the bone formation mechanism promoted by these nanoparticles.Polyethylenimine-modified porous silica nanoparticles(PPSNs)were synthesized by an ethyl ether emulsion approach.Scanning electron microscope images and transmission electron microscope images showed that the PPSNs were dendritic,with porous structure and good dispersibility.The size of PPSNs was 110-230 nm.The positive charge of PPSNs was determined by dynamic light scattering,which could theoretically be combined with negative charge of the plasmids.The effects of PPSNs on apoptosis of MC3T3-E1 cells were detected with flow cytometry.It was found that PPSNs caused cells apoptosis in a concentration-dependent manner.But the cell survival rate was still above 80%when reached a higher concentration than control group,suggesting their good biocompatibility.In the ELISA experiment,MC3T3-E1 cells were transfected with PPSNs,BMP-2 plasmid(pBMP-2),PPSNs loading BMP-2 plasmid(PPSNs/pBMP-2).The content of BMP-2 protein in pBMP-2 group was not detected in serum.It was considering that the plasmid was digested by lysosomes after entering the cells.Therefore,the experiment was divided into three groups,including a control group and experimental groups(PPSNs group,PPSNs/pBMP-2 group).Next,the intracellular autophagosomes were observed by transmission electron microscopy.A number of autophagosomes could be detected in the experimental groups,which were higher than the control group.Acridine Orange staining was used to mark the structures of acid vesicle organelles.Microscopy images showed that there were more acid vesicles in the experimental groups than the control group.Western Blot observed the changes of autophagy-related protein levels,indicating that the conversion rate form LC3-I to LC3-II increased in the experimental group and the PPSNs/pBMP-2 group's increased significantly.These results confirmed that PPSNs and PPSNs/pBMP-2 stimulated the process of autophagy in MC3T3-E1 cells.Finally,we detected the expression of osteogenic related genes ALP and Coll of groups treated with autophagy inhibitor,3-Methyladenine.The results of Real-Time PCR showed the expression of ALP and Coll decreased in the autophagy inhibitor-treated group.But in the PPSNs/pBMP-2 group it was still higher than control group,which mainly indicated that both PPSNs stimulated autophagy and pBMP-2 expression could promote osteoblasts differentiation.In a summary,PPSNs and PPSNs/pBMP-2 could promote osteoblast differentiation through autophagy.