Silver Nanoparticle-Loaded TiO₂ Nanotubes Promote Osteo-Immune Response Via Autophagy Of Macrophages And Scavenging Of Reactive Oxygen Species

The endosseous biomaterials used in bone regeneration are often impaired by host response.Especially macrophage-related immune responses,play a central role in the bone healing process.To take advantage of the immune responses,fabricating osteo-immunomodulatory biomaterials is a promising strategy.In this study,we investigated the influences of silver nanoparticle-loaded TiO₂ nanotubes（Ag@TiO₂-NTs）on macrophages behavior and osteo-immune microenvironment.We found that the surface modification provided important cues for macrophages behavior such as cell adhesion,shape and polarization.Ag@TiO₂-NTs are able to polarize macrophages into M2 phenotypes and create an osteo-immune microenvironment.Next,we explored the mechanisms between nanotopography and macrophages polarization.We found Ag@TiO₂-NTs have prominent ROS scavenging capabilities.However,autophagy are able to inhibit ROS production,so we assumed that Ag@TiO₂-NTs are capable to activate autophagy in adherent macrophages,and then scavenge ROS within macrophages,therefore modulating the macrophages polarization to create an osteo-immune microenvironment.To test our hypothesis,we use qPCR,western blot and ELISA to evaluate M1/M2 makers such as iNOS,TNF-α,ARG and TGF-βexpression,and autophagy level such as p62,Beclin-1 and LC3B.After that we evaluated the ROS production in cells.Results showed that macrophages grown on Ag@TiO₂-NTs expressed more M2 makers（ARG,TGF-β）,less M1 makers（iNOS,TNF-α）,higher level of autophagy and few ROS production than control groups.To further affirm our hypothesis,we used3-Methyladenine and rapamycin to regulate the level of autophagy,and found that macrophages with activated autophagy level tend to polarize into M2phenotypes.Then,we evaluated the osteo-immune environment constructed by Ag@TiO₂-NTs in vitro and in vivo,the co-culture model stimulated MC3T3-E1 cells to express osteogenic gene（Runx2,ALP,OCN and OPG）and produce extracellular matrix deposition.And the animal experiments demonstrated that Ag@TiO₂-NTs have stimulated new bone formation by creating an osteo-immune microenvironment.In summary,our study demonstrated that Ag@TiO₂-NTs are able to polarize macrophages into M2phenotypes and modulate osteo-immune microenvironment,and we clarified that this property results from the promoted autophagy level and the enhanced reactive oxygen species scavenging within macrophage.