Simvastatin Promotes Osteogenesis And Angiogenesis Through Mediating Immunology Of Senescent Macrophages

As a kind of bone implant material,titanium and its alloys have been widely used in clinical practice,and have a high implant success rate for young individuals,but still have a high risk of failure for the elderly.The main reason is that the osteogenic activity in the elderly is compromised,resulting poor osseointegration between the implant and bone tissue.As the main effector cells of the immune response,macrophages play an important role in the process of osseointegration.However,the decreased autophagy level of senescent macrophages leads to the imbalance of homeostasis,which impairs their timely transformation from pro-inflammatory M1 to pro-healing M2 phenotype,resulting in a chronic inflammatory microenvironment around the implants and therefore inhibiting the process of osseointegration.In this study,simvastatin is proposed to be used as a drug to promote autophagy of senescent macrophages to rebuild intracellular homeostasis,which in turn promote the timely transformation of macrophages from M1 to M2 phenotype.M2macrophages can create an immune microenvironment conducive to osteogenesis and angiogenesis,accelerate the process of osseointegration,and improve the implantation success rate of titanium implants for elderly individuals.In the present work,we firstly established senescent models of macrophages,endothelial cells and bone marrow mesenchymal stem cells.Then,we investigated the effects of different concentration of simvastatin on the biological behaviour of senescent macrophages and their mediated immune microenvironment on osteogenesis and angiogenesis.Finally,we explored the feasibility of loading simvastatin on titanium surface to mediate the function of senescent macrophages.The specific research results are as follows:（1）When simvastatin concentration was lower than 0.1μM,the proliferation and activity of senescent macrophages increased with the increase of simvastatin concentration,and the oxidative stress level within the cells decreased,while the cells were polarized to pro-healing M2 phenotype.With the increase of drug concentration,the level of cellular autophagy was increased.High concentration of the drug（100μM）induced apoptosis of senescent macrophages.（2）The supernatant obtained by stimulating senescent macrophages with different concentrations of simvastatin could enhance the migration,NO synthesis and angiogenesis of senescent endothelial cells,and reduced their oxidative stress level.When the concentration of simvastatin was 0.1μM,the supernatant showed the best effect on angiogenesis of senescent endothelial cells.（3）When senescent bone marrow mesenchymal stem cells were incubated with the supernatant derived from senescent macrophages stimulated by simvastatin of different concentrations,the secretion of type I collagen and the level of extracellular matrix mineralization were enhanced,while the level of oxidative stress was reduced.The optimal concentration of simvastatin in promoting osteogenic differentiation of senescent bone marrow mesenchymal stem cells was 0.1μM.（4）The chitosan/gelatin coating could be used as a sustained release layer for simvastatin on the surface of titanium implants.Proliferation and activity of senescent macrophages were closely related to drug loading.When the drug loading on titanium surface was 4.18566×10^-5mg/cm²,the senescent macrophages showed the highest proliferation rate.In conclusion,simvastatin is an effective and safe drug for promoting autophagy of senescent macrophages and thus promoting their transformation from pro-inflammatory M1to pro-healing M2 phenotype,creating a favorable immune microenvironment for osteogenesis and angiogenesis.The drug can also be loaded onto titanium surface through chitosan/gelatin coating and the loading can promote the functions of senescent macrophages.