The Interaction Between Biodegradable Mg-Nd-Zn-Zr Alloy And Macrophage

Biodegradable Mg-based alloys are promising candidates for use in biomedical applications such as cardiovascular stent or scaffold due to favorable mechanical properties,biodegradability,and good biocompatibility.However,foreign body response(FBR)is one of most important adverse effects for Mg-based alloy applicaition in clinic.Macrophage,as an innate immune cell,has two isoforms—pro-inflammatory type(M1)and anti-inflammatory type(M2).The former is activated by exogenous material to induce an inflammaory response,while the latter is regulated by exogenous/endogenous factors to produce an anti-inflammatory response for tissue repair.Due to the plasticity of macrophage,it also plays an important role in FBR.In addition,one of important reasons why biomaterials failure after implantation is an inflammatory response triggered by M1 macrophage.As it was known that the degradable products from Mg-based alloy composite of magnesium salts,OH^-and hydrogen,few knows,however,the effects of biodegradable Mg-based alloys on macrophage.In addition,the metabolism of degradable particles(DPs)of Mg-based alloy is still to be elucidated.In this study,we used a biodegradable Mg-Nd-Zn-Zr alloy(detonated JDBM)to in vitro analysis the interaction of Mg-based alloy and macrophage by 4aspects as follow:1.The bioadapatibility of JDBM extract on macrophage.2.The potential mechanism by which JDBM inhibited the inflammation of macrophage.3.The immunoregulation of JDBM on macrophage.4.The metabolism and phagocytotic pathway of macrophage on the degradable products of JDBM.The bioadapatibility of JDBM extract on macrophage firstly were firstly studied and results demonstrated the various concentration of extracts(10%extract,20%extract,50%extract,and 100%extract)lead to diverse effects.Specifically,the low concentration extracts(10%extract and 20%extract)had no impact on the cytoxicity,morphology and function(migration,proliferation,differentiation and phagocytic capacity,etc)of macrophage,while the rest concentrations(50%extract and 100%extracts)resulted in partial or complete damage of above mentioned phenomenons due to an apoptotic or necrotic dependent cytotoxicity.Interestingly,our study showed an anti-inflammatory effect of extract on the lipopolysaccharide(LPS)-induced macrophage and noted the Mg²⁺concentration of extracts(not alkalinity)was major cause of above results.Together,these results offered an experimental foundation to explore the bioeffects of Mg-based alloy on the macrophage in depth.Secondarily,based on the anti-inflammatory effect of Mg-based alloy,this study explored the potential molecular mechanism by which extracts inhibited LPS induced inflammatory response.It was found that the Mg²⁺derived from 15%extract inhibited the LPS-induced tumor necrosis factor(TNF-?)and interleukin(IL)-6 expression but did not result in cytoxicity.Mechanically,Mg²⁺effectively inhibited the activity of LPS-induced Toll like 4(TLR-4)-Myeloid differentiation factor 88(MYD88)-Nuclear factor kappa beta(NF-k B)/Mitogen-activated protein kinase(MAPK)and the overexpression of LPS-triggered mitogen-activated protein kinase(ROS).However,Mg²⁺did not inhibit LPS bind to TLR-4 receptor but activated Transient receptor potential melastatin7(TRPM7)-phosphatidylinositol3-kinase(PI3K)-AKT1 signaling pathway to suppress the LPS inflicted inflammation.Furthermore,the anti-inflammatory capacity of Mg²⁺had to rely on its constant existence.Together,these results elucidated the molecular mechansim over the anti-inflammatory ability of Mg-based alloy.Then,in terms of the anti-inflammatory effect of Mg-based alloy,this study explored the immunoregulation of extract to vascular smooth muscular cells(VSMCs)function by macrophage.Firstly,the effects of 5%extract and 15%extract on the polarization and expression of VSMCs relative cytokines—e.g.,matrix metalloproteinase(MMP),Platelet derived growth factor(PDGF),transforming growth factor(TGF)and vascular endothelial growth factor(VEGF),etc—of macrophage were analysed.The results showed that Mg²⁺of extracts reversed the tendency of LPS-induced M1 polarization and inhibited these cytokines expression,while induced M2 polarization.Then,the immunoregulatory effects of extract on VSMCs function were detected with indirect method(conditional medium,CM and Transwell).It was found that the extract inhibited the effects of LPS-induced of macrophage on the migration,phenotype switch and proliferation of VSMCs but did not resulted in cytoxicity.Together,these results suggested that Mg-based alloy could regulated VSMCs function via immunoregulation of macrophage.Finally,because Mg-based alloy will be degraded into micro/nano-scale degradable products(DPs)in vivo,which are not as same as extracts in physicochemical characteristics,it is essential to explore relationship between macrophage and DPs.This study revealed that DPs resulted in an apoptotic dependent cytoxicity on macrophage in a dose manner.Additionally,by means of transmission electron microscope(TEM)and microscopy observation,we found no larger than 10?m of DPs could be internalized into cell by heterophagy instead of autophagy and kept into the phagolysosome for degradation.Finally,it was found that the DPs slightly activated a ROS-induced inflammatory response due to the possible endoplasmic reticulum(ER)stress.Together,these results showed the Mg-based alloy degradable behavor and metabolism in the macrophage.In a nutshell,this study systematically disclosed the bioadapatibility of Mg-based alloy on macrophage.Then,based on the anti-inflammatory effect of Mg-based alloy,this study disclosed an anti-inflammatory mechanism of Mg-based alloy on the LPS-induced inflammation.These studies indicated the degradable products of Mg-based alloy could inhibit inflammation and attenuated the impact of FBR.Next,in terms of anti-inflammatory capacity of Mg-based alloy,this study explored the effects of Mg-based alloy to VSMCs by immunoregulation of macrophage in the context of LPS stimulation,which demonstrated a unique advantage for Mg-basd cardiovascular stent.Finally,this study elucidated the degradable pathway and metabolism of DPs within macrophage,which enlarged the knowledge over the metabolism of biodegradable Mg-based alloy in vivo.Altogether,this study systematically analyzed the interaction between Mg-based alloy and macrophage in vitro and all of results offered new theories and guidelines for biodegradable Mg-based alloy application in the future.