Effects Of Soluble Degradation Products Of Magnesium And Its Alloys On The Phenotype Of Vascular Smooth Muscle Cells

Under normal physiological condition,vascular smooth muscle cells（VSMC）are low growth and low synthesis contractile phenotype.When the abnormal microenvironment of the blood vessels（such as atherosclerosis）,the contraction of VSMC to a variety of phenotypic changes.Stent implantation is one of the main methods for the treatment of atherosclerosis.Magnesium based metal materials have become the representative materials for the new generation of vascular stents due to their excellent mechanical properties and biological safety.However,due to the rapid degradation of magnesium,a large number of degradation products have a significant impact on the vascular microenvironment,and thus affect the vascular tissue cells（especially VSMC）.Previous studies on the effects of magnesium degradation products on VSMC mainly focused on the study of cell viability,proliferation and apoptosis,and ignored the role of the phenotypic changes.In view of the significant plasticity of VSMC and the different phenotypes of VSMC are involved in the formation and development of arterial plaques to varying degrees,Using pure magnesium and AZ31 magnesium alloy extracts with contraction of vascular smooth muscle cells were co cultured,explored the possible mechanism on the cellular and molecular effects of magnesium based metal soluble degradation products on the phenotypic transformation of VSMC and the phenotype transformation mediated.Firstly,we used collagenase and trypsin compound enzymes digestion to obtain rat thoracic aortic smooth muscle cells.The method is simple and easy to operate,and has good repeatability,good cell viability and high purity.Secondly,the corrosion behavior of pure magnesium and AZ31 magnesium alloy in DMEM-F12 medium containing 20%FBS was investigated by means of SEM,polarization curve,EIS and ion content and pH of the extract.The results showed that the corrosion rate of pure magnesium was higher than AZ31 magnesium alloy,corrosion process produces large amounts of Mg²⁺ and AZ31 magnesium alloy pH is higher than pure magnesium after corrosion.However,the corrosion of pure magnesium is lamellar,the longer the time,the more smooth and delicate,the AZ31 magnesium alloy is the point corrosion,the longer the corrosion pits,the more uneven.Then,four kinds of extracts were used to treat vascular smooth muscle cells,and the changes of cell proliferation,migration and apoptosis were observed.The results showed that pure magnesium extract could significantly promote the growth of co cultured vascular smooth muscle cells,proliferation and migration of cells were increased,and the apoptosis was inhibited;extraction time,liquid ratio and the reaction was positively related to accounting for these cells.The extract of AZ31 slightly inhibited the proliferation and migration of vascular smooth muscle cells.ICC,QRT-PCR and WB were used to investigate the effect of the extract on the phenotype of co cultured VSMC.The results showed that the expression levels of Myh11,α-SMA,Smtn,Sm22a and so on in the cells of two kinds of materials were significantly decreased after treatment with the extracts of the kinds of materials,and the decrease depends on the extraction time and liquid ratio change,show that soluble magnesium based metal material degradation material weakening of vascular smooth muscle ability to maintain cell contraction type traits;on the other hand,a series of inflammatory cell phenotype genes（including Vcam1,ILIB,Ccl2,Ccl20）the expression level of the overall increase,showed that the extract treated vascular smooth muscle cell inflammatory phenotype was significantly enhanced.After comparing two kinds of magnesium based metal materials,We used MgS04（2.5,5,25,and 50 20%FBS）mmol/L containing DMEM-F12 medium to treat vascular smooth muscle cells.To investigate the effects of Mg²⁺ on the expression of contractile marker genes and proteins as well as inflammatory phenotype genes.The results showed that with the increase of Mg²⁺ content,the expression of VSMC reduced molecular marker gene was decreased and the expression of molecular marker gene was enhanced.It is suggested that the release of soluble Mg²⁺ from magnesium based metal corrosion may partly mediate the change of VSMC from contractile to inflammatory phenotype.Finally,we analyzed the effects of the extract and the single magnesium ion on the expression of two key transcription factors Myocardin and Klf4 in co cultured contractile VSMC.The results showed that the mRNA and protein levels of Myocardin,a promoter of the VSMC gene,were decreased with the treatment time,with the decrease of treatment time and the concentration of treatment;However,the expression of Klf4 was significantly increased.These results indicated that the release of magnesium ions from the magnesium based metal could induce the reversal of the expression of two transcription factors and the formation of the contractile VSMC to the inflammatory phenotype.