Effects And Mechanism Of New Magnesium Alloys On Osteoblast Adhesion And Proliferation

Objective:（1）To study the effect and mechanism of new magnesium alloy on osteoblast adhesion;（2）To study the effect and mechanism of new magnesium alloy on osteoblast proliferation.Methods:（1）Preparation of magnesium alloy and titanium alloy leaching solution:Calcμlate the volume of cμlture medium required to prepare the leaching solution according to ISO10993-12 standard.Both magnesium alloy and titanium alloy need to be placed in an μltrasonic cleaner for 30 min before use.And high temperature and high pressure sterilization,and then placed in 10% fetal bovine blood MEM-α cell cμlture medium,respectively,at 37 ℃,5% CO2 incubator for 24 hours to prepare magnesium alloy,titanium alloy extract.The leaching solution was diluted with MEM-α to four concentrations of 25%,50%,75%,and 100%,respectively.（2）To study the effect and effect of the new magnesium alloy on the adhesion of osteoblasts: After the mouse MC3T3-E1 cells were cμltured to the third passage,the new magnesium alloy was investigated by staining with rhodamine-containing phalloidin,DAPI and alkaline phosphatase.and titanium alloys on MC3T3-E1;the effects of fibronectin,type I collagen,integrin α2,and FAK on the adhesion of new magnesium alloys and titanium alloys in the process of osteoblast adhesion were determined by Western blot and quantitative PCR;by Western blot The expression of key proteins in the FAK-Aktm TOR signaling pathway was determined by the method,and the specific inhibitor of the FAK-Akt-m TOR signaling pathway,Wortmannin,was added to the magnesium alloy extract to further study the effect of the FAK-Akt-m TOR signaling pathway on osteoblasts.effects of biological behavior.（3）To study the effect and mechanism of the new magnesium alloy on the proliferation of osteoblasts: determine the effect of the new magnesium alloy and titanium alloy on the proliferation of osteoblasts by CCK-8method;determine the new magnesium alloy and titanium alloy by Western blot and quantitative PCR Influence of FAK and CLUT3 in the process of osteoblast proliferation;the expression of key proteins of ERK signaling pathway was determined by Western blot method,and the specific inhibitor of ERK signaling pathway SCH772984 was added to the magnesium alloy extract to further To study the effect of ERK signaling pathway on the biological behavior of osteoblasts.Resμlts:（1）The effect and mechanism of the new magnesium alloy on the adhesion of osteoblasts:（1）The staining resμlts of phalloidin containing rhodamine,DAPI and alkaline phosphatase showed that compared with titanium alloy,the new magnesium alloy can promote MC3T3-E1 The adhesion of MC3T3-E1 cells also increased with the increase of the concentration of the extract,which was statistically significant（P<0.05）.（2）The protein and m RNA expressions of fibronectin,collagen type I,integrin α2,FAK were determined by Western blot and quantitative PCR.The resμlts showed that compared with the titanium alloy extract group,the histone and m RNA expressions of the new magnesium alloy extract were higher than those of the titanium alloy extract group.Significantly increased,with statistical significance（P<0.05）.（3）The expression of m TOR m RNA during the adhesion process of MC3T3-E1 cells was detected by quantitative PCR and the protein expressions of pAKT and p-m TOR were detected by Western blot.The resμlts showed that compared with the MEM-α group,the related genes and The protein expression was increased;after the addition of Wortmannin,a specific inhibitor of the FAK-Akt-m TOR signaling pathway,the adhesion of the new magnesium alloy extract to MC3T3-E1 cells was reversed.（2）The effect and mechanism of magnesium alloy on osteoblast proliferation:（1）MC3T3-E1 cells were cμltured with different concentrations of new magnesium alloy extract and titanium alloy extract for 1,3,5,and 7 days.The proliferation activity of MC3T3-E1 cells was detected by the method.The resμlts showed that the new magnesium alloy leaching solution promoted the proliferation of MC3T3-E1 cells better than the titanium alloy leaching solution（P<0.05）.The proliferation activity of MC3T3-E1 cells increased with the immersion The concentration of the extract increased with the increase of the concentration of the extract,and reached a peak on the 5th day of the 50% concentration of the extract,which was statistically significant（P<0.05）.Proliferative activity decreased.（2）Western blot and quantitative PCR were used to detect the expression of FAK and GLUT3 protein and m RNA during the proliferation process.The resμlts showed that the new magnesium alloy extract coμld significantly promote the expression of FAK and GLUT3 protein and m RNA,with statistical significance（P<0.05）.（3）The expression of ERK m RNA during the adhesion process of MC3T3-E1 cells was detected by quantitative PCR and the protein expressions of p-ERK was detected by Western blot.The resμlts showed that compared with the MEM-α group,the related genes and the protein expression increased;after adding SCH772984,a specific inhibitor of the ERK signaling pathway,the effect of the new magnesium alloy extract on promoting the proliferation of MC3T3-E1 cells was reversed.Conclusion:（1）The new magnesium alloy is better than titanium alloy in promoting the adhesion of MC3T3-E1,and promotes the adhesion of MC3T3-E1 through the FAK-Akt-m TOR signaling pathway.（2）The effect of the new magnesium alloy in promoting the proliferation of MC3T3-E1 is better than that of the titanium alloy,and it promotes the proliferation of MC3T3-E1 through the ERK signaling pathway.