Effects Of Mg-2Zn-0.5nd Alloy On The Adhesion And Intracellular BMP-2/FoxO1 Activity Of Rat Skeletal Muscle Cells And Its Molecular Mechanism

Objective:Mg alloys have been extensively studied in recent years due to their excellent properties,such as close density and elastic modulus to those of nature bone,high specific strength and biodegradation.However,the degradation of magnesium alloy in physiological environment is too fast,and the hydrogen produced during the degradation process hinders the healing of tissue,which limits its clinical application.One of the strategies to improve the corrosion resistance of magnesium alloys is to design new magnesium alloys.The institute of metal research,Chinese Academy of Sciences has developed a novel magnesium alloy,Mg-2Zn-0.5Nd alloy,whose microstructure and mechanical properties have been investigated.On the basis,we will further observe the biocompatibility and corrosion resistance of Mg-2Zn-0.5Nd alloy in vivo and in vitro.Rat skeletal muscle cells are commonly used in cell toxicity experiments,which have the advantages of easy culture,stable passage,rapid growth and so on.In this experiment we will study the effects of Mg-2Zn-0.5Nd alloy on adhesion of rat skeletal muscle,and further study its effects on BMP-2 and FoxO1activity in skeletal muscle of rats,and explore its molecular mechanism.At the same time,since the rat has the advantages of fast growth and good breeding performance,the Mg-2Zn-0.5Nd alloy was implanted into the muscle of the rats in vivo,and the corrosion of Mg-2Zn-0.5Nd alloy and the condition of muscle tissue were observed.Methods:This study was carried out in two aspects:in vivo and in vitro experiments.In vitro experiments,the rats were divided into 4 groups:control group,317L alloy group,Ti-6Al-4V alloy group and Mg-2Zn-0.5Nd alloy group.The control group used the glass,while other groups used the alloy materials as mentioned above.The rat skeletal muscle cells were cultured in 24 hole culture plate.Put different alloy into corresponding culture plate,after 2h,6h and 12h respectively,the adhesion rate was calculated.The rat skeletal muscle cells were inoculated with 6 hole culture plate,and the alloy material was placed into it,each group of cells were stained with fluorescence after 24h.According to the standard of international organization for Standardization?ISO10993-5:2009?,we prepared three kinds of extract liquid:317L alloy,Ti-6Al-4V alloy and Mg-2Zn-0.5Nd alloy.Rat skeletal muscle cells were cultured with different kinds of extract liquid.Cell proliferation was detected by CCK-8 assay after 1d,3d and 5d.We extracted the protein after culturing with the extract liquid for 24h,and determined the protein concentration.The expression of BMP-2,AKT,p-AKT,mTOR,p-mTOR,FoxO1,p-FoxO1,P38 and p-P38 were detected by Western blot,and the images were analyzed with Quantity One.SPSS19.0 statistical software was used for statistical analysis.The measurement data were expressed as mean±standard deviation,and the data were tested for normality and homogeneity of variance.The comparison between groups was analyzed by one-way ANOVA.The LSD-t test was used to compare each other.P<0.05 indicated that the difference was statistically significant.In vivo experiment:40 healthy male SD rats were divided into four groups:control group,317L alloy group,Ti-6Al-4V alloy group and Mg-2Zn-0.5Nd alloy group.Both hind limbs were selected as the experimental site,and 40 rats were divided into four groups.The 317L alloy group,Ti-6Al-4V alloy group and Mg-2Zn-0.5Nd alloy group were implanted with corresponding metal wires.After intraperitoneal anesthesia,the 50ml syringe needle was inserted into the limbs of the rats,and the materials were inserted into the muscle tissue along the needle tube.The concentration of serum Mg²⁺was measured in 2 and 4 weeks after operation.The peripheral tissue was removed after 4 weeks.Paraffin embedding,HE staining,and the tissue was observed under an optical microscope.Immunohistochemical staining,microscopic observation,and measured the average gray value of BMP-2 positive reaction products with Quantimet970 image analysis system.SPSS19.0 statistical software was used for statistical analysis.The measurement data were expressed as mean±standard deviation,and the data were tested for normality and homogeneity of variance.The comparison between groups was analyzed by one-way ANOVA.The LSD-t test was used to compare each other.P<0.05 indicated that the difference was statistically significant.The rats were sacrificed after 4 weeks,and the electron microscope was used to observe the corrosion degree.Results:In vitro experiment:The adhesion rate of Mg-2Zn-0.5Nd group was higher than other three groups?P<0.05?,and there were no significant differences among the control group,317L alloy group and Ti-6Al-4V alloy group?P>0.05?.Fluorescence microscopy showed that the skeletal muscle cells were attached to the surface of material in each group after 24h,and the cell adhesion number of Mg-2Zn-0.5Nd alloy group was higher than other three groups?P<0.05?.The results showed that Mg-2Zn-0.5Nd could promote the adhesion of rat skeletal muscle cells.Cell proliferation of Mg-2Zn-0.5Nd alloy group was significantly higher than other groups at 1day,3 day and 5 day?P<0.05?.There were no significant differences among the control group,317L group and Ti-6Al-4V alloy group?P>0.05?.The results showed that Mg-2Zn-0.5Nd could increase the proliferation of rat skeletal muscle cells.After culturing rat skeletal muscle cells with corresponding alloys for 24 hours,we found that BMP-2 in Mg-2Zn-0.5Nd alloy group was significantly higher than other groups?P<0.05?,and there were no significant differences among the control group,317L alloy group and Ti-6Al-4V alloy group?P>0.05?.There are a large number of FoxO1 in skeletal muscle,which has an effect on the adhesion and proliferation of skeletal muscle cells.In this study,we investigated the effects of Mg-2Zn-0.5Nd on the activation of FoxO1 protein.The expression of p-FoxO1 protein was increased in Mg-2Zn-0.5Nd alloy group and Ti-6Al-4V alloy group,but not in the 317L alloy group.There were no significant effects on the expression of t-FoxO1 in the 317L alloy group,Mg-2Zn-0.5Nd alloy group and Mg-2Zn-0.5Nd alloy group.t-mTOR protein was expressed in all groups,but the expressions of t-mTOR were not significantly different in the control group,317L alloy group,Ti-6Al-4V alloy group and Mg-2Zn-0.5Nd alloy group?P>0.05?.p-mTOR is the active form of mTOR,and the expression of p-mTOR were gradually enhanced in the four groups in turn,the control group,317L alloy group,Ti-6Al-4V alloy group and Mg-2Zn-0.5Nd alloy group.However,there were no significant difference between the normal control group,317L alloy group and Ti-6Al-4V alloy group?P>0.05?.There were significant differences between Mg-2Zn-0.5Nd alloy group and other three groups?P<0.05?.There was no significant difference in the expression of t-AKT in each group.p-AKT is the active form of AKT.p-AKT protein was significantly increased in the Mg-2Zn-0.5Nd group compared with 317L alloy or Ti-6Al-4V alloy?P<0.05?.These results suggested that Mg-2Zn-0.5Nd might affect rat skeletal muscle cells via AKT-mTOR pathway.In order to further study how the Mg-2Zn-0.5Nd alloy affect cell adhesion,the expression of P38 protein in each group was detected by Western blot method.The expression of p-P38 and t-P38 protein in 4 groups were not significantly different.These data suggested that the P38/MAPK pathway might not be involved in the effects of Mg-2Zn-0.5Nd on the adhesion of rat skeletal muscle cells.In vivo:The rats were sacrificed after 4 weeks,HE staining was used to observe surrounding tissue.The morphology of the muscle was good in control group,there was no tissue necrosis and local inflammatory cell.In the 317L alloy,Ti-6Al-4V alloy and Mg-2Zn-0.5Nd alloy group,there was no tissue necrosis,and the proliferation of multinucleated giant cells and fibroblasts was observed.After 4 weeks,The expression of BMP-2 in skeletal muscle satellite cells in control group,317L alloy group,Ti-6Al-4V alloy group and Mg-2Zn-0.5Nd alloy group was positive.According to the image analysis system,the positive expression of BMP-2 in skeletal muscle satellite cells of Mg-2Zn-0.5Nd group was more than that of normal control group,317L alloy group and Ti-6Al-4V alloy group,and the difference was statistically significant?P<0.05?.After 4 weeks of operation,the alloy was observed by scanning electron microscope,we found that the 317L alloy,Ti-6Al-4V alloy and Mg-2Zn-0.5Nd alloy were with good continuity.Uneven surface material could be seen on the surface of317L alloy.There was no obvious corrosion on the surface of Ti-6Al-4V alloy.The corrosion also occurred on the surface of Mg-2Zn-0.5Nd alloy.However,compared with 317L alloy,Mg-2Zn-0.5Nd alloy showed uniform corrosion degeneration.Conclusion:Mg-2Zn-0.5Nd could promote the adhesion of rat skeletal muscle cells through AKT-mTOR pathway and BMP-2 protein.At the same time,it has good cell compatibility.Mg-2Zn-0.5Nd could promote the expression of BMP-2 in skeletal muscle satellite cells in vivo,and had good biocompatibility.