Proteomic Study On Biocompatibility Of Domestic Porous Tantalum To MG63 Cells

Objectives Proteomic iTRAQ technique was used to screen differentially expressed proteins from porous tantalum materials and MG63 cells.ELISA,Western-blot and realtime fluorescent quantitative PCR were used to detect the expression of differentially expressed proteins.To elucidate the molecular mechanism of the effect of porous tantalum on the biological effect of osteoblasts,and to provide experimental and theoretical basis for the clinical application of porous tantalum materials.Methods 1 observation on the morphology and microstructure of domestic porous tantalum materials.2 Grouping of domestic porous tantalum materials and MG63 cells before and after culture;Blank group(MG63 cells and conventional medium culture),Tantalum extract group(MG63 cells and tantalum extract culture),Tantalum stents group(MG63 cells co-cultured with tantalum stents and conventional medium),Three groups of cells were observed by inverted phase contrast microscope.3 FITC-labeled ghost pen cyclic peptide staining was used to observe the composite culture of domestic porous tantalum and MG63 cells.4 CCK-8 assay was used to detect cell proliferation in the three groups.5 The iTRAQ quantitative experiment of proteomics was used to screen the differentially expressed proteins among the three groups.6 The effect of domestic porous tantalum on the secretion of different proteins in MG63 cells was determined by ELISA.7Western blot analysis of the effect of domestic porous tantalum on the expression of different proteins in MG63 cells;8.Real-time fluorescence quantitative PCR method was used to detect the effect of domestic porous tantalum on the mRNA expression of differential protein in MG63 cells.Results 1 The domestic porous tantalum support material is a sheet material with a diameter of 1.5cm and a thickness of 0.2cm.It is hard and dark gray in texture,with a bumpy and shiny surface.2 Cell morphology was observed by inverted phase contrast microscope: MG63 cells adhered to the wall in the blank group and tantalum extract group,showing a long fusiform shape,while the light-impermeable scaffold materials were surrounded by cell adherent growth in tantalum stent group.3 FITC-labeled ghostpen-ring peptide staining showed that MG63 cells grew on the surface and inside of porous tantalum material at the initial stage of culture.Cells adhered to the material scaffold and grew continuously.Over time,cells gradually covered the material surface completely,and the micropore inside the material was gradually filled.MG63 cells gradually grew from the surface of porous tantalum material to the inside.Finally,MG63 cells covered the whole material.Its package.4 CCK-8 method was used to detect cell proliferation.The results showed that there was no significant difference in the proliferation of MG63 cells between different groups at the same time point(P > 0.05);there was significant difference in cell proliferation with the prolongation of cell culture time at different time points(P < 0.05),indicating that domestic porous tantalum materials had no significant effect on the proliferation of MG63 cells,and the number of cells increased with the prolongation of time in the same group.5 Proteomics iTRAQ quantitative experiment screened three different proteins: alpha-2 macroglobulin,glutamyl transaminase 2,calcium binding protein A4.The expression of alpha-2 macroglobulin in tantalum extract group was 3.699 times higher than that in blank group,the expression of glutamyl transaminase 2 in tantalum extract group was 1.764 times higher than that in blank group,and the expression of calcium binding protein A4 in tantalum scaffold group was 1.622 times higher than that in blank group.6 The results of ELISA showed that at the same time,A2 M secretion in Tascaffold group was higher than that in blank group and Ta extract group(P < 0.05),A2 M secretion in Ta extract group was higher than that in blank group(P < 0.05),TGM2 secretion in Ta scaffold group was higher than that in blank group and Ta extract group(P< 0.05),and the difference was statistically significant(P < 0.05).The secretion of TGM2 in the extract group was higher than that in the blank group(P < 0.05);the secretion of S100A4 in the tantalum scaffold group was lower than that in the blank group and the tantalum extract group(P < 0.05);there was no significant difference between the tantalum extract group and the blank group(P > 0.05).7 Western-blot analysis showed that the expression of A2 M in Ta scaffold group was higher than that in blank group and Ta extract group(P < 0.05).The expression of TGM2 in Ta scaffold group was higher than that in blank group on the 5th,7th and 9th day(P < 0.05).The expression of TGM2 in Ta scaffold group was higher than that in other two groups(P < 0.05).The expression of S100A4 in Ta stent group was lower than that in other two groups(P < 0.05).On the 3rd,7th and 9th day,the expression of S100A4 in Ta stent group was lower than that in blank group(P < 0.05).8 Real-time fluorescence quantitative PCR results showed that the relative expression of A2 M mRNA in Ta scaffold group was higher than that in blank group and Ta extract group on the 7th and 9th day(P < 0.05).At four time points,the relative expression of A2 M mRNA in Ta extract group was higher than that in blank group(P < 0.05).On the 5th,7th and 9th day,the expression of TGM2 mRNA in Ta scaffold group was higher than that in blank group(P < 0.05).The relative expression of TGM2 mRNA was higher than that of blank group and tantalum extract group(P < 0.05).On the 5th,7th and 9th day,the relative expression of TGM2 mRNA in tantalum extract group was higher than that in blank group(P < 0.05),and the relative expression of S100A4 in tantalum scaffold group was lower than that in blank group and tantalum extract group(P < 0.05).The relative expression of S100A4 in the extract group was lower than that in the blank group(P < 0.05).Conclusions 1 The growth and proliferation of domestic porous tantalum cells were good after co-culture with MG63 cells in vitro,which confirmed that domestic porous tantalum had good biocompatibility.2 Proteomics iTRAQ quantitative experiment screened differentially expressed proteins before and after co-culture of domestic porous tantalum and MG63 cells.The expression of alpha-2 macroglobulin and glutamyltransferase A2 increased,while the expression of calcium binding protein A4 decreased.It was suggested that the threedimensional structure of domestic porous tantalum was conducive to cell adhesion,migration and mineralization,and had a good bone-promoting effect.Figure 34;Table 14;Reference 119.