| Objectives To study the effects of the domestic porous tantalum scaffold modified by RGD peptide on the adhesion,growth and proliferation of human MG-63 osteoblast,and to explore the mechanism of COL-I,Integrinβ1 and FAK cell signaling pathway in cell growth and proliferation.To provide theoretical basis for the clinical application of enhanced surface bioactivity of domestic porous tantalum scaffold materials.Methods 1.The Morphological characteristics of domestic porous tantalum materials were observed under gross and scanning electron microscopy;2.The growth and proliferation of MG-63 cells were observed under the phase contrast microscope;3.The porous tantalum modified by RGD polypeptide were prepared and co-cultured with MG-63 cell;4.Groups of experiment: Porous Tantalum Group with RGD polypeptide: Porous tantalum scaffolds modified by RGD peptide were co-cultured with MG-63 cells;Porous Tantalum Group: Porous tantalum scaffolds were co-cultured with MG-63 cells;Blank Group: MG-63 osteoblasts were cultured alone;5.The adhesion of MG-63 cells on the porous tantalum scaffold was observed by inverted phase contrast microscope and SEM;6.The growth and proliferation of MG-63 cells in each group were detected by CCK-8;7.The expression of COL-I,Integrinβ1 and FAK protein in MG-63 cells were detected by immunocytochemical staining;8.The expression of COL-I,Integrinβ1 and FAK protein in MG-63 cells were detected by Western-blot;9.The expression of COL-I,Integrinβ1 and FAK m RNA in MG-63 cells were detected by RQ-PCR.Results 1.The gross appearance of Tantalum stent material is disc shape sheet,the color is gray hard and texture is hard;The surface of Tantalum stent material is rough and surface is luster;Uniform distribution of porous tantalum stent,a sponge,as small as a tip;SEM showed abundant micropores evenly,inside and outside the hole between Unicom presents three-dimensional porous structure,small space beam is microporous structure;2.MG-63 cells were observed under the inverted phase contrast microscope daily.Most of the cells were long spindle at the early stage of inoculation,and a small number of cells were triangular and round.With the prolongation of culture time,the number of cells increased,and the morphology of them was similar to that of long spindle or polygonal stellate.In the later stage of culture,cytoplasm was rich and full of shape,fused and aggregated to form monolayer cells,which covered the bottom of the bottle;3.CCK-8 assay: MG-63 cells continue to multiply,the OD value will continue to rise.The proliferation of MG-63 cells in porous tantalum with RGD polypeptides group at all time points was significantly different from that in the porous tantalum group and the blank group(P<0.05).The cell proliferation activity of porous tantalum with RGD polypeptide group was significantly higher than that in the porous tantalum group and the blank group.Compared with the blank group,the difference between the porous tantalum group and the blank group was statistically significant(P<0.05).The cell proliferation activity in the porous tantalum group was higher than that in the blank group;4.The SEM results showed: with the co culture time,MG-63 cells gradually attachment and adhesion on surfaces of porous tantalum scaffolds,and then extended pseudopodia grow into internal pore of porous tantalum,and mutually connected pieces,until completely cover the entire extracellular matrix of porous tantalum stent;5.The results of immunocytochemical staining showed that:On the fifth day,COL-I,Integrinβ1 and FAK were expressed in different degrees in the cell membrane and pulp of MG-63 cells of each group,which showed brown yellow granules.Statistical analysis showed that the expression of Integrinβ1and FAK in porous tantalum with RGD polypeptide group was significantly higher than that in porous tantalum group and blank group,and the difference was statistically significant(P < 0.05).The expression of COL-I in the porous tantalum with RGD polypeptide group was higher than that in the porous tantalum group and the blank group,but the difference was not statistically significant(P > 0.05);6.Western-blot detection showed that: The expression of COL-I and Integrinβ1 in MG-63 cells of porous tantalum with RGD polypeptide was significantly higher than that in porous tantalum group and blank group,and the difference was statistically significant(P < 0.05);7.RQ-PCR detection showed that:The expression of COL-I,Integrinβ1 and FAK m RNA in MG-63 cells of porous tantalum group with RGD polypeptide was significantly higher than that in porous tantalum group and blank group,and the difference was statistically significant(P < 0.05).Compared with the blank group,the expression of COL-I,Integrinβ1 and FAK m RNA increased significantly in the porous tantalum group,and the difference was statistically significant(P < 0.05).Conclusions 1 The biocompatibility of domestic porous tantalum modified by RGD polypeptide was improved,which was suitable for the growth of MG-63 cells on the porous tantalum scaffold;2.The domestic porous tantalum modified by RGD peptide has obvious cell adhesion;3.Domestic porous tantalum modified by RGD polypeptide can promote the expression of adhesion protein Integrinβ1 in MG-63 cells,and activate COL-I,Integrinβ1 and FAK signal transduction pathways,and promote MG-63 cell adhesion,growth and proliferation. |
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