Influence Of Expression Of ITGβ1/Vinculin/F-actin Signaling Pathway On MG63 Cells And Cell Proliferation In Platelet Lysate Combined With Domestic Porous Tantalum

Objectives To investigate the effect of platelet lysate(PL)and domestic porous tantalum scaffold constructs for the proliferation of MG63 cells and expression of ITGβ1/Vinculin/F-actin signaling pathway.Methods There are four experimental groups including Blank group(A):MG63 cells.PL with MG63 cells(B):3%,5%,7%,10% PL co-cultured MG63 cells(CCK-8 method was used to screen the optimal volume fraction group).Porous tantalum with MG63 cells(C):domestic porous tantalum scaffold co-cultured with MG63 cells.MG63 cells with PL and porous tantalum(D):PL and porous tantalum scaffold co-cultured MG63 cells.1 Scanning electron microscopy(SEM)was used to observe the surface morphology of domestic porous tantalum and PL-porous tantalum-MG63 cell complex.2 CCK-8 method was used to detect the proliferation of MG63 cells.3 Real-time fluorescence quantitative PCR(q PCR),immunocytochemical staining and Western-blot were used to detect The expression of ITGβ1,Vinculin,F-actin mRNA and proteinin MG63 cells.Results 1 MG63 cells resuscitation on the first day,the appearance is spherical,adherent growth after 24 h.The cells are elongated fusiform or polygonal,cytoplasm protrude from the cell.On the third day.The cells grow rapidly and larger,some pseudopods meet each other,the cells grow into pieces.The morphology is not easy to distinguish.2 Scanning electron microscopy(SEM)showed that tiny particles of 20-50μm were observed on the surface and cross section of porous tantalum scaffolds.The microstructures were uniformly distributed.The pore size was 400-600μm in diameter.The three-dimensional structures were interconnected.Electron microscopy showed that MG63 cells pseudopodia can be observed in the scaffold material,merge into pieces.The secretion of cell matrix covered in the structure of the surface and the gap.3 The results of CCK-8 study showed that PL of 7% in volume fraction can the most quickly promote the proliferation of MG63 cells,and screened as the PL group.With the prolongation of culture time,all the 4 groups of cells adhered、grew well and then stable amplification.The statistical analysis showed that the cell proliferation of PL group and porous tantalum group were better than that of A group,the difference was significant(P<0.05),but there was no difference between the B group with C group(P>0.05).Compared with other three groups,PL-porous tantalum group showed the best cell proliferation activity with statistical significance(P<0.05).4 Immunocytochemical staining showed that the expression of various factors in PL group and porous tantalum group was significantly higher than that in blank control group at the 5th day after MG63 cell culture,the difference was significant(P<0.05),but there was no difference between the B group with C group(P>0.05).The detection factor expression of PL-porous tantalum group was higher than the other three groups,the difference was statistically significant(P<0.05).5 Quantitative real-time PCR results showed that the m RNA expression of various detection factors in PL group and porous tantalum group was significantly higher than that in control group on the 5th day(P<0.05).The expression of F-actin and Vinculin in PL group were higher than that in porous tantalum group.The expression level of ITGβ1 in porous tantalum group was higher than that in B group,but the difference was not statistically significant(P>0.05).The m RNA expression of PLporous tantalum group was significantly higher than the A B C groups,the difference was statistically significant(P<0.05).6 Western-blot results showed that the expression of ITGβ1,Vinculin and F-actin protein in PL and porous tantalum group were higher than that in blank control group on the 5th day(P<0.05).The expression of ITGβ1,F-actin protein in the PL group was higher than that in the porous tantalum group.The expression of Vinculin protein in the porous tantalum group was higher than that in the PL group,but the difference was not statistically significant(P>0.05).The various factors protein expression of PL-porous tantalum group was significantly higher than the other groups,the difference was statistically significant(P<0.05).Conclusions PL and domestic porous tantalum material can synergistically promote the proliferation of MG63 cells,up-regulate the m RNA and protein expression of ITGβ1/ Vinculin/F-actin signaling pathway.The activation of this signaling pathway may contribute to the proliferation、differentiation and adhesion of MG63 cells.Thus,PL can be used as an auxiliary drug for bone graft repair materials to repair bone defects,it can promote bone ingrowth,and provide a scientific theoretical basis for PL combined with porous tantalum for the treatment of bone fracture and bone defect.