钛合金表面不同沟脊宽度对成纤维细胞的影响研究

Effects of ridge-modified titanium alloy surface on activity of fibroblasts

目的观察钛合金片(简称钛片)表面沟槽化修饰对成纤维细胞黏附、增殖及分化的影响,研究成纤维细胞在不同沟脊宽度(简称脊宽)钛片表面的生物学特点。方法制备脊宽分别为50、80、100、150和200μm的沟槽化修饰钛片,5个实验组沟宽均为50μm,沟深均为10μm;无沟槽的钛片作为对照组。将成纤维细胞接种各组钛片后,于不同时间通过扫描电镜(SEM)观察细胞的黏附状态,采用细胞增殖活性检测(CCK-8)和细胞免疫荧光染色检测细胞的增殖情况,通过黏着斑蛋白染色观察沟脊宽度对细胞黏附部位的影响,通过免疫荧光观察沟脊宽度对α-平滑肌肌动蛋白(α-SMA)表达的作用。结果SEM显示接种48 h后,细胞黏附于钛片的沟脊部位;脊宽较小组(50~150μm),细胞形态细长,并沿沟脊方向延伸生长。CCK-8提示不同脊宽对细胞增殖无显著影响,6组间差异无统计学意义(P>0.05)。细胞荧光染色显示细胞可顺沟脊方向有序排列,其中50μm组细胞长轴与沟脊的延伸方向一致性最好,6组间差异有统计学意义(P<0.05)。黏着斑蛋白荧光染色检测发现细胞黏着斑蛋白分泌集中于沟脊部位,半定量分析显示50μm组黏着斑蛋白分泌最多,6组间比较差异有统计学意义(P<0.05)。α-SMA检测结果显示脊宽对成纤维细胞的成肌分化有调节作用,其中50μm组α-SMA表达最强,6组间比较差异有统计学意义(P<0.05)。结论钛片表面沟脊修饰可对成纤维细胞的排列、黏附和成肌分化产生影响。当脊宽50μm时,细胞排列极性更强、黏附相关蛋白分泌更多、成肌分化趋势更显著。

Objective To characterize the biological activity of fibroblasts on the surface of titanium alloy sheets with different ridge widths by investigating the effects of ridge widths on the adhesion,proliferation and differentiation of fibroblasts.Methods Five groups of titanium sheets with ridge widths of 50μm,80μm,100μm,150μm and 200μm were prepared,with all the groove depths being 10μm.The titanium sheets with no ridges were taken as a control group.After fibroblasts were incubated on the sheets,states of their adhesion were observed by scanning electron microscopy(SEM)at different time points.CCK-8 cell proliferation test and immunofluorescence staining were used to observe proliferation and shape of the cells.The effects of ridge widths on adhesion of fibroblasts were evaluated by Vinculin immunofluorescence staining,and the effects of ridge widths on expression ofα-smooth muscle actin(α-SMA)by immunofluorescence.Results SEM showed that the cells adhered to the ridges on the titanium sheets 48 hours after inoculation.In the groups with smaller ridge widths(from 50μm to 150μm),the cells were slender in shape and grew along the ridge direction.CCK-8 indicated that different ridge widths had no significant effect on the proliferation of fibroblasts between the 6 groups(P>0.05).Immunofluorescence staining showed that the cells arranged in an orderly direction along the ridges;the long axis of the cells in the 50μm group showed the best consistency with the extending direction of the ridge,with significant differences among the 6 groups(P<0.05).The Vinculin test found that the secretion of cell adhesion protein was concentrated in the ridge and semi-quantitative analysis showed that the 50μm group had the most Vinculin secretion,with significant differences among the 6 groups(P<0.05).Theα-SMA test showed that the ridge width had a regulatory effect on the myogenic differentiation of fibroblasts,and the 50μm group had the strongest expression ofα-SMA,with significant differences among the 6 groups(P<0.05).Conclusions Modification of ridges on the surface of titanium sheets may affect arrangement,adhesion and myogenic differentiation of fibroblasts.The ridges of 50μm in width may lead to stronger polarized arrangement of fibroblasts,more secretion of adhesion-related protein and more pronounced myogenic differentiation of fibroblasts.