钛表面贻贝蛋白包被对人真皮成纤维细胞粘附和增殖的影响

摘要

目的:观察人真皮成纤维细胞在两种贻贝足丝蛋白提取物Usun-afix和BD CELL-TAKTM试剂包被钛表面的粘附和增殖状况,研究贻贝蛋白包被法是否能够用于经皮种植体表面处理.方法:分别制备光滑钛片和两种贻贝蛋白包被的钛片样本,选取P3代人真皮成纤维细胞接种到3种培养基底上:①不做处理的光滑钛表面(对照组);②Usun-afix包被的钛表面;③BD CELL-TAKTM包被的钛表面.MTT法检测1、3、5d细胞在3种钛表面的增殖数量;利用DiI-AcLDL荧光染色结合MTT法检测15、30 min和1、2、4h时间点细胞的粘附状况.结果:培养1、3、5d,成纤维细胞在3种表面活性均较好,3组之间无明显统计学差异(P＞0.05);DiI-AcLDL荧光染色显示,在培养15、30 min和1h各时间点,Usun-afix和BD CELL-TAKTM包被钛表面粘附细胞数目明显多于光滑钛表面,在1、2、4h各时间点,两包被组与对照组相比有更好的细胞伸展形态;不同时间点MTT检测显示,两包被组在接种15、30min和1、2h各时间点的吸光度值均明显高于对照组(P＜0.05),在4h时,虽两包被组OD值比对照组略高,但无统计学差异(P＞0.05);两包被组在各个时间点的促粘附效果相似,差异均无统计学意义(P＞0.05).结论:成纤维细胞接种早期(＜4 h),可更容易粘附到两种贻贝足丝蛋白提取物Usun-afix和BD CELL-TAKTM试剂包被的钛表面,而贻贝蛋白对成纤维细胞的增殖能力没有明显的促进效果.%AIM; To investigate the effect of titanium surfaces coated with Usun-afix and BD CELL-TAKTM rnon the proliferation and adhesion of human skin-derived fibroblasts. METHODS: Human skin-derived fibroblasts were seeded on three different titanium surfaces: group 1, bare smooth titanium (control group) ; group 2, titanium surface coated with Usun-afix; and group 3, titanium surface coated with BD CELL-TAKTM.The proliferation effects were evaluated by MTT assays in 5 days of culture . The adhesion effects were measured by DiI-AcLDL staining and MTT assays when cells were allowed to attach for 15 min,30 min, 1 h, 2 h and 4 h. RESULTS: Cell proliferation results indicated that there was no significant difference among the samples at day 1,3,5 (P > 0. 05). Cell-adhesion analyses revealed that cell attachment rates of groups 2 and 3 were higher than those of control group (P < 0. 05) at 15 min,30 min, 1 h, 2 h time points, however there was no significant difference at 4 h time point (P >0.05). There was no significant difference between groups 2 and 3 at any time point (P >0. 05). Dil-AcLDL staining results showed that the morphologies of cells grown on protein-coated surfaces were better at 1 h,2 h,4 h time points compared with those of control group. CONCLUSION: Human skin-derived fibroblasts can adhere to titanium surface coated with mussel adhesive protein better than to normal surface within 4 hours after seeding. And these mussel adhesive proteins have no positive effect on cell proliferation.