［摘要］ 目的：通过观察精氨酸甘氨酸天冬氨酸（Arg-Gly-Asp polypeptedes，RGD）多肽表面修饰多孔钽对MG63成骨样细胞-多孔钽结合界面形态变化以及相关成骨因子表达的影响，评价RGD修饰多孔钽对钽骨界面骨整合所起的重要作用。方法：直径10 mm、厚3 mm的多孔钽片通过表面吸附法分别将3种质量浓度的RGD溶液（1 g/L、5 g/L、10 g/L）吸附于多孔钽表面。取第2代MG63成骨样细胞与钽RGD复合培养成为成骨细胞/钽/RGD复合物，分为4组：未修饰Ta组（对照组），1 g/L成骨细胞/Ta/RGD组，5 g/L成骨细胞/Ta/RGD组及10 g/L成骨细胞/Ta/RGD组。扫描电子显微镜观察多孔钽及复合物外观特征、成骨细胞与Ta/RGD界面的相互作用，测定成骨细胞在Ta/RGD材料上的黏附率，免疫细胞化学染色检测各组成骨蛋白骨钙蛋白（osteocalcin，OC）、纤维连接蛋白（fibronectin，FN）及丝状肌动蛋白（filamentous actin, F-actin）的表达。结果：扫描电子显微镜观察，成骨细胞在RGD修饰后多孔钽界面铺展并向孔隙内伸延，分泌细胞外基质覆盖于多孔钽微孔内及界面。24 h、48 h及72 h各组成骨细胞在Ta/RGD材料界面的黏附率均高于未修饰组（对照组），其中5 g/L成骨细胞/Ta/RGD组在48 h的黏附力最强［(68.07±3.80) vs. (23.40±4.39)，P<0.05］。免疫细胞化学染色发现，成骨细胞与Ta/RGD复合培养48 h成骨细胞OC、FN及F-actin的阳性表达均高于未修饰组，其中5 g/L成骨细胞/Ta/RGD组高于10 g/L组及1 g/L组［OC：(18.08±0.08) vs. (15.14±0.19)，P<0.05；(18.08±0.08) vs. (14.04±0.61)，P<0.05。FN：(24.60±0.98) vs. (15.90±0.53)，P<0.05；(24.60±0.98) vs. (15.30±0.42)，P<0.05。F-actin：(29.20±1.31) vs. (24.50±1.51)，P<0.05；(29.20±1.31) vs. (16.92±0.40)，P<0.05］。细胞骨架蛋白F-actin呈丝状分布于胞质，沿细胞胞突纵向排列，其蛋白表达高于OC及FN。结论：RGD多肽有利于成骨细胞在多孔钽界面黏附、铺展及细胞骨架蛋白的重组，从而促进成骨细胞多孔钽界面改建及骨整合。
Objective： To investigate the effects of the Arg-Gly-Asp polypeptedes (RGD) peptides-modified porous tantalum surface on osteoblasts morphology and expressions of osteogenesis factors, and to evaluate RGD peptides promotes junctura ossium of tantalumbone interface in vivo. Methods: RGD peptides of different concentrations (1 g/L, 5 g/L, and 10 g/L) were loaded to porous tantalum slices with a diameter of 10 mm and a thickness of 3 mm by physical absorption. The 3rd generation of MG63 cells were co-cultured with tantalum and divided into 4 groups: Ta-cells (control) group, 1 g/L cells/Ta/RGD group, 5 g/L cells/Ta/RGD group, and 10 g/L cells/Ta/RGD group. Porous tantalum compo-sites and osteoblasts-tantalum interface were observed by scanning electron microscopy. The adhesion rate of osteoblasts was detected and immunocytochemistry was used to detect the expressions of filamentous actin (F-actin), osteocalcin (OC) and fibronectin (FN). Results: The scanning electron microscope (SEM) revealed that osteoblasts distributed on the surface of porous tantalum and secreted extracellular matrix on outside and inner of micro-pores. The osteoblasts adhesion rate on porous tantalum modified with RGD was higher than that in the unmodified porous tantalum at the end of 24, 48, and 72 hours. The best adhesion effect was got in 5 g/L cells/Ta/RGD group at hour 48 ［(68.07±3.80) vs. (23.40±4.39), P<0.05］. The results of immunocytochemistry showed that the expressions intensity of F-actin, OC and FN in osteoblasts on porous tantalum modified groups with RGD were stronger than that in the unmodified groups, and the expressions of 5 g/L cells/Ta/RGD group were significantly higher than those in the 10 g/L group and 1 g/L group ［OC: (18.08±0.08) vs. (15.14±0.19), P<0.05; (18.08±0.08) vs. (14.04±0.61), P<0.05. FN: (24.60±0.98) vs. (15.90±0.53), P<0.05; (24.60±0.98) vs. (15.30±0.42), P<0.05. Factin: (29.20±1.31) vs. (24.50±1.51), P<0.05; (29.20±1.31) vs. (16.92±0.40), P<0.05］. Correspondingly F-actin in osteoblasts was showed in longitudinal arrangement, and the expressions intensity was stronger than those OC and FN. Conclusion: The RGD peptides is beneficial to enhance adhesion of osteoblast, spreading and reorganization of cytoskeleton on porous tantalum surface and improve the interface morphology, further promoting osteoblasts-tantalum conjunctive interface osseointegration.
