精氨酸-甘氨酸-天冬氨酸多肽表面修饰多孔钽对软骨细胞黏附、增殖和分泌功能的促进作用

目的:研究精氨酸-甘氨酸-天冬氨酸(RGD)多肽表面修饰多孔钽(Ta)对软骨细胞黏附、增殖和分泌功能等生物学行为的影响,探讨RGD/软骨细胞/多孔钽复合物作为软骨组织工程支架材料修复软骨缺损、促进软骨再生的可行性。方法:通过物理吸附的方法将不同浓度RGD肽及Ⅱ型胶原(ColⅡ)吸附于多孔钽表面,分为Ta-RGD组、Ta-ColⅡ组、Ta-RGD/ColⅡ0.2 g·L^-1组、Ta-RGD/ColⅡ1.0 g·L^-1组、Ta-RGD/ColⅡ5.0 g·L^-1组、Ta-RGD/ColⅡ10.0 g·L^-1组及纯Ta组。分离培养新西兰幼兔关节软骨细胞,取第2代细胞种植于修饰前后多孔钽使其成为RGD/软骨细胞/多孔钽复合物;扫描电镜观察RGD/软骨细胞/多孔钽复合物形貌特征以及软骨细胞生长状态,沉淀法检测多孔钽修饰前后软骨细胞黏附率,MTT法检测多孔钽修饰前后软骨细胞的增殖水平,羟脯氨酸法测定软骨细胞分泌功能。结果:多孔钽经RGD修饰后各组软骨细胞黏附率均高于修饰前(P<0.05),其中黏附效果最好的是4 h的Ta-RGD/ColⅡ5.0 g·L^-1组,同时各组间黏附率比较差异有统计学意义(P<0.05)。扫描电镜下各组软骨细胞在修饰后的多孔钽表面生长状态良好,细胞在多孔钽表面及孔隙内生长,分泌细胞外基质覆盖于多孔钽表面;MTT检测,多孔钽经RGD修饰后各组软骨细胞增殖水平均高于修饰前(P<0.05),其中增殖效果最好的是13 d的Ta-RGD/ColⅡ1.0 g·L^-1组;羟脯氨酸检测,Ta-RGD/ColⅡ1.0 g·L^-1组羟脯氨酸水平高于其他各组(P<0.05)。结论:RGD多肽可有效加强软骨细胞在多孔钽表面及孔隙内的黏附及增殖,对软骨细胞的分泌有促进作用。

Promotion effect of RGD peptides-grafted porous tantalum on adhesion,proliferation and secretion of chondrocytes in vitro

Objective To study the effects of arg-gly-Asp (RGD) peptides coated porous tantalum(Ta) on the adhesion, proliferation and secretion of chondrocytes, and to investigate the feasibility of RGD/chondrocyte/porous tantalum composites for chondral defect repair and regeneration. Methods RGD peptides with different concentrations were loaded to the porous tantalum(Ta) surface by physical absorption and divided into 7 groups:Ta-RGD, Ta-ColⅡ, Ta-RGD/ColⅡ0.2 g·L^-1, Ta-RGD/ColⅡ 1.0 g·L^-1, Ta-RGD/ColⅡ5.0 g·L^-1, Ta-RGD/ColⅡ10.0 g·L^-1 and Pure Ta groups.The articular chondrocytes from immature rabbits were cultured and the 2rd generation chondrocytes were seeded onto porous tantalum.The morphology of RGD/chondrocyte/porous tantalum composites was observed by scanning electron microscopy(SEM).The adhesion rate of chondrocytes was detected by precipitation process, the proliferation level of chondrocytes was measured by MTT assay, and the changes of hydroxyproline level of chondrocytes were checked. Results The adhesion rate of chondrocytes in the porous tantalum modified with RGD peptides was higher than that in the unmodified porous tantalum(P<0.05).The best cell adhesion effect was gained in Ta-RGD/ColⅡ5.0 g·L^-1 group at 4 h and there were significant differences between various groups (P<0.05).The SEM found that the chondrocytes growed best on the surface and inner of porous tantalum and secreted extracellular matrix.The Results of MTT showed that the proliferation level of chondrocytes in the porous tantalum modified with RGD peptides was higher than that in the unmodified porous tantalum(P<0.05) and the best effect was gained in Ta-RGD/ColⅡ1.0 g·L^-1 group at 13 d.The determination of hydroxyproline showed that the hydroxyproline level of chondrocytes in Ta-RGD/ColⅡ1.0 g·L^-1 group was significantly higher than those in other groups(P<0.05). Conclusion The porous tantalum modified with RGD peptides is advantageous to enhance chondrocyte adhesion and proliferation on the surface and inner of porous tantalum and promots the secretion of chondrocytes.