从牛软骨中提取Ⅱ型胶原蛋白及初步构建软骨支架

Extracting collagen type Ⅱ from bovine cartilage and preliminary construction of cartilage scaffolds

建立了一种材料来源广泛、操作步骤简单的提取II型胶原蛋白的新方法,以牛软骨为原料,预处理后再经胃蛋白酶消化、盐析、透析、冷冻干燥得到II型胶原蛋白纯品,其纯度经SDSPAGE电泳后利用凝胶成像系统分析为98.25%。以制备的II型胶原蛋白纯品、透明质酸(HA)和硫酸软骨素(CS)为原料,按CII∶HA∶CS为9∶1∶1、7∶1∶1、5∶1∶1、3∶1∶1的比例混合,通过真空冷冻干燥初步构建了人工软骨支架。综合比较四组软骨支架的形态、交联度、孔隙率、孔径、吸水率及降解率各项参数,确定比例9∶1∶1为所构建软骨支架的最优比例,其孔隙率为(86.63±0.67)%,孔径为(83.33±7.20)μm,吸水率为(684.52±8.57)%,降解率为(26.73±1.88)%。

Collagen type II （CII） was commonly used to repair injured cartilage. In this study, a method was developed for extracting collagen type II. Bovine cartilage, as raw material, was pretreated, then di- gested with pepsin, salted out with NaC1 and dialyzed with deionized water. Finally, the CII sample was obtained by vacuum freeze drying. Its purity was analyzed by SDS-PAGE and a gel imaging system to be 98.25%. Then, four ratios of collagen type II, hyaluronic acid （HA） and chondroitin sulfate （CS） were used to construct scaffolds with EDC/NHS as crosslinking agent. The morphology, crosslink degree, po- rosity, pore diameter, absorption rate and degradation rate were determined to evaluate properties of four scaffolds and the ratio 9： 1：1 was optimal. In this study, the optimal cartilage scaffold＇ s porosity, pore diameter, absorption rate and degradation rate were （86.63 ± 0.67） %, （83.33 ± 7.20） μm, （684.52 ± 8.57） % and （26.73 ± 1.88） % , respectively.