改良以聚羟基乙酸为支架同种异体组织工程化塑形软骨的构建

Improvement upon construction of tissue-engineered allogeneic cartilage molded with polyglycolic acid as the scaffold

目的优化组织工程技术构造塑形同种异体软骨组织的方法.方法酶消化法获取乳兔关节和肋软骨细胞并进行体外培养,传代时适当延长胰蛋白酶作用时间.收集第3-4代培养细胞用于实验.将聚羟基乙酸(PGA)塑形后接种软骨种子细胞构建成细胞-PGA复合物,植入成兔皮下(在材料塑形和复合物种植过程中改良传统方法).于种植后不同时间点取材进行大体和组织学观察,评价软骨形态和再生情况并与改良前方法比较.结果(1)培养细胞传代时适当延长胰酶作用时间所获软骨细胞活性为(92±2)%,传统方法为(93+2)%(P＞0.05).(2)种植后4周,标本呈乳白色,有一定撑力;组织学观察见软骨细胞不成熟,基质含量低,周边有炎细胞浸润.8和12周时,标本呈瓷白色,软骨组织基本成熟,炎细胞浸润不明显.16周时,外观与12周一样,软骨细胞生长良好,新形成的软骨组织内未见血管生长,无炎细胞浸润.结论调整培养细胞传代时胰酶作用时间收获的软骨种子细胞在有免疫力动物体内成软骨效应良好,无明显免疫排斥;PGA塑形和复合物皮下种植方法的改良有利于同种异体组织工程化预定形态软骨组织的形成.

Objective To improve the method for constr uc ting allogeneic molded cartilage by means of tissue engineering techniques. Met hods The chondrocytes from the rib and articular cartilage of infant rabbits wer e harvested by typeⅡcollage-nase digestion, followed by in vitro cell culture f or 3 to 4 passages. The chondrocytes were then prepared into cell suspension an d seeded onto 'C'-and 'O'-shaped pre-molded polyglycolic acid (PGA) scaffol ds form chondrocyte-PGA composites, which were subsequently cultured in vitro f or 7 to 10 d before implanted subcutaneously into adult rabbits. Improvement was made upon conventional shaping and implantation procedures. Morphological obse rvation and cartilage regeneration assess-ment were conducted at different time points following the implantation, in comparison with the observation by convent ional shaping and implantation methods. Results During in vitro cell culture, t he rate of viable chondrocytes in the final cell suspen-sion was (92±2)% after well-controlled prolongation of digestion trypsin, similar to the viable cell ra te (93±2)% by traditional procedures (P>0.05). Gross observation found milk-wh ite, newly generated cartilage which had good flexibility 4 weeks after implant ation, and after 8 weeks and later, the cartilage took on the color of porcelain-white. Histological examination showed a few inflammatory cells around the new ly generated immature cartilage 4 weeks after implantation, and the inflammation a-bated when the newly generated cartilage acquired similar histological proper ties to that of the original cartilage 8 weeks post-operatively and later. After 16 weeks, no blood vessel or capillaries were visible within the new cartilage. Conclusion The chondrocyte viability is not affected when the cells are treate d with well-controlled prolonged digestion with trypsin during in vitro cell cul ture. Improved PGA scaffolds shaping and the implantation procedure facilitate t he regeneration of the cartilage after the implantation of the composites.