猪来源关节软骨脱细胞支架的生物安全性研究

目的评价猪来源关节软骨脱细胞支架的生物安全性。方法取猪四肢关节软骨,分别制备脱细胞和未脱细胞软骨支架。在人软骨细胞中分别加入浓度为100%、50%、25%的脱细胞软骨支架浸提液无血清DMEM培养液与支架表面积按1ml:(3～6)cm2比例混合,37℃静置72h],计算相对增殖率,判定其体外细胞毒性。0.9%NaCl与支架表面积按1ml:(3～6)cm2比例混合,37℃静置72h制备脱细胞软骨支架浸提液,分别用于全身急性毒性实验(小白鼠)、溶血实验、热原检测实验、皮内刺激实验(大白兔),分别观察其体内毒性、溶血程度、热原和刺激情况。在大白兔体内分别埋植脱细胞软骨支架与未脱细胞软骨支架,观察炎症反应和细胞免疫情况。结果猪脱细胞软骨支架浸提液对细胞生长无抑制作用,无细胞毒性。注射支架浸提液后小白鼠一般情况良好,无全身急性毒性反应。支架浸提液溶血程度为1.3%,无溶血作用。支架浸提液无热原存在且原发刺激指数为0分,皮内刺激实验阴性。脱细胞软骨支架与未脱细胞软骨支架相比免疫原性小,无炎症反应和淋巴细胞浸润。结论猪来源关节软骨脱细胞支架具有良好的生物相容性,将有可能成为理想的天然生物材料,具有良好的应用前景。

Research of biological safty of porcine articular cartilage extracellular matrix derived scaffold

Objective Evaluate the biological safety of porcine articular acellular cartilage scaffold. Methods Porcine articular cartilage was used to prepare acellular and non-acellular cartilage scaffold, Add 100%, 50% and 25% acellular cartilage scaffold leaching fluid DMEM culture solution and surface area of scaffold were mixed according to 1 ml: (3 - 6) cm~2, 37 ℃, 72 h], RGR was valued to determine its vitro cytotoxicity. After acellular cartilage scaffold leaching fluid were prepared 0.9% NaCl and surface area of scaffold were mixed according to 1 ml: (3 - 6) cm~2, 37 ℃, 72 h], the biocompatility was evaluated both in vivo and in vitro, including long term subcutaneous implantation test, cytotoxic test, acute systemic toxicity test, hemolysis test, intradermal test, pyrogenic test. Results Porcine acellular cartilage scaffold leaching liquid has no inhibitory effect on cell growth. After the injection of leaching fluid, rats showed no acute systemic toxicity. The leaching fluid has no hemolysis, the degree of hemolysis was 1.3%. Biologic assays were pyrogen free and primary stimulation index was 0. Compared with non-acellular cartilage scaffold, acellular cartilage scaffold showed low immunogenicity, no inflammatory response and no lymphocyte infiltration. Conclusions Porcine acellular cartilage scaffold had good biocompatibility. It will he an ideal natural biological material and has good application prospects.