软骨基质来源定向结构支架复合软骨细胞体外构建组织工程软骨的研究

目的：探讨采用软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞，在体外静态培养条件下生成组织工程软骨的可能性。方法：制备牛关节软骨细胞外基质材料，利用温度梯度热诱导相分离技术构建具备垂直定向孔道结构的软骨支架，同时采用传统冷冻干燥方法制备非定向支架，检测两组支架的力学性能；提取兔关节软骨细胞，分别接种两组支架，体外静态培养2周及4周后取材，对构建的组织工程软骨进行组织切片染色、生物化学分析及生物力学检测。结果：定向软骨支架的压缩弹性模量数值明显高于非定向软骨支架，体外培养时定向支架上种子细胞在3-9d内增殖高于非定向支架，差异有统计学意义（P〈0．05）；体外静态培养4周后形成的两组新生组织工程软骨进行软骨特异性染色均呈阳性，在定向组新生软骨切片中在垂直方向上可见大量呈规则平行排列的粗大胶原纤维，两组新生软骨的生物化学检测包括总DNA、总GAG及总胶原含量差异无统计学意义（P〉0．05）。定向组织工程软骨压缩弹性模量在2周及4周时均高于非定向组织工程软骨，差异有统计学意义（P〈0．05）。但两组组织工程软骨上述指标均显著低于正常关节软骨（P〈0．05）。结论：软骨细胞外基质材料制备的定向结构软骨支架复合软骨细胞，在体外静态培养条件下能够成功生成具有定向纤维结构的组织工程软骨，并可以有效促进新生软骨组织力学性能的提升，在软骨组织工程中具有良好的应用前景。

Cartilage Tissue Engineering Using Oriented Cartilage Extracellular Matrix-derive Scaffold Combined with Chondrocytes in Vitro

Objective： To fabricate an oriented cartilage extracellular matrix （ECM）-derived scaffold combined with chondrocytes for enhancement of the biomechanical property of tissue-engineered （TE） cartilage in vitro. Methods： An oriented cartilage ECM-derived scaffold was fabricated composed of microtubules arranged in parallel in vertical section. Mechanical property of oriented and non-oftented scaffold was determined by measurement of Young＇s modulus. Oriented and non-oriented scaffolds were seeded with chondrocytes. Cell-scaffold constructs were cultured for 2 weeks and 4 weeks in vitro, after which biochemical, histological and biomechanical properties were evaluated. Results： The compressive modulus of oriented scaffolds was higher than that of a typical non-oriented scaffold （P 〈 0.05）. Proliferation in the oriented scaffold group was higher than in the non-oriented scaffold group from day 3 to day 9 （P 〈 0.05）. At 4 weeks, all samples stained positive for safi＇anin O, toluidine blue, and collagen type II. Oriented-structure constructs contained numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells aligned along the fibers. Total DNA, glycosaminoglycans and collagen contents increased with time and these values were similar in the two groups （P 〉 0.05 ）. The Young＇s modulus of the TE cartilage was higher for the oriented scaffold compared with the non-oriented scaffold after implantation for 2 weeks and 4 weeks （P 〉 0.05 ）. Total DNA, GAG, collagen and Young＇s modulus in the two experimental groups at both time points were lower compared with native cartilage （P〉0.05）. Conclusion： These results indicate that oriented ECM-derived scaffolds enhance the biomechanical property of TE cartilage in vitro and thus represent a promising approach to cartilage tissue engineering.