丝素蛋白无纺网支架体外构建组织工程化软骨

Construction of tissue-engineered cartilage using silk fibroin nets and chondrocytes

目的:探讨丝素蛋白无纺网作为耳廓软骨细胞外支架构建组织工程化软骨的可行性。方法:取成年新西兰兔耳廓软骨,酶消化法获取软骨细胞,体外培养扩增后取原代细胞接种到制备好的丝素蛋白无纺网支架上,体外复合培养。每天倒置显微镜下观察细胞黏附、生长、增殖情况,于复合培养第3、7、10天时取材进行扫描电镜观察;于复合培养第1、2、4、6周取材进行组织学评价,同时取材通过RT-PCR方法检测复合物上软骨细胞Ⅱ型胶原mRNA的表达情况。结果:48 h后软骨细胞已黏附于丝素蛋白纤维上,72 h后细胞开始分泌少量细胞外基质;电镜下观察显示软骨细胞及分泌的薄层细胞外基质主要分布于支架材料表面。组织学观察显示2周时支架浅层已有一定厚度软骨组织,可见少量类软骨陷窝,4周和6周时支架浅层软骨组织增厚,软骨陷窝增多,但支架内部软骨细胞数目少,细胞呈星形或梭形,细胞分泌的基质也很少。RT-PCR检测显示在各检测时间点均有Ⅱ型胶原mRNA的表达。结论:兔耳廓软骨细胞在丝素蛋白无纺网支架上已初步形成软骨样组织,但培养条件应进一步优化。

Objective：To evaluate the feasibility of non-woven silk fibroin net as a biocompatible scaffold of the auricular chondrocytes for construction of tissue-engineered cartilage. Methods：Chondrocytes were harvested from auricular cartilage by trypsin and type Ⅱ collagenase digestion,and expanded in culture flasks.The chondrocytes（P1） were seeded into non-woven silk fibroin nets,and incubated in CO2 incubator.The adhesion,proliferation and growth of cells were observed under inverted microscope every day and the microstructure of the constructs was observed by using scanning electron microscope（SEM） at 3,7 and 10 d after coculture.The constructs were harvested and analyzed by histology at 1,2,4 and 6 weeks after coculture,and the mRNA expression of type Ⅱ collagen was examined using RT-PCR. Results：The scaffold was covered with chondrocytes at 48 h and these cells began to secrete extracellular matrix（ECM） at 72 h after coculture.The surface of scaffold was covered with chondrocytes and ECM under SEM.Stent shallow already had some cartilage tissue and a little lacunae at 2 weeks.The cartilage tissue thickened and lacunae increased,but the certer of stent had a little cells,which were star or spindle type and secreted a little ECM at 4 and 6 weeks.The mRNA expression of type Ⅱ collagen was detected by RT-PCR at all time-points. Conclusions：Cartilage-like tissues can form at non-woven silk fibroin net with the auricular chondrocytes,but culture conditions should be further optimized.