组织工程培养人关节软骨的实验研究

The purpos of this study was to verify the ability of PLGA scaffolds to support the growth of human chondrocytes in reconstructing human articular cartilage by tissue engineering, and to identify the effect of human transforming growth factorβ2(TGF-β2) on articular chondrocytes and mesenchymal progenitor cells in monolayer culture with the use of gene transfection technology. Free chondrocyte isolated from adult articular cartilage were seeded onto sponge PLGA scaffolds after expansion by culture. The complex of cell-scaffold were then cultured ex vivo and transplanted to subcutaneous of nude mouse respectively. A recombinant of pcDNA3.1(+)/TGF-β2 was constructed and then transferred by liposome to chondrocytes and mesenchymal progenitor cells. Histological staining, scanning electron microscopy, RT-PCR, Western-Blotting and Immunohistochemistry analysis were performed to verify the interactions between cells and biomaterial and to evaluate the expression of TGF-β2 and cartilage associated genes and proteins. The results showed that articular chondrocytes were well distributed and adhered to PLGA scaffolds and maintained their characteristics of articular cartilage. After cultured in vivo for 8 weeks the cell-seeded scaffolds grew into hyaline cartilage-like tissue. This manifested that the PLGA scaffolds can be suitable carriers for tissue engineering of articular cartilage. Transfection of pcDNA3.1(+)/TGF-β2 was able to provide transient and persistent expression in chondrocytes and mesenchymal progenitor cells. The de-differentiation of chonrocytes could be inhibited by TGF-β2 transfection, and the differentiation of human marrow-derived mesenchymal progenitor cells into chondrocyte in monolayer culture was shown be feasible and can be induced by TGF-β2. This may suggest good potential in amplifying chondro-lineage progenitors from bone marrow for cartilage repair, as well as in studying the mechanisms involved in early chondrogenesis combined with gene transfer techniques.

本实验拟通过组织工程方法，以多孔聚乳酸和聚乙醇酸为载体吸附人软骨细胞，以过体外培养和裸鼠体内培养，获得软骨组织。对所得软骨进行组织学、免疫组织化学、基质和纤维成定量及生物力学检测，并与正常关节软骨对照，以期获得接近正常关节软骨的组织工程软骨，为临床治疗关节软骨操作和病变准备移植材料。