Xie Aiguo, Xue Jixin, Shen Gan, Nie Lanjun
Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong UniversityNo.639 Zhizaoju Road, Huangpu District, Shanghai 200011, P. R. China.
Department of Hand and Plastic Surgery, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical UniversityNo.109, Xueyuan West Road, Wenzhou 325027, Zhejiang, P. R. China.
Am J Transl Res. 2017 Jul 15;9(7):3487-3498. eCollection 2017.
Cartilage tissue engineering provides a new method in the treatment of cartilage defects, and adipose derived stem cells seem to be an ideal seed cell in cartilage tissue engineering because of its characteristics. However, ossification after in vivo implantation of tissue engineered cartilage remains a challenge. Thrombospondin-1 which has been reported to have an inhibitory effect on angiogenesis, may play an important role in inhibiting the ossification of tissue engineered cartilage constructed by adipose derived stem cells. Therefore, the effect of thrombospondin-1 in inhibiting the ossification of tissue engineered cartilage was evaluated in this study. Lentivirus vectors carrying thrombospondin-1 cDNA were transfected into adipose derived stem cells, and the transfected cells were used in the experiments. The expression of thrombospondin-1 was evaluated by quantitative reverse transcriptase-polymerase chain reaction and western blot, and the effects of thrombospondin-1 over-expression on angiogenesis were analyzed by angiogenesis assays. The quality of tissue engineered cartilage and the degree of ossification were assessed by biomechanical and molecular biology methods. The results showed that thrombospondin-1 infected cells have a high expression of thrombospondin-1 in mRNA and protein level, which inhibited the tube formation of endothelial cells, indicating the anti-angiogenic effects. Gene expression analyses in vitro showed that thrombospondin-1 inhibits the osteogenic differentiation of adipose derived stem cells significantly, and the results of in vivo study revealed that thrombospondin-1 significantly inhibits the expression of osteogenic genes. Compared to that in the control group, tissue engineered cartilage constructed by thrombospondin-1 transfected adipose derived stem cells in vivo showed a higher GAG content and lower compressive modulus, which indicating lower level of ossification. In conclusion, the current study indicated that the anti-angiogenic factor thrombospondin-1 suppresses the osteogenic differentiation of adipose derived stem cells in vitro, and inhibits ossification of tissue engineered cartilage constructed by adipose derived stem cells in vivo.
软骨组织工程为软骨缺损的治疗提供了一种新方法，脂肪来源干细胞因其特性似乎是软骨组织工程中理想的种子细胞。然而，组织工程化软骨体内植入后的骨化仍然是一个挑战。据报道，血小板反应蛋白-1对血管生成有抑制作用，可能在抑制脂肪来源干细胞构建的组织工程化软骨骨化中发挥重要作用。因此，本研究评估了血小板反应蛋白-1在抑制组织工程化软骨骨化中的作用。将携带血小板反应蛋白-1 cDNA的慢病毒载体转染到脂肪来源干细胞中，并将转染后的细胞用于实验。通过定量逆转录-聚合酶链反应和蛋白质免疫印迹法评估血小板反应蛋白-1的表达，并通过血管生成试验分析血小板反应蛋白-1过表达对血管生成的影响。通过生物力学和分子生物学方法评估组织工程化软骨的质量和骨化程度。结果表明，血小板反应蛋白-1感染的细胞在mRNA和蛋白质水平上有高表达的血小板反应蛋白-1，其抑制了内皮细胞的管腔形成，表明具有抗血管生成作用。体外基因表达分析表明，血小板反应蛋白-1显著抑制脂肪来源干细胞的成骨分化，体内研究结果显示，血小板反应蛋白-1显著抑制成骨基因的表达。与对照组相比，体内由血小板反应蛋白-1转染的脂肪来源干细胞构建的组织工程化软骨显示出更高的糖胺聚糖含量和更低的压缩模量，这表明骨化水平较低。总之，当前研究表明，抗血管生成因子血小板反应蛋白-1在体外抑制脂肪来源干细胞的成骨分化，并在体内抑制脂肪来源干细胞构建的组织工程化软骨的骨化。
