Human Cartilage Tissue Engineering with Pluronic and Cultured Chondrocyte Sheet

Despite many outstanding research works on cartilage tissue engineering, actual clinical application is not quite successful because of the absorption and progressive distortion of tissue engineered cartilage. We have developed a new method of cartilage tissue engineering comprising chondrocyte mixed Pluronic F-127 and cultured chondrocyte cell sheet which entirely cover the cell-Pluronic complex. We believe the addition of cultured chondrocyte cell sheet enhances the efficacy of chondrogenesis in vivo. Human ear cartilage piece was enzymatically dissociated and chondrocyte suspension was acquired. Chondrocytes were cultured and expanded as the routine manner. Cultured chondrocytes were plated in high-density monolayer and cultured with Chondrogenic media in 5% CO2 incubator. After 3 weeks of culture, chondrocyte cell sheet was formed and complete single sheet of chondrocyte could be harvested by gentle manipulation of culture plate with a cell scraper. Chondrocyte-Pluronic complex was established by mixing 1x 106 cells with 0.5 of Pluronic F- 127. Chondrocyte-Pluronic complex was completely covered with a sheet of cultured chondrocyte. The completed tissue engineered constructs were implanted into the subcutaneous tissue pocket of nude mice on the back. Tissue engineered constructs without cultured cell sheet were used as control. Samples were harvested at 8 weeks postoperatively and they were subjected to histological analysis and assayed for glycosaminoglycan (GAG), and type II collagen. Grossly, the size of cartilage specimen of cultured chondrocyte cell sheet covered group was larger than that of the control. On histologic examination, the specimen of cultured chondrocyte cell sheet covered group showed lacunae-containing cells embedded in a basophilic matrix. The chondrocyte cell sheet covered group specimen resembled mature or immature cartilage. The result of measurement of GAG and type II collagen of cartilage specimen of cultured chondrocyte sheet covered group was higher than that of the control. In conclusion, the new method of cartilage tissue engineering using chondrocyte cell sheet seems to be an effective method providing higher cartilage tissue gain and reliable success rate for cartilage tissue engineering.