Research On Chondrocytes Cultured In Bioreactor And Redifferention Effect Of Collagen Type II To Dedifferentiated Rabbit Chondrocytes

Objective: Research On Chondrocytes Cultured In Bioreactor And Redifferention Effect Of Collagen Type II To Dedifferentiated Rabbit ChondrocytesMethods: 1 Chondrocytes was harvested from tibio-femoral joints of New Zealand white rabbit.after culured in vitro, Chondrocytes was implanted in agarose scaffold. Cyclical loading in the bioreactor with a 20% strain magnitude at a frequency of 1Hz for half an hours a day. Live-Dead Cell Staining,Alcian Blue staining and GAG measuring was performed in day 7,14d. the mRNA expression of collagen type I,collagen typeⅡ,GAG was tested at day 14. 2 cartilage tissue engineering. Methods Cartilage was harvested under sterile conditions from tibio-femoral joints of 7-month-old New Zealand white rabbit. The rabbit articular chondrocytes were subcultured in vitro to the 7th generation (named P1-P7). Dediferentiated rabbit chondrocytes were chosen by RT-PCR, real-time PCR, and 1, 9-dimethylmethylene blue (DMMB) assay.Then dedif erentiated rabbit chondrocytes were treated with various concentrations (0, 0.5%, 1.0%, and 1.5%) of exogenous collagen type II. The redif erentiation of dedif erentiated chondrocytes was measured by RT-PCR and real-time PCR, and the glycosaminoglycan content was determined by DMMB assay.Results: 1 In control group Alcian blue stainings in day 7 was negative and Apparent blue dyeing was observed at day 14. proteoglycan secretions was less quantity day 7 and large quantity at day 14. significant differences between the loading group and control group. RealTime Quantitative PCR was performed at day 14. collagen type I mRNA concentrations in loading group was lower than control group significantly(P<0.05). The mRNA expression of collagen typeⅡ,GAG in loading group were higher than control group(P<0.05). 2 The glycosaminoglycan content of P1-P7 chondrocytes were (12.20±0.17), (11.20±0.24), (11.18±0.16), (10.89±0.50), (8.73±0.19), (9.39±0.32), and (8.18±0.20)μg, respectively, showing no significant diference (P > 0.05) among P2, P3, and P4, and showing significant differences (P < 0.05) among other generations. The mRNA of collagen type I, collagen type II, and aggrecan expressed at P4-P7, showing no significant diference in the mRNA expression of collagen type I (P > 0.05) and significant differences in the mRNA expressions of collagen type II and aggrecan (P < 0.05) among P4-P7. The glycosaminoglycan content at concentrations of 0, 0.5%, 1.0%, and 1.5% were (8.20±0.16), (14.61±0.33), (13.93±0.25), and (19.59±0.46)μg, showing significant differences among different concentrations (P < 0.05). With exogenous collagen type II concentrations increased, the mRNA expressions of collagen type II and aggrecan gene were up-regulated gradually, but collagen type I gene was down-regulated, showing significant differences (P <0.05). Conclusion Collagen type II can promote rediferentiation and activation of dedifferentiated rabbit chondrocytes.Conclusion: 1 The rolling compression loading bioreactor had positive effect on collagen typeⅡ,GAG, negative effect on collagen type I. It could support the chondrocytic phenotype. 2 Type II collagen can promote redifferentiation and activation of dedifferentiated rabbit chondrocytes.