Effects Of BFGF And Collagen On The Construction Of Tissue-engineered Cartilage In Vitro

Objective:(1) To study the effects of basic fibroblast growth factor (bFGF)on theproliferation and differentiation of growth-plate chondrocytes cultured in monolayer, and search for the optimal concentration of bFGF applied in pellet culture in vitro; (2)In order to improve the culture condition of tissue-engineered cartilage regeneration. We incubated growth-plate chondrocytes in pellet culture, and compared with the effect of bFGF and collagen, alone and in combination.Methods:(1)Growth-plate chondrocytes were isolated from rabbit costal cartilage andcultured in monolayer. bFGF was added in medium at different concentrations. (2)Cells growth was detected by WST-8; The secretion of GAGs was observed by species stained with Toluidine Blue O; The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method; ALP activity was measured by modified enzyme dynamical method. (3)Growth-plate chondrocytes were incubated in pellet culture. bFGF was added in medium based on the result of previous search. Collagen was applied with or without bFGF. (4)The histological structure of neocartilage was observed by species stained with H&E; Immunohistochemical analysis was performed to detect the expression of collagen I and collagen II; The secretion of GAGs was observed by species stained with Toluidine Blue O; The DNA content of neocartilage was measured by hoechst33258 dye; The production of matrix was estimated from the determination of hydroxyproline content and Alcian Blue method.Resultsrln monolayer culture system:?Chondrocytes dedifferentiated with passage, losttheir specific shape and acquired fibroblast-like shape. ?The cultured chondrocytes in medium with bFGF at the concentrations of 5-100 ng/mL were significantly more than those in the control group, which reached peak at the concentration of 25 ng/mL; The synthesis of collagen was inhibited by bFGF at the concentrations above 50 ng/mL; The activity of ALP was suppressed by bFGF at the concentrations above 2.5 ng/mL; The production of PG was not affected by bFGF compared with the control group.In pellet culture system:(3)Chondrocytes formed cartilage-like tissue with distinct differentiated zones and regions. ?The DNA content of neocartilage was significantly enhanced by bFGF and collagen; The production of collagen and PG was promoted by collagen. The production of matrix was not affected by bFGF; The expression of collagen I rose along with culture, while the expression of collagen II demonstrated opposite direction to collagen I .bFGF and collagen could suppress the expression of collagen I while they could decelerate the decrease of collagen II ;The combination of bFGF and collagen had synergistic effects on the DNA content of neocartilage, the production of collagen and the maintenance of phenotype.Conclusion:?Pellet culture could be helpful to cartilage regeneration as it maintainedchondrocyte phenotype and enhanced matrix production. (DbFGF could stimulate the proliferation of chondrocyte, it suppressed differentiation of chondrocyte at the concentrations above 2.5 ng/mL. ?Collagen could stimulate the proliferation and the matrix production of chondrocyte, it also had a positive effect on the maintenance of cell phenotype. ?The combination of bFGF and collagen had synergistic effects on the maintenance of chondrocyte phenotype and the regeneration of cartilage.