Abstract
In this study, there are there major parts including fabricating collagen type II scaffold, various glycosaminoglycans effect on chondrocytes, and serum-free media design for chondrocytes. In first part, we successfully isolated collagen type II from bovine trachea and crosslinked by genipin. Crosslinking by genipin indeed reinforced the scaffold intensity and physico-chemical characteristics. In second part, our aim is to investigate the effect of various glycosaminoglycans into medium cultured with genipin-crosslinked collagen type II scaffolds. Our results suggest that chondrocytes cultured within genipin-crosslinked scaffolds may show better tendency for differentiation by adding certain dose of GAGs (10μg/ml CS-A, 50μg/ml CS-B, 50μg/ml CS-C, 5μg/ml HS, and 500kDa Hyaluronan). On transcription level we show that treatment with CS-A, CS-C, and 500kDa HA lead to a significant up-regulation of collagen type II and aggrecan expression. These indicate that supplement with GAGs into medium are potent for promoting excreting ECM of normal cartilage. Although non-depolymerization CS-A and CS-C act as good addition of cultured medium, CS-C6 (hexasaccharides of CS-C) may be more powerful because it can inhibit further degradation due to catabolic activities, in the meantime heighten the anabolic ability. In the last part, seven kinds of growth factors which are most often used for chondrocytes growth and differentiation are selected. The 27-3 fractional factorial design is adopted here to determine what growth factors are required in the chondrogenic culture. There were five growth factors left to determine concentration in the serum-free medium along the steepest ascent path. Applicable serum-free and growth factor-containing medium for the chondrocytes is developed, which is composed of DMEM containing 10.26 ng/ml FGF-2, 10.30 ng/ml EGF, 10.42 ng/ml TGF-β1, 53.00 ng/ml BMP-2, and 11.01 ng/ml VEGF. Finally, we conclude that our serum-free medium including glycosaminoglycans and growth factors are functionally substituted for animal serum.