The Role of Growth Factors to Rabbit Chondrocytes and Subtypes of Collagen in Three Dimensional High Density Culture

ABSTRACT

Cartilage is commonly used autogenous material for aesthetic and reconstructive surgery and major donor sites of cartilage are ear, nasal septum, and rib. As the cartilage correlates with ossification and can be used for joint reconstruction. Many growth factors influencing growth and differentiation of chondrocytes have been reported, and matrix composition produced by chondrocytes may vary in types and quantity according to culture duration. Initially the chondrocytes in culture aggregate, then secrete type I collagen. Type II collagen is produced during differentiation process, and synthesis of type X collagen is the last step. In this study, chondrocytes were isolated from ear cartilage of the New Zealand white rabbit weighing 400 gm. We performed high density culture using penicylinder and pellet method. The cells were polygonal in morphology and viable under the inverted microscope. This experiment was designed to evaluate the effect of IGF-I, TGF- p, and b- FGF on the synthesis of collagen in chondrocyte culture. Optimal concentration of growth factors was determined using H-thymidine incorporation into DNA. After the addition of optimal concentration of each growth factors in experimental groups, the uptake of H-proline was measured. Only IGF-I showed a statistically significant increase of collagen synthesis. We observed how subtypes of collagen were influenced by growth factors in two culture methods and by differing the addition timing of growth factors. SDS-PAGE was adopted for subtyping of collagen. All subtypes of collagen were found in both culture methods and all growth factors facilitated the production of type II and type X collagen and may be devoted to the differentiation of chondrocytes. Immunohistochemical staining for type I, and type II collagen was examined to confirm the above result. In pellet culture, type II collagen was stained densely in response to the addition of three kinds of growth factors. The results of penicylinder culture showed similar outcome to those from pellet cultured group. From the above results, we concluded as follows; First, IGF-I generally influence the synthesis of type I and II collagen. Second, TGF beta increased the synthesis of collagen. Third, b-FGF increased the synthesis of type II and type X collagen. We concluded that IFG-I is the only growth factor which is effective regardless of culture duration and method. TGF- beta and b-FGF, which are potent mitogen, facilitate the secretion of collagen.