ABSTRACT
Primary cultures of embryonic chick chondrocytes inoculated at high densities (5 – 6 × 106 cells/25 cm2 Falcon Flask) with adequate pH control, grow rapidly and multilayer with 5 – 8 cell layers forming a uniform thick approximately 50 – 80 μ m dense cartilage matrix. Cultures inoculated at lower densities tended to form nodular aggregates of chondrocytes secreting a typical cartilage matrix with the internodular areas occupied by elongated, vacuolated, fibroblast-like cells secreting little matrix. Ultrastructurally, using ruthenium red during fixation, early cultures revealed rounded cells surrounded by a collagen matrix of small diameter (150 nm) collagen fibres to which were irregularly attached electron-dense granules approximately 20 nm diameter. Fine filaments of 1 nm diameter linked the electrondense granules to one another, to collagen fibres and to the cell membrane. In older cultures larger cells were observed with ultrastructural features similar to hypertrophic chondrocytes seen in vivo. Electron-dense material composed of fine needle-like structures was observed in the matrix associated with these cells. These observations were interpreted as early calcification occurring in vitro in relation to hypertrophic cells.
The molecular weight of the proteoglycans and the spectrum of glycosaminoglycans synthesized were similar to that of normal embryonic chick cartilage, demonstrating maintenance of chondrocyte differentiation in this system.
This study demonstrates that it is possible by using high density cultures with adequate nutrient media and pH control to maintain chondrocytes in a differentiated state using the criteria of ultrastructural morphology and specificity of the matrix they synthesize.
