Study on the mechanism of terminal differentiation and calcification of cultured mandibular chondrocytes

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During the term of the year of 1993, we studied expression of differentiated phenotype of rabbit mandibular chondrocytes in culture. The experiments done yielded the following results.[Microscopic and Histological Analyzes] Rabbit mandibular chondrocytes took flat spindle-like shape resembling fibroblasts in appearance by the day 6 of culture. On the day 10, however, they changed their shape into rounded one characteristic to differentiated chondrocytes. Then the cells produced and secreted out tremendous amount of cartilage-matrix molecules to became hypertrophic by the day 16 of culture. Alcian blue staining became intense by the day 13, and then gradually declined. In contrast, alizarin red staining could be detectable on the day 16, and then increased to become fully positive by the day 27 or later.[Transmission Electron Microscopic Analyzes] While a large amount of type I collagen fiber accumulated in the pericellular space of the cells during the first week of culture, type II collagen fiber was recognized as a major component of interterritorial matrix. The cells after culture for two weeks in vitro appeared to be at the initial calcifying stage. After four weeks, the cells became hypertrophic, but no transdifferentiation into osteoblasts occurred.[Expression of ALPase Activity] The cells expressed a low level of ALPase activity as long as bFGF was present in the culture. When bFGF was withdrawn from the culture at confluence on the day 5 of culture, ALPase activity began to increase. The enzyme activity reached a maximal level on the day 15, and then gradually decreased.[Northern Blot] Expression of type II collagen and ALPase mRNAs reached maximum on the day 7 of culture, and then decreased. Expression of type I collagen mRNA gradually increased time-dependently to become maximal on the day 12. During culture of the cells for four weeks, no expression of type X collagen, osteopontin or osteonectin mRNA was detectable.