Abstract: The ATDC5 cell line exhibits the multistep chondrogenic differentiation observed during endochondral bone formation. However, it takes up to two months to complete the process of cell expansion, insulin addition to promote differentiation and further changes in culture conditions effectively to induce hypertrophy. We sought to produce consistent chondrogenesis with significant hypertrophic differentiation with simpler conditions in a more practical time period. By adding ascorbate, the prechondrogenic proliferation phase was shortened from 21 to 7 days, with production of cartilaginous nodules during the chondrogenic phase, after insulin addition, that were greater in number and larger in size. Immunohistochemistry indicated much greater matrix elaboration and the mRNA expression of sox9, aggrecan and collagen type II were all significantly increased earlier and to a much higher degree when compared with controls. Moreover, there was a robust induction of hypertrophy: Col10a1, Runx2 and Mmp13 were all induced within 7-10 days. In conclusion, addition of ascorbate to ATDC5 cultures shortened the prechondrogenic proliferation phase, produced earlier chondrogenic differentiation, heightened gene expression and robust hypertrophic differentiation, abrogating the need for extended culture times and the changes in culture conditions. This simple modification considerably enhances the practicality of this cell line for studies of chondrogenesis.

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