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Background  A double-network (DN) gel, which was composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) and poly-(N,N’-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated whether DN gel induced chondrogenic differentiation of ATDC5 cells in a maintenance medium without insulin, and whether supplementation of hyaluronic acid enhanced the chondrogenic differentiation effect of DN gel.    Methods  ATDC5 cells were cultured on the DN gel and the polystyrene (PS) dish in maintenance media without insulin for 21 days. Hyaluronic acid having a molecular weight of approximately 800 kDa was supplemented into the medium so that the concentration became 0.01, 0.1, or 1.0 mg/mL. The cultured cells were evaluated using immunocytochemistry for type-2 collagen and real time PCR for gene expression of type-2 collagen, aggrecan, and Sox9 at 7 and 21 days of culture.    Results  The cells cultured on the DN gel formed nodules and were stained with an anti-type-2 collagen antibody, and expression of type-2 collagen and aggrecan mRNA was significantly greater on the DN gel than on the PS dish surface (p < 0.05) in the hyaluronic acid-free maintenance medium. Hyaluronic acid supplementation of a high concentration (1.0 mg/mL) significantly enhanced expression of type-2 collagen and aggrecan mRNA in comparison with culture without hyaluronic acid at 21 days (p < 0.05).    Conclusions  The DN gel induced chondrogenic differentiation of ATDC5 cells without insulin. This effect was significantly affected by hyaluronic acid, depending on the level of concentration. There is a high possibility that hyaluronic acid plays an important role in the in vivo hyaline cartilage regeneration phenomenon induced by the DN gel.

Hyaluronic acid enhances the effect of the PAMPS/PDMAAm double-network hydrogel on chondrogenic differentiation of ATDC5 cells