Implantation of PAMPS/PDMAAm double-network (DN) gel can induce hyaline cartilage regeneration in the osteochondral defect. However, it is a problem that the volume of the regenerated cartilage tissue is gradually reduced at 12 weeks. This study investigated whether intra-articular administration of hyaluronic acid (HA) increases the volume of the cartilage regenerated with the DN gel at 12 weeks. A total of 48 rabbits were used in this study. A cylindrical osteochondral defect created in the bilateral femoral trochlea was treated with DN gel (Group DN) or left without any implantation (Group C). In both Groups, we injected 1.0 mL of HA in the left knee, and 1.0 mL of saline solution in the right knee. Quantitative histological evaluations were performed at 2, 4, and 12 weeks, and PCR analysis was performed at 2 and 4 weeks after surgery. In Group DN, the proteoglycan-rich area was significantly greater in the HA-injected knees than in the saline-injected knees at 12 weeks (P = 0.0247), and expression of type 2 collagen, aggrecan, and Sox9 mRNAs was significantly greater in the HA-injected knees than in the saline-injected knees at 2 weeks (P = 0.0475, P = 0.0257, P = 0.0222, respectively). The intra-articular administration of HA significantly enhanced these gene expression at 2 weeks and significantly increased the volume of the hyaline cartilage regenerated by implantation of a DN gel at 12 weeks. This information is important to develop an additional method to increase the volume of the hyaline cartilage tissue in a potential cartilage regeneration strategy using the DN gel.

BackgroundA 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.MethodsATDC5 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.ResultsThe 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).ConclusionsThe 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.