Abstract
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.
Highlights
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
We have recently demonstrated that articular cartilage repair is feasible using a synthetic Poly-(2-Acrylamido-2methylpropanesulfonic acid) (PAMPS)/ PDMAAm double-network (DN) hydrogel, which consists of poly-(2-acrylamido-2-methylpropanesulfonic acid)
The effect of the PAMPS/PDMAAm DN gel on chondrogenic differentiation of ATDC5 cells At 7 days, mRNA expression of type-2 collagen and aggrecan was significantly greater in culture on the DN gel than in culture on the PS dish (p < 0.0001 and p < 0.0001, respectively) (Figure 1)
Summary
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. We have recently demonstrated that articular cartilage repair is feasible using a synthetic PAMPS/ PDMAAm double-network (DN) hydrogel, which consists of poly-(2-acrylamido-2-methylpropanesulfonic acid). The single-network PAMPS gel, which is a component gel of the DN gel, differentiates chondrogenic ATDC5 cells into chondrocytes even in an insulin-free medium [4] These results imply that chondrogenic differentiation of ATDC5 cells is induced by the culture microenvironment modified by gel components as well as insulin supplementation. Engler et al [6] reported that the elasticity of the substrate on which the cultured cells are attached directs stem cell differentiation It is not known whether the PAMPS/PDMAAm DN gel induces chondrogenic differentiation of ATDC5 cells in a maintenance medium without insulin
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