Kitamura Nobuto, Kurokawa Takayuki, Fukui Takaaki, Gong Jian P, Yasuda Kazunori
Department of Sports Medicine and Joint Surgery, Graduate School of Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan.
BMC Musculoskelet Disord. 2014 Jul 6;15:222. doi: 10.1186/1471-2474-15-222.
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.
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.
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).
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.
由聚（2-丙烯酰胺-2-甲基丙烷磺酸）和聚（N，N'-二甲基丙烯酰胺）（PAMPS/PDMAAm）组成的双网络（DN）凝胶在体外和体内均具有诱导软骨形成的潜力。本研究调查了DN凝胶在无胰岛素的维持培养基中是否能诱导ATDC5细胞的软骨分化，以及补充透明质酸是否能增强DN凝胶的软骨分化效果。
将ATDC5细胞在无胰岛素的维持培养基中培养于DN凝胶和聚苯乙烯（PS）培养皿上21天。向培养基中补充分子量约为800 kDa的透明质酸，使其浓度达到0.01、0.1或1.0 mg/mL。在培养7天和21天时，使用免疫细胞化学方法检测Ⅱ型胶原蛋白，并通过实时PCR检测Ⅱ型胶原蛋白、聚集蛋白聚糖和Sox9基因的表达，对培养的细胞进行评估。
在无透明质酸的维持培养基中，培养在DN凝胶上的细胞形成结节，并用抗Ⅱ型胶原蛋白抗体染色，DN凝胶上Ⅱ型胶原蛋白和聚集蛋白聚糖mRNA的表达明显高于PS培养皿表面（p < 0.05）。与无透明质酸培养相比，在21天时补充高浓度（1.0 mg/mL）透明质酸可显著增强Ⅱ型胶原蛋白和聚集蛋白聚糖mRNA的表达（p < 0.05）。
DN凝胶在无胰岛素的情况下可诱导ATDC5细胞的软骨分化。这种作用受透明质酸的显著影响，取决于其浓度水平。透明质酸很有可能在DN凝胶诱导的体内透明软骨再生现象中发挥重要作用。
