Inagaki Yusuke, Kitamura Nobuto, Kurokawa Takayuki, Tanaka Yasuhito, Gong Jian P, Yasuda Kazunori, Tohyama Harukazu
Department of Sports Medicine and Joint Surgery, Graduate School of Medicine, Hokkaido University, Kita-15 Nishi-7, Sapporo 060-8638, Japan.
BMC Musculoskelet Disord. 2014 Sep 27;15:320. doi: 10.1186/1471-2474-15-320.
Recently, several animal studies have found that spontaneous hyaline cartilage regeneration can be induced in vivo within a large osteochondral defect by implanting a synthetic double-network (DN) hydrogel, which is composed of poly-(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly-(N,N'-dimethyl acrylamide) (PDMAAm), at the bottom of the defect. However, the effect of hydrogel on hyaline cartilage regeneration remains unexplained. The purpose of this study was to investigate the chondrogenic differentiation of C3H10T1/2 cells on PAMPS/PDMAAm DN gel.
C3H10T1/2 cells of 1.0 × 105 were cultured on PAMPS/PDMAAm DN gel in polystyrene tissue culture dishes or directly on polystyrene tissue culture dishes. We compared cultured cells on PAMPS/PDMAAm DN gel with those on polystyrene dishes by morphology using phase-contrast microscopy, mRNA expression of aggrecan, type I collagen, type II collagen, Sox 9 and osteocalcin using real-time RT-PCR, and local expression of type II collagen using immunocytochemistry.
C3H10T1/2 cells cultured on the PAMPS/PDMAAm DN gels formed focal adhesions, aggregated rapidly and developed into large nodules within 7 days, while the cells cultured on the polystyrene surface did not. The mRNA levels of aggrecan, type I collagen, type II collagen, Sox 9 and osteocalcin were significantly greater in cells cultured on the PAMPS/PDMAAm DN gel than in those cultured on polystyrene dishes. In addition, C3H10T1/2 cells cultured on PAMPS/PDMAAm DN gel expressed more type II collagen at the protein level when compared with cells cultured on polystyrene dishes.
The present study showed that PAMPS/PDMAAm DN gel enhanced chondrogenesis of C3H10T1/2 cells, which are functionally similar to mesenchymal stem cells. This suggests that mesenchymal stem cells from the bone marrow contribute to spontaneous hyaline cartilage regeneration in vivo in large osteochondral defects after implantation of PAMPS/PDMAAm DN gels.
最近，多项动物研究发现，通过在大的骨软骨缺损底部植入由聚（2-丙烯酰胺-2-甲基丙烷磺酸）（PAMPS）和聚（N，N'-二甲基丙烯酰胺）（PDMAAm）组成的合成双网络（DN）水凝胶，可在体内诱导透明软骨自发再生。然而，水凝胶对透明软骨再生的影响仍不清楚。本研究的目的是研究C3H10T1/2细胞在PAMPS/PDMAAm DN凝胶上的软骨形成分化。
将1.0×105个C3H10T1/2细胞接种于聚苯乙烯组织培养皿中的PAMPS/PDMAAm DN凝胶上，或直接接种于聚苯乙烯组织培养皿上。我们通过相差显微镜观察形态、实时逆转录聚合酶链反应检测聚集蛋白聚糖、I型胶原、II型胶原、Sox 9和骨钙素的mRNA表达以及免疫细胞化学检测II型胶原的局部表达，比较PAMPS/PDMAAm DN凝胶上培养的细胞与聚苯乙烯培养皿上培养的细胞。
在PAMPS/PDMAAm DN凝胶上培养的C3H10T1/2细胞形成粘着斑，迅速聚集并在7天内形成大的结节，而在聚苯乙烯表面培养的细胞则没有。在PAMPS/PDMAAm DN凝胶上培养的细胞中，聚集蛋白聚糖、I型胶原、II型胶原、Sox 9和骨钙素的mRNA水平显著高于在聚苯乙烯培养皿上培养的细胞。此外，与在聚苯乙烯培养皿上培养的细胞相比，在PAMPS/PDMAAm DN凝胶上培养的C3H10T1/2细胞在蛋白质水平上表达更多的II型胶原。
本研究表明，PAMPS/PDMAAm DN凝胶增强了功能上类似于间充质干细胞的C3H10T1/2细胞的软骨形成。这表明，骨髓来源的间充质干细胞在植入PAMPS/PDMAAm DN凝胶后，有助于大的骨软骨缺损在体内自发形成透明软骨。
