Effects of culture on PAMPS/PDMAAm double-network gel on chondrogenic differentiation of mouse C3H10T1/2 cells: in vitro experimental study.
Effects of culture on PAMPS/PDMAAm double-network gel on chondrogenic differentiation of mouse C3H10T1/2 cells: in vitro experimental study.
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DOI：
10.1186/1471-2474-15-320
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发表时间：
2014-09-27
影响因子：
2.3
通讯作者：
Tohyama H
中科院分区：
文献类型：
--
作者：
Inagaki Y;Kitamura N;Kurokawa T;Tanaka Y;Gong JP;Yasuda K;Tohyama H
最近，一些动物研究发现，通过在缺损底部植入由聚-（2-丙烯酰胺基-2-甲基丙磺酸）（PAMPS）和聚-（N，N '-二甲基丙烯酰胺）（PDMAAm）组成的合成双网（DN）水凝胶，可以在体内在大的骨软骨缺损内诱导自发的透明软骨再生。然而，水凝胶对透明软骨再生的影响仍然无法解释。本研究的目的是研究C3 H10 T1/2细胞在PAMPS/PDMAAmDN凝胶上的软骨分化。将1.0 × 105的C_3H_（10）T_1/2细胞在聚苯乙烯组织培养皿中的PAMPS/PDMAAm DN凝胶上培养或直接在聚苯乙烯组织培养皿上培养。我们比较了在PAMPS/PDMAAm DN凝胶上培养的细胞与在聚苯乙烯培养皿上培养的细胞的形态学（相差显微镜）、聚集蛋白聚糖、I型胶原、II型胶原、Sox 9和骨钙素的mRNA表达（实时荧光定量PCR）以及II型胶原的局部表达（免疫细胞化学）。C3 H10 T1/2细胞在PAMPS/PDMAAm DN凝胶上培养7天后形成局灶性粘连，迅速聚集并形成大结节，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，培养在PAMPS/PDMAAm DN凝胶上的C3 H10 T1/2细胞在蛋白水平上表达更多的II型胶原，与聚苯乙烯培养皿上的细胞相比。目前的研究表明，PAMPS/PDMAAm DN凝胶增强C3 H10 T1/2细胞的软骨形成，其功能类似于间充质干细胞。这表明来自骨髓的间充质干细胞有助于在植入PAMPS/PDMAAm DN凝胶后在大的骨软骨缺损中体内自发的透明软骨再生。本文的在线版本（doi：10.1186/1471-2474-15-320）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1186/1471-2474-15-320) contains supplementary material, which is available to authorized users.
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