Effects of culture on PAMPS/PDMAAm double-network gel on chondrogenic differentiation of mouse C3H10T1/2 cells: in vitro experimental study.

Yusuke Inagaki,Takayuki Kurokawa,Kazunori Yasuda,Jian P Gong,Yasuhito Tanaka,Harukazu Tohyama,Nobuto Kitamura

doi:10.1186/1471-2474-15-320

Yusuke Inagaki, Takayuki Kurokawa + Show 5 more

Open Access

https://doi.org/10.1186/1471-2474-15-320

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Abstract

BackgroundRecently, 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.MethodsC3H10T1/2 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.ResultsC3H10T1/2 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-320) contains supplementary material, which is available to authorized users.

Highlights

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
Real-time RT-polymerase chain reaction (PCR) analysis analysis of variance (ANOVA) showed that expression of aggrecan, type I, type II collagens, Sox9 and osteocalcin genes was significantly greater in the cells cultured on the PAMPS/PDMAAm DN gels than on polystyrene dishes
The present study showed that C3H10T1/2 cells cultured on PAMPS/PDMAAm DN gels formed focal adhesions, aggregated rapidly and developed into large nodules within 7 days, while cells cultured on the polystyrene surface did not form any nodules during the same period

Summary

Introduction

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. The purpose of this study was to investigate the chondrogenic differentiation of C3H10T1/2 cells on PAMPS/PDMAAm DN gel. We found that spontaneous hyaline cartilage regeneration can be induced in vivo within a large osteochondral defect in the rabbit at 4 weeks by implanting a synthetic double-network (DN) hydrogel composed of poly-(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) and poly-(N,N’-dimethyl acrylamide) (PDMAAm) at the bottom of the defect, leaving a 1.5- to 3.5-mm deep vacant space within the defect [3,4]. Kwon et al reported that changing the ratio of AMPS and DMAAm could modulate the proliferation and differentiation of chondrogenic ATDC5 cells [5]

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