Introduction

Cartilage regeneration have been attracted attentions because of the poor ability of cartilage tissues to regenerate. Three-dimensional (3D) culture of chondrocytes is considered to be advantageous for cartilage regeneration. Although it is plausible that maturation of the constructs before transplantation positively affects the chondrogenesis, matured constructs after cultures for longer periods do not necessarily result in effective cartilage regeneration. In this study, we compared different types of culture media including growth factors which are clinically available. We prepared differentiation medium containing insulin-like growth factor-1 (IGF-1), proliferation medium containing fibroblast growth factor-2 (FGF-2) and insulin, and combination of them, and compared their efficacies on chondrogenesis when used in 3D culture of engineered cartilage constructs.

Methods

Cartilage constructs were fabricated by auricular chondrocytes and atelocollagen, and they were 3D-cultured with four types of media: control medium, differentiation medium, proliferation medium, and combination medium. After 3 weeks of culture, the constructs were analyzed for cell number, gene and protein expressions and mechanical properties. The constructs were also transplanted into nude mice. After 8 weeks, the degree of cartilage regeneration was evaluated. Constructs manufactured with canine auricular chondrocytes were subjected to autologous transplantation into beagles and examined for cartilage regeneration.

Results

During 3D culture, remarkably high gene expression of type II collagen was detected in the construct cultured with the differentiation medium whereas cell apoptosis were suppressed in the proliferation medium. When transplanted into nude mice, the constructs 3D-cultured in the proliferation medium produced abundant cartilage matrices. In autologous implantation model, the construct cultured in the proliferation medium again showed better chondrogenesis than those in other media.

Conclusions

The present study indicates that 3D culture with the proliferation medium maintains the cell viability to potentiate the subsequent cartilage regeneration. Here, we propose that not only differentiation but also high cell viability accompanied by proliferation factors should be taken into account to improve cartilage regeneration.

中文翻译：

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耳廓软骨细胞三维培养中的增殖培养基促进体内有效的软骨再生。

介绍

由于软骨组织的再生能力差，软骨再生引起了人们的关注。软骨细胞的三维 (3D) 培养被认为有利于软骨再生。尽管移植前构建体的成熟对软骨形成有积极影响是合理的，但培养较长时间后成熟的构建体不一定会导致有效的软骨再生。在这项研究中，我们比较了不同类型的培养基，包括临床可用的生长因子。我们制备了含有胰岛素样生长因子-1（IGF-1）的分化培养基、含有成纤维细胞生长因子-2（FGF-2）和胰岛素的增殖培养基，以及它们的组合，

方法

软骨构建体由耳廓软骨细胞和去端胶原蛋白制成，并用四种培养基进行 3D 培养：对照培养基、分化培养基、增殖培养基和组合培养基。培养 3 周后，分析构建体的细胞数量、基因和蛋白质表达以及机械性能。还将构建体移植到裸鼠中。8周后，评估软骨再生程度。用犬耳软骨细胞制造的构建体进行自体移植到比格犬中并检查软骨再生。

结果

在 3D 培养过程中，在用分化培养基培养的构建体中检测到非常高的 II 型胶原基因表达，而在增殖培养基中细胞凋亡受到抑制。当移植到裸鼠体内时，在增殖培养基中 3D 培养的构建体产生了丰富的软骨基质。在自体植入模型中，在增殖培养基中培养的构建体再次表现出比其他培养基中更好的软骨形成。

结论

本研究表明，使用增殖培养基进行 3D 培养可维持细胞活力，从而增强随后的软骨再生。在这里，我们建议不仅要考虑分化，还要考虑伴随增殖因子的高细胞活力，以改善软骨再生。