In vitro formation of bilayer composite graft from chondrocytes and bone-marrow-derived stromal cells

近年来，利用胶原凝胶和三维聚合物等支架材料在体外培养软骨组织方面取得了较好的效果。然而，一些患者渴望一种新的骨软骨植入技术，而不需要人工或动物组织衍生的支架。本研究的目的是建立体外形成软骨细胞和骨髓基质细胞的双层复合移植物，用于骨软骨植入。本研究采用藻酸盐回收软骨细胞法(ARC法)，这是一种新型的两步法无支架软骨组织培养体系。将兔骨髓基质细胞高密度接种于组织培养片上，然后用ARC法进行三维软骨细胞培养。取上软骨层和下骨髓细胞层的双层组织，进行生化和组织学检测。随着骨髓细胞数目的增加，形成的组织中蛋白多糖含量降低，而其胶原含量变化不明显。培养4周的复合移植物足够坚硬，可以用钳子夹住，而且比普通的软骨细胞制成的ARC组织要坚硬。组织的软骨层用藏红花-O和抗II型胶原抗体染色良好。裸鼠皮下移植后4周仍保持双层结构，骨髓细胞层未见骨化。这项研究首次表明，在体外形成骨软骨复合移植物是可能的，而不使用支架。对于临床应用，可能还需要进一步的研究，特别是在骨髓细胞层实现适当的骨形成。

Recently, several strategies using scaffold, such as collagen gel and three-dimensional polymer, have been developed for the production of cartilaginous tissue in vitro. However, some patients desire a new technique of osteochondral implantation without artificial or animal-tissue-derived scaffold. The purpose of this study is to establish in vitro formation of bilayer composite graft from chondrocytes and bone-marrow-derived stromal cells for the use of osteochondral implantation. The alginate-recovered-chondrocyte method(ARC method), which is a novel two-step scaffold-free culture system to produce cartilaginous tissue, was used in this study. Stromal cells from rabbit bone marrow were seeded in high density on the tissue culture insert, followed by three-dimensional chondrocyte culture using the ARC method on the insert. Obtained bilayer tissue, consists of upper cartilage layer and lower bone marrow cell layer, were examnined with both biochemical and histological analysis. When cell number of bone marrow cells was increased, proteoglycan content of the formed tissue was decreased, whereas its collagen content did not change significantly. The composite graft cultured for 4 weeks was hard enough to be held with forceps, and stiffer than regular ARC tissue that was simply made of chondrocytes. The cartilage layer of the tissue was stained well with Safranin-O and anti-type II collagen antibody. The bilayer structure was maintained at 4 weeks after transplantation under the skin of athymic nude mice, while ossification was not observed in the layer of bone marrow cells. This study showed that, for the first time, in vitro formation of osteochondral composite graft was possible without using scaffold. For the clinical application, farther investigation may be needed, especially to achieve proper bone formation in the marrow cell layer.