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制备软骨组织的研究进展迅速。然而，一些患者需要一种新的骨软骨植入技术，而不需要人工或动物组织来源的支架。本研究的目的是建立软骨细胞和骨髓源性基质细胞在体外形成的双层复合移植物用于骨软骨植入。本研究采用海藻酸盐回收软骨细胞法（ARC法），这是一种新型的两步无支架培养系统来产生软骨组织。将兔骨髓基质细胞高密度植入组织培养植入物上，利用ARC法在植入物上进行三维软骨细胞培养。获得由上软骨层和下骨髓细胞层组成的双层组织，进行生化和组织学分析。随着骨髓细胞数量的增加，形成的组织中蛋白多糖含量降低，而胶原蛋白含量变化不显著。培养4周的复合移植物足够坚硬，可以用镊子夹住，比单纯由软骨细胞组成的常规ARC组织更硬。红花素- o和抗II型胶原抗体对组织软骨层进行染色。裸鼠皮肤下双分子层结构在移植后4周保持不变，骨髓细胞层未见骨化。本研究首次实现了骨软骨复合材料在体外不使用支架而形成骨软骨复合材料的可能性。对于临床应用，可能需要进一步的研究，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.