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

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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.