This study investigated whether a three-dimensional (3D) fibrin gel-polyurethane scaffold composite can provide an environment for chondrogenesis of human bone marrow mesenchymal stem cells (hMSCs) that is as supportive as pellet culture, which is an established model for evaluating chondrogenesis. Pellet culture was carried out in serum-free medium in the absence or presence of transforming growth factor beta 1 (TGF-beta1) and dexamethasone. hMSCs were seeded into a fibrin gel-biodegradable polyurethane scaffold at cell densities of 2 x 10(6), 5 x 10(6), and 10 x 10(6) cells per scaffold and cultured in serum-free medium supplemented with TGF-beta1 and dexamethasone. With comparable proteoglycan synthesis and type I and type X collagen gene expression levels, scaffolds seeded with 5 x 10(6) cells expressed higher type II collagen and aggrecan gene transcripts than pellets on day 14. The deposition of proteoglycan and type II collagen was detected on the top layer of scaffolds seeded with 10 x 10(6) cells and was more evenly distributed in the scaffolds seeded with 5 x 10(6) cells. The scaffold composite culture system shows chondrogenesis of hMSCs comparable with that of pellet culture. Initial cell seeding density influences the ability and process of hMSC chondrogenesis. This study founded a basic system for cartilage neo-tissue formation in vitro.