Owing to of the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease. Chondroitin sulfate (CS) and hyaluronan (HA) are the components of the cartilage extracellular matrix (ECM) and are known to influence the proliferation and differentiation of chondrocytes. Scaffolds composed of type-if collagen, CS, and HA may create an environment that can preserve the normal phenotype of cells to promote regeneration of cartilage-like constructs. In this investigation, we prepared and characterized 3-dimensional type-II collagen scaffolds both with and without HA and CS. Porous composite scaffolds fabricated by freeze-drying showed interconnected pores with mean diameters of 140 30 pm and porosities of 92-95% after cross-linking with genipin. After a 14-day in vitro culture, morphologically round chondrocytes were found to be uniformly distributed throughout the sponges. Expression of genes of aggrecan, type-II-collagen and cartilage oligomeric matrix protein (COMP) was statistically and significantly increased on scaffolds with CS and HA than those without CS and HA. Furthermore, there was a markedly greater accumulation of proteoglycans (PGs) on the scaffolds with CS and HA. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.