Biological integration of cartilage grafts with subchondral bone remains a significant clinical challenge. We hypothesize that interaction between osteoblasts and chondrocytes is important in regenerating the osteochondral interface on tissue-engineered osteochondral grafts. We describe here a sequential co-culturing model which permits cell-cell contact and paracrine interaction between osteoblast and chondrocytes in 3-D culture. This model was used to determine the effects of co-culture on the phenotypic maintenance of osteoblasts and chondrocytes. It was found that while chondrocytes synthesized a type II collagen and glycosaminoglycan (GAG) matrix, GAG deposition was significantly lower in co-culture. Alkaline phosphatase activity was maintained in osteoblasts, but cell-mediated mineralization in co-culture was markedly lower compared to osteoblast controls. These results collectively suggest that interactions between osteoblasts and chondrocytes modulate cell phenotypes, and the importance of these interactions on osteochondral interface regeneration will be explored in future studies. (c) 2005 Elsevier Inc. All rights reserved.