Phenotypic stability of articular chondrocytes in vitro: The effects of culture models, bone morphogenetic protein 2, and serum supplementation

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the st ability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone m orphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type TT, aggrecan, (V+C)(-) fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of pr ocollagen type IT and induction of procollagen type I expression. The aggre gate and pellet culture models supported matrix protein gene expression pro files more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 si gnificantly increased the expression of procollagen type IZ and aggrecan in monolayer cultures; however, other models showed comparatively little resp onse. Similarly, serum supplementation significantly down-regulated procoll agen type ZI and aggrecan expression in monolayer cultures but had less eff ect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These r esults suggest that the influence of exogenous BMP-2 and serum on expressio n of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organizatio n. Finally, the analyses of fibronectin expression suggest that V and C reg ion alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.