Cells, signaling molecules and three-dimensional (3D) scaffolds are the major contributors to the in vitro regeneration of cartilage. This study evaluates the differentiation of mesenchymal stem cells to chondrocytes, in a 3D semi-interpenetrating polymer network (semi-IPN) scaffold that gives an appropriate niche for chondrogenic differentiation. The 3D semi-IPN scaffold poly(vinyl alcohol) and poly(caprolactone) mimics the properties of extracellular matrix of native cartilage. The chondrogenic differentiation of mesenchymal stem cells on the 3D scaffolds is carried out by supplementing signaling molecules like TGFbeta1, TGFbeta3, and BMP2 individually and in two different combinations. The results indicate that each growth factor supplement or combinations showed a different influence on cell morphology, overall distribution of cells, and secretion of cartilage specific molecules. We conclude from our results, that a combination of TGFbeta3 and BMP2 promotes better differentiation of mesenchymal stem cells to chondrocytes in our scaffold. This study hence points out that an appropriate combination of 3D scaffolds and signaling molecules are required in the differentiation and maintenance of the chondrogenic phenotype during in vitro regeneration of cartilage tissue. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.