A double-chamber bioreactor has been developed to generate a tissue-engineered osteochondral composite (TEOC). However, a TEOC generally requires two types of cells (i.e. chondrogenic and osteogenic cells). Therefore, the capacity of mesenchymal stem cells (MSCs) as a single-cell source to work within a double-chamber bioreactor and biphasic scaffolds for generating TEOC was investigated. Compared with static culture, the double-chamber bioreactor not only can promote faster cellular proliferation, indicated by the PicoGreen dsDNA assay, SEM and confocal imaging, but also can trigger efficient chondrogenic and osteogenic differentiation of MSCs in biphasic scaffolds simultaneously, evidenced by gene expression. Thus MSCs are promising as the ideal single-cell source and the double-chamber bioreactor is an advanced culture system to generate TEOC.