In this study, transgenic effects of combination of transforming growth factor (TGF) beta 3 and shRNA silencing type I collagen (Coil) on chondrogenesis of synovium-derived mesenchymal stem cells (SMSCs) were evaluated. SMSCs were infected with recombinant adenoviruses encoding TGF beta 3 (Ad-TGF beta 3) and/or anti-Col I shRNA (Ad-shRNA) separately, simultaneously (Ad-combination), or conjugately (Ad-double, mediated by one vector encoding both). The transduced SMSCs were encapsulated in alginate hydrogel and cultured for 30 days in chondrogenic medium. The expression of cartilaginous extracellular matrix components was investigated by quantitative real-time RT-PCR (qRT-PCR) and histological staining. qRT-PCR showed an up-regulation in chondrocytes marker genes such as type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP) in Ad-TGF beta 3, Ad-double, and Ad-combination groups on day 30. Whereas, Ad-TGF beta 3 treatment induced significant elevation in Coil, which could be largely resisted by anti-Col I shRNA functionality. Histological and immunohistochemical staining results were consistent with our qRT-PCR data. These results demonstrate that the application of combinational adenoviral vector-mediated transgenic TGF beta 3 and shRNA targeting Col 1 possesses the potential in promoting the chondrogenic differentiation of SMSCs as well as inhibiting the formation of fibrocartilage. Biotechnol. Bioeng. 2010;106: 818-828. (C) 2010 Wiley Periodicals, Inc.