Mesenchymal stem cells (MSCs) can differentiate into nucleus pulposus (NP) cells upon being co-cultured with NP cells. Important growth factors and morphogens secreted by MSCs during the differentiation process also enhance the biological properties of NP cells. In this study, the interactions between human NP cells and MSCs co-cultured in different cell-ratio (100% NP, 75% NP with 25% MSCs, 50% NP with 50% MSCs, 25% NP with 75% MSCs, and 100% MSCs) in a three-dimensional gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold were examined. Results showed that the cell proliferation was increased when NP and MSCs were co-cultured. Real-time PCR and irnmunohistochemical staining revealed that all co-culture groups produced type II collagen which represent normal NP cells but not type I collagen secreted by degenerated NP cells. FADD expression, which modulates cell survival and extracellular matrix homeostasis, was maintained in a stable status for co-cultured groups. The cultures containing 75% NP cells with 25% MSCs showed high level of collagen production and glycosaminoglycan content. Moreover, 75% NP cells with 25% MSCs had upregulated SOX9 that contributes to the improvement in type II collagen mRNA expression and protein production. These findings showed the NP/MSC cell-ratio influenced the cell functions dramatically. The co-culture of NP/MSC cells in a bioactive scaffold is a promising treatment for intervertebral disc diseases. (C) 2012 Published by Elsevier Ltd.