SYNOPSIS
Previously we reported a successful three-dimensional cartilage regeneration using an RWV (rotation wall vessel) bioreactor from human mesenchymal stem cells (hMSCs)(Sakai et al. J Orthop Res. 27, 2009), which is useful for better un-derstanding the initiation mechanism of chondrogenic differentiation of hMSCs and discovering its associated proteins. Entire proteome of hMSCs and spherical car-tilaginous constructs by RWV culture was comparatively analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization/ time-of-flight/mass spectrometry (MALDI-TOF-MS/MS) to determine candidate proteins associated with early chondrogenesis. Among them, there found a greatly up-regulation of COL6A1 and COL6A2 proteins on day 10, which encode matrix proteins with great changes at early stage of chondrogenesis in parallel with the increase of SOX9. A specific antibody against to the extracellular protein COL6 effectively postponed the progress of early chondrogenesis, fol-lowed by the successive reduction of extracellular COL6 in spite of a temporary increase in genes expression and up-regulation of SOX9 phosphorylation on the early stage of antibody neutralization. It suggested that the extracellular matrix protein COL6 might be a potential key molecule to involve in early events of chondrogenesis via regulating or influencing the expression of SOX9.
Key words: MSCs, chondrogenesis, Type VI collagen, comparative proteomics