The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells

Chondroitin/dermatan sulphate (CS/DS) sulphation motifs on cell and extracellular matrix proteoglycans (PGs) within stem/progenitor cell niches are involved in modulating cell phenotype during the development of many musculoskeletal connective tissues. Here, we investigate the importance of CS/DS chains and their motifs in the chondrogenic differentiation of bone marrow mesenchymal stem cells (bMSCs), using p-nitrophenyl xyloside (PNPX) as a competitive acceptor of CS/DS substitution on PGs. Comparison of cultures grown in control chondrogenic medium, with those grown in the presence of PNPX showed that PNPX delayed the onset of chondrogenesis, characterised by cell rounding and aggregation into spheroidal beads. PNPX reduced gene expression of SOX-9, aggrecan and collagen type II, and caused reduced levels of collagen type II protein. PNPX-treated cultures also showed delayed expression of a native CS/DS sulphation motif epitope recognised by antibody 6C3. This epitope appeared associated with a range of PGs, particularly biglycan, and its close association was lost after PNPX treatment. Overall our data show that perturbation of PG glycosylation with CS/DS GAGs using PNPX significantly delays the onset of chondrogenic differentiation of bMSCs, highlighting the importance of CS/DS during the initial stages of chondrogenesis. The delayed expression of the CS/DS sulphation motif recognised by 6C3 suggests that this motif, in particular, may have early involvement in chondrogenesis. The mechanism(s) by which CS/DS chains on PGs contribute to early chondrogenic events is unknown; however, they may be involved in morphogenetic signalling through the capture and cellular presentation of soluble bioactive molecules (e.g. growth factors).