Abstract
Introduction
Novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) have been shown to be putative biomarkers of progenitor/stem cell sub-populations (Hayes et al., 2007; Dowthwaite et al., 2005). Also, recent studies show that unique CS sulphation motifs are localized in putative stem/progenitor cell niches at sites of incipient articular cartilage & other musculoskeletal tissues (Hayes et al., 2011), which indicates their potential importance in cell differentiation during development. In this study, we investigated the importance of CS in the differentiation of bone marrow stem cells to the chondrogenic phenotype in vitro using p-nitrophenyl xyloside (PNPX) as a competitive inhibitor of CS substitution on matrix PGs.
Methods
Bovine bone marrow stem cells (BMSCs) were isolated from 7-day-old cow hock joints and cultured as monolayer for 4 weeks with chondrogenic medium ± 0.25mM PNPX. DMMB assay, real-time PCR, Western Blotting & immunohistochemistry (IHC) were used to analysis the chondrogenic markers. The expression and distribution of structural CS proteoglycans (CS-PGs) were analysed by immunofluorescent staining combined with confocal microscopy scanning.
Results
BMSCs cultured in chondrogenic medium started to aggregate and form mini-cell beads in 3 days and these mini cell beads clustered together to form a large single alcian blue positive cartilaginous cell bead in 2-4 weeks, indicative of the chondrogenesis. In contrast, there was an apparent delay in the cell bead formation in the BMSCs cultured with PNPX. Moreover, PNPX significantly inhibited or delayed the expression of chondrogenic markers including aggrecan, SOX-9 & type II collagen gene and/or protein expression. Furthermore, IHC analyses showed that a decreased expression of native CS sulphation epitopes in chondrogenic media + PNPX, suggesting the importance of their role in allowing the chondrogenic differentiation to occur.
Discussion
These results suggest that CS sulphation motifs play an important role in the differentiation of BMSCs into chondrocytes. The precise mechanism is not known, but CS sulphation motifs may be involved in the growth factor presentation needed for cell differentiation that leads to cell aggregation and extracellular matrix-cell interactions during chondrogenesis.
