Influence of biglycan on osteogenesis and matrix and mineral quality

Previous reports have demonstrated a role of biglycan (BGN) in bone morphogenetic protein (BMP)- induced osteoblast differentiation and function in vitro. In addition to, it was shown that the removal of glycosaminoglycans (GAGs) of BGN further stimulated in vitro osteoblast differentiation as determined by alkaline phosphatase activity. Objectives: To investigate whether the presence or absence of GAGs regulates known early to intermediate osteogenic markers, collagen maturation, matrix mineralization and mineral crystallinity. Methods: MC3T3-E1 cells were cultured in osteogenic media and received either glycanated BGN (G-BGN) or non-glycanated BGN (NG-BGN). Gene expression of Cbfa1/Runx2, osterix, and bone sialoprotein was examined at 7 days of culture by qRT-PCR. Cultures were differentiated to 14 days with ascorbic acid and stained with Picrosirius red (PSR) to visualize collagen organization and maturation. To assess matrix mineralization, cultures were differentiated in mineralization media with BGN forms with and without addition of recombinant BMP-2 for 21 days and stained with Alizarin Red. To quantify mineral crystallinity, cultures were differentiated 21 days and analyzed by Fourier transform infrared spectroscopy (FTIR). Results: Cbfa1/Runx2 gene expression increased in BGN-treated cultures with NG-BGN showing a more significant increase than G-BGN. Gene expression of osterix and bone sialoprotein was suppressed by G-BGN treatment while cultures treated with NG-BGN behaved in the opposite manner showing a robust increase compared to untreated control. PSR staining showed decreased amounts of mature collagen fibrils in G-BGN cultures. Alzarin red staining revealed increased matrix mineralization associated with BGN treated cultures compared to control with greater staining observed in NG-BGN treated cultures. FTIR demonstrates increased crystallinity associated with BGN treated cultures. Conclusions: These results confirm a regulatory role of BGN in bone formation particularly suggesting how GAGs of BGN selectively modulate multiple aspects of osteoblast differentiation, matrix mineralization and maturation.