Glycosaminoglycans and their sulfate derivatives differentially regulate the vitality and gene expression of osteocyte-like cell lines

Collagen and glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major components of bone structure, and collagen-GAG composites are currently being evaluated for a wide range of clinical applications. We produced native and sulfate-modified GAGs and investigated their molecular and cellular effects on osteocytes, fundamental cellular regulators of bone remodeling. GAGs were tested for their effects on viability, necrosis, apoptosis and regulation of gene expression in the murine MLO-Y4 and the rat UMR 106 – 01 cell lines, which both display properties of primary osteocytes. Native and sulfated GAGs were non-toxic and incorproated by osteocytic cells. In MLO-Y4 cells, sulfation of GAGs led to a significant inhibition of osteocyte apoptosis (-35%, p < 0.01 for highly sulfated CS and -42%, p < 0.001 for highly sulfated HA, respectively), indicating dependence on the sulfation degree and not the monosaccharide composition. Cell vitality and proliferation were not affected. In UMR 106 – 01 cells, treatment with highly sulfated HA reduced the RANKL/OPG ratio by 58% compared to its native form (p < 0.05), whereas highly sulfated CS led to 60% reduction of the RANKL/OPG ratio in comparison to its native product (p < 0.05). The expression of SOST, the gene encoding sclerostin, was reduced by 50% and 45%, respectively, by highly sulfated HA and CS compared to their native forms (p = 0.06 and p < 0.05). Furthermore, the expression of BMP-2, a marker of osteoblast differentiation was doubled after treatment with highly sulfated HA in comparison to its native form (p < 0.05). A similar trend was seen for sulfated CS. In conclusion, highly sulfated GAGs inhibit ostocyte apoptosis in vitro and promote an osteoblast-supporting gene expression profile. Whether this translates into enhanced osteoblast activity remains to be investigated.