Osteogenic differentiation enhances the MC3T3-E1 secretion of glycosaminoglycans with an affinity for basic fibroblast growth factor and bone morphogenetic protein-2

Abstract

Introduction: It is generally recognized that a wide variety of morphogens and growth factors bind to the glycosaminoglycans (GAG) of proteoglycans (PG) to affect their bioavailability to ligands. Many growth factors involving in osteogenic differentiation require the GAG side chains to facilitate their interaction to the cell surface receptors and the biosynthesis of osteogenic proteins. The objective of this study is to investigate the secretion of GAG from MC3T3-E1 pre-osteoblasts of a murine bone calvaria during the osteogenic differentiation. Methods: When MC3T3-E1 cells were cultured in the differentiation medium (DM) containing bone morphogenetic protein (BMP)-2, the alkaline phosphatase activity, calcium content and the amount of basic fibroblast growth factor (bFGF)- or BMP-2-bound sulfated GAG were determined. Moreover, the disaccharide analysis of the GAG was performed. Results: When MC3T3-E1 cells were cultured in the differentiation medium (DM) containing bone morphogenetic protein (BMP)-2, the alkaline phosphatase activity and calcium content were significantly enhanced compared with those of the BMP-2-free DM and normal medium with or without BMP-2. Significantly higher amount of GAG secreted was detected for cells cultured in the DM containing BMP-2, in contrast to other culture conditions. The GAG secreted had an affinity for BMP-2 and basic fibroblast growth factor (bFGF). The disaccharide analysis of GAG demonstrated that the percentage of ΔHexA α1,4GlcNSO3 and ΔHexA (2-OSO3) α1,4GlcNSO3 increased, but that of ΔHexA α1,4GlcNSO3(6-OSO3) decreased (ΔHexA: unsaturated uronic acid residue, GlcNSO3: N-sulfated glucosamine, ΔHexA (2-OSO3): unsaturated uronic acid 2-sulfate residue, GlcNSO3(6-OSO3): N-sulfated glucosamine 6-sulfated). Conclusion: It was found that the osteogenic differentiation allowed cells to enhance the secretion of GAG with an affinity for BMP-2 and bFGF. Keywords: Osteogenic differentiation, Glycosaminoglycans, Basic fibroblast growth factor, Bone morphogenetic protein-2, Secretion, Disaccharide