The inhibition of glycosaminoglycan incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars

Abstract

The extracellular matrix (ECM) contains a variety of complex macromolecules including proteoglycans (PGs) and glycosaminoglycans (GAGs). PG consists of a protein core with covalently attached carbohydrate side chains called GAGs. Several PGs, including versican, biglycan, decorin and syndecan are involved in odontogenesis while the role of GAGs in those PGs in this process remains unclarified. The purpose of this study was to investigate the influence of GAGs on tooth development. The mandibular first molars at early bell stage were cultivated with or without 4-methylumbelliferyl-β-d-xyloside (Xyl-MU). The cultured tooth germs were metabolically labelled with [³⁵S] Na₂SO₄, then PGs in tooth germs and cultured medium were extracted separately and analyzed by gel filtration. Morphological changes were evaluated on days 2, 4, 6, and histological changes were examined by hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). Related proteins and genes of cytodifferentiation were further examined by immunohistochemistry (IHC) and quantitive real-time PCR (qPCR) respectively. Meanwhile, BrdU incorporation assay was used to explore the effect of Xyl-MU on the cell proliferation of cultured tooth germs. The results demonstrated that the incorporation of GAGs to PGs in cultured tooth germs was heavily inhibited by Xyl-MU. Accompanied by the inhibition of GAGs incorporation, Xyl-MU altered tooth morphogenesis and delayed the differentiation of ameloblasts and odontoblasts. Proliferation of inner enamel epithelium (IEE) was also inhibited. Therefore, we draw a conclusion that the inhibition of GAGs incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

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