Enamel matrix proteins regulate hypoxia-induced cellular biobehavior and osteogenic differentiation in human periodontal ligament cells

Abstract

Hypoxia is a crucial microenvironment for inflamed periodontal tissue and periodontal wound healing. Enamel matrix proteins (EMPs) potentially can promote the formation of new periodontium. The effects of EMPs on periodontal ligament cells under hypoxia, however, remain unclear. We investigated the effects of EMPs on cellular biobehavior and osteogenic differentiation of human periodontal ligament cells (hPDLCs) under hypoxia. Under cobalt chloride (CoCl2)-induced hypoxia, cellular biobehavior of hPDLCs, including proliferation, attachment, spreading, and migration with or without EMPs, was evaluated by 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), cell counting, spreading area measurement and wound scratch assay. The osteogenic activity of hPDLCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining (ARS). The expressions of osteogenic genes including runt related transcription factor 2 (Runx2), ALP, osteocalcin (OCN) and collagen type I (Col-I) were detected using real time quantitative PCR, western blot and immunocytochemistry assays. The biobehavior and osteogenic differentiation of hPDLCs were inhibited significantly under hypoxia. EMPs have no effect on cell proliferation under mimicked hypoxia. EMPs partly reversed the inhibitory effects of hypoxia, however, for other cellular biobehavior including attachment, spreading and migration, and markedly up-regulated osteogenic differentiation activities including ALP, mineralization ability and the expressions of osteogenic genes such as Runx2, ALP, osteocalcin, and collagen type I in hPDLCs under hypoxia. EMPs attenuate the hypoxic injury to cellular biobehavior and osteogenic differentiation in hPDLCs under hypoxia.