关键词: Emdogain, 成骨细胞, 内皮细胞, 直接共培养, 间接共培养, 细胞增殖, 细胞分化, 细胞交联

Abstract: PURPOSE: To evaluate the effect of emdogain on proliferation and differentiation of osteoblasts and endothelial cells under different co-culture conditions, and to assess their correlationship. METHODS: Human umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG63s) were directly and indirectly co-cultured in 12-well cell culture plate at a ratio of 2∶1. The cells were treated by emdogain of different concentrations (0, 0.1, 1, 10, 50 μg/mL) for 72 h. Cell proliferation was measured by cell counter in combination with flow cytometry. Direct co-cultured HUVECs and MG63s were separated by fluorescence-activated cell sorting. The expression level of alkaline phosphatase(ALP), collagen type I(Col-1), vascular endothelial growth factor (VEGF) in MG63s, and vascular endothelial growth factor receptors Flt-1 and KDR, Von Willebrand factor (vWF), endothelial cell protein C receptor (EPCR), E-selectin, and angopoietin-2(Ang-2) in HUVECs were measured by real-time quantitative polymerase chain reaction (qPCR). Statistical analysis was performed using SPSS 24 software package. RESULTS: Under direct and indirect co-culture condition, the effect of emdogain on MG63s and HUVECs proliferation was in a concentration dependent manner. The proliferation rate of cells was reduced with the increase of emdogain concentration. The inhibitory effect of emdogain on MG63s and HUVECs proliferation was the most prominent at the concentration of 50 μg/mL. The expression of ALP and Col-1 in MG63s was enhanced with the increase of emdogain concentration. The highest level of ALP and Col-1 was observed at the concentration of 50 μg/mL, and had significant difference compared with that in other groups (P<0.01). Under conditions of same emdogain concentration, the ALP and Col-1 level in directly co-cultured MG63s was significantly higher than that in indirectly co-cultured MG63s (P<0.01). Apart from the group of 10 μg/mL, VEGF level was significantly higher under direct co-culture condition than under indirect co-culture condition (P<0.01). The gene expression level of Flt-1, KDR, vWF and E-selectin in direct co-cultured HUVECs with 50 μg/mL emdogain was significantly higher than that in indirect co-cultured HUVECs with the same emdogain concentration, as well as that in other groups of direct co-culture (P<0.01). Under conditions of indirect co-culture, EPCR and Ang-2 level was significantly higher than that of direct co-culture (P<0.05). CONCLUSIONS: The effect of emdogain on proliferation and differentiation of co-cultured MG63s and HUVECs depends on its concentration, 50 μg/mL could be the key concentration. Under the condition of direct co-culture, 50 μg/mL emdogain inhibits the proliferation of MG63s and HUVECs, whereas promotes the differentiation of MG63s and HUVECs simultaneously. This effect is stronger than that under other conditions, cell-to-cell communication may play an important role.

Key words: Emdogain, Osteoblasts, Endothelial cells, Direct co-culture, Indirect co-culture, Cell proliferation, Cell differentiation, Cell communication