Figure 3.

Co-treatment of BMP2, BMP4, and TGFβ-1 induces ameloblastic cytodifferentiation in the gingival epithelial cells. (A) Microscopic observation of cellular morphology of cells treated with cytokines for 7 days. a, cells without treatment; b, cells treated with BMP4; c, cells co-treated with BMP4 and BMP2; d, cells treated with TGFβ-1; e, cells co-treated with BMP4, BMP2, and TGFβ-1. (B) Relative mRNA expressions of amelogenic and osteogenic markers in cells treated with cytokines. mRNA expression was analyzed by qPCR as described in the Materials and Methods. See also Table S1. a, expression of amelogenin; b, expression of enamelin; c, expression of ameloblastin; d, expression of bone sialoprotein; e, expression of osteocalcin; f, expression of osteopontin; g, expression of KLK4; h, expression of MMP20. 1, control without treatment; 2, treatment with BMP2; 3, treatment with BMP4; 4, co-treatment with BMP4 and BMP2; 5, treatment with TGFβ-1; 6, co-treatment with BMP4, BMP2, and TGFβ-1. The data were originated from average value of 3 individual cultures. Statistical significance of *P < 0.1, **P < 0.01, or ***P < 0.001 was determined by using Student t-test. (C,D) Estimation of alkaline phosphatase activity and mineralization efficiency. Epithelial cells were incubated in KGM containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 5 μM dexamethasone for 7~14 days after ameloblastic cytodifferentiation. The data were originated from average value of 2 individual cultures. Statistical significance of *P < 0.1, **P < 0.01, or ***P < 0.001 was determined by using Student t-test. 1, without treatment; 2, co-treatment with BMP4 and BMP2; 3, treatment with TGFβ-1; 4, co-treatment with BMP4, BMP2 & TGFβ-1; 5, control of buffer only.