Inhibitory effect of EMD on gingival epithelial cells

Objectives: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding the effects of purified EMD fractions on the proliferation of gingival epithelial cells. We evaluated the influence of EMD on epithelial cell in vitro. Methods: The lyophilized EMD was dissolved in 0.1% trifluoroacetic acid (TFA). HPLC was carried out using a Waters system C18 column. Epithelial cell line, GE-1 was cultured with EMD and the purified sample, and the cell viability was examined by WST-1 assay. Bioactive fraction was eluted with a gradient of 70% acetonitrile in 0.1% TFA. The mechanism of suppression of cell growth was examined by observing change in nuclear morphology and an increase in the proportion of fragmented DNA. We determined the percentage of apoptotic GE-1 cells by examining hypodiploid DNA using a flow cytometer. Results: Purified EMD were found to suppress the proliferation of GE-1 cells by WST-1 assay, when GE-1 cells were cultured with EMD and the purified fractions for 48 h. When being cultured with EMD and the purified fraction for 24-48 h, the percentage of cells with hypodiploid DNA increased. To confirm the nature of apoptotic cell death in the presence of EMD preparations, we analyzed with a combination of Annexin Vand PI. The results indicate that GE-1 cells undergo apoptosis. Conclusions: In our previous study, we purified some fractions that induced osteoclast formation in mouse marrow cultures. In this study, we purified other biological active fractions in EMD that inhibited the proliferation of epithelial cells. These results suggest that EMD has a beneficial effect in down-growth of epithelial cells, periodontal wound healing and regeneration.