Purified fraction of EMD inhibits the proliferation of 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 examined the mechanisms by which EMD inhibited the proliferation of epithelial cells. Methods: The lyophilized EMD was dissolved in 0.1% trifluoroacetic acid (TFA). HPLC was carried out using a POROS/HQ20 column and Waters system C18 column. Epithelial cell line, GE-1 was cultured with EMD or its 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 characteristics of the cells were 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 by a flow cytometer. Results: Purified EMD fractions were found to suppress the proliferation of GE-1 cells by WST-1 assay. When cultured with EMD or its purified fraction for 24-48 h, the percentage of cells with hypodiploid DNA increased. To confirm the property of apoptotic cell death in the presence of EMD preparations, we analyzed with a combination of AnnexinⅤand PI. The results indicate that GE-1 cells occur apoptosis. From the result of the silver stain and LC-MS/MS, the protein inhibition of proliferation seemed to be ameloblastin. 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. Interestingly, our results suggest that ameloblastin has a valuable effect in inhibition of down-growth of epithelial cells.