The effects of enamel matrix derivative on human gingival fibroblasts in vitro study: Proliferation, differentiation and cytokines expression
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Objectives. Enamel matrix derivative (Emdogain ® , EMD) is used clinically for periodontal regeneration, where it stimulates cementum formation and promotes gingival healing. Several studies have provided histologic and radiographic evidence of periodontal regeneration with the use of Emdogain ® . However, the mechanism of action of EMD in periodontal wound healing process is not well understood. Therefore, the purpose of this study was to evaluate the effects of EMD on proliferation and differentiation of human gingival fibroblasts as well as gene and cytokine expression in these cells. Methods. HGF cells were harvested from gingival tissue of healthy patients. The cells were seeded into six well plates and were exposed to different concentrations of EMD at 5, 25, 50 and 100μgm/ml in α-MEM media for 3 days. Cultures without EMD served as the control. Alkaline phosphatase activity and cell viability/proliferation were assayed using biochemical spectrometric analyses at 3, 5 and 7 days. Total mRNA was extracted for reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the expression of several different markers of these cells. Cytokine expression was analyzed using commercially available kits (Proteoplex). The data were analyzed by ANOVA with comparisons between control and EMD treated groups. Results. Our results showed that EMD at a concentration of 25-50μgm/ml increased cell viability as evident from the MTT assay and EMD at a concentration of 100μgm/ml enhanced alkaline phosphatase activity of HGF significantly when compared to the controls (HGF only). This activity of EMD was noticeable only on days 3 and 5 of the experiments. On day 7, no comparable differences were observed between the groups. RT-PCR showed the expression collagen on day 7. Although the alkaline phosphatase gene was expressed at all three time frames (3, 5, & 7) no appreciable adjunctive effect of EMD was noticed. Expression of BSP or cbfa genes was not evident. A cytokine assay demonstrated the expression of IL-7, IL-8, IL-12 and INF-g at 3 and 5 days respectively. No other cytokines were significantly expressed. Conclusion. EMD increases cell proliferation, alkaline phosphatase activity and collagen expression in HGF with a relatively higher concentration of EMD (50-100μgm/ml) required to elicit this effect. Also evident was the limited time frame 3 and 5 days, during which EMD elicited significant effects on HGF. On day 7, there was no difference between the control and the EMD groups. The studies on cytokine expression are suggestive of an involvement of some cytokines in the mechanism of its action, but future studies are necessary to further delineate the role of each of the cytokines noted.