A comparison of the role of protein kinase C in enamel matrix derivative and transforming growth factor-beta stimulated human gingival fibroblasts
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Aim. EMD has been advocated as a periodontal regenerative material, but its mechanism of action remains unclear. Recent studies provide evidence of the presence of several growth factors namely, TGF-β & BMPs, which are thought to play a role in the mechanism of action of EMDs. Hence, the aims of this study are to compare the effect of enamel matrix derivative and transforming growth factor - beta on human gingival fibroblasts proliferation and differentiation. In addition, the role of a PKC dependent pathway in these cells differentiation and proliferation will be investigated. Finally, the dose response effect of TGF-β on HGF will be evaluated. Methods. HGFs were cultured in α-minimum essential medium (α-MEM) with 10% fetal calf serum) and allowed to attach for 24 hours. Then, EMD 25μg/ml, 50μg/ml & TGF-β 10ng/ml were added and general PKC inhibitor 40 nM & compared to the negative control in terms of cell proliferation via a tritiated thymidine assay at 24 hours. The differentiation of cells within the treatment groups was also investigated by an alkaline phosphatase assay at 72 & 96 hours. An alkaline phosphatase assay was also run at 72 hours for the different TGF-β concentrations of 0.1, 1, 10 & 100ng/ml. Results. TGF-β & EMD showed a significant uptake of tritiated thhymidine by treated HGF; meaning increased mitogenic effect when compared to control (p<0.5). The results of the alkaline phosphatase assay show no significant effect of EMD 50μg/ml when compared to the control, an inconsistent effect was observed for EMD 25 μg/ml, and a significant decrease with TGF-β 10ng/ml. PKC inhibitor decreased the enzyme activity for both Emdogain® concentrations significantly. Finally, the different concentrations of TGF-β resulted in an insignificant increase in the enzyme activity. Conclusion. Within the limits of this study, it is clear that the enhanced soft tissue healing observed clinically after application of Emdogain® is the result of increased cellular proliferation rather than differentiation. A PKC signaling pathway might be involved in the action of HGF cells stimulated by Emdogain®, although the proliferation of these cells is not dependent on this pathway. The results do not exclude the possibility that TGF-β is an active component of EMD. Nevertheless, this effect seems to be partial and governed by the presence of other growth factors and EMPs.