Role of Protein kinase C in enamel matrix derivative (Emdogain) induced human periodontal ligament fibroblast proliferation and differentiation
Chiang, Chuen Chie
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Background. Enamel matrix derivative (EMD) or Emdogain ® has been used clinically as an adjunct to mucogingival surgeries to promote regeneration of lost or destroyed periodontal tissue. Numerous clinical studies have documented the success of Emdogain as a resorbable, implantable, osteoconductive and cementoconductive material. However, very limited information is available regarding the precise mechanism of action of EMD on pluripotent mesenchymal cells. To improve the understanding of the possible modes of action of EMD, in vitro effects of EMD on cells participating in periodontal regeneration have been conducted. Earlier studies have shown an increase in the expression of Protein kinase C (PKC), a secondary signal transduction pathway, by human periodontal fibroblasts at various concentrations of EMD. PKC are a family of cytosolic enzymes, with at least 12 known isoforms, that have profound effects on cell activity by regulating important cellular functions such as growth, proliferation and differentiation. Specifically, PKC-eta has been shown to be up regulated in EMD induced human periodontal ligament fibroblast (HPLF) proliferation and differentiation. Objective. The aim of our study was to determine whether Emdogain (EMD) induces proliferation and differentiation of human PDL fibroblasts through the PKC pathway. A PKC-eta specific inhibitor NPC 15437, was used to inhibit the expression of PKC-eta and 1 its effects on control groups and EMD treated HPLF cells. In addition, studies comparing the inhibitory effect of a general PKC inhibitor, Staurosporine, as opposed to NPC 15437 were carried out. Materials and methods. Human periodontal ligament fibroblasts were grown in culture plates in the presence of Emdogain. PKC-eta inhibitor, NPC 15437 at two different concentrations of 10 -6 and 10 -8 M, or the general PKC inhibitor, Staurosporine (40nM), was added to the culture plates after 24 hours. Time points of 24, 48 and 72 hours of inhibitor incubation were investigated. Negative controls were also assessed. Cell count assay was used to evaluate proliferation of HPLF under the different conditions. MTT assay was conducted to evaluate cell activity while alkaline phosphatase activity was calculated to assess cell differentiation. The data from the groups were statistically analyzed by ANOVA including post hoc analysis. Results. Our results showed that the PKC inhibitors affected the proliferation of the periodontal fibroblasts by a significant decrease in their metabolic activity. Cell proliferation measured by the tritiated thymidine assay showed decreased cell numbers of EMD stimulated fibroblasts in presence of Staurosporine. The PKC-eta inhibitor decreased the alkaline phosphatase levels at the 48 hour time period while the general inhibitor had a greater effect on the untreated HPLFs and 5μg/ml EMD at 72 hour incubation. Conclusion. Protein kinase C does seem to play a role in the mechanism of action of Emdogain on the growth and differentiation behavior of HPLF's. Inhibition of the specific pathway influences the response of the PDL fibroblasts by way of a decrease in cellular activity, proliferation as well as early osteogenic marker ALP.