EMD in Bone and Pulp Cell Mediated Mineralization

Objectives: EMD (enamel derivative proteins) have been successfully used for bone regeneration in implant studies. However, although some studies have shown potential for pulp capping, results were never as consistent. This study had the purpose to observe some underlying intracellular mechanisms and their possible relationship with the different responses from bone and pulp tissues to this material.
Methods: This study used primary human dental pulp cell (DPC) and osteoblast (OB) cultures, stimulated by EMD. BMP-2, a well-known mineralization agent, was used as control. Initially, cells were exposed to different concentrations of both substances to determine an optimal concentration for further experiments. After the optimal concentration was defined (50ng/ml of BMP-2 and 100µg/ml of EMD), cells were treated and the differentiation was confirmed by the expression of DMP-1 and DSPP for DPC and OPN and BSP for OB. Exposed cells were also analyzed by western blotting, to assess the effects of both substances on the activation of Smad, Wnt and MAPKinase pathways. A functional assay with Alizarin Red was also done to confirm the mineralization effects.
Results: Both cell lines were properly stimulated by both substances as observed by the alkaline phosphatase and gene expression assays. However, OB always presented higher ALP activity. As expected, BMP-2 clearly activated the Smad pathway on both cell types, while EMD did not promote changes in Smad 4 concentrations and only activated Smad 2/3 in DPC. No substance induced the activation of the Wnt pathway in any cell type, whereas OB had MAPKinase activation with both BMP-2 and EMD. The functional assay confirmed that both cells are active in producing mineralization nodules, more evidently for EMD treated OB.
Conclusions: It can be suggested that, due to the fact that EMD promotes MAPKinase activation only in osteoblasts, this pathway is key to induce consistent and predictable mineralization. This can explain, at least partially, the efficacy of EMD for implant use (bone regeneration), while not as adequate for pulp capping.