Enamel Matrix Derivative Subfractions Have Direct Effects on Osteoblasts

Objective: Porcine fetal tooth germ enamel matrix derivative (EMD) is a protein complex enriched in amelogenins, which corresponds to the bioactive constituents of Emdogain® (Institut Straumann AG). Analysis of EMD by high performance liquid chromatography revealed the presence of three main components: (1) a 20 kDa protein, (2) two proteins of 12 and 9 kDa, and (3) a 5 kDa peptide. Two of these components (20 kDa, 5 kDa) have been purified and characterized. The 20 kDa protein corresponds to the full length amelogenin protein and the 5 kDa peptide is the N-terminal part of this protein. The aim of the present study was to examine the effect of these two EMD components on osteoblasts. Cell responses were compared to the effects of the EMD complex and to recombinant human amelogenin (rhAmel) over-expressed in Escherichia coli. Methods: Confluent cultures of MG63 cells and normal human osteoblasts were treated with EMD, rhAmel, or 20 kDa protein (0.01-100µg/ml) or 5 kDa peptide (0.1-250µg/ml) for 24 hours. Effects on DNA content and alkaline phosphatase specific activity (ALP), and osteocalcin, osteoprotegerin, and vascular endothelial growth factor levels in the conditioned media were determined. Results: The 5 kDa peptide reduced DNA content of MG63 cells in a dose-dependent manner and increased alkaline phosphatase and osteocalcin with peak increases at 10 mg/ml. The peptide also increased OPG and VEGF in a dose-dependent manner. The effects of the 5 kDa peptide were similar to those of the 20 kDa protein, EMD, and rhAmel. Moreover, normal human osteoblasts responded in a similar manner to MG63 cells. Conclusions: These results indicate that the 5 kDa component of Emdogain possesses osteogenic activities and that the osteogenic effects of amelogenin may be due the N-terminal region of the protein. AR052102, the ITI Foundation, and Institut Straumann.