Background: Porcine enamel matrix derivative (EMD) has a clinical use in facilitating periodontal healing by enhancing the regeneration of alveolar bone, cementum, and periodontal ligament. The mechanism of clinical use has not been elucidated, but in vitro studies suggest that EMD may enhance healing, in part, by stimulating angiogenesis. This study analyzes the effect of EMD on the production of blood vessels in the chorioallantoic membrane (CAM) of the developing chicken egg.
Methods: Various amounts of EMD ranging from 15 to 125 ng/5 microl were pipetted onto a 3-mm diameter x 2-mm thick bioabsorbable hemostatic gelatin and placed onto the surface of the CAM. Recombinant human amelogenin and a purified 5-kDa protein fraction derived from EMD were also tested at various amounts ranging from 15 to 62 ng/5 microl. A mixture of fibroblast growth factor and vascular endothelial growth factor served as the positive control. The negative control was 0.9% saline. A histologic examination of the interface of the gelatin and CAM was performed, evaluating for new blood-vessel formation on an ordinal scale of 0 to 3. Non-parametric statistical analyses were applied to compare test groups with the negative controls.
Results: CAM treated with EMD displayed moderate vascularity as indicated by a maximal score of 3.0 +/- 0.05 (mean +/- SEM). This compared favorably to the degree of vascularity stimulated by the mixture of fibroblast growth factor and vascular endothelial growth factor, which had a score of 3.0 +/- 0.05. Interestingly, the stimulation of angiogenesis by EMD was significant only at the lowest amounts tested. At the higher amounts, vascularity was reduced and not significantly different from the negative control. Vascularity was also increased by recombinant human amelogenin as indicated by a maximal score of 2.9 +/- 0.14. By contrast, there was only mild vascularity in sections treated with the negative control as indicated by a score of 1.7 +/- 0.4. The vascularity of the 5-kDa EMD-protein fraction was not different from the negative-control group (2.5 +/- 0.5 versus 1.7 +/- 0.4, respectively).
Conclusions: EMD stimulates angiogenesis in the CAM model. As a heterogeneous mixture of extracellular matrix components, EMD may have multiple biologic functions, but it is likely that at least part of the explanation for its observed positive clinical effects may be the stimulation of angiogenesis. The fact that vascularity was also increased by recombinant human amelogenin raises the possibility that this 28.9-kDa protein may be the source of the angiogenic activity because it is the predominant protein of the EMD mixture. These results, taken together with results from previously reported in vitro studies, suggest that EMD may increase angiogenesis directly and/or indirectly at the wound-healing site.