Enamel matrix derivative (EMD) has been shown to enhance both soft tissue healing and regeneration of the periodontium, but the mechanisms of this action are still unknown. It is assumed that amelogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate specific cellular effects of EMD with different molecular weight fractions following size exclusion chromatography.
Methods and Materials
Freshly dissolved EMD was fractionated by gel filtration and forty-five 7ml fractions collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on differentiation of osteoprogenitor cells (C2C12) and proliferation and differentiation of human microvascular endothelial cells (HMVEC). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after pre-incubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels co-polymerized with denatured type I collagen to determine gelatinolytic activities in each fraction.
EMD fractionated into three major protein peaks following size exclusion chromatography with Sephadex G-100. Peak I was associated with the column void volume, while peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with both peak II and peak III for the microvascular cells. Differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak II. The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than amelogenin or other unknown proteins. Gelatinolytic activities were detected in the early fractions of peaks 1 and 2 of gel fractionated EMD.
The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), while effects on proliferation and angiogenesis are associated with lower molecular weight species present in peak II and peak III. Finally, unheated EMD displays gelatinolytic activities that are also detectable following size exclusion separation of EMD constituents. The masses of these activities were consistent with those reported for latent and active MMP-20.