Abstract
Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2–5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.
Highlights
The goal of regenerative periodontal therapy is the reconstitution of the lost periodontal structures [1,2,3]
enamel matrix derivative (EMD) significantly increased COL1 expression, a larger increase was observed in MC3T3-E1 pre osteoblast cell-line compared to the other cell types (Fig. 2A)
This effect was further supported by OC mRNA data where almost a 4 fold increase was observed for MC3T3-E1 cells treated with EMD compared to its control whereas less than a 2 fold increase was observed in mature osteoblast cell lines including ROS and MG63 cells and primary HAO (Fig. 2C)
Summary
The goal of regenerative periodontal therapy is the reconstitution of the lost periodontal structures (i.e. the new formation of root cementum, periodontal ligament and alveolar bone) [1,2,3]. Initial in vitro studies demonstrated that PDL cells grown on dentin slices were unable to form cementum without specific EMPs demonstrating the critical importance of EMPs during cementogenesis [19] These proteins have since been formulated into an enamel matrix derivative (EMD) for clinical application (Emdogain, Institut Straumann AG, Basel, Switzerland). The major components of EMD are amelogenins a family of hydrophobic proteins derived from different splice variants and controlled by post-secretory processing from a single gene that account for more than 95% of the total protein content [20] These proteins self-assemble into supramolecular aggregates that form an insoluble extracellular matrix that function to control the ultrastructural organization of the developing enamel crystallites [20]. While a large number of studies have demonstrated that EMD promotes both cell growth (proliferation) and differentiation [21,22,23,24,25,26,27,28,29,30,31,32,33,34], others have failed to show any additional benefit towards differentiation [35,36,37,38] and a small number have demonstrated an inhibitory effect on either cell proliferation or differentiation [39,40,41]
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