Background: Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown.
Methods: Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools.
Results: One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-µg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin�leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides.
Conclusions: These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.
Enamel matrix derivative (EMD) is a regenerative protein extract derived from the developing tooth buds of 6-month-old piglets. Clinically, the commercially available EMD has demonstrated relevant benefits for both hard and soft tissue and is used for a myriad of regenerative purposes. In randomized controlled clinical trials, EMD has demonstrated significant clinical attachment gain, periodontal disease reduction, and radiographic bone gain compared to controls in the treatment of intrabony defects.1 In vivo studies with EMD demonstrated new cementum, alveolar bone, and periodontal ligament (PDL) formation.1,2 Additionally, Cochran et al.3 showed new alveolar bone formation, PDL formation, and cementum in all specimens treated with EMD.
Although EMD is in wide clinical use, questions still arise about the substance or substances within it that are responsible for the clinical effects. EMD is enriched with several abundant enamel matrix proteins, which have been subjected to a wide variety of experiments.4 A family of hydrophobic low-molecular-weight proteins known as amelogenins makes up a major part of EMD.5 The remaining part is composed of ameloblastin (also known as sheathlin or amelin), enamelin, tuftelin, enamelysin (matrix metalloproteinase-20), and enamel matrix serum proteinase 1 (EMSP-1, also known as kallikrein-4).6-12 EMD has been shown to stimulate bone-related proteins and gene expression in in vitro cultures of PDL cells13 and in osteoblast models14 and, more recently, in osteoblasts, bone marrow stromal cells, and PDL cell models in vitro.15 In its clinical use, propylene glycol alginate is added to enhance EMD precipitation, exposing the PDL cells and osteoblast and cementoblast stem cells to the EMD, allowing for matrix�cell interactions.16 This precipitation may mimic the enamel matrix protein deposition by Hertwig�s epithelial root sheath on the peripheral dentin during the original formation of the periodontium, leading to the formation of new cementum and PDL cells.
In this series of studies, the authors evaluate the osteogenic and potential angiogenic properties of EMD at various concentrations along with size-fractionated pools of EMD, using the in vivo calvarial injection assay for osteogenesis.17 This simple assay simulates activation of a periosteal bone surface and can be used quantitatively to evaluate any compound or protein mix on bone formation processes.
The hypothesis is that a unique combination of biologically active materials within EMD gives it the capacity to stimulate new bone formation. Therefore, the objectives of these investigations are: 1) to determine the osteoinductive potential of different protein-peptide pools of EMD compared to unfractionated (complete) EMD; 2) to determine if there is a dose response when testing a known osteoinductive EMD protein pool; 3) to determine the mechanisms of how EMD complete protein or purified components of EMD protein stimulate new bone formation in this model; and 4) to determine the specific protein composition of the major osteoinductive EMD pools.
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