Publications by authors named "Megan K Pugach"

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization , as observed in enamel rods.

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Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein.

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Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness.

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The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.

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Amelogenin is the most abundant enamel protein involved in enamel mineralization. Our goal was to determine whether all three regions of amelogenin (N-terminus, C-terminus, central core) are required for enamel formation. Amelogenin RNA is alternatively spliced, resulting in at least 16 different amelogenin isoforms in mice, with M180 and LRAP expressed most abundantly.

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Purpose: The purposes of this study were to: (1) investigate adhesion through shear bond strength (SBS) testing of a resin composite bonded with a self-etching bonding system (SEB) to amelogenesis imperfecta (AI)-affected deproteinized mouse enamel or dentin; and (2) compare wild-type (WT), amelogenin null (AmelxKO), and matrix metalloproteinase-20 null (Mmp20KO) enamel and dentin phenotypes using micro-CT and nanoindentation.

Methods: Enamel incisor surfaces of WT, AmelxKO, and Mmp20KO mice were treated with SEB with and without sodium hypochlorite and tested for SBS. Incisor dentin was also treated with SEB and tested for SBS.

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A primary goal of enamel research is to understand and potentially treat or prevent enamel defects related to amelogenesis imperfecta (AI). Rodents are ideal models to assist our understanding of how enamel is formed because they are easily genetically modified, and their continuously erupting incisors display all stages of enamel development and mineralization. While numerous methods have been developed to generate and analyze genetically modified rodent enamel, it is crucial to understand the limitations and challenges associated with these methods in order to draw appropriate conclusions that can be applied translationally, to AI patient care.

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RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up-regulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene-regulatory sequences.

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The amelogenin proteins are required for normal enamel development, and the most abundant amelogenins expressed from alternatively spliced mRNAs are M180 and leucine-rich amelogenin protein (LRAP). The X-Chromosomal Amelogenin (Amelx) null [knockout (KO)] mouse has an enamel defect similar to human X-linked amelogenesis imperfecta. The disorganized enamel layer in KO mice is 10-20% of the thickness of wild-type (WT) enamel and lacks prismatic structures.

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Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoA(DN)) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoA(DN) fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy.

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Objective: To determine the effect of dentin smear layers created by various abrasives on the adhesion of a self-etching primer (SE) and total-etch (SB) bonding systems.

Methods: Polished human dentin disks were further abraded with 0.05 micro m alumina slurry, 240-, 320- or 600-grit abrasive papers, # 245 carbide, # 250.

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