The transmembrane proteins cdon and boc are implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration suggest that cdon/boc may play additional functions in regulating directed cell movements. We use novel and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon;boc mutant embryos. We further show that this migration phenotype is associated with defects to the differentiation of slow-twitch muscle cells, and the loss of a Col1a1a containing extracellular matrix, suggesting that neural crest defects may be a secondary consequence to defects in mesoderm development. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and suggest that the zebrafish can be used to study the function of hedgehog receptor paralogs.
OBJECTIVES/GOALS: Our objective is to investigate the antibacterial and regenerative properties of a novel AHA dental coating for the prevention and treatment of deep caries (cavities). Further, we aim to investigate and compare these properties through in vivo murine models and assessment on human saliva samples and human pulpal cells collected from clinical samples. METHODS/STUDY POPULATION: In vitro antibacterial studies were performed by collecting and culturing human salivary bacteria with AHA substrates and quantifying survival of cariogenic Sm (S. mutans). In vivo, C57BL/6 mice were treated with AHA composite fillings, infected with Sm clinical isolates, and fed a high sucrose diet with cavity formation assessment after 6 weeks. To evaluate regeneration, mice were similarly given composite with AHA or MTA (standard of care) upon pulpal exposure with regeneration quantified by microCT and histological analysis of dentin bridge formation, ALP production, and odontoblast migration. In vitro, AHA substrates were cultured with MC3T3-E1 pre-osteoblast cells and dental pulp stem cells obtained from clinical samples over 21 days, with mineralization and ALP assessed indicating osteogenesis. RESULTS/ANTICIPATED RESULTS: In vivo studies have shown the reduction of cavity formation in mice treated with AHA as well as dentin regeneration upon pulpal exposure using microCT and histological image analysis. Coinciding with these findings, AHA substrates eradicated cariogenic Sm in human saliva samples and single species cultures in vitro. Further, preliminary results in vitro have shown increased mineralization of MC3T3-E1 cells when cultured with AHA substrates in comparison to uncoated substrates. We anticipate similar increased mineralization as well as increased ALP production of human pulpal stem cells from clinical samples when cultured with AHA substrates, suggesting osteogenesis. Further, we anticipate increased odontoblast migration and ALP production upon additional analysis of in vivo tissue samples. DISCUSSION/SIGNIFICANCE: This work will elucidate the antibacterial and regenerative properties of AHA dental coatings. These results further support the translation of AHA coatings into the clinic as a novel therapeutic for the prevention and treatment of dental decay, which may overcome the limitations associated with the current treatments.
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