This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The formation of enamel in the teeth of vertebrates, including rodents and man, is mediated by specific cells, the ameloblasts, that synthesize and secrete a class of small proteins, the amelogenins, into the extracellular matrices of this tissue. At locations close to the points of secretion along the so-called Tomes' processes of the ameloblasts, the amelogenins have been proposed to undergo assembly into two- or three-dimensional arrays of nanospheres, ~20-25 nm in diameter. These arrays are thought to initiate the deposition of enamel, long ribbon-like crystallites chemically identified as apatite (a calcium phosphate) and themselves organized in three-dimensions. The assembly of nanospheres and their interactions wtih calcium and phosphate ions (in the extracellular matrices of the ameloblasts) leading to nucleation and growth of enamel mineral are poorly defined and understood. Conventional electron microscopy and atomic force microscopy have been used previously to help characterize these putative events, but both techniques have been limited in their utility and the interpretation of their resulting data. The principal reasons for the limitations lie in the use of sample preparation methods that alter enamel crystal composition or structure and, in the case of conventional electron microscopy, images that are two-dimensional rather that three. High voltage electron microscopy and tomographic imaging are technical approaches that hold promise of providing novel three-dimensional information on the assembly of nanospheres, their interaction with nascent enamel crystallites, and the elaboration of the mineral ribbons. Combined with anhydrous sample preparation, including the use of ethylene glycol or cryofixation that does not interfere with mineral composition or structure, high voltage microscopy and tomographic reconstruction of collected images should be extremely powerful in circumventing the uncertain techniques of analysis that have been applied earlier. In addition to investigations of normal enamel specimens obtained from mice as well accepted models for human tooth structure, two available mutant animals will also be examined. These mice contain known genetic alterations in the structure of their constituent amelogenin proteins and, as a result, they should mediate enamel crystallites that are different from those determined by normal amelogenins. In these studies of mouse mutants, high voltage and tomography will be applied to identify putative changes in their ameloblast nanosphere assembly and enamel mineral organization. In the previous reporting period, Dr. Landis visited the RVBC to review archived data from past years.
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