STUDY OF THE FORMATION OF ENAMEL CRYSTALLITES BY AMELOGENINS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。包括啮齿动物和人类在内的脊椎动物牙齿中釉质的形成是由特定的成釉细胞介导的，成釉细胞合成并分泌一类小蛋白质，即釉原蛋白，进入该组织的细胞外基质。在靠近沿着成釉细胞的所谓Tomes'过程的分泌点的位置处，已经提出釉原蛋白经历组装成直径约20-25 nm的纳米球的二维或三维阵列。这些阵列被认为是启动釉质的沉积，长带状微晶化学鉴定为磷灰石（磷酸钙），并在三维空间中组织起来。纳米球的组装及其与钙离子和磷酸根离子（在成釉细胞的细胞外基质中）的相互作用导致釉质矿物质的成核和生长的定义和理解很少。传统的电子显微镜和原子力显微镜以前已被用来帮助表征这些推定的事件，但这两种技术已被限制在其效用和解释其产生的数据。限制的主要原因在于使用的样品制备方法，改变釉质晶体的组成或结构，并在传统的电子显微镜的情况下，图像是二维的，而不是三个。高压电子显微镜和断层成像的技术方法，有望提供新的三维信息的组装纳米球，它们与新生的釉质微晶的相互作用，以及制定的矿物带。 与无水样品制备相结合，包括使用乙二醇或不干扰矿物成分或结构的冷冻固定，高压显微镜和所收集图像的断层重建应该是非常强大的，以规避先前应用的不确定的分析技术。除了研究从小鼠获得的正常牙釉质样本以及公认的人类牙齿结构模型外，还将检查两种可用的突变动物。这些小鼠在其组成釉原蛋白的结构中含有已知的遗传改变，因此，它们应该介导不同于由正常釉原蛋白确定的釉质微晶。在这些小鼠突变体的研究中，高电压和断层扫描将被应用于确定其成釉细胞纳米球组装和釉质矿物质组织的推定变化。在上一个报告期内，Landis博士访问了RVBC，以审查过去几年的存档数据。

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.