STUDY OF THE FORMATION OF ENAMEL CRYSTALLITES BY AMELOGENINS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。包括啮齿动物和人类在内的脊椎动物牙齿中牙釉质的形成是由特定的细胞(成釉细胞)介导的，成釉细胞合成并分泌一类小蛋白--釉原蛋白--进入该组织的细胞外基质。在靠近成釉细胞所谓Tomes‘s突起分泌点的位置，釉原蛋白被认为可以组装成直径约20-25 nm的二维或三维纳米球阵列。这些阵列被认为开始沉积釉质，化学上被识别为磷灰石(一种磷酸钙)的长条带状微晶，并以三维方式组织起来。纳米球的组装及其与钙和磷离子的相互作用(在成釉细胞的细胞外基质中)导致釉质矿物的成核和生长，目前尚不清楚和了解。传统的电子显微镜和原子力显微镜以前曾被用来帮助描述这些假定的事件，但这两种技术在其实用性和对结果数据的解释方面都受到了限制。限制的主要原因在于使用了改变釉质晶体成分或结构的样品制备方法，在常规电子显微镜的情况下，图像是二维的而不是三维的。高压电子显微镜和层析成像是一种技术手段，有望为纳米球的组装、它们与新生的釉质微晶的相互作用以及矿物带的细化提供新的三维信息。与无水样品制备相结合，包括使用不干扰矿物成分或结构的乙二醇法或冷冻固定法，高压显微镜和采集图像的断层扫描重建在规避早先应用的不确定分析技术方面应该是极其强大的。除了对从小鼠身上获得的正常牙釉质样本以及公认的人类牙齿结构模型进行调查外，还将检查两种可用的突变动物。这些小鼠在其组成的釉原蛋白的结构中存在已知的遗传变化，因此，它们应该调节不同于正常釉原蛋白的釉质微晶。在这些对小鼠突变体的研究中，高压和断层扫描将被用来确定它们的成釉细胞纳米球组装和釉质矿物组织的假定变化。在上一个报告所述期间，兰迪斯博士访问了加拿大皇家VBC，审查了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.