Enamel matrix 3D organization and maturation stage ion flow

牙釉质基质 3D 组织和成熟阶段离子流

• 批准号: 9304187
• 负责人: Felicitas B Bidlack
• 金额: $ 50.16万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304187
• 关键词: Acidity; Address; Advanced Development; Amelogenesis Imperfecta; Animals; Architecture; Bacteria; Belief; Biological; Biomimetics; Carbonates; Characteristics; Clinical Treatment; Crystal Formation; Crystallization; Data; Defect; Dental Enamel; Development; Diffusion; Dimensions; Disease; Durapatite; Electron Microscopy; Electrons; Enamel Formation; Excision; Fluorescence Microscopy; Genes; Goals; Growth; Helium; Human; Hydroxyapatites; Image; Imagery; In Situ; In Vitro; Ions; Knock-in; Knock-out; Knockout Mice; Knowledge; Label; Length; Light; Magnesium; Mass Spectrum Analysis; Microscopy; Minerals; Modeling; Molds; Movement; Mus; Mutate; Natural regeneration; Outcome Study; Phase; Phenotype; Play; Post-Translational Protein Processing; Proteins; Recombinants; Research; Resolution; Role; Sampling; Scanning Electron Microscopy; Shapes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Techniques; Testing; Thick; Thinness; Tissue Engineering; Tooth Tissue; Tooth structure; Tweens; Width; ameloblastin; amelogenin; base; calcium phosphate; enamel matrix proteins; enamelin; high resolution imaging; improved; in vivo Model; innovation; inorganic phosphate; insight; kallikrein 4; mineralization; mouse model; mutant; nanometer; prevent; protein distribution; sound; spatiotemporal; sugar; three dimensional structure; tomography; ultra high resolution

The goal of this project is to advance our understanding of enamel formation by overcoming an apparent impasse in the field. Although the need for multi-protein studies is clearly recognized, in vitro studies have heavily focused on amelogenin as the key enamel matrix protein. However, the two other matrix proteins ameloblastin and enamelin occur in much lower abundance in the matrix and have extensive posttranslational modifications. Their low abundance precludes purification from animal teeth and attempts to express these proteins in recombinant form have failed because they are not properly post-translationally modified. This has resulted in a limited view of enamel formation because in vitro studies have difficulty addressing the role ameloblastin and enamelin play in enamel formation. Mouse models have shown that both ameloblastin and enamelin are critical for enamel formation and that no enamel forms if either protein is absent. Therefore, a critical need exists to better understand ameloblastin and enamelin function in enamel development. We address this problem by proposing three specific aims and working hypotheses. 1) Enamelin and/or ameloblastin facilitate the conversion of amorphous calcium phosphate (ACP) to hydroxyapatite (HAP) and their cleavage products become part of a support structure for growing crystallites. 2) In the absence of amelogenin, the enamelin and/or ameloblastin proteins still regulate the mineral phase. 3) The inability to remove matrix proteins from Klk4 knockout enamel prevents proper ion movement that then compromises enamel maturation. We will test these hypotheses by characterizing the matrix architecture in situ relative to mineral phase at the mineralization front in wild type and amelogenin ablated mice. Likewise, we will analyze pH, expression levels of ion exchangers and their protein levels, and compare the enamel matrix composition between Klk4 KO mouse versus wild type using mass spectrometry LC-MS/MS. Taking advantage of recent advances in microscopy techniques, we can now perform in situ fluorescence microscopy of specifically labeled enamel matrix proteins correlated with scanning electron microscopy in undermineralized samples. This allows visualization of both protein and crystallites and to extend the scale of analytical resolution from micrometers to nanometers for the study of protein and mineral phase. Importantly, we will be able to address questions of protein distribution around crystallites within prisms and identify mineral phase by further extending the analytic resolution to an atomic scale through in situ atom probe analyses. To examine and visualize the three dimensional organization of matrix and developing crystallites in situ with unprecedented depth of field, we will perform high resolution helium ion microscopy. The realization of this project will substantially advance the field of enamel research by integrating the distribution of all three structural matrix proteins relative to forming mineral into a new concept of enamel formation. This information provides the basis for understand how hierarchically organized composite materials form, which is necessary knowledge for tissue engineering efforts focused on regenerating functional tooth tissues.

该项目的目的是通过克服明显的 田野中的僵局。尽管清楚地认识到对多蛋白质研究的需求，但体外研究已经大大 专注于氨基蛋白酶作为关键搪瓷基质蛋白。但是，其他两个基质蛋白ameloboblastin 并且搪瓷素出现在矩阵中的丰度较低，并且具有广泛的翻译后修饰。 它们的低丰度排除了动物牙齿的纯化，并试图表达这些蛋白质 重组形式失败了，因为它们在翻译后修改未正确。这导致了 牙釉质形成的有限视图是因为体外研究难以解决氨基蛋白细胞蛋白和 搪瓷在搪瓷形成中发挥作用。鼠标模型表明，氨基蛋白细胞蛋白和搪瓷蛋白都是至关重要的 对于搪瓷形成，如果没有任何一种蛋白质，则不会形成搪瓷。因此，存在关键需求 更好地了解酰基细胞蛋白和搪瓷蛋白在搪瓷发育中的功能。我们通过 提出三个特定的目标和工作假设。 1）搪瓷和/或杏仁蛋白促进转化 磷酸无形钙（ACP）至羟基磷灰石（HAP）及其裂解产物成为A的一部分 生长结晶的支持结构。 2）在没有氨基蛋白酶的情况下，搪瓷蛋白和/或蛋白蛋白 蛋白质仍调节矿物相。 3）无法从KLK4敲除搪瓷中删除基质蛋白 防止适当的离子运动，从而损害搪瓷成熟。我们将通过 表征野生型矿物质阵线处的矩阵结构相对于矿物相对于矿物相，野生型和 氨基蛋白蛋白消融的小鼠。同样，我们将分析pH，离子交换器及其蛋白质的表达水平 级别，并比较使用质量与野生型之间的搪瓷基质组成 光谱LC-MS/MS。利用显微镜技术的最新进展，我们现在可以执行 特定标记的牙釉质基质蛋白与扫描电子相关的原位荧光显微镜 不突破样品中的显微镜。这允许可视化蛋白质和微晶，并延伸 从微米到纳米的分析分辨率的规模，用于研究蛋白质和矿物相。 重要的是，我们将能够解决棱镜内蛋白质分布的问题 通过进一步将分析分辨率通过原位原子探针扩展到原子量表来识别矿物相 分析。检查和可视化矩阵的三维组织，并在 原位具有前所未有的景深，我们将执行高分辨率氦离子显微镜。实现 该项目将通过整合这三个的分布来大大推动搪瓷研究领域 结构基质蛋白相对于形成矿物的结构蛋白，成为牙釉质形成的新概念。此信息 为了解层次结构组织的复合材料表格提供了基础，这是必要的 组织工程工作的知识集中于再生功能性牙周组织。