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.

这个项目的目标是通过克服一种明显的牙釉质形成 田野上的僵局。尽管多蛋白质研究的必要性已被清楚地认识到，但体外研究在很大程度上 重点研究了釉原蛋白作为关键的釉质基质蛋白。然而，另外两种基质蛋白成釉蛋白 而釉蛋白在基质中的丰度要低得多，并具有广泛的翻译后修饰。 它们的低丰度排除了从动物牙齿中纯化这些蛋白质的可能性，并试图在 重组形式之所以失败，是因为它们没有经过适当的翻译后修改。这导致了一种 对釉质形成的看法有限，因为体外研究很难解决成釉蛋白和成釉蛋白的作用 釉质蛋白在釉质形成中起作用。小鼠模型表明成釉蛋白和釉质蛋白都是至关重要的。 对于釉质的形成，如果没有任何一种蛋白质，就不会形成釉质。因此，迫切需要 更好地了解成釉蛋白和釉质蛋白在釉质发育中的作用。我们通过以下方式解决这一问题 提出三个具体目标和工作假设。1)釉蛋白和/或成釉蛋白促进转化 无定形磷酸钙(ACP)到羟基磷灰石(HAP)及其切割产物成为 生长微晶的支撑结构。2)在没有釉原蛋白的情况下，釉蛋白和/或成釉蛋白 蛋白质仍然控制着矿物相。3)不能从Klk4基因敲除牙釉质中去除基质蛋白 阻止适当的离子移动，从而影响牙釉质的成熟。我们将通过以下方式检验这些假设 野生型与矿化前沿矿物相的原位基质构筑特征 釉原蛋白消融小鼠。同样，我们将分析pH值、离子交换器及其蛋白质的表达水平 水平，并比较Klk4KO小鼠和野生型小鼠的釉质基质组成 利用显微技术的最新进展，我们现在可以进行 扫描电子相关的特殊标记釉质基质蛋白的原位荧光显微镜研究 矿化程度较低的样品中的显微镜。这允许蛋白质和微晶的可视化，并扩展 从微米到纳米的分析分辨率，用于蛋白质和矿物相的研究。 重要的是，我们将能够解决棱镜和棱镜内微晶周围的蛋白质分布问题 通过原位原子探头将分析分辨率进一步扩展到原子尺度来识别矿物相 分析。检查和可视化基质和发育中的微晶的三维组织 现场，以前所未有的景深，我们将进行高分辨率氦离子显微镜。的实现。 这个项目将通过整合这三个领域的分布，大大推进牙釉质研究领域的发展。 结构基质蛋白形成矿物质形成牙釉质的新概念。此信息 为理解按层次组织的复合材料是如何形成的提供了基础，这是必要的 组织工程学方面的知识主要集中在再生功能性牙齿组织上。