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个明显的， 在现场的僵局。虽然多蛋白研究的必要性已被明确认识，但体外研究仍存在严重的局限性。 专注于釉原蛋白作为关键的釉基质蛋白。然而，另外两种基质蛋白成釉蛋白 和釉蛋白以低得多的丰度存在于基质中，并具有广泛的翻译后修饰。 它们的低丰度妨碍了从动物牙齿中纯化并试图在哺乳动物中表达这些蛋白质。 重组形式失败，因为它们没有被适当地后修饰。这导致 由于体外研究难以阐明成釉蛋白的作用， 釉质蛋白在釉质形成中起作用。小鼠模型表明，成釉蛋白和釉蛋白都是关键的 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蛋白在釉质发育中的作用。我们通过以下方式解决这个问题： 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）转化为羟基磷灰石（HAP）及其裂解产物成为 用于生长微晶支撑结构。2)在不存在釉原蛋白的情况下，釉蛋白和/或成釉蛋白 蛋白质仍然调节矿物质相。3)不能从Klk 4敲除釉质中去除基质蛋白 阻止了适当的离子运动，从而损害了釉质的成熟。我们将测试这些假设， - 表征野生型中矿化前沿处相对于矿物相的原位基质结构， 釉原蛋白消融小鼠。同样，我们将分析pH值，离子交换剂及其蛋白质的表达水平 水平，并比较Klk 4 KO小鼠与野生型之间的釉质基质组成， 光谱法LC-MS/MS。利用显微镜技术的最新进展，我们现在可以进行 釉基质特异性标记蛋白的扫描电镜原位荧光显微镜观察 显微镜下观察矿化不足的样品。这允许蛋白质和微晶的可视化， 用于蛋白质和矿物相研究的从微米到纳米的分析分辨率尺度。 重要的是，我们将能够解决蛋白质分布在棱柱内微晶周围的问题， 通过原位原子探针将分析分辨率进一步扩展到原子尺度来识别矿物相 分析。为了检查和可视化基质的三维组织和发展中的微晶， 现场与前所未有的景深，我们将进行高分辨率氦离子显微镜。实现 该项目将通过整合所有三个方面的分布， 结构基质蛋白相对于形成矿物质成为釉质形成的新概念。这些信息 为理解分层组织的复合材料如何形成提供了基础，这是必要的 组织工程学的知识集中在功能性牙齿组织的再生上。