Protein-Mineral Interactions During Initial Stages of Enamel Formation

牙釉质形成初始阶段的蛋白质-矿物质相互作用

• 批准号: 8244215
• 负责人: Felicitas B Bidlack
• 金额: $ 14.93万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8244215
• 关键词: Address; Amelogenesis; Animals; Antibodies; Applications Grants; Biomedical Research; Biomimetics; Charge; Cleaved cell; Collaborations; Dental Enamel; Dentin; Development; Electron Beam; Enamel Formation; Exhibits; Extracellular Matrix Proteins; Family suidae; Fluorescence Microscopy; Fluorescent Dyes; Future; Goals; Helium; Hydroxyapatites; Image; Imagery; Imaging Techniques; In Situ; In Vitro; Incisor; Ions; Knock-out; Label; Laser Microscopy; Length; Location; Manufacturer Name; Mediating; Microscope; Microscopy; Minerals; Mus; Pattern; Peptides; Phase; Phosphorylation; Process; Property; Proteins; Protocols documentation; Recombinants; Regulation; Relative (related person); Research; Resolution; Role; Sampling; Shapes; Specimen; Staging; Surface; Techniques; Testing; Time; Tissues; Tooth structure; Work; amelogenin; base; biomineralization; bone; calcium phosphate; design; enamel matrix proteins; enamelin; experience; fluorescence imaging; in vivo; innovation; insight; leucine-rich amelogenin peptide; light microscopy; mineralization; multi-photon; nanometer; prevent; research study; self assembly; tissue regeneration; ultra high resolution

DESCRIPTION (provided by applicant): This project addresses research questions pertinent to the very initial stages of tooth enamel formation and the mechanisms of protein-mediated control of spatial organization and phase of forming calcium phosphate, the mineral in our bones and teeth. We propose a biomimetic approach to test the working hypothesis that phosphorylated full-length amelogenin specifically regulates the initial formation of parallel arrays of apatitic crystals in vivo. Furthermore, this project aims to develop the use of Helium Ion Microscopy (HIM) for the high-resolution visualization and analysis of both mineral and organic phase in samples from: (i) in vitro mineralization studies and (ii) developing teeth from mice. Due to the novelty of the analytical approach of HIM for the study of biomineralization, we will work in close collaboration with the microscope manufacturer Carl Zeiss, NTS LLC., to fully exploit the outstanding and unique features of HIM for biomedical research. The Specific Aims are 1) To test the hypothesis that the full-length amelogenin forms unique protein-mineral assemblies that regulate the structural organization and result in parallel arrays of apatitic crystals in vitro similar to those seen in developing enamel. More specifically, we will apply HIM analyses to samples from in vitro mineralization experiments using mixtures of full-length and cleaved forms of pig amelogenin to critically test this hypothesis using various substrate surfaces which may influence protein assembly. Recombinant (rP172 and rP147) amelogenins will first be used to optimize HIM protocols for simultaneous characterization of both protein and mineral phases at sub-nanometer resolution to then also study the analogous native proteins (P173 and P148). 2) To optimize the use of fluorescent markers with HIM analyses to test the hypothesis that full-length and cleaved amelogenins do not co-localize on mineral phases formed in the presence of their mixture, but rather each protein exhibits a distinctive self-assembly pattern and distribution relative to each other and the mineral phase. Specifically, we will apply fluorescent markers with HIM analyses a) first for in vitro samples from mineralization experiments comprised of different labeled enamel matrix proteins/peptides (rP172, rP147, LRAP) and mineral phases, and then b) for developing mouse enamel incisors examined during the secretory stage of amelogenesis. In particular, we will use primary and secondary antibodies for the specific labeling of enamel matrix proteins and/or their degradation products to allow for the identification and location of full-length and cleaved amelogenins and/or other enamel matrix proteins (LRAP, enamelin) in vitro and in situ. These studies will again be carried out in collaboration with Carl Zeiss, SMT. Achieving these goals will elucidate fundamental mechanisms of protein-guided mineralization and the role of specific enamel matrix proteins in regulating crystal shape and alignment during amelogenesis. The innovative use of HIM for fluorescence imaging introduces a new imaging technique into biomineralization studies for direct visualization of protein-mineral relationships in both wild type and knock out animals. PUBLIC HEALTH RELEVANCE: This project studies processes relating to the structural organization of both enamel matrix and mineral during the very initial stages of tooth enamel formation. In particular, it is our goal to elucidate mechanisms of protein-mediated control of the spatial organization and phase of forming calcium phosphate mineral. To realize this goal, we propose the use of helium ion microscopy for sub-nanometer visualization and analysis of both mineral and organic phase. This new microscopy technique is ideally suited for studies of tooth enamel formation because it reveals surface topography and information about material properties at sub-nanometer resolution of organic and mineral phases at the same time. The results from this work will provide new insights into the mechanisms of protein-guided mineralization of calcium phosphate, the mineral in mammalian bone and teeth. A better understanding of these processes will aid our efforts to develop new materials and treatments for diseased mineralized tissues.

描述（由申请人提供）：该项目涉及与牙釉质形成的最初阶段有关的研究问题，以及蛋白质介导的空间组织控制机制和形成磷酸钙的阶段，磷酸钙是我们骨骼和牙齿中的矿物质。我们提出了一种仿生方法来测试磷酸化全长釉原蛋白在体内特异性调节磷灰石晶体平行阵列的初始形成的工作假设。此外，该项目旨在开发氦离子显微镜（HIM）的使用，以高分辨率可视化和分析样品中的矿物和有机相：（i）体外矿化研究和（ii）小鼠牙齿发育。由于HIM用于生物矿化研究的分析方法的新奇，我们将与显微镜制造商Carl Zeiss，NTS LLC.密切合作，充分利用HIM的突出和独特功能进行生物医学研究。具体目的是：1）检验全长釉原蛋白形成独特的蛋白质-矿物组装体的假设，所述蛋白质-矿物组装体调节结构组织并在体外产生类似于发育中的釉质中所见的平行排列的磷灰石晶体。更具体地说，我们将应用HIM分析的样本在体外矿化实验中使用的全长和切割形式的猪釉原蛋白的混合物，严格测试这一假设，使用各种基板表面，可能会影响蛋白质的组装。重组（rP 172和rP 147）釉原蛋白将首先用于优化HIM方案，以亚纳米分辨率同时表征蛋白质和矿物质相，然后还研究类似的天然蛋白质（P173和P148）。2)为了优化荧光标记物与HIM分析的使用，以检验以下假设：全长和切割的釉原蛋白不共定位于在其混合物存在下形成的矿物质相上，而是每种蛋白质相对于彼此和矿物质相表现出独特的自组装模式和分布。具体而言，我们将应用荧光标记物与HIM分析a）首先用于来自矿化实验的体外样品，所述体外样品由不同标记的釉质基质蛋白/肽（rP 172、rP 147、LRAP）和矿物相组成，然后B）用于在釉质形成的分泌阶段期间检查的发育中的小鼠釉质切牙。特别是，我们将使用一抗和二抗特异性标记釉基质蛋白和/或其降解产物，以允许在体外和原位鉴定和定位全长和切割的釉原蛋白和/或其他釉基质蛋白（LRAP，釉蛋白）。这些研究将再次与Carl Zeiss，SMT合作进行。实现这些目标将阐明蛋白质引导的矿化的基本机制和特定的釉质基质蛋白在调节晶体形状和排列在釉质形成过程中的作用。HIM荧光成像的创新使用为生物矿化研究引入了一种新的成像技术，用于直接可视化野生型和敲除动物中的蛋白质-矿物质关系。 公共卫生相关性：本项目研究在牙齿釉质形成的最初阶段，釉质基质和矿物质的结构组织相关的过程。特别是，这是我们的目标是阐明蛋白质介导的控制的空间组织和形成磷酸钙mineral. To实现这一目标的机制，我们建议使用氦离子显微镜的亚纳米可视化和分析的矿物和有机相。这种新的显微镜技术非常适合牙釉质形成的研究，因为它揭示了表面形貌和材料性能的信息，在亚纳米分辨率的有机和矿物相在同一时间。这项工作的结果将为哺乳动物骨骼和牙齿中的矿物质磷酸钙的蛋白质引导矿化机制提供新的见解。更好地了解这些过程将有助于我们努力开发新材料和治疗疾病矿化组织。