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）：本项目旨在研究牙釉质形成的最初阶段，以及蛋白质介导的控制磷酸钙（骨骼和牙齿中的矿物质）形成的空间组织和阶段的机制。我们提出了一种仿生方法来测试磷酸化全长淀粉原蛋白特异性调节体内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）从小鼠发育的牙齿。由于HIM分析方法在生物矿化研究中的新颖性，我们将与显微镜制造商卡尔蔡司，NTS LLC.密切合作，充分利用HIM在生物医学研究中的突出和独特的特点。具体目的是：1)验证全长淀粉原蛋白形成独特的蛋白质-矿物质组合的假设，这些蛋白质-矿物质组合调节结构组织，并在体外形成类似于发育中的牙釉质中所见的磷灰石晶体的平行排列。更具体地说，我们将对体外矿化实验的样品进行he分析，使用全长和裂解形式的猪淀粉原的混合物，使用可能影响蛋白质组装的各种底物表面来严格检验这一假设。重组（rP172和rP147）淀粉原蛋白将首先用于优化HIM方案，在亚纳米分辨率下同时表征蛋白质和矿物相，然后研究类似的天然蛋白（P173和P148）。2)优化荧光标记与HIM分析的使用，以验证全长和裂解淀粉原蛋白在其混合物存在时不会在矿物相上共定位的假设，而是每种蛋白质相对于彼此和矿物相表现出独特的自组装模式和分布。具体来说，我们将在HIM分析中应用荧光标记a)首先用于矿化实验中不同标记的牙釉质基质蛋白/肽（rP172, rP147， LRAP）和矿物相的体外样品，然后b)用于在成釉发生分泌阶段检测的发育中的小鼠牙釉质门牙。特别是，我们将使用一抗和二抗对搪瓷基质蛋白和/或其降解产物进行特异性标记，以便在体外和原位鉴定和定位全长和裂解的淀粉原蛋白和/或其他搪瓷基质蛋白（LRAP，搪瓷蛋白）。这些研究将再次与卡尔蔡司，SMT合作进行。实现这些目标将阐明蛋白质引导矿化的基本机制以及特定釉质基质蛋白在成釉发育过程中调节晶体形状和排列的作用。HIM荧光成像的创新应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的蛋白质-矿物质关系。