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

基本信息

批准号：
8435404
负责人：
Felicitas B Bidlack
金额：
$ 14.33万
依托单位：
ADA FORSYTH INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2014-12-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8435404
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

项目摘要

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.

描述（由申请人提供）：该项目解决了与牙釉质形成的最初阶段以及蛋白质介导的空间组织控制机制和磷酸钙（骨骼和牙齿中的矿物质）形成阶段相关的研究问题。我们提出了一种仿生方法来测试磷酸化全长牙釉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，该项目旨在开发使用氦离子显微镜（HIM）对来自以下样品中的矿物和有机相进行高分辨率可视化和分析：（i）体外矿化研究和（ii）小鼠牙齿发育。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。具体目标是 1) 测试以下假设：全长牙釉蛋白形成独特的蛋白质-矿物质组件，调节结构组织并在体外产生类似于牙釉质发育中所见的平行磷灰石晶体阵列。更具体地说，我们将使用全长和切割形式的猪牙釉蛋白混合物对体外矿化实验的样品进行 HIM 分析，以使用可能影响蛋白质组装的各种基质表面严格检验这一假设。重组（rP172 和 rP147）牙釉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) 通过 HIM 分析优化荧光标记的使用，以测试以下假设：全长和切割的牙釉蛋白不会共定位在其混合物存在下形成的矿物相上，而是每种蛋白质都表现出独特的自组装模式以及相对于彼此和矿物相的分布。具体来说，我们将应用荧光标记进行 HIM 分析，a) 首先用于来自矿化实验的体外样品，这些样品由不同标记的牙釉质基质蛋白/肽（rP172、rP147、LRAP）和矿物相组成，然后 b) 用于检查发育中的小鼠牙釉质门牙在釉质形成的分泌阶段。特别是，我们将使用一抗和二抗来特异性标记牙釉质基质蛋白和/或其降解产物，以识别和定位全长和切割的釉原蛋白和/或其他牙釉质基质蛋白（LRAP、牙釉质）体外和原位。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矿化的基本机制以及特定牙釉质基质蛋白在釉质形成过程中调节晶体形状和排列的作用。 HIM 在荧光成像中的创新应用将一种新的成像技术引入生物矿化研究中，以直接可视化野生型和基因敲除动物中的蛋白质-矿物质关系。