MATRIX BASED MINERAL ENAMEL-BIOMIMETICS

基于基质的矿物釉质仿生学

基本信息

批准号：
9902380
负责人：
Janet M. Oldak
金额：
$ 46.15万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2023-04-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9902380
关键词：
Affect Ameloblasts Amelogenesis Animal Model Binding Biocompatible Materials Biological Biological Models Biomimetics Cell Culture Techniques Cell membrane Cell-Matrix Junction Cells Chemical Models Chemicals Complex Crystal Formation Crystallization Data Dental Dental Enamel Dental Materials Enamel Formation Event Exons Extracellular Matrix Future Generations Genetic Goals Growth Hydroxyapatites In Situ In Vitro Investigation Knowledge Liposomes Mediating Minerals Molecular Morphology Mutation NMR Spectroscopy Outcome Pathologic Pattern Point Mutation Procedures Process Property Proteins Publishing Resolution Structure Transmission Electron Microscopy ameloblastin amelogenin base biomineralization calcium phosphate cell motility design enamel matrix proteins gene product in vivo insight malformation migration mineralization mouse model mutant nanoscale novel overexpression polarized cell restorative material

项目摘要

Project Summary / Abstract To achieve the long-term goal of restoring dental enamel, it is necessary to understand the fundamental chemical and biological principles of extracellular matrix assembly and the manner they control mineral nucleation and growth. There is still a large gap in our understanding of the underlying molecular mechanisms by which enamel matrix proteins assemble and interact with cells to control nucleation and oriented growth of hydroxyapatite crystals, and possibly cell movement and polarization. This is particularly true of ameloblastin protein, which is the focus of our proposed study. The goal of this proposal is therefore to advance our understanding of ameloblastin’s structure and function through a systematic investigation of its interactions with different targets. We hypothesize that the highly organized carbonated hydroxyapatite crystals in enamel continuously grow and form prismatic structures by means of complex ameloblastin-cell, ameloblastin- amelogenin, and ameloblastin-mineral interactions. Two major aims are proposed to systematically examine the above hypothesis by applying in vitro chemical models, cell culture and animal models for amelogenesis. Aim I: To investigate ameloblastin-cell membrane interactions and identify the interacting domains using in vitro synthetic liposomes and ameloblast-like cell culture model systems. We will design several mouse models with point mutations in the interacting domains identified, and examine the consequence of these mutations on enamel prismatic structure, ameloblast morphology, and the attachment of the cells to the matrix. We hypothesize that ameloblastin interacts with ameloblast cells via a domain in the sequence encoded by exon 5 and functions to anchor the mineralizing extracellular matrix to the enamel-forming cells affecting cell polarization, migration, and the formation of Tomes’ processes. Aim II: To investigate ameloblastin-amelogenin interactions on the nanoscale in vivo and in vitro, and to identify their interacting domains using solution NMR spectroscopy. We will study the dynamics of calcium phosphate mineralization events on the nanoscale when ameloblastin is combined with amelogenin, using high resolution in situ atomic force (AFM) and Cryo- transmission electron microscopy (TEM). We hypothesize that amelogenin and ameloblastin form hetereomolecular entities that are functional during different stages of amelogenesis to control crystal formation. We anticipate to gain more insight into the structure, assembly properties and function of ameloblastin. We will define a novel cell- membrane- binding domain on ameloblastin protein. Novel protein- protein-interacting domains on the ameloblastin and amelogenin sequences will be identified. The effect of ameloblastin combined with amelogenin on mineralization will be elucidated and mineral-binding domains will be identified. These studies will advance understanding of the molecular function of ameloblastin in enamel biomineralization and will contribute to our efforts to fabricate synthetic enamel.

项目概要/摘要要实现修复牙釉质的长期目标，有必要了解牙釉质的根本细胞外基质组装的化学和生物学原理及其控制矿物质的方式我们对成核和生长的潜在分子机制的理解仍然存在很大差距。牙釉质基质蛋白通过其组装并与细胞相互作用来控制成核和定向生长羟基磷灰石晶体，以及可能的细胞运动和极化，对于成釉细胞尤其如此。蛋白质，这是我们提出的研究的重点，因此该提案的目标是推进我们的研究。通过对其相互作用的系统研究来了解成釉细胞的结构和功能我们捕获了牙釉质中高度组织化的碳酸羟基磷灰石晶体。通过复杂的成釉细胞、成釉细胞不断生长并形成棱柱结构提出了系统研究釉原蛋白和成釉细胞-矿物质相互作用的两个主要目标。上述假说通过应用体外化学模型、细胞培养和动物模型进行釉质形成。目标 I：研究成釉细胞膜相互作用并确定用于体外合成脂质体和成釉细胞样细胞培养模型系统我们将设计几种小鼠模型。确定相互作用域中的点突变，并检查这些突变的结果牙釉质棱柱结构、成釉细胞形态以及细胞与基质的附着。培养发现成釉细胞通过外显子 5 编码序列中的一个结构域与成釉细胞相互作用以及将矿化细胞外基质锚定到影响细胞的牙釉质形成细胞的功能极化、迁移和 Tomes 过程的形成目标 II：研究成釉素-釉原蛋白。体内和体外纳米尺度的相互作用，并使用溶液核磁共振识别它们的相互作用域我们将研究纳米级磷酸钙矿化事件的动力学。利用高分辨率原位原子力 (AFM) 和冷冻技术将成釉素与釉原蛋白结合在一起我们勇敢地发现了釉原蛋白和成釉细胞。在釉质形成的不同阶段发挥作用以控制晶体的异分子实体我们期望对结构、组装特性和功能有更多的了解。我们将在新蛋白上定义一个新的细胞膜结合结构域。将鉴定成釉蛋白和釉原蛋白序列上的蛋白质相互作用结构域的影响。将阐明成釉细胞与釉原蛋白结合对矿化的影响，并且将阐明矿物质结合域这些研究将促进对牙釉质中成釉细胞的分子功能的了解。生物矿化并将有助于我们制造合成牙釉质的努力。