MATRIX BASED MINERAL ENAMEL-BIOMIMETICS

项目摘要/摘要 为了实现修复牙釉质的长期目标，有必要了解 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 成核和生长。在我们对潜在的分子机制的理解上仍有很大的差距。 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。成釉蛋白尤其如此。 蛋白质，这是我们建议研究的重点。因此，这项提议的目标是推进我们的 通过系统研究成釉蛋白的相互作用了解成釉蛋白的结构和功能 有不同的目标。我们推测牙釉质中高度组织化的碳化羟基磷灰石晶体 通过复合型成釉蛋白细胞、成釉蛋白、成釉蛋白细胞的持续生长形成棱柱状结构。 成釉蛋白和成釉蛋白与矿物质的相互作用。提出了两个主要目标来系统地检查 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研究。 目的I：研究成釉蛋白与细胞膜的相互作用并确定相互作用区域。 体外合成脂质体和造釉细胞样细胞培养模型系统。我们将设计几个鼠标模型 识别相互作用区域中的点突变，并检查这些突变对 釉质棱柱状结构，成釉细胞形态，以及细胞与基质的附着。我们 假设成釉蛋白通过外显子5编码序列中的一个结构域与成釉细胞相互作用 以及将矿化的细胞外基质锚定到影响细胞的釉质形成细胞上的功能 极化、迁移和Tomes过程的形成。目的II：研究成釉蛋白-釉原蛋白 体内外纳米尺度的相互作用及其相互作用结构域的溶液核磁共振鉴定 光谱学。我们将在纳米尺度上研究磷酸钙矿化事件的动力学 成釉蛋白与成釉蛋白结合，利用高分辨率的原位原子力(AFM)和冷冻-凝胶渗透技术，使成釉蛋白与成釉蛋白结合。 透射电子显微镜(TEM)。我们推测釉原蛋白和成釉蛋白形成 在釉质形成的不同阶段起作用以控制晶体的杂大分子实体 队形。我们期望对其结构、组装特性和功能有更深入的了解 成釉蛋白。我们将在成釉蛋白上定义一个新的细胞膜结合区。新型蛋白质-- 成釉蛋白和成釉蛋白序列上的蛋白质相互作用结构域将被识别。的影响 成釉蛋白和成釉蛋白在矿化中的联合作用将被阐明，矿物结合结构域将被阐明 被指认出来。这些研究将促进对成釉蛋白在釉质中的分子功能的理解。 生物矿化，并将有助于我们制造合成釉质的努力。

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.