MATRIX BASED MINERAL ENAMEL-BIOMIMETICS

项目概要/摘要 要实现恢复牙釉质的长期目标，有必要了解其根本 细胞外基质组装的化学和生物学原理以及它们控制矿物质的方式 成核和生长。我们对潜在的分子机制的理解仍有很大的差距 釉质基质蛋白通过其组装并与细胞相互作用以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，这是我们研究的重点。因此，本提案的目标是推动我们的 通过系统研究成釉蛋白的相互作用，了解其结构和功能 不同的目标。我们假设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，成釉蛋白， 成釉蛋白和成釉蛋白-矿物质相互作用。提出了两个主要目标，以系统地研究 应用体外化学模型、细胞培养和动物模型进行成釉，证实了上述假设。 目的一：研究成釉细胞蛋白与细胞膜的相互作用，并鉴定其相互作用结构域。 体外合成脂质体和成釉细胞样细胞培养模型系统。我们将设计几个鼠标模型 与点突变的相互作用领域确定，并检查这些突变的后果， 釉质棱柱结构、成釉细胞形态和细胞与基质的附着。我们 假设成釉蛋白通过外显子5编码序列中的结构域与成釉细胞相互作用 并起到将矿化细胞外基质锚于釉质形成细胞的作用 极化、迁移和Tomes过程的形成。目的二：研究成釉细胞蛋白-釉原蛋白 在体内和体外的纳米级的相互作用，并确定其相互作用域使用溶液NMR 谱我们将研究纳米级磷酸钙矿化事件的动力学， 使用高分辨率原位原子力（AFM）和低温冷冻， 透射电子显微镜（TEM）。我们假设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异分子实体 阵我们期望获得更多的了解的结构，组装性能和功能， 成釉蛋白我们将在成釉蛋白上定义一个新的细胞膜结合结构域.新型蛋白质- 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 将阐明成釉蛋白与釉原蛋白结合对矿化的作用， 被识别。这些研究将进一步了解成釉蛋白在釉质中的分子功能 生物矿化，并将有助于我们努力制造合成釉质。

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.