A New Concept of Amelogenin-guided Mineralization in Enamel

描述（由申请人提供）：牙釉质是人体中最坚硬、矿化程度最高的组织，由一种独特的磷灰石纳米纤维组织组成，宽度只有50纳米，长度只有几微米到毫米。它的结构是蛋白质引导的磷灰石晶体沿其c轴在三维有机框架中的单轴生长过程的结果，该过程与推进矿化协调水解，转化为几乎完全由矿物组成的组织。虽然釉质基质蛋白（尤其是淀粉原蛋白）的自组装作用已被广泛认为是控制釉质结构发育的关键因素，但目前基于淀粉原蛋白纳米球形成的模型在球形结构引导最初带状磷灰石晶体各向异性生长并转变为致密矿化结构的能力方面存在显著局限性。淀粉原蛋白是一种疏水蛋白，约占釉质基质蛋白的90%。最近，我们发现重组人全长淀粉原蛋白（rH174）形成17 nm宽的条带，在几天内生长到几微米长的条带。这种条带具有自我排列的能力，并形成束，类似于釉质棒中排列的磷灰石晶体的外观。条带的形成需要钙离子和磷酸盐离子的存在，这表明离子桥的发展和驱动自组装过程。这种条带的合成在水-油乳液体系中得到动力学增强，但条带也在无油环境中生成，通常在磷酸钙溶液中培养3至5天内形成。虽然rH174的纳米带含有钙和磷酸盐，但它们并不直接促进磷灰石的结晶，而是似乎稳定了一种无定形矿物。然而，取向磷灰石在由淀粉原裂解产物rH146制成的纳米带上形成，这表明全长蛋白的加工可能导致磷灰石从无定形转变为结晶。这一提议是基于一个假设，即淀粉原纳米带是牙釉质发育过程中与生物相关的超分子结构，纳米带的水解需要实现定向磷酸钙矿化。这一假设将通过以下两个具体目的进行检验：1。在体外诱导淀粉原纳米带上生长定向磷酸钙晶体2. 为了证明在体外观察到的淀粉原纳米带是体内发育的牙釉质的主要超分子结构。

DESCRIPTION (provided by applicant): Enamel, the hardest and most mineralized tissue in the human body, is comprised of a unique organization of apatite nanofibers of only 50 nm width but several micrometers to millimeters in length. Its structure is the result of a protein-guided uniaxial growth process of apatite crystals along their c-axes in a three-dimensional organic framework that hydrolyzes in coordination with advancing mineralization to transform into a tissue almost entirely comprised of mineral. While the role of self-asembly of enamel matrix proteins, in particular amelogenin, has widely been recognized as a crucial factor in controlling structure development of enamel, the current model based on the formation of amelogenin nanospheres has significant limitations with regards to the ability of a spherical structure guidin the anisotropic growth of initially ribbon-like apatite crystals and their transformation into a compact mineralized structure. Amelogenin is a hydrophobic protein which comprises about 90% of the enamel matrix proteins. Recently we discovered that the recombinant human full-length amelogenin protein (rH174) forms ribbons of 17 nm width, which grow over a period of days to several micrometer in length. Such ribbons have the ability to self-align and to form bundles which resemble the appearance of aligned apatite crystallites in an enamel rod. Ribbon formation requires the presence of both calcium and phosphate ions suggesting that ion bridges develop and drive the self-assembly process. Synthesis of such ribbons was kinetically enhanced in a water-oil emuslion system but ribbons were also generated in an oil-free environment and commonly formed within 3 to 5 days of incubation in calcium phosphate solutions. While nanoribbons of rH174 contain calcium and phosphate, they do not directly promote apatite crystllization, but instead appear to stabilize an amorphous mineral. Oriented apatite formed however on nanoribbons made from an amelogenin cleavage product, rH146, indicating that the processing of the full- length protein might induce a transformation from amorphous to crystalline apatite. This proposal is based on the hypothesis that amelogenin nanoribbons are the biologically relevant supramolecular structures in developing enamel and hydrolysis of nanoribbons is required to enable oriented calcium phosphate mineralization. This hypothesis will be tested through the following two specific aims: 1. To induce oriented calcium phosphate crystal growth on amelogenin nanoribbons in-vitro; 2. To demonstrate that amelogenin nanoribbons, as observed in-vitro, are the predominant supramolecular structure of developing enamel in-vivo.