A New Concept of Amelogenin-guided Mineralization in Enamel

说明(申请人提供)：牙釉质是人体内最坚硬和矿化程度最高的组织，由一种独特的磷灰石纳米纤维组成，其宽度仅为50纳米，但长度为几微米至几毫米。它的结构是蛋白质引导的磷灰石晶体在三维有机框架中沿着其c轴单轴生长过程的结果，该有机框架在推进矿化的同时进行水解，转变为几乎完全由矿物组成的组织。尽管釉质基质蛋白，特别是釉原蛋白的自溶作用已被广泛认为是控制牙釉质结构发展的关键因素，但目前基于釉原蛋白纳米球形成的模型在指导最初带状磷灰石晶体的各向异性生长及其向致密矿化结构转变的能力方面存在明显的局限性。釉原蛋白是一种疏水性蛋白，约占釉质基质蛋白的90%。最近我们发现，重组人全长釉原蛋白(RH174)可形成宽度为17 nm的条带，这些条带可以在几天内生长到几微米长。这种带子具有自我对准的能力，并形成类似于珐琅棒中排列的磷灰石微晶外观的束状物。条带的形成需要钙离子和磷酸根离子的同时存在，这表明离子桥发展并驱动了自组装过程。这种带的合成在水-油体系中得到了动力学促进，但也在无油的环境中生成了带，通常在磷酸钙溶液中孵育3-5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结晶，而是似乎稳定了一种无定形矿物。然而，在釉原蛋白裂解产物rH146制成的纳米带上形成了定向磷灰石，这表明全长蛋白质的加工可能会诱导从无定形磷灰石到晶状磷灰石的转变。这一建议是基于这样的假设，即釉原蛋白纳米带是发育中的釉质中具有生物学意义的超分子结构，需要纳米带的水解才能实现定向磷酸钙矿化。这一假说将通过以下两个特定目的来验证：1.体外诱导定向磷酸钙晶体在釉原蛋白纳米带上生长；2.证明体外观察到的釉原蛋白纳米带是体内发育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.