Recombinant Amelogenin Matrices for Apatite Nanofibers

描述（由申请人提供）：牙釉质通过蛋白质控制的矿化和降解过程形成，具有纳米级精度，新技术和人类工程可能能够模仿。独特的珐琅质微观结构是蛋白质引导的高度各向异性磷灰石晶体在三维有机框架中生长的结果，该框架是由淀粉原蛋白自组装产生的，淀粉原蛋白在推进矿化的协调下水解转化为几乎完全由矿物质组成的组织。该研究的总体目标是通过设计一种重组蛋白基质框架来生成类似于牙釉质晶体的纳米纤维磷灰石，该框架可以引导晶体生长，并且易于酶消化。克隆牙釉质细胞外基质的蛋白质和蛋白酶为模拟牙釉质形成的生物化学过程提供了一个人工环境。新的滴定电子设备允许精确添加纳升的矿化溶液，以维持离子微环境在长时间内保持恒定水平，类似于体内过程。因此，我们提出通过人体工程可以实现成釉细胞的两个主要功能，即基质蛋白和蛋白酶的表达和提供以及离子浓度的精确控制，从而为我们提供模拟牙釉质形成的能力。假设纤维状磷灰石纳米晶体可以通过重组蛋白基质的自组装及其酶降解与羟基磷灰石晶体从恒定组成的饱和溶液中生长的协调而产生。这一假设将通过以下具体目标进行测试：(1)确定使淀粉原蛋白自组装成为超分子框架的物理化学和生化参数；(2)在成核表面诱导磷灰石结晶，同时伴有淀粉原蛋白超分子自组装；(3)在矿化过程中，MMP-20和/或丝氨酸蛋白酶逐渐降解蛋白质支架，模拟牙釉质成熟。本项目将有助于进一步了解牙釉质的生物矿化，包括一些牙釉质基质蛋白和蛋白酶在分子水平上的功能。所获得的方法和知识将为类似的仿生方法提供基础，以了解牙本质，骨，壳和其他钙化组织的矿化。重要的是，理解对这一复杂过程的纳米级控制将为开发矿化组织修复的新方法提供基础。

DESCRIPTION (provided by applicant): Dental enamel forms through a protein controlled mineralization and degradation process with a nanoscale precision that new technologies and human engineering may be able to mimic. The unique enamel microstructure is a result of protein-guided growth of highly anisotropic apatite crystals in a three-dimensional organic framework generated by the self-assembly of amelogenin proteins that hydrolyze in coordination with an advancing mineralization to transform into a tissue almost entirely comprised of mineral. The overall objective of the proposed research is to generate nanofibrous apatite similar to crystals in enamel through the design of a recombinant protein matrix framework that guides crystal growth and is susceptible to enzymatic digestion. Cloning of proteins and proteases of the extracellular matrix of enamel provides the opportunity to generate an artificial environment that can mimic the biochemistry of the forming enamel. New titration electronics allow the accurate addition of nanoliters of mineralizing solutions to maintain an ionic microenvironment at constant levels over long periods of time similar to the in-vivo process. Thus, we propose that two major activities of ameloblast cells, e.g. the expression and provision of matrix proteins and proteases and the precise control over ionic concentration can be achieved through human engineering, providing us with the ability to mimic enamel formation. The hypothesis is that fibrous apatite nanocrystals can be generated by the coordination of the self-assembly of a recombinant protein matrix and its enzymatic degradation with the growth of hydroxyapatite crystals from a saturated solution of constant composition. This hypothesis will be tested by the following specific aims: (1) To determine the physicalchemical and biochemical parameters that enable self-assembly of amelogenin proteins into a supramolecular framework; (2) To induce apatite crystallization on nucleating surfaces in synchronization with amelogenin supramolecular self-assembly and (3) To mimic enamel maturation by gradual degradation of the protein scaffold by MMP-20 and/or serine proteases while mineralization proceeds. This project will result in an improved understanding of biomineralization in dental enamel including the functions of some of the enamel matrix proteins and proteases at the molecular level. The methodology and knowledge gained will provide the basis for similar biomimetic approaches to understand mineralization in dentin, bone, shells and other calcified tissues. Importantly, comprehending nanoscale control over this complex process will provide a basis for development of novel methods for mineralized tissue repair.