Recombinant Amelogenin Matrices for Apatite Nanofibers

描述(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，具有纳米级的精度，新技术和人类工程学可能能够模仿。这种独特的釉质微结构是在蛋白质引导下在三维有机框架中生长高度各向异性磷灰石晶体的结果，这种生长是由釉原蛋白自组装产生的，釉原蛋白在推进的矿化过程中进行水解，转化为几乎完全由矿物组成的组织。拟议研究的总体目标是通过设计一种指导晶体生长并对酶消化敏感的重组蛋白质基质框架，生成类似于牙釉质中晶体的纳米纤维磷灰石。克隆牙釉质细胞外基质的蛋白质和蛋白酶提供了创造人工环境的机会，这种环境可以模拟形成牙釉质的生物化学。新的滴定电子设备允许准确地添加纳米级矿化溶液，以与体内过程类似，在很长一段时间内保持恒定的离子微环境。因此，我们认为成釉细胞的两个主要活动，即基质蛋白和蛋白酶的表达和提供以及对离子浓度的精确控制可以通过人类工程来实现，从而为我们提供模拟釉质形成的能力。假设纤维状磷灰石纳米晶可以通过重组蛋白基质的自组装及其酶降解与从恒定组成的饱和溶液中生长的羟基磷灰石晶体的协同作用而产生。这一假说将通过以下特定目标得到验证：(1)确定使釉原蛋白能够自组装到超分子框架中的物理化学和生化参数；(2)与釉原蛋白超分子自组装同步地诱导成核表面的磷灰石结晶；以及(3)通过在矿化过程中逐渐降解蛋白支架来模拟釉质成熟。该项目将有助于更好地了解牙釉质中的生物矿化作用，包括在分子水平上一些釉质基质蛋白和蛋白水解酶的功能。所获得的方法和知识将为类似的仿生方法理解牙本质、骨、贝壳和其他钙化组织的矿化提供基础。重要的是，了解纳米尺度对这一复杂过程的控制将为开发新的矿化组织修复方法提供基础。

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.