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。独特的釉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，所述三维有机框架由自组装产生。该研究的总体目标是通过设计一种纳米纤维状磷灰石，这种纳米纤维状磷灰石类似于牙釉质中的晶体。重组蛋白质基质框架，其引导晶体生长并且对酶消化敏感。釉质细胞外基质的蛋白质和蛋白酶的克隆提供了产生可以模拟形成釉质的生物化学的人工环境的机会。新的滴定电子设备允许精确添加纳升的矿化溶液，以保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，我们提出成釉细胞的两个主要活动，例如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实现，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体可以通过重组蛋白基质的自组装及其酶促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调来产生。本研究将从以下几个方面对这一假说进行验证：（1）确定釉原蛋白自组装成超分子结构的物理化学和生物化学参数;（2）诱导磷灰石在成核表面上结晶，与釉原蛋白超分子自组装同步;（3）通过MMP-20和/或MMP-20逐渐降解蛋白质支架来模拟釉质成熟。或丝氨酸蛋白酶。该项目将导致对牙釉质中生物矿化的更好理解，包括在分子水平上的一些釉质基质蛋白和蛋白酶的功能。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，骨骼，贝壳和其他钙化组织中的矿化。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.