ENAMEL PROTEIN IN TISSUE SPECIFIC BIOMINERALIZATION

本提案的总体目标是了解 釉质基因产物（即，釉原蛋白、釉蛋白和釉质 蛋白酶），以促进对过程的理解， 釉质发生，创造一个战略，以产生釉质生物陶瓷作为一个 新的牙科修复生物材料，并提高诊断的 各种类型的釉质发生。 我们假设这些图案 在阴离子釉蛋白（例如簇绒蛋白）结构内的蛋白质用于 成核初始结晶相，并且釉原蛋白内的基序 蛋白质聚集体调节蛋白质的大小、方向和模式。 生长釉质晶体 我们进一步预测，蛋白酶（例如， 钙依赖性金属蛋白酶）降解蛋白质并促进 从矿化的细胞外基质中除去水和蛋白质。 为了解决这一假设，已经制定了以下具体目标： （1）确定釉原蛋白和釉蛋白的功能 在体外成釉细胞培养的小鼠成釉过程中， 转染的成牙本质细胞培养和小鼠磨牙器官培养 使用反义抑制策略的系统。 (2)确定 釉蛋白使磷酸八钙（OCP）成核的功能，或 羟基磷灰石（HAP）。 将对重组牙釉质蛋白进行评价 作为OCP或HAP的体外成核剂。 (3)来确定函数 釉原蛋白调节OCP或HAP晶体的大小和形态 体外生长 重组釉原蛋白将作为调节剂进行评价 用于体外晶体生长和生长模式。 后续 使用定点诱变的蛋白质工程方法将 能够识别OCP或HAP晶体所需的基序 沿着c轴生长以及晶体生长的模式。 （四） 为了确定釉质蛋白酶的功能，以调节两种结构， 性能和釉质蛋白的逐步去除， 来调节晶体的形成 我们建议分离并表征 釉质蛋白酶基因产物。 (5)制备搪瓷HAP晶体 体外形成。 牙釉质替代疗法是一种策略， 重组釉质蛋白基序以制造釉质生物陶瓷， 一种可能的牙釉质修复生物材料 这些实验 拟议的功能研究的逻辑延伸，以确定如何 釉质蛋白调节釉质OCP和HAP晶体的形成。

The overall objective of this proposal is to understand the function of enamel gene products (i.e., amelogenins, enamelins and enamel proteinases), to advance the understanding of the process of amelogenesis, to create a strategy to produce an enamel bioceramic as a novel dental restorative biomaterial, and to improve the diagnosis of the various types of amelogenesis imperfecta. We hypothesize the motifs within the anionic enamelin protein (e.g.tuftelin) structure serve to nucleate the initial crystalline phase, and that motifs within amelogenin protein aggregates regulate the size, orientation and patterns of the growing enamel crystals. We further predict that proteases (e.g. calcium-dependent metalloproteinases) degrade proteins and facilitate the removal of water and protein from the mineralizing extracellular matrix. To address this hypothesis, the following Specific Aims have been formulated: (1) To determine the function of amelogenins and enamelins during mouse amelogenesis in in vitro ameloblast cell culture, in transfected odontoblast cell cultures and mouse molar tooth organ culture systems using antisense inhibition strategies. (2) To determine the function of enamelins to nucleate octacalcium phosphate (OCP) or hydroxyapatite (HAP) in vitro. Recombinant enamelins will be evaluated as nucleators for OCP or HAP in vitro. (3) To determine the function of amelogenins to regulate the size and patterns of OCP or HAP crystal growth in vitro. Recombinant amelogenins will be evaluated as regulators for the crystal growth and patterns of growth in vitro. Subsequent protein engineering approaches using site-directed mutagenesis will enable the identification of the motifs required for OCP or HAP crystal growth along the c-axis as well as the patterns of crystal growth. (4) To determine the function of enamel proteases to regulate both structural properties and the stepwise removal of enamel proteins and subsequently to regulate crystal formation. We propose to isolate and characterize the enamel protease gene product. (5) To fabricate enamel HAP crystal formation in vitro. Enamel replacement therapy is a strategy to use recombinant enamel protein motifs to fabricate an enamel bioceramic as a possible dental enamel restorative biomaterial. These experiments are a logical extension of the proposed functional studies to define how enamel proteins regulate enamel OCP and HAP crystal formation.