Enamel biomineralization; the role of pH cycling

项目摘要/摘要 矿化牙釉质是人体内最坚硬的组织，其适当的形成对保护牙釉质至关重要 并终生保持牙齿健康。釉质生物矿化是由上皮源性的 成釉细胞。成釉细胞首先分泌釉质基质蛋白，然后这些蛋白被水解并被 羟基磷灰石晶体在成熟过程中。在成熟过程中，牙釉质基质的pH值 在大约80%的成熟阶段周期性地调节为酸性，其余的 大约20%的时间保持中立。成熟期的成釉细胞也会周期性地 随着基质pH的循环，改变细胞的形态和功能。当pH循环被基因打乱时 突变体或环境因素导致成釉细胞调节失调，釉质成熟失调， 导致釉质矿化不足，形成不良的牙釉质。然而，驱动基质pH循环的机制 成釉细胞的调控相互作用知之甚少。在初步研究中，我们发现了釉原蛋白 能以pH依赖的方式不同地影响细胞的形态和黏附；成釉细胞的调节 与局部黏附-P13K-AKT信号通路的变化相关。此外，我们确定了可能的 基质pH对pH依赖性多功能酶转谷氨酰胺酶2功能的贡献 釉质基质成熟过程中釉原蛋白水解酶的调控。在这些拟议的研究中，我们将解决 釉质基质pH调节成釉细胞功能、蛋白质结构和基质的中心假说 矿化作用。我们将使用体外和体内模型，内吞作用、钙转运、 基质水解和pH，以验证我们的中心假设与以下具体目标。1)确定 细胞外pH对成熟期成釉细胞黏附和调控的影响 PH和钙对整合素介导的焦点黏附-PI3K-AKT-mTOR信号通路的影响 3)测定基质pH对成釉细胞转谷氨酰胺酶2活性的影响。 成熟期釉质基质。这些研究将使我们能够更好地了解牙釉质的病因 低矿化，它们将使我们能够应用这一知识来降低牙釉质缺陷的风险。 此外，这些研究的结果将有助于我们确定釉质策略的长期目标。 组织生物工程、修复，以及利用釉质蛋白治疗的目的。

PROJECT SUMMARY/ABSTRACT Mineralized enamel is the hardest tissue in the human body, and its proper formation is critical for the protection and maintenance of healthy teeth for a lifetime. Enamel biomineralization is directed by the epithelial-derived ameloblasts. Ameloblasts secrete enamel matrix proteins first, which are then hydrolyzed and replaced by hydroxyapatite crystals in the maturation process. During the maturation process, the pH of the enamel matrix modulates periodically to be acidic in approximately 80% of the maturation stage, with the remaining approximately 20% of the time remaining neutral. Maturation stage ameloblasts also modulate periodically along with the matrix pH cycling, changing cell morphology and functions. When pH cycling is disrupted by gene mutants or environmental factors that dysregulate ameloblast modulation, enamel maturation is dysregulated, resulting in hypomineralized, poorly formed enamel. However, the mechanisms that drive both matrix pH cycling and ameloblast modulation interactions are poorly understood. In preliminary studies, we found amelogenins can differentially affect cell morphology and adhesion in a pH-dependent manner; and ameloblast modulation is associated with changes in focal adhesion-P13k-AKT signaling pathways. Furthermore, we identified possible contributions of matrix pH to the function of the pH-dependent multifunctional enzyme, transglutaminase 2, in regulating amelogenin hydrolysis in the maturation of enamel matrix. In these proposed studies we will address the central hypothesis that enamel matrix pH regulates ameloblast function, protein structure, and matrix mineralization. We will use both in vitro and in vivo models with alterations in endocytosis, calcium transport, matrix hydrolysis and pH, to test our central hypothesis with the following specific aims. 1) To determine the effect of extracellular pH on adhesion and modulation of maturation stage ameloblasts; 2) To determine the effect of pH and calcium on integrin-mediated focal adhesion - PI3K-AKT-mTOR signaling pathways of ameloblasts; 3) To determine the significance of matrix pH on Transglutaminase 2 activity in the maturation stage enamel matrix. These studies will allow us to better understand the etiology of enamel hypomineralization, and they will allow us to apply this knowledge to reduce the risk of enamel defects. Furthermore, the findings from these studies will contribute to our long-term goal to identify strategies for enamel tissue bioengineering, repair, and the use of enamel proteins therapeutic purposes.