Ameloblast Differentiation and Amelogenesis: Next-Generation Models to Define Key Mechanisms and Factors Involved in Biological Enamel Formation

摘要 釉质形成是一个生物学过程，通过该过程，高度特化的釉质器官上皮细胞被称为 成釉细胞将大量的钙离子和釉质蛋白转运到分泌釉质中 基质和制造具有特殊结构和机械性能的生物材料：牙釉质 （Pandya and Diekwisch 2018）。最近的研究已经确定了牙齿中的一些钙通道 基底成釉细胞方面的成釉细胞（Nurbaeva et al. 2015，Lacruz 2017）。不过有 在分泌型成釉细胞的离子和蛋白质运输机制上， 极和整个成釉细胞体。为了支持本申请，我们开发了 三种高度创新的模式，将解决知识差距，并促进我们对 生理学和病理学釉质发生，包括（i）条件网格蛋白缺失小鼠模型，（ii） 成釉细胞3D生物反应器细胞培养模型，和（iii）新的液体细胞原子分辨率成像 用于囊泡和细胞外釉质基质的生命原位成像的技术。建立 网格蛋白敲除模型代表了囊泡运输研究领域的重大进展， 这是研究包被囊泡在釉质形成过程中功能的有力工具。网格蛋白包被的囊泡是 在最丰富的细胞囊泡中，网格蛋白的丢失与严重的 通常为致死表型（罗宾逊2015）。在这里，我们提出了令人兴奋的初步数据表明， 在釉质形成过程中网格蛋白的缺失导致釉质棱柱结构和晶体密度的改变。 我们的3D生物反应器釉质发生模型标志着釉质研究的另一个里程碑，因为它促进了 延长釉原蛋白分泌细胞的繁殖，克服了传统2D 成釉细胞培养技术。第三，我们的原子分辨率液体室模型有助于 前所未有的囊泡内容物和天然釉质基质的原位成像， 在釉质形成的最早阶段识别基质/矿物簇。关于RFA-DE- 19-004我们现在已经设计了一个研究计划来开发和优化这些模型系统（UG 3 阶段），并验证其生理相关性和有用性，以了解机制， 牙釉质发育和疾病在UH 3阶段。

Abstract Amelogenesis is a biological process by which highly specialized enamel organ epithelial cells called ameloblasts transport substantial amounts of calcium ions and enamel proteins into the secretory enamel matrix and manufacture a biomaterial of exceptional structural and mechanical properties: tooth enamel (Pandya and Diekwisch 2018). Recent studies have identified some of the calcium channels in dental enamel cells at the basal ameloblast aspect (Nurbaeva et al. 2015, Lacruz 2017). However, there is remarkably little agreement on the mechanisms of ion and protein trafficking at the secretory ameloblast pole and throughout the ameloblast cell body. In support of the present application we have developed three highly innovative models that will address knowledge gaps and advance our understanding of physiological and pathological amelogenesis, including (i) a conditional clathrin deletion mouse model, (ii) an ameloblast 3D bioreactor cell culture model, and (iii) a new liquid cell atomic resolution imaging technology for life in situ imaging of vesicular and extracellular enamel matrices. Establishment of a clathrin knockout model represents significant progress in the area of vesicular trafficking research and a powerful tool to study the function of coated vesicles during amelogenesis. Clathrin-coated vesicles are among the most abundant cellular vesicles, and loss of clathrin has been associated with severe and usually lethal phenotypes (Robinson 2015). Here we present exciting preliminary data demonstrating that clathrin depletion during amelogenesis resulted in altered enamel prism structure and crystal density. Our 3D bioreactor amelogenesis model marks another milestone in enamel research as it promoted the propagation of elongated amelogenin secreting cells, overcoming shortcomings of traditional 2D ameloblast cell culture technology. Third, our atomic resolution liquid chamber model facilitates unprecedented in situ imaging of vesicular contents and native enamel matrix, allowing for the identification of matrix/mineral clusters at the earliest stages of amelogenesis. In response to RFA-DE- 19-004 we have now designed a research plan to develop and optimize these model systems (UG3 phase) and to validate their physiological relevance and usefulness for understanding mechanisms of enamel development and disease during the UH3 phase.