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

摘要 釉质发生是一种生物过程，高度专门化的釉器上皮细胞被称为 成釉细胞将大量的钙离子和釉质蛋白输送到分泌性釉质中。 制作具有特殊结构和机械性能的生物材料：牙釉质 (潘迪亚和迪克维希2018年)。最近的研究已经确定了牙齿中的一些钙通道 成釉细胞底部的成釉细胞(Nurbaeva等人)。2015年，Lacruz 2017)。然而，还有 关于离子和蛋白质在分泌性成釉细胞中的转运机制，人们几乎没有达成一致意见。 极和整个成釉细胞胞体。为了支持本应用程序，我们开发了 三个高度创新的模式将解决知识差距并促进我们对 生理性和病理性成釉，包括(I)条件性分子筛蛋白缺失小鼠模型，(Ii) 成釉细胞三维生物反应器细胞培养模型和(Iii)一种新的液体细胞原子分辨率成像 泡状和细胞外牙釉质基质的原位生命成像技术。设立一个 细胞骨架蛋白基因敲除模型代表了囊泡运输研究领域的重大进展和 是研究包被囊泡在成釉过程中功能的有力工具。笼状蛋白包被的囊泡 在最丰富的细胞囊泡中，胞膜蛋白的丢失与严重的和 通常是致命的表型(Robinson 2015)。在这里，我们展示了令人兴奋的初步数据 釉质发生过程中的蛋白缺失会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牙釉质研究的又一个里程碑，它促进了 细长釉原蛋白分泌细胞的增殖，克服了传统2D的缺点 成釉细胞培养技术。第三，我们的原子分辨率液室模型有助于 史无前例的囊泡内容物和天然釉质基质的原位成像，允许 在釉质形成的早期阶段鉴定基质/矿物团。回应RFA-DE- 我们现在已经设计了一项研究计划来开发和优化这些模型系统(UG3 阶段)，并验证它们的生理学相关性和对理解 UH3期的釉质发育和疾病。

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.