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

成釉细胞分化和成釉细胞：定义生物牙釉质形成涉及的关键机制和因素的下一代模型

• 批准号: 10460290
• 负责人: Tom Diekwisch
• 金额: $ 18.87万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460290
• 关键词: 3-Dimensional; Address; Agreement; Ameloblasts; Amelogenesis; Animal Model; Area; Biocompatible Materials; Biological; Biological Models; Biological Process; Bioreactors; Calcium Channel; Calcium ion; Cell Culture Techniques; Cell Lineage; Cells; Clathrin; Clathrin-Coated Vesicles; Coated vesicle; Communities; Crystallization; DNA Sequence Alteration; Data; Defect; Dental Enamel; Development; Disease; Enamel Formation; Enamel Organ; Epithelial Cells; Event; Growth; Health; Image; Imaging technology; Inherited; Ion Transport; Ions; Knock-out; Knowledge; Life; Liquid substance; Minerals; Modeling; Pathologic; Phase; Phenotype; Physiological; Process; Proteins; Research; Resolution; Structure; Study models; Technology; Testing; Toxic Environmental Substances; Validation; Vesicle; amelogenin; cellular imaging; combat; density; design; effectiveness validation; extracellular; human disease; in situ imaging; innovation; insight; mechanical properties; mouse model; new technology; next generation; novel; protein transport; response; tool; trafficking; vesicle transport

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.

抽象的 釉质生成是高度专业化的釉质器官上皮细胞（称为釉质器官上皮细胞）的生物过程。 成釉细胞将大量的钙离子和牙釉质蛋白运输到分泌性牙釉质中 基质并制造具有特殊结构和机械性能的生物材料：牙釉质 （Pandya 和 Diekwisch 2018）。最近的研究已经确定了牙科中的一些钙通道 基底成釉细胞方面的牙釉质细胞（Nurbaeva et al. 2015，Lacruz 2017）。然而，有 关于分泌性成釉细胞的离子和蛋白质运输机制的共识很少 极和整个成釉细胞体。为了支持当前的应用，我们开发了 三个高度创新的模型将解决知识差距并增进我们对 生理和病理性釉质形成，包括（i）条件性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，以及 (iii) 新的液体细胞原子分辨率成像 囊泡和细胞外牙釉质基质的生命原位成像技术。设立一个 网格蛋白敲除模型代表了囊泡运输研究领域的重大进展和 研究釉质形成过程中涂层囊泡功能的有力工具。网格蛋白包被的囊泡是 网格蛋白的损失与严重和严重的细胞囊泡有关。 通常是致命的表型（Robinson 2015）。在这里，我们提供令人兴奋的初步数据证明 牙釉质发生过程中网格蛋白的消耗导致牙釉质棱柱结构和晶体密度的改变。 我们的 3D 生物反应器釉质生成模型标志着釉质研究的另一个里程碑，因为它促进了 延长的牙釉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体室模型有助于 对囊泡内容物和天然牙釉质基质进行前所未有的原位成像，允许 在釉质形成的最早阶段鉴定基质/矿物质簇。回应 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（UG3 阶段）并验证其生理相关性和对于理解机制的有用性 UH3 阶段的牙釉质发育和疾病。