Ameloblast Differentiation In Vitro: A Step Closer to Enamel Engineering

描述（由申请人提供）：牙釉质是由上皮来源的成釉细胞形成的矿化组织。牙釉质器官上皮细胞与牙间充质细胞相互作用导致成釉细胞分化，成釉细胞控制着牙釉质基质的合成。成釉细胞功能和分化的研究受到原代培养中细胞分离和维持的困难以及我们对成釉细胞生物学的有限知识的限制。这些研究的目的是在培养中表征成釉细胞谱系并研究它们的分化。我们假设成釉细胞的局部微环境中存在的因素将指导它们的分化，并提出以下具体目标。特异性目的：从体外单细胞集落中分离、扩增和维持原代未分化成釉细胞系细胞。目的2：诱导成釉细胞系细胞向成熟成釉细胞分化。釉质器官上皮细胞将由单细胞克隆，其特征是上皮标记细胞角蛋白14，以及牙本质唾液蛋白（DSP）和淀粉原蛋白（amelogenin）等前成釉细胞标记。成釉细胞分化是由细胞间充质细胞、细胞外基质蛋白、生长因子和钙的相互作用诱导的。早期分化的分泌性成釉细胞的特征是成釉原蛋白、成釉酶和MMP-20的上调。成熟成釉细胞的特征是klikin - 4 （KLK4）、amelotin和Ae2的上调。确定驱动成釉细胞分化的因素将使我们更好地了解正常和病理成釉细胞生物学，以及这些细胞如何设计形成釉质基质。牙釉质的再生需要了解控制成釉细胞（牙釉质形成）谱系细胞扩张和分化的因素。在这一建议中，我们提出确定关键因素，直接成釉细胞谱系的命运决定，因为他们形成釉质基质。

DESCRIPTION (provided by applicant): Enamel is a mineralized tissue formed by the epithelially derived ameloblasts. The reciprocal interactions between epithelial cells of the enamel organ and dental mesenchymal cells lead to the differentiation of ameloblasts, which govern the synthesis of the enamel matrix. Studies of ameloblast function and differentiation have been limited by difficulties in isolation and maintenance of the cells in primary culture, as well as our limited knowledge of ameloblast biology. The goal of these proposed studies is to characterize ameloblast lineage cells in culture and to study their differentiation. We hypothesize that factors present in the local microenvironment of the ameloblasts will direct their differentiation and propose the following specific aims. Specific Aim1: To isolate, expand and maintain primary undifferentiated ameloblast lineage cells from single-cell colonies in vitro. Specific aim 2: To induce differentiation of ameloblast lineage cells to mature ameloblasts. Cells from the enamel organ epithelium will be cloned from single cells, characterized by the epithelial marker cytokeratin 14, and markers of preameloblasts including dentin sialoprotein (DSP) and amelogenin. Ameloblast differentiation will be induced by cell/cell interactions with dental mesencymal cells, extracellular matrix proteins, growth factors and calcium. The early-differentiated secretory ameloblasts will be characterized by upregulation of amelogenin, ameloblastin, and MMP-20. Mature ameloblasts will be characterized by upregulation of kallikrein 4 (KLK4), amelotin and Ae2. The identification of factors that drive ameloblast differentiation will allow us to better understand normal and pathological ameloblast biology, and how these cells engineer the formation of the enamel matrix. The regeneration of tooth enamel requires an understanding of the factors that control expansion and differentiation of ameloblast (enamel forming) lineage cells. In this proposal we propose to identify key factors that direct ameloblast lineage cell fate decisions as they form an enamel matrix.