Ameloblast Differentiation In Vitro: A Step Closer to Enamel Engineering

性状（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。成釉器上皮细胞和牙间充质细胞之间的相互作用导致成釉细胞的分化，成釉细胞控制釉基质的合成。成釉细胞功能和分化的研究一直受到限制的困难，在分离和维护的细胞在原代培养，以及我们有限的知识成釉细胞生物学。这些拟议的研究的目标是表征成釉细胞谱系细胞在文化和研究他们的分化。我们假设成釉细胞局部微环境中存在的因素将指导其分化，并提出以下具体目标。目的1：从单细胞集落中分离、扩增和维持原代未分化成釉细胞系细胞。具体目标2：诱导成釉细胞谱系细胞分化为成熟成釉细胞。来自成釉器官上皮的细胞将从单细胞克隆，其特征在于上皮标记物细胞角蛋白14和成釉细胞的标记物，包括牙本质唾液蛋白（DSP）和釉原蛋白。成釉细胞分化将通过与牙间质细胞、细胞外基质蛋白、生长因子和钙的细胞/细胞相互作用来诱导。早期分化的分泌型成釉细胞的特征在于釉原蛋白、成釉蛋白和MMP-20的上调。成熟成釉细胞的特征在于激肽释放酶4（KLK 4）、釉素和Ae 2的上调。识别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性成釉细胞生物学，以及这些细胞如何设计釉基质的形成。牙釉质的再生需要了解控制成釉细胞（釉质形成）谱系细胞扩增和分化的因素。在这个建议中，我们提出确定关键因素，直接成釉细胞谱系细胞的命运决定，因为他们形成釉基质。

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.