Mineralization Studies Related to Oral Biology

描述（由申请方提供）：拟定研究的重点是在牙齿和骨骼矿化中起起始作用的基质囊泡（MV）。MV是亚微观的、细胞外的、膜包埋的颗粒，其充当牙本质、生长板软骨和发育中的骨中的钙化的初始位点。我们的实验室参与了MV的首次鉴定、分离和表征。我们和其他人提供的证据表明，MV磷酸酶，包括碱性磷酸酶（ALP）和ATP酶参与MV矿化。此外，这些磷酸酶被整合到MV膜下的矿物质首先出现，这表明在启动钙化的MV膜的组件的关键作用。我们的研究是针对增加了解的机制，MV启动生物矿化。我们将研究组成型MV磷酸酶，如碱性磷酸酶（ALP），ATP酶，核苷酸三磷酸焦磷酸水解酶（NTPPase）和无机焦磷酸酶（PPiase）的相互作用，在调节矿化分离MV从大鼠生长板，并在促进或抑制体内MV矿化使用正常与ALP和/或NTPPase基因失活的转基因小鼠。由于程序性细胞死亡已被认为是生长板中MV生物发生的必要先决条件，我们将研究软骨细胞凋亡在培养的大鼠生长板软骨细胞产生矿化能力MV中的作用。继我们的初步观察，分离的大鼠MV含有大量的骨形态发生蛋白（BMP），我们将测试的假设，细胞外BMP，驻留在MV的生长板软骨的形态发生信号，调节新骨形成后，其释放的软骨基质吸收在干骺端和次级骨化中心的载体。 具体目的：1）进一步研究MV磷酸酶在MV启动的生物矿化中的作用。2)研究遗传诱导的低磷酸酶和/或低焦磷酸酶对突变小鼠中MV引发的生物矿化的影响。3)通过软骨细胞质膜出芽研究MV生物发生的机制，以及MV生物发生是否受程序性细胞死亡（凋亡）的调节。4)研究以确认BMP在分离的大鼠生长板MV中的存在，并确定基质囊泡BMP是否可以促进软骨-骨分化。这是一个基本的研究机制，牙齿和骨骼形式的矿化开始。基质囊泡钙化的新知识可应用于广泛的主题，包括发生异常钙化的特定疾病。

DESCRIPTION (provided by applicant): The proposed study is focused on matrix vesicles (MVs) which play an initiating role in the mineralization of teeth and bones. MVs are submicroscopic, extracellular, membrane-invested particles that serve as the initial site of calcification in dentin, growth plate cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We and others have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), and ATPase are involved in MV mineralization. Furthermore, these phosphatases are integrated into the MV membrane beneath which mineral first appears, suggesting a critical role for components of the MV membrane in initiating calcification. Our study is directed toward an increased understanding of the mechanism by which MVs initiate biomineralization. We will examine the interaction of constitutive MV phosphatases, e.g. alkaline phosphatase (ALP), ATPase, nucleotide triphosphate pyrophosphohydrolase (NTPPase) and inorganic pyrophosphatase (PPiase), in regulating the mineralization of isolated MVs from rat growth plate, and in promoting or inhibiting in vivo MV mineralization using normal versus transgenic mice with ALP and/or NTPPase gene inactivation. Since programmed cell death has been suggested as a necessary precondition for MV biogenesis in growth plate, we will examine the role of chondrocyte apoptosis in the generation of mineralization-competent MVs by cultured rat growth plate chondrocytes. Following up on our preliminary observation that isolated rat MVs contain significant amounts of bone morphogenetic proteins (BMPs) we will test the hypothesis that extracellular BMPs, residing in the MVs of growth plate cartilage are carriers of morphogenetic signals that regulate new bone formation following their release by cartilage matrix resorption in metaphyses and secondary ossification centers. Specific Aims: 1) a further characterization of the role MV phosphatases in regulating MV initiated biomineralization. 2) Studies of the effect of genetically induced hypophosphatasia and/or hypopyrophosphatasia on MV-initiated biomineralization in mutant mice. 3) A study of the mechanism of MV biogenesis by chondrocyte plasma membrane budding and whether MV biogenesis is regulated by programmed cell death (apoptosis). 4) Studies to confirm the presence of BMPs in isolated rat growth plate MVs, and to determine whether matrix vesicle BMPs can promote chondro- osseous differentiation. This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.