Mineralization Studies Related to Oral Biology

描述(申请人提供)：建议的研究集中在基质囊泡(MVS)，它在牙齿和骨骼的矿化中起着启动作用。MVS是亚显微的、细胞外的、膜包裹的颗粒，是牙本质、生长板软骨和发育中的骨中钙化的初始位置。本实验室参与了MVS的首次鉴定、分离和鉴定。我们和其他人已经提供了包括碱性磷酸酶(ALP)和ATPase在内的MV磷酸酶参与MV矿化的证据。此外，这些磷酸酶被整合到MV膜中，矿物首次出现在MV膜下，这表明MV膜的成分在启动钙化过程中起着关键作用。我们的研究旨在加深对MVS启动生物矿化的机制的了解。我们将利用ALP和/或NTPPase基因失活的正常小鼠和转基因小鼠，研究构成MV磷酸酶的相互作用，如碱性磷酸酶(ALP)、ATPase、核苷酸三磷酸焦磷酸水解酶(NTPPase)和无机焦磷酸酶(PPiase)在调节大鼠生长板分离的MV矿化中的相互作用，以及在体内促进或抑制MV矿化的作用。由于细胞程序性死亡被认为是生长板MV生物发生的必要前提，我们将研究软骨细胞凋亡在培养的大鼠生长板软骨细胞生成矿化活性MVS中的作用。根据我们的初步观察，分离的大鼠MVs含有大量的骨形态发生蛋白(BMPs)，我们将检验这一假说，即位于生长板软骨MVs中的细胞外BMPs是形态发生信号的载体，通过干骺端和次级骨化中心的软骨基质吸收释放它们，调节新骨的形成。 具体目的：1)进一步表征MV磷酸酶在MV启动的生物矿化中的调控作用。2)基因诱导的低磷和/或低焦磷酸对MV启动的突变小鼠生物矿化的影响。3)研究了软骨细胞质膜萌发的生物发生机制，以及细胞程序性死亡(细胞凋亡)是否调控了MV的生物发生。4)证实BMPs在体外培养的大鼠生长板MVS中的存在，并确定基质囊泡BMPs是否具有促进软骨-骨分化的作用。这是对牙齿和骨骼矿化启动机制的基础性研究。基质囊泡钙化的新知识可以应用于广泛的主题，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.