Mineralization Studies Related to Oral Biology

描述（由申请人提供）：拟议的研究重点是基质囊泡（MVs），它在牙齿和骨骼的矿化中起初始作用。MVs是亚微观的、细胞外的、膜包裹的颗粒，在牙本质、生长板软骨和发育中的骨中起初始钙化作用。我们的实验室参与了mv的首次鉴定、分离和表征。我们和其他人已经提供了证据，证明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矿物质首先出现的中压膜中，这表明中压膜成分在启动钙化过程中起着关键作用。我们的研究旨在增加对mv启动生物矿化的机制的理解。我们将研究组成MV磷酸酶的相互作用，例如碱性磷酸酶（ALP）， atp酶，核苷酸三磷酸焦磷酸水解酶（NTPPase）和无机焦磷酸酶（PPiase），在调节从大鼠生长板中分离的MV矿化，以及在ALP和/或NTPPase基因失活的正常小鼠和转基因小鼠体内促进或抑制MV矿化。由于程序性细胞死亡被认为是生长板中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能力MV中的作用。根据我们的初步观察，分离的大鼠MVs中含有大量的骨形态发生蛋白（BMPs），我们将验证生长板软骨MVs中的细胞外BMPs是形态发生信号的载体，这些信号在骺端和继发性骨化中心被软骨基质吸收释放后调节新骨的形成。

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.