Extracellular remodeling in bone development and repair

描述（由申请人提供）：软骨内成骨是骨骼发育和成人骨骼再生的关键事件。在这两种情况下，破骨细胞和破软骨细胞侵蚀肥厚软骨，肥厚软骨细胞死亡，血管生长，随后骨祖细胞募集，骨和骨髓腔形成。这些事件需要细胞外蛋白水解，是正常骨形成和修复所必需的。本研究的目的是阐明由基质金属蛋白酶（MMPs）控制的分子和细胞机制，协调软骨内骨形成和再生的事件。在之前的资助期内，我们表明MMP-9的蛋白质水解是软骨内骨形成事件的关键决定因素。MMP-9和血管内皮生长因子（VEGF）在生长板发育和骨骼再生过程中通过重叠通路调节肥大软骨重塑、血管生成和骨形成程序。我们现在建议使用遗传，分子和细胞生物学方法来进一步表征这两种MMPs如何单独或共同调节软骨内骨发育程序。我们将在小鼠中使用靶向和条件突变来评估表达MMP-9和MMP-13（胶原酶-3）的不同细胞类型在软骨重塑、成骨细胞和破骨细胞功能、血管募集和骨骼重塑中的相对作用，使用形态计量学和生化方法结合细胞和器官型培养。从这些研究中，我们将能够确定骨骼生长板形态发生的步骤。我们将重点关注肥大软骨细胞的终末分化、细胞外基质重塑、血管募集和骨重塑的调节。然后，我们将使用MMP突变小鼠和不稳定和稳定骨折作为工具来评估机械环境如何决定野生型和突变小鼠骨骼再生期间的再生反应。然后，我们将在这些分析的基础上确定MMPs在发育和骨骼修复过程中的生理基础。这些研究将使我们深入了解MMPs、细胞外基质蛋白和血管生成调节因子在软骨内骨形成中的新功能和重要功能。软骨内成骨过程在骨关节炎和顽固性硬组织病变中被错误调节。更全面地了解血管生成的核心作用及其在这些过程中通过蛋白水解的调节可能对治疗干预和某些类型的不愈合骨折的治疗发展具有重要意义。

DESCRIPTION (provided by applicant): Endochondral ossification is a pivotal event in skeletal development and in adult skeletal regeneration. In both, osteoclasts and chondroclasts erode hypertrophic cartilage, hypertrophic chondrocyte die, blood vessels grow in followed by recruitment of osteoprogenitor cells and bone and bone marrow cavity formation. These events require extracellular proteolysis and are essential for normal bone formation and repair. The goal of this proposal is to elucidate the molecular and cellular mechanisms, controlled by matrix metalloproteinases (MMPs), that coordinate the events in endochondral bone formation and regeneration. In the previous funding period we showed that proteolysis by MMP-9 is a critical determinant of the events in endochondral bone formation. MMP-9 and vascular endothelial growth factor (VEGF) act in overlapping pathways to regulate the program of hypertrophic cartilage remodeling, angiogenesis and bone formation in growth plate development and skeletal regeneration. We now propose to use genetic, molecular and cell biological approaches to further characterize how these two MMPs, each separately and together, regulate the program of endochondral bone development. We will use targeted and conditional mutations in mice to evaluate the relative roles various cell types expressing MMP-9 and MMP-13 (collagenase-3) to cartilage remodeling, osteoblast and osteoclast function, vascular recruitment and skeletal remodeling, using morphometric and biochemical approaches coupled with cell and organotypic cultures. From these studies we will be able to determine the steps in skeletal growth plate morphogenesis. We will focus on terminal differentiation of hypertrophic chondrocytes, extracellular matrix remodeling, regulation of vascular recruitment and bone remodeling. We will then use the MMP mutant mice and unstable and stabilized fractures as tools to evaluate how the mechanical environment determines the regenerative response during in skeletal regeneration in wild type and mutant mice. We will then build on these analyses to determine the physiologic substrates of the MMPs during development and skeletal repair. These studies will give us insights into novel and important functions for the MMPs, extracellular matrix proteins and angiogenic regulators in endochondral bone formation. The endochondral ossification processes are misregulated in osteoarthritis and recalcitrant hard tissue lesions. A fuller understanding of the central role of angiogenesis and its regulation by proteolysis in these processes may have implications for therapeutic interventions and the development of therapy for certain types of non-healing fractures.