Extracellular remodeling in bone development and repair

描述(申请人提供)：软骨内骨化是骨骼发育和成人骨骼再生的关键事件。在这两种情况下，破骨细胞和软骨破骨细胞侵蚀肥大的软骨，肥大的软骨细胞死亡，血管生长，随之而来的是骨祖细胞的招募以及骨和骨髓腔的形成。这些事件需要细胞外的蛋白分解，对正常的骨形成和修复是必不可少的。这项建议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。在之前的资助阶段，我们已经证明了基质金属蛋白酶-9的蛋白分解是软骨内骨形成事件的关键决定因素。在生长板发育和骨骼再生过程中，基质金属蛋白酶-9和血管内皮生长因子(VEGF)相互重叠，共同调控肥大软骨重塑、血管生成和骨形成。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是如何单独和共同调节软骨内骨发育的程序的。我们将在小鼠中使用靶向性和条件性突变，结合细胞和器官培养，使用形态计量学和生化方法，评估不同类型细胞表达基质金属蛋白酶-9和基质金属蛋白酶-13(胶原酶-3)对软骨重塑、成骨细胞和破骨细胞功能、血管募集和骨骼重塑的相对作用。通过这些研究，我们将能够确定骨骼生长板形态发生的步骤。我们将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，我们将使用基质金属蛋白酶突变小鼠和不稳定和稳定的骨折作为工具，评估机械环境如何决定野生型和突变小鼠骨骼再生过程中的再生反应。然后，我们将在这些分析的基础上确定基质金属蛋白酶在发育和骨骼修复过程中的生理底物。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成骨中的新的和重要的功能。在骨关节炎和顽固性硬组织病变中，软骨内成骨过程被错误地调节。更全面地了解血管生成的核心作用及其在这些过程中的蛋白降解调节，可能对某些类型的非愈合性骨折的治疗干预和治疗的发展具有重要意义。

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.