INTEGRIN/ECM INTERACTIONS IN BONE FORMATION AND REMODELING

细胞与细胞外基质和相邻细胞(固态环境)的相互作用对组织发育过程中的形态发生以及组织的动态平衡和一生中的重塑至关重要。这些实验室以前使用成骨细胞培养的研究表明，整合素与纤维连接蛋白的相互作用在成骨细胞分化和存活中都发挥着关键作用。这项拟议研究的总体目标是使用转基因小鼠模型来检验这些观察结果的生理学相关性。在一个这样的模型中，在转基因小鼠的成熟成骨细胞中表达了整合素β1亚单位的截短形式。对表型的初步表征表明，在出生后的幼年动物中，成骨细胞的骨基质沉积存在缺陷，成骨细胞的骨重建过度。在目标1中，将通过组织结构、基质和矿物质质量的分析，以及与项目6合作的先进成像技术，在骨组织水平上进一步表征这种表型。还将在细胞水平上通过确定转基因表达对从野生型和转基因小鼠分离的成骨细胞的黏附、迁移和存活的影响来表征这种表型。最后，将与项目2合作研究转基因对生物力学特性和修复能力的影响。目标2的研究将集中于纤维连接蛋白在体内成骨细胞分化和存活中的作用。N端70 kDa的基质组装和胶原结合域将在转基因小鼠的成骨细胞中表达。该结构域可抑制未成熟原代成骨细胞的分化，并可诱导成熟成骨细胞的凋亡。表型将使用目标1所示的方法和相互作用来表征。此外，将在与项目2合作的牵张成骨模型中测试完整的纤维连接蛋白或纤维连接蛋白片段加速或干扰骨骼修复的能力。由于目前还没有动物模型来测试纤维连接蛋白及其整合素受体在成骨细胞功能和骨重建中的作用，这些研究应该提供与骨骼修复和骨骼退行性疾病相关的新的见解。

Cell interactions with extracellular matrix and with neighboring cells (the solid state environment) are critical for tissue morphogenesis during development, and for tissue homeostasis and remodeling throughout life. Previous studies from these laboratories using osteoblastic cells in cultured, have shown that integrin interactions with fibronectin play critical roles in both osteoblast differentiation and survival. The overall goal of the proposed studies is to test the physiologic relevance of these observations using transgenic mouse models. In one such model a truncated form of the integrin beta 1 subunit has been expressed in mature osteoblasts of transgenic mice. Preliminary characterization on the phenotype has revealed defective bone matrix deposition by osteoblasts and excessive bone remodeling by osteoblasts in post-natal young animals. In Aim 1, this phenotype will be characterized further at the bone tissue level by analysis of tissue architecture and matrix and mineral quality, using histomorphometry, and advanced imaging technologies in collaboration with project 6. The phenotype will also be characterized at the cellular level by determining the effects of transgene expression on the adhesion migration and survival of osteoblasts isolated from wildtype and transgenic mice. Finally, effects of this transgene on the biomechanical properties and repair capabilities will be studied in collaboration with Project 2. Studies in Aim 2 will focus on the role of fibronectin in osteoblast differentiation and survival in vivo. The N-terminal 70 kDa matrix assembly and collagen binding domain will be expressed in osteoblasts of transgenic mice. This domain was shown previously to suppress the differentiation of cultured immature primary osteoblasts and to trigger apoptosis of mature cultured osteoblasts. The phenotype will be characterized using the approaches and interactions indicated for Aim 1. In addition, the ability of intact fibronectin or fragments of fibronectin to accelerate or interfere with skeletal repair will be tested in a distraction osteogenesis model in collaboration with Project 2. Since there are currently no animal models that test the roles of fibronectin and its integrin receptors in osteoblast function and bone remodeling, these studies should provide novel insights with relevance to skeletal repair and skeletal degenerative disorders.