INTEGRIN/ECM INTERACTIONS IN BONE FORMATION AND REMODELING

细胞与细胞外基质和邻近细胞（固态环境）的相互作用对于发育过程中的组织形态发生以及整个生命过程中的组织稳态和重塑至关重要。这些实验室先前对培养成骨细胞的研究表明，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起着关键作用。拟议研究的总体目标是使用转基因小鼠模型测试这些观察结果的生理学相关性。在一个这样的模型中，整合素β 1亚基的截断形式已在转基因小鼠的成熟成骨细胞中表达。表型的初步表征揭示了出生后幼龄动物成骨细胞有缺陷的骨基质沉积和成骨细胞过度的骨重塑。在Aim 1中，这种表型将在骨组织水平上进一步表征，通过分析组织结构、基质和矿物质量，使用组织形态计量学，以及与项目6合作的先进成像技术。表型还将在细胞水平上通过确定转基因表达对野生型和转基因小鼠分离的成骨细胞的粘附、迁移和存活的影响来表征。最后，该转基因对生物力学特性和修复能力的影响将与项目2合作研究。Aim 2的研究将集中在纤维连接蛋白在成骨细胞分化和体内存活中的作用。在转基因小鼠的成骨细胞中，n端70 kDa基质组装和胶原结合域将得到表达。该结构域抑制培养的未成熟原代成骨细胞的分化，并引发成熟培养成骨细胞的凋亡。表型将使用Aim 1中指出的方法和相互作用进行表征。此外，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将在与Project 2合作的牵张成骨模型中进行测试。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塑中的作用，这些研究应该为骨骼修复和骨骼退行性疾病提供新的见解。

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.