OSTEOGENESIS AND REPAIR IN LIVING INTACT BONE

描述（由申请人提供）：骨形成和修复是一个复杂的过程，涉及基因表达的改变，伴随着表达血管生成、基质合成和矿化所必需的蛋白质和酶的细胞的增加。我们的长期目标是深入了解在活的、完整的骨的生理条件下成骨途径的调控。该提议的假设是，由于动态和环境依赖性调节，许多分子骨形成途径在体外甚至原位被隐藏或与体内不同。在一种创新的方法中，我们将把活体分子成像与传统的死后分析结合起来。成像将提供生理条件下骨骼生物学的非侵入性观察，而离体分析将允许验证体内发现以及体内和体外分析之间的相关性。鉴于转录程序启动和控制成骨，第一个具体目标是直接解决我们对骨愈合过程中血管生成基因表达如何调节的不完整理解。利用创新的转基因成像报告基因小鼠，我们将验证在骨缺损愈合过程中发生血管内皮生长因子（VEGF）受体2和VEGF表达的动态模式，以及转录因子早期生长反应基因1 （EGR-1）是这些血管生成转录途径的体内调节剂的假设。第二个目标侧重于骨前体细胞增殖，这一事件通常先于骨基质和矿物质的形成。目前的模型将细胞增殖描述为骨科生物材料新生骨形成的关键。然而，这种生物反应的体内时间、强度和特异性仍不清楚。使用一种新的基于成像的增殖测量方法，我们建议在体内环境中测试生物材料表面是否会引起原代人骨前体细胞的差异增殖反应。我们的第三个目标将提供原理证明，即可以使用小成像分子和临床相关成像范例直接在活的完整骨骼中检测和测量酶促骨细胞活性。总之，所提出的研究将作为未来实验的模型，这些实验将利用成像技术以及传统的离体方法来分析骨形成途径。公共卫生相关性：本跨学科项目计划研究生理条件下控制活骨成骨的生物学。为了开始建立成骨转录、细胞增殖和骨细胞活性在体内的相关性，我们的三个目标将分别引入和验证转基因成像报告基因小鼠、基于成像的增殖测量和创新的小成像分子策略。此外，我们将利用转基因动物研究骨愈合过程中的血管生成转录，并利用体内增殖测量来研究与新生骨形成相关的生长反应。

DESCRIPTION (provided by applicant): Bone formation and repair are complex processes involving alterations in gene expression with a concomitant increase of cells expressing proteins and enzymes essential for angiogenesis, matrix synthesis, and mineralization. Our long range goal is to gain insight into the regulation of osteogenic pathways under physiological conditions in living, intact bone. The hypothesis underlying this proposal is that due to dynamic and milieu-dependent regulation, many molecular bone formation pathways are concealed or different in vitro or even in situ as compared to in vivo. In an innovative approach, we will combine molecular imaging in vivo with conventional post mortem analysis. Imaging will offer non-invasive observation of bone biology under physiological conditions, while ex vivo analysis will allow for validation of the in vivo findings and for correlation between in vivo and in vitro analyses. Given that transcriptional programs initiate and govern osteogenesis, the first specific aim is directed to address our incomplete understanding of how angiogenic gene expression is regulated during bone healing. Utilizing innovative transgenic imaging reporter gene mice, we will test the hypothesis that a dynamic pattern of vascular endothelial growth factor (VEGF) receptor 2 and VEGF expression occurs during bone defect healing and that the transcription factor early growth response gene 1 (EGR-1) is an in vivo regulator of these angiogenic transcription pathways. The second aim focuses on bone precursor cell proliferation, an event that typically precedes bone matrix and mineral formation. Current models describe cell proliferation as crucial for de novo bone formation on orthopaedic biomaterials. However, the in vivo timing, magnitude, and specificity of this biological response remain unclear. Using a novel imaging-based proliferation measurement, we propose to test, in an in vivo environment, whether biomaterial surfaces provoke differential proliferative responses in primary human bone precursor cells. Our third aim will provide proof-of-principle that enzymatic bone cell activity can be detected and measured directly in living, intact bone using a small imaging molecule and a clinically relevant imaging paradigm. Together, the proposed studies will serve as model for future experiments which utilize imaging technology in addition to traditional ex vivo methods for the analysis of bone formation pathways. PUBLIC HEALTH RELEVANCE: This interdisciplinary project plans to investigate the biology that controls osteogenesis in living bone under physiological conditions. To begin to establish the relevance of osteogenic transcription, cell proliferation and bone cell activity in vivo, our three aims will introduce and validate transgenic imaging reporter gene mice, imaging-based proliferation measurement and an innovative small imaging molecule strategy, respectively. In addition, we will utilize the transgenic animals to study angiogenic transcription during bone healing and take advantage of the in vivo proliferation measurement to investigate the growth response associated with de novo bone formation.