In vivo Imaging of Growth Plate Dynamics

描述（由申请人提供）：儿童的骨伸长是通过长骨末端软骨生长板的软骨内成骨而发生的。母体R01题为“生长板动力学的体内成像”，资助了一个使用多光子显微镜观察活体小鼠生长板软骨中分子运输和细胞动力学的项目。这些研究提出了关于生长软骨内氧水平的具体问题，以及它们与软骨分化过程中的细胞代谢和导致干骺端内皮细胞血管侵入软骨的信号级联反应的关系。尽管50多年来的实验数据表明，生长板细胞外基质（ECM）中存在氧气梯度，在软骨细胞分化中起重要作用，但根据方法和实验系统的不同，确定的测量结果仍然不确定，结果相互矛盾。导致生长板软骨ECM中氧气输送和运动不明确的原因是，研究人员无法直接绘制体内该区域内和周围的氧气浓度。我们的BIRT提案描述了一个高度跨学科的努力，以生产基于钌配合物的氧敏感荧光粉，它是明亮的，敏感的和稳定的。专为多光子显微镜设计，它们将在体内产生相对深入小鼠生长软骨及其周围的氧张力的快速微米分辨率图像。候选传感器不仅要测试其光物理特性，还要测试它们在组织中的相互作用和功能。探针的发展将适用于共聚焦和多光子显微镜，以及潜在的整个动物“分子成像”系统。我们的目标是将微环境O2水平与软骨细胞分化的特定阶段联系起来，并将ECM内的O2梯度与三种不同周围血管途径的O2可用性水平联系起来。为获得BIRT奖而组建的多学科团队包括：一位具有设计氧传感探针经验的化学家，一位具有设计无毒输送系统技能的纳米技术专家，一位具有使用多光子显微镜进行辐射测量和体内成像系统专业知识的物理学家，以及一位专注于研究出生后动物软骨细胞分化级联的细胞动力学的生物学家。

DESCRIPTION (provided by applicant): Bone elongation in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. The parent R01 entitled In vivo Imaging of Growth Plate Dynamics funds a project using multiphoton microscopy to visualize molecular transport and cellular dynamics in growth plate cartilage of living mice. These studies have led to specific questions concerning oxygen levels within growth cartilage, and their relationship both to cellular metabolism during chondrogenic differentiation and to signaling cascades leading to vascular invasion of cartilage from metaphyseal endothelial cells. Although for more than fifty years experimental data have suggested that there is an O2 gradient within the growth plate extracellular matrix (ECM) that has an essential role in chondrocytic differentiation, definitive measurements still are inconclusive with conflicting results depending upon the methodology and the experimental system. What contributes to the ambiguity concerning O2 delivery to, and movement within, the growth plate cartilage ECM is that researchers are unable to directly map O2 concentrations within and around the region in vivo. Our BIRT proposal describes a highly interdisciplinary effort to produce oxygen-sensitive phosphors based on ruthenium complexes that are bright, sensitive and stable. Tailored for multiphoton microscopy, they will yield rapid micron-resolved images of O2 tension relatively deep into and around the growth cartilage of mice in vivo. Candidate sensors will be tested not only for their photophysical properties, but also for how they interact and function within tissue. Probe developments will be applicable both to confocal and multiphoton microscopy, and potentially whole animal "molecular imaging" systems. Our goal is to relate microenvironmental O2 levels to specific stages of chondrocytic differentiation, and to correlate O2 gradients within the ECM with O2 availability levels in three different surrounding vascular routes. The multidisciplinary team assembled for this BIRT award combines the expertise of a chemist with experience in designing oxygen-sensing probes, a nanotechnologist with skills in designing non-toxic delivery systems, a physicist with expertise in using multiphoton microscopy for radiometric measurements and in vivo imaging systems, and a biologist whose research has focused on cellular dynamics of the chondrocytic differentiation cascade in the postnatal animal.