In vivo Imaging of Growth Plate Dynamics

描述（由申请人提供）：儿童的骨延长通过长骨末端软骨生长板的软骨内骨化发生。题为生长板动力学体内成像的父R01资助了一个项目，该项目使用多光子显微镜来可视化活小鼠生长板软骨中的分子运输和细胞动力学。这些研究导致了有关生长软骨内氧水平的具体问题，以及它们与软骨形成分化过程中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的关系。虽然超过50年的实验数据表明，有一个O2梯度内的生长板细胞外基质（ECM），在软骨细胞分化的重要作用，明确的测量仍然是不确定的，根据不同的方法和实验系统的相互矛盾的结果。导致生长板软骨ECM内O2输送和运动不明确的原因是研究人员无法直接绘制体内区域内和周围的O2浓度。我们的BIRT提案描述了一个高度跨学科的努力，以生产基于钌络合物的氧敏感磷光体，该磷光体明亮，灵敏且稳定。为多光子显微镜量身定制，它们将产生相对深入小鼠体内生长软骨及其周围的O2张力的快速微米分辨率图像。候选传感器不仅要测试其生物物理特性，还要测试其在组织内的相互作用和功能。探针的发展将适用于共聚焦和多光子显微镜，并可能整个动物的“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相关联，并将ECM内的O2梯度与三种不同周围血管途径中的O2可用性水平相关联。为这个BIRT奖组装的多学科团队结合了化学家的专业知识，具有设计氧传感探针的经验，具有设计无毒输送系统技能的纳米技术专家，具有使用多光子显微镜进行辐射测量和体内成像系统的专业知识的物理学家，以及一位生物学家，其研究重点是出生后动物软骨细胞分化级联的细胞动力学。

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.