IN VIVO IMAGING OF GROWTH PLATE DYNAMICS

描述（由申请人提供）：儿童的骨生长通过长骨末端软骨生长板中的软骨内骨化发生。在过去的十年中，转基因构建体的分析已成为研究软骨细胞分化级联反应中作用的内分泌、旁分泌和自分泌调节因子的最有效方法。然而，尽管在描绘定义骨骼生长及其调节电路的细胞和分子事件方面取得了重大进展，但对内分泌（和潜在的旁分泌）调节剂需要到达生长板软骨细胞的血管通路的生理学以及这些分子如何在空间异质性生长板基质内移动知之甚少。这在很大程度上反映了缺乏在活体动物的真实的时间条件下研究这些问题的方法。这对于研究生长板尤其具有挑战性，生长板具有三种概念上不同的血液供应：骨骺，干骺端和软骨膜（包括环形血管和丛）。 我们将采用一种新的方法来解决这些问题，利用多光子显微镜（MPM）的小鼠近端胫骨生长板的多小时在体内成像。我们的实验系统是用绿色荧光蛋白连接到胶原II启动子（Col II/GFP）的麻醉的4-5周龄转基因小鼠。具体目标概述如下： 通过直接成像IV注射的荧光示踪剂（从葡聚糖开始，但转移到已知的软骨细胞分化的生物学重要调节剂）的到达，检验不同大小的分子进入生长板的递送速率不同的假设。 检验不同分子量的示踪剂通过不同血管途径到达生长板并在生长板内以不同基质亚室行进的假设。对于骨骺血管系统，分析将包括次级骨化中心完全发育之前的时间点。 对干骺端软骨-骨连接处的细胞更新事件进行真实的实时成像，作为与骨延长率数据相关的“内部时钟”。 这些分析与理解生长异常有关，例如创伤伴过早闭合和骨膜破坏导致过度生长。详细的功能知识的血管路线的生长板，生长板内的分子的运动，是必不可少的，因为考虑到靶向治疗剂的生长板。

DESCRIPTION (provided by applicant): Bone growth in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. In the last decade, analysis of transgenic constructs has become the single most powerful approach for the study of endocrine, paracrine and autocrine regulators acting during the chondrocytic differentiation cascade. However, despite major advances in delineating the cellular and molecular events that define skeletal growth and its regulatory circuitry, relatively little is known about the physiology of the vascular access that endocrine (and potentially paracrine) regulators require to reach growth plate chondrocytes, and how these molecules move within the spatially heterogeneous growth plate matrix. To a large extent this reflects a lack of methods to study these questions under real time conditions in living animals. This is particularly challenging for studying the growth plate, which has three conceptually different blood supplies: epiphyseal, metaphyseal, and perichondrial (including a ring vessel and a plexus.) We will apply a novel approach to these questions by utilizing multiphoton microscopy (MPM) for multi-hour in vivo imaging of the murine proximal tibial growth plate. Our experimental system is anesthetized 4-5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter (Col II/GFP). The specific aims are summarized as: To test the hypothesis that the rate of delivery into the growth plate varies for molecules of different size, by directly imaging the arrival of IV injected fluorescent tracers (starting with dextrans but moving to known biologically significant regulators of chondrocytic differentiation.) To test the hypothesis that tracers of different molecular weight reach the growth plate through different vascular routes and travel within the growth plate in different matrix subcompartments. For the epiphyseal vasculature the analysis will include time points prior to full development of the secondary center of ossification. To image cellular turnover events at the metaphyseal chondroosseous junction in real time, as an "internal clock" for correlation with data on rates of bone elongation. These analyses are relevant to the understanding of growth aberrations such as trauma with premature closure and disruption of the periosteum leading to overgrowth. Detailed functional knowledge of vascular routes to the growth plate, and movement of molecules within the growth plate, is essential as thought is given to targeting therapeutic agents to the growth plate.