IN VIVO IMAGING OF GROWTH PLATE DYNAMICS

描述（由申请人提供）：儿童骨骼生长通过长骨末端软骨生长板的软骨内成骨发生。在过去的十年中，转基因构建体的分析已经成为研究在软骨细胞分化级联过程中作用的内分泌、旁分泌和自分泌调节因子的唯一最有力的方法。然而，尽管在描述定义骨骼生长及其调控回路的细胞和分子事件方面取得了重大进展，但对于内分泌（和潜在的旁分泌）调节剂到达生长板软骨细胞所需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中移动，我们所知相对较少。这在很大程度上反映了缺乏在活体动物的实时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，因为生长板有三个概念上不同的血液供应：骨骺、干骺和软骨膜（包括环状血管和神经丛）。

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.