Axon Guidance by Critical Cues - Engineering Nerve Growth In Vitro and Observing From Afar

1134166 Hoffman-Kim智力优点：神经在受伤后无法正确重新连接，目前的医疗实践无法成功控制神经再生的过程。拟议中的研究试图通过量化指导线索如何单独和组合促进轴突生长来解决这个问题。这些知识对于理解神经发育和促进神经再生至关重要。工作假设是定向轴突生长需要多种线索，这些线索必须在局部细胞环境水平上得到明确定义和协调。为了验证这一假设，需要制造新的平台来研究神经元的生长。这些平台将（1）以可控和可量化的方式提供线索组合;（2）提供一种方法来测试它们的分层和合作互动，特别是在细胞牵引力水平上。凭借神经发育、再生、生物材料和微加工方面的跨学科专业知识，该小组设计了一套这样的平台，并通过初步数据表明，它们有能力向神经元提供生物化学和地形学指导线索。这些平台使创新实验成为可能，这些实验将测试细胞地形和生化指导线索的组合如何促进神经突生长，以及神经突如何在生长过程中施加牵引力。目的是将定向轴突生长与特定数量和比率的线索联系起来，从而改变对神经系统精确连接如何形成的理解，以及在损伤后重新连接这些连接的策略。研究将确定哪些细胞地形特征编码导航神经元的关键指导信息，随后，地形和生化指导线索如何相互作用影响神经突生长，在合作和竞争平台上进行测试。此外，神经牵引力将在神经引导期间通过多个线索来确定。神经突的生长将采用定制设计的图像分析方法和具有相位对比和激光扫描共聚焦光学的延时显微镜在图案化材料上进行表征。条件将由与地形特征的接触限制生长神经突可以定向的角度来确定。总体目标是开发神经系统损伤后轴突可被引导的方法。拟议的多学科实验量化了生长中的轴突如何对特定的地形和生化刺激做出反应，因此它们将为开发新的管道、支架和基于生物材料的一般策略以再生神经组织提供关键信息。更广泛的影响：该项目将产生多个更广泛的影响。它将为更大的工程和科学界，特别是生物医学工程，神经生物学和细胞生物学界提供一套多功能，可定制的平台，用于测试与多种细胞和组织类型相关的各种类型的信息，包括机械，地形和生化线索。 它还将产生新的框架，用于概念化局部细胞环境中的细胞行为，在这种环境中，可以解构和重组环境以促进理解。通过该项目，参与生物医学工程研究的女本科生和研究生人数将增加，因为PI在培训女生物医学工程师方面有着良好的记录。此外，该项目还包括一个推广方案，以加强普罗维登斯公立学校中学生对生物医学工程研究的了解。迫切需要提高中学的科学成绩，这种技术，工程和探究式学习的注入为应对这一挑战提供了理想的策略。视频会议将使大量的中学生能够在真实的时间内与其他无法进入的环境中的实验进行互动，之后由受过训练的研究生以及教师实验室访问进行一系列综合课堂模块。这个密集的计划将增加中学科学课程，培养生物医学工程专业的学生向不同背景的观众解释科学和工程概念，为教师提供实习机会，并激励中学生在教育的关键时刻考虑科学和工程职业。

1134166Hoffman-KimIntellectual Merit: Nerves fail to reconnect properly after injury and current medical practice is unable to successfully control the process of nerve regeneration. The proposed research seeks to attend to this problem by quantifying how guidance cues, both individually and in combination, promote axon growth. This knowledge is central to understanding nerve development and promoting nerve regeneration. The working hypothesis is that directed axon growth requires multiple cues, which must be well-defined and coordinated at the level of the local cellular environment. To test this hypothesis will necessitate the fabrication of new platforms upon which to study neuronal growth. These platforms will (1) deliver combinations of cues in a controllable and quantifiable manner; and (2) provide a means by which to test their hierarchical and cooperative interactions, specifically at the level of cellular traction forces. With interdisciplinary expertise in nerve development, regeneration, biomaterials, and microfabrication, the group has designed a set of such platforms, and has shown with preliminary data that they have the capability to deliver biochemical and topographical guidance cues to neurons. These platforms make possible innovative experiments that will test how combinations of cell-topographical and biochemical guidance cues promote neurite growth, and how neurites exert traction forces during growth. The objective is to correlate directed axon growth to specific quantities and ratios of cues, thus transforming the understanding of how the precise connections of the nervous system form as well as strategies to rewire these connections after injury.Investigations will determine which cellular topographical features encode critical guidance information to a navigating neuron, and subsequently, how topographical and biochemical guidance cues interact to influence neurite growth, testing on both cooperative and competitive platforms. Further, neuronal traction forces will be determined during nerve guidance by multiple cues. Neurite growth will be characterized on patterned materials with custom designed image analysis approaches, and time-lapse microscopy with phase contrast and laser scanning confocal optics. Conditions will be determined by which contact with topographical features limits the set of angles over which growing neurites can orient. The overarching goal is to develop methods by which axons can be guided following injury to the nervous system. The proposed multidisciplinary experiments quantify how growing axons respond to specific topographical and biochemical stimuli, and as such they will provide critical information with which to develop new conduits, scaffolds, and general biomaterial-based strategies to regenerate nerve tissue.Broader Impacts: This project will have multiple Broader Impacts. It will provide to the larger engineering and scientific communities, in particular the Biomedical Engineering, Neurobiology and Cell Biology communities, a set of versatile, tailorable platforms with which to test a wide variety of types of information of relevance to multiple cell and tissue types, including mechanical, topographical and biochemical cues. It will also generate new frameworks with which to conceptualize cell behaviors in the local cellular environment, where the environment can be deconstructed and reassembled to advance understanding. Through this project, the numbers of female undergraduate and graduate students involved in Biomedical Engineering research will be increased as the PI has a strong record of training female biomedical engineers. Further, this project includes an Outreach Program to enhance the exposure of middle school students in the Providence Public Schools to biomedical engineering research. There is an urgent need to improve middle school science performance, and this infusion of technology, engineering, and inquiry-based learning provides an ideal strategy to address this challenge. Video conferencing will allow large numbers of middle school students to interact in real time with experiments in otherwise inaccessible environments, following a series of curriculum-integrated classroom modules by trained graduate students as well as teacher-laboratory visits. This intensive program will augment the middle school science curriculum, train biomedical engineering students to explain science and engineering concepts to audiences with diverse backgrounds, provide teachers with internship opportunities, and inspire middle school students, at critical points in their education, to consider science and engineering careers.