Microenivironment Dimensionality Modulates Neuronal Signaling

微环境维度调节神经信号

基本信息

批准号：
7742254
负责人：
Jennie B Leach
金额：
$ 32.43万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE COUNTY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7742254
关键词：
Adhesives Architecture Behavior Biochemical Biocompatible Biocompatible Materials Biological Biopolymers Cell Adhesion Molecules Cell Survival Cell-Matrix Junction Cells Cellular Morphology Clinical Controlled Environment Cues Cytoskeleton Data Diffusion Engineering Environment Extracellular Matrix Fibronectins Future Gel Goals Hypoxia In Vitro Integrins Investigation Knowledge Laboratories Laboratory Study Laminin Ligands Measurement Measures Methods Morphology Mus Nerve Neurites Neurobiology Neurons Normal tissue morphology Outcome Oxygen PTK2 gene Peptides Performance Pharmacologic Substance Physiology Play Process Property Reporter Role Signal Pathway Signal Transduction Signaling Molecule Spinal Ganglia Spinal cord injury Stroke System Testing Tissues Translating Vinculin Work Wound Healing base cell type density design improved in vivo neuronal survival neurophysiology next generation novel physical property public health relevance relating to nervous system repaired research study response scaffold success three dimensional structure tool two-dimensional

项目摘要

DESCRIPTION (provided by applicant): Translating information from two-dimensional (2D) culture into three-dimensional (3D) systems has been a major hurdle in the use of biopolymers for tissue repair applications. In order to design improved culture environments and responsive architectures for neuronal repair, our goal is to advance the understanding of how neurons respond to 3D environments. We hypothesize that 3D culture 1) imposes changes in matrix ligand organization that directly alter neuronal behavior by modulating 1 integrin-cytoskeletal signaling and 2) imposes changes in dissolved oxygen profiles. Therefore substrate dimensionality is a critical factor for neuronal survival and re-establishment of functional connectivity required for the success of cell-based neural therapies. To test this hypothesis, we will first investigate the roles of 1 integrin, vinculin, FAK and pFAK in DRG neurite outgrowth in 3D laminin culture scaffolds (Aim 1). We will then optimize the 3D culture scaffolds to maximize neurite outgrowth and determine whether the type of 1 integrin ligands impacts integrin signaling during neurite outgrowth in 3D scaffolds (Aim 2). Finally, we will determine how oxygen concentration impacts neuronal survival and outgrowth in 3D culture by applying novel oxygen-sensing microparticles to directly measure spatial and temporal dissolved oxygen profiles (Aim 3). Our preliminary studies indicate that 3D culture imposes changes in 1 integrin signaling that result in altered neurite outgrowth. To study this effect in more detail, we have established two novel tools to provide quantitative data in a physiologically relevant 3D system. First, we have developed a 3D culture system with controllable physical and biochemical material properties. Second, we have developed novel fluorescent oxygen-sensing microparticles to detect spatial and temporal changes in dissolved oxygen content. The microparticles demonstrate sensing performance comparable to traditional electrochemical probes, but are biocompatible and allow rapid, automated and non-invasive measurements local to cells and without consuming oxygen. Based on these studies, we will use cellular and environmental markers of neural morphology and dissolved oxygen to design a system that recapitulates tissue physiology. Our studies will delineate key signaling mechanisms to provide a biological basis for testing new 3D nerve repair therapies. Moreover, the adaptability of the proposed tunable synthetic gels allows for the addition of other biomolecules, pharmaceuticals, reporter constructs and cell types. Thus, the tunable synthetic gels will have broad utility towards investigations of permissive/inhibitory matrix cues as well as neuronal-glial interactions in normal and diseased states. The proposed project will provide new fundamental knowledge about neuronal response to 3D microenvironments and will enable the improved design of future biomaterials-based approaches for neural repair. PUBLIC HEALTH RELEVANCE: Much of our current understanding of neurobiology relies on disrupted tissues, laboratory studies in artificial environments, and clinical observations. We hypothesize that the next generation of nerve repair therapies relies on the design of materials that better replicate the three-dimensional structure and physiology of native tissues. The goals of this proposed work is to advance the understanding of neuronal response to three-dimensional environments and to provide new improved materials and tools to study and repair neurons.

描述（由申请人提供）：将二维（2D）培养的信息转化为三维（3D）系统一直是使用生物聚合物进行组织修复应用的主要障碍。为了设计改进的文化环境和神经元修复的响应式体系结构，我们的目标是促进对神经元如何响应3D环境的理解。我们假设3D培养1）施加基质配体组织的变化，该组织通过调节1个整合素 - 细胞骨架信号和2）施加溶解的氧气谱的变化，直接改变神经元行为。因此，底物维度是神经元存活和重建基于细胞神经疗法所需的功能连接性的关键因素。为了检验这一假设，我们将首先研究3D层粘连蛋白培养支架中DRG神经突生长中1个整合素，Vinculin，Fak和Pfak的作用（AIM 1）。然后，我们将优化3D培养基支架，以最大化神经突生长，并确定1个整合素配体的类型是否会影响3D支架中神经突生长过程中整联蛋白信号传导（AIM 2）。最后，我们将通过应用新型的氧气感应微粒直接测量空间和颞溶解的氧谱（AIM 3）来确定氧浓度如何通过应用新颖的氧气微粒来影响3D培养的神经元存活和产物（AIM 3）。我们的初步研究表明，3D培养物施加了1个整合素信号传导的变化，从而导致神经突生长发生变化。为了更详细地研究这种效果，我们建立了两个新型工具，以在生理相关的3D系统中提供定量数据。首先，我们开发了具有可控物理和生化材料特性的3D培养系统。其次，我们开发了新型的荧光氧气微粒，以检测溶解氧含量的空间和时间变化。微粒表现出与传统电化学探针相当的感应性能，但具有生物相容性，并且可以快速，自动化和非侵入性测量，而无需消耗氧气。基于这些研究，我们将使用神经形态的细胞和环境标志物并溶解氧气来设计一种概括组织生理学的系统。我们的研究将描述关键信号传导机制，以提供测试新的3D神经修复疗法的生物学基础。此外，提出的可调合成凝胶的适应性允许添加其他生物分子，药物，报告基因构建体和细胞类型。因此，可调节的合成凝胶将在正常状态和患病状态下的允许/抑制基质提示以及神经元相互作用方面具有广泛的实用性。拟议的项目将提供有关对3D微环境的神经元反应的新基本知识，并将能够改善未来基于生物材料的神经修复方法的设计。公共卫生相关性：我们当前对神经生物学的大部分理解都依赖于组织中断，人工环境中的实验室研究和临床观察结果。我们假设下一代神经修复疗法依赖于更好地复制天然组织的三维结构和生理学的材料的设计。这项拟议工作的目标是提高对神经元对三维环境的反应的理解，并提供新的改进的材料和工具来研究和修复神经元。