Microenivironment Dimensionality Modulates Neuronal Signaling

描述(申请人提供)：将二维(2D)培养的信息转换为三维(3D)系统一直是生物聚合物用于组织修复应用的主要障碍。为了设计更好的培养环境和神经元修复的响应性架构，我们的目标是促进对神经元如何对3D环境做出响应的理解。我们假设，3D培养1)通过调节整合素-细胞骨架信号，改变基质配体的组织结构，直接改变神经元的行为；2)改变溶氧谱。因此，底物维度是神经元存活和重建功能连接的关键因素，这是基于细胞的神经治疗成功所必需的。为了验证这一假设，我们将首先研究1整合素、纽蛋白、FAK和pFAK在3D层粘连蛋白培养支架中DRG突起生长中的作用(目标1)。然后，我们将优化3D培养支架以最大化轴突生长，并确定1整合素配体的类型是否影响3D支架中轴突生长过程中的整合素信号(目标2)。最后，我们将通过应用新型氧气感应微粒直接测量空间和时间的溶解氧分布来确定氧浓度如何影响3D培养中神经元的存活和生长(目标3)。我们的初步研究表明，3D培养会导致1整合素信号的改变，从而导致轴突生长的改变。为了更详细地研究这种影响，我们建立了两个新的工具来提供与生理相关的3D系统中的定量数据。首先，我们开发了一种物理和生化材料特性可控的3D培养系统。其次，我们开发了新型荧光氧敏微粒来检测溶解氧含量的空间和时间变化。这种微粒表现出与传统电化学探针相当的传感性能，但具有生物兼容性，允许在细胞局部进行快速、自动化和非侵入性的测量，而不消耗氧气。在这些研究的基础上，我们将使用神经形态和溶解氧的细胞和环境标记物来设计一个概括组织生理学的系统。我们的研究将描述关键的信号机制，为测试新的3D神经修复疗法提供生物学基础。此外，建议的可调合成凝胶的适应性允许添加其他生物分子、药物、报告结构和细胞类型。因此，这种可调节的合成凝胶将在研究允许/抑制基质线索以及正常和疾病状态下的神经元-神经胶质相互作用方面具有广泛的用途。拟议的项目将提供关于神经元对3D微环境的反应的新的基础知识，并将使未来基于生物材料的神经修复方法的改进设计成为可能。公共卫生相关性：我们目前对神经生物学的大部分理解依赖于破坏的组织、人工环境中的实验室研究和临床观察。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的材料的设计。这项拟议工作的目标是促进对神经元对三维环境的反应的理解，并为研究和修复神经元提供新的改进材料和工具。

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.