Mechanisms for Axonal Guidance Using Living Tissue Engineered Scaffolds

 描述（由申请人提供）：神经创伤和神经退行性疾病每年影响数百万人。一种常见的病理是长距离连接的丧失，特别是连接中枢神经系统区域或传递外周信号的轴突。这种轴突变性可能导致永久性缺陷。由于中枢和外周神经系统中长距离轴突连接缺乏自发再生能力，研究人员正在开发组织工程结构来逆转创伤或神经退行性疾病的影响。成功的应用包括整合工程活组织，以直接恢复失去的功能或创造更合适的 再生环境。为了促进轴突再生，我们利用新型组织工程神经移植物（TENG），其由通过“拉伸生长”产生的长的对齐的轴突束组成，这是一种在定制的机械生物反应器中复制的自然机制，以在短时间内产生前所未有长度的轴突。轴突束作为神经再生的活支架。在先前的啮齿动物和猪的研究中，TENG中的活轴突束已经被认为是指导再生轴突的“引导路径”，再生的宿主轴突直接沿着移植的TENG轴突生长，证明了直接轴突介导的轴突再生（AMAR）。然而，负责这一现象的分子介质仍然未知，但对于进一步增强这一技术至关重要。因此，在我的研究任期内，我打算阐明主要负责AMAR的分子介质，通过开发一个体外试验床，并利用创新的体内轴突再生范例，系统地阐明主要驱动AMAR的细胞因子。具体来说，我假设，阿曲他克林信号-轴突表面线索和伴随的可溶性因子的亲密介绍相结合-驱动AMAR。将通过免疫组织化学、共聚焦显微镜、超分辨率显微镜以及啮齿动物功能恢复的电生理学分析来检验这一假设。确定AMAR中涉及的精确阿曲他克林信号传导广泛适用于改善外周以及中枢神经系统修复和再生，从而最终改善一系列创伤性损伤或神经退行性疾病后的神经恢复。

 DESCRIPTION (provided by applicant): Neurotrauma and neurodegenerative diseases affect millions of people annually. A common pathology is the loss of long-distance connections, specifically axons connecting regions of the central nervous system or relaying peripheral signals. This axonal degeneration may result in permanent deficits. Due to the lack of spontaneous regenerative capability of long-distance axonal connections in the central and peripheral nervous system, researchers are developing tissue engineered constructs to reverse the effects of trauma or neurodegenerative disease. Successful application involves the integration of engineered living tissue to directly restore lost function or create a more suitable environment for regeneration. To facilitate axon regeneration, we utilize novel tissue engineered nerve grafts (TENGs) comprised of long, aligned axonal tracts generated by "stretch-growth", a natural mechanism that is replicated in custom mechano-bioreactors to generate axons of unprecedented lengths in a short period of time. The axonal tracts serve as a living scaffold for neuroregeneration. In previous rodent and swine studies, the living axonal tracts in TENGs have been seen to serve as "guidance paths" to direct regenerating axons, with regenerating host axons growing directly along transplanted TENG axons, demonstrating direct axon mediated axon regeneration (AMAR). However, the molecular mediators responsible for this phenomenon remain unknown, yet are crucial to further enhance this technology. Therefore, during my fellowship tenure, I intend to elucidate the molecular mediators primarily responsible for AMAR by developing an in vitro test bed and utilizing an innovative in vivo axon regeneration paradigm to systematically elucidate the cellular factors primarily driving AMAR. Specifically, I hypothesiz that juxtacrine signaling - a combination of axon-surface cues and concomitant intimate presentation of soluble factors - drives AMAR. This hypothesis will be tested through immunohistochemistry, confocal microscopy, super resolution microscopy, as well as electrophysiological analyses for functional recovery in rodents. Determining the precise juxtacrine signaling involved in AMAR is broadly applicable to improving peripheral as well as central nervous system repair and regeneration, thus ultimately improving neurological recovery following a range of traumatic injuries or neurodegenerative diseases.