Nondestructive, High Resolution Imaging Platform For Tissue Regeneration Research

描述(申请人提供)：组织的发展和再生是高度复杂和动态的，涉及发育中和/或损伤组织内细胞及其周围的细胞外基质的广泛重塑。尽管组织工程技术在研究再生方面发展迅速，但我们仍然无法以微创的方式实时监测动态生物过程，这显著降低了这些技术的临床相关性。大多数可用的评估方法都是静态的，需要在固定的时间点牺牲实验样本。因此，对新技术的需求尚未得到满足，这些新技术将对开发和再生过程进行非破坏性和动态监测。生物学中的光学成像大致可分为弹道成像和扩散成像。荧光粉的组合 而具有光电子学和计算技术的生物发光探测器导致了光学分子成像工具的发展，使人们能够随着时间的推移可视化复杂的生命系统中的生物相互作用。然而，尽管光学分子成像具有巨大的潜力，但由于组织混浊，导致光的强烈散射和吸收以及有限的穿透深度，它尚未被用作组织再生研究的使能技术，需要直接观察组织。我们最近发表了几篇开创性的手稿，描述了通过结合光纤技术和图像重建方法开发间接、非破坏性的细胞水平成像仪器，以及使用荧光标记细胞组合生成生物工程成熟的和血管化的骨骼肌组织。本研究的目的是利用生物工程骨骼肌模型，开发并验证一种可广泛应用于组织再生研究的新型光学分子层析成像平台。我们假设1)光学成像、光子传输建模和图像重建将允许对生物工程肌肉组织结构进行非侵入性(间接)动态分析，2)不同荧光探针的断层扫描将改进对由多种细胞类型组成的生物工程肌肉结构的检查。我们将通过开发一种配备光纤成像系统的多孔组织培养皿来检验这些假设。我们将首先测试成像系统产生荧光标记细胞光学模体的能力，然后使用成像系统在体外评估肌肉前体细胞和内皮细胞的组织和分化为多细胞骨骼肌组织的能力。这些研究有可能推动从2D培养模型中细胞功能的静态分析向3D组织的系统分析的范式转变。实现这项提案中提出的目标将建立一种构建和成像3D复合生物工程组织的新技术，并提高我们对组织发育和再生机制的理解。

DESCRIPTION (provided by applicant): Tissue development and regeneration is highly complex and dynamic, involved with extensive remodeling of cells and the extracellular matrix surrounding them inside the developing and/or injured tissues. Despite the rapid development of tissue engineering technologies to study regeneration, a major barrier still exists in our inabilit to monitor dynamic biological processes in a minimally invasive real-time fashion, which significantly reduces the clinical relevance of these techniques. Most available assessment methods are static, requiring sacrifice of experimental samples at fixed time points. Therefore, there is an unmet need for new technologies that will provide non-destructive and dynamic monitoring of the development and regeneration processes. Optical imaging in biology can be broadly classified as either ballistic imaging or diffusive imaging. The combination of fluorescent and bioluminescent probes with optoelectronics and computing techniques has led to the development of optical molecular imaging tools that allow the visualization of biologic interactions in complex, living systems over time. However, despite the great potential of optical molecular imaging, it has not yet been harnessed as an enabling technology for tissue regeneration research, due to tissue turbidity, resulting in strong scatter and absorption of light and limited penetration depth, requiring direct view of the tissue. We have recently published several seminal manuscripts describing the development of an indirect, non-destructive, cellular-level imaging instrument through a combination of fiber optic technology and an image reconstruction approach and generation of bioengineered mature and vascularized skeletal muscle tissue using combinations of fluorescently labeled cells. These achievements serve as the motivation for the current proposal, which aims to utilize the model of bioengineered skeletal muscle to develop and validate a novel optical molecular tomography platform, which could be broadly used for tissue regeneration research. We hypothesize that 1) optical imaging, photon transport modeling, and image reconstruction will allow for the non- invasive (indirect), dynamic analysis of bioengineered muscle tissue constructs~ and 2) tomography of distinct fluorescent probes will improve the examination of developing bioengineered muscle constructs, comprised of multiple cell types. We will test these hypotheses by developing a multiwell tissue culture dish equipped with fiber-based imaging system. We will first test the capacity of the imaging system to generate optical phantoms of fluorescently labeled cells and subsequently use the imaging system to assess the organization and differentiation of muscle progenitor and endothelial cells into a multicellular skeletal muscle tissue in vitro. These studies have the potential to drive a paradigm shift from static assays of cellular function in 2D culture models towards systematic analyses of 3D tissues. Achieving the goals set forth in this proposal will establish a novel technology to construct and image 3D composite bioengineered tissues and improve our understanding of tissue development and regeneration mechanisms.