Integrated Platform to Construct and Image 3-D Perfused Vascular Network Within Thick Matrix

智能优点：组织工程的主要挑战之一是由于缺乏血管灌流而无法生长厚组织。此外，许多成体干细胞的一个关键特征是它们接近体内的血管系统。血管细胞不仅形成输送营养和氧气的管道，而且还提供线索来控制周围组织中的细胞表型。因此，构建具有生理灌流功能的血管网络对于干细胞和组织工程领域都是至关重要的。这项提议的目标是创造一个灌流的、具有功能的、成分和生理参数可控的血管周围利基环境。我们的方法是使用我们实验室开发的一种新的生物制造技术，在水凝胶中建立一个三维灌流功能血管通道。这些制作的血管通道提供了一个真实的体外模型系统，用于研究3D基质中灌流的血管通道周围的毛细血管形态发生。同时，对于如此厚的3-D矩阵(2~3 mm)，缺乏评估血管通道的三维结构和功能以及观察血管网络生长的成像手段。为了解决这个问题，我们还在开发和改进一种新的成像技术，称为层流光学层析成像(Lot)。LOT能够以高灵敏度获得深度分辨率高达几毫米的三维定量图像。为了充分发挥Lot在厚组织成像方面的潜力，促进其在组织工程研究中的应用，我们建议将这两个学科(三维成像和三维组织打印)合并到一个新的平台中来研究厚基质中的血管形成：具体目标1：利用一种新的细胞打印技术在三维基质中创建一个灌流的、相互连接的血管系统。具体目标2：集成用于厚组织成像的高分辨率、多光谱分子层流光学层析成像平台。具体目标3：验证Lot在血管结构和流体流动的多色成像中的作用；从基质特性和培养参数方面研究合成结构的成熟过程和功能。这项研究将在干细胞、组织工程和生物医学光学领域产生重大的智力优势：(1)通过将一种新的细胞打印技术整合到血管形成过程中，这项研究将带来一种新的方法来总结和研究体外血管周围的生态位。通过研究其在3D灌流环境下的构型和成熟过程，本研究将确定3D血管系统实现其功能的关键因素。(2)本研究将整合一个新的基于CCD的LOT系统，并将针对大数据集开发专门的重建算法。这将建立一种新的高分辨率介观荧光成像技术，为进行三维组织工程构建的功能和分子成像提供新的方法。(3)更广泛地说，这项工作将确定一套打印结构的设计原则，以支持周围组织的体外生长，从而为未来更厚的组织的生物丰富奠定基础。广泛的影响：本研究在研究、社会和教育方面具有广泛的影响：(1)拟议的研究活动将产生与三维血管组织形成相关的基础知识，以及获得组织功能分子成像的新技术。这两个进展对于设计厚组织结构和理解三维基质内细胞相互作用的复杂生物学都是至关重要的，三维基质对整个领域变得越来越重要。(2)拟议的教育部分将培养本科生/研究生掌握最新的技术、实验技能和创造性思维，这些在不断增长的生物医学工程领域是不可或缺的。(3)拟议的研究和教育活动还将通过几个针对科学和工程领域代表性不足的群体的拟议计划，以及针对纽约科技谷地区学生的广泛推广计划，对K-12教育产生广泛影响。通过展示工程师如何解决社会挑战的活动，我们的学生将更有可能从事科学和工程方面的职业。

PI: Guohao DaiCBET ID: 1263455Intellectual Merit: One of the major challenges in tissue engineering is the inability to grow thick tissues due to lack of vascular perfusion. In addition, a key feature of many adult stem cell niches is their proximity to the vasculature in vivo. Vascular cells not only form conduits to deliver nutrient and oxygen, but also provide cues to control the cellular phenotype in the surrounding tissues. Therefore, building a functional vascular network with physiological perfusion is critical to both fields of stem cells and tissue engineering. The objective of this proposal is to create a perfused, functional perivascular niche with controllable components and physiological parameters. Our approach is to build a 3-D perfused functional vascular channel within hydrogels using a new bio-fabrication technology developed in our lab. These fabricated vascular channels provide a realistic in vitro model system to investigate capillary morphogenesis around the perfused vascular channel within the 3-D matrix. Meanwhile, the imaging modalities to assess the 3-D structures and functions of the vascular channels and to observe the outgrowth of vascular networks is lacking for such thick 3-D matrices (2~3mm). To overcome this problem, we are also developing and improving a new imaging technique called Laminar Optical Tomography (LOT). LOT is able to obtain depth-resolved 3-D quantitative images to depths of several millimeters with high sensitivity. To achieve the full potential of LOT in imaging thick tissue and promote its application in tissue engineering research, we propose to merge these two disciplines (3-D imaging & 3-D tissue printing) in a new platform to study the vasculature formation in thick matrix: Specific Aim 1: Create a perfused, interconnected vasculature within 3-D matrix utilizing a novel cell printing technology. Specific Aim 2: Integrate a high-resolution, multi-spectral molecular laminar optical tomography platform for thick tissue imaging. Specific Aim 3: Validate the LOT in multi-color imaging of vascular structures and fluid flow; Study the maturation process and functionalities of the synthesized structure with regard to matrix properties and culture parameters. The proposed research will generate significant intellectual merits in the fields of stem cells, tissue engineering and biomedical optics: (1) By integrating a novel cell printing technology into the vasculature formation process, this research will lead to a new method to recapitulate and study perivascular niches ex vivo. Through studying its patterning and maturation process under a 3-D perfused environment, this research will define critical factors for the 3-D vasculature to achieve its functions. (2) This research will integrate a new CCD based LOT system and will develop specialized reconstruction algorithm for large data sets. This will establish a new high-resolution mesoscopic fluorescence imaging technique, which will provide a new method to perform functional and molecular imaging of 3-D tissue engineered construct. (3) More generally, this work will identify a set of design principles for the printed structures to support the growth of the surrounding tissues in vitro, and thereby lay the foundation to biofabricate thicker tissues in the future.Broader Impacts: This research has broad impacts in research, society, and education: (1) The proposed research activities will generate fundamental knowledge related to 3-D vascular tissue formation as well as new technologies to obtain functional molecular imaging of the tissues. Both of these advancements will be critical for engineering thick tissue construct and understanding the complex biology of cell interactions within 3-D matrix, which has become increasingly important to the whole field. (2) The proposed educational components will educate undergraduate/graduate students with up-to-date technologies, experimental skills, and creative thinking that are indispensable in the growing field of biomedical engineering. (3) The proposed research and education activities will also have a broad impact on K-12 education through several proposed programs for underrepresented groups in science and engineering, as well as extensive outreach programs targeting students from throughout New York's Tech Valley region. Through activities demonstrating how engineers can solve societal challenges, our students will be more likely to pursue career in science and engineering.