Vascularization of polymeric tissue beds

描述（由申请人提供）：该提案题为聚合组织床的血管化，旨在解决再生医学的广泛挑战领域，以及工程组织中血管网络的具体挑战主题：11- eb -101。组织工程和再生医学领域进展的主要障碍是组织工程结构缺乏有效的血管化，无法及时输送氧气和营养物质，并清除废物。为了解决这一重大挑战，提出的研究旨在通过使用可以探测材料参数的细胞平台和可以调整以增强血管生成的模块化合成平台来阐明指导血管生成和血管生成的机制。总体目标是通过调整细胞平台来增强归巢和重塑，以及有利于血管生成的聚合物平台来创建血管化的组织床。中心假设是，有利于血管形成的组织工程结构需要具备以下能力：1)提供内皮细胞特异性附着；2)局部组织重塑；3)通过细胞因子信号传导招募内皮细胞。为了验证这一假设，我们提出了四个具体目标：(1)设计胚胎干细胞衍生的内皮细胞的细胞附着和基质重塑功能。我们计划过表达α - v和β -3，（¿v¿3）或α -5和β -1（¿5¿1）整合素，以增强细胞在ecm模拟聚合物网络中的附着能力，并过表达MMP1以增强细胞的组织重塑能力。(2)设计一种基于聚乙二醇的ecm模拟水凝胶，该水凝胶包含整合素结合肽（- rgd -或- crretawac -具有高亲和力和选择性的- v¿3和- 5¿1），用于细胞附着；一种胶原酶敏感肽- gpqgiagq -用于EC介导的生物降解，以及为血管生成因子（VEGF）的控制释放提供储存库的包裹纳米颗粒。(3)整合和调整细胞和聚合物平台，以实现最佳的血管生成和血管生成反应，由血管计数和3D血管重建决定。表达不同水平的¿v´3和¿5´1整合素的克隆将用具有一系列肽配体密度的模拟ecm水凝胶进行测试。表达不同水平MMP1的克隆将用不同PEG分子量的水凝胶进行测试。VEGF分泌的影响将通过EC祖细胞迁移的定量测量来评估。(4)为了评估细胞和聚合物平台在体内的预血管化，优化后的支架将被植入小鼠皮下血管化模型，并与Matrigel阳性对照进行比较，并通过体内成像技术（生物发光和PET）进行监测。本研究的成功完成将对在工程组织结构中发展血管网络所需的要求产生根本性的见解，并为促进临床转化为工程组织奠定基础。

DESCRIPTION (provided by applicant): This proposal, entitled Vascularization of Polymeric Tissue Beds, seeks to address the broad Challenge Area of (11) Regenerative Medicine, and the specific Challenge Topic of vascular networks in engineered tissues: 11-EB-101. The major impediment to progress in the field of tissue engineering and regenerative medicine is the lack of effective vascularization in tissue-engineered constructs capable of timely delivery of oxygen and nutrients, and removal of waste products. To address this major challenge, the proposed research seeks to elucidate mechanisms that direct vasculogenesis and angiogenesis by using a cellular platform that can probe the material parameters, and a modular synthetic platform that can be tuned to enhance vasculogenesis. The overall goal is to create a prevascularlized tissue bed by tuning a cellular platform to enhance homing and remodeling, and a polymeric platform that is conducive for vasculogenesis. The central hypothesis is that a tissue engineered construct conducive to vascularization requires the capacity 1) to provide endothelial cell specific attachment, 2) for local tissue remodeling, and 3) to recruit endothelial cells through cytokine signaling. To investigate this hypothesis, we propose four specific Aims: (1) to engineer cellular attachment and matrix remodeling functions into embryonic stem cell derived endothelial cells. We plan to overexpress alpha-V and beta-3, (¿v¿3) or alpha-5 and beta-1 (¿5¿1) integrins, to enhance the cell's capacity for attachment in an ECM-mimetic polymer network, and overexpress MMP1 to enhance the cell's capacity for tissue remodeling. (2) To engineer a PEG-based ECM-mimetic hydrogel, which incorporates integrin binding peptides (-RGD- or -CRRETAWAC- which has high affinity and selectivity for ¿v¿3 and ¿5¿1) for cell attachment; a collagenase-sensitive peptide -GPQGIAGQ- for EC mediated biodegradation, and entrapped nanoparticles that provide a reservoir for controlled release of angiogenic factors (VEGF). (3) To integrate and tune the cellular and polymeric platforms, to achieve optimal vasculogenic and angiogenic responses, as determined by vessel counts and 3D vessel reconstruction. Clones expressing varying levels of ¿v¿3 and ¿5¿1 integrins will be tested with ECM-mimetic hydrogels with a range of peptide ligand density. Clones expressing varying levels of MMP1 will be tested with hydrogels varying in PEG molecular weight. The effect of VEGF secretion will be evaluated by quantitative measures of EC progenitor migration. (4) To evaluate prevascularization of the cellular and polymeric platforms in vivo, optimized scaffolds will be implanted into a mouse subcutaneous model of vascularization and compared with Matrigel positive controls, and monitored by in vivo imaging techniques (bioluminescence and PET). Successful completion of this research will generate fundamental insights into the requirements needed for developing vascular networks in engineered tissue constructs and lay a basic foundation for facilitating clinical translation into engineered tissues. PUBLIC HEALTH RELEVANCE: Tissue engineering is an approach that seeks to augment or replace the function of failing organs, and offers the potential for major beneficial impact on human health. However, the major impediment to progress in this field is the lack of effective vascularization of the tissue-engineered constructs capable of timely delivery of oxygen and nutrients and removal of waste products. To address this major challenge, the proposed research seeks to evaluate the mechanisms that direct angiogenesis in parallel with using a cellular platform that can probe the material parameters, and a modular synthetic platform that can be tuned to improve vasculogenesis. Successful completion of the research will generate fundamental insights into the requirements needed for developing vascular networks in engineered tissue constructs and facilitate translation into clinical applications.