Vascularization of polymeric tissue beds

描述(由申请人提供)：这项题为聚合物组织床的血管化的提案寻求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发展的主要障碍是缺乏能够及时输送氧气和营养物质以及清除废物的组织工程构造中的有效血管形成。为了应对这一重大挑战，这项拟议的研究试图通过使用可以探测材料参数的细胞平台和可以调整以促进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，以及创建有利于血管生成的聚合平台，从而创建预血管化的组织床。中心假设是，有利于血管形成的组织工程化构建需要1)提供内皮细胞特异性附着，2)局部组织重塑，3)通过细胞因子信号征募内皮细胞。为了研究这一假设，我们提出了四个具体目标：(1)将细胞附着和基质重塑功能工程到胚胎干细胞来源的内皮细胞中。我们计划过表达α-V和β-3或α-5和β-1整合素，以增强细胞附着在类似ECM的聚合物网络中的能力，并过度表达MMP1以增强细胞的组织重塑能力。(2)设计一种基于聚乙二醇类ECM的水凝胶，它包含用于细胞附着的整合素结合肽(-RGD-或-CRRETAWAC-对3和5具有高亲和力和选择性)，用于EC介导的生物降解的胶原酶敏感肽-GPQGIAGQ，以及包裹的纳米颗粒，为血管生成因子(VEGF)的可控释放提供储存库。(3)整合和调整细胞和聚合物平台，以获得最佳的血管生成和血管生成反应，如血管计数和3D血管重建所决定的。表达不同水平的v 3和v 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的克隆将用不同分子量的聚乙二醇水凝胶进行测试。血管内皮生长因子分泌的影响将通过EC祖细胞迁移的定量指标来评估。(4)为了评估细胞和聚合物平台在体内的预血管形成，将优化后的支架植入小鼠皮下血管形成模型，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。这项研究的成功完成将对在工程化组织结构中发展血管网络所需的需求产生基本的见解，并为促进临床转化为工程化组织奠定基础。 与公共健康相关：组织工程学是一种寻求增强或取代衰竭器官功能的方法，并提供了对人类健康产生重大有益影响的潜力。然而，这一领域进展的主要障碍是缺乏能够及时输送氧气和营养并清除废物的组织工程构建物的有效血管化。为了应对这一重大挑战，拟议的研究试图评估指导血管生成的机制，同时使用可以探测材料参数的细胞平台和可以调整以改善血管生成的模块化合成平台。这项研究的成功完成将对在工程化组织结构中开发血管网络所需的需求产生基本的见解，并促进将其转化为临床应用。

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.