Engineering vascular replacements for strength and elasticity

工程血管替代物的强度和弹性

基本信息

批准号：
8186353
负责人：
ROBERT B VERNON
金额：
$ 55.79万
依托单位：
BENAROYA RESEARCH INST AT VIRGINIA MASON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-15 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8186353
关键词：
Address Adherent Culture Adult Affinity Animal Model Animals Apoptosis Area Arteries Biocompatible Biocompatible Materials Biological Assay Blood Vessels Caliber Carotid Arteries Cell Count Cells Collagen Collagen Type I Coronary Artery Bypass Deposition Dermal Development Devices Dialysis patients Elastic Fiber Elastic Tissue Elasticity Elastin Endothelial Cells Engineering Engraftment Exhibits Extracellular Matrix Fibroblasts Fibronectins Foundations Future Goals Gold Immune response In Vitro Insecta Laboratories Lead Limb structure Mechanics Medial Mediating Membrane Methods Modeling Necrosis Patients Performance Phase Physiological Population Dynamics Production Proliferating Property Proteins Proteoglycan Protocols documentation Public Health RNA Splicing Rat-1 Rattus Recombinants Research Rubber Shunt Device Signaling Molecule Smooth Muscle Myocytes Staging Stenosis Stress Structure System Techniques Testing Time Tissue Engineering Tissues Translations Transplantation Tropoelastin Tube Tunica Adventitia Variant Vascular Graft Work base crosslink density graft failure in vivo injured meetings novel novel strategies operation resilience response scaffold success vascular smooth muscle cell proliferation versican

项目摘要

DESCRIPTION (provided by applicant): The development of biologically-based tissue-engineered blood vessels (TEBVs) that combine vascular cells with biocompatible extracellular matrix (ECM) scaffolds shows promise, but is problematic in two key areas, which will be addressed in this application: 1) the absence of elastin and 2) excessive maturation times in vitro. In native blood vessels, the rubber-like protein elastin provides resilience and limits vascular smooth muscle cell (VSMC) proliferation. Unfortunately, adult VSMCs synthesize little or no elastin, hence TEBVs incorporating VSMCs from adult patients (to limit tissue rejection) would be deficient in elastin, which can lead to stenosis and mechanical failure of the graft. The Wight Laboratory has discovered that splice variant 3 of the ECM proteoglycan versican (V3) can induce VSMCs in vitro and in vivo to produce and assemble elastin. In the present application, we propose that purified, recombinant V3 (rV3) can be used to stimulate elastic fiber formation within TEBVs as they mature in vitro. In regard to excessive maturation times, many ECM-based scaffold materials are mechanically weak, which requires that TEBVs mature for months in vitro before they are strong enough to engraft safely. To address this problem, the Vernon Laboratory has developed novel, ECM-based scaffolds (microgrooved collagen membranes - MGCMs) that are mechanically strong and induce seeded cells to align uniaxially on the grooves within 24-48 h. MGCM sheets populated with aligned vascular cells have been successfully converted into tubes. We propose to combine this method of TEBV fabrication with rV3-mediated elastogenesis to create strong, elastic TEBVs that will mature in vitro in a relatively short time. This application has 3 Specific Aims: In Aim 1, rat rV3 will be produced using an Sf9 insect cell expression system, then purified and tested initially for elastogenic capacity on rat VSMC monolayer cultures. Subsequently, MGCM-scaffolded TEBVs, populated with rat VSMCs (for media) and rat dermal fibroblasts (for adventitia), will be exposed to the rV3 during their maturation in vitro. In Aim 2, we will evaluate the structural, mechanical, and physiological performance of the TEBVs created in Aim 1 (with native arteries as the "gold standard" for performance) according to the following criteria: 1) cell orientation and population dynamics; 2) composition and organization of the ECM produced by the cells; 3) mechanical properties, including stress- strain responses and burst-strength; and 4) vasoresponse. Finally, in Aim 3, robust, candidate TEBVs will be populated with endothelial cells to produce a non-thrombogenic lining and transplanted into rats to evaluate their performance in vivo. Endpoints will include TEBV patency, integrity, mechanical properties, endothelialization, thrombogenicity, vasoresponse, and host immune responses. In summary, the work proposed in this application represents the next stage of our extensive preliminary studies of the elastogenic properties of V3 and of methods to fabricate TEBVs. We believe that this work will make significant progress toward the goal of an engineered vascular replacement that functions like a native blood vessel. PUBLIC HEALTH RELEVANCE: Efforts to create small (<5 mm)-diameter tissue-engineered blood vessel (TEBV) replacements for diseased and injured arteries have met with limited success. Utilizing novel approaches, we propose to combine cells with natural structural and signaling molecules to create small-diameter TEBVs with a strength and elasticity like that of native arteries. With approximately 600,000 coronary bypass operations performed per year in the USA and a need for readily-available vascular shunts for dialysis patients and vascular grafts for limbs, successful development of small-diameter TEBV replacements would have a major impact on public health.

描述（由申请人提供）：将血管细胞与生物相容性细胞外基质（ECM）支架相结合的基于生物的组织工程血管（TEBV）的开发显示出前景，但在两个关键领域存在问题，这将在本报告中解决应用：1) 缺乏弹性蛋白，2) 体外成熟时间过长。在天然血管中，橡胶状弹性蛋白提供弹性并限制血管平滑肌细胞 (VSMC) 增殖。不幸的是，成人 VSMC 合成很少或不合成弹性蛋白，因此纳入成人患者 VSMC（以限制组织排斥）的 TEBV 缺乏弹性蛋白，这可能导致移植物狭窄和机械故障。怀特实验室发现，ECM 蛋白聚糖多功能蛋白聚糖 (V3) 的剪接变体 3 可以在体外和体内诱导 VSMC 产生和组装弹性蛋白。在本申请中，我们提出纯化的重组V3（rV3）可用于刺激TEBV在体外成熟时内部弹性纤维的形成。由于成熟时间过长，许多基于 ECM 的支架材料机械性能较弱，这需要 TEBV 在体外成熟数月才能足够坚固以安全植入。为了解决这个问题，Vernon 实验室开发了新型 ECM 支架（微槽胶原膜 - MGCM），其机械强度高，可诱导种子细胞在 24-48 小时内在凹槽上单轴排列。填充有排列整齐的血管细胞的 MGCM 片已成功转化为管。我们建议将这种 TEBV 制造方法与 rV3 介导的弹性生成相结合，以创建坚固、有弹性的 TEBV，并在相对较短的时间内在体外成熟。该应用有 3 个具体目标：在目标 1 中，将使用 Sf9 昆虫细胞表达系统产生大鼠 rV3，然后进行纯化并初步测试大鼠 VSMC 单层培养物的弹性生成能力。随后，装有大鼠 VSMC（用于培养基）和大鼠真皮成纤维细胞（用于外膜）的 MGCM 支架 TEBV 将在其体外成熟过程中暴露于 rV3。在目标 2 中，我们将根据以下标准评估目标 1 中创建的 TEBV 的结构、机械和生理性能（以天然动脉作为性能的“黄金标准”）：1）细胞方向和群体动态； 2）细胞产生的ECM的组成和组织； 3）机械性能，包括应力应变响应和爆裂强度； 4) 血管反应。最后，在目标 3 中，将用内皮细胞填充强大的候选 TEBV，以产生非血栓形成的内层，并移植到大鼠体内以评估其体内性能。终点包括 TEBV 通畅性、完整性、机械特性、内皮化、血栓形成、血管反应和宿主免疫反应。总之，本申请中提出的工作代表了我们对 V3 的弹性特性和制造 TEBV 的方法进行广泛初步研究的下一阶段。我们相信这项工作将在实现像天然血管一样功能的工程血管替代物的目标方面取得重大进展。公共健康相关性：为患病和受伤的动脉制造小直径（<5毫米）的组织工程血管（TEBV）替代品的努力取得了有限的成功。利用新颖的方法，我们建议将细胞与天然结构和信号分子结合起来，创造出具有与天然动脉相似的强度和弹性的小直径 TEBV。美国每年进行约 600,000 例冠状动脉搭桥手术，透析患者需要现成的血管分流器和四肢血管移植物，成功开发小直径 TEBV 替代品将对公众健康产生重大影响。