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）：基于生物学的组织工程血管（TEBV）的发展，将血管细胞与生物相容性的细胞外基质（ECM）脚手架相结合，但在两个关键领域中是有希望的，但在此应用中会解决这两个关键领域，这将在此应用中解决：1）弹性蛋白和2）过量的Vitro时代。在天然血管中，橡胶样蛋白弹性蛋白具有弹性，并限制了血管平滑肌细胞（VSMC）增殖。不幸的是，成年VSMC几乎没有或根本没有弹性蛋白，因此，将成年患者的VSMC（以限制组织排斥）的TEBV缺乏弹性蛋白，这可能导致移植的狭窄和机械失败。 Wight实验室发现，ECM蛋白聚糖versican（V3）的剪接变体3可以在体外和体内诱导VSMC，以产生和组装弹性蛋白。在本应用中，我们提出纯化的重组V3（RV3）可用于刺激TEBV内的弹性纤维形成，它们在体外成熟。关于过度的成熟时间，许多基于ECM的脚手架材料在机械上很弱，这要求TEBV在体外成熟数月，然后才能安全地植入。为了解决这个问题，弗农实验室已经开发了新型的基于ECM的支架（微胶凝胶原膜-MGCMS），它们在机械上很强，并诱导种子细胞在24-48 h内单轴上对齐。填充有排列血管细胞的MGCM板已成功转化为管。我们建议将这种TEBV制造方法与RV3介导的弹性发生相结合，以产生强，弹性的TEBV，这些弹性TEBV会在相对较短的时间内在体外成熟。该应用具有3个特定的目的：在AIM 1中，将使用SF9昆虫细胞表达系统产生大鼠RV3，然后最初对大鼠VSMC单层培养物进行纯化和测试。随后，在其体外成熟期间，将在RV3上暴露于RV3。在AIM 2中，我们将根据以下标准评估AIM 1（本地动脉作为绩效的“金标准”）在AIM 1中创建的TEBV的结构，机械和生理性能：1）细胞方向和种群动态； 2）细胞产生的ECM的组成和组织； 3）机械性能，包括应力应变反应和爆发强度； 4）血管响应。最后，在AIM 3中，稳健的候选TEBV将被内皮细胞填充，以产生非紧密的衬里并移植到大鼠中以评估其在体内的性能。终点将包括TEBV通畅性，完整性，机械性能，内皮化，血栓形成性，血管响应和宿主免疫反应。总而言之，本应用程序中提出的工作代表了我们对V3的弹性特性和制造TEBV的方法的广泛初步研究的下一个阶段。我们认为，这项工作将取得重大进展，朝着像本地血管一样起作用的工程血管置换。公共卫生相关性：创建小型（<5毫米） - 直径的组织工程血管（TEBV）替代患病和受伤的动脉的替代者的成功率有限。利用新方法，我们建议将细胞与天然结构和信号分子相结合，以形成像天然动脉一样强度和弹性的小直径TEBV。每年在美国进行大约60万名冠状动脉搭桥手术，并且需要透析患者容易获得的血管分流和四肢血管移植物，因此成功开发了小直径的TEBV替代品将对公共卫生产生重大影响。