Engineering vascular replacements for strength and elasticity

描述（由申请人提供）：结合血管细胞和生物相容性细胞外基质（ECM）支架的基于生物的组织工程血管（tebv）的发展显示出前景，但在两个关键领域存在问题，这将在本申请中解决：1)缺乏弹性蛋白和2)体外成熟时间过长。在天然血管中，橡胶样蛋白弹性蛋白提供弹性并限制血管平滑肌细胞（VSMC）的增殖。不幸的是，成人VSMCs合成很少或不合成弹性蛋白，因此结合成人患者VSMCs的tebv（以限制组织排斥）将缺乏弹性蛋白，这可能导致移植物狭窄和机械失效。Wight实验室发现ECM蛋白聚糖（V3）的剪接变体3可以诱导体外和体内VSMCs产生和组装弹性蛋白。在目前的应用中，我们提出纯化的重组V3 （rV3）可用于刺激tebv体外成熟时弹性纤维的形成。关于成熟时间过长，许多基于ecm的支架材料在机械上很弱，这需要tebv在体外成熟几个月才能足够强大到可以安全植入。为了解决这个问题，弗农实验室开发了一种新型的基于ecm的支架（微沟槽胶原膜- MGCMs），这种支架具有机械强度，可在24-48小时内诱导种子细胞在沟槽上单轴排列。填充有排列的血管细胞的MGCM片已成功转化为管。我们建议将这种TEBV制造方法与rv3介导的弹性发生相结合，以制造出在体外相对较短时间内成熟的强弹性TEBV。在目标1中，大鼠rV3将使用Sf9昆虫细胞表达系统产生，然后纯化并初步测试大鼠VSMC单层培养物的弹性能力。随后，mgcm支架的tebv，填充大鼠VSMCs（培养基）和大鼠真皮成纤维细胞（外膜），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)细胞取向和种群动态；2)细胞产生的ECM的组成和组织；3)力学性能，包括应力应变响应和破裂强度；4)血管反应。最后，在Aim 3中，健壮的候选tebv将填充内皮细胞以产生非血栓形成的内膜，并移植到大鼠体内以评估其在体内的表现。终点将包括TEBV通畅、完整性、机械特性、内皮化、血栓形成性、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弹性性能和制造tebv方法的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品的目标方面取得重大进展。

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.