Engineering vascular replacements for strength and elasticity

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）支架联合收割机组合的基于生物学的组织工程化血管（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）增殖。不幸的是，成人VSMC合成很少或不合成弹性蛋白，因此并入来自成人患者的VSMC的TEBV（以限制组织排斥）将缺乏弹性蛋白，这可导致移植物的狭窄和机械故障。Wight实验室已经发现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（rV 3）可用于刺激TEBV内的弹性纤维形成，因为它们在体外成熟。关于过度的成熟时间，许多基于ECM的支架材料在机械上较弱，这需要TEBV在体外成熟数月，然后才足够坚固以安全地植入。为了解决这个问题，弗农实验室开发了新型的基于ECM的支架（微槽胶原膜-MGCM），其机械强度高，并诱导接种细胞在24-48小时内在槽上单轴排列。已成功地将填充有对齐的血管细胞的MGCM片材转换成管。我们建议将这种TEBV制造方法与rV 3介导的弹性发生联合收割机相结合，以产生将在相对短的时间内在体外成熟的坚固的、有弹性的TEBV。本申请具有3个特定目的：在目的1中，将使用Sf 9昆虫细胞表达系统生产大鼠rV 3，然后纯化并初步检测大鼠VSMC单层培养物的弹性蛋白生成能力。随后，将用大鼠VSMC（用于培养基）和大鼠真皮成纤维细胞（用于外膜）填充的MGCM支架的TEBV在其体外成熟期间暴露于rV 3。在目标2中，我们将评估目标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“金标准”）：1）细胞取向和群体动力学; 2）由细胞产生的ECM的组成和组织; 3）机械性能，包括应力-应变响应和破裂强度;（4）血管反应。最后，在目标3中，稳健的候选TEBV将用内皮细胞填充以产生非血栓形成衬里，并移植到大鼠中以评估其体内性能。终点将包括TEBV通畅性、完整性、机械性能、内皮化、血栓形成、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的弹性生成性质和制造TEBV的方法的广泛初步研究的下一阶段。我们相信，这项工作将取得重大进展的目标，工程血管替代，功能像一个天然的血管。

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.