Engineering vascular replacements for strength and elasticity

描述(申请人提供)：将血管细胞与生物相容的细胞外基质(ECM)支架相结合的基于生物的组织工程血管(TEBV)的发展前景看好，但在两个关键领域存在问题，这两个领域将在本申请中解决：1)弹性蛋白的缺乏和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很少或根本不合成弹性蛋白，因此将成年患者的VSMCs加入TEBV中(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故障。鬼魂实验室发现，细胞外基质蛋白多糖V3的剪接变异体3可以在体外和体内诱导血管平滑肌细胞产生和组装弹性蛋白。在目前的应用中，我们建议可以使用纯化的重组V3(RV3)来刺激TEBV在体外成熟时形成弹性纤维。至于过多的成熟时间，许多基于ECM的支架材料机械性能较弱，这需要TEBV在体外成熟数月后才能足够坚固，才能安全植入。为了解决这个问题，弗农实验室开发了基于ECM的新型支架(微槽胶原膜-MGCM)，这种支架具有机械强度，可以诱导种子细胞在24-48小时内在凹槽上单轴排列。填充了排列好的血管细胞的MGCM薄片已成功转化为管状。我们建议将这种制造TEBV的方法与rV3介导的弹性形成相结合，以创造出在相对较短的时间内将在体外成熟的强大的、有弹性的TEBV。这项应用有三个特定的目标：在目标1中，将使用Sf9昆虫细胞表达系统生产大鼠rV3，然后进行纯化，并在大鼠VSMC单层培养上初步测试其弹性能力。随后，以大鼠VSMCs和大鼠真皮成纤维细胞(外膜)填充的MGCM支架TEBV在体外成熟过程中将暴露于rV3。在目标2中，我们将根据以下标准评估在目标1中创建的TEBV的结构、机械和生理性能(以天然动脉为性能的“黄金标准”)：1)细胞取向和群体动力学；2)细胞产生的细胞外基质的组成和组织；3)机械性能，包括应力-应变响应和破裂强度；以及4)血管响应。最后，在目标3中，健壮的候选TEBV将填充内皮细胞以产生非血栓形成的衬里，并移植到大鼠体内以评估其体内性能。终点将包括TEBV的通畅性、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的弹性特性和制造TEBV的方法的广泛初步研究的下一阶段。我们相信，这项工作将朝着工程化血管置换的目标取得重大进展，使其功能类似于天然血管。 公共卫生相关性：为患病和受伤的动脉创造小直径(&lt；5 mm)组织工程血管(TEBV)替代物的努力取得的成功有限。利用新的方法，我们建议将细胞与天然的结构和信号分子结合起来，创造出具有天然动脉那样强度和弹性的小直径TEBV。由于美国每年约有60万例冠状动脉旁路手术，透析患者需要容易获得的血管分流和四肢血管移植物，成功开发小直径TEBV替换术将对公众健康产生重大影响。

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.