Preclinical Assessment of a Compliance Matched Biopolymer Vascular Graft

项目摘要 心血管疾病是全球主要的死亡原因，目前占全球的32%。 在新冠肺炎大流行开始之前报告的所有死亡人数中。心血管疾病的增加 日益突出的是对心脏搭桥术(CABG)的需求水平越来越高，这现在是 世界上最常见的心脏手术。尽管冠状动脉旁路移植术对自体组织的干预被视为 作为最有效的治疗方案之一，他们的失败率仍然高达42.8%，只有50%到 60%的患者在10年后仍保持通畅。血管工程学研究试图开发出组织 无细胞或细胞化结构形式的工程化血管移植(TEVG)是一种替代解决方案。 尽管研究取得了几十年的进展，但很少有TEVG达到临床研究，而且没有 目前正在使用的经临床批准的TEVGs。在这里，我们试图增进我们对电视节目是如何 在三种不同的蛋白质转化生长亚型刺激下被宿主细胞渗透 因子Beta，以及这种治疗对TEVGs细胞重塑的影响。 目的1：研究天然血管组织基质细胞在TEVG上的迁移和增殖 也就是说，在3D培养系统中加载了顺应性匹配和转化生长因子β亚型。我们将检验这一假设 含有转化生长因子β-2载药微粒的顺应性匹配的TEVGs可以调节移植的速率 天然血管组织细胞在邻近TEVG上的迁移和增殖 然后利用3D培养系统提供两种可供选择的转化生长因子β亚型。天然的大鼠动脉组织将是 将其置入3D培养系统中，与符合条件的TEVGs相邻。脱细胞的 TEVG最初将被制造成携带载有转化生长因子β异构体(转化生长因子β-1， -2、-3)，以提供可控释放的转化生长因子β从TEVGs的内膜。我们会 评估细胞在TEVG支架上的早期和晚期迁移和增殖作为培养的功能 时间和转化生长因子β亚型释放通过选定的释放速率和剂量。我们将对这些结果进行量化 使用专门的3D双轴TEVG培养系统的多光子活体成像。 目的2：评价顺应性匹配的转化生长因子β异构体负载血管内皮细胞的细胞重构 通过活体内双光子成像。研究顺应性的后续ECM重塑 携带转化生长因子β异构体的匹配TEVGs在3D培养中，我们将利用移植的活体成像 原代培养的大鼠腹主动脉与培养的TEVGs相邻。在这个补充目标中，我们将利用 双光子成像系统通过二次谐波通过胶原/弹性蛋白含量量化ECM重塑 产生(SHG，胶原蛋白)和双光子激发荧光(2PEF，弹性蛋白)信号。我们将评估 相对SHG和2PEF水平对支架基质重塑的影响 转化生长因子β亚型交付。 目的3：研究渐进性双向生物力学刺激对转化生长因子β异构体的影响 通过评估TEVGs的细胞迁移、增殖和重塑来加载TEVGs 浸润性基质细胞。我们将利用我们定制的生物反应器的另一个功能(双轴生物力学 加载)对TEVG施加受控的脉动压力和轴向拉伸，以确定这是否 生物力学刺激与转化生长因子β异构体传递协同作用，以增强细胞活性。和以前一样， 天然的大鼠主动脉组织将与TEVG相连，这一次暴露于一种渐进的 双轴生物力学调理情景。TEVGs的细胞迁移、增殖和重塑 将像以前一样用我们的活体内双光子成像系统进行评估，TEVG重建将是 由整个支架不同位置的相对倍频和SPEF信号产生确定为 在此之前。我们将评估这些措施作为培养时间和双向生物机械负荷水平的函数。 结合转化生长因子β异构体传递来确定每种方法如何独特地影响细胞重塑 结果。

PROJECT ABSTRACT Cardiovascular diseases represent the leading cause of death worldwide and are currently responsible for 32% of all reported deaths before the start of the COVID-19 pandemic. The increase in cardiovascular disease prominence has placed increasing levels of demand for cardiac bypass grafting (CABG), which is now the most common cardiac surgery in the world. Despite CABG interventions with autologous tissues are viewed as one of the most effective treatment options, their failure of rate remains as high as 42.8%, with only 50% to 60% maintaining patency after 10 years. Vascular engineering research has sought to develop tissue engineered vascular grafts (TEVG) in the form of acellular or cellularized constructs as an alternative solution. Despite decades of research advancements, very few TEVGs have reached clinical studies, and there are no clinically approved TEVGs currently in use. Here we seek to advance our understanding of how TEVGs are infiltrated by the host’s cells when stimulated by three different isoforms of the protein Transforming Growth Factor Beta, and the impact this treatment can have on cellular remodeling of the TEVGs. Aim 1: Investigate native vascular tissue stromal cell migration onto and proliferation within a TEVG that is compliance matched and TGFβ isoform loaded in a 3D culture system. We will test the hypothesis that compliance matched TEVGs containing TGFβ2 loaded microparticles can modulate the rates of explanted native vascular tissue cellular migration and proliferation onto an adjoined TEVG at significantly higher levels than delivery of two alternative TGFβ isoforms utilizing a 3D culture system. Native rat arterial tissues will be canulated and placed into the 3D culture system adjoined to a compliance matched TEVGs. The acellular TEVGs will initially be manufactured to carry PLGA microparticles (MPs) loaded with TGFβ isoforms (TGFβ-1, -2, -3, respectively) to provide a controlled release of TGFβ from the intimal layer of the TEVGs. We will assess early and late term cellular migration and proliferation onto the TEVG scaffolds as a function of culture time and TGFβ isoform release across a selection of release rates and dosages. We will quantify these results using multiphoton intravital imaging of the specialized 3D biaxial TEVG culture system. Aim 2: Assess the cellular remodeling of the compliance matched TGFβ isoform loaded TEVGs through intravital 2-photon imaging. To investigate the subsequent ECM remodeling of the compliance matched TEVGs carrying TGFβ isoform loaded MPs in 3D culture, we will utilize intravital imaging of explanted native rat abdominal aortas adjoined to TEVGs in culture as before. In this supplemental aim, we will utilize the 2-photon imaging system to quantify ECM remodeling via collagen/elastin content though second harmonic generation (SHG, collagens) and 2-photon excited fluorescence (2PEF, elastin) signals. We will assess scaffold matrix remodeling by relative SHG and 2PEF levels as a function of culture time and the respective TGFβ isoform delivered. Aim 3: Investigate the impact of progressive biaxial biomechanical stimulation of the TGFβ isoform loaded TEVGs by assessing cellular migration, proliferation, and remodeling of the TEVGs by infiltrating stromal cells. We will utilize another feature of our custom-built bioreactors (biaxial biomechanical loading) to impose controlled pulsatile pressures and axial stretches upon the TEVGs to determine if this biomechanical stimulation synergizes with the TGFβ isoform delivery to enhance cellular activity. As before, the native rat aorta tissues will be adjoined to the TEVGs, this time exposed to a selection of progressive biaxial biomechanical conditioning scenarios. Cellular migration, proliferation, and remodeling of the TEVGs will be assessed with our intravital 2-photon intravital imaging system as before, and TEVG remodeling will be determined by the relative SHG and SPEF signal generation at various locations throughout the scaffolds as before. We will assess these measures as a function of culture time and levels of biaxial biomechanical loading in combination with TGFβ isoform delivery to determine how each uniquely impacts cellular remodeling outcomes.