Preclinical Assessment of a Compliance Matched Biopolymer Vascular Graft

顺应性匹配的生物聚合物血管移植物的临床前评估

• 批准号: 10731964
• 负责人: Jonathan Pieter Vande Geest
• 金额: $ 12.51万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-15 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10731964
• 关键词: 3-Dimensional; Acute; Adult; Animal Model; Anti-Inflammatory Agents; Aorta; Autologous; Bilateral; Biomechanics; Biopolymers; Bioreactors; Blood Vessels; Bypass; COVID-19 pandemic; Cardiac; Cardiac Surgery procedures; Cardiovascular Diseases; Cause of Death; Cd68; Cell Proliferation; Cells; Cessation of life; Clinical; Clinical Research; Clinical Treatment; Collagen; Coronary Artery Bypass; Custom; Cytoskeleton; Deposition; Development; Diameter; Disease; Elastin; Elements; Engineering; Exhibits; Exposure to; Extracellular Matrix; Failure; Fluorescence; Gelatin; Generations; Heart Diseases; Hyperplasia; Image; Implant; In Vitro; Individual; Infiltration; Intervention; Legal patent; Location; Macrophage; Maintenance; Measures; Mechanics; Modeling; Myosin Heavy Chains; Organ Transplantation; Outcome; Performance; Phenotype; Play; Process; Production; Proliferating; Protein Isoforms; Rattus; Reporting; Research; Role; Saphenous Vein; Sheep; Signal Transduction; Site; Smooth Muscle Myocytes; Sprague-Dawley Rats; Stretching; Stromal Cells; System; TGFB1 gene; TGFB3 gene; Testing; Thrombosis; Time; Tissues; Transforming Growth Factor Beta 2; Transforming Growth Factor beta; Tropoelastin; United States; Vascular Diseases; Vascular Graft; Vascular Smooth Muscle; Woman; Work; abdominal aorta; calponin; cell motility; conditioning; controlled release; dosage; effective therapy; graft failure; imaging system; improved; in vivo; intravital imaging; manufacture; mechanical properties; men; multi-photon; multiphoton imaging; parent project; particle; pre-clinical; pre-clinical assessment; preimplantation; pressure; recruit; response; scaffold; second harmonic; synergism; three dimensional cell culture; two-photon; vascular smooth muscle cell migration; vascular tissue engineering

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.

项目摘要 心血管疾病是全球死亡的主要原因，目前占32%。 在COVID-19大流行开始之前报告的所有死亡病例中。心血管疾病的增加 心脏旁路移植术（CABG）的需求日益增加，这是目前最重要的手术。 最常见的心脏手术尽管使用自体组织的CABG干预被视为 最有效的治疗方案之一，其失败率仍高达42.8%，只有50%， 10年后60%保持通畅。血管工程研究试图开发组织 工程血管移植物（TEVG）以无细胞或细胞化构建体的形式作为替代解决方案。 尽管几十年来的研究进展，很少有TEVG已经达到临床研究， 目前正在使用的临床批准的TEVG。在这里，我们寻求推进我们对TEVG如何 当被转化生长蛋白的三种不同亚型刺激时， 因子β，以及这种治疗对TEVG的细胞重塑的影响。 目的1：研究天然血管组织基质细胞在TEVG上的迁移和增殖 即顺应性匹配和TGFβ同种型加载在3D培养系统中。我们将检验这个假设 含有TGFβ2负载的微粒的顺应性匹配的TEVG可以调节肿瘤的发生率， 天然血管组织细胞迁移和增殖到相邻的TEVG上，水平显著较高 相比于利用3D培养系统递送两种替代性TGFβ同种型。将天然大鼠动脉组织 插管并置于与顺应性匹配的TEVG邻接的3D培养系统中。脱细胞 TEVG最初将被制造成携带载有TGFβ同种型（TGFβ-1， -2，-3）以提供TGFβ从TEVG的内膜层的受控释放。我们将 评估早期和晚期细胞迁移和增殖到TEVG支架上作为培养的函数 时间和TGFβ同种型的释放，在选择的释放速率和剂量。我们将量化这些结果 使用专门的3D双轴TEVG培养系统的多光子活体成像。 目的2：评估顺应性匹配的TGFβ同种型负载的TEVG的细胞重构 通过活体双光子成像研究顺应性的后续ECM重塑 在3D培养中，我们将利用携带TGFβ同种型负载MP的TEVG的活体成像， 如前所述，在培养物中，将天然大鼠腹侧肌与TEVG邻接。在本补充目标中，我们将利用 2-通过二次谐波通过胶原蛋白/弹性蛋白含量量化ECM重塑的光子成像系统 产生（SHG，胶原）和2-光子激发的荧光（2 PEF，弹性蛋白）信号。我们将评估 支架基质重塑通过相对SHG和2 PEF水平作为培养时间的函数， 输送TGFβ亚型。 目的3：研究TGFβ亚型的渐进双轴生物力学刺激的影响 通过评估TEVG的细胞迁移、增殖和重塑， 浸润基质细胞。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），以在TEVG上施加受控的脉动压力和轴向拉伸， 生物力学刺激与TGFβ同种型递送协同作用以增强细胞活性。和以前一样， 将天然大鼠主动脉组织与TEVG邻接，这次暴露于选择的渐进性的 双向生物力学调节场景。TEVG的细胞迁移、增殖和重塑 将像以前一样用我们的活体2光子活体成像系统进行评估，TEVG重塑将被 通过在整个支架的各个位置处的相对SHG和SPEF信号产生来确定， 以前我们将评估这些措施作为培养时间和双轴生物力学负荷水平的函数 结合TGFβ亚型递送，以确定每种亚型如何独特地影响细胞重塑 结果。