Tissue engineering of small diameter vascular graft

描述（由申请人提供）：生产不含合成材料的真正稳定的人造血管的目的需要内皮细胞和平滑肌细胞以及成纤维细胞侵入和生长到支架中。这可以在体内从植入后的邻近组织或循环细胞实现，或者通过培养的细胞在体外实现。这种移植物不应诱导可能损伤其壁、为长期动脉瘤形成设置场景或触发急性血栓形成的实质性炎症反应。基于上述组织工程学原理和我们的初步研究结果，我们提出了一个中心假设，即通过脱细胞、肝素共价连接、肝素结合生长因子和内皮祖细胞（EPC）接种等方法，可以从猪颈动脉中组织工程化出一种新型的小直径血管移植物，这种移植物可以保持其力学性能和天然顺应性，降低宿主免疫反应，并具有良好的生物相容性。提供抗凝表面;加速血管细胞生长和重塑，从而在体内保持长期效力。下面提出了三个具体目标来检验我们的中心假设：目标1：确定脱细胞肝素化猪颈动脉移植物（D-H移植物）的机械性能、自然顺应性、宿主免疫反应和抗凝性能。我们将测试D-H移植物可以保持其机械性能和自然顺应性，提供抗凝表面，并减少宿主免疫反应的假设。包括体外和动物模型。将在犬（异种环境）和猪（同种异体环境）中使用D-H移植物进行颈动脉旁路手术。目的2：研究bFGF与D-H移植物结合对移植物血管细胞生长和再增殖的影响。我们将测试这一假设，即碱性成纤维细胞生长因子结合D-H移植物可能会促进血管细胞的生长和移植物的再生，从而加速血管愈合和重塑。在体外研究bFGF的结合和释放特性以及对细胞增殖和抗凝作用的影响。将研究bFGF结合的D-H移植物的体内性能。目的3：研究内皮祖细胞的增殖分化特性及其与碱性成纤维细胞生长因子（bFGF）结合的D-H移植物的应用。我们将检验bFGF结合的D-H移植物可能增强EPC增殖和分化的假设，与未接种的移植物相比，EPC接种的bFGF结合的D-H移植物可能具有更好的愈合和重塑特征。将从狗或猪外周血中分离和表征EPC。研究bFGF和血流动力学对EPC分化和增殖的影响。将研究EPC接种的bFGF结合的D-H移植物的体内性能。这项研究代表了一个多学科的方法，包括组织工程，细胞和分子生物学，动物模型。该方案的成功将直接标志着组织工程化小直径血管移植物的临床应用。

DESCRIPTION (provided by applicant): The aim to produce a truly stable artificial blood vessel containing no synthetic material requires invasion and in growth of endothelial cell and smooth muscle cells as well as fibroblasts into the scaffold. This can be achieved either in vivo from the adjacent tissue or circulating cells after implantation, or in vitro by means of cultivated cells. Such graft should not induce substantial inflammatory reactions that could either damage its wall, setting the scene for long term aneurysm formation or trigger acute thrombosis. Based on such tissue engineering principles and our preliminary data, we propose a central hypothesis that a novel small diameter vascular graft can be tissue-engineered from the porcine carotid artery by decellularization, heparin covalent linkage, and heparin binding growth factors, and endothelial progenitor cell (EPC) seeding; and this graft may maintain its mechanical property and natural compliance; reduce host immune response; provide anticoagulation surface; and accelerate vascular cell growth and remodeling, thereby maintaining a long term potency in vivo. Three specific aims are proposed below to test our central hypothesis: Aim 1: To determine the mechanical property, natural compliance, host immune response, and anticoagulation property of decellularized-heparinized porcine carotid artery grafts (D-H grafts). We will test the hypothesis that D-H grafts may maintain their mechanical property and natural compliance, provide anticoagulation surface, and reduce host immune response. Both in vitro and animal models are included. Carotid artery bypass surgery using D-H grafts will be performed in dogs (xenogenic setting) and in pigs (allogenic setting). Aim 2: To determine the effect of bFGF binding to D-H grafts on vascular cell growth and repopulation of the grafts. We will test the hypothesis that bFGF binding to D-H grafts may promote vascular cell growth and repopulation of the grafts, thereby accelerating vascular healing and remodeling. Characteristics of bFGF binding and release and effect on cell proliferation and anticoagulation will be investigated in vitro. In vivo performance of bFGF bound D-H grafts will be studied. Aim 3: To characterize cell proliferation and differentiation of EPC and its application with bFGF bound D-H grafts. We will test the hypothesis that bFGF bound D-H grafts may enhance EPC proliferation and differentiation, and EPC seeded bFGF bound D-H grafts may have better healing and remodeling characteristics as compared to un-seeded grafts. EPC will be isolated and characterized from dog or pig peripheral blood. The effect of bFGF and hemodynamics on EPC differentiation and proliferation will be investigated. In vivo performance of EPC seeded bFGF bound D-H grafts will be studied. This study represents a multidisciplinary approach including tissue engineering, cellular and molecular biology, and animal models. Success of this proposal will directly indicate the clinical applications of tissue engineered small diameter vascular grafts.