Engineered Artery Growth in Vitro, Cell-Remodeled Fibrin

描述(由申请人提供)： 这个项目的最终目标是一种完全生物的人工动脉(生物人工动脉)，适合作为小直径的血管移植物，例如用于冠状动脉搭桥术。我们将在两项重大发现的基础上再接再厉：(1)当新生儿平滑肌细胞(SMC)被包裹在非粘附棒周围形成管状的纤维蛋白凝胶中时，SMC收缩棒周围的凝胶，导致纤维蛋白原纤维和SMC周向排列。当SMC随后降解纤维蛋白时，它们会产生广泛的交联型胶原和弹性纤维，这些纤维也是周向排列的。这种重建使构建的结构具有接近动脉组织的拉伸机械性能；(2)从血液中培养的内皮细胞可以在不到6周的时间内扩增到10A8细胞，这种扩张可能发生在循环中的骨髓来源的血管母细胞。在具体目标1中，我们将比较用新生SMC(大鼠、猪和人)制备的改型纤维蛋白凝胶管在静态培养条件下的性能，这些凝胶管受到循环扩张，传递出我们在胶原凝胶管中发现的有望促进组织生长的机械信号，并控制培养介质的跨壁流动，最大限度地减少可溶性因子的梯度，潜在地导致组织增加和接近均匀的组织生长。在目标2中，我们将研究生物人工动脉的特性，包括上述重塑的纤维蛋白/SMC结构以及使用上述血液长出内皮细胞(BOECs)的传统晚期种植管状结构生成的内皮，包括它们在生理剪应力下脉动流动中的黏附强度和它们的激活状态。一旦这些构建物在体外得到验证，我们将继续在动物模型中测试其活性、通畅性和血液相容性。生物人工动脉将被植入大鼠的主动脉，首先在同基因环境中，然后在“同种异体”环境中(关于SMC；将使用自体BOECs)。随后将在猪身上进行类似的同种异体研究。我们还将从人的SMC和BOECs中构建生物人工动脉，因此这些结果应该与未来的临床研究直接相关。

DESCRIPTION (provided by applicant): The ultimate goal of this project is a completely biological artificial artery (bioartificial artery) that is suitable as a small diameter vascular graft, such as for use in coronary bypass. We will build upon our two major discoveries: (1) when neonatal smooth muscle cells (SMCs) are entrapped in fibrin gel formed as a tube around a nonadhesive rod, the SMCs contract the gel around the rod, causing the fibrin fibrils and SMCs to become circumferentially aligned. As the SMCs subsequently degrade the fibrin, they produce extensive cross-linked collagen and elastic fibers, which also are circumferentially aligned. This remodeling provides the construct with tensile mechanical properties approaching values of arterial tissue; (2) endothelial cells cultured from blood can be expanded to 10A8 cells in less than six weeks, the expansion occurring from a putative circulating marrow-derived angioblast. In Specific Aim 1, we will compare the properties of remodeled fibrin gel tubes prepared with neonatal SMCs (rat, pig, and human) under the static culture condition used to date with tubes subject to cyclic distension, imparting a mechanical signal expected to increase tissue growth as we found in collagen gel-based tubes, and controlled transmural flow of culture medium, minimizing gradients of soluble factors and potentially leading to increased and near-uniform tissue growth. In Aim 2, we will study the properties of bioartificial arteries, the remodeled fibrin/SMC constructs described above plus an endothelium generated using traditional late-stage seeding of the tubular construct with the blood outgrowth endothelial cells (BOECs) noted above, including their adhesion strength in pulsatile flow at physiological shear stress and their state of activation. Once such constructs have been validated in vitro we will proceed with testing the viability, patency, and hemocompatibility properties in animal models. The bioartificial artery will be implanted into the aorta of the rat, first in the syngeneic setting and subsequently in the "allogeneic" setting (with respect to the SMCs; autologous BOECs will be used). A similar allogeneic study will subsequently be performed in the pig. We will also fabricate bioartificial arteries from human SMC and BOECs, so that these results should be directly relevant for a future clinical study.