Abstract
Shortage of autologous blood vessel sources and disadvantages of synthetic grafts have increased interest in the development of tissue-engineered vascular grafts. However, tunica media, which comprises layered elastic laminae, largely determines arterial elasticity, and is difficult to synthesize. Here, we describe a method for fabrication of arterial grafts with elastic layer structure from cultured human vascular SMCs by periodic exposure to extremely high hydrostatic pressure (HP) during repeated cell seeding. Repeated slow cycles (0.002 Hz) between 110 and 180 kPa increased stress-fiber polymerization and fibronectin fibrillogenesis on SMCs, which is required for elastic fiber formation. To fabricate arterial grafts, seeding of rat vascular SMCs and exposure to the periodic HP were repeated alternatively ten times. The obtained medial grafts were highly elastic and tensile rupture strength was 1451 ± 159 mmHg, in which elastic fibers were abundantly formed. The patch medial grafts were sutured at the rat aorta and found to be completely patent and endothelialized after 2.5 months, although tubular medial constructs implanted in rats as interpositional aortic grafts withstood arterial blood pressure only in early acute phase. This novel organized self-assembly method would enable mass production of scaffold-free arterial grafts in vitro and have potential therapeutic applications for cardiovascular diseases.
Highlights
Biological tissue-engineered blood vessels, which possess elasticity and withstand arterial blood pressure, have considerable potential to improve the outcome for patients with cardiovascular disease
Pressurization (PHP) on stress fiber formation and fibronectin (FN) fibrillogenesis in human umbilical artery smooth muscle cells using a custom-made pressurization system (Supplemental Fig. 1), because FN fibrillogenesis is the critical step for several scaffold extracellular matrix (ECM) and organogenesis[8]
When human umbilical arterial SMCs (hUASMCs) were exposed to various degrees of pressurization at 0.002 Hz for 24 h, the setting of PHP 110 to 180 kPa significantly increased stress fiber formation and FN fibrillogenesis; these changes were not observed in hUASMCs cultured under 101 kPa (Fig. 1a–c)
Summary
Biological tissue-engineered blood vessels, which possess elasticity and withstand arterial blood pressure, have considerable potential to improve the outcome for patients with cardiovascular disease. A recent study demonstrated implantable scaffold-free tubular graft composed of multicellular spheroids[2], a method for the fabrication of artificial scaffold-free elastic arterial media has not yet been established This is partially because it is difficult to generate the elastic structure of arterial media, i.e., multiple layers of helically arranged smooth muscle cells (SMCs) surrounded by elastic fiber components. We previously demonstrated that arterial media-mimetic constructs, consisting of either human umbilical arterial SMCs (hUASMCs) or rat neonatal aortic SMCs, were grown in layered cell culture[3, 4] When they were pulled under tension, they were readily ruptured, indicating that elastic layer structure was incomplete and that tensile strength was not sufficient. In support of this idea, hydrostatic pressure has been reported to correlate to synthesis of extracellular matrix (ECM) proteins, such as collagens, elastin, and sulfated glycosaminoglycans[5,6,7]
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