Collagen and Elastin based Tissue Engineered Vascular Grafts

摘要

Cardiovascular disease is the leading cause of death worldwide, accounting for 29% of all global deaths and is set to rise to 23 million deaths a year by 2030 (World Health Organisation, 2012). Arterial bypassing, both peripheral and coronary, is usually performed with autologously harvested vessels. However, the quantity available is often very limited as well as the vessels of elderly patients often suffering from thrombus, aneurysm formation or arthrosclerosis in high pressure arterial sites. The shortcomings of autografts has led to a substantial amount of research being directed towards tissue engineered vascular grafts (TEVGs) (Kakisis et al., 2005). Currently available artificial grafts for small diameter vasculature (mm) suffer from poor patency rates due to thrombosis, aneurysm formation, and a compliance mismatch, which often stems from the inherent properties of synthetic polymers.The primary goal of the research presented in this thesis was to develop a small diameter tissue engineered vascular graft (TEVG) using the natural polymers collagen and elastin, coupled with dynamic mechanical conditioning. In this context, the aim was to develop a collagen-elastin composite scaffold with optimised intrinsic physiochemical characteristics which displayed the capacity to support smooth muscle cells in vitro while also displaying suitable viscoelastic properties. Subsequent investigation focused on emulating the anatomical architecture of native vessels using this novel collagen-elastin composite, and examining in vitro maturation through dynamic conditioning in a custom designed pulsatile bioreactor.In the study presented in Chapter 2 of this thesis, elastin addition to a porous collagen scaffold was shown to play a major role in altering its biological and mechanical response. The addition of elastin improved the viscoelastic characteristics with a higher degree of cyclical strain recovery and creep resistance, which indicates the biomaterial may possess sufficient recoil to be utilised for long-term cyclical distension with reduced aneurysm risk. Additionally, the gene expression and proliferation data suggested that the presence of elastin resulted in a more contractile smooth muscle cell (SMC) phenotype, in the absence of any exogenous stimulation. This biomaterial platform was deemed to possess great potential for cardiovascular tissue engineering and was amenable to multiple fabrication methods.In Chapter 3, this biomimetic collagen-elastin composite was subsequently fabricated into a physiologically relevant bilayered tubular architecture. The bilayered scaffold consisted of a porous outer layer with an optimised microarchitecture to support SMCs, while the inner layer consisted of a dense film designed to increase the overall scaffold mechanical properties and present a suitable surface for future endothelial seeding. The properties of the dense luminal lining were shown to be highly controllable via crosslinking, which enabled the modification of the mechanical properties, degradation resistance, and inflammatory profile. These bilayered tubular scaffolds were ultimately considered highly suitable for further investigation as a TEVG.In Chapter 4, a novel pulsatile flow bioreactor system was developed which was capable of recreating the complex haemodynamic environment in vitro. The system was capable of applying physiological fluid shear stresses, cyclical strain and pulsatile pressure to mounted constructs. The flexible design allowed the mounting of variable diameter constructs and was designed to be utilised to examine the effect of mechanical stimulation on the in vitro maturation of the bilayered tubular collagen-elastin TEVGs described. In the final chapter (Chapter 5), the effect of TEVG architecture, crosslinking, and dynamic conditioning on the maturation of the grafts was examined in the custom pulsatile bioreactor from Chapter 4. Specifically, bilayered scaffolds coupled with EDAC crosslinking displayed far greater mechanical properties than single layered scaffolds and DHT crosslinking respectively. Furthermore, the application of dynamic conditioning resulted in further increases in the TEVG mechanical properties as a result of increased cell density, improved collagen circumferential alignment, and an apparent increase in vessel wall density.Collectively, this study has led to the development of a composite bilayered tubular scaffold with optimised intrinsic physiochemical characteristics to support smooth muscle cells in vitro while subsequently displaying suitable viscoelastic properties for sustained dynamic conditioning in a custom designed pulsatile bioreactor.