Electrochemical Fabrication of Elastin-Containing Bi-Layered Scaffold for Vascular Tissue Engineering Applications
Nguyen, Thuy - Uyen Ky
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Lack of a viable vascular graft for the replacement of diseased small diameter blood vessels (< 4 mm) has encouraged the development of a self-repairing tissue engineered vascular graft (TEVG) as a promising alternative source. Although many different fabrication approaches have been devised to generate TEVGs using both natural and synthetic biomaterials, a functional TEVG that can serve as an effective replacement of diseased small-diameter blood vessels (< 4 mm) is still elusive mainly due to concerns associated with thrombosis and intimal hyperplasia. One approach to improve the clinical outcome of vascular grafts is to develop a 3D biomimetic scaffold that provides the essential physicochemical cues (i.e., compositional, topographical) to modulate cellular response (i.e., cell phenotype, endothelialization, de novo elastic matrix production) and thereby aid in tissue repair and remodeling. This approach entails the development of a novel 3D scaffold that provides an aligned collagen platform to guide smooth muscle cell (SMC) orientation and insoluble elastic matrix (i.e., native form of elastin) to promote SMC contractility and thereby prevent overproliferation and hence alleviate concerns with intimal hyperplasia. In addition, the scaffold must comprise of a smooth luminal side to support the formation of a continuous endothelium that is essential to prevent thrombosis. Most existing biofabrication methodologies to generate vascular scaffolds (e.g., extrusion, electrospinning) have limited scope because of the inability to incorporate insoluble elastic matrix within the aligned collagen framework due to the disparity in the size of the needle and insoluble elastin particles. Therefore, there is a need for an alternative biofabrication strategy to generate a 3D biomimetic scaffold with both structural similarity (i.e., aligned collagen) and compositional similarity (i.e., presence of insoluble elastic matrix) with native vessels. The overarching goal of this dissertation is to assess the feasibility of an electrochemical fabrication technique to develop a functional insoluble-elastin containing biomimetic TEVG for the replacement of diseased small diameter blood vessels. Towards this goal, the work performed in the current study entailed three main tasks: 1) to assess the effects of insoluble elastic matrix incorporation on the mechanical properties of electrochemically aligned collagen (ELAC) fibers and smooth muscle cell (SMC) response; 2) to determine the feasibility of the electrochemical fabrication technique for the generation of a biomimetic elastin-containing 3D bi-layered vascular scaffold; and 3) to design a dynamic perfusion-based co-culture system to simultaneously culture SMCs and endothelial cells (ECs). In the first task, two different types of elastin, soluble and insoluble, were incorporated into ELAC fibers, and the effect of elastin incorporation on fiber mechanics and SMC response was investigated. Results showed that incorporation of elastin (both soluble and insoluble) decreases the mechanical strength and stiffness of ELAC fibers (p <0.05). However, real-time polymerase chain reaction (PCR) results showed a significant upregulation of contractile markers (-smooth muscle actin (-SMA), and calponin; p < 0.05) in SMCs cultured on insoluble elastin incorporated ELAC fibers compared to those with soluble elastin and the control (collagen only) suggesting that insoluble elastin is capable of inducing contractility in SMCs. In the second task, the electrochemical fabrication technique was modified to generate an insoluble elastin containing 3D bi-layered scaffold and the effect of electrochemical process parameters on scaffold morphology and mechanics was assessed. Further, SMCs were statically seeded on the outer surface of the scaffold, and the effect of insoluble elastin incorporation on SMC phenotype was assessed. Lastly, EC functionality on the scaffold lumen surface was investigated. Polarized light microscopy and confocal images revealed that insoluble elastic matrix incorporation maintains the overall alignment of collagen fibrils in ELAC, and this aligned topography guides SMC orientation on the outer layer of the scaffold. Real-time PCR results showed that insoluble elastic matrix incorporation triggers higher expression of contractile markers (-SMA, calponin, and smooth muscle myosin heavy chain; p < 0.05) in SMCs. Further, immunofluorescence results showed that ECs express functional marker (eNOS) when cultured on the scaffold. In the third task, a perfusion flow bioreactor culture system was designed in which SMCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in a dynamic culture environment, and their viability was assessed. Live/dead assay results showed that cell viability was maintained post-culture and indicating that the designed reactor system has the potential to be used to validate the functionality of the electrochemically fabricated elastin-containing bi-layered scaffold. Overall, the results from the current study suggest that the electrochemical fabrication technique is a viable method for the generation of a functional TEVG with potential to be used as a vascular graft for the replacement of diseased small diameter vessels.