With 500,000 coronary artery bypass procedures performed each year in the United States, and only one-third of patients possessing suitable autologous grafts, there is a clinical need for tissue engineered blood vessels (TEBVs). The overall goal of this project was to develop a one- step approach to rapidly produce entirely cell- derived tubular tissue constructs without scaffold materials. To achieve this goal, we developed "centritubing"-- a system based on applying centrifugal force to suspended cells to create a tube-shaped cellular aggregate. Briefly, rat aortic smooth muscle cells were injected into cylindrical polycarbonate spinning chambers and then spun to apply centrifugal force, which pelletted the cells on the inner wall of the chamber. After three days in culture with growth medium, the cells remodeled to form tissue tubes. In previous work we have shown, in principle, that centritubing produces tubular constructs, however tissue tube production was not consistently achieved. The first objective of this study was to develop modifications to the centritubing device that would lead to consistent lumen diameter, rapid cellular aggregation into a tube construct, and an improved success rate of tube formation. The second objective was to investigate cellular parameters that contribute to tubular tissue construct formation using centritubing. Prior to changes in manufacturing of the centritubing device and culture system, the success rate of centritubing was inconsistent. After these changes, the success rate of tubular construct formation improved to 85% (11/13). Noteworthy modifications to the centritubing device included the addition of a central mandrel as a substrate for tissue contraction, development of a smoother seeding surface, and manufacture of a reusable culture chamber. The results of this study support the proof of concept for centritubing as a device for rapid production of tubular tissue constructs and provide insight for future progress using the centritubing methodology.

Despite efforts by clinicians and scientists world-wide, coronary artery disease remains to be the leading cause of morbidity and mortality in industrialized nations. Development of a tissue engineered coronary by-pass graft with low thrombogenicity and immune responses, suitable mechanical properties, and a capacity to remodel to their environment could have a significant impact on the treatment of coronary artery disease. While many methods for the tissue engineering of blood vessels have been developed, one promising approach is the self-assembly method. Using autologous cells that produce an endogenous extracellular matrix (ECM), the potential for therapeutic success is high due to biocompatibility. However, despite these advantages, improvements can be made which will give the grafts an even higher rate of patency. This dissertation presents a study of the characterization of the biaxial mechanical properties of self-assembly tissue engineered blood vessels (SA-TEBV), as well as developing a framework for fabrication strategies of SA-TEBV. Native arteries are exposed to multiaxial mechanical loads, including (a pulsatile) blood pressure that causes the vessel to cyclically distend circumferentially, blood flow that induces a shearing load along the luminal surface, and an axial extending load; the latter is relieved upon excision, causing the vessel to retract. These mechanical loads introduce intramural wall stresses and flow induced wall shear stresses that play a key role in mechano-biological signaling and tissue homeostasis. Until now, the mechanical properties of SA-TEBV have only been characterized in the circumferential direction (i.e. burst pressure and circumferential elastic modulus). The objective of this work is to characterize the biaxial mechanical properties of SA-TEBV to quantify their mechanical behavior and local intramural stresses under physiological loading. The work will show that while the global mechanical response of the SA-TEBV is similar to that of native arteries (and potentially sufficient), the local intramural stresses (using the current fabrication techniques) differ greatly from native coronary arteries. Therefore, a novel approach to fabricate the self-assembly derived tissue sheets is developed and tested which utilizes biaxial mechanical stimulation to alter the microstructure, thereby controlling their mechanical response.