On the Decellularization and Recellularization of Tissue Engineered Heart Valves

Abstract

The overall objective of this thesis was to fill critical knowledge gaps for the development of the tissue engineered heart valve (TEHV). Heart valve tissue engineering is a promising solution for the currently unmet need of an ideal prosthetic heart valve. However, the TEHV still faces a number of challenges ranging from scaffold creation to the lack of consistent recellularization. Therefore, the progression of this thesis was designed around the pathway leading to the THEV and focuses on addressing some of the key challenges. There are the three principal steps in TEHV development: scaffold formation through decellularization, seeding a patient specific cell population, and conditioning through culture in a bioreactor. The current body of work followed these steps with three aims focused on valve decellularization, cell seeding, and bioreactor conditioning, respectively. The first aim provided a detailed investigation into the species-specific effects of decellulzation on human and ovine aortic heart valves. The tested multi-detergent decellularization process lead to cell free scaffolds for both human and ovine heart valves; however, decellularization was found to more greatly affect ovine tissue than human tissue. The next aim of this thesis investigated the ability for recellularization, and the potential mechanism by which recellularization occurred for four cell populations: MNCs, low dose MSCs, high dose MSCs, and VICs. The higher dose of MSCs showed the greatest cellular infiltration compared to MNCs or VICs and maintained a phenotype similar to native cells. The final aim of the thesis was to develop bioreactor-conditioning protocols to increase the recellularization of cell seeded valves. Detailed investigations into bioreactor conditioning were performed individually for valves seeded with MNCs and MSCs. Bioreactor conditioning for MNC seeded valves focused on the duration of bioreactor culture and found three weeks of culture resulted in proliferation of the MSC sub-population and partial recellularization. The investigation into bioreactor conditioning for MSC seeded valves focused on non-physiologic parameters of chamber pressure and hypoxic conditioning to increase recellularization into the distal leaflet. Hypoxic conditions greatly increased the infiltration of seeded cells while cyclic negative pressure conditioning better maintained stem cell gene markers. The goal driving this thesis was the clinical application of a TEHV and the described methodologies were developed to be translatable to the clinic. The current body of work offers real, obtainable advances for the field of heart valve tissue engineering.