Strategies to optimize engineered tissue towards native human aortic valves

unclear to what extent mechanical loading affects the collagen fibril morphology. To determine if local stresses affect the collagen fibril morphology (i.e. fibril diameter, its distribution, and fibril density), these parameters were investigated with transmission electron microscopy in adult human aortic valve leaflets. The mechanical behavior of human aortic valves was implemented in a computational model to predict the stress distribution in the valve leaflet during the diastolic phase of the cardiac cycle. The results showed that large tissue stress was associated with larger average fibril diameter, lower fibril density and wider fibril size distribution compared with low stress locations in the leaflets. These findings provide insight in the effect of mechanical loading on the collagen ultrastructure, and are valuable to optimize mechanical conditioning protocols for heart valve tissue engineering. The second objective of this thesis was to improve the mechanical properties of engineered tissues towards values of healthy native human aortic valves, which was considered an objective bench-mark for tissue engineering. Two strategies were adopted in the tissue engineering protocol to achieve this. The first approach involved modification of the scaffold design to provide sufficient mechanical support to engineered tissues. The currently used scaffold material is a mesh of rapid degrading polyglycolic acid coated with poly-4-hydroxybutyrate (PGA-P4HB). This material degrades within weeks, and does not provide mechanical integrity after implantation. As some tissue engineering applications do require a prolonged period of mechanical support by the scaffold, the feasibility of a slow degrading polymer scaffold of electrospun poly-"-caprolactone (PCL) was evaluated for cardiovascular tissue engineering, and compared with the PGA-P4HB scaffold. After optimization of the electrospun PCL scaffold, proper cell ingrowth and extracellular matrix biosynthesis were observed, while retaining elastic properties and mechanical integrity. PCL scaffolds appeared a promising alternative to PGA-P4HB scaffolds, specifically for tissue engineered blood vessels and the wall of an engineered heart valve, where prolonged mechanical support of the scaffold may be desired. As a second approach, the growing engineered tissues were biochemically stimulated to enhance tissue formation and strengthen the tissue. After an evaluation of biochemical factors known to promote protein synthesis, hypoxia and insulin were chosen for the experiments. A physiologically relevant oxygen tension, being lower than currently used in tissue engineering approaches, and insulin supplements were applied to the growing heart valve tissues to enhance their strength. Both insulin and hypoxia were associated with enhanced matrix production and improved mechanical properties, however, a synergistic effect was not observed. Although the amount of collagen and cross-links in the engineered tissues were still lower than native adult human aortic valves, tissues cultured under hypoxic conditions reached native human aortic valve values of tissue strength and stiffness after four weeks of culture, and were up to twice the values of the normoxic controls. These results strongly indicate that oxygen tension is a key parameter to achieve native-based bench-mark values of tissue strength in engineered heart valves. Engineered tissues, based on rapid degrading scaffolds, of such strength have not been achieved up to now. These findings bring the potential use for systemic applications a step closer, and can be considered an important improvement in heart valve tissue engineering. Heart valve replacement is a common treatment of end-stage valvular diseases to restore functionality of the valve. Although conventional valve replacements by mechanical or biological prosthesis offer prosperous function, they are associated with risks that limit their success. An important shortcoming of all prosthetic valves is their inability to grow, adapt and repair, which is particularly relevant for treatment of pediatric and adolescent patients. The lack of these features in current prosthesis drives the multidisciplinary approach of tissue engineering as a promising technique to create living heart valve substitutes. The concept of tissue engineering is based on seeding autologous cells onto a carrier of biodegradable material (the scaffold). This construct of cells and scaffold is stimulated to grow and develop in a mimicked physiological environment. Implantations of tissue engineered valves have been performed successfully at the pulmonary position in animal models. However, engineered valves did not possess sufficient mechanical integrity for implantation at the aortic position. Therefore, a major challenge in tissue engineering is to create tissue structures that resemble properties of native tissues to ensure durable functioning and in-vivo survival. For future human applications, one of the most important questions is: "How good is good enough for in vivo survival of tissue engineered heart valves?". The first objective of this work was to define qualitative and quantitative bench-marks for tissue engineered heart valves to determine when these valves qualify for implantation in patients. In this thesis these bench-marks were based on mechanical and structural characteristics of healthy human adult aortic valve leaflets. In native aortic valves, the collagen fiber architecture is the most prominent matrix component responsible for sustaining the load under high pressure conditions. Therefore, knowledge about the function of collagen in relation with the mechanical behavior of native heart valve tissue was an important research focus in the process to define bench-marks for tissue engineering. The relation between mechanical properties and collagen organization was investigated on a global and local scale in human adult aortic valve leaflets. Mechanical properties obtained by tensile tests of the leaflets were correlated to the amount of collagen and cross-links. Collagen cross-links, but not the collagen amount, appeared highly correlated to tissue stiffness in human heart valve leaflets. With these findings, the relevance of collagen cross-links for the mechanical integrity of engineered tissues should be given particular attention. Furthermore, in heart valve tissue, it remained