Mechano-control of tissue properties in engineered cardiovascular constructs

Currently used heart valve and coronary artery replacement strategies all have specific drawbacks. Their main drawback is the inability to grow, repair and remodel in response to the changing physiological environment. Tissue engineering is a novel treatment strategy, which aims to overcome the problems associated with currently used replacement strategies. It intends to develop autologous and living replacement tissues, which are tailor-made for individual recipients. Despite early successes, however, tissue engineers face major challenges, when creating tissues with a primarily biomechanical function. The most successful approach so far in cardiovascular tissue engineering has been the use of bioresorbable scaffolds seeded with autologous cells. Recently, engineered heart valves, cultured using this technique, were successfully implanted as pulmonary valve replacements in sheep and after 20 weeks showed development of tissue properties comparable to native. In addition, heart valve leaflets were cultured that showed potential for placement at the aortic position. Similar to heart valve tissue engineering, strong tissue engineered blood vessels have been created using this scaffold based technique. Nevertheless, the mechanical behaviour of engineered human heart valve leaflets was more linear, much stiffer and less anisotropic compared to the mechanical behaviour of native porcine leaflets and thus requires improvement. Tissue mechanical behaviour is strongly coupled to the collagen organisation, which in turn is related to the mechanical loading condition of the tissue. The challenge in cardiovascular tissue engineering is to create strong and functional tissues in the shortest period of time possible. By systematically investigating the influence of mechanical loading on collagen architecture, an improved understanding of the effect of certain mechanical loading protocols on tissue organisation was obtained. A fluorescent collagen probe (CNA35) was developed in order to relate mechanical loading to changes in microstructure. This probe was applied to a variety of samples including native cardiovascular tissue, engineered cardiovascular tissue and a real-time study of collagen synthesis in monolayer culture. This was done in order to optimise the application of this probe and to demonstrate its potential for collagen remodelling studies. The probe showed enhancement in contrast compared to imaging with second harmonic generation and allowed imaging of immature collagen fibrils. In addition, a model system was developed in order to allow repetitive long term straining of polyglycolic acid based engineered cardiovascular constructs. The system was used to address the effect of different strain magnitudes on the properties of engineered cardiovascular constructs. The different continuous dynamic strain magnitudes resulted in decreased levels of collagen production, but increased levels of hydroxylyslypyridinoline (HP) crosslink fraction. This suggested a compensatory mechanism of cells in which they produced collagen with different intrinsic mechanical properties in order to resist the effect of mechanical straining more effectively. In addition, the microstructure of the strained engineered cardiovascular constructs showed a striking difference in cell and collagen orientation between the superficial layers and the deeper layers. The same setup was used to compare continuous and intermittent loading protocols in order to investigate if intermittent loading further optimised the culture conditions for engineered cardiovascular constructs. Intermittent dynamic loading significantly increased the production of collagen per cell and the fraction of HP crosslinks per collagen triple helix, whereas no differences were observed among the different intermittent loading protocols. Furthermore, uniaxal, biaxial and equibiaxial loading conditions were applied to engineered cardiovascular constructs in order to relate mechanical loading conditions to differential effects on the tissue composition and the collagen architecture. The various loading conditions resulted in differences in collagen production and cross-link fraction and, more specifically, biaxial loading conditions introduced biaxial mechanical properties. In addition, the orientation analysis revealed differences in the trend of the orientation angle as a function of the penetration depth depending on the loading condition. With these experimental techniques, a structural investigation of the remodelling processes, that occur during mechanical loading, can be performed. Ultimately, a detailed understanding of the effect of mechanical loading on tissue properties in developing engineered tissues is obtained, resulting in mechanocontrol of tissue properties.