Advanced X-ray-based 3D microstructural characterization of the heart and heart valves

The heart has a complex and highly heterogeneous 3D structural organization, in which heart valves play a crucial role by maintaining the unidirectional blood flow throughout the heart. However, the microstructure of the heart and heart valves is not fully understood yet or has been inconsistently described in the literature. For this reason, and to counteract the limitations of classical 2D histology, ex vivo X-ray microfocus computed tomography (microCT) has been suggested to image the heart and its constituents in a non-destructive way, with a high and isotropic spatial resolution. To improve the limited discrimination of soft tissue constituents using X-rays, microCT is combined with contrast-enhancing staining agents (CESAs), and corresponds to contrast-enhanced computed tomography (CECT). This thesis aims at optimizing microCT-based techniques to investigate the heart and heart valve microstructure in 3D. First, using a non-destructive CESA (Hf-WD POM), CECT enabled to visualize fibrosis in case of hypertrophy, and the coronary blood vessels and heart valves in entire murine hearts in 3D. However, classical CECT does not allow to visualize individual muscle fibers in the myocardium. Cryo-CECT, a technique in which the sample is frozen and imaged in cryogenic conditions, has thus been optimized and applied to hypertrophic murine hearts. It was possible to distinguish individual muscle fibers in the myocardium and to compute their orientation and thickness. Then, human calcified aortic valve cusps were investigated using microCT to determine the volume fraction, size and density composition of the calcifications. Finally, CECT and cryo-CECT were applied to the porcine mitral valve apparatus to assess its microstructure. Blood vessels, adipocytes and the extracellular matrix fibers were visualized in the leaflet and in the chorda tendinea.