Non-destructive 3D characterization of the blood vessel wall microstructure in different species and blood vessel types using contrast-enhanced microCT and comparison with synthetic vascular grafts.

To improve the current treatment for vascular diseases, such as vascular grafts, intravascular stents, and balloon angioplasty intervention, the evaluation of the native blood vessel microstructure in full 3D could be beneficial. For this purpose, we used contrast-enhanced X-ray microfocus computed tomography (CECT): a combination of X-ray microfocus computed tomography (microCT) and contrast-enhancing staining agents (CESAs) containing high atomic number elements. In this work, we performed a comparative study based on staining time and contrast-enhancement of 2 CESAs: Monolacunary and 1:2 Hafnium-substituted Wells-Dawson polyoxometalate (Mono-WD POM and Hf-WD POM, respectively) for imaging of the porcine aorta. After showing the advantages of Hf-WD POM in terms of contrast enhancement, we expanded our imaging to other species (rat, porcine, and human) and other types of blood vessels (porcine aorta, femoral artery, and vena cava), clearly indicating microstructural differences between different types of blood vessels and different species. We then showed the possibility to extract useful 3D quantitative information from the rat and porcine aortic wall, potentially to be used for computational modeling or for future design optimization of graft materials. Finally, a structural comparison with existing synthetic vascular grafts was made. This information will allow to better understand the in vivo functioning of native blood vessels and to improve the current disease treatments. STATEMENT OF SIGNIFICANCE: Synthetic vascular grafts, used as treatment for some cardiovascular diseases, still often fail clinically, potentially because of a mismatch in mechanical behaviour between the native blood vessel and the graft. To better understand the causes of this mismatch, we studied the full 3D microstructure of blood vessels. For this, we identified Hafnium-substituted Wells-Dawson polyoxometalate as contrast-enhancing staining agent to perform contrast-enhanced X-ray microfocus computed tomography. This technique allowed to show important differences in the microstructure of different types of blood vessels and in different species, as well as with that of synthetic grafts. This information can lead to a better understanding of the functioning of blood vessels and will allow to improve current disease treatments, such as vascular grafts.

Cryogenic contrast-enhanced microCT enables nondestructive 3D quantitative histopathology of soft biological tissues.

Biological tissues comprise a spatially complex structure, composition and organization at the microscale, named the microstructure. Given the close structure-function relationships in tissues, structural characterization is essential to fully understand the functioning of healthy and pathological tissues, as well as the impact of possible treatments. Here, we present a nondestructive imaging approach to perform quantitative 3D histo(patho)logy of biological tissues, termed Cryogenic Contrast-Enhanced MicroCT (cryo-CECT). By combining sample staining, using an X-ray contrast-enhancing staining agent, with freezing the sample at the optimal freezing rate, cryo-CECT enables 3D visualization and structural analysis of individual tissue constituents, such as muscle and collagen fibers. We applied cryo-CECT on murine hearts subjected to pressure overload following transverse aortic constriction surgery. Cryo-CECT allowed to analyze, in an unprecedented manner, the orientation and diameter of the individual muscle fibers in the entire heart, as well as the 3D localization of fibrotic regions within the myocardial layers. We foresee further applications of cryo-CECT in the optimization of tissue/food preservation and donor banking, showing that cryo-CECT also has clinical and industrial potential.

Ensemble learning and test-time augmentation for the segmentation of mineralized cartilage versus bone in high-resolution microCT images.

High-resolution non-destructive 3D microCT imaging allows the visualization and structural characterization of mineralized cartilage and bone. Deriving statistically relevant quantitative structural information about these tissues, however, requires automated segmentation procedures, mainly because manual contouring is user-biased and time-consuming. Despite the increased spatial resolution in microCT 3D volumes, automatic segmentation of mineralized cartilage versus bone remains non-trivial since they have similar grayscale values. Our work investigates how reliable 2D segmentation masks can be predicted automatically based on a (set of) convolutional neural network(s) trained with a limited number of manually annotated samples. To do that, we compared different strategies to select the 2D samples to annotate and considered ensemble learning and test-time augmentation (TTA) to mitigate the limited accuracy and robustness resulting from the small number of annotated training samples. We show that, for a fixed amount of annotated image samples, 2D microCT slices to annotate should preferably be selected in distinct 3D volumes, at regular intervals, rather than being grouped in adjacent slices of a same 3D volume. Two main lessons are drawn regarding the use of ensembles or TTA instead of a single model. First, ensemble learning is shown to improve segmentation accuracy and to reduce the mean and standard deviation of the absolute errors in cartilage characteristics obtained with different initializations of the neural network training process. In contrast, TTA appears to be unable to improve the model's robustness to unlucky initializations. Second, both TTA and ensembling improved the model's confidence in its predictions and segmentation failure detection.

A Review of Ex Vivo X-ray Microfocus Computed Tomography-Based Characterization of the Cardiovascular System.

Cardiovascular malformations and diseases are common but complex and often not yet fully understood. To better understand the effects of structural and microstructural changes of the heart and the vasculature on their proper functioning, a detailed characterization of the microstructure is crucial. In vivo imaging approaches are noninvasive and allow visualizing the heart and the vasculature in 3D. However, their spatial image resolution is often too limited for microstructural analyses, and hence, ex vivo imaging is preferred for this purpose. Ex vivo X-ray microfocus computed tomography (microCT) is a rapidly emerging high-resolution 3D structural imaging technique often used for the assessment of calcified tissues. Contrast-enhanced microCT (CE-CT) or phase-contrast microCT (PC-CT) improve this technique by additionally allowing the distinction of different low X-ray-absorbing soft tissues. In this review, we present the strengths of ex vivo microCT, CE-CT and PC-CT for quantitative 3D imaging of the structure and/or microstructure of the heart, the vasculature and their substructures in healthy and diseased state. We also discuss their current limitations, mainly with regard to the contrasting methods and the tissue preparation.