Quantification of Collagen Fiber Morphologies in Human Arterial Walls

The arterial wall consists of three major microstructural components, collagen, elastin, and smooth muscle cells; all of which play important roles in the cardiovascular function of health and disease. From a biomechanical perspective, collagen with its high tensile strength constitutes the most important component, endowing the arterial wall with strength and load resistance. Changes in the mechanical properties of a healthy wall play a role in pathological and degenerative processes such as increased wall stiffness, atherosclerosis, or enlargement of intracranial aneurysms. To that end we studied the fundamental role of collagen in intramural healing through the remodeling processes of arterial or aneurysmal dissections in an apolipoprotein-E null mouse model. A meaningful quantification of morphological collagen data in healthy arteries is fundamental to a better understanding of the underlying mechanical principles governing the biomechanical response of the vessel wall. Furthermore, such data can be utilized for modeling of the cardiovascular system and to increase our understanding of disease progression. Towards obtaining such data we started with the quantification of collagen fiber angles in the human thoracic and abdominal aorta and common iliac artery, using a well established experimental approach based on measuring (by hand) individual fiber angles from histological sections through polarized light microscopy in combination with a universal stage. The study yielded mean data for each of the three individual layers of the arterial wall, which was quantified using a dispersion model. In trying to overcome several limitations inherent to this traditional method, we began to develop an automatic approach for the quantification of collagen fiber distributions from 2D images. Not only were we able to speed up the measuring process, but the new method also enabled us to assess and evaluate entire fiber distributions among varying lengthscales using statistical approaches, which has implications for numerical modeling, e.g., on determining appropriate mesh densities. Finally, we hoped to move beyond a two-dimensional quantification of collagen structures by trying to obtain image stacks throughout the entire thickness of a human arterial wall. The major experimental challenge to overcome was the limited application of optical techniques to assess the orientation of collagen throughout approximately 800− 1500 μm of non-transparent wall tissue. We succeeded by developing a novel approach which combines a new sample preparation method with optical tissue clearing and subsequent imaging using second-harmonic generation microscopy. To extract and quantify the morphological collagen structures from image stacks we advanced our 2D image analysis approach, now yielding a 3D distribution of amplitudes (1◦ resolution) representing the orientations of the collagen fibers throughout the tissue. With that we can now identify isotropic regions in the tissue where no preferred fiber orientations are observed and quantify regions of anisotropy by calculating structural parameters which can directly be utilized in numerical codes using fiber-reinforced constitutive laws.