A method for the quantification of the pressure dependent 3D collagen configuration in the arterial adventitia

Collagen plays an important role in the response of the arterial wall to mechanical loading and presumably has a load-bearing function preventing overdistension. Collagen configuration is important for understanding this role, in particular in mathematical models of arterial wall mechanics. In this study a new method is presented to image and quantify this configuration. Collagen in the arterial adventitia is stained with CNA35, and imaged in situ at high resolution with confocal microscopy at luminal pressures from 0 to 140 mm Hg. The images are processed with a new automatic approach, utilizing techniques intended for MRI-DTI data. Collagen configuration is quantified through three parameters: the waviness, the transmural angle and the helical angle. The method is demonstrated for the case of carotid arteries of the white New Zealand rabbit. The waviness indicated a gradual straightening between 40 and 80 mm Hg. The transmural angle was about zero indicating that the fibers stayed within an axial-circumferential plane at all pressures. The helical angle was characterized by a symmetrical distribution around the axial direction, indicating a double symmetrical helix. The method is the first to combine high resolution imaging with a new automatic image processing approach to quantify the 3D configuration of collagen in the adventitia as a function of pressure. (C) 2012 Elsevier Inc. All rights reserved.

Vascular physiology

Sylvain Roy

This thesis aims at better understanding the role played by vascular smooth muscle cells (VSM) in the biomechanics of the arterial wall. Current models assume that under maximal vasodilation VSM carry no load, or the so-called "active stress" is zero. Biomechanical tests on arterial segments, where VSM cells were either chemically removed or structurally weakened have shown that VSM are playing a determinant role in the biomechanical properties of the arterial wall and carry important load even when maximally dilated. Based on these results a new model of the arterial wall biomechanics is proposed that takes into account the passive structure of the arterial wall constituents. The entire thesis consists of three distinct studies, either published or submitted and under review for publication. The first paper presents the effects of decellularization on the biomechanical properties of porcine common carotid arteries. The decellularization was performed by a detergent-enzymatic procedure, which preserves the extracellular matrix scaffold. Rupture tests were conducted. Inner diameter and wall thickness were measured by echotracking during pressure inflation from 10 to 145 mmHg. Distensibility and incremental elastic modulus were computed. At 10 mmHg, mean inner diameter of decellularized arteries was substantially higher than controls, whereas both decellularized and control arteries reached the same internal diameter value at 145 mmHg. Removing cellular elements from media lead to changes in arterial dimensions. The incremental elastic modulus increased, and the collagen fibers engaged more rapidly during inflation, thus yielding a stiffer vessel. Distensibility and compliance were therefore significantly lower in decellularized vessels, reaching a factor three reduction in the physiological range of pressures. The conclusion of this paper is that decellularization yields vessels able to withstand high inflation pressures with, however, markedly different geometrical and biomechanical properties. The second paper proposes an interpretation of the data collected in the experiments conducted in the first paper. This initial work on decellularized arteries revealed the existence of significant residual stresses within the arterial wall, which are released upon chemical removal of vascular smooth muscle in normal arteries causing substantial radial expansion. Hence, the often-used Hill's model describing active and passive stresses within the wall does not hold true, because the existence of prestresses precludes the fundamental assumption of zero active stress when the vascular smooth muscle is inactive. A new mathematical model based on a modified Hill's model was developed, where the total wall elastin is partitioned in two parts: one in-parallel to vascular smooth muscle and collagen and one connected in-series with vascular smooth muscle. Based on experimental evidences, compressive prestresses were assumed to exist on the parallel elastic component and tensile prestresses on the series elastic component. Further, it was assumed that the elastic constants of elastin and collagen and the statistical description of collagen engagement are not affected by decellularization. Excellent fits of the pressure-diameter curves of normal and decellularized arteries were obtained. The model predicts that the majority of elastin is in-series with the vascular smooth muscle (74±8%) and thus only about one-fourth of elastin acts in parallel to the vascular smooth muscle. The conclusion of this paper is that correct biomechanical modeling of the arterial wall requires the knowledge of the zero stress state of both the series and parallel elastic components. The third and last paper analyzes the effects of cytoskeleton destruction, after administration of Cytochalasin D, on the biomechanical properties of porcine common carotids. Compared to untreated, maximally dilated controls, Cytochalasin D-treated arteries have shown a marked increase in compliance in the elastin-dominated, 10-150 mmHg, pressure range. After weakening the vascular smooth muscle stress-bearing cytoskeleton by Cytochalasin D the artery would expand, reaching a new equilibrium state. This study brings further evidence that VSM is under tension, even when it is under zero load and at maximal vasodilation. This residual tension was released upon partial destruction of the cytoskeleton with Cytochalasin D. From a biomechanical standpoint, this means that the Zero Stress States of the in series and parallel elastic components are substantially different.

EPFL2008

Biomechanics of Vascular Wall

Rana Saitta-Rezakhaniha

This thesis contributes to the field of biomechanics of vascular wall. The focus is particularly on the microstructure of vascular elastin and collagen constituents and their contribution to the macroscopic mechanical behavior of the wall. The analysis is done in the framework of continuum mechanics. The work characterizes structural features of elastin and collagen fibers using microscopy techniques and introduces these features to constituent-based constitutive models. The models are applied to the experimental data, derived from inflation-extension tests, to predict the gross mechanical behavior of the tissue. The developed constitutive models could be further used to study in detail the mechanics of vascular tissue in health and disease. The thesis is presented in form of an introduction, four chapters (corresponding to four papers) and a conclusion. The introduction provides the motivation for this thesis as well as the background on vascular wall structure and mechanics. A brief description of the imaging techniques used is presented. Also, structural constitutive modeling of vascular wall is briefly discussed with particular attention to the modeling of elastin and collagen constituents. The first paper focuses on anisotropic properties of elastin in veins. We show that earlier constituent-based strain energy functions (SEFs), where elastin is modeled as an isotropic material, fail in describing accurately the tissue response to inflation-extension loading. We hypothesize that these shortcomings are partly due to unaccounted anisotropic properties of elastin. We extend the previously developed biomechanical model in our laboratory (Zulliger et al., 2004, J. Biomech., 37(7): 989-1000 (2004)) to account for elastin anisotropy and present an anisotropic strain energy function for elastin with one family of fibers in the longitudinal direction. The model is validated using experimental data from inflation-extension tests on rabbit facial veins. The tissue is tested under a fully relaxed smooth muscle state, for longitudinal stretch ratios ranging from 100% to 130% of the in vivo length. The model with the anisotropic elastin fits well the data for a wide range of longitudinal stretch ratios. The main finding of this paper is that the anisotropic description of elastin is required for a full 3-D characterization of the biomechanics of the venous wall. The second paper addresses the role of elastin in anisotropic properties of arteries with particular attention to the structural organization of elastin. A constituent-based model including an anisotropic elastin, with one family of fibers in the circumferential direction, is presented. Micro-structural imaging, based on electron microscopy techniques, is used to support this anisotropy. Inflationˆextension tests, on intact and elastase-treated arteries, provide a data set to validate the model and to study the effect of elastin removal. We show that the SEF, with an anisotropic elastin part, characterizes more accurately the mechanical properties of the arterial wall as compared to models with simply an isotropic elastin. Transmission electron microscopy (TEM) and serial block-face scanning electron microscopy (SBF-SEM) show interlamellar elastin fibers in the circumferential direction and therefore support the nature of the assumed anisotropy. The model predicts an earlier engagement of collagen in elastase-treated arteries compared to the intact arteries and thus suggests a clear functional interaction between the elastin and collagen component that is often neglected in constituent-based SEFs. The third paper presents a structural constitutive model of the vascular wall which integrates both waviness and orientational dispersion of collagen fibers. We extend the model of Zulliger et al., which already accounts for collagen waviness, to include orientational distribution of collagen. We study the effect of parameters related to the orientational distribution on macro-mechanical behavior of the tissue during inflation-extension tests. The model is further applied to the experimental data from rabbit facial veins. The model accurately fits the experimental data of veins, but it does not improve the quality of the fit compared to the one without dispersion. We show that the orientational dispersion of collagen fibers can be compensated by a less abrupt and shifted to higher strain collagen engagement pattern. This should be taken into consideration when the model is used to fit experimental data and model parameters are used to study structural modifications of collagen fiber network in physiology and disease. In the fourth paper, we measure and quantify the waviness and orientational distributions of collagen in rabbit carotids. Quantification of collagen orientation distributions at the zero stress state of arteries is needed to develop realistic and precise biomechanical models. Using the fluorescence collagen marker CNA38-OG488 and confocal laser scanning microscopy, we visualize collagen fibers in adventitia of rabbit common carotids ex vivo. To get the properties related to the zero stress sate, the arteries are cut open along their longitudinal axes. We use semi-automatic and automatic techniques to measure parameters related to the waviness and fiber orientation. We show that the straightness parameter (i.e. the ratio between the distances of endpoints of a fiber to the fiber length) is distributed with a beta distribution. The shape of the probability density distribution does not depend on the mean angle orientation of fibers. In addition, our measurements reveal four axially symmetric families of fibers with mean orientations of 0°, 90°, 43° and -43° and circular standard deviations of 40°, 47°, 37° and 37°, with respect to the axial direction, respectively. To the best of our knowledge, this is the first study focusing on structural properties of collagen in the zero stress state and quantifying fiber waviness. The results of this study can be used to develop more precise structural models of the adventitia including waviness and orientational dispersion of fibers. The conclusion section summarizes the main results of the thesis, presents the improvements made over previous studies, and proposes future perspectives of this work.

EPFL2010

Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy

Aristotelis Agianniotis, Alessandra Griffa, Daniel Sage, Jelle Tymen Christiaan Schrauwen, Nikolaos Stergiopulos, Michaël Unser

Mechanical properties of the adventitia are largely determined by the organization of collagen fibers. Measurements on the waviness and orientation of collagen, particularly at the zero-stress state, are necessary to relate the structural organization of collagen to the mechanical response of the adventitia. Using the fluorescence collagen marker CNA38-OG488 and confocal laser scanning microscopy, we imaged collagen fibers in the adventitia of rabbit common carotid arteries ex vivo. The arteries were cut open along their longitudinal axes to get the zero-stress state. We used semi-manual and automatic techniques to measure parameters related to the waviness and orientation of fibers. Our results showed that the straightness parameter (defined as the ratio between the distances of endpoints of a fiber to its length) was distributed with a beta distribution (mean value 0.72, variance 0.028) and did not depend on the mean angle orientation of fibers. Local angular density distributions revealed four axially symmetric families of fibers with mean directions of 0°, 90°, 43° and −43°, with respect to the axial direction of the artery, and corresponding circular standard deviations of 40°, 47°, 37° and 37°. The distribution of local orientations was shifted to the circumferential direction when measured in arteries at the zero-load state (intact), as compared to arteries at the zero-stress state (cut open). Information on collagen fiber waviness and orientation, such as obtained in this study, could be used to develop structural models of the adventitia, providing better means for analyzing and understanding the mechanical properties of vascular wall.