A multiaxial computer-controlled organ culture and biomechanical device for mouse carotid arteries

Much of our understanding of vascular mechanotransduction has come from studies using either cell culture or in vivo animal models, but the recent success of organ culture systems offers an exciting alternative. In studying cell-mediated vascular adaptations to altered loading, organ culture allows one to impose well-controlled mechanical loads and to perform multiaxial mechanical tests on the same vessel throughout the culture period, and thereby to observe cell-mediated vascular adaptations independent of neural and hormonal effects. Here, we present a computer-controlled perfused organ culture and biomechanical testing device designed for small caliber (50-5000 micron) blood vessels. This device can control precisely the pulsatile pressure, luminal flow, and axial load (or stretch) and perform intermittent biaxial (pressure-diameter and axial load-length) and functional tests to quantify adaptations in mechanical behavior and cellular function, respectively. Device capabilities are demonstrated by culturing mouse carotid arteries for 4 days.     

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.     

Fundamental role of axial stress in compensatory adaptations by arteries

Arteries exhibit a remarkable ability to adapt to diverse genetic defects and sustained alterations in mechanical loading. For example, changes in blood flow induced wall shear stress tend to control arterial caliber and changes in blood pressure induced circumferential wall stress tend to control wall thickness. We submit, however, that the axial component of wall stress plays a similarly fundamental role in controlling arterial geometry, structure, and function, that is, compensatory adaptations. This observation comes from a review of findings reported in the literature and a comparison of four recent studies from our laboratory that quantified changes in the biaxial mechanical properties of mouse carotid arteries in cases of altered cell-matrix interactions, extracellular matrix composition, blood pressure, or axial extension. There is, therefore, a pressing need to include the fundamental role of axial wall stress in conceptual and theoretical models of arterial growth and remodeling and, consequently, there is a need for increased attention to evolving biaxial mechanical properties in cases of altered genetics and mechanical stimuli.     

Evolving biaxial mechanical properties of mouse carotid arteries in hypertension

Quantifying the time course of load-induced changes in arterial wall geometry, microstructure, and properties is fundamental to developing mathematical models of growth and remodeling. Arteries adapt to altered pressure and flow by modifying wall thickness, inner diameter, and axial length via marked cell and matrix turnover. To estimate particular biomaterial implications of such adaptations, we used a 4-fiber family constitutive relation to quantify passive biaxial mechanical behaviors of mouse carotid arteries 0 (control), 7-10, 10-14, or 35-56 days after an aortic arch banding surgery that increased pulse pressure and pulsatile flow in the right carotid artery. In vivo circumferential and axial stretches at mean arterial pressure were, for example, 11% and 26% lower, respectively, in hypertensive carotids 35-56 days after banding than in normotensive controls; this finding is consistent with observations that hypertension decreases distensibility. Interestingly, the strain energy W stored in the carotids at individual in vivo conditions was also less in hypertensive compared with normotensive carotids. For example, at 35-56 days after banding, W was 24%, 39%, and 47% of normal values at diastolic, mean, and systolic pressures, respectively. The energy stored during the cardiac cycle, W(sys)-W(dias), also tended to be less, but this reduction did not reach significance. When computed at normal in vivo values of biaxial stretch, however, W was well above normal for the hypertensive carotids. This net increase resulted from an overall increase in the collagen-related anisotropic contribution to W despite a decrease in the elastin-related isotropic contribution. The latter was consistent with observed decreases in the mass fraction of elastin.     

Constitutive modeling of mouse carotid arteries using experimentally measured microstructural parameters

Changes in the local mechanical environment and tissue mechanical properties affect the biological activity of cells and play a key role in a variety of diseases, such as cancer, arthritis, nephropathy, and cardiovascular disease. Constitutive relations have long been used to predict the local mechanical environment within biological tissues and to investigate the relationship between biological responses and mechanical stimuli. Recent constitutive relations for soft tissues consider both material and structural properties by incorporating parameters that describe microstructural organization, such as fiber distributions, fiber angles, fiber crimping, and constituent volume fractions. The recently developed technique of imaging the microstructure of a single artery as it undergoes multiple deformations provides quantitative structural data that can reduce the number of estimated parameters by using parameters that are truly experimentally intractable. Here, we employed nonlinear multiphoton microscopy to quantify collagen fiber organization in mouse carotid arteries and incorporated measured fiber distribution data into structurally motivated constitutive relations. Microscopy results demonstrate that collagen fibers deform in an affine manner over physiologically relevant deformations. The incorporation of measured fiber angle distributions into constitutive relations improves the model's predictive accuracy and does not significantly reduce the goodness of fit. The use of measured structural parameters rather than estimated structural parameters promises to improve the predictive capabilities of the local mechanical environment, and to extend the utility of intravital imaging methods for estimating the mechanical behavior of tissues using in situ structural information.     

Biaxial biomechanical properties of the nonpregnant murine cervix and uterus

From a biomechanical perspective, female reproductive health is an understudied area of research. There is an incomplete understanding of the complex function and interaction between the cervix and uterus. This, in part, is due to the limited research into multiaxial biomechanical functions and geometry of these organs. Knowledge of the biomechanical function and interaction between these organs may elucidate etiologies of conditions such as preterm birth. Therefore, the objective of this study was to quantify the multiaxial biomechanical properties of the murine cervix and uterus using a biaxial testing set-up. To accomplish this, an inflation-extension testing protocol (n = 15) was leveraged to quantify biaxial biomechanical properties while preserving native matrix interactions and geometry. Ultrasound imaging and histology (n = 10) were performed to evaluate regional geometry and microstructure, respectively. Histological analysis identified a statistically significant greater collagen content and significantly smaller smooth muscle content in the cervix as compared to the uterus. No statistically significant differences in elastic fibers were identified. Analysis of bilinear fits revealed a significantly stiffer response from the circumferentially orientated ECM fibers compared to axially orientated fibers in both organs. Bilinear fits and a two-fiber family constitutive model showed that the cervix was significantly less distensible than the uterus. We submit that the regional biaxial information reported in this study aids in establishing an appropriate reference configuration for mathematical models of the uterine-cervical complex. Thus, may aid future work to elucidate the biomechanical mechanisms leading to cervical or uterine conditions.     

Numerical simulation of local blood flow in the carotid and cerebral arteries under altered gravity

A computational fluid dynamics (CFD) approach was presented to model the blood flows in the carotid bifurcation and the brain arteries under altered gravity. Physical models required for CFD simulation were introduced including a model for arterial wall motion due to fluid-wall interactions, a shear thinning fluid model of blood, a vascular bed model for outflow boundary conditions, and a model for autoregulation mechanism. The three-dimensional unsteady incompressible Navier-Stokes equations coupled with these models were solved iteratively using the pseudocompressibility method and dual time stepping. Gravity source terms were added to the Navier-Stokes equations to take the effect of gravity into account. For the treatment of complex geometry, a chimera overset grid technique was adopted to obtain connectivity between arterial branches. For code validation, computed results were compared with experimental data for both steady-state and time-dependent flows. This computational approach was then applied to blood flows through a realistic carotid bifurcation and two Circle of Willis models, one using an idealized geometry and the other using an anatomical data set. A three-dimensional Circle of Willis configuration was reconstructed from subject-specific magnetic resonance images using an image segmentation method. Through the numerical simulation of blood flow in two model problems, namely, the carotid bifurcation and the brain arteries, it was observed that the altered gravity has considerable effects on arterial contraction/dilatation and consequent changes in flow conditions.     

A comparison of age-related changes in axial prestretch in human carotid arteries and in human abdominal aorta

Biomechanics and Modeling in Mechanobiology - It is known that large arteries in situ are subjected to significant axial prestretch. This prestretch plays an important physiological role in...     

Biaxial mechanical properties of the human thoracic and abdominal aorta,common carotid,subclavian, renal and common iliac arteries

The biomechanics of large- and medium-sized arteries influence the pathophysiology of arterial disease and the response to therapeutic interventions. However, a comprehensive comparative analysis of human arterial biaxial mechanical properties has not yet been reported. Planar biaxial extension was used to establish the passive mechanical properties of human thoracic (TA, \(n=8\) ) and abdominal (AA, \(n=7\) ) aorta, common carotid (CCA, \(n=21\) ), subclavian (SA, \(n=12\) ), renal (RA, \(n=13\) ) and common iliac (CIA, \(n=16\) ) arteries from 11 deceased subjects ( \(54\pm 21\)  years old). Histological evaluation determined the structure of each specimen. Experimental data were used to determine constitutive parameters for a structurally motivated nonlinear anisotropic constitutive model. All arteries demonstrated appreciable anisotropy and large nonlinear deformations. Most CCA, SA, TA, AA and CIA specimens were stiffer longitudinally, while most RAs were stiffer circumferentially. A switch in anisotropy was occasionally demonstrated for all arteries. The CCA was the most compliant, least anisotropic and least frequently diseased of all arteries, while the CIA and AA were the stiffest and the most diseased. The severity of atherosclerosis correlated with age, but was not affected by laterality. Elastin fibers in the aorta, SA and CCA were uniformly and mostly circumferentially distributed throughout the media, while in the RA and CIA, elastin was primarily axially aligned and concentrated in the external elastic lamina. Constitutive modeling provided good fits to the experimental data for most arteries. Biomechanical and architectural features of major arteries differ depending on location and functional environment. A better understanding of localized arterial mechanical properties may support the development of site-specific treatment modalities for arterial disease.     

Biaxial anisotropy of dog carotid artery: estimation of circumferential elastic modulus

Experiments were performed to study biaxial anisotropy in relaxed canine carotid arteries subjected to a wide range of longitudinal lengths and transmural pressures. In order to encompass the range of vessel lengths which occurs in situ, 120 carotid arteries were studied at 20%, 40%, 60% and 80% extension relative to retracted vessel length. These studies were performed in systematically randomized order. At each length, the vessels were pressurized in steps up to 200 mmHg transmural pressure, or until the traction force fell to zero. Results showed that longitudinal extension markedly increased the longitudinal elastic modulus, but had only a slight effect on the circumferential modulus. At physiological pressures, the vessels were nearly isotropic at about 70% longitudinal extension, but were anisotropic at shorter lengths. Estimates of the anisotropic circumferential modulus by a number of simplified methods revealed that use of a 'pressure-elastic modulus' (Ep) underestimated the anisotropic modulus by 80%, but was extremely consistent. Therefore when suitably corrected, Ep could be used to reliably estimate the anisotropic modulus of carotid arteries over a wide range of pressures and vessel lengths.     

Diameter-dependent axial prestretch of porcine coronary arteries and veins

The pressure-diameter relation (PDR) and the wall strain of coronary blood vessels have important implications for coronary blood flow and arthrosclerosis, respectively. Previous studies have shown that these mechanical quantities are significantly affected by the axial stretch of the vessels. The objective of this study was to measure the physiological axial stretch in the coronary vasculature; i.e., from left anterior descending (LAD) artery tree to coronary sinus vein and to determine its effect on the PDR and hence wall stiffness. Silicone elastomer was perfused through the LAD artery and coronary sinus trees to cast the vessels at the physiologic pressure. The results show that the physiological axial stretch exists for orders 4 to 11 (> 24 μm in diameter) arteries and orders -4 to -12 (>38 μm in diameter) veins but vanishes for the smaller vessels. Statistically, the axial stretch is higher for larger vessels and is higher for arteries than veins. The axial stretch λ(z) shows a linear variation with the order number (n) as: λ(z) = 0.062n + 0.75 (R(2) = 0.99) for artery and λ(z) = -0.029n + 0.89 (R(2) = 0.99) for vein. The mechanical analysis shows that the axial stretch significantly affects the PDR of the larger vessels. The circumferential stretch/strain was found to be significantly higher for the epicardial arteries (orders 9-11), which are free of myocardium constraint, than the intramyocardial arteries (orders 4-8). These findings have fundamental implications for coronary blood vessel mechanics.     

Origin of axial prestretch and residual stress in arteries

The structural protein elastin endows large arteries with unique biological functionality and mechanical integrity, hence its disorganization, fragmentation, or degradation can have important consequences on the progression and treatment of vascular diseases. There is, therefore, a need in arterial mechanics to move from materially uniform, phenomenological, constitutive relations for the wall to those that account for separate contributions of the primary structural constituents: elastin, fibrillar collagens, smooth muscle, and amorphous matrix. In this paper, we employ a recently proposed constrained mixture model of the arterial wall and show that prestretched elastin contributes significantly to both the retraction of arteries that is observed upon transection and the opening angle that follows the introduction of a radial cut in an unloaded segment. We also show that the transmural distributions of elastin and collagen, compressive stiffness of collagen, and smooth muscle tone play complementary roles. Axial prestresses and residual stresses in arteries contribute to the homeostatic state of stress in vivo as well as adaptations to perturbed loads, disease, or injury. Understanding better the development of and changes in wall stress due to individual extracellular matrix constituents thus promises to provide considerable clinically important insight into arterial health and disease.     

Mechanical identification of layer-specific properties of mouse carotid arteries using 3D-DIC and a hyperelastic anisotropic constitutive model

The role of mechanics is known to be of primary order in many arterial diseases; however, determining mechanical properties of arteries remains a challenge. This paper discusses the identifiability of the passive mechanical properties of a mouse carotid artery, taking into account the orientation of collagen fibres in the medial and adventitial layers. On the basis of 3D digital image correlation measurements of the surface strain during an inflation/extension test, an inverse identification method is set up. It involves a 3D finite element mechanical model of the mechanical test and an optimisation algorithm. A two-layer constitutive model derived from the Holzapfel model is used, with five and then seven parameters. The five-parameter model is successfully identified providing layer-specific fibre angles. The seven-parameter model is over parameterised, yet it is shown that additional data from a simple tension test make the identification of refined layer-specific data reliable.     

An experimental study on the ultimate strength of the adventitia and media of human atherosclerotic carotid arteries in circumferential and axial directions

Atherosclerotic plaque may rupture without warning causing heart attack or stroke. Knowledge of the ultimate strength of human atherosclerotic tissues is essential for understanding the rupture mechanism and predicting cardiovascular events. Despite its great importance, experimental data on ultimate strength of human atherosclerotic carotid artery remains very sparse. This study determined the uniaxial tensile strength of human carotid artery sections containing type II and III lesions (AHA classifications). Axial and circumferential oriented adventitia, media and intact specimens (total=73) were prepared from 6 arteries. The ultimate strength in uniaxial tension was taken as the peak stress recorded when the specimen showed the first evidence of failure and the extensibility was taken as the stretch ratio at failure. The mean adventitia strength values calculated using the first Piola–Kirchoff stress were 1996±867 and 1802±703 kPa in the axial and circumferential directions respectively, while the corresponding values for the media sections were 519±270 and 1230±533 kPa. The intact specimens showed ultimate strengths similar to media in circumferential direction but were twice as strong as the media in the axial direction. Results also indicated that adventitia, media and intact specimens exhibited similar extensibility at failure, in both the axial and circumferential directions (stretch ratio 1.50±0.22). These measurements of the material strength limits for human atherosclerotic carotid arteries could be useful in improving computational models that assess plaque vulnerability.     

Active and passive properties of carotid arteries]

A mathematical model of carotid arteries is constructed from the known experimental data. Passive properties of the vascular wall are characterized by an alternating module of elasticity, the active ones by the specific power of muscle contraction. Its maximum value (0,023 n/m-3) is shown to be reached with intravascular pressure 190 mm Hg. The dependence of inner radius on the power of muscle contraction is studied at different values of intravascular pressure. It is shown that theactive properties of carotid arteries are essentially determined by their passive properties and depend on the stretching of the vessel wall.     

Biomechanical properties of decellularized porcine common carotid arteries

To analyze the effects of decellularization on the biomechanical properties of porcine common carotid arteries, decellularization was performed by a detergent-enzymatic procedure that preserves extracellular matrix scaffold. Internal diameter, external diameter, and wall thickness were measured by optical microscopy on neighboring histological sections before and after decellularization. Rupture tests were conducted. Inner diameter and wall thickness were measured by echo tracking during pressure inflation from 10 to 145 mmHg. Distensibility and incremental elastic modulus were computed. At 10 mmHg, mean diameter of decellularized arteries was 5.38 mm, substantially higher than controls (4.1 mm), whereas decellularized and control arteries reached the same internal diameter (6.7 mm) at 145 mmHg. Wall thickness decreased 16% for decellularized and 32% for normal arteries after pressure was increased from 10 to 145 mmHg. Decellularized arteries withstood pressure >2,200 mmHg before rupture. At 145 mmHg, decellularization reduced compliance by 66% and increased incremental elastic modulus by 54%. Removal of cellular elements from media led to changes in arterial dimensions. Collagen fibers engaged more rapidly during inflation, yielding a stiffer vessel. Distensibility was therefore significantly lower (by a factor of 3) in decellularized than in normal vessels: reduced in the physiological range of pressures. In conclusion, decellularization yields vessels that can withstand high inflation pressures with, however, markedly different geometrical and biomechanical properties. This may mean that the potential use of a decellularized artery as a scaffold for the creation of xenografts may be compromised because of geometrical and compliance mismatch.