Arteries are rarely loaded in only one direction. As blood pressure changes through the cardiac cycle, the arterial wall experiences a combination of circumferential and longitudinal loading, with collagen, elastin, smooth muscle cells, and other components of the extracellular matrix contributing differently as the tissue deforms.
That makes measuring “arterial stiffness” less straightforward than it might first appear.
A recent study published in Extreme Mechanics Letters looked at this problem in the descending thoracic aorta after intermittent hypobaric hypoxia (IHH). The researchers combined aortic biaxial testing, stress relaxation, digital image correlation, and histology to look for changes that might not be apparent from a single stiffness measurement. A CellScale BioTester 5000 was used for the mechanical testing.
Most of the elastic response remained surprisingly similar between the groups. There were, however, differences elsewhere. Chronic exposure was associated with lower longitudinal stiffness at high stretch and reduced cell nuclei density, while several measures of viscoelastic behavior shifted without reaching statistical significance.
It is a useful example of why vascular biomechanics research often involves looking beyond one modulus or one loading direction.
Why Use Aortic Biaxial Testing?
An artery is mechanically anisotropic. Its response depends on the direction in which it is loaded, and that response becomes increasingly nonlinear as the tissue stretches.
This matters when choosing how to test it.
A conventional uniaxial tensile test stretches a specimen along one axis. That can still provide useful information, but the authors point out that deformation in a single preferred direction may also cause fiber realignment that does not fully represent the tissue’s in situ architecture. With biaxial testing, loads can instead be applied along two axes at the same time.
For an aortic specimen, those axes can correspond to the circumferential and longitudinal directions of the vessel.
The distinction is particularly relevant when researchers are interested in aortic anisotropy or want to compare circumferential and longitudinal mechanical properties. Previous research has used similar approaches to investigate how axial pre-stretch affects carotid artery biaxial testing, and BioTester systems have been used more broadly in vascular biomechanics research, at the University of Nebraska, for example.
In this study, the question was slightly different. The researchers wanted to know whether repeated exposure to hypobaric hypoxia altered the passive biaxial mechanics of the aortic wall.
Why Use Aortic Biaxial Testing the Aorta After Intermittent Hypobaric HypoxiaTesting?
Intermittent hypobaric hypoxia occurs when periods of reduced atmospheric oxygen alternate with periods at normal atmospheric pressure. The authors note that this type of exposure is relevant to people who repeatedly travel between high-altitude occupational environments and lower elevations.
They studied adult Wistar rats divided into three groups, with six animals in each.
The control animals remained under normobaric conditions. An acute IHH group experienced four cycles during the final month of life, while a chronic IHH group experienced ten cycles over the final three months.
Each cycle consisted of four days under hypobaric hypoxia at approximately 428 torr, corresponding to an altitude of roughly 4,600 m, followed by four days under normobaric normoxia. All animals were studied at six months of age.
After the descending thoracic aorta was removed, the researchers did something useful from a mechanical testing standpoint: they did not rely on a single type of measurement.
They looked at the predominantly elastic response with aortic biaxial testing, then examined time-dependent behavior using stress relaxation. Adjacent tissue was also analyzed histologically.
That combination allowed changes in the mechanical response to be considered alongside changes in the tissue itself.
How the Researchers Performed Aortic Biaxial Testing
The descending thoracic aorta was opened longitudinally and cut into approximately 6 × 6 mm square specimens. The circumferential and longitudinal directions were identified so that each could be tracked during testing.
The luminal surface was given a fine black speckle pattern for digital image correlation, or DIC. The specimens were then mounted in a BioTester 5000 using BioRakes along their four edges.
The tissue was not tested dry. Throughout the experiment, it remained immersed in calcium-free Krebs-Ringer solution at 37°C. Maintaining soft biological samples in fluid and near physiological temperature can be an important part of hydrated mechanical testing, particularly when the goal is to characterize tissue under conditions closer to its normal environment.
Equal actuator displacements were applied along the two axes while forces were measured in the circumferential and longitudinal directions using 2.5 N load cells. For the monotonic tests, displacement increased at 1.5 mm/min.
The resulting stress-stretch curves were nonlinear, which is typical of many collagenous soft tissues. Rather than describing those curves with a single stiffness value, the researchers separated them into low- and high-stretch behavior.
Measuring Low-Stretch and High-Stretch Aortic Stiffness
The initial slope, E1, described stiffness in the lower-stretch region. E2 described the slope at higher stretches. The transition between these regions was characterized using a knee stretch, λc, and corresponding knee stress, Sc.
That distinction becomes important later in the study because the mechanical changes associated with chronic hypoxia did not appear equally across the entire stress-stretch curve.
DIC Adds Local Strain Measurement to Aortic Biaxial Testing
There is another detail in the experiment worth looking at.
The BioTester actuators provide a measure of how far the specimen is being displaced globally. But displacement at the grips and deformation in the middle of a soft tissue specimen are not necessarily identical.
The researchers therefore used images from the camera integrated with the BioTester and analyzed the speckled tissue surface using DaVis software. A central region of interest covering approximately 35% of a reference area was selected for DIC analysis. Choosing the central region helped reduce the influence of the rake boundaries on the measured deformation.
The resulting circumferential and longitudinal stretch fields were nearly homogeneous within this region, while shear remained close to zero. Differences between DIC-derived strain and actuator-derived strain stayed below 10% over the progression of the test.
For soft tissue testing, that comparison is useful. It provides a check on what is happening locally in the tissue rather than assuming actuator motion translates directly into specimen strain.
What Did Aortic Biaxial Testing Show?
At first glance, perhaps less changed than might be expected.
The stress-stretch curves remained nonlinear in both directions, and most of the elastic parameters showed no statistically significant differences among the control, acute IHH, and chronic IHH groups.
Circumferentially, the researchers did not detect significant differences between groups in the elastic parameters they evaluated. The low-stretch longitudinal stiffness was also relatively similar.
One result did stand out.
The high-stretch longitudinal stiffness, E2, differed significantly between groups. Chronic IHH animals had a lower longitudinal E2 than controls. The overall group comparison gave p = 0.03, with the chronic IHH versus control comparison reaching p = 0.04.
The difference was not observed between the control and acute IHH groups, and the knee parameters remained similar.
So it would be misleading to describe the chronic hypoxia group simply as having a “softer aorta.” What changed was more specific: the longitudinal high-stretch portion of the mechanical response was reduced, while most of the passive biaxial elastic behavior remained relatively stable.
The tissue also retained its directional mechanical behavior rather than losing its anisotropy.
This is one reason aortic biaxial testing can provide more information than reducing the entire response to a single stiffness number.
Stress Relaxation Looks at a Different Part of Aortic Mechanics
Elastic stiffness was only one part of the experiment.
Arterial tissue is also viscoelastic, meaning its response depends partly on time. If the tissue is stretched and then held at a fixed deformation, the measured stress does not simply remain at its initial value. It gradually falls.
The researchers investigated this using a biaxial stress relaxation protocol.
Before the relaxation test, each specimen went through ten loading and unloading cycles to precondition the tissue. It was then stretched at a nominal strain rate of 15%/s to a target stretch of λ = 1.60 and held at that deformation for 400 seconds.
The stress dropped rapidly at first and then more slowly as the hold continued.
To describe that behavior, the researchers calculated the degree of stress decay, a characteristic relaxation time, peak stress at the start of relaxation, and accumulated energy dissipation. In other words, they were looking at how much stress was lost, how quickly it was lost, and how much mechanical energy was dissipated during the process.
This type of measurement is part of the broader problem of viscoelastic testing, where the same material can behave differently depending on loading history and time. Other approaches to characterizing this behavior have also been explored in CellScale-related research, including work on viscoelasticity testing using the sigma-dot method.
Chronic Hypoxia Showed Trends Toward Greater Stress Relaxation
The stress relaxation results were less clear-cut than the longitudinal E2 result.
In the circumferential direction, the mean effective viscoelastic fraction increased progressively from the control group to acute IHH and then chronic IHH. Accumulated energy dissipation followed a similar pattern.
But neither difference reached statistical significance.
The longitudinal measurements likewise did not show significant differences in stress decay, peak stress, or characteristic relaxation time.
That distinction matters. The data suggest a possible shift toward greater relaxation and energy dissipation with chronic exposure, particularly circumferentially, but this study cannot establish those changes as group differences.
The authors discuss them as trends rather than confirmed effects.
Still, the relaxation measurements add something that the monotonic aortic biaxial testing alone would not show. Two tissues can have broadly similar quasi-static stress-stretch responses while behaving somewhat differently when deformation is maintained over time.
Looking at the Aortic Wall Itself
An adjacent portion of each descending thoracic aorta was used for histology.
The researchers quantified cell nuclei density, elastic fiber area, and collagen area. They also measured intima-media thickness, media-to-lumen ratio, and cross-sectional area. H&E staining was used for nuclei, Orcein for elastic fibers, and Picrosirius Red for collagen.
Here, one statistically significant difference emerged.
Nuclei density decreased in the chronic IHH group compared with controls. Mean collagen and elastin measurements also varied between groups, but those differences did not reach statistical significance.
The authors considered these structural measurements alongside the mechanical data. High-stretch stiffness is associated with collagen recruitment, so the reduced longitudinal E2 may reflect changes in how collagen fibers contribute at larger deformations. That does not necessarily mean a simple change in collagen quantity. Fiber orientation, waviness, recruitment, and interactions between extracellular matrix components can all affect the mechanical response.
There is also an important limitation here: the histological analysis did not provide corresponding longitudinal structural data that could directly explain the directional mechanical result. The authors note this limitation themselves.
Aortic Biaxial Testing Shows Why "Stiffness" Is Not Always One Number
One thing that stands out in this study is how easy it would have been to tell a different story with fewer measurements.
Looking only at much of the quasi-static elastic data might suggest that chronic intermittent hypobaric hypoxia had little mechanical effect on the descending thoracic aorta.
Looking only at longitudinal high-stretch stiffness would suggest a measurable reduction.
Looking at the stress relaxation data adds possible changes in time-dependent behavior, although the group differences were not statistically significant. Histology adds another layer again, with reduced nuclei density despite relatively modest changes in the measured passive elastic response.
None of those observations cancels the others.
Instead, the aortic wall appears to have undergone some structural remodeling while preserving much of its passive biaxial mechanical behavior under the conditions tested. The changes that were detected were relatively specific rather than a broad shift in arterial stiffness.
That is a useful consideration beyond this particular hypoxia model. Cardiovascular soft tissues such as arteries, pericardium, and heart valve leaflets are direction-dependent and nonlinear. Researchers have similarly used biaxial testing to examine porcine pericardium for heart valve design and the mechanical properties of heart valve leaflets.
The measurement chosen can affect which part of that mechanical behavior becomes visible.
How the BioTester Was Used in This Study
The BioTester 5000 sat at the center of the mechanical portion of the experiment, but the way it was used is more interesting than the instrument name itself.
The researchers mounted approximately 6 × 6 mm aortic specimens along four edges using rakes. Two orthogonal axes corresponded to the circumferential and longitudinal directions of the artery. The system then applied displacement simultaneously along both axes while forces were measured independently in each direction.
The bath kept the samples submerged in Krebs-Ringer solution at 37°C throughout testing.
Images acquired with the BioTester’s integrated camera were used for the DIC analysis. Rather than using grip displacement alone to estimate deformation, the researchers measured circumferential and longitudinal stretches from the central region of the specimen.
The same mounted specimens could then be taken through different loading protocols. Slow planar biaxial loading was used to examine the predominantly elastic stress-stretch response, while the ramp-and-hold protocol provided the stress relaxation measurements.
In practice, that meant force, displacement, local deformation, loading direction, and time-dependent behavior could all be considered within the same mechanical testing framework.
Using the BioTester for Biaxial Soft Tissue Testing
The CellScale BioTester is used for planar biaxial mechanical testing of soft tissues and biomaterials. Four actuators apply loads along two perpendicular axes, allowing researchers to investigate materials whose mechanical response changes with direction.
That includes arterial and other vascular tissues, but also heart valves, pericardium, skin, fascia, membranes, and engineered soft tissues where a uniaxial test may not capture the full material response.
Specimens can be mounted with BioRakes, as they were in this study, and tested in a temperature-controlled fluid bath. Image-based measurements can be used to track deformation within a selected region of the specimen rather than relying only on actuator displacement.
Depending on the research question, the BioTester can be used for monotonic planar biaxial loading, cyclic testing, and time-dependent protocols such as stress relaxation. Its role is specifically tensile and planar biaxial mechanical testing rather than compression.
For arterial biomechanics, that makes it possible to examine circumferential and longitudinal behavior simultaneously. As the IHH study shows, the value of doing so is not necessarily that every experiment produces a large directional difference. Sometimes the more interesting observation is that most of the mechanical response remains stable while one portion of the loading curve, or another aspect of tissue behavior, begins to change.
Source Publication
Fabián Álvarez-Carrasco, Enzo Brito, Álvaro Navarrete, Cristian C. Pozo, Andrés Utrera, Simón De Pablo, Claudio García-Herrera, Carlos Godoy-Guzmán, Emilio A. Herrera, Passive biaxial mechanics and viscoelastic relaxation of the thoracic aorta in intermittent hypobaric hypoxia, Extreme Mechanics Letters, Volume 86, 2026, 102491, ISSN 2352-4316,
https://doi.org/10.1016/j.eml.2026.102491.
