J. Biomedical Science and Engineering, 2010, 3, 645-652 JBiSE
doi:10.4236/jbise.2010.37088 Published Online July 2010 (http://www.SciRP.org/journal/jbise/).
Published Online July 2010 in SciRes. http://www.scirp.org/journal/jbise
Effect of freezing on the passive mechanical properties of
arterial samples
Jorge O. Virues Delgadillo1, Sebastien Delorme2, Rouwayda El-Ayoubi2, Robert DiRaddo2, Savvas G.
1Department of Chemical & Biological Engineering, University of British Columbia, Vancouver, Canada;
2Industrial Materials Institute, National Research Council of Canada, Boucherville, Canada.
Email: hatzikir@interchange.ubc.ca; Sebastien.Delorme@imi.cnrc-nrc.gc.ca
Received 19 December 2009; revised 18 January 2010; accepted 27 January 2010.
Little mechanical data is available on human arteries
because of the difficulty of testing artery samples
often obtained from autopsy, while arteries are still
considered “fresh”. Various solutions mimicking the
physiological environment have been used to pre-
serve artery samples from harvesting to testing. Cry-
opreservation might provide a means to preserve the
mechanical properties of arteries for days or weeks
after harvesting. The objective of this study is to inves-
tigate the effect of several preservation methods, in-
cluding simplified cryopreservation methods, on the
passive mechanical properties of arteries. Eighteen
fresh cruciform samples were mechanically tested.
Samples were divided in three groups based on pres-
ervation medium and freezing method: isotonic saline
solution, Krebs-Henseleit buffer solution with dime-
thyl sulfoxide (DMSO), and dipped in liquid nitrogen.
In each group, half of the samples were stored at
-20 and the other half at -80. Two months later,
all the tissues were thawed at 4 and mechanical
tests were repeated. Preservation of arteries for two
months in Krebs solution with DMSO (at -20 or at
-80) or in isotonic saline solution at -20 were the
methods that least changed the mechanical properties
of the arteries.
Keywords: Thoracic Aorta; Mechanical Testing; Physio-
logical Solution; Cryopreservation; Cryoprotective Agent
The most common method for investigating the me-
chanical behavior of soft tissues consists of conducting
mechanical tests on animal tissue explants, i.e., har-
vested within a day after the death of the animal [1-3].
There is however few data on in vitro mechanical be-
havior of human arterial tissues, in part because of the
logistical difficulty of performing mechanical tests on
fresh human tissues. Some studies [4-12] have reported
testing human tissues several days after death, assuming
that mechaniccal properties of the tissues were preserved
by refrigerating and by using chemical solutions mim-
icking the physiological environment, such as saline,
Ringer’s, Tyrode’s, Hank’s, Krebs and variations based
on these solutions. Cryopreservation methods [13] de-
veloped to preserve artery samples for re-implantation
could also provide a means to preserve arteries for days
and weeks before mechanical testing.
Artery mechanical properties have been shown to de-
pend on the relative proportion and arrangement of the
arterial wall constituents such as collagen, elastin and
smooth muscle cells [14,15], as well as on the integrity
of the elastin and collagen fibers [16,17]. Smooth mus-
cle cells contribute to structural, mechanical and func-
tional changes in the arterial wall through several proc-
esses, including cell growth, elongation and reorganiza-
tion of cells, and alteration of extracellular matrix com-
position [18]. The viability of smooth muscle cells, as
well as the integrity of elastin and collagen fibers con-
tribute to the arterial wall mechanical behaviour [16,17].
Damage caused to smooth muscle cells by ice formation
and fragmentation of extracellular matrix fibers might
affect the mechanical properties of the artery. Cryopro-
tective agents (CPA), such as dimethyl sulfoxide
(DMSO), have been used in several studies to protect
the cells from cryoinjury [19-21]. These CPAs are typi-
cally added to the storing solution in order to reduce ice
formation in both intra- and extracellular space by pre-
venting water movement out of the tissue [21]. The ef-
fect of cryopreservation has been investigated with re-
spect to cell viability [22,23] and histological changes
[24-28], but little is known about the effect of cryopre-
servation on mechanical properties of the arterial wall.
It has been found [29,30] that major arteries can with-
stand freezing and thawing without subsequent rupture.
J. O. V. Delgadillo et al. / J. Biomedical Science and Engineering 3 (2010) 645-652
Copyright © 2010 SciRes. JBiSE
For instance, Pascual et al. [30] observed that slow
thawing minimizes fissure and crack propagation com-
pared to rapid thawing if the tissue was frozen at -196.
They also observed that slow and rapid thawing does not
significantly change the structure of the vessel frozen at
-80. Fresh and cryopreserved behaviour of arteries has
been investigated using inflation [31] and uniaxial tests
[32,33]. Blondel et al., [31] and Venkatasubramanian
et al., [33] observed significant stiffening of femoral
arteries cryopreserved at -80 and -150 respectively
compared to fresh arteries. Adham et al., [32] observed
no difference in high strain modulus of aortas preserved
at +4 for 1 month compared to cryopreservation with
DMSO at -135 for 4 months.
The goal of the present study was to evaluate the ef-
fect of several conservation methods on the passive bi-
axial mechanical properties of arteries, regardless of cell
viability, for the purpose of delayed mechanical testing.
The preservation methods investigated include freezing
for two months at either -20 or -80, in the presence
of isotonic saline solution or Krebs-Henseleit solution
with DMSO, or without immersing in solutions but
dipped in liquid nitrogen. Because some of these meth-
ods are simplified adaptations of cryopreservation tech-
niques, cell viability is not expected to be maintained in
the samples. This study describes the storing conditions
that best preserve the passive mechanical properties of
2.1. Experimental Setup
Eighteen thoracic aortas were harvested within the day
of death of pigs from a local slaughterhouse and cleaned
of remaining connective tissue. One cruciform-shaped
sample (55-by-55 mm) was cut out from each aorta for
equibiaxial testing (Figure 1(a)). The average thickness
of all tested specimens, measured with a vernier caliper,
was 2.3 ± 0.2 mm. During transport and preparation,
samples were stored in isotonic saline solution at 4
for up to 8 hours prior to testing.
Cruciform samples were mechanically tested on a
planar biaxial test bench (ElectroForce® LM1, Bose
Corporation, Minnetonka, MN), shown in Figure 1(b)
and capable of applying a peak force of 200 N over a
displacement range of 12 mm per actuator. Samples
were mounted in horizontal configuration inside a saline
bath heated at body temperature (37). Grip clamps
were used to attach the four tabs of the cruciform sample
to the arms extending from the actuators over the top of
the bath, accordingly to a previously described method
[34]. As well as being technically less difficult to set-up
Figure 1. Sample dimension in millimetres for biaxial testing
and (b) biaxial test bench used for cruciform sample clamped
with grips.
than using square samples attached with hooks, and elimi-
nating the need for videoextensometry, the cruciform sam-
ple method offers the advantage of stretching the samples
to higher stretch ratios before failure than square samples
with hooks [34]. The experimental data obtained from
cruciform samples can be used in further studies (e.g.
using inverse modeling) to obtain for example, which
material parameters of the constitutive equation selected
are most sensitive to freezing effects.
Equibiaxial testing was done by applying a 12 mm
displacement (which corresponds to an average nominal
stretch ratio of 1.55) on each one of the four grips, cre-
ating a non uniform strain distribution in the sample. The
nominal stretch ratio was calculated using the distance
between facing grips. The 1.55 stretch ratio was selected
based on earlier experiments [34] because it allows cap-
turing the nonlinear part of the stress-stretch curve, while
avoiding rupture of the samples. Equibiaxial testing cre-
ated a non uniform strain distribution in the cruciform
sample. Triangular displacement wave forms were ap-
plied at a deformation rate of 110%/s, which corre-
sponds to a frequency of 1.0 Hz (60 cycles per secod).
Displacements were applied for 20 cycles. The first 10
J. O. V. Delgadillo et al. / J. Biomedical Science and Engineering 3 (2010) 645-652
Copyright © 2010 SciRes. JBiSE
cycles were used for pre-conditioning. The force-stretch
data was averaged over the last 10 cycles.
After the 18 fresh samples had been tested biaxially,
they were randomized into three groups for storing:
Group I, samples were put in a polypropylene tube filled
with isotonic saline solution; Group II, samples were put
in a polypropylene tube filled with Krebs-Henseleit so-
lution, supplemented by 1.8 M DMSO; and group III,
samples were dipped in liquid nitrogen and then put in a
polypropylene tube without any solution. For each group,
half of the samples were stored at -20 and the other
half at -80. Samples in group II were stored at 4 for
20 minutes prior to freezing as proposed by Oliver [35].
Three samples were stored under each of the six differ-
ent storage conditions, as shown in Table 1.
After two months, the samples were thawed at 4
for 24 hours and rinsed in isotonic saline solution at
body temperature for one minute. The same mechanical
testing procedure and conditions were then repeated.
2.2. Statistical Analysis
Medians and ranges (percentile between 75%-25%) were
calculated for circumferential and axial force-stretch
curves. Thawed/fresh force ratio (max max
Thawed Fresh
FF) at
1.55 stretch ratio were compared between all groups
using the ANOVA Krustal-Wallis statistic test for in-
dependent variables with a 0.05 level of significance
(0.05p). The p value quantifies the probability of
concluding that the storing condition used has an effect
on the mechanical properties when in reality it did not.
Krustal-Wallis test (non-parametric) was selected because
the small sample size does not allow verifying the hypo-
thesis of normal distribution, which is required to perform
a Student’s t-test. A statistical power analysis was also
used to know the probability of falsely rejecting the null
hypothesis using a threshold of 80%.
3.1. Experimental Results
The medians of the force-stretch curves in the circum-
ferential and axial directions for fresh specimens (18n
are plotted in Figure 2. The difference in mechanical
behavior below a stretch ratio of 1.5 is small.
The medians of the force-stretch curves for the sam-
ples in group I, II and III are presented in Figures 3, 4
and 5 respectively. In groups I, II and III, preserved
samples appeared to be stiffer than fresh samples in the
circumferential direction, but these differences were not
statistically significant. Preserved sample forces in the
axial direction appeared to be similar to fresh samples.
Figures 6(a) and 6(b) shows the Thawed/fresh ratios
(max max
Thawed Fresh
FF) of the circumferential forces measured
at the maximum stretch ratio (1.55
) for all the
Table 1. Number of samples for each storage condition.
Thawed Tissue
Storage Temperature
-20 -80
I 3 3
II 3 3
III 3 3
Figure 2. Medians of the force-stretch behavior of fresh cruci-
form samples tested at 110%/s (18n). Open triangles and
closed circles represent axial and circumferential mean forces,
respectively. The percentile of the data points is also shown.
samples stored at -20 and -80, respectively. The load-
ing forces in the circumferential direction of all thawed
samples (Groups, I, II and III) were almost two times
higher than the loading forces of fresh samples (median
of the ratio maxmax 2.0
Thawed Fresh
FF). On the other hand,
difference in axial forces between fresh and thawed tis-
sue was lower than 40% for all groups (Figures 6(c) and
6(d) shows axial results at -20 and -80 °C, respectively).
The medians, percentiles (75%-25%) and the ANOVA
Krustal-Wallis test p values are also included in Fig-
ure 6.
This study investigated the influence of freezing in dif-
ferent solutions on the mechanical properties of arterial
wall. Results were reported in terms of force vs. stretch
ratio rather than stress vs. stretch ratio. This is due to the
fact that stress and strain distributions in a cruciform
sample subjected to biaxial extension experiment are not
homogeneous, even when an equibiaxial stretch is ap-
plied. It was shown in [34] that the highest and lowest
stresses can be found near the curved boundaries and
near the center of the sample, respectively.
J. O. V. Delgadillo et al. / J. Biomedical Science and Engineering 3 (2010) 645-652
Copyright © 2010 SciRes. JBiSE
Figure 3. Medians of cruciform samples tested at a deforma-
tion rate of 110%/s, and stored in saline solution (3n): Arte-
rial wall behavior in (a) circumferential and (b) axial direc-
The differences observed in the mechanical behavior
(i.e. medians of force data points) of thawed samples were
not significant (0.64)p, independently of the storing
medium used (saline, Krebs with DMSO and dipping in
liquid nitrogen). In order to confirm that indeed there is no
differences in fresh and thawed behavior; a statistical
power analysis (http://www.dssresearch.com/toolkit) was
performed using the force data points obtained at 1.55
stretch ratio for each group between the fresh and the
thawed specimens. The probability of rejecting a false
null hypothesis (i.e. the storing condition used has no
effect on the mechanical properties when in reality it
might have an effect) and thus minimizing the occur-
rence of a β error (which occurs if it is concluded that
Figure 4. Medians of cruciform samples tested at a deforma-
tion rate of 110 %/s, and stored in Krebs solution with di-
methyl sulfoxide, DMSO (3n
): Arterial wall behavior in (a)
circumferential and (b) axial directions.
there is no difference in fresh and thawed specimen
behavior when in reality there might be a difference)
increases as the statistical power increases. Statistical
powers between 13% and 93% were obtained in circum-
ferential direction. In axial direction, the power was
lower than 47% and higher than 5%. The statistical po-
wer obtained for mostly all groups was not high enough
to verify how significant the difference was between
thawed and fresh tissue. In addition, the software used to
obtain the power assumes that the data is parametric.
Non-parametric analysis will likely estimate a slightly
less power. Future studies with a higher sample size may
further support the results obtained in this work. The
observed lack of significant differences in this study was
J. O. V. Delgadillo et al. / J. Biomedical Science and Engineering 3 (2010) 645-652
Copyright © 2010 SciRes. JBiSE
Figure 5. Medians of cruciform samples tested at a deforma-
tion rate of 110 %/s, dipped in liquid nitrogen and stored in air
(3n): Arterial wall behavior in (a) circumferential and (b)
axial directions.
likely due to the small number of samples tested per
storing group (3n). However, these results are con-
sistent with findings reported by Adham et al. [32] and
by Venkatasubramanian et al. [33], who reported no sig-
nificant difference between mechanical behavior of fresh
artery samples and of samples cryopreserved with a
cryoprotective agent such as DMSO, with up to 13 sam-
ples in each group.
The thawed samples appeared to be stiffer than the
fresh samples at high stretch ratios. The differences in
the load force-stretch curves might be the result of a
modification in structure due to crosslinking or a change
in fiber alignment. Elder et al. [36] stated that the in-
creased tissue stiffness after cryopreservation may be
related to the thermal change (i.e. drop of temperature)
that occurs during preservation, catalyzing the thermal
crosslinking of collagen fibers within the extracellular
matrix. Collagen fibers are mainly oriented in the cir-
cumferential direction [37], thus the collagen matrix
would develop stiffer interconnections in the circumfer-
ential than in axial direction during freezing. After thaw-
ing, the collagen matrix might have been reinforced in
the circumferential direction, which is reflected in the
stiffer response observed.
In this study the effect of freezing on cell injury and
functionality was not investigated. However previous
studies [38-40] have shown that contractile function of
both endothelial and smooth muscle cells were preserved
in arteries of a variety of species following freezing and
thawing in DMSO solutions. DMSO reduces the mass
transport of water and solutes throughout cell membrane
while freezing. When water moves out of the cytoplasm,
DMSO dissolves the suspended electrolytes, reducing
the harmful effects of high solute concentration. DMSO
interacts and partially replaces water molecules in such a
way that the freezing point in the solution is lowered
during cooling and the intracellular ice is reduced. Ice
crystal formation and growth is prevented when the cry-
oprotectant-water mixture solidifies in a glass-like struc-
ture; and thus, preserving cell viability and mechanical
properties. Song et al. [38] found that the maximum
concentration needed to prevent damage in the tissue is
15% (wt/wt). In the present study, the DMSO concentra-
tion was 1.8 M, i.e. < 12% wt/wt. This concentration is
expected to maintain cell viability within the tissue.
Evidence has shown that after thawing, both biochemical
and functional activities of arterial tissue cryopreserved
at low temperatures in Krebs solution containing 1.8 M
of DMSO were comparable to fresh tissues [41].
Freezing and storing arteries in saline solution do not
conserve the mechanical properties. As described by
Lovelock [42], during the freezing process, the volume
of liquid water in the cytoplasm decreases due to ex-
tracellular ice formation, leading to cellular dehydration.
If the dehydration is too severe, the high electrolyte
concentration inside the cytoplasm could result in cell
death [43]. DMSO reduces the mass transport of water
and solutes through the cell membrane while freezing.
Ice crystal formation and growth is prevented when the
cryoprotectant-water mixture solidifies in a glass-like
structure, thus preserving cell viability and mechanical
properties. The concentration of DMSO used in this
study is expected to maintain cell viability within the
tissue, albeit this study was not focused on freezing ef-
fects on cell viability. The better results obtained with
DMSO suggest that cell death might play a role in
changes of tissue stiffness, although this study could not
unveil the exact mechanism involved. Also, during thaw-
J. O. V. Delgadillo et al. / J. Biomedical Science and Engineering 3 (2010) 645-652
Copyright © 2010 SciRes. JBiSE
(a) (b)
(c) (d)
Figure 6. Comparison of Thawed/Fresh force ratios per storage group at the maximum stretch ratio applied (1.55
): (a, b)
circumferential and (c, d) axial direction max max
Thawed Fresh
FF ratios of samples stored at -20 and -80.
ing, the deformation of the cytoplasm due to volume in-
crease could have permanently modified the orientation of
the intracellular fibers, and as a result permanently chang-
ed the mechanical behaviour of the cells. Therefore our
results suggest that, as concluded by Venkatasubramanian
et al. [33], the changes in mechanical properties could be
due to cell loss, to damage to the extracellular matrix or
to a combination of both. Stachecki et al. [44] suggested
that the reduction or removal of sodium from the storing
solution is of primary importance to freeze cells effi-
ciently, and proved that it is possible to replace sodium
with a bigger molecular size ion, which encounters more
obstacles to penetrate the cell membrane.
Dipping the arterial sample in liquid nitrogen could
not preserve the mechanical properties of the specimens
evaluated in this study. When exposing the specimens to
a very rapidly decreasing temperature, ice crystal forma-
tion might have occurred faster than cell dehydratation,
resulting in intracellular ice formation [45] and cell death.
The differences observed between fresh and thawed -
samples suggest that the formation of intracellular ice
crystals might have and effect on tissue stiffness. Vitri-
fication is an alternative to obtain an amorphous glassy
state matrix and thus minimizing ice nucleation and
growth. However, it requires high concentrations of cry-
oprotective agents [46,47]. For example, Song et al. [48]
were able to avoid crystallization by using a combination
of CPAs (DMSO, formamide and 1,2-propanediol) and
rapidly cooling the vessel to -196. Jiménez Rios and
Rabin [49] have improved cell viability by using a spe-
cific freezing rate, pressurized liquid nitrogen, and spe-
cific type and concentration of CPA. Non-toxic cryopro-
J. O. V. Delgadillo et al. / J. Biomedical Science and Engineering 3 (2010) 645-652
Copyright © 2010 SciRes. JBiSE
tectants, like glyerol [50] or fetal bovine serum [51]
could also be used.
In the present study, we have found that the mechanical
properties of arteries were not significantly changed af-
ter preserving arteries for two months in Krebs solution
with DMSO (at -20 or at -80) or in isotonic saline
solution at -20. However, the preservation approach
taken here needs to be improved in order to maintain cell
viability and as a result determine the best conditions to
preserve the mechanical properties of the arterial wall.
Selection of the optimal preservation method might be
obtained by performing a study where the variables to be
adjusted are slow cooling rate and step-wise DMSO
loading, and therefore minimize changes to the arterial
microstructure by the reduction of thermal stresses within
the frozen tissue. Further studies are required to clarify
the impact of cryopreservation on extracellular matrix
architecture to help tailor an optimized approach in order
to preserve the mechanical properties of arteries.
This research was possible thanks to the PhD scholarship given by the
Mexican Council of Science and Technology (Consejo Nacional de
Ciencia y Tecnología), CONACYT. The authors would also like to
thank Marc-Andre Rainville for his guidance and assistance in sample
preparation and mechanical testing.
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