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Introduction
Understanding the biomechanical behavior of the
connective tissue is crucial in several fields of medi-
cine, such as Orthopedics. Intensive research is car-
ried out through experimental protocols involving
laboratory animals and in particular rodents. The
study of tendon biomechanics in small-size labora-
tory animals is characterized as a highly demanding
procedure exhibiting considerable technical dif-
ficulties. There are two main technical challenges
that have to be met. The first one is the problem of
rigidly gripping the tendon without damaging it or
altering its mechanical behaviour. Without this basic
requirement it is impossible to conduct an accurate
and reliable experiment. The second challenge is the
reduction of the forces and the displacements im-
posed to stresses and strains, respectively. The ad-
vantage of this reduction is that the latter quantities
do not depend on the morphometry of the specific
specimens but they rather characterize the material
of the tendon itself, eliminating thus the interspeci-
men variability of the results.
For the problem of rigidly gripping a tendon at high
loads without damaging it, several methodologies
have been proposed over the last years. The most
widely used ones are: a) conventionally gripping
using compression and b) clamping the tendon us-
ing rapid freezing. The compression of the specimen
(using for example serrated grips) alters the biome-
chanical characteristics of the examined tissue (Rie-
mersa & Schamhardt, 1982). In addition slippage is
not always avoided even in cases of relatively long
tendons (Cheung & Zhang, 2006). Especially in the
case of very short tendons (like the Achilles tendon
of rats) the above mentioned difficulties are intensi-
fied rendering the method practically inapplicable.
On the other hand the rapid freezing technique re-
sults in a more efficient gripping of the tissue with
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
Published in the Scandinavian Journal of Laboratory Animal Science - an international journal of laboratory animal science
The Fracture Stress of Rat Achilles Tendons
by P. E. Chatzistergos1, S. I. Tsitsilonis2, A. S. Mitousoudis1, D. N. Perrea2, A. B. Zoubos3 & S. K. Kourkoulis1,*
1Unit of Biomechanics, Department of Mechanics, School of Applied Mathematical and Physical Sciences,
National Technical University of Athens, Zografou Campus, Greece
2Department of Experimental Surgery and Surgical Research «N. S. Christeas», Medical School,
University of Athens, Greece
3Department of Orthopaedic Surgery, University of Athens, School of Medicine, Athens, Greece
Summary
For the determination of the fracture stress of soft tissues both the fracture force and the cross sectional
area are required. For short tissues these prerequisites are difficult experimental tasks. The determination
of the fracture force necessitates proper gripping without damaging the tissues or altering their properties.
In order to meet this challenge the rapid-freezing technique was employed, modified to ensure that the
tendon was not frozen. On the other hand an accurate value of the cross sectional area of short soft tissues
is difficult to be obtained using conventional techniques. In this context a novel procedure is proposed here
based on the histologically-measured cross-sectional area of the dehydrated tendon after the biomechanical
testing. Combination of these solutions permitted the performance of tension tests for rat Achilles tendons
and calculation of their fracture stress. The values of the Achilles tendon failure stress, as estimated above,
exhibited considerably lower scattering compared to those of the fracture forces.
149
*Correspondence: S. K. Kourkoulis
Unit of Biomechanics, Department of Mechanics, School
of Applied Mathematical and Physical Sciences, National
Technical University of Athens, Zografou Campus,
Theocaris building, 157-73 Attiki, Greece
Tel +30 210 7721263
Fax +30 210 7721302
E-mail stakkour@central.ntua.gr
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
150
reduced compression of the specimen (Riemersa &
Schamhardt, 1982; Sharkey et al., 1995; Wieloch
et al., 2004). This technique was introduced by
Riemersa & Schamhardt in 1982 and was further
developed by many others. According to them the
clamped part of the tendon and the metal clamp are
rapidly frozen using liquid CO2. Rapid freezing is
widely used today with relatively good results. The
greatest disadvantage of rapid freezing emanates
from the technical difficulty to accurately restrict
freezing in a pre-defined portion of the tissue. This
difficulty is even greater in the case of short tis-
sues such as the Achilles tendons of rats. Since the
freezing of the tissue causes significant alterations
to its mechanical properties, a failure to control the
freezing can undermine the reliability of the study.
In order to overcome the above mentioned difficul-
ty, an alternative clamping technique, using rapid
freezing, is described in the present paper for the
experimental study of the mechanical behaviour of
bone-Achilles tendon-muscle units of rats.
Concerning the problem of reducing the forces
measured to reliable stress estimations, the different
procedures found in the literature could be separat-
ed into three main categories according to the time
and frequency of the cross-sectional area measure-
ments. Some researchers measure the cross-section-
al area before the biomechanical test, either using
a calliper (Nakagaki et al., 2007; Teramoto & Luo,
2008) or other custom-made devices (Butler et al.,
1984; Loitz et al., 1989; Wu et al., 2004). Further-
more others take several real-time measurements
of the cross-sectional area of the tendon during the
loading using indenter probes connected to high
resolution displacement transducers (Derwin et al.,
1994; Huang et al., 2004; Soslowsky et al., 1994;
Soslowsky et al., 2000). Finally some researchers
estimate the cross-sectional area after the end of the
test, based on the percentage of collagen in the ten-
don (Oxlund et al., 1984). For the purposes of the
present study a novel post-fracture indirect method
for the estimation of the fracture stress of rat Achil-
les tendons was followed, according to which the
cross-sectional area of the dehydrated tendon is
identified as an “effective” cross-sectional area (at
least for tensile loads).
Materials and Methods
Animals
Male Wistar rats, aged 4 months, were studied, com-
ing from the same breeder (National Research Cent-
er of Natural Sciences “Dimokritos”). The animals
were supplied by van in filter boxes and quarantined
for 2 weeks in the Central Animal Laboratory of
the Department of Experimental Surgery and Sur-
gical Research of the University of Athens. The
experimental protocol was carried out according
to Greek legislation regarding ethical and experi-
mental procedures (Presidential Decree 160/1991,
in compliance to the EEC Directive 86/609, and
Law 2015/1992, and in conformance with the Eu-
ropean Convention “for the protection of vertebrate
animals used for experimental or other scientific
purposes, 123/1986”).
Husbandry
The animals were housed, in an open system, two per
cage, in polycarbonate cages of dimensions: 480 x
265 x 210 mm, floor area: 940 cm2 (2154F, Tecniplast,
Italy). Wooden, dust-free, litter was used for bed-
ding, with no pretreatment (Scobis-Uno, Italy). The
conditions in the animal house were 15 air changes/
hour, with regulated environmental temperature at
22±2 ºC, regulated relative humidity 55±10% and
artificial light/dark at 06.00/18.00, using fluorescent
lighting ca. 300 lux. All animals were acclimatized to
the laboratory conditions for one week period prior
to the experiment. The animals were fed ad libitum
a commercial pelleted food (510K, Greek Animal
Food Industry, Greece), with no pretreatment, the
nutrient contents of which are described in Table 1,
and had free access to mains water.
Experimental procedure
The experiments were carried out from November
11 to November 14, 2009 from 10.00 to 14.00, us-
ing 22 rats, with no time interval between sampling
and processing.
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
The animals were euthanized with an overdose of
ether and a skin incision at the site of gastrocnemius
muscle was elaborated. The hind limb was disartic-
ulated at the knee joint and the skin and fascia were
removed, in order to expose the muscle-tendon-
bone unit. The tibia was removed at the ankle, and
the forefoot was removed from the midfoot, thus
leaving the midfoot and the hindfoot attached to the
Achilles tendon. Special surgical care was taken in
order to avoid injury of the Achilles tendon, while
separating the soleus and flexor digitorum superfi-
cialis muscle-tendon units.
Experimental set-up
For the purpose of the present study a modified
cryo-jaw was used and was based on that proposed
by Wieloch et al. (2004). The clamping device con-
sists of two separate parts. The first is a pincers-
like clamp for bone fixation, while the second is
the modified cryo-jaw which comprises of four
parts (Fig.1). Parts A and C are identical and, when
combined with part B, they form a cavity inside
which the muscle of the bone-tendon-muscle unit
is placed. The specimen is placed in such way that
only the muscle is in contact with the surfaces of
the jaw. Part D is the cup for the refrigerant (Liquid
Nitrogen, L-N). The cryo-jaw is fixed at the inferior
site of the load frame and the L-N cup is covered
with an aluminium foil sheet to avoid freezing of the
tendon from the evaporated L-N.
In order to estimate the volume of L-N needed to
freeze entirely the muscle but not the tendon, a
number of preliminary tests were performed. The
muscle of the specimen was placed inside the cavity
of the cryo-jaw which was then fixed on the load
frame (MTS MiniBionix 858, MTS Systems Corp.,
Eden Prairie, MN, USA). The bone was clamped
with the pincers-like clamp (Fig. 2). A T-type ther-
mocouple probe was inserted inside the tendon tis-
sue at the transition area between the tendon and the
muscle giving real time temperature measurements.
Finally a volume of L-N was poured inside the cup,
the drop of the temperature of the tendon was re-
corded and axial displacement was imposed to the
pincers-like clamp at a rate of 1 mm/min. The axial
force exerted on the specimen was measured using
a 50 kgr Instron Tensile Load Cell (Instron, Canton,
MA) and the measurements were recorded at a rate
of 10Hz. These preliminary tests revealed that pour-
ing 75cm3 of L-N into the cryo-jaw was enough to
freeze most of the muscle to a satisfactory degree
in order to withstand loads until failure without any
noticeable slippage. At the same time the tendon re-
151
Table 1. The measured maximum forces, cross-sec-
tional areas and estimated failure stresses.
Number
of
specimen
Maxi-
mum
force
Cross-
sectional
area
Failure
stress
(N) (mm2) (MPa)
1 47.2 2.61 18.1
2 45.2 2.86 15.8
3 30.3 1.90 15.9
4 45.5 3.73 12.2
5 35.2 2.48 14.2
6 46.6 2.55 18.3
Mean
value 41.7 2.69 15.7
Standard
deviation 7.1 0.60 2.3
Relative
error (%) 17.1 22.35 14.8
Figure 1. The pincers-like clamp for bone fixation
(left) and the modified cryo-jaw (right).
152
mained unfrozen with a temperature drop of about
10 ºC. During the biomechanical tests, the room
temperature was kept constant at 25±2 ºC.
The aforementioned procedure, without the use of
thermocouple, was followed for the estimation of
the tensile strength of the Achilles tendon of Wistar
rats: Six bone-tendon-muscle units were harvested
from the right feet of six rats. The units were first
submitted to biomechanical testing according to
the previously described procedure. After the test-
ing they were submitted to histological analysis and
their cross sectional area was determined.
Histological Fixation
Achilles Tendons were fixed in 3% glutaraldehyde
in 0.1 M phosphate buffer (pH 7.2), post-fixed in
1% osmium tetroxide (OsO4), sequentially dehy-
drated in a series of upgrading concentrations of
ethanol followed by propylenoxide and embedded
in a Spurr resin mixture. Sections were stained with
toluidine blue for light microscopy (Fig.3).
Measurement of the “Effective” Cross-sectional
Area
After the fixation, cross-sections of the Achilles
tendons, 10μm thick, were obtained at the site of
the rupture provoked by the biomechanical test-
ing (Fig.3). Special care was taken to ensure that
the whole cross-sectional area of the tendon would
appear in each section and that the sections would
be absolutely transverse. From each Achilles ten-
don ten consecutive cross-sections were cut and
they were examined using light microscopy. Digital
photographs were obtained for each section at a 10-
fold magnification. The area of each one of the ten
sections was measured using MatLab (MathWorks,
Natick, MA, USA) and their mean value was as-
sessed for each specimen.
The measured cross-sectional area of each tendon
was used for the assessment of the maximal sus-
tained stress of the tendon, based on the fact that,
when the tendon is subjected to quasi-static ten-
sion, its mechanical behaviour is not affected by its
internal fluid pressure (Herzog, 2007). In addition
the tensile bearing capacity of the liquid phase of
the extracellular matrix of the tendon is obviously
negligible and therefore it can be excluded from the
stress assessment procedure. The cross-sectional
area of the dehydrated tendon corresponds to the
area of the solid components of the tendon, most of
which is collagen. Generally, the collagen content
is over 75% of the dry weight of a tendon and up to
99% in cases of extremity tendons (Nordin et al.,
2001). Besides collagen the area of solid compo-
nents consists also of ground substance and a small
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
Figure 2. The experimental set-up.
Figure 3. A characteristic cross-sectional section of
a rat Achilles tendon after the biomechanical test.
153
amount of elastin. Since these components are the
only ones considered capable of bearing loads their
cross-sectional area can be considered as an “effec-
tive” cross-sectional area of the tendon (Aeff). So the
tensile fracture strength, ft of the tendon is assessed
by the relation:
where Fmax is the maximum force recorded during
the tensile test.
Results
The temperature measurements in the preliminary
tests showed that the tendons were not frozen in
any way, as the lowest temperature recorded in the
musculo tendi no us junction was 15±2 ºC. This ex-
cludes the possibility that the biomechanical prop-
erties of the tendons were affected to such a degree
that would render the results unreliable.
The rapid freezing of the muscle alone provided the
necessary fixation force capable to sustain signifi-
cant tension loads up to the fracture of the tendon
without slippage and without affecting the mechani-
cal behaviour of the “gage-length” of the tendon. In
Fig.4 a characteristic load-displacement curve from
the present series of experiments is shown. The
three portions, typical for soft tissues under tension,
are clearly distinguished: An initial non-linear re-
gion (“toe” region) is followed by an almost linear
one which eventually leads to a sudden drop of the
load due to the failure of the tendon.
The average maximum tensile force (Fmax) was
measured equal to 41.7N with a standard deviation
of 7.1N, corresponding to a relative error equal to
17.1%. On the other hand the cross-sectional area
of the tendons (Aeff) after the biomechanical test
were found equal to 2.69±0.60mm2. Following the
procedure previously described, the average failure
stress was estimated to 15.7MPa with a standard
deviation of 2.3MPa and a relative error equal to
14.8%, almost 13.5% lower than the respective er-
ror of the fracture forces. The results for all speci-
mens are presented in detail in Table 1.
Discussion
Using small laboratory animals for the elabora-
tion of experimental research protocols is of great
value for the comprehension of the biomechanical
behaviour of soft tissues such as the tendons. The
fact that these studies cannot be easily performed
in vivo, renders the design of a reliable apparatus
for in vitro testing very important, provided that the
biomechanical characteristics of the examined tis-
sue are minimally affected.
It is well known that one of the most important
parameters that could significantly affect the bio-
mechanical properties of a soft biological tissue
during an in vitro test is the boundary conditions
imposed to the specimen. In particular, in the case
of the Achilles tendon, gripping is one of the most
challenging and important experimental parameters
mainly due to its extremely small size.
Usually gripping a specimen in a tensile test is
achieved by compressing it between the plates of a
clamping devise generating high frictional forces.
But in the case of tendons, which are soft, wet, col-
lagenous tissues, strong frictional forces are diffi-
cult to be developed because the friction coefficient
between the tissue and the material of the clamping
devise is very low. One way to increase frictional
forces is by increasing the compression force ex-
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
Figure 4. A characteristic Load-Displacement curve
from a tensile test of a Wistar rat Achilles tendon.
(1) The “toe region”, (2) “linear” region, (3) and (4)
“failure” regions.
max
=
teff
F
fA
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
154
erted on the specimen inside the clamping device.
However in this case higher compression of the ten-
don leads to dehydration and formation of a water
film between the surfaces of the tissue and the grip
leading in turn to a significant decrease of the coef-
ficient of friction. Additionally, high compression
force could damage the tissue and alter its mechani-
cal properties. Alternatively increased frictional
forces can be obtained by modifying the surfaces of
the clamping device in order to increase the friction
coefficient. Typical example is the study by Viidik,
where the metallic surfaces of the grips that were
in contact with the tissue were covered with differ-
ent layers of textiles and paper, but with rather poor
results (Viidik, 1966). Relatively good results can be
found in the literature for serrated jaw clamps. For
example in the study by Cheung et al. (2006) bovine
tendons were loaded up to 3000N before slippage
occurred. Even with significant high compression
of the tissue the jaws weren’t able to grip the tendon
until failure.
It is clear from the literature that increased compres-
sion force on the tendon is inevitable in clamping
devices using friction. Unfortunately compression
of the specimen can cause large deformation of the
clamped part, as well as changes in the initial length
of a number of fibres. These alterations can produce
an uneven distribution of the load among the fibres.
Additionally, since the friction between adjacent
fibres is lower than the respective one between su-
perficial fibres and the clamp, a relative slippage of
the inner fibres can occur without being noticed. In
this case the fibres that lay near the surfaces of the
tendon will carry higher loads than the inner ones
producing this way a misleading force vs. displace-
ment curve.
An alternative gripping technique is the one based
on rapid freezing. This technique was introduced by
Riemersa et al. (1982), who froze both the speci-
men and the clamping device with the use of liquid
CO2. They observed that rapid freezing resulted in a
strong gripping of the tendon to the jaws. However
this mainly occurs with much bigger tendons, com-
pared to that of rats (e.g. horse tendons). In the case
of small laboratory animal tendons this methodol-
ogy cannot offer sufficient grip of the tendon, as the
contact surface is limited.
Wieloch et al. (2004) proposed a modification of the
device of Riemersa et al. (1982). Their main idea
was to freeze the muscle instead of the tendon, in an
appropriately shaped cavity of the cryo-jaw, achiev-
ing adequate holding of muscle-tendon units. The
methodology described in the present paper is based
on the same principle, but also explores some im-
portant aspects that had not been studied previously.
The use of a thermocouple at the muscle-tendon
junction confirmed the fact that the tendon was not
frozen; therefore its biomechanical attributes were
not substantially changed. In addition the methodol-
ogy proposed by Wieloch et al. (2004) was elaborat-
ed for biomechanical testing at a loading rate equal
to 1000 mm/min. However at such a loading rate the
tests can not be characterized as quasi-static ones
but rather they resemble dynamic testing. The dis-
placement rate used in the present study was much
lower (1 mm/min) resulting in a quasi-static biome-
chanical testing of the soft tissue.
However the main contribution of the present study
is the proposal of an innovative technique for the
assessment of the fracture stress of tendons based
on the histologically-measured cross-sectional area
of the tendon. The direct and constant measurement
of cross-sectional area during the experimental pro-
cedure (e.g. using laser micrometers or miniature
LVDTs) is extremely difficult and perhaps unreli-
able due to the very small size of the specimens
and sophisticated mechanisms are required for the
parallel transfer of the measuring devices with the
continuously elongated tendon. On the other hand
the measurement of the initial cross-sectional area
of the tendon cannot offer reliable stress data, since
it is known that during the biomechanical testing the
cross-sectional area of the tendon, which is a hy-
perelastic material, changes dramatically. Therefore
the histological measurement of the “effective” area
after the rupture of the tendon appears to be closer
to the actual cross-sectional area at the moment of
fracture.
Scand. J. Lab. Anim. Sci. 2010 Vol. 37 No. 3
155
Another significant parameter of the experimental
procedure is the preconditioning of the specimen.
However the nature of the influence of precondition-
ing on the biomechanical behaviour of a soft tissue
is not clear. While it was traditionally considered to
be an experimental artifact, recent studies suggest
that it is a physical property of the tendon and there-
fore an experimental parameter itself (Maganaris,
2003; Maganaris et al., 2002). In the present meth-
odology preconditioning was not elaborated due to
the risk of de-freezing of the muscle and slippage of
the specimen.
The limitations of the methodology proposed here
emanate mainly from the nature and the geometri-
cal characteristics of the tested specimens. First of
all the strain of the tendon was not determined. The
small dimensions of the specimens rendered the uti-
lization of optical strain measurement techniques
very difficult and the displacement of the load-frame
could not be accepted as elongation without second
thoughts. In any case this issue remains available for
further study.
Finally, according to the proposed methodology, the
measured cross-sectional area corresponds only to
the moment of failure. Therefore, it can not be used
for the reduction of the entire set of load measure-
ments to the corresponding stresses, but only for
the reduction of the maximum force to the fracture
stress.
The above observations render the proposed meth-
odology ideal for comparative biomechanical
studies of soft tissues in small laboratory animals
considering the difficulties encountered using such
specimens.
Acknowledgements
The authors would like to thank Professor C. Fas-
seas, Department of Agricultural Biotechnology,
Agricultural University of Athens, Greece for his
great assistance and for supplying us with the nec-
essary equipment for the elaboration of microscopy
studies.
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