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Fascicles from energy-storing tendons
show an age-specific response to cyclic
fatigue loading
rsif.royalsocietypublishing.org
Research
Cite this article: Thorpe CT, Riley GP, Birch
HL, Clegg PD, Screen HRC. 2014 Fascicles from
energy-storing tendons show an age-specific
response to cyclic fatigue loading. J. R. Soc.
Interface 11: 20131058.
http://dx.doi.org/10.1098/rsif.2013.1058
Received: 15 November 2013
Accepted: 4 December 2013
Subject Areas:
biomedical engineering
Keywords:
fascicle, structure– function, micromechanics,
confocal microscopy, ageing
Chavaunne T. Thorpe1,2, Graham P. Riley3, Helen L. Birch4, Peter D. Clegg2
and Hazel R. C. Screen1
1
Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary University of London,
Mile End Road, London E1 4NS, UK
2
Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool,
Leahurst Campus, Neston CH64 7TE, UK
3
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
4
Institute of Orthopaedics and Musculoskeletal Science, University College London, Stanmore HA7 4LP, UK
Some tendons, such as the human Achilles and equine superficial digital
flexor tendon (SDFT), act as energy stores, stretching and recoiling to increase
efficiency during locomotion. Our previous observations of rotation in
response to applied strain in SDFT fascicles suggest a helical structure,
which may provide energy-storing tendons with a greater ability to extend
and recoil efficiently. Despite this specialization, energy-storing tendons are
prone to age-related tendinopathy. The aim of this study was to assess the
effect of cyclic fatigue loading (FL) on the microstructural strain response of
SDFT fascicles from young and old horses. The data demonstrate two independent age-related mechanisms of fatigue failure; in young horses, FL caused low
levels of matrix damage and decreased rotation. This suggests that loading
causes alterations to the helix substructure, which may reduce their ability
to recoil and recover. By contrast, fascicles from old horses, in which the
helix is already compromised, showed greater evidence of matrix damage
and suffer increased fibre sliding after FL, which may partially explain the
age-related increase in tendinopathy. Elucidation of helix structure and the precise alterations occurring owing to both ageing and FL will help to develop
appropriate preventative and repair strategies for tendinopathy.
1. Introduction
Author for correspondence:
Chavaunne T. Thorpe
e-mail: [email protected]
Tendons provide an attachment from muscle to bone, transferring force generated by muscle contraction to the skeleton and facilitating movement. The
ability to withstand large unidirectional forces is provided by their structure;
tendons are hierarchical fibre-composite materials, in which type I collagen
molecules are grouped together, forming subunits of increasing diameter, the
largest of which is the fascicle [1]. At the larger hierarchical levels, the collagen
is interspersed with a predominantly non-collagenous matrix [2]. Historically,
many assumed that the structure of all tendons was very similar, but there
is recent evidence demonstrating numerous compositional, structural and
organizational specializations according to tendon function [3– 9].
Tendons can broadly be divided into two categories depending on their
function, those that act purely to position the limb for locomotion and those
that act as elastic springs during locomotion, storing energy and thus reducing
the energetic cost of locomotion [10,11]. Energy-storing tendons are often subjected to high forces and are more compliant than positional tendons to allow
the elongation required for maximal energy storage and return [3,6,12]. Therefore, energy-storing tendons must withstand large, repetitive stresses and
strains during exercise. In the Achilles tendon, which is the predominant
energy store in humans, strains in excess of 10% have been recorded during
hopping exercise [13]. The energy-storing equine superficial digital flexor
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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2.1. Sample collection and preparation
Forelimbs distal to the carpus were collected from half- to fullthoroughbred horses aged 3 – 6 years (n ¼ 10, young (skeletally
mature) group) and 17 – 20 years (n ¼ 10, old (geriatric) group
[31]), euthanized at a commercial equine abattoir. Only tendons
that had no evidence of previous tendon injury at post-mortem
examination were included in the study. The SDFT was dissected
free from the limbs from the level of the carpus to the
2.2. Fascicle creep tests to failure
Preliminary tests were carried out to assess the cyclic creep
response of fascicles from young and old SDFTs (n ¼ 4 tendons
per age group, replicates ¼ 10 – 12 fascicles per tendon). Each fascicle was secured in a custom-made chamber system [34] at a
resting grip-to-grip length of 10 mm. Fascicles were maintained
in DMEM for the duration of the experiment. A displacement
of 0.2 mm was applied to remove any slack from the samples
prior to testing, and reported strains were established relative
to this consistent starting condition. Half of the fascicles were
tested to failure at a speed of 1 mm s21, and force– displacement
data were recorded at a frequency of 100 Hz (WINTEST software,
Bose ElectroForce 5500 Biodynamic test instrument, Gillingham,
UK). The average failure stress was calculated for the fascicles
from each tendon. The remainder of the fascicles were subjected
to cyclic FL to 50% of their predicted failure stress, with a median
stress of 25% and a minimum force of 0.2 N using a sinusoidal
waveform at a frequency of 1 Hz until failure. Force – displacement data were recorded at a frequency of 100 Hz, recording
data from 30 consecutive cycles in every 300 cycles. In addition,
every 10th maximum and minimum reading was recorded. Per
cent strain was calculated from maximum displacement data
and the resultant data were used to plot creep curves to failure.
2.3. Effect of fatigue loading on fascicle extension
mechanisms
2.3.1. Fatigue loading
Fascicles from a subset of young and old SDFTs were analysed to
assess the effect of FL on fascicle extension mechanisms (n ¼ 6
tendons per age group, replicates ¼ 6 – 8 fascicles per tendon).
Half of the fascicles acted as unloaded controls, while the
remaining fascicles were subjected to cyclic FL for 1800 cycles
at a frequency of 1 Hz using the cyclic loading protocol described
above. After 1800 cycles had been completed, fascicles were
removed from the chambers and the fibre-level response to
applied strain was assessed.
2.3.2. Assessment of fibre-level response to incrementally
applied strain
Fascicles in both the control and FL groups were stained with
the collagen stain 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein
hydrochloride (5-DTAF) at a concentration of 2 mg ml21 in
0.1 M sodium bicarbonate buffer, pH 9 for 20 min. Following
staining the fascicles were washed in two changes of DMEM
2
J. R. Soc. Interface 11: 20131058
2. Material and methods
metacarpophalangeal joint, wrapped in tissue paper dampened
with phosphate-buffered saline and stored frozen at 2208C
wrapped in aluminium foil within 24 h of animals’ death. It
has previously been shown that one freeze– thaw cycle does
not affect tendon mechanical properties [32]. On the day of testing, the tendons were allowed to thaw at room temperature and
fascicles (8 –12 fascicles per tendon, approx. 25 mm in length,
diameter of 0.2– 0.4 mm) were isolated from the mid-metacarpal
region of the tendon by cutting with a scalpel longitudinally
through the tendon using previously established protocols
[33,34]. Fascicle hydration was maintained by storing the fascicles on tissue paper dampened with Dulbecco’s modified
eagles medium (DMEM) prior to testing. Fascicle diameter was
measured continuously at a single angle along a 10 mm region
in the mid-portion of the fascicle using a laser micrometer [6].
The smallest diameter recorded was used to estimate fascicle
cross-sectional area, assuming a circular cross section. All experiments were performed at room temperature. Fascicles were
observed carefully during each experiment to ensure that only
one fascicle was being tested.
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tendon (SDFT) has a similar function to the human Achilles
[3,7,14,15] and experiences similar high strains, which can
reach 16% during galloping [16]. However, the mechanisms
that enable these high levels of extension in energy-storing
tendons are yet to be determined.
Previous analysis of the microstructural strain response in
tendon fascicles has demonstrated that, in general, fascicle
extension is dominated by sliding between the collagen
fibre components rather than fibre extension [17 –20]. However, the majority of these studies were performed on
fascicles from rat tail tendon, which has a purely positional
function. Our recent work comparing the microstructural
strain response of fascicles from the energy-storing SDFT
and positional common digital extensor tendon (CDET) has
demonstrated a novel mechanism for elongation involving
rotation of the fascicle; this rotation occurs to a much greater
extent in the SDFT fascicles than in CDET fascicles in
response to applied strain [7]. This rotation suggests the presence of helical substructures within energy-storing tendons.
Further, our previous results demonstrate that SDFT fascicles
are better able to recoil and recover following loading than
CDET fascicles, suggesting that these helical substructures
may provide a mechanism for efficient extension and recoil [7].
Despite these adaptations, energy-storing tendons are
particularly prone to injury, which is thought to occur as a
result of accumulation of microdamage within the tendon
matrix rather than acute injury [21]. In humans, the prevalence of Achilles injury is reported as 3% in the general
population under 45 years of age [22], increasing to
15– 56% in elite athletes, depending on the type of sport
undertaken [22,23]. Injury to the SDFT in athletic horses is
also common, with SDFT pathology reported in 11–24% of
thoroughbred racehorses [24,25] and shows a similar
initiation and progression to human Achilles tendinopathy
[26,27]. Further, tendons become more prone to injury with
increasing age, with regards to both the human Achilles
[22,28] and equine SDFT [29,30]. This age-related predisposition to injury is not well understood. However, our previous
work has demonstrated that ageing results in decreased
rotation in SDFT fascicles under monotonic loading and
that this is accompanied by decreased recovery and increased
hysteresis [7]. These findings suggest that ageing results in
alterations to the helix substructure, resulting in a decreased
ability to recoil and recover, which may predispose aged tendons to injury. Therefore, the aim of this study was to assess
how cyclic fatigue loading (FL) affects the extracellular matrix
microstructural response in SDFT fascicles from young and
old horses. We hypothesized that FL would result in alterations in tendon fascicle extension mechanisms and that
these alterations would show age-specific differences, with
greater changes in samples from aged individuals.
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3
(a)
d2
J. R. Soc. Interface 11: 20131058
y + Dy
d1
(b)
x + Dx
qx
Figure 1. Images showing grid bleached onto fascicles stained with 5-DTAF at 0% applied strain, with loading direction indicated by arrows (a), and grid deformation at 4% applied strain in control samples (b). The pixel lines generated during image analysis have been superimposed on to image (b). Schematic (c) showing
the four parameters used to quantify grid deformation: fibre extension (x-strain), transverse strains ( y-strain), fibre sliding (d1 þ d2) and grid rotation (ux).
for 20 min, and secured in a custom-made tensile testing rig [17],
at a resting grip-to-grip length of 10 mm. Fascicles were maintained in DMEM for the duration of the experiment. Previous
experiments have shown that 5-DTAF does not affect fascicle
material properties [7].
Each fascicle was viewed under the laser scanning confocal
microscope (TCS SP2, Leica Microsystems GmbH, Wetzlar,
Germany) using a 20 objective (HC PL Fluotar, Nikon,
Kingston-Upon-Thames, UK). Fascicle alignment and orientation
was checked under brightfield settings to ensure that fibres were
aligned with the loading direction and no gross twisting of the
sample had occurred. The grips were slowly moved apart and
the sample monitored visually until a small amount of tension
was applied, which was signified by the fascicle slightly lifting
off the base of the rig [35]. This corresponded to a load of
approximately 0.1 N (range: 0.05– 0.15 N). A grid was then
photobleached at a depth of approximately 20 – 25 mm within
the fascicle, using a 488 nm krypton – argon laser. The grid
encompassing a series of 2 mm thick lines, bleached in the central
region of the fascicle away from the gripping regions, to create a
grid of four squares, each 50 50 mm2 (figure 1a), covering
approximately half of the fascicle width. The laser intensity
was then reduced to the imaging range, and the sample
imaged in the same focal plane with the same objective lens at
a resolution of 2048 2048 pixels2, with each pixel measuring
0.18 0.18 mm2. A focal plane of 20 – 25 mm within the fascicle
was chosen as images at this depth had the greatest clarity.
The fascicle was then strained to 2% at a rate of 1% s21 and the
grid was refocused, before imaging again (figure 1b). Incremental
strains of 2% were applied in this manner up to a maximum of
10%, and the grid was imaged at each strain increment. Previous
experiments have established that at macrolevels, applied strains
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(c)
are distributed evenly across the fascicle [7]. There was a hold
period of approximately 1 min before imaging at each increment, while the focal plane was located; it has previously been
shown that this is sufficient time for complete stress relaxation
to occur [7,17].
2.3.3. Image analysis
Images from the confocal experiments were processed using the
analysis software IMAGEJ (1.34s, National Institute of Health,
USA) as described previously [7]. The grid deformation parameters calculated are shown in figure 1c. In order to calculate
the local x- and y-strains, the points where the lines in the
x- and y-planes crossed were marked, and the image was thresholded and skeletonized; with the resulting image consisting
of nine pixels representing the grid corners. The coordinates of
these pixels were exported to Microsoft Excel for data analysis.
In order to quantify the local strains within the grid, the following parameters were calculated from the grid corner coordinates:
— local strain (%) in x-direction (100Dx/x): percentage strain in
x-direction (with axis of loading); and
— local strain (%) in y-direction (100Dy/y): percentage strain in
y-direction ( perpendicular to loading axis).
Poisson’s ratios (v) were calculated at each strain increment
using the following equation:
v¼
local y-strain
:
applied strain
In order to calculate the maximum deviation of the vertical
gridline ( y-axis), images were thresholded, despeckled and
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The distribution of the data was tested using a D’Agostino –
Pearson test for normality (GraphPad Prism). Data with a
normal distribution were analysed using a two-way ANOVA
followed by Bonferroni post hoc tests to assess the effect FL on
microstructural strain response and determine whether this
was altered with ageing. Data that were not normally distributed
were analysed using Kruskal – Wallis tests followed by Dunn’s
multiple comparison post hoc analysis (GraphPad Prism).
Statistical significance was set at p , 0.05.
3. Results
3.1. Fascicle failure and creep properties
There was no difference in average fascicle diameter or
failure stress in samples from young and old horses (diameter: 0.26 + 0.03 mm versus 0.26 + 0.04 mm; failure stress:
65.39 + 27.84 MPa versus 61.80 + 29.22 MPa). These values
are likely to be lower than the true failure stress, owing to
the slow test speed relative to the in vivo loading rate, and
the presence of stress concentrations at the grips [36]. However, these values can be used to determine an appropriate
stress for fatigue testing. Concerning fatigue testing, the
number of cycles to failure was significantly lower in fascicles
from aged horses, decreasing from 16 825 + 6104 cycles in
young horses to 4062 + 927 cycles in old horses ( p ¼ 0.02,
figure 2).
3.2. Effect of fatigue loading on response to
incremental strains in young samples
FL caused mild damage to the tendon matrix in fascicles from
young horses, characterized by the presence of mildly kinked
fibres throughout the matrix (figure 3a), which were observed
in the fluorescently stained fascicles under the scanning
confocal microscope.
3.2.1. Microstructural strain response
In agreement with previous studies [7,17], local longitudinal strains, representing fibre extension, were consistently
smaller than overall applied strain. The amount of fibre
extension did not differ between FL and control samples
(figure 4a). In control samples from young horses, large compressive strains were observed perpendicular to the axis of
loading. There was a trend towards decreasing transverse
strain with FL but this was not significant (figure 4b). Therefore, the resulting Poisson’s ratios were larger in control
samples than that in FL samples, although this only reached
4
% strain
20
10
0
5000
10 000
cycles
15 000
20 000
Figure 2. Typical creep curves to failure, showing maximum strain (%)
plotted against cycle number, for SDFT fascicles from young (dashed line)
and old (solid line) horses.
significance at 6% applied strain ( p , 0.05, figure 4c). A small
amount of between fibre sliding (d1 þ d2) was seen in the
majority of samples, but FL had no effect on the degree of
fibre sliding measured in young samples (figure 4d). Levels
of grid rotation were similar to those recorded previously
in unloaded SDFT fascicles [7]. FL resulted in a significant
decrease in rotation at all strain increments above 2% in
samples from young horses ( p , 0.05, figure 4e).
3.3. Effect of fatigue loading on response to
incremental strains in old samples
Damage to the fascicle matrix was more obvious after FL in
old samples than in young (figure 3a), with a greater
number of fibre kinks observed. In addition, the majority of
FL samples from old animals exhibited widening of the
interfibre space, and in a few cases fibre discontinuities
were observed.
3.3.1. Microstructural strain response
The amount of fibre extension was similar to that seen in
samples from young horses, and FL had no significant
effect on the amount of fibre extension observed (figure 5a).
In agreement with our previous findings [7], transverse
strain was decreased in control samples from aged individuals compared with young ( p , 0.02). FL appeared to
result in a further reduction in transverse strain in aged
samples, although this was not significant (figure 5b). Correspondingly, there was a trend towards decreased Poisson’s
ratios in FL samples from aged individuals, but this was
only significant at 2% applied strain (figure 5c, p , 0.05).
Levels of fibre sliding were similar between samples in the
young and old control groups. Fibre sliding showed a significant increase after FL in old samples at all strain increments
(figure 5d, p , 0.05). In agreement with previous results,
ageing resulted in decreased rotation in control samples [7].
However, there was no reduction in rotation after FL in
these aged samples (figure 5e).
4. Discussion
The data support the hypothesis, showing that FL results in
altered extension mechanisms in SDFT fascicles from both
J. R. Soc. Interface 11: 20131058
2.4. Statistical analysis
30
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skeletonized in IMAGEJ to generate a single pixel trace of the left
most gridline in the y-plane. Any noise was removed from the
image and the coordinates of the line exported to Origin (OriginLab, Northampton, USA). A straight line was fitted between the
start and endpoints of this line. The maximum deviations of the
gridline from the straight line in either direction were also calculated, and the sum of these deviations (d1 þ d2, figure 1c) used to
provide a measure of fibre sliding.
In order to quantify grid rotation, images were processed in
IMAGEJ (as described above) to generate a single pixel trace of the
bottom horizontal gridline (x-axis) and the coordinates of this
line were exported to Origin. A linear line of best fit was fitted
to the coordinates, and the angle of this line relative to the
x-axis (ux, figure 1c) was calculated.
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fatigue loaded
control
fatigue loaded
old
J. R. Soc. Interface 11: 20131058
old
young
(b)
5
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control
young
(a)
Figure 3. Typical confocal images of control and fatigue-loaded SDFT fascicles from young and old horses at (a) 0% and (b) 10% applied strain. Fatigue-induced
damaged is characterized by fibre kinks (indicated by arrows); these kinks were more prominent in aged samples. Widening of the interfibre space was also observed
in samples from aged individuals (indicated by circles). Scale bar, 25 mm.
young and old horses, and that these alterations are age
specific, with greater evidence of matrix damage after FL in
old samples. While ageing did not cause alterations in
observed tendon fascicle failure properties, it did result in a
deterioration of fatigue resistance, suggesting that aged
samples are less resilient to cyclic loading. This decrease in
cycles to failure was accompanied by greater alterations in
microstructural strain response after a bout of cyclic loading.
Several studies of the effect of fatigue on tendon properties have been carried out in a variety of species, from
small to large animal models [34,37,38]. While species such
as the mouse and rat provide a simple system to understand
basic mechanisms of damage, it is not clear how data from
these studies translate to human tissues, particularly in
terms of energy-storing tendons. The horse is an excellent
large animal in which to study tendon structure– function
relationships, as the equine SDFT is an extreme example of
an energy store, with a function very similar to that of the
Achilles [15]. Indeed, the horse is one of a few species that
suffers from naturally occurring tendon injuries, the
initiation, progression and age-related susceptibility of
which is comparable to that seen in Achilles tendinopathy
[15,22,27,29]. Elucidation of structure– function relationships
in the equine SDFT are therefore likely highly relevant to
understanding human Achilles function in heath, disease
and ageing.
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2.0
1.5
1.0
0.5
0
2
4
6
8
applied strain (%)
10
(c)
5
0
2
2
4
8
6
*
10
–5
–10
–15
1
0
2
4
6
8
applied strain (%)
(e)
line angle (°)
10
8
6
4
2
0
**
4
12
2
6
8
4
applied strain (%)
10
10
*
3
*
2
**
1
0
2
4
6
8
applied strain (%)
10
Figure 4. (a) Longitudinal strain, (b) transverse strain, (c) Poisson’s ratio, (d ) fibre sliding and (e) grid rotation at increasing strain increments in control (filled circle)
and fatigue-loaded (open circle) SDFT fascicles from young horses. Data are displayed as mean + s.e.m. Statistical significance: *p , 0.05; **p , 0.01.
Previous studies have shown that cyclic loading causes
damage to the tendon matrix, which becomes evident
before any significant changes in tendon mechanical properties are observed [39]. Low-level fatigue damage is
characterized by the presence of kinked fibre deformations
[39 –42]. As the level of fatigue damage increases, the
number of fibre kinks also becomes larger; this is
accompanied by widening of the interfibre space. Highlevel fatigue damage is characterized by severe matrix
disruption, fibre thinning, angulations and fibre discontinuities [37,40,43]. In this study, similar changes were observed
and it is evident that the fatigue damage generated by
cyclic loading is more severe in SDFT fascicles from older
horses. FL samples from young horses exhibited low-level
fatigue damage, with the presence of a few kinked fibres.
In aged samples, FL induced more notable damage, with
evidence of disruption between fibres in the majority of
samples, and severe matrix damage and fibre discontinuities
in some samples.
While several studies have investigated the effect of FL on
matrix appearance [39,40], alterations in mechanical properties [34,39,41] and cell response [34,38,44], to the authors’
knowledge, this study is the first to present data regarding
the effect of FL on tendon fascicle microstructural strain
response. Our results show that in samples from young
horses, levels of fibre extension were not altered by FL,
suggesting that fibre integrity was maintained throughout
the test procedure. There were also no alterations in the
amount of fibre sliding measured after FL. However, we
have previously established that, while fibre sliding governs
fascicle extension in tendons with a purely positional function [7], in the energy-storing SDFT, extension appears to
be dominated by the ‘unwinding’ of helical substructures,
resulting in grid rotation [7]. This rotation is associated
with a greater ability to recoil and recover efficiently [7]. In
this study, we observed a significant decrease in rotation
after FL in samples from young horses, suggesting that FL
causes alterations to the helix structure. This is somewhat
supported by the trend for decreased Poisson’s ratios
observed in samples from young horses; while the large
Poisson’s ratios observed may be owing to exudation of
fluid from the fascicles [17], finite-element modelling has
shown that the presence of helical substructures within
tendon predicts large Poisson’s ratios [45]. While the specific
alterations that occur to the helix during FL are as yet
unknown, it is possible that some of the characteristic
sample lengthening observed during creep may occur as a
result of straightening of the coil of the helix, such that
helix pitch angle decreases. This has important implications
for tendon fatigue resistance, as alterations to the helix may
decrease the ability of SDFT fascicles to extend and recoil efficiently, which is likely to increase the risk of microdamage
occurring to the tendon matrix.
One limitation of this study is the methods used to calculate fascicle cross-sectional area. Previous studies have
demonstrated that fascicle cross section within the equine
SDFT appears irregular [46] such that assuming a circular
cross section may lead to errors when calculating the stress
to apply during FL. This may partially account for the
larger variation seen in the microstructural strain response
in fatigue-loaded samples.
When assessing the effect of FL on fascicle microstructural
strain response, it is also important to consider the gripping
methods and sample length used, as well as the values of
stress and strain that were applied. Gripping the samples
during FL resulted in some damage and flaring at the fascicle
ends, which may have contributed to the alterations seen in
microstructural strain response. However, owing to the
increase in sample length during FL, fascicle ends were
gripped in a previously unclamped region when secured in
the confocal rig for evaluation of strain response, minimizing
any gripping artefact in FL samples. Furthermore, the large
aspect ratio (approx. 33) of fascicles ensured that any flaring
near the gripping sites did not persist into the central region
J. R. Soc. Interface 11: 20131058
–1
(d )
fibre sliding (mm)
6
applied strain (%)
Poisson’s ratio
2.5
local transverse strain (%)
(b)
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local longitudinal strain (%)
(a)
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2.5
2.0
1.5
1.0
(c)
applied strain (%)
5
0
2
4
6
8
10
–5
–10
0.5
0
2
4
6
8
10
applied strain (%)
10
**
(e)
*
***
line angle (°)
10
**
8
6
*
***
4
4
3
2
1
2
0
2
6
8
4
applied strain (%)
10
0
2
4
6
8
applied strain (%)
10
Figure 5. (a) Longitudinal strain, (b) transverse strain, (c) Poisson’s ratio, (d) fibre sliding and (e) grid rotation at increasing strain increments in control (filled
square) and fatigue-loaded (unfilled square) SDFT fascicles from old horses. Data are displayed as mean + s.e.m. Statistical significance: *p , 0.05; **p , 0.01.
of the fascicle where the grid was bleached, and so is unlikely
to have had a significant effect on the results. However, the
use of a control group that had been exposed to very low
levels of cyclic loading would have ensured any observed
differences were owing to FL rather than gripping of the
samples during the loading procedure. The effect of sample
length also needs to be taken into account. In this study,
the grip-to-grip distance was 10 mm. Our previous work
has shown that the end effects generated by such a grip-togrip length artificially augment fascicle strains and should
subsequently be corrected for [33]. Using our previous
methods [6] and data from SDFT fascicle failure strains, the
appropriate correction factor was calculated as 0.65. This
results in a maximum applied strain of 6.5%, which is still
more than three times the local longitudinal strain measured
within the grids at this strain increment, showing that strain
is distributed throughout the tendon matrix by mechanisms
other than fibre extension.
It is also important to consider how the stress and
strain applied during the fatigue test may relate to in vivo
levels. In this study, FL samples were subjected to cyclic loading to 50% of their predicted failure stress. Data from
previous studies suggest that the SDFT may experience
in vivo stresses as high as 90% of their in vitro failure stress
and strain during maximal exercise [3,16,47,48]. However,
our previous results suggest that, in the SDFT as a whole,
initial tendon extension is facilitated by sliding between adjacent fascicles [6], such that fascicles are likely to be exposed to
lower stresses and strains than the whole tendon. Peak in vivo
fascicle stresses are yet to be determined, so it is difficult to
compare the stresses applied in this study with the in vivo
stress environment. However, it is evident that the loads
applied were high enough to induce mild to moderate
damage within the samples.
Though several studies have investigated alterations in
tendon mechanical properties as a function of ageing
[14,49 –52], few have assessed the effect of FL on aged
tendon. Pike et al. [53] applied cyclic fatigue tests to ovine
flexor and extensor tendons, and demonstrated that the
time to rupture increased from birth to maturation. However,
this study did not include tendons from aged individuals. A
more recent study by Kietrys et al. [54], using an in vivo rat
overuse model has shown that repetitive loading in aged
individuals resulted in greater tendon inflammation and
reduced limb agility compared with young tendons that
had undergone the same loading regime [54]. These findings,
combined with the data presented in this study, indicate that
maturation results in an increased ability of tendons to resist
repetitive loading, whereas ageing causes a decrease in fatigue resistance. The confocal images presented in figure 3
show evidence of more extensive damage within the matrix
if FL samples are from old animals. While none of the tendons tested had gross evidence of pathology, it is possible
that microdamage may have been present within the matrix
of some aged samples prior to in vitro FL. Indeed, previous
studies indicate that there are several mechanisms that predispose aged tendons to microdamage; it has been shown
that partially degraded collagen accumulates in aged
SDFTs, likely owing to an accumulation of non-enzymatic
cross-linking rendering the collagen more resistant to proteolytic enzyme activity [5]. In addition, a decreased capacity for
tendon extension through the interfascicular matrix may
expose fascicles from aged tendons to higher levels of strain
[14], increasing the risk of microdamage occurring to the
fascicle matrix.
In addition to altering the microstructural strain response,
FL and subsequent microdamage within the tendon matrix is
also likely to result in alterations to cell behaviour. In this
study, experiments were performed on tissues that had
previously been frozen, and as such cell response could not
be studied. However, it has previously been reported that
cyclic loading of viable tendon explants in vitro results in
increased cell death and production of prostaglandin-E2
and collagenases at high magnitudes of applied load [55].
J. R. Soc. Interface 11: 20131058
4
6
8
applied strain (%)
(d )
12
fibre sliding (µm)
2
1
–1
–15
0
7
2
Poisson’s ratio
local transverse strain (%)
(b)
rsif.royalsocietypublishing.org
local longitudinal strain (%)
(a)
Downloaded from http://rsif.royalsocietypublishing.org/ on July 31, 2017
2.5
2.0
1.5
1.0
0.5
0
4
6
8
10
–5
–10
–15
1
0
2
4
6
8
applied strain (%)
10
–1
10
(e)
4
8
6
4
2
0
2
applied strain (%)
2
8
2
6
8
4
applied strain (%)
10
3
2
1
0
2
4
6
8
applied strain (%)
10
Figure 6. (a) Longitudinal strain, (b) transverse strain, (c) Poisson’s ratio, (d) fibre sliding and (e) grid rotation in control (filled circle) and fatigue-loaded (unfilled
circle) fascicles from young SDFTs, and control (filled square) and fatigue-loaded (unfilled square) fascicles from old SDFTs. To allow easy visual comparison of all
datasets, error bars have been removed.
young energy-storing tendons
old energy-storing tendons
(a)
(c)
fibres
fascicles
q>
q<
strain applied
strain applied
apply fatigue loading
(b)
(d )
q<
q→0
strain applied
strain applied
Figure 7. Schematic showing the combined effects of FL and ageing on the levels of grid rotation and how this relates to helix substructure. (a) In SDFT fascicles
from young horses, extension occurs owing to helix unwinding, resulting in sample rotation. (b) FL of these fascicles results in a reduction in helix-related rotation.
(c) In SDFT fascicles from aged horses, the integrity of the helix is already compromised, and the fascicle response to loading is similar to that seen after FL in young
samples. (d) Fascicles from aged tendon therefore have little helical capacity to manage the loading response, and FL results in increased levels of fibre sliding.
(Online version in colour.)
It has been suggested that this degenerative cascade is
owing to understimulation of the cells as a result of
damage to the collagen fibres [56]. Further, injured tendons
often exhibit cell rounding [21], which is likely to be the
result of an altered mechanical environment. While any
age-related alterations to cell behaviour as a result of FL are
yet to be determined, it is likely that the greater levels of
damage observed with FL in aged samples in this study
would result in greater alterations in cell response, further
increasing the risk of subsequent injury.
J. R. Soc. Interface 11: 20131058
4
6
8
applied strain (%)
(d )
10
5
line angle (°)
2
fibre sliding (µm)
0
(c)
Poisson’s ratio
local transverse strain (%)
(b)
rsif.royalsocietypublishing.org
local longitudinal strain (%)
(a)
Downloaded from http://rsif.royalsocietypublishing.org/ on July 31, 2017
in terms of the greater variability seen in matrix appearance
and the increased levels of fibre sliding.
5. Conclusion
Data accessibility. Mechanical test data and confocal images are available
from the Dryad digital repository: doi:10.5061/dryad.8b35s.
Funding statement. This work was supported by a project grant ( prj/
752) from the Horserace Betting Levy Board.
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9
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The age-specific alterations in microstructural strain
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less resistant to cyclic loading. When aged SDFT fascicles
are then subjected to FL in vitro, there is subsequently little
helical capacity to manage the loading response, which
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