Kristin S. Miller
Brianne K. Connizzo
Louis J. Soslowsky1
McKay Orthopaedic Research Laboratory,
University of Pennsylvania,
424 Stemmler Hall, 36th and Hamilton Walk,
Philadelphia, PA, 19104-6081
Effect of Preconditioning and
Stress Relaxation on Local
Collagen Fiber Re-Alignment:
Inhomogeneous Properties of
Rat Supraspinatus Tendon
Repeatedly and consistently measuring the mechanical properties of tendon is important
but presents a challenge. Preconditioning can provide tendons with a consistent loading
history to make comparisons between groups from mechanical testing experiments. How-
ever, the specific mechanisms occurring during preconditioning are unknown. Previous
studies have suggested that microstructural changes, such as collagen fiber re-alignment,
may be a result of preconditioning. Local collagen fiber re-alignment is quantified
throughout tensile mechanical testing using a testing system integrated with a polarized
light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side
of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corre-
sponding mechanical properties are measured. Local circular variance values are com-
pared throughout the mechanical test to determine if and where collagen fiber
re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local
circular variance values, optical moduli and optical transition strain values. Although
the largest amount of collagen fiber re-alignment was found during preconditioning, sig-
nificant re-alignment was also demonstrated in the toe and linear regions of the mechani-
cal test. No significant changes in re-alignment were seen during stress relaxation. The
insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a
more disorganized collagen fiber distribution throughout all mechanical testing points
compared to the tendon midsubstance. This study identified a correlation between colla-
gen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment
may be a potential mechanism of preconditioning and merits further investigation. In par-
ticular, the conditions necessary for collagen fibers to re-orient away from the direction
of loading and the dependency of collagen reorganization on its initial distribution must
be examined. [DOI: 10.1115/1.4006340]
Keywords: collagen fiber re-alignment, inhomogeneous nonlinear tendon mechanics,
preconditioning, polarized light, supraspinatus tendon
Cyclic preconditioning is a commonly accepted initial compo-
nent of most tendon mechanical testing protocols. Preconditioning
provides tendons with a consistent “history,” and stress-strain
results become repeatable, allowing for rigorous evaluation and
comparison. Protocols frequently involve the repeated stretching
of a sample to a sub-failure load to produce a repeatable mechani-
cal response [1–4]. The internal structure of tendon changes in
response to each loading cycle. After applying repeated cycles, a
steady state is reached at which no further changes will occur
unless the cycling routine is changed .
While it is widely accepted that preconditioning is important,
changes that occur during preconditioning are not well under-
stood. Micro-structural alterations, such as re-arrangement of col-
lagen fibers, is one proposed mechanism of preconditioning [5–7].
Recently, a strong correlation between changes in collagen fiber
alignment and changes in the mechanical response of ligament
during cyclic tensile preconditioning has been reported .
The correlation found between reduced force response during pre-
conditioning and change in fiber alignment after preconditioning
suggests that viscoelastic effects and microstructural reorganiza-
tion both contribute to the time-and-history dependence of me-
chanical properties . However, the dependence of collagen
fiber re-alignment during preconditioning on tendon location has
not yet been examined. Additionally, collagen fiber re-alignment
during stress relaxation or a tensile ramp-to-failure following pre-
conditioning has not been examined in tendon.
Additionally, recent studies have shown that collagen fiber re-
alignment varies by tendon location [8,9]. The tendon-to-bone
insertion site of the supraspinatus tendon experiences higher
strains and has been shown to have a more disorganized fiber dis-
tribution compared to the tendon midsubstance [8,10–12]. There-
fore, the objective of this study was to locally measure collagen
fiber re-alignment and corresponding mechanical properties
throughout tensile mechanical testing to address mechanisms of
preconditioning and stress relaxation as well as tissue nonlinearity
and inhomogeneity in the rat supraspinatus tendon model. We
hypothesized that fiber re-alignment will be greatest in the toe
region of the ramp-to-failure test but that a change in circular var-
iance will also occur during preconditioning despite the small
loads used in the mechanical testing protocol. Additionally, we
hypothesize that the collagen fiber distribution will become more
Contributed by the Bioengineering Division of ASME for publication in the
JOURNAL OF BIOMECHANICAL ENGINEERING. Manuscript received September 23, 2011;
final manuscript received February 24, 2012; accepted manuscript posted March 14,
2012; published online March 26, 2012. Editor: Michael Sacks.
Journal of Biomechanical EngineeringMARCH 2012, Vol. 134 / 031007-1 Copyright V
C2012 by ASME
disorganized throughout the stress relaxation test and that the me-
chanical properties and initial collagen fiber alignment will be
greater in the midsubstance location of the tendon compared to
the tendon-to-bone insertion site.
2.1 Sample Preparation. This study was approved by the
University of Pennsylvania IACUC. Twenty-two Sprague–Daw-
ley rats were sacrificed, and supraspinatus tendons (SST) were
removed for mechanical testing. All soft tissue was removed from
around the tendon, leaving the supraspinatus muscle-tendon unit
attached to the humerus. The supraspinatus muscle was removed.
Verhoeff stain lines were placed on the tendons denoting the
insertion site and tendon midsubstance for optical strain analysis
to denote regions for alignment analysis and to define the gauge
section. The first stain line was placed at the tendon-to-bone inser-
tion site on the humerus as described previously , and the
remaining stain lines were placed at 2, 4, 7, and 9 mm from the
insertion site. The cross-sectional area at each location was meas-
ured using a noncontact laser device with a resolution of 2 lm
. The humerus was embedded in a holding fixture with the use
of polymethylmethacrylate (PMMA). After the PMMA had set,
the humeral head was sanded down to prevent the bone from
impeding light from passing through the tendon insertion site for
polarized light analysis. A second coating of PMMA was applied
to prevent failure at the growth plate. The holding fixture was
inserted into a custom testing fixture. The proximal end of the
tendon was glued between two pieces of sandpaper and placed in
custom grips for tensile testing. All samples had an initial
gauge length of 7 mm and had an average width and thickness of
3 mm?0.4 mm.
2.2 Mechanical Testing and Data Analysis. Samples were
placed in a 37 deg phosphate buffered saline bath and loaded in a
tensile testing system (Instron, Norwood, MA) integrated with a
polarized light setup, consisting of a linear backlight (Dolan-
Jenner, Boxborough, MA), 90 deg-offset rotating polarizer sheets
(Edmund Optics, Barrington, NJ) on either side of the test sample,
and a digital camera (Basler, Exton, PA) (Fig. 1) . Prior to
testing, the encoder embedded in the stepper motor (Fig. 1) (Lin
Engineering, Santa Clara, CA) that rotates the polarizer sheets
was initialized by resetting the encoder value with the polarizer
sheets set at a position corresponding to 0 deg of angular rotation.
To determine biomechanical properties, tensile testing along the
long axis of the tendon was performed using a 100 N load cell
with a resolution of 0.001 N for all tests.
This study utilizes an established uniaxial mechanical testing
protocol for rat rotator cuff tendon mechanical testing to examine
and compare tendon mechanical properties at specific locations
and in response to different treatments . At several points
throughout the mechanical testing protocol, sets of 14 images
were acquired as the polarizers rotated through a 125 deg range
for measurement of fiber alignment during loading as previously
described . Initially, samples were preloaded to a nominal load
(0.01 N) and then preconditioned under load control for 10 cycles
between 0.1 N and 0.5 N. Preconditioning cycles were performed
at an average frequency of 0.2260.03 Hz and cycled between
0.9860.06% and 1.1360.03% grip-to-grip strain. Precondition-
ing is performed before the stress relaxation and ramp-to-failure
tests to provide all samples with a consistent loading history to
allow for more consistent and simplified mathematical interpreta-
tion of mechanical properties. The preconditioning loads used in
this study were selected to ensure that the tendons would not be
damaged during preconditioning and were selected based on pilot
studies with intact and injured and repaired rat SSTs. The loads
are low to enable the preconditioning protocol to be applied for
both injured and intact tendon to compare their mechanical prop-
erties across studies. One 14 image alignment map was acquired
both before preconditioning (but after the preload was adminis-
tered) and immediately following the 10th cycle of precondition-
ing to determine if the spread of the collagen fiber distribution
changed during preconditioning (that collagen fiber re-alignment
occurred) (Fig. 2; points 1 and 2, respectively).
After returning to the lower load limit of 0.1 N following the
10th cycle of preconditioning, a 300 s hold was applied to allow
the tissue to equilibrate before the stress relaxation test. In addi-
tion to the alignment map taken immediately following the 10 pre-
conditioning cycles, an additional alignment map was acquired at
the end of the 300 s hold to determine if collagen fiber alignment
changed over the 300 s time course (Fig. 2; point 3). Next, to
measure viscoelastic properties, samples were then subjected to a
relative ramp of a 0.42 mm grip-to-grip displacement at a rate of
0.35 mm/s, followed by a 600 s hold to reach an equilibrium load.
A series of alignment maps was taken every 5 s during the stress
relaxation test to determine if the initial 0.42 mm grip-to-grip
showing polarized light and imaging system: light source,
rotating cross-polarized sheets, stepper motors, and camera
Angled side-view of the tendon tensile testing setup
the mechanical testing protocol. Fourteen-image alignment
maps were taken for alignment analysis at (1) before precondi-
tioning, (2) after preconditioning, (3) after preconditioning fol-
lowing a 300 s hold, (4) after the SR displacement, (5) during
SR, (6) after SR following a return to zero displacement, (7) at
the transition strain, and (8) at a point in the linear-region.
Load-time graph of a representative sample undergoing
031007-2 / Vol. 134, MARCH 2012Transactions of the ASME
displacement as well as the subsequent 600 s hold affected the
collagen fiber distribution.
Immediately following the 600 s hold, samples were returned
to zero displacement by being displaced 0.42 mm at a rate of
?0.35 mm/s. To measure toe-region properties during the ramp-
to-failure, samples were returned to zero-displacement via dis-
placement control; therefore some samples maintained a nominal
load during the 60 s hold. Pilot studies demonstrated that a return
to zero displacement was more consistent and repeatable than a
return to zero load. An additional alignment map was taken fol-
lowing the return to zero displacement (Fig. 2; point 6). Following
the 60 s hold, a ramp to failure was applied at a rate of 0.21 mm/s.
Alignment map images were taken every 5 s during the ramp-to-
failure. Images were also obtained every 5 s for optical strain
analysis during the ramp-to-failure.
A custom MATLAB program (Matlab, Natick, MA) was used to
optically track strain lines during the ramp-to-failure as previously
described . Stress was calculated as force divided by initial
area. A pilot study examined the best model to fit the load-to-fail-
ure data and determined the most accurate way to compare the
optical strains measured to fiber re-alignment. A structural fiber
recruitment model , an exponential model, and a bilinear fit
model were all used on a subset of data. The pilot study deter-
mined that the bilinear fit model provided the most consistent and
accurate representation of the data and therefore was implemented
for this study. A bilinear curve fit was applied to the optical
stress-strain data to quantify optical transition stress, optical tran-
sition strain, and the moduli in the toe and linear regions from the
optical stress-strain data. Following the bilinear fit analysis, points
representing the toe and linear regions of the optical stress-strain
curve were selected to examine how the collagen fiber distribu-
tions changed during the toe and linear regions of the optical
stress-strain curve. Alignment maps from the optical transition
strain (calculated from the bilinear fit) and a point in the linear-
region were selected for fiber alignment analysis.
Fiber alignment was calculated from the image sets as
described . Briefly, images of the tendon surface were divided
into rectangular areas (30-wide?30-long¼900 areas). Pixel
intensities were summed by area per image and plotted against
angle of polarizer rotation. A sine wave was fitted to the intensity-
angle data to determine the angle corresponding to minimum pixel
intensity, which represents the average direction of the area’s
collagen fiber alignment. A limitation of the crossed polarizer
method is that fiber angles can only be calculated within a 90 deg
range (645 deg to the predominant fiber direction) rather than the
entire possible range of orientations. To overcome this limitation,
fibers were assumed to re-align in the direction of loading as pre-
viously described . Circular variance (VAR), a measure of the
distribution of collagen fiber alignment (from Eq. 26.17 in Zar
et al.), was calculated at several points throughout the mechanical
test [17,18]. VAR was calculated for fiber distributions before pre-
conditioning (BP), immediately after preconditioning (AP), fol-
lowing a 300 s hold before stress relaxation (300h), immediately
after the SR displacement (SRdisp), at the end of the SR test (SR),
after the SR test following a return to zero displacement (zero), at
a point representing the toe-region of the optical stress-strain
curve, and at a point representing the linear-region of the optical
stress-strain curve (Fig. 2). Fiber re-alignment throughout the
mechanical test was evaluated by comparing VAR values at two
mechanical testing points. Collagen fiber re-alignment was said to
occur during that region of the mechanical test if the difference of
fiber distributions was found to be statistically significant. Fiber
re-alignment during preconditioning was evaluated by comparing
VAR values before and after preconditioning. Similar methods
were used to determine fiber re-alignment during the 300 s hold
following preconditioning, during the SR, after a return to zero
displacement following SR, as well as in the toe and linear regions
of the stress-strain curve. Mean angle values were calculated for
collagen fiber distributions at each location for all mechanical test
2.3 Statistical Analysis. Shapiro–Wilk tests indicated non-
normally distributed data for VAR values. As a result,
alignment (decreased VAR) during preconditioning, the dis-
placement for SR in the toe and linear regions for midsubstance
and insertion site. A decrease in alignment (increased VAR)
was noted following the return to zero displacement for both
locations. (*P<0.0125, **P<0.0025, ***P<0.000255sig.).
Circular variance (VAR) values demonstrate increasing
of the optical stress-strain curve. Insertion site linear modulus
is lower than the midsubstance indicating inhomogeneous me-
chanical properties of the rat SST. (***P<0.000255sig.).
Linear modulus values obtained from the linear region
tion point (intersection of toe and linear regions of the optical
stress-strain curve) is shown for both locations. A higher local,
optical strain is necessary for the insertion site of the tendon to
transition to the linear region than for the tendon midsub-
Local optical strain values required to reach the transi-
Journal of Biomechanical EngineeringMARCH 2012, Vol. 134 / 031007-3
nonparametric statistical tests were used for evaluating fiber re-
alignment. Changes in fiber alignment (Friedman test) were com-
pared for tendon location (midsubstance versus insertion) and for
the mechanical test region (preconditioning, stress-relaxation, toe
and linear region). Bonferroni corrections were used for multiple
comparisons to remain conservative in our data interpretation
and significance (P<0.0125¼0.05/4) was determined using
Wilcoxon signed-rank post hoc tests. VAR data are presented
as median6interquartile ranges, and statistics are paired compar-
isons. Mechanical parameters were evaluated with parametric
statistics. Changes in parameters were compared for tendon
location (midsubstance versus insertion). Data are presented as
VAR values and mean angle were examined at several stages of
the mechanical test. Similar results were found for re-alignment
behavior and mean angle changes at each location. During precon-
ditioning, VAR values demonstrate significant re-alignment under
tension (decreasing VAR). Additionally, the mean angle shifted
an average of 4 deg in the midsubstance and 5.5 deg at the inser-
tion site location during preconditioning. During the 300 s hold
following preconditioning, no significant changes in re-alignment
were found at either location. After the initial displacement was
performed for the SR test, a decrease in VAR (increase in organi-
zation) was found at both locations. However, no significant
changes in re-alignment were found throughout the 600 s
stress relaxation test for either location (Fig. 3). Following the
stress relaxation test, the sample was returned to zero displace-
ment, and a more disorganized collagen fiber distribution was
found at both locations. Finally, the collagen fiber distribution
became more organized (decrease in VAR) throughout the ramp
to failure. Significant differences in collagen fiber distributions
were found at both the toe and linear regions at both locations
Locally, VAR values were significantly different (less organ-
ized at the insertion site compared to the midsubstance) at
all mechanical testing points. Linear-region moduli were signifi-
cantly greater in the midsubstance than in the insertion site
(1.6?greater, Fig. 4), and a trend was present for the toe-region
moduli. Additionally, moduli values demonstrated that both the
insertion site and midsubstance locations were highly nonlinear
(?10?linear/toe-region ratio). Optical transition strain (deter-
mined from the optical stress-strain data) was higher for the inser-
tion site than for the midsubstance location (Fig. 5).
This study found a correlation between cyclic preconditioning
and early re-alignment of collagen fibers regardless of location.
Contradictory to our hypothesis, the largest amount of collagen
fiber re-alignment occurred during preconditioning (Figs. 3 and
6). Additionally, the largest shift in mean angle also occurred
during preconditioning. While previous work has noted some col-
lagen fiber re-alignment during preconditioning, the large shift in
alignment seen at such small loads in the present study was
surprising [1,19]. These findings suggest that the re-alignment
of collagen fibers may be an underlying mechanism of precondi-
tioning as well as a potential explanation for the increase in
tendon strength seen after preconditioning in highly aligned ten-
dons. This result supports previous studies examining the
post-preconditioning mechanical response of patellar and Achilles
tendons [6,20]. The midsubstance location of the rat SST consists
mainly of parallel oriented fibers that can be gradually re-oriented
toward the direction of loading during preconditioning cycles.
Additionally, a vector correlation algorithm has recently been
used in ligament to detect microstructural changes in collagen
fiber alignment during preconditioning and found a strong correla-
tion between collagen fiber rotation and changes in force . The
mean angle shift toward 90 deg found in the present study during
preconditioning supports the concept that fibers re-align in the
direction of loading and could account for the changes in mechan-
ical response noted post-preconditioning [1,21–23]. Additionally,
no changes in alignment were found following the 300 s hold
applied post-preconditioning (Fig. 3). These findings indicate that
holding the tendon at a constant load for 300 or less seconds may
not signal a decrease in collagen fiber alignment with short recov-
ery times. It is possible that the response of collagen fibers
depends not only on the amount of relaxation time but also the ini-
tial displacement and loading history. After the 300 s hold was
performed, a 0.42 mm grip-to-grip displacement was performed.
Following the displacement, VAR values decreased, demonstrat-
ing an increase in collagen fiber organization at both locations.
Next, a 600 s stress relaxation test was performed. Contrary to
our hypothesis, no change in collagen fiber re-alignment was
found during the stress relaxation test (Figs. 3 and 6). Interest-
ingly, crimp analysis in rabbit medial collateral ligament revealed
no changes in collagen fiber crimp behavior before and after stress
relaxation tests, while fibers were recruited during creep tests
, supporting the idea that the microstructural mechanisms of
creep and stress relaxation are different . This finding together
with the lack of collagen fiber re-alignment during stress relaxa-
tion in the present study suggests that a shift in the structural orga-
nization of collagen fibers may not be responsible for stress
Following the stress relaxation test, samples were returned to
zero displacement, and a decrease in collagen fiber organization
(increased VAR) was found compared to the distribution imaged
at the end of the stress relaxation test at both the midsubstance
and insertion site locations. This finding suggests that returning to
a reference point (marked by a decrease in both load and displace-
ment) may cause a decrease in collagen fiber alignment at both
locations allowing the tendon’s collagen fibers to re-orient away
displacement for the stress relaxation test, followed by a decrease in alignment after returning to zero-displacement and
increases in toe-region (comparing return to zero and transition) and linear-region (comparing transition and linear-region).
(*P<0.0125, **P<0.0025, ***P<0.000255sig.).
Midsubstance histograms for a representative sample show increasing alignment during preconditioning, during the
031007-4 / Vol. 134, MARCH 2012Transactions of the ASME
from the direction of loading. This supports previous work in
which a tissue-equivalent demonstrated a recovery of fiber align-
ment during a stress-free return to zero-displacement but not dur-
ing a return to zero-force, suggesting that fiber re-alignment away
from the direction of loading occurred only after a return to an
un-stretched length during unloading . While the majority of
fiber re-alignment occurred during preconditioning in our study,
significant re-alignment was also found throughout the tensile
ramp-to-failure test in both the toe and linear regions of the optical
stress-strain curve for both the midsubstance and insertion site
locations (Fig. 3). Additionally, no significant differences in colla-
gen fiber organization were found between the two “rest” points
(after the 300 s hold and following the return to zero displace-
ment) at either locations (data not shown).
Further, this study reports the inhomogeneous, nonlinear me-
chanical properties and fiber alignment of the rat SST. After
examining collagen fiber re-alignment throughout the entire
mechanical testing protocol, similar patterns in collagen fiber re-
alignment were found for fiber distributions between the midsub-
stance and insertion site locations at all mechanical testing points,
indicating that the tendon functions as a continuous unit. Further-
more, local differences in mechanical properties and collagen
fiber alignment, at all regions of the mechanical loading profile,
demonstrate that the rat SST is inhomogeneous. The lower moduli
(Fig. 4) and higher VAR values (signifying increased disorganiza-
tion) found at the insertion site compared to the midsubstance
location demonstrate that the multiaxial loads experienced at the
SST insertion may affect both the structure and mechanics of the
tissue. Additionally, a higher strain was required for the insertion
site to reach the linear region than the tendon midsubstance
(Fig. 5). This suggests that the more disorganized insertion site
may require more microstructural changes before it is able to tran-
sition to the linear region of the stress-strain curve, such as colla-
gen fiber crimp, which is believed to explain the toe-region of the
stress-strain curve [25–29].
This study is not without limitations. First, the crossed polarizer
method used in this study can only measure fibers 645 deg of the
tendon long axis (90 deg total range), instead of the full 180 deg
range that would contain all possible fiber orientations. While
other methods are available to determine collagen fiber orienta-
tion, we selected the crossed polarizer method as it is the simplest
method to provide the data necessary for analysis to test our study
hypotheses. An angle value correction was applied based on the
assumption that fibers must reorient toward the direction of load-
ing. Second, the observations of collagen fiber re-alignment
behavior made in this study were for one specific mechanical test-
ing protocol. Despite this limitation, this study provides insight
into the understanding of tendon fiber re-alignment throughout the
mechanical testing protocol in addition to examining potential
mechanisms of preconditioning and stress relaxation. While colla-
gen fiber re-alignment at the low loads seen during the applied
preconditioning protocol may be a function of the mechanical
testing protocol, imposed uniaxial boundary conditions, or re-
moval of the native tension in the tendon, it is still important to
understand what effects or influence the mechanical testing proto-
col and experimental design set up itself has on the measured me-
chanical properties because these are commonly used in the
biomechanical testing literature and interpreting such data for
clinical relevance. Preload and specific preconditioning protocols
are not consistently reported in tendon literature. The results of
this study suggest that the mechanical testing protocol itself may
have implications on the consistency and repeatability of the me-
chanical properties measured; therefore, it is important to report
the potential effects of preconditioning protocols, their implica-
tions on interpretations of mechanical properties, and relating
future ex vivo work to the physiologic condition. Further, this
study provides valuable data regarding the specific mechanical
and organizational properties of the rat SST.
Future studies in our laboratory will investigate if the amount
of collagen fiber re-alignment is dependent on the preconditioning
protocol as well as studies to investigate if local changes in colla-
gen fiber crimp are present to account for the differences noted in
transition strain. It is possible that the insertion site is more
crimped, requiring an increased strain to reach the linear region as
noted in this study. Further studies are necessary to evaluate struc-
tural changes throughout a variety of mechanical testing protocols
to determine if the changes observed are a result of the strain rate,
amount of load versus the type of loading (load control, displace-
ment control). Finally, the amount of time and conditions neces-
sary for collagen fiber orientation to return to its original
orientation is currently unknown. Future studies incorporating
longer rest intervals are necessary to determine what conditions
are necessary for fiber alignment to return to re-align away from
the direction of loading.
The authors would like to acknowledge Elizabeth Feeney,
David P. Beason, and Joseph J. Sarver for assistance and the NIH/
NIAMS for financial support.
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