The document summarizes a study that characterized the biomechanical properties and regional strain patterns of murine Achilles tendons. The following key points were reported: 1. Murine Achilles tendons exhibited significant regional variations in tissue strain, with the highest strain observed at the insertion region and the lowest in the distal midsubstance region. 2. At peak forces seen in other animal models, the insertional strain in murine Achilles tendons was significantly greater than both the distal and proximal midsubstance regions. 3. Future work will focus on using this testing method to observe the biomechanical response and regional strain variations in mutant mouse models lacking tendon transcription factors, to study tendon injury models.

1. Kallenbach, Jacob G.1 ; Gilday, Steve D.1 ; Shearn, Jason T.1
INTRODUCTION RESULTS
METHODS
1Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA
Murine Achilles Tendon Biomechanical Properties
and Regional Strain Patterns
• Injuries involving the Achilles tendon (AT) occur in 5.5 to 10 individuals per 100,000 each year in
North America resulting in significant pain, discomfort, and disability[4]. Elucidating the
mechanisms of injury and healing process behind many treatments and rehabilitation protocols
is inconclusive[1].
• Because current clinical tests cannot evaluate Achilles tendon mechanical properties in vivo,
there is vast clinical importance of utilizing methods to test the Achilles tendon at normal
physiological levels in diseased and injured animal models through fatigue and failure loading.
Objective
• Understand the mechanical properties of healthy murine Achilles tendon tissues in order to relate
to structural changes seen before injury, during healing, and after injury.
Hypothesis
• Normal, un-operated murine Achilles tendons will exhibit significant regional variations in
tissue strain.
DISCUSSION
1. The purpose of this study was to characterize native adult murine Achilles tendon mechanical and structural
properties by designing and validating a repeatable testing mechanism and analysis method. Regional strain
variations were found to be statistically different between insertion, distal midsubstance and proximal midsubstance in
the murine strain C57BL/6J AT.
2. At peak in vivo forces seen in rabbit patellar tendon[5] and in goat patellar tendon[6], insertional strain in the murine
Achilles tendon is significantly greater than both distal and proximal midsubstances. Increased strain at the insertional
AT is clinically important in AT injuries as it may lead to insertional AT tendinopathy, which is a chronic condition
causing pain, impairment, and swelling at the insertion[3].
3. Future work will focus this testing and analysis protocol on various mutant mouse models, who have missing tendon
specific transcription factors, to observe the biomechanical response and regional strain variations in an injury model.
REFERENCES
[1] Freedman BR. J Biomech. 2014. [2] Dyment NA. Journal of Orthopaedic Research. 2012. [3] Hu CT. Operative Techniques in Orthopaedics. 2008. [4]
Suchak AA. Foot and Ankle International. 2005. [5] Juncosa N. J Biomech. 2003. [6] Korvick DL. J Biomech. 1996.
ACKNOWLEDGEMENTS
I thank Dr. Christy Holland, Dr. Daria Narmoneva, and Cindi Gooch for their technical assistance and Jessica Arble for moral support.
50μm
C57BL/6J AT Average Structural and Material Curves
Figure 3: Average murine AT Structural and Material Curves. Average curve of Load vs. Displacement (A). Average stress-strain curve for normal murine
Achilles tendons (B). Error bars indicate standard error margin (SEM); n=12.
Animal model
• Adult C57BL/6J male mice at 18-20 week old (n=12, Fig 1).
• Mice displayed normal tendon morphology with before dissection (Fig 1).
Achilles Tendon Dissection and Biomechanical Testing
• After examining the gross morphological appearance, the murine AT was isolated by dissecting
away the tibia, fibula, tarsal, and metatarsal bones leaving the calcaneus bone intact (Fig 1A,B).
After removal of the gastrocnemius musculature, the calcaneus and AT whole sample was
secured and mounted in a materials testing system (100R Test Resources).
• Once mounted, tendons were submerged in 37C phosphate buffered saline and preloaded to
0.02N. Samples were preconditioned at 25 cycles, 0-1% strain, 3 microns/sec, and failed in
uniaxial tension at a rate of 3 microns/sec [2] while recording displacement and load (Fig 1C,D).
Figure 1: Murine Achilles Tendon Dissection and Experimental Setup. Male murine Achilles Tendon (The Jackson Laboratory)
(A). Murine Achilles tendon total length (B). Grip to grip mounting of AT with calcaneus embedded in bottom grip (C). Materials
testing system full experimental setup (D).
Optical Strain Analysis
• After dissection and before mounting, 6-0 silk suture saturated in Verhoeff’s stain was used to
create two local strain marks on the AT 1/3 the total distance up from the calcaneus and a
second 2/3 of the total distance up from the calcaneus (Fig 2A).
• Once mounted, high resolution digital images were taken in the sagittal and frontal planes to
calculate initial AT dimensions. Images of the posterior surface of the AT were captured at 5
second intervals throughout the failure test to optically measure regional tissue strains and
assess failure location (Fig 2B-D).
• Regional tissue strains were computed by optically tracking applied stain lines for each
specimen using the MTrack2 plugin for ImageJ (Fig 2B-D). Raw displacement data was utilized
to calculate and plot tensile strain in the insertion, distal midsubstance, and proximal
midsubstance as a function of load.
Figure 2: Optical Strain Analysis. Achilles tendon specimens were marked with two stain lines and loaded into the tensile testing system
(A). Local tissue strain is calculated utilizing with high resolution images (B). Thresholded 8-bit binary grayscale image of centroid (C).
Frame by frame displacement of centroid paths tracked by MTrack2 plugin for ImageJ (D).
C57BL/6J AT Regional Strain Outcomes in Murine Achilles Tendons
Figure 4: Regional Strain Variations. Normal murine Achilles tendons exhibited distinct regional variations in tissue strain at all levels greater than 0.25N. At
failure, insertion reached a maximum strain of 49.21% +/- 7.15 (mean+/-SEM) compared to both distal 10.18% +/- 5.25 and proximal 39.82% +/- 7.35
midsubstances (A). At 21% and 40% of normal AT failure force, normal tendons showed increased local strain in the insertion region compared to both distal
and proximal midsubstances (B). Curves represent average of 12 specimens; error bars indicate SEM and statistical significance * p<0.05.
A CB D
{
{
{Proximal
Midsubstance
Distal
Midsubstance
Insertion
Calcaneus
A DCB
0
10
20
30
40
50
60
0 1 2 3 4 5 6 7
OpticalStrain(%)
Load (N)
Insertion
Distal Midsubstance
Proximal Midsubstance
*
*
* *
* *
* * * * * * * * *
* * * * * * * * *
0
10
20
30
40
50
60
21% of Failure Load 40% of Failure Load 100% of Failure Load
OpticalStrain(%)
Insertion
Distal Midsubstance
Proximal Midsubstance
*
*
*
*
*
0
2
4
6
8
10
0 0.5 1 1.5 2
Load(N)
Grip-to-Grip Displacement (mm)
C57BL6
0
2
4
6
8
10
0 4 8 12 16 20 24 28 32
Stress(MPa)
Strain (%)
C57BL6
AT
DISSECTION
TENDON MOUNTED IN
SYSTEM
FROM FAILURE TEST
Age (wk)
Length
(mm)
Avg CSA
(mm^2)
Ult Load (N)
Stiffness
(N/mm)
Disp at Ult
Load (mm)
Ult Stress
(MPa)
Modulus
(MPa)
% Strain at Ult
Stress
Mean 19.250 5.312 0.819 7.194 10.640 1.610 9.082 74.095 30.501
SD 0.783 0.444 0.172 0.946 3.713 0.436 1.880 40.320 8.219
SEM 0.226 0.128 0.050 0.273 1.072 0.126 0.543 11.639 2.373
Table 1: Average Mouse AT Data. Average structural and material properties of normal mouse AT (n=12).
C57BL/6J AT Specimen Structural and Material Average Data
A B
*