WATCH THE VIDEO: http://bit.ly/2nl7Nx6 In this webinar presented by Aurora Scientific, Matthew Borkowski and Dylan Sarver discuss how to characterize the structural and functional properties of tendon. Specifically, Mr. Borkowski describes the engineering behind the multi-purpose Aurora Scientific Dual Mode Lever — a fast actuator and sensitive force transducer in one — and how this device can be used to study connective tissue. Following, Mr. Sarver discusses his current research focused on sex-related differences in the structural and functional status of Tendon, from macromolecular structural properties to transcriptomic, proteomic, and cell biology of resident tendon fibroblasts. He explains why tendon research is important, reviews methodology for investigating tendon structure and function, and discusses research findings supporting sex-related differences in tendon. Key topics covered during this webinar will include… - How to use The Dual-Mode Lever to perfom demanding stress/strain assays - What is a tendon, and why is tendon research important - How to characterize the structural and functional status of tendon - Case Study: investigating sex-related differences in tendon

1. How To Study Structural and
Functional Properties of Tendon
Experts show how to study the mechanical properties of
tendon and connective tissue samples by completing
stress/strain assays using a novel Dual-Mode Lever System

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3. Matthew Borkowski
Sales and Support Manager,
Aurora Scientific Inc.
mattb@aurorascientific.com
Equipment for Mechanical Testing
of Tendon and Connective Tissue

4. About Aurora Scientific
• Aurora has built instruments for a
variety of researchers across many
different fields
• Equipment designed for one
experiment often finds a niche in
another un-related field
Muscle Physiology
Environmental &
Mine Monitoring
Neuroscience
Material Science

5. • The Dual Mode Lever is our
Flagship instrument –
designed to measure the
active properties of muscle
• Its high performance relative
to cost make it ideal for
measuring passive properties
of connective tissue
Dual Mode Lever
System
Muscle
Tendon
and
Ligament Artificial
Muscle
About Aurora Scientific

6. How Does
it Work ?
Learn More Online >>

7. Dual Mode Lever
System
• Instrument design based upon a
servo motor with a fast position
response time
• Force derived from the position
detector of the motor and the
electrical current drawn
• Can be used in unique ‘Force
control mode’ – hold a constant
force 309C Dual Mode Lever System -20N

8. • System has very precise resolution of position
and force
• Step response time of position is also very fast;
on the order of milliseconds.
• Opens up the possibility of going beyond
traditional stress/strain assays to look at how
connective tissue responds to a changing load

9. • Lever systems range in size to accommodate
small ligaments up to tendons from rats and
larger animals.
• Single attachment to the instrument simplifies
experimental setup design.
• Lever systems are often paired with
experimental apparatus and software.

10. Experimental
Apparatus
• Temperature controlled bath
for various tissue types
• Fine positioning of transducer
relative to the sample
• Customized clamps and
accessories designed to make
experiments easy
Experimental Chamber shown with
10N Dual Mode Lever System

11. System Control & Software

12. • All experiments performed with our customized acquisition
software (DMC) and saved in an open format
• Stretches, slacks and instantaneous tensile force all fully
controlable and synchronized through software
• Library of standard protocols allows for infinite customization
• Straightforward to use. Once sample attached, load protocol and
begin
System Control & Software

13. Data Analysis
• Software suite for performing
visualization and analysis
• Automatic High Throughput
data analysis module: Fast
scripts to automate stress/strain
• Available programming service
for customer specific features
and free updates released
regularly

14. What Standard Experiments
Can we Perform?
✓ Stress vs Strain rate
✓ Yield Stress
✓ Stiffness
✓ Elastic Modulus
✓ Stress Relaxation
✓ Material Creep
✓ Unique, constant tension
protocols
✓ And many more!

15. Dylan Sarver, ATC
Research Associate,
University of Michigan,
Department of Orthopaedic Surgery
dcsarver@umich.edu
Sex Based Differences in Tendon
Composition and Mechanical Properties

16. Tendon
Structure
• Tendon is organized
similar to muscle
• Made of fibrillar and
network collagens
• Largely hypocellular
tissue

17. Collagen Structure
Collagen - Type I and III
+
Collagen - Type IV and VI
(Ortega and Werb, 2002)(Hardin et al, 2012)
Collagen Fiber
=

18. Tendon Structure

19. Fibroblasts
Tendon Composition

20. Force Transmission
Physiological
Loading
Positive Adaptations:
Energy storage
Peak stress and strain
Tendon CSA
Fibroblast Density
Healthy Tendon
40x HE
40x Polarized HE
Tendon Function and AdaptationAchillesTendonFunction

21. Healthy Tendon Tendinosis
Soslowsky 2000
2014 Orthocentre
 Tendinitis
 Tendinosis
Tendinopathies
 Tendon Rupture

22. Epidemiological Studies Show:
• Achilles tendon rupture more common in males
• Females have increased burden associated with
tendon injury
Why?
? ?
?
?
Sex Differences in Tendinopathy

23. To Evaluate Sex-based Differences in Tendon
• Mechanics
• Cell Biology
• Proteomics
• Gene Expression
?
vs.
Study Purpose

24. • RNA Isolation
• Mechanics
• Histology
• Proteomics
• RNA Isolation
• Histology
• Proteomics
• Fibroblasts
• Collagen
Methods

25. 0.0
0.1
0.2
0.3
0.4
AchillesTendonCSA(mm2)
*
Male
Female
• Males have great Achilles CSA
• Females have a greater Cell Density
• Values are mean±SD
• significant difference (P<0.05)
0
500
1000
1500
2000
2500
CellDensity(cells/mm2)
*
Histology

26. Mechanics Overview
Tendon
Calcaneus
Stationary Hook
Servo Motor & Force
Transducer
0.00 0.02 0.04 0.06 0.08 0.10
0
2
4
6
8
Strain
Stress(MPa)
0 50 100 150 200 250 300
0
20
40
60
80
100
Cycle Number
%ofInitialLoad
A
B
0.00 0.02 0.04 0.06 0.08 0.1
0
2
4
Strain
Stress(MP
0 50 100 150 200 250 30
0
20
40
60
80
100
Cycle Number
%ofInitialLoad
Male Female
B

27. Cross-Sectional Area

28. Measure
CSA
Tendon Fixation

29. Tendon
Fixation

30. Plantaris Tendons:
- 10% stretch (Left) 20% stretch (Right)
- 300 stretches
Mechanics Overview

31. Mechanics Overview

32. 0.00 0.02 0.04 0.06 0.08 0.10
0
2
4
6
8
Strain
Stress(MPa)
80
100
d
A
B
0.00 0.02 0.04 0.06 0.08 0.10
0
2
Strain
Stre
0 50 100 150 200 250 300
0
20
40
60
80
100
Cycle Number
%ofInitialLoad
Male Female
B
Mechanics Overview
0.00 0.02 0.04 0.06 0.08 0.10
0
2
4
Strain
Stress
0 50 100 150 200 250 300
0
20
40
60
80
100
Cycle Number
%ofInitialLoad
Male Female
B

33. 1 20 50 100 300
0
2000
4000
6000
8000
10000
Cycle Number
Stress(kPa)
Male
Female
Values are mean±SD.
* significant difference (P<0.05)
0.00
0.02
0.04
0.06
0.08
0.10
PlantarisTendonCSA(mm2)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Length(mm)
1 20 50 100 300
0
100
200
300
400
500
600
Cycle Number
Load(mN)
Mechanics

34. 1 20 50 100 300
0
25
50
75
100
125
150
175
200
Cycle Number
TangentModulus(MPa)
Male
Female
1 20 50 100 300
0
25
50
75
100
125
150
Cycle Number
EnergyLoss(µJ/mg)
Mechanics

35. 0
1 10
Nor
0
1 108
2 108
3 108
NormalizedAbundance
Biglycan
0
1 106
2 106
3 106
NormalizedAbundance
Versican
0
1 107
2 107
3 107
4 107
5 107
NormalizedAbundance
Tenascin A
*
0
Nor
0
1 108
2 108
3 108
4 108
NormalizedAbundance
Decorin
0
2 108
4 108
6 108
8 108
1 109
NormalizedAbundance
COMP
0
1 108
2 108
3 108
4 108
NormalizedAbundance
Tsp4
0
Nor
0
2 108
4 108
6 108
NormalizedAbundance
Fmod
0
1 107
2 107
3 107
4 107
NormalizedAbundance
Finc
*
0
1 106
2 106
NormalizedAbundance
Tnmd
0
Nor
0
2 107
4 107
6 107
8 107
NormalizedAbundance
Lumican
0
2 106
4 106
6 106
8 106
NormalizedAbundance
Periostin
*
Male
Female
E F G H
I J K L
M N O
0
1 109
2 109
3 109
4 109
NormalizedAbundance
Col1a1
0
1 108
2 108
3 108
NormalizedAbundance
Biglycan
2 106
3 106
dAbundance
Versican
0
1 109
2 109
3 109
NormalizedAbundance
Col1a2
0
1 108
2 108
3 108
4 108
NormalizedAbundance
Decorin
6 108
8 108
1 109
dAbundance
COMP
0
1 108
2 108
3 108
NormalizedAbundance
Col3a1
0
2 108
4 108
6 108
NormalizedAbundance
Fmod
2 107
3 107
4 107
dAbundance
Finc
*
0
1 108
2 108
3 108
NormalizedAbundance
Col12a1
0
2 107
4 107
6 107
8 107
NormalizedAbundance
Lumican
4 106
6 106
8 106
dAbundance
Periostin
*
A B C D
E F G H
I J K L
* P<0.05
Protein

36. • Female Achilles tendons have smaller CSA and
higher cell density than male
• Non-destructive mechanical testing shows no differences
between sexes
• Proteome of male and female tendons are very similar in
structural proteins, but differ in Fibronectin, Periostin, and
Tenascin C abundance
Conculsions

37. Further Detail on
Sex Differences in
Tendon
Sarver, D. C., Kharaz, Y. A., Sugg, K. B.,
Gumucio, J. P., Comerford, E. and Mendias,
C. L. (2017), Sex differences in tendon
structure and function. J. Orthop. Res..
doi:10.1002/jor.23516
Gene Expression
• Whole Tendon
• Cell culture
Tendons play a critical role in the transmission of forces between muscles and
bones, and chronic tendon injuries and diseases are among the leading causes of
musculoskeletal disability. Little is known about sex-based differences in tendon
structure and function. Our objective was to evaluate the mechanical properties,
biochemical composition, transcriptome, and cellular activity of plantarflexor
tendons from 4 month old male and female C57BL/6 mice using in vitro
biomechanics, mass spectrometry-based proteomics, genome-wide expression
profiling, and cell culture techniques. [Read More]
Sex Differences in Tendon Structure and Function
Dylan C. Sarver, Yalda Ashraf Kharaz, Kristoffer B. Sugg, Jonathan P. Gumicio, Eithne Comerford,
Christopher L. Mendias

38. Thank you!
Mendias Lab
• Chris Mendias
• Jon Gumucio
• Kris Sugg
• James Markworth
• Chris Ciric
• Andrew Noah
• Jeff Talarek
Brooks Lab
• Sue Brooks
• Dennis Claflin
• Carol Davis
University of Liverpool
• Eithne Comerford
• Yalda Ashraf Kharaz

39. Matthew Borkowski,
Sales Manager,
Aurora Scientific Inc.
mattb@aurorascientific.com
Dylan Sarver,
Research Associate,
University of Michigan
dcsarver@umich.edu
Thank You!
If you have questions for the presenters please contact
them by email.
For additional information on the solutions presented in
this webinar please visit www.aurorascientific.com