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How To Study Structural and Functional Properties of Tendon

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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

Published in: Science
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How To Study Structural and Functional Properties of Tendon

  1. 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
  2. 2. InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in the sharing and distribution of scientific information regarding innovative technologies, protocols, research tools and laboratory services. JOIN FOR FREE AT WWW.INSIDESCIENTIFIC.COM
  3. 3. Matthew Borkowski Sales and Support Manager, Aurora Scientific Inc. mattb@aurorascientific.com Equipment for Mechanical Testing of Tendon and Connective Tissue
  4. 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. 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. 6. How Does it Work ? Learn More Online >>
  7. 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. 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. 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. 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. 11. System Control & Software
  12. 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. 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. 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. 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. 16. Tendon Structure • Tendon is organized similar to muscle • Made of fibrillar and network collagens • Largely hypocellular tissue
  17. 17. Collagen Structure Collagen - Type I and III + Collagen - Type IV and VI (Ortega and Werb, 2002)(Hardin et al, 2012) Collagen Fiber =
  18. 18. Tendon Structure
  19. 19. Fibroblasts Tendon Composition
  20. 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. 21. Healthy Tendon Tendinosis Soslowsky 2000 2014 Orthocentre  Tendinitis  Tendinosis Tendinopathies  Tendon Rupture
  22. 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. 23. To Evaluate Sex-based Differences in Tendon • Mechanics • Cell Biology • Proteomics • Gene Expression ? vs. Study Purpose
  24. 24. • RNA Isolation • Mechanics • Histology • Proteomics • RNA Isolation • Histology • Proteomics • Fibroblasts • Collagen Methods
  25. 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. 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. 27. Cross-Sectional Area
  28. 28. Measure CSA Tendon Fixation
  29. 29. Tendon Fixation
  30. 30. Plantaris Tendons: - 10% stretch (Left) 20% stretch (Right) - 300 stretches Mechanics Overview
  31. 31. Mechanics Overview
  32. 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. 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. 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. 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. 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. 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. 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. 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

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