Ex vivo Mechanical Loading of Tendon

Summary

A new in vitro system for simultaneously loading four tendons in culture is described.

Abstract

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37°C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

Protocol

1. Supplement  media CO₂-independent media with Fetal Bovine serum, l-glutamine, antibiotics, and ascorbic acid
2. Aliquot 40 ml into 50 ml centrifuge tubes (1 for each tendon)
3. Aliquot 10 ml into 15 ml centrifuge tubes (1 for each tendon)
4. Sterilize surgical tools, autoclave sheets, tweezers and torque wrench in autoclave
5. Sterilize clamps and force micrometer tips by soaking in 70% ethanol overnight
6. Harvest tendons from the rear paws of the rabbit. Rabbit should have been euthanized no more than 2 hours ago.
7. Place tendons in 15 ml centrifuge tube filled with media
8. Lay out a sterilized autoclave sheet to establis....

Discussion

The video presents a new system for the simultaneous loading of up to four live tendons in tissue culture. Typical loading durations are 2 to 48 hours. After loading, the tendons are removed and processed for gene expression. The system allows for the investigation of the effect of loading pattern on changes in genes associated with degenerative changes in the tendon. These experiments can provide some insight into mechanisms of injury associated with repetitive loading. They also provide insight into loads that can.......