Name: Ex vivo Mechanical Loading of Tendon (Video) | JoVE Journal
Uploaded: 2007-05-28T00:00:00.000+00:00
Duration: 11 min 36 s
Description: Learn ex vivo mechanical loading of tendons using a multi-actuator system for bioengineering research. This method enables precise cyclic loading to study tendon disorders and injury mechanisms.

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<ol>
<li>Supplement &nbsp;media CO2-independent media with Fetal Bovine serum, l-glutamine, antibiotics, and ascorbic acid</li>
<li>Aliquot 40 ml into 50 ml centrifuge tubes (1 for each tendon)</li>
<li>Aliquot 10 ml into 15 ml centrifuge tubes (1 for each tendon)</li>
<li>Sterilize surgical tools, autoclave sheets, tweezers and torque wrench in autoclave</li>
<li>Sterilize clamps and force micrometer tips by soaking in 70% ethanol overnight</li>
<li>Harvest tendons from the rear paws of the rabbit. Rabbit should have been euthanized no more than 2 hours ago.</li>
<li>Place tendons in 15 ml centrifuge tube filled with media</li>
<li>Lay out a sterilized autoclave sheet to establish a sterile field</li>
<li>Measure cross-sectional area of tendon <ol style="margin-top: 0in">
<li>Place micrometer tips on micrometer, align properly</li>
<li>Grip tendon by the ends</li>
<li>Fit thinnest tendon section in groove</li>
<li>Have assistant place 50g weight</li>
<li>Wait 30 seconds, read thickness (CSA = thickness x 1.3)</li>
</ol></li>
<li>Place tendon back in 15 ml centrifuge tube</li>
<li>Repeat with each tendon to be loaded</li>
<li>Remove actuator from base</li>
<li>Cut two 15mm by 40mm strips and one 3mm by 5mm piece from sterile gauze for each tendon</li>
<li>Roll each strip tightly and place between the grooves of the distal clamp</li>
<li>Remove tendon from centrifuge tube and place on clamp, the distal end of the tendon on the distal clamp and the fibrocartilage region of the tendon on the proximal clamp</li>
<li>Place 3x5 gauze piece on top of the distal end of the tendon, and secure the clamp</li>
<li>Secure proximal clamp, ensuring tendon remains aligned between the clamps and with enough slack that it is not under tension when clamps are tightened. Tighten clamps to 1N-m</li>
<li>Place 50ml centrifuge tube with media in base</li>
<li>Place actuator in base</li>
<li>Repeat with each tendon to be loaded</li>
<li>Place base over water bath</li>
<li>Loading Protocol <ol style="margin-top: 0in">
<li>Precondition tendons</li>
<li>Apply 0.5N load, measure tendon length (this is the gauge length)</li>
<li>Apply cyclic loading</li>
</ol></li>
<li>Remove actuator from base</li>
<li>Release tendon from clamps</li>
<li>Process tendon</li>
</ol>

<ol><li>Asundi, K., Kursa, K., Lotz, J. C., Rempel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+system+for+applying+cyclic+loads+to+connective+tissues+under+displacement+or+force+control%5BTitle%5D)+AND+%22Ann+Biomed+Eng%22%5BJournal%5D)">In vitro system for applying cyclic loads to connective tissues under displacement or force control.</a> Ann Biomed Eng. 35, 1188-1195 (2007).</li>
<li>Asundi, K. R., Rempel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cyclic+loading+inhibits+expression+of+MMP-3+but+not+MMP-1+in+an+in+vitro+rabbit+flexor+tendon+model%5BTitle%5D)+AND+%22Clin+Biomech%22%5BJournal%5D)">Cyclic loading inhibits expression of MMP-3 but not MMP-1 in an in vitro rabbit flexor tendon model.</a> Clin Biomech. , In Press Forthcoming.</li>
<li>Asundi, K. R., Rempel, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MMP-1%2C+IL-1%CE%B2+and+COX-2+mRNA+Expression+is+Modulated+by+Static+Load+in+Rabbit+Flexor+Tendon%5BTitle%5D)+AND+%22Ann+Biomed+Eng%22%5BJournal%5D)">MMP-1, IL-1β and COX-2 mRNA Expression is Modulated by Static Load in Rabbit Flexor Tendon.</a> Ann Biomed Eng. , In Press Forthcoming.</li>
</ol>

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...

ex vivo mechanical loading of tendon

Ex vivo Mechanical Loading of Tendon

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&deg;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.

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

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

My name's David Rempel. I'm a faculty in the Department of Bioengineering at u at the University of California at Berkeley. The experiment you're gonna see involves stretching tendons and really the purpose is to try to underline the, understand the underlying mechanism to tendon disorders at the wrist and elbow.These tendon disorders are common among athletes and workers who do repetitive work with their hands. Krish Sundi, one of the doctoral students in our laboratory, will demonstrate several features of the system, specifically the measurement of the cross-sectional area of the tendon. He'll load the tendons into the actuators.He'll demonstrate the system loading the tendon cyclically over a 24 hour period and then remove the tendon from the system. Okay, so this is the loading system that we've designed and built. As you can see, it consists of six actuators.These are high precision actuators, which are capable of applying displacement of one micrometer in series with the actuator is a load cell to record loads applied to tendons. The in series with the load cell is, are these clamps which extend away far enough so that they can be submerged in media in order to keep tendons viable. This entire system would then be placed in a water bath to maintain the media at 37 degrees Celsius.What I'm gonna show you right now is how I would go about loading a tendon into this system. The first step we need to do is measure the cross-sectional area of the tendon so that we can apply the appropriate stresses. Can you pass me the tube with the tendon?Now, these tendons have been harvested from a New Zealand white rabbit. They have, they're just been brought over, so they're still fresh within less than two hours of have the rabbit being euthanized. A portion of the tendon whose cross-sectional area that I'm measuring is along the thinnest part of the tendon.This is because this is the point where the greatest stresses will be, will be resulting because of the low, smallest cross-sectional area. Can you now put the weight on top of the piston? This now we wait 30 seconds in order to read the, the thickness read 1.3.Can you lift the piston and replace the tendon back in the media? And I'm going to unmount dismount the actuator in which I'm going to load the tendon. So now I'm going to mount a tendon into the clamps.Here we can have a closeup of the actuator. You can see the, the actuator itself, the load cell, and the clamps, which extend away These pads cause pads. I would slice approximately half an inch to three quarters inch wide and approximately one and a half inches long.Then take these gauze pads and dip them in the media in order to wet them. It makes the rolling process easier And place these rolled gauze strips In the grooves of these serpentine clamps. I'm now going to remove the tendon from the media.This end is the insertion site of the tendon of the flexor tendon into the end of the, the, the digit of the rabbit. This region here is a fiber cartilage region. The tendon is placed in such a position such that the fiber cartilage region is clamped within the proximal clamp.The tenant is then clamped, and again, the screws are tied into one newton meter. Then the excess tendon is cut, so now the actuator is secured into the brace and the entire tendon and clamps are submerged. In modified media, The same procedure would be used to mount tendons on subsequent actuators.As you can see, we have a total of six, so we can do this with six individual tendons. This entire system is then lifted and placed into a water bath in order to maintain the media at 37 degrees. So now we're gonna apply a cyclic load to the tendon between 0.5 and five megapascals.The software we use is lab view and it's custom written. The first thing we do is to precondition the tendon. We apply 20 cycles of cyclic load between 0.5 megapascals and two megapascals.As for the thickness measured, use it using the four supplied micrometer to put that in. It will cal automatically calculate the cross-sectional area. The program will then instruct the actuator to increase the displacement until a two megapascal load is applied to the tendon.Here what we're following is the purple trace. Once a two megapascal load is applied to the tendon, the program will then apply a 0.5 megapascal load to the to the tendon. And once these two loads are found, the program will sit cycle between these two loads, updating the position continuously between these two desired loads.This loading is being done at 10 megapascals per second at a frequency of 0.5 hertz. Once we have preconditioned the tendon, we then seek a 0.5 newton load. We define the length of the tendon under a 0.5 Newton load as the gauge length of the tendon.Once the program has found this load, we read the position of the actuator and this position, which reads currently six eight hundred and twenty five, is the gauge length of tendon. After we've recorded the gauge length of the tendon, we then return to the cyclic loading program, input our desired values of five megapascals and 0.5 megapascals, input the total number of cycles we wanna load it to, and again, run through the same program in order to, in order to cyclically load the tendon between 0.5 and five megapascals. Again, this loading is done at 10 megapascals per second with a frequency of 0.5 hertz.So after being cyclically loaded, typically for 24 hours, the actuator is then removed from the system and from here the tendon can be cut and processed as desired. Well, you've just seen our tendons that were harvested sterly from a rabbits. The process you've saw seen was a semi sterile process, but it led to a cyclical loading of the tendons over a 24 hour period.The tendons are subsequently analyzed using R-T-P-C-R for gene expression. We're also analyzing the tendons for other changes, structural changes, other histologic markers. Ultimately, the purpose of this experiment is determine what effect different loading patterns have on basically the underlying mechanisms of injury to the tendons that might be associated with tendinopathies and tendonitis.

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Learn ex vivo mechanical loading of tendons using a multi-actuator system for bioengineering research. This method enables precise cyclic loading to study tendon disorders and injury mechanisms.

Ex vivo Mechanical Loading of Tendon

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