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Claire Hillery
05-26-2002, 09:49 PM
I would like to sincerely thank everyone who responded. Some of the ideas put forward were extremely interesting and I will be getting in touch with some of you in the near future. Thank you again for your help.

Regards

Claire Hillery
Royal London Hospital
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Original question:
I am currently trying to clamp an animal ligament, called ligamentum nuchae. I an encountering a great deal of trouble as the ligament is extremely slipperly and easily deformed. f I clamp the material really tightly it will deform at the grips, slip and still break at the clamps. I was wondering and hoping that someone might have experience in clamping this or a similar material. I will be performing fatigue tests on this material, so cryogenic clamps would not really but suitable (too expensive to upkeep).

Additionally, the material I am using is fibrous and of an unusual shape, so it is necessary for me to cut it into a standard size and shape. By doing this I am probably cutting some fibres, what is the effect of this on my sample?

In a few weeks time I will gladly post the answers I receive.

Thanks
Claire Hillery
Royal London Hospital

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Hello Claire,

A very simple approach to clamping tissue samples is wrap your tissue with
folded-sandpaper before clamping (i.e., rough on both the tissue side and
the clamp side). From my experience, this does not always work, but it is
very easy, inexpensive and it can help tremendously sometimes.

As for cutting your sample, this is obviously a problem. I'm sure you'll
get many good responses.

Good luck,

Lisa Friis
lfriis@ku.edu
------------


we usually cut a section of the bone around the ends of the ligament and
clamp those at both ends to avoid slipping problems.

daniel smolinski
uni of western australia
dsmol@cyllene.uwa.edu.au
---------

Claire,
One approach that people have used is to section the bone around the
insertion point and clamp your apparatus to the bone, using the natural
attachment between the bone and the ligament. This also avoids having to cut
fibers of the tendon. You could measure the geometry of the sample and
factor that into your calculations, rather than force the sample to assume a
particular geometry. Just and idea...
Chris

PolettoC@ninds.nih.gov
----------
Hi Claire -
would it be possible to glue the ligament using cyanoacrylate (superglue) -
I have heard of this being used for clamping soft tissue tests, but don't
know whether it would work in your case.

Regards,
Ciaran Simms


Trinity College, dublin
csimms@tcd.ie
---------
Dear Claire,
A student here in the Dublin Institute of Technology is attempting to do something similar, with tendons. She dipped each end of the tendon into liquid nitrogen and then clamped it. However she had to keep applying the liquid nitrogen throughout the test so this might be impractical for fatigue testing. The books listed below describe different clamping techniques, such as drying the ends of the sample.
Biomechanics - Materials: a Practical Approach, IRL Press (Oxford University Press), Vincent, J. F. V.,
Biomechanics - Structures and Systems, A Practical Approach. (A.A. Biewener - Editor)
Regards,
John Hanley
Dublin Institute of Technology
johnfhanley@yahoo.com

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I do not recommend cutting the ligament into a standard size and shape as this will have great, and variable, effects on the resulting strength. In addition, the only method I have successfully used to attach a ligament or tendon to a testing machine is through the use of a freeze clamp. We normally use dry ice to freeze the clamp, but you can use a continuous stream of liquid nitrogen if the mechanical test will take a long time to complete.

Catherine.G.Ambrose@uth.tmc.edu
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Dear Claire,

I developed a dry ice freeze clamping methodology in 1996 while at Johns Hopkins and used it quite successfully on quadriceps and hamstrings work. It reliably held loads exceeding 5000N. It was mentioned in my 1997 ORS abstract page 260.

All you do is make up some thin aluminium plates (1/4 to 1/8 inch thick) and put groves on the inside
face. I used V shaped groves that fit into one another with a depth of about 1/8 inch. Not too sharp or you can cut the ligament. Then using 4 bolts tighten the ligament between the plates only ever so slightly. This is not what holds the ligament, so don't tighten it very much at all. Then, take a small block of dry ice, and place it one one of the outside faces of the clamp. I designed some small spring loaded plastic
containers that both held the ice in place, insulated it, and pressed it against the face of the clamp. With this done, you are set! We had one end of the clamp with a hinge joint so that we could attach it to the
actuator, but any interface will do. The ice block (1inch x 0.5 inch x inch lasted about 15minutes and then we just put another in. The frozen clamp could hold more than 5000N, but we never did test it to failure.
Once the clamp unfroze and the ligament just slipped right out, but that was our fault. The freezing never had any bad effects on the ligament and we were always very happy with the design. You could use muscle as well as ligament in the clamp, and aside from the small amount of extra weight that the clamp added to the system, it was perfect. Try it out, I highly recommend using dry ice, and the left overs are great for the grad student parties.

It cost us about 50 cents a pound and we usually got 10 pounds for a complete day of testing. We cut it into blocks using a regular band saw, but watch out that you give the band saw a rest every minute or so, of you will freeze the blade and it will fracture.

A picture of these freeze clamps in operation can be found on the Johns
Hopkins Biomechanics Lab Web site.
http://www.biomech.jhu.edu/ under Projects then Knee Simulation

Good luck,
John
jdesjar@CLEMSON.EDU
----------

Hi Claire

I have some experience in clamping tendons and know how difficult they can be. I too did fatigue tests and the most successful way myself and former collegues had of clamping even quite thick sheep plantaris tendon was to air dry the ends. To do this, I wrapped all but 1-2 cm (depends on type of clamp used - do you have a choice here?) of the tendon in saline soaked tissue paper, then in cling film. The protruding ends were then left to air dry, which in the case of small tendons would only be a matter of minutes. The trick is to then clamp the sample with the minimum amount of dried material on the test side of the clamp. with practice i could get this down to less that 1 mm for the smaller samples. Too much and the stiffness of the sample will be affected, too little, and the sample would rupture at the clamp as before. refs you may find useful for this include Wang and Ker (1995), J. exp Biol 198, 831-845 and Ker et al., 2000, J. exp biol 203, 1317-1327.

I haven't done any testing on ligamentum nuchae myself, but I know that air dring has been used with some success. The thickness of the sample is the critical bit. As for cutting your sample, you will be cutting through some fibres, thus the tensile properties will be affected. However, in ligament the fibres are in a mesh like arrangement, unlike tendon where they are in a linear arrangement, so it is hard to know by how much.

Hope this is of some use. If you want more information, then please get in touch

regards
Anna Pike
a.pike@coventry.ac.uk
--------
Dear Claire,

the easiest way to improve grip (though not the best one) is to glue some sandpaper on the surfaces of your clamps.

Or you can embed the extremities of your specimen in some resin (PMMA or other). It's much easier to hold the extremities then.

In all cases, your specimen is more likely to fail near the extremities as any constraint you use, is a stress concentrator (the second option above is definitely better from this point of view). If the original tissue is wide enough, you might try to obtain some dog-bone specimens like those used in standard material testing. Then you can avoid failure at the extremities.

Best regards

Luca
cristofolini@tecno.ior.it
-----
Consider using freeze clamps. Neil Sharkey, from the University of California, Davis, published some work using freeze clamps several years ago.

Good luck,

Doug
------

Instead of gripping the ligament tissue, you may want to grip the bone to which it's attached, if possible. The bone ends can be gripped using Steinmann pins or potted in a cup using PMMA or a low melting-point alloy like Ceralow, then arranged in their original anatomic orientation.

Jim Funk
University of Virginia Center for Applied Biomechanics
1011 Linden Ave.
Charlottesville, VA 22902
Phone: 434-296-7288
Fax: 434-296-3453
------

Claire

I run the biomechanics lab at the Royal Vet College here in London - we've clamped a variety of tendons from a variety of animals - in fact we are now successfully clamping individual tendon fascicles

We use two basic techniques for clamping - cryo-clamps and drying the ends

Edward
e.draper@ic.ac.uk
-----
Hello Claire,
I had seen your note on the Biomch-L list and may have a suggestion for you.

Please point your browser to this URL
http://www.enduratec.com/accessory.cfm/acc/36
to see a set of thermo-electrically chilled grips designed for the type of work you had described.

Best,

Scott Anderson
Scott.Anderson@enduratec.com

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Hello Ms. Hillery,

We use sand paper to increase the friction on the grip interface. We also try to tighten each piece at the same degree. We've had some success with this for tensile testing, though occasionally we still get slippage (say, 1 out of 10 pieces, I think that's pretty good).

Our specimens are 300 um thick, around 3-4 mm wide, and up to 2 cm long. We don't use serpentine frozen grips simply because we don't have them.

My two cents on the fibrous nature of your tissue is that I do think it will matter. According to the structure/function relationship of tissues, we believe that the collagen fibers provide tensile strength to the tissue. Thus, the direction of testing is very important - I am assuming that you will be pulling your tissues along an axis parallel to the insertion points. If you do it in any perpendicular axis you will find significantly reduced strength. This is because when you are pulling parallel to the axis of insertion points you will be pulling on the collagen fibers length wise, and when perpendicular you will be pulling fibers apart from each other. The analogy is the difference between pulling a rope or pulling the individual fibers in it apart. Fatigue testing is more interesting when we get to see how long it takes before collagen fibers break down, as opposed to how long it takes to separate one fiber from another.

Fitting the rope analogy to your situation, you can see that, if you cut a really thick rope at an angle and pull it not all the force will be applied towards pulling the fibers, but some will be used to pull the fibers apart from each other. Since the forces holding the fibers together is significantly less than that which holds an individual fiber together you will see decreased mechanical properties. Likewise, if you already have cuts in the fibers of the rope (nicks in the rope), you will get decreased mechanical properties.

What we do, then, is several things. We cut the tissue and do electron microscopy on it to check on the thickness of the collagen fibers, so that we know we cannot make tensile specimens thinner than several diameters of a collagen fiber. We also take our specimens on a microtome so that we get as accurate a thickness as possible to minimize variations. After we take our specimens we take histological sections to ensure that our specimens do not have knife marks on them (stress concentration points).

It's lunch time, and I gave a long-winded response - I hope it was
helpful.

Jerry Hu
Musculoskeletal Bioengineering Laboratory
Rice University
Department of Bioengineering - MS 142
P.O. Box 1892, Houston, Texas 77251-1892
Tel: (713) 348-6393
Fax: (713) 348-5877
email: ragnarok@rice.edu

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Dear Claire,
Your prolem is a common one. You might, and probably, have already have referred to the work of Woo (Wu? - I'm sorry I'm not at the office to confirm the spelling of the name) who performed a number of experiments on mechanical testing of biological tissues. When I had to do some experiments on gripping slippery and fragmentous material I used grips that increased the compressive force on the sample with increasing tension of the sample. Such grips can be constructed from metal cyclinders with a tread pattern on the curved surface. The trick is to mount the cylinders eccentrically so that as the sample is pulled through there is just enough force to grip it. It might work for you. See badly drawn diagram in document attached.
Adam
Adam Shortland PhD, MIPEM, SRCS
One Small Step Gait Laboratory,
Guy's Hospital
London
UK
adam.shortland@virgin.net
----
Claire,

Search the literature for CO2 grips - these freeze the ligament, but have
no affect on the gauge length region

Cheers
Best Regards,

Dr. Mark Gillies

m.gillies@unsw.edu.au
----
Claire

Have you tried a search of the BIOMCH-L archives as this topic has been raised a couple of times before. Have you considered cryo-clamping where you freeze-clamp the ends of the specimen with liquid nitrogen. I would suggest a medline search. End effects will always affect your results but can be minimised by calculating the cross-sectional area of the middle of the specimen (there are techniques for this) and using noncontact exstensometry considering the displacement of two points near the middle of the specimen. This is for Young's Modulus and these techniques are expensive and difficult. Life is easier if you just want force displacement. There are also commercially available clamps which tighten when slip occurs, but end failure is also difficult with this method. Our group has also sutured ligamentous structures and used fixation methods on the sutures. Might not be appropriate for your specimen. We all want mid-substance failure!!!!!!

Best of luck

Dan Barker
Dan.Barker@rgh.sa.gov.au
---
Ahh, the age old question of biomaterials!! I had to do a study on nerve tension a couple of years ago with the same problem. There is no answer as far as I am aware but would love to hear of other's ideas.

Some ideas to look at though:
1. putting super glue or the like on the ends to make them a bit more rigid
and easier to deal with.
2. use gravity to your advantage with a pulley system (so the deformation
might occur between the pulleys or posts rather than at the clamp site).
3...can't think of a number 3!

Good Luck!!
You'll need it!

Carrie
c.newbold@medoto.unimelb.edu.au
---
Hi Claire:
Have you checked out some of the papers by Helio Schechtman? He used self-tightening clamps in his fatigue tests of human tendons - these might be helpful in your application.
Hope this helps,
Michelle

Michelle L. Zec
MD/PhD Program, University of Calgary
Joint Injury and Arthritis Research Group
e-mail: mlzec@ucalgary.ca



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