Name: biomechanical testing of murine tendons
Uploaded: 2019-10-15T14:30:00
Description: Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

The study was supported by the NIH / NIAMS (R01 AR055580, R01 AR057836).

Animal studies were approved by Columbia University Institutional Animal Care and Use Committee. Mice used in this study were of a C57BL/6J background and were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). They were housed in pathogen-free barrier conditions and were provided food and water ad libitum.
1. Development of custom-fit 3D printed fixtures for gripping bone
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	<li>Bone image acquisition and 3D bone model construction
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		<li>Dissect the bone of interest in pr...

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S., Connizzo, B. K., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+re-alignment+in+a+neonatal+developmental+mouse+supraspinatus+tendon+model.">Collagen fiber re-alignment in a neonatal developmental mouse supraspinatus tendon model.</a> Annals of Biomedical Engineering. 40, 1102-1110 (2012).</li><li>Cong, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+role+of+subacromial+impingement+in+rotator+cuff+tendinopathy:+Development+and+analysis+of+a+novel+murine+model.">Evaluating the role of subacromial impingement in rotator cuff tendinopathy: Development and analysis of a novel murine model.</a> Journal of Orthopaedic Research. 36, 2780-2788 (2018).</li><li>Thomopoulos, S., Birman, V., Genin, G. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiscale+structural+and+mechanical+effects+of+mouse+supraspinatus+muscle+unloading+on+the+mature+enthesis.">The multiscale structural and mechanical effects of mouse supraspinatus muscle unloading on the mature enthesis.</a> Acta Biomaterialia. 83, 302-313 (2019).</li><li>Killian, M. L., Thomopoulos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleraxis+is+required+for+the+development+of+a+functional+tendon+enthesis.">Scleraxis is required for the development of a functional tendon enthesis.</a> FASEB Journal. 30, 301-311 (2016).</li><li>Schwartz, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+Alters+Mechanical+Properties+and+Collagen+Fiber+Re-Alignment+in+Multiple+Mouse+Tendons.">Diabetes Alters Mechanical Properties and Collagen Fiber Re-Alignment in Multiple Mouse Tendons.</a> Annals of Biomedical Engineering. 42, 1880-1888 (2014).</li><li>Eekhoff, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionally+Distinct+Tendons+From+Elastin+Haploinsufficient+Mice+Exhibit+Mild+Stiffening+and+Tendon-Specific+Structural+Alteration.">Functionally Distinct Tendons From Elastin Haploinsufficient Mice Exhibit Mild Stiffening and Tendon-Specific Structural Alteration.</a> Journal of Biomechanical Engineering. 139, 111003 (2017).</li><li>Mikic, B., Bierwert, L., Tsou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achilles+tendon+characterization+in+GDF-7+deficient+mice.">Achilles tendon characterization in GDF-7 deficient mice.</a> Journal of Orthopaedic Research. 24, 831-841 (2006).</li><li>Sikes, K. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ibuprofen+differentially+affects+supraspinatus+muscle+and+tendon+adaptations+to+exercise+in+a+rat+model.">Ibuprofen differentially affects supraspinatus muscle and tendon adaptations to exercise in a rat model.</a> American Journal of Sports Medicine. 44, 2237-2245 (2016).</li><li>Galasso, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+of+Life+and+Functional+Results+of+Arthroscopic+Partial+Repair+of+Irreparable+Rotator+Cuff+Tears.">Quality of Life and Functional Results of Arthroscopic Partial Repair of Irreparable Rotator Cuff Tears.</a> Arthroscopy - Journal of Arthroscopic and Related Surgery. 33, 261-268 (2017).</li><li>Sarver, D. 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Murine animal models are commonly used to study tendon disorders, but characterization of their mechanical properties is challenging and uncommon in the literature. The purpose of this protocol is to describe a time efficient and reproducible method for tensile testing of murine tendons. The new methods reduced the time required to test a sample from hours to minutes and eliminated a major gripping artifact that was a common problem in previous methods.
Several steps described in this protocol...

Tendon disorders are common and highly prevalent among the aging, athletic, and active populations1,2,3. In the United States, 16.4 million connective tissue injuries are reported each year4 and account for 30% of all injury-related physician office visits3,5,6,7,8. The most commonly affected sites include the rotator cuff, Achilles tendon, and patellar tendon9. Although a variety of non-operative and operative treatments have been explored, including anti-inflammatory drugs, rehabilitation, and surgical repair, outcomes remain poor, with limited return to function and high rates of failure5,6. These poor clinical outcomes have motivated basic and translational studies seeking to understand tendinopathy and to develop novel treatment approaches.
Tensile biomechanical properties are the primary quantitative outcomes defining tendon function. Therefore, laboratory characterization of tendinopathy and treatment efficacy must include a rigorous testing of tendon tensile properties. Numerous studies have described methods to determine the biomechanical properties of tendons from animal models such as rats, sheep, dogs, and rabbits10,11,12. However, few studies have tested the biomechanical properties of murine tendons, primarily due to the difficulties in gripping the small tissues for tensile testing. As murine models have numerous advantages for mechanistically studying tendinopathy, including genetic manipulation, extensive reagent options, and low cost, development of accurate and efficient methods to biomechanically test murine tissues is needed.
In order to properly test the mechanical properties of tendons, the tissue must be gripped effectively, without slipping or artifactual tearing at the grip interface or fracturing of the growth plate. In many cases, particularly for short tendons, the bone is gripped on one end and the tendon is gripped on the other end. Bones are typically secured by embedding them in materials such as epoxy resin13 and polymethylmethacrylate14,15. Tendons are often placed between two layers of sandpaper, glued with cyanoacrylate, and secured using compression clamps (if the cross section is flat) or in a frozen medium (if the cross section is large)15,16,17. These methods have been applied to biomechanically test murine tendons, but challenges arise due to the small size of the specimens and the compliance of the growth plate, which never ossifies18. For example, the diameter of the murine humeral head is only a few millimeters, thus making gripping of the bone difficult. Specifically, tensile testing of murine supraspinatus tendon-to-bone samples often results in failure at the growth plate rather than in the tendon or at the tendon enthesis. Similarly, biomechanical testing of the Achilles tendon is challenging. Although the Achilles tendon is larger than other murine tendons, the calcaneus is small, making gripping of this bone difficult. The bone can be removed, followed by gripping the two tendon ends; however, this precludes the testing of the tendon-to-bone attachment. Other groups report gripping the calcaneus bone using custom-made fixtures19,20, anchoring by clamps21, fixing in self curing plastic cement22 or using a conical shape slot22, yet these prior methods remain limited by low reproducibility, high gripping failure rates, and tedious preparation requirements.
The objective of the current study was to develop an accurate and efficient method for tensile biomechanical testing of murine tendons, focusing on the supraspinatus and Achilles tendons as examples. Using a combination of 3D reconstructions from native bone anatomy, solid modeling, and additive manufacturing, a novel method was developed to grip the bones. These fixtures effectively secured the bones, prevented growth plate failure, decreased specimen preparation time, and increased testing reproducibility. The new method is readily adaptable to test other murine tendons as well as tendons in rats and other animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose</td>
			<td >Fisher Scientific</td>
			<td >BP160-100</td>
			<td >Dissovle 1g in 100 ml ultrapure water to make 1% agarose&#160;</td>
		</tr>
		<tr >
			<td >Bruker microCT&#160;</td>
			<td >Bruker BioSpin Corp</td>
			<td >Skyscan 1272&#160;</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >ElectroForce&#160;</td>
			<td >TA Instruments</td>
			<td >3200</td>
			<td>Testing platform</td>
		</tr>
		<tr >
			<td >Ethanol 200 Proof</td>
			<td >Fisher Scientific</td>
			<td >A4094</td>
			<td>Dilute to 70% and use as suggested in protocol</td>
		</tr>
		<tr >
			<td >Fixture to attach grips</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Kimwipes</td>
			<td >Kimberly-Clark</td>
			<td >&#160;S-8115</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >MicroCT CT-Analyser (Ctan)</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >MilliQ water (Ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>100 ml&#160;</td>
		</tr>
		<tr >
			<td >Meshmixer</td>
			<td >Autodesk</td>
			<td >http://www.meshmixer.com/</td>
			<td >Free engineering software used by authors to refine mesh</td>
		</tr>
		<tr >
			<td >Objet EDEN 260VS&#160;</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Precision Prototyping</td>
		</tr>
		<tr >
			<td >Objet Studio</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Used by authors with 3D printer</td>
		</tr>
		<tr >
			<td >PBS - Phosphate-Buffered Saline</td>
			<td >ThermoFisher Scientific</td>
			<td >10010031</td>
			<td >2.5 L of 10% PBS&#160;</td>
		</tr>
		<tr >
			<td >S&#38;T Forceps</td>
			<td >Fine Science Tools</td>
			<td >00108-11</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Blade - #11</td>
			<td >Fine Science Tools</td>
			<td >10011-00</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Handle - #3</td>
			<td >Fine Science Tools</td>
			<td >10003-12</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >SkyScan 1272</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >Skyscan CT-Vox</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SkyScan NRecon</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SolidWorks CAD</td>
			<td >Dassault Syst&#232;mes</td>
			<td >SolidWorks Research Subsription</td>
			<td>Solid modeling computer-aided design used by authors</td>
		</tr>
		<tr >
			<td >SuperGlue</td>
			<td >Loctite</td>
			<td >234790</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >Testing bath</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Thin film grips&#160;</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >VeroWhitePlus</td>
			<td >Stratasys LTD</td>
			<td >NA</td>
			<td>3D printing material used by authors</td>
		</tr>
		<tr >
			<td >WinTest&#160;</td>
			<td >WinTest Software</td>
			<td ></td>
			<td>Used by authors to collect data</td>
		</tr>
	</tbody>
</table>

biomechanical testing of murine tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

3D-printed fixtures were used to test 8-week old murine supraspinatus and Achilles tendons. All mechanically tested samples failed at the enthesis, as characterized by microCT scans, visual inspection, and video analysis after tensile tests. A one-to-one comparison of the previous and current methods for supraspinatus tendon testing in our laboratory is shown in Figure 3. In the previous method28,29,30</s...

Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

Biomechanical Testing of Murine Tendons

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

The protocol describes an efficient and reproducible method for biomechanical testing of mouse tendons through reusable custom-fit 3D printed fixtures. The new methods reduce the time required to test the sample from hours to minutes and eliminate a major gripping artifact that was a common problem in previous methods. This method is not limited to the supraspinatus and Achilles tendons.It can easily be adapted to testing other mouse tendons and tendons from larger animals. To ensure reproducibility and efficiency of the approach, multiple cycles of design prototyping and validation may be needed for fixtures not described here. To begin, perform a microcomputed tomography scan of the entire bone in the agarose gel.After reconstruction into cross-section images, select the command File Open to open the file dataset. Open the dialog file Preferences and select the Advanced tab. Use the adaptive rendering algorithm to construct the 3D models which provide smoother surface detail.Use 10 as the locality parameter which defines the distance in pixels to the neighboring point used for finding the object border. Minimize tolerance to 0.1 to decrease the file size. To specify the volume of interest, manually select two images to set as the top and bottom of the selected VOI range, then move to the second page, Region of Interest.Manually select the region of interest on a single cross-section image. Repeat selecting the ROI for every 10 to 15 cross-section images. Next, move to the third page, Binary Selection.On the Histogram menu, click From Dataset to show the histogram distribution of brightness from all images of the dataset. Also on the Histogram menu, click the Create a 3D model file menu. Save a 3D model of the bone in STL file format.In the Meshmixer software, import mesh and select All to edit. Choose Produce from the Tool Set edit, then select Triangle Budget from the tool set Reduce target. Reduce the tricount and accept changes.Resave the newly-reduced file in STL format by choosing Export As.To design a supraspinatus tendon humeral bone, use a solid-modeling computer-aided design program to create a custom-fit model of humerus-gripping fixture. Open the STL format file of the humerus bone in a solid-modeling program and save as a part file in SLDPRT format. Then open the Part file to manually create three anatomically relevant planes, for example, sagittal, coronal, and transverse.Click on File New to create the solid block component part. Click on File New to create an assembly model with two components, the solid block and either the right or left humerus bone. Define the orientation of the bone within the block to ensure the angle between the tendon and bone to be 180 degrees.Ensure that the entire bone volume fits inside the block. In the Assembly window, select the block and click Edit Component from the Assembly toolbar. Click Insert, Features, Cavity.Select Uniform Scaling and enter 0%as the value to scale in all directions. Suppress the bone part and save the assembly as a part. Open the cylinder with cavity part.Create a sketch, ensure to only leave 0.5 millimeters above the humeral head. From Features, select Extruded Cut. Cut the assembly along the sagittal plane to create two symmetrical components that fit the bone anteriorly and posteriorly.Cut the assembly part along the sagittal plane to create two symmetrical components that fit the bone anteriorly and posteriorly. It's critical to ensure the humeral head to prevent growth plate failure and to define a tight clearance that avoids disengaging of the humerus from the model during testing. Proceed as described in the manuscript to finish the posterior component and anterior component.Save both components as separate part files. Now press Insert Pattern, Mirror, and select Mirror to create 3D mirror models for each component of the fixture for the opposite limb. Select the front face as a mirror face plain.Select the part as the body to mirror. Select the mirror plane and create a sketch that includes all material. From Features, select Extruded Cut to remove the original part and to keep only mirrored part.Click on the bottom of the part and use as a plane to make a sketch. Click on Sketch Text and add L or R.This adds an etch on the bottom of the fixtures to distinguish between the left and right sides. Save all fixture parts in STL standard file format in preparation for 3D printing.To dissect the supraspinatus muscle-tendon, humerus bone specimen, first position the euthanized mouse in a prone position and make an incision in the skin from above the elbow of the forepaw towards the shoulder. Use forceps to carefully remove the skin with blunt dissection so that the musculature of the shoulder is visible. Remove the tissue surrounding the humerus until the bone is exposed.Hold the humerus with forceps and carefully remove the deltoid and trapezius muscles to expose the coracoacromial arch. Identify the AC joint and carefully separate the clavicle from the acromion with a scalpel blade. Taking care not to damage the supraspinatus tendon and its bony attachment, remove the muscle from its scapular attachment using a scalpel blade.Detach the humeral head from the glenoid and lacerate the joint capsule and the infraspinatus subscapularis and teres minor tendons. Disarticulate the elbow joint to separate the humerus from the ulna and radius. Isolate the humerus-supraspinatus tendon muscle specimen and clean off excess soft tissues on the humerus and humeral head.To dissect the Achilles tendon, the calcaneus bone, position a euthanized mouse in a prone position. Taking care not to damage the Achilles tendon and its bony attachment, remove the skin with blunt dissection so that the musculature around the ankle and knee joints is exposed. Using a scalpel blade, starting at the Achilles tendon-calcaneus attachment, carefully detach the gastrocnemius muscle from its proximal attachments.Carefully disarticulate the calcaneus from the various adjacent bones and isolate the Achilles tendon-calcaneus specimen and clean off excess soft tissues. To determine the cross-sectional area of the tendon, insert the bone upside down to suspend the specimen in the cryotube filled with agarose gel with the bone in the agarose gel and the tendon and muscle outside. After the scan using microcomputed tomography, gently remove the muscle from the tendon using a scalpel blade.Insert the bone into the 3D printed fixture and attach them to the testing grids. Insert and glue the tendon between a folded thin tissue paper and clamp it using thin film grips. Insert the sample and the grips into a testing bath of PBS at 37 degrees Celsius and perform a mechanical test.In this study, using reusable 3D printed fixtures to grip bone, specimen preparation time was reduced from hours to minutes. Representative load deformation curves for tensile testing of supraspinatus tendon using current methods eliminated major gripping artifact such as growth plate failure. The mechanical properties of supraspinatus and Achilles tendons demonstrate a significant effect of sex based on unpaired T tests with P value less than 0.05.Micro CT successfully measured the cross-sectional area along the length of supraspinatus tendon along the length of Achilles tendon. It's important to remember that each anatomic site has specific design criteria necessary for effective gripping. Therefore, the design for each fixture should be adapted accordingly.In addition to tensile testing to failure, these fixtures can be used for cyclic loading tests, which provide information about tendon fatigue and viscoeleastic properties. Characterizing the mechanical properties of murine tendons is uncommon in the literature due to a number of reasons, including the difficulty in gripping these small tissues, tedious and time-consuming specimen preparation methods, and growth plate fractures. This protocol presents a time-efficient and reproducible method for testing mouse tendons that has eliminated artifactual grip failures and has tripled the number of specimens that can be tested in a day.

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