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
<ol>
	<li>Bone image acquisition and 3D bone model construction
	<ol>
		<li>Dissect the bone of interest in pr...

<ol>
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K., Sarver, J. J., Iozzo, R. V., Birk, D. E., 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=Effect+of+Age+and+Proteoglycan+Deficiency+on+Collagen+Fiber+Re-Alignment+and+Mechanical+Properties+in+Mouse+Supraspinatus+Tendon.">Effect of Age and Proteoglycan Deficiency on Collagen Fiber Re-Alignment and Mechanical Properties in Mouse Supraspinatus Tendon.</a> Journal of Biomechanical Engineering. 135, 021019 (2013).</li><li>Beason, D. P., 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=Hypercholesterolemia+increases+supraspinatus+tendon+stiffness+and+elastic+modulus+across+multiple+species.">Hypercholesterolemia increases supraspinatus tendon stiffness and elastic modulus across multiple species.</a> Journal of Shoulder and Elbow Surgery. 22, 681-686 (2013).</li><li>Miller, K. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Interfaces+and+Attachments+in+Biology.">Structural Interfaces and Attachments in Biology.</a> Infection and Immunity. 35, (2013).</li><li>Boivin, G. P., 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=Biomechanical+properties+and+histology+of+db/db+diabetic+mouse+Achilles+tendon.">Biomechanical properties and histology of db/db diabetic mouse Achilles tendon.</a> Muscles, Ligaments and Tendons Journal. 4, 280-284 (2014).</li><li>Ansorge, H. L., Adams, S., Birk, D. E., Soslowsky, L. 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B., Melrose, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achilles+and+tail+tendons+of+perlecan+exon+3+null+heparan+sulphate+deficient+mice+display+surprising+improvement+in+tendon+tensile+properties+and+altered+collagen+fibril+organisation+compared+to+C57BL/6+wild+type+mice.">Achilles and tail tendons of perlecan exon 3 null heparan sulphate deficient mice display surprising improvement in tendon tensile properties and altered collagen fibril organisation compared to C57BL/6 wild type mice.</a> PeerJ. 6, 5120 (2018).</li><li>Probst, A., 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=A+new+clamping+technique+for+biomechanical+testing+of+tendons+in+small+animals.">A new clamping technique for biomechanical testing of tendons in small animals.</a> Journal of Investigative Surgery. 13, 313-318 (2000).</li><li>Talan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature+of+C57BL/6J+mice+with+age.">Body temperature of C57BL/6J mice with age.</a> Experimental Gerontology. 19, 25-29 (1984).</li><li>Newton, M. 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=The+influence+of+testing+angle+on+the+biomechanical+properties+of+the+rat+supraspinatus+tendon.">The influence of testing angle on the biomechanical properties of the rat supraspinatus tendon.</a> Journal of Biomechanics. 49, 4159-4163 (2016).</li><li>Schwartz, A. G., Lipner, J. H., Pasteris, J. D., Genin, G. M., 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=Muscle+loading+is+necessary+for+the+formation+of+a+functional+tendon+enthesis.">Muscle loading is necessary for the formation of a functional tendon enthesis.</a> Bone. 55, 44-51 (2014).</li><li>Gimbel, J. A., Van Kleunen, J. P., Williams, G. R., Thomopoulos, S., 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=Long+durations+of+immobilization+in+the+rat+result+in+enhanced+mechanical+properties+of+the+healing+supraspinatus+tendon.">Long durations of immobilization in the rat result in enhanced mechanical properties of the healing supraspinatus tendon.</a> Journal of Biomechanical Engineering. 129, 400-404 (2006).</li><li>Freedman, B. R., Sarver, J. J., Buckley, M. R., Voleti, P. B., 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=Biomechanical+and+structural+response+of+healing+Achilles+tendon+to+fatigue+loading+following+acute+injury.">Biomechanical and structural response of healing Achilles tendon to fatigue loading following acute injury.</a> Journal of Biomechanics. 47, 2028-2034 (2014).</li><li>Deymier, A. 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. G., Long, F., 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=Enthesis+fibrocartilage+cells+originate+from+a+population+of+Hedgehog-responsive+cells+modulated+by+the+loading+environment.">Enthesis fibrocartilage cells originate from a population of Hedgehog-responsive cells modulated by the loading environment.</a> Development. 142, 196-206 (2015).</li><li>Bell, R., Taub, P., Cagle, P., Flatow, E. L., Andarawis-Puri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+model+of+supraspinatus+tendon+insertion+site+healing.">Development of a mouse model of supraspinatus tendon insertion site healing.</a> Journal of Orthopaedic Research. 33, 25-32 (2014).</li><li>Connizzo, B. K., Bhatt, P. R., Liechty, K. W., 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=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. J., 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=Knockout+of+hyaluronan+synthase+1,+but+not+3,+impairs+formation+of+the+retrocalcaneal+bursa.">Knockout of hyaluronan synthase 1, but not 3, impairs formation of the retrocalcaneal bursa.</a> Journal of Orthopaedic Research. 36, 2622-2632 (2018).</li><li>Wang, V. M., Banack, T. M., Tsai, C. W., Flatow, E. L., Jepsen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+in+tendon+and+knee+joint+biomechanics+among+inbred+mouse+strains.">Variability in tendon and knee joint biomechanics among inbred mouse strains.</a> Journal of Orthopaedic Research. 24, 1200-1207 (2006).</li><li>Wang, V. M., 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=Murine+tendon+function+is+adversely+affected+by+aggrecan+accumulation+due+to+the+knockout+of+ADAMTS5.">Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5.</a> Journal of Orthopaedic Research. 30, 620-626 (2011).</li><li>Zhang, K., 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=Tendon+mineralization+is+progressive+and+associated+with+deterioration+of+tendon+biomechanical+properties,+and+requires+BMP-Smad+signaling+in+the+mouse+Achilles+tendon+injury+model.">Tendon mineralization is progressive and associated with deterioration of tendon biomechanical properties, and requires BMP-Smad signaling in the mouse Achilles tendon injury model.</a> Matrix Biology. 52-54, 315-324 (2016).</li><li>Rooney, S. 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.

Research

JoVE Journal

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...