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 preparation for 3D model creation and 3D bone grip printing; the humerus and the calcaneus are used as examples in the current protocol. 
		NOTE: Detailed instructions to dissect bone-tendon-muscle specimens for mechanical testing are provided in step 2.1.1. The following steps should be followed to isolate bones for the purpose of creating 3D-printed bone grips.
		<ol>
			<li>Dissection of the humerus: Euthanize a mouse per IACUC-approved procedure. Remove upper extremity skin, remove all muscles over the humerus, disarticulate the elbow and glenohumeral joints, and carefully remove all connective tissues attached to the humerus.</li>
			<li>Dissection of the calcaneus: Euthanize a mouse per IACUC-approved procedure. Remove lower extremity skin, disarticulate the Achilles tendon-calcaneus joint and joints between calcaneus and other foot bones, and carefully remove all connective tissues attached to the calcaneus.</li>
		</ol>
		</li>
		<li>Perform a microcomputed tomography scan of the entire bone, e.g., scan the humerus and calcaneus samples. 
		NOTE: Depending on the scanner used, the settings will be different. For the scanner used in the current study (Table of Materials), the recommended settings are: scan at an energy of 55 kVP, Al 0.25 filter, at a resolution of 6 &#956;m.
		<ol>
			<li>Mix agarose powder in ultrapure water and microwave for 1-3 min until the agarose is completely dissolved. It is helpful to microwave for 30-45 s, stop and swirl, and then continue towards a boil. Fill cryotubes up to three-quarters full with agarose. Let the agarose cool for about 5-10 min.</li>
			<li>Insert bone into the agarose gel (this will prevent movement artifacts during scanning). Insert a cryotube with bone into the scanner. 
			NOTE: For the scanner used in the current study, a 16-position automatic sample changer was used for all scans. This scanner can automatically select magnification according to a sample&#8217;s size and shape.&#160;</li>
		</ol>
		</li>
		<li>Reconstruct microcomputed tomography scan projection images into cross-section images. Use recommended parameters for the experimenter&#8217;s scanner/software combination. 
		NOTE: For the program used in the current study (Table of Materials) it is recommended to use the following reconstruction parameters: Smoothing: 0-2, Beam Hardening Correction: 45, Ring Artefact Reduction: 4-9 and to reconstruct slices in 16-bit TIFF format.</li>
		<li>Create a 3D&#160;model and save into a standard STL format compatible with most 3D printers and rapid prototyping. For the program used in the current study (Table of Materials), do the following:
		<ol>
			<li>Select the command File &#62; Open to open the file dataset. Open the dialog File &#62; Preferences and select the Advanced tab.</li>
			<li>Use the adaptive rendering algorithm to construct the 3D models. This algorithm minimizes the number of facet triangles and provides smoother surface detail. Use 10 as the locality parameter; this parameter defines the distance in pixels to the neighboring point used for finding the object border. Minimize tolerance to 0.1 to decrease file size. 
			NOTE: After opening the dataset, the images are shown in the &#8220;Raw Images&#8221; page.</li>
			<li>To specify the volume of interest (VOI), manually select two images to set as the top and bottom of the selected VOI range.</li>
			<li>Move to the second page, Region of Interest. Manually select the region of interest on a single cross section image. 
			NOTE: The selected region will be highlighted in red (i.e., the humerus cross-sectional area).</li>
			<li>Repeat the previous step every 10&#8211;15 cross-section images.</li>
			<li>Move to the third page Binary Selection. On the histogram menu, click From Dataset. The histogram distribution of brightness from all images of the dataset will be shown. Also on the histogram menu, click the Create a 3D Model file menu.</li>
		</ol>
		</li>
		<li>Save a 3D model of the bone in STL file format.</li>
		<li>Refine the mesh: Manipulate the mesh to reduce the size of the STL file and make it compatible with any solid modeling computer-aided design program. For the program used in the current study (Table of Materials), follow the steps below:
		<ol>
			<li>Import mesh and select all to edit. Choose Reduce&#160;from the toolset Edit. Then, select&#160;Triangle Budget from the toolset Reduce Target. Reduce the Tri Count and accept changes.&#160;Resave the newly reduced file in STL format by choosing Export as&#8230;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Design of custom-fit bone fixtures
	<ol>
		<li>Supraspinatus tendon-humeral bone
		<ol>
			<li>Use a solid modeling computer-aided design program to create a custom-fit model of humerus gripping fixture (Figure 1, Supplemental Files). 
			NOTE: The program used in the current study is listed in the Table of Materials.</li>
			<li>Open the STL format file of the humerus bone in a solid modeling program and save as a part file. 
			NOTE: For the software used in the current study (Table of Materials), the 3D bone object was saved in SLDPRT format.</li>
			<li>Open the part file and manually create three anatomically relevant planes (i.e., sagittal, coronal, transverse).
			<ol>
				<li>Manually define the sagittal plane to cut through the supraspinatus tendon attachment at the greater tuberosity. Ensure that the 3D block contains the sagittal plane as a plane of symmetry. To achieve this, add or cut material from the block if needed. 
				NOTE: This plane of symmetry ensures that when the specimen is inserted into the fixtures the tendon and tendon attachment are located in the central axis of the fixture.</li>
			</ol>
			</li>
			<li>Measure dimensions of the bone along each of the three planes (i.e., height, width, length).</li>
			<li>Measure the dimensions of the mechanical testing grips where the 3D printed fixture will be attached.</li>
			<li>Begin by designing a solid block part (e.g., a solid cylinder).
			<ol>
				<li>Ensure that each dimension of the block is at least 5 mm greater than the dimensions of the humerus.</li>
				<li>Account for design constraints from mechanical testing grips (i.e., ensure that the 3D printed fixture can be assembled and disassembled freely in the mechanical testing grips).</li>
			</ol>
			</li>
			<li>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 (i.e., the angle between the tendon and bone). Ensure that the entire bone volume fits inside the block.</li>
			<li>Create a cavity in the block using the humerus bone as the mold. If using the software specified in the Table of Materials, follow the following steps:
			<ol>
				<li>Insert the design part (humerus) and the mold base (cylinder block) into an interim assembly. In the assembly window, select the block, and click Edit Component from the Assembly toolbar.</li>
				<li>Click Insert &#62; Features &#62; Cavity. Select Uniform Scaling and enter 0% as the value to scale in all directions.</li>
			</ol>
			</li>
			<li>Suppress the bone part and save the assembly as a part.</li>
			<li>Open part (cylinder with cavity). Cut the part along the sagittal plane to create two symmetrical components that fit the bone anteriorly and posteriorly (e.g., two half cylinders, as seen in Figure 1). 
			NOTE: Two components are designed that fit the bone anteriorly and posteriorly. The anterior component includes a half spherical-shaped cavity extended from the anterior side of the humeral head up to the supraspinatus tendon attachment. The posterior component cavity is shaped like the posterior part of the humerus (i.e., posterior side of the humeral head, deltoid tuberosity, and medial and lateral epicondyle).</li>
			<li>Save each component as a separate file part.</li>
			<li>For the anterior component, ensure that the humeral head is embedded in the cavity of the part by defining appropriate tolerances. 
			NOTE: In the current study, using the software specified in the Table of Materials, it is suggested to follow the steps below:
			<ol>
				<li>Create a revolved cut to smooth the mesh geometry of the cavity. Create a sketch for the cut by emulating the cavity geometry and adding a locational clearance. 
				NOTE: The clearance allows for free assembly and disassembly between the bone and the anterior component.</li>
			</ol>
			</li>
			<li>Modify the posterior component to imitate the cavity geometry to create a cut that adds clearance, as described above for the anterior component.</li>
			<li>Make a cut in the transverse plane starting from the top of the posterior component up to the crest of the greater/lesser tubercle. 
			NOTE: As seen in Figure 1 and Figure 2, the posterior component includes a cut that creates an opening at the tendon attachment.</li>
			<li>Create a snug fit between the two components to allow for free assembly and disassembly. 
			NOTE: A hole-shaft fit with a loose running clearance was created for the fixtures in the current study.</li>
			<li>Create 3D mirror models for each component of the fixture for the opposite limb (i.e., left or right).</li>
			<li>Add an etch on the bottom of the fixtures to distinguish between the left and right sides.</li>
			<li>Save all fixture parts in STL standard file format in preparation for 3D printing.</li>
		</ol>
		</li>
		<li>Achilles tendon-calcaneus bone
		<ol>
			<li>Follow the same steps as described above for supraspinatus-humeral head fixture. 
			NOTE: Only one set of fixtures is necessary for the Achilles-calcaneal, since the anatomy of the left and right calcaneus bones is nearly symmetrical.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Biomechanical testing of murine tendons
<ol>
	<li>Specimen preparation and cross-sectional area measurement
	<ol>
		<li>Dissect the muscle-tendon-bone of interest in preparation for tensile mechanical testing. In the current study, supraspinatus muscle - tendon - humerus bone specimens (N=10, 5 male, 5 female) and gastrocnemius muscle - Achilles tendon-calcaneus bone specimens (N=12, 6 male, 6 female) were isolated from 8 week old C57BL/6J mice.
		<ol>
			<li>Dissection of the supraspinatus muscle - tendon - humerus bone specimen
			<ol>
				<li>Euthanize a mouse per IACUC-approved procedure. Position the mouse in a prone position. Make an incision in the skin from above the elbow of the forepaw towards the shoulder.</li>
				<li>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 and can be held securely with forceps.</li>
				<li>Hold the humerus with forceps and carefully remove the deltoid and trapezius muscles to expose the coracoacromial arch. Identify the acromioclavicular joint and carefully separate the clavicle from the acromion with a scalpel blade.</li>
				<li>Taking care not to damage the supraspinatus tendon and its bony attachment, remove the muscle from its scapular attachment using a scalpel blade. Taking care not to damage the supraspinatus tendon and its bony attachment, detach the humeral head from the glenoid; using a scalpel blade, lacerate the joint capsule and the infraspinatus, subscapularis, and teres minor tendons.</li>
				<li>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.</li>
			</ol>
			</li>
			<li>Dissection of the Achilles tendon - calcaneus bone sample
			<ol>
				<li>Euthanize a mouse per IACUC-approved procedure. Position the 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.</li>
				<li>Using a scalpel blade, starting at the Achilles tendon - calcaneus attachment, carefully detach the gastrocnemius muscle from its proximal attachments.</li>
				<li>Carefully disarticulate the calcaneus from the various adjacent bones. Isolate the Achilles tendon - calcaneus specimen and clean off excess soft tissues.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Determine the cross-sectional area of the tendon using microcomputed tomography. 
		NOTE: For the scanner used in the current study (Table of Materials), the recommended settings are: scan at an energy of 55 kVP, Al 0.25 filter, at a resolution of 5 &#956;m.
		<ol>
			<li>Mix agarose powder in ultrapure water and microwave for 1-3 min until the agarose is completely dissolved. It is helpful to microwave for 30-45 s, stop and swirl, and then continue towards a boil. Fill cryotubes up to three-quarters full with agarose. Let the agarose cool for about 5-10 min.</li>
			<li>Suspend the specimen in the cryotube by inserting the bone upside down. 
			NOTE: Only the bone should be in the agarose gel. The tendon and muscle should be suspended outside.</li>
		</ol>
		</li>
		<li>After the scan, gently remove muscle from tendon using scalpel blade. Insert the specimen into the 3D-printed fixture. 
		NOTE: The grips are reusable for each test. Do not use glue or epoxy in the fixture; the bone is held in a press fit.</li>
		<li>Insert and glue the tendon between a folded thin tissue paper (2 cm x 1 cm) and clamp the construct using thin film grips. Attach the 3D printed fixture with the specimen into the testing grips.</li>
		<li>Insert the sample and the grips into a testing bath of phosphate buffered saline (PBS) at 37 &#176;C (i.e., mouse body temperature23).</li>
	</ol>
	</li>
	<li>Tensile testing
	<ol>
		<li>Perform tensile mechanical test on a material testing frame. 
		NOTE: For the testing frame used in the current study (Table of Materials), the recommended protocol is:
		<ol>
			<li>Define the gauge length as the distance from the tendon attachment to the upper grip.</li>
			<li>Precondition with 5 cycles between 0.05 N and 0.2 N.</li>
			<li>Hold for 120 s.</li>
			<li>Use a tension to failure of 0.2%/s.</li>
		</ol>
		</li>
		<li>Collect load-deformation data.</li>
		<li>Calculate the strain as the displacement relative to the initial gauge length of the tendon.</li>
		<li>Calculate the stress as the force divided by the initial tendon cross-sectional area (as measured from microCT).</li>
		<li>If interested in viscoelastic behavior, perform a stress relaxation prior to the tension test to failure and use the data to calculate parameters such as A, B, C, tau1, and tau2 from the quasilinear viscoelastic model24.</li>
		<li>From the load deformation curve, calculate the stiffness (slope of linear portion of curve), the maximum force, and the work to yield (the area under the curve up to yield force).
		<ol>
			<li>Identify the linear portion by choosing a window of points in the load-deformation curve that maximizes the R2 value for a linear least squares regression25.</li>
			<li>Determine the stiffness as the slope of the linear portion of the load-displacement curve25,26.</li>
		</ol>
		</li>
		<li>From the stress strain curve, calculate the modulus (slope of linear portion of curve), the strength (maximum stress), and the resilience (area under the curve up to yield stress). 
		NOTE: Using the RANSAC algorithm, the yield strain (x-value) is defined as the first point when the y-fit has deviated more than 0.5% of the expected stress value (y-value). Yield stress is the corresponding y-value of the yield strain. 
		NOTE: In addition to the monotonic tensile loading to failure described in the current study, cyclic loading can provide important information about tendon fatigue and/or viscoelastic properties. For example, Freedman et al. reported fatigue properties of the murine Achilles tendons27.</li>
		<li>After completion of tensile testing, perform a microcomputed tomography scan of the entire bone, e.g., scan the humerus and calcaneus samples. 
		NOTE: For the scanner used in the current study (Table of Materials), the recommended settings are: scan at an energy of 55 kVP, Al 0.25 filter, at a resolution of 6 &#956;m.
		<ol>
			<li>Repeat steps 1.1.2.1&#8211;1.1.2.2.</li>
		</ol>
		</li>
		<li>Repeat step 1.1.3.</li>
		<li>Use a 3D visualization program compatible with the scanner to create a volume-rendered 3D model of the scanned object. 
		NOTE: The program used in the current study is listed in the Table of Materials.</li>
		<li>Determine the failure mode and failure site area by inspecting the 3D object.</li>
	</ol>
	</li>
	<li>Statistical analysis: Show all sample results as mean &#177; standard deviation (SD). Make comparisons between groups using student&#8217;s t-tests (two-tailed and unpaired). Set significance as p &#60; 0.05. 
	NOTE: The statistical software used in the current study is listed in the Table of Materials.</li>
</ol>

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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. 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=Sex+differences+in+tendon+structure+and+function.">Sex differences in tendon structure and function.</a> Journal of Orthopaedic Research. 35, 2117-2126 (2017).</li><li>Razmjou, H., 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=Disability+and+satisfaction+after+Rotator+Cuff+decompression+or+repair:+A+sex+and+gender+analysis.">Disability and satisfaction after Rotator Cuff decompression or repair: A sex and gender analysis.</a> BMC Musculoskeletal Disorders. 12, 66 (2011).</li></ol>

The authors have nothing to disclose.

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><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&amp;nbsp;</td></tr><tr><td>Bruker microCT&amp;nbsp;</td><td>Bruker BioSpin Corp</td><td>Skyscan 1272&amp;nbsp;</td><td>Used by authors</td></tr><tr><td>ElectroForce&amp;nbsp;</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>&amp;nbsp;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&amp;nbsp;</td></tr><tr><td>MilliQ water (Ultrapure water)</td><td>Millipore Sigma</td><td>QGARD00R1 (or related purifier)</td><td>100 ml&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>S&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>SolidWorks CAD</td><td>Dassault Syst&amp;egrave;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&amp;nbsp;</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&amp;nbsp;</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, ...

Research

JoVE Journal

Bioengineering

13.1K Views.  Columbia University. Het protocol beschrijft een efficiënte en reproduceerbare methode voor biomechanische testen van muispezen door middel van herbruikbare custom-fit 3D-geprinte armaturen. De nieuwe methoden verminderen de tijd die nodig is om het monster te testen van uren tot minuten en elimineren een belangrijk aangrijpend artefact dat een veelvoorkomend probleem was in eerdere methoden. Deze methode is niet beperkt tot de supraspinatus en achillespezen.Het kan gemakkelijk worden aangepast aan het te...

Video: Biomechanical Testing of Murine Tendons

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 ...

Biology

Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue remodeling. Based on this interest, several analytical techniques have been developed to measure local tissue strains within tendon explants during loading. However, in several cases, the accuracy and sensitivity of these techniques have not been reported, and none of the algorithms are publicly available. This has made it difficult for the more widespread measurement of local tissue strains in tendon explants. Therefore, the objective of this paper was to create a validated analysis tool for measuring local tissue strains in tendon explants that is readily available and easy to use. Specifically, a publicly available augmented-Lagrangian digital image correlation (ALDIC) algorithm was adapted for measuring 2D strains by tracking the displacements of cell nuclei within mouse Achilles tendons under uniaxial tension. Additionally, the accuracy of the calculated strains was validated by analyzing digitally transformed images, as well as by comparing the strains with values determined from an independent technique (i.e., photobleached lines). Finally, a technique was incorporated into the algorithm to reconstruct the reference image using the calculated displacement field, which can be used to assess the accuracy of the algorithm in the absence of known strain values or a secondary measurement technique. The algorithm is capable of measuring strains up to 0.1 with an accuracy of 0.00015. The technique for comparing a reconstructed reference image with the actual reference image successfully identified samples that had erroneous data and indicated that, in samples with good data, approximately 85% of the displacement field was accurate. Finally, the strains measured in mouse Achilles tendons were consistent with the prior literature. Therefore, this algorithm is a highly useful and adaptable tool for accurately measuring local tissue strains in tendons.

Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation

This paper describes an open-source digital image correlation algorithm for measuring local 2D tissue strains within tendon explants. The accuracy of the technique has been validated using multiple techniques, and it is available for public use.

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue ...

Engineering

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Two-Photon Microscopy for the Study of Tendons

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence.
SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.

This article outlines the process of preparing, setting up, and imaging tendons using multiphoton microscopy. Additionally, it covers the application of SHG for analyzing collagen fibril alignment and the creation of a 3D representation of tendons. This methodology proves highly valuable in characterizing tendon cells and their ECM during injury and development.

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in ...

Developmental Biology

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Medicine

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Cyclic Mechanical Loading for Inducing Tendinopathy in Rat Achilles Tendon

A Passive Ankle Dorsiflexion Testing System for an In Vivo Model of Overuse-induced Tendinopathy

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery time. This protocol describes a testing system that cyclically applies mechanical loads via passive dorsiflexion to the rat Achilles tendon. The custom-written code consists of pre- and post-cyclic loading measurements to assess the effects of the loading protocol along with the feedback control-based cyclic fatigue loading regimen. 
We used 25 Sprague-Dawley rats for this study, with 5 rats per group receiving either 500, 1,000, 2,000, 3,600, or 7,200 cycles of fatigue loads. The percentage differences between the pre- and post-cyclic loading measurements of the hysteresis, peak stress, and loading and unloading moduli were calculated. The results demonstrate that the system can induce varying degrees of damage to the Achilles tendon based on the number of loads applied. This system offers an innovative approach to apply quantified and physiological varying degrees of cyclic loads to the Achilles tendon for an in vivo model of fatigue-induced overuse tendon injury.

Author Spotlight: Integrating Mechanical and Biological Analysis in Tendinopathy Research 

This protocol presents a testing system used to induce quantifiable and controlled fatigue injuries in a rat Achilles tendon for an in-vivo model of overuse-induced tendinopathy. The procedure consists of securing the rat's ankle to a joint actuator that performs passive ankle dorsiflexion with a custom-written MATLAB script.

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery ...

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

Using Q Suture to Enhance Resistance to Gap Formation and Tensile Strength of Repaired Flexor Tendons

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q suture, an alternative to peripheral sutures, is presented for the use in tendon repair. Its effects on gap formation and tensile strength of the repaired tendons were compared with conventional running peripheral sutures. Three 2-strand sutures and three 4-strand sutures were used in repairing porcine tendons. The time required for performing 2Q and running sutures were recorded. The repaired tendons were subjected to a cyclic loading test, and the cycle number, during which a 2-mm gap was formed, was determined. After the cyclic loading, the gap size at the tendon ends and the ultimate strength of the repaired tendons were measured. Augmentation with the Q sutures reduced the number of tendons showing 2-mm gaps at tendon ends during cyclic loading. With addition of Q sutures 2-strand sutures significantly increased the ultimate strength of the repaired tendons and 4-strand sutures decreased the gap distance at the repair site of tendons. The time required for performing 2Q sutures was significantly less than that for running sutures. Therefore, we conclude that the Q suture is efficient in enhancing the tensile resistance and tendon repair strength and can be an alternative to conventional peripheral sutures.

Here, we present a &#8220;Q&#8221; suture technique that can be performed in tendon repair and its effects on the gap formation and tensile strength of the repaired tendons. Q suture is shown to be efficient in enhancing the tensile resistance and tendon repair strength.

Peripheral epitendinous sutures are believed to enhance core suture strength in tendon repair and decrease the risk of gapping between tendon ends. Here Q ...

Biomechanical Testing of Murine Tendons

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