We would like to acknowledge our funding supports: the Joe Fallon Research Fund, the Dr. Louis Meeks BIDMC Sports Medicine Trainee Research Fund, and an intramural grant (AN), all from BIDMC Orthopaedics, along with support from the National Institutes of Health (2T32AR055885 (PMW)).

This study was conducted per Institutional Animal Care and Use Committee (IACUC) approval at Beth Israel Deaconess Medical Center. Animals were anesthetized using 5% isoflurane for induction and 2.5% for maintenance, and care was taken to avoid hypothermia.
1. Setting up the testing system
<ol>
	<li>Control passive ankle rotation by a stepper motor to apply consistent rotation and torque. Control the stepper motor with a microcontroller. Use the inputs from the 3D position a...

Cyclic Mechanical Loading for Inducing Tendinopathy in Rat Achilles Tendon

<ol>
	<li>Kaux, J. F., Forthomme, B., Goff, C. L., Crielaard, J. M., Croisier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+opinions+on+tendinopathy.">Current opinions on tendinopathy.</a> J Sports Sci Med. 10 (2), 238-253 (2011).</li><li>Maffulli, N., Longo, U. G., Kadakia, A., Spiezia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achilles+tendinopathy.">Achilles tendinopathy.</a> Foot Ankle Surg. 26 (3), 240-249 (2020).</li><li>Teunis, T., Lubberts, B., Reilly, B. T., Ring, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+and+pooled+analysis+of+the+prevalence+of+rotator+cuff+disease+with+increasing+age.">A systematic review and pooled analysis of the prevalence of rotator cuff disease with increasing age.</a> J Shoulder Elbow Surg. 23 (12), 1913-1921 (2014).</li><li>von Rickenbach, K. J., Borgstrom, H., Tenforde, A., Borg-Stein, J., McInnis, K. 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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=Achilles+tendon+injuries.">Achilles tendon injuries.</a> Curr Opin Rheumatol. 13 (2), 150-155 (2001).</li><li>Silbernagel, K. G., Hanlon, S., Sprague, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+clinical+concepts:+conservative+management+of+Achilles+tendinopathy.">Current clinical concepts: conservative management of Achilles tendinopathy.</a> J Athl Train. 55 (5), 438-447 (2020).</li><li>Tayfur, 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=Are+landing+patterns+in+jumping+athletes+associated+with+patellar+tendinopathy?+A+systematic+review+with+evidence+gap+map+and+meta-analysis.">Are landing patterns in jumping athletes associated with patellar tendinopathy? A systematic review with evidence gap map and meta-analysis.</a> Sports Med. 52 (1), 123-137 (2022).</li><li>Malliaras, P., Cook, J. L., Kent, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+ankle+dorsiflexion+range+may+increase+the+risk+of+patellar+tendon+injury+among+volleyball+players.">Reduced ankle dorsiflexion range may increase the risk of patellar tendon injury among volleyball players.</a> J Sci Med Sport. 9 (4), 304-309 (2006).</li><li>Backman, L. J., Danielson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+range+of+ankle+dorsiflexion+predisposes+for+patellar+tendinopathy+in+junior+elite+basketball+players:+a+1-year+prospective+study.">Low range of ankle dorsiflexion predisposes for patellar tendinopathy in junior elite basketball players: a 1-year prospective study.</a> Am J Sports Med. 39 (12), 2626-2633 (2011).</li><li>Glazebrook, M. A., Wright, J. R., Langman, M., Stanish, W. D., Lee, J. 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G., 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=Increased+serum+and+musculotendinous+fibrogenic+proteins+following+persistent+low-grade+inflammation+in+a+rat+model+of+long-term+upper+extremity+overuse.">Increased serum and musculotendinous fibrogenic proteins following persistent low-grade inflammation in a rat model of long-term upper extremity overuse.</a> PLoS One. 8 (8), e71875 (2013).</li><li>Fung, 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=Early+response+to+tendon+fatigue+damage+accumulation+in+a+novel+in+vivo+model.">Early response to tendon fatigue damage accumulation in a novel in vivo model.</a> J Biomech. 43 (2), 274-279 (2010).</li><li>Ueda, Y., 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=Molecular+changes+to+tendons+after+collagenase-induced+acute+tendon+injury+in+a+senescence-accelerated+mouse+model.">Molecular changes to tendons after collagenase-induced acute tendon injury in a senescence-accelerated mouse model.</a> BMC Musculoskelet Disord. 20 (1), 120 (2019).</li><li>Bloom, E., 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=Overload+in+a+rat+in+vivo+model+of+synergist+ablation+induces+tendon+multi-scale+structural+and+functional+degeneration.">Overload in a rat in vivo model of synergist ablation induces tendon multi-scale structural and functional degeneration.</a> J Biomech Eng. 145 (8), 081003 (2023).</li><li>Williamson, P. 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This study presents a method to cyclically load the rat Achilles tendon with a passive ankle dorsiflexion system for an in-vivo overuse-induced tendinopathy model. The importance of the system lies in its ability to isolate the Achilles tendon, apply quantifiable loads without surgically accessing the tendon, and measure in-vivo tendon properties. 
In 2010, Fung et al. presented a rat patellar tendon fatigue model with a custom-built testing system14. ...

As tendons connect muscle to bone and experience daily repetitive motions throughout their lifetime, they are highly prone to overuse injuries that are painful and limiting and result in impaired mechanical function, affecting 30-50% of the population1. Tendinopathies are chronic conditions considered overuse injuries due to repetitive fatigue motions and inadequate healing to pre-injury levels. Both upper and lower extremities are commonly affected, including the rotator cuff, elbow, Achilles tendon, and patellar tendon2,3,4,5. Achilles tendinopathy is common in activities involving running and jumping, especially athletes involved in track and field, middle- and long-distance running, tennis, and other ball sports, affecting 7-9% of runners6,7. Injuries from running and jumping may also cause limited ankle dorsiflexion, which is a risk factor for Achilles and patellar tendinopathies8,9,10. Thus, there is a need for a better assessment and characterization of tendinopathy, which this study can provide as a rat model of passive ankle dorsiflexion for overuse Achilles tendon injuries.
Previous work using small animal models has been aimed at studying the development and markers of tendinopathy. These include treadmill exercise, repetitive reaching, direct tendon loading, collagenase injections, surgery, and in vitro studies11,12,13,14,15,16. Although the literature has benefited from the identification of damage markers from employing these tendinopathy models, limitations include loading the tendon in non-physiologically relevant joint motions, as in the case of direct loading of the tendon, not directly measuring applied loads, such as for treadmill studies, and not using physiological overuse, as in the case for collagenase injections, among others. To that end, this study aimed to develop a system that noninvasively applies quantified loads to the Achilles tendon with the application for overuse-induced tendinopathy studies to fill the gaps in previously developed small animal models for tendinopathy. We performed a pilot study to demonstrate that the system induces reproducible changes in mechanical properties over a range of loading cycles. This system enables physiologically relevant motion and loading to induce overuse while simultaneously quantifying and measuring the forces applied to and experienced by the tendon during the loading regimen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/32&#39;&#39; Aluminum beads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D digitizing pen</td>
			<td>Polhemus, Vermont, NH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D electromagnetic positioning and orientation sensor</td>
			<td>Polhemus, Vermont, NH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5% isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Customized device: 1) Assembly, sensors, 3D printed animal bed and ankle mount actuator</td>
			<td></td>
			<td></td>
			<td>Assembled as described in manuscript</td>
		</tr>
		<tr>
			<td>MATLAB code</td>
			<td>MATLAB, Natick, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcontroller</td>
			<td>Ivrea, Italy</td>
			<td>Arduino UNO, Rev3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Nose cone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel and scalpel holder</td>
			<td></td>
			<td></td>
			<td>No. 11 scalpel</td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Charles River Laboratories, Wilmington, MA, USA</td>
			<td></td>
			<td>11-13 weeks old</td>
		</tr>
		<tr>
			<td>Stepper driver</td>
			<td>SparkFun Electronics, Niwot, CO 80503</td>
			<td>DM542T</td>
			<td></td>
		</tr>
		<tr>
			<td>Stepper motor</td>
			<td>SparkFun Electronics, Niwot, CO 80503</td>
			<td>23HE30-2804S</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Torque sensor assembly</td>
			<td>Futek Inc., Irvine, CA, USA&#160;</td>
			<td>FSH03985, FSH04473, FSH03927</td>
			<td></td>
		</tr>
		<tr>
			<td>Water heating pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

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

Our work addresses the mechano biological factors involved in the initiation and progression of overuse induced tendinopathy. The questions we are trying to answer include what factors of exercise, such as frequency, magnitude, and duration of load affect the tendon's response to injury. Current tools such as mechanical measures, histology, and biological assays to evaluate outcome measures are centered and limited to end stage tissue harvesting.With this model, we can explore in vivo continuous measurements with tools such as ultrasound and MRI In the field, in vivo models load the tendon and physiologically relevant manners, while ex vivo models allow for the measurement of biological responses to injuries. Our protocol combines these two measures for direct measurement of tendon stresses and strains during injuries and the associated biological changes. We've established an adaptive system to induce tendon injuries, which allows for simultaneous exploration of mechanical and biological factors.Having established this model, future directions include assessing different parameters, therapeutic avenues, and rehabilitation measures.

With the increasing number of applied cycles, there was a greater reduction in in vivo tendon mechanical properties. There was a significantly lower reduction in hysteresis and the loading and unloading moduli for the 500-cycle group in comparison to the 3,600 and 7,200 cycle groups (p &#60; 0.05) (Figure 2). While there was a significant reduction in peak stress per cycle from the 500 cycle to the 3,600 cycle group, there was no significant reduction between the 500 and 7,200 cycle...

Watch this Scientific Journal Video about Integrating Mechanical and Biological Analysis in Tendinopathy Research at JoVE.com

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 ...