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 and orientation system to mark the degrees of rotation. Use the outputs from the torque sensor to provide feedback control for increasing the angle of dorsiflexion if the limit to threshold is not reached.</li>
	<li>To begin, connect the microcontroller, torque sensor, 3D electromagnetic positioning, and orientation system to a computer and the power supply. Control the custom-built system using an in-house developed MATLAB code (Figure 1). Download the MATLAB code files from GitHub and follow specific instructions on running the code from the GitHub page instructions (https://github.com/Nazarian-Lab/PassiveAnkleDorsiflexionSystem).</li>
	<li>Open MATLAB with the code files. Open the PDImfc software to connect the 3D electromagnetic positioning and orientation system to the MATLAB program. Click Connect | Continuous P&#38;O | StartSockExport(). Keep the application open in the background.</li>
</ol>
2. Ex-vivo and post-mortem
<ol>
	<li>Euthanize six 13-week-old Sprague-Dawley rats via CO2 inhalation and a secondary method of euthanasia via thoracotomy. Dissect the right Achilles tendon with the calcaneus and myotendinous junction intact. Freeze at -20&#176; C to perform mechanical testing at a later time. After the tendon is thawed, fine dissected, and prepared for mechanical testing, perform&#160;tensile loading to failure to obtain the ultimate tensile strength (UTS) of the tendon (Preload to 0.1N, Preconditioning for 10 cycles from 0.1 - 1 N, Ramp to failure at a constant displacement of 0.1 mm/s). Use 15% of the UTS as an input for the system to perform preconditioning for a later step, as described in step 3.4.</li>
	<li>Euthanize another group of five animals with the same procedure for moment arm and strain measurements. Perform an X-ray of the left leg with the ankle in 90&#176; dorsiflexion next to a ruler as a reference. Open the X-ray image in Fiji, using the ruler in the image as a reference, measure the tendon moment arm from the center of rotation of the ankle joint to the back of the ankle to be used as an input in the MATLAB code to convert the input force for preconditioning described in step 2.1 to the corresponding torque value as well as conversion between outputted torque and force for data analysis.</li>
	<li>Immobilize the left hindlimb by taping two splints to place the knee in full extension. Lightly dorsiflex the ankle by pushing on the toes to ensure that the ankle rotation occurs due to the isolated tendon rather than involving surrounding soft tissues and is in tension. If not in tension or if there is movement in the knee, retape the splint.</li>
	<li>Expose the tendon by removing the skin around the Achilles tendon. Place glue on a 1/32-inch aluminum bead, place it on the free tendon closest to the myotendinous junction of the Achilles tendon, and use a cotton swab with saline to remove excess glue. Repeatedly apply saline to the Achilles tendon throughout the remaining procedure to ensure moistness of the tissue.</li>
	<li>Measure the cross-sectional area of the tendon using a digital caliper prior to applying any load. Assume the tendon is an ellipse and measure the width and thickness in triplicates.</li>
	<li>Place the rat on the full-body platform in a prone position. Secure the ankle onto the joint actuator with a zip tie around the ankle and another around the toes, and secure the knee split with two zip ties. Rotate the axle so that the ankle is at full plantarflexion.</li>
	<li>Connect the digitizing pen of the 3D electromagnetic position and orientation system to the computer and turn on the power supply.</li>
	<li>Run the system code (described in further detail in Step 3) for the specified number of cycles (in this study, six euthanized rats received 7,200 cycles).</li>
	<li>At 0, 500, 1,000, 2,000, 3,600, and 7,200 cycles for the ex vivo strain measurements, pause the cyclic loading regimen and measure the length of the tendon from the calcaneus to the aluminum bead at increments of 5&#176; from 0 to 40&#176; of dorsiflexion (the limit of actuation due to physical constraints of the system) using the 3D digitizing pen in triplicates in alternating fashion.</li>
	<li>Calculate the tendon strain at varying angles using the lengths obtained from step 2.9, where the initial length is at 0&#176; dorsiflexion for each cycle number. Perform a linear fit to obtain the relationship between dorsiflexion angle and strain at each cycle number. Use this relationship to convert raw angle data to strain for data analysis.</li>
	<li>Calculate the tendon cross-sectional area using a digital caliper at 40&#176; dorsiflexion assuming incompressibility (constant volume) with the tendon length measurements at 0&#176; and 40&#176; and the measured cross-sectional area at 0&#176;. Use this cross-sectional area at each number of cycles to convert force to stress for data analysis (stress = force / cross-sectional area).</li>
</ol>
3. Mechanical loading protocol
<ol>
	<li>For this section of the study, we used 25 female Sprague-Dawley 11-week-old rats, with 5 rats each randomly allocated to receive 500, 1,000, 2,000, 3,600, or 7,200 cycles of fatigue loading. 
	NOTE: The preconditioning, initial calibration, and pre- and post-measurement take around 15 min to run, and the cyclic fatigue loading regimen takes 1 second per cycle. Thus, the longest time the rat is under anesthesia is around 2 h, which was performed under IACUC-approved protocols.</li>
	<li>Connect the microcontroller, torque sensor, 3D electromagnetic positioning, and orientation system to a computer and the power supply. Control the custom-built system using the in-house developed MATLAB code (Figure 1).</li>
	<li>Turn on the computer and open MATLAB with the code files. Open the PDImfc software to connect the 3D electromagnetic positioning and orientation system to the MATLAB program. Click Connect | Continuous P&#38;O | StartSockExport(). Keep the application open in the background.</li>
	<li>Induce anesthesia with 5% isoflurane through inhalation in an induction chamber. Following induction, secure the animal onto the full-body platform with a water-based heating element attached to maintain temperature and sustain anesthesia with 2.5% isoflurane via a nose cone attachment. Use a wet ointment on the eyes to prevent dryness during anesthesia.</li>
	<li>Place the rat on the full-body platform in a prone position. Secure the ankle onto the joint actuator with a zip tie around the ankle and another around the toes, and secure the knee split with two zip ties. Rotate the axle so that the ankle is at full plantarflexion. 
	NOTE: Ensure the zip ties do not cause constriction or lesions, take care in tightening, and if needed, place gauze in between the zip tie and the skin for a layer of protection.</li>
	<li>For the following steps involving running the system&#39;s code, click Run on MATLAB for each section of the code corresponding to the specific loading test.</li>
	<li>Cycle the ankle 50 times to 15% of the ultimate tensile stress based on the value of the ultimate tensile stress of the Achilles tendon from ex vivo pull to failure tests as measured based on step 2.1.</li>
	<li>Perform an initial calibration of the tendon by dorsiflexing it three times to 12&#176;. Use the slope of the linear region of the loading region of the hysteresis curve to calculate the exponential region of the curve.</li>
	<li>Incrementally dorsiflex the ankle at increasing angles until either the exponential region of the curve is obtained by calculating the slope of the peak of the loading region of the curve (calculated using the in-house developed MATLAB code) or until it is rotated to 40&#176;, whichever occurs first.</li>
	<li>At the final obtained angle, perform five cyclic mechanical measurements as a preloading baseline.</li>
	<li>Perform the cyclic fatigue loading regimen for a specified number of cycles (in this study, for either 500, 1,000, 2,000, 3,600, or 7,200 cycles).</li>
	<li>Every 50 cycles, calculate the slope of the loading portion of the hysteresis curve (calculated through the in-house developed MATLAB code) to ensure it is still in the exponential region. Increase the dorsiflexion angle by 1&#176; unless it is already at 40&#176; until this exponential region is achieved.</li>
	<li>After the cyclic loading regimen is complete, perform five cyclic mechanical measurements as post loading measurements at the initially chosen angle to measure tendon mechanical properties.</li>
	<li>Remove the zip ties and splint. Return the animal to the recovery chamber. The animal is not left unattended until it has regained sufficient consciousness, followed by which it is returned to its cage. Monitor the animals daily for any adverse clinical signs and if present, administer buprenorphine at a dose of 1.2 mg/kg subcutaneously once every 72 h or perform early euthanasia. Euthanize the animals following 7 days of cage activity via CO2 inhalation and a secondary means of euthanasia via thoracotomy. 
	&#8203;NOTE: The cyclic loading application and mechanical measurements were obtained with a custom-made jig consisting of a torque sensor, 3D-printed ankle joint actuator and animal bed, a 3D electromagnetic position and orientation system, and a stepper motor rotating a shaft to achieve dorsiflexion, as previously reported by our group17. This system is controlled by a MATLAB script mentioned in step 1.2. The torque sensor and the position and orientation system capture torque and position data throughout the system&#39;s loading protocol.</li>
</ol>
4. Data analysis
<ol>
	<li>Load the pre- and post-measurement data separately into MATLAB.</li>
	<li>Convert the torque to stress based on the measured moment arm from step 2.2 and the cross-sectional area measured at the specified number of loads applied obtained from step 2.11 using equations (1) and (2): 
	<img alt="Equation 1" src="/files/ftp_upload/65803/65803eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
	<img alt="Equation 2" src="/files/ftp_upload/65803/65803eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;(2)</li>
	<li>Convert the angle to strain based on the conversion obtained from step 2.10.</li>
	<li>Calculate the average hysteresis (area between the loading and unloading curves), peak stress (maximum stress value of the cycle) and loading and unloading moduli (linear fit of the last 50% of the loading and the first 60% of the unloading curves) for the pre- and post- measurement cycles.</li>
	<li>Calculate the percentage change in the mechanical properties from step 4.4 between the pre- and post-measurement cycles.</li>
</ol>

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. C. <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:+evaluation,+rehabilitation,+and+prevention.">Achilles tendinopathy: evaluation, rehabilitation, and prevention.</a> Curr Sports Med Rep. 20 (6), 327-334 (2021).</li><li>Aicale, R., Oliviero, A., Maffulli, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+Achilles+and+patellar+tendinopathy:+what+we+know,+what+we+can+do.">Management of Achilles and patellar tendinopathy: what we know, what we can do.</a> J Foot Ankle Res. 13 (1), 59 (2020).</li><li>Jarvinen, T. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+achilles+tendons+in+an+overuse+rat+model.">Histological analysis of achilles tendons in an overuse rat model.</a> J Orthop Res. 26 (6), 840-846 (2008).</li><li>Carpenter, J. E., Flanagan, C. L., Thomopoulos, S., Yian, E. H., 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=The+effects+of+overuse+combined+with+intrinsic+or+extrinsic+alterations+in+an+animal+model+of+rotator+cuff+tendinosis.">The effects of overuse combined with intrinsic or extrinsic alterations in an animal model of rotator cuff tendinosis.</a> Am J Sports Med. 26 (6), 801-807 (1998).</li><li>Gao, H. 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. 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=A+passive+ankle+dorsiflexion+testing+system+to+assess+mechanobiological+and+structural+response+to+cyclic+loading+in+rat+Achilles+tendon.">A passive ankle dorsiflexion testing system to assess mechanobiological and structural response to cyclic loading in rat Achilles tendon.</a> J Biomech. 156, 111664 (2023).</li><li>Oliveira, L. F., Peixinho, C. C., Silva, G. A., Menegaldo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+passive+mechanical+properties+estimation+of+Achilles+tendon+using+ultrasound.">In vivo passive mechanical properties estimation of Achilles tendon using ultrasound.</a> J Biomech. 49 (4), 507-513 (2016).</li></ol>

The authors have no conflicts of interest to declare.

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><tbody><tr><td>1/32&#039;&#039; 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&amp;nbsp;</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&amp;nbsp;</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.

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

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.

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 ...

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Research

JoVE Journal

Bioengineering

786 Views.  Beth Israel Deaconess Medical Center, Harvard Medical School. Ons werk richt zich op de mechanobiologische factoren die betrokken zijn bij het ontstaan en de progressie van door overmatig gebruik geïnduceerde tendinopathie. De vragen die we proberen te beantwoorden, zijn onder meer welke factoren van lichaamsbeweging, zoals frequentie, omvang en duur van de belasting, van invloed zijn op de reactie van de pees op blessures. De huidige hulpmiddelen zoals mechanische metingen, histologie en biologische tests om uitkomstmaten te evalueren, zijn gecentreer...

Video: Auteur in de schijnwerpers: integratie van mechanische en biologische analyse in onderzoek naar tendinopathie 

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

Medicine

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described to assess MGT. Still, a standardized examination procedure is missing. Literature argues for weightbearing examination but the degree of knee flexion needed to eliminate the restraining effect of the M. gastrocnemius on ankle dorsiflexion (ADF) is unknown. This manuscript investigates the effect of knee flexion on ankle dorsiflexion and provides a detailed description of a standardized examination protocol. Examination on 20 healthy individuals revealed, that 20&#176; of knee flexion is sufficient to fully eliminate the influence of the M. gastrocnemius on ADF. This builds the prerequisite for a standardized examination for MGT. Non-weightbearing and weightbearing examination of ADF has to be conducted with the knee fully extended and at least 20&#176; flexed. Two investigators should conduct non-weightbearing testing with the subject in supine position. In order to obtain reliable results, the axis of the fibula should be marked. One examiner can conduct weightbearing examination with the subject in lunge stance. Isolated MGT is present if ADF is impaired with the knee fully extended and knee flexion results in a significant ADF increase. The herein presented standardized examination is the prerequisite for future studies aiming at establishing norm values.

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Isolated Musculus gastrocnemius tightness is a common cause for foot and ankle pathologies. Currently no standardized examination procedure exists. This manuscript demonstrates that 20 degree of knee flexion eliminates the restraining effect of the M. gastrocnemius on ankle dorsiflexion and presents a video description of a standardized examination protocol.

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described ...

Autologous Microfractured and Purified Adipose Tissue for Arthroscopic Management of Osteochondral Lesions of the Talus

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several studies have focused their attention on mesenchymal stem cells derived from adipose tissue. Adipose-derived stem cells (ADSCs) exhibit morphological characteristics and properties similar to other mesenchymal cells, and are able to differentiate into several cellular lines. Moreover, these cells are also widely available in the subcutaneous tissue, representing 10 - 30% of the normal body weight, with a concentration of 5,000 cells per gram of tissue.
In the presented technique, the first step involves harvesting ADSCs from the abdomen and a process of microfracture and purification; next, the surgical procedure is performed entirely arthroscopically, with less soft tissue dissection, better joint visualization, and a faster recovery compared with standard open procedures. Arthroscopy is characterized by a first phase in which the lesion is identified, isolated, and prepared with microperforations; the second step, performed dry, involves injection of adipose tissue at the level of the lesion.
Between January 2016 and September 2016, four patients underwent arthroscopic treatment of osteochondral lesion of the talus with microfractured and purified adipose tissue. All patients reported clinical improvement six months after surgery with no reported complications. Functional scores at the latest follow-up are encouraging and confirm that the technique provides reliable pain relief and improvements in patients with osteochondral lesion of the talus.

The aim of this study is to report a protocol for the arthroscopic treatment of osteochondral lesions of the talus using microfractured and purified adipose-derived stem cells.

In recent years, regenerative techniques have been increasingly studied and used to treat osteochondral lesions of the talus. In particular, several ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

Neuroscience

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

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 ...

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 ...

A Mouse Model of Ankle-Subtalar Complex Joint Instability

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and can eventually lead to post-traumatic osteoarthritis (PTOA) in the long term. However, due to the complexity of the injury mechanism and the clinical manifestations, such as ecchymosis, hematoma, or tenderness in the lateral foot, there is no clinical consensus on diagnosing and treating ASCJ instability. Since the musculoskeletal structure of the bones and ligaments of the mouse hindfoot is comparable to that of humans, an animal model of ASCJ instability in mice was established by the transection of ligaments around the ASCJ. The model was well-validated through a series of behavioral tests and histological analyses, including a balance beam test, a footprint analysis (an assessment of exercise level and balance ability in mice), a thermal nociception assessment (an assessment of foot sensory function in mice), micro-computed tomography (CT) scanning, and section staining of the articular cartilage (an assessment of articular cartilage damage and degeneration in mice). The successful establishment of a mouse model of ASCJ instability will provide a valuable reference for clinical research on the injury mechanism and result in better treatment options for ankle sprain.

The ankle-subtalar complex joint (ASCJ) is the core of the foot and plays a key role in balance control in daily activities. Sports injuries often lead to instability in this joint. Here, we describe a mouse model of ligament transection-induced instability of the ASCJ.

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and ...

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

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