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 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.
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.
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
2. Ex-vivo and post-mortem
3. Mechanical loading protocol
4. Data analysis
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 < 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 groups. There was a consistent percentage decrease in hysteresis, peak stress, and loading and unloading moduli for the 3,600 and 7,200 cycle groups. Hematoxylin and eosin- and Masson's Trichrome stained images of tendon samples verified higher levels of microstructural damage with higher cycles of dorsiflexion with more rounded cells, hypercellularity, fiber disruption, and fiber crimping (Figure 3). The results in this paper are shown to demonstrate that higher cycles of dorsiflexion cause increased levels of damage to the Achilles tendon.
Figure 1: Passive ankle dorsiflexion testing system. (A) Power supply, (B) microcontroller, (C) stepper motor, (D) torque sensor, (E) 3D electromagnetic positioning and orientation sensor, (F) 3D printed ankle mount, (G) 3D printed animal bed, (H) 3D printed nose cone holder. Please click here to view a larger version of this figure.
Figure 2: Representative cyclic loading stress-strain curves. Hysteresis curves at 0, 500, 1,000, 2,000, 3,600, and 7,200 cycles. The arrow indicates decreasing peak stress with an increasing number of cycles. Please click here to view a larger version of this figure.
Figure 3: Representative histologically stained images of tendon samples. Hematoxylin and Eosin (left) and Masson's Trichrome (right) stained images of tendons for 500, 1,000, 2,000, 3,600, and 7,200 cycle groups for this study demonstrated that increasing the number of cycles applied results in more rounded cells, hypercellularity (stars), fiber disruption, and fiber crimping (arrows). Please click here to view a larger version of this figure.
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. Their study presented a method of directly loading the patellar tendon by exposing the tendon. While this method also applied quantifiable fatigue loads to the tendon, the direct application of loads may introduce an additional inflammatory wound healing response to the skin incision and subsequent closure. With our method, the noninvasively applied loads ensure that any measured biological response is entirely due to the loading protocol rather than any external factors.
A critical component of this loading protocol is the feedback-control loop. By checking the slope of the hysteresis loading curve and increasing the dorsiflexion angle, if necessary, the system continuously fatigues the Achilles tendon. Knee splinting is a critical step since it ensures that the dorsiflexion only strains the tendon instead of moving the knee and other surrounding soft tissue. To check whether the splinting is done correctly, manually actuate the ankle after splinting to feel for a stiff tendon and monitor the hysteresis curves produced prior to the cyclic loading step.
One of the limitations of this study is that the strain values are relatively large. However, they are comparable to passive dorsiflexion of human Achilles tendons and could be caused by the elongation of the Achilles tendon and the gastrocnemius muscle18. Another limitation is that the conversions between torque and stress are limited to ex vivo measured average tendon cross-sectional area and moment arm around the ankle joint, which may vary between animals.
The pathology and early stages of chronic tendinopathy are yet to be elucidated. Along with age and other risk factors, overuse is a major contributing factor to the development of chronic tendinopathy. Reproducible overuse injuries can be simulated with multiple applications of fatigue cyclic loading bouts through our system. Further, the noninvasiveness of this system allows for the assessment of biological and structural changes in tendon damage and healing responses over long periods to understand critical biomarkers in tendinopathy.
The authors have nothing to disclose.
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)).
1/32'' Aluminum beads | |||
2.5% isoflurane | |||
3D digitizing pen | Polhemus, Vermont, NH, USA | ||
3D electromagnetic positioning and orientation sensor | Polhemus, Vermont, NH, USA | ||
5% isoflurane | |||
Customized device: 1) Assembly, sensors, 3D printed animal bed and ankle mount actuator | Assembled as described in manuscript | ||
MATLAB code | MATLAB, Natick, MA, USA | ||
Microcontroller | Ivrea, Italy | Arduino UNO, Rev3 | |
Nose cone | |||
Scalpel and scalpel holder | No. 11 scalpel | ||
Sprague-Dawley rats | Charles River Laboratories, Wilmington, MA, USA | 11-13 weeks old | |
Stepper driver | SparkFun Electronics, Niwot, CO 80503 | DM542T | |
Stepper motor | SparkFun Electronics, Niwot, CO 80503 | 23HE30-2804S | |
Straight forceps | |||
Torque sensor assembly | Futek Inc., Irvine, CA, USA | FSH03985, FSH04473, FSH03927 | |
Water heating pad |