Achilles tendinopathy is a debilitating condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Effective therapies remain elusive, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of symptomatic human tendinopathy. Post-injury analgesia is often achieved with non-steroidal anti-inflammatory drugs such as ibuprofen; however, there is increasing evidence that NSAID usage may interfere with the healing process. The deposition of aggrecan/hyaluronan (HA)-rich matrix within the tendon body and surrounding peritenon impede tendon healing and result in compromised biomechanical properties. Herein, we present work investigating chemical, biological, and mechanical loading approaches to treating Achilles tendinopathy in a murine model. Our previously established TGF-ß1-induced murine model of Achilles tendinopathy was used to investigate the cellular mechanism by which ibuprofen (chemical) therapy might lead to a worsening of tendon pathology, potentially by interfering with the native inflammation phase of tendon healing. We conclude that the use of ibuprofen for pain relief during inflammatory phases of tendinopathy has detrimental effects on the turnover of a pro-inflammatory HA matrix produced ain response to soft-tissue injury, thus preventing the switch to cellular responses associated with functional matrix remodeling and eventual healing. We examined the therapeutic potential of a recombinant human hyaluronidase, rHuPH20 (biologic, FDA approved for reducing HA accumulation in tumors) in a novel Achilles tendinopathy and retrocalcaneal bursitis injury model. The potential of rHuPH20 to effectively clear the pro-inflammatory, HA-rich matrix within the retrocalcaneal bursa (RCB) and tendon strongly supports the future refinement of injectable glycosidase preparations as potential treatments to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse. Finally, we developed a novel mouse model of hind limb muscle loading (mechanical) designed to achieve a tissue-targeted therapeutic exercise. When applied to a murine Achilles tendinopathy model, muscle loading led to a significant improvement in Achilles tendon biomechanical outcome measures, with a decrease in cross-sectional area and an increase in material properties, compared to untreated animals. Our model facilitates the future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries. Overall, these findings enhance our understanding of the mechanisms of injury and treatment in Achilles tendinopathy injuries. / Doctor of Philosophy / Achilles tendinopathy is a chronic, overuse condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Therapies are limited, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Drugs such as ibuprofen are commonly prescribed at the onset of injury to treat pain. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of human tendinopathy; however, the reasons driving the healing are not well understood. Characteristics of the disease include pain, increased tendon size, and disorganization of tendon fiber structure. Here, we present work investigating chemical, biological, and mechanical loading approaches to treating Achilles tendinopathy in a mouse model. Our mouse model of Achilles tendinopathy was used to investigate how ibuprofen (chemical) therapy might lead to a worsening of tendon by potentially interfering with the inflammation phase of tendon healing. We conclude that the use of ibuprofen for pain relief during inflammatory phases of tendinopathy has negative effects on the turnover of matrix produced in response to injury, affecting the transition to the next phase in the tendon healing response. We examined the potential of a recombinant human hyaluronidase, rHuPH20 (biologic, FDA approved for reducing HA accumulation in tumors) in a novel Achilles tendinopathy and retrocalcaneal bursitis injury model. The potential of rHuPH20 to effectively clear the proinflammatory, HA-rich matrix within the retrocalcaneal bursa (RCB) and tendon strongly supports the future refinement of injectable treatments as a potential to protect or regenerate tendon tissue by reducing inflammation and scarring in the presence of bursitis or other inducers of damage such as mechanical overuse. Finally, we developed a mouse model of hind limb muscle loading (mechanical) based on physical therapy exercises. This model led to an improvement in biomechanical measures compared to untreated animals. The model allows for investigation of the underlying mechanisms in which physical therapy promotes healing of Achilles tendon injuries. Overall, these findings enhance our understanding of the mechanisms of injury and treatment in Achilles tendinopathy injuries.

Tendinopathies are common, painful, and debilitating injuries that can be challenging to treat. Current treatment methods are limited to surgery, nonsteroidal anti-inflammatory drugs, dry needling, and injectable therapeutics, platelet rich plasma and corticosteroids. Unfortunately, these existing treatments display poor long-term outcomes and have an increased risk of reinjury. Additionally, the healing mechanism for injured tendons forms scar tissue which is characterized by disrupted extracellular matrix rather than complete injury resolution. These structural changes impact the mechanical properties of tendon, reducing their capacity to transfer and store energy, making them inferior to uninjured tendons. The reduced mechanical properties increase the risk of rupture, exacerbating this debilitating disease and decreasing quality of life. Physical therapy (eccentric loading) decreases the symptoms of tendinopathy and restores Achilles tendon functionality. However, the mechanism by which these mechanical stimulations induce healing is poorly understood. There is a clinically relevant motivation to better understand the healing cascade in response to eccentric exercises. We aim to identify and characterize the effects of eccentric rehabilitative muscle loading on the Achilles tendon and gastrocnemius muscle complex using our preclinical TGF-ß1-induced murine model of Achilles tendinopathy. To accomplish our objective, we tested three muscle loading magnitudes (50%, 75%, and 100% body weight), over three treatment durations (1, 2, and 4 weeks) to determine their effects on tendon healing. Age-matched injured/untreated and naïve groups accompanied each loading magnitude and duration period. The functional biomechanical properties, morphological adaptations, transcriptomic response, and muscle strength of the Achilles tendon were assessed. Injured/untreated tendons had a significantly increased cross-sectional area compared to naïve and all loading groups at 2 and 4 weeks. Maximum stress and elastic modulus of injured/untreated tendons were significantly lower compared to naïve and all loading groups after 4 weeks. Gastrocnemius muscle strength was maintained over time as loading magnitude increased. Force output was lower after 2 weeks at 100% body weight loading compared to the naïve group, then recovered to naïve levels after 4 weeks. Histological findings included increased cross-sectional area, matrix disorganization, and increased cellular density of injured/untreated tendons. The transcriptomic evaluation revealed several patterns of expression among exercised groups. Biological processes associated with exercised groups revealed genes responsible for inflammation, extracellular matrix organization, and cell to cell signaling. Overall, eccentric muscle loading improved tendon geometry and material properties compared to naïve levels and improved muscle strength over time. Morphological evaluation also showed improvements in cross-sectional area, and collagen orientation, and cell appearance after 2 and 4 weeks of eccentric loading. Similarly, the transcriptomic changes showed an effect from exercise and upregulation of genes essential for extracellular matrix organization, inflammatory regulation, and cell to cell signaling. / Doctor of Philosophy / Tendons are connective tissues that join muscle to bone to facilitate movement. Tendons are prone to injury because of their consistent use for daily activities. The Achilles tendon is particularly at risk for injury since it is responsible for walking, running, and jumping, making it susceptible to overuse. Current treatment methods such as surgery and pain relief medication can provide immediate symptomatic relief, but have limited long-term success. Physical therapy provides relief of symptoms of chronic Achilles tendinopathy and improves the tendon healing response. Eccentric-based exercises (lengthening of the muscle while it contracts) are known as 'heel drops' and have been the most successful physical therapy technique to improve Achilles tendon healing. However, the way these rehabilitative exercises facilitate healing is poorly understood. It is difficult to determine the exact methods of healing because the required frequency and amount of exercise varies between patients, and recovery times can take weeks or months. In this research, we aim to better characterize how different 'heel drop' routines improve tendon healing, providing a foundation for determining the intensity and duration of rehabilitative exercises that can be applied for better clinical outcomes. To examine the effects of different eccentric-based loading profiles, we used a previously developed mouse model of chronic Achilles tendinopathy and customizable muscle stimulation device to simulate human physical therapy exercises at different intensity levels: full body weight (100%), assisted body weight (75%), and half body weight (50%). Prior to beginning treatments, we induced a tendon injury that mimics human injury and measured muscle strength before any exercise was performed. Mice were then introduced to muscle loading treatments that mimic clinical exercise routines: 3 sets of 10 heel drops, performed twice a week for 1-, 2- or 4-weeks. After the final day of exercise, muscle strength was measured again to see how the heel drop exercises impacted the muscle tissue. Tendons were collected and the mechanical properties, histologic changes, and transcriptomic adaptations were evaluated. Eccentric-based exercises improved the mechanical properties injured tendons and improved the architecture compared to injured/untreated controls. Injured tendons without treatment had inferior tendon mechanical properties and inferior structural changes. We also saw improved tissue changes and upregulation of genes responsible for tendon healing after exercise compared to naïve or injured/untreated mice. Our research demonstrates that performing consistent eccentric-based exercises for 2 or more weeks positively impacts tendon healing.