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China, 2The Hong Kong Jockey Club Sports Medicine and Health Sciences Centre, ... University of Hong Kong, Hong Kong SAR, China, 4Centre of Sports and ...
Scand J Med Sci Sports 2011: 21: 3–17 doi: 10.1111/j.1600-0838.2010.01164.x

& 2010 John Wiley & Sons A/S

Review

What are the validated animal models for tendinopathy? P. P. Y. Lui1,2,3, N. Maffulli4, C. Rolf5, R. K. W. Smith6 1

Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China, 2The Hong Kong Jockey Club Sports Medicine and Health Sciences Centre, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China, 3Program of Stem Cell and Regeneration, School of Biomedical Science, The Chinese University of Hong Kong, Hong Kong SAR, China, 4Centre of Sports and Exercise Medicine, Barts and The London School of Medicine and Dentistry Mile End Hospital, London UK, 5Department of Orthopaedics, Clintec, Karolinska Institutet, Stockholm, Sweden, 6Department of Veterinary Clinical Sciences, The Royal Veterinary College, Herts, UK

Corresponding author: Pauline Po Yee Lui, PhD, Prince of Wales Hospital, Room No. 74025, 5/F Clinical Sciences Building, Shatin, Hong Kong SAR, China. Tel: 1852 2632 3072, Fax: 1852 2646 3020, E-mail: [email protected] Accepted for publication 25 May 2010

Chronic tendinopathy refers to a broad spectrum of pathological conditions in tendons and their insertion, with symptoms including activity-related chronic pain. To study the pathogenesis and management strategies of chronic tendinopathy, studies in animal models are essential. The different animal models in the literature present advantages and limitations, and there is no consensus regarding the criteria of a universal tendinopathy animal model. Based on the review of literature and the discussion in the International Symposium on Ligaments and Tendons-X, we concluded that established clinical, histopathological and functional characteristics of human

tendinopathy were all important and relevant criteria to be met, if possible, by animal models. As tendinopathy is a progressive, multifactorial tendon disorder affecting different anatomical structures, it may not be realistic to expect a single animal model to study all aspects of tendinopathy. Staging of tendinopathy over time and clearer definition of tendinopathies in relation to severity and type would enable realistic targets with any animal model. The existing animal models can be used for answering specific questions (horses for courses) but should not be used to conclude the general aspects of tendinopathy neither in animals nor in human.

Tendinopathy is a chronic painful tendon disorder that is prevalent among athletes and sedentary subjects. Tendon ‘‘overuse’’ injuries has been claimed to account for 30–50% of all sports-related injuries (Kannus, 1997), and almost half of all occupational illnesses in the United States (United States, Bureau of Labor Statistics, 1995). Commonly affected tendons from sports activities are the Achilles, patellar, rotator cuff and medial/lateral elbow tendons. In this context, we deliberately disregard a number of wellknown collagen disorders and systemic diseases such as osteogeneiss imperfecta, where alterations and mechanical weakening of tendons and ligaments are typical (Gautieri et al., 2009). The lifetime cumulative incidence of Achilles tendinopathy in elite endurance athletes is about 10 times higher than that in sedentary persons (Kujala et al., 2005). The incidence of lateral epicondylar tendinopathy in general practice in some countries is 4–7% per year, with an annual incidence of 1–3% in the general population (Allander, 1974; Chard & Hazleman, 1989). However, this does not mean that tennis per se causes these painful conditions (Chard & Hazleman, 1989). It should be noted that up to 30% of severe Achilles tendinopathy cases requiring surgery are not related

to physical activity (Rolf & Movin, 1997). Inactive patients with tendinopathy might have some underlying risk factors, which were not clear at present. Recently, Gaida et al. (2009) reported that subjects with chronic painful Achilles tendinopathy were associated with dyslipidemia, which was a characteristic of insulin resistance, suggesting a possible metabolic involvement in mid-portion Achilles tendinopathy as it often affects middle-aged, non-active and overweight individuals (Rolf & Movin, 1997; Fahlstrom et al., 2003; Holmes & Lin, 2006; Frey & Zamora, 2007). The association between tendinopathy and being overweight (Fahlstrom et al., 2003; Holmes & Lin, 2006; Frey & Zamora, 2007), hip ratio (Gaida et al., 2004; Shiri et al., 2006) or waist circumference (Shiri et al., 2006; Malliaras et al., 2007) has also been reported. Therefore, factors (both intrinsic and extrinsic) could affect the progression of tendinopathy in addition to overuse. Even among elite level athletes, a slightly elevated waist circumference was reported to dramatically increase the risk of tendon abnormality (Malliaras et al., 2007). In addition, direct trauma to a tendon may play a role in the development of tendinopathy (Garau et al., 2008).

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Lui et al. Despite its prevalence, the underlying pathogenesis of tendinopathy is poorly understood, and management usually only aims to remove the signs and symptoms. The presence of a validated animal model would enable in-depth studies on the etiology, molecular mechanisms and potential treatments of tendinopathy, as animals are more homogeneous and are easier to control than humans. As we can better control the variation in animal experiments, it would be possible to isolate the effect of a single factor. Tissue specimens can be obtained easily and at early time points before the onset of symptoms, which is not possible in humans. The availability of preclinical data is definitely essential before the potential treatment modalities are studied in clinical trials according to the Food and Drug Administration requirements. Animal models are therefore indispensable for tendinopathy research. However, the use of animals for experimentation also raises many concerns about the welfare of the animals. To make both scientific and animal welfare decision, experiments must have the clear objective of improving the welfare of man and/or animals, and the researchers need to keep constantly animal welfare at the forefront to ensure humane treatment of all animals. It is necessary to carefully study the possible behavioral changes of the animal such as pain, stress and discomfort, which may be related to the scientific interests at hand, but more to the animals’ welfare. The principles of three Rs (replacement, reduction and refinement) in animal studies should be observed (Uvarov, 1985; Baumans, 2005). Several animal models are currently being used to study the pathogenesis and potential interventions in tendinopathy. These models are established either according to the frequently hypothesized etiological factor or injection of chemicals reported in tendinopathy. Each of these animal models has its own advantages and limitations, and the models are often criticized for not showing similarities to clinical tendinopathy. The lack of consensus on appropriate animal models for the study of tendinopathy hinders the future development in this area. With the availability of new findings on the pathogenesis of tendinopathy, the animal models presently in use need to be re-evaluated. A research forum with clinical and basic experts in tendinopathy was held as part of an associative program in the International Symposium on Ligaments and Tendons-X, which provided valuable insight to the authors in preparing this review after a review of the literature. This review aims to summarize the clinical definition of tendinopathy and its pathogenesis, from which to reassess the current animal models and propose improvement of the existing animal models. It is hoped that by reaching a consensus among the clinical and basic scientists, the agreed appropriate animal mod-

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els can be used to further advance developments in this field. Definition of tendinopathy Clinical and functional presentation Chronic tendinopathy can affect virtually all tendons, but is more prevalent in the Achilles, patellar, rotator cuff and medial/lateral elbow tendons (Maffulli et al., 2003). Clinically, the affected tendon presents with chronic activity-related tendon pain with various degrees of incapacity, tenderness and localized swelling. Hypoechogenic regions and increase in tendon thickness with loss of the imaging signals typical of a well-aligned collagen fiber are often observed at ultrasonography (US) (Movin et al., 1998b). Tendon enlargement and heterogeneity, with focal high T1 and T2 signal, are typical findings at the tendon insertion or mid-substance in magnetic resonance images (MRI) (Movin et al., 1998a, b). Neovascularization evident on Doppler and color Doppler US may correlate with pain and function (Reiter et al., 2004; Cook et al., 2005; Zeisig et al., 2010), although it should be kept in mind that neovascularity is a normal response associated with healing and to physical activity and training, and not necessarily a sign of pathology (Malliaras et al., 2008), which is consistent with other reports showing that the amount of Doppler activity was unrelated to the degree of pain (Khan et al., 1999; van Snellenberg et al., 2007; Malliaras et al., 2008; Pfirrmann et al., 2008; Lewis et al., 2009). Calcium deposits, although not always observed, are sometimes observed at a later stage of the development of the disorder and in a sub-set of tendinopathy called calcifying tendinopathy. The presence of calcification worsens the clinical manifestation of tendinopathy with an increase in rupture rate (Jim et al., 1993), slower recovery times (Hashimoto et al., 2003) and a higher frequency of post-operative complications (Aina et al., 2001). Whether preceding tendinopathy will predispose the tendon to rupture is a topic of debate, although there appears to be more evidence in support of the association (Arner et al., 1959; Kannus & Jozsa, 1991; Jarvinen et al., 1997; Maffulli et al., 2000; Tallon et al., 2001; Zafar et al., 2009; Kongsgaard et al., 2010). Histological evaluation of acutely ruptured Achilles tendons revealed a greater degree of established degeneration than seen in chronic painful tendons caused by overuse, suggesting that the histopathological changes in tendinopathic tendons were less advanced than those found in ruptured tendons (Tallon et al., 2001). On the other hand, Kongsgaard et al. (2010) reported that fibril morphology was abnormal in tendinopathy but there was no difference in tendon mechanical properties, which might predispose the tendon to rupture.

Tendinopathy animal model Histopathology Histologically, tendinopathic tissue shows a defective healing response with increase in cellularity, vascularity, matrix disturbance with increase in proteoglycan deposition, particularly the oversulfated form, extracellular matrix degradation, rounding of cell nuclei and acquisition of chondrocyte phenotypes, occasional adipose and bony metaplasia and changes in the expression profiles of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (Riley et al., 1996; Fu et al., 2002a, b, 2007; Jones et al., 2006; Maffulli et al., 2006a, b; Karousou et al., 2008, 2010). Because of the degradation and change in the composition of the extracellular matrix (Xu & Murrell, 2008), it is hypothesized that the affected tendon is weakened and hence is predisposed to rupture or re-injury, although the presence of degenerative changes does not always lead to symptoms. Although there can be degenerative changes in the extracellular matrix, tendinopathy is not a typical degenerative disorder, as the tenocytes isolated from the patellar tendon, and other tendons, including damaged ones, show high metabolic activity (Rolf et al., 2001), tendinopathy should be more regarded as a defective healing response to accumulated micro-injuries that the tendon tissue, for unknown reasons, is unable to repair effectively (Rees et al., 2009). The tenocytes, while active, may display chondrocyte-like morphology with cell rounding (Maffulli et al., 2006a, b) and the expression of chondrocyte markers (Fu et al., 2007). Although the tendinopathic samples displayed increased cellularity, increased apoptotic cell death was also observed (Lian et al., 2007). There is usually a lack of infiltration of inflammatory cells under histological evaluation of chronically diseased specimens (Khan et al., 1999). This observation is consistent with the clinical observation of failed response to non-steroidal anti-inflammatory drug and other physiotherapy protocols, which aim to reduce inflammation in long term (Andres & Murrell, 2008). However, a recent study showed that mast cells are present in patellar tendinopathy and associated with micro-vessels, particularly in patients with longstanding symptoms (Scott et al., 2008). Because it was difficult to obtain early clinical samples, the presence of inflammation at the early stages or before the onset of symptoms in human tendinopathy cannot therefore be ruled out (Abate et al., 2009). The definition of ‘‘inflammation’’ is, however, controversial in this essence. While the infiltration of inflammatory cells may be absent, the presence of pro-inflammatory cytokines and neuropeptides in the tendon midsubstances and their production in healthy tendon explants, healthy tenocytes and human tendinopathy samples have been well documented (Alfredson &

Lorentzon, 2002; Fu et al., 2002b; Yang et al., 2005; Flick et al., 2006). Therefore, the debate on whether the condition is a ‘‘tendinitis’ or a tendinopathy’’ more reflects the different definition of an inflammatory disease, similar to the debate that has raged for many years in the cartilage field where, most recently, osteoarthrosis has been replaced with the older term of osteoarthritis. It is considered highly plausible that while cell-mediated inflammation may not be a dominant factor in tendinopathy, the involvement of inflammatory cytokines may be central to the progressive degenerative nature of the disease. As pain affecting the physical performance is the major complaint of patients suffering from chronic tendinopathy, studies on the origin of chronic tendon pain are essential for effective management. Neuropeptides and other factors released by stimulated cells or nerve endings such as substance P, CGRP, tyrosine hydroxylase and a1-adrenoreceptor (Lian et al., 2006; Andersson et al., 2007, 2008; Danielson et al., 2007a), acetylcholine production machineries (Danielson et al., 2007b) and glutamate (Alfredson & Lorentzon, 2002) in or around the tendon might influence matrix turnover and tendon pain (Scott & Bahr, 2009). Increased cyclooxygenase-2 (COX-2) expression and PGE2 production may also contribute to the painful response (Fu et al., 2002b).

Assessment criteria of tendinopathy animal models To be considered as a valid animal model of tendinopathy, the model needs to consistently replicate the clinical, histopathological and functional characteristics of tendinopathy in humans. Different authors have used slightly different histopathological scoring systems for the evaluation of their animal models (Backman et al., 1990; Soslowsky et al., 1996, 2000; Movin et al., 1997b; Messner et al., 1999; Sullo et al., 2001; Scott et al., 2007; Glazebrook et al., 2008). These scoring systems focused on histopathology only. The clinical and functional aspects of tendinopathy including hypoechogenicity, crosssectional enlargement and altered longitudinal fiber pattern in US imaging, high T1 and T2 signals in MRI and activity-related tendon pain need to be evaluated using other appropriate techniques. In conclusion, we recommended the use of the following parameters to define tendinopathy: (1) organizational parameters based on histopathology: (a) hypercellularity; (b) hypervascularity; (c) loss of matrix organization or collagen fragmentation; (d) rounding of cell nuclei or acquisition of chondrocyte phenotype/markers such as proteoglycan accumulation; (2) compositional parameters based on the objective evaluation of gene and protein analysis of the extracellular matrix;

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Lui et al. (3) clinical parameters: (a) long-term (equivalent to 6 months in human) defective healing; (b) hypoecogenicity, increased crosssectional area and altered longitudinal fiber pattern in US imaging, or high T1 and T2 signals in MRI; and (4) functional-related parameter such as gait changes or activity-related tendon pain.

However, as tendinopathy is a progressive multifactorial tendon disorder affecting different anatomical structures and as there is at present no clinical staging of the disorder as in other common diseases, it was difficult to use only one set of criteria to define the disorder and hence evaluate the animal models. The animal models may be useful for reproducing some, but not all aspects of human tendinopathy. If no single animal model can fully reproduce the human condition, several animal models may have to be used in a concerted effort to study the different aspects, clinically, histopathologically and functionally, of human tendinopathy. Current animal models Naturally occurring tendinopathy Horses and dogs naturally develop tendinopathy when trained and raced (Dowling et al., 2000; Fransson et al., 2005). There are obvious stark anatomical differences between quadripeds and bipeds, especially in the forelimbs where biomechanical considerations are different, but individual animal species have naturally occurring strain-induced injuries of specific tendons (not always at the equivalent anatomical site) with similarities to specific human tendinopathies, such as superficial digital flexor tendon injury in the horse and Achilles tendinopathy in man. These injuries result in activity-related gait changes, US and MRI changes, altered composition and histopathological changes with similarities to human tendinopathy (Dowling et al., 2000). However, they are frequently not practical animal models for tendinopathy given their large size and high costs. Induced tendinopathy models The most popular and convenient animal species for induced tendinopathy are rats and rabbits. They are less costly and more easily available compared with the large animals. Moreover, more biological tools are available for study compared with the large animals. Rabbits have the advantage of having larger tendons, which provide larger samples for analysis and are easier to manipulate during surgical operation. However, they are less tough compared with rats, and can die easily after surgery or from diarrhea. Special care needs to be provided to rabbits used as the animal model. While rats and rabbits are

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popular animal models, better understanding about their behavior and physiology is essential as these animals may use different mechanisms for balancing, locomotion and pain perception compared with human. Induced animal models fall into two categories. Mechanical loading is the most frequently reported extrinsic factor for tendinopathy, and therefore many current animal models have been developed based on mechanical overloading of tendon. A second category of model involves the introduction of chemicals reported in human tendinopathic samples into normal animal tendon. Mechanical overloading Mechanical overloading is the most popular method to induce tendinopathy in animals, as the use of the pathology-inducing factor would facilitate the translation of research findings to clinical application. Three methods, including (1) forced treadmill running, (2) tendon loading via artificial muscle stimulation and (3) direct repetitive tendon stretching via an external loading device have been used to induce tendinopathy in animals, with forced treadmill running being the most popular method. Forced treadmill running

To develop tendinopathy, the animal is forced to run on a treadmill to mimic tendon overuse in human. This method has been used to produce tendinopathy in the supraspinatus tendon and Achilles tendon of rats (Soslowsky et al., 2000; Huang et al., 2004; Perry et al., 2005; Szomor et al., 2006; Archambault et al., 2007; Scott et al., 2007; Glazebrook et al., 2008; Jelinsky et al., 2008; Millar et al., 2008, 2009). Increased cellularity, collagen disorganization, changes in cell morphology and larger cross-section area similar to tendinopathy were observed in the rat supraspinatus tendon after treadmill running up to week 16 (Soslowsky et al., 2000). In addition, a decrease in elastic modulus and maximum stress started at week 4 and remained consistently below those of the control group for up to week 16 in the same study (Soslowsky et al., 2000). Another study reported increased cellularity, glycosaminoglycan (GAG) content and collagen fiber disorganization in rat supraspinatus tendons after forced treadmill running (Scott et al., 2007). Chondrocyte-like cells exhibiting chondrocytic phenotypes were observed in different studies with the same treadmill running protocol (Archambault et al., 2007; Scott et al., 2007). Glutamate signaling and stress response genes related to excitotoxicity and apoptosis were also reported to be significantly up-regulated with treadmill running (Molloy et al., 2006). The increased amounts

Tendinopathy animal model and activity of IGF-1 have been demonstrated in rat superspinatus tendons after forced treadmill running (Scott et al., 2007). Some studies have described a general lack of inflammation (Archambault et al., 2007; Scott et al., 2007; Glazebrook et al., 2008) while other have shown an increased expression of pro-inflammatory genes 5-lipoxygenase-activating protein and COX-2 (Perry et al., 2005) or perturbation of the inflammatory genes (Molloy et al., 2006). While forced treadmill running uses naturally generated cyclical loading which is believed to be the major extrinsic risk factor for tendinopathy, it shows variable success in producing the pathological features of tendinopathy, as it can be difficult to force all rodents to run sufficiently fast or intensely to reach the level necessary for overuse. Some researchers have reported conducting a training period similar to what happens in human studies to acclimatize the rats to running at the desired final speed and duration, replacing non-cooperative animals to reduce variations in the study (Szomor et al., 2006; Scott et al., 2007). Tendons healed with rest within as short as 2 weeks after running for 2 or 4 weeks (Jelinsky et al., 2008), and hence the model may not be reproducible and consistent with the defective healing response observed in clinical tendinopathy. The time required for the production of tendinopathy in this animal model is also long and labor intensive. Artificial muscle stimulation

As tendons transmit the contractile forces of the muscle to the skeleton for motion or stabilization of different skeletal segments, muscle stimulation has been used to induce tendinopathy in the flexor digitorum profundus (FDP) tendon of rabbits and the Achilles tendon of rabbits and rats by electrical stimulation of muscles via surface electrodes (Archambault et al., 1997, 2001; Messner et al., 1999; Nakama et al., 2005, 2006). Increase in tear size, tear area and tear density compared with the contralateral, unloaded tendon was reported (Nakama et al., 2005). However, no tenderness, lameness, nodules, swelling and reduction in the range of motion and gross claw flexion strength were noted (Nakama et al., 2005). Tendon degradation and the development of tendinopathic changes have also been reported in rabbit Achilles tendons as a result of the combined treatment with active muscle stimulation and passive ankle joint movements (Backman et al., 1990). However, tendinopathy did not develop in other studies using a lower rate of muscle stimulation without passive joint movements for a longer period of time (Archambault et al., 1997, 2001; Messner et al., 1999).

Direct repetitive tendon stretching

Another method to induce direct tendon overload is by stretching the tendon repetitively and directly with a single application of sub-failure cyclic load using an external device (Sun et al., 2008; Fung et al., 2010). Histology showed no inflammation up to 3 days post-stretching (Fung et al., 2010). There was interfiber space widening and severe matrix disruption with major fiber angulations, thinning and discontinuities. This was also accompanied with a marked increase in the mRNA expression of collagen types I, III and V in the tendon overload model, while only collagen type I increased in the tendon laceration model (Fung et al., 2010). Another study by the same group using the same animal model reported an increased MMP-13 and IL-1b expression with high strain of tendon stretching (Sun et al., 2008). However, as the follow-up time in these studies was short, whether the micro-structural changes would ultimately be repaired remains to be elucidated. Unlike treadmill running, where the induced loading likely varies among animals, both the artificial muscle stimulation and direct repetitive tendon stretching methods have the advantage of applying a direct controlled load to the tendon on anesthetized animals. Variations from animal compliance, muscle fatigue and stress of animals during running are eliminated, theoretically producing more consistent results. Some authors suggest using the contralateral limb as the control. However, as the loading pattern on the contralateral limb may be changed as a result of tendon overload, the use of the contralateral limb as control for comparison, as in intratendinous injection of chemicals, should be taken with caution. Both methods require anesthesia, and in the latter method the tendon is also exposed surgically for mechanical stretching. Overuseinduced tendinopathy is commonly regarded as the result of chronic overload and repetitive defective healing processes. The use of a single intervention in the direct repetitive tendon stretching method to induce tendon damage (similar to the strategy used in some experiments using intratendinous injection of chemicals) therefore does not mimic the more gradual course of development of overuse-induced tendinopathy. These two methods to establish tendinopathy animal models are less well characterized compared with treadmill running. Because of the limited understanding in the histopathology, clinical and imaging presentation of these two methods, much research needs to be performed to validate the usefulness of these methods to produce tendinopathy animal models. Limited success in some tendons for mechanical overloading methods

The use of mechanical overload for the development of tendinopathy has not shown universal success in

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Lui et al. different tendons. Tendinopathy was successfully produced in the supraspinatus tendon, but with variable success in the Achilles tendon using treadmill running (Huang et al., 2004; Glazebrook et al., 2008). One study reported no changes in the Achilles tendons of rat after treadmill running based on gross observation, geometric measurements and mechanical testing analyses (Huang et al., 2004) using the same exercise protocol that produced injuries in the supraspinatus tendon (Soslowsky et al., 2000). However, with the same treatment time and duration, other authors reported the successful production of histological changes typical of Achilles tendinopathy in a rat model (Glazebrook et al., 2008). Different assessment methods were used in these two studies, and hence they are difficult to compare. Of note was that the direction of inclination used was also different in these two studies. Glazebrook et al. (2008) suggested that uphill, rather than downhill, running might increase the likelihood of damage to the Achilles tendon. It should be noted that the supraspinatus tendinopathy model reported in the literature was induced by downhill running. The potential injury mechanism in supraspinatus tendinopathy might be different from that of other tendinopathies as it might be induced by compression, while other tendinopathies were most likely to be induced by overstrain in exercise-induced tendinopathy. The uphill model used by Glazebrook et al. (2008) more closely mimicked the potential mechanism of an exercise-induced Achilles tendon injury. However, this model has not been reproduced by others, and therefore needs confirmation. Artificial stimulation of the muscle successfully produced tendinopathy in the FDP tendon only (Nakama et al., 2005, 2006), but failed at the Achilles tendon in rabbits and rats (Archambault et al., 1997, 2001; Messner et al., 1999). Electrical stimulation of the triceps surae muscle to induce Achilles tendon injury in rats produced inflammation with increased vascularity and neural elements of the epi- and paratendon, but tendon alterations typical of chronic tendinopathy did not develop in 11 weeks (Messner et al., 1999). Archambault et al. (1997, 2001) also loaded rabbit Achilles tendon with repetitive eccentric exercise for 66 h of cumulative loading. However, they found no signs of injury at histology, although there was an increase in the mRNA expression of collagen types I, III, MMPs and IL-1b and a decrease in the expression of IGF-II. Direct mechanical stretching of tendon using an external device is feasible only to the patella tendon, which has a bony attachment for clamping, and hence allows stretching without directly damaging the tendon substance. A vast majority of tendinopathy cases are related to mechanical loading. The use of mechanical over-

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loading to produce tendinopathy in animals is therefore useful for testing the hypothesis that chronic mechanical loading induces the development of tendon disorder and has the ‘‘etiological’’ advantage that may facilitate the translation of research findings to clinical application for the management of exercise-induced tendinopathy in man. However, mechanical loading is possibly not the only predisposing factor (etiology) leading to tendinopathy. There are also a significant proportion of patients who are not regular exercisers (Rolf & Movin, 1997; Maffulli et al., 2006a, b), and hence the use of mechanical overloading models cannot represent this group of patients. There is still a lack of evidence on the validity of artificial muscle stimulation and direct repetitive tendon stretching methods for the successful production of overuse-induced tendinopathy in animals. The success rate varied, and few reports directly assess the animal models based on the key histopathological, clinical and imaging observations of tendinopathy in human. Further evaluation of the forced treadmill running method on other important characteristics of clinical tendinopathy, including extracellular matrix disturbance, long-term defective healing, hypechogenicity in US imaging or high T1 and T2 signal in MRI imaging and activity-related tendon pain, should be performed to further understand the animal model. Strategies to increase the compliance of animals to the overuse regimen in forced treadmill running are essential to ensure the reproducibility and hence the validity of the animal model.

Intratendinous injection of chemicals As alterations in the functional responses of tenocytes as a result of overload have been suggested as the mechanisms leading to the loss of matrix organization, hypercellularity and hypervascularity, intratendinous injection of chemicals that have been observed in clinical samples of tendinopathy had been used for the establishment of tendinopathy animal models. Chemicals that have been used to produce animal models include collagenase, cytokines, PGE1, PGE2 and fluoroquinolone. They all are less labor intensive and produce more consistent tendon damage compared with forced treadmill running, but suffer from the induction of an acute injury to the tendon, which is not usually the case for tendinopathy. While the injection models may be able to duplicate a specific developmental process of tendinopathy, they cannot mimic the entire process. Depending on the objectives of the study, this may not prevent their use for studying disease pathogenesis, prevention and treatment, given that we understand their limitations. We cannot always identify

Tendinopathy animal model ‘‘the first cause’’ for prevention as sometimes ‘‘the first cause’’ is unpreventable. If strategies that can interfere with the key developmental stages of tendinopathy are also considered to be useful and they are required to be studied, then injection models that can mimic the key histopathological features of tendinopathy and can provide a more consistent and feasible alternative for studying the potential treatments should also be considered as valuable. An experiment needs to be reproducible before it can be valid, and the use of injection models can potentially produce more consistent tendon damage. Collagenase injection As both repeated mechanical stimulation (Yang et al., 2005) and clinical tendinopathy samples (Fu et al., 2002a) showed increased an MMPs expression, enzyme-induced tendon injury is inevitably involved in conditions labeled as ‘‘overuse tendinopathies.’’ Intratendinous injection of collagenase therefore has been widely used to produce lesions in the flexor digitorum superficialis tendon, deep digital flexor tendon, Achilles tendon, patellar tendon and supraspinatus tendon in horse, rabbit and rat (Soslowsky et al., 1996; Stone et al., 1999; Hsu et al., 2004; Dahlgren et al., 2006; Lui et al., 2009). Intratendinous injection of collagenase in the rabbit patellar tendon demonstrated increased angiogenesis, hypercellularity and fibrosis around the tendon at 4 weeks (Stone et al., 1999). At 16 weeks, myxoid changes, focal fibrosis and collagen-bundle disarray

with persistent increase in cellularity were noted (Stone et al., 1999). There was increase in crosssectional area at 4 but not at 16 weeks. There was also a significant decrease in collagen content at week 4 and increase in cross-linking at week 16 (Stone et al., 1999). The tendon stiffness, but not the ultimate load of the injured group decreased compared with the control group (Stone et al., 1999). Another study also reported reproducing key features of tendinopathy in a collagenase-induced patellar tendon injury rat model (Fu et al., 2009; Lui et al., 2009, 2010a, 2010b). They reported (1) hypevascularity; (2) hypercellularity; (3) acquisition of chondrocyte phenotype; (4) loss of matrix organization; (5) stable defective healing from week 8 to week 32; (6) tendon swelling and hypoechogenicity in US imaging; (7) extracellular matrix disturbance with sustained expression of proteoglycans and collagen type III/I ratio; and (8) expression of substance P and CGRP, which positively correlates with activityrelated tendon pain in animal gait analysis up to week 16 in their animal model. In addition, intratendinous focal bone formation was observed starting at week 12 and up to week 91 (Fig. 1) (Fu et al., 2009; Lui et al., 2009, 2010a, 2010b). The animal model was reported to be stable and reproducible. Regarding the mechanical properties of the tendon after collagenase injection, some studies reported that the collagenase-injected tendons did not regain ultimate tensile strength compared with normal controls in the rabbit patellar model (Stone et al., 1999; Hsu et al., 2004) and rat Achilles tendon model

Fig. 1. Intratendinous injection of collagenase reproduces key features of tendinopathy including (a) gross similarity to human tendinopathy with tendon swelling; (b) hypercellularity and hypervascularity (arrows); (c) ectopic chondrogenesis (arrowheads) and calcification (CR); (d) calcification as seen in vivaCT (arrow); (e) loss of matrix organization; (f) hypoechogeneity in ultrasound imaging; (g) sustained expression of (i) decorin, (ii) biglycan, (iii) fibromodulin and (iv) aggrecan; (h) expression of (i) substance P and (ii) CGRP (arrowheads: chondrocyte-like cells; arrows: tendon fibroblasts) and (i) increase in pain and double stance duration (*Po0.050 compared with the saline control in post hoc comparison). The magnification of (b–c), (g–h) are the same. (panel h(iii) was from Lui et al., 2010b while panels i and j were from Lui et al., 2010a).

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Lui et al. (Chen et al., 2004) while others reported lower stiffness and stress as well as higher strain in equine flexor tendon after collagenase injection (Dowling et al., 2002a, b). The expression of collagen type III was reported to be higher and this contributed to the decreased mechanical properties in the collagenase group compared with that in the control group (Silver et al., 1983; Williams et al., 1984b; Dahlgren et al., 2002, 2005a, b). There were conflicting reports on the presence of pyridinoline, an index of tendon crosslinks and maturity (Silver et al., 1983; Stone et al., 1999; Marsolais et al., 2003; Hsu et al., 2004). Similar to clinical tendinopathy, changes in the expression of GAG were reported in the collagenase model (Yamamoto et al., 2002). Some authors are against the injection of collagenase and inflammatory substances to produce an animal model of tendinopathy, with the view that the injury induced by collagenase and inflammatory substances lacks similarity with naturally occurring disease in that it is acute and severe, and inflammation is present at the early stage of injury (Silver et al., 1983; Williams et al., 1984a). Chronic overuse tendon injury could involve quite different mechanisms from acute injury (Archambault et al., 1995). Some researchers, in fact, induced acute tendon lesions using collagenase (Dahlgren et al., 2006). However, as discussed above, the absence of inflammation in the established pathology does not rule out the involvement of inflammation in the development of the condition at the early stages. Leukocytes were reported in the first 3 days after injury, returning to normal values after 7 days in rat Achilles tendon after collagenase injection (Marsolais et al., 2001, 2003). However, infiltration of inflammatory cells was not detected in a rat patellar tendon injury model (Lui et al., 2009) and rat supraspinatus tendon injury model (Soslowsky et al., 1996) 2 and 4 weeks, respectively, after collagenase injection. Another common criticism of the collagenaseinduced injury animal model is the repair of injured tendon, which was not consistent with the defective healing response of tendinopathy. Dahlgren et al. (2005b, 2006) described the injury after central collagenase injection in the large horse’s superficial digital flexor tendon as core lesions with extracellular matrix damage and loss of spindle-shaped fibroblasts with the lesion, grossly similar to naturally occurring tendinopathy in this species. The damaged region of the tendon was then filled with an amorphous and acellular tissue in 1–2 weeks and mature fibrous tissue at week 24. On occasion, islands of nondigested tendon can reside within the treated area making analysis more problematical. However, a more even loss of matrix organization rather than core damage was reported in Lui et al.’s (2009) study.

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As transient healing was observed at week 8 and defective healing developed afterwards, the changes observed after week 8 were likely not due to the acute injury of collagenase. Rather, the damaged extracellular matrix and altered matrix composition could lead to greater sensitivity of tendon to force and subsequently increased its susceptibility to damage, resulting in a ‘‘vicious cycle’’ that leads to tendinopathy (Lui et al., 2009). The presence of ectopic calcification has been reported only in the animal model described by Lui et al. (2009) using collagenase. Of note is that the follow-up time of all the other studies was relatively short compared with this one. Most reports on collagenase-induced tendon injury only followed the animals for 8 weeks, and calcifications were also not present at week 8 in Lui et al.’s (2009) study. These conflicting results might also arise from differences in experimental protocols and in the species used. Although most clinical tendinopathies are not calcifying, all clinical pathological cases usually had received treatments before presenting to the physicians, and the conservative or drug treatments received by the patients might modify the course of defective healing in tendinopathy. This is supported by the effect of dexamethasone, triamcinolone and platelet-rich plasma on modulating the osteogenic/chondrogenic differentiation of ligament and tendon fibroblasts (Murata et al., 2004; de Mos et al., 2009). Injection of cytokines Stone et al. (1999) injected cytokines and cell-activating factor (CAF) into the rabbit patellar tendon to establish tendinopathy. No inflammatory cells were seen at week 4. There was only a transient increase in cellularity, and the tendon matrix appeared intact after injection. While there was a significant decrease in the ultimate load at 16 weeks, there was no change in tendon cross-sectional area. There was no difference in collagen content and cross-linking with the controls. The injection of cytokines, therefore, only modeled a reversible mild injury with no marked alteration in the extracellular matrix. This was different from the result of collagenase injection in the same study, which is more consistent with the typical picture of tendinopathy. As the cytokine and CAF mixture is ill defined and is not produced by tendon fibroblasts under mechanical loading, the validity of using the cytokine and CAF mixture for the development of tendinopathy is questioned. Intratendinous injection of prostaglandins This is based on the increased expression of COX-2 and PGE2 in clinical samples of tendinopathy (Fu

Tendinopathy animal model et al., 2002b), the increased production of PGE2 during exercise (Langberg et al., 1999; Zhang & Wang, 2010) and the increased production of COX1, -2 and PGE2 in human tendon fibroblasts after repetitive mechanical loading in vitro (Almekinders et al., 1993; Wang et al., 2003). Repeated intratendinous injection of PGE2 was reported to lead to focal hypercellularity and matrix degeneration in the patellar tendon of rabbits similar to those seen in tendinopathy (Khan et al., 2005). Although PGE2 is a known inflammatory mediator, there is no evidence of inflammatory cells within the tendon substance 1 week after PGE2 injection (Khan et al., 2005). The fact that PGE2 production was increased by physical activity does not necessarily imply that it plays a role in the pathogenesis of tendinopathy, but it could provide a mechanistic link between mechanical loading and tendinopathy and overexpression of COX-2 and PGE2 were observed in clinical samples of tendinopathy (Fu et al., 2002b). Another research group injected PGE1 into the peritendon around the rat Achilles tendon, and reported inflammatory responses followed by degenerative changes including increased vascularity, cellularity and fiber disorganization around and within the tendon (Sullo et al., 2001). One limitation of this study, recognized by the authors, is the lack of a long-term follow-up, and hence the natural history of alterations following PGE1 injection is not known. Also, the production of PGE1 in tendinopathy has not been demonstrated. The clinical applicability of this model is therefore unknown, except to point out that tendinopathic changes can likely be produced by any pro-inflammatory cytokine provided that sufficient quantities are applied for long enough. Use of fluoroquinolone Fluoroquinolone antibiotics have been implicated in the etiology of tendinopathy and subsequent tendon rupture, particularly in the presence of other associated risk factors, such as steroid therapy or systemic diseases (Khaliq & Zhanel, 2003). The Achilles tendon is the tendon most commonly affected by tendinopathy after fluoroquinolone use, but other tendons, such as the biceps brachii, supraspinatus and extensor pollics longus can also be affected (van der Linden et al., 2001). In fluoroquinolone-associated tendinopathy, hypercellularity, increased GAG content, ‘‘mucoid’’ degeneration, with high signal intensity on T1- and T2-weighted images and hypoechogenic areas in US imaging and an increase in tendon thickness, were observed (Astrom et al., 1996; Movin et al., 1997a). These changes were similar to those seen in overuse conditions in athletes. Therefore, fluoroquinolones may be used as agents to produce tendinopathy in animals. Fluoroquinolones

may alter the function of the tenocytes, with an excess production of the non-collagenous extracellular matrix and a subsequent change in the cell to matrix ratio (Astrom et al., 1996). The use of pefloxacin in a mouse model demonstrated a change in proteoglycan synthesis in the Achilles tendon (Simonin et al., 2000). Oxidative damage was also found, suggesting the involvement of reactive oxygen species. Edema and increased mononuclear cells were reported in the tendon sheath of the rat Achilles tendon after a single oral administration of fluoroquinolone in another study (Kashida & Kato, 1997). Detachment of tenocytes from the extracellular matrix, a decrease in tendon fibril diameter and an increase in fibril separation in Achilles tendon were reported 4 weeks after a single dose of fleroxacin in rats (Shakibaei & Stahlmann, 2001). There were decreases in the amount of collagen type I, elastin, fibronectin and b1 integrin in Achilles tendon of dogs treated with ciprofloxacin orally for 5 days (Shakibaei et al., 2001). Although fluoroquinolone antibiotics may induce tendinopathy, they are generally not involved in most cases of tendinopathy and neither do they induce tendinopathy in every case. The general value based on the use of fluoroquinolone for the establishment of tendinopathy animal model therefore may be questioned. In summary, intratendinous injection of chemicals for the induction of tendinopathy has the advantages of higher reproducibility, lower cost and potentially a defined dose–response relationship compared with forced treadmill running. The limitation of injection models is that the injury is acute, and they may not mimic the ‘‘first cause’’ but the pathological features of tendinopathy. The injection of cytokines and prostaglandins has been used only in specialist laboratories, and therefore there is still limited evidence about their validity. Fluoroquinolone antibiotics are generally not involved in the majority cases of tendinopathy, and hence the use of fluoroquinolones for the establishment of tendinopathy animal model is questionable. There are comparatively more reports on the collagenase-induced tendinopathy model. Although variation exists, some research groups have demonstrated the model to reproduce many key features of clinical tendinopathy including matrix degeneration, increased cellularity, increased vascularity and defective healing, hypoechogencity in US imaging and activity-related tendon pain. The severity of injury can be easily controlled with reproducible results in contrast to treadmill running. Longer-term effects after collagenase injection should be looked for to avoid any changes that may possibly result from the acute injury induced by the injection of collagenase. Table 1 shows the comparison of different methods to establish an animal model of tendinopathy.

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12 Artificial muscle stimulation

Direct repetitive tendon stretching

Lower cost

Labor intensive

High cost Does not require Requires anesthesia anesthesia Does not require surgery Repeated injuries to tendon

Reproducibility

Time for model establishment Cost Need for anesthesia Need for surgery Single or repeated injuries

Function test not reported

Availability of function test outcome

Function test used but no difference reported

Restricted success in certain anatomical locations

Generalizability

Function test not reported

Cytokines

Fluoroquinolone

Production of PGE1 in tendinopathy not demonstrated

Short follow-up time after injury induction; effect on defective healing unknown

Short follow-up time after injury induction; effect on defective healing unknown

A single injury to A single or tendon repeated injuries to tendon Have been studied in Have been studied in only one anatomical location various common anatomical locations of tendinopathy Function test not reported Function test used and difference reported in some studies

No defective tendon healing but a reversible mild injury

Not reproduced elsewhere except in the specialist laboratory Not used intentionally to produce tendinopathy animal model

Does not require anesthesia

Long-term defective healing reported in some studies

PGE1

Cytokine and cell Mechanisms not activating factor clear except (CAF) used were tendinopathy was not well-defined observed in and not justified people with fluoroquinolone by the disease use pathogenesis Lack of good evidence of consistency to clinical tendinopathy

PGE2

Requires surgery Does not require surgery A single injury to tendon Repeated injuries to tendon

Variable success due to More reproducible tendon damage in each animal individual animal variation Tendon healing after rest Long-term defective healing not reported reported

Less labor intensive

Widely studied by different groups

Lack of good evidence of Lack of good evidence of Good histological, clinical and consistency to clinical consistency to clinical functional evidence tendinopathy tendinopathy of consistency to clinical tendinopathy Widely studied by Have been studied by Not reproduced several groups elsewhere except in the different groups specialist laboratory

Popularity

Reversibility

Collagenase

Based on the exercise as the commonly reported external etiological factor and Expression observed in tendinopathy hence facilitate the translation of research findings to clinical application for the and may be involved in the progression management of overuse-induced tendinopathy; however, non-overuse-induced of disease tendinopathy such as those in the sedentary subjects due to other causes cannot be represented by these models

Level of evidence Good histological evidence of consistency to clinical tendinopathy

Justification for use

Forced treadmill running

Table 1. Comparison of different methods for the establishment of animal model of tendinopathy

Lui et al.

Tendinopathy animal model Limitation of animal models and further improvement of animal models There is no perfect animal model. A clear delineation of the validity of existing animal models and the development of new animal models are essential in parallel with an enhanced understanding of the pathogenesis of human tendinopathy. However, we are limited at the moment about how they should be improved or developed given that the staging and our understanding of tendinopathy were not clear. In view of such limitations, we must above all focus on an achievable and relevant target for each of the animal models (‘‘horses for courses’’ which means that a racehorse performs best on a racecourse specifically suited to it) and should not use the data to conclude general aspects of tendinopathy neither in animals nor humans. It is likely that several different models will be necessary to test specific hypotheses. While structural tendon alterations over time and throughout a healing process are easy to study, they may not be necessarily directly relevant for clinical management. From clinical and management perspectives, the major complaint is pain in man. The source and mechanism of this pain are not always directly associated with the severity of histopathologic alterations and in most cases we do not have a confirmative explanation. Treatments are instead based on the removal of the abnormal signs of the diseases. For example, the presence of persistent hypervascularity is abnormal, and can be related to the pain in tendinopathy. Therefore, there is the tendency to treat this obvious finding with sclerosing agents against the hypervascularity although it is not known whether the link is cause or effect. A major problem with tendinopathy is activityrelated tendon pain. A functional test to measure the painful response during loading is therefore a relevant aspect of validation of a tendinopathy animal model. Some animal models may be limited in the sense that we cannot ask the animal ‘‘how painful it is’’ and test its athletic performance. It is therefore difficult to monitor the outcome of various treatments. We can instead indirectly monitor the animal’s behavior, which we relate to pain such as gait pattern as a clinically relevant outcome. Few studies on tendinopathy animal models have assessed activity-related tendon pain (Keg et al., 1994; Davidson et al., 1997; Messner et al., 1999; Clayton et al., 2000a, b; Meershoek et al., 2002). Changes in gait pattern are easier to observe in large animal such as horses than small animals like mice and rats, where the study of activity-related changes in gait pattern required the use of highly sensitive equipment. Fu et al. (2009) has established a method to measure the gait changes produced by patellar tendon pain in rats after collagenase injection using high-speed CCD digital video camera and motion analysis software.

A positive relationship between tendon pain and double stance duration was reported and was reversible with the administration of buprenorphrine (Fu et al., 2009). We recommended the measurement of activity-related tendon pain, if possible, in the establishment of the animal model and studies on the effect of potential interventions of tendinopathy, although we need to be aware of the difference in our evaluation of pain and locomotion between human and animals. At present, there are no validated animal models for all the tendinopathies observed in human. Continuous establishment of validated animal models for tendinopathies at different anatomical locations is essential. Mouse is an attractive species for the establishment of animal model of human diseases given its lower cost, short life cycle and availability. The availability of transgenic animal provides a useful tool for understanding the pathophysiology of tendinopathy although the tendon matrix’s response to insult may be very different from larger animals and humans. Certainly, ossification is a much more frequent finding in the mouse. The use of mouse for the development of animal model of tendinopathy is also limited by its small size and hence difficulty in manipulation including intratendinous injection, histology, biomechanical test, functional test and sample amount. There are at present only two studies from the same group reporting the repeated mechanical loading of patellar and Achilles tendons in mouse by the forced treadmill running method (Szczodry et al., 2009; Zhang & Wang, 2010). Zhang and Wang’s (2010) study was limited by loading the tendons with only one bout of intensive running, and it was unlikely to cause tendinopathy. Both studies did not characterize the animal model with respect to features of human tendinopathy. Szczodry et al. (2009), in fact, reported an anabolic effect of their running protocol. Therefore, there is at present no validated mouse model of tendinopathy. It is necessary to carefully study the behavioral changes of the animal such as pain, stress and discomfort during the establishment and the use of the animal models. Some models may generate more pain and discomfort than the other models. For example, the forced treadmill running method might induce more stress to the animal due to repeated daily running. This is primarily to ensure a humane treatment of animals but may also be benefited for the better understanding of the disease and the effect of different interventions on human in the future.

Conclusion A validated animal model is a useful tool for the investigation of etiology, pathogenesis and/or therapeutic interventions of tendinopathy, particularity

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Lui et al. at the early asymptomatic stage, which is not possible in humans. Clinical, histopathological and functional characteristics of human tendinopathy are all important and relevant criteria to be met, if possible, by animal models. While structural and metabolic changes were considered feasible to reproduce, subjective painful responses and functional performance are more difficult to evaluate in smaller animals. The current animal models are not perfect. Comparatively, there were more consistent findings with the forced treadmill running and collagenase injection protocols. As tendinopathy is a progressive multifactorial tendon disorder affecting different anatomic structures, it may not be realistic to expect using a single animal model to study all aspects of tendinopathy. Staging of tendinopathy over time and clearer definition of tendinopathy in relation to severity and type are suggested to enable realistic targets with any animal model. The existing animal models can only be used for answering specific questions (horses for courses) and should not be used to conclude general aspects of tendinopathy in animals or humans. Several different models may be necessary depending on the achievable target of the existing models. The use of mechanical overloading is more relevant for testing the hypothesis that chronic mechanical loading causes the development of tendon disorder while intratendinous injection of chemicals such as collage-

nase would be more relevant for investigating tendon healing or defective healing process after the chemical insult. There is no animal model for all the common pathologies or anatomical sites of human tendinopathy. A clear delineation of the validity of existing animal models or development of new animal models is essential in parallel with an enhanced understanding of the pathogenesis of human tendinopathy. However, we are limited at the moment about how they should be improved or developed given that the staging and our understanding of the disorder are not clear. Further studies are essential with careful attention to choosing relevant outcome parameters. As activity-related pain is a key problem of tendinopathy, pain assessment is a relevant parameter in the assessment of the animal model and therapeutic efficacy of different interventions. Key words: tendinopathy, animal models, assessment.

Acknowledgements This work was supported by resources donated by the Hong Kong Jockey Club Charities Trust and the Restructuring and Collaboration Fund from University Grant Council. We would also like to thank the participants in the research forum of the associative program of ISLT-X 2010 for the discussion and comments.

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