The rotator cuff tendinopathy is one of the most common shoulder problems leading to full-thickness rotator cuff tendon tear and, eventually, to degenerative arthritis. Recent research on rotator cuff tendon degeneration has focused on its relationship to cell death. The types of cell death known to be associated with rotator cuff tendon degeneration are apoptosis, necrosis, and autophagic cell death. The increased incidence of cell death in degenerative tendon tissue may affect the rates of collagen synthesis and repair, possibly weakening tendon tissue and increasing the risk of tendon rupture. The biomolecular mechanisms of the degenerative changes leading to apoptotic cell death in rotator cuff tenofibroblasts have been identified as oxidative-stress-related cascade mechanisms. Furthermore, apoptosis, necrosis, and autophagic cell death are all known to be mediated by oxidative stress, a condition in which ROS (reactive oxygen species) are overproduced. Lower levels of oxidative stress trigger apoptosis; higher levels mediate necrosis. Although the signaltransduction pathway leading to autophagy has not yet been fully established, ROS are known to be essential to autophagy. A neuronal theory regarding rotator cuff degeneration has been developed from the findings that glutamate, a neural transmitter, is present in increased concentrations in tendon tissues with tendinopathy and that it induces rat supraspinatus tendon cell death. Recent studies have reported that hypoxia involved in rotator cuff tendon degeneration. Because antioxidants are known to scavenge for intracellular ROS, some studies have been conducted to determine whether antioxidants can reduce cell death in rotator cuff tendon-origin fibroblasts. The first study reported that an antioxidant has the ability to reduce apoptosis in oxidative-stressed rotator cuff tenofibroblasts. The second study reported that antioxidants have both antiapoptotic effects and antinecrotic effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The third study reported that an antioxidant has antiautophagic-cell-death effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The fourth study reported that glutamate markedly increases cell death in rotator cuff tendonorigin fibroblasts. The glutamate-induced cytotoxic effects were reduced by an antioxidant, demonstrating its cytoprotective effects against glutamate-induced tenofibroblast cell death. The fifth study reported that hypoxia significantly increases intracellular ROS and apoptosis. The hypoxia-induced cytotoxic effects were markedly attenuated by antioxidants, demonstrating their cytoprotective effects against hypoxia-induced tenofibroblast cell death. In conclusion, antioxidants have cytoprotective effects on tenofibroblasts exposed in vitro to an oxidative stressor, a neurotransmitter, or hypoxia. These cytoprotective effects result from antiapoptotic, antinecrotic, and antiautophagic actions involving the inhibition of ROS formation. These findings suggest that antioxidants may have therapeutic potential for rotator cuff tendinopathy. Further studies must be conducted in order to apply these in vitro findings to clinical situations

Stem cells represent an exciting biological therapy for the management of many musculoskeletal tissues that suffer degenerative disease and/or where the reparative process results in non-functional tissue (‘failed healing’). The original hypothesis was that implanted cells would differentiate into the target tissue cell type and synthesise new matrix. However, this has been little evidence that this happens in live animals compared to the laboratory, and more recent theories have focussed on the immunomodulatory effects via the release of paracrine factors that can still improve the outcome, especially since inflammation is now considered one of the central processes that drive poor tendon healing. Because of the initial ‘soft’ regulatory environment for the use of stem cells in domestic mammals, bone and fat-derived stem cells quickly established themselves as a useful treatment for naturally occurring musculoskeletal diseases in the horse more than 20 years ago (Smith, Korda et al. 2003). Since the tendinopathy in the horse has many similarities to human tendinopathy, we propose that the following challenges and, the lessons learnt, in this journey are highly relevant to the development of stem cells therapies for human tendinopathy:. Source – while MSCs can be recovered from many tissues, the predominant sources for autologous MSCs have been bone and fat. Other sources, including blood, amnion, synovium, and dental pulp have also been commercialised for allogenic treatments. Preparation – ex vivo culture requires transport from a licensed laboratory while ‘minimally manipulated’ preparations can be prepared patient-side. Cells also need a vehicle for transport and implantation. Delivery – transport of cells from the laboratory to the clinic for autologous ex vivo culture techniques; implantation technique (usually by ultrasound-guided injection to minimise damage to the cells (or, more rarely, incorporated into a scaffold). They can also be delivered by regional perfusion via venous or arterial routes. Retention – relatively poor although small numbers of cells do survive for at least 5 months. Immediate loss to the lungs if the cells are administered via vascular routes. Synovially administered cells do not engraft into tendon. Adverse effects – very safe although needle tracts often visible (but do not seen to adversely affect the outcome). Allogenic cells require careful characterisation for MHC Class II antigens to avoid anaphylaxis or reduced efficacy. Appropriate injuries to treat – requires a contained lesion when administered via intra-lesional injection. Intrasynovial tendon lesions are more often associated with surface defects and are therefore less appropriate for treatment. Earlier treatment appears to be more effective than delayed, when implantation by injection is more challenging. Efficacy - beneficial effects shown at both tissue and whole animal (clinical outcome) level in naturally-occurring equine tendinopathy using bone marrow-derived autologous MSCs Recent (licenced) allogenic MSC treatment has shown equivalent efficacy while intra-synovial administration of MSCs is ineffective for open intra-synovial tendon lesions. Regulatory hurdles – these have been lighter for veterinary treatments which has facilitated their development. There has been greater regulation of commercial allogenic MSC preparations which have required EMA marketing authorisation

Adipose tissue releases several bioactive peptides and hormones, like adipokines that promote a low inflammatory systemic state. Inflammation, affecting the tendon homeostasis, could play a role in tendon disease development as well as in the healing process. Obese patients show a dysregulated level of adipokines and considering the higher mechanical demand, this relates to higher incidence of tendinopathies among these subjects. A systematic review was performed searching PubMed, Embase and Cochrane Library databases. Inclusion criteria were studies of any level of evidence published in peer-reviewed journals reporting clinical or preclinical results. Evaluated data were extracted and critically analysed. PRISMA guidelines were applied, and risk of bias was assessed, as was the methodological quality of the included studies. We excluded all the articles with high risk of bias and/or low quality after the assessment. After applying the previously described criteria, we included 12 articles assessed as medium or high quality. Leptin, others adipokines and in general changes in the hormones delicate equilibrium affect the tendon either qualitatively and/or quantitatively. The evidence still lacks consensus on their role which is probably involved in both anabolic and catabolic pathways. The role of adipokines in the structure and healing of tendons is still debated. Further studies are needed to clarify the relation between deregulated levels of adipokines and the development of tendinopathy. A better understanding of the molecular interactions could allow us to individuate future therapeutic targets

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies

Tendinopathies represent the 45% of the musculoskeletal lesions and they are a big burden in clinics. Indeed, despite the relevant social impact, both the pathogenesis and the development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. Indeed, current treatment for tendinopathy are mainly symptomatic, and they present a high rate of pathology re-occurrence. In this contest, the development of an efficient in vivo model of acute tendinopathy, focused on the choice of the most appropriate species and strategy to induce the disease, would allow a better understanding of the pathology progression throughout its phases. Then, the purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies. 40 Sprague Dawley rats were randomly divided into two groups, treated by injection of collagenase type I within the Achilles tendon at 1 mg/mL (low dose, LD) or 3 mg/mL (high dose, HD). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days by H&E staining. Our results showed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater fatty degeneration and neovascularization with respect to the lower dose. These changes are time-dependent, thus resembling the tendinopathy development in humans. Indeed, the acute phase occurred from day 3 to day 15, while from day 15 to 45 it progressed towards the proliferative phase, displaying a degenerative appearance associated with a precocious remodeling process. The model represents a good balance between feasibility, in terms of reproducibility and costs, and similarity with the human disease. Moreover, the present model contributes to improve the knowledge about tendinopathy development, and then it could be useful to design further pre-clinical studies, in particular in order to test innovative treatments for tendinopathy

Obese patients show a higher incidence of tendon-related pathologies. These patients present a low inflammatory systemic environment and a higher mechanical demand which can affect the tendons. In addition, inflammation might have a role in the progression of the disease as well as in the healing process. A systematic review was performed by searching PubMed, Embase and Cochrane Library databases. Inclusion criteria were studies of any level of evidence published in peer-reviewed journals reporting clinical or preclinical results. Evaluated data were extracted and critically analysed. PRISMA guidelines were applied, and risk of bias was assessed, as well as the methodological quality of the included studies. We excluded all the articles with high risk of bias and/or low quality after the assessment. Due to the high heterogeneity present among the studies, a metanalysis could not be done. Thus, a descriptive analysis was performed. After applying the previously described criteria, thirty articles were included, assessed as medium or high quality. We analysed the data of 50865 subjects, 6096 of which were obese (BMI over 30 accordingly to the WHO criteria). The overall risk of re-tear after surgery is about the 10% more than normal BMI subjects. The rupture risk fluctuates in the studies without showing a significant trend. Obese subjects have a higher risk to develop tendinopathy and a worse outcome after surgery as confirmed in several human studies. The obesity influence on tendon structure and mechanical properties may rely on the fat tissue endocrine proprieties and on hormonal imbalance. Clinicians should consider obesity as a predisposing factor for the development of tendinopathies and for a higher risk of complications in patients who underwent surgical repair of tendons

Tendon-related pathologies such as tendinopathy represent a relevant clinical and socioeconomic issue. The most innovative and conservative therapeutic approaches are meant to stimulate the intrinsic healing capability of the tissue. In this study, the use of pulsed electromagnetic fields (PEMFs) was investigated in a rat model of Achilles tendinopathy as a potential therapy. Achilles tendinopathy was chemically induced in eighty-six Sprague Dawley rats by injecting collagenase Type I within the tendon fibers. Fifty-six of them were stimulated with PEMFs (8 hours/day, 1.5 ± 0.2 mT; 75 Hz), divided in different experimental groups basing on the starting-time of PEMFs exposure (after 0, 7, 15 after Collagenase injection) and its duration (7, 15 or 30 days). Thirty animals were left unstimulated (CTRL group). According to the different time points, explanted tendons were evaluated through histological and immunohistochemical analyses in term of matrix deposition, fiber re-organization, neovascularization and inflammatory reaction. The most effective PEMF stimulation was demonstrated in the 15 days of treatment. However, when PEMF were applied immediately after the collagenase injection, no significant therapeutic results were found. On the contrary, when PEMF were applied after 7 and 15 days from the collagenase injection, they promoted the deposition of extracellular matrix and tendon fiber re-organization, reducing both the inflammatory reaction and vascularization, with significant differences compared to the CTRL group (p<0.05). Therefore, these results suggest an effective activity of PEMFs stimulation that provides a satisfying restoration of the damaged tissue, although the most performing protocol of application still needs to be identified

Alarmins- also referred to as damage associated molecular patterns (DAMPS)- are endogenous molecules mobilized in response to tissue damage known to activate the innate immune system and regulate tissue repair and remodelling. The molecular mechanisms that regulate inflammatory and remodelling pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. S100A8 and S100A9 are low molecular weight calcium binding proteins primarily released by activated phagocytes in an inflammatory setting and also secreted as a heterodimeric complex that exhibits cytokine like functions. Based on our previous investigations we sought evidence of S100A8/A9 expression in human tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate inflammatory mediators and matrix regulation in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. S100A8/A9 expression was analysed at transcript and protein level using quantitative RT-PCR and immunohistochemistry, respectively. Primary human tenocytes were cultured from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The in vitro effect of recombinant human S100 A8/A9 on primary human tenocytes was measured using quantitative RT-PCR and ELISA. Immunohistochemistry of tendinopathic tissues demonstrated the presence of S100 A8/A9 in diseased tissues compared to control tissue. In addition, early pathological diseased tissue indicated greater S100A9 expression compared with established diseased pathology. These findings were reflected by data obtained at transcript level from diseased tissues. Recombinant human S100A8, A9 and A8/A9 complex led to significant increase in expression of inflammatory mediators, including IL-6 in vitro. Further analysis via quantitative RT-PCR demonstrated recombinant S100A8, A9 and A8/A9 complex treatment on tenocytes, in vitro, had no direct effect on the expression of genes involved in matrix remodelling. The presence of S100A8 and S100A9 in early tendinopathic lesions suggests expression is upregulated in response to cellular damage. S100A8 and S100A9 are endogenous ligands of Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE). These receptors have known regulatory effects on immune mediated cytokine production. We propose S100A8 and S100A9 as active alarmins in the early stages of tendinopathy and thus targeting of its downstream signalling may offer novel therapeutic approaches in the management of human tendon disorders

Achilles tendinopathy is classically defined as a tendinosis devoid of an inflammatory cell population. However, recent literature suggests inflammation as a mediator in the pathogenesis. These finding were mainly based on semi-quantative immunohistochemistry. We therefore used flow cytometry to obatain a more accurate identification and quantification of the different cell types involved. Thirty-two samples were obtained from twelve patients with chronic tendinopathic lesions undergoing Achilles tendon surgery. Samples obtained from three patients with hemiplegia requiring surgical release due to spastic Achilles tendons served as control. We used two panels to identify the myeloid and lymphoid population targeting the following markers: CD45, CD3, CD8, CD4, CD19, CD11b, CD56, CD14, CD16, Vα7.2, 6b11, CD161, TCRγδ. To assess the presence of fibroblasts CD90 was targeted. The mean count of CD45+ hematopoietic cells in the tendinopathic samples was significantly higher than in the control samples, respectively 13.27% and 3.24% of the total cell count (P<0.001). The mean fraction of CD3+ cells present in the complete cell population was significantly higher in pathological samples than in control samples, respectively 1.70% and 0.37% (P<0.05). Presence of CD19+ B cells was not reported. The mean fraction of γδ T cells was significantly higher in tendinopathic samples compared to blood samples of the same patient and consisted of 12.9% and 5.8% γδ T cells respectively (P<0.05). These findings support an inflammatory cell infiltration in midportion Achilles tendinopathy that show similarities to enthesitis in SpA. This implies a potential target to investigate in novel treatment modalities

Summary Statement. The peripheral neuronal phenotype is significantly altered in rotator cuff tendinopathy (RCT) with a clear upregulation of the Glutaminergic system being present in disease. Introduction. Shoulder pain is the third most frequent cause of chronic musculoskeletal pain in the community and is usually caused by rotator cuff tendinopathy (RCT). The central and peripheral nervous system play an important role in both tissue homoeostasis and tendon healing. The Glutaminergic system is of key importance in driving the peripheral and central neuronal changes which increase the body's sensitivity to pain (1, 2). No study to date has investigated the role of the glutaminergic system in human RCT. We hypothesised that the peripheral neuronal phenotype would be altered in RCT, and would vary according to disease stage as measured by size of tear. The term ‘peripheral neuronal phenotype’ is used to refer to refer to specific characteristics of the peripheral nervous system, neuronal mediators and the receptors for these mediators in peripheral tissue. Methods. Rotator cuff tendon specimens were obtained from 64 patients undergoing the surgical repair of rotator cuff tears. Control supraspinatus tendon was obtained from 10 patients undergoing surgery for anterior instability using an ultrasound guided biopsy technique. Patients with rotator cuff tears were divided into 2 groups: the small/medium group (≤ 3cm size) and the large/massive group (>3cm size). The tendon tissue was histologically stained using Haematoxylin and Eosin, and immunohistochemically stained with primary antibodies visualised using 3, 3′-diaminobenzidine (DAB). Image analysis was performed blindly by 2 observers using Image-J to quantify the amount of DAB positive staining. Data was non-parametric in distribution and Mann-Whitney U tests were carried out using SPSS with significance levels set at a minimum of p<0.025. Results. There were significant changes in the peripheral neuronal phenotype in RCT. The Glutaminergic system was significantly up-regulated with an increase in Glutamate and changes in several related receptors in disease versus control (p<0.01). The standard deviation in nuclei count and mean cell nuclear area were both increased in disease (p<0.01) compared to controls. Tendon vascularity and cell proliferation were reduced in disease vs control (p<0.01). There were no significant correlations between pain scores and the peripheral tissue markers. Discussion/Conclusion. The peripheral neuronal phenotype is significantly altered in rotator cuff tendinopathy (RCT) with clear changes in the Glutaminergic system in disease. These findings are novel and improve our understanding of pain and tissue healing in RCT, potentially providing novel therapeutic targets

Introduction. Achilles tendinopathy (AT) is a highly prevalent injury in athletes and non-athletes with an unknown aetiology. Genetic risk factors have been a recent focus of investigation. The aim of this systematic review was to determine which loci have been linked with mid-portion AT and could potentially be used as biomarkers in tendinopathy risk models or as preventative or therapeutic targets. Materials and Methods. Eight electronic bibliographic databases were searched from inception to April 2015 for cross-sectional, prospective cohort and case-control studies that included empirical research investigating genes associated with mid-portion AT. Potential publications were assessed by two independent reviewers (AAC and PRJ) for inclusion and quality. Quality was evaluated using a validated scale. Results. Twelve candidate gene studies and three pathway-based genetic association studies that investigated genetic risk factors for AT were identified. According to Ariëns's criteria, there was strong evidence for the COL5A1 gene. There was some evidence for 6 of the other genes investigated: COL5A3, TNC, CASP8, MIR608, GDF5, MMP3 and TIMP2 genes. There was inconclusive evidence for the following genes: COL3A1, COL5A2, COL11A1, COL11A2, COL12A1, COL1A1, COL27A1, COL14A1, COMP, THBS2, ADAMTS2, ADAMTS5, ADAMTS14, ADAM12, TGFβ1, IL-1β, IL-1RN, IL-6, NOS2 and NOS3. There was some evidence for combinations of functional variants of different genes and pseudohaplotypes constructed from many functional variants. The quality of included studies varied (3/9 to 7/9), and the average quality assessment score was 5.5/9 (61%). Discussion. There are genetic differences between subjects with and without AT. To further elucidate these findings, prospective studies are needed to investigate the increased risk associated with specific genetic findings. Modifying training loads or preventative exercise may be used to mitigate increased risk, although it needs to be highlighted that a genetic association does not necessarily mean an individual will develop Achilles tendinopathy. Gene therapy may have a role in tendon healing, but further research is necessary to develop risk models and establish the most advantageous genes to transfer

Introduction. The COL5A1 gene encodes for the α1 chain of type V collagen, a minor fibrillar collagen that is an important regulator of collagen fibrillogenesis. Several polymorphisms, including rs12722 (C/T), within the 3′-UTR of COL5A1 are associated with chronic Achilles tendinopathy and other musculoskeletal soft tissue injuries as well as exercise-related phenotypes. It is hypothesised that polymorphisms within the 3′-UTR regulate the amount of the α1(V) chain synthesised and type V collagen production. This in turn influencing the mechanical properties of tendons and other musculoskeletal soft tissues. In our laboratories, two major functional forms, namely the T- and C-allelic forms of the COL5A1 3′-UTR, were identified and associated predominately with severe chronic Achilles tendinopathy and healthy asymptomatic control individuals, respectively. Materials and Methods. To further investigate the functional differences between the two major 3′-UTR functional forms as well as to start mapping the regions which are responsible for the tendinopathic phenotype, skin biopsies from donors having a known genotype at rs12722 and primary fibroblast cell lines were established in order to quantify COL5A1 and COL1A1 expression levels in a pilot study. Lastly, in preliminary RNA EMSAs, biotinylated C- and T-allelic RNA probes for a specific 57bp functional region within the 3′-UTR were incubated with either fibroblast nuclear or cytoplasmic protein extracts to investigate putative distinguishing RNA:RBP complex formation. Results. An overall higher relative mRNA expression of both COL5A1 (p<0.001) and COL1A1 (p=0.0015) were observed in primary skin fibroblasts from donors having a rs12722 TT genotype compared to donors with a CC genotype. A unique RNA:RBP complex was also identified with the C-allelic probe. Discussion. These novel results have important implications for our understanding of the proposed role of type V collagen in the aetiology of tendon and other musculoskeletal soft tissue injuries, as well as, other exercise-related phenotypes

Summary Statement. ASTM therapy is commonly used to treat Achilles tendinopaty. However, there was no report to evaluate the biomechanical effects, especially the dynamic viscoelasticity. We have shown that ASTM treatment was biomechanically useful for chronic Achilles tendinopathy in an animal model. Introduction. Achilles tendinopathy is a common chronic overuse injury. Because Achilles tendon overuse injury takes place in sports and there has been a general increase in the popularity of sports activities, the number and incidence of Achilles tendon overuse injury has increased. Augmented Soft Tissue Mobilization (ASTM) therapy is a modification of traditional soft tissue mobilization and has been used to treat a variety of musculoskeletal disorders. ASTM therapy is thought to promote collagen fiber realignment and hasten tendon repair. It might also change the biomechanical behavior of the injured tendon, especially the dynamic viscoelasticity. The purpose of this study is to evaluate the effect of ASTM therapy in a rabbit model of Achilles tendinopathy by quantifying dynamic biomechanical properties and histologic features. Patients & Methods. The hind limbs of 12 rabbits were used, and 24 Achilles tendons were injected with collagenase to produce tendon injury. One hind limb of each animal was then randomly allocated to receive ASTM therapy, while the other received no treatment and served as a control. ASTM was performed on the Achilles tendon for 3 minutes on postoperative days 21, 24, 28, 31, 35, and 38. The Achilles tendons were harvested 10 days after the last treatment. Specimens were examined with dynamic viscoelasticity and light microscopy. Results. The mean±SD cross-sectional area for the treated and untreated tendons was 12.30±5.47 mm. 2. and 9.57±8.36 mm. 2. , respectively. The difference between the treated and untreated tendons was statistically significant (P<.01). At all dynamic loading frequencies, the storage modulus in the untreated tendons tended to be higher than that in the treated tendons. At 0.1 Hz and 10 Hz, in the untreated tendons was significantly higher than that in the treated tendons (P=.05). The loss modulus was significantly lower in the treated tendons than in the untreated tendons (P<.05). There was no significant difference in tan δ between the treated and untreated tendons. HE stain showed that the untreated tendon fiber was wavy and kinking and displayed a disordered collagen arrangement. In contrast, the tendon fiber was well aligned in the treated tendons. In the immunohistochemically stained specimens, the type III collagen showed higher color intensity in the untreated tendons than in the treated tendons. Discussion/Conclusion. We have shown that ASTM was a biomechanically useful treatment for chronic Achilles tendinopathy. Biomechanical and histologic data showed the treated Achilles tendons had better biomechanical function and histologic outcomes than the untreated tendons

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff. We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis). The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears. These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment

Introduction. The exact mechanisms leading to tendinopathies and tendon ruptures remain poorly understood while their occurrence is clearly associated with exercise. Overloading is thought to be a major factor contributing to the development of tendon pathologies. However, as animal studies have shown, heavy loading alone won't cause tendinopathies. It has been speculated, that malfunctioning adaptation or healing processes might be involved, triggering tendon tissue degeneration. By analysing the expression of the entirety of degrading enzymes (degradome) in pathological and non-pathological, strained and non-strained tendon tissue, the aim of this study was to identify common or opposite patterns in gene regulation. This approach may generate new targets for future studies. Materials and Methods. RNA was extracted from different tendon tissues: normal (n=7), tendinopathic (n=4) and ruptured (n=4) Achilles tendon; normal (n=4) and tendinopathic (n=4) posterior tibialis tendon; normal hamstrings tendon with or without subjection to static strain (n=4). The RNA was reverse transcribed, then pooled per group The expression of 538 protease genes was analysed using Taqman low-density array quantitative RT-PCR. To be considered relevant, changes had to be at least 4fold and measurable at a level below 36 Cts. Results. In general, there was little common regulation when exercised was compared with pathological tissue. The expression of PAMR1 and TNFαIP3 was upregulated with exercise (169-fold and 78-fold), Achilles tendinopathy (9724-fold and 7-fold) and Achilles tendon rupture (1809-fold and 10-fold), while DDI1, PSMB11 and PSH2 which were down-regulated with exercise were upregulated with Achilles pathology. Discussion. The newly found targets may deliver insights into the initiation and progression of tendon pathologies: PAMR1, a regeneration associated muscle protease which has been shown to be downregulated in Duchenne muscular dystrophy and upregulated in regenerating muscle fibers, might also be involved in tendon regeneration; TNFαIP3, which negatively regulates the NF-κB/pro-inflammatory pathway, could have anti-inflammatory function in tendon regeneration. PSMB11 and PSH2 are for the first time shown to be expressed in tendon and regulated in tendon pathology. Using this approach we were able to generate new targets and to add information on function, regulation and expression sites of recently identified proteins

The objective was to seek evidence of hypoxia in early human tendinopathy and thereafter, to explore mechanisms whereby tissue hypoxia may regulate apoptosis, inflammatory mediators and matrix regulation in human tenocytes. Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Markers of hypoxia were quantified by immunohistochemical methods. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The impact of hypoxia upon tenocyte biology ex vivo was measured using quantitative RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of HIF 1a, Bcl-2 and clusterin (hypoxic and apoptotic markers) was detected in subscapularis tendon samples compared to both matched torn samples and non matched control samples (p<0.01). Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (p<0.001), altered matrix regulation (p<0.01) with increased production of Collagen type III operating through a MAPK dependent pathway. Finally, hypoxia increased expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. Hypoxia promotes expression of proinflammatory cytokines, key apoptotic mediators and drives matrix component synthesis towards a collagen type III profile by human tenocytes. We propose hypoxic cell injury as a critical pathophysiological mechanism in early tendinopathy offering novel therapeutic opportunities in the management of tendon disorders

Summary. Treatment of equine naturally occurring over-strain tendinopathy with mesenchymal stem cells suspended in bone marrow supernatant resulted in significant improvements compared to saline treated tendons in the normalisation of biomechanical, morphological, and compositional parameters with no adverse effects. Introduction. Tendon injuries are a common age-related degenerative condition where natural repair involves scarification, resulting in a functionally inferior tissue. 1. that frequently re-injures. Naturally-occurring human and equine tendinopathy possess many similarities. 2. making the horse a good clinically-relevant model. A multitude of treatments are used but few have a strong evidence base. Regenerative approaches using mesenchymal stem cells (MSCs) to improve outcome are supported by clinical data demonstrating reduced re-injury rates in racehorses. 3. We therefore hypothesised that implantation of autologous MSCs into injured equine tendons would result in a tissue more closely resembling normal tendon matrix than the fibrous scar tissue formed subsequent to natural repair. The aim of this controlled experimental study was to assess the biomechanical, histological and compositional parameters following MSCs implantation into naturally injured tendons. Methods. This study was carried out following informed consent from the owners of donated horses and under Institutional Ethics and Welfare Committee approval and UK Home Office Licences. Thoroughbred and Thoroughbred cross horses aged between 5 and 15 (mean 7.8±3.0) years, suffering career-ending severe superficial digital flexor tendinopathy (SDFT) within the metacarpal region of the forelimb of less than 2 months duration (average 30 days) were recruited. Autologous bone marrow derived MSCs were expanded and 1×10. 7. MSCs in 2ml of citrated bone marrow supernatant were injected into the central defect of the damaged SDFT as described. 2. in 6 horses. The control group (6 horses) received an identical volume of isotonic saline. The horses then entered a standardised ascending exercise rehabilitation regimen of walking (3 months) and walking combined with trotting (next 3 months) after which horses were euthanised. Treated and contralateral SDFTs were recovered and analysed for mechanical, histological (blinded), and compositional parameters. Results. The treated tendons exhibited statistically significant improvements in all parameters compared to saline-injected control tendons towards that of normal tendons and those in the contralateral limbs. Specifically, in comparison to saline-treated tendons, MSC treated tendons had significantly improved elasticity (p<0.05), lower (improved) histological scoring of organisation (p<0.001) and crimp pattern (p<0.05), lower cellularity from histological scoring (p<0.002) and DNA content (p<0.05), reduced vascularity (p<0.03), water content (p<0.05), GAG content (p<0.05). Total collagen content was unaltered between groups and interestingly, while tissue-linked fluorescence (indicator of collagen age) was similar between treatment and control groups, both were significantly lower than the contralateral limb (p<0.014), which suggested similar amounts of new collagen in the injured limbs. A higher remodelling rate, exemplified by increased MMP-13 activity, was also demonstrated for the control group compared to the MSC treated group and contralateral limbs (p<0.02). Conclusion. This data support the hypothesis that the injection of MSC with BM supernatant results in a tissue more like normal tendon matrix rather than the fibrous scar tissue formed after natural injury and repair. The mechanism of this effect is not clear but the improved tissue elasticity, reduced vascularity, cell content and GAGs is more consistent with an immunomodulatory role for MSCs with a subsequent reduction in fibrosis than a true regenerative effect

Summary Statement. The effects of local glucocorticoid on tendon appear broadly negative and this supports the emerging clinical evidence which points toward significant long term harms associated with this treatment modality. Introduction. The use of locally administered glucocorticoid is widespread in the treatment of painful tendinopathy. Despite evidence of short term benefit, the emerging evidence points toward significant long term harms associated with this method of treatment, including an increased risk of recurrence, rupture and worsened clinical outcomes (1, 2). Our primary purpose was to summarise the known effects of locally administered glucocorticoid on tendon tissue and tendon cells. Methods. We conducted a systematic review of the scientific literature using the PRISMA and Cochrane guidelines of the Medline database using specific search criteria. Only studies analysing the effects of locally administered glucocorticoid on tendon tissue or tendon cells with adequate controls were included. Specific attention was paid to histological and biomechanical findings. Inclusion was agreed upon by two independent researchers after review of abstracts or full text. The search yielded 4424 results, of which 42 met the inclusion criteria. The final 42 articles consisted of 13 human in vitro studies, 15 animal in vivo studies and 14 animal in vitro studies. Results. Due to study heterogeneity, statistical pooling or meta-analysis of data was not possible. The results are therefore described qualitatively. Histologically, there was a loss of collagen structure (5 studies) and an increase in collagen necrosis (4 studies). The proliferation and viability of fibroblasts was reduced (11 studies). An increased inflammatory cell infiltrate was shown in 3 animal in vivo studies, while an increased fibroblast infiltrate was seen in 2 studies. Fibroblast migration was reduced in 2 in vitro studies. Collagen synthesis was reduced in 13 studies. An increased ratio of type 3 to type 1 Collagen was shown in 2 studies. Apoptosis was unaffected in 2 studies. 19 studies investigated the mechanical properties of tendon. Of these 7 showed deterioration in mechanical properties, 4 showed an improvement and 8 showed no difference. Discussion/Conclusion. Overall it is clear that the local administration of glucocorticoid has significant negative effects on tendon cells in vitro, such as reduced cell viability, cell proliferation and collagen synthesis. There is increased collagen disorganisation and necrosis as shown by animal in vivo studies. The mechanical effects are equivocal. This review supports emerging clinical evidence showing significant long term harms associated with glucocorticoid injections. There is clearly a significant need for better designed human trials with appropriate blinding and control arms to investigate the effects of glucocorticoid on both clinical outcomes and characteristics of tendon tissue

Introduction. Numerous risk factors have been identified for patellar tendinopathy (PT), often in small population studies. The aim was to use an online questionnaire internationally to generate a large database and identify significant risk factors. Materials and Methods. Subjects were recruited from England, Spain and Italy with the questionnaire available in all three languages, with the questionnaire previously having been validated by Morton et al. (2014) as to be suitable for self-administration. The questionnaire can be viewed at: . http://patellartendinopathyquestionnaire.blogspot.co.uk/. (English), . http://tendinopatiarotuliana.blogspot.co.uk/. (Spanish) and . http://tendinopatiarotulea.blogspot.co.uk/. (Italian). All data was anonymised and password protected. 825 data sets were collected with 23.4% having clinically diagnosed PT. Results. Eight risk factors were included in the analysis based on a purposeful selection procedure: gender, hours of training, hamstring flexibility, previous patellar tendon rupture, previous knee injury, current/previous back pain, family history and age. To be female was found to be positively associated with PT, suggesting being female is protective (odds ratio (OR) = 0.70, 95% CI: 0.49–1.00, p=0.05). As hours of training increased the association with PT became stronger so that training >20 hours a week had a very significant OR of 8.94 (95%CI: 4.68–17.08, p<0.05), the most significant OR calculated. There was an association between a previous knee injury and PT (OR=2.10, 95% CI: 1.45–3.04, p<0.05) and having self-reported flexible hamstrings suggested some protection from PT (OR=0.61, 95% CI0.38–0.97, p=0.04). There was a trend towards association for back pain (OR=1.45, 95% CI: 0.99–2.14, p=0.06) and a family history of tendon problems (OR=1.51, 95% CI: 0.96–2.37, p=0.08). Discussion. Risk factors have been identified that are potentially modifiable in order to inform prevention and rehabilitation programmes; future research is required to establish causal relationships. Certain risk factors require investigation as they are not currently recognised in the literature

Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation