Kim Wheway

Inflammation activation and resolution in human tendon disease.

Resolution of inflammatory pathway gene expression signatures in diseased tendons correlates with reduced tendon pain. Tending toward resolution Inflammation activation and resolution pathways remain poorly defined in tendon disease. In new work, Dakin et al. investigate shoulder tendons from patients before and after surgery. Diseased tendons showed different inflammation gene and protein signatures in early-stage disease compared to advanced-stage disease. The researchers identified pathways implicated in the resolution of tendon pain after surgical treatment. Investigation of inflammation activation pathways in cultured stromal cells derived from human diseased tendons revealed that the stromal cells may have been primed for inflammation. The authors also identified a stable metabolite of aspirin that may be therapeutically beneficial for the resolution of tendon inflammation. Improved understanding of the role of inflammation in tendon disease is required to facilitate therapeutic target discovery. We studied supraspinatus tendons from patients experiencing pain before and after surgical subacromial decompression treatment. Tendons were classified as having early, intermediate, or advanced disease, and inflammation was characterized through activation of pathways mediated by interferon (IFN), nuclear factor κB (NF-κB), glucocorticoid receptor, and signal transducer and activator of transcription 6 (STAT-6). Inflammation signatures revealed expression of genes and proteins induced by IFN and NF-κB in early-stage disease and genes and proteins induced by STAT-6 and glucocorticoid receptor activation in advanced-stage disease. The proresolving proteins FPR2/ALX and ChemR23 were increased in early-stage disease compared to intermediate- to advanced-stage disease. Patients who were pain-free after treatment had tendons with increased expression of CD206 and ALOX15 mRNA compared to tendons from patients who continued to experience pain after treatment, suggesting that these genes and their pathways may moderate tendon pain. Stromal cells from diseased tendons cultured in vitro showed increased expression of NF-κB and IFN target genes after treatment with lipopolysaccharide or IFNγ compared to stromal cells derived from healthy tendons. We identified 15-epi lipoxin A4, a stable lipoxin isoform derived from aspirin treatment, as potentially beneficial in the resolution of tendon inflammation.

Platelet-Rich Plasma Injection With Arthroscopic Acromioplasty for Chronic Rotator Cuff Tendinopathy A Randomized Controlled Trial

Background: Platelet-rich plasma (PRP) has been proposed to augment tendon healing through improving tissue structure during the initial repair phase. Purpose: To investigate both the clinical and tissue effects of the coapplication of PRP injection with arthroscopic acromioplasty (AA) in patients with chronic rotator cuff tendinopathy. Design: Randomized controlled trial; Level of evidence, 1. Methods: The study comprised 60 randomized patients diagnosed with rotator cuff tendinopathy (55% women) aged between 35 and 75 years. Patients were randomized to AA alone or in combination with an injection of autologous PRP into the subacromial bursa (AA + PRP). Efficacy of treatment was assessed by analysis of patient-reported outcomes up to 2 years after treatment (Oxford Shoulder Score [OSS]) and by analysis of tendon biopsy specimens taken 12 weeks after treatment. Results: There was no significant difference in the OSS between AA alone and AA + PRP at any time point in the study. From 12 weeks onward, there was a significant increase in the OSS for both groups compared with their baseline scores (P < .001). Bonar scoring determined no significant change in tissue structure with the coapplication of PRP compared with surgery alone. The number of blood vessels and tendon cellularity were significantly decreased in tissue biopsy specimens taken from PRP-treated patients. The expression of p53-positive apoptotic cells increased after AA + PRP but decreased after AA alone. Conclusion: Arthroscopic acromioplasty significantly improves long-term clinical outcomes up to 2 years. The coapplication of PRP did not affect clinical outcomes. PRP significantly alters the tissue characteristics in tendons after surgery with reduced cellularity and vascularity and increased levels of apoptosis. Clinical Relevance: The coapplication of PRP did not improve clinical outcomes and may have potential deleterious effects on healing tendons. Registry Number: ISRCTN 10464365

Comparison of transforming growth factor beta expression in healthy and diseased human tendon.

BackgroundDiseased tendons are characterised by fibrotic scar tissue, which adversely affects tendon structure and function and increases the likelihood of re-injury. The mechanisms and expression profiles of fibrosis in diseased tendon is understudied compared to pulmonary and renal tissues, where transforming growth factor (TGF)β and its associated superfamily are known to be key drivers of fibrosis and modulate extracellular matrix homeostasis. We hypothesised that differential expression of TGFβ superfamily members would exist between samples of human rotator cuff tendons with established disease compared to healthy control tendons.MethodsHealthy and diseased rotator cuff tendons were collected from patients presenting to an orthopaedic referral centre. Diseased tendinopathic (intact) and healthy rotator cuff tendons were collected via ultrasound-guided biopsy and torn tendons were collected during routine surgical debridement. Immunohistochemistry and quantitative real-time polymerase chain reaction were used to investigate the protein and gene expression profiles of TGFβ superfamily members in these healthy and diseased tendons.ResultsTGFβ superfamily members were dysregulated in diseased compared to healthy tendons. Specifically, TGFβ-1, TGFβ receptor (R)1 and TGFβ R2 proteins were reduced (p < 0.01) in diseased compared to healthy tendons. At the mRNA level, TGFβ R1 was significantly reduced in samples of diseased tendons, whereas TGFβ R2 was increased (p < 0.01). BMP-2, BMP-7 and CTGF mRNA remained unchanged with tendon disease.ConclusionsWe propose that downregulation of TGFβ pathways in established tendon disease may be a protective response to limit disease-associated fibrosis. The disruption of the TGFβ axis with disease suggests associated downstream pathways may be important for maintaining healthy tendon homeostasis. The findings from our study suggest that patients with established tendon disease would be unlikely to benefit from therapeutic TGFβ blockade, which has been investigated as a treatment strategy in several animal models. Future studies should investigate the expression profile of fibrotic mediators in earlier stages of tendon disease to improve understanding of the targetable mechanisms underpinning tendon fibrosis.

Persistent stromal fibroblast activation is present in chronic tendinopathy

BackgroundGrowing evidence supports a key role for inflammation in the onset and progression of tendinopathy. However, the effect of the inflammatory infiltrate on tendon cells is poorly understood.MethodsWe investigated stromal fibroblast activation signatures in tissues and cells from patients with tendinopathy. Diseased tendons were collected from well-phenotyped patient cohorts with supraspinatus tendinopathy before and after sub-acromial decompression treatment. Healthy tendons were collected from patients undergoing shoulder stabilisation or anterior cruciate ligament repair. Stromal fibroblast activation markers including podoplanin (PDPN), CD106 (VCAM-1) and CD248 were investigated by immunostaining, flow cytometry and RT-qPCR.ResultsPDPN, CD248 and CD106 were increased in diseased compared to healthy tendon tissues. This stromal fibroblast activation signature persisted in tendon biopsies in patients at 2–4 years post treatment. PDPN, CD248 and CD106 were increased in diseased compared to healthy tendon cells. IL-1β treatment induced PDPN and CD106 but not CD248. IL-1β treatment induced NF-κB target genes in healthy cells, which gradually declined following replacement with cytokine-free medium, whilst PDPN and CD106 remained above pre-stimulated levels. IL-1β-treated diseased cells had more profound induction of PDPN and CD106 and sustained expression of IL6 and IL8 mRNA compared to IL-1β-treated healthy cells.ConclusionsWe conclude that stromal fibroblast activation markers are increased and persist in diseased compared to healthy tendon tissues and cells. Diseased tendon cells have distinct stromal fibroblast populations. IL-1β treatment induced persistent stromal fibroblast activation which was more profound in diseased cells. Persistent stromal fibroblast activation may be implicated in the development of chronic inflammation and recurrent tendinopathy. Targeting this stromal fibroblast activation signature is a potential therapeutic strategy.

Up-regulation of Glutamate in Painful Human Supraspinatus Tendon Tears

Background: Pain related to rotator cuff tendinopathy is a common problem, but little is known regarding the origin and cause of pain from the tendon substance. No study to date has looked at the association between tissue changes and patient outcomes. Purpose: To describe the peripheral neuronal phenotype in painful rotator cuff tears and to determine correlations between tissue changes and clinical outcome measures. Study Design: Controlled laboratory study. Methods: Tissue samples of the supraspinatus were taken from patients undergoing surgery to repair a rotator cuff tendon tear. Patients were classified as having small/medium or large/massive tears. Control tissue was obtained from patients undergoing surgery for posttraumatic shoulder instability. Immunohistochemical techniques were performed using antibodies to known nociceptive and neuronal markers as well as general tissue structural markers. Results: There was no correlation between tissue changes and patient-reported outcomes. A significant increase in the expression of glutamate was seen in tendon tears. There were differences in the expression of metabotropic and ionotropic glutamate receptors. Expression changes were also observed for markers of the sensory and autonomic systems; however, no differences were found in neurotrophins. Conclusion: Glutamate and the glutaminergic system play a key role in painful human tendon tears; however, the exact role is still uncertain, as glutamate is highly involved in both pain and metabolic pathways. Clinical Relevance: This study has identified a number of markers that could be potential therapeutic targets.

Chronic inflammation is a feature of Achilles tendinopathy and rupture

Background Recent investigation of human tissue and cells from positional tendons such as the rotator cuff has clarified the importance of inflammation in the development and progression of tendon disease. These mechanisms remain poorly understood in disease of energy-storing tendons such as the Achilles. Using tissue biopsies from patients, we investigated if inflammation is a feature of Achilles tendinopathy and rupture. Methods We studied Achilles tendon biopsies from symptomatic patients with either mid-portion tendinopathy or rupture for evidence of abnormal inflammatory signatures. Tendon-derived stromal cells from healthy hamstring and diseased Achilles were cultured to determine the effects of cytokine treatment on expression of inflammatory markers. Results Tendinopathic and ruptured Achilles highly expressed CD14+ and CD68+ cells and showed a complex inflammation signature, involving NF-κB, interferon and STAT-6 activation pathways. Interferon markers IRF1 and IRF5 were highly expressed in tendinopathic samples. Achilles ruptures showed increased PTGS2 and interleukin-8 expression. Tendinopathic and ruptured Achilles tissues expressed stromal fibroblast activation markers podoplanin and CD106. Tendon cells isolated from diseased Achilles showed increased expression of pro-inflammatory and stromal fibroblast activation markers after cytokine stimulation compared with healthy hamstring tendon cells. Conclusions Tissue and cells derived from tendinopathic and ruptured Achilles tendons show evidence of chronic (non-resolving) inflammation. The energy-storing Achilles shares common cellular and molecular inflammatory mechanisms with functionally distinct rotator cuff positional tendons. Differences seen in the profile of ruptured Achilles are likely to be attributable to a superimposed phase of acute inflammation and neo-vascularisation. Strategies that target chronic inflammation are of potential therapeutic benefit for patients with Achilles tendon disease.

Differential expression of alarmins—S100A9, IL-33, HMGB1 and HIF-1α in supraspinatus tendinopathy before and after treatment

Background Alarmins, endogenous molecules released on tissue damage have been shown to play an important role in inflammatory musculoskeletal conditions including fracture repair andrheumatoid arthritis. However, the contribution of alarmins to the pathogenesis of tendon disease is not fully understood. Methods We investigated expression of alarmin proteins (S100A9, high-mobility group box 1 (HMGB1) and interleukin-33 (IL-33) and hypoxia-inducible factor 1α (HIF-1α), a subunit of an oxygen sensitive transcription factor, in three cohorts of human supraspinatus tissues: healthy (n=6), painful diseased (n=13) and post-treatment pain-free tendon samples (n=5). Tissue samples were collected during shoulder stabilisation surgery (healthy) or by biopsy needle (diseased/treated). Immunohistochemistry was used to investigate the protein expression of these factors in these healthy, diseased and treated tendons. Kruskal-Wallis with pairwise post hoc Mann-Whitney U tests were used to test for differences in immunopositive staining between these tissue cohorts. Additionally, costaining was performed to identify the cell types expressing HIF-1α, S100A9, IL-33 and HMGB1 in diseased tendons. Results Immunostaining showed HIF-1α and S100A9 were increased in diseased compared with healthy and post-treatment pain-free tendons (p<0.05). IL-33 was reduced in diseased compared with healthy tendons (p=0.0006). HMGB1 was increased in post-treatment pain-free compared with healthy and diseased tendons (p<0.01). Costaining of diseased tendon samples revealed that HIF-1α, S100A9 and IL-33 were expressed by CD68+ and CD68− cells, whereas HMGB1 was predominantly expressed by CD68− cells. Conclusions This study provides insight into the pathways contributing to the progressionand resolution of tendon disease. We found potential pro-inflammatory and pathogenic roles for HIF-1α and S100A9, a protective role fornuclear IL-33 and a potentially reparative function for HMGB1 in diseased supraspinatus tendons.

ROTATOR CUFF TENDINOPATHY: IMMUNOHISTOCHEMICAL CHANGES ACROSS THE SPECTRUM OF PATHOLOGY

Introduction The tissue changes associated with spectrum of rotator cuff tendinopathy are poorly understood. The aim of this study was to describe the tissue characteristics of the onset and progression of rotator cuff tendinopathy by using a novel, ultrasound-guided biopsy technique to obtain tissue samples from the supraspinatus tendon in several accurately phenotyped groups representing the spectrum of rotator cuff tendinopathy from normal, healthy tissue through to patients with a full thickness tear of the supraspinatus tendon. Methods Participants were grouped using ultrasound or arthroscopic diagnosis of cuff integrity and subdivided by the treatments being undertaken. Control—under 35 years, no history of rotator cuff tendinopathy. Injection—patients over 35 years with a history of rotator cuff tendinopathy without a full thickness tear undergoing subacromial injection of glucocorticoid. SAD—patients over 35 years with a history of rotator cuff tendinopathy without a full thickness tear undergoing arthroscopic subacromial decompression due to failed glucocorticoid injection therapy. FTT—patients over 35 years with a full thickness rotator cuff tear undergoing rotator cuff repair surgery. Samples were taken under ultrasound guidance from the supraspinatus tendon. The biopsies were obtained in clinic under local anaesthetic for the injection group and in the operating theatre for the surgical and control groups. The tissue samples were wax embedded, sectioned and stained using the following markers: H&E to assess cellularity; CD34 to identify vascular endothelial tissue; CD45 Leucocyte common antigen to identify white cells as a marker of inflammation; MIB-1 to identify proliferating cells; Active Caspase-3 to identify cells undergoing apoptosis. Slides were imaged using a Zeiss Axio Imager M1 light microscope. Analysis used computer software to calculate the cellular density from the H&E images and the proportion of positively stained tissue from the immunohistochemistry images. Results Table 1 details the composition of the study groups. The quantitative histological analyses (figure 1) demonstrate a significant reduction in vascularity and cell proliferation in line with progression of pathology. There was no significant change in cellularity. A significant increase in inflammatory cells and apoptotic cells was seen in early tendinopathy represented by the injection group compared to controls. Figure 1. Histological analyses (*p<0.05 t-test vs control group). Table 1. Group sizes and mean age Group No Mean Age Control 16 23.3 Injection 15 50.7 SAD 27 52.2 FTT 13 58.2 Discussion This study has demonstrated significant changes in tissue characteristics across the progressive stages of rotator cuff pathology from a unique group of tissue samples obtained consistently from a focal area of the supraspinatus tendon in normal and tendinopathic tissue using a novel ultrasound guided biopsy method. The results suggest an early inflammatory component to the condition in patients with clinical signs of impingement but without tearing of the tendon. As the tendinopathy progresses to require subacromial decompression and eventually rotator cuff repair due to full thickness tearing of the supraspinatus tendon, the viability and healing potential of the tissue deteriorates significantly with reduced vascularity and proliferative activity within the tendon tissue.

SHORT TERM TISSUE RESPONSE TO CURRENT TREATMENTS FOR ROTATOR CUFF TENDINOPATHY

Introduction In the past it has not been possible to evaluate the impact of current treatments upon tendinopathic rotator cuff tissue due to an inability to sample the tendon tissue before and after treatment. Serial tissue sampling pre- and post-intervention allows assessment of current therapies for the condition and may direct the development of novel future therapeutic options. The aim of this study was evaluate the short-term rotator cuff tendon tissue response to Glucocorticoid injection (GCI), Subacromial Decompression (SAD) and Rotator Cuff Repair (RCR). Methods Patients with a history of rotator cuff tendinopathy were recruited into groups defined by the treatments being undertaken and the structural integrity of the rotator cuff was assessed using ultrasound or arthroscopic diagnosis. Three groups were recruited: GCI—patients with a history of rotator cuff tendinopathy without a full thickness tear undergoing subacromial injection of glucocorticoid. SAD—patients with a history of rotator cuff tendinopathy without a full thickness tear undergoing arthroscopic subacromial decompression due to failed glucocorticoid injection therapy. RCR—patients with a full thickness rotator cuff tear undergoing rotator cuff repair surgery. Biopsies of the supraspinatus tendon were taken on the day of intervention, prior to treatment, and repeated at initial follow up 7-weeks post-intervention. These paired samples were then analysed to evaluate the tissue response to the intervention undertaken. Samples were taken under ultrasound guidance from the supraspinatus tendon. The tissue samples were wax embedded, sectioned and stained using the following markers: CD34 to identify vascular endothelial tissue; CD45 Leucocyte common antigen to identify white cells as a marker of inflammation; MIB-1 to identify proliferating cells; Active Caspase-3 to identify cells undergoing apoptosis. Slides were imaged using a Zeiss Axio Imager M1 light microscope. Analysis used computer software to calculate the cellular density from the H&E images and the proportion of positively stained tissue from the immunohistochemistry images. Results Table 1 details the composition of the study groups. The immunohistochemical results (figure 1 at the top of next page) show a comparison of tissue characteristics on the day of intervention (pre-) and 7-weeks post-intervention. A significant increase in vascularity is seen in the RCR group at 7-weeks post intervention. There is a significant increase in inflammatory cells post treatment in the SAD and RCR groups. Proliferation is significantly reduced post treatment in the GCI group. No difference was seen in apoptosis after any of the interventions. Figure 1. Histological analyses (*p<0.05 paired t test Pre vs Post). Table 1. Group Sizes Group Paired Samples GCI 11 SAD 13 RCR 9 Discussion This study has shown changes in tissue characteristics in response to treatment. The results show increased vascularity in the RCR group, supporting a theory of a healing response. Both surgical groups showed increased inflammation within the tissue, which may simply reflect the early post-operative sampling time point. Perhaps the most significant finding was the reduction in proliferation within the tendon tissue in response to glucocorticoid injection supporting the theory of the negative effects of steroid treatment in tendinopathy. Further work is needed to define other biomarkers of tissue response and to investigate the tissue effects of treatment at other time points post-intervention.

27 The Neurohistology Of Painful And Pain-free Rotator Cuff Tendons

Introduction The relationship between rotator cuff tendon structure and pain symptoms is imperfect and icompletely understood.2 The inability to gather tendon-matched and age-matched control tissue from live donors has been a significant limiting factor in many previous studies.1 The aim of this study was to determine whether there were neurohistological differences between painful and pain-free rotator cuff tendons. Methods Supraspinatus tendon specimens were obtained by ultrasound guided biopsy from 9 patients with painful rotator cuff tendinopathy (RCT) resistant to conservative management (painful group) and 9 pain-free patients at over 6 years following subacromial decompression (SAD) (pain-free group). Pain symptoms were measured using the validated Oxford Shoulder Score (OSS). Structural tendon integrity was assessed ultrasonographically. Basic histological staining and immunohistochemistry was performed. Mann-Whitney U tests were used to test for differences between groups. Results The groups were similar in terms of age, sex and structural tendon abnormality. The painful group consisted of 7 males and 2 females, the pain-free group of 6 males and 3 females. The mean age of the painful group was 51 years and that of the pain-free group was 52 years. The median OSS in the painful group was 32 (range 23 to 36) and this was significantly lower (p = 0.0002) than the median OSS in the pain-free group (all 48). There were two partial thickness tears in both groups and no full thickness tears. There were no significant differences between groups in terms of cellularity, vascularity, proliferation and hypoxia inducible-factor 1α expression. The leucocyte count (% of CD45 positive cells) and macrophage count (% of CD206 positive cells) were increased in the painful group versus pain-free (p = 0.038 and 0.0004 respectively). The expression of metabotropic glutamate receptor 7 (mGluR7) was reduced in the painful group versus pain-free (p = 0.002), while the expression of the Kainate 1 receptor was increase in the painful group (p = 0.003). PGP 9.5 (a nerve marker) and Lactate Dehydrogenase (LDH) expression were increased in the painful group versus pain-free (p = 0.008 and 0.007 respectively). There were no significant differences in glutamate, the inotropic glutamate receptor (NMDAR1) and the metabotropic glutamate receptors (mGluR1 and 2) between groups. Discussion This study has shown that specific characteristics of tendon neurohistology are associated with the presence of shoulder pain. This provides strong evidence that the rotator cuff tendon is of key importance in the symptomatology of RCT. The mechanism behind these tendon differences is unclear. These findings are novel and improve our understanding of pain in RCT, and may help provide novel therapeutic targets. References 1 Dean BJ, Franklin SL, et al. A systematic review of the histological and molecular changes in rotator cuff disease. Bone Joint Res. 2012;1(7):158–166 2 Yamaguchi K, D. K. et al. The demographic and morphological features of rotator cuff disease. A comparison of asymptomatic and symptomatic shoulders. JBJS (Am). 2006;88(8):1699–1704