Stephanie G. Dakin

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.

Are inflammatory cells increased in painful human tendinopathy? A systematic review

Background The role of inflammation in tendinopathy has historically been a subject of significant controversy. Our primary aim was to determine whether inflammatory cell numbers were increased in painful human tendinopathy versus healthy control tendons. Our secondary aim was to assess whether the inflammatory cells had been linked with symptoms or disease stage. 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 measuring inflammatory cells using specific markers in tissue from human patients with the clinical diagnosis of tendinopathy were included. Inclusion was agreed on by 2 independent researchers on review of abstracts or full-text using specific predetermined criteria. The search yielded 5 articles in total. Results There were increased numbers of macrophages (4 studies) and mast cells (3 studies) in tendinopathic versus healthy control tissues. One study demonstrated increased numbers of T cells in tendinopathic tissue versus healthy control tendons. There were reduced numbers of T cells (1 study), macrophages (2 studies) and mast cells (2 studies) in torn tendon versus intact tendinopathic tissue. Conclusions The existing evidence supports the hypothesis that increased numbers of inflammatory cells are present in pathological tendons. The lack of high-quality quantitative studies in this area demonstrates a clear need for future research to better understand the role of inflammation in tendinopathy.

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

Biocompatibility of implantable materials: An oxidative stress viewpoint

Oxidative stress occurs when the production of oxidants surpasses the antioxidant capacity in living cells. Oxidative stress is implicated in a number of pathological conditions such as cardiovascular and neurodegenerative diseases but it also has crucial roles in the regulation of cellular activities. Over the last few decades, many studies have identified significant connections between oxidative stress, inflammation and healing. In particular, increasing evidence indicates that the production of oxidants and the cellular response to oxidative stress are intricately connected to the fate of implanted biomaterials. This review article provides an overview of the major mechanisms underlying the link between oxidative stress and the biocompatibility of biomaterials. ROS, RNS and lipid peroxidation products act as chemo-attractants, signalling molecules and agents of degradation during the inflammation and healing phases. As chemo-attractants and signalling molecules, they contribute to the recruitment and activation of inflammatory and healing cells, which in turn produce more oxidants. As agents of degradation, they contribute to the maturation of the extracellular matrix at the healing site and to the degradation of the implanted material. Oxidative stress is itself influenced by the material properties, such as by their composition, their surface properties and their degradation products. Because both cells and materials produce and react with oxidants, oxidative stress may be the most direct route mediating the communication between cells and materials. Improved understanding of the oxidative stress mechanisms following biomaterial implantation may therefore help the development of new biomaterials with enhanced biocompatibility.

Differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of pain in human rotator cuff tendinopathy

IntroductionThe relationship between peripheral tissue characteristics and pain symptoms in soft tissue inflammation is poorly understood. The primary aim of this study was to determine immunohistochemical differences in tissue obtained from patients with persistent pain and patients who had become pain-free after surgical treatment for rotator cuff tendinopathy. The secondary aim was to investigate whether there would be differences in glutaminergic and inflammatory gene expression between disease-derived and healthy control cells in vitro.MethodsSupraspinatus tendon biopsies were obtained from nine patients with tendon pain before shoulder surgery and from nine further patients whose pain had resolved completely following shoulder surgery. Histological markers relating to the basic tendon characteristics, inflammation and glutaminergic signalling were quantified by immunohistochemical analysis. Gene expression of glutaminergic and inflammatory markers was determined in tenocyte explants derived from painful rotator cuff tendon tears in a separate cohort of patients and compared to that of explants from healthy control tendons. Dual labelling was performed to identify cell types expressing nociceptive neuromodulators.ResultsTendon samples from patients with persistent pain demonstrated increased levels of metabotropic glutamate receptor 2 (mGluR2), kainate receptor 1 (KA1), protein gene product 9.5 (PGP9.5), CD206 (macrophage marker) and CD45 (pan-leucocyte marker) versus pain-free controls (p <0.05). NMDAR1 co-localised with CD206-positive cells, whereas PGP9.5 and glutamate were predominantly expressed by resident tendon cells. These results were validated by in vitro increases in the expression of mGluR2, N-methyl-D-aspartate receptor (NMDAR1), KA1, CD45, CD206 and tumour necrosis factor alpha (TNF-α) genes (p <0.05) in disease-derived versus control cells.ConclusionsWe conclude that differences in glutamate receptors and inflammatory cell numbers are associated with the resolution of shoulder pain in rotator cuff tendinopathy, and that disease-derived cells exhibit a distinctly different neuro-inflammatory gene expression profile to healthy control cells.

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.

Translating Regenerative Biomaterials Into Clinical Practice.

Globally health care spending is increasing unsustainably. This is especially true of the treatment of musculoskeletal (MSK) disease where in the United States the MSK disease burden has doubled over the last 15 years. With an aging and increasingly obese population, the surge in MSK related spending is only set to worsen. Despite increased funding, research and attention to this pressing health need, little progress has been made toward novel therapies. Tissue engineering and regenerative medicine (TERM) strategies could provide the solutions required to mitigate this mounting burden. Biomaterial‐based treatments in particular present a promising field of potentially cost‐effective therapies. However, the translation of a scientific development to a successful treatment is fraught with difficulties. These barriers have so far limited translation of TERM science into clinical treatments. It is crucial for primary researchers to be aware of the barriers currently restricting the progression of science to treatments. Researchers need to act prospectively to ensure the clinical, financial, and regulatory hurdles which seem so far removed from laboratory science do not stall or prevent the subsequent translation of their idea into a treatment. The aim of this review is to explore the development and translation of new treatments. Increasing the understanding of these complexities and barriers among primary researchers could enhance the efficiency of biomaterial translation. J. Cell. Physiol. 230: 36–49, 2016.

Profibrotic mediators in tendon disease: a systematic review

BackgroundTendon disease is characterized by the development of fibrosis. Transforming growth factor beta (TGF-β), bone morphogenic proteins (BMPs) and connective tissue growth factor (CTGF) are key mediators in the pathogenesis of fibrotic disorders. The aim of this systematic review was to investigate the evidence for the expression of TGF-β, BMPs and CTGF along tendon disease progression and the response of tendon cells to these growth factors accordingly.MethodWe conducted a systematic screen of the scientific literature using the Medline database. The search terms used were “tendon AND TGF-β,” “tendon AND BMP” or “tendon AND CTGF.” Studies of human samples, animal tendon injury and overuse models were included.ResultsThirty-three studies were included. In eight studies the expression of TGF-β, BMPs or CTGF was dysregulated in chronic tendinopathy and tendon tear patient tissues in comparison with healthy control tissues. The expression of TGF-β, BMPs and CTGF was increased and showed temporal changes in expression in tendon tissues from animal injury or overuse models compared with the healthy control (23 studies), but the pattern of upregulation was inconsistent between growth factors and also the type of animal model. No study investigated the differences in the effect of TGF-β, BMPs or CTGF treatment between patient-derived cells from healthy and diseased tendon tissues. Tendon cells derived from animal models of tendon injury showed increased expression of extracellular matrix protein genes and increased cell signaling response to TGF-β and BMP treatments compared with the control cells (two studies).ConclusionThe expression of TGF-β, BMPs and CTGF in tendon tissues is altered temporally during healing in animal models of tendon injury or overuse, but the transition during the development of human tendon disease is currently unknown. Findings from this systematic review suggest a potential and compelling role for TGF-β, BMPs and CTGF in tendon disease; however, there is a paucity of studies analyzing their expression and stimulated cellular response in well-phenotyped human samples. Future work should investigate the dynamic expression of these fibrotic growth factors and their interaction with tendon cells using patient samples at different stages of human tendon disease.

The macrophage marker translocator protein (TSPO) is down-regulated on pro-inflammatory 'M1' human macrophages.

The translocator protein (TSPO) is a mitochondrial membrane protein, of as yet uncertain function. Its purported high expression on activated macrophages, has lent utility to TSPO targeted molecular imaging in the form of positron emission tomography (PET), as a means to detect and quantify inflammation in vivo. However, existing literature regarding TSPO expression on human activated macrophages is lacking, mostly deriving from brain tissue studies, including studies of brain malignancy, and inflammatory diseases such as multiple sclerosis. Here, we utilized three human sources of monocyte derived macrophages (MDM), from THP-1 monocytes, healthy peripheral blood monocytes and synovial fluid monocytes from patients with rheumatoid arthritis, to undertake a detailed investigation of TSPO expression in activated macrophages. In this work, we demonstrate a consistent down-regulation of TSPO mRNA and protein in macrophages activated to a pro-inflammatory, or ‘M1’ phenotype. Conversely, stimulation of macrophages to an M2 phenotype with IL-4, dexamethasone or TGF-β1 did not alter TSPO expression, regardless of MDM source. The reasons for this are uncertain, but our study findings add some supporting evidence for recent investigations concluding that TSPO may be involved in negative regulation of inflammatory responses in macrophages.