The role of inflammation and cytokines in the pathogenesis of tendinopathy

Tendon disorders - tendinopathies - are the primary reason form musculoskeletal consultation in primary care in the UK and the US. The molecular pathophysiology of tendinopathy remains difficult to interpret while inflammation and its role in tendinopathy have been historically ignored due to its absence in human surgical specimens. This thesis explores the role of inflammation in human tendinopathy and dissects potential molecular pathways involved in its initiation and perpetuation. Firstly I characterize inflammatory cell subtypes within a model of human tendinopathy highlighting a distinct inflammatory infiltrate particularly of mast cells and macrophages. Hypoxia and hypoxic cell death have been a long considered aetiology of tendon degeneration. In this thesis I demonstrate that hypoxia related proteins are present in early tendinopathy biopsies and thereafter in mechanistic studies demonstrate that hypoxia regulates inflammatory and apoptotic mediators in tendon cells associated with a significant shift in collagen matrix synthesis. The cytokines, interleukins 17 and 33 are emerging inflammatory mediators known to play key roles in fibroblast biology. I explored the cellular sources of IL- 17A in human tendinopathy with experiments revealing the majority of IL17A colocalised to mast cells. Moreover IL-17A induced proinflammatory cytokines and apoptosis in vitro and again resulted in a significant switch in collagen extracellular matrix production. IL 33 is a new member of the IL-1 superfamily that signals through the ST2 receptor. Herein I demonstrate that IL-33 expression is up regulated in human tendinopathic biopsies whilst rhIL-33 promotes proinflammatory cytokine release and significantly shifts matrix production toward a collagen III phenotype. WT mice undergoing a tendon injury model showed significant up regulation of IL-33 and ST2 while ST2-/- mice exhibit a reduced collagen response and biomechanical tendon strength at early time points post injury Addition of rh-IL33 increased type III collagen production and reduced the biomechanical strength of WT tendons. Furthermore mechanistic investigations has highlighted a key role for the microRNA 29 family in the modulation of collagen regulation in tendinopathy but also in controlling IL-33 induced changes as a direct target of sST2. Based on these experiments I propose IL-33 as an important and influential alarmin in early tendon injury and tendinopathy, which may be influential in the balance between reparation and degeneration in tendon disease.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A Strathclyde cluster model for gait kinematic measurement using functional methods : a study of inter-assessor reliability analysis with comparison to anatomical models

A major source of error in reliability of gait analysis arises from the palpation of anatomical landmarks (ALs). The purpose of this study was to investigate whether less reliance on manually identifying ALs could improve inter-assessor reliability of joint kinematics compared to two anatomical models. It was hypothesised that the Strathclyde Functional Cluster Model (SFCM), in which the hip, knee and ankle joint centres and knee and ankle flexion axes were determined by functional methods, would obtain greater inter-assessor reliability. Ten able-bodied participants and seven assessors were recruited. Each participant completed three trials conducted by different assessors on non-consecutive days. Agreement and inter-assessor reliability between the models were compared and analysed, whilst factor effects of assessor experience and body mass index (BMI) were investigated. The SFCM obtained excellent agreement with anatomical models for all sagittal angles and hip ab/adduction angle, and it showed slightly higher inter-assessor reliability with smaller variations in the knee and ankle. The assessor experience was not a significant factor, but the BMI had a significant effect on the inter-assessor reliability. The results demonstrate that the SFCM may be more beneficial for less experienced assessors

Routine clinical motion analysis : comparison of a bespoke real-time protocol to current clinical methods

Motion analysis (MA) hardware has recently become more accessible; however, protocols have not developed in conjunction. Routine clinical assessment mostly relies on unreliable observational methods. This study aimed to develop an MA protocol for routine clinical use and compare kinematics and reliability to the gold-standard. Ten participants completed 10 over ground walks with a comprehensive marker set (bespoke and gold-standard). Inter/intra-assessor reliability was also compared. Results demonstrated comparable kinematics. Reliability of the bespoke model was lower than the gold standard but higher than observational methods. The bespoke model can be recommended for routine clinical use to assess patient progress and function

From lab to clinic : towards a virtual reality platform for routine clinical rehabilitation

Background: Recent advances in technology have led to the widespread commercialisation of virtual reality (VR). Many researchers have investigated the use of VR for rehabilitation; however few have extended VR use into routine clinical practice. This is mainly due to systems being too complex and time consuming or too simple to provide necessary information regarding patient function. Aim: Develop a VR platform that can provide an objective measure of patient function and can be integrated into clinical practice with minimal disruption to routine care. Method: Motion analysis is currently the gold standard for non-invasive measurement of human movement and therefore was implemented in this study to provide an objective analysis of function. A bespoke, cluster based protocol was developed and used to create an avatar and three feedback scenarios for standard orthopaedic rehabilitation exercises (step up, sit to stand, weight transfer). A cohort study was carried out in a hospital clinic with 15 control and 15 intervention orthopaedic rehabilitation patients to assess the effectiveness of feedback and the integration of the system into routine practice. Results/Findings: Visual feedback was successfully delivered using motion capture with minimal disruption to routine practice. Further, provision of feedback may have a positive effect on knee sagittal RoM, although larger scale studies are required to confirm these findings. Discussion and Conclusion: Use of a motion analysis protocol which was designed for purpose allowed integration of the system into routine practice. There was minimal disruption to patient care and use of the system may improve functional outcome of orthopaedic rehabilitation patients

Alarmins in frozen shoulder: a molecular association between inflammation and pain

Background: The pathophysiological mechanisms behind proliferation of fibroblasts and deposition of dense collagen matrix in idiopathic frozen shoulder remain unclear. Alarmins (also known as danger signals) are endogenous molecules that are released into the extracellular milieu after infection or tissue injury and that signal cell and tissue damage. Purpose: To investigate whether the presence of alarmins is higher in patients with idiopathic frozen shoulder than in control subjects. Study Design: Controlled laboratory study. Methods: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients with unstable shoulders (control). The samples were stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry using antibodies against alarmin molecules including high-mobility group protein B1 (HMGB1), interleukin 33, S100A8, S100A9, and the peripheral nerve marker PGP9.5. Immunoreactivities were rated in a blinded fashion from “none” to “strong.” Immunohistochemical distribution within the capsule was noted. Before surgery, patient-ranked pain frequency, severity, stiffness, and the range of passive shoulder motion were recorded and statistically analyzed. Results: Compared with control patients, patients with frozen shoulder had greater frequency and severity of self-reported pain (P = .02) and more restricted range of motion in all planes (P < .05). H&E-stained capsular tissue from frozen shoulder showed fibroblastic hypercellularity and increased subsynovial vascularity. Immunoreactivity of alarmins was significantly stronger in frozen shoulder capsules compared with control capsules (P < .05). Furthermore, the expression of the alarmin molecule HMGB1 significantly correlated (r > 0.9, P < .05) with the severity of patient-reported pain. Conclusion: This study demonstrates a potential role for key molecular danger signals in frozen shoulder and suggests an association between the expression of danger molecules and the pain experienced by patients

Development of a bespoke movement analysis protocol for routine clinical use in orthopaedics

Assessment of progress and outcome of orthopaedic interventions are often carried out using subjective observational methods. These may not be the most accurate or sensitive way to assess patient progress (Ong et al. 2008). In contrast, motion capture is currently the gold-standard for measuring human movement (Gage 1993; Cook et al. 2003) and the equipment cost is decreasing, making routine clinical use a possibility. However, current movement analysis protocols and setups are not suited for routine clinical use as they are time consuming and complex. Therefore, the aim of this study was to develop a protocol which could be easily adopted by the orthopaedic community and provide more sensitive outcome measures in routine clinical practice. A bespoke, cluster based marker model (CM) was developed. Kinematics were calculated using the Grood and Suntay (1983) method and the kinematic output was compared to the current clinical gold-standard (Vicon Plug in Gait; PiG). Ten healthy volunteers wore a comprehensive marker set comprised of CM and PiG and performed 10 over-ground walking trials. Hip and knee flexion, abduction and rotation were compared along with ankle dorsi/plantar flexion. T-tests determined any significant difference between models. The cluster based marker set was quick and easy to apply. When comparing the kinematic output between CM and PiG, there were some small but statistically significant differences. Differences were more likely to occur in rotations out with the sagittal plane. CM provides a kinematic output comparable to that of the current clinical gold-standard. Differences in output may be due to different methods for estimating joint centres and calculating kinematics. In conclusion, CM is tailored for clinical use and should be considered the preferred option in routine clinical practice. Using the methods described, a gait test can be conducted in 10 minutes in the clinic by a physiotherapist or nurse

Targeting danger molecules in tendinopathy: the HMGB1/TLR4 axis

Objectives: To seek evidence of the danger molecule, high-mobility group protein B1 (HMGB1) expression in human tendinopathy and thereafter, to explore mechanisms where HMGB1 may regulate inflammatory mediators and matrix regulation in human tendinopathy. Methods: 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. Markers of inflammation and HMGB1 were quantified by reverse transcriptase PCR (RT-PCR) and immunohistochemistry. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon anterior cruciate ligament reconstruction and used through passage 3. In vitro effects of recombinant HMGB1 on tenocyte matrix and inflammatory potential were measured using quantitative RT-PCR, ELISA and immunohistochemistry staining. Results: Tendinopathic tissues demonstrated significantly increased levels of the danger molecule HMGB1 compared with control tissues with early tendinopathy tissue showing the greatest expression. The addition of recombinant human HMGB1 to tenocytes led to significant increase in expression of a number of inflammatory mediators, including interleukin 1 beta (IL-1β), IL-6, IL-33, CCL2 and CXCL12, in vitro. Further analysis demonstrated rhHMGB1 treatment resulted in increased expression of genes involved in matrix remodelling. Significant increases were observed in Col3, Tenascin-C and Decorin. Moreover, blocking HMGB1 signalling via toll-like receptor 4 (TLR4) silencing reversed these key inflammatory and matrix changes. Conclusion: HMGB1 is present in human tendinopathy and can regulate inflammatory cytokines and matrix changes. We propose HMGB1 as a mediator driving the inflammatory/matrix crosstalk and manipulation of the HMGB1/TLR4 axis may offer novel therapeutic approaches targeting inflammatory mechanisms in the management of human tendon disorders

Development of a motion analysis protocol for use in routine clinical care

There is widespread agreement that motion analysis is currently the gold standard for measuring human movement in a non-invasive manner [1]. Current commercially available systems, such as Vicon Plug in Gait (PiG, Vicon Motion Systems, Oxford, UK) have been developed over a number of years and are capable of providing a biomechanical analysis which is robust enough to dictate complex treatment plans, such as multi-level surgery [1]. However, due to the vast capabilities of PiG, it is a time consuming and technically complex protocol to deliver. Additionally, there are currently limited options for delivering motion capture using other protocols which vastly limits the use of motion analysis in other aspects of clinical care, such as outpatient rehabilitation. Cluster based marker sets may provide a faster and less technically complex alternative to models such as PiG; however these are currently not commercially available and have thus far been restricted to research environments. Therefore, the aim of this study was to develop a bespoke cluster based motion analysis protocol (Strathclyde Cluster Model; SCM) capable of calculating lower limb kinematics which could be implemented in routine clinical care in order to expand the use of motion analysis beyond research and complex clinical cases. Further aims included an assessment of the kinematic output and reliability of SCM in comparison to PiG. SCM is a motion analysis protocol which has been developed for routine clinical use, such as outpatient rehabilitation and therefore application of markers and participant calibration is quicker and easier than current commercial alternatives. Further, kinematic output and reliability are comparable between SCM and the current clinical gold standard. Therefore, SCM is a suitable alternative for providing an objective assessment of function and outcome in routine clinical practice

Effects of visual feedback on orthopaedic rehabilitation

Currently, functional outcome following total knee arthroplasty (TKA) is not fully restored, with the majority of TKA patients exhibiting lower functional outcome scores than their healthy counterparts [1]. A contributing factor to limited functional outcome may be the nature of the rehabilitation provided as there is still some controversy regarding the most appropriate methods for rehabilitation delivery [2]. Providing patients with visual feedback during rehabilitation has had a positive effect in other patient populations such as stroke survivors [3] and therefore may also improve the efficacy of orthopaedic rehabilitation. The aim of this study was to develop a visual feedback tool based on real time data from 3D motion capture for routine clinical use. Further aims included determining if provision of augmented feedback was acceptable to patients and whether it had a positive effect on functional outcome. Visual feedback using motion analysis was successfully delivered in a routine clinical environment and was widely acceptable to patients. Further, provision of visual feedback appeared to lead to improved knee range of motion in the sagittal plane in comparison to control patients. However, larger scale studies are required to confirm these positive effects

Characterization of Histone Modifications in Late-Stage Rotator Cuff Tendinopathy

The development and progression of rotator cuff tendinopathy (RCT) is multifactorial and likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in RCT, including the role of histone modification, is not well established. Using chromatin immunoprecipitation sequencing, differences in the trimethylation status of H3K4 and H3K27 histones in late-stage RCT compared to control were investigated in this study. For H3K4, 24 genomic loci were found to be significantly more trimethylated in RCT compared to control (p < 0.05), implicating genes such as DKK2, JAG2, and SMOC2 in RCT. For H3K27, 31 loci were shown to be more trimethylated (p < 0.05) in RCT compared to control, inferring a role for EPHA3, ROCK1, and DEFβ115. Furthermore, 14 loci were significantly less trimethylated (p < 0.05) in control compared to RCT, implicating EFNA5, GDF6, and GDF7. Finally, the TGFβ signaling, axon guidance, and regulation of focal adhesion assembly pathways were found to be enriched in RCT. These findings suggest that the development and progression of RCT is, at least in part, under epigenetic control, highlighting the influence of histone modifications in this disorder and paving the way to further understand the role of epigenome in RCT