Background: Cardiac surgery with cardiopulmonary bypass (CPB) can lead to a spectrum of post-operative complications as a result of activation of systemic inflammatory responses. Cellular injury can lead to the release of damage-associated molecular patterns (DAMPs) such as mitochondria DNA (mtDNA), which act as a ligand activating leukocytes and endothelial cells via innate immunity receptors such as Toll-like receptor 9 (TLR9). However, the contributions of DAMPs to inflammatory responses to CPB are unknown. Aim: This study is to identify the DAMPs and associated receptors that drive systemic inflammatory responses to surgery, which may lead to the identification of novel anti-inflammatory interventions such as TLR antagonists with subsequent translation of our findings to the clinical field. Additionally, Post-operative atrial fibrillation (POAF) is frequent complication in cardiac surgery, which may contribute to the inflammatory response. We aimed to identify a possible predictor for POAF, which may lead to its prevention or early management. Methods: Sixty-six patients undergoing CABG were recruited, 44 with on-pump and 22 with off-pump CABG (OPCAB) to compare the effect of CPB. To identify the effect of ischemic heart disease (IHD) on the mtDNA release. We recruited a separate group of 22 patients undergoing aortic valve replacement (AVR) with normal coronary angiogram. Blood samples were taken at different time-points to CPB. Quantitative PCR was generated to quantify mtDNA concentration (Chapter 3). Pro-inflammatory biomarkers such as interleukines, interferons, MAP kinases , NF-κB and other biomarkers were assessed by PCR array (Chapter 5). Both mtDNA and the proinflmmatory biomarker were preoperatively compared to assess of the development of POAF (Chapter 6). Additionally, we used different animal models experiments, to establish the effect of surgery and CPB on TLR9 signalling and to test the effect of blocking TLR9. Specifically, we performed sternotomy in mouse and rat models respectively and performed sternotomy with CPB in the pig model (Chapter 4). Results: mtDNA was significantly higher in patients with IHD than those without (p < 0.01). CABG with CPB led to a significant elevation in serum mtDNA levels compared to OPCAB (p < 0.001 at peak of CPB time). The potential downstream activation of TLR9 also showed significant elevation in all cytokines and chemokines utilizing the TLR9 signalling pathway (p varies from < 0.001 - < 0.05) but not those using other signalling pathway (p > 0.05). Blocking the TLR9 in mice has significantly reduced the production of proinflammatory cytokine (IL-6). INF-α and mtDNA were the only independent predictors for POAF development. Conclusion: Elevation in circulating mtDNA level is related to the extent of the IHD. CPB can influence the release of circulating mtDNA and proinflammatory cytokines production via signalling the TLR9, which may contribute to the initiation of a sterile systemic inflammatory response. The mtDNA and INF- α were independent predictors for development of POAF, which are in agreement with the inflammatory theory that relates the inflammation to the POAF development.

Spinal cord injury (SCI) is a medical condition that occurs as a result of trauma, sickness or brain injury and as a result the connection between the brain and some part of the body is lost. This will result in tremendous changes in the daily life of the individual affected. Secondary complications arising from SCI are of very importance as well since some of them if left unattended, will result in life treating conditions. Functional electrical stimulation (FES) is one of the methods developed by researchers in order to get paralysed limbs functional with the help of electrical stimulation once again but the issue of muscle fatigue is limiting the efficiency of this method. In this thesis the primary and secondary matters arising from SCI are discussed and a CAD-based VN4D humanoid model integrated with Matlab/ Simulink, for use as a platform for analysis, design and evaluation of the developed strategies and approaches in this research is developed. Muscle models using different methodologies are developed in order to represent the reaction of the paralysed muscle to electrical stimulation and their output torque once moved. It was decided that the best model to represent the muscle is the proposed ANFIS model which was then integrated into the VN4D model. One of the main limitations of FES which is rapid muscle fatigue is studied in depth and the stimulation parameters which are having the greatest impact on the muscle fatigue are identified. It was determined that frequency modulation results in faster muscle fatigue therefore the controlled parameter is set to be pulse width after an experiment on 15 SCI individuals. Different control methodologies, including PID, fuzzy, adaptive neuro-fuzzy and iterative learning control (ILC) to move the simulated paralyzed model previously build using VN4D are explained and has been suggested that fuzzy and PID resulted in fatigue happening later once compared with adaptive and iterative learning control. This is made possible by the study of the trend of changes in pulse width and the amount of energy induced to the muscles. While all four control methods are showing great results once compared to a reference trajectory, adaptive and ILC controllers have induced greater amount of energy to the muscles resulting in faster fatigue. Two practical FES exercise activities including FES leg extension exercise and FES rowing are designed and controlled in a feedback control setting. It is realised that by employing mechanical facilitators in an FES activity a smoother performance is derived and less fatigue is induced in the muscles resulting in the individual being able to exercise for a longer period of time without damaging any tissue or bone. In summary, this work resulted in development of a new generalised accurate muscle model which was then used to display that how the issue of premature fatigue is affecting the performance of an FES exercise machine and how the control parameters and the choice of control strategy will result in different amount of energy induced in the muscles which in return results in fatigue showing in the earlier cycles of the movement. Further experiments are carried out on a group of spinal cord injured individuals and two FES exercise facilities, FES leg extension exercise machine and FES rowing machine, are further developed.

This work examines the use of a small number of naturally-derived materials as scaffolds, specifically for bone tissue engineering. Damaged bone is typically replaced with grafts, and second only to blood, bone is the most transplanted tissue [1]. The ability to produce autologous grafts from patients’ own cells using an engineered scaffold would therefore be extremely valuable. This thesis reports the design and use of microparticle cell carriers for bone tissue engineering applications, as potential injectable or mouldable units. The suitability of silk fibroin (SF) and gelatin (G) blends as biomaterials was tested in two-dimensional cultures, using 3T3 fibroblasts and rat MSCs (rMSCs). The blends (25:75, 50:50 and 75:25) were shown to be biocompatible and with appropriate mechanical properties for bone tissue engineering with Young’s moduli between 36 and 59 MPa. The SF/G blends supported the osteodifferentiation of rMSCs at levels equivalent to tissue culture plastic. Although SF alone did not strongly support cell attachment, the cells that did adhere showed high levels of osteodifferentiation, measured by osteopontin expression. The inclusion of gelatin significantly improved cell attachment while retaining the ability of the SF/G blends to support osteodifferentiation. Novel microparticles were created from the same SF/G blends in a reproducible, controllable manner using a flow focussing device assembled from commercially available fittings. Cell behaviour on the 3D scaffolds mimicked the results observed on 2D films: cell adhesion was significantly improved by the addition of gelatin with the seeding efficiency of 3T3 fibroblasts increasing from 25% on SF microparticles to 69 – 81% on the blended microparticles. Osteogenic differentiation of rMSCs was observed on SF/G 25:75 microparticles in both basal and osteogenic medium, and osteopontin expression was shown to be slightly higher than for rMSCs grown on commercially available Cultispher-S microparticles. Although attempts were made to mould the particles into larger 3D structure, successful preliminary results could not be repeated. Adapting the flow focussing device from liquid-liquid to a liquid-air system allowed the rapid production of oxidised alginate microparticles, blended with extracellular matrix proteins, and with encapsulated cells. These microparticle scaffolds were also shown to support cell viability (75% of rMSCs were viable after 7 days) and osteogenic differentiation. With the potential to easily encapsulate different proteins or ECM extracts, alginate-blended microparticles are potentially useful tools for culture of different cell types. Finally, the culture of microparticles within a hollow fibre bioreactor, with the hollow fibre mimicking blood capillaries, was shown to improve cell viability compared to a non-perfused control: 20 times more 3T3 fibroblasts were harvested from the bioreactor than from the control. This showed the potential for using the hollow fibre bioreactor to rapidly produce large, viable, tissue engineered constructs in vitro.

Introduction: The research undertaken in this thesis was to inform OPTIMISE, a randomised controlled trial of goal directed haemodynamic therapy (GDHT) versus usual care in high-risk patients undergoing gastrointestinal surgery. The trial involved a complex intervention of cardiac output monitored administration of fluids and inotropic drugs during the perioperative period. Uncertainty exists regarding: 1. Whether the choice of fluid therapy could have influenced the outcome of the trial. 6% hydroxyl-ethyl starch (HES) has been associated with risk of death and acute kidney injury (AKI) in critically ill patients. 2. Whether he availability or provision of critical care beds is associated with improved surgical outcome and thus could have influenced the outcome of the trial. The trial intervention has traditionally been administered in a critical care setting, and this may have a bearing on outcome. 3. The trial intervention itself could have been associated with increased cardiac complications. Concerns remain regarding the administration of inotropic agents outwith traditional indications. Methods: 1. A meta-analysis was undertaken comparing perioperative use of 6% HES solutions to any comparator. 2. Surgical activity, population demographics and critical care provision in the UK were examined using large administrative databases. 3. A UK-wide cohort of noncardiac high-risk surgical patients admitted to intensive care was generated by combining data held by the Scottish Intensive Care Audit Group (SICSAG) and the Intensive Care National Research and Audit Centre (ICNARC) for the calendar year 2009. 4. Using this data, advanced statistical modelling techniques were used to test the association between critical care bed provision and outcome after high-risk surgery. 5. Measurement of postoperative 5th generation highly sensitive troponin (HST) release was undertaken in a subgroup of trial participants, in order to determine if the intervention was associated with increased myocardial necrosis. Logistic regression was undertaken to test if preoperative measurement of HST was associated with risk of death or major adverse cardiac events (MACE). Results: The principal findings of this thesis were: 1. In a meta-analysis of 1567 patients from 19 clinical trials comparing perioperative administration of 6% HES solutions versus any comparator no difference was observed in 30-day mortality arms (p=0.91, 12=0%; FEM: RD 0.00, 95% CI -0.02, 0.02) or AKI (p=0.62,12=0%; FEM: RD -0.01, 95% CI -0.04, 0.02) was observed. 2. Significant variation exists in ICU bed provision within the UK. 3. In an epidemiological study of 16 147 patients admitted to ICU following surgery in the UK, significant variation in acute hospital mortality was observed (OR 1.42; 95% CI: 1.29, 1.62). This did not appear to be accounted for by severity of illness, other patient-level factors or ICU bed provision. 4. Using HST we were unable to detect any difference in myocardial injury or infarction between GDHT and usual care groups. Preoperative HST measurement did not predict those at risk of perioperative death or MACE. Conclusion: Use of 6% HES in the trial intervention was unlikely to have affected trial outcome. Significant regional variation exists in outcome after surgery in the UK, which cannot be account for by patient level-factors or ICU bed provision. The trial intervention in OPTIMISE was unlikely to have caused increased incidence of myocardial infarction or necrosis. In this study preoperative measurement of 5th generation HST did not appear to predict those at risk of death at 30 or 180 days or MACE.

Introduction: Almost a decade ago, there was a call to establish patient safety reporting systems that would operate at local, regional and national levels; it was envisaged that these would help healthcare professionals and organisations to learn from mistakes and lead to the development of interventions aimed at mitigating against these errors. This policy call led to the creation of the National Reporting and Learning System (NRLS). It however remains unclear whether reporting systems result in safer care. Specialties such as orthopaedics pose a high potential risk of iatrogenic harm, and this clinical area therefore represents a useful exemplar in which to study the opportunities offered by this national repository of errors to improve the safety of orthopaedic care provision. Aims: The aims of this thesis were to: • understand the opportunities offered by the NRLS to ascertain the frequency, types and causes of errors in orthopaedic surgery • develop the risk prediction potential of the system • offer critical reflections on the role of reporting systems for improving the care received by orthopaedic patients. Methods: Data on orthopaedic entries over the time period 2005-2008 were extracted from the National Patient Safety Agency's NRLS. Given the high volume of orthopaedic error reports, an approach was developed to prioritise areas most likely to result in patient harm. This approach was used to select four key areas, and examples of work undertaken to reduce the harm associated with orthopaedic surgery in these areas are presented. A detailed assessment of all orthopaedic deaths was also undertaken using an inductive approach of content analysis. A key aspect of this thesis was the creation of the Orthopaedic Error Index for hospitals, which allows a national assessment of the relative safety of provision of orthopaedic surgery. It uses existing principles of benchmarking to identify outlier hospitals where a large proportion of harm occurs compared to other hospitals. Results: There were 48,971 free-text reports of orthopaedic errors made available for analyses. These reports were grouped into 15 categories, which have been used since inception of the NRLS. A method of prioritising these categories of errors was developed which yielded an odds ratio of the most harmful category of errors compared to the others; these included errors associated with implementation of care and on-going monitoring/review [OR = 2.55 (95% CI 2.49, 2.62)]; self-harming behaviour [OR = 1.60 (95% CI 1.30, 1.96)]; infection control [OR= 1.50 (95% CI 1.41, 1.61)]; treatment, procedure [OR= 1.31 (95% CI 1.22, 1.42)]; and patient accidents [OR = 1.02 (95% CI 0.99, 1.05)]. In each of these error categories, where possible, topics were selected where there was a paucity of national guidelines on delivering safer orthopaedic care. All the deaths (n = 257) were also reviewed (2005-2009). Four main thematic categories emerged: (1) stages of the surgical journey - 62% of deaths occurred in the post-operative phase; (2) causes of patient death - 32% were related to severe infections; (3) reported quality of medical interventions - 65% of patients experienced minimal or delayed treatment; and (4) skills of healthcare professionals - 44% of deaths had a failure in non-technical skills. A single error could have multiple themes, hence all errors did not add up to 100%. National alerts were then produced to mitigate risks associated with the use of digital tourniquets, hip cement, and slips, trips and falls. Data from 155 hospitals were used to create an Orthopaedic Error Index (OEI) which was normally distributed. The mean OEI was 7.09/year (SD 2.72); five hospitals were identified as outliers, lying three standard deviations above the mean OEI. This is the first time that a direct measure of patient safety has been created and used. Discussion: Reporting systems such as the NRLS offer a potentially important approach for orthopaedic surgeons to better understand the safety considerations of their work. This work has shown that content analyses and prioritisation of errors can be beneficial for large databases and can alert orthopaedic surgeons to practices of unsafe care. Subsequent solutions to mitigate against these errors can furthermore be developed. It is also possible to use the NRLS for risk prediction and identify, earlier on, any hospitals that have significant variation in the severity and propensity of errors. It is hoped that this work will catalyse efforts by a few in orthopaedic surgery to recognise that unsafe care is a problem and needs to be better understood and appropriate solutions developed.

Atherosclerosis is now widely viewed as an inflammatory disease. Intraplaque inflammation drives the progression and destabilisation of atherosclerotic lesions, converting chronic stable asymptomatic lesions into acute lesions with ensuing clinical sequelae, including acute coronary syndrome, transient ischaemic attack or stroke. There is currently no clinical imaging technique available to assess the degree of inflammation associated with plaques. The purpose of this work is to develop and utilise novel in vivo magnetic resonance imaging (MRI) methodologies to assess inflammation in atherosclerotic plaques. This thesis describes the development of antibody-conjugated iron oxide particles targeted against endothelial adhesion molecules in order to act as a contrast agent for MRI of inflammation in atherosclerosis. This study aims at visualising and characterising atherosclerosis using targeted iron oxide particles as an MRI probe for detecting inflamed plaque disease in both human atherosclerotic tissue and an apolipoprotein E-deficient (ApoE-/-) mouse model. This study is comprised of four main experimental stages. The initial in vitro feasibility study confirmed MRI detection of activated endothelial cells using anti-E-selectin antibody and anti-VCAM-1 antibody conjugated superparamagnetic iron oxide particles (SPIO). Subsequent ex vivo studies demonstrated MRI detection and characterisation of inflammatory markers on human atherosclerotic plaques using anti-VCAM-1 antibody and anti-E-selectin antibody conjugated SPIO with confirmatory immunohistochemistry. Further, the ex vivo in situ stage consisted of MRI detection of atherosclerotic lesions in the aortic root of ApoE-/- mice using a new improvised version of iron oxide particles – the dual antibody-conjugated microparticles of iron oxide (MPIO) against VCAM-1 and P-selectin. The final in vivo stage involved detection and characterisation of atherosclerotic lesions in the aortic root and aortic arch of ApoE-/- mice by in vivo MRI using the dual-targeted MPIO. Following this, an animal model (ApoE-/- mouse) with focal atherosclerotic lesions in carotid arteries was developed by means of peri-arterial cuff placement to allow in vivo molecular MRI using these probes. The in vitro cellular model of endothelial inflammation demonstrated stimulated bovine aortic endothelial cells were detectable on MRI using targeted SPIO as a contrast agent, confirmed by immunocytochemistry. Inflammation of human atherosclerotic plaques was similarly detectable by ex vivo MRI. Further, we have demonstrated that the MR contrast effect induced by endothelial-bound dual-ligand MPIO quantitatively tracked with macrophage content within the aortic root lesions of ApoE-/- mice by in vivo MRI. In the final in vivo mouse carotid stage, we have utilised the shear stress modifying cuff to generate both stable and rupture-prone lesions in the murine carotid artery. Using dual-targeted MPIO, we have subsequently identified these high-risk inflamed carotid plaque lesions by in vivo MRA. The in vivo MRI combined with dual-targeted MPIO approach will potentially allow real time in vivo characterisation of plaque vulnerability, leading to accurate risk stratification in individual patients, thereby contributing a personalised approach to the management of carotid atherosclerotic disease in the future.

Introduction: Heart Transplantation (HTx) remains the standard treatment for patients with advanced chronic heart failure (ACHF). Over the last 20 years, despite rising donor numbers, practitioners have observed a decline in the numbers of adult HTx in the UK. Due to this decline and due to increasing waiting times for HTx more patients are requiring a ventricular assist device (VAD) as a bridge to heart transplantation (BTT). This work aims to evaluate VAD practice in the UK and to describe outcomes, which include survival and quality of life. Methods: A national audit study was undertaken to collect VAD data. The data was recorded in a database and analysed. An audit of quality of life was also undertaken and all adult HTx centres participated. Quality of life (QoL) data was collected from patients who were being medically treated for ACHF; patients who had received a VAD and patients who had received a heart transplant. Results: 247 patients received VADs within the study period. The use of 3rd-gen devices increased over time. The median duration of support increased from 141 days (interquartile range 80 to 253 days) to 578 days (lower quartile 204 days). Survival improved with device generation (p=0.003). At 1-year, 50.0% of patients receiving a 1st generation device were alive (95% CI 34.9 to 63.3%) compared to 76.9% of patients receiving a 3rd generation device (95%CI 68.0 to 83.6%). 386 patients completed QoL questionnaires. Patients after HTx reported the best QoL; patients with LVADs reported better QoL scores than patients being assessed for HTx and patients listed for HTx on medical therapy. Conclusions: VAD implantation has improved and increased, and has become a credible option for some patients awaiting HTx. Quality of life for patients with VADs is better than patients being treated with maximal medical therapy.

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