Introduction. Free tissue transfer is the 'gold standard' of surgical care for patients requiring composite tissue reconstruction when local options are unavailable or unsuitable. It is a form of autologous transplant wherein composite tissue is harvested from a distant site and used to reconstruct the primary defect. The flap is rendered ischaemic following transection of its vascular pedicle until successful anastomosis with the recipient vessels is completed. Ischaemia depletes cellular ATP, lowers pH and strains cellular homeostatic mechanisms. The only way to halt the inevitable progression to cell death is by reperfusion. However, reperfusion per se initially worsens the injury through the influx of inflammatory cells and mediators. This biphasic injury is named ischaemia reperfusion injury (IRI) and is characterized by microcirculatory dysfunction primarily mediated by oxidative stress. This can lead to inadequate perfusion and ultimately tissue necrosis. IRI occurs in all transplants, is unavoidable and has no treatment. Preconditioning is an intervention performed before a known event that improves the outcome of that event. The elective nature of transplants permits such interventions to be executed. Haem-­oxygenase 1 (HO-­1) is a cytoprotective enzyme that is up-­regulated in response to diverse stressors including oxidative stress. Haem arginate (HA) is a potent inducer of this enzyme. Pharmacological preconditioning with HA has been shown to reduce IRI and improve clinical outcome in models of visceral IRI. Aim: to investigate whether HA could be used to improve outcome in myocutaneous flaps. Objectives: (1) to establish a reliable model of myocutaneous IRI (2) to assess the effects of pharmacological preconditioning with HA on clinical outcome measures and (3) to investigate the mechanisms underlying the effects of HA preconditioning demonstrated in the in vivo model by in vitro work. Methods. An in situ transverse rectus abdominis myocutaneous (TRAM) flap was developed. Forty male, Lewis rats were randomly assigned to receive IV: Control (NaCl); HA; HA + tin mesoporphyrin (SnMP, an HO-­1 inhibitor) and SnMP alone. Laser Doppler imaging (LDI) scans were performed to assess perfusion. Clinical outcome was assessed by percentage area flap necrosis and perfusion. In vitro adult human epidermal keratinocytes (HEKa) were treated in: Control medium; HA; SnMP and Desferrioxamine (DF) or combinations thereof. MTT and VialightTM plus ATP assays were used to assess cytotoxicity. Intracellular reactive oxygen species (ROS) concentration was determined by flow cytometry (CMH2DCFDA assay). Statistical analysis was performed by one-­way analysis if variance (ANOVA) followed by Tukey's test. Results. In vivo preconditioning with HA increased HO-­1 protein expression and level of bioactivity. This bioactivity was successfully inhibited by SnMP. In the skin, HO-­1 up-­regulation occurred in macrophages. HA based treatments resulted in significantly worse necrosis at 48 h: Control vs HA (p = 0.01). HA based treatments significantly decreased perfusion at: 24 h (Control vs HA, p = 0.0002) and 48 h (Control vs HA, p = 0.04). By contrast, SnMP did not affect either clinical outcome measure. In vitro preconditioning with HA was cytotoxic and increased intracellular ROS: both were reversed by co-­administration of DF but not SnMP. Conclusion. In contrast to data from visceral models, HA preconditioning proved deleterious in myocutaneous flaps. This is most likely due to the generation of ROS by free haem independent of HO-­1 up-­regulation.

We recognise consciousness in ourselves and in those around us. Consciousness is the essence of our existence, who and what we are, but we are willing and able to let go of it daily during sleep, which we welcome and associate with rest, recovery and well being, knowing that consciousness will return reliably, when we are ready. Yet we cannot define this thing or process which makes us "us". We do not understand how it is constructed from the activity in our brains, how it is deconstructed by sleep, drugs or disease, or how it can be reconstructed by waking or recovery. Our ignorance renders us reliant on inadequate means of measuring consciousness, dependent on movement for its detection. Propofol is an intravenous anaesthetic drug with the capacity to safely, rapidly and reliably produce sedation and anaesthesia, providing an ideal model of unconsciousness for study. Functional magnetic resonance imaging (fMRI) provides a non-invasive means of measuring activity within the brain. EEG is a convenient broad measure of neuronal activity. This thesis exploits the advantages of each of these techniques, fMRI and EEG, first separately and then together, to link highly informative, spatially specific fMRI observations to convenient, reproducible electrophysiological surface measurements. A safe and reliable model of unconsciousness suitable for fMRI interrogation is first developed and explored. Changes in the spatial extent and interregional correlation of neuronal activity when subjects become unresponsive show that the functional connectivity of the striatum is specifically impaired as perception fails. Disruption of the brain’s internal temporal frame of reference impairs the synthesis of perceptions from their fragments. The second experimental chapter specifically examines the behaviour of sleep oscillations during ultraslow increases and decreases in the depth of sedation with propofol. Functional activity shows that the brain is intensely active despite loss of consciousness and reveals measurable transitions in neuronal activity. Combined simultaneous EEG/FMRI then shows that these transitions reflect stepwise changes in the processing of experience and a shift from externally modulated thalamocortical signaling to an internal dialogue.

No description available.

It is predicted that the percentage of persons over 50 years of age affected by bone diseases will double by 2020 (Navarro et al., 2008). Clearly this represents a need for permanent, temporary or biodegradable orthopaedic devices that are designed to substitute or guide bone repair. Polymeric medical devices are widely used in orthopaedic surgery and play a key role in fracture fixation and in areas of orthopaedic implant design. Initial uncertainty regarding the adequacy of polymeric materials to withstand functional stresses obliged clinicians to implement these biomaterials in non-load-bearing applications such as fixation of the maxillofacial skeleton. Strategies to guide bone repair, have included topographical modification of these devices in an attempt to regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved the field of surface modification and, in particular, nanotechnology has allowed the development of experimental nanoscale substrates for the investigation into cell-nanofeature interactions. This thesis is concerned with the study of nanotopographical structures on osteoblast adhesion and mesenchymal stem cell (MSC) function, with an aim to improving the functionality of orthopaedic craniomaxillofacial devices. In this study primary human osteoblast (HOBs) were cultured on nanoscale topographies fabricated by lithographic and phase separation techniques in poly(methyl methacrylate) (pMMA). Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions investigated via immunocytochemistry with scanning electron microscopy. To investigate the effects of these substrates on cellular function 1.7 K microarray analysis was employed to study the changes in gene profiles of enriched MSC populations cultured on these nanotopographies. Nanotopography differentially affected the formation of adhesions in HOBs and induced significant changes in genetic expression of MSCs on experimental substrates. Nanopit type topographies fabricated by electron beam lithography were shown to inhibit directly the formation of large adhesion complexes in HOBs and induce significant down-regulation of canonical signalling and functional pathways in MSCs. Nanocrater and nanoisland type topographies fabricated by polymer demixing however reduced adhesion formation and induced up-regulation of osteospecific pathways. Nanogrooved topographies fabricated by photolithography influenced HOB adhesion formation and MSC osteospecific function in a manner dependant on the groove width. The findings of this study indicate that nanotopographical modification significantly modulates both osteoblast adhesion and MSC function, implicating topographical modification as a viable strategy to enhance orthopaedic device functionality.

Solid organ transplantation is the gold standard treatment for a variety of conditions that result in organ failure. However, despite considerable advances in clinical transplantation in recent decades, the almost ubiquitous requirement of life-long immunosuppression of transplant recipients persists and is complicated by graft loss to rejection in the long term and multiple serious adverse effects that are frequently life limiting. Helminths currently infect more than one quarter of the world’s population and it is now well established that their success as parasites is the result of active immunomodulation of the host immune response. Whilst this primarily secures ongoing survival of the parasites, in some cases helminth-induced immunomodulation can be beneficial to the infected host and is not associated with the adverse sequelae of pharmacological immunosuppression. An emerging body of evidence suggests that harmful immune responses to alloantigens can be suppressed by helminths, but little mechanistic data exists and the active immunomodulators involved have remained hitherto unidentified. The hypothesis behind this thesis is that the model intestinal nematode, Heligmosomoides polygyrus, produces immunomodulatory molecules that can suppress responses to allo- and auto-antigens in animal models of transplantation and autoimmunity, and that some of these molecules could potentially be exploited as novel therapeutic agents. Full-thickness skin grafting was performed between fully-allogeneic mouse strains (BALB/c to C57BL/6). Recipient mice infected with H. polygyrus immediately prior to transplantation showed significantly prolonged allograft survival. Likewise, protection from allograft rejection could be replicated in recipient mice in which H. polygyrus excretory-secretory products (HES) (isolated from culture of adult worms) were delivered by continuous infusion via surgically implanted osmotic minipumps. A number of potential mechanisms underlying allograft protection were identified including induction of CD4+CD25+Foxp3+ regulatory T cells (Treg) and suppression of Th1 and Th17 effector CD4+ T cell phenotypes. H. polygyrus and HES were further shown to ameliorate disease in murine (pMOG) experimental autoimmune encephalomyelitis and colitis induced by T cell transfer. In addition to expansion of Treg, H. polygyrus-mediated protection against EAE was found to be almost completely lost in IL-4 receptor deficient mice, indicating a protective role of Th2 immune responses in this context. Finally, the mechanisms of action of the newly-identified TGF-β mimic, TGM, contained within HES were investigated. Despite bearing no sequence homology or structural resemblance to TGF-β, TGM was shown to act through the TGF-β receptor complex to induce Treg in human and mouse CD4+ T cells in vitro and to suppress murine allogeneic skin graft rejection in vivo. TGM may represent the origin of a safe, effective and long-overdue novel alternative to current immunosuppression therapy.

No description available.

Limb lengthening procedures use fixation devices to extend the constantly regenerating bone and surrounding soft tissues. Automated devices have been developed that aim to provide a more gradual tissue extension, resulting in better quality of treatment for the patient. Benefits include pain reduction and probable enhanced tissue outcomes. The development of one such new smart lengthening device is described. An integrated numerical model of tissue mechanics during lengthening is presented. It represents the mechanical environment in which the devices extend. The mechanism of the automated device is also modelled using Matlab software and validation was achieved through experimental testing. Validation of the tissue model includes the design of an experimental hydraulic system with the ability to control the peak loads and relaxation over time. A simplified mechanobiological model for the longer term healing effects is proposed. Calibration of the tissue model to clinical data allows for direct comparison of the load and extension of identical tissues, one being lengthened by a traditional device, the other an automated device. This simulation can be extended to include a range of lengthening rates and frequencies of distraction alongside various patient dependent tissue properties. The models also provide the opportunity to assess the effects of iterative changes to the device parameters (such as stiffness) on its performance as well as analyse the effect that these changes have on tissue extension and loading. Use of these models to optimise the device design alongside optimisation of the extension regime can result in improved device design and consequently improved patient outcomes.

Numerous testing regimes have been used in vitro to assess the fatigue behaviour of acrylic bone cements. While some attempts have been made to introduce an optimal protocol that measures the fatigue life of bone cement under similar stress conditions to those exist in vivo, the effects of specific testing variables such as test specimen specification and stress parameters are still questionable. These factors can be important since inconsistency in results have been reported regarding the precise effects of other variables such as the mixing method of cement components and the resultant porosity. For a given series of testing variables; namely, specimen cross sectional shape, surface production method and stress type and level (herein collectively termed testing regime), this study investigates the effect of each variable on both the fatigue life and the fatigue crack propagation properties (fatigue behaviour) of bone cement. Testing was constantly performed in 37˚C saline under stress-controlled conditions at a frequency of 3Hz (2Hz for the CT specimens). All specimens were produced after vacuum mixing of the cement components and soaked in 37˚C saline for 1- 6 weeks. Specimens were manufactured with two cross sectional shapes: rectangular (ISO 527-2) and circular (ASTM F2118), using two production methods: direct moulding or machining. Two different bone cements were used: SmartSet GHV and CMW1. For each specimen type, at least 10 specimens were fatigued to failure at a maximum stress of 20 MPa applying either fully reversed tension-compression (R= –1) or tension-tension (R= 0.1) loading, followed by Weibull analysis. For the fully reversed loading only, at least 5 specimens were tested for each group at other three levels: ±12.5, ±15 and ±30 MPa and the four stresses were compared using S-N curves. Behaviour of fatigue cracks were assessed based on the cyclic stress-strain responses. CT specimens were used to measure the crack growth rates in the two cements. The findings of this study have emphasised the important role of the set of a testing regime variables included in testing and identified the influence of each testing variable on the fatigue behaviour of bone cement. Machining of test specimens and applying high stress levels, in particular, can lead to irrelevant findings when considering the in vivo conditions, depending also on the cement composition. While these “inappropriate” testing variables can be considered as possible reasons for the variations in fatigue results reported in previous work, it is suggested to consider the effects of these variables in future work.

This thesis presents an in-depth ethnographic mapping of deceased donation in a Catalan hospital. A unique site in terms of leading edge technoscientific practices, high rates of donation and its consolidated specialised team of transplant coordinators (TCs). The thesis situates donation as an embedded medical practice and traces the practicalities and specificities of making donation a possibility at the hospital. The empirical accounts offer a distinctive contribution that complements and challenges existing social sciences literature about donation. The latter have predominantly focused on donation as a controversial practice through highlighting the emotional experiences of donors’ families and individual medical practitioners involved. This empirical investigation mobilises, and further develops, STS material semiotics tools to provide an account of donation enacted as both procurement and healthcare. Ethnographic insights illustrate the shifting processes of mutual inclusion and exclusion that underpin the trajectory of integrating donation as a routinized hospital practice, along the recurring set of enduring tensions. This is achieved by following the work of TCs along the stages of donor detection, evaluation, maintenance, consent request and organ extraction. Crucially, the analytical focus decenters the individual actors’ perspectives, broadening the scope of the inquiry and making visible the complex sociomaterial arrangements that take place, inside and outside the hospital, which are rendered as a gradual process of assembling donations. Families’ consent to donation is essential but it is decentered, it is neither that which starts a donation process nor the only factor that contributes to the assembling of a donation process. Unlike available anthropological and sociological studies of donation this work is not about documenting the reductionist transition from patient to donor, whole to parts, person to thing and denouncing the fall from subject to object reified in donation practices. The emphasis here is on tracing the overlap between donors as patients, thus the analysis shows the shifting enactments of the embedded donor/patient configuration, which includes the donor/body, donor/person and donor/corpse figures simultaneously along the donation process. The intervention of bodies as active entities is examined through a speculative and pragmatic elucidation on the situated and relational enactments of responsive bodies and organs. This thesis contributes to contemporary re/articulations of materiality and agency through the lens of distributed joint action and entangled actors from a nonanthropomorphic stance. The research also contributes to current policy debates in the UK, and in Scotland in particular, that propose to tackle the national problem of low donation rates with a legislative move to an opt-out system for donation. It offers robust empirical evidence to contest the dominant organ shortage problematisation that is reduced to the legal polarity of either opting in or out of donation. I suggest that questions about increasing donation rates cannot be restricted to the domain of individual choice as this excludes the situated medical practices that enable the choice of donation in the first place.

Mycophenolate mofetil (MMF) is widely used to minimise acute rejection following solid organ transplantation as it inhibits inosine monophosphate dehydrogenase (IMPDH) and thereby reduces lymphocyte activation. The effects of MMF and azathioprine on renal allograft outcome were examined by analysis of the national transplant database held at National Health Service (NHS) Blood and Transplant, Stoke Gifford, Bristol, UK. In a paired kidney analysis, MMF treated patients had a 3 year death censored graft survival of 91% (n=217) contrasts to 97% (n=231) in azathioprine treated patients (p=0.07) with an increased acute rejection rate in the first year after transplantation (44 v 31%, n=105 v 74, p<0.01). In a further study, 13% (n=71) of patients were found to be taking less than 1 g of MMF which was associated with a 3-fold increased risk of graft failure and inferior graft function up to 36 months. One strategy to improve graft outcome would entail targeting MMF dose according to pre-transplant IMPDH activity, which is known to display wide variability between patients, in order to maximize efficacy and minimize toxicity. Therefore, it was decided to measure pre-transplant IMPDH activity and to investigate associations with renal allograft outcome and MMF dose tolerated after transplantation. IMPDH activity was measured by detection of generated XMP by a validated HPLC method in the peripheral mononuclear cells of 55 patients waiting for renal transplantation and was found to exhibit a 4-fold variation of IMPDH activity. Black males had significantly increased IMPDH activity contrasts to Black females (p=0.01). Within the first year of transplantation, 71% (n=12) patients required a reduction in MMF dose. There was no association between pre-transplant IMPDH activity and MMF dose achieved at 1 year or MMF associated side effects or eGFR up to 36 months. It was proposed that the inter-individual variability of IMPDH activity may be associated with genetic polymorphisms and therefore sequencing of the exons of IMPDH I and II was undertaken. Two novel single nucleotide polymorphisms (SNPs), Leu244Leu and Ala285Thr, were identified in the IMPDH I gene. Patients with these variants did not exhibit differential IMPDH activity. Genotyping for established intronic SNPs was undertaken in our patient cohort as well as a random sample of 1040 recipients from the Collaborative Transplant Study DNA bank based at the University of Heidelberg, Germany. The presence of these SNPs did not increase the risk of rejection or affect graft function or MMF dose tolerated at 1 year after transplantation and there was no association between pre-transplant IMPDH activity, 5 year graft and patient survival and genotype. In our study, MMF treatment did not result in improved renal allograft outcomes in comparison to azathioprine therapy. Furthermore, we suggest that measurement of pre-transplant IMPDH activity or genotyping of the IMPDH enzymes is unlikely to assist in optimizing MMF dose and renal allograft outcome.