No description available.

Wounds present an ideal environment for the growth of bacteria as they are usually moist and warm. The impact of bacteria on wound healing and developing infection is debatable and only partially understood. Some clinicians believe that the number of bacteria is a crucial factor in determining whether the wound is likely to heal. Others argue that the presence of specific pathogens and their interactions are the main cause of non-healing wounds. Also, the methodology of wound culture has been prone to controversy for many years. Most diagnostic laboratories use conventional microbiological techniques to indicate if there are pathogens in a wound. Some specialists still argue that greater than 105 organisms per gram of tissue is diagnostic for infection. Introduction of new molecular techniques have shown that only a small percentage of bacteria are identified and they grown in biofilms, which makes sampling difficult. In this project, the aspect of sample collection and transport was investigated as well as the conventional and molecular approaches for bacteria identification and quantification. Four different swab transport systems were tested for their ability to maintain viability of the most common wound bacteria during transport and their performance with molecular methods in order to establish the best swab transport devices for further testing on patients. The most satisfactory results were achieved with Sigma dry swabs and this swab remained the best choice for further in vivo studies involving both conventional and molecular techniques of bacteria identification and quantification. The semi-quantitative swab and biopsy culture was compared with quantitative culture to establish the best method for bacteria culture. Our findings demonstrated a statistically strong significant correlation between semi-quantitative and quantitative swab and biopsy methods and use of semi-quantitative count as a cost effective method compared to quantitative serial dilutions. However, when time is important rapid methods should be employed thus Real-Time PCR (RT-PCR) assay was developed for the direct and rapid detection of MRSA and compared with conventional methods. The diagnostic values of the RT-PCR assay for the detection of mecA and femB genes were as follows: sensitivity 83.3%, specificity 88.5%, PPV 62.5% and NPV 95.8%. Quantitative analysis revealed that the average difference between the MRSA counts obtained using the RT-PCR and conventional culture results was 0.61 log. These findings show the potential of the RT-PCR assay in rapid detection and quantification of MRSA. Development of a RT-PCR assay for MRSA detection was the first step in developing a multiplex RT-PCR assay for chronic wound samples. In further studies, a DGGE-sequencing method was developed for the analysis of the diversity of microflora in chronic wounds and healthy feet and compared with conventional methods. DGGE-sequencing allowed identification of a number of strains not detected by culture techniques with 43% of the DGGE fragments representing organisms not cultured from the wound from which they had been amplified. This highlights the fact that a significant proportion of the resident microflora was not able to be analysed by culture. Development of PCR-DGGE sequencing and investigation of the diversity of microflora in chronic wounds allowed us to select the panel of microorganisms for the further development of multiplex RT-PCR assay for the rapid detection of bacteria in chronic wounds.

Following injury, fibroblasts migrate into the wound bed, where they play essential roles in effective wound healing through matrix deposition and contraction. Fibroblast migration relies on cell-matrix and cell-cell contacts, both of which are regulated by transmembrane receptors that are differentially regulated during wound healing. The fibronectin receptor syndecan-4 is increased upon wounding, which promotes fibroblast migration through activation of small GTPases and the recycling of integrin a5~1. Members of the Eph family of receptor tyrosine kinases, which regulate cellcell contacts, are also differentially regulated upon wounding. Little is known about whether there is a relationship between the syndecan-4 and Eph receptors, and how this might regulate the balance between cell -cell and cell-matrix contacts, and therefore fibroblast migration and clustering. Using transient knockdown and genetic knockout models, this project demonstrates that syndecan-4 downregulates EphA2, a receptor that mediates cell-cell repulsion, through a PKCa- and Fyn-dependent mechanism. In syndecan-4 knockout mouse embryonic fibroblasts, elevated EphA2 expression increases sensitivity to the EphA2 ligand ephrinA 1 and increases the rate and frequency of cell-cell repulsion when compared to wild type, demonstrating that the relationship between syndecan-4 and EphA2 has a direct effect on fibroblast clustering. Importantly, the findings are translated in vivo; EphA2 expression is increased in syndecan-4 knockout mouse wounds compared to wild type. This may contribute to the healing defect previously observed in syndecan-4 knockout mice 1. We hypothesise that the balance between syndecan-4 and EphA2 signalling regulates the switch between fibroblast clustering or scattering, and therefore coordinates the initiation and resolution of healing.

Memory disturbances following a trauma are a characteristic feature of posttraumatic stress disorder. Despite alcohol’s frequent involvement in real-life traumatic events, our understanding of its contribution to trauma-related symptoms is unclear. The research in this thesis aimed to determine the way in which alcohol intoxication during a traumatic experience might influence memory for the event. Experiment 1 showed that alcohol impaired recognition associated with recollection with greater reductions as dose increased (0, 0.4, 0.6, 0.8g/kg); in contrast, recognition associated with familiarity was preserved. Experiments 2 and 3 utilised an analogue trauma film to examine how low (0.4g\kg) and high (0.8g/kg) doses of alcohol affected intrusive imagery and explicit memory for the footage. Alcohol during encoding resulted in a dose-dependent inverted U-shaped curve on intrusive imagery, with increased intrusions only following a low dose. Explicit memory for the footage was reduced in a dose-dependent linear manner. In addition, experiment 3 concurrently assessed same- and shifted-view object location recognition to determine the mechanisms that might underpin alcohol’s effects on trauma memory. Results showed that a low dose of alcohol selectively impaired shifted-view recognition, thought to rely on an allocentric representation. However, same-view recognition was preserved, suggesting a spared egocentric representational system. In contrast, the high dose disrupted both same- and shifted-view recognition, suggesting a global disruption in both memory systems. Experiment 4 examined the effects of alcohol (0.4/kg) on contextual fear acquisition and extinction and both same- and shifted-view recognition. Fear acquisition was unaffected by alcohol, whilst extinction learning was impaired with persistent conditioned responses throughout extinction. Alcohol-induced reductions in extinction learning were highly correlated with decreases in shifted-view recognition, supporting the role of contextual encoding in extinction. The findings of these studies suggest that alcohol dose-dependently influences trauma memories and this could result in a distinct set of trauma-related symptoms.

Early identification of infection is imperative in the management of chronic wounds in preventing limb threatening events. There is a clear requirement for the development of in situ sensors that can monitor the healing progress of chronic wounds and identify the early onset of infection, providing the clinician with a more detailed picture of the wound dynamics. A variety of carbon composite materials were employed for use in wound monitoring technologies. The mechanical flexibility of the polyethylene and polycarbonate films are ideal for incorporation within existing dressing materials and could be produced in bulk at relatively low cost, a pre-requisite given the frequency with which dressings need to be replaced. Surface modification of the films through laser ablation and electrochemical anodisation was required to enhance the sensor's electroanalytical performance and improve both the selectivity and sensitivity towards uric acid - a key wound biomarker used to assess the wound physiology through measuring both the wound pH and wound severity. A preliminary assessment of the films performance in simulated wound fluid and defibrinated horse blood was conducted. A prototype smart bandage was designed, based on interfacing the carbon film to a portable potentiostat, and the response to urate and potential interferences assessed. The sensing strategies developed were adapted in order to facilitate the monitoring of central venous catheters which are also subject to the complications of infection. Carbon fibre filaments were selected as the core substrates for the sensing electrode due to their dimensional characteristics and as such could be easily integrated within existing catheter architectures.

It is estimated that the UK has more than 19 million people aged over 50, with about 9.4 million aged 65 or more [1]. In 2000, fragility fractures represented an estimated cost of £1.8 billion [2] to the UK. Depending on the severity, treatment can include surgical intervention where plates, screws and wires are used to stabilise the fracture. However, fracture fixations can fail, leading to two or more interventions, increasing costs and trauma to patients. The mechanical test performed on these fixations represented the fracture as flat line or a gap between the fractured bones [3, 4]. Recent research has shown that the geometry of the fracture have an impact in the response of the system, proving that a more close representation of the fracture is still needed in order to correctly assess and test the designs of fracture fixations. The aim of this work was therefore to characterise real bone fractures in terms of its shape and surface properties. Transverse fractures were successfully generated in ten porcine femurs, using a specially designed three-point rig. Post-fracture the specimens were scanned in a µCT machine (XtremeCT, Scanco Medical AG, Brüttisellen, Switzerland) with a resolution of 82 microns. Various metrics were captured from each specimen, including length of fracture and range of feature sizes. Mechanical testing was performed of the fractured bones under axial load and rotational displacement. The torsional results were compared with the results obtained from the computational simulations, for each specimen and agreement was observed. The experimental results showed that the torsional behaviour for transverse fractures reached a plateau after 5 degrees of rotational displacement, and the average rotational stiffness was found to be 1.64 ±0.77 Nm/degree. Torsional behaviour in the FE models was particularly sensitive to changes in the elastic modulus of the specimen, coefficient of friction between the bone and the geometry of the fracture. Future work is still needed. An investigation of the effects of the microdamage caused by the mechanical test can be useful and the inclusion of locking plates in the models could be interesting.

No description available.

1. The prime aim of this study was to investigate the structure and cellularity of human granulation tissue, by the taking of punch biopsies from clinical wounds, a method not previously reported in wound research. It was intended both to further the understanding of wound biology, and to provide baseline information for clinical trials of therapeutic agents, such as dressing materials and growth factors. 2. To evaluate the validity of the universally used animal models of wound healing, and the extent to which data from them can be extrapolated to the human wound. 3. To relate histological criteria to the progress of wound healing as observed clinically, in both normally healing and unhealthy wounds. This might allow diagnosis of difficult healing problems to be made by biopsy, and also has relevance to wound biology in providing circumstantial evidence of cell function. 4. To compare conventional and immunocytochemical staining techniques in identification of cell types in human granulation tissue. 5. As a particular area of interest, to clarify the role of the myofibroblast in wound contraction by identification of a suitable marker for this cell in human granulation tissue, and by examining the temporal relationship between myofibroblast presence and wound contraction.

Scaphoid fracture union depends upon early diagnosis and adequate management and is considered to be adversely influenced by proximal scaphoid avascularity and carpal instability. There is, however, controversy on various aspects in the diagnosis and management of this injury. Studies investigating some of these aspects are reported in this thesis. With regard to the initial diagnosis of this fracture this thesis demonstrated that the fracture was usually visible on the 'Ziter' or the posterio-anterior radiographic view while radiographic evaluation of soft tissue swelling was of less value than that suggested by current literature. Moreover, the second week radiograph did not provide reliable evidence of a fracture in those suspected on clinical examination alone. Normal lines in an intact scaphoid could be mis-interpreted as a fracture. With regard to management, the fracture healed equally well in a Colles' cast: thumb immobilisation did not appear to be neccessary. The incidence of non-union was around 12% and most were symptomatic. A further 25%, in whom the fracture site was easily identified but appeared to have healed, formed a distinct clinical group and many had symptoms. The reliability and reproducibility of radiographic signs of union were poor. Therefore, a real time ultrasonographic method of demonstrating movement at the scaphoid fracture site was developed. Proximal avascularity was more common than that suggested by radiographic appearances alone while carpal instability was uncommon within two years of injury. Based on these observations a rational approach to management of scaphoid fractures was proposed.

Cutaneous wound healing is orchestrated by a number of physiological pathways which ultimately lead to re-formation of skin integrity and the production of functional scar tissue. Matrix metalloproteinases (MMPs) act to control the remodelling of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and as such have a role to play in the healing of infected wounds. Of particular interest in this thesis is Pseudomonas aeruginosa elastase, which has been reported in previous studies to induce degradation of host proteins including proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. This evidence, along with recent data demonstrating the presence of P. aeruginosa biofilms in equine wounds, led to the hypothesis that P. aeruginosa proteases play a role in the non-healing of equine wounds and that the wound environment has an influence on the pathogenicity of P. aeruginosa in terms of protease expression. In this thesis I identify that isolates of P. aeruginosa obtained from equine wounds display a highly variable protease expression and biofilm forming potential (BFP). Furthermore, environmental changes made to P. aeruginosa cultures including changes to the initial culture pH (i.e. changes to the initial pH of the media in a non-buffered system) and depletion of oxygen in vitro were shown to alter protease expression. Further experimentation has shown that the biofilm mode of growth causes changes in the proteolytic profiles of these isolates. A further study was conducted to investigate the effects of exoproducts derived from P. aeruginosa in addition to purified P. aeruginosa elastase on the viability, growth and MMP/TIMP mRNA expression of equine dermal fibroblasts in vitro. Preliminary findings demonstrated that P. aeruginosa exoproducts, including purified elastase exert deleterious effects on both normal skin fibroblasts and granulation tissue fibroblasts in a concentration-dependent manner. Immunohistochemistry analysis of wound tissue revealed the presence of bacterial clusters disseminated throughout the wound bed and this bacterial staining occurred in conjunction with moderate staining of MMPs -2, -3 and -13 throughout the tissue. Furthermore, whilst data is preliminary, QRT-PCR analysis showed that mRNA of each of the MMPs and TIMPs of interest were expressed in normal skin fibroblasts and granulation tissue fibroblasts to a higher level than GapDH, with cells from different equine donors responding differently in terms of MMP/TIMP mRNA expression when treated with P. aeruginosa conditioned media and P. aeruginosa elastase. My results also identified the potential MMP modulating capacity of a novel foam dressing which incorporates polyphosphate. The dressing was effectively able to reduce the levels of MMP-2 and MMP-9 in both their active and latent forms and the dressing demonstrated its potential in the regulation of P. aeruginosa derived proteases. In light of the results from these investigations, it is proposed that in future work the proteolytic phenotype and BFP of infecting bacteria should be considered when investigating the clinical nature of chronically infected wounds.