Research has shown that the installation of Impact Absorbing Playground Surfaces (LAPS) in play areas in Cardiff has reduced the amount of serious head injuries. However, even though head injuries have been greatly reduced in severity, arm fractures have not reduced in severity or frequency. This confirms that the current strategies for the assessment and prevention of head injury potential have been shown to be highly successful, yet totally ineffective for preventing upper limb fractures. A need therefore exists to develop a method for the assessment of playground surfaces for their ability to alleviate arm fractures. The force required to fracture the arm during a fall in a playground is currently unknown. The overall aims of the study were to investigate the forces that are produced in the arm during a fall onto the outstretched hand at a non-injurious level and to then utilize the data to aid in the development of a mechanical arm fracture model that could be used, alongside a British Standard head impact testing device, to additionally test LAPS for their ability to alleviate arm fractures. A study was conducted, using motion analysis equipment, to investigate the forces produced during a fall onto the outstretched hand. Thirty-five volunteers of mixed gender, age and weight were dropped through a height of 3 and 5 cm onto force plates, and from 5 cm onto a domestic surface and a playground surface. A further study was conducted to assess the mechanism of the arm prior to and during impact using high-speed video (HSV) equipment. Electromyography (EMG) equipment was also used to record the activation of certain muscles throughout the falls. These tests were completed with five volunteers at heights of 1 and 5 cm onto force plates, and further tests were conducted on one volunteer at heights of 10 and 20 cm, and two volunteers from standing height. The results showed that falls onto the outstretched hand produce an impact force characterised by an initial force peak, Fl, followed by two further force peaks, F2 and F3. The magnitude of force peak Fl was found to be generally higher than the subsequent force peaks F2 and F3. Force peak F3 was not prevalent in all force time curves and it was suggested from the results that when it did occur it was not significantly different to force peak F2. A positive linear relationship was found between force peaks Fl and F2 (as Fl increases, F2 increases). The increase of force peak F2 was not as great as the increase of force peak Fl, confirming the suggestion that the magnitude of force peak Fl is generally greater than F2. Gender, fall height and effective mass were found to be important factors when assessing the impact forces found during a fall onto the outstretched hand. The mechanism of the arm prior to and during impact was found to be similar regardless of the fall height and falls were found to not generally occur onto a fully extended elbow. A shock wave was produced in the arm on impact with a surface and the initiation of arm muscles in response to a fall was found to be due to motor vestibular reflexes and not an intentional cerebral decision as to when was best to arrest the fall. Using the information from the experimental fall studies, a computer model was developed to enable falls from a higher height, such as those that occur during a playground fall, to be investigated. The model was developed and a series of simulations were performed at heights of 1 and 2 cm. Despite an overestimation of the magnitude of force peak Fl, the computer model was found to correlate well with the results found from the experimental studies. Force peak Fl was influenced by fall height and force peak F2 was influenced by effective mass (in male volunteers). Data from the experimental falls and computer modelling conducted in this study, along with data from a playground injury study can be used to further develop and validate an arm fracture model to investigate playground injuries and also for application to domestic falls and non-accidental injury investigation for a range of ages.

A complication associated with wound healing is wound infection. The diagnosis of infection in chronic wounds can be a difficult clinical decision. Signs and symptoms used to diagnose infection can often be masked by factors relating to the host, chronic inflammation and the tissue damage, associated with chronic wounds. This Study aimed to determine whether there is a measurable biochemical host response that could serve as potential biomarkers of chronic wound infection, providing an alternative diagnostic tool to aid the Clinician. An in vitro model further investigated the expression of markers from neutrophils, in response to bacterial supernatant from Pseudomonas aeruginosa and Staphylococcus aureus commonly associated with chronic wounds and their infections. Chronic venous leg ulcer and diabetic foot ulcer wound fluids were collected, wound microflora assessed, and a variety of host factors, including serine proteases, matrix metalloproteinase, their inhibitors and cytokines/growth factors, IL-ip, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, 11-12 p40, IL-12p70 and IL-13, angiogenin, IFN-y, TGF-pi, VEGF, TNF-a, TNF-r2, ICAM-1 and IP-10, analysed. No significant differences (p>0.1) in the activities/levels of host factors were observed between non-infected and infected wounds, based on clinical diagnosis. Significant differences (p<0.05) in a variety of these host factors were observed in these ulcer types, upon defining infection by wound microbial bioburden, the number of genera or bacterial species. Of these factors, cytokines were found to distinguish on two of the three defining parameters. Specifically, a number of cytokines were found to be significantly elevated in venous leg ulcer wounds, IL-lp, TNF-a, TNFr2 and ICAM-1, with an additional set of cytokines, IL-2, IL-5, IL-12p40, IL-12p70, IFN-y and TGF-P,, significantly decreased within diabetic foot ulcer wounds. in vitro, neutrophils were treated with Pseudomonas aeruginosa or Staphylococcus aureus 'acellular' supernatants, at mid-log or stationary growth phases, for 4 and 24h. Significant increases (p<0.05) in the levels of proteases and cytokines were observed from neutrophils, treated with both mid-log and stationary phase supernatants, from both species. These increases were both neutrophil donor- and growth phase-dependent. A larger cytokine response was induced from neutrophils stimulated with stationary phase, compared with mid-log phase supernatants. Combined with the increased expression of virulence factors, including bacterial enzymes, this Study suggests that bacterial growth is an important feature of chronic wound infection.

When cutaneous injury involves disruption of the basement membrane, keratinocytes must disassemble their complex attachment to it, enabling lateral migration over a wound surface consisting of a new provisional matrix. Integrin cell adhesion molecules have already been characterised as prime mediators of this process in experimental human wounds. However, very little is known about the role played by integrins as well as other epidermal adhesion molecules such as syndecans in more complex chronic wounds. In this study, a human model of rapidly healing surgically induced wounds and pilonidal excision wound tissue was used to establish the previously documented expression of epidermal adhesion molecules in the acute healing process. Chronic venous leg ulcer wound tissue was then taken as representing the process of chronic wounding and used to investigate its effect on these adhesion molecules. Differences were found between the expression of adhesion molecules in acute and chronic wounds. In particular as pi expression was decreased in chronic wound epidermis. As a result, a tissue explant culture system was developed to investigate the possibility of modifying its expression on chronic wound keratinocytes. Characterisation of venous leg ulcer explant tissue within this system highlighted the inherent heterogeneity of the tissue, leading to a large degree of variability in the levels of analytes such as IL-8 measured within it. Pilot experiments with two biopsies suggested that aspi expression could be up regulated on chronic wound keratinocytes in response to an inflammatory stimulus. However, further development of this system will be required to reduce the variability within it, before real changes in cell behaviour in response to exogenous stimuli can be demonstrated. Given the difficulties in extrapolating data from animal to human tissue, development of models such as this could prove invaluable in trying to understand the complexities of chronic wounds.

The host response to a severe burn injury is characterised by exaggerated systemic inflammation. Early clinical manifestations include shock, respiratory failure, renal failure and immunosuppression. The signalling pathways that propagate the inflammatory response are unclear but have traditionally been thought to involve overspill of proinflammatory cytokines. The importance of microvesicles, sub-cellular membrane-bound particles, is increasingly being recognised in the context of intercellular communication. Although circulating microvesicles are elevated in proinflammatory states such as sepsis, their relevance to the post-burn inflammatory response has not previously been evaluated. We hypothesised that circulating microvesicles play a crucial role in propagating the post-burn inflammatory response. Our overall aims were to 1) optimise protocols for the processing and analysis of plasma samples for microvesicle content; 2) characterise the circulating microvesicle profile associated with severe burn injury; 3) develop in vitro techniques to assess microvesicle production and function. The major findings of this work were that microvesicles derived from leukocytes, neutrophils, monocytes and endothelial cells were significantly elevated within 24 hours of burn injury. Microvesicle levels fell rapidly and were significantly decreased by day two post-injury. Total leukocyte- and neutrophil-derived microvesicles were significantly higher in non-survivors of burn injury as compared to survivors. In vitro studies demonstrated that neutrophil microvesicle release could be elicited by incubation with opsonised heat-killed cells. Pilot analysis of burn patient samples, using an endothelial co-culture assay, suggested that microvesicles may regulate the innate immune response to burn injury. These findings indicated that circulating microvesicles are an important component of the post-burn inflammatory response. Their precise activity is likely to be subtype-specific but the association of neutrophil-derived microvesicles with patient outcome alludes to a key role in burn pathophysiology.

In recent decades, improvised explosive devices have been one of the main causes of injuries due to blast effects to military personnel as well as civilians. Such injuries are very complex, with multiple types of injuries happening at once. To understand the nature of such injuries, it is important to be able to re-create the blast waves, isolating their different time-dependent effects (e.g. initial accelerations by shock waves, ballistic impacts, etc.). The shock tube is a versatile apparatus that can generate these elements of blasts in laboratory environment. The project aims to deepen the current understanding about the shock tube by characterising it over a range of conditions such as diaphragm breakage, and to measure the evolution of the pressure generated. Then, based on these characterisations, additional adjustments and adaptors are introduced to adapt the performance of the shock tube to specific purposes, especially blast injury and mitigation studies. Experiments were per- formed on an air-driven shock tube system with Mylar and aluminium diaphragms of various thicknesses, and with different lengths of driver section. Single-diaphragm and double-diaphragm configurations were employed, as were open or closed tube configurations. The arrangement was designed to enable high-speed photography and pressure measurements. Overall, the results from the shock tube are highly reproducible, and show that diaphragm burst pressure is the most influential factor on the output pressure pulses. The diaphragm burst pressure is shown to be linearly related to its thickness in the range studied. Com- paring single and double diaphragm systems, both produce similar effects but the latter provides more control over the generation of blast waves. The output blasts were also characterised against different locations, orientations and sizes of sample mountings. The thesis also reports studies on interactions between produced blast waves and various structures, for both biological sample mounting and blast mitigation purpose. It shows that the shock tube system can allow studies of blast effects on biological samples (e.g. osteoblast and Schwann cell cultures), and blast mitigating properties of different materials and structures (e.g. perforated sheets, reticulated foams). Finally, a computational fluid dynamic simulation for the blast generation in the shock tube has been developed, which gives reasonable agreements with experimental data. Improved versions of the simulation will be coupled with structural program to model interactions between blast waves and different geometries and materials.

C-reactive protein (CRP) is a homopentameric acute-phase inflammatory protein that exhibits elevated expression during inflammation. CRP can be found in its pentameric form (nCRP) or monomeric form (mCRP). To date there has been little research investigating the effects of these CRP isoforms in wound healing processes and age-related impaired healing, which is known to have an altered inflammatory response. The study investigated CRP isoform localisation in murine wounds (detected by immunohistochemistry) and circulating blood (measured by immunoblotting) using models of acute healing in young adults and age-related impaired healing (n=3). Using in vitro inflammatory cell assays developed with the monocytic U937 cell line this study investigated the effects of CRP isoforms in nitric oxide release (detected by an immunoassay), phagocytosis (measured by a bacterial recovery assay), apoptosis (detected via DNA fragmentation) and cytokine secretion (measured by a multiplex immunoassay) in both monocyte and macrophage-like cells (n=6). Pharmacological inhibitors were used to determine the potential CRP-induced pathways or mechanisms. Both isoforms were localised to sites of inflammation, implying that they may play an active inflammatory role during wound repair. Moreover, the ratio of CRP isoforms (mCRP:nCRP) reflected the degree of inflammation, with higher values indicating elevated mCRP localisation and pronounced inflammation in the model of age-related impaired healing. In in vitro inflammatory cell assays, nCRP exhibited a more anti-inflammatory effect leading to significantly (P=0.01) increased phagocytosis, increased apoptosis, decreased nitric oxide (NO) production, and reduced overall pro-inflammatory cytokine production in monocytes and macrophages. The mCRP isoform significantly (P < 0.001) increased NO production and overall levels of pro-inflammatory cytokines, thus confirming its pro-inflammatory nature indicated in the literature. This research highlighted that estrogen mediated CRP-induced responses in a cell type- and status-specific manner, being pro-inflammatory in circulating cells (monocytes) but anti-inflammatory in wound tissue cells (mature activated macrophages). The profile of CRP isoforms shifted in age-related (estrogen-deprived) impaired healing to relatively higher levels of mCRP compared to nCRP, suggesting impaired healing in the elderly may result from the dissociation of nCRP to mCRP following the decline in estrogen with increasing age. In conclusion, this study has provided novel data showing that both isoforms of CRP may differentially mediate inflammatory responses during wound healing and may play key roles in age-related impaired healing and may ultimately provide potential therapeutic strategies for the treatment of chronic wounds in the elderly.

No description available.

Cutaneous wound repair occurs as a continuous process in both space and time; however, studies of healing mechanisms and outcomes frequently generate spatially and temporally sparse datasets. We propose a range of techniques that allow the size, cellular processes and scar tissue properties of wounds to be measured and predicted at high spatial and temporal resolution. A non-invasive wound imaging system is shown to provide reliable measurements of wound diameter, perimeter and surface area, but is less reliable in producing 3D metrics such as volume and depth. Wound size and time post healing have a combined effect on reliability, with more reliable measurements obtained at earlier timepoints. A semi-automated pipeline is found to be appropriate for determining the cellular composition of the wound space, but cannot be applied to areas of healthy epidermis due to the close packing of keratinocytes. A range of mathematical models are employed to predict cell numbers within the wound space. An extended domain, partial differential equation model with spatial control of cell proliferation and migration is found to best recapitulate the cellular dynamics observed in vivo. However, if epidermal stratification is to be incorporated, an agent-based description may be preferable. Finally, we formulate a model system that can predict the alignment of collagen fibres and fibroblasts over continuous orientation space. Parameter sets that include large shear forces (which may result from elongated wound geometries or interventions such as suturing) can produce skewed distributions of orientation that cannot be established using discontinuous approaches. Together, this suite of computational approaches provides a powerful set of tools with which the mechanisms of cutaneous wound healing can be investigated, quantified and elucidated.

In mammals, repair of bone fractures is believed to be achieved by local activation and differentiation of osteogenic progenitor cells. However, lineage-tracing studies after fin amputation have demonstrated that bone regenerates from mature osteoblasts in the zebrafish fin through a process of partial dedifferentiation, migration and proliferation. Thus, this raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, or a process found more generally to repair bone in this animal. These studies used tail amputation which is a more severe form of damage than a bone fracture. Nevertheless, Sousa and colleagues (2012) proposed a novel crush assay for adult bones in zebrafish fin rays which showed promising results and was also developed for this project. The first objective of the thesis was to go beyond to find differences in zebrafish compared to what is already known in the mammalian fracture model during the different healing stages (inflammation, repair, remodeling). Interestingly, my analysis showed no remarkable differences at the cellular or molecular level in comparison to mammalian fracture repair. Notably, reactive oxygen species (ROS) production, which are one of the first signals to be induced after damage, depend on the lesion type in adult zebrafish. IL1β cytokine is induced early after bone damage and neutrophils are recruited at the fracture site after few hours as well. Both seem to induce directly or indirectly osteoclast recruitment. Osteoclasts participate early but also remained active after several days. Moreover, to determine whether osteoblasts dedifferentiation is restricted to appendage regeneration, efforts were done to establish the Cre-Lox system in adult zebrafish. As a first step, a set of six double transgenic lines were created to enable tracking of bone cells in vivo. Afterwards, different tamoxifen-induced Cre-recombination strategies were implemented but no successful results were obtained. Further research is needed to get positive results. The knowledge generated in this research can contribute to set the basis for the development of further studies of bone repair in zebrafish that can complement the ones performed in classical models.

No description available.