Within the five experimental chapters of this doctoral thesis (i) the eubacterial diversity of the microbiota of chronic wounds and healthy skin was investigated, (ii) biofilm formation and associated coaggregation interactions of wound and skin-associated bacterial isolates was examined, (iii) formulation of media which reproduced some aspects of the nutritional conditions of wounds and healthy skin were developed, (iv) novel wound biofilm models were developed and validated and (v) microbial population interactions associated with healthy skin and chronic wounds were investigated using a novel model system. (i) The microbial diversity of chronic wounds and contralateral skin swabs was investigated using culture, denaturing gradient gel electrophoresis (DGGE) and microscopy. Intrapersonal analysis identified that non-infected wounds had a proportionally higher incidence of bacteria which were identified on contralateral healthy skin according to DGGE analysis when compared to infected wounds indicating that taxonomically distinct consortia are associated with infection. Microcolonies and putative biofilms structures were identified in both culture-defined infected and non-infected wounds indicating that the presence of biofilms may not be linked to infection. (ii) By assessing pair-wise combinations of skin and wound-associated bacteria, the role of coaggregation in the formation of wound polymicrobial communities was assessed using a quantitative spectrophotometric assay. Aggregation interactions were weak or not detectable, apart from those associated with Corynebacterium xerosis. This bacterium produced a high autoaggregation score (c. 50%). The limited coaggregation interactions suggest that coaggregation may be comparatively unimportant in the development of wound biofilms. (iii) In order to facilitate the development of biofilm models specific to chronic wounds, the formulation of representative growth media is important in order to reproduce the in situ nutrient environment. Therefore complex, artificial sweat and serum media broadly reflective of the nutrient availability in wounds and healthy skin were developed and validated based upon their ability to support realistic phenotypes (assessed by proteomics) and the growth of a range bacterial isolates. Developed media maintained the sessile growth the test bacteria and produced broadly similar proteomic profiles to foetal calf serum. (iv) Two novel model systems were developed to study cross-sectional population interactions and to investigate longitudinal population development of wound consortia and biofilm formation. A fine celled foam (FCF) multi-well wound model and a multiple membrane FCF model maintained dynamic steady state of axenic and mixed populations of bacteria associated with chronic wounds and supported the development of biofilms. (v) The FCF multi-well wound model was used to investigate population interactions in environments broadly reflective of healthy skin and wounds. When grown in artificial sweat prior colonisation with Staphylococcus saprophyticus resulted in a significant reductions in methicillin resistant Staphylococcus aureus (99%) and P. aeruginosa (75%) whilst prior colonisation by C. xerosis resulted in a significant reduction in P. aeruginosa (91%) only. However no significant reductions in pathogenic bacteria were noted in artificial serum indicating colonisation resistance could be simulated in the model and the outcome of immigration was markedly influenced by the species of established bacterium and nutrient availability.

Yes / Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation.

Abstract The expression intensities of cytokeratins and tight junction proteins were determined on re-epithelization. Experimental blister wound healing was studied in patients with diabetes mellitus and in patients with obstructive jaundice. Suction blisters were induced on healthy volunteers, and the healing blisters were biopsied at different time points. Cytokeratin expression and the tight junction proteins ZO-1 and occludin were studied immunohistochemically. Blisters were induced on 17 patients with diabetes and 11 control subjects, and the healing process was followed indirectly by measuring water evaporation and blood flow in the wounds. Microvascular reactivity in the diabetic patients was also studied by using non-immunologic contact irritants. Wound healing, skin collagen synthesis and serum levels of procollagen propeptides were studied in 24 patients with obstructive jaundice caused by neoplastic pancreaticobiliary obstruction and in 17 control patients with the corresponding condition without jaundice. Cytokeratin expression was altered in healing epidermis. In the suprabasal layer, K10 was replaced by K14 and, most likely, by K16. K18 keratin, which is not present in normal epidermis, was found in the basal and suprabasal layers. Thus, there was a shift towards lower molecular weight cytokeratins, which is a reflection of immaturity, and probably towards motility. The tight junction proteins ZO-1 and occludin were expressed in the migrating epidermal sheet, where they apparently form an early barrier. Enhanced expression was seen in the hyperproliferative zone of the wound edge. The diabetic patients showed slower restoration of the epidermal barrier and a weaker initial inflammatory response. Obstructive jaundice and its resolution had no effect on healing. Skin collagen synthesis was decreased in jaundiced patients, and it increased slightly after drainage. Serum type III collagen propeptide levels were elevated in patients with biliary obstruction and dropped after drainage. The elevated levels may be related to the increased synthesis due to fibrosis. As a conclusion, diabetes mellitus impairs epidermal wound healing, while obstructive jaundice does not.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os receptores β1- e β2-adrenérgicos estão presentes em inúmeras células que participam do processo de reparo como fibroblastos, queratinócitos, células inflamatórias e células endoteliais. Diversos trabalhos demonstram que estes receptores modulam o processo de reparo tecidual. Entretanto, nenhum trabalho demonstrou se o bloqueio destes receptores compromete o reparo de úlceras de pressão. O objetivo deste estudo foi avaliar o efeito do bloqueio dos receptores β1- e β2-adrenérgicos no reparo de úlceras de pressão em camundongos, para isto utilizamos um modelo não invasivo de lesão por isquemia e reperfusão. No presente estudo, utilizamos animais que foram tratados por gavagem com propranolol (um antagonista não seletivo dos receptores β1- e β2-adrenérgicos). A administração do antagonista teve início um dia antes do início dos ciclos de isquemia e reperfusão e se manteve diariamente até a eutanásia. Para desenvolver a úlcera de pressão, um par de magnetos foi aplicado no dorso dos animais previamente depilado. O período de permanência do magneto é caracterizado como período de isquemia, enquanto sua retirada é caracterizada como período de reperfusão. Os ciclos de isquemia e reperfusão foram repetidos duas vezes, e ao final do último ciclo, duas úlceras circulares foram criadas no dorso dos animais. Os animais foram mortos 3, 7, 14 e 21 dias após a lesão. Após o último ciclo de isquemia, o fluxo sanguíneo da área comprimida foi nulo, 7 horas após a compressão o fluxo sanguíneo estava elevado, com níveis superiores ao da pele normal. Após 24 e 48 horas, o fluxo sanguíneo estava reduzido e abaixo dos níveis da pele normal. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou os níveis de peróxidos lipídicos 3 dias após a lesão, comprometeu a migração dos queratinócitos, levando a um aumento da proliferação epitelial, resultando em uma re-epitelização atrasada. O retardo na formação da neo-epiderme induzido pelo bloqueio destes receptores prejudicou a remoção do tecido necrótico. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou o número de células inflamatórias (neutrófilos e macrófagos), os níveis proteicos de elastase neutrofílica 3 dias após a lesão e reduziu os níveis proteicos de MCP-1 3 dias após a lesão e os níveis proteicos de MMP-12 7 dias após a lesão. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou a proliferação celular e apoptose no tecido conjuntivo 7 dias após a lesão e aumentou a densidade de vasos sanguíneos 14 e 21 dias após a lesão. O bloqueio dos receptores β1- e β2-adrenérgicos retardou a diferenciação miofibroblástica e reduziu os níveis proteicos de TGF-β 1/2/3 3 dias após a lesão e a contração da lesão. Vinte e um dias após a lesão, o bloqueio dos receptores β1- e β2-adrenérgicos aumentou a espessura da neo-epiderme e a expressão de tenascina-C em fibroblastos e reduziu a deposição de colágeno. Em conclusão, o bloqueio dos receptores β1- e β2-adrenérgicos atrasa o reparo tecidual em úlceras de pressão. / β1- and β2-adrenergic receptors are present in several cells that participate in tissue repair such as fibroblasts, keratinocytes, inflammatory cells and endothelial cells. Several studies demonstrate that these receptors modulate cutaneous wound healing. However, neither study demonstrated if the blockade of theses receptors compromises the cutaneous wound healing of pressure ulcers. Thus, the aim of this study was to evaluate the effect of blockade of β1- and β2-adrenergic receptors in wound healing of pressure ulcer in mice using a noninvasive model of lesion induced by ischemia and reperfusion. In this study, animals were also treated with propranolol (a nonselective antagonist of β1- and β2-adrenergic receptors). Antagonism administration began one day before ischemia-reperfusion cycles and daily maintained until euthanasia. In order to develop a pressure ulcer, a shaved skin was placed between a pair of magnet disks. The period of magnet placement is characterized as ischemia period, whereas release period as reperfusion period. Two cycles of ischemia and reperfusion were performed, and after last cycle, two circular ulcers per animal were created in the animal dorsum. Animals were killed 3, 7, 14 and 21 days after wounding. After last ischemia cycle, blood flow of compressed area was nulled, 7 hours after compressed area underwent reperfusion during subsequent hours reaching blood flow 98% superior of normal skin. After 24 and 48 hours, blood flow of compressed area was again reduced when compared with normal skin. Blockade of β1- and β2-adrenergic receptors increased the levels of lipid peroxydes 3 days after wounding, compromised keratinocyte migration leading to an increase epithelial proliferation and impairment in the re-epithelialization. The impairment in the neo-epidermis formation induced by β1- and β2-adrenergic receptor blockade delayed the necrotic tissue loss. Blockade of β1- and β2-adrenergic receptors increased the inflammatory cell number (macrophages and neutrophils), protein levels of neutrophil elastase 3 days after wounding and reduced protein levels of MCP-1 3 days after wounding and protein levels of MMP-12 7 days after wounding. Blockade of β1- and β2-adrenergic receptors increased cell proliferation and apoptosis in connective tissue 7 days after wounding and blood vessel density 14 and 21 days after wounding. Blockade of β1- and β2-adrenergic receptors impaired myofibroblastic differentiation and reduced protein levels of TGF-β 1/2/3 3 days after wounding and lesion contraction. Twenty-one days after wounding, β1- and β2-adrenergic receptor blockade increased neo-epidermis thickness and tenascin-C-positive fibroblast number and reduced collagen deposition. In conclusion, blockade of β1- and β2-adrenergic receptors delays cutaneous wound healing of pressure ulcers.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os receptores β1- e β2-adrenérgicos estão presentes em inúmeras células que participam do processo de reparo como fibroblastos, queratinócitos, células inflamatórias e células endoteliais. Diversos trabalhos demonstram que estes receptores modulam o processo de reparo tecidual. Entretanto, nenhum trabalho demonstrou se o bloqueio destes receptores compromete o reparo de úlceras de pressão. O objetivo deste estudo foi avaliar o efeito do bloqueio dos receptores β1- e β2-adrenérgicos no reparo de úlceras de pressão em camundongos, para isto utilizamos um modelo não invasivo de lesão por isquemia e reperfusão. No presente estudo, utilizamos animais que foram tratados por gavagem com propranolol (um antagonista não seletivo dos receptores β1- e β2-adrenérgicos). A administração do antagonista teve início um dia antes do início dos ciclos de isquemia e reperfusão e se manteve diariamente até a eutanásia. Para desenvolver a úlcera de pressão, um par de magnetos foi aplicado no dorso dos animais previamente depilado. O período de permanência do magneto é caracterizado como período de isquemia, enquanto sua retirada é caracterizada como período de reperfusão. Os ciclos de isquemia e reperfusão foram repetidos duas vezes, e ao final do último ciclo, duas úlceras circulares foram criadas no dorso dos animais. Os animais foram mortos 3, 7, 14 e 21 dias após a lesão. Após o último ciclo de isquemia, o fluxo sanguíneo da área comprimida foi nulo, 7 horas após a compressão o fluxo sanguíneo estava elevado, com níveis superiores ao da pele normal. Após 24 e 48 horas, o fluxo sanguíneo estava reduzido e abaixo dos níveis da pele normal. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou os níveis de peróxidos lipídicos 3 dias após a lesão, comprometeu a migração dos queratinócitos, levando a um aumento da proliferação epitelial, resultando em uma re-epitelização atrasada. O retardo na formação da neo-epiderme induzido pelo bloqueio destes receptores prejudicou a remoção do tecido necrótico. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou o número de células inflamatórias (neutrófilos e macrófagos), os níveis proteicos de elastase neutrofílica 3 dias após a lesão e reduziu os níveis proteicos de MCP-1 3 dias após a lesão e os níveis proteicos de MMP-12 7 dias após a lesão. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou a proliferação celular e apoptose no tecido conjuntivo 7 dias após a lesão e aumentou a densidade de vasos sanguíneos 14 e 21 dias após a lesão. O bloqueio dos receptores β1- e β2-adrenérgicos retardou a diferenciação miofibroblástica e reduziu os níveis proteicos de TGF-β 1/2/3 3 dias após a lesão e a contração da lesão. Vinte e um dias após a lesão, o bloqueio dos receptores β1- e β2-adrenérgicos aumentou a espessura da neo-epiderme e a expressão de tenascina-C em fibroblastos e reduziu a deposição de colágeno. Em conclusão, o bloqueio dos receptores β1- e β2-adrenérgicos atrasa o reparo tecidual em úlceras de pressão. / β1- and β2-adrenergic receptors are present in several cells that participate in tissue repair such as fibroblasts, keratinocytes, inflammatory cells and endothelial cells. Several studies demonstrate that these receptors modulate cutaneous wound healing. However, neither study demonstrated if the blockade of theses receptors compromises the cutaneous wound healing of pressure ulcers. Thus, the aim of this study was to evaluate the effect of blockade of β1- and β2-adrenergic receptors in wound healing of pressure ulcer in mice using a noninvasive model of lesion induced by ischemia and reperfusion. In this study, animals were also treated with propranolol (a nonselective antagonist of β1- and β2-adrenergic receptors). Antagonism administration began one day before ischemia-reperfusion cycles and daily maintained until euthanasia. In order to develop a pressure ulcer, a shaved skin was placed between a pair of magnet disks. The period of magnet placement is characterized as ischemia period, whereas release period as reperfusion period. Two cycles of ischemia and reperfusion were performed, and after last cycle, two circular ulcers per animal were created in the animal dorsum. Animals were killed 3, 7, 14 and 21 days after wounding. After last ischemia cycle, blood flow of compressed area was nulled, 7 hours after compressed area underwent reperfusion during subsequent hours reaching blood flow 98% superior of normal skin. After 24 and 48 hours, blood flow of compressed area was again reduced when compared with normal skin. Blockade of β1- and β2-adrenergic receptors increased the levels of lipid peroxydes 3 days after wounding, compromised keratinocyte migration leading to an increase epithelial proliferation and impairment in the re-epithelialization. The impairment in the neo-epidermis formation induced by β1- and β2-adrenergic receptor blockade delayed the necrotic tissue loss. Blockade of β1- and β2-adrenergic receptors increased the inflammatory cell number (macrophages and neutrophils), protein levels of neutrophil elastase 3 days after wounding and reduced protein levels of MCP-1 3 days after wounding and protein levels of MMP-12 7 days after wounding. Blockade of β1- and β2-adrenergic receptors increased cell proliferation and apoptosis in connective tissue 7 days after wounding and blood vessel density 14 and 21 days after wounding. Blockade of β1- and β2-adrenergic receptors impaired myofibroblastic differentiation and reduced protein levels of TGF-β 1/2/3 3 days after wounding and lesion contraction. Twenty-one days after wounding, β1- and β2-adrenergic receptor blockade increased neo-epidermis thickness and tenascin-C-positive fibroblast number and reduced collagen deposition. In conclusion, blockade of β1- and β2-adrenergic receptors delays cutaneous wound healing of pressure ulcers.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctorate of Philosophy. / Senecio serratuloides is widely used for wound healing in South Africa but minimal information regarding its efficacy is available. Furthermore toxic pyrrolizidine alkaloids may be present. The following investigation sought firstly to evaluate the efficacy and safety of Senecio serratuloides in a porcine wound model; secondly to assess for a potential mechanism and finally isolate and identify fractions in in-vitro assays. Assessment of Efficacy and Safety Materials and Methods: Deep partial thickness and full thickness wounds were created on 9 pigs. Treatment included an occlusive dressing (negative control), activated carbon, or the Senecio preparation. Wounds were monitored using photographic documentation, pH measurement and histological analysis (skin thickness and collagen content). Toxicity was monitored on blood and liver samples. Results and Discussion: Efficacy of Senecio serratuloides was established with a significantly thicker epidermis, maximal at day 7 post-operative, 2 days before the controls. Effects on collagen content was negligible with no toxicity detected. Mechanistic investigation Materials and Methods: Wound fluid was analysed for IL-10, IL-12, IL-1β, IL-6, IL-8, TNF-α using flow cytometry based assays. Tyrosine phosphorylation and cellular proliferation was assessed using dual immunofluorescence staining. Results and Discussion: IL-1β levels were significantly greater in the Senecio treatment. Tyrosine phosphorylation increased to day 9 post-operative where it stabilised in all groups. In the same period, cellular proliferation was sustained in the Senecio treated wounds but not in the controls. Keratinocyte proliferation was identified as the target for in-vitro assays. Extraction, Isolation and Partial Identification using In-vitro Proliferation Assays. Materials and Methods: The plant was fractionated using solid phase extraction cartridges. Keratinocytes were grown under standard conditions in 96-well plates. Cellular proliferation was assessed spectrophotometrically using a resazurin dye technique. Active fractions were analysed using gas chromatography and mass spectrometry. Results and Discussion: Identified fractions increased the rate of proliferation by 300- 400%. Potential lead compounds were identified. Importantly, pyrrolizidine alkaloids could not be detected. Conclusion Senecio serratuloides is efficacious in treating deep partial thickness wounds without inducing liver toxicity. Sustained keratinocyte proliferation linked to tyrosine phosphorylation may be an underlying mechanism. Although successful, in-vitro detection of active fractions requires further characterisation.

<p>Future long-duration space missions will take humans farther from the support resources of Earth than ever before. These missions will require microgravity surgical technologies in the case of an emergency that necessitates medical intervention. This experiment integrated three different surgical technologies for testing in weightlessness on parabolic flights: a surgical containment dome, a multi-function surgical wand, and a microgravity blood-air separator. Two fluid loops were utilized: one in which the surgical wand, containment dome, and a wound model were used to provide a realistic mixture of blood simulant and air to the blood-air separator. The other fluid loop used prescribed mixture ratios of air and blood to test the performance of the separator under varying conditions. The results of this experiment showed that the multi-functional surgical tool and dome functioned as designed. In addition, each separator successfully separated the blood and air from the mixture, allowing for future blood transfusion. With this demonstration, each system used in this experiment qualifies as technology readiness level 6. Advancing the technology readiness level of these technologies further will require long duration zero-g testing on-orbit before inclusion in authentic space mission emergency surgical strategy. </p>