Thesis (Ph.D.), Faculty of Health Sciences, University of the Witwatersrand, 2009 / In recent years, there has been a growing interest in natural and traditional medicines for the treatment of wounds. Attempts to find agents that promote wound-healing and that are affordable, effective and non-toxic have a long history. In South Africa, hundreds of different indigenous plants are used to treat wounds and burns. The merits of relatively few of these have been scientifically evaluated. Bulbine natalensis and Bulbine frutescens of the Asphodelaceae family are indigenous to southern Africa and are widely used as a skin remedy. This study aimed to investigate the in vitro and in vivo effect of Bulbine natalensis and Bulbine frutescens on cutaneous wound healing. In vitro cell culture study: In vitro studies were carried out on dermal fibroblasts and human keratinocytes cultured under standard conditions using Iscove’s Modified Eagles Medium (MEM) and Dulbecco’s MEM respectively. Confluent cultures of both cell lines were treated with varying concentrations of the leaf extracts of B. frutescens and B. natalensis. These cultures were subjected to the MTT, WST-1 and BrdU assays to determine the cytotoxicity and proliferation effect of the extracts. In addition, migration of cells across a score was analysed over a 48 hour period. In vivo animal study: Excisional and incisional wounds were created on the back of 12 domestic pigs. Mirror imaged wounds were created as control wounds. The excisional wounds were biopsied at days 2, 4, 7, 10 and 16 and the incisional wounds were biopsied at day 16. The rate of closure of the wounds was also recorded. Each excisional wound was analyzed for its biochemical composition by estimating the total amount of protein, DNA, collagen and hexosamine that was present in the wound tissue. The wound healing process was documented histologically (using haematoxylin and eosin and a Mallory’s trichrome stain) and immunohistochemically (using anti- α smooth muscle actin, vascular endothelial growth factor WitsETD and transforming growth factor β receptors I and II). The incisional wounds were used to test tensile strength of the healed wounds using a tensiometer.In the in vitro studies, neither extract caused cytotoxicity to either the fibroblast or keratinocyte cells. Cell proliferation was greater than 100% at 0.1-5 and 100-300 μg/ml for Bulbine natalensis and at 0.1μg/ml for Bulbine frutescens. There was no significant difference in the effects of the two leaf extracts on cell proliferation. The biochemical analysis of the wound tissue showed a significant increase in the collagen, protein and total DNA content of both B frutescens and natalensis treated wounds when compared to the untreated wounds. There was no significant difference in the hexosamine content of both B. frutescens- and B. natalensis-treated and untreated wounds. Analysis of the wound tissue displayed an increase rate of closure of the wound tissue treated with B. frutescens and B. natalensis when compared to the untreated wounds. Full re-epithelialisation of both treated wounds occurred earlier than in the untreated wounds. These findings have important implications for the use of these extracts to treat wound healing.

No description available.

"May 2004" / Bibliography: leaves 172-184. / xvi, 186 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D.Nurs.)--University of Adelaide, Dept. of Clinical Nursing, 2004

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The mechanisms responsible for the healing of corneal surface wounds are the subject of biological controversy. In particular, the role and source of the regulatory chemical epidermal growth factor (EGF) is an area of intense debate. In the first part of this thesis, we propose a reaction-diffusion model which focuses on the stimulus for increased mitotic and migratory activity due to secretion of EGF. A detailed numerical study of various possible models, with parameter values based on biological data, reveals that, for realistic healing times, EGF must be released by the underlying layers of the cornea, in addition to the tear film source. The model exhibits travelling wave solutions and further analysis elucidates the interaction and role of the parameters in determining the speed of healing. Furthermore, we consider the effect of topical application of EGF and investigate the effect of curvature of the eye. We show that our model is consistent with many of the key features of corneal wound healing. Adult dermal wounds, in contrast to foetal wounds, heal with the formation of scar tissue. A crucial factor in determining the nature of the healed tissue is the ratio of collagen 1 to collagen 3, which indicates the fibril diameter. We develop a reaction-diffusion model which focuses on the stimulus for collagen synthesis due to the secretion of the different isoforms of the regulatory chemical transforming growth factor β (TGFβ). Numerical simulations of the model without diffusion lead to a value of this ratio consistent with that of healthy tissue for the foetus but corresponding to scarring in the adult. The model equations evolve to waves moving into the wound, but addition of TGFβ only has a transient effect on the final collagen levels. We investigate this effect by developing a caricature model. The model indicates that the main source of the fibroblasts is the underlying subcutaneous tissue and we determine key parameters which explain the difference between adult and foetal wound healing. Furthermore we make clinically testable predictions on the effects that topical application of various chemicals will have on scar formation.

A pele tem como principal função manter o isolamento do meio interior com o exterior. Quando esse órgão sofre uma lesão o organismo tenta reparar essa agressão ao promover a substituição do tecido por meio de um processo complexo dividido em fases. O ácido hialurônico está presente em todas as fases dessa reparação tecidual. Contudo a sua presença em adultos reduz cerca de 50% comparado ao recém-nascido, o que reduz a regeneração tecidual drasticamente. Este trabalho teve como objetivo analisar o efeito de uma pomada utilizando veículo comercial composto de lanolina e vaselina manipulada com ácido hialurônico, extraído da crista do frango, no processo cicatricial de feridas cutâneas em dorso de ratos. Para atingir o objetivo desse trabalho o ácido hialurônico foi extraído e purificado da crista do frango e foi acrescido ao veículo comercial composto de 10% lanolina e 90% vaselina. A pomada obtida foi analisada por meio de um reômetro de tensão controlada. Para os estudos in vivo, foram separados aleatoriamente 28 animais em 4 grupos. Em cada animal do grupo efetuou-se duas lesões em seu dorso, uma para a aplicação da pomada, com ou sem ácido, e a outra lesão para controle. A pomada foi aplicada diariamente durante 7 e 14 dias conforme protocolo estabelecido. Os resultados mostraram que as propriedades viscoelásticas da pomada, quando acrescentou o ácido hialurônico, tiveram pequenas alterações, porém não de forma significativa, o que possibilitou sua utilização. Como resultados dos testes in vivo, o grupo tratado com a pomada com ácido hialurônico durante 7 dias, obteve menor média da área da lesão com 0,07 cm² contra 0,11 cm² do seu controle, 0,09 cm² do grupo tratado com a pomada placebo e 0,10 cm² do controle do placebo. Ainda, os grupos tratados com a pomada com AH durante 7 e 14 dias obtiveram uma maior média na porcentagem de colágeno, e o grupo tratado com a pomada com AH por 14 dias, resultou em uma melhor cicatrização da lesão. / The skin has as main function to maintain the insulation of the inner tissues of the body from the external environment. When this organ is injured, the organism attempts to repair the damage with a tissue replacement, in a complex process divided into several stages. Hyaluronic acid is present in all phases of this process, although its expression in adults is reduced to approximately 50% compared to newborn individuals, leading to drastically reduced tissue regeneration. This study aimed to analyze the effect of an ointment composed of a commercial vehicle of lanolin and vaseline, manipulated with chicken crest-extracted hyaluronic acid, in skin wounds healing in rats. Hyaluronic acid was extracted and purified from chicken crests, and the extract was added to the commercial vehicle composed of 10% lanolin and 90% vaseline. The obtained ointment was analyzed using a controlled stress rheometer. For in vivo studies, 28 rats were randomly separated into four groups. Each animal was injured twice in the back, one lesion destined to the ointment application, and the other lesion as a control injury. The ointment was applied for 7 to 14 days according to the protocol established. Results showed that the viscoelastic properties of the ointment mixed with hyaluronic acid changed slightly, but not significantly, allowing its use in vivo. The group of animals treated with the ointment for seven days showed lower average lesion area, with 0.07 cm² against the control area of 0.11 cm². The animals treated with a placebo ointment showed an average lesion area of 0.09 cm² against a 0.10 cm² area of controls. The animals treated with the ointment with hyaluronic acid for 7 and 14 days had a higher average percentage of collagen, and the group treated for 14 days exhibited an improved wound healing.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In cats, cutaneous healing presents qualitative and quantitative differences in comparison with another species, that seems predispose them to healing problems. Wounds that heal by first intention present less resistance, rate of contraction and epithelization. In the second intention healing, the inflammatory phase is bland and the proliferative phase has slow appearing and progression. Considering these aspects, it becomes important to search ways to promote and conduce cutaneous wound healing in cats. The aims of this study were standardize a technique to obtain autologous platelet-rich plasma (PRP) in cats and evaluate your effect on cutaneous healing of experimentally produced wounds in this species. The experiment was developed in two stages: three manual protocols to produce autologous platelet-rich plasma in cats were compared in the first stage. Fifteen adult healthy indoor cats, randomly divided into three groups, were utilized. Five samples of 4.5 ml of jugular blood were collect, with a vacuum system, sequentially in citrated tubes from each cat. The samples were processed using double centrifugation protocols (P): P1 (113g 10 minutes; 652g 5 minutes); P2 (400g 10 minutes; 800g 10 minutes); P3 (400g 10 minutes; 600g 10 minutes). Platelet concentration obtained in PRP obtained in different protocols and the ratio of PRP and platelet counts were compared by t-test. The feasibility of the collect method to the species and ease of execution of techniques was evaluated too. In the second stage, ten healthy adult indoor cats were used. In each animal were surgically produced four circular skin defects, with a diameter of 1 cm, on left and right sides of trunk dorsal midline. The lesions on one side were treated with fluid PRP, obtained of animal itself, according to the best protocol defined in the first stage of the experiment. The control defects in the opposite side were filled with NaCl 0,9% sterile solution. Three observers evaluated the wounds, every three days, for 28 days. The presence of crusts, pain, swelling, exudate, granulation tissue and the color were considered. The results were statistically evaluated by Mann-Whitney rank test. The area of each lesion was analyzed by morphometry every seven days and the data compared by t-test. Skin fragments with normal and scar tissue, from two more cranial lesions (treated and control), were obtained weekly. The fragments were stained with hematoxylin-eosin (HE) and Masson's trichrome, and qualitatively evaluated for the presence of crust, ulcer, acanthosis, fibroplasia, collagen deposition and neovascularization. These data were analyzed using the Mann-Whitney rank test. The collect method was satisfactory and simplified the procedure. Two techniques (P2 and P3) were effective to produce PRP, yielding concentrations higher than 1x106 platelets.μL-1. The P3 protocol was statistically higher in capacity increase, and was selected for use in experimental wounds. In the second stage, the techniques for blood collection, and the selected protocol, were satisfactory. The use of PRP in liquid form, deposited directly on the lesion, made it easy to use. Clinically, there was a statistically significant difference (p = 0.025) and higher exudation in wounds treated to three days. In morphometric analysis we noticed a greater contraction in wounds treated with PRP, especially of the first seven days (p = <0.001). Histopathological evaluation was not significant in any compared parameters. Therefore, it is concluded that the sampling method is efficient and P2 and P3 protocols can be considered feasible to obtain PRP from cats. The protocol P3 was superior in the platelet concentration ability, producing a better quality PRP. Activation of PRP liquid by tissue injury is possible. The increased exudation suggests enhancement of the inflammatory process in treated wounds. Therapeutic use of liquid PRP improves contraction capacity on experimental wounds in cats, despite the absence of histological evidence by way the evaluation adopted. / A cicatrização cutânea em gatos possui diferenças qualitativas e quantitativas quando comparada à de outras espécies, fato que parece predispor a problemas. Feridas que cicatrizam por primeira intenção apresentam menor resistência, taxa de contração e epitelização. Já na cicatrização por segunda intenção, a fase inflamatória é branda e a fase proliferativa apresenta surgimento e progressão lenta. Tendo em vista esses aspectos, torna-se importante a busca de meios que ajudem a favorecer e conduzir o reparo de feridas cutâneas em gatos. Este estudo objetivou a padronização de uma técnica para obtenção de plasma rico em plaquetas (PRP) autólogo em gatos e a avaliação de sua ação sobre a cicatrização de feridas cutâneas produzidas experimentalmente nessa espécie. Ele desenvolveu-se em duas etapas distintas: na primeira foram comparados três protocolos manuais para produção de PRP em gatos. Utilizaram-se 15 animais adultos hígidos e domiciliados, divididos aleatoriamente em três grupos. Cinco amostras de 4,5ml de sangue foram colhidas da jugular de cada animal, por sistema a vácuo, sequencialmente, em tubos com citrato de sódio. As amostras de cada grupo foram processadas por meio de protocolos de dupla centrifugação: P1 (113g 10 minutos; 652g 5 minutos); P2 (400g 10 minutos; 800g 10 minutos); P3 (400g 10 minutos; 600g 10 minutos). As concentrações plaquetárias obtidas no PRP dos diferentes protocolos e a relação entre o PRP e contagem de plaquetas foram comparadas por teste-t. A viabilidade do modo de coleta a vácuo para a espécie e a facilidade de execução das técnicas também foram avaliadas. Na segunda etapa, foram utilizados dez gatos adultos hígidos, domiciliados. Em cada animal foram produzidos, cirurgicamente, quatro defeitos circulares, pareados, com diâmetro de 1 cm, na pele, nas laterais esquerda e direita da linha média dorsal do tronco. As lesões de um lado foram tratadas com PRP líquido, obtido do próprio animal, de acordo com o melhor protocolo definido na primeira etapa do experimento. Os defeitos do lado oposto, considerados controle, foram preenchidos com solução de NaCl 0,9% estéril. As feridas foram avaliadas por três observadores, a cada três dias, por 28 dias. Considerou-se a presença de crostas, dor, edema, exsudato, tecido de granulação e a coloração. Os resultados foram avaliados estatisticamente pelo teste de Mann-Whitney em ranks. A área de cada lesão foi analisada por morfometria a cada sete dias e os dados comparados pelo teste-t. Também, semanalmente, foram obtidos fragmentos de pele contendo tecido normal e cicatricial a partir das duas lesões (tratada e controle) mais craniais. Os fragmentos foram corados por hematoxilina-eosina (HE) e tricrômico de Masson, e avaliados qualitativamente quanto a presença de crosta, úlcera, acantose, fibroplasia, colagenização e neovascularização. Esses dados foram analisados pelo teste de Mann-Whitney em ranks. O sistema de coleta de sangue a vácuo mostrou-se satisfatório e simplificou o procedimento. Duas das técnicas (P2 e P3) mostraram-se factíveis para a produção do PRP, proporcionando concentrações superiores a 1x106 plaquetas.μl-1. O protocolo P3 mostrou-se estatisticamente superior na capacidade de incremento, e foi selecionado para uso nas feridas experimentais. Na segunda etapa, a técnica para coleta de sangue e o protocolo selecionado, mostraram-se satisfatórios. A utilização do PRP na forma líquida, depositado diretamente sob a lesão, tornou fácil sua utilização. Clinicamente, houve diferença estatisticamente significativa (p=0,025) e maior exsudação nas feridas tratadas no dia três. Na análise morfométrica, notou-se maior contração nas feridas tratadas com PRP, principalmente nos primeiros sete dias (p=<0,001). Na avaliação histopatológica semi-quantitativa não houve significância em nenhum dos parâmetros comparados. Logo, conclui-se que o método de coleta é eficiente, e podem-se considerar exequíveis as técnicas para obtenção do PRP em gatos, a partir dos protocolos P2 e P3. O protocolo P3 demonstra superior eficiência na capacidade de concentrar plaquetas, gerando um PRP de melhor qualidade. É possível a ativação do PRP líquido por meio da lesão tecidual. O aumento da exsudação sugere intensificação do processo inflamatório nas feridas tratadas. O uso terapêutico do PRP na forma líquida melhora a capacidade de contração em feridas produzidas experimentalmente em gatos, apesar da ausência de evidências histopatológicas pela avaliação adotada.

A pele tem como principal função manter o isolamento do meio interior com o exterior. Quando esse órgão sofre uma lesão o organismo tenta reparar essa agressão ao promover a substituição do tecido por meio de um processo complexo dividido em fases. O ácido hialurônico está presente em todas as fases dessa reparação tecidual. Contudo a sua presença em adultos reduz cerca de 50% comparado ao recém-nascido, o que reduz a regeneração tecidual drasticamente. Este trabalho teve como objetivo analisar o efeito de uma pomada utilizando veículo comercial composto de lanolina e vaselina manipulada com ácido hialurônico, extraído da crista do frango, no processo cicatricial de feridas cutâneas em dorso de ratos. Para atingir o objetivo desse trabalho o ácido hialurônico foi extraído e purificado da crista do frango e foi acrescido ao veículo comercial composto de 10% lanolina e 90% vaselina. A pomada obtida foi analisada por meio de um reômetro de tensão controlada. Para os estudos in vivo, foram separados aleatoriamente 28 animais em 4 grupos. Em cada animal do grupo efetuou-se duas lesões em seu dorso, uma para a aplicação da pomada, com ou sem ácido, e a outra lesão para controle. A pomada foi aplicada diariamente durante 7 e 14 dias conforme protocolo estabelecido. Os resultados mostraram que as propriedades viscoelásticas da pomada, quando acrescentou o ácido hialurônico, tiveram pequenas alterações, porém não de forma significativa, o que possibilitou sua utilização. Como resultados dos testes in vivo, o grupo tratado com a pomada com ácido hialurônico durante 7 dias, obteve menor média da área da lesão com 0,07 cm² contra 0,11 cm² do seu controle, 0,09 cm² do grupo tratado com a pomada placebo e 0,10 cm² do controle do placebo. Ainda, os grupos tratados com a pomada com AH durante 7 e 14 dias obtiveram uma maior média na porcentagem de colágeno, e o grupo tratado com a pomada com AH por 14 dias, resultou em uma melhor cicatrização da lesão. / The skin has as main function to maintain the insulation of the inner tissues of the body from the external environment. When this organ is injured, the organism attempts to repair the damage with a tissue replacement, in a complex process divided into several stages. Hyaluronic acid is present in all phases of this process, although its expression in adults is reduced to approximately 50% compared to newborn individuals, leading to drastically reduced tissue regeneration. This study aimed to analyze the effect of an ointment composed of a commercial vehicle of lanolin and vaseline, manipulated with chicken crest-extracted hyaluronic acid, in skin wounds healing in rats. Hyaluronic acid was extracted and purified from chicken crests, and the extract was added to the commercial vehicle composed of 10% lanolin and 90% vaseline. The obtained ointment was analyzed using a controlled stress rheometer. For in vivo studies, 28 rats were randomly separated into four groups. Each animal was injured twice in the back, one lesion destined to the ointment application, and the other lesion as a control injury. The ointment was applied for 7 to 14 days according to the protocol established. Results showed that the viscoelastic properties of the ointment mixed with hyaluronic acid changed slightly, but not significantly, allowing its use in vivo. The group of animals treated with the ointment for seven days showed lower average lesion area, with 0.07 cm² against the control area of 0.11 cm². The animals treated with a placebo ointment showed an average lesion area of 0.09 cm² against a 0.10 cm² area of controls. The animals treated with the ointment with hyaluronic acid for 7 and 14 days had a higher average percentage of collagen, and the group treated for 14 days exhibited an improved wound healing.

The diploma thesis if focused on the study of bioactive hydrogél and nanofiber wound dressings composed of natural biopolymers, which were functionalized by active compounds in the form of analgesic, antibiotics and enzymes. Hydrogél wound dressings were constituted from alginate and chitosan and nanofibers were created from polyhydroxybutyrate. The following 7 active compounds were selected to be added to the wound dressings: ampicillin, streptomycin, ibuprofen, papain, bromelain, collagenase and trypsin. In the theoretical part the structure of the skin and types of wound injuries were described. This part also talks about types of wound dressing and their applications, as well as treatment of skin wounds using enzymes and compounds with analgesic and antimicrobial properties. In addition, this section describes safety assays, in particular cytotoxicity assays on human cells. At the beginning of the experimental part, the process of preparation of hydrogél wound dressing was optimised. Subsequently, the dressings were enriched with active compounds and the rate of gradual releasing of the substances into model environment was monitored. In the case of enzymes, their proteolytic activity was also tested after their incorporation to the wound dressings. Furthermore, the prepared bioactive wound dressings were analyzed for possible cytotoxic effect on human keratinocytes. Finally, the wound dressing with combined content of active substances was created and also characterized for the rate of substance release, proteolytic activity and cytotoxicity. Antimicrobial activity of this wound dressings, against two selected strains of microorganisms: Escherichia coli and Staphylococcus epidermidis, was also evaluated.

The diploma thesis is focused on the study of fibrous wound dressings prepared by electrospinning method from natural biopolymers. Three active ingredients were added to the dressings: ampicillin, ibuprofen and collagenase, which are responsible for relieving pain, reducing the risk of infection and selectively removing necrotic tissue in the wound. The theoretical part describes the therapeutic dressings currently available on the market and the most common methods of nanofiber production. The experimental part evaluates the optimization of the preparation of gelatin, alginate and chitosan fibrous wound dressings, which were subsequently enriched with active substances and their gradual release into the model environment was determined spectrophotometrically. Antimicrobial effects against E.coli and S. epidermidis strains andantifungal activity against C. glabrata yeast were monitored. Finally, two cytotoxicity tests on the human keratinocyte cell line HaCaT confirmed the safety of the prepared products, which can serve as bioactive skin dressings in the future.