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The starting point of this investigation was the clinical observation that raw areas of the body resurfaced by split-thickness skin grafts contract markedly, whereas those covered by thick grafts (e.g. Wolfe grafts) contract little or not at all. The reason for this behaviour is unknown. The present work was an attempt to investigate this behaviour using fibroblasts cultured in a hydrated collagen lattice (FHCL) as a laboratory model for wound contraction in vivo. Sequential extracts of normal human dermis were prepared in water, 0.15M sodium chloride, 1M sodium chloride, 0.15M citrate buffer (pH 3.5) and 6M urea, and their effects on FHCL contraction were examined. Only citrate buffer dermal extract had a marked inhibitory effect on FHCL contraction. The effect was reversible, concentration-dependent and lasted throughout the course of the 96 hours' experiments. There was no toxic effect on the cells although their proliferation within the lattices was also inhibited. Furthermore the extracts from the deeper parts of the dermis have more inhibitory effect on the FHCL contraction than those from the more superficial layers. Preliminary attempts to characterise the citrate buffer dermal extract biochemically showed the presence of protein and proteoglycans or glycosaminoglycans with a range of molecular weights showed by gel electrophoresis, and the presence of collagen was excluded by amino acid analysis. This study shows that there is a biochemical factor (factors) present in the dermis which inhibits the ability of fibroblasts to cause lattice contraction. This factor (s) can be extracted by citrate buffer. The inhibitory effect of the citrate buffer dermal extract on lattice contraction is due to inhibition of both fibroblasts proliferation and migration.

No description available.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2017-10-18T18:04:12Z No. of bitstreams: 2 Dissertação - Ana Carolina Barros da Rosa Pedroso - 2017.pdf: 4185666 bytes, checksum: 974e13fd85577c264019ad51d76003b6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-19T10:29:32Z (GMT) No. of bitstreams: 2 Dissertação - Ana Carolina Barros da Rosa Pedroso - 2017.pdf: 4185666 bytes, checksum: 974e13fd85577c264019ad51d76003b6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-19T10:29:32Z (GMT). No. of bitstreams: 2 Dissertação - Ana Carolina Barros da Rosa Pedroso - 2017.pdf: 4185666 bytes, checksum: 974e13fd85577c264019ad51d76003b6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-30 / The economic relevance of equine breeding in Brazil indicates the importance of the development of new economically viable treatments to promote tissue healing. Skin grafts and Platelet Rich Plasma (PRP) are available alternatives for improving the wound healing process in these animals. This study aimed to establish a simple method for autologous PRP production, as well as to evaluate its action on cutaneous wounds containing skin autografts in horses. Eight healthy horses were used, ranging in age from 3 to 18 years and mean live weight of 350 kg. For PRP obtention, a double centrifugation protocol was used. Two 6 x 6 cm wounds were performed on each side of the gluteal region. After the wound was filled by granulation tissue, punch autografts were obtained, with fragments harvested from the neck. Left-sided wounds were treated with PRP (GT) and right-sided wounds were not treated (GC). The protocol used to obtain PRP resulted in platelet concentration, on average, three times higher than that of whole blood. In the evaluation of PRP action in the healing of autografts, macroscopic and microscopic variables were considered. There was no significant difference between the groups (p> 0.05) for the variables inflammatory infiltrate, young fibroblasts, collagenization, epidermal thickness, macroscopic and microscopic integration of the autografts and retraction of the wound edges. However, neovascularization was significantly greater (p = 0.0191) in the PRP group, on the 14 th day after insertion of the autografts. It is concluded that the double centrifugation method results in PRP with adequate platelet concentration for therapeutic use. As for the action of PRP, this favors the process of skin repair with autografts in horses, since it increases the neovascularization in the initial phase of wound healing. Moreover, PRP seems to positively influence the integration of skin autografts and retraction of the wound edges, which is of fundamental importance for the reduction of hospitalization time and consequently of the therapeutic costs. / A relevância econômica da criação de equinos no Brasil denota a importância do desenvolvimento de novos tratamentos, economicamente viáveis, para promover a cicatrização tecidual. Os enxertos de pele e o Plasma Rico em Plaquetas (PRP) são alternativas disponíveis para a melhoria do processo de cicatrização nesses animais. Este estudo teve por objetivo estabelecer um método simples para produção de PRP autólogo, assim como avaliar sua ação sobre feridas cutâneas contendo autoenxertos de pele em equinos. Foram utilizados oito equinos hígidos, com idade variando entre três e 18 anos e peso vivo médio de 350 kg. Na obtenção do PRP, foi utilizado protocolo em dupla centrifugação. Foram realizadas duas feridas de 6 x 6 cm, em cada um dos lados da região glútea. Após o preenchimento da ferida por tecido de granulação, foram realizados autoenxertos do tipo punch, com fragmentos colhidos do pescoço. As feridas do lado esquerdo receberam tratamento com PRP (GT) e as feridas do lado direito não receberam tratamento (GC). O protocolo utilizado na obtenção do PRP resultou em concentração plaquetária, em média, três vezes acima daquela do sangue total. Na avaliação da ação do PRP na cicatrização dos autoenxertos, foram consideradas variáveis macroscópicas e microscópicas. Não houve diferença significativa entre os grupos (p>0,05) quanto às variáveis infiltrado inflamatório, fibroblastos jovens, colagenização, espessura da epiderme, integração macroscópica e microscópica dos autoenxertos e retração das bordas da ferida. Contudo, a neovascularização foi significativamente maior (p=0,0191) no grupo tratado com o PRP, no 14º dia após a inserção dos autoenxertos. Conclui-se que o método de dupla centrifugação resulta em PRP com concentração plaquetária adequada ao uso terapêutico. Quanto à ação do PRP, este favorece o processo de reparo da pele com autoenxertos em equinos, já que aumenta a neovascularização na fase inicial da cicatrização da ferida. Ainda, o PRP parece influenciar positivamente a integração dos autoenxertos de pele e a retração das bordas da ferida, o que é de fundamental importância para a redução do tempo de internação dos animais e, consequentemente, dos custos terapêuticos.

INTRODUCTION. La guérison rapide des sites donneurs des greffes cutanées favorise la survie des victimes de brûlures graves (>50 % de superficie brûlée). La mortalité élevée de ces patients est attribuable au fait que la superficie des brûlures excède celle de la peau saine. Des cultures épithéliales autologues (CEA) sont des feuillets de kératinocytes produits en culture à partir de la peau du patient. Cette étude a évalué l’effet des CEA sur l'épithélialisation des sites donneurs chez les grands brûlés. MÉTHODES. Tous les patients recevant des CEA ont été prospectivement inclus. Les plaies des sites donneurs ont été recouvertes de CEA, sauf pour une région contrôle randomisée de 7 x 7 cm. Des biopsies faites sur la greffe de peau ont permis de contrôler la profondeur des plaies sur les sites donneurs. Il y avait deux types de contrôles, avec gaze non adhérente trempée dans le milieu de culture ou dans le salin. L’épithélialisation était quantifiée globalement (% d’épithélialisation par photographie) et histologiquement (par biopsie au poinçon) à simple insu. La guérison des zones de contrôle et CEA était comparée par analyse de variance et par le test de Student. RÉSULTATS. Entre 2008 et 2009, 6 patients furent recrutés avec un total de 11 sites donneurs. Ces patients avaient en moyenne 43.5 ans, 56 % de superficie brûlée, 45% de brûlure pleine épaisseur, 66% avaient une brûlure d’inhalation, 75 jours de séjour. Il n’y a aucune corrélation entre le pourcentage d’épithélialisation et l’épaisseur du prélèvement des greffes (Pearson 0.19). Le score photographique est significativement influencé par le traitement (CEA vs Contrôle; p = 0,039) et par le jour postopératoire (p < 0,001). Le temps moyen pour atteindre un score photographique de guérison pour les zones contrôles fut de 10.2 jours contre 8.6 jours pour le CEA (p = 0,021). A l’évaluation histologique, les sites donneurs traités par le milieu de culture ont évolué aussi favorablement que ceux traités par des feuillets de CEA. CONCLUSION. L’utilisation de CEA sur les sites donneurs semble accélérer leur épithélialisation chez les victimes de brûlures graves. Cet effet est probablement le résultat d’une stimulation de la réépithélialisation innée de la plaie, plutôt que par une adhérence des feuillets de kératinocytes cultivés à la surface de la plaie. / RATIONALE: Prompt healing of split thickness skin graft donor sites is primordial to the survival of severely burned patients. Increased mortality of patients with >50 % TBSA is attributable to the limited availability of donor sites. This study evaluated the effect of Cultured Epithelial Autograft (CEA) application on skin graft donor site healing. METHODS: All burn patients receiving CEA were prospectively included. Donor site wounds were covered with CEA except a randomly designated 7x7 cm control region. Autograft biopsies were taken to document graft harvest thickness. One half of the controls were covered with non-adherent gauze soaked in the culture media and the other controls only received a non-adherent gauze dressing. Epithelialization was objectively evaluated by scoring blinded photographs with an analogue scale. Punch biopsies of the donor sites were evaluated histologically. Repeated measures ANOVA and T-test were used. RESULTS: Between 2008 and 2009, 6 patients were enrolled for a total of 11 donor sites. The patients averaged 43.5 years, 56 % TBSA, 45 % FT-TBSA, 66 % had inhalation injury and mean length of stay was 75 days. As expected, dermatome settings and autograft thickness measured by microscope did not correlate (Pearson 0.19). There was no correlation between the percentage of epithelialization of the punch biopsies of the donor sites and the thickness of the harvest. Photographic score was significantly influenced by its treatment CEA vs Control (p=0.039) and by postoperative day (p<0.001). Mean time to healing was 8.6 days for CEA compared to 10.2 days for controls (p=0.021). Infection was noted on only one donor site. On histologic analysis, the control sites dressed with gauze soaked in the culture media healed as nicely and promptly as the CEA sheet treated region. CONCLUSION: Use of CEA on donor sites appears to stimulate epithelialization. This effect is probably mediated by stimulation of local wound healing processes rather than by engraftment of keratinocytes from the CEA sheets.

INTRODUCTION. La guérison rapide des sites donneurs des greffes cutanées favorise la survie des victimes de brûlures graves (>50 % de superficie brûlée). La mortalité élevée de ces patients est attribuable au fait que la superficie des brûlures excède celle de la peau saine. Des cultures épithéliales autologues (CEA) sont des feuillets de kératinocytes produits en culture à partir de la peau du patient. Cette étude a évalué l’effet des CEA sur l'épithélialisation des sites donneurs chez les grands brûlés. MÉTHODES. Tous les patients recevant des CEA ont été prospectivement inclus. Les plaies des sites donneurs ont été recouvertes de CEA, sauf pour une région contrôle randomisée de 7 x 7 cm. Des biopsies faites sur la greffe de peau ont permis de contrôler la profondeur des plaies sur les sites donneurs. Il y avait deux types de contrôles, avec gaze non adhérente trempée dans le milieu de culture ou dans le salin. L’épithélialisation était quantifiée globalement (% d’épithélialisation par photographie) et histologiquement (par biopsie au poinçon) à simple insu. La guérison des zones de contrôle et CEA était comparée par analyse de variance et par le test de Student. RÉSULTATS. Entre 2008 et 2009, 6 patients furent recrutés avec un total de 11 sites donneurs. Ces patients avaient en moyenne 43.5 ans, 56 % de superficie brûlée, 45% de brûlure pleine épaisseur, 66% avaient une brûlure d’inhalation, 75 jours de séjour. Il n’y a aucune corrélation entre le pourcentage d’épithélialisation et l’épaisseur du prélèvement des greffes (Pearson 0.19). Le score photographique est significativement influencé par le traitement (CEA vs Contrôle; p = 0,039) et par le jour postopératoire (p < 0,001). Le temps moyen pour atteindre un score photographique de guérison pour les zones contrôles fut de 10.2 jours contre 8.6 jours pour le CEA (p = 0,021). A l’évaluation histologique, les sites donneurs traités par le milieu de culture ont évolué aussi favorablement que ceux traités par des feuillets de CEA. CONCLUSION. L’utilisation de CEA sur les sites donneurs semble accélérer leur épithélialisation chez les victimes de brûlures graves. Cet effet est probablement le résultat d’une stimulation de la réépithélialisation innée de la plaie, plutôt que par une adhérence des feuillets de kératinocytes cultivés à la surface de la plaie. / RATIONALE: Prompt healing of split thickness skin graft donor sites is primordial to the survival of severely burned patients. Increased mortality of patients with >50 % TBSA is attributable to the limited availability of donor sites. This study evaluated the effect of Cultured Epithelial Autograft (CEA) application on skin graft donor site healing. METHODS: All burn patients receiving CEA were prospectively included. Donor site wounds were covered with CEA except a randomly designated 7x7 cm control region. Autograft biopsies were taken to document graft harvest thickness. One half of the controls were covered with non-adherent gauze soaked in the culture media and the other controls only received a non-adherent gauze dressing. Epithelialization was objectively evaluated by scoring blinded photographs with an analogue scale. Punch biopsies of the donor sites were evaluated histologically. Repeated measures ANOVA and T-test were used. RESULTS: Between 2008 and 2009, 6 patients were enrolled for a total of 11 donor sites. The patients averaged 43.5 years, 56 % TBSA, 45 % FT-TBSA, 66 % had inhalation injury and mean length of stay was 75 days. As expected, dermatome settings and autograft thickness measured by microscope did not correlate (Pearson 0.19). There was no correlation between the percentage of epithelialization of the punch biopsies of the donor sites and the thickness of the harvest. Photographic score was significantly influenced by its treatment CEA vs Control (p=0.039) and by postoperative day (p<0.001). Mean time to healing was 8.6 days for CEA compared to 10.2 days for controls (p=0.021). Infection was noted on only one donor site. On histologic analysis, the control sites dressed with gauze soaked in the culture media healed as nicely and promptly as the CEA sheet treated region. CONCLUSION: Use of CEA on donor sites appears to stimulate epithelialization. This effect is probably mediated by stimulation of local wound healing processes rather than by engraftment of keratinocytes from the CEA sheets.

Le développement de nouvelles stratégies vaccinales et le traitement de nombreuses pathologies dermatologiques font partis des enjeux majeurs des années à venir. L'étude et la compréhension des mécanismes de réponses immunitaires cutanées sont ainsi essentielles. Les cellules de Langerhans épidermiques et les cellules dendritiques dermiques jouent un rôle central dans l'immunité adaptative spécifique à un pathogène, et dans l'immunité innée, de par leur fonction de présentation des antigènes (APC), mais également d'activateur et de régulateur de l'action des autres cellules immunitaires. Durant ce travail de recherche, nous avons mis au point et testé plusieurs modèles d'étude des mécanismes de l'immunité cutanée. Un premier modèle de " souris humanisées " a été mis au point, à partir de souris immunodéprimées NSG HLA-A2 tg et de greffes de peau humaine. Une fois le modèle techniquement mis au point, nous avons vérifié de la conservation et de la fonctionnalité des APC. La faible conservation des APC au-delà de plusieurs semaines, nous a conduit à mettre au point un deuxième modèle de " souris humanisées " qui bénéficiaient d'injections intra-hépatiques de progéniteurs hématopoïétiques humains. La colonisation en APCs cutanées humaines n'étant pas suffisante, il a fallu injecter aux souris des APCs provenant du même sang que les progéniteurs hématopoïétiques afin d'obtenir une réponse immunitaire suffisante. La faible concentration en APC cutanées de ce modèle, la lourdeur et le coût de mise au point sont des limites non négligeables pour l'utilisation de ce support d'étude. Ces souris humanisées sont d’excellents modèles d’étude, mais ils présentent chacun leurs limites. Lorsque cela est possible il est ainsi plus simple d’utiliser des explants cutanés frais. Nous avons quantifié les APC cutanées en fonction du site d’origine (abdomen, seins…) et de l’âge du donneur de ces explants issus de chirurgie plastique. Sur les 21 explants testés, nous n’avons pas retrouvé de différence significative dans la concentration en APC quel que soit le site et l’âge du donneur. Ces 3 modèles offrent des possibilités d’étude des mécanismes de l’immunité cutanée, nous les utilisons actuellement au sein du laboratoire pour des tests vaccinaux, et pour l’étude du rôle de l’IL-32 produite par les kératinocytes impliqués dans l’activation des cellules de Langerhans. / The development of new vaccination strategies and the treatment of many dermatological pathologies are among the major challenges of the years to come. The study and understanding of the mechanisms of cutaneous immune responses are thus essential. Epidermal Langerhans cells and dermal dendritic cells play a central role in pathogen-specific adaptive immunity, and in innate immunity, by their antigen presenting function (APC), but also as an activator and regulator of the action of other immune cells.During this research, we developed and tested several models for the study of the mechanisms of cutaneous immunity. A first model of "humanized mice" was developed from immunosuppressed NSG HLA-A2 tg mice and human skin grafts. Once the model was technically developed, we verified the conservation and functionality of the APCs. The low PCA retention beyond several weeks, led us to develop a second model of "humanized mice" that benefited from intrahepatic injections of human hematopoietic progenitors. Since colonization in human skin APCs was not sufficient, it was necessary to inject the APCs from the same blood as the hematopoietic progenitors to obtain a sufficient immune response. The low concentration of skin APCs in this model, the cumbersomeness and the cost of development are not insignificant limits for the use of this study support.These humanized mice are excellent models of study, but they each have their limitations. Where possible, it is thus easier to use fresh skin explants. We quantified the cutaneous APCs according to the site of origin (abdomen, breasts ...) and the age of the donor of these explants resulting from plastic surgery. Of the 21 explants tested, there was no significant difference in APC concentration at any site and age of the donor.These three models offer possibilities for studying the mechanisms of skin immunity, we are currently using them in the laboratory for vaccine tests, and for studying the role of IL-32 produced by the keratinocytes involved in l activation of Langerhans cells.

Epidermal wound healing requires the coordinated involvement of complex cellular and biochemical processes. In the case of epidermal wounds associated with burns, the healing process may be less than optimal and may take a significant amount of time, possibly resulting in infection and scarring. An innovative method to assist in the repair of the epidermis (the outer layer of skin) is to use an aerosolised apparatus. This method involves taking skin cells from an area of the patient's undamaged skin, culturing the cells in a laboratory, encouraging them to rapidly proliferate, then harvesting and separating the cells from each other. The cells are then sprayed onto the wound surface. We investigate this novel treatment strategy for the healing of epidermal wounds, such as burns. In particular, we model the application of viable cell colonies to the exposed surface of the wound with the intent of identifying key factors that govern the healing process. Details of the evolution of the colony structure are explored in this two-dimensional model of the wound site, including the effect of varying the initial population cluster size and the initial distribution of cell types with different proliferative capacities. During injury, holoclones (which are thought to be stem cells) have a large proliferative capacity while paraclones (which are thought to be transient amplifying cells) have a more limited proliferative capacity. The model predicts the coverage over time for cells that are initially sprayed onto a wound. A detailed analysis of the underlying mathematical models yields novel mathematical results as well as insight into phenomena of healing processes under investigation. Two one-dimensional systems that are simplifications of the full model are investigated. These models are significant extensions of Fisher's equation and incorporate the mixed clonal population of quiescent and active cells. In the first model, an active cell type migrates and proliferates into the wound and undergoes a transition to a quiescent cell type that neither migrates nor proliferates. The analysis yields the identification of the key parameter constraints on the speed of the healing front of the cells on this model and hence the rate of healing of epidermal wounds. Approximations for the maximum cell densities are also obtained, including conditions for a less than optimal final state. The second model involves two active cell types with different proliferative capacity and a quiescent cell type. This model exhibits two distinct behaviours: either both cell types coexist or one of them dies out as the wound healing progresses leaving the other cell type to fill the wound space. Conditions for coexistence are explored.