Skin is a dynamic and complex organ that relies on the interaction of different cell types,biomacromolecules and signaling molecules. Upon injury, a cascade of events occurs to quickly restore the skin’s integrity. Depending on the size and severity of the wound, a dressing is used to provide a temporary barrier to protect from dehydration, microorganisms and debris. Current wound dressings however, cannot accelerate wound healing beyond the natural rate, require frequent dressing changes, and cannot be easily removed without triggering additional pain ortissue destruction. Insulin, a peptide used to treat Type 1 diabetes, has been reported to improve the recovery of severe burn wounds. Yet, no one has successfully demonstrated a convenient and effective insulin delivery vehicle that can be used to accelerate burn wound healing. Poly(lactic-co-glycolic acid) microparticles, were shown to release bioactive insulin for a period of 25 days, stimulating human keratinocyte migration in vitro. A wound dressing made from poly(ethylene glycol) and alginate was formulated incorporating the insulin-loaded poly(lactic-co-glycolic acid) microparticles. Bioactive insulin release was achieved for nearly 3 weeks, along with favourable water handling and physical properties conducive for wound healing. Finally, in vivo testing confirmed that a constant dose of insulin from alginate-PEG sponge dressings loaded with 0.125mg, or 0.04mg/cm2 insulin, with dressing changes every 3 days, was sufficient to significantly improve wound healing by 25%, as compared to an alginate- PEG sponge dressing without insulin. Insulin releasing alginate-PEG sponge dressings are therefore, an effective method of improving burn wound healing and may serve as a delivery vehicle platform to incorporate other therapeutic molecules in the future. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-12-20 17:50:47.872

Wounding of the cornea generates lateral electric fields (EFs) and initiates the expression of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF; Chiang et al., 1992; Wilson et al., 1999a). Therefore, these biologically generated EFs and endogenous growth factors may be of particular significance during wound healing. In the presence of an EF (150 mV/mm) cultured corneal epithelial cells (CECs) oriented perpendicular, directed cathodally and migrated at an enhanced rate. The induction times, induction thresholds and response patterns for these behaviours in increasing field strengths, indicated that they may operate through separate and parallel pathways. The application of either HGF or KGF enhanced the rate of CEC migration but neither affected the extent of CEC orientation or directionality. The distribution of HGF receptors (HGFR) was found to be exclusive to the cell body in the presence of an EF, the receptors accumulated cathodally. Moreover, the asymmetrical accumulation of HGFR in the presence of an EF correlated with the direction of CEC migration. The application of both HGF and an EF activated extracellular-signal regulated kinase (ERK) a mitogen-activated protein kinase. Furthermore, in the presence of an EF the observed ERK activation was greater in the cathodal facing half of the CECs. Inhibition of ERK reduced the extent of HGF and EF-enhanced CEC migration rate but did not alter EF-induced CEC cathodal directionality or perpendicular orientation. The HGF- and EF-enhancement of CEC migration rate may involve the activation of ERK and with the downstream liberation of leukotrienes and phosphorylation of MLCK, would culminate in actin contraction and polymerisation respectively. The potential clinical relevance for this work would concern the topical application of HGF and exogenous application of EFs to corneal epithelial wounds in order to augment healing in patients where this process is slow or deficient.

Dermal wound healing involves a myriad of highly regulated and sophisticated mechanisms, which are coordinated and carried out via several specialized cell types. The dominant players involved in this process include platelets, neutrophils, macrophages and fibroblasts. These cells play a vital role in the repair of the wound by orchestrating tasks such as forming a fibrin clot to stanch blood flow, removing foreign organisms and cellular debris, depositing new collagen matrix and establishing the contractile forces which eventually bridge the void caused by the initial infraction.\\[5pt] \indent Our current understanding of these mechanisms has been primarily based upon animal models. Unfortunately, these models lack insight into pathologic conditions, which plague human beings, such as keloid scar or chronic ulcer formation. Consequently, investigators have proposed a number of {\it in vivo} techniques to study wound repair in humans in order to overcome this barrier. One approach, which has been devised to increase our level of understanding of these chronic conditions, involves the cutaneous placement of a small cylindrical structure within the appendage of a human test subject.\\[5pt] \indent Researches have designed a variety of these implantable structures to examine different aspects of wound healing in both healthy subjects and individuals that experience some trauma related condition. In each case, several implants are surgically positioned at multiple locations under sterile conditions. These structures are later removed at distinct time intervals at which point they are histologically analyzed and biochemically assayed to deduce the presence of biological markers involved in the repair process. Implantable structures used in this way are often referred to as Human Implantable Models or Systems.\\[5pt] \indent Clinical studies with implantable models open up tremendous opportunities in fields such as biomathematics because they provide an experimentally controlled setting that aids in the development and validation of mathematical models. Furthermore, experiments carried out with implants greatly simplify the mathematics required to describe the repair process because they minimize the modeling of complex features associated with healing such as wound geometry and the evolution of contractile forces.\\[5pt] \indent In this work, we present a notional mathematical model, which accounts for two fundamental processes involved in the repair of an acute dermal wound. These processes include the inflammatory response and fibroplasia. Our system describes each of these events through the time evolution of four primary species or variables. These include the density of initial damage, inflammatory cells, fibroblasts and deposition of new collagen matrix. Since it is difficult to populate the equations of our model with coefficients that have been empirically derived, we fit these constants by carrying out a large number of simulations until there is reasonable agreement between the time response of the variables of our system and those reported by the literature for normal healing. Once a suitable choice of parameters has been made, we then compare simulation results with data obtained from clinical investigations. While more data is desired, we have a promising first step toward describing the primary events of wound repair within the confines of an implantable system.

Macrophages are key cells during inflammation and repair. Their activity is highly varied and requires precise regulation. The characterisation of cues coordinating macrophage functions has focussed on chemical and biological soluble mediators. Little is known about their responses to physical stimuli, in particular electric fields (EF) that are generated naturally in wounded tissue and infected tissue. Importantly, EFs are known to accelerate wound healing and limit infection but the mechanisms of this remain poorly understood. To address this gap in understanding, this study tested how key properties of human monocyte-derived macrophages are regulated by applied EFs equivalent to physiological EF strengths generated naturally. Using live-cell video microscopy, we show macrophage migration is directed anodally by EFs as low as 5 mV/mm and is EF-strength dependent, with effects peaking around 300 mV/mm. In contrast, monocytes, as macrophage-precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that EFs significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils and the nominal opportunist pathogen Candida albicans, all of which engage different classes of surface receptors. These EF-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium and actin polarization. EFs also selectively modulate cytokine production and augment effects of conventional polarising stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the co-ordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically-applied EFs in conditions where macrophage activity is crucial.

Orientador: Lucilene Delazari dos Santos / Coorientador: Luciana Patricia Fernandes Abbade / Resumo: Introdução: Úlceras venosas crônicas atingem até 4% da população mundial >65 anos, causando impacto socioeconômico, principalmente relacionado à diminuição da mobilidade e autoestima. O exsudato destas lesões, pode ser útil na identificação dos fatores envolvidos na reparação tecidual. Objetivos: Identificar proteínas expressas no exsudato de úlceras venosas, agrupando-as de acordo com suas principais funções, e correlancionando-as com variaveis clínicas e epidemiológicas. Métodos: Estudo clínico do tipo transversal, descritivo e analítico envolvendo trinta e sete úlceras de 28 pacientes. Todos os pacientes foram submetidos à questionário clínico-epidemiológico auto descritivo, análise de área e a coleta de exsudato das úlceras. Fluidos das lesões foram submetidos à digestão tríptica em solução e sequenciados por espectrometria de massas para identificação do perfil proteômico. A análise multivariada entre dados clínicos e expressão proteica do exsudato foi explorada por escalonamento multidimensional, a partir da distância euclidiana entre as variáveis. Resultados: A maioria dos pacientes era do sexo feminino (62%), com idade média de 70(±10.1) anos, relatando adesão à compressão e ao repouso, histórico de varizes primárias e hipertensão arterial sistêmica, apresentando tecido desvitalizado no leito da ferida e tempo de evolução >10 anos. Foram identificadas 74 proteínas no exsudato, agrupadas de acordo com sua principal função na cicatrização. O perfil proteômico evidenciou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: not available / Mestre

Orientador: Rosemary Adriana Chiérici Marcantonio / Resumo: O objetivo desse estudo foi avaliar o efeito de uma membrana de látex natural sobre a cicatrização de feridas no palato duro provenientes da remoção de enxerto gengival livre. Vinte e quatro pacientes participaram desse estudo e foram divididos aleatoriamente em 2 grupos de acordo com o tratamento utilizado para proteger o leito doador: Grupo controle (GC): A ferida foi recoberta com placa acrílica associada ao cimento cirúrgico (n=14); Grupo Látex (GL): A ferida foi recoberta com placa acrílica associada a membrana de látex natural (n=10). Foram realizadas tomadas fotográficas padronizadas das regiões das feridas nos períodos de baseline, 3, 7, 15 e 30 dias após o procedimento cirúrgico. Um examinador cego e calibrado realizou avaliação clínica, levando se em consideração os parâmetros: 1) fechamento de ferida; 2) área de superfície epitelizada por meio da utilização da água oxigenada aplicada na região; 3) Avaliação do auto relato de sensação dolorosa por meio da aplicação da escala de dor VAS. Os resultados obtidos mostraram que em ambos os grupos, houve diminuição gradativa da área da ferida cirúrgica, sendo que a partir dos 15 dias essa era inexistente para todos os pacientes avaliados e em relação à dor houve uma redução significativa da sensibilidade dolorosa relatada pelos pacientes do grupo látex em relação ao grupo controle. O uso da membrana de látex não promoveu efeito adicional a cicatrização, apresentando os mesmos resultados clínicos que a utilização de cimento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this project was to evaluate the effect of a natural latex membrane in wound healing on donor sites of free gingival grafts. Twenty-four patients were used in this study and were randomly divided in 2 groups according with the treatment used to protect the donor site: Control group (CG) – Donor site was covered with an acrylic plate associated with surgical cement (n=14); Látex group (LG) – Donor site was covered with acrylic plate associated with a natural latex membrane and surgical cement (n=10). At baseline, 3, 7, 15 and 30 days after surgery, standardized photos of the wound were taken. A blind and calibrated examiner made the clinical evaluation by considering the parameters: 1) total wound area; 2) epithelized surface area through the use of hydrogen peroxide applied in the region; 3) Evaluation of self-report of pain sensation through the application of the VAS pain scale. The achieve results showed that both groups had a gradual decrease in the area of the surgical wound, and from 15 days this was non-existent for all patients evaluated and on the pain avaliation we found that the patients in latex group had significant reduction in reported pain when compared to the control group. The use of natural latex membrane didn’t promote any additional effect for wound healing, showing the same clinical results as the use of surgical cement with the addition of a lower pain report by the patients. / Mestre

The Ectodysplasin/Ectodysplasin receptor (Eda/Edar) signalling pathway is critical during development for the formation of skin appendages. However, its roles during adulthood are only recently being elucidated. Adult appendages, such as hair follicles (HFs), are known to become activated to respond to cutaneous injury. However, the HF houses distinct cell populations that display differing capacities to participate and persist in re-epithelialisation. We show, contrary to previous findings, that the best-characterised stem cell (SC) niche within the HF (the bulge) does not respond to injury during the earliest stages of wound healing. We propose that bulge SCs are prevented from participating in early repair as a protection mechanism against tumourigenesis. Despite the bulge niche not participating in early repair, we found the upper HF outer root sheath (ORS) to respond rapidly to injury. Our investigation into the role of Eda/Edar signalling during wound healing revealed that activation of the pathway was able to specifically induce proliferation within this portion of the HF. We further demonstrate a number of roles for the Eda/Edar pathway during adult wound healing, including, surprisingly, influencing several wound responses within the dermis. Specifically, an absence of Eda/Edar signalling in Tabby mice results in delayed wound healing, whereas acute activation of the pathway in wild-type (WT) mice can stimulate re-epithelialisation and enhance wound repair. These effects also translate to a model of human wound healing, where activation of Eda/Edar signalling accelerates re-epithelialisation and increases peri-wound proliferation. RNA-seq analysis reveals diverse gene regulation in the presence/absence of Eda/Edar signalling. Overall, these findings suggest that manipulation of the Eda/Edar pathway may represent an attractive potential therapeutic for enhancement of wound repair, potentially through maximising the natural growth capacity of peri-wound HFs.

Patients who suffer from severe joint destruction caused by arthritis often undergo total joint arthroplasty (TJA). A major limitation of this treatment and common long-term complication is the development of aseptic loosening of the prosthesis in as many as 20% of patients. The current paradigm to explain aseptic loosening proposes that wear debris generated from the prosthesis initiates a macrophage-mediated inflammatory response by resident macrophages, leading to osteoclast activation and bone resorption at the implant interface. This can then lead to the development of a peri-prosthetic fracture. The principal aim of fracture healing is to restore the bone to its original form and strength. However, this ultimate goal can be altered if the healing is impaired. This impairment may be due to bone disease (osteoporosis) or even the introduction of a foreign material such as PE wear debris that could have migrated from the articulating surface to the fracture site. A standard closed unilateral fracture of the right femur was performed in both normal and oestrogen deficient rats following fixation with a k-wire. Ceridust (PE wear debris) was combined with hyaluronic acid and saline and injected directly into the fracture site. Femurs were assessed using radiographs, histology and immunohistochemistry. Histological analysis revealed that complete remodelling was achieved in all control groups by 6 weeks post-fracture with mechanical strength returning to normal values. The mechanical properties of the fractures were not influenced by the presence of PE wear debris in the dose and timing examined. Histology and immunohistochemistry however, did reveal a local effect of the presence of PE wear debris. The histology adjacent to the PE particles was inferior to the controls but did not manifest itself in a reduction in the mechanical properties except in the oestrogen deficient bone at 6 weeks post-fracture. The levels of MMP-1 and TNF-?? correlated to the presence of PE particles. In this thesis, I have shown the mechanism by which bone remodelling in fracture healing could be retarded due to the presence of PE wear debris, by increased matrix degradation in both normal and oestrogen deficient animals.

Transglutaminase II (TG2) is a complex protein with five different reported activities. Increases in TG2 expression and TGase activity have previously been observed during wound healing in rat studies; however, it has been unclear whether these phenomena were directly involved in the healing process or if they were simply a by-product of it. The aims of this thesis were, thus, to determine if TG2 plays a role in wound healing in vivo and to elucidate the mechanism of any effects TG2 may have at the cellular level. TG2 ablation resulted in delayed wound healing. To gain mechanistic insight into this abnormality, primary fibroblast cultures from TG2-knockout and wildtype mouse embryos were analysed. TG2-null fibroblasts displayed decreased adhesion and integrin signalling during initial stages of adhesion. Intriguingly, TG2-null cells showed faster activation of Rac1 and RhoA in response to adhesion. Long-term adhesion of TG2-null fibroblasts resulted in increased basal phosphorylation of FAK and number of paxillin-stained focal adhesions, enhanced PI3-kinase signalling, faster actin dynamics and altered activation of p44/42 MAPK. These results are indicative of futile cycling of intracellular signalling pathways resulting from reduced focal adhesion turnover in the TG2-knockout fibroblasts. Rescue experiments demonstrated that TG2-mediated effects on cell adhesion occurred in the extracellular environment and that neither GTP-binding nor TGase activity is required for these effects. Results further showed that a ???compact??? conformation of TG2 was not required for this role of TG2. Interestingly, addition of recombinant TG2 to the extracellular environment increased cell spreading of TG2-null cells to a level far greater than that seen in wildtype cells, which did not increase their spreading in response to exogenous TG2. Demonstration of faster activation of the small GTPases in the TG2-null MEFs, and the apparent inhibition of exogenous TG2???s extracellular effects on cell spreading by endogenous protein in the wildtype cells, provide tantalising evidence for a role for intracellular TG2 in regulating activation of the small GTPases to promote efficient fibroblast migration. This work identifies TG2 as a facilitator of efficient wound closure through extracellular effects on integrin-mediated signalling and intracellular effects on activation of the small GTPases.

Thesis (M. D. S.)--University of Hong Kong, 2003. / Title proper from title frame. Also available in printed format.