Dissertations / Theses on the topic 'Antimicrobial biomaterials for wound healing'

Chronic skin wounds are a major socioeconomic burden in developed societies, affecting specially elder and diabetic people. It is estimated that 1 to 2% of the population will suffer a chronic wound in their lifetime and, as global population ages and adopts a sedentary lifestyle, the incidence of these wounds will continue its upward trend. Chronic injuries are characterized for presenting a complicated and diverse pathophysiology that make them resistant to current therapies. For this reason, novel therapeutic strategies based on the release of growth factors and the use of tissue engineered constructs are being investigated and show promising results. However, very few biologically based products reach the market, mainly due to regulatory, economic and stability constraints, amplifying the need for easily translational novel treatments. Recently, inorganic biomaterials known as bioceramics have been acknowledged for their wound healing and vascularization capability, mainly due to their ion release. Based on this concept, the present thesis project was dedicated to investigate the potential application of novel bioceramics on wound healing and soft tissue regeneration. More specifically we focused on the role of the calcium ion and its release from newly designed bioceramics to stimulate wound healing and blood vessel formation in both in vitro and in vivo systems. Although it is known that calcium affects all the phases of wound healing, the concentrations and release profile that can improve the healing process has not been described. For this reason, we evaluated the effect of different concentrations of extracellular calcium in vitro on dermal fibroblast, a crucial cell type in the skin and the healing process, and found stimulation of relevant biological responses at specific concentrations. In addition, we compared whether similar effects could be obtained with the ion release of newly designed bioceramic particles containing equivalent calcium concentrations. Interestingly, while stimulating most of the effects, the ion release inhibited some responses triggered by calcium alone that are not desired in the context of chronic wound healing. Then, we investigated the cellular mechanism mediating some of the responses stimulated by calcium, focusing on the implication of the calcium-sensing receptor (CaSR). Several agonists of the receptor stimulated similar effects than calcium, suggesting the relevance of the CaSR on fibroblasts behavior, and opening a window to the design of novel bioceramics that release CaSR-agonists. In order to test the healing capability of the above mentioned bioceramic particles in vivo, they were incorporated in a mat of poly(lactic acid) (PLA) fibers. This novel dressing was applied on a model of chronic wounds in vivo, and compared with a mat of particle-free PLA and to a frequently used commercially available dressing. We found that the PLA-bioceramic mat accelerated wound closure and increased vasculature at the injured site at initial time-points. Thus, improved healing was achieved with the newly designed dressing. Finally, a different composite material was synthesized combining bioceramic particles and human mesenchymal stromal cells (hMSC) in a degradable hydrogel, and its vasculogenic potential was evaluated in soft tissue. This material supported hMSC survival and stimulated the release of the angiogenic factor IGF-1 from these cells in vitro. In addition, when implanted in soft tissue of immunocompromised mice, the composite construct improved hMSC survival and generated a more mature vasculature at the site of implantation. In conclusion, this thesis shows that calcium-releasing bioceramics can successfully contribute to the treatment of chronic wounds and soft tissue regeneration.
Las heridas crónicas tienen un gran impacto socioeconómico sobre los países desarrollados, afectando especialmente a personas en edad avanzada y diabéticos. Se estima que entre el 1 y 2% de la población sufrirá una herida crónica a lo largo de su vida y, con el envejecimiento de la población y el aumento del sedentarismo, la incidencia de estas heridas seguirá una tendencia ascendente. Las heridas crónicas presentan una patofisiología complicada y diversa que las hace resistentes a las terapias actuales. Por esta razón, se están desarrollando nuevos productos mediante ingeniería de tejidos basados en el uso de factores de crecimiento y células. Sin embargo, la translación de estas terapias a la clínica es muy complicada por cuestiones regulatorias, económicas y de estabilidad del producto, por lo que hay una gran necesidad de nuevos tratamientos que puedan llegar más fácilmente al mercado. Recientemente, se ha descubierto que los biomateriales inorgánicos llamados biocerámicos pueden estimular la curación de heridas y la vascularización, principalmente a través del efecto de los iones que liberan. Partiendo de esta idea, este proyecto de tesis se ha centrado en investigar el uso potencial de nuevos biocerámicos en curación de heridas y la regeneración de tejido blando. Más concretamente, nos hemos centrado en el rol del ión calcio y su liberación de nuevos biocerámicos para estimular la curación de heridas y la formación de vasos sanguíneos in vitro e in vivo. A pesar de que el calcio afecta en todas las fases de la curación de una herida, las concentraciones y perfil de liberación que pueden mejorar el proceso de curación no han sido descritos. Por ello, evaluamos el efecto de diferentes concentraciones de calcio extracelular en fibroblastos dermales, un tipo celular esencial en el proceso de curación, y encontramos estimulación de diferentes respuestas biológicas a concentraciones específicas. Además, comparamos si se podían obtener efectos similares mediante el producto iónico liberado de unas nuevas partículas biocerámicas con concentraciones equivalentes de calcio. Curiosamente, el producto iónico inhibió algunos efectos estimulados por el calcio en solución que no son deseables en un contexto de tratamiento de heridas crónicas. Entonces, quisimos indagar en el mecanismo celular a través del cual el calcio estimula a los fibroblastos, centrándonos en la implicación del receptor sensor de calcio (CaSR). Varios agonistas de este receptor estimularon respuestas parecidas al calcio, mostrando la relevancia del CaSR sobre el comportamiento de los fibroblastos, y abriendo una ventana al diseño de nuevos biocerámicos con libración de agonistas del CaSR. Por otro lado, quisimos probar la capacidad curativa de las partículas biocerámicas usadas sobre los fibroblastos incorporándolas en fibras de ácido poliláctico. Este nuevo apósito generado se aplicó sobre un modelo de heridas crónicas in vivo, y su efecto se comparó con un apósito de PLA sin partículas y con un apósito comercial. El apósito de PLA-biocerámico aceleró la curación de las heridas además de estimular la formación de vasos sanguíneos a tiempos tempranos, con lo que se consiguió una mejora en la curación. Finalmente, se sintetizó un biomaterial implantable combinado partículas biocerámicas, células madre mesenquimales adultas (hMSC) y un hidrogel sintético degradable, con el objetivo de evaluar su capacitat vasculogénica en tejidos blandos. In vitro, el material mantuvo la superviencia de las células encapsuladas y se aumentó la liberación del factor angiogénico IGF-1. Además, al implantarse en tejido blando de ratones immunodeprimidos, el material con biocerámico mejoró la supervivencia de las hMSC y estimuló la maduración de la vasculatura en el sitio de implantación

The objective of this study was to determine if topical antimicrobials silver sulfadiazine and povidone-iodine ointment increase rates of healing of equine distal limb wounds that heal by second intention. Second, to determine the effect of bandaging with these topical antimicrobials. Six healthy adult horses were used to create thirty-six, 2.5 cm2 standardized full-thickness metacarpal/tarsal skin wounds. Each wound was exposed to a single treatment: 1.0 % silver sulfadiazine cream bandaged (SSD-B), 1.0 % silver sulfadiazine slow release matrix bandaged (SDX-B), 1.0% silver sulfadiazine slow release matrix not bandaged (SDX-NB), povidone-iodine ointment bandaged (PI-B), untreated control bandaged (C-B) and untreated control not bandaged (C-NB) until healing. Wound area, granulation tissue area and perimeter were measured with planimetry software from digital images obtained at each observation. Exuberant granulation tissue was excised when present. The days until healing, rate of healing parameter, rates of contraction and epithelialization were compared among groups using pair-wise analysis of least square means. The healing parameters and mean days to healing did not statistically differ between groups. Analysis of percent wound contraction and rate of epithelialization between groups was similar. Mean number of days to healing ranged from 83 (PI-B and C-B) to101 (SSD-B). All bandaged wounds produced exuberant granulation tissue requiring excision compared to none of the unbandaged. The identified rates of epithelialization and wound contraction found insignificant differences between antimicrobial treated versus untreated wounds. Similarly, rates of epithelialization and wound contraction found insignificant differences between bandaged versus unbandaged wounds. Topical povidone-iodine and silver sulfadiazine did not increase rates of healing under bandage. The 1.0% silver sulfadiazine slow release matrix not bandaged (SDX-NB) adhered well to dry wounds. Silver sulfadiazine slow-release matrix provides does not impede wound healing and provides good adherence to dry wounds not amenable to bandaging.
Master of Science

Despite our understanding of the importance of the 3D environment on the behaviour of virtually every cell, most studies are still performed within 2D engineered cell culture devices. In this project, the main goal was to design and engineer tunable three-dimensional (3D) extracellular matrix (ECM)-mimicking scaffolds made of collagen and fibronectin (namely the two major building blocks of the ECM) that recapitulate the ECM structural and mechanical properties essential for wound healing and cancer research. Two different methods were implemented to fabricate 3D scaffolds. First, 3D collagen scaffolds with a ‘porous’ structure (fabricated by a previous student via an ice-templating technique) were used. It was shown that, by increasing collagen concentration to 1.25 wt.%, homogenous scaffolds with interconnected pores (needed for cell invasion through the entire scaffold) were obtained. Fibronectin (Fn) was then incorporated using thermal and mechanical gradients to modify protein content and tune scaffolds microarchitecture. The effect of Fn coating of the collagen underlying structure on cell behaviour such as cell adhesion, invasion and matrix deposition was studied. Results showed that overall more cells adhered to Fn-coated scaffolds with respect to pure collagen scaffolds. Furthermore, our findings indicated that cells were also able to sense the conformation of the Fn coating (as assessed by Fluorescence Resonance Energy Transfer, FRET) since they deposited a more compact ECM on compact Fn coating while a more unfolded and stretched ECM was deposited on unfolded Fn coating. Second, 3D more complex physiologically relevant scaffolds with a ‘fibrillar’ structure were fabricated via a cold/warm casting technique. Pure collagen scaffolds were first generated: in cold-cast scaffolds, clear thin and long collagen fibers were observed while warm-cast scaffolds were denser and comprised shorter collagen fibers. The effect of both collagen concentration and casting temperature on scaffolds’ microstructure was studied. Our results indicate a preponderant effect of temperature. We further engineered dual-protein fibronectin-collagen fibrillar scaffolds by incorporating Fn fibers using thermal gradient. Clear Fn fibers were observed in some conditions. FRET assessment of Fn fibers also showed significant difference of Fn conformation. In this more advanced casting technique, cells were initially embedded into the scaffolds, which provided a more homogeneous cell distribution and a better tissue-mimicking setting. In each case, the effect of resulting ECM properties was tested via cell viability assays. Our data indicate that cells were viable after 72 hours, they could proliferate inside the scaffolds and were able to spread in some conditions. Collectively, our 3D ECM-mimicking scaffolds represent a new tunable platform for biological and biomaterial research with many potential applications in tissue engineering and regenerative medicine. Investigating cell behaviour in 3D ECM-mimicking environment will provide valuable insights to understand cancer progression and approaches to limit the progression and ultimately prevent metastasis.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Dec. 11, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry Analytical Chemistry." Discipline: Chemistry; Department/School: Chemistry.

Screening of ethnomedicinal plants for antimicrobial activity among plants used in Ghanaian folklore medicine to treat infectious conditions was conducted on Alstonia boonei (Apocynaceae), Balanites aegyptiaca (Balatinaceae), Bidens pilosa (Compositae), Distemonanthus benthamianus (Leguminosae), Funtumia elastica (Apocynaceae), Kigelia africana (Bignonaceae), Mitracarpus villosus (Rubiaceae), Parquetina nigrescens (Asclepiadaceae) and Penianthus zenkeri (Menispermaceae). The selection of the plants was based on ethnomedicinal use and literature search and the aim was to investigate the scientific basis of their use in treating infectious diseases. It was, in addition, aimed at selecting the plant with the most potent activity for detailed bioactivity and phytochemical studies. The methanolic extracts of the plants showed antimicrobial activity with an MIC range of 0.5 - 2.0mg/ml against at least three of the test bacteria (Gram-positives and Gram-negatives) and the yeast-like fungi, Candida albicans, used. F. elastica leaf was selected for further bioactivity studies based on its relatively more potent antimicrobial activity. Bioactivity-guided phytochemical investigation of F. elastica, used to treat infections (including dysentery and gonorrhoea), wound and whooping cough, lead to isolation of eight compounds designated as C2RPA, C2RP1, C2RP2, C2RP3, C2RP4, C2RP5, C2RP6 and C2RP7. Three of them were characterized as (-)epicatechin [C2RP1], taxifolin-3ß-O-glucopyranoside [C2RP2] and the rare A-type proanthocyanidin, epicatechin-(4->8; 2-ºO--'7)-epicatechin (4-+8)-epicatechin (proanthocyanidin trimer) [C2RPA] using spectroscopic methods. This is the first report on isolation and characterization of these compounds from F. elastica. In addition, other compounds obtained as mixtures, and characterized to comprise hydrocarbons including hexadecane, octadecane, eicosane, heneicosane, tetracosane, octacosane, nonacosane and heptacosanew ere also identified. Epicatechin, the proanthocyanidin trimer, C2RP4 and C2RP5, showed antimicrobial activity against the Gram-positive bacteria and C. albicans with MIC range of 31- 500gg/m1; C2RP5 exhibiting the highest antimicrobial activity with an MIC of 31 μg/ml against Stapylococcus aureus. These compounds also exhibited an ability to destroy biofilm formed by MRSA using the standard Calgary MBECTM HTP Assay. Fractions of the crude extract and two isolates containing mainly taxifolin-3ß-Oglucopyranoside and proanthocyanidin trimer each exhibited anti-quorum sensing activity by inhibiting the QS controlled phenotypic purple violacein production by Chromobacterium violaceum. Furthermore, the extracts and the compounds isolated also showed an excellent antioxidant activity with ECso values ranging from 3.88μg (for proanthocyanidin trimer) to 24.59μg (for C2RP7). Also, taxifolin-3Q-Oglucopyranoside stimulated proliferation of fibroblast by 19.5% at a concentration of 4μg/ml. The study on F. elastica leaf has shown that in addition to its ability to arrest the growth of micro-organisms, the plant may have other novel mechanisms of attenuating bacterial pathogenicity by way of interfering with quorum sensing signals and formation of biofilm. Furthermore, the anti-oxidant activity and the ability to stimulate growth of fibroblast, coupled with the antimicrobial activity, may suggest that F. elastica may be useful in wound management. The study therefore shows that plants such as F. elastica may be useful in the management of infectious diseases and wound as purported in folklore medicine.

As society draws closer to the post-antibiotic era and the pipeline for alternatives dries, there is an urgent need for the development of novel antimicrobial therapies that do not promote bacterial resistance, particularly for immunocompromised chronic wound patients. Antimicrobial peptides (AMPs), including human-derived LL37, show considerable promise as broad spectrum alternatives that also have wound healing properties; however, few have been clinically implemented as novel antimicrobials due to their cytotoxicity stemming from a poor understanding of their mechanisms and low stability in vivo. It has been suggested that tethering, or attaching AMPs onto surfaces, is a viable strategy of delivering bioactive AMPs to surfaces while reducing cytotoxicity and improving stability. Thus, we designed new chimeric versions of LL37 with collagen-binding domains (CBD), derived from collagenase (cCBD-LL37) and fibronectin (fCBD-LL37) for non-covalent tethering onto collagen, a prevalent biopolymer in commercially available wound dressings and scaffolds. Our overall hypothesis was that CBDs would mediate stable tethering of broadly active, non-cytotoxic CBD-LL37 onto collagen-based scaffolds. We first studied the loading, release and bioactivities (e.g. antimicrobial activity and cytotoxicity) of each CBD-LL37 on commercially available 100% collagen type I PURACOL® wound scaffolds. We found that both cCBD-LL37 and fCBD-LL37 bound highly to collagen, were active against relevant wound pathogens, demonstrated stable activity after 14 days of release, and were not cytotoxic to human fibroblasts. The addition of different CBDs onto LL37 also markedly altered their soluble bioactivities. Using similar methods, we then studied the loading, release and bioactivity of each CBD-LL37 on a commercially available FIBRACOL® wound scaffolds, comprised of 90% collagen type I and 10% calcium alginate biopolymers. We found that both cCBD-LL37 and fCBD-LL37 also bound highly to and retained on collagen for 14 days, but were only active against Gram-negative P. aeruginosa. This suggested that the presence other biopolymers in addition to collagen, which is common among commercial wound dressings, could cause significant differences in binding, retention and bioactivities of CBD-LL37. To better understand how CBD modification affected CBD-LL37 structure leading to different bioactivities, we studied the CBD sequence-, peptide structure-, concentration-, time-, and bilayer composition-dependent interactions of soluble CBD-LL37 and compared these findings with the properties of unmodified LL37. Using Molecular Dynamics (MD) simulations, circular dichroism (CD) spectroscopy, quartz crystal microbalance with dissipation (QCM-D), and fluorescent bilayer imaging we determined the structural basis behind CBD alterations in bioactivities. MD and CD, in addition to other intrinsic CBD properties (helicity, amphiphilicity, charge) we hypothesized that cCBD-LL37 utilized similar mechanisms as unmodified LL37 while fCBD-LL37 demonstrated based primarily on surface adsorption. We used QCM-D and Voigt-Kelvin viscoelastic modeling to determine the time- and concentration-dependent interactions of unmodified LL37 with model mammalian lipid bilayers, the mechanisms of which are still controversial in literature despite being widely studied. These results were used to propose a model for the interaction mechanism of LL37 with zwitterionic bilayers that aligned with its bioactive concentrations. LL37 adsorbed at concentrations where it is immunomodulatory until reaching a threshold which corresponded with its antimicrobial concentrations. The threshold was correlated to lipid bilayer saturation, after which LL37 formed transmembrane pores. We observed collapse of the bilayer into a rigid proteolipid film at concentrations higher than the reported cytotoxic threshold of LL37. The mechanistic and structural information for each CBD-LL37 and unmodified LL37 provided a baseline for QCM-D and Voigt-Kelvin viscoelastic modeling to further elucidate the time-, concentration-, lipid composition- and CBD sequence-dependent basis behind the observed bioactivities of cCBD-LL37 and fCBD-LL37. We found that similar to LL37, cCBD-LL37 demonstrated pore formation mechanisms likely due to their similar charges, structural content and amphiphilicity. fCBD-LL37 demonstrated time-dependent, adsorption-based mechanism likely due to its anchoring aromatic residues, low charge, and low amphiphilicity. Knowledge gained from this study allowed mechanistic predictions of two newly designed hypothetical CBD-LL37 peptides. Results from this study contribute to a better understanding of a new class of antimicrobial, non-cytotoxic therapies based on collagen-tethered CBD-LL37, bringing it closer to clinical implementation in chronic wound applications and demonstrate the viability of biopolymer tethering as a platform toward using AMPs to quench the resistance crisis.

Ganoderma lucidum, a saprophytic bracket fungus native to Queensland’s tropical rainforests, was traditionally used to heal wounds and ensure smooth tissue regeneration. Its pharmaceutical formulations such as teas, decoctions and capsules are claimed to cover a range of diseases, however, the alleged pharmacological properties of G. lucidum have not yet been fully scientifically validated. This study has demonstrated G. lucidum’s in vitro role as a wound healing agent as a result of its concomitant and/or predominant antibacterial, anti-inflammatory and regenerative properties. This research will contribute to a development of a novel therapeutic for the treatment of chronic non-healing wounds.

The goal of this research was to develop a system of individualized medicine that could be applied to dermal wounds serving as a wound dressing and synthetic extracellular matrix while delivering stem cells to the wound bed. First, fabrication parameters for electrospinning polymer fibers were determined. This involved evaluating fiber morphology with respect to polymer selection and solution concentration. Next, construct fabrication was examined to produce an integrated void space, or cargo area, suitable to maintain stem cells. In vitro studies to ensure stem cell viability and phenotype were conducted, and results supported the notion that cells could be administered to the wound site through construct pre-seeding. Lastly, in vivostudies were conducted to evaluate the construct as an applied biomaterial and as a cellular delivery device. Wound closure and quality were assessed, and neo-vascularization quantified. This project will provide insight into the tissue engineering field regarding cell-based therapies and dermal wound healing.

This project has made an important contribution towards understanding the medicinal potential of three Australian plants. The identification of novel and previously isolated wound healing compounds from the methanolic extracts of the target plants demonstrate that Australian natives are a rich source of bioactive constituents, which may have a therapeutic role for the future treatment of chronic non-healing wounds.

Chronic wounds are a significant burden to national health services worldwide and a potential reason for patients’ declining quality of life. As such, this project focused on assessing the therapeutic effect of two Australian native plants, Alocasia macrorrhizos and Syncarpia hillii for their potential in chronic wound healing, through investigating their antibacterial, anti-inflammatory, and regenerative properties.

Skin wound dressings are commonly used to stimulate and improve the repair of the injured tissue. In a healthy organism, the wound healing process is a highly articulate cascade of events that take place sequentially but with an overlap. Commercially available patches and sterile gauzes are commonly used to separate the wound from the external environment and avoid bacterial contaminations. Although constituting a physical barrier that protects the wound area, these systems do not possess some pivotal characteristics that can effectively guide the healing process. In the last decades, the interest in exploiting the properties of natural-based polymers (e.g. biocompatibility, non-toxicity, biodegradability) for wound dressing fabrication has been growing. Natural polymers are a promising class of materials able to improve the healing process and protect the wound against bacterial infections. In particular, plant-based biopolymers have been shown promising features that make them ideal for the fabrication of nano/micro-structured scaffolds for wound healing applications. More detailed considerations about it will be discussed in Chapter 1. This doctoral thesis aimed to develop and fabricate plant-based wound dressings, mainly composed of zein, an alcohol-soluble protein extracted from corn, and pectin, the most abundant polysaccharide placed in the plant cell wall. In the literature, there are some studies in which zein and pectin were mixed to produce micro/nanoparticles. Nonetheless, little is known about their blends used for the production of other types of micro and nanocomposites, such as films and nano/microfibers. In the first project, discussed in Chapter 2, zein and pectin were used to fabricate composite polymeric films. The samples were chemically and physically analyzed. The degradation rate of the samples was monitored over time after their immersion in various buffers with different pH values. The presence of the pectin inside the composite films led to a faster degradation rate, as also demonstrated by thermogravimetric analyses. This trend was also confirmed by monitoring the release of a hydrophilic model compound loaded inside the constructs, which resulted in a higher release for the samples containing pectin. Moreover, the films showed the ability to inhibit the growth of three skin pathogens (Staphylococcus aureus, Escherichia coli, and Candida albicans), demonstrating an excellent antimicrobial activity. Finally, in vitro biocompatibility was confirmed on a representative class of skin cells via MTS assay and through cell morphology inspection by confocal microscopy. In a second project, discussed in Chapter 3, plant-based microfibrous scaffolds were fabricated through vertical electrospinning, starting from various zein/pectin formulations. These systems were loaded with various concentrations of a bioactive molecule, Vitamin C (VitC), evaluating its release from the 3D scaffolds over time in parallel to its antioxidant properties and collagen synthesis stimulation activity. Pectin's ability to crosslink in the presence of Ca2+ was exploited by immersing the samples in a calcium chloride solution. In this way, non-crosslinked and crosslinked samples were obtained, characterized, and compared. The main comparisons were made in terms of degradation rate and water uptake ability. These analyses highlighted the ability of the crosslinked samples to behave as a hydrogel when immersed in an aqueous solution, resulting in a more susceptibility toward a tested protease. In vitro biocompatibility was confirmed on two representative skin cell populations through MTS assay, confocal microscopy investigation, and direct plating test. Subsequently, the ability of the VitC to stimulate the synthesis of collagen in fibroblasts was confirmed by RT-PCR. Finally, the samples that showed better biocompatibility and gene expression characteristics were selected for the in vivo test on a mouse model of skin burns. The investigation outcomes demonstrated the ability of our newly designed construct to significantly reduce the inflammatory cytokine in the wound area. Lastly, the general conclusions of this thesis study and some considerations for future steps are summarized in Chapter 4.

Chronic ulcers are an important and costly medical issue, causing their sufferers a large amount of pain, immobility and decreased quality of life. The common pathology in these chronic wounds is often characterised by excessive proteolytic activity, leading to the degradation of both the extracellular matrix, as well as key factors critical to the ulcer's ability to heal. As matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), it was hypothesised that this specific proteolytic activity was directly related to an ulcer's chronic nature. Although previous studies have identified elevated proteases in CWF, many have reported contradictory results and therefore the precise levels and species of MMPs in CWF are poorly understood. The studies reported herein demonstrate that MMP activity is significantly elevated in CWF compared with acute wound fluid (AWF). In particular, these studies demonstrate that this proteolytic activity can be specifically attributed to MMPs and not another class of proteases present in wound healing. Furthermore, it is shown that MMP-9 is the predominant protease responsible for matrix degradation by CWF and is an indicator of the clinical status of the wound itself. Moreover, MMP-9 can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and also tethered to a synthetic biocompatible hydrogel compromised of aqueous poly (2-hydroxy methacrylate) PHEMA synthesised in the presence of poly(ethylene glycol) (PEG). Together, these results highlight the potential use of a tethered MMP inhibitor as an improved ulcer treatment to inhibit protease activity in the wound fluid, while still allowing MMPs to remain active in the wound bed where they perform vital roles in the activation of growth-promoting agents and immune system regulation.

Chronic ulcers are an important and costly medical issue, causing their sufferers a large amount of pain, immobility and decreased quality of life. The common pathology in these chronic wounds is often characterised by excessive proteolytic activity, leading to the degradation of both the extracellular matrix, as well as key factors critical to the ulcer's ability to heal. As matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), it was hypothesised that this specific proteolytic activity was directly related to an ulcer's chronic nature. Although previous studies have identified elevated proteases in CWF, many have reported contradictory results and therefore the precise levels and species of MMPs in CWF are poorly understood. The studies reported herein demonstrate that MMP activity is significantly elevated in CWF compared with acute wound fluid (AWF). In particular, these studies demonstrate that this proteolytic activity can be specifically attributed to MMPs and not another class of proteases present in wound healing. Furthermore, it is shown that MMP-9 is the predominant protease responsible for matrix degradation by CWF and is an indicator of the clinical status of the wound itself. Moreover, MMP-9 can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and also tethered to a synthetic biocompatible hydrogel compromised of aqueous poly (2-hydroxy methacrylate) PHEMA synthesised in the presence of poly(ethylene glycol) (PEG). Together, these results highlight the potential use of a tethered MMP inhibitor as an improved ulcer treatment to inhibit protease activity in the wound fluid, while still allowing MMPs to remain active in the wound bed where they perform vital roles in the activation of growth-promoting agents and immune system regulation.

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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
As queimaduras de um modo geral apresentam altas incidências no Brasil e no mundo, culminando, em muitos casos, em necessidade de atenção hospitalar. Muitas alternativas terapêuticas tem sido propostas no sentido minimizar o sofrimento dos pacientes que são acometidos por este tipo de agressão, dentre elas está a membrana de celulose microbiana, detentora de propriedades peculiares que a tomam um material altamente promissor na aceleração do processo cicatricial bem como a terapia Laser de baixa potencia, conhecidamente, eficiente na biomodulação de processos biológicos. Levando em consideração a escassez de estudos experimentais que contemplem as queimaduras, em especifico, as de segundo grau, bem como a associação da laserterapia a membrana de celulose microbiana no tratamento destas, o presente estudo teve como objetivos estabelecer um modelo experimental capaz de reproduzir uma queimadura de segundo grau e avaliar histológicamente do processo de reparo dessas lesões induzidas em dorso de ratos e submetidas a tratamento com membrana de celulose associada ao Laser AIGalnP. 660nm, 40mW. Para este fim foram utilizados 64 ratos Wistar albinus machos divididos em 4 grupos: Controle: sem tratamento (n=16); Gí:Tratado com Laser (n=16); Gll: Tratado com Membrana (n=16) e GIH: Tratado com Membrana e Laser (n=16). Uma queimadura quadrada padronizada de 4cm^foi irKiuzida no dorso dos ratos de todos os grupos. Após a realização da queimadura foi aplicada a membrana de celulose microbiana; nos grupos pré-estabelecidos o protocolo de laserterapia foi executado, imediatamente após a queimadura e a cada 48 horas com a dosimetria de 20J/cm^ por sessão, até o sacrifício, que se deu em 24h, 3, 7 e 14 dias. Os espécimes foram fixados em formalina tamponada e processados histológicamente pelas colorações H/E, Picrosírius e Orceína, sendo posteriormente realizada a análise microscópica e estatística dos dados (testes de Kolmogorov Smirnov e Exato de Fisher). Ao final da análise dos resultados apesar de não haverem diferenças relevantes estatisticamente, foi observada um aumento da quantidade de vasos neoformados e edema de graduação intensa, no grupo tratado apenas com Laser em especial no sétimo dia experimental e uma redução marcante das fibras elásticas a medida que o experimento evoluía, em todos os grupos estudados. Um resultado interessante verificado neste estudo foi a presença de adipócitos na derme reticular superficial. Dessa forma concluímos que: O modelo proposto foi capaz de reproduzir a queimadura de segundo grau; Independente da modalidade de tratamento empregada a queimadura de segundo foi por si só capaz de induzir a redução das fibras elásticas na pele de ratos; A agressão representada pela queimaduras de segundo grau parece ter sido responsável pelo surgimento de células adiposas na derme até o sétimo dia experimental.
In general, there are great incidences of cutaneous burns in Brazil and worldwide, and very often, there is a need for the hospitalization of patients. In the last few years, many alternative therapies have been proposed to minimize the suffering of patients who are affected by this type of injury. Among them is the microbial cellulose membrane which holds unique properties making it a promising material in accelerating the healing process. On the other hand, the low-level-laser therapy is known as an efficient modulator of biological systems. Taking literature into account, there are very few experimental studies focusing on cutaneous bums, pariiculariy the second-degree related and also the associatton of laser therapy and the microbiane cellulose membrane in the treatments of these lesions. Therefore, the present study is aimed at establishing an experimental model for cutaneous second degree burns and to histologically evaluate the healing process of these kind of lesions induced in the dorsal surface of wistar rats subjected to treatment with micobial microbiane cellulose membrane associated or not with AIGalnP l-aser, 660nm, 40mW. For that purpose, we used 64 male \Mstar albinos rats placed into four groups: Control: with no treatment (n=16), Gl: Treated with laser (n=16), Gil: Treated with membrane (n=16), and Gill: Treated with laser and membrane (n=16). A standard 4 cm^ bum was inflicted on the back skin of all the rats in each group. According to the experimental groups, the microbiane cellulose membrane was applied to the burnt area of the animals and the established Laser protocol was immediately run after the surgical procedures, and later on every 48 hours with a dose of 20J/cm‘. The animals/specimen were sacrificed under C0> atmosphere at 24 hours, three, seven and 14 days. The skin samples were fixed in buffered formalin, embedded in parafin and histological slides with 4 ¡j thick were prepared. These slides were stained in HE, Sirius Red and Orcein and analysed by light microscopy. Data were statistically analysed (Kolmogorov Smirnov and Fisher’s Exact tests). Despite the absence of statistical significance (maybe due to the small number of rats per group) the results showed an higher number of newly formed blood vessels and exacerbated edema after seven days in the group treated only with Laser All groups exhibited a reduction of elastic fibers which became more evident at the end of the experiments. One interesting result was the presence of addipose cells inside the reticular demnis in all the groups. We concluded that: The proposed experimental nmdel was able to reproduce a second degree skin bum; The second degree bum induced the reducing of the elastic fibers; The injury represented by the bum might be responsable for the presence of adipocytes in the denmis till the seventh experimental day.

In this project, the therapeutic effects of two medicinal plants were assessed for their potential in treatment of chronic wounds. Currently, chronic wounds impose a heavy socio-economic burden on healthcare systems and societies world-wide. As such, the antibacterial, anti-inflammatory effects and human skin cell regenerative properties of the plant extracts and their isolated bio-active compounds, inflicts high potential to develop novel therapeutics for the treatment of chronic wounds.

Mucoadhesive drug delivery systems which are being used from 1980’s to avoid first pass metabolism of drugs, commercially exist for only systemic drug delivery with fast erosion times (15-60 min), that may not be appropriate for local oral disorders. The goal of this research was to develop and characterize mucoadhesive films with flexibility of carrying different drugs and proteins and provide sustained release for local treatment of oral disorders. Mucoadhesive films composed of polyvinylpyrrolidone (PVP) and carboxymethlycellulose (CMC) were formulated with imiquimod, an immune response modifier. Problems such as solubilization of imiquimod to increase drug loading, uniformity in films and total amount of drug released into supernatants were addressed by use of acetate buffer after investigating multiple methods. Subsequently, other relevant properties of mucoadhesive systems, such as adhesion (shear, pull-off), tensile properties, swelling profiles, transport kinetics, and subsequent changes in release profiles as a function of film composition were characterized. The potential of the system for local retention of imiquimod, determined in oral mucosa of hamsters showed time dependent decrease in imiquimod amount through 12 hours, with no traces of drug in blood. Further testing in humans revealed that the residence time of the mucoadhesive films depended on the application site, increasing in the order of tongue < cheek < gingiva. In parallel, mucoadhesive films loaded with epidermal growth factor (EGF) were developed to promote treatment of oral mucosal wounds. Bioactivity was tested in vitro on buccal tissues by creating a wound followed by application of films. Although EGF-loaded films did not accelerate wound healing, but rather elicited a hyperparakeratotic response. In vitro buccal tissues may not be appropriate for testing the effects of EGF in wound healing without incorporation of other biochemical factors. Overall, a mucoadhesive system capable of delivering bioactive small molecules and proteins in sustained manner was developed in this work. A thorough understanding of the system properties was achieved to further tune for future applications. In vitro studies and in vivo studies in hamsters and humans clearly showed the potential and usefulness of the system to translate in to clinic for treatment of oral precancerous lesions.

Chronic wounds are a costly and growing problem to global health care systems and affect the productivity of patients and their quality of life. In all cultures, people have used medicinal plants for the treatment and alleviation of various health issues. This project has shown a new potential role for the primary extracts and isolated compounds of C. terminalis derived-kino and the primary extracts of H. tiliaceus (L.) leaves as wound healing agents in vitro, and hence, potentially paves the way for future novel therapeutics for the treatment of chronic non-healing wounds.

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Neste trabalho foram avaliadas as atividades angiogênica e antimicrobiana do óleo essencial da Curcuma longa (açafrão). O óleo essencial foi extraído pelo método da hidrodestilação por arraste a vapor, a partir do pó dos rizomas do açafrão. A atividade angiogênica foi avaliada pelo método experimental da membrana corioalantóide, e o tratamento estatístico realizou-se por meio da análise de variância (Kruskal-Wallis) e por comparação múltipla (método de Dunn) do grupo teste com os grupos dos controles, considerando o nível de significância de p<0,05. A atividade antimicrobiana do óleo essencial foi avaliada para bactérias gram negativa Escherichia coli e gram positiva Staphylococcus simulans. No teste realizado com a membrana corioalantóide, o óleo essencial do açafrão produziu aumento da rede vascular superior ao controle negativo e inibidor (p<0,05), não apresentou diferença estatística quanto comparada ao controle indutor (p>0,05). A avaliação da atividade antimicrobiana demonstrou que não houve formação do halo de inibição para o óleo essencial do açafrão.

There is interest in the pharmaceutical area in the use of sponges, produced with biopolymers, as matrices for the controlled release of drugs. The biopolymers for pharmaceutical applications must have physicochemical and appropriate mechanical and biocompatibility. Alginate is a biopolymer, obtained from the alginic acid, which has been largely studied due to its ability to incorporate and promote the controlled release of drugs. Gelatin is a protein obtained by hydrolysis of collagen and has been used in obtaining produce hydrogels and sponges with therapeutic properties. Usnic acid (UA) is the most abundant metabolite of the lichen Cladonia substellata has proven antimicrobial, anti-inflammatory, analgesic and healing activities. In this context, the aim of this work was to physicochemical characterization and evaluate the effect of alginate-gelatin sponges, containing usnic acid on wound healing in rats. The sponges were prepared from gelatin, alginate and incorporated with usnic acid in concentrations of 1 and 5% (m/v) and freeze dried. For material characterization were used thermogravimetry/derivative thermogravimetry (TG/DTG), differential scanning calorimetry (DSC), infrared absorption spectroscopy (FTIR), scanning electron microscopy (SEM) and mechanical properties tests. The biological assay was performed with 36 rats divided into three groups: without treatment (CTR); treated with inert sponge (ALG/GEL); and treated with the sponge containing usnic acid 5% (AU 5%). After 7 and 14 days, six animals from each group were submitted to euthanasia for histological analysis. The average content of usnic acid was 60.03 and 97.38% for sponges in 1 and 5%, respectively. DSC analysis results show the absence of endothermic peak of UA pure, indicating that the drug was incorporated into the polymer system. TG/DTG curves corroborate with the DSC data and showed that the incorporation of usnic acid has not changed the thermal behavior of sponges. The FTIR spectra of sponges showed a sum corresponding to the event of usnic acid, alginate and gelatin bands, indicating the presence of these compounds in the system. The analysis by SEM showed that the incorporation of different concentrations of UA, in sponges promotes changes in microscopic structure, resulting in different mechanical behaviors. The biological assays in 7 days, showed an inflammatory reaction in the marginal and central portions of the wound, but ALG/ GEL and AU 5% groups, promoted an acceleration in the formation of granulation tissue. In 14 days, in the AU 5% group, was a clear reduction of the inflammatory component even in the central portion of the wound. Additionally, there was a marked reduction in vascular component, when compared to the CTR group with a homogeneous pattern of colagenization type III in the entire length of the scar. In conclusion, the incorporation of usnic acid has not changed the thermal stability of sponges and, in addition, the use of alginate-gelatin sponge and promotes the wound healing and the incorporation of the drug eases wound contraction and a homogeneous deposition of collagen, avoiding the formation of hipertrophic scars.
Existe um interesse na área farmacêutica no uso de esponjas, produzidas com biopolímeros, como matrizes para a liberação controlada de fármacos. Os biopolímeros para aplicações farmacêuticas devem possuir propriedades físico-químicas e mecânicas adequadas, além de serem biocompatíveis. O alginato é um biopolímero, obtido a partir do ácido algínico, que tem sido largamente estudado devido a sua capacidade de incorporar compostos e promover a liberação controlada de fármacos. A gelatina é uma proteína obtida pela hidrólise do colágeno e vem sendo empregada na obtenção de hidrogéis e esponjas com propriedades terapêuticas. O ácido úsnico (AU) é o metabólito mais abundante do líquen Cladonia substellata e possui comprovada atividade antimicrobiana, anti-inflamatória, analgésica e cicatrizante. Neste contexto, o objetivo deste trabalho foi caracterizar físico-quimicamente e avaliar o efeito de esponjas de alginato-gelatina, contendo ácido úsnico sob a cicatrização em ratos. As esponjas foram preparadas a partir do alginato e gelatina, incorporadas com ácido úsnico, nas concentrações de 1 e 5% (m/v) e liofilizadas. Para caracterização do material foram utilizadas a análise termogravimétrica/termogravimétrica derivada (TG/DTG), a calorimetria exploratória diferencial (DSC), espectroscopia de absorção na região do infravermelho (FTIR), microscopia eletrônica de varredura (MEV) e testes de propriedades mecânicas. O ensaio biológico foi realizado com 36 ratos divididos em três grupos: sem tratamento (CTR); tratados com esponja inerte (ALG/GEL); e tratados com esponja contendo ácido úsnico a 5% (AU 5%). Após 7 e 14 dias, seis animais de cada grupo foram eutanasiados para análise histológica. O teor médio de ácido úsnico foi de 60,03 e 97,38% nas esponjas de 1 e 5%, respectivamente. Os resultados da análise de DSC mostram a ausência do pico endotérmico referente ao AU puro, indicando que o fármaco foi incorporado ao sistema polimérico. As curvas TG/DTG corroboram com os dados do DSC e mostraram que a incorporação do ácido úsnico não alterou o perfil geral da decomposição térmica das esponjas. Os espectros FTIR das esponjas mostraram um somatório de eventos correspondentes às bandas do ácido úsnico, alginato e gelatina, indicando a presença dos referidos compostos no sistema. A análise por MEV mostrou, que a incorporação de diferentes concentrações de AU, nas esponjas promove alterações na estrutura microscópica, resultando em diferentes comportamentos mecânicos. Os ensaios biológicos em 7 dias, mostram uma reação inflamatória nas porções centrais e marginais da ferida, porém os grupos ALG/GEL e AU 5%, promoveram uma aceleração na formação de tecido de granulação mais maduro. Em 14 dias, no grupo AU 5%, foi nítida a redução do componente inflamatório mesmo na porção central da ferida. Adicionalmente, observou-se uma marcante redução na rede vascular, quando comparado ao grupo CTR, acompanhada de um padrão homogêneo de colagenização tipo III em toda a extensão da cicatriz. Em conclusão, a incorporação do ácido úsnico não alterou a estabilidade térmica das esponjas e, além disso, o uso de esponja de alginato e gelatina favorece a cicatrização de feridas e a incorporação do fármaco facilita a contração da ferida e uma deposição homogênea de colágeno, evitando a formação de cicatrizes antiestéticas.

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Mestrado Acadêmico em Ciências do Cuidado em Saúde
Este estudo teve como objetivo analisar a presença de cepas de Staphylococcus aureus nas lesões cutâneas crônicas tratadas com hidrogel a 2% e placa de poliuretano. Para tal, foi realizada pesquisa descritiva, com abordagem quantitativa, tendo como campo de pesquisa o Ambulatório de Reparo de Feridas do Hospital Universitário Antônio Pedro (HUAP/UFF) e a Policlínica Comunitária da Engenhoca (PCE), ambos localizados na cidade de Niterói, RJ. A análise microbiológica foi realizada no Laboratório de Controle Microbiológico da Faculdade de Farmácia da Universidade Federal Fluminense (UFF) e no Instituto de Microbiologia Paulo de Góes da Universidade Federal do Rio de Janeiro (UFRJ). A coleta de dados foi realizada em duas etapas, a primeira relacionada ao exame clínico com a identificação do paciente e descrição clínica das lesões e a segunda relacionada à coleta do espécime clínico utilizando-se como instrumento de coleta o swab. Todas as cepas de S. aureus analisadas foram identificadas por MALDI-TOF e a suscetibilidade a antimicrobianos foi determinada pelo teste de disco-difusão em meio sólido, seguindo as normas recomendadas do CLSI (Clinical and Laboratory Standards Institute). A reação em cadeia da polimerase (PCR) foi empregada na detecção do gene mecA e genes lukF-PV e lukS-PV. A diversidade clonal foi verificada pelo PFGE (pulsed-field gel electrophoresis). O S. aureus foi detectado em 82,9% (29/35) dos pacientes em uso de hidrogel e em 100% (8/8) dos pacientes em uso de placa de poliuretano. Os pacientes em uso de placa de poliuretano com prata apresentaram mais sinais clínicos de infecção quando comparados aos pacientes em uso de hidrogel a 2%, principalmente pela presença do exsudato purulento. A razão de prevalência demonstrou que os pacientes que usaram a placa de poliuretano com prata tiveram pelo menos 3,6 vezes maior chance de apresentarem infecção nas feridas quando comparados com os pacientes em uso de hidrogel. A maioria dos S. aureus identificados, em ambos os campos de pesquisa, apresentou resistência à penicilina, meticilina, eritromicina e clindamicina. Em cinco pacientes (10 cepas- 20%) foi observada amplificação para o gene mecA, demonstrando a colonização por MRSA. Não foi observada amplificação para os genes lukF-PV e lukS-PV. Embora tenha sido detectada grande diversidade genética entre as cepas analisadas, o mesmo padrão se repetiu entre os S. aureus coletados em dois momentos diferentes nos mesmos pacientes. No entanto, as amostras 1 e 32/32* apresentaram o mesmo padrão de fragmentação de DNA pelo PFGE. Através da razão de prevalência, determinou-se que os pacientes com MSSA têm 16 vezes mais chance de ter infecção em feridas quando comparados aos pacientes com MRSA. Assim, o tratamento com hidrogel a 2% ou placa de poliuretano não interferiu na colonização por S. aureus. Além disso, verificamos que o uso de placa de poliuretano com prata não é indicado para feridas infectadas quando o paciente possuir como comorbidades hipertensão arterial e insuficiência venosa crônica.
This study aimed to analyze the presence of Staphylococcus aureus in chronic wounds treated with hydrogel 2% and polyurethane plate. To do this, was done a descriptive study with a quantitative approach. The field research was the Wound Repair Clinic at the University Hospital Antônio Pedro (HUAP / UFF) and the Community Polyclinic of the Engenhoca, both located in the city of Niterói, RJ. The microbiological analysis was conducted at the Laboratory of Microbiological Control of the Faculty of Pharmacy of the University Federal Fluminense (UFF) and the Institute of Microbiology Paulo de Góes of the University Federal of the Rio de Janeiro (UFRJ). Data collection was carried out in stages, the first related to the clinical examination with the patient identification and clinical description of the wounds and the other step related to the collection of the clinical specimen using the swab. All strains of S. aureus analyzed were identified by MALDI-TOF and susceptibility to antibiotics was determined by disk diffusion test on solid medium following the standards recommended by CLSI (Clinical and Laboratory Standards Institute). The polymerase chain reaction (PCR) was used in the detection of the mecA gene and lukF-PV and luks-PV genes. The clonal diversity was verified by PFGE (pulsed-field gel electrophoresis). The S. aureus was detected in 82.9% (29/35) of patients using hydrogel and in 100% (8/8) the patients using polyurethane plate. Patients using polyurethane plate with silver presented more clinical signs of infection when compared to patients using hydrogel 2%, mainly by the presence of purulent exudate. The prevalence ratio showed that patients who used polyurethane plate with silver had at least 3.6 times more chance to have infection in the wound when compared to patients using hydrogel. Most S. aureus identified in both research fields presented resistance to penicillin, methicillin, erythromycin and clindamycin. Five patients (10 strains - 20%) were observed amplification for the gen mecA demonstranting colonization by MRSA. There was no amplification for lukF-PV and lukS-PV genes. Although was detected a big genetic diversity among the strains analyzed, the same pattern repeated among the S. aureus collected at two different times from the same patients. However, samples 1 and 32/32* showed the same fragmentation pattern by PFGE. Through the prevalence ratio identified that patients with MSSA had 16 times more likely to have infection in wounds when compared to patients with MRSA. Thus, the treatment with hydrogel 2% or polyurethane board did not interfered in the colonization by S. aureus. In addition, we perceived that the use of polyurethane plate with silver is not indicated for infected wounds when patients had comorbidities such as hypertension and chronic venous insufficiency.

Currently, biopolymers have aroused great interest mainly in the food, pharmaceutical, cosmetic, and also in the biomedical fields, due to the intelligent and versatile properties that present, namely, biocompatibility, biodegradability, and non-toxicity. In addition to these properties, many polymers have vital characteristics that assist in wound healing processes and, when incorporated into nanoparticles of elements such as silver (Ag), gold (Au), and selenium (Se), further increase the effectiveness of these polymers. Therefore, the main objective of this study was to determine the best conditions for the preparation of biocomposites for application in the treatment of wounds, using different extracellular bacterial polysaccharides (EPSs) (EPS1, EPS2, EPS3, EPS4, and FucoPol) as reducing and stabilizing agents for the synthesis of silver nanoparticles (AgNP), gold (AuNP) and selenium (SeNP). EPSs were characterized as to the composition in sugars and substituting groups, because, depending on the composition, they can interfere in some way in the properties of each one. EPS1 is composed mainly of mannose (56.1% mol), EPS2 and EPS3 are composed mainly of glucose, 67% mol and 39.1% mol, respectively, EPS4 is composed mainly of glucuronic acid (55.9% mol) and FucoPol by fucose (34.9 mol%). Biocomposites were also characterized by UV-vis spectroscopy, FTIR, TGA, XRD, DLS, Zeta potential and ICP. Cytotoxicity and wound healing effect of EPSs and EPS/NPs biocomposites were analyzed in human keratinocyte cells (HaCaT cell line). The samples were tested using a polymer concentration of 62.5 - 1000 mg/L and none of the samples showed cytotoxicity, except for the EPS3/Ag+ biocomposite when tested at a polymer concentration above 125 mg/L, with a content Ag+ above 6.9 mg/L (decreased cell viability by ~91.9%). On the other hand, among all EPSs and their functionalized materials, FucoPol and the biocomposite FucoPol/AgNP exhibited a greater capacity to promote keratinocyte migration in vitro. Although EPS1 did not present wound healing effect, the biocomposites EPS1/AgNP (100 mg/L) and EPS1/AuNP (500 mg/L) showed to promote the migration of HaCaT cells. The results obtained suggest that both FucoPol and the biocomposite FucoPol/AgNP, as well as the biocomposites EPS1/AgNP and EPS1/AuNP, may have potential application in dermo-cosmetics, mainly in wound healing mechanisms.
Atualmente, os biopolímeros têm despertado grande interesse principalmente na área alimentar, farmacêutica, cosmética e também na área biomédica, devido às propriedades inteligentes e versáteis que apresentam, nomeadamente, biocompatibilidade, biodegradabilidade e não toxicidade. Além dessas propriedades, muitos polímeros possuem características vitais que ajudam em processos de cicatrização de feridas e quando incorporados com nanopartículas de elementos como a prata (Ag), ouro (Au) e selénio (Se), aumentam ainda mais a eficácia desses polímeros. Assim sendo, o principal objetivo deste estudo foi determinar as melhores condições de preparação de biocompósitos para aplicação em tratamento de feridas, utilizando diferentes polissacáridos bacterianos extracelulares (EPSs) (EPS1, EPS2, EPS3, EPS4 e FucoPol) como agentes redutores e estabilizadores para a síntese de nanopartículas de prata (AgNP), ouro (AuNP) e selénio (SeNP). Os EPSs foram caracterizados quanto à composição em açúcares e grupos substituintes, pois, dependendo da composição, podem interferir de certa forma nas propriedades apresentadas por cada um. O EPS1 é composto principalmente por manose (56.1 %mol), o EPS2 e o EPS3 são compostos principalmente por glucose, 67 %mol e 39.1 %mol respectivamente, o EPS4 é composto maioritariamente por ácido glucurónico (55.9 %mol) e o FucoPol por fucose (34.9 %mol). Os biocompósitos também foram caracterizados por espectroscopia UV-vis, FTIR, TGA, XRD, DLS, potencial Zeta e por ICP. A citotoxicidade e o efeito de cicatrização de feridas dos EPS e dos biocompósitos EPSs/NPs foram analisados em células de queratinócitos humanos (linha celular HaCaT). As amostras foram testadas usando uma concentração de polímero de 62.5-1000 mg/L e nenhuma amostra apresentou citotoxicidade, excepto para o biocompósito EPS3/Ag+ quando testado em uma concentração de polímero acima de 125 mg/L, com um teor de Ag+ acima de 6.9 mg/L (diminuição da viabilidade cellular em ~ 91.9%). Por outro lado, entre todos os EPSs e seus materiais funcionalizados, o FucoPol e o biocompósito FucoPol/AgNP exibiram uma maior capacidade de promover a migração in vitro dos queratinócitos. Embora o EPS1 não tenha apresentado efeito cicatrizante, os biocompósitos EPS1/AgNP (100 mg/L) e EPS1/AuNP (500 mg/L) mostraram promover a migração de células HaCaT. Os resultados obtidos sugerem que tanto o FucoPol e o biocompósito FucoPol/AgNP, bem como os biocompósitos EPS1/AgNP e EPS1/AuNP podem ter potencial aplicação na dermocosmética, sobretudo nos mecanismos de cicatrização de feridas.

碩士
國立臺北科技大學
生物科技研究所
97
Skin, one of the largest organs in human body, covers whole body surface and functions as a first line of defense mechanisms against the invasion of foreign microbes. Once the integrity of skin is disrupted by an open wound, the protective barrier is lost and the wounded area could provide an entry site for the foreign microorganisms. Microbial infection will perturb and delay the progression of normal wound healing and causes the chronicity of cutaneous wound. Severe infection could cause extensive tissue necrosis, amputation, systemic sepsis, and death. In general, most of commercial wound dressings deal with either wound infection or wound healing. To control infection and improve wound healing simultaneously, we constructed a dressing combined with collagen and silver compounds. Collagen is a major component of connective tissues and plays an important role in normal wound healing, especially in the granulation phase. The advantages of collagen include high biocompatibility, non-cytotoxicity, low immunogenicity, biodegradability, and bioresorption. Collagen is also attractive to be a flexible drug delivery system. The silver compounds have powerfully antimicrobial activity against a broad spectrum of microbes, including common wound pathogens. We also establish the in vitro antimicrobial tests and an in vivo infected wound model to assay the antimicrobial efficacy of this dressing. In summary, we demonstrated the dressing comprised of both collagen and silver compounds not only effectively controlled the infection but also significantly promoted the wound healing.

“Surgery is a profession defined by its authority to cure by means of bodily invasion. The brutality and risks of opening a living person's body have long been apparent, the benefits only slowly and haltingly worked out”, says Atul Rawande on reviewing 200 yrs. of Surgery as a specialty in NEJM. My research focuses on working out these benefits, specifically looking at reduction of scar tissue formation in ENT, Abdominal & Spine surgery. Scar tissue formation is an outcome of healing process that can be excessive due to inflammation or infection and thereby has the ability to curtail the benefits or warrant revision surgery. Multiple strategies have been tested and employed thus far and none have given favourable results without causing additional harm or economic burden in health care costs. I propose to use a hydrogel synthesized by combining Chitosan and Dextran aldehyde -Chitin is an exoskeleton extracted polymer and Dextran Aldehyde a sugar, with added noveldrugs Deferiprone and Gallium Protoporphyrin providing additional anti scaring and antibiotic properties which could potentially augment the healing properties of the gel. I have conducted 3 types of studies. There are 2 animal studies and a Phase 1 Human clinical trial. The animal studies are an abdominal surgery rat model and a spine surgery sheep model. These studies show the safety and efficacy of this chitogel-drug combination at various dosages and illustrate the healing benefits of gel-drug combination.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2021

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

Sutures and staples have long been considered the gold standard for tissue wound repair and re-constructive surgeries. Their usage can often result in foreign-body inflammation, infection, excessive scarring, as well as air and fluid leakage in procedures involving the lungs, blood vessels, dura mater, and urethra. With the advent of bioadhesives, there are now several alternative techniques available that limit these adverse effects. Although the majority of these techniques are minimally invasive and provide sufficient wound closure, they can lack flexibility, and present a risk of cytotoxicity. Sutureless procedures for wound repair and closure have recently integrated nano-structured devices to improve their efficacy and clinical outcome. Gecko-inspired adhesives, for example rely mostly on van der Waals forces to adhere to a surface. This adherence is challenged by the moist environment of the tissue during wound closure and significantly compromises the bonding strength.

Impaired wound healing and the resulting chronic wounds may cause significant morbidity and mortality. In these pathogenic wound environments, the ratio of inflammatory and anti-inflammatory cytokines is highly biased to the pro-inflammatory side. While the inflammatory process is an essential step in healthy wound healing, chronic wounds remain in a constant self-sustaining state of inflammation. Thus, decreased cell proliferation, reduced matrix deposition and delayed wound closure are the results. Although various cytokine-based therapies have shown promising results on skin regeneration in preliminary studies, their overall clinical use has been considerably limited by the short half-life time of the signaling molecules due to rapid dilution and degradation in the protease-rich chronic wound environment. In this work, we explored the ability of starPoly(ethylene glycol)-GAG hydrogels to modulate the hallmarks of chronic wound development, such as the prolonged inflammation, increased cell influx and delayed proliferative phase. Therefore, different strategies were developed to shape the cytokine levels in the wound towards a more pro-regenerative direction, finally promoting the natural repair process in chronic skin wounds. By biomimetically utilizing the interactions between cytokines and the tissue ECM in a GAG-based biohybrid hydrogel, we could engineer the concentrations of various signaling factors involved in the regulation of the repair process. More in detail, we utilized customized functionalized starPEG-GAG hydrogels to (1) reduce the extensive levels of inflammatory chemokines by scavenging them via GAG component of the hydrogel and thus diminish immune cell influx in a mouse wound model; (2) locally deliver the immunomodulatory IL-4 and IL-10 to shift the signaling balance into the pro-regenerative direction and thus resolve inflammation and (3) administer pre-conjugated TGF-β to enhance myofibroblast differentiation and extracellular matrix deposition. We believe that the presented hydrogel platform may become a promising tool in the management of cytokines in regenerative applications, which can be translated towards the clinical use for the treatment of chronic wounds and other diseases characterized by uncontrolled inflammation.:1 introduction 1.1 Motivation 1.2 Current state of biomaterial-based concepts in dermal wound healing 1.3 Objective 2 fundamentals 2.1 The physiological process of wound healing 2.1.1 The role of macrophages in wound healing 2.1.2 The role of fibroblasts in wound healing 2.1.3 The role of cytokines and their interaction with the ECM 2.2 The pathophysiology of chronic wounds 2.3 Strategies for treatment of chronic wounds 2.4 Biomaterials in medicine 2.4.1 Polymers in medicine 2.4.2 Mechanical properties 2.4.3 Cellular adhesion 2.4.4 Interaction with cytokines 2.4.5 Scaffold degradability 2.4.6 StarPEG-GAG hydrogels as potential material in wound healing 3 materials & methods 3.1 Preparation of hydrogels 3.1.1 Functionalization of glass surfaces 3.1.2 Hydrogel formation with EDC - NHS chemistry 3.1.3 Hydrogel formation with thiol - maleimide chemistry 3.1.4 Rheometric measurement of hydrogel discs 3.1.5 Characterization of cytokine uptake and release 3.2 Culture of human & murine cells 3.2.1 Isolation and differentiation of murine dermal fibroblasts 3.2.2 Isolation & differentiation of murine macrophages 3.2.3 Culture of human & murine cell lines 3.3 In vitro methods 3.3.1 Enzyme-linked immunosorbent assay (ELISA) 3.3.2 Bead-based multiplex immunoassay 3.3.3 Live/Dead Staining 3.3.4 Crystal violet staining 3.3.5 Cell proliferation assay 3.3.6 RNA extraction & analysis 3.3.7 cDNA synthesis 3.3.8 Quantitative real time rt-PCR 3.4 Statistical analysis 3.5 Software use 4 scavenging inflammatory chemokines to control immune cell influx in the wound 4.1 Results 4.1.1 Engineering heparin-based hydrogels to scavenge chemokines 4.1.2 Heparin-based hydrogels reduce migration of immune cells 4.1.3 Heparin-based hydrogels decrease wound immune cell influx and inflammatory signaling 4.2 Discussion 5 promotion of regenerative macrophage polarizationin inflammatory environments 5.1 Results 5.1.1 Reversible complexation of IL-4 & IL-10 to starPEG-heparin gels 5.1.2 Stabilizing effects of starPEG-heparin gels on IL-4 5.1.3 IL-4 & IL-10-laden starPEG-heparin hydrogels modulate macrophage polarization 5.1.4 IL-4-laden starPEG-heparin induce collagen deposition in dermal fibroblasts 5.2 Discussion 6 modulation of human dermal fibroblast proliferation and differentiation 6.1 Results 6.1.1 Reversible complexation of TGF-b to starPEG heparin gels 6.1.2 Cell attachment, spreading and proliferation 6.1.3 Matrix deposition by fibroblasts grown on starPEG-heparin hydrogels 6.1.4 Degradation of starPEG-heparin hydrogels 6.1.5 TGF-b-laden starPEG-heparin that efficiently induces myofibroblast differentiation 6.2 Discussion 7 general discussion 7.1 Summary and conclusion 7.2 Future perspective Appendix 8 supplementary materials & methods 9 declaration of authorship 10 publications and conference contributions bibliography list of figures list of tables nomenclature selbstständigkeitserklärung

Aloe vera is found in the Northern Africa and the Mediterranean areas while Aloe ferox is found in Southern Africa. Aloe ferox and Aloe vera prepared by different methods have been shown to possess the following properties: Stimulatory effects on different cell types (e.g human fibroblasts, rat adrenal cells, calf pulmonary artery endothelial cells etc.), wound healing, antimicrobial, antioxidant, antidiabetics etc. In this study solvent extracted gel powders and whole leaf juice of Aloe ferox and Aloe vera prepared specifically without bitter components were tested. The aim was to assess if the samples could be used orally for therapeutic purposes with regards to wound healing, antimicrobial and antioxidant properties avoiding the laxative effects of the bitter components. The following were used: Human lymphocytes cells to determine cytotoxicity effects, chicken fibroblasts cells for potential wound healing properties, Candida albicans, Staphylococcus aureus and Pseudomona aeruginosa microorganisms for antimicrobial properties and ORAC, DPPH, TEAC and chemiluminescence assays for antioxidant properties. Most of the results obtained were contrary to the bulk of the literature available about these beneficial plants’ extract. Bitter components have been reported to stimulate different cell types and to have antimicrobial and antioxidant properties. Thus the removal has been suggested as the main reason why the effects of the tested extracts did not correspond to much of the reported literature. From the results obtained from various aspects of this study it could be concluded that the removal of bitter components contributed to the apparently contradictory results. From this study it might be concluded that the four Aloe extract samples tested could not be used therapeutically for wound healing, antimicrobial or antioxidant properties. However they could still be effective for cosmetics purposes as obtained from the literature.
Dissertation (MSc)--University of Pretoria, 2014.
gm2014
Pharmacology
unrestricted

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
O comprometimento da barreira cutânea origina um conjunto de etapas de cicatrização, nomeadamente: homeostase, fase inflamatória, fase proliferativa e fase de remodelação que, uma vez perturbadas, poderão levar à instalação de uma ferida crónica. São vários os fatores que, por aumentarem o tempo e a intensidade da fase inflamatória, podem despontar a cronicidade de uma ferida. De entre os principais fatores que desencadeiam o processo de cronicidade, a presença de microrganismos é a mais importante. Os géneros que mais se encontram envolvidas na colonização de feridas são: Staphylococcus, Pseudomonas, Enterobacter, Enterococcus e Finelgodia. Contudo, esta colonização é, na maioria dos casos, provocada por mais do que um patogéneo, sendo que estes, muitas vezes, interagem entre si através de biofilmes. Os biofilmes são mais difíceis de debelar do que as bactérias no estado planctónico por motivos relacionados com a aquisição de genes de resistência e aumento da tolerância às terapias antimicrobianas, quer por ação direta das bactérias no hospedeiro, quer por ação de estratégias que se revelam subterapêuticas, devido à dificuldade em chegar ao local de ação e em penetrar no biofilme. No que diz respeito ao diagnóstico de feridas crónicas, apesar de não existirem ainda testes padrão, é recomendado o uso concomitante de vários ensaios de natureza diferente (morfológicos, microbiológicos e moleculares) para que se consiga uma aproximação do real o mais rigorosa possível. Além disso, dada a importância de um bom diagnóstico para instituir uma terapêutica adequada, estão em estudo métodos baseados em wound blotting e transcriptómica que parecem mostrar-se promissores a detetar com maior precisão e exatidão a composição de um biofilme. Atualmente, a abordagem terapêutica a feridas crónicas deve ser multifatorial, baseada no melhor diagnóstico possível. De início deve fazer-se o desbridamento da ferida, o controlo da humidade e de patologias de base associadas e incluir antibióticos e antissépticos que sejam ativos contra o biofilme, assim como inibidores do QS.
When cutaneous barrier is compromised, there are some steps to cicatrize the wound, namely: homeostasis, inflammatory phase, proliferative phase and remodeling phase. If these steps are disturbed, a chronic wound can appear. Many factors can increase the time and intensity of the inflammatory phase, which helps a wound to become chronic. The presence of infection is the most important reason to increase chronicity. The genus that are most involved in wound colonization are Staphylococcus, Pseudomonas, Enterobacter, Enterococcus e Finelgodia. However, this colonization is, in most cases, caused by more than one pathogen and these interact with each other through biofilms. Biofilms are more difficult to treat then planktonic bacteria because of gain of resistance genes and increase tolerance to antimicrobial therapies, either by direct action of the bacteria in the host or by the action of strategies that are revealed as sub-therapeutic, by the difficulty to enter into the place of action and to penetrate the biofilm. For diagnosis of chronic wounds, even without gold-standard tests, is recommended the concurrent use of many different assays (morphologic, microbiologic and molecular) to helps get the diagnostic the closer as possible to the real. Besides that, given the importance of a good diagnostic to found right therapeutic, there are some methods under study based on wound blotting and transcriptomic. These methods seem promising to detect with greater precision and accuracy the biofilm composition. Nowadays, chronic wound therapeutic must be multifactorial, based on the best as possible diagnosis. It should start with the wound debridement, moisture control and keep primary pathologies under control and include antibiotics or antiseptics specific chosen for biofilms, as well as QS inhibitors.

As feridas crónicas são um problema de saúde relevante em todo o mundo. Para garantir a cicatrização é necessário controlar fatores como a infeção a relevância dos biofilmes e o papel destes na cicatrização. Os biofilmes funcionam como sistemas biológicos formados por comunidades de microrganismos agregados, organizados e funcionais embebidos numa matriz exopolissacarídica. A formação de um biofilme ocorre geralmente em várias etapas consecutivas. A resistência antimicrobiana tornou-se uma das mais eminentes ameaças para a saúde global e uma preocupação crescente para os especialistas em saúde. Muitas infeções tornaram-se resistentes aos antimicrobianos usados para as tratar, resultando em elevadas taxas de mortalidade. Uma vez que, as resistências individuais são já um problema universal, é possível inferir que o paradigma séssil é ainda mais problemático, pois representa um somatório de resistências que permite ao biofilme ser substancialmente mais resistente à ação de antimicrobianos do que as células que estão em estado planctónico. A fraca penetração e difusão dos antimicrobianos através da matriz polisacarídica, a forma específica como os microorganismos se organizam no biofilme são algumas das hipóteses explicativas para o aumento da resistência individual e coletiva (biofilmes) aos antimicrobianos. O diagnóstico e a terapêutica das infeções causadas por biofilme é um processo difícil, daí haver um reforço na importância da prevenção. Deste modo, as feridas crónicas apresentam-se como um desafio à qualidade de vida dos doentes, à abordagem efetuada pelos profissionais de saúde e aos recursos despendidos pelas instituições de saúde no seu tratamento. Assim sendo, é relevante propor um algoritmo síntese que possa ser facilitador na gestão, por parte dos profissionais de saúde, dos biofilmes nas feridas. Como se explica a existência de feridas crónicas? Qual a importância do paradigma séssil para explicar os sinais e sintomas que caracterizam as infeções crónicas? Que dinâmica de investigação existe atualmente para apresentar soluções para este grave problema de saúde pública?
Chronic wounds are a major health problem all around the world. To ensure healing is necessary to control factors such as infection, the relevance of biofilms and their role in healing. Biofilmes can be defined as biological systems formed by communities of aggregated, organized and functional cells embedded in exopolimeric matrix. The biofilm formation usually occurs in several stages. The antimicrobial resistance has become one of the most eminent threats to global health and a rising concern for healthcare specialists. Many common infections are becoming resistant to the antimicrobial drugs used to treat them, resulting in higher mortality rates. Once the individual resistances are already a universal problem, it can be inferred that the sessile paradigm is even more problematic because it is a summation of resistances allowing the biofilm to be substantially more resistant to the action of antimicrobials than those cells grown planctonically. The low penetration and dissemination of antimicrobial agents through the polysaccharide matrix and the specific way in which the microorganisms are organized in biofilms are some of the hypotheses proposed to explain the increasing individual and collective (biofilms) antimicrobial resistance. The diagnosis and treatment of biofilm infections caused by biofilms is a difficult process, that’s why prevention is really important. This way, chronic wounds appear as a challenge to the patients’ quality of life, the approach made by health professionals and resources spent by health institutions in their treatment. Therefore, it is important to propose a synthesis algorithm that can be a help in the management of biofilms in wounds by health professionals. How do you explain the existence of chronic wounds? How important is the sessile paradigm to explain the signs and symptoms that characterize chronic infections? What dynamic research currently exists to provide solutions to this serious public health problem?