Novel silk fibroin/elastin wound dressings

Silk fibroin (SF) and elastin (EL) scaffolds were successfully produced for the first time for the treatment of burn wounds. The self-assembly properties of SF, together with the excellent chemical and mechanical stability and biocompatibility, were combined with elastin protein to produce scaffolds with the ability to mimic the extracellular matrix (ECM). Porous scaffolds were obtained by lyophilization and were further crosslinked with genipin (GE). Genipin crosslinking induces the conformational transition from random coil to β-sheet of SF chains, yielding scaffolds with smaller pore size and reduced swelling ratios, degradation and release rates. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to obtain scaffolds suitable for biological applications. Wound healing was assessed through the use of human full-thickness skin equivalents (EpidermFT). Standardized burn wounds were induced by a cautery and the best re-epithelialization and the fastest wound closure was obtained in wounds treated with 50SF scaffolds; these contain the highest amount of elastin after 6 days of healing in comparison with other dressings and controls. The cytocompatibility demonstrated with human skin fibroblasts together with the healing improvement make these SF/EL scaffolds suitable for wound dressing applications.We would like to acknowledge FCT - Portuguese Foundation for Science and Technology for the scholarship conceded to Andreia Vasconcelos; European FP6 project Lidwine, contract no. NMP2-CT-2006-026741 and PEst-C/BIA/UI4050/2011

The use of Keratin in biomedical applications

Keratins are naturally derived proteins that can be fabricated into several biomaterials morphologies including films, sponges and hydrogels. As a physical matrix, keratin biomaterials have several advantages of both natural and synthetic materials that are useful in tissue engineering and controlled released applications. Like other naturally derived protein biomaterials, such as collagen, keratin possess amino acid sequences, similar to the ones found on extracellular matrix (ECM), that may interact with integrins showing their ability to support cellular attachment, proliferation and migration. The ability of developing biomaterials that mimic ECM has the potential to control several biological processes and this is the case for keratin which has been used in a variety of biomedical applications due to its biocompatibility and biodegradability. This review describes the progress to date towards the use of keratin in the field of wound healing, tissue engineering and drug delivery applications, with highlight to reports of particular relevance to the development of the underlying biomaterials science in this area

Obtenção de tecidos de poliéster de baixo peso por tratamento enzimático

Dissertação de mestrado em Química Têxtil.A produção de tecidos de poliéster de baixo peso é obtida pela remoção química da parte celulósica de uma mistura poliéster/algodão. O processo convencional consiste no tratamento das misturas com uma solução de ácido sulfúrico a 75% (v/v). Os efeitos negativos dos processos químicos podem ser evitados utilizando processos enzimáticos. Neste trabalho estudou-se a acção de uma celulase total comercial sobre um tecido 100% algodão e uma mistura poliéster/algodão (34/66%), com o objectivo de obter a degradação total da celulose. A actividade da celulase foi medida em termos de perda de peso total das amostras, e foi estudado também a influência da dosagem enzimática, acção mecânica e tempo de tratamento na actividade enzimática. A hidrólise total da celulose do tecido 100% algodão foi atingida após 6 horas de tratamento com uma dosagem enzimática e acção mecânica elevadas. Na mistura de poliéster/algodão obteve-se uma hidrólise máxima de 55% após 9 horas de tratamento com uma dosagem enzimática e acção mecânica elevadas. Verificou-se que a flexão, a força e alongamento à ruptura diminuem com o aumento da perda de peso, e o coeficiente de atrito aumenta com o aumento da perda de peso total das amostras.The production of light weight polyester fabrics is achieved by chemical removal of cellulose on a polyester/cotton blend. The traditional process consists on the blend treatment with a sulfuric acid solution 75% (v/v). The negative effects of the chemical processes can be avoided by enzymatic treatments. In the present work, the mode of action of a total commercial cellulose was investigated towards a cotton fabric and polyester/cotton blend (34/66%) to achieve the total hydrolysis of cellulose. The cellulase activity was measured in terms of total weight loss, and we also studied the influence of enzyme dosage, mechanical action and treatment time on cellulase activity. The total hydrolysis of cellulose from the cotton fabric was accomplished after 6 hours of treatment with high level of mechanical action and enzyme dosage. On the polyester/cotton blend we obtained a maximum hydrolysis of 55% after 9 hours of treatment with high level of mechanical action and enzyme dosage. It was observed that the bending and breaking strength of samples decreases, and that the friction coefficient increases with the increase of total weight loss

Protein matrices for wound dressings

Tese de doutoramento em Engenharia TêxtilFibrous proteins such as silk fibroin (SF), keratin (K) and elastin (EL) are able to mimic the extracellular matrix (ECM) that allows their recognition under physiological conditions. The impressive mechanical properties, the environmental stability, in combination with their biocompatibility and control of morphology, provide an important basis to use these proteins in biomedical applications like protein-based wound dressings. Along time the concept of wound dressings has changed from the traditional dressings such as honey or natural fibres, used just to protect the wound from external factors, to the interactive dressings of the present. Wounds can be classified in acute that heal in the expected time frame, and chronic, which fail to heal because the orderly sequence of events is disrupted at one or more stages of the healing process. Moreover, chronic wound exudates contain high levels of tissue destructive proteolytic enzymes such as human neutrophil elastase (HNE) that need to be controlled for a proper healing. The aim of this work is to exploit the self-assemble properties of silk fibroin, keratin and elastin for the development of new protein materials to be used as wound dressings: i) evaluation of the blending effect on the physical and chemical properties of the materials; ii) development of materials with different morphologies; iii) assessment of the cytocompatibility of the protein matrices; iv) ultimately, study the ability of the developed protein matrices as wound dressings through the use of human chronic wound exudate; v) use of innovative short peptide sequences that allow to target the control of high levels of HNE found on chronic wounds. Chapter III reports the preparation of silk fibroin/keratin (SF/K) blend films by solvent casting evaporation. Two solvent systems, aqueous and acidic, were used for the preparation of films from fibroin and keratin extracted from the respective silk and wool fibres. The effect of solvent system used was studied by evaluating the physical-chemical properties of the resulting films. It was shown that SF and K are able to establish intermolecular interactions when mixed and, that the mechanical properties and the biological degradation can be tuned by the blend composition. In Chapter IV, SF/K films were further used to serve as a platform for the release of HNE inhibitors peptides. Bowman-Birk inhibitor (BBI) based peptide was incorporated onto the SF/K films that were consequently incubated with porcine pancreatic elastase (PPE) as a model for HNE, to monitor the decrease in activity. The results indicated that swelling properties, degradation and release rates are dependent on the amount of keratin present in the blend. Furthermore, no cytotoxicity was observed in the presence of mouse fibroblasts, which makes these SF/K films suitable candidates for interactive wound dressings with a specific goal – controlling high levels of HNE. The next step of the work, Chapter V, reports for the first time blends of silk fibroin with elastin (SF/EL) for the production of scaffolds. These were prepared by lyophilization technique and crosslinked with a natural and low toxic agent, genipin. The crosslink allows the control of the scaffolds morphology, such as pore size and porosity, which in turns, modulates the ex vivo degradation rates, by a human chronic wound exudate, and the release rates of model compounds. In addition, no cytotoxicity was observed for SF/EL samples, with and without genipin, by human skin fibroblasts. Thus, the high porosity observed for SF/EL scaffolds, allowing the growth and cellular attachment, together with their biocompatibility provide fitting characteristics for wound dressings. Chapter VI, describes the design of two elastase inhibitors peptides based on the reactive site-loop of the BBI protein in order to control the high levels HNE. To a known peptide sequence, modifications were made at both N- and C-terminal. Inhibition kinetics analysis indicated that these peptides are competitive inhibitors for HNE and PPE and, that the inhibitory potency can be regulated by the introduced modifications. Additionally, these peptides showed no toxicity with human skin fibroblasts and, were also effective in reducing the HNE activity found in a human chronic wound exudate, which allow them to be applied to those wounds. The motivation for this thesis was to combine the excellent properties of silk fibroin with other proteins. Blending allows modulating the physical-chemical properties of the resulting materials such as mechanical strength, swelling, morphology, degradation and release rates. Silk fibroin is widely characterized in the literature for the production of biomaterials, but this work is the first that successfully evaluates the blends silk fibroin/keratin (SF/K) and silk fibroin/elastin (SF/EL) for their application as wound dressings.Proteínas fibrosas como a fibroína da seda, a queratina e a elastina têm a vantagem de conseguir imitar a matriz extracelular, permitindo o seu reconhecimento em condições fisiológicas. As excelentes propriedades mecânicas destas proteínas, a estabilidade química juntamente com a biocompatibilidade e controlo da morfologia, são importantes pressupostos para o uso destas proteínas em aplicações biomédicas. Um exemplo é o tratamento de feridas com compressas baseadas nestas proteínas. As compressas usadas nas feridas sofreram grandes mudanças ao longo do tempo, partindo das tradicionais baseadas em mel e fibras naturais, que eram usadas apenas para cobrir e proteger as feridas de factores externos, até às compressas interactivas do presente. As feridas podem ser classificadas em agudas, que curam no tempo previsto e crónicas, que não cicatrizam porque o processo de cura é interrompido. Estas feridas podem ainda ser diferenciadas pelo tipo de exudado que produzem. Nas feridas crónicas, o exudado contém níveis elevados de enzimas proteolíticas como a elastase humana dos neutrófilos (HNE) e as metaloproteases de matriz (MMP), que precisam de ser controlados para um melhor processo de cicatrização. O objectivo deste trabalho é explorar as excelentes propriedades da fibroína, queratina e elastina para o desenvolvimento de novos materiais proteícos para serem usados como compressas. Para tal estudou-se: i) avaliação do efeito das misturas nas propriedades físicas e químicas dos materiais; ii) desenvolvimento de materiais com diferentes morfologias; iii) avaliação da toxicidade dos materiais; iv) avaliação das propriedades dos materiais desenvolvidos usando um exudado obtido de feridas crónicas; v) desenvolvimento de péptidos inibidores para controlar os elevados níveis de HNE nas feridas crónicas. No Capítulo III é descrita a preparação de membranas baseadas na mistura fibroína e queratina (SF/K) por evaporação de solvente. Foram utilizados dois sistemas de solventes, aquoso e ácido, para a preparação dos filmes a partir da fibroína e queratina extraídas das respectivas fibras têxteis, seda e lã. Estudou-se o efeito dos solventes usados nas propriedades físico-químicas das membranas resultantes. Verificou-se que a fibroína e a queratina estabelecem interacções intermoleculares e, as propriedades mecânicas e a degradação podem ser moldadas através da composição da mistura. No Capítulo IV, as membranas desenvolvidas foram posteriormente utilizadas para a libertação de inibidores da elastase. Péptidos baseados na proteína inibidora Bowman- Birk (BBI) foram incorporados nas membranas de fibroína e queratina verificando-se que após incubação com a elastase há uma diminuição da actividade da mesma. Os resultados demonstram que as propriedades de inchamento, degradação e libertação dependem da quantidade de queratina presente na mistura. Adicionalmente, os materiais não apresentam toxicidade na presença de fibroblastos de ratinho. Os resultados indicam que estas membranas são candidatas adequados para o uso em compressas interactivas com uma actividade específica – controlar os elevados níveis de HNE. O próximo passo do trabalho, Capítulo V, reporta pela primeira vez, o desenvolvimento de estruturas esponjosas baseadas na mistura de fibroína e elastina (SF/EL). Estes materiais foram preparados por liofilização e fixados com um agente de fixação natural e de baixa toxicidade, a genipina. Este passo de fixação permite controlar a morfologia dos materiais como o tamanho dos poros e porosidade, que têm influência directa na degradação pelo exudado e na libertação de compostos modelo. Observou-se também, que as estruturas esponjosas não apresentam toxicidade para fibroblastos humanos de pele, mesmo depois da fixação com genipina. Consequentemente, a biocompatibilidade demonstrada juntamente com a porosidade observada nestes materiais, que permitem a adesão e crescimento de células, são características importantes para o uso destes em compressas para feridas. O Capítulo VI descreve o desenvolvimento de dois péptidos inibidores da elastase baseados no centro activo da proteína BBI de forma a controlar os elevados níveis de HNE presentes nas feridas crónicas. Foram introduzidas modificações nos terminais C e N de uma sequência conhecida. Estudos cinéticos de inibição indicam que estes péptidos são inibidores competitivos para a elastase de diferentes fontes, humana e porcina, e que a capacidade inibitória depende das modificações introduzidas. Adicionalmente, os péptidos não são tóxicos na presença de fibroblastos humanos de pele sendo eficazes na redução da actividade da elastase presente num exudado de uma ferida crónica. Estes resultados indicam que os péptidos desenvolvidos podem ser usados no tratamento das mesmas. A motivação deste trabalho partiu das excelentes propriedades da fibroína, queratina e elastina. O desenvolvimento de novos materiais baseados em misturas destas proteínas permite moldar as propriedades finais como a força mecânica, inchamento, morfologia, degradação e libertação de compostos. O uso da fibroína em biomateriais está vastamente descrito na literatura. Contudo, este trabalho descreve pela primeira vez, com sucesso, o uso das misturas fibroína/queratina e fibroína/elastina para a sua aplicação em compressas para feridas.The research described in this thesis was financially supported by Fundação para a Ciência e Tecnologia by means of a PhD grant (SFRH/BD/41603/2007)

Biodegradable materials based on silk fibroin and keratin

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles

Comportamento sísmico de paredes de alvenaria de fachada : uma breve revisão

O trabalho proposto surge no âmbito do estudo do comportamento sísmico de uma tipologia de parede de fachada, fachada ventilada. Pelo facto de existir uma panóplia considerável de de sistemas construtivos de fachadas ventiladas, apresenta-se neste trabalho algumas tipologias mais representativas em Portugal, sendo caracterizadas ao nível dos materiais utilizados, tipologia e espaçamento de ligadores, regulamentação existente e aplicação em obra. Também se apresenta um conjunto de estudos efetuados do comportamento estático e dinâmico deste sistema construtivo e seus componentes. Este estudo teórico pretende ser o ponto de partida para o desenvolvimento de uma campanha experimental e numérica que visa a caracterização destas paredes a ações sísmicas e do vento, com vista a obtenção de um conjunto de recomendações técnicas de melhoria do comportamento sísmico e do vento.Fundação para a Ciência e a Tecnologia (FCT

Kind Inference for the FreeST Programming Language

We present a kind inference algorithm for the FREEST programming language. The input to the algorithm is FREEST source code with (possibly part of) kind annotations replaced by kind variables. The algorithm infers concrete kinds for all kind variables. We ran the algorithm on the FREEST test suite by first replacing kind annotation on all type variables by fresh kind variables, and concluded that the algorithm correctly infers all kinds. Non surprisingly, we found out that programmers do not choose the most general kind in 20% of the cases.Comment: In Proceedings PLACES 2023, arXiv:2304.0543

Caracterização experimental da deterioração de alvenaria de tijolo a ciclos de gelo-degelo

Dado que as paredes de tijolo face à vista estão diretamente expostas às condições ambientais, decidiu-se avaliar a degradação da alvenaria de tijolo em relação aos ciclos de gelo-degelo ciclos. Além da alvenaria de tijolo, a argamassa utilizada na construção de da alvenaria (face à vista e de enchimento) foi também submetida a vários ciclos de gelo-degelo. A avaliação da possível degradação em argamassa foi realizada com base nas propriedades físicas e mecânicas e na inspeção visual.Given that the brick veneer walls are directly exposed to environmental conditions, it was decided to evaluate the decay of brick veneer masonry to freeze and thaw cycles. Besides brick masonry, mortar used in the construction of brick veneer and infill wall was also submitted to several freeze and thaw cycles. The assessment of possible decay on mortar specimens was carried out based on physical and mechanical properties and on visual inspection.The authors acknowledge the support of the Portuguese Foundation for Science and Technology (FCT), through the financing of the research project SEVen - Development of Sustainable Ceramic Brick Masonry Veneer Walls for Building Envelops (PTDC/ECICON/30876/2017)