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Antimicrobial resistance (AMR) has become a global concern as many bacterial species have developed resistance to commonly prescribed antibiotics, making them ineffective to treatments. One type of antibiotics, gallium(III) compounds, stands out as possible candidates due to their unique “Trojan horse” mechanism to tackle bacterial growth, by substituting iron(III) in the metabolic cycles of bacteria. In this study, we tested three polysaccharides (carboxymethyl cellulose (CMC), alginate, and pectin) as the binding and delivery agent for gallium on three bacteria (Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) with a potential bioresponsive delivery mode. Two types of analysis on bacterial growth (minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)) were carried out while iron(III)-loaded polysaccharide samples were also tested for comparison. The results suggested that gallium showed an improved inhibitory activity on bacterial growth, in particular gallium(III)-loaded carboxymethyl cellulose (Ga-CMC) sample showing an inhibiting effect on growth for all three tested bacteria. At the MIC for all three bacteria, Ga-CMC showed no cytotoxicity effect on human dermal neonatal fibroblasts (HDNF). Therefore, these bioresponsive gallium(III) polysaccharide compounds show significant potential to be developed as the next-generation antibacterial agents with controlled release capability.

Paper fluidics is based on patterning hydrophilic paper with channels bounded by hydrophobic barriers. Fluids move along channels by capillarity. Several methods are available for patterning paper, with different costs/resolutions. Paper patterning for microfluidics also used the embossing technique to design open-channel microfluidic devices, fabricated by compressing the sheet of paper with the help of 3D plastic printing moulds. These approaches are adopted in this work to develop a multiwell-microplate paper-based microfluidic, aiming the creation of organs-on-chip, combining complexity and miniaturization. Bacterial Cellulose (BC) represents a source of highly pure and biocompatible cellulose, with huge technological potential in many fields - biomedical, composites, textiles, food and cosmetics textiles - but currently still rather underexploited [Gama et al, 2916; Klemm et al, 2018]. This work describes a novel approach towards the development of a nanostructured and multifunctional cellulose-based device for the continuous culture of animal cells and tissues. A multilayered system of modified BC (hydrophobized and electroconductive) was used to assemble the skin-on-chip, a microfluidic platform, using the lab-on-paper technology intended to mimic vascularization, with controlled flow, to introduce external stimuli, such as electrical or mechanical, and to support multicellular growth. This chip serves a multifactorial purpose, aiming the control of each part that make up the overall complex 3D system, including dynamic control of physical, chemical and gaseous gradients, ensure mimetic vascularization, introduce favourable stimuli and co-culture of skin cells. This model sustains cell growth and allow real time and in a high throughput manner to assess cellular phenomena, such as cell-cell crosstalk, paracrine factor exchange, ECM production, as well as tissue homeostasis in the presence of chemical, mechanical, electrical and biological stimuli, and also kinetics of substance delivery on/through the skin. BC hydrophobization was achieved using a new strategy for the surface modification of BC through the combination of oxygen plasma deposition and silanization with trichloromethyl silane. The combined use of the two techniques modifies both the surface roughness and energy and therefore maximizes the hydrophobic effect obtained. These modified membranes were characterized by SEM, water contact angle measurements, FTIR-ATR and XPS, and its cytotoxic potential was investigated using both indirect and direct contact studies with cells. Importantly, this surface modification revealed no short-term cytotoxic effects on L929 and hDNFs cells. This material was used for the construction of a BC-based well plate for cell culture, which can be supplied continuously with culture medium in long term studies (ranging from days to weeks), using a two-layered 3D full-thickness skin equivalent consisting of an epidermal and a dermal tissue layer, cultivated in alginate scaffolds, that can be maintained and studied as a skin surrogate on the SkinChip [Maia et al, 2014]., info:eu-repo/semantics/publishedVersion

Submitted by Ana Costa (apcosta@porto.ucp.pt) on 2021-05-25T11:39:30Z No. of bitstreams: 1 Tese_FilipaSerodio_pdfa.pdf: 3551230 bytes, checksum: c3fc6182892d57d3514eca10db272233 (MD5) Approved for entry into archive by Maria Helena Ribeiro (helena.ribeiro@lisboa.ucp.pt) on 2021-06-09T10:14:24Z (GMT) No. of bitstreams: 1 Tese_FilipaSerodio_pdfa.pdf: 3551230 bytes, checksum: c3fc6182892d57d3514eca10db272233 (MD5) Made available in DSpace on 2021-06-09T10:14:24Z (GMT). No. of bitstreams: 1 Tese_FilipaSerodio_pdfa.pdf: 3551230 bytes, checksum: c3fc6182892d57d3514eca10db272233 (MD5) Previous issue date: 2020-05-26

Skin is the largest organ of the human body, being crucial on its protection from foreign objects and microorganisms thus playing an important role for the homeostasis of the organism. It is composed by three layers, epidermis, dermis and hypodermis that together form the outmost layer of the body. Once this layer is damaged, human life could be at risk. The lack of viable donors leads to the urgency of developing skin substitutes. One of the strategies proposed by Tissue Engineering for the treatment of skin lesions is the development of skin substitutes made by natural or synthetic polymer or even a blend of both. Silk fibroin is a natural polymer with good biocompatibility and profiles already explored in the literature. Scaffolds based on this natural polymer have attracted great attention on the last decades due to its characteristics for tissue engineering (TE) applications. In the present work, silk fibroin based scaffolds were obtained through electrospinning processing technique, using silk fibroin solutions with a concentration of 12% (w/v) and formic acid as spinning solvent. Silk fibroin electrospun matrices were further treated with methanol at different concentrations and incubation times (50% (v/v), 75% (v/v), 100% (v/v) with a time length of 10 and 60 minutes each). The physico-chemical properties of silk fibroin were investigated, as well as human fibroblast cellular behavior (cytotoxicity and adhesion assays). The results demonstrated that the electrospun membranes treated with different methanol concentrations present β-sheet conformation, high water vapor transmission rate, similar to the control group, which is regarded as a crucial parameter for wound dressing. Cytotoxicity of the silk fibroin electrospun meshes was evaluated using hDNFs showing normal metabolic activity. We were able to successfully produce silk fibroin electrospun matrices that demonstrated no cytotoxic effect and presented suitable characteristics for the use in the context of skin regeneration. A pele é o maior órgão humano, crucial na proteção do corpo humano de objetos prejudiciais e microrganismos. Figurando como principal responsável pela homeostasia do organismo, é composto por três camadas, epiderme, derme e hipoderme, as quais em conjunto formam o revestimento mais externo do corpo. Quando danificada a vida humana pode estar em risco. A falta de dadores viáveis leva à necessidade de desenvolver substitutos para a pele. Neste contexto, a Engenharia de Tecidos, surge como uma tecnologia promissora para o tratamento de lesões a nível cutâneo através do desenvolvimento de substitutos produzidos a partir de polímeros naturas, sintéticos ou uma mistura dos dois. Fibroína de seda é um polímero natural, que apresenta elevada biocompatibilidade e biodegradabilidade. Scaffolds com base neste polímero natural, têm atraído considerável atenção nas últimas décadas devido às suas características para aplicações em Engenharia de Tecidos. No presente trabalho, scaffolds à base de fibroína de seda foram obtidos através do método de electrofiação. Com este objectivo, foram utilizadas soluções de fibroína de seda com uma concentração de 12% (w/v) em ácido fórmico. Matrizes de fibroína de seda, obtidas através do método de electrofiação, foram posteriormente tratadas com metanol em diferentes concentrações (50% (v/v), 75% (v/v),100% (v/v)) durante 10 e 60 minutos. As propriedades físico-químicas da fibroína de seda foram estudadas e o seu comportamento celular investigado através de ensaios de cito-toxicidade e de adesão celular. Os resultados demonstraram que, após o tratamento com metanol, as membranas apresentavam conformação β-folheada, taxas de transmissão de vapor de água elevadas, semelhantes ao grupo de controlo, figurando como um parâmetro fundamental no tratamento de feridas. A cito-toxicidade das membranas de fibroína de seda foi avaliada através da utilização de hDNFs, apresentando boa atividade metabólica. Conseguiu-se produzir, de forma bem sucedida, matrizes de fibroína de seda através do método de electrofiação, que não demonstraram efeitos cito-tóxicos e apresentavam características apropriadas para o uso no contexto de regeneração da pele.

Doutoramento em Ciência e Engenharia de Materiais A presente dissertação teve como foco principal o desenvolvimento de estruturas 3D porosas (scaffolds) para regeneração óssea por robocasting. Esta técnica de fabrico aditivo permite a produção de scaffolds com morfologia e estrutura predefinidas e sem a necessidade de maquinagem subsequente, podendo ser usada para o fabrico de implantes personalizados com estrutura interna semelhante à do osso que se pretende substituir. Como materiais de partida, foram utilizados pós de fosfatos de cálcio bifásicos (hidroxiapatite + β-fosfato tricálcico) não dopados, e dopados com diferentes iões (Sr, Ag, Cu, Mg e Zn), obtidos por precipitação em meio aquoso. A substituição parcial de iões Ca pelos iões dopantes induziu alterações na composição de fases cristalinas e resultou em partículas com características morfológicas distintas e em materiais com desempenhos biológicos diferentes. Os pós foram dispersos em meio aquoso com a ajuda de aditivos de processamento adequados (um dispersante e um ligante) de modo a obter suspensões com elevada concentração de sólidos, requisito essencial para o fabrico de componentes por robocasting. O tamanho e a morfologia das partículas e a concentração dos aditivos de processamento desempenharam os papéis cruciais no comportamento reológico das suspensões. A adição de agente floculante modificou drasticamente as propriedades reológicas do sistema e permitiu a obtenção de pastas com comportamento viscoelástico adequado para o processo de extrusão. Os scaffolds com diferentes tamanhos de poro foram produzidos pela deposição de um filamento com 410 μm de diâmetro. A resistência à compressão dos scaffolds sinterizados a 1100°C foi comparável ou mesmo superior à do osso esponjoso. Scaffolds com diferentes tamanhos de poro (120-500 μm) foram testados in vitro usando células estaminais mesenquimais humanas (hMSCs). Pretendeu-se avaliar a influência do tamanho dos poros na adesão celular inicial, na atividade metabólica e no potencial osteogénico. Os resultados obtidos mostraram que os poros com maior dimensão proporcionam condições favoráveis para a diferenciação osteogénica das hMSCs. Além disso, os fosfatos de cálcio co-dopados com Sr e Zn melhoraram o desempenho biológico, incluindo adesão, atividade metabólica e proliferação das hMSC. A última parte da tese foi dedicada à preparação de scaffolds não sinterizados incorporando um fármaco (levofloxacina) de forma a obter componentes com capacidade de regeneração óssea e de tratamento local de infeções. A pasta utilizada foi obtida através da dispersão dos pós de CaP numa solução de quitosano na presença do fármaco e de um agente de reticulação (genipin). Os scaffolds 3D à base de fosfatos de cálcio produzidos por robocasting no âmbito desta tese revelaram-se muito promissores para aplicações na engenharia de tecidos, particularmente regeneração de tecido ósseo e administração de fármacos. The main focus of this thesis was the development of novel 3D porous scaffolds for bone regeneration by robocasting. This additive manufacturing technique allows the production of scaffolds with predefined morphology and structure without the need for subsequent machining and can be used for the manufacture of customized implants with an internal structure similar to that of the bone to be replaced. Biphasic calcium phosphates (hydroxyapatite + β-tricalcium phosphate), undoped and doped (with Sr, Ag, Cu, Mg and Zn), obtained through aqueous precipitation process were used as starting powders. The partial substitution of Ca ions by doping ions induced changes in the crystalline phase assemblages and resulted in particles with distinct morphological characteristics and in materials with different biological outcomes. The powders were dispersed in aqueous medium with the aid of suitable processing additives (a dispersant and a binder) in order to obtain suspensions with high solids loading, an essential requirement for the manufacture of components by robocasting. The size and morphology of the particles and the concentration of the processing additives played crucial roles in the rheological behaviour of the suspensions. The addition of a flocculating agent drastically modified the rheological properties of the systems and allowed obtaining pastes with viscoelastic behaviour suitable for the extrusion process. The scaffolds with different pore sizes were produced by the controlled deposition of filaments with 410 μm in diameter. The compressive strength of scaffolds sintered at 1100 ° C was comparable or even superior to that that of spongy bone. Scaffolds with different pore sizes (120-500 μm) were tested in vitro using human mesenchymal stem cells (hMSCs). The aim was to evaluate the influence of pore size on initial cell adhesion, metabolic activity and osteogenic potential. The results showed that larger pores provide favourable conditions for osteogenic differentiation of hMSCs. In addition, calcium phosphates codoped with Sr and Zn improved the biological performance, including adhesion, metabolic activity and proliferation of hMSC. The last part of the thesis was devoted to the preparation of sintering-free scaffolds incorporating a drug (levofloxacin) in order to obtain components with capacity for bone regeneration and local treatment of infections. The paste used was obtained by dispersing the CaP powders in a solution of chitosan in the presence of the drug and a crosslinking agent (genipin). The 3D scaffolds produced by robocasting in the frame of this thesis program revealed to be very promising for tissue engineering applications, particularly bone tissue regeneration and drug delivery.

Doutoramento em Ciência e Engenharia de Materiais Submitted by Cristina Santos (cmaria@ua.pt) on 2018-03-02T11:01:07Z No. of bitstreams: 1 Catarina Marques_Tese PhD.pdf: 55290948 bytes, checksum: 9f627ce80836497dbb9f2b21206fcbca (MD5) Made available in DSpace on 2018-03-02T11:01:07Z (GMT). No. of bitstreams: 1 Catarina Marques_Tese PhD.pdf: 55290948 bytes, checksum: 9f627ce80836497dbb9f2b21206fcbca (MD5) Previous issue date: 2017-12-21

Tissue Engineering creates strategies capable of interacting with cells, to promote tissue regeneration. Cell adhesion is essential in cell communication and regulation, and cell-scaffold interactions occurring at the surface of the material are dependent on the biomolecules adsorbed. This knowledge promotes the biofunctionalization of biomaterials to enhance cellular response. Molecular Imprinting (MI) is an alternative to molecular recognition phenomena present in living systems, such as the antibody-antigen bonding, and has been proposed as a biofunctionalization method to improve scaffolds selectivity and recognition. The principle of MI is the polymerization of monomers and crosslinkers in the presence of the template molecule of interest and the subsequent removal of the template. The crosslinker enables the formation of specific cavities, which enables the material to recognize the template. The challenging in MI is the incorporation of biomacromolecules important for the wound healing. The imprinting of biomacromolecules holds drawbacks associated to the high molecular weight and complex structure. This work proposes a MI system based on methacrylated alginate imprinted with the model protein bovine serum albumin (BSA), using photo-polymerization. A non-imprinted polymer acting as control was also prepared by photo-polymerization without BSA. The effect of two crosslinkers and a monomer on mechanical properties of polymer discs was studied and the results revealed an improvement of the mechanical properties of crosslinked polymers. The template removal and rebinding capacity were also characterized for molecularly imprinted discs. The template removal showed results below 70%, most likely due to the amount of methacrylated alginate (4% w/v) and the bulk imprinting. Results concerning the capacity of the molecularly imprinted material to recognize BSA showed higher results when comparing to non-imprinted polymers. However, the system still needs to be improved, concerning the template removal and rebinding capacity. Still, it enabled the understanding of the effect of different crosslinkers on MI.