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- Layer-by-Layer Deposition of Antibacterial Polyelectrolytes on Cotton FibresPublication . Gomes, Ana; Mano, João F.; Queiroz, João; Gouveia, Isabel C.The introduction of molecules with biological properties on textile materials is essential for a number of biotechnological applications. With the purpose of testing new processes applied to textiles, in this study, we present the first results on the feasibility of using the Layerby-Layer (LbL) deposition process in natural fibers such as cotton, with natural polyelectrolytes like chitosan (CH) and alginic acid sodium salt (ALG), the durability of CH/ALG multilayer on cotton were evaluated. The increase of negative charges to the substrate cotton was made with NaBr and TEMPO, to ensure the success of the process of LbL. Three characterization methods to assess electrostatic LbL deposition were performed: the contact angle between a liquid (water) and the sample surface, in order to characterize the wettability of the samples with the different layers of CH and ALG; dyeing of the CH/ALG assembled cotton fabric with cationic methylene blue that shows regular changes in terms of color depth (K/S value), which indicate that the surface were alternately deposited with CH and ALG layers and, finally, the analysis by infrared spectroscopy using Fourier Transform with Attenuated Total Reflection (ATR-FTIR), to assess the changes in the interaction between CH and ALG deposited on cotton samples.
- In Vivo High-Content Evaluation of Three-Dimensional Scaffolds BiocompatibilityPublication . Oliveira, Mariana; Ribeiro, MP.; Miguel, Sónia; Neto, Ana; Coutinho, Paula Isabel Teixeira Gonçalves; Correia, Ilídio Joaquim Sobreira; Mano, João F.While developing tissue engineering strategies, inflammatory response caused by biomaterials is an unavoidable aspect to be taken into consideration, as it may be an early limiting step of tissue regeneration approaches. We demonstrate the application of flat and flexible films exhibiting patterned high-contrast wettability regions as implantable platforms for the high-content in vivo study of inflammatory response caused by biomaterials. Screening biomaterials by using high-throughput platforms is a powerful method to detect hit spots with promising properties and to exclude uninteresting conditions for targeted applications. High-content analysis of biomaterials has been mostly restricted to in vitro tests where crucial information is lost, as in vivo environment is highly complex. Conventional biomaterials implantation requires the use of high numbers of animals, leading to ethical questions and costly experimentation. Inflammatory response of biomaterials has also been highly neglected in high-throughput studies. We designed an array of 36 combinations of biomaterials based on an initial library of four polysaccharides. Biomaterials were dispensed onto biomimetic superhydrophobic platforms with wettable regions and processed as freeze-dried three-dimensional scaffolds with a high control of the array configuration. These chips were afterward implanted subcutaneously in Wistar rats. Lymphocyte recruitment and activated macrophages were studied on-chip, by performing immunocytochemistry in the miniaturized biomaterials after 24 h and 7 days of implantation. Histological cuts of the surrounding tissue of the implants were also analyzed. Localized and independent inflammatory responses were detected. The integration of these data with control data proved that these chips are robust platforms for the rapid screening of early-stage in vivo biomaterials' response.
- Layer-by-layer deposition of antimicrobial polymers on cellulosic fibers: a new strategy to develop bioactive textilesPublication . Gomes, Ana; Mano, João F.; Queiroz, João; Gouveia, Isabel C.In recent years, there has been an increase of infectious diseases caused by different microorganisms and the development of antibiotic resistance. In this way, the search for new and efficient antibacterial materials is imperative. The main polysaccharides currently used in the biomedical and pharmaceutical domains are chitin and its derivative chitosan (CH) and alginates (ALG). In this study, a simple technique of Layer by Layer (LbL) of applying polycation CH and polyanion ALG was used to prepare CH/ALG multilayers on cotton samples via the electrostatic assembly with success. The CH/ALG cotton samples (functionalized) were investigated for their antibacterial properties towards Staphylococcus aureus and Klebsiella pneumonia using the international standard method JIS L 1902:2002. The antibacterial activity of the functionalized samples was tested in terms of bacteriostatic and bactericidal activity, and results showed that the samples exhibited a bacteriostatic effect on the two bacteria tested, as expected. In addition, samples with five layers (CH/ALG/CH/ALG/CH) were more effective in inhibiting bacterial growth. This new coating for cellulosic fibers is a new strategy and may open new avenues for the development of antimicrobial polymers with potential application in health-care field.
- Bioinspired multilayer membranes as potential adhesive patches for skin wound healingPublication . Sousa, Maria Peixoto; Neto, Ana; Correia, Tiago R.; Miguel, Sónia P.; Matsusaki, Michiya; Correia, I.J.; Mano, João F.Bioinspired and adhesive multilayer membranes are produced using the layer-by-layer (LbL) assembly of chitosan (CHT), alginate (ALG) and hyaluronic acid modified with dopamine (HA-DN). Freestanding multilayer membranes without DN are also produced as a control. The success of the synthesis of HA-DN was confirmed using UV-visible spectroscopy. Scanning electron microscopy images indicate that the surface of the DN-containing membranes is more porous than the control ones; they also present a higher average thickness value for the same number of CHT/ALG/CHT/HA(-DN) tetralayers (n = 100). Also, water uptake, mechanical strength and adhesion are enhanced with the introduction of DN moieties along the nano-layers. Besides, human dermal fibroblast viability, enhanced adhesion and proliferation were confirmed by immunofluorescence assays and by measuring both the metabolic activity and DNA content. Moreover, in vivo assays with such kinds of DN-containing multilayer membranes were performed; the application of these membranes in the treatment of dermal wounds induced in Wistar rats results in the highest decrease of inflammation of rat skin, compared with the control conditions. Overall, this investigation suggests that these mussel-inspired freestanding multilayer membranes may enhance either their mechanical performance or cellular adhesion and proliferation, leading to an improved wound healing process, being a promising material to restore the structural and functional properties of wounded skin.
- Incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applicationsPublication . Gomes, Ana; Mano, João F.; Queiroz, João; Gouveia, Isabel C.A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, peptides are the most widespread resulting in a new generation of antimicrobial agents with higher specific activity. In the present study we have developed a new strategy to obtain antimicrobial wound-dressings based on the incorporation of antimicrobial peptides into polyelectrolyte multilayer films built by the alternate deposition of polycation (chitosan) and polyanion (alginic acid sodium salt) over cotton gauzes. Energy dispersive X ray microanalysis technique was used to determine if antimicrobial peptides penetrated within the films. FTIR analysis was performed to assess the chemical linkages, and antimicrobial assays were performed with two strains: Staphylococcus aureus (Gram-positive bacterium) and Klebsiella pneumonia (Gram-negative bacterium). Results showed that all antimicrobial peptides used in this work have provided a higher antimicrobial effect (in the range of 4 log-6 log reduction) for both microorganisms, in comparison with the controls, and are non-cytotoxic to normal human dermal fibroblasts at the concentrations tested.