Papers by Author: João F. Mano

Abstract: In the present work, chitosan (CHI) and elastin-like recombinamers (ELRs) were used to conceive nanostructured thin films driven by sequential electrostatic layer-by-layer (LbL), a simple and versatile technique that discards the use of harmful reagents. Two similar ELRs were engineered to contain negatively charged aminoacids and organized and a single monoblock or a triblock. The buildup of the films was monitored in real time using a quartz-crystal microbalance with dissipation monitoring (QCM-D). Wettability transitions were observed from a moderate hydrophobic surface to an extremely wettable upon increasing the temperature to 50 °C, accompanied by topography changes at the nanoscale as assessed by atomic force microscopy (AFM). Furthermore, the dependence on time for the surface molecular rearrangement was studied for the films with each ELR. The potential of this technology may stimulate the development of devices and biomaterials for biomedical applications in the near future, such as surfaces with tunable and patterned cell adhesion, while the use of ELRs will allow developing polypeptides with biological significance.

Abstract: In this study a new P-free system applied to the production of Bioactive Glass Nanoparticles (BG-NPs) is proposed, in order to evaluate the effect of suppressing this component, in the bioactivity capability of the materials. The BG-NPs, based on both ternary (SiO2-CaO-P2O5) and binary (SiO2-CaO) systems, were prepared via a sol-gel method. The morphology and composition of the BG-NPs were studied using FTIR and SEM. New composite membranes were produced combining chitosan and the BG-NPs. The bioactive character of the prepared biodegradable membranes was accessed in vitro by analyzing the capability for apatite formation onto the surface after being immersed in simulated body fluid (SBF). EDX and SEM were used to confirm the bioactivity of the materials.

Abstract: A number of combinations of biodegradable polymers and bioactive ceramics have been used for orthopaedic applications including in hard tissue regeneration. Ideally, composites aimed to be used in orthopaedic applications should combine adequate mechanical properties and bioactivity. Chitosan (CTS) has been widely used for biomedical applications, namely in tissue regeneration or drug delivery. In this sense, membranes of chitosan and chitosan with Bioglass® (BG) were prepared by solvent casting and characterised using Scanning Electron Microscopy. In vitro bioactivity tests were performed in the composite membranes, namely by monitoring their capability to induce the precipitation of apatite upon immersion in simulated body fluid (SBF). The results showed that the addition of BG promoted the deposition of an apatite-like layer. The deposition of apatite could influence the mechanical performance of the material. Therefore, in order to follow this biomineralization, the viscoelastic properties of these composite membranes (immersed in SBF) were evaluated. The change in the storage modulus (E’) and the loss factor (Tan δ) were measured as a function of immersion time using non-conventional dynamic mechanical analysis (DMA) tests, in which the samples were kept in wet conditions and at 37°C during the measurements. The mechanical properties of the chitosan membranes were improved by the addition of BG particles. An increase on the storage modulus was observed by the composite membranes while for the pure chitosan membranes the storage modulus was stable up to 7 days. Clear changes were detected in the composite membranes that contrasted with pure chitosan (CTS) membranes that exhibit stable viscoelastic properties up to 7 days. In addition, this work showed that sample characterization in the hydrated state can be useful to predict the mechanical performance of composites under meaningful physiological conditions.

Abstract: Surfaces and biointerfaces are recurrent when materials are used in bio-related applications. Bio-inspired concepts have been implemented in this field and gained increasing interest. In this context, stimuli-responsive surfaces have found applicability in a variety of subjects, including switchable surfaces to control protein immobilization or cell adhesion, porous membranes for controlled drug delivery applications, substrates and scaffolds for tissue engineering applications, biosensors, or membranes for bioseparation. Moreover, self-assembly strategies and hierarchical structures often found in Nature could inspire scientist and engineers to propose new solutions in the design and fabrication of surfaces with special features, such as wettability within extreme ranges, improved adhesion, optical and mechanical properties and specific bioactivity. In this work some aspects related to the use of stimuli-responsive and biomimetic surfaces in the field of biomedicine and biotechnology are highlighted and some examples are presented.

Abstract: Chitosan membranes were subjected to a pre-treatment in a double diffusion system, with a calcium solution in one chamber and a phosphate solution in the other chamber. Both chambers were separated by the chitosan membrane and subject to three mineralization periods (5, 10 and 15 minutes). After this pre-treatment the bioactivity of the different calcium phosphate coatings formed was tested for different periods of immersion time, 7, 14 and 21 days at room temperature and 37°C, in acellular simulated body fluid (1.0x). The results obtained demonstrated that the calcium phosphate coatings formed during the pre-treatment process are bioactive. It was found that the calcification is effective just in the side of the membrane exposed to the calcium solution chamber. This enabled to develop membranes with asymmetric osteoinductive properties that can be useful in different orthopedic applications.

Abstract: Bone marrow cells are a potential source to induce different lineage cells which can be used to rebuild or replace damaged tissues using a Tissue Engineering (TE) approach. However, TE strategies usually require the use of a material to support the development of a biological tissue. Beta-polyvinylidene fluoride (β-PVDF) is a biocompatible, thermoplastic with piezo-electrical properties that has been shown to provide a good cellular attachment and therefore might present advantageous properties as a scaffold material for cell seeding/culturing. The present study describes the characterization of β-PVDF membranes as a support material for growth and differentiation of goat marrow cells (GMCs) into osteoblasts, leading to the formation of substitutes for tissue regeneration. The obtained results suggest that β-PVDF piezoelectric properties influence cellular behavior. β- PVDF membranes not only enhance GMCs adherence and proliferation but also improve differentiation towards the osteogenic phenotype both in static and dynamic culture conditions. Furthermore, β-PVDF membranes exhibit very promising properties, suggesting that this material provides adequate support for the seeding and the development of undifferentiated cells towards a desired phenotype.

Abstract: An elastin-like polymer (ELP) was designed by genetic engineering techniques to mimic the extracellular matrix (ECM). The precise control of the polymer sequence offered many advantages that allowed the definition of distinct and specific domains. The cell attachment domain present in the sequence enhances the cell adhesion. Hexamethylene diisocyanate, a lysine-targeted crosslinker, was used to crosslink the purified polymer. The produced matrices presented an adequate mechanical performance and the morphological analysis by scanning electron microscopy show a homogeneous porous structure, ranging from nanometers to few micrometres. The biological tests will be assessed in future work. These results show the big potential of the ELPs in biomedical applications, especially in the development of systems for tissue engineering and drug release.

Abstract: Articular cartilage has an inadequate natural rebuilding capacity. Tissue engineering has shown to have potential to provide an effective alternative to engineer the damaged cartilage. In this study, an integrated porous bi-layered scaffold was developed aiming to mimic the requirements of cartilage and underlying subchondral bone. The osteochondral approach explored in this work was to include a common polymeric component in both cartilage and bone components, which maximised the integration at the interface by mean of a melt-based processing route. A blend of starch and poly(Llactic acid),PLLA, was used in the cartilage side, which was found to possess an adequate water uptake capability. For the bone region, to induce bioactivity, PLLA had been reinforced with hydroxyapatite (HA) and bioactive glass (BG). The surfaces of the constructs were investigated as a function of soaking time in a simulated body (SBF) fluid using scanning electron microscopy (SEM) and FTIR. The SEM – FTIR indicated a bone-like apatite formation and the surface coverage by apatite layer increased with increasing soaking time, whereas the cartilage-layer did not exhibit the formation of any apatite like layer.

Abstract: In this study, it is shown that it is possible to develop 3D-porous bilayer hydroxyapatite/chitosan scaffolds by means of combining a sintering and a freeze-drying technique. Scanning electron microscopy (SEM/EDS) studies revealed that the scaffolds possess a well-defined orientation and anisotropic porosity, with pore size ranging between 50-350 µm. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) patterns evidenced the formation of crystalline hydroxyapatite. Moreover, the compression tests revealed that these scaffolds have adequate mechanical properties for being used in tissue engineering of osteochondral defects.

Abstract: In this work, the structural evolution and damage of PET during stretching is assessed by wide- and small-angle X-ray scattering (respectively, WAXS and SAXS) experiments in specimens pre-deformed at different strain levels (ex-situ characterization). Injection moulded PET rectangular tensile specimens were stretched (at 2 mm/min) into the plastic domain in a universal test machine at different strain levels at room temperature. The structure of the central zone of the deformed specimens was then characterized by WAXS and SAXS experiments using an X-ray synchrotron source. PET was initially (before stretching) amorphous. A strong molecular orientation in the stretching direction is quickly developed for the initial plastic deformation levels, evidenced by strong equatorial WAXS reflections. This orientation rapidly levels off, remaining constant during further stretching. The WAXS patterns are accompanied with no reflections on SAXS, evidencing a local ordering phenomenon, typical of nematic liquid-crystalline structures. The SAXS patterns evidence the occurrence of some voiding in the cold drawing regime just after yielding. These anisotropic voids are oriented perpendicular to the stretching direction, as in a craze-like structure. The void size drastically increases at the onset of the strong strain hardening behaviour.