Maria P. Sousa

Patterned superhydrophobic paper for microfluidic devices obtained by writing and printing

This work outlines inexpensive patterning methodologies to create open-air microfluidic paper-based devices. A phase-separation methodology was used to obtain biomimetic superhydrophobic paper, hierarchically composed by micro and nano topographies. Writing and printing are simple actions that can be used to pattern flat superhydrophobic paper with more wettable channels. In particular, inkjet printing permits controlling the wettability of the surface by changing the darkness of the printed regions. The difference between capillary forces provides the possibility to control and drive liquid flows through the open path lines, just by titling the piece of paper. Additionally, maintaining a continuous flow, it is possible to direct the liquid at different volumetric rates in a horizontal position along non-linear channel paths printed/written over the surface.

Superhydrophobic Paper in the Development of Disposable Labware and Lab-on-Paper Devices

Traditionally in superhydrophobic surfaces history, the focus has frequently settled on the use of complex processing methodologies using nonbiodegradable and costly materials. In light of recent events on lab-on-paper emergence, there are now some efforts for the production of superhydrophobic paper but still with little development and confined to the fabrication of flat devices. This work gives a new look at the range of possible applications of bioinspired superhydrophobic paper-based substrates, obtained using a straightforward surface modification with poly(hydroxybutyrate). As an end-of-proof of the possibility to create lab-on-chip portable devices, the patterning of superhydrophobic paper with different wettable shapes is shown with low-cost approaches. Furthermore, we suggest the use of superhydrophobic paper as an extremely low-cost material to design essential nonplanar lab apparatus, including reservoirs for liquid storage and manipulation, funnels, tips for pipettes, or accordion-shaped substrates for liquid transport or mixing. Such devices take the advantage of the self-cleaning and extremely water resistance properties of the surfaces as well as the actions that may be done with paper such as cut, glue, write, fold, warp, or burn. The obtained substrates showed lower propensity to adsorb proteins than the original paper, kept superhydrophobic character upon ethylene oxide sterilization and are disposable, suggesting that the developing devices could be especially adequate for use in contact with biological and hazardous materials.

Non-monotonic cell differentiation pattern on extreme wettability gradients

In this study, we propose a methodology to obtain a family of biomimetic substrates with a hierarchical rough topography at the micro and nanoscale that span the entire range of wettability, from the superhydrophobic to the superhydrophilic regime, through an Ar-plasma treatment at increasing durations. Moreover, we employ the same approach to produce a superhydrophobic-to-superhydrophilic surface gradient along centimetre-length scale distances within the same sample. We characterize the biological activity of these surfaces in terms of protein adsorption and cell response, using fibronectin, a major component of the extracellular matrix, and C2C12 cells, a myoblast cell line. Fibronectin conformation, assessed via binding of the monoclonal antibody HFN7.1, exhibits a non-monotonic dependence on surface wettability, with higher activity on hydrophilic substrates (WCA = 38.6 ± 8.1°). On the other hand, the exposition of cell-binding epitopes is diminished on the surfaces with extreme wetting properties, the conformation being particularly altered on the superhydrophobic substrate. The assessment of cell response via the myogenic differentiation process reveals that a gradient surface promotes a different response with respect to cells cultured on discrete uniform samples: even though in both cases the same non-monotonic differentiation pattern is found, the differential response to the various wettabilities is enhanced along the gradient while the overall levels of differentiation are diminished. On a gradient surface cells are in fact exposed to a range of continuously changing stimuli that foster cell migration and detain the differentiation process.

Antibacterial bioadhesive layer-by-layer coatings for orthopedic applications

In this study, thin LbL films were produced by combining the adhesive properties of the hyaluronic acid-dopamine conjugate with the bioactivity and bactericidal properties of silver doped bioactive glass nanoparticles. The build-up of these films was investigated by quartz crystal microbalance with dissipation monitoring. LbL coatings were then constructed on a glass substrate for further characterization. We found that these antimicrobial bioinspired films display enhanced adhesive strength. In vitro bioactivity tests were performed by immersing them in simulated body fluid solution for 14 days where the constructed films promoted the formation of a bone-like apatite layer. From microbiological assays, it was found that coatings containing silver doped nanoparticles exhibited a remarkable antibacterial effect against Staphylococcus aureus and Escherichia coli cultures. Finally, in vitro cellular behavior tests showed enhanced cell adhesion, proliferation and viability for these antibacterial bioadhesive films. Therefore, the constructed thin films showed promising properties and evidenced great potential to be used as coatings for orthopedic implants.

High performance free-standing films by layer-by-layer assembly of graphene flakes and ribbons with natural polymers

In this work, novel free-standing (FS) films based on chitosan, alginate and graphene oxide (GO) were developed through layer-by-layer assembly. First, GO was synthesized from graphite and multi-walled carbon nanotubes using a modified Hummers method, yielding oxidized graphene flakes (o-GFs) and oxidized graphene nanoribbons (o-GNRs), respectively, which were then characterized. Then FS films were produced and their morphological, thermal and mechanical properties, as well as the o-GF and o-GNR dispersion along the films were assessed. Their degradation and swelling profiles as well as their biological behavior were evaluated. Graphite and nanotubes were successfully oxidized and exfoliated forming stable suspensions that could be combined with chitosan (CHI) and alginate (ALG) solutions by layer-by-layer processing. The addition of o-GFs and o-GNRs resulted in rougher, hydrophilic FS films with significantly improved mechanical properties relative to CHI/ALG films. The presence of o-GFs or o-GNRs did not affect the thermal stability and the addition of o-GFs resulted in films with enhanced cytocompatibility. The results demonstrate the high potential of the GO reinforced films for biomedical applications, in particular o-GF films, for wound healing, and cardiac and bone engineering applications.

Elastic chitosan/chondroitin sulfate multilayer membranes.

Freestanding multilayered films were obtained using layer-by-layer (LbL) technology from the assembly of natural polyelectrolytes, namely chitosan (CHT) and chondroitin sulfate (CS). The morphology and the transparency of the membranes were evaluated. The influence of genipin (1 and 2 mg ml(-1)), a naturally-derived crosslinker agent, was also investigated in the control of the mechanical properties of the CHT/CS membranes. The water uptake ability can be tailored by changing the crosslinker concentration that also controls the Youngs modulus and ultimate tensile strength. The maximum extension tends to decrease upon crosslinking with the highest genipin concentration, compromising the elastic properties of CHT/CS membranes: nevertheless, when using a lower genipin concentration, the ultimate tensile stress is similar to the non-crosslinked one, but exhibits a significantly higher modulus. Moreover, the crosslinked multilayer membranes exhibited shape memory properties, through a simple hydration action. The in vitro biological assays showed better L929 cell adhesion and proliferation when using the crosslinked membranes and confirmed the non-cytotoxicity of the developed CHT/CS membranes. Within this research work, we were able to construct freestanding biomimetic multilayer structures with tailored swelling, mechanical and biological properties that could find applicability in a variety of biomedical applications.

Cell-Adhesive Bioinspired and Catechol-Based Multilayer Freestanding Membranes for Bone Tissue Engineering

Mussels are marine organisms that have been mimicked due to their exceptional adhesive properties to all kind of surfaces, including rocks, under wet conditions. The proteins present on the mussel’s foot contain 3,4-dihydroxy-l-alanine (DOPA), an amino acid from the catechol family that has been reported by their adhesive character. Therefore, we synthesized a mussel-inspired conjugated polymer, modifying the backbone of hyaluronic acid with dopamine by carbodiimide chemistry. Ultraviolet–visible (UV–Vis) spectroscopy and nuclear magnetic resonance (NMR) techniques confirmed the success of this modification. Different techniques have been reported to produce two-dimensional (2D) or three-dimensional (3D) systems capable to support cells and tissue regeneration; among others, multilayer systems allow the construction of hierarchical structures from nano- to macroscales. In this study, the layer-by-layer (LbL) technique was used to produce freestanding multilayer membranes made uniquely of chitosan and dopamine-modified hyaluronic acid (HA-DN). The electrostatic interactions were found to be the main forces involved in the film construction. The surface morphology, chemistry, and mechanical properties of the freestanding membranes were characterized, confirming the enhancement of the adhesive properties in the presence of HA-DN. The MC3T3-E1 cell line was cultured on the surface of the membranes, demonstrating the potential of these freestanding multilayer systems to be used for bone tissue engineering.

Multilayered membranes with tuned well arrays to be used as regenerative patches

Membranes have been explored as patches in tissue repair and regeneration, most of them presenting a flat geometry or a patterned texture at the nano/micrometer scale. Herein, a new concept of a flexible membrane featuring well arrays forming pore-like environments to accommodate cell culture is proposed. The processing of such membranes using polysaccharides is based on the production of multilayers using the layer-by-layer methodology over a patterned PDMS substrate. The detached multilayered membrane exhibits a layer of open pores at one side and a total thickness of 38±2.2µm. The photolithography technology used to produce the molds allows obtaining wells on the final membranes with a tuned shape and micro-scale precision. The influence of post-processing procedures over chitosan/alginate films with 100 double layers, including crosslinking with genipin or fibronectin immobilization, on the adhesion and proliferation of human osteoblast-like cells is also investigated. The results suggest that the presence of patterned wells affects positively cell adhesion, morphology and proliferation. In particular, it is seen that cells colonized preferentially the well regions. The geometrical features with micro to sub-millimeter patterned wells, together with the nano-scale organization of the polymeric components along the thickness of the film will allow to engineer highly versatile multilayered membranes exhibiting a pore-like microstructure in just one of the sides, that could be adaptable in the regeneration of multiple tissues. STATEMENT OF SIGNIFICANCE Flexible multilayered membranes containing multiple micro-reservoirs are found as potential regenerative patches. Layer-by-layer (LbL) methodology over a featured PDMS substrate is used to produce patterned membranes, composed only by natural-based polymers, that can be easily detached from the PDMS substrate. The combination of nano-scale control of the polymeric organization along the thickness of the chitosan/alginate (CHT/ALG) membranes, provided by LbL, together with the geometrical micro-scale features of the patterned membranes offers a uniqueness system that allows cells to colonize 3-dimensionally. This study provides a promising strategy to control cellular spatial organization that can face the region of the tissue to regenerate.

Biomedical films of graphene nanoribbons and nanoflakes with natural polymers

Graphene and its derivatives are promising as reinforcement for polymer nanocomposites. Additionally to their inherent outstanding mechanical properties, these nanoparticles may be functionalized to enhance their compatibility with the polymeric matrix and provide specific chemical and physical properties. In this work, new freestanding films (FS) based on chitosan (CHI), alginate (ALG) and functionalized graphene were developed using the layer-by-layer assembly. Suspensions of functionalized graphene nanoflakes (f-GF) and nanoribbons (f-GNR) were prepared from expanded graphite (EG) and multi-walled carbon nanotubes (MWNTs), respectively. The graphene nanoflakes and MWNTs were covalently functionalized using a 1,3-dipolar cycloaddition reaction that allowed the nanoparticles exfoliation. f-GNR and f-GF suspensions were characterized to demonstrate that graphene nanoflakes and MWNTs were successfully functionalized and exfoliated. Then, the layer-by-layer deposition of CHI, ALG and both types of functionalized graphene was investigated and FS films were produced. The morphology, thermal and mechanical characteristics of the produced FS films were assessed. Their degradation and swelling profiles as well as their biological behavior were evaluated. The incorporation of f-GF resulted in smoother films while the incorporation of f-GNR resulted in rougher films. When compared with the CHI/ALG bi-component films. Both graphene containing films remained hydrophobic. The graphene incorporation in the multilayered FS was estimated to be 1.7 wt% for f-GF and 2.5 wt% for f-GNR. The presence of functionalized graphene did not affect the thermal stability of the films, it increased the storage modulus and the dynamic mechanical response at 1 Hz and 37 °C, and decreased the electrical resistivity. The biological assays revealed cytocompatibility towards L929 cells when both f-GF and f-GNR were incorporated in the CHI/ALG matrix. In conclusion, these new f-GF and f-GNR reinforced FS films present great potential for use in biomedical applications such as films for wound healing or cardiac and bone engineering.