Ana Rita C. Duarte

© 2014 American Institute of Chemical Engineers. Significance: Natural deep eutectic solvents (NADES) are defined as a mixture of two or more solid or liquid components, which at a particular composition present a high melting point depression becoming liquids at room temperature. NADES are constituted by natural molecules and fully represent the green chemistry principles. For these reasons, the authors believe that the submitted manuscript is a highly valuable contribution to the field of green chemistry and chemical engineering. For the first time, the possibility to use NADES as enhancers of supercritical fluid technology is revealed.

The design and production of structures with nanometer-sized polymer films based on layer-by-layer (LbL) are of particular interest for tissue engineering since they allow the precise control of physical and biochemical cues of implantable devices. In this work, a method is developed for the preparation of nanostructured hollow multilayers tubes combining LbL and template leaching. The aim is to produce hollow tubes based on polyelectrolyte multilayer films with tuned physical-chemical properties and study their effects on cell behavior. The final tubular structures are characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), microscopy, swelling, and mechanical tests, including dynamic mechanical analysis (DMA) in physiological simulated conditions. It is found that more robust films could be produced upon chemical cross-linking with genipin. In particular, the mechanical properties confirms the viscoelastic properties and a storage and young modulus about two times higher. The water uptake decreases from about 390{%} to 110{%} after the cross-linking. The biological performance is assessed in terms of cell adhesion, viability, and proliferation. The results obtained with the cross-linked tubes demonstrate that these are more suitable structures for cell adhesion and spreading. The results suggest the potential of these structures to boost the development of innovative tubular structures for tissue engineering approaches.

Green technology actively seeks new solvents to replace common organic solvents that present inherent toxicity and have high volatility, leading to evaporation of volatile organic compounds to the atmosphere. Over the past two decades, ionic liquids (ILs) have gained enormous attention from the scientific community, and the number of reported articles in the literature has grown exponentially. Nevertheless, IL "greenness" is often challenged, mainly due to their poor biodegradability, biocompatibility, and sustainability. An alternative to ILs are deep eutectic solvents (DES). Deep eutectic solvents are defined as a mixture of two or more components, which may be solid or liquid and that at a particular composition present a high melting point depression becoming liquids at room temperature. When the compounds that constitute the DES are primary metabolites, namely, aminoacids, organic acids, sugars, or choline derivatives, the DES are so called natural deep eutectic solvents (NADES). NADES fully represent green chemistry principles. Can natural deep eutectic solvents be foreseen as the next generation solvents and can a similar path to ionic liquids be outlined? The current state of the art concerning the advances made on these solvents in the past few years is reviewed in this paper, which is more than an overview on the different applications for which they have been suggested, particularly, biocatalysis, electrochemistry, and extraction of new data. Citotoxicity of different NADES was evaluated and compared to conventional imidazolium-based ionic liquids, and hints at the extraction of phenolic compounds from green coffee beans and on the foaming effect of NADES are revealed. Future perspectives on the major directions toward which the research on NADES is envisaged are here discussed, and these comprised undoubtedly a wide range of chemically related subjects. © 2014 American Chemical Society.

In this work, blends of starch and poly-$ε$-caprolactone (PCL) doped with different concentrations of 1-butyl-3-methylimidazolium acetate ([BMIM]Ac) or 1-butyl-3-methylimidazolium chloride ([BMIM] Cl) were studied. The blends were characterized by mechanical analysis, infra-red spectroscopy (FTIR), differential scanning calorimetry (DSC) and dielectric relaxation spectroscopy (DRS), evaluating the IL doping effect. The samples were subjected to supercritical carbon dioxide foaming and the morphology of the structures was assessed. DSC shows a single glass transition and melting endotherm for foamed and unfoamed samples, having no effect upon IL doping, and DRS shows increased molecular mobility for blends with higher IL concentrations, and some hindrance for lower ones. The conductivity for SPCL doped with 30{%} [BMIM] Cl, before and after foaming, is comparable to the conductivity of the IL but exhibits more stable conductivity values, opening doors for applications as self-supported conductive materials. © 2014 the Partner Organisations.

Some approaches have been developed in our group to investigate the role of novel ionic liquids as process and property modifiers of natural-based polymers. In our previous work, we proposed the use of ionic liquids as plasticizing agents for the creation of porous structures from a semi-crystalline natural-based polymer. The current work intended to complement the previous studies, evaluating the ability of ionic liquid (IL) to plasticize polymers such as blends of starch-poly-lactic acid (SPLA) and its effect on supercritical fluid foaming process (SCF) and providing more insights on the mechanisms involved. For this purpose, blends of starch with poly (lactic) acid, with different ratios of starch and poly-lactic acid of 50:50 and 30:70 were modified and processed using 1-butyl-3-methylimidazolium chloride ([bmim]Cl). Supercritical fluid foaming was studied at different soaking times (1, 3 and 6h) using carbon dioxide at 20.0MPa and 40°C. The blends were characterized by different techniques, such as infra-red spectroscopy, differential scanning calorimetry and compression and tensile mechanical analysis. The morphology of the foamed structures was analyzed by scanning electron microscopy and micro-computed tomography. The results suggest that after 3h of soaking time an equilibrium state of carbon dioxide into the bulk samples is attained, yielding structures with 6{%} and 15{%} of porosity, for SPLA70 and SPLA50 respectively. The solubility of carbon dioxide within the matrices was studied for the same conditions and the results demonstrate a higher sorption degree in the samples doped with ionic liquid. Sorption and desorption diffusion coefficients of supercritical CO 2 in the SPLA matrix were determined for the raw polymer and for the SPLA doped with [bmim]Cl. It was found that the lower desorption diffusion coefficients are related with the higher porosity obtained by the foaming process. © 2013.

Marine biomaterials are a new emerging area of research with significant applications. Recently, researchers are dedicating considerable attention to marine-sponge biomaterials for various applications. We have focused on the potential of biosilica from Petrosia ficidormis for novel biomedical/industrial applications. A bioceramic structure from this sponge was obtained after calcination at 750 °C for 6 h in a furnace. The morphological characteristics of the three-dimensional architecture were evaluated by scanning electron microscopy (SEM) and microcomputed tomography, revealing a highly porous and interconnected structure. The skeleton of P. ficidormis is a siliceous matrix composed of SiO2, which does not present inherent bioactivity. Induction of bioactivity was attained by subjecting the bioceramics structure to an alkaline treatment (2M KOH) and acidic treatment (2M HCl) for 1 and 3 h. In vitro bioactivity of the bioceramics structure was evaluated in simulated body fluid (SBF), after 7 and 14 days. Observation of the structures by SEM, coupled with spectroscopic elemental analysis (EDS), has shown that the surface morphology presented a calcium-phosphate CaP coating, similar to hydroxyapatite (HA). The determination of the Ca/P ratio, together with the evaluation of the characteristic peaks of HA by infrared spectroscopy and X-ray diffraction, have proven the existence of HA. In vitro biological performance of the structures was evaluated using an osteoblast cell line, and the acidic treatment has shown to be the most effective treatment. Cells were seeded on bioceramics structures and their morphology; viability and growth were evaluated by SEM, MTS assay, and DNA quantification, respectively, demonstrating that cells are able to grow and colonize the bioceramic structures. © 2014 American Chemical Society.

© 2014 American Chemical Society. Engineering metabolically demanding tissues requires the supply of nutrients, oxygen, and removal of metabolic byproducts, as well as adequate mechanical properties. In this work, we propose the development of chitosan (CHIT)/alginate (ALG) freestanding membranes fabricated by layer-by-layer (LbL) assembly. CHIT/ALG membranes were cross-linked with genipin at a concentration of 1 mg·mL {\textless} sup {\textgreater} -1 {\textless} /sup {\textgreater} or 5 mg·mL {\textless} sup {\textgreater} -1 {\textless} /sup {\textgreater} . Mass transport properties of glucose and oxygen were evaluated on the freestanding membranes. The diffusion of glucose and oxygen decreases with increasing cross-linking concentration. Mechanical properties were also evaluated in physiological-simulated conditions. Increasing cross-linking density leads to an increase of storage modulus, Young modulus, and ultimate tensile strength, but to a decrease in the maximum hydrostatic pressure. The in vitro biological performance demonstrates that cross-linked films are more favorable for cell adhesion. This work demonstrates the versatility and feasibility of LbL assembly to generate nanostructured constructs with tunable permeability, mechanical, and biological properties.

An alternative, green method was used to develop chitin-based biocomposite (ChHA) materials by an integrated strategy using ionic liquids, supercritical fluid drying, and salt leaching. ChHA matrices were produced by dissolving chitin in 1-butyl-methylimidazolium acetate along with salt and/or hydroxyapatite particles and then subsequent drying. The ChHA composite formed had a heterogeneous porous microstructure with 65{%}-85{%} porosity and pore sizes in the range of 100-300 $μ$m. The hydroxyapatite was found to be well distributed within the composite structures and had a positive effect in the viability and proliferation of osteoblast-like cells, in vitro. Our findings indicate that these ChHA matrices have potential applications in bone tissue engineering. © The Author(s) 2013.

Chitin agglomerated scaffolds were produced and functionalized using the green chemistry principles and clean technologies. Such combination enabled the functionalization of chitin microparticles prepared through dissolution of the polymer in ionic liquids, followed by of the application of a sol-gel method. Finally, the 3D constructs were moulded and dried using a supercritical assisted agglomeration method. Structural and morphological characterization is presented using scanning electronic microscopy (SEM) and micro-computed tomography ([small micro]-CT). An evaluation of the bioactive behavior of the matrices was made by immersing them in simulated body fluid (SBF) for up to 21 days. The potential of such matrices as drug delivery systems was evaluated after the incorporation of dexamethasone into the matrices during drying in supercritical assisted agglomeration. The findings suggested that the morphological features such as porosity, interconnectivity and pore size distribution of the matrices can be tunned by changing particle size, chitin concentration and the pressure applied during moulding. Chitin microspheres were modified by siloxane and silanol groups, providing a bioactive behavior; the apatite formation was shown to be dependent on the amount and arrangement of silanol groups. Furthermore, in vitro drug release studies showed that dexamethasone was sustainably released. All findings suggest that this strategy is a feasible and advantageous process to obtain chitin-based 3D structures with both functional and structural characteristics that make then suitable for regenerative medicine applications.

The possibility to fabricate marine collagen porous structures crosslinked with genipin under high pressure carbon dioxide is investigated. Collagen from shark skin is used to prepare pre-scaffolds by freeze-drying. The poor stability of the structures and low mechanical properties require crosslinking of the structures. Under dense CO 2 atmosphere, crosslinking of collagen pre-scaffolds is allowed for 16 h. Additionally, the hydrogels are foamed and the scaffolds obtained present a highly porous structure. In vitro cell culture tests performed with a chondrocyte-like cell line show good cell adherence and proliferation, which is a strong indication of the potential of these scaffolds to be used in tissue cartilage tissue engineering. The development of an optimized processing technique for the preparation of stable structures from marine origin collagen is described. The samples are processed under a dense carbon dioxide atmosphere that promotes crosslinking and enhances the morphology of the 3D architectures obtained. © 2013 WILEY-VCH Verlag GmbH {&} Co. KGaA, Weinheim.

One of the major scientific challenges that tissue engineering and regenerative medicine (TERM) faces to move from benchtop to bedside regards biomaterials development, despite the latest advances in polymer processing technologies. A variety of scaffolds processing techniques have been developed and include solvent casting and particles leaching, compression molding and particle leaching, thermally induced phase separation, rapid prototyping, among others. Supercritical fluids appear as an interesting alternative to the conventional methods for processing biopolymers as they do not require the use of large amounts of organic solvents and the processes can be conducted at mild temperatures. However, this processing technique has only recently started to receive more attention from researchers. Different processing methods based on the use of supercritical carbon dioxide have been proposed for the creation of novel architectures based on natural and synthetic polymers and these will be unleashed in this paper. © 2013 Elsevier B.V. All rights reserved.

One of the major scientific challenges that tissue engineering and regenerative medicine (TERM) faces to move from benchtop to bedside regards biomaterials development, despite the latest advances in polymer processing technologies. A variety of scaffolds processing techniques have been developed and include solvent casting and particles leaching, compression molding and particle leaching, thermally induced phase separation, rapid prototyping, among others. Supercritical fluids appear as an interesting alternative to the conventional methods for processing biopolymers as they do not require the use of large amounts of organic solvents and the processes can be conducted at mild temperatures. However, this processing technique has only recently started to receive more attention from researchers. Different processing methods based on the use of supercritical carbon dioxide have been proposed for the creation of novel architectures based on natural and synthetic polymers and these will be unleashed in this paper. © 2013 Elsevier B.V. All rights reserved.

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10 6 cells/mL) were encapsulated in GG hydrogels (1.5{%}) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×10 6 cells/mL) were encapsulated in GG hydrogels (1.5{%}) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner. © Copyright 2012, Mary Ann Liebert, Inc.

A new generation of scaffolds capable of acting not only as support for cells but also as a source of biological cues to promote tissue regeneration is currently a hot topic of in bone Tissue Engineering (TE) research. The inclusion of growth factor (GF) controlled release functionalities in the scaffolds is a possible strategy to achieve such goal. Platelet Lysate (PL) is an autologous source of GFs, providing several bioactive agents known to act on bone regeneration. In this study, chitosan-chondroitin sulfate nanoparticles loaded with PL were included in a poly(d,l-lactic acid) foam produced by supercritical fluid foaming. The tridimensional (3D) structures were then seeded with human adipose-derived stem cells (hASCs) and cultured in vitro under osteogenic stimulus. The osteogenic differentiation of the seeded hASCs was observed earlier for the PL-loaded constructs, as shown by the earlier alkaline phosphatase peak and calcium detection and stronger Runx2 expression at day 7 of culture, in comparison with the control scaffolds. Osteocalcin gene expression was upregulated in presence of PL during all culture period, which indicates an enhanced osteogenic induction. These results suggest the synergistic effect of PL and hASCs in combinatory TE strategies and support the potential of PL to increase the multifunctionality of the 3D hybrid construct for bone TE applications. © 2012 Elsevier B.V. All rights reserved.

In this work, the ability to foam semi-crystalline natural-based polymers by supercritical fluid technology is evaluated. The application of this technique to natural polymers has been limited due to the fact that they are normally semi-crystalline polymers, which do not plasticize in the presence of carbon dioxide. This can be overcome by the use of plasticizers, such as glycerol, which is a commonly used plasticizer, or ionic liquids, which have recently been proposed as plasticizing agents for different polymers. Following the green chemistry principles, the main aim is, hereafter, the design and development of new 3D architectures of natural-based polymers, combining ionic liquids (IL) and supercritical fluid (SCF) technology. A polymeric blend of starch, one of the most abundantly occurring natural polymers, and poly-$ε$-caprolactone, a synthetic polymer, which is a biodegradable aliphatic polyester commonly used in an array of biomedical applications (SPCL), was processed by supercritical fluid foaming, at different operating conditions, namely pressure (10.0 up to 20.0 MPa), temperature (35 up to 60 °C) and soaking time (30 min up to 3 h). The ionic liquid tested in this work was 1-butyl-3-methylimidazolium acetate ([bmim]Ac). The interactions between SPCL and [bmim] Ac or glycerol were analysed by Fourier transform infrared spectroscopy, differential scanning calorimetry and by mechanical tests, using both tensile and compressive modes. Morphological analysis, porosity, interconnectivity and pore size distribution of the matrixes were evaluated and the morphology was analyzed by scanning electron microscopy and by micro-computed tomography. To our knowledge the use of ionic liquids as foaming agents is reported here for the first time. The results obtained suggest that this approach can further promote the development of composite polymer-IL materials, particularly for catalysis, chromatography, extraction and separation purposes. © The Royal Society of Chemistry 2012.

A marine derived polysaccharide, ulvan, extracted from green algae, was combined with poly-d, l-lactic acid (PDLLA) in order to produce a novel scaffold for bone tissue engineering applications. Three dimensional (3D) scaffolds of PDLLA loaded with ulvan particles were originally prepared by subcritical fluid sintering with carbon dioxide at 40°C and 50 bar. Prepared matrixes were characterized in order to validate their suitability to be used as scaffolds for bone tissue regeneration. Characterization included micro-computed tomography, mechanical compression testing, water uptake and degradation testing, and cytotoxicity assays. In addition, ulvan particles loaded with dexamethasone, were also dispersed within the PDLLA matrix and the respective release profile from the samples was evaluated. Prepared PDLLA scaffolds enriched with ulvan particles demonstrated appropriate physicochemical and cytocompatible features to be used for the envisaged applications. On the other hand, the release of dexamethasone from ulvan particles embedded within the PDLLA matrix revealed that the designed systems can be potentially suitable for localized drug delivery. These results further contribute to the establishment of ulvan as a potential novel biomaterial. © 2012 Elsevier B.V. All rights reserved.

The aim of this study was to evaluate the possibility of preparing chitosan porous matrixes using supercritical fluid technology. Supercritical immersion precipitation technique was used to prepare scaffolds of a natural biocompatible polymer, chitosan for tissue engineering purposes. The physicochemical and biological properties of chitosan make it an excellent material for the preparation of drug delivery systems and for the development of new biomedical applications in many fields from skin to bone or cartilage. Supercritical assisted phase inversion experiments were carried out and the effect of different organic solvents on the morphology of the scaffolds was assessed. Chitosan scaffold morphology, porosity and pore size were evaluated by SEM and micro-CT. A thermodynamic analysis of the process was carried out and insights on the solubility parameter and Flory-Huggins interaction parameters are given. The preparation of a highly porous and interconnected structure of a natural material, chitosan, using a clean and environmentally friendly technology constitutes a new processing technology for the preparation of scaffolds for tissue engineering using these materials. © 2011 Elsevier B.V.

The application of green chemistry principles in the processing of materials for advanced technologies is a steadily increasing field of research. In this work porous chitin-based materials were developed by combining the processing of chitin using ionic liquids (ILs) as a green solvent together with the use of supercritical fluid technology (SCF) as clean technology. Chitin was dissolved in 1-butyl-3-imidazolium acetate, followed by regeneration of the polymer in ethanol in specific moulds. The IL was removed using Soxhlet extraction and successive steps of extraction with SCF using carbon dioxide/ethanol ratios of 50/50 and 70/30. The developed porous chitin-based structures (ChIL) can be classified as mesoporous materials, with very low density and high porosity. The cytotoxicity of ChIL extracts was investigated using L929 fibroblast-like cells, and the results demonstrated that the produced materials have extremely low cytotoxicity levels. Therefore, the findings suggest that the porous chitin structures may be potential candidates for a number of biomedical applications, including tissue engineering. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A completely new strategy for cell culture focusing on the design of three-dimensional (3D) smart surfaces by supercritical fluid technology has been developed. This approach might overcome the limitations on cell expansion and proliferation of currently existing techniques. An alternative technology, based on supercritical carbon dioxide, was used to polymerize poly(N- isopropylacrylamide) (PNIPAAm) and to foam poly(d,l-lactic acid) (P D,L LA), creating a thermosensitive 3D structure which has proven to have potential as a substrate for cell growth and expansion. We demonstrated that the thermosensitive matrices promoted cell detachment, thus P D,L LA scaffolds have the potential to be used as substrates for cell growth and expansion avoiding enzymatic and mechanical methods of cell harvesting. The harvested cells were replated to evaluate their viability, which was not compromised. A major advantage of this technology is the fact that the prepared materials can be recovered and reused. Therefore, the same substrate can be recycled and reused for different batches. An indirect impact of the technology developed is related to the field of biotechnology, as this novel technology for cell expansion can be applied to any adherent cell cultures. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

We present some selected results indicating the feasibility of preparing therapeutic finished ophthalmic articles, namely commercially available soft contact lenses, using a supercritical solvent impregnation (SSI) technique. Several commercial soft contact lenses were tested and, among these, four lenses were selected for more complete studies: Nelfilcon A (FocusDailies®, CIBA Vision), Omafilcon A (Proclear® Compatibles, CooperVision), Methafilcon A (Frequency® 55, CooperVision) and Hilafilcon B (SofLens® 59 Comfort, Bausch {&} Lomb). Supercritical carbon dioxide (scCO2) was the chosen supercritical fluid and two ophthalmic drugs were tested: flurbiprofen (a NSAID, hydrophobic) and timolol maleate (an anti-glaucoma drug, hydrophilic). The effects of operational pressure, of impregnation duration and of the addition of a cosolvent (ethanol) were studied on the overall drug loading yields. Depending on the experiment, we employed pressures from 9 up to 16 MPa and impregnation times from 30 up to 180 min. Temperature was kept constant and equal to 313 K. The employed depressurization rates were kept low and between 0.1 and 0.2 MPa/min. Results are discussed in terms of the employed operational conditions and taking in consideration all the possible interactions between supercritical fluids, drugs, cosolvents and the polymers which compose the employed hydrogel contact lenses. In vitro drug release experiments were carried out in order to evaluate the resulting drug release profiles. Obtained results were also compared with drug-loaded contact lenses obtained by conventional drug "soaking" in aqueous solutions. Results also proved that SSI can be considered as a viable, efficient and safe alternative for the impregnation of drugs, including those of hydrophobic character or presenting low aqueous solubility, into commercial soft contact lenses. SSI proved to be a "tunable" process since the variation of the employed operational conditions indicated that it is possible to control the amount of impregnated drug. In the end, the ophthalmic articles were recovered undamaged and without the presence of harmful solvent residues. This method also permits to process already prepared commercial contact lenses, without interfering with their manufacture methods and, after processing, store them for future use. © 2010 Elsevier B.V. All rights reserved.

Starch-based polymers have been proposed for different tissue engineering applications due to their inherent properties. In this work, a polymeric blend of starch-poly-(?-caprolactone) (SPCL) was processed using supercritical fluid technology, namely, by supercritical assisted phase inversion. As SPCL is a biodegradable polymer, the matrices produced are susceptible of undergoing enzymatic degradation upon implantation in the human body. In vitro assessment of the enzymatic degradation of SPCL was carried out in different buffer solutions containing a-amylase and/or lipase. The effect of the presence ofthese enzymes was studied by monitoring different parameters in order to characterise both bulk and the surface of the scaffolds. As regards to bulk analysis, weight loss of the samples incubated for 1, 3, 7, 14 and 21 days was determined, further differential scanning calorimetry was carried out. The morphology of the scaffolds after these periods was analysed by micro-computed tomography (?-CT) and surface chemistry was characterised by infra-red spectroscopy and contact angle measurements. Results suggest that SPLC scaffolds undergo bulk degradation, which is typically characterised by hydrolysis of chemical bonds in the polymer chain at the centre of the matrix, resulting in a highly porous material. ? 2010 Elsevier Ltd. All rights reserved.