Ana Rita C. Duarte

The fluid phase behaviour for the binary systems carbon dioxide+cyclobutanone and propane+cyclobutanone has been determined experimentally, using Cailletet equipment. For both the systems bubble points have been determined for a number of isopleths covering the whole mole fraction range. Additionally, for the binary system carbon dioxide+cyclobutanone dew points and critical points could be observed for a number of overall-compositions rich in carbon dioxide. The temperature and pressure range were, respectively, from 278 to 369K and from 0.1 to 14.0MPa. Correlation of the experimental data of both systems has been performed using the Soave-Redlich-Kwong (SRK) equation of state. Satisfactory results have been achieved using only one binary interaction parameter. © 2003 Elsevier B.V. All rights reserved.

Phase equilibrium measurements of single and mixed organic clathrate hydrates with hydrogen were determined within a pressure range of 2.0-14.0. MPa. The organic compounds studied were furan, 2,5-dihydrofuran, tetrahydropyran, 1,3-dioxolane and cyclopentane. These organic compounds are known to form structure II clathrate hydrates with water. It was found that the addition of hydrogen to form a mixed clathrate hydrate increases the stability compared to the single organic clathrate hydrates. Moreover, the mixed clathrate hydrate also has a much higher stability compared to a pure hydrogen structure II clathrate hydrate. Therefore, the organic compounds act as promoter materials. The stabilities of the single and mixed organic clathrate hydrates with hydrogen showed the following trend in increasing order: 1,3-dioxolane {\textless} 2,5-dihydrofuran {\textless} tetrahydropyran {\textless} furan {\textless} cyclopentane, indicating that both size and geometry of the organic compound determine the stability of the clathrate hydrates. © 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.

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.

© 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.

Marine sponges are a rich source of natural bioactive compounds. One of the most abundant valuable products is collagen/gelatin, which presents an interesting alternative source for pharmaceutical and biomedical applications. We have previously proposed an innovative green technology for the extraction of collagen/gelatin from marine sponges based in water acidified with carbon dioxide. In this work, we have optimized the process operating conditions toward high yields and collagen quality as well as to reduce extraction procedure duration and energy consumption. The process extraction efficiency is higher than 50{%}, corresponding to a yield of approximately 10{%} of the sponge dry mass, obtained for mild operating conditions of 10 bar and 3 h. The extracted material was characterized by scanning electron microscopy (SEM), rheology, Fourier transformed infrared spectroscopy (FTIR), circular dichroism (CD), amino acid analysis, and SDS-PAGE. The extracts were found to be composed of highly pure mixtures of co...

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.

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.

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 the tissue engineering (TE) field, the concept of producing multifunctional scaffolds, capable not only of acting as templates for cell transplantation but also of delivering bioactive agents in a controlled manner, is an emerging strategy aimed to enhance tissue regeneration. In this work, a complex hybrid release system consisting in a three-dimensional (3D) structure based on poly(d,l-lactic acid) (PDLLA) impregnated with chitosan/chondroitin sulfate nanoparticles (NPs) was developed. The scaffolds were prepared by supercritical fluid foaming at 200 bar and 35 °C, and were then characterized by scanning electron microscopy (SEM) and micro-CT. SEM also allowed to assess the distribution of the NPs within the structure, showing that the particles could be found in different areas of the scaffold, indicating a homogeneous distribution within the 3D structure. Water uptake and weight loss measurements were also carried out and the results obtained demonstrated that weight loss was not significantly enhanced although the entrapment of the NPs in the 3D structure clearly enhances the swelling of the structure. Moreover, the hybrid porous biomaterial displayed adequate mechanical properties for cell adhesion and support. The possibility of using this scaffold as a multifunctional material was further evaluated by the incorporation of a model protein, bovine serum albumin (BSA), either directly into the PDLLA foam or in the NPs that were eventually included in the scaffold. The obtained results show that it is possible to achieve different release kinetics, suggesting that this system is a promising candidate for dual protein delivery system for TE applications. © 2010 Elsevier B.V.

© 2017 Elsevier B.V. Porous polymeric materials are studied in tissue engineering, because they can act as support for cell proliferation and as drug delivery vehicles for regeneration of tissues. Hydrogel foaming with supercritical CO 2 is a suitable alternative for the creation of these structures, since it avoids the use of organic solvents and high temperature in the processing. In this work, $\beta$-glucans were used as raw materials to create hydrogels due to their easily gelation and biological properties. The enhancement of porosity was generated by a fast decompression after keeping the hydrogels in contact with CO 2 . The effect of the processing conditions and type of $\beta$-glucan in the final properties was assessed regarding morphological and mechanical properties. Finally, the ability of these materials to sustainably deliver dexamethasone was evaluated. The scaffolds had good morphology and provided a controlled release, thus being suitable to be used as scaffolds and drug delivery vehicles.

Polysaccharide aerogels are a good alternative as carriers for drug delivery, since they allow high loading of the active compounds in matrices that are non-toxic, biocompatible and from a renewable feedstock. In this work, barley and yeast $\beta$-glucans aerogels were produced by gelation in aqueous solution, followed by solvent exchange and drying with supercritical CO2. First, viscoelastic properties and melting profile of the hydrogels were determined. Then, the obtained aerogels were analyzed regarding morphology, mechanical properties and behavior in physiological fluid. Both in the hydrogels and in the aerogels, big differences were observed between barley and yeast $\beta$-glucans due to their different chain structure and gelation behavior. Finally, impregnation of acetylsalicylic acid was performed at the same time as the drying of the alcogels with supercritical CO2. The release profile of the drug in PBS was analyzed in order to determine the mechanism governing the release from the $\beta$-glucan matrix. 2017 Elsevier Ltd. All rights reserved.

A laboratory-scale reactor system is built and operated to measure the kinetic of formation for single and mixed carbon dioxide-tetrahydrofuran hydrates. The T-cycle method, which is used to collect the kinetic data, is briefly discussed. For single carbon dioxide hydrate, the induction time decreases with the increase of the initial carbon dioxide pressure up to 2.96. MPa. Beyond this pressure, the induction time is becoming relatively constant with the increase of initial carbon dioxide pressure indicating that the liquid phase is completely supersaturated with carbon dioxide. Experimental results show that the inclusion of tetrahydrofuran reduces the induction time required for hydrate formation. These observations indicate hydrate nucleation process and onset growth are more readily to occur in the presence of tetrahydrofuran. In contrast, the presence of sodium chloride prolongs the induction time due to clustering of water molecules with the ions and the salting-out effects. It is also shown that the degree of subcooling required for hydrate formation is affected by the presence of tetrahydrofuran and sodium chloride in the hydrate forming system. The presence of tetrahydrofuran in the hydrate system significantly reduces the amount of carbon dioxide uptake. The apparent rate constant, k, for those systems are reported. © 2010.

This paper presents a novel approach toward the production of hybrid alginate–lignin aerogels. The key idea of the approach is to employ pressurized carbon dioxide for gelation. Exposure of alginate and lignin aqueous alkali solution containing calcium carbonate to CO2at 4.5 MPa resulted in a hydrogel formation. Various lignin and CaCO3concentrations were studied. Stable hydrogels could be formed up to 2:1 (w/w) alginate-to-lignin ratio (1.5 wt{%} overall biopolymer concentration). Upon substitution of water with ethanol, gels were dried in supercritical CO2to produce aerogels. Aerogels with bulk density in the range 0.03–0.07 g/cm3, surface area up to 564 m2/g and pore volume up to 7.2 cm3/g were obtained. To introduce macroporosity, the CO2induced gelation was supplemented with rapid depressurization (foaming process). Macroporosity up to 31.3 ± 1.9{%} with interconnectivity up to 33.2 ± 8.3{%} could be achieved at depressurization rate of 3 MPa/min as assessed by micro-CT. Young's modulus of alginate–lignin aerogels was measured in both dry and wet states. Cell studies revealed that alginate–lignin aerogels are non-cytotoxic and feature good cell adhesion making them attractive candidates for a wide range of applications including tissue engineering and regenerative medicine.

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.

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.

Aerogels are a special class of ultra-light porous materials with growing interest in biomedical applications due to their open pore structure and high surface area. However, they usually lack macroporosity, while mesoporosity is typically high. In this work, carbon dioxide induced gelation followed by expansion of the dissolved CO{\textless}inf{\textgreater}2{\textless}/inf{\textgreater} was performed to produce hybrid calcium-crosslinked alginate-starch hydrogels with dual meso- and macroporosity. The hydrogels were subjected to solvent exchange and supercritical drying to obtain aerogels. Significant increase in macroporosity from 2 to 25{%} was achieved by increasing expansion rate from 0.1 to 30 bar/min with retaining mesoporosity (BET surface and BJH pore volume in the range 183-544m{\textless}sup{\textgreater}2{\textless}/sup{\textgreater}/g and 2.0-6.8cm{\textless}sup{\textgreater}3{\textless}/sup{\textgreater}/g, respectively). In vitro bioactivity studies showed that the alginate-starch aerogels are bioactive, i.e. they form hydroxyapatite crystals when immersed in a simulated body fluid solution. Bioactivity is attributed to the presence of calcium in the matrix. The assessment of the biological performance showed that the aerogels do not present a cytotoxic effect and the cells are able to colonize and grow on their surface. Results presented in this work provide a good indication of the potential of the alginate-starch aerogels in biomedical applications, particularly for bone regeneration.

© 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.

Electrospun structures were proposed as scaffolds owing to their morphological and structural similarities with the extracellular matrix found in many native tissues. These fibrous structures were also proposed as drug release systems by exploiting the direct dependence of the release rate of a drug on the surface area. An osteogenic differentiation factor, dexamethasone (DEX), was incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (5, 10, 15 and 20 wt.{%} polymer), in a single-step process. The DEX incorporated into the polymeric carrier is in amorphous state, as det ermined by DSC, and does not influence the typical nanofibers morphology. In vitro drug release studies demonstrated that the dexamethasone release was sustained over a period of 15 days. The bioactivity of the released dexamethasone was assessed by cultivating human bone marrow mesenchymal stem cells (hBMSCs) on 15 wt.{%} DEX-loaded PCL NFMs, under dexamethasone-absent osteogenic differentiation medium formulation. An increased concentration of alkaline phosphatase and deposition of a mineralized matrix was observed. Phenotypic and genotypic expression of osteoblastic-specific markers corroborates the osteogenic activity of the loaded growth/differentiation factor. Overall data suggests that the electrospun biodegradable nanofibers can be used as carriers for the sustained release of growth/differentiation factors relevant for bone tissue engineering strategies. © 2010 Elsevier Ltd.

Fast-dissolving delivery systems (FDDS) have received increasing attention in the last years. Oral drug delivery is still the preferred route for the administration of pharmaceutical ingredients. Nevertheless, some patients, e.g. children or elderly people, have difficulties in swallowing solid tablets. In this work, gelatin membranes were produced by electrospinning, containing an encapsulated therapeutic deep-eutectic solvent (THEDES) composed by choline chloride/mandelic acid, in a 1:2 molar ratio. A gelatin solution (30{%} w/v) with 2{%} (v/v) of THEDES was used to produce electrospun fibers and the experimental parameters were optimized. Due to the high surface area of polymer fibers, this type of construct has wide applicability. With no cytotoxicity effect, and showing a fast-dissolving release profile in PBS, the gelatin fibers with encapsulated THEDES seem to have promising applications in the development of new drug delivery systems.

© 2017 American Chemical Society. Recent findings have reported the reason why some living beings are able to withstand the huge thermal amplitudes between winter and summer in their natural habitats. They are able to produce metabolites decreasing deeply the crystallization temperature of water, avoiding cell disrupture due to the presence of ice crystals and overcoming osmotic effects. In vitro, the possibility to cool living cells and tissues to cryogenic temperatures in the absence of ice can be achieved through a vitrification process. Vitrification has been suggested as an alternative approach to cryopreservation and could hereafter follow an interesting biomimetic perspective. The metabolites produced by these animals are mostly sugars, organic acids, choline derivatives, or urea. When combined at a particular composition, these compounds form a new liquid phase which has been defined as Natural Deep Eutectic Solvents (NADES). In this review, we relate the findings of different areas of knowledge from evolutive biology, cryobiology, and thermodynamics and give a perspective to the potential of NADES in the development of new cryoprotective agents.