Ana Rita C. Duarte

© 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.

Supercritical fluid impregnation was tested to prepare a new scaffold loaded with a bioactive compound. Dexamethasone is used in osteogenic media to direct the differentiation of stem cells towards the osteogenic lineage. Dexamethasone was impregnated in chitosan scaffolds at different operating conditions, in order to optimize the impregnation process. Pressure and temperature affect the carbon dioxide density and influence the swelling of the polymer and the drug solubility in the fluid phase, therefore these are two important parameters that were studied in this work. Chitosan sponges prepared by freeze drying were impregnated with the active compound at pressures from 8.0 up to 14.0 MPa and temperatures from 35 up to 55 °C. The effect of the impregnation contact time (3 h and 6 h) was also evaluated. From the experiments performed we can conclude that the yield of impregnation is lower when increasing pressure and temperature. The contact time will mainly influence the amount of drug impregnated in the scaffold and for higher contact times the impregnation yield is also higher. Scanning electron microscopy shows particles of dexamethasone in the bulk of the scaffold, which confirms the feasibility of the supercritical fluid impregnation technology for the preparation of delivery devices. The loading capacity of the scaffolds was determined by spectroscopic analysis and the highest loading was achieved for the experiments performed at 8.0 MPa and 35 °C. Furthermore, in vitro drug release studies were carried out and the results show that dexamethasone was sustainably released. Supercritical fluid impregnation proved to be feasible for the preparation of a drug delivery system for bone tissue engineering purposes. © 2008 Elsevier Ltd. All rights reserved.

Ethylcellulose/methylcellulose blends were produced using different precipitation techniques and impregnated with naproxen, a non-steroidal anti-inflammatory drug (NSAID). Solvent-evaporation technique was used not only for the preparation of ethylcellulose/methylcellulose microspheres but also to encapsulate naproxen. Supercritical fluid (SCF) impregnation was also performed to prepare naproxen loaded microspheres. The microspheres, impregnated by the SCF technique, were prepared both by solvent-evaporation and by a supercritical antisolvent (SAS) process. In vitro release profiles at pH 7.4 and 1.2, of naproxen-loaded microspheres were evaluated and the results were modelled Fick's law of diffusion and Power law. Miscrospheres prepared by supercritical antisolvent have a higher loading capacity and present a slower release profile. The systems studied present a release mechanism controlled by drug diffusion which complies Fick's law of diffusion. © 2005 Elsevier B.V. All rights reserved.

The supercritical antisolvent (SAS) technique was used to prepare ethyl cellulose/methyl cellulose blends, two biocompatible polymers commonly used as drug carriers in controlled delivery systems. Ethyl cellulose is widely used as a drug carrier. The drug release of the delivery devices can be controlled to some extent by addition of a water-soluble or water swellable polymer, such as methyl cellulose. This leads to the solubility enhancement of poorly water-soluble molecules. SAS experiments were carried out at different operational conditions and microspheres with mean diameters ranging from 5 to 30 $μ$m were obtained. The effect of CO2 and liquid flow, temperature and pressure on particle size and particle size distribution was evaluated. The microspheres were precipitated from a mixture of dichloromethane (DCM) and dimethylsulfoxide (DMSO) (4:1 ratio). The best process conditions for this mixture were according to our study 40°C and 80 bar. © 2006 Elsevier B.V. All rights reserved.

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.

The aim of this study was to evaluate the possibility of preparing starch-based porous matrixes using supercritical fluid technology. Supercritical immersion precipitation technique was used to prepare scaffolds of a polymeric blend of starch and poly(l-lactic acid) for tissue engineering purposes.Immersion precipitation experiments were carried out at different operational conditions and highly porous and interconnected scaffolds were obtained. Two organic solvents, dichloromethane and chloroform were tested, and from the results obtained chloroform was the more favourable for the process. The effect of polymer solution concentration (5 up to 20 wt{%}), temperature (35 up to 55 °C) and pressure (100 up to 200 bar) in the SPLA (50:50 wt{%}) membrane morphology, porosity and interconnectivity was evaluated. All the conditions tested were in the region of total miscibility between the organic solvent and carbon dioxide. Additionally, a blend with a different starch-poly(l-lactic acid) ratio (30:70 wt{%}) was tested. Bicontinuous structures were formed indicating that the L-L demixing process that governs the phase inversion is the spinodal decomposition. © 2008 Elsevier B.V. All rights reserved.

The aim of this study was to develop a new process for the production of bioactive 3D scaffolds using a clean and environmentally friendly technology. The possibility of preparing composite scaffolds of Bioglass?? and a polymeric blend of starch and poly(l-lactic acid) (SPLA50) was evaluated. Supercritical phase-inversion technique was used to prepare inorganic particles loaded starch-based porous composite matrixes in a one-step process for bone tissue engineering purposes. Due to their osteoconductive properties some glasses and ceramics are interesting materials to be used for bone tissue engineering purposes; however their poor mechanical properties create the need of a polymeric support where the inorganic fraction can be dispersed. Samples impregnated with different concentrations of Bioglass?? (10 and 15{%} wt/wt polymer) were prepared at 200??bar and 55????C. The presence of Bioglass?? did not affect the porosity or interconnectivity of the polymeric matrixes. Dynamic mechanical analysis has proven that the modulus of the SPLA50 scaffolds increases when glass particles are impregnated within the matrix. In vitro bioactivity studies were carried out using simulated body fluid and the results show that a calcium-phosphate layer started to be formed after only 1??day of immersion. Chemical analysis of the apatite layer formed on the surface of the scaffold was performed by different techniques, namely EDS and FTIR spectroscopy and X-ray diffraction (XRD). The ion concentration in the simulated body fluid was also carried out by ICP analysis. Results suggest that a bone-like apatite layer was formed. This study reports the feasibility of using supercritical fluid technology to process, in one step, a porous matrix loaded with a bioactive material for tissue engineering purposes. ?? 2009 Elsevier B.V. All rights reserved.

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.

Equilibrium solubility of acetazolamide, a carbonic-anhydrase inhibitor, in supercritical carbon dioxide in the presence of a cosolvent was measured by a static analytical method for three mole fractions of ethanol (5, 7.5, and 10) {%} at 313.0 K from (13.0 to 21.0) MPa and at 323.0 K from (13.0 to 21.0) MPa for a mole fraction of 5{%} ethanol The presence of a cosolvent (ethanol) was essential for the solubilization of the bioactive compound in supercritical carbon dioxide. The results obtained are useful for the design of supercritical processes with this drug. Experimental solubility data were correlated with two enhanced density-based models (Chrastil, I. Solubility of Solids in Supercritical Gases. J. Phys. Chem. 1982, 86, 3016-3021; Santiago, J. M.; Teja, A. S. The solubility of solids in supercritical fluids. Fluid Phase Equilib. 1999, 158-160, 501-510).

Carbon dioxide solubility in a natural biodegradable polymer, namely poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and the diffusion coefficients are reported. Equilibrium solubility of dense carbon dioxide in PHBV was studied by a gravimetric method in a temperature range from 308 to 313 K and a pressure range from 10.0 to 15.0 MPa. The copolymer presented Fickian behavior and Fick's diffusion model was applied to determine the amount of carbon dioxide present in the samples after a predermined exposure time as well as the diffusion coefficients. Diffusion coefficients for the sorption under supercritical (sc) conditions and desorption at ambient conditions were determined and compared. To evaluate the influence of the HV content in the amount of maximum sorption degree of the polymer, different samples of PHBV copolymers were tested and the sorption curves are here presented. © 2006 Elsevier B.V. All rights reserved.

Equilibrium solubility of flurbiprofen, a nonsteroidal antiinflammatory agent, in supercritical carbon dioxide was measured by a static analytical method in the pressure range from (8.0 to 25.0) MPa, at temperatures of (303.0, 313.0, and 323.0) K. The cosolvent effect of ethanol in the solubility of the bioactive compound in supercritical carbon dioxide was investigated at 18 MPa and 313 K. The results obtained have a potential application in supercritical processes for this drug. Experimental solubility data were correlated with an empirical density-based Chrastil model.

Mass sorption and diffusion coefficients in one acrylate biocompatible copolymer contacted with supercritical (sc) carbon dioxide are reported. Equilibrium solubility of dense carbon dioxide in poly(methylmethacrylate-co-ethylhexylacrylate-co-ethyleneglycoldimethacr ylate) (P(MMA-EHA-EGDMA)) was studied by a gravimetric method in a temperature range from 308 to 323 K and a pressure range from 10.0 to 20.0 MPa. The cross-linked copolymer presented Fickian behavior and Fick's diffusion model was applied to determine the amount of carbon dioxide present and the diffusion coefficients. Diffusion coefficients for the sorption under supercritical conditions and desorption at ambient conditions were determined and compared. Samples of P(MMA-EHA-EGDMA) with different thickness were used for comparison of the maximum sorption degree. Polymerization conditions were also varied in order to evaluate the influence of the molecular weight of the copolymer in the CO2 sorption process. To investigate the possibility of impregnating this acrylate copolymer with an anti-inflammatory drug, a preliminary experiment was performed. © 2005 Elsevier B.V. All rights reserved.

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.

The micronization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from organic solutions using supercritical antisolvent (SAS) technique has been successfully achieved. SAS experiments were carried out at different operational conditions and microspheres with mean diameters ranging from 3 to 9 $μ$m were obtained. The effect of CO2 and liquid flow, temperature and pressure on particle size and particle size distribution was evaluated. The microspheres were precipitated from a dichloromethane (DCM) solution. The best process conditions for this mixture were, according to our study, 40 °C, 100 bar, 1 mL min-1 liquid flow and 10 L min-1 carbon dioxide flow. Experiments with polymers containing different HV percentages were carried out. The powders obtained became more spherical as the HV content decreased. © 2006 Elsevier B.V. All rights reserved.

Supercritical fluid impregnation was tested to prepare a new ophthalmic drug delivery device. Poly(methylmethacrylate-co-ethylhexylacrylate-co-ethyleneglycoldimethacr ylate), P(MMA-EHA-EGDMA) has been proposed by Mariz [M. Mariz, Preparação de uma lente intra-ocular dotada de um sistema de libertação controlada de fármaco, Master Thesis, Universidade de Coimbra, 1999] as a promising matrix to be used for intraocular delivery of anti-inflammatory drugs used in eye surgery. This matrix was successfully impregnated with flurbiprofen, a non-steroidal anti-inflammatory agent. The success of the impregnation was evaluated by scanning electron microscopy (SEM) analysis and also by in vitro drug release studies. The effect of some operating parameters was evaluated, namely, pressure and contact time. The operating pressure will influence both the solubility of the drug in the supercritical fluid but also the sorption degree of the polymeric matrix in the presence of carbon dioxide. The solubility of the drug in carbon dioxide and the sorption degree are reported in previous studies. A comparison between the batch and the semi-continuous impregnation process is also presented. The supercritical fluid impregnation proved to be feasible for the preparation of a new ophthalmic drug delivery system. The drug release profiles suggest that the drug can be released up to three months, which is a major advantage for the prevention of the inflammatory response after ophthalmic surgery. © 2007 Elsevier B.V. All rights reserved.

Herein the preparation of molecularly imprinted polymers (MIPs) using supercritical fluid technology is evaluated. Poly(diethylene glycol dimethacrylate), polyDEGDMA, was synthesised in supercritical carbon dioxide (scCO2) using a carboxylic acid end-capped perfluoropolyether oil as stabiliser. Polymerisations were carried out in the presence of different concentrations of two different template drug molecules, salicylic acid and acetylsalicylic acid. Results suggest that molecular imprinted polymers were successfully prepared by supercritical polymerisation and then impregnated with the template in order to prepare controlled release systems. © 2006 Elsevier B.V. All rights reserved.

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.

Over the past several years, the definition of a scaffold for tissue engineering has changed dramatically, from a material that acts only as an inert structural support for cell attachment to serving as a more complex and dynamic environment for tissue development. This paper is a review on the existing and on the new emerging techniques based on supercritical fluid technology for the preparation of scaffolds and particles for tissue engineering applications. Supercritical fluid technology has already proven to be feasible for many pharmaceutical applications and is now emerging as an alternative to conventional materials' processing methods for the preparation of three-dimensional structures and injectable particles suitable to be used in regenerative medicine. The basic principles underlying each technique are here presented as well as the advantages and disadvantages of each process. The state of the art is reviewed and the major conclusions of the studies reported in the literature are discussed.

In this work, a starch-based polymer, namely a blend of starch-poly(epsilon-caprolactone) was processed by supercritical assisted phase inversion process. This processing technique has been proposed for the development of 3D structures with potential applications in tissue engineering applications, as scaffolds. The use of carbon dioxide as non-solvent in the phase inversion process leads to the formation of a porous and interconnected structure, dry and free of any residual solvent. Different processing conditions such as pressure (from 80 up to 150 bar) and temperature (45 and 55 degrees C) were studied and the effect on the morphological features of the scaffolds was evaluated by scanning electron microscopy and micro-computed tomography. The mechanical properties of the SPCL scaffolds prepared were also studied. Additionally, in this work, the in vitro biological performance of the scaffolds was studied. Cell adhesion and morphology, viability and proliferation was assessed and the results suggest that the materials prepared are allow cell attachment and promote cell proliferation having thus potential to be used in some for biomedical applications.

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.

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.