Ana Rita C. Duarte

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 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.

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.

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.

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.

In this experimental phase equilibrium study, we show for the first time that it is possible to stabilize structure sH of hydrogen clathrate hydrate with the help of some selected promoters. It was established that the formation pressures of these systems are significantly higher than that of structure sII of hydrogen clathrate hydrate when tetrahydrofuran (THF) is used as a promoter. Although no experimental evidence is available yet, it is estimated that the hydrogen storage capacity of structure sH can be as high as 1.4 wt {%} of H 2 , which is about 40{%} higher compared to the hydrogen storage capacity in structure sH. © 2008 American Chemical Society.

The possibility of preparation of ophthalmic drug delivery systems using compressed anti-solvent technology was evaluated. Eudragit RS 100 and RL 100 were used as drug carriers, acetazolamide was the model drug processed. Compressed anti-solvent experiments were carried out as a semi-continuous or a batch operation from a liquid solution of polymer(s) + solute dissolved in acetone. Both techniques allowed the recovery of composite particles, but the semi-continuous operation yielded smaller and less aggregated populations than the batch operation. The release behaviour of acetazolamide from the prepared microparticles was studied and most products exhibited a slower release than the single drug. Moreover, the release could be controlled to some extent by varying the ratio of the two Eudragit used in the formulation and by selecting one or the other anti-solvent technique. Simple diffusion models satisfactorily described the release profiles. Composites specifically produced by semi-continuous technique have a drug release rate controlled by a diffusion mechanism, whereas for composites produced by the batch operation, the polymer swelling also contributes to the overall transport mechanism. © 2006 Elsevier B.V. All rights reserved.

The possibility of preparation of ophthalmic drug delivery systems using compressed anti-solvent technology was evaluated. Eudragit RS 100 and RL 100 were used as drug carriers, acetazolamide was the model drug processed. Compressed anti-solvent experiments were carried out as a semi-continuous or a batch operation from a liquid solution of polymer(s) + solute dissolved in acetone. Both techniques allowed the recovery of composite particles, but the semi-continuous operation yielded smaller and less aggregated populations than the batch operation. The release behaviour of acetazolamide from the prepared microparticles was studied and most products exhibited a slower release than the single drug. Moreover, the release could be controlled to some extent by varying the ratio of the two Eudragit used in the formulation and by selecting one or the other anti-solvent technique. Simple diffusion models satisfactorily described the release profiles. Composites specifically produced by semi-continuous technique have a drug release rate controlled by a diffusion mechanism, whereas for composites produced by the batch operation, the polymer swelling also contributes to the overall transport mechanism. © 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.

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.

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.

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.

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).