Generation of chitosan nanoporous structures for tissue engineering applications using a supercritical fluid assisted process

In recent years, considerable attention has been given to chitosan-based materials and their applications in the field of tissue engineering. However, the techniques proposed until now for the formation of chitosan scaffolds present some limitations such as: they are very time-consuming, use organic solvents, have difficulties in the obtainment and preservation of various levels of porosity and the 3-D structure. In this work, a new SC-CO₂ assisted process for the production of chitosan scaffolds is proposed; it consists of three steps: formation of a chitosan hydrogel by thermally induced phase separation; substitution of water with a suitable solvent; drying of the gel using SC-CO₂. Using this process, we produced chitosan nanostructured networks with filaments diameters around 50 nm, without any collapse of the gel nanostructure, characterized by a high porosity (>91%) and high compressive modulus (150 kPa).     

Comparison of different extraction methods for the extraction of major bioactive flavonoid compounds from spearmint (Mentha spicata L.) leaves

Different bioactive flavonoid compounds including catechin, epicatechin, rutin, myricetin, luteolin, apigenin and naringenin were obtained from spearmint (Mentha spicata L.) leaves by using conventional soxhlet extraction (CSE) and supercritical carbon dioxide (SC-CO₂) extraction at different extraction schemes and parameters. The effect of different parameters such as temperature (40, 50 and 60 °C), pressure (100, 200 and 300 bar) and dynamic extraction time (30, 60 and 90 min) on the supercritical carbon dioxide (SC-CO₂) extraction of spearmint flavonoids was investigated using full factorial arrangement in a completely randomized design (CRD). The extracts of spearmint leaves obtained by CSE and optimal SC-CO₂ extraction conditions were further analyzed by high performance liquid chromatography (HPLC) to identify and quantify major bioactive flavonoid compounds profile. Comparable results were obtained by optimum SC-CO₂ extraction condition (60 °C, 200 bar, 60 min) and 70% ethanol soxhlet extraction. As revealed by the results, soxhlet extraction had a higher crude extract yield (257.67 mg/g) comparing to the SC-CO₂ extraction (60.57 mg/g). Supercritical carbon dioxide extract (optimum condition) was found to have more main flavonoid compounds (seven bioactive flavonoids) with high concentration comparing to the 70% ethanol soxhlet extraction (five bioactive flavonoids). Therefore, SC-CO₂ extraction is considered as an alternative process compared to the CSE for obtaining the bioactive flavonoid compounds with high concentration from spearmint leaves.     

Enzymatic saccharification of lignocellulosic materials after treatment with supercritical carbon dioxide

Lignocellulosic materials, such as agricultural residues, are abundant renewable resources for bioconversion to sugars. The sugar cane bagasse was studied here to obtain simple sugars for the production of alcohols and other chemicals. The crystalline structure of cellulose and the lignin that physically seals the surrounding cellulose fibers makes enzymatic hydrolysis difficult by preventing the contact between the cellulose and the enzyme. Two different samples of sugar cane (bagasse pulp and skin) were used and compared with microcrystalline cellulose (Avicel). The investigated samples were pretreated with SC-CO₂ explosion before hydrolysis. The experiments were conducted at 12, 14 and 16 MPa at a temperature of 60 °C. In this process, particles of celluloses within the size range from 0.25 to 0.42 mm were placed in defined amounts inside the experimental vessel, CO₂ was injected and let stand for 5 and 60 min. The explosion pretreatment of cellulosic materials by SC-CO₂ was performed in an apparatus of a static type with 300 ml of volume. The hydrolysis reaction using cellulose enzyme was carried at 55 °C for 8 h. After the pretreatment, the glucose yield increased in 72% to the bagasse sample. The SC-CO₂ pretreatment together with alkali increased the glucose yield in 20% as compared with alkali only. X-ray, microscopy and thermal analysis were used to investigate the effect of the pretreatment.     

Supercritical carbon dioxide and solvent extraction of 2-acetyl-1-pyrroline from Pandan leaf: The effect of pre-treatment

Pandan (Pandanus amaryllifolius Roxb.) leaf is a source of natural flavoring widely used in South-east Asia. The major compound contributing to the characteristic flavor of Pandan is 2-acetyl-1-pyrroline (2AP). This highly volatile compound also contributes significantly to the flavor of aromatic rice such as basmati and jasmine rice. As the consumer requirement for use of natural flavors, extraction of components from natural sources has been sought. In this study, supercritical carbon dioxide (SC-CO₂) and solvent extraction of components from Pandan leaves were performed. Experimental parameters included particle size and drying method (oven and freeze drying). Results indicated that the initial value of moisture content and particle size of Pandan leaves had the greatest effect on the total yield and 2AP concentration of the extracts. Almost 80% of water in Pandan leaves can be removed by drying. Yields of supercritical extracts were 10 times lower when compared to the hexane extracts. The total yield of extracts was increased up to 50% with decreasing particle size of Pandan leaves. Extraction of coarsely ground freeze-dried Pandan leaves by SC-CO₂ obtained the highest yield (0.88 ± 0.06%) followed by oven dried (0.38 ± 0.09%) and fresh leaves (0.34 ± 0.01%). The 2AP was identified by GC-MS and analyzed by GC-FID. Supercritical and hexane extracts of pre-treated Pandan leaves were found to have a small quantity of 2AP ranging between 0.04 ± 0.01 and 0.45 ± 0.01 ppm. Grinding pre-treatment was the best method for both SC-CO₂ and hexane extractions while the freeze drying method was the best for SC-CO₂.     

Preparation of liposomes entrapping essential oil from Atractylodes macrocephala Koidz by modified RESS technique

A modified technique of rapid expansion of supercritical solutions (RESS) was applied to incorporate essential oil extracted from Atractylodes macrocephala Koidz into liposomes. In the modified RESS process, both the liposomal materials and the essential oil were dissolved in the mixture of supercritical carbon dioxide (SC-CO₂)/ethanol and then the solution was sprayed into an aqueous medium through a coaxial nozzle to form liposomes suspension. The encapsulation performance of liposomes could be controlled by changing expansion processing conditions such as pressure, temperature of SC-CO₂ and the amount of ethanol. The entrapment efficiency, drug loading and average particle size of liposomes were found to be 82.18%, 5.18% and 173 nm, respectively, under the optimum conditions of at a pressure of 30 MPa, a temperature of 338 K and a ethanol mole fraction in SC-CO₂ [(x(CH₃CH₂OH)] of 15%. The formed liposomes appeared as double-layered colloidal spheres with a uniform and narrow particle size distribution. The physicochemical properties of liposomes including entrapment efficiency, dissolution rate and stability were complied with the provisions of Chinese pharmacopoeia. All these results indicate that the modified RESS technique is an innovative way for self-assembly of liposomes incorporation of multi-components extracted from Chinese traditional medicines in the SC-CO₂.     

Preparation in supercritical CO2 of porous poly(methyl methacrylate)-poly(l-lactic acid) (PMMA-PLA) scaffolds incorporating ibuprofen

Partially biodegradable porous scaffolds incorporating bioactive molecules prepared by clean techniques posses an enormous interest in tissue engineering applications. Poly(methyl methacrylate)-poly(l-lactic acid) (PMMA-PLA) blends were submitted to CO₂ supercritical conditions (P = 160-260 bar, T = 60 °C) after certain time and then rapidly depressurized to obtain porous structures that have been related with the supercritical parameters and to the polymer blend composition. In some cases ibuprofen was also incorporated to the formulations previously to the CO₂ treatment and studied the appropriate conditions for avoiding its extraction in SCCO₂. Scaffolds purity, thermal transitions, swelling and degradation behaviour, and the ibuprofen release were also studied to determine the appropriate scaffolds with a desired porosity for cell seeding. Cell culture was performed on the selected porous scaffolds using human fibroblast examined by scanning electron microscopy (SEM).     

Solid-state supercritical CO2 foaming of PCL and PCL-HA nano-composite: Effect of composition, thermal history and foaming process on foam pore structure

In this work we investigated the solid-state supercritical CO₂ (scCO₂) foaming of poly(?-caprolactone) (PCL), a semi-crystalline, biodegradable polyester, and PCL loaded with 5 wt% of hydroxyapatite (HA) nano-particles.In order to investigate the effect of the thermal history and eventual residue of the crystalline phase on the pore structure of the foams, samples were subjected to three different cooling protocols from the melt, and subsequently foamed by using scCO₂ as blowing agent. The foaming process was performed in the 37-40 °C temperature range, melting point of PCL being 60 °C. The saturation pressure, in the range from 10 to 20 MPa, and the foaming time, from 2 to 900 s, were modulated in order to control the final morphology, porosity and pore structure of the foams and, possibly, to amplify the original differences among the different samples.The results of this study demonstrated that by the scCO₂ foaming it was possible to produce PCL and PCL-HA foams with homogeneous morphologies at relatively low temperatures. Furthermore, by the appropriate combination of materials properties and foaming parameters, we prepared foams with porosities in the 55-85% range, mean pore size from 40 to 250 μm and pore density from 10⁵ to 10⁸ pore/cm³. Finally, we also proposed a two-step depressurization foaming process for the design of bi-modal and highly interconnected foams suitable as scaffolds for tissue engineering.     

Continuous production of fatty acid methyl esters from corn oil in a supercritical carbon dioxide bioreactor

Continuous production of fatty acid methyl esters (FAMEs) from corn oil was studied in a supercritical carbon dioxide (SC-CO₂) bioreactor using immobilized lipase (Novozym 435) as catalyst. Response surface methodology (RSM) based on central composite rotatable design (CCRD) was employed to investigate and optimize the reaction conditions: pressure (11-35 MPa), temperature (35-63 °C), substrate mole ratio (methanol:corn oil 1-9) and CO₂ flow rate (0.4-3.6 L/min, measured at ambient conditions). Increasing the substrate mole ratio increased the FAME content, whereas increasing pressure decreased the FAME content. Higher conversions were obtained at higher and lower temperatures and CO₂ flow rates compared to moderate temperatures and CO₂ flow rates. The optimal reaction conditions generated from the predictive model for the maximum FAME content were 19.4 MPa, 62.9 °C, 7.03 substrate mole ratio and 0.72 L/min CO₂ flow rate. The optimum predicted FAME content was 98.9% compared to an actual value of 93.3 ± 1.1% (w/w). The SC-CO₂ bioreactor packed with immobilized lipase shows great potential for biodiesel production.     

Reactive infiltration processing (RIP) of ultra high temperature ceramics (UHTC) into porous C/C composite tubes

A novel reactive infiltration processing (RIP) technique was employed to infiltrate porous carbon fibre reinforced carbon (C/C) composite hollow tubes with ultra high temperature ceramic (UHTC) particles such as ZrB₂. The C/C composite tubes had initial porosity of ∼60% with a bimodal (10 μm and 100 μm) pore size distribution. A slurry with 40-50% ZrB₂ solid loading particles was used to infiltrate the C/C tubes. Our approach combines in situ ZrB₂ formation with coating of fine ZrB₂ particles on carbon fibre surfaces by a reactive processing method. A Zr and B containing diphasic gel was first prepared using inorganic-organic hybrid precursors of zirconium oxychloride (ZrOCl₂·8H₂O), boric acid, and phenolic resin as sources of zirconia, boron oxide, and carbon, respectively. Then commercially available ZrB₂ powder was added to this diphasic gel and milled for 6 h. The resultant hybrid slurry was vacuum infiltrated into the porous hollow C/C tubes. The infiltrated tubes were dried and fired for 3 h at 1400 °C in flowing Ar atmosphere to form and coat ZrB₂ on the carbon fibres in situ by carbothermal reaction. Microstructural observation of infiltrated porous C/C composites revealed carbon fibres coating with fine nanosized (∼100 nm) ZrB₂ particles infiltrated to a depth exceeding 2 mm. Ultra high temperature ablation testing for 60 s at 2190 °C suggested formation of ZrO₂ around the inner bore of the downstream surface.     

Polymeric microparticles prepared by supercritical antisolvent precipitation

This work has as main objective the development of a model process to obtain microparticles of an acrylate-methacrylate copolymer (Eudragit L100^® and Eudragit EPO^®) using supercritical carbon dioxide (SC-CO₂) as antisolvent (GAS). After studying the behaviour of the copolymers in SC-CO₂ at different operation conditions (pressure, temperature and presence of ethanol (EtOH)), efforts were invested in the optimization of Eudragit EPO^® precipitation from an organic solution using carbon dioxide as antisolvent in batch mode. After loading the precipitation vessel with a fixed quantity of the copolymer dissolution, the SC-CO₂ has been added until the pressure of operation has been reached. Three process parameters, namely, solution nature, presence of surfactants and organic solvent removal step, have been evaluated. Microparticles with mean diameter from about 2 to 12 μm are obtained.     

Development of macroporous nanocomposite scaffolds of gelatin/bioactive glass prepared through layer solvent casting combined with lamination technique for bone tissue engineering

In this study, macroporous bioactive nanocomposite scaffolds were developed using cross-linked gelatin and bioactive glass (BaG) nanoparticles. First, BaG nanoparticles were synthesized via sol–gel method and characterized. Then, macroporous nanocomposites were prepared through layer solvent casting combined with freeze-drying and lamination techniques. This research has developed a new composition to produce a new bioactive nanocomposite which is porous with three-dimensional (3D) inter-connected microstructure, pore sizes are 200–500 μm, porosity are 72–86% and BaG nanoparticles are dispersed evenly among cross-linked gelatin matrices. It is mentionable that in this study, we have reported the formation of chemical bonds between BaG nanoparticles and gelatin for the first time. Finally, the in vitro cytocompatibility of the nanocomposite scaffolds was tested using SaOS-2 cell line.     

Synthesis and characterization of nanosized-silica gels formed under controlled conditions

Silica gel has been synthesized by the refluxing of rice husk ash with 1 M NaOH and subsequently adjusting the pH using 1 M H₂SO₄. The high purity of the silica gel has been found to be dependent on: (i) reflux time, (ii) water loading by addition of boiling DI water to the silica gel prior to titration with 1 M H₂SO₄ and (iii) rinse time on removal of impurities prior to drying. The surface area, pore size and volume of the silica gel were measured; XRD peaks of Na₂SO₄ impurity were absent after rinsing > 4 times. FTIR spectra showed that all the silica gels made by different schedules had a similar functional composition. The advantages of the present synthesis route are (i) a cost reduction due to the absence of pre-treatment for the rice husks before calcination below 700 °C, and (ii) the formation of a pure high surface area mesoporous amorphous silica gel.     

Biodiesel production from supercritical carbon dioxide extracted Jatropha oil using subcritical hydrolysis and supercritical methylation

This study investigates supercritical carbon dioxide (SC-CO₂) extraction of triglycerides from powdered Jatropha curcas kernels followed by subcritical hydrolysis and supercritical methylation of the extracted SC-CO₂ oil to obtain a 98.5% purity level of biodiesel. Effects of the reaction temperature, the reaction time and the solvent to feed ratio on free fatty acids in the hydrolyzed oil and fatty acid esters in the methylated oil via two experimental designs were also examined. Supercritical methylation of the hydrolyzed oil following subcritical hydrolysis of the SC-CO₂ extract yielded a methylation reaction conversion of 99%. The activation energy of hydrolysis and trans-esterified reactions were 68.5 and 45.2 kJ/mole, respectively. This study demonstrates that supercritical methylation preceded by subcritical hydrolysis of the SC-CO₂ oil is a feasible two-step process in producing biodiesel from powdered Jatropha kernels.     

Lithium ion conductivity of molecularly compatibilized chitosan-poly(aminopropyltriethoxysilane)-poly(ethylene oxide) nanocomposites

Films of composites of chitosan/poly(aminopropyltriethoxysilane)/poly(ethylene oxide) (CHI/pAPS/PEO) containing a fixed amount of lithium salt are studied. The ternary composition diagram of the composites, reporting information on the mechanic stability, the transparence and the electrical conductivity of the films, shows there is a window in which the molecular compatibility of the components is optimal. In this window, defined by the components ratios CHI/PEO 3:2, pAPS/PEO 2:3 and CHI/PEO 1:2, there is a particular composition Li_x(CHI)₁(PEO)₂(pAPS)_1.2 for which the conductivity reaches a value of 1.7 × 10⁻⁵ S cm⁻¹ at near room temperature. Considering the balance between the Lewis acid and basic sites available in the component and the observed stoichiometry limits of formed polymer complexes, the conductivity values of these products may be understood by the formation of a layered structure in which the lithium ions, stabilized by the donors, poly(ethylene oxide) and/or poly(aminopropyltriethoxysilane), are intercalated in a chitosan matrix.     

Electrochemical insertion of sodium into graphite in molten sodium fluoride at 1025 °C

The electrochemical insertion of sodium into graphite was studied in molten sodium fluoride at 1025 °C. The results obtained evidenced two mechanisms for sodium insertion into graphite: sodium intercalation between the graphite layers and sodium sorption into the porosity of the material. Subsequent internal rearrangement of inserted sodium occurred, via transference from the pores towards the intercalation sites. In addition, the intercalation compound was found to undergo a fast decomposition process (k = 2.55 × 10⁻⁹ mol s⁻¹). X-ray diffraction analysis was used to confirm the formation of a high stage compound (Na_0.1C₈), the composition of which was consistent with compositions observed in the case of chemical vapor and electrochemical insertion of sodium, during experiments in the sodium perchlorate-ethylene cabonate electrolyte.     

Preparation of CEL2 glass-ceramic porous scaffolds coated with chitosan microspheres that have a drug delivery function

The present work focused on the preparation of CEL2 bioactive glass (SiO₂–P₂O₅–CaO–MgO–K₂O–Na₂O) scaffolds loaded with chitosan microspheres. Chitosan microspheres, with a mean particle size of 0.55 μm ± 0.25 μm and loaded with acetaminophen, were obtained through the water-in-oil single emulsion solvent evaporation method and were adhered to the surface of the scaffolds by a simple dip-coating technique. The characterization of the microsphere-loaded scaffolds, before and after immersion in simulated body fluid (SBF), was performed by scanning electron microscopy, X-ray diffraction, and infrared spectroscopy. In vitro bioactivity was performed for 21 days. The glass-ceramic microsphere-loaded scaffolds showed more than 70% interconnected porosity and an average compressive strength of 1.2 ± 0.43 MPa after immersion in SBF. They also showed the formation of a hydroxyapatite layer from the first day of immersion in SBF, demonstrating their high bioactivity. The microspheres were shown to be homogeneously dispersed on the scaffold surfaces. After 120 hours, the biologic tests showed good fibroblast cell proliferation onto the scaffolds. The encapsulated drug in the microspheres was released by diffusion in a sustained manner (90% and 99% in 200 hours). The results suggest that scaffolds have a promising role in applications of bone tissue engineering.     

Synthesis of (α- and β-)Si3N4/Si2N2O into silicon particulate porous preforms by hybrid system CVI and direct nitridation

An investigation on the microstructure and mechanical properties of Si/Si₃N₄/Si₂N₂O porous ceramic composites, synthesized in a multi-step approach via hybrid precursor system chemical vapor infiltration (HYSYCVI) and direct nitridation (DN) has been conducted. Particulate silicon porous preforms were infiltrated in subsequent stages S1-1, S1-2 (both at 1300 °C for 70 min in high purity nitrogen (HPN) using Na₂SiF₆ as solid precursor) and S2 (1350 °C for 120 min in ultra high purity nitrogen (UHPN)). Chemical reactions that account for the formation of Si₂N₂O and Si₃N₄ are proposed. Results show that the microstructure of the composites was influenced by atmosphere type and processing stage, affecting kind, morphology and size (including nanometric size) of nitrides formed. Porous composites (43% porosity) with modulus of rupture (MOR) ≈ 43 ± 3.5 MPa (evaluated in four-point bending tests) and elastic modulus (E) ≈ 29 GPa (determined by the pulse-echo ultrasonic method) are routinely obtained.     

Novel Fe2O3-doped glass /chitosan scaffolds for bone tissue replacement

In this study quaternary bioglass system (BG) SiO₂–CaO–Na₂O–P₂O₅ doped with Fe₂O₃ was prepared by the sol–gel method. Furthermore, 3D scaffolds were designed through blending Fe₂O₃ -doped bioglass with chitosan to obtain various compositions of scaffolds by the freeze-drying technique. The thermal behavior, morphological properties, porosity (%), mechanical properties and physicochemical properties of BG and scaffolds were evaluated by DSC/TGA, TEM, SEM, liquid displacement method, universal testing machine, XRD and FTIR. In addition, the in vitro bioactivity of the prepared scaffolds was studied in phosphate buffer saline (PBS) through the determination of PBS ions concentrations, as well as the degradation and the observation of precipitated calcium phosphate layer by SEM coupled with EDX and FTIR behavior. The cell viability of the prepared scaffolds was conducted against Baby Hamster Kidney fibroblasts (BHK-21) cell line. The presence of Fe₂O₃ decreased the T_g (from 513 to 390?°C) and the size decreased (from 20.89 to 50.81–13.92–27.87?nm). The scaffolds porosity (%) decreased upon Fe₂O₃ doping but the mechanical strength increased. Cell viability results for the designed scaffolds demonstrated acceptable cell viability compared with normal cells. Therefore, the designed scaffolds are promoted as regenerated materials that can be used for bone tissue replacement.     

Systematic approach to treat chronic osteomyelitis through ceftriaxone-sulbactam impregnated porous β-tri calcium phosphate localized delivery system

Present investigation deals with in vitro and in vivo experimentation to treat chronic osteomyelitis, using pure β-tri calcium phosphate porous scaffolds. A novel approach was given to treat such infections using the scaffolds and drug combinations consisting of ideal antibiotics. In vitro studies include variation of porosity with interconnectivity, pore-drug interfacial studies by SEM-EDAX and drug elution studies both in contact with PBS and SBF at ca. 37 °C. In vivo trials were based on experimental osteomyelitis in rabbit model in tibia by Staphylococcus aureus. Characterizations included histopathology, radiology and estimation of drug in both bone and serum for 42 days by HPLC and subsequent bone-biomaterial interface by SEM. Samples having 60-65% porosity with average pore size ca. 55 μm and higher interconnectivity (22-113 μm), high adsorption efficiency (ca. 79%) of drug showed prolonged, sustained release of the drugs considered being sufficient to treat chronic osteomyelitis with desirable bone formation.     

In vitro biodegradable and mechanical performance of biphasic calcium phosphate porous scaffolds with unidirectional macro-pore structure

In this study, porous biphasic calcium phosphate (BCP) scaffolds were fabricated by a freeze–gel casting technique using a tertiary-butyl alcohol (TBA) based slurry. After sintering, unidirectional macropore channels of scaffolds aligned regularly along the TBA ice growth direction were tailored simultaneously with micropores formed in the outer wall of the pore channels. The synthesized porous BCP scaffolds (two different sintering temperatures) exhibit compressive strength of 46.8 MPa for 43.0% porosity and 33.1 MPa for 45.9% porosity, respectively. After immersion in Hank's balanced salt solution (HBSS) for 1, 2, 4, 8 weeks, a precipitation started to be formed with individual small granules on the scaffolds surface. In the case of BCP scaffolds sintered at 1200 °C, β-TCP were slowly degraded with increasing the immersing time; on the other hand, α-TCP (from BCP scaffolds sintered at 1300 °C) was extremely degraded within 1 week of immersing. This behavior could be due to a fast hydrolysis (dissolution–reprecipitation) as a phase transformation from α-TCP to brushite or apatite compared to the β-TCP. After immersion in HBSS, overall the compressive strength of the scaffolds reduced by the gradual degradation in biological environment solution. This behavior is consistent with the degradation behavior of scaffolds after immersion in HBSS.