Design of bioactive and biomimetic scaffolds based on chitosan-alginate polyelectrolyte complexes for tissue engineering.

The replacement and regeneration of biological tissues by fabricating three-dimensional functionalized constructs that can improve material interaction with cells is an important challenge of tissue engineering. In this study, bioactive and biomimetic scaffolds based on chitosan-alginate polyelectrolyte complexes (PECs) were fabricated by freeze-drying method and then crosslinked with CaCl2. Various chitosan-alginate (CS-AL) molar ratios were used to obtain PECs with different structural and mechanical properties. The CS1-AL2.3 scaffold showed to possess the best mechanical properties (8 MPa) and good pore morphology with an average size of 100-150 µm. After the crosslinking process, a less porous structure but with higher elastic modulus (30 MPa) was obtained. To make matrix bioactive and biomimetic, the CS1-AL2.3 system was first functionalized with 3,4-dihydroxyhydrocinnamic acid (HCAF) and then with PySO3 or Heparin to introduce groups/molecules mimicking the extracellular matrix. While the antioxidant properties of the scaffolds containing HCAF improved by 3 orders of magnitude, compared to the non-functionalized matrix, the introduction of sulfonic groups into the bioactive scaffold made the structure more porous and hydrophilic with respect to the heparinized one also favoring the penetration and proliferation of fibroblasts into the scaffold. These results indicate the potential of these novel systems for tissue engineering.

Molecularly Imprinted Polymers Based on Chitosan for 2,4-Dichlorophenoxyacetic Acid Removal.

The development of low-cost and eco-friendly materials for the removal of pollutants from water is one of the main modern challenges. For this purpose, molecularly imprinted polymers were prepared under optimized conditions starting from chitosan (CS), chemically or ionically modified with glycidyl methacrylate (GMA) or itaconic acid (ITA), respectively. 2,4-Dichlorophenoxyacetic acid (2,4-D) was used as a template, obtaining the CS_GMA and CS_ITA series. The influence of the template concentration on the MIPs' (molecularly imprinted polymers) morphology, thermal behaviour and swelling ability, as well as on the 2,4-D removal capacity, were analyzed. The amount of the template used for the imprinting, together with the different permeability of the matrices, were the key factors driving the analyte uptake process. Despite the good performance shown by the non-imprinted CS_GMA sample, the best results were obtained when CS_GMA was imprinted with the highest amount (5%) of template (CS_GMA_5). This system was also more efficient when consecutive adsorption experiments were carried out. In addition, CS_GMA_5 had a desorption efficiency of 90-100% when a low pesticide concentration was used. These findings suggest that the presence of imprinted cavities could be useful in improving the performance of sorbent materials making CS_GMA_5 a possible candidate for 2,4-D removal.

Application of a Low Transition Temperature Mixture for the Dispersive Liquid-Liquid Microextraction of Illicit Drugs from Urine Samples.

The use of psychoactive substances is a serious problem in today's society and reliable methods of analysis are necessary to confirm their occurrence in biological matrices. In this work, a green sample preparation technique prior to HPLC-MS analysis was successfully applied to the extraction of 14 illicit drugs from urine samples. The isolation procedure was a dispersive liquid-liquid microextraction based on the use of a low transition temperature mixture (LTTM), composed of choline chloride and sesamol in a molar ratio 1:3 as the extracting solvent. This mixture was classified as LTTM after a thorough investigation carried out by FTIR and DSC, which recorded a glass transition temperature at -71 °C. The extraction procedure was optimized and validated according to the main Food and Drug Administration (FDA) guidelines for bioanalytical methods, obtaining good figures of merit for all parameters: the estimated lower limit of quantitation (LLOQ) values were between 0.01 µg L-1 (bk-MMBDB) and 0.37 µg L-1 (PMA); recoveries, evaluated at very low spike levels (in the ng-µg L-1 range), spanned from 55% (MBDB) to 100% (bk-MMBDB and MDPV); finally, both within-run and between-run precisions were lower than 20% (LLOQ) and 15% (10xLLOQ).

Chitosan-Graphene Oxide Composite Membranes for Solid-Phase Extraction of Pesticides.

Solid-phase extraction (SPE) coupled to LC/MS/MS analysis is a valid approach for the determination of organic micropollutants (OMPs) in liquid samples. To remove the greatest number of OMPs from environmental matrices, the development of innovative sorbent materials is crucial. Recently, much attention has been paid to inorganic nanosystems such as graphite-derived materials. Graphene oxide has been employed in water-purification processes, including the removal of several micropollutants such as dyes, flame retardants, or pharmaceutical products. Polysaccharides have also been widely used as convenient media for the dispersion of sorbent materials, thanks to their unique properties such as biodegradability, biocompatibility, nontoxicity, and low cost. In this work, chitosan-graphene oxide (CS_GO) composite membranes containing different amounts of GO were prepared and used as sorbents for the SPE of pesticides. To improve their dimensional stability in aqueous medium, the CS_GO membranes were surface crosslinked with glutaraldehyde. The composite systems were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, swelling degree, contact angle, and mechanical measurements. As the GO content increased, a decrease in surface homogeneity, an improvement of mechanical properties, and a reduction of thermal stability of the CS-based membranes were observed. The increased dimensional stability in water, together with the presence of high GO amounts, made the prepared composite membranes more efficacious than the ones based just on CS in isolating and preconcentrating different hydrophilic/hydrophobic pollutants.

Chitosan scaffolds with enhanced mechanical strength and elastic response by combination of freeze gelation, photo-crosslinking and freeze-drying.

In this study, a new scaffold fabrication method based on the combination of a series of stabilization processes was set up to obtain chitosan scaffolds with improved mechanical properties for regeneration of load-bearing tissues. Specifically, thermally induced phase separation (TIPS) of chitosan solutions was used to obtain an open structure which was then stabilized by freeze-gelation and photo cross-linking. Freeze-gelation combined with freeze-drying permitted to obtain a porous structure with a 95 µm-mean pore size suitable for osteoblast cells' housing. Photo-crosslinking improved by ca. three times the scaffold compressive modulus, passing from 0,8 MPa of the uncrosslinked scaffolds to 2,2 MPa of the crosslinked one. Hydrated crosslinked scaffolds showed a good elastic response, with an 80% elastic recovery for at least 5 consecutive compressive cycles. The herein reported method has the advantage to not require the use of potentially toxic cross-linking agents and may be extended to other soft materials.

Preparation and Characterization of Chitosan-Coated Manganese-Ferrite Nanoparticles Conjugated with Laccase for Environmental Bioremediation.

Bioremediation with immobilized enzymes has several advantages, such as the enhancement of selectivity, activity, and stability of biocatalysts, as well as enzyme reusability. Laccase has proven to be a good candidate for the removal of a wide range of contaminants. In this study, naked or modified MnFe2O4 magnetic nanoparticles (MNPs) were used as supports for the immobilization of laccase from Trametes versicolor. To increase enzyme loading and stability, MNPs were coated with chitosan both after the MNP synthesis (MNPs-CS) and during their formation (MNPs-CSin situ). SEM analysis showed different sizes for the two coated systems, 20 nm and 10 nm for MNPs-CS and MNPs-CSin situ, respectively. After covalent immobilization of laccase by glutaraldehyde, the MNPs-CSin situ-lac and MNPs-CS-lac systems showed a good resistance to temperature denaturation and storage stability. The most promising system for use in repeated batches was MNPs-CSin situ-lac, which degraded about 80% of diclofenac compared to 70% of the free enzyme. The obtained results demonstrated that the MnFe2O4-CSin situ system could be an excellent candidate for the removal of contaminants.

Preparation and Characterization of TPP-Chitosan Crosslinked Scaffolds for Tissue Engineering.

Scaffolds are three-dimensional porous structures that must have specific requirements to be applied in tissue engineering. Therefore, the study of factors affecting scaffold performance is of great importance. In this work, the optimal conditions for cross-linking preformed chitosan (CS) scaffolds by the tripolyphosphate polyanion (TPP) were investigated. The effect on scaffold physico-chemical properties of different concentrations of chitosan (1 and 2% w/v) and tripolyphosphate (1 and 2% w/v) as well as of cross-linking reaction times (2, 4, or 8 h) were studied. It was evidenced that a low CS concentration favored the formation of three-dimensional porous structures with a good pore interconnection while the use of more severe conditions in the cross-linking reaction (high TPP concentration and crosslinking reaction time) led to scaffolds with a suitable pore homogeneity, thermal stability, swelling behavior, and mechanical properties, but having a low pore interconnectivity. Preliminary biocompatibility tests showed a good osteoblasts' viability when cultured on the scaffold obtained by CS 1%, TPP 1%, and an 8-h crosslinking time. These findings suggest how modulation of scaffold cross-linking conditions may permit to obtain chitosan scaffold with properly tuned morphological, mechanical and biological properties for application in the tissue regeneration field.

Hyaluronic Acid Reduces Bacterial Fouling and Promotes Fibroblasts' Adhesion onto Chitosan 2D-Wound Dressings.

Wound healing is a dynamic process that can be seriously delayed by many factors including infectious complications. The development of dressings with intrinsic wound healing activity and/or releasing bioactive compounds may help with addressing such an issue. In this study, hyaluronic acid (HA) at different percentages (1-35%) was used to modify chitosan (CS) biological and physico-chemical properties in order to obtain 2D-matrices able to promote healing and protect from infection. HA incorporation in the CS matrix decreased film transparency and homogeneity, but improved film water uptake and surface wettability. The water vapor transmission rate (WVTR) increased up to a 5% HA content, where it reached the highest value (672 g/m2 day), and decreased for higher HA contents. At all of the tested HA concentrations, HA affected mechanical properties providing matrices more flexible than pure CS with benefit for wound care. Pure CS films permitted S. epidermidis adhesion and biofilm formation. That was not true for CS/HA matrices, where HA at concentrations equal to or greater than 5% was able to avoid S. epidermidis adhesion. Fibroblasts adhesion also took benefit from the HA presence in the film, especially at 5% content, where the best adhesion and proliferation was found.

Graphene Oxide Oxygen Content Affects Physical and Biological Properties of Scaffolds Based on Chitosan/Graphene Oxide Conjugates.

Tissue engineering is a highly interdisciplinary field of medicine aiming at regenerating damaged tissues by combining cells with porous scaffolds materials. Scaffolds are templates for tissue regeneration and should ensure suitable cell adhesion and mechanical stability throughout the application period. Chitosan (CS) is a biocompatible polymer highly investigated for scaffold preparation but suffers from poor mechanical strength. In this study, graphene oxide (GO) was conjugated to chitosan at two weight ratios 0.3% and 1%, and the resulting conjugates were used to prepare composite scaffolds with improved mechanical strength. To study the effect of GO oxidation degree on scaffold mechanical and biological properties, GO samples at two different oxygen contents were employed. The obtained GO/CS scaffolds were highly porous and showed good swelling in water, though to a lesser extent than pure CS scaffold. In contrast, GO increased scaffold thermal stability and mechanical strength with respect to pure CS, especially when the GO at low oxygen content was used. The scaffold in vitro cytocompatibility using human primary dermal fibroblasts was also affected by the type of used GO. Specifically, the GO with less content of oxygen provided the scaffold with the best biocompatibility.

Synthesis, Characterization, and Bacterial Fouling-Resistance Properties of Polyethylene Glycol-Grafted Polyurethane Elastomers.

Despite advances in material sciences and clinical procedures for surgical hygiene, medical device implantation still exposes patients to the risk of developing local or systemic infections. The development of efficacious antimicrobial/antifouling materials may help with addressing such an issue. In this framework, polyethylene glycol (PEG)-grafted segmented polyurethanes were synthesized, physico-chemically characterized, and evaluated with respect to their bacterial fouling-resistance properties. PEG grafting significantly altered the polymer bulk and surface properties. Specifically, the PEG-grafted polyurethanes possessed a more pronounced hard/soft phase segregated microstructure, which contributed to improving the mechanical resistance of the polymers. The better flexibility of the soft phase in the PEG-functionalized polyurethanes compared to the pristine polyurethane (PU) was presumably also responsible for the higher ability of the polymer to uptake water. Additionally, dynamic contact angle measurements evidenced phenomena of surface reorganization of the PEG-functionalized polyurethanes, presumably involving the exposition of the polar PEG chains towards water. As a consequence, Staphylococcus epidermidis initial adhesion onto the surface of the PEG-functionalized PU was essentially inhibited. That was not true for the pristine PU. Biofilm formation was also strongly reduced.