Chitosan/copper nanocomposites: Correlation between electrical and antibacterial properties.

Correlation between electrical and antibacterial properties of chitosan/copper nanocomposites (CS/CuNPs) is investigated. We aim at achieving the minimum CuNPs concentration in a CS-matrix while keeping high antibacterial activity. UV-vis, TEM and XRD measurements confirms the formation of polygonal metallic CuNPs (ca. 30-50 nm). Interactions between NH2/OH groups of CS and CuNPs were determined by FTIR and XPS suggesting Cu chelation-induced mechanism during the CuNPs formation. DC electrical conductivity measurements reveals a percolation threshold at CuNPs volumetric concentration of ca. 0.143%. Antibacterial assays against Gram-positive bacteria and DC measurements helps correlate the antibacterial potency to the electron transfer between the negatively charged bacteria and CuNPs. Our study suggests that nanocomposite's maximum antibacterial activity is obtained below the electrical percolation threshold at extremely low CuNPs concentrations; this fact may prove useful in the design of nontoxic nanocomposites for biomedical applications.

Fabrication and in vitro behavior of dual-function chitosan/silver nanocomposites for potential wound dressing applications.

We report the synthesis and in vitro evaluation of dual-function chitosan-silver nanoparticles (CTS-AgNPs) films with potential applications as wound dressings. We attempted to formulate nanocomposite films with appropriate AgNPs concentrations to simultaneously display antibacterial activity and suitability for cell culture. Nanocomposites were obtained by CTS-mediated in situ chemical reduction of AgNO3. Circular-shape AgNPs (sizes ca. 7-50 nm) well distributed within the CTS matrices were obtained in concentrations from 0.018 to 0.573 wt%. Efficacy (bacteriostatic and bactericidal properties) of CTS-AgNPs films to decrease planktonic and biofilm bacterial growth was AgNPs concentration- and bacteria strain-dependent. Films showed significant antibacterial activity against Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus. Antibacterial activity against S. epidermidis was moderated. Films suitability for cell culture was characterized using primary human fibroblasts (HF). HF displayed cell viability higher than 90% and the characteristic fusiform morphology of adhered fibroblast upon culture on films with AgNPs concentration ≤ 0.036 wt%. HF cultured on these films also showed positive expression of tropoelastin, procollagen type I and Ki-67, characteristic proteins of extracellular matrix and proliferative cells, respectively. In vitro assays demonstrated that cytocompatibility/antibacterial properties decreased/increased as silver concentration increased, suggesting that CTS-AgNPS nanocomposite films with ≈0.04-0.20 wt% might be considered as potential temporary dual-function wound dressings.

Correction: Porous monoliths synthesized via polymerization of styrene and divinyl benzene in nonaqueous deep-eutectic solvent-based HIPEs.

[This corrects the article DOI: 10.1039/C5RA02374B.].

Combined antibacterial/tissue regeneration response in thermal burns promoted by functional chitosan/silver nanocomposites.

We report the combined antibacterial/tissue regeneration responses to thermal burns promoted by functional chitosan/silver nanocomposites (CS/nAg) with ultralow silver content (0.018wt.%, 7-30nm). Our approach allows one to produce CS/nAg nanocomposites without silver nanoparticles (nAg) agglomeration, with bactericide potency higher than 1wt.% of nAg (ca. 10nm) content and, promoting the healing process in controlled thermal burns. CS/nAg films exhibit high antibacterial activity against S. aureus and P. aeruginosa after 1.5h of incubation, demonstrating the bacterial penetration into hydrated films and their interaction with nAg. Additionally, exceptional healing of induced thermal burns was obtained by increasing myofibroblasts, collagen remodeling, and blood vessel neoformation. These factors are associated with epiderma regeneration after 7days of treatment with no nAg release. Our results corroborate the controlled synthesis of nAg embedded in CS matrix with combined antibacterial/biocompatibility properties aiming to produce functional nanocomposites with potential use in wound dressing and health care applications.

(Chitosan-g-glycidyl methacrylate)-xanthan hydrogel implant in Wistar rats for spinal cord regeneration.

This work reports the results of in vivo assays of an implant composed of the hydrogel Chitosan-g-Glycidyl Methacrylate-Xanthan [(CTS-g-GMA)-X] in Wistar rats. Degradation kinetics of hydrogels was assessed by lysozyme assays. Wistar rats were subjected to laminectomy by cutting the spinal cord with a scalpel. After the surgical procedure, hydrogels were implanted in the injured zone (level T8). Somatosensory evoked potentials (SEPs) obtained by electric stimulation onto periphery nerves were registered in the corresponding central nervous system (CNS) areas. Rats implanted with the biomaterials showed a successful recovery compared with the non-implanted rats after 30days. Lysozyme, derived from egg whites, was used for in vitro assays. This study serves as the basis for testing the biodegradability of the hydrogels (CTS-g-GMA)-X that is promoted by enzymatic hydrolysis. Hydrogels' hydrolysis was studied via lysozyme kinetics at two pH values, 5 and 7, under mechanical agitation at 37°C. Results show that our materials' hydrolysis is slower than pure CTS possibly due to the steric hindrance imposed by the GMA grafting of functionalization. This hydrolysis helps degrade the biomaterial and at the same time it provides support for spinal cord recovery. Combination of these results may prove useful in the use of these hydrogels as scaffolds for cells proliferation and their application as implants in living organisms.

Preparación y Caracterización de Nanocompositos Quitosano-Cobre con Actividad Antibacteriana para aplicaciones en Ingeniería de Tejidos / Preparation and characterization of Copper Chitosan Nanocomposites with Antibacterial Activity for Applications in Tissue Engineering

RESUMEN: El presente trabajo describe la preparación de nanocompositos formulados a partir de quitosano (QS)/nanopartículas de cobre (nCu) con características antibacterianas y aplicación potencial en ingeniería de tejidos. Para ello, se prepararon nanocompositos mediante mezclado en solución asistido con ultrasonido con el objetivo de incrementar la dispersión de la carga nanométrica en el biopolímero. El análisis de FTIR demostró que la presencia de nCu en la matriz de QS favorece la interacción del nCu con los grupos amino/hidroxilo de la molécula del QS. Se determinó mediante UV-Vis que los nanocompositos QS/nCu presentan absorción asociada con la presencia de nanopartículas y la posible liberación de iones Cu2+ en medio líquido. Mediante AFM se determinó que el QS hidratado forma una malla con microporos, que puede favorecer la penetración de bacterias en el nanocomposito y su interacción con las nCu. Finalmente, se determinó el efecto antibacteriano del material al contacto con la bacteria Staphylococcus aureus, en donde se presenta una actividad antibacteriana superior al 90% entre los 90 y 180 min de interacción. Dichos resultados sugieren que es posible obtener nanomateriales antibacterianos biocompatibles para su posible aplicación en ingeniería tisular.

ABSTRACT: The Present work describes the preparation of nanocomposites based on chitosan (QS)/copper nanoparticles (nCu) with antibacterial properties and potential application in tissue engineering. For this purpose, nanocomposites were prepared by solution blending with ultrasound assisted, aiming to increase the nanoparticles dispersion in the biopolymer. FTIR analyses demonstrates that nCu supported in QS increase their interaction of nanoparticles with amine/hydroxyl groups of QS molecule. UV-Vis analyses demonstrates that QS/nCu nanocomposites have an absorption signal associated with the presence of nanoparticles and the possible Cu2+ ions release in liquid media. AFM analyses shown that hydrated QS form a mesh with micro pores, improving the bacterial penetration and the direct contact with nCu. This behavior was corroborated by antibacterial assays, where QS/nCu nanocomposites shown an antibacterial activity higher than 90% between 90-180 minutes of interaction. Our results suggest that is possible to obtain combined antibacterial/biocompatible nanomaterials with potential application in tissue engineering.

Development of meniscus substitutes using a mixture of biocompatible polymers and extra cellular matrix components by electrospinning.

Despite the significant advances in the meniscus tissue engineering field, it is difficult to recreate the complex structure and organization of the collagenous matrix of the meniscus. In this work, we developed a meniscus prototype to be used as substitute or scaffold for the regeneration of the meniscal matrix, recreating the differential morphology of the meniscus by electrospinning. Synthetic biocompatible polymers were combined with the extracellular matrix component, collagen and used to replicate the meniscus. We studied the correlation between mechanical and structural properties of the polymer blend as a function of collagen concentration. Fibers were collected on a surface of a rapidly rotating precast mold, to accurately replicate each sectional morphology of the meniscus; different electro-tissues were produced. Detailed XRD analyses exhibited structural changes developed by electrospinning. We achieved to integrate all these electro-tissues to form a complete synthetic meniscus. Vascularization tests were performed to assess the potential use of our novel polymeric blend for promising meniscus regeneration.

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.

Controlled release of lidocaine hydrochloride from polymerized drug-based deep-eutectic solvents.

This work takes advantage of the transformation of lidocaine hydrochloride into deep-eutectic solvents (DESs) - ionic liquid analogues - to incorporate polymerizable counterparts into DESs, such that polymer-drug complexes are synthesized by free-radical frontal polymerization without the use of a solvent. DESs are formed through hydrogen bonding of an ammonium salt with a hydrogen-bond donor (HBD). It is demonstrated that lidocaine hydrochloride - as the ammonium salt - is able to form DESs with acrylic acid and methacrylic acid. The properties of DESs allow frontal polymerization in the bulk with full conversion achieved in a one-pot synthesis, yielding monoliths of polymers loaded with a high concentration of drug. In in vitro experiments, the sustained release of the drug takes place in a controlled manner triggered by the pH, ionic strength and solubility of the drug in the medium. Such control is owed to the swelling of polymers as well as to the specific interactions between the drug and the polymers already established in the DES precursor. Finally, it is noteworthy that different monomers (such as HBD) and crosslinkers can be used, thus expanding the possibilities of drug delivery systems for transdermal technologies by exploiting the DES chemistry.

Mechanical and structural response of a hybrid hydrogel based on chitosan and poly(vinyl alcohol) cross-linked with epichlorohydrin for potential use in tissue engineering.

The development and characterization of a hybrid hydrogel based on chitosan (CS) and poly(vinyl alcohol) (PVA) chemically cross-linked with epichlorohydrin (ECH) is presented. The mechanical response of these hydrogels was evaluated by uniaxial tensile tests; in addition, their structural properties such as average molecular weight between cross-link points (Mcrl), mesh size (DN), and volume fraction (v(s)) were determined. This was done using the equivalent polymer network theory in combination with the obtained results from tensile and swelling tests. The films showed Young's modulus values of 11 ± 2 MPa and 9 ± 1 MPa for none irradiated and ultraviolet (UV) irradiated hydrogels, respectively. The cell viability was assessed using Calcein AM and Ethidium homodimer-1 assay and environmental scanning electron microscopy. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan thiazolyl blue formazan (MTT Formazan assay) results did not show cytotoxic effects; this was in good agreement with nuclear magnetic resonance and fourier transform infrared spectroscopies; their results did not show traces of ECH. This indicated that after the crosslinking process, there was no free ECH; furthermore, any possibility of ECH release in the construct during cell culture was discarded. The CS-PVA-ECH hybrid hydrogel allowed cell growth and extracellular matrix formation and showed adequate mechanical, structural, and biological properties for potential use in tissue engineering applications.

Effect of doping in carbon nanotubes on the viability of biomimetic chitosan-carbon nanotubes-hydroxyapatite scaffolds.

This work describes the preparation and characterization of biomimetic chitosan/multiwall carbon nanotubes/nano-hydroxyapatite (CTS/MWCNT/nHAp) scaffolds and their viability for bone tissue engineering applications. The cryogenic process ice segregation-induced self-assembly (ISISA) was used to fabricate 3D biomimetic CTS scaffolds. Proper combination of cryogenics, freeze-drying, nature and molecular ratio of solutes give rise to 3D porous interconnected scaffolds with clusters of nHAp distributed along the scaffold surface. The effect of doping in CNT (e.g. with oxygen and nitrogen atoms) on cell viability was tested. Under the same processing conditions, pore size was in the range of 20-150 µm and irrespective on the type of CNT. Studies on cell viability with scaffolds were carried out using human cells from periosteum biopsy. Prior to cell seeding, the immunophenotype of mesenchymal periosteum or periosteum-derived stem cells (MSCs-PCs) was characterized by flow cytometric analysis using fluorescence-activated and characteristic cell surface markers for MSCs-PCs. The characterized MSCs-PCs maintained their periosteal potential in cell cultures until the 2nd passage from primary cell culture. Thus, the biomimetic CTS/MWCNT/nHAp scaffolds demonstrated good biocompatibility and cell viability in all cases such that it can be considered as promising biomaterials for bone tissue engineering.

Synthesis and characterization of a hybrid (chitosan-g-glycidyl methacrylate)-xanthan hydrogel.

This work reports the synthesis and characterization of a new material obtained by mixing the hybrid natural-synthetic chitosan-g-glycidyl methacrylate (CTS-g-GMA) biopolymer and xanthan gum (X). All materials were characterized by infrared spectroscopy (FTIR), X-ray diffraction, and thermal analysis (DSC and TGA) and the results were contrasted with those of the precursor materials. The swelling index of the hydrogels decreases when the GMA mass percentage increases. The X-ray diffraction patterns show that the hybrid hydrogels are amorphous in contrast to chitosan (CTS), which is semi-crystalline. FTIR analysis confirms the existence of physical interactions among constituents. Rheological properties, η, G', and G", were determined as a function of flow allowing one to conclude that (CTS-g-GMA)-X behaves as physical hydrogel. Additionally, we report viability of fibroblasts when cultured onto the synthesized hydrogels. This study shows that these hydrogels support cell viability and have potential for use in biomedical engineering applications.

Hybrid natural-synthetic chitosan resin: thermal and mechanical behavior.

Poly(methyl methacrylate) (PMMA) is one of the most commonly plastics used as dental-base material, due to its good biological compatibility and mechanical properties. Chitosan has wide application in chemical, biochemical and biomedical fields of research. In this work, chitosan (CTS) was functionalized with glycidyl methacrylate (GMA), to ease a further reaction with MMA. The resulting co-polymer was finally blended with PMMA and poly(butyl acrylate) PBA which works as a damper, the polymers were cured by UV to obtain the final resin. Characterization of UV-cured resins was carried out by thermal measurements, X-ray diffraction, atomic force microscopy (AFM), micro and nanoindentation, water absorption and elution in water. As a result a higher thermal stability of the final resin compared with the precursor co-polymer ((CTS-GMA)-g-PMMA) was obtained. The resin presented roughness in the nanometer scale and nanoparticles embedded in the acrylic matrix producing a tough material. However, XRD measurements show that all materials are in an amorphous state. Values of hardness and elastic modulus results were very near to those of the dentine. The results of elution in water of the tested resin samples show them as clinically acceptable as a dental base material.

[Effects of cooking and steep time of nixtamalized corn grain (sea mayz L.) on the physicochemical, rheological, structural and textural characteristics of grain, mass and tortillas]. / Efecto del tiempo de cocimiento y reposo del grano de maíz (Zea mayz L.) nixtamalizado, sobre las características fisicoquímicas, reológicas, estructurales y texturales del grano, masa y tortillas de maíz.

The corn tortilla is elaborated using corn grain masa processed by the traditional nixtamalization method, which consists of two steps: cooking and steeping. In these steps the physicochemical and structural properties are strongly affected, resulting in changes in the textural characteristics of the tortilla produced. In this work the effects of cooking and steeping time on the moisture content, amylographic profiles, crystallinity, weigh loss from masa to tortilla, starch damage, rollability, elasticity and cutting force for grain, masa and corn tortillas, were evaluated. The milling of the nixtamalized grain and the cooking of tortilla conditions were the same in all treatments. All tortillas samples showed a good rollability. The results show that the moisture content of corn grain was increased up to 42 g/100 g during the total cooking time (45 min), and it further increased to 52-53 g/100 g after when the cooked grain was steeped for 4 h. All evaluated parameters showed high correlation coefficients with the texture properties of tortillas. The starch damage was the variable with the best correlation among all evaluated parameters. The correlation coefficients between starch damage and moisture content, weigh loss and maximum viscosity for corn grain, masa and tortillas were larger than 0.92 (p < 0.01). The best tortillas were produced using nixtamal with the follow characteristics: moisture content of nixtamal, 42-44 g/100 g and tortilla, 43-44 g/100 g; adhesiveness of masa, 30-50 g; maximum viscosity of nixtamal, 860-880 cp and tortilla 490-510 cp; starch damage of nixtamal, 14 g/100 g and tortilla, 35-37 g/100 g, and weigh loss of tortilla during cooking, 16 g/100 g.