Inmaculada Aranaz

Improvement of Porous β-TCP Scaffolds with rhBMP-2 Chitosan Carrier Film for Bone Tissue Application

Ceramic materials are osteoconductive matrices extensively used in bone tissue engineering approaches. The performance of these types of biomaterials can be greatly enhanced by the incorporation of bioactive agents and materials. It is previously reported that chitosan is a biocompatible, biodegradable material that enhances bone formation. In the other hand, bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive factor. In this work we coated porous beta-tricalcium phosphate (beta-TCP) scaffolds with recombinant human BMP-2 (rhBMP-2) carrier chitosan films and studied how they could modify the ceramic physicochemical properties, cellular response, and in vivo bone generation. Initial beta-TCP disks with an average diameter of 5.78 mm, 2.9 mm thickness, and 53% porosity were coated with a chitosan film. These coating properties were studied by X-ray diffraction, Fourier transform-infrared analysis, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). Treatment modified the scaffold porous distribution and increased the average hardness. The biocompatibility did not seem to be altered. In addition, adhered C2C12 cells expressed alkaline phosphatase activity, related to cell differentiation toward osteogenic lineage, due to the incorporation of rhBMP-2. On the other hand, in vivo observations showed new bone formation 3 weeks after surgery, a much shorter time than control beta-TCP ceramics. These results suggest that developed coating improved porous beta-TCP scaffold for bone tissue applications and added osteoinductive properties.

Chitosan Gelation Induced by the in Situ Formation of Gold Nanoparticles and Its Processing into Macroporous Scaffolds

This work describes a simple synthetic route to induce chitosan (CHI) gelation by the in situ formation of gold nanoparticles (AuNPs). AuNPs were obtained by thermal treatment (e.g., 40 and 80 °C) of CHI aqueous solutions containing HAuCl(4) and in the absence of further reducing agents. The CHI hydrogels resulting after AuNP formation were submitted to unidirectional freezing and subsequent freeze-drying via ISISA (ice-segregation-induced self-assembly) process for the preparation of CHI scaffolds. The study of AuNP-CHI scaffolds by SEM and confocal fluorescence microscopy revealed a morphological structure characteristic of the hydrogel nature of the samples subjected to the ISISA process. Interestingly, not only the morphology but also the dissolution and swelling degree of the resulting CHI scaffolds were strongly influenced by the strength of the hydrogels obtained by the in situ formation of AuNP. We have also studied the catalytic activity AuNP-CHI scaffolds in the reduction of p-nitrophenol. The negligible dissolution and low swelling degree obtained in certain AuNP-CHI scaffolds allowed them to be used for more than four cycles with full preservation of the reaction kinetics.

Urea assisted hydroxyapatite mineralization on MWCNT/CHI scaffolds

Urea assisted hydroxyapatite (HAp) mineralization was performed on scaffolds composed of a major fraction of multiwall carbon nanotubes (MWCNT, 85 wt.%) and a minor one of chitosan (CHI, 15 wt.%). The MWCNT/CHI scaffolds were synthesized through a cryogenic process (so called ISISA, ice segregation induced self-assembly) that allowed the achievement of macroporous monoliths whose structure resembled a chamber-like architecture in the form of interconnected MWCNT/CHI sheets arranged in parallel layers crossed by pillars. The mineralized architectures were composed of flower like hydroxyapatite (HAp) crystalline clusters of ca. 1 µm, homogeneously distributed throughout the internal surface of the scaffold macrostructure. HAp mineralized MWCNT/CHI scaffolds were characterized by X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning and transmission electron microscopy (SEM and TEM, respectively). Calibrated energy dispersion X-ray spectroscopy (EDS) and selected-area electron diffraction (SAED) were also performed in the transmission electron microscope to further HAp characterization. Preliminary in vitro experiments demonstrated the suitability of HAp mineralized MWCNT/CHI scaffolds for bone tissue growth.

Preparation of Chitosan Nanocompositeswith a Macroporous Structure by Unidirectional Freezing and Subsequent Freeze-Drying

Chitosan is the N-deacetylated derivative of chitin, a naturally abundant mucopolysaccharide that consists of 2-acetamido-2-deoxy-β-d-glucose through a β (1→4) linkage and is found in nature as the supporting material of crustaceans, insects, etc. Chitosan has been strongly recommended as a suitable functional material because of its excellent biocompatibility, biodegradability, non-toxicity, and adsorption properties. Boosting all these excellent properties to obtain unprecedented performances requires the core competences of materials chemists to design and develop novel processing strategies that ultimately allow tailoring the structure and/or the composition of the resulting chitosan-based materials. For instance, the preparation of macroporous materials is challenging in catalysis, biocatalysis and biomedicine, because the resulting materials will offer a desirable combination of high internal reactive surface area and straightforward molecular transport through broad “highways” leading to such a surface. Moreover, chitosan-based composites made of two or more distinct components will produce structural or functional properties not present in materials composed of one single component. Our group has been working lately on cryogenic processes based on the unidirectional freezing of water slurries and/or hydrogels, the subsequent freeze-drying of which produce macroporous materials with a well-patterned structure. We have applied this process to different gels and colloidal suspensions of inorganic, organic, and hybrid materials. In this review, we will describe the application of the process to chitosan solutions and gels typically containing a second component (e.g., metal and ceramic nanoparticles, or carbon nanotubes) for the formation of chitosan nanocomposites with a macroporous structure. We will also discuss the role played by this tailored composition and structure in the ultimate performance of these materials.

Singular thermosensitivity of polymethyl methacrylate/poly-N-isopropylacrylamide conetworks prepared by a facile synthetic route

A novel and facile route to prepare acrylic-based conetworks consisting of polymethacrylate and polyacrylamide chains is described. This method uses amine–succinimide coupling chemistry and sequential polymerization, and it is described using NIPA and MMA monomers as model components. The thermosensitivity in water, related to the NIPA component, has been found to be very different among the conetwork and the reference network of the crosslinked random copolymer.

Short-Chain Chitin Oligomers: Promoters of Plant Growth

Chitin is the second most abundant biopolymer in nature after cellulose, and it forms an integral part of insect exoskeletons, crustacean shells, krill and the cell walls of fungal spores, where it is present as a high-molecular-weight molecule. In this study, we showed that a chitin oligosaccharide of lower molecular weight (tetramer) induced genes in Arabidopsis that are principally related to vegetative growth, development and carbon and nitrogen metabolism. Based on plant responses to this chitin tetramer, a low-molecular-weight chitin mix (CHL) enriched to 92% with dimers (2mer), trimers (3mer) and tetramers (4mer) was produced for potential use in biotechnological processes. Compared with untreated plants, CHL-treated plants had increased in vitro fresh weight (10%), radicle length (25%) and total carbon and nitrogen content (6% and 8%, respectively). Our data show that low-molecular-weight forms of chitin might play a role in nature as bio-stimulators of plant growth, and they are also a known direct source of carbon and nitrogen for soil biomass. The biochemical properties of the CHL mix might make it useful as a non-contaminating bio-stimulant of plant growth and a soil restorer for greenhouses and fields.

Controlled formation of the anhydrous polymorph of ciprofloxacin crystals embedded within chitosan scaffolds: study of the kinetic release dependence on crystal size

This work describes a simple procedure for preparation of chitosan (CHI) scaffolds containing ciprofloxacin (CFX) anhydrous crystals. The capability of CHI scaffolds to control the CFX crystal size and to preserve the anhydrous crystal habit even after long term storage was useful for the development of drug delivery systems (e.g., CFX/CHI scaffolds) exhibiting a novel way to control the kinetic release, that is, by the solubility/hydration ratio of CFX anhydrous crystals depending on their crystal size rather than by typical mechanisms based on swelling, hydration and/or erosion of the polymer acting as carrier.

Pseudo-double network hydrogels with unique properties as supports for cell manipulation

Pseudo-double network hydrogels based on vinylpyrrolidone and anionic methacrylic units were prepared, for the first-time, via a simple one step radical polymerization procedure using thermal or photoinitiation. These networks showed improved mechanical properties, in the hydrated state, compared with their single network cousins and were capable of hosting cells to confluence. Rapid cell detachment can be induced through simple mechanical agitation and the cell sheets can be transplanted easily without the need for a cell superstrate. The results reported in this work suggest that these hydrogels could be used as support systems for cell manipulation and are candidates to compete with the conventionally used thermoresponsive cell platforms based on poly-N-isopropylacrylamide (pNIPAm).

Green Synthesis of Hierarchically Structured Silver-Polymer Nanocomposites with Antibacterial Activity

The in situ formation of silver nanoparticles (AgNPs) aided by chondroitin sulfate and the preparation of a hierarchically structured silver-polymer nanocomposite with antimicrobial activity is shown. Green synthesis of AgNPs is carried out by thermal treatment (80 and 90 °C) or UV irradiation of a chondroitin sulfate solution containing AgNO3 without using any further reducing agents or stabilizers. Best control of the AgNPs size and polydispersity was achieved by UV irradiation. The ice-segregation-induced self-assembly (ISISA) process, in which the polymer solution containing the AgNPs is frozen unidirectionally, and successively freeze-drying were employed to produce the chondroitin sulfate 3D scaffolds. The scaffolds were further crosslinked with hexamethylene diisocyanate vapors to avoid water solubility of the 3D structures in aqueous environments. The antimicrobial activity of the scaffolds was tested against Escherichia coli. The minimum inhibitory concentration (MIC) found for AgNPs-CS (chondroitin sulfate) scaffolds was ca. 6 ppm.

Macroporous Calcium Phosphate/Chitosan Composites Prepared via Unidirectional Ice Segregation and Subsequent Freeze-Drying

Calcium phosphate chitosan-based composites have gained much interest in recent years for biomedical purposes. In this paper, three-dimensional calcium phosphate chitosan-based composites with different mineral contents were produced using a green method called ice segregation induced self-assembly (ISISA). In this methodology, ice crystals were used as a template to produce porous structures from an aqueous solution of chitosan (CS) and hydroxyapatite (Hap) also containing acetic acid (pH = 4.5). For better characterization of the nature of the inorganic matter entrapped within the resulting composite, we performed either oxygen plasma or calcination processes to remove the organic matter. The nature of the phosphate salts was studied by XRD and NMR studies. Amorphous calcium phosphate (ACP) was identified as the mineral phase in the composites submitted to oxygen plasma, whereas crystalline Hap was obtained after calcination. SEM microscopy revealed the formation of porous structures (porosity around 80–85%) in the original composites, as well as in the inorganic matrices obtained after calcination, with porous channels of up to 50 µm in diameter in the former case and of up to 20 µm in the latter. The biocompatibility of the composites was assessed using two different cell lines: C2C12GFP premyoblastic cells and MC3T3 preosteoblastic cells.