Nanoarchitectonics and Biological Properties of Nanocomposite Thermosensitive Chitosan Hydrogels Obtained with the Use of Uridine 5'-Monophosphate Disodium Salt.

Currently, an important group of biomaterials used in the research in the field of tissue engineering is thermosensitive chitosan hydrogels. Their main advantage is the possibility of introducing their precursors (sols) into the implantation site using a minimally invasive method-by injection. In this publication, the results of studies on the new chitosan structures in the form of thermosensitive hydrogels containing graphene oxide as a nanofiller are presented. These systems were prepared from chitosan lactate and chitosan chloride solutions with the use of a salt of pyrimidine nucleotide-uridine 5'-monophosphate disodium salt-as the cross-linking agent. In order to perform the characterization of the developed hydrogels, the sol-gel transition temperature of the colloidal systems was first determined based on rheological measurements. The hydrogels were also analyzed using FTIR spectroscopy and SEM. Biological studies assessed the cytotoxicity (resazurin assay) and genotoxicity (alkaline version of the comet assay) of the nanocomposite chitosan hydrogels against normal human BJ fibroblasts. The conducted research allowed us to conclude that the developed hydrogels containing graphene oxide are an attractive material for potential use as scaffolds for the regeneration of damaged tissues.

Influence of Injection Application on the Sol-Gel Phase Transition Conditions of Polysaccharide-Based Hydrogels.

Polysaccharide matrices formed via thermoinduced sol-gel phase transition are promising systems used as drug carriers and minimally invasiveness scaffolds in tissue engineering. The strong shear field generated during injection may lead to changes in the conformation of polymer molecules and, consequently, affect the gelation conditions that have not been studied so far. Chitosan (CS) and hydroxypropyl cellulose (HPC) sols were injected through injection needles (14 G-25 G) or sheared directly in the rheometer measuring system. Then the sol-gel phase transition conditions were determined at 37 °C using rheometric, turbidimetric, and rheo-optical techniques. It was found that the use of low, respecting injection, shear rates accelerate the gelation, its increase extends the gelation time; applying the highest shear rates may significantly slow down (HPC) or accelerate gelation (CS) depending on thixotropic properties. From a practical point of view, the conducted research indicates that the use of thin needles without preliminary tests may lead to an extension of the gelation time and consequently the spilling of the polymeric carrier before gelation. Finally, an interpretation of the influence of an intensive shear field on the conformation of the molecules on a molecular scale was proposed.

Influence of Glycerophosphate Salt Solubility on the Gelation Mechanism of Colloidal Chitosan Systems.

Recently, thermosensitive chitosan systems have attracted the interest of many researchers due to their growing application potential. Nevertheless, the mechanism of the sol-gel phase transition is still being discussed, and the glycerophosphate salt role is ambiguous. The aim of the work is to analyze the possibility of the exclusive use of a non-sodium glycerophosphate salt and to determine its impact on the gelation conditions determined by rheological and turbidimetric measurements as well as the stability of the systems by measuring changes in the Zeta potential value. It was found that ensuring the same proportions of glycerophosphate ions differing in cation to amino groups present in chitosan chains, leads to obtaining systems significantly different in viscoelastic properties and phase transition conditions. It was clearly shown that the systems with the calcium glycerophosphate, the insoluble form of which may constitute additional aggregation nuclei, undergo the gelation the fastest. The use of magnesium glycerophosphate salt delays the gelation due to the heat-induced dissolution of the salt. Thus, it was unequivocally demonstrated that the formulation of the gelation mechanism of thermosensitive chitosan systems based solely on the concentration of glycerophosphate without discussing its type is incorrect.

An Investigation of the Sol-Gel Transition of Chitosan Lactate and Chitosan Chloride Solutions via Rheological and NMR Studies.

In recent years, intensive research has been carried out on the use of hydrogels obtained from natural polymers, mainly chitosan. These products are increasingly replacing solutions based on synthetic materials in medicine. This publication presents the results of studies on the sol-gel transition of chitosan solutions as the base material for the preparation of thermosensitive hydrogels for potential applications in tissue engineering. The measurements were carried out for systems consisting of chitosan lactate and chitosan chloride solutions using ß-glycerol phosphate disodium salt pentahydrate and uridine 5'-monophosphate disodium salt as the cross-linking agents. The sol-gel transition point of the solutions was determined based on the rheological measurements in the cone-plate configuration of the rotational rheometer and experiments performed using the method of nuclear magnetic resonance. The obtained results showed a significant influence of the cross-linking agent on the course of the sol-gel transition of chitosan salt solutions, and the systems that consisted of chitosan lactate seemed to be especially interesting for biomedical applications.

Thermoinduced aggegation of chitosan systems in perikinetic and orthokinetic regimes.

Thermoresponsive colloidal chitosan systems forming the polymer structure in situ are an example of promising solutions in tissue engineering as an injectable scaffolds or drug carriers. Their application method, and thus shearing, may affect the aggregation process in accordance with the colloidal engineering approach. The aim of the study is to compare the kinetics of chitosan aggregation in the perikinetic regime (limited by Brownian motions) with the orthokinetic process carried out under the influence of an external shear field. The research was carried out using static multiple light scattering (S-MLS) and rheometric measurement techniques coupled with small-angle light scattering (Rheo-SALS). It has been found that the introduction of an external shear field (orthokinetic regime) accelerates the aggregation of chitosan systems. Simultaneously, the rotational measurements can even lead to spontaneous gelation, most likely caused by changes in the conformation of chitosan molecules, their deformation and ordering along the shear field.

Injectability of Thermosensitive, Low-Concentrated Chitosan Colloids as Flow Phenomenon through the Capillary under High Shear Rate Conditions.

Low-concentrated colloidal chitosan systems undergoing a thermally induced sol-gel phase transition are willingly studied due to their potential use as minimally invasive injectable scaffolds. Nevertheless, instrumental injectability tests to determine their clinical utility are rarely performed. The aim of this work was to analyze the flow phenomenon of thermosensitive chitosan systems with the addition of disodium ß-glycerophosphate through hypodermic needles. Injectability tests were performed using a texture analyzer and hypodermic needles in the sizes 14G-25G. The rheological properties were determined by the flow curve, three-interval thixotropy test (3ITT), and Cox-Merz rule. It was found that reducing the needle diameter and increasing its length and the crosshead speed increased the injection forces. It was claimed that under the considered flow conditions, there was no need to take into account the viscoelastic properties of the medium, and the model used to predict the injection force, based solely on the shear-thinning nature of the experimental material, showed very good agreement with the experimental data in the shear rate range of 200-55,000 s-1. It was observed that the increase in the shear rate value led to macroscopic structural changes of the chitosan sol caused by the disentangling and ordering of the polysaccharide chains along the shear field.

The effects of sucrose on the sol-gel phase transition and viscoelastic properties of potato starch solutions.

In this paper, starch pastes in the form of solutions and gels were investigated to determine viscoelastic properties and sol-gel phase transition temperatures using rheological methods. The gelatinization process was carried out at a temperature of 95 °C with the use of a pressureless starch cell with a stirrer. Starch pastes obtained were used to determine rheological properties under isothermal conditions (in the temperature range of 45-25 °C) by a cone-plate measurement system. The viscoelastic behavior of the tested medium was confirmed. The range of gelation temperatures was determined and the influence of several factors was discussed, e.g. the effect of sucrose addition and cooling rate on the phenomenon of sol-gel phase transition. In addition, mechanical properties of the obtained starch gel structures were determined using a fractional rheological model.

The Application of Small-Angle Light Scattering for Rheo-Optical Characterization of Chitosan Colloidal Solutions.

In the recent studies on chitosan hydrogels, it was found that understanding both rheological and structural properties plays an important role in their application. Therefore, a combination of two independent techniques was applied to investigate micro- and macroscopic properties of chitosan colloidal system. Studies on viscous properties, as well as the sol-gel phase transition process, were performed using rheological methods coupled with the small angle light scattering (SALS) technique. Based on the anisotropy of scattering patterns obtained during rotational shear tests, it was found that the chitosan solution reveals two different behaviors delimited by the critical value of the shear rate. Below a critical value, chitosan clusters are deformed without breaking up aggregates, whereas after exceeding a critical value, chitosan clusters apart from deformation also breakup into smaller aggregates. The values of the radius of gyration determined by applying the Debye function allow one to state that with an increase of chitosan concentration, molecule size decreases. An analysis of the light scattering data from the temperature ramp test showed that with an increase of temperature, the level of polymer coil swelling increases. Simultaneously, the supply of thermal energy leads to a neutralization of the charge of chitosan chains. As a consequence, the formation of intermolecular links occurs and a gel structure is formed.

Rheo-Kinetic Study of Sol-Gel Phase Transition of Chitosan Colloidal Systems.

Chitosan colloidal systems, created by dispersing in aqueous solutions of hydrochloric acid, with and without the addition of disodium ß-glycerophosphate (ß-NaGP), were prepared for the investigation of forming mechanisms of chitosan hydrogels. Three types of chitosan were used in varying molecular weights. The impacts of the charge and shape of the macromolecules on the phase transition process were assessed. The chitosan system without the addition of ß-NaGP was characterized by stiff and entangled molecules, in contrast to the chitosan system with the addition of ß-NaGP, wherein the molecules adopt a more flexible and disentangled form. Differences in molecules shapes were confirmed using the Zeta potential and thixotropy experiments. The chitosan system without ß-NaGP revealed a rapid nature of phase transition-consistent with diffusion-limited aggregation (DLA). The chitosan system with ß-NaGP revealed a two-step nature of phase transition, wherein the first step was consistent with reaction-limited aggregation (RLA), while the second step complied with diffusion-limited aggregation (DLA).

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor.

It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle-enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·108 L-1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sn at shear rate about 0.05 s-1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 µm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5-fold.