Gellan Gum Aqueous Solutions Research Articles

Development of Gellan Gum-Based Microparticles/Hydrogel Matrices for Application in the Intervertebral Disc Regeneration

Low back pain is one of the most reported medical conditions associated to intervertebral disc (IVD) degeneration. Nucleus pulposus (NP) is often regarded as the structure where IVD degeneration begins. Gellan gum (GG)-based hydrogels for acellular and cellular tissue engineering strategies have been developed for finding applications as NP substitutes. The innovative strategy is based on the reinforcement of the hydrogel matrix with biocompatible and biodegradable GG microparticles (MPs), which are expected to improve the mechanical properties, while allowing to tailor its degradation rate. In this study, several GG MP/hydrogel disc formulations were prepared by means of mixing high acyl GG (0.75% (w/v)) and low acyl GG (2% (w/v)) GG aqueous solutions at different ratios, namely, 75%:25% (v/v), 50%:50% (v/v), and 25%:75% (v/v), respectively. The GG MP size was measured using a stereo microscope, and their dispersion within the hydrogel matrix was evaluated by means of staining the MPs with Toluidine Blue-O. The developed GG MPs/hydrogel discs were physicochemically characterized by Fourier-transform infrared spectroscopy and (1)H-nuclear magnetic resonance spectroscopy. The swelling behavior and degradation rate were assessed by immersion in a phosphate buffer saline for 14 days. The morphology and mechanical behavior were investigated by scanning electron microscopy and dynamic mechanical analysis, respectively. The mechanical properties of the hydrogel disc were improved by mixing the gels with the MPs. In addition, the possible cytotoxicity of the leachables released by MPs/hydrogel discs was screened in vitro, using a mouse lung fibroblast cell line (L929 cells). To investigate the encapsulation efficacy of L929 cells into the GG MPs/hydrogel discs, cells were stained with DAPI blue/Texas Red-Phalloidin and observed by confocal microscopy, after 24, 48, and 72 h of culturing. A cell viability assay was also performed using Calcein AM staining. The cell culture studies demonstrated that MPs/hydrogel discs are noncytotoxic over L929 cells. It was also demonstrated that L929 cells can be successfully encapsulated into the GG MPs of different formulations, remaining viable after 72 h of culturing. This study showed that GG hydrogel matrices reinforced with cell-loaded MPs could be a candidate strategy for NP regeneration.

Critical behavior at sol–gel transition in gellan gum aqueous solutions with KCl and CaCl 2 of different concentrations

The sol–gel transition in aqueous gellan gum solutions with added KCl or CaCl 2 upon cooling was investigated using rheology measurements. The gelation temperature T gel (gel point) and the relaxation critical exponent n were successfully determined according to the Winter's criterion. With increasing salt concentration, T gel increased while n decreased. The scaling for the zero shear viscosity η 0 in the sol phase and the equilibrium modulus G e in the gel phase were established against the relative distance ɛ ( ɛ = | T − T gel|/ T gel) from the gel point, which gave the critical exponents k and z. No universal critical exponent n was found for this sol–gel transition because this gelation belonged to the cross-linking of existing macromolecules and the concept of random connectivity failed. The fractal dimension d f calculated from n without hydrodynamic interaction nor excluded volume suggested a denser filling structure in the critical gel at higher salt concentration.

Concentration dependence of critical exponents for gelation in gellan gum aqueous solutions upon cooling

The sol–gel transition in aqueous gellan gum solutions induced upon cooling was investigated by rheology measurements. The gelation temperature was determined from the crossover point of storage and loss moduli, i.e., G′ = G′′ ( T c) and from the Winter’s criterion ( T gel), respectively, which increased with gellan concentration. T gel was higher than T c and the difference became larger as the gellan concentration got higher. The relaxation critical exponent n was estimated with the Winter’s method and the self-similarity was observed from the critical gel. The scaling for the zero-shear viscosity η 0 before the gel point and the equilibrium modulus G e after the gel point was established against the relative distance ε from the gel point over the gellan concentration C g of 1.0–2.5 wt%, giving the critical exponents k and z. The critical exponent n calculated from k and z agrees well with n from the Winter’s criterion. However, no universal n was found for the gelation in aqueous gellan gum solutions, indicating that this gelation should be classified into the cross-linking category for the physical gelation. The critical exponent n decreased with increasing C g for the gellan gum solution. The fractal dimension d f calculated from n with the screened hydrodynamic interaction and the excluded volume effect suggested a denser structure in the critical gel with higher C g.

Gelation of gellan gum aqueous solutions studied by polarization modulation spectroscopy.

Circular birefringence (CB, or optical rotation) and linear birefringence (LB) were measured for gellan gum aqueous solutions with and without salt to examine the gelling system in the helical structure as well as in the orientation. It was found that gelling samples with salt show nonzero LB values, whereas LB is zero for the samples without salt even in the gel state. This difference can be explained by the thermal deformation of the system containing anisotropic aggregations of helices formed with the shielding effect of the added salt on the intramolecular and intermolecular electrostatic repulsions. Considering that the presence of LB in the system affects the estimation of CB, we developed an original procedure of the CB measurement to eliminate the contribution of LB. It was shown that our methods for eliminating the contribution of LB can improve the CB measurement for the gellan gum gel. The temperature dependence of [alpha] for the samples with salt in the gel state is quite different from that for the samples without salt, suggesting that the aggregates of helices in the samples containing a high concentration of salt form a supramolecular structure that contributes to CB.

Thermodynamics and structural aspect of the gelling process in the gellan gum/metal salt aqueous solutions

Gellan gum undergoes gelation by forming domains composed of associated double helices. Here the further random aggregation of associated double helices seems necessary for the formation of the network in the case of Na-gellan with adding NaCl. Gellan gum aqueous solutions were prepared with or without adding various concentrations of NaCl, and their gel-sol transitions were observed by the differential scanning calorimetry (DSC) and the small-angle X-ray scattering (SAXS). The DSC endothermic peaks are attributed to the dissociation of the ordered domains, and the network dissociation in the case of gel samples. The SAXS results are analyzed in terms of the molecular models of associated double helices.

Non-Newtonian flow behaviour of gellan gum aqueous solutions

Rheological properties of gellan gum solutions with and without salt have been monitored using oscillatory measurements and steady-shear viscosity measurements. The steady-shear viscosity measurements indicated that gellan gum solutions showed a wide Newtonian plateau when gellan gum molecules took a coil conformation, and that the shear-thinning behaviour became more conspicuous with conformational change of gellan gum molecules from coiled to helical, and the range of the Newtonian plateau became limited only to very low shear rates. When gellan gum solutions showed rheological behaviour as a dilute or concentrated polymer solution, these systems obeyed the Cox–Merz superposition of steady-state viscosity and dynamic viscosity. As gellan gum solutions formed a weak gel, the Cox–Merz rule was not valid; however, the deviation from this superposition was less significant than that of xanthan gum solutions which also show weak-gel behaviour.

Effects of glucose, mannose and konjac glucomannan on the gel–sol transition in gellan gum aqueous solutions by rheology and DSC

Abstract Effects of glucose, mannose and konjac glucomannan with different molecular weights on the sol–gel transition in gellan gum aqueous solutions were studied by dynamic viscoelastic measurement and differential scanning calorimetry. Temperature dependence of the loss shear modulus G ″ for a 1% gellan gum solution without sugars showed a one-step-like change at a certain temperature, which was attributed to the helix–coil transition temperature T hc . On addition of sugar, storage shear modulus G ′ and loss shear modulus G ″ increased and T hc shifted to higher temperatures. With progressive addition of sugar, the cross-over of G ′ and G ″ was observed at a lower temperature than T hc , which was attributed to the sol–gel transition T sg . Moreover, in the presence of sufficient sugar, both moduli did not decrease remarkably during heating up to 60 °C, although both moduli showed a steep increase around 50 °C during cooling process. In the presence of sugar of fairly low concentration, DSC measurement showed that both exothermic and endothermic peak temperatures, T s and T m , slightly shifted to lower temperatures, and that exothermic and endothermic enthalpies, Δ H s and Δ H m , significantly decreased. In contrast, rheological measurement showed that elastic moduli and the relaxational strength Δ G significantly increased by the addition of a sugar of fairly low concentration. However, further addition of sugar shifted T s and T m to higher temperatures and increased Δ H as well as elastic moduli. In the presence of sufficient sugar, both cooling and heating DSC curves became broader; however, many small peaks were observed at both higher and lower temperatures. Gellan with sufficient sugar formed various junction zones with different thermal stabilities during cooling, and then these junction zones became markedly heat resistant. In the presence of konjac glucomannan with relatively lower molecular weight, the effective concentration of gellan gum increases by immobilising water molecules in gellan gum solutions, so that konjac glucomannan shifts the helix– coil transition temperature in gellan gum molecules to higher temperatures.

Small angle X-ray characterization of gellan gum containing a high content of sodium in aqueous solution

A temperature dependence of small-angle X-ray scattering (SAXS) profiles was observed from the aqueous solutions of bacterial polysaccharide gellan gum at various concentrations. The sample is a water-soluble sodium salt type and is provided by Kelco Division of Merk, Co. for use by the Gellan Gum Working Party organized by the Gel Research Group of the Society of Polymer Science and Technology. The cross-sectional radius of gyration R G, c evaluated by crosssectional Guinier plots indicates that gellan gum consists mainly of single chains at higher temperatures. On cooling, R G, c was observed to increase to the value corresponding to the gellan double helix at lower concentrations when no gelation takes place. At higher concentration where a gel is formed, a much larger R G, c was obtained, suggesting the further association of double helices. The observed SAXS profiles were analyzed in terms of the broken rod-like model where the rod thickness and the interference due to the repulsive interaction between domains was taken into account. The calculated scattering function describes well the SAXS profile from gellan gum aqueous solutions.

Rheological and thermal studies of gel-sol transition in gellan gum aqueous solutions

The rheological and thermal properties of sodium-form gellan gum solutions with and without sodium chloride, potassium chloride, calcium chloride and magnesium chloride were studied by dynamic viscoelastic measurement and differential scanning calorimetry. Temperature dependence of the loss modulus G″ for gellan gum solutions of lower concentrations without salt showed one step-like change at a certain temperature, however, that for concentrated gellan gum solutions (> 2.0%) showed two step-like changes. The higher temperature process was attributed to the helix-coil transition, and was found in between the exothermic and endothermic peak temperatures T s and T m observed in cooling and heating DSC curves, while the lower temperature process was attributed to the sol-gel transition. Temperature dependence of G″ for gellan gum solutions of higher concentrations (> 3.2%) showed a large hysteresis, moreover, the temperature at which G″ showed the second step decrease in the heating process shifted to higher temperatures with increasing concentration of gellan gum. The cooling or heating DSC curves for gellan gum solutions of lower concentrations showed a single exothermic or endothermic peak, and both T s and T m shifted to higher temperatures, and both exothermic and endothermic enthalpies increased with increasing concentration of gellan gum. However, for a gellan gum solution with a concentration higher than 3.2%, two endothermic peaks were observed on heating while the cooling curve showed only one exothermic peak. The lower temperature endothermic peak corresponds to the first step decrease of G″ in the heating process, and the higher temperature endothermic peak corresponds to the second step decrease of G″. The viscoelastic behavior of gellan gum solutions was influenced much more strongly by divalent cations than by monovalent cations. DSC heating curves for a 1% gellan gum solution in the presence of sufficient monovalent cations showed multiple endothermic peaks, however, these multiple peaks were observed at significantly higher temperatures than those for a gellan gum solution of high concentration (3.2%) without salts. The endothermic peaks in the presence of sufficient divalent cations in the heating DSC curve were too broad to be resolved from the baseline, however, many small peaks were observed at higher temperatures and the largest endothermic peak was observed at a temperature higher than 100 °C. Therefore, although divalent cations promote the formation of thermally stable junction zones much more strongly than monovalent cations, the gellan gum gels containing divalent cations may also consist of physical junction zones of hydrogen bonds because these junction zones can be unzipped on heating to 120 °C.

Viscoelastic properties of Ca 2+ — and Na +-gellan gum aqueous solutions

Viscoelastic properties of gellan gum aqueous solutions under forces of the order of 10 −6-10 −8 N and displacements of 10 −3-10 −4 m were studied using a magnetic rheometer constructed in our laboratory. The concentration of Ca 2+ in the gellan gum has a strong effect upon the mechanical behaviors of the solutions: (i) solutions of Specimen 1 (Ca 2+ 0.5 wt%, C p = 1.15%) showed negative thixotropy; (ii) abrupt decrease in viscosity was observed in the narrow temperature range (±1 °C) for the solution of Specimen 2 (Ca 2+ 0.1 wt%, C p = 1–3 wt%) and Specimen 3 (Ca 2+ < 0.01%, C p = 2–3%), while rigidity was almost constant in the same temperature range; (iii) the solution of Specimen 3 shows unstable mechanical behavior, and this may be attributed to the inhomogeneity of the order of ~10~ −4 m in solution. A small amount of Ca 2+, as in the case of Specimen 2, removes this inhomogeneity from gellan gum aqueous solutions.

Influence of Ca2+-ion upon viscoelastic properties of gellan gum aqueous solutions

Abstract Viscoelastic properties of gellan gum aqueous solutions [San-Eigen F.F. I. (Osaka), Lot No. 62058A] were observed in order to study the influence of Ca 2+ -ions upon the solution properties. The results were (i) the solution of Sample 1 (Ca 2+ ~ 0.5%) showed narrowing of the oscillating amplitude of the ball after ~10 cycles (this is known as negative thixotropy); (ii) abrupt decreases in viscosity were observed for the solution of Sample 2 (Ca 2+ ~ 0.1%) in the narrow temperature range of ± 0.5 °C; (iii) unstable behavior of oscillating ball was found for the solution of Sample 3 (Ca 2+ ~ 0.00%), and this may be attributed to the inhomogeneity in the solution of the order of 10 −4 m, which was removed after stirring for one week.

Viscoelastic properties of gellan gum aqueous solutions

The magnetic rheometer which was recently designed in our laboratory was used to observe the viscoelastic properties of hydrocolloid gellan gum aqueous solutions. It was found that the mechanical properties of these solutions depended strongly upon the time and the temperature after dissolving gellan gum in water, and that negative thixotropy was found when the specimen stood for 25–100 h after dissolving.

Osmotic pressure measurements for gellan gum aqueous solutions

Osmotic pressure measurements for aqueous solutions of a gellan gum and standard samples, such as three kinds of pullulans and a dextran, were made to determine number average molecular weights Mn and osmotic second viral coefficients A 2 using a Hewlett-Packard high speed membrane osmometer. From the measurements of the standard samples, some technical problems for the osmometry of aqueous solutions were discussed. It was found that the values of Mn obtained for the standard samples were close to but somewhat smaller than the Mw values determined by sedimentation equilibrium measurements. Osmometry of the polyelectrolyte of gellan gum was carried out at 40°C in tetramethyl ammonium nitrate solution using the sample converted into tetramethyl ammonium salt. The Mn values of the sample obtained were 5.4 × 10 4 and 5.5 × 10 4 in 0.05 and 0.075 mol/dm 3 tetramethyl ammonium nitrate solutions respectively. The A 2 values obtained were rather large due to the Donnan effect.