Rheological study of genipin cross-linked chitosan hydrogels

This paper reports the rheological behavior of chitosan solutions that have been cross-linked with different amounts of genipin, at body temperature and physiological pH. The effect of the cross-linker loading on the rheological properties of hydrogels has been evaluated. The oscillatory time sweep method was used to monitor the dynamic viscoelastic parameters during in situ (i.e., in the rheometer) gelation experiments, enabling the determination of the gelation time. The stress and frequency sweeps were employed to measure G' of the cured hydrogels. It was found that the solutions of chitosan cross-linked with genipin, under physiological conditions, could form relatively strong elastic gels when compared to those of pure chitosan. Moreover, the gelation time obtained from the crossover of G' and G' was in excellent agreement with the value obtained from the Winter-Chambon criterion. A significant reduction on this parameter was achieved even at low genipin concentrations. This behavior suggests that these formulations are able to be produced in situ and thus constitute promising matrices for cells and bioactive molecule encapsulations.     

Interaction of liposome with immobilized chitosan during main phase transition

It has been recently demonstrated that chitosan in aqueous solution alters the phase behavior and structure of a phospholipid bilayer (Fang, N.; et al. Biomacromolecules 2001, 2, 1161-1168). Until now, the physical driving forces between chitosan and the phospholipid bilayer upon their initial encounter remains unknown. In this study, confocal reflectance interference contrast microscopy (C-RICM), phase contrast microscopy and bioadhesion modeling are concurrently applied to probe the interaction of phospholipid vesicle with immobilized chitosan at various temperatures, pH, and osmotic stress. First, the successful immobilization of chitosan on amino-silanized glass is indicated by the increases in both the degree of vesicle deformation and adhesion energy of vesicles adhering on chitosan modified substrate in comparison with those on amino-silanized glass. Second, the phase transition of a phospholipid bilayer does not modulate the adhesion strength at the chitosan-biomembrane interface at pH 7.4. With increase of the degree of protonation on the chitsoan backbone at pH 4, the adhesion energy is increased by 5-fold for vesicles of all sizes compared to that in pH 7.4. Furthermore, pH reduction amplifies the thermal-induced response of larger vesicles on the immobilized chitosan layer. Interestingly, a moderate increase of osmotic stress maximizes the degree of vesicle deformation and adhesion energy at 23 degrees C and dampens the effect of phase transition on vesicle adhesion. Overall, this study demonstrates the quantitation of chitosan-biomembrane interactions that will be critical for future applications of chitosan in biological systems.     

Heat-induced transfer of protons from chitosan to glycerol phosphate produces chitosan precipitation and gelation

Recently, chitosan dissolved in solutions containing glycerol phosphate (GP) were found to undergo a sol-gel transition when heated and the proposed gelling mechanism was based on increasing hydrophobic interactions with temperature. Subsequently, an investigation of ionization and precipitation behavior of chitosan, including dependencies on temperature, added salt, and fraction of deacetylated monomers (fD) was performed. This latter study revealed important differences in the temperature dependence of pKa of chitosan versus GP and led us to propose an alternative hypothesis for the mechanism of gelation in chitosan-GP systems whereby heat induces transfer of protons from chitosan to glycerol phosphate thereby neutralizing chitosan and allowing attractive interchain forces to form a physical gel. To investigate this specific molecular thermogelling mechanism, temperature ramp experiments on dilute chitosan-GP solutions were performed. Chitosans with fD of 0.72 and 0.98 were used to prepare solutions with a range of molar ratios of GP to chitosan glucosamine monomer of 1.25 to 10 and with 0 or 150 mM added monovalent salt. Light transmittance measurements were performed simultaneously to indicate precipitation in these dilute systems as a surrogate for gelation in concentrated systems. Measured temperatures of precipitation ranged from 15 to 85 degrees C, where solutions with less GP (used in a disodium salt form) had lower precipitation temperatures. A theoretical model using acid-base equilibria with temperature dependent pKa's, including the electrostatic contribution from the polyelectrolyte nature of chitosan, was used to calculate the degree ionization of chitosan (alpha, the fraction of protonated glucosamine monomer) as a function of temperature and showed a significant decrease in alpha with increased temperature due to proton transfer from chitosan to GP. This heat-induced proton transfer from chitosan to GP was experimentally confirmed by 31P NMR measurements during temperature ramp experiments since the chemical shift of 31P of GP is an indicator of its level of protonation. By assuming average temperature independent values of alpha p that were calculated from measured T(p), the model was able to accurately predict measured temperatures of precipitation (T(p)) of all chitosan-GP mixtures. The resulting alpha(p) were temperature independent but increased with increased chitosan fD and with increased salt. Measurements and theory revealed that T(p) can be adjusted in a predictable manner by changing the chitosan-GP molar ratio and thereby systematically tailored to obtain a large range of precipitation temperatures. Finally, similar temperature ramp experiments using inorganic phosphate and MES in place of GP demonstrated that the temperature-induced precipitation of chitosan also occurs with these buffers, confirming that the key feature of the buffer used with chitosan is its ability to absorb heat-stimulated release of chitosan protons and facilitate chitosan neutralization. A theoretical expression for the variation of chitosan ionization degree with temperature in a system composed of two titratable species (chitosan and buffer) was derived and allowed us to establish the required characteristics of the buffer for efficient heat-stimulated proton transfer between a chitosan and the buffer. These results provide a useful explanation for the mechanism of heat-induced gelation of chitosan-based systems that could be exploited for numerous practical applications.     

Improved blood biocompatibility of composite film of chitosan/carbon nanotubes complex

Single-walled carbon nanotubes are novel molecular-scale wires having excellent anti-adhesion properties with regard to platelets. On the other hand, chitosan is a partially de-acetylated derivative of chitin that has a critical role in cell attachment and growth. The aim of this study was to investigate how carbon nanotubes improve the blood biocompatibility of chitosan film. We prepared composite films with various concentrations of chitosan/carbon nanotubes (CS/CNTs) (1.3–6.3 wt%). The sample surfaces were characterized by Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and contact angle measurements. The surface characterization revealed that the surface of the CS/CNTs composite film became more hydrophobic with increasing amounts of CNTs. Cell attachment tests using bovine aortic endothelial cells (BAECs) indicated that CS/CNTs composite films retained their cell adhesion ability. The blood compatibility of the CS/CNTs composite films was evaluated using the blood platelet adhesion and activation tests in vitro. Platelet adhesion results confirmed that platelet adhesion and the formation of a platelet network were inhibited on composite films with higher concentrations of CNTs (5.1 wt%). Our experimental results show that the novel composite film containing CS/CNTs possesses two paradoxical characteristics, namely, good adherence of endothelial cells and minimum adherence and activation of platelets, making this film a promising antithrombogenic material for use in the biomedical field.     

Synthesis and physicochemical and dynamic mechanical properties of a water-soluble chitosan derivative as a biomaterial

The physicochemical and rheological properties of a water-soluble chitosan (WSC) derivative were characterized in order to facilitate its use as a novel material for biomedical applications. The WSC was prepared by conjugating glycidyltrimethylammonium chloride (GTMAC) onto chitosan chains. Varying the molar ratio of GTMAC to chitosan from 3:1 to 6:1 produced WSCs with a degree of substitution (DS) that ranged from 56% to 74%. The WSC with the highest DS was soluble in water up to concentrations of 25 g/dL at room temperature. An increase in the polymer concentration gradually increased both the pH and conductivity of the WSC solutions. The rheological properties of the WSC solutions were found to be dependent on the salt and polymer concentrations as well as the DS value. In the absence of salt, the rheological behavior of the WSC was found to be typical of that for a polyelectrolyte in the dilute solution regime. However, the addition of salt decreased the viscosity of the polymer solution due to the reduction of electrostatic repulsions by the positively charged trimethylated ammonium groups of the WSC. In the concentrated regime, the viscosity of the WSCs was found to follow a power-law expression. The lowest DS WSC had the more favorable viscoelastic properties that were attributed to its high molecular weight, as confirmed by the stress relaxation spectra and intrinsic viscosity measurements. The effect of DS on the degree of interaction between WSC and the lipid egg phosphatidylcholine was investigated by FTIR analysis. Overall, the lower DS WSC had enhanced rheological properties and was capable of engaging in stronger intermolecular physical interactions.     

In vivo evaluation of in situ polysaccharide based hydrogel for prevention of postoperative adhesion

In this paper, the carboxymethyl chitosan/oxidized dextran hydrogel was developed and its potency application in the prevention of postoperative adhesion was investigated. The developed hydrogel showed porous and interconnected interior structure with pore size about 250μm, which was sensitive to lysozymic solution (1.5μg/ml) with almost complete degradation after 4 weeks of in vitro incubation. In vivo study suggested that the developed hydrogel showed the great capacity on the prevention of postoperative adhesions in rat model. According to the result of histopathological examination, it clearly showed that the mesothelial cell layer of abdominal wall and cecum were completely recovered after 7 days of surgery in 3% carboxymethyl chitosan/oxidized dextran hydrogel group, while obvious adhesion between abdominal wall and cecum was observed as treatment with saline solution or 3% carboxymethyl chitosan solution after 1 day of surgery. All these results suggested that the developed biodegradable hydrogel might have potential application in the prevention of postoperative adhesion.     

Novel starch/chitosan blending membrane: Antibacterial,permeable and mechanical properties

Antibacterial membranes were prepared from a mixture of hydrolyzed starch and chitosan. Glycerin was incorporated in the membranes to as plasticizer agent. The effects of component ratio on the mechanical and permeable properties of the prepared membranes were investigated. The elongation-at-break and water vapor transmission rate of starch/chitosan blending membranes were largely improved compared with each single component due to the interaction formed between the hydroxyl groups of starch and the amino ones of chitosan, which was confirmed by FT-IR characterizations. With the help of optical microscope, the influence of component ratio on the morphologies of starch/chitosan membranes was systematically investigated. It comes to a conclusion that extreme low or high starch content will cause an asymmetric membrane surface. To prove the antibacterial activity of obtained membranes, Escherichia coli (E. coli) was chosen as the target bacteria via optical density method. The resulted starch/chitosan membranes exhibited an outstanding antibacterial activity against E. coli.     

Flow properties of N-(carboxymethyl) chitosan aqueous systems in the sol and gel domains

The flow behaviour and the viscoelastic properties of N-(carboxymethyl) chitosan aqueous systems in the sol and gel domains have been investigated by means of dynamic, steady and transient shear techniques. For polymer concentrations Cp up to 1%, a typical response of moderately concentrated polymer solutions was observed under continuous and oscillatory shear conditions. No time-dependent properties were detected during transient shear experiments. On the other hand, for all the samples with Cp greater than 1%, the rheological properties were more similar to those of a weak gel system. The continuous shear flow behaviour was of the plastic type and the viscoelastic quantities G' and G" were parallel to each other and slightly dependent on the frequency of oscillation omega. Stress overshoots were observed during transient shear experiments, and the kinetics of the structural breakdown and build-up processes were found to be dependent both on the polymer concentration and the applied shear rate.     

Adhesion and growth of HUVEC endothelial cells on films of enzymatically functionalized chitosan with phenolic compounds

Human Umbilical Vein Endothelial Cell (HUVEC) growth on chitosan films and its enzymatically functionalized derivatives films with ferulic acid (FA) and ethyl ferulate (EF) was assessed by evaluating cell adhesion, morphology and cell viability. The results indicated that chitosan derivative films improved protein adsorption properties compared to chitosan films. The HUVEC cell morphology showed well attachment and spread phenotype on chitosan derivative films compared to those growing on chitosan films which did not spread and remained round. Evaluation of cell viability revealed improvement of cell adhesion on chitosan derivative films compared to chitosan film depending on the quantity of oxidized phenols grafted on chitosan. In addition, FA-/EF-chitosan films allowed almost similar cell adhesion. Furthermore, cell adhesion was increased with the film thickness. These results suggested that the oxidized phenols grafting on chitosan is a promising process to enhance cell adhesion, growth and creating useful functional biomaterials.     

Hemagglutination and intestinal adherence properties of clinical and environmental isolates of non-O1 Vibrio cholerae

Hemagglutination and intestinal adherence properties of non-O1 Vibrio cholerae were studied in vitro. No definite correlation between the cell-associated hemagglutinin titers and the intestinal adhesion indices was noted. Sugar- and glycoprotein-mediated inhibition data also indicated differences between the hemagglutination and adherence processes in respect to the receptor structures. Intestinal adherence of most V. cholerae strains could be inhibited to various extents by N-acetyl D-glucosamine. This observation provides a likely explanation for the ecological behavior of these organisms, which are known to associate themselves with chitinous (chitin:homopolymer of N-acetyl D-glucosamine) surfaces of zooplankton. The absence of any significant difference between the intestinal adherence indices of clinical and environmental isolates suggests that intestinal adhesion may be an essential but not sufficient prerequisite for colonization by and subsequent expression of pathogenicity of these microorganisms.     

Hemagglutination and intestinal adherence properties of clinical and environmental isolates of non-O1 Vibrio cholerae.

Hemagglutination and intestinal adherence properties of non-O1 Vibrio cholerae were studied in vitro. No definite correlation between the cell-associated hemagglutinin titers and the intestinal adhesion indices was noted. Sugar- and glycoprotein-mediated inhibition data also indicated differences between the hemagglutination and adherence processes in respect to the receptor structures. Intestinal adherence of most V. cholerae strains could be inhibited to various extents by N-acetyl D-glucosamine. This observation provides a likely explanation for the ecological behavior of these organisms, which are known to associate themselves with chitinous (chitin:homopolymer of N-acetyl D-glucosamine) surfaces of zooplankton. The absence of any significant difference between the intestinal adherence indices of clinical and environmental isolates suggests that intestinal adhesion may be an essential but not sufficient prerequisite for colonization by and subsequent expression of pathogenicity of these microorganisms.     

Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.     

Stability of adhesion clusters and cell reorientation under lateral cyclic tension

This work is motivated by experimental observations that cells on stretched substrate exhibit different responses to static and dynamic loads. A model of focal adhesion that can consider the mechanics of stress fiber, adhesion bonds, and substrate was developed at the molecular level by treating the focal adhesion as an adhesion cluster. The stability of the cluster under dynamic load was studied by applying cyclic external strain on the substrate. We show that a threshold value of external strain amplitude exists beyond which the adhesion cluster disrupts quickly. In addition, our results show that the adhesion cluster is prone to losing stability under high-frequency loading, because the receptors and ligands cannot get enough contact time to form bonds due to the high-speed deformation of the substrate. At the same time, the viscoelastic stress fiber becomes rigid at high frequency, which leads to significant deformation of the bonds. Furthermore, we find that the stiffness and relaxation time of stress fibers play important roles in the stability of the adhesion cluster. The essence of this work is to connect the dynamics of the adhesion bonds (molecular level) with the cell's behavior during reorientation (cell level) through the mechanics of stress fiber. The predictions of the cluster model are consistent with experimental observations.     

Dynamic and extensional properties of starch in aqueous dimethylsulfoxide

The effect of starch composition and concentration on the rheological properties of starch in a mixed solvent, water–DMSO, was investigated in dynamic shear and extensional mode. High amylose corn starch containing 70% amylose and 30% amylopectin, common corn starch containing 25% amylose and 75% amylopectin, and waxy corn starch containing about 99% amylopectin were used in this study. Concentrations of 2, 4, 6, and 8% (w/v) in 10% water-90% DMSO (v/v) were used for each starch type. An increase in the amylopectin content of starch from 30 to 99% caused a change in behavior from semidilute solution to viscoelastic solid at a concentration of 8% (w/v). At a concentration of 2%, an increase in the amylopectin content of starch from 30 to 99% caused a change from Newtonian to incipient gel-like behavior. Behavior at intermediate concentrations of 4 and 6% (w/v) varied from semidilute to critical gel-like with increasing amylopectin content. A power-law relaxation was observed for all concentrations of common and waxy corn starches with the slope decreasing with increase in concentrations. A 2% waxy corn starch solution displayed extension thinning behavior, while a 2% high amylose corn starch solution displayed Newtonian behavior.     

Fluorescence recovery after photobleaching as a probe of diffusion in starch systems

The diffusion coefficients of dextran probes of various molecular weights in starch solutions over a wide concentration range were carried out using fluorescent recovery after photobleaching (FRAP), combined with a confocal microscope and tracer probe diffusion. The technique is simple to implement and can be carried out using increasingly common microscopy apparatus, giving access to a wide variety of new structural and kinetic information. The data can be rationalized in terms of the effects of probe molecular weight and on matrix starch concentration and structure. This provides a new tool to investigate the behavior of systems where starch is an ingredient that contributes to the processing and textural properties of food.     

Microstructural imaging and characterization of the mechanical, chemical, thermal, and swelling properties of starch-chitosan blend films

Chitosan has wide range of applications as a biomaterial, but barriers still exist to its broader use due to its physical and chemical limitations. The present study evaluated the properties of the polymeric blend films obtained from chitosan and potato starch by the casting/solvent evaporation method. The swelling properties of the different films studied as a function of pH showed that the sorption ability of the blend films increased with the increasing content of starch. Fourier transform infrared (FTIR) analyses confirmed that interactions were present between the hydroxyl groups of starch and the amino groups of chitosan in the blend films while the x-ray diffraction (XRD) studies revealed the films to exhibit an amorphous character. Thermogravimetric analyses showed that in the blend films, the thermal stability increased with the increasing starch content and the stability of starch and chitosan powders reduced when they were converted to film. The differential scanning calorimetry (DSC) studies revealed an endotherm corresponding to water evaporation around 100 degrees C in all the films and an exotherm, corresponding to the decomposition in the chitosan and blend films. Scanning electron microscopy (SEM) observations indicated that the blend films were less homogenous and atomic force microscopy (AFM) studies revealed the chitosan films to be smooth and homogenous, while the starch films revealed characteristic granular pattern. The blend films exhibited an intermediate character with a slight microphase separation. The starch-chitosan blend films exhibited a higher flexibility and incorporation of potato starch into chitosan films improved the percentage elongation.     

Effect of Ionic Strength on Initial Interactions of Escherichia coli with Surfaces, Studied On-Line by a Novel Quartz Crystal Microbalance Technique

A novel quartz crystal microbalance (QCM) technique was used to study the adhesion of nonfimbriated and fimbriated Escherichia coli mutant strains to hydrophilic and hydrophobic surfaces at different ionic strengths. This technique enabled us to measure both frequency shifts (Deltaf), i.e., the increase in mass on the surface, and dissipation shifts (DeltaD), i.e., the viscoelastic energy losses on the surface. Changes in the parameters measured by the extended QCM technique reflect the dynamic character of the adhesion process. We were able to show clear differences in the viscoelastic behavior of fimbriated and nonfimbriated cells attached to surfaces. The interactions between bacterial cells and quartz crystal surfaces at various ionic strengths followed different trends, depending on the cell surface structures in direct contact with the surface. While Deltaf and DeltaD per attached cell increased for nonfimbriated cells with increasing ionic strengths (particularly on hydrophobic surfaces), the adhesion of the fimbriated strain caused only low-level frequency and dissipation shifts on both kinds of surfaces at all ionic strengths tested. We propose that nonfimbriated cells may get better contact with increasing ionic strengths due to an increased area of contact between the cell and the surface, whereas fimbriated cells seem to have a flexible contact with the surface at all ionic strengths tested. The area of contact between fimbriated cells and the surface does not increase with increasing ionic strengths, but on hydrophobic surfaces each contact point seems to contribute relatively more to the total energy loss. Independent of ionic strength, attached cells undergo time-dependent interactions with the surface leading to increased contact area and viscoelastic losses per cell, which may be due to the establishment of a more intimate contact between the cell and the surface. Hence, the extended QCM technique provides new qualitative information about the direct contact of bacterial cells to surfaces and the adhesion mechanisms involved.     

Adhesion and viability of two enterococcal strains on covalently grafted chitosan and chitosan/kappa-carrageenan multilayers

Chitosans are natural aminopolysaccharides, whose low cytotoxicity suggests their potential use for nonadhesive, antibacterial coatings on biomaterials implant surfaces. Here, the antiadhesive behavior and ability to kill bacteria upon adhesion ("contact killing") of chitosan coatings were evaluated for two strains of Enterococcus faecalis, isolated from clogged biliary stents. Chitosan coatings covalently grafted or applied as chitosan/kappa-carrageenan multilayers were characterized by ellipsometry, scanning force microscopy (SFM), X-ray photoelectron spectroscopy (XPS), and electrokinetic measurements. Decreases in initial bacterial deposition rates and the number of bacteria adhering in a more advanced state of the adhesion process were observed on both types of modified surfaces, with more pronounced effects on highly hydrated multilayers. Adhesion of negatively charged enterococci was slightly enhanced on chitosan-terminated multilayers, but antibacterial effect was absent on kappa-carrageenan-terminated multilayers. Thus, the efficacy of multilayers remains an interesting interplay between the promoting effect of cationically charged groups on adhesion of negatively charged bacteria and, on the other hand, their antibacterial effects.