Ultrastructure of hybrid chitosan-glycerol phosphate blood clots by environmental scanning electron microscopy.

Chitosan-based polymers have been extensively studied for biomedical applications. Recently, liquid solutions of chitosan in a glycerol phosphate buffer (chitosan-GP) with physiological pH and osmolality were mixed with autologous blood to form hybrid chitosan-GP/blood implants that improved the repair of articular cartilage lesions in a large animal model. The mixture of chitosan-GP and blood forms a viscous liquid, which solidifies in minutes via normal blood coagulation as well as chitosan-mediated mechanisms. Here we have examined the ultrastructure of these chitosan-GP/blood clots as well as regular blood clots and chitosan-GP gels, the latter produced by heating. Both unfixed and fixed samples of chitosan-GP/blood clots, regular blood clots, and chitosan-GP gels were investigated by environmental scanning electron microscopy (ESEM) in conjunction with energy dispersive X-ray spectrometry (EDS), the former permitting direct observation of the ultrastructure in hydrated conditions simulating the natural state. By examination of unfixed specimens using ESEM we found that chitosan formed a network structure in both chitosan-GP gels and chitosan-GP/blood clots; however this structure was altered by aldehyde fixation to produce artifactual aggregates of chitosan microparticles. We were also able to identify chitosan in chitosan-GP/blood clots by washing samples in low concentration NaCl solutions followed by local EDS analyses to identify excess chloride versus sodium, and thus presence of cationic chitosan in analyzed features. Additional results indicated that the majority of glycerol phosphate diffuses freely from chitosan-GP gels (by EDS of phosphorus) and that hyperosmotic paraformaldehyde-based fixatives (i.e. 4% w/v) significantly disturb erythrocyte morphology in fixed whole blood clots.

Cytocompatible gel formation of chitosan-glycerol phosphate solutions supplemented with hydroxyl ethyl cellulose is due to the presence of glyoxal.

To deliver and retain viable repair cells in a surgically prepared cartilage lesion, we previously developed an adhesive in situ-gelling cell carrier by suspending cells in a solution of hydroxyethyl cellulose (HEC), which was then mixed with chitosan-glycerol phosphate to form a chitosan-GP/HEC gel. The purpose of this study was to elucidate the mechanism of gelation to maximally control gel time and viability of encapsulated cells. We analyzed the role of osmolality, pH, gelation temperature, gel shrinkage, and HEC. A chitosan-GP solution at pH 6.8 with cytocompatible osmotic pressure (419 mOsm/kg) was achieved by lowering disodium GP concentration from 370 to 135 mM. This solution was still thermogelling but only at 73 degrees C. We next discovered that glyoxal, a common additive in ether cellulose manufacturing, was responsible for chitosan gelation. Monolayer cells survived and proliferated in up to 1 mM of glyoxal, however only a very narrow range of glyoxal concentration in chitosan-GP/HEC, 0.1-0.15 mM, permitted gel formation, cell survival, and cell proliferation. Chitosan gels containing HEC required slightly less glyoxal to solidify. Chitosan-GP/HEC loaded with viable chondrocytes formed an adhesive seal with ex vivo mosaic arthroplasty defects from sheep knee joints. In mosaic arthroplasty defects of live sheep, bleeding occurred beneath part of the hydrogel carrier, and the gel was cleared after 1 month in vivo. These data indicate that chitosan-GP/HEC is suitable as an adhesive and injectable delivery vehicle for clinical orthopedic applications involving single use treatments that guide acute cartilage repair processes.

Pseudo-thermosetting chitosan hydrogels for biomedical application.

To prepare transparent chitosan/beta-glycerophosphate (betaGP) pseudo-thermosetting hydrogels, the deacetylation degree (DD) of chitosan has been modified by reacetylation with acetic anhydride. Two methods (I and II) of reacetylation have been compared and have shown that the use of previously filtered chitosan, dilution of acetic anhydride and reduction of temperature in method II improves efficiency and reproducibility. Chitosans with DD ranging from 35.0 to 83.2% have been prepared according to method II under homogeneous and non-homogeneous reacetylation conditions and the turbidity of chitosan/betaGP hydrogels containing homogeneously or non-homogeneously reacetylated chitosan has been investigated. Turbidity is shown to be modulated by the DD of chitosan and by the homogeneity of the medium during reacetylation, which influences the distribution mode of the chitosan monomers. The preparation of transparent chitosan/betaGP hydrogels requires a homogeneously reacetylated chitosan with a DD between 35 and 50%.

Pseudo-thermosetting chitosan hydrogels for biomedical application.

To prepare transparent chitosan/beta-glycerophosphate (betaGP) pseudo-thermosetting hydrogels, the deacetylation degree (DD) of chitosan has been modified by reacetylation with acetic anhydride. Two methods (I and II) of reacetylation have been compared and have shown that the use of previously filtered chitosan, dilution of acetic anhydride and reduction of temperature in method II improves efficiency and reproducibility. Chitosans with DD ranging from 35.0 to 83.2% have been prepared according to method II under homogeneous and non-homogeneous reacetylation conditions and the turbidity of chitosan/betaGP hydrogels containing homogeneously or non-homogeneously reacetylated chitosan has been investigated. Turbidity is shown to be modulated by the DD of chitosan and by the homogeneity of the medium during reacetylation, which influences the distribution mode of the chitosan monomers. The preparation of transparent chitosan/betaGP hydrogels requires a homogeneously reacetylated chitosan with a DD between 35 and 50%.

Effects of steam sterilization on thermogelling chitosan-based gels.

A new thermogelling chitosan-glycerophosphate system has been recently proposed for biomedical applications such as drug and cell delivery. The objectives of this work were to characterize the effect of steam sterilization on the in vitro and in vivo end performances of the gel and to develop a filtration-based method to assess its sterility. Autoclaving 2% (w/v) chitosan solutions for as short as 10 min resulted in a 30% decrease in molecular weight, 3-5-fold decrease in dynamic viscosity, and substantial loss of mechanical properties of the resulting gel. However, sterilization did not impair the ability of the system to form a gel at 37 degrees C. The antimicrobial activity of chitosan against several microorganisms was evaluated after inoculation of chitosan solutions and removal of the cells by filtration. It was found that, although chitosan was bacteriostatic against the heat sterilization bioindicator Bacillus stearothermophilus, the bacteria could rapidly grow after separation from the chitosan solution by filtration. This indicated that B. stearothermophilus is an adequate strain to validate a heat sterilization method on chitosan preparations, and accordingly this strain was used to assess the sterility of chitosan solution following a 10 min autoclaving time.

Novel injectable neutral solutions of chitosan form biodegradable gels in situ.

A novel approach to provide, thermally sensitive neutral solutions based on chitosan/polyol salt combinations is described. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells and therapeutic proteins; they form monolithic gels at body temperature. When injected in vivo the liquid formulations turn into gel implants in situ. This system was used successfully to deliver biologically active growth factors in vivo as well as an encapsulating matrix for living chondrocytes for tissue engineering applications. This study reports for the first time the use of polymer/polyol salt aqueous solutions as gelling systems, suggesting the discovery of a prototype for a new family of thermosetting gels highly compatible with biological compounds.

Characterization of thermosensitive chitosan gels for the sustained delivery of drugs.

The aim of this study was to investigate the physical properties of a chitosan/glycerophosphate (GP) thermosensitive solution which gels at 37 degrees C and evaluate the in vitro release profiles of different model compounds. The gelation rate was dependent on the temperature and on the chitosan deacetylation degree. The solution containing 84%-deacetylated chitosan could be stored 3 months at 4 degrees C without apparent change in viscosity. The in vitro release profiles of the model compounds depended on the presence of GP in the chitosan solution, on their molecular weight and on the presence of lysozyme in the release media. They were not affected by the electrostatic charge of the model compound when present at low concentrations. During the first 4 h, the release was accompanied by a substantial loss of the gel weight which was mainly attributed to the leaching of water and excess GP. Scanning electron micrographs revealed that the solutions yield gels with a highly porous structure after 24 h of exposure to a continuous flow of phosphate buffered saline. These results indicate that the chitosan/GP thermosensitive solutions gel rapidly at body temperature, can remain in the sol state at 4 degrees C and can sustain the delivery of macromolecules.