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.