Chitosan-glycerol-phosphate (GP) gels release freely diffusible GP and possess titratable fixed charge

Chitosan-glycerol-phosphate (GP) solutions undergo a sol-gel transition upon heating. A model of heat-induced transfer of protons from chitosan to GP in solution has been proposed to explain this sol-gel transition. This model suggests that reduced inter-chain electrostatic repulsion by a decreased protonation of positively charged amino groups - via proton transfer to GP(2-) thereby reduced to GP(-) - would allow chitosan polymer chains to precipitate together and form a solid hydrogel structure. The hypothesis that GP has the single role of acting as a proton acceptor and not as a divalent electrostatic cross-linker of chitosan amine groups suggests that it should freely diffuse out of the gel after the gel formation. We found indeed that GP is free to diffuse and the experimental diffusion profile corresponds to a free diffusion model indicating that it plays no role in electrostatic cross-linking. Finally since chitosan amine groups in the gel are not bound to GP, we examined whether they are titratable in the gel. We show that chitosan in the hydrogel indeed possesses titratable amine groups with significant fixed charge up to +80 mM and follows the same ionization behavior as chitosan in solution. The results of these studies are significant in light of the current and future biomedical applications of this system, including its recent clinical approval as a biomaterial for cartilage repair.