Biological evaluation of chitosan salts cross-linked to genipin as a cell scaffold for disk tissue engineering

Degenerative disc disease has been implicated as a major component of spine pathology. However, although biological repair of the degenerate disk would be the ideal treatment, there is no universally accepted scaffold for tissue engineering of the intervertebral disk. To help remedy this, we investigated the gelation kinetics of various concentrations (2.5 to 10%) of two water-soluble chitosan chlorides (low molecular weight Protasan UP CL113 and high molecular weight Protasan UP CL213) and two chitosan glutamates (low molecular weight Protasan UP G113 and high molecular weight Protasan UP G213). Various concentrations (5 to 20%) of genipin, a naturally occurring cross-linking reagent used in herbal medicine and in the fabrication of food dyes, were used to prepare cross-linked chitosan hydrogels. The results show that 2.5% Protasan UP G213 cross-linked to 5% genipin was the best candidate. This formulation gelled fastest at 37 degrees C, and maintained 95% viability of encapsulated cultured disk cells. The gel did not produce an inflammatory reaction when injected subcutaneously into C57BL/6 mice and is therefore biocompatible. Most importantly, when injected into the degenerated nucleus pulposus of human cadaveric intervertebral disk, the gel flowed into the clefts without leakage. This study demonstrates that 2.5% Protasan UP G213 cross-linked to 5% genipin might be a promising scaffold for disk tissue engineering.