Enzyme-mediated fast injectable hydrogels based on chitosan–glycolic acid/tyrosine: preparation, characterization, and chondrocyte culture

In this study, water-soluble chitosan–glycolic acid (GA)/tyrosine (Tyr) conjugates (denoted as CH–GA/Tyr) are designed and prepared for fast, in situ formation of hydrogels by tyrosinase or horseradish peroxidase (HRP)-mediated crosslinking under physiological conditions. These CH–GA/Tyr conjugates with a phenol group and primary amine in tyrosine residue are readily obtained by conjugating GA and N-tert-butoxycarbonyl (Boc)–tyrosine to native chitosan and then deprotection of the Boc group. The CH–GA20/Tyr14 with a degree of substitution (DS, defined as the number of substituted NH2 groups per 100 glucopyranose rings of chitosan) of GA of 20 and the DS of Tyr of 14 displays higher water solubility with a maximum pH value of 14.0 when compared to native chitosan, CH–GA20 and phloretic acid-coupled CH–GA20. Both tyrosinase and HRP can effectively crosslink CH–GA/Tyr conjugates to form hydrogels. HRP can mediate a faster gelation process than tyrosinase. The gelation time of the CH–GA/Tyr hydrogels may be adjusted from seconds to minutes by modulating the concentration of the enzymes/conjugates and DS of Tyr residue. Rheological analysis results show that these hydrogels are elastic and that the HRP-crosslinked hydrogels have higher storage modulus as compared to the tyrosinase-crosslinked hydrogels. The tyrosinase-crosslinked hydrogels have lower cytotoxicity as compared to the HRP-crosslinked hydrogels when NIH/3T3 cells are encapsulated in these hydrogels. Further, the tyrosinase-based hydrogels can maintain high survival of chondrocytes over 14 days. The results of this study show that the tyrosinase-crosslinked chitosan-based hydrogels have high potential for cartilage tissue engineering.