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

Abstract

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.