Dual Functionalization of Gelatin for Orthogonal and Dynamic Hydrogel Cross-Linking

Abstract

Gelatin based hydrogels are widely used in biomedical fields owing to its abundance of bioactive motifs that support cell adhesion and matrix remodeling. While inherently bioactive, unmodified gelatin exhibits temperature-dependent rheology and solubilizes at body temperature, making it unstable for three-dimensional (3D) cell culture. Therefore, the addition of chemically reactive motifs is required to render gelatin-based hydrogels with highly controllable crosslinking kinetics and tunable mechanical properties that are critical for 3D cell culture. This article provides a series of methods toward establishing orthogonally crosslinked gelatin-based hydrogels for dynamic 3D cell culture. In particular, we prepared dually functionalized gelatin macromers amenable for sequential, orthogonal covalent crosslinking. Central to this material platform is the synthesis of norbornene-functionalized gelatin (GelNB), which forms covalently crosslinked hydrogels via orthogonal thiol-norbornene click crosslinking. Using GelNB as the starting material, we further detail the methods for synthesizing gelatin macromers susceptible to hydroxyphenylacetic acid (HPA) dimerization (i.e., GelNB-HPA) and hydrazone bonding (i.e., GelNB-CH) for on-demand matrix stiffening. Finally, we outline the protocol for synthesizing a gelatin macromer capable of adjusting hydrogel stress-relaxation via boronate ester bonding (i.e., GelNB-BA). The combinations of these orthogonal chemistries affords a wide range of gelatin based hydrogels as biomimetic matrices in tissue engineering and regenerative medicine applications.