Injectable gelatin-based hydrogel with tuneable mechanical properties and osteogenic potential

Hydrogels based on chemically modified gelatin offer a promising platform for bone-regenerative applications due to their biocompatibility, biodegradability, and capacity for controlled network formation. In this study, dual-functionalised gelatin-methacryloyl-norbornene (gel-MA-NB) derivatives were developed to enable orthogonal chain-growth and step-growth crosslinking, allowing independent modulation of hydrogel mechanics and architecture. Two formulations with distinct degrees of substitution were developed using three crosslinking techniques (single, simultaneous vs. two-step dual-crosslinking with thiolated poly(ethylene glycol) as crosslinker). Subsequently, their mechanical properties, injectability, and biological performance were assessed. Rheological characterisation demonstrated that storage modulus strongly depended on methacrylamide and norbornene functionalisation with dual-crosslinked hydrogels exhibiting an increased stiffness compared to single crosslinked variants. Injectability testing showed differences between formulations and crosslinking techniques, with single crosslinked hydrogels requiring the lowest injection force. Biological evaluation using mesenchymal stromal cells showed high cytocompatibility across all conditions. Early and late osteogenic differentiation potential were affected by both formulation and crosslinking technique: single crosslinked hydrogels with the highest amount of -MA groups, characterized by lower stiffness, supported the most pronounced alkaline phosphatase activity and calcium deposition over time. Furthermore, we showed that normoxic culture conditions supported metabolic activity and osteogenic differentiation more than hypoxia. Together, these findings highlight the capacity of gel-MA-NB hydrogels to serve as a tuneable and injectable biomaterial platform for bone regeneration. Careful selection of the degree of substitution and crosslinking technique enables precise control over mechanical performance, injectability, and osteogenic differentiation potential, underscoring the translational relevance of this biomaterial for bone-regenerative applications.