Publications

Hydrogels are attractive scaffolds for regenerative medicine, yet few systems combine robust mechanical performance, antimicrobial functionality, and controlled bioactivity. Here, we engineered chitosan-based hydrogels crosslinked via strain-promoted azide-alkyne cycloaddition (SPAAC) using 4-arm PEG-DBCO and functionalized with NeoNectin, a de novo-designed protein exhibiting subnanomolar affinity and high specificity for integrin alpha 5 beta 1. Rheological analysis confirmed stable hydrogel formation with an elastic modulus of similar to 0.1 kPa, within the physiological range of bone marrow and typically associated with maintenance of mesenchymal stem cells (MSCs) in an undifferentiated state, enabling evaluation of integrin-specific biochemical cues in an inhibitory mechanical environment. Antimicrobial assays confirmed intrinsic bactericidal activity against Staphylococcus aureus and Pseudomonas aeruginosa, mitigating infection risks associated with implantation. Encapsulated human MSCs maintained high viability across all groups, validating cytocompatibility of the SPAAC crosslinking. Importantly, only covalently immobilized NeoNectin promoted sustained proliferation, increased expression of osteogenic genes, and significantly enhanced alkaline phosphatase activity, despite the low stiffness of the hydrogel matrix. These findings demonstrate that alpha 5 beta 1-specific signaling can override mechanical cues and drive osteogenic differentiation within ultra-soft environments. Overall, NeoNectin-functionalized SPAAC hydrogels provide a multifunctional platform that integrates antimicrobial properties, mechanical stability, and cellinstructive signaling for bone regeneration and broader tissue engineering applications.

JTD Keywords: Binding, Chitosan, Controlled-release, De novo protein design, Films, Hydrogel, In-vivo, Ir, Peg, Spaac reaction