Extracellular Vesicle Crosslinkers Constructing Hydrogels with Stress-Relaxation and Bioactive Protein Modification

Abstract

Extracellular vesicle (EV)-incorporated hydrogels have emerged as promising scaffolds for tissue repair due to their ability to present biological cues. However, the encapsulation efficiency and distribution of EVs within hydrogels still require improvement to enhance tissue healing outcomes. In this study, a novel approach is developed that uses EVs as crosslinkers for hydrogel formation, ensuring that EVs are present at every crosslinking point and thereby achieving both functional incorporation and uniform distribution of EVs. Amphiphilic molecules with various functional groups are successfully inserted into the EV membrane, enabling crosslinking with hydrogel macromers, which is versatile for multiple crosslinking chemistries. EV-crosslinked hydrogels exhibited faster stress relaxation properties due to EV stretchability compared to hydrogels crosslinked with traditional elastic polymers, promoting enhanced cell spreading and proliferation. Additionally, it is demonstrated that EV crosslinkers could present proteins throughout the hydrogel network while maintaining their biological activity. Using VEGF-loaded EV crosslinkers, induced endothelial cell clustering and sprouting are successfully, indicating early angiogenic responses. These results underscore the potential of EV-crosslinked hydrogels for tissue engineering and regenerative medicine, offering tunable mechanical properties and the capacity for effective protein delivery.