Gelatin microspheres reinforced by o-nitrobenzyl–vinyl coupling for 3D cell culture and high-fidelity extrusion printing

Microfluidics enables the fabrication of highly uniform gelatin methacryloyl (GelMA) hydrogel microspheres with broad potential in biomedical applications. However, these GelMA microspheres rely on a single-network architecture generated by free-radical polymerization that is intrinsically defect-rich and structurally heterogeneous, resulting in poor mechanical robustness under shear and compression and thereby limiting practical use. Existing reinforcement strategies require non-homologous additives or multi-step crosslinking schemes that compromise biocompatibility or disrupt microfluidic processing. Here, we address this limitation by reinforcing GelMA microspheres at the microstructural level through partial substitution with o-nitrobenzyl–modified gelatin (GelNB). The underlying NB–vinyl coupling reaction strengthens the hydrogel network without altering the native gelatin formulation, introducing auxiliary reagents, or interfering with droplet-based microfluidic fabrication. The resulting GelNB microspheres exhibit a 3.13-fold increase in compressive strength compared with GelMA counterparts and withstand shear deformation of up to 50% without structural damage. Importantly, they retain inherent cell-adhesive motifs, cytocompatibility, and degradability. They function as degradable microcarriers for 3D cell culture and enable high-fidelity extrusion printing of complex architectures. This strategy broadens the utility of gelatin microspheres for biofabrication, regenerative engineering, and therapeutic delivery.