Fabrication of cross-scale biomimetic 3D hydrogel scaffolds with thermo-mechanical optimized properties via femtosecond laser dual-wavelength dual-pulse regulation

The mechanical properties and structural integrity of tissue engineering scaffolds demand high precision to replicate native extracellular matrix (ECM) cross-scale architecture. Femtosecond laser two-photon polymerization enables precise control of hydrogel scaffolds. Still, single-wavelength processing cannot simultaneously achieve high stiffness, low thermal damage, and cross-scale structures. This study proposes a dual-wavelength dual-pulse spatiotemporal synergy strategy. The results indicate that dual-wavelength processing reduces the polymerization threshold to 0.2 mW, which is 1/30 of 800 nm processing. More importantly, this strategy breaks through the single-wavelength fabrication limit, which only allows 3 layers at 400 nm or 10 layers at 800 nm, and achieves a homogeneous 20-layer structure. The scaffold exhibits an average compressive modulus of 148 kPa, higher than 27.7 kPa from single 800 nm and 88 kPa from single 400 nm. Concurrently, the scaffold surface exhibits uniform nanoscale topography of approximately 40 nm, forming cross-scale architectures spanning nanometers to millimeters. Cellular experiments show that these high-modulus scaffolds with nanotopography induce the strongest actin fluorescence in L929 cells, demonstrating that the dual-wavelength scaffolds provide a more favourable microenvironment for cell adhesion and cytoskeletal organisation. This study achieved the synergistic fabrication of mechanically reinforced cross-scale hydrogel cell scaffolds with minimal thermal damage, offering promising applications in tissue engineering.