Precision 3D Bioprinting of Cell-Proliferative Hydrogel Scaffolds via Oxygen-Inhibition-Free Dual-Pulse Femtosecond Laser Cross-Linking

In recent years, femtosecond laser-based three-dimensional (3D) printing has provided a high-precision solution for fabricating cross-scale biomimetic cell scaffolds. However, the collapse and thermal damage inherent to single-pulse laser processing severely limit the efficiency of hydrogel photo-cross-linking and the structural stability, resulting in fabricated scaffolds that struggle to effectively support cell growth. This study proposes a dual-pulse sequence femtosecond laser regulation strategy that optimizes photo-cross-linking kinetics through staged energy input, aiming to achieve high-performance fabrication of 3D cellular scaffolds for tissue engineering. The results demonstrate that single-pulse laser processing leads to structural collapse at heights exceeding 60 μm due to oxygen inhibition and bubble interference, while the dual-pulse sequence (up to approximately 34% reduction in total energy) significantly suppresses oxygen inhibition by locally depleting oxygen and generating free radicals in a stepwise manner. This approach enables high-resolution printing of 3D scaffolds with heights of 60 μm, markedly improving structural integrity and stability. Mathematical modeling further reveals the synergistic mechanism between the dynamic evolution of oxygen concentration and free radical kinetics under dual-pulse regulation. Biological validation demonstrates that the extract from dual-pulse fabricated scaffolds exhibits no cytotoxicity. Furthermore, comparative experiments with direct cell seeding on scaffolds show that dual-pulse scaffolds maintain their morphology significantly better than single-pulse scaffolds in the cell culture environment, and the cells on them exhibit stronger fluorescence signals, confirming that dual-pulse scaffolds provide a more favorable microenvironment for cell growth. This study provides theoretical support and technical innovation for the cross-scale fabrication of complex biomimetic hydrogel structures.