Preparation of tunable stiffness bioink

This study provides an experimental and theoretical basis for the precise design of bioink stiffness and is expected to promote the application of 3D bioprinting in complex tissue repair. We tackled key material challenges in 3D bioprinting by designing a tunable-stiffness bioink for complex tissue engineering. To address the limitation of traditional bioinks’ single mechanical properties, a three-component system (amino-Gel, sulfhydryl-CS, aldehyde-DAC) was developed, forming a Schiff base-thioacetal double-crosslinked network. Through orthogonal and 27 supplementary experiments, the effects of multi-component ratio on hydrogel stiffness were explored. ANOVA showed DAC is critical for Young’s modulus (F-value much higher than others), with concentration positively correlated to modulus (e.g., Gel = 3, CS = 1: DAC 0.2→0.6, modulus 11.11→21.86 kPa), and CS-DAC synergistic crosslinking. Rubber elasticity theory calculations confirmed that DAC linearly increases crosslinking density (0.2-0.6 DAC: 1.49→2.94 mol/m 3 , R 2 = 0.92) to regulate stiffness. This provides a basis for precise bioink stiffness design, aiding 3D bioprinting in complex tissue repair.