The mechanical properties of the extracellular matrix (ECM), including stiffness and stress relaxation, play an instructive role in regulating cellular behaviors. Given the inherent heterogeneity of tissue viscoelasticity in vivo, the precise influence of viscoelastic variations on cellular behaviors remains incompletely understood. To study these mechanobiological responses in vitro, gradient hydrogels have been developed to mimic the mechanical heterogeneity of native ECM. However, current fabrication strategies, including photomask polymerization and diffusion-based methods, face several limitations, such as complex operation, narrow material applicability, and compromised biocompatibility due to harsh reaction conditions. To address this challenge, we developed a programmable syringe pump system (PSPS) comprising three syringe units to fabricate a gradient viscoelastic hydrogel (GoHG) at a 600 μm scale. The hydrogel was synthesized through dynamic imine crosslinking between oxidized hyaluronic acid (oxi-HA) and gelatin, with precise spatial control over viscoelastic properties achieved by independently modulating the flow rates of individual injection units. This strategy enabled precise control over hydrogel composition, resulting in continuous stiffness gradients (19~47 kPa) and stress relaxation gradients (6~62 s) while maintaining excellent cytocompatibility (>95%). The results on the effect of viscoelastic gradient on cell spreading behavior indicate that different cell types exhibited distinct responses to the viscoelastic gradient. Fibroblasts (NIH-3T3) displayed enhanced spreading behavior in regions with higher stiffness (28.5 kPa) and slower stress relaxation (34.7 s), whereas myoblasts (C2C12) showed larger spreading area in areas with lower stiffness (23.6 kPa) and faster stress relaxation (30.0 s), highlighting cellular specificity to mechanical cues. These findings underscore the importance of viscoelasticity as a critical regulator of cell behavior. In conclusion, the PSPS-based fabrication method offers significant advantages, including simplicity, scalability, and broad material compatibility, making it a versatile platform for mechanobiology research. Furthermore, this approach advancing our understanding of cell-ECM mechanical interactions and shows great potential for tissue engineering applications, where mimicking native ECM mechanics is essential for guiding cell function and tissue regeneration.

Key words: programmable syringe pump system, viscoelasticity, gradient hydrogel, cell spreading