Gradient in Motion: Mechanical Stimulation Of Cells Cultured on Melt Electrowritten Scaffolds

Tissue engineering often relies on scaffolds to support cell growth and proliferation. To accurately mimic damaged tissues, complex, supportive structures are required. One promising approach is Melt Electrowriting (MEW), a high-precision 3D printing technique that enables the fabrication of intricate scaffolds with exceptional control over structure and mechanics. In this study, we designed and printed gradient scaffolds comprising three distinct zones-square, rhombus, and radial-seamlessly connected with unprecedented precision achieving relatively small pore sizes, relevant for cells bridging. Mechanical testing revealed that Young's modulus of the gradient scaffold corresponds to the average modulus of the three individual zones. Under applied force. Human dermal fibroblasts cultured on the gradient scaffolds exhibited zone-specific alignment, influenced by both scaffold design and mechanical forces. Under dynamic stimulation (7% cyclic tensile strain for 10 days, following 14 days of initial static culture), cell alignment was preserved, whereas in continuous static culture, alignment was lost after 24 days. The unique deformation responses of each scaffold zone to tensile strain played a key role in directing cell organization. This study demonstrates that a single applied force on the MEW-fabricated, precisely designed and printed gradient scaffold in xy-plane can induce distinct, zone-specific cellular responses. This occurs because the scaffold's region-dependent deformation modes generate localized mechanical cues, leading to varied cell behaviors across the zones. Our findings open new avenues for advanced tissue engineering applications, particularly interesting for musculoskeletal repair, where scaffold design and mechanical stimulation can be locally tailored to enhance tissue regeneration. STATEMENT OF SIGNIFICANCE: This study introduces a gradient scaffold fabricated using Melt Electrowriting (MEW), a precise 3D printing method. The scaffold features three smoothly connected zones (square, rhombus, and radial) with distinct structures and mechanical properties. For the first time, we show how these gradient scaffolds guide human cell alignment and response under mechanical strain, mimicking how tissues behave in the body. We show that under a single applied load, different mechanical cues emerge for each zone, guiding localized cell behaviour. Importantly, only when dynamic mechanical forces were applied, cells maintained alignment, highlighting the importance of mechanical stimulation in tissue design. These findings have broad implications for improving scaffold design in tissue regeneration, particularly in load-bearing tissues like muscle or enthesis.