Development of collagenous filaments with tuneable mechanical properties using a 3D bioprinter and molecular crowding

Engineering collagen constructs that replicate the mechanical strength and biological functionality of load-bearing tissues like tendon remains a significant challenge. This study establishes a scalable and accessible biofabrication method that com-bines the precision of a commercial 3D bioprinter with the physicochemical principles of molecular crowding. Using an acidic collagen solution in combination with a hypertonic polyethylene glycol bath, we systematically investigate how modulating pulling speed and nozzle diameter dictates the final filament properties. Our results demonstrate that faster pulling speeds and smaller nozzle diameters produce thinner, more densely packed filaments. High-resolution scanning electron microscopy and polarised light microscopy reveal a tuneable transition in surface morphology, from a disordered isotropic state to a highly ordered phase, quantitatively confirmed by a sharp in-crease in birefringence. Critically, we demonstrate the preservation of collagen's native, periodic 66 nm d-banding, an essential motif for cell interaction. This structural refinement translates to a dramatic enhancement of mechanical performance, with the strongest filaments achieving an ultimate tensile strength of 0.64 GPa and a Young's modulus of 8.9 GPa, values that surpass native tendon. Further-more, the fabricated filaments are highly biocompatible and bio-instructive, promoting robust alignment and elongation of mesenchymal stem cells. This work provides a direct link between bioprocess parameters and the resulting hierarchical structure, offering a versatile platform for fabricating high-performance, biomimetic materials for regenerative medicine.