Engineering collagen hydrogels in reduced gravity through kinetic and crowding control.

Collagen self-assembly underlies extracellular matrix architecture, yet how gravitational forces influence collagen fibrillogenesis remains poorly understood. Here, we investigated simulated microgravity as a physical regulator of type I collagen hydrogel formation using a random positioning machine. Rat-tail and bovine type I collagen were assembled under graded simulated gravity conditions and characterized by real-time turbidity monitoring, bright-field and confocal microscopy, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and fibroblast culture. Simulated microgravity delayed collagen nucleation and prolonged overall assembly while promoting the formation of thicker, more spatially heterogeneous fibril networks in both collagen sources. Despite these architectural changes, post-exposure Fourier transform infrared and circular dichroism analyses indicated preservation of the native triple-helical secondary structure, indicating that gravitational unloading primarily perturbs higher-order assembly rather than molecular integrity. Macromolecular crowding with Ficoll 400 partially restored network uniformity and reduced microgravity-induced fibril thickening, while attenuating architecture-associated fibroblast contractile activation. Together, these findings establish a mechanistic link between gravitational unloading, fibrillogenesis kinetics, and collagen network architecture. This work identifies gravity as a tunable physical parameter for engineering collagen hydrogels and highlights microgravity-enabled control of extracellular matrix organization for biomaterials design and space-based biofabrication.