Fibronectin mediates bidirectional elasticity changes in cell-laden collagen scaffolds

The extracellular matrix (ECM) provides cells with structural support and mechanical cues that regulate fundamental cellular processes. While ECM mechanics is known to influence cell behaviour, the reciprocal effect, how cells modify ECM mechanical properties, remains poorly understood, particularly in three-dimensional (3D) environments. Here, we engineered tuneable 3D collagen-fibronectin scaffolds with distinct microarchitectures (cold-cast: thick fibres, large pores; warm-cast: thin fibres, small pores) to investigate how embedded fibroblasts affect scaffold elasticity. We found that fibronectin is essential for cells to alter scaffold mechanics: in collagen-only scaffolds, cells induced structural changes but no mechanical change. Strikingly, in collagen-fibronectin scaffolds, cells softened cold-cast but stiffened warm-cast scaffolds, a bidirectional response demonstrating that cell-induced elasticity changes are critically dependent on matrix microarchitecture. Pair distribution function analysis showed that cells disrupt fibre connectivity in cold-cast scaffolds but preserve network organisation in warm-cast scaffolds, with implications for tissue engineering and disease.