Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel’s initial elastic modulus, cell-adhesion-ligand density and degradation. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.

The effectiveness of stem-cell therapies has been hampered by cell death and limited control over fate1. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype2–4. Stem cell behavior can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials5–7, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem-cell behaviors in situ.

The rate of stress relaxation of adhesion substrates potently regulates cell fate and function in vitro, and here we tested whether it could regulate bone formation in vivo by implanting alginate gels with differing rates of stress-relaxation carrying human mesenchymal stem cells into rat calvarial defects. After three months, the rats that received fast-relaxing hydrogels (t1/2 ~ 50s) showed significantly more new bone growth than those that received slow-relaxing, stiffness-matched hydrogels. Strikingly, substantial bone regeneration resulted from rapidly relaxing hydrogels even in the absence of transplanted cells. Histological analysis revealed that the new bone formed with rapidly relaxing hydrogels was mature and accompanied by extensive matrix remodeling and hydrogel disappearance. This tissue invasion was found to be prominent after just two weeks and the ability of stress relaxation to modulate cell invasion was confirmed with in vitro analysis. These results suggest that substrate stress relaxation can mediate scaffold remodeling and thus tissue formation, giving tissue engineers a new parameter for optimizing bone regeneration.

Wound contraction is an ancient survival mechanism of vertebrates that results from tensile forces supporting wound closure. So far, tissue tension was attributed to cellular forces produced by tissue‐resident (myo‐)fibroblasts alone. However, difficulties in explaining pathological deviations from a successful healing path motivate the exploration of additional modulatory factors. Here, it is shown in a biomaterial‐based in vitro wound healing model that the storage of tensile forces in the extracellular matrix has a significant, so‐far neglected contribution to macroscopic tissue tension. In situ monitoring of tissue forces together with second harmonic imaging reveal that the appearance of collagen fibrils correlates with tissue contraction, indicating a mechanical contribution of tensioned collagen fibrils in the contraction process. As the re‐establishment of tissue tension is key to successful wound healing, the findings are expected to advance the understanding of tissue healing but also underlying principles of misregulation and impaired functionality in scars and tissue contractures.