Subtle Regulation of Scaffold Stiffness for the Optimized Control of Cell Behavior

The rigidity of extracellular matrices can impact cell fate, guide tissue development, and initiate tumor formation. Scaffolds such as hydrogels with tunable levels of stiffness have been developed to control cell adhesion, migration, and differentiation, providing suitable microenvironments for different tissue outcomes. However, studies of cell-material interactions are largely confined to biomaterials with stiffness values that are coarsely regulated, so refinements in sensitive cellular responses and optimal stiffness values that determine cell fate remain elusive. Here, a freezing temperature, as a tunable regulating factor, was introduced to freeze-drying processes to form silk fibroin (SF) scaffolds with refined control of stiffness values. Due to this control of intermediate structural conformations of SF, the scaffolds exhibited differences in stiffness values to permit refined assessments of impact on cell behavior on cell-friendly surfaces. Both <i>in vitro</i> and <i>in vivo</i> results with these scaffolds exhibited gradually changeable cell migration and differentiation outcomes, as well as differences in tissue ingrowth, demonstrating the sensitivity of cellular responses to such refined mechanical cues. The optimal vascularization capacity of these SF scaffolds was in the 3-7.4 kPa range, suggesting a key range to develop bioactive biomaterials. Systematic fine regulation of scaffold rigidity based on the present strategy provides a platform for an improved understanding of cell-material interactions and also for generating optimized microenvironments for tissue regeneration.