(616f) Stiffening Hydrogels to Probe Stem Cell Response to Dynamic Mechanics

Stem cells respond to microenvironmental cues towards their decisions to spread, migrate, and differentiate, and these cues can be incorporated into materials for regenerative medicine applications.  In the last decade, matrix mechanics alone has been implicated in regulating cell functions including spreading, migration, proliferation and differentiation.  With this in mind, a variety of natural and synthetic hydrogels have been used in vitro to mimic the elasticity of native tissues.  Despite helping to develop this important field and gather valuable information, these substrates primarily exhibit static mechanics and lack the dynamic nature of many cellular processes such as development, fibrosis and cancer.  Thus, an area of interest in vitro is temporal manipulation of matrix elasticity to better understand and develop strategies to control these biological processes.  In this work, we developed a sequential crosslinking process, i.e. initial gelation via addition reaction followed by secondary crosslinking through light-initiated radical polymerization, to fabricate hyalunoric acid hydrogel substrates that stiffen immediately when desired and in the presence of cells.  We demonstrated the utility of this model system by investigating the short-term (hours) and long-term (days to weeks) human mesenchymal stem cell (hMSC) response to dynamic stiffening from 3 to 30 kPa.  Short-term studies revealed a significant increase in both mean cell area (from 400 to 1500 um²) and average traction (from 1 to 10 kPa) within 4 hours immediately after substrate stiffening.  Long-term studies in a biopotential differentiation media, which supports adipogenic (soft, 3kPa) and osteogenic (stiff, 30kPa) differentiation, showed that hMSCs differentiated based on stiffening time point, i.e. how long they were culture on a substrate of a specific stiffness. Specifically, adipogenesis was favored when stiffening was performed later times, whereas osteogenesis was favored stiffening at early times.