(567c) Controlling Stem Cell Differentiation with Stiffening Hydrogels

Stem cells respond to many microenvironmental cues towards their decisions to spread, migrate, and differentiate and these cues can be incorporated into materials to control stem cells for regenerative medicine.  As an example, matrix mechanics alone has been shown to regulate stem cell morphology, spreading, migration, proliferation and differentiation.  Although many hydrogel systems have been explored with tunable mechanics by controlling the degree of crosslinking (e.g., polyacrylamide gels), these systems primarily exhibit static mechanical properties.  However, the native tissue environment is often dynamic and these dynamic changes in matrix mechanics play critical roles in many biological events such as during tissue development and regeneration, wound healing, fibrosis and tumor progression.  In this work, we investigated the effect of in situ matrix stiffening on human mesenchymal stem cell (hMSC) behavior in vitro on methacrylated hyaluronic acid (MEHA) substrates.  MEHA (modified with RGD ligands) was designed to allow sequential crosslinking via Michael-type addition using a dithiol crosslinker (initial gel formation) and then radical photopolymerization (temporal stiffening) via UV exposure, where the secondary step could be performed with adhered cells present. Hydrogel mechanics (from ~3kPa to ~100kPa) were tuned precisely by controlling the initial methacrylate consumption during addition crosslinking, and following UV exposure time.  In particular, we investigated the hMSC response to substrate stiffening from soft (~3kPa) to stiff (~30kPa) in terms of spreading, traction forces (via dynamic traction force microscopy), migration, proliferation and differentiation (using a mixed adipogenic/osteogenic bipotential media).  We found that hMSCs respond to stiffening by increases in cell area, traction forces, migration and proliferation, particularly on a time scale of hours. We also observed that hMSCs differentiate based on how long they are on a substrate of a specific mechanics – e.g., more adipogenic the longer the cell is on the softer substrate and more osteogenic the longer the cell is on the stiffer substrate.  After 14 days of culture ~98% of the cells stained positive for adipogenic markers on soft substrates and ~98% stained positive for osteogenic markers on stiff substrates.  When the soft substrate was stiffened at day 7, ~45% of the cells were adipogenic and the remaining ~55% were osteogenic when investigated at day 14.