Micro-mechanical viscoelastic properties of crosslinked hydrogels using the nano-epsilon dot method

Engineering materials that recapitulate pathophysiological mechanical properties of native tissues in vitro is of interest for the development of biomimetic organ models. To date, the majority of studies have focused on designing hydrogels for cell cultures which mimic native tissue stiffness or quasi-static elastic moduli through a variety of crosslinking strategies, while their viscoelastic (time-dependent) behavior has been largely ignored. To provide a more complete description of the biomechanical environment felt by cells, we focused on characterizing the micro-mechanical viscoelastic properties of crosslinked hydrogels at typical cell length scales. In particular, gelatin hydrogels crosslinked with different glutaraldehyde (GTA) concentrations were analyzed via nano-indentation tests using the nano-epsilon dot method. The experimental data were fitted to a Maxwell Standard Linear Solid model, showing that increasing GTA concentration results in increased instantaneous and equilibrium elastic moduli and in a higher characteristic relaxation time. Therefore, not only do gelatin hydrogels become stiffer with increasing crosslinker concentration (as reported in the literature), but there is also a concomitant change in their viscoelastic behavior towards a more elastic one. As the degree of crosslinking alters both the elastic and viscous behavior of hydrogels, caution should be taken when attributing cell response merely to substrate stiffness, as the two effects cannot be decoupled.