| The design and implementation of extracellular matrix (ECM)-mimetic hydrogels for tissue engineering (TE) applications requires an intensive understanding of cell-material interactions, including matrix remodeling and stem cell differentiation. However, the influence of microenvironmental cues, e.g., matrix biodegradability, on cell behavior in vitro has not been well studied in the case of direct cell encapsulation within 3-dimensional (3D) hydrogels. To address these issues, a facile sequential crosslinking technique was developed that provides spatial and temporal control of 3D hydrogel degradability to investigate the importance of material design on cell behavior. Specifically, hydrogels were synthesized from hyaluronic acid (HA) macromers in a sequential process: (1) a primary Michael-type addition crosslinking using cell adhesive and matrix metalloprotease (MMP)-degradable oligopeptides to consume a portion of total reactive groups and resulting in "-UV" hydrogels permissive to cell-mediated degradation, followed by (2) a secondary, light initiated free-radical crosslinking to consume remaining reactive groups and "switch" the network to a non-degradable structure ("+UV") via the addition of non-degradable kinetic chains.;Using this approach, we demonstrated control of encapsulated hMSC spreading by varying the crosslink type (i.e., the relative hydrogel biodegradability), including with spatial control. Upon incubation with bipotential soluble differentiation factors, these same degradation-mediated spreading cues resulted in an hMSC differentiation fate switch within -UV versus +UV environments. Follow-up studies demonstrated that degradation-mediated traction generation, rather than matrix mechanics or cell morphology, is the critical biophysical signal determining hMSC fate. Sequentially crosslinked HA hydrogels were also studied for the capacity to support remodeling by in vivo and ex vivo tissues, including with spatial control, toward tissue engineering (e.g., neovascularization) applications.;In total, the work presented here highlights sequential crosslinking as a versatile platform technology affording processing capabilities to better mimic dynamic features of native microenvironments, including spatial patterning and temporal alteration of hydrogel degradability, toward both basic studies of cell behavior and TE applications. |
