Multi-step functionalization of hydrogels through mechano- and photo-responsive linkages.

Patterning soft materials with cell adhesion motifs can be used to emulate the structures found in natural tissues. While patterning in tissue is driven by cellular assembly, patterning soft materials in the laboratory most often involves light-mediated chemical reactions to spatially control the presentation of cell binding sites. Here we present hydrogels that are formed with two responsive crosslinkers-an anthracene-maleimide adduct and a disulfide linkage-thereby allowing simultaneous or sequential patterning using force and UV light. Hydrogels were formed using poly(ethylene glycol)-based crosslinkers, yielding homogeneous single networks where the mechanical properties can be controlled with crosslinker content. Compression with a PDMS stamp inked with a cysteine-terminated peptide leads to (1) force-mediated retro-Diels Alder revealing a pendant maleimide and (2) subsequent Michael-type addition of the peptide. Successful functionalization was verified through monitoring anthracene fluorescence and via cell adhesion to the immobilized peptides. The material was further functionalized using UV light to open the disulfide bond in the presence of a maleimide-terminated peptide, thereby allowing a second immobilization step. Sequential derivatization was demonstrated by adding a second cell type, yielding patterns of multiple cell populations. In this way, force and light serve as complementary triggers to create geometrically structured heterotypic cell cultures for next-generation bioassays and materials for tissue engineering.