| Epithelial cells reside on specialized extracellular matrices that provide instructive cues to regulate and support cell function. We have previously demonstrated that substrate topography with dimensions similar to the native extracellular matrix (submicrometer and nano-scale features) significantly impacts corneal epithelial proliferation and migration. In this work, we included additional instructive cues by incorporating specific peptide ligands into topographically nano-patterned synthetic hydrogels. Peptides RGD and AG73 were conjugated to monodisperse poly(ethylene glycol) spacers that separated the peptide from the monomeric functionality which reacts during hydrogel polymerization. These PEG-peptide conjugates were then copolymerized with PEG diacrylate to form an inert hydrogel network decorated with peptide ligands for cell interactions. The efficient, systematic study of multiple instructive cues (peptide, peptide concentration, topographic dimensions), however, was also contingent on the development of enhanced throughput platforms. Towards this goal, we developed a hydrogel array platform to systematically and rapidly evaluate combinations of the two different peptide motifs and a range of nano-scale topographic dimensions. Elastomeric stencils with arrays of millimeter-scale regions were used to spatially confine hydrogel precursor solutions on elastomeric stamps with nano-scale patterns generated by soft lithography. The resulting topographically and peptide-functionalized hydrogel arrays were used to characterize single cell behavior, including morphology, proliferation, and migration. We showed that epithelial cell migration speed and persistence was governed by both the biochemical and topographical cues of the underlying substrate. We also developed a wound healing assay for these hydrogel arrays to study collective epithelial cell migration. Similar to single-cell studies, collective cell migration and wound healing rates were faster on topographic surfaces with the addition of AG73. The strategy and synthetic techniques developed here for well-defined hydrogel arrays functionalized with both peptides and topographic features provides unparalleled control for investigating synergistic interactions of both physical and chemical cues. |
