Figure 2. Cytocompatible, biochemical patterning within preformed click hydrogels.

(a) The thiol-ene reaction mechanism provides a means to quantitatively couple sulfhydryls (-SH) with vinyl functionalities (-C=C) in the presence of light. (b) Upon swelling into the material, relevant thiol-containing biomolecules are covalently affixed to the hydrogel network at varying concentrations by altering the dosage of exposed light (intensity and exposure time). (c) A Live/Dead stain at 24 hours after photolithographic patterning of 3T3s indicates a predominantly viable population (live cells are shown in green, while dead cells are red) and that the patterning process is cytocompatible. (d) The thiol-ene reaction is confined to user-defined regions in space using photomasks to introduce three different fluorescently-labeled peptide sequences within the gel, a process that can be repeated at desired times and spatial locations to introduce additional biochemical cues. (e) By controlling the focal point of the laser light in three-dimensions using a confocal microscope, micron-scale spatial patterning resolution is achieved. Values in (b) are reported as mean ± SD (n=5). The image in (c) represents a 200 µm confocal projection. The images in (d) and (e) represent confocal micrographs of fluorescently-tagged peptides patterned within the networks. Scale bar = 100 µm for (c), 100 µm for (d), and 50 µm for (e).