Journal: Biomaterials Science

Article Title: Design of thiol–ene photoclick hydrogels using facile techniques for cell culture applications † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4bm00187g Click here for additional data file.

doi: 10.1039/c4bm00187g

Figure Lengend Snippet: Biochemical cues spatially patterned within hydrogels. (A) To create patterns of biochemical cues, hydrogels are polymerized off-stoichiometry ([SH] > [alloc]) and incubated with excess pendant RGDS or AF488RGDS peptide. Gels were irradiated through photomasks printed with black lines or squares for 1 and 5 minutes (left). Samples are subsequently analyzed with fluorescent light or Ellman's reagent to determine the modification of free thiols with pendant biochemical cues (right). (B) Gels (10 wt% with respect to PEG) were polymerized with 2.2 mM LAP for 1 and 5 minutes off stoichiometry (2 mM free thiol at preparation). After equilibrium swelling, the initial free thiol concentration in these gels were 1.13 ± 0.09 and 0.97 ± 0.10 mM, respectively, as determined by Ellman's assay. Only 0.01 ± 0.01 and 0.003 ± 0.003 mM free thiol remained after adding the RGDS tether indicating the efficient coupling of the model biochemical cue to the hydrogel network. (C) Following the setup shown in (A), arbitrary patterns (squares, 1600 μm 2 ; lines of different thickness, 200–1000 μm) of a fluorescent peptide (AF488RGDS) were created within pre-formed hydrogels and imaged on a confocal microscope for analysis. Resolution of the pattern is observed in the x -, y -, and z -planes indicating selective coupling to only regions of the gel that were exposed to light (scale bar, 200 μm). (D) As a quick and inexpensive alternative to fluorescence, a non-fluorescent pendant peptide (RGDS) was photopatterned (lines of different thickness) into pre-formed hydrogels. Ellman's reagent was directly applied to the top of these gels to identify regions lacking the pendant peptide (yellow) with resolution in the x - and y -planes over short times (<5 min) (scale bar, 1 mm).

Article Snippet: Viability was quantified at 3 days post-encapsulation with a LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Invitrogen), and gels were imaged with a confocal microscope (Zeiss 510 NLO).

Techniques: Incubation, Irradiation, Modification, Concentration Assay, Microscopy, Fluorescence