Shape‐Morphing Photoresponsive Hydrogels Reveal Dynamic Topographical Conditioning of Fibroblasts

Abstract The extracellular environment defines a physical boundary condition with which cells interact. However, to date, cell response to geometrical environmental cues is largely studied in static settings, which fails to capture the spatiotemporally varying cues cells receive in native tissues. Here, a photoresponsive spiropyran‐based hydrogel is presented as a dynamic, cell‐compatible, and reconfigurable substrate. Local stimulation with blue light (455 nm) alters hydrogel swelling, resulting in on‐demand reversible micrometer‐scale changes in surface topography within 15 min, allowing investigation into cell response to controlled geometry actuations. At short term (1 h after actuation), fibroblasts respond to multiple rounds of recurring topographical changes by reorganizing their nucleus and focal adhesions (FA). FAs form primarily at the dynamic regions of the hydrogel; however, this propensity is abolished when the topography is reconfigured from grooves to pits, demonstrating that topographical changes dynamically condition fibroblasts. Further, this dynamic conditioning is found to be associated with long‐term (72 h) maintenance of focal adhesions and epigenetic modifications. Overall, this study offers a new approach to dissect the dynamic interplay between cells and their microenvironment and shines a new light on the cell's ability to adapt to topographical changes through FA‐based mechanotransduction.


Materials and Methods
Measuring thickness of SBS layers: Glass slides were spin-coated with SBS in toluene according to the protocol described in the main text.Random scratches in the SBS layer were made using a razor blade, and the distance between the glass slide and SBS layer were measured using optical profilometry (Sensofar Plµ 2300 with a 20×, 0.45 NA Nikon objective).Data was processed using Plu Optical Imaging Profiler 2.41 software.
Live/Dead viability assay: nhDF cells were seeded on hydrogel constructs and topography was induced as described in the main text.Prior to, or 1h after inducing topography, cells were washed with PBS and stained with CalceinAM (1.5 µM, 17783, Merck Life Science NV) and propidium iodide (3 µM, P4864, Merck Life Science NV) in PBS.Samples were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 25 min.After incubation, the staining solution was removed, and the samples were washed with PBS and stored in PBS at 37 °C to prevent the sample from drying out.Stained cells were imaged using a Leica TCS SP5 confocal microscope with a 10×, 0.4 NA objective.
Live-cell imaging.Brightfield images of fibroblasts on hydrogels were taken every 15 minutes using a CytoSMART Lux (Axion BioSystems) live-cell imaging system with a 5× objective that was placed inside a humidified incubator (37 °C, 5% CO 2 ).Table S1.Surface distances were calculated before and after masked light actuation (90 µm wide lines) using optical profilometry data.The surface distance after actuation was calculated by using the Pythagorean theorem and by using the equation that corresponds to a fitted parabola on the data.Movie 1. Live-cell imaging of fibroblasts on SBS-Sp-pNIPAM hydrogels after 1 round of actuation (1× 5h 90 µm wide grooves).

Figure S1 .
Figure S1.Stiffness of uncoated Sp-pNIPAM hydrogels, as measured with nanoindentation.Mean ± SD; each dot represents one hydrogel (n = 5 hydrogels).Each hydrogel was indented at 5 randomly selected positions and the averaged value was plotted.

Figure S2 .
Figure S2.Thickness of the SBS layer measured using optical profilometry.A) Height profile.B) Measured thickness.Mean ± SD, n = 3 different samples measured at at least 3 random positions.

Figure S3 .
Figure S3.Representative UV-vis spectrum of SBS-Sp-pNIPAM hydrogel in cell culture medium.The spiropyran isomerizes spontaneously to the protonated merocyanine after illumination with blue light (455 nm).

Figure S6 .
Figure S6.The surface contour length between two arbitrary chosen points was measured before and after actuation.

Figure S13 .
Figure S13.3D representation of SBS-Sp-pNIPAM hydrogels after 15 min illumination with a 150 µm open line mask in an humidified atmosphere (37 °C).The fluorescence signal of the pronated merocyanine was detected using confocal microscopy, and z-stacks were converted into a 3D surface plot using ImageJ.

Figure S14 .
Figure S14.Cell orientation on flat and dynamic hydrogel constructs, where 0° indicates parallel alignment and 90° indicates perpendicular alignment with the induced grooves (shaded areas).A Kolmogorov-Smirnov test showed a significant difference in the distribution of the orientation data between dynamic and flat samples.

Figure S18 .
Figure S18.Orientation of the nucleus on flat and dynamic hydrogel constructs.nhDFs were subjected to two rounds of actuation (90 µm wide grooves, 2x 5h).