Fig. 6.

Collagen fiber assembly on PAAm substrates. (a) Representative fluorescence images are shown for OG-Col immobilized on 5.8 kPa PAAm gels with various 2PCA-acrylamide content. Scale bars = 100 μm. The reaction was analyzed by fluorescence intensity of OG-Col immobilized on substrates with or without 10 mM piperazine-2PCA (S5) (b) and 40 mM benzylalkoxyamine (benzyl-ONH₂) treatment (c). The fluorescence intensity of 0% 2PCA-PAAm without excess 2PCA or benzylalkoxyamine treatment (F₀) was subtracted from the fluorescence intensity (F) of the individual samples. Bars represent mean ± SEM; *, p < 0.05 with respect to PAAm of corresponding 2PCA content; N ≥ 6 gels per condition. (d) Representative SEM (d) and DIC (e) images are shown for 50 μg/mL unlabeled collagen immobilized on 5.8 kPa 0.01% 2PCA-PAAm gels or sulfo-SANPAH activated PAAm gels. SEM scale bars = 0.5 μm. DIC scale bars = 20 μm. DIC inset scale bars = 4 μm. (f) Movie stills showing U2OS RFP-LifeAct behavior on OG-Col immobilized PAAm substrates. 50 μg/mL collagen was conjugated to 5.8 kPa 0.0001% 2PCA-PAAm or sulfo-SANPAH activated PAAm before U2OS cell seeding (t = 0). PAAm with low 2PCA content is shown to highlight interactions between the cells and collagen fibers. Fluorescence time-lapse imaging of LifeAct-tag RFP (red) and OG-Col (green) started 7 h after cell seeding. Fluorescence contrast of sulfo-SANPAH activated PAAm substrate is enhanced to show the lack of fiber formation. Time stamp is in hours:minutes. Scale bars = 20 μm.