Fig. 3: 3D collagen scaffold system recapitulates observations seen in the porcine tissue.

a Freshly isolated fibroblasts were seeded into 3D collagen scaffolds and either restrained (no strain, NS, gray) or subjected to strain (S, blue) or strain + 10 μM FAKI (S + FAKI, red). b, c We used titanium oxide dots (inner 9 circles) to track and quantify the exact imposed strains on the collagen scaffolds, shown by gross photography (top row) and a schematic (bottom row). Scaffolds were pinned at the arms to enforce the strain (outer circles; 2 circles per arm) (****p = 0.0001). Scale bar: 1 cm. d Alpha smooth muscle actin (αSMA) myofibroblast protein expression in fibroblasts cultured in all three conditions was quantified by immunofluorescence staining. (NS: n = 4 independent collagen scaffolds; S and S + FAKI: n = 5 independent collagen scaffolds per group; *p < 0.0462). DAPI = blue, αSMA = red. Scale bar: 140 µm. e Alignment of fibroblasts within 3D stretch culture system that underwent uniaxial restraint (no strain), 10% uniaxial strain only, or 10% uniaxial strain + FAKI was quantified using a previously published algorithm to analyze fibroblasts immunostained for phalloidin (green, n = 5; ****p = 0.0001, ***p = 0.0005)²⁶. Scale bar: 140 µm. f Contraction in vitro assay using collagen scaffolds to quantify remodeling of the ECM environment (****p = 0.0001). Scale bar: 1 cm. g Pharmacological unloading (FAKI treatment) and mechanical unloading create similar decreases in YAP expression. Control (grey) & FAKI (purple) loaded (n = 5 independent collagen scaffolds; *p = 0.0258); control (light gray) & FAKI (light purple) unloaded (n = 2 independent collagen scaffolds). Scale bar: 140 µm. Statistical comparisons were made by using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests (c–g). Each datapoint represents an independent collagen scaffold. All data represent mean ± SEM. Representative images are shown from similar images across all experiments.