229 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment Stiffness increased but stress relaxation remained comparable when fibroblasts were in fibrotic hydrogels Lastly, we characterized mechanical properties of both empty and fibroblast-seeded control and IPF hydrogels after 7 or 14 days of culture. Stiffness (Figure 5A and 5B) and viscoelastic stress relaxation (Figure 5E and 5F) behavior of hydrogels were measured using a Low-Load Compression Tester (LLCT). Empty IPF hydrogels were stiffer than empty control hydrogels on both day 7 (p = 5.57 x 10-6) and day 14 (p = 4.93 x 10-22) (Supplementary Tables 33&34, also shown as the dotted line in Figure 5C and 5D for day 7 and 14, respectively), reflecting previous reports [12]. When control fibroblasts were seeded in IPF hydrogels, they stiffened the hydrogels significantly more than they stiffened control hydrogels on day 7 (Figure 5C, p = 0.002). Similar to control fibroblasts, IPF fibroblasts also increased stiffness of IPF hydrogels more than they stiffened control hydrogels (Figure 5D, p = 0.002). The responses of control and IPF fibroblasts in IPF hydrogels compared to control hydrogels were comparable. However, on day 14 only control fibroblasts increased the stiffness of IPF hydrogels compared to control hydrogels beyond the already existing stiffness differences (Figure 5D, p = 9.22 x 10-5). Modulation of IPF hydrogels compared to control hydrogels by control fibroblasts on day 14 was significantly different from how IPF fibroblasts modulated the two types of hydrogels (p = 0.027). Viscoelastic stress relaxation was the other mechanical parameter analyzed after 7 and 14 days of culture of control and IPF fibroblasts in control and IPF hydrogels. The time to reach 50% stress relaxation was measured in seconds and compared between the groups (Figure 5E). IPF hydrogels relaxed significantly slower than control hydrogels both on day 7 (p = 1.47 x 10-12) and day 14 (p = 5.82 x 10-14) (Supplementary Tables 35&36, also shown as the dotted line in Figure 5G and 5H for day 7 and 14, respectively). Seeding of either control or IPF fibroblasts into the hydrogels did not significantly change stress relaxation beyond the intrinsic differences that were already present on day 7 (Figure 5G). On day 14, however, only control fibroblasts caused a slower relaxation of IPF hydrogels compared to control hydrogels, in addition to the existing differences (Figure 5H, p = 0.033). IPF fibroblasts did not change the relaxation properties of the hydrogels beyond the existing differences between control and IPF. 9
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