225 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment The analysis of average fiber length illustrated that, intrinsically, IPF hydrogels had shorter fibers on average, compared to control hydrogels (Supplementary Tables 23&24, also shown as the dotted line in Figure 3A and 3D for day 7 and 14, respectively). When control fibroblasts were seeded in IPF hydrogels, the average fiber length was further decreased in IPF hydrogels on day 7 (Figure 3A, p = 7.44 x 10-5), while this modulation was not detected on day 14 (Figure 3D). IPF fibroblasts, on the other hand, increased the average fiber length in IPF hydrogels compared to control hydrogels only on day 14 (Figure 3D, p = 1.26 x 10-4). These modulations by control and IPF fibroblasts were significantly different from each other both on day 7 (p = 6.30 x 10-5) and day 14 (p = 2.00 x 10-5). The number of individual fibers (number of endpoints) was higher in IPF hydrogels compared with control hydrogels on day 7, but this difference was not detected on day 14 (Supplementary Tables 25&26, also shown as the dotted line in Figure 3B and 3E for day 7 and 14, respectively). When control fibroblasts were seeded into these hydrogels, we found more endpoints in IPF hydrogels compared to control hydrogels on day 7, above the existing intrinsic difference between these two hydrogels (Figure 3B). This modulation, however, was not observed on day 14 (Figure 3E). In contrast, IPF fibroblasts did not change the number of endpoints in IPF hydrogels compared to control hydrogels on either day 7 or day 14. These differential responses between control and IPF fibroblasts, differed significantly on both day 7 (p = 0.005) and day 14 (p = 5.56 x 10-4) (Figure 3B and 3E, respectively). We also investigated the number of branchpoints as a measure of the number of fibers that had connections with other fibers. IPF hydrogels had more branchpoints than control hydrogels on day 7 (p = 0.007) while this intrinsic difference was not detectable on day 14 (Supplementary Tables 27&28, also shown as the dotted line in Figure 3C and 3F for day 7 and 14, respectively). When either control or IPF fibroblasts were seeded in these hydrogels, no additional changes in the number of branchpoints induced by fibroblasts were detected in either control or IPF hydrogels on day 7 (Figure 3C). However, on day 14 both control (p = 5.57 x 10-6) and IPF fibroblasts (p = 2.13 x 10-5) strongly decreased the number of branchpoints in IPF hydrogels but not in control hydrogels (Figure 3F). 9
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