223 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment Fibroblasts change collagen organization only in IPF hydrogels We then investigated whether fibroblasts induced changes in collagen organization and whether this was different in fibrotic versus control hydrogels using TWOMBLI. This analysis revealed the changes triggered in fibrotic ECM (Figure 2). The first parameter we studied was the percentage area occupied by high density matrix, which describes the collagen fiber organization at a global level (Figure 2A). Intrinsic differences between control and IPF hydrogels were not detected on day 7, but were clearly visible on day 14 with IPF hydrogels having more high density matrix than control hydrogels (Supplementary Tables 19&20, also denoted as the dotted lines in Figure 2B-C). When we seeded fibroblasts in these hydrogels, we found that control fibroblasts further decreased the percentage of high density matrix in IPF hydrogels compared to control hydrogels below the already existing differences, both on day 7 and day 14 (Figure 2B and 2C, respectively). On the other hand, IPF fibroblasts decreased the percentage of high density matrix of IPF hydrogels only on day 14 (Figure 2C). The differential modulation of the hydrogels by control and IPF fibroblasts was significantly different (p = 5.12 x 10-6 for day 7 and p = 0.027 for day 14), indicating that not only hydrogel type but also the fibroblast origin contributes to dysregulated collagen organization in IPF. We then characterized the degree of fiber alignment within the hydrogels (Figure 2D). We did not detect any intrinsic differences between control and IPF hydrogels with respect to fiber alignment (Supplementary Tables 21&22). When control fibroblasts were seeded in each type of hydrogel, they did not change the percentage fiber alignment in IPF hydrogels compared to control hydrogels at any time point (Figure 2E and 2F). However, when seeding IPF fibroblasts, we found fiber alignment was greater in IPF hydrogels compared to control hydrogels on both day 7 and day 14 (Figure 2E and 2F, p = 4.00 x 10-3 for day 7 and p = 2.57 x 10-6 for day 14). The differential modulation of the fiber alignment by control and IPF fibroblasts was significantly different on both days (p = 5.00 x 10-3 for day 7, p = 7.64 x 10-4 for day 14). Fibroblasts modify individual collagen fibers differently in fibrotic compared to control ECM In addition to global fiber organization, individual fiber structure could also be altered by the seeded fibroblasts. Individual fiber structure was therefore assessed using TWOMBLI. Three individual fiber structural parameters were analyzed: average fiber length (AFL, μm), number of endpoints per 1000 μm fiber total length, and number of branchpoints per 1000 μm fiber total length (Figure 3). 9
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