227 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment Fibrotic microenvironment triggered differential regulation of fiber curvature by fibroblasts We then moved on to characterize changes in the curvature of the collagen fibers in empty and fibroblast-seeded ECM-derived hydrogels as another parameter for comparing responses of control and IPF fibroblasts to their microenvironment. The curvature of fibers was analyzed with respect to low and high curvature windows (Figure 4A). Low curvature windows capture the individual waviness (periodicity) of the fibers (more micro-scale) while higher curvature windows detect the global changes in the fiber shapes (changes in the peak height of the curves). These parameters are useful for describing the topographical arrangement of the fibers within the ECM hydrogel. Intrinsic differences between IPF and control hydrogels in the low curvature window were present in day 7 samples (p = 0.004), while they were not apparent for the high curvature windows (Supplementary Tables 29&30, respectively, also shown as the dotted lines in Figure 4B). Control fibroblasts seeded in IPF hydrogels increased the fiber periodicity in low curvature windows (p = 0.001, Figure 4B) while they did not change the curvature heights in the higher curvature windows. IPF fibroblasts did not induce any periodicity changes (as measured in the low curvature windows); however, these fibroblasts reduced the peak heights of the fiber curves (as measured in the high curvature windows) in IPF hydrogels beyond the existing differences between IPF and empty hydrogels (p = 0.049, Figure 4B). Both low and high curvature window responses of control and IPF fibroblasts to fibrotic hydrogels differed from each other (p = 3.2 x 10-4 for low and p = 0.006 for high curvature windows). Empty hydrogels analyzed on day 14 revealed intrinsic differences between IPF and control hydrogels in low (p = 1.15 x 10-11), and high (p = 0.026) curvature window samples (Supplementary Tables 31&32, respectively, also shown as dotted line in Figure 4C). The existing differences between the IPF and control hydrogels were decreased by both control (p = 3.57 x 10-4) and IPF (3.01 x 10-10) fibroblasts in low curvature window. While both groups of fibroblasts decreased the fiber curvature periodicity in IPF hydrogels compared to control hydrogels, their responses significantly differed from each other as well (p = 7.74 x 10-4). On the other hand, only IPF fibroblasts responded to fibrotic hydrogels in measurements in the high curvature window (p = 0.002). Even though only IPF fibroblasts altered the fiber curvature height, there were no apparent differences in the responses of IPF and control fibroblasts in this setting. These data indicate that IPF fibroblasts altered the topographical arrangement of the collagen fibers within their 3D microenvironment to a greater extent than the control fibroblasts. 9
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