221 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment RESULTS Collagen presence in control and IPF hydrogels is similar and does not change upon seeding with either control or IPF fibroblasts We first assessed interactions between a fibrotic microenvironment and fibroblasts on collagen presence in the lung ECM-derived hydrogels by analyzing the collagen content at day 7 and 14. PicroSirius Red (PSR)-stained sections of empty or fibroblastseeded control and IPF hydrogels were analyzed using an automated image analysis that compared three different levels of staining strength: weak, moderate and strong staining (Figure 1A). In all three categories, we did not detect intrinsic differences in the percentage of PSR-stained area between control and IPF hydrogels without fibroblasts (Supplementary Tables 1-6, also shown as the dotted line in Figure 1B-G for each separate analysis). When fibroblasts were seeded in these hydrogels, we found no differences in percentage area positive for collagen content induced by either control or IPF fibroblasts at day 7 or 14 (Figure 1B-G). Similarly, the mean intensities of staining within the weak, moderate and strong PSR-staining was comparable in all groups (Supplementary Figure 1, Supplementary Tables 7-12). We also examined the distribution of (GAG) in all groups by staining adjacent sections with Alcian Blue. Comparable to collagen content, pecentage area of GAG content was similar in control and IPF empty hydrogels, and this did not change in hydrogels seeded with control or IPF fibroblasts (Supplementary Figure 2, Supplementary Tables 13-18). 9
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