233 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment hydrogels compared with physically crosslinked hydrogels [26]. While it was out of the scope of this study, it is important to recognize that a role of proteoglycans in collagen crosslinking and fiber organization has been previously reported [27], and their presence might also play a role in the collagen organization measured in this model. The organization of individual collagen fibers is also an important element for determining cellular responses to the microenvironment in which they reside [11]. With respect to the individual fiber organization parameters that were analyzed in our study, control and IPF fibroblasts responded differently from each other, with the exception of the alterations to the number of branchpoints. The opposite responses elicited by the fibroblasts to fibrotic and control hydrogels highlights that not only the origin of the microenvironment but also the origin of fibroblasts plays a role in dictating the collagen organization. Fiber curvature (collagen topographical arrangement), as both an individual and global fiber parameter, was differentially regulated by control and IPF fibroblasts seeded in fibrotic hydrogels compared to control hydrogels. It is intriguing that these changes coincided with the changes in the mechanical parameters initiated by the fibroblasts. In particular, control fibroblasts had an exaggerated response to the fibrotic microenvironment, resulting in an increased stiffness compared to control hydrogels. While the decrease in high density matrix and increase in stiffness do not seem to go hand-in-hand, fibroblasts might be realigning the fibers that are dissociated from the high density matrix in a manner that leads to the increased stiffness. The lack of changes in the peak height of the collagen fiber curves in control fibroblast-seeded hydrogels could also be one of the key factors playing a role in increased stiffness in these hydrogels. Together with previous reports showing the influence of fiber curvature amplitude and wavelength on fibroblast migration and polarization [28], investigating the influence of fibrotic ECM curvature on fibroblasts might reveal new insights into how fibroblast-ECM interactions are regulated by the physical state of the matrix structure. The stress relaxation behavior of the empty and fibroblast-seeded hydrogels showed that the changes in high density matrix and stiffness do not strongly influence the stress relaxation capacity of the fibers. It would be of interest to further investigate the altered stress relaxation of the fibers with respect to activation of the fibroblasts, as previously pre-stress in ECM fibers has been shown to release stored TGF-β from the ECM [29, 30]. Regardless, a recent report indicates that a microenvironment with slow stress capacity hinders cellular migration of mesenchymal spheroids [31]. How cellular migration plays a role in the profibrotic activation of fibroblasts with respect to organization of ECM requires further studies. While our study establishes the interplay between the native microenvironment and the fibroblast responses in 3D, it has a few limitations. Human lung ECM (both 9
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