232 Chapter 9 Collagen amount and organization are known to be drastically altered in IPF; higher amounts of collagen with an increased disorganization of the fiber structure have been consistently documented [8, 11, 13]. In our hydrogel system, fibroblasts did not induce changes in collagen amount between empty and fibroblast-seeded hydrogels, suggesting that these fibroblasts did not deposit detectable new collagen in this model and timeframe. A recent study reported an increase in protein levels of collagen types VII, X and XIV, detected using mass spectrometry, by control fibroblasts cultured in spheroid form with presence of IPF lung ECM, compared to non-IPF ECM [20]. These data provide further evidence, supporting prior reports [11, 14, 21, 22] that the IPF ECM provides a pro-fibrotic signal for fibroblasts. While our data appear to contrast the previous studies, the use of mass spectrometry may provide additional sensitivity that would enable a more penetrating investigation of the collagen changes. In our model system, the fibroblast-induced differences in high density matrix and collagen fiber alignment as directed by the type of environment (hydrogel) in which the cells were grown, implies that the lack of detectable changes in total collagen amount does not necessarily reflect a lack in pro-fibrotic responses by fibroblasts. Increased fiber density was previously proposed as a mechanism for triggering activation of fibroblasts [15]. Therefore, in a fibrotic microenvironment, with higher amounts of dense fibers [23, 24], greater fibroblast responses would be expected. Interestingly, control fibroblasts appeared to have opposite responses at different time points, with an initial increase in high density matrix after 7 days but a subsequent decrease after 14 days. These differences in time might reflect how naïve fibroblasts are imprinted by a fibrotic microenvironment as time passes so their responses to fibrotic environment are changed. In IPF fibroblasts, the decrease in high density matrix with a concomitant increase in fiber alignment, points at exaggerated responses of these fibroblasts, when confronted with fibrotic ECM. These specific responses of IPF fibroblasts suggest that the origin of both microenvironment and fibroblasts play a crucial role in determining the organizational changes of collagen fibers. Enhanced collagen crosslinking is recognized to be enhanced in IPF lung tissues [9, 25]. Our results showing decreased amounts of high density matrix in fibrotic ECM modulated by both types of fibroblasts in day 14 samples do not initially seem to be in concert with previous reports showing enhanced fibroblast activation by increased ECM crosslinking in IPF [25]. Further research is required to examine if the high density matrix in this hydrogel model is composed of crosslinked collagen fibers or if it is non-covalent aggregations of fibers. The latter may have different cellular signaling implications than the highly cross-linked ECM in IPF tissue, parallel to the previous reports showing different levels of myofibroblast activation in chemically crosslinked
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