234 Chapter 9 control and IPF) hydrogels were variable between experimental runs. Although we generated a combined batch of ECM derived from 7 different donors to minimize this variation, sample-to-sample variation was still present in our results. Empty hydrogels harvested at each of the assessment time points also reflected this variation. However, we accounted for this variation during our analyses and only compared empty hydrogels to fibroblast-seeded hydrogels within the same time point and the same experimental run. Even with this comparison, it is not possible to rule out the fact that spontaneous fiber reorganization still continues during the course of 14 day cell culture in empty and fibroblast-seeded hydrogels. Another potential limitation of the study is related to collagen detection: as the starting weights of control and IPF lung ECM powders were the same, and as the majority of the remaining proteins within the dECMs were collagens, detecting small changes that might have been induced by the fibroblasts may not have been possible with the methodologies used in this study. While investigating changes in different collagens individually and also other ECM proteins was outside of the scope of this project, it is not possible to rule out that specific collagens may have been altered by the fibroblasts more than the others, or that other ECM proteins including proteoglycans may have been involved. Lastly, viscoelastic stress relaxation of the control and IPF lung ECM-derived hydrogels is difficult, if at all possible, to resemble native tissue (a recognized limitation of this model [12, 32]), as opposed to the stiffness values that do recapitulate the patterns seen in lung tissues. The model described in this study also provides opportunities for further research. Although our current model is based on fibroblast-ECM interactions, introducing other cell types such as epithelial cells, circulating immune cells or other mesenchymal cells would help mimicking the complex interplay between these cells and ECM during IPF or other fibrotic lung diseases. Moreover, this model system can greatly advance investigating cell responses following treatment with Nintedanib or Pirfenidone, which were initially performed on cell-derived matrices [33]. While our study focused on IPF, it has important implications for understanding the interplay between cells and their fibrotic environment in many other fibrotic diseases and even cancer, reflecting the remodeled ECM associated with tumors. Understanding how activation of fibroblasts occurs in a fibrotic microenvironment has the potential to reveal additional intervention possibilities for the treatment of diseases involving fibrotic responses. Future studies utilizing this model could investigate if the fibroblast responses differ in the presence of antifibrotic treatments that are currently approved for pulmonary fibrosis.
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