293 General discussion and future perspectives domains [23]. Using individual ECM components such as collagen type I or hyaluronic acid as ECM substitutes in in vitro models have advanced our understanding of the influence dictated by the choice of material in such models (as outlined in Chapter 6). More recently, generating hydrogels from the decellularized lung ECM itself has been described and has paved the way for a multitude of opportunities for mimicking in vivo complexity of ECM in in vitro conditions (as reviewed in Chapter 6 and discussed in Chapter 7). Lung ECM-derived hydrogels provide both most of the diversity of ECM biochemical composition as seen in vivo and some of the mechanical properties of the native lung tissue [24, 25]. While these properties are important, modifying them in a controlled and cell-friendly manner was not previously explored. In Chapter 8, I addressed this unmet need in the field and applied UV-visible light triggered crosslinking of fibers through Ruthenium crosslinking in porcine lung ECM-derived hydrogels. By doing so, I successfully kept the biochemical composition intact while altering mechanical properties to be able to study the influence of altered mechanics in chronic lung diseases such as IPF. I verified the successful application of fiber crosslinking in our model by showing fibrotic mechanical properties such as stiffness and stress relaxation as well as altered fiber characteristics, which include fiber curvature, fiber alignment and percentage area covered by dense fibers. As a result of such changes in the fiber organization in the hydrogels, fibroblasts seeded on crosslinked lung ECM-derived hydrogels responded in a manner similar to the responses of pro-fibrotic fibroblasts isolated from IPF lungs, demonstrated through increased alpha-smooth muscle actin (αSMA) expression and increased nuclear area. These characterizations are in concert with previously published studies examining the role of altered stiffness in fibroblast responses [26-29]. While my initial setup was two-dimensional for proof-of-principle purposes, applying the same methodology in three-dimensional cultures with the presence of cells can also be performed while retaining cell viability [30], although responses of different cells types (such as lung epithelial or endothelial cells) have yet to be characterized. The applied fiber crosslinking in my study was through linking tyrosine amino acids, [31] while the natural fiber crosslinking in collagens in ECM takes place through lysyl oxidases and transglutaminases acting on lysine and glutamine amino acids [32]. Involvement of both of these enzymes for fiber crosslinking in the context of IPF has been previously shown. The comparison of how the enzymatic crosslinking differs from the Ruthenium-induced crosslinking, and how their potential differences influence cellular responses remains unexplored. In addition to the enzymatic crosslinking for the arrangement of fibers, the collagen organization is also regulated through other ECM components such as proteoglycans and FACITs. Inclusion of collagen type XIV that I described in Chapter 3 in the development of improved models of fibrotic mechanics generated in Chapter 8 would certainly be worthy of investigation. 10
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