202 Chapter 8 lack the chemical crosslinks and the cellular components present in intact and native ECM. This is most likely why the stress relaxation and Young’s modulus of the hydrogels differs from intact tissue. Introducing the crosslinks back into the hydrogel as well as tissue specific cells who will remodel their local environment over time can possibly be the factor to bridge the gap that still exists between hydrogel models and native tissue. Stress relaxation has been found to be an important mechanism that can regulate cellular fate and behaviour [36]. Ruthenium crosslinked LdECM hydrogels had a lower and more complex stress relaxation than uncrosslinked LdECM hydrogels, similar to that of IPF human lung compared to normal human lung [6]. To date, attributing individual Maxwell elements to specific components of a hydrogel such as water, small molecules, cells, or type of crosslinks formed in the ECM remains difficult in absence of a dedicated systematic study [37]. However, the fourth Maxwell element (with a relaxation time constant of ~100 s) for Ru-LdECM hydrogels required to describe their relaxation profile can be attributed to the secondary ECM network formed through the ruthenium crosslinking since this is the only difference between the two tested hydrogels. A similar difference in stress relaxation was found between control and fibrotic human lung ECM-derived hydrogels, showing three Maxwell elements in control hydrogels and 4 Maxwell elements in fibrotic hydrogels [6]. While the differences in species might complicate comparisons between porcine and human lung-derived hydrogels with respect to mechanical properties, the presence of a fourth Maxwell element in a Ru-LdECM hydrogels suggests that these hydrogels were able to resemble the stress relaxation behaviour of fibrotic lung ECM-derived hydrogels. Ruthenium crosslinking of ECM hydrogels led to a more dense ECM network reflective of tissue changes in fibrotic diseases. Crosslinking the ECM fibres together in LdECM hydrogels resulted in lower fibre lengths and more dense matrix packed areas. In fibrotic lung diseases like IPF, topography and organization of the ECM are altered, as recently summarized elsewhere [38]. Through post-translational modifications and crosslinking of the collagen network by the lysyl oxidase (LO) family of enzymes, lung ECM in IPF has been reported to be more mature and organized, compared to non-IPF lung ECM [4, 39]. The denser matrix with a high degree of crosslinking is a key feature of fibrotic lung disease and protects the ECM from proteolysis [40]. The overall organization of the ECM in IPF is decreased when compared to normal lungs, a characteristic which was also present in the Ru-LdECM hydrogels as seen in the lower alignment [41]. Similar values in normalized numbers of endpoints and branchpoints suggest that fibre integrity was not affected during the crosslinking
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