199 An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition Ruthenium crosslinking of ECM hydrogel promotes differentiation of fibroblasts to myofibroblasts Fibroblasts seeded on Ru-LdECM hydrogels had higher expression of α-SMA when compared to LdECM hydrogel-seeded fibroblasts (Figure 7). In addition to the stronger expression of α-SMA, the organization of the cytoskeleton was altered in the fibroblasts seeded on the Ru-LdECM hydrogels (Figure 7B, lower row). When these images were quantified using ImageJ, fibroblasts seeded on Ru-LdECM hydrogels had significantly higher α-SMA expression per nuclei (p < 0.0001) compared with the fibroblasts seeded on LdECM hydrogels (Figure 8A). These myofibroblast-like characteristics were also accompanied by a change in the nuclear morphology. At day 7, the nuclei in the fibroblasts on Ru-LdECM hydrogels had an altered morphology as illustrated by the higher area (p < 0.0001, Mann-Whitney test) with an increased circularity (p < 0.0001, Mann-Whitney test) compared with the fibroblasts seeded on LdECM hydrogels (Figure 8, B and C). DISCUSSION In this study we describe a model that enables modulation of the mechanical properties of an ECM without changing the composition. Using this model, we illustrated that by modulating the crosslinks between ECM fibres, the stiffness and stress relaxation properties of the ECM-derived hydrogels were altered. The crosslinking influenced the ECM fibre characteristics with a higher percentage of high density matrix and lower percentage of alignment being evident within the hydrogels treated with ruthenium. Fibroblasts grown on the surface of the crosslinked hydrogels displayed more myofibroblast-like characteristics. The features of this model illustrate that it would provide an innovative research tool for investigating the importance of biomechanical changes in fibrotic diseases. The increase in stiffness caused by ruthenium crosslinking in the LdECM hydrogels is similar to the increase in stiffness seen in fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) [15]. Booth et al. measured the stiffness of whole non-IPF and IPF human lungs before and after decellularization and found that the fibrotic regions of the IPF lung often reached a stiffness of 100 kPa or more, a vast increase compared to that of normal lung which has an average stiffness of 1.96 kPa [15]. The increased stiffness of IPF human lung was still present in hydrogels when compared to hydrogels generated from control human lungs, albeit proportionally reduced when compared to the intact lung tissue [6]. Recreating the (patho)physiological stiffness 8
RkJQdWJsaXNoZXIy MTk4NDMw