203 An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition and existing branches were crosslinked. The shorter fibres in crosslinked hydrogels might be explained with the increased curvature in these samples: the effect of crosslinking on curvature of fibres with respect to the fibre length was prominent in shorter fibres (<40 µm) while longer fibres did not have differences among the two groups. These observations indicate that the ruthenium-crosslinking mainly influences the shorter fibres and decreases the average fibre length. Together with the mechanical characterization data, these results show that the mechanical properties were altered through the changes in the alignment and density of the matrix (HDM) in the crosslinked hydrogels. One of the most important components of the fibrotic microenvironment is the (myo) fibroblasts and their responses to the altered ECM. In our model, the cells remained viable and the fibroblasts seeded on crosslinked hydrogels lost their spindle-like morphology that we observed on the native LdECM hydrogels. This observation is in parallel with previously reported studies showing the effect of stiffness on human lung fibroblasts [42]. As a response to the altered mechanical properties in Ru-LdECM hydrogels, the fibroblasts have displayed more myofibroblast-like characteristics.. Increased α-SMA expression and altered organization have been previously reported in lung fibroblasts as a response to increasing stiffness of their environment [42, 43]. Parallel to these previous studies, IPF fibroblasts have been reported to have higher levels of α-SMA expression both at gene [44] and protein [45] levels, compared with non-disease control lung-derived fibroblasts. In addition, another study reported that myofibroblast differentiation was not triggered by an isolated increase in the fibre density of synthetic 3D culture while keeping the stiffness of the environment constant [46]. This observation is also in concert with our results as both stiffness and the fibre organization are altered in Ru-LdECM hydrogels. The accompanying changes in the nuclear morphology was also in agreement with the influence of increased stiffness [47]. As reviewed by Wang et al., nuclear mechanotransduction as a response to a stiffer microenvironment (such as in fibrosis) has yet to be completely understood; however, such a change can result in altered gene regulation or nuclear transportation of cytoplasmic factors [48]. Another implication of the altered nuclear morphology due to increased stiffness was shown to influence the differentiation of mesenchymal stromal cells [49]. These demonstrated changes on the seeded fibroblasts in our study suggest that the biomechanical properties of the fibrotic microenvironment were replicated in our model. Our study utilizes a crosslinking strategy on native ECM-hydrogels using a ruthenium complex and sodium persulfate, as described recently by Kim et al. [25]. While this innovative approach has its advantages, our study has also some limitations. First of 8
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