201 An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition observed in fibrotic diseases. Ruthenium crosslinking relies on the crosslinking of the tyrosine amino acids and the potential for employing this strategy on the ECMderived hydrogels has recently been demonstrated by Kim et al. [25]. The ruthenium crosslinking of the ECM in our model allows us to recreate the (patho)physiological mechanical environment in a fibrotic lung. This method can most likely be adapted to generate tissue specific fibrotic environments that are representative of many organ microenvironments. Figure 8: Comparison of the fibroblasts seeded on LdECM and Ru-LdECM hydrogels. α-SMA and DAPI-stained fluorescent images of the fibroblasts were analysed using the ImageJ and CellProfiler software to compare the α-SMA expression per nuclei, the nuclear area and circularity. A) Quantification of the α-SMA expression per cell nuclei imaged in the fluorescent images. B) Comparison of nuclear area of fibroblasts seeded on LdECM and Ru-LdECM. C) Nuclear circularity of the fibroblasts seeded on LdECM and Ru-LdECM hydrogels. For panel A, each data point represents the quantification of the images generated from different randomized regions (n=5 per hydrogel) from different hydrogels (n=5 total). Applied statistical test: Mann-Whitney test. For panels B and C, each data point represents measurement on an individual nucleus for the respective characteristic, in total from 5 different randomized regions on the fluorescent images of DAPI staining for each sample (n=5). Applied statistical test: Mann-Whitney test. α-SMA: alpha-smooth muscle actin, LdECM: Lung-derived ECM Hydrogels, Ru-LdECM: Ruthenium-crosslinked Lung-derived ECM Hydrogels Different rheology measuring techniques will yield slightly different results and have specific benefits. A study by Polio et al. used cavitation rheology, micro-indentation, tensile testing and small amplitude oscillatory shear rheometry on porcine lung and found that each technique resulted in a different Young’s modulus [35]. A previous study shows the difference in viscoelastic properties of human lung ECM hydrogels and intact (patho)physiological counterparts where the hydrogels had a lower stiffness and higher total relaxation [6]. The first Maxwell element was the main contributor to the stress relaxation in hydrogels whereas with lung tissue this was more equally divided amongst the elements. ECM hydrogels are reconstituted solutions of decellularized and enzyme-digested ECM proteins and therefore they 8
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