196 Chapter 8 Increased density and decreased alignment of fibres in lung ECM hydrogels after ruthenium crosslinking Ru-LdECM hydrogels had a denser fibre network compared to LdECM hydrogels (Figure 5A). The average fibre length was shorter in Ru-LdECM than LdECM hydrogels (p = 0.0026, paired t-test). While the normalized numbers of endpoints and branchpoints did not differ between LdECM and Ru-LdECM hydrogels, the percentage of area with high density matrix (HDM) was greater in Ru-LdECM hydrogels compared with LdECM hydrogels (p = 0.0146, paired t-test). Alignment of the fibres in RuLdECM hydrogels was lower than LdECM hydrogels (p = 0.0048, paired t-test) (Figure 5B-G). The differences in the curvature of the fibres with different length were compared in LdECM and Ru-LdECM hydrogels. Curvature of the fibres with shorter length (< 40 µm) were higher in the crosslinked hydrogels, suggesting that shorter fibres were more bent in Ru-LdECM hydrogels while curvature of the longer fibres was not different between these two groups (Table 1). Ruthenium crosslinking does not affect fibroblast viability but induces altered morphology After 1 day of culture, no dead cells were observed and the fibroblasts were viable on both types of hydrogels (Figure 6). The viability of the fibroblasts did not change over a 7-day culture period (Supplementary Figure 4). On both gels the fibroblasts appeared to be lying flat on the surface of the hydrogels; however, the fibroblasts on LdECM hydrogels display a more spindle-shaped morphology, while fibroblasts on RuLdECM gels are were more hypertrophic and display more protrusions. Suggesting a more migratory phenotype for fibroblasts on Ru-LdECM hydrogels. At day 7, a fully confluent monolayer was present on both control and crosslinked hydrogels with no differences in viability.
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