204 Chapter 8 all, assessing the amounts of excess (unreacted) ruthenium and sodium persulfate remaining in the hydrogels was not possible with our current methodology; however, our future research is looking into further optimizing the amount of ruthenium and sodium persulfate in the reaction. In this study, we have seeded the fibroblasts on top of the hydrogels (2D) instead of seeding them within the hydrogel network. Although a 3D environment would represent the physiological situation in the body, a 2D culture system was preferred in this study to ensure proper visualization of the cell viability and morphology. Our study reports a model for examining the influence of biomechanical changes of the fibrotic microenvironment without investigating any gene and protein output from the fibroblasts . Although the influence of a fibrotic biomechanical microenvironment on fibroblasts have been shown to promote a pro-fibrotic phenotype both in gene and protein levels (as reviewed in [5, 50]), such investigations are beyond the scope of this study. Lastly, the power of Maxwell modelling of the stress relaxation profiles of the native and crosslinked ECM hydrogels has not been completely realized and seems to remain as a mathematical exercise. The reason is that unlike other research areas e.g. microbial biofilms where relaxation constants has been linked to the composition [37], for hydrogels this systematic study is not yet available. With that, such modelling still proves useful in terms of analysing the altered stress relaxation behaviour. CONCLUSION This study demonstrates the mechanical characterization of an in vitro ECMbased fibrosis model for advancement of investigations on effects of a fibrotic microenvironment on the cells. The next step for this model is to investigate how changes in the stiffness or viscoelastic relaxation can instruct the cells for further profibrotic responses, especially in a 3-dimensional setting. In addition, fibre characteristics analysis revealed that the changes in the fibre organization (alignment, density, curvature) accompany the altered pattern in the viscoelastic stress relaxation behaviour. More research on the influence of these altered fibre characteristics on the profibrotic activation of different cells (fibroblasts, macrophages…) have yet to be explored. Overall, this study shows the preparation and the characterization of an in vitro fibrosis model. Such advanced in vitro models for fibrosis research will improve our understanding on de-coupling the mechanical changes from the biochemical changes taking place in fibrosis.
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