Fibrosis is a complex chronic disease that can occur in various tissues following injury. A key initial event in the healing of injuries or the development of fibrosis is the activation of fibroblasts and their subsequent differentiation into their functional form, the myofibroblast. Fibrosis is also a common consequence of exposure to nanoparticles in polluted air. After inhalation, healthy lungs are unable to remove nanoparticles, leading to their accumulation in the alveoli. This characteristic, combined with their large surface area and high reactivity, results in chronic lung inflammation and fibroblast activation even after a single exposure to nanoparticles. These particles are highly heterogeneous, both in terms of their surface reactivity and their size and shape, which necessitates the identification of the potential to induce inflammation or fibrosis for individual nanoparticles to ensure safety. To this end, we developed a method using confocal microscopy, which allows us to monitor phenotypic changes in living fibroblasts and thereby detect differentiation into myofibroblasts as a result of exposure to various factors. We used the MLg cell line as an in vitro model of lung fibroblasts, in which we fluorescently labeled the nucleus, membranes, and actin. As a positive control for fibroblast activation, we selected transforming growth factor beta TGF-β1.
After determining the appropriate concentrations of all fluorescent probes, we identified the minimal time after exposure to TGF-β1 at which a statistically significant difference was observed between the negative and positive control, as well as determining a suitable collagen I-coated glass substrate onto which MLg cells were seeded. We monitored numerous phenotypic characteristics of the cells, particularly differences between the negative and positive control, and selected the most frequently observed variables: presence of stress fibers, visible focal adhesion complexes (FAK), cell area, binucleation, »projections« (lamellipodia), and presence of thin projections or filaments. Among all the variables in the set, the most sensitive indicators were found to be the presence of FAK and the cell area or its increase. In order to simplify the automation of the method, we decided to monitor only the differences in the cell surface. By monitoring cell surface area, we observed the appearance of a second population of cells with a larger surface area after addition of TGF-β1, thereby evaluating TGF-β1 as a suitable addition for a positive control. We then conducted a triplicate of biological repetitions of the experiment containing a negative and positive control, as well as the nanomaterials MWCNT401, TiO2NT and "Diesel" nanoparticles. The results obtained suggest that exposure of fibroblasts to nanoparticles in vitro is not sufficient to induce their differentiation into myofibroblasts.The established method of monitoring phenotypic changes as a result of exposure to nanoparticles could, by including the proposed improvements, be developed into a validated method for the prediction of fibrosis or the determination of the toxicity various nanoparticles in the lung. The proposed method would enable a faster and cheaper prediction of the impact of nanoparticles on the development of lung fibrosis, while reducing the need for test animals for these purposes.
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