An experimental model for fibroblasts to myofibroblasts transition on mechano-mimetic polyacrylamide hydrogels

Myofibroblasts are considered the cells primarily responsible for the formation of a transient scar required to restore the integrity and preserve the function of injured tissues. They derive mainly from the differentiation of fibroblasts in the extracellular matrix (ECM), promoted by the combined action of pro-fibrogenic factors, particularly transforming growth factor (TGF)-β1 and of mechanical stimuli in the damaged microenvironment. They exhibit the features of both collagen-synthetically active fibroblasts and smooth muscle cells showing de novo expression of α-smooth muscle actin (sma) and, although they are not regarded as electrically excitable cells, peculiar biophysical properties and trans-membrane ion currents typical of smooth muscle cells. As compared to fi broblasts, diff erentiated myofibroblasts are larger, have a polygonal shape and secrete higher amounts of collagen. In physiological conditions, once the tissue regeneration has accomplished, the scar will be degraded and myofibroblasts progressively disappear. By contrast, the generation of myofibroblasts and their persistence in an activated functional state are recognized as “core cellular mechanisms” of pathological fibrosis. High-throughput in vitro models of the fi broblast-myofibroblast transition appear mandatory for a comprehensive study of myofibroblast biology and fibrosis mechanisms, for the identification of smart therapeutic targets and novel antifibrotic therapy screening. Here we generated three mechano-mimetic substrates of polyacrylamide (PA) hydrogels by free radical polymerization with an accurate tailoring of water content and crosslinking degree to modulate the mechanical properties. The bulk compression modulus ranged from 29 to 1 kPa. NIH/3T3 fibroblasts were cultured on glass coverslips (control) and on PA hydrogels for 48 h in proliferation medium or in low serum condition in absence or presence of TGF-β1 and morpho-functionally analysed for myofibroblast differentiation. Preliminary results indicate that the optimal myofibroblastic differentiation occurs when the cells are cultured on PA hydrogels with a bulk compression modulus around 1 kPa in low serum condition plus TGF-β1, as judged by the confocal immunofluorescence analysis of α-sma expression, the cell shape evaluation and by the electrophysiological analysis of the biophysical and conductive properties. Notably, cells cultured on PA hydrogels in the absence of TGF-β1 showed stiff ness-dependent differences in morpho-functional properties suggesting that mechanical stimuli especially from “tissue-soft” substrates are sufficient to induce a myofibroblastic phenotype acquisition. Experiments are ongoing to evaluate the involvement of stretch activated channels in the different cell responses and the potential cross-talk between mechano-transduction and TGF-β1 mediated pathways.