Mechanical stimulation of cells : Dynamic behavior of cells on cyclical stretched substrates

Abstract

Besides the biochemical factors in the environment, physical factors can also influence biological processes in tissues or in single cells. For, example the mechanical stimulation of cells can regulate their proliferation, apoptosis or the expression of genes within them. Previous studies concerning the influence of cyclical strain on cells adhering to flexible substrates showed that the cells attempt to reorient themselves to be perpendicular to the stretch direction. This behavior has been described qualitatively, but no systematic, quantitative studies of this phenomenon have yet been undertaken. Furthermore, the cells were only observed prior to and following stretch. Studies of cellular dynamics during the cyclical stretch are lacking. In the present study, our aim was to both observe and quantitatively describe the dynamics of the cellular reaction by means of a biophysical model. We therefore developed a new stretching system which allows live-cell observations during the stretching experiments. The behavior of different cell types was investigated, according to a variety of different parameters such as stretching frequency, stretching amplitude, or cell density. As a model system, we used two types of fibroblasts: rat embryonic fibroblasts (REF52) and primary human fibroblasts (HDF) taken from donors of various ages. We observed that the perpendicular reorientation of the cells occurs at an exponential rate over time. Accordingly, we employed a simple mathematical model to determine how long it characteristically took for the cells to reorient themselves in response to the various mechanical parameters. Our results demonstrated a previously unknown characteristic biphasic cellular behavior which depended on the stretching frequency. Both REF52 and HDF fibroblasts were found to reorient faster, until a certain threshold frequency was reached. In this regime the characteristic reorientation time decreased by a power law, as the frequency increased (characteristic time ~ fn). Above this threshold frequency, the characteristic time ceased to decrease. When the cells were stretched with higher frequencies than this threshold frequency, a saturation of the characteristic time was reached. All tested cell types displayed this biphasic behavior. Cell-specific differences, however, were observed in the reaction kinetics and in the threshold frequencies. The REF52 cells already began to react at a frequency which is approximately 10 times lower compared to the HDF1 cells, in general they reoriented themselves faster than the HDF1 cells at all frequencies. Furthermore, we demonstrated that older HDF cells reoriented themselves faster than young HDF cells. When we increased the cell density to a confluent cell layer, we also observed a power law dependent decrease in the characteristic reorientation time, when the frequency increased. Compared with the single cells, however, a plateau of saturation of the characteristic reorientation time could not be observed. Furthermore, the confluent cells reacted approximately twice as fast as the single cells. Activation of cell-cell contacts involved in mechanotransduction in addition to focal contacts may constitute one possible explanation for this observation. When the stretching amplitude was varied, the characteristic reorientation time was found to decrease, along with an increase in amplitude. However, in contrast to the frequency variation, in this case we observed a linear decrease. The different reaction characteristics resulting from variations in the stretch frequency and the stretch amplitude (power law-dependent and linear) suggested that the inserted energy, the reorientation process depends on can not be described as a simple product of frequency and amplitude. Fluorescence microscopy was used to observe the dynamics of focal adhesion contacts during cyclical stretch. We determined that focal adhesions reoriented themselves faster, compared to the entire cell. Our investigations showed for the first time the reaction dynamics of cells during cyclical mechanical stretch. We thereby determined an interesting general reaction characteristic which was found to be dependent on the stretch frequency, and involved cell-specific thresholds. The molecular mechanisms underlying these observations will be further investigated in future studies.