The effect of combined cyclic mechanical stretching and microgrooved surface topography on the behavior of fibroblasts.

Abstract

Under the influence of mechanical stress, cultured fibroblasts have a tendency to orient themselves perpendicular to the stress direction. Similar cell alignment can be induced by guiding cells along topographical clues, like microgrooves. The aim of this study was to evaluate cell behavior on microgrooved substrates, exposed to cyclic stretching. We hypothesized that cellular shape is mainly determined by topographical clues. On basis of earlier studies, a 10-microm wide square groove, and a 40-microm wide V-shaped groove pattern were used. Smooth substrates served as controls. Onto all substrates fibroblasts were cultured and 1-Hz cyclic stretching was applied (0, 4, or 8%) for 3-24 h. Cells were prepared for scanning electron microscopy, immunostaining of filamentous actin, alignment measurements, and PCR (collagen-I, fibronectin, alpha1- and beta1-integrins). Results showed that cells aligned on all grooved surfaces, and fluorescence microscopy showed similar orientation of intracellular actin filaments. After 3 h of stretch, cellular orientation started to commence, and after 24 h the cells had aligned themselves almost entirely. Image analysis showed better orientation with increasing groove depth. Statistical testing proved that the parameters groove type, groove orientation, and time all were significant, but the variation of stretch force was not. Substrates with microgrooves perpendicular to the stretch direction elicit a better cell alignment. The expression of beta1-integrin and collagen-I was higher in the stretched samples. In conclusion, we can maintain our hypothesis, as microgrooved topography was most effective in applying strains relative to the long axis of the cell, and only secondary effects of stretch force were present.