Early spreading events of fibroblasts on microgrooved substrates

We investigated the "contact guidance" phenomenon, shortly after cell attachment. For this purpose polystyrene substrates were produced, either smooth, or equipped with micogrooves (depth 0.5 micrometer, width 1-10 micrometer). On these substrates, fibroblasts were cultured for 15, 30, 45, 60, 120, or 240 min. Subsequently, they were studied with light microscopy, scanning electron microscopy, confocal laser scanning microscopy, and digital image analysis. Up to 1 h, cell attachment on the grooved substrates was impaired. Further, cells oriented to the direction of the microgrooves. This orientation was established fastest on the narrow grooves. After 30 min, cells showed abundant membrane extensions in all directions. Well-formed actin filaments were not present in the cell body at timepoints before 4 h. Furthermore, cells on smooth surfaces exhibited less filaments. The addition of cytochalasin-B only caused a delay of cell attachment and spreading. From these experiments, we conclude that a well-formed cellular actin cytoskeleton is no prerequisite for the occurrence of contact guidance. Actin microfilaments in the lamellipodia, and the interplay between the lamellipodium and extracellular matrix molecules seem to be the determining factor in the establishment of contact guidance.