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This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and micro-grooved substrata (groove depth: 1 mum, width: 1, 2, 5, 10 microm), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and alpha(1)- and beta(1)-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces.

Adhesion is a process that can be divided into three separate stages: (1) cell attachment, (2) cell spreading, and (3) the formation of focal adhesions and stress fibers. With each stage the adhesive strength of the cell increases. De-adhesion can be defined as the process involving the transition of the cell from a strongly adherent state, characterized by focal adhesions and stress fibers, to a state of intermediate adherence, represented by a cell that is spread, but that lacks stress fibers terminating at adhesion plaques. We propose that this modification of the structural link between the actin cytoskeleton and the extracellular matrix results in a more malleable cellular state conducive for dynamic processes such as cytokinesis, mitogenesis, and motility. Anti-adhesive proteins, including thrombospondin, tenascin, and SPARC, rapidly signal de-adhesion, potentially mediating proliferation and migration during development and wound healing. Intracellular signaling molecules involved in the regulation of de-adhesion are only beginning to be identified. Interestingly, many of the same signaling proteins recognized to play important roles during the process of adhesion have also been found to act during de-adhesion. Characterization of the precise mechanisms by which these signals modulate adhesive structures and the cytoskeleton will further our understanding of the regulation of adhesive strength and its function in cellular physiology.