Examination of membrane rupture as a mechanism for mammalian cell detachment from fibronectin-coated biomaterials.

Synthetic biomaterials intended for the reconstruction of tissues and organs must be capable of sustaining adhesive contact with adjacent cells and tissues under mechanical and hydrodynamic stresses. To facilitate this adhesion, extracellular matrix proteins or peptide sequences are frequently immobilized to the biomaterial interface. These ligands enhance cell attachment by raising the number of cell receptor/ligand interactions, but consequently they may alter the mechanism of cell detachment. In particular, as the cell membrane is more strongly immobilized to the substratum, the tendency for cell detachment to involve membrane rupture may increase. To test this hypothesis, cells were fluorescent stained with a membrane dye, allowed to attach to fibronectin-coated model substrates for 30 min, and then subjected to a spatially dependent range of shear stress for 5 min (28-220 dyn/cm2) using a radial-flow chamber. Phase-contrast and fluorescent images were analyzed to determine the probability for cell detachment and the area of fluorescent debris left by detaching cells as a function of fibronectin concentration, magnitude of shear stress, and time. It was found at all concentrations of fibronectin that the majority of detaching cells left membrane fragments, the mean size of these fragments was independent of shear stress, and the shape independent of the direction of flow. However, mean fragment area increased with concentration of fibronectin and decreased with duration of shearing flow. We postulate that the area of debris reflects the extent of cell attachment prior to the application of shear and that adhesive complexes can disassemble at the onset of flow.