The study of cell-surface interactions is important for several different applications. While the effects of surfaces on cell proliferation, migration, growth and genes expression are accessible via traditional microscopy and labeling techniques, the determination of cell adhesion remains a major challenge. In the context of the European project Newbone, working on development of a new generation of non-metallic biomimetic bone implants, cell adhesion is also at the center of the preoccupations. The objective is not only to observe modifications of adhesion properties on different surfaces but mainly to develop some strategies of cell adhesion measurements in order to understand the mechanisms responsible of cell adhesion modulations. Atomic force microscopy, and particularly force spectroscopy are used to directly measure the cell adhesion. The JPK Nanowizard II is a bioAFM allowing to manipulate, to stretch and to detach cells with a z-range of 100 μm.
Single cell force spectroscopy can be used with the cell on the cantilever, on the surface or both to quantify cell-cell adhesion forces. Force distance curves provide information about cell adhesion properties and cell viscoelasticity properties. Capturing the cell on the cantilever at a specific position requires a prefunctionalized cantilever depending on the molecules exposed at the cell surface to obtain the highest affinity.
A) Schematic single cell force spectroscopy measurement and B) a force distance curve acquired during the approach (steps I-II) and the retracting step (steps III-IV). In the initial phase of the approach there is no contact between the cell and the surface (step I). Then the cell is pressed onto the surface until a pre-set maximal force is attained. During this phase the elastic response of the cell can be observed (step II). The position of the cantilever is held constant for a given contact time. Information on different mechanical parameters can be obtained from the retraction: the work of detachment, the number, amplitude and position of the unbinding events corresponding to single proteins or proteins complexes and finally the maximal force needed to detach the cell from the surface (step III). At the end, there is no physical contact between the cell and the surface (step IV).
Binding proteins and/or protein complexes at the cell membrane remain similar both in number and in their binding properties during all the phases of the cell cycle. Rounding up and reduced adhesion of cells during mitosis is not associated with large differences in the binding proteins in the cell membrane. Instead, a reorganization of the cytoskeleton is the origin of the rounding up, while increased stiffness and reduced contact area are probably the cause of the reduced adhesion of the cells.
Histograms showing the number of unbinding events of human osteoblasts on a glass surface after an applied force of 900 pN for 1 s. The number of unbinding events was measured for unsynchronized cells and synchronized cells in G1, S and G2M phases. Between 4 and 23 unbinding events were observed in each force distance curve independently of the cell cycle phase.
The development of a method that allows de-adhesion measurements for longer times.
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