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The influence of topographic microstructures on the initial adhesion of L929 fibroblasts studied by single-cell force spectroscopy.

Elter P, Weihe T, Lange R, Gimsa J, Beck U - Eur. Biophys. J. (2010)

Bottom Line: Spherical cells exhibited a significantly lower Young's modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere.The process was found to be independent of the applied contact force for values between 100 and 1,000 pN.The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate.

View Article: PubMed Central - PubMed

Affiliation: Department for Interface Science, Institute for Electronic Appliances and Circuits, University of Rostock, Albert-Einstein-Str. 2, 18059 Rostock, Germany. patrick.elter@uni-rostock.de

ABSTRACT
Single-cell force spectroscopy was used to investigate the initial adhesion of L929 fibroblasts onto periodically grooved titanium microstructures (height ~6 μm, groove width 20 μm). The position-dependent local adhesion strength of the cells was correlated with their rheological behavior. Spherical cells exhibited a significantly lower Young's modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere. While in contact with the planar regions of the substrate, the cells started to adapt their shape through slight ventral flattening. The process was found to be independent of the applied contact force for values between 100 and 1,000 pN. The degree of flattening correlated with the adhesion strength during the first 60 s. Adhesion strength can be described by fast exponential kinetics as C₁[1-exp(-C₂·t] with C₁ = 2.34 ± 0.19 nN and C₂ = 0.09 ± 0.02 s⁻¹. A significant drop in the adhesion strength of up to 50% was found near the groove edges. The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate. Our results explain the role of the substrate's topography in contact guidance and suggest that rheological cell properties must be considered in cell adhesion modeling.

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Force–distance curves of an L929 fibroblast at different positions on the microstructured substrate. The data originate from the same cell used in Fig. 5. The contact time was 10 s, the contact force was 250 pN, and the retraction speed was 5 μm/s. a Curve measured on top with primarily horizontal contact area. b Curve measured near the flanks with maximal vertical contact area. A long plateau with sawtooth shape was frequently observed for this position. Inset Magnification of the plateau. c Local adhesion energy (area under the force–distance curve) for different positions on the grooved microstructure. The groove is located in the center of the diagram (see Fig. 5b for the profile). The hatched bars show the local adhesion energy (mean ± SD, left scale); the solid line represents the calculated local contact area (right scale). The arrows denote the positions near the groove flanks where a high adhesion energy but a low adhesion force was found
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Fig6: Force–distance curves of an L929 fibroblast at different positions on the microstructured substrate. The data originate from the same cell used in Fig. 5. The contact time was 10 s, the contact force was 250 pN, and the retraction speed was 5 μm/s. a Curve measured on top with primarily horizontal contact area. b Curve measured near the flanks with maximal vertical contact area. A long plateau with sawtooth shape was frequently observed for this position. Inset Magnification of the plateau. c Local adhesion energy (area under the force–distance curve) for different positions on the grooved microstructure. The groove is located in the center of the diagram (see Fig. 5b for the profile). The hatched bars show the local adhesion energy (mean ± SD, left scale); the solid line represents the calculated local contact area (right scale). The arrows denote the positions near the groove flanks where a high adhesion energy but a low adhesion force was found

Mentions: Flattening of the cell near the edge will allow the establishment of a contact area with the side-walls of the groove (Fig. 5d, dotted line). The breaking of specific bonds in the horizontal or vertical directions may provide different contributions to the total adhesion. Therefore, the individual force–distance curves were analyzed for possible contributions from the side-walls. Figure 6 shows the force–distance curves for a position on top of a plateau with maximal horizontal contact area and for a position near the groove flanks with maximal vertical contact area. Both curves exhibit an initial peak in the retraction curve, which obviously resulted from nonspecific interactions between the cell and the substrate, as well as a rupture of the specific bonds connected to the horizontal contact area. However, a subsequent plateau with sawtooth shape was frequently observed, which was significantly extended for the positions with the maximal vertical contact area and exhibited a length which roughly corresponds to the groove depth (Fig. 6b). The results can be interpreted in two ways. First, the cell was still in contact with the side-walls for the first 1.2 s of the retraction period (5 μm/s retraction speed and 6 μm groove depth). Hence, new bonds could be formed during retraction, leading to the sawtooth shape of the curve, which was determined by additional increases of the cell–substrate interaction and the subsequent breakage of the bonds. Second, cell membranes are frequently considered to be a continuous fluid in which membrane proteins are free to move by diffusion (Zagyansky and Edidin 1976; Swaisgood and Schindler 1989; Hirata et al. 2005; Singer and Nicolson 1972) as long as no focal contacts are formed (Duband et al. 1988). When the cell was retracted in the vertical direction, the bonds to the horizontal plateaus of the substrate would be loaded almost simultaneously, whereas the receptors attached to the side-walls would be exposed to a smaller force because the lipids of the cell membrane might be able to slide around them. Hence, their full contribution to the measured adhesion strength was observed only when the respective receptors reached the leading edge of the cell: each time a receptor reached the leading edge, the cell–substrate interaction increased, followed by the subsequent rupture of the bond; this process results in the observed sawtooth structure. Figure 6c displays the local adhesion energy (area under the force–distance curve) for different positions on the grooved microstructure. The data originate from the same cell used in Fig. 5. In contrast to the maximal adhesion force, the adhesion energy is also sensitive to the tip–sample separation distance of the individual unbinding events. For positions near the groove flanks (Figure 6c, arrows) the adhesion energy decreases less than the adhesion force (Fig. 5c), which leads to a significantly broadened peak in the center of the diagram. The smaller decrease of the adhesion energy indicates a shift of some of the unbinding events to greater distances for these positions. The results support our interpretation that some unbinding events occur at greater tip–sample separations due to the contact with the side-walls and do not contribute with their full magnitude to the maximal adhesion force. Nevertheless, a missing contribution of the side-walls to the measured maximal adhesion strength cannot be the only reason for its decrease near the flanks, because the total number of unbinding events decreased near the flanks (Fig. 5c, black bars) and the contact area to the side-walls is too small to explain the full effect (only ~20% vertical contact area versus ~50% decrease of adhesion, see Fig. 5c and d). Hence, the total contact area must decrease due to the limited elasticity of the cell.Fig. 6


The influence of topographic microstructures on the initial adhesion of L929 fibroblasts studied by single-cell force spectroscopy.

Elter P, Weihe T, Lange R, Gimsa J, Beck U - Eur. Biophys. J. (2010)

Force–distance curves of an L929 fibroblast at different positions on the microstructured substrate. The data originate from the same cell used in Fig. 5. The contact time was 10 s, the contact force was 250 pN, and the retraction speed was 5 μm/s. a Curve measured on top with primarily horizontal contact area. b Curve measured near the flanks with maximal vertical contact area. A long plateau with sawtooth shape was frequently observed for this position. Inset Magnification of the plateau. c Local adhesion energy (area under the force–distance curve) for different positions on the grooved microstructure. The groove is located in the center of the diagram (see Fig. 5b for the profile). The hatched bars show the local adhesion energy (mean ± SD, left scale); the solid line represents the calculated local contact area (right scale). The arrows denote the positions near the groove flanks where a high adhesion energy but a low adhesion force was found
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045512&req=5

Fig6: Force–distance curves of an L929 fibroblast at different positions on the microstructured substrate. The data originate from the same cell used in Fig. 5. The contact time was 10 s, the contact force was 250 pN, and the retraction speed was 5 μm/s. a Curve measured on top with primarily horizontal contact area. b Curve measured near the flanks with maximal vertical contact area. A long plateau with sawtooth shape was frequently observed for this position. Inset Magnification of the plateau. c Local adhesion energy (area under the force–distance curve) for different positions on the grooved microstructure. The groove is located in the center of the diagram (see Fig. 5b for the profile). The hatched bars show the local adhesion energy (mean ± SD, left scale); the solid line represents the calculated local contact area (right scale). The arrows denote the positions near the groove flanks where a high adhesion energy but a low adhesion force was found
Mentions: Flattening of the cell near the edge will allow the establishment of a contact area with the side-walls of the groove (Fig. 5d, dotted line). The breaking of specific bonds in the horizontal or vertical directions may provide different contributions to the total adhesion. Therefore, the individual force–distance curves were analyzed for possible contributions from the side-walls. Figure 6 shows the force–distance curves for a position on top of a plateau with maximal horizontal contact area and for a position near the groove flanks with maximal vertical contact area. Both curves exhibit an initial peak in the retraction curve, which obviously resulted from nonspecific interactions between the cell and the substrate, as well as a rupture of the specific bonds connected to the horizontal contact area. However, a subsequent plateau with sawtooth shape was frequently observed, which was significantly extended for the positions with the maximal vertical contact area and exhibited a length which roughly corresponds to the groove depth (Fig. 6b). The results can be interpreted in two ways. First, the cell was still in contact with the side-walls for the first 1.2 s of the retraction period (5 μm/s retraction speed and 6 μm groove depth). Hence, new bonds could be formed during retraction, leading to the sawtooth shape of the curve, which was determined by additional increases of the cell–substrate interaction and the subsequent breakage of the bonds. Second, cell membranes are frequently considered to be a continuous fluid in which membrane proteins are free to move by diffusion (Zagyansky and Edidin 1976; Swaisgood and Schindler 1989; Hirata et al. 2005; Singer and Nicolson 1972) as long as no focal contacts are formed (Duband et al. 1988). When the cell was retracted in the vertical direction, the bonds to the horizontal plateaus of the substrate would be loaded almost simultaneously, whereas the receptors attached to the side-walls would be exposed to a smaller force because the lipids of the cell membrane might be able to slide around them. Hence, their full contribution to the measured adhesion strength was observed only when the respective receptors reached the leading edge of the cell: each time a receptor reached the leading edge, the cell–substrate interaction increased, followed by the subsequent rupture of the bond; this process results in the observed sawtooth structure. Figure 6c displays the local adhesion energy (area under the force–distance curve) for different positions on the grooved microstructure. The data originate from the same cell used in Fig. 5. In contrast to the maximal adhesion force, the adhesion energy is also sensitive to the tip–sample separation distance of the individual unbinding events. For positions near the groove flanks (Figure 6c, arrows) the adhesion energy decreases less than the adhesion force (Fig. 5c), which leads to a significantly broadened peak in the center of the diagram. The smaller decrease of the adhesion energy indicates a shift of some of the unbinding events to greater distances for these positions. The results support our interpretation that some unbinding events occur at greater tip–sample separations due to the contact with the side-walls and do not contribute with their full magnitude to the maximal adhesion force. Nevertheless, a missing contribution of the side-walls to the measured maximal adhesion strength cannot be the only reason for its decrease near the flanks, because the total number of unbinding events decreased near the flanks (Fig. 5c, black bars) and the contact area to the side-walls is too small to explain the full effect (only ~20% vertical contact area versus ~50% decrease of adhesion, see Fig. 5c and d). Hence, the total contact area must decrease due to the limited elasticity of the cell.Fig. 6

Bottom Line: Spherical cells exhibited a significantly lower Young's modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere.The process was found to be independent of the applied contact force for values between 100 and 1,000 pN.The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate.

View Article: PubMed Central - PubMed

Affiliation: Department for Interface Science, Institute for Electronic Appliances and Circuits, University of Rostock, Albert-Einstein-Str. 2, 18059 Rostock, Germany. patrick.elter@uni-rostock.de

ABSTRACT
Single-cell force spectroscopy was used to investigate the initial adhesion of L929 fibroblasts onto periodically grooved titanium microstructures (height ~6 μm, groove width 20 μm). The position-dependent local adhesion strength of the cells was correlated with their rheological behavior. Spherical cells exhibited a significantly lower Young's modulus (<1 kPa) than that reported for spread cells, and their elastic properties can roughly be explained by the Hertz model for an elastic sphere. While in contact with the planar regions of the substrate, the cells started to adapt their shape through slight ventral flattening. The process was found to be independent of the applied contact force for values between 100 and 1,000 pN. The degree of flattening correlated with the adhesion strength during the first 60 s. Adhesion strength can be described by fast exponential kinetics as C₁[1-exp(-C₂·t] with C₁ = 2.34 ± 0.19 nN and C₂ = 0.09 ± 0.02 s⁻¹. A significant drop in the adhesion strength of up to 50% was found near the groove edges. The effect can be interpreted by the geometric decrease of the contact area, which indicates the inability of the fibroblasts to adapt to the shape of the substrate. Our results explain the role of the substrate's topography in contact guidance and suggest that rheological cell properties must be considered in cell adhesion modeling.

Show MeSH
Related in: MedlinePlus