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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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Rheological properties of L929 fibroblasts on a titanium substrate. a Vertical compression [mean ± standard deviation (SD)] for a round L929 fibroblast with radius of 9.2 μm. The line indicates the vertical compression according to the Hertz model for an elastic sphere (Eq. 2 with E = 267 Pa, v = 0.5, and R = 9.2 μm). b Distribution of the Young’s moduli (rel. counts ± Poisson error) for 24 cantilever-attached cells. c Distribution of the cell radii for 80 cells (rel. counts ± Poisson error). d Height decrease of the cell (mean ± SD, cell radius 9.2 μm) during the contact period for different contact forces. e Maximal adhesion strength (mean ± SD) for different contact forces. The results in (a), (d), and (e) originate from the same cell
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Fig3: Rheological properties of L929 fibroblasts on a titanium substrate. a Vertical compression [mean ± standard deviation (SD)] for a round L929 fibroblast with radius of 9.2 μm. The line indicates the vertical compression according to the Hertz model for an elastic sphere (Eq. 2 with E = 267 Pa, v = 0.5, and R = 9.2 μm). b Distribution of the Young’s moduli (rel. counts ± Poisson error) for 24 cantilever-attached cells. c Distribution of the cell radii for 80 cells (rel. counts ± Poisson error). d Height decrease of the cell (mean ± SD, cell radius 9.2 μm) during the contact period for different contact forces. e Maximal adhesion strength (mean ± SD) for different contact forces. The results in (a), (d), and (e) originate from the same cell

Mentions: In the initial experiments, the rheological behavior of the fibroblasts was characterized in the planar regions of the titanium-coated substrate using SCFS. To this end, the vertical compression (the distance between the contact point and the measured height at the desired contact force in the approach curve corrected for cantilever deflection) was analyzed for different contact forces. Figure 3a presents the results for a round, cantilever-attached fibroblast with radius of 9.2 μm. The data point at 0 N was obtained by assuming that zero force will result in zero compression. With increasing contact force, the vertical compression shows a nonlinear increase, which can be explained by the Hertz model (Hertz 1881) prediction of a 2/3-power behavior for the elastic compression of a sphere between two planes. The relationship between the compression α of a hemisphere and the contact force F is given by2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \alpha = \left( {\frac{3}{4}} \right)^{2/3} F^{2/3} \left( {{\frac{{1 - v^{2} }}{E}}} \right)^{2/3} \left( {\frac{1}{R}} \right)^{1/3} $$\end{document}where R is the radius of the cell, E is its Young’s modulus, and v is the Poisson ratio. We must note that the Hertz model is related to elastic materials, whereas cells are viscoelastic. The agreement of the data with the 2/3-power behavior of the Hertz model indicates that the initial compression phase of an SCFS cycle is primarily governed by elastic effects. This finding is understandable considering the high compression rate of 5 μm/s. Clearly, the resulting compression time was too short for significant viscoelastic relaxation of the cell. Neglecting the viscoelastic relaxation during compression and assuming both a Poisson ratio of 0.5 (Wu et al. 1998) and equal compression of the cellular hemispheres on the cantilever and substrate sides, the Young’s modulus can be estimated by an error-weighted linear regression of α3/2 versus F. The resulting value of 267 ± 89 Pa is significantly lower than the data obtained for the center of spread fibroblasts (Wu et al. 1998) but is consistent with other reports on round cells (Bacabac et al. 2008; Rosenbluth et al. 2006; Guilak et al. 1999); for example, an elastic constant between 200 and 263 Pa was found for partially adherent round MLO-Y4 osteocytes, which increased to 4.3 kPa during the spreading process (Bacabac et al. 2008). The distribution of the Young’s moduli and the cell radii for multiple cells is displayed in Figs. 3b and c, respectively. Almost all of the round cells were found to have Young’s modulus below 1 kPa. Nevertheless, their elastic properties and radii differed considerably from cell to cell.Fig. 3


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)

Rheological properties of L929 fibroblasts on a titanium substrate. a Vertical compression [mean ± standard deviation (SD)] for a round L929 fibroblast with radius of 9.2 μm. The line indicates the vertical compression according to the Hertz model for an elastic sphere (Eq. 2 with E = 267 Pa, v = 0.5, and R = 9.2 μm). b Distribution of the Young’s moduli (rel. counts ± Poisson error) for 24 cantilever-attached cells. c Distribution of the cell radii for 80 cells (rel. counts ± Poisson error). d Height decrease of the cell (mean ± SD, cell radius 9.2 μm) during the contact period for different contact forces. e Maximal adhesion strength (mean ± SD) for different contact forces. The results in (a), (d), and (e) originate from the same cell
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Fig3: Rheological properties of L929 fibroblasts on a titanium substrate. a Vertical compression [mean ± standard deviation (SD)] for a round L929 fibroblast with radius of 9.2 μm. The line indicates the vertical compression according to the Hertz model for an elastic sphere (Eq. 2 with E = 267 Pa, v = 0.5, and R = 9.2 μm). b Distribution of the Young’s moduli (rel. counts ± Poisson error) for 24 cantilever-attached cells. c Distribution of the cell radii for 80 cells (rel. counts ± Poisson error). d Height decrease of the cell (mean ± SD, cell radius 9.2 μm) during the contact period for different contact forces. e Maximal adhesion strength (mean ± SD) for different contact forces. The results in (a), (d), and (e) originate from the same cell
Mentions: In the initial experiments, the rheological behavior of the fibroblasts was characterized in the planar regions of the titanium-coated substrate using SCFS. To this end, the vertical compression (the distance between the contact point and the measured height at the desired contact force in the approach curve corrected for cantilever deflection) was analyzed for different contact forces. Figure 3a presents the results for a round, cantilever-attached fibroblast with radius of 9.2 μm. The data point at 0 N was obtained by assuming that zero force will result in zero compression. With increasing contact force, the vertical compression shows a nonlinear increase, which can be explained by the Hertz model (Hertz 1881) prediction of a 2/3-power behavior for the elastic compression of a sphere between two planes. The relationship between the compression α of a hemisphere and the contact force F is given by2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \alpha = \left( {\frac{3}{4}} \right)^{2/3} F^{2/3} \left( {{\frac{{1 - v^{2} }}{E}}} \right)^{2/3} \left( {\frac{1}{R}} \right)^{1/3} $$\end{document}where R is the radius of the cell, E is its Young’s modulus, and v is the Poisson ratio. We must note that the Hertz model is related to elastic materials, whereas cells are viscoelastic. The agreement of the data with the 2/3-power behavior of the Hertz model indicates that the initial compression phase of an SCFS cycle is primarily governed by elastic effects. This finding is understandable considering the high compression rate of 5 μm/s. Clearly, the resulting compression time was too short for significant viscoelastic relaxation of the cell. Neglecting the viscoelastic relaxation during compression and assuming both a Poisson ratio of 0.5 (Wu et al. 1998) and equal compression of the cellular hemispheres on the cantilever and substrate sides, the Young’s modulus can be estimated by an error-weighted linear regression of α3/2 versus F. The resulting value of 267 ± 89 Pa is significantly lower than the data obtained for the center of spread fibroblasts (Wu et al. 1998) but is consistent with other reports on round cells (Bacabac et al. 2008; Rosenbluth et al. 2006; Guilak et al. 1999); for example, an elastic constant between 200 and 263 Pa was found for partially adherent round MLO-Y4 osteocytes, which increased to 4.3 kPa during the spreading process (Bacabac et al. 2008). The distribution of the Young’s moduli and the cell radii for multiple cells is displayed in Figs. 3b and c, respectively. Almost all of the round cells were found to have Young’s modulus below 1 kPa. Nevertheless, their elastic properties and radii differed considerably from cell to cell.Fig. 3

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