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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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Illustration of the experiment. a Schematic setup: the adhesion strength is mapped for different positions perpendicular to the grooves on the microstructure. b Force–distance curve of an L929 fibroblast on a planar titanium substrate (10 s contact time, 500 pN contact force, 5 μm/s retraction speed). Sections (1)–(3) correspond to the approach of the cell to the substrate, while sections (4)–(7) correspond to the retraction. (1) Baseline of the free cell, (2) contact point, (3) height at the contact force, (4) reduced height after the contact period, (5) maximal adhesion strength, (6) rupture events of receptors/tethers, (7) baseline of the free cell. Inset phase-contrast image of an L929 fibroblast attached to a tipless Arrow TL-1 cantilever
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Fig1: Illustration of the experiment. a Schematic setup: the adhesion strength is mapped for different positions perpendicular to the grooves on the microstructure. b Force–distance curve of an L929 fibroblast on a planar titanium substrate (10 s contact time, 500 pN contact force, 5 μm/s retraction speed). Sections (1)–(3) correspond to the approach of the cell to the substrate, while sections (4)–(7) correspond to the retraction. (1) Baseline of the free cell, (2) contact point, (3) height at the contact force, (4) reduced height after the contact period, (5) maximal adhesion strength, (6) rupture events of receptors/tethers, (7) baseline of the free cell. Inset phase-contrast image of an L929 fibroblast attached to a tipless Arrow TL-1 cantilever

Mentions: Silicon wafer pieces (1 × 1 cm2) with thickness of 500 μm were microstructured using deep reactive-ion etching (Center for Microtechnologies ZFM, Chemnitz, Germany). A periodically grooved topography (Fig. 1a) with plateau and groove width of 20 μm and step height of ~6 μm was fabricated on the chip and sputter-coated with 50 nm titanium to provide a uniform, biocompatible (Bogner et al. 2006) surface chemistry. The dimensions of the topography were selected such that the cells and the surface structures were of similar size and were in the range where contact guidance has been observed in a number of other studies (Scheideler et al. 2003; Brunette 1986). Moreover, the length of a full topographical period perpendicular to the grooves did not exceed the lateral scanning limits of the AFM (100 × 100 μm2), and only the cell (and not the cantilever) came into contact with the substrate. Each sample had a small planar area in one corner for reference measurements. Overview images of the samples were made using field-emission scanning electron microscopy (Supra 25; Zeiss, Germany), and cross-sectional profiles were recorded by AFM (see below) in intermittent contact mode using NCH cantilevers (doped silicon, 42 N/m spring constant; NanoWorld, Neuchâtel, Switzerland).Fig. 1


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)

Illustration of the experiment. a Schematic setup: the adhesion strength is mapped for different positions perpendicular to the grooves on the microstructure. b Force–distance curve of an L929 fibroblast on a planar titanium substrate (10 s contact time, 500 pN contact force, 5 μm/s retraction speed). Sections (1)–(3) correspond to the approach of the cell to the substrate, while sections (4)–(7) correspond to the retraction. (1) Baseline of the free cell, (2) contact point, (3) height at the contact force, (4) reduced height after the contact period, (5) maximal adhesion strength, (6) rupture events of receptors/tethers, (7) baseline of the free cell. Inset phase-contrast image of an L929 fibroblast attached to a tipless Arrow TL-1 cantilever
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045512&req=5

Fig1: Illustration of the experiment. a Schematic setup: the adhesion strength is mapped for different positions perpendicular to the grooves on the microstructure. b Force–distance curve of an L929 fibroblast on a planar titanium substrate (10 s contact time, 500 pN contact force, 5 μm/s retraction speed). Sections (1)–(3) correspond to the approach of the cell to the substrate, while sections (4)–(7) correspond to the retraction. (1) Baseline of the free cell, (2) contact point, (3) height at the contact force, (4) reduced height after the contact period, (5) maximal adhesion strength, (6) rupture events of receptors/tethers, (7) baseline of the free cell. Inset phase-contrast image of an L929 fibroblast attached to a tipless Arrow TL-1 cantilever
Mentions: Silicon wafer pieces (1 × 1 cm2) with thickness of 500 μm were microstructured using deep reactive-ion etching (Center for Microtechnologies ZFM, Chemnitz, Germany). A periodically grooved topography (Fig. 1a) with plateau and groove width of 20 μm and step height of ~6 μm was fabricated on the chip and sputter-coated with 50 nm titanium to provide a uniform, biocompatible (Bogner et al. 2006) surface chemistry. The dimensions of the topography were selected such that the cells and the surface structures were of similar size and were in the range where contact guidance has been observed in a number of other studies (Scheideler et al. 2003; Brunette 1986). Moreover, the length of a full topographical period perpendicular to the grooves did not exceed the lateral scanning limits of the AFM (100 × 100 μm2), and only the cell (and not the cantilever) came into contact with the substrate. Each sample had a small planar area in one corner for reference measurements. Overview images of the samples were made using field-emission scanning electron microscopy (Supra 25; Zeiss, Germany), and cross-sectional profiles were recorded by AFM (see below) in intermittent contact mode using NCH cantilevers (doped silicon, 42 N/m spring constant; NanoWorld, Neuchâtel, Switzerland).Fig. 1

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