Adhesion response of filopodia to an AFM lateral detachment force and functional changes after centrifugation of cells grown on nanoporous titanium

Abstract

Cells sense and respond to mechanical cues from the surrounding substrate through filopodia. Regulation of cellular biomechanics operates at the nanoscale. Therefore, a better understanding of the relationship between filopodia and nanoscale surface features is highly relevant for the rational design of implant surfaces. The objective of this work was to determine the biomechanical contribution of filopodia and their nanoprotrusions to the adhesive interaction of cells with nanostructured surfaces. We have also analyzed the functional changes of entire cells subjected to an external force. MC3T3-E1 osteogenic cells were cultured on polished (Ti-Control) and nanotextured titanium discs (Ti-Nano). An AFM approach was used to measure the lateral detachment force of filopodia. Filopodia on Ti-Nano exhibited higher resistance to a lateral detachment force, which indicates that they adhere to the surface with more strength. SEM analysis revealed a restructuration of the cell membrane in response to centrifugation, being more evident on Ti-Nano. Fluorescence labeling also highlighted a difference in the mitochondrial footprint, a cellular compartment that provides energy for cellular processes. Together, these results show for the first time that surface topography can change the adhesive interaction of a subcellular structure that is fundamental in sensing physico-chemical surfaces features.

Keywords: AFM; Centrifugation; Filopodia; Mechanotransduction; Nanotopography; Titanium.

Graphical abstract

Fig. 1

(A) Image from the optical camera showing the top view of the AFM cantilever scanning a cell. (B) SEM micrograph of the lateral view of the pyramidal silicon nitride tip. (C) Schematic representation of the lateral view showing the direction of the compression and lateral forces applied to move the cell. (D) Schematic representation of the disc arrangement for the centrifugation assay.

Fig. 2

Scanning electron micrographs of the (A) smooth polished Ti surface and (B) nanoporous topography created by the oxidative chemical treatment. (C) Size distribution of the nanopores (n ​= ​100).

Fig. 3

AFM images of filopodia on (A) Ti-Control and (C) Ti-Nano showing the probed regions (A, C) before and (B, D) after increasing the deflection setpoint of the cantilever. In all AFM images arrows represent the direction of the cell body. (E) Quantitative analysis of the lateral force required to detach or break the filopodium on both surfaces obtained after calculation. Dots represent individual data points. Error bars represent the standard deviations, ∗ indicates statistically significant differences (p ​< ​0.05).

Fig. 4

(A) Count from fluorescence microscopy images of cells stained with DAPI (blue) for nuclei and rhodamine/phalloidin (red) for actin. (B) Enlargement of the area outlined by the white square in A. (C) Nuclei maps generated using Image J to automatically calculate (D) the cell number. (E) Number of cells on the polished (Ti-Control) and nanoporous (Ti-Nano) surfaces before and after centrifugation. Dots represent individual data points. Error bars represent the standard deviation. The results show no statistical differences.

Fig. 5

Fluorescence microscopy images of cells stained with DAPI (blue) for nuclei and rhodamine/phalloidin (red) for actin attached on Ti-Control and Ti-Nano (A, C) before and (B, D) after centrifugation. (E) Cells map generated using Image J to automatically calculate the cell area, incomplete cells were excluded from data. Some cells showed regions of peripheral membrane folding (white ovals). (F) The cell areas on Ti-Control and Ti-Nano surfaces before and after centrifugation show no statistical differences under all conditions. Dots represent individual data points. Error bars represent the standard deviations.

Fig. 6

Representative SEM images of cells attached on (A–D) Ti-Control and (E–H) Ti-Nano (A, E) before and (B, C, D, F, G, H) after centrifugation. The distribution of filopodia is represented with arrowheads. High-resolution images of filopodium on (D) Ti-Control and (F) Ti-Nano after centrifugation. (H) Nanoscale protrusions emanating from a filopodium attached to the Ti-Nano surface (arrows).

Fig. 7

Representative fluorescence micrographs of cells stained with DAPI (blue) for nuclei, rhodamine/phalloidin (red) for actin, and MitoTracker Green (green) for mitochondrial network attached on Ti-Control and Ti-Nano (A, C) before and (B, D) after centrifugation. (E) The surface occupied by the mitochondria. Dots represent individual data points. Error bars represent the standard deviations, ∗ indicates statistically significant differences (p ​< ​0.05).

Fig. 8

Immunofluorescence images of cells stained with DAPI (blue) for nuclei, rhodamine/phalloidin (red) for actin, and Anti-vinculin (green) for FAs attached on Ti-Control and Ti-Nano, (A, C) before and (B, D) after centrifugation. (E) Number of FAs. Dots represent individual data points. Error bars represent the standard deviations. The results show no statistical differences.