Three-dimensional traction forces of Schwann cells on compliant substrates

Abstract

The mechanical interaction between Schwann cells (SCs) and their microenvironment is crucial for the development, maintenance and repair of the peripheral nervous system. In this paper, we present a detailed investigation on the mechanosensitivity of SCs across a physiologically relevant substrate stiffness range. Contrary to many other cell types, we find that the SC spreading area and cytoskeletal actin architecture were relatively insensitive to substrate stiffness with pronounced stress fibre formation across all moduli tested (0.24-4.80 kPa). Consistent with the presence of stress fibres, we found that SCs generated large surface tractions on stiff substrates and large, finite material deformations on soft substrates. When quantifying the three-dimensional characteristics of the SC traction profiles, we observed a significant contribution from the out-of-plane traction component, locally giving rise to rotational moments similar to those observed in mesenchymal embryonic fibroblasts. Taken together, these measurements provide the first set of quantitative biophysical metrics of how SCs interact with their physical microenvironment, which are anticipated to aid in the development of tissue engineering scaffolds designed to promote functional integration of SCs into post-injury in vivo environments.

Keywords: Schwann cell; cell mechanics; traction force microscopy.

© 2014 The Author(s) Published by the Royal Society. All rights reserved.

Figures

Figure 1.

SCs perform a range of functions in the body. (a) During development, SCs sort axons based on their diameter and (b) deposit myelin on larger axons to increase conduction velocity of action potentials. (c) SCs aid axon regeneration by migrating into an injury site after a small injury, or into a transplanted nerve guidance channel (NGC) after a large injury. Once there, SCs proliferate and align. NGCs can also be pre-seeded with exogenous SCs to enhance regeneration. (Online version in colour.)

Figure 2.

Substrate stiffness affected morphological distribution of SCs in vitro. (a) Maximum projection confocal micrographs of representative SCs exhibiting unpolarized, bipolar and multipolar morphologies (bottom to top) stained with phalloidin for actin, and TO-PRO-3 iodide for nuclei. Scale bar represents 20 µm. (b) Bar plot of the percentage occurrence of unpolarized (black), bipolar (grey) and multipolar (white) morphology occurrence per stiffness shows that the bipolar phenotype is most prevalent across all elastic moduli; 0.24 (n = 298), 1.70 (n = 382) and 4.80 kPa (n = 156). (Online version in colour.)

Figure 3.

SC spreading area was similar across elastic moduli. Box and whiskers plot of SC spreading area for the three tested elastic moduli; 0.24 (n = 143), 1.70 (n = 252) and 4.80 kPa (n = 120). Solid bands: medians, top and bottom boxes: upper and lower quartiles, whiskers: standard deviations, and dots: fifth and 95th percentiles. Representative confocal micrographs of SCs for each elastic modulus are shown. Scale bar represents 20 µm.

Figure 4.

SCs exhibited a mature actin cytoskeleton, including stress fibres across all moduli. Maximum projection confocal micrographs of representative SCs cultured on elastic moduli of 0.24, 1.70 and 4.80 kPa that were stained with phalloidin for actin, and TO-PRO-3 iodide for nuclei. White box shows a two-times zoomed in image to highlight the stress fibres. Scale bars represents 20 µm. (Online version in colour.)

Figure 5.

SC displacements and tractions varied across substrate stiffness. Surface contour maps of the magnitude of (a–c) the three-dimensional displacement field , (d–f) the displacement gradient and (g–i) the traction field (|T|) vector for each elastic modulus; 0.24, 1.70 and 4.80 kPa. The cell outline (white) is superimposed onto the contour maps to show its position with respect to each field. Scale bar represents 20 µm. (Online version in colour.)

Figure 6.

SC displacements, tractions and strain energies varied with elastic moduli. Bar plots of the (a) root-mean-squared displacements magnitude (uRMS), (b) root-mean-squared tractions (TRMS), (c) maximum displacement gradient magnitude and (d) strain energy (U) as a function of elastic modulus; 0.24 (n = 143), 1.70 (n = 252) and 4.80 kPa (n = 120). The uRMS and TRMS values are split into their respective shear components and normal components (black and grey, respectively). Geometric mean (μg) and geometric mean absolute deviation (MAD) are shown. *p < 0.05 by one-way ANOVA on ranks across elastic moduli, and **p < 0.001 by t-test between normal and shear components, respectively.

Figure 7.

SC-induced normal tractions played a significant role in the SC traction profile and exhibited an alternating dipole pattern across substrate stiffness. (a–c) Vector field of the SC applied shear tractions and (e–f) contour map of the SC applied normal tractions for the three tested substrate stiffnesses. The colour bar represents the magnitude of the respective component and the reference vector shows a shear traction magnitude of 250, 350 and 800 Pa for E = 0.24, E = 1.70, E = 4.80 kPa, respectively. The cell outline (white) is superimposed to show its position with respect to the tractions. Scale bar represents 20 μm. (Online version in colour.)

Figure 8.

Finite-element simulation approximates the experimentally observed dipole and out-of-plane SC displacement behaviour. (a) Schematic cartoon showing how SC might transmit force at an adhesion plaque of discrete size. Intracellular actin filaments contract with a force F, inducing dipole such as displacements (u) and rotational moments about the adhesion plaque. (b) Contour map of the FEM results along the symmetry plane showing the normal displacements induced in the deformed configuration of the PA gel (E = 0.24 kPa). The adhesion plaque is modelled as a rigid plate that is firmly bonded to the PA gel with a uniform shear traction of magnitude 180 Pa applied to the top surface of the FA plaque. (c) Experimentally measured out-of-plane SC displacement field. Scale bar represents 5 μm. (d) Plot of the mean normal and shear maximum displacement ratios from our experimental measurements and our finite-element analysis across elastic moduli. (Online version in colour.)