Elucidating the signal for contact guidance contained in aligned fibrils with a microstructural-mechanical model

Abstract

Despite its importance in physiological processes and tissue engineering, the mechanism underlying cell contact guidance in an aligned fibrillar network has defied elucidation due to multiple interdependent signals that such a network presents to cells, namely, anisotropy of adhesion, porosity and mechanical behaviour. A microstructural-mechanical model of fibril networks was used to assess the relative magnitudes of these competing signals in networks of varied alignment strength based on idealized cylindrical pseudopods projected into the aligned and orthogonal directions and computing the anisotropy of metrics chosen for adhesion, porosity and mechanical behaviour: cylinder-fibre contact area for adhesion, persistence length of pores for porosity and total force to displace fibres from the cylindrical volume as well as network stiffness experienced upon cylinder retraction for mechanical behaviour. The signals related to mechanical anisotropy are substantially higher than adhesion and porosity anisotropy, especially at stronger network alignments, although their signal to noise (S/N) values are substantially lower. The former finding is consistent with a recent report that fibroblasts can sense fibril alignment via anisotropy of network mechanical resistance, and the model reveals this can be due to either mechanical resistance to pseudopod protrusion or retraction given their signal and S/N values are similar.

Keywords: aligned fibril networks; cell–matrix interaction; contact guidance.

Figure 1.

Illustration of Dunn's hypotheses for the sensing mechanism of cell contact guidance in aligned fibrils, adapted from Thrivikraman et al. 2021 [8]. A cell with two pseudopods is shown in an aligned fibre network. (a) The aligned network presents a higher porosity, P, to the cell in the direction of alignment (P_||) versus perpendicular to fibre alignment (P_⊥). (b) The aligned network offers more adhesion sites, S, shown in green, in the direction of alignment (S_||) versus against it (S_⊥). (c) The aligned network has a higher fibre stiffness, E, in the direction of alignment (E_||) compared to perpendicular to alignment (E_⊥). Note this figure is not drawn to scale for clarity; a pseudopod is much larger than the typical pore size in the standard in vitro models (collagen and fibrin gels) and interacts with many interconnected fibrils simultaneously, not single fibrils.

Figure 2.

Computational model set-up. (a) Cells were imaged and then analysed in ImageJ to determine average pseudopod lengths and widths. (b) The resulting measurements were used to model cell pseudopods as rigid cylinders, while the surrounding ECM was modelled with a discrete network model. (c) Pseudopods were placed into fibre networks parallel to network alignment (i), and perpendicular to network alignment (ii). (d) The initial state of the model with the cylinder placed within the network, resulting in fibres initially passing through it. (e) Intersecting fibres are discretized. (f) The resulting network configuration after discretized fibres are pushed out.

Figure 3.

Predicted anisotropy metrics. Plots of (a) chemical anisotropy, L_c,x/L_c,y, (b) mechanical anisotropy measured through the ratio of pseudopod pushing force, F_p,x/F_p,y, (c) mechanical anisotropy measured through stiffness sensed via pseudopod retraction, K_x/K_y and (d) structural anisotropy, D_x/D_y as a function of network alignment, Ω_xx. Spearman's rank correlation coefficient ρ is reported for each plot (a–d), with significance set to p < 0.05. (e) All signals (chemical, mechanical via pushing force, mechanical via retraction force and structural anisotropy) plotted against network alignment, shown as power series fits of the data. (f) Logarithmic signal to noise ratios (log base 10 of the average anisotropy signal divided by standard deviation of the signal) versus binned network alignment intervals for all anisotropy metrics.

Figure 4.

Predicted anisotropy metrics for Voronoi networks. Plots of (a) chemical anisotropy, (b) mechanical anisotropy measured as the pseudopod pushing force, (c) mechanical anisotropy measured as stiffness sensed via pseudopod retraction and (d) structural anisotropy, as a function of network alignment, Ω_xx. Spearman's rank correlation coefficient ρ is reported for each, with significance set to p < 0.05.