Myofibroblasts slow down defect recombination dynamics in mixed cell monolayers

TL;DR

This study investigates how myofibroblasts reshape defect dynamics in dense fibroblast–myofibroblast monolayers using an active-nematic framework. By inducing myofibroblasts with TGF-$\beta$1 and tracking $+rac{1}{2}$ and $-rac{1}{2}$ topological defects, the authors show that increasing MF content acts as quenched disorder, slowing defect recombination and reducing global alignment on uniaxial substrates, while leaving the steady-state defect density largely unchanged. MF display a preferential affinity for $-rac{1}{2}$ defects and, conversely, fibroblasts favor $+rac{1}{2}$ defects, with MF–defect interactions persisting on LCE fiber substrates and over hundreds of microns. The work links mechanical defect landscapes to cell fate signaling (e.g., YAP activity) and proposes a simple, quantitative assay based on defect dynamics and alignment as a diagnostic readout for fibrotic progression, while contributing to the broader understanding of quenched disorder in dry active nematics.

Abstract

Cellular organization and mechanotransduction pathways are crucial regulators of tissue morphogenesis, whereas their dysregulation contributes to pathologies. Overactive fibroblasts, or myofibroblasts, are key drivers of fibrosis, yet how their presence alters collective cellular ordering remains unclear. Inspired by the analogy between liquid crystals and cells, we investigate how topological defects influence reorganization in dense monolayers of co-cultured fibroblasts and myofibroblasts. Owing to steric interactions, these elongated cells exhibit local order; topological defects, where alignment is disrupted, have been postulated to serve as mechanical centers. In this study, we examine how the incorporation of contractile myofibroblasts impacts defect relaxation. The behavior is reminiscent of active nematics with quenched disorder: myofibroblast concentration modulates the disorder strength; increasing their fraction slows defect recombination. Higher myofibroblast concentrations similarly reduce the overall cell alignment on microgrooved surfaces, as myofibroblasts interfere with monolayer reorganization. This observation highlights the potential of a simple, quantitative assay for diagnosing disease progression. Furthermore, we found that myofibroblasts preferentially localize at negatively charged -1/2 defects, compared to fibroblasts, which tend to be localized in +1/2 defects. Consequently, the slowdown of recombination dynamics can be partially attributed to the reduced velocity of the more mobile +1/2 defects. Our study suggests that myofibroblasts can exploit negatively charged defects by avoiding regions of compressive stress and evading apoptosis. Combining live-cell imaging and immunofluorescence studies, this work provides insights into the role of topological defects in fibrotic disease progression.

Figures (5)

• Figure 1: (a) Fluorescent micrographs of mixed fibroblast (magenta) and myofibroblast (green) monolayers, where the percentage of myofibroblasts is systematically increased, mimicking the progression of fibrotic diseases. The scale bars are 500 $\mu$m. (b-c) Schematics of topological defects with charges $\pm\frac{1}{2}$. (d) Defect detection in a real tissue monolayer, where the detected director field is plotted as an overlay in yellow, the cores of the defects are denoted in red ($+\frac{1}{2}$ defects) and blue ($-\frac{1}{2}$ defects), respectively. The inset shows a zoomed-in view of both types of defects. The scale bars are 500 $\mu$m.
• Figure 2: (a) The density of topological defects decreases over time and eventually plateaus at a finite value. Solid lines indicate the exponential fit. (b) Dependence of the decay constant ($\tau_D$) on myofibroblast concentration $\Phi_\mathrm{MF}$. (c) Time sequence images showing the annihilation of defect pairs with opposite topological charges in both low and high concentrations of myofibroblasts. The scale bars are 100 $\mu$m. (d) Dependence of steady-state defect density ($\rho_\infty$) on myofibroblast concentrations $\Phi_\mathrm{MF}$. (e) The average velocity of all defects ($v_\mathrm{defect}$) across myofibroblast concentrations $\Phi_\mathrm{MF}$.
• Figure 3: (a) Schematic of the rubbing protocol used to align cells, with cells orienting along the rubbing direction (denoted by double-sided arrows). (b) Polar distribution of director field angles. (c) Distribution of variation angle $\delta \theta$. (d) Spatial correlation of the nematic order parameter, where the color denotes the orientation angle, illustrated by the color wheel (above). The scale bars are 500 $\mu$m.
• Figure 4: (a-b) Cell number density for (a) fibroblasts and (b) myofibroblasts. (c) Combined density plot for fibroblasts (magenta) and myofibroblasts (green). (d) Counts of fibroblasts and myofibroblast-enriched regions that coincide with $\pm \frac{1}{2}$ defects (n = 202 defects). (e) Spatial velocity map obtained from particle image velocimetry. (f) Box and whisker plots of velocities at $\pm \frac{1}{2}$ defect sites (from n$_{+1/2}$ = 15 defects and n$_{-1/2}$ = 15 defects, p = 0.038). The scale bars are 500 $\mu$m in panels (a-c) and (e).
• Figure 5: Cells cultured on LCE fibers with patterns of topological charges (a) +$\frac{1}{2}$, (b)- $\frac{1}{2}$, (c) +1, and (b) -1. Column 1 shows the merged fluorescent micrographs with HDF (magenta), actin (green), and nuclei (blue). The scale bars are 200 $\mu$m. Column 2 shows the cell density map overlaid with the underlying director field and the defects. Column 3 shows the local myofibroblast concentration $\phi_\mathrm{MF}$ within an annulus at a distance $|\mathbf{r}-\mathbf{r}_\mathrm{defect}|$ from the center of the defect. The dashed lines denote the sample average of myofibroblast concentration. The shaded area denotes the range where one phenotype is in excess near the defect core.