DOI: 10.5061/dryad.08kprr59c
title: Data rom: Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment

description: The ability of cells to organize into tissues with proper structure and
 function requires the effective coordination of proliferation, migration,
 polarization, and differentiation across length scales. Skeletal muscle is
 innately anisotropic; however, few biomaterials can emulate mechanical
 anisotropy to determine its influence on tissue patterning without
 introducing confounding topography. Here, we demonstrate that substrate
 stiffness anisotropy coordinates contractility-driven collective cellular
 dynamics resulting in C2C12 myotube alignment over millimeter-scale
 distances. When cultured on mechanically anisotropic liquid crystalline
 polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively
 polarize in the stiffest direction. Cellular coordination is amplified
 through reciprocal cell-ECM dynamics that emerge during fusion, driving
 global myotube-ECM ordering. Conversely, myotube alignment was restricted
 to small local domains with no directional preference on mechanically
 isotropic LCNs of the same chemical formulation. These findings provide
 valuable insights for designing biomaterials that mimic anisotropic
 microenvironments and underscore the significance of stiffness anisotropy
 in orchestrating tissue morphogenesis.
description: # Stiffness anisotropy coordinates supracellular contractility driving
 long-range myotube-ECM alignment
 [https://doi.org/10.5061/dryad.08kprr59c](https://doi.org/10.5061/dryad.08kprr59c) Dataset contains replicate data for all figures presented in the main text and supplemental materials. ## Description of the data and file structure Each figure has an associated .xlsx file with all replicate data and annotations if applicable. Fig. 1B: Elastic modulus parallel and orthogonal to mLCN nematic director derived from the initial linear regime of stress-strain curves. Stiffness anisotropy ratio and difference is calculated from the mean of repeated tensile tests. Fig. 1E/F: Myotube orientation-order parameter (*S*) and nematic correlation length (µm) of myotubes after 5 days of differentiation on isotropic and aligned substrates. Fig. 2B: Myoblast migration speed (µm/hr) and normalized migration ± 10˚ from nematic director on mLCNs through confluence. Fig. 2C/D: (C) Temporal evolution of cell density (cells/mm2), orientation-order parameter (*S*), spatial disorder (%), velocity correlation length (µm), and cellular speed (µm/hr) on mLCNs and iLCNs. Cells highlighted in green indicate maximum cell density for timeseries alignment. Cells highlighted in red were excluded from analysis due to stage motion artifacts or inability to link PIV analysis to subsequent frame.  Fig. 2F: Temporal evolution of orientation-order parameter (*S*), nematic correlation length (µm), and velocity correlation length (µm) on mLCNs. Cells highlighted in red were excluded from analysis due to stage motion artifacts or inability to link PIV analysis to subsequent frame.  Fig. 3A: Angular frequency of actin and fibronectin alignment on mLCNs after reaching confluence. Fig. 3B: Angular frequency of myosin and fibronectin alignment on mLCNs after 5 days of differentiation. Fig. 3C: Angular frequency of myosin and laminin alignment on mLCNs after 5 days of differentiation. Fig. 3D: Angular frequency of myosin and collagen IV alignment on mLCNs after 5 days of differentiation. Fig. 4A/B: Temporal evolution of orientation-order parameter (S), nematic correlation length (µm), velocity correlation length (µm), and cellular speed (µm/hr) on mLCNs ± blebbistatin treatment. Cells highlighted in red were excluded from analysis due to stage motion artifacts or inability to link PIV analysis to subsequent frame.  Fig. 4C: Angular frequency of fibronectin alignment after 3.5 days of differentiation on mLCNs ± blebbistatin treatment Fig. S1E: Representative stress-strain data from tensile testing of mLCN-0.5, -0.75, and -1.0. Fig. S1F: Contact angle measurements on mLCN-0.5, mLCN-0.75, and mLCN-1.0. Fig. S1G: Line profiles of AFM height maps on mLCN-0.5, mLCN-0.75, and mLCN-1.0. Fig. S2B: Cumulative angular frequency (10˚ bins) of C2C12 myoblast alignment on glass, mLCN-0.5, -0.75, and -1.0 after 24, 48, and 72 hr of growth. Fig. S2C: Cell density (cells/mm2) on glass, mLCN-0.5, -0.75, and -1.0 after 24, 48, and 72 hr of growth. Fig. S2D: Average cell area (µm2) on glass, mLCN-0.5, -0.75, and -1.0 after 72 hr of growth. Fig. S2E: Average cell aspect ratioon glass, mLCN-0.5, -0.75, and -1.0 after 72 hr of growth. Fig. S3A: Angular frequency of myotube alignment on isotropic and monodomain LCNs, glass and NanoSurface after 3 and 5 days of differentiation. Fig. S3B/C: Myotube orientation-order parameter (*S*) and nematic correlation length (µm) isotropic and monodomain LCNs, glass and NanoSurface after 3 and 5 days of differentiation. Fig. S4A: Myotube peak alignment offset to the nematic director of mLCNs and groove direction of NanoSurface substrates after 3 and 5 days of differentiation. Fig. S4C: Angular frequency of myosin and laminin alignment in each layer of myotubes on mLCNs after 5 days of differentiation. Fig. S4D: Intensity frequency of myosin and laminin as a function of height from the bottom of the mLCN after 5 days of differentiation. Fig. S4F: Angular frequency of myosin and laminin alignment in each layer of myotubes on NanoSurface after 5 days of differentiation. Fig. S4G: Intensity frequency of myosin and laminin as a function of height from the bottom of the NanoSurface after 5 days of differentiation. Fig. S5: Cumulative angular frequency (10˚ bins) of C2C12 myoblast cell division angle throughout the proliferative phase on mLCNs and iLCNs. Fig. S8: Number of nuclei per myotube after 3.5 days of differentiation on mLCNs +/- blebbistatin treatment.