Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment

Abstract

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 importance of stiffness anisotropy in orchestrating tissue morphogenesis.

Fig. 1.. mLCN stiffness anisotropy drives C2C12 myotube ordering.

(A) Illustration of mLCN and iLCN network structure and mechanical anisotropy (n indicates nematic director of mLCN). (B) Elastic modulus parallel and orthogonal to mLCN nematic director derived from the initial linear regime of stress-strain curves. Stiffness anisotropy ratio (E_||/E_⊥) and difference (E_|| − E_⊥) is calculated from the mean of repeated tensile tests. N = 4, 3, 6, 3, 3, and 3 (top to bottom); line indicates mean, all replicates shown. (C) Illustration of C2C12 growth and differentiation on mLCNs. Created with BioRender.com. (D) Representative images of myotubes stained for myosin II heavy chain (MF-20, gray) after 5 days of differentiation on monodomain (left) and isotropic (right) LCN-1.0. Scale bars, 1000 μm. (E) Myotube orientation-order parameter (S) and (F) nematic correlation length (μm) of myotubes after 5 days of differentiation on isotropic and aligned substrates (isotropic versus monodomain for LCNs, glass coverslip versus NanoSurface substrate for glass). N = 3, 3, 4, 3, 4, 5, 3, and 3 (top to bottom); the box limits extend from the 25th to 75th percentiles; the horizontal line indicates the median value; the whiskers extend by 1.5× the interquartile range; all replicates including outliers are shown. Statistical analysis was performed with one-tailed (isotropic versus monodomain) or two-tailed (monodomain versus monodomain) unpaired Student’s t test with Welch’s correction, with significance claimed at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

Fig. 2.. Stiffness anisotropy coordinates collective cellular dynamics across multiple length scales.

(A) Representative images of C2C12 nuclei (top) and actin (bottom) on mLCNs at t_a = 12, 27, 42, 57, and 75 hours (left to right). Scale bars, 200 μm. (B) Myoblast migration speed (μm/hour; left axis) and normalized migration ±10° from nematic director (right axis) on mLCNs. Dashed line indicates approximate density at which myoblasts achieved confluence. N = 12 independent fields of view across three mLCNs; data are represented as means ± SEM. (C) Top to bottom: Temporal evolution of cell density (cells/mm²), orientation-order parameter (S), spatial disorder (%), and velocity correlation length (μm) on mLCNs and iLCNs. Dashed lines indicate approximate time at which myoblasts achieved confluence (left) and initiated fusion (right). N = 5 and 4 independent fields of view across three mLCNs and two iLCNs, respectively; data are represented as means ± SEM. (D) Velocity correlation length (μm) as a function of cellular speed (μm/hour) after confluence on mLCNs. N is the same as in (C); data are represented as means ± SEM. (E) Representative images (top) and inset (bottom) of C2C12 actin on mLCNs at t_a = 60, 90, and 120 hours (left to right). Scale bars, 1000 μm (top) and 200 μm (bottom). Red circle indicates location of a contractile defect. (F) Top to bottom: Temporal evolution of orientation-order parameter (S), nematic correlation length (μm), and velocity correlation length (μm) on mLCNs. N = 3 mLCNs; data are represented as means ± SEM.

Fig. 3.. Nascent ECM alignment develops in parallel with myotube alignment.

(A) Representative maximum intensity projections of actin (left) and fibronectin (middle) on mLCNs after reaching confluence. Scale bars, 100 μm. Corresponding frequency distribution plot (right) of actin and fibronectin alignment. N = 3 mLCNs; data are represented as means ± SD. (B) Representative maximum intensity projections of myosin II heavy chain (left) and fibronectin (middle) on mLCNs after 5 days of differentiation. Scale bars, 200 μm. Corresponding frequency distribution plot (right) of myosin and fibronectin alignment. N = 2 mLCNs; data are represented as means ± SD. (C and D) Frequency distribution plots of myosin and (C) laminin, or (D) collagen IV alignment after 5 days of differentiation. N = 5 mLCNs for (C) and 3 mLCNs for (D); data are represented as means ± SD.

Fig. 4.. Reciprocal cell-ECM dynamics mediate collective cellular flows on mLCNs.

(A) Top to bottom: Temporal evolution of orientation-order parameter (S), nematic correlation length (μm), and velocity correlation length (μm) on mLCN ± blebbistatin treatment. Dashed lines indicate timing of daily medium changes, with the first addition of blebbistatin occurring at t_a = 12 hours. N = 2 mLCNs for each condition; data are represented as means ± SEM. (B) Velocity correlation length (μm) as a function of cellular speed (μm/hour) on mLCN ± blebbistatin treatment. N is the same as in (A); data are represented as means ± SEM. Arrow indicates first addition of blebbistatin. (C) Representative maximum intensity projections (left) of myosin II heavy chain (top) and fibronectin (bottom) after 3.5 days of differentiation on mLCN ± blebbistatin treatment. Scale bars, 50 μm. Corresponding frequency distribution plot (right) of fibronectin alignment after 3.5 days of differentiation on mLCN ± blebbistatin treatment. N is the same as in (A); data are represented as means ± SD.

Fig. 5.. Spatiotemporal evolution of collective cellular dynamics that drive C2C12 myoblast and myotube alignment with the nematic director of mLCNs.

Created with BioRender.com.