Functionalized Multi-Walled Carbon Nanotube Enhanced Myogenic Differentiation for Aligned Topography-Induced Skeletal Muscle Engineering

Abstract

Skeletal muscle engineering utilizing bio-activators and myogenic cells to regenerate tissues for volumetric muscle loss offers a promising alternative to tissue grafts. Modified biointerfaces with aligned micro-scale topography and electroconductivity are critical for directing cellular behavior toward functional muscle constructs. This study modified polydimethylsiloxane (PDMS) with aligned surface topography and functionalized multi-walled carbon nanotubes (fCNTs), creating a conductive scaffold (0.11 µScm^-1 vs original 0.51 nScm^-1) with regulated hydrophilicity (76 ± 2° vs original 50 ± 10° in water contact angle) and enhanced protein absorption. The fCNT-wrinkled surfaces maintained >90% cell viability while promoting aligned myotube formation. Specifically, fCNT integration with aligned topography increased myotube length from 303.74 ± 27.61 µm to 441.63 ± 10.27 µm and elevated fusion index to 40.43% ± 2.67% within three differentiation days. Immunostaining confirmed enhanced myogenic maturation through improved cell alignment and nuclei organization. These biophysical modifications synergistically accelerated myoblast differentiation while maintaining cytocompatibility by combining electrical conductivity, optimized wettability, and directional cues. The demonstrated capacity to physiologically mimic native muscle microenvironments highlights this strategy's potential for improving muscle regeneration therapies through precise control of surface-electrotopographical properties.

Keywords: coating; electrochemical impedance spectroscopy; multi‐walled carbon nanotube; muscle tissue engineering; myoblasts; topography.

Figure 1

Schematic illustration of the coating methods. After flat and wrinkled PDMS was obtained by following the procedure optimized by our group, the PDMS surface was air‐plasma treated by a plasma oven to obtain a negatively charged PDMS surface. After oxidation, spraying the solution of positively charged fCNT solution on the oxidized PDMS slides to complete the spray‐coated procedure, a) overnight and rinse in water to remove the extra fCNT on the surface to get a spray‐coated sample, b). The dip coating method c) was followed by immersing the oxidized PDMS slides into the solution of positively charged fCNT overnight and washing with water to remove the extra fCNT on the surface to get the dip‐coated sample d). The samples were observed by optical microscope (scale bar, 50 µm) and camera (scale bar, 2 mm).

Figure 2

Surface property. a) SEM morphology of fCNT‐coated PDMS surface and without fCNT‐coated PDMS surface at low (up, 20 µm) and high (below, 1 µm) magnification. b) AFM images of fCNT‐coated PDMS surface and fCNT‐coated wrinkled PDMS surface, micro‐wrinkle size was 9.67 ± 0.32 µm in wavelength and 1.00 ± 0.08 µm in amplitude. c) Hydrophilicity property is measured by water contact angle on different surfaces. d) Schematic sketch of the PDMS cast on the microelectrodes and the corresponding equivalent circuit. Bode plots of the impedance modulus e) and phase f). Data are shown as mean ± standard deviation (SD) (***p < 0.001); three independent experiments were performed.

Figure 3

Live/Dead cell viability assay of V49 myoblast after 24 h cultured on fCNT‐coated surface and without fCNT‐coated surface. The cytoplasm of live cells emitted green fluorescence when stained with Calcein‐AM. The nuclei of dead cells emitted red fluorescence when stained with PI. The scale bar is 100 µm; 3 independent experiments were performed. Data are shown as mean ± standard deviation (SD). There is no significant difference between the groups.

Figure 4

Cell morphology of myoblasts. a) Typical immuno‐fluorescence images of V49 myoblast after 24 h grown on TCP, fCNT‐coated surfaces, and without fCNT‐coated surfaces. Cell nuclei were stained with DAPI (blue), F‐actin was stained with TRITC‐labeled phalloidin (red), and vinculin was stained with green. b) Quantitative analysis of Area per cell, c) Cell length, d) Cell width, e)Cell aspect ratio, and (f) FA area per cell. The scale bar is 50 µm. Data are shown as mean ± standard deviation (SD) (n = at least 100 cells), *p < 0.05, **p < 0.01, ***p < 0.001). Three independent experiments were performed. The yellow solid arrows indicate the direction of the micro‐wrinkle surface.

Figure 5

Fabrication of myotubes after 3 days of differentiation. a) Schematic illustration of the myogenic differentiation process. The cells were grown 2 days in proliferation medium, followed by starting to induce multiple myoblasts to fuse to myotubes, a multinucleated structure, in the presence of differentiation medium, and observed the fabrication of myotubes by staining nucleus and myosin heavy chain (a widely used myogenic differentiation marker) for 3 days and 8 days in differentiation medium. b) Typical immuno‐fluorescence images of V49 myoblast after 3 days of differentiation grown on TCP, fCNT‐coated surface, and without fCNT‐coated surface. The cell nuclei were stained with DAPI (blue), and Myotubes were stained with myosin heavy chain (green). c) Quantitative analysis of Myotube diameter, d) Myotube length, e)Myotube number, f) The total myotube area, g) The fusion index, which was calculated as the percentage of total nuclei inside the myotubes. h) Myosin fluorescence relative intensity normalized by the mean values of the total myotube area. The scale bar is 100 µm. Data are shown as mean ± standard deviation (SD) (n = at least 50 myotubes, *p < 0.05, **p < 0.01, ***p < 0.001); three independent experiments were performed. The yellow solid arrows indicate the direction of the micro‐wrinkle surface.