Micropatterning-Induced Alignment and Differentiation of Primary Skeletal Myoblasts on Plasma-Modified Substrates

Abstract

Reproducing the highly aligned architecture of native skeletal muscle remains a major challenge in skeletal muscle tissue engineering. In this study, a plasma-assisted microchannel-flowed plasma (µCFP) micropatterning technique was used to fabricate alternating hydrophilic and hydrophobic regions for guiding the alignment and differentiation of primary skeletal myoblasts derived from rat hind limb muscles. Micropatterns with widths of 20 µm, 200 µm, and 1000 µm were generated using octadecyl trichlorosilane (OTS) and 3-aminopropyltriethoxysilane (APTES) surface modification. Primary myoblasts isolated from rat skeletal muscle were cultured on these patterned substrates and evaluated for alignment, differentiation, fusion index, maturation index, myotube diameter, actin cytoskeleton organization, and spontaneous twitching activity. Micropatterned substrates significantly enhanced myoblast alignment and promoted the formation of elongated multinucleated myotubes compared with non-patterned controls. Orientation order parameter and two-dimensional fast Fourier transform (2D FFT) analyses confirmed improved directional organization of myotubes and actin cytoskeleton. The findings demonstrate that plasma-assisted micropatterning provides effective contact-guidance cues for regulating skeletal muscle cell organization and differentiation in vitro.