Combined substrate micropatterning and FFT analysis reveals myotube size control and alignment by contact guidance

Abstract

Most important evaluating criteria for in vitro skeletal muscle models include the extent of differentiation and the degree of alignment in the tissue model. Substrate micropatterning is considered as an effective tool as it recreates in vivo like cellular microenvironment and helps in understanding the fundamental concepts and mechanisms underlying myogenesis. However, the influence of micropatterning based contact guidance cues over satellite cell alignment and myotube formation needs to be explored and studied further. In the present work, we demonstrate the regulation of myotube size control and alignment through the substrate micropatterning. For this purpose, primary myoblast cells (i.e., satellite cells) isolated from rat hind limb muscle were characterized and cultured for a period of 14 days on micropatterned glass substrates processed by the microchannnel flowed plasma process. Several characteristic parameters of muscle differentiation, including the fusion index, maturation index, and average width of the myotubes were quantified. The functional behavior of cultured myotubes exhibiting spontaneous contractions was assessed through kymograph to determine the twitch frequency. In addition, we evaluated the degree of alignment of myotubes on micropatterned substrates through examining orientation order parameter and two-dimensional fast Fourier transform analysis. Altogether, the outcomes reveal that the contact guidance cues arising due to micropatterning of the substrates could be a key regulator for controlling the size and degree of alignment of myotubes during the myogenesis process.contact guidance, microchannel flowed plasma, micropatterning, muscle differentiation, myogenesis, myotube alignment, orientation order parameter

Cell contact guidance via sensing anisotropy of network mechanical resistance

“…Contact guidance on 2D substrata like parallel grooves or adhesive ligand stripes has been intensively studied (recent examples include refs. [4][5][6] because in these experiments, the guidance field can be precisely defined and easily manipulated. However, it is the case of aligned fibrils that has the preponderance of physiological relevance.…”

Emulsion-templated microparticles with tunable stiffness and topology: Applications as edible microcarriers for cultured meat

“…S5 ). At length scales of a single cell, F-actin alignment can be an indicator of cellular elongation or stress fiber formation [ 53 , 54 ], and at length scales of myotubes, F-actin alignment correlates with myotube alignment [ 69 ], which is a key feature of skeletal muscle. To quantify F-actin alignment, we calculated a parameter termed the orientation index (OI) that is derived from the Fourier transform power spectrum of images to quantify actin alignment at different length scales: as actin alignment increases, OI → 100; as the fiber network becomes more diffuse and randomly oriented, OI → 0.…”