Abstract
Tissue engineering of skeletal muscle aims to replicate the parallel alignment of myotubes on the native tissue. Directional topography gradients allow the study of the influence of topography on cellular orientation, proliferation, and differentiation, resulting in yield cues and clues to develop a proper in vitro environment for muscle tissue engineering. In this study, we used a polydimethylsiloxane‐based substrate containing an aligned topography gradient with sinusoidal features ranging from wavelength (λ) = 1,520 nm and amplitude (A) =176 nm to λ = 9,934 nm and A = 2,168 nm. With this topography gradient, we evaluated the effect of topography on human myoblasts distribution, dominant orientation, cell area, nuclei coverage, cell area per number of nuclei, and nuclei area of myotubes. We showed that human myoblasts aligned and differentiated irrespective of the topography section. In addition, aligned human myotubes showed functionality and maturity by contracting spontaneously and nuclei peripheral organization resembling natural myotubes.
Highlights
Skeletal muscle is one of the tissues of the body with regenerative capacity
The images were analyzed with Image J to determine the nuclear area (DAPI), myotube diameter, and percentage of area covered by cells after 3 days in proliferation medium (PM) and 2 and 5 days in differentiation medium (DM)
Myoblasts aligned to the topography in all sections of the gradient during adhesion and proliferation (Figure 2d, PM) these cells had a random distribution on the tissue culture polystyrene (TCP) and flat PDMS controls
Summary
Skeletal muscle is one of the tissues of the body with regenerative capacity. After skeletal muscle injury, the endogenous muscle stem cells, satellite cells, are activated to recover the lost myofibers (Juhas, Ye, & Bursac, 2016). Cast ECM‐based hydrogels with embedded myoblasts and fixed at their termini were investigated for their propensity to build up pulling tension (Madden, Juhas, Kraus, Truskey, & Bursac, 2015) In these 3D systems, alignment of myotubes occurred, yet these were randomly scattered in the gels without formation of full size muscle fibers. Model substrates consisting of linearly aligned topography nanometer to micrometer‐sized gradients in polydimethylsiloxane (PDMS, silicone rubber) are useful to investigate biological features such as adhesion, proliferation, morphology, and differentiation of (stem) cells (Zhou et al, 2017, 2015a) These 2D systems, more than 3D systems, add to understand the role between the topography and muscle formation (Duffy et al, 2016). We hypothesized that primary human myoblasts adhere and proliferate in a preferred surface topography, whereas this promotes fusion, maturation, and alignment of myotubes
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