Milliscale Substrate Curvature Promotes Myoblast Self‐Organization and Differentiation

Abstract

Biological tissues comprise complex structural environments known to influence cell behavior via multiple interdependent sensing and transduction mechanisms. Yet, and despite the predominantly nonplanar geometry of these environments, the impact of tissue‐size (milliscale) curvature on cell behavior is largely overlooked or underestimated. This study explores how concave, hemicylinder‐shaped surfaces 3–50 mm in diameter affect the migration, proliferation, orientation, and differentiation of C2C12 myoblasts. Notably, these milliscale cues significantly affect cell responses compared with planar substrates, with myoblasts grown on surfaces 7.5–15 mm in diameter showing prevalent migration and alignment parallel to the curvature axis. Moreover, surfaces within this curvature range promote myoblast differentiation and the formation of denser, more compact tissues comprising highly oriented multinucleated myotubes. Based on the similarity of effects, it is further proposed that myoblast susceptibility to substrate curvature depends on mechanotransduction signaling. This model thus supports the notion that cellular responses to substrate curvature and compliance share the same molecular pathways and that control of cell behavior can be achieved via modulation of either individual parameter or in combination. This correlation is relevant for elucidating how muscle tissue forms and heals, as well as for designing better biomaterials and more appropriate cell–surface interfaces.

Review on Porous Scaffolds Generation Process: A Tissue Engineering Approach

“…10 These concepts are starting to gain attention since they explain certain phenomena that can affect the complete scaffold, like a failure in cell attachment due to high curved struts 11 or due to high shear stress at the wall of pore size, 12 different rates of migration in curved surfaces, 13 and differentiation due to mechanical cues. 14 Due to the difficulty in controlling the pore at a cellular scale with current fabrication methods, mechanobiology has been studied mostly with computer-in silico models 15 or evaluated in single blocks and curved substrates, 16,17 using wavy landscapes, 18 microcurved channels, 19 or semispherical wells with different radii of curvature. 13,20 There are 524 articles on lens.org that relate scaffolds and mechanobiology in the past five years, and only 138 also include the term porous.…”

“…Due to the difficulty in controlling the pore at a cellular scale with current fabrication methods, mechanobiology has been studied mostly with computer- in silico models or evaluated in single blocks and curved substrates, , using wavy landscapes, microcurved channels, or semispherical wells with different radii of curvature. , …”

Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges

“…[358] Similarly, C2C12 myoblasts expressed more undifferentiated cell markers when grown on planar substrates or on half-cylinders with diameters above 15-35 mm and more markers of early and late-stage myogenic differentiation when the substrate diameter was below 10-20 mm, as well as more consistent alignment on substrates with small diameter curvatures. [359] Lithography, embossing, and micromolding have also been used to generate anisotropy within tissues. Anisotropy was successfully produced in thin films by casting a biopolymer solution using a laser-etched mold with appropriately-sized ridges, leading to better tissue organization and higher expression of muscle differentiation-associated markers compared to isotropic controls when these films were seeded with myoblasts.…”

“…[ 358 ] Similarly, C2C12 myoblasts expressed more undifferentiated cell markers when grown on planar substrates or on half‐cylinders with diameters above 15–35 mm and more markers of early and late‐stage myogenic differentiation when the substrate diameter was below 10–20 mm, as well as more consistent alignment on substrates with small diameter curvatures. [ 359 ]…”

Vascularizing the brain in vitro

“…These substrates have the main role of mimicking the BM, which has a thickness ranging from 20 to 200 nm ( Thomsen et al., 2017 ). Different substrate-related parameters were investigated and proved to have a great impact on cellular behavior, including nanoscale ordering ( Dalby et al., 2007 ), substrates chemistry ( Anderson et al., 2004 ; Nasir et al., 2021 ), substrate stiffness, and strain ( Leipzig and Shoichet, 2009 ; Yeung et al., 2005 ), cell shapes, and substrate curvature ( Baptista et al., 2019 ; Callens et al., 2020 ; Connon and Gouveia, 2021 ). Therefore, choosing an adequate substrate material and structure is fundamental to constructing the in vitro BBB model.…”