Geometry-dependent regulation of myogenic and osteogenic differentiation on microgeometry polystyrene substrates

Abstract

Advances in microfabrication technology have enabled precise control of surface geometry, which strongly influences cellular behavior, including adhesion, alignment, and differentiation. However, previous studies have employed diverse substrate materials and fabrication conditions, making it difficult to rigorously evaluate the pure geometric effects of the microstructures. Consequently, variations in physicochemical and mechanical properties, such as surface chemistry and stiffness, have confounded the interpretation of geometry-specific effects. To clarify the influence of microgeometry on cell behavior, particularly cell differentiation, stripe- and mesh-patterned polystyrene substrates were used to systematically investigate the relationship between surface geometry and cell behavior. Human mesenchymal stem cells (hMSCs) and C2C12 myoblasts were seeded on the substrates, and their adhesion morphology and alignment were observed using calcein-AM staining. Osteogenic and myogenic differentiation were subsequently induced, and the expression of differentiation markers was analyzed by immunostaining and RT-qPCR. In hMSCs, osteogenic differentiation was promoted in geometries that facilitated intercellular contact, whereas it was suppressed in highly confined geometries, such as stripes and meshes with greater ridge heights. In C2C12 myoblasts, a clear enhancement of myogenic differentiation was observed on striped substrates, where cells exhibited elongated morphologies aligned with the grooves, accompanied by an elevated expression of myogenin and dystrophin. These findings indicate that the differentiation-promoting or differentiation-suppressive effects of microgeometry are cell type-dependent and are governed by cellular alignment, intercellular interactions, and adhesion morphology. The insights gained from this study may contribute to the rational design of next-generation regenerative scaffolds and highlight the potential applications of microgeometric substrates in drug-screening platforms.

Keywords: C2C12 myoblasts; Cell alignment; Cell differentiation; Human mesenchymal stem cells; Microfabrication; Surface geometry; Tissue engineering..