Spatiotemporal organized patterns of cell surface-associated and extracellular matrix (ECM)-embedded molecules play important roles in the development and functioning of tissues. ECM proteins interact with the surface of bioscaffold polymers and influence material-driven control of cell differentiation., Using X-ray phase-contrast micro computed tomography (microCT), we visualized the three-dimensional (3D) image of ECM organization after in vitro seeding of bone marrow-derived human and murine mesenchymal stem cells (MSCs) induced to myogenic differentiation, labelled with iron oxide nanoparticles, and seeded onto polyglycolic acid-polylactic acid scaffolds. X-ray microCT enabled us to detect with high spatial resolution the 3D structural organization of ECM within the bioscaffold and how the presence of cells modified the construct arrangement. Species-specific differences between the matrix produced by human and murine cells were observed. In conclusion, X-ray synchrotron radiation microCT analysis appeared to be a useful tool to identify the spatiotemporal pattern of organization of ECM fibers within a bioscaffold.