Skeletal muscle has a well-organized tissue structure comprised of aligned myofibers and an encasing extracellular matrix (ECM) sheath or lamina, within which reside satellite cells. We hypothesize that the organization of skeletal muscle tissues in culture can affect both the structure of the deposited ECM and the differentiation potential of developing myotubes. Furthermore, we posit that cellular and ECM cues can be a strong determinant of myoblast fusion and morphology in 3D tissue culture environments. To test these, we utilized a thermoresponsive nanofabricated substratum to engineer anisotropic sheets of myoblasts which could then be transferred and stacked into multilayered tissues. Within such engineered tissues, we found that myoblasts rapidly sense topography and deposit structurally organized ECM proteins. Furthermore, the initial tissue structure was found to exert significant control over myoblast fusion and eventual myotube organization. These results highlight the importance of ECM structure on myoblast fusion and organization, and provide insights into substrate-mediated control of myotube formation in the development of novel, more effective, engineered skeletal muscle tissues. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1543-1551, 2018.

We report a simple and versatile method for in vitro fabrication of scaffold-free tissue-engineered constructs with predetermined cellular alignment, by combining magnetic cell levitation with thermoresponsive nanofabricated substratum (TNFS) based cell sheet engineering technique. The TNFS based nanotopography provides contact guidance cues for regulation of cellular alignment and enables cell sheet transfer, while magnetic nanoparticles facilitate the magnetic levitation of the cell sheet. The temperature-mediated change in surface wettability of the thermoresponsive poly(N-isopropylacrylamide), PNIPAM, substratum enables the spontaneous detachment of cell monolayers, which can then be easily manipulated through use of a ring or disk shaped magnet. Our developed platform could be readily applicable to production of tissue-engineered constructs containing complex physiological structures for the study of tissue structure-function relationships, drug screening, and regenerative medicine.