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Polyelectrolyte Multilayer Films of Controlled Stiffness Modulate Myoblast Cell Differentiation

Authors

  • Kefeng Ren,

    1. Dynamique des Interactions Membranaires Normales et Pathologiques UMR 5235, CNRS, Université Montpellier II et I, cc 107 Montpellier, 34 095 (France)
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  • Thomas Crouzier,

    1. Dynamique des Interactions Membranaires Normales et Pathologiques UMR 5235, CNRS, Université Montpellier II et I, cc 107 Montpellier, 34 095 (France)
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  • Christian Roy,

    1. Dynamique des Interactions Membranaires Normales et Pathologiques UMR 5235, CNRS, Université Montpellier II et I, cc 107 Montpellier, 34 095 (France)
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  • Catherine Picart

    Corresponding author
    1. Dynamique des Interactions Membranaires Normales et Pathologiques UMR 5235, CNRS, Université Montpellier II et I, cc 107 Montpellier, 34 095 (France)
    • Dynamique des Interactions Membranaires Normales et Pathologiques UMR 5235, CNRS, Université Montpellier II et I, cc 107 Montpellier, 34 095 (France)
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  • This work has been supported by the ‘Association Françaize contre les Myopathies’ (AFM, grant no. 12671), by the ‘Association pour la Recherche sur la Cancer’ (grant no. 7918), by the ‘Fondation Recherche Médicale’ (grant no. INE20061108297), by ‘Agence Nationale pour la Recherche’ (grant ANR-06-NANO-006), and by the NIH (R21 grant) via a subcontract to C.P. (no. 544168A). The authors thank Cécile Gauthier-Rouvière (CRBM, Montpellier) for providing C2C12 cells and for fruitful discussions, and Fabrice Mérézègue and Philippe Montcourrier (CRBM, Montpellier) for their technical help with the SEM experiments. Dennis Discher, Adam Engler (University of Pennsylvania, Philadelphia), and Michel Pucéat (Généthon, Evry, France) are acknowledged for their fruitful advice. CP is a Junior Member of the ‘Institut Universitaire de France’ whose support is gratefully acknowledged. KR is indebted to the CNRS for providing a post-doctoral fellowship and TC thanks the AFM for a PhD fellowship. Supporting Information is available online from Wiley InterScience or from the author.

Abstract

In addition to their chemical properties and topographical features, the mechanical properties of gels have been recently demonstrated to play an important role in various cellular processes, which include cell attachment, proliferation, and differentiation. In this work, multilayer films made of poly(L-lysine)/hyaluronan (PLL/HA) of controlled stiffness have been used to investigate the effects of the mechanical properties of thin films on skeletal muscle cell (C2C12 cells) differentiation. Prior to differentiation, the cells need adhere and proliferate in a growth medium. Stiff films (E0 > 320 kPa) promote the formation of focal adhesions and organization of the cytoskeleton as well as an enhanced proliferation, whereas soft films are not favorable for cell anchoring, spreading, or proliferation. C2C12 cells were then switched to a low serum-containing medium to induce cell differentiation, which was also greatly dependent on film stiffness. Although myogenin and troponin T expressions are only moderately affected by the film stiffness, the morphology of the myotubes exhibit striking stiffness-dependent differences. Soft films allow differentiation only for a few days and the myotubes are very short and thick. Cell clumping followed by aggregate detachment is observed after ∼2 to 4 d. On stiffer films, significantly more elongated and thinner myotubes are observed for up to ∼2 weeks. Myotube striation is also observed but only for the stiffer films. These results demonstrate that film stiffness strongly modulates adhesion, proliferation and differentiation, each of these processes having its own stiffness requirement.

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