3-D in vitro model of early skeletal muscle development

Authors

  • U. Cheema,

    1. Tissue Repair and Engineering Centre, University College London, RFUCMS, Institute of Orthopaedics, RNOH, Stanmore, Middlesex, United Kingdom
    2. Department of Surgery, Royal Free and University College Medical School, London, United Kingdom
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  • S.-Y. Yang,

    1. Department of Surgery, Royal Free and University College Medical School, London, United Kingdom
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  • V. Mudera,

    1. Tissue Repair and Engineering Centre, University College London, RFUCMS, Institute of Orthopaedics, RNOH, Stanmore, Middlesex, United Kingdom
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  • G.G. Goldspink,

    1. Department of Surgery, Royal Free and University College Medical School, London, United Kingdom
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  • R.A. Brown

    Corresponding author
    1. Tissue Repair and Engineering Centre, University College London, RFUCMS, Institute of Orthopaedics, RNOH, Stanmore, Middlesex, United Kingdom
    • Tissue Repair & Engineering Centre, University College London, RFUCMS, Institute of Orthopaedics, RNOH, Stanmore, Middlesex, HA7 4LP, UK
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Abstract

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse c2c12 myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0–7 days). This identified a transient surge in both IGF-1 Ea and MGF expression on day 3 of the developing construct. This peak of IGF-1 Ea and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 Ea stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 Ea on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues. Cell Motil. Cytoskeleton 54:226–236, 2003. © 2003 Wiley-Liss, Inc.

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