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Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle

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Abstract

Skeletal muscle lost through trauma or disease has proven difficult to regenerate due to the challenge of differentiating human myoblasts into aligned, contractile tissue. To address this, we investigated microenvironmental cues that drive myoblast differentiation into aligned myotubes for potential applications in skeletal muscle repair, organ-on-chip disease models and actuators for soft robotics. We used a 2D in vitro system to systematically evaluate the role of extracellular matrix (ECM) protein composition and geometric patterning for controlling the formation of highly aligned myotubes. Specifically, we analyzed myotubes differentiated from murine C2C12 cells and human skeletal muscle derived cells (SkMDCs) on micropatterned lines of laminin compared to fibronectin, collagen type I, and collagen type IV. Results showed that laminin supported significantly greater myotube formation from both cells types, resulting in greater than twofold increase in myotube area on these surfaces compared to the other ECM proteins. Species specific differences revealed that human SkMDCs uniaxially aligned over a wide range of micropatterned line dimensions, while C2C12s required specific line widths and spacings to do the same. Future work will incorporate these results to engineer aligned human skeletal muscle tissue in 2D for in vitro applications in disease modeling, drug discovery and toxicity screening.

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Abbreviations

ECM:

Extracellular matrix

LAM:

Laminin

FN:

Fibronectin

Col I:

Collagen I

Col IV:

Collagen IV

SkMDCs:

Skeletal muscle derived cells

μCP:

Microcontact printed

MFI:

Myotube fusion index (nuclei/myotube)

PDMS:

Polydimethylsiloxane

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Acknowledgments

Financial support from the National Institutes of Health Director’s New Innovator Award (DP2HL117750) to Adam Feinberg and the John and Claire Bertucci Fellowship from the Carnegie Institute of Technology to Rebecca Duffy. The lab of Johnny Huard at the University of Pittsburgh graciously provided initial aliquots of Cook Myosite human SkMDC and Cook MyoSite Inc. provided assistance with human SkMDC lot selection. We thank Lucas Friedman for providing assistance with image analysis.

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Authors and Affiliations

  1. Regenerative Biomaterials and Therapeutics Group, Department of Biomedical Engineering, Carnegie Mellon University, 700 Technology Dr., Pittsburgh, PA, 15219, USA

    Rebecca M. Duffy, Yan Sun & Adam W. Feinberg

  2. Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China

    Yan Sun

  3. Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA, 15219, USA

    Adam W. Feinberg

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  1. Rebecca M. Duffy
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Correspondence to Adam W. Feinberg.

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Associate Editor Akhilesh K Gaharwar oversaw the review of this article.

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Duffy, R.M., Sun, Y. & Feinberg, A.W. Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle. Ann Biomed Eng 44, 2076–2089 (2016). https://doi.org/10.1007/s10439-016-1592-8

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