Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle
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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.
KeywordsSkeletal muscle Tissue engineering Extracellular matrix Microcontact printing
- Col I
- Col IV
Skeletal muscle derived cells
Myotube fusion index (nuclei/myotube)
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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