Abstract
Adult newts possess the ability to completely regenerate organs and appendages. Immediately after limb loss, the extracellular matrix (ECM) undergoes dramatic changes that may provide mechanical and biochemical cues to guide the formation of the blastema, which is comprised of uncommitted stem-like cells that proliferate to replace the lost structure. Skeletal muscle is a known reservoir for blastema cells but the mechanism by which it contributes progenitor cells is still unclear. To create physiologically relevant culture conditions for the testing of primary newt muscle cells in vitro, the spatio-temporal distribution of ECM components and the mechanical properties of newt muscle were analyzed. Tenascin-C and hyaluronic acid (HA) were found to be dramatically upregulated in the amputated limb and were co-expressed around regenerating skeletal muscle. The transverse stiffness of muscle measured in situ was used as a guide to generate silicone-based substrates of physiological stiffness. Culturing newt muscle cells under different conditions revealed that the cells are sensitive to both matrix coating and substrate stiffness: Myoblasts on HA-coated soft substrates display a rounded morphology and become more elongated as the stiffness of the substrate increases. Coating of soft substrates with matrigel or fibronectin enhanced cell spreading and eventual cell fusion.
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Acknowledgments
The authors would like to thank Prof. Ken Shull and Katie Otim, of the Department of Materials Science and Engineering at Northwestern University, for assistance with mechanical testing. This research was funded by the NIH, DARPA and the Searle Foundation.
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Calve, S., Simon, HG. (2010). Extracellular Control of Limb Regeneration. In: Garikipati, K., Arruda, E. (eds) IUTAM Symposium on Cellular, Molecular and Tissue Mechanics. IUTAM Bookseries, vol 16. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-3348-2_22
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DOI: https://doi.org/10.1007/978-90-481-3348-2_22
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