Résumé :
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There is increasing agreement that 3D culture of skeletal muscle satellite cells and myoblasts holds great promise to investigate pathophysiological mechanisms and to test therapeutic strategies for neuromuscular disorders. However, the construction of functional, tissue-engineered skeletal muscles (TESM) containing more than a few layers of mature muscle cells remains challenging. Obvious concerns relate to incomplete myotube differentiation, impaired nutrient diffusion and limited cell viability in bioartificial constructs. Because the Arg-Gly-Asp (RGD) motif of fibronectin plays a critical role in cell-extracellular matrix interactions through a specific interaction with integrins ?5?1, we hypothesized that TESM development could be improved by coupling RGD peptides to collagen scaffold. The method used for the RGD-collagen scaffold coupling allows i) high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; ii) spectroscopic quantification of RGD coupling; iii) a spacer arm of 36 angstroms, a length reported as optimal for RGD peptide presentation and favorable for integrin receptor clustering and subsequent activation. A nonfunctional RGE peptide was used as control. Collagen-cross-linked RGD or RGE scaffolds were seeded with immortalized human myoblasts (hMS, clone 9) and incubated in proliferative and then differentiation medium. Morphological analysis demonstrated that both collagen scaffolds have the ability to support hMS cell proliferation and differentiation. However, the presence of RGD improved cell attachment by activating ?5 integrin signalling pathway, enhanced proliferation and cell migration within the matrix, and enhanced differentiation of myotubes. In conclusion, we report a novel method of engineering a highly effective and stable mature tissue-engineered human skeletal muscle based on collagen matrix cross-linked to RGD peptides. It should provide a useful tool for basic research and for identifying new therapeutic strategies in human myopathies.
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