Skeletal muscle regenerative approach combining stem cells and 3D bioprinting

Skeletal muscles own a remarkable self-repair and regenerative capacity in response to acute injuries, such as exercise-induced damage or disease. However, when muscle loss becomes irreversible, lesions are so severe that they impair muscle functionality. Thus, 3D bioprinting approaches are becoming more wide-spread with the important task to reproduce in a three-dimensional way the proper fiber organization and finally the functional skeletal muscle tissue. In this context, hydrogels are suitable scaffolds to mimic extracellular matrix and support tissue functions. In this study, we present a new strategy for the generation of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is built on a pneumatic extrusion-based 3D bio-plotter (INCREDIBLE+) for high resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we use a bioink with a specific alginate/fibrinogen-based hydrogel (CELLINK) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 14 till 28 days of culture, the encapsulated myoblasts started fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation and sarcomere genesis. These results were confirmed by immunofluorescence and qRT-PCR analysis, revealing the expression of specific myogenic differentiation markers including MyHC, MyoD and MCK respectively. Finally, we demonstrate that myoblast bioprinting allows to design 3D multicellular assemblies with characteristic compartmentalization of the encapsulated myogenic cells. Our preliminary results demonstrate myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid method for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for further clinical applications