Abstract
In this work, the influence of mechanical stiffness and geometrical confinement on the 3D culture of myoblast-laden gelatin methacryloyl (GelMA) photo-crosslinkable hydrogels was evaluated in terms of in vitro myogenesis. We formulated a set of cell-laden GelMA hydrogels with a compressive modulus in the range 1 ÷ 17 kPa, obtained by varying GelMA concentration and degree of cross-linking. C2C12 myoblasts were chosen as the cell model to investigate the supportiveness of different GelMA hydrogels toward myotube formation up to 2 weeks. Results showed that the hydrogels with a stiffness in the range 1 ÷ 3 kPa provided enhanced support to C2C12 differentiation in terms of myotube number, rate of formation, and space distribution. Finally, we studied the influence of geometrical confinement on myotube orientation by confining cells within thin hydrogel slabs having different cross sections: (i) 2,000 μm × 2,000 μm, (ii) 1,000 μm × 1,000 μm, and (iii) 500 μm × 500 μm. The obtained results showed that by reducing the cross section, i.e., by increasing the level of confinement—myotubes were more closely packed and formed aligned myostructures that better mimicked the native morphology of skeletal muscle.
Highlights
Skeletal muscle (SM) is a highly dynamic and plastic tissue able to modify its intrinsic size or strength following electric impulse, mechanical loading, or diet
We investigate the influence of two parameters— namely, hydrogel stiffness and geometrical confinement—on the in vitro differentiation of C2C12 myoblasts encapsulated in gelatin methacryloyl (GelMA) hydrogels
Since our first goal was to evaluate the influence of hydrogel stiffness on myoblast differentiation, before encapsulating the myogenic precursor, we explored two different variables that influence hydrogel stiffness: (i) the concentration of GelMA in the precursor hydrogel solutions and (ii) the UV crosslinking time
Summary
Skeletal muscle (SM) is a highly dynamic and plastic tissue able to modify its intrinsic size or strength following electric impulse, mechanical loading, or diet. SM accounts for about 30–45% of body weight, being the most abundant among human body tissues (Buckingham and Montarras, 2008). This tissue can self-repair relatively small damages resulting from tears, small lacerations, strains, or toxins via a three-stage process that involves demolition, repair, and remodeling of myofibers. Tissue engineering holds great promise for the fabrication of artificial muscles to be used for in vitro studies and for the replacement of diseased or injured muscle tissue (Bach et al, 2004; Levenberg et al, 2005). Due to its structural complexity, engineering a functional muscle tissue in vitro still represents a daunting task. Two of the most challenging aspects consist in attaining (i) a proper 3D organization of myotubes into highly packed and aligned structures
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