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Engineering Muscle Networks in 3D Gelatin Methacryloyl Hydrogels: Influence of Mechanical Stiffness and Geometrical Confinement

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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.

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Gelatin methacryloyl stiffness affecting C2C12 myogenesis as observed by phase contrast microscopy up to 14 days. Scale bars: 100 µm.
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Figure 3: Gelatin methacryloyl stiffness affecting C2C12 myogenesis as observed by phase contrast microscopy up to 14 days. Scale bars: 100 µm.

Mentions: Cell-laden hydrogels were timely observed by phase contrast microscopy up to 14 days, and the results are summarized in Figure 3. At lower concentrations (3–4% w/v), C2C12 rapidly spread within the hydrogel matrices revealing the formation of short myotubes already 48 h after polymerization, whereas at higher concentrations, cells presented limited spreading (6% w/v) or even almost exclusively round shape (8% w/v). At later time points—7/14 days after polymerization—C2C12 encapsulated into 3% GelMA hydrogel displayed a remarkable amount of myotubes forming a 3D-entangled network homogeneously distributed in the hydrogel volume. Similarly, 4% GelMA hydrogel supported a pronounced formation of myotubes, comparable to the one obtained with 3% GelMA, albeit with a slightly less homogeneous distribution. On the contrary, at higher GelMA concentrations (6–8%), C2C12 presented a remarkable detriment in myogenic differentiation, with minor myotube formation generally localized in clusters.


Engineering Muscle Networks in 3D Gelatin Methacryloyl Hydrogels: Influence of Mechanical Stiffness and Geometrical Confinement
Gelatin methacryloyl stiffness affecting C2C12 myogenesis as observed by phase contrast microscopy up to 14 days. Scale bars: 100 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383707&req=5

Figure 3: Gelatin methacryloyl stiffness affecting C2C12 myogenesis as observed by phase contrast microscopy up to 14 days. Scale bars: 100 µm.
Mentions: Cell-laden hydrogels were timely observed by phase contrast microscopy up to 14 days, and the results are summarized in Figure 3. At lower concentrations (3–4% w/v), C2C12 rapidly spread within the hydrogel matrices revealing the formation of short myotubes already 48 h after polymerization, whereas at higher concentrations, cells presented limited spreading (6% w/v) or even almost exclusively round shape (8% w/v). At later time points—7/14 days after polymerization—C2C12 encapsulated into 3% GelMA hydrogel displayed a remarkable amount of myotubes forming a 3D-entangled network homogeneously distributed in the hydrogel volume. Similarly, 4% GelMA hydrogel supported a pronounced formation of myotubes, comparable to the one obtained with 3% GelMA, albeit with a slightly less homogeneous distribution. On the contrary, at higher GelMA concentrations (6–8%), C2C12 presented a remarkable detriment in myogenic differentiation, with minor myotube formation generally localized in clusters.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus