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

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.

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C2C12 cells encapsulated into 4% GelMA at different degrees of geometrical confinement. (A–F) Phase contrast micrographs of C2C12 captured 24 h (A–C) and 72 h (D–F) after cross-linking. From left to right: 2,000 μm × 2,000 μm; 1,000 μm × 1,000 μm; 500 μm × 500 μm. Black arrow in (F) indicates myostructure shrinkage occurring in the thinner structure. (G–L) Immunofluorescence (IF) against myosin heavy chain (MHC) (red) (G–I) following 14 days culture; nuclei were counterstained by DAPI (blue) (J–L). Scale bars: 100 µm. (M–O) Myotube orientation distribution plots (with 0° corresponding to the direction of the major axis of symmetry of each hydrogel structure) calculated from IF micrographs (MHC signal).
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Figure 5: C2C12 cells encapsulated into 4% GelMA at different degrees of geometrical confinement. (A–F) Phase contrast micrographs of C2C12 captured 24 h (A–C) and 72 h (D–F) after cross-linking. From left to right: 2,000 μm × 2,000 μm; 1,000 μm × 1,000 μm; 500 μm × 500 μm. Black arrow in (F) indicates myostructure shrinkage occurring in the thinner structure. (G–L) Immunofluorescence (IF) against myosin heavy chain (MHC) (red) (G–I) following 14 days culture; nuclei were counterstained by DAPI (blue) (J–L). Scale bars: 100 µm. (M–O) Myotube orientation distribution plots (with 0° corresponding to the direction of the major axis of symmetry of each hydrogel structure) calculated from IF micrographs (MHC signal).

Mentions: C2C12 cells were successfully retained within the three different hydrogel strings as confirmed by phase contrast micrographs showing a regular round shape (Figures 5A–C). After 3 days of culture, the structures revealed a remarkable compaction (Figures 5D–F) most likely due to C2C12 differentiation and myotube formation, particularly highlighted in the hydrogel with the smallest cross section (arrow in Figure 5F). Fourteen days after cell encapsulation, IF analyses were performed to evaluate the organization of MHC positive myotubes (Figures 5G–L). Micrographs showed a considerable parallel orientation for the myostructure cultured in the smallest (500 μm × 500 μm) and middle (1,000 μm × 1,000 μm) hydrogel structures (Figures 5H,I), while in the largest ones (2,000 μm × 2,000 μm), parallel organization was just partially achieved (Figure 5G), revealing the significant importance of proper geometrical confinement of myogenic precursors during the SM differentiation process. This can be better appreciated from the myotube orientation distribution plots (Figures 5M–O). In all cases, myotube orientation distribution was peaked along the hydrogel string axis. However, by reducing the hydrogel cross section, the distribution plots appeared increasingly sharp (Figures 5N,O), confirming that the geometrical confinement plays a significant role in myotube alignment.


Engineering Muscle Networks in 3D Gelatin Methacryloyl Hydrogels: Influence of Mechanical Stiffness and Geometrical Confinement
C2C12 cells encapsulated into 4% GelMA at different degrees of geometrical confinement. (A–F) Phase contrast micrographs of C2C12 captured 24 h (A–C) and 72 h (D–F) after cross-linking. From left to right: 2,000 μm × 2,000 μm; 1,000 μm × 1,000 μm; 500 μm × 500 μm. Black arrow in (F) indicates myostructure shrinkage occurring in the thinner structure. (G–L) Immunofluorescence (IF) against myosin heavy chain (MHC) (red) (G–I) following 14 days culture; nuclei were counterstained by DAPI (blue) (J–L). Scale bars: 100 µm. (M–O) Myotube orientation distribution plots (with 0° corresponding to the direction of the major axis of symmetry of each hydrogel structure) calculated from IF micrographs (MHC signal).
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Figure 5: C2C12 cells encapsulated into 4% GelMA at different degrees of geometrical confinement. (A–F) Phase contrast micrographs of C2C12 captured 24 h (A–C) and 72 h (D–F) after cross-linking. From left to right: 2,000 μm × 2,000 μm; 1,000 μm × 1,000 μm; 500 μm × 500 μm. Black arrow in (F) indicates myostructure shrinkage occurring in the thinner structure. (G–L) Immunofluorescence (IF) against myosin heavy chain (MHC) (red) (G–I) following 14 days culture; nuclei were counterstained by DAPI (blue) (J–L). Scale bars: 100 µm. (M–O) Myotube orientation distribution plots (with 0° corresponding to the direction of the major axis of symmetry of each hydrogel structure) calculated from IF micrographs (MHC signal).
Mentions: C2C12 cells were successfully retained within the three different hydrogel strings as confirmed by phase contrast micrographs showing a regular round shape (Figures 5A–C). After 3 days of culture, the structures revealed a remarkable compaction (Figures 5D–F) most likely due to C2C12 differentiation and myotube formation, particularly highlighted in the hydrogel with the smallest cross section (arrow in Figure 5F). Fourteen days after cell encapsulation, IF analyses were performed to evaluate the organization of MHC positive myotubes (Figures 5G–L). Micrographs showed a considerable parallel orientation for the myostructure cultured in the smallest (500 μm × 500 μm) and middle (1,000 μm × 1,000 μm) hydrogel structures (Figures 5H,I), while in the largest ones (2,000 μm × 2,000 μm), parallel organization was just partially achieved (Figure 5G), revealing the significant importance of proper geometrical confinement of myogenic precursors during the SM differentiation process. This can be better appreciated from the myotube orientation distribution plots (Figures 5M–O). In all cases, myotube orientation distribution was peaked along the hydrogel string axis. However, by reducing the hydrogel cross section, the distribution plots appeared increasingly sharp (Figures 5N,O), confirming that the geometrical confinement plays a significant role in myotube alignment.

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