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

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.

Keywords: C2C12 differentiation; gelatin methacryloyl; geometrical confinement; hydrogel stiffness; skeletal muscle.

Figure 1

(A) Scheme of the synthetic route for the synthesis of gelatin methacryloyl (GelMA) and (B) ¹H-NMR spectrum of GelMA in which the peaks relative to hydrophobic alkyl side chains of valine (Val), leucine (Leu), and isoleucine (Ile) and methacrylamide substituent groups of lysine (Lys) and hydroxylysine (Hly) amino acids are shown.

Figure 2

Stress–strain curves for different values of gelatin methacryloyl (GelMA) concentration (3, 4, 6, and 8% w/v) and UV cross-linking time [4 min (A) and 5 min (B)].

Figure 3

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

Figure 4

Scheme of the micromolding process for the fabrication of gelatin methacryloyl (GelMA) hydrogels: a U-shaped glass capillary is embedded within the hydrogel to act as a supporting frame for the hydrogel strings having different cross sections.

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).