3D bioprinting of alginate/gelatin hydrogels with tunable mechanical properties for skeletal muscle regeneration

Mechanically tunable double-network (DN) hydrogels are emerging as versatile biomaterials for soft tissue engineering, yet achieving precise control over their architecture and mechanics remains challenging. Here, we develop soft ionically crosslinked double-network hydrogels composed of sodium alginate (Alg) and gelatin (Gel) for three-dimensional (3D) skeletal muscle tissue engineering. Calcium ion (Ca²⁺) crosslinking of the Alg generated entrapped Gel microphases, producing mechanically reinforced hydrogels with interconnected microporous structures. Three compositions of Alg/Gel (Alg:Gel = 1:0, 1:0.25, and 1:0.50 w/w) were fabricated, in which increasing Gel content significantly modulated hydrogel properties. The compressive modulus increased from 14.8 kPa to 23.7 kPa, while the Alg:Gel (1:0.25) formulation exhibited the highest tensile strength of 194 kPa. The storage (G') and loss (G") moduli also increase with gelatin incorporation and exhibit maxima of 43.5 kPa (G'), and 9.6 kPa (G") for Alg/Gel (1:0.50). Developed Alg/Gel hydrogels were explored as bioinks for 3D bioprinting, enabling fabrication of mechanically stable 3D complex structures with high shape fidelity. C2C12 myoblasts encapsulated in 3D bioprinted Alg/Gel hydrogels exhibited robust metabolic activity, cell viability (>90%), and enhanced proliferation. Furthermore, the Alg/Gel hydrogels supported enhanced expression of MyoD, MyoG, myosin heavy chain (MYH), driving efficient myogenic differentiation and formation of multinucleated myotubes. Together, the tunable mechanics, microporosity, viscoelastic relaxation, and gelatin-mediated bioactivity position Alg/Gel double-network hydrogels as a promising bioink platform for 3D bioprinted skeletal muscle regeneration.