Fabrication and characterization of a multifunctional alginate–gelatin–fibrinogen hydrogel for potential muscle tissue reconfiguration <i>in vitro</i>

In recent years, Matrigel-fibrinogen-thrombin (MFT) hydrogels have gained prominence in muscle tissue regeneration and biohybrid robotics owing to their remarkable bioactivity. Nevertheless, addressing their disadvantages-the instability of Matrigel, inadequate mechanical strength, complex fabrication processes, and limited structural tunability of MFT hydrogels-while maintaining biocompatibility remains challenging. In this study, sodium alginate and gelatin are used to formulate an alginate-gelatin-fibrinogen hydrogel that avoids the operational complexity associated with the initial cross-linking of thrombin and the instability of Matrigel. Notably, the alginate-gelatin-fibrinogen hydrogel showed significant shear-thinning behavior and exhibited good printability for different structures. This approach overcomes the critical limitations of extrusion printing using MFT hydrogels to fabricate muscle tissue structures with different requirements. We also used a novel quality assessment method proposed in our previous study, which incorporates the relative mean width (<i>Rel.X̄</i>) and relative standard deviation (<i>Rel.SD</i>) of extruded filaments to quantitatively evaluate printing quality. This approach enables the realization of an ideal printing boundary. In addition, a comparison with the Young's modulus of MFT hydrogels revealed that alginate-gelatin-fibrinogen hydrogels crosslinked with calcium chloride possessed significantly improved mechanical properties. The dual-crosslinking mechanism achieved <i>via</i> enzymatic and/or ionic methods resulted in flexible tunability of the modulus and porosity of the material. Experimental findings using C2C12 myoblast cells grown on the alginate-gelatin-fibrinogen hydrogel surface demonstrate that this biomaterial facilitates 3D bioprinting of anatomically complex muscle tissue constructs and biohybrid robotic systems. Overall, this study provides a novel strategy for the application in muscle tissue construction, repair, and bio-robotics through designing alginate-gelatin-fibrinogen composite hydrogels.