Development and characterization of bioinks for 3D bioprinting of in vitro skeletal muscle constructs

Abstract The use of 3D bioprinting to construct in vitro skeletal muscle models presents a promising approach; however, selecting an optimal bioink remains a common challenge. This study focuses on the development and characterization of bioinks for extrusion-based 3D bioprinting, specifically targeting the creation of accurate skeletal muscle models. By exploring various compositions of alginate, gelatin, fibrinogen, and nanofiber cellulose, we evaluate these formulations based on printability and their support for the growth and differentiation of C2C12 myoblast cells. While alginate provided a strong, stable matrix for printing scaffolds embedded with C2C12 cells, it did not effectively promote cell growth and differentiation. The addition of fibrinogen to alginate enhanced cell growth and differentiation but was limited mainly to the scaffold surfaces, even with the inclusion of gelatin as a sacrificial ink. Notably, replacing alginate with nanofiber cellulose (NFC) alongside fibrinogen significantly improved cell growth and differentiation, leading to the formation of mature myotubes. Cell distribution was observed both inside and on the surfaces of the scaffolds, indicating effective spatial cell distribution. Furthermore, the scaffolds were tailored to form skeletal muscle bundles anchored between PDMS pillars for contractility testing. Upon exposure to electrical stimulation, the cells displayed measurable displacement, demonstrating contractile function. These findings offer valuable insights into optimizing bioink formulations that promote myoblast growth and differentiation into skeletal muscle in vitro , with potential applications in future neuromuscular disease modeling.