Printable ADA-GEL-based composite inks containing Zn-doped bioactive inorganic fillers for skeletal muscle biofabrication

Bioprinting shows significant promise in advancing medical treatments by offering patient-specific solutions for repairing skeletal muscle tissues. Zinc (Zn) is one of the key ions in the human body involved in the development of myogenic cells. This study investigates the integration of Zn-doped bioactive inorganic fillers (BIFs) into alginate-dialdehyde-gelatin (ADA-GEL) as composite ink for bioprinting applications. BIFs were mesoporous calcium-silicate-based bioactive glass nanoparticles with nominal composition: 80% SiO₂-X% CaO-Y% ZnO (X = 20, 18, 15, or 10; Y = 0, 2, 5, or 10; mol%). Ion release profiles confirmed that the addition of Zn ions prevented the burst release of Si and Ca ions. The incorporation of BIFs, particularly at higher dopant concentrations, significantly affected the hydrogel swelling and mechanical properties. With increasing concentration of Zn ions (to 5 mol%), the hydrogels exhibited greater dimensional swelling after 24 h of incubation in aqueous solutions, while all compositions lost weight over time after the initial swelling phase. Indirect cell studies demonstrated that 0.1 wt% BIFs extracts, obtained after 24 h-incubation in a cell culture medium, were cytocompatible with C2C12 cells. Furthermore, bioprinted C2C12 cells encapsulated in the bioinks showed a clear increase in cell number after seven days of culture. In particular, cells in the composite inks containing Zn-BIFs exhibited higher spreading, elongation, and alignment than those in pure ADA-GEL, indicating the biological activity provided by the Zn-BIF particles. This study introduces therefore an effective formulation of ADA-GEL-based composite bioinks enriched with Zn-containing nanoparticles for skeletal muscle tissue engineering applications.