Optimizing Gelatin Methacryloyl for Craniofacial Muscle Regeneration: Material Design and Application

Gelatin methacryloyl (GelMA) hydrogels are widely used in tissue engineering because of their tunable mechanical and biological properties. However, many studies have arbitrarily selected key synthesis parameters, such as methacrylic anhydride (MA) concentration, (lithium phenyl-2 4 6-trimethyl-benzoyl phosphinate) LAP concentration, GelMA content, UV exposure time, and reaction duration, without clear justification. This study aimed to systematically optimize GelMA hydrogel fabrication and evaluate the mechanical and biological performances of the resulting hydrogels for craniofacial muscle tissue engineering. Hydrogels were synthesized following a standardized protocol, and the reaction progress was confirmed via proton nuclear magnetic resonance (¹H-NMR). The swelling ratio, degradation behavior, compressive strength, and metabolic activity (AlamarBlue assay using C2C12 myoblasts) were assessed. Statistical analysis was performed using independent t-tests and one-way ANOVA with Tukey's post hoc test (p < 0.05). The results showed that small variations in MA concentration and reaction time significantly affected the hydrogel properties. Higher GelMA concentrations (10-20%) enhanced the mechanical strength but reduced the biological activity. LAP ≥ 0.5% and prolonged UV exposure lowered metabolic activity, whereas 0.1% LAP with 1-2 min of UV exposure provided an optimal balance. These findings provide a reproducible framework for GelMA fabrication and establish a foundation for developing tailored biomaterials for muscle tissue engineering.