Preparation of gelatin methacryloyl hydrogels

Gelatin methacryloyl (GelMA) macromers were synthesized according to a previously described method (28). Briefly, to obtain a 10 weight percentage (wt %) gelatin aqueous solution, 10 g gelatin (type A, 300 bloom; Sigma-Aldrich, St. Louis, MO) was dissolved in 100 mL of phosphate-buffered saline (PBS) at 60°C under stirring. Then, 8 mL methacrylic anhydride (Sigma-Aldrich) was added into the gelatin solution at a rate of 0.5 mL/min under stirring at 50°C. After a reaction in the dark for 4 h, the products were diluted with fivefold PBS (50°C) and then dialyzed against Milli-Q water for 7 days at 40°C using a dialysis membrane (12–14 kD molecular weight cut-off; Spectrum Laboratory, Cary, NC) to remove salts and excess free methacrylic anhydride. The products were then lyophilized for 2 days to obtain white porous foam.

To obtain GelMA hydrogels with different stiffness, different concentrations of GelMA macromers (10, 12, and 14 wt %) and photoinitiator 2-hydroxy-1-(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959; Sigma-Aldrich) (1 wt %) were dissolved in PBS at 50°C. The solution was added into a six-well plate (Thermo Fisher Scientific, Waltham, MA), forming a hydrogel substrate with a diameter of ∼35 mm and a thickness of ∼1 mm. The construct was exposed to 365 nm ultraviolet light (CL-1000; Funakoshi, Tokyo, Japan) at a distance of 20 cm for 1 min with an irradiation intensity of 6.9 mW/cm² (Fig. 1).

The stiffness of GelMA hydrogels was determined by BOSE ELF 3200 dynamic mechanical analyzer (BOSE). Hydrogel samples were incubated in PBS at 37°C for 24 h, and their stiffness was then measured at room temperature. Five samples of each group were measured for the calculation of means and SDs.