3.2.1. Fabrication of GelMA Hydrogel

(a) Synthesis of GelMA: Methacrylated gelatin was prepared as described earlier (Figure 1a) [25,31]. Concisely, 10% (w/v) of gelatin solution was prepared in phosphate-buffered saline (PBS) at 50 °C. Then, MA was added until the target volume was reached at a rate of 0.5 mL/min to the gelatin solution under continuous stirring at 50 °C. The reaction mixture was allowed to react for 3 h. The degree of methacrylation on gelatin was determined by varying the concentration of MA. After that, the reaction was stopped by adding an excess of warm PBS (40 °C). The reaction mixture was dialyzed against distilled water using 12–14 kDa cutoff dialysis tubing for 5 days at 40 °C to remove unreacted materials. At this point, the obtaining solution was lyophilized and the attained GelMA was stored at 4 °C until further use.

(b) Estimation of Degree of Methacrylation on Gelatin: The degree of methacrylation of lysine groups modified on the gelatin was determined by using the Habeeb method [45]. Briefly, 200 μg/mL (w/v) of freeze-dried GelMA was dissolved in 0.1 M sodium bicarbonate (pH 8.5). Next, 0.25 mL of TNBS solution (0.01% (w/v)) was added to 0.5 mL of each sample solution. The obtaining samples were nurtured at 37 °C for 3 h. Then, the reaction was stopped by adding 0.25 mL of 10% sodium dodecyl sulfate (SDS) and stabilized by adding 0.125 mL of 1 N hydrogen chloride (HCl). An optical density (OD) of specimens were measured using a UV/vis spectrophotometer at 335 nm. The degree of methacrylation was deliberated by comparing the amount of unreacted amino derivatives on gelatin and GelMA.

(c) Fabrication of GelMA Hydrogel: Pristine GelMA hydrogel was fabricated as follows. Initially, freeze-dried GelMA (10% w/v) was dissolved in PBS containing 0.5% (w/v) of Irgacure 2959 as a photoinitiator. Then, the precursor solution was placed in poly(dimethylsiloxane) (PDMS) molds and subsequently exposed to UV irradiation (wavelength 360–480 nm) for different time intervals (1 to 10 min) to form the pristine GelMA hydrogel with different crosslinking densities. Then, the GelMA hydrogel was placed into PBS, and the temperature was decreased to 37 °C to occur physical gelation. To prepare the DOX-loaded GelMA hydrogel, 5 mg/mL of DOX was added to the above solution (pristine GelMA) before the photo-cross-linking step. DOX loaded hydrogels represented as GelMA/DOX.