Methacrylic anhydride

Gelatin methacrylate (GelMA) was synthesized according to previously published methods^{31 (link)–33 (link)}. Briefly, Type A porcine skin gelatin (Sigma-Aldrich; 300 bloom) was mixed at 10% (w/v) in phosphate buffered saline (PBS; Thermo Fisher Scientific) at 50°C for 20 minutes. Methacrylic anhydride (Sigma-Aldrich) was then added at 0.6 g of Methacrylic anhydride / g gelatin under rigorous stirring for one hour. The reactants were then diluted 2x with PBS to stop the reaction. The mixture was centrifuged and the pellet was discarded. The supernatant was dialyzed against deionized water at 50°C using dialysis cassettes (10kDa MWCO, Thermo Fisher Scientific) for at least 3 days. The deionized water was changed twice a day to remove salts and excess methacrylic acid. The dialyzed GelMA was then lyophilized for at least 3 days and store at −80°C until further use.

For in vivo procedures, gelatin methacrylate (GelMA) was purchased (Sigma, 900496). For in vitro procedures, GelMA was synthesized by dissolving Type A porcine skin gelatin (Sigma, G1890) at 10% (w/v) in 1x Dulbecco’s phosphate buffered saline without calcium at 50C for 1 h. Then, methacrylic anhydride (Sigma) was added dropwise to a final volume ratio of 1:4 methacrylic anhydride:gelatin solution. This resulted in GelMA with a degree of substitution of 80%. The solution was stirred at 50 C for 1 h, and then diluted 5X with DPBS. The resulting mixture was dialyzed for 3-4 d against distilled water with frequent water replacement in 12-14 kDa molecular weight cutoff tubing (Spectrum Labs). The dialyzed solution was lyophilized, and the resulting GelMA was stored at −20 C freezer until use.

Gelatin methacrylate was synthesized according to an earlier protocol.^{[56 (link)]} Briefly, a 10% w/v gelatin (porcine skin, Sigma) solution in phosphate buffered saline (PBS) was prepared and heated to 60 °C with constant mixing. After the gelatin was dissolved completely, the temperature was reduced to 50 °C and allowed to reach steady state. After the solution temperature reached 50 °C, methacrylic anhydride (Sigma) was slowly added to the solution to achieve a 5:1 volumetric ratio of gelatin solution : methacrylic anhydride solution. The typical basis reaction volume consisted of 50 ml gelatin solution. Subsequently, the mixture was allowed to react for one hour at 50 °C with continual mixing. After one hour, warmed PBS (40 °C) heated in a secondary beaker was added at a 4:1 volume ratio to the gelatin-methacrylic anhydride solution to deactivate the reaction. Subsequently, the resulting mixture was added to 10 kDa dialysis tubing and the tubing was placed in reverse osmosis water and allowed to dialyze for one week. To ensure effective separation, the dialysis solution was replaced with fresh solution daily. Following the dialysis procedure, the gelatin mixture was lyophilized until dry.

N-Methacrylation of chitosan is the method that has been adopted in our lab after it has been used and characterized by Li et al. [10 (link)] based on a previous method by Yu et al. [17 (link)] and it is summarized in Fig. 1. Briefly, chitosan (Protosan UP B80/20, NovaMatrix, Drammen, Norway) was dissolved in 2% (v/v) acetic acid at a final concentration of 3 wt%. Methacrylic anhydride (Sigma-Aldrich, St. Louis, MO, USA) was then added at 1 : 0.4 molar ratio (chitosan : Methacrylic anhydride) and allowed to mix magnetically for 3+ h at RT. After complete dissolution, the mixture was dialyzed (12–14 kDa dialysis tubing, Spectrum Labs, Waltham, MA, USA) against distilled water for 72 hours. Following dialysis, samples were freeze-dried and stored at −20 °C until use.