2.2. Gelatin methacryloyl (GelMA) synthesis and CHX/GelMA formulation

The GelMA synthesis was carried out as described previously [30,31]. Type-A gelatin (10 % w/v) was solubilized into DPBS with the help of a heating plate at 50 °C. Next, 8 mL of methacrylic anhydride (MA) was carefully dropped into the solution and left to react for 2 h under stirring conditions. To interrupt the functionalization, 100 mL of DPBS was added at 40 °C. To remove the unreacted monomers, the solution was poured into 12–14 kDa dialysis tubing and dialyzed in DI water at 45 ±5 °C for 1 week. The water was changed twice a day. After 7 days, the solution was filtered and freeze-dried at Labconco FreeZone 2.5 L (Labconco Corporation, Kansas City, MO, USA) for 5 days, then stored at − 20 °C until further use. The chemical characterization of GelMA was demonstrated by NMR spectra using DMSO at 35 °C at a frequency of 500 MHz and using a Varian INOVA NMR spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA) with a single axis gradient inverse probe.

CHX solutions at different concentrations (0.12, 0.5, 1 %, 2 %, and 5 %) were prepared by dissolving 20 % chlorhexidine digluconate (Lot #BCBM3595V, Sigma-Aldrich) solution in H₂O. Lyophilized GelMA was then dissolved into each solution at 15 % (w/v). After dissolving, the photoinitiator (LAP) was added 0.05 % (w/v) at 50 °C under stirring (240 rpm) conditions. Pure GelMA was used as a control (15 % GelMA, hereafter referred as GelMA group).