Quantifying Methacrylation Degree in Gelatins

To determine the degree of substitution, the alternation in the amounts of primary amino functional groups of gelatins before and after methacrylation was quantitatively measured as suggested by a previous report [32 (link)]. To this end, a ninhydrin test (Sigma-Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s instructions. Both gelatin and GelMa were dissolved in deionized water, after which ninhydrin solution was dripped into the solutions and allowed to react for 10 min in an oven. An amount of 500 μL of pure ethanol was used to stop the reaction, and the absorbance was measured at a wavelength of 570 nm. Glycine, with its known wavelength among the various amino groups, was used as the comparison group. In addition, 1H NMR was used to determine for the degree of substitution. Both gelatin and GelMa were dissolved in deuterium oxide (Sigma-Aldrich) and analyzed using a 400 MHz NMR spectrometer (JEOL Co. Ltd., Tokyo, Japan). The degree of substitution was calculated using the following formula [33 (link)]: $\mathrm{Methacrylation}\mathrm{degree}(\%)=\frac{\mathrm{Number}\mathrm{of}\mathrm{methacrylate}\mathrm{groups}}{\mathrm{Number}\mathrm{of}\mathrm{amine}\mathrm{group}\mathrm{on}\mathrm{unreacted}\mathrm{polymers}}\times 100$