• Predicted ¹H NMR spectra & amino acids of gelatin/GelMA to calculate DS accurately • Applied DOE with full factorial design to rigorously validate new GelMA DS models • Validated GelMA DS equations via DOE and full factorial design for rigorous assessment • Developed a 2nd-order model using MAA/FAs to predict high-DS GelMA for synthesis control • Found non-linear DS behavior in high-DS GelMA, unlike linear trends at moderate levels Bioresorbable scaffolds are emerging as promising alternatives for regenerative medicine. Gelatin, a key structural component of many human tissues, and its methacrylated derivative, gelatin methacryloyl (GelMA), offer tunable mechanical and biochemical properties for tissue engineering. Accurate characterization and optimized functionalization are essential for their clinical application. This study presents a novel linear algebra–based method to predict the amino acid composition of gelatin from 1 H-NMR chemical shift regions, and conversely. The model was validated using experimental high-performance liquid chromatography (HPLC) and 1 H-NMR analyses of bovine-derived gelatin type B. Highly methacrylated gelatin was synthesized via a modified six-step methacrylic anhydride (MAA) addition protocol in CB buffer, reducing the MAA-to-free amine (MAA/FAs) molar ratio from 1.86 to 1.2 for a degree of substitution (DS) of ∼0.3 mmol/g. This modification increased methacrylation efficiency and enabled high DS levels. New internal standards, including methyl and vinyl proton signals of methacryloyl and methacrylate groups, were introduced in the ¹HNMR spectrum to improve the accuracy of DS determination. Evaluation using a complete factorial design of experiments (DOE) with varying MAA/FAs ratios identified the S 5 region (3.4-3.7ppm) and the methacrylate methyl signal as the most reliable DS indicators for highly methacrylated GelMA. Quadratic regression analysis revealed a reduction in methacrylation efficiency at higher DS values. Mechanistic analysis indicated predominant amine substitution at low DS, with hydroxyl substitution becoming more significant at high DS. Together, these advances enhance the characterization and functionalization of GelMA scaffolds, supporting the development of mechanically optimized bioresorbable materials for regenerative medicine.
Hosseinzadeh et al. (Fri,) studied this question.