Designing Tunable GelMA Hydrogels by Integrating Mammalian and Non-Mammalian Gelatins

Modulating the physical crosslink architecture of gelatin methacryloyl (GelMA) hydrogels without altering total polymer concentration or introducing exogenous components remains a central challenge in biomaterial design. Here, we present a source blending strategy in which porcine skin gelatin (PG) and salmon skin gelatin (SG), two gelatins with markedly different proline and hydroxyproline contents, are combined at seven compositional ratios (PG weight fractions 0–1.0) and subsequently functionalized to GelMA under standardized conditions (8% v/v methacrylic anhydride, 60 °C, 3 h). Near-complete degrees of substitution (95–98%) were achieved across all formulations, as confirmed by both TNBS and ¹H-NMR analyses. In the parent gelatin mixtures, increasing PG fraction progressively increased viscosity, elastic modulus (G′), gelation temperature (Tgel), and compression modulus at 4 °C, with DSC revealing independent SG (0–15 °C) and PG (20–40 °C) endothermic transitions that suggest partial hindrance of PG triple-helix formation by high SG fractions. These composition-dependent trends were preserved after functionalization to GelMA, albeit with attenuated physical crosslinking due to steric impairment by the methacrylate groups. Photocrosslinked GelMA hydrogels fabricated after pre-incubation at 4 °C exhibited systematically higher compression moduli and lower swelling degrees with increasing PG content, demonstrating that the PG/SG ratio provides an effective means for independently tuning hydrogel mechanics and mesh architecture. In vitro release assays using Rhodamine 6G further demonstrated that pre-incubation at 4 °C prior to photocrosslinking effectively modulates transport kinetics in SG-PG GelMA hydrogels. This strategy delayed characteristic release times and constrained Weibull shape parameters to the anomalous-transport regime (0.75 < β < 1), where diffusion is governed by network chain relaxation. This effect was most pronounced in the 0.4SG:0.6PG formulation, where lower SG content permitted unhindered triple-helix formation, as corroborated by DSC and compression studies. Ultimately, adjusting the pre-incubation temperature and gelatin source combination provides a straightforward, processing-additive-free strategy to achieve programmable release profiles via controlled matrix tortuosity.