This study explores the molecular structuring of salmon gelatin (SG) with controlled molecular weight produced from salmon skin, and its relationship with its thermal and rheological properties. SG was produced under different pH conditions to produce samples with well-defined high (SGH), medium (SGM), and low (SGL) molecular weight. These samples were characterized in terms of their molecular weight (MW, capillary viscometry), molecular weight distribution (electrophoresis), amino acid profile, and Raman spectroscopy. These results were correlated with thermal (gelation energy) and rheological properties. SGH presented the higher MW (173 kDa) whereas SGL showed shorter gelatin polymer chains (MW < 65 kDa). Raman spectra and gelation energy suggest that amount of helical structures in gelatin is dependent on the molecular weight, which was well reflected by the higher viscosity and G′ values for SGH. Interestingly, for all the molecular weight and molecular configuration tested, SG behaved as a strong gel (tan δ < 1), despite its low viscosity and low gelation temperature (3–10 °C). Hence, the molecular structuring of SG reflected directly on the thermal and viscosity properties, but not in terms of the viscoelastic strength of gelatin produced. These results give new insights about the relationship among structural features and macromolecular properties (thermal and rheological), which is relevant to design a low viscosity biomaterial with tailored properties for specific applications.

For biomedical applications, gelatin is usually modified with methacryloyl groups to obtain gelatin methacryloyl (GelMA), which can be crosslinked by a radical reaction induced by low wavelength light to form mechanically stable hydrogels. The potential of GelMA hydrogels for tissue engineering has been well established, however, one of the main disadvantages of mammalian-origin gelatins is that their sol-gel transitions are close to room temperature, resulting in significant variations in viscosity that can be a problem for biofabrication applications. For these applications, cold-water fish-derived gelatins, such as salmon gelatin, are a good alternative due to their lower viscosity, viscoelastic and mechanical properties, as well as lower sol-gel transition temperatures, when compared with mammalian gelatins. However, information regarding GelMA (with special focus on salmon GelMA as a model for cold-water species) molecular conformation and the effect of pH prior to crosslinking, which is key for fabrication purposes since it will determine final hydrogel’s structure, remains scarce. The aim of this work is to characterize salmon gelatin (SGel) and salmon methacryloyl gelatin (SGelMA) molecular configuration at two different acidic pHs (3.6 and 4.8) and to compare them to commercial porcine gelatin (PGel) and methacryloyl porcine gelatin (PGelMA), usually used for biomedical applications. Specifically, we evaluated gelatin and GelMA samples’ molecular weight, isoelectric point (IEP), their molecular configuration by circular dichroism (CD), and determined their rheological and thermophysical properties. Results showed that functionalization affected gelatin molecular weight and IEP. Additionally, functionalization and pH affected gelatin molecular structure and rheological and thermal properties. Interestingly, the SGel and SGelMA molecular structure was more sensitive to pH changes, showing differences in gelation temperatures and triple helix formation than PGelMA. This work suggests that SGelMA presents high tunability as a biomaterial for biofabrication, highlighting the importance of a proper GelMA molecular configuration characterization prior to hydrogel fabrication.

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.

Modulating the physical crosslink architecture of gelatin methacryloyl (GelMA) hydro-gels without altering total polymer concentration or introducing exogenous components remains a central challenge in biomaterial design. Here we report 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 across seven compositional ratios (PG weight fractions 0–1.0) and functionalized to GelMA under standardized conditions (8% v/v methacrylic anhydride, 60 °C, 3 h). Near-complete de-grees of substitution (95–98%) were achieved across all formulations, confirmed by both TNBS and ¹H-NMR. 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) en-dothermic 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 is an effective lever for independently tuning hydrogel mechanics and mesh architecture. In vitro release assays using Rhodamine 6G confirmed that pre-incubation at 4°C prior photocrosslinking serves as a effective way to modulate 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 thermally induced structural restriction 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.