Understanding the Molecular Conformation and Viscoelasticity of Low Sol-Gel Transition Temperature Gelatin Methacryloyl Suspensions.

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.

Anatase Incorporation to Bioactive Scaffolds Based on Salmon Gelatin and Its Effects on Muscle Cell Growth.

The development of new polymer scaffolds is essential for tissue engineering and for culturing cells. The use of non-mammalian bioactive components to formulate these materials is an emerging field. In our previous work, a scaffold based on salmon gelatin was developed and tested in animal models to regenerate tissues effectively and safely. Here, the incorporation of anatase nanoparticles into this scaffold was formulated, studying the new composite structure by scanning electron microscopy, differential scanning calorimetry and dynamic mechanical analysis. The incorporation of anatase nanoparticles modified the scaffold microstructure by increasing the pore size from 208 to 239 µm and significantly changing the pore shape. The glass transition temperature changed from 46.9 to 55.8 °C, and an increase in the elastic modulus from 79.5 to 537.8 kPa was observed. The biocompatibility of the scaffolds was tested using C2C12 myoblasts, modulating their attachment and growth. The anatase nanoparticles modified the stiffness of the material, making it possible to increase the growth of myoblasts cultured onto scaffolds, which envisions their use in muscle tissue engineering.

Rheological and Structural Study of Salmon Gelatin with Controlled Molecular Weight.

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.

Exploiting the natural poly(3-hydroxyalkanoates) production capacity of Antarctic Pseudomonas strains: from unique phenotypes to novel biopolymers.

Extreme environments are a unique source of microorganisms encoding metabolic capacities that remain largely unexplored. In this work, we isolated two Antarctic bacterial strains able to produce poly(3-hydroxyalkanoates) (PHAs), which were classified after 16S rRNA analysis as Pseudomonas sp. MPC5 and MPC6. The MPC6 strain presented nearly the same specific growth rate whether subjected to a temperature of 4 °C 0.18 (1/h) or 30 °C 0.2 (1/h) on glycerol. Both Pseudomonas strains produced high levels of PHAs and exopolysaccharides from glycerol at 4 °C and 30 °C in batch cultures, an attribute that has not been previously described for bacteria of this genus. The MPC5 strain produced the distinctive medium-chain-length-PHA whereas Pseudomonas sp. MPC6 synthesized a novel polyoxoester composed of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate). Batch bioreactor production of PHAs in MPC6 resulted in a titer of 2.6 (g/L) and 1.3 (g/L), accumulating 47.3% and 34.5% of the cell dry mass as PHA, at 30 and 4 °C, respectively. This study paves the way for using Antarctic Pseudomonas strains for biosynthesizing novel PHAs from low-cost substrates such as glycerol and the possibility to carry out the bioconversion process for biopolymer synthesis without the need for temperature control.

Edible Scaffolds Based on Non-Mammalian Biopolymers for Myoblast Growth.

In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 µm diameter) and biocompatibility, permitting myoblast cell adhesion (~40%) and growth (~24 h duplication time). The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.