An advanced biphasic porous and injectable scaffold displays a fine balance between mechanical strength and remodeling capabilities essential for cartilage regeneration.

An important challenge in tissue engineering is the regeneration of functional articular cartilage (AC). In the field, biomimetic hydrogels are being extensively studied as scaffolds that recapitulate microenvironmental features or as mechanical supports for transplanted cells. New advanced hydrogel formulations based on salmon methacrylate gelatin (sGelMA), a cold-adapted biomaterial, are presented in this work. The psychrophilic nature of this biomaterial provides rheological advantages allowing the fabrication of scaffolds with high concentrations of the biopolymer and high mechanical strength, suitable for formulating injectable hydrogels with high mechanical strength for cartilage regeneration. However, highly intricate cell-laden scaffolds derived from highly concentrated sGelMA solutions could be deleterious for cells and scaffold remodeling. On this account, the current study proposes the use of sGelMA supplemented with a mesophilic sacrificial porogenic component. The cytocompatibility of different sGelMA-based formulations is tested through the encapsulation of osteoarthritic chondrocytes (OACs) and stimulated to synthesize extracellular matrix (ECM) components in vitro and in vivo. The sGelMA-derived scaffolds reach high levels of stiffness, and the inclusion of porogens impacts positively the scaffold degradability and molecular diffusion, improved fitness of OACs, increased the expression of cartilage-related genes, increased glycosaminoglycan (GAG) synthesis, and improved remodeling toward cartilage-like tissues. Altogether, these data support the use of sGelMA solutions in combination with mammalian solid gelatin beads for highly injectable formulations for cartilage regeneration, strengthening the importance of the balance between mechanical properties and remodeling capabilities.

A novel porous hydrogel based on hybrid gelation for injectable and tough scaffold implantation and tissue engineering applications.

Although there have been many advances in injectable hydrogels as scaffolds for tissue engineering or as payload-containing vehicles, the lack of adequate microporosity for the desired cell behavior, tissue integration, and successful tissue generation remains an important drawback. Herein, we describe an effective porous injectable system that allowsin vivoformation of pores through conventional syringe injection at room temperature. This system is based on the differential melting profiles of photocrosslinkable salmon gelatin and physically crosslinked porogens of porcine gelatin (PG), in which PG porogens are solid beads, while salmon methacrylamide gelatin remains liquid during the injection procedure. After injection and photocrosslinking, the porogens were degraded in response to the physiological temperature, enabling the generation of a homogeneous porous structure within the hydrogel. The resultant porogen-containing formulations exhibited controlled gelation kinetics within a broad temperature window (18.5 ± 0.5-28.8 ± 0.8 °C), low viscosity (133 ± 1.4-188 ± 16 cP), low force requirements for injectability (17 ± 0.3-39 ± 1 N), robust mechanical properties after photo-crosslinking (100.9 ± 3.4-332 ± 13.2 kPa), and favorable cytocompatibility (>70% cell viability). Remarkably,in vivosubcutaneous injection demonstrated the suitability of the system with appropriate viscosity and swift crosslinking to generate porous hydrogels. The resulting injected porous constructs showed favorable biocompatibility and facilitated cell infiltration for desirable potential tissue remodeling. Finally, the porogen-containing formulations exhibited favorable handling, easy deposition, and good shape fidelity when used as bioinks in 3D bioprinting technology. This injectable porous system serves as a platform for various biomedical applications, thereby inspiring future advances in cell therapy and tissue engineering.

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.

The impact of gender and peripheral blood parameters on the characteristics of L-PRF membranes.

Aim: The purpose of this study was to evaluate the impact of gender and peripheral blood parameters on the characteristics of Leucocyte-and Platelet-Rich Fibrin (L-PRF) membranes and to describe histologically three different zones of L-PRF membranes. Methods: Blood was collected from twenty healthy donors (10 men and 10 women). Peripheral blood parameters including leucocyte and platelet counts, and fibrinogen levels were recorded. L-PRF membranes were prepared to quantify the release of growth factors (PDGF, VEGF, BMP-2, and BMP-9) at 1, 2, 3 and 7 days and for histological examination. Three zones within each L-PRF membrane (face, body, and tail) were analysed separately, quantifying the area of leucocytes, platelets, and fibrin in percentage. The Young's modulus of the membranes was also considered (during tensile and compression tests). Results: Women had significantly higher fibrinogen levels in their peripheral blood, and a higher release of BMP-9, whereas men showed a significantly higher Young's modulus in compression tests. The histology revealed significant differences in cellular content and fibrin concentration between the 3 areas, with the face being biologically the richest. Conclusion: Several factors influenced the final characteristics of L-PRF membranes. These need to be taken into consideration when interpreting the results of research, but especially in clinical practice.

Rheological and Thermal Study about the Gelatinization of Different Starches (Potato, Wheat and Waxy) in Blend with Cellulose Nanocrystals.

The goal of this work was to analyze the effect of CNCs on the gelatinization of different starches (potato, wheat and waxy maize) through the characterization of the rheological and thermal properties of starch-CNC blends. CNCs were blended with different starches, adding CNCs at concentrations of 0, 2, 6 and 10% w/w. Starch-CNC blends were processed by rapid visco-analysis (RVA) and cooled to 70 °C. Pasting parameters such as pasting temperature, peak, hold and breakdown viscosity were assessed. After RVA testing, starch-CNC blends were immediately analyzed by rotational and dynamic rheology at 70 °C. Gelatinization temperature and enthalpy were assessed by differential scanning calorimetry. Our results suggest that CNCs modify the starch gelatinization but that this behavior depends on the starch origin. In potato starch, CNCs promoted a less organized structure after gelatinization which would allow a higher interaction amylose-CNC. However, this behavior was not observed in wheat and waxy maize starch. Insights focusing on the role of CNC on gelatinization yielded relevant information for better understanding the structural changes that take place on starch during storage, which are closely related with starch retrogradation. This insight can be used as an input for the tailored design of novel materials oriented towards different technological applications.

Effect of Adding Curcumin on the Properties of Linseed Oil Organogels Used as Fat Replacers in Pâtés.

Beeswax-based organogels were formulated with linseed oil and curcumin according to a statistical design to increase the oxidative stability of spreadable meat products (pâté) where these organogels (OGCur) were incorporated as fat substitutes. The organogels obtained under optimal conditions (9.12% beeswax, 0.54% curcumin) showed a mechanical strength similar to pork backfat determined by back extrusion and high oil binding capacity (OBC; over 90%). The incorporation of curcumin at this concentration did not lead to any change in the arrangement of the crystal network, OBC, and mechanical, thermal, or rheological properties of the organogels. Beeswax organogels with and without curcumin, with a ß' orthorhombic subcell structure, showed a predominant elastic behavior and a melting event wider and shifted to lower temperatures than pure beeswax, suggesting a plasticizer effect of the oil in the wax crystals. The oxidative stability of the organogels under accelerated oxidation conditions increased due to the incorporation of curcumin. A decrease in the curcumin content was found from day 4 at 60 °C, together with a significantly lower formation of both peroxides and malonaldehyde. When pork backfat was partially or totally replaced by OGCur in pâtés, a noticeable protective effect of curcumin against lipid oxidation was found during chilled storage.

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.

Cold-adaptation of a methacrylamide gelatin towards the expansion of the biomaterial toolbox for specialized functionalities in tissue engineering.

Tissue regeneration is witnessing a significant surge in advanced medicine. It requires the interaction of scaffolds with different cell types for efficient tissue formation post-implantation. The presence of tissue subtypes in more complex organs demands the co-existence of different biomaterials showing different hydrolysis rate for specialized cell-dependent remodeling. To expand the available toolbox of biomaterials with sufficient mechanical strength and variable rate of enzymatic degradation, a cold-adapted methacrylamide gelatin was developed from salmon skin. Compared with mammalian methacrylamide gelatin (GelMA), hydrogels derived from salmon GelMA displayed similar mechanical properties than the former. Nevertheless, salmon gelatin and salmon GelMA-derived hydrogels presented characteristics common of cold-adaptation, such as reduced activation energy for collagenase, increased enzymatic hydrolysis turnover of hydrogels, increased interconnected polypeptides molecular mobility and lower physical gelation capability. These properties resulted in increased cell-remodeling rate in vitro and in vivo, proving the potential and biological tolerance of this mechanically adequate cold-adapted biomaterial as alternative scaffold subtypes with improved cell invasion and tissue fusion capacity.

Design of a biodegradable UV-irradiated gelatin-chitosan/nanocomposed membrane with osteogenic ability for application in bone regeneration.

Guided bone regeneration membranes are used in oral surgery to protect the site of a lesion exposed to connective tissue invasion which, in turn, prevents new bone formation. Although non-degradable and degradable materials have been applied in clinical treatments, biodegradable membranes have the advantage that they do not require a secondary surgical procedure to be removed. However, they have a very low mechanical strength. As biodegradable membranes, biomaterials based on gelatin-chitosan have gained importance in clinical applications due to their unique properties. Gelatin contains RGD-like sequences, promoting cell adhesion/migration, and it can be blended with chitosan, which allows the immobilization of nanoparticles. In this work, we designed a new gelatin-chitosan polymeric membrane which contains hydroxyapatite and titania nanoparticles as two very well-documented osteoconductive materials. UV radiation was used as a non-toxic cross-linking agent to improve the thermophysical/mechanical characteristics and to control the biodegradability of the nanocomposed membrane. The microstructure, thermophysical and mechanical properties of the UV-irradiated material were studied by scanning electron microscopy, differential scanning calorimetry and Young's modulus, respectively. The in vitro biocompatibility of the new nanocomposite was evaluated by cell adhesion and proliferation assays. The osteoconductive ability was determined by an alkaline phosphatase production assay using mouse embryonic fibroblast (MEF) cells. The results show a homogeneous material with an appropriate distribution of nanoparticles. Cross-linking by UV radiation improved the mechanical and biological performance of the membrane. The presence of two osteoconductive nanoparticles, such as titania and hydroxyapatite, increased the osteogenic potential of the gelatin-based material in vitro, which confers a biological function, in addition to functioning as a physical barrier. The material obtained herein represents a good alternative to current guided bone regeneration membranes, with high potential for use in oral/orthopaedic applications in patients.

About the endothermal transitions of galactomannans: A multi-analytical DSC, LF-1H NMR and DMA study.

The thermal transitions of biopolymers were subject of great discussion in the 90s due to their relevance in structure development during processing and stability on storage. In the present work two galactomannans, vinal gum and guar gum, were evaluated by DSC, DMA and LF-1H NMR in order to compare them, establishing their potential operational application range and promoting the use of the non-conventional VG in foods or other products. Three endothermal transitions appeared when heating the samples in the DSC: one at temperatures -90 to -10 °C (LTT), other around 50 °C (MTT) and a third one between 50 and 100 °C (HTT). Both LTT and HTT showed water content dependence and low ΔCp values, which difficulted the assignment of a glass transition. MTT appeared as an enthalpic relaxation independent on frequency or on water content. This transition was related to changes in mechanical properties and with the stabilization of proton mobility.

Reduction of enthalpy relaxation in gelatine films by addition of polyols.

The aim of this study was to evaluate the effect of plasticisers with different molecular weights (glycerol and sorbitol) on the structural relaxation kinetics of bovine gelatine films stored under the glass transition temperature (Tg). Plasticisers were tested at weight fractions of 0.0, 0.06 and 0.10. Films conditioned in environments under ∼44% relative humidity gave moisture contents (w/w) in the range 0.14-0.18. The enthalpy relaxation (ΔH) was determined using differential scanning calorimetry (DSC). Samples used had Tg values in the range 24-49 °C. After removing the thermal history (30 °C above Tg, 15 min), samples were isothermally stored at 10 °C below Tg for between 2 and 80 h. The addition of plasticisers induced a significant reduction in the rate of structural relaxation. The linearisation of ΔH by plotting against the logarithm of ageing time showed a reduction in the slope of samples plasticised with both polyols. The reduction in relaxation kinetics may be related to the ability of polyols to act as enhancers of molecular packing, as recently reported using positron spectroscopy (PALS). However, a direct correlation between the relaxation kinetics and the plasticiser's molecular weight could not be established, suggesting that this phenomenon may be governed by complex molecular gelatin-plasticiser-water interactions.

Efecto del etiquetado calórico sobre la elección de un menú: estudio preliminar en Santiago de Chile / Effect of calorie labeling on menu selection: a preliminary study in Santiago, Chile

Folate is an essential nutrient because mammals lack biological activity to synthesize. It different factors generate folate deficiency. Recent studies have identified that the C677T variant of the enzyme methylene tetrahydrofolate reductase (MTHFR), can play a role in serum folate concentrations (SFC) and red cell folate (RCF). The aim of this rewiev was to actualice some generalities of folate metabolism, factors related to its deficiency, biochemical indicators used to assess the nutritional status of folate and role of the C677T polymorphism of the MTHFR enzyme on the cycle of folate and methionine. It is necessary to design studies with representative samples corroborating the effect of polymorphisms on biochemical indicators of nutritional status of folate and determine the dose-response effect and contribute to modify the nutritional recommendations with the necessary scientific evidence.

El 62% de la población chilena presenta sobrepeso (dato OMS). Publicar calorías en menús de restaurantes podría ayudar a controlar este problema. El objetivo fue estudiar el efecto de la entrega de información calórica en la elección de almuerzos típicos. La metodología tuvo un enfoque cuantitativo, con encuesta on line de diseño transversal, estructurada, con preguntas abiertas y cerradas, y con escala tipo Lykert. Se obtuvo 227 respuestas válidas. Los encuestados seleccionaron un almuerzo de 3 elementos, antes y después de exponer información calórica. Los resultados mostraron que el 49% de los encuestados reduce en promedio 292 kcal, (39,2% del total de calorías en menú) al considerar la información calórica. Nuestros resultados sugieren que aproximadamente para la mitad de los encuestados, la información fue útil en la selección de menú. Esta información podría ayudar a profesionales de la Salud a crear conciencia, facilitando a los consumidores elecciones más saludables.

Stress Transfer Quantification in Gelatin-Matrix Natural Composites with Tunable Optical Properties.

This work reports on the preparation and characterization of natural composite materials prepared from bacterial cellulose (BC) incorporated into a gelatin matrix. Composite morphology was studied using scanning electron microscopy and 2D Raman imaging revealing an inhomogeneous dispersion of BC within the gelatin matrix. The composite materials showed controllable degrees of transparency to visible light and opacity to UV light depending on BC weight fraction. By adding a 10 wt % fraction of BC in gelatin, visible (λ = 550 nm) and UV (λ = 350 nm) transmittances were found to decrease by ∼35 and 40%, respectively. Additionally, stress transfer occurring between the gelatin and BC fibrils was quantified using Raman spectroscopy. This is the first report for a gelatin-matrix composite containing cellulose. As a function of strain, two distinct domains, both showing linear relationships, were observed for which an average initial shift rate with respect to strain of -0.63 ± 0.2 cm(-1)%(-1) was observed, followed by an average shift rate of -0.25 ± 0.03 cm(-1)%(-1). The average initial Raman band shift rate value corresponds to an average effective Young's modulus of 39 ± 13 GPa and 73 ± 25 GPa, respectively, for either a 2D and 3D network of BC fibrils embedded in the gelatin matrix. As a function of stress, a linear relationship was observed with a Raman band shift rate of -27 ± 3 cm(-1)GPa(-1). The potential use of these composite materials as a UV blocking food coating is discussed.

Improvement of biomaterials used in tissue engineering by an ageing treatment.

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60% (scaffold without ageing treatment) to 80%. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.

Designing a gelatin/chitosan/hyaluronic acid biopolymer using a thermophysical approach for use in tissue engineering.

Cell culture on biopolymeric scaffolds has provided treatments for tissue engineering. Biopolymeric mixtures based on gelatin (Ge), chitosan (Ch) and hyaluronic acid (Ha) have been used to make scaffolds for wound healing. Thermal and physical properties of scaffolds prepared with Ge, Ch and Ha were characterized. Thermal characterization was made by using differential scanning calorimetry (DSC), and physical characterization by gas pycnometry and scanning electron microscopy. The effects of Ge content and cross-linking on thermophysical properties were evaluated by means of a factorial experiment design (central composite face centered). Gelatin content was the main factor that affects the thermophysical properties (microstructure and thermal transitions) of the scaffold. The effect of Ge content of the scaffolds for tissue engineering was studied by seeding skin cells on the biopolymers. The cell attachment was not significantly modified at different Ge contents; however, the cell growth rate increased linearly with the decrease of the Ge content. This relationship together with the thermophysical characterization may be used to design scaffolds for tissue engineering.