Optimization of Ink Composition and 3D Printing Process to Develop Soy Protein-Based Scaffolds.

Inks based on soybean protein isolate (SPI) were developed and their formulations were optimized as a function of the ink heat treatment and the content of other biopolymers to assess the effects of protein-polysaccharides and protein-protein interactions. First, the rheological behavior of the inks was analyzed in relation to the polyvinyl alcohol (PVA) concentration employed (20, 25, and 30 wt%) and, as a result of the analysis, the ink with 25 wt% PVA was selected. Additionally, sodium alginate (SA) and gelatin (GEL) were added to the formulations to improve the viscoelastic properties of the inks and the effect of the SA or GEL concentrations (1, 2, and 3 wt%) was studied. All inks showed shear thinning behavior and self-supporting abilities. Among all the 3D printed scaffolds, those with higher SA (3 wt%) or GEL (2 and 3 wt%) content showed higher shape fidelity and were selected for further characterization. Texture profile analysis demonstrated that the scaffolds prepared with previously heat-treated inks containing 3 wt% GEL showed the highest strength. Additionally, these scaffolds showed a higher water-uptake capacity profile.

Valorization of pectins from coffee wastes for the development of pectin-chitosan films.

Coffee wastes are underused materials, largely available in coffee producing regions, which can be used to obtain pectins for the development of films for packaging. Coffee residual water (CRW) provided a phenolic and protein rich-pectic fraction (CRWP), which has 49 % uronic acid. This pectic fraction was used for the development of films with chitosan (Chit). Additionally, pectins extracted from coffee pulp with acid, Coffea arabica pectin (CAP), hot water-soluble pectic fraction (HWSP), and chelating agent-soluble pectic fraction (CSP), were used to develop pectin-chitosan films. Flow and viscoelastic properties of film forming solutions were assessed, showing better characteristics for the pectins from the pulp over those from the residual water. The different composition of the pectin fractions allowed to relate film properties with their structural features and Fourier transform infrared (FTIR) spectroscopy showed interactions between pectin and chitosan in the films. Results showed that CAP-Chit and CSP-Chit films were transparent, hydrophobic, and had the best mechanical properties. These results demonstrate that coffee residual wastes have the potential to provide pectins that can be used for the development of films.

Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.

Biodegradable and biocompatible collagen-based hybrid materials for force sensing applications.

With the aim of replacing synthetic macromolecules by biological macromolecules for advanced applications, collagen films were produced with two different ionic liquids (ILs), choline dihydrogen phosphate ([Ch][DHP]) and choline serinate ([Ch][Seri]), added in order to modulate the electrical responses. The films were prepared by casting, varying IL content between 0 and 6 wt%. The morphology and thermal properties of the resulting films were found to be independent of both IL type and content. However, the highest direct curret (d.c.) electrical conductivity (1.4 × 10-8 S·cm-1) was achieved for collagen films containing 3 wt% [Ch][DHP]. Furthermore, it was demonstrated that IL/collagen films were non-cytotoxic, with cell activity values exceeding 70 %. These collagen films were proven to be suitable for force sensing applications, displaying excellent sensitivity and stability upon repeated testing.

Soy protein/ß-chitin sponge-like scaffolds laden with human mesenchymal stromal cells from hair follicle or adipose tissue promote diabetic chronic wound healing.

Chronic wounds are a worldwide problem that affect >40 million people every year. The constant inflammatory status accompanied by prolonged bacterial infections reduce patient's quality of life and life expectancy drastically. An important cell type involved in the wound healing process are mesenchymal stromal cells (MSCs) due to their long-term demonstrated immunomodulatory and pro-regenerative capacity. Thus, in this work, we leveraged and compared the therapeutic properties of MSCs derived from both adipose tissue and hair follicle, which we combined with sponge-like scaffolds (SLS) made of valorized soy protein and ß-chitin. In this regard, the combination of these cells with biomaterials permitted us to obtain a multifunctional therapy that allowed high cell retention and growing rates while maintaining adequate cell-viability for several days. Furthermore, this combined therapy demonstrated to increase fibroblasts and keratinocytes migration, promote human umbilical vein endothelial cells angiogenesis and protect fibroblasts from highly proteolytic environments. Finally, this combined therapy demonstrated to be highly effective in reducing wound healing time in vivo with only one treatment change during all the experimental procedure, also promoting a more functional and native-like healed skin.

Sustainable Collagen Composites with Graphene Oxide for Bending Resistive Sensing.

This work reports on the development of collagen films with graphene oxide nanoparticles (GO NPs), aiming toward the development of a new generation of functional sustainable sensors. For this purpose, different GO NP contents up to 3 wt % were incorporated into a collagen matrix, and morphological, thermal, mechanical and electrical properties were evaluated. Independently of the GO NP content, all films display an increase in thermal stability as a result of the increase in the structural order of collagen, as revealed by XRD analysis. Further, the inclusion of GO NPs into collagen promotes an increase in the intensity of oxygen characteristic absorption bands in FTIR spectra, due to the abundant oxygen-containing functional groups, which lead to an increase in the hydrophilic character of the surface. GO NPs also influence the mechanical properties of the composites, increasing the tensile strength from 33.2 ± 2.4 MPa (collagen) to 44.1 ± 1.0 MPa (collagen with 3 wt % GO NPs). Finally, the electrical conductivity also increases slightly with GO NP content, allowing the development of resistive bending sensors.

Combination of 3D printing and electrospinning to develop chitin/gelatin/PVA scaffolds.

In this study, novel scaffolds based on natural polymers were developed by combining 3D printing (3DP) and electrospinning (ES) techniques. ES ink was prepared with gelatin and poly(vinyl alcohol) (PVA), while 3DP ink was prepared with gelatin and chitin. Different biopolymers were used to confer unique properties to each ink and obtain a multilayered scaffold suitable for tissue regeneration. First, gelatin is able to exhibit the characteristics needed for both inks since gelatin chains contain arginineglycine-aspartic (RGD) motifs, an important sequence in the promotion of cell adhesion, which gives gelatin an improved biological behavior in comparison to other polymers. Additionally, PVA was selected for ES ink to facilitate gelatin spinnability, and chitin was incorporated into 3DP ink as reinforcement to provide mechanical support and protection to the overall design. In this work, chitin was extracted from fruit fly pupae. The high extraction yield and purity of the chitin obtained from the fruit fly pupae confirmed that this pupa is an alternative source to produce chitin. Once the chitin was characterized, both inks were prepared and rheological analysis was carried out in order to confirm the shear thinning behavior required for additive manufacturing processes. The combination of 3DP and ES processes resulted in porous scaffolds, which were proven biocompatible, highlighting their potential for biomedical applications.

Agar/gelatin hydro-film containing EGF and Aloe vera for effective wound healing.

In the current study, we produced a hydro-film dressing for the treatment of chronic wounds. The hydro-film structure was composed of gelatin cross-linked with citric acid, agar and Aloe vera extract (AV); additionally epidermal growth factor (EGF) was loaded to promote wound healing. Due to the excellent hydrogel-forming ability of gelatin, the obtained hydro-film was able to swell 884 ± 36% of its dry weight, which could help controlling wound moisture. To improve gelatin mechanical properties, polymer chains were cross-linked with citric acid and agar, reaching an ultimate tensile strength that was in the highest range of human skin. In addition, it showed a slow degradation profile that resulted in a remaining weight of 28 ± 8% at day 28. Regarding, biological activity, the addition of AV and citric acid provided the ability to reduce human macrophage activation, which could help reverse the permanent inflammatory state of chronic wounds. Moreover, loaded EGF, together with the structural AV of the hydro-film, promoted human keratinocyte and fibroblast migration, respectively. Furthermore, the hydro-films presented excellent fibroblast adhesiveness, so they could be useful as provisional matrices for cell migration. Accordingly, these hydro-films showed suitable physicochemical characteristics and biological activity for chronic wound healing applications.

Valorization of agar production residue as a filler in soy protein hydrogels for 3D printing.

Cellulose-containing residue from agar production was incorporated as a filler into soy protein-based hydrogels and revalorized without further purification. Rheological assessment of these hydrogels was carried out in order to confirm their shear-thinning behavior and their suitability for 3D printing. It was observed that all hydrogels behaved as weak gels, which are suitable for 3D printing and have good printability and shape fidelity. The addition of cellulose did not cause chemical crosslinking but physical interactions, which led to morphological changes, thereby promoting hardness and shape recovery of the 3D-printed products. The hydrogel with the highest residue content (8 wt %) showed the highest value (78%) in shape recovery. Furthermore, the physicochemical characterization of these 3D-printed products revealed that although they have high swelling capacity, they preserve their integrity in wet conditions. These results suggested the potential of the 3D-printed products developed using residues without further purification to promote circular economy, increasing the efficiency in resources utilization.

Collagen-Laponite Nanoclay Hydrogels for Tumor Spheroid Growth.

The extracellular matrix (ECM) plays an important regulatory role in the development and progression of tumoral tissue. Its functions and properties are crucial in determining tumor cell behavior such as invasion, migration, and malignancy development. Our study explores the role of collagen type I in cancer development and spread using engineered tumor models like multicellular spheroids grown in collagen-based hydrogels to simulate early tumor formation. We employ microfluidic techniques to test the hypothesis that (i) adding Laponite nanoclay to collagen hydrogels modifies mechanical and rheological properties and (ii) changing the stiffness of the collagen microenvironment affects tumor spheroid growth. Our findings support our theories and suggest the use of ECM components and engineered tumor models in cancer research, offering a biocompatible and biomimetic method to tailor the mechanical properties of conventional collagen hydrogels.

Sustainable Collagen Blends with Different Ionic Liquids for Resistive Touch Sensing Applications.

Considering the sustainable development goals to reduce environmental impact, sustainable sensors based on natural polymers are a priority as the large im plementation of these materials is required considering the Internet of Things (IoT) paradigm. In this context, the present work reports on sustainable blends based on collagen and different ionic liquids (ILs), including ([Ch][DHP], [Ch][TSI], [Ch][Seri]) and ([Emim][TFSI]), processed with varying contents and types of ILs in order to tailor the electrical response. Varying IL types and contents leads to different interactions with the collagen polymer matrix and, therefore, to varying mechanical, thermal, and electrical properties. Collagen/[Ch][Seri] samples display the most pronounced decrease of the tensile strength (3.2 ± 0.4 MPa) and an increase of the elongation at break (50.6 ± 1.5%). The best ionic conductivity value of 0.023 mS cm-1 has been obtained for the sample with 40 wt % of the IL [Ch][Seri]. The functional response of the collagen-IL films has been demonstrated on a resistive touch sensor whose response depends on the ionic conductivity, being suitable for the next generation of sustainable touch sensing devices.

Tumour growth: An approach to calibrate parameters of a multiphase porous media model based on in vitro observations of Neuroblastoma spheroid growth in a hydrogel microenvironment.

To unravel processes that lead to the growth of solid tumours, it is necessary to link knowledge of cancer biology with the physical properties of the tumour and its interaction with the surrounding microenvironment. Our understanding of the underlying mechanisms is however still imprecise. We therefore developed computational physics-based models, which incorporate the interaction of the tumour with its surroundings based on the theory of porous media. However, the experimental validation of such models represents a challenge to its clinical use as a prognostic tool. This study combines a physics-based model with in vitro experiments based on microfluidic devices used to mimic a three-dimensional tumour microenvironment. By conducting a global sensitivity analysis, we identify the most influential input parameters and infer their posterior distribution based on Bayesian calibration. The resulting probability density is in agreement with the scattering of the experimental data and thus validates the proposed workflow. This study demonstrates the huge challenges associated with determining precise parameters with usually only limited data for such complex processes and models, but also demonstrates in general how to indirectly characterise the mechanical properties of neuroblastoma spheroids that cannot feasibly be measured experimentally.

Effect of sterilization processes on alginate/gelatin inks for three-dimensional printing.

309Sterilization is a crucial step in the process of developing bioinks for tissue engineering applications. In this work, alginate/gelatin inks were subjected to three sterilization methods: ultraviolet (UV) radiation, filtration (FILT), and autoclaving (AUTO). In addition, to simulate the sterilization effect in a real environment, inks were formulated in two different media, specifically, Dulbecco's Modified Eagle's Medium (DMEM) and phosphate-buffered saline (PBS). First, rheological tests were performed to evaluate the flow properties of the inks, and we observed that UV samples showed shear thinning behavior, which was favorable for three-dimensional (3D) printing. Furthermore, the 3D-printed constructs developed with UV inks showed better shape and size fidelity than those obtained with FILT and AUTO. In order to relate this behavior to the material structure, Fourier transform infrared (FTIR) analysis was carried out and the predominant conformation in protein was determined by deconvolution of the amide I band, which confirmed that the prevalence of a-helix structure was greater for UV samples. This work highlights the relevance of sterilization processes, which are essential for biomedical applications, in the research field of bioinks.

A Cross-Sectional Study on the Associations between Depression and Anxiety, Medication Use for These Diseases and Physical Activity Level in Spanish People with Hypertension.

Hypertension (HTN) has a high prevalence in the overall population, affecting people's mental health. Physical Activity (PA) has proven to be an effective tool to improve mental health. This study analyzed the associations between Depression and Anxiety prevalence, medication use for these disorders (antidepressants and anxiolytics) and Physical Activity Level (PAL) in people with HTN. A cross-sectional study was conducted with data from the Spanish National Health Survey 2017 (ENSE2017) with a final sample of 3228 individuals over 15 years of age with HTN who resided in Spain. Data normality was assessed through the Kolmogorov-Smirnov test. Associations between Depression and Anxiety prevalence, antidepressant and anxiolytic use and PAL were studied using a chi-square test. Possible differences between Depression and Anxiety prevalence and medication use according to the PAL were analyzed with a z-test for independent proportions. Depression or Anxiety and antidepressant and anxiolytic use odds ratios (OR) were calculated for every PAL group, taking the "Very Active" group as a reference. Risk factors were evaluated using multiple binary logistic regression. Dependency relationships were found between Depression and Anxiety prevalence, antidepressant and anxiolytic use and PAL (p < 0.001). The Inactive group displayed the highest prevalence and medication use according to their PAL (p < 0.05). Higher ORs for Depression or Anxiety and pharmacological treatments used were also found in the Inactive group compared to the other PAL groups.

Whey Protein Films for Sustainable Food Packaging: Effect of Incorporated Ascorbic Acid and Environmental Assessment.

The management of food waste and by-products has become a challenge for the agri-food sector and an example are whey by-products produced in dairy industries. Seeking other whey valorisation alternatives and applications, whey protein films for food packaging applications were developed in this study. Films containing different amounts (0, 5, 10, and 15 wt%) of ascorbic acid were manufactured via compression-moulding and their physicochemical, thermal, barrier, optical, and mechanical properties were analysed and related to the film structure. Additionally, the environmental assessment of the films was carried out to analyse the impact of film manufacture. Regarding physicochemical properties, both FTIR and water uptake analyses showed the presence of non-covalent interactions, such as hydrogen bonding, between whey protein and ascorbic acid as band shifts at the 1500-1700 cm-1 region as well as a water absorption decrease from 380% down to 240% were observed. The addition of ascorbic acid notably improved the UV-Vis light absorbance capacity of whey protein films up to 500 nm, a relevant enhancement for protecting foods susceptible to UV-Vis light-induced lipid oxidation. In relation to the environmental assessment, it was concluded that scaling up film manufacture could lead to a reduction in the environmental impacts, mainly electricity consumption.

Magnetically responsive chitosan-pectin films incorporating Fe3O4 nanoparticles with enhanced antimicrobial activity.

Chitosan-pectin films with iron oxide (Fe3O4) magnetic nanoparticles were prepared by solution casting in order to produce biopolymer based magnetically active materials. Infrared (FTIR) spectra indicated physical interactions between the matrix and nanoparticles, corroborated by differential scanning calorimetry (DSC) results. In addition, thermal characterization suggested that the interactions between chitosan, pectin and the nanoparticles resulted in a less compact structure, influencing the film mechanical properties. Regarding vibrating-sample magnetometry (VSM) and electrical analysis, chitosan-pectin films with Fe3O4 nanoparticles showed ferrimagnetic behavior, with an increase of the dielectric constant as the nanoparticle concentration increased. Furthermore, films displayed enhanced antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) bacteria. Therefore, chitosan-pectin films with Fe3O4 magnetic nanoparticles provide promising results for active and intelligent food packaging applications.

Hybrid 3D Printed and Electrospun Multi-Scale Hierarchical Polycaprolactone Scaffolds to Induce Bone Differentiation.

Complex scaffolds composed of micro- and nano-structures are a key target in tissue engineering and the combination of sequential 3D printing and electrospinning enables the fabrication of these multi-scale structures. In this work, dual 3D printed and electrospun polycaprolactone (PCL) scaffolds with multiple mesh layers were successfully prepared. The scaffold macro- and micro-porosity were assessed by optical and scanning electron microscopy, showing that electrospun fibers formed aligned meshes within the pores of the scaffold. Consequently, the hydrophilicity of the scaffold increased with time, enhancing cell adhesion and growth. Additionally, compression tests in back and forth cycles demonstrated a good shape recovery behavior of the scaffolds. Biological results indicated that hybrid PCL scaffolds are biocompatible and enable a correct cell culture over time. Moreover, MC3T3-E1 preosteoblast culture on the scaffolds promoted the mineralization, increased the alkaline phosphatase (ALP) activity and upregulated the expression of early and late osteogenic markers, namely ALP and osteopontin (OPN), respectively. These results demonstrate that the sequential combination of 3D printing and electrospinning provides a facile method of incorporating fibers within a 3D printed scaffold, becoming a promising approach towards multi-scale hierarchical scaffolds capable of guiding the osteogenic differentiation.

Sialyltransferase Inhibitor Ac53FaxNeu5Ac Reverts the Malignant Phenotype of Pancreatic Cancer Cells, and Reduces Tumor Volume and Favors T-Cell Infiltrates in Mice.

Hypersialylation is a feature of pancreatic ductal adenocarcinoma (PDA) and it has been related to tumor malignancy and immune suppression. In this work, we have evaluated the potential of the sialyltransferase inhibitor, Ac53FaxNeu5Ac, to decrease tumor sialoglycans in PDA and to revert its malignant phenotype. Sialoglycans on PDA cells were evaluated by flow cytometry, and the functional impact of Ac53FaxNeu5Ac was assessed using E-selectin adhesion, migration, and invasion assays. PDA tumors were generated in syngeneic mice from KC cells and treated with Ac53FaxNeu5Ac to evaluate tumor growth, mice survival, and its impact on blocking sialic acid (SA) and on the tumor immune component. Ac53FaxNeu5Ac treatment on human PDA cells decreased α2,3-SA and sialyl-Lewisx, which resulted in a reduction in their E-selectin adhesion, and in their migratory and invasive capabilities. Subcutaneous murine tumors treated with Ac53FaxNeu5Ac reduced their volume, their SA expression, and modified their immune component, with an increase in CD8+ T-lymphocytes and NK cells. In conclusion, Ac53FaxNeu5Ac treatment weakened PDA cells' malignant phenotype, thereby reducing tumor growth while favoring anti-tumor immune surveillance. Altogether, these results show the positive impact of reducing SA expression by inhibiting cell sialyltransferases and open the way to use sialyltransferase inhibitors to target this dismal disease.

Sustainable Sheep Wool/Soy Protein Biocomposites for Sound Absorption.

The wool fibers of the Latxa sheep breed were combined with a soy protein isolate (SPI) matrix to develop sustainable biocomposites with acoustic properties, adding value to Latxa sheep wool, which is currently considered a residue. Samples with 7, 10, 15, and 20 wt % wool were prepared by freeze drying in order to develop porous structures, as shown by SEM analysis. Additionally, XRD analysis provided the evidence of a change toward a more amorphous structure with the incorporation of wool fibers due to the interactions between the soy protein and keratin present in wool fibers, as shown by the relative intensity changes in the FTIR bands. The biocomposites were analyzed in a Kundt's tube to obtain their sound absorption coefficient at normal incidence. The results showed an acoustic absorption coefficient that well-surpassed 0.9 for frequencies above 1000 Hz. This performance is comparable to that of the conventional synthetic materials present in the market and, thus, sheep wool/SPI biocomposites are suitable to be used as acoustic absorbers in the building industry, highlighting the potential of replacing not only synthetic fibers but also synthetic polymers, with natural materials to enhance the sustainability of the building sector.