Design and functional testing of a multichamber perfusion platform for three-dimensional scaffolds

Perfusion culture systems are widely used in tissue engineering applications for enhancing cell culture viability in the core of three-dimensional scaffolds. In this work, we present a multichamber confined-flow perfusion system, designed to provide a straightforward platform for three-dimensional dynamic cell cultures. The device comprises 6 culture chambers allowing independent and simultaneous experiments in controlled conditions. Each chamber consists of three parts: a housing, a deformable scaffold-holder cartridge, and a 7 mL reservoir, which couples water-tightly with the housing compressing the cartridge. Short-term dynamic cell seeding experiments were carried out with MC3T3-E1 cells seeded into polycaprolactone porous scaffolds. Preliminary results revealed that the application of flow perfusion through the scaffold favored the penetration of the cells to its interior, producing a more homogeneous distribution of cells with respect to dropwise or injection seeding methods. The culture chamber layout was conceived with the aim of simplifying the user operations under laminar flow hood and minimizing the risks for contamination during handling and operation. Furthermore, a compact size, a small number of components, and the use of bayonet couplings ensured a simple, fast, and sterility-promoting assembling. Finally, preliminary in vitro tests proved the efficacy of the system in enhancing cell seeding efficiency, opening the way for further studies addressing long-term scaffold colonization

Effects of Drying Methods and Temperatures on the Quality of Chestnut Flours

The demand for chestnut flour is growing because of its use in gluten-free products. Previous studies have correlated the quality of chestnut flours to the drying temperature and technology applied. This work is a novel study on the role of the traditional drying method with a wood fire in a "metato" building for flour compared with a food dryer at 40 degrees C or 70 degrees C. The contents of antioxidants, total polyphenols and sugars were determined as well as the presence of toxic volatiles or aflatoxins. The flour, resulting from the traditional method, presented lower polyphenol content and antioxidant power compared to the others. The content of the sugars was similar to the flours obtained after drying with hot air, both at 40 degrees C and 70 degrees C. The toxic volatile molecules, furfural, guaiacol, and o-cresol, were found. There was no correlation between the aflatoxin content and the presence of damage in chestnut fruits. The traditional method should not be abandoned since it confers a pleasant smoky taste to the product, but it is necessary to regulate the level and steadiness of temperature. Future research needs to be directed to the quantification of harmful volatile compounds and their correlation with the quantity of smoke emitted by the wood fire

The plot thickens: the emerging role of matrix viscosity in cell mechanotransduction

Cell mechanotransduction is an area of intense research focus. Until now, very limited tools have existed to study how cells respond to changes in the extracellular matrix beyond, for example, mechanical deformation studies and twisting cytometry. However, emerging are a range of elastic, viscoelastic and even purely viscous materials that deform and dissipate on cellular length and timescales. This article reviews developments in these materials, typically translating from 2D model surfaces to 3D microenvironments and explores how cells interact with them. Specifically, it focuses on emerging concepts such as the molecular clutch model, how different extracellular matrix proteins engage the clutch under viscoelastic‐stress relaxation conditions, and how mechanotransduction can drive transcriptional control through regulators such as YAP/TAZ

Sensory profiling and consumer acceptability of new dark cocoa bars containing Tuscan autochthonous food products

A new set of cocoa bars named Toscolata® were developed containing top-quality extra virgin olive oil, dried apples cultivars, and chestnut flour. The present work has been conducted to define the sensory profile of these products through tasting by trained experts and consumers to study the acceptability, preference, and quality perception. The four sensorial profiles of the bars differed in the level of persistence, bitterness, aromaticity, acidity, astringency, and tastiness. In particular, the sour attribute could be traced to the presence of dried apple. Bars containing apple and chestnut flour obtained higher acceptance ratings, compared to those with extra virgin olive oil. The bar with chestnut flour was preferred by consumers who considered it to be sweeter due to the presence of natural sugars, which lowered the bitter sensation of cocoa. These results showed that the selection of the preferred bar by consumers was mainly based on the level of bitterness and, in particular, elderly consumers expressed a strong preference for the sweetest product. As far as we know, this is the first study comparing the results of a panel of expert tasters with that of consumers in the tasting of dark chocolate

Pasta Enriched with Carrot and Olive Leaf Flour Retains High Levels of Accessible Bioactives after In Vitro Digestion

The aim of this research was to evaluate the levels of antioxidants and polyphenols in pasta enriched with either carrot or olive leaf flours after simulating gastrointestinal digestion. Pasta samples were prepared with fixed amounts of carrot and olive leaf flours (15% and 6% of the total mixture, respectively). We measured the antioxidant capacity and polyphenol content at different stages of the pasta production process, starting from the initial flour to the cooked pasta, and tested samplesoftheliquid componentandsolidwasteresultingfromthedigestionprocess. Theantioxidant activity was measured by the FRAP method, while the polyphenol content was measured by the Folin–Ciocalteu method. Vitamin E contents were measured by HPLC. The pasta enriched with carrot (1.26 0.05 mmol/100 g) and olive leaf (2.9 0.07 mmol/100 g) exhibited higher antioxidant power compared to the unenriched pasta (0.8 0.1 mmol/100 g). The polyphenol content followed a similar trend, with values of 131.23 3.08 for olive flour-enriched pasta, 79.15 1.11 for carrot flour-enriched pasta, and 67.5 1.39 for the wheat-only pasta. The pasta samples maintained their antioxidant and polyphenol levels even after undergoing the simulated digestion process. Significantly, the liquid component of the pasta with olive leaf flours had the highest levels of antioxidants and polyphenols during all stages of the digestion process. According to the results of this study, pasta enriched with carrot and olive leaf flours shows promising potential for improving nutritional and functional properties by increasing antioxidant and polyphenol content. The samples were also evaluated by a sensory panel, which showed that fortification modified the perception of some organoleptic attributes without affecting the overall taste of the pasta

Functionalization of PLLA with Polymer Brushes to Trigger the Assembly of Fibronectin into Nanonetworks

[EN] Poly-l-lactic acid (PLLA) has been used as a biodegradable polymer for many years; the key characteristics of this polymer make it a versatile and useful resource for regenerative medicine. However, it is not inherently bioactive. Thus, here, a novel process is presented to functionalize PLLA surfaces with poly(ethyl acrylate) (PEA) brushes to provide biological functionality through PEA's ability to induce spontaneous organization of the extracellular matrix component fibronectin (FN) into physiological-like nanofibrils. This process allows control of surface biofunctionality while maintaining PLLA bulk properties (i.e., degradation profile, mechanical strength). The new approach is based on surface-initiated atomic transfer radical polymerization, which achieves a molecularly thin coating of PEA on top of the underlying PLLA. Beside surface characterization via atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle to measure PEA grafting, the biological activity of this surface modification is investigated. PEA brushes trigger FN organization into nanofibrils, which retain their ability to enhance adhesion and differentiation of C2C12 cells. The results demonstrate the potential of this technology to engineer controlled microenvironments to tune cell fate via biologically active surface modification of an otherwise bioinert biodegradable polymer, gaining wide use in tissue engineering applications.The authors acknowledge the EPSRC (EP/P001114/1) and MRC (MR/S005412/1) funding. The authors also acknowledge the EPSRC funding as part of the Doctoral Training Centre EP/F500424/1. This work was also funded by a grant from the UK Regenerative Medicine Platform. X-ray photoelectron spectroscopy was conducted by the National EPSRC XPS Users' Service (NEXUS), Newcastle.Sprott, MR.; Ferrer, G.; Dalby, MJ.; Salmerón Sánchez, M.; Cantini, M. (2019). Functionalization of PLLA with Polymer Brushes to Trigger the Assembly of Fibronectin into Nanonetworks. Advanced Healthcare Materials (Online). 8(3):1-12. https://doi.org/10.1002/adhm.201801469S11283A. J. Rincon Lasprilla G. A. Rueda Martinez B. H. Lunelli J. E. Jaimes Figueroa A. L. Jardini R. Maciel Filho Chem. Eng. Trans 2011 985Khan, F., Tanaka, M., & Ahmad, S. R. (2015). Fabrication of polymeric biomaterials: a strategy for tissue engineering and medical devices. Journal of Materials Chemistry B, 3(42), 8224-8249. doi:10.1039/c5tb01370dXu, F. J., Yang, X. C., Li, C. Y., & Yang, W. T. (2011). Functionalized Polylactide Film Surfaces via Surface-Initiated ATRP. Macromolecules, 44(7), 2371-2377. doi:10.1021/ma200160hKhan, F., & Tanaka, M. (2017). Designing Smart Biomaterials for Tissue Engineering. 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Cocoa Bar Antioxidant Profile Enrichment with Underutilized Apples Varieties

The impact of dried apples (Malus x domestica Borkh.) addition on improving the antioxidant characteristics of dark chocolate was evaluated. The antioxidant activity was measured through DPPH scavenging activity and showed an increase in the cocoa bar with 'Nesta' dry apple (17.3% vs. 46.8%) in comparison to cocoa mass. The 15 polyphenols analyzed by UHPLC-ESI-MS/MS indicated great variability among the apple varieties. Quercetin was detected in the highest concentrations (ranged from 753.3 to 1915.5 mu g g(-1)), while the lowest were for kaempferol 7-O-glucoside, measured only in 'Mora' and 'Nesta' cocoa bars (from 0.034 to 0.069 mu g g(-1), respectively). P-coumaric acid, trans-ferulic acid, and chlorogenic acid contribute largely to the antioxidant activity in cocoa bars. Principal component analysis shows that a cocoa bar with the addition of 'Nesta' dry apple differ from others due to its higher content of polyphenols (1614 +/- 61.8 mg gallic acid equivalents per 100 g). In conclusion, data confirm that cocoa bars with dry apples might be considered as a polyphenol-enriched food

Material-driven fibronectin assembly for high-efficiency presentation of growth factors

Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness

Drought Stress Affects the Response of Italian Local Tomato (Solanum lycopersicum L.) Varieties in a Genotype-Dependent Manner

Drought stress is one of the most severe conditions for plants, especially in the face of the emerging problem of global warming. This issue is important when considering economically relevant crops, including the tomato. For these plants, a promising solution is the valorization of local agrobiodiversity as a source of genetic variability. In this paper we investigated how six Italian tomato varieties react to a prolonged period of water depletion. We used a multidisciplinary approach, from genetics to plant physiology and cytology, to provide a detailed overview of the response of plants to stress. The varieties analyzed, each characterized by a specific genetic profile, showed a genotype-specific response with the variety 'Fragola' being the most resistant and the variety 'Pisanello' the most susceptible. For all the parameters evaluated, 'Fragola' performed in a manner comparable to that of control plants. On the contrary, 'Pisanello' appeared to be more affected and showed an increase in the number of stomata and a drastic increase in antioxidants, a symptom of acute oxidative stress. Our work suggests the existence of a valuable reservoir of genetic biodiversity with more drought-tolerant tomato genotypes opening the way to further exploitation and use of local germplasm in breeding programs

Engineered microenvironments for synergistic VEGF - integrin signalling during vascularization

We have engineered polymer-based microenvironments that promote vasculogenesis both in vitro and in vivo through synergistic integrin-growth factor receptor signalling. Poly(ethyl acrylate) (PEA) triggers spontaneous organization of fibronectin (FN) into nanonetworks which provide availability of critical binding domains. Importantly, the growth factor binding (FNIII12-14) and integrin binding (FNIII9-10) regions are simultaneously available on FN fibrils assembled on PEA. This material platform promotes synergistic integrin/VEGF signalling which is highly effective for vascularization events in vitro with low concentrations of VEGF. VEGF specifically binds to FN fibrils on PEA compared to control polymers (poly(methyl acrylate), PMA) where FN remains in a globular conformation and integrin/GF binding domains are not simultaneously available. The vasculogenic response of human endothelial cells seeded on these synergistic interfaces (VEGF bound to FN assembled on PEA) was significantly improved compared to soluble administration of VEGF at higher doses. Early onset of VEGF signalling (PLCγ1 phosphorylation) and both integrin and VEGF signalling (ERK1/2 phosphorylation) were increased only when VEGF was bound to FN nanonetworks on PEA, while soluble VEGF did not influence early signalling. Experiments with mutant FN molecules with impaired integrin binding site (FN-RGE) confirmed the role of the integrin binding site of FN on the vasculogenic response via combined integrin/VEGF signalling. In vivo experiments using 3D scaffolds coated with FN and VEGF implanted in the murine fat pad demonstrated pro-vascularization signalling by enhanced formation of new tissue inside scaffold pores. PEA-driven organization of FN promotes efficient presentation of VEGF to promote vascularization in regenerative medicine applications