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- Identification and quantification of stilbenes (piceatannol and resveratrol) in passiflora edulis by-productsPublication . Krambeck, Karolline; Oliveira, Ana; Santos, Delfim; Pintado, Maria Manuela; Baptista Silva, João Baptista; Lobo, José Manuel Sousa; Amaral, Maria HelenaRecently, studies on the by-products from the food industry, such as passion fruit seeds, have significantly increased, as these can have an added value, due to their properties, such as potential antioxidant activity. This study was conducted to determine the presence of piceatannol and resveratrol in various extracts of passion fruit (Passiflora edulis) seeds from Madeira Island and a commercial passion fruit oil was used as reference. The commercial oil and the extracts that were obtained by traditional Soxhlet method with ethanol and acetone did not reveal the presence of the two stilbenes, piceatannol and resveratrol. However, the extracts that were obtained by the ultrasound method showed significant amounts of piceatannol and resveratrol when compared with the commercial oil. The presence of these compounds indicates that this oil could have potential application in cosmetic and pharmaceutical industries, due to their proven antioxidant and anti-aging properties.
- Development of composite scaffolds based on cerium doped-hydroxyapatite and natural gums-biological and mechanical propertiesPublication . Santos, Marcus Vinicius Beserra dos; Rocha, Lorenna Bastos Nogueira; Vieira, Ewerton Gomes; Oliveira, Ana Leite; Lobo, Anderson Oliveira; Carvalho, Maria Acelina Martins de; Osajima, Josy Anteveli; Silva-Filho, Edson CavalcantiHydroxyapatite (HAp) is a ceramic material composing the inorganic portion of bones. Ionic substitutions enhance characteristics of HAp, for example, calcium ions (Ca2+) by cerium ions (Ce3+). The use of HAp is potentialized through biopolymers, cashew gum (CG), and gellan gum (GG), since CG/GG is structuring agents in the modeling of structured biocomposites, scaffolds. Ce-HApCG biocomposite was synthesized using a chemical precipitation method. The obtained material was frozen (–20 °C for 24 h), and then vacuum dried for 24 h. The Ce-HApCG was characterized by X-Ray diffractograms (XRD), X-ray photoemission spectra (XPS), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), and energy dispersive spectroscopy (EDS). XRD and FTIR showed that Ce-HApCG was successfully synthesized. XRD showed characteristic peaks at 2θ = 25.87 and 32.05, corresponding to the crystalline planes (0 0 2) and (2 1 1), respectively, while phosphate bands were present at 1050 cm−1 and 1098 cm−1, indicating the success of composite synthesis. FESEM showed pores and incorporated nanostructured granules of Ce-HApCG. The mechanical test identified that Ce-HApCG has a compressive strength similar to the cancellous bone’s strength and some allografts used in surgical procedures. In vitro tests (MTT assay and hemolysis) showed that scaffold was non-toxic and exhibited low hemolytic activity. Thus, the Ce-HApCG has potential for application in bone tissue engineering.
- New prospects in skin regeneration and repair using nanophased hydroxyapatite embedded in collagen nanofibersPublication . Ribeiro, Nilza; Sousa, Aureliana; Cunha-Reis, Cassilda; Oliveira, Ana Leite; Granja, Pedro L.; Monteiro, Fernando J.; Sousa, SusanaThis study reflects an exploitation of a composite matrix produced by electrospinning of collagen and electrospraying of nanophased hydroxyapatite (nanoHA), for skin regeneration applications. The main goal was to evaluate the effect of nanoHA, as source of localized calcium delivery, on human dermal fibroblasts, keratinocytes, and human mesenchymal stem cells (hMSCs) growth, proliferation, differentiation, and extracellular matrix production. This study revealed that calcium ions provided by nanoHA significantly enhanced cellular growth and proliferation rates and prevented adhesion of pathogenic bacteria strains typically found in human skin flora. Moreover, hMSCs were able to differentiate in both osteogenic and adipogenic lineages. Rat subcutaneous implantation of the membranes also revealed that no adverse reaction occurred. Therefore, the mechanically fit composite membrane presents a great potential to be used either as cell transplantation scaffold for skin wound regeneration or as wound dressing material in plastic surgery, burns treatment or skin diseases.
- High efficient strategy for the production of hydroxyapatite/silk sericin nanocompositesPublication . Veiga, Anabela; Castro, Filipa; Oliveira, Ana L.; Rocha, FernandoBACKGROUND: Sericin (SS) induces nucleation of bone-like hydroxyapatite (HAp) when used as an organic matrix. HAp/SS nanocomposites have been conventionally synthesized through precipitation in stirred tank reactors (STs). Despite its simplicity, this process is time consuming and presents difficulties in scale-up. In our study, HAp/SS nanocomposites were successfully synthesized in a ST and in a meso-oscillatory flow reactor (meso-OFR), to compare the efficiency of both reactors and to study HAp mineralization using SS as a template. RESULTS: The production of stable HAp, indicated by pH stabilization, was achieved after 180 min in the ST and after 30 min in the meso-OFR. X-ray diffraction and Fourier transform infrared analyses showed that the particles obtained in both reactors are HAp/SS nanocomposites with low crystallinity. Scanning electron microscopy evidenced the formation of rod- and plateshaped nanoparticles and revealed that the presence of SS led to the production of larger particles. The latter observation was confirmed by laser diffraction. Additionally, increasing SS concentration resulted in the formation of more plate-like particles. CONCLUSIONS: Precipitation is more efficient in the meso-OFR, HAp/SS being obtained four times faster. The presence and concentration of SS led to differences in the size and morphology of the synthesized particles, suggesting a critical role of SS in the mineralization process. This work reports a new approach for the manufacture of high-added-value nanocomposites with similar characteristics to biological bone and the results of a study of the influence of SS as an organic component in HAp nucleation. Further, the use of this protein and technology leads to significant waste minimization.
- Antimicrobial properties of gallium (III)-and iron (III)-loaded polysaccharides affecting the growth of escherichia coli, staphylococcus aureus, and pseudomonas aeruginosa, in vitroPublication . Best, Mark G.; Cunha-Reis, Cassilda; Ganin, Alexey Y.; Sousa, Aureliana; Johnston, Jenna; Oliveira, Ana L.; Smith, David G. E.; Yiu, Humphrey H. P.; Cooper, Ian R.Antimicrobial resistance (AMR) has become a global concern as many bacterial species have developed resistance to commonly prescribed antibiotics, making them ineffective to treatments. One type of antibiotics, gallium(III) compounds, stands out as possible candidates due to their unique “Trojan horse” mechanism to tackle bacterial growth, by substituting iron(III) in the metabolic cycles of bacteria. In this study, we tested three polysaccharides (carboxymethyl cellulose (CMC), alginate, and pectin) as the binding and delivery agent for gallium on three bacteria (Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) with a potential bioresponsive delivery mode. Two types of analysis on bacterial growth (minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)) were carried out while iron(III)-loaded polysaccharide samples were also tested for comparison. The results suggested that gallium showed an improved inhibitory activity on bacterial growth, in particular gallium(III)-loaded carboxymethyl cellulose (Ga-CMC) sample showing an inhibiting effect on growth for all three tested bacteria. At the MIC for all three bacteria, Ga-CMC showed no cytotoxicity effect on human dermal neonatal fibroblasts (HDNF). Therefore, these bioresponsive gallium(III) polysaccharide compounds show significant potential to be developed as the next-generation antibacterial agents with controlled release capability.
- Impact of food matrix components on nutritional and functional properties of fruit-based productsPublication . Oliveira, Ana; Amaro, Ana L; Pintado, ManuelaNowadays, worldwide consumers seek delightful and appealing foods that are simultaneously safer, nutritional and healthier. Fruit-based products are largely consumed due to, in part, the consumer’s perception of these products as healthy and convenient perceived as similar or even with better health benefits than the raw fruits that originated them. However, it is of paramount importance to understand the impact of the food matrix components on nutritional and functional properties of fruit-based products. Isolated compounds, or fruit extracts are used more often than food products in interaction studies. These approaches do not consider the interferences of food matrix components and/or the impacts of food processing on bioactivity and bioaccessibility of the active compounds. In fact, the bioaccessibility of bioactive compounds can be different in diverse food matrices and this review aims to provide an integrated approach to the field of matrix interactions.
- Tumor growth suppression induced by biomimetic silk fibroin hydrogelsPublication . Yan, Le-Ping; Silva-Correia, Joana; Ribeiro, Viviana P.; Miranda-Gonçalves, Vera; Correia, Cristina; Morais, Alain da Silva; Sousa, Rui A.; Reis, Rui M.; Oliveira, Ana L.; Oliveira, Joaquim M.; Reis, Rui L.Protein-based hydrogels with distinct conformations which enable encapsulation or differentiation of cells are of great interest in 3D cancer research models. Conformational changes may cause macroscopic shifts in the hydrogels, allowing for its use as biosensors and drug carriers. In depth knowledge on how 3D conformational changes in proteins may affect cell fate and tumor formation is required. Thus, this study reports an enzymatically crosslinked silk fibroin (SF) hydrogel system that can undergo intrinsic conformation changes from random coil to β-sheet conformation. In random coil status, the SF hydrogels are transparent, elastic, and present ionic strength and pH stimuli-responses. The random coil hydrogels become β-sheet conformation after 10 days in vitro incubation and 14 days in vivo subcutaneous implantation in rat. When encapsulated with ATDC-5 cells, the random coil SF hydrogel promotes cell survival up to 7 days, whereas the subsequent β-sheet transition induces cell apoptosis in vitro. HeLa cells are further incorporated in SF hydrogels and the constructs are investigated in vitro and in an in vivo chick chorioallantoic membrane model for tumor formation. In vivo, Angiogenesis and tumor formation are suppressed in SF hydrogels. Therefore, these hydrogels provide new insights for cancer research and uses of biomaterials.
- Silk-based anisotropical 3D biotextiles for bone regenerationPublication . Ribeiro, Viviana P.; Silva-Correia, Joana; Nascimento, Ana I.; Morais, Alain da Silva; Marques, Alexandra P.; Ribeiro, Ana S.; Silva, Carla J.; Bonifácio, Grata; Sousa, Rui A.; Oliveira, Joaquim M.; Oliveira, Ana L.; Reis, Rui L.Bone loss in the craniofacial complex can been treated using several conventional therapeutic strategies that face many obstacles and limitations. In this work, novel three-dimensional (3D) biotextile architectures were developed as a possible strategy for flat bone regeneration applications. As a fully automated processing route, this strategy as potential to be easily industrialized. Silk fibroin (SF) yarns were processed into weft-knitted fabrics spaced by a monofilament of polyethylene terephthalate (PET). A comparative study with a similar 3D structure made entirely of PET was established. Highly porous scaffolds with homogeneous pore distribution were observed using micro-computed tomography analysis. The wet state dynamic mechanical analysis revealed a storage modulus In the frequency range tested, the storage modulus values obtained for SF-PET scaffolds were higher than for the PET scaffolds. Human adipose-derived stem cells (hASCs) cultured on the SF-PET spacer structures showed the typical pattern for ALP activity under osteogenic culture conditions. Osteogenic differentiation of hASCs on SF PET and PET constructs was also observed by extracellular matrix mineralization and expression of osteogenic-related markers (osteocalcin, osteopontin and collagen type I) after 28 days of osteogenic culture, in comparison to the control basal medium. The quantification of convergent macroscopic blood vessels toward the scaffolds by a chick chorioallantoic membrane assay, showed higher angiogenic response induced by the SF-PET textile scaffolds than PET structures and gelatin sponge controls. Subcutaneous implantation in CD-1 mice revealed tissue ingrowth's accompanied by blood vessels infiltration in both spacer constructs. The structural adaptability of textile structures combined to the structural similarities of the 3D knitted spacer fabrics to craniofacial bone tissue and achieved biological performance, make these scaffolds a possible solution for tissue engineering approaches in this area.
- Rapidly responsive silk fibroin hydrogels as an artificial matrix for the programmed tumor cells deathPublication . Ribeiro, Viviana P.; Silva-Correia, Joana; Gonçalves, Cristiana; Pina, Sandra; Radhouani, Hajer; Montonen, Toni; Oliveira, Joaquim M.; Oliveira, Ana L.Timely and spatially-regulated injectable hydrogels, able to suppress growing tumors in response to conformational transitions of proteins, are of great interest in cancer research and treatment. Herein, we report rapidly responsive silk fibroin (SF) hydrogels formed by a horseradish peroxidase (HRP) crosslinking reaction at physiological conditions, and demonstrate their use as an artificial biomimetic three-dimensional (3D) matrix. The proposed SF hydrogels presented a viscoelastic nature of injectable hydrogels and spontaneous conformational changes from random coil to β-sheet conformation under physiological conditions. A human neuronal glioblastoma (U251) cell line was used for screening cell encapsulation and in vitro evaluation within the SF hydrogels. The transparent random coil SF hydrogels promoted cell viability and proliferation up to 10 days of culturing, while the crystalline SF hydrogels converted into β-sheet structure induced the formation of TUNEL-positive apoptotic cells. Therefore, this work provides a powerful tool for the investigation of the microenvironment on the programed tumor cells death, by using rapidly responsive SF hydrogels as 3D in vitro tumor models.
- Effect of cerium-containing hydroxyapatite in bone repair in female rats with osteoporosis induced by ovariectomyPublication . Vieira, Ewerton; Silva, Marcos; Maia-Filho, Antonio; Ferreira, Daniel; Figuerêdo-Silva, José; Rovaris, Karla; Fialho, Ana Cristina; Leite-Oliveira, Ana; Oliveira, André L. Menezes de; Fonseca, Maria Gardênnia da; Osajima, Josy A.; Silva-Filho, Edon C. daOsteoporosis is a public health problem, with bone loss being the main consequence. Hydroxyapatite (HA) has been largely used as a bioceramic to stimulate bone growth. In our work, a cerium-containing HA (Ce-HA) has been proposed and its effects on the antimicrobial and bone-inducing properties were investigated. The synthesis of the materials occurred by the suspension–precipitation method (SPM). The XRD (X-ray Diffraction) confirmed the crystalline phase, and the Rietveld refinement confirmed the crystallization of HA and Ce-HA in a hexagonal crystal structure in agreement with ICSD n◦ 26205. Characterizations by FT-IR (Fourier Transform Infrared Spectroscopy), XPS (X-ray Photoemission Spectroscopy), and FESEM-EDS (Field Emission Scanning Electron Microscope-Energy Dispersive X-ray Spectroscopy) confirmed the presence of cerium (Ce3+ and Ce4+ ). The antibacterial activity of Has was evaluated against Staphylococcus aureus 25,923 and Escherichia coli 25,922 strains, which revealed that the material has antimicrobial properties and the cytotoxicity assay indicated that Ce-containing HA was classified as non-toxic. The effects of Ce-HA on bone repair, after application in bone defects in the tibia of female rats with osteoporosis induced by ovariectomy (OVX), were evaluated. After 15 and 30 days of implantation, the samples were analyzed by Raman, histology and X-ray microtomography. The results showed that the animals that had the induced bone defects filled with the Ce-HA materials had more expressive bone neoformation than the control group.
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