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- 3D printing of new biobased unsaturated polyesters by microstereo-thermal-lithographyPublication . Gonçalves, Filipa A. M. M.; Costa, Cátia S. M. F.; Fabela, Inês G. P.; Farinha, Dina; Faneca, Henrique; Simões, Pedro N.; Serra, Arménio C.; Bártolo, Paulo J.; Coelho, Jorge F. J.New micro three-dimensional (3D) scaffolds using biobased unsaturated polyesters (UPs) were prepared by microstereo-thermal-lithography (μSTLG). This advanced processing technique offers indubitable advantages over traditional printing methods. The accuracy and roughness of the 3D structures were evaluated by scanning electron microscopy and infinite focus microscopy, revealing a suitable roughness for cell attachment. UPs were synthesized by bulk polycondensation between biobased aliphatic diacids (succinic, adipic and sebacic acid) and two different glycols (propylene glycol and diethylene glycol) using fumaric acid as the source of double bonds. The chemical structures of the new oligomers were confirmed by proton nuclear magnetic resonance spectra, attenuated total reflectance Fourier transform infrared spectroscopy and matrix assisted laser desorption/ionization-time of flight mass spectrometry. The thermal and mechanical properties of the UPs were evaluated to determine the influence of the diacid/glycol ratio and the type of diacid in the polyester's properties. In addition an extensive thermal characterization of the polyesters is reported. The data presented in this work opens the possibility for the use of biobased polyesters in additive manufacturing technologies as a route to prepare biodegradable tailor made scaffolds that have potential applications in a tissue engineering area.
- 3D-printed multisampling holder for microcomputed tomography applied to life and materials science researchPublication . Vasconcelos, Isabel; Franco, Margarida; Pereira, Mário; Duarte, Isabel; Ginjeira, António; Alves, NunoThe aim of this work was to design, fabricate, test and validate a 3D-printed multisampling holder for multi-analysis by microcomputed tomography. Different raw materials were scanned by microcomputed tomography. The raw material chosen was used to fabricate the holder by 3D printing. To validate the multisampling holder, five teeth were filled with a high density-material and scanned in two ways: a single and a multisampling scan mode. For each tooth, the root canal filling volume, porosity volume, closed pore volume, and open pore volume were calculated and compared when the same tooth was scanned in the two sampling scan mode. ABSplus P430™ allowed a high transmission value (84.3 %), and then it was the polymeric material selected to fabricate the holder. In a single sampling scan mode, the scan duration for scanning five teeth was 87.42 min, contrasting with 21.51 min for a multisampling scan mode, which scanned five teeth at the same time. The scan duration time and the cost using a multisampling holder represented a reduction of 75 % and the data volume generated represented a reduction of 60 %. Comparing the two scan modes, the results also showed that the difference of root canal filling volume, porosity volume, closed pore volume, and open pore volume was not statistically significant (p > .05). The multisampling holder was validated to do multi-analysis by microcomputed tomography without significant loss of quantitative accuracy data, allowing a reduction in scan duration time, imaging cost, and data storage.
- Bioactive Electrospun Fibers of Poly(ε-Caprolactone) Incorporating α-Tocopherol for Food Packaging ApplicationsPublication . Dumitriu, Raluca P.; Stoleru, Elena; Mitchell, Geoffrey R.; Vasile, Cornelia; Brebu, MihaiAntioxidant activity is an important feature for food contact materials such as packaging, aiming to preserve freshness and retard food spoilage. Common bioactive agents are highly susceptible to various forms of degradation; therefore, protection is required to maintain functionality and bioavailability. Poly(ε-caprolactone) (PCL), a biodegradable GRAS labeled polymer, was used in this study for encapsulation of α-tocopherol antioxidant, a major component of vitamin E, in the form of electrospun fibers. Rheological properties of the fiber forming solutions, which determine the electrospinning behavior, were correlated with the properties of electrospun fibers, e.g., morphology and surface properties. Interactions through hydrogen bonds were evidenced between the two components. These have strong effect on structuration of macromolecular chains, especially at low α-tocopherol amounts, decreasing viscosity and elastic modulus. Intra-molecular interactions in PCL strengthen at high α-tocopherol amounts due to decreased solvation, allowing good structural recovery after cease of mechanical stress. Morphologically homogeneous electrospun fibers were obtained, with ~6 µm average diameter. The obtained fibers were highly hydrophobic, with fast release in 95% ethanol as alternative simulant for fatty foods. This induced good in vitro antioxidant activity and significant in vivo reduction of microbial growth on cheese, as determined by respirometry. Therefore, the electrospun fibers from PCL entrapping α-tocopherol as bioactive agent showed potential use in food packaging materials.
- Effect of process parameters on the morphological and mechanical properties of 3D Bioextruded poly(ε‐caprolactone) scaffoldsPublication . Domingos, M.; Chiellini, F.; Gloria, A.; Ambrosio, L.; Bártolo, P.; Chiellini, E.Purpose – This paper aims to report a detailed study regarding the influence of process parameters on the morphological/mechanical properties of poly(1-caprolactone) (PCL) scaffolds manufactured by using a novel extrusion-based system that is called BioExtruder. Design/methodology/approach – In this study the authors focused investigations on four parameters, namely the liquefier temperature (LT), screw rotation velocity (SRV), deposition velocity (DV) and slice thickness (ST). Scaffolds were fabricated by employing three different values of each parameter. Through a series of trials, scaffolds were manufactured varying iteratively one parameter while maintaining constant the other ones. The morphology of the structures was investigated using a scanning electron microscope (SEM), whilst the mechanical performance was assessed though compression tests. Findings – Experimental results highlight a direct influence of the process parameters on the PCL scaffolds properties. In particular, DV and SRV have the highest influence in terms of road width (RW) and consequently on the porosity and mechanical behaviour of the structures. Research limitations/implications – The effect of process and design parameters on the biological response of scaffolds is currently under investigation. Originality/value – The output of this work provides a major insight into the effect of process parameters on the morphological/mechanical properties of PCL scaffolds. Moreover, the potential and feasibility of this novel extrusion-based system open a new opportunity to study how structural features may influence the characteristics and performances of the scaffolds, enabling the development of integrated biomechanical models that can be used in CAD systems to manufacture customized structures for tissue regeneration.
- Influence of Aloe vera on water absorption and enzymatic in vitro degradation of alginate hydrogel filmsPublication . Pereira, Rúben F.; Carvalho, Anabela; Gil, M.H.; Mendes, Ausenda; Bártolo, Paulo J.This study investigates the influence of Aloe vera on water absorption and the in vitro degradation rate of Aloe vera-Ca-alginate hydrogel films, for wound healing and drug delivery applications. The influence of A. vera content (5%, 15% and 25%, v/v) on water absorption was evaluated by the incubation of the films into a 0.1 M HCl solution (pH 1.0), acetate buffer (pH 5.5) and simulated body fluid solution (pH 7.4) during 24 h. Results show that the water absorption is significantly higher for films containing high A. vera contents (15% and 25%), while no significant differences are observed between the alginate neat film and the film with 5% of A. vera. The in vitro enzymatic degradation tests indicate that an increase in the A. vera content significantly enhances the degradation rate of the films. Control films, incubated in a simulated body fluid solution without enzymes, are resistant to the hydrolytic degradation, exhibiting reduced weight loss and maintaining its structural integrity. Results also show that the water absorption and the in vitro degradation rate of the films can be tailored by changing the A. vera content.
- Influence of commensal bacteria on the proteolytic and antigenic profiles of INFOGEST-like digested wheat gliadinPublication . Pereira-Costa, Flávio; Domingues, Vanessa S.; Roque, Ana; Almeida, Zaida L.; Cruz, Pedro F.; Cordeiro, Rachel; Trindade, Daniela; Moura, Carla; Melo, Joana B.; Pereira, Sónia G.; Vaz, Daniela C.Introduction: Celiac disease (CeD) is a chronic autoimmune enteropathy developed by genetically predisposed individuals when exposed to gluten. Gluten gliadins, along with gut microbiota, may influence CeD onset and progression through mechanisms that remain unclear. Methods: Gliadin-degrading bacterial isolates obtained from CeD patients, and their 1st-degree relatives’ stool and blood were identified (Bacillus tropicus, Enterococcus faecalis, Micrococcus sp., Cronobacter sakazakii, Pseudomonas aeruginosa, and Serratia marcescens) and used in an INFOGEST-like protocol to simulate gliadin digestion after 4 h (digested gliadin, d-gliadin). The d-gliadin digesta were analyzed by fast protein liquid chromatography (FPLC), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), fluorescence spectroscopy, and polyclonal and monoclonal (R5 and G12) enzyme-linked immunosorbent assays (ELISA). Results and discussion: In the absence of the bacterial isolates, gliadin is poorly digested and self-assembles within 1 day into intermediate and large protein oligomers/aggregates, enriched in β-sheet structure (FTIR amide I band between 1,600 and 1,700 cm−1) and able to bind thioflavin T and Congo red. Conversely, in the presence of the bacterial isolates, gliadin is further digested, leading to an increase in protein fragments. After 4 h, the P. aeruginosa, C. sakazakii, and B. tropicus d-gliadin digesta presented a mixture of d-gliadin peptides and aggregates that showed higher antigenicity (associated with the exposure of the 5-amino acid QQPFP and 6-amino acid QPQLPY epitopes, present in the 25-mer and 33-mer, respectively) than control digestions (without bacteria), while E. faecalis led to lower antigenicity. In turn, within 24 h of incubation, all bacterial isolates led to the formation of undigested material with lower antigenicity, either due to fewer 33-mers and 25-mers in solution, or to fragment aggregation into amorphous material, not exposing antigenic sequences. Hence, intestinal flora may enhance or diminish the antigenicity of gliadin, thereby modulating the immunogenic response to gliadin/gluten.
- MSCs Conditioned Media and Umbilical Cord Blood Plasma Metabolomics and CompositionPublication . Pereira, Tiago; Ivanova, Galya; Caseiro, Ana Rita; Barbosa, Paula; Bártolo, Paulo Jorge; Santos, José Domingos; Luís, Ana Lúcia; Maurício, Ana Colette; Ivanovic, ZoranHuman mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium - CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by 1H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture.