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- Biomechanical performance of hybrid electrospun structures for skin regenerationPublication . Dias, J.R.; Baptista-Silva, S.; Sousa, A.; Oliveira, A. L.; Bártolo, P. J.; Granja, P. L.Wound dressings made by electrospun nanofibers have been demonstrating great potential to regenerate skin tissue as compared to the conventional membrane products available in the market. Until today most of the developed dressings have only demonstrated the capability to regenerate the dermis or epidermis. In this study we propose new hybrid electrospun meshes combining polycaprolactone and gelatin. Several approaches, multilayer, coating and blend were stablished to investigate the most appropriate hybrid structure with potential to promote skin regeneration in its full thickness. The structures were evaluated in terms of physico-chemical properties (porosity, water vapor permeability, contact angle and swelling degree) and according to its mechanical and biological performance. Multilayer and blend structures demonstrated to fit most of native skin requirements. However, looking to all the performed characterization we considered multilayer as the most promising hybrid structures, due its high porosity which contributed to an ideal water vapor permeability rate and good mechanical and biological properties. Based on this multilayer structure is a promisor wound dressing.
- Advanced face mask filters based on PCL electrospun meshes dopped with antimicrobial MgO and CuO nanoparticlesPublication . Ferreira, Carolina A. M.; Guerreiro, Sara F.C.; Valente, Joana F. A.; Patrício, Tatiana M.F.; Alves, Nuno; Mateus, Artur; Dias, Juliana R.The pandemic situation caused by coronavirus clearly demonstrated the need for alternatives able to protect the respiratory tract and inactivate the infectious agents. Based on this, antibacterial face-mask filters of polycaprolactone (PCL) dopped with magnesium oxide (MgO) and copper oxide (CuO) nanoparticles (NPs) were produced using an electrospinning technique. A morphological analysis of electrospun meshes evaluated the success of nanoparticles’ incorporation as well as the average fibers’ diameters (481 +- 272 nm). The performance of electrospun nanofibers was also assessed in terms of tensile strength (0.88 +- 0.25 MPa), water vapor permeability (11,178.66 +- 35.78 g.m-2.day-1), stability under wet conditions and antibacterial activity according to the standard guidelines. The filters showed structural stability up to 2 h of washing and improved antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for optimized concentrations of MgO and CuO NPs. Overall, electrospun meshes with antibacterial activity were successfully developed for advanced filtering applications.
- In situ enabling approaches for tissue regeneration: Current challenges and new developmentsPublication . Dias, Juliana R.; Ribeiro, Nilza; Baptista-Silva, SaraIn situ tissue regeneration can be defined as the implantation of tissue-specific biomaterials (by itself or in combination with cells and/or biomolecules) at the tissue defect, taking advantage of the surrounding microenvironment as a natural bioreactor. Up to now, the structures used were based on particles or gels. However, with the technological progress, the materials’ manipulation and processing has become possible, mimicking the damaged tissue directly at the defect site. This paper presents a comprehensive review of current and advanced in situ strategies for tissue regeneration. Recent advances to put in practice the in situ regeneration concept have been mainly focused on bioinks and bioprinting techniques rather than the combination of different technologies to make the real in situ regeneration. The limitation of conventional approaches (e.g., stem cell recruitment) and their poor ability to mimic native tissue are discussed. Moreover, the way of advanced strategies such as 3D/4D bioprinting and hybrid approaches may contribute to overcome the limitations of conventional strategies are highlighted. Finally, the future trends and main research challenges of in situ enabling approaches are discussed considering in vitro and in vivo evidence.
- Continuous production of highly tuned silk/calcium-based composites: exploring new pathways for skin regenerationPublication . Veiga, Anabela; Magalhaes, Rui; Duarte, Marta M.; Dias, Juliana R.; Alves, Nuno M.; Costa-Pinto, Ana Rita; Castro, Filipa; Rocha, Fernando; Oliveira, Ana L.Calcium plays an important role in barrier function repair and skin homeostasis. In particular, calcium phosphates (CaPs) are well established materials for biomedical engineering due to their biocompatibility. To generate biomaterials with a more complete set of biological properties, previously discarded silk sericin (SS) has been recovered and used as a template to grow CaPs. Crucial characteristics for skin applications, such as antibacterial activity, can be further enhanced by doping CaPs with cerium (Ce) ions. The effectiveness of cell attachment and growth on the materials highly depends on their morphology, particle size distribution, and chemical composition. These characteristics can be tailored through the application of oscillatory flow technology, which provides precise mixing control of the reaction medium. Thus, in the present work, CaP/SS and CaP/SS/Ce particles were fabricated for the first time using a modular oscillatory flow plate reactor (MOFPR) in a continuous mode. Furthermore, the biological behavior of both these composites and of previously produced pure CaPs was assessed using human dermal fibroblasts (HDFs). It was demonstrated that both CaP based with plate-shaped nanoparticles and CaP-SS-based composites significantly improved cell viability and proliferation over time. The results obtained represent a first step towards the reinvention of CaPs for skin engineering.
- Experimental validation of a computational fluid dynamics model using micro-particle image velocimetry of the irrigation flow in confluent canalsPublication . Pereira, Mário Rito; Silva, Gonçalo; Semião, Viriato; Silvério, Vânia; Martins, Jorge N.R.; Pascoal-Faria, Paula; Alves, Nuno; Dias, Juliana R.; Ginjeira, AntónioAim: This study aimed to experimentally validate a computational fluid dynamics (CFD) model, using micro-particle image velocimetry (micro-PIV) measurements of the irrigation flow velocity field developed in confluent canals during irrigation with a side-vented needle. Methodology: A microchip with confluent canals, manufactured in polydimethylsiloxane was used in a micro-PIV analysis of the irrigation flow using a side-vented needle placed 3 mm from the end of the confluence of the canals. Velocity fields and profiles were recorded for flow rates of 0.017 and 0.1 ml/s and compared with those predicted in CFD numerical simulations (using a finite volume commercial code –FLUENT) for both laminar and turbulent regimes. Results: The overall flow pattern, isovelocity and vector maps as well as velocity profiles showed a close agreement between the micro-PIV experimental and CFD predicted data. No relevant differences were observed between the results obtained with the laminar and turbulent flow models used. Conclusions: Results showed that the laminar CFD modelling is reliable to predict the flow in similar domains.
- A biorefinery approach to the biomass of the seaweed Undaria pinnatifida (Harvey Suringar, 1873): obtaining phlorotannins-enriched extracts for wound healingPublication . Ferreira, Carolina A. M.; Félix, Rafael; Félix, Carina; Januário, Adriana P.; Alves, Nuno; Novais, Sara C.; Dias, Juliana R.; Lemos, Marco F.L.Brown seaweeds are recognized sources of compounds with a wide range of properties and applications. Within these compounds, phlorotannins are known to possess several bioactivities (e.g., antioxidant, anti-inflammatory, and antimicrobial) with potential to improve wound healing. To obtain phlorotannins enriched extracts from Undaria pinnatifida, a biorefinery was set using lowcost industry-friendly methodologies, such as sequential solid–liquid extraction and liquid–liquid extraction. The obtained extracts were screened for their antioxidant and antimicrobial activity against five common wound pathogens and for their anti-inflammatory potential. The ethanolic wash fraction (wE100) had the highest antioxidant activity (114.61 +- 10.04 mmol mg-1 extract by Diphenyl-1-picrylhydrazyl (DPPH) and 6.56 +-1.13mMeq. Fe II mg-1 extract by and Ferric Reducing Antioxidant Power (FRAP)), acting efficiently against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria, and showing a nitric oxide production inhibition over 47% when used at 0.01 ug mL-1. NMR and FTIR chemical characterization suggested that phlorotannins are present. Obtained fraction wE100 proved to be a promising candidate for further inclusion as wound healing agents, while the remaining fractions analyzed are potential sources for other biotechnological applications, giving emphasis to a biorefinery and circular economy framework to add value to this seaweed and the industry.
- Optimization of phycobiliprotein pigments extraction from red algae Gracilaria gracilis for substitution of synthetic food colorantsPublication . Pereira, Tatiana; Barroso, Sónia; Mendes, Susana; Amaral, Renata A.; Dias, Juliana R.; Baptista, Teresa; Saraiva, Jorge A.; Alves, Nuno; Gil, Maria M.The extraction of phycobiliprotein (PBP) pigments from red algae Gracilaria gracilis was optimized using maceration, ultrasound-assisted extraction (ultrasonic water bath and ultrasonic probe), high pressure-assisted extraction, and freeze-thaw. The experimental conditions, namely homogenization time (t1), buffer concentration (C), treatment time (t2), biomass: buffer ratio (R), and pressure (P), were optimized using Response Surface Methodology (RSM). The yield of phycoerythrin (PE) extracted, determined spectroscopically, was used as the response variable. Maceration was the most efficient extraction method yielding 3.6 mg PE/g biomass under the optimal conditions (t1 = t2 = 10 min; C = 0.1 M; R = 1:50). Scanning Electron Microscopy (SEM) analysis of the biomass before and after the cell disruption treatments revealed a more efficient cell wall rupture with maceration.
- Box-Behnken Design a Key Tool to Achieve Optimized PCL/Gelatin Electrospun Mesh, Macromolecular Materials and EngineeringPublication . Guerreiro, S.F.C.; Valente, J.F.A.; Dias, J.R.; Alves, N.Hybrid electrospun nanofibers of polycaprolactone (PCL)/gelatin are considered as drug-delivery systems for increasing the treatment efficacy in superficial (skin) wounds. Continuous delivery of therapeutic agents, skin extracellular matrix similarity, management of wound exudate, and antimicrobial barrier effect are the major advantages of electrospun nanofibers in skin applications. Additionally, combining the favorable properties of PCL and gelatin, regarding their biocompatibility, biodegradability and mechanical performance have been revealed promising parameters to be considered for blend in hybrid structures. However, the usual optimization protocol of nanofibers’ production in electrospinning is based on the observation of one-variable-attime being this methodology expensive and time-consuming. Therefore, in this research work, a statistical model based on four input variables namely, the flow rate, the needle-working distance, the applied voltage, and the ratio of PCL in the solution, is developed to predict the behavior of nanofibers. The performance of nanofibers is monitored by measurements of fiber’s diameter, mesh’s thickness, and mesh’s permeability. Overall, the model showed to be statistically significant (p-value < 0.05) and an independent analysis validated the predicted response for optimal condition. Finally, a delivery study is performed to evaluate the electrospun mesh performance as a drug carrier.
- Fast decellularization process using supercritical carbon dioxide for trabecular bonePublication . Duarte, Marta M.; Ribeiro, Nilza; Silva, Inês V.; Dias, Juliana R.; Alves, Nuno M.; Oliveira, Ana L.Decellularization is a process that consists on the removal of immunogenic cellular material from a tissue, so that it can be safely implanted as a functional and bioactive scaffold. Most decellularization protocols rely on the use of harsh chemicals and very long washing processes, leading to severe changes in the ultrastructure and loss of mechanical integrity. To tackle these challenges, supercritical carbon dioxide (scCO2) is herein proposed as an alternative methodology for assisting decellularization of porcine trabecular bone tissue and is combined, for the first time, with Tri(n-butyl) phosphate (TnBP). Histological and DNA analysis revealed that both TnBP and scCO2 were able to extract the DNA content from the scaffolds, being this effect more pronounced in treatments that used TnBP as a co-solvent. The combined protocol led to a decrease in DNA content by at least 90%, demon- strating the potential of this methodology and opening new possibilities for future optimizations.
- Composite Central Face Design—An Approach to Achieve Efficient Alginate MicrocarriersPublication . Valente, J.F.A.; Dias, J.R.; Sousa, A.; Alves, N.Microparticulated drug delivery systems have been used as promising encapsulation systems for protecting drugs for in vitro and in vivo applications, enhancing its stability, providing an increased surface to volume ratio, reducing adverse effects, and hence an improvement in bioavailability. Among the studied microparticles, there is a rising interest in the research of alginate microparticles for pharmaceutical and biomedical fields confirming its potential to be used as an effective matrix for drug and cell delivery. Moreover, calcium alginate has been one of the most extensively forming microparticles in the presence of divalent cations providing prolonged drug release and suitable mucoadhesive properties. Regarding the above mentioned, in this research work, we intended to produce Ca-alginate micro-vehicles through electrospraying, presenting high encapsulation efficiency (EE%), reduced protein release across the time, reduced swelling effect, and high sphericity coefficient. To quickly achieve these characteristics and to perform an optimal combination among the percentage of alginate and CaCl2, design of Experiments was applied. The obtained model presented to be statistically significant (p-value < 0.05), with a coefficient of determination of 0.9207, 0.9197, 0.9499, and 0.9637 for each output (EE%, release, swelling, and sphericity, respectively). Moreover, the optimal point (4% of alginate and 6.6% of CaCl2) was successfully validated.
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