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Electrical stimulation (ES) has been tested clinically for over 40 years to promote bone healing, mainly as an adjunct to standard fracture treatments. However, the biological mechanisms by which ES promotes bone healing and the osteogenic commitment of bone progenitors cells remain poorly described.

In a recent study published in Scientific Reports, iBB researchers (João C. Silva, Fábio Garrudo and Frederico Ferreira) in collaboration with Sofia Fernandes (IBEB-Faculdade de Ciências-Universidade de Lisboa) and João Meneses, Nuno Alves and Paula Pascoal-Faria (CDRSP-Politécnico de Leiria), study the effect of five different ES protocols on the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs). A numerical finite element method (FEM) model of the culture platform was employed to characterize the system and predict the magnitude/distribution of the electric fields generated by the different ES protocols. In vitro cell culture studies showed that all the ES protocols did not impair cell viability and morphology and supported the osteogenic differentiation of hBM-MSCs. Our results evidenced relevant differences when considering the applied protocol operation mode (potential versus current controlled), including the choice of stimulus duration and period. They also suggest an improved performance of the applied current-controlled protocol (STIM3 OM) in promoting hBM-MSCs osteogenic differentiation as shown by a more efficient in vitro mineralization and higher expression of the late osteogenic marker genes. Overall, this work emphasizes the critical role of numerical modelling in selecting and optimizing ES parameters to improve MSC-based osteogenesis in vitro, a step forward towards the development of novel therapeutic strategies for bone regeneration.

This study was developed under the scope of the FCT funded projects “InSilico4OCReg” (PTDC/EME-SIS/0838/2021) and “OptiBioScafold” (PTDC/EME-SIS/4446/2020).

The production of piezoelectric constructs in order to address osteoporotic-related fractures holds significant promise. Such scaffolds could be used to mimic the native piezoelectric features of bone as well as assisting electrical stimulation-based therapies, which have been found to accelerate bone repair. In an article recently published in the journal Science and Technology of Advanced Materials, iBB researchers Frederico Barbosa, João Silva, Fábio Garrudo, Marta Carvalho, Paola Alberte, Leonor Resina and Frederico Ferreira, in collaboration with colleagues from the University of Nottingham and the Universitat Politècnica de Catalunya, developed novel hydroxyapatite-filled PVDF-TrFE nanofibers with enhanced piezoelectrical properties and osteogenic potential.

Magnetic hydrogels have recently been drawing a lot of attention in the scientific community due to their remote controllability and high biocompatibility. By combining these materials with a 3D bioprinting technique, it is possible to fabricate intricate structures responsive to an external magnetic stimulus, thus allowing the tuning of the constructs properties to better recapitulate the microarchitecture of native tissues. In this article recently published in the International Journal of Bioprinting, iBB researchers Duarte Almeida, Paola Sanjuan-Alberte, João C. Silva and Frederico Ferreira provided an overview of the current state of the art of magnetic hydrogels, exploring the production of the magnetic components and their introduction in the hydrogels, and emphasizing the current research made on the applications of these materials for tissue engineering strategies.

Biosurfactants, particularly mannosylerythritol lipids (MELs), have excellent biochemical properties and a wide range of potential applications. However, for use at an industrial level, it is necessary to develop more sustainable production processes, both in terms of the environment and economy. In this regard, the study “Production of mannosylerythritol lipids using oils from oleaginous microalgae: two sequential microorganism culture approach” led by iBB team, with Tiago Coelho, Miguel Nascimento, Nuno Faria, Frederico Fereira, together with LNEG team, Alberto Reis and Luísa Gouveia, was just published in Microorganisms journal. It reports, for the first time, the production of MELs using oils produced from microalgae. The bio-oil was extracted from Neochloris oleoabundans and evaluated for its use as a sole carbon source or as a co-substrate strategy, using D-glucose as an additional carbon source, on Moesziomyces spp. cultures to support cell growth and induce the production of MELs. Both M. antarcticus and M. aphidis were able to grow and produce MELs using algae-derived bio-oils as carbon sources and, interestingly, there were no significant differences in productivity when oils from microalgae or vegetable oils were used as carbon sources. These results provide new perspectives for the production of MELs in systems that combine different microorganisms.

CellAgri Portugal, the Portuguese Association for Cellular Agriculture Development, is a recently created independent non-profit organization, headquartered at IST-TagusPark.

The mission of CellAgri Portugal is to facilitate and encourage the cooperative development of cellular_agriculture in Portugal as well as to inform the public about the industrial, scientific, and technological developments in the field involving the Portuguese community worldwide.

Joaquim Cabral is the president of CellAgri Portugal and Carlos Rodrigues is the executive director. CellAgri Portugal also counts with the iBB researchers Frederico Ferreira, Ana Fernandes-Platzgummer, Paola Alberte, and the iBB PhD students Diana Marques and Hélder Tavares.

Lupanine is used as a building block in the synthesis of sparteine, a chiral selector in drug synthesis. This alkaloid is found in wastewaters derived from the debittering process that makes lupin beans edible. In a recently published work, carried out by researchers of the Faculty of Sciences and Faculty of Pharmacy from the University of Lisbon, and Teresa Esteves, Frederico Ferreira, Flávio Ferreira and Ana Mota from BERG-iBB, a computational chemistry approach was taken to design molecularly imprinted polymers (MIPs) selecting itaconic acid, a biobased building block, as a functional monomer that can provide higher affinities for lupanine. In this work, lupanine was concentrated from lupin bean wastewater by nanofiltration, extracted with ethyl acetate, and purified using the synthesized MIP, which was able to selectively recognize lupanine and improve its purity to 88%, with 82% recovery of the alkaloid, showing the potential application of this strategy to render the industrial process more sustainable.

Carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals and there is growing interest in producing them through microbial processes. While many CAs are intermediates of microbial central carbon metabolism, and therefore envisaging their production in a host of choice is relatively straightforward; for other CAs this approach is difficult. This could be because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). In a review published in Journal of Fungi, Nuno Mira’s team (iBB) reviewed the synthetic biology approaches that have been made towards enabling the production of non-natural CAs in microbes, with a large emphasis on metabolic retrobiosynthesis methodologies. Additionally, the authors also reviewed the pathway prospecting studies towards microbial levulinic, poly-lactic and methacrylic acid production, as two case-studies where there is a need to bridge the gap between natural CAs and their non-natural industrial derivatives.

Cassava (Manihot esculenta) cultivation is of great importance in many economies, particularly in Colombia. About 630,000 tons of C-rich cassava waste is produced annually and applications to high value products, applying the circular economy concept, must be developed. A recent publication in Journal of Polymers and the Environment assesses the potential use of cassava peel for the production of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. A copolymer of P3HB-3HV was produced and processed into electrospun meshes of random and aligned microfibers, allowing the development of structures that can be applied in the context of tissue engineering. This work involved Manuela Fonseca, Frederico Ferreita and Teresa Cesário form BERG-IBB and has been done in collaboration with researchers from the University of Antioquia, Medellin-Colombia.

Removal of genotoxic impurities (GTI) from active pharmaceutical ingredients (API) is mandatory to deliver safe medicines, but to achieve GTI at ultra-low limit (<1.5 µg/day) implies significative API loss. A recent paper published in the journal Membranes by Flávio Ferreira, Leonor Resina, Teresa Esteves and Frederico Ferreira from BERG-iBB, propose the use of a mathematical model to predict whether adsorption or membrane process is suitable for API purification. Moreover, in cases where none of them have an acceptable API loss, a hybrid process combining both processes was developed, reducing API loss from 24.76% to 9.76%. Furthermore, economic and environmental analyses were also performed.

Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, costly and not very versatile. In a recent study published in Biotechnology Journal, researchers from SCERG-iBB working with colleagues from the Polytechnic Institute of Leiria and Rensselaer Polytechnic Institute (USA) developed a novel, cost-effective and customizable perfusion bioreactor fabricated by additive manufacturing (AM) to study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly (ε-caprolactone) (PCL) scaffolds. Results suggest that the chondrogenic differentiation of hBMSCs was enhanced in cell-scaffold constructs cultured under perfusion and highlights the potential of customizable AM platforms for developing more reliable in vitro models and improved personalized cartilage repair strategies.

The regulation of angiogenesis could be very helpful in the development of therapeutic approaches for wound healing and anticancer therapy. Recently, Dra. Filipa Pires from Instituto de Telecomunicações in collaboration with IBB (Dr. João C Silva and Dr. Frederico C. Ferreira) and FCT-UNL (Dr. Carla Portugal) researchers, designed magnetic heparinized hydrogels to modulate the anti-inflammatory and angiogenic activity of mesenchymal stem cells (MSCs), aiming to accelerate tissue healing. The results evidenced that the application of a static magnetic field guides MSC alignment and affects the angiogenic and anti-inflammatory character of the MSCs secretome. Particularly, the outcomes showed that the hydrogels containing less heparin and magnetic nanoparticles (0.25%) display a low pro-inflammatory character, ensuring an MSCs secretome with enhanced pro-angiogenic properties, which showed to be able to enhance wound contraction and cell migration rate compared to non-heparinized hydrogels.

The repair of bone defects remains an important challenge in current orthopaedic clinical practice. Novel approaches, including electrical stimulation-based therapies, have been gaining traction due to their encouraging results in both in vivo and in vitro settings. The development of electroconductive scaffolds to assist this type of therapy holds significant promise, as such constructs could be used to guide electrical stimuli directly to the damaged site, therefore increasing the effectiveness of the regeneration process. In an article recently published in the journal International Journal of Molecular Sciences, iBB researchers Frederico Barbosa, João Silva, Fábio Garrudo and Frederico Ferreira, in collaboration with colleagues from Instituto de Telecomunicações (IT) and CERENA, developed mineralized and electroconductive PEDOT:PSS-based nanofibers with bone-like features. This project was developed under the scope of Frederico Barbosa’s PhD thesis, supervised by Frederico Ferreira and João Silva.

The irreversible progressive nature of degenerative osteochondral defects, affecting both articular cartilage and the subchondral bone, is a leading cause for joint disease and disability among adults, resulting in severe limitations for people to perform daily activities and represents a heavy economic burden for society. Current treatments do no provide adequate long-term solutions and innovative tissue engineering strategies are regarded as a promising alternative to improve clinical outcomes. A key aspect that needs to be considered is the integration between native tissue and the tissue engineered construct. In this recent article resulting from a collaboration between researchers from iBB and CDRSP-Politécnico de Leiria, published in the Journal Polymers, a new approach to design and manufacture curved scaffolds to mimic osteochondral tissue geometry was presented. The curvature of the sphere was proposed as a template and a procedure was developed for an automated design of scaffolds with explicitly defined curvatures. Fused filament fabrication (3D-Printing) was used for scaffolds’ manufacturing. A Shape fidelity analysis implemented through micro-CT and SEM imaging validated the maximum curvature printability limit predicted from CAD modeling and confirmed the suitability of fused filament fabrication to manufacture curvature-featuring scaffolds. Additionally, a mechanical analysis was conducted with printed scaffolds and through finite element analysis to determine scaffold mechanical properties and identify the regions more susceptible to higher loads.

The use of Mannosylerythritol lipids (MELs), a group of biosurfactants with excellent biochemical properties, in bioremediation in marine and terrestrial environments has been envisioned. However, knowledge regarding their ecotoxicity is limited, and the current production costs are too high to make them competitive in the surfactant market. In an effort to facilitate the use of MELs for marine bioremediation purposes, the team from 2BRG/iBB studied MELs production using seawater in medium formulation as reported on “Towards Mannosylerythritol Lipids (MELs) for Bioremediation: Effects of NaCl on M. antarcticus Physiology and Biosurfactant and Lipid Production; Ecotoxicity of MELs”, published in Journal of Marine Science and Engineering. The cells were exposed to different levels of NaCl during fermentation and the effects of increased salinity on the cells and their performance were monitored. In addition, the cells were briefly exposed to osmotic shock by introducing pure NaCl into the broth to measure their physiological responses. Although the overall effect of NaCl in the medium was negative, cells produced more lipases in these stress conditions. Furthermore, the changes triggered by osmotic shock caused changes in the cell surface and affected their hydrophobicity, reducing the levels of MELs adsorbed to the cells, which in turn led to an increase in the formation of MEL-rich beads. Marine-level salinity (3.5%) was found to be sufficient to enable the production of MELs under non-sterile conditions and to inhibit the introduction of bacterial contaminants. Finally, the toxicity levels of MELs in a model marine organism and plant model were lower than those of other biosurfactants and a commercial chemical dispersant used for bioremediation.

This publication has as first-author the (now former) PhD student Petar Kekovic of the MIT-Portugal Program in Bioengineering - and as a corresponding author Frederico Ferreira and Nuno Faria.  Additionally, the co-authors and a team from Sintef – Roman Netzer, Umer Farooq and Ariadna Szczybelski submitted a patent application for formulation of a novel MEL based oils spill response agent, reference number 117583.

As current treatment options for cartilage degeneration remain ineffective, tissue engineering has emerged as an exciting approach to create cartilage substitutes. In particular, hydrogels seem to be suitable candidates for this purpose due to their high biocompatibility and customizability, being able to be tailored to fit the biophysical properties of native cartilage. Moreover, these hydrogel matrices can be combined with conductive materials in order to simulate the natural electrochemical properties of articular cartilage. In this recent review published in the journal Gels, iBB researchers Filipe Miguel (MSc student in Biotechnology), Frederico Barbosa (PhD student in Bioengineering), Prof. Frederico Ferreira and Dr. João Silva highlight the most common conductive materials combined with hydrogels and their diverse applications, and discuss the current state of research on the development of electrically conductive hydrogels for cartilage tissue engineering.

Understanding the mechano–biological coupling mechanisms of biomaterials for tissue engineering is of major importance to assure proper scaffold performance in situ. Therefore, it is of paramount importance to establish correlations between biomaterials, their processing conditions, and their mechanical behaviour, as well as their biological performance. In a collaborative work between CDRSP-Politécnico de Leiria and SCERG-iBB  (João C. Silva and Frederico Ferreira), it was possible to infer a correlation between the addition of different concentrations of graphene nanoparticles (GPN) in three-dimensional poly(ε-caprolactone) (PCL)-based scaffolds, their extrusion-based processing parameters, and the lamellar crystal orientation observed in the different scaffolds through small-angle X-ray scattering experiments. Moreover, in vitro cell culture studies performed at SCERG-iBB demonstrated the suitability and potential of these novel 3D PCL/GPN scaffolds for tissue engineering applications. The results of this study were just published in Polymers.

"This week in the Portuguese newspaper "Público" you will find a summary of the Algae2Fish project, where you can read about the reasons behind the project and its perspectives for the next two years. The team, composed by professor Frederico Ferreira, Dr. Paola Sanjuan Alberte, Dr. Carlos Rodrigues, and Msc Diana Marques, with the support of The Good Food Institute, is currently working to produce the first cell-cultured sea bass fillet!"

The project Algae2Fish led by Frederico Ferreira from SCERG-iBB has received funding from the Global Food Institute with the aim of developing a boneless cultivated seabass fillet. 3D printing technology will be used to produce scaffolds that will give the fillet structure, replicating the fibrous texture of fish. The scaffolds will be formed using material from algae and plants, with the algae contributing valuable omega-3 fatty acids such as those found in conventional fish. Read more about the project here.

Ana Isabel de Vila-Santa Braga Campos will be defending her PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Friday the 16th of April 2021, at 15:00 am (https://videoconf-colibri.zoom.us/j/81314376867). During the last years, and under the supervision of Nuno Mira and Frederico Ferreira from iBB, and Kristala Prather from MIT, Ana Isabel investigated how pathways for the synthesis of levulinic and itaconic acids can be engineered in yeasts. The title of her thesis is “Synthetic biology approaches to foster yeasts as hosts for the production of carboxylic acids and their derivatives: emphasis on levulinic and itaconic acids”.

João Carlos Silva and Frederico Castelo Ferreira from SCERG-iBB are guest-editing a special issue for the open-access journal “Polymers” entitled “Advanced Polymeric Scaffolds for Stem Cell Engineering and Regenerative Medicine”. Polymer scaffolds play a crucial role in tissue engineering and regenerative medicine applications since they can closely mimic the architecture of a native extracellular matrix (ECM) and improve the biological performance of cells both in vitro and in vivo. This Special Issue welcomes full research papers, communications and reviews on recent exciting developments of polymeric scaffolds for tissue engineering and regenerative medicine applications.