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The bone is a complex and dynamic structure subjected to constant stress and remodeling. Due to the worldwide incidence of bone disorders, engineered bone tissues have emerged as solution for bone grafting, which require sophisticated scaffolding architectures while keeping high mechanical performance. However, the conjugation of bone-like scaffold architecture with efficient mechanical properties is still a critical challenge for biomedical applications.  In this sense, the present study is focused on the development of silk fibroin (SF) scaffolds crosslinked with horseradish peroxidase and mixed with zinc (Zn) and strontium (Sr)-doped β-tricalcium phosphate (ZnSr.TCP) to mimic bone structures. The ZnSr.TCP-SF hydrogels were tuned in programmable ice-templating parameters, and further freeze-dried, to obtain 3D scaffolds with controlled pore orientation. The results showed interconnected channels in the ZnSr.TCP-SF scaffolds that mimic the porous network of the native subchondral bone. The architecture of the scaffolds was characterized by microCT and showing tunable pore size according to freezing temperatures (-196 ºC: ~80.2 ± 20.5 µm; -80 ºC: ~73.1 ± 20.5 µm; -20 ºC: ~104.7 ± 33.7 µm). The swelling ratio, weight loss, and rheological properties were also assessed, revealing that the scaffolds were able to keep their integrity and morphology after aqueous immersion. Thus, the ZnSr.TCP-SF scaffolds made of aligned porous structure were developed as affordable candidates for future applications in clinical osteoregeneration and in vitro bone tissue modelling., FCT for the financial support of the Hierarchitech project (M435 ERA.NET/0001/2014) and for the distinctions attributed to S.P. (CEECIND/03673/2017) and C. 436 G. (SFRH/BPD/94277/2013). R. F. Canadas is also thankful to FCT for the doctoral scholarship 437 (SFRH/BD/92565/2013), Fundo Europeu de Desenvolvimento Regional (FEDER), and Programa 438 Operacional Competitividade e Internacionalização (POCI) for funding the Young Researcher 439 Contract (B-Liver Project, PTDC/EMD-EMD/29139/2017)

Osteoarthritis is a degenerative joint disease characterized by the progressive deterioration of articular cartilage, synovial inflammation and changes in periarticular and subchondral bone, being a leading cause of disability. Conventional treatments present several side effects and can involve the use of painkillers and non-steroidal anti-inflammatory drugs. In this sense, researchers have shifted the focus of new therapeutics to biomolecular agents and/or their combination with cells. However, the efficacy of these molecules is dependent on their metabolization which can differ according to the administration route and delivery approach. Consequently, tissue engineering strategy comprising the use of biomaterials can provide support for neo-tissue osteochondral repair/regeneration whilst conferring proper mechanical and functional features as well as protecting biomolecular agents from premature degradation. The current strategies and challenges used in biomaterials functionalization with peptides that can mimic ECM proteins or other natural soluble biomolecules, important to induce the complex interactions between cells and the ECM, are discussed herein. Many opportunities for treating OA are being explored by means of peptide-biofunctionalization of biomaterials which can be designed to be recognizable, induce differentiation, prevent infection, degrade at an intended rate or act as drug delivery systems for controlled release or even as simple triggers of cell behavior., The authors would like to acknowledge the financial supports from the Portuguese Foundation for Science and Technology for the M-ERA.NET/0001/2014 project and for the funds provided under the program Investigador FCT 2012 and 2015 (IF/00423/ 2012 and IF/01285/2015), and European Commission via H2020- MSCA-RISHE programme (BAMOS, Grant No. 734156).

A Kefiran-based scaffold was developed using a freeze gelation technique, and its potential use for both drug delivery system and tissue engineering applications were investigated. This scaffold showed, through micro-computed tomography and scanning electron microscopy, high porosity (82.3 ± 4.4%), thick pore walls (13.4 ± 0.7 μm), aerogel form with foam-like structure, and elastic behaviour (δ = 16 ± 0.7°). Moreover, the Kefiran scaffold degradation showed a delayed profile for over 28 days. The developed scaffold allowed a slow and sustained diclofenac release over two weeks, and the human Adipose-derived Stem Cells, cultured onto the Kefiran scaffold, were metabolically active after 72 h. Therefore, our research suggests that Kefiran cryogel could be a potential candidate for drug delivery of controlled bioactive molecules and tissue engineering scaffolding., Hajer Radhouani, Cristiana Gonçalves and F. Raquel Maia were supported by the Fundação para a ciência e a Tecnologia (FCT) from Portugal, with references CEECIND/00111/2017, SFRH/BPD/94277/2013 and SFRH/BPD/117492/2016, respectively. Diana Bicho was supported through the M-ERA-NET/0001/2014 project (FCT). Joaquim. M. Oliveira would like to thank the FCT for the fund provided under the program Investigador FCT 2015 (IF/01285/2015). This work was funded by the R&D Project KOAT – Kefiran exopolysaccharide: Promising biopolymer for use in regenerative medicine and tissue engineering, with reference PTDC/BTMMAT/29760/2017 (POCI-01-0145-FEDER-029760), financed by FCT and co-financed by European Regional Development Fund (FEDER) and Operational Programme for Competitiveness and Internationalisation (POCI).

Influenza viruses cause annual epidemics and occasional pandemics and thus represent a significant public health problem together with considerable economic consequences. As current influenza virus vaccines do not provide the best immunological protection, plasmid DNA vaccines have been seen as a potential alternative due to the stimulation of both B- and T-cell responses without the presence of any infectious agent and because they are easily produced, highly stable and safe. From this standpoint, several downstream methods have been proposed to obtain high quantities of pharmaceutical grade supercoiled plasmid DNA. This work describes a rapid process, based on ion exchange chromatography using an ethylenediamine (EDA) monolith for the purification of an experimental DNA influenza vaccine. The purification process allowed a significant reduction of the host contaminants, such as proteins, RNA and genomic DNA and the DNA vaccine recovery with a purity degree of 97.1% and a step yield of 47%. Finally, both transfection efficiency of the influenza vaccine and its cytotoxicity in two types of eukaryotic cell lines were tested in order to compare with the same plasmid DNA purified with a commercial kit.

PEX1 and PEX6 are two members of the ATPases associated with diverse cellular activities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a bona fide substrate of the PEX1-PEX6 complex. The authors thank Britta Moellers (Ruhr-Universität Bochum, Germany) for providing plasmids and recombinant protein for the generation of the anti-PEX6 antibody. This work was funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020 –Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020 – Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (NORTE-01-0145-FEDER-000008). A.G.P, T.F., D.B., A.F.D., A.B.B. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.

The osteochondral tissue represents a complex structure composed of four interconnected structures, namely hyaline cartilage, a thin layer of calcified cartilage, subchondral bone, and cancellous bone. Due to the several difficulties associated with its repair and regeneration, researchers have developed several studies aiming to restore the native tissue, some of which had led to tissue-engineered commercial products. In this sense, this chapter discusses the good manufacturing practices, regulatory medical conditions and challenges on clinical translations that should be fulfilled regarding the safety and efficacy of the new commercialized products. Furthermore, we review the current osteochondral products that are currently being marketed and applied in the clinical setting, emphasizing the advantages and difficulties of each one.

In embryonic development, pure cartilage structures are in the basis of bone-cartilage interfaces. Despite this fact, the mature bone and cartilage structures can vary greatly in composition and function. Nevertheless, they collaborate in the osteochondral region to create a smooth transition zone that supports the movements and forces resulting from the daily activities. In this sense, all the hierarchical organization is involved in the maintenance and reestablishment of the equilibrium in case of damage. Therefore, this interface has attracted a great deal of interest in order to understand the mechanisms of regeneration or disease progression in osteoarthritis. With that purpose, in vitro tissue models (either static or dynamic) have been studied. Static in vitro tissue models include monocultures, co-cultures, 3D cultures, and ex vivo cultures, mostly cultivated in flat surfaces, while dynamic models involve the use of bioreactors and microfluidic systems. The latter have emerged as alternatives to study the cellular interactions in a more authentic manner over some disadvantages of the static models. The current alternatives of in vitro mimetic models for bone-cartilage interface regeneration are overviewed and discussed herein.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the “plunger” - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the “barrel” - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the “barrel” is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model. We would like to thank Dr. Marc Fransen (KU Leuven) for his critical reading of the manuscript. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional, funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministerio da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “Themolecular mechanisms of peroxisome biogenesis” (PTDC/BEX-BCM/2311/2014), and through Norte 2020–Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008).” T.F., T.A.R., A.F.D., A.B.B., and D.B. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu.

Gellan gum is a polysaccharide polymer used in the pharmaceutical and food industries due to its gelling ability. In this work, it was intended to innovatively develop a new chromatographic matrix based on this polymer. This new chromatographic approach takes into account properties like the porosity and hydrophilicity of gellan gels as well as its anionic nature in order to study the interactions established with three model proteins. The versatility of interactions of this new matrix was also tested applying hydrophobic interaction chromatography conditions. Binding/elution studies with three model proteins were performed using gellan cross-linked with calcium. These studies established the elution patterns of the studied biomolecules, separating simple mixtures of proteins with different interaction profiles. Finally, it was made experiments with nucleic acids in hydrophobic conditions with the same purposes, proving the versatility of interactions in the gellan matrix. The structure of the formulation applied in the chromatographic steps was evaluated by confocal laser microscopy. This technique showed the microscopic organization of the gels correlating it with its macroscopic porosity and permeability. Due to the necessity of the presence of calcium in the chemical environment of the stationary phase it was studied the stability that 3 different ions (calcium, copper and zinc) conferred to the matrix. Of the three studied ions it was copper who gave the biggest stability to the matrix. These preliminary results show a new potential application of gellan gum as a chromatography matrix, taking advantage of its natural charge without the necessity of functionalizing it with specific ligands.

DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids

Affinity chromatography represents a sole technique in purification of different biomolecules. The specific recognition between affinity ligands and target biomolecules has a major role in the specificity of the process. Therefore, choosing the right ligand is a crucial step for the development of a successful purification system. This work describes the application of the DNA intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as a chromatographic affinity ligand for the specific separation and purification of supercoiled plasmid DNA (pDNA). The support was prepared by coupling DAPP onto an epoxy-activated Sepharose matrix, using mild conditions and resulting in a ligand density of 0.15 mmol DAPP/g derivatized Sepharose. The characterization of DAPP-Sepharose support in terms of dynamic binding capacity was achieved by studying the effect of plasmid DNA concentration and flow rate on pDNA adsorption. The maximum capacity value of 336.75 μg pDNA/mL gel was obtained at 1 mL/min with a pDNA concentration of 150 μg/mL. Moreover, the values did not vary significantly with the variation of flow rate. In addition, the DAPP-Sepharose showed a high affinity towards pDNA as quantified by the respective dissociation constant (Kd = 2.29 ± 0.195 × 10−7 M). The support was also tested for the purification of two plasmid molecules with different sizes (pVAX1-LacZ and pCAMBIA-1303, with 6.05 kbp and 12.361 kbp, respectively) from clarified Escherichia coli lysate solutions. Total retention of all lysate components was achieved without any added salt to the eluent buffer. The selective elution of impurities and supercoiled pDNA was accomplished simply by the addition of small amounts of salt to the buffer solution. The yield for pCAMBIA-1303 was 65% and for pVAX1-LacZ was 94%, with all host impurity levels in accordance with the requirements established by the regulatory agencies.

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.

In recent years, researchers have made several efforts to fight highly contagious respiratory disease caused by influenza virus. One of the best options for reducing the impact of this virus infection is DNA vaccination, therefore it is necessary a large quantity of highly pure plasmid DNA (pDNA) [1, 2]. Thus, in this work it is proposed the production and purification of pDNA expressing influenza virus hemaglutinin protein. For the purification strategy, monoliths are chosen because of their high binding capacity and the excellent mass transfer properties. Agmatine was the ligand of choice, once this amino acid derivative showed to be successful not only in the purification of sc pDNA isoforms but also from complex lysates. The results showed that agmatine is a multifaceted ligand to purify the sc pDNA influenza vaccine under the requirements of the regulatory agencies. In vitro experiments revealed that sc pDNA was able to transfect fibroblast cells and to produce hemaglutinin protein, as proved by immunochemistry analysis with mouse monoclonal anti HA H5N1 IgG primary antibody. The effect of plasmid transfection on cell viability was over 90% as demonstrated with activity of lactate dehydrogenase resazurin assays. In conclusion, our collective approach provides a valuable choice for the efficient isolation of sc pDNA hemaglutinin vaccine which can in near future prevent influenza infection.

The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.

Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.