Direct electrochemical generation of catalytically competent oxyferryl species of classes i and p dye decolorizing peroxidases

This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of H2O2 or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from Bacillus subtilis (BsDyP; Class I) and Pseudomonas putida (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH. Interestingly, the electrocatalytic efficiencies obtained at pH 7.6 are very similar to the values recorded for regular catalytic ABTS/H2O2 assays at the optimal pH of the enzymes, ca. 3.7. These findings pave the way for the design of DyP-based electrocatalytic reactors operable in an extended pH range without the need of harmful reagents such as H2O2.Fil: Scocozza, Magali Franca. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Martins, Lígia O.. Universidade Nova de Lisboa; PortugalFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentin

Shape fidelity and sterility assessment of 3D printed polycaprolactone and hydroxyapatite scaffolds

AbstractPolycaprolactone (PCL) and hydroxyapatite (HA) composite are widely used in tissue engineering (TE). They are fit to being processed with three-dimensional (3D) printing technique to create scaffolds with verifiable porosity. The current challenge is to guarantee the reliability and reproducibility of 3D printed scaffolds and to create sterile scaffolds which can be used for in vitro cell cultures. In this context it is important for successful cell culture, to have a protocol in order to evaluate the sterility of the printed scaffolds. We proposed a systematic approach to sterilise 90%PCL-10%HA pellets using a 3D bioprinter before starting the printing process. We evaluated the printability of PCL-HA composite and the shape fidelity of scaffolds printed with and without sterilised pellets varying infill pattern, and the sterility of 3D printed scaffolds following the method established by the United States Pharmacopoeia. Finally, the thermal analyses supported by the Fourier Transform Infrared Spectroscopy were useful to verify the stability of the sterilisation process in the PCL solid state with and without HA. The results show that the use of the 3D printer, according to the proposed protocol, allows to obtain sterile 3D PCL-HA scaffolds suitable for TE applications such as bone or cartilage repair

Myoblast 3D bioprinting to burst in vitro skeletal muscle differentiation

Skeletal muscle regeneration is one of the major areas of interest in sport medicine as well as trauma centers. Three-dimensional (3D) bioprinting (BioP) is nowadays widely adopted to manufacture 3D constructs for regenerative medicine but a comparison between the available biomaterial-based inks (bioinks) is missing. The present study aims to assess the impact of different hydrogels on the viability, proliferation, and differentiation of murine myoblasts (C2C12) encapsulated in 3D bioprinted constructs aided to muscle regeneration. We tested three different commercially available hydrogels bioinks based on: (1) gelatin methacrylate and alginate crosslinked by UV light; (2) gelatin methacrylate, xanthan gum, and alginate-fibrinogen; (3) nanofibrillated cellulose (NFC)/alginate-fibrinogen crosslinked with calcium chloride and thrombin. Constructs embedding the cells were manufactured by extrusion-based BioP and C2C12 viability, proliferation, and differentiation were assessed after 24 h, 7, 14, 21, and 28 days in culture. Although viability, proliferation, and differentiation were observed in all the constructs, among the investigated bioinks, the best results were obtained by using NFC/alginate-fibrinogen-based hydrogel from 7 to 14 days in culture, when the embedded myoblasts started fusing, forming at day 21 and day 28 multinucleated myotubes within the 3D bioprinted structures. The results revealed an extensive myotube alignment all over the linear structure of the hydrogel, demonstrating cell maturation, and enhanced myogenesis. The bioprinting strategies that we describe here denote a strong and endorsed approach for the creation of in vitro artificial muscle to improve skeletal muscle tissue engineering for future therapeutic applications

The role of molecular crowding in long-range metalloprotein electron transfer: Dissection into site- and scaffold-specific contributions

Here we report the effect of molecular crowding on long-range protein electron transfer (ET) and disentangle the specific responses of the redox site and the protein milieu. To this end, we studied two different one-electron redox proteins that share the cupredoxin fold but differ in the metal center, T1 mononuclear blue copper and binuclear CuA, and generated chimeras with hybrid properties by incorporating different T1 centers within the CuA scaffold or by swapping loops between orthologous proteins from different organisms to perturb the CuA site. The heterogeneous ET kinetics of the different proteins was studied by protein film electrochemistry at variable electronic couplings and in the presence of two different crowding agents. The results reveal a strong frictional control of the ET reactions, which for 10 Å tunnelling distances results in a 90% drop of the ET rate when viscosity is matched to that of the mitochondrial interior (ca. 55 cP) by addition of either crowding agent. The effect is ascribed to the dynamical coupling of the metal site and the milieu, which for T1 is found to be twice stronger than for CuA, and the activation energy of protein-solvent motion that is dictated by the overall scaffold. This work highlights the need of explicitly considering molecular crowding effects in protein ET.Fil: Zitare, Ulises Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Szuster, Jonathan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Scocozza, Magali Franca. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Espinoza Cara, Andrés Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Leguto, Alcides José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Morgada, Marcos Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentin

Bioprinting for osteosarcoma model: Methodological aspects and experimental applications

The study aims at using the bioprinting technique to create an in vitro 3D construct of osteosarcoma, as an alternative model for studies related to Boron Neutron Capture Therapy (BNCT)

Comparative analysis of different hydrogels for the bioprinting of 3D in vitro skeletal muscle models

In this study we demonstrated an application of 3D Bioprinting using different commercially available hydrogels (CELLINK AB, Sweden) with the aim to identify the most suitable biomaterial for the proliferation and differentiation of murine muscle cells (C2C12)

Electrochemical Actuation of a DyP Peroxidase: A Facile Method for Drastic Improvement of the Catalytic Performance

Dye decolorizing peroxidases (DyP) have attracted interest for applications such as dye-containing wastewater remediation and biomass processing. So far, efforts to improve operational pH ranges, activities, and stabilities have focused on site-directed mutagenesis and directed evolution strategies. Here, we show that the performance of the DyP from Bacillus subtilis can be drastically boosted without the need for complex molecular biology procedures by simply activating the enzyme electrochemically in the absence of externally added H2O2. Under these conditions, the enzyme shows specific activities toward a variety of chemically different substrates that are significantly higher than in its canonical operation. Moreover, it presents much broader pH activity profiles with the maxima shifted toward neutral to alkaline. We also show that the enzyme can be successfully immobilized on biocompatible electrodes. When actuated electrochemically, the enzymatic electrodes have two orders of magnitude higher turnover numbers than with the standard H2O2-dependent operation and preserve about 30% of the initial electrocatalytic activity after 5 days of operation-storage cycles.Fil: Scocozza, Magali Franca. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Vieyra, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Battaglini, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Martins, Ligia O.. Universidade Nova de Lisboa; PortugalFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentin

Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities

The intertwined adoption of synthetic biology and 3D bioprinting has the potential to improve different application fields by fabricating engineered living materials (ELMs) with unnatural genetically-encoded sense & response capabilities. However, efforts are still needed to streamline the fabrication of sensing ELMs compatible with field use and improving their functional complexity. To investigate these two unmet needs, we adopted a workflow to reproducibly construct bacterial ELMs with synthetic biosensing circuits that provide red pigmentation as visible readout in response to different proof-of-concept chemical inducers. We first fabricated single-input/single-output ELMs and we demonstrated their robust performance in terms of longevity (cell viability and evolutionary stability >15 days, and long-term storage >1 month), sensing in harsh, non-sterile or nutrient-free conditions compatible with field use (soil, water, and clinical samples, including real samples from Pseudomonas aeruginosa infected patients). Then, we fabricated ELMs including multiple spatially-separated biosensor strains to engineer: level-bar materials detecting molecule concentration ranges, multi-input/multi-output devices with multiplexed sensing and information processing capabilities, and materials with cell-cell communication enabling on-demand pattern formation. Overall, we showed successful field use and multiplexed functioning of reproducibly fabricated ELMs, paving the way to a future automation of the prototyping process and boosting applications of such devices as in-situ monitoring tools or easy-to-use sensing kits

Prediction of the mechanical response of a 3D (bio)printed hybrid scaffold for improving bone tissue regeneration by structural finite element analysis

: Scaffolds for bone tissue engineering should be osteoinductive, osteoconductive, biocompatible, biodegradable, and, at the same time, exhibit proper mechanical properties. The present study investigated the mechanical properties of a coprinted hybrid scaffold made of polycaprolactone (PCL) and an alginate-based hydrogel, which was conceived to possess a double function of in vivo bio-integration (due to the ability of the hydrogel to release lyosecretome, a freeze-dried formulation of mesenchymal stem cell secretome with osteoinductive and osteoconductive properties) and withstanding loads (due to the presence of polycaprolactone, which provides mechanical resistance). To this end, an in-silico study was conducted to predict mechanical properties. Structural finite element analysis (FEA) of the hybrid scaffold under compression was performed to compare the numerical results with the corresponding experimental data. The impact of alginate inclusion and infill patterns on scaffold stiffness was investigated. Results show an increase in mechanical properties by changing the scaffold infill pattern (linear: 145.38±28.90 vs. honeycomb: 278.96±50.19, mean and standard deviation, n = 8), while alginate inclusion does not always impact the mechanical performance of the hybrid scaffold (stiffness: 145.38±28.90 vs. 195.42±38.68 N/mm, with vs without hydrogel inclusion, respectively). This is confirmed by FEA analysis, in which a good correspondence between experimental and numerical stiffness is shown (142±28.94 vs. 117.18, respectively, linear scaffold with hydrogel inclusion). In conclusion, the computational framework is a valid tool for predicting the mechanical performance of scaffolds and is promising for future clinical applications in the maxillofacial field

Three-Dimensional Bioprinted Controlled Release Scaffold Containing Mesenchymal Stem/Stromal Lyosecretome for Bone Regeneration: Sterile Manufacturing and In Vitro Biological Efficacy

Recently, 3D-printed scaffolds for the controlled release of mesenchymal stem cell (MSC) freeze-dried secretome (Lyosecretome) have been proposed to enhance scaffold osteoinduction and osteoconduction; coprinting of poly(ε-caprolactone) (PCL) with alginate hydrogels allows adequate mechanical strength to be combined with the modulable kinetics of the active principle release. This study represents the feasibility study for the sterile production of coprinted scaffolds and the proof of concept for their in vitro biological efficacy. Sterile scaffolds were obtained, and Lyosecretome enhanced their colonization by MSCs, sustaining differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralized matrix detected by alizarin red was significantly higher for the Lyosecretome scaffolds. The amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the Lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with the Lyosecretome scaffolds after 14 and 28 days. Overall, these results prove the role of MSC secretome, coprinted in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC secretome are now available for in vivo pre-clinical tests of bone regeneration