Positive biocompatibility of several graphene derivatives with dopaminergic cells at long term culture

The emerging carbon nanomaterial graphene (G) and its oxidized derivative graphene oxide (GO) have recently gained considerable attention in biomedical applications such as cancer therapy or biosensors. It has for example been demonstrated that G has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells (Li et al., 2013). This way, G could acts as a physical support or scaffold to promote axonal sprout as a “deceleration” support for the DA cells derived from neural stem cells. Since GO in its multilayer form and with multiples carboxilate and epoxy groups seems to shows interesting biological properties (Yang et al., 2013) the aim of the present work has been to test different graphene derivatives searching for the best scaffold to be used in stem cell differentiation. For this purpose we have tested the cytotoxicity of GO and reduced GO, and specifically its biocompatibility with SN4741, a dopaminergic cells line derived from mouse substance nigra, measuring the effect in the cells at long term culture. The cells were cultured in Dulbecco’s modified Eagle’s medium 10% FCS (Gibco) to about 80% confluence. Cells were incubated applying 1.000 cells in 96-well microliter plates with graphene using three chemically different types of GO as powders and films: 1) GO, which is hydrophilic; 2) partially reduced GO (PRGO) which is hydrophobic and 3) fully reduced GO (FRGO), also hydrophobic, in five concentrations: 1 mg/ml; 0.1 mg/ml; 0.05 mg/ml; 0.02 mg/ml and 0.01 mg/ml, in each type of graphene. Cells were cultured with GO and cell viability was determined after 24 hours, 1 week and 2 weeks using the MTT assay (Roche) and cytotoxicitity was determined by the lactate dehydrogenase (LDH) (Roche) assay measured at 560nm. The results demonstrated positive biocompatibility between the G-derivatives and SN4741 cells. We conclude that the use of our G-derivative scaffolds can enhance the neural differentiation towards neurons (TH positive) providing a cell growth microenvironments and appropriate synergistic cell guidance cues. This findings demonstrated that biocompatibility of scaffolds is a pre-requisite for generation of successful clinical application of graphene. It could offer a platform for neural stem cells and a promising approach for neural regeneration in the research of neurological diseases like PD. Long-term studies on the biological effects of graphene will now be performed for the development of therapeutic treatment as the goal. (Refs: Li N., Zhang Q, Gao S. et a., 2013, Nature/Sci Rep. 3:1604. doi: 10.1038/srep01604; Yan K., Li Y., Tan X., et al., 2013, Small., 9(9-10): 1492-1503)1. Universidad de Malaga. Campus de Excelencia Internacional Andalucia Tech, España. 2. The Norwegian Research Council (grant nº 215086, Oslo, Noruega. 3. Karolinska Institute Reasearch Fund, Estocolmo, Suecia

Graphene derivative scaffolds facilitate in vitro cell survival and maturation of dopaminergic SN4741 cells

The emerging carbon nanomaterial Graphene (G), in the form of scaffold structure, has an efficient bioconjugation with common biomolecules and activates cell differentiation of neuronal stem cells, providing a promising approach for neural regeneration. We propose the use of G as a scaffold to re-address the dopaminergic (DA) neurons and the residual axons from dead or apoptotic DA neurons in Parkinson´s disease (PD). G could act as a physical support to promote the axonal sprout as a “deceleration” support for the DA cells derived from neural stem cells or DA direct cell conversion, allowing the propagation of nerve impulses. We cultured a clonal substantia nigra (SN) DA neuronal progenitor cell line (SN4741) in presence of G as scaffold. This cell line derived from mouse embryos was cultured in Dulbecco’s modified Eagle’s medium/10% FCS to about 80% confluence. Cells were incubated in three chemically different G derivatives and two different presentation matrixes as powder and films: 1) G oxide (GO); 2) partially reduced GO (PRGO) which is hydrophobic; and 3) fully reduced GO (FRGO). Cell viability was determined using the MTT assay after adding the following G concentrations: 1mg/ml; 0.1mg/ml; 0.05mg/ml; 0.02mg/ml and 0.01mg/ml, in each type of GO. To study cellular morphology and assessment of cell engraftment into GO films (GO film, PRGO film, FRGO film), we analyzed the immunostaining of the anti-rabbit neuron-specific DNA-binding protein (NeuN) antibody, the anti-rat Beta-3-tubulin antibody in combination with the mitochondrial marker mouse anti-ATP synthase antibody, and the anti-rabbit DCX as immature neuronal marker. Hoechst label was used as nuclei marker. Reactive oxidative species (ROS) were measured by flow cytometry to study the influence of G on the cell redox-state. With this purpose, cells were loaded with dihydroethidium. The mitochondrial membrane potential after JC-1 incubation was studied by flow cytometry. Our results show an increase of survival and metabolism (30-40%) at low concentrations of PRGO and FRGO (0.05-0.01 mg/ml) respect to the higher concentration (1 mg/ml), while no changes were seen in the GO group. LDH concentration was measured in the supernatant using a COBAS analyzer showing a neuroprotective action at low concentrations. Furthermore, either PRGO film or FRGO film show an increase in the effective anchorage capacity to nest into the G matrix and in the maturation of the SN 4741 cells. We conclude that the use of G scaffolds in the research of neurological diseases like PD could offer a powerful platform for neural stem cells, direct cell conversion techniques and neural tissue engineering.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Norwegian Research Council (grant nº 215086

The graphene oxide species induce a different biological response in SN4741 Parkinson cell line

Introduction: Graphene Oxide (GO)has recently emerged as a reliable material to create scaffolds for the neural tissue because of its biocompatibility, electroconductive and physicochemical properties. Graphene is a 2-dimensional material consisting of rings of carbon atoms with an excellent electrical conductivity originating in the sp2 hybridized carbons network. Nevertheless, there is not a consensus which kind of graphene oxide is most useful of benefit.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Graphene oxide and reduced derivates, as powder or film scaffolds, differentially promote dopaminergic neuron differentiation and survival.

Emerging scaffold structures made of carbon nanomaterials, such as graphene oxide (GO) have shown efficient bioconjugation with common biomolecules. Previous studies described that GO promotes the differentiation of neural stem cells and may be useful for neural regeneration. In this study, we examined the capacity of GO, full reduced (FRGO), and partially reduced (PRGO) powder and film to support survival, proliferation, differentiation, maturation, and bioenergetic function of a dopaminergic (DA) cell line derived from the mouse substantia nigra (SN4741). Our results show that the morphology of the film and the species of graphene (GO, PRGO, or FRGO) influences the behavior and function of these neurons. In general, we found better biocompatibility of the film species than that of the powder. Analysis of cell viability and cytotoxicity showed good cell survival, a lack of cell death in all GO forms and its derivatives, a decreased proliferation, and increased differentiation over time. Neuronal maturation of SN4741 in all GO forms, and its derivatives were assessed by increased protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), the glutamate inward rectifying potassium channel 2 (GIRK2), and of synaptic proteins, such as synaptobrevin and synaptophysin

Additive Manufactured Scaffolds for Bone Tissue Engineering: Physical Characterization of Thermoplastic Composites with Functional Fillers

Thermoplastic polymer–filler composites are excellent materials for bone tissue engineering (TE) scaffolds, combining the functionality of fillers with suitable load-bearing ability, biodegradability, and additive manufacturing (AM) compatibility of the polymer. Two key determinants of their utility are their rheological behavior in the molten state, determining AM processability and their mechanical load-bearing properties. We report here the characterization of both these physical properties for four bone TE relevant composite formulations with poly(ethylene oxide terephthalate)/poly(butylene terephthalate (PEOT/PBT) as a base polymer, which is often used to fabricate TE scaffolds. The fillers used were reduced graphene oxide (rGO), hydroxyapatite (HA), gentamicin intercalated in zirconium phosphate (ZrP-GTM) and ciprofloxacin intercalated in MgAl layered double hydroxide (MgAl-CFX). The rheological assessment showed that generally the viscous behavior dominated the elastic behavior (G″ > G′) for the studied composites, at empirically determined extrusion temperatures. Coupled rheological–thermal characterization of ZrP-GTM and HA composites showed that the fillers increased the solidification temperatures of the polymer melts during cooling. Both these findings have implications for the required extrusion temperatures and bonding between layers. Mechanical tests showed that the fillers generally not only made the polymer stiffer but more brittle in proportion to the filler fractions. Furthermore, the elastic moduli of scaffolds did not directly correlate with the corresponding bulk material properties, implying composite-specific AM processing effects on the mechanical properties. Finally, we show computational models to predict multimaterial scaffold elastic moduli using measured single material scaffold and bulk moduli. The reported characterizations are essential for assessing the AM processability and ultimately the suitability of the manufactured scaffolds for the envisioned bone regeneration application.The work was supported by a Horizon 2020 Research and Innovation Programme grant from the European Union, called the FAST project (grant no. 685825, project website: http:// project-fast.eu). The authors acknowledge the support of the FAST project consortium for the various aspects of this wor

Graphite Oxide: An Interesting Candidate for Aqueous Supercapacitors

A graphite oxide, obtained on a large scale at low cost as an intermediate in the graphene production, achieves specific capacitances (159 Fg−1 in H2SO4 and 82 Fg−1 in (C2H5)4NBF4 in acetonitrile) that compete with those of activated carbons and largely surpass the values obtained with graphene nanoplatelets. More promising, the high electrode density leads to volumetric capacitances of 177 and 59 F cm−3 in the aqueous and the organic electrolytes, respectively, which are above most data reported for carbons. In the aqueous electrolyte, the graphite oxide stands out on energy density when compared to graphene nanoplatelets and on power capability if compared to an activated carbon commercialized for supercapacitors, whereas in the organic electrolyte, the limited interlayer spacing restricts the mobility of the larger ions into the expanded graphitic structure. This study also illustrates that the specific surface of carbons measured by standard gas adsorption may not be a relevant parameter as it does not always match the electrochemically active area involved in the energy storage.Financial support to T.A.C. from EU 7FP (Project Electrograph- 266391) and MICINN (project MAT 2011-25198) is gratefully acknowledged. Financial support for graphene oxide development was obtained from the Research Council of Norway, through grant No 73146. The authors thank the Instituto Universitario de Investigación en Nanociencia de Aragón-Universidad de Zaragoza for the HRTEM images.Peer reviewe

Introduction to EU-H2020 project WEARPLEX: wearable multiplexed biomedical electrodes

WEARPLEX is a multidisciplinary research and innovation action with the overall aim to integrate printed electronics with flexible and wearable textile-based biomedical multi-pad electrodes. It aims to answer the growing need for user-friendly electrodes for pervasive measurement of electrophysiological signals and application of electrical stimulation. It focuses on the development of the printable electronics and manufacturing processes for stretchable textile based multi-pad electrodes with integrated logic circuits that enable a significant increase in the number of electrode pads (channels) and facilitate the creation of new products in the sectors of medical electronics and life-style. The advanced printed electronics integrated in WEARPLEX electrodes will allow the individual pads to be connected in arbitrary configurations to the output leads of the electrode. Therefore, the pads will be flexibly organized into several virtual electrodes of arbitrary position, shape and size that can be connected to any standard multi-channel recording and stimulation system. In addition, software methods will be developed for automatic calibration of these virtual electrodes, to detect stimulation/recording hotspots and adjust the virtual electrodes accordingly. Therefore, the WEARPLEX project will lead to a new generation of smart electrodes that will be able to adapt simultaneously to the user (wearable and stretchable garment), recording/stimulation scenario (movement type and target muscles) and recording/stimulation system (number of channels). This is a paradigm shift in designing the recording and stimulation systems, as the switching electronics is shifted from the custom-made stimulator/recording device to the smart electrode, leading to a universal solution compatible with any system. WEARPLEX is funded under the EU Horizon2020 call ICT-02-2018, the work program is over 3 years starting on the 1st of January 2019; this abstract is therefore an opportunity to introduce the project to the community and discuss research directions and early results for the project. The individual components of the project are at TRL2-4 with the goal of improving and combining them to achieve TRL6-7 industrial demonstrators by the end of the project.<br/

Graphene derivatives as scaffold for ex vivo survival and maturation of dopaminergic SN4741 cells.

El Congreso de la Sociedad Española de Ciencias Fisiológicas se celebra bianualmente y es el foro más adecuado para el intercambio científico entre investigadores españoles que trabajan en el campo de la Fisiología humana, animal, celular y vegetal.Carbon nanomaterial Graphene (G) can form a three-dimensional porous structure with efficient bioconjugation and cell differentiation properties, providing a promising scaffold for neural regeneration. Aims: To study this putative new application of G, we cultured a clonal substantia nigra dopaminergic neuronal progenitor cell line (SN4741) in presence of G as scaffold. Methods: Cells were cultured in DMEM/10% FCS to about 80% confluence and incubated with different concentrations (0.001 to 1 mg/ml) of three chemically different G derivatives (G oxide (GO); partially reduced GO (PRGO) and fully reduced GO (FRGO)) and two different presentation matrixes as powder and films. Cell viability was measured by the MTT assay. To study cellular characterization, morphology and assessment of cell engraftment into G films, we analyzed the immunostaining of the neuronal marker NeuN, the anti-rat Beta-3-tubulin antibody, and the anti-rabbit DCX as immature neuronal marker. Reactive oxidative species (ROS) and the mitochondrial membrane potential after JC-1 incubation were measured by flow cytometry. Lactate dehydrogenase was measured in the culture supernatant. Results: We found similar increase of survival and metabolism (30-40%) at low concentrations of PRGO and FRGO (0.05-0.01 mg/ml) compared with the higher concentration (1 mg/ml), no changes were seen in the GO group. PRGO or FRGO films showed an increased in the effective anchorage capacity to nest into the G matrix and in the maturation of the dopaminergic SN4741 cells. Conclusions: G scaffolds could offer a powerful platform for neural stem cells, direct cell conversion techniques and neural tissue engineering.Universidad de Málaga, Campus de Excelencia Internacional Andalucía Tec