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The use of metallic nanostructures in the fabrication of bioelectrodes (e.g., neural implants) is gaining attention nowadays. Nanostructures provide increased surface area that might benefit the performance of bioelectrodes. However, there is a need for comprehensive studies that assess electrochemical performance of nanostructured surfaces in physiological and relevant working conditions. Here, we introduce a versatile scalable fabrication method based on magnetron sputtering to develop analogous metallic nanocolumnar structures (NCs) and thin films (TFs) from Ti, Au, and Pt. We show that NCs contribute significantly to reduce the impedance of metallic surfaces. Charge storage capacity of Pt NCs is remarkably higher than that of Pt TFs and that of the other metals in both morphologies. Circuit simulations of the electrode/electrolyte interface show that the signal delivered in voltage-controlled systems is less filtered when nanocolumns are used. In a current-controlled system, simulation shows that NCs provide safer stimulation conditions compared to TFs. We have assessed the durability of NCs and TFs for potential use in vivo by reactive accelerated aging test, mimicking one-year in vivo implantation. Although each metal/morphology reveals a unique response to aging, NCs show overall more stable electrochemical properties compared to TFs in spite of their porous structure.

Selenium is an essential trace element with a key behavior in human biology and a valuable additive in different industrial applications. When introduced into the nanoscale, selenium nanomaterials—from particles to wires, rods, and other shapes—have shown remarkable properties. They have been explored as potential therapeutics in various oxidative stress, and inflammation-mediated disorders like arthritis, cancer, and diabetes, among many others. Moreover, selenium nanoparticles constitute an attractive carrier platform to transport a diverse array of drugs to the site of action, using a synergetic effect that takes advantage of the inherent antimicrobial, anticancer, and antioxidant properties of these nanomaterials. Still, many of these features are linked to how the nanomaterials are synthesized, as it happens with many other elemental particles. In this regard, using living organisms, biomolecules, and other natural resources to produce selenium-based nanomaterials offers significant advantages compared to traditionally synthesized nanostructures, such as improved performance, lack of toxic by-products in the final product, and the employment of environmentally friendly and cost-effective techniques. This chapter covers some of the most exciting and updated research on the green synthesis of selenium nanomaterials and their use in medicine.

This publication contains the original data supporting our results concerning the application of biphasic pulsed electrical stimulation on MG-63 cells. We aim to discriminate between effects of the electric field and electrochemically conditioned medium. The dataset consists of the original output files, excel sheets to consolidate the information for the experiments as well as Readme.txt files with most important information concerning the data. For additional information on the material and methods as well as results see the corresponding research article.

We have developed a platform that combines nanostructured Pt electrodes with a microgrooved-compartmentalized cell culture module, for electrical stimulation of neural cells. We demonstrated that nanostructuration significantly improves electrochemical properties of the bioelectrodes, including charge storage capacity. Moreover, nanostructuration has positive effect on conserving the delivered signal. We also, established a methodology for simulating stimualtion conditions before experimental work. Future work will be focused on culturing neural cells in this platform, to expand their axonal compartment within the grooves, and testing the effect of electrical stimulation on growth and survival of the axons.

INTRODUCTION Electrical stimulations (ES), with various intensity, frequency, duration, signal shape, etc. are used to enhance tissue regeneration. However, it is not yet clear which combination of parameters is efficient in each application. A relevant approach to refine these sets of parameters is to reveal the significant electrochemical features linked to the biophysical responses [1,2]. Here, we characterized the electrochemical features of different in vitro ES systems and regimes in biologically relevant conditions. METHODS We characterized three different systems: miniaturized devices with electrode plates, coated with either Pt thin film (TF) or Pt nanocolumns (NC)[4]; and an 8-well plate with L-shaped Pt wires (W). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were measured using a potentiostat. Chronoamperometry was carried out for different ES regimes. RESULTS Impedance is significantly reduced when NC is used. The NC effective capacitance is larger than that of TF due to its increased electrochemically active area. The safe limits are between -0.7 V and 1.0 V for both NC and TF. Redox peaks were observed in the same potential ranges for both morphologies. Accumulated charge was considerably larger for both morphologies in the potential regions were oxidation of Pt and adsorption-desorption of H2 occur, showing its dependency on the applied potential ranges. Moreover, charge per pulse was around two orders of magnitude larger for NC than for TF in every region. DISCUSSION AND CONCLUSIONS The applied electrical signals are not equal to what cells sense due to the electric double layer at the interface and its characteristics are extractable from EIS. Chronoamperometry provides the accumulated charge in the system. The sum of charges passed to the extracellular fluid highly depends on electrode material, operation voltage range, and signal shape. Finally, even being in the safe operating voltage range, faradaic reactions [3], res

Pulsed electrical stimulation at 25 mV/mm emerges as a potent physical approach for inducing neural differentiation in neuroblastoma cells, even in the absence of the critical growth factor BDNF. Our study demonstrates that the effectiveness of this method is dose-dependent, with the frequency and duty cycle significantly influencing the morphological expression of neurite outgrowth. Moreover, these parameters also exhibit a meaningful impact on the production yield of EVs and their protein cargo. In summary, our findings underscore the viability of fine-tuning of cell behavior and cell secretion through the regulation of electrical stimulation parameters.

Background Electrical stimulation is used for enhanced bone fracture healing. Electrochemical processes occur during the electrical stimulation at the electrodes and influence cellular reactions. Our approach aimed to distinguish between electrochemical and electric field effects on osteoblast-like MG-63 cells. We applied 20 Hz biphasic pulses via platinum electrodes for 2 h. The electrical stimulation of the cell culture medium and subsequent application to cells was compared to directly stimulated cells. The electric field distribution was predicted using a digital twin. Results Cyclic voltammetry and electrochemical impedance spectroscopy revealed partial electrolysis at the electrodes, which was confirmed by increased concentrations of hydrogen peroxide in the medium. While both direct stimulation and AC-conditioned medium decreased cell adhesion and spreading, only the direct stimulation enhanced the intracellular calcium ions and reactive oxygen species. Conclusion The electrochemical by-product hydrogen peroxide is not the main contributor to the cellular effects of electrical stimulation. However, undesired effects like decreased adhesion are mediated through electrochemical products in stimulated medium. Detailed characterisation and monitoring of the stimulation set up and electrochemical reactions are necessary to find safe electrical stimulation protocols.

Pluripotent stem cell-derived organoid models of the central nervous system represent one of the most exciting areas in in vitro tissue engineering. Classically, organoids of the brain, retina and spinal cord have been generated via recapitulation of in vivo developmental cues, including biochemical and biomechanical. However, a lesser studied cue, bioelectricity, has been shown to regulate central nervous system development and function. In particular, electrical stimulation of neural cells has generated some important phenotypes relating to development and differentiation. Emerging techniques in bioengineering and biomaterials utilise electrical stimulation using conductive polymers. However, state-of-the-art pluripotent stem cell technology has not yet merged with this exciting area of bioelectricity. Here, we discuss recent findings in the field of bioelectricity relating to the central nervous system, possible mechanisms, and how electrical stimulation may be utilised as a novel technique to engineer “next-generation” organoids.

Electrical stimulation (ES) has been employed in nume-rous biomedical applications such as neural stimulation, neural interfacing and tissue regeneration. The biological complexity of in vivo conditions encouraged many researchers to evaluate the role of ES via in vitro devices1. Among the several ES delivery methods, direct coupling is the one that fairly mimics implantable devices. The electrochemical properties of in vitro ES devices and the characteristics of the delivered electrical field remain broadly understudied. Electrode materials have a critical role in determining the impedance (Z) and charge injection capacity (CIC) of the system. A small CIC and high Z require larger potentials to inject a significant amount of charge, which may corrode the electrode or hydrolyse the electrolyte. In addition, electrochemical reactions result in by-products such as reactive oxygen species (ROS) or changes in the pH, which are harmful for the cells2. Nano-structuration is shown to improve the electrochemical properties of bioelectrodes. In this study, we defined biologically safe limits of ES delivered by both Pt thin films (TF) and nanostructured (NC) electrodes incorporated in an in vitro ES device. To this end, we performed comprehensive electrochemi-cal characterization in biologically relevant conditions.

[ES] Electrodos nanoestructurados para la estimulación eléctrica de células en cultivo, dispositivos, sistemas y procedimientos asociados. La presente invención se refiere a un electrodo (1) de estimulación eléctrica celular, adaptado para su inserción en una placa (2) miniaturizada con mini-pocillos (3) de cultivo celular, que comprende al menos un material dispuesto como sustrato (4) y un material eléctricamente conductor dispuesto sobre dicho sustrato (4). Ventajosamente, el material eléctricamente conductor está formado por nanopilares (5) metálicos, depositados sobre el sustrato (4) a través de deposición con ángulo de incidencia (GLAD) con pulverización catódica por magnetrón. La invención se refiere, asimismo, a diferentes dispositivos, sistemas, usos y procedimientos asociados a dichos electrodos (1)., [EN] Nanostructured electrodes for the electrical stimulation of cells in culture, devices, systems and associated procedures. The present invention refers to an electrode (1) for cellular electrical stimulation, adapted for insertion in a miniaturized plate (2) with mini-wells (3) for cell culture, comprising at least one material arranged as substrate (4) and an electrically conductive material arranged on said substrate (4). Advantageously, the electrically conductive material is formed by metallic nanopillars (5), deposited on the substrate (4) through deposition with angle of incidence (GLAD) with magnetron sputtering. The invention also refers to different devices, systems, uses and procedures associated with said electrodes (1).

Neural implants are increasingly investigated to assist patients with defects or pathologies in nervous system. There is a growing research about developing new biomaterials to improve the effectiveness and biocompatibility of neural implants. For example, inflammatory responses to implantation of these devices, also called foreign body reactions (FBR), are still a challenge for their performance. During FBR, immune cells such as monocytes and macrophages are recruited and activated, leading to several consequences. One of those is the increased amount of reactive oxygen species (ROS) due to differentiation and phagocytosis of macrophages. ROS affects the performance of neural implants in the long-term by increasing corrosion and delamination. Reactive accelerated aging tests (RAAT) are used as a tool to test the durability of bioelectrodes and neural implants, mimicking the presence of ROS in vivo. In RAAT, an H2O2 solution with high temperature can mimic several years of implantation during a few weeks due to the increased reaction rate provided by temperature. A RAAT protocol for testing neural implants has been recently approved by FDA. In this work, we have built a RAAT setup based on this protocol. We have studied different parameters that affect the stability of the system and we have employed RAAT to analyse the durability of different flat and nanostructured metallic layers.

Bioelectricity facilitates sending and receiving information to/from the body to provide controlling and feedbacking ºystems to regulate, and monitor biological microenvironment. Also, numerous studies showed electrical stimulation (ES) promotes proliferation, migration and secretion of pro-regenerative factors in the cells and accelerates tissue regeneration, and wound healing. Moreover, ES has a great potential to be adapted as a digital medicine and/or smart therapeutic. Growing research on the use of ES in biological systems, promotes development of in vitro platforms to study the role and applications of ES in contact with cells and tissues. Up to now several in vitro ES devices has been established, such as: devices for measuring electrical impedance of cells that is used to evaluate specific biological activities; devices for inducing specific cell responses by means of low voltage ES (e.g., cell migration, differentiation); devices for stimulating and/or recording electrical activity of electroactive cells in CMOS-based multi-electrode arrays (MEAs) and/or multipurpose setups such as lab-on-the-chip. Nevertheless, quality of electrical signals, reproducibility of experiments, increasing of throughput and accuracy of performance of current in vitro platforms are yet to be improved. We believe, advances in biomaterials technology, computational modelling, microelectronics, microfabrication, and electrochemistry have opened new doors to meet these needs. Here we present the evolution of in vitro platforms for electrical stimulation and recording for different proposes and introduce our innovative device for low voltage direct electrical stimulation. We discussed design, fabrication and characterization of this platform and its benefits for conducting high throughput and controlled experiments within a standard cell culture vessel.

Exogenous electrical fields have been explored in regenerative medicine to increase cellular expression of pro-regenerative growth factors. Adipose-derived stem cells (ASCs) are attractive for regenerative applications, specifically for neural repair. Little is known about the relationship between low-level electrical stimulation (ES) and ASC regenerative potentiation. In this work, patterns of ASC expression and secretion of growth factors (i.e., secretome) were explored across a range of ES parameters. ASCs were stimulated with low-level stimulation (20 mV/mm) at varied pulse frequencies, durations, and with alternating versus direct current. Frequency and duration had the most significant effects on growth factor expression. While a range of stimulation frequencies (1, 20, 1000 Hz) applied intermittently (1 h × 3 days) induced upregulation of general wound healing factors, neural-specific factors were only increased at 1 Hz. Moreover, the most optimal expression of neural growth factors was achieved when ASCs were exposed to 1 Hz pulses continuously for 24 h. In evaluation of secretome, apparent inconsistencies were observed across biological replications. Nonetheless, ASC secretome (from 1 Hz, 24 h ES) caused significant increase in neurite extension compared to non-stimulated control. Overall, ASCs are sensitive to ES parameters at low field strengths, notably pulse frequency and stimulation duration.

The use of electricity in interaction with biological systems for therapeutic and diagnostic purposes become a prominent research topic. Electrodes are key components of electrical stimulation and recording devices for in vivo and in vitro usages. Bioelectrodes should present excellent electroconductivity to be able to transfer the electrical current efficiently (charge mobility inside the device). Moreover, they need to provide excellent electroactivity to convert electronic current to ionic current at the electrode/extracellular fluid (ECF) interphase. The use of nanostructures in fabrication of bioelectrodes is beneficial, since nanostructures offer superior effective surface area that decreases impedance (Z) and increases charge storage capacity (CSC) of the electrodes. Low Z prevents irreversible faradic reactions that are harmful for biological systems (safe stimulation), and decreases thermal noises that interfere recording bioelectrical signals. Higher CSC ensures effective charge transfer (effective stimulation)1. Here we developed highly pure metallic nanostructured electrodes from Ti, Au and Pt and characterized their electrochemical properties. Electrical stimulation (ES) modes for both in vitro and in vivo applications were simulated. Finally, we present the design of an in vitro ES platform incorporating nanostructured electrodes.

The capability of electrical stimulation (ES) in promoting bone regeneration has already been addressed in clinical studies. However, its mechanism is still being investigated and discussed. This study aims to investigate the responses of macrophages (J774A.1) and preosteoblasts (MC3T3-E1) to ES and the faradic by-products from ES. It is found that pH of the culture media was not significantly changed, whereas the average hydrogen peroxide concentration was increased by 3.6 and 5.4 µM after 1 and 2 hr of ES, respectively. The upregulation of Bmp2 and Spp1 messenger RNAs was observed after 3 days of stimulation, which is consistent among two cell types. It is also found that Spp1 expression of macrophages was partially enhanced by faradic by-products. Osteogenic differentiation of preosteoblasts was not observed during the early stage of ES as the level of Runx2 expression remains unchanged. However, cell proliferation was impaired by the excessive current density from the electrodes, and also faradic by-products in the case of macrophages. This study shows that macrophages could respond to ES and potentially contribute to the bone formation alongside preosteoblasts. The upregulation of Bmp2 and Spp1 expressions induced by ES could be one of the mechanisms behind the electrically stimulated osteogenesis.