Electrochemical control of bone microstructure on electroactive surfaces for modulation of stem cells and bone tissue engineering.

Controlling stem cell behavior at the material interface is crucial for the development of novel technologies in stem cell biology and regenerative medicine. The composition and presentation of bio-factors on a surface strongly influence the activity of stem cells. Herein, we designed an electroactive surface that mimics the initial process of trabecular bone formation, by immobilizing chondrocyte-derived plasma membrane nanofragments (PMNFs) on its surface for rapid mineralization within 2 days. Moreover, the electroactive surface was based on the conducting polymer polypyrrole (PPy), which enabled dynamic control of the presentation of PMNFs on the surface via electrochemical redox switching, further resulting in the formation of bone minerals with different morphologies. Furthermore, bone minerals with contrasting surface morphologies had differential effects on the differentiation of human bone marrow-derived stem cells (hBMSCs) cultured on the surface. Together, this electroactive surface showed multifunctional characteristics, not only allowing dynamic control of PMNF presentation but also promoting the formation of bone minerals with different morphologies within 2 days. This electroactive substrate could be valuable for more precise control of stem cell growth and differentiation, and further development of more suitable microenvironments containing bone apatite for housing a bone marrow stem cell niche, such as biochips/bone-on-chips.

Mechanistic and Kinetic Studies of the Ring Opening Metathesis Polymerization of Norbornenyl Monomers by a Grubbs Third Generation Catalyst.

The mechanism of ring-opening metathesis polymerization (ROMP) for a set of functionalized norbornenyl monomers initiated by a Grubbs third generation precatalyst [(H2IMes)(pyr)2(Cl)2Ru═CHPh] was investigated. Through a series of 12C/13C and 1H/2H kinetic isotope effect studies, the rate-determining step for the polymerization was determined to be the formation of the metallacyclobutane ring. This experimental result was further validated through DFT calculations showing that the highest energy transition state is metallacyclobutane formation. The effect of monomer stereochemistry (exo vs endo) of two types of ester substituted monomers was also investigated. Kinetic and spectroscopic evidence supporting the formation of a six-membered chelate through coordination of the proximal polymer ester to the Ru center is presented. This chelation and its impact on the rate of polymerization are shown to vary based on the monomer employed and its stereochemistry. The combination of this knowledge led to the derivation of a generic rate law describing the rate of polymerization of norbornene monomers initiated by a Grubbs third generation catalyst.

Exchanged cations and water during reactions in polypyrrole macroions from artificial muscles.

The movement of the bilayer (polypyrrole-dodecylbenzenesulfonate/tape) during artificial muscle bending under flow of current square waves was studied in aqueous solutions of chloride salts. During current flow, polypyrrole redox reactions result in variations in the volumes of the films and macroscopic bending: swelling by reduction with expulsion of cations and shrinking by oxidation with the insertion of cations. The described angles follow a linear function, different in each of the studied salts, of the consumed charge: they are faradaic polymeric muscles. The linearity indicates that cations are the only exchanged ions in the studied potential range. By flow of the same specific charge in every electrolyte, different angles were described by the muscle. The charge and the angle allow the number and volume of both the exchanged cations and the water molecules (related to a reference) between the film to be determined, in addition to the electrolyte per unit of charge during the driving reaction. The attained apparent solvation numbers for the exchanged cations were: 0.8, 0.7, 0.6, 0.5, 0.5, 0.4, 0.25, and 0.0 for Na(+), Mg(2+), La(3+), Li(+), Ca(2+), K(+), Rb(+), and Cs(+), respectively.

Effect of the electrolyte concentration and substrate on conducting polymer actuators.

The effect of the electrolyte concentration (NaCl aqueous electrolyte) on the dimensional variations of films of polypyrrole doped with dodecylbenzenesulfonate PPy(DBS) on Pt and Au wires was studied. Any parallel reaction that occurs during the redox polymeric reaction that drives the mechanical actuation, as detected from the coulovoltammetric responses, was avoided by using Pt wires as substrate and controlling the potential limits, thus significantly increasing the actuator lifetime. The NaCl concentration of the electrolyte, when studied by cyclic voltammetry or chronoamperometry, has a strong effect on the performance as well. A maximum expansion was achieved in 0.3 M aqueous solution. The consumed oxidation and reduction charges control the fully reversible dimensional variations: PPy(DBS) films are faradaic polymeric motors. Parallel to the faradaic exchange of the cations, osmotic, electrophoretic, and structural changes play an important role for the water exchange and volume change of PPy(DBS).

Preliminary profile of the gut microbiota from amerindians in the Brazilian amazon experiencing a process of transition to urbanization.

The Yanomami are one of the oldest indigenous tribes in the Amazon and are direct descendants of the first people to colonize South America 12,000 years ago. They are located on the border between Venezuela and Brazil, with the Venezuelan side remaining uncontacted. While they maintain a hunter-gatherer society, they are currently experiencing contact with urbanized populations in Brazil. The human gut microbiota of traditional communities has become the subject of recent studies due to the Westernization of their diet and the introduction of antibiotics and other chemicals, which have affected microbial diversity in indigenous populations, thereby threatening their existence. In this study, we preliminarily characterized the diversity of the gut microbiota of the Yanomami, a hunter-gatherer society from the Amazon, experiencing contact with urbanized populations. Similarly, we compared their diversity with the population in Manaus, Amazonas. A metabarcoding approach of the 16 S rRNA gene was carried out on fecal samples. Differences were found between the two populations, particularly regarding the abundance of genera (e.g., Prevotella and Bacteroides) and the higher values of the phyla Bacteroidetes over Firmicutes, which were significant only in the Yanomami. Some bacteria were found exclusively in the Yanomami (Treponema and Succinivibrio). However, diversity was statistically equal between them. In conclusion, the composition of the Yanomami gut microbiota still maintains the profile characteristic of a community with a traditional lifestyle. However, our results suggest an underlying Westernization process of the Yanomami microbiota when compared with that of Manaus, which must be carefully monitored by authorities, as the loss of diversity can be a sign of growing danger to the health of the Yanomami.

Electrochemistry of carbon nanotubes: reactive processes, dual sensing-actuating properties and devices.

Single-walled carbon nanotubes (SWCNT) embedded in a non-electroactive polymer are electrochemically characterized. The increasing voltammetric maximums obtained with rising temperature or electrolyte concentration point to a chemical nature of the processes. The chemical kinetic control of the processes is corroborated by its empirical chemical kinetics: the initial reaction rates are obtained from the chronoamperometric responses to potential steps. The activation energy of the reaction includes information about the structural state of the SWCNT before the potential step. Under constant current the potential evolution (chronopotentiometric response) and consumed electrical energy at any time change as a function of (are sensors of) the experimental temperature or the electrolyte concentration. The reactive material, or any device based on this material, senses these working variables, and shows dual and simultaneous actuating-sensing properties.

Biohybrid Variable-Stiffness Soft Actuators that Self-Create Bone.

Inspired by the dynamic process of initial bone development, in which a soft tissue turns into a solid load-bearing structure, the fabrication, optimization, and characterization of bioinduced variable-stiffness actuators that can morph in various shapes and change their properties from soft to rigid are hereby presented. Bilayer devices are prepared by combining the electromechanically active properties of polypyrrole with the compliant behavior of alginate gels that are uniquely functionalized with cell-derived plasma membrane nanofragments (PMNFs), previously shown to mineralize within 2 days, which promotes the mineralization in the gel layer to achieve the soft to stiff change by growing their own bone. The mineralized actuator shows an evident frozen state compared to the movement before mineralization. Next, patterned devices show programmed directional and fixated morphing. These variable-stiffness devices can wrap around and, after the PMNF-induced mineralization in and on the gel layer, adhere and integrate onto bone tissue. The developed biohybrid variable-stiffness actuators can be used in soft (micro-)robotics and as potential tools for bone repair or bone tissue engineering.

Usefulness and applicability of the revised dengue case classification by disease: multi-centre study in 18 countries.

BACKGROUND: In view of the long term discussion on the appropriateness of the dengue classification into dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), the World Health Organization (WHO) has outlined in its new global dengue guidelines a revised classification into levels of severity: dengue fever with an intermediary group of "dengue fever with warning sings", and severe dengue. The objective of this paper was to compare the two classification systems regarding applicability in clinical practice and surveillance, as well as user-friendliness and acceptance by health staff. METHODS: A mix of quantitative (prospective and retrospective review of medical charts by expert reviewers, formal staff interviews), semi-quantitative (open questions in staff interviews) and qualitative methods (focus group discussions) were used in 18 countries. Quality control of data collected was undertaken by external monitors. RESULTS: The applicability of the DF/DHF/DSS classification was limited, even when strict DHF criteria were not applied (13.7% of dengue cases could not be classified using the DF/DHF/DSS classification by experienced reviewers, compared to only 1.6% with the revised classification). The fact that some severe dengue cases could not be classified in the DF/DHF/DSS system was of particular concern. Both acceptance and perceived user-friendliness of the revised system were high, particularly in relation to triage and case management. The applicability of the revised classification to retrospective data sets (of importance for dengue surveillance) was also favourable. However, the need for training, dissemination and further research on the warning signs was highlighted. CONCLUSIONS: The revised dengue classification has a high potential for facilitating dengue case management and surveillance.

Molecular and epidemiologic characterization of the diphtheria outbreak in Venezuela.

In 2016, Venezuela faced a large diphtheria outbreak that extended until 2019. Nasopharyngeal or oropharyngeal samples were prospectively collected from 51 suspected cases and retrospective data from 348 clinical records was retrieved from 14 hospitals between November 2017 and November 2018. Confirmed pathogenic Corynebactrium isolates were biotyped. Multilocus Sequence Typing (MLST) was performed followed by next-generation-based core genome-MLST and minimum spanning trees were generated. Subjects between 10 and 19 years of age were mostly affected (n = 95; 27.3%). Case fatality rates (CFR) were higher in males (19.4%), as compared to females (15.8%). The highest CFR (31.1%) was observed among those under 5, followed by the 40 to 49 age-group (25.0%). Nine samples corresponded to C. diphtheriae and 1 to C. ulcerans. Two Sequencing Types (ST), ST174 and ST697 (the latter not previously described) were identified among the eight C. diphtheriae isolates from Carabobo state. Cg-MLST revealed only one cluster also from Carabobo. The Whole Genome Sequencing analysis revealed that the outbreak seemed to be caused by different strains with C. diphtheriae and C. ulcerans coexisting. The reemergence and length of this outbreak suggest vaccination coverage problems and an inadequate control strategy.

Membrane-targeted synergistic activity of docosahexaenoic acid and lysozyme against Pseudomonas aeruginosa.

Antimicrobial polypeptides, including lysozymes, have membrane perturbing activity and are well-documented effector molecules of innate immunity. In cystic fibrosis, a hereditary disease with frequent lung infection with Pseudomonas aeruginosa, the non-esterified fatty acid DA (docosahexaenoic acid), but not OA (oleic acid), is decreased, and DA supplementation has been shown to improve the clinical condition in these patients. We hypothesized that DA may, either alone or in conjunction with lysozyme, exert antibacterial action against Ps. aeruginosa. We found that DA and lysozyme synergistically inhibit the metabolic activity of Ps. aeruginosa, in contrast with OA. Electron microscopy and equilibrium dialysis suggest that DA accumulates in the bacterial membrane in the presence of lysozyme. Surface plasmon resonance with live bacteria and differential scanning calorimetry studies with bacterial model membranes reveal that, initially, DA facilitates lysozyme incorporation into the membrane, which in turn allows influx of more DA, leading to bacterial cell death. The present study elucidates a molecular basis for the synergistic action of non-esterified fatty acids and antimicrobial polypeptides, which may be dysfunctional in cystic fibrosis.

A Biomimetic Approach to Increasing Soft Actuator Performance by Friction Reduction.

While increasing power output is the most straight-forward solution for faster and stronger motion in technology, sports, or elsewhere, efficiency is what separates the best from the rest. In nature, where the possibilities of power increase are limited, efficiency of motion is particularly important; the same principle can be applied to the emerging biomimetic and bio-interacting technologies. In this work, by applying hints from nature, we consider possible approaches of increasing the efficiency of motion through liquid medium of bilayer ionic electroactive polymer actuations, focusing on the reduction of friction by means of surface tension and hydrophobicity. Conducting polyethylene terephthalate (PET) bilayers were chosen as the model actuator system. The actuation medium consisted of aqueous solutions containing tetramethylammonium chloride and sodium dodecylbenzenesulfonate in different ratios. The roles of ion concentrations and the surface tension are discussed. Hydrophobicity of the PET support layer was further tuned by adding a spin-coated silicone layer to it. As expected, both approaches increased the displacement-the best results having been obtained by combining both, nearly doubling the bending displacement. The simple approaches for greatly increasing actuation motion efficiency can be used in any actuator system operating in a liquid medium.

Conjugated Polymer Actuators and Devices: Progress and Opportunities.

Conjugated polymers (CPs), as exemplified by polypyrrole, are intrinsically conducting polymers with potential for development as soft actuators or "artificial muscles" for numerous applications. Significant progress has been made in the understanding of these materials and the actuation mechanisms, aided by the development of physical and electrochemical models. Current research is focused on developing applications utilizing the advantages that CP actuators have (e.g., low driving potential and easy to miniaturize) over other actuating materials and on developing ways of overcoming their inherent limitations. CP actuators are available as films, filaments/yarns, and textiles, operating in liquids as well as in air, ready for use by engineers. Here, the milestones made in understanding these unique materials and their development as actuators are highlighted. The primary focus is on the recent progress, developments, applications, and future opportunities for improvement and exploitation of these materials, which possess a wealth of multifunctional properties.

Artificial Muscles Powered by Glucose.

Untethered actuation is important for robotic devices to achieve autonomous motion, which is typically enabled by using batteries. Using enzymes to provide the required electrical charge is particularly interesting as it will enable direct harvesting of fuel components from a surrounding fluid. Here, a soft artificial muscle is presented, which uses the biofuel glucose in the presence of oxygen. Glucose oxidase and laccase enzymes integrated in the actuator catalytically convert glucose and oxygen into electrical power that in turn is converted into movement by the electroactive polymer polypyrrole causing the actuator to bend. The integrated bioelectrode pair shows a maximum open-circuit voltage of 0.70 ± 0.04 V at room temperature and a maximum power density of 0.27 µW cm-2 at 0.50 V, sufficient to drive an external polypyrrole-based trilayer artificial muscle. Next, the enzymes are fully integrated into the artificial muscle, resulting in an autonomously powered actuator that can bend reversibly in both directions driven by glucose and O2 only. This autonomously powered artificial muscle can be of great interest for soft (micro-)robotics and implantable or ingestible medical devices manoeuvring throughout the body, for devices in regenerative medicine, wearables, and environmental monitoring devices operating autonomously in aqueous environments.

Type I Collagen-Derived Injectable Conductive Hydrogel Scaffolds as Glucose Sensors.

The advent of home blood glucose monitoring revolutionized diabetes management, and the recent introduction of both wearable devices and closed-loop continuous systems has enormously impacted the lives of people with diabetes. We describe the first fully injectable soft electrochemical glucose sensor for in situ monitoring. Collagen, the main component of a native extracellular matrix in humans and animals, was used to fabricate an in situ gellable self-supporting electroconductive hydrogel that can be injected onto an electrode surface or into porcine meat to detect glucose amperometrically. The study provides a proof-of-principle of an injectable electrochemical sensor suitable for monitoring tissue glucose levels that may, with further development, prove clinically useful in the future.

Electrospun silk fibroin scaffolds coated with reduced graphene promote neurite outgrowth of PC-12 cells under electrical stimulation.

Novel approaches to neural research require biocompatible materials capable to act as electrode structures or scaffolds for tissue engineering in order to stimulate or restore the functionality of damaged tissues. This work offers promising results that indicate the potential use of electrospun silk fibroin (SF) scaffolds coated with reduced graphene oxide (rGO) in this sense. The coated material becomes conductor and electroactive. A complete characterisation of SF/rGO scaffolds is provided in terms of electrochemistry, mechanical behaviour and chemical conformation of fibroin. The excellent biocompatibility of this novel material is proved with cultures of PC-12 cells. The coating with rGO improved the adhesion of cells in comparison with cells growing onto the surface of pure SF scaffolds. Also, the use of SF/rGO scaffolds combined with electrical stimulation promoted the differentiation into neural phenotypes reaching comparable or even superior levels to those obtained by means of the traditional treatment with neural growth factor (NGF).

Asymmetric Bilayer Muscles: Cooperative Actuation, Dynamic Hysteresis, and Creeping in NaPF6 Aqueous Solutions.

Three bilayer muscles [polypyrrole-paraphenolsulfonic acid/polypyrrole-dodecylbenzensulfonic acid (PPy-HpPS/PPy-DBS) asymmetric bilayer, PPy-HpPS/tape, and PPy-DBS/tape] were characterized during potential cycling in NaPF6 aqueous solutions. In parallel, the angular displacement of the muscle was video-recorded. The dynamo-voltammetric (angle-potential) and coulo-dynamic (charge-potential) results give the reaction-driven ionic exchanges in each PPy film. Electrochemical reactions drive the exchange of anions from the PPy-HpPS layer and cations from the PPy-DBS layer. This means that both layers from the asymmetric bilayer follow complementary volume changes (swelling/shrinking or shrinking/swelling), owing to complementary ionic exchanges (entrance/expulsion) driven by the bilayer oxidation or reduction. The result is a cooperative actuation; the bending amplitude described by the asymmetric bilayer muscle is one order of magnitude larger than those attained from each of the conducting polymer/tape muscles. The cooperative actuation almost eliminates creeping effects. A large dynamical hysteresis persists, which can be attributed to an irreversible reaction of the organic acid components at high overpotentials.

Fabrication of electrospun silk fibroin scaffolds coated with graphene oxide and reduced graphene for applications in biomedicine.

Silk fibroin and graphene are both promising biomaterials described in the bibliography. Hybrid scaffolds combining their properties could be attractive for tissue engineering applications. In this work, a new methodology to produce electrospun fibroin scaffolds coated with graphene materials is provided. The mechanical, electrical and electrochemical properties of the materials attained were characterised. The fibre diameters were measured (from 3.9 to 5.2 µm). The samples coated with reduced grapheme were electronic conductors and electroactive in liquid electrolytes, showing maximum oxidation and reduction (around−0.4 V peak). The chronoamperometric responses showed a reduction shoulder, pointing to the entrance of balancing cations from the solution by nucleation­relaxation: the reaction induced structural changes in the graphene. In order to check the biocompatibility of the materials, they were seeded with L929 fibroblasts. The excellent biocompatibility of silk fibroin meshes was maintained after coating with graphene, being the proliferation results equal in all the treatments 7 days after the seeding (Tukey, p N 0.05).The conductive and electroactive properties of meshes coated with reduced graphene allow the potential application of local electric fields or local ionic currents to cell cultures, biological interfaces or animal models without host response.

Deep Reduced PEDOT Films Support Electrochemical Applications: Biomimetic Color Front.

Most of the literature accepts, despite many controversial results, that during oxidation/reduction films of conducting polymers (CPs) move from electronic conductors to insulators. Thus, engineers and device's designers are forced to use metallic supports to reoxidize the material for reversible device work. Electrochromic front experiments appear as main visual support of the claimed insulating nature of reduced CPs. Here, we present a different design of the biomimetic electrochromic front that corroborates the electronic and ionic conducting nature of deep reduced films. The direct contact PEDOT metal/electrolyte and film/electrolyte was prevented from electrolyte contact until 1 cm far from the metal contact with protecting Parafilm(®). The deep reduced PEDOT film supports the flow of high currents promoting reaction induced electrochromic color changes beginning 1 cm far from the metal-polymer electrical contact and advancing, through the reduced film, toward the metal contact. Reverse color changes during oxidation/reduction always are initiated at the film/electrolyte contact advancing, under the protecting film, toward the film/metal contact. Both reduced and oxidized states of the film demonstrate electronic and ionic conductivities high enough to be used for electronic applications or, as self-supported electrodes, for electrochemical devices. The electrochemically stimulated conformational relaxation model explains those results.

Biomimetic intracellular matrix (ICM) materials, properties and functions. Full integration of actuators and sensors.

The electrochemistry of conducting polymers, and other organic compounds, originates biomimetic (intracellular matrix, ICM, reactive gels) materials, properties and devices here reviewed. One reaction changes several properties (multifunctionality): one device can integrate different actuators (artificial muscles, batteries, smart windows) and sensors (temperature, concentration, mechanical). Actuating (current and charge) and sensing (potential) magnitudes are present in the two connecting wires, and can be read by the computer, at any working time mimicking brain-organs dialog. The theoretical description of any multi-functional device envisages intelligent gel robots. The kinetic magnitudes of the reaction become a function of the conformational structure: predictive structural, chemical and biochemical kinetics are emerging.

Biomimetic dual sensing-actuators: theoretical description. Sensing electrolyte concentration and driving current.

Here we present the theoretical (electrochemical and polymeric) description of chronopotentiometric responses (under driven constant current) from reacting conducting polymers both, as films or taking part of electrochemical devices, that sense driving current and electrolyte concentration during reactive actuation. The attained sensing-actuation equations describe the potential, or the consumed electrical energy, evolution as a function of working and environmental variables: driving current, temperature, electrolyte concentration, or mechanical conditions. Good agreement between theoretical and experimental results is attained here by using polypyrrole films under flow of different currents or in different electrolyte concentrations. Being a general theoretical description, any reactive device based on the electrochemistry of conducting polymers or carbon based reactive compounds is expected to sense working and environmental conditions being described by those equations as tactile artificial muscles do. Only two connecting wires contain actuating (current) and sensing (potential) signals that are detected, simultaneously and at any actuating time, by the computer as mammalians brains do.