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Real-Time Nitric Oxide and Inflammation Sensing in 2D Osteoarthritis Models: Microsensor Design and Application
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Nitric oxide (NO) is a gaseous molecule that has a central role in signaling pathways involved in numerous physiological processes (e.g., vasodilation, neurotransmission, inflammation, apoptosis, and tumor growth). Due to its gaseous form, NO has a short half-life, and its physiology role is concentration dependent, often restricting its function to a target site. Providing NO from an external source is beneficial in promoting cellular functions and treatment of different pathological conditions. Hence, the multifaceted role of NO in physiology and pathology has garnered massive interest in developing strategies to deliver exogenous NO for the treatment of various regenerative and biomedical complexities. NO-releasing platforms or donors capable of delivering NO in a controlled and sustained manner to target tissues or organs have advanced in the past few decades. This review article discusses in detail the generation of NO via the enzymatic functions of NO synthase as well as from NO donors and the multiple biological and pathological processes that are modulated by NO. The methods for incorporation of NO donors into diverse biomaterials including physical, chemical, or supramolecular techniques are summarized. Then, these NO-releasing platforms are highlighted in terms of advancing treatment strategies for various medical problems.
Osteoarthritis (OA) is one of the most common musculoskeletal degenerative diseases and contributes to heavy socioeconomic burden. Current pharmacological and conventional non-pharmacological therapies aim at relieving the symptoms like pain and disability rather than modifying the underlying disease. Surgical treatment and ultimately joint replacement arthroplasty are indicated in advanced stages of OA. Since the underlying mechanisms of OA onset and progression have not been fully elucidated yet, the development of novel therapeutics to prevent, halt, or reverse the disease is laborious. Recently, small molecules of herbal origin have been reported to show potent anti-inflammatory, anti-catabolic, and anabolic effects, implying their potential for treatment of OA. Herein, the molecular mechanisms of these small molecules, their effect on physiological or pathological signaling pathways, the advancement of the extraction methods, and their potential clinical translation based on in vitro and in vivo evidence are comprehensively reviewed.
In the field of regenerative medicine, considerable advances have been made from the technological and biological point of view. However, there are still large gaps to be filled regarding translation and application of mesenchymal stromal cell (MSC)-based therapies into clinical practice. Indeed, variables such as cell type, unpredictable donor variation, and expansion/differentiation methods lead to inconsistencies. Most protocols use bovine serum (FBS) derivatives during MSC expansion. However, the xenogeneic risks associated with FBS limits the use of MSC-based products in clinical practice. Herein we compare a chemically defined, xenogeneic-free commercial growth medium with a conventional medium containing 10% FBS and 5 ng/ml FGF2. Furthermore, the effect of a fibronectin-coated growth surface was investigated. The effect of the different culture conditions on chondrogenic commitment was assessed by analyzing matrix deposition and gene expression of common chondrogenic markers. Chondrogenic differentiation potential was similar between the FBS-containing αMEM and the chemically defined medium with fibronectin coating. On the contrary, the use of fibronectin coating with FBS-containing medium appeared to reduce the differentiation potential of MSCs. Moreover, cells that were poorly responsive to in vitro chondrogenic stimuli were shown to improve their differentiation potential after expansion in a TGF-β1 containing medium. In conclusion, the use of a xenogeneic-free medium provides a suitable alternative for human bone marrow MSC expansion, due the capability to maintain cell characteristic and potency. To further improve chondrogenic potential of BMSCs, priming the cells with TGF-β1 during expansion is a promising strategy.
In osteoarthritis (OA), inhibition of excessively expressed pro-inflammatory cytokines in the OA joint and increasing the anabolism for cartilage regeneration are necessary. In this ex-vivo study, we used an inflammatory model of human OA chondrocytes microtissues, consisting of treatment with cytokines (interleukin 1β (IL-1β)/tumor necrosis factor α (TNF-α)) with or without supplementation of six herbal compounds with previously identified chondroprotective effect. The compounds were assessed for their capacity to modulate the key catabolic and anabolic factors using several molecular analyses. We selectively investigated the mechanism of action of the two most potent compounds Vanillic acid (VA) and Epimedin C (Epi C). After identification of the anti-inflammatory and anabolic properties of VA and Epi C, the Ingenuity Pathway Analysis showed that in both treatment groups, osteoarthritic signaling pathways were inhibited. In the treatment group with VA, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was inhibited by attenuation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) phosphorylation. Epi C showed a significant anabolic effect by increasing the expression of collagenous and non-collagenous matrix proteins. In conclusion, VA, through inhibition of phosphorylation in NF-κB signaling pathway and Epi C, by increasing the expression of extracellular matrix components, showed significant anti-inflammatory and anabolic properties and might be potentially used in combination to treat or prevent joint OA.
Extracorporeal shock waves (ESWTs) are “mechanical” waves, widely used in regenerative medicine, including soft tissue wound repair. Although already being used in the clinical practice, the mechanism of action underlying their biological activities is still not fully understood. In the present paper we tried to elucidate whether a proinflammatory effect may contribute to the regenerative potential of shock waves treatment. For this purpose, we exposed human foreskin fibroblasts (HFF1 cells) to an ESWT treatment (100 pulses using energy flux densities of 0.19 mJ/mm ² at 3 Hz), followed by cell analyses after 5 min, up to 48 h. We then evaluated cell proliferation, reactive oxygen species generation, ATP release, and cytokine production. Cells cultured in the presence of lipopolysaccharide (LPS), to induce inflammation, were used as a positive control, indicating that LPS-mediated induction of a proinflammatory pattern in HFF1 increased their proliferation. Here, we provide evidence that ESWTs affected fibroblast proliferation through the overexpression of selected cytokines involved in the establishment of a proinflammatory program, superimposable to what was observed in LPS-treated cells. The possibility that inflammatory circuits can be modulated by ESWT mechanotransduction may disclose novel hypothesis on their biological underpinning and expand the fields of their biomedical application.
A joint is the point of connection between two bones in our body. Inflammation of the joint leads to several diseases, including osteoarthritis, which is the concern of this review. Osteoarthritis is a common chronic debilitating joint disease mainly affecting the elderly. Several studies showed that inflammation triggered by factors like biomechanical stress is involved in the development of osteoarthritis. This stimulates the release of early-stage inflammatory cytokines like interleukin-1 beta (IL-1 β ), which in turn induces the activation of signaling pathways, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κ B), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), and mitogen-activated protein kinase (MAPK). These events, in turn, generate more inflammatory molecules. Subsequently, collagenase like matrix metalloproteinases-13 (MMP-13) will degrade the extracellular matrix. As a result, anatomical and physiological functions of the joint are altered. This review is aimed at summarizing the previous studies highlighting the involvement of inflammation in the pathogenesis of osteoarthritis.
Nitric oxide (NO) is an essential signaling molecule within biological systems and is believed to be involved in numerous diseases. As a result of NO’s high reaction rate, the detection of the concentration of NO, let alone the presence or absence of the molecule, is extremely difficult. Researchers have developed multiple assays and probes in an attempt to quantify NO within biological solutions, each of which has advantages and disadvantages. This review highlights many of the current NO sensors, from those that are commercially available to the newest sensors being optimized in research labs, to assist in the understanding and utilization of NO sensors in biological fields.
The environmental monitoring has been one of the priorities at the European and global scale due to the close relationship between the environmental pollution and the human health/socioeconomic development. In this field, the biosensors have been widely employed as cost-effective, fast, in situ, and real-time analytical techniques. The need of portable, rapid, and smart biosensing devices explains the recent development of biosensors with new transduction materials, obtained from nanotechnology, and for multiplexed pollutant detection, involving multidisciplinary experts. This review article provides an update on recent progress in biosensors for the monitoring of air, water, and soil pollutants in real conditions such as pesticides, potentially toxic elements, and small organic molecules including toxins and endocrine disrupting chemicals.
This study investigated the mechanisms and kinetics of nitric oxide (NO) generation by derivatives of Piloty’s acid (NO-donors) under physiological conditions. In order to qualitatively and quantitatively measure NO release, electron paramagnetic resonance (EPR) was carried out with NO spin trapping. In addition, voltammetric techniques, including cyclic voltammetry and constant potential amperometry, were used to confirm NO release from Piloty’s acid and its derivatives. The resulting data showed that Piloty’s acid derivatives are able to release NO under physiological conditions. In particular, electron-withdrawing substituents favoured NO generation, while electron-donor groups reduced NO generation. In vitro microdialysis, performed on PC12 cell cultures, was used to evaluate the dynamical secretion of dopamine induced by the Piloty’s acid derivatives. Although all the studied molecules were able to induce DA secretion from PC12, only those with a slow release of NO have not determined an autoxidation of DA itself. These results confirm that the time-course of NO-donors decomposition and the amount of NO released play a key role in dopamine secretion and auto-oxidation. This information could drive the synthesis or the selection of compounds to use as potential drugs for the therapy of Parkinson’s disease (PD).
The supplementation of culture medium with fetal bovine serum (FBS, also referred to as 'fetal calf serum') is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where (a) regulatory aspects, (b) the serum dilemma, (c) alternatives to FBS, (d) case-studies of serum-free in vitro applications, and (e) the establishment of serum-free databases, were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated, despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with above mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.
Enzyme-based chemical biosensors are based on biological recognition. In order to operate, the enzymes must be available to catalyze a specific biochemical reaction and be stable under the normal operating conditions of the biosensor. Design of biosensors is based on knowledge about the target analyte, as well as the complexity of the matrix in which the analyte has to be quantified. This article reviews the problems resulting from the interaction of enzyme-based amperometric biosensors with complex biological matrices containing the target analyte(s). One of the most challenging disadvantages of amperometric enzyme-based biosensor detection is signal reduction from fouling agents and interference from chemicals present in the sample matrix. This article, therefore, investigates the principles of functioning of enzymatic biosensors, their analytical performance over time and the strategies used to optimize their performance. Moreover, the composition of biological fluids as a function of their interaction with biosensing will be presented.
Applying soluble nitric oxide (NO) donors is the most widely used method to expose cells of interest to exogenous NO. Because of the complex equilibria that exists between components in culture media, the donor compound and NO itself, it is very challenging to predict the dose and duration of NO cells actually experience. To determine the actual level of NO experienced by cells exposed to soluble NO donors, we developed the CellNO Trap, a device that allows continuous, real-time monitoring of the level of NO adherent cells produce and experience in culture without the need to alter cell culturing procedures. Herein, we directly measured the level of NO that cells grown in the CellNO Trap experienced when soluble NO donors were added to solutions in culture wells and we characterized environmental conditions that effected the level of NO in in vitro culture conditions. Specifically, the dose and duration of NO generated by the soluble donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and the diazeniumdiolate diethyltriamine (DETA/NO) were investigated in both phosphate buffered saline (PBS) and cell culture media. Other factors that were studied that potentially affect the ultimate NO level achieved with these donors included pH, presence of transition metals (ion species), redox level, presence of free thiol and relative volume of media. Then murine smooth muscle cell (MOVAS) with different NO donors but with the same effective concentration of available NO were culturedand it was demonstrated that the cell proliferation ratio observed does not correlate with the half-lives of NO donors characterized in PBS, but do correlate well with the real-time NO profiles measured under the actual culture conditions. This data demonstrates the dynamic characteristic of the NO and NO donor in different biological systems and clearly illustrates the importance of tracking individual NO profiles under actual biological conditions.
Cytokines induce nitric oxide synthesis by endothelial cells, macrophages and polymorphonuclear leucocytes, indicating a role for nitric oxide in inflammatory processes. Nitric oxide production was therefore measured indirectly as nitrite in serum and synovial fluid samples from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) together with serum samples from healthy volunteers matched for age and sex. Serum nitrite concentrations in patients with RA and OA were significantly higher than in controls. In both disease groups synovial fluid nitrite was significantly higher than serum nitrite, implying nitric oxide synthesis by the synovium. Serum and synovial fluid nitrite concentrations in RA were also significantly higher than those in OA. These data show increased nitric oxide production in RA and OA and suggest a role for nitric oxide as an inflammatory mediator in rheumatic diseases.
Nitric oxide (NO) is an ubiquitous signaling molecule of intense interest in many physiological processes. Nitric oxide is a highly reactive free radical gas that is difficult to deliver with precise control over the level and timing that cells actually experience. We describe and characterize a device that allows tunable fluxes and patterns of NO to be generated across the surface upon which cells are cultured. The system is based on a quartz microscope slide that allows for controlled light levels to be applied to a previously described photosensitive NO-releasing polydimethylsiloxane (PDMS). Cells are cultured in separate wells that are either NO-releasing or a chemically similar PDMS that does not release NO. Both wells are then top coated with DowCorning RTV-3140 PDMS and a polydopamine/gelatin layer to allow cells to grow in the culture wells. When the waveguide is illuminated, the surface of the quartz slide propagates light such that the photosensitive polymer is evenly irradiated and generates NO across the surface of the cell culture well and no light penetrates into the volume of the wells where cells are growing. Mouse smooth muscle cells (MOVAS) were grown in the system in a proof of principle experiment, whereby 60% of the cells were present in the NO-releasing well compared to control wells after 17 h. The compelling advantage of illuminating the NO-releasing polymers with the waveguide system is that light can be used to tunably control NO release while avoiding exposing cells to optical radiation. This device provides means to quantitatively control the surface flux, timing and duration of NO cells experience and allows for systematic study of cellular response to NO generated at the cell/surface interface in a wide variety of studies.
The present paper aims to miniaturize a graphite-epoxy and synthetic zeolite-modified graphite-epoxy composite macroelectrode as a quasi-microelectrode aiming in vitro and also, envisaging in vivo simultaneous electrochemical detection of dopamine (DA) and ascorbic acid (AA) neurotransmitters, or DA detection in the presence of AA. The electrochemical behavior and the response of the designed materials to the presence of dopamine and ascorbic acid without any protective membranes were studied by cyclic voltammetry and constant-potential amperometry techniques. The catalytic effect towards dopamine detection was proved for the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode, allowing increasing the sensitivity and selectivity for this analyte detection, besides a possible electrostatic attraction between dopamine cation and the negative surface of the synthetic zeolite and electrostatic repulsion with ascorbic acid anion. Also, the synthetic zeolite-modified graphite-epoxy composite quasi-microelectrode gave the best electroanalytical parameters for dopamine detection using constant-potential amperometry, the most useful technique for practical applications.
In this study we present the real-time monitoring of three key brain neurochemical species in conscious rats using implantable amperometric electrodes interfaced to a biotelemetric device. The new system, derived from a previous design, was coupled with carbon-based microsensors and a platinum-based biosensor for the detection of ascorbic acid (AA), O2 and glucose in the striatum of untethered, freely-moving rats. The miniaturized device consisted of a single-supply sensor driver, a current-to-voltage converter, a microcontroller and a miniaturized data transmitter. The redox currents were digitized to digital values by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC), and sent to a personal computer by means of a miniaturized AM transmitter. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption and good linear response in the nanoampere current range. The in-vivo results confirmed previously published observations on striatal AA, oxygen and glucose dynamics recorded in tethered rats. This approach, based on simple and inexpensive components, could be used as a rapid and reliable model for studying the effects of different drugs on brain neurochemical systems.
Reports that globular proteins could enhance the interference blocking ability of the PPD (poly(o-phenylenediamine) layer used as a permselective barrier in biosensor design, prompted this study where a variety of modifying agents were incorporated into PPD during its electrosynthesis on Pt-Ir electrodes. Trapped molecules, including fibrous proteins and β-cyclodextrin, altered the polymer/modifier composite selectivity by affecting the sensitivity to both H2O2 (signal molecule in many enzyme-based biosensors) and the archetypal interference species, ascorbic acid. A comparison of electrochemical properties of Pt and a Pt-Ir alloy suggests that the benefits of the latter, more rigid, metal can be exploited in PPD-based biosensor design without significant loss of backward compatibility with studies involving pure Pt.
A miniaturized biotelemetric device for the amperometric detection of brain tissue oxygen is presented. The new system, derived from a previous design, has been coupled with a carbon microsensor for the real-time detection of dissolved O(2) in the striatum of freely moving rats. The implantable device consists of a single-supply sensor driver, a current-to-voltage converter, a microcontroller, and a miniaturized data transmitter. The oxygen current is converted to a digital value by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC). The digital data is sent to a personal computer using a six-byte packet protocol by means of a miniaturized 434 MHz amplitude modulation (AM) transmitter. The receiver unit is connected to a personal computer (PC) via a universal serial bus. Custom developed software allows the PC to store and plot received data. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption, and good linear response in the nanoampere current range. The in vivo results confirmed previously published observations on oxygen dynamics in the striatum of freely moving rats. The system serves as a rapid and reliable model for studying the effects of different drugs on brain oxygen and brain blood flow and it is suited to work with direct-reduction sensors or O(2)-consuming biosensors.
The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.
Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage + cytokines + endotoxin. The superinduction of PGE2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor HN(G)-monomethyl-L-arginine monoacetate inhibited OA cartilage NO production by > 90%, but augmented significantly (twofold) the spontaneous production of PGE2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE2 production. Cytokine + endotoxin stimulation of OA explants increased PGE2 production above the spontaneous release. Addition of L-NMMA further augmented cytokine-induced PGE2 production by at least fourfold. Inhibition of PGE2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows (a) superinduction of PGE2 due to upregulation of COX-2, and (b) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE2. These studies, together with others, also suggest that PGE2 may be differentially regulated in normal and OA-affected chondrocytes.
Biomedical research is undergoing a paradigm shift towards approaches centred on human disease models owing to the notoriously high failure rates of the current drug development process. Major drivers for this transition are the limitations of animal models, which, despite remaining the gold standard in basic and preclinical research, suffer from interspecies differences and poor prediction of human physiological and pathological conditions. To bridge this translational gap, bioengineered human disease models with high clinical mimicry are being developed. In this Review, we discuss preclinical and clinical studies that benefited from these models, focusing on organoids, bioengineered tissue models and organs-on-chips. Furthermore, we provide a high-level design framework to facilitate clinical translation and accelerate drug development using bioengineered human disease models.
Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of medications that are routinely been used across the world. Their analgesic, anti-inflammatory, and antipyretic effects have all been well-documented. Moreover, they are been deliberated to have a protective role against various critical diseases such as cancer and cardiovascular diseases. However, the data presented by numerous studies in past have signified the adverse effects of NSAIDs due to overdosing on various systems such as cardiovascular, gastrointestinal, hepatic, renal, neural, etc. Despite substantial studies representing the mechanism behind the clinical risk of NSAIDs, there are very few reviews that have collated comprehensive records of various toxicities caused by overdosing on NSAIDs. As a result, we have presented a comprehensive overview of existing information on NSAIDs in this review. In addition to that, we have concentrated on presenting our understanding of various organ-based toxicities caused due to NSAID's prolonged use/overdosage.
Fracture healing is a complex physiologic process, relying on the crucial interplay between biological and mechanical factors. It is generally assessed using imaging modalities, including conventional radiology, CT, MRI and ultrasound (US), based on the fracture and patient features. Although these techniques are routinely used in orthopaedic clinical practice, unfortunately, they do not provide any information about the biomechanical status of the fracture site.
Therefore, in recent years, several non-invasive techniques have been proposed to assess bone healing using ultrasonic wave propagation, changes in electrical properties of bones and callus stiffness measurement.
Moreover, different research groups are currently developing smart orthopaedic implants (plates, intramedullary nails and external fixators), able to provide information about the fracture healing process. These devices could significantly improve orthopaedic and trauma clinical practice in the future and, at the same time, reduce patients’ exposure to X-rays.
This study aims to define the role of traditional imaging techniques and emerging technologies in the assessment of the fracture healing process.
Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti-inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL-1β-stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL-6, COX-2, PGE2, iNOS and NO in IL-1β-stimulated OA chondrocytes. Further, Wogonin exhibits potent chondroprotective potential by switching the signaling axis of matrix degradation from catabolic towards anabolic ends and inhibited the expression, production and activities of matrix degrading proteases including MMP-13, MMP-3, MMP-9, and ADAMTS-4 in OA chondrocytes, and blocked the release of s-GAG and COL2A1 in IL-1β-stimulated OA cartilage explants. Wogonin also elevated the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and inhibited the IL-1β-mediated depletion of COL2A1 and proteoglycan content in the matrix of cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs or NF-κB activation. Instead, Wogonin induced mild oxidative stress through the generation of ROS and depletion of cellular GSH, thereby modulating the cellular redox leading to the induction of Nrf2/ARE pathways through activation of ROS/ERK/Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis in OA chondrocytes. Molecular docking studies revealed that Wogonin can disrupt KEAP-1/Nrf-2 interaction by directly blocking the binding site of Nrf-2 in the KEAP-1 protein. Genetic ablation of Nrf2 using specific siRNA, significantly abrogated the anti-inflammatory and chondroprotective potential of Wogonin in IL-1β-stimulated OA chondrocytes. Our data indicates that Wogonin exerts chondroprotective effects through the suppression of molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate and Wogonin as a therapeutic agent for the management of OA.
The presence of biological interferents in physiological media necessitates chemical modification of the working electrode to facilitate accurate electrochemical measurement of nitric oxide (NO). In this study, we evaluated a series of self-terminating electropolymerized films prepared from one of three isomers of phenylenediamine (PD), phenol, eugenol, or 5-amino-1-naphthol (5A1N) to improve the NO selectivity of a platinum working electrode. The electrodeposition procedure for each monomer was individually optimized using cyclic voltammetry (CV) or constant potential amperometry (CPA). Cyclic voltammetry deposition parameters favoring slower film formation generally yielded films with improved selectivity for NO over nitrite and l-ascorbate. Nitric oxide sensors were fabricated and compared using the optimized deposition procedure for each monomer. Sensors prepared using poly-phenol and poly-5A1N film-modified platinum working electrodes demonstrated the most ideal analytical performance, with the former demonstrating the best selectivity. In simulated wound fluid, platinum electrodes modified with poly-5A1N films proved superior with respect to the NO sensitivity and detection limit.
We have demonstrated spontaneous nitric oxide (NO) production by primary synovial cultures from rheumatoid (RA) and osteoarthritis patients. Increased NO production followed addition of staphylococcal enterotoxin B. Immunochemical double staining with specific anti-human inducible NO synthase (iNOS) and nonspecific esterase (NSE), or anti-CD68 (markers for tissue macrophages) showed that although many lining layer cells in RA synovium expressed iNOS, most (approximately 90%) were NSE- and CD68-, with only a minor population (approximately 10%) which were iNOS+, CD68+/NSE+. These data demonstrate the capacity for high output of NO by human synovial tissue and show that, although human macrophages can express high levels of iNOS, the majority of cells expressing iNOS are fibroblasts. We also report that synoviocytes, and macrophage cell lines, cultured with the NO donor, S-nitroso-acetyl penicillamine, produced high concentrations of tumor necrosis factor (TNF)-alpha. These results suggest that NO may mediate pathology in RA through the induction of TNF-alpha production.
An organic molecule - O-phenylene diamine (OPD) is selected as aldehyde sensing material. It is studied for selectivity to aldehyde vapors both by experiment and simulation. A chemiresistor based sensor for detection of aldehydes vapor is fabricated. O-phenylene diamine - carbon black composite is used as the sensing element. The amine groups in the OPD would interact with the carbonyl group of the aldehydes. The selectivity and cross sensitivity of the OPD-CB sensor to VOCs-aldehyde, ketone and alcohol are studied. The sensor shows good response to aldehydes compared to other VOC's. The higher response for aldehyde is attributed to the interaction of the carbonyl oxygen of aldehydes with –NH2 group of OPD. The surface morphology of the sensing element is studied by scanning electron microscopy. The OPD-CB sensor is responsive to 10 ppm of formaldehyde. The interaction of the VOCs with the OPD-CB nanocomposite is investigated by molecular dynamics studies. The interaction energies of the analyte with OPD-CB nanocomposite were calculated. It is observed that the interaction energies for aldehydes are higher than for other anlaytes. Thus the OPD-CB sensor shows selectivity to aldehydes. The simulated radial distribution function is calculated for O-H pair of analyte and OPD which further supports that the amine groups are involved in the interaction. These results suggest that it is important and easy to identify appropriate sensing materials based on understanding of analyte interaction properties.
A new carbon ascorbate oxidase-based sensor-biosensor system (SB) was coupled to a dual-channel telemetric device, for on line simultaneous electrochemical detection of ascorbic acid (AA) and antioxidant capacity in Hamlin, Sanguinello and Moro orange varieties. The electrocatalytic performances of the SB were investigated by cyclic voltammetry and amperometric techniques. The phenol composition of orange juice of each variety, and the cyclic voltammetries of the most represented phenols, were provided. The in vitro calibrations were performed in PBS (pH 5.6), applying a constant potential of +500 mV. A standard mixture of phenols, based on orange juice composition, was used as reference material for studying SB behavior. SB works at an applied potential of +500 mV, in a concentration range comprised between the LOD (0.26 μM) and 20 μM. In this concentration range, limiting the data acquisition time to two minutes, the problems of electrode passivation due to phenols polymerization were overcome. AA calibration showed that the biosensor registered statistically lower currents than the sensor since the enzyme oxidized AA before it reached the electrode surface. Standard mixture calibration showed that currents registered by sensor and biosensor did not statistically differ. The difference between sensor and biosensor AA registered currents was used to calculate an AA selectivity index and, consequently, to determine the AA content and the antioxidant capacity in the juices. The novelty of the SB is its ability to distinguish between AA and phenols contribution to antioxidant capacity. The obtained results were in accordance with reference methods.
A new telemetry system for the simultaneous detection of extracellular brain glucose and lactate, and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat-I/V converter, a microcontroller unit (MCU), a signal transmitter, and a miniaturized micro-vibration sensor. Although based on simple and inexpensive components, the bio-telemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal micro-vibrations. The device was characterized and validated in vitro before in-vivo experiments. The biosensors were implanted in the striatum of freely-moving animals and the bio-telemetric device fixed to the animals' head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behaviour in awake, untethered animals.
In attempts to improve the permselectivity of poly-o-phenylenediamine (PoPD) for biosensor applications, we investigated the influence of applied electropolymerization potential on the permeability properties of the non-conducting, non-ionic form of PoPD deposited on Pt–Ir wire electrodes in neutral media, using fixed potential amperometry and cyclic voltammetry. The analytes chosen were hydrogen peroxide (the signal transduction molecule in many oxidase-based biosensors) and ascorbic acid (AA, the archetypal interference species in biological applications of biosensors). The permselectivity (S%) of Pt/PoPD, expressed as the percentage interference by AA in hydrogen peroxide calibrations carried out at +0.7V versus SCE, showed three distinct ranges: very poor (∼70%) for mild anodic applied polymerization potentials (
Continuous cell lines (CCLs) engage in 'wasteful' glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phenotype, but instead can shift their metabolism toward increased oxidative metabolism as nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the development of novel strategies to force cultures into desirable metabolic phenotypes, by combining fed-batch feeding strategies with direct metabolic engineering of host cells.
Calibration of nitric oxide (NO) sensor is a key step to measurement of NO. Currently there are three methods to calibrate electrochemical NO sensors. However, there are many problems associated with these techniques, especially for calibration of microsensors. This article describes a novel method for calibration of NO microsensors, which has the advantage of being simple, reproducible, wide range and suitable for all kind of NO electrochemical sensors, especially for the commercial available integrated NO microsensors such as ISO-NOP30. This method is based on quantitative release of NO from decomposition of SNAP trigged by Cu+. Using of monovalant copper, as described in the present study, represents a major methodological advancement in the quantitative release of NO from SNAP. The kinetic decomposition of SNAP, effect of Cu+ concentration, pH of the solution, temperature and oxygen concentration in the solution on the release of NO are discussed. Compared with other calibration methods, this method is simple, convenient, reliable, accurate.
Chemically modified electrodes exhibit unique behavior that can greatly benefit electrochemical sensing. In this article we review the development and applications of modified electrodes for chemical sensing. After a general introduction, different strategies for surface modification, as employed in chemical sensing, are discussed in detail. Special attention is given to permselective coatings, which offer controlled access at the sensor surface or the surface immobilization of biological entities that specifically recognize the analyte. Better, understanding of these modified electrodes is achieved by using high resolution surface characterization techniques. Future prospects are evaluated.
The discoveries made in the 1980s that NO could be synthesized by mammalian cells and could act as physiological messenger and cytotoxic agent had elevated the importance of its detection. The numerous properties of NO, that enable it to carry out its diverse functions, also present considerable problems when attempting its detection and quantification in biological systems. Indeed, its total free concentration in physiological conditions has been established to be in nanomolar range. Thus, detection of nitric oxide remains a challenge, pointing out the difficult dual requirements for specificity and sensitivity. Exception made for the electrochemical techniques, most of the approaches (namely UV-visible spectroscopy, fluorescence, electron paramagnetic resonance spectroscopy) use indirect methods for estimating endogenous NO, relying on measurements of secondary species such as nitrite and nitrate or NO-adducts. They also suffer from allowing only ex situ measurements. So, the only strategies that allow a direct and in vivo detection of NO are those based on the use of ultramicroelectrodes. The reality is that surface electrode modification is needed to make the ultramicroelectrode material selective for NO. Therefore, the design of modified electrode surfaces using organized layers is very attractive and provides the ideal strategy. This review addresses a global description of the various approaches that have involved chemically modified microelectrodes specially designed for the electrochemical detection of NO in biological media. Selected significant examples of applications in biological tissues are also reported in order to highlight the importance of this approach in having new insights into the modulatory role of NO in physiology and pathophysiology.
Osteoarthritis (OA) is caused by both biochemical and mechanical factors. While the mechanisms that underlie the disease are not completely understood, investigators have characterized a number of catabolic and protective factors that have a role in the disease process. Nitric oxide (NO) and its redox derivatives appear to have a number of different functions in both normal and pathophysiological joint conditions. Until recently, NO was considered a catabolic factor that was responsible for perpetuating the OA disease process by mediating the expression of proinflammatory cytokines, inhibiting the synthesis of collagen and proteoglycans and inducing apoptosis. However, recent studies suggest that NO and its redox derivatives may also have protective effects on cartilage. This review will summarize the literature on the effects of NO on cartilage and chondrocytes as well as discuss some evidence that suggests potential protective effects of NO and/or its derivatives on other cell types. More research is needed to elucidate the role of NO and its derivatives on both normal and osteoarthritis cartilage.
Endogenous nitric oxide (NO) appears to modulate many physiological and pathophysiological processes. In order to obtain direct evidence for NO functions in vivo, we have developed 4,5-diaminofluorescein (DAF-2) as a novel fluorescent indicator for NO. Green-fluorescent triazolofluorescein formed by the reaction of NO and DAF-2 affords high sensitivity for NO (detection limit: 5 nM). Membrane-permeable DAF-2 diacetate (DAF-2 DA) was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner. This imaging method should be useful for studies of the dynamic biological actions of NO at the molecular level with fine temporal and spatial resolution.
Nitric oxide is an important bioregulatory molecule, being responsible, for example, for activity of endothelium-derived relaxing factor (EDRF). Acute hypertension, diabetes, ischaemia and atherosclerosis are associated with abnormalities of EDRF. Nitric oxide is thought to be a retrograde messenger in the central nervous system. The technology is not yet available for rapid detection of NO released by a single cell in the presence of oxygen and/or nitrite, so the release, distribution and reactivity of endogenous NO in biological systems cannot be analysed. Here we describe a porphyrinic microsensor that we have developed and applied to monitoring NO release in a microsystem. We selectively measured in situ the NO released from a single cell with a response time of less than 10 ms. The microsensor consists of p-type semiconducting polymeric porphyrin and a cationic exchanger (Nafion) deposited on a thermally sharpened carbon fibre with a tip diameter of approximately 0.5 microns. The microsensor, which can be operated in either the amperometric or voltammetric mode, is characterized by a linear response up to 300 microM and a detection limit of 10 nM. Nitric oxide at the level of 10(-20) mols can be detected in a single cell.
Nitric oxide (NO) is a small, gaseous, paramagnetic radical with a high affinity for interaction with ferrous hemoproteins such as soluble guanylate cyclase and hemoglobin. Interest in NO measurement increased exponentially with the discovery that NO or a related compound is the endothelium-derived relaxing factor (EDRF). In addition to being a potent endogenous vasodilator, NO has a role in inflammation, thrombosis, immunity, and neurotransmission. Measurement of NO is important as many of its effects (e.g., vasodilatation, inhibition of platelet aggregation) are similar to those of other substances produced by the endothelium, such as prostacyclin. NO is formed in small amounts in vivo and is rapidly destroyed by interaction with oxygen, making measurement difficult. A computerized search of the past five year's literature found NO measurements reported in fewer than 50 of 955 articles dealing with EDRF. Inhibitors of NO synthesis such as the arginine analogs or agents that inactivate NO, such as reduced hemoglobin, are commonly used as specific probes for NO, in vivo and in vitro; however, none of the NO inhibitors is completely specific. The most widely used assays use one of three strategies to detect NO: 1) NO is "trapped" by nitroso compounds, or reduced hemoglobin, forming a stable adduct that is detected by electron paramagnetic resonance (EPR) (detection threshold approximately 1 nmol); 2) NO oxidizes reduced hemoglobin to methemoglobin, which is detected by spectrophotometry (detection threshold approximately 1 nmol); 3) NO interacts with ozone producing light, "chemiluminescence" (detection threshold approximately 20 pmol). These assays can be performed to exclusively detect NO, or by adding acid and reducing agents to the sample, can measure NO and related oxides of nitrogen such as nitrite. Several new amperometric microelectrode assays offer the potential to measure smaller amounts of NO (10(-20) M), permitting NO measurement in intact issues and from single cells. This review describes the pharmacology and toxicology of NO and reviews the major techniques for measuring NO in biological models.
The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green-fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.
Nitric oxide is a gaseous, free radical which plays a role as an intracellular second messenger and a diffusable intercellular messenger. To obtain direct evidence for NO functions in vivo, we have designed and synthesized diaminofluoresceins (DAFs) as novel fluorescent indicators for NO. The fluorescent chemical transformation of DAFs is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. The N-nitrosation of DAFs, yielding the highly green-fluorescent triazole form, offers the advantages of specificity, sensitivity, and a simple protocol for the direct detection of NO (detection limit 5 nM). The fluorescence quantum efficiencies are increased more than 100 times after the transformation of DAFs by NO. Fluorescence detection with visible light excitation and high sensitivity enabled the practical assay of NO production in living cells. Membrane-permeable DAF-2 diacetate (DAF-2 DA) can be used for real-time bioimaging of NO with fine temporal and spatial resolution. The dye was loaded into activated rat aortic smooth muscle cells, where the ester bonds are hydrolyzed by intracellular esterase, generating DAF-2. The fluorescence in the cells increased in a NO concentration-dependent manner.
The steady-state concentration and thus the biological effects of NO are critically determined not only by its rate of formation, but also by its rate of decomposition. Bioreactivity of NO at physiological concentrations may differ substantially from that suggested by in vitro experiments. The charge neutrality and its high diffusion capacity are hallmarks that characterize NO bioactivity. Reactive oxygen derived species are major determinants of NO breakdown. Biotransformation of NO and its related N-oxides occurs via different metabolic routes within the body. S-Nitrosothiols formed upon reaction of NO with redox-activated thiols represent an active storage pool for NO. The major oxidative metabolites represent nitrite and nitrate, the ratio of both is determined by the microenvironmental redox conditions. In humans, circulating nitrite represents an attractive estimate of regional endothelial NO formation, whereas nitrate, with some caution, appears useful in estimating overall nitrogen/NO turnover. Within the near future, more specific biochemical tools for diagnosis of reduced NO bioactivity will become available. Increasing knowledge on the complex metabolism of NO in vivo will lead to the development of new therapeutic strategies to enhance bioactivity of NO via modulation of its metabolism.
Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.
The membrane-permeating indicator DAF-FM DA is transformed by intracellular esterases into the highly water-soluble dye DAF-FM, which traps NO produced by NO synthase (NOS) to yield a highly fluorescent triazole compound in cells (see schematic diagram). Monitoring with a fluorescence microscope should allow direct identification of intracellular production and location of NO.
Nitric oxide (NO) has complex and diverse functions in physiological and pathophysiological phenomena. The mechanisms of many events induced by NO are now well defined, so that a fundamental understanding of NO biology is almost established. Accumulated evidence suggests that NO and oxygen radicals such as superoxide are key molecules in the pathogenesis of various infectious diseases. NO biosynthesis, particularly through expression of an inducible NO synthase (iNOS), occurs in a variety of microbial infections. Although antimicrobial activity of NO is appreciated for bacteria and protozoa, NO has opposing effects in virus infections such as influenza virus pneumonia and certain other neurotropic virus infections. iNOS produces an excessive amount of NO for long periods, which allows generation of a highly reactive nitrogen oxide species, peroxynitrite, via a radical coupling reaction of NO with superoxide. Thus, peroxynitrite causes oxidative tissue injury through potent oxidation and nitration reactions of various biomolecules. NO also appears to affect a host's immune response, with immunopathological consequences. For example, overproduction of NO in virus infections in mice is reported to suppress type 1 helper T-cell-dependent immune responses, leading to type 2 helper T-cell-biased immunological host responses. Thus, NO may be a host response modulator rather than a simple antiviral agent. The unique biological properties of NO are further illustrated by our recent data suggesting that viral mutation and evolution may be accelerated by NO-induced oxidative stress. Here, we discuss these multiple roles of NO in pathogenesis of virus infections as related to both non-specific inflammatory responses and immunological host reactions modulated by NO during infections in vivo.