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Cardiovascular diseases are currently the most common cause of death in developed countries. Due to lifestyle and environmental factors, this problem is only expected to increase in the future. Reactive oxygen species (ROS) are a key player in the onset of cardiovascular diseases but also have important functions in healthy cardiac tissue. Here, the interplay between ROS generation and cardiac mechanical forces is shown, and the state of the art and a perspective on future directions are discussed. To this end, an overview of what is currently known regarding ROS and mechanosignaling at a subcellular level is first given. There the role of ROS in mechanosignaling as well as the interplay between both factors in specific organelles is emphasized. The consequences at a larger scale across the population of heart cells are then discussed. Subsequently, the roles of ROS in embryogenesis, pathogenesis, and aging are further discussed, exemplifying some aspects of mechanoregulation. Finally, different models that are currently in use are discussed to study the topics above.
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Metals are widely utilized as implant materials for bone fixtures as well as stents. Biodegradable versions of these implants are highly desirable since patients do not have to undergo a second surgery for the materials to be removed. Attractive options for such materials are zinc silver alloys since they also offer the benefit of being antibacterial. However, it is important to investigate the effect of the degradation products of such alloys on the surrounding cells, taking into account silver cytotoxicity. Here we investigated zinc alloyed with 1 % of silver (Zn1Ag) and how differently concentrated extracts (1 %-100 %) of this material impact human umbilical vein endothelial cells (HUVECs). More specifically, we focused on free radical generation and oxidative stress as well as the impact on cell viability. To determine free radical production we used diamond-based quantum sensing as well as conventional fluorescent assays. The viability was assessed by observing cell morphology and the metabolic activity via the MTT assay. We found that 1 % and 10 % extracts are well tolerated by the cells. However, at higher extract concentrations we observed severe impact on cell viability and oxidative stress. We were also able to show that quantum sensing was able to detect significant free radical generation even at the lowest tested concentrations.
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Aleaciones , Supervivencia Celular , Células Endoteliales de la Vena Umbilical Humana , Nanodiamantes , Estrés Oxidativo , Zinc , Humanos , Aleaciones/química , Supervivencia Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Nanodiamantes/química , Plata/toxicidad , Plata/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Radicales Libres/metabolismo , Ensayo de Materiales/métodos , Implantes Absorbibles/efectos adversosRESUMEN
Cumulus granulosa cells (cGCs) and mural granulosa cells (mGCs), although derived from the same precursors, are anatomically and functionally heterogeneous. They are critical for female fertility by supporting oocyte competence and follicular development. There are various techniques used to investigate the role of free radicals in mGCs and cCGs. Yet, temporospatial resolution remains a challenge. We used a quantum sensing approach to study free radical generation at nanoscale in cGCs and mGCs isolated from women undergoing oocyte retrieval during in vitro fertilization (IVF). Cells were incubated with bare fluorescent nanodiamonds (FNDs) or mitochondria targeted FNDs to detect free radicals in the cytoplasm and mitochondria. After inducing oxidative stress with menadione, we continued to detect free radical generation for 30 min. We observed an increase in free radical generation in cGCs and mGCs from 10 min on. Although cytoplasmic and mitochondrial free radical levels are indistinguishable in the physiological state in both cGCs and mGCs, the free radical changes measured in mitochondria were significantly larger in both cell types, suggesting mitochondria are sites of free radical generation. Furthermore, we observed later occurrence and a smaller percentage of cytoplasmic free radical change in cGCs, indicating that cGCs may be more resistant to oxidative stress.
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Diamond-based T1 relaxometry is a new technique that allows nanoscale magnetic resonance measurements. Here we present its first application in patient samples. More specifically, we demonstrate that relaxometry can determine the free radical load in samples from arthritis patients. We found that we can clearly differentiate between osteoarthritis and rheumatoid arthritis patients in both the synovial fluid itself and cells derived from it. Furthermore, we tested how synovial fluid and its cells respond to piroxicam, a common nonsteroidal anti-inflammatory drug (NSAID). It is known that this drug leads to a reduction in reactive oxygen species production in fibroblast-like synoviocytes (FLS). Here, we investigated the formation of free radicals specifically. While FLS from osteoarthritis patients showed a drastic decrease in the free radical load, cells from rheumatoid arthritis retained a similar radical load after treatment. This offers a possible explanation for why piroxicam is more beneficial for patients with osteoarthritis than those with rheumatoid arthritis.
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Artritis Reumatoide , Osteoartritis , Humanos , Líquido Sinovial , Membrana Sinovial/patología , Piroxicam/uso terapéutico , Células Cultivadas , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Osteoartritis/diagnóstico por imagen , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , Fibroblastos/patologíaRESUMEN
Nanodiamonds are increasingly popular in biomedical applications, including optical labelling, drug delivery and nanoscale sensing. Potential new applications are in studying infertility or labelling sperm cells. However, for these applications, it is necessary that nanodiamonds are inert and do not alter sperm properties. In this article, we assessed the biocompatibility of nanodiamonds in detail. We investigated different sizes and concentrations of nanodiamonds and sperm preparation methods. We evaluated if the metabolic activity, membrane integrity, morphology and formation of reactive oxygen species were altered. These parameters were tested for sperm cells in their uncapacitated and capacitated states. Unfortunately, FNDs are not universally biocompatible. Generally, cells in the capacitated state are more prone to stress. Additionally, larger particles and lower concentrations are tolerated better than smaller and higher concentrated particles.
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A broad spectrum of biomaterials has been explored in order to design cardiovascular implants of sufficient hemocompatibility. Most of them were extensively tested for the ability to facilitate repopulation by patient cells. It was shown that stiffness, surface roughness, or hydrophilicity of polyelectrolyte films have an impact on adhesion, proliferation, and differentiation of cells. At the same time, it is still unknown how these properties influence cell functionality and as a consequence interactions with blood components under dynamic conditions. In this study, we aimed to determine the impact of chemical cross-linking of Chitosan (Chi) and Chrondroitin Sulphate (CS) on endothelium-blood cross-talk. We have found that the morphology of the endothelium monolayer was not altered by changes in coating properties. However, free radical generation by endothelial cells varied depending on the elastic properties of the coating. Simultaneously, we have observed a significant decrease in the level of adhering and circulating active platelets as well as aggregates when the endothelium monolayer was formed on stiffer films than on the other coating variants. Moreover, the same type of films has promoted significantly higher adhesion of blood morphotic elements when they were not functionalized by endothelium. The observed changes in hemocompatibility indicate the importance of a design of coatings that will promote cellularization in vivo in a relatively short time and which will regulate cell function.
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Materiales Biocompatibles , Células Endoteliales , Humanos , Polielectrolitos , Adhesión Celular , Endotelio , Materiales Biocompatibles/farmacología , Materiales Biocompatibles/química , Propiedades de SuperficieRESUMEN
Relaxometry is a technique which makes use of a specific crystal lattice defect in diamond, the so-called NV center. This defect consists of a nitrogen atom, which replaces a carbon atom in the diamond lattice, and an adjacent vacancy. NV centers allow converting magnetic noise into optical signals, which dramatically increases the sensitivity of the readout, allowing for nanoscale resolution. Analogously to T1 measurements in conventional magnetic resonance imaging (MRI), relaxometry allows the detection of different concentrations of paramagnetic species. However, since relaxometry allows very local measurements, the detected signals are from nanoscale voxels around the NV centers. As a result, it is possible to achieve subcellular resolutions and organelle specific measurements.A relaxometry experiment starts with polarizing the spins of NV centers in the diamond lattice, using a strong laser pulse. Afterward the laser is switched off and the NV centers are allowed to stochastically decay into the equilibrium mix of different magnetic states. The polarized configuration exhibits stronger fluorescence than the equilibrium state, allowing one to optically monitor this transition and determine its rate. This process happens faster at higher levels of magnetic noise. Alternatively, it is possible to conduct T1 relaxation measurements from the dark to the bright equilibrium by applying a microwave pulse which brings NV centers into the -1 state instead of the 0 state. One can record a spectrum of T1 at varying strengths of the applied magnetic field. This technique is called cross-relaxometry. Apart from detecting magnetic signals, responsive coatings can be applied which render T1 sensitive to other parameters as pH, temperature, or electric field. Depending on the application there are three different ways to conduct relaxometry experiments: relaxometry in moving or stationary nanodiamonds, scanning magnetometry, and relaxometry in a stationary bulk diamond with a stationary sample on top.In this Account, we present examples for various relaxometry modes as well as their advantages and limitations. Due to the simplicity and low cost of the approach, relaxometry has been implemented in many different instruments and for a wide range of applications. Herein we review the progress that has been achieved in physics, chemistry, and biology. Many articles in this field have a proof-of-principle character, and the full potential of the technology still waits to be unfolded. With this Account, we would like to stimulate discourse on the future of relaxometry.
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Diamante , Nanodiamantes , Diamante/química , Nitrógeno/química , Nanodiamantes/química , Fluorescencia , TemperaturaRESUMEN
Acetaminophen overdoses cause cell injury in the liver. It is widely accepted that liver toxicity is initiated by the reactive N-acetyl-para-aminophenol (APAP) metabolite N-acetyl-p-benzoquinone imine (NAPQI), which first depletes glutathione and then irreversibly binds to mitochondrial proteins and nuclear DNA. As a consequence, mitochondrial respiration is inhibited, and DNA strands break. NAPQI also promotes the oxidative stress since glutathione is one of the main free-radical scavengers in the cell. However, so far it is unknown where exactly free radicals are generated. In this study, we used relaxometry, a novel technique that allows nanoscale magnetic resonance imaging detection of free radicals. The method is based on fluorescent nanodiamonds, which change their optical properties based on their magnetic surrounding. To achieve subcellular resolution, these nanodiamonds were targeted to cellular locations, that is, the cytoplasm, mitochondria, and the nucleus. Since relaxometry is sensitive to spin noise from radicals, we were able to measure the radical load in these different organelles. For the first time, we measured APAP-induced free-radical production in an organelle-specific manner, which helps predict and better understand cellular toxicity.
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Acetaminofén , Nanodiamantes , Acetaminofén/toxicidad , Citosol/metabolismo , Glutatión , Mitocondrias/metabolismo , Radicales Libres/metabolismo , MacrófagosRESUMEN
Here, we present multifunctional fluorescent nanodiamonds (FNDs) for simultaneous drug delivery and free radical detection. For this purpose, we modified FNDs containing nitrogen vacancy (NV) centers with a diazoxide derivative. We found that our particles enter cells more easily and are able to deliver this cancer drug into HeLa cells. The particles were characterized by infrared spectroscopy, dynamic light scattering, and secondary electron microscopy. Compared to the free drug, we observe a sustained release over 72 h rather than 12 h for the free drug. Apart from releasing the drug, with these particles, we can measure the drug's effect on free radical generation directly. This has the advantage that the response is measured locally, where the drug is released. These FNDs change their optical properties based on their magnetic surrounding. More specifically, we make use of a technique called relaxometry to detect spin noise from the free radical at the nanoscale with subcellular resolution. We further compared the results from our new technique with a conventional fluorescence assay for the detection of reactive oxygen species. This provides a new method to investigate the relationship between drug release and the response by the cell via radical formation or inhibition.
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Nanodiamantes , Dispersión Dinámica de Luz , Células HeLa , Humanos , Microscopía Fluorescente , Nanodiamantes/química , Nitrógeno/químicaRESUMEN
The blood compatibility of self-assembled monolayers (SAMs) of oligoproline, a nonionic antifouling peptide, was investigated using the cone-and-plate assay imitating arterial blood flow conditions. End-capped oligoprolines composed of 6 and 9 proline residues (Pro6 and Pro9) and a Cys residue were synthesized for preparing SAMs (Pro-SAMs) on Au-sputtered glass. The surface of Pro-SAMs indicated hydrophilic property with a smooth topology. The adsorption of blood components and the adhesion of blood cells, including leukocytes and platelets, were strongly suppressed on Pro-SAMs. Moreover, Pro9-SAM did not trigger the activation of platelets (i.e., the conformational change of GPIIb/IIIa and P-selectin (CD62P) expression on platelets and the formation of aggregates). Our results demonstrate that Pro9-SAM completely inhibited acute thrombogenic responses and the activation of platelets under dynamic conditions.
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Plaquetas , Selectina-P , Adsorción , Prolina , Propiedades de SuperficieRESUMEN
Free radicals play a major role in sperm development, including maturation and fertilization, but they are also linked to infertility. Since they are short-lived and reactive, they are challenging to detect with state of the art methodologies. Thus, many details surrounding their role remain unknown. One unknown factor is the source of radicals that plays a role in the sperm maturation process. Two alternative sources have been postulated: First, the NADPH-oxidase system embedded in the plasma membrane (NOX5) and second, the NADH-dependent oxidoreductase of mitochondria. Due to a lack of localized measurements, the relative contribution of each source for capacitation remains unknown. To answer this question, we use a technique called diamond magnetometry, which allows nanoscale MRI to perform localized free radical detection. With this tool, we were able to quantify radical formation in the acrosome of sperm heads. This allowed us to quantify radical formation locally in real time during capacitation. We further investigated how different inhibitors or triggers alter the radical generation. We were able to identify NOX5 as the prominent source of radical generation in capacitation while the NADH-dependent oxidoreductase of mitochondria seems to play a smaller role.
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Acrosoma , Capacitación Espermática , Masculino , Humanos , NAD/metabolismo , Semen , Espermatozoides/metabolismo , Radicales Libres/metabolismo , Imagen por Resonancia Magnética , Oxidorreductasas/metabolismoRESUMEN
Degradable polymers are widely used in the biomedical fields due to non-toxicity and great biocompatibility and biodegradability, and it is crucial to understand how they degrade. These polymers are exposed to various biochemical media in medical practice. Hence, it is important to precisely follow the degradation of the polymer in real time. In this study, we made use of diamond magnetometry for the first time to track polymer degradation with nanoscale precision. The method is based on a fluorescent defect in nanodiamonds, which changes its optical properties based on its magnetic surrounding. Since optical signals can be read out more sensitively than magnetic signals, this method allows unprecedented sensitivity. We used a specific mode of diamond magnetometry called relaxometry or T1 measurements. These are sensitive to magnetic noise and thus can detect paramagnetic species (gadolinium in this case). Nanodiamonds were incorporated into polylactic acid (PLA) films and PLA nanoparticles in order to follow polymer degradation. However, in principle, they can be incorporated into other polymers too. We found that T1 constants decreased gradually with the erosion of the film exposed to an alkaline condition. In addition, the mobility of nanodiamonds increased, which allows us to estimate polymer viscosity. The degradation rates obtained using this approach were in good agreement with data obtained by quartz crystal microbalance, Fourier-transform infrared spectroscopy, and atomic force microscopy.
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Nanodiamantes , Diamante , Magnetometría , Nanodiamantes/química , Poliésteres , PolímerosRESUMEN
Free-radical generation is suspected to play a key role in cardiovascular diseases. Another crucial factor is shear stress. Human umbilical vein endothelial cells (HUVECS), which form the lining of blood vessels, require a physiological shear stress to activate many vasoactive factors. These are needed for maintaining vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone. Additionally, blood clots form at regions of high shear stress within a blood vessel. Here, we use a new method called diamond magnetometry which allows us to measure the dynamics of free-radical generation in real time under shear stress. This quantum sensing technique allows free-radical detection with nanoscale resolution at the single-cell level. We investigate radical formation in HUVECs in a microfluidic environment under different flow conditions typically found in veins and arteries. Here, we looked into free-radical formation before, during, and after flow. We found that the free-radical production varied depending on the flow conditions. To confirm the magnetometry results and to differentiate between radicals, we performed conventional fluorescent reactive oxygen species (ROS) assays specific for superoxide, nitric oxide, and overall ROS.
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Nanodiamantes , Células Endoteliales de la Vena Umbilical Humana , Humanos , Óxido Nítrico , Especies Reactivas de Oxígeno , Estrés MecánicoRESUMEN
Successful delivery of fluorescent nanodiamonds (FNDs) into the cytoplasm is essential to many biological applications. Other applications require FNDs to stay within the endosomes. The diversity of cellular uptake of FNDs and following endosomal escape are less explored. In this article, we quantify particle uptake at a single cell level. We report that FNDs enter into the cells gradually. The number of internalized FNDs per cell differs significantly for the cell lines we investigated at the same incubation time. In HeLa cells we do not see any significant endosomal escape. We also found a wide distribution of FND endosomal escape efficiency within the same cell type. However, compared with HeLa cells, FNDs in HUVECs can easily escape from the endosomes and less than 25% FNDs remained in the vesicles after 4 h incubation time. We believe this work can bring more attention to the diversity of the cells and provide potential guidelines for future studies.
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Nanodiamantes , Endosomas , Colorantes Fluorescentes , Células HeLa , HumanosRESUMEN
To date 15% of couples are suffering from infertility with 45-50% of males being responsible. With an increase in paternal age as well as various environmental and lifestyle factors worsening these figures are expected to increase. As the so-called free radical theory of infertility suggests, free radicals or reactive oxygen species (ROS) play an essential role in this process. However, ROS also fulfill important functions for instance in sperm maturation. The aim of this review article is to discuss the role reactive oxygen species play in male fertility and how these are influenced by lifestyle, age or disease. We will further discuss how these ROS are measured and how they can be avoided during in-vitro fertilization.
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Infertilidad Masculina , Fertilización In Vitro , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Masculino , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Espermatozoides/metabolismoRESUMEN
The main purpose of the research was to analyze the influence of surface modification of the cobalt-based alloy used in dental prosthetics by applying zirconium oxide (ZrO2) layers using the ALD (Atomic Layer Deposition) method. The samples were made using the DMLS (Direct Metal Laser Sintering) technique, and their surfaces were prepared in accordance with the principles of removable partial dentures (RPDs). A 50 nm-thick zirconium oxide coating was applied to the prepared substrates. This paper deals with the issues of prosthetic stomatopathy, which is a complex of pathological changes occurring in approx. 40% of the Polish population using removable dentures. Often, these changes, occurring on the mucosa, are related to improper performance, allergic reactions or the multiplication of bacteria on the surface of partial dentures. An innovative method of surface modification was proposed, together with the analysis of its influence on the physicochemical properties of the alloy and the adhesion of bacteria to the surface.
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The phenomenon of superior biological behavior observed in titanium processed by an unconventional severe plastic deformation method, that is, hydrostatic extrusion, has been described within the present study. In doing so, specimens varying significantly in the crystallographic orientation of grains, yet exhibiting comparable grain refinement, were meticulously investigated. The aim was to find the clear origin of enhanced biocompatibility of titanium-based materials, having microstructures scaled down to the submicron range. Texture, microstructure, and surface characteristics, that is, wettability, roughness, and chemical composition, were examined as well as protein adsorption tests and cell response studies were carried out. It has been concluded that, irrespective of surface properties and mean grain size, the (101Ì 0) crystallographic plane favors endothelial cell attachment on the surface of the severely deformed titanium. Interestingly, an enhanced albumin, fibronectin, and serum adsorption as well as clearly directional growth of the cells with preferentially oriented cell nuclei have been observed on the surfaces having (0001) planes exposed predominantly. Overall, the biological response of titanium fabricated by severe plastic deformation techniques is derived from the synergistic effect of surface irregularities, being the effect of refined microstructures, surface chemistry, and crystallographic orientation of grains rather than grain refinement itself.
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Osteoblastos , Titanio , Cristalografía , Propiedades de Superficie , HumectabilidadRESUMEN
Surface functionalization of materials to improve their hemocompatibility is a challenging problem in the field of blood-contacting devices and implants. Polyelectrolyte multilayer films (PEMs), which can mimic functions and structure of an extracellular matrix (ECM), are a promising solution to the urgent need for functional blood-contacting coatings. The properties of PEMs can be easily tuned in order to provide a scaffold with desired physico-chemical parameters. In this study chitosan/chondroitin sulfate (Chi/CS) polyelectrolyte multilayers were deposited on medical polyurethane. Afterwards PEMs were modified by chemical cross-linking and nanoparticles introduction. Coatings with variable properties were tested for their hemocompatibility in the cone-plate tester under dynamic conditions. The obtained results enable the understanding of how substrate properties modulate PEMs interaction with blood plasma proteins and the morphotic elements.
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PURPOSE: The aim of the work was to create an appropriate substrate for organ transplantation using bioactive tissue-based scaffold populated by cells of the graft recipient. The purpose of the modeling was to investigate the mechanical effects of wave loading of aortic and pulmonary tissue material. METHODS: The biological properties of tissues of aortic and pulmonary valves were modified by the process of decellularization. The host cells were removed by various physical methods with focus on minimal degradation of the extracellular matrix. Thus, the decellularization process was controlled by histological methods. The tissue decellularization process was simulated by finite element modelling. RESULTS: The mechanical results represented by a displacement at the center of the sample were coherent and the heterogeneity of the distribution of the caves on the surface of the samples was confirmed, both by experiment and in the simulation by the alternate occurrence of local minima and maxima. The latter results from the uneven removal of cells from the effect of the wave causing decellularization were also predicted by the numerical model. Laser radiation had a destructive effect on the components of the extracellular matrix (e.g., collagen and elastic fibers), mainly depending on the fluence and number of pulses in a single exposure. CONCLUSIONS: The differences between the valve tissue materials were shown, and the impact of the process of decellularization on the properties of the tissues was analyzed. It should be emphasized that due to low absorption and high scattering, laser radiation can deeply penetrate the tissue, which allows for effective decellularization process in the entire volume of irradiated tissue.