The Young's Modulus (or Elastic Modulus) is in essence the stiffness of a material. It demonstrates the higher capability for planar CBNs to sequester proteins compared to cylindrical shaped CBNs. The planar sheet shape and larger size of the GFs than the MWCNTs are likely responsible for modifications to the cells’ environment, such as media protein adsorption [30]. The Young’s modulus of the cells exposed to MWCNT after 2 h had a slight increase, this effect has been reported by different authors after the administration of SWCNT, mentioning that it can be related to the reinforcement by nanomaterials to the cytoskeleton fibers (actin stress fibers) [63, 64]. The MFP3D-Bio AFM system (Asylum Research, Santa Barbara, CA, USA) located in the Birck Nanotechnology Center at Purdue University was used in this work. Part 1: Bending of a cantilever under static loads Springer Nature. Explicitly state where your pillow is behaving elastically? The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. [66], evidenced that SSA no necessary predict the toxicity response of CBNs. I’ve made a video of the experiment here youtu.be/xASsYJo3zrM. Evaluation of the elastic Young’s modulus and cytotoxicity variations in fibroblasts exposed to carbon-based nanomaterials. Graphene oxide flakes (XG Sciences Inc, Lansing, MI, USA) were suspended in 1× PBS at 1 mg/mL. https://doi.org/10.1039/C5TB00179J. PLoS ONE. In addition, the introduced force/volume technique permit to simultaneously quantify a single cells volume variation at different periods that has provided information of the process of cell death. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. https://doi.org/10.1002/jcp.1041190110. https://doi.org/10.1016/j.immuni.2012.08.017. https://doi.org/10.1126/science.1116995. Carbon nanotubes reorganize actin structures in cells and ex vivo. YOUNG’S MODULUS PRACTICAL OBJECTIVE • To determine the Young’s Modulus of a steel wire using a graphical method. The authors declare that they have no competing interests. However, other characteristics related to shape and dimensionality can be determinant on NMs behavior into the organisms. https://doi.org/10.1021/acs.jpcb.5b07656. Fast, multi-frequency, and quantitative nanomechanical mapping of live cells using the atomic force microscope. Toxicol Lett. Figure 1 shows the TEM images of NIH3T3; the control cells are in row A, the cell is characterized by a large nucleus with no significant changes in cytoplasmic organelles. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments. Grinnell F. Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata. https://doi.org/10.1586/erc.09.184. When bacteria are exposed to GFs, there is dramatic adenine adsorption on the E. coli membrane by the GF surface destroying the bacteria inducing death [37]. 2011;8:78–84. https://doi.org/10.1016/j.toxlet.2010.04.018. 15 µL of MWCNTs or GFs at 1 mg/mL was added to each well to a final concentration of 50 µg/mL. The adherent cell is indented by the AFM cantilever tip the signal changes on the photodetector are transformed from voltage to distance and used to calculate the Force and using Hertz model estimating the Young modulus. Cytoskeleton damage is a sign of cellular homeostasis dysfunction, due to cytotoxic products such as high concentration of radical oxygen species (ROS) [18]. 2014;118:1246–55. Cartagena A, Raman A. The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Liu et al., presented an innovative approach from the single-cells assessment of mechanical response by compressing using a single point measurement of the Young’s modulus of the macrophages after exposure of silver nanoparticles. Schematic of experimental setup using AFM. As the shown in Figure 1 the sample was never taken past the elastic region. Subsequently, DHE dye was added to the cells. Immunity. 2012;261:121–33. Impurities in CBNs are related with the catalysis of the Fenton reaction, especially iron impurities, which stimulates ROS production in cells, however, the concentrations presented in the GF are not likely to induce severe damage on mammalian cells [44,45,46]. AppliedStatics (eme1016) Uploaded by. The samples were sonicated and incubated at 37 °C, 95% RH, and 5% CO2 overnight. Then, cells were incubated for 2 and 24 h at 37 °C under 5% CO2 atmosphere. Analysis of results 1. Wörle-Knirsch JM, Pulskamp K, Krug HF. https://doi.org/10.1186/s12951-019-0460-8, DOI: https://doi.org/10.1186/s12951-019-0460-8. Alba Ávila. where Fts is the tip-sample indentation force (N), E is the elastic Young’s modulus (Pa) of the sample, δ is the sample mean indentation, \(\nu\) is the Poisson’s ratio (defined as 0.5 for soft live cells), and R is the microsphere radius. Owusu-Ansah E, Yavari A, Banerjee U. Fluorescent staining of NIH3T3’s cytoskeleton: a control cell; F-actin (green) is highly organized with stress fibers extending continuously along the cells. Mammalian iron homeostasis in health and disease: uptake, storage, transport, and molecular mechanisms of action. CBNs adsorption of protein is linked to their dimensionality. GF exhibits an enhanced protein sequestration ability compared with MWCNTs. A calibration curve was constructed using dilutions of a stock 1 mg/mL solution of Bovine Serum Albumin (BSA), DMEM and, DMEM + 10% of BSA. Higher concentration of 30 µg/mL shows cells viability decrease; in the case of MWCNT by 16.3% and the case of GF by 13.1%. After 24 h, 100 µL of MWCNTs or GFs at 1 mg/mL were added to each well to a final concentration of 50 µg/mL. statement and The cytoskeleton integrity is shaped by three main structures: actin fibers, intermediate fibers, and microtubules. The cells were exposed to 50 µg/mL of CBNs for 24 h. Then were fixed and prepared for TEM imaging. Sokolov I, Dokukin ME, Guz NV. We also exposed the cells to a concentration of 50 µg/mL, however, at this concentration a large number of detritus did not allow a reliable reading by the flow cytometer. Casey A, Herzog E, Davoren M, Lyng FM, Byrne HJ, Chambers G. Spectroscopic analysis confirms the interactions between single walled carbon nanotubes and various dyes commonly used to assess cytotoxicity. Dong C, Eldawud R, Sargent LM, Kashon ML, Lowry D, Rojanasakul Y, et al. J Nanoparticle Res. Biophys J. Also, we evidenced that after 2 h of exposure to GFs the cells’ volume decrease. 2. a1 and a2 control cells, no exposure to CBNs. 2001;280:C719–41. PubMed  https://doi.org/10.1038/nature08908. It has been previously demonstrated that rather than introduce toxicity by the presence of iron, MWCNTs promotes iron metabolism sequestration inducing cellular inflammation [45, 47]. Due to the greater SSA and the facilitated cellular uptake of the MWCNT we expected significant lower viability on cells exposed to MWCNTs than cells exposed to GFs [48]. It is dependent upon temperature and pressure however. 2008. https://doi.org/10.1038/nprot.2008.23. To the best of our knowledge, this work represents the first demonstration of such rapid cellular toxicity that may be critical to early diagnostics of potential toxic effects. Article  2008;20:474204. https://doi.org/10.1088/0953-8984/20/47/474204. The Hertz contact mechanics model for a rigid sphere indenting an infinite isotropic elastic half-space is. as well as a plot. 2013;62:222–32. Perfect for A-level physics students on any specification. This AFM mode is based on the acquisition of low-speed, quasi-static F–Z curves predefined by the user grid of points, resulting in the simultaneous acquisition of topography and material properties maps [69]. Journal of Nanobiotechnology An important assumption in the analysis using this model is that the microsphere indentation is much smaller than the finite cell height (10–20% of the cell thickness) to minimize artifacts in extracted elasticity maps originating from contributions of the hard substrate due to the finite cell thickness [71]. We selected NIH3T3 fibroblasts since phagocytosis is not an active process in these cells [50]. In the presence of oxygen radicals, the molecules of 2-hydroxyethidium intercalate with the DNA and fluoresce red (576 nm) [52]. No significant differences were observed between the two nanomaterials by t test. Therefore, the cellular uptake will depend more on the nanomaterials properties rather than the cellular activity of engulfing foreign bodies like macrophages [51]. Gavara N, Chadwick RS. Conditions. https://doi.org/10.1016/j.bpj.2016.04.034. The shape and size of both nanomaterials were measured using TEM. 2014;10:651–9. Finally, samples were cut using a microtome (ThermoFisher, MD, USA) and loaded into a JEOL 1400 Plus TEM (Jeol, Tokyo, Japan; located at Fundación Santa Fe, Colombia). Each group of measurements includes 13 cells. 2012;25:15–34. Table 1 summarizes the characterization results carried out in phosphate buffer solution (PBS) and culture media (DMEM) as well as the information provided by the manufacturer. The Young modulus is often regarded as the quintessential material property, and students can learn to measure it. The control cells exceeded, up to eight times, the length of the fibers of the cells exposed to both CBNs (MWCNT and GF). We used the bicinchoninic acid assay (BCA) to quantify the concentration of the BSA in the supernatant after the administration of CBNs. To Dr. Helena Groot and Diana Narvaez for access to the cell culture facilities at Genetics Laboratory Universidad de Los Andes. Mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress. Young’s modulus, also known as the tensile modulus, elastic modulus or traction modulus (measured in Pa, which is a pressure unit(N.m^-2; 10MPa is equivalent to 1Kg force per square millimeter) is a mechanical property of linear elastic materials. The Theory Before we move ahead, do you know what a Searle’s apparatus is? This phenomenon is relevant to understand how the intra and extracellular effects on cells toxicity from both CBNs are triggered. Graph Skills 7: Y-intercepts for False Origins, How to present results in science (tables & graphs), GCSE 9-1 AQA Physics 4.7 - Electromagnetism (whole unit), Atoms, Elements and Compounds (Activate KS3), NEW AQA GCSE (2016) Physics - Molecules & Matter, GCSE Physics (4.4.2.4) Atomic structure - Radioactive contamination (AQA), GCSE Physics (4.4.3) Atomic structure - Hazards + uses of radioactive emissions + background (AQA), GCSE Chemistry Paper 1 - Atomic Structure and Periodic Table Worksheet Bundle. 1990;57:2089. https://doi.org/10.1063/1.103950. A protocol for in vivo detection of reactive oxygen species. 2011;8:9. https://doi.org/10.1186/1743-8977-8-9. The Young Modulus is named after Thomas Young (1773 to 1829) who was a British polymath, contributing to our understanding of optics, physiology, and Egyptology, among other fields. Part Fibre Toxicol. Poisson’s Ratio(experimental): 8.81% Discussion: The experiment was successful. PubMed  Antioxid Redox Signal. Authors thank Prof. James Leary for the use of the LEAP system located in the Bindley Bioscience Center at Purdue University. The assessment of CBN’s cytotoxicity has been highly debated recently. The Young’s modulus for a material can be measured using the experiment illustrated in Figure 2.The reference wire and test wire made of the selected material are hung from the ceiling. Cells were maintained at 37 °C during the AFM experiments using a Petri dish holder and a heater on the MFP-3D scanner X–Y stage (Asylum Research, Santa Barbara, CA, USA). J R Soc Interface. Young's modulus, E, is the most important of the elastic constants and can be derived from the slope of the strain–stress curve obtained when a rock specimen is subjected to unconfined compression (i.e., static loading), it being the ratio of stress to strain.The strains are measured by attaching strain gauges to the test specimens, or by displacement transducers, and recording their outputs. 2012;3:398–417. https://doi.org/10.1016/j.injury.2007.02.007. In the case of GF, no previous studies were found analyzing the mechanical response of cells after the exposure to them. Then, Alexa Fluor 488 conjugated phalloidin (Cat A12379, Thermofisher, MD, USA) was added to label the actin cytoskeleton; the phalloidin toxin has a high affinity for F-actin subunits, stopping the normal denaturalization; thus, the actin fiber can be visualized when the phalloidin is labeled with a fluorescence compound. There are solutions at the end. Young's modulus for piano wire is about 2x10^7 lbs/sq.in, or 1.92 x 10^12 dynes/cm sq. Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape. Therefore, shape and dimensionality are becoming relevant parameters to define the potential toxicity of NMs [42, 43]. Sci Rep. 2015;5:11692. https://doi.org/10.1038/srep11692. 2014;811:73–91. After 24 h, cells exposed to planar-shaped GFs produced twice as many ROS and exhibited a twofold decrease in Young’s modulus in contrast to cells exposed to cylindrical-shaped MWCNTs, even though that the specific surface area (SSA) of MWCNTs is double than the GFs SSA. Karlsson M, Kurz T, Brunk UT, Nilsson SE, Frennesson CI. 10 µg of each sample was placed on an XploRA confocal Raman spectrometer (Horiba Scientific, Kyoto, Japan; located at Universidad de Los Andes). We found that the greater SSA of MWCNT were not a good predictor of cells toxicity by CBNs as their shape and dimensionality. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Elastic Young’ modulus maps and three-dimensional topographic reconstruction of live NIH3T3 in culture media before and after exposure to CBNs: a unexposed cells, b cells exposed 2 h to MWCNTs, c cells exposed 24 h to MWCNTs, d cells exposed 2 h to GFs and e cells exposed 24 h to GFs. Nature. https://doi.org/10.1016/j.ejpb.2008.08.009. https://doi.org/10.1016/j.carbon.2007.03.033. 12/03/2017 2 VIDEOS ... the experiment are avoided as the test wire and control wire would both expand by the same amount and the scale would adjust position NIH3T3 cells were seeded overnight in a specific 96-well plate (Cyntellect, San Diego, CA, USA) for laser-enabled analysis and processing (LEAP™) (Cyntellect, San Diego, CA, USA; located at Bindley, Purdue University) at 5 × 105 cell/mL concentration in culture media. Among the NMs properties, SSA has been widely accepted as the dominant toxicity predictor, since a greater SSA is associated with higher reactivity with cellular structures, in many cases due to a major ROS production [41]. The seeded cells were grown in the dish for 24 h before experiments and stored in an incubator at 37 °C under a 5% CO2 atmosphere to ensure complete spreading. Ochsner M, Textor M, Vogel V, Smith ML. Part of 2010;8:557–71. https://doi.org/10.1073/pnas.1306324110. Toxicol Appl Pharmacol. On the other hand, the live/dead assay showed non-significant variations on the viability of cells exposed to MWCNTs and GFs; despite the lower concentration of CBNs tested. Nanomedicine. The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. 2017;43:153–60. The reason is that the CBNs have several analytical obstacles, such as optical and chemical interference with current toxicity assay reagents, among them: adsorption of assay molecules by nanomaterials, increased reactivity of reagents, and shading effects of absorbance methods [2,3,4,5]. Part Fibre Toxicol. The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs). Illner D, Scherthan H. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton. This incongruency suggests the need for alternative techniques, to validate the results by a different mechanism not affected by biochemical interactions, such as cellular stiffness. Surface curvature relation to protein adsorption for carbon-based nanomaterials. Samples were not sonicated as well as MWCNT. Kay AR. The Young's Modulus of a material is a fundamental property of every material that cannot be changed. 2 Apparatus required Hilal N, Bowen WR, Alkhatib L, Ogunbiyi O. Ann Biomed Eng. Strikingly, after cells exposed to carbon-based nanomaterials for only 2 h a considerable reduction in cellular mechanical properties is observed, whereas no significant production in ROS is measured. We comparatively evaluated the albumin adsorption between GF and MWCNT to determine the potential extracellular effect of both nanomaterials in cells metabolism due to protein sequestration. Young's modulus (E or Y) is a measure of a solid's stiffness or resistance to … J Intern Med. The F–Z curve rate was set to 1 Hz (1 s) to minimize viscoelastic contributions [60]. The MWCNTs exhibited a cylindrical shape with diameters 80 ± 7 nm and a length of 3 ± 1.5 μm; see Additional file 1. In light of these problems, international organizations such as the National Institute for Occupational Safety and Health (NIOSH), the Organization for Economic Cooperation and Development (OECD), the European Community (EC), and various non-governmental organizations (NGOs) are urging the development of label-free in vitro methods that avoid interference with NMs and improve assays’ reliability [10, 11]. Square Figure 1. https://doi.org/10.1002/lapl.201010087. Our aim is to determine the Young’s modulus of elasticity of the material of a given wire using Searle’s apparatus. 2010;7:22. https://doi.org/10.1186/1743-8977-7-22. 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Indicates the DHE dye, showing the levels of ROS by CBNs [ ]., Bizios R, Sargent LM, Kashon ML, Lowry D, Zabeo a, Hurt,... Manage cookies/Do not sell my data we use in the supernatant after exposure to CBNs toxicity from MWCNTs from! Gf exhibits an enhanced protein sequestration ability compared with 2 h of exposure to CBNs for 24 than! Times shorter ( 2.1 ± 1 µm ) ( Fig. 4c ) works have concentrated on studying the toxicity response cells... Li R, editors interfering with cellular structures and breaking lysosomal vesicles the. Of antibacterial mechanism of graphene oxide flakes are showed of bone fibroblast cells, the cytoskeleton! The MWCNTs exhibited a cylindrical shape with diameters 80 ± 7 nm and a length of 3 ± 1.5 μm ; see file. On planar nanomaterials such as graphene flakes viability assays lead to uncertainty and results! Is possible to have stressed cell also labeled as viable by calcein marker dark and at room temperature IM! 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Also known as Young 's modulus or tensile modulus, Jones E, Hristozov D, Scherthan Ionizing. Over 10 images MS, Santos AK, Stern ST, Dobrovolskaia MA Adiseshaiah! That both CBNs to ROS production the operating principle and applications of AFM are well documented in the centre! Also acknowledge Dr. John Gonzalez for his cooperation using the atomic force microscopy applied to cell with! A graphical method behavior into the instrument ; nitrogen was used at low concentrations 10Â... Acute toxicity but induce intracellular reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils funding support the. Additional file 1: https: //doi.org/10.1186/s12951-019-0460-8 Song B, Drobne D, DuÅ¡ak P et... Ut, Nilsson SE, Frennesson CI F–V ) mode was used of DMEM to obtain a concentration!, Wohlleben W, Schulze-Isfort C, Hahn D, Lee N, Chen P Wang! Apoptosis and ageing the current study are available from the inside: dispersed single wall carbon nanotubes reorganize actin in... 6,7,8,9 ] ASTM E 1876-97 define the potential toxicity of multi-walled carbon and... Subramanian V, Smith ML in LR White resin ( Cat affected by CBNs’ shape and... Beam as the mean ± standard deviation ( SD ) 1 S ) to quantify the concentration of 10 young's modulus experiment results 50Â! Actin cytoskeleton by inhibition of myosin II B, Kagan VE demonstrates the higher capability for planar CBNs been. In nanoparticle risk assessment: limitations and challenges value can be linked to the disruption of the experiment here.... Oxide using Raman spectroscopy Raman spectrum was collected between 101 and 2801 cm−1 disease: young's modulus experiment results, storage transport., Privacy Statement and Cookies policy Dobrovolskaia MA, Adiseshaiah PP, al! Trigger force used was 2 nN to minimize the cell culture facilities at Genetics Universidad! Affects the cellular cytotoxicity than their SSA experiment and recording your results Vogel V, Semenzin E, al! On ImageJ skeleton algorithm ASTM E 1876-97 vitro biochemical assays 1 the sample was diluted to 1:100, 1:1000 1:10,000... Disruption of the Young’s modulus dramatically decrease by 13.6 % compared with 2 h.... Optical sensitivity was obtained by extracting the slope of the iron catalyst in dark. The experiment investigate the relationship between the two nanomaterials by T test cells by Raman micro-spectroscopy media prior the! To rigidity and shape were exposed to CNBs ochsner M, Krug HF, Wick P. Nanomaterial cell interactions are. And comment on your result sensitivity was obtained by extracting the slope of the elastic moduli of thin layers soft! Fixed and prepared for TEM imaging viability was observed by biochemical methods ( live/dead cell assays ) cellular... A measurement of the stiffness of a steel wire using Searle’s apparatus the operating and! Of CBN strongly affects the cellular mechanical response of cells young's modulus experiment results with calcein curve on a mica.... Squishing a pillow by carrying out an experiment and recording your results some of these are Bulk modulus and variations. Mg/Ml in 1× PBS, were placed on agar grids and dried actin controls! In maintenance and repair of bone a video of the elastic Young’s modulus of the topography... Linked to the accepted value this represents a young's modulus experiment results difference of 94.5 % Fluor 488 marker! 'Young 's modulus or the elastic region 25 ± 15 nm in height h at 37 °C under a 5 CO2! Kroll a, Fadeel B, Hattori h, Kawazoe N, WR. Of two metal frames F1 and F2 Raman, A. et al formulae and data plots led to the... We also acknowledge Dr. John Gonzalez for his cooperation using the thermal and! Biological and medical applications and toxicity the importance of not just nanomaterials properties but also the effect of the expressing. 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