Sakai J, Li J, Subramanian KK, Mondal S, Bajrami B, Hattori H, et al. Protoc Exch. Integrating physiological regulation with stem cell and tissue homeostasis. The shape is critical for protein absorption, which may be enhanced on planar nanomaterials such as graphene flakes. Inherent characterization of nanomaterials, as well as the host response and metabolic conditions, is required to identify the relevant properties related to nanomaterials toxicity; otherwise, the results are meaningless [38, 39]. Subsequently, calcein-AM green dye (Life Technologies, Carlsbad, CA, USA) and propidium iodine (PI) (Life Technologies, Carlsbad, CA, USA) were added to stain the cells for 30 min in the dark at room temperature. After 2 h of exposure, fibroblasts exposed to GFs exhibited a fast-mechanical regulation, a 10% decrease in modulus and after 24 h, the modulus had decreased by 20%. The cells are exposed to CBN, the presence of these materials in the media induce ROS production, protein adsorption, and F-actin polymerization disruption. 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. J Cell Physiol. 2012;3:398–417. The length of the actin fibers can be used as a descriptor for the cytoskeleton disruption that explains the changes on cells Young’s modulus induced by the exposure of CBNs, at a concentration of 50 µg/mL. Is your pillow an elastic material? It is dependent upon temperature and pressure however. Oberdörster G. Safety assessment for nanotechnology and nanomedicine: concepts of nanotoxicology. https://doi.org/10.1186/s12951-019-0460-8, DOI: https://doi.org/10.1186/s12951-019-0460-8. 2006;6:1261–8. https://doi.org/10.1089/ars.2011.4271. Part Fibre Toxicol. Our experimental protein absorption results demonstrated higher adsorption rates for GFs than for MWCNTs, which led to a significant decrease of the availability of proteins for cell homoeostasis, increasing the ROS production after 24 h of exposure to 32% for cells exposed to GF and 17% for cells exposed to MWCNTs. CAS  3. CAS  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]. https://doi.org/10.1111/risa.12394. Also, we evidenced that after 2 h of exposure to GFs the cells’ volume decrease. Cells exposed to GFs resulted in shorter actin fibers after 24 h than the fibers in cells exposed to MWCNT. Braakhuis HM, Park MV, Gosens I, De Jong WH, Cassee FR. 2015;10:2423–50. In: Puleo D, Bizios R, editors. c The cell exposed to MWCNTs for 24 h; the F-actin is dispersed around the cytoplasm. They found a correlation between the filamentous actin cytoskeleton disruption and the cells’ Young’s modulus decrease on cells that had phagocytose the nanoparticles [58, 59]. To Dr. Helena Groot and Diana Narvaez for access to the cell culture facilities at Genetics Laboratory Universidad de Los Andes. Discher DE, Janmey P, Wang Y-L. Tissue cells feel and respond to the stiffness of their substrate. J Funct Biomater. Holt BD, Shams H, Horst TA, Basu S, Rape AD, Wang Y-L, et al. Results 1. CBN uptake by NIH3T3. Kay AR. 2014;276:12–24. The F-actin measurement indicated a time depending disruption as well as dimensionality relation. The conventional approaches to assess the potential cytotoxic effects of nanomaterials (NMs) mainly rely on in vitro biochemical assays. L9774, Sigma-Aldrich, USA). However, the quantitative connections related to the ROS production and the cell mechanical properties with the CBNs’ dimensionality, such as the cylindrical MWCNT with one dimension out the nanoscale against planar GF with two dimensions, remains poorly understood. Terms and Conditions, A concentration-dependent decrease of cell viability is observed. Article  Article  Especially, techniques that will monitor changes measuring intracellular properties that are needed to understand NMs fate into the cells, the interaction with the cytoskeleton components and other organelles. 2015;3:3983–92. By contrast, at longer exposure times (24 h), the cells’ metabolic stress was induced by ROS production, and cytoskeleton damage, which led to an increase in cell volume, probably as result of sodium/potassium pumps dysfunction [67]. C. How to Calculate Young’s Modulus. https://doi.org/10.1111/ropr.12129. Zimmer CC, Liu YX, Morgan JT, Yang G, Wang K-H, Kennedy IM, et al. Figure 5 shows the Young’s modulus maps and the 3D reconstruction of the cells’ topography. The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Shvedova AA, Pietroiusti A, Fadeel B, Kagan VE. 2007;45:1425–32. Clearly show your work and justify your answer. Subsequently, DHE dye was added to the cells. The elastic Young’s modulus of fibroblast cells after exposure to GFs significantly decreases by ~ 20% than the control cells, whereas cells exposed to MWCNTs decreased ~ 10%. Chem Eng Res Des. 2010;30:1634–44. J Intern Med. Nanomedicine. In both cases, no major variation on the cell viability was observed by biochemical methods (live/dead cell assays). Evaluation of cytotoxicity, biophysics and biomechanics of cells treated with functionalized hybrid nanomaterials. Mrs Wilkins shows you how to determine the Young Modulus of a metal wire.00:00 Experiment set up04:30 Reading Vernier scale05:12 Plotting graph & analysis 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]. 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]. https://doi.org/10.1007/978-94-017-8739-0_5. b The cell exposed to GF for 24 h; the F-actin integrity is compromised and is only observed somewhat organized close to the nucleus. Mesarič T, Baweja L, DraÅ¡ler B, Drobne D, Makovec D, DuÅ¡ak P, et al. The excess of ROS results in additional glutathione residues to the actin monomers during the polymerization process of F-actin [19]. 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. Young's Modulus Experiment Lab report for Youngs Modulus Experiment. 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. We used the standard force–volume AFM mode to generate topographic, and elasticity map sought to determine the cell’s stiffness (Young’s modulus) of untreated and CBN-treated (MWCNT and GF) fibroblasts after 2 and 24 h of exposure, to 50 µg/mL concentration, Fig. 5. 2010;62:1–16. Bai H, Chen P, Fang H, Lin L, Tang GQ, Mu GG, et al. Hilal N, Bowen WR, Alkhatib L, Ogunbiyi O. We found that the greater SSA of MWCNT were not a good predictor of cells toxicity by CBNs as their shape and dimensionality. We extracted the cell elasticity offline for each recorded F–Z curve by initially converting to force–indentation (F–δ) and subsequently fitting with the Hertz contact mechanics model [70]. J Am Chem Soc. Risk assessment of the carbon nanotube group. https://doi.org/10.2217/nnm.11.88. Reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils. Nat Nanotechnol. Illner D, Scherthan H. Ionizing irradiation-induced radical stress stalls live meiotic chromosome movements by altering the actin cytoskeleton. It is a measure of the stiffness of a material; however, in practice, other properties of materials, scientists and engineers are often interested in, such as yield stress, have more influence on the selection of materials for a particular purpose. In particular, shorter stress fibers are associated with reduced cell adhesion and motility [54], which are critical cellular processes for cell division and migration. The ability to adsorb proteins by planar CBNs has been extensively reported [36]. Exposure assessment approaches for engineered nanomaterials. Gourlay CW, Ayscough KR. Colony forming efficiency and microscopy analysis of multi-wall carbon nanotubes cell interaction. Young’s Modulus or Elastic Modulus or Tensile Modulus, is the measurement of mechanical properties of linear elastic solids like rods, wires, etc. After 24 h of cell culture, CBNs (MWCNTs or GFs) were added to the medium at a final concentration of 50 g/mL. Nano Lett. Nature. A protocol for in vivo detection of reactive oxygen species. Angew Chem Int Ed Engl. We used TR400PB soft microcantilevers (Olympus, Tokyo, Japan) with a nominal spring constant of 0.09 N/m, modified by attachment of a 5 µm borosilicate glass microsphere close to the tip (Novascan, Ames, IA, USA). Young's modulus (E or Y) is a measure of a solid's stiffness or resistance to … Stress is given by force over area (F/A) and strain is given by the change in length over initial length (Δ L/L). 2002;13:33–7. Therefore, cytoskeleton’s structures disruptions can be associated with cells metabolic stress. CBNs adsorption of protein is linked to their dimensionality. 1 μL of the supernatant of each sample was diluted to 1:100, 1:1000 and 1:10,000 with deionized water. J Intern Med. 2014;16:2153. https://doi.org/10.1007/s11051-013-2153-9. https://doi.org/10.2217/nnm.15.65. https://doi.org/10.1016/j.carbon.2007.03.033. This worksheet is ideal for home learning since it has an accompanying video explanation of the experiment, the analysis of results and solutions to all questions are included in the document. PubMed  Questions 1. The Young's Modulus of a material is a fundamental property of every material that cannot be changed. AC was responsible for AFM experimentation, AFM analysis, document preparation, biological experimentation. Biological interactions of graphene-family nanomaterials: an interdisciplinary review. The Hertz contact mechanics model for a rigid sphere indenting an infinite isotropic elastic half-space is. It is probably related to the water displacement outside the cells by osmotic pressure due to the high affinity of proteins such as albumin to be adsorbed by GFs increasing the oncotic pressure. Nakanishi J, Morimoto Y, Ogura I, Kobayashi N, Naya M, Ema M, et al. 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. For 20 gauge copper wire the following experimental results produced a Young's Modulus constant of 6063 MPa, or 6.063 GPa. 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 experimentally determined value for Young’s Modulus is compared to the theoretical values of Young’s Modulus for different types of wires and therefore the material of the wire is concluded from that. J Trace Elem Med Biol. J Phys Chem C. 2011;115:23323–8. We measured the actin stress fiber length using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) by segmenting the continuous line in the actin image representing stress fibers. Google ScholarÂ. 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]. Part 1: Bending of a cantilever under static loads Modulus of Elasticity Report Page 1 PURPOSE The purpose of this experiment was to determine the variation in the modulus of elasticity (E) of three (3)different classes of concrete (a normal concrete [Class S] and two high performance concretes [Class HP- London WC1R 4HQ. Dimensionality controls cytoskeleton assembly and metabolism of fibroblast cells in response to rigidity and shape. The NMs properties can also interfere with the reagents of the biochemical and optical assays leading to skewed interpretations and ambiguous results related to the NMs toxicity. Yue H, Wei W, Yue Z, Wang B, Luo N, Gao Y, et al. 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). When compared to the accepted value this represents a percent difference of 94.5%. Cytotechnology. One hundred microliters of the MWCNT suspension was added to 2 mL of DMEM (Gibco, Waltham, Massachusetts, USA) to obtain a final concentration of 50 µg/mL. Fluorescent staining of NIH3T3’s cytoskeleton: a control cell; F-actin (green) is highly organized with stress fibers extending continuously along the cells. We emphasize that the in vitro toxicity assessment is improved by considering the measurement of mechanical properties in living cells exposed to carbon-based nanomaterials, here demonstrated to overcome the interference of biochemical reagents of the current cytotoxicity kits. Authors thank Prof. James Leary for the use of the LEAP system located in the Bindley Bioscience Center at Purdue University. However, MWCNTs seems to exert more intracellular effects interfering with cellular structures and breaking lysosomal vesicles increasing the ROS production [26]. 100 µL of GF suspension was added to 2 mL of DMEM to obtain a final concentration of 50 µg/mL. Figure 1: Young’s Modulus Comparison. [59], study the effect SiO2 NP on the cell’s membrane stiffness, cells exposed to NPs evidenced a concentration-dependent decrease of cell membrane modulus. The incorporation of carbon-based nanomaterials in novel and everyday technologies requires to overcome its potential toxic effects by its beneficial properties; it is particularly true for those technologies in intimate contact with biological structures [1]. Mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress. It is more evident when the adsorbed proteins are calculated considering the SSA; in the case of MWCNT, its protein adsorption regarding the SSA is 3.04 µg/m2 while the GF had protein adsorption of 33.5 µg/m2. The TEM images of the nanomaterials were carried out only in PBS. Graphene oxide flakes (XG Sciences Inc, Lansing, MI, USA) were suspended in 1× PBS at 1 mg/mL. Evaluation of the elastic Young’s modulus and cytotoxicity variations in fibroblasts exposed to carbon-based nanomaterials. Risk Anal. https://doi.org/10.1016/j.toxlet.2010.04.018. 2006;84:282–92. All authors read and approved the final manuscript. 2010;267:89–105. This observation suggests that the GF depletes the protein availability for cell growth, faster than MWCNT; leading to metabolic stress and ROS production [32,33,34,35]. Cells were fixed with 3% formaldehyde for 30 min at room temperature. 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]. Samples were not sonicated to simulate a direct biological contact under normal NMs exposure in the environment. The LEAP images are presented for cells exposed to MWCNT and GF after 12 and 24 h measuring ROS production by DHE dye and live cells by Calcein dye. The slide with its slits is most commonly purchased from a manufacturer who provides a measured value for the slit separation distance - the d value in Young's equation. Secondary to the glutathionylation, the F-actin cytoskeleton no longer possesses straight filaments across the cytoplasm, but instead an aggregation of actin monomers. https://doi.org/10.1039/C2IB20117H. Cells were seeded in culture media overnight on 5 cm diameter Petri dishes at a concentration of 2.5 × 106 cell/mL. The data collected in the video can be used to complete a table and graph. Next, the cells were fixed using 3.7% formaldehyde for 10 min at room temperature and permeabilized in 0.2% Triton X-100 for 2 min. Local viscoelastic properties of live cells investigated using dynamic and quasi-static atomic force microscopy methods. 2010;5:e9445. https://doi.org/10.1205/cherd05053. Part Fibre Toxicol. https://doi.org/10.1016/j.jtemb.2017.01.005. J Mater Chem B. Both the wires should be supported from same rigid support. The increased production of ROS by CBNs can be linked to the biomechanical changes on fibroblast, specifically to lower Youngs modulus. Carbon nanotubes disrupt iron homeostasis and induce anemia of inflammation through inflammatory pathway as a secondary effect distant to their portal-of-entry. The concentrations of these structures change on different cells depending on their biological function. However, the introduced analysis of the length modifications in the actin networks once exposed to CBNs and their estimation of cell’s Young modulus indicates a deleterious effect on the mechanical stability of living cells that was opposite to the results of the viability assays. The modulus variations correlated with the generation of ROS and the viability assays demonstrated the weak reliability of the evaluation of cytotoxicity based only on one endpoint with traditional biochemical assays: live/dead. 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. It is a measure of the stiffness of a material that is independent of the particular sample of a substance. Altogether, the changes in mechanical properties and the length of F-actin fibers confirmed that disruption of the F-actin cytoskeleton is a major consequence of cellular toxicity. This behavior has been previously demonstrated via molecular dynamics simulations, where π–π stacking with the aromatic residues of proteins showed to be reduced in cylindrical nanostructures, limiting the adsorption [30, 31, 36, 65]. 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