Biological Effects of Clinically Relevant CoCr Nanoparticles in the Dura Mater: An Organ Culture Study
"> Graphical abstract
"> Figure 1
<p>Images of histological sections of the isolated porcine dura-mater tissue at Day 0 (<b>A</b>) and after seven days in organ culture (<b>B</b>) stained with H&E. Immunohistochemical staining of the porcine dura mater with antibodies to fibronectin (<b>C</b>,<b>D</b>), collagen I (<b>F</b>,<b>G</b>), collagen II (<b>I</b>,<b>J</b>) at Day 0 and Day 7 respectively. Isotypes controls for fibronectin (<b>E</b>), collagen I (<b>H</b>) and collagen II (<b>K</b>) antibodies. Tissue viability over a period of 7 days in organ culture as determined by MTT assay (<b>L</b>). Results are presented as OD at 570 nm per mg of wet weight of tissue. Data is presented as the mean (<span class="html-italic">n</span> = 3) ± 95% confidence limits. Data were analysed by one-way analysis of variance and individual differences between group means determined by the T-method. * indicates significant difference (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 2
<p>(<b>A</b>) Representative image of cobalt-chrome nanoparticles that were generated using a pin-on-plate tribometer. Images were captured by FEGSEM; (<b>B</b>) Size distribution of cobalt-chrome nanoparticles. The size distribution of the cobalt-chrome nanoparticles was determined from SEM images taken from different locations on the filter membrane and analysed using Image Pro-Plus imaging software; (<b>C</b>) Effects of CoCr nanoparticles on the viability of dura-mater at 0 and 7 days of culture. Porcine dura mater tissue was cultured in the absence (control) and presence of CoCr nanoparticles at an estimated dose of 5 and 50 µm<sup>3</sup> per epithelial cell and viability determined by the MTT assay. Data are expressed as the mean (<span class="html-italic">n</span> = 3) ± 95% confidence limits. The data were analysed by ANOVA which revealed no significant variation between control and CoCr-treated tissues at day 0 or day 7. Images of dura mater tissue exposed to cobalt-chrome nanoparticles at an estimated dosage of 0 (<b>D</b>), 5 (<b>E</b>) and 50 (<b>F</b>) µm<sup>3</sup> per epithelial cell for a period of 7 days and stained with H&E. Tissues sections shown are orientated so that the outer epithelial layer is closest to the top of the section.</p> "> Figure 3
<p>TEM images across the dura mater tissue that was exposed to CoCr nanoparticlesfor a period of seven days. (<b>A</b>–<b>C</b>) Images of the control dura mater from the epithelial region (<b>A</b>), inner collagen region (<b>B</b>) and the basal side of the dura mater close to arachnoid mater (<b>C</b>); (<b>D</b>–<b>F</b>) Images of the dura mater exposed to an estimated dose of 5 µm<sup>3</sup> of CoCr particles per epithelial cell, from the epithelial region (<b>D</b>), inner collagen region (<b>E</b>), and the basal side of the dura mater close to arachnoid mater (<b>F</b>); (<b>G</b>–<b>I</b>) Images of the dura mater exposed to an estimated dose of 50 µm<sup>3</sup> of CoCr particles per epithelial cell, from the epithelial region (<b>G</b>), inner collagen region (<b>H</b>), and the basal side of the dura mater close to arachnoid mater (<b>I</b>). Single continuous black arrows indicate the dural epithelial cells. Single dotted black arrow indicated the dural fibroblast cells. Black circles indicated disruption of the collagen layer.</p> "> Figure 4
<p>Pattern of cytokine and other mediator release during the exposure of porcine dura mater to CoCr nanoparticles for a period of 0–7 days. The dura mater organ culture was exposed to estimated doses of 0 (control), 5 and 50 µm<sup>3</sup> of CoCr nanoparticles per epithelial cell. The factors that were investigated were IL-8 (<b>A</b>), IL-1β (<b>B</b>), TNF-α (<b>C</b>), IL-6 (<b>D</b>), LBT-4 (leukotriene B4) (<b>E</b>), IL-33 (<b>F</b>), ECP (eosinophil chemotactic protein, eotaxin, CCL-11) (<b>G</b>), and tenascin C (<b>H</b>). Data is expressed as the mean (<span class="html-italic">n</span> = 3) ±95% confidence limits. Data were analysed by one-way analysis of variance and individual differences between group means determined by the T-method. * indicates significantly difference between control and CoCr-treated tissue (* <span class="html-italic">p</span> < 0.05).</p> "> Figure 5
<p>Images of dura mater exposed for seven days to CoCr nanoparticles and stained for the presence of matrix metalloproteinases and TIMP-1 by immunhistochemistry. Images of section of control dura mater tissue (<b>A</b>,<b>D</b>,<b>G</b>,<b>J</b>,<b>M</b>), dura mater exposed to an estimated dose of 5 µm<sup>3</sup> CoCr nanoparticles per epithelial cell (<b>B</b>,<b>E</b>,<b>H</b>,<b>K</b>,<b>N</b>) and dura mater exposed to an estimated dose of 50 µm<sup>3</sup> CoCr nanoparticles per epithelial cell (<b>C</b>,<b>F</b>,<b>I</b>,<b>L</b>,<b>O</b>). The tissues were stained for MMP-1 (<b>A</b>–<b>C</b>), MMP-3 (<b>D</b>–<b>F</b>), MMP-9 (<b>G</b>–<b>I</b>), MMP-13 (<b>J</b>–<b>L</b>), TIMP-1 (<b>M</b>–<b>O</b>).</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Histological Characterisation and Viability Assessment of Dura Mater in Organ Culture
2.2. Cobalt-Chrome Alloy Nanoparticle Generation and Characterisation
2.3. Exposure of the Porcine Dura Mater to Cobalt-Chrome Nanoparticles
2.4. Proinflammatory Cytokine Response of the Dura Mater Exposed to Cobalt-Chrome Nanoparticles
2.5. MMP and TIMP-1 Expression in Dura Mater after Exposure to Cobalt-Chrome Nanoparticles
CoCr Nanoparticle Treatment (µm3 per epithelial cell) | ||||||
---|---|---|---|---|---|---|
5 µm3 | 50 µm3 | |||||
Type of MMP/TIMP | Epithelial cells | Fibroblasts | Extracellular matrix | Epithelial cells | Fibroblasts | Extracellular matrix |
MMP-1 | ++ | + | −−− | + | + | −−− |
MMP-3 | ++ | + | + | + | +/− | −−− |
MMP-9 | +++ | ++ | ++ | +++ | ++ | +++ |
MMP-13 | + | +/− | +/− | ++ | ++ | ++ |
TIMP-1 | +++ | +/− | −−− | + | +/− | −−− |
3. Experimental Section
3.1. Aseptic Isolation of the Dura Mater from Pigs
3.2. Tissue Processing for Histology and H & E Staining
3.3. Immunohistochemistry
3.4. CoCr Nanoparticle Production and Characterisation
3.5. Maintenance and Treatment of Dura Mater with CoCr Nanoparticles
3.6. Determination of the Effects of Cobalt-Chromium Nanoparticles on Tissue Viability
3.7. Determination of the Effects of Cobalt-Chromium Nanoparticles on Cytokine and Other Mediator Secretion
3.8. Immunohistochemical Staining of the Tissue for MMPs and TIMP-1 Following Treatment with CoCr Nanoparticles
3.9. Transmission Electron Microscopy (TEM) of the Treated vs. Untreated Tissue
3.10. Statistical Analysis
4. Conclusions
Acknowledgments
Conflicts of Interest
References
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Papageorgiou, I.; Abberton, T.; Fuller, M.; Tipper, J.L.; Fisher, J.; Ingham, E. Biological Effects of Clinically Relevant CoCr Nanoparticles in the Dura Mater: An Organ Culture Study. Nanomaterials 2014, 4, 485-504. https://doi.org/10.3390/nano4020485
Papageorgiou I, Abberton T, Fuller M, Tipper JL, Fisher J, Ingham E. Biological Effects of Clinically Relevant CoCr Nanoparticles in the Dura Mater: An Organ Culture Study. Nanomaterials. 2014; 4(2):485-504. https://doi.org/10.3390/nano4020485
Chicago/Turabian StylePapageorgiou, Iraklis, Thomas Abberton, Martin Fuller, Joanne L. Tipper, John Fisher, and Eileen Ingham. 2014. "Biological Effects of Clinically Relevant CoCr Nanoparticles in the Dura Mater: An Organ Culture Study" Nanomaterials 4, no. 2: 485-504. https://doi.org/10.3390/nano4020485