A comprehensive biological evaluation of ceramic nanoparticles as wear debris
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BASIC SCIENCE
Nanomedicine: Nanotechnology, Biology, and Medicine
7 (2011) 975 – 982
Research Article nanomedjournal.com
A comprehensive biological evaluation of ceramic nanoparticles as
wear debris
Y.F. Zhang, MPhila , Y.F. Zheng, PhDa,⁎, L. Qin, PhDb,c
a
State Key Laboratory for Turbulence and Complex System and Department of Advanced Materials and Nanotechnology,
College of Engineering, Peking University, Beijing, China
b
Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong
c
Translational Medicine Research & Development Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzheng, China
Received 6 October 2010; accepted 18 April 2011
Abstract
Patients are exposed internally to nanoparticles (NPs) by wear mechanisms associated with total joint arthroplasty. This tissue-specific
retention implies that the biological evaluation of NPs shall be integrative and niche targeting. Here, we report that ceramic zirconia and
silicon nitride NPs interfere with MG63 cells' function and remarkably stimulate the secretion of TNF-α in RAW264.7 cells. However,
alumina NPs significantly promote the alkaline phosphatase (ALP) activity of MG63 cells at low concentration and do not show irritation to
macrophages. In this study, we prove that ceramic materials at nanoscale are bioactive to cells. These findings also suggest that a more
rational paradigm for the biosafety evaluation of NPs than is currently in place is needed before their clinical applications.
From the Clinical Editor: In this study, the authors demonstrate that ceramic nano materials associated with normal wear-and-tear of joint
prostheses at nanoscale are bioactive to cells.
© 2011 Elsevier Inc. All rights reserved.
Key words: Ceramic nanoparticles; Wear debris; Biosafety; Orthopedics
Periprosthetic osteolysis is emerging as the major cause of be as bioinert as initially perceived.6,7 In the periprosthetic
failure in the long-term durability of total hip prosthesis.1,2 The membrane of loosened ceramic prostheses, clinicians found not
tissue membranes obtained from patients with aseptic loosening only abundant ceramic wear particles, including alumina and
provided the evidence of foreign-body reaction accompanying zirconia particles, but also Ti6Al4V alloy, and pure Ti particles
wear particles.3 Currently, the osteolysis has mainly been were found with the presence of histiocytic foreign-body
attributed to the biological responses to the repetitive exposure reaction.8,9 As with metal and polyethylene particles, ceramic
of wear particles at the bone-implant interface.4 particles might be phagocytosed by macrophages. Although some
In 1972, Boutin first used ceramic material in total hip authors considered the ceramics, especially zirconia particles, as
arthroplasty (THA) and not only attributed superior mechanical the major cause of osteolysis around ceramic implants, it remains
properties to it but also fewer wear particles, which made ceramics unclear whether the particle dose or composition determined the
attractive in orthopedics.5 Currently, the ceramic articulate tissue's biological responses around the implant.9
components have been widely applied in THA. However, Due to less accurate techniques used in periprosthetic tissue
loosened ceramic prostheses retrieved from patients associated digestion and particles' extraction, previous studies mainly
with extensive osteolysis suggests that ceramic bearings might not focused on wear particles with sizes ranging from submicron to
micron meters.10,11 With laser capture microdissection (LCM)
This work is supported by National High Technology Research and and transmission electron microscope (TEM), Ingham et al found
Development Program of China (863 Program) under grant number more tiny alumina wear particles (5–90 nm) accompanied by
2011AA030101, National Natural Science Foundation of China (No. tissue necrosis as evidenced by presence of macrophages in
51041004), and Program for New Century Excellent Talents in University
tissues around the alumina-on-alumina prosthesis.12 Considering
(NCET-07-0033).
⁎Corresponding author. Department of Advanced Materials and the fact that differently sized particles had discriminating effects
Nanotechnology, College of Engineering, Peking University, No. 5 Yi-He- on osteoblast function and proliferation,13 the particle size must
Yuan Road, Hai-Dian District, Beijing 100871, CHINA. be considered an important parameter. Moreover, it was proven
E-mail address: yfzheng@pku.edu.cn (Y.F. Zheng). that cobalt–chromium (CoCr) NPs caused more free radicals and
1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.nano.2011.04.005
Please cite this article as: Y.F. Zhang, Y.F. Zheng, L. Qin, A comprehensive biological evaluation of ceramic nanoparticles as wear debris. Nanomedicine:
NBM 2011;7:975-982, doi:10.1016/j.nano.2011.04.005976 Y.F. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 975–982
induced more DNA damage than the micron-sized particles did.14 the experimental NPs were examined with TEM microscope
Other studies also pointed out that NPs (b100 nm) had more (TEM, FEI Tecnai F30, Eindhoven, The Netherlands), and
radical interference to cells in vitro than larger ones (N100 nm) scanning electron microscope (SEM, AMRAY 1910FE, Bed-
from metal alloy, polyethylene and ceramics.14,15 ford, Massachusetts). The energy-dispersive x-ray spectroscope
In the microenvironment between bone and prosthesis, wear (EDX 9100, EDAX, Mahwah, New Jersey) was used to analyze
particles could directly interact with osteoblasts and suppress the particle composition. The crystallization of NPs was checked
osteoblastic activity. Although fibroblasts stimulated by wear by x-ray diffraction (XRD, Rigaku-D/maxrB diffractometer, FEI
particles might participate in the periprosthetic fibrogenesis and Tecnai F30) operated at 40kV and 100 mA at room temperature.
enhance the synthesis of metalloproteinases (MMPs), MMPs The characterization showed that all experimental particles
could affect the attachment and synthetic activity of osteoblasts coincided with the description of the manufacturer.
and eventually cause bone loss.16 In addition, when macro-
phages phagocytosed wear particles, they released many pro-
inflammatory and pro-osteoclastogenic cytokines, such as tumor Cells and Methods
necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 for
promoting osteoclastogenesis.17 Several studies demonstrated Cell culture
that wear particles interacted with many types of osteo-related Human osteoblast-like cell lines MG-63 (American Type
cells and impaired the well-balanced coupling between bone Culture Collection, Rockville, Maryland), mouse fibroblast cell
formation and resorption.11,13,18 Accordingly, this study was lines L929 (Cell Bank of Shanghai Institutes for Biological
designed to investigate whether the nanosized ceramic particles Sciences, Chinese Academy of Science, China) and Murine
had any negative effect on bone-remolding processes that in turn macrophagic cell lines RAW264.7 (American Type Cell Collec-
attributed to aseptic loosening. Unlike many other studies tion) were cultured in monolayer in Dulbecco modified eagle
emphasizing the cytotoxicity of NPs, we aimed to develop medium (DMEM) (GIBCO-BRL, Grand Island, New York)
protocols for systemic evaluation of NPs' biological effects in a containing 10 % fetal bovine serum (FBS) (Hyclone Laboratories,
special bio-microenvironment. Logan, Utah) and supplemented with penicillin (50 μg/ml, Gibco-
In this study, three kinds of ceramic NPs, i.e., alumina BRL) and streptomycin (50 μg/ml, Gibco-BRL) in a humidified
(Al2O3), zirconia (ZrO2), and silicon nitride (Si3N4), which are atmosphere of 95% air and 5% carbon dioxide at 37°C. Cells were
commonly used in orthopedic fields,19 were evaluated in vitro. subcultivated using 0.25% trypsin-(1 mML) EDTA (Gibco) after
Human steoblast-like cell lines' (MG63) viability and prolifer- reaching 70%–90% confluence. Cells were counted in a
ation were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- hemocytometer, and the cell viability was determined by the
diphenyltetrazolium bromide) assay. MG63 cells' function and exclusion Trypan Blue (Sigma-Aldrich) method.
macrophage RAW264.7 cells' irritation were determined For the MTT assays and analysis of the total intracellular
separately by ALP activity assay and enzyme-linked immuno- protein (TCP) content, the cells were seeded in 24-well plates
sorbent assay (ELISA) kit. In all experiments specified below, (Corning, Lowell, Massachusetts) at a density of 1.0 × 104
we used pure titanium (Ti) particles as control for comparison. cells per well in 500 μL culture medium. After 12 hours, all
kinds of experimental particles were freshly dispersed in the
Methods cell culture medium and diluted to appropriate concentrations
(5-500 μg/mL). MG-63 and L929 cells were cultured in media
Materials containing particles for 24, 48 and 72 hours. Culture media
Alumina nanopowders (40–50nm, 99.5%, Catalog #44932; without particles served as the control in each experiment.
Alfa Aesar, Inc., Ward Hill, Massachusetts) and commercially For ALP activity analysis, the MG63 cells were plated at
pure titanium particles (b20 μm, with average diameter of 4.50 50,000 cells per well in 24-well plates (Corning) in 500 μL
μm, 93%, Catalog #00681, Alfa Aesar, Inc.), silicon nitride culture medium per well (medium supplemented with 50 mg/mL
nanopowders (b50 nm, N98%, Catalog #634581), and zirconia ascorbic acid (Sigma) and 10 mM β-glycerophosphate). At 24
(IV) nanopowders (b50 nm, Catalog #544760) were purchased hours the media were replaced with experimental media
from Sigma-Aldrich (St. Louis, Missouri). The experimental NPs containing dilutions of the particle stock solutions, and the
were autoclaved at 135°C for 3 hours and then treated with 70% cells were cultured for an additional 48 hours.
ethanol for 48 hours at 20°C followed by washing in phosphate For the measurement of cytokine levels by ELISA, RAW
buffered saline (PBS) to remove endotoxin. The particles' 264.7 cells (2 × 106 cells per well in a 6-well plate) were incubated
endotoxin level was lower than 0.1 EU/mL, as determined by a for 24 hours to reach confluence, then removed the media and
commercial detection kit (Chromogenic endpoint TAL with a replaced with 2 ml experimental media containing different
Diazo coupling kit, Xiamen Houshiji, Fujian, China). concentrations of experimental particles for another 24 hours.
Separate wells were challenged with lipopolysaccharide (LPS)
Dispersion and characterization of NPs 5EU/ml (Xiamen Houshiji, Fujian, China) as positive control.
All experimental NPs were dispersed in ultrapure, filtrated Cytotoxicity
water (Millipore, S185, resistance N18.2 MΩ) and deagglomer-
ated by sonication (Shanghai Branson Ultrasonics Co. Ltd., MTT assays were used to evaluate the proliferation of MG63
Shanghai, China). The geometric size and surface morphology of and L929 cells cultured in media containing differentY.F. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 975–982 977
concentrations (5–500 μg/mL) of the evaluated experimental polyclonal antibodies specific for the mouse TNF-α. An enzyme-
particles. Mitochondrial enzymes in metabolically active cells linked polyclonal antibody specific for the mouse TNF-α was
decomposed the tetrazolium salt into a colored formazan added to the wells and left to react for 2 hours, followed by a final
product. Cells in the 24-well plates were treated with culture wash to remove any unbound antibody-enzyme reagent. The
media containing different concentrations of particles for 24, 48 intensity of the color detected at 450 nm (correction wavelength
and 72 hours. After each time point, the medium was aspirated. 570 nm) was measured after addition of a substrate solution and
The cells were gently rinsed with phosphate-buffered saline was proportional to the amount of TNF-α produced.
solution (1000 μL). Fresh serum-free medium (450 μL) was
added to each well as well as a MTT stock solution (50 μL, 5 mg/ Uptake of NPs by MG63 and their subcellular localization
mL). Cells were incubated for 2 hours at 37°C. The medium was MG63 cells were plated in 100 mm culture dish (1.0 × 107
removed, 500 μL of 5% SDS was added to each well and the cells per well) and incubated at 37°C for 24 hours. At the end of
plates incubated for a further 24 hours at 37°C. Following the incubation period, the cells were treated with experimental
incubation, 200 μL of culture medium was aspirated from each NPs (300 μg/mL) for 6 hours. Cells were observed by TEM.
well and the optical density was measured at 570 nm (correction
wavelength 630 nm). At least three independent experiments Statistical analysis
were carried out for statistical analysis.
The data were expressed as the mean ± standard deviation
Total intracellular protein content (SD). Student's t test was used for comparison, with P values of
less than 0.01 being considered significant.
At the end of 72 hours' incubation, MG63 and L929 cells were
lysed using 0.5% (v/v) Triton X-100 and twice freeze-thawed
cycles. Total protein content in the cell lysates was determined Results
spectrophotometrically using a commercially available kit (Sigma-
Aldrich) and following manufacturer's instructions. For this Cytotoxic assay analysis and decreased viability
purpose, aliquots of each protein-containing supernatant were The cytotoxic assay (MTT) on Al2O3, ZrO2 and Si3N4 NPs with
incubated with a solution of copper sulfate and bicinchoninic acid MG63 osteoblast-like cells at 24, 48 and 72 hours' incubation time
at 37°C for 30 minutes. Light absorbance of these samples was points were performed, with Ti microparticles as control (Figure 1).
measured at 570 nm (correction wavelength 630 nm). A significant dose-dependent increase was shown in the absorbance
ALP activity of Si3N4 NPs, and the ZrO2 NPs group revealed a dose-dependent
decrease from 15 ppm to 150 ppm at 24 hours. When incubation
ALP activity in the osteoblast cell layer was measured using time extended to 48 hours and 72 hours, the absorbance at all
the assay by measuring the release of p-nitrophenol from p- concentrations was close to or exceeded that of the control level. At
nitrophenylphosphate at pH 10.2 as described by Barbara et al.20 the highest dose (500 ppm) of ZrO2 NPs, the absorbance
When the cells were cultured for 48 hours with media significantly exceeded the control. In comparison with Si3N4 and
containing various dilutions of the experimental particle solutions, ZrO2 NPs, Al2O3 NPs did not show significant effect on cellular
the media was aspirated, the cells were washed three times with viability. In contrast, pure Ti microparticles showed disciplinary
PBS, and 500 μl of 0.5% (v/v) Triton X-100 was added to each dose-dependent decrease in the absorbance, and the suppression of
well. The 24-well culture plate was freeze-thawed twice, the cell cellular viability eased over the entire incubation time.
lysates were sonicated for 1 minute on ice and each well mixed with Because the increasing absorbance obtained in the presence
a Titre-tek pipettor. The cell lysate was centrifuged and a volume of of Si3N4 and ZrO2 NPs could be related to the increase in cellular
100 μl supernatant cell lysate was tested, 50% of the cell lysate was viability or the possible increase in the cell number, the total
used for determination of cell total protein content and another 50% intracellular proteins of MG63 at 72 hours were also measured as
cell lysate was used for detection of ALP activity. an index of cell proliferation (Figure 2). A slight decrease in
In addition, p-Nitrophenol was produced in the presence of intracellular protein content was found in both Ti microparticles
ALP and the absorbance was measured with a microplate reader and ZrO2 and Al2O3 NP groups (statistically significant at 150
at 405 nm (correction wavelength 630 nm). The change in the and 500 ppm), whereas it remained stable in both the Si3N4 NP
rate of absorbance was directly proportional to the ALP activity. group and the control group. Interestingly, to L929 cells (Figure
Data were normalized by the total cell protein measured with a S1in the Supplementary Materials), Si3N4 NPs could obviously
commercial kit (Sigma-Aldrich). decrease the TCP of L929 cells in a dose-dependent manner at 72
hours, but it has no effect on the TCP of MG63 cells.
Cytokine levels of macrophages Both in MTT and TCP assay, Al2O3 NPs had no detachable
The level of tumor necrosis factor-α (TNF-α) was determined adverse effect on MG63 cells and L929 cells in various
by a commercially available kit (R&D Systems, Abingdon, concentrations at any time point.
United Kingdom). The assay was performed according to the ALP activity
manufacturers' instructions. Briefly, cell culture supernatants
were collected immediately after the treatment and spun at The increase in ALP activity reflects maturation of osteoblast
13,000g for 10 minutes to remove any particulates. The medium differentiation. As shown in Figure 3, ZrO2 and Si3N4 NPs
was added to a 96-well plate precoated with affinity-purified caused notable increases (P b 0.01) in the intrinsic ALP activity978 Y.F. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 975–982
Figure 1. MTT assay showing the cytotoxicity of (A) Al2O3 NPs; (B) ZrO2 NPs; (C) Si3N4 NPs and (D) pure Ti microparticles to MG63 cells at different
concentrations. Experiments were carried out with 24, 48, 72 h incubation time with MG63 cells. *P b 0.01 in comparison with controls (n = 4).
of MG63 cells at high concentrations (especially at 500 ppm). In We also tested the TNF-α expression of RAW264.7 cells
contrast, Al2O3 NPs obviously increased ALP activity of MG63 stimulated by different sized Al2O3 particles. All sizes of Al2O3
cells at low doses (15 ppm) and decreased at high doses. particles were found to have negative effect on this assay (Figure
We also tested the effect of differently sized Al2O3 particles on S3, Supplementary Materials).
the ALP activity of MG63 cells (Figure S2 in the Supplementary
Materials). The results showed that Al2O3 (b1 μm) increased the Cellular vesicles formation and change observed by TEM
ALP activity of MG63 cells in a dose-dependent manner and
Al2O3 (b10 μm but larger N1 μm) did not have such property. TEM image of MG63 cells treated with 300 μg/mL Al2O3,
This indicated that the sizes of the particles might have different ZrO2 and Si3N4 NPs for 6 hours showed the intracellular
contributions to osteoblast function and the smallest Al2O3 vesicles containing phagocyted NP agglomerates (Figure 5).
particles were more potent to increase ALP activity of MG63 cells The cell membrane began phagocyting and NPs were
at low concentrations (15 ppm). observed in Figure 5, A. Most of the particles phagocyted
In comparison with Ti microparticles, ceramic NPs signifi- by the cell were agglomerating and included into a vesicle or
cantly promoted the ALP activity of MG63 cells at either low or lysosome; only a few NPs were free in the different sites of
high doses. the cell. It could be clearly observed that the individual and/or
agglomerates of NPs were internalizing and particles wrapped
Tumor necrosis factor-α (TNF-α) production in RAW264.7 cells in the endosome and/or lysosomal vesicles ultimately were
translocated to the perinuclear region of the cell (Figure 5, A
The production levels of TNF-α released in the supernatant of and 5, G).
the cultures at 24 hours for the various particles and two Cells that phagocyted Si3N4 and ZrO2 NPs (especially Si3N4)
concentrations are shown in Figure 4. The RAW264.7 cells showed rich mitochondria and ectatic endoplasmic reticulum
incubated with 500 ppm ZrO2, Si3N4 NPs had almost three times (ER) in cytoplasm (Figure 5, C-5, D). In the cells with
more production of TNF-α than the control. Even at a lower phagocyted Si3N4 NPs, the mitochondria were swollen, partial
concentration (100 ppm), cells challenged by ZrO2 and Si3N4 crista fragmentation and endoplasmic reticulum dilation were
NPs significantly elevated TNF-α production. also found (Figure 5, E_5, F).Y.F. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 975–982 979
Figure 2. Total intercellular protein content curve of MG63 cells treated with Figure 3. ALP activity of MG63 cells treated with Al2O3, Si3N4, ZrO2 NPs
Al2O3, Si3N4, ZrO2 NPs and pure Ti particles at different concentrations and pure Ti particles at three different concentrations during 48 h. *P b 0.01
during 72 h. *P b 0.01 in comparison with controls. in comparison with controls.
Discussion
This study demonstrated that ceramic NPs were bioactive to
cells. With MG63 cells, all ceramic NPs showed no significant
toxicity as tested up to 72 hours. The increases in absorbance
(MTT assay) observed in Si3N4 and ZrO2 NPs were not due to
the increased proliferation of cells. These results were in
agreement with that reported by Di Virgilio et al21 Furthermore,
our TEM observation showed the increasing number of
mitochondria in cells that phagocyted Si3N4 and ZrO2 NPs.
This explained the increase in absorbance observed in Si3N4 and
ZrO2 NPs as the MTT salt was mainly reduced by dehydroge-
nases in mitochondria.22 The cell respiratory burst caused by
NPs could increase the reduction of MTT salt. From the TEM
images of cells that phagocyted Si3N4 NPs, we found the
swelling mitochondria, partial crista fragmentation and endo- Figure 4. TNF-α amount of RAW264.7 cells treated with Al2O3, Si3N4, ZrO2
plasmic reticulum dilation. This implied dysfunction in cellular NPs and pure Ti particles at low and high concentrations during 24 h. *P b
0.05, **P b 0.01 in comparison with negative controls.
energy production and protein synthesis.
Referring to the report by others that CoCr NPs induced more
cell DNA damage than the larger CoCr particles,14 our findings the cytokines secreted by macrophages, TNF-α was considered
suggest that ceramic NPs were less toxic than alloy NPs were. to be the most direct and important marker for bone resorption.17
Notably, Si3N4 NPs decreased the TCP of L929 cells in a dose- In our study, Al2O3 particles of all three sizes (b100 nm, b1 μm,
dependent manner but not to MG63 cells (refer to the and b10 μm) (see Supplementary Materials Figure S3) did not
Supplementary Materials, Figure S1). This phenomenon indi- irritate RAW264.7 cells. However, noticeable irritation of
cated that the biological effect of these NPs was strongly RAW264.7 cells by Si3N4 and ZrO2 NPs was observed, and
dependent on the cell types, and the discriminating effects might these activities were found stronger than that of Ti microparti-
inhibit the formation of fibrous tissue around the prosthesis. cles. This result indicated that ZrO2 and Si3N4 NPs might be
With increase in concentrations of Si3N4 and ZrO2 NPs, the more irritating to macrophages than Ti microparticles. The
ALP activity of MG63 cells increased. However, Al2O3 NPs chemical composition of NPs might play an important role in cell
already significantly increased the ALP activity of MG63 cells and tissue response.
at low concentration (15 ppm). We also observed that the ALP We proved that ceramic NPs, especially ZrO2 and Si3N4 NPs,
activity of MG63 cells treated with Ti microparticles was lower were bioactive to osteoblast and macrophage-like cells. This
than that treated by ceramic NPs. This observation might concerned the application of these two kinds of ceramic materials
indicate the superiority of ceramics in osteogenesis in in orthopedics. As Al2O3 NPs did not show significant irritating
comparison with metals. effect on osteoblast-like cells and macrophage-like cells, the
Ingham et al found that the particles with different sizes biological reaction observed in the ceramic periprosthetic
(nanometers to several hundred micrometers) appeared in all membrane might be induced by particles of other compositions,
kinds of implant bearings in vivo.12,23 The smaller ultra-high such as polyethylene and metal alloy.
molecular weight polyethylene (UHMWPE) particles (0.1– The internalization of the NPs was an energy-dependent
1.0 μm) were more reactive than particles above the 1 μm. Of process and controlled by multiple mechanisms, including980 Y.F. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 975–982
Y.F. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 975–982 981
24
clathrin-caveolae-mediated endocytosis and macropinocytosis. because alumina might remain as one of good options for
A large number of nanoparticles' internalization into cell needed orthopedic applications, the potential concerns regarding in vivo
sufficient energy and proteins and it would facilitate the cells to applications of silicon nitride and zirconia nanomaterials should
enhance the function of mitochondria and ribosome. The be addressed experimentally.
swelling mitochondria, partial crista fragmentation and endo-
plasmic reticulum dilation observed during this study (see Figure
5, C-D) might be explained by a mechanism known as
“supercompensation.” Considering that NPs themselves did not Appendix A. Supplementary data
have biomolecule surface structures, we would speculate that
NPs might promote the ALP synthesis in MG63 cells in an Supplementary data associated with this article can be found,
indirect way. In a recent work by Bhabra et al, CoCr NPs were in the online version, at doi:10.1016/j.nano.2011.04.005.
found to be able to cause DNA damage without significant cell
death across a cellular barrier, and the damage was mediated by a
novel mechanism involving adenosine triphosphate (ATP) References
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Figure 5. (A) TEM images of MG63 cells at 37°C (incubation with Al2O3 NPs for 6 h), Arrows pointing to the process of internalization at the surface associated
with actin rearrangement near the plasma membrane and extension into the extracellular space; (B) TEM images of MG63 cells at 37°C (incubation with ZrO2
NPs for 6 h); (C) TEM images of MG63 cells at 37°C (incubation with Si3N4 NPs for 6 h); (D) TEM images of MG63 cells at 37°C (incubation with ZrO2 NPs
for 6 h), bold arrow pointing to NPs, thin arrows pointing to an increasing number of mitochondria and endoplasmic reticulum (ER); (E) TEM images of MG63
cells at 37°C (incubation with Si3N4 NPs for 6 h), Arrows pointing to swelling mitochondria with partial crista fragmentation and endoplasmic reticulum (ER)
dilation; (F) TEM images of MG63 cells at 37°C (incubation with Si3N4 NPs for 6 h), thin arrows pointing to endoplasmic reticulum (ER) dilation; (G) TEM
images of MG63 cells at 37°C (incubation with NPs for 6 h), arrows pointing to the process of internalization at the surface associated with actin rearrangement
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