BioscienceHorizons
Volume 5 2012 10.1093/biohorizons/hzs006
Research article
Varied storage conditions on the cytotoxic
potential of cobalt chrome nanoparticles when
cultured with L929 fibroblasts
Samuel James Collins*
Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
*Corresponding author: Email: [email protected]
Supervisor: Prof. Eileen Ingham, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Association of ultra-high-molecular-weight polyethylene wear with osteolysis, leading to late aseptic loosening, has resulted
in increased interest in alternative bearing prostheses. Alternative prostheses with cobalt-chrome bearing surfaces are now
used more frequently, but research is needed to determine potential long-term biological effects of cobalt-chrome wear. The
biological reactivity of cobalt-chrome particles may alter due to passivation and the storage of these particles in the laboratory; therefore, before any research can be carried out with these particles, an optimum storage protocol must be developed.
This study aimed at determining any effects of the storage medium on the biological reactivity of cobalt-chrome wear. The
viability of L929 cells was assessed following culture with clinically relevant cobalt-chrome particles stored in phosphatebuffered saline, in serum and dry at using condition the 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay.
Storage of these cobalt-chrome nanoparticles (100 and 50 µm3 cell−1) in serum resulted in a significantly greater reduction in
cell viability compared with dry stored particles at the same dose, indicating that the storage of cobalt-chrome wear influences the biological reactivity of the particles. Therefore, it is suggested that studies investigating effects of cobalt-chrome
wear particles should store them in serum prior to use in laboratory studies, these particles display the highest level of cytotoxicity and are how the particles are presented in vivo.
Keywords: cobalt-chrome wear, storage, cytotoxicity, metal hip prosthesis, cobalt-chrome nanoparticles, fibroblasts
Submitted in September 2012; accepted in September 2012
Introduction
Osteoarthritis is a painful, debilitating disease that results in
aching joints and restriction to mobility. Around 8%–15% of
the population suffer from osteoarthritis, and this figure is
expected to double by 2031 (Rat et al., 2006). Pharmacological
treatments only delay the time before the need for surgical
intervention. Replacing the arthritic joint with a prosthetic
implant has revolutionized patients’ lives. To this day, the
most popular total-hip prostheses, consists of either a ceramic
or metal femoral head articulating within an acetabular cup
of ultra-high-molecular-weight polyethylene (UHMWPE),
this system follows the low-frictional-torque principles of
Charnley and Cuic (1973). However, there have been
­ roblems associated with prostheses containing UHMWPE,
p
due to the generation of UHMWPE wear. There is considerable evidence that UHMWPE wear stimulates a macrophage
response that leads to the induction of osteolysis within the
surrounding bone. Osteolysis leads to aseptic loosening
which requires revision surgery.
With around 500 000 hip prostheses implanted worldwide (Ingham and Fisher, 2000) and a younger population
suffering from osteoarthritis, there is need for a longer-lasting
prosthesis. This has led to an increase in metal-on-metal
bearings used in joint prostheses. These bearings were
thought to produce fewer wear particles and thus minimize
associated osteolysis (Sieber, Rieker, and Kottig, 1999).
© The Author 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution,
and reproduction in any medium, provided the original work is properly cited.
1
Research article
However, due to the short time period, cobalt-chrome (CoCr)
has been in clinical use, little data have been gathered relating
to the long-term effects of CoCr wear. Cobalt-chrome wear
particles have the ability to be disseminated throughout the
body due to their nanometre size (Brown, Fisher, and Ingham,
2006). Particles have been found in the liver, spleen and
lymph nodes (Langkamer et al., 1992; Case et al., 1994), and
thus it is important to determine the effects of these particles
on a cellular level and in these systems.
Concerns have been raised about the potential of CoCr
wear from metal-on-metal prostheses to evoke cytotoxic
responses (Ingham and Fisher, 2000). Doorn et al. (1996)
reported the presence of necrobiotic tissue within periprosthetic tissue of patients with failed McKee–Farrar metal-onmetal prostheses, indicating potential cytotoxic effects of
CoCr wear. Savarino et al. (1999) reported an increase in
serum concentration of cobalt and chromium ions in patients
with failed prostheses. Patients with implants containing
CoCr components were shown by Granchi et al. (2003) to
have reduced levels of T-lymphocytes which was suggested to
be a cytotoxic effect of cobalt and chromium ions. Recent
in vitro studies have been carried out using CoCr particles of
clinical significance. Germain et al. (2003) showed a reduction in cell viability of L929 fibroblasts following treatment
with 50 and 5 µm3 CoCr particles per cell.
Brown, Fisher and Ingham (2006) proposed that cobalt and
chromium ions may lead to DNA damage and genotoxicity.
Cobalt and chromium ions are known carcinogens, and Lison
et al. (2001) have reported these ions to possess genotoxic ability, showing cobalt (II) ions to have in vitro genotoxic activity.
Doherty et al. (2001) reported an increase in DNA damage in
tissue from around metal-on-metal p
­ rostheses. The mechanism
by which chromium is able to exert its genotoxic effects are
Bioscience Horizons • Volume 5 2012
shown in Fig. 1. Chromium (VI) is known to enter the cell
(Connett and Wetterhahn, 1983; Kortenkamp, Beyersmann
and Obrien, 1987) and is reduced by internal reductants forming chromium (III) along with free radicals. Chromium (III)
and its free radicals then induce DNA damage and chromium
(III) can potentially induce mutagenesis (Singh et al., 1998).
Metal prostheses are able to undergo corrosion within the in
vivo setting. Jacobs, Gilbert and Urban (1998) reported that
the metal alloys used in the manufacturing of metal-on-metal
prostheses are able to undergo passivation. Passivation is a process where a passive layer of oxide forms over the surface of the
metal reducing the oxidation rate and consequently leading to
a reduction in corrosion. This reduction in corrosion is due to
the prevention of electron transport and also metal ions across
the oxide layer (Jacobs, Gilbert and Urban, 1998).
It has been demonstrated that around 22% and 50% of
wear in the biotribocorrosion system is associated with corrosion and the rest with mechanical mechanisms (Yan et al.,
2006). Yan et al. (2006) reported the damage to the material
consists of mechanical damage to the passive layer followed
by electrochemical repassivation.
It was reported by Germain et al. (2003) that sonication
damages the oxide layer which leads to metal ion release.
Understanding the potential biological effects of CoCr
wear involves culturing cells with the particles in vitro or testing the CoCr wear in laboratory animals. Since CoCr wear
may alter their biological reactivity as a result of passivation,
the storage of the particles and nature of the medium in which
they are presented to cells may affect their biological reactivity. Therefore, in order to determine any effects of the storage
medium on the biological reactivity of CoCr wear, the viability of L929 cells was assessed following culture with CoCr
wear stored in phosphate-buffered saline, in serum and dry.
Materials and Methods
Cell culture medium contained 500 ml Dulbecco’s Modified
Eagle Medium (Lonza), 10% (v/v) foetal calf serum (FCS,
Lonza), 2 mM l-glutamine (Lonza), 50 U ml−1 penicillin
(Lonza) and 50 U ml−1 streptomycin (Lonza). This is subsequently referred to as cell culture medium (CCM), which was
stored at 4°C until required.
Analysis of cobalt-chrome nanoparticles
Analysis of cobalt-chrome nanoparticles using Quanta 200F
FEGSEM.
Figure 1. ​The mechanism by which chromium (Cr) enters the cell. Cr6+
passes and undergoes reduction into Cr5+ which undergoes further
reduction. The reduction of Cr5+ produces Cr3+ and free radicals which
are then able to induce DNA damage.
2
Particles were sonicated and filtered onto 0.1 µm polycarbonate filters (Whatman International) before being viewed
using the Quanta 200F FEGSEM (FEI) and the images were
analysed using Image Pro Plus.
X-ray dispersive analysis (energy-dispersive X-ray) of
cobalt-chrome nanoparticles
Bioscience Horizons • Volume 5 2012
Energy-dispersive X-ray (EDX) analysis of the nanoparticles was conducted during field-emission gun-scanning electron microscope (FEGSEM) analysis.
Cobalt-chrome nanoparticle stock solution
A stock solution (1 mg ml−1) of cobalt-chrome nanoparticles
was made by Dr Chris Brown using distilled water. Both the
pin and plate used to generate the nanoparticles were made
from medical grade wrought CoCr alloy (according to ASTM
F1537) with high carbon content.
Cell culture maintenance
L929 fibroblast cell culture
L929 fibroblasts (ECACC) were cultured in CCM (10 ml) in
75 cm2 tissue culture flasks (Fisher) at 37°C. Once the fibroblasts reached 70% confluence, the CCM from the flask was
discarded. The cells were then rinsed twice with 10 ml of
Dulbecco’s Phosphate-Buffered Saline (DPBS) (without calcium and magnesium, Lonza) and treated with trypsin–EDTA
(Lonza) for 10 min at 37°C. Once the cells detached, 10 ml of
CCM was added. The cell suspension was then centrifuged
(150g) for 10 min. The percentage cell viability was determined using Trypan blue (Sigma) dye exclusion and the cells
were diluted in CCM to 1 × 105 cells ml−1. The cell suspension (10 ml) was then added to sterile 75 cm2 tissue culture
flasks and incubated at 37°C.
Culture of L929 cells at different seeding
densities over 5 days
Percentage cell viability was determined by Trypan blue dye
exclusion and L929 cells were then seeded into 96-well plates
at seeding densities of 1 × 104, 5 × 104, 1 × 105, 5 × 105 and
1 × 106 cells ml−1. Cell suspension (100 µl) was added to
6 wells; this was carried out for four plates. The plates were
then incubated at 37°C for 0, 1, 2 and 5 days before cell viability was determined using the standard 3-[4, 5-Dimethylthiazol2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay.
Culture of L929 cells with cobalt-chrome
nanoparticles
Seeding of L929 fibroblasts into 96-well plates
Percentage cell viability was determined using Trypan blue
dye exclusion. Cells were then seeded at a density of
1 × 105 cells ml−1 into each well. The cell suspension (100 µl)
was then added to sterile clear flat-bottomed 96-well plates
(Fisher) and incubated at 37°C for 24 h.
Culture of L929 cells with cobalt-chrome nanoparticles.
L929 cells were seeded into 96-well plates (see the section
‘Seeding of L929 fibroblasts into 96-well plates’) and were
cultured with CoCr nanoparticles at a volume of 500, 100,
50, 5, 0.5, 0.05 and 0.005 µm3 cell−1. The particle suspensions were made from a 1 mg ml−1 stock and were sonicated
before being cultured with the cells. The negative control was
Research article
L929 cells with 200 µl of CCM and the positive control was
­camptothecin (2 µg ml−1, Invitrogen) as this is cytotoxic and
kills all cells. Each CoCr nanoparticle dose (100 µl) and the
positive control (100 µl) were added to the corresponding
wells. Each treatment consisted of six repeats and the cells
were cultured with the particles for 0, 1, 2 and 5 days at
37°C. The cell viability was determined using the MTT assay
at day 0, 1, 2 and 5.
Culture of L929 cells with PBS, serum and dry stored
cobalt-chrome nanoparticles
The CoCr nanoparticle stock (333 µl) was pippetted into
each of three eppendorf tubes. These were centrifuged
(19 000g) for 10 min using the Microcentaur. The supernatant was then removed and each of the metal pellets were
resuspended in either 1 ml of DPBS (without calcium and
magnesium), 1 ml of FCS or left to dry. These were then
stored at room temperature for a period of 3 h. Following the
storage period, the eppendorfs (Fisher) were centrifuged
(19 000g) for 10 min and the supernatant was removed and
the pellet was resuspended in CCM to make particle suspensions of 100 µm3 CoCr nanoparticles per cell, 50 µm3 CoCr
nanoparticles per cell and 5 µm3 CoCr nanoparticles per cell
stored in DPBS, serum and dry. These particles were then cultured with L929 cells seeded into 96-well plates (see the section ‘Seeding of L929 fibroblasts into 96-well plates’). The
negative control was L929 cells with 200 µl of CCM and the
positive control was camptothecin (2 µg ml−1). Each CoCr
nanoparticle dose (100 µl) and the positive control (100 µl)
was added to the corresponding wells. Each treatment consisted of six repeats and the cells were cultured with the particles for 0, 1, 2 and 5 days at 37°C. The cell viability was
determined using the MTT assay at day 0, 1, 2 and 5.
The above was repeated using particles that were stored
for 24 h and 8 days before culturing with the L929 cells.
MTT assay
The culture supernatant was removed and the cells were resuspended twice with DPBS (without calcium and magnesium).
MTT solution (200 µl of 0.5 mg ml−1, made from 1:10 dilution
of stock MTT with CCM) was added and the plates were incubated for 3.5 h at 37°C in 5% (v/v) CO2 in air. Following the
incubation period, the MTT solution was discarded and 200 µl
of DMSO was added to each well to solubilize the formazan
crystals and left for 15 min and the absorbance at 570 nm was
read spectrophotometrically with a refrence taken at 630 nm.
The results were expressed as the mean absorbance at 570–
630 nm, ±95% confidence limits (CLs).
Statistical analysis
Data are expressed as mean ± 95% CLs. Data were analysed
by two-way analysis of variance. Individual differences
between group means were calculated by the T-method to
determine the minimum significant difference (MSD,
p < 0.05).
3
Research article
Bioscience Horizons • Volume 5 2012
Results
Characterization of cobalt-chrome nanoparticles
The CoCr particles were filtered onto a 0.1 µm pore-sized polycarbonate filter and FEGSEM images of the nanoparticles are
shown in Fig. 2. The FEGSEM image was used to determine the
size of the particles (Fig. 3). Analysis identified that the particles
had a mode size of 40–49 nm with a high frequency of particles
in the 30–39 and 50–59 nm size range. The EDX spectroscopy
was used to confirm the chemical characteristics of the suspected CoCr nanoparticles; the EDX spectrum of the nanoparticles is presented in Fig. 4. The EDX analysis identified peaks
indicating the presence of carbon, oxygen, platinum, cobalt and
Figure 4. ​EDX analysis of CoCr nanoparticles. CoCr nanoparticles were
imaged using FEGSEM and subjected to EDX analysis.
chromium within the sample. It was concluded that the
nanoparticles were CoCr (according to ASTM F1537).
Determination of the optimal seeding
density of L929 cells
Prior to investigating the effect of storage of CoCr nanoparticles on L929 cell viability, it was necessary to determine the
optimal seeding density of L929 cells.
Figure 2. ​The FEGSEM image of isolated CoCr nanoparticles. CoCr
nanoparticles were generated using a flat pin-on-plat tribometer, then
isolated onto a 0.1 µm pore-sized polycarbonate filter before being
visualized at 100.72K× magnification.
L929 cells were seeded into 96-well plates and growth was
monitored by MTT conversion over 5 days. Cells seeded at
1 × 104 cells mL−1 had a long lag phase whereas cells seeded at
5 × 105 and 1 × 106 cells mL−1 showed a small increase in MTT
conversion from day 0 to 5. Cells seeded at 5 × 104 cells mL−1
showed linear growth and cells seeded at 1 × 105 cells mL−1
showed a normal growth curve.
It was determined that the optimal seeding density of
L929 cells per well was of 1 × 105 cells mL−1. This was determined from the steady growth of L929 cells from day 0 to 2
and subsequent stationary phase seeded at 1 × 105 cells mL−1,
shown in Fig. 5.
Determination of the doses of cobaltchrome nanoparticles
Prior to investigating the effect of storage of CoCr nanoparticles on L929 cell viability, it was necessary to determine the
doses of CoCr nanoparticles that would allow the changes in
MTT conversion in either direction.
Figure 3. ​Size of cobalt chromium nanoparticles. The FEGSEM image
was used alongside Image Pro Plus® 4.5.1 to determine the length of
the nanoparticles.
4
The cells’ only control is the control where only cells are
seeded. This is to show the normal growth without any factors
that may inhibit growth. The camptothecin-positive control
showed a decrease in MTT conversion from day 0 to 5. Culture
of L929 cells with 500 and 50 µm3 CoCr nanoparticles per cell
for 0 days resulted in a significant (p < 0.05) reduction in cell
viability compared with the negative ­control. One day culture
with 500, 100, 50, 5, 0.5 and 0.05 µm3 CoCr nanoparticles
per cell resulted in a significant (p < 0.05) reduction in cell
Bioscience Horizons • Volume 5 2012
Research article
Table 1. ​Effects of CoCr nanoparticles on L929 growth
Day
Dose of CoCr nanoparticles per cell (µm3)
500
100
50
5
0.5
0.05
0
*
1
*
*
*
*
*
*
2
*
*
*
*
*
*
5
*
*
*
0.005
*
The data obtained during the growth of L929 cells in the presence of CoCr
nanoparticles was analysed by two-way analysis of variance. Individual differences between means were compared by the T-method.
*Significant (p < 0.05) reduction in MTT conversion compared with cells only
control at that time point.
Figure 5. ​Growth of L929 cells seeded at 1 × 104 (blue), 5 × 104 (red),
1 × 105 (green), 5 × 105 (purple) and 1 × 106 (black) cells ml−1 into 96well plates at a volume of 100 µl well−1 over a 5 day period. Growth
was determined by MTT conversion. Data are expressed as the mean
(n = 4) ± 95% CL. Each plate was used once, either day 0, 1, 2 or 5 to
give a mean (n = 4, 1st and 6th well ignored for top and tailing).
Figure 6. ​The effect of 0.005 µm3 (red), 0.05 µm3 (green), 0.5 µm3
(purple), 5 µm3 (blue), 50 µm3 (orange), 100 µm3 (light blue) and
500 µm3 (pink) CoCr nanoparticles per cell on the growth of L929 cells
over 5 days compared with a negative (black) and camptothecin
positive (light green) control. Growth was determined by MTT
conversion. Data are expressed as the mean (n = 6) ± 95% CL. CoCr
nanoparticles were stored in distilled water.
viability compared with the negative control. This result was
also seen following 2 days of culture. Five-day culture with
500, 100 and 50 µm3 CoCr nanoparticles per cell resulted in a
significant (p < 0.05) reduction in cell viability compared with
the negative control. These data can be seen in Fig. 6 and the
significance of these data is presented in Table 1.
Culture of L929 with cobalt-chrome
nanoparticles stored in PBS, serum or dry
It was determined to culture L929 cells with 100, 50 and
5 µm3 CoCr nanoparticles per cell stored either in PBS, serum
or dry for varying time periods.
Three-hour storage of cobalt-chrome nanoparticles
There was no significant difference seen between the cell viability after 0 days culture of L929 cells with any of the CoCr
nanoparticles. Following 1-day culture, a significant (p < 0.05)
difference was seen in cell viability between the negative control and 100 µm3 cell−1 CoCr nanoparticles stored dry but no
significant difference was recorded between the negative control and 5 µm3 cell−1 CoCr nanoparticles stored dry. This significant difference was also seen in cells treated with
50 µm3 cell−1 CoCr nanoparticles stored dry, 100 µm3 cell−1
CoCr nanoparticles stored in PBS, 100 µm3 cell−1 CoCr
nanoparticles stored in serum and 50 µm3 cell−1 CoCr
nanoparticles stored in serum. However, no significant difference was seen between the negative control and 50 µm3 or
5 µm3 cell−1 of CoCr nanoparticles stored in PBS or 5 µm3 cell−1
CoCr nanoparticles stored in serum. Two-day culture resulted
in a significant (p < 0.05) difference in cell viability between
the negative control and all CoCr nanoparticle treatments.
100 µm3 cell−1 CoCr nanoparticles stored dry, 100 µm3 cell−1
CoCr nanoparticles stored in PBS, 100 µm3 cell−1 CoCr
nanoparticles stored in serum and 50 µm3 cell−1 CoCr
nanoparticles stored in serum resulted in a significant
(p < 0.05) reduction in cell viability following 5-day culture.
However, no significant difference was seen between the negative control and 50 µm3 cell−1 CoCr nanoparticles stored dry
or in PBS or 5 µm3 cell−1 of CoCr nanoparticles stored in any
method. These data can be seen in Fig. 7, and the significance
of these data compared with the negative control are presented in Table 2.
To determine any difference between the mode of storage
and toxicity of the nanoparticles, the data for particles stored
in serum, PBS and dry were compared (Table 3). This indicated that particles (100 µm3 and 50 µm3 cell−1) stored in
serum or PBS were more toxic than dry stored particles.
Particles (100 µm3 cell−1) stored in serum were also more
toxic than PBS stored particles at 5 days.
Twenty-four-hour storage of cobalt-chrome nanoparticles
There was no significant difference seen between cell viability
after 0-day culture of L929 cells with any of the CoCr
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Research article
Bioscience Horizons • Volume 5 2012
Figure 7. ​The effect of 100 µm3 (A, top left), 50 µm3 (B, top right) and 5 µm3 (C, bottom left) per cell of CoCr nanoparticles stored in PBS (green),
serum (purple) or dry (red) for a period of 3 h on the viability of L929 cells over 5 days compared with a negative (black) and camptothecin
positive (blue) control. Absorbance data presented as mean (n = 6) ± 95% CL.
Table 2. ​Effects of CoCr nanoparticles stored in PBS, serum or dry for 3 h on L929 growth
Day
Dose of CoCr nanoparticles per cell
100 µm3 dry
50 µm3 dry
5 µm3 dry
100 µm3 PBS
50 µm3 PBS
5 µm3 PBS
100 µm3 serum
50 µm3 serum
5 µm3 serum
0
1
*
*
2
*
*
5
*
*
*
*
*
*
*
*
*
*
*
*
*
*
The data obtained during the growth of L929 cells in the presence of CoCr nanoparticles (stored for 3 h) were analysed by two-way analysis of variance. Individual
differences between means were compared by the T-method.
*Significant (p < 0.05) reduction in MTT conversion compared with cells only control at that time point.
nanoparticles. Following 1-day culture, a significant
(p < 0.05) difference was seen in cell viability between the
negative control and 5 µm3 cell−1 CoCr nanoparticles stored
dry. This significant difference was also seen in cells treated
with 5 µm3 cell−1 CoCr nanoparticles stored in PBS, but not
5 µm3 cell−1 of CoCr nanoparticles stored in serum. No significant difference was recorded between the negative control
and 100 µm3 cell−1 CoCr nanoparticles or 50 µm3 cell−1 CoCr
nanoparticles stored in any method. Two-day culture resulted
6
in a significant (p < 0.05) difference in cell viability between
the negative control and 100 µm3 cell−1 CoCr nanoparticles
stored in PBS, 50 µm3 cell−1 CoCr nanoparticles stored in
PBS, 100 µm3 cell−1 CoCr nanoparticles stored in serum and
50 µm3 cell−1 CoCr nanoparticles stored in serum. No significant difference was seen between any volume of the dry
stored particles or 5 µm3 cell−1 CoCr nanoparticles stored in
either PBS or serum. 100 µm3 cell−1 CoCr nanoparticles
stored in PBS and 100 µm3 cell−1 CoCr nanoparticles stored
Bioscience Horizons • Volume 5 2012
Research article
in serum resulted in a significant (p < 0.05) reduction in cell
viability following 5-day culture but no significant difference
was seen between the negative control and 50 µm3 cell−1
CoCr nanoparticles or 5 µm3 cell−1 CoCr nanoparticles
stored in these methods. However, there was no significant
difference recorded between the negative control and any
Table 3. ​Effects of CoCr nanoparticles stored in PBS, serum or dry for
3 h on L929 growth
Day
Serum vs. PBS
PBS vs. dry
50
5
100
50
*
*
5
100
50
*
*
*
*
5
0
1
2
5
*
*
In order to determine any difference between the mode of
storage and toxicity of the nanoparticles, the data for particles stored in serum, PBS and dry were compared (Table 5).
This indicated that particles (100 µm3 and 50 µm3 cell−1)
stored in serum or PBS were more toxic than dry stored particles.
Eight-day storage of cobalt-chrome nanoparticles
Serum vs. dry
Dose of CoCr nanoparticles per cell (µm3)
100
volume of CoCr nanoparticles stored dry. These data can be
seen in Fig. 8 and the significance of these data compared
with the negative control is presented in Table 4.
The data obtained during growth of L929 cells in the presence of CoCr nanoparticles (stored for 3 h) were analysed using two-way analysis of variance.
Individual differences between means were compared by the T-method.
*Significant (p < 0.05) decrease in MTT conversion.
There was no significant difference seen between cell viability
after 0-day culture of L929 cells with any of the CoCr
nanoparticles. Following 1-day culture, a significant
(p < 0.05) difference was seen in cell viability between the
negative control and 5 µm3 cell−1 CoCr nanoparticles stored
dry. This significant difference was also seen in cells treated
with 100 µm3 and 5 µm3 cell−1 CoCr nanoparticles stored in
PBS as well as cells treated with 50 µm3 and 5 µm3 cell−1
CoCr nanoparticles stored in serum. However, no significance was seen between the negative control and
100 µm3 cell−1 CoCr nanoparticles stored dry or in serum.
No significance was also seen between the negative control
Figure 8. ​The effect of 100 µm3 (A, top left), 50 µm3 (B, top right) and 5 µm3 (C, bottom left) per cell of CoCr nanoparticles stored in PBS (green),
serum (purple) or dry (red) for a period of 24 h on the viability of L929 cells over 5 days compared with a negative (black) and positive (blue)
control. Absorbance data are presented as mean (n = 6) ± 95% CL.
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Research article
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Bioscience Horizons • Volume 5 2012
Table 4. ​Effects of CoCr nanoparticles stored in PBS, serum or dry for 24 h on L929 growth
Day
Dose of CoCr nanoparticles per cell
100 µm3 dry
50 µm3 dry
5 µm3 dry
100 µm3 PBS
50 µm3 PBS
5 µm3 PBS
100 µm3 serum
50 µm3 serum
5 µm3 serum
0
1
**
**
2
*
5
*
*
*
*
*
Data obtained from the growth of L929 cells in the presence of CoCr nanoparticles (stored for 24 h) were analysed by two-way analysis of variance. The individual
differences between means were determined by the T-method.
*Significant (p < 0.05) reduction in MTT conversion compared with cells only control at that time point.
**Significant (p < 0.05) increase in MTT conversion compared with cells only control at that time point.
Table 5. ​Effects of CoCr nanoparticles stored in PBS, serum or dry for
24 h on L929 growth
Day
Serum vs. PBS
PBS vs. dry
Serum vs. dry
Dose of CoCr Nanoparticles per cell (µm3)
100
50
5
100
50
5
100
50
5
0
1
2
*
*
5
*
*
*
These data obtained during the growth of L929 cells with CoCr nanoparticles
(stored for 24 h) were analysed by two-way analysis of variance. Individual
differences between means were compared by the T-method.
*Significant (p < 0.05) decrease in MTT conversion.
and 50 µm3 cell−1 CoCr nanoparticles stored either dry or in
PBS. Two-day culture resulted in a significant (p < 0.05) difference in cell viability between the negative control and
100 µm3 and 50 µm3 cell−1 CoCr nanoparticles stored dry, in
PBS and in serum. No significance was recorded between the
negative control and 5 µm3 cell−1 CoCr nanoparticles stored
in any method. 50 µm3 cell−1 CoCr nanoparticles stored in
serum resulted in a significant (p < 0.05) reduction in cell
viability following 5-day culture. No significance was seen
between the negative control and any of the other nanoparticle volumes stored in any method. These data can be seen in
Fig. 9 and the significance of these data compared with the
negative control are presented in Table 6.
To determine any difference between the mode of storage
and toxicity of the nanoparticles, the data for particles stored
in serum, PBS and dry were compared (Table 7). This indicated that particles (100 µm3 and 50 µm3 cell−1) stored in
serum were more toxic than dry-stored particles. Particles
(100 µm3 cell−1) stored in serum were also more toxic than
PBS-stored particles at 1 day.
Discussion
The revival of interest in CoCr metal-on-metal prostheses
came after the association of metal-on-UHMWPE prostheses
8
with osteolysis, which leads onto late aseptic loosening
(Ingham and Fisher, 2000). Schmalzried, Jasty and Harris
(1992) demonstrated the presence of UHMWPE particles
associated with macrophages in areas of bone loss. Tipper
et al. (2000) reported that the majority of UHMWPE particles generated by the Charnley prosthesis were between 0.1
and 0.5 µm in length. In vitro studies by Green et al. (1998)
have reported an increased likelihood of phagocytosis of
UHMWPE particles (0.3–10 µm) by murine peritoneal macrophages. Activation of macrophages occurs following
phagocytosis of UHMWPE particles (Brown, Fisher and
Ingham, 2006) which leads to the release of TNF-α and IL-1β
which have been reported by Green et al. (1998) to have bone
resorption capacity. Doorn et al. (1998) reported the mean
size of CoCr particles recovered from tissue as 81 nm with
the majority of particles <50 nm in size. These particles are
therefore outside the 0.3–10 µm phagocytic range which has
led to CoCr wear having little association with osteolysis
(Brown et al., 2007). However, there has been an indication
that CoCr wear particles led to a reduction in cell viability
(Germain et al., 2003; Papageorgiou et al., 2007).The aim of
this project was therefore to determine whether varying the
storage conditions of CoCr nanoparticles altered the effect of
the CoCr nanoparticles on cell viability. Since CoCr wear
particles may alter their biological reactivity as a result of
passivation, the storage of the particles and nature of the
medium in which they are presented to cells may affect their
biological reactivity.
The use of L929 murine fibroblast cells as a model to
assess the effect of CoCr nanoparticles on cell viability was
selected since these cells have been used previously to study
the toxicity of CoCr nanoparticles (Papageorgiou et al.,
2007). Papageorgiou et al. (2007) also showed the presence
of CoCr nanoparticles within the cells using transition electron microscopy, indicating that these particles are taken into
the cell.
The CoCr nanoparticles were generated using a flat pinon-plate tribometer which produces particles similar to those
found in vivo (Leslie et al., 2008). The particles used within
this study had a mode size of 40–49 nm with particles r­ anging
Bioscience Horizons • Volume 5 2012
Research article
Figure 9. ​The effect of 100 µm3 (A, top left), 50 µm3 (B, top right) and 5 µm3 (C, bottom left) per cell of CoCr nanoparticles stored in PBS (green),
serum (purple) or dry (red) for a period of 8 days on the viability of L929 cells over 5 days compared with a negative (black) and positive (blue)
control. Absorbance data are presented as mean (n = 6) ± 95% CL.
Table 6. ​Effects of CoCr nanoparticles stored in PBS, serum or dry for 8 days on L929 growth
Day
Dose of CoCr nanoparticles per cell
100 µm3 dry
50 µm3 dry
5 µm3 dry
100 µm3 PBS
50 µm3 PBS
5 µm3 PBS
100 µm3 serum
50 µm3 serum
5 µm3 serum
0
1
2
**
*
*
*
*
**
*
5
**
*
**
*
*
The data obtained during the growth of L929 cells in the presence of CoCr nanoparticles were analysed by two-way analysis of variance. Individual differences
between means were compared by the T-method.
*Significant (p < 0.05) reduction in MTT conversion compared with cells only control at that time point.
**Significant (p < 0.05) increase in MTT conversion compared with cells only control at that time point.
in size from 30 to 79 nm. Doorn et al. (1998) reported that
CoCr wear retrieved from tissue surrounding metal-on-metal
prostheses had a range of 51–116 nm with a mean size of
81 nm. Brown et al. (2007) reported that the mean size of
CoCr particles generated in vitro was lower than 50 nm
under all conditions. From these data it can be concluded
that the CoCr wear used whilst conducting this study were
clinically relevant as the mode size of these particles fell into
the size ranges of CoCr wear particles reported both in vivo
(Doorn et al., 1998) and in vitro (Brown et al., 2007). The
size of these particles were of importance as it has been stated
by Ingham and Fisher (2000) that for studies investigating
the effects of metal wear particles in vitro to be of relevance,
the nanometer size of the particles must be considered.
EDX analysis of these particles confirmed that they were
composed of the elements such as cobalt and chromium and
therefore these particles were identified as CoCr n
­ anoparticles
9
Research article
Bioscience Horizons • Volume 5 2012
Table 7. ​Effects of CoCr nanoparticles stored in PBS, serum or dry for 8
days on L929 growth
Day
Serum vs. PBS
PBS vs. dry
Serum vs. dry
Dose of CoCr nanoparticles per cell (µm3)
100
50
5
100
50
5
100
50
5
0
1
2
5
*
*
*
*
These data were obtained during the growth of L929 cells in the presence of
CoCr nanoparticles (stored for 8 days) and were analysed by two-way analysis
of variance. Individual differences between means were compared by the
T-method.
*Significant (p < 0.05) decrease in MTT conversion.
with no contaminating factors. Similar to Brown, Fisher and
Ingham (2006), this study used endotoxin-free particles to
ensure that this factor was unable to influence the results
obtained. This approach of using endotoxin-free particles
was not undertaken by Trindade et al. (2001), which may
have influenced the results obtained as Brown, Fisher and
Ingham (2006) provided conflicting evidence using endotoxin-free particles.
The use of the MTT assay was employed to indirectly
determine cell viability at specific time points. The MTT
assay assesses mitochondrial function by the detection of
enzyme (succinate dehydrogenase) levels within the cell
(Liang and Godley, 2003). It is a cheap and effective way of
measuring cell viability which has been used in prior studies
(Papageorgiou et al., 2007).
It has previously been reported by Mahendra et al. (2009)
that second-generation metal-on-metal prostheses are associated with tissue necrosis, indicating a cytotoxic effect of
CoCr wear. It was decided to investigate whether altering
storage conditions influenced the particles cytotoxicity as it is
known that CoCr wear have altered biological reactivity as a
result of passivation.
Preliminary investigations revealed that the optimal seeding density of L929 cells was 1 × 105 cells mL−1.
Furthermore, it was revealed that CoCr nanoparticle volumes from 0.05 µm3 cell−1 up to 500 µm3 cell−1 were cytotoxic to L929 cells following 1- and 2-day culture. It has
previously been shown that CoCr nanoparticles reduce cell
viability of L929 murine fibroblasts as Brown, Fisher and
Ingham (2006) reported particle doses of 5 µm3 cell−1 or
above resulted in a significant reduction in cell viability. In
preliminary studies, a range of CoCr particle doses were
therefore tested up to 500 µm3 cell−1. From this preliminary
investigation, it was determined to investigate the effect of
storage on 100 µm3 CoCr nanoparticles per cell, 50 µm3
CoCr nanoparticles per cell and 5 µm3 CoCr nanoparticles
per cell as these dose volumes had the potential to reduce or
10
increase their cytotoxicity in response to their storage conditions.
It was demonstrated that the higher dose volumes of CoCr
nanoparticles, 50 and 100 µm3 cell−1 resulted in decreased
cell viability over the 5-day culture period, indicating the
potential cytotoxic effects of these particles. The ability of
CoCr particles to induce cytotoxic effects at these dose volumes has previously been reported (Germain et al., 2003;
Brown, Fisher and Ingham, 2006; Papageorgiou et al., 2007)
and the results obtained here were in agreement with the
published literature.
In this study, it was demonstrated, for the first time, that
storage of CoCr nanoparticles in serum led to an increase in
their in vitro toxicity when compared with those stored dry
and in some cases those stored in PBS. It is therefore plausible
that the storage of the particles may affect their biological
reactivity when cultured with cells in vitro and potentially
this effect may be seen when CoCr particles are released
in vivo due to the bodily fluids consisting of similar components as the serum. However, although a difference has been
reported between the cytotoxicity of particles stored in serum
compared with those stored in PBS, these differences are
small in magnitude and the bigger conclusion to be drawn is
that ‘wet’-stored CoCr particles are more cytotoxic than
‘dry’-stored CoCr particles. The reason for this is probably
because particles stored in dry conditions are not undergoing
corrosion and hence metal ions are not being released.
The data also suggested that a longer period of storage
resulted in a reduced toxic effect of the CoCr particles.
However, this observation requires substantiation in studies
designed to test the effects of particles stored for varying
durations in the same experiment since the design of the
experiments presented here was focused on direct comparison of the effects of storage in different conditions.
Culturing of L929 cells with CoCr nanoparticles has been
shown to cause a reduction in cell viability indicating a cytotoxic effect. This effect is greater for particles stored in serum
compared with the dry-stored particles. However, the cytotoxic effects demonstrated may be a result of either particle
internalization or release of metal ions into the medium. It
has been demonstrated by Germain et al. (2003) that cobalt
and chromium ions are released into medium and that sonication enhances the release of these metal ions. It has been
reported by Tkaczyk et al. (2010) that chromium (III) ions
are able to associate with FCS allowing diffusion across the
membrane into macrophages. It is therefore possible to suggest that the L929 murine fibroblasts in this investigation
could be functioning in a similar way. It is plausible that
metal ions have the ability to produce cytotoxic effects within
the cell and that storage of the CoCr particles in serum aided
the internalization of the metal ions released.
It cannot be determined whether the cytotoxic effects of
the CoCr particles were as a result of the particle ­internalization
or the release of ions into the surrounding medium.
Bioscience Horizons • Volume 5 2012
Although this studyis unable to determine the cause of
cytotoxicity associated with CoCr wear, it has presented evidence to support the hypothesis that the storage conditions of
CoCr wear alters biological reactivity.
Conclusion
It can be concluded from the results presented in this report
that the storage of CoCr nanoparticles in serum may lead to
an increased cytotoxic effect on L929 cells. Therefore, it
would be advisable for future experiments investigating
the effects of CoCr wear to store them in serum prior to the
investigation. This is because they have been shown to be the
most cytotoxic form but also they are presented in serum
within the in vivo setting and therefore this storage condition
would mimic this situation more accurately.
Within the clinical setting, these results have shown that
CoCr nanoparticles at doses >0.05 µm3 cell−1 can result in
significantly reduced cell viability. However, it is still
unknown whether the cause of cytotoxicity is due to particle
internalization or metal ion release. Therefore, it is advisable
that future development of metal-on-metal prostheses seeks
to reduce the rate of wear to enable a reduced loss of cell
viability.
Acknowledgements
I thank Professor Eileen Ingham and Dr Chris Brown for
their support in the project.
Funding
This work was supported by the NIHR (National Institute
for Health Research) as a part of collaboration with the
LMBRU (Leeds Musculoskeletal Biomedical Research Unit),
by EPSRC, by the Leeds Centre of Excellence in Medical
Engineering funded by the Wellcome Trust and EPSRC,
WT088908/z/09/z.
Author biography
I have recently graduated with a 1st class BSc (Hons) in
Human Physiology from the University of Leeds, where I had
a specific interest in cardiac physiology, aquaporins and associated pathology. During my studies I had an interest in
embryology, specifically congenital heart malformations and
what can lead to these and how they are treated. I will be
studying medicine at the University of Birmingham from
September 2012 and hope to pursue a career in Accident and
Emergency Medicine.
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