CYTOTOXICITY OF METAL IONS RELEASED BY AN ALUMINIUM
BRONZE DENTAL MATERIAL.
SYNERGIC EFFECT OF THE MIXTURE OF IONS.
Claudia Grillo1, María L. Morales2, María V. Mirífico 1,2*,
Mónica Fernández Lorenzo de Mele 1,2*
1
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA, CCT La Plata-CONICET),
Facultad de Ciencias Exactas, Departamento de Química,
Universidad Nacional de La Plata, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina
2
Facultad de Ingeniería, Áreas Departamentales Ingeniería Química y Mecánica,
Universidad Nacional de La Plata, Calle 47 y 1, 1900 La Plata, Argentina
E-mail: mirifi@inifta.unlp.edu.ar - mmele@inifta.unlp.edu.ar
Abstract. Aluminium bronzes have appeared as economical substitutes of
conventional gold rich alloys to fabricate crowns and bridges. However, a very low
corrosion resistance and possible cytotoxic effects on surrounding cells were reported.
The aim of this work is to study the dissolution of an aluminium bronze and the
cytotoxic effects of the ions released on osteoblastic cell line. Conventional
electrochemical techniques and atomic absorption spectroscopy to obtain the average
concentration (AC) of ions were employed to study the corrosion process. The
cytotoxicity was evaluated by a) ions released by the metal alloy immersed in the cell
culture and b) salts of the metal ions. Results showed that the AC of the metal ions
released can not be predicted on the basis of the overall composition of the alloy.
Cytotoxic effects in cells of the vicinity of the metal were observed. Importantly,
synergistic effects were found when Al-Zn ion combinations and mixtures of the ions
at 9 x AC concentrations were employed. These results were interpreted considering
the synergistic effects and a diffusion controlled mechanism that, in the metal
surroundings, yields to concentration levels several times higher than the AC value.
Keywords: Cytotoxicity, Metal ion, Dental alloy, Metallic salt
1.
INTRODUCTION
Despite expressed doubts as to their suitability (Stoffers et al, 1987) copper base
alloys have been used as dental casting alloys for fixed prosthesis in the U.S., Japan,
South America and some countries of Eastern Europe for more than 25 years. (Tibballs
and Erimescu, 2006; Eschler et al, 2003) According to dental technicians and dental
suppliers their popularity is due to their good mechanical strength, high elastic modulus,
low density, its bright yellow color with strong resemblance gold and its low cost
relative to noble metal alloys. (Ardlin et al, 2009) Clinical studies have shown that
corrosion of crowns and posts of such alloys causes staining and inflammatory
processes in the surrounding gum tissue and blue-green pigment in the roots of teeth.
(Arvidson et al, 1980; McGuiness et al, 1987) Furthermore, high levels of toxic
elements were found after static immersion testing in cell cultures (Ardlin et al, 2009).
Thus, the widespread use of copper based alloys is a cause of great concern due
to the undesirable effects found on surrounding cells by metals ions released from these
alloys (Cu, Ni, Zn, Al). (Tibballs and Erimescu, 2006; Ardlin et al, 2009; Arvidson et
al, 1980; Arvidson and Wróblewski, 1978).
Biocompatibility of dental alloys is strongly related to their metal ions release.
(Elshahawy et al, 2009). The biological effects of these metal ions are significantly
different and depend of several factors such as oral environment, exposure time, nature
and amount of release metal cations (not generally proportional to the abundance of
each metal in the alloy), etc. (Schmalz et al, 1998)
Different sources of ions have been employed in cytotoxic studies: a) the
extracts obtained from the dissolution of a metallic sample ex situ and then added to the
cell culture (extract contact: ExtC) (Wataha et al, 2001; Granchi et al, 1998;
Bumgardner et al, 2002); b) the dissolution of metal samples in situ (direct contact: DC)
(Wataha et al, 1992; Bumgardner and Lucas, 1995; Grill et al, 2000; Grill et al, 1997;
Kapanen et al, 2002; Kapanen et al, 2001); c) the metal salts added to the cell culture
(ES). (Huk et al, 2004; Fleury et al, 2006; Craig and Hanks, 1990; Merrit et al, 1984;
Schmalz et al, 1998; Wataha et al, 2000; Wataha et al, 2002).
One of the copper based alloys widely employed is an aluminium-bronze
commercially available as Ventura Orcast PLUS®. It is featured to be used for fixed
dental prosthesis like crowns and bridges covered with resins but it is also employed in
endodontical posts.
The aim of this work is to study the cytotoxic effects of the ions released by the
Orcast PLUS® casting alloy as a response to the increasing demand of control of dental
materials. The highly toxic environment provoked by the release of ions is also a
suitable area of study to assess an additional purpose: to elucidate the cause of the
different behaviour of cells in relation to a) ions released by a metal alloy immersed in
the cell culture (in situ) and b) the presence of equivalent amounts of salts of the metal
ions added to the cell culture. For this reason, the effect of a disc of the copper based
dental casting alloy on the surrounding cells and the variation of the cytotoxic effect
with distance between cells and the source of ions were assessed in this work.
Conventional electrochemical techniques were employed to study the corrosion process
and the concentration of the ions released was measured by atomic absorption
spectroscopy. The cytotoxic effect on osteoblastic cells of individual ions, some
combinations of two ions and the mixtures of all the ions of the alloy components were
also analyzed to investigate possible synergic effects.
2.
MATERIALS AND METHODS
2.1.
Cells culture
Rat osteosarcoma derived cells (UMR 106 line) was originally obtained from
American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were grown
as monolayer in Falcon T-25 flasks with 10 ml D-MEM culture medium (GIBCO-BRL,
Los Angeles, USA) supplemented with 10% inactivated fetal calf serum (Natocor,
Carlos Paz, Córdoba, Argentina), 50 IU/ml penicillin and 50μg/ml streptomycin sulfate
(complete culture medium) at 37º C in a 5% CO2 humid atmosphere. Cells were
counted in an improved Neubauer haemocytometer and viability was determined by the
exclusion Trypan Blue (Sigma, St. Louis, MO, USA) method; in all cases viability was
higher than 95%.
2.2.
Copper based alloy and metal ions released
The copper based alloy (CuBA) used in the assays was Orcast PLUS ® (Cu
81.5%, Al 7%, Ní 4.5%, Fe 3%, Mn 2% and Zn 2%) (Madespa S.A., Toledo, Spain).
Cylindrical casting copper and CuBA electrodes, casting CuBA discs for
experiments with cells and casting CuBA samples for atomic absorption spectroscopy
were made by lost wax casting process (Macchi, 2000). With this objective a wax
pattern with the suitable shapes for each purpose was formed.
To assess the metal ions release square shape sheets of the casting copper alloy
of ca. 49 cm2 of geometrical area were immersed in synthetic saliva (SS: NaCl 0.40 gr/l,
KCl 0.40 gr/l, CaCl2 0.80 gr/l, NaH2PO4 0.16 g/l, urea 1.00 g/l, KSCN 0.16 g/l; pH =
4.77) (200 mL) for 24 h, at 37o C. After this period, the metal ions concentration was
measured by flame atomic absorption spectrophotometry. Each measurement was
repeated three times in independent experiments.
2.3.
Corrosion tests
The electrochemical experiments were performed in a conventional undivided
gastight glass cell with dry nitrogen gas inlet and outlet. The working electrode was an
Orcast PLUS® bar encapsulated in Teflon, with an exposed geometrical circular area of
0.1256 cm2, the counter-electrode was a 2 cm2 Pt foil and as reference a saturated
calomel electrode (sce) (to which all potentials reported are referred) was used.
A computer controlled PAR 273A potentiostat was employed for experiments.
The potentiodynamic measures were performed in nitrogen deareated SS (20 mL) and in
nitrogen deareated culture medium (D-MEM, pH=7) (20 mL).
Prior to each electrochemical measurement, the working electrodes were
prepared according to previous reports (Grillo et al, 2009). Potentiodynamic
polarization curves of CuBA and copper electrodes exposed to the synthetic saliva were
obtained in the conventional way. Potentiodynamic scans were made at 5 mVs-1 sweep
rate (v) and started at -0.580 V in the anodic direction to +0.050 V. Prior to each
measurement the electrode was subjected to a cathodic pre-treatment by holding it
potentiostatically at -0.580 V for 60 s, to reduce oxide film possibly formed in air. Each
potentiodynamic experiment was repeated at least three times to check the
reproducibility and the polarization curves were repetitive.
2.4
Metal ion solutions for cytotoxicity assays
Cytotoxicity tests caused by the metal ions were made using the corresponding
salts dissolved in the culture medium. The concentration of the salts corresponds to the
average concentration level reached after 8h of immersion in saliva (AC8h), measured
by atomic absorption spectroscopy. This AC8h value was calculated based on the
atomic absorption spectroscopy analysis and assuming that a sample of 3 cm2 of
geometric area was exposed to 1 mL of synthetic saliva during 8 h (sleeping period).
Concentrations corresponding to 15, 30 and 60 times higher than this minimum level
were also assayed.
The evaluation of cytotoxicity of metal ions was made by exposures to solutions
of different concentrations of the corresponding metal salts obtained from Merck
Chemical Co. (Darmstadt, Germany). The metal salts included, copper (CuCl2.6 H2O),
aluminum (AlCl3), nickel (NiCl2.6H2O), iron (FeCl2. 6 H2O) manganese (MnCl2.4H2O)
and zinc (ZnCl2) salts.
2.5
Evaluation of the effect of metal ions released from the aluminium bronze
by Acridine Orange staining
For this set of experiments 4.5 x 104 cells were seeded in Petri dish (100 mm
diameter) and grown at 37ºC in 5% CO2 humid atmosphere in complete culture medium
(D-MEM, pH=7), for 24 h. Then, the medium was removed, a 4 mm diameter CuBA
alloy disc was added in the center of each Petri dish, and immediately fresh medium
was incorporated. Cells were grown under these conditions during different periods: 3,
24 and 48 h. UMR 106 cell culture without the alloy disc were used as negative
controls. To facilitate the analysis of cytotoxic effects as a function of the distance from
the source of ions, the area with cells was divided in regions A1, A2, B and C according
to Fig. 1. After exposure periods, adherent cells were stained with Acridine Orange dye
(Sigma, St Louis, MO, USA) and immediately after, they were examined by
fluorescence microscopy (Olympus BX51, Olympus Corp., Tokyo, Japan) equipped
with appropriated filter, connected to an Olympus DP71 (Olympus Corp., Tokyo,
Japan) color video camera. The images were taken immediately after opening the
microscope shutter to the computer monitor.
Fig. 1. Scheme of the regions of the Petri dish and the disc of Orcast PLUS® copper
based alloy in the center. The radii (r) of the regions are: rA1 = 0.9 cm; rA2 = 1.4 cm;
rB = 2.4 cm; rC = 3.4 cm.
2.6.
Evaluation of the colony formation (CF)
Colony formation or clonogenic assay is an in vitro cell survival assay based on
the ability of a single cell to grow into a colony (Franken et al, 2006). For this analysis
50 cells/Petri dish were grown at 37° C in 5% CO2 humid atmosphere in complete
culture medium in presence of an alloy disc in the center of each place. An additional
cell culture without alloy disc was used as negative control. After incubation for 7 days
the colonies of acceptable size were taken into account for scoring. They were fixed
with methanol: acetic acid (3:1) and stained with Acridine orange. The enumeration and
classification of colonies (cell clusters), was made under fluorescence microscopy with
a 40x objective (Olympus BX51, Olympus Corp., Tokyo, Japan). Two experiments
were performed in independent trials to assess reproducibility.
2.7.
Determination of cytotoxicity of metal ions by Neutral Red assay
Metals ions cytotoxicity was estimated in UMR-106 cells by
Neutral Red (NR) assay (Borenfreud and Puerner, 1984). This assay measures cellular
transport based on the dye uptake by living cells. Absorbance change is directly
proportional to the number of viable cells.
For this analysis 2.7 x 103 cells/well were cultured in 96 multi-well plate in
complete culture medium for 4 h. Then, the cell were grown in presence of the different
metal ions concentrations during 24 h. The NR uptake assay was performed according
to previous reports (Grillo et al, 2009). Ethanol (7%) was used as positive control.
Cytotoxicity percentage was calculated as [(A–B) /A] × 100, where A and B are the
absorbance of control and treated cells, respectively. Each experiment was repeated in
two independent assays every one including 16 wells, that is 32 wells for each
concentration tested. Data were analyzed using one-way ANOVA test and multiple
comparisons were made using p values corrected using the Bonferroni method.
3.
RESULTS
3.1.
Corrosion test
Potentiodynamic polarization curves for CuBA immersed in synthetic saliva
solution (SS, pH = 4.77) and in cell culture medium (CCM, pH = 7.0) (Fig. 2a) showed
similar electrochemical response immediately after the immersion, in the potential zone
that includes the open circuit potentials (-0.250 V) and up to ca. -0.050 V.
Fig. 2a. Typical potentiodynamic polarization curves for Orcast PLUS® copper based
alloy immediately after immersion in deaerated (——) synthetic saliva solution (SS, pH
= 4.77) and (— •) culture medium (CCM, pH = 7.0), at 37° C. Potentiodynamic
polarization curve for copper, the metal base of alloy, is included for comparison: (- - -):
SS, pH = 4.77, (• • •): CCM, pH = 7.0.
When the electrode had previously been immersed in SS (Fig. 2b) and CCM
(Fig. 2c), and then the polarization curves were recorded, corrosion current markedly
diminished in both media indicating that the dissolution process decreases with time,
after an initial strong electrodissolution process.
Fig. 2b. Typical potentiodynamic polarization curves for Orcast PLUS® copper based
alloy measured (——) immediately, after (- - -) 30 min, and (•••) 60 and 1080 min of
immersion in deareated and quiet artificial saliva, at 37° C.
Fig. 2c. Typical potentiodynamic polarization curves for Orcast PLUS ® copper based
alloy measured (——) immediately, after (— —) 60 min, (- - - -) 180 min, and (•••••••)
1440 min of immersion in deaerated and quiet culture medium, at 37° C.
With the aim of comparison, polarization curves measured with pure copper
were also included in Fig. 2a. In this case current intensity values corresponding to SS
(pH = 4.77) are lower than those of CCM (pH = 7.0) revealing a marked effect of pH
and organic substances present in CCM on copper dissolution. In the case of CuBA the
electrochemical response is more complex and the influence of the medium composition
is not so evident.
3.2.
Effects metal ions released by CuBA on the number of living cells as a
function of the distance from the metal
The influence of the distance from the source of metal ions on cell viability was
measured by epifluorescence microscopy after Acridine Orange staining. These assays
showed (gray bars, Fig. 3) that after 3 h exposure those cells which were close to the
metal surface (Region A1) were severely altered (p < 0.001). For longer distances
(regions A2, B, C) a slight decrease in the number of living cells to 85% of the control
value was detected indicating that they were less affected by the metal ions released by
the copper alloy.
Fig. 3. Viability of cells by of the Acridine Orange staining after 3, 24 and 48 h
exposure to CuBA. Variation with the distance from CuBA disc. Regions A1, A2, B
and C according to Fig. 1.
3.3.
Effect of metal ions released by CuBA on the number living cells as a
function of exposure time
The effect of possible high local concentration of ions in the vicinity of the
metallic alloy was also investigated after exposures to the metal for complete growth
periods. In this case both, cell duplication and the accumulation of ions released by the
biomaterial occur simultaneously.
After 24 h and 48 h, epifluorescence microscopy images revealed that the
deleterious effect on the growing cells is again more notorious in region A1 (Fig. 3)
where strong reductions to 30 and 5% in the number of cells related to the control value
were detected, respectively. Interestingly, higher number of cells than in the case of 3 h
assay was found in the zones A2, B, C after 24 h (24 h data, Fig. 3), indicating that cells
were able to duplicate during this period. Conversely, new young cells were severely
altered by the metal ions during the following growth period (48 h data, Fig. 3). This
resulted in a marked decrease (close to 60% of the control value) of the number of cells
after a 48 h period in regions A2, B, C.
3.4.
Effect of metal ions released by CuBA on colony forming units
Colony forming units assays showed that, in the vicinity of the alloy (region
A1), after 7 days of exposure to the metal ions released from the alloy disc, a drastic
decrease in the number of colonies was observed with respect to the control experiment
without the metal (Table 1). Additionally, microscopic observations revealed that the
average diameters of the colonies corresponding to the control and those grown with
CuBA were 112.21 ± 5.45 μm and 80.83 ± 4.95 μm. This result indicates an important
effect of the ions released by CuBA on the size and number of the colonies. In Table 1
colonies with diameters in the 95 μm to 150 μm were considered as large colonies while
those with shorter diameters as small colonies.
Type of colony
Small colonies
Large colonies
Total
% Disc surroundings
(A1 region)
10.46 (0.40)
6.23 (0.71)
16.69 (1.11)
% Out of A1region*
59.56 (1.21)
23.75 (0.81)
83.31 (2.02)
*
Standard Errors are between brackets
Table 1. Enumeration of colonies within region A1 and out of A1 according to their
sizes after 7 days of exposure to the metal disc.
3.5.
Measurements of the amount of ions released from the metal surface
Results presented in Fig. 3 and Table 1 show a notorious temporal and spatial
dependent effect of the mixture of metal ions released by the copper alloy on the cell
growth. The different components of the alloy (Cu, Al, Ni, Fe, Mn, Zn) may be
involved in this deleterious action. In order to quantify the metal ions released by the
copper alloy, atomic absorption measurements were performed (Table 2).
Interestingly, the release of a particular element showed in Table 2 is not related
with its atomic percent in the alloy. Thus, the % of copper and aluminium in the alloy
are 81.5%, and 7%, respectively, however a lower level of copper ions than aluminium
ions (0.175 and 9.797 μg/cm2, respectively) was detected in the synthetic saliva where
the CuBA was immersed during 24 h (Table 2).
Metal ion
Copper
Aluminium
Nickel
Iron
Manganese
Zinc
Concentration of the metal ion released by
CuBA in synthetic salivaa
mg/L
µg/cm2
0.041 ± 0.014
0.175 ± 0.068
2.384 ± 1.958
9.797 ± 7.923
0.088 ± 0.015
0.376 ± 0.078
0.113 ± 0.076
0.493 ± 0.341
0.071 ± 0.015
0.306 ± 0.078
0.102 ± 0.022
0.439 ± 0.112
Composition of
CuBA (%w/w)
81.5
7.0
4.5
3.0
2.0
2.0
a
Detection limits (mg/L): Cu=0.005; Al=0.024; Ni=0.006; Fe=0.006; Mn=0.008; Zn=0.007
Table 2. Concentration of the metal ions released by CuBA in synthetic saliva, after 24
h of immersion at 37° C.
3.6.
Cytotoxicity of metal ions by Neutral Red assays: Effect of the ions from
metal salts
Lysosomal activity (Neutral Red assay) in UMR-106 cell line after treatments
with the ions of each of the alloy components was assayed.
For these experiments the minimum concentration tested (AC8h) for each ion
was proportional to the average concentration measured by atomic absorption
spectrophotometry, referred to a 3 cm2 dental alloy (as a source of metal ions release)
that was exposed to 1 mL of saliva during 8 h (sleeping period). Assays with AC8h
concentration with single ions did not show any decrease in the lysosomal activity.
Similarly, the mixture with all the metal ions did not show any effect. It could be
inferred that AC8h of both, individual ions or the total mixture, is below the cytotoxic
threshold value and consequently does not affect the cellular activity.
However, these results are in disagree with the reduction in the number of living
cells that were observed in region A1, close to the metal, shown in Fig. 3. Probably the
concentration of ions in the region A1 may be several times higher than that of the bulk.
In addition, experiments using metal ions levels several times higher than AC8h
were performed. The results show that no significant effects were found for single Mn,
Fe, Ni, Cu and Zn ions, when the concentrations employed were 30 times higher than
the average (30 x AC8h). In the case of Al, the solubility limit impeded the use of
concentrations higher than 9x AC8h
Experiments with higher concentrations (60 x AC8h) only showed weak
deleterious action for Mn and strong for Zn (p < 0.001) (Fig. 4). A slight increase in
lysosomal activity was found in the case of Fe.
140
120
% Control
100
80
60
40
20
0
Control
Mn
Fe
Ni
Cu
Zn
Control +
Metal Ions
Fig. 4. Cytotoxicity of metal ions at 60 fold minimum concentration tested (60 x AC8h)
by Neutral Red assay.
In case of Zn ions, Fig. 5 shows that when different concentrations between 9 x
AC8h and 60 x AC8h were assayed only concentrations ≥ 36 x AC8h markedly affect
the lysosomal activity of cells.
Fig. 5. Effect of Zn ions on the lysosomal activity of cells at concentrations in the 9 x to
60 x AC8h.
In the case of Al ions a slight reduction in lysosomal activity, ca. 80 - 90 % of
the control, in Al 4.5 - 7.5 x AC8h concentration range was detected but a reduction to
ca. 40% was observed for 9 x AC8h (Fig. 6).
Fig. 6. Effect of Al ions on the lysosomal activity of cells at concentrations in the 4.5 x
(Al4.5) to 9 x AC8h (Al9) range.
3. 7.
Synergistic effect of mixtures of ions
In Table 3 the effect of the single Zn and Al ions and their combinations are
compared. When Al-Zn combination was used (Fig. 7) the reduction in the lysosomal
activity was found even in the case of mixtures Al-Zn of 6 x AC8h (90%). It worth
mentioning that 6 x AC8h is below the cytotoxic level, when single ions are used (Figs.
5 and 6). In the case of 9 x AC8h Al-Zn mixtures values close to 50 % of the control
were found (Fig. 7). Interestingly, single ions at 7.5 – 15 x AC8h range did not show
any cytotoxic effect. However the mixture of all the ions at 9x AC8h concentrations
showed a reduction in the lysosomal activity to ca. 20 % of the control value (Table 3).
% Lysosomal activity after exposure to different ions*
Ions
(Al)
(Zn)
(Mixture Al-Zn)
(Mixture of all
the ions)
-----95.21 (0.65)
100.35 (1.58)
87.14 (4.97)
67.43 (2.25)
49.13 (1.98)
------------20.69 (1.63)
Concentration
94.32 (3.07)
6xAC8h
84.08 (4.01)
7.5xAC8h
44.59 (5.75)
9Xac8h
*
Standard Errors are between brackets
Table 3. Lysosomal activity of cells after exposure to single Zn and Al ions and their
combinations.
Fig. 7. Effect of Al and Zn ions on the lysosomal activity of cells.
4.
DISCUSSION
4.1
Aluminium bronzes as dental materials
Aluminium bronzes have appeared as economical substitutes of conventional
gold rich alloys to fabricate crowns and bridges. (Eschler et al, 2003; Carvalho and
Matson 1990) However, previous reports have shown a very low corrosion resistance
and possible cytotoxic effects on surrounding cells. (Ardlin et al, 2009) Consequently,
the widespread use of these dental alloys is a cause of great concern.
Like other alloys, the corrosion rate of aluminium bronzes is dependent on their
composition. However, it is well known that an alloy does not necessarily release
elements in proportion to its composition. (Wataha, 2000; Tibballs and Erimescu, 2006)
The difference in the corrosion rate of each component according to the composition of
the copper-based alloys (Cu, Al, Mn, Fe and Ni in case of Orcast and NPG alloys) as
well as the influence of the electrolyte composition (Elshahawy et al, 2009; Tibballs
and Erimescu, 2006; Ardlin et al, 2009) yielding to different cytotoxic response have
been previously shown. (Bumgardner et al, 2002; Ardlin et al, 2009) In agreement with
previous results our data show that in the case of Orcast alloy the higher ion release was
found for Al.
As it was expected, our electrochemical data showed that the dissolution rate of
CuBA is different from that of pure copper, being dependent on the pH and composition
of the electrolyte (SS or CCM). Additionally the anodic current intensities were also
dependent on the exposure period: immediately after the immersion current intensities
were higher than after the different immersion periods in the electrolyte.
In agreement with clinical tests (Arvidson et al, 1980; Arvidson and
Wróblewski, 1978) the high initial release rate of metal ions from CuBA resulted in
cytotoxic effects on the surrounding cells. These results seem to disagree with data that
show no cytotoxic effect when the cells were exposed to single ions (except for Al) at
concentrations in the AC8h to 30 x AC8h range. These data could be interpreted on the
basis of a process controlled by the diffusion of ions. The study of the distribution of
metal ion concentration in the different zones in order to describe and simulate
qualitative the space/time variation of ion concentration is being carried out in our
laboratory.
4.2.
Synergistic cytotoxic effects of the combination of metal ions
When the cytotoxicity of single ions were tested, Al cations showed a high
cytotoxic effect for 9 x AC8h. A similar effect was shown in case of Zn when 36 x
AC8h. Cytotoxic effects of Al ions have been previously reported. (Eisenbarth et al,
2004; Kopaci et al, 2002; Lima et al, 2007; Urania et al, 2001) However, to the best of
our knowledge possible synergistic effect of some combinations of the ions released by
aluminum bronze has not been analyzed.
Our assays with Al-Zn combinations show synergistic effects when
concentrations between 6 x and 9 x AC8h were used. Accordingly, the uptake of each
ion (Al or Zn) may be interfered by the other yielding to a significant higher cytotoxic
effect (synergism). Urania et al. (Urania et al, 2001) showed that when results obtained
with concentrations of metal ions used singly or in combination were compared they
observed that Zn was accumulated in cells at a significant higher concentration when
used in combination with Cu. Additionally, a marked decrease in cell viability and
protein content was found for this mixture. On the other hand, Fe and Zn can also
interfere in other ions uptake processes (Mantha et al, 2011; Flemming and Trevors,
1989).
It was suggested that the synergism of the mixture Cu + Zn was evident as the
redoxactive metal Cu could enhance the Zn absorption in a living system (Stauber and
Florence, 1990; Moriwaki et al, 2008). Furthermore this combination of Cu + Zn might
also play a main role in the mixtures with other ions. In addition, synergic effects of
mixtures of some metal ions (Cu, Fe, Ni, Cr ions) on oxidative DNA damage mediated
by a Fenton-type reduction have been previously identified (Xu et al., 2011).
Xu et al. (2011) made toxicological assays with individual, binary, ternary and
quaternary mixture of four heavy metals (Cu, Pb, Zn and Cd) on embryos. Their results
showed that in most of the binary combinations, the interactions were synergistic.
Accordingly, our results revealed that when the osteoblastic cells were exposed to the
overall mixture of ions at 9 x AC8h a higher cytotoxic effect than in the case of Zn-Al
combination was found. Considering that each individual ion (except Al) did not show
any cytotoxic effect up to 30 x AC8h concentration value, these results clearly indicate
the dramatic influence of the simultaneous presence of several ions at concentrations
close to 9 x AC8h on osteoblastic cell viability.
Consequently, our results on cytotoxic effects in the vicinity of metal discs at
AC values lower than toxic level can be interpreted considering that the cells in this
region may be exposed, as a result of concentration gradients, to mixtures of ions that,
even nontoxic as single ions, are cytotoxic in mixtures, due to synergistic effects. The
difference in response to ions released from the alloys and salt solutions representing
the ions released from the alloys reported by other authors (Messer and Lucas, 1999)
may also be interpreted following this scheme.
4.3.
Cellular response to metal discs within the cell culture and culture media
with metallic salts.
At this point it is interesting to complement the interpretation of some apparent
inconsistencies detected by Messer and Lucas (1999) in the results of biocompatibility
assays. They found that cellular functions were not similarly altered in response to ions
released from the alloys and to their salts. They highlighted that salt solutions cannot be
easily used to represent alloy cytotoxicity because ionic release from alloys is a
complex process with a dose-time dependence. It should also be considered that when
the concentration of the ions released is evaluated by atomic absorption spectroscopy
the concentration of the samples measured for this analysis is the corresponding average
concentration value, AC of the original concentration gradient. When salts or extracts
are used to simulate the effect of ion release in cultures, the concentration is uniform
and similar to the corresponding AC. There are no concentration gradients in these
cases. Thus, Schmalz et al (1998) demonstrated that the results of experiences with salts
and extracts were only slightly different. This concentration levels may be below the
toxicity threshold and consequently no effect on cells are found. However, in
experiments with discs, concentrations close to the discs are high and time dependent,
reaching values markedly higher than the AC value, and exciding citotoxic threshold
levels, mainly in the case of mixtures with synergistic effects. Thus, although the AC
value measured by atomic absorption spectrophotometry is below the toxic threshold
value, cytotoxic effects may be found near the metal disc leading to the decrease in cell
viability.
In the oral environment, changes in quantity and quality of saliva, diet, oral
hygiene, polishing of the alloy, distribution of occulsal fouces, and burshing can also
influence the rate of ions release. Although the release of copper, aluminium, nickel,
manganese and iron remains far below the upper tolerable human intake levels (Lopez
Alías, 2006) they may cause cytotoxic effects locally.
Overall, different criteria have been applied when the cytotoxicity of metal ions
is assessed. Some authors have suggested that complete materials should be used to
evaluate the cytotoxicity of dental materials. Others consider that even such assays can
assess the total cytotoxicity of a dental alloy, the evaluation of the toxicity of individual
components is impeded. Importantly, present study demonstrated that experiments with
dental alloys and the use of mixture of ions are highly applicable to evaluate possible
synergic effects of ions and also space/time variation in cytotoxicity.
CONCLUSIONS
Results showed that the concentrations of the metal ions released by CuBA
(average concentration: AC) cannot be predicted on the basis of the overall composition
of this alloy. Higher concentrations than that of the copper base metal were found for
Al, Zn, Ni and Fe released ions. An important cytotoxic effect (reduction in cell
viability and in the size of colonies) was observed for cells in the surroundings of the
metal disc. Experiments with individual salts of the metal ions at concentrations levels
similar to those released by a 3 cm2 alloy after 8h (AC8h) exposure and with the
mixture of all the salts did not show any effect on the cells. However aluminum, zinc
and manganese ions at concentrations 6x, 36x and 60xAC8h, respectively, affected
lysosomal activity. Importantly, a synergistic effect was found when Al-Zn mixtures of
6x, 7.5x, 9xAC8h were used. A stronger synergism was observed when the mixture of
all ions at 9xAC8h concentrations was used, demonstrating that cell viability was also
affected by the simultaneous presence of more than two ions. Cytotoxic effects in the
vicinity of cell discs (zone A1) with the average AC levels of each ion lower than the
cytotoxic threshold values could be interpreted through a diffusion controlled
mechanism that could yield to concentration gradients, with concentrations levels
several times higher than the AC value in the metal surroundings.
The existence of concentration gradients may also contribute to the
interpretation of apparent discrepancies reported by other authors when two different
sources of metal ions are used: multiple ion salt solutions and samples of the dental
alloys. The importance of the evaluation of the synergic cytotoxic effect of mixtures of
ions when cytotoxic effects of alloys are analyzed is also highlighted.
ACKNOWLEDGMENTS
This work was supported by grants from: Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) (PIP 0847), Agencia Nacional de Promoción
Científica y Técnica (ANPCyT) (PICT 2010-BID 1779), Universidad Nacional de La
Plata (UNLP), and Facultad de Ingeniería UNLP, Áreas Departamentales de Ingeniería
Química y Mecánica (11- I129 and 11- I133).
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