Oral Keratinocyte Responses to Nickel-based Dental Casting Alloys In Vitro C. M. WYLIE AND A. J. DAVENPORT School of Metallurgy and Materials, University of Birmingham Edgbaston, Birmingham, B15 2TT, UK

P. R. COOPER AND R. M. SHELTON* Biomaterials Unit, School of Dentistry, University of Birmingham St Chads Queensway, Birmingham, B4 6NN, UK

ABSTRACT: Adverse reactions of oral mucosa to nickel-based dental casting alloys are probably due to corrosion metal ion release. We exposed H400 oral keratinocytes to two Ni-based dental alloys (Matchmate and Dsign10) as well as NiCl2 (1–40 mg/mL Ni2þ). Alloy derived Ni2þ media concentrations were determined. Direct culture on both alloys resulted in inhibited growth with a greater effect observed for Dsign10 (higher ion release). Indirect exposure of cells to conditioned media from Dsign10 negatively affected cell numbers (64% of control by 6 days) and morphology while Matchmate-derived media did not. Exposure to increasing NiCl2 negatively affected cell growth and morphology, and the Granulocyte-macrophage colony-stimulating factor (GM-CSF) transcript was significantly up-regulated in cells following direct and indirect exposure to Dsign10. NiCl2 exposure up-regulated all cytokine transcripts at 1 day. At day 6, IL-1b and IL-8 transcripts were suppressed while GM-CSF and IL11 increased with Ni2þ dose. Accumulation of Ni2þ ions from alloys in oral tissues may affect keratinocyte viability and chronic inflammation. KEY WORDS: cell viability, dental alloy, gene expression, inflammation, metal ion release, metal ion toxicity.

*Author to whom correspondence should be addressed. E-mail: [email protected] Figures 1–5 appear in color online: http://jba.sagepub.com

JOURNAL OF BIOMATERIALS APPLICATIONS Volume 25 — September 2010 0885-3282/10/03 0251–17 $10.00/0 DOI: 10.1177/0885328209349870 ß The Author(s), 2010. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav

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INTRODUCTION

N

ickel-based alloys are relatively inexpensive and are therefore frequently used in dentistry in metal–ceramic crowns. Other advantages include an increased modulus of elasticity, which enables thinner sections to be used clinically, resulting in less sound tooth destruction during crown preparation. The thermal expansion coefficient of Ni-based alloys also compares well with that of conventional veneering porcelain, enabling maintenance of intimate bonding during firing between the metal and ceramic crown thereby preventing veneer cracking [1,2]. To enhance the workability and longevity following placement of Ni-based alloys, a variety of other metals are frequently incorporated. Chromium (Cr) is added to promote the formation of a stable oxide layer increasing corrosion resistance. Molybdenum (Mo) is also often combined promoting resistance to pitting and crevice corrosion [3,4]. While the addition of beryllium (Be) improves alloy castability, it significantly decreases corrosion resistance since a chromium-depleted NiBe eutectic phase is subsequently formed [5]. Notably, regardless of the level of Cr and Mo in the alloy, increase in the Be content to levels above 0.6 wt% results in attack of the NiBe eutectic phase in artificial saliva [6]. Proteins in saliva can further affect corrosion with studies demonstrating accelerated corrosion reactions in the presence of proteins [7–9]. Owing to the release of metal ions from Ni-based alloys via corrosion mechanisms, concerns remain regarding their biocompatibility following placement. Nickel is the main element released during corrosion of Ni-based alloys and is detectable in patients’ saliva and accumulates within tissues adjacent to dental cast alloys [10,11]. Notably, Ni is also considered to be the most allergenic of all metallic elements [12]. The other major alloying elements, Cr and Mo, are released at much lower concentrations and are considered less toxic [13]. Intraoral adverse reactions described in response to Ni-based dental materials include gingival inflammation, swelling and erythema, mucosal pain, and lichenoid reactions [14–16]. The biological interaction between Ni-based alloys and the oral tissues is considered to occur via mechanisms involving cytotoxicity, subcytotoxic effects, allergic reactions, and effects on bacterial adhesion [14]. Cell culture systems are commonly used to test for cytotoxic and subcytotoxic effects of Ni-based alloys. However, the data obtained from these experiments are inconsistent since researchers commonly use different cell types and culture conditions [17,18]. In addition, a variety

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of cytotoxicity and subcytotoxic effects are analyzed, including changes in cellular morphology, proliferation rate, viability, protein synthesis, and ATP production [13,19–23]. Studies have also considered the induction of proinflammatory mediators following cellular exposure to Ni as adverse reactions to Ni-based alloys exhibit an inflammatory and immunological component [24,25]. While human oral keratinocytes are key structural cells within the oral cavity, are one of the first cell types to encounter dental alloys and are important in regulating the immune response, limited data exists with regards to their response to corrosion products derived from Nibased dental casting alloys. It is likely that their cellular response may significantly contribute to the chronic adverse reaction seen in certain patients. The purpose of the current in vitro investigation was therefore to assess how two commercially available dental alloys released Ni2þ, and how this affected oral keratinocyte viability and inflammatory mediator gene expression. MATERIALS AND METHODS

Sample Preparation Two commercially available Ni–Cr alloys, Matchmate NP (Davis Schottlander and Davis Ltd., Letchworth, Herts, UK), and Williams Dsign10 (Ivoclar Vivadent Ltd., Meridian South, Leicester, UK) were used (Table 1). Disc-shaped specimens (12 mm diameter and 1.5 mm thickness) were cast using a centrifugal casting machine (Motorcast, Degussa, Germany) according to manufacturers’ instructions. Heat treatment was performed to simulate porcelain firing and consisted of 10108C for 5 min and removal plus additional cycles of 9808C, 9708C, and 9808C for 1 min prior to cooling slowly. A surface finish equivalent to the clinical condition was achieved by sand blasting the disc-shaped specimens with aluminim oxide (BasicMaster, Renfert GmbH, Germany) followed by three polishing Table 1. Alloy elemental composition as provided by the manufacturers. wt% Matchmate Dsign10

Ni

Cr

Mo

Al

Si

Fe

62.2 75.4

25 12.6

9.5 8

– 3.3

3.3 0.2

– 0.5

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stages using a slow speed handpiece with rubber polishing wheels as recommended for nonprecious alloys as follows: (i) Polysoft A, (ii) Dedeco Blue, and (iii) Sunlite (Bracon, UK). Samples were cleaned with a cotton mop and ultrasonically with ethanol. To minimize bacterial contamination, samples were placed in 100% ethanol for 30 min, rinsed in sterile distilled water, and air dried in a sterile environment. Cell Culture H400 human oral keratinocytes (passages 11–18) [26] were cultured in DMEM supplemented with fetal calf serum (10%), hydrocortisone (0.5 mg/ mL), L-glutamine (4 mM), and HEPES buffer (2.5%) without antibiotics. The dose response to Ni2þ was determined for H400 cells by the addition of nickel chloride solution to the cultures. Concentrated solutions of NiCl2 were prepared and filter sterilized, and subsequently added to cultures to obtain final concentrations of 0–40 mg/mL Ni2þ. Cell numbers were determined using the 2-vinylpyridine, 1-(4,5dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay [27] with a standard curve relating cell numbers to absorbance at 570 nm initially determined (data not shown). Morphology was examined using either phase contrast microscopy (not requiring fixation) or scanning electron microscopy (SEM) following fixation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and dehydration through a graded series of ethanol and critical point drying from CO2. For direct dental alloy exposure, H400 cells were seeded at 8  103 cells/cm2 per well directly onto the disc-shaped specimens contained in 24-well culture plates and incubated at 378C in a 5% CO2 humidified environment. Thermanox tissue culture plastic was used as a control substrate and had an equivalent surface area for cell growth (1.1 cm2). RNA was subsequently extracted from cultured cells for gene expression analysis (see below). For indirect dental alloy exposure, H400 cells were exposed to alloy extracts in culture medium. Disc-shaped specimens were immersed for 3 days or 6 weeks in culture medium (1 mL) giving a surface area to volume ratio of 2.8 cm2/mL. Cells were seeded at 8  103 cells/cm2 into a 24-well culture dish containing normal growth medium. Prior to adding the conditioned medium, the cells were allowed to attach and proliferate for 24 h. To avoid degradation of L-glutamine in exposed media, this supplement was added (at 4 mM concentration) to the extract medium immediately prior to keratinocyte exposure. Cells were exposed directly and indirectly to a 3-day extract solution, derived from both Ni-based alloys (Matchmate NP and Dsign10), for up to 6 days of culture. RNA was

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subsequently extracted from cultured cells for gene expression analysis (see below). Quantitation of Nickel Release The cell culture medium (1 mL) exposed to alloys, for a period up to 6 weeks, was analyzed for Ni2þ using inductively coupled plasma atomic emission spectroscopy (ICP-AES; Optima 4300 DV, PerkinElmer Instruments, USA). Samples were diluted to 20 mL with nitric acid to minimize protein and salt interference, giving detection limits of 0.04 mg/mL. Calibration standard solutions were prepared and analyzed with NiCl2 (0–3 mg/mL Ni2þ) in culture medium with nitric acid to resolve the sample concentrations. Gene Expression Analyses One microgram of isolated RNA (RNeasy kit, Qiagen, UK) was oligodT reverse transcribed (Omniscript kit, Qiagen, UK). Fifty nanograms of cDNA were used to seed 25 mL polymerase chain reactions (PCRs) and products amplified using the RedTaq PCR system (Sigma, UK). Primers were designed from GenBank sequences using Primer3 software (http:// frodo.wi.mit.edu/cgi-bin/primer3/primer3; Table 2). A typical amplification cycle, of 958C for 20 s, 60.58C for 20 s, and 728C for 20 s, was performed using a GeneAmp 2700 PCR system (Applied-Biosystems, UK). Following cycling, 6 mL of the PCR mix was removed and products visualized on 1.5% agarose gels containing 0.5 mg/mL ethidium bromide. Gels were illuminated using ultraviolet light and images captured with Table 2. Details of PCR assays. Gene Interleukin-1beta Interleukin-8 Granulocyte macrophage colony stimulating factor Interleukin-11

Symbol

Accession Number

Sequence

IL1B

NM_000576.2 F-TCC AGG GAC AGG ATA TGG AG R-TCC TGC TTG AGA GGT GCT GA IL8 NM_000584.2 F-TAG CAA AAT TGA GGC CAA GG R-GGA CTT GTG GAT CCT GGC TA GMCSF M11220.1 F-ACT ACA AGC AGC ACT GCC CT R-CTT CTG CCA TGC CTG TAT CA

IL11

NM_000641.2 F-AGC TGC AAG GTC AAG ATG GT R-GAC AGG GTT TTG CCA TGT CT

Product (bp) 292 204 303

311

All DNA sequences are shown in the 50 to 30 orientation. Tm ¼ Annealing temperature (8C); pb ¼ base pairs; (F) ¼ Forward primer (R) ¼ Reverse primer.

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EDAS 120 software (Kodak, UK). Scanned gel images were imported into AIDA image analysis software (FUJI, UK) and the volume density of amplified products calculated and normalized against the GAPDH housekeeping gene control values. Statistical Analysis All experimental investigations were performed in triplicate and statistical significance ( p50.05) was determined by a one-way analysis of variance (ANOVA) and a post hoc Tukey test compared with control cultures using Excel software (Microsoft, USA). RESULTS

Cellular and Molecular Response of Oral Keratinocytes to Nickel Ions H400 cells exposed to increasing Ni2þ concentrations (540 mg/mL) for up to 6 days in culture indicated that concentrations greater than 5 mg/mL altered cellular morphology and caused a dose- and timedependent decrease in the number of viable cells and therefore decreased proliferation (Figure 1). Notably, even at low Ni2þ concentrations (1 mg/mL), cell proliferation was decreased after 6 days of exposure, although no morphological changes were apparent (Figure 1). (a)

Cell viability (% control)

100 80 60 40 1 day 2 days 3 days 6 days

20 0

0

10

20

30

40

Nickel concentration (μg/mL)

Figure 1. (a) Graph of percentage of viable human oral keratinocytes relative to controls exposed to increasing concentrations of nickel ions (0–40 mg/mL) in cultures of up to 6 days (n ¼ 3). (b) SEM micrographs showing the morphology of H400 human oral keratinocytes exposed to different concentrations of nickel ions for 6 days. (i) 0 mg/mL (control), (ii) 1 mg/mL, (iii) 5 mg/mL, (iv) 10 mg/mL, (v) 20 mg/mL, and (vi) 40 mg/mL.

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(i)

25μm

100μm

100μm

25μm

100μm

25μm

100μm

25μm

(v)

(ii)

25μm

100μm

(vi)

(iii)

25μm

100μm

Figure 1. (Continued) (a) (i) 0

(b) (i)

2+ Ni (µg/mL)

20

0

GAPDH

GAPDH

IL-1β

IL-1β

IL-8

IL-8

GM-CSF

GM-CSF

IL-11

IL-11

(ii) 1.6

(ii) 1.8

IL-1ß IL-8 GMCSF IL-11

1.4 1.2 1.0 0.8 0.6

1

5

10

20

IL-1ß IL-8 GMCSF IL-11

1.6 Relative intensity

Relative intensity

2+ Ni (µg/mL) 1 5 10

1.4 1.2 1.0 0.8 0.6

0.4 0

5 10 15 Nickel concentration (µg/mL)

0.4

20

(iii)

0

5 10 15 Nickel concentration (µg/mL)

20

(iii)

Nickel concentration (µg/mL) 1 5 10 20

IL-1β

+ + +

IL-8

GMCSF

+

+ + + +

IL-11

+ +

Nickel concentration (µg/mL) 1 5 10 20

IL-1β

+

IL-8

GMCSF

IL-11

+

+ + + +

+ + +

Figure 2. Cytokine gene expression in keratinocytes exposed to increasing nickel ion concentrations for (a) 1 day and (b) 6 days. (i) Representative agarose gel image, (ii) Graphical representation of expression levels relative to GAPDH expression, and (iii) Table showing significant differences (‘þ’ symbols) for the three PCR replicates determined by one-way ANOVA ( p50.05).

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Analysis of cytokine gene expression in H400 cells exposed to Ni2þ, in general, indicated that transcription was dose dependently up-regulated following 1 day exposure (Figure 2(a)). However, following 6 days of culture, a more complex pattern of expression was evident, as while Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-11 exhibited a similar up-regulated profile, IL-1b and IL-8 showed marked down-regulation with increasing Ni2þ dose used (Figure 2(b)). Cellular and Molecular Response of Oral Keratinocytes Following Direct and Indirect Exposure to Nickel Alloys Direct cell material exposure demonstrated that H400 cells grown on the surface of both alloys exhibited significantly decreased cell numbers relative to control conditions. Notably, after 6 days, oral

Cell viability (% control)

(a) 120 100 80

(b) ∗ ∗

∗ ∗

(i)

∗ Control

∗‡

Matchmate

60

Dsign10

40

100µm

25µm

100µm

25µm

100µm

25µm

20 (ii) 0 1

Relative intensity

(c)

3 6 Exposure time (days)

1.4 1.2

*

1.0

(iii)

0.8 0.6 0.4 0.2 0.0 IL-1β

IL-8

GMCSF

IL-11

Control Matchmate Dsign10

Figure 3. (a) Viability of human oral keratinocytes seeded directly on the surface of the alloys for up to 6 days (n ¼ 3). *Indicates statistical difference with controls and z indicates statistical difference between sample groups. (b) SEM micrographs showing the morphology of H400 human oral keratinocytes cultured for 6 days directly on substrates. (i) Thermanox control, (ii) Matchmate, and (iii) Dsign10. (c) Cytokine (IL-1b, IL-8, GMCSF, IL-11) gene expression in oral keratinocytes cultured directly on the surface of Ni-based alloys for 6 days. Histogram shows relative expression levels between samples normalized to GAPDH are shown. *Indicates statistical significance for three PCR replicates determined by one-way ANOVA ( p50.05).

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keratinocytes grown directly on the Dsign10 substrate also demonstrated decreased cell numbers compared with cells grown on the Matchmate alloy (Figure 3(a)). High-resolution SEM images indicated no apparent change in cellular morphology when H400 cells were cultured on control Thermanox or either Ni2þ alloy (Figure 3(b)). Direct exposure of H400 cells to dental alloys had minimal affect on cytokine gene expression, although GM-CSF was statistically significantly upregulated due to cell growth on Dsign10 for 6 days (Figure 3(c)). To determine whether components leached from the nickel alloys influenced oral keratinocyte behavior, H400 cells were exposed indirectly to conditioned media obtained by immersion of alloy discs for 3 days. Indirect exposure of H400 cells to Matchmate (with lower Ni2þ release – Figure 4) did not influence morphology (Figure 5(a)) or proliferation (data not shown), while the higher release of Ni2þ from Dsign10 affected both, causing a significant decrease in cell numbers after 3 days exposure (85% control), which further decreased (64% control) after 6 days. Oral keratinocytes exposed to Dsign10 after 6 days demonstrated regions of inhibited cell growth and with apparently more Matchmate Dsign10 2.0

Total nickel release (μg/mL)

1.8 1.6 1.4 1.2 1.0 0.8

∗

0.6 0.4

∗

0.2 0.0 0

6

Pre-treatment time (weeks) Figure 4. Graph demonstrating concentration of nickel ion release, as determined by ICP-AES, from the alloys into culture medium after 3 days, comparing the initial release with that after 6 weeks pretreatment in culture medium. *Indicates statistical significant nickel release after 6 weeks compared with initial release.

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(a)

ET AL.

(i)

100µm (ii)

(iii)

100µm

100µm

(b) (i) 1.4

∗

Relative intensity

Relative intensity

(ii) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

∗

1.2 1.0 0.8 0.6 0.4 0.2 0.0 IL-1β

IL-8 Control

GMCSF

IL-11 Matchmate

IL-1β

IL-8

GM-CSF

IL-11

Dsign10

Figure 5. (a) Phase contrast micrographs of human oral keratinocytes exposed indirectly for 6 days on substrates. (i) Thermanox control, (ii) Matchmate, and (iii) Dsign10. (b) Cytokine gene expression for unexposed (C ¼ Control) and exposed keratinocytes to Ni-based alloys (M ¼ Matchmate, D ¼ Dsign10) for (i) 1 day and (ii) 6 days. Histograms of relative expression levels normalized to GAPDH. *Indicates statistical differences for PCR replicates (n ¼ 3) determined by one-way ANOVA ( p50.05).

rounded morphologies than controls. Notably, Ni2þ release, as determined by ICP-AES, was initially higher for both alloys, although after 6 weeks of pretreatment, Ni2þ release was significantly decreased for both alloys (Figure 4) and did not influence proliferation rates (data not shown). H400 cells exposed to conditioned media exhibited minimal changes in gene expression for the cytokines analyzed. Data, however, indicated, similar to that obtained for direct alloy exposure, that GM-CSF was statistically significantly up-regulated at both 1 and 6 days following exposure to conditioned media obtained using Dsign10 (Figure 5(b)). DISCUSSION

In general, both dental alloys tested were found to significantly affect cell viability and minimally affect cytokine expression in cultured oral

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keratinocytes. In addition, the dental alloy Dsign10, which released greater amounts of Ni2þ into culture media, exhibited the greatest cellular and molecular affects. It was, however, notable that following direct exposure of oral keratinocytes to the Ni-based alloys, cellular morphology appeared only minimally affected and therefore potentially alloy surface topography may be important in enabling initial cell adhesion, although prolonged exposure may subsequently have more deleterious affects on the cells. Indications were that the Ni2þ ions released by the Ni-based alloys were generally responsible for the cellular and molecular effects observed. This was supported by the data demonstrating that the oral keratinocyte response to increasing concentrations of Ni2þ resulted in a dose- and time-dependent reduction in cell viability and an initial dose-dependent increase in cytokine expression. Interestingly, however, the cytokine expression profile observed in the later 6-day time point appeared more complex with both up- and down-regulation occurring following increasing Ni2þ dose, suggesting that chronic exposure may be important in modulating the tissue inflammation observed in vivo. The detection by ICP-AES of Ni2þ release into culture media even following 6 weeks pretreatment suggests that the accumulation of Ni2þ within oral cells and tissues may be important in the prolonged adverse reactions observed in vivo, especially in conjunction with the increased corrosive environment found within the oral cavity. Notably, Ni-based alloy stability is reduced in acidic environments, such as crevices, leading to an increase in metal ion release [28]. In addition, while saliva has a pH buffering capacity, it may form a thin film of moisture on the Ni-based alloy surface, enhancing oxygen access to the metal outside the crevice subsequently leading to greater differential aeration and increased corrosion within the crevice [29]. It is evident from the data presented here that Dsign10 is more susceptible to corrosion and is thus more likely to continually release increased levels of metal ions into the oral environment. The cellular and molecular mechanisms by which corrosion products released from Ni-based alloys cause adverse reactions is not yet fully elucidated. Similar work by Bumgardner et al. [20] demonstrated that some Ni-based dental alloys did not directly influence cell viability or morphology but rather the corrosion products decreased cellular proliferation rates by interfering with cellular energy metabolism. In addition, while metal ion release into a culture medium can be relatively low prolonged, low-dose exposures have been shown to suppress cell activity [30,31]. Subsequently, it has therefore been argued that monitoring metabolic activity may provide a more appropriate

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methodology for assessing the potential of adverse reaction to Ni-based dental alloys [32]. It is possible that associated decreased cell proliferation rates caused by Ni-based dental alloys placed in vivo as opposed to direct cytotoxic effects may limit the oral tissues ability for repair. The proinflammatory cytokines studied here are involved in the host’s inflammatory and immune response and in particular regulate immune cell maturation and chemoattraction. Importantly, these cytokines have also been described as being involved in several epithelial pathologies [33–36]. The present study indicated that oral keratinocyte expression of GM-CSF identified a gene sensitive to transcriptional control in response to both direct and indirect exposure to the Ni-based dental alloy Dsign10 (Figures 3 and 5). Significantly, this data corroborated that obtained by the Dsign10 Ni2þ release analysis and that from the NiCl2 exposure of the oral keratinocytes. Ni2þ release data for Dsign10 indicated that 1.5 mg/mL was present in the media after 3 days exposure, while the only transcript differentially regulated by the oral keratinocytes at below 5 mg/mL Ni2þ exposure was GM-CSF. In addition, GM-CSF, along with IL-11, were the only cytokines clearly up-regulated in both a dose- and time-dependent manner at both the 1 and 6 day time points (Figure 1). Previous analysis has demonstrated that GM-CSF is an important stimulator of dendritic cell maturation, which is a key regulator of allergic reactions [37]. Recent work has also shown that skin component cells, including keratinocytes, up-regulate GM-CSF in response to Ni2þ haptens [38]. Another study investigating cytokine production in reconstructed human epidermis in response to Ni2þ suggested that measurement of IL-1a and IL-8, in conjunction with the MTT assay, might enable identification of irritant and sensitizing agents [39]. GM-CSF expression may also provide an appropriate measure for the potential for adverse reactions to Ni-based alloys, although further work is still required in this area. The underlying mechanisms explaining how Ni-based alloys or Ni2þ exert their cellular and molecular affects remains unclear. Several groups have, however, demonstrated that Ni2þ ions enter cells [40–42] and bind with several biological components [43], affecting cellular functions, morphology, and ultrastructure [31,32,44]. In addition, Ni is a known redox active metal able to impair cellular defense mechanisms against peroxidation [45,46]. Subsequently, Trombetta et al. [47] have shown that Ni2þ affect the cellular redox equilibrium within the cell potentially leading to subcellular damage mediated by reactive oxygen species. It is therefore conceivable that the redox sensitive transcription

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factor, NF-kB, may play a role in the cellular response to nickel especially as this key intracellular signaling molecular is involved in cellular stress response and in particular responsible for regulating cytokine production and apoptosis [48]. Indeed, recent work by Gazel et al. [49] using microarrays to analyze the molecular response of reconstructed human epidermis to Ni2þ identified involvement of this pathway. It is also interesting to speculate that due to the bacterial colonization of the oral cavity, synergistic effects occur between Toll-like receptor (TLR) signaling pathways and those invoked by Ni2þ subsequently contributing to the adverse reactions observed. Indeed, TLRinduced cytokeratin expression changes which are reported to occur in white patch lesions are also similar to those previously reported during in vitro analysis of H400 cell exposure to oral bacteria [48,50]. It is, however, likely that while Ni2þ release initially affects the oral keratinocyte, the perpetuation of the chronic inflammatory cycle may also include additional affects on recruited immune and inflammatory cells. Indeed, one aspect of immune cell function that appears to have been overlooked with regards to the subcytotoxic effect of nickel is that of macrophage phagocytosis. Indeed, Ni2þ can inhibit this process [51] and such an affect may subsequently result in failed debridement of apoptotic cell debris and/or failed removal of bacteria. Disruption of such an important immune cell mechanism has the potential to result in a localized inflammatory reaction, which cannot be resolved [52]. Therefore, determining the combined effect of Ni2þ on both keratinocytes and recruited immune cells may be important in understanding the associated pathologies. While this study has focused on Ni2þ as being the major hapten released from Ni-based alloys, it is also important to be aware that other alloy components are, at least in vitro, reported to cause cytotoxic effects. Indeed, recent studies have shown that chromium is highly cytotoxic potentially having a greater affect than nickel in studies using healthy and patient volunteer primary and passaged keratinocytes [53]. It is therefore important to be aware that in vivo, a mixture of different metal ions is present in the tissues and that synergistic as well as antagonistic effects may occur between the alloying elements [54]. CONCLUSIONS

While dental alloy Ni2þ release into culture medium is relatively low, the prolonged contact of the Ni alloy with the oral epithelium may contribute to the chronic inflammatory reactions seen in some patients. The present study indicates that accumulation of Ni2þ released from

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Ni-based alloys in culture medium can reduce oral keratinocyte proliferation, alter morphology, and affect cytokine gene expression. Higher early release of Ni2þ compared with later stages may indicate a greater risk for adverse reactions immediately following the placement of crowns. These risks can be minimized by selecting alloys with relatively low Ni2þ release. ACKNOWLEDGMENT

We would like to thank Professor Stephen Prime, University of Bristol, for the gift of the H400 oral epithelial cell line and advice on its growth and maintenance. REFERENCES 1. Leinfelder, K.F. An Evaluation of Casting Alloys Used for Restorative Procedures, J. Am. Dent. Assoc., 1997: 128: 37–45. 2. McLean, J.W. (1979). The Science and Art of Dental Ceramics. The Nature of Dental Ceramics and Their Clinical Use, Chicago, Quintessence Publishing, Vol. 1. 3. Friend, W.Z. (1980). Corrosion of Nickel and Nickel-Base Alloys, New York, John Wiley and Sons. 4. Geis-Gerstorfer, J. and Weber, H. In Vitro Corrosion Behaviour of Four Ni–Cr Dental Alloys in Lactic Acid and Sodium Chloride Solutions, Dent. Mater., 1987: 3: 289–295. 5. Baran, G.R. The Metallurgy of Ni–Cr Alloys for Fixed Prosthodontics, J. Prosthet. Dent., 1983: 50: 639–649. 6. Pan, J., Geis-Gerstorfer, J., Thierry, D. and Leygraf, C. Electrochemical Studies of the Influence of Beryllium on the Corrosion Resistance of Ni–25Cr–10Mo Cast Alloys for Dental Applications, J. Electrochem. Soc., 1995: 142: 1454–1458. 7. Clark, G.C. and Williams, D.F. The Effects of Proteins on Metallic Corrosion, J. Biomed. Mater. Res., 1982: 16: 125–134. 8. Endo, K. Chemical Notification of Metallic Implant Surfaces with Biofunctional Proteins (Part 2). Corrosion Resistance of a Chemically Modified NiTi Alloy, Dent. Mat. J., 1995: 14: 199–210. 9. Omanovic, S. and Roscoe, S.G. Electrochemical Studies of the Adsorption Behaviour of Bovine Serum Albumin on Stainless Steel, Langmuir, 1999: 15: 8315–8321. 10. Garhammer, P., Hiller, K.A., Reitinger, T. and Schmalz, G. Metal Content of Saliva of Patients With and Without Metal Restorations, Clin. Oral Investig., 2004: 8: 238–242. 11. Garhammer, P., Schmalz, G., Hiller, K.A. and Reitinger, T. Metal Content of Biopsies Adjacent to Dental Cast Alloys, Clin. Oral Investig., 2003: 7: 92–97.

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