COPYRIGHT 2002 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY.

. NO PART OF THIS ARTICLE MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

Elements Released from Dental Casting Alloys and Their Cytotoxic Effects

Ahmad S. Al-Hiyasat, BDS, MScD, PhDa Omar M. Bashabsheh, BDS, MScb Homa Darmani, BSc, FIMLS, PhDc

Purpose: This in vitro study investigated the element release from seven commercially available dental casting alloys and tested their cytotoxic effects. Materials and Methods: The casting alloys tested were one high-noble alloy (Bioherador N) and six base-metal alloys, including four Ni-Cr alloys (Remanium CS, Heranium NA, Wiron 99, CB Soft), one Co-Cr alloy (Wirobond C), and one Cu-based alloy (Thermobond). Ten specimens from each alloy were prepared in the form of disks, and each of the seven dental casting alloys (10 disks per group) were conditioned in distilled water at 37C for either 72 or 168 hours. The conditioning media were analyzed for element release, and the cytotoxic effects were assessed on Balb C fibroblasts using MTT assay. Results: Element release was greater at 168 hours of conditioning than at 72 hours. The extract from the high-noble alloy showed the least amount of element release (only Zn), with no cytotoxic effects. The greatest amount of element release was detected in the Cu-based alloy Thermobond and the Ni-Cr alloy CB Soft; their extracts were significantly more toxic than all the other alloy extracts. The cytotoxic effects of the other Ni-Cr alloy extracts were not statistically significantly different from the high-noble alloy extract. However, the Co-Cr alloy (Wirobond C) extract was significantly more cytotoxic than the high-noble alloy extract. Conclusion: Element release from casting alloys is proportional to the conditioning time. The content of Cr and Mo in the alloy protects the alloy from dissolution, while the Cu content makes it more susceptible to corrosion and dissolution, rendering it more cytotoxic. Int J Prosthodont 2002;15:473478.

lement release from dental casting alloys into the oral cavity is of clinical concern and considered to be a potential health problem to the dental patient.1,2 Previous reports have detected these elements in tongue scrapings and in saliva.3 During the past 20 years, many types of alloys have been developed with formulations that are fundamentally different from traditional gold-based alloys.4 These low-gold or no-gold

aAssistant Professor, Department of Restorative Dentistry, Faculty of Dentistry, Jordan University of Science and Technology, Irbid, Jordan. bDental Practitioner, Ministry of Health, Jordan. cAssociate Professor, Department of Applied Biological Sciences, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan.

Reprint requests: Dr Ahmad S. Al-Hiyasat, Department of Restorative Dentistry, Faculty of Dentistry, Jordan University of Science and Technology, PO Box 3030, Irbid 22110, Jordan. Fax: + 02-7278962. e-mail: hiyasat@just.edu.jo

alloys have been introduced to dental work as a result of an increase in the price of gold.5 This change has led to a widespread interest in what have been classified as base-metal alloys, which are also referred to as nonprecious or non-noble metal alloys.6 Therefore, at present, clinicians and dental laboratory workers choose dental alloys from among many different alloy compositions, and they must consider the biologic safety of the alloys before they make a decision. The most important factor in determination of the biologic safety is corrosion, since the systemic and local toxicity and carcinogenicity of an alloy all result from elements in the alloy being released into the mouth during corrosion.7 Previous corrosion studies have reported that alternative alloys (low gold or no gold) release more elements than traditional highgold alloys.2,810 The adverse tissue reactions of the gingiva and the periodontium close to dental casting alloys are thought to depend on the amount of

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released elements available, which is a function of corrosion rates.11 Indeed, the occurrence of a local toxic effect is not well documented, but it poses a higher risk, primarily because local tissue is exposed to much greater concentrations of released metal ions.7 The aim of this study was to evaluate the elemental release from six commercially available, predominantly base-metal alloys with different compositions and one high-noble alloy and to investigate the cytotoxicity of the released elements using a well-established in vitro fibroblast model.

were autoclaved at a temperature of 150C for 60 minutes. Analysis of Elements Released The 20 disks of each type of alloy were divided into two groups of 10. Each group of the seven dental casting alloys was then transferred to 11.5 mL of distilled water and left at 37C for either 72- or 168-hour conditioning periods. Following this, the conditioning media were aspirated; 200 L was used for the cytotoxicity test, and the remaining volume (11.3 mL) was used for element release analysis. The conditioning media were analyzed for the presence of Ni, Cr, Co, Cu, Zn, and Mo using an Inductively Coupled Plasma Atomic Emission Spectrophotometer (ICPAES; PerkinElmer). Triplicate absorbance readings per element were made for each sample. Each reading was used to determine the mean concentration of the different elements in parts per billion (ppb) released from the alloys. The detection limit for each element and wavelength of detection are summarized in Table 2. Cell Culture and Cytotoxicity Testing Balb C 3T3 mouse fibroblasts, clone A31 (European Collection of Cell Culture) were maintained in Dulbeccos Modified Eagle Medium (DMEM; Life Technologies) supplemented with 5% fetal bovine serum, 5% newborn calf serum, 100 units/mL penicillin and streptomycin, and 0.25 g/mL amphotericin B at 37C in a humidified incubator with 5% CO2. The cytotoxic effect of the conditioning media was assessed on Balb C fibroblasts as follows. Briefly, 200 L of cell suspension (5 105 cells/200 L) was added to each well of a 96-well plate, and the cells were incubated for 2 hours at 37C with 5% CO2. After the incubation period, 20-L aliquots of conditioning medium were added to the cells. For each alloy, 10 replicates were used at each of the two different conditioning times. For controls, an equivalent volume of distilled water was added to 10 wells containing Balb C fibroblasts. The cells were incubated for 24 hours at 37C with 5% CO2, and cytotoxicity of the extracts (conditioning media) was assessed using the MTT assay. Briefly, MTT was added in amounts equal to 10% (20 L each) of the culture media volume. The cells were incubated at 37C for 3 hours, and then the resulting formazan crystals were dissolved by adding an equivalent volume of solubilization solution (0.1 M HCl in anhydrous isopropanol). The absorbance was measured at 580 nm using an ELISA plate reader (Multiskan Plus EFIAB, Titrek). DMEM without cells was used as a blank.

Materials and Methods

Seven cast alloys were used in this study to determine the elements released from them into distilled water as the conditioning medium, and to assess the cytotoxic effects of the conditioning medium. The materials consisted of one high-noble alloy, four basemetal nickel-chromium (Ni-Cr) alloys, one base-metal cobalt-chromium (Co-Cr) alloy, and one copperbased alloy (Table 1). Twenty specimens from each type of casting alloy were constructed in the form of disks 5 mm in diameter and 3 mm in thickness. To produce disks with a reproducible size and shape, a silicone mold was used to produce the wax pattern of the casting alloy specimens. The wax pattern was produced using inlay wax (Bego). The wax was then sprued, invested, and cast in the phosphate-bonded investment material Belavest (Bego) using a conventional lost-wax technique. All processes of investment, burn out, and casting were according to the manufacturers instructions. The specimens were sandblasted using 250-m aluminum oxide to remove the investment material. The sprues were then removed, and the specimens were finished and polished using finishing stone burs in the sequence brown, green, and pink (Bego), and then black followed by green rubber polishing wheels (Bego). Finally, iron oxide rouge polishing compound (Bego) was used. Each type of alloy specimen was finished and polished separately using separate instruments. All processes of finishing and polishing were carried out in a similar way to simulate the preparation of the cast-metal alloys for clinical cases. The disks were soaked in a detergent solution for 5 minutes and then scrubbed using a soft bristle brush. They were rinsed under tap water for 5 minutes and under distilled water for another 5 minutes. The disks were then placed into 95% ethanol and cleaned by sonication for 5 minutes, removed and placed in sterile distilled water, and cleaned by sonication for another 5 minutes. Finally, the disks

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Table 1
Alloy

Dental Casting Alloys Used

Type High noble (Au-Pt) Base metal (Ni-Cr) Base metal (Ni-Cr) Base metal (Ni-Cr) Base metal (Co-Cr) Base metal (Ni-Cr) Base metal (Cu-Al) Composition (wt%)* Au: 86.2; Pt: 11.2; Zn: 1.5; Ta: 0.3; Ru: 0.4; Mn: 0.1 Ni: 61; Cr: 26; Mo: 11; Si: 1.5 (Fe, Ce, Al, Co < 1) Ni: 59.3; Cr: 24; Mo: 10 (Fe, Mn, Ta, Si, No < 2) Ni: 65; Cr: 22.5; Mo: 9.5; No: 1; Si: 1; Fe: 0.5; Ce: 0.5; C: 0.02 Co: 65; Cr: 26; Mo: 6; W: 0.5; Si: 1; Fe: 0.5; Ce: 0.5; C: 0.02 Ni: 72.8; Cr: 4.9; Cu: 12.3; others: 10 Cu: 87.5; Al: 8.5 (Si, Fe, and others < 4)

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Bioherador N, Heraeus Kulzer Remanium CS, Dentaurum Heranium NA, Heraeus Kulzer Wiron 99, Bego Wirobond C, Bego CB Soft, Hatykeyama Dental Thermobond, Dentecan
*As listed by manufacturer.

Data and Statistical Analysis The mean concentrations of elements released from the alloy specimens were presented in ppb, with a detection limit of 10 ppb. Cytotoxicity of alloy extracts was determined by measuring cell viability (by MTT) relative to controls (100% cell viability = no toxicity). Analysis of variance (ANOVA) was used to analyze the data for cytotoxicity of the casting alloys. Followup comparisons between the groups were carried out using Tukeys multiple comparison test (at 95% confidence interval, = .05) and a paired t test.

Table 2 ICPAES Parameters for Element Release Analysis*

Element Nickel Copper Chromium Cobalt Molybdenum Zinc Wavelength (nm) 341.476 324.754 267.617 228.616 203.844 213.856 Correlation coefficient 0.9999 0.9981 0.9996 0.9994 0.9997 0.9985

*All elements had a detection limit of 10 ppb and required a linear calibration equation.

Results
Element Release Table 3 shows the element release from dental casting alloys for the two conditioning times (72 and 168 hours) measured by ICPAES. Overall, at both conditioning times Thermobond had the highest amount of element release (Cu) followed by CB Soft (Ni and Cu), whereas the amount of element release from all the other base-metal alloys was within a much smaller range. The high-noble alloy Bioherador N showed the least amount of element release (Zn). Cytotoxicity of Alloy Extracts Figure 1 illustrates the mean percentage of cell viability relative to the control for the extracts of the alloys at the two conditioning times. The least degree of cytotoxicity was observed with the extracts of Bioherador N and the most with the extract of Thermobond at both conditioning times. There was considerable increase in cytotoxicity of the 168-hour extract compared to the 72-hour extract for CB Soft and Thermobond, while the change in cytotoxicity for all the other alloy extracts was negligible. Statistical analysis of the cytotoxicity of extracts using two-way ANOVA showed that both the type of the alloy and the conditioning time had a statistically significant effect (P < .001). The interaction between the type of the alloy and the conditioning time also was significant (P < .001), indicating that the cytotoxicity of the extracts was different from one type of alloy to another in relation to the conditioning time. Comparison between the alloy extract groups (Tukey) revealed that the cytotoxicity of the extract from Remanium CS, Heranium NA, and Wiron 99 at 72 and 168 hours was not significantly different from the extracts of Bioherador N. However, the cytotoxicity of the extract from Wirobond C, CB Soft, and Thermobond was significantly greater than Bioherador N. Moreover, the extracts from Thermobond and CB Soft for both conditioning times were significantly more toxic than the extracts from all the other alloys. Indeed, the extract from Thermobond was significantly more toxic than the extract from CB Soft at 168

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Table 3 Element Release in ppb (Mean Standard Deviation) from the Casting Alloys in the Conditioning Medium (Distilled Water) After 72 and 168 Hours of Conditioning
Alloy Bioherador N Remanium CS Heranium NA Wiron 99 Wirobond C CB Soft Thermobond Conditioning time (h) 72 168 72 168 72 168 72 168 72 168 72 168 72 168 Zn 134 4.5 167 6.5 Ni 196 6.4 231 3.8 208 2.3 246 4.1 214 4.3 262 4.3 458 11 710 13 Co 193 6.0 278 8.2 Cu 507 17 886 18 2450 55 3750 62 Cr 16 1.1 12 1.4 Mo 24 1.8 32 1.8 21 1.3 25 1.6 27 1.3 37 1.3 25 2.6 68 3.1 Total element release 134 167 220 263 229 271 241 315 218 358 965 1596 2450 3750

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= not detected or below detection limit (10 ppb).

120 72 h 100 168 h

Cell viability (%)

80

60

40

20

Remanium CS

Heranium NA

Fig 1 Percentage of cell viability for the extracts of casting alloys after 72 and 168 hours of conditioning (100% cell viability = no toxicity).

hours of conditioning. All the differences in cytotoxicity between the extracts of the other types of alloys were not statistically significant at either extraction period, except for the Wirobond C extract at 168 hours, which became significantly more toxic than Remanium CS. The extracts of each alloy at the two conditioning times were compared to each other using a t test paired comparison to determine the effect of the period of extraction on cytotoxicity. The differences in cytotoxicity of the extracts at 72- and 168-hour conditioning periods for Bioherador N, Remanium CS,

Bioherador N

Heranium NA, Wiron 99, and Wirobond C were not statistically significant (P > .05). However, for CB Soft and Thermobond, there was a statistically significant increase in the cytotoxicity of extracts for the 168-hour conditioning time compared to that at 72 hours (P = .003 and P < .001, respectively).

Discussion
The construction of the alloy specimens was carried out to simulate the technique used to produce dental cast restorations in dental laboratories. Cleaning and

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Thermobond

Wiron 99

Wirobond C

CB Soft

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sterilization of the specimens were performed according to previous reports.1,4,12,13 The shape and dimensions of the alloy specimens (disks) have been commonly used for in vitro tests.1,4,5,13,14 The ratio of surface area of the specimens to volume of conditioning medium agrees with the ISO dental standard.14 Different types of conditioning media, such as cell culture medium, artificial saliva, saline, and dilute acids, have been used in previous investigations.4,5,9,15,16 All these media contain various minerals or organic constituents that may have an effect on the corrosion susceptibility of the alloys and thus influence the element release. In the present study, distilled water was used as a conditioning medium to avoid such effects on the alloy and also to avoid interference with the ICPAES analysis for element release. It should be mentioned that this study is part of an ongoing work that will also investigate the effect of different conditioning media on element release. Element Release The results of ICPAES showed an increase in the levels of release of metal elements in relation to conditioning time. The increase in release is associated with the corrosion susceptibility of alloys. The limited or absent Cr and Mo content in the alloys Thermobond and CB Soft resulted in higher amounts of element release than in Remanium CS, Heranium NA, and Wiron 99. The latter alloys had more Cr and Mo in their compositions. Thus, they are more corrosion resistant.17,18 The release of Ni from CB Soft was observed to be two to three times greater than that of the other Ni-Cr alloys. This might be due to CB Softs low content of Cr and absence of Mo and also its content of Cu. The presence of Cu makes the alloy more corrosion susceptible and more liable to tarnish.13 Earlier investigations have suggested that only Ni-Cr alloys containing 16% to 27% Cr develop an adequate protective oxide layer.18 In addition to Cr, Mo plays an active role in the formation of this protective oxide layer. Thus, an increasing concentration of Cr and Mo on the surface layer may synergistically lower dissolution rates of metals.17 CB Soft contains only 4.9% Cr by weight, and the absence of Mo makes this alloy more susceptible to corrosion in comparison to Remanium CS, Heranium NA, and Wiron 99, in which the content of Cr is 26%, 24%, and 22.5% respectively, and Mo is 11%, 10%, and 9.5%, respectively. Cr release from the Ni-Cr alloys and Co-Cr alloy (Wirobond C) used in this study was very low, in most cases below the detection limit, in comparison with the release of Mo and Ni. Other studies have also reported similarly low Cr release in artificial saliva, lactic acid, and saline solutions.15,16 The reason for

the increased release of Mo and Ni from Ni-Cr alloys compared with Cr is not known and needs further investigation.19 High amounts of Cu were released from CB Soft and Thermobond alloys. The release of Cu from these alloys was most probably due to the high corrosion susceptibility of Cu resulting in greater release. Other studies have also shown that Cu is released in high concentrations from alloys.1,11 The Cu-based alloy Thermobond showed a very high amount of Cu release, and this agrees with other investigations that have shown that Cu-based alloys tarnish more and are more corrosion prone than Ni- or Au-based alloys.20 The high-noble alloy Bioherador N showed release only of Zn. This agrees with other investigations that have also shown that only Zn is detected from highnoble alloys in conditioning media, while the release of noble elements like Au and Pt is below the detection limit.11,21 The differences in the amounts of element release in the present study in comparison with other studies were most probably due to the conditioning medium that was used. In the present study, distilled water was used as a neutral medium, while other studies used different types of conditioning media varying in pH, salt, and protein content, which may result in greater element release from the surface of the alloy. Cytotoxicity of Conditioning Media (Extract) For the two conditioning times, the extracts of the high-noble alloy Bioherador N and the Ni-Cr alloys Heranium NA, Remanium CS, and Wiron 99 showed a slight cytotoxic effect when compared to the control. The differences between these groups were not statistically significant. This may suggest that conditioning in water does not accelerate the release of elements from Ni-Cr alloys. However, the cytotoxicity of elements released from the Co-Cr alloy (Wirobond C) was slightly higher than the Ni-Cr alloys, which may suggest that the cytotoxic effect of Co is greater than that of Ni. The greater cytotoxicity of extracts of CB Soft and Thermobond is most probably due to the presence of high amounts of Cu, although there was also a high amount of Ni released from CB Soft. These two elements were reported in another study to have moderate cytotoxic reaction.22 Other investigations have reported that a concentration of 4.0 g/mL of Cu reduces succinic dehydrogenase (SDH) activity to 50%.1 In the current study, the Thermobond extracts contained concentrations of Cu ranging between 2.45 g/mL (at 72 hours of conditioning) and 3.75 g/mL (at 168 hours of conditioning), which we found to be enough to cause a cytotoxic effect and reduce the SDH activity to 80.07% and 65.88%, respectively.

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In agreement with our findings, other investigations have also reported that the extracts of high-noble alloys, base-metal alloys (Ni-Cr), and pure Cu conditioned in culture media for 3 or 7 days are not cytotoxic, except in the case of the pure Cu.11 Finally, it is clear that the element release from the casting alloys and their cytotoxic effects depend on the composition of dental alloys and the conditioning time.

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Conclusions
This in vitro study investigated the element release from dental casting alloys into distilled water and the cytotoxicity of these extracts. Under the conditions of this study, the following conclusions have been drawn: 1. Overall, the amount of element release increased with increasing conditioning time. 2. The high-noble alloy (Bioherador N) showed the least element release, and its extract showed little, if any, cytotoxic effect. 3. The release of Ni and Mo from the Ni-Cr alloys Remanium CS, Heranium NA, and Wiron 99 was similar, and the cytotoxic effects of their extracts were not significantly different from the extracts of the high-noble alloy Bioherador N. 4. Co and Mo were detected in the extract of the CoCr alloy (Wirobond C), and the extract from this alloy was significantly more toxic than that of the high-noble alloy. 5. A relatively greater amount of Ni was detected in the extract of the Ni-Cr alloy (CB Soft) in addition to a greater amount of Cu. The extract from this alloy was significantly more cytotoxic than the extracts from all the other types of alloys, except for the Cu-based alloy (Thermobond). 6. The extract of the Cu-based alloy contained a remarkably greater amount of Cu, and the cytotoxic effect of this extract was significantly higher than the extracts from all the other types of alloys. 7. The element release from cast alloys and their cytotoxic effects depend on the composition of the alloys.

Acknowledgment
This study was supported by the Deanship of Research at Jordan University of Science and Technology, grant 51/2000.

References
1. Wataha JC, Malcolm CT, Hanks CT. Correlation between cytotoxicity and element release by dental casting alloys. Int J Prosthodont 1995;8:914.

2. Wataha JC, Malcolm CT. Effect of alloy surface composition on release of elements from dental casting alloys. J Oral Rehabil 1996;23:583589. 3. Stenberg T. Release of cobalt from cobalt chromium alloy constructions in the oral cavity of man. Scand J Dent Res 1982;90: 472479. 4. Wataha JC, Lockwood PE, Nelson SK, Bouillaguet S. Long-term cytotoxicity of dental casting alloys. Int J Prosthodont 1999;12: 242248. 5. Nelson SK, Wataha JC, Lockwood PE. Accelerated toxicity testing of casting alloys and reduction of intraoral release of elements. J Prosthet Dent 1999;81:715720. 6. Shillingburg HT, Hobo S, Whitesett LD, Jacobi R, Brackett SE. Fundamentals of Fixed Prosthodontics, ed 3. Chicago: Quintessence, 1997:365383. 7. Wataha JC. Biocompatibility of dental casting alloys: A review. J Prosthet Dent 2000;83:224234. 8. Lappalainen R, Yli-Urpo A. Release of elements from some gold alloys and amalgams in corrosion. Scand J Dent Res 1987;95: 364368. 9. Johansson BI, Lemons JE, Hao SQ. Corrosion of dental copper, nickel, and gold alloys in artificial saliva and saline solutions. Dent Mater 1989;5:324328. 10. Mezger PR, Stols AL, Vrijhoel MM, Greener EH. Metallurgical aspects and corrosion behavior of yellow low-gold alloys. Dent Mater 1989;5:350354. 11. Schmalz G, Langer H, Schweikl H. Cytotoxicity of dental alloy extracts and corresponding metal salt solutions. J Dent Res 1998;77: 17721778. 12. Wataha JC, Lockwood PE. Release of elements from dental casting alloys into cell culture medium over 10 months. Dent Mater 1998;14:158163. 13. Wataha JC, Craig RG, Hanks CT. The release of elements from dental casting alloys into cell culture medium. J Dent Res 1991;70: 10141018. 14. Vuilleme MN. Ion release from dental alloys: A key to the interpretation of biocompatibility tests. In: Biocompatibility, Allergies and Resistance to Corrosion: 8 Years of Research. Neuchatel, Switzerland: Metalor, 1996:919. 15. Tai Y, De Long R, Goodkind RJ, Douglas WH. Leaching of nickel, chromium and beryllium ions from base metal alloys in an artificial oral environment. J Prosthet Dent 1992;68:692697. 16. Geis-Gerstorfer G, Weber H. In vitro corrosion behavior of four Ni-Cr dental alloys in lactic acid and sodium chloride solutions. Dent Mater 1987;3:289295. 17. Brune D. Mechanism and kinetics of metal release from dental alloys. Int Endod J 1988;21:135142. 18. Bumgardner GD, Lucas LC. Surface analysis of nickel-chromium dental alloys. Dent Mater 1993;9:252259. 19. Bumgardner JD, Lucas LC. Cellular response to metallic ions released from nickel-chromium dental alloys. J Dent Res 1995;74: 15211527. 20. Kaga M, Seale NS, Hanawa T, Ferracane JL, Waite DE, Okabe T. Cytotoxicity of amalgam alloys and their element and phases. Dent Mater 1991;11:6872. 21. Plumb JA. Cell sensitivity assay. In: Brown R, Bger-Brown U (eds). Cytotoxic Drug Resistance Mechanisms. Totowa, NJ: Humana Press, 1999:2530. 22. Wataha JC. Cytotoxicity test to screen biological performance of casting alloys. In: Biocompatibility, Allergies and Resistance to Corrosion: 8 Years of Research. Neuchatel, Switzerland: Metalor, 1996:2732.

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