Neoplastic transformation of cells by soluble but not particulate forms of metals used in orthopaedic implants

Biomaterials 19 (1998) 751 — 759

Neoplastic transformation of cells by soluble but not particulate forms of metals used in orthopaedic implants A. Doran!, F.C. Law!, M.J. Allen",*, N. Rushton! !University of Cambridge Orthopaedic Research Unit, Addenbrooke+s Hospital, Hills Road, Cambridge CB2 2QQ, UK "Department of Orthopedic Surgery, SUNY-Health Science Center, 750 East Adams Street, Syracuse, NY 13210, USA Received 28 October 1996; revised 27 August 1997

Abstract Recent developments in cell culture techniques have made it possible to study the cellular mechanisms involved in carcinogenesis and to apply these methods as screening tools in vitro. This study investigated and compared the ability of the metals most commonly used in orthopedic implants to induce toxicity and neoplastic transformation in the C3H10T1 mouse fibroblast cell line. Eight metals 2 (cobalt, chromium, nickel, iron, molybdenum, aluminium, vanadium and titanium) and their alloys (stainless steel, cobalt-chrome alloy and titanium alloy) were tested, both as soluble salts and as solid particles. There were marked differences between the various metals in terms of both toxicity and transforming ability. Significant increases in the incidence of cell transformation were seen with soluble forms of cobalt, chromium, nickel and molybdenum but not with iron, aluminium, vanadium or titanium. For most of the metals, transforming ability was directly related to toxicity, although this correlation did not hold for either molybdenum or vanadium. The physical form of the metal was critically important in determining its effects, and transformation occurred only with soluble metal salts. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Metals; Soluble ions; Particles; C3H10T1; Neoplasia; Transformation 2

1. Introduction Modern implant alloys are chosen both for their mechanical properties and high degree of biocompatibility. However, over time, the processes of corrosion and wear lead to the release of metallic debris and soluble metal ions [1—3]. The recent trend towards the use of metalon-metal and uncemented, porous-coated implants, along with their application in younger patients, has led to concern over the possibility of greater exposure of patients to metal degradation products over longer periods of time. Chromium and nickel are recognized carcinogens in the industrial environment [4, 5], and a number of metals have been shown to produce malignant tumours in experimental animals [6—9]. Recent reports of malignant tumors in association with joint replacements in human patients [10] have led to calls for the establishment of a central registry for implant-related tumors [11, 12]. * Corresponding author. Tel.: (315) 464 4245; fax: (315) 464 6638; e-mail: allenm@hscyr. edu. 0142-9612/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII S 0 1 4 2 - 9 6 1 2 ( 9 7 ) 0 0 2 0 9 - 3

Epidemiological studies of patients with total joint replacements have provided conflicting evidence of a difference in cancer susceptibility compared to that of the general population [13—17]. Animal studies have also produced mixed results, with some researchers reporting no association between implants and tumour formation [18—20], while others suggest an increased incidence of tumour formation either adjacent to, or distant from, an implant [21, 22]. Since the animal studies have failed to produce a consensus on this issue, attention has turned to the possibility of developing in vitro assays for studying the relationship between implant materials and neoplastic transformation. A number of screening tests have been developed for investigating the cellular mechanisms of carcinogenesis in vitro. In 1973, Reznikoff et al. [23] reported the establishment and characterization of a permanent mouse fibroblast cell line termed C3H10T1 clone 8. Under nor2 mal conditions, these cells are highly sensitive to postconfluence inhibition of division. However, exposure to polycyclic aromatic hydrocarbons, such as 3-methylcholanthrene and 7,12-dimethylbenz[a]-anthracene,

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induces neoplastic transformation of the cell line [24]. The transformed foci are easily distinguished in vitro since they form foci of deeply staining cells which stand out against the faintly stained background cell monolayer. Three types of transformed focus have been reported (Figs. 1—3). Cells from type I foci do not produce tumors when inoculated into irradiated syngeneic mice and are not considered to be fully transformed. Approximately 50% of type II foci and 80% of type III foci produce sarcomas when inoculated into mice; both type II and type III foci are considered to have undergone malignant transformation [25]. This study investigated the ability of the metallic constituents of surgical alloys (cobalt-chromium alloy, titanium alloy and stainless steel) to induce neoplastic transformation and cytotoxicity in C3H10T1 mouse embryo 2 fibroblasts in vitro. Metals were tested both as soluble salts and as solid particles. The aims of the study were (1) to determine whether transformation could be induced by particulate or soluble metals and (2) to establish the relative risks of the individual metals in terms of toxicity and carcinogenicity.

Fig. 2. The type II focus consists of several layers of deeply staining cells (a). The edge of the focus is well defined and stands out against the background monolayer (b). Giemsa stain, original magnification ]40 (a) and ]100 (b).

2. Materials and methods 2.1. Cells and cell culture

Fig. 1. Type I foci show an increase in cell density and staining (a). There is little or no multi-layering, the edge of the focus is poorly defined and blends into the background monolayer (b). Giemsa stain, original magnification ]40 (a) and ]100 (b).

C3H10T1 cells were purchased at passage 8 from the 2 American Type Culture Collection (Rockville, MD). Cells were grown in Eagle’s Basal Medium with Earle’s Salts (EBME; ICN Flow, Thame, UK), supplemented with 10% (v/v) foetal calf serum (Gibco BRL, Paisley, UK) and 292.3 mg ml~1 L-glutamine (ICN Flow, Thame, UK). Cultures were grown at 37°C in a humidified atmosphere of 5% CO in air. 2 For routine cell passage, C3H10T1 cells were seeded at 2 a density of 5]104 cells per 75 cm2 flask and maintained in the exponential growth phase by subculturing before they reached confluence. Cultures were discarded at passage 13 to avoid possible increases in the rate of spontaneous cell transformation [26]. All of the serum batches used in this study were prescreened according to IARC guidelines to ensure that they supported optimal plating efficiency, population doubling times and induction of morphological transformation in the C3H10T1 cell line [26]. 2

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ferrous chloride (FeCl ) were purchased from BDH Ltd 2 (Upminster, UK). Sodium molybdate (Na MoO ) and 2 4 aluminium chloride (AlCl ) were purchased from Sigma 3 Chemicals Ltd. (Poole, UK). Titanium chloride (TiCl ) 3 and sodium chromate (Na CrO ) were purchased from 2 4 Aldrich Chemical Co. (Gillingham, UK). All of the metal salts used in this study were '99% pure, as certified by their manufacturers. Stock solutions of metal salts were prepared and sterilized by filtration through 0.22 lm nylon filters. The desired test concentration was achieved by serial dilution, the final dilution being made in complete medium.

Fig. 3. The type III focus is multilayered and deeply basophilic (a). The cells at the edge of the focus tend to be rather spindle-shaped, randomly orientated and appear to invade the surrounding monolayer (b). Giemsa stain, original magnification ]40 (a) and ]100 (b).

Plating efficiency was determined by seeding cells at a density of 200 cells per well in 4-well polystyrene tissue culture plates. Cells were then grown for 10 days, with a change of medium after the first 120 h. Cells were fixed in methanol, stained with Giemsa and examined under a dissecting microscope. Colonies of over 50 cells were counted, and the percentage plating efficiency derived by dividing this number by the total number of cells seeded. In each case, the colony count was the average of four wells. For the construction of growth curves, cells were seeded at an initial density of 5]103 cells per well in 24-well plates. At 24 h intervals, three wells were trypsinized and the number of cells in each well counted using a haemocytometer. Growth curves were then constructed by plotting the average cell count against time on semilogarithmic graph paper. 2.2. Preparation of metals 2.2.1. Soluble salts Cobalt chloride (CoCl ), chromium chloride (CrCl ), 2 3 nickel chloride (NiCl ), vanadyl chloride (VOCl ) and 2 2

2.2.2. Particulate metals Cobalt and aluminium particles ((25 lm) were purchased from Kochlite Ltd. (Haverhill, UK). Chromium ((2 lm), titanium ((150 lm), iron ((60 lm), 316L stainless steel ((45 lm) and titanium—aluminium— vanadium alloy particles ((45 lm) were purchased from Goodfellow Ltd. (Cambridge, UK). Cobalt—chromium alloy particles ((104 lm) were a kind gift from Howmedica Inc. (Rutherford, USA). Molybdenum ((8 lm), vanadium ((45 lm) and nickel particles ((3 lm) were purchased from Aldrich Chemical Co. (Gillingham, UK). Since it has been shown that C3H10T1 cells are ca2 pable of internalizing particles by phagocytosis [27, 28], and since the metals in periprosthetic tissues are predominantly 0.1—5 lm in size [29, 30], particles of 5 lm or less were used in this study. Suspensions of particles of the desired size were prepared by sonication of the metal powders in acetone, allowing the larger particles to sink to the bottom of the test tube. Smaller particles remaining in suspension in the upper fraction were then removed with the acetone, which then evaporated. Particle size was confirmed by microscopy using an eyepiece graticule. Particles were sterilized in dry heat at 150°C for 2 h. Suspensions of metal particles were prepared in complete medium. 2.3. Experimental design The C3H10T1 cell transformation assay was carried 2 out according to the guidelines of the IARC working group [25]. The dose levels for the test metals were selected following preliminary cytotoxicity tests. Cultures were exposed to different concentrations of metals for 48 h and the plating efficiency determined 10 d later. The plating efficiency in the treated groups was expressed as a percentage of that in the negative controls to give a relative plating efficiency (RPE). The RPE results provided an accurate measure of the cytotoxicity of the test material and were used to determine an appropriate range of concentrations for use in the transformation assays. When possible, the highest test dose was selected to give 80—90% toxicity and the lowest was the maximal non-toxic dose, with 2—3 intermediate doses added.

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Concentrations of test compounds were prepared immediately prior to use. The negative control for tests with soluble metals was double—distilled water. The positive control was 3-methylcholanthrene (3-MCA; Sigma Chemicals, Poole, UK) at a final concentration of 2.5 lg ml~1. Each assay consisted of two limbs, which were carried out concurrently. Cells were seeded at a density of 200 cells per 60 mm well in four replicates for cytotoxicity studies, and at 2000 cells per 60 mm well for transformation studies. Seeding was carried out the day before treatment to allow settling and adherence of the cells. Cells were then exposed to test compounds or controls for 48 h, after which the medium was aspirated and replaced with fresh medium containing no test material. Cytotoxicity assays on cultures were carried out as described above and the results expressed as RPE values. Cells treated for transformation studies were maintained in culture for six weeks, after which they were fixed with methanol and stained with Giemsa. The plates were coded and scored blind for the presence of type II and type III foci. Foci that appeared intermediate in character (type I/II or type II/III) were assigned to the less aggressive category. The total numbers of type II and type III foci for each treatment group were recorded. The transformation incidence for each treatment was calculated as the number of wells with foci divided by the total number of wells scored. Statistically significant differences between test and control groups were determined using the Fishers exact probability test at p(0.05.

Table 1 Results for cobalt chloride in the C3H10T1 assay. The relative plating 2 efficiency (RPE) provides a measure of the cytotoxicity of the test material. Note that the transformation incidence is calculated by dividing the number of wells containing type II or III foci (rather than the total number of type II or III foci) by the total number of test wells seeded (approximately n"40 for most concentrations). CoCl (lg ml~1) 2

0.1 1.0 5.0 10.0 20.0 Water 3-MCA

RPE (%)

78.8 47.9 5.0 1.0 0.2 100.0 78.0

Focus type II

III

2 5 11 13 17 1 80

0 0 0 0 0 0 25

Transformation incidence

2/40 5/40 11/40 (p(0.005) 12/39 (p(0.001) 13/40 (p(0.001) 1/40 34/40

Table 2 Results for nickel chloride in the C3H10T1 assay 2 NiCl (lg ml) 2

1.0 10.0 20.0 40.0 60.0 Water 3-MCA

RPE (%)

104.5 43.0 6.7 0.2 0.5 100.0 78.0

Focus type II

III

1 9 15 8 8 1 80

0 0 0 0 0 0 25

Transformation incidence

1/40 8/40 (p(0.05) 10/40 (p(0.005) 6/39 6/40 1/40 34/40

3. Results 3.1. Soluble metal salts The result given for each salt is the combination of two assays, giving a maximum of 40 wells at each concentration in the transformation assay. Four metals demonstrated both toxicity and transforming ability. Cobalt chloride (Table 1) demonstrated dose-dependent toxicity and transformation. The incidence of transformation became statistically significant at 5 lg ml~1 and remained significant throughout the test range. All of the transformed foci were of the type II morphology. Nickel chloride (Table 2) was only slightly less toxic than cobalt chloride. Statistically significant increases in transformation incidence were seen at 10 and 20 lg ml~1, but not at 40 and 60 lg ml~1. As with cobalt chloride, all of the transformed foci were type II. Chromium chloride displayed dose-dependent toxicity (Table 3), but induced significant transformation only at very high concentrations (1200 lg ml~1 as compared with 5 lg ml~1 for cobalt chloride). At the highest concentration tested, chromium chloride precipitated out of

Table 3 Results for chromium chloride in the C3H10T1 assay 2 CrCl (lg ml~1) 3

300 600 900 1200 Water 3-MCA

RPE (%)

104.5 71.9 53.8 35.6 100.0 78.0

Focus type II

III

5 2 4 13 1 80

0 0 0 2 0 25

Transformation incidence

5/40 2/40 4/40 8/39 (p(0.05) 1/40 34/40

solution and formed aggregates in the medium. This precipitation was associated with extensive cell death and detachment of the monolayer. Despite this, the transformation incidence was significantly higher than in the control. Sodium chromate was clearly the most toxic of all the metal salts (Table 4). Significant transformation occurred only at the most toxic dose (0.5 lg ml~1) but was very marked.

A. Doran et al. / Biomaterials 19 (1998) 751—759 Table 4 Results for sodium chromate in the C3H10T1 assay 2 Na CrO 2 4 (lg ml~1)

0.005 0.010 0.050 0.100 0.500 Water 3-MCA

RPE (%)

93.6 93.2 69.4 45.7 0.2 100.0 78.0

Focus type II

III

0 2 0 3 12 1 80

1 0 0 0 0 0 25

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Table 5 Results for sodium molybdate in the C3H10T1 assay 2 Transformation incidence

1/40 2/40 0/40 3/36 10/36 (p(0.005) 1/40 34/40

Sodium molybdate (Table 5) was tolerated in high concentrations (up to 500 lg ml~1) without any sign of toxicity. A significant transformation incidence was reached at 10 lg ml~1, with 9 of 40 wells producing foci. Higher concentrations of sodium molybdate did not, however, induce cellular transformation. Vanadyl chloride (Table 6) was only slightly less toxic than sodium chromate. Although foci were seen at 0.1 lg ml~1, the transformation incidence did not reach statistical significance at this or any other concentration. Three metal salts showed both minimal toxicity and no significant transformation effect. Ferrous chloride (Table 7) produced very little toxicity at concentrations of up to 200 lg ml~1. Transformed foci developed sporadically at all concentrations but the incidence failed to reach statistical significance. At concentrations above 200 lg ml~1 the ferrous chloride precipitated out of solution and denatured the culture medium. Aluminium chloride displayed mild toxicity (Table 8) and although foci were seen, the transformation incidence did not reach significance at any concentration. Similar results were seen with titanium chloride (Table 9), which showed very little toxicity and was in fact the only metal salt to produce a clear negative result in the transformation assay.

Na MoO 2 4 (lg ml~1)

1.0 10.0 100.0 500.0 Water 3-MCA

RPE (%)

100.1 96.9 97.6 102.7 100.0 78.0

Focus ype II

III

6 8 2 4 1 80

0 3 0 0 0 25

Transformation Incidence

6/40 9/40 (p(0.01) 0/40 3/36 1/40 34/40

Table 6 Results for vanadyl chloride in the C3H10T1 assay 2 VOCl (lg ml~1) 2

0.10 0.50 1.00 5.00 Water 3-MCA

RPE (%)

87.9 52.8 35.4 0.0 100.0 78.0

Focus type II

III

1 0 0 0 2 52

1 0 0 0 0 16

Transformation incidence

1/40 2/40 0/40 3/36 2/40 26/39

Table 7 Results for ferrous chloride in the C3H10T1 assay 2 FeCl (lg ml~1) 2

10.0 50.0 100.0 200.0 Water 3-MCA

RPE (%)

98.4 92.5 90.8 90.2 100.0 82.7

Focus type II

III

4 6 4 6 2 50

1 0 0 1 0 21

Transformation incidence

3/40 6/40 4/40 6/36 2/40 31/40

3.2. Particulate metals In contrast with the soluble salts, particulate metals failed to induce significant increases in transformation incidence in any of the assays. In fact, in the majority of cases no foci were seen at all (results not shown). There were, however, differences in toxicity between the various metal particles tested (Fig. 4). Cobalt particles were toxic at concentrations above 0.5 lg ml~1. Nickel and vanadium particles became markedly toxic at 50 lg ml~1 and chromium particles became toxic at 100 lg ml~1. The remaining pure metals (molybdenum, aluminium, iron and titanium) showed marked toxicity only at the highest concentration tested (500 lg ml~1).

Table 8 Results for aluminium chloride in the C3H10T1 assay 2 AlCl (lg ml~1) 3

1.0 10.0 100.0 500.0 Water 3-MCA

RPE (%)

80.9 82.5 92.3 92.1 100.0 78.0

Focus type II

III

6 2 1 2 1 44

0 0 0 0 0 27

Transformation incidence

4/40 2/40 1/40 2/40 1/40 28/39

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Table 9 Results for titanium chloride in the C3H10T1 assay 2 Na CrO 2 4 (lg ml~1)

10.0 50.0 100.0 500.0 Water 3-MCA

RPE (%)

90.3 82.6 91.2 79.9 100.0 86.5

Focus type II

III

0 2 0 0 2 14

1 0 0 0 0 25

Transformation incidence

0/40 0/40 0/40 0/36 2/40 25/39

As might be expected, the three alloys (stainless steel, cobalt—chromium and titanium alloy) showed patterns of toxicity which were intermediate between those of their least and most toxic constituents (Fig. 4). The tests with particulate metals were designed to be performed with the same 48 h exposure time as was used for the metal salt assays. In practice, however, it proved impossible to remove all of the particles off the cell monolayer at the end of the exposure period. The results from both the cytotoxicity and the transformation studies with particulate metals therefore reflect a period of continuous exposure of the cells to metal particles.

4. Discussion It is now recognized that periprosthetic tissues and fluids from patients with metallic implants may contain significant amounts of both particulate and soluble metal

[3, 31—33]. Particulate metal debris has been implicated in the development of chronic inflammation [34, 35], bone resorption [36, 37], infection [38] and neoplasia [22] in periprosthetic tissues. In addition to these local effects, particulate metal debris released from arthroplasty implants may be disseminated to local and regional lymph nodes [39], as well as more distant sites [40]. These collections of debris, scattered throughout the body, represent a vast potential reservoir of soluble metal ions, which can be released either locally or into the systemic circulation. Potential complications of these metal ions might include toxicity, hypersensitivity reactions, neurological disorders, metabolic bone disease and even neoplasia [41, 42]. In this study, we investigated the potential of particulate and soluble metals to induce toxicity and transformation in vitro. As has been reported previously, particulate forms of cobalt, and nickel demonstrated significant toxicity, while chromium displayed only mild toxicity [43, 44]. Titanium, aluminium, iron and molybdenum were relatively non-toxic [45]. Our results with soluble metals support earlier findings from studies in which macrophages [46, 47], fibroblasts [43, 48] and osteogenic cells [49, 50] were exposed to solutions of metal ions. Cobalt, chromium (III), chromium (VI) and nickel ions exhibited dose-dependent toxicity, while the remaining soluble metals showed minimal adverse effects. Much less attention has been paid to the role of particulate and soluble implant metals on cell transformation. In previous studies with C3H10T1 cells, transformation 2 has been reported with particulate forms of nickel [51] and chromium [28]. We could not repeat these findings

Fig. 4. The cytotoxicity of particulate metal alloys and their elemental constituents for C3H10T1/2 cells in vitro. Results are expressed in terms of plating efficiency, relative to that of the control (no metal added): (a) cobalt—chromium alloy; (b) titanium alloy; (c) stainless steel.

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Fig. 4. Continued

in our study. One possible explanation for this may be that these earlier studies used insoluble crystalline forms of chromium and nickel (e.g.; nickel subsulphide), rather than metallic compounds. It is well recognized that phagocytosis rates for crystalline compounds are often much higher than those for metallic compounds [52], so it is likely that the intracellular concentrations of nickel and chromium ions are much higher in cells when cry-

stalline particles are used. Since we were interested only in the effects of wear and corrosion products from metal implants, we focused attention on particles with a similar size and chemistry to those reported in vivo [29, 30, 34]. Under these conditions, there was no evidence of cell transformation with any particulate metal or alloy. In contrast with the negative results with particulate metals and alloys, soluble forms of cobalt, nickel,

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chromium and vanadium produced positive results in our C3H10T1 transformation assays. Similar results have 2 been reported in studies on cobalt, chromium and nickel in other cell culture systems [53—56], and there appears to be good correlation between these in vitro findings and those from animal studies, in which it has been shown that soluble nickel and hexavalent chromium salts can be potent carcinogens [6]. The other metals of orthopedic interest have received relatively little attention. DiPaulo and Casto [53] found no evidence of transformation with iron, aluminium and titanium, and Sabbioni et al. [57] found that vanadium produced transformation in its pentavalent form (ammonium vanadate) but not in its tetravalent form (vanadyl sulphate). The clinical significance of these results is unclear. The mechanisms involved in the release, distribution and biological interactions of corrosion and wear products around metallic implants are complex and only partially understood. Techniques such as atomic absorption spectroscopy [3] and neutron activation analysis [31] make it possible to quantify total metal concentrations in biological tissues and fluids, but cannot differentiate between the particulate and soluble fractions in this total. In addition, the metal ions released from an implant by corrosion may exist in different oxidation states, and although the oxidation states of particulate (solid) corrosion products may be identified by X-ray photoelectron spectroscopy, this technique cannot be used to analyse soluble metal ions. Recently, Merritt and Brown [58] have described a novel technique for differentiating between chromium (III) and chromium (VI). Using this technique, they have shown that both forms of chromium ion are released from corroding stainless steel and cobalt—chromium alloys in vitro and in vivo. Under physiological conditions, chromium (III) ions are likely to predominate, but chromium (VI) is known to be the more biologically active [42]. This is borne out by the fact that toxicity and transformation in our C3H10T1 assays were 2 always more significant with chromium (VI) than with chromium (III). The results from this study do not allow us to draw definite conclusions about the risk of neoplastic complications from metallic implants. However, the C3H10T1 system does provide a valuable assay for grad2 ing the relative risks of different metals and alloys, in terms of both toxicity and transformation in vitro. Under the conditions of this study, it is clear that some of the metals used in orthopaedic prostheses are potentially more hazardous than others. The greatest risks, both for toxicity and carcinogenicity, were associated with cobalt, chromium and nickel. The lowest risks were associated with iron, aluminium and titanium. Vanadium was potentially harmful because of its cytotoxicity, and molybdenum showed evidence of possible carcinogenicity. On the basis of these results, the safest alloy in current use

would appear to be titanium alloy. None of its constituent metals caused transformation and only vanadium, a minor element of the alloy, was toxic. Both stainlesssteel and cobalt—chromium alloy contain appreciable amounts of metals which showed evidence of both transforming ability and toxicity.

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