Si ratio on genotoxicity of germanium-containing glass ionomer cements

Materials Letters 168 (2016) 151–154

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Effect of Ge/Si ratio on genotoxicity of germanium-containing glass ionomer cements Caitlin M. Pierlot a,n, Lauren Kiri a, Daniel Boyd a,b,nn a b

Department of Applied Oral Sciences, Dalhousie University, Canada School of Biomedical Engineering, Dalhousie University, Canada

art ic l e i nf o

a b s t r a c t

Article history: Received 18 August 2015 Received in revised form 18 November 2015 Accepted 27 December 2015 Available online 29 December 2015

Glass ionomer cements have been discussed across broad and progressive applications from dentistry to orthopedics. However, in orthopedic applications, adverse patient outcomes that have been clinically linked to the release of aluminum from conventional alumino-silicate GICs, demonstrate a critical need for the development of new aluminum-free materials. Accordingly, the present study uses an Ames Screen to evaluate the preliminary genotoxicity profiles of a series of promising new germanium-containing, aluminum-free GICs. The results herein reveal that in addition to the already demonstrated superior handling characteristics and suitable mechanical strength, these cements are non-mutagenic to bacteria strains Salmonella typhimurium (TA98) and Escherichia coli (WP2uvrA). The results of this study provide strong positive evidence regarding the genotoxicity of these novel GICs, warranting further investigation into the biocompatibility of these materials for numerous in vivo applications. & 2016 Elsevier B.V. All rights reserved.

Keywords: Glass Ionomer Polyalkenoate Cement Germanium Genotoxicity

1. Introduction Glass ionomer cements (GICs) have been discussed extensively in the literature as potential bone cements due to their inherent attributes suitable for vertebroplasty and kyphoplasty: intrinsic radiopacity, negligible setting exotherm, bioactivity, and customization potential [1–3]. Unfortunately conventional aluminumcontaining GICs (used in dentistry) are contraindicated for orthopedic applications [4] due to concerns relating to the local (connective tissue and bone) toxic deposition and systemic toxicity of aluminum (Al) [5–7]. Accordingly, significant work has been undertaken over the past decade to produce an Al-free GIC that adequately balances desirable injectability profiles with appropriate strength characteristics – all with little success. However, in 2013 Dickey et al. reported, for the first time, a series of germanium (Ge) inclusive Al-free GICs with clinically viable handling characteristics and mechanical properties [1]. This series of Gecontaining cements are the first Al-free GICs that decouple the setting reaction from final strength and are therefore the only vertebroplasty/kyphoplasty-compatible GICs documented to date. Crucially, however, the genotoxicity profile of Ge-based n Corresponding author. Department of Applied Oral Sciences, Dalhousie University, Canada. nn Corresponding author at: Dalhousie University, Department of Applied Oral Sciences and School of Biomedical Engineering, 5981 University Avenue, Halifax, NS, Canada B3H 4R2. Tel.: þ1 902 494 6347. E-mail addresses: [email protected] (C.M. Pierlot), [email protected] (D. Boyd).

http://dx.doi.org/10.1016/j.matlet.2015.12.120 0167-577X/& 2016 Elsevier B.V. All rights reserved.

materials, including cements, is currently unknown. Furthermore, animal studies have shown evidence of renal toxicity associated with the intake of germanium dioxide (GeO2) [8], demonstrating the potential health risk of Ge-based products for human use as well. The objective of the current study was thus to investigate the genotoxicity of a series of Ge-based GICs, by way of an Ames screen, to determine whether (i) the presence of Ge, and/or (ii) the ratio between Ge and Si, has any influence on the genotoxicity of this class of GICs.

2. Experimental details Five glass compositions (Table 1) were synthesized, ground, and sieved to a particle size r45 mm [1]. Each respective glass transition temperatures (Tg) was measured (Table 1) and each powder was annealed at Tg–30 °C, as per the procedures of Dickey et al. [1]. All cements were prepared by hand-mixing processed glass powder with aqueous (50-wt%) PAA at a P:L ratio of 2.0:1.5 [1]. After mixing for 60 s, cement disks were prepared by filling Teflon disk molds (d¼ 15 mm, t ¼1 mm) to excess with each GIC (n ¼3). Once filled, the molds were covered with acetate, clamped between two stainless steel plates, and incubated in a dry environment at 37 °C for 2 h. After 2 h, the mold assemblies were broken down, cement flash was removed, and the flat ends of each disk were ground flat using wet 800 grit silicon carbide paper. Each sample was immersed in 10 ml tissue culture water (37 °C, 2 Hz)

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3. Results and discussion

Table 1 Glass compositions (mol. fraction) and glass transition temperatures (Tg). Glass

SiO2

GeO2

ZnO

CaO

Na2O

ZrO2

Tg (°C)

G1 G2 G3 G4 G5

0.48 0.36 0.24 0.12 0.00

0.00 0.12 0.24 0.36 0.48

0.36 0.36 0.36 0.36 0.36

0.11 0.11 0.11 0.11 0.11

0.025 0.025 0.025 0.025 0.025

0.025 0.025 0.025 0.025 0.025

654.9 633.7 619.6 601.0 590.0

for 72 h [9]. Incubating solutions were sterile filtered (0.2 μm), and stored at 4 °C until further analysis. GICs produced using the glass powders will herein be referred to by their glass label (G1-5). Genotoxicity testing (Ames Assay) was performed by NAMSA (Northwood, OH, USA), using the standard plate incorporation assay method [10] for all test articles (including tissue culture water for the genotoxicity baseline of the extraction vehicle). Test articles included (i) undiluted extracts for each of G1-G5, as well as (ii) diluted extracts for G5 (GIC containing highest concentration of Ge) – diluted by 7  , 70  , and 700  using tissue culture water, producing a total of 3 diluted versions of the G5-GIC extracts: G5DIL1, G5-DIL2, and G5-DIL3. One strain each of Salmonella typhimurium (TA98) and Escherichia coli (WP2uvrA) was used to detect mutagens causing frame-shift mutations and missense base-pair substitutions respectively, with tester strain genotypes confirmed prior to testing. This study was conduced in the presence and absence of S9 metabolic activation, to distinguish between direct and indirect mutagens (where present). For the assay, molten top agar was supplemented with histidine–biotin solution or tryptophan solution for the S. typhimurium or E. coli tester strains, respectively. Separate tubes containing 2.0 mL of supplemented molten top agar were inoculated with 0.1 mL of culture for the two tester strains, and 0.1 mL of each undiluted extract respectively. A 0.5 mL aliquot of sterile water for injection or S9 homogenate was added when necessary. The mixture was poured across triplicate Minimal E plates, which were then incubated at 37 °C for 2 days. Parallel testing was also conducted with a negative control (saline) and four positive controls (Table 2) – quantity of spontaneous revertants of controls were validated based on historical data collected at the testing facility. Following the incubation period, the revertant colonies on each plate were recorded and compared to negative control plates. Any extract demonstrating a statistically significant 2-fold or greater increase in the number of mean revertants over the means obtained from the negative control, was identified as a “potential mutagen”. Using the extracts prepared for each GIC, 1 ml of each cement extract was diluted to 5 ml using 2% (v/v) HNO3. Calibration standards were prepared using 2% (v/v) HNO3 from standard solutions of 1000 ppm Si, Ge, Zn, Ca, Na, and Zr (Perkin Elmer Atomic Spectroscopy Standards, Waltham, USA). The concentration of each ionic species was measured for each cement extract via ICP optical emission spectroscopy (Perkin Elmer Optima 8000, Waltham, USA) by comparing dilute extract concentrations to the calibration curves using WinLab32 ICP software (n ¼3 per extract). Tissue culture water was used as the control. Table 2 Positive control summary. Positive control

Concentration

S9

Tester strain

Benzo[a]pyrene 2-Nitrofluorene

2.5 μg/plate 5.0 μg/plate

Presence Absence

TA98

2-Aminoanthracene Methyl methanesulfonate

20 μg/plate 3.25 mg/plate

Presence Absence

WP2uvrA

The results of genotoxicity testing are summarized in Table 3. In all cases where genotoxicity was successfully assessed (i.e. all extracts excluding TA98 -S9 for G2-G5), the number of revertants for each cement extract were not significantly different from each other nor from the negative controls. All genotoxicity assays demonstrated non-mutagenic responses to bacterial strains TA98 and WP2uvrA ( o2 fold increase in revertants compared to negative control). Both positive and negative controls for each tester strain exhibited a characteristic number of spontaneous revertants. Each positive control mean exhibited at least a 3-fold (655x) increase over the respective negative control mean for both tester strains. At the initial dilution, G2-G5 yielded a toxic response (Tox) that prevented evaluation of mutagenic response for tester strain TA98 in the absence of metabolic activation, however follow-up serial dilutions of G5, assayed with TA98 in the presence of S9, were non-mutagenic (Neg). The Ge ion release levels at 72 h for each GIC are provided, for completeness, in Fig. 1. It is clear from the data that concentrations of Ge did not exceed 150 ppm for any GIC. The concentrations of Si, Zn, Ca, Na, and Zr did not exceed 150 ppm, 40 ppm, 8 ppm, 80 ppm, and 0.4 ppm, respectively. It can be concluded that the ions released did not correlate with a genotoxic response for either bacteria strain. Interestingly, although extensively used in the clinical setting, the biocompatibility profiles (particularly cytotoxicity and genotoxicity) of conventional GICs are not well documented nor understood. In contrast to the preliminary biocompatibility results presented in this study, many conventional GICs, particularly resin-modified GICs (RM-GICs) are cytotoxic, displaying mild to severe cytotoxicity [11–18] depending on the experimental conditions (e.g. concentration, setting mechanism, etc.). Genotoxicity studies, which can provide important links to carcinogenicity, are significantly less commonplace even though they are generally simple and inexpensive when performed in vitro, as completed in the current study. Genotoxicity testing of biomaterials is indicated in the case of all materials where the genotoxicity profile has not already been well established [9]. This is largely the case for any GIC, even though numerous GIC-based materials are currently in clinical use. Only a handful of studies have assessed (and published) genotoxic potential of conventional GICs, through a variety of experimental techniques, but with conflicting and sometimes unclear conclusions: RM-GICs (OptiBand, Band-lok, Vitrebond) and resin-composites (RCs; Variolink II and Panavia) have been shown to induce mild to severe genetic damage via comet assay [11,14], SCE and chromosomal aberration test [12,15], CHO mammalian cell gene mutation tests [19], and Ames assays [19,20]. Conventional GICs (Ketac Molar, Ketac Cem, Ketac Endo, Fuji, and others) have a less clear genotoxic profile under similar testing: comet assay – mildly genotoxic [14], SCE and chromosomal aberration – mildly genotoxic [12,15], and Ames assays – ranging from non-mutagenic to an unclear response [16,20–22]. Although conventional GICs are widely used for dental applications, their genotoxicity profiles, and thus their potential for biocompatibility in regard to their intended applications, are largely unknown. It is, however, known that conventional aluminosilicate GICs have been associated with several fatal cases of aluminum-induced encephalopathy, as well local connective tissue and bone toxicity resulting in impaired osteoblastic function [4–7]. It is clear then that these conventional GICs, while well tolerated in dental applications, pose a significant biocompatibility risk elsewhere in the human body. The inclusion of Ge into the glass network may mitigate this issue, producing Al-free GICs that provide (i) clinically viable handling characteristics for percutaneous orthopedic procedures, (ii) sufficient mechanical properties

C.M. Pierlot et al. / Materials Letters 168 (2016) 151–154

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Table 3 Genotoxicity of cement extracts. Extract

TA98

WP2uvrA

Absence of S9

Presence of S9

Absence of S9

Presence of S9

Rev.

Res.

Rev.

Res.

Rev.

Res.

Rev.

Res.

G1 G2 G3 G4 G5 DIL1 DIL2 DIL3

197 1 – – – – 22 72 20 72 21 72

Neg Tox Tox Tox Tox Neg Neg Neg

267 1 217 1 237 3 217 1 207 1 – – –

Neg Neg Neg Neg Neg – – –

91 73 877 4 977 3 887 5 957 3 – – –

Neg Neg Neg Neg Neg – – –

967 2 927 4 977 5 967 4 867 2 – – –

Neg Neg Neg Neg Neg – – –

Negative control

21 75

237 2

857 7

74 78

Positive control

11477 214

960 7 18

1093 7 62

432 7 37

Note: Values are mean number of revertants (rev.) 7 SE of each cement extract. Mutagenic response (Res.) is indicated as either negative/non-mutagenic (Neg; o 2-fold increase in revertants compared to negative control), positive/potential mutagen (Pos; Z 2-fold increase in revertants compared to negative control), or cytotoxic (Tox; indicating that the genotoxicity could not be assessed at that dilution). Initially tested cement extracts (G1, G2, G3, G4, and G5) are undiluted. Serial dilutions of G5 (DIL1, DIL2, and DIL3) are diluted by 7  , 70  , and 700  using tissue culture water.

150

Ge Release (ppm)

10

5

100

50

5 D B

3

4 D B

D

D B

B

Ca Release (ppm)

40

20

8 6 4 2

0.8

60 40 20

5 D B

3

4 D B

D

D B

B

80

Zr Release (ppm)

1.0

B

1 D

5 D B

4 D B

3 D B

2 D B

1 D B

100

2

0

0

0.6 0.4 0.2

5 D B

4 D B

3 D B

2 B D

5 B D

4 B D

3 B D

2 B D

1 D B

1

0.0

0

D

Zn Release (ppm)

B

1 D

5 D B

4 D B

3 D B

2 D B

1 D B

10

60

Na Release (ppm)

2

0

0

B

Si Release (ppm)

15

Fig. 1. (a) silica, (b) germanium, (c) zinc, (d) calcium, (e) sodium, and (f) zirconium release from Ge-containing GICs.

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for the stabilization of vertebral fractures, and (iii) a non-genotoxic biocompatibility profile with regard to the present study. Limitations

 The extract time period (72 h) was selected based on recognized





international standards [9]; however, the authors recognize this time period may not encompass the entire burst release of the ionic species in question. Full ion release profiling may prove beneficial prior to future biocompatibility assessments to ensure maximum ion levels are assessed. The authors caution the in vitro test setup utilized in this study may affect both genototixicty results and ion release behavior. Though it can be concluded the conditions examined in this study resulted in non-genotoxic outcomes, this study comprises an initial biocompatibility screen of these cements and is not intended to predict full in vivo performance. With regards to the genotoxicity test, it is important to note (i) the inherent differences between prokaryotes and eukaryotes particularly in the metabolism of metal ions may limit the extrapolation of data and (ii) only one strain of S. typhimurium (TA98) was used for this work, while conventionally TA100, TA1535, and TA1537 are used in addition to TA98 [23].

[2] [3] [4] [5]

[6] [7] [8] [9]

[10] [11]

[12]

[13]

4. Conclusion This study adds a substantial contribution to the limited body of published investigations regarding the genotoxicity of modern GICs intended for use as implantable biomaterials, and, for the first time, probes the biological performance of a novel, Al-free, Gecontaining injectable GIC. The results presented herein demonstrate that this germanium-silicate GIC series can be considered as non-mutagenic to S. typhimurium tester strain TA98 and to E. coli tester strain WP2uvrA, a reassuring result that warrants further biocompatibility testing of these materials.

[14]

[15]

[16]

[17] [18]

[19]

Acknowledgments The authors wish to acknowledge the ACOA Atlantic Innovation Fund (Grant number: 197820) for funding support.

[20]

[21]

[22]

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