How much do resin-based dental materials release? A meta-analytical approach

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

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Review

How much do resin-based dental materials release? A meta-analytical approach K.L. Van Landuyt a,∗ , Tim Nawrot b,c , B. Geebelen d , J. De Munck a , J. Snauwaert e , K. Yoshihara a , Hans Scheers c , Lode Godderis c,f , P. Hoet c , B. Van Meerbeek a a

Leuven BIOMAT Research Cluster, Department of Conservative Dentistry, School of Dentistry, Oral Pathology and Maxillo-Facial Surgery, Catholic University of Leuven, Kapucijnenvoer 7, B-3000 Leuven, Belgium b Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium c Occupational, Environmental and Insurance Medicine, Catholic University of Leuven, Kapucijnenvoer 35/5, B-3000 Leuven, Belgium d Faculty of Engineering and Science, Catholic University of Leuven, Kasteelpark Arenberg 1, B-3001 Heverlee Belgium e Department of Chemistry, Catholic University of Leuven, Celestijnenlaan 200D, B-3001 Heverlee, Belgium f Idewe, External Service for Prevention and Protection at Work, Interleuvenlaan 58, B-3001 Heverlee, Belgium

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objectives. Resin-based dental materials are not inert in the oral environment, and may

Received 20 December 2010

release components, initially due to incomplete polymerization, and later due to degrada-

Received in revised form

tion. Since there are concerns regarding potential toxicity, more precise knowledge of the

24 February 2011

actual quantity of released eluates is necessary. However, due to a great variety in analyti-

Accepted 9 May 2011

cal methodology employed in different studies and in the presentation of the results, it is still unclear to which quantities of components a patient may be exposed. The objective of this meta-analytical study was to review the literature on the short- and long-term release

Keywords:

of components from resin-based dental materials, and to determine how much (order of

Composites

magnitude) of those components may leach out in the oral cavity.

Biocompatibility

Methods. Out of an initial set of 71 studies, 22 were included. In spite of the large statisti-

Systemic toxicity

cal incertitude due to the great variety in methodology and lack of complete information

Resin based dental materials

(detection limits were seldom mentioned), a meta-analytical mean for the evaluated eluates

Release

was calculated. To relate the amount of potentially released material components with the

HPLC

size of restorations, the mean size of standard composite restorations was estimated using

LC

a 3D graphical program.

GC

Results. While the release of monomers was analyzed in many studies, that of additives,

MS

such as initiators, inhibitors and stabilizers, was seldom investigated. Significantly more

Elution

components were found to be released in organic than in water-based media. Resin-based

Leaching

dental materials might account for the total burden of orally ingested bisphenol A, but

Quantity

they may release even higher amounts of monomers, such as HEMA, TEGDMA, BisGMA and

Identification

UDMA. Compared to these monomers, similar or even higher amounts of additives may

Eluate

elute, even though composites generally only contain very small amounts of additives.

∗

Corresponding author. Tel.: +32 16 33 75 87; fax: +32 16 33 27 52. E-mail address: [email protected] (K.L. Van Landuyt). 0109-5641/$ – see front matter © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2011.05.001

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d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

A positive correlation was found between the total quantity of released eluates and the volume of extraction solution. Significance. There is a clear need for more accurate and standardized analytical research to determine the long-term release from resin-based materials. Several guidelines for standardization are proposed. © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4.

1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Search strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Inclusion/exclusion criteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Re-calculation of the average released quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Customized database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Determination of the dimensions of average teeth and restorations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Systematic review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Average volume and surface of standardized restorations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Appendix calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

In spite of 150 years’ worth of good clinical performance, the use of amalgam as a tooth filling material remains controversial. The most common allegations against amalgam are environmental pollution and possible hazardous health effects due to release and systemic uptake of mercury [1–3]. The ongoing discussion about the safety of amalgam has also led to an increased focus on the safety of resin-based restorative materials [4]. The use of resin-based materials in dentistry is nowadays ubiquitous, and during the past decades composite restorations have proved to be a satisfying alternative for amalgam to restore traumatized and decayed teeth [5]. Resin-based dental materials generally consist of a polymer matrix and inorganic filler particles that are attached to the resin matrix through a siloxane coupling [6]. The most common resins used in dentistry are (meth)acrylates [7], but recently, new resin systems, such as ormocers (polysiloxane backbone with methacrylate sidebranches) and siloranes (silorane ringopening system) have been introduced [8]. Despite their growing popularity, there are concerns that resin-based materials may be toxic based on the fact that they may release components [9]. Three main routes of systemic intake of chemical substances released by resin-based restorations have been postulated: the first through ingestion of released compounds in the gastro-intestinal tract, the second through diffusion to the pulp through the dentinal tubules [9,10], and the third via uptake of volatile components in the

724 725 725 725 725 725 725 730 730 730 731 734 735 742 742 744

lungs [11,12]. The last route is of special importance for the dental practitioner and the dental personnel, while the first and second route are more relevant for the patient. Resin-based materials may release unpolymerized monomers, additives and filler components in the oral environment after placement of the restoration. Even though the patient may come into contact with large amounts of uncured monomers during the placement of the composite restoration, the release of unpolymerized monomers after polymerization causes most concerns in literature. Under clinical circumstances with a short curing time of usually not more than 40 s, and a temperature around 37 ◦ C in the oral cavity, composites are never polymerized to a full extent as the propagation of the crosslinking reaction drastically reduces the mobility of the monomers [13]. As a result, not only unbound substances, like additives, but also uncured monomers can leach out. Depending on the resin-based material, the degree of conversion can vary between 50 and 70% [14–16]. The maximum degree of conversion is reached only after 24 h due to a post-cure process (‘in-the-dark’ polymerization), which signifies that the polymerization rate immediately after light-curing may be even lower (30–40%) [15–18]. Filler leachability encompasses both release of complete filler particles after hydrolysis of the filler-matrix siloxane bond, and the release of filler components, such as SiO2 , Ba, Sr, Na due to hydrolysis and ion-exchange mechanisms [19–22]. Release of filler components has mainly been associated with progressive wear of composites; however little is known regarding possible health effects.

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Intra-oral degradation processes may induce additional release of components from resin-based restorations [23]. First, mechanical [24], hydrolytic and enzymatic [25] degradation may result in chain scission and release of polymeric breakdown products in the form of monomeric or oligomeric molecules. Most of these degradation products have probably not yet been identified [26]. Second, aging of composite materials may also lead to more porosities due to an interplay of mechanical swelling and water sorption and chemical/enzymatic degradation [27], and thus result in increased release of unpolymerized monomers that were initially trapped in the polymer network [24]. In literature, there are many indications that release of monomers and of some additives are potentially dangerous and might have compromising local or even systemic effects [28–30]. Apart from the well-documented allergenicity of monomers [31–34], several ingredients have been shown to be cytotoxic [35–40], genotoxic and mutagenic [42–47], and toxic to the reproductive system [48–52]. Besides the identification of hazards, risk assessment requires an accurate knowledge of the amounts of released compounds [53]. In spite of many analytical studies, the lack of standardized methodologies for quantification and of uniformity in presenting the results hinders correct interpretation of the quantities of released eluates. In other words, it is still unclear to what amount of specific components a patient may be exposed. This makes risk assessment of possible health hazards due to resin-based dental materials problematic. The objective of this study was to review peer-reviewed international literature on the unintended release of ingredients in the oral environment. Since there have been very few in vivo studies [41,54], only in vitro studies were included in this review. The quantities measured in studies that quantified the amount of released ingredients, were converted to a common unit. The main purpose was to gain knowledge on the total quantity of compounds that can be released by resin-based dental materials in the oral cavity.

2.

Materials and methods

2.1.

Search strategies

Using different online databases (PubMed, Web of Science and Embase), the international literature available until January 2010 was searched for papers that reported on the elution process of dental resin-based materials. The used keywords were: ‘resin-based’, ‘elution’, ‘eluate’, ‘dental composite’, ‘HPLC’, ‘LC’, ‘LC–MS’, ‘quantification’, ‘release’, ‘substances’, ‘ingredients’, ‘components’. Besides database searches, several papers (42%) were found by means of references in other papers.

2.2.

725

quantified data could be re-calculated to a molar quantity (mol) per volume or surface area of the tested specimens. Depending on the units used, this implied that the volume of the extraction solvent and the dimensions of the tested resinbased material needed to be mentioned. (4) In spite of the lack of standardization of the measurement intervals, most often researchers still measured the release after a 24-h time interval. It was therefore decided to include only papers that analyzed the released components after a 24-h or longer time interval. Studies that measured the release after several minutes were not included [56,57]. (5) Papers in which a relative (semi-quantitative) quantification was performed, could not be included [58–60]. (6) Studies in which released substances were measured in vivo [41], or in which the release and migration of components through the dentin were measured [10,61] were also not included. For those papers in which not all necessary information had been provided in the paper, attempts were made to obtain necessary information by contacting (email, letter) the corresponding author or co-authors. Papers with missing information that could not be recovered in this way, were not included. These inclusion criteria are listed in Table A1.

2.3.

Re-calculation of the average released quantity

Release data were expressed in a plethora of units (␮g/ml, ppm, mmol/ml, mg/g, wt/wt%, ␮g/cm2 ) (Table 1). Therefore data were re-calculated and expressed in mol per volume of the resin sample and mol per surface area of the resin sample. The calculations are shown in Appendix calculations). Since most researchers analyzed the incubation solution at 24 h, this was taken as a reference for comparison between the different studies included. For periods longer than 24 h, the total cumulative value of release for a compound was computed. The calculations can be found in Appendix A. Sometimes the quantitative data had only been displayed in a graph, without mentioning the absolute values [62]. If the absolute values could not be obtained through correspondence with the (co-)author, the graph was scanned and the values were measured using a graphical program (Coreldraw 10, Corel Corporation, Ottawa, ON, Canada). This was only done when the graph proved to be accurate enough, which was the case in the following studies with reference numbers [63–65].

2.4.

Customized database

A three-dimensional customized database was made to collect all information and variables per study. FileMaker Pro 9 (Filemaker Inc, Santa Clara, CA, USA) was used to design a three-dimensional database ((1) study, (2) tested resin-based material and (3) eluate) in which the included studies and data were gathered, and in which the calculations were performed.

Inclusion/exclusion criteria 2.5.

Data have been included only from studies (1) in which the release of components was quantified in vitro by incubating a sample of resin-based material in a solvent for a certain period of time (at least 24 h), (2) in which there was no preincubation period (so that all possible eluates were quantified after polymerization of the sample) [55], and (3) in which the

Statistical analysis

A weighted geometrical mean concentration of each eluate over all studies was calculated using a meta-analysis approach. A random-effects meta-analytical method that combined the concentration estimates from different studies was used to take into account the inter-study heterogeneity

726

Table 1 – Studies that met the inclusion criteria and could be included in the quantitative review. Authors

Title

Journal

Resin-based material

Analytical method

Units

Quantified eluates

Incubation medium

1

Al-Hiyasat AS, Darmani H, Milhem MM [95]

Clin Oral Investig 2005;9:21–5

Composite filling material

HPLC

␮g/ml

Bis-EMA BisGMA BPA TEGDMA UDMA

Culture medium without serum

2

Darmani H, Al-Hiyasat AS, Milhem MM [119]

Quintessence Int 2007;38:789–95

Composite filling material

HPLC

␮g/ml

Hamid A, Hume WR [94]

Dent Mater 1997;13:98–102

Sealants

HPLC

nmol/mm2 pmol/mm2

4

Imazato S, Horikawa D, Nishida M, Ebisu S [125]

J Biomed Mater Res B: Appl Biomater 2009;88:378–86

Composite filling material RM-GI cements

HPLC

␮g/ml

5

Imazato S, Horikawa D, Ogata K, Kinomoto Y, Ebisu S [64]

J Biomed Mater Res A 200615;76:765–72

Composite filling material RM-GI cements

HPLC

␮g/ml

BisGMA TEGDMA MMA HEMA

Distilled water

6

Kawai K, Takaoka T [63]

Am J Dent 2002;15:149–52

RM-GI

HPLC

␮g/ml

HEMA

Distilled water

7

Manabe A, Kaneko S, Numazawa S, Itoh K, Inoue M, Hisamitsu H, Sasa R, Yoshida T [68]

Effects of monomers eluted from dental resin restoratives on osteoblast-like cells Responses of MC3T3-E1 cells to three dental resin-based restorative materials, Fluoride, hydrogen ion and HEMA release from light-cured GIC restoratives Detection of bisphenol-A in dental materials by gas chromatographymass spectrometry

BPA BisGMA TEGDMA Bis-EMA UDMA TEGDMA BisGMA BPA BHT CQ UDMA TMA HEMA TEGDMA MMA HEMA

Dulbelco’s modified eagles medium without serum 96% ethanol/water

3

Cytotoxicity evaluation of dental resin composites and their flowable derivatives Cytotoxicity of dental composites and their leached components A study of component release from resin pit and fissure sealants in vitro

Dent Mater J 2000;19:75–86

Composite filling material Sealants

GC/MS

ng/mg

BPA

Phosphate buffer solution

Distilled water d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Distilled water

Mazzaoui SA, Burrow MF, Tyas MJ, Rooney FR, Capon RJ [72]

9

Michelsen VB, Moe G, Skalevik R, Jensen E, Lygre H [67]

10

Michelsen VB, Moe G, Strom MB, Jensen E, Lygre H [82]

11

Miletic V, Santini A, Trkulja I [132]

12

Moharamzadeh K, Van Noort R, Brook IM, Scutt AM [65]

Long-term quantification of the release of monomers from dental resin composites and a resin-modified glass ionomer cement Qualification of organic eluates from polymerized resin-based dental restorative materials by use of GC/MS

J Biomed Mater Res 2002;63:299–305

Composite filling material RM-GI

Electrospray ionization/MS

mmol/mm2

TEGDMA HEMA UDMA BisGMA BPA BisDMA

Water 50% Ethanol/water 75% Ethanol/water

J Chromotography B 2007;850:83–91

Composite filling material

GC/MS

␮g/mm2

Ringer’s solution 100% Ethanol

Quantitative analysis of TEGDMA and HEMA eluted into saliva from two dental composites by use of GC/MS and tailor-made internal standards Quantification of monomer elution and carbon-carbon double bonds in dental adhesive systems using HPLC and micro-Raman spectroscopy HPLC analysis of components released from dental composites with different resin compositions using different extraction media

Dent Mater 2008;24:724–731

Composite filling material

GC/MS

␮g/cm2

HEMA MEHQ CQ BHT DMABEE TEGDMA TMPTMA HMBP TIN P HEMA TEGDMA

J Dent 2009;37:177–184

Adhesives

HPLC

ppm/mg

HEMA TEGDMA BisGMA

75% Ethanol/water

J Mater Sci Mater Med 2007;18:133–7

Composite filling material

HPLC

mg/ml

TEGDMA BisGMA UDMA

Water Saline Dulbelco’s modified eagle medium Dulbelco’s modified eagle medium with serum Artificial saliva

Passive human saliva

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

8

727

728

Table 1 – (Continued) Title

Journal

Resin-based material

Analytical method

Units

Quantified eluates

Incubation medium

13

Nalc¸aci A, Ulusoy N, Atakol O [127]

Oper Dent 2006;31:197–203

Composite filling material

HPLC

ppm

TEGDMA BisGMA

HPLC-grade methanol

14

Ortengren U, Langer S, Göransson A, Lundgren T [100]

Eur J Oral Sci 2004;112:530–7

Composite filling material

Solid phase micro extraction/GC/MS

␮g/sample

MA MMA HQ EGDMA TEGDMA BPA

citratephosphate buffer (McIlvanes standard buffer solution)

15

Pelka M, Distler W, Petschelt A [71]

Time-based elution of TEGDMA and BisGMA from resin composite cured with LED, QTH and high-intensity QTH lights Influence of pH and time on organic substance release from a model dental composite: a fluorescence spectrophotometry and gas chromatography/mass spectrometry analysis Elution parameters and HPLC-detection of single components from resin composite

Clin Oral Investig 1999;3:194–200

Composite filling material

HPLC

nmol/ml

Water HCL

16

Polydorou O, Hammad M, König A, Hellwig E, Kümmerer K [74] Polydorou O, König A, Hellwig E, Kümmerer K [70]

Dent Mater 2009;25:1090–5

Core composites

HPLC–MS/MS

␮g/ml

Eur J Oral Sci 2009;117:68–75

Composite filling material

LC–MS/MS

␮g/ml

TEGDMA MA TMA BPA BisGMA CQ Quantacure BEA Irgacure 651 HMPB BHT BisGMA TEGDMA UDMA1 UDMA2 BPA BisGMA TEGDMA UDMA1 UDMA2 BPA

17

Release of monomers from different core build-up materials Long-term release of monomers from modern dentalcomposite materials

75% Ethanol/water

75% Ethanol/water

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Authors

18

19

20

Tabatabaee MH, Mahdavi H, Zandi S, Kharrazi MJ [124]

21

Yap AU, Han VT, Soh MS, Siow KS [133]

22

Yap AU, Lee HK, Sabapathy R [101]

Elution of monomers from two conventional dental composite materials Determination of bisphenol A and related aromatic compounds released from bis-GMA-based composites and sealants by high performance liquid chromatography HPLC analysis of eluted monomers from two composite resins cured with LED and halogen curing lights Elution of leachable components from composites after LED and halogen light irradiation Release of methacrylic acid from dental composites

Dent Mater 2007;23:1535–41

Composite filling material

LC–MS/MS

␮g/ml

Environ Health Perspect 2000;108:21–7

Composite filling material Sealants

HPLC/GC–MS

␮g/ml

J Biomed Mater Res B: Appl Biomater 2009;88:191–6

Composite filling material

HPLC

Oper Dent 2004;29:448–53

Composite filling material

Dent Mater 2000;16:172–9

BPA BisGMA UDMA TEGDMA BPA EBPA PBPA BADGE BisGMA Bis-DMA

75% Ethanol/water

␮g/l

BisGMA TEGDMA

Water Passive human saliva

HPLC

ppm

BisGMA TEGDMA

Acetonitrile

CE (capillary electrophoresis) UV detector

ppm

MA

Artificial saliva

Water

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Polydorou O, Trittler R, Hellwig E, Kümmerer K [69] Pulgar R, Olea-Serrano MF, Novillo-Fertrell A, Rivas A, Pazos P, Pedraza V, Navaquantitatives JM, Olea N [75]

Abbreviations: BADGE: bispenol A diglycidyl ether; Bis-DMA: bisphenol A dimethacrylate; Bis-EMA: ethoxylated bisphenol A glycol dimethacrylate; BisGMA: bisphenol A diglycidyl methacrylate; BHT: butylated hydroxytoluene; BPA: bisphenol A; CQ: camphorquinone; DMABEE: ethyl 4-(diethylamino)benzoate; EBPA: bisphenol A ethoxylate; EGDMA: triethylene glycoldimethacrylate; HEMA: 2-hydroxyethyl methacrylate; HMPB: 2-hydroxy-4-methoxybenzophenone; HQ: hydroquinone monomethyl ether; Irgacure 651: 2,2-dimethoxy-1,2-diphenyl-ethan-1-on; MA: methacrylic acid; MEHQ: 4-methoxyphenol or monoethyl ether hydroquinone; MMA: methyl methacrylate; Quantacure BEA: 2-n-butoxyethyl-4-dimethyl-aminobenzoat; PBPA: bisphenol A propoxylate; RM-GI: resinmodified glass ionomer; TEGDMA: triethylene glycol dimethacrylate; TIN P: 2-(2-hydroxy-5-methylphenyl) benzotriazole; TMA: 3-(trimethoxysilyl)propyl methacrylate; TMPTMA: trimethylolpropane trimethacrylate; UDMA: urethane dimethacrylate; UV-absorber: 2-hydroxy-4-methoxybenzophenone.

729

730

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

[66]. To include measurements below the detection limit, they were substituted by the value of the detection limit was inserted for quantification. When the limit of detection was not mentioned and could not be retrieved from the authors, an estimated mean concentration was imputed equal to the lowest measured concentration in the other studies (Table A2). These analyses were performed separately for concentrations before and after 24 h, and for concentrations per surface and per volume. Due to the right-skewed distribution of the concentrations in most situations, the analyses were performed on log-transformed values. However, results are reported after back-transformation to the original scale. Since the studyspecific means are log-transformed, the mean over the studies obtained after back-transformation is a geometric mean. A z-test (significance level of 5%) was used to compare the weighted means of the most frequently analyzed compounds (BPA, BisGMA, TEGDMA, UDMA and HEMA) between waterbased and organic incubation media. Spearman correlation test was performed to evaluate correlations between (1) the release and the sample surface and volume, (2) between the release and the volume of the incubation solution, (3) between the release and the pH of the incubation solution, (4) between the release and the presence of an oxygen inhibition layer, and (5) between the leached quantity of a compound and its molecular mass. The Spearman correlation test was performed with Statistica 10.0 (StatSoft, Tulsa, OK, USA).

2.6. Determination of the dimensions of average teeth and restorations Standardized polymer teeth (Frasaco, Tettnang, Germany), which are used for dental education, were optically scanned (3Shape D200, 3Shape, Copenhagen, Denmark) to obtain three-dimensional digital information in the form of a triangulated pointcloud. By use of the 3-matic software (Materialise NV, Leuven, Belgium), the total surface area and the volume of each tooth crown was computed. In addition, the surface area exposed to the oral environment and the volume of typical restorations was determined to serve as an example.

3.

Results

3.1.

Systematic review

In total, 71 research papers (3 review papers, 6 studies assessing the release qualitatively and 62 studies determining the release quantitatively) were found dealing with the topic of release from resin-based materials. Twenty-two papers could be included in the quantitative review, representing 716 separate data on 25 different eluates (Table 1). Typically, a flat cylindrical-shaped resin-based specimen was incubated in a solvent after polymerization, and after a certain period the quantity of pre-determined eluates in the solvent was analyzed. In the majority of studies, the release of ingredients was analyzed by high performance liquid chromatography (HPLC). Fewer studies utilized GC/MS

(gas chromatography/mass spectrometry) or LC/MS (liquid chromatography/mass spectrometry) (Table 1). Standard calibration curves were usually made by analyzing known concentrations of purchased ingredients, which were used for further quantification of the eluates from the immersed resin-based sample. Alternatively, library databases of spectra of known compounds were used in studies that employed mass spectroscopy [67]. Only a minority of authors mentioned a detection limit for quantification of the analyzed molecules (Table A3), which severely hindered the statistical analysis. Some authors also measured the release from uncured samples [68–70], but these results were not included in the database for meta-analysis. To determine the effect of the surface area on the release, Pelka et al. [71] determined the release from pulverized samples. These results were also not included in the database. All compounds that were analyzed are listed in Table 2. The monomers bisphenol A diglycidyl methacrylate (BisGMA), 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) and the contaminant bisphenol A (BPA) were most frequently analyzed. Initiators, co-initiators, inhibitors, silane-coupling agents and possible degradation products were seldom evaluated. Different kinds of resin-based dental materials were investigated. Most frequently, the eluates of resin-based direct cavity filling materials were analyzed, but core composites, adhesives, resin-based cements and sealants were also assessed. There was great variety in chemical composition among the tested resin-based dental materials (Table 1). Regarding monomer/polymer system, they contained either methacrylates, poly-acid modified methacrylates (so-called ‘compomers’), siloranes, polyacrylic acid (so-called ‘glass ionomer’) or ormocers. Most frequently, the release was determined within one week (short-term); only few studies researched the long-term release: Mazzaoui et al. [72] after 3 months, Ortengren et al. [73] after 6 months, Polydorou et al. [70] after 12 months. Irrespective of the duration of incubation of the samples, a total cumulative mean value for total release for each eluate was calculated in each study. However, the included studies also made use of different methodologies to measure the long-term release. Usually, the samples were left undisturbed until the predetermined moment for analysis of the incubation solution, but in some studies, the incubation solution was renewed (‘refreshed’) in between [69,70,74]. The solvents that were used to incubate the sample could be divided in two main groups: (1) water or aqueous mixtures, such as cell culture media, human or artificial saliva or water-based buffer solutions; and (2) organic solvents, usually ethanol- or methanol-based, including their aqueous mixtures (Table 1). In the majority of the studies, an incubation medium belonging to the first group was used. The amount of incubation solvent also varied, ranging from 0.1 ml to 10 ml. Apart from the detection limit for quantification, which was only mentioned in few studies [67,69,70,74,75], the studies often also lacked information on specific details regarding the material and methods, such as the size of the resin-based samples, the curing light and the applied light-curing energy.

731

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Table 2 – Analyzed molecules, their molecular mass and function. Trivial name

Chemical name

Molecular mass

Function

Evaluated in studies

Number of records

BADGE

Bisphenol A diglycidyl ether

340.45

1/22

28

BHT Bis-DMA

Butylated hydroxytoluene Bisphenol A dimethacrylate

220 364

3/22 1/22

20 27

Bis-EMA

Ethoxylated bisphenol A glycol dimethacrylate Bisphenol A diglycidyl methacrylate Bisphenol A

452

Bisphenol A related compound Inhibitor Bisphenol A related compound Monomer

2/22

14

512.59

Monomer

15/22

125

228.29

11/22

94

Camphorquinone Ethyl 4-(dimethylamino)benzoate Bisphenol A ethoxylate

166 193

Contaminant (derivative?) Initiator Co-initiator

3/22 1/22

20 8

1/22

28

198

1/22

3

130.14

Monomer

7/22

41

228.25

UV-absorber

2/22

19

110.1 256.3

Inhibitor Initiator

1/22 1/22

3 11

MA

Ethyleneglycol dimethacrylate 2-hydroxyethyl methacrylate 2-hydroxy-4methoxybenzophenone Hydroquinone 1,2-Diphenyl-2,2dimethoxyethanone Methacrylic acid

Bisphenol A related compound Monomer

86

3/22

18

MEHQ MMA PBPA

4-Methoxyphenol Methylmethacrylate Bisphenol A propoxylate

1/22 3/22 1/22

8 9 28

Quantacure BEA

2-n-butoxyethyl-4dimethyl-aminobenzoat 3-(Trimethoxysilyl)propylmethacrylate Triethylene glycol dimethacrylate 2-(2-Hydroxy-5methylphenyl)benzotriazole Trimethylolpropane trimethacrylate Diurethane dimethacrylate, different isomers and molecules possible

Monomer, degradation product Inhibitor Monomer Bisphenol A related compound Photoinitiator

1/22

11

2/22

12

286.32

Silane coupling monomer Monomer

17/22

108

225.1

UV-stabilizer

1/22

8

338.2

Monomer

1/22

8

470.56/498

Monomer

8/22

65

BisGMA BPA CQ DMABEE EBPA

EGDMA HEMA HMBP HQ Irgacure 651

TMA (silane GF 31) TEGDMA TIN P (drometrizole) TMPTMA UDMA

3.2.

316

124.14 100.12 344

248.35

Release

In Tables 3 and 4, the geometrical mean together with the minimum and maximum of the released quantities are shown, expressed in mol per surface (␮mol/mm2 ) and mol per volume (␮mol/mm3 ). The maximum measured value was sometimes more than 100,000 times larger than the minimum, indicating a lot of variation regarding the released quantities. This is partly due to large variation between the different studies regarding the type of resin-based material that was analyzed, but also due to difference in measuring techniques and type of

analysis used. The variation between different studies is also illustrated in Figs. 1 and 2 in which the mean 24-h release values per study are depicted for water-based and organic solutions. In general, comparable or lower quantities of BPA were released compared to the analyzed monomers (Tables 3 and 4, Figs. 3 and 4). Among the monomers, HEMA was released the most, followed by TEGDMA, UDMA and BisGMA. These differences among the monomers were most clear when evaluating the released amount per volume (Fig. 4). Very small amounts of BisGMA were released in water-based

24 h release

Eluate n

% >DL

Total cumulative release

Geometric Lower C.I. mean

Upper C.I.

Minimum

Maximum

n

% >DL

Geometric Lower C.I. mean

Upper C.I.

Minimum

Maximum

/ / / 0.00027 0.01752 / / 0.00233* 0.32660 / 0.00273* / 1.68978 / 0.18061 / 0.04493 / / / /

/ / / 0.00015 0.00097 / / / 0.09007 / / / 0.71642 / 0.06420 / 0.00985 / / / /

/ / / 0.00050 0.31510 / / / 1.18427 / / / 3.98560 / 0.50805 / 0.20495 / / / /

/ / / 0.00006 0.00001 / / 0.00003 0.02613 / 0.00120 / 0.20621 / 0.00026 / 0.00010 / / / /

/ / / 0.42000 67.00000 / / 0.00670 134.00000 / 0.00482 / 114.12273 / 1.29442 / 22.63160 / / / /

5 / 10 19 19 5 4 3 15 4 3 / 7 4 9 / 29 4 1 4 16

20% / 40% 84% 47% 80% 75% 100% 47% 25% 100% / 100% 100% 44% / 69% 25% 0% 25% 44%

0.03506 / 0.35742 0.07320 0.01600 0.20372 0.56001 0.00617* 0.37647 / 0.01874* / 2.84550 0.24699 0.00307 / 0.15296 0.02221 / 0.57599 0.08158

0.02706 / 0.29278 0.03989 0.00301 0.09106 0.33531 / 0.11599 / / / 0.21473 0.04891 0.00192 / 0.07584 / / / 0.03669

0.04541 / 0.43632 0.13434 0.08520 0.45580 0.93529 / 1.22187 / / / 37.70792 1.24735 0.00489 / 0.30850 / / / 0.18143

0.07955 / 0.29108 0.03862 0.00000 0.07530 0.36269 0.00094 0.65314 / 0.02891 / 5.98550 0.06444 0.00037 / 0.04715 0.02221 / 0.57599 0.12000

0.07955 / 0.61538 2.08841 124.00000 0.57831 1.00518 0.01074 / 0.75844 0.31537 / 126.46032 2.09441 3.64911 / 159.72000 0.02221 / 0.57599 8.91040

0.21090 / 0.30227 0.10013 0.55063 1.62824 0.40463 1.33249 / 0.32558 0.18672 0.48221 1.17133 0.07694 /

0.02003 / 0.17895 0.00340 0.12880 0.55167 0.09580 0.33077 / 0.07326 0.12105 0.08440 0.18468 0.03768 /

2.22080 / 0.51056 2.94098 2.35404 4.80565 1.70901 5.36783 / 1.44688 0.28802 2.75494 7.42903 0.15708 /

0.03182 / 0.04000 0.00509 0.13554 1.86528 1.61365 7.18983 / 0.08055 0.08870 4.93114 9.81668 0.01000 /

5.22273 / 21.65858 .00000 2.04819 3.36788 475.00000 7.18983 / 0.86999 19.49526 4.93114 9.81668 0.95978 /

/ 4 35 35 / / 10 / / / 35 / / 29 /

/ 75% 100% 34% / / 20% / / / 77% / / 48% /

/ 21.07339 3.48943 0.10816 / / 0.18630 / / / 0.31228 / / 0.31907 /

/ 2.86967 1.24740 0.06610 / / 0.08514 / / / 0.12090 / / 0.06429 /

/ 154.75222 9.76119 / / / 0.40763 / / / 0.80660 / / 1.58360 /

/ 16.19718 0.17000 0.00785 / / 399.00000 / / / 0.26569 / / 0.09000 /

/ 367.24597 94.78309 399.00000 / / 630.00000 / / / 242.85607 / / 268.00497 /

n is the number of measurements, including those that were below the detection limit and those that were replaced by an estimated mean concentration that was equal to the lowest measured concentration in the other studies. The minimum and maximum are the minimum and maximum mean values that were measured. ∗ Only one measurement; no standard deviation; could not be included in the meta-analysis. The average was calculated.

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Water-based incubation solution 7 0% BHT / / Bis-DMA / / Bis-EMA 27 44% Bis-GMA 15 20% BPA 7 0% CQ / / DMABEE 100% EGDMA 3 15 60% HEMA 0% HMBP 6 3 100% HQ 0% 6 Irgacure 651 100% MA 13 / MEHQ / 67% MMA 6 0% 6 Quantacure BEA 32 70% TEGDMA / / TIN P 6 0% TMA / / TMPTMA 11 0% UDMA Organic incubation solution 4 75% BHT / / Bis-EMA 41 85% BisGMA 25 32% BPA 100% CQ 4 4 75% DMABEE 14 43% HEMA 4 25% HMBP MA / / MEHQ 4 100% 78% TEGDMA 45 4 25% TIN-P TMPTMA 4 25% UDMA 25 40% 0% UDMA1 9

732

Table 3 – Released quantity of ingredients from resin-based dental material per surface area of the resin-based sample, expressed in nmol/mm2 of the resin sample.

Table 4 – Released quantity of ingredients from resin-based dental material per volume of the resin-based sample, expressed in nmol/mm3 of the resin sample. Eluate

24 h release n

% >DL

Geometric Lower C.I. mean

Upper C.I.

Minimum

Maximum

n

% >DL

Geometric Lower C.I. mean

Upper C.I.

Minimum

Maximum

0.61228 / 0.24275 / 0.00143 0.00242 / 0.26101 0.00995* 2.28512 / 0.01165 / 2.12483 0.00046* 1.49764 / 0.20550 / /

0.32498 / 0.15461 / 0.00032 0.00170 / 0.13803 / 0.35120 / / / 0.86944 / 0.46627 / 0.01939 / /

1.16604 / 0.38116 / 0.00640 0.00343 / 0.49354 / 14.86813 / / / 5.19289 / 4.81040 / 2.17804 / /

0.09179 / 0.00993 / 0.00010 0.00000 / 0.07252 0.00014 0.74541 / 0.00514 0.00000 0.28870 0.00113 0.69698 / 0.00018 / /

10.93521 / 3.90911 / 0.88960 0.11493 / 1.52127 0.02858 99.35260 / 0.02056 / 486.92365 1.81219 8.51087 / 31.68424 / /

/ 1 / 10 13 14 1 / 3 6 / 3 / 7 9 / / 19 / 11

/ 0% / 40% 71% 47% 0% / 100% 33% / 100% / 100% 56% / / 79% / 55%

/ / / 0.58115 0.07651 0.00108 / / 0.02630* 18.18137 / 0.08000 / 18.18137 0.01348 / / 3.14800 / 1.59422

/ / / 0.52475 0.03766 0.00075 / / / 1.47279 / / / 1.47279 0.00546 / / 2.10210 / 1.00985

/ / / 0.64360 0.15541 0.00154 / / / 224.44670 / / / 224.44670 0.03329 / / 4.71431 / 2.51674

/ / / 0.52394 0.22107 0.00000 / / 0.00400 97.83614 / 0.01230 / 0.83797 0.00158 / / 0.23831 / 1.92112

/ / / 0.80000 2.71494 0.00959 / / 0.04580 98.25195 / 1.34600 / 539.56405 3.28420 / / 240.40001 / 11.58351

/ 0.57136 0.01065 123.59886 2.51396 0.96373 /

/ 0.33137 0.00509 58.56945 2.16048 0.70264 /

/ 0.98515 0.02230 230.83011 2.92527 1.32186 /

/ 0.23318 0.00706 31.66192 0.12320 0.73255 /

/ / 0.03532 374.23772 27.07675 1.33303 /

4 30 19 5 25 19 9

75% 97% 32% 100% 100% 32% 0%

29.71748 7.58582 0.00174 126.07264 5.67454 / /

4.32755 2.50953 / 60.99033 2.11938 / /

204.07127 22.93039 / 260.60407 15.19334 9.87277 /

21.05634 0.59975 0.01091 31.66192 0.36901 5.63798 /

477.41976 123.21801 0.40826 374.23772 315.71289 348.40646 /

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Water-based incubation solution 28 64% BADGE 7 0% BHT 27 44% Bis-DMA / / Bis-EMA 50 38% Bis-GMA 41 73% BPA 7 0% CQ 28 32% EBPA EGDMA 3 100% 75% HEMA 8 0% HMBP 6 3 100% HQ 6 0% Irgacure 651 13 100% MA 6 67% MMA 28 14% PBPA 6 0% Quantacure BEA 25 92% TEGDMA 6 0% TMA 6 0% UDMA Organic incubation solution Bis-EMA / / BisGMA 36 83% BPA 15 27% 5 100% HEMA TEGDMA 31 100% UDMA 15 20% UDMA1 9 0%

Total cumulative release

n is the number of measurements, including those that were below the detection limit and those that were replaced by an estimated mean concentration that was equal to the lowest measured concentration in the other studies. The minimum and maximum are the minimum and maximum mean values that were measured. ∗ Only one measurement; no standard deviation; could not be included in the meta-analysis. The average was calculated.

733

734

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Fig. 1 – Mean 24-h release (␮mol/mm2 ) per study in water-based incubation solutions, illustrating the variety between the different studies. The y-axis is logarithmic. The red dot is the geometric mean. Ingredients not included in the table were not analyzed by the mentioned authors. The percentage of measurements above the detection limit (%>DL) is given. Abbreviations: DL: detection limit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

media. Compared to the monomers, similar or even higher amounts of additives, such as butylated hydroxytoluene (BHT), 4-methoxyphenol (MEHQ), camphorquinone (CQ), ethyl 4-(diethylamino)benzoate (DMABEE), 2-(2-hydroxy-5methylphenyl) benzotriazole (TIN-P) and trimethylolpropane trimethacrylate (TMPTMA) were set free. In most cases, the release in organic solvents was higher than in water or in a water-based solution (Figs. 3 and 4). However, due to the large confidence intervals, this difference was not always statistically significant. Nevertheless, a statistically significant difference between the released amount of BPA, BisGMA and HEMA in water-based and organic media could be found. There were proportionally more measurements below the detection limit in water-based solutions than in organic solvents for BisGMA, HEMA, TEGDMA and UDMA. Spearman correlation test showed that there was a weak, but significant (positive) correlation between the released amount of each eluate and the total surface area of the resinbased specimen (rs = 0.23), and between the released amount of each eluate and the amount of solution in which the

resin-based specimen was immersed (rs = 0.14). There was no significant correlation between the released amount of each eluate and the total volume of the resin-based sample, or the pH of the incubation solution. No correlation could be found between the released amount and the presence or absence of an oxygen-inhibition layer. There was a trend for compounds with a high molecular mass to leach less, but this correlation was not significant.

3.3. Average volume and surface of standardized restorations The calculated average of the total crown surface area (mm2 ) and total crown volume (mm3 ) for superior teeth are shown in Table 5. As a restoration not always involves the total tooth crown, estimates of some typical restorations are also shown. Combined with the release results as shown in Tables 3 and 4, these data allow calculation of the possible release according to the size of a restoration.

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

735

Fig. 2 – Mean 24-h release (␮mol/mm2 ) per study in organic incubation solutions, illustrating the variety between the different studies. The y-axis is logarithmic. The red dot is the geometric mean. Ingredients not included in the table were not analyzed by the mentioned authors. The percentage of measurements above the detection limit (%>DL) is given. Compared to the release in water-based solutions, a clear trend to higher amounts of released compounds can be observed. Abbreviations: DL: detection limit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

4.

Discussion

Several methods are currently available for quantification of the release of components from dental resin-based materials. Gravimetrical measurement of a composite sample before and after extraction of components is the least expensive method [17,19,73,76,77] and indicated that depending on the extracting solvent, up to 10–11 wt% can be extracted from resin-based restorative materials [17,78–80]. To determine the individual release of separate compounds, sophisticated analytical methods should be used. High performance liquid chromatography (HPLC), liquid chromatography (LC), gas chromatography (GC) are based on the separation of dissolved components. When these techniques use a classical detector, they cannot identify compounds. This implies that identification and quantification of eluates can only be per-

formed after calibration with standards, and consequently only purchasable compounds can be quantified. GC requires the ingredients to be vaporized and to be stable at high temperatures [81] and is more suitable for analysis of low molecular weight compounds, whereas HPLC and LC are more applicable for analysis of high molecular weight compounds [82]. Mass spectrometry (MS) on the other hand does give information on the molecular mass of the compound and allows detection and quantification of compounds based on their ionization, and computation of the mass-to-charge ratio. For identification, library databases of spectra of known compounds can be consulted. MS can be used as sophisticated detector in conjunction with one of the abovementioned analyses and allows also detection of degradation products. Other chemical methods, such as infrared spectroscopy [83], and Fourier transform infrared spectroscopy [84,85] have been used for analysis of extracts from dental composites, but

736

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Fig. 3 – Statistical comparison between the releases of BPA, BisGMA, HEMA, TEGDMA and UDMA in water-based or organic incubation solutions, expressed per surface as ␮mol/mm2 of the resin sample. A red asterisk indicates a statistically significant difference (p < 0.05) between the release in water-based and organic solutions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

are nowadays regarded as outdated as the interpretation of the spectrogram is difficult and not molecule-specific. In the studies included in this review, HPLC was most frequently used, which must be explained by the wide availability of this technique and the fact that the equipment is less expensive than that for LC/MS or GC/MS. The detection limit of LC/MS and GC/MS is, however, in general lower than that of HPLC. The objective of this literature review was to gather the results of as many studies as possible to gain information on the order of magnitude of the ingredient concentrations released. Even though many extraction protocols can be followed to determine the elution from resin-based materials, most researchers analyzed the released substances in vitro by immersing a standardized block of the resin-based material in a certain amount of solvent for a specified period of time and by subsequently analyzing the incubation solution. While this simple test set-up hardly resembles the clinical situation, which is typically characterized by a rapid and continuous flow and removal of saliva [86], it can give an indication on the quantities of components that may be released. A meta-analytical approach was preferred as a meta-analytical weighed mean takes into account the number of measurements. Out of 68 studies that measured release of components from resin-based materials, only 22 could be included. Besides missing information, studies often could not be included

because they assessed the release in a qualitative way (detected or not detected) [73,87,88] or in a semi-quantitative manner. For example, the released quantities were often expressed relative to an internal standard, usually caffeine [58–60,80,89], but sometimes also in optical density (peak area under curve of chromatogram) [27,81,90,91,49,92]. Self-evidently, semi-quantitative data may allow comparison between the different tested materials that were tested in that particular study, but cannot be used for comparison between studies. Several different problems were encountered performing the meta-analysis. The first issue was that the released quantities needed to be converted to a common unit, as the many different presentations of the results and the use of different units hindered comparative interpretation of the released quantities (Table 1). Most frequently, the release is expressed as a concentration, but some researchers already presented their results per surface area or volume [67,72,82,93,94]. It was therefore first decided to express the released amount not in mass, but in a molar quantity (mol). Since different monomers differ from each other in molecular mass, quantities in the form of mass cannot be compared. As ‘Mol’ is a measure for the number of molecules (1 mol = 6.02214 × 1023 ), it was judged more suitable for comparison of the released quantities of different monomers and substances. Secondly, expressing the quantity per surface area or volume allows referring to in vivo restorations and estimation of released quantities based on

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

737

Fig. 4 – Statistical comparison between the releases of BPA, BisGMA, HEMA, TEGDMA and UDMA in water-based or organic incubation solutions, expressed per volume as ␮mol/mm3 of the resin sample. A red asterisk indicates a statistically significant difference (p < 0.05) between the release in water-based and organic solutions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

the average size of the restoration. In addition, the association between release and surface area or volume could easily be investigated. The second problem was the great diversity and heterogeneity between the studies, which is also reflected in the large variation in results between different studies, as shown in Figs. 1 and 2. Due to lack of standardization, the studies not only differed regarding the tested resin-based materials, but also regarding the test methodology and the test set-up. There were numerous differences regarding the shapes and sizes of the tested resin-based samples, the volume of extraction liquid that was used and the testing period. The results could be presented as cumulative or non-cumulative. There were also differences regarding the moment of immersion of the sample; some authors preferred to wait for 24 h to allow for a post-irradiation cure [95]. Another important issue was the fact that studies often failed to describe their study parameters completely. In particular the fact that the limit of detection/quantification was most often not mentioned severely hindered the statistical analysis. Including measurements below the detection limit as zero would lead to a bias to lower mean release values, while not including them would definitively lead to a bias to higher mean release values. Hence, it was decided to replace undetected measurements by the limit of detection, on the condition that an exact limit of detection/quantification was given in the materials and methods of the study. This

approach may result in an overestimation of the release of each compound, which should, however, be regarded as more convenient since the results of this review study may be used for risk assessments. Unfortunately, the majority of studies did not mention the detection limit (only 5 out of 22, Table A3), and therefore an estimate of the limit of detection was made by determining the lowest measured quantity in other studies to replace undetected measurements. Thanks to this strategy, the results of studies that otherwise needed to be excluded could be included, even though it is clear that this entailed a lot of uncertainty. Yet, the detection limits that were mentioned for the eluates were more or less in correspondence with the lowest measured mean value. It is generally known that the results of a (meta-analytical) review are as reliable as the included studies and that only methodologically sound studies should be included. Considering the abovementioned weaknesses of the included studies, the results of this meta-analysis should not be regarded as exact but rather as estimates, especially due to the uncertainty of the imputed quantities for the measurements below the detection limit. Yet, the merit of this study lies in the fact that it gives an order of magnitude of released quantities based on real-size restorations. This information has so far not been available in scientific literature. In this review, data on 25 eluates could be gathered. The release of monomers was most often measured, whereas the release of additives and/or initiators or inhibitors was seldom

738

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

Table 5 – Mean total exposed crown surface area (SA) and volume (Vol) of average teeth, as computed by three-dimensional software. The surface area and volume of some typical restorations is given as illustration.

evaluated. Beside the filler particles (60–80 wt%), monomers make out the proportionally largest part of the composition of dental composites (20–40 wt%) and may thus represent the largest risk for biotoxic effects upon absorption. Few researchers investigated the release of additives and initiators/inhibitors [96]. This must not only be explained by the small quantities that are usually used (1–3 wt%), but also by the fact that researchers are often oblivious of the exact ingredients of composites. Manufacturers of dental materials are very reluctant to reveal the components in their products due to commercial protectionism and there is to date no legislation obliging them to disclose all ingredients. A product’s MSDS (Material Safety Data Sheet) should give information on all its ingredients with a concentration above 1% [79]. Unfortunately, the MSDS has several times been shown to be incomplete, even for ingredients with a concentration above 1%, and can thus not be considered as very reliable [34,97]. In addition, resin-based materials may contain substances that were not meant to be included, like impurities from the synthesis of the monomers [80,98,99] or degradation products [24,25,27,87]. With the exception of bisphenol A, the studies included in this study seldom evaluated the release of degradation products. Pelka et al. [71], Ortengren et al. [100] and Yap et al. [101] analyzed the release of MA, which may elute from composites upon hydrolysis of the ester group in monomers [102,103]. Pulgar et al. [75] analyzed the release of bisphenol-A-related compounds, such as Bispenol A diglycidyl ether (BADGE), bisphenol A ethoxylate (EBPA) and bisphenol A propoxylate (PBPA). The origin of these compounds has not yet

been fully elucidated, but there are indications that some of them may be degradation products [41,24]. Oysaed et al. [104] evaluated the release of formaldehyde, which was hypothesized to be derived from degradation of methacrylates. Because the mean values as given in Tables 3 and 4 still cannot be related to the oral situation, the total tooth surface area and volume of the superior dentition was estimated by computation using three-dimensional digital models of average-sized teeth (Table 5). Combining the results of the meta-analysis and the three-dimensional models should allow calculation of an approximate exposure of patients to xenobiotics originating from dental materials, and estimation of so-called ‘worst-case’ exposure scenarios. To give an example, the potential mean release of BisGMA in a waterbased solvent from a mesio-distal restoration of a molar after 24 h can be estimated to be between 0.3 and 1.7 nmol (calculated from mean release per volume or surface respectively) In an organic solvent, the mean 24-h release can be estimated between 1.4 and 9.5 nmol (calculated from mean release per volume or surface respectively). Higher amounts will be found when the maximum measured value is used for this calculation. The most notorious compound that may leach from resinbased materials is no doubt BPA, which is known to act as a estrogen–receptor agonist and thus may cause so-called ‘endocrine disruption’ [54,105–107]. Composites may contain BPA as an impurity from the synthesis process of BisGMA [98,108], but there are also indications that BPA may be released from composites following degradation of BisGMA

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

[41,109]. In literature, there is much controversy about the amount of BPA that may be released and the pharmacokinetics of this xeno-estrogen [56,68,110,111]. Especially the relevance of the released amounts of BPA from dental materials is up to debate, as BPA is omnipresent in epoxyresins and polycarbonates that are used as food containers [112–114]. Based on the results of this meta-analysis, it can be computed that one full crown restoration of a molar (with surface area 573 mm2 ) may release in the worst case (release per surface – organic solution – maximum measured value (231 nmol/mm2 )) 132.36 ␮mol after 24 h (however, measured for Scotchbond MultiPurpose [72], a dental adhesive that is never used in large quantities) or on average 57.38 nmol (release per surface – organic solution –geometric mean (0.1 nmol/mm2 )). Typical BPA migration levels from BPA-based food-contact materials are assumed to be less than 10 ␮g/kg food (=43.8 nmol) [115–117], indicating that the 24-h release of BPA from dental materials may be relevant in patients with multiple large restorations and that resin-based dental materials may represent an relevant source of BPA next to contaminated food, especially in case of large and/or multiple restorations. However, the Tolerable Daily Intake proposed by EFSA (European Food Safety Agent) was determined to be 0.05 mg BPA/kg bodyweight/day, which is equal to a limit of 220 nmol/kg bodyweight/day. This indicates that the levels of BPA released from resin-based dental materials may be safe [115,118], but more research is warranted, especially regarding the safety of composite restorations in children. The amounts of released monomers are in the same range or even higher in comparison with the released amounts of BPA. By expressing the released quantities in mol, the released quantity of each eluate can easily be compared (Tables 3 and 4). Among the eluted monomers, the monomer that is released in the highest quantity is HEMA. This must be explained by its small dimension and low molecular weight. In spite of its relative hydrophilicity, a higher amount of HEMA is released in organic solutions than in water-based solutions. Second most released monomer is TEGDMA, while BisGMA is released in relatively lower concentrations, especially in water-based solutions. The high molecular weight of BisGMA, its voluminous dimensions and its low solubility in water explain the difference in released amounts. In none of the included studies, UDMA could be detected in a water-based solution after 24 h. Yet, it was striking that additives were also released in relatively high concentrations, in particular in organic solutions, in spite of the small concentrations that are used in resin-based materials. Even though the released averages for additives must be interpreted with care since they were usually based on the measurements of few studies, their release may be explained by the fact that additives are not bound to the resin matrix. Despite the significant heterogeneity of the studies included in the review, the obtained database revealed some correlations. First, this study confirmed that the type of extracting solution plays an important role. For the majority of the tested eluates, there was a higher release in an organic solvent than in a water-based solution (Figs. 3 and 4). This corroborates with previous research [17,67,72,76,78,80,119,120]. In water-based solutions, the release of UDMA was so small

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that it could not be measured after 24 h. There were also significantly fewer measurements below the detection limit in organic solvents than in water-based solutions. The difference between the two kinds of solutions must be explained by the higher solubility of monomers, which are often rather hydrophobic, in organic solvents. Thanks to their high dipole moment but relatively small dielectric constant, alcoholic solutions are good solvents for methacrylate cross-linking monomers [121]. In addition, ethanol and ethanol/water solutions better penetrate methacrylate polymers, leading to more sorption, swelling and plasticization [72,78,120,122,123]. Consequently, the larger pores allow more unbound substances to elute from the polymer network. Even though an alcoholic extraction solution is clinically not relevant, it may allow evaluation of extraction of all possibly extractable compounds. Several authors [75,100] tested the effect of the pH of the incubation solution on the release, and observed that the release of components was pH dependent. They found that depending on the specific tested eluate, there was in general a higher release in either acidic or alkaline solutions. In addition, Pelka et al. [71] showed that HCl induced the release of MA from a dental composite, as a result of hydrolysis of the ester in methacrylates. In this review, no significant correlation could be observed between the pH of the incubation solution and the release. This is most probably due to the fact that there the majority of measurements included in the database had been done in an extracting solution with a neutral pH. This review also allowed assessing the relevance of the surface area and the volume of the cured resin-based material. Several authors already indicated that the release depends to a large extent on the actual surface area that is exposed [57,94]. Pelka et al. [71] observed significantly more release of TEGDMA in pulverized composite samples. Most included studies used flat cylindrical-shaped samples with a relatively large surface area and a small volume. This review confirmed that there is a weak but significant correlation between the exposed surface of the tested sample and the release, while no significant correlation could be found for the volume. A plausible physical explanation for the association between the release and the surface area may be the fact that released components originate from the surface of the samples, while unbound compounds inside the polymer sample will be trapped in the crosslinked polymer and can thus not leach (Fig. 5). Probably, the correlation between the surface area of the sample and the release would have been stronger if the authors had mentioned the real exposed surface area. Due to the great variety in test set-ups, the exposed surface area of the samples in the included studies was not always equal to their total surface area. Most researchers let their sample rest in the incubation solution, with the consequence that the bottom side of the sample was not in contact with the extraction solution. In contrast, some researchers used a seesaw, a magnetic stirrer or sonication to continuously agitate the sample in the solution [67,71,75,82,107], and Tabatabaee et al. [124] punctured their samples and let them hang in the solution using a small string. Anyhow, these results indicate that in future studies, more attention should be given to the actual exposed surface area of the samples. There was also a correlation between the release and the volume of extraction solution. This correlation signifies

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Fig. 5 – Figure illustrates the correlation between the total surface area of the specimen and the release. Two resin-based samples are incubated in an extraction solution. They have the same volume x, but the square-shaped specimen has the greatest surface area (Y > y). The results of this meta-analysis showed that the sample with the largest surface area will release more compounds.

that significantly more components were released when a sample was put in a larger volume of extraction solution, irrespective of the kind of solvent (Fig. 6). From a physicochemical point of view, the release must be regarded as a chemical reaction that will arrest when a chemical equilibrium is achieved [86]. In a smaller volume of solution, this chemical balance will be reached earlier than in a larger volume (Fig. 6). This indicates that the used test set-ups with a block of composite immersed in a solvent are probably not very clinically relevant, as the oral environment represents a unique situation with a continuous flow of saliva [86]. In the mouth, the elution of compounds may thus be much larger than expected, since an equilibrium can never be reached due to the removal of the eluates with saliva. Several researchers observed that the largest release took place in the first hours and days (asymptotically) [10,17,62,64,125–127], but these results may also be confounded by the fact that an equilibrium had been reached between the concentration of dissolved eluates and the remainder unbound molecules in the resin block. Especially to test long-term release, a test protocol that takes into account the continuous removal of fluid, for instance by refreshing the extraction solution at equal time intervals, is preferred. Polydorou et al. [70] tested the oneyear release of composites by putting a block of resin in 75%

ethanol and removing (and refreshing) the solvent after 24 h, 7 days, 28 days and 1 year. Analysis after the set time intervals showed that the composites released similar amounts of monomers (especially BisGMA), even after one year and they concluded that composites may still release high concentrations of monomers after long periods. However, it is likely that even larger amounts of eluates will be measured when a set-up with continuous removal of the solvent is used. Most studies included in this review did not refresh the elution solvent, and thus presented cumulative released quantities. Based on the previously found correlation between the volume of the extraction solution and the actual released quantity, and on the fact that there was no standardized longterm period for evaluation, it was decided not to take the actual period of immersion into account, but rather to calculate a total cumulative release for each eluate (Tables 3 and 4) irrespective of the tested period. It is clear that the cumulative results should be interpreted with care because they represent measurements in very diverse circumstances. Together with the paucity of studies that investigated long-term release, these findings indicate that only little is known about the long-term release from resin-based materials and that more long-term research is warranted.

Fig. 6 – Release of compounds from resin-based samples must be regarded as a chemical reaction that will arrest when an equilibrium is reached and the extraction solution is saturated with eluates. As a consequence, more ingredients will be released in larger volumes of extraction solutions.

d e n t a l m a t e r i a l s 2 7 ( 2 0 1 1 ) 723–747

The presence of an oxygen-inhibition layer was previously shown to play an important role in the release of components: higher releases were observed when the surface of cured resin-based specimens was covered by an oxygeninhibition layer [75]. Because the composite samples were covered by a glass plate during light-curing in the majority of the included studies, no correlation between the presence of an oxygen-inhibition layer and the release could be demonstrated. Based on the results of this literature review, several guidelines for standardization may be suggested: 1. The diversity in expressing the released quantities and the use of different units hindered this review. To allow unequivocal interpretation and comparison between different studies, it is recommended to express quantitative release data in standardized units. In this study, it was decided to present the results in ␮mol/mm2 or ␮mol/mm3 , depending on the data (surface area and/or volume of the sample) that were available. By expressing the release per surface area or volume, the quantitative data can also be related to teeth and the oral situation. 2. Studies that express the release from composites semiquantitatively, only give information on the order of magnitude of the released components in an indirect way. In addition, the release results of these studies cannot be compared with other studies. In view of future metaanalyses, it is recommendable to express absolute values of release. 3. Release of compounds from polymerized dental resinbased materials is dependent on the surface area exposed to the incubation solution. The exact surface area should therefore be described in the materials and methods. By expressing the released amounts per surface area, they can be easily related to dental restorations. 4. Special care should be taken to ascertain accurate test methodology. Analyses that utilize mass spectroscopy should be preferred [128]. It is always recommended to check the validity of the results by internal standards. Caffeine is used frequently as internal standard [58–60,89,129]. In a first study, Michelsen et al. used diethyl phthalate [67] as internal standard, but in a more recent study from the same researchers [82], they used deuterium analogs of the analyzed monomers. Analyses with low detection limits are preferred. 5. The limits for detection/quantification of each analyzed eluate are essential for the interpretation of the results, and should therefore always be mentioned. Compounds that could not be detected, may still have been released [91], but in concentrations below the detection limit. It would thus not be correct to assume that they are not released from resin-based materials [56]. Wataha et al. [91] showed that in spite of negative HPLC measurements, sufficient ingredients were released to cause in vitro cytotoxity. Undetected compounds should also not be counted in as zero [72]. 6. Contamination may lead to false-positive detection of compounds, and great care should be taken to avoid any contamination. Especially the use of polymer-based mate-

7.

8.

9.

10.

11.

741

rials, such as plastic containers or instruments, and disposable gloves should be avoided, as they themselves may release ingredients (for example phthalates may originate from polyethylene [81,96]). Glass vials and metal instruments are preferred [67]. When human saliva is used as elution medium, it should originate from persons without dental restorations, and, as described by Michelsen et al. [82], any other contamination of the saliva by dental hygiene products, smoking, or intake of beverages and foods should be avoided. To determine the long-term release, it is recommended to refresh the extraction solution after predetermined equal time intervals, in order to simulate the continuous flow of the saliva. Depending on the used volume of extraction solution, saturation of the solution with eluates will occur sooner or later. Leaving the sample after the saturation point will not result in more leaching. Too often, the materials and methods failed to mention necessary information, such as the volume of incubation solution, the percentage of solvent in case of dilutions [57], the pH of the solution, the size of the samples and the number of tested samples [68], the volume of extraction solvent [130]. Often it was also not clear whether the results were presented cumulatively or not. Imitation of the clinical situation is preferable. For example, most studies incubated their specimens at a constant temperature of 37 ◦ C, although some authors do not mention the temperature of the incubation solution [75,94,131] or store their samples at room temperature [69,70,74]. Some researchers advocated a post-irradation cure, signifying that they waited for a period (usually 24-h) before immersing their samples [76,95]. The clinical relevance of such a post-irradiation curing period is up to debate. The conversion of methacrylate-based materials may still increase dramatically during the post-irradiation period, and thus may reflect in lower release rates. It is recommended that the CAS-numbers and molecular masses of the analyzed molecules are always mentioned in the materials and methods. Polydorou et al. [128] showed that the use of trivial names and abbreviations of chemicals may lead to confusion. Especially when the trivial name is used for a group of several different molecules, which is the case for UDMA, the use of the CAS number along with the exact molecular mass is indispensable. The immersion time between the different release studies varied from 5 min to one year. The majority of the studies tested the immersion after 24 h, which was therefore taken as reference incubation time in this study. As a result, some studies could not be included in this review [57]. It is therefore recommended to have a standardized incubation time of 24 h to anticipate future meta-analytical review.

To conclude, this review tried to gather available data in the literature on the release of components from resin-based dental materials, and to determine the order of magnitude of the released substances. The results of this review study must, however, be interpreted with caution, as they are based on studies that were very heterogeneous regarding study set-up, and that also showed some methodological flaws. In analyti-

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cal release research, the limits of detection should always be mentioned. Preferentially, the exact composition of the tested material should be known, but this requires disclosure of the composition by the manufacturers. Most information could be found on the release of monomers, while information on the released quantities of additives is scarce. There is certainly need for more accurate research to determine the long-term release from composite materials.

Acknowledgments Kirsten L. Van Landuyt has been appointed as post-doctoral research fellow of the Research Foundation – Flanders (FWO). This research was supported by FWO grants G.0496.10, KAN 2010 1.5.128.10 and KAN 1.5.158.09.N.00 (‘Krediet aan Navorsers’).

Appendix A. Appendix calculations

Mol/surface area [mol/m2 ]

Mol/volume [mol/m3 ]

Concentration (C) [g/l]

C [g/l] * Volume solvent [l] * 1/Mm [mol/g] * 1/ surface area tested specimen [1/m2 ]

C [g/l] * Volume solvent [l] * 1/Mm [mol/g] * 1/ volume tested specimen [1/m3 ]

Molar concentration (M) [mol/l]

M [mol/l] * Volume solvent [l] * 1/ surface area tested specimen [1/m2 ]

M [mol/l] * Volume solvent [l] * 1/ volume tested specimen [1/m3 ]

Concentration in ppm (parts per million)

((ppm/1.000.000) * Dsol [g/m3 ]§ ) * Volume solvent [l] * 1/Mm [mol/g] * 1/ surface area tested specimen [1/m2 ]

((ppm/1.000.000) * Dsol [g/m3 ]§ ) * Volume solvent [l] * 1/Mm [mol/g] * 1/ volume tested specimen [1/m3 ]

Mass per surface area [g/m2 ]

Mass per surface area [g/m2 ] * 1/Mm [mol/g]

could not be determined

Mass per total mass of tested specimen (meluate /mtot ) [g/g]$

could not be determined Mol/volume [mol/m3 ] = meluate /mtot [g/g] * 1/Mm [mol/g] * Dcomp [g/m3 ] When the released amount of an eluate had been measured after different time periods, all released amountswere added. To calculate the standard deviation, the following equation was used:



(SDi2 )

i=all ime periods

Polydorou et al. [69] provided the released concentrations in logarithmic values. The concentration was determined by calculating the inverse log. The standard deviation of a logarithmic value was determined by following equation: 1 2 Var(log (x)) = Var(x) ∗ 2 With var being the variance, which is (SD) (average(x)∗ln (10))

Abbreviations: C: concentration; Mm: Molecular mass; M: molar concentration; ppm: parts per million; Dsol : density solvent; Dcomp : density tested composite specimen; m: mass, Var: variance. $ The results of the studies with reference number [120,123,134–138] could not be re-calculated, as the density of the tested composite could not be retrieved from the manufacturer, or the tested composites were not commercially available.

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Table A1 – Inclusion criteria check list. • Was the study performed in vitro? In-vivo studies cannot be included. • Was the release at 24 h measured? • Is the release expressed in absolute release data? Papers with relative or semi-quantitative data cannot be included as their results cannot be used for recalculation. • Was the surface, volume or weight of the tested specimens measured? • Was there a pre-incubation period? If so, the study cannot be included, since the compounds released during the pre-incubation period will not have been quantified.

Table A2 – The lowest measured quantity in the database per eluate. Eluate

Lowest measured amount/surface (␮mol/mm2 )

Lowest measured amount/vol (␮mol/mm3 )

BADGE BHT Bis-DMA Bis-EMA BisGMA BPA CQ DMABEE EBPA EGDMA HEMA HMBP HQ Irgacure 651 MA MEHQ MMA PBPA Quantacure BEA TEGDMA TIN P TMA TMPTMA UDMA UDMA1

/ 3.18182E−05 / 0.00029108 5.51985E−08 5.08511E−06 0.000135542 0.001865285 / 2.67938E−08 2.61257E−05 0.000758439 1.20462E−06 / 0.000206214 8.05542E−05 0.001294423 / / 9.88202E−08 0.004931142 / 0.009816677 0.00001 /

9.17903E−05 / 9.92983E−06 0.000523944 9.93573E−08 2.73775E−07 / / 7.25211E−05 1.1432E−07 0.000745414 / 5.13973E−06 / 0.0002887 / 0.001812192 0.000696974 / 1.77876E−07 / / / 0.000732555 /

Table A3 – Limits of detection/quantification, presented as ␮mol/mm2 or ␮mol/mm3 of the resin sample. Study Pulgar et al., 2000 [75]

Polydorou et al., 2007 [69]

Polydorou et al., 2009 [74]

Polydorou et al., 2009 [70]

Tabatabaee et al., 2009 [124]

BADGE Bis-DMA BisGMA BPA EBPA PBPA BisGMA BPA TEGDMA UDMA BisGMA BPA TEGDMA UDMA UDMA1 BisGMA BPA TEGDMA UDMA UDMA1 BisGMA TEGDMA

(␮mol/mm2 )

(␮mol/mm3 )

1.00E−04 1.00E−06 1.00E−04 1.00E−04 1.00E−05 1.00E−07 1.00E−05 1.00E−05 1.00E−05 1.00E−05 1.00E−07 1.00E−05 1.00E−05 1.00E−05 1.00E−06 1.00E−06

1.00E−05 1.00E−05 1.00E−05 1.00E−07 1.00E−05 1.00E−04 1.00E−04 1.00E−06 1.00E−03 1.00E−04 1.00E−05 1.00E−07 1.00E−05 1.00E−05 1.00E−05 1.00E−05 1.00E−07 1.00E−05 1.00E−05 1.00E−05 1.00E−06 1.00E−05

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