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resin obturation

  1. 1. Critical Reviews in Oral Biology & Medicine Biocompatibility of Resin-Modified Filling Materials W. Geurtsen CROBM 2000 11: 333 DOI: 10.1177/10454411000110030401 The online version of this article can be found at: Published by: On behalf of: International and American Associations for Dental Research Additional services and information for Critical Reviews in Oral Biology & Medicine can be found at: Email Alerts: Subscriptions: Reprints: Permissions: >> Version of Record - Jan 1, 2000 What is This? Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission.
  2. 2. BIOCOMPATIBILITY OF RESIN-MODIFIED FILLING MATERIALS W. Geurtsen Department of Conservative Dentistry & Periodontology, Medical University Hannover, Carl-Neuberg-Str. 1, D-30539 Hannover, Germany; ABSTRACT: Increasing numbers of resin-based dental restorations have been placed over the past decade. During this same period, the public interest in the local and especially systemic adverse effects caused by dental materials has increased significantly It has been found that each resin-based material releases several components into the oral environment. In particular, the comonomer, triethyleneglycol di-methacrylate (TEGDMA), and the 'hydrophilic' monomer, 2-hydroxy-ethyl-methacrylate (HEMA), are leached out from various composite resins and 'adhesive' materials (e.g., resin-modified glass-ionomer cements IGICsl and dentin adhesives) in considerable amounts during the first 24 hours after polymerization. Numerous unbound resin components may leach into saliva during the initial phase after polymerization, and later, due to degradation or erosion of the resinous restoration. Those substances may be systemically distributed and could potentially cause adverse systemic effects in patients. In addition, absorption of organic substances from unpolymerized material, through unprotected skin, due to manual contact may pose a special risk for dental personnel. This is borne out by the increasing numbers of dental nurses, technicians, and aentists who present with allergic reactions to one or more resin components, like HEMA, glutaraldehyde, ethyleneglycol di-methacrylate (EGDMA), and dibenzoyl peroxide (DPO). However, it must be emphasized that, except for conventional composite resins, data reported on the release of substances from resin-based materials are scarce. There is very little reliable information with respect to the biological interactions between resin components and various tissues. Those interactions may be either protective, like absorption to dentin, or detrimental, e.g., inflammatory reactions of soft tissues. Microbial effects have also been observed which may contribute indirectly to caries and irritation of the pulp. Therefore, it is critical, both for our patients and for the profession, that the biological effects of resin-based filling materials be clarified in the near future, Key words. Resin-based materials, biocompatibility, leaching, adverse effects. (I) Introduction The long-lasting, ongoing controversy on the hazards for patients due to mercury exposure from amalgam has also increased the public interest in possible adverse effects caused by other dental restorative materials, like composite resins and resinous pit and fissure sealants. Therefore, a thorough scientific evaluation of the biological behavior of each of those materials must be performed. This analysis should be based upon detailed data reported on the release of components from these materials and their local and systemic 'interactions with various tissues Schmalz and Arenholt--Bindslev, 1998). It has been found that, due to degradation and/or corrosion, several components are leached out from each resin-IDased dental material into the oral environment. This, in turn. may cause adverse local and/or systemic effects Geurtsen, 1998). In addition, direct interactions at the interface between a restoration and the oral tissues may also influence biocompatibility. For example, it has been reported that plaque and gingival indices as well as probing depths adjacent to 5- to 6-year-old direct composite restorations were significantly higher compared with those at non -restored sites (Peumans et cl., 1998). RevOrl 33 0 1111 5~2 I I 3) .3 3 3- 3 5 5 22000) 0 1 G To begin with, this review article will focus on the most important parameters which characterize the biocompatibility of a resin-based restorative material. This will be followed by a critical evaluation of reports dealing with the dispersal of substances from various resinous materials (resin composites, resin-modified GlCs, polyacid-modified composite resins l'compomers'l, dentin adhesives, and pit and fissure sealants). Finally, the biological effects of those materials and the substances released by them will be examined, and possible clinical consequences will be discussed. (11) Determination of Biocompatibility of Resin-based Materials Various factors determine the biocompatibility of a resin-based material, particularly the amount and nature of leachable components as well as surface structure of the final restoration. Several authors have reported a correlation between increased plaque accumulation and gingival irritation adjacent to resin restorations and roughness and marginal adaptation of the filling (Sanchez-Sotres et al., 1969; Peumans et al., 1998). In contrast to the local irritation caused by BolMe Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 333
  3. 3. Figure la. Human primary gingrival fibroblast monolayer, nonincubated control culture. Cells reveal normal morphology. Original magnification 40x. Figure l b. 'Direct contact test' with a polymerized resin-modified GIC (lonosealTM). The non-cytotoxic material did not induce any cellular alteration. Cells have grown close to the specimen. Original magnification 40x. Figure lc. 'Direct contact test' with a cured specimen of a resin modified GIC (VitrebondTM). No viable cells are visible due to the highly cytotoxic material. Original magnification 40x. 334 surface parameters, released substances can induce local effects in oral tissues (pulp, gingiva, oral mucosa) as well as adverse systemic reactions. The nature of such responses is either allergic or toxic (Hume and Gerzina, 1996). In general, two mechanisms may result in the segregation of components from resinous dental materials. After polymerization, unbound monomers and additives are extracted by solvents, e.g., saliva and/or dietary solvents, especially during the first 24 hrs. Thus, monomerpolymer conversion is a very important aspect of the biocompatibility of a resin restoration. But leachable substances may also be generated by erosion and degradation over time (Ferracane, 1994, 1995; Geurtsen, 1998). Resin degradation and erosion may be caused by photo, thermal, mechanical, or chemical influences. For example, it has been found that salivary esterases can degrade the surfaces of composite resins, which may then result in the liberation of methacrylic substances (GCpferich, 1996). The nature of the matrix monomer(s) can also significantly influence the release of components. It has been reported that UDMA-based composite resin IUDMA = urethane di-methacrylate; 1,6-bis(methacrylyloxy-2ethoxycarbonylamino)-trimethylhexaneI is less water-soluble than materials containing Bis-GMA ("Bowen monomer", bisphenol-A-glycidyl methacrylate) (Pearson and Longman, 1989). Prior to clinical application, each resin-based restorative material must be thoroughly investigated for biocompatibility by means of numerous standardized tests. This structured approach was first addressed by Autian (1974), who suggested three consecutive steps or levels: (I) screening tests (non-specific toxicity), (11) usage tests in animals (specific toxicity), and (111) clinical studies with humans. Today, a battery of in vitro and in vivo tests has been recommended by the International Standards Organization for the 'Biological evaluation of medical devices' (ISO, Geneve, Switzerland, 1992-1997). Those standards include guidelines for sample preparation, tests for cytotoxicity, genotoxicity, carcinogenicity, and reproductive toxicity as well as tests to evaluate local reactions after implantation, irritation, sensitization, and systemic toxicity. Furthermore, pre-clinical and clinical evaluation as well as risk analysis and risk management are defined (Schmalz, 1997). (A) CYTOTOXICITY TESTS Cell cultures derived from animals and humans have been used during the past 30 years for the determination of cytotoxic effects caused by resin-based restorative materials Permanent cell lines and primary cells, derived mainly from oral tissues, have Cr0 Rev Orcil Biol Med Crit Rev Orol Bid Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 333 355 120001 1 1(3):333-3 55 (2000)
  4. 4. been used (Figs. 1-3) (Ratanasathien et al., 1995; Geurtsen et cil., 1998b, 1999a). Generally, permanent cells which have been cultured for years show homogeneous morphology and physiology In contrast, primary cell cultures which may originate from target tissues reveal a limited life span and are heterogeneous. Thus, an in vivo situation is generally better-simulated by primary cultures. In addition to the use of primary cells derived from oral tissues, artificial pulp chambers have been created. The rationale for the use of these devices is to reproduce interactions (e.g., absorption) between released substances and dentin and to determine the significant toxic influences of those components on the pulp. These systems have, in common, dentin as a diffusion or adsorption barrier ('in vitro pulp chamber tests') (Hanks et cil., 1988; Schmalz et al, 1999). At present, three-dimensional (3-D) cell cultures with one cell type or two different cell lines have been developed (Figs. 3a, 3b). Those cell cultures more closely resemble the various tissues with their complex organization of extracellular matrix and different cell types. Therefore, interactions between resin substances and extracellular matrix may be better reproduced, in the future, by the application of 3-D cell cultures. On the other hand, it must be emphasized that the more complex In vivo-like reactions in such 3-D cell cultures may be more difficult to interpret than responses observed in simple "2-D monolayer" cultures. Various 'biological endpoints' are used for the investigation of cytotoxic effects due to solid specimens or extracts from polymerized samples, e.g., *evaluation of enzyme activity, *membrane integrity and cell metabolism (DNA-, RNA-, and protein synthesis), *alteration of cell morphology and cytoskeleton by light- and electron microscopy, and *determination of cell growth inhibition and of ED90 I= effective dose which causes a 50% reduction of cell proliferation) (Geurtsen, 1988; Geurtsen and Leyhausen, 1997). Substances liberated from resin-based dental materials can also interact chemically and osmotically with the membranes of suspended erythrocytes and eventually lead to the release of hemoglobin. The total hemolytic activity due to segregated substances and surface effects is assessed for evaluation of the cytotoxicity of resin-based materials and various individual substances, e.g., Bis-GMA (Fujisawa et cial, 1978, Stanford, 1980). In addition, interactions between hydrophobic resin components and liposomes have been investigated for determination of the detrimental solubilizing effects on the lipid bilayer of cell membranes (Fujisawa et cia, 1988). I1 1(3):333-3 5 5 (2000) fl3j 333 355 120001 Figure 2a. Sub-confluent human primary osteoblast-like cells derived from alveolar bone, non-incubated control, cells with normal morphology. Original magnification 40x. Figure 2b. Osteoblast-like cells 24 hrs after incubation with the aqueous extract of a PMMA-bone cement (Palacos.m). The cytocompatible eluate did not cause any cellular injuries. Original magnification 40x. Figure 2c. Osteoblast-like cells 24 hrs after treatment with water el ates of a resin-modified GIC (VitrebondTM). The eluate was extremely cytotoxic; no viable cells are visible. Original magnification 40x. Downloaded from at BIOLOGICAL LABS LIBRARY on December 27,Med For personal use only. No other uses without permission. Cr0 Rev Orcil B!ol 2013 Crit Rev Orcal Biol Med 335 335
  5. 5. (B) MICROBIAL TESTS The purpose of microbial tests is to determine whether a material may inhibit or stimulate the growth of microorganisms. Furthermore, studies have been undertaken to evaluate whether bacteria may degrade resinous materials intra-orally. Micro-organisms originating from the oral cavity and other sites have been used. Most authors, however, have used cariogenic micro-organisms (e g, mutans streptococci like S. sobrinus, and lactobacilli) for the evaluation of microbial effects of resin-based materials (Updegraff et al., 1971; Orstavik and HenstenPettersen, 1978; Hansel et al., 1998). (C) DETERMINATION OF GENOTOXICITY/MUTAGENICITY AND CARCINOGENICITY Genotoxicity and carcinogenicity are very important parameters that can have an impact on the systemic compatibility of a resin-based material. Genotoxicity means the presence of a DNA-reactive substance which may be mutagenic or carcinogenic (Leyhausen et al., 1995). Due to the extremely hazardous consequences, genotoxicity/mutagenicity and carcinogenicity are of very high public interest. Therefore, each material must be thoroughly evaluated by several in vitro and in vivo tests, which are described in the OECD guidelines for the testing of chemicals (Heil et al., 1996). In vitro assays for the evaluation of genotoxicity can be differentiated into procaryotic and eucaryotic tests. Figure 3a. Primary human gingival fibroblasts after three-dimensional growth in a polyester mesh. The culture reveals a more 'tissue-like' structure rather than a monolayer (Fig. 1 a). HE staining. Original magnification 750x. Figure 3b. 3-D culture of primary human gingival fibroblasts grown for 4 wks (SEM). Fibroblasts grow between the mesh fibers and exhibit a tissue-like organization. Original magnification 3000x. Bar, 100 ,m. (Figs. 3a and 3b courtesy of Dr. Hillmann, Hannover, Germany) The application of various biological endpoints or the evaluation of different parameters greatly influences the results of cytotoxicity studies For this reason, it is difficult to compare the results from different investigations On the other hand, similar results obtained from a wide range of cell culture tests may provide a powerful tool for characterization of the effects of resin components on host tissues. 336 Important bacterial tests are the classic Ames test and the recently developed uvnu-test. Recommended eucaryotic tests are the chromosomal aberration test, the hypoxanthine-guanine phosphoribosyltransferase assay (HPRT), the sister chromatid exchange, and the DNA synthesis inhibition test (DIT) (Geurtsen and Leyhausen, 1997, Stea et al., 1998, Schweikl and Schmalz, 1999). Since it is difficult to discriminate between bactericidal or cytotoxic effects and genotoxicity, the procaryotic or eucaryotic test system cannot be the only basis for the determination of DNA-damaging properties of an individual substance or a resin-based restorative material. Consequently, different test systems must be used for a thorough monitoring of genotoxicity, e.g., procaryotic and eucaryotic tests supplemented by an in vivo assay. A rapid and simple in vivo test for genotoxicity is the Alkaline Filter Elution assay (AFE) with viable freshwater clams Dreissenci polymorpho and Corbicula fluminea. This method allows for the quick determination of DNA strand breaks induced by the exposure of the animals to genotoxic substances (Heil et al 1996). (D) IMPLANTATION TESTS IN ANIMALS Non-specific local histocompatibility of restorative resin materials is evaluated in vivo by means of implantation Crit Rev Oral Biol Medl Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 1 (30333-3 55 (2000)
  6. 6. tests in animals, e.g., rabbits and rats. Pure specimens are implanted intramuscularly, suocutaneously, and into bones. Alternatively, fenestrated or non-fenestrated polyethylene tubes which contain the test material are implanted for the elimination of possible effects due to different surface structures of individual specimens. Tissue reactions are determined macro- and microscopically as well as histochemically (Autian, 1970, 1974; Burpee et al, 1978; Wennberg et al., 1983; Henderson et al., 1987). Differences in how resin materials are used clinically is. the implantation of these materials into rodent soft tissues must be consideredl when results from implant tests are evaluated. (E) SYSTEMIC TOXICITY Rodents are used mainly for the determination of adverse systemic effects to resin-based restorative materials. Various aspects can be investigated, including acute oral toxicity (LD50 = lethal dose for 50% of the test animals after oral uptake), acute inhalation toxicity, reproductive toxicity, alterations in systemic organs, and cardiovascular effects (Geurtsen, 1988). It should be recognized that resin-based materials release components in relatively small amounts. Therefore, acute oral toxicity is of less value for the assessment of the biocompatibility of resin-based dental materials. (F) TESTS FOR EVALUATION OF SENSITIZATION AND IRRITATION According to ISO guidelines for the biological evaluation of medical devices applied in dentistry, resin-based materials must also be tested for skin irritation and sensitization. For an evaluation of skin irritation, test materials are applied to shaved dorsal skin. Final assessment of skin reactions is performed 72 hrs after baseline. For the maximization method (sensitization), test solutions are injected into skin adjacent to the neck (Clemmensen, 1985; Altuna and Freeman, 1987; Katsuno et al., 1998). (G) PULP STUDIES (H) CLINICAL STUDIES Clinical studies are necessary for the final assessment of the biological behavior of resin-based materials, especially of their long-term biocompatibility. These clinical trials may be performed only with those materials which have revealed a sufficient biocompatibility in pre-clinical screening and usage tests. Various 'biological' parameters are evaluated in clinical investigations pulp reactions, compatibility with gingiva/periodontium! irritation of the oral mucosa, and plaque accumulation (Samaha, 1982; Dunkin and Chambers, 1983; Geurtsen and Schoeler, 1997). (111) Toxicity of Individual Resin Components IN ANIMALS AND HUMANS In contrast to implantation tests, pulp studies are performed for the determination of specific local reactions. Due to the specific application of the resinous material, these studies are referred to as "usage tests". Resinbased materials, as well as single components, have been investigated (Dalleske et al., 1978; Stanley et al., 1979). The experimental animals used in these investigations include rats, dogs, pigs, and monkeys (Tyas and Browne 1977;, McCluggage et al., 1980; Schmalz, 1981). A few studies have been performed with human teeth, obtained from adolescents, which had to be extracted for orthodontic reasons (Plant and lones, 1976; Tyas and Browne, 1977, Dalleske et al. 1978; Elbaum et al., 1992; Dejoux et al, 1993i! Due to ethical concerns about human 1 experiments, especially with children and adolescents, there are only a few pulp studies involving humans (Goracci et al., 1995). It has been found that the in livo toxicity of the resinous test materials may be modified by various factors. Toxic effects induced by released components can be reduced by interaction with dentin. However, adverse reactions induced by resin components can also be enhanced, e.g., by pre-existing caries lesions, acid-etching of dentin ('total-etch technique'), or bacterial proliferation in the gap between the filling and the cavity wall (Fiore-Donno and Baume, 1966; Eriksen and Leidal, 1979; Torstenson et al., 1982). Transferability of usage tests performed by various researchers or with different species is limited due to technical, physiological, or morphological factors, etc. For example, histological technique and subjective evaluation of the pulpal conditions may differ considerably among authors. In addition, regenerative potential of the pulp or the number and diameter of dentinal tubules may vary with different species (Tyas and Browne, 1977, Geurtsen, 1988 Schilke et al., 1999). These important factors must be considered when results of various pulp studies, especially with different species, are appraised or compared (A) CYTOTOXICITY TESTS Numerous individual ingredients of resin-based materials have been tested for cytotoxicity in various cell culture systems with diverse biological endpoints. As a result, it is difficult to compare data obtained in the various reported studies. Ratanasathien et al. 11995) observed interactive effects-synergism, additive effects, and antagonism-of four important resin components in Balb/c 3T3 cultures. In addition, increased exposure time of the cells to the tested materials resulted in an increased cytotoxicity I'toxic ranking' Bis-GMA > urethane dimethacrylate (UDMA) > triethyleneglycol dimethacrylate (TEGDMA) >>> 2-hydroxy-ethyl-methacry- 355i20i0iCrlfRevOralBio Me 33 11(3 Crit Rev Oral Biol Med 1(3).333-355 (2000) Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 337
  7. 7. TABLE 1 Substances Identified in Methanol Extracts and Aqueous Eluates (heavy-type) of Composite Resins, Polyacid-modified Composite Resins, Resin-modified Glass-ionomer Cements, and ED50 Concentrations (Geurtsen, 1998; Geurtsen et al., 1 998a,b, 1999; *according to 0ysaed et aL, 1988) Abbr. M [u] ED50 [mM] Allergy Mut. Compound 512 480 452 364 470 0.08-0.14 Yes Yes2 "Bowen monomer"; bisphenol-A-glycidyl methacrylate Yes Yes2 "Propoxylated bisphenol-A-di-methacrylate" "Ethoxylated bisphenol-A-di-methacrylate" "Bisphenol-A-dimethacrylate" Yes Yes3 (Co)monomers Bis-GMA Bis-PMA Bis-EMA Bis-MA UDMA UPGMA HEGDMA PEGDMA TEGDMA TEGMMA TEGMAA TEEGDMA DEGDMA EGDMA GDMA DDDMA HDMMA HDDMA PDDMA BDDMA MBDDMA 1/2 DBDDMA 1/2 PRDMA HPMA DMTCDDA BEMA SIMA SYHEMA 1/2 TMPTMA HEMA MMA MAA Initiators CQ BL DMBZ DPICI DBPO BPE 338 968 418 374 286 218 272 330 242 198 142 310 186 254 240 226 258 384 212 144 304 176 248 166 338 130 100 86 166 210 256 316 242 198 n.d. 0.21-0.78 0.10-0.16 0.06-0.47 n.d. n.d. n.d. 0.12-0.26 Yes Yes4 n.d. n.d. n.d. 0.07-0.18 0.46-2.31 Yes Yes n.d. > 5.0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 1.93-4.10 No n.d. n.d. n.d. 1.77-2.52 5.0 > Yes Yes No n.d. 2.17-2.40 0.68-2.02 0.24-0.34 0.0465 0.43-3.80 0.92-2.42 1 ,6-bis(methocryloyloxy-2- ethoxycarbonylamino)-2,4,4-trimethylhexane; urethane di-methacrylate "Urethane bisphenol-A-di-methacrylate" Hexaethyleneglycol di-methacrylate Pentaethyleneglycol di-methacrylate Yes'12 Yes2'3 Yes Triethyleneglycol di-methacrylote Triethyleneglycol mono-methacrylate Triethyleneglycol methacrylate acrylate Tetraethyleneglycol di-methacrylate Diethyleneglycol di-methacrylate Ethyleneglycol di-methacrylate Glycidyl methacrylate 1,1 0-Decanediol di-methacrylate 1,6-Hexamethylene monomethacrylate 1,6-Hexanediol di-methacrylate 1,5-Pentanediol di-methacrylate 1,,4-Butanediol di-methacrylate BDDMA-methanol-adduct 1/2 BDDMA-auto-adduct 1/2 1,2-Propanediol di-methacrylate Hydroxypropylmethacrylate Bis(acryloxymethyl)tricyclo[5.2. 1 .02 6]decane Benzyl methacrylate 3-Trimethoxysilane propylmethacrylate 1 /2-Cyclohexene methacrylate Trimethylolpropane tri-methacrylate 2-Hydroxy-ethyl-methacrylate Methyl methacrylate Methacrylic acid Camphoroquinone Benzil Dimethoxybenzoin Diphenyliodoniumchloride Dibenzoyl peroxide Benzoicacid phenylester Crit Rev Oral Biol Med Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. (2000) 1 1(3):333-355 (2000)
  8. 8. Mut. Compound M [u] ED50 [mM] 0.31-0.59 0.31-0.48 0.39-1.47 1.25-2.86 2.30-4.25 CEMA DMABEE DMABBEE DMABEHE DMAEMA DEMAEEA 213 241 177 269 135 195 181 117 165 160 193 265 277 157 313 Photostabilizer HMBP TINP TIN326 TIN350 TIN328 228 225 315 323 351 0.44-3.07 n.d. n.d. n.d. n.d. 2-Hydroxy-4-methoxy benzophenone 2(2'-Hydroxy-5'-methylphenyl) benzotriazole Tinuvin 326 Tinuvin 350 Tinuvin 328 Inhibitors HQME BHT MBP MBEP 124 220 312 368 n.d. 0.16-0.20 n.d. n.d. Hydroqu inone-monomethyl-ether 2,6-Di-t-butyl-4-methyl phenol Plasticizer DCHP DEHP 330 390 0.69-0.85 n.d. Dicyclohexyl-phthalate Bis(2-ethylhexyl) phthalate 2.14-2.81 1.99-5.96 n.d. 1.83-3.49 1.74-3.17 n.d. n.d. 1.17-2.53 n.d n.d n.d. n.d. Benzoic-acid-methylester Triethylene-glycol Abbr. Co-initiators DMDDA DMTDA DIPA THA DMPT DHEPT DCHA DEAE DMAPE Reaction/decomposition products 136 BME 150 TEG 62 EG 168 DICH 108 BEA 132 MMMA 30 FA* 182 CA 168 HC 1/2 112 CIB 156 BRB 204 IB 1.30-3.58 Allergy Yes2 Yes Yes' 2.35-4.48 2.61-4.17 2.78 - > 5.00 n.d. 1.22-1.26 n.d. n.d. n.d. n.d. Dimethyl-dodecylamine Dimethyl-tetradecylamine 2,6-Diisopropyl-aniline Trihexylamine Dimethyl-p-toluidine Dihydroxy-ethyl-p-toluidine Dicyclo-hexylamine Diethyl-amino-ethanol Yes' Yes2 2-(4-Dimethylaminophenol)ethanol N-(2-Cyanoethyl-)N-methylanilin 4-N,N-Dimethylaminobenzoic acid ethyl ester 4-N,N-Dimethylaminobenzoic acid butyl ethoxy ester 4-N,N-Dimethylaminobenzoicacid 2-ethylhexyl ester N,N-Dimethyl aminoethyl methacrylate N,N-(Bisethylmethacrylate)-2-ethoxyethylamine 2,2'-Methylene-bis(6-t-butylphenol) 2,2'-Methylene-bis(6-t butyl-4-ethylphenol) Ethylene-glycol 1,6-Di isocyanato-hexane Benzyl alcohol Methyl-methacrylate-methanol adduct Formaldehyde (0ysaed et al., 1988) Yes Yes'2 Camphoric anhydride ? 7 Chlorine benzene (from DPICI) Bromine benzene (from DPICI) iodine benzene (from DPICI) 2(3)-endo-Hydroxyepicamphor Contaminants TPP TPSb Triphenyl-phosphane 0.32-0.45 262 Yes23 Triphenyl-stibane 0.09-0.10 352 Mut. = results from genotoxicity/mutagenicity studies (data from 1 umu test, 2DIT, 3AFE assay; Heil et al., 1996; 4HPRT assay; Schweikl and Schmalz, 1999). Allergy = resin components which may cause hypersensitivity/allergy in humans (Jordan et aL, 1979; Kanerva et al., 1 994b, 1995; Richter and Geier, 1996; Richter, 1996) 11(3):333-355 (2000) Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 339
  9. 9. Biomass production of L. acidophilus after incubation with Bis4GMA and UDMA for 40 h 120 100 0 ow 0 0 go - 40 20 I I 0 11c 00 A I -J r a I I Bis-GMA a N I_ L:'J*I UDMA Biomass production of L. aiophilus after incubation with EGDMA and TEGDMA for 40 h 140 (DPICL), and the contaminant triphenyl-stibane (TPSb) exhibited severe cytotoxic effects. Within the groups of (co)monomers and (co)initiators, high or moderate cytotoxic reactions were observed, whereas the evaluated decomposition/reaction products caused only moderate or slight effects (Table 1). The most important photo-initiator, camphoroquinone, which was found in significant amounts in aqueous extracts from resinbased materials, revealed moderate cytotoxic effects. This was confirmed by Atsumi et al. (1998) with permanent human submandibular-duct cells. Analysis of the data from various reports, taken together, indicates that, for most of the severely cytotoxic resin components, less toxic alternatives are available. In particular, the cytotoxicity of TEGDMA may be of great clinical interest. This relatively hydrophilic comonomer is released from many resin-based materials in considerable amounts (Spahl and Budzikiewicz, 1994; Geurtsen, 120 Blomass production of S. sobrinus after incubation with BIs.GMA and UDMA for 10 h in0 0 a 0 600 . v e -W B EGDMA TEGDMA 0 0 Figure 4a. Bis-GMA and UDMA significantly inhibited growth of L. 0 acidophilus (Hansel et al., 1998). Figure 4b. In contrast to base monomers, the relatively hydrophilic comonomers EGDMA and TEGDMA significantly promoted proliferation of L. acidophilus (Hansel et al., 1998). late (HEMA)I. These authors concluded that exposure time as well as interactions between and among various released substances significantly influence biocompatibility. The high cytotoxicity of the monomers Bis-GMA, UDMA, and TEGDMA was corroborated by other studies (Hanks et al., 1991; Lehmann et al., 1993; Yoshii, 1997). It has been reported that TEGDMA can induce lipid peroxidation of microsomes. Furthermore, this comonomer also exhibited surfactant-like potency which may cause detrimental solubilization of the lipid bilayer of cell membranes (Terakado et al., 1984; Fujisawa et al., 1988). Geurtsen et al. ( 1998a) investigated cytotoxic effects of 35 single monomers and additives of composite resins in permanent 3T3 cells and three primary human oral fibroblast cultures (Table 1). ED50 values varied significantly, from 0.0465 mM to > 5 mM. The tested inhibitor 2,6-di-t-butyl-4-methyl phenol (BHT), the photostabilizer 2-hydroxy-4-methoxy benzophenone (HMBP), the initiator diphenyliodonium chloride 340 A BIs4MA UDMA Biomass production of S. sobrinus after incubation with EGDMA and TEGDMA for 10 h 140 100..................i........., .. ........ I Il 0 ........ o0 .40 -! 0 B 011Id,IiI I Ii J." I I 20 EGDMA TEGDMA Figure 5a. In contrast to L. acidophilus, proliferation of S. sobrinus not affected by Bis-GMA (Hansel et al., 1998). was Figure 5b. EGDMA and TEGDMA also enhanced growth of S. sobrinus (Hansel et al., 1998). Crit Rev Oral Biol Med Crit Rev Oral Biot Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. fl(3)333 355 11(3):333-355 (2000)
  10. 10. 1998; Spahi et al., 1998). Due to its high cytotoxic potency, this substance may significantly contribute to adverse local and systemic effects. The liberation of TEGDMA from resin restorations, therefore, should be minimized or prevented Furthermore, it must be emphasized that there is no cell line which is consistently more sensitive compared with others (Geurtsen et al., 1998a; MacDougall et al, 1998). Thus, each substance must be comprehensively screened for cytotoxicity by rneans of different permanent and primary cell cultures. The application of an Ii vitro pulp chamber device (dentin barrier assay) may help to simulate complex intra-oral interaction which can significantly modify cytotoxic effects in vivo (B) MICROBIAL EFFECTS There is currently a great controversy over whether pulpal irritation due to a resin restoration is mainly caused by bacteria proliferating between the cavity wall and the filling lBrannstrbm and Vojinovic, 1976; Qvist, 1993). Solid- as well as liquid-phase systems have been used to detect whether single-resin components can influence microbial growth (Updegraff et al., 1971; Hansel et cl., 1998). Proliferation of mutans streptoccoci (S. sobrinus) and lactobacilli (L. acidophilus) was either inhibited, promoted, or not influenced in a dose-dependent manner by the substance that was tested. Bis-GMA did not alter growth of S. sobrinus, whereas proliferation of L. acidophilus was significantly inhibited. UDMA reduced proliferation of both cariogenic pathogens. Generally, only very small amounts of these hydrophobic substances are released into an aqueous environment. Taken together, those large base monomers should not have microbial effects. The water-soluble comonomers EGDMA (ethylene glycol di-methacrylate) and TEGDMAA, however, promoted proliferation of S. sobrinus and L acidophilus (Figs. 4a, 4b, 5a 5b) iHansel et al., 1998). Analysis of these data clearly shows that the severely cytotoxic comonomers EGDMA and TEGDMA, which are present in considerable amounts in aqueous extracts of numerous resin-based materials, may significantly promote growth of cariogenic pathogens (Spahl et al 1998). Thus, cytotoxic properties and m icrobial growth promotion of these comon .mers may contribute to pulpal injury. (C) In ViVO STUDIES The LD,o concentrations (acute oral toxicity) of several substances have been evaluated in experimental animal studies The LD50 (per I kg body weight) was 950 mg for dibenzoyl-peroxide and 8400 mg for methyl-methacrylate (Deichmann. 941 1 Geurtsen, 1988). Due to the relatively low concentrations of components released from polymerized resin restorations in the oral cavity, acute oral (systemic) toxicity is not of great value in the assessment 11(3)):3 33-3 55 12000o) I 1 333 355 .OOOi of the biocompatibility of those materials. It has been reported that methyl methacrylate (MMA) may be teratogenic and can cause adverse cardiovascular effects in animals (Phillips et al., 1971, Singh et al., 1972; Karlsson et al., 1995). In addition, subcutaneous polymethyl-methacrlylate implants induced malignant tumors in rodents (Oppenheimer et al., 1955). Very few experiments have been performed to assess the acute local (pulp) toxicity of individual resin substances. It was found that the degree of pulpal alterations induced by those components correlates with cavity depth (Stanley, 1992). Two substances (dibenzoyl peroxide, 2-hydroxy-4-methoxy-benzophenone) caused pulpal inflammation in teeth of monkeys when applied to cavities without a protective lining (Stanley et a)., 1979)- (IV) Conventional and Polyacid-modified Composite Resins (A) COMPOSITION AND LEACHING OF COMPONENTS Conventional composite resins contain a polymerizable organic matrix, inorganic reinforcing fillers, and a silane coupling agent which bridges the organic and inorganic components (Ferracane, 1995). The organic matrix consists of several (co)monomers, e.g., Bis-GMA, UDMA, ethyleneglycol di-methacrylate compounds ( EGDMA, D/TEGDMA, etc.), and various additives ((co)initiators, stabilizer, inhibitorl (Table 1). In general, all organic ingredients of a composite resin are extractable by organic solvents, like methanol, after polymerization. A few components, however, are also leached into an aqueous medium. In particular, considerable amounts of TEGDMA may be released by polymerized composite resin into water. Bis-GMA, UDMA, EGDMA DEGDMA 1,6-hexanediol di-methacrylate, methyl methacrylate, camphorquinone, 4-N,N-dimethylamino-benzoic acid ethyl ester, and various other substances have been identified in minor concentrations in aqueous extracts (Geurtsen, 1998; Spahl et al., 1998). Several composite resins liberated formaldehyde into water over a long time period (11 5 days) This degradation product was especially noted in extracts from specimens with an unpolymerized oxygen-inhibited surface layer. The amount of extracted formaldehyde was partly in the order of magnitude which was found to cause local allergic reactions (Oysaed and Ruyter, 1988; Koch and Staehle, 1997). Inorganic substances (ions) like silicon, boron, sodium, and barium are also released from filler particles in trace amounts (0ysed et a)., 1988). 'Compomers' (polyacid-modified composite resins) have been developed to combine the fluoride release of glass-ionomer cements (GICs) with the mechanical prop- Crit Rev Oral Biol Med Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 341 341
  11. 11. erties of composite resins. Thus, these materials are composed of an ion-releasing glass, mainly calcium-aluminum-fluoro-silicate glass, and a polymerizable organic matrix. The fillers are partially silanized to couple the glass with the polymer network. In addition to conventional monomers (Bis-GMA, UDMA), the organic matrix of compomers contains bi-functional monomers with two carboxylic groups and two double-bond functions, e.g., TCB (tetracarboxylic butyl methacrylate), which is synthesized from butane-tetracarboxylic acid and 2hydroxyethyl methacrylate (HEMA) (Attin and Buchalla, 1998; Meyer et al., 1998). The bi-functional monomers can react with methacrylates by radical polymerization and by acid-base reaction to bring about release of ions from the glass in the presence of water. However, compomers do not contain water. Thus, no significant acid-base neutralization occurs during polymerization. Those materials, therefore, are much closer to composite resins than to glass-ionomer cements (Meyer et al., 1998). There are very few data on the release of substances from 'compomers'. The aqueous eluates of polyacidmodified composite resins were analyzed by gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC). Ethylene glycol compounds (comonomers) and the hydrophilic monomer HEMA were the chief constituents identified in these aqueous extracts (Geurtsen et at., 1998b; Hamid et al., 1998). In addition, the following decomposition products of base monomers and several co-initiators were found: N-(2-cyanoethyl)-N-methylaniline (CEMA), 4N,N-dimethyl amino benzoic acid ethylester (DMABEE), and N,N-dimethyl amino ethyl methacrylate (DMAEMA) (Geurtsen et al., 1998b). Due to the ion-leaching glass fillers, compomers may also release fluoride, especially during the first few days after polymerization. But it must be emphasized that those materials leach out considerably smaller amounts of fluoride than do conventional and resin-modified glass-ionomer cements (Attin and Buchalla, 1998; Geurtsen et at., 1998c, 1999b). (B) CYTOTOXICITY Solid specimens and extracts of polymerized samples of composite resins have been tested in various cell cultures. The results of those studies have varied significantly, depending on the product tested (Lampert and Heidemann, 1980; Tronstadt and Wennberg, 1980). It was reported that polymerized specimens of one hybrid-type composite resin were moderately cytotoxic during a fouryear period in permanent and primary human oral cell cultures. Non-polymerized samples of this product, however, were severely cytotoxic and revealed genotoxic effects (Geurtsen, 1987a, 1988). Aqueous eluates of several hybrid-type composite resins induced only moderate cytotoxic reactions (Geurtsen, 1987b). 342 Nakamura et al. (1985) tested various consecutive extracts of light-curing products over six weeks in HeLa cells. The first eluates caused moderate to severe cellular alterations which were not observed with later extracts. In contrast, set material of light-cured and chemically cured composite resins revealed slight or no toxic effects when tested in an artificial pulp chamber made up of dentin slices placed between the materials and the cell cultures (Hanks et al., 1988). Polymerized composite resin separated from the extraction medium by etched dentin had toxic potency higher than that of specimens placed on unetched dentin (Hume, 1985). This observation indicates that the application of the 'total-etch technique' may be associated with a higher risk for pulp irritation due to composite resin restorations, even in combination with dentin bonding (Gerzina and Hume, 1996). Mohsen et al. (1998) investigated the influence of various parameters on the cytotoxic potential of several light-curable UDMA-based composite resins. Cytotoxicity decreased with increasing curing time and higher aging time. A reduction of cytotoxicity was also observed when polished samples without an oxygen-inhibited surface layer were tested. Furthermore, cell viability increased when specimens were extracted with water, ethanol, or other organic solvents prior to cell culture experiments (Mohsen et al., 1998; Rathbun et al., 1991). The cytotoxic potency of polyacid-modified composite resins varied considerably depending on the product tested (Pertot et al., 1997; Geurtsen et al., 1998b). Aqueous eluates of two polyacid-modified composite resins induced moderate injuries in permanent 3T3 fibroblast cultures, whereas one product (DyractTM Cem, De Trey Dentsply) caused severe cytotoxic effects. The severely toxic extract contained a very high concentration of TEGDMA, whereas the two moderately cytotoxic composite resins released only small amounts of comonomers, like HEMA, and various ethylene glycol compounds. In summary, then, cytotoxicity of composite resins varies depending on the product tested and especially upon the quantity of leachable components. An optimum polymerization, therefore, is of high importance for the cytocompatibility of those materials. Furthermore, extractable amounts of components should be reduced, e.g., by the use of less-water-soluble components. Severely cytotoxic substances should be replaced by less toxic alternatives, if available. (C) MICROBIAL EFFECTS Fresh specimens of several composite resins inhibited microbial growth (0rstavik and Hensten-Pettersen, 1978). But other authors found no significant influence on the proliferation of micro-organisms (Updegraff et al., 1971). Recently, it was reported that the water extract of a hybrid-type composite resin promoted the growth of Grit Rev Oral Biol Med Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. (2000) 11(3):333-355 (2000)
  12. 12. treated dentin layers (Horsted ct al), 1986; Quist et al., 1989, Fujitani et at., 1992). When one considers the permeability of dentin, especially after acid pre-treatment, and the release of cytotoxic and hydrophilic the caries pathogen L. acidophilus. It was found that this eluate contained a high amount of TEGDMA. Aqueous extracts of another composite resin with a low concentration of TEGDMA, however, inhibited growth of this micro-organism. Thus, the release of high concentrations of TEGDMA may stimulate proliferation of cariogenic bacteria within marginal gaps which then may contribute to recurrent caries and pulpal irritation (Figs. 4a, 4b, 5a, 5b, 6a, 6b). Release of microbial growth-stimulating substances, therefore, should be minimized or prevented (Hansel et al., 1998). Alternatively, antibacterial monomers (like methacryloyloxydodecylpyridinium bromide, MDPB) or fillers, e.g., ApacideriM, based on apatite containing silver and zinc, have been added to composite resins. These experimental materials significantly inhibited proliferation of cariogenic bacteria (Syafiuddin et al., 1997. Imazato et a)., 1998c). (D) In ViVO STUDIES Local, non-specific toxicity of polymerized composite resins was determined after implantation into various tissues, e.g, muscle and bone. Generally, composite resins caused only slight to moderate tissue alterations which decreased over time (Chan et a)., 1972; Howden and Silver, 1980, Wennberg et al., 1983). Numerous pulp studies have been performed for the evaluation of local specific reactions. The results of these usage tests have yielded contradictory results. An absence of or only slight pulp irritations were reported by Retief et al. ( 1973) and Suzuki et al. ( 1995). Other investigators, however, observed moderate to severe reactions (Tronstadt and Spangberg, 1974; Valcke et al., 1980). It has been observed that several co-factors influence the pulp toxicity of composite resins. A protective cavity lining coupled with an increased thickness in remaining dentin reduced the risk of pulpal inflammation (Quist, 1975; Dalleske et al., 1978) On the other hand, previous damage to the pulp due to deep caries lesions and acid-etching of dentin on the cavity floor enhanced toxicity (Fiore-Donno and Baume, 1966; Stanley et a)., 1 975; Eriksen and Leidal, 1979). Several authors have noted a correlation between pulpal irritations of resin-restored teeth and bacteria (and their by-products), whereas material toxicity per se was of no significance (Brannstrom and Vojinovic, 1976; Torstenson et a/., 1982; Cox, 1992, Cox and Suzuki, 1994). It should be recognized, however, that comonomers released from a resin filling into marginal clefts may stimulate microbial growth and consequently may, at least indirectly, contribute to pulpal irritation caused by those bacteria (Hansel et a)., 1998). However, pulpal inflammation was also found in resin-restored teeth without bacterial microleakage, especially when low-viscous bonding agents had been applied to thin acid-pre111)31333 355 (2000) 100 33-355 120001 Cril Ree A B Figure 6a. This is a 20-year-old patient presenting with an upper right first molar with an occlusal amalgam restoration and a composite resin filling. The amalgam restoration reveals marginal gaps; the resin filing shows only a slight marginal discoloration. Figure 6b. The restorations were removed without excavation of carious dentin. The amalgam cavity is caries-free, whereas extensive recurrent caries is visible beneath the composite restoration. This caries recurrence may have been caused by the growth of cariogenic micro-organisms due to release of comonomers, e.g., EGDMA or TEGDMA (Hansel et a/., 1998). Oral Biol Med Crit Rcv Oral Biol Mcd Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 343
  13. 13. (co)monomers from polymerized composite resin, it is reasonable to suggest that those substances can contribute significantly to pulp injuries (Hume, 1985; Hamid et al., 1996; Eick et al., 1997; Marshall et al., 1997; Abou Hashieh et al., 1998; Gale and Darvell, 1999). On the other hand, it must be emphasized that the barrier function of dentin may protect the pulp from toxic influences. Thus, for example, diffusion of monomers decreases significantly with thicker dentin, and the penetration of bacterial by-products is also reduced by dentin (Pissiotis and Spangberg, 1992; Hamid et al., 1996; Hamid and Hume, 1997a). Several retrospective and prospective studies on the clinical long-term performance of composite resins have been reported (Mjbr et al., 1990; Wilder et al., 1991; Roberts et al., 1992). In general, evaluation of the fillings included determination of pulpal conditions. A four-year clinical study with 1209 Class I and Class 11 restorations revealed long-lasting post-operative pain or pulpitis as a cause for failure in only four cases. Glass-ionomer cement was applied as a base in all cavities. Additionally, areas with thin dentin were covered with a calcium hydroxide cement (Geurtsen and Schoeler, 1997). A meta-analysis of 16 long-term clinical studies of posterior composite resin fillings also indicated no increased risk of pulpal irritation (El-Mowafy et alc., 1994). In summary, there is sufficient evidence that various factors associated with composite resin restorations possess tissue-damaging potency. Furthermore, pulp inflammation/necrosis as a consequence of a composite resin filling is probably due to the interaction of several tissue-irritating parameters rather than to a single detrimental factor. Usage tests as well as long-term clinical studies indicate that composite resins do not pose a special risk for the dental pulp if they are properly applied. (V) Resin-based Pit and Fissure Sealants (A) COMPOSITION AND RELEASE OF SUBSTANCES Resinous pit and fissure sealants consist of an unfilled or filled organic matrix. Like composite resins, the matrix of dental sealants is composed of a mixture of various (co)monomers (mainly Bis-GMA, UDMA, and TEGDMA) and additives. Some sealants also contain fluoridereleasing components. Either clear, tinted, or opaque auto-polymerizing and visible-light-curable products are available (Waggoner and Siegal, 1996). Public concern about the safety of resin sealants was greatly increased by the report of Olea et al. (1996). Using the HPLC technique, these authors reported that the potentially estrogenic substance bisphenol-A was released from fissure sealants in vivo. It must be emphasized that this observation was mainly based on one 344 female patient who had received fissure sealants two years earlier. Bisphenol A, however, is rapidly released from polymerized sealants and is quickly excreted (Ashby, 1997). Thus, it is more than unlikely that dental sealants can serve as a long-term intra-oral source for the leaching of bisphenol A. Olea et al. (1996) also reported that resin oligomers may liberate bisphenol-A after storage in alkaline (pH 13) or acidic (pH 1) media at 100°C for 30 min. But these conditions do not exist in vivo. In contrast to the findings by Olea et al. (I1996), using HPLC or GC/MS methodology, a number of authors reported that the product investigated by Olea et al. (1996), and other sealants as well, did not release bisphenol-A into water or ethanol. TEGDMA, however, was released from several sealants (Hamid and Hume, 1997b; Nathanson et al., 1997; Geurtsen et al., 1999a). Interestingly, TEGDMA was not identified by Olea et al. in their analysis. This strongly suggests that Olea et al. (1996) may have misinterpreted TEGDMA peaks as indicative of bisphenol-A. Due to different mass spectra and retention times, bisphenol-A and TEGDMA can be easily distinguished from each other by GC/MS. Accordingly, no bisphenol-A was found with GC/MS (Geurtsen et al., 1999a). In addition to TEGDMA, monomers (like Bis-GMA, UDMA), comonomers (mainly ethylene glycol compounds), and additives [e.g., camphorquinone and DMABEE (4-N,N-Dimethylaminobenzoic acid ethyl ester)l leach out from sealants in minor quantities (Hamid and Hume, 1997b; Nathanson et al., 1997; Spahl et al., 1998; Geurtsen et al., 1999a). (B) BIOCOMPATIBILITY Few in vitro studies have been published about the cytotoxicity of resinous pit and fissure sealants. There are no in vivo data available except for one case report of an allergic reaction to the sealants (see below) (Hallstrbm, 1993). Water extracts of two sealants were investigated in human embryonic lung fibroblast cultures. It was observed that both materials were more cytotoxic than PMMA (polymethyl methacrylate) acrylic controls (Rawls et al., 1992). Aqueous eluates of four sealants caused moderate-to-severe inhibition of cell growth in 3T3 cultures. Considerable cytotoxic effects in permanent 3T3 fibroblasts were caused by one product which leached high amounts of TEGDMA into de-ionized water (Geurtsen et al., 1999a). In general, only sparse results on biocompatibility and release of substances from these materials are available. These limited data as well as the similarities in composition and degradation compounds indicate that leaching and biological behavior of resin-based pit and fissure sealants should be comparable with that of composite resins. Grit Rev Oral Biol Crit Rev Oral Biol Med Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. (2000) 11(3):333-355 (2000)
  14. 14. (B) BIOCOMPATIBILITY OF DENTIN ADHESIVES (VI) Dentin Adhesives (A) COMPOSITION (1) In vitro studies AND RELEASE OF SUBSTANCES Rakich et al. (1998) investigated the effects of four components of dentin bonding agents on the mitochondrial activity (MTT assay) of macrophages which are important in wound healing and inflammatory reactions. HEMA, 4methacryloxyethyl-trimellitic anhydride 14-META), BisGMA, and UDMA were evaluated. The cytocompatibility varied significantly: HEMA (best compatibility) > 4META >>> Bis-GMA > UDMA. A similar ranking of toxicity was found in experiments with Balb/c 3T3 mouse fibroblasts (Ratanasathien et al., 1995). Aqueous extracts of five adhesives were tested in 3T3 fibroblast cultures. The cytotoxic potency of those products varied considerably. The most toxic material, which almost completely inhibited cell growth, released TEGDMA in high amounts. Two materials released high concentrations of HEMA and reduced cell proliferation considerably. Additionally, two products liberated small quantities of (co)monomers (EGDMA, TEGDMA, or HEMA) that affected cell growth only slightly (Geurtsen et l., 1999c). The influence of dentin on the cytotoxicity of four dentin adhesives was evaluated with L 929 fibroblasts. Each product was tested using dentin slices with high and low hydraulic conductance. Generally, all adhesives were significantly more cytotoxic when slices with high hydraulic conductance were applied (Abou-Hashieh et al., 1998). These observations confirm that the nature and quantity of released substances, as well as the barrier function of dentin, decisively influence the cytotoxic potential of dentin adhesives. Some products release high quantities of (co)monomers (TEGDMA, HEMA) which may cause pulpal alterations, especially when applied in areas with a thin dentin layer. Thus, only good cytocompatible dentin adhesives are recommended as a pulp protective cavity lining on thicker dentin layers. Members of the group of dentin adhesives differ considerably in their chemical composition, mode of action, and clinical handling. Dentin adhesives are mainly used to improve the bonding between dentin and composite resins. In addition, dentin bonding agents are applied as cavity liners and for treatment of hypersensitive teeth with exposed root surfaces (Cox and Suzuki, 1994; lain et al., 1997; Schuckar and Geurtsen, 1997) Depending on the product, dentin adhesives may consist of one to three components ('bottles'). Conditioners (etchants) containing acid (e.g., phosphoric acid, maleic acid) or EDTA are used for partial or total removal of the dentin smear layer and for the structural modification of the binding substrate (Eick et al., 1997). The dissolved smear layer and conditioner are removed by thorough rinsing with water. This results in the opening of most orifices of the dentinal tubules. Primers consist of a 'hydrophilic' monomer (e.g., HEMA) or a mixture of monomers dissolved in solvents like water, ethanol, or acetone. Primers are applied to expand or re-expand the conditioned demineralized collagen network and to increase the wettability of the hydrophilic substrate for the hydrophobic components of the bonding agent. Furthermore, some dentin primers (e.g., SyntaciM) contain glutaraldehyde for stabilization of demineralized collagen fibers. B3onding agents are mainly unfilled mixtures of various hydrophobic and/or hydrophilic co)monomers (e.g., Bis-GMA, UDMA, TEGDMA, HEMA) and additives. They are used to bind the conditioned and/or primed dentin surface with composite resin. Several moderr products combine conditioner and primer ('self-conditioning primers') or primer and bonding agent into one bottle. Very little is known about the release of components from dentin adhesives. Nor-polymerized specimens of various dentin bonding agents were extracted by acetone. Gas chromatography/mass spectrometry (GC/MS) analysis revealed that the eluates predominantly contained ethylene-glycol methacrylates, such as EGDMA and D/TEGDMA, as well as smaller amounts of H-EIMA and MMA (methyl methacrylate) (Kanerva et al., 1994a). Geurtsen et al. ( 1999c) analyzed aqueous extracts of five modern dentin adhesives by GC/MS The adhesives analyzed released several substances, predominantly EGDMA, TEGDMA, and HEMA, into de-ionized water. All materials released camphoroquinone Additionally, four adhesives also released other (co)initiators, e ., 4-N,N -dimethylaminobenzoic acid ethyl ester (DMABEE) and diphenyliodoniumchloride (DPICI). I1 1(31-333-355 i2OM l(3i 333 355 2000 (2) Microbial effects Several micro-organisms are associated with caries lesions, especially mutans streptococci (S. mutans S sobrinus) and lactobacilli (Emilson and Krasse, 1985). Bacteria and their by-products may also cause pulpal irritation. Therefore, dentin adhesives with antimicrobial properties would be of great clinical advantage for prophylaxis of recurrent caries as well as for prevention of pulpal alterations due to microbial effects. The experimental incorporation of the monomer, methacryloyloxy dodecylpyridinium bromide, into a selfetching primer significantly increased the antimicrobial potency, even after polymerization (Imazato et al., 1998a,b). A screening of seven adhesives revealed antimicrobial effects of conditioners/etchants (EDTA, phosphoric acid). Antibacterial properties were also observed with primers Med Biol Crit Rev Oral Biot Med Rev Oral Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 345
  15. 15. containing maleic acid or glutaraldehyde. The other primers and most bonding agents, however, did not show any antimicrobial property (Emilson and Bergenholtz, 1993). Similar observations with other self-conditioning primers have been reported (Imazato et al., 1998c). A polymerizing dentin adhesive containing glutaraldehyde (SyntacTM) had a strong antibacterial effect when tested on eight microbial cultures. But set and aged materials were not tested (Fraga et al., 1996). It can be concluded that most dentin adhesives exhibit antimicrobial effects only in the initial phase after application. Thus, generally speaking, no anti-cariogenic and pulp-protecting effect due to long-term antimicrobial potency of dentin adhesives can be expected. (3) In vivo studies Dentin bonding agents may irritate the dental pulp by three mechanisms: (I) by the conditioner/etchant, (11) direct cytotoxic effects due to released substances, and (111) by bacteria or microbial by-products. Additionally, it must be appreciated that dentin conditioning/etching ('total-etch technique') can significantly increase transport of released organic substances through dentin. Thinner dentin layers may enhance pulpward diffusion of monomers as well as the total leaching of substances from polymerized dentin adhesive (Hamid et al., 1996; Hamid and Hume, 1997a). Interestingly, it was observed that diffusion of HEMA and TEGDMA, in particular, through dentin is not prevented by positive hydrostatic pressure (Gerzina and Hume, 1995). The application of a HEMA-containing bonding resin in combination with a composite resin reduced TEGDMA diffusion through dentin only slightly in comparison with a composite resin that was used without an adhesive. But the dentin adhesive-composite resin combination resulted in additional pulpward diffusion of considerable quantities of released HEMA. Thus, dentin adhesives that leach high amounts of the cytotoxic (co)monomer may cause pulp alteration, especially when the 'total-etch technique' is used. No pulp-protective effects due to reduced diffusion of TEGDMA leached from composite resin can be expected. Several studies on the response of the pulp to dentin adhesives have been performed. The results of these studies are not easily comparable because of the differences in the composition of the adhesives and the techniques used. The application of a three-component product, according to the principles of the total-etch technique, did not cause significant pulpal irritation of monkey teeth over a 90-day period (Inokoshi et al., 1998). Similar observations were made by White et al. (1994) in adult rhesus monkeys. Evidence was presented that irreversible pulpal inflammation is not induced when dentin adhesives are applied in cavities with a thick dentin layer, particularly in caries-free dentin preparations extending just inside the dento-enamel junction (Goracci et al., 346 1995; Pameijer and Stanley, 1995; Gilpatrick et al., 1996). Several investigators have compared the reactions of exposed and non-exposed pulps after the application of dentin adhesives in combination with dentin acid-etching. No differences were found between exposed or nonexposed pulps and between the adhesives and the Ca(OH)2 group, which generally served as a control (Akimoto et al., 1998; Cox et al., 1998; Tarim et al., 1998). But detrimental effects of the total-etch technique with dentin adhesives in vital-pulp-capping of monkey teeth were reported by Pameijer and Stanley (1998). Interestingly, these authors also observed that micro-organisms were present in a large percentage of vital and non-vital teeth. Thus, bacteria had no significant influence on the reaction of the pulps after application of dentin adhesives. Additionally, it was found that pulps directly capped with dentin adhesive may develop a sub-acute foreign body response due to impacted resin particles. The authors concluded that the long-term effect of resin particles in pulpal tissue needs further clarification (Gwinnett and Tay, 1998). These contradictory results clearly show that there are insufficient data for a reasonable conclusion on the effects of dentin adhesives on the pulp. In particular, it must be noted that the abovementioned histological pulp studies covered a period of only about three months. Consequently, the long-term, in vivo, biological effects are not known. Although dentin adhesives may be of benefit to reduce marginal microleakage and to prevent pulpal hypersensitivity in cavities with a relatively thick dentin layer, dentin in deep cavities should be covered by a cavity liner for pulp protection. This is corroborated by in vitro observations indicating that HEMA and TEGDMA may cross dentin even under positive hydrostatic pressure. Exposed pulps should be directly capped with Ca(OH-)2 until there is enough scientific evidence in support of the long-term clinical efficiency and biocompatibility of various dentin adhesives. (VII) Resin-modified Glass-ionomer Cements (A) COMPOSITION AND LEACHING OF SUBSTANCES Resin-modified glass-ionomer cements (rmGIC) contain ion-releasing glass particles, water-soluble polyacrylic acids, light-curable monomers (e.g., HEMA), and additives. Various products are composed of photo-curable side-chains linked to water-soluble polymeric acids. In contrast to polyacid-modified composite resins (compomers), rmGICs contain water and thus exhibit an acidbase reaction with salt formation and a free-radical polymerization (Kakaboura et al., 1996; Nicholson, 1998). Due to their ease of application in comparison with conventional GICs, resin-modified systems are being increasingly used clinically, especially in pediatric dentistry Crit Rev Oral Biol Med Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. I1(3):333 355 11(3):333-355 (2000)
  16. 16. (Vaikuntam, 1997). Several investigators evaluated the release of fluoride from rmGlCs )Forss, 1993; Ulukapi et al., 1996; Friedl et al., 1997; Geurtsen et al., 1998c). The release of fluoride varied significantly, depending on the product tested and the extraction medium. In general, less fluoride leached from rmGlCs than from conventional GICs (Geurtsen, 1998). Leaching of organic components from various rmGICs into distilled water was investigated by Hamid et al. 11998). HEMA was the only compound detected in the water extracts by HPLC. Aqueous eluates of two rmGlCs were analyzed with GC/MS (Geurtsen et al., 1998b). Both products released HEMA into de-ionized water. In addition, one material (Vitrebond'IMm) released the initiator, diphenyliodoniumchloride (DPICI), which was also present in one dentin adhesive (SyntaciM) (Geurtsen et al., 1999c). DPICI was identified by its GC-decomposition products chlorine-, iodine- and bromine benzene. It might be possible, however, that these highly toxic substances are generated during polymerization and are thereafter released into an aqueous environment. Our knowledge about the quality and amount of leachable components from rmGlCs is insufficient. Besides (co)monomers, (co)initiators, etc., reaction or degradation products, e.g., benzenes, are released which may then cause adverse local and/or systemic effects. These leachable reaction or degradation products, therefore, should be identified as soon as possible. (B) BIOCOMPATIBILITY OF RESIN-MODIFIED GICs The cytocompatibility of various conventional and resinmodified GICs was evaluated in cultures of primary human osteoblasts. All materials, except for one product, exhibited good cytocompatibility. Viable osteoblasts grew inito contact with the specimens. One resin-modified GIC (Vitremer"'Nl), however, was very cytotoxic. The authors concluded that these adverse reactions might be due to release of high amounts of HEMA (Oliva et a)., 1996). Comparable observations were reported by Consiglio et il, ('1998). Two rmGlCs (VitrebondiM and Vitremeri'( reduced protein synthesis of human gingival fibroblasts by 00°o, whereas the four tested conventional GlCs caused significantly less inhibition of protein synthesis. Severe cytotoxicity of Vitremert'N in perma nent 3T3 fibroblasts was also found by Kan et al. (1997). The other rmGIC (Fu'ji 11 LCiSII caused no or only slight cellulal alterations. Finally, the detrimental cellular effects of Vitrebond'%- and the mild cytotoxicity of Fuji 11 LC I' were corroborated in monolayers of 3T3 fibroblasts, primary human gingival fibroblasts, human peripheral lymphocytes, and in dentin barrier tests with fibroblasts (Leyhaulsen et al., 1998, Geurtsen et al., 1998c; Stea et a)., 1998, Schmalz tot a)., 1999) (Figs. Ia-c, 2a-c). It appears that the deleterious effects of Vitrebond'im may be partly due to the release of the highly cytotoxic initiator, diphenyliodoniumchloride, and/or its decomposition products (Ceurtsen et a)., 1998c). It is noteworthy that the abovementioned in vitro studies produced fairly similar results, even though different cell types and biological endpoints were used. Besides its cytotoxic effect, fresh Vitremerii' also revealed moderate to strong antibacterial effects in cultures of mutans streptoccoci and lactobacilli (Fraga et al., 1996, Fried) et al., 1997). Direct and indirect genotoxic reactions were caused by Vitrebond"'5' in procaryotic and eucaryotic in vitro assays, whereas other resin-modified and conventional GICs induced only questionable indirect genotoxicity or no such effect. These in vitro results were confirmed by in vivo assays (Heil et a), 1996; Stea etal., 1998). Taken together, there is strong evidence that some resin-modified GICs may be genotoxic and severely cytotoxic due to release of various substances, e.g., HEMA and DPICI. Consequently, rmClCs may cause adverse local and/or systemic effects. Thus, leaching of those components must be minimized or prevented. (VIII) Allergic Reactions Caused by Resin-modified Filling Materials The allergic or sensitizing potential of resin materials and components was evaluated in various in vivo studies, e.g., maximization tests with guinea pigs. A marked allergic potential was exhibited by impurities of the matrix monomer Bis-GMA (B)jrkner et al. 1984; B)brkner, 1984b). Aliphatic urethane (meth)acrylates, for dental application, were more potent sensitizers than aromatic urethane acrylates and (meth)acrylated aliphatic urethane (Bj6rkner, 1984a). HEMA also revealed a marked allergic effect. From 60 to 100% of tested guinea pigs reacted to high concentrations of this monomer in the maximization test (Clemmensen, 1985). In addition, mutual cross-sensitivity to methacrylates was found. Guinea pigs sensitized to various methacrylates also responded strongly to methacrylates which had not been used for sensitization (Chung and Giles, 1977). Similar observations have also been made in humans (Kanerva et al., 1992b; Richter and Geier, 1996). Skin reactions were provoked by some bonding systems in guinea pigs and Macaca mulatta monkeys (Altuna and Freeman, 1987; Katsuno et al., 1998). Patients, and especially dental personnel, are increasingly exhibiting local and systemic allergic reactions to components of resinous dental materials (Kanerva et al., 1994a,b; Richter and Geier, 1996; Tschernitschek et al., 1998). It must be emphasized, however, that the number of individuals allergic to resin34 11(3 3~ 35~2001 1 1(3)333-355 (2000) Cr Re Ora Bil Me Crit Rev Orail Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 347
  17. 17. based dental materials is still low. In addition, etiologic factors other than dental materials may also induce intra-oral contact allergy, e.g., ingredients in food, beverages, and oral hygiene products (De Rossi and Greenberg, 1998). Kallus and Mjor (1991) investigated the incidence of adverse reactions to dental materials. Two verified longstanding effects to resin-based materials (dentures) were found in 13,325 patients. Only eight patients out of a total number of 31 1 persons who had been referred to a dental hospital for possible allergic reactions to dental materials suffered from a verified allergy to methacrylates (Tschernitschek et al., 1998). Adverse reactions to a Bis-GMA-based fissure sealant in a six-year-old girl have been described by Hallstrom (1993). One day after application of the material, the patient developed multiple allergic reactions: asthma, blisters on the gingiva adjacent to the resin sealants, swellings, and rashes on various areas of the body (arms, palms, legs, lips, ears). The symptoms completely disappeared within nine days after removal of the sealants. A verified extra-oral hypersensitivity to composite resin was observed in one patient (Nathanson and Lockhart, 1979). Other patients revealed an allergic contact dermatitis to composite resin. The reactions were caused by Bis-GMA and benzoyl peroxide (Carmichael et al., 1997; Kanerva et al., 1989). Seventeen oral lichenoid lesions on the mucosa in contact with composite resin restorations were described by Lind (1998). Total remission of the lichenoid reactions was observed after replacement of the resin fillings in four cases and partial remission in another five patients. The author suggested that contact of the oral mucosa with formaldehyde derived from the resin restoration may have been the main etiologic factor in these cases. There are several reports in the medical and dental literature regarding hypersensitivity or allergic reaction of patients to MMA (Kaaber, 1990; Richter and Geier, 1996; Hochman and Zalkind, 1997) (Table 1). Dental personnel often have manual contact with resinous materials. Many acrylates rapidly penetrate latex or vinyl gloves (Kanerva et al., 1993). Monomer vapors can be inhaled through face masks. Thus, devices like gloves and face masks provide only incomplete protection. Taken together, the incidence of allergy to resinbased materials in dental professionals (dentists, nurses, dental technicians) has increased. Lonnroth and Shahnavaz (1998b) found that 16-17% of the dental personnel in their study were hypersensitive to dental materials, in comparison with 0.5%-2% in the control group. Hand dermatitis and symptoms on the fingers were significantly more frequent among dental professionals than in the control group (Lonnroth and Shahnavaz, 1998a). There are some resin components which are especially important with respect to allergic reactions in 348 dental personnel. Dental nurses showed a high incidence of allergic reactions to glutaraldehyde and benzoyl peroxide, whereas dental technicians were frequently hypersensitive to HEMA, EGDMA, MMA, benzoyl peroxide, TEGDMA, and dihydroxy-ethyl-p-toluidine (Schnuch and Geier, 1994). Other investigators have reported that dental nurses and dentists developed allergic contact dermatitis to HEMA caused by handling of dentin adhesives (Kanerva et al., 1992a, 1994a). Six nurses and one dentist had positive patch tests to composite resins. Four of those individuals were hypersensitive to Bis-GMA, and three reacted to TEGDMA; two patients were also hypersensitive to MMA. All of these dental nurses had to stop the practice of their profession (Kanerva et al., 1989). It is very likely that the use of resin-based materials in dentistry will significantly increase in the future. Concomitantly, the incidence of allergies to dental resin components will probably also increase considerably, among patients and especially among dental personnel. The proper handling of those products, therefore, is of considerable importance. Specifically, contact with unpolymerized material must be avoided. In addition, adequate ventilation of rooms for processing of resinbased materials is recommended, to minimize the concentration of monomer vapors in the air. (IX) Genotoxicity of Resinbased Restorative Materials There is little information on the genotoxic effects of individual resin components. Hensten-Pettersen et al. (1978) screened single ingredients of composite resins (initiators, inhibitors, UV-absorbers) in the classic Ames test. None of the tested components revealed mutagenic activity. Miller et al. (1986), however, found weak mutagenic reactions due to one co-initiator. No mutagenicity was observed with Bis-GMA and UDMA in the Ames test (Geurtsen, 1988). Fourteen mono-substances were investigated by means of the umu test (procaryotic, in vitro), the DIT (DNA-synthesis inhibition test; eucaryotic, in vitro), and the AFE assay (in vivo) (Table 1) (Heil et al., 1996). Interestingly, almost all tested resin components were genotoxic in at least one test system. The effects ranged from 'borderline' to 'strong positive'. No positive results were observed with the monomers BEMA (benzyl methacrylate) and HEMA, whereas Bis-GMA (umu, AFE) and UDMA (AFE) were genotoxic in one or two assays. Recently, it has been reported that TEGDMA induced large DNA sequence deletions in the hprt gene of V79 cells. The authors concluded that the induction of such DNA sequence deletions might be common for acrylates and methacrylates (Schweikl and Schmalz, 1999). Mutagenic effects in the Salmonella typhimurium mutagenicity test system ('Ames test') were produced by glutaraldehyde as well as by dimethyl sulfoxide Crit Rev Oral Biol Med Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 1 1(3):333-355 11(3):333-355 (2000)
  18. 18. (DMSO) and physiologic saline eluates of a dentin adhesive. Two other products exhibited no mutagenicity. It was concluded that the mutagenic effects of one of those dentin adhesives (SyntacFmv) were induced by the genotoxic ingredient, glutaraldehyde (Schweikl et al., 1994). Dose-dependent mutagenicity was found in Ames tests with unpolymerized rnaterial of two dentin adhesives. Genotoxic effects were also caused by monomeric solutions used in orthodontics for cementation. However, no specific component was identified as the causative agent in those studies (Athas et al., 1979; Li et al., 1990). Scarce data are available about the genotoxicity of composite resins. DMSO and cell culture medium extracts of two hybrid-type composite resins were investigated in various assays. Genotoxic effects were produced by the DMSO eluates of one composite resin (SonoCem]N1) in the eucaryotic DIT (Heil et al., 1996) (Table 2). A resin-modified glass-ionomer cement (Vitrebond[M) exhibited strong genotoxic action. Extracts of this rmGlC induced sister chromatid exchange in human peripheral lymphocytes in vitro. Genotoxicity was verified by three different in vitro and in vivo assays: the procaryotic umu test, the eucaryotic DIT, and the in vivo NFE assay. Analysis of water extracts of this GIC indicates that leaching of the initiator, DPIGI, may contribute to this strong genotoxic action (Heil et al 1996; Stea et al., 1998) (Table 2). Taken together, it is clear that there are insufficient data documenting the genotoxicity of resin-based dental materials. In light of the evidence of the genotoxic potential of some of these materials, it is important that the research in this area be given sericus consideration. (X) Conclusions and Summary The biocompatibility of a resin-based dental restorative material is predominantly determined by the amount and nature of released organic substances. The cytotoxic potential of these components varies significantly. TABLE 2 Mutagenicity of Aqueous Eluates and DMSO Extracts from Two Composite Resins (cr) and Two Resin-modified Glass-ionomer Cements (GIC) Product Aqueous Extract DMSO Extract HerculiteTM (cr) No Sono-CeMTM (cr) lonosea ITM (GIC) VitrebondTM (GIC) No No Yes2 No Yes'-3 Yes' n.d.4 Results from umu test, IDIT, 3AFE assay IHeil et al., 1996); 4not determined. 000 3133355 (3):3 3 3- 3 5 5 (2000) Additionally, interactive effects of secreted organic and/or inorganic substances may occur, e.g., synergism, additive effects, and antagonism. Resin-based restorations release several (co)monomers (like ethylene glycol compounds, HEMA) and additives (mainly initiating substances) into the oral cavity which may cause adverse local and systemic effects in patients, e.g., lichenoid reactions of the oral mucosa or allergic reactions. There are sufficient data demonstrating that the liberated substances may diffuse pulpward through dentin in high quantities (TEGDMA HEMA), particularly through thin dentin layers or after acid-etching of dentin. It appears that high quantities of these components may result in pulpal inflammation. Although it is of considerable significance, it is not known if substances leached from resinous materials enter the blood stream, and whether they are transformed in the liver and/or accumulate in other tissues. Manual contact between organic components and unprotected skin during application of resin-based materials poses an increased risk for allergic reactions in dental personnel. There is strong evidence that some ingredients of resin-based materials (e.g., glutaraldehyde), as well as aqueous extracts of various products, are genotoxic in vitro and in vivo. Thus, a high priority should be given to clear identification of the genotoxic components in resin-based materials. Taken together, one can conclude that resin-based restorations may cause adverse local and systemic effects. Therefore, a careful handling of resin-based dental materials is critical in order to minimize adverse effects, e.g., optimum polymerization, cavity lining in areas with deep dentin, and no direct skin contact. The increasing usage of tooth-colored fillings in the posterior region as well as the development and clinical application of new adhesive techniques (ceramic inlays and crowns, etc.) indicate that the use of resinous materials will significantly increase in the future. This, in turn, will very likely increase the incidence of local and systemic side-effects. The following aspects, therefore, should be primary aims of future research *Development of in vitro biocompatibility tests which better simulate in vivo conditions *Determination of those chemical-biological interactions which are critical for the biocompatibility of resinous materials *Investigation of the systemic distribution, metabolism, and/or accumulation of leached components in the human body *Identification of severely cytotoxic and/or genotoxic compounds and replacement of these substances by better biocompatible components *Development of resinous materials with high monomer-polymer conversion and low amounts of leachable substances Cri Re Ora Bil Me Crit Rev Oral Biol Med Downloaded from at BIOLOGICAL LABS LIBRARY on December 27, 2013 For personal use only. No other uses without permission. 34 349
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