Effects of one versus two applications of an unfilled, all-in-one adhesive on dentine bonding

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1 Journal of Dentistry 30 (2002) Effects of one versus two applications of an unfilled, all-in-one adhesive on dentine bonding Edna L. Pashley a, Kelli A. Agee a, David H. Pashley a, Franklin R. Tay b, * a Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA , USA b Paediatric Dentistry and Orthodontics, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR, People s Republic of China Received 27 March 2001; revised 31 October 2001; accepted 5 November 2001 Abstract Objectives. This study examined the null hypothesis that there is no difference between the effect of a one versus two-layer applications of Prompt L-Pop (3M ESPE, Seefeld, Germany) to sound, abraded human coronal dentine. Methods. In group I, the mixed adhesive was applied for 15 s, and light-cured for 10 s. In group II, after light-curing the first layer, the adhesive was re-applied and light-cured. Specimens bonded with a hybrid composite were sectioned into beams for microtensile bond strength evaluation. Additional teeth from each group were bonded similarly using a lining composite for transmission electron microscopy examination of the resin dentine interfaces, and nanoleakage evaluation using ammoniacal silver nitrate. Results. A significant difference ( p, 0.001) was detected between microtensile bond strengths in the two groups. Stained, demineralized sections revealed 3 5 mm thick hybrid layers in both groups. An electron-lucent layer between 7 and 20 mm thick was present between the adhesive and the overlying composite. This layer was absent from the interfaces after removal with ethanol before composite placement. The use of a single application in group I resulted in the direct contact of the electron-lucent layer with the dentine surface and tubular orifices. In unstained, undemineralized sections used to evaluate nanoleakage, silver deposits were found mostly in the hybrid layer in group II, but throughout the entire adhesive layer in group I. Conclusion. Bonding of this unfilled all-in-one adhesive to dentine may be improved by application of a second adhesive layer after lightcuring the first layer. This ensures that the exposed dentine surface and dentinal tubules are coated with adhesive that is adequately polymerized. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: All-in-one adhesive; Air inhibition; Dentine bonding; Bisacylphosphine oxide photoinitiator 1. Introduction Dentine adhesives are currently available as three-step, two-step and single-step systems depending on how the three cardinal steps of etching, priming and bonding to tooth substrates are accomplished or simplified [1]. Two-step systems are sub-divided into the self-priming adhesives that require a separate etching step, and the self-etching primers that require an additional bonding step [2]. The recently introduced all-in-one adhesives further combined these three bonding procedures into a single-step application. Irrespective of their packaging designs, these systems are supplied as two-component assemblies to maintain adequate shelf-lives. They are mixed together immediately before use, and the mixture of hydrophilic and hydrophobic resin * Corresponding author. Tel.: þ ; fax: þ address: kfctay@hknet.com (F.R. Tay). components is then applied to the tooth substrate. Of the six commercially available all-in-one adhesives, Prompt L-Pop (3M ESPE, Seefeld, Germany), Etch and Prime 3.0 (Degussa AG, Hanau, Germany), AQ Bond (Sun Medical, Kyoto, Japan) are unfilled versions, whereas One-Up Bond F (Tokuyama, Tokyo, Japan), Reactmer Bond (Shofu, Kyoto, Japan), and Xeno CF Bond (Sankin, Tokyo, Japan) are filled versions that contain fluoride-releasing glass fillers or monomers. Manufacturers of unfilled dentine adhesives often recommend application of at least two coats of adhesive to ensure that etched dentine is adequately covered, as oxygen inhibition of thin, unfilled adhesive layer can result in defective bond formation [3 5]. Thicker unfilled adhesive layers also contribute to greater reduction in polymerization shrinkage stress and the extent of microleakage in cavities that are subsequently restored with resin composites [6]. All-in-one adhesives and self-priming /02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S (02)

2 84 Table 1 Unfilled all-in-one adhesive used in the study Adhesive Composition Lot number ph Water, methacrylated phosphoric acid-hema esters, BAPO intiator a stabilizer, fluoride complex Parabenes FW Prompt L-Pop (composite and compomer version) (3M ESPE, Seefeld, Germany) Bisacylphosphine oxide (e.g. Bis-(2,6-dichlorbenzoyl)-(4-butylphenyl)-phosphine oxide or bis-(2,6-dichlorobenzoyl)-(4-n-propyl phenyl)-phosphine oxide) [10] replaces camphorquinone/tertiary amine as photoinitiator/accelerator. a adhesives differ from self-etching primers in that the latter utilize additional strength-conveying resin layers to couple resin composites to the primed tooth substrates. Recent studies showed that some unfilled all-in-one adhesives exhibited relatively low bond strengths when compared with two-step self-etching systems [7,8]. The manufacturer instructions in Etch and Prime 3.0 and AQ Bond recommend the application of a second coat of adhesive to improve their bonding efficacy. A recent study also reported that bonding of Prompt L-Pop may similarly be improved with additional layers of adhesive [9]. Prompt L-Pop is a unique light-curable adhesive in that it utilizes bisacylphosphine oxide (BAPO) as a photoinitiator that does not require a tertiary amine accelerator as in conventional camphorquinone-containing systems [10]. This study correlated the microtensile bond strengths (mtbs) of Prompt L-Pop achieved after one or two-layers of adhesive application, with differences in ultrastructure and nanoleakage within the bonded interfaces as observed by transmission electron microscopy (TEM). To avoid the possibility of inadvertent mineral dissolution with an acidic silver nitrate (AgNO 3 ) tracer solution, we employed a modified silver-staining technique that consisted of the use of a basic version of ammoniacal AgNO 3 (ph 9.5) [11]. The null hypothesis tested was that there is no difference between the location of the air inhibition layer and the mtbs and extent of silver uptake within the resin dentin interfaces after one or two-layer application of this selfetching, all-in-one adhesive. 2. Materials and methods 2.1. Tooth preparation Bonding was performed on the occlusal surfaces of deep coronal dentine of 14 human third molars that were stored in 0.5% chloramine T and used within 1 month following extraction. The occlusal enamel was removed using a slowspeed saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA) under water lubrication. On the dentine surface 180-grit silicon carbide paper was used under running water to create a smear layer of clinically relevant thickness [12]. The bonding surfaces were examined under a stereoscopical microscope (Nikon SMZ10, Tokyo, Japan) to ensure that they were free of retained enamel. The teeth were randomly divided into two groups Bonding procedures The new composite/compomer version of Prompt L-Pop (Table 1) was used in the experiments. A new blister pack was used for each tooth. Each pack was activated by squeezing and emptying the liquid out of the red blister into the yellow blister. The activated liquid mixture was then emptied into the green section of the blister pack and then

3 85 applied to the dentine specimens using the pre-packaged, disposable applicator. In group I, the all-in-one adhesive was liberally applied to the entire dentine surface and agitated for 15 s. The liquid was then gently air-dried and spread into a homogenous, slightly shiny film. Each bonded surface was examined under 10 magnification to check that the exposed dentin was adequately covered with adhesive. As the BAPO photoinitiator in Prompt L-Pop has a lower peak absorption wavelength (383 nm; range nm) than camphorquinone (468 nm; range m) [13], it may not be polymerized completely using plasma ARC lights or lasers. The adhesive-coated surfaces were light-cured for 10 s using a conventional halogen light-activation unit (Variable Intensity Polymeriser, Bisco, Schamburg, IL, USA) equipped with a cut-off filter in the range of nm, and with the intensity set at 500 mw/cm 2. In group II, after light-curing the first layer, a second layer of adhesive was re-applied and light-cured in a similar manner mtbs evaluation Two adhesive-coated teeth from each group were used for this part of the experiment. Resin composite build-ups were performed using a light-cured hybrid composite (Filtek Z250, 3M ESPE, St Paul, MN, USA) in five 1 mm increments that were light-activated separately. After storage in distilled water at 37 8C for 24 h, each tooth was sectioned occluso-gingivally into serial slabs using an Isomet saw under water lubrication. They were further sectioned into mm 2 composite-dentine beams, according to the technique for the non-trimming version of the microtensile bond test reported by Shono et al. [14]. Two teeth from each group yielded beams for bond strength evaluation. Specimens were stressed to failure under tension in a Bencor Multi-T device (Danville Engineering, San Ramon, CA, USA) using a universal testing machine (Model 4440; Instron Inc., Canton, MA, USA) at a crosshead speed of 1 mm/min. Beams with premature bond failure were assigned a null bond strength value and were included in the compilation of the mean TBS. The bond strength data was statistically analyzed with Student s t-test using SigmaStat Version 2.03 (SPSS, Chicago, IL, USA). Statistical significance was set in advance at the 0.05 probability level Ultrastructural examination of resin dentine interfaces Three adhesive-coated tooth specimens from each group were examined. In two of the three teeth, a lining resin composite (Protect Liner F, Kuraray Co. Ltd, Osaka, Japan) was applied after light-curing the final layer of adhesive. It was added in three 1 mm increments to the bonded interface and light-cured individually for 40 s each. The use of a lining composite that contains colloidal silica particles and pre-polymerized fillers facilitates subsequent TEM specimen preparation. For the remaining tooth in each group, the air inhibition layer on the surface of the cured adhesive was removed with lint-free gauze that was saturated with 100% ethanol prior to placement of the lining composite. This additional control enabled us to interpret some of the features present in the two experimental groups. Demineralized ultrathin sections of the bonded specimens were prepared according to the TEM protocol described in Tay et al. [15]. Sections were double-stained with uranyl acetate and Reynold s lead citrate and examined with a TEM (Philips EM208S, Eindhoven, The Netherlands) operating at 80 kv. Digitized images were recorded using the charge couple device (CCD) camera (Bioscan, Model 792, Gatan Inc., Pleasanton, CA, USA) attached to the microscope. Five digitized images from each group were imported into image analysis software (NIH Image 1.60, Scion Corp., Frederick, MD, USA). The thickness of the hybrid layers and the electron-lucent layers were measured from two positions in each micrograph and the pooled data from each group were statistically analyzed in the manner previously described Silver-staining technique An ammoniacal AgNO 3 solution was first prepared by dissolving 25 g of AgNO 3 crystals (Sigma Chemical Co., St Louis, MO, USA) in 25 ml of 28 wt% aqueous ammonium hydroxide (NH 4 OH; Sigma Chemical Co.) in the presence of ambient laboratory light. This created a suspension of black silver particles. Additional 28% NH 4 OH was used to titrate the black solution, until the solution slowly became clear as the ammonium ions complexed the silver into diammine silver ions ([Ag(NH 3 ) 2 ] þ ). This solution was diluted to 50 ml with distilled water to achieve a 50 wt% solution (ph 9.5). The last two teeth from each group were bonded similarly using the lining composite described previously. One 0.9 mm thick slab was prepared from each tooth by sectioning perpendicularly to the bonded interface with the use of an Isomet saw under water lubrication. These slabs were coated with fast-setting nail varnish applied 1 mm from the bonded interfaces. Without allowing these slabs to be grossly dehydrated, they were immersed immediately in the 50 wt% ammoniacal AgNO 3 solution for 24 h. The silver-stained slabs were rinsed in distilled water and placed in photo-developing solution for 8 h under a fluorescent light to facilitate reduction of the silver ions into metallic silver particles within potential voids along the bonded interfaces [16] as well as in the polymerized resins [17]. Two 1 mm wide rectangular beams were sectioned from each silver-stained slab, from which nm thick undemineralized TEM sections were prepared according to the TEM protocol described in Tay et al. [15]. They were collected on single slot, carbon- and formvar-coated copper grids (Electron Microscopy Sciences, Fort Washington, PA, USA) and examined without further staining.

4 86 Table 2 Microtensile bond strengths (mtbs) and thickness of the hybrid layer and air-inhibition layer after one- or two-layer application of Prompt L-Pop Layers of adhesive Bond strength measurement Thickness of hybrid layer (mm)** Thickness of air inhibition layer (mm)** mtbs (MPa)* Number of specimens tested Number of premature failure One layer (group I) 14.2 ^ 7.2 a ^ ^ 5.2 Two layers (group II) 29.7 ^ 5.7 b ^ ^ 4.9 *Values are means ^ standard deviation for beams with average cross-sectional area of 0.81 mm 2. Beams with premature failure during specimen preparation were included as null bond strengths in the calculation of mean bond strength. Groups identified with different superscript letters are significantly different ( p, 0.001). **Values represent means ^ standard deviation of 10 measurements obtained from five micrographs. Groups identified with the same superscript symbols are not significantly different ( p. 0.05). 3. Results Statistical analysis of the tensile bond strength data (Table 2) revealed significant difference between one and two layers of adhesive application ( p, 0.001). Stained, demineralized sections of resin dentine interfaces from group I (single adhesive layer) are shown in Figs. 1 and 2, and those from group II (two adhesive layers) are shown in Fig. 3. Both groups contained 3 5 mm thick hybrid layers in which smear layers were completely dissolved by the adhesive. Between the adhesive and the overlying composite was a 7 20 mm thick electron-lucent layer in both groups. There was no statistical significant difference in the thickness of these two entitles in both groups ( p. 0.05; Table 2). The location of this electron-lucent layer varied according to whether one or two layers of adhesive were applied before composite placement. The electron-dense (black) adhesive layer was considerably thinner in group I, to the extent that there were always some areas in every specimen where there was direct contact of the electron-lucent layer with the dentine surface and tubular orifices. This electronlucent layer could not be mistaken as smear layer remnants as the former was absent from the control specimen in both groups, when the cured adhesive layer was first wiped with ethanol before placement of the lining composite. Unstained, undemineralized sections of specimens that were immersed in ammoniacal silver nitrate are shown in Fig. 4. Heavy silver deposits were observed along the entire adhesive layer as well as the hybrid layer in group I. In contrast, silver deposits were present only in some areas of the hybrid layer, and were generally absent from the adhesive layer in group II. 4. Discussion The electron-lucent layer in Prompt L-Pop could be readily discerned ultrastructurally by comparing experimental and control specimens in both groups. For group I experimental specimens, bonded interfaces in which the exposed dentine was covered with even a very thin film of adequately polymerized adhesive (Fig. 1) did not appear to be very much different from group II experimental specimens that were covered with thick polymerized adhesive layers (Fig. 3). The drop in bond strength and the occurrence of extensive silver staining in the adhesive interface (Fig. 4) in the group I are thus attributable to the location of an electron-lucent layer directly on dentine surfaces and within tubular orifices (Fig. 2). This leads to a rejection of the null hypothesis. Although the effect of adhesive thickness on bond strength [18] or fracture toughness [19] is equivocal and probably system specific, it is important that exposed dentine is adequately covered by an adhesive to achieve optimal bonding results [20,21]. Incomplete coverage of

5 87 exposed dentine has also been encountered to a certain extent with the use of unfilled self-priming adhesives. Like the all-in-one adhesive examined in this study, the bulk of these adhesives consist of the solvents that are used to dissolve the resin components. They lack additional solventfree resin layers such as those utilized in three-step and selfetching primer systems. The low resin concentration and/or low viscosity of some of these self-priming adhesives could result in the resin composites displacing thin oxygeninhibited adhesive layers from the surface of the hybrid layers and their penetration into dentinal tubules [22]. Such a scenario may be further aggravated when these adhesives are used on clinically relevant cavities in which an adhesive layer of even thickness is difficult to achieve. This is due to pooling of the adhesive along internal cavity line angles, leaving behind only thin adhesive layers along cavity walls [23,24]. Although the benefit of filled versus unfilled selfpriming adhesives has not been substantiated in a recent clinical trial [25], incorporation of microfillers or nanofillers in these adhesives to increase their viscosity does allow sufficient bulk of resin to be retained with a single layer application [6,26,27]. It is known that vinyl resin monomers that polymerize via a free radical addition polymerization mechanism may be inhibited by oxygen, which is an effective free radical scavenger [28]. For resin compositions that utilize photoinitator systems such as camphorquinone and a tertiary amine, oxygen quenches the excited triplet state of camphorquinone and also reacts with the free amine radicals and monomer propagation radicals, producing peroxy radicals that result in premature chain termination [29]. With volatile monomers such as methyl methacrylate that have significant vapor pressure, the uncured monomers on the surface of an otherwise polymerized adhesive evaporate relatively quickly, exposing the hard polymeric surface beneath. However, when an uncured comonomer composition contains less volatile vinyl monomers, the presence of oxygen inhibits surface curing and leaves a residual layer of uncured monomer. The thickness of the air inhibition layer depends on the resin viscosity, which affects the rate of oxygen diffusion [30]. Increased concentration of HEMA in a comonomer blend also resulted in an increased thickness of the air inhibition layer [31]. Normally, placement of a second adhesive layer or resin composite over an air Fig. 1. Stained, demineralized TEM micrographs of experimental and control specimens in group I (one adhesive layer) showing regions where thin electron-dense adhesive layers could be recognized on the dentine surfaces. (a) Experimental specimen with a thick electron-lucent layer between the unfilled, electron-dense adhesive layer and the lining composite. (b) Thinning of this adhesive layer to a point where it disappeared from the surface of the hybrid layer. (c) Control specimen with the electron-lucent layer removed with ethanol. The electron-dense adhesive layer was in direct contact with the lining composite. (C: lining composite; IL: inhibited electron-lucent layer; Pointer: adhesive layer; H; hybrid layer; D: laboratory demineralized intertubular dentine).

6 88 Fig. 2. Stained, demineralized TEM micrographs of experimental and control specimens in group I (one adhesive layer) showing regions where thin adhesive layers were absent from the dentine surfaces. (a) Experimental specimen in which a thick electron-lucent layer was in direct contact with the surface of the hybrid layer and tubular orifices. The electron-dense adhesive could only be found inside the dentinal tubules. Collagen fibrils along the surface of the hybrid layer exhibited a shag carpet appearance. (b) Control specimen with the electron-lucent layer removed with ethanol. The lining composite was in direct contact with the surface of the hybrid layer as well as the polymerized adhesive within the dentinal tubules. Collagen fibrils along the surface of the hybrid layer were probably collapsed after removal of the unpolymerized adhesive. (C: lining composite; IL: inhibited electron-lucent layer; Pointer: adhesive layer; H; hybrid layer; D: laboratory demineralized intertubular dentine). inhibition layer seals the uncured resins from a continuous supply of atmospheric oxygen. There is a competition between scavenging of free radicals by the remaining oxygen and the generation of new free radicals by the resin composite initiator (kinetic effect). When the generation offree radicals is fast and abundant, polymerization will proceed. The inhibition layer, in turn, is either displaced or absorbed into the overlying adhesive or composite layer [30]. We speculate that the electron-lucent layer between the bottom of the composite and the top of the adhesive is a layer of air-inhibited, poorly polymerized oligomers. This layer in Prompt-L-Pop bonded specimens is unique in two aspects. Firstly, this layer is unusually thick, (although the mean thickness of the electron-lucent layer in the two groups was not significantly different despite their different locations along the resin dentine interfaces). The electronlucent layer in Prompt L-Pop is also unusual in that it exists almost as a separate entity that contains very little resin composite fillers. Normally, the resin matrix of a composite should intermix with the air inhibition layer, as it is adapted into the uncured adhesive monomers. Adhesives containing phosphoric acid esters such as 2,2,6-6 tetraacryloxyloxymethyl-4, 8-dioxa-9-oxo-11-undecyl phosphoric acid (PENTA; e.g. Prime and Bond NT, Dentsply DeTrey, Konstanz, Germany) and 10-methacryloyloxy methacrylate (MDP; e.g. Clearfil SE Bond, Kuraray, Osaka, Japan) generally have high affinities for uranyl acetate and lead citrate and appear fairly electron-dense under TEM examination [15,32]. The air inhibition layers disappear in these systems when a similar layer of lining composite is placed on top of the cured adhesive layers. Our observation of an undulating surface at the base of the composite layer in specimens bonded using Prompt L-Pop suggests that the composite did intermix with the top portion of the uncured adhesive monomers. The fact that very little composite microfillers were present in what we observed ultrastructurally as the electron-lucent layer suggests that it was only partially polymerized, and had attained sufficient rigidity to resist the blending of the resin composite. It is also possible that the electron-lucent layer was due to a phase separation and that its composition differs from that of the underlying adhesive. Thus, we speculate the existence of three interconnected zones (layers) in the light-activated Prompt L-Pop adhesive layer: a totally-uncured air-inhibited top layer, a partiallycured middle layer and a completely-cured bottom layer. The totally uncured top layer intermixed with the composite as it was applied on top of the adhesive and was not recognizable ultrastructurally. It is possible that the adhesive interface that exhibited extensive silver staining in Fig. 4(a) was the ultrastructural manifestation of the partially cured middle layer. Our TEM data confirmed the micro-raman spectroscopic results reported by Wang et al. [33] that Prompt-L-Pop was incompletely polymerized on dentine. Incomplete polymerization of the adhesive resin within the hybrid layer probably explained why bond strength was low when one coat of adhesive was used. Thus, multiple coats of adhesive application are recommended for optimal results to be achieved with this all-in-one adhesive. Acknowledgements We thank Amy Wong of the Electron Microscopy Unit,

7 89 Fig. 4. Unstained, undemineralized TEM micrographs of experimental specimens after immersion in ammoniacal AgNO3 solution. (a) A group I specimen showing extensive silver deposition within the adhesive as well as the hybrid layer. (b) A group II specimen with silver deposition within some parts of the hybrid layer. Silver deposits were absent from the adhesive. (C: lining composite; A: unstained adhesive in which the electron-lucent layer could not be discerned; H: unstained hybrid layer; U: undemineralized intertubular dentine; T: dentinal tubule). Fig. 3. Stained, demineralized TEM micrographs of experimental and control specimens in group II (two adhesive layers). (a) Experimental specimen in which a mm thick adhesive layer was observed on the surface of the hybrid layer. (b) A higher magnification view from another specimen showing an intermixed zone that was formed by the dispersion of some microfillers from the lining composite within the electron-lucent layer of the adhesive. (c) Control specimen with the electron-lucent layer removed with ethanol. A 12 mm thick polymerized adhesive layer was still present between the surface of the hybrid layer (pointer) and the lining composite. (C: lining composite; IL: inhibited electron-lucent layer; A: adhesive layer; H: hybrid layer; D: laboratory demineralized intertubular dentine).

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