Effect of Dentin Conditioning Time on Nanoleakage
Objectives: To study the nanoleakage pattern in the dentin hybrid layer by using different dentin adhesives. The null hypotheses tested in this study were: 1) dentin conditioning time does not affect nanoleakage within the hybrid layer; 2) the type of dentin adhesive used does not affect nanoleakage. Methods: Standardized Class V cavities were prepared in 30 intact human molars on the buccal and lingual surfaces. The specimens were randomly assigned to 2 total-etch dentin adhesives (OptiBond SOLO Plus [OPS, Kerr] and One-Step [ONS, BISCO Inc]) and 2 self-etch dentin adhesives (Clearfil SE Bond [CSE, Kuraray] and Adper Prompt L-Pop [APL, 3M ESPE]). The specimens were etched or conditioned for 15 seconds, 30 seconds or 60 seconds. Upon restoration of the Class V cavities with the proprietary resin composite, the specimens were isolated with nail polish except for a 2.0-mm rim around the restoration, and they were immersed in 50 wt% ammoniacal silver nitrate solution (pH=9.5) for 24 hours followed by 8 hours of immersion in photo-developing solution to reduce the silver ions to metallic silver. The specimens were fixed, dehydrated and processed for FESEM and TEM. Silver penetration was measured along the cervical wall, and data were analyzed with Kruskal-Wallis non-parametric tests at a significance level of 95%. Results: There were no statistically significant differences among the experimental groups for the factor “conditioning time” (p>0.926). There were significant differences for the variable “dentin adhesive” (p<0.0001). The least amount of nanoleakage within the hybrid layer occurred with CSE, while ONS resulted in the greatest penetration of silver ions. The adhesives OPS and APL ranked in the intermediary subset. Under TEM, all adhesives resulted in some degree of nanoleakage within the hybrid layer. Both spotted/reticular and water-tree nanoleakage patterns were observed. Significance: Longer conditioning times did not increase nanoleakage within the hybrid layer. Nanoleakage varied with the type of adhesive used.SUMMARY
INTRODUCTION
The ultimate goal of a bonded restoration is to attain an intimate adaptation of the restorative material with the dental structure.1 This is difficult to achieve, as the bonding process is different for both enamel and dentin, because dentin is more humid, more dynamic and more organic than enamel.2
The treatment of dentin with acids, followed by the permeation of hydrophilic monomers into small spaces created within the dentin collagen network, results in resin-enveloped collagen fibrils and the formation of a resin-dentin interdiffusion zone, which was first described in 1982 as the hybrid layer.3 This improved sealing may result in decreased post-operative sensitivity, better marginal fit, and it may even act as an elastic buffer that compensates for polymerization shrinkage stress during contraction of the restorative composite.4–11
A continuous, fairly rapid turnover of adhesive materials has occurred within the last 2 decades.12–13 Multi-step adhesive systems include complicated, time-consuming and technique-sensitive application procedures. The newest simplified adhesive systems are preferred by clinicians, because they are easier to apply. Research has shown that dentin must be treated with an acidic agent to form a hybridized structure.14–19 Despite of different classifications of adhesive systems, current adhesion strategies depend on how the adhesive system interacts with the modified dentin surface—total-etch or self-etch.20
Total-etch adhesives involve a separate etching and rinsing step. Multi-bottle total-etch adhesives have 3 different steps: acid-etching, followed by priming and the application a fluid resin. Even the most popular “1-bottle” systems, which combine the primer and adhesive resin into 1 solution, usually need more than 1 application to achieve an acceptable micromechanical interlocking of monomers into the micro-retentive collagen network left by etching.21–23
Self-etch adhesives consist of non-rinsing acidic monomers that simultaneously condition and prime dentin and enamel. Self-etch adhesives may be classified according to the number of application steps or according to their pH and, consequently, their ability to demineralize dentin and enamel—mild, moderate or aggressive self-etch adhesives.13,24 The new “all-in-one” self-etch adhesives combine etchant, primer and bonding resin into a single solution. Self-etch adhesives have the potential to form a hybrid layer and seal dentin.20 With these systems, the collagen fibrils are not completely deprived of hydroxyapatite; this is in contrast with total-etch adhesives.25–26 Mild self-etching (pH≃2) adhesives feature a submicron hybrid layer with less pronounced resin tag formation, while strong self-etching adhesives (pH≃1) result in an interfacial ultra-morphology resembling that produced typically by total-etch adhesives with the formation of abundant resin tags.
Several silver dyes have been used to test the sealing ability of dentin adhesives—silver nitrate,27–29 silver methenamine30 and, more recently, ammoniacal silver nitrate.31 It has been shown that the hybrid layer is somewhat porous and is accessible to a silver dye, even in the absence of marginal gap.32 This type of leakage is a result of penetration paths through the network of interfibrillar spaces with a size in the range of a few nanometers.32–34 As this phenomenon may be independent of microleakage, the term “nanoleakage” was introduced.32 Recently, other studies have shown that the difference in molecular weight of the monomer may play an important role in reducing or increasing nanoleakage.26,34 The presence of filler in the dentin adhesive might result in more nanoleakage if the filler particles occlude the diffusion channels among the collagen fibrils, modifying the gradient of resin penetration.34 Additionally, the dentin adhesive solvent may have an effect on this differential resin penetration, because the water chasing ability of the organic solvent may open more space for the precipitation of monomers into the nano-channels of the demineralized dentin area.35
This project studied the degree of nanoleakage in the dentin hybrid layer, using different dentin adhesives as a function of conditioning time. The null hypothesis is two-fold: 1) dentin conditioning time does not affect nanoleakage; 2) there is no difference in nanoleakage among the different adhesives.
METHODS AND MATERIALS
Sixty extracted, caries-free and unrestored human molars stored in an aqueous solution of 0.5% chloramine at 4°C for 1 month after extraction were used. Standardized Class V cavities were prepared on the buccal and lingual surfaces of the teeth using a high-speed handpiece with a #245 carbide bur (Midwest/Dentsply Professional Division West, Milford, DE, USA) under water spray coolant. The dimensions of the preparations were 3.0 mm mesiodistally, 3.0 mm occluso-cervically and 2.0 mm in depth. The cervical cavosurface margin was located below the cementum-enamel junction. Each bur was used for 5 preparations, then discarded. All preparations were checked under 20× magnification for possible cracks or fissures, which could compromise the result.
The specimens were randomly and equally assigned to 4 different dentin adhesive systems: 2 total-etch adhesives and 2 self-etch adhesives, as listed in Table 1. For each adhesive, the dentin was conditioned for 15, 30 or 60 seconds, which resulted in 3 subgroups for each adhesive (4×3, n=5).
For the total-etch adhesives, the dentin was etched with the respective manufacturer's phosphoric acid gel (OptiBond Solo Plus, OPS–37.5% phosphoric acid gel, Gel Etchant, Kerr, Orange, CA, USA and One-Step, ONS–32% phosphoric acid, Uni-Etch, BISCO Inc, Schaumburg, IL, USA). After rinsing the gel for 10 seconds, the excess moisture was removed by “blot-drying” the dentin with a damp cotton pellet to obtain a visibly hydrated, shining surface without excessive moisture.7 The adhesive was then applied using a disposable brush, as per the manufacturer's instructions, and light-cured for 20 seconds (XL2500, 3M ESPE, St Paul, MN, USA). The preparations were restored with each manufacturer's recommended resin composite (Table 1), which was inserted in 3 increments from the cervical margin to the occlusal cavosurface margin. Each increment was light-cured for 40 seconds with a curing distance of 0.5 mm and a light intensity of 540mW/cm2 that was constantly monitored.
For the self-etch adhesive Clearfil SE Bond (CSE, Kuraray), the acidic primer was applied to the entire preparation for 15, 30 or 60 seconds using a disposable brush. The solution was left in place for 20 seconds, followed by a short, oil-free air blast to remove the excess solvent. The bonding agent was applied over the conditioned surface with a disposable brush and light-cured for 20 seconds. For the self-etch adhesive Adper Prompt L-Pop (APL, 3M ESPE), the adhesive was applied by scrubbing with a disposable brush on the preparation for 15, 30 or 60 seconds. Then, a gentle oil-free air stream was applied to the entire preparation and light-cured for 20 seconds. The preparation was restored as previously described for the total-etch adhesives.
All specimens were stored in distilled water at 37°C for 24 hours and finished with discs of decreasing abrasiveness (Sof-Lex Pop-On, 3M ESPE). The specimens were coated with 2 layers of nail polish, except for a 2.0-mm rim around the restoration, to allow for contact of the tracing agent with the margins of the restoration. The specimens were then immersed in an aqueous solution of 50 wt% ammoniacal silver nitrate (pH=9.5) for 24 hours, followed by 8 hours in a photo-developing solution in order to permit reduction of the diammine silver ions to metallic silver grains.31 This solution was used to prevent the possibility of artifactual dissolution of remnant calcium phosphate salts along resin-tooth interfaces with the use of a mildly acidic silver nitrate solution.31
The specimens were retrieved from the photo-developing solution and washed in running water for 1 minute. The nail polish was removed and the specimens immediately immersed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 for 12 hours at 4°C.36 After fixation, the specimens were rinsed with 20mL of 0.2 M sodium cacodylate buffer at pH 7.4 for 1 hour, with 3 changes, followed by distilled water for 1 minute. The specimens were dehydrated in ascending grades of ethanol as follows: 25% for 20 minutes, 50% for 20 minutes, 75% for 20 minutes, 95% for 30 minutes and 100% for 60 minutes.36
Specimen Preparation for Field-emission SEM
After the final ethanol step, the specimens were cross-sectioned through the center of the restoration with a water-cooled, low speed diamond disk (Isomet 1000, Buehler Ltd, Lake Bluff, IL, USA) to a 1.0 mm width. Half of the specimen was metallurgically polished to high gloss with a polishing apparatus (Struers, Ballerup, Denmark) with waterproof silicon carbide papers of decreasing abrasiveness (600-, 800- and 1200-grit) followed by soft tissue disks with increasingly fine diamond suspensions of 2 μm and 1 μm (Buehler Ltd). The specimens were ultrasonicated in 100% ethanol for 10 minutes, thoroughly dried and demineralized in 0.5% silica-free phosphoric acid for 1 minute. The specimens were then dried by immersion in hexamethyldisilazane ([CH3]3SiNHSi[CH3]3, HMDS, Electron Microscope Sciences, Fort Washington, PA, USA) for 10 minutes, placed on filter paper inside a covered glass vial and air dried at room temperature.36 The specimens were sputter-coated with 10 Å platinum and observed under a Field Emission Scanning Electron Microscope with a backscattered detector (S-4700, Hitachi High Technologies America, Inc, Pleasanton, CA, USA) at an accelerating voltage of 8.0 kV and a working distance of 11.0 to 13.0 mm. The length of silver penetration along the preparation wall was analyzed using Quartz PCI 4.00 software (Quartz Imaging Corporation, Vancouver, BC, Canada). Nanoleakage was calculated as a percentage of dye penetration into the total preparation wall length (N= [p/L] 100×, where N= nanoleakage through the hybrid layer of the cervical wall, p= length of silver penetration within the hybrid layer [μm] and L= total length of the cervical wall).32 The data were submitted to a Kruskal-Wallis non-parametric statistical analysis at p<0.05 (SPSS 11.0, SPSS Inc, Chicago, IL, USA).
Specimen Preparation for TEM
The remaining half of the specimen was sectioned in a small “stick” 2.5-mm long and a cross-section of 1.0 mm2 under a water-cooled, slow speed diamond saw (Isomet 1000, Buehler Ltd). The sticks were fixed, dehydrated and embedded in 50% propylene oxide/50% MedCast epoxy resin (Ted Pella Inc) in a Pelco Infiltron rotator (Ted Pella Inc) at 6 rpm. After 6 hours, the specimens were immersed in 100% epoxy resin at room temperature and placed under vacuum for 12 hours to allow for resin penetration into the specimen. They were then embedded in molds with fresh epoxy resin and polymerized for 12 hours at 65°C in an oven. The resulting resin-embedded specimen blocks were left at room temperature for 24 hours, trimmed to expose the interface and sectioned in an 8800 Ultratome III (LKB, Bromma, Sweden) ultra-microtome equipped with a Material Sciences Type II diamond knife (Micro Star Technologies Inc, Huntsville, TX, USA). Then, ultra-thin unstained 90±10-nm thick sections were made, and localization of the metallic silver grains within the hybrid layer was observed under the TEM (Jeol 1200, JEOL USA Inc, Peabody, MA, USA) using an accelerating voltage of 80 kV.
RESULTS
The percentage of silver penetration length for each adhesive in function of conditioning time is shown in Figure 1. No statistically significant differences were found among the experimental groups for the factor “conditioning time” (Table 2, p>0.926). There were significant differences for the variable “dentin adhesive” (Table 3p<0.0001). The least amount of nanoleakage within the hybrid layer was obtained with CSE, while ONS resulted in the greatest dye penetration. The remaining adhesives (OPS and APL) ranked in the intermediary subset and resulted in statistically similar nanoleakage scores (p>0.617).
Citation: Operative Dentistry 31, 4; 10.2341/05-86
TEM analysis showed that the hybrid layer was incompletely infiltrated, regardless of the adhesive used. Both spotted/reticular and water-tree nanoleakage patterns were observed in most of the specimens. The reticular nanoleakage pattern can be defined as small, discontinuous silver clusters within the hybrid layer or dentin tubules.31 The reticular pattern occurred most probably where water was not totally removed in the adhesive layer or within the collagen fibrils. Silver deposits of the reticular pattern were observed for all groups, especially for OPS (Figures 2C, 3B and 3D). Other adhesive systems also presented some reticular pattern, but no correlation was found in function of etching times (ONS [Figure 6C], CSE [Figure 9B] and APL [Figures 10D and 11C]). Furthermore, incomplete resin infiltration within the exposed collagen fibrils was found for most of the specimens. Spotted silver deposits occurred within the interfibrillar spaces of demineralized dentin. OPS revealed a discrete spotted pattern for 15 seconds of etching (Figure 2C); in contrast, an extensive spotted pattern was displayed for longer etching times (Figure 3D). Silver-infiltrated interfibrillar spaces were found for all specimens restored with ONS (Figures 4C, 5C and 6C). Water-tree silver deposits were observed at the adhesive-hybrid layer interface (Figures 4C and 5C).
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
The least extensive silver penetration within the hybrid layer was found for CSE. The spotted pattern was noticeably found for 15 seconds of self-etch primer application (Figure 7C). However, silver clusters were present within the adhesive and at the adhesive-hybrid layer interface (Figures 7C and 8C).
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
TEM evaluation of non-stained and non-decalcified specimens also revealed heavy silver uptake into the demineralized portion of the APL hybrid layer and the adhesive-hybrid layer interface (Figures 10B and 10D). Both the spotted (Figure 11C) and the continuous reticular pattern (Figures 10B, 10D and 11C) were evident for this all-in-one adhesive.
Citation: Operative Dentistry 31, 4; 10.2341/05-86
Citation: Operative Dentistry 31, 4; 10.2341/05-86
DISCUSSION
There are 4 possible theoretical mechanisms of bonding to tooth structures: penetration of resin and subsequent formation of resin tags, precipitation of substances on the dentin surface to which monomers can bond mechanically or chemically, chemical bonding to the inorganic component (hydroxyapatite) and chemical bonding to the organic components of dentin.11,37 Dentin structural modification is required for any bonding procedure.38 The application of acids or acidic monomers to dentin results in total or incomplete removal of the smear layer.4,15–16,39
The demineralization of dentin during acid-etching is a complex process restricted to the outermost dentin surface layer.39,40–42 After this procedure, 3 layers can be distinguished: first, a superficial smeared collagen layer; second, an intermediate, densely packed fibrilar layer and third, a deeper area with some scattered mineral crystals and a few randomly exposed collagen fibrils.36 The size of the porosities in dentin has been estimated to be about 10–50 nm and are filled with water or dentinal fluids.43 The 3 layers of decalcification were found for the groups G1 (Gel Etchant, Kerr, 37.5% phosphoric acid) and G2 (Uni-Etch, BISCO 32% phosphoric acid), even with differences in the concentration of phosphoric acid used.
Despite the fact that no significant difference was found among the experimental groups, increasing the etching time increased the depth of dentin decalcification.40 The main decalcification was located at the peritubular area,44 with less acid penetration at the inter-tubular aspect.40–41 The deepest demineralization occurred with the phosphoric acid conditioners groups (G1 and G2). This was expected, due to their pH (Gel Etchant, Kerr, pH 0.1; Uni-Etch, BISCO, pH 0.32),45 in contrast to group G3 acidic primer (Clearfil SE Bond, Kuraray, pH 1.9)24 and group G4, the “all-in-one” self-etch adhesive (Adper Prompt L-Pop, 3M ESPE, pH 1.0).46
Dentin decalcification is limited by the buffer properties of hydroxyapatite.47–48 As the acid components used in dentin adhesives (phosphoric acid or acidic primers) are hypertonic,39 dentin moisture is absorbed, which dilutes and reduces penetration of the acid.45 For CSE, increasing conditioning time did not affect the depth of decalcification (from 1.2 μm to 1.4 μm), which is in contrast with ADP, which displayed decalcification (ranging from 1.9 μm to 3.5 μm) similar to the total-etch adhesives tested (1.9 μm to 3.8 μm). As the depth of decalcification increases with the decrease in pH,40 it would be more difficult for the hydrophilic monomers to penetrate into deeper areas of the decalcified dentin.49–50 However, a correlation between the different pH and depth of decalcification was not observed regarding the percentage of nanoleakage within the hybrid layer, as stated in Figure 1. The results obtained in this study are not in agreement with Dörfer and others,51 which observed that shorter etching times might result in deeper dye penetration.
The more hydrophilic the monomers, the better they penetrate within the spaces of the minuscule collagen channels of etched dentin. The porosities found in etched dentin resemble small channels, with estimated dimensions of 10–50 nm that are filled with water or liquids proceeding from the pulp.43 This humidity may compete with the resin monomer diffusion, reducing wetness of the collagen fibrils and the mineralized portion. The resin monomers are then diluted and precipitate.43–54 As a result, gaps with nanometric dimensions appear within the hybrid layer, jeopardizing the quality of the dentin sealing.51,54–55 Increasing etching time does not imply an increase in the number of channels for adhesion, nor does it imply an increase in the tubular fluid proceeding outward from the pulp.41 Therefore, the ability to seal may be more related to the application technique and the hydrophilicity of the adhesive than the etching time.56–57
For OPS, with etching times of 15 and 60 seconds, spotted and reticular silver deposits were found and located predominantly at the medium and basal third of the hybrid layer (Figures 2A, 2B, 2C, 3C and 3D). For an etching time of 30 seconds, silver deposits were observed at the top and base of the hybrid layer (Figures 3A and 3B). A complete infiltration of the resin monomers within decalcified dentin did not occur, resulting in porosities where the metallic silver grains lodged, resulting in a spotted pattern of nanoleakage (Figures 2C and 3D). If one considers the microscopic size of the penetration path created by dentin etching, along with the molecular weight of the monomers, the adhesive system would theoretically be able to fill the decalcified dentin. The paths created by dentin etching are 20 to 30 nm around the collagen fibrils and 40 to 50 nm at the superficial dentin.43 Nevertheless, not all filler particles in OPS have a size compatible with the dimensions of the diffusion channels. For instance, the fumed silica particles are about 40-nm wide; whereas, the glass fillers have a diameter of 600 nm.58 The fillers with high molecular weight may obliterate the diffusion paths among the collagen fibrils, making it difficult for low molecular weight monomers to penetrate completely, which modifies the gradient of resin penetration.26,34,49 Water may be trapped in these areas and may be not fully removed from the penetration paths, explaining the presence of the reticular pattern (Figures 2C, 3B and 4D).31 Thus, the dentin adhesive solvent may have a direct effect on this differential resin penetration, because the water-chasing ability of the solvent may leave more space for precipitation of the monomers into the collagen inter-fibril channels. The presence of the remaining water may also lead to an incomplete polymerization of the adhesive,59 particularly with ethanol-based adhesives, due to the ability of ethanol to form hydrogen bonds with water.31
The spotted and reticular pattern of nanoleakage within the hybrid layer was also observed for APL (Figures 10A through 11C). In all-in-one, self-etch adhesives, the hydrophobic and hydrophilic resin components may react before polymerization starts.20 However, the water, which was present in the adhesive composition, as well as within dentin, may not be completely removed, resulting in the polymers fractioning during their reaction.60 Consequently, the silver uptake with Adper Prompt L-Pop (3M ESPE) might represent areas of minimal conversion inside the polymeric matrix due to incomplete solvent removal.26
ONS displayed the higher degree of nanoleakage among the adhesives tested in this project (Figures 4A through 6C). The predominant pattern observed was the spotted type, especially at the basal area of the hybrid layer, as well as “water-tree” silver deposits (Figures 4C and 5C). The reticular-type pattern was observed only for ONS, with 60 seconds of etching (Figure 6C). Acetone-based adhesives possess an intrinsic ability to remove water due to acetone being a highly volatile solvent.31 However, acetone-based adhesives are extremely sensitive to dentin moisture.7 A separation of the adhesive resin phase components might occur during acetone evaporation, resulting in microporosities inside the resin matrix, where silver grains can lodge.60 An induced, outward movement of water during etching may fill the basal region of the etching dentin, competing with and diluting the resin monomers.
Less porosity in the hybrid layer was obtained with CSE (Figures 7A through 9B). Among the different adhesives studied, Clearfil SE Bond (Kuraray) has a relatively mild pH of 1.9, which produces intense intertubular microporosity with the residual smear layer in the tubules and on the dentin surface. The relatively high pH resulted in less dentin decalcification regardless of conditioning time, which contrasted with other, more aggressive adhesives. Few specimens featured nanoleakage within the hybrid layer; however, when it did occur, spotted-pattern nanoleakage was located at the junction between the hybrid layer and the adhesive layer (Figures 7C and 8C) and at the transition between non-demineralized dentin and the hybrid layer (Figures 7C and 9B). For the self-etch adhesive CSE, 2 steps are necessary to achieve adhesion to dentin: application of an acidic primer and placement of a bonding agent. After application of the acidic primer and before bonding, it is necessary to use an air stream to remove the excess solvent from dentin. However, a small amount of water may be trapped, because HEMA reduces water evaporation, resulting in the development of different forms of hydrogels.59 Thus, the hybrid layer may display areas where an incomplete polymerization reaction has occurred, as well as the formation of porous poly(HEMA) hydrogels.60 As a consequence, the application technique may have influenced the quality of the hybrid layer.57 Therefore, the nanoleakage associated with CSE may not be necessarily a result of the failed interdiffusion of hydrophilic monomers within the collagen network, but, instead, from areas where residual water competed with the adhesive polymerization reaction.
The presence of “water-tree” deposits of silver was observed at the hybrid layer and the adhesive layer junction (Figures 4C, 5C, 10B and 10D). “Water-tree” represents an area where a certain volume of water is retained, causing incomplete polymerization of the adhesive.61 It has been speculated that this pattern of silver uptake may produce degradation of the bonded surface due to hydrolysis61 over time.62 However, the full nature of this degradation and how the bonding integrity is affected are not yet completely understood. Other studies have shown that thermocycling, long-term storage and load cycling do not influence nanoleakage.63–65 Nevertheless, at the collagen fibril level, water degradation may produce disarrangement of the collagen network and widening of the interfibrillar space,66 which can interfere with the quality of bonding.
The first null hypothesis was accepted—different etching times did not necessarily result in an increase in silver uptake within the hybrid layer. The second null hypothesis was rejected—different adhesives resulted in different levels of nanoleakage.
CONCLUSIONS
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Increasing the conditioning time did not increase nanoleakage for each of the adhesives tested.
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Clearfil SE Bond (Kuraray), a self-etch adhesive system, resulted in the least penetration of silver nitrate within the hybrid layer.
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TEM images resulted in more accurate observations of nanoleakage within the hybrid layer.
Percentage of silver penetration along the cervical wall according to different conditioning times.
Secondary electron Fe-SEM image of specimen restored with OPS/15 seconds. Magnification = 5000×. Figure 2B: Same field of Figure 2A, revealing the backscattered Fe-SEM image of silver deposits (white arrow) within the hybrid layer (HL) and within the resin tags (black arrow) with OPS/15 seconds. (D-dentin). Magnification = 5000×. Figure 2C: TEM image of the same specimen of Figure 2A (OPS/15 seconds) displaying reticular pattern (white arrow) and spotted pattern (circle). (T-tubule). Magnification = 10000×.
Backscattered Fe-SEM image of specimen conditioned for 30 seconds with OPS exhibiting nanoleakage (white arrow) within the hybrid layer (HL).(A-Adhesive, D-Dentin) Magnification = 3000×. Figure 3B: TEM view of the same specimen (OPS/30 seconds) with reticular pattern of nanoleakage (black arrow). Magnification = 15000×. Figure 3C: Backscattered Fe-SEM image of extensive silver deposits (white arrow) at the basal part of the hybrid layer (HL) with OPS/60 seconds (HL,A,D). Magnification = 5000×. Figure 3D: TEM view of the same specimen (OPS/60 seconds) showing extensive silver penetration at the basal part of the hybrid layer (HL) with reticular and spotted pattern of nanoleakage (white arrow). Magnification = 10000×.
Secondary electron Fe-SEM image of specimen restored with ONS/15 seconds. (HL–hybrid layer, D–dentin, R–resin). Magnification = 5000×. Figure 4B: Backscattered Fe-SEM image of silver deposits (white arrow) at the basal third of the hybrid layer (HL) with ONS/15 seconds. (R–resin, D-dentin). Magnification = 5000×. Figure 4C: TEM view of the same specimen (ONS/15 seconds) with spotted pattern of nanoleakage (circle) within the hybrid layer (HL) and at the hybrid layer (HL)/adhesive layer (A) interface (white arrow, D-dentin). Magnification = 7500×.
Secondary electron Fe-SEM image of specimen restored with ONS/30 seconds. (HL–hybrid layer, D–dentin, R–resin). Magnification = 5000×. Figure 5B: Backscattered Fe-SEM image of extensive silver deposits along the hybrid layer (HL) length (white arrow) with ONS/30 seconds. Magnification = 5000×. Figure 5C: TEM view of the same specimen (ONS/30 seconds) revealing spotted pattern (circle) within the hybrid layer (HL) and water-trees at the hybrid layer (HL)/adhesive layer (A) interface (white arrow). Magnification = 10000×.
Secondary electron Fe-SEM image of specimen restored with ONS/60 seconds (HL–hybrid layer, D–dentin, *–resin tags). Magnification = 5000×. Figure 6B: Same site backscattered Fe-SEM image of extensive silver deposits at the basal part of the hybrid layer (HL) (white arrow), especially among the resin tags (*) with ONS/60 seconds(D-dentin). Magnification = 5000×. Figure 6C: TEM image showing spotted pattern (white arrow) and reticular pattern along the hybrid layer (HL) with ONS/60 seconds (D-dentin). Magnification = 10000×.
Secondary electron Fe-SEM image of specimen restored with CSE/15 seconds. (HL–hybrid layer, D–dentin, G–gap). Magnification = 5000×. Figure 7B: Backscattered Fe-SEM image of silver deposits at the top of the hybrid layer (HL) (white arrows) and within the resin tags with CSE/15 seconds (D-dentin). Magnification = 5000×. Figure 7C: TEM image showing silver clusters (white arrow) at the hybrid layer (HL)/adhesive (A) interface with CSE/15 seconds. Spotted pattern of nanoleakage within the hybrid layer (HL) is also noted (R-resin, D-dentin). Magnification = 15000×.
Secondary electron Fe-SEM image of specimen restored with CSE/30 seconds (HL–hybrid layer, D–dentin, R–resin). Magnification = 5000×. Figure 8B: Backscattered Fe-SEM image showing no silver deposits within the hybrid layer (HL) with CSE/30 seconds (R-resin, D-dentin). Magnification = 5000×. Figure 8C: TEM image revealing silver clusters at the hybrid layer (HL)/adhesive (A) interface in the same specimen (CSE/30 seconds) (G–gap, R-resin, D-dentin). Magnification = 5000×.
Backscattered Fe-SEM image showing no silver deposits within the hybrid layer (HL) with CSE/60 seconds (R-resin, D-dentin). Magnification = 5000×. Figure 9B: TEM showing silver deposits (white arrow) along the hybrid layer (HL) with CSE/60 seconds (G-gap, R-resin, D-dentin). Magnification = 15000×.
Fe-SEM (backscattered) image of silver deposits in the whole length of the hybrid layer (HL) (white arrow) with APL/15 seconds. Magnification = 5000×. Figure 10B: TEM view of the same specimen (APL/15 seconds) revealing water-trees (white arrow) at the hybrid layer (HL)/adhesive (A) interface (G-gap, D-dentin). Magnification = 10000×. Figure 10C: Fe-SEM (backscattered) image of silver deposits observed within the hybrid layer (HL) (white arrow), and within the resin tags with APL/30 seconds )R-resin, D-dentin). Magnification = 5000×. Figure 10D: TEM image showing reticular pattern within the hybrid layer (HL) and water-trees (white arrow) at hybrid layer (HL)/adhesive (A) interface for the same specimen (APL/30 seconds). Magnification = 10000×.
Secondary electron Fe-SEM of specimen restored with APL/60 seconds (HL–hybrid layer, D–dentin, A–adhesive layer). Magnification = 5000×. Figure 11B: Same site Fe-SEM (backscattered) image of silver deposits within the hybrid layer (HL) (white arrow) with APL/60 seconds (A-adhesive, D-dentin). Magnification = 5000×. Figure 11C: TEM image for the same specimen (APL/60 seconds) displaying reticular and spotted silver deposits along the hybrid layer (HL), and water-trees at the hybrid layer (HL)/adhesive (A) interface. (T–tubule, D-dentin). Magnification = 10000×.
Contributor Notes