Editorial Type:
Article Category: Research Article
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Online Publication Date: 01 Jan 2008

Adhesion to Er:YAG Laser-prepared Dentin After Long-term Water Storage and Thermocycling

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Page Range: 51 – 58
DOI: 10.2341/07-30
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SUMMARY

This in vitro study evaluated the microtensile bond strength of a resin composite to Er:YAG-prepared dentin after long-term storage and thermocycling. Eighty bovine incisors were selected and their roots removed. The crowns were ground to expose superficial dentin. The samples were randomly divided according to cavity preparation method (I–Er:YAG laser and II–carbide bur). Subsequently, an etch & rinse adhesive system was applied and the samples were restored with a resin composite. The samples were subdivided according to time of water storage (WS)/number of thermocycles (TC) performed: A) 24 hours WS/no TC; B) 7 days WS/500 TC; C) 1 month WS/2,000 TC; D) 6 months WS/12,000 TC. The teeth were sectioned in sticks with a cross-sectional area of 1.0-mm2, which were loaded in tension in a universal testing machine. The data were subjected to two-way ANOVA, Scheffé and Fisher's tests at a 5% level. In general, the bur-prepared group displayed higher microtensile bond strength values than the laser-treated group. Based on one-month water storage and 2,000 thermocycles, the performance of the tested adhesive system to Er:YAG-laser irradiated dentin was negatively affected (Group IC), while adhesion of the bur-prepared group decreased only within six months of water storage combined with 12,000 thermocycles (Group IID). It may be concluded that adhesion to the Er:YAG laser cavity preparation was more affected by the methods used for simulating degradation of the adhesive interface.

INTRODUCTION

The Er:YAG laser has been specially advocated to prepare cavities1–9 for the preservation of healthy dental structure10–11 due to its highly efficient absorption in both water and hydroxyapatite without causing thermal damage to surrounding tissues.12–13 Its efficacy on caries removal,1014–16 surface pre-treatment1317–19 and in increasing enamel acid resistance20–22 has also been emphasized. Compared to rotary cutting instruments, cavity preparation using Er:YAG laser takes more time,23–24 but its advantages include low noise and vibration, eliminating, in most cases, the need for local anesthesia.23

With regard to the adhesion process, Er:YAG laser irradiation on tooth structure has been described as favorable, because it leads to a rough microretentive surface11725 with a good definition of open dentinal tubules, preservation of tubular structure626 and without smear layer formation.61326 However, bond strength tests of contemporary adhesive systems to the Er:YAG-irradiated tooth have been primarily performed over short storage periods,35–61727 which make durability of such bonds difficult to predict.

In an attempt to mimic the natural aging process of a dental restoration, thermocycling protocols28–32 and the water storage of bonded specimens33–34 have been suggested as efficient methods to provide in vitro simulation of in vivo conditions. Thermocycling has been the most used method to stress the adhesive interface,31 while water storage has been shown to reduce bond strength, even after a short period of storage, indicating that bonds degrade over time.35–36

Considering that the Er:YAG laser is a promising alternative method for cavity preparation and that, to date, no available data have described the adhesion of resin composite to the lased-surface after a long-term degradation process, this study evaluated the microtensile bond strength of a resin composite to Er:YAG-prepared dentin after long-term storage and thermocycling.

METHODS AND MATERIALS

Specimen Preparation

Eighty bovine incisors, freshly extracted and stored in a 0.5% chloramine solution at 4°C for 48 hours, were selected and cleaned with a scaler and water/pumice slurry in dental prophylatic cups. The teeth were sectioned using a water-cooled diamond saw in a sectioning machine (Minitrom, Struers A/S, Copenhagen, Denmark) to obtain one fragment per tooth from the median region of the crown with surface dimensions of at least 8×8 mm, totaling 80 samples.

The fragments were ground in a water-cooled polishing machine (Politriz DP-9U2, Struers A/S, Copenhagen, DK-2610, Denmark) with 400- and 600-grit silicon carbide (SiC) papers (Buehler Ltd, Lake Bluff, IL, USA) to expose the superficial dentin surface. The surface area for adhesion was delimited32 using a piece of adhesive tape with a rectangular or square area of 36 mm2.

The specimens were randomly assigned to two groups of equal size, according to the cavity preparation method: Er:YAG laser irradiation (Group I) and Carbide bur preparation (Group II). The Er:YAG laser device was the Fotona (Fidelis, Fotona Medical Lasers, Ljubljana, Slovenia), emitting a wavelength of 2.94 μm. The spot size at the interacting surface was about 0.5 mm2, which corresponds to a 0.8-mm spot diameter. In both cavity preparation methods, just the tooth surface was prepared, thus, a cavity was not formed.

The parameter settings used included a 260-mJ energy, a 4-Hz frequency and a 59 J/cm2 energy density. The laser beam was delivered on a non-contact and focused mode with a fine water mist of 1.5 mL/minute.37 The 12-mm irradiation distance19 was standardized by a custom designed apparatus consisting of two parts: a holder to fix the laser handpiece in such a way that the laser beam was delivered perpendicular to the specimen surface at a constant working distance from the target site and a semi-adjustable base, upon which the plexiglass plate and fragment were attached. The irradiation time was 30 seconds.

The remaining samples (bur group) were prepared for 10 seconds with a #245 carbide bur (KG Soresen, Barueri, SP, Brazil) using a high-speed handpiece (Dabi-Atlante, Ribeirão Preto, SP, Brazil) with water/air spray. The high-speed handpiece was fixed in a milling machine (MPC, ElQuip, São Carlos, SP, Brazil) in which the movement of the axle was monitored by digital comparing clocks, supplying a precision of 0.01 mm in the cavities' dimension. New burs were used after every five preparations.

After cavity preparation, bonding procedures were performed according to the manufacturer's instructions: Surfaces were treated with a 37% phosphoric acid gel (Etching gel, 3M Dental Products, St Paul, MN, USA) for 15 seconds, rinsed for 15 seconds and gently dried with absorbing paper to keep a moist surface. Then, an etch and rinse adhesive system (Single Bond 2 Adper, 3M Dental Products) was applied for 15 seconds; the remaining solvent was evaporated using a brief, gentle dry air jet and light-cured for 10 seconds (XL-3000, 3M Dental Products). Then, a hybrid resin composite block (Filtek Z-250, 3M/ESPE Dental Products), 4-mm high, was built onto the bonding surface, following the incremental technique. Each layer of composite (1 mm thick) was cured for 20 seconds using a visible light-curing unit with an output of 450-mW/cm2.

The restored specimens were kept in distilled water at 37°C and were divided into four subgroups (n=10) according to the time of water storage (WS) and the number of thermocycles (TC) used to simulate adhesive interface degradation (Table 1).

Table 1 Experimental Design and Distribution of Groups
Table 1

Thermocycling was conducted in a thermocycling machine (MPC, ElQuip, São Carlos, SP, Brazil). Each cycle consisted of water baths of 5°C and 55°C, with a dwell time of 30 seconds and a transfer time of seven seconds. After each cycle, the specimens were stored in distilled water at 37°C. Five hundred thermocycles per week were administered, totaling 2,000 thermocycles at the end of one-month and 12,000 thermocycles after the six-month period.

μTBS Testing

After the pre-determined storage/thermocycling time for each subgroup, the teeth were sectioned perpendicular to the bonding surface to create multiple beam-shaped sticks with a cross-sectional surface area of approximately 1.0 mm2 (± 0.2 mm).

The cross-sectional area of each stick was measured with a digital caliper (Mitutoyo, Tokyo, Japan). The sticks were attached to a metallic testing device using a cyanoacrilate adhesive (Super Bonder Gel, Henkel Ltda, SP, Brazil) and subjected to tensile stress in a universal testing machine (MEM-2000 model, EMIC, São José dos Pinhais, PR, Brazil) at a crosshead speed of 0.5 mm/minute and a 50N load cell until fracture. The bond strength values were reported in MPa and derived by dividing the imposed force (in N) at the time of fracture by the bond area (mm2). When the specimens failed before actual testing, the μTBS was determined from the specimens that survived processing. The comparison was done using the mean of each tooth (10 sticks per tooth). The means and standard deviations were calculated and the data were analyzed by two-way ANOVA, Scheffé test and Fisher's test at a 0.05 significance level. The fractured specimens were observed at a magnification of 80× using a stereo-microscope (Leica S6D Stereozoom, Leica Mycrosystems AG, Switzerland) to assess the failure modes, which were classified as adhesive if it occurred at the substrate/adhesive interface, cohesive if it occurred in the material or the substrate and mixed if it involved both the interface and material. Bond failure sites were not statistically analyzed.

A schematic illustration presenting the experimental design of the microtensile bond strength test is shown in Figure 1.

Figure 1. Schematic illustration. (A) the roots were removed from bovine incisors; (B) sectioning of crowns to obtain specimens 8×8×2 mm (C), which were ground to expose superficial dentin; (D) delimitation of the dentin bonding site (6×6 mm2); (E) cavity preparation device (Er:YAG laser + Carbide Bur); (F) resin composite restoration; (G) thermocycling and water storage of the specimens; (H) sectioning samples into 1×1 mm sticks (I); (J) sticks were loaded in tension; (K) types of fracture that were analyzed under SEM.Figure 1. Schematic illustration. (A) the roots were removed from bovine incisors; (B) sectioning of crowns to obtain specimens 8×8×2 mm (C), which were ground to expose superficial dentin; (D) delimitation of the dentin bonding site (6×6 mm2); (E) cavity preparation device (Er:YAG laser + Carbide Bur); (F) resin composite restoration; (G) thermocycling and water storage of the specimens; (H) sectioning samples into 1×1 mm sticks (I); (J) sticks were loaded in tension; (K) types of fracture that were analyzed under SEM.Figure 1. Schematic illustration. (A) the roots were removed from bovine incisors; (B) sectioning of crowns to obtain specimens 8×8×2 mm (C), which were ground to expose superficial dentin; (D) delimitation of the dentin bonding site (6×6 mm2); (E) cavity preparation device (Er:YAG laser + Carbide Bur); (F) resin composite restoration; (G) thermocycling and water storage of the specimens; (H) sectioning samples into 1×1 mm sticks (I); (J) sticks were loaded in tension; (K) types of fracture that were analyzed under SEM.
Figure 1 Schematic illustration. (A) the roots were removed from bovine incisors; (B) sectioning of crowns to obtain specimens 8×8×2 mm (C), which were ground to expose superficial dentin; (D) delimitation of the dentin bonding site (6×6 mm2); (E) cavity preparation device (Er:YAG laser + Carbide Bur); (F) resin composite restoration; (G) thermocycling and water storage of the specimens; (H) sectioning samples into 1×1 mm sticks (I); (J) sticks were loaded in tension; (K) types of fracture that were analyzed under SEM.

Citation: Operative Dentistry 33, 1; 10.2341/07-30

RESULTS

The mean values and standard deviations of the microtensile bond strength (μTBS) are presented in Table 2 and graphically represented in Figure 2.

Table 2 Microtensile Bond Strength Means (Mpa) and Standard Deviations in Each Experimental Group
Table 2
Figure 2. Box-plot of dentin microtensile bond strengths (μTBS). The box represents the spread of data. The central vertical line represents the median. The “whiskers” extend to the minimum and maximum value.Figure 2. Box-plot of dentin microtensile bond strengths (μTBS). The box represents the spread of data. The central vertical line represents the median. The “whiskers” extend to the minimum and maximum value.Figure 2. Box-plot of dentin microtensile bond strengths (μTBS). The box represents the spread of data. The central vertical line represents the median. The “whiskers” extend to the minimum and maximum value.
Figure 2 Box-plot of dentin microtensile bond strengths (μTBS). The box represents the spread of data. The central vertical line represents the median. The “whiskers” extend to the minimum and maximum value.

Citation: Operative Dentistry 33, 1; 10.2341/07-30

Analysis of the data revealed that there were significant differences when the cavity preparation methods were compared (p<0.05). In general, the bur-prepared group displayed higher μTBS than the laser-treated group.

The interaction between cavity preparation method and water storage/thermocycling procedures demonstrated that there was no statistical difference (p>0.05) among Groups IA, IB, IIA, IIB and IIC (Table 2), which showed the highest μTBS values. The lowest μTBS were found in the Er:YAG laser-prepared samples stored in water for six-months with 12,000 thermocycles (Group ID), and this value was statistically different from the other groups (p<0.05). The laser-prepared group, in which the restorations were stored in water for one-month with 2,000 thermocycles (Group IC) and the bur-treated group, in which the restorations were stored in water for six-months with 12,000 thermocycles (Group IID), presented intermediate μTBS values, with no statistical difference between them.

The analysis of failure after the microtensile test revealed that the adhesive failure mode was predominantly observed (62.1%) in the Er:YAG lased-fractured specimens. Mixed failure was found in 19.3%, cohesive failure within substrate in 5.7% and cohesive failure within material in 12.9%. The bur-treated group showed 22.9% of adhesive failures, 22.9% of mixed failures, 28.5% of cohesive failures within substrate and 25.7% of cohesive failure within the material (resin composite). The fracture pattern of the specimens is presented in Figure 3.

Figure 3. Fracture types.Figure 3. Fracture types.Figure 3. Fracture types.
Figure 3 Fracture types.

Citation: Operative Dentistry 33, 1; 10.2341/07-30

DISCUSSION

Currently, the microtensile bond strength test (μTBS) has been described as one of the most important laboratory tests used to evaluate the performance of restorative systems,31–32 with the advantage of allowing for the evaluation of small areas and different substrates.38

Using this methodology, the current study disclosed that cavity preparation with Er:YAG laser negatively affected μTBS when compared to the bur-prepared group. This result is probably due to the morphology and topography of Er:YAG laser-irradiated dentin.8 It has been demonstrated that microexplosions caused during irradiation leave a scaly surface,1518 represented by a porous layer of melted minerals within microfissures, which may be partially infiltrated by the adhesive system.18 In addition, Er:YAG laser irradiation is selective for organic tissue ablation, thus leaving less collagen to be exposed and hybridized.318 On the basal part of the laser-modified layer, remnant denatured collagen fibrils fused and poorly attached to the underlying dentin were found, which probably restricted acid diffusion418 and, consequently, adhesive system penetration. Even though separate phosphoric acid conditioning could have eliminated this surface modified layer, Er:YAG laser irradiation has been shown to initiate subsurface damage on dentin, which is presented with microcracks just below the hybrid layer31839 and is probably not achievable by acid etching. Thus, a weakened substrate without resin reinforcement underneath may be formed.3 These facts could explain the predominance of the adhesive fracture pattern on the lased group in the current research.

In this study, the degradation of resin-dentin bonds by the storage of specimens in water combined with thermocycling produced negative effects on microtensile bond strength after the one-month period of water storage with 2,000 thermocycles and six months of water storage with 12,000 thermocycles in the Er:YAG-prepared groups (IC and ID) and after six months of water storage with 12,000 thermocycles in the bur prepared Group (IID). Chemical reactions have been responsible for the degradation of resin-dentin bonds over time and, consequently, the decrease in bond strength, including the loss of stability of the adhesive systems33 and the extraction of resin-material from the hybrid layer.40 Also, a fall in bond strength has been ascribed to the hydrolysis of the adhesive and collagen fibrils at the base of the hybrid layer,3341–42 thereby weakening the physical properties of the resin-dentin bond.43 This process is accelerated by heat and repetitive contraction/expansion stresses generated at the tooth-resin interface during thermocycling.44–45

These findings may be better explained by the representative SEM images (Figure 4) where the methods used to simulate the degradation of the adhesive interface (water storage and thermocycling procedures) promoted less morphological alterations on the adhesive interface of the bur-prepared group (Figure 4A, 4C, 4E and 4G), with a more uniform, thick hybrid layer at baseline and over all periods of water storage and thermo-cycling. This may justify the more stable μTBS values obtained for this group. In the Er:YAG-laser prepared group, numerous funnel-shaped tags along the interface can be observed, exhibiting lateral branches. Resin tags may be primarily responsible for adhesion in this group (Figure 4B and 4D). However, morphological changes in the Er:YAG-laser prepared group were more evidenced. In Figure 4F, a gap can be observed over the one-month period of water storage and the 2,000 thermocycles in the Er:YAG-laser prepared samples. Even though the μTBS values for this group were similar to the bur-prepared group with 12,000 thermocycles (IID), gap formation was not found in Group IID (Figure 4G). Therefore, the analysis of representative SEM images may support the contention that Er:YAG treatment promotes morphological changes in the dentin adhesive interface that negatively affect bond durability.

Figure 4. Representative SEM images of the adhesive interface after cavity preparation: carbide bur (A,C,E,G) and Er:YAG (B,D,F,H). Periods of water storage and the number of thermocycles: A and B—24 hours/None; C and D seven days/500; E and F one month/2,000; G and H six months/12,000. Gaps are visible throughout the adhesive interface of Er:YAG laser prepared groups after one month/2,000 and six months/12,000 (arrows).Figure 4. Representative SEM images of the adhesive interface after cavity preparation: carbide bur (A,C,E,G) and Er:YAG (B,D,F,H). Periods of water storage and the number of thermocycles: A and B—24 hours/None; C and D seven days/500; E and F one month/2,000; G and H six months/12,000. Gaps are visible throughout the adhesive interface of Er:YAG laser prepared groups after one month/2,000 and six months/12,000 (arrows).Figure 4. Representative SEM images of the adhesive interface after cavity preparation: carbide bur (A,C,E,G) and Er:YAG (B,D,F,H). Periods of water storage and the number of thermocycles: A and B—24 hours/None; C and D seven days/500; E and F one month/2,000; G and H six months/12,000. Gaps are visible throughout the adhesive interface of Er:YAG laser prepared groups after one month/2,000 and six months/12,000 (arrows).
Figure 4 Representative SEM images of the adhesive interface after cavity preparation: carbide bur (A,C,E,G) and Er:YAG (B,D,F,H). Periods of water storage and the number of thermocycles: A and B—24 hours/None; C and D seven days/500; E and F one month/2,000; G and H six months/12,000. Gaps are visible throughout the adhesive interface of Er:YAG laser prepared groups after one month/2,000 and six months/12,000 (arrows).

Citation: Operative Dentistry 33, 1; 10.2341/07-30

Figure 5. Surface morphology following cavity preparation.Figure 5. Surface morphology following cavity preparation.Figure 5. Surface morphology following cavity preparation.
Figure 5 Surface morphology following cavity preparation.

Citation: Operative Dentistry 33, 1; 10.2341/07-30

Also, after six months of water storage with 12,000 TC, a larger gap can be seen on the adhesive interface, showing that the adhesive interface in the Er:YAG-laser prepared group was more affected by the methods used for simulating its degradation.

Resin bonding durability in Er:YAG laser cavity preparations after thermocycling procedures and long-term water storage is being described for the first time in this study. Most reports3571827 that evaluated adhesion in Er:YAG laser-irradiated tooth structure have performed bond strength tests after 24 hours of water storage without thermocycling. Oliveira and others8 have evaluated the effect of 1,400 thermocycles on the microtensile bond strength of adhesive system to Er:YAG irradiated dentin, but long-term water storage was not performed.

The Er:YAG-laser prepared specimens that debonded after six-months, combined with 12,000 thermocycles, yielded the lowest μTBS mean, which was statistically different from the other groups. The effects of Er:YAG laser irradiation, such as fusion of the collagen fibrils and restriction of resin diffusion into the subsurface dentin,418 may result in an adhesive interface with failed areas and gaps in which hydrolytic degradation could be facilitated. Furthermore, during Er:YAG laser irradiation on dentin, the amount of water is decreased, which later can be partly restored by water uptake.3 This reduction in water content during Er:YAG laser irradiation probably decreases diffusion of adhesive resin and elimination of the solvent. Hydrolytic degradation in Er:YAG-laser prepared dentin may also be enhanced by the two-step etch and rinse adhesive system, as used in this study, wherein the remaining solvent kept in the interface may create non-homogenous regions with uncontrollable voids or increase polymer chain mobility, thus making polymers more susceptible to water sorption.46

It seems appropriate to emphasize that further research is required to confirm these results, in which other types of adhesives, such as self-etching systems and other methods that simulate degradation of the adhesive interface, including pH cycling and mechanical loading, should be performed.

CONCLUSIONS

  • Bond strength values obtained in bur-prepared samples were similar to Er:YAG laser values in terms of initial periods of evaluation (no cycling/24 hours water storage and 500 thermocycles/one week water storage), but they were higher after two months of water storage within 2,000 thermocycles and six months of water storage within 12,000 thermocycles.

  • From the one-month water storage and 2,000 thermocycles point in time, performance of the tested adhesive system to Er:YAG laser irradiated-dentin was negatively affected.

  • Adhesion of the bur-prepared group decreased after six months of water storage combined with 12,000 thermocycles.

  • Adhesion to the Er:YAG laser cavity preparation was more affected by the methods used to simulate degradation of the adhesive interface.

Acknowledgments

This manuscript is part of a thesis submitted by the first author to the Faculty of Dentistry of Ribeirão Preto, University of São Paulo (USP) in partial fulfillment of the requirements for the Master's degree in Restorative Dentistry. This study was supported by Capes (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (grant #477923/2004/6). The authors thank the Institute of Physics of São Carlos (IFSC - USP) and P Vanderley S Bagnato for putting the Er:YAG laser at their disposal and Nick Bewick for English review.

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Copyright: Copyright: © 2008 This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2008
Figure 1
Figure 1

Schematic illustration. (A) the roots were removed from bovine incisors; (B) sectioning of crowns to obtain specimens 8×8×2 mm (C), which were ground to expose superficial dentin; (D) delimitation of the dentin bonding site (6×6 mm2); (E) cavity preparation device (Er:YAG laser + Carbide Bur); (F) resin composite restoration; (G) thermocycling and water storage of the specimens; (H) sectioning samples into 1×1 mm sticks (I); (J) sticks were loaded in tension; (K) types of fracture that were analyzed under SEM.

Figure 2
Figure 2

Box-plot of dentin microtensile bond strengths (μTBS). The box represents the spread of data. The central vertical line represents the median. The “whiskers” extend to the minimum and maximum value.

Figure 3
Figure 3

Fracture types.

Figure 4
Figure 4

Representative SEM images of the adhesive interface after cavity preparation: carbide bur (A,C,E,G) and Er:YAG (B,D,F,H). Periods of water storage and the number of thermocycles: A and B—24 hours/None; C and D seven days/500; E and F one month/2,000; G and H six months/12,000. Gaps are visible throughout the adhesive interface of Er:YAG laser prepared groups after one month/2,000 and six months/12,000 (arrows).

Figure 5
Figure 5

Surface morphology following cavity preparation.

Contributor Notes

*Flávia Lucisano Botelho do Amaral, DDS, MS, University of São Paulo, School of Dentistry of Ribeirão, Preto, Department of Operative Dentistry, São Paulo, Brazil

Vivian Colucci, DDS, MS, University of São Paulo, Ribeirão Preto School of Dentistry, Department of Operative Dentistry, São Paulo, Brazil

Aline Evangelista de Souza-Gabriel, DDS, MS, University of São Paulo, Ribeirão Preto School of Dentistry, Department of Operative Dentistry, São Paulo, Brazil

Michelle Alexandra Chinelatti, DDS, MS, University of São Paulo, Ribeirão Preto School of Dentistry, Department of Operative Dentistry, São Paulo, Brazil

Regina Guenka Palma-Dibb, DDS, MS, PhD, associate professor, University of São Paulo, Ribeirão Preto School of Dentistry, Department of Operative Dentistry, São Paulo, Brazil

Silmara Aparecida Milori Corona, DDS, MS, PhD, associate professor, University of São Paulo, Ribeirão Preto School of Dentistry, Department of Operative Dentistry, São Paulo, Brazil

*Reprint request: Av do Café, S/N, Monte Alegre, CP: 14040-904, Ribeirão Preto, SP, Brazil; e-mail: flbamaral@gmail.com
Received: 15 Feb 2007
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