ORIGINAL ARTICLE

Nanoleakage in primary teeth prepared by laser irradiationor bur

Fatih Öznurhan & Ayşegül Ölmez

Received: 8 May 2012 /Accepted: 3 September 2012 /Published online: 12 September 2012# Springer-Verlag London Ltd 2012

Abstract The aim of this in vitro study was to analyzehybrid layer and nanoleakage of composite resin resto-rations in cavities prepared by either Er,Cr:YSGG laseror bur, followed by acid etching in primary teeth. Tenextracted primary molar teeth were randomly allocatedinto two groups consisting of ten cavities according tosurface treatment regimen: Er,Cr:YSGG laser + acidetching(group 1) and bur + acid etching(group 2).Restorations of all samples were completed. Then, teethwere sectioned and immersed to ammoniacal silver ni-trate solution. After polishing, hybrid layer thicknesseswere examined under scanning electron microscopy(SEM) and ion analysis was carried out with SEM/energy dispersive X-ray spectroscopy preparation interms of nanoleakage. Hybrid layer thickness and theamount of silver ions were assessed for the acid-etchedgroups. The collected data were analyzed with indepen-dent sample t test and Spearman’s rank correlation. Ingroups 1 and 2, the mean hybrid layer thicknesses were4.25±1.41 and 5.24±1.07 μm and the silver ion percen-tages were 10.97±13.81 and 22.79±21.62 %, respectively.Although no significant correlation was observed between theincrease of hybrid layer thickness and the amount of silverions, more silver ions were observed in group 2 (p<0.05).According to the results of this study, acid-etched cavities

prepared with laser promoted better results when comparedto the acid-etched cavities prepared with bur.

Keywords Primary teeth . Er,Cr:YSGG laser .

Nanoleakage . Acid etching . Hybrid layer

Introduction

The Er,Cr:YSGG laser has been used in dentistry for manydifferent purposes with the advantage of cutting both softand hard tissues. The advantages of using lasers on hardtissues is that it reduces pain, makes less noise, does notvibrate, and eliminates, in most cases, the need of anes-thetics [1–13]. The laser is capable of removing the smearlayer and opening the dentin tubules [4–6, 14, 15] and alsohas an antibacterial effect [16]. According to some authors,the use of laser device can easily eliminate the acid etchingprocedure [3, 15, 17, 18]. Nevertheless, other authors rec-ommend the use of the laser in combination with acidetching to have a better adhesion to dental hard tissues[17, 19].

Adhesion to dental hard tissues can be achieved by theuse of bonding agents that promote a micromechanical inter-locking with both enamel and dentin. Bonding to dentindepends on the infiltration of synthetic adhesive monomersinto a biological, collagen-rich substrate to form a hybridlayer. The evolution of adhesive systems, designed toachieve the bond of the adhesive to the dental structure,has improved the clinical performance of restorations be-cause of a better adaptation to the cavity walls and a hybridlayer formation [20, 21]. Consequently, there has been adecrease in microleakage and secondary caries. However,even the latest generation of adhesives has not eliminatedmicroleakage completely [16].

Sano et al. [22] described nanoleakage as the penetrationof fine silver particles through the hybrid layer, due to the

F. Öznurhan (*)Department of Pediatric Dentistry, Faculty of Dentistry,Cumhuriyet University School of Dentistry,58140, Kampüs,Sivas, Turkeye-mail: fatihozn@hotmail.com

A. ÖlmezDepartment of Pediatric Dentistry, Faculty of Dentistry,Gazi University School of Dentistry,82.sok Emek,Ankara, Turkeye-mail: aysegul@gazi.edu.tr

Lasers Med Sci (2013) 28:1099–1105DOI 10.1007/s10103-012-1204-2

incomplete penetration of bonding resins into the area decal-cified by the acids, forming a weak zone more susceptible tohydrolysis and microleakage over time [16, 22–24]. Thiskind of leakage may allow the penetration of bacterialproducts and dentinal or oral fluid along the interface, whichmay result in hydrolytic breakdown of either the adhesiveresin or collagen within the hybrid layer, thereby compro-mising the stability of the resin–dentin bond [25]. Nano-leakage is different from microleakage and nanoleakagecould be seen in the absence of gap formation [26].

Microleakage of the cavities with composite resin fillingsprepared by Er,Cr:YSGG laser has also been investigated.Some authors suggested that there was no significant differencein microleakage between the cavities prepared by Er,Cr:YSGGlaser and those prepared by the conventional method [15].

The composition and micromorphology of the dentin inprimary teeth have been shown to differ from permanent teeth.A comparative analysis of the dentin hardness indicated thatthe central area of the coronal dentin is considerably harder inpermanent teeth. It was concluded that permanent dentin ismore highly mineralized, based on the fact that hardness isrelated to the degree of mineralization. The concentrations ofcalcium and phosphorus in both peritubular and intertubulardentin are lower in primary than in permanent teeth. Evalua-tion of the dentinal micromorphology also indicates potentialdifferences between primary and permanent teeth. Comparedwith permanent teeth, primary teeth presented a lower con-centration and a smaller diameter of dentinal tubules at adistance of 0.4 to 0.5 mm from the pulpal surface [27].

There are few studies regarding the effects of Er,Cr:YSGG lasers on primary teeth. Cehreli et al. [28] showedthat Er,Cr:YSGG laser pretreatment did not influence theresistance to microleakage of bonded fissure sealant appli-cation in primary teeth. Hossain et al. [15] compared thesurface alterations of the enamel and dentin as well as thedegree of microleakage in cavities prepared by Er,Cr:YSGGlaser irradiation with etched bur cavities in human primaryteeth and found that laser cavity surface facilitated a goodadhesion with the restorative materials and the acid etch stepcan be easily avoided with the laser treatment.

On the contrary to these studies, Gutknecht et al. [17]examined microleakage of composite fillings in Er,Cr:YSGGlaser-prepared class II cavities and recommended the additionaluse of etching after Er,Cr:YSGG laser preparation as it is usedin the classical cavity preparation technique in permanent teeth.Ergucu et al. [19] compared the microleakage of compositeresin restorations using two different dentin adhesive systemsand two different modes of cavity preparation, a high-speedhandpiece and an Er,Cr:YSGG laser, and recommend addition-al acid etching after Er,Cr:YSGG laser preparation. Laser irra-diation affects adversely the adhesion of resin to the dentin butacid etching following laser irradiation could increase the ten-sile bond strength and reduce the microleakage [17, 19, 29].

To date, nanoleakage in the Er,Cr:YSGG laser-preparedcavities in primary teeth has not been a subject of any study.Therefore, the aim of this in vitro study was to analyze thethickness of hybrid layer and degree of nanoleakage ofcomposite resin restorations in cavities prepared by eitherEr,Cr:YSGG laser or bur following with acid etching inprimary teeth. The null hypothesis established for this studywas that there is no difference between Er,Cr:YSGG and burpreparation in terms of hybrid layer formation and nano-leakage regardless of cavity preparation procedure.

Materials and methods

Ten human mandibular primary molars, free of caries andother macroscopic defects, extracted for orthodontic reasonswere collected and stored in sterile saline solution at 4 °C forup to 1 month.

Cavity preparations

Standard class V cavities (3×2×1.5 mm) were prepared onbuccal surfaces with the gingival margins located at 1 mmabove the cementoenamel junction with Er,Cr:YSGG hydro-kinetic laser system (Waterlase MD, Biolase Technology Inc.,San Clemente, CA, USA) operating at a wavelength of2,780 nm and having a pulse duration of 140–200 μs with arepetition rate of 20 Hz. The power output was set at 6.0W forthe enamel and 3.5 W for dentine preparation. Air and waterspray of the handpiece was adjusted to a level of 85–90 % airand 80–85%water for 6.0W, and 70% air and 65%water for3.5 W to prevent cavity surfaces from overheating. The laserbeam was aligned perpendicular to the buccal surfaces in non-contact mode at 1.5–2 mm distance, in accordance with themanufacturer’s instructions. After the preparation, the cavitieswere etched with 35 % phosphoric acid (3M ScotchbondTM

Etchant Delivery System, 3M ESPE, St Paul, USA) for 15 s,rinsed with distilled water, and then gently dried.

The lingual surfaces of the teeth were used for the burpreparation group; standard class V cavities with the samestandard dimensions and location were prepared with dia-mond bur (Shofu Inc., Kyoto, Japan). Then, cavities onlingual surfaces were acid-etched, rinsed with distilled wa-ter, and then gently dried. Thereby, two study groups wereobtained consisting of ten cavities in each:

Group 1: Laser and acid etchingGroup 2: Bur and acid etching

Adhesive system and bonding procedures

After preparation, the cavities were dried and bonding system(AdperTM Single Bond (SB), 3M ESPE, St Paul, USA) was

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applied with brush to the cavities, gently air thinned for 5 s andlight cured for 10 s. After application of the adhesive system,the cavities were restored with composite resin (FiltekTM

Z250, 3M ESPE, St Paul, USA) in two increments that werelight-activated separately for 20 s with Hilux Ultra DentalCuring Light (600 mWcm−2, 450–520 nm, Benlioglu, Tur-key), according to the manufacturer’s instructions. The finalfinishing was achieved by using flexible polishing disks. Afterpolishing, the teeth were embedded into acrylic resin. Afterthe polymerization of acrylic resin, the teeth were sectioned(Mecatome T201 Presi, France), first mesiodistally and thenbuccolingually to obtain 1×1-mm sticks from the center of thecavities. One buccal and one lingual stick were obtained fromthe center of the cavities and the sticks were prepared fornanoleakage test and scanning electron microscopy/energydispersive X-ray spectroscopy (SEM-EDX).

Teeth preparation for nanoleakage test

The sticks obtained from the two groups were covered withtwo layers of nail polish with the exception of the restorationareas and 1 mm outside margins of the restorations. Thecovered sticks were immersed in 50 wt% ammoniacal silvernitrate solution (pH04.2) for 24 h in a dark chamber accord-ing to Tay et al. [30], washed with running water for 5 min,placed in photodeveloping solution for 8 h under a fluorescentlight to reduce the silver or diamine silver ions into metallicsilver grains, and then washed with running water for 5 min.The samples were stored in distilled water for 1 month at roomtemperature and the solution was altered weekly.

Scanning electron microscopy/energy dispersive X-rayspectroscopy preparation

The sticks were embedded into acrylic resin prior to polishing.The sticks were polished with wet 600-, 1,200-, and 2,400-gritsilicon carbide papers to create a uniform surface and finishedwith diamond polishing paste (Sparkle, Pulpdent, Watertown,MA, USA). The specimens were conditioned with 5 %

phosphoric acid for 5 s and then immersed in ethanol solution(70 %) for 10 s. The specimens were coated with a thin layerof gold (sputtering) and analyzed in SEM (Jeol 6060, Japan)by backscattered electron images. The amount of silver nitratewithin the hybrid layer in each stick measured with SEM-EDX in three measurements from the bottom of the cavity atequal distances was obtained from each teeth regions of thesticks, by this means, 30 measurements were evaluated foreach group The thickness of the hybrid layer was scanned andmeasured. In these regions, the silver nitrate uptake wasmeasured as a percentage and the thickness of the hybrid layeras micrometer, and the data were collected at Statistical Pack-ages for Social Sciences 15.0 for Windows. Since Shapiro–Wilk test indicated the normal distribution of data, parametrictest was used for comparing the thickness of the hybrid layerbetween groups. Thus, the data were analyzed with indepen-dent sample t test and Spearman’s rank correlation at theconfidence interval of 95 % (Fig. 1).

Results

The resin–dentin interfaces in two groups were analyzedwith SEM-EDX. Silver deposition was observed at the

Fig. 1 a The black arrowsshow the buccal and palatinal/lingual cavities. b The teethwere sectioned firstmesiodistally (1) and thenbuccolingually (2) to obtain 1×1-mm sticks from the center ofthe cavities. c The stick shapecould be seen and the blackarrows show the threemeasurements taken at equaldistances from the bottom of thecavity

Fig. 2 Image of laser and acid-etch group (×3,500). The Ag ions couldbe seen in hybrid layer. CR composite resin, D dentin, H hybrid layer

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bottom of the hybrid layer and spotted pattern of nanoleak-age was described. When these two groups were analyzedwith line scan, Si, C, and Ca element peaks were detected.

In group 1, the hybrid layer could be observed andmeasured with SEM-EDX. The mean thickness of hybridlayer in group 1 was 4.25 μm, and that in group 2 was5.24 μm (Figs. 2 and 3). The mean silver ion percentage ingroup 1 was 10.97 %, and that in group 2 was 22.79 %(Figs. 4 and 5)

Data analysis of groups 1 and 2 is shown in Table 1. Thethickness of the hybrid layer and the silver ion percentagesin group 2 were significantly higher than group 1 (p<0.005). Spearman’s rank correlation indicated that therewas no correlation between the thickness of hybrid layerand silver ion percentage (p>0.005).

Discussion

The present study analyzed the thickness of hybrid layer aswell as the degree of nanoleakage determined by the amount

of silver ions within hybrid layer in the cavities prepared byEr,Cr:YSGG laser or bur. The cavities prepared with laserpromoted better results when compared to cavities preparedwith bur.

The development of hybrid layer, an intermixture ofdentin collagen and diffusible components of the adhesivesystem, is a determinant of the clinical outcome of restora-tions bonded to dentin [31]. The ideal hybrid layer should belarge enough to allow for a stable interlocking of the adhe-sive resin around the exposed collagen fibers. On the otherhand, the demineralized zone should not be excessivelydeep because the primer and adhesive resin may not flowamong all the exposed fibers and completely embed them[27]. Less microleakage, better and long life restorations canbe obtained with bonding systems and hybrid layer [32–34].

Acid etching is a crucial step in adhesive restorations.Although Usumez et al. [35], Basaran et al. [36], and Sunget al. [14] found that there were no statistical differencesbetween acid etching and laser etching, Gutknecht et al.[17], Ergucu et al. [19], and Lee et al. [29] recommendedthe use of acid etching technique after the cavity preparationwith laser. Consistent with these findings, we preferred acidetching procedure prior to bonding agent application to lasercavities.

Adhesive systems of different composition and applica-tion modes present different nanoleakage patterns. Potentialwater-binding domains within hybrid layers and adhesivelayers in resin–dentin interfaces are traced by ammoniacalsilver nitrate. Differences in hydrophilicity and water con-tent have an important role in nanoleakage patterns pre-sented by adhesive systems [37]. SB showed nanoleakagein all parts of the hybrid layer. Yuan et al. [38] foundreticular and spotted pattern of nanoleakage in their study.In the present study, silver deposition was observed at thebase of the hybrid layer and spotted type of nanoleakagewas seen. The authors described that SB had some porositiesand this may possibly explain the observation of silver ionswithin the hybrid layer in the present study. In addition, the

Fig. 4 The Ag ion percentageof laser and acid-etch group inFig. 2

Fig. 3 Image of bur and acid-etch group (×2,000).The Ag ions couldbe seen in the dentin side and line scan analysis showed Ag ion peakswhile analyzing dentin (Fig. 5). CR composite resin, D dentin, Hhybrid layer

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authors claimed that the best results were obtained with SB,since SB showed better hybridization quality. Reis et al. [37]found that the total-etch adhesive SB presented a high degreeof nanoleakage. Even though reports have demonstrated highbond strengths for SB, a greater penetration of silver has alsobeen reported for this system compared with a two-step self-etching system. In another study, Reis et al. [39] reported thatthe two-step, acetone-based, etch-and-rinse adhesive Primeand Bond presented lower silver deposition than the water/ethanol-based SB after storage in water for 6 months. How-ever, the samples were stored in distilled water only for1 month in the present study to provide a reliable accumula-tion of silver particles. Hashimoto et al. [40] reported that theuse of multiple applications of adhesives also allows moretime for the removal of water by inward diffusion of adhesivemonomers and subsequent solvent evaporation from the inter-fibrillar spaces. Therefore, adhesive was applied only in onelayer in the present study. SB is a total-etch fifth-generationadhesive system. With BISGMA, it has hydrophobic charac-teristic, and with ethanol and water, it has hydrophilic charac-teristics. It was thought that the best results may be found withSB, and therefore, SB was preferred.

In agreement with Yuan et al. [38], line scan analysesshowed that the silver ions were in the resin–dentin inter-face. Si, C, and Ca peaks were seen in line scan and Si and Cpeaks were attributed to the composite resin whereas Capeak was due to the dentin [40].

In group 1, hybrid layer could be seen in most parts of thecavities. In the cavities, three locations were selected andmeasured in terms of hybrid layer thickness and the amountof the silver ions. In agreement with Schein et al. [41], whenacid etching was performed on dentin, degradation of

collagen matrix led to the diffusion of the adhesive. Inhybrid layer studies, the sections should be vertical to thecavity floor to observe hybrid layer. In lased cavities, thecavity surfaces were irregular making it impossible to havevertical sections. Therefore, this might be one of the possi-ble explanations why hybrid layer could not be seen in someparts of the cavities.

In the selected locations of the cavities, hybrid layer andthe percentage of silver ions were measured. In the presentstudy, hybrid layer was thinner in group 1 than group 2.Ablation affect exerted by the laser system results with thedecrease of the collagen content, and this might account forthe occurrence of less hybridization.

In many studies, it was suggested that mean hybrid layerthickness ranges from 2 to 8.15 μm [27, 31, 42–44]. In thepresent study, the mean hybrid layer thickness was found5.24 μm in group 2. Li et al. [25] found that the hybrid layerwas approximately 3–4 μm thick. The mean hybrid layerthickness in group 2 was greater than this result. However, itis reported that hybrid layer in primary teeth is thicker thanpermanent teeth [45–49]. The thickness of hybrid layer isdifferent from 1.50 to 7.28 μm, and this diversity and thequality of the hybrid layer are related with pH of the etchant,dentine’s organic and inorganic portion, the ability of theresin monomer to flow into the demineralized dentin, andchemophysiological and morphological characteristics ofthe dentine [50]. But when laser was used with acid etching,it is also different from these results and the laser has sideeffect to hybrid layer. De Munck et al. [51] observed thatcavities prepared by laser appeared less receptive to adhe-sive procedures than conventional bur cavities. The authorsstated that after acid etching laser-conditioned dentin, the

Fig. 5 The Ag ion percentageof bur and acid-etch group inFig. 3

Table 1 Maximum measurements, minimum measurements, and means of hybrid layer and the percentages of Ag in groups 1 and 2

Groups Hybrid layer (μm) min Hybrid layer (μm) max Hybrid layer (μm) mean Ag (%) min Ag (%) max Ag (%) mean

Group 1 (n010) 1.50 7.28 4.25±1.41 0.73 62.05 10.97±13.81

Group 2 (n010) 2.80 7.10 5.24±1.07 0.77 81.90 22.79±21.62

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hybridization effectiveness is compromised because of theselective ablation of organic tissue, leading to less collagenleft to be exposed and consequently to be hybridized. Stud-ies showed that laser irradiation can negatively influence thedentin/adhesive system interface, hampering the hybrid lay-er formation [52, 53]. Other studies proposed the applicationof acid etching after cavity preparation with laser irradiation[54]. The results of this study verified a decrease in nano-leakage with the use of Er,Cr:YSGG laser for cavity prepa-ration and dentin pretreatment when combined with acidetching, so the null hypothesis had to be rejected.

Conclusion

In the present study, nanoleakage was evaluated in laser andbur cavities in primary teeth and the best results were seen ingroup 1 in which laser preparation was combined with acidetching. Further research is necessary to better explain thedentinal changes induced by the use of the laser in primaryteeth.

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