Effects of zirconia nanoparticles addition to experimental adhesives on radiopacity and microhardness

Aim: To evaluate the radiopacity and microhardness (KHN) of experimental dental adhesives (EX). The experimental adhesive resins of the present study were formulated based on the simplified adhesive system Ambar (FGM). Methods: Five EX with different concentrations of zirconia nanoparticles [0(EX0), 15(EX15), 25(EX25), 30(EX30) e 50%(EX50)] were incorporated in a UDMA/HEMA adhesive (control). Adper Single Bond TM 2 (SB, 3M ESPE) was used as a commercial reference. For the radiopacity (n=5), KHN (n=5), adhesive specimens were fabricated using a stainless steel mold. Data were submitted to one-way ANOVA and Tukey´s test ( α =0.05). Results: The filler addition on the EX showed radiopacity similar to enamel and higher than SB. The EX25, EX35 and EX50 showed higher KHN values when compared to the commercial SB. EX25, EX35 and EX50 showed higher KHN values when compared to the commercial SB. Conclusions: The results of the present investigation suggest that the addition of zirconia nanoparticles seems to be a good alternative to produce radiopaque adhesives with increased microhardness.


Introduction
The radiopacity of adhesive materials is clinically relevant, mainly in technique-sensitive restorations, where evaluation of the tooth/restorative interface is critical 1,2 . Unfortunately, the great majority of the commercial adhesives available are radiolucent 3 and they cannot be clearly detected in radiographs 4 when applied in layers thicker than 40 µm. This is a pertinent clinical concern since adhesive layers should be distinguished from marginal gaps with potential for secondary caries.
The radiopacity of esthetic restorative materials has been established as an important requirement, improving the radiographic diagnosis. To the extent of our knowledge, the flowable composites 5,6 , composite resins 7 or resin cements 6,8 available are radiopaque and only bonding agents are still radiolucent 9 . The development of radiopaque adhesive systems can avoid inappropriate replacements 10,11 due to misinterpretations in the diagnosis of secondary caries 2,10,12 and detection of gaps near the restoration 4,12 . Thus, the studies should focus on  Recently, there has been a great interest in the application of nanotechnology in resin-based materials 13,14 to produce dental composite materials with increased hardness 7,13,15 . These promising findings led investigators to evaluate the effects of filler addition on the mechanical properties [16][17][18][19][20] as well as bonding features of adhesives 18,22 . However few of them incorporated nano-sized filler particles into the adhesive formulations 13,15 and evaluated the role of nanofillers on the material's radiopacity 18,20 . Therefore, the aim of the present investigation was to evaluate the incorporation of varied concentrations of zirconia nanofiller into a two-step etch-and-rinse adhesive on the material´s radiopacity and microhardness.

Material and methods
This research project was approved by the Institutional Review Board from the local Dental School under protocol # 28/2010.
The experimental adhesive resins from the present study were formulated using the simplified adhesive system Ambar (FGM Dental Products, Joinville, SC, Brazil) as base. This material was specifically formulated for this study without any filler content. The detailed composition of this adhesive system, as provided by the manufacturer, can be seen in Table 1. The simplified etch-and-rinse commercial adhesive system (Adper Single Bond TM 2; 3M ESPE, St. Paul, MN, USA) was used as reference (Table 1).
Using a circular stainless steel mold, five specimens measuring 5.0 mm in diameter and 1.0 mm thick were prepared for each material. The adhesive was dispensed in the mold until complete filling. All visible air bubbles trapped in the adhesive solution were carefully removed. The solvent was evaporated by gentle air blowing from a dental syringe for 40 s. Each specimen was polymerized for 80 s with a visiblelight curing unit (VIP; Bisco Inc., Schaumburg, IL, USA) with a power density 450 mW/cm 2 . Enamel and dentin specimens were obtained from 1.0-mm thick longitudinal sections of human third molars previously stored in 0.5% thymol and used within 6 months after extraction. Slices were prepared using a low-speed diamond blade (Isomet 1000; Buehler, Lake Bluff, IL, USA) mounted in a cutting machine under water cooling.
A total of five radiographs were made. Each radiograph was taken with one specimen of each experimental condition and the enamel-dentin slice positioned on the digital sensor. An digital radiography was then taken with an exposure time of 0.2 s. The radiographic position was standardized: the radiographic central beam focusing in a 90° angle with the surface of the image receptor, at a 30 cm focus-object distance and parallelism between the sensor and the specimens with the Heliodent Vario machine (Sirona, Bensheim, Germany). The digital radiopacity (% white) was measured by pixels counting using the UTHSCSA ImageTool 3.0 software (Department of Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, TX, USA).
Data for each property was subjected to one-way ANOVA.

Effects of zirconia nanoparticles addition to experimental adhesives on radiopacity and microhardness
Post-hoc multiple comparisons were performed using Tukey's test at a significance level of 5%.

Results
One-way ANOVA detected statistically significant difference among groups (Table 2, p<0.001). All experimental bonding adhesives showed radiopacity similar to enamel, except for EX0 and SB. These two adhesives showed radiopacity similar to the dentin substrate. For KHN oneway ANOVA detected significant differences between groups (

Discussion
It is noteworthy to mention that the monomeric composition of the commercially available two-step etchand-rinse adhesive Ambar (FGM Dental Products, Brazil) was employed. This simplified adhesive contains nanofillers, which were especially removed by the manufacturer preparing the evaluated experimental adhesives. Therefore, the experimental adhesives contained only the filler loading added by the authors.
The radiopacity of esthetic restorative materials has been established as an important requirement, improving radiographic diagnosis 1,2,24 . Radiolucent areas around restorations may result from either a halo effect or the radiographic density of the adhesives. Therefore, the use of an adhesive with radiopaque fillers can avoid inappropriate replacements 10,11 due to misinterpretations in the diagnosis of secondary caries 2,10,12 . For adequate assessment of the restoration quality both at baseline and in recall examinations, radiographic evaluations are very useful and for such, dental materials should be sufficiently radiopaque to be detected against a background of sound and caries-affected enamel and dentin substrate, and thus allow correct evaluation of the presence of secondary caries, marginal defects, contour of restoration, and contact with adjacent teeth, cement overhangs and interfacial gaps 1,2,9 . To the extent of the authors' knowledge only one study investigated the impact of filler addition on the adhesive radiopacity 22 and this is therefore a novel study that attempts to investigate this issue. The results of the present study showed that the addition of zirconia nanoparticles yielded enamel radiopacity to the experimental adhesives evaluated. Even the smallest percentage of zirconia nanoparticles evaluated in this study (15%) produced a radiopaque experimental adhesive similar to the radiopacity of the enamel substrate. Similarly Schulz et al. also observed increased adhesive radiopacity after inclusion of agglomerated Ta 2 O 5 /SiO 2 nanoparticles.
Dental adhesives are intricate mixtures of components and they are designed to bond composite resins to enamel and dentin 25,26 . Irrespective of the number of bottles, an adhesive system typically contains resin monomers, curing initiators, inhibitors, stabilizers, solvents and inorganic filler. Each one of these components has a specific function 25 . Although the addition of filler to adhesives has shown to be beneficial 20 this finding is not consensual in the literature 20,27 .
The addition of filler particles to composite resins is made in an attempt to improve the mechanical properties of dental composites 13,25,28,29 . Similarly, the present investigation showed that the addition of filler loading higher equal to or higher than 25% allowed the increase in the microhardness of the experimental adhesives. A recent study also demonstrated that the addition of hydroxyapatite nanoparticles 30 and niobium pentoxide 31 also produced an adhesive layer with increased microhardness and other superior properties.
The commercial SB adhesive showed the lowest KHN. This means that the attainment of higher microhardness is not dependent only on the filler loading. An adhesive system with adequate and balanced monomer composition blend may also lead to the production of a polymeric material with increased properties. Compared to the commercial SB adhesive, the unfilled experimental adhesive (EX0) showed higher KNH and such difference may be attributed to composition differences in monomer blends.
The addition of filler loading in moderate concentrations (EX25 and EX35) produced radiopaque materials. Compared to the commercial SB adhesive, the unfilled experimental adhesive (EX0) with higher KNH that were either improved or remained unchanged compared to the unfilled version. The results of the present investigation suggest that the addition of zirconia nanoparticles seems to be a good alternative to produce radiopaque adhesives with increased microhardness. Further studies should focus on the enamel and dentin bonding strength of adhesive systems with zirconia nanoparticles. by the National Council for Scientific and Technological Development (CNPq) under grants 301937/2009-5 and 301891/2010-9. The authors of this study are very grateful to Iverson E. Woyceichoski for the help in the radiographs and Luiz Lima Junior for the help in microhardness test.