The impact of artificially caries-affected dentin on bond strength of multi-mode adhesives.

AIM: The aim of this study is to evaluate the impact of dentin condition on bond strength of multi-mode adhesive systems (MMAS) to sound and artificially induced caries-affected dentin (CAD).
METHODS: Flat dentin surfaces of 112 bovine incisors were assigned to 16 subgroups (n = 7) according to the substrate condition (sound and CAD- pH-cycling for 14 days); adhesive systems (Scotchbond Universal, All-Bond Universal, Prime and Bond Elect, Adper Single Bond Plus and Clearfil SE Bond) and etching strategy (etch-and-rinse and self-etch). All systems were applied according to the manufacturer's instructions, and resin composite restorations were built. After 24 h of water storage, specimens were sectioned (0.8 mm2) and submitted to the microtensile test. STATISTICAL ANALYSIS: Data (MPa) were analyzed using three-way analysis of variance and Tukey's test (α = 0.05).
RESULTS: MMAS presented similar bond strength values, regardless etching strategy in each substrate condition. Bond strength values were lower when MMAS were applied to CAD in the etch-and-rinse strategy.
CONCLUSION: The etching strategy did not influence the bond strength of MMAS to sound or CAD, considering each substrate separately. However, CAD impact negatively on bond strength of MMAS in etch-and rinse mode.

INTRODUCTION

Following the trend to obtain straightforward materials and techniques universal or multi-mode adhesives systems were developed to be used both in etch-and-rinse or self-etch approaches. This versatility allows the professional decide the most appropriate protocol for each clinical situation.[1] The multi-mode adhesive systems (MMAS) have been assessed in the past years,[123456] and it has been reported that their in vitro performance is dependent on the adhesive strategy, especially regarding enamel bonding.[7] However, for dentin, there is no consensus on the literature about the influence of etching strategy and the long-term performance of multi-mode adhesives. Besides that, caries-affected dentin (CAD), a clinically relevant substrate[8] has not been considered in in vitro evaluations of MMAS. Considering that, the differences in composition and microstructure of CAD compared to sound one and the consequently lower bond strength values found to this substrate in studies with previous adhesive systems[91011] it seems reasonable to include CAD on MMAS' evaluations.

A recent study[12] showed that the immediate bond strength of a universal adhesive system was not influenced by the etching approach either by the dentin condition (artificially caries-affected primary or permanent dentin). However, this study assessed only one multi-mode adhesive system, and a gap remains about the performance of universal adhesives on clinically relevant substrates as CAD.

Therefore, the aim of this in vitro study was to evaluate the influence of dentin condition– sound or artificially-induced caries-affected on bond strength performance of three MMAS, using two etching strategies. The null hypotheses tested were as follows: (1) different MMAS present similar bond strength irrespective of the etching mode, and (2) the bond strength of MMAS is not influenced by dentin condition.

METHODS

One hundred and twelve freshly extracted bovine incisors were used in this study. They were selected and stored previously in chloramine at 4°C for a maximum of 30 days.

Tooth preparation

The root was removed using a diamond disc in a high-speed handpiece. The buccal surfaces were ground under water-cooling using a 100-grit SiC paper in a grinder-polisher machine (EcoMet 250, Buehler; Illinois, USA) obtaining flat dentin surfaces, then manually polished using 600-grit SiC paper under water for the 60s rendering a standardized smear layer.[13]

Artificial caries induction– artificially caries-affected dentin

The teeth were randomly assigned into two groups according to the substrate (sound and artificially CAD). Teeth allocated to sound dentin were immersed in distilled water only, and those of CAD group were exposed to artificial caries induction with a pH-cycling model.[1214] They were individually submitted to immersion for 8 h in 10 ml of demineralizing solution (2.2 mM CaCl2, 2.2 mM NaH2PO4, 50 mM acetic acid, adjusted pH of 4.8 with 1M KOH) and for 16 h in the same volume of remineralizing solution (1.5 mM CaCl2, 0.9 mM NaH2PO4, 0.15 mM KCl with adjusted pH of 7.0). Solutions' pH was confirmed and replaced every cycle for 14 days and at each interval, specimens were washed with deionized water and dried with paper towels.

Bonding and restorative procedures

Teeth from each substrate (sound dentin and CAD) were randomly reallocated (RANDOM. ORG, Randomness and Integrity Services Ltd.; Dublin, Ireland) to 8 subgroups according to the adhesive system and etching strategy (n = 7). Three multi-mode adhesives were evaluated as follows: Scotchbond Universal (SU), all-bond universal (AB) and Prime and Bond Elect (PB). All materials were applied in either self-etch (SE) or etch-and-rinse (ER) protocols. As control groups for each strategy, a two-step etch-and-rinse Adper Single Bond Plus (SB) and a two-step self-etch Clearfil SE Bond (CS) were used. All adhesive systems were applied strictly according to the manufacturer's instructions [Table 1].

Table 1

Adhesive systems (manufacturers and batch number), composition and application mode*

After hybridization, a composite block (10 mm × 7 mm × 6 mm) (Filtek Z250, EA2, 3M ESPE; St. Paul, MN, USA) was build up in three increments individually light cured for 20 s using a led curing unit (Emitter B, Schuster; Santa Maria, RS, Brazil), with an output of 600 mW/cm2, monitored with a radiometer. All specimens were stored in distilled water at 37°C for 24 h.

Microtensile bond strength (the microtensile bond strength)

Specimens were sectioned in two axes under water-cooled diamond saw in a cutting machine (Labcut 1010, Extec Co; Enfield, CT, USA) by a single blinded operator, obtaining stick shaped specimens (approximately 0.8 mm2) measured individually with a digital caliper (Carbografite; Petrópolis, RJ, Brazil).

Specimens were fixed to metallic devices (Odeme Medical and Dental; Joaçaba, SC, Brazil) with cyanoacrylate glue (Three Bond Super Gel, ThreeBond Ind. E Com. Ltda.; Diadema, SP, Brazil) and submitted by a single and blinded operator to microtensile test in a universal testing machine (EMIC; Sao José dos Pinhais, PR, Brazil) at a crosshead speed of 1 mm/min until fracture. Specimens that failed before the test, were recorded as pretesting failures (PTF). For PTF, a mean between zero and the lower bond strength obtained was calculated, and hence, a value of 4 MPa was attributed and included in the bond strength means.[4] All fractured specimens were observed under ×40 stereoscope (Discovery. v20, Zeiss; Gottingen, Germany) to identify and classify the type of failure as adhesive/mixed or cohesive (dentin or resin).

Statistical analysis

For the analysis, the tooth was the experimental unit, and hence, the mean values of all specimens from each tooth were calculated, and the average value of the 7 teeth was calculated. The sample size was previously estimated considering a coefficient of variation of 20% and a power of 80%. Three factors were considered for analysis: adhesive system (AB, PB, SU, SB, and CS), bond strategy (ER and SE), and dentin condition. Normality of data was confirmed using Kolmogorov–Smirnov test. Three-way analysis of variance and a post hoc Tukey's test were performed to compare bond strength values at a significance level of 0.05. Statistical analysis was performed using a statistical software package (Minitab, Minitab Inc.; State College, PA, USA).

RESULTS

All the microtensile bond strength (μTBS) data are presented in Table 2. Statistically significant cross-interaction among the three main factors was found (P = 0.029).

Table 2

Bond strength mean in MPa (standard deviation) (tested sps/premature failures) to each experimental group

In general, all adhesive systems assessed used in ER strategy produced higher values in sound dentin compared to artificially CAD. For SE strategy, the μTBS values were similar between sound and artificially CAD, except those obtained with SU, which were higher in sound dentin.

All multi-mode systems presented similar μTBS values, regardless of the etching strategy in each substrate condition. The same was not valid for adhesive systems considered as controls, as SB showed higher values than CS in sound dentin.

Adhesive/mixed failure pattern was predominant for all experimental groups [Figure 1]. Cohesive failures (resin or dentin) seemed to be more frequent in sound dentin.

Figure 1

Fracture type distribution per experimental groups and substrates (sound– SND and caries affected dentin-DEM)

DISCUSSION

According to the obtained results, the versatility concept of multi-mode adhesives that allows the clinicians to choose either etch-and-rinse or a self-etch protocol seems to be accurate, considering each dentin condition separately. Nonetheless, bond strength values for all adhesive systems tested in the etch-and-rinse strategy were significantly higher in sound dentin when compared to artificially induced CAD. These findings indicate different interaction patterns in each substrate since sound and demineralized dentin present differences in structural characteristics. CAD is more porous than sound dentin due to the mineral loss in the demineralization process.[9] The thicker hybrid layer has been found in demineralized dentin,[10] which suggests that the increase in porosity allows a great diffusion of primers and monomers.

For MMAS, the etching strategy did not affect bond strength values considering each substrate condition separately. Other studies have also found no differences on immediate bonding of universal adhesive systems using etch-and-rinse and self-etch protocols in sound dentin.[126] Similarly, the etching strategy did not influence on bond strength to artificially induced CAD, as observed previously in artificially induced CAD in the primary[1215] and permanent teeth.[12]

The study findings are partially in accordance with previous studies[91116] that found the improved bond strength of adhesive systems to sound dentin. Higher bond strength values were found to sound dentin when compared to artificially induced CAD, only when MMAS were used in etch-and-rinse mode. The aggressiveness of etch-and-rinse strategy in an artificially induced CAD may produce a thicker layer of demineralization[10] and probably incomplete infiltration by resin components.[16]

Multi-mode adhesives used in the self-etch mode were not influenced by substrate condition since almost all systems presented similar bond strength values. Only the SU produced higher bond strength values in SE strategy in sound dentin compared to artificially induced CAD. CS, AB and PB (both in SE strategy) presented similar bond strength on both substrates. The performance of these adhesives in both substrates assent with other studies,[1115] that showed no differences on sound and artificially CAD for self-etch adhesives. Moreover, the less acidity of self-etch adhesives when applied on CAD may reduce the depth difference between conditioned area and monomer infiltration.[15]

The composition of multi-mode adhesives may play an important role in bond strength values. A recent systematic review revealed that acidic monomers of multi-mode adhesives might have an influence on bond strength values.[7] Rosa et al.,[7] found that ER strategy enhances the performance of ultra-mild adhesives. A reasonable explanation for that behavior is that ultra-mild adhesives in SE approach could not be able to properly conditioning and preparing the dentin substrate.[7] However, there were no significant differences between strategies, supporting the main purpose of MMAS: Versatility to be used in both strategies without compromising the performance, at least in immediate evaluation.

Considering the adhesives used as controls, SB performed better than CS on the sound dentin. Other studies[1718] already showed such behavior for both adhesive systems on the sound substrate. A possible explanation for the decrease the performance of self-etch gold standard is the fact that this was the only adhesive tested which manufacturer does not recommend the active application. Studies[1920] revealed that active application enhances immediate and long-term bond strength of self-etch adhesives. The fact that CS is available on the market for a long time could explain manufacturer's instruction without active application. However, this issue does not impair the clinical performance of CS, as the clinical effectiveness of CS on noncarious cervical lesions remained excellent even after 13 years of follow-up.[21]

The lower mineral content of artificially induced CAD makes them similar to natural CAD, a clinically relevant substrate. Several studies using a similar methodology to create artificially CAD found that this substrate present demineralization on intertubular dentin and an opening of dentinal tubules.[22] These findings support the use of the pH-cycling method to induce artificially CAD for testing the adhesive interfaces.[23] In addition, a study[24] that investigated an artificially CAD model for in vitro bonding studies reported that the μTBS values to CAD were lower than those obtained for sound dentin, but were not influenced by the type of caries. These findings support the use of pH cycling as a suitable methodology to induce artificially CAD avoiding the great variability of natural caries.

Although the pH-cycling model for artificial caries induction is supported on the literature, we should consider that, for in vitro studies, it is not possible to simulate all the physiological changes that occur in a clinical situation, such as the response of dentin-pulp complex like dentin sclerosis and tertiary dentin formation. It is likely that these pulp defense responses cause micromorphological changes in dentin, and their effect on adhesion establishment cannot be completely assessed in an in vitro study design.

Bovine teeth are considered a viable substrate for adhesive in vitro investigations. A recent systematic review with meta-analysis[25] supported the use of bovine teeth on bond strength studies of adhesive systems. Thereby, the use of bovine teeth for in vitro studies as an alternative to human teeth seems advantageous, since bovine teeth are obtained easily and aplenty, unlike human teeth.

Considering obtained results, we accepted the first null hypothesis, since adhesive systems showed similar performance for both strategies on each substrate condition individually. The second null hypothesis was rejected since the bond strength of the MMAS were influenced by substrate condition when applied in etch-and-rinse mode. Long-term studies are necessary and already are being developed by our study group to investigate the bond strength behavior of these multi-mode adhesives on different substrate condition.

CONCLUSION

The etching strategy does not influence the bond strength of multi-mode adhesives to sound or artificially CAD, considering each substrate separately. The dentin condition has a great influence on the immediate performance of multi-mode adhesives applied in etch-and-rinse mode– artificially induced CAD presented lower values than sound dentin.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.