Introduction

Poor penetration of simplified adhesive systems within the collagen layer and a hydrolytic degradation of the monomers can make the collagen exposed to enzymatic degradation ^1,2,3,4, which may cause margin discoloration, microleakage and secondary caries ³.

Various alternatives have been applied to improve the bonding interface longevity: insertion of multi-layered adhesive ⁵, a device that improves adhesive infiltration into the collagen layer ⁶; longer application and polymerization times ⁽⁵, which modify application of the protocol, by introducing additional steps leading to possible mistakes and increasing chair time.

On the other hand, some researches advocate that an increase in the adhesive system mechanical properties improves the quality of bonded interfaces ⁷. Therefore, some studies added colloidal silica particles ⁷ and barium borosilicate to the adhesive matrix ³. In addition to encouraging such interfaces to act as filler reinforcement, it would also be interesting that these particles induce mineral deposits on the interfaces and protect exposed collagen ⁸. Therefore, there is growing interest in the development of experimental materials associated with bioactive glass (45S5) that induces adhesive interface re-mineralization by releasing phosphate and calcium ions ^{1,9,10,11,12,13}. An ion release leads to an increase in pH, which may would cause inhibition of bacterial growth and action of enzymes that degrade collagen ^8,14,15.

The silicate glass has low chemical stability, which leads to high ion release in short periods, which can be solved by replacing Si with Nb ^16,17. In some materials, the presence of Nb increases the biocompatibility ⁽¹⁶, radiopacity ² and chemical stability of bioactive glass ⁽¹⁸. Recent studies have shown promising results in the deposition of ions at the dentin interface ⁽⁴ and the formation of 'bioactive smear layers' ⁽¹⁷.

The incorporation of bioactive particles into experimental adhesives is reported to be a promising option to induce re-mineralization ^10,11,12, since it would not require extra steps to insert the bioactive glass into adhesive layer. The formulation of a new adhesive seems to be another challenge, because factors, such as pH of the mixture and proportion of acid monomers is very problematic ¹². No one could demonstrate a consensus for the best formulation, or ever used a commercial adhesive without inorganic particles to insert bioactive ones.

Besides, the literature advocates particle silanization because the presence of silane produces interaction between inorganic and organic matrixes and uniform stress distribution ¹⁹. Silanization prevents cluster formation in the adhesive layer and compromise of the cohesive strength of adhesive systems ¹⁹. This is why this study included an experimental group with silanized NbG micro-filler.

The objective of this study was to characterize the bioactive glass (NbG) and evaluate the impact of adding NbG microfiller particles, with and without silane, on the degrees of conversion and microtensile bond strength, by using two different etch-and-rinse adhesive systems. The tested null hypothesis was that the inclusion of NbG in the composition of the commercial adhesives would induce: (i) no effect on the degrees of conversion and (ii) no effect on microtensile bond strength.

Material and Methods

Degree of Conversion

The DC was evaluated with infrared attenuated by total Fourier spectroscopy reflection (FTIR/ATR, Nexus 470, Nicolet, Thermo Scientific, Waltham, MA, USA). Two drops of each adhesive were applied on the spectrum reader. The first spectrum reading obtained by the transmission method (32 scans with a 4 cm⁻¹ resolution) was taken 10 s later. A new readout was taken after 20 s of specimen polymerization (Optilux 501, Kerr Dental, Kerr Corp., Orange, CA, USA) at 600 mW/cm².

The ratio between unpolymerized adhesive absorption bands of carbon double bonds - aliphatic (1639 cm⁻¹⁾ and aromatic (1609 cm⁻¹⁾ - is used to calculate degrees of conversion.

Percentage calculation formula ³:

Microtensile Bond Strength

Forty eight freshly extracted human third molars were studied in accordance with the local Research Ethics Committee rules (No. 27 777/CAE 13264413.9.0000.5086). The exposed dentin surface was obtained from horizontal sections of the occlusal enamel layer, by using a constantly cooled cutting machine (Isomet 1000, Buehler, Lake Bluff, IL, USA). Next, the surface was sanded for 60 s with #120 silicon paper. Dentin surfaces were magnified 40× with an optical microscope to assess the absence of enamel.

The dentin surface was etched with 37% H₃PO₄ (15 s), rinsed for 15 s, and kept the dentin slightly moist, so that all adhesives were applied on the moist substrate ²¹; two layers of the adhesive system were applied, and soft air jets were blown on the samples for 10 s, followed by polymerization (Optilux 501, Kerr, Orange, CA, USA) at 600 mW/cm² for 20 s. Table 1 shows the application mode, composition, manufacturer and lot number of the used materials.

Table 1 Material, manufacturer, composition and application mode of adhesive systems* 

*Composition provided by the manufacturers. Bis-GMA: Bisphenol A-glycidyl methacrylate; BPDM: biphenyldicarboxylic-acid dimethacrylate; HEMA: 2-hydroxyethyl methacrylate; PENTA: Dipentaerythritol penta acrylate monophosphate.

After the adhesive procedure, a 6.0 mm high composite resin restoration (Lis, FGM, Joinville, SC, Brazil) was performed in three 2-mm increments of and light polymerized for 40 s.

After mounting the tooth in the cutting machine device (Isomet 1000), the bond interface was placed perpendicularly to the blade. Two sequences of longitudinal and perpendicular cross sections were cut to obtain specimens of approximately 0.8 mm². The cross-sectional area was measured and recorded using digital calipers (Absolute Digimatic, Mitutoyo, Tokyo, Japan). After this, the specimens were randomly divided into two groups that were tested at different time intervals: immediately after preparation (24 h) and after 6 months of storage at 37 °C.

Each specimen was fixed with cyanoacrylate gel glue to a Geraldeli testing jig (Odeme, Joaçaba, SC, Brazil), and then coupled to a universal testing machine (Instron 3342, Norwood, MA, USA). Tests were performed at 1.0 mm/min and the results were expressed in MPa.

Results

Characterization of NbG

Particle size analyses indicated that most obtained micro-fillers were between 0.1 and 100 μm (Fig. 1). The average size obtained was 10.42 μm (Fig. 1A). Figure 1B shows a different size of filler, some smaller than 10 μm and nanometric size.

Figure 1 (A) NbG particle size analysis. The Silas device found micro-filler between 0.1 to 100 μm and average size was 10.42 μm. (B) SEM showed images of NbG micro-filler smaller than 10 μm. 

Degree of Conversion

The DC results for all adhesive systems are presented in Table 2. There was no significant difference between the control groups and the experimental groups (p>0.05).

Table 2 Mean and standard deviations of degree conversion (%) and microtensile bond strength values (MPa) obtained for the different experimental groups 

Total number of beams (tested stick/pre-load failure) and percentage distribution of failure mode after microtensile bond strength testing [Failure % AM/CD/CR]. Different letters after mean values represent statistically significant differences between groups (p<0.05).

Microtensile Bond Strength

The average cross-sectional area of specimens ranged from 0.79 to 0.83 mm². The statistical analysis found no interaction between the main factors (p=0.988). Table 2 shows microtensile bond strength values of adhesive systems tested in the different time intervals (immediately and after 6 months). The addition of NbG micro-fillers did not affect bond strength of the tested adhesives (p<0.05). Bond strength values were reduced in all the tested adhesives (p<0.05) over six months.

The fracture pattern shows a prevalence of adhesive-mixed fractures for all groups that were tested (Table 2). Figures 2A and 2B reveal adhesive-joint fractures for OS and PB control groups where exposure of dentinal tubules and adhesive layer area is seen.

Figure 2 Representative specimen of control group: SEM evaluation of adhesive/mixed fracture of commercial adhesive systems (A) One Step (Immediate) (B) Prime & Bond 2.1 (Six months). Both had extensive areas of exposed dentinal tubules. 

Figure 3 (A, B and C) shows fractures between the adhesive and composite resin of the OSNbG group, where considerable particle dispersal in the resin matrix of the adhesive material was identified. There were some larger micro-filler particles (white arrow 3C) and in some areas, free spaces revealed the NbG pull-out load (white arrow 3B).

Figure 3 Representative specimen of OSNbG group: Immediately debonded specimens. (A) SEM evaluation of mixed fracture between composite resin and adhesive system. (B) Same image denotes NbG micro-filler size diversity: some>±10 μm distributed in the adhesive layer with particles pulled out of the resin matrix (white arrow). (C) Aggregates formed by possibly smaller particles incorporated into the resin matrix (black arrow); larger particles loose in the adhesive layer (white arrow). 

Figure 4 (A, B and C) depicts micro-fillers dispersed in the resin matrix of the PBNbGS group adhesive layer; the lower amount of micro-filler dispersed in both the matrix and at the entrance of the dentinal tubules.

Figure 4 Representative specimen of PBNbGS group after six months: (A) SEM evaluation of mixed fracture with silanized NbG, exposure of dentinal tubules (white arrow) and composite resin (black arrow). (B) Large volume of micro-filler distributed in the adhesive layer. (C) Micro-filler at the dentinal tubule entrances. 

Discussion

addition of bioactive glass micro-filler in the adhesives may contribute to release of Ca²⁺ and PO₄ ^-3 ions, and provide the re-mineralization of unprotected collagen ^11,12. The presence of bioactive glass in an adhesive systems may contribute to increase the pH ², thus inhibiting bacterial growth and blocking MMP action ^3,15.

In this study, an experimental bioactive glass with Nb was used ¹⁶. Tamai et al. ¹³ showed that Nb-based bioactive glass is able to increase the activity of alkaline phosphatase and promote osseointegration. The amount of calcified tissue was directly proportional to the concentration of dissolved Nb ions ¹³. The NbG stimulated bone growth in the tibia of rabbits demonstrating high bioactivity ⁽¹⁶. Carvalho et al. ⁴ recently demonstrated that experimental gutta-percha containing NbG micro-fillers was able to deposit precipitates along the root dentin interface that increased bond strength of 'bioactive gutta', showing the bioactivity of this new material ⁴. Another important clinical advantage of NbG added to adhesive systems is the possibility of obtaining a radiopaque material. A similar study demonstrated that with this concentration (40%w/w), it was possible to obtain radiopacity, making it possible to distinguish the restorative material from enamel and dentin, in addition to allowing the diagnosis of caries lesions ²².

In this study, the DC values found for the two control groups (PB and OS) were in agreement with values in the literature ^21,23. Leitune et al. ² found no difference in the DC when incorporating Nb₂O micro-filler into an experimental adhesive system. Similarly, the addition of bioglass (45S5) did not affect the DC of composite material ¹⁵.

On the other hand, a high ionic release of NbG may somehow neutralize the action of acidic monomers (BPDM and PENTA) and decrease the conversion of monomers. Reduced DCs make adhesive layers more permeable and susceptible to degradation ⁷. However, the addition of NbG did not affect the DCs of PB and OS adhesive systems, which is a favorable result, as it could compromise mechanical properties. Leitune et al. relate that when niobium pentoxide was incorporated to experimental adhesives, a slight increase in the reaction rate of the DC was observed ², which suggests that the presence of NbG could compensate any decrease of DC. Thus, the null hypothesis of this study was accepted (i).

The incorporation of glass into the tested adhesive systems was unable to prevent the reduction in bond strength after 6 months of storage. A reduction in μTBS values was observed in all groups, thus the null hypothesis was rejected (ii).

However, comparing the microtensile values in the groups in which the particles were silanized, it is possible to observe that the values were not changed. This could be confirmed by the fracture mode, from which it was possible to observe that adhesive-mixed layer prevailed in all groups.

The SEM images revealed that NbG-added glass groups clearly showed cohesive cracks in the adhesive system layer (Fig. 3). An ultimate tensile strength test should have been performed to evaluate the effect of the addition of bioactive filler particles on the cohesive strength of adhesive systems, however with the PB adhesive system, it was impossible to obtain specimens for this test, because after polymerization, the hour-glass shaped specimens showed moisture and softness.

Other studies have also not been successful with the incorporation of bioglass (45S5) into adhesive systems, as the presence of a bioactive glass in dental adhesives did not prevent the decrease in bond strength after 3 months ^11,12. Profeta ¹⁰ et al. observed maintenance of µTBS values after 6 months, but this was found in the group that used an adhesive and added 45S5 associated with the application of 45S5 solution to rewet the dentine ⁽¹⁰. It was also hypothesized that bioactive micro-filler particles may have reacted with water to release calcium hydroxide that increased the alkalinity of the surrounding environment and the alkaline pH may have slowed the activity of MMPs.

Moreover, adhesive systems containing 45S5 glass are capable of increasing nanohardness and the modulus of elasticity of the adhesive interface, suggesting dentin re-mineralization ⁽¹². Some studies detected mineral crystals deposited in the adhesive interface layer, which reduced nanoleakage ^(10,12.

In this study, it was possible to detect bioactive NbG nano-fillers in the dentinal tubule entrances (Fig. 4). However, further studies are still required to confirm the influence of NbG nano-filler deposition within the hybrid layer by another test.

At this time, many researchers are looking for adhesive material able to re-mineralize hybrid layers and completely restore the mechanical properties of mineral-depleted dental collagen structures within resin-bonded interfaces by biomimetic apatite formation. The greatest challenge of these researchers is to insert demineralized bioactive glasses smaller than 40 nm between collagen fibrils, since this is the size required to fit into this network ²⁴. However, the presence of the NbG micro-filler next to the collagen network may provide ions (Ca²⁺ and PO₄ ^-3) to inhibit enzymatic degradation, bacterial growth and re-mineralize collagen.

The incorporation of experimental NbG into commercial adhesives at 40% concentration maintained the DC values unaltered. However, it was unable to prevent a reduction in bonding strength after 6 months.