Resin-dentin bond stability of etch-and-rinse adhesive systems with different concentrations of MMP inhibitor GM1489

Abstract Enzymatic degradation of the hybrid layer can be accelerated by the activation of dentin metalloproteinases (MMP) during the bonding procedure. MMP inhibitors may be used to contain this process. Objective To evaluate the degree of conversion (DC%), dentin bond strength (µTBS) (immediate and after 1 year of storage in water), and nanoleakage of an experimental (EXP) and a commercial (SB) adhesive system, containing different concentrations of the MMP inhibitor GM1489: 0, 1 µM, 5 µM and 10 µM. Methodology DC% was evaluated by FT-IR spectroscopy. Dentin bond strength was evaluated by µTBS test. Half of beams were submitted to the µTBS test after 24 h and the other half, after storage for 1 year. From each tooth and storage time, 2 beams were reserved for nanoleakage testing. Data were analyzed using ANOVA and Tukey’s test to compare means (α=0.05). Results All adhesive systems maintained the µTBS after 1 year of storage. Groups with higher concentrations of inhibitor (5 µM and 10 µM) showed higher µTBS values than groups without inhibitor or with 1 µM. The nanoleakage values of all groups showed no increase after 1 year of storage and values were similar for SB and EXP groups, in both storage periods. The inhibitor did not affect the DC% of the EXP groups, but the SB5 and SB10 groups showed higher DC% values than those of SB0 and SB1. Conclusions The incorporation of GM1489 in the adhesive systems had no detrimental effect on DC%. The concentrations of 5 µM GM1489 for SB and 5 µM or 10 µM for EXP provided higher μTBS than groups without GM1489, in the evaluation after 1 year of storage; whereas the concentration of inhibitor did not affect adhesive systems nanoleakage.


Introduction
Advances in adhesive Dentistry have led to increased immediate bond strength of resin composites to dentin, but the resin-dentin bonds are not as durable as resin-enamel bonds. Degradation of the hybrid layer may occur at various levels and stages, 1,2 as well as degradation of the unprotected collagen fibrils 2 because of its incomplete permeation by dentin adhesive or by elution of unreacted monomers and oligomers. These unprotected fibrils are prone to proteolytic degradation by metalloproteinases (MMP), 3 which are a family of endogenous proteolytic enzymes capable of degrading all components of the extracellular matrix. 4 The MMP 1,2,3,8,9 and 20 have been identified in dentin and saliva. However, the collagenase MMP 8 and gelatinases MMP 2 and 9 have been identified as being key enzymes in the process of degradation of the collagen matrix in dentin. 5 The MMP present in dentin are inactive and can be activated by changes in pH during caries lesion progression 5 or in adhesive protocol. 6,7 Other proteolytic enzymes that act on dentin are the cysteines-cathepsins, which seem to act on caries progression and degradation of the hybrid layer. 8,9 Possibly, cysteines-cathepsins and MMP act synergistically in an enzymatic cascade of degradation of the collagen matrix. 8 Some studies have shown that MMP inhibitors such as Chlorhexidine, Batimastat, Galardin, and EDTA can improve the integrity and stability of the resindentin bond when used as a dentin pretreatment, before resin infiltration. 4,10,11 However, the results regarding the use of MMP inhibitors in dentin bonding are still controversial and in few studies, the inhibitor was incorporated into the adhesive system. [12][13][14] Chlorhexidine is the most frequently investigated MMP inhibitor, and it is capable of reducing dentinresin degradation when added to experimental 14,15 and commercial 16-18 adhesive systems, in addition to decreasing the gelatinolytic activity of MMP. 18,19 However, this inhibitor is susceptible to leaching in a short period of time, which interrupts the inactivation of the MMP, promoting degradation of the exposed collagen fibrils at the adhesive interface and decreasing the resin-dentin bond strength. 20,21 Other MMP inhibitors such as Batimastat and Galardin have been incorporated into adhesive systems. 13,14,22 In previous study, Galardin and Batimastat were able to inhibit the MMP of dentin, but they were not capable of maintaining the µTBS after three months of storage. 13 On the other hand, another study showed that Batimastat, Chlorhexidine and GM1489, in experimental adhesive, were capable of maintaining the µTBS after 12 months of storage, different from Control and Galardin; 14 Batimastat and GM1489 also maintained resin-dentin bond stability after 12 months for superficial and deep dentin, different from Chlorhexidine and the control group. 22 Another broad-spectrum synthetic MMP inhibitor, , which has inhibitory action on MMP 1,2,3,8 and 9 have been used in the medical field. 23,24 It has shown promising results regarding resin-dentin bonding stability when added to experimental and commercial total etch adhesive system. 14,22 However, there is still little information available about this inhibitor.
Therefore, this study aims to evaluate the influence of different GM1489 concentrations (0, 1 µM, 5 µM and 10 µM) on the stability of bond strength to dentin, nanoleakage and degree of conversion of commercial and experimental adhesive systems. The hypotheses tested were: 1) higher concentrations of GM1489 could preserve the dentin bond strength after 1 year of storage, 2) higher concentrations of GM1489 could reduce the nanoleakage at the adhesive interface, and 3) higher concentrations of GM1489 could not affect the degree of conversion of adhesive systems.

Methodology
Synthesis of the experimental adhesive systems The experimental adhesive system (EXP) was formulated as in previous study, 14  of GM1489 in each adhesive system, they were homogenized at 2400 rpm for 2 min. 14 Figure 1 shows the tested groups.  da Silva, et al. 14 (2015). The occlusal dentin of the teeth was exposed using a cut machine (IsoMet 1000, Buëhler, Lake Bluff, IL, USA) and the peripheral enamel was removed using a diamond bur (#4138, KG Sorensen, Cotia, SP, Brazil). The smear layer of dentin was standardized with 600-grit SiC papers (Arotec, Cotia, SP, Brazil) in politriz (DPU 10, Struers, Denmark) for 1 minute. After preparation of the dentin surfaces, the teeth were divided into eight groups (n=6) according to the adhesive system tested Nanoleakage After storing (immediate or 1 year), two beams of each tooth were prepared for the nanoleakage test as previously described. 22 The beams received two layers of nail varnish up to 1 mm from the bonding interface on both sides and were individually immersed in 50

Degree of conversion (DC%)
Increments of each adhesive system were inserted into a Teflon mold (0.785 mm 3 ) positioned onto a crystal, using attenuated total reflection mode of the FT-IR spectrometer (Alpha-P/Platinum ATR Module, Bruker Optics GmbH, Ettlingen, Germany) and the spectra between 1600 and 1800 cm -1 were recorded with the spectrometer operating with 40 scans, at a resolution of 4 cm -1 . 14 Afterwards, the increments were light-cured for 20 seconds with an irradiance of 650 mW/cm² (DEMI, Kerr Corporation, Middleton, WI, USA) and the spectra were recorded exactly as it was performed for the unpolymerized increments. Each adhesive system was evaluated in triplicate (n=3). The DC% was estimated from the ratio between the integrated area of absorption bands of the aliphatic C=C bond (1638 cm -1 ) to that of the C=O bond (1720 cm -1 ), used as an internal standard, which were obtained from the polymerized and unpolymerized increments, 14 using the following equation: where R = integrated area at 1638 cm -1 / integrated area at 1720 cm -1

Statistical analysis
The obtained data were analyzed using Statgraphics Centurion XVI software (STATPOINT Technologies Inc, Warrenton, VA, USA). Initially, the normal distribution of errors and homogeneity of data variances were checked using Shapiro-Wilk's and Levene's test, respectively. 14,22 Based on these preliminary analyses, the DC% was evaluated by two-way ANOVA (GM1489 concentration and adhesive system) and Tukey's HSD post hoc test. Nanoleakage and μTBS data were analyzed using three-way ANOVA (GM1489 concentration, adhesive system and storage time) and Tukey's HSD post hoc test for multiple comparisons.
The analyses were performed at a significance level of 5%.

Results
The µTBS results are shown in Table 1      The results of DC% are presented in Figure 6. Two-way ANOVA detected a statistical significance for the independent factors adhesive (p=0.0000) and inhibitor concentration (p=0.0000) as well as for the interaction between these two factors (p=0.0022).
The incorporation of the inhibitor GM 1489 did not affect the DC% of experimental adhesive systems.
Whereas, the addition of 5 µM or 10 µM of GM1489 to SB showed significant increase in DC%.

Discussion
In this study, a simple formulation of an adhesive blend was used based on that by da Silva, et al. 14 (2015). This occurred because the commercial adhesive systems available did not detail the promoter and as demineralizing monomer. 25 By reacting with water, 4-META monomer is hydrolyzed to 4-MET, which is able to establish an ionic bonds with Ca 2+ in hydroxyapatite. [26][27][28] This chemical interaction during the formation of the hybrid layer may improve the durability of the adhesive restorations. 1 GM1489 was chosen because it is a MMP inhibitor of broad spectrum that has not been extensively studied in Dentistry. Also, in a previous study, this inhibitor has maintained the bond of an experimental adhesive system to dentin after 12 months of storage when used in the 5 µM concentration. 14 GM 1489 is an acetohydroxamic acid that contains the critical metal ligand group and a complex heterocyclic structure, which could favor its chelation potential. 14,22 Similarly to Galardin, the GM1489 can bind to the active site of MMP, chelating the zinc ion that is located in the catalytic domain of MMP. 19 GM1489 presents the following in vitro inhibitory constants (K i ): MMP 1=0.2 nM, MMP 2=500 nM, MMP 3=20 µM, MMP 8=100 nM, and MMP 9=100 nM. Therefore, it was reasonable to claim that lower concentrations of GM1489 could inhibit the activity of MMP 2, 8 and 9, thereby preventing the degradation of hybrid layer over time.
This was the reason for testing the 1 µM concentration in this study. The concentration of 5µM was based on the results by da Silva, et al. 14 (2015) and the higher 10 µM concentration as a function of the inhibitory constant for MMP3. The commercially available adhesive system (SB) was used to evaluate whether a different adhesive composition would influence the effectiveness of GM1489 on dentin bond stability.
Indeed, when 10 µM GM1489 was used with the experimental adhesive system, the µTBS values were the highest in both time intervals of evaluation, but without significant difference from 5 µM. The initial  µTBS was also increased by use of 5 µM and 10 µM of GM1489 for the experimental adhesive system, but the same did not occur with SB. On the other hand, for SB the greatest µTBS after 1 year of storage occurred when 5 µM GM1489 was used, but without significant difference from 1 µM and 10 µM. Thus, the second research hypothesis that higher concentrations of GM1489 would preserve the bond strength to dentin after 1 year of storage was partially accepted. These results showed that the composition of adhesive systems could influence the optimal concentration of GM1489 required to improve the dentin bond strength.
Moreover, the µTBS did not decrease after 1 year of storage, for all groups. This may indicate that longer time of storage may be required to highlight the effect of GM1489 on resin-dentin bond preservation, although its use has caused increase of dentin µTBS.
In the immediate µTBS test, adhesive failures were predominant, which could indicate the reliability of the test and demonstrate that the bond interface had In general, for polymer-based restorative materials, a high degree of conversion is the first step for the development of clinically-welcomed physicomechanical properties. 29,30 Specifically for adhesive systems, this property is directly related to the efficacy of the bond to dentin. 31 For example, if an adhesive polymer presents a poor degree of conversion, unreacted monomers in the hybrid layer may leach out over time, thereby creating porosity in its structure that may increase its permeability. This plethora of phenomena favor the hybrid layer degradation, reducing its sealing ability, which might jeopardize the service life of adhesive restorations. 3,32 In the present study, the DC% of experimental adhesives ranged from 98.32% to 99.34%, values that nicely agree with previous studies evaluating commercially available and experimental adhesive systems. [33][34][35] Most probably, these high values of DC% were influenced by the chemical structure of the monomers used in the adhesive formulations tested here. First, TEGDMA is an aliphatic monomer with high flexibility that increases the adhesive system reactivity. 35 Second, the "solvent-like" behavior of HEMA may allow a reduction in the adhesive blend viscosity, favoring its reaction with the C=C bonds of long chains even after these are entrapped into the polymer network. 36 The DC% of SB was statistically lower than those of the experimental adhesives ( Figure   6). This result may be explained by the absence of TEGDMA and by the lower concentration of HEMA (5-15%) in SB composition, which could have affected its viscosity and, consequently, its DC%. Different from the experimental adhesive systems, in which the incorporation of GM1489 had no influence on DC%, for SB, the formulations with 5 µM and 10 µM GM1489 presented statistically higher DC% ( Figure 6). As GM1489 has no polymerizable groups in its structure it was hypothesized that, in these SB formulations, GM1489 could have acted as a spacer, increasing the distance between the monomer and polymer chains during the polymerization reaction. This behavior could have slightly increased the gelation phase of polymerization, thereby allowing more mobility to the terminal C=C bonds to find new polymerizable groups, 37 positively affecting the DC%. These findings led to the partial acceptance of the third research hypothesis established for the present study.
The second research hypothesis that higher concentrations of GM1489 would be able to reduce the nanoleakage at the interface of adhesive systems was rejected since the nanoleakage results showed no differences among groups for SB and for EXP, in both evaluation times (immediate and after 1 year of storage). Although the nanoleakage values were higher after 1 year of storage, a significant increase in nanoleakage was observed for EXP0 only. However, a trend towards decrease in nanoleakage after 1 year of storage was observed when 10 µM of the inhibitor was used, for both adhesives ( Figure 5).
These nanoleakage results could be correlated with the DC%, since SB showed higher DC% with 5 µM or 10 µM of inhibitor. High DC% of adhesive systems can contribute to the stability of the resin-dentin bond and lower nanoleakage expression. 29, 38,39 As was done in the present study, some authors incorporated the MMPs inhibitors into the adhesive system to evaluate their properties, such as µTBS and micropermeability/nanoleakage. [13][14][15]17 All studies showed a trend towards conservation of the hybrid layer in groups with incorporation of the MMP inhibitors. 2020;28:e20190499 8/10 The study of Silva, et al. 14 (2015) was the first about the use of GM1489 in dentistry, and as was shown in this study, they demonstrated promising results for this inhibitor, which maintained the µTBS stability after 1 year of water storage (similar to chlorhexidine and BB94) and showed a clinically acceptable degree of conversion and lower water sorption than the commercial control without the inhibitor.

Conclusions
Within the limitations of this study, it could be concluded that 5 µM or 10 µM GM1489 concentrations for experimental adhesive and 5 µM for commercial adhesive should be the choice for the improvement of dentin bonding. Moreover, the DC% of adhesive systems and the nanoleakage were not jeopardized by GM1489.