Experimental etch-and-rinse adhesive systems containing MMP-inhibitors: Physicochemical characterization and resin-dentin bonding stability
Abstract
Objectives: To evaluate the degree of conversion (DC%), water sorption (WS), solubility (SO) and the resin– dentin bonding stability (mTBS) of experimental (EXP) etch-and-rinse adhesive systems containing MMP-inhibitors: Galardin-GAL, Batimastat-BAT, GM1489-GM1 and chlorhexidine diacetate-CHX. Methods: DC% was measured using FT-IR spectroscopy, while WS and SO were calculated based on ISO4049. Thirty-six human molars were wet ground until the occlusal dentin was exposed. The adhesive systems were applied and resin composite buildups were incrementally constructed. After 24 h immersion in distilled water at 37 ◦C, the specimens were cut into resin-dentin beams with a cross-
sectional area of 1 mm2. The mTBS was evaluated after 24 h, 6 months and 12 months of water storage at 37 ◦C. Adper Single Bond 2 (SB2) was used as a commercial control. The data were analyzed using ANOVA and Tukey’s HSD test.
Results: SB2 presented the highest DC% (p < 0.05). CHX presented the lowest WS (p < 0.05). GM1, GAL and BAT presented similar WS when compared with SB2 (p > 0.05). SO was found to be not significant (p > 0.05). All adhesive systems maintained mTBS stability after 6 months of water storage. Only BAT, GM1 and CHX maintained mTBs stability after 12 months of water storage.
Conclusions: The experimental adhesive systems with GM1489 and chlorhexidine diacetate presented the best physicochemical properties and preserved resin–dentin bonding stability after 12 months of water storage.
Clinical significance: GM1489 could be suitable for inclusion as an MMP-inhibitor in etch-and-rinse adhesive systems to maintain resin-dentin bonding stability over time.
1. Introduction
The quality of the hybrid layer is crucial for the long-term stability of resin-dentin bonding and the clinical longevity of adhesive restorations involving dentin as a substrate [1,2]. This acid-resistant layer is a biocomposite formed from a scaffold of collagen fibrils exposed after acid conditioning, few remaining apatite crystallites and a mix of hydrophilic and hydrophobic methacrylate monomers polymerized in situ [3]. This means that both structures, i.e., collagen fibrils and adhesive polymer, must be resistant to the harsh conditions present in the oral environment [4]. However, vast scientific information shows that resin-dentin bonding obtained with contemporary adhesives systems is prone to degradation over time [5–8].
Previous studies have been shown that, even after polymeriza- tion, adhesive polymers behave as semi-permeable membranes that allow water movement across the adhesive layer [9,10]. Additionally, it is well established that water movement is predominantly influenced by the hydrophilicity of the monomers present in the adhesive systems [11] and by the degree of conversion reached after polymerization [12]. Poor polymerization results in greater permeability of the adhesive polymer and may jeopardize its mechanical properties [13], while also decreasing its bond strength to dentin [14].
On the other hand, both the hydrolytic effect of water and the proteolytic action of the host-derived matrix metalloproteinases (MMP) play a crucial role on the degradation of resin-dentin bonding strength over time [15]. MMPs are endogenous Zn2+ and Ca2+ dependent proteases synthesized during dentinogenesis, and remaining a latent state within the dentin extracellular matrix [16,17]. At least four MMPs are found in human dentin: MMP-8 (collagenase), MMP-2 and -9 (gelatinases), and stromelysis-1 (MMP-3) [16,17]. Scientific studies have shown that dentin demineralization produced by adhesive systems can activate different MMPs [5,18–20], starting a degradation mechanism of collagen fibrils unprotected by the adhesive monomers [7,15,21,22].
In an attempt to overcome this shortcoming, several studies have used MMP-inhibitors; such as Batimastat, benzalkonium chloride, chlorhexidine, doxycycline, EDTA, glutaraldehyde, Galardin, MDPB and epigallocatechin-3 gallate (EGCG) [1,16,21,23–30]; as an additional step for dentin treatment or added to commercially available adhesive systems. However, the results obtained with these strategies are not linear, showing neutral, positive or negative effects on resin-dentin bonding stability. These dissimilar results encourage the development of new studies in this field. Therefore, the purpose of this study was to synthesize experimental etch-and-rinse adhesive systems containing MMP-inhibitors (GM1489, Galardin, Batimastat and chlorhexidine diacetate) and to evaluate their DC%, water sorption, solubility, and resin-dentin bonding stability over a period of twelve months. The null hypotheses tested were: (1) there would be no difference in DC% among adhesives containing different MMP-inhibitors, (2) there would be no difference in water sorption among adhesives containing different MMP- inhibitors, (3) there would be no difference on solubility among adhesives containing different MMP-inhibitors, and (4) there would be no difference on resin-dentin bonding stability over a period of 12 months among adhesives containing different MMP- inhibitors.
2. Materials and methods
2.1. Synthesis of the experimental adhesive systems
Five experimental etch-and-rinse adhesive systems were synthesized. The HEMA, TEGDMA and 4-META (Essthec, Inc., Essington, PA, USA) were used as received. Camphorquinone and ethyl N,N-dimethyl-4aminobenzoato—EDMAB (Aldrich Chemical Company, Inc., Milwaukee, WI, USA) were incorporated as photosensitizer and reducing agent, respectively. All the compo- nents were weighed using an analytical balance (AUW 220D, Shimadzu, Tokyo, Japan) and mixed in a dual centrifuge (150.1 FVZ SpeedMixer DAC, FlackTek Inc., Herrliberg, Germany). The mono- mers were first mixed with acetone and water. Then, the photosensitizer and reducing agent were added and the mixtures centrifuged at 1300 rpm for 1 min. The MMP-inhibitors Galardin (GAL), Batimastat (BAT) and GM1489 (GM1), at a concentration of 5 mM [23], and chlorhexidine diacetate (CHX) at a concentration of 2 wt.% [8], (Table 1) were incorporated in four experimental adhesive. Finally, the mixtures were homogenized at 2400 rpm for 2 min. The experimental (EXP) adhesive system without any MMP inhibitor was used as control and the adhesive system, Adper Single Bond 2 (SB2), was used as a commercial control group. The characteristics of the adhesive systems are depicted in Table 2.All specimens in the present study were light cured using an LED unit (Bluephase, IvoclarVivadent, Schan, Liechtenstein), at an irradiance of 1600 mW/cm2.
2.2. Degree of conversion (DC%)
Increments of each adhesive system were inserted into a teflon mold (0.785 mm3) positioned onto an ATR crystal 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 and at a resolution of 4 cm—1. Afterwards, the increments were light-cured for 20 s and the spectra were recorded exactly as performed for the unpolymerized increments. The DC% was calculated 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,where R = integrated area at 1638 cm—1/integrated area at 1720 cm—1.
2.3. Water sorption and solubility
Before building up the specimens, the solvent from all adhesive systems was allowed to evaporate. The adhesive systems were dispensed into a container (5.0 cm in diameter and 1.0 cm in depth) on an analytical balance with a precision of 0.01 mg (AUW 220D, Shimadzu, Tokyo, Japan), which was protected from ambient light to prevent premature polymerization. The initial mass was recorded and the specimens remained on the analytical balance until reaching mass equilibrium.
Disk-shaped specimens were prepared using an aluminum mold (1 mm thick and 6 mm in diameter) [31]. A micropipette was used to dispense the adhesive systems directly into the mold. After filling the mold to excess, all visible air bubbles were carefully removed using a hypodermic needle. After a polyester strip and glass slide were placed on top of the mold, the disks were light cured for 20 s. The disks were removed from the mold and the discs were light cured from the bottom surface for 20 s. The top and bottom surfaces of all disks were manually polished using 4000- grit SiC abrasive paper (Arotec, Cotia, SP, Brazil) to eliminate any surface irregularities. Six disks were produced for each experi- mental adhesive system and SB2 Water sorption and solubility were determined based on the ISO 4049 Standard (2000) [32], although the specimens’ dimen- sions and periods of immersion were extended until a constant mass was achieved [31].
Immediately after polymerization, the disks were placed in a desiccator containing dehydrated silica gel and transferred to an oven at 37 1 ◦C (Q316B15, Quimis, Rio de Janeiro, Brazil). The disks were weighed daily using an analytical balance with a precision of 0.01 mg (AUW 220D, Shimadzu, Tokyo, Japan) until a constant mass was attained (m1) (three consecutive days with a mass variation less than 0.01 mg).
The thickness and diameter of each disk were measured at four points using a digital caliper (MPI/E-101, Mitutoyo, Tokyo, Japan), and the volume (V) was calculated in mm3. The specimens were then individually placed in sealed glass vials containing 10 mL of distillate water at 37 ◦C. The immersion media were renewed every 7 days to prevent the proliferation of fungi and bacteria. The disks were repeatedly weighed at 24 h intervals until a constant mass was attained (m2) (three consecutive days with a mass variation less than 0.01 mg). Before weighing, the specimens were gently wiped with soft absorbent paper. The specimens were again put in a desiccator containing fresh silica gel, kept in a pre-conditioning oven at 37 ◦C (Q316B15, Quimis, Rio de Janeiro, Brazil), and weighed daily until a constant mass was obtained (m3). Sorption (WS) and solubility (SO) in mg/mm3 were calculated using the following formulae: where m1 is the disk mass (mg) after drying, m2 is the disk mass (mg) at the equilibrium uptake (maximum sorption), m3 is the mass (mg) of the re-dried disk, and V is the disk volume (mm3).
2.4. Microtensile bond strength (mTBS) measurement
Thirty-six extracted, caries free, human third molars (Ethical Committee Approval HUAP 840.168) were disinfected in 0.5% chloramine solution for 7 days, stored in distillated water and used within six months after extraction. The occlusal dentin of the teeth was exposed using a cut machine (IsoMet 1000, Buëhler, Lake Bluff, IL, USA) and the peripheral enamel removed using a diamond bur (#4138, KG Sorensen, Cotia, SP, Brazil). The smear layer of dentin was standardized using 600-grit SiC paper (Arotec, Cotia, SP, Brazil). After preparation of the dentin surfaces, the teeth were divided into six groups (n = 6) according to the adhesive system used (Table 2).
Dentin surfaces were etched with 37% phosphoric acid for 15 s (Condac37, FGM, Joinville, SC, Brazil), rinsed with distillate water for 30 s and blot dried with absorbent paper. The adhesive systems were applied (Table 2) and five increments of 1 mm thick resin composite (TPH3, shade A3, Dentsply, Petrópolis, RJ, Brazil) were horizontally added to the bonded surfaces and individually light cured for 40 s.
After storage in distilled water at 37 ◦C for 24 h, the teeth were longitudinally sectioned in both the mesio-distal and buccal- lingual directions, across the bonded interfaces (IsoMet 1000, Buëhler, Lake Bluff, IL, USA) to obtain beams with a cross-sectional area of approximately 1 mm2. Each tooth provided 18 to 20 beams. These beams were divided into three groups, according to the time of storage in distilled water (37 ◦C): immediate, 6 months and 12 months. After each period of storage, the beams had their adhesive interfaces cross-sectional area measured with a digital caliper (MPI/E-101, Mytutoyo; Tokyo, Japan) and were individually fixed to a microtensile device (ODMT03d, Odeme Biothecnology, Joaçaba, SC, Brazil) using cyanoacrylate glue (Superbonder Gel, 3 M, São Paulo, Brasil) and loaded under tension using a universal testing machine (EMIC DL 2000, São José dos Pinhais, SP, Brazil) at a crosshead speed of 0.5 mm/min until failure occurred. The mTBS (MPa) was obtained by dividing the load at failure (N) by the cross- sectional area of the beam (mm2). Each failed beam was evaluated with a stereomicroscope at 40x magnification (SZ40, Olympus, Tokyo, Japan) and the mode of failure was classified as: adhesive (failures at the adhesive interface), cohesive (failures occurring mainly within dentin or resin composite), or mixed (mixture of adhesive and cohesive failure within the same fractured surface).
2.5. Statistical analysis
The obtained data were analyzed using Statgraphics 5.1 Soft- ware (Manugistics, Rockville, MD, USA). Initially, the normal distribution of errors and the homogeneity of variances of the data were checked using Shapiro-–Wilk’s and Levene’s tests, respec- tively. Based on these preliminary analyses, the DC, water sorption and solubility data were analyzed using one-way ANOVA and Tukey’s HSD post hoc test and the mTBS data were analyzed using two-way ANOVA and Tukey’s HSD post hoc test [33]. The analyses were performed at a significance level of a = 0.05.
3. Results
The results of DC%, WS and SO are summarized in Table 3. With respect to DC%, one-way ANOVA detected statistically significant differences among adhesive systems (p < 0.05), with SB2 presenting the highest DC% (p < 0.05). No differences in DC % were found among the experimental adhesives (p > 0.05). Regarding WS, one-way ANOVA showed a statistical significance among adhesive systems (p < 0.05). The results of Tukey’s HSD test showed that CHX presented the lowest WS, but was not statistically different from GM1. The groups GM1, GAL and BAT presented similar WS to SB2 (p > 0.05). The highest WS was presented by EXP (p < 0.05). No statistical significance was found for solubility (p = 0.2593). 4. Discussion Differently from previous studies, in which MMP-inhibitors were incorporated in commercially available adhesive systems [6,23,30], the present study choose to add these substances to an experimental standard formulation. This was done to analyze the real effects of the MMP-inhibitors, avoiding the influence of unknown ingredients present in commercially available adhesive systems, as well as their contents, on the present results. The experimental adhesive system was based on a simple adhesive with the necessary characteristics to produce a good interaction with dentin. HEMA is a monofunctional high-hydrophilic mono- mer able to act as an adhesion-promotion agent while improving the miscibility between the hydrophobic and hydrophilic compo- nents of the adhesive mixture [2,34]. TEGDMA, a hydrophobic low molecular-weight monomer, was used as cross-linking agent. 4-META was chosen as a functional monomer because it improves the adhesive wettability and establishes a chemical bond with the Ca2+ in dentin, thereby improving the resin–dentin bond strength [2,35–37]. Three of the four inhibitors used in the present study (Table 1) have already proven activity against dentin MMPs. Incorporated inside commercially available adhesive systems, Batimastat, a peptide-like analogue of collagen, has shown effectivity against MMP-1 and-2 and in maintaining bond stability over time [23]. Galardin, a hydroxamic acid analog, presents a collagen-like structure, capable of binding to the MMP active sites, and a hydroxamate domain, which may chelate Zn2+ and inhibit the collagen-catalytic effect of the MMPs [5,23]. Chlorhexidine is the most well documented dentin MMP-inhibitor. It has been demonstrated that, when applied as a pre-conditioner or incorporated into adhesive systems, chlorhexidine can preserve the integrity of the demineralized collagen matrix and reduce the resin–dentin bond strength degradation over time [1,6,8,25–30]. Instead of digluconate, chlorhexidine diacetate was used in the current study because previous studies have shown that it preserved the resin-dentin bonding stability and did not interfere with water sorption, solubility or the degree of conversion of adhesive systems and resin blends [6,29,38,39]. GM 1489, an analog of Galardin, acetohydroxamic acid, is a potent broad-range MMP inhibitor that has also a molecular structure with functional groups capable of combining with specific sites of MMPs (Table 1). Although its molecule contain the critical metal ligand group but without the amino acid side chains necessary for binding to the MMPs, it also presents a more complex heterocyclic structure than Galardin, which could favor its chelation potential. It is possible that compared to other MMP inhibitors used here this structure could increase the MMP-inhibition capacity of this substance. To the best of our knowledge, this is the first time that this MMP- inhibitor is tested in adhesive dentistry field. Since no differences were found in DC% among the experimental adhesive systems, the first null hypothesis was accepted. The values of DC%, ranging from 69.0 to 69.5% (Table 3), agree with other data presented by commercially available adhesive systems [14], and are compatible with this clinical application. It is possible that the fact that all of the MMP-inhibitors used in the current study were powders could have influenced this result. Stanislawc- zuk et al. [6] showed that the addition of chlorhexidine diacetate (powder) in concentrations ranging from 0.01 to 0.2 wt.% did not influence the degree of conversion of commercially available adhesive systems. According to those authors, the low concen- trations of chlorhexidine diacetate powder did not interfere with the well-balanced monomer/solvent ratio of the adhesive systems tested. Moreover, Cadenaro et al. [39] demonstrated that there was no difference in the degree of conversion of resin blends modified with 1 and 5 wt.% of chlorhexidine diacetate. Therefore, the current authors hypothesized that the powders of the MMP-inhibitors did not chemically react with any molecule of the experimental (EXP) adhesive formulations, not interfering with the propagation of the polymerization reaction and remaining entrapped between linear chains after adhesive polymer network formation. Instead of simple physicochemical properties, water sorption and solubility of adhesive systems involve a chain of phenomena that may influence their clinical behavior. Firstly, the polymer chains suffer relaxation due to the swelling caused by the absorbed water. This process induces a reduction in the frictional forces between collateral polymer chains, causing plasticizing of the adhesive polymer network. After that, the non-reacted monomers entrapped in the nanovoids inside the adhesive polymer network can be released to the surrounding medium, creating nanovoids in the material structure. It is well accepted that these phenomena reflect a degradation mechanism of the material, which could weaken the adhesive polymer network and the hybrid layer [11,40,41]. Although it is not easy to directly relate these phenomena to the degradation of the resin-dentin bonding, some scientific evidences can be used to establish a reasonable link between them. Analyzing experimental adhesive systems based on carboxylic monomers, Tanaka et al. [42] showed that more water sorption resulted in a larger decrease of resin-dentin bonding strength. Moreover, Ito et al. [14] indicated that the adhesive systems with higher water sorption presented the lowest values of mTBS. Based on these studies, the current authors submitted the water sorption and mTBS data of the experimental etch-and-rinse adhesive systems tested in the present study to linear regression analysis (r = —0.844/mTBS = 43.61–0.095WS). This strong negative correlation clearly shows that water sorption influenced the mTBS. The present results showed that the highest water sorption was presented by EXP (169.7 mg/mm3), followed by GAL (147.1 mg/mm3) and BAT (136.1 mg/mm3). On the other hand, CHX (100.0 mg/mm3) and GM1 (116.0 mg/mm3) presented similar and the lowest water sorption. Therefore, the second hypothesis, that there would be no difference in water sorption among adhesives containing different MMP-inhibitors, was rejected. Since all experimental adhesive systems had the same monomer composi- tion, it was speculated that the different polar characteristics of the MMP-inhibitors influenced the water sorption in the present study. In fact, the four MMP-inhibitors have polar groups (——NH, ——OH, ——C=O) that could establish hydrogen bonds with water molecules: Galardin (8), Batimastat (7), GM1489 (7) and Chlorhexidine diacetate (10), (Table 1) [11,43]. Thus, it was hypothesized that the way in which these inhibitors were entrapped within the polymer network could have influenced the number of polar sites available for hydrogen bonding with water, thereby contributing to the differences observed in the water sorption phenomenon. On the other hand, it was unexpected that chlorhexidine diacetate, with 10 polar groups in its molecule, presented the lowest value of solubility. The only reasonable explanation for this finding could be the reduction of the available –NH groups prone to hydrogen bonds with water due to the intramolecular hydrogen bonding among –NH groups present in the biguanide groups at the aliphatic core of the chlorhexidine diacetate molecule. Alternatively, no statistical differences in solubility among the experimental adhesive systems were found, with values ranging from 15.7 to 22.8 mg/mm3 (Table 3). These results support the acceptance of the third null hypothesis. Most probably, this behavior was influenced by the similarities in DC% reached by all of the experimental adhesive systems (Table 2). Theoretically, similar DC% would indicate that the adhesive systems have a similar number of non-reacted monomers that could be lixiviate from the polymer networks, thereby influencing their solubilities [14]. It is reasonable to speculate that the MMP-inhibitors were strongly entrapped within the polymer network and could not be dislodged by the sorbed water, consequently there was no interference with the solubility of the adhesive systems. 5. Conclusions Within the limitations of the present study, it was concluded that incorporation of Batimastat, GM1489 and Chlorhexidine diacetate in etch-and rinse adhesive systems may contribute to a decrease in the degradation of resin-dentin bonding after twelve months of water storage without impairing the physicochemical properties of the materials. Further biochemical investigations are needed to reinforce the role GM 1489 plays on BB-94 dentin MMP inhibition.