Background: A decline in pH and dissolution of the inorganic content of the dental tissues are followed by exposure of the organic portion of the tooth, which, in dentin, is largely composed of collagen fibres. These unprotected fi bres are then degraded by metalloproteinases and cysteine cathepsins, proteolytic enzymes present in dentin. We evaluated the influence of protease inhibitors on the bond strength of a self-etch adhesive system to caries-affected dentin. Eighty permanent third molars were selected for the study. Dentinal caries were induced artificially by the microbial method and the teeth were divided in four groups: G1 — application of Clearfil SE Bond adhesive system (CL); G2 — 2% chlorhexidine (CLX) + CL; G3 — sodium bicarbonate (BIC) + CL; G4 — BI + CLX + CL. Bond strength was assessed immediately and at six months. During the six months, the specimens were stored in distilled water. Microtensile bond strength testing was performed. On immediate testing, there was no significant difference in bond strength across the control, BIC, and CLX groups. The combination of BIC + CLX, however, led to an immediate, significant reduction in bond strength. After six months, bond strength was reduced in all groups. The highest bond strength was obtained in the control group (P < 0.05). Most fractures were adhesive, both immediately and at six months. We concluded that the cavity pretreatment with 2% CLX or 2% BIC did not have an immediate negative impact on bond strength of the Clearfil SE Bond system. After specimens were stored for six months in water, their bond strength of specimens was reduced in all groups. This reduction was the greatest in the groups exposed to the inhibitors.
The dentinal caries plaque biofilm has a complex structure and produces acids that degrade the inorganic matrix of enamel, dentin, and cementum [
The longevity of adhesive restorations may be compromised by the presence of metalloproteinases. Research has sought to characterize inhibitors of these enzymes. These include 2% chlorhexidine and 17% EDTA [
Self-etching adhesive systems contain acid monomers in their composition. These adhesives systems are capable of forming chemical bonds with the calcium in hydroxyapatite. The stability of these bonds over time is greater than that of conventional adhesive systems, which require phosphoric acid etching prior to application [
The present study was approved by the Research Ethics Committee of PUC- Campinas (protocol number 1.203.078).
Eighty unerupted permanent third molars were selected at the Dental Clinic of PUC-Campinas. The patients included were male and female and the ages ranged from 20 and 30 years.
Sample size calculation was performed by ANOVA, with a minimum difference between treatment means = 0.23, standard error = 0.25, number of treatments = 4, and statistical power = 0.80. The minimum number of teeth per group was thus calculated as 15.
The inclusion criteria were:unerupted permanent third molars with complete coronal and radicular formation observed in periapical radiography. The permanent third molars included didn’t have done coronal section during extraction and after extraction, the teeth included didn’t have visible cracks or fractures under 10X magnification.
The selected teeth were stored in 0.5% chloramine-T (LabCenter, São Paulo, Brasil) at 37˚C up the begining of research (Marchesi et al., 2014) [
The specimens were sealed with epoxy resin (Araldite, São Bernardo do Campo, Brazil) and nail polish (Colorama, São Paulo, Brazil) throughout, except for the dentin surface. The specimens were esterilizated. With the purpose of simulating caries-affected dentin, the teeth were placed in sterile test tubes with brain heart infusion (BHI) broth plus 0.5% yeast extract, 0.5% glucose, and 1% sucrose (all supplies obtained from LabCenter, São Paulo, Brazil). Streptococcus mutans type strain ATCC® 25175™ (Fundação André Tosello, Campinas, Brazil), standardized to 0.5 MacFarland turbidity, was added to the BHI medium. The samples were incubated at 37˚C for 14 days in anaerobic jars with gas-generating envelopes (LabCenter, São Paulo, Brazil) in an atmosphere containing 85% nitrogen (N2), 10% carbon dioxide (CO2), and 5% hydrogen (H2), and stored in a bacteriological incubator. During this 14-day period, BHI broth (LabCenter) was replaced every 24 hours [
Sterile curettes (SS White, Rio de Janeiro, Brazil) were used to remove infected dentin from all specimens. The criteria used for removal were those suggested by Banerjee and Schwendicke [
For bond strength testing, experimental groups were randomly divided (http://www.random.org) as follows (n = 20) (
Group 1: Clearfil SE Bond adhesive system (Kuraray Noritake Dental; Sakazu, Kurashiki, Okayama, Japan), applied in accordance with manufacturer instructions.
Material | Primary components | pH | Mode of application |
---|---|---|---|
Clearfil SE Bond adhesive system (Kuraray Noritake Dental; Sakazu, Kurashiki, Okayama, Japan) Batch No. Primer: 9N0168 Adhesive: 9U0269 | Primer: 10-MDP, HEMA, hydrophilic methacrylate, dl-camphorquinone, N,N-dihydroxyethyl-p-toluidine, water. Bond: 10-MDP, bis-GMA, HEMA, hydrophobic methacrylate, dl-camphorquinone, N,N-dihydroxyethyl-p-toluidine, silanized colloidal silica | 2.0 | 1) Active application of primer to the dry dentin surface for 20 s 2) Gentle air-jet drying for 5 s 3) Application of bonding agent 4) Gentle air-jet drying for 5 s 5) Light-curing for 10 s |
Sodium bicarbonate (Biodinâmica Química e Farmacêutica LTDA; Ibiporã, Paraná, Brazil) Batch No.: 321/15 | Sodium bicarbonate (NaHCO3) and flavouring agents | 8.0 | Dilution: 200 mg sodium bicarbonate in 10 mL normal saline 1) Active application to the dry dentin surface for 60 s 2) Removal of excess solution |
Chlorhexidine (FGM Dentscare LTDA; Joinville, Santa Catarina, Brazil) Batch No.: 050815 | 2% chlorhexidine digluconate, deionized water, volatile surfactant | 5.5 - 7 | 1) Active application to the dry dentin surface for 60 s 2) Removal of excess solution |
Filtek Z350 XT (3M ESPE; St Paul, MN, USA) Batch No.: 1535700494 | Silane-treated ceramic filler, bisphenol A-glycidyl methacrylate (bis-GMA), bisphenol A-polyethylene glycol diether dimethacrylate (bis-EMA), silane-treated silicon dioxide filler, silane-treated silica/zirconia filler, diurethane dimethacrylate, polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate (TEG-DMA), 2,6-di-tert-butyl-p-cresol (BHT), pigments | _ | 1) Composite resin build-up technique 2) Light-curing for 40 s |
Chemical structures: chlorhexidine (A) and sodium bicarbonate (B).
Group 2: active application of 10 µL 2.0% chlorhexidine (FGM Dentscare LTDA; Joinville, Santa Catarina, Brazil) for 60 seconds, followed by application of Clearfil SE Bond (Kuraray Noritake Dental; Sakazu, Kurashiki, Okayama, Japan) in accordance with manufacturer instructions.
For groups 3 and 4, 200 mg sodium bicarbonate (Biodinâmica, Ibiporã, Brazil) was diluted in 10 mL normal saline to obtain a 2% sodium bicarbonate solution, which was applied to the remaining caries-affected tissue.
Group 3: active application of 10 μL 2.0% sodium bicarbonate (Biodinâmica, Ibiporã, Brazil) for 60 seconds, followed by application of Clearfil SE Bond (Kuraray Noritake Dental; Sakazu, Kurashiki, Okayama, Japan) in accordance with manufacturer instructions.
Group 4: active application of 10 μL 2.0% sodium bicarbonate (Biodinâmica, Ibiporã, Brazil) for 60 seconds, followed by active application of 10 µL 2.0% chlorhexidine (FGM Dentscare LTDA; Joinville, Santa Catarina, Brazil) for 60 seconds, and application of Clearfil SE Bond (Kuraray Noritake Dental; Sakazu, Kurashiki, Okayama, Japan) in accordance with manufacturer instructions.
The application and removal of excess of sodium bicarbonate and chlorhexidine were done with microbrush. The removal excess of protease inhibitors was made until the affected dentin didn’t have moisture.
After application of the adhesive system, a Z350 composite resin build-up (3M ESPE, St. Paul, MN, USA) was made on the dentin surface of each specimen. Each layer of the build-up was approximately 1 mm thick. Layers were oriented parallel to one another and perpendicular to the long axis of the tooth, and were light-cured for 20 seconds. The intensity of the curing light (Gnatus, Ribeirão Preto, Brazil) was 1400 mW/cm2, as measured by a radiometer (Curing Light, Brasília, Brazil) at the start of the experiments in each group.
Then, 10 specimens from each group were subjected to immediate microtensile bond strength testing, while the remaining 10 specimens were stored in distilled water for 6 months and then tested [
The teeth were embedded in acrylic and wax (Kota, Cotia, Brazil). Each tooth- adhesive-composite block was sectioned with a diamond disk (Buehler, Illinois, USA), in parallel planes in the buccolingual direction, along the long axis of the tooth. The sections were cut sequentially along the mesiodistal plane to obtain beam-shaped specimens with a cross-sectional area of approximately 1 mm2.
Each specimen was visualized under a stereo microscope (Opton, Cotia, Brazil) at 40X magnification to verify that the adhesive interface was perpendicular to the long axis of the specimen.
Before microtensile bond strength testing, the width and thickness of each specimen were measured with a digital caliper (0.01 mm resolution; Mitutoyo, Suzano, Brazil). The adhesive interface was perpendicular to the long axis of the tensile forces. The assay was performed on a universal testing machine (Instron, São José dos Pinhais, Brazil). Traction was applied to the specimen at a crosshead speed of 0.5 mm/min until fracture occurred. At the time of fracture, the applied load in newtons (N) was recorded and the specimen was examined under a stereo microscope at 40X magnification to ascertain the fracture mode (interface, cohesive in dentin, or cohesive in resin). Only those specimens in which adhesive/mixed-mode fracture at the interface (i.e., debonding) occurred were taken into account for calculation of bond strength. To do so, the fracture load of each specimen was divided by the bond area. Microtensile bond strength tests were performed immediately after specimen preparation and 6 months later.
For statistical analysis, the results of testing were converted from newtons to megapascals (MPa), and each tooth was considered a distinct sampling unit. After testing, all specimens were examined under a stereo microscope (Opton, Cotia, Brazil) at 40X magnification to ascertain the fracture type and percentage as described by Lenzi et al. [
Score 1: Adhesive/mixed-mode fracture;
Score 2: Cohesive fracture in dentin alone;
Score 3: Cohesive fracture in composite resin alone;
Score 4: Premature fracture.
For the analysis of fracture mode, each fractured stick was considered as the sampling unit.
The results were analyzed in the Biostat 4.0 program. D’Agostino’s test for normality revealed that the sample was nonparametric. Therefore, the Kruskal- Wallis test followed by the Student-Newman-Keuls procedure and the Mann- Whitney U test were used for analyses.
The longevity of adhesive restorations may be compromised by the presence of metalloproteinases and chlorhexidine was described in literature with a metalloproteinases inhibitor in association with convencional adhesives systems. Sodium bicarbonate is a basic substance that can inactivate the cathepsin precursors of endogenous proteases, and could thus act as an indirect metalloproteinase inactivator. Furthermore, it is biocompatible and readily available to dental practitioners. Within this context, the present study sought to assess the influence of protease inhibitors on the bond strength of weak self-etch adhesive systems.
On immediate testing, there was no significant difference in bond strength between the control and chlorhexidine groups (P > 0.05). Application of sodium bicarbonate was not associated with a significant difference as compared with control, chlorhexidine only, or the combination of bicarbonate and chlorhexidine (P > 0.05). The combination of bicarbonate and chlorhexidine, however, led to a significant reduction in bond strength as compared with the other groups (P < 0.05) (
There were no significant differences in fracture mode across groups (P > 0.05) (
At six months, bonding strength was greatest in the control group, with statistically significant differences in relation to all others (P < 0.05). There was no significant difference in bond strength across the bicarbonate, chlorhexidine, and bicarbonate + chlorhexidine groups (P > 0.05) (
There were no significant differences in fracture mode across groups (P > 0.05) (
Control | Bicarbonate | Chlorhexidine | BIC + CLX | |
---|---|---|---|---|
Median (IQR) | 16.7 (16.7)A | 14.3 (10.7)AB | 20.3 (15.1)A | 8.3 (8.3)B |
Mean (SD) | 19.3 (11.9) | 15.5 (8.3) | 21.7 (16.3) | 12.3 (11.9) |
P-value (KW) | 0.0337 |
Different letters denote P < 0.05.
Control | Bicarbonate | Chlorhexidine | BIC + CLX | |
---|---|---|---|---|
Median (IQR) | 1.0 (0.00)A | 1.00 (3.00)A | 1.0 (3.00)A | 1.00 (3.00)A |
Mean (SD) | 1.63 (1.21) | 1.96 (1.38) | 1.98 (1.38) | 1.90 (1.35) |
P-value (KW) | 0.3270 |
Same letter denotes P > 0.05.
Control | Bicarbonate | Chlorhexidine | BIC + CLX | |
---|---|---|---|---|
Median (IQR) | 2.5 (1.4)A | 1.3 (1.1)B | 1.3 (1.6)B | 0.9 (0.7)B |
Mean (SD) | 2.5 (1.2) | 1.6 (1.0) | 1.9 (1.8) | 1.1 (1.0) |
P-value (KW) | 0.0035 |
Different letters denote P < 0.05.
Six months after specimen preparation, bond strength was significantly weaker across all groups (P < 0.0001) (
The contemporary restorative dentistry is based on so-called ultraconservative preparations. Removal only of infected dentin tissue, with preservation of caries-affected dentin that is still amenable to structural repair, has been advocated [
Control | Bicarbonate | Chlorhexidine | BIC + CLX | |
---|---|---|---|---|
Median (IQR) | 1.00 (0.00)A | 1.00 (3.00)A | 1.00 (3.00)A | 1.00 (4.00)A |
Mean (SD) | 1.46 (0.94) | 1.81 (1.26) | 1.85 (1.27) | 1.98 (1.39) |
P-value (KW) | 0.1056 |
Same letter denotes P > 0.05.
Control | Bicarbonate | ||||
---|---|---|---|---|---|
Before | Six months | Before | Six months | ||
Median (IQR) | 16.7 (16.7)A | 2.5 (1.4)B | 14.3 (10.7)A | 1.3 (1.1)B | |
Mean (SD) | 19.3 (11.9)A | 2.5 (1.2)B | 15.5 (8.3)A | 1.6 (1.0)B | |
P-value (Mann-Whitney) | <0.0001 | <0.0001 | |||
Chlorhexidine | BIC + CLX | ||||
Before | Six months | Before | Six months | ||
Median (IQR) | 20.3 (15.1)A | 1.3 (1.6)B | 8.3 (8.3)A | 0.9 (0.7)B | |
Mean (SD) | 21.7 (16.3)A | 1.5 (1.8)B | 12.3 (11.9)A | 1.1 (1.0)B | |
P-value (Mann-Whitney) | <0.0001 | <0.0001 | |||
Different letters denote P < 0.05.
When using adhesive systems that require phosphoric acid etching, inhibitors have beneficial effects on the durability of bonding to dentin, as they preserve the collagen fibers present largely in the organic dentin portion of the tooth [
Kim and Shin [
In our sample, pretreatment with 2% chlorhexidine or 2% sodium bicarbonate did not have an immediate negative effect on the bond strength of the Clearfil SE Bond self-etch adhesive system to caries-affected dentin. This corroborates the findings of Sacramento et al. [
In the present study, pretreatment of caries-affected dentin with 2% sodium bicarbonate did not interfere with the immediate bond strength of the Clearfil SE Bond system. This may be explained by the lack of any immediate interference of sodium bicarbonate with the effects of the functional acid monomer or with the organic acids present in the primer. Another hypothesis is incorporation of sodium bicarbonate particles into the hybrid layer and smear layer [
After 6 months, bond strength was diminished in all groups. This disagrees with the findings of Gunaydin et al. [
A high rate of premature fracture was observed in all groups, both immediately after specimen preparation and after 6 months. This may be attributed to the low cohesive strength of caries-affected dentin, and is consistent with the findings of Yoshiyama et al. [
Taking into account the limitations inherent to an in vitro experiment, the present study demonstrated that there is no place for protease inhibitors during adhesive restorations performed with composite resin and the Clearfil SE Bond adhesive system. Further studies in a similar line of research are needed to contribute to the evidence base on this topic.
Cavity pretreatment with the protease inhibitors 2% chlorhexidine or sodium bicarbonate did not have an immediate negative impact on bond strength of the Clearfil SE Bond system. At 6 months, however, bond strength was reduced in all groups. This reduction was the greatest in the groups exposed to the inhibitors.
The authors have no financial relationships relevant to this article to disclose.
The authors have no conflicts of interest to disclose.
Grandizoli, D.R.P. and Pinheiro, S.L. (2018) Influence of Protease Inhibitors on Bond Degradation of Self- Etch Adhesive Systems to Caries-Affected Dentin: An in Vitro Study. Advances in Bio- logical Chemistry, 8, 15-28. https://doi.org/10.4236/abc.2018.81002