Abstract
Objectives
This in situ study aimed to investigate whether the dentin treatment with MMPs inhibitor (CHX 2%) could influence the development of secondary caries wall lesions in different dentin-composite interfaces.
Material and methods
For 21 days, 15 volunteers wore a modified-occlusal splint loaded with dentin-composite samples treated or not with CHX and restored according 4 different interface conditions: Bonding (B = samples restored with complete adhesive procedure), no bonding (NB = restored with composite resin without adhesive procedure), 100 μm (no adhesive procedure and with intentional gap) and 100 μm + B (adhesive material on composite side and intentional gap). Eight times per day, the splint with samples was dipped in a 20% sucrose solution for 10 min. Before and after caries development, samples were imaged with T-WIM and lesion depth (LD) and mineral loss (ML) were calculated.
Results
Linear mixed effect analysis showed that dentin treatment with CHX did not significantly affect the caries lesion progression (LD and ML; p ≤ 0.797). Dentin wall lesions were observed in the 100 μm and 100 μm + B groups independently of MMP inhibitor treatment.
Conclusion
The treatment of dentin with MMP inhibitor was not able to slow down the secondary caries wall lesion development in this in situ study.
Significance
The dentin treatment with 2% CHX did not prevent secondary caries wall lesion initiation.
1
Introduction
Dental work in general practice consists of a significant proportion of placing and replacing restorations. Secondary caries has been shown to be the most common reason for posterior restoration failure [ ].
Over time, dynamics in the oral cavity, e.g. masticatory forces, enzymatic activity of dentin (proteinases), and biofilm activity may negatively affect the quality of the restoration interface leading to marginal gaps or defects. In the presence of cariogenic plaque and fermentation products this can result in secondary caries development [ ]. Therefore, the good sealing between dentin and restorative material is the main focus to prevent secondary caries and to prolong the lifetime of composite restorations [ ].
Several studies have shown that enzymatic activity in dentin contributes significantly to adhesive interface degradation [ ]. This phenomenon can be attributed to host-derived enzymes such as Matrix Metalloproteinases (MMPs) which are Zn +2 − and Ca +2 −dependent endopeptidases and are considered to be mainly responsible for degrading collagenous dentin proteins[ ]. The MMPs are also claimed to be involved during progression of dentin caries lesions, where they are responsible for breakdown of the collagenous organic matrix of dentin after demineralization occasioned by acid from bacteria metabolism [ ].
The activity of MMPs on dentin substrate can be retarded by use of inhibitors. Several studies have demonstrated increase of bonding strength and reduced interfacial degradation over time when MMP inhibitors are used during adhesive procedure (e.g., chlorhexidine – CHX, galardin, hesperidin, etc.) [ ]. Recently, exogenous MMPs inhibitors were reported to reduce the degradation of human dentin matrix (acid-demineralized dentin) in situ [ ] and retard the caries process in rats [ ]. Similarly, other in situ studies have been showing that gels or solutions delivering MMPs inhibitions including CHX are able to prevent dental demineralization caused by erosion [ ]. These observations suggest that approaches against organic matrix degradation might also be useful in caries prevention [ ].
A number of studies have investigated the effect of CHX as MMP inhibitor on bonding stability when there is a good marginal seal [ ]. To the authors’ knowledge there are no previous studies evaluating the effect of this MMP inhibitor on secondary caries development at interfaces with a compromised marginal seal due to defects at the interface (e.g. gaps). Therefore, the aim of this in situ study was to investigate whether the dentin treatment with MMP inhibitor (CHX 2%) could influence the development of secondary caries wall lesions in different interfaces, including bonded and non-bonded conditions and interfacial gaps. The hypothesis of this study was that MMP inhibitor would reduce secondary caries wall lesion progression.
2
Material and methods
2.1
Study design
This was a mono-centre in situ study with a split mouth design. The protocol and design of this study were submitted and approved by an Ethical Committee Board (CMO code NL 56622.091.16). Independent variables were dentin treatment applied or not (2% CHX, MMP inhibitor) and interface conditions whereas the outcome variables were mineral loss (ML) and lesion depth (LD).
2.2
Sample size calculation
Since a split mouth design would be used, the equation for sample size calculation was applied: n = f(α,β)*σ 2 /(μ1-μ2) 2 [ ]. Using a power of 90%, significance level of 5% and considering the outcomes from a previously published study [ ], the following parameters were used: the average between the SD from no gap and smallest gap size (σ = 26.5); average of lesion progression in dentin samples restored with composite and with wall lesion development (μ1 = 48.5 μm); and difference on lesion progression lower than 50%, which would not be clinically meaningful (μ2 = 24.3 μm). The sample size needed was 13 volunteers. Considering a drop-out rate of 20%, the final sample required was 16 volunteers.
2.3
Volunteers
Sixteen volunteers with good general health (5 men and 11 women, mean age = 28.4 years) were recruited within the Campus of Radboud University (Nijmegen, The Netherlands). All the volunteers agreed and signed the written informed consent. Exclusion criteria were active caries, periodontitis (DPSI > 2), ASA > 2, and the wearing of orthodontic or a removable prosthetic appliance in the mandibular jaw.
2.4
Sample preparation
Thirty-two sound human molars were collected and ground flat with 220-grit Sic paper until complete enamel removal and dentin exposure. The roots were cut off with a diamond blade (Buehler diamond wafering blade nr.11-4244) and the remaining crowns were perpendicularly cut in 64 dentin bars with fixed width of 3.2 mm and various lengths. Subsequently the dentin bars were manually ground with 400-grit Sic paper to a height of 2.0 mm ( Fig. 1 A–B) and sterilized with ethylene oxide (Isotron Nederland B.V., Venlo, The Netherlands) [22]. One dentin-composite sample was created by two dentin bars that were attached to each other with a thin layer of composite (0.5 mm) fixed with self-etching primer and bonding agent on the pulpal side (ClearFil SE Bond, Kuraray, Okayama, Japan; CSE) ( Fig. 1 C). In each dentin-composite sample, four slots were made parallel to the dentin tubule with a 0.12 cylindrical bur with a depth of 1.9 mm. Self-etching primer was applied on the dentin wall of each produced slot. Subsequently, 2% CHX solution was applied with a disposable syringe for 60 s on dentin samples assigned to pre-treatment ( Fig. 1 D–F).
Two slots were filled with resin composite (AP-X PLT, color A2, Clearfil, Kuraray, Okayama, Japan) and a gap was created by placing a matrix of 100 μm of thickness between the dentin and the composite. One of these slots received a layer of bonding material on side of metal matrix creating an interface where bonding was located on resin composite side of the gap (100 μm + B). From the remaining two slots, one was filled completely with composite, but no adhesive was used (no bonding; NB) and the other slot was restored with composite and adhesive procedure (Bonding; B − control group; CSE) ( Fig. 1 G). Resin composite and bonding agents were activated according to manufactures’ instructions using a Bluephase ® 20i light curing (Ivoclar Vivadent Ltda). Those dentin-composite samples treated with MMP inhibitor were immersed in 2 ml of neutralizing solution (2 x; D/E Neutralizing Broth, Acumedia, Michigan, USA) for 10 s and followed by immersion in distilled water (10 s) to inactivate the antibacterial effect of MMP inhibition solution used and therefore to avoid crossed effects in the study. The same protocol was used to dentin-composite samples not treated with MMP inhibitor, but distilled water was used instead of neutralizing solution.
Each volunteer received a modified-occlusal splint for mandibular jaw ( Fig. 1 H) with buccal flanges holding four embedded metal slots of 20 mm × 3.2 mm × 2.5 mm. Only the two upper slots were used for this study. Thirty-two dentin-composite samples were placed at left or right side alternately per volunteer considering the treatment applied (n = 16/treatment). Positions of different composite-dentin interface conditions (B, NB, 100 μm and 100 μm + B) were changed per volunteer (mesial to distal). The sequence was manually generated using computer software (Excel Program).
2.5
Experimental protocol
The occlusal splints were worn 24 h per day for 21 days (3 weeks), and were only removed during eating, drinking or oral hygiene. During these periods the device remained in physiological salt solution. Volunteers were instructed to dip the splint in 20% sucrose solution eight times per day (10 min). The intervals between sucrose dippings were at least 1 h. They received a diary to record the exact moments of sucrose exposure. After the dipping in sucrose, the splint was rinsed with tap water and replaced in the mouth. All volunteers used fluoride toothpaste (1450 ppm; Colgate Caries Protection, Colgate-Palmolive-Company, The Netherlands) and were asked to apply the fluoride toothpaste slurry on the samples once a day (2 min) when they brushed their teeth. The volunteers were instructed not to clean or brush the samples. Instructions were given both orally and in writing by a researcher involved in the study.
2.6
Transversal wavelength independent microradiography (T-WIM)
T-WIM images were made at baseline (T0) and after 21 days (T21). The microradiographs were taken using 45 kV, 40 mA and 8 s of x-ray exposure. A step wedge with the same absorption coefficient as tooth material (94% Al/6% Zn alloy) was used for quantitative measurement of LD and ML. After x-ray exposure, films were developed (10 min), fixed (7 min), rinsed and dried. Digital images of each sample were recorded with a light microscope (Leica Microsystems, Germany) with a magnification of × 10 and a CMOS camera (Canon EOS 50D, Japan). The T-WIM images were edited using the method of Kuper et al. [ ]. From each sample the wall lesions in the dentin facing the gaps were measured using a software program developed in our laboratory at a fixed area 400 μm under the surface. Baseline measurements (T0) were subtracted from measurements taken after 21 days (T21), in order to estimate true LD and ML. The subtracted values were used in the statistical analysis. Actual gap sizes were measured on gaps from baseline T-WIM images using the same software program as described elsewhere [ ].
2.7
Statistical analysis
The effect of MMP inhibitor on LD and ML was analysed using linear mixed-effects models. Absolute differences between averages of LD and ML considering the effect of MMP inhibitor were entered into the model as fixed effect. More complex mixed-effects models were tested to verify the effect of added factors such as location of the gap (more distal or mesial) and interface conditions. The created models were compared among them by ANOVA. As there was not an improvement in the models by addition of factors (p > 0.05), a simple mixed-effect model was used. All tests were conducted using R statistical program with the significant level set as 5%.
2
Material and methods
2.1
Study design
This was a mono-centre in situ study with a split mouth design. The protocol and design of this study were submitted and approved by an Ethical Committee Board (CMO code NL 56622.091.16). Independent variables were dentin treatment applied or not (2% CHX, MMP inhibitor) and interface conditions whereas the outcome variables were mineral loss (ML) and lesion depth (LD).
2.2
Sample size calculation
Since a split mouth design would be used, the equation for sample size calculation was applied: n = f(α,β)*σ 2 /(μ1-μ2) 2 [ ]. Using a power of 90%, significance level of 5% and considering the outcomes from a previously published study [ ], the following parameters were used: the average between the SD from no gap and smallest gap size (σ = 26.5); average of lesion progression in dentin samples restored with composite and with wall lesion development (μ1 = 48.5 μm); and difference on lesion progression lower than 50%, which would not be clinically meaningful (μ2 = 24.3 μm). The sample size needed was 13 volunteers. Considering a drop-out rate of 20%, the final sample required was 16 volunteers.
2.3
Volunteers
Sixteen volunteers with good general health (5 men and 11 women, mean age = 28.4 years) were recruited within the Campus of Radboud University (Nijmegen, The Netherlands). All the volunteers agreed and signed the written informed consent. Exclusion criteria were active caries, periodontitis (DPSI > 2), ASA > 2, and the wearing of orthodontic or a removable prosthetic appliance in the mandibular jaw.
2.4
Sample preparation
Thirty-two sound human molars were collected and ground flat with 220-grit Sic paper until complete enamel removal and dentin exposure. The roots were cut off with a diamond blade (Buehler diamond wafering blade nr.11-4244) and the remaining crowns were perpendicularly cut in 64 dentin bars with fixed width of 3.2 mm and various lengths. Subsequently the dentin bars were manually ground with 400-grit Sic paper to a height of 2.0 mm ( Fig. 1 A–B) and sterilized with ethylene oxide (Isotron Nederland B.V., Venlo, The Netherlands) [22]. One dentin-composite sample was created by two dentin bars that were attached to each other with a thin layer of composite (0.5 mm) fixed with self-etching primer and bonding agent on the pulpal side (ClearFil SE Bond, Kuraray, Okayama, Japan; CSE) ( Fig. 1 C). In each dentin-composite sample, four slots were made parallel to the dentin tubule with a 0.12 cylindrical bur with a depth of 1.9 mm. Self-etching primer was applied on the dentin wall of each produced slot. Subsequently, 2% CHX solution was applied with a disposable syringe for 60 s on dentin samples assigned to pre-treatment ( Fig. 1 D–F).
