Marcela Rocha de Oliveira Carrilho

In vivo Preservation of the Hybrid Layer by Chlorhexidine

Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-infiltrated dentin. This study tested the hypothesis that interfacial degradation of resin-dentin bonds may be prevented or delayed by the application of chlorhexidine (CHX), a matrix metalloproteinase inhibitor, to dentin after phosphoric acid-etching. Contralateral pairs of resin-bonded Class I restorations in non-carious third molars were kept under intra-oral function for 14 months. Preservation of resin-dentin bonds was assessed by microtensile bond strength tests and TEM examination. In vivo bond strength remained stable in the CHX-treated specimens, while bond strength decreased significantly in control teeth. Resin-infiltrated dentin in CHX-treated specimens exhibited normal structural integrity of the collagen network. Conversely, progressive disintegration of the fibrillar network was identified in control specimens. Auto-degradation of collagen matrices can occur in resin-infiltrated dentin, but may be prevented by the application of a synthetic protease inhibitor, such as chlorhexidine.

Chlorhexidine Preserves Dentin Bond in vitro

Loss of hybrid layer integrity compromises resin-dentin bond stability. Matrix metalloproteinases (MMPs) may be partially responsible for hybrid layer degradation. Since chlorhexidine inhibits MMPs, we hypothesized that chlorhexidine would decelerate the loss of resin-dentin bonds. Class I preparations in extracted third molars were sectioned into two halves. One half was customarily restored (etch-and-rinse adhesive/resin composite), and the other was treated with 2% chlorhexidine after being acid-etched before restoration. Specimens were stored in artificial saliva with/without protease inhibitors. Microtensile bond strengths and failure mode distribution under SEM were analyzed immediately after specimens’ preparation and 6 months later. With chlorhexidine, significantly better preservation of bond strength was observed after 6 months; protease inhibitors in the storage medium had no effect. Failure analysis showed significantly less failure in the hybrid layer with chlorhexidine, compared with controls after 6 months. In conclusion, this in vitro study suggests that chlorhexidine might be useful for the preservation of dentin bond strength.

Bonding BisGMA to Dentin—a Proof of Concept for Hydrophobic Dentin Bonding

The use of TEGDMA as a diluent comonomer in the formulation of hydrophobic adhesives for ethanol wet-bonding is a concern, due to its leaching potential, higher water sorption, and bio-incompatibility. This study tested the hypothesis that hydrophobic bonding to acid-etched dentin may be accomplished with the use of ethanol-solvated BisGMA only. Phosphoric-acid-etched, oxalate-occluded, deep coronal dentin bonded under 20 cm water pressure with experimental BisGMA adhesives by ethanol wet-bonding exhibited tensile strengths that were not significantly different from that achieved with OptiBond FL bonded according to the manufacturer-recommended protocol, with similar acid-/base-resistant hybrid layers, resin tags, and nanoleakage distribution. Ethanol replacement of water-saturated dentin produced wider interfibrillar spaces, more extensive shrinkage of the collagen fibrils, and narrower hybrid layers. Experimental BisGMA adhesives provide the proof of concept that relatively hydrophobic resins may be coupled to acid-etched dentin by increasing its hydrophobic characteristics via ethanol replacement. They should be further optimized before clinical application.

Host-derived loss of dentin matrix stiffness associated with solubilization of collagen.

Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding procedures and are thought to contribute to the progressive degradation of resin-dentin bonds over time. The purpose of this study was to evaluate the changes in mechanical, biochemical, and structural properties of demineralized dentin treated with or without chlorhexidine (CHX), a known MMP-inhibitor. After demineralizing dentin beams in EDTA or phosphoric acid (PA), the baseline modulus of elasticity (E) of each beam was measured by three-point flexure. Specimens were pretreated with water (control) or with 2% CHX (experimental) and then incubated in artificial saliva (AS) at 37 degrees C for 4 weeks. The E of each specimen was remeasured weekly and, the media was analyzed for solubilized dentin collagen at first and fourth week of incubation. Some specimens were processed for electron microscopy (TEM) immediately after demineralization and after 4 weeks of incubation. In EDTA and PA-demineralized specimens, the E of the control specimens fell (p < 0.05) after incubation in AS, whereas there were no changes in E of the CHX-pretreated specimens over time. More collagen was solubilized from PA-demineralized controls (p < 0.05) than from EDTA-demineralized matrices after 1 or 4 weeks. Less collagen (p < 0.05) was solubilized from CHX-pretreated specimens demineralized in EDTA compared with PA. TEM examination of control beams revealed that prolonged demineralization of dentin in 10% PA (12 h) did not denature the collagen fibrils.

Immunohistochemical identification of MMP-2 and MMP-9 in human dentin: Correlative FEI-SEM/TEM analysis.

Matrix metalloproteinases (MMPs) are a family of peptidases trapped within mineralized dentin matrix and involved with degradation of the extracellular matrix components in hybrid layers and caries. Despite their identification through indirect evidences and biochemical assays, MMP-2 and -9 have not been localized within the human dentin extracellular organic matrix. Thus, this study aimed to assess the localization and distribution of MMP-2 and -9 in human dentin organic matrix by employing a correlative field emission in-lens-scanning electron microscopy (FEI-SEM) and transmission electron microscopy (TEM) immunohistochemical approach. Dentin specimens were submitted either to a preembedding or to a postembedding immunolabeling technique using primary monoclonal antibodies anti-MMP-2 and anti-MMP-9 and exposed to a secondary antibody conjugated with gold nanoparticles. MMP-2 and -9 labelings were identified in the demineralized dentin matrix as highly electron-dense gold particles dispersed on the collagen fibrils. Correlative FEI-SEM/TEM observations confirmed that MMP-2 and MMP-9 are endogenous components of the human dentin organic matrix and revealed the three-dimensional relationship between these proteinases and the collagen fibrils, showing that both antibodies yielded a similar labeling pattern. In conclusion, the results of the study contribute to reveal distinct distribution pattern of gelatinases and support the hypothesis that these enzymes are intrinsic constituents of the dentin organic matrix after decalcification.

The effect of initial irrigation with two different sodium hypochlorite concentrations on the erosion of instrumented radicular dentin

OBJECTIVEnThis study evaluated the effects of different NaOCl concentrations and contact times on removal of the organic phase from mineralized dentin with and without the adjunctive use of EDTA, and the effect of NaOCl concentrations on canal wall erosion after the use of EDTA as the final active irrigant.nnnMETHODSnDentin powders were immersed in 5.25% or 1.3% NaOCl for different contact periods and then rinsed with 17% EDTA for 2 min. Before and after the use of 17% EDTA as the final rinse, the NaOCl-treated dentin powders were examined using ATR-FT-IR spectroscopy to analyze the relative loss of organic and inorganic components. Scanning (SEM) and transmission electron microscopy (TEM) were used to examine the erosion of instrumented canal walls irrigated with 5.25% NaOCl/EDTA or 1.3% NaOCl/EDTA.nnnRESULTSnCompared with 1.3% NaOCl, less intact collagen remained within the subsurface of the mineralized dentin powder after the use of 5.25% NaOCl, irrespective of subsequent rinsing with 17% EDTA. Canal wall erosion was apparent only under SEM when root canals were irrigated 5.25% NaOCl followed by 17% EDTA. Under TEM examination, subsurface erosion extended 10-15 microm beneath the sealer-bonded dentin surface after the use of 5.25% NaOCl for 20 min.nnnCONCLUSIONnThe superficial destructive effect of NaOCl on mineralized dentin is irreversible and is present irrespective of whether EDTA is subsequently employed as the final active irrigant. The EDTA removes the collagen-depleted apatite phase to expose the underlying cause of destruction that is morphologically perceived as canal wall erosion.

An All-in-One Adhesive Does Not Etch beyond Hybrid Layers

Continuous etching of aggressive all-in-one adhesives occurs in wet dentin tubules after polymerization of the adhesives. This study challenged the hypothesis that unpolymerized acidic monomers from an aggressive all-in-one self-etching adhesive continue to etch beyond dentin hybrid layers. Dentin surfaces bonded with Adper Prompt L-Pop were sectioned into 0.3-mm-thick slabs. Some of the slabs were stored in water (pH 6.8) or glycine buffer (pH 11.1) for six weeks and then examined by CLSM, SEM, and TEM. The rest were immersed in a biomimetic remineralizing medium for up to 4 months. Morphologic analysis indicated no difference in demineralization thickness between the two 6-week storage groups. However, increased permeability and loss of integrity occurred along the base of the hybrid layers in the glycine buffer group, but not in the water storage group. These findings were also confirmed by the results of biomimetic remineralization along the bases of those hybrid layers.

Microtensile bond strength of adhesive systems to dentin with or without application of an intermediate flowable resin layer

This study evaluated the effect of flowable composite resin application on the microtensile bond strength (microTBS) of adhesive systems to dentin. Occlusal surfaces of human third molars were ground to obtain flat dentin surfaces. The crown of each tooth was sectioned occluso-gingivally into four quarters with a water-cooled diamond saw. One of the following adhesive systems was applied to dentin surface in each quarter of the same tooth, following manufacturers instructions: Scotchbond Multipurpose, Single Bond Adper Prompt and Clearfil SE Bond. Experimental and control groups received the same treatment, except for the fact that a layer of flowable composite (Filtek Flow) was placed and light-cured on top of the adhesive layer in the specimens of the experimental groups. Resin composite (Filtek Z250) crown buildups were then made on the bonded surfaces and incrementally light-cured for 20 s. The restored teeth were stored in water at 37 degrees C for 24 h. Each tooth quarter was serially cut in a longitudinal direction in order to obtain several bonded sticks (0.9-mm(2) in cross-section). Maximal microtensile stress (in MPa) at failure was recorded using a universal testing machine. Data were analyzed by two-way ANOVA and Tukeys test at 5% significance level. Placement of a low-viscosity resin to the hybridized dentin increased the microTBS for all tested adhesive systems. However, such increase was significant only for Clearfil SE Bond (p<0.05). When the restorations were placed without low-viscosity resin, Clearfil SE Bond presented higher microTBS than Adper Prompt (p<0.05). For the groups treated with flowable composite, Clearfil SE Bond produced the highest microTBS means to dentin. In conclusion, the effect of the addition of an intermediate flowable composite layer on microTBS to dentin was material-dependent and resulted in an increased adhesion for all tested materials, though with significance only for Clearfil SE Bond.

Effect of sodium hypochlorite on the bond strength of an adhesive system to superficial and deep dentin

The objective of this study was to evaluate the bond strength to superficial (SU) and deep (D) dentin, accessed via apical (DA) or occlusal (DO), using One-Step adhesive system applied according to the manufacturers instructions (C) or following deproteinization with 10% sodium hypochlorite (H) for 60s, after acid etching. Three sound extracted human molars were prepared for each experimental condition. Restorations were performed using Z100 in 2mm increments, each one being light-cured for 40s. Teeth were longitudinally sectioned to obtain stick-shaped specimens with a cross-sectional area of 0.8mm², which were submitted to bond strength test (0.6mm/min). Results obtained after statistical analysis using a two-way ANOVA (substrate vs. surface treatment) and Tukeys test were: SU (35.4 ± 12.3), DO (26.5 ± 8.5), DA (26.1 ± 10.2) following conventional surface treatment, and SU (28.82 ± 12.7), DO (24.3 ± 8.3) and DA (23.5 ± 8.5), after surface treatment using sodium hypochlorite. The interaction of the factors was not significant (p>0.05). However, the main factors were significant (p<0.05). Mean bond strength values in superficial dentin were statistically superior to deep dentin (p<0.05), although no statistically significant difference was observed between the two via of access to deep dentin (SU>DO=DA). The conventional surface treatment resulted in higher bond strength values than the sodium hypochlorite treatment (p<0.05). It was concluded: 1) bond strength values were higher in superficial dentin, 2) no difference was found between the two deep substrate preparations, and 3) the application of sodium hypochlorite following dentin acid etching may reduce bond strengths.

Effect of oxalate desensitizer on the durability of resin-bonded interfaces.

Potassium oxalate desensitizers were previously shown to effectively reduce the immediate permeability of resin-bonded dentin. The current study evaluated whether the effect of the combined application of oxalate with etch-and-rinse adhesives interferes with the durability of resin-dentin bonds when using etch-and-rinse adhesives. The bond strength of resin-bonded dentin specimens composed of two-step or three-step etch-and-rinse adhesives (Single Bond, One-Step and Scotchbond Multi-Purpose, respectively) was tested immediately (24 hours) and after 12 months of water storage. The adhesives were used either according to the manufacturers instructions (control groups) or after treating acid-etched dentin with a potassium oxalate gel (BisBlock, BISCO, Inc). The treatment of dentin with potassium oxalate was shown to negatively affect the baseline bond strength of resin-bonded dentin specimens, regardless of the adhesive used (p < 0.05). After storage, the bond strength of the resin-bonded interfaces was significantly reduced for all the tested groups (p < 0.001). Nevertheless, the rate of decreasing bond strength was significantly lower for oxalate-treated specimens than for the controls (p < 0.05).