Phosphoric acid concentration affects dentinal MMPs activity.

OBJECTIVES: To evaluate whether the concentration of phosphoric acid (PA) has an effect on the proteolytic activity of sound human demineralized dentin. It is hypothesized that the activity of matrix-bound and extracted enzymes depends on the PA concentration used to demineralize dentin. METHODS: One-gram aliquots of mid-coronal human dentin powder were demineralized with 1wt%, 10wt% and 37wt% PA. Concentrations of released calcium were measured for each set of demineralization. Extracted MMP-2 was immunologically identified by western blot and its activity was determined by conventional gelatin zymography. Analysis of released hydroxyproline (HYP) and in situ zymography were performed to evaluate the activity of insoluble, bound-matrix enzymes. RESULTS: The amount of released calcium from dentin powder treated with 37wt% PA was significantly higher (p≤0.05) than that obtained by dentin demineralization with 10wt% and 1wt% PA. Expression and activity of endogenous enzymes, extracted from or bound to dentin matrix, were detected for all samples regardless of the PA concentration. However, the expression and activity of extracted MMP-2 were significantly higher when dentin was treated with 10wt% PA (p<0.05), followed by 1wt% and 37wt% PA. Similarly, the highest concentration of released HYP (i.e. meaning higher percentage of collagen degradation) and the highest activity in in situ zymography were observed when dentin samples were treated with 10wt% PA (p<0.05). CONCLUSIONS: It was confirmed that PA does not denature endogenous enzymes of dentin matrices, but it may somehow modulate the expression and activity of these enzymes in a concentration-dependent manner. CLINICAL SIGNIFICANCE: Endogenous proteases have been identified and suggested to be responsible for the digestion of dentin matrix when activated by the acidic components of dental adhesives. Proteolytic activity of dentinal MMPs showed to be dependent on phosphoric acid concentration. The clinically-used concentration (37%) does not inhibit MMPs activity, but slows it.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells.

OBJECTIVES: To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. METHODS: Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm(2)) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney's tests (p < 0.05). RESULTS: Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. SIGNIFICANCE: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.

Abundance of MMPs and cysteine cathepsins in caries-affected dentin.

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metalloproteases (MMPs) and cysteine cathepsins (CTs). Since this relationship has not been fully established, we hypothesized that the abundance of MMPs and CTs in caries-affected dentin must be higher than in intact dentin. To test this premise, we obtained 5 slices (200 µm) from 5 intact teeth and from 5 caries-affected teeth (1 slice/tooth) and individually incubated them with primary antibodies for CT-B, CT-K, MMP-2, or MMP-9. Negative controls were incubated with pre-immune serum. Specimens were washed and re-incubated with the respective fluorescent secondary antibody. Collagen identification, attained by the autofluorescence capture technique, and protease localization were evaluated by multi-photon confocal microscopy. The images were analyzed with ZEN software, which also quantitatively measured the percentages of collagen and protease distribution in dentin compartments. The abundance of the test enzymes was markedly higher in caries-affected than in intact dentin. CT-B exhibited the highest percentage of co-localization with collagen, followed by MMP-9, MMP-2, and CT-K. The high expression of CTs and MMPs in caries-affected teeth indicates that those host-derived enzymes are intensely involved with caries progression.

Tooth bleaching increases dentinal protease activity.

Hydrogen peroxide is an oxidative agent commonly used for dental bleaching procedures. The structural and biochemical responses of enamel, dentin, and pulp tissues to the in vivo bleaching of human (n = 20) premolars were investigated in this study. Atomic force microscopy (AFM) was used to observe enamel nanostructure. The chemical composition of enamel and dentin was analyzed by infrared spectroscopy (FTIR). The enzymatic activities of dental cathepsin B and matrix metalloproteinases (MMPs) were monitored with fluorogenic substrates. The amount of collagen in dentin was measured by emission of collagen autofluorescence with confocal fluorescence microscopy. The presence of Reactive Oxygen Species (ROS) in the pulp was evaluated with a fluorogenic 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) probe. Vital bleaching of teeth significantly altered all tested parameters: AFM images revealed a corrosion of surface enamel nanostructure; FTIR analysis showed a loss of carbonate and proteins from enamel and dentin, along with an increase in the proteolytic activity of cathepsin-B and MMPs; and there was a reduction in the autofluorescence of collagen and an increase in both cathepsin-B activity and ROS in pulp tissues. Together, these results indicate that 35% hydrogen peroxide used in clinical bleaching protocols dramatically alters the structural and biochemical properties of dental hard and soft pulp tissue.

Effects of potassium oxalate on knoop hardness of etch-and-rinse adhesives.

The objective of this study was to determine whether the hardness of etch-and-rinse adhesives may be affected by the pretreatment of acid-etched dentin with potassium oxalate desensitizer. Unerupted human third molars were cut into crown segments by removing the occlusal enamel and roots. The pulp chamber of these crown segments was connected to a syringe barrel filled with phosphate-buffered saline so that the moisture of dentin was maintained during the bonding procedures. Three etch-and-rinse adhesives-two two-step systems (Adper Single Bond 2 [SB], One-Step [OS]) and one three-step system (Adper Scotchbond Multi-Purpose [MP])-were applied to acid-etched dentin that had been treated (experimental groups) or not (control groups) with potassium oxalate (BisBlock). The Knoop hardness (KHN) of adhesives was taken at different sites of the outer surface of the adhesive-bonded dentin. The KHN of the three tested adhesives applied to acid-etched dentin treated with potassium oxalate was significantly lower than that exhibited by the respective controls (not treated with oxalate; p<0.05). Regardless of the adhesive, the treatment with potassium oxalate reduced the adhesives' KHN (p<0.05), with the OS system exhibiting the lowest KHN compared with the MP and SB systems.

Chlorhexidine inhibits the activity of dental cysteine cathepsins.

The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2' of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.

Effect of air-drying on the solvent evaporation, degree of conversion and water sorption/solubility of dental adhesive models.

This study evaluated protocols to eliminate acetone from dental adhesives and their effect on the kinetic of water sorption and percent of conversion of these adhesives. Experimental methacrylate-based adhesives with increasing hydrophilicity (R2, R3, R5) were used as reference materials. Primer-like solutions were prepared by addition of 50 wt% acetone. Acetone elimination was measured gravimetrically before and after: a spontaneous evaporation, an application of air-drying at room temperature or application of 40°C air-drying. Protocols were performed from 15 to 60 s. Specimens of adhesive/acetone mixtures were photo-activated and tested for degree of conversion, water sorption and solubility. Data were analyzed by ANOVA and Bonferroni's tests (α = 0.05). Complete acetone elimination was never achieved, but it was significantly greater after the 40°C air-drying application. Higher acetone elimination was observed for the least hydrophilic adhesive. Longer periods for acetone evaporation and heated air-stream can optimize polymerization and reduce the water sorption/solubility of adhesive system models.

Finite element analysis of stress distribution in intact and porcelain veneer restored teeth.

UNLABELLED: The aim of this study was to investigate the stress distribution generated in a veneer restoration of an upper central incisor compared to intact teeth using the finite element analysis after applying a lingual buccal loading at the incisal edge. METHODS: Two models were developed: one model contained enamel, dentine, cementum, periodontal ligament, cortical and trabecullar bones, and the other model was a veneer restoration; both models were developed using MSC/Nastran software (MacNeal-Schwendler Corporation, Los Angeles, CA, USA) as the pre- and post-processor. A 10-N load was applied at the incisal edge from the lingual to the buccal side to simulate oral conditions in this area (protrusion). RESULTS: Von Mises stresses were then analysed for three different regions: A-B (enamel elements under the veneer or second enamel layer), A'-B' (buccal enamel and/or veneer element layer) and C-D (lingual enamel elements layer). A higher stress mode was observed for both models at the lingual cervical region. CONCLUSIONS: The presence of a veneer restoration on the incisors is a good alternative to mimic the behaviour of enamel under protrusion loading conditions. The use of veneers to replace enamel during rehabilitations is recommended.

Cysteine cathepsins in human carious dentin.

Matrix metalloproteinases (MMPs) are important in dentinal caries, and analysis of recent data demonstrates the presence of other collagen-degrading enzymes, cysteine cathepsins, in human dentin. This study aimed to examine the presence, source, and activity of cysteine cathepsins in human caries. Cathepsin B was detected with immunostaining. Saliva and dentin cysteine cathepsin and MMP activities on caries lesions were analyzed spectrofluorometrically. Immunostaining demonstrated stronger cathepsins B in carious than in healthy dentin. In carious dentin, cysteine cathepsin activity increased with increasing depth and age in chronic lesions, but decreased with age in active lesions. MMP activity decreased with age in both active and chronic lesions. Salivary MMP activities were higher in patients with active than chronic lesions and with increasing lesion depth, while cysteine cathepsin activities showed no differences. The results indicate that, along with MMPs, cysteine cathepsins are important, especially in active and deep caries.

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.

Chlorhexidine Inhibits the Proteolytic Activity of Root and Coronal Carious Dentin in vitro.

The purpose of this study was to evaluate the effect of chlorhexidine on the proteolytic activity of carious coronal and root dentin collected from patients. Sound dentin from freshly extracted human teeth was used as a control. Dentin fragments were mixed with a synthetic substrate for proteolytic enzymes (N-benzoyl-DL-arginine-naphthylamide--BANA) and the suspensions mixed with either 0.12% chlorhexidine digluconate or distilled water. These mixtures were incubated for 18 h at 37 degrees C, color was developed by the addition of 0.1% Fast Garnet and their optical density was recorded spectrophotometrically. BANA hydrolysis measured by the optical density of incubated specimens was detected in all tested groups, but was significantly higher for carious than for sound dentin (p < 0.05). The proteolytic activity was reduced for carious coronal and root dentin by chlorhexidine (p < 0.05; 50 and 30%, respectively). Chlorhexidine also reduced the proteolytic activity in sound root dentin (p < 0.05; 20%). Conversely, changes in the proteolytic activity of sound coronal dentin were not observed in the presence of chlorhexidine. The reduction in proteolytic activity by chlorhexidine was significantly higher in carious coronal dentin than in carious root dentin (p < 0.05). In conclusion, part of the effect of chlorhexidine in controlling caries progression in humans may be due to a decrease in the proteolytic activity of carious coronal and root dentin. Because of the prolonged incubation time in the present study, similar results may be obtained clinically with prolonged dentin exposure to chlorhexidine, e.g. chlorhexidine-containing varnishes.

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.

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.

Interaction of resin-modified glass-ionomer cements with moist dentine.

OBJECTIVES: The objective of this study was to report on a novel phenomenon that occurs when resin-modified glass-ionomer cements (RMGICs) are bonded to moist human dentine. METHODS: Dentine surfaces from extracted third molars were abraded with 180-grit SiC paper. Ten teeth were prepared for each of the two RMGICs tested (Fuji II LC, GC Corp. and Photac-Fil Quick, 3M ESPE). RMGIC buildups were made according to the manufacturers' instructions. After storage at 37 degrees C, 100% humidity for 24 h, the bonded specimens were cut occlusogingivally into 0.9 x 0.9 mm beams. Dentine surfaces bonded with the two RMGICs were examined along the fractured RMGIC/dentine interfaces. Additional beams fractured within the RMGICS and at 3 mm away from the interfaces were used as controls. The fractured beams were examined using scanning electron microscopy (SEM), field emission-environmental SEM (FE-ESEM) and transmission electron microscopy (TEM). RESULTS: SEM and FE-ESEM revealed numerous solid spherical bodies along the RMGIC/dentine interfaces. By contrast, no spherical bodies could be identified within the RMGIC fractured 3 mm distant from the bonded interface. TEM and energy dispersive X-ray analyses performed on carbon-coated ultrathin sections showed that these solid spherical bodies consisted of a thin aluminum and silicon-rich periphery and an amorphous hydrocarbon core within the air voids of the original resin matrix. CONCLUSION: The spherical bodies probably represent a continuation of GI reaction and poly(HEMA) hydrogel formation that results from water diffusion from the underlying moist dentine. Their existence provides evidence for the permeation of water through RMGIC/dentine interfaces.

Effect of resin hydrophilicity and water storage on resin strength.

This study evaluated the change in the ultimate tensile strength (UTS) of five polymerised resin blends of increasing hydrophilicity, after ageing in distilled water or silicon oil. Resin blocks were prepared from each resin blend by dispensing the uncured resin into a flexible, embedding mould, containing multiple cavities. The resins were polymerised in the moulds under nitrogen at 551.6 kPa and light-activated at 125 degrees C for 10 min. After dry ageing for 24 h at 37 degrees C, the middle third of each resin specimen was trimmed into an 'I' shape. Fifteen control specimens were randomly selected from each resin blend for baseline UTS evaluation. The UTS of the experimental specimens were determined after 1, 3, 6 and 12 months of ageing in water or oil. The UTS of each group of resins at different storage periods in water or oil were analysed using the Friedman multiple ANOVA on ranks and Dunn's multiple comparison tests at 95% confidence level. Significant reduction (p < 0.01) in UTS was observed in Groups II-V resins after 12-month storage in water, while the most hydrophobic Group I resin showed no significant change (p > 0.05) in the same period. The percentage reduction in UTS increased with the hydrophilicity of the resin blends. Long-term water storage of hydrophilic resin blends such as those employed in dentine adhesives, resulted in a marked reduction in their mechanical strength that may compromise the durability of resin-dentine bonds.

Re-attachment of anterior fractured teeth: fracture strength using different techniques.

Fracture of anterior teeth by trauma is a common problem in children and teenagers. Complex metal-ceramic crowns with considerable loss of remaining sound structure are no longer necessary due to adhesive techniques, such as composite restorations and re-attachment techniques. This study compared the fracture strength of sound and restored anterior teeth using a resin composite and four re-attachment techniques. A "one bottle" adhesive system (One-Step, BISCO) and a dual cure resin cement (Duo-Link, BISCO) were applied. Thirty-five sound permanent lower central incisors were fractured by an axial load applied to the buccal area and randomly divided into five groups. The teeth were restored as follows: 1) bonded only = just bonding the fragment; 2) chamfer-group = after bonding, a chamfer was prepared on the enamel at the bonding line and filled with composite; 3) overcontour group = after bonding, a thin composite overcontour was applied on the buccal surface around the fracture line; 4) internal dentinal groove = before bonding, an internal groove was made and filled with a resin composite; 5) resin composite group = after a bevel preparation on the enamel edge, the adhesive system was applied and the fractured part of the teeth rebuilt by resin composite. Restored teeth were subjected to the same loading in the same buccal area. Fracture strength after restorative procedure was expressed as a percentage of the original fracture strength and the results analyzed by Kruskal-Wallis statistical analysis. The mean percentages of fracture strength were: Group 1: 37.09%, Group 2: 60.62%, Group 3: 97.2%, Group 4: 90.54% and Group 5: 95.8%. It was concluded that the re-attachment techniques used in Groups 3 and 4, as well as the composite restored group (Group 5), were statistically similar and reached the highest fracture resistance, similar to the fracture resistance of sound teeth.

Clinical evaluation of posterior composite restorations: 6-year results.

PURPOSE: To evaluate the wear resistance of resin restorations (Z100; Tetric; Charisma) in posterior teeth (Class I and II) after 6 years. MATERIAL AND METHODS: One operator placed 103 restorations in 13 patients. Each patient had at least three restorations with three different resin-based composites. All restorations were made using rubber dam isolation and the cavity design was restricted to the elimination of carious tissue. Deeper cavities were covered with calcium hydroxide and/or glass ionomer cement and in shallow and medium cavities only an adhesive system was used. Each composite was placed according to the manufacturer's instructions. In Class II cavities the resin placement followed the Krejci et al or Opdam et al technique, according to the cavity size. One week later, the restorations were finished/polished and stone dies were immediately built from the impressions. Eleven patients attended the 6-year recall, and 90 restorations were evaluated based on new stone dies. The 6-year stone dies were compared with the baseline ones by two examiners. The evaluation was based on the modified Mahler et al. criteria and Busato et al, using six different scores. The scores were statistically analyzed. RESULTS: A total of 87% of the restorations were analyzed after 6 years. Only 15% (6 for Tetric and 7 for Charisma) of the 90 evaluated restorations had been already replaced. No statistical difference was found in the wear rate of the composites used in this study. None of the patients complained of any symptom after the placement of the composite (baseline data) nor after 6 years.

Microtensile bond strengths of one-bottle dentin adhesives.

PURPOSE: To investigate the in vitro performance of three one-bottle adhesives by measuring their dentin microtensile strength. MATERIALS AND METHODS: Extracted human molars had the dentin exposed on the occlusal surface. The teeth were randomly assigned to three dentin adhesives: Group 1 - Prime & Bond 2.1; Group 2 - Prime & Bond NT; and Group 3 - Experimental (unfilled NT). All materials were applied as recommended by the manufacturer. A high-density resin composite (Surefil) was packed on the bonding area in five increments. Specimens (sticks) were obtained using a diamond disc and were stored in distilled water for 24 hrs. The microtensile bond strengths were measured with a universal testing machine. RESULTS: Group 1: 54.3+/-15.6 MPa; Group 2: 62.0+/-17.6 MPa; Group 3: 54.6+/-16.8 MPa. Although no statistically significant difference was found between pairs of means, Prime&Bond NT showed a tendency for higher bond strengths.