Direct and transdentinal biostimulatory effects of grape seed extract rich in proanthocyanidin on pulp cells.

AIM: To evaluate the potential biostimulatory effects of grape seed extract (GSE) on a primary culture of human pulp cells. METHODOLOGY: Human molars were used to obtain the primary pulp cell culture and 0.5-mm dentine discs. For GSE direct exposure, dose-response (0.0065-6.5%) and time response (1-60 min of contact) were examined. For transdentinal exposure, 0.65% of GSE was tested for 24 h. Cellular metabolism, nitric oxide and collagen production, and cell morphology alterations were assessed at periods of 24 and 72 h. After cell differentiation and direct exposure to GSE, the total protein production (TP), alkaline phosphatase activity (ALP) and formation of mineralization nodules (MN) were assessed. The results were analysed by parametric tests or non-parametric tests (α = 0.05). RESULTS: The lower concentration of GSE tested (0.0065%) was associated with an increase in cellular metabolism, a reduction in the production of nitric oxide and an increase in extracellular matrix synthesis (collagen). Distinct behaviours were observed for the different concentrations, without a reduction of cellular metabolism >10% compared with the control, either when applied directly or transdentinally. SEM revealed no significant change in cell morphology, except for the positive control (H2 O2 ). There was no difference in TP, ALP or MN between the control group and the group exposed to GSE. CONCLUSIONS: Treatment with grape seed extract, even at the highest concentration and longest period, caused neither direct nor transdentinal cytotoxic effects on human pulp cells. Grape seed extract components may play a biostimulatory role and protect dental pulp cells when in direct contact.

Effect of gamma irradiation on ultimate tensile strength of enamel and dentin.

The effect of gamma irradiation therapy on the ultimate tensile strength (UTS) of enamel and dentin in relation to prism orientation, dentin tubule orientation, and location is unknown. It was hypothesized that tubule and prism orientation, location, and irradiation have an effect on the UTS of dental structures. Forty human third molars were used, half of which were subjected to 60 Gy of gamma irradiation, in daily increments of 2 Gy. The specimens were evaluated by microtensile testing. Results showed that irradiation treatment significantly decreased the UTS of coronal and radicular dentin and of enamel, regardless of tubule or prism orientation. With or without irradiation, enamel was significantly stronger when tested parallel to its prismatic orientation. Coronal and radicular dentin of non-irradiated specimens presented significantly higher UTS when tested perpendicularly to tubule orientation. However, when the teeth were irradiated, the influence of tubule orientation disappeared, demonstrating that irradiation is more harmful to organic components.

Influence of intracoronal bleaching agents on the ultimate strength and ultrastructure morphology of dentine.

OBJECTIVE: To evaluate the effects of intracoronal bleaching on ultimate tensile strength (UTS) of sound and etched dentine and its ultrastructure morphology. METHODOLOGY: Bovine dentine specimens with (e) or without previous etching with 37% phosphoric acid for 15 s were used for the intracoronal bleaching experiments. Teeth were randomly assigned to five treatments (n = 10): (C) control--no bleaching, (SP) sodium perborate, (CP) 35% carbamide peroxide, (25% HP) 25% hydrogen peroxide and (35% HP) 35% hydrogen peroxide. Bleaching was performed four times within a 72 h interval and afterwards, dentine pulp chamber blocks were obtained. The blocks were sectioned in 0.7 mm-thick slices and these were trimmed to reduce the inner dentine to a dumbbell shape with a cross-sectional area of 0.8 mm(2). Specimens were tested with the microtensile method (0.5 mm min(-1)) and data were analysed (two-way ANOVA-Tukey test, P < 0.05). Additional teeth were prepared for transmission electron microscopy (TEM) to evaluate dentine ultramorphology. RESULTS: The mean values of the UTS (SD) in MPa for sound dentine were: C = 48.3(8.5)a, SP = 34.6 (8.2)b, CP = 32.9 (8.9)b, 25% HP = 28.0(4.6)b, 35% HP = 26.4(6.6)b, and pre-etched dentine: Ce = 38.9(13.8)a, SPe = 31.3 (9.3)ab, CPe = 28.4 (6.2)ab, 25% HPe = 30.0 (7.9)ab, 35% HPe = 19.9(4.6)b. Significant differences between the means are indicated by the letters. TEM observations exhibited demineralization areas for all bleaching treatments. CONCLUSION: Bleaching decreased dentine UTS after treatment. Pre-etched not-bleached dentine (Ce) presented UTS similar to pre-etched bleached dentine, except for 35% HPe. The decrease of UTS of bleached dentine could be attributed to ultrastructural alterations such as loss of inorganic components.

Effects of natural cross-linkers on the stability of dentin collagen and the inhibition of root caries in vitro.

PURPOSE: To evaluate the effects of dentin collagen modifications induced by various cross-linkers on the stability of collagen matrix and the inhibition of root caries. MATERIALS AND METHODS: The following cross-linkers were tested: 5% glutaraldehyde (GA), 0.5% proanthocyanidin (PA), 0.625% genipin (GE). In the first experiment, cross-linker-treated demineralized human root dentin was digested with bacterial collagenase, centrifuged, and the supernatants were subjected to amino acid analysis to determine collagen content. The residues were analyzed by SDS-PAGE and hydroxyproline analysis. In the second experiment, bovine root surfaces were conditioned with phosphoric acid, treated with the cross-linkers, incubated with Streptococcus mutans and Lactobacillus acidophilus for 1 week and the root caries inhibition was evaluated with confocal microscopy. Lastly, the ability of the bacteria to colonize the root surface was evaluated. In this experiment slabs of bovine root were treated with the cross-linkers and incubated in a suspension of S. mutans and L. acidophilus. The slabs were washed, resuspended in water, glucose was added, and the pH measured. RESULTS: While all collagen was digested with collagenase in the control groups, only a small proportion was solubilized in the GA-, PA-, and GE-treated groups. The root caries was significantly inhibited by treatment with PA or GA. Drops in pH in the cross-linker-treated groups were essentially the same as in the untreated group. CONCLUSION: Naturally occurring cross-linkers, especially PA, could be used to modify root dentin collagen to efficiently stabilize collagen and to increase its resistance against caries.

Interfacial ultramorphology of single-step adhesives: nanoleakage as a function of time.

The aim of this study was to examine the effectiveness of single-step self-etching adhesives in preventing nanoleakage over a 90-day water-storage period, and analyse the ultramorphological characteristics of resin-dentin interfaces. Three single-step self-etching adhesives were evaluated: Adper Prompt L-Pop - LP (3M ESPE), iBond - iB (Heraeus Kulzer), and Clearfil Tri-S Bond - S3 (Kuraray). Bonded specimens were sectioned into 0.9-mm thick slabs and stored in water for 1, 60 or 90 days. After the storage periods, a silver tracer solution was used to reveal nanometer-sized spaces and evidence of degradation within resin-dentin interfaces. Epoxy resin-embedded sections were prepared, and the interfaces observed with the TEM. Nanoleakage patterns were compared among adhesives and storage periods using image analysis software. Data were statistically analysed by two-way anova and Tukey test. Nanoleakage was observed in all resin-dentin interfaces produced by the single-step self-etching adhesives. Results showed that LP presented the lowest silver deposition means at 1 day. However, after 60 and 90 days, the area of silver deposition significantly increased for LP. iB presented intense silver deposition after 1 day and a small increase after 90 days. S3 presented the lowest silver deposition means after 60 and 90 days of water-storage.

Removal of noncollagenous components affects dentin bonding.

The structural integrity of fibrillar type I collagen is critical for effective dentin bonding. Since most noncollagenous matrix components in dentin are closely associated with collagen, we hypothesized that they may also contribute to dentin bonding. To test this hypothesis, bovine dentin was acid-etched, treated with chondroitinase ABC (C-ABC), endo-beta-galactosidase (Endo-beta), or trypsin. Controls were prepared in the same manner but without the enzymes. All control and experimental specimens were then bonded with One-Step. Bond strength data were analyzed by one-way ANOVA and Fisher's PLSD test (p < 0.05). When dentin was treated with C-ABC or trypsin, bond strengths significantly decreased for the rewetted groups (p < 0.05). The treatment with Endo-beta showed no effects on bond strengths (p > 0.05). When the treated dentin surfaces were observed under SEM, the C-ABC and trypsin treated groups revealed significant loss of collagen fibril architecture. The results indicate that chondroitin sulfate glycosaminoglycans and trypsin-digestible noncollagenous proteins play roles in maintaining the open dimensions of the collagen fibril scaffold, which is essential for optimal dentin bonding.

Effect of blood contamination with 1-step self-etching adhesives on microtensile bond strength to dentin.

This study evaluated the effect of blood contamination and decontamination methods on the microtensile bond strength of 1-step self-etching adhesive systems to dentin contaminated after adhesive application and light curing. Three commercially available "all-in-one" adhesives (One Up Bond F, Xeno III and Adper Prompt L-Pop) and 1 resin composite (Clearfil AP-X) were used. Third molars that had been stored in distilled water with 0.5% thymol at 4 degrees C were ground with #600 SiC paper under running water to produce a standardized smear layer. The specimens were randomly divided into groups according to the 3 adhesive systems. The adhesive systems were used under 3 conditions: no contamination, which was the control (C); contamination of the light-cured adhesive surface with blood and reapplication of adhesive (Contamination 1) and contamination of the light-cured adhesive surface with blood, then washing, drying and reapplication of the adhesive (Contamination 2). Following light curing of the adhesive, the resin composite was placed in 3 increments up to a 5-mm-thick layer on the bonded surface. All specimens were stored in distilled water at 37 degrees C for 24 hours. The microtensile bond strength was measured using a universal testing machine (EZ test), and data were analyzed by 1-way ANOVA followed by the Duncan test to make comparisons among the groups (p=0.05). After debonding, 5 specimens were selected from each group and examined in a scanning electron microscope to evaluate the modes of fracture. For all adhesives, contamination groups showed lower bond strength than the control (p<0.05). There was no statistically significant difference among the control groups (p>0.05). For Xeno III and Adper Prompt L-Pop, contamination group #2 showed the lowest bond strength among the groups (p<0.05). For One Up Bond F, contamination group #2 showed higher bond strength than contamination group #1 but showed no statistical significance between them (p>0.05).

Effect of saliva contamination on the microshear bond strength of one-step self-etching adhesive systems to dentin.

This study evaluated the effect of saliva contamination and decontamination methods on the dentin bond strength of one-step self-etching adhesive systems. Three commercially available "all-in-one" adhesives (One Up Bond F, Xeno III and Adper Prompt) and one resin composite (Filtek Z-250) were used. Third molars stored in distilled water with 0.5% thymol at 4 degrees C were ground with #600 SiC paper under running water to produce a standardized smear layer. The specimens were randomly divided into groups according to contamination methods: no contamination, which was the control (C); contamination of the adhesive surface with fresh saliva before light curing (A) and contamination of the adhesive surface with fresh saliva after light curing (B). Each contamination group was further subdivided into three subgroups according to the decontamination method: A1-Saliva was removed by a gentle air blast and the adhesive was light-cured; A2-Saliva was rinsed for 10 seconds, gently air-dried and the was adhesive light-cured; A3-Saliva was rinsed and dried as in A2, then the adhesive was re-applied to the dentin surface and light-cured; B1-Saliva was removed with a gentle air blast; B2-Saliva was rinsed and dried; B3-Saliva was rinsed, dried and the adhesive was re-applied and light cured. Tygon tubes filled with resin composite were placed on each surface and light cured. All specimens were stored in distilled water at 37 degrees C for 24 hours. Microshear bond strength was measured using a universal testing machine (EZ test), and data were analyzed by one-way ANOVA followed by the Duncan test to make comparisons among the groups (p<0.05). After debonding, five specimens were selected and examined in a scanning electron microscope to evaluate the modes of fracture. The A2 subgroup resulted in the lowest bond strength. For One Up Bond F and Adper Prompt, there was no significant difference between subgroup A1 and the control, and subgroup A3 and the control (p>0.05). Bond strengths of all B groups were significantly lower compared to the controls (p<0.05). For Xeno III, A1 subgroup showed the greatest decrease in bond strength as compared to the control (p<0.05). On the other hand, it showed more resistance to salivary contamination after adhesive curing. There was no statistically significant difference among the control groups (p>0.05).

Collagen cross-linking and ultimate tensile strength in dentin.

Several studies have indicated differences in bond strength of dental materials to crown and root dentin. To investigate the potential differences in matrix properties between these locations, we analyzed upper root and crown dentin in human third molars for ultimate tensile strength and collagen biochemistry. In both locations, tensile strength tested perpendicular to the direction of dentinal tubules (undemineralized crown = 140.4 +/- 48.6/root = 95.9 +/- 26.1; demineralized crown = 16.6 +/- 6.3/root = 29.0 +/- 12.4) was greater than that tested parallel to the tubular direction (undemineralized crown = 73.1 +/- 21.2/root = 63.2 +/- 22.6; demineralized crown = 9.0 +/- 3.9/root = 16.2 +/- 8.0). The demineralized specimens showed significantly greater tensile strength in root than in crown. Although the collagen content was comparable in both locations, two major collagen cross-links, dehydrodihydroxylysinonorleucine/its ketoamine and pyridinoline, were significantly higher in the root (by ~ 30 and ~ 55%, respectively) when compared with those in the crown. These results indicate that the profile of collagen cross-linking varies as a function of anatomical location in dentin and that the difference may partly explain the site-specific tensile strength.

Effect of thermal and mechanical load cycling on microtensile bond strength of a total-etch adhesive system.

To evaluate the effect of thermal and mechanical cycles on dentin bond strength to cervical margins of Class II restorations, 80 box-type Class II cavities were prepared on the surfaces of bovine incisors. The cavities were restored with Single Bond (3M-ESPE) and Z-250 composite (3M-ESPE) according to manufacturer's instructions. The incisors were divided into four groups: G1-Control, G2- Thermal cycling (2,000 cycles, 5 degrees C-55 degrees C), G3- Mechanical cycling (100,000 cycles; 50N) and G4- Thermal and mechanical cycling (2,000 cycles 5 degrees C-55 degrees C/100,000 cycles; 50N). The restorations were sectioned perpendicular to the cervical bonded interface into 0.7 +/- 0.2 mm-thick slabs. The slabs were further trimmed at the interface to 1.4 +/- 0.2 mm with a fine diamond bur to produce a cross-sectional surface area of 1 mm2. All specimens were then subjected to microtensile bond testing. Means and standard deviations were expressed in MPa. The bond strength data were analyzed by one-way ANOVA and Fisher's PLSD test (p<0.05). Fracture mode analysis was performed using SEM. Bond strengths were significantly lower when thermal and mechanical cycling were performed [G4-2.41 (8.57)] when compared to the other groups [G1-28.15 (14.03); G2-27.60 (10.14); G3-27.59 (8.67)]. No differences were observed among Groups 1, 2 and 3. Interfacial fracture of the control (G1) and thermocycling (G2) groups mainly occurred between the deepest portion of the adhesive resin and the top layer of the demineralized dentin (Interphase). Mixed failure was predominant and increased when mechanical cycling was applied (G3 and G4).

Dimensional changes and ultimate tensile strengths of wet decalcified dentin applied with one-bottle adhesives.

OBJECTIVES: The objectives of this study were to test the null hypothesis that no dimensional changes in wet decalcified dentin matrices will occur during the application of one-bottle adhesives, and to evaluate the ultimate tensile strengths (UTS) of resin-infiltrated dentin matrices using the microtensile test. METHODS: Dentin disks 0.2 mm thick were prepared from mid-coronal dentin of human unerupted third molars. They were completely decalcified in 0.5 M EDTA (pH 7.4) for 5 days at 25 degrees C and then placed in the bottom of an aluminum well to permit use of the LVDT portion of a thermal mechanical analyzer. Changes in matrix height in response to the application of Single Bond, One-Step or Prime & Bond NT were measured along with the UTS of resin-infiltrated specimens that were compared using a one-way ANOVA and Fisher's PLSD test. RESULTS: All one-bottle adhesives produced a gradual, progressive shrinkage of the decalcified matrix of 26-33%. The shrinkage produced by Single Bond was significantly greater (p<0.05) than that produced by the other adhesives. The UTS of One-Step was significantly higher (<0.05) than that of Prime & Bond NT (42.0+/-13.6 vs 29.7+/-1.9 MPa, respectively), with Single Bond producing intermediate UTS (34.3+/-7.4 MPa). SIGNIFICANCE: If the durability of resin-dentin bonds depends upon the size of interfibrillar spaces for both diffusion channels and resin uptake, then adhesive formulations should be designed to minimize matrix shrinkage during resin infiltration.

Effects of mechanical properties of adhesive resins on bond strength to dentin.

OBJECTIVES: The purpose of this study was to evaluate the relationship between the micro-tensile bond strength to dentin and mechanical properties of the cured adhesive resins. METHODS: Coronal dentin surfaces of extracted human teeth were treated with four commercial self-etching priming systems (Clearfil SE Bond; UniFil Bond; Tokuso Mac-Bond II; and Imperva Fluoro Bond) and bonded with a resin composite. After 24h storage in water at 37 degrees C, the bonded specimens were trimmed and subjected to micro-tensile bond strength testing at a cross-head speed of 1mm/min. Debonded surfaces were observed under a FE-SEM. For testing mechanical properties, 0.7-mm thick slabs of each adhesive resin were prepared, light-cured, and stored dry at the room temperature for 24h. After trimming, ultimate micro-tensile strength was measured. The nano-hardness and Young's modulus were also evaluated using cured adhesives that were prepared in the same manner as described above. RESULTS: The micro-tensile bond strengths to dentin and ultimate micro-tensile strengths of the resins were not significantly different among all systems (P>0.05). However, the nano-hardness and Young's modulus of Clearfil SE Bond and Imperva Fluoro Bond adhesive resins were significantly higher than those of UniFil Bond and Tokuso Mac-Bond II resins (P<0.05). The micro-tensile bond strength significantly correlated with the ultimate micro-tensile strength of the resins (r(2)=0.77; P<0.05), but was not correlated with the nano-hardness or Young's modulus (P>0.05). SEM observation of the debonded surfaces revealed a mixed type of fracture with a combination of interfacial and cohesive failure within the adhesive resin. SIGNIFICANCE: The four self-etching priming systems exhibited similar dentin bond strengths, which also correlates with the ultimate strength of the adhesive resins.