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 2009 The Authors. Journal compilation  2009 Eur J Oral Sci

Eur J Oral Sci 2009; 117: 587–596 Printed in Singapore. All rights reserved

European Journal of Oral Sciences

Influence of chlorhexidine digluconate concentration and application time on resin–dentin bond strength durability

Alessandro D. Loguercio1, Rodrigo Stanislawczuk1, Luceli G. Polli1, Jully A. Costa1, Milton D. Michel2, Alessandra Reis1 1

Department of Restorative Dentistry, School of Dentistry, Universidade Estadual de Ponta Grossa, PR, Brazil; 2Department of Mechanical Engineering, School of Mechanical Engineering, University Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil

Loguercio AD, Stanislawczuk R, Polli LG, Costa JA, Michel MD, Reis A. Influence of chlorhexidine digluconate concentration and application time on resin–dentin bond strength durability. Eur J Oral Sci 2009; 117: 587–596.  2009 The Authors. Journal compilation  2009 Eur J Oral Sci Although it is known that chlorhexidine application may preserve resin–dentin bonds from degradation, the lowest optimal concentration and application time have yet to be established. This study evaluated the effects of different concentrations of chlorhexidine digluconate and different application times on the preservation of resin– dentin bonds formed using two etch-and-rinse adhesives. In experiment 1, after acid etching, the occlusal demineralized dentin was rewetted either with water or with 0.002, 0.02, 0.2, 2, or 4% chlorhexidine for 60 s. In experiment 2, the surfaces were rewetted with water, or with 0.002% or 2% chlorhexidine for 15 or 60 s. After this, both adhesives and composite resin were applied and light-cured. Bonded sticks (0.8 mm2) were tested under tension (0.5 mm min)1) immediately or after 6 months of storage in water. Two bonded sticks from each tooth were immersed in silver nitrate and analyzed quantitatively using scanning electron microscopy. Reductions in microtensile bond strengths and higher silver nitrate uptake were observed for both adhesives when the rewetting procedure was performed with water. Stable bonds were maintained for up to 6 months under all chlorhexidine conditions tested, irrespective of the chlorhexidine concentration and application time. The use of 0.002% chlorhexidine for 15 s seems to be sufficient to preserve resin–dentin interfaces over a 6month period.

Recent studies have pointed out that dentin bondstrength values measured immediately after formation of the bond do not always correlate with long-term bond stability (1), as degradation throughout the dentin bonding interface occurs rapidly (i.e. within 6 months) (2, 3). Bonding is created by impregnating the dentin substrate with blends of resin monomers, and the stability of the bonded interface relies on the creation of a compact and homogenous hybrid layer. In the etchand-rinse strategy, after preliminary etching to demineralize the substrate, resin monomers impregnate the porous etched substrate (4, 5) and thus stable bonds may be achieved if the etched substrate is fully infiltrated by the adhesive (6). However, when incompletely infiltrated zones occur along the bottom of hybrid layers within the acid-etched dentin, a decreasing gradient of resin monomer diffusion, with denuded collagen fibrils (7–9), is likely to occur. This region is susceptible to hydrolytic degradation in the long term, leading to reductions in bond strength (10). Moreover, the elution of unpolymerized monomers from the hybrid layer in contact with oral fluids is responsible for the accelerated process of bond interface degradation. This leaves more collagen fibrils within the hybrid layer exposed and susceptible to the degradation

Alessandro D. Loguercio, Universidade Estadual de Ponta Grossa – Mestrado em Odontologia, Rua Carlos Cavalcanti, 4748, Bloco M, Sala 64A – Uvaranas, Ponta Grossa, Paran 84030-900, Brazil Telefax: +55–42–32203741 E-mail: [email protected] Key words: adhesive system; application time; chlorhexidine concentration; dentin; longevity Accepted for publication May 2009

process (11). The combined degradation by hydrolysis of resin and/or collagen weakens the physical properties of the resin–dentin bond interface (11). Studies have revealed the contribution of host-derived proteinases, responsible for the breakdown of collagen matrices, in the pathogenesis of dentin caries (12, 13), periodontal disease (14), and in the degradation of dentin bonding (15). It has been reported that mineralized dentin contains collagenolytic and gelatinolytic enzyme activities derived from the action of matrix metalloproteinases (MMPs) (16–18). Both in vitro and in vivo studies have shown that 2% chlorhexidine digluconate (CHX), applied for 60 s on demineralized dentin, postpones the resin–dentin degradation of adhesive interfaces, when compared with interfaces to which no CHX is applied (19–22). Despite these advantages, the use of 2% CHX for 60 s demands more chair-time during the adhesive procedure and this contrasts with the cliniciansÕ needs for simplification (23). It is also worthwhile mentioning that CHX solutions with concentrations (close to 0.01%) are highly cytotoxic to cultured cells (24, 25). Obviously, when CHX solution is applied as a cavity cleanser there is enough dentin structure to act as a barrier and protect the pulp cells against the cytotoxic effects of dental materials or rinsing

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solutions. However, no data have so far been published concerning the capacity of CHX molecules to diffuse through dentin tubules and reach the pulpal cells. The use of very low concentrations of CHX has been shown to strongly inhibit the collagenolytic activity of pure MMPs (26) and dentin matrix-bound MMPs (27). Specific host-derived MMPs, responsible for the breakdown of the collagen matrices (MMP-2, MMP-8, and MMP-9), can be inhibited by low concentrations of CHX (0.0001–0.02%) (26). Fortunately, no cytotoxic effect on odontoblast-like cells has been reported for CHX at these concentrations (25). However, to the best of the authorÕs knowledge, no study has so far evaluated the effects of CHX concentrations on the durability of resin–dentin bonding. The application time of MMP inhibitors is still a matter to be addressed. In a recent investigation, Stanislawczuk et al. (28) showed that when 2% CHX containing phosphoric acid was applied for 15 s the durability of the resin–dentin bonds was preserved. This seems to indicate that even a short period of CHX in contact with the demineralized dentin appears to be sufficient to inhibit the action of specific host-derived proteinases. Therefore, the ideal situation would be to apply CHX at a low concentration, and also for a short period of time, as part of the strategy to simplify the bonding protocol. Therefore, the aim of this study was to evaluate the effect of different concentrations of CHX and different application times on the durability of the dentin bonds. To do so, the study was divided into two experiments. The objective of the first experiment was to evaluate the early and 6-month resin–dentin bond strength and silver nitrate uptake of two-step etch-and-rinse adhesives rewetted with different CHX concentrations (0.002, 0.02, 0.2, 2, and 4%) applied for 60 s. The second experiment examined the early and 6-month resin–dentin bond strength and the silver nitrate uptake (SNU) pattern of two-step etch-and-rinse adhesives after rewetting the demineralized dentin with two CHX concentrations (0.002 and 2%) applied for two different application times (15 and 60 s). It was hypothesized that the lowest concentration of CHX applied for only 15 s would be

sufficient to prevent reductions in resin–dentin bond strength and deposition of SNU within adhesive and hybrid layers after 6 months of water storage.

Material and methods Preparation of teeth

One-hundred and twenty extracted, caries-free human third molars were used. The teeth were collected after obtaining the patientÕs informed consent. The Ponta Grossa State University Review Board approved this study. Teeth were disinfected in 0.5% chloramine, stored in distilled water, and used within 6 months of extraction. A flat and superficial dentin surface was exposed on each tooth after wet grinding the occlusal enamel on #180-grit silicon carbide (SiC) paper. The enamel-free, exposed dentin surfaces were further polished on wet #600-grit SiC paper for 60 s to standardize the smear layer. Microtensile bond strength (lTBS) test – Two different solvent-based, etch-and-rinse adhesive systems were tested: Adper Single Bond (SB; 3M ESPE, St Paul, MN, USA), an ethanol/water-based system; and Prime & Bond 2.1 (PB; Dentsply De Trey, Konztanz, Germany), an acetone-based system (Table 1). For the first experiment, 60 teeth were divided into 12 groups (n = 5) according to the combination of the main factors ÔadhesiveÕ (two levels) and Ôrewetting solutionÕ (six levels). The exposed dentin surfaces were conditioned with the respective phosphoric acids of each adhesive system for 15 s, rinsed off (15 s), air-dried (Table 1), and rewetted either with water (control group) or with aqueous solutions of 0.002, 0.02, 0.2, 2, or 4% CHX (Fleming drugstore, Ponta Grossa, PR, Brazil). All rewetting procedures were performed for 60 s. Afterwards, the adhesive systems were applied in accordance with the manufacturerÕs directions (Table 1). The aim of this first experiment was to select two extreme CHX concentrations that had beneficial effects on the preservation of the resin–dentin bonds over a period of 6 months.

Table 1 Adhesive systems: composition, groups, and application mode Adhesive systems Prime & Bond 2.1 (PB) (Dentsply De Trey, Konstanz, Germany) Adper Single Bond (SB) (3M ESPE, St Paul, MN, USA)

Composition

Application mode*

1. Caulk Tooth Conditioner Gel 34% phosphoric acid 2. Adhesive – UDMA, Bis-GMA, PENTA, butylated hydroxitoluene, 4-ethyl dimethyl aminobenzoate, cetilamine hydrofluoride, initiator, and acetone 1. Scotchbond Etchant: 37% phosphoric acid 2. Adhesive – Bis-GMA, HEMA, dimethacrylates, polyalkenoic acid copolymer, initiators, water, and ethanol

a, b, c, d, e1, f, g

a, b, c, d, e2, f, g

*a, acid-etch (15 s); b, rinse (15 s); c, air-dry (30 s); d, dentin rewetted with water or chlorhexidine digluconate (CHX) solution for 15 or 60 s; e1, two coats of adhesive were applied for 20 s; e2, one coat of adhesive was applied for 10 s; f, air-dry for 10 s at 20 cm; g, light-cure (10 s; 600 mW cm)2). Bis-GMA, bisphenol A diglycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; PENTA, dipentaerythritol pentacrylate monophosphate; UDMA, urethane dimethacrylate.

Chlorhexidine on dentin adhesion

For the second experiment, 60 teeth were divided into 12 groups (n = 5) according to the combination of the main factors ÔadhesiveÕ (two levels), Ôrewetting solutionÕ (three levels), and Ôapplication timeÕ (two levels). The dentin surfaces were acid etched with phosphoric acid, rinsed off, air-dried, and rewetted either with water (control group) or with aqueous solutions of 0.002 or 2% CHX (Fleming drugstore). In this case, all rewetting procedures were performed for 15 or 60 s. Afterwards, thee adhesive systems were applied according to the manufacturerÕs instructions. For both experiments, the light-curing procedure was performed by means of a quartz-tungsten-halogen light (VIP; Bisco, Schaumburg, IL, USA; 600 mW cm)2) for the recommended time (10 s). Resin-composite build-ups (Opallis; FGM, Joinville, SC, Brazil) were placed on the bonded surfaces (three increments of approximately 1.5 mm each) and individually light activated for 40 s each. All bonding procedures were carried out by a single operator, and at 24C and 50% relative humidity. After the bonded teeth had been stored in distilled water at 37C for 24 h, they were longitudinally sectioned in both ÔxÕ and ÔyÕ directions across the bonded interface using a diamond saw in a Labcut 1010 machine (Extec, Enfield, CT, USA), under water cooling at 300 r.p.m., to obtain bonded sticks with a cross-sectional area of approximately 0.8 mm2. The number of prematurely debonded sticks (D) per tooth during specimen preparation was recorded. The remaining dentin thickness (RDT) was measured with a caliper and recorded (Absolute Digimatic, Mitutoyo, Tokyo, Japan). The cross-sectional area of each stick was measured with the digital caliper to the nearest 0.01 mm and recorded for subsequent calculation of the lTBS. The bonded sticks that originated from the same teeth were randomly divided and assigned to be tested immediately, or after 6 months of storage in distilled water at 37C. The storage solution was not changed and its pH was monitored monthly. Each bonded stick was attached to a jig in the universal testing machine (Emic, Sa˜o Jose´ dos Pinhais, PR, Brazil) with cyanoacrylate resin (Zapit; Dental Ventures of North America, Corona, CA, USA) and subjected to a tensile force of 0.5 mm min)1. The failure modes were evaluated at 400· magnification (microhardness HMV-2; Shimadzu, Tokyo, Japan) and classified as cohesive (failure exclusively within the dentin or composite; C), adhesive (failure at resin/dentin interface; A), or adhesive/mixed (failure at the resin–dentin interface that included cohesive failure of the neighboring substrates; A/M). SNU measurement under scanning electron microscopy – For both experiments, the amount of SNU into the hybrid layer and the adhesive layer was measured. Two bonded sticks from each tooth for each storage period were coated with two layers of nail varnish applied up to within 1 mm of the bonded interfaces. The specimens were rehydrated in distilled water for 10 min before immersion in the tracer solution for 24 h. Ammoniacal silver nitrate was prepared according to the protocol

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previously described by Tay et al. (29). The sticks were placed in ammoniacal silver nitrate in the dark for 24 h, rinsed thoroughly in distilled water, and immersed in photo developing solution for 8 h under fluorescent light to reduce silver ions into metallic silver grains within voids along the bonded interface. All sticks were wet-polished with 600 SiC paper to remove the nail varnish. After this, the specimens were placed inside an acrylic ring, which was attached to a double-sided adhesive tape, and embedded in epoxy resin. After the epoxy resin had set, the thickness of the embedded specimens was reduced to approximately half by grinding with SiC papers under running water. Specimens were polished with a 1,000-grit SiC paper and 1 and 0.25 lm diamond paste (Buehler, Lake Bluff, IL, USA) using a polishing cloth. Specimens were ultrasonically cleaned and demineralized in a 50% phosphoric acid solution for 3 s followed by immersion in 1% NaOCl for 10 min. Next, specimens were air-dried for 24 h, mounted on aluminum stubs, and sputtered with gold (Sputter Coater IC 50; Shimadzu, Tokyo, Japan). Resin–dentin interfaces were analyzed using a scanning electron microscope (SSX-550; Shimadzu) operated in the back-scattering electron model. The working distance was 10 mm and the accelerating voltage (ACCV) was 15 Kv. Three pictures were taken of each specimen. The first picture was taken in the center of the stick. The other two pictures were taken 0.3 mm to the left and right of the first picture. As two sticks per tooth were evaluated and a total of five teeth were used for each experimental condition, 30 images were evaluated for each group. They were all taken by a technician who was blinded to the experimental conditions under evaluation. The relative percentage of SNU within the adhesive and hybrid layer areas was measured in all pictures using the uthscsa ImageTool 3.0 software (Department of Dental Diagnostic Science at The University of Texas Health Science Center, San Antonio, TX, USA) by an author blind to the test and control samples. First of all, the total area of the adhesive layer plus the hybrid layer was recorded. Then the area occupied by the silver nitrate deposits was delimitated by a software tool, summed, and the relative ratio between the total area vs. the impregnated areas was calculated to give the percentage of SNU within each specific bonding interface. The experimental unit in this study was the tooth half, because half of the sample was tested immediately and the other half was tested after 6 months. The lTBS values of all sticks from the same tooth half were averaged for statistical purposes. The prematurely debonded specimens were included in the tooth half mean. The average value attributed to specimens that failed prematurely during preparation was arbitrary and corresponded to approximately half of the minimum lTBS that could be measured in this study (ca. 5.0 MPa). For SNU (%), the mean SNU of all pictures originated from sticks from the same tooth half was averaged for statistical purposes. The SNU of every test group was expressed as the mean of the five tooth halves used per group and is expressed in per cent.

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Before submitting the data for analysis using the appropriate statistical test, the Kolmogorov–Smirnov test was performed to assess whether the data followed a normal distribution, and the Barlett¢s test for equality of variances was performed to determine if the assumption of equal variances was valid (30). After observing the normality of the data distribution and the equality of the variances, the lTBS (MPa) and SNU (%) data were subjected to appropriate statistical analysis. In the first experiment, the lTBS data and SNU were subjected to a two-way repeated-measures analysis of variance (chlorhexidine concentration vs. storage time) for each adhesive. The repeated measure was the storage time. The post hoc test (TukeyÕs test at a = 0.05) was used for pairwise comparisons. In the second experiment, the lTBS and SNU data were subjected to a three-way repeated-measures analysis of variance (chlorhexidine concentration vs. application time vs. storage time) for each adhesive. The repeated measure was the storage time. A post hoc test (TukeyÕs test at a = 0.05) was used for pairwise comparisons.

Results The mean cross-sectional area ranged from 0.78 to 1.02 mm2 and no significant difference among groups was detected (P > 0.05). The RDT for all specimens ranged from 2.7 to 3.3, indicating that the interfaces were located in mid-dentin (31). lTBS

For the first experiment, the cross-product interaction CHX concentration vs. storage time for each adhesive system was statistically significant (P < 0.05). Significant reductions in the lTBS were observed in the control group (i.e. when water was used instead of CHX as the rewetting agent) (Table 2). The two etch-and-rinse adhesives tested had approximately the same overall failure mode rates under similar experimental conditions (Table 3). For the second experiment, only the cross-product CHX concentration vs. storage time for both adhesives

was statistically significant (P < 0.05). Significant reductions in resin–dentin bond-strength values were observed for both adhesives when the demineralized dentin was rewetted with water, irrespective of the application time. By contrast, rewetting with CHX in the different concentrations and application times yielded stable resin–dentin bonds after 6 months of water storage (Table 4). The two etch-and-rinse adhesives tested had approximately the same overall failure mode rates under similar experimental conditions (Table 5). SNU

In experiment 1, the cross-product interaction CHX concentration vs. storage time was statistically significant for both adhesive systems (P < 0.05). For both adhesives, a lower percentage of SNU immediately after treatment was observed for the interfaces treated with CHX, irrespective of its concentration, when compared with the control group immediately after treatment (P = 0.01) (Table 6). After 6 months of water storage, a significantly higher SNU was observed for both adhesives under the experimental conditions; however, this uptake was much less pronounced for the CHX groups when compared with the controls (P = 0.001) (Table 6). Observe in Table 6 that among the different CHX concentrations used, 4% CHX showed the highest SNU, which differed statistically from that of the lower CHX concentrations (P = 0.01). In experiment 2, only the cross-product CHX concentration vs. storage time for each adhesive system was statistically significant (P < 0.05). For both adhesives, a lower percentage of SNU immediately after treatment was observed for the interfaces treated with CHX, irrespective of its concentration, when compared with the control group immediately after treatment (P = 0.01) (Table 7). After 6 months of water storage, significantly higher SNU was observed for both adhesives under the experimental conditions; however, this uptake was much less pronounced for the CHX-treated groups than for the controls (P = 0.001) (Table 7). Representative back-scattering scanning electron microscopy images captured at the resin–dentin interfaces from experiment 2 are depicted in Figs 1–4.

Table 2 Overall microtensile bond strength means (in MPa) and the respective standard deviations obtained in each experimental condition in experiment 1* Prime & Bond 2.1 Conditions Control 0.002% CHX 0.02% CHX 0.2% CHX 2% CHX 4% CHX

Immediate 32.0 28.2 29.9 30.9 34.2 26.7

± ± ± ± ± ±

3.2 A 3.4 A 5.3 A 4.7 A 5.1 A 4.7 A,B

Adper Single Bond 6 months

21.3 25.1 30.1 27.4 31.3 21.1

± ± ± ± ± ±

2.4 3.2 4.2 4.6 4.1 3.5

B A,B A A A B

Immediate 34.1 35.2 30.2 34.2 32.4 26.3

± ± ± ± ± ±

4.6 6.2 4.3 4.1 6.1 4.2

a a a a a a,b

6 months 24.2 31.1 27.3 36.2 28.3 24.3

± ± ± ± ± ±

5.4 5.3 5.1 4.0 3.5 4.2

b a a,b a a b

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter are not significantly different (P > 0.05). CHX, chlorhexidine digluconate.

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Chlorhexidine on dentin adhesion Table 3

Number and percentage of specimens (%) according to fracture pattern mode and the premature debonded specimens from each experimental condition from experiment 1* Prime & Bond 2.1 Immediate

Pattern fracture Control 0.002% CHX 0.02% CHX 0.2% CHX 2% CHX 4% CHX

A/M 24 38 38 35 39 30

C

(75.0) (77.6) (67.9) (87.5) (75) (88.2)

0 9 0 0 1 0

Adper Single Bond 6 months

Debonded

A/M

C

Immediate Debonded

(0) 8 (25.0) 34 (80.9) 0 (0) (18.4) 2 (4.0) 33 (71.7) 5 (10.9) (0) 18 (32.1) 36 (75.0) 11 (22.9) (0) 5 (12.5) 32 (94.1) 0 (0) (2.5) 9 (22.5) 36 (94.4) 0 (0) (0) 4 (11.8) 25 (76) 0 (0)

8 8 1 2 2 6

(19.1) (17.4) (2.1) (5.9) (5.6) (24)

A/M 26 36 26 32 37 38

C

(81.2) (73.5) (46.4) (80) (84.1) (77.6)

0 7 0 3 2 4

6 months

Debonded

A/M

(0) 6 (18.8) 29 (69) (14.3) 6 (12.2) 35 (76.1) (0) 30 (53.6) 36 (75) (7.5) 5 (12.5) 30 (88.2) (4.5) 5 (11.4) 34 (97.1) (8.2) 7 (14.2) 33 (70.2)

C 3 5 7 2 0 7

Debonded

(7.2) 10 (23.8) (10.9) 6 (13) (14.6) 5 (10.4) (5.9) 2 (5.9) (0) 1 (2.9) (14.9) 7 (14.9)

*A/M, adhesive/mixed fracture mode; C, cohesive fracture mode; CHX, chlorhexidine digluconate; Debonded, premature debonded specimens.

Table 4 Overall microtensile bond strength means (in MPa) and the respective standard deviations obtained in each experimental condition from experiment 2* Prime & Bond 2.1 Immediate Application time

15 s

Adper Single Bond 6 months

60 s

Immediate

15 s

60 s

15 s

6 months

60 s

15 s

60 s

Control 28.3 ± 4.3 A 32.4 ± 5.4 A 20.1 ± 4.2 B 21.2 ± 3.8 B 39.2 ± 5.4 a 41.5 ± 6.4 a 27.9 ± 6.2 b 25.4 ± 4.1 b 0.002% CHX 25.7 ± 2.4 A,B 29.2 ± 3.4 A 23.2 ± 4.1 A,B 27.0 ± 3.6 A,B 41.4 ± 4.8 a 43.2 ± 6.1 a 37.2 ± 6.1 a 40.1 ± 3.7 a 2% CHX 33.1 ± 6.5 A 31.3 ± 5.1 A 27.3 ± 4.2 A,B 28.1 ± 4.4 A 43.5 ± 4.1 a 41.2 ± 4.2 a 40.1 ± 5.7 a 37.6 ± 3.3 a

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter are not significantly different (P > 0.05). CHX, chlorhexidine digluconate.

Table 5 Number and percentage of specimens (%) according to fracture pattern mode and the premature debonded specimens of each experimental condition from experiment 2* Prime & Bond 2.1 Immediate Pattern fracture

A/M

C

Debonded

Adper Single Bond 6 months

A/M

Control (15 s) 27 (69.2) 02 (5.1) 10 (25.7) 38 (84.5) Control (60 s) 35 (85.4) 2 (4.9) 4 (9.7) 37 (84.1) 0.002% CHX (15 s) 42 (75.0) 0 (0) 14 (25.0) 39 (79.6) 0.002% CHX (60 s) 38 (82.6) 1 (2.2) 7 (15.2) 33 (82.5) 2% CHX (15 s) 41 (87.2) 2 (4.3) 4 (8.5) 39 (88.6) 2% CHX (60 s) 36 (94.7) 2 (5.3) 0 (0) 40 (88.9)

C 2 3 3 3 4 0

(4.4) (6.8) (6.1) (7.5) (9.1) (0)

Immediate

Debonded 5 4 7 4 1 5

(11.1) (9.1) (14.3) (10.0) (2.3) (11.1)

A/M 26 33 32 39 35 38

(74.3) (76.7) (80.0) (83.0) (85.4) (90.5)

C 2 7 6 2 1 3

(5.7) (16.3) (15.0) (4.3) (2.4) (7.1)

6 months

Debonded 7 3 2 6 5 1

(20.0) (7.0) (5.0) (12.7) (12.2) (2.4)

A/M 35 36 40 36 42 37

(85.4) (87.8) (95.2) (92.3) (85.7) (90.2)

C 4 2 1 0 3 4

(9.7) (4.9) (2.4) (0) (6.1) (9.8)

Debonded 2 3 1 3 4 0

(4.9) (7.3) (2.4) (7.7) (8.2) (0)

*A/M, adhesive/mixed fracture mode; C, cohesive fracture mode; CHX, chlorhexidine digluconate; Debonded, premature debonded specimens.

Discussion The results of this study confirm previously published findings that resin–dentin interfaces bonded with simplified etch-and-rinse adhesives can degrade after short periods of time (2, 3). One mechanism of degradation proposed in the literature is the incomplete impregnation of resin into the collagen network and hybrid layer itself (1, 11).

It has recently been speculated that the autodegradation of dentin collagen fibrils is responsible for such findings (15). This mechanism is initiated from beneath the bonded interface, with the breakdown of aciddemineralized collagen matrices by host-derived MMPs. The low, but persistent, endogenous collagenolytic activity can be completely inhibited by the use of protease inhibitors (15). Chlorhexidine digluconate has the potential to minimize the reductions in the resin–dentin

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Loguercio et al. Table 6

Overall mean percentage of silver nitrate uptake (%) within hybrid layer and adhesive layer and the respective standard deviations (in MPa) obtained in each experimental condition from experiment 1* Prime & Bond 2.1 Conditions

Immediate

Control 0.002% CHX 0.02% CHX 0.2% CHX 2% CHX 4% CHX

26.3 12.1 12.2 10.3 9.8 14.6

± ± ± ± ± ±

4.6 3.3 4.2 2.9 3.2 5.6

Adper Single Bond 6 months

B,C A A A A A

42.3 24.6 25.4 22.1 27.8 32.4

± ± ± ± ± ±

5.6 4.3 4.1 3.8 3.9 4.6

Immediate

D B B B B,C C

24.5 10.1 11.1 9.6 12.4 16.2

± ± ± ± ± ±

3.6 2.3 3.5 2.7 4.3 3.6

6 months

b,c a a a a a

38.1 22.9 21.4 22.5 24.5 27.4

± ± ± ± ± ±

6.5 4.2 2.4 3.2 5.4 3.6

d b b b b,c c

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter are not significantly different (P > 0.05). CHX, chlorhexidine digluconate.

Table 7 Overall mean percentage of silver nitrate uptake within hybrid layer and adhesive layer and the respective standard deviations (MPa) obtained in each experimental condition from experiment 2* Prime & Bond 2.1 Immediate Application time Control 0.002% CHX 2% CHX

Adper Single Bond 6 months

Immediate

6 months

15 s

60 s

15 s

60 s

15 s

60 s

15 s

60 s

28.7 ± 3.4 B 12.3 ± 2.6 A 11.2 ± 2.5 A

26.3 ± 4.2 B 12.6 ± 2.3 A 10.2 ± 5.2 A

39.5 ± 6.3 C 24.1 ± 3.9 B 25.1 ± 3.5 B

40.2 ± 5.4 C 23.6 ± 2.8 B 26.5 ± 3.8 B

18.3 ± 3.7 b 10.3 ± 1.8 a 10.4 ± 2.3 a

22.4 ± 4.2 b 9.8 ± 2.5 a 10.2 ± 3.2 a

45.3 ± 6.4 c 21.3 ± 5.6 b 26.7 ± 4.3 b

42.4 ± 3.6 c 24.3 ± 4.8 b 22.1 ± 3.8 b

*Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letter are not significantly different (P > 0.05). CHX, chlorhexidine digluconate.

15 s

Adper Single Bond

A

B

C

D

E

F

Fig. 1. Representative back-scattered scanning electron microcopy images of the resin–dentin interfaces bonded with Adper Single Bond immediately after treatment (A–C) or 6 months after treatment (D–F), in which the rewetting time was 15 s. In the samples tested immediately after treatment, only a few areas of silver nitrate uptake were observed within the hybrid layer (A–C), mainly in the group in which dentin was rewetted with water (white stains indicated by the white pointer, panel A). After 6 months, silver nitrate uptake was higher in the hybrid layer only for the control group (water) (white pointer, panel D). In panels E and F, only a small amount of silver nitrate was taken up in the hybrid layer (white stains indicated by the white pointer, panel E) (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

Chlorhexidine on dentin adhesion

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Adper Single Bond

A

B

C

D

E

F

Fig. 2 Representative back-scattered scanning electron microcopy images of the resin–dentin interfaces bonded with Adper Single Bond immediately after treatment (A–C) or 6 months after treatment (D–F), in which the rewetting time was 60 s. In the samples tested immediately after treatment, only a few areas of silver nitrate uptake in the hybrid layer (panels A–C) were observed (white stains indicated by the white pointer, panel A). After 6 months, silver nitrate uptake was highest in the hybrid layer and across the entire thickness of the adhesive layer (white stains indicated by the white pointer, panel D). After 6 months, in the 0.002% and 2% chlorhexidine digluconate (CHX) groups (panels E and F, respectively) only a small amount of silver nitrate was taken up (white stains indicated by white pointer, panel E). (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

Prime & Bond 2.1

A

B

C

D

E

F

Fig. 3 Representative back-scattered scanning electron microcopy images of the resin–dentin interfaces bonded with Prime & Bond 2.1 immediately after treatment (A–C) or 6 months after treatment (D–F), after rewetting for 15 s. In the samples tested immediately after treatment, a considerable amount of silver nitrate uptake within the hybrid layer was observed in the control group (water) (white stains indicated by the white pointer, panel A), compared with the groups rewetted with 0.002% and 2% chlorhexidine digluconate (CHX) (panels B and C, respectively). In panel A, signs of phase separation can be observed (black star). After 6 months, the silver nitrate uptake was higher in the hybrid layer only for the control group (water) (white stains indicated by white pointer, panel D). In panels E and F, only a small amount of silver nitrate uptake was seen in the hybrid layer (white stains indicated by the white pointer, panel F) (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

bond strengths commonly observed for simplified conventional adhesives after long-term water storage and also to preserve the morphological properties of hybrid layers by inhibiting host-derived proteases (19–22, 28). Therefore, one might consider that preventing the degradation of these incompletely resin-infiltrated collagen fibrils and the hybrid layer by MMPs in durability studies is an important issue to investigate, because this could be the key to increasing the durability of restorations that involve dentin bonding. While some in vitro

and in vivo studies have suggested that the application of 2% CHX solution, a MMP inhibitor, to acid-etched dentin for 60 s could minimize the degradation of the dentin bond over time, the fibrillar network of the hybrid layers formed without pretreatment with CHX solution depicts a significant and progressive disintegration (19–22, 28). This was confirmed to some extent in the present investigation. Significant reductions in the lTBS and higher SNU were seen within the adhesive and hybrid layers after 6 months of water storage in the

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A

B

C

D

E

F

Fig. 4 Representative back-scattered scanning electron microcopy images of the resin–dentin interfaces bonded with Prime & Bond 2.1 immediately after treatment (A–C) or 6 months after treatment (D–F), when the rewetting was applied for 60 s. In the samples tested immediately after treatment, no signs of silver nitrate uptake (panels B and C), in comparison with a small amount of silver nitrate uptake within the hybrid layer, was observed in the control group (water) (white stains indicated by the white pointer, panel A). After 6 months, the groups rewetted with chlorhexidine digluconate (CHX) (0.002 and 2%, respectively in panels E and F) showed preservation of the hybrid layer. Only a small amount of silver nitrate uptake was seen in panel E (white stains indicated by white pointer). The highest amount of silver penetration occurred in the hybrid layer in the group in which demineralized dentin was rewetted with water (white stains indicated by a white pointer, panel D) (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

control groups (rewetted with water) for both adhesive systems tested. However, contrasting findings were observed for CHX-treated groups. Very few silver nitrate deposits were seen within the hybrid layer after 6 months and no degradation of the resin–dentin bond strengths was detected. Interestingly, the good performance of CHX (in terms of resin–dentin bond strengths) in the preservation of the dentin bonds over time was independent of its concentration. This is in agreement with the conclusion previously reached by Gendron et al. (26), who found that extremely low concentrations of CHX (around 0.01%) can inhibit protease activity. It has been demonstrated that pure MMP-2, MMP-8, and MMP-9 can be inhibited by CHX concentrations of 0.0001, 0.02, and 0.002%, respectively, through proteolytic action (26). Recently, Carrilho et al. (27) evaluated the collagenolytic activity of the dentin-bound MMPs and showed that the collagenolytic activity of demineralized dentin was significantly reduced by pretreatment with 2% CHX in comparison with non-treated specimens. Gendron et al. (26) proposed two different mechanisms of action involved in MMP inhibition: a chelating mechanism for the inhibition of MMP-2 and MMP-9; and the interaction of CHX with the essential sulfhydryl groups and/or cysteine present in MMP-active sites in the case of MMP-8. Therefore, it seems that 0.002% CHX is sufficient to prevent host-derived MMPs from degrading exposed collagen fibrils, at least during the time-period evaluated in the present investigation (i.e. 6 months). The use of low CHX concentrations (0.002%) conveys a distinct advantage in that it is not toxic to human periodontal

cells (24) or odontoblast-like cells (25); therefore, this low CHX concentration can be regarded as a potential chemical agent for use as a cavity cleanser. The high substantivity of CHX may explain why the application time did not have a significant effect for either the 0.002 or 2% CHX solutions. The term ÔsubstantivityÕ has been much more correlated in the literature to the residual antibacterial activity of CHX in human dentin (32, 33) than to the inhibitory effect of CHX on MMP activity. Chlorhexidine digluconate is one of the most commonly used antimicrobial agents because it retains a therapeutic effect for a prolonged period of time. The substantivity of CHX is related to the release of positively charged molecules from CHXtreated surfaces and its ability to adsorb onto surfaces of the oral cavity (33, 34). Theoretically, this can also occur in the demineralized exposed collagen fibrils, and is the explanation for the bonds being preserved after longterm water exposure. One may suggest that CHX is likely to bind to collagen fibrils at a very fast rate, and thus even short periods of time, such as 15 s, seem to be sufficient to guarantee such binding. This hypothesis was raised on the basis of the results of a recent investigation (28). A 2% CHX solution containing phosphoric acid was able to maintain the stability of the resin–dentin bond of two simplified etchand-rinse adhesives after 6 months of water storage, suggesting that only a few seconds of contact with CHX might be sufficient to inhibit the MMP activity, corroborating the findings of the present investigation. Several studies have so far evaluated the effect of CHX in the immediate and long-term durability of dentin bonds (19–22), but among them only one has measured the amount of silver nitrate deposits within the hybrid

Chlorhexidine on dentin adhesion

layer (28). It was previously hypothesized that CHX could reduce the amount of SNU after 6 months but that improvements would not be expected immediately after treatment. Immediately after treatment with CHX, very few silver nitrate deposits were observed within the hybrid layer compared with the control, as shown by Stanislawczuk et al. (28). As CHX has cationic properties it can bind to sites containing collagen, as hypothesized by Carrilho et al. (22) and possibly to calcium at the bottom at the hybrid layer, reducing the nanopores within the hybrid layer to which silver nitrate can deposit. However, this still deserves further investigation. One cannot rule out the fact that despite the advantages of using 2% CHX, its use for 60 s after acid etching includes another bonding step during the restorative procedure and this works against the cliniciansÕ need for simplification (23). In this research, the application time of CHX was reduced (15 s) and this reduction in application time did not jeopardize the benefits of CHX in the preservation of the dentin bonds. Therefore, the results of the present investigation suggest that a low concentration of CHX, applied for 15 s, is sufficient to preserve dentin bonds for at least 6 months under the laboratory conditions of this study. However, further in vivo studies are needed to clarify whether the use of a 0.002% CHX solution for 15 s is able to preserve resin–dentin bonds after long-term function. Acknowledgements – This study was partially supported by CNPq grants 473101/2006-8 and 305870/2004-1. The authors would like to thank FGM Dental Products for the donation of the composite resin used in this study.

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