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Exposed Collagen in Resin Bonds to Caries-affected

Dentin After Dentin Treatment with Aqueous and

Alcoholic Chlorhexidine Solutions

Hérica Adad Riccia / Débora Lopes Salles Scheffelb / Matheus Racy Mariussoc / Denise Madalena Palomari Spolidoriod / Carlos Alberto de Souza Costae / Josimeri Heblingf

Purpose: To evaluate the effect of saturation of demineralized dentin with aqueous and alcoholic excipients of chlorhexidine (CHX) on the exposure of collagen fibrils in resin-dentin bonds in sound and caries-affected dentin.

Materials and Methods: Flat midcoronal dentin surfaces were prepared from 24 noncarious molars, and ar-tificial caries was induced in half of the sample. For each substrate, the surfaces were assigned to 4 groups (n = 3) according to the saturation solution of the dentin: water, ethanol, 1% CHX aqueous or alcoholic solution. Infected dentin was removed by abrasive papers. After acid etching, the dentin surface was saturated with each solution for 60 s followed by application of Single Bond. The specimens were processed for Goldner’s trichrome staining and the thickness of the exposed collagen zone (ECZ) at the resin/dentin interfaces was measured under optical microscopy. Data were analyzed statistically by Kruskal-Wallis and Mann-Whitney tests ( = 0.05).

Results: Regardless of the saturation solution, caries-affected dentin presented a thicker ECZ at the bottom of the hybrid layer than did sound dentin. For both substrates, 100% ethanol had a negative influence on collagen exposure in comparison with water, but the same was not observed for the CHX alcoholic solution. CHX solu-tions did not differ significantly from each other or from their respective solvents.

Conclusion: The saturation of phosphoric acid-demineralized dentin with either CHX aqueous or alcoholic solu-tions did not affect the exposure of collagen fibrils in the resin-dentin bonds produced in sound and caries-affected dentin. A thicker zone of exposed collagen was found in hybridized caries-affected dentin compared to noncarious dentin.

Keywords: dental caries, dentin bonding agents, dentin, ethanol, chlorhexidine.J Adhes Dent 2014; 16: 21–28. Submitted for publication: 25.10.12; accepted for publication: 22.05.13doi: 10.3290/j.jad.a30716

a PhD Student, Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, University Estadual Paulista (UNESP), Araraquara, SP, Bra-zil. Performed the experiments in partial fulfillment of requirements for PhD, wrote manuscript.

b PhD Student in Pediatric Dentistry, Department of Orthodontics and Pedi-atric Dentistry, Araraquara School of Dentistry, University Estadual Paulista (UNESP), Araraquara, SP, Brazil. Helped perform the experiments.

c Undergraduate Student, Department of Orthodontics and Pediatric Den-tistry, Araraquara School of Dentistry, University Estadual Paulista (UNESP), Araraquara, SP, Brazil. Helped perform experiments in partial fulfillment of requirements for DDS degree.

d Associate Professor, Department of Physiology and Pathology, Araraquara School of Dentistry, University Estadual Paulista (UNESP), Araraquara, SP, Brazil. Supervised experiment on microbiological development of the dentin lesions.

e Full Professor, Department of Physiology and Pathology, Araraquara School of Dentistry, University Estadual Paulista (UNESP), Araraquara, SP, Brazil. Con-tributed substantially to discussion, proofread manuscript, responsible for the Goldner’s trichrome method and photomicrographs.

f Associate Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara School of Dentistry, University Estadual Paulista (UNESP), Ara-raquara, SP, Brazil. Experimental design, co-wrote and proofread manuscript, conducted statistical analysis.

Correspondence: Professor Josimeri Hebling, Faculdade de Odontologia de Araraquara – UNESP, Rua Humaitá, 1680, Araraquara, SP, Brazil 14.801-903. Tel: +55-16-3301-6334, Fax: +55-16-3301-6329. e-mail: jhebling@foar.unesp.br

Collagen fibrils remain exposed after the establish-ment of resin-dentin bonds because the depth of

dentin demineralization exceeds the capacity of infiltra-tion of resin monomers.30,37 This is due to a series of factors such as demineralization protocol, gradual reduction of the interfibrillar spaces towards the miner-alized dentin zone, the high molecular weight and low hydrophilicity of some resin monomers present in adhe-sive systems,31 moisture gradient of dentin32 which al-lows the occurrence of phase separation,31 and type of functional group in the adhesive.14 In addition to these immediate factors, collagen fibrils can be exposed due to long-term polymer hydrolytic degradation6 and release of uncured residual monomers and low molecular weight polymers.

Greater collagen exposure at the base of the hybrid layer has been demonstrated for resin-dentin bonds pro-duced in caries-affected dentin.36 The lower mineraliza-tion and greater porosity of the intertubular dentin in the carious substrate favors the diffusion of acid agents, in-creasing the depth of demineralization.5,36

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The resin-dentin bond components are subjected to hydrolytic and enzymatic degradation. Enzymatic degrada-tion is mediated by a group of enzymes that represent a family of metal-dependent endopeptidases capable of degrading extracellular matrix components – the dentin matrix metalloproteinases (MMPs). MMPs can originate both from dentin17,34 and from saliva.34 More recently, it was found that dentin cystein cathepsins also degrade the exposed collagen.28 These endogenous proteases are released from dentin upon demineralization caused by the carious process34 or acid-etching step of the bonding protocol,17 and are directly involved in the degradation of the organic content of dentin.

CHX has an inhibitory effect on MMPs3,17 and dentin cysteine cathepsins;28 it also has a good substantivity, binding to demineralized dentin.2 Although CHX binding is of electrostatic and reversible nature, it remains after resin-dentin bonding,10 which may be responsible for the long-term efficacy of CHX as an MMP inhibitor in resin-dentin bonds.12

The use of 2% CHX aqueous solution as a bonding primer has been shown to have an effective immediate1,20 and long-term1,7,21 action on decelerating the loss of resin-dentin bonds. Moreover, it has been previously suggested that CHX could increase dentin wettability in the same way it does for enamel.18 However, CHX has a protective effect only against the degradation of the organic compo-nent (collagen fibrils) exposed at the adhesive interface, while the resin portion remains susceptible to hydrolytic degradation. Therefore, recent research has focused on attempts to improve the stability of the resin component by infiltrating the acid-etched dentin with essentially hydro-phobic monomers, such as bis-GMA.9,11,22,24,29,33 This condition can be achieved by replacing the water in the demineralized collagen matrix with ethanol, in a technique known as “ethanol wet-bonding”.16 It would be tempting to infer that the incorporation of CHX into the ethanol used to saturate the acid-etched dentin would result in a joint effect to protect both components of the resin-dentin bond – polymer and collagen fibrils – rendering the interface more resistant to degradation. That assumption, however, still needs to be investigated and a start would be to evaluate whether an alcoholic CHX solution would interfere in any way with the adhesive system’s infiltration of the acid-etched dentin.

Exposed collagen in resin-dentin bonds can be identi-fied using Goldner’s trichrome staining. This technique was used for the first time for this purpose by Spencer et al30 and was later validated by other authors.4,35 Collagen fibrils that are not completely encapsulated by adhesive remain unprotected after bonding and are freely available for reaction with the stains as well as easy to identify by their dark red color under microscopy. To date, however, only two studies have used this technique for analysis of resin-dentin bonds produced in caries-affected dentin.5,25

Using Goldner’s trichrome staining, the aim of this study was to evaluate the effect of saturation of demin-eralized dentin with CHX aqueous and alcoholic solutions on collagen exposure in resin-dentin bonds in sound and caries-affected dentin. The null hypothesis tested was

that the saturation of the acid-etched dentin with CHX, irrespective of the excipient used, has no effect on the amount of collagen exposed in resin-dentin bonds pro-duced on both sound and caries-affected dentin.

MATERIALS AND METHODS

Selection of Teeth

Noncarious human third molars were collected from the Human Tooth Bank of the School of Dentistry of Ara-raquara, UNESP, after approval of the research protocol by the institutional Ethics Committee (process #61/09). The teeth were examined macroscopically after removal of surface-adhered debris and copious water rinsing. Twenty-four teeth with intact crowns and no hypoplastic areas were selected for the study and stored in a 0.1% thymol solution at 4°C until the moment of use.

Preparation of Dentin Specimens

Flat midcoronal dentin surfaces were prepared by grind-ing the occlusal third of the teeth on wet 320-grit silicon carbide (SiC) paper in a polishing machine (Buehler; Lake Bluff, IL, USA) at 500 rpm. Grinding was completed when enamel-free dentin surfaces were obtained as con-firmed by examination under a stereomicroscope (Model SZX7; Olympus, São Paulo, SP, Brazil) at 10 to 40X magnification.

Induction of Artificial Caries Lesion

In half of the teeth (n = 12), one of the roots was per-forated in the apical region with a round diamond bur, and an orthodontic wire was transfixed through the perforation so that the teeth could be held immersed in a cariogenic solution.13,25 The teeth were rendered waterproof with one layer of quick-setting epoxy adhe-sive (Araldite, Ciba Especialidades Químicas; São Paulo, Brazil) and one layer of an acid-resistant nail polish (Colorama, CEIL; São Paulo, SP, Brazil), leaving exposed only the dentin surface, and were then sterilized with ethylene oxide. A cariogenic solution was prepared with 3.7 g brain-heart infusion (BHI) broth (Becton Dickinson; Sparks, MD, USA), 2 g sucrose (Synth, LabSynth; São Paulo, SP, Brazil), 1 g glucose (Synth, LabSynth), and 0.5 g yeast extract (Becton Dickinson) for every 100 ml of distilled water. The solution was autoclaved at 121°C for 20 min prior to inoculation with a reference strain of Streptococcus mutans (ATCC 25175; Tropical Culture Collection, Andre Tosello Research Foundation; Campi-nas, SP, Brazil) as 2% of a test tube containing 5 ml of culture at 108 cfu/ml. The sterile teeth were suspended in the cariogenic solution (25 ml/tooth) and the sets were incubated in microaerophilia at 37°C (candle jar system) for 14 days. During this period, the cariogenic solution was renewed at 48-h intervals, without inocu-lating new microorganisms. After the incubation period, the biofilm formed on tooth surfaces was removed with gauze and the coating materials (epoxy adhesive and nail polish) were removed with a scalpel blade; then the teeth were abundantly rinsed in deionized water. The re-

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sulting dentin was darker in color and softer, as felt with the tip of an explorer held without pressure. In addition, two distinct layers of carious dentin are produced by this method: an outer layer that resembles the infected den-tin of natural lesions and an inner layer similar to the affected dentin.13

Removal of Caries-infected Dentin

The softened, infected carious dentin was removed by gently grinding the specimens on 320-grit SiC paper, thus preserving the inner caries-affected layer and obtaining a flat dentin surface. The limit for removal of carious tissue was established by visual examination and tactile inspec-tion with the use of an excavator applied without pres-sure. The caries-affected dentin surface should appear darkened and felt to be slightly hard when contacted with the instrument. One experienced and previously trained operator performed this procedure.

An approximately 0.5-mm-thick layer of dentin was removed from the noncarious specimens (n = 12) by grinding wet on 320-grit SiC paper in the polishing ma-chine at 500 rpm in order to compensate for the dentin thickness removed from the carious specimens. Then, all specimens (sound and carious) were hand polished with 320-grit SiC paper for 15 s in order to produce a standard-ized smear layer.

Adhesive Procedures

The specimens were randomly assigned according to the type of substrate (sound or caries-affected) and the saturation solution (deionized water, 1% CHX aqueous solution, anhydrous ethanol and 1% CHX alcoholic solu-tion). The smear-covered dentin was etched with 35% phosphoric acid (Ultradent Products; South Jordan, UT, USA) for 15 s, rinsed with water for 15 s and blot dried with absorbent paper leaving a moist surface. Then, 20 l of the saturation solution were passively applied on this surface and left undisturbed on dentin for 60 s. Excess was blotted with absorbent paper to obtain a surface with a moist appearance. The commercial brands, manufacturers, and main components of the

materials used in the study are presented in Table 1. The 1% CHX aqueous and alcoholic solutions were pre-pared in the proportions described in Table 1.

Next, two successive layers of Single Bond 2 (3M ESPE; St Paul, MN, USA) adhesive system were applied on den-tin, each one slightly air thinned for 5 s at a distance of 10 cm for solvent evaporation. The adhesive layers were photoactivated together for 10 s with a light-curing unit (Optilux 500, Kerr; Danbury, CT, USA), with a minimum irradiance of 500 mW/cm2, as checked with a curing radiometer. Four additional layers of the adhesive system were applied and photoactivated after application of the last layer in order to enable cutting the specimens in a microtome. The teeth were maintained in 100% relative humidity at 37°C for 24 h.

Histological Procedures

The teeth were cut in a precision cutting machine (Isomet 1000; Buehler, Lake Bluff, IL, USA) to obtain two to three blocks (2 mm thick x 2 mm wide x 5 mm long) per tooth. The blocks were fixed in a 10% formalin solution for 48 h and slightly demineralized in a 10% Morse solution for 48 h without agitation. Next, the blocks were washed in running tap water for 24 h, neu-tralized in a 5% sodium sulfate solution for the same time, washed with water again for 24 h, dehydrated in a series of increasing ethanol solutions (70% to 100%), cleared in xylol, and embedded in paraffin under vac-uum. Approximately 5- m-thick serial sections were cut from the blocks with a microtome (820 Spencer Micro-tome, American Optical; Buffalo, NY, USA) and stained with Goldner’s trichrome.30 In this staining technique, green indicates mineralized dentin, beige indicates the adhesive layer, orange indicates the collagen-resin hy-bridized layer, and dark red indicates exposed collagen.

Optical Microscopy Analysis

One section from each block was randomly chosen and analyzed under 400 X magnification (Olympus BX51 and Camedia C5060 camera; Tokyo, Japan). Using the UTH-SCSA Image Tool (The University of Texas Health Science

Table 1 Commercial brands, main components, and manufacturers of the materials used in the study

Commercial brand (batch number) Main components Manufacturer

Single Bond 2 (N190766BR)

Bis-GMA, HEMA, diurethane dimethacrylate, polyalkenoic acid copolymer, camphorquinone, water, ethanol and glyc-erol dimethacrylate 1.3, 10% by weight of silica nanopar-ticles

3M ESPE; St Paul, MN, USA

Etchant(B622Z)

35% phosphoric acid Ultradent; South Jordan, UT, USA

1% CHX aqueous solution(prepared at laboratory)

Powder: chlorhexidine diacetateLiquid: deionized water (0.25 g/25 ml)

Powder: Sigma-Aldrich; St Louis, MO, USA

1% CHX alcoholic solution(prepared at laboratory)

Powder: chlorhexidine diacetateLiquid: Anhydrous ethanol (0.19 g/25 ml)

Powder: Sigma-Aldrich Anhydrous ethanol: J.T. Baker–Mallinckrodt; Xalostoc, México

Bis-GMA: bisphenol-A-glycidyl dimethacrylate; HEMA: 2-hydroxyethyl methacrylate.

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Table 2 Thickness of the exposed collagen zone ( m) at the base of hybrid layers formed in acid-etched noncari-

ous and caries-affected dentin saturated with water, 1% chlorhexidine (CHX) aqueous solution, anhydrous ethanol,

and 1% CHX alcoholic solution

Saturation solution Substrate

Noncarious dentin Caries-affected dentin

Deionized water 2.26 (1.87-2.30)* [7] A,a 10.01 (8.76-10.99) [9] AB,b

CHX aqueous solution 2.62 (2.26-2.78) [6] AB,a 7.04 (4.67-8.01) [6] A,b

Anhydrous ethanol 2.91 (2.63-3.23) [6] B,a 14.83 (13.56-16.17) [9] B,b

CHX alcoholic solution 2.65 (2.30-2.84) [6] AB,a 9.47 (7.95-14.39) [9] AB,b

* All values are given as medians (25th/75th). [Number of specimens in the group]. Groups identified with same uppercase letters in columns and lowercase letters in rows do not differ statistically significantly (Mann-Whitney test, p > 0.05).

Fig 1 Representative photomicrographs of caries-affected dentin hybridized with Single Bond 2 after acid-etched dentin saturation with (a) water, (b) water/1% CHX, (c) ethanol and (d) ethanol/1% CHX, stained with Goldner’s trichrome, 400X magnification. At the top of the images in beige is the adhesive system, mineralized dentin is green, the hybridized dentin orange, and the exposed col-lagen layer is bright red.

a b

c d

20 μm 20 μm

20 μm 20 μm

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Fig 2 Representative photomicrographs of sound dentin hybridized with Single Bond 2 after acid-etched dentin saturation with (a) water, (b) water/1% CHX, (c) ethanol and (d) ethanol/1% CHX, stained with Goldner’s trichrome, 400X magnification. At the top of the images in beige is the adhesive system, mineralized dentin is green, the hybridized dentin orange, and the exposed collagen layer is bright red.

a b

c d

Center; San Antonio, TX, USA) software, three measure-ments of the thickness of the exposed collagen zone (ECZ) at the base of the hybrid layer were made in each section by demarcating one central and two equidistant points (one to the right and one to the left). The measurements were repeated by the same calibrated operator within a one-week interval and the means were calculated.

Statistical Analysis

The intraclass correlation coefficient (ICC) was used to determine the intra-examiner agreement for the two ECZ thickness measurements (the response variable) on the resin-dentin bonds. The data were non-normally distributed and were subjected to the Kruskal-Wallis and Mann-Whitney tests, considering substrate and satura-tion solution as variable factors. A significance level of 5% was set for all statistical analyses.

RESULTS

A high intra-examiner correlation (ICC = 0.98) was ob-tained for the measurements of ECZ thickness at the base of the hybrid layer. Therefore, the six measure-ments were averaged. ECZ thickness data are presented in Table 2 as medians and 25th/75th percentiles, ac-cording to the substrate and saturation solution.

Red-stained exposed collagen fibrils were identified in all resin-dentin bonds (Figs 1 and 2). Regardless of the saturation solution, caries-affected dentin (Fig 1) pre-sented significantly thicker ECZ at the bottom of the hybrid layer than did sound dentin (Fig 2) (Table 2, rows).

For both types of substrates, saturation of dentin with ethanol increased the thickness of the ECZ compared with saturation with water, which was not observed when the acid-etched dentin was saturated with the

20 μm 20 μm

20 μm 20 μm

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CHX alcoholic solution; in this case, values equivalent to dentin saturation with water were obtained (Table 2, columns). The CHX solutions did not differ significantly from each other or from their respective solvents. There-fore, it may be stated that the aqueous and alcoholic excipients of CHX had no negative influence on the infiltration of demineralized dentin by Single Bond 2. In other words, there was no statistically significant dif-ference in the ECZ thickness in the resin-dentin bonds produced when the substrate was pretreated with CHX solutions (Table 2, columns).

DISCUSSION

The advent of contemporary dental adhesive technol-ogy and bioactive restorative materials allied with a more conservative operative philosophy for treatment of carious lesions resulted in resin-dentin bonds being established in cavities in which sound and caries-af-fected dentin tissue coexist. Compared with noncarious dentin, caries-affected dentin has less mineral content and more porosities in addition to the narrowing and ultimately obliteration of the dentinal tubules due to de-position of intratubular dentin.36,39 However, while the characteristics and alterations of caries-affected dentin interfere directly in the quality of resin-dentin bonds, the majority of studies on adhesion have used sound dentin for testing adhesive interfaces.19,36

A microbiological protocol for induction of artificial car-ies was used in the present study. This method intended to simulate the carious process by using bacterial strains and reproducing some characteristics of natural dentin carious lesions, such as color, collagen degradation, and the presence of two distinct layers. The outer layer is simi-lar to the infected dentin tissue, has a softened consist-ency and is easily removed, exposing a harder inner layer, which is similar to caries-affected dentin.13

After acid etching, a demineralized dentin zone is cre-ated and should ideally be completely infiltrated by ad-hesive resin monomers. However, due to a number of factors that include size and molecular weight of mono-mers, presence of moisture in the substrate,31,32 and long-term polymer hydrolytic degradation,6 unprotected collagen fibrils remain at the base of the hybrid layer. This exposed collagen zone permits the ingress of fluids and bacterial byproducts, comprising the well-known phe-nomenon of nanoleakage.26 This nanoleakage pathway favors the hydrolytic and enzymatic degradation of the resin-dentin bond components, ultimately causing their functional failure.

In the present study, exposed collagen fibrils were identified at the resin-dentin bonded interface regardless of the saturation solution and type of substrate. How-ever, the caries-affected dentin presented a significantly thicker ECZ at the bottom of the hybrid layer than did sound dentin. A similar result was reported by Haj-Ali et al5 using the same histological staining method. This could be explained by the fact that the increased porosity of the intertubular dentin and the lower mineral content

of caries-affected dentin36,39 facilitate the diffusion of the acid agent; on the other hand, buffering of the acid agent by dissolved minerals is reduced because of the lower mineral content of caries-affected dentin compared with sound dentin. Nevertheless, using the same method for induction of artificial caries lesion and phosphoric-acid concentration of the present study, Sanabe et al25 did not find a statistically significant difference in the ECZ thickness formed in sound and caries-affected dentin. The only difference between the methodologies that could explain the divergent results was the fact that those au-thors used a low-speed round bur to remove the infected dentin, while in the present study, a 320-grit SiC paper was employed. Grinding on an abrasive paper permits better control during carious tissue removal, resulting in a flat caries affected-dentin surface; in contrast, the bur removes carious tissue more roughly, making it difficult to identify the caries-affected/sound-dentin interface. Thus, the adhesive procedures could have been done predomi-nately on almost sound dentin in the Sanabe et al25 study.

Ethanol wet-bonding is a new approach to dentin bond-ing with etch-and-rinse adhesive systems based on the concept of water replacement in inter- and intrafibrillar spaces by ethanol in order to create a hydrophobic envi-ronment for hydrophobic resin monomers to better infil-trate the matrix.16 The infiltration of hydrophobic mono-mers into an ethanol-saturated demineralized collagen matrix decreases water sorption, resin plasticization, and the hydrolytic cleavage of collagen by enzymes,23 creating more durable bonds.8

The present study evaluated whether the saturation of acid-etched dentin with ethanol prior to adhesive system application would facilitate monomer infiltration to re-duce the ECZ thickness at the bottom of the hybrid layer. However, saturation of sound and caries-affected dentin with ethanol seems to have impaired monomer infiltration compared with water, since thicker ECZs were observed for hybridized acid-etched ethanol-saturated dentin.

The negative effect of ethanol on monomer infiltra-tion observed in the present study could be explained by the results of Osorio et al,15 who evaluated different protocols of water replacement in the collagen matrix by ethanol after acid etching. These authors observed that application of absolute ethanol on the dentin surface for 1 min produced collapse and shrinkage of collagen fibrils, reducing the infiltration of the mineral-depleted collagen matrix by resin monomers. Conversely, when a sequence of ascending ethanol concentrations was used, there was no collapse of the collagen network, and less collagen fi-bril shrinkage occurred. In addition, a gradual replacement of the water in the acid-etched dentin by ethanol results in improved bond strength of Single Bond to both sound and caries-affected dentin when compared to water or to a single application of 100% ethanol for 20 s.9

Single Bond 2, used in the present study, is a sim-plified etch-and-rinse adhesive system that contains a blend of hydrophobic and hydrophilic monomers and has water and ethanol as solvents. Hydrophobic monomers are more resistant to hydrolytic degradation, but their larger molecular structures limit their penetration into

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the interfibrillar spaces, which is even more critical when the collagen matrix collapses due to dentin saturation with absolute ethanol.15 Moreover, the consequences of introducing ethanol into the acid-etched dentin with the solvents already present in the adhesive system are as yet unknown in terms of quality of the polymer and hybrid layer formed. The quality of hybrid layers produced by Single Bond containing 30% ethanol was better than when 50% ethanol was incorporated in the same adhe-sive.38

Regarding the ECZ thickness, no significant difference was found between the CHX aqueous solution and wa-ter, between the CHX alcoholic solution and anhydrous ethanol, or between the CHX solutions for both types of dentin substrate. Thus, the null hypothesis was accepted that the saturation of the acid-etched dentin with CHX, irrespective of the excipient used, has no effect on the amount of collagen exposed in resin-dentin bonds pro-duced on both sound and caries-affected dentin. However, despite the lack of effect on the amount of remaining col-lagen exposed, the use of CHX should still be considered, since the organic component of collagen is prone to both hydrolytic and enzymatic degradation over time. CHX has a recognized positive effect on MMP and dental cysteine cathepsin28 inhibition and consequently on extending the longevity of resin-dentin bonds demonstrated in both in vitro1 and in vivo studies.7,21 Although the durability of the effect of CHX on dentin protease inhibition when used adjunctively in the bonding protocol has not yet been de-termined, it has been demonstrated that CHX has an exceptional substantivity on dentin.2 Besides, when in-corporated into the resin-dentin bond, the molecules of CHX are trapped by both the adhesive layer on the top and the adhesive tags formed inside the dentinal tubules on the bottom, rendering its removal from the interface rather difficult. Some in vivo studies have demonstrated a favorable effect of CHX on retarding resin-dentin bond degradation for up to 14 to 18 months of function in the oral cavity.7,21

Although the goal of ethanol wet bonding is to im-prove the infiltration of demineralized collagen matrix by more hydrophobic monomers, it has been demonstrated that the efficacy of this technique might be affected by the chemical composition of the adhesive27 and the ethanol replacement protocol.15 Further studies are ne-cessary to establish parameters that can ensure the ef-ficacy of ethanol wet bonding for clinical use. Moreover, the safety of ethanol use in vital teeth has not yet been investigated.

CONCLUSION

Within the limits of this study, it may be concluded that hybridized caries-affected dentin contains more unpro-tected collagen at the base of the hybrid layer than does noncarious dentin, and that the application of CHX aque-ous and alcoholic solutions does not reduce the amount of collagen exposed.

ACKNOWLEDGMENTS

The authors acknowledge the excellent collaboration of Mrs. Juli-ana Pirola and Mr. José Antonio Sampaio Zuanon, technicians at the Laboratory of Experimental Pathology and Biomaterials of the School of Dentistry of Araraquara/UNESP, in the preparation of the microscopic slides stained with Goldner’s trichrome. We also acknowledge the funding agencies CNPq (grant #305204/2010-6) and FAPESP (grant #2010/09802-4 and 2010/20495-6) for finan-cial support.

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Clinical relevance: Although CHX renders hybrid layers more resistant to degradation, it does not have any effect on the amount of collagen fibrils ex-posed after the resin-dentin bond is produced in both noncarious and caries-affected dentin. However, a remarkably higher amount of collagen is left unpro-tected in caries-affected dentin, making bonding to that substrate more challenging.

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