Basic Research—Technology

Effects of Irrigation Solutions on Dentin Wettability andRoughnessXiaoli Hu, DDS, MS,* Junqi Ling, DDS, MS, PhD,* and Yan Gao, DDS, MS†

Abstract

Introduction: Irrigation solutions are used to disinfectthe root canal, flush out debris, and dissolve pulp tissue.It is hypothesized that irrigation solutions might influ-ence the physicochemical properties of human rootcanal dentin including the wettability and roughness.Methods: The roots of 20 human permanent anteriorteeth were sectioned longitudinally into dentin slicesand randomly divided into 4 groups with different treat-ments as follows: (1) 17% ethylenediaminetetraaceticacid (EDTA) for 10 minutes, (2) 5.25% NaOCl for10 minutes, (3) 3% H2O2 for 10 minutes, and (4) distilledwater as control. Wettability was evaluated by contactangle measurement. Atomic force microscopy wasused to measure the surface roughness (Ra). Results:Water contact angles significantly decreased from54.35 degrees (control) to 22.04 and 46.35 degrees onthe dentin treated by 5.25% NaOCl and 3% H2O2,

respectively, with the wettability increasing accordingly(both P < .05). The roughness of dentin surfaces (Ra)was increased from 85.39 (control) to 121.90 and140.49 nm by 5.25% NaOCl and 17% EDTA, respec-tively (both P < .05). Conclusions: These findingsindicated that all the irrigation solutions could alterthe physicochemical characteristics of dentin surfacesand might influence bacterial adhesion. (J Endod2010;36:1064–1067)

Key WordsAtomic force microscopy, irrigation, roughness, wetta-bility

From the *Department of Operative Dentistry andEndodontics and †Department of VIP, Guanghua College ofStomatology, Sun Yat-sen University, Guangzhou, China.

Address requests for reprints to Junqi Ling, PhD, Professor,Department of Operative Dentistry and Endodontics, GuanghuaCollege of Stomatology, Sun Yat-sen University, Guangzhou,China. E-mail address: lingjq@mail.sysu.edu.cn.0099-2399/$0 - see front matter

Copyright ª 2010 American Association of Endodontists.doi:10.1016/j.joen.2010.03.007

1064 Hu et al.

The success of root canal therapy depends on the quality of several factors, includingthe instrumentation, irrigation, disinfection, and 3-dimensional obturation of the

root canal (1). Mechanical and chemical effects of the irrigant solutions are crucialduring irrigation. Mechanical effects are generated by the flow and backflow ofirrigant solutions in the root canal (2). The irrigant solutions with antimicrobialeffects demonstrate superior effectiveness in bacteria elimination (3). Sodiumhypochlorite (NaOCl), hydrogen peroxide (H2O2), and combination of NaOCl andH2O2 have been widely used in endodontic therapy for many years because of theirantimicrobial properties (4). Ethylenediaminetetraacetic acid (EDTA) solutions havebeen used to assist in removing the smear layer (5, 6). NaOCl dissolves proteins andremoves other organic components from biologic materials. In recent studies,researchers found that collagen exposed on the dentin surface can be quicklyremoved by NaOCl in a deproteination process (7).

Irrigation with H2O2, NaOCl, or EDTA significantly decreased the microhardness ofroot canal dentin (8, 9). NaOCl and EDTA also increased the roughness of dentin (9).Irrigation solutions might also change other surface properties of radicular and coronaldentin such as wettability, which might influence the adhesion of bacteria (10) and theinteraction between the dentin and restorative materials (11). Because wettability of thesurface is a crucial factor for adhesion, it is of interest to examine the effect of irrigationon dentin surface wettability. Wettability is strongly dependent on chemical composi-tion, roughness, and hydration state and could be influenced by tubule density (12).Currently, there is no report about the effect of irrigation on dentin wettability.

Roughness is an important factor in adhesion and has been related to wettability inthe Wenzel equation. Wenzel equation applies to equilibrium angles on rough surfacesand is expressed as follows (13): r = cos q1/cos q2, where r is the ratio of the actual toapparent or projected area, and the contact angles q1 and q2 refer to the roughenedsurface and smooth surface contact angles, respectively.

Roughness can be measured by different instruments such as a stylus profilom-eter, a computerized roughness tester, or an atomic force microscope (9, 14).Atomic force microscopy (AFM) emerged as a powerful imaging tool in 1986(15). AFM works by scanning a very tiny tip mounted at the end of a flexiblemicrocantilever in gentle touch with the sample in a raster fashion. AFM has someadvantages. It can work in air and liquid, requires little or no sample preparation,and provides high-resolution imaging of 3-dimensional surface topography (16).Because a sample does not require extensive preparation to be analyzed by AFM,it can be mapped many times, acting as its own control. Furthermore, becauseAFM is software-driven, a high mathematical accuracy of surface characterizationmight be obtained, and image properties (such as roughness, peaks, etc) can bedetermined quantitatively. Although studies involving AFM have been conducted ina number of fields of dentistry (17–20), no reports are available describing theapplication of this technique to roughness measurement of dentin after irrigation.

Therefore, the 2-fold aim of this study was to (1) evaluate changes in averageroughness (Ra) after irrigation by using AFM and (2) determine dentin wettability bycontact angle measurement.

Materials and MethodsPreparation of Dentin Specimens

Twenty human fully developed permanent anterior teeth were used in this study.They were recently extracted for periodontal reasons from male and female patients

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TABLE 1. Means and Standard Deviations of Water Contact Angles of DentinSurfaces for Treatment and Control Groups (n = 10/group)

GroupWater contact angle (degrees)(mean ± standard deviation)*

17% EDTA 54.99 � 4.06a

5.25% NaOCl 22.04 � 3.07b

3% H2O2 46.35 � 4.58c

Control 54.35 � 4.25a

*Different letters in the column indicate a statistically significant difference (P < .05). Same letters in

the column indicate the differences are not significant (P > .05).

TABLE 2. Means and standard deviations of roughness values of dentinsurfaces for treatment and control groups (n = 10/group)

GroupRoughness values,

Ra (nm) (mean ± SD)*

17% EDTA 140.49 � 28.09b

5.25% NaOCl 121.90 � 27.63b,c

3% H2O2 99.22 � 31.11a,c

Control 85.39 � 26.45a

*Different letters in the column indicate a statistically significant difference (P < .05). Same letters in

the column indicate the differences are not significant (P > .05).

Basic Research—Technology

ranging in age from 30–45 years. The teeth were selected on thebasis of dimension, similarity in morphology, and absence of anycrack or carious defects, especially within the root portion. Debrisand soft tissue remnants on the root were removed with a sharpscalpel. All teeth were stored in phosphate-buffered saline at 4�Cuntil used.

The tooth crowns were removed at the cementoenamel junctionby using a high-speed bur under water coolant. Each root wassectioned longitudinally into 2 parallel dentin slices by a Microslice2 (Metals Research Limited, Cambridge, England). Forty dentin sliceswere ground smooth through 400-, 600-, 800-, and 1200-grit polish-ing papers under distilled water to remove any surface scratches. Ateach step, dentin samples were rinsed and ultrasonicated in purifiedwater.

The 40 dentin samples were randomly divided into 4 groups of10 samples each and prepared as follows: group 1, treatment with17% EDTA (Sigma-Aldrich Inc, St Louis, MO) for 10 minutes; group2, treatment with 5.25% NaOCl (Sigma-Aldrich Inc) for 10 minutes;group 3, treatment with 3% H2O2 (Sigma-Aldrich Inc) for 10 minutes;and group 4 (controls), treated with deionized and distilled water for10 minutes.

Dentin samples were rinsed copiously by the deionized anddistilled water after immersion into the above irrigating solutions.

Contact Angle MeasurementsWater contact angles (q) were measured by sessile drop tech-

nique on dentin surfaces by using a VCA Optima Surface AnalysisSystem (AST Products, Billerica, MA). Two drops (0.0002 mL/drop) of distilled water were deposited on each dentin surface witha microsyringe. Images were captured immediately after depositionwithin a microvideo system. The acquired images were analyzed byVCA-2500XE software (AST Products), and contact angles wereprovided. The values were averaged to produce 1 water contact anglefor each dentin specimen.

AFM AnalysisA Digital Instruments Nanoscope atomic force microscope

(Veeco, Santa Barbara, CA) was operated in contact mode to imagethe dentin surfaces by using pyramidal tips made of Si3N4 (DNP-20;Veeco). It was controlled with the aid of the included softwareV614r1. Three fields of view of 10 � 10 mm2 for each sample wereobtained, and the roughness (Ra) was measured. The values wereaveraged to produce a single Ra value for each dentin specimen.

Statistical AnalysisWater contact angles and roughness values were statistically

analyzed by one-way analysis of variance, followed by a post hocTukey test for pair-wise comparison. Statistical significance was setat P <.05.

ResultsThe means and standard deviations of water contact angles of

dentin surfaces for the treatment and control groups are listed inTable 1. The values for contact angles were significantly reduced afterthe dentin samples were treated with 5.25% NaOCl and 3% H2O2.Hence, wettability was increased (P < .05). Seventeen percent EDTAdid not influence the wettability of root dentin surface after 10 minutes(P > .05).

The means and standard deviations of roughness values (Ra) ofdentin surfaces for the treatment and control groups are listed inTable 2. The roughness values were significantly increased by 5.25%

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NaOCl and 17% EDTA (P < .05). There was no effect on the roughnessof root dentin surface by 3% H2O2 (P > .05). Figure 1 shows the AFMimages for typical dentin surfaces after treatment with 17% EDTA,5.25% NaOCl, 3% H2O2, and distilled water (control).

DiscussionWettability is one of the most important physicochemical proper-

ties. Contact angle measurements determine the wettability of thesubstratum. Low contact angles are an indication of high surface freeenergy, whereas high contact angles indicate low surface free energy(21). Interfacial surface free energy has been postulated as a drivingforce for initial adhesion of microorganisms to solid surface.

To date there is no report about the effect of irrigation solutions onthe wettability of dentin surfaces. Our study indicated that both 3% H2O2

and 5.25% NaOCl significantly increased the wettability of dentinsurfaces. Some groups have found that hydrophilic materials weremore resistant to bacterial adhesion than hydrophobic materials(22, 23). However, An et al (24) found that hydrophobic UHMWPEdisks attracted much less Staphylococcus epidermidis comparedwith the hydrophilic metal surface. Furthermore, there were significantincreases in adhesion force of Enterococcus faecalis to NaOCl- andEDTA-treated dentin, whereas NaOCl alone did not significantly influ-ence the adhesion force (25). Hence, adhesion of bacteria is not justrelated to surface wettability but might also relate specifically with thespecies of bacteria.

It is well-known that H2O2 is a strong oxidant and is also acidic.Hydrogen peroxide affects the inorganic components of dentinthrough acidic demineralization and attacks the organic-rich intertub-ular dentin by collagen denaturation (26). It is likely that theincreased wettability of the dentin surface is the result of this acidicproperty of H2O2. In support of this view, Rosales et al (12) reportedthat the values of water contact angles of superficial dentin werereduced significantly after their specimens were etched by 10% phos-phoric acid for 15 seconds, thus increasing wettability. Because of theenlargement of the fluid-filled dentin tubules by the acid-etchingaction, the volume of water significantly increased. Consequently,this highly hydrated surface became more hydrophilic (27). Our

Effect of Irrigation on Dentin Wettability and Roughness 1065

Figure 1. AFM images of 10 � 10 mm area of dentin surfaces after treatment with (A) 17% EDTA, (B) 5.25% NaOCl, (C) 3% H2O2, and (D) distilled H2O(control).

Basic Research—Technology

study also found that 5.25% NaOCl increased the wettability of dentinsignificantly. Toledano et al (28) found that contact angles on deepdentin etched by 35% phosphoric acid and then deproteinized by5% NaOCl were lower than those of etched only dentin. Morhonine(14.3% EDTA containing a surfactant 0.084% cetrimide) spreadrapidly over the dentin surfaces, resulting in the inability to measurethe contact angle of these surfaces after conditioning with NaOCl solu-tion (29). NaOCl is frequently used to clean, disinfect, and nonspecif-ically deproteinize the root canal environment during endodontictreatment. Recently, NaOCl was used to remove the collagen fibrils(30). The removal of collagen results in dentin surfaces similar tothose of etched enamel (31, 32). An increase in wettability afterNaOCl treatment can be explained because deproteination leads toa hydrophilic surface (33).

In our study, NaOCl also increased the roughness of dentin. Thisfinding that increased roughness resulted in a decrease in contactangle is in agreement with the Wenzel equation (13). The roughnessof dentin (Ra) after irrigation was evaluated by AFM. Ra valuesincreased significantly after treatment by 5.25% NaOCl and 17%EDTA. This increase agrees with the findings of Ari et al (9), whoused a computerized roughness tester (Mitutoyo Surftest Analyser;Matsuzawa Seiki Co, Ltd, Tokyo, Japan) to measure the surface rough-ness after treatment with the same irrigation solutions. The chelatingaction of EDTA softens root canal wall by the dissolution of its inor-ganic components. Dentin tubules became patent and the surfaceroughness increased as a result of the smear layer removal by EDTA(1). The organic-dissolving properties of NaOCl on the collagencomponents of dentin (34) might explain how this irrigation solutionincreased the surface roughness. Rough surfaces could be a clinicalbenefit as in the case of micromechanical bonding of the adhesivematerials that need the irregularities on the surface. However, roughsurfaces will promote bacterial adhesion, which might lead to plaqueformation (35).

All the irrigation solutions in this study influenced the physico-chemical properties of dentin surfaces, such as wettability and rough-ness. Hence, the effects of the changes of physicochemical propertieson the bacterial adhesion and restorative adhesive procedure shouldbe further studied. Due consideration should be given to these effectsas the irrigants are used during root canal treatment.

1066 Hu et al.

AcknowledgmentsThis study was supported by Key Clinical Program of the

Ministry of Health, China.

References1. Eldeniz AU, Erdemir A, Belli S. Effect of EDTA and citric acid solutions on the micro-

hardness and the roughness of human root canal dentin. J Endod 2005;31:107–10.2. Siqueira JF Jr, Lima KC, Magalhaes FA, Lopes HP, de Uzeda M. Mechanical reduction

of the bacterial population in the root canal by three instrumentation techniques.J Endod 1999;25:332–5.

3. Bystrom A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodiumhypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55:307–12.

4. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98.5. Cury JA, Bragotto C, Valdrighi L. The demineralizing efficiency of EDTA solutions on

dentin: I—influence of pH. Oral Surg Oral Med Oral Pathol 1981;52:446–8.6. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root

canal irrigation regimens. J Endod 1987;13:147–57.7. Di Renzo M, Ellis TH, Sacher E, Stangel I. A photoacoustic FTIRS study of the chem-

ical modifications of human dentin surfaces: II—deproteination. Biomaterials2001;22:793–7.

8. Saleh AA, Ettman WM. Effect of endodontic irrigation solutions on microhardness ofroot canal dentine. J Dent 1999;27:43–6.

9. Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solutionson the microhardness and the roughness of root canal dentin. J Endod 2004;30:792–5.

10. Pringle JH, Fletcher M. Influence of substratum wettability on attachment of fresh-water bacteria to solid surfaces. Appl Environ Microbiol 1983;45:811–7.

11. Baier RE. Principles of adhesion. Oper Dent 1992;(Suppl 5):1–9.12. Rosales JI, Marshall GW, Marshall SJ, et al. Acid-etching and hydration influence on

dentin roughness and wettability. J Dent Res 1999;78:1554–9.13. Wenzel RN. Resistance of solid surfaces to wetting by water. Ind Eng Chem 1936;28:

988–94.14. Tholt de Vasconcellos B, Miranda-Junior WG, Prioli R, Thompson J, Oda M. Surface

roughness in ceramics with different finishing techniques using atomic force micro-scope and profilometer. Oper Dent 2006;31:442–9.

15. Binning G, Quate CF, Gerber C. Atomic force microscopy. Phys Rev Lett 1986;56:930–3.

16. Marshall GW Jr, Balooch M, Kinney JH, Marshall SJ. Atomic force microscopy ofconditioning agents on dentin. J Biomed Mater Res 1995;29:1381–7.

17. Eliades G, Palaghias G, Vougiouklakis G. Effect of acidic conditioners on dentinmorphology, molecular composition and collagen conformation in situ. Dent Mater1997;13:24–33.

18. Farina M, Schemmel A, Weissmuller G, Cruz R, Kachar B, Bisch PM. Atomic forcemicroscopy study of tooth surfaces. J Struct Biol 1999;125:39–49.

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19. Saeki K, Marshall SJ, Gansky SA, Marshall GW. Etching characteristics of

dentin: effect of ferric chloride in citric acid. J Oral Rehabil 2001;28:301–8.

20. Oliveira SS, Pugach MK, Hilton JF, Watanabe LG, Marshall SJ, Marshall GW Jr. Theinfluence of the dentin smear layer on adhesion: a self-etching primer vs a total-etchsystem. Dent Mater 2003;19:758–67.

21. Duncan-Hewitt W. Hydrophobic surfaces. In: Doyle R, Rosenberg M, eds. Microbialcell surface hydrophobicity. Washington, DC: American Society for MicrobiologyPress; 1990:39–73.

22. Hogt AH, Dankert J, de Vries JA, Feijen J. Adhesion of coagulase-negative staphylo-cocci to biomaterials. J Gen Microbiol 1983;129:2959–68.

23. Ludwicka A, Jansen B, Wadstrom T, Pulverer G. Attachment of staphylococci tovarious synthetic polymers. Zentralbl Bakteriol Mikrobiol Hyg A 1984;256:479–89.

24. An YH, Bradley J, Powers DL, Friedman RJ. The prevention of prosthetic infectionusing a cross-linked albumin coating in a rabbit model. J Bone Joint Surg Br1997;79:816–9.

25. Kishen A, Sum CP, Mathew S, Lim CT. Influence of irrigation regimens on theadherence of Enterococcus faecalis to root canal dentin. J Endod 2008;34:850–4.

26. Lewinstein I, Hirschfeld Z, Stabholz A, Rotstein I. Effect of hydrogen peroxide andsodium perborate on the microhardness of human enamel and dentin. J Endod1994;20:61–3.

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27. Aguilar-Mendoza JA, Rosales-Leal JI, Rodriguez-Valverde MA, Cabrerizo-Vilchez MA.Effect of acid etching on dentin wettability and roughness: self-etching primersversus phosphoric acid. J Biomed Mater Res B Appl Biomater 2008;84:277–85.

28. Toledano M, Osorio R, Perdigao J, Rosales JI, Thompson JY, Cabrerizo-Vilchez MA.Effect of acid etching and collagen removal on dentin wettability and roughness.J Biomed Mater Res 1999;47:198–203.

29. Iwanami M, Yoshioka T, Sunakawa M, Kobayashi C, Suda H. Spreading of root canalirrigants on root dentine. Aust Endod J 2007;33:66–72.

30. Habelitz S, Balooch M, Marshall SJ, Balooch G, Marshall GW Jr. In situ atomic forcemicroscopy of partially demineralized human dentin collagen fibrils. J Struct Biol2002;138:227–36.

31. Sakae T, Mishima H, Kozawa Y. Changes in bovine dentin mineral with sodiumhypochlorite treatment. J Dent Res 1988;67:1229–34.

32. Tanaka J, Nakai H. Application of root canal cleaning agents having dissolvingabilities of collagen to the surface treatment for enhanced bonding of resin to dentin.Dent Mater J 1993;12:196–208.

33. Attal JP, Asmussen E, Degrange M. Effects of surface treatment on the free surfaceenergy of dentin. Dent Mater 1994;10:259–64.

34. Gordon TM, Damato D, Christner P. Solvent effect of various dilutions of sodiumhypochlorite on vital and necrotic tissue. J Endod 1981;7:466–9.

35. Quirynen M, Bollen CM. The influence of surface roughness and surface-free energyon supra- and subgingival plaque formation in man: a review of the literature. J ClinPeriodontol 1995;22:1–14.

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