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J OURNALF ENDOCONTICSCopyright 0 2000 by The American Association of Endodontists

Printed in U.S.A.VOL.26, No. 6, J UNE 000

SCI ENTl FIC ARTICLES

Antimicrobial Substantivity of Chlorhexidine-TreatedBovine Root Dentin

Richard Komorowski, DDS, Helen Grad, MSc, PHM, Xiao Yu Wu, PhD, and Shimon Friedman, DMD

Previous studies have demonstrated antimicrobialsubstantivity in root canal dentin up to 7 days after

treatment with chlorhexidine. This in vitro studyassessed the antimicrobial substantivity of chlo-rhexidine-treated bovine root dentin over a periodof 21 days. Sixty standardized bovine root sectionswere randomly divided into three equal groups,and their canals immersed in one of the followingsolutions: (i) sterile saline; (ii) 2.5% NaOCI; or (iii)0.2% chlorhexidine (CHX). Half the specimens ineach group were treated with the solution for 5 minand the other half for 7 days. After solutions wereremoved, the specimens were incubated at 37C inBrain Heart Infusion broth containing Enferococ-

cus faecalis (ATCC 29212). A fresh inoculum wasadded to the broth every other day over a 21-dayperiod. The canals were then enlarged with sterileburs, and the dentin shavings collected and cul-tured for the presence of cultivable bacteria in thedentinal tubules. Specimens treated with CHX for 7days demonstrated significantly less dentin colo-nization by E. faecalis than the other specimens.CHX has potential as an intracanal medicament, ifit can be applied for a period of at least 7 days.

The role of bacteria in the developme nt of apical periodontitis hasbeen well established (1). A major goal of endodontic therapy is,therefore, to eliminate bacteria fromthe root canal system. Rootcanal asepsis is attempted by means of mechanical instrumenta-tion, chemical irrigation, and interappointmen t intracanal medica-tion (2). Despite these procedures, bacteria, particularlyEntero-coccus faecalis, may persist within the root canal system, andsustain the presence of apica l periodontitis (3-5). Apical periodon-titis can also develop subsequent to treatment, due to bacterialcontamination of the filled root canal system resulting from coro-nal leakage. Recent animal studies have demonstrated develop-

ment of apical periodontitis after bacterial inoculation of the pulp

chambers in endodontically treated teeth (6). It seems that cur-rently used root can al filling materials do not effectively protect theroot canal system against bacterial challenges that may occurduring the long-term function of the endodontically treated tooth.

Several researchers have pointed to the potential advantage ofchlorhexidine gluconate (CHX ) as an antimicrobial medicament inendodontic therapy (7-18). CH X is a broad-spectrum antimicrobialagent (19) that can be. used effectively as an irrigant (7-12),disinfect the dentinal tubules (13-15), and adsorb on to the dentin(7). Root dentin treated w ith CHX seems to acq uire antimicrobialsubstantivity (7, 16, 17). This effect has been co nfirmed to extendat least 7 days; when CHX was used as an intracanal medicamentin bovine roots for 1 wk, the root dentin demonstrated inhibition ofcolonization by E. faecalis for a period of 7 days (16).

The authors are unaware of studies in which the antimicrobialsubstantivity of CHX-treated root dentin was tested for periodslonger than 1 wk. Therefore, the aim ofthis study was to assess theantimicrobial substantivity of CHX-treated bovine root dentin overa period of 21 days.

MATERIALS AN D METHODS

The study methodology was basedon that of previous studies(13, 14, 16). Bovine incisors were placed in0.5% NaOCl for 24hrto attain surface disinfection. Using a water-cooled diamond sawrotating at 700 rpm, the apical5 mm and clinical crowns of theteeth were resected, the cementum was removed from the remain-

ing roots, and each root was cut perpendicular to its long axis into5 mm discs. Each root disc specimen had an external diameter of-7 mm. The internal diameter was then standardized by enlargingthe root canal with anI S 0 033 round bur.The specimens were keptin water during all procedures to avoid dehydration. Organic andinorganic debris, including the smear layer, were removed byimmersing the discs in an ultrasonic bath with 17%EDTA oblowed by 5.25% NaOCl for5 min. Absence of smear layer wasconfirmed by scanning electron microscopic examinationo f acontrol specimen. The specimens were placed in testtubes con-taining Brain Heart infusion (BHI) broth and autoclavedthreetimes at 121C. After each sterilization cycle of30 min th e discswere incubated for 24hr at 37C in ai r to confirm sterility. The

sterilized specimens were immersed for 15 min inan ultrasonic31 5

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316 Komo rows ki et al. Journ al of Endodontics

TABLE . Optical density of broth incubated with shavings obtained from different depths of circumpulpal dentin in bovine rootdiscs

Treatment Mean Optical Density (SD)/Dentin Depth (mm)

Time 0.1 0.2 0.35 0.45Solution

Saline 5 min 0.72 (0.12) 0.76 (0.07) 0.76 (0.10) 0.84 (0.06)7 days 0.53 (0.09) 0.69 (0.14) 0.77 (0.16) 0.67 (0.21)

5.25% NaOCl 5 min 0.64 (0.13) 0.66 (0.10) 0.65 (0.09) 0.73 (0.10)7 days 0.69 (0.25) 0.59 (0.17) 0.66 (0.21) 0.86 (0.26)

0.2% CHX 5 min 0.65 (0.16) 0.62 (0.07) 0.69 (0.08) 0.71 (0.08)7 days 0.010 0.003) 0.015 (0.005) 0.01 0 (0.005) 0.01 0 0.005)

The canals were immersed with test solutions for either 5 min or 7 days, and then inoculated with E. faecalis for 21 days.

bath containing fresh BHI broth to enhance penetration of the brothinto the dentinal tubules. They were then blotteddry with sterilepaper, coated externally with nail polish, and mounted on stickywax in Petri dishes.

A total of 60 specimens were randomly assigned to three equalgroups. Under aseptic conditions, the root canal of each specimenwas immersed in 10 ml of one of the following solutions: (i) sterilesaline (positive control); (ii) 5.25% NaOC l; or (ii) 0.2% CHX . Thesolutions were introduced with a sterile syringe and 25 gaugeneedle placed in the center of the root canal. Excess solution wasremoved from the top surface with a sterile suction tip.In half thespecimens in each group,the solutions were left in place f or5 min.In the other half, the solutions were left in place for7 days. Thelatter specimens were kept in an incubator at 37"C, and the solu-tions were replenished daily.

At the termination of the treatment period ofthe specimens, thetest solutions were removed with a sterile suction tip, and thespecimens blotted dry, xternally with sterile paper and internallywith sterile paper points. The root ca nal of each specimen w as thenfilled entirely with an overnight suspension ofE. fueculis (ATCC29212) in BHI broth. BHI broth was added daily, and freshinoculum was added daily every second day for a period of 21days. The discs were incubated at 37C. Sam ples of the broth weretaken from the canals of the specimens at regular intervals toconfim the viability and purity of the inoculum.

After 21 days the specimens were removed from the Petridishes, thoroughly rinsed with sterile water, and blotteddry withsterile paper. Sterile round burs-IS0 sizes 035, 037,040, and042-were used sequentially to enlarge the canals and thus removedentin shavings ranging in depth from 0.1 to 0.45 m m. Th e dentinshavings obtained with each bur were collected in a separate testtube containing 3 ml of fresh BH I broth, and incubated for 24hrat 37C. The optical density of the broth, proportional to thenumber of viable bacteria present in the dentin shavings, wasmeasured in a spectrophotometer at 540nm. When the brothbecame turbid, indicating bacterial growth, the culture w as exam-ined for purity of E. fueculis.

The mea n optical density and standard deviation were calculatedfor each treatment solution, period and dentin de pth, and statisti-cally analyzed using Mann-WhitneyU est. Two-tailed p valuesof

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Vol. 26, No. 6, June 2000

of the root canal, as shown in bovine roots imga ted with0.2% and1% CHX for 20 and 40 min (7), and in huma n teeth irrigated withseveral milliliters of2% CHX (17). Otherstudies (9, 11). however,have failed to corroborate these results. We considered irrigationofthe root canal for 20 to 40 min to be impractical, as it wouldconsiderably prolong treatment, particularly in multirooted teeth.We, therefore, used a treatment period of 5 min to simulateimgation. In addition, the period of bacterial challenge inthis

study was extended to3 wk. Under these experimental conditions,irrigation with0.2% CHX failed to induce noticeable antimicrobialsubstantivity.

In contrast to the short-term treatment, as in im gatio n, treatmentwith CHX for 7 days did induce antimicrobial substantivity, cor-roborating the report by Heling et al. (16). Such prolong applica-tion also may help eliminate any residual bacteria that wouldotherwise persist within the root canal system. Elimination ofresidual bacteria would improvethe prognosis of treatment(3, 4).In endodontically treated teeth with persisting apical periodontitisthe canals are frequently infected withE. fueculis (4,5), in contrastto untreated infected teeth where E. fueculis is uncommon (4).Apparently, E. fuecalis can adapt well to the substrate-scant envi-

ronment of the filled root canal system, where it can sustainmonoinfections (5). It can resist mechanical debridement and thecommonly used antibacterial medicaments suchas calcium hy-droxide (2 , 13). Because of its characteristics,E. fueculis is diffi-cult to eradicate during retreatment of teeth w ith persistent a picalperiodontitis (4), and its presence is associated with a poorerprognosis of suc h teeth(4). E. fuecalis is, however, susceptible toCH X (19). A prolonged applicationof CHX may, therefore, proveto be advantageous, particularly when used in retreatment cases.

Another mechanism by which endodontically treated teeth candevelop apical periodontitis is by ingress of bacteria at any timesubsequent to treatment (6). The risk for bacterial ingress is par-ticularly high when a breakdown occurs inthe coronal seal, re-

sulting in infection of the pulp chamber. Our group has clearlydemonstrated the ability of bacteria present inthe pulp chambers ofendodontically treated teeth to ingress the canals and cause ap icalperiodontitis within weeks or months (6). It canbe speculated thatthe antimicrobial substantivity acquired by the root dentin aftertreatment with CHX could inhibit reinfection of the canal subse-quent to treatment.To do so, the antimicrobial substantivity shouldextend for a very long time, ideally, the lifetimeof the tooth. Theduration of this effect is unknown a t present. Results ofthe presentstudy demonstrated, however, that the antimicrobial substantivityof CHX-treated bovine dentin was as effective after3 wk asobserved earlier for 1 wk (16). Future experiments should targetlonger time periods to assess the duration of this effect.

To effectively rem ain in the canal for7 days, CHX must be usedas an intracanal medicament. Assuming that CHX could notbe leftin the canal in plain liquid form, attempts have been reported todispense it from controlled- or sustained-release devices (14, 16,18). In these devices, CHX is contained in a polymer sheath that,when placed in a liquid environment, gradually dissolves andreleases the CHX.Although the applicability of such devices totheroot canal of human teeth remains tobe proven, their efficacy inthe release of CHX has been show n both in agar plates(18) and inbovine roots (14, 16). Most importantly, the use of such a deviceresulted in antimicrobial substantivity ofthe bovine root dentin,confirmed over a periodof 7 days (16). In sum mary, it seems that

ChlorhexidinerTreatedRoot Dentin 317

CHX merits use an intracand medicament in endodontics.Convenient and predictable me ans of delivery shouldbe developedto allow its placemen t and retention in the root canal f or periodsofat least 1 wk.

The authors thankfully acknowledge the assistance obtained from Profes-

Drs. Komorowski and Friedman are affiliated with the Department of End-odontics. Faculty of Dentistry; Dr. Grad is affiliated with the Department ofPharmacology, Faculty of Medicine, Faculty of Dentistry; and Dr. Wu is affil-iated with the Faculty of Pharmacy, University of Toronto, Toronto, Ontario,Canada. Address requests for reprints to Dr. Richard Komorowski. Depart-ment of Endodontics, Faculty of Dentistry, University of Toronto, 124 EdwardStreet, Toronto, Ontario M5G 1G6, Canada.

sors Adele Csima (statistical analysis) and Richard Ellen (microbiology).

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