Vol. 99 No. 5 May 2005

ENDODONTOLOGY Editor: Larz S. W. Spangberg

Effect of rotary or manual instrumentation, with or without a calcium

hydroxide/1% chlorhexidine intracanal dressing, on the healing of

experimentally induced chronic periapical lesions

Andiara De Rossi, DDS, MSc,a Lea A. B. Silva, DDS, PhD,b Mario R. Leonardo, DDS, PhD,c LenaldoB. Rocha, DDS, MSc,d and Marcos A. Rossi, MD, PhD,e Ribeirao Preto and Araraquara, BrazilUNIVERSITY OF SAO PAULO AND STATE UNIVERSITY OF SAO PAULO

Objective. To evaluate the healing of experimentally induced chronic periapical lesions in dogs at 30, 75, and 120 days after

root canal instrumentation with rotary NiTi files or manual K-files, with or without a calcium hydroxide/1% chlorhexidine

paste intracanal dressing.

Study design. The second, third, and fourth mandibular premolars and the second and third maxillary premolars of 5 dogs (12 to

18 months of age, weighing 8 to 15 kg) were selected for treatment (a total of 82 root canals). After pulp removal, the root canals

were left exposed to the oral cavity for 7 days to allow microbial contamination, after which the root canals were sealed with

ZOE cement until periapical lesions were confirmed with radiography. Group I and II teeth were instrumented with manual

K-files using the crown-down technique. In group III and IV teeth, NiTi rotary files were used. The apical delta was perforated

by using #20 to #30 K-files at the length of the tooth, thus creating a standardized apical opening. The apical stop was enlarged

to size 70, with 2.5% sodium hypochlorite irrigation at each file change. Teeth in groups II and IV were dressed with calcium

hydroxide (Ca(OH)2)/1% chlorhexidine (CHX) paste for 15 days before root filling. Group I and III teeth did not receive an

intracanal dressing. The access openings of the teeth were permanently restored with silver amalgam condensed on a glass

ionomer cement base. Pairs of standardized periapical radiographs were taken at the beginning of the treatment (0 days) and at

30, 75, and 120 days after filling.

Results. There was no significant difference in the rate of radiographic healing of the periapical lesions between manual and

rotary instrumentation. Radiographs taken at 120 days showed that the treatment with Ca(OH)2/1% CHX paste resulted in

a significant reduction in mean size of the periapical lesions in comparison to single-session treatment. These findings were also

true for histologic observations.

Conclusion. The findings support the hypothesis that, regardless of the instrumentation technique (manual or rotary), the

use of an intracanal dressing is important in the endodontic treatment of dog’s teeth with experimentally induced chronic

periapical lesions.

(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:628-36)

Soon after the introduction of nickel-titanium (NiTi)endodontic manual instruments in 1988,1 NiTi rotaryfile systems became well known. The super elastic

This work was supported by grants from the Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP- 01/06745-0; 01/08438-0;

01/14059-0).aPostgraduate student of Experimental Pathology, Department of

Pathology, Ribeirao Preto School of Medicine, University of Sao

Paulo. Recipient of a Scholarship from FAPESP (01/04732-9).bProfessor, Department of Pediatric Dentistry, Ribeirao Preto School

of Dentistry, University of Sao Paulo.cProfessor, Department of Endodontics, Araraquara School of

Dentistry, State University of Sao Paulo.

628

property of NiTi, coupled with advanced file design,allows efficient instrumentation to be carried out inless time than it takes with traditional manual

dPostgraduate student of Experimental Pathology, Department of

Pathology, Ribeirao Preto School of Medicine, University of Sao Paulo.eProfessor, Department of Pathology, Ribeirao Preto School of

Medicine, University of Sao Paulo.

Received for publication May 3, 2004; returned for revision Jun 17,

2004; accepted for publication Jul 18, 2004.

Available online 8 December 2004.

1079-2104/$ - see front matter

� 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.tripleo.2004.07.018

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Volume 99, Number 5 De Rossi et al 629

instrumentation techniques, with minimal risk ofledging or transporting of the root canals.2 Nevertheless,evidence suggests that rotary and manual instrumenta-tion techniques are similarly ineffective in removingintracanal bacteria, eliminating some but not all bacteriafrom the root canal system.3-7 Because, even aftercareful biomechanical preparation, bacteria may sur-vive, grow, and multiply inside root canal systems or onapical biofilm, the use of an effective, antibacterial,intracanal dressing in teeth with periapical lesions is stillrecommended to reduce endodontic microbiota, therebyfavoring periapical tissue healing.3,7

In recent years, chlorhexidine gluconate (CHX) hasemerged as an effective antimicrobial medication inendodontic therapy.8-11 It has a broad spectrum of ac-tivity against gram-positive and gram-negative bacteriaand is able to adsorb to dental tissue and mucousmembrane with prolonged gradual release at therapeuticlevels. When used as a root canal treatment, CHX hasbeen shown to be more effective than calcium hydroxide(Ca(OH)2) in eliminating Enterococcus faecalis in-fection inside dentinal tubules.8 On the other hand,Ca(OH)2 mediates the neutralization of bacterial endo-toxin (LPS)12,13 and stimulates periapical hard-tissuehealing.14,15

To our knowledge, there have been no studies of theeffect of Ca(OH)2 paste in combination with CHX usedas an intracanal dressing to aim a higher spectrum ofactivity against bacteria inside the root canal and tofavor periapical tissue repair. This study evaluates theperiapical healing process in terms of the instrumenta-tion technique used, with or without an intracanaldressing with Ca(OH)2/1% CHX paste, at 30, 75, and120 days after endodontic treatment of teeth withchronic periapical lesions.

MATERIAL AND METHODSAll animal procedures performed in this study

conformed to protocols reviewed and approved by theAnimal Care Committee of the University of Sao Paulo(Protocol #2002.1.121.53.0).

The second, third, and fourth mandibular premolarsand the second and third maxillary premolars of 5 dogs(12 to 18 months of age, weighing 8 to 15 kg) wereselected for treatment (a total of 82 root canals). Theanimals were anesthetized intravenously with sodiumthiopental (30 mg/kg body weight; Thionembutal;Abbott Laboratories, Sao Paulo, Brazil), and standard-ized radiographs were taken using customized stents.16

Crown access was created on the occlusal surface withspherical carbide burs. After pulp removal, the rootcanals were left exposed to the oral cavity for 7 days toallow microbial contamination. Access openings werethen sealed with quick-setting zinc oxide-eugenol

cement (Pulposan, SS White Artigos Dentarios; Riode Janeiro, Brazil). After 30 days, radiographs weretaken at 15-day intervals until radiolucent imagesindicating chronic periapical lesion were obtained,which normally occurred after 60 days.

The 4 experimental protocols were used in eachanimal. After isolation with a rubber dam anddisinfection of the operative field with 2% CHX, thezinc oxideeeugenol cement was removed and eachquadrant received 1 of the treatments, in an alternatingmanner. Instrumentation in group I and group II teethwas carried out using manual K-files (DentsplyMaillefer Instruments; Ballaigues, Switzerland) usingthe crown-down technique. In group III and IV teeth,NiTi rotary files (GT System; Dentsply MailleferInstruments) were used in the crown-down mode forextensive root canals with large apical diameters.17 Inall tooth groups, the working length was determined to 2mm short of the radiographic apex by using #30 K-files.Using the odontometric measurement, the apical deltawas perforated by using #20 to #30 K-files at the lengthof the tooth, thus creating a standardized apical opening.In all techniques, the apical stop was enlarged withK-files up to size 70, with 2.5% sodium hypochloriteirrigation (3.6 mL) at each file change, using a syringeand a 27-gauge needle (Terumo; Tokyo, Japan). Thesame operator performed all procedures.

After irrigation, the root canals were dried byaspiration and sterile absorbent paper points, filled with14.3% buffered EDTA (pH 7.4; Odahcan-HerpoProdutos Dentarios; Petropolis, Brazil), which wasagitated with a K-file for 3 min, then irrigated withsaline and dried. In groups II and IV, the canal dressingwith Ca(OH)2/1% CHX paste was applied. For this pastepreparation, 0.48 mL of the CHX at 10% was added to4.8 g of Ca(OH)2 paste (Calen; composition: 2.5 gCa(OH)2, 0.5 g zinc oxide p.a., 0.05 g colophony, 1.75mL polyethylene glycol 400; SS White ArtigosDentarios) in a laminar air flow, homogenized and keptin sterile tubettes. The paste was applied during rootcanal using an ML syringe (SS White Artigos Dentarios)with a 27-gauge needle, up to the working length, whichwas marked on the needle with a rubber stop. Sterilecotton pledget was placed in the pulp chamber, andthe access cavity was filled with quick-setting zincoxideeeugenol cement.

Fifteen days later, under saline irrigation, thisintracanal dressing was removed with a #70 K-file tothe working length and with a #30 K-file to the length ofthe tooth. The canals were dried and filled with EDTAfor 3 min, irrigated with saline, and then redried. Rootcanal filling was performed with gutta-percha cones andAH Plus sealer (Dentsply De Trey; Konstanz,Germany). Master gutta-percha cones were chosen in

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630 De Rossi et al May 2005

a diameter corresponding to the last instrument used forbiomechanical preparation (confirmed through radio-graphs), coated with sealer along their entire length(including the tip) and placed in the root canal to theworking length. Active lateral condensation was per-formed with the aid of finger spreaders (DentsplyMaillefer).

Group I and III teeth did not receive intracanaldressing. After biomechanical preparation, the rootcanals were filled in 1 step using the same method as thatused in group II and IV teeth.

The crown openings of the 4 groups were perma-nently restored with silver amalgam (Velvalloy; SSWhite Artigos Dentarios), which was condensed ona glass ionomer cement base (Vitremer; 3M DentalProducts, St Paul, Minn).

Fig 1. Rates (6 SEM) of percent reduction of the periapicallesions as revealed as radiolucent areas from: group I, manualinstrumentation and root canal filling in 1 session; group II,manual instrumentation and Ca(OH)2/1% CHX paste intra-canal dressing and root canal filling in 2 sessions; group III,rotary instrumentation and root canal filling in 1 session; andgroup IV, rotary instrumentation and Ca(OH)2/1% CHX pasteintracanal dressing and root canal filling in 2 sessions; at 30,75, and 120 days after filling. *P \ .05 in comparison togroups I and III.

Radiographic studyPairs of standardized periapical radiographs were

taken at the beginning of the treatment (0 days) and at30, 75, and 120 days after filling. A Heliodent dentalx-ray machine (Siemens; Erlanger, Germany) was usedwith exposure factors set at 60 kV, 10 mA, and 0.4 s.Ultraspeed periapical film (Eastman Kodak; Rochester,NY) was used, and the radiographs were processed bythe time/temperature method.

The images were digitized through an opticalscanning process for transparencies (Scanjet 7450C,Hewlett-Packard; Palo Alto, Calif). The images weretransferred to the Image J program (version 1.28 u,National Institutes of Health, Washington DC) for mea-surement and recording of results. For each image pair,the radiolucent areas of periapical lesion were de-lineated and measured. Delineation was performed onthe radiograph image to exclude tooth structure butinclude the area of rarefaction. Investigators weremasked as to which groups the teeth belonged to.Evaluation consisted of observing the differencebetween the appearance of the radiolucent area beforethe treatment (0 days) and during the experimentalperiods (at 30, 75, and 120 days). The percent ratereduction of the periapical lesion was calculated.

Histopathologic studyAll animals were killed with an anesthetic over-

dose, and the maxillae and mandibles were removed.The teeth were separated and individually fixed in2.5% glutaraldehyde in cacodylate buffer withsaccharose for 24-48 hours at room temperature.The specimens were then washed and demineralizedwith EDTA in a microwave oven. Subsequently, theroots were washed in running water for 24 hours,dehydrated in ascending concentrations of ethanol,cleared in xylol and embedded in paraffin. Seriallongitudinal sections 6-mm thick were cut in mesiodistalorientation, stained with hematoxylin and eosin, andexamined under light microscopy. A skilled observermasked to the treatment groups made the examination ofthe periapical lesion.

Statistical analysisData are expressed as mean 6 standard error of

the mean (SEM). Statistical analyses were performed by1-way analysis of variance (ANOVA), followed by theTukey test (to correct for multiple comparisons). A levelof significance of 5% was chosen to denote thedifference between group means. Data were analyzedusing a GraphPad Prism statistic program (GraphPadSoftware; San Diego, Calif).

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Fig 2. Radiographic images representative of the reduction in mean rates of the periapical lesions from: group I, manualinstrumentation and root canal filling in 1 session; group II, manual instrumentation and Ca(OH)2/1% CHX paste intracanaldressing and root canal filling in 2 sessions; group III, rotary instrumentation and root canal filling in 1 session; and group IV, rotaryinstrumentation and Ca(OH)2/1% CHX paste intracanal dressing and root canal filling in 2 sessions; at 30, 75, and 120 days afterfilling.

RESULTSRadiographic study

Rates of percent reduction of the periapical lesion asrevealed by radiolucent areas from all groups at days 30,75, and 120 after root canals filling are presented in Fig1. Radiographs taken at 30, 75, and 120 days show thatthe manual and rotary instrumentation techniques didnot significantly affect the healing of the periapicallesions. At 30 and 75 days, although the intracanaldressing with Ca(OH)2/1% CHX paste resulted inmarked dispersion of the rate of reduction seen in theradiographic images of the periapical lesion, the imageswere similar to those of both groups that were treated inonly 1 session. However, radiographs taken at 120 daysshowed that the treatment with Ca(OH)2/1% CHX paste

induced a significant reduction in mean rates of theperiapical lesions in comparison with single-sessiontreatment.

Radiographic images representative of the reductionin mean rates of the periapical lesions from all groupsare shown in Fig 2.

Histopathologic study30 and 75 days. One-session treatment (groups I and

III): Periapical lesion and inflammatory infiltratecomposed of neutrophils, histiocytes, xanthomatoushistiocytes, plasma cells, and lymphocytes and markededema. Surface resorption areas (Howship’s lacunae)were evident, similar to those seen on the cementumsurface (Figs 3 and 4).

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632 De Rossi et al May 2005

Two-session treatment (groups II and IV): Twodifferent basic patterns: one mainly showing cicatricialreparative tissue characterized by proliferation offibroblasts, small vessels, and increased connectivetissue, and the other showing an inflammatory infiltrate

Fig 3. Representative views of the periapical lesions at 30 and75 days after root canal filling from groups subjected to handinstrumentation or rotary instrumentation and root canalfilling in 1 session (A) and from groups subjected to handinstrumentation or rotary instrumentation combined withintracanal dressing with Ca(OH)2/1% CHX and root canalfilling in 2 sessions characterized by presence (B) or absence(C) of cicatricial repair (hematoxylin-eosin; original magni-fication 3194).

similar to that of teeth filled in one session. Betweenthese 2 basic patterns, most specimens presenteda mixed periapical lesion (Figs 3 and 4).

120 days. One-session treatment (groups I andIII): Periapical lesion and inflammatory infiltratecomposed of neutrophils, histiocytes, xanthomatoushistiocytes, plasma cells, and foreign-body giant cells.Surface resorption areas (Howship’s lacunae) wereevident, similar to those seen on the cementum surface(Fig 5).

Two-session treatment (groups II and IV): Markedreduction in the size of the periapical alterations,characterized by cicatricial reparative tissue mostlycomposed of fibroblasts interspersed with collagenfibers and numerous microvessels. Microfoci ofhistiocytes and plasma cells were occasionally seeninterspersed with neutrophils (Fig 5).

DISCUSSIONBecause bacteria and their products play an essential

role in the initiation, propagation, and persistence ofperiapical disease, elimination of bacteria from rootcanals seems to be a logical objective for successfulendodontic treatment.18 In addition, the presence ofbacterial biofilm on the external surfaces of the root apexin teeth with nonvital pulp and chronic periapical lesionsis associated with endodontic treatment failure.19 Theprognosis of healing or progression of disease after rootcanal treatment is important to patient management andassessment of treatment efficacy. Radiographs, despitetheir limitations, are an important part of root canaltreatment and have being used in numerous diagnosticand prognostic studies to determine the success ofnewer endodontic techniques and materials.12,14,20,21

The accuracy of radiographic diagnosis of such resorp-tion depends on several factors22,23 and can be verifiedconclusively only through examination of histologicsections of the affected teeth.24

The procedure used in the present study canconsistently induce periapical lesions in the teeth ofdogs. Briefly, the teeth are submitted to pulp removaland the cavities are left open for a week for con-tamination and then sealed for 60 days. At this time, theradiographic images reveal radiolucent periapical areascombined with morphologic changes to the periapicalregion, characterized by root and bone resorption,bacteria, and dense inflammatory infiltrate composedof mononuclear cells, plasma cells, and neutrophils(paper in preparation).

Our radiographic and histopathologic findings clearlyshow that, at 30, 75, and 120 days after root canal filling,using manual or rotary instrumentation techniques didnot significantly affect the healing of the experimentallyinduced periapical lesions. Both the manual and

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Fig 4. Representative views of the periapical lesions at 30 and 75 days after root canal filling from group I, manual instrumentationand root canal filling in 1 session, and group III, rotary instrumentation and root canal filling in 1 session (A-C), and from group II,manual instrumentation and Ca(OH)2/1% CHX paste intracanal dressing and root canal filling in 2 sessions, and group IV, rotaryinstrumentation and Ca(OH)2/1% CHX paste intracanal dressing and root canal filling in 2 sessions (D-F). A-C, Inflammatoryinfiltrate composed of neutrophils (fine arrows), histiocytes (arrowhead), xanthomatous histiocytes (empty arrows), plasma cells(traced arrows), and lymphocytes (dense arrows) and marked edema. D-F, Cicatricial reparative tissue characterized byproliferation of fibroblasts, small vessels (empty arrows), increased connective tissue, and inflammatory infiltrate composed ofneutrophils (arrows), histiocytes (arrowheads), xanthomatous histiocytes (asterisks), plasma cells, and lymphocytes associatedwith marked edema (squares). (Hematoxylin-eosin; original magnification A-C 3732; D 3660; E 3534; F 3660.)

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Fig 5. Representative views of the periapical lesions at 120 days after root canal filling from groups subjected to handinstrumentation or rotary instrumentation and root canal filling in 1 session (A) and from groups subjected to hand instrumentationor rotary instrumentation combined with intracanal dressing with Ca(OH)2/1% CHX and root canal filling in 2 sessions (B).C, Periapical lesion from 1-session treatment and the inflammatory infiltrate composed of neutrophils, histiocytes, xanthomatoushistiocytes, plasma cells, and lymphocytes. D, Foreign-body giant cells (arrows). E-G, Periapical lesion from 2-session treatmentwith inflammatory infiltrate composed of a cicatricial reparative tissue containing mostly fibroblasts (dense arrows), numerousmicrovessels (traced arrows), and little lymphocytes (fine arrows), plasma cells (arrowheads), and histiocytes (empty arrows).G, Occasional inflammatory infiltrate composed of histiocytes and plasma cells interspersed with neutrophils (asterisks).(Hematoxylin-eosin; original magnification A, B 3156; C 3988; D 31590; E 3915; F 31079; G 3813.)

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rotary techniques followed by intracanal dressing withCa(OH)2/1% CHX paste resulted in similar (radiolog-ically confirmed) healing at 30 and 75 days after rootcanal filling in comparison with both groups treated insingle sessions. However, the intracanal dressing re-sulted in marked dispersion of the values of the rate ofpercent reduction of the periapical radiolucent lesions.In addition, the histopathologic evaluation, rangingfrom complete absence of healing to maximum healing,reflected this dispersion of the values of periapicalradiolucent lesions. On the other hand, at 120 days, theintracanal dressing induced a significant reduction in themean area of periapical radiolucent lesions and a parallel(histopathologically confirmed) healing of the experi-mentally induced periapical lesions in comparison withteeth treated in 1 session only.

Many methods have been described to measure thesize of periapical radiolucencies. New methods, such asthe semiautomated one used in this study, can be used toincrease the amount of information offered by theradiographs, thus making the evaluation more objec-tive.14,21,25 Conventional radiographs were digitized tomake the periapical radiolucencies more visible, similarto what has been done in other studies.25,14 Some of theadvantages of using the digitized images are the abilityto alter brightness, contrast, and gray-scale density,thereby allowing better viewing of the periapical lesion.

The suggestion that NiTi rotary instrumentation isfaster and results in fewer procedural errors haspopularized its use. However, endodontic treatment ofteeth with pulp necrosis and concomitant periapicallesion should be directed toward elimination ofmicroorganisms from the root canal and apical biofilm.It has been demonstrated that, even when the higheststandards are maintained, endodontic treatment failurecan still occur as a consequence of persistent infection.19

Several studies have demonstrated that the NiTi rotaryinstrumentation seems to be inadequate for completedisinfection of root canals in 1 session without the aid ofan intracanal dressing.3-7 Thus, in addition to bio-mechanical preparation, intracanal medication has beenclaimed to be a valuable adjunct in the elimination ofbacteria from both the root canal system and the apicalbiofilm. Our results reinforce this concept. Single-session filling has been recommended by endodontists26

who believe that, in teeth with periapical lesions, theroot canal biomechanical preparation provides con-ditions for repair after endodontic treatment.

Several substances have been recommended for useas intracanal dressings. Among dressings for endodontictreatment of teeth without pulp vitality and with chro-nic periapical lesions, Ca(OH)2 has been widelyrecommended and used because of its proven antibac-terial properties,7,20 periapical tissue healing stimula-

tion,14,15 and capacity to detoxify bacterial LPS.12,13

The dissociation of Ca(OH)2 into calcium and hydroxylions is responsible for the alkalinization of the cavity,which is not conducive to bacterial development andproliferation. Aqueous vehicles have rapid ionic dis-sociation and consequently short-term action. In teethwith periapical lesions, the use of Ca(OH)2 pastes, typi-cally combined with either polyethylene glycol or watervehicles, has been reported to produce better results withpolyethylene glycol 400, which permits slower libera-tion of hydroxyl ions, maintaining its action for a longerperiod.15 This type of Ca(OH)2 paste is known as Calenpaste and its prolonged action may explain the positiveresults obtained by using it in combination with CHX at120 days.

In conclusion, our findings support the hypothesisthat, regardless of the instrumentation technique chosen(manual or rotary), the use of an intracanal dressing isimportant in the endodontic treatment of dog’s teethwith experimentally induced chronic periapical lesions.However, owing to the fact that Ca(OH)2 paste com-bined with 1% CHX produced better results only at120 days, its use in a clinical situation should be furtherinvestigated during the endodontic treatment of teethwith pulp necrosis and periapical lesions.

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Reprint requests:

Andiara De Rossi

Postgraduate student of Experimental Pathology

Department of Pathology

Ribeirao Preto School of Medicine

University of Sao Paulo 14049-900—Ribeirao Preto, S.P.

Brazil

andiaraderossi@bol.com.br