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    1x2 visits 2012 1x2 visits 2012 Document Transcript

    • Clinical ResearchOne- versus Two-visit Endodontic Treatment of Teethwith Apical Periodontitis: A Histobacteriologic StudyJorge Vera, DDS,* Jos F. Siqueira, Jr, DDS, MSc, PhD,† Domenico Ricucci, MD, DDS,‡ eSimona Loghin, DDS,‡ Nancy Fern ndez, DDS,§ Belina Flores, DDS,k aand Alvaro G. Cruz, DDS, MSc¶AbstractIntroduction: This study analyzed the in vivo micro- an antibacterial interappointment agent is necessary to maximize bacterial reductionbiological status of the root canal systems of mesial before filling. (J Endod 2012;-:1–13)roots of mandibular molars with primary apical perio-dontitis after 1- or 2-visit endodontic treatment. Key WordsMethods: Mesial root canals were instrumented by Calcium hydroxide, endodontic infection, endodontic treatment, 1-visit endodontics,using either a combination of K3 and LightSpeed instru- sodium hypochloritements (mesiobuccal canals) or the ProTaper system(mesiolingual canals), with 5% NaOCl irrigation.Patency files were used. Smear layer was removed,and a final rinse with 5 mL of 2% chlorhexidine was T he microbiological goals of the endodontic treatment of teeth with apical periodon- titis are to reduce the microbial bioburden to levels compatible with periradicular tissue healing and to prevent microbial recolonization of the treated canal. The formerperformed. In the 2-visit group (7 roots, 14 canals), can be attained by antimicrobial measures involving chemomechanical procedurescanals were medicated with calcium hydroxide for 1 and intracanal medication, whereas the latter is a role for root canal obturation.week and then obturated by using the continuous One of the most controversial issues in endodontics is whether an interappointmentwave of compaction technique. In the 1-visit group (6 medication is really needed to improve disinfection and then enhance treatmentroots, 12 canals), canals were immediately obturated outcome (1).after chemomechanical procedures. Teeth were Several clinical studies have evaluated the intracanal antimicrobial activity ofextracted 1 week after root canal instrumentation and chemomechanical preparation by using NaOCl as the irrigant in concentrations rangingprocessed for histobacteriologic analysis. Results: In from 0.5%–5%, but most of them demonstrated that 40%–60% of the canals still exhibitthe 1-visit group, no case was completely free of detectable cultivable bacteria (2–12). Because residual bacteria have been shown tobacteria; residual bacteria occurred in the main root adversely affect treatment outcome (4, 13), the use of an interappointmentcanal (5 of 6 cases), isthmus (5 of 6), apical ramifications intracanal medication has been recommended to supplement the antibacterial effects(4 of 6), and dentinal tubules (5 of 6). In the 2-visit of chemomechanical procedures and maximize bacterial reduction (3, 5, 7, 9, 10,group, 2 cases were rendered bacteria-free; residual 14). Calcium hydroxide is arguably the most commonly used intracanal medication,bacteria were found in the main canal only in 2 cases but its effectiveness in significantly increasing the number of culture-negative root(none of them with persistent dentinal tubule infection), canals after chemomechanical preparation has been demonstrated to be inconsistentin the isthmus (4 of 7 cases), and in ramifications (2 of (3, 5, 15).7). The 2 instrumentation techniques performed simi- There are many studies showing that root canals that cultured negative beforelarly. When filling material was observed in ramifica- filling have a greater potential of improved outcome (4, 16, 17). Thus, the choicetions, it was usually intermixed with necrotic tissue, for clinical protocols that predictably lead to negative cultures as demonstrated bydebris, and bacteria. Conclusions: The 2-visit protocol the literature has been recommended (18). Nevertheless, culture has limitations,by using an interappointment medication with calcium because it has low sensitivity, many endodontic bacterial species remain to be cultivated,hydroxide resulted in improved microbiological status and the method used for sampling only shows the microbiological conditions of theof the root canal system when compared with the 1- main canal (19). The histobacteriologic approach used in previous studies (20–22)visit protocol. Residual bacteria were more frequent has the great advantage of providing information about the spatial location ofand abundant in ramifications, isthmuses, and dentinal residual bacteria, including those present in areas distant from the main canal, suchtubules when root canals were treated without an as tubules, isthmuses, and ramifications, which are difficult to treat and sample.interappointment medication. Apical ramifications and Therefore, this method has great potential to provide information about clinicalisthmuses were never completely filled. The use of protocols that are better suited to control the infection in the root canal system. From the *Department of Endodontics, University of Tlaxcala, Tlaxcala, Mexico; †Department of Endodontics, Faculty of Dentistry, Estcio de S University, Rio de a aJaneiro, Rio de Janeiro, Brazil; ‡Private Practice, Cetraro, Italy; §Private Practice, Monterrey, Mexico; kDepartment of Endodontics, University of Veracruz–UAV, Veracruz, ¶Mexico; and Department of Dental Clinics, University of Guadalajara, Guadalajara, Mexico. Address requests for reprints to Dr Domenico Ricucci, Piazza Calvario, 7, 87022 Cetraro (CS), Italy. E-mail address: dricucci@libero.it0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists.doi:10.1016/j.joen.2012.04.010JOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 1
    • Clinical Research The present histobacteriologic study was undertaken to analyze experiment. All clinical procedures were carried out by 1 experiencedthe in vivo microbial status of the middle and apical segments of the endodontist (J.V.).root canal system of mesial roots of human mandibular molars withprimary apical periodontitis after 1- or 2-visit endodontic treatment. 2-visit GroupTwo different nickel-titanium rotary instrumentation techniques were This group included the mesial canals of 7 mandibular molars.used for both groups, and calcium hydroxide was used in the 2-visit After local anesthesia, the cusps were cut down, carious dentin wastreatment protocol. excavated, and the tooth was isolated with a rubber dam. Thorough disinfection of the tooth and the rubber dam was done by using 5% Material and Methods NaOCl, and the endodontic access cavity was prepared with sterileClinical Procedures high-speed carbide burs. Next, the access cavity and the distal canal Teeth selected for this study were human mandibular molars were profusely irrigated with 5% NaOCl, calcium hydroxide was placedwith necrotic pulps and radiographic evidence of apical periodontitis at the entrance of the distal canal, and the distal aspect of the accessthat were extracted for reasons not related to this study (nonrestor- cavity was separated from the mesial one by building a mechanicalability because of extensive caries/crown fractures or periodontal barrier with LC Block-out resin (Ultradent, South Jordan, UT).disease). The study protocol was approved by the institutional review After exploration with a scouting #10 K-file, the 2 separate mesialboard of Tlaxcala University, Mexico, and informed consent was canals were coronally flared by using Gates-Glidden burs sizes 3 andobtained from the individuals participating in the study after the clin- 2, along with copious irrigation with 5% NaOCl. The working lengthical procedures were thoroughly explained. Thirteen teeth were (WL) was determined with the aid of an electronic apex locator (Elementsselected and randomly assigned to either of 2 experimental groups Diagnostic Unit; Sybron Endo, Orange County, CA) at the 0.0 reading ofby using a coin toss. Only the 2 mesial canals were included in the the device with a #10 K-file. Mesio-angled and/or disto-angledFigure 1. Two-visit group. (A) Tooth #30 with massive buccal carious lesion involving the pulp chamber floor in 56-year-old woman. (B) Postobturation radio-graph taken with mesiodistal angulation. Note the excess material extruded from the lingual canal. (C) Radiograph of mesial root taken at 90 angle. The rootfractured obliquely during extraction. (D) Mesial root in the clearing agent. Obturation material is visible in a space connecting the 2 canals. The pathologic peri-apical tissue remained attached to the root tip at extraction. (E) Cross-cut section taken from the middle third at the level of line 2 in (D). No isthmus is present atthis level between the 2 canals (Taylor modified Brown & Brenn stain; original magnification. Â16). (F) Section taken from the apical third at the level of line 1 in(D). An isthmus containing obturation material (black) and connecting uninterruptedly the 2 mesial canals is now visible. Presence of particles of obturationmaterial on the cut dentin is artifactual, as these float in the Canada balsam and are displaced from the root canal space (original magnification, Â25). (G) Sectiontaken at the level of line 3 in (D), encompassing only the lingual root tip and canal. A moderate quantity of filling material has been forced into the periapicalpathologic tissue (original magnification, Â16). (H) Detail from (G) (original magnification, Â50). Note: no bacteria were found at any level in this specimen.2 Vera et al. JOE — Volume -, Number -, - 2012
    • Clinical ResearchTABLE 1. Microbiological Status and Location of Residual Microorganisms in the Middle and Apical Root Canal System of Teeth with Apical Periodontitis afterTreatment in 1 or 2 Visits Specimen MBC MLC IST DT* AR Overall† Remarks 1 visit DLH À + + + + + Extraradicular biofilm OP À À + + À + VCG À + À À À + RPC + À + + + + Canals confluent IM + + + + + + Canals confluent EGH + + + + + + Canals confluent; extraradicular biofilm 2 visits VG + + À À + + Extraradicular biofilm CA À + + + À + MT À À + À À + EM À À + À + + MFG À À À À À À EN À À + À À + Canals confluent NC À À À À À ÀAR, apical ramifications; DT, dentinal tubules; IST, isthmus; MBC, mesiobuccal canal; MLC, mesiolingual canal.*Residual dentinal tubule infection denotes bacterial presence in dentin surrounding the main root canal and the isthmus area.† Presence (+) or absence (À) of microorganisms in 1 or more of the 5 anatomic locations examined.radiographs were taken to visualize and confirm the presence of distinct mL of 2% chlorhexidine (Consepsis). A patency file was placed 1mesiobuccal and mesiolingual canals. All mesiobuccal canals were in- mm beyond the WL to check for patency before filling.strumented by using K3 rotary instruments (Sybron Endo) until a size The root canals were obturated with gutta-percha and Pulp Canal40/.06 reached the WL, after which, further enlargement of the apical Sealer (Sybron Endo) by using the continuous wave of compaction tech-portion of the canal was completed by using LightSpeed instruments nique (23). For this, the Elements Obturation System (Sybron Endo)from size 42.5 to size 60 (Sybron Dental Specialties, Orange, CA). All me- was set on the down-pack mode at 200 C, and the Buchanan Heatsiolingual canals were instrumented by using the ProTaper system Plugger F.06 (Sybron Endo) was used 3–5 mm short of the WL.(Dentsply/Maillefer, Ballaigues, Switzerland) up to the F2 instrument Compaction of the gutta-percha mass was achieved by using the(25/.08). Copious irrigation with 5% NaOCl (5 mL after each instrument Buchanan Hand Plugger #1 (Sybron Endo). Backfill was done by inject-used) was done throughout the instrumentation procedure. A 27-gauge ing thermoplasticized gutta-percha from the obturator cartridge of theside-vented needle (Endo-Eze; Ultradent) was used to deliver the irrigant, Elements Obturation System set on the backfill mode at 100 C and byinitially to the deepest possible penetration of the needle and then to using a 23-gauge needle. After compaction of the injected gutta-a distance of 2 mm from the WL. Special care was taken to instrument percha, excess material was removed from the pulp chamber, andand irrigate the canals with NaOCl for a total period of about 45 minutes. the access cavities were sealed with IRM. Subsequent to the use of every instrument, a #10 K-file was used to Immediately thereafter the teeth were carefully extracted. A roundmaintain patency of the apical foramen by taking it 1 mm beyond the WL. sterile carbide bur size ¼ was used to prepare a shallow groove on theThe root canals were rinsed with 5 mL of 17% ethylenediaminetetraace- buccal surface of the exposed root to allow recognition of the mesio-tic acid (EDTA) (Smear Clear; Sybron Endo), followed by 5 mL of sterile buccal canal and orientate the tooth processing. The teeth weresaline solution. Chemomechanical procedures were completed by per- immersed immediately in a chilled fixative solution consisting of 4%forming a final rinse with 5 mL of 2% aqueous chlorhexidine solution paraformaldehyde (Merck, Darmstadt, Germany) in phosphate buffer(Consepsis; Ultradent). The canals were then dried with sterile paper (776 mOsm, pH 7.2). The patients were given oral and written post-points. extraction instructions. All canals from the 2-visit group were subsequently medicated witha freshly prepared paste of calcium hydroxide (Sultan, Fenix, NJ) insterile saline for a period of 1 week. The pH of the calcium hydroxide TABLE 2. Summary of Microbiological Status of Teeth Treated in 1 or 2 Visits and Detailed in Table 1paste was measured before application and was found to be abovepH 12.5. The paste was spun into the canals with the aid of a lentulo 1 visit 2 visitsspiral. Care was taken to properly fill the root canal with the calciumhydroxide paste without any radiographically visible air bubbles. The Microbiological status n % n %paste was condensed at the canal orifice level with the aid of a sterile Total number of treated teeth 6 100 7 100cotton pellet. Another sterile cotton pellet moistened in absolute alcohol Teeth exhibiting residual 6 100 5 71 microorganismswas used to clean the pulp chamber walls from calcium hydroxide resi- Mesiobuccal canals with residual 3 50 1 14dues. Access cavies were closed with intermediate restorative material microorganisms(IRM) (Dentsply DeTrey GmbH, Konstanz, Germany). Mesiolingual canals with residual 4 67 2 29 After a period of 1 week, the patient was anesthetized, rubber dam microorganisms Teeth with residual microorganisms in 5 83 4 57was applied, the operative field was disinfected as above, and the root isthmusescanals were reinstrumented and obturated. The procedures consisted Teeth with residual microorganisms in 5 83 0 0of repeating instrumentation with the last instrument operated at the dentinal tubules surrounding theWL with 10 mL of 5% NaOCl as the irrigant. Thereafter, the canals main canalswere rinsed with 10 mL of EDTA, followed by 5 mL of sterile saline Teeth with residual microorganisms in 4 67 2 29 apical ramificationssolution. A final disinfecting rinse was performed once again with 5JOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 3
    • Clinical ResearchFigure 2. One-visit group. (A) Severely broken-down mandibular right first molar in 30-year-old woman. Caries has involved the furcation, and apical perio-dontitis lesions are present on both roots. (B) Postobturation radiograph. (C) Radiograph of mesial root after extraction. (D) Specimen immersed in clearingagent before embedding in paraffin. The root canal filling is slightly overextended apically. (E) Cross-cut section taken from the middle third at the level of line 1 in(D). Both canals appear well-prepared, with dentin removed circumferentially and a round shape of similar diameter at this level. Note the wide isthmus harboringobturation material connecting the 2 canals and the lingual extension of the lingual canal (arrow) (Taylor modified Brown & Brenn stain; original magnification,Â16). (F) Detail of lingual canal. A large mass of necrotic debris colonized by bacteria occupies the central part of the canal lumen, embedded in the obturationmaterial. High-power view from center of this mass shows amorphous material (likely food remnants) surrounded by heavy concentration of bacterial profiles(original magnification, Â50; inset, Â400). (G) Detail of isthmus in (E) (original magnification, Â50). (H) Magnification of area indicated by upper arrow in(G). Bacterial biofilm covers the irregularity in dentin wall of the isthmus (original magnification, Â400). (I) Magnification of area on opposite isthmus wallindicated by lower arrow in (G). In addition to biofilm present on dentin wall, bacteria are also colonizing dentinal tubules (original magnification, Â400).1-visit Group Controls This group included the mesial canals of 6 mandibular molars that Four healthy mandibular third molars with vital pulps andwere treated in 1 visit. These canals were treated exactly as described for extracted because of pericoronitis were processed histologicallythose in the 2-visit group, except that they were instrumented and filled to serve as negative controls. Four untreated mandibular molarsat the same appointment. No interappointment dressings with calcium with necrotic pulps and radiographic evidence of apical periodon-hydroxide were used. The teeth were extracted in accordance with the titis were extracted because of the reasons explained above andprocedures described above 7 days after the intracanal procedures served as positive controls.without reentering the canal.4 Vera et al. JOE — Volume -, Number -, - 2012
    • Clinical ResearchFigure 2. (continued). (J) Section taken from apical third approximately at level of line 2 in (D) where the 2 canals join. Note that the size of the preparation ofthe lingual canal, showing an extension, is considerably smaller than that of the buccal canal (original magnification, Â50). (K) Magnification of confluence areaindicated by upper left arrow in (J). Amorphous material and necrotic debris heavily colonized by bacteria (original magnification, Â400). (L) Magnification ofarea indicated by lower arrow in (J). Abundance of bacteria colonizing debris packed onto dentin wall at confluence between the 2 canals. This histologic picturecorresponds to the dark area in the apical third, indicated by arrow in (D) (original magnification, Â400). (M) Magnification of area of dentin wall indicated byupper right arrow in (J). Debris and bacteria fill the irregularity and appear packed by the obturation material (original magnification, Â400). (N) Cross-cutsection from apical third, not far from that shown in (J). Buccal portion of root dentin (original magnification, Â50). (O) Magnification of more external dentinindicated by left arrow in (N). Few tubules show bacteria in reduced numbers (original magnification, Â400). (P) Magnification of inner dentinal area indicatedby right arrow in (N). Bacterial colonization of dentinal tubules is heavier at this level (original magnification, Â400). (Q) Overview of cross-cut section passingthrough apical ramification shown in (D) (original magnification, Â16). (R) Detail of portion of ramification more proximal to the root canal. High-power viewshows bacterial biofilm intermixed with the obturation material squeezed into this area (original magnification, Â100; inset, Â400).Tissue Processing removed from the paraffin blocks and re-embedded upside down to After fixation for a minimum period of 1 week, the mesial roots obtain cross-cut serial sections from the root tip in a coronal direction;were separated from the teeth with a diamond disk under water 300–600 sections were cut for each root tip. To prevent detachment ofspray just beyond the root canal orifices, and radiographs were the apical small tissue during the bath in acetone-containing solutions,taken in a mesiodistal projection. Demineralization of the roots all sections from the root tips were placed on special polarized slideswas carried out in an aqueous solution consisting of a mixture of (Superfrost Plus; Gerhard Menzel GmbH, Braunschweig, Germany).22.5% (vol/vol) formic acid and 10% (wt/vol) sodium citrate for Thus, sections were divided according to the 3 regions analyzed: middle4 weeks, with the end point being determined radiographically. third, apical third, and root tip.All specimens were washed in running tap water for 48 hours, de- Slides were stained with the Taylor modified Brown and Brennhydrated in ascending grades of ethanol, and cleared in xylene. technique for bacteria (24). The accuracy of the bacterial stainingPhotographs of the mesial and distal views of the cleared roots method was tested by using the protocol described by Ricucci andwere taken while immersed in xylene. Bergenholtz (25). Slides were examined under the light microscope With a sharp razor blade the roots were arbitrarily cross-sectioned, by 2 evaluators (D.R., S.L.). Evaluations were performed separately,under adequate magnification, approximately at the transition between and whenever disagreement occurred, it was resolved by jointthe coronal and the middle thirds and between the middle and the apical discussion. The following were specifically looked for in the histo-thirds. The middle and apical segments were finally infiltrated and logic examination: presence of residual bacteria in the main canal,embedded separately in paraffin (melting point 56 C) according to stan- isthmuses, ramifications, and dentinal tubules (around the maindard procedures. With the microtome set at 4-5 mm, 200 consecutive canals or the isthmus area); presence of debris; type of tissue incross-cut sections were taken respectively of the middle and apical thirds apical ramifications as well as the presence of bacteria, debris,in an apical direction for all specimens. The apical segments were then and filling material therein.JOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 5
    • Clinical ResearchFigure 3. One-visit group. (A) Tooth #19 with destructive carious process in 35-year-old man. Caries has destroyed the furcation. (B) Postobturation radiograph.Considerable amount of obturation material has been extruded through a ramification. (C) Radiograph of mesial root. There is confluence of the 2 canals. (D) Distalview of cleared specimen. Obturation material did not remain attached to the root at extraction, but 2 apical ramifications ‘‘filled’’ by obturation material can beseen. (E) Cross-cut section from middle third taken at level of line 1 in (D). Overview shows a wide and short isthmus connecting the 2 canals (Taylor modifiedBrown & Brenn stain; original magnification, Â25). (F) Detail of isthmus. Necrotic debris heavily colonized by bacteria and surrounded by remnants of obturationmaterial (original magnification, Â100; inset, Â400). (G) Detail from lingual canal wall showing colonization of dentinal tubules (original magnification, Â100;inset, Â400). (H–J) Sequence of sections encompassing the whole course of the most apical ramification, cut in a coronal direction, and taken approximately 60sections from each other (original magnification, Â16). (K and L) Progressive magnifications of content of ramification in (I). Thick bacterial biofilm with highdensity of filamentous forms and surrounded by obturation material can be seen (original magnification, Â100 and Â400). (M) Section passing at the periodontalligament end of the more coronal ramification in (D) (original magnification, Â16). (N) Detail of ramification in (M). Obturation material is enmeshed in thickbacterial biofilm (original magnification, Â100). (O) High-power view of area indicated by arrow in (N). Biofilm fills the irregularities of dentin wall (originalmagnification, Â400).6 Vera et al. JOE — Volume -, Number -, - 2012
    • Clinical Research Results depths. These bodies were faintly stained, reddish or bluish, but without All specimens were available for analyses after the histobacterio- the typical appearance profile of bacterial cells (Fig. 6C and D).logic processing. Although some occasional sections were lost duringthe cutting process, neighboring sections allowed the full picture to Debrisbe seen. Some artifacts were observed in a few cases because of partial In the 1-visit group, debris was always present in varying amountsdetachment of the sections from the slides during the staining proce- in the isthmuses and in most cases in the root canals at different levels. Itdures. This involved only sections taken in the middle and apical thirds was observed as masses of amorphous material including dentin shav-and not sections taken from the root tip, which were processed on ings, sometimes apparently embedded in the obturation materialspecial slides. Another common artifact was the displacement of the (Fig. 2E and F) or packed onto the instrumented root canal wallsobturation material remnants onto the cut dentin surface by the micro- (Fig. 2J–M) and sometimes layering the entire circumference of thetome blade during cutting or because of their tendency to float on the canal. In some instances, necrotic masses were apparently free frommounting medium. However, these artifacts have not significantly inter- bacteria, but in some cases they were heavily infected (Figs. 2E andfered with the overall histologic assessment. F, J–M and 3E and F). Histologic sections confirmed the presence of mesiobuccal and Debris was also present in the isthmuses of the 2-visit group, moremesiolingual canals in all 13 teeth included in this study. In 4 cases frequently in the most apical part of the canals (Fig. 5G–I). In some(3 from the 1-visit and 1 from the 2-visit group), the 2 mesial canals cases, at the root tip the canal lumen in cross section appearedshowed confluence toward the apices. Isthmuses connecting the 2 completely filled with debris, which was densely packed apical to themesial canals were observed in all mesial roots. Although at some levels root canal filling (Fig. 7D and E).these communications appeared sometimes incomplete (Fig. 1E), thehigh number of sections taken allowed the observation of complete Biofilm on the External Root Surfacecommunication between the 2 main canals (Fig. 1F). Ramifications In 3 cases (2 from the 1-visit group and the other from the 2-visit(apical and/or lateral) were also present in all 13 roots, varying in group), a bacterial biofilm was observed beyond the root canal spacenumber and size, and were particularly concentrated in the apical third. on the external apical root surface (Table 1; Figs. 4J and K and 7DIn 10 cases (3 from the 1-visit group and all 7 cases from the 2-visit and F). One of these teeth was extracted because of periodontal disease,group), the overfilling observed radiographically and in the cleared but the apical biofilm was clearly independent of the periodontal bio-specimens was confirmed histologically; small to moderate amount film. In 1 case a consistent portion of the buccal apical profile wasof obturation material was present beyond the main apical foramen covered by a thick bacterial biofilm with abundance of extracellular(Fig. 1G and H). matrix (Fig. 7D and F). All cases of extraradicular biofilm also showed Sections that stained positive for microorganisms demonstrated residual bacteria in some intraradicular areas (Fig. 4E–I).various different bacterial morphotypes, including cocci, rods, and fila-mentous forms. Microorganisms were found in the middle and/orapical third of 11 of the 13 root canal treated specimens (85%) (all Apical Ramifications6 roots of the 1-visit group and 5 of the 7 roots of the 2-visit group) Apical ramifications were present in varying numbers in all 6 cases(Tables 1 and 2). Microbial distribution was different from case to of the 1-visit group. In all these cases, debris was observed within thecase and between groups (Tables 1 and 2). ramification. Of the 6 cases, residual bacteria were seen in 4 (Figs. 2Q and R and 3H–O) and vital tissue in 4; obturation material was observed within some ramifications from all cases. Combinations ofMicrobiological Status these observations were observed in all cases. In the 1-visit group, no tooth had its root canal system rendered Likewise, all 7 cases of the 2-visit group showed apical ramifica-bacteria-free. Only in 1 case were residual bacteria not observed in tions in varying numbers (Fig. 8). All of them had debris. Bacteriathe main root canals at the 3 levels. However, in this specific case, were present in 2 cases and vital tissue in 2 cases; obturation materialresidual bacteria were present in the isthmus and within dentinal was seen in some ramifications from 3 cases. Combinations of thesetubules. In 2 cases, bacteria were not seen in the main canals at the observations occurred in all cases.middle and apical thirds, but they occurred in sections from the roottip. Bacteria were observed only in the lingual canal (ProTaper) in 2 Controlsspecimens, only in the buccal canal (K3/LightSpeed) in 1 specimen,and in both canals in 2 cases (Table 1). Microorganisms were observed In the negative controls, the pulp tissue at the middle and apicalin the isthmuses from 5 of 6 specimens (Figs. 2E–I, 3E and F, and 4H root segments was normal, and no bacteria were detected either inand I) and in dentinal tubules from 5 of 6 cases (Figs. 2I and N–P, 3G, the root canal or in the periradicular tissues. Positive controls showedand 4F). This intratubular infection involved tubules of the dentin bacterial colonization of the main canal, usually forming biofilm struc-surrounding the main canals (Figs. 2N–P, 3G, and 4E and F) and the tures adhered to the main root canal walls. Some bacterial cells wereisthmus area (Fig. 2G–I). Bacterial persistence into tubules was also enmeshed in the necrotic tissue in the main canal. Bacterial bio-observed at varying depths, sometimes reaching more than two thirds films were seen in ramifications and isthmuses, and dentinal tubularof the dentin thickness (Fig. 2N and O). invasion was invariably observed, especially in dentin underneath bio- In the 2-visit group, 2 teeth had their root canal systems rendered films (data not shown).free of bacteria. Residual bacteria were not observed in the main rootcanals of 5 of the 7 roots. Bacteria were, however, demonstrated in the Discussionisthmuses in 4 of the 7 cases (Fig. 5D) (Table 1). No bacteria were The ideal microbiological goal of the endodontic treatment of teethseen in the dentinal tubules surrounding the main canals from all 7 roots. with apical periodontitis is total eradication of the infection, but this canTypical bacterial profiles within tubules were identified only in 1 spec- rarely be reached by using the currently available techniques andimen, which showed a few bacterial cells in the dentin surrounding the substances (2, 26–28). This was confirmed by the present study, inisthmus. In 2 specimens, some bodies were seen within tubules at varying which only 2 cases showed complete absence of bacteria afterJOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 7
    • Clinical ResearchFigure 4. One-visit group. (A) Tooth #19 with complicated crown fracture in 73-year-old man. (B) Postobturation radiograph. (C) Radiograph of mesial rootafter extraction. (D) Cleared specimen. (E) Cross-cut section taken at middle third at level of line 1 in (D). Root canal walls of lingual canal appear well-prepared(Taylor modified Brown & Brenn stain; original magnification, Â50). (F) High magnification from lingual canal wall. Bacterial colonization of some dentinaltubules (original magnification, Â400). (G) Distal root canal wall of lingual canal. Bacteria entrapped between obturation material and dentin wall (originalmagnification, Â400). (H) Cross-cut section taken from apical third at level of line 2 in (D). Buccal canal connected to a wide isthmus (original magnification,Â25). (I) High-power view of area of isthmus indicated by arrow in (H). Debris heavily colonized by bacteria (original magnification, Â400). (J) Section taken atlevel of line 3 in (D), encompassing only the lingual canal. The foramen ends on the distal aspect of the apex (original magnification, Â50). (K) High-power viewshows biofilm on opposite sides of apical constriction, extending to external root surface (original magnification, Â400).treatment (both cases were treated in 2 visits). Considering the high studies demonstrating that bacteria in primary infections are usuallysuccess rate of the endodontic treatment, it is reasonable to assume organized in biofilm communities (21, 29). There are manythat what is clinically achievable and possibly sufficient for an optimal advantages of the biofilm lifestyle; one of them is the ability to protectoutcome is maximal reduction of the microbial bioburden to levels that the community members from the effects of antimicrobial therapeuticare compatible with periradicular tissue healing (18). The present find- measures, especially when the community is not mechanicallyings indicate that the root canal systems of teeth treated in 2 visits with 1- affected by instruments and the irrigant flow. Histobacteriologicweek calcium hydroxide interappointment medication had an improved analyses of teeth with post-treatment disease reveal biofilms associatedmicrobiological status when compared with teeth treated in a single visit. with many cases, especially in those areas inaccessible to treatment (21, Bacteria arranged in biofilms were commonly seen on untouched 22). This represents strong indirect evidence that bacterial biofilmsroot canal walls of treated teeth as well as in isthmuses and ramifications remaining undisturbed in those difficult-to-reach areas can jeopardize(Figs. 2E–H, J–M, Q, and R, 3E–O, 4G–K, and 5D). These findings, the endodontic treatment outcome, and efforts should be directedalong with those from the positive controls, are in agreement with toward improving disinfection of those anatomic complexities.8 Vera et al. JOE — Volume -, Number -, - 2012
    • Clinical ResearchFigure 5. Two-visit group. (A) Tooth #30 in 64-year-old woman with large distal radicular caries. (B) Postobturation radiograph. (C) Specimen after clearing.The 2 canals show confluence. (D) Cross-cut section taken at middle third [line 1 in (C)]. The 2 canals appear well-prepared, and there is a continuous isthmusconnecting them. Inset shows high-power view of area of isthmus indicated by the arrow. Bacterial biofilm covers an irregular area (Taylor modified Brown &Brenn stain; original magnification, Â16; inset, Â400). (E) Section taken at transition between middle and apical thirds [line 2 in (C)] (original magnification,Â16). (F) Magnification of the lingual canal. Canal walls are clean, and no bacteria can be seen (original magnification, Â50). (G) Section taken at level of line 3 in(C), apically to confluence (original magnification, Â16). (H) Detail of the common canal. Large part of canal circumference is covered by debris (original magni-fication, Â50). (I) High-power view of area indicated by arrow in (H). Debris is covered by remnants of obturation material. No stainable bacteria can be observed(original magnification, Â400). The anatomic complexity of the apical root canal system is one of calcium hydroxide medication. The present findings confirm that itthe most important factors limiting the attainment of proper disinfection requires time for the therapeutic antibacterial procedures to affect(30). In infected root canals associated with apical periodontitis, bacteria located in anatomic complexities and then maximize bacterialbacteria can be located not only in the lumen of the main canal but reduction to levels that cannot be achieved in 1 visit.also in recesses, dentinal tubules, isthmuses, lateral canals, and apical Data from many studies using culture also confirm the advantageramifications. In these areas, they are usually protected from the effects of using intracanal medication in reducing the bacterial load and theof chemomechanical procedures. This is because it is physically impos- number of cases with positive culture because of residual bacteriasible for instruments to reach most of these areas, and the antibacterial (3, 5, 10). This is interesting because the method of samplingirrigants are left to act in the canal for only a short time, which is usually commonly used provides information of the bacteriologic conditionsinsufficient for them to diffuse and reach effective concentrations to kill in the main canal, an area where instruments and irrigants arebacteria harbored in anatomic complexities (31) (Figs. 2I–R, 3E–O, expected to act. Nonetheless, even in the main canal there are areasand 4E–I). This has been previously demonstrated (26) and was that remain uninstrumented (32, 33) (Fig. 2J and M). The additionalevidently confirmed in the present study for the 1-visit group; residual benefit of using an intracanal medication for reduction of bacteria inbacteria were seen in the isthmuses (5 of 6 cases), apical ramifications the main canal was confirmed by this histobacteriologic study. Although(4 of 6 cases), and dentinal tubules (5 of 6 cases). The main reason to in the 1-visit group residual bacteria were found in the root canals of 5use an interappointment medication is to allow time for the medication of the 6 teeth, in the 2-visit group only 2 teeth showed residual bacteriato diffuse and reach bacteria in those areas inaccessible to instruments in the main canal. In addition to the effects of the medication per se, theand irrigants. Our results indicate that this strategy is really effective in reinstrumentation with the last apical instrument and the final rinse withreducing the bacterial bioburden in the whole system, because residual NaOCl and chlorhexidine in the second visit might have contributed tobacteria were only seen in the isthmuses of 4 cases and in ramifications the improved disinfection of the main canals.of 2 cases in the 2-visit group and usually in lower abundance than in the Two instrumentation protocols were used in the present study. Ourteeth treated in 1 visit. No residual dentinal tubule infection was found in results demonstrated that both performed equally well in terms of clean-the middle and apical thirds of the main root canal after 1 week of ing and disinfecting the main canal. This is curious if one considers thatJOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 9
    • Clinical ResearchFigure 6. Two-visit group. Tooth #30 in 39-year-old woman with 8-mm-deep periodontal pocket caused by fracture on the pulp chamber floor. (A) Clearedmesial root. Obturation material is present in the communications between the 2 canals. (B) Cross-cut section taken from middle third at level of line 1 in(A). Overview shows that the canals are well-prepared at this level, and there is an incomplete isthmus (Taylor modified Brown & Brenn stain; original magni-fication, Â16). (C) Buccal canal. Some tubules exhibit red-stained structures (original magnification, Â100). (D) High magnification demonstrates that thesestructures do not have the appearance of usual bacterial profiles (original magnification, Â400). (E) Cross-cut section taken at apical third at level of line 2in (A). An extension of the buccal canal and an incomplete isthmus are present (original magnification, Â16). (F) Detail of buccal extension (original magni-fication, Â100). (G) High magnification shows that the tissue in the extension is partly vital. Absence of bacteria (original magnification, Â400). Area of the isthmusindicated by arrow in (E) is magnified in the inset. Obturation material and debris. Absence of bacteria (original magnification, Â400).the size of apical preparation differed in the 2 protocols. Some previous patency files for comparison was not included in the present investiga-studies demonstrated that the larger the apical preparation, the larger tion. Further studies are required to help elucidate the importance, ifthe bacterial reduction (34–36). This is because larger preparations any, of using patency files to improve apical disinfection and cleaning.increase the chances for incorporating recesses and irregularities, Evidence of extraradicular infection was observed in 3 cases. In allwhich might harbor bacteria, in addition to removing more infected those cases, infection was primarily organized as biofilms adhered todentin. However, this was not apparently confirmed in the present the external apical root surfaces (Figs. 4J and K and 7D and F), whichstudy, suggesting that the level of enlargement achieved in the smallest were beyond the reaches of instruments, irrigants, and intracanal medi-preparations (ProTaper group, apical file dimensions 25/.08) along cation. Whether these remaining extraradicular biofilms can remainwith copious irrigation with 5% NaOCl was sufficient to favor bacterial active after root canal treatment to the point of jeopardizing the treat-elimination from the mesial canals of mandibular molars. ment outcome cannot be answered by the present study. However, It is also worth pointing out that apical patency was performed in reports suggest that the presence of a biofilm on the external apicalall cases with a #10 K-file, but debris and bacteria were seen in various root surface might be implicated in the failure of apical periodontitiscases in the apical canal (Figs. 5H and I and 7D and E). Debris and/or to heal (38, 39).bacteria were present in the main foramina in 8 of the 13 cases. More All mesial roots analyzed showed apical ramifications. Obturationspecifically in the 1-visit group, debris and bacteria were present in 3 material was observed in ramifications from all cases treated in 1 visitcases and only debris in another case. In the 2-visit group, debris and in 3 cases of the 7 teeth treated in 2 visits. Although this mightand bacteria were present in 1 case and only debris in 3 other cases. suggest that calcium hydroxide interferes with ‘‘filling’’ ramifications,Although this might suggest that patency files are not as effective in the main question is whether such a ‘‘filling’’ means adequate sealinghelping disinfection and cleaning as suggested (37), any further discus- and disinfection. In fact, residual bacteria were more commonly seension on this topic would not be appropriate because a group with no in ramifications from the 1-visit group than in the 2-visit group10 Vera et al. JOE — Volume -, Number -, - 2012
    • Clinical ResearchFigure 7. Two-visit group. (A) Tooth #30 in 39-year-old woman with crown fracture. Large apical periodontitis lesions on both roots. (B) Postobturation radio-graph. (C) Mesial root after clearing. (D) Section passing at root tip. The lingual canal (right) ends on the distal side, and there is a slight overfilling, with obturationmaterial beyond the foramen (Taylor modified Brown & Brenn stain; original magnification, Â25). (E) Detail of buccal canal in (D). Its lumen appears filled withdebris densely packed (original magnification, Â100). (F) Magnification of area of radicular profile indicated by arrows in (D). The root is covered by thickbiofilm, filling irregularities of cementum. Extracellular component is predominant. Inset shows high-power view of area indicated by arrow (original magnifi-cation, Â1000).(Figs. 2Q and R and 3H–O). Debris was present in ramifications from showed virtually no percentage difference (45) or even 10% moreall cases treated regardless of the group. In most cases in which the success for 1-visit treatment (46, 47). Data available have beenfilling material was observed in ramifications, it was intermixed with limited and did not allow precise conclusions about superiority of 1necrotic tissue and debris. In some cases, bacteria were also present method over the other (48). However, virtually all these studies werealong with the filling material (Figs. 2Q and R and 3H–O). Voids plagued by a small sample size. A very recent study with a larger numberwere usually observed. Therefore, thorough filling, seal, and disinfec- of cases demonstrated a significantly better outcome for teeth whosetion cannot be expected by squeezing the filling material into ramifica- canals were treated in 2 visits with calcium hydroxide as interappoint-tions. These findings are in line with previous reports (20, 40). ment medication as compared with 1-visit treatment (49). This is in line The optimal outcome depends on the ability of treatment proce- with the better disinfection observed in the present study for the 2-visitdures to restore healthy conditions by creating favorable conditions group and is consistent with the notion that root canal disinfection isto healing (41). The best root canal environmental conditions that integral to success.are highly conducive to periradicular healing are represented by One argument in favor of treating infected root canals in 1 visit isabsence of microorganisms, a condition similar to that found in vital that residual bacteria surviving treatment are entombed by obturationcases that have a very high potential for success. Therefore, clinicians and die because a source of nutrients is denied (46, 50). Thisare encouraged to pursue treatment protocols that predictably control argument might be valid to bacteria remaining on untouched canalthe root canal infection and allow for establishment of an environment walls or within dentinal tubules (18). However, bacteria remaining inthat is favorable to healing. Studies comparing the success rate of the the very apical part of the root canal (Fig. 2J–M), in apical ramificationsendodontic treatment of teeth with apical periodontitis performed in (Figs. 2Q and R and 3H–O), and in lateral canals can maintain long-1 or more visits revealed that 2 or more visits with calcium hydroxide standing infections. These bacteria are in direct contact with the perira-as the intracanal medication offer a success rate that is 10%–20% dicular tissues, which are a sustainable source of nutrients. The simplehigher than 1-visit treatment (4, 17, 42–44). However, other studies fact that bacteria can be found in the main root canal of many cases withJOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 11
    • Clinical Research conduct, and cases are hard to find, select, and collect. As a conse- quence of the small sample size, statistical analysis was not really useful. Molars were selected because they are, by and large, the teeth most diffi- cult to treat. However, this is different from the material analyzed in several previous microbiological studies, which usually used single- rooted teeth. Research has been conducted to disclose procedures that are able to expedite bacterial elimination in the root canal system and then opti- mize single-visit disinfection (55). Approaches such as performing a final rinse with chlorhexidine have been suggested to supplement the antimicrobial effects of chemomechanical procedures. Although ex vivo (56) and in vivo studies (57) have shown that a final rinse with chlorhexidine improves disinfection, the present study revealed that these effects were not satisfactory enough to obviate the need for an interappointment medication with calcium hydroxide. Ultrasonic activation of NaOCl is another supplementary approach that has the potential to improve disinfection, but the literature has shown inconclu- sive results about its effectiveness (56, 58). Studies are underway that use the present methodology to evaluate the antimicrobial clinical efficacy of ultrasonic activation of NaOCl. In conclusion, this study demonstrated that the 2-visit protocol with an interappointment medication with calcium hydroxide resulted in improved microbiological status of the root canal system when compared with a single-visit protocol. No histobacteriologic difference was observed between minimal apical preparation and apical enlarge- ment. Residual bacteria were more commonly observed and in higherFigure 8. Two-visit group. Tooth #30 in 34-year-old woman (EM, Table 1). abundance in ramifications, isthmuses, and dentinal tubules of teeth(A) Cross-cut section taken close to root tip. Periapical pathologic tissue re-mained attached to buccal radicular portion (left). The buccal canal ends treated without interappointment medication. Apical ramificationsanticipated on the distal aspect. Overview shows numerous cross-cut tiny rami- and isthmuses were never completely filled, and the filling material,fications (arrowheads). One of them, with larger diameter, shows obturation when forced to these areas, was usually interspersed with debris,material. Arrows indicate termination of some ramifications to periodontal necrotic tissue, and bacteria. The present results reinforce the conceptligament (Taylor modified Brown & Brenn stain; original magnification, that current instruments, irrigants, and techniques cannot predictablyÂ25). (B) Section taken approximately 100 sections coronally to the one disinfect the root canal system in a single visit and the use of an antibac-in (A). The 2 main canals appear well-prepared at this level. Abundance of terial interappointment agent is necessary to maximize bacterial reduc-ramifications with varying diameters (original magnification, Â25). tion before filling.post-treatment disease (51, 52) indicates that entombment is not Acknowledgmentsreliable. Findings from this and previous studies (13, 22, 53, 54)suggest that even bacteria sequestered in the root canal by obturation The authors are grateful to Ramachandran Nair for themight derive nutrients from debris and tissue remnants until a more insightful discussions concerning the study protocol.sustainable source of nutrients is established. The authors deny any conflicts of interest related to this study. A great advantage of the method used in this study to evaluate theantimicrobial performance of clinical protocols is that it provides infor- Referencesmation as to the anatomic localization of residual bacteria. This gives 1. Bergenholtz G, Sp angberg L. Controversies in endodontics. Crit Rev Oral Biol Meda clear idea of the deficiencies of the different approaches and allows 2004;15:99–114.for further development of strategies to enhance disinfection. When 2. Bystr€m A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA o in 60 cases of endodontic therapy. 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Simon JH. The apex: how critical is it? Gen Dent 1994;42:330–4. J Endod 2011;37:496–501.31. Siqueira JF Jr. Strategies to treat infected root canals. J Calif Dent Assoc 2001;29: 57. Zamany A, Safavi K, Sp angberg LS. The effect of chlorhexidine as an endodontic 825–37. disinfectant. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:578–81.32. Peters OA, Sch€nenberger K, Laib A. Effects of four Ni-Ti preparation techniques on o 58. Carver K, Nusstein J, Reader A, Beck M. In vivo antibacterial efficacy of ultrasound root canal geometry assessed by micro computed tomography. Int Endod J 2001;34: after hand and rotary instrumentation in human mandibular molars. J Endod 2007; 221–30. 33:1038–43.JOE — Volume -, Number -, - 2012 1- versus 2-visit Endodontics 13