Case Report/Clinical Techniques

Infection in a Complex Network of Apical Ramifications asthe Cause of Persistent Apical Periodontitis: A Case ReportMichael Arnold, Dipl Stom,* Domenico Ricucci, MD, DDS,† and Jos�e F. Siqueira, Jr, DDS, MSc, PhD‡

Abstract

Introduction: This article reports a case of persistentapical periodontitis lesion in a mesiobuccal root ofa maxillary molar subjected to single-visit endodontictreatment. Methods: The treatment protocol followedendodontic standards including using nickel-titaniuminstruments with working length ending 0.5-mm shortof the apex, establishment and maintenance of apicalforamen patency, irrigation with 5% NaOCl, smear layerremoval, a final rinse with and ultrasonic agitation ofchlorhexidine, and filling by the vertical compactiontechnique. Even so, the lesion in the mesiobuccal rootbecame larger in size after follow-up examination at 1year 6 months, and periradicular surgery was performed.Radiographic control after 11 months showed that peri-radicular healing was almost complete. The root apexand the lesion were analyzed histologically and histo-bacteriologically. Results: The lesion was diagnosedas a ‘‘pocket cyst,’’ and no bacteria were noted extrara-dicularly. The cause of continued disease was a heavybacterial biofilm infection located in an intricatenetwork of apical ramifications. Bacteria were alsoobserved on the walls of one of the mesiobuccal canalspacked between the obturation material and the rootcanal wall. Conclusions: This case report reinforcesthe need for treating the infected root canal as a complexsystem that possesses anatomic intricacies in whichbacteria can spread and remain unaffected by treatmentprocedures. (J Endod 2013;39:1179–1184)

Key WordsApical delta, endodontic treatment, posttreatmentapical periodontitis, root canal infection, treatmentoutcome

From the *Private Practice, Dresden, Germany; †PrivatePractice, Cetraro, Italy; and ‡Department of Endodontics,Faculty of Dentistry, Est�acio de S�a University, Rio de Janeiro,Rio de Janeiro, Brazil.

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 2013 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.04.036

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Primary and posttreatment apical periodontitis lesions are primarily caused bymicrobial infection of the root canal system. The successful outcome of endodontic

treatment depends on thorough disinfection of the root canal system (1). Although ithas been suspected that persistent infection can be related to resistant and more robustmicrobial species present in the canal (2, 3), there is ample evidence that the main (ormost common) reason for bacterial persistence after treatment is the fact that infectioncan spread to areas of the root canal system that remain unaffected by instruments andantimicrobial substances used for irrigation or medication (4, 5). These include notonly untouched walls of the main canal (6) but also areas distant from the main canal,such as lateral canals (7, 8), apical ramifications (9, 10), isthmuses (6, 11), anddentinal tubules (8, 12). The present case report is about a persistent posttreatmentapical periodontitis lesion caused by infection established in a complex apical rootcanal anatomy.

Case ReportA 51-year-old male patient was referred to an endodontic specialist by his general

dentist who had initiated root canal treatment of tooth #14. The patient presented to hisdentist for a routine checkup, and a review of his medical history was noncontributory.The patient reported that more than 15 years earlier a prosthetic restoration wasperformed in his upper left jaw, consisting of a bridge to replace teeth #11 and #13.A recurrent deep caries lesion was diagnosed in tooth #14, and the patient declaredno symptoms. A radiograph showed large periradicular radiolucencies on themesiobuccal (MB) and palatal roots as well as a minor radiolucency on the distobuccalroot of tooth #14; the root canals appeared consistently narrowed (Fig. 1A). Noradiographic signs of periodontal disease were observed. The diagnosis of pulpnecrosis was made, and root canal treatment was indicated. An access cavity wasprepared through the existing restoration. The general dentist was not able to locatethe orifices and negotiate the canals, so the patient was referred to an endodontist.

At the examination performed by the endodontist, the buccal mucosa did not showany pathological changes. On the palatal side, the gingival margins were heavilyinflamed, and bleeding occurred on probing. A carious lesion was detected on thepalatal side, apical to the restoration margin, and the tip of a probe could penetratethe pulp chamber. The periodontal probing depth both mesially and distally was4 mm. The tooth was not tender to percussion (vertical and lateral) or palpation(buccal and palatal). On the bases of the existing diagnostic radiograph and clinicalexamination, the diagnosis of pulp necrosis with apical periodontitis lesions wasconfirmed for tooth #14, and root canal treatment was scheduled.

One week later, the bridge was sectioned, and the crown on tooth #14 wasremoved. After rubber dam isolation, carious tissue was excavated with low-speedround burs under water spray. The crown was then restored with composite(Tetric EvoFlow; Ivoclar Vivadent, Ellwangen, Germany) after acid etching and bondingapplication (Optibond FL; Kerr, Ratstatt, Germany). With the aid of an operatingmicroscope, the calcified tissue in the pulp chamber was removed, and 4 root canalorifices (2 in the MB root [MB1 and MB2]) were evident. The orifices appearedobstructed by calcified tissue (Fig. 1B–D). The root canals were located by cautiouslyremoving the calcified tissue with ultrasonic diamond tips. After preparing theorifices of MB1 and MB2, an isthmus connecting the 2 canals was evident (Fig. 1C).The MB portion of this isthmus, which appeared patent, was opened with a prebent#25 ultrasonic file (Irri K-Files; VDW, M€unchen, Germany) (Fig. 1D). Under

Apical Delta Infection and Treatment Failure 1179

Figure 1. (A) A radiograph of tooth #14 taken during a routine checkup by a general dentist showing periradicular radiolucencies. (B–D) A sequence of photo-graphs showing the negotiation of MB1 and MB2 and the isthmus connecting the 2 canals as performed by an endodontist. (E) Gutta-percha cone selection and (F)postobturation radiograph. (G) A 6-month follow-up radiograph. The radiolucencies on the distobuccal and palatal roots had considerably decreased whereas thaton the mesial root remained the same size. (H) An 18-month follow-up radiograph. The lesion on the MB root became larger. Apical surgery was scheduled. (I–K)Cone-beam computed tomographic scans showing the extent of the lesion and its relationship with the maxillary sinus floor. (L and M) After elevating a mucoper-iosteal flap, the cortical bone covering the pathologic tissue was carefully mobilized to create an access to the MB periradicular area. (N) The resected root end, (O)the prepared root-end cavity, and (P) filling with MTA. (Q) A follow-up radiograph taken 11 months after surgery.

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Figure 2. (A) A mesial view of the apical biopsy including the MB root tip and the surrounding pathologic tissue in their original relationship. (B) A view of theresected surface. A cavity can be observed in the soft tissue. (C) A section taken on a buccolingual plane encompassing the apical portion of the MB canal, a largeramification, and the very apical portion of MB2 (arrow). The overview reveals that the lesion is a ‘‘pocket cyst’’ with its lumen in direct continuity with the rootcanal space through the wide apical ramification (hematoxylin-eosin, original magnification�8). (D) A detailed view of the area from the cyst wall demarcated bythe rectangle in C. Stratified squamous epithelium with an arcading structure can be seen. The subepithelial connective tissue is infiltrated by inflammatorycells (original magnification �50). (E and F) Progressive magnifications of the basal layer of the epithelial wall in D. The epithelium is infiltrated mostly bypolymorphonuclear leukocytes (arrowheads in F) (original magnification�400 and�1,000). (G) A section taken at a short distance from that shown in C (Taylormodified Brown and Brenn, original magnification�8). (H) A detailed view of the root tip. MB1 bifurcates into 2 apical ramifications filled by a biofilm. The exit ofa wide ramification can be seen on the left profile as well as the entrance of a large ramification more coronally (original magnification�25). The area indicated bythe arrow is magnified in Figure 3E. (I) A detailed view of the 2 ramifications in H (original magnification �100).

Case Report/Clinical Techniques

microscopic observation, the isthmus seemed to end at the middle thirdof the MB root canal system.

The working length (WL) was established 0.5 mm short of theapical foramen using the electronic apex locator from the VDWGold inte-grated system. The root canals were prepared with rotary nickel-titaniumfiles (ProFile [Maillefer, Ballaigues, Switzerland] in combination withGTX [Dentsply Tulsa Dental Specialties, Tulsa, OK] and FlexMaster[VDW]). A #08 K-file (Maillefer) was used to ensure patency of the apicalforamen by taking it 1 mm beyond the WL. The fourth canal (MB2) wasfound to have an independent course and foramen.

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The buccal root canals were instrumented apically up to #35.04and the palatal canal up to #45.04 using ProFile and FlexMaster rotaryinstruments in a step-down approach. During instrumentation, thecanals were thoroughly irrigated with 5% NaOCl. Finally, all canalswere irrigated with 10% citric acid to remove the smear layer followedby a final rinse with 2% chlorhexidine. These solutions were activatedwith ultrasonics for about 20 seconds each. The root canals weredried with sterile paper points and filled with gutta-percha and2Seal (VDW) using Schilder’s vertical compaction technique(Fig. 1E and F). Subsequently, the tooth was restored with a DTLight

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Case Report/Clinical Techniques

SL post (VDW) and composite (Tetric EvoFlow). Finally, a temporarycrown was placed.

At the first follow-up visit 6 months later, the tooth was asymptom-atic. Percussion and palpation tests yielded normal responses. A newpermanent crown was inserted by the general dentist. The radiographshowed that the radiolucencies on the palatal and distobuccal rootshad consistently reduced, whereas the lesion on the buccal root hadremained the same size, even though the corticated margin was nolonger visible (Fig. 1G).

The patient returned after 1 year 6 months because 1 weekpreviously he had noted buccal swelling and an active sinus tract inthis region. At examination, no pathological signs were present. Thesinus tract was no longer present, and there was no tenderness topalpation and percussion. Periodontal probing did not reveal pockets.A periapical radiograph showed that although the lesions on the palataland distobuccal roots had apparently healed, the radiolucency on theMB root had become larger (Fig. 1H). Cone-beam computedtomographic imaging showed a large radiolucency associated mostlywith the MB root apex, with corticated margins (Fig. 1I–K) andelevation of the maxillary sinus floor (Fig. 1I). The case was regardedas root canal treatment failure, and apical surgery was scheduled for1 week later.

Oral disinfection was performed before treatment by rinsing with0.2% chlorhexidine (Chlorhexamed; GlaxoSmithKline Healthcare,B€uhl, Germany) for 15 minutes. After anesthesia, a full-thicknessperiosteal flap with 1 vertical incision was elevated. The MB root wasimmediately visible (Fig. 1L), and the thin buccal bone in correspon-dence to the lesion was carefully removed (Fig. 1L) to improve theaccess to the apical periodontitis lesion (Fig. 1M). An attempt wasmade to obtain the resected root tip and the surrounding pathologicsoft tissue in their original relationship. The root tip was first resectedapproximately 3 mm short of the apex with a diamond bur cooled bysterile saline solution. Subsequently, the soft tissue was carefullyenucleated from the bone crypt with smooth microelevators. Thecut surface of the MB root showed no signs of cracks, fractures, oraccessory root canals at observation under 16� magnification(Fig. 1N). The root canal filling material was removed 3-mm deepwith ultrasonic tips. MB1 and MB2 canals were joined in the last3 mm in a single root-end cavity by using ultrasonic preparation(Fig. 1O). After disinfection with 10% citric acid and 2% chlorhexidine,the root-end cavity was filled with mineral trioxide aggregate (ProRootMTA; Dentsply, Konstanz, Germany) (Fig. 1P). The postoperativeradiograph showed optimal root-end filling. Eleven months aftersurgery, the tooth was asymptomatic, and a periapical radiographshowed that healing was almost complete. The cavity was filled by newlyformed bone, and only residual widening of the periodontal ligamentspace around the MB root could be observed (Fig. 1Q).

The patient gave consent for histologic examination. The biopsyspecimens, consisting of the root tip with the soft pathological tissue(Fig. 2A and B) and 2 buccal cortical bone fragments, were immediatelyimmersed in 10% neutral buffered formalin solution and sent to thelaboratory for histologic and histobacteriologic processing.

Tissue ProcessingThe specimens were kept in fixative for 5 days. Demineralization

was performed by immersion in an aqueous solution consisting ofa mixture of 22.5% (vol/vol) formic acid and 10% (wt/vol) sodiumcitrate for 3 weeks. The endpoint was determined radiographically.The specimens were washed in running water for 48 hours, dehydratedin ascending grades of ethanol, cleared in xylene, infiltrated, andembedded in paraffin (melting point 56�C) according to standard

1182 Arnold et al.

procedures. With the microtome set at 4–5 mm, longitudinal serialsections of the apex with the surrounding pathologic tissue were takenon a buccolingual plane until the specimen was exhausted. Particularcare was taken to obtain sections encompassing the foramen(ina) inconjunction with the pathologic periradicular tissue. Approximately500 sections were cut in total for the apical biopsy. Sections of thebone fragments were also cut on a buccolingual plane to observe thecontact area between the apical periodontitis lesion and the corticalbuccal bone. Sections were stained with a modified Brown and Brenntechnique for staining bacteria (13, 14). Selected slides werestained with hematoxylin-eosin. Slides were examined under the lightmicroscope.

Histopathologic and Histobacteriologic ObservationsMicroscopically, sections of the MB root tip revealed the presence

of a complex root canal system in the apical third, with an apical deltacharacterized by an intricate network of ramifications (Figs. 2C andG–Iand 3F). Numerous foramina could be observed in the serial sectionsending at the geometrical top and on the palatal aspect of the roottip. The ramification ending on the palatal aspect exhibited a large diam-eter and ended in a cavity surrounded by epithelium (Fig. 2C,G, andH).The lesion had the characteristics of a ‘‘pocket cyst’’ with an epitheliallining adhered to the apical structure apically and palatally to form anepithelial collar. The lumen of the cyst cavity was in direct continuitywith the root canal space through the major apical ramification(Fig. 2C and G).

The epithelial lining had the characteristics of stratified squamousepithelium. It was thick and irregular with ridges that branched to forman arcading structure (Fig. 2C and D). The ridges of proliferatingepithelium enclosed islands of granulomatous tissue (Fig. 2D). Theseislands of connective tissue as well as the subepithelial connective tissuewere well vascularized and infiltrated by many mononuclear inflamma-tory cells (Fig. 2D). Inflammation was milder toward the pseudocap-sule, where only collagen bundles with some scattered chronicinflammatory cells could be observed. Epithelium was infiltrated bynumerous polymorphonuclear leukocytes (Fig. 2E and F). The lumenof the cyst was apparently empty with some necrotic tissue debris andblood remnants (Fig. 2C and G). Bacteria were not observed in thecyst lumen or on the cyst wall.

In some sections, the main MB canal (MB1) in the very apicalportion bifurcated into 2 symmetric ramifications whose lumina werecompletely filled by thick biofilms (Fig. 2H and I). The biofilm ineach ramification was clearly demarcated from the inflammatoryreaction and exhibited a certain amount of extracellular matrix in whichcoccoidal forms dominated (Fig. 3A and B).

The lumen of the large ramification communicating with the cystcavity was also clogged by a thick bacterial biofilm with abundantextracellular matrix, but bacterial filamentous forms were dominantin this location (Fig. 3C and D). Bacteria were also observed on thewalls of MB1 packed between the obturation material and the root canalwall (Fig. 3E). Other sections disclosed additional ramifications, allexhibiting bacterial colonization (Fig. 3F). Sections of the bonefragments showed both marrow and compact bone with characteristicsof normality, except for some scattered osteoclasts (data not shown).

DiscussionIn the large majority of teeth with apical periodontitis, microbial

infection is present not only in the main root canal but also propagatesto variations of the internal anatomy of the system, including dentinaltubules, recesses, isthmuses, lateral canals, furcal canals, and apicalramifications (15–18). Bacterial biofilms are observed in the apical

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Figure 3. (A and B) A high-power view of the 2 apical ramifications. They are clogged with a thick biofilm in which round bacterial morphotypes dominate(original magnification �400; Inset �1,000). (C) A detailed view of the left ramification in Figure 2H (original magnification �100). (D) Magnification ofthe foraminal area showing a biofilm with abundant extracellular matrix in which filamentous forms are predominant (original magnification �400; inset�1,000). (E) A high-power view of the area from the left canal wall indicated by the arrow in Figure 2H. A bacterial biofilm can be observed between theroot canal wall and the obturation material (original magnification�100). (F) Another section: the exits of 4 ramifications can be discerned (original magnification�50).

Case Report/Clinical Techniques

part of the root canal system in about 80% of teeth with apicalperiodontitis (18). The older the infectious process, as inferred bylesion size or histopathologic diagnosis of a cyst, the more complexthe infection in the apical canal is (18). Consequently, a complexinfection lodged in a complex anatomy poses a great challenge tocontrol and helps explain the lower success rate for teeth with apicalperiodontitis when compared with teeth with no disease (19–21)and teeth with large lesions when compared with teeth with smalllesions (22–25).

In this case report, bacteria persisting after treatment werearranged in biofilm structures located in an intricate network of apicalramifications that remained apparently unaffected by treatment. It isnoteworthy that treatment was performed based on optimal standards,including apical preparation with reasonably large nickel-titanium

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instruments and at an adequate WL (0.5 mm short), establishmentandmaintenance of apical foramen patency throughout the procedures,copious irrigation with highly concentrated (5%) NaOCl, smear layerremoval, and a final rinse with and ultrasonic agitation of chlorhexidine.These approaches worked well for the other canals of the same tooth,leading to healing of the lesions on the distobuccal and palatal roots asrevealed radiographically. The reason why the lesion on the MB rootpersisted after treatment is probably a more complex anatomy of thisroot, making single-visit disinfection less effective, but a definiteestablishment of the different tissue reaction cannot be establishedbecause only the MB root was available for histopathologic analysis(for obvious ethical reasons).

It becomes clear that all these antimicrobial procedures, exceptfor the patency files, were physically precluded from contacting the

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bacterial biofilms in the apical ramifications. The effects of the smallpatency file, allegedly restricted to only 1 of the ramifications or themain apical foramen, were apparently negligible. In addition to keepingthe apical foramen patent, patency files are also expected to disruptapical biofilms mechanically (26) and chemically by carrying irrigantsolution to the very apical canal or improving delivery of irrigants tothat region (27). If these effects actually occurred to some degree,histobacteriologic sections revealed that the apical biofilm apparentlyrecovered from such disturbances, at least morphologically, becausethe biofilms seemed undisturbed in all ramifications.

Treatment was performed in a single visit, and whether or not aninterappointment medication would have improved prognosis can onlybe speculated at this time. The main reason to apply a medicationbetween appointments of treatment of a tooth with apical periodontitisis to allow time for the antimicrobial substance (usually a calciumhydroxide paste) to diffuse and reach bacteria persisting unaffectedin remote areas of the root canal system (28). Studies have shownimproved disinfection when an intracanal medication is used betweenvisits (5, 29, 30). However, it is unknown whether a single dressing withcalcium hydroxide in an inert vehicle would have helped very much ina complex case like the one reported here. This is because this alkalinesubstance has low solubility, and as it diffuses through organic orinorganic tissues, its pH is reduced and may not reach enoughmagnitude to kill bacteria in ramifications (31). Multiple changes ofcalcium hydroxide dressings or an association of this substance withanother antimicrobial medication may be required to improve theeffects of the medication on bacteria located in areas distant from themain canal or protected by organic/inorganic tissues (30, 32–34).

In conclusion, this case report reinforces the need for treating theinfected root canal as a system that possesses anatomic intricacies inwhich bacteria can spread and remain unaffected by treatmentprocedures. It is a challenge for the specialty to develop strategiesand find substances that predictably disinfect the complex root canalsystem to the point of achieving infection control that is compatiblewith periradicular tissue healing. It seems advisable that in cases withlongstanding pulp necrosis and large apical periodontitis lesions theclinician makes the patient aware of the difficulties of obtaininga bacteria-free root canal system and discusses the possibilities of treat-ment and the projected prognosis as supported by scientific evidence.

AcknowledgmentsThe authors deny any conflicts of interest related to this study.

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