histologic and histobacteriologic observations of failed revascularization/revitalization therapy: a...

8/13/2019 Histologic and Histobacteriologic Observations of Failed Revascularization/Revitalization Therapy: A Case Report

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Histologic and Histobacteriologic Observations of FailedRevascularization/Revitalization Therapy: A Case Report Louis M. Lin, BDS, DMD, PhD,* Emi Shimizu, DDS, PhD,* Jennifer L. Gibbs, MAS, DDS, PhD,*

Simona Loghin, DDS,† and Domenico Ricucci, MD, DDS †

Abstract

Introduction: Mechanical debridement plays an impor-tant role in eliminating intracanal bacteria, such as bio-film on the canal walls and bacteria in the dentinaltubules. Mechanical debridement is not recommendedfor root canal disinfection in revascularization/revitaliza-tion therapy. Here we report a failed revascularization/revitalization case, which could be due to inadequateroot canal disinfection without mechanical removal of biofilm and bacteria in dentinal tubules. Methods: A

6-year-old boy had a traumatic injury to tooth #9, whichwas avulsed and replanted within 40 minutes. The toothsubsequently developed a local swelling in the periapi-cal area. The patient was referred to the PostgraduateEndodontic Clinic for revascularization/revitalizationtherapy on tooth #9. The treated tooth remained asymp-tomatic for 16 months and then developed pain andlocal periapical swelling. The oral surgeon extractedthe revascularized/revitalized tooth. On request, the ex-tracted tooth was processed for histologic and histobac-teriologic examination. Results: The tissue in the canalwas completely destroyed. Most bacteria were observedin the apical portion and not in the coronal portion of the

canal and formed biofilm on the canal walls and pene-trated into the dentinal tubules. Conclusions: On thebasis of histobacteriologic observations, the failure of re-vascularized/revitalized tooth could be due to inadequateroot canal disinfection without mechanical debridement.It may be important to perform mechanical debridementas part of the revascularization/revitalization therapy todisrupt thebiofilm on thecanal wallsand remove bacteriain the dentinal tubules because revascularization/revitali-zation therapy is able to increase thickening of the canalwalls. (J Endod 2014;40:291–295)

Key WordsImmature permanent tooth, reinfection, revasculariza-tion/revitalization, root canal disinfection

Bacterial infectionis the primary cause of pulpal and periapical disease (1).Whentheroot canal is infected, bacteria colonize the canal walls as biofilm (2, 3) and

penetrate into the dentinal tubules (4, 5) and lateral/accessory canals (6) as well asisthmuses(7).Thenumberanddepthofdentinaltubulesinvadedbybacteriaarehigherand deeper in the teeth of young than of old people (8, 9). Bacteria in biofilm firmly attached to the canal walls and in the canal dentinal tubules are very hard toeliminate by canal irrigants and intracanal medicaments during root canal therapy (10–14). In addition, biofilm can evade the host’s innate and adaptive defensemechanisms and resist antimicrobial chemotherapy (15).

Similar to open flap debridement for marginal periodontitis, root canal therapy requires mechanical instrumentation or debridement to remove biofilms, canal wallscontaminated by bacteria toxins, and bacteria in the dentinal tubules (16, 17). Thecurrent protocol used to control root canal infection of immature permanent teeth with infected necrotic pulp in revascularization/revitalization procedures is by irrigation with sodium hypochlorite and intracanal medication with calciumhydroxide or triple antibiotics without mechanical debridement (18). Regardless of apexification or revascularization/revitalization procedures, effective control of root canal infection is the key to the success of both treatments. Infection/inflammationprevents tissue regeneration and stem cell differentiation (19) and therefore has tobe under control for wound healing to take place. It has been suggested that control of root canal infection in revascularization/revitalization procedures might have to bemore thorough than that of regular root canal therapy for pulp tissue regeneration

to occur (20). No failures of revascularization/revitalization cases have been reportedby using sodium hypochlorite irrigation and triple antibiotics intracanal medication without mechanical debridement in human (21) and in animal studies (22–24), which are of short-termobservations. These studies give the impression that the clinical outcome of revascularization/revitalization therapy is a 100% success.

We have experienced failed revascularization/revitalization cases by using current root canal disinfection protocol and had to re-treat the failed cases with apexificationprocedures. The purpose of the present case report is to describe a failed immaturepermanent tooth with infected necrotic pulp and acute apical abscess after revascular-ization/revitalization procedures by histologic and histobacteriologic examination. Thefailure of the present case could be causedby an inadequate control of intracanal infec-tion by using sodium hypochlorite irrigation and calcium hydroxide as well as tripleantibiotics paste intracanal dressing without mechanical debridement of the infected

canal.Althoughthis is a singlecasereport, we hope that it will call attentionto reevaluatethe effectiveness of root canal disinfection protocol of revascularization/revitalizationprocedures in vivo to improve the success of revascularization/revitalization therapy.

Case Report A 6-year-old boy was referred to the Postgraduate Endodontic Clinic at New York

University College of Dentistry from a university-affiliated City Hospital for treatment of tooth #9. The patient’s medical history was not contributory. Tooth #9 was avulsedand replanted with rigid splint by an oral surgeon at the City Hospital within 40 minutesof avulsion approximately 4 months prior. The general dentist at the City Hospital performedinstrumentation of the root canal space because the patient developeda local-ized swelling in the periapical area of tooth #9 about 2 months after tooth replantation.

From the *Department of Endodontics, New York Univer-sity College of Dentistry, New York, New York; and †Privatepractice, Cetraro, Italy.

Address requests for reprints to Dr Domenico Ricucci,Piazza Calvario, 7, 87022 Cetraro (CS), Italy. E-mail address:[email protected]/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.08.024

Case Report/Clinical Techniques

JOE — Volume 40, Number 2, February 2014 Failing Revascularization/Revitalization Therapy 291

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The dentist irrigated the canal with 2% chlorhexidine (Steris Co, Mentor,OH), filled the canal with Calasept (Nordiska Dental, Angelholm,Sweden), and closed the access cavity with a cotton pellet and interme-diate restorative material (IRM) (Dentsply International, Milford, DE).The patient and his mother were advised to visit the PostgraduateEndodontic Clinic at New York University College of Dentistry forcontinuing treatments.

At the initial visit, clinical examination showed that tooth #9 was

asymptomatic and had a class I mobility. There was no swelling or sinustract associated withtooth#9. Thetoothwasnottender to percussionandpalpation. It did not respond to pulp tests with cold, heat, and electricpulp tester. No deep periodontal pockets were present. Periapical radio-graph revealed that tooth #9 was an immature permanent tooth. It hada wide canal space and an open apex with incompletely formed root.The periapical area of the tooth had a localized radiolucent lesion(Fig. 1 A). The clinical diagnosis of tooth #9 was previously initiatedroot canal therapy and asymptomatic apical periodontitis. Treatment options and outcomes including revascularization/revitalization, apexifi-cation, no treatment, and extraction were carefully explained to the

patient and his mother. They decided to keep the tooth and agreed tohave revascularization/revitalization therapy. Informed consent wasobtained. Even though tooth #9 was avulsed and replanted, we deter-mined that it was reasonable to perform revascularization/revitalizationprocedures recommended by the American Association of Endodontists(18, 25) because our concept of revascularization is derived fromthe studies of revascularization of replanted immature teeth in animals(26, 27).

At the second treatment visit under local infiltration with lidocainecontaining 1:100,000 epinephrine (Novocol Pharmaceutical, Cam-bridge, ON, Canada), the tooth was isolated with rubber dam. TheIRMtemporaryfillingand thecottonpelletwere removed fromthe accesscavity. The working length was estimated from the radiograph, and Cal-aseptinthecanalwasgentlyremovedbyusinga#80handfileinconjunc-tion with irrigation with copious amounts of 5.25% sodium hypochloritesolution (Sultan Healthcare, Hackensack, NJ). The canal was dried withsterile paper points and filled with calcium hydroxide mixed with salinesolution to a pasty consistence to the apical third of the canal with plug-gers. The access cavity was closed with a cotton pellet and IRM.

Figure 1. ( A) Diagnostic radiograph. ( B ) Radiograph taken after completion of revascularization/revitalization procedures and restoration of access cavity. ( C )Radiograph taken 16 months after treatment, immediately before extraction. ( D) Crosscut section taken from middle third of the root (hematoxylin-eosin; original magnification,16). ( E ) Detailed view of left radicular contour in ( D) (original magnification, 50). ( F ) Magnification of area indicated by arrows in ( D). Theresorptive defect has been repaired by calcified tissue resembling bone (original magnification, 50). (G ) Section not distant from that shown in ( D) (Taylormodified Brown and Brenn technique; original magnification,16). ( H ) High-power view of area of the root canal wall indicated by left arrow in (G). A bacterial biofilm is present (original magnification, 400). ( I ) High-power view of area of the root canal wall indicated by right arrow in (G). Small bacterial aggregate.Bacteria are colonizing some dentinal tubules (original magnification, 400).


292 Lin et al. JOE — Volume 40, Number 2, February 2014



Two weeks later at the third treatment visit, the tooth was asymp-tomatic. It was isolated with rubber dam under local anesthesia. TheIRMand cottonpellet were removed from theaccess cavity. The calciumhydroxide was gently flushed out of the canal with copious amounts of 5.25% sodium hypochlorite irrigation. The canal was dried and filled with triple antibiotics paste (metronidazole 500 mg/mL, ciprofloxacin500 mg/mL, minocycline 200 mg/mL; HM Compounding, Brooklyn,NY), because we did not initiate the root canal treatment and wanted

to be sure that the root canal infection was under control beforecompletion of revascularization/revitalization procedures. At the fourth treatment visit 2 weeks after the third visit, the tooth

was asymptomatic. A local infiltration with Carbocaine without epineph-rine(NovocolPhamaceutical) wasgiven to the patient. The tooth was iso-lated with rubber dam, and IRM together with the cotton pellet wasremoved from the access cavity. The canal was again irrigated withcopious amounts of 5.25% sodium hypochlorite solution and dried with paper points. Under a surgical microscope, a sterile #60 handfile was used to induce periapical bleeding into the canal up to thecoronal area. A 3-mm thickness of mixture of ProRoot mineral trioxideaggregate (MTA) (Dentsply Tulsa Dental, Tulsa, OK) and saline solution was placed over partially coagulated blood slightly below the cementoe-namel junction and covered with a moist cotton pellet and IRM. One

week later, IRM and cotton pellet were removed and replaced withbonded resin after MTA had set (Fig. 1 B ). The setting of MTA wasconfirmed with an endodontic explorer under the surgical microscope.The patient and his mother were instructed to go back to the City Hospital for follow-up examinations. The general dentist at the City Hospital wasalso informed that revascularization/revitalization of tooth #9 wascompleted and required to have periodic follow-up examinations.

Tooth #9 had been asymptomatic at 2 follow-up examinations,each at 6-month intervals at the City Hospital. The patient developedsymptoms of pain and local swelling at the periapical area of tooth#9 approximately 16 months after revascularization/revitalizationprocedures. Radiograph did not show evidence of increased thickeningof the canal walls and continued root development (Fig. 1C ). Besides

the sign and symptom, the patient and his mother also did not likethe discoloration of the crown caused by MTA and decided to havethe tooth removed. The oral surgeon at the City Hospital extracted thetooth. The patient’s general dentist at the City Hospital informed usabout theextraction. With permission of theCity Hospital andthe patient andhis mother, we were able to obtain theextracted tooth for histologicand histobacteriologic examination. Although the reason for the failure was not clear-cut, we had carefully followed the treatment protocol rec-ommended for revascularization/revitalization therapy. The extractedtooth was processed for histologic and histobacteriologic examination.

Tissue Processing

Immediately after extraction, the specimen was fixed in a 10%neutral buffered formalin solution. The crown was separated from theroot with a diamond disk under water cooling. The root was demineral-ized in an aqueous solution consisting of a mixture of 22.5% (vol/vol)formic acid and 10% (wt/vol) sodium citrate for 3 weeks. The end point was determined radiographically. At the end of the demineralizationprocess theroot wasseparated transversally into2 portions (one coronal and one apical) with a sharp razor blade. The 2 radicular segments were washedin running water for 48 hours,dehydrated in ascending gradesof ethanol,cleared in xylene, and infiltratedand embedded in paraffin. Withthe microtome set at 4–5 mm, approximately 200 consecutive crosscut sections were taken for thecoronal portion in a coronal direction. Longi-tudinal serial sections were cut for the apical portion on a buccolingual plane until the specimen was exhausted.Every fifth slide was stained with

hematoxylin-eosin for screening purposes and assessment of tissuesformed in the canals. Selected slides were stained with the Taylor modi-fied Brown and Brenn technique for bacteria. Slides were examinedunder the light microscope.

Histologic and Histobacteriologic ObservationsCrosscut sections of the coronal portion, passing approximately

through the middle third of the root, stained with hematoxylin-eosin,

showed extensive dentin resorption altering the radicular contour(Fig. 1 D). In some areas a calcified tissue resembling cementum wascovering the resorbed dentin. Fragments of periodontal ligament withno inflammatory cells could be seen attached to this tissue ( Fig. 1 E ).In other areas the resorptive defects appeared to have repaired almost completely, with a calcified tissue exhibiting a lamellar structure andthus resembling bone (Fig. 1 F ). The root canal was apparently empty, with remnants of an amorphous material (likely MTA). Bacterial stain-ing revealed bacterial aggregates on the root canal walls (Fig. 1G–I ).Bacteria could also be seen colonizing some dentinal tubules (Fig. 1 I ).

Longitudinal sections passing through the center of the canal andforamen showed that the apical canal was filled with necrotic debris. Nopredentin was present, and no cells aligning the dentin walls could be

seen (Fig. 2 A). Bacterial staining revealed that the necrotic mass fillingthe foramen was a thick bacterial biofilm exhibiting different bacterial concentrations (Fig. 2 B ). Bacterial cells were predominant over theextracellular matrix in the foraminal area, where the biofilm extendedbeyondthe root canal limits, faced with a concentration of polymorpho-nuclear leukocytes (Fig. 2C and D). Bacterial concentration was lesspronounced more coronally, where the extracellular matrix dominated(Fig. 2 B ). Cocci, rods, and filamentous bacteria were recognized. Thebacterial biofilm appeared well-adapted to the wall irregularities, andthe subjacent dentinal tubules were heavily colonized (Fig. 2C and D).

DiscussionThe key to the success of revascularization/revitalization therapy is

to effectively control the root canal infection. If infection persists in theroot canal, not only regeneration but also repair would not occur in thepulp-periapical tissue complex, even though stem cells might survive inthe inflamed periapical tissues (28).

When the extracted tooth #9 was examined under the surgical microscope, thecoronal seal by thebonded resin appeared to be intact.The MTA seal, visible after separation of the crown, was also intact.Histologically, the tissue in the canal space was completely disinte-grated. The majority of bacteria were observed in the apical portionand not in the coronal portion of the canal. The bacteria formedbiofilmon the canal walls and penetrated into the dentinal tubules. MTA hasbeen shown to have an excellent sealing ability (29). On the basis of these findings, the possibility of coronal bacterial leakage could be

ruled out as the cause of root canal reinfection. Accordingly, the effec-tiveness of root canal disinfection protocol of revascularization/revital-ization procedures to eliminate bacteria in the infected root canal wasquestioned.

In the present case, it is possible that vital tissue might be able togrow into the canal space from the periapical area after revasculariza-tion/revitalization procedures because bacteria, which were not killedby root canal disinfection, were likely to be in the lateral/accessory canals or dentinal tubules and possibly in some biofilm on the canal walls in the apical portion of the canal. The current root canal prepa-ration protocol of infected mature or immature teeth for root canal therapy or apexification, including mechanical debridement, antisepticirrigation, andintracanal medication, is not ableto completely eliminatebacteria in the isthmuses, lateral/accessory canals, or dentinal tubules.





The bacteria remaining in these areas inaccessible to root canal prep-

aration canslowly proliferate andreinfect theroot canal (6, 7, 30); they are also not reachable by the host’s innate and adaptive immunedefenses. In addition, the tissue in the root canal lacks collateral circulation to effectively deliver cellular and humoral defensecomponents to the canal. Therefore, it is impossible for tissue torecover in the canal if it gets infected and not treated (31).

Radiographically, the periapical lesion initially showed process of healing after revascularization/revitalization procedures. During thisperiod of time, the bacteria in the dentinal tubules or lateral/accessory canals might be in the stationary phase or perhaps in balance with thedefense mechanisms of thenewlyformed tissuein the canal. Thepatient developed the clinical symptom of pain andthe sign of apical swelling of the involved tooth 16 months after revascularization/revitalizationprocedures. This could be due to increased growth of bacteria in the

dentinal tubules, lateral/accessory canals, or biofilm on the canal walls,

which overwhelmed the defenses of the newly formed tissue, reinfectedthe rootcanal, and caused tissue destruction. Small numbers of bacteria that contaminate or colonize the vital tissue will be killed by the host’sdefenses.It is not easy forintracanal bacteria to obtain nutrientsin well-obturatedteeth becauseof the lackof communication between the canal and the periapical tissues (32). However, in revascularized/revitalizedteeth, bacteria in the lateral/accessory canals or dentinal tubules caneasily receive nutrients from the tissue fluid. Furthermore, it is not known whether the defense capability of the newly formed tissue inthe canal after revascularization/revitalization therapy is similar tothat of normal pulp tissue.

It is questionable that the current root canal disinfection protocol of revascularization/revitalization procedures that lacks mechanical debridement of biofilm on the canal walls is able to effectively eliminate

Figure 2. ( A) Longitudinal section encompassing the wide canal and foramen (hematoxylin-eosin; original magnification, 16). ( B ) Section proximal to that in( A), stained with Taylor modification of Brown and Brenn technique. The canal is filled by necrotic debris, heavily colonized by bacteria (original magnification,16). (C ) Detailed view of area demarcated by rectangle in ( B). A thick bacterial biofilm takes adherence to the apical canal wall and extends beyond the root canal limits (original magnification, 100). ( D) High-power view of area demarcated by rectangle in (C). The biofilm lies on a thin residual dentin wall. Accu-mulation of polymorphonuclear leukocytes (original magnification, 400).


294 Lin et al. JOE — Volume 40, Number 2, February 2014



most intracanal bacteria in vivo. In this case report, bacteria werepresent mostly in theapical area andnot in thecoronalarea of thecanal.It appears that the root canal disinfection protocol of revascularization/ revitalization procedures was able to eliminate most bacteria in thecoronal area but not in the apical area of the infected root canal.Mechanical debridement plays an important role in root canal therapy.It has been demonstrated that mechanical instrumentation in conjunc-tion with saline irrigation alone is able to significantly reduce the

number of bacteria in infected root canals (33–35).The chelating agent EDTA has been recommended in revasculariza-tion procedures (18, 25). EDTA has weakantimicrobial activity (14, 36)but is capable of inhibiting biofilm formation (36). EDTA can deminer-alize the dentin and expose the dentin matrix and also cause release of growth factors from the dentin matrix reservoir (24, 37, 38).Therefore, EDTA may encourage dentin-pulp regeneration and enhancethe attachment of newly formed tissue to the canal walls (24, 39).However, it is not known whether EDTA is able to completely disrupt biofilm firmly attached to the canal walls in the complex root canal system.

Revascularization/revitalization therapy has been shown to be ableto increase thickening of the canal walls; therefore, it would be morebeneficial to disrupt biofilm and to remove the hard tissue of the canal

walls contaminated by bacterial toxins as well as bacteria in the dentinal tubules by performing a certain degree of mechanical debridement during revascularization/revitalization procedures. This shouldenhance the attachment of the newly formed tissue on the canal walls.This procedure is similar to guided tissue regeneration in regenerativeperiodontal therapy to mechanically remove bacterial plaque on theroot surface and bacterial toxin contaminated cementum in the peri-odontal pockets.

Infection prevents regeneration, repair, and stem cell functions. A sterile microenvironment is likely required for tissue regenerationincluding pulp tissue in immature permanent teeth with infectednecrotic pulp after revascularization/revitalization therapy. The successrate of revascularization/revitalization therapy should be evaluated in

long-term clinical outcome studies. Also, the effectiveness of current root canal disinfection protocol for revascularization/revitalizationprocedures must be determined to improve the success of revascular-ization/revitalization therapy.

Acknowledgments

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

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