clinical, radiographic, and histological observation of a human...

Case Report/Clinical Techniques

Clinical, Radiographic, and Histological Observationof a Human Immature Permanent Tooth with ChronicApical Abscess after Revitalization TreatmentEmi Shimizu, DDS, PhD,* Domenico Ricucci, MD, DDS,

‡Jeffrey Albert, DDS,

†

Adel S. Alobaid, DDS,†Jennifer L. Gibbs, MAS, DDS, PhD,

†George T.-J. Huang, DDS, MDS, DSc,

§

and Louis M. Lin, BDS, DMD, PhD†

Abstract
Introduction: Revitalization procedures have beenwidely used for the treatment of immature permanentteeth with apical periodontitis. The treatment proce-dures appear to be capable of encouraging continuedroot development and thickening of the canal walls.The nature of tissues formed in the canal space andat the root apex after revitalization has been shownhistologically in several animal studies; similarstudies in humans were recently reported. Methods:A 9-year-old boy had a traumatic injury to his upperanterior teeth. Tooth #9 suffered a complicated crownfracture with a pulp exposure, which was restoredwith a composite resin. The tooth developed a chronicapical abscess. Revitalization procedures were per-formed on tooth #9 because it was an immature perma-nent tooth with an open apex and thin canal walls.Twenty-six months after revitalization, the tooth hada horizontal crown fracture at the cervical level andcould not be restored. The tooth was extracted andprocessed for routine histological and immunohisto-chemical examination to identify the nature of tissuesformed in the canal space. Results: Clinically and radio-graphically, the revitalization of the present case wassuccessful because of the absence of signs and symp-toms and the resolution of periapical lesion as well asthickening of the canal walls and continued root devel-opment. The tissue formed in the canal was well-mineralized cementum- or bone-like tissue identifiedby routine histology and immunohistochemistry. Nopulp-like tissue characterized by the presence of polar-ized odontoblast-like cells aligning dentin-like hardtissue was observed. Conclusions: The tissues formedin the canal of revitalized human tooth are similar tocementum- or bone-like tissue and fibrous connectivetissue. (J Endod 2013;39:1078–1083)
From the Departments of *Basic Science and Craniofacial BiolPractice, Rome, Italy; and §Department of Bioscience Research, Col

Address requests for reprints to Dr Louis M. Lin, Department of EE-mail address: [email protected]/$ - see front matter

Copyright ª 2013 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.04.032

1078 Shimizu et al.

Key WordsApical periodontitis, bone-like tissue, cementum-like tissue, immature permanenttooth, revitalization

Since the report of Iwaya et al (1) that a type of treatment termed ‘‘revascularization’’was applied to treat human immature permanent tooth with apical periodontitis and

sinus tract and resulted in not only resolution of apical periodontitis but also thickeningof the root canal walls and continued root development, many human immaturepermanent teeth with apical periodontitis have been treated with revascularizationprocedures instead of traditional apexification procedures (2). It has been discussedthat the term ‘‘revitalization’’ is more appropriate to describe such clinical treatment;therefore, revitalization will be used herein (3, 4). The nature of tissues formed inthe root canals of immature teeth with apical periodontitis after revitalization wasdescribed histologically as mineralized tissue resembling cementum or bone andperiodontal ligament-like tissue in several animal studies (5–8). The nature of thetissue formed in the root canals of human revitalized teeth was recently reported(9–11). We report clinical, radiographic, and histological observation of a humanimmature permanent tooth with chronic apical abscess after revascularizationtreatment.

Case ReportDiagnosis and Treatment Planning Visit

A 9-year old boy was referred from the Postgraduate Pediatric Clinic to the Post-graduate Endodontic Clinic at New York University College of Dentistry for the evaluationof tooth #9. The patient had a history of trauma to the upper anterior teeth that occurredfrom a fall approximately 3months ago. The patient’s chief complaint was ‘‘I hit my frontteeth 3 months ago, and now I have an infection.’’ The patient did not report any symp-toms. Clinical examination showed that tooth #9 had a complicated crown fracture witha pulp exposure, which was restored with a composite resin. A sinus tract was present inthe apical area of tooth #9. The tooth was not sensitive to percussion and palpation. Italso did not respond to Endo Ice (Coltene/Whaledent Inc, Cuyahoga, OH) or an electricpulp tester (Vitality Scanner; SybronEndo, Glemdora, CA). Radiographic examinationrevealed a well-circumscribed large periapical radiolucent lesion measuring approxi-mately 8� 8 mm around the apex of tooth #9 (Fig. 1A). The immature tooth had thincanal walls and an open apex (Fig. 1A). The clinical diagnosis for tooth #9 was pulpalnecrosis and chronic apical abscess. Treatment options including revitalization,

ogy and †Endodontics, New York University College of Dentistry, New York, New York; ‡Privatelege of Dentistry, University of Tennessee, Health Science Center, Memphis, Tennessee.ndodontics, New York University College of Dentistry, 345 East 24th Street, New York, NY 10012.

JOE — Volume 39, Number 8, August 2013

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_surname

Delta:1_given name

Delta:1_surname

Delta:1_given name

mailto:[email protected]

http://dx.doi.org/10.1016/j.joen.2013.04.032

Figure 1. (A) Preoperative radiograph: tooth #9 exhibits incomplete formation of the root. Periapical radiolucency is present. (B) Postoperative radiograph: theperiapical radiolucent area appears to be larger than the preoperative lesion, with sharp margins. (C) Follow-up radiograph taken 12 months after revitalization: theperiapical radiolucency has resolved with only slight thickening of the periodontal ligament around the root apex. The canal space is reduced in size and thethickness of the canal walls is increased. (D) The patient presented after 25 months because the crown was mobile. A complete crown fracture was diagnosed,and the tooth could not be restored. Clinical view after removal of the fractured crown. (E) Radiograph taken before removal of the fractured crown. Thickening ofthe root canal walls increased further. The periapical lesion completely resolved.


mineral trioxide aggregate apexification, and calcium hydroxide apex-ification were discussed with the mother and child, and after discussionthe decision was made to perform a revitalization procedure.

First Treatment VisitAt the first treatment appointment, access to the pulp cavity was

obtained under rubber dam isolation without administration of localanesthetic. Purulent exudate and bleeding were noted in the canal. Aworking length radiograph was taken and recorded at 23 mm. Thecanal was gently irrigated with copious amounts of 2.6% sodiumhypochlorite (Sultan Healthcare, Hackensack, NJ) and dried with paperpoints. Calcium hydroxide (Henry Schein, Melville, NY) mixed withsaline solution was used as an intracanal medication and placed intothe apical portion of the canal. The access was closed with a sterilecotton pellet followed by an intermediate restorative material (DentsplyInterntional, Milford, DE).

Second Treatment VisitEleven days after the initial treatment, tooth #9 was asymptomatic,

and the sinus tract had closed. Local infiltration with 3% mepivacainewithout vasoconstrictor was administered. The access cavity wasreopened after isolation with a rubber dam. The root canal was irrigatedwith copious amounts of 2.6% sodium hypochlorite and dried withpaper points. Bleeding was induced into the canal up to the coronal


third by irritating the periapical tissues with a #30 K-file. Mineraltrioxide aggregate (Dentsply Tulsa Dental, Johnson City, TN) mixedwith saline solution was placed on the top of the partially coagulatedblood clot. The access was sealed with a light-cured composite restora-tion (Amelogen Plus; Ultradent, South Jordan, UT) (Fig. 1B).

Recall VisitsAt the 12-month recall appointment, the tooth was asymptomatic.

There was radiographic evidence of resolution of the periapical lesionand thickening of the canal walls (Fig. 1C). Thus, this case was both anendodontic success in that the periapical lesion had healed and a revi-talization success because there was obvious increased thickening ofthe canal walls and continued root development. Twenty-six monthsafter the completion of the revitalization treatment, the patient presentedto the Postgraduate Endodontic Clinic with a horizontal crown fractureat the cervical level of tooth #9 (Fig. 1D), and the tooth was determinedto be nonrestorable. Radiographically, the tooth showed completeresolution of the periapical lesion and marked thickening of the canalwalls as well as closure of the root apex (Fig. 1E). Tooth #9 wasextracted and processed for routine histological and immunohisto-chemical examination.

The primary mouse monoclonal antibodies were used againsthuman bone sialoprotein (BSP), dentin sialoprotein, and neurofilament200 N52 (LifeSpan Biosceiences, Inc, Seattle, WA). The avidin-biotin

Chlorhexidine-based Endodontic Treatment 1079

complex staining kit was obtained from Santa Cruz Biotechnology, Inc(Santa Cruz, CA). The routine histological and immunohistochemicalstaining procedures were described in our previous study (9).
Quantification of Radiographic Changes in the RootLength and Area at the 12-month Recall

Images were analyzed using ImageJ (National Institutes of Health,Bethesda, MD). The preoperative and 3-month recall films were alignedusing the TurboReg Plug-In tool. (Unfortunately, the 12-month follow-up film was too elongated to use for comparison.) After alignment, theradiographic root width was measured by outlining the total root widthand canal width at 3 discreet points (1midroot, 1 at the apical third, and

Figure 2. (A) Section passing approximately at the center of the root canal. A minmagnification �16). (B) Detail of the apical portion in A. An island of soft tissueinset shows magnification of the ramification indicated by the lower arrow. Its l(C) A high-power view of the apical soft tissue in B. Vital connective tissue with fibrobmagnification �400). (D) Magnification of the area indicated by the upper arrowapically by a cementum-like tissue, with some osteoblast-like lacunae. Increased roo�400). (E) A high-power view of the area of the root canal wall indicated by thetubules (tubules are cut transversally by the microtome blade), area with less�400). (F) A high-power view of the area of the root canal wall indicated by the rig(cut obliquely by the microtome blade), area with only few tubules, and calcified

1080 Shimizu et al.

1 in the apical fifth). The root length was measured by drawing a linefrom the most mesial aspect of the cemento-enamel junction (CEJ) aswell as the most distal aspect of the CEJ to the center of the most apicalportion of the root canal space. All measurements were completed twice,and the values were averaged. The percentage change in the measure-ments from the preoperative to the recall film was then calculated.

A comparison of the preoperative and postoperative films showedthat a quantifiable increase in the radiographic area of the root wasobserved subsequent to the procedure. At the 3-month recall, weobserved minimal change (mean = 2.3% increase, average changemeasured from the mesial and distal CEJ) in root length. However,we were able to measure more significant increases in root width(mean = 10.4% increase, average change measured at 3 discreet

eralized tissue fills the apical portion of the canal (hematoxylin-eosin; originalis present in the calcified tissue apically (original magnification �25). The

umen contains uninflamed connective tissue (original magnification �400).lasts and abundance of collagen fibers. Absence of inflammatory cells (originalin B. The calcified tissue filling the apical canal is irregular and is demarcatedt length is caused by deposition of cementum-like tissue (original magnificationleft arrow in A. From left to right: area with high concentration of dentinaltubules, and calcified tissue with no dentin tubules (original magnificationht arrow in A. From right to left: area with high concentration of dentin tubulestissue with no dentin tubules (original magnification �400).


points). Importantly, these findings suggest that the case should beconsidered a successful revitalization procedure because of thequantification of increased root width.
Histological and Immunohistochemical ObservationsHistologically, the most apical portion of the canal was filled with

newly formed mineralized tissue (Fig. 2A and B). Some uninflamed vitalconnective tissue was noted enclosed in the mineralized tissue (Fig. 2B

Figure 3. (A) Same section as that in Figure 2. Middle third portion of the canal. T�16). (B) Detail of the coronal portion of the canal in A. The canal is partly occupiepower view of the area of the left root canal wall indicated by the left arrow in B. Frotubular dentin, and necrotic debris and calcified tissue with no tubules (originatissue indicated by the right arrow in B. Dystrophic calcification. Small lacunaeapproximately 50 sections from that shown in A. The apical canal appears totallyforamina area in E. Vital tissue on the left. Necrotic debris on the right (original mby the arrow. Two neutrophilic leukocytes can be recognized (original magnificat


and C). The newly formed mineralized tissue appeared to resemblecementum-like tissue with incremental lines and osteocyte-like lacunae(Fig. 2D). The demarcation between the canal dentin and the newlyformed mineralized tissue could be easily recognized because of theabsence of dentinal tubules in the latter tissue (Fig. 2E and F). Theincreased root length was caused by apical deposition of cementum-like tissue (Fig. 2B and D). In the midportion of the canal, the canalspace was partially filled with mineralized tissue with pockets ofnecrotic debris (Fig. 3A, B, and D). Necrotic tissue was also present

he canal appears empty at this level (hematoxylin-eosin, original magnificationd by a newly formed calcified tissue (original magnification�50). (C) A high-m left to right: highly tubular dentin, calciotraumatic line and transition to a lessl magnification �400). (D) A high-power view of the area of the calcifiedcontaining necrotic debris (original magnification �400). (E) Section takenobliterated by calcified tissue (original magnification �16). (F) Detail of theagnification �100). The inset shows high magnification of the area indicatedion �400).


between the canal dentin and the mineralized tissue (Fig. 3C). Thereappeared to be a change of pattern in dentin formation before pulpunderwent necrosis, probably because of the history of trauma(Fig. 3C). The apical canal appears obliterated by calcified tissue(Fig. 3E and F). No pulp-like tissue characterized by the presence ofpolarized odontoblast-like cells aligning the dentin-like tissue wasobserved. Immunohistochemical staining was only positive for BSP(Fig. 4A–C).
DiscussionIn all animal studies, the tissues formed in the canal spaces after

revitalization procedures of immature teeth are described asmineralizedtissue and periodontal ligament-like tissue (5–8). The mineralizedtissue resembles either cementum or bone. The tissues formed in thecanal space of the present human case after revitalization proceduresare similar to those observed in animal studies (5–8) and the tissuesformed in the canal spaces of a previous human case report (11).Continued root development was caused by apical deposition ofcementum-like tissue without dentin, which inferred likely survival ofHertwig’s epithelial root sheath (HERS) before completion of rootformation. It is HERS, and not apical papilla, that regulates root devel-opment (12, 13). The apical papilla in the present case probably didnot survive chronic apical abscess; otherwise, HERS would induceprogenitor/stem cells in the ectomesenchymal apical papilla to

Figure 4. Immunohistochemical staining: the tissue is positive for BSP. (A) Low mamagnification�20). (B) High magnification of upper square in A. Junction betweenHigh magnification of lower square in A. The junction between the original apic(arrow). The increased root length is caused by the deposition of newly formed c

1082 Shimizu et al.

differentiate into root odontoblasts and produce root dentin (12, 14).It has been shown that HERS is capable of inducing progenitor/stemcells in the ectomesenchymal dental follicle to differentiate intocementoblasts during root development (13). The cellular and molec-ular biology of revitalization is still not fully understood (15).

Immunohistochemical staining was positive for BSP. The mineral-ized tissue formed in the canal space in the present case most likely wasnot dentin because of the absence of a tubule-like structure and mightbe well-mineralized cementum- or bone-like tissue. Although nervefibers were not detected immunohistochemically, this does not meanthat nerve fibers were not present because vital connective tissue andblood vessels were observed in the canal of the revitalized tooth. Nervesplay an important role in maintaining tissue vitality, such as the controlof blood flow (16), the response to injury (17), and the regulation ofimmune system (18).

Based on the present case and a previous case report (11) as wellas animal studies (3–6), in situ regeneration of the pulp tissue in theteeth with necrotic pulp and apical periodontitis after revitalizationtreatment did not occur. This is probably because of the completedestruction of the parenchymal cells of the pulp tissue and the apicalpapilla as predicted previously (4). It has not been shown thatin situ regeneration of tissues or organs is possible if the parenchymalcells were completely destroyed, at least in the revitalization of immaturepermanent teeth with apical periodontitis. However, in 1 study,pulp-like tissue was observed in revitalized tooth (10).

gnification of the section similar to B (immunohistochemical staining, originalthe canal dentin and newly formed bone-like tissue in the canal (arrows). (C)al root dentin and newly deposited cementum-like tissue after revitalizationementum-like tissue. C, cementum-like tissue; D, dentin.


Regeneration of the dentin-pulp complex in the teeth with total
necrotic pulp and apical periodontitis likely requires tissue engineeringusing a cell-based approach that includes the use of autologous pulptem/progenitor cells, scaffold, and morphogens (growth factors)(19–21) (ideally, in a sterile root canal system). The exogenouslyintroduced cells may be a heterogeneous or subpopulation of stem/progenitor cells. The current non–cell-based approach withoutintroducing exogenous cells would result in repair rather thanregeneration (22, 23). Some investigators are testing the use ofchemotactic factors to induce homing by recruiting stem cells fromother area of tissues into the root canal space for pulp regeneration(24). So far, no convincing pulp regeneration data have been reportedin revascularization/revitalization of immature permanent teeth withapical periodontitis.

ConclusionBased on clinical and radiographic observations, revitalization of

the present case was successful because of the absence of signs andsymptoms and the resolution of apical periodontitis as well as thick-ening of the canal walls and continued root development. The tissuesformed in the canal space are similar to cementum- or bone-like tissueand fibrous connective tissue.

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

References1. Iwaya SI, Ikawa M, Kubota M. Revascularization of an immature permanent tooth

with apical periodontitis and sinus tract. Dent Traumatol 2001;17:185–7.2. Bose R, Nummikoski P, Hargreaves K. A retrospective evaluation of radiographic

outcome in immature teeth with necrotic root canal system treated with regenerativeendodontic procedures. J Endod 2009;10:1343–8.

3. Huang GTJ, Lin LM. Letter to the editor: comments on the use of the term ‘‘revas-cularization.’’ J Endod 2008;34:511.

4. Huang GT. Apexification: the beginning of its end. Int Endod J 2009;42:855–66.5. Thibodeau B, Teixeira F, Yamauchi N, et al. Pulp revascularization of immature dog

teeth with apical periodontitis. J Endod 2007;33:700–5.6. Bezerra da Silva LA, Nelson-Filho P, Bezerra da Silva RA, et al. Revascularization

and periapical repair after endodontic treatment using apical negative pressure


irrigation versus conventional irrigation plus triantibiotic intracanal dressing indog’s teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Radiol Endod2010;109:313–9.

7. Wang X, Thibodeau B, Trope M, et al. Histological characterization of regeneratedtissues in canal space after the revitalization/revascularization procedure ofimmature dog teeth with apical periodontitis. J Endod 2010;36:56–63.

8. Yamauchi N, Nagaoka H, Yamauchi S, et al. Immunohistolocal charaterization ofnewly formed tissues after regenerative procedure in immature dog teeth.J Endod 2011;37:1636–41.

9. Shimizu E, Jong G, Partridge N, et al. Histological observation of a human immaturepermanent tooth with irreversible pulpitis after revascularization/regenerativeprocedure. J Endod 2012;38:1293–7.

10. Torabinejad M, Faras H. A clinical and histological report of a tooth with an openapex treated with regenerative endodontics using platelet-rich plasma. J Endod2012;38:864–8.

11. Gabriela M, Ricucci D, Gibbs JL, et al. Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod 2003;39:138–44.

12. Zeichner-David M, Oishi K, Su Z, et al. Role of Hertwig’s epithelial root sheath cellsin tooth root development. Dev Dyn 2003;228:651–63.

13. Sonoyama W, Seo B-M, Yanaza T, et al. Human Hertwig’s epithelial root sheath cellsplay crucial roles in cementum formation. J Endod 2007;86:594–9.

14. Thomas HF. Root formation. Int J Dev Biol 1995;39:231–7.15. Lin LM, Rosenberg PA. Repair and regeneration in endodontics. Int Endod J 2011;

44:889–906.16. Olgart L. Neural control of pulp blood flow. Crit Rev Oral Biol Med 1996;7:159–71.17. Byers MR, Narhi MV. Dental injury models: experimental tools for understanding

neuroinflammatory interaction and polymodal nociceptorfunction. Crit Rev OralBiol Med 1999;10:4–39.

18. Kin NW, Sanders VM. It takes nerve to tell T and B cells what to do. J Leukocyte Biol2006;79:1093–104.

19. Nakashima M, Akamine A. The application of tissue engineering to regeneration ofpulp and dentin in endodontics. J Endod 2005;31:711–8.

20. Iohara K, Imabayashi K, Ishizaka R, et al. Complete pulp regeneration after pulpec-tomy by transplantation of CD 105+ stem cells-derived factor-1. Tissue Eng Part A2011;17:1911–20.

21. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novoregeneration of dental pulp with newly deposited continuous layer of dentin in anin vivo model. Tissue Eng Part A 2010;16:605–15.

22. Bartold PM, McCulloch CAG, Narayanan AS, et al. Tissue engineering: a newparadigm for periodontal regeneration based on molecular and cellular biology.Periodontology 2000 2000;24:253–69.

23. Ivanovski S, Grnthos S, Shi S, et al. Stem cells in the periodontalligament. Oral Dis2006;12:358–63.

24. Kim JY, Xin X, Moioli EK, et al. Regeneration of dental-pulp-like tissue bychemotaxis-induced homing. Tissue Eng Part A 2010;16:3023–31.


http://refhub.elsevier.com/S0099-2399(13)00344-0/sref1





























































clinical, radiographic, and histological observation of a human...

Documents