Australian Dental Journal

The ofcial journal of the Australian Dental Association

CASE REPORT

Australian Dental Journal 2010; 55: 446452 doi: 10.1111/j.1834-7819.2010.01268.x

Regenerative endodontics biologically-based treatment for immature permanent teeth: a case report and review of the literature
A Thomson,* B Kahler*
*School of Dentistry, The University of Queensland, Brisbane.

ABSTRACT
Background: A paradigm shift in the treatment of immature, necrotic teeth has occurred with biologically-based principles and regenerative endodontic protocols replacing traditional apexication procedures. Preliminary research suggests that stem and progenitor cells from the pulp and or periodontium contribute to continued root development when regenerative procedures are followed. Methods: A mandibular premolar tooth with a chronic periapical abscess was irrigated with sodium hypochlorite with minimal instrumentation and then dressed with tri-antibiotic paste consisting of ciprooxacin, metronidazole and amoxicillin. At a subsequent visit a blood clot was evoked in the canal by irritating periapical tissues and the canal sealed with mineral trioxide aggregate, glass ionomer cement and composite resin. Results: Resolution of apical periodontitis and the draining sinus, continued root maturation and apical closure occurred over an 18-month period. The tooth became responsive to pulp sensibility testing. Conclusions: It is important that dentists recognize the potential of regenerative endodontics in the treatment of necrotic, immature teeth. Initial management should involve irrigation with sodium hypochlorite only. Intra-canal medicaments, such as calcium hydroxide, are contraindicated as they inhibit further root growth. This report uses a variation of the triantibiotic paste currently recommended for regenerative procedures that avoided the discolouration of the crown associated with current protocols. Regenerative endodontics with continued root growth may reduce the risk of fracture and premature tooth loss associated with traditional apexication procedures where the root remains thin and weak.
Keywords: Regenerative endodontics, revascularization, tri-antibiotic paste. Abbreviation: MTA = mineral trioxide aggregate. (Accepted for publication 25 January 2010.)

INTRODUCTION Revascularization of a necrotic pulp following a traumatic incident such as avulsion in an immature tooth with an open apex is well established and a desirable outcome as it allows further development of the root.1 In this clinical scenario, the pulp is necrotic but not infected and can act as a matrix to allow revascularization of the tooth. Optimal replantation techniques, prompt replantation to minimize the risk of infection, and an open apex greater than 1.1 mm increases the chances of revasularization.2 Recently, a number of case series and case reports have described continued root development and even responses to pulp sensibility testing in treated necrotic immature teeth with infected root canal systems, including those with draining sinus tracts.39 These regenerative endodontic
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procedures and revasularization of the root canal in teeth with infected root canal systems have been termed a paradigm shift in the treatment protocol for endodontic management.7,10 Traditionally, the clinical protocol for immature teeth with infected root canal systems was termed apexication and involved placing calcium hydroxide as an intra-canal medicament to rstly eliminate the intra-radicular infection and then induce an apical barrier over time.11 There was a requirement for multiple visits and a lengthy time period (average 12 months) before a root lling could be completed.12 More recently, an alternative apexication protocol involved placement of mineral trioxide aggregate (MTA) which acts as an articial barrier on which a hard tissue barrier forms.13 An advantage of this technique is it is generally completed in one or two
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Pulp regeneration appointments.14 However, apexication techniques with either placement of calcium hydroxide or MTA have in general not allowed continued root maturation. Hence, there is a greater risk of fracture as a consequence of thin dentinal walls and premature loss of the tooth.15 Regenerative procedures generally advocate the placement of a tri-antibiotic paste to eliminate the intra-radicular infection which is regarded as a mandatory requirement for success.39 The tri-antibiotic paste in these studies consists of a combination of ciprofoxacilin, metronidazole and minocycline which has been shown to reliably eliminate bacteria in infected root dentine whereas the respective drugs used alone have only substantially reduced but did not eliminate the bacteria.16 This combination of drugs has also been shown to be effective at eliminating bacteria in the deep layers of root canal dentine.17 However, a disadvantage of this technique is discolouration of the crown of the tooth presumably due to the minocycline.9 Ledermix (Lederle Pharmaceutical, GMBH Wolfratshausen, Germany), an intra-canal medicament containing tetracycline, has been shown to cause greater discolouration in immature teeth than mature teeth.18 Sato et al. studied the antibacterial efficacy of a variety of antibiotic combinations and found that amoxycillin, used as the third antibiotic, was also as effective as minocycline when used in combination with ciprofloxacin and metronidazole in eliminating bacteria.17 However, there are no known reports of regenerative protocols using a tri-antibiotic paste which includes in its formulation amoxycillin, and whether this combination also results in crown discolouration. The inherent risk of sensitization and allergy to penicillin would be a contraindication in some patients.19 This case report describes an endodontic regenerative procedure using a combination of ciprooxacin, metronidazole and amoxycillin for a mandibular left second premolar diagnosed with pulpal necrosis, an infected root canal system and chronic periapical abscess. CASE REPORT A 12-year-old Caucasian female patient presented to the Kingston School Dental Clinic for evaluation of her mandibular left second premolar. The medical history was non-contributory. There was no history of pain or discomfort with the tooth and her only complaint was that of intermittent bad breath, whilst her mother was concerned about some swelling of the gingiva adjacent to the mandibular left second premolar. An intra-oral examination revealed the tooth to be intact with no signs of caries without any history of trauma. A large draining sinus was present in the buccal gingiva (Fig 1). Sensibility testing of the mandibular left second premolar was non-responsive to both cold and
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Fig 1. Clinical photograph of teeth in the posterior segment of the left mandible. Gingival swelling and pointed abscess is evident adjacent the lower second premolar.

electric pulp sensibility testing, whilst the remaining teeth in the lower left arch were all responsive. Periodontal probing conrmed normal attachment levels with no probing depths greater then 3 mm. Mobility of the tooth was in the physiologic range. No crown discolouration was noted (Fig 1). Radiographically, the mandibular left second premolar had an increase in the periodontal ligament space, incomplete root formation and a diffuse periapical radiolucency 5 mm x 3 mm in size (Fig 2). No carious lesion was evident. A gutta-percha point was placed in the sinus tract and illustrated the relationship between the lesion and the periapical lucency (Fig 3). A diagnosis of pulpal necrosis, an infected root canal system and chronic periapical abscess was made on the basis of the clinical and radiographic examination. It was considered that the aetiology of the infection was an occlusal tubercle that had fractured, allowing bacterial contamination of the pulp. Pulpal regeneration was regarded as the optimal treatment choice considering the stage of root development, the thin dentinal wall maturation and the wide open apex. A comprehensive discussion of the risks, complications and alternative treatment options was undertaken and parental consent obtained. Local anaesthesia was administered and the mandibular left second premolar isolated with rubber dam. Access preparation was made utilizing an operating
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A Thomson and B Kahler Endo, Orange, CA, USA) for length conrmation. The le was placed carefully so as to limit any damage to the canal walls. The canal was then irrigated with 30 mL of 1% sodium hypochlorite 2 mm from the working length prior to drying the canal with paper points. The tri-antibiotic paste consisted of a powder of 20 mg each of metronidazole, ciprooxacin and amoxicillin mixed with 1 mL of sterile water. The paste was carefully introduced into the canal with a lentulo spiral root canal ller attempting to minimize placement in the coronal portion of the tooth and lled to the level of just below the cemento-enamel junction. The access cavity was sealed with 4 mm of Cavit (ESPE, Seefeld, Germany) and 2 mm of Fuji IX (GC, IL, USA). The tooth was reviewed three weeks later. The patient reported some pain three days postoperatively which required oral analgesics (Ibuprofen), but had been asymptomatic since that time. Intraorally, the draining sinus had resolved. However, due to the episode of pain it was decided to redress the tooth. Under rubber dam isolation, the canal was irrigated with 20 mL of sodium hypochlorite, which was ultrasonically agitated with a size 10 K-le for one minute after each 5 ml was administered. The canal was then dried and redressed with the tri-antibiotic paste combination mentioned earlier utilizing the same placement technique. The patient was recalled after a further three weeks reporting that the tooth had been asymptomatic for that entire time. A periapical radiograph was taken and revealed some Cavit had been dislodged apically in the root canal. Under rubber dam isolation, the canal was irrigated with 10 mL of sodium hypochlorite with ultrasonic agitation for one minute every 5 mL of solution. With the infection and foreign material controlled, the regenerative process was commenced. After the canal was dried with paper points, a D11T NiTi hand spreader (Dentsply Tulsa Dental, TN, USA) was used to irritate the apical tissue until bleeding occurred apically in the root canal space so as to create a biological scaffold for the regenerative process (Fig 4). Over a 15-minute time period, the blood was allowed to clot to a level 3 mm below the cementoenamel junction. Then 3 mm of ProRoot white MTA (Dentsply Tulsa Dental, TN, USA) was placed with the aid of a Lee block (San Francisco, USA) and Buchannan Pluggers (Sybron Endo, Orange, CA, USA). The access cavity was sealed with 3 mm of Fuji IX and 2 mm of acid etched composite resin (Fig 5). The tooth remained asymptomatic over an 18-month follow-up period. Clinically, the tooth was responsive to electric pulp sensibility testing while earlier testing throughout the follow-up period had been inconclusive. No tenderness to percussion or palpation could be discerned. Radiographically, osseous healing of the periapical lesion was evident as well as root maturation
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Fig 2. Periapical radiograph illustrating a periapical radiolucency associated with the lower left mandibular second premolar. Thin dentinal wall at the apex of the root which exhibits a wide open apex is noted. There is no evidence of caries.

Fig 3. A radiographic image of a gutta-percha point placed in the draining sinus tract that traces to the periapical radiolucency.

microscope and a single, wide canal was visualized and drainage of haemorrhagic, purulent exudate was obtained. Working length was estimated with an apex locater (Sybron Endo, Orange, CA, USA) and a periapical radiograh taken with a size 15 K-le (Sybron
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Pulp regeneration

Fig 4. Clinical picture of the tooth where bleeding has been evoked by irritating the periapical tissues with a D11T instrument.

Fig 6. Radiographic image taken at the six-month follow-up appointment. Resolution of the periapical radiolucency and some closure of the apex are evident.

Fig 5. Radiographic image following placement of MTA to a level 3 mm below the cemento-enamel junction. The access cavity is lled with glass ionomer cement and composite resin. The radio-opaque material at the apical border is Cavit.

(Figs 2, 6, 7 and 8). The appearance of the mandibular left second premolar showed no obvious change in shade or colour (Fig 9). DISCUSSION The promise and potential of regenerative endodontic therapies in necrotic teeth was rst explored by
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Nygaard-stby in 1961 with limited success.20 Histological analysis demonstrated that a functional pulpdentine complex was not routinely induced. Current and future research into regenerative endodontics is focused on tissue engineering principles including root canal revascularization, postnatal stem cell therapy, pulp implantation, scaffold implantation, injectable scaffold delivery, three-dimensional cell printing and gene delivery.21 The challenge for the clinician is to recognize the potential of these new therapies and where appropriate incorporate them into everyday practice. The current report and others demonstrate that regenerative endodontics is a viable treatment option which has been described as a paradigm shift that allows for continued root development, a return of vitality and health in formerly necrotic immature teeth.10 Hargreaves et al. outline a number of common factors that are consistent findings and important observations in these reports.22 Firstly, the presence of a wide, open apex is most likely a physical requirement for tissue ingrowth. Secondly, because of the young age of the patients there may be a high stem cell regenerative potential. Thirdly, care is required not to instrument the canal walls and sodium hypochlorite has proved to be an effective irrigant. Fourthly, calcium hydroxide is not recommended as an intra-canal medicament.22 There is a concern that calcium hydroxide
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A Thomson and B Kahler

Fig 9. Clinical photograph of teeth in the posterior segment of the left mandible taken at the 18-month follow-up appointment. When compared to Fig 1 there is no obvious change in shade or colour.

Fig 7. Radiographic image taken at the 18-month follow-up appointment. Continued root maturation and resolution of the periapical radioluceny is evident.

Fig 8. Periapical lm taken at the 18-month follow-up appointment.

may kill viable pulp cells, including stem and progenitor cells in the apical papilla, considered crucial for further root maturation.4 In a study where calcium hydroxide was used, thickening of the dental wall only occurred apically to the level of where the medicament was placed.7 Fifthly, the use of a tri-antibiotic paste consisting of ciprooxacin, metronidazole and minocycline is effective for eliminating bacteria from the infected root canal system. Finally, initiation of a blood
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clot may act as a protein scaffold that allows tissue regeneration.22 Not listed by Hargreaves et al., but another consistent observation of these reports, is the use of MTA to complete the coronal seal which has known biological conductive properties to ensure an adequate coronal seal.23 While these case reports have been labelled as either pulpal revascularization and or regenerative endodontics with continued root development and apical closure, it is unknown what tissue has indeed been regenerated. Some authors have suggested that there may be remaining viable pulpal tissue in necrotic teeth that initiates the regenerative process.4,24 Indeed, the resilience of pulpal tissue survival in necrotic and or pulpless teeth has long been known.25,26 However, a recent study on dog teeth has shown that the ingrowth of tissue is more likely to originate from the periodontal ligament consisting of cementum, bone, and dentinelike material rather than pulp tissue.27,28 These authors suggest that there is approximately a 30% chance of pulp tissue re-entering the pulp space.28 Further histological research on dogs by this group has reported that the canal dentinal walls were thickened by the apposition of cementum-like tissue they termed intra-canal cementum.29 Despite these limitations of knowledge, these case reports show evidence of the success of regenerative endodontic procedures but the literature to date has only been low levels of evidence; case series and case reports. There have been only a few published reports that address success and failure of the regenerative procedure.5,7,8,30 Jung et al. examined the outcomes of
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Pulp regeneration nine teeth, five of which had some residual vital tissue so treatment consisted of irrigation with sodium hypochlorite and tri-antibiotic paste was placed as a medicament but no irritation to induce bleeding of the periapical tissues was performed.5 In the remaining four teeth, as no vital tissue could be discerned the protocol was the same except a blood clot was evoked. In all nine teeth, patients were asymptomatic with resolution of apical periodontis and draining sinuses when present over a 15 year follow-up period. However, in one of the teeth with no vital pulp remnants, there was no observable narrowing of the root canal space despite resolution of the apical periodontitis. Ding et al. examined 12 teeth that commenced regenerative endodontics that included irrigation with 5.25% sodium hypochlorite and medication with triantibiotic paste.8 Six patients dropped out of the study as a consequence of pain or failure to induce bleeding after canal disinfection and were treated with an apexication procedure with a MTA barrier technique. A further three patients failed to attend recall appointments. The remaining three patients exhibited complete root development with the teeth responsive to pulp sensibility testing. Cheuh et al. irrigated four teeth with 2.5% sodium hypochlorite but used calcium hydroxide placed over an induced blood clot without medication with tri-antibiotic paste.7 Whilst all four teeth demonstrated further root maturation and apical closure, these authors noted that this phenomenon only occurred apically to the calcium hydroxide and concluded that the use of calcium hydroxide was contraindicated as potential progenitor cells may be eliminated. Shah et al. attempted regenerative endodontics with a different protocol.27 Fourteen teeth were irrigated with 2.5% sodium hypochlorite and 3% hydrogen peroxide and formocresol was used as an inter-appointment medicament rather than tri-antibiotic paste. Over a follow-up period of 0.5 to 3.5 years, radiographic resolution was considered good to excellent in 93% (13 14) of teeth. Thickening of the dentinal walls was evident in 57% (8 14) of cases and increased root length was observed in 71% (10 14) of cases. There is an obvious need for randomized clinical trials and further studies to evaluate clinical outcomes and suggest a standardized approach. The American Association of Endodontists has commenced a database so that clinicians can supply details of their regenerative endodontic cases, evaluate the different approaches and determine guidelines that have optimal outcomes.31 Interestingly, ultrasonic irrigation is not generally reported in the listed case reports39 as ultrasonic agitation of the irrigant has been shown to enhance the cleaning and disinfection of the canal.32 In this report, ultrasonication was used during the second treatment session as the patient had experienced some discomfort which may have been related to the lack of mechanical
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preparation of the canal as outlined in the protocol. It is important to use ultrasonics with minimal endosonic ling to avoid damage to the canal walls. Ding et al. reported two cases where pain was experienced following placement of the antibiotic paste and the regenerative endodontic procedure abandoned in favour of traditional apexification techniques.8 In that report, endosonic irrigation was not performed but these authors suggested that this practice be recommended in future clinical trials. This report suggests this recommendation has merit as treatment successfully included ultrasonic activation of the irrigant. This case reported on a successful regenerative endodontic procedure utilizing a slight variation of the tri-antibiotic pasted developed by Hoshino and colleagues16 as amoxicillin was used instead of minocycline. In this case, there was no observable colour change that is often associated with minocycline. However, amoxicillin use would not be indicated in patients with known allergy to penicillin. An alternative approach to discolouration was taken by Reynolds et al.9 where the dentinal tubules of the crown were rst etched with 35% phosphoric acid (Ultra-Etch, Ultradent, South Jordan, UT, USA) and then sealed with SingleBond (3M, Minneapolis, MN, USA) and owable composite (PermaFlo DC, Ultradent, South Jordan, UT, USA) before placement of the tri-antibiotic paste. These authors also recommended backlling of the tri-antibiotic paste with a 20G needle to reduce the risk of coronal placement and potential discolouration.9 SUMMARY The immature teeth featured in the majority of the quoted case reports were either maxillary incisors where necrosis had developed following trauma or premolar teeth where the evaginatus had fractured, allowing bacterial invasion of the root canal system.39 This case report and others suggest that indeed there is a paradigm shift in the endodontic management of these teeth. Regenerative endodontic procedures allow for resolution of apical peridontitis and associated draining sinus tracts and continued root maturation. Furthermore, this may reduce the risk of root fracture associated with the thin roots in teeth treated by traditional apexication procedures. Therefore, it is important that dentists recognize the new protocols and the importance of not instrumenting the canal walls or applying medicaments such as calcium hydroxide which have been shown to be detrimental to the outcome. In some cases, the inability to generate bleeding or pain may necessitate the use of traditional apexication procedures, such as lling the apical third of the canal with MTA. This alternative therapy should be outlined to the patient and their guardians before embarking on regenerative endodontic procedures as
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A Thomson and B Kahler part of informed consent and advice on the inherent risks of the procedure. This case report described a successful regenerative endodontic procedure where amoxicillin was used instead of minocycline to reduce the risk of discolouration of the tooth. Further guidelines on optimal outcomes should be released by the American Association of Endodontists in the future as this and other case reports are evaluated. REFERENCES
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18. Kim ST, Abbott PV, McGinley P. The effects of Ledermix paste on discolouration of immature teeth. Int Endod J 2000;33:233 237. 19. Athanassiadis B, Abbott PV, Walsh LJ. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Aust Dent J 2007;52 (1 Suppl):S64 S82. 20. Nygaard-stby B. The role of the blood clot in endodontic therapy: an experimental histological study. Acta Odontol Scand 1961;79:333349. 21. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod 2007;33:377390. 22. Hargreaves KM, Geisler T, Henry M, Wang Y. Regeneration potential of the young permanent tooth: what does the future hold? J Endod 2008;34:S51S56. 23. Fischer EJ, Arens DE, Miller CH. Bacterial leakage of mineral trioxide aggregate as compared with zinc-free amalgam, intermediate restorative material, and Super-EBA as a root-end lling material. J Endod 1998;24:176179. 24. Huang GT, Sonoyama W, Liu Y, Liu H, Wang S, Shi S. The hidden treasure in apical papilla: the potential role in pulp dentine regeneration and bioroot engineering. J Endod 2008;34:645 651. 25. Heithersay GS. Stimulation of root formation in incompletely developed pulpless teeth. Oral Surg Oral Med Oral Pathol 1970;29:620630. 26. Lin L, Shovlin F, Skribner J, Langeland K. Pulp biopsies from the teeth associated with periapical radiolucency. J Endod 1984;10:436448. 27. Thibodeau B, Teixeira F, Yamauchi M, Caplan DJ, Trope M. Pulp revascularization of immature dog teeth with apical periodontitis. J Endod 2007;33:680689. 28. Ritter AL, Ritter AV, Murrah V, Sigurdsson A, Trope M. Pulp revascularization of replanted immature dog teeth after treatment with minocycline and doxycycline assessed by laser Doppler owmetry, radiography and histology. Dent Traumatol 2004;20:7584. 29. Wang X, Thibodeau B, Trope M, Lin LM, Huang GT. Histologic characterization of regenerated tissues in canal space after the revitalization revascularizaion procedure of immature dog teeth with apical periodontitis. J Endod 2010;36: 5663. 30. Shah N, Logani A, Bhaskar U, Aggarwal V. Efcacy of revasularization to induce apexication apexogenesis in infected, nonvital, immature teeth: a pilot clinical study. J Endod 2008;34: 919925. 31. American Association of Endodontists. Available from: http:// www.aae.org. 32. Gutarts R, Nusstein J, Reader A, Beck M. In vivo debridement efcacy of ultrasonic irrigation following hand-rotary instrumentation in human mandibular molars. J Endod 2005;31:166 170.

Address for correspondence: Dr Bill Kahler School of Dentistry The University of Queensland 200 Turbot Street Brisbane QLD 4000 Email: w.kahler@uq.edu.au
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