Case Report/Clinical Techniques

Drawbacks and Unfavorable Outcomes of Regenerative Endodontic Treatments of Necrotic Immature Teeth: A Literature Review and Report of a Case Ali Nosrat, DDS, MS,* Negar Homayounfar, DDS, MS,† and Kaveh Oloomi, DDS, MS‡

Abstract Introduction: Endodontic treatment of immature reatment of necrotic immature teeth is very challenging in . Immature necrotic teeth is challenging. Recently a biologically Troots are weak, short, and more susceptible to fracture. It is difficult to perform che- based treatment called regenerative endodontic treat- momechanical debridement and create an effective apical seal by using conventional ment was introduced. Although regenerative en- endodontic treatment methods (1). Historically, multiple-visit apexification was the treat- dodontic treatment causes root development, there ment of choice (2). Although this method was successful (3), it had several disadvantages are several drawbacks and unfavorable outcomes that including long-term treatment, increased root brittleness, and increased risk of should be addressed. This article describes regenerative root fracture because of long-term presence of calcium hydroxide inside the endodontic treatment of 2 maxillary central incisors with space (4). Apical barrier technique was introduced as a replacement for apexification poor root development outcomes. Methods: A healthy with calcium hydroxide (5). In the apical barrier technique a barrier material is placed 14-year-old female patient was referred. The patient at the apex to facilitate obturation procedure. Considering its sealing ability (6),biocom- had history of an impact trauma 6 years before the first patibility (7), hard-tissue induction potential (8), and the ability to set in the presence of visit. Clinical and radiographic examinations revealed moisture (9), mineral trioxide aggregate (MTA) is the material of choice for the apical that maxillary central incisors were immature and barrier technique. Clinical studies have shown high success rates of this method (10). necrotic with symptomatic apical periodontitis. After However, none of the aforementioned methods can promote root development. local anesthesia, rubber dam isolation, and access cavity Recently, a biologically based treatment called regenerative endodontic treatment preparation each tooth was irrigated with 20 mL of was introduced (11). This approach is based on the presence of osteo/odonto progen- NaOCl 5.25% and received triple antibiotic dressing itor stem cells in the apical papilla that are resistant to the infection and necrosis caused (ciprofloxacin, metronidazole, minocycline) for 4 weeks. by proximity to periodontal blood supply (12). In this treatment, the ideal goal is to In the next visit, after eliminating antibiotic dressing, prepare an appropriate environment inside the root canal space (ie, absence of bleeding was induced inside the canals, and then the bacteria and necrotic tissue, presence of a scaffold and a tight coronal seal) coronal thirds of the canals were sealed with mineral that promotes repopulation of these stem cells, regeneration of pulp tissue, and trioxide aggregate. A week later, both teeth were continuation of root development (13). The treatment procedure begins with chemical permanently restored. Results: In clinical and radio- disinfection by copious irrigation of the root canal space with NaOCl (14), combination graphic follow-ups, both teeth were functional, periapi- of NaOCl/chlorhexidine (15, 16) or NaOCl/hydrogen peroxide (17), followed by place- cal lesions were healed, and the apices formed. ment of an intracanal medicament at the first visit. Several medicaments like triple However, the roots were not developed. After 6 years, antibiotic mixture (metronidazole, ciprofloxacin, and minocycline) (18), calcium because of moderate discoloration and caries, both hydroxide (19), and formocresol (20) have been used successfully. At the next visit, teeth received root canal therapy and were permanently which should be at least 1 week after the initial session (21) or more (14), in the restored with casting dowel core and full crown restora- absence of clinical signs of inflammation, the clinician removes the intracanal medica- tions. Conclusions: Criteria for case selection in ment and induces bleeding inside the root canal space by irritating the periradicular regenerative endodontic treatments should be deter- tissue. After clot formation, the clinician seals the root canal space by placing an mined. (J Endod 2012;38:1428–1434) MTA plug over the blood clot (18). There are several case studies that demonstrate successful clinical and radiographic outcome for this treatment approach in single- Key Words rooted (15, 22) and molar teeth (23, 24). Dental trauma, immature teeth, MTA, regenerative However, there are several drawbacks and unfavorable outcomes that are not endodontics, root development, triple antibiotic paste, addressed yet. The purpose of this study was to present these drawbacks and their unfavorable outcome

From the *Iranian Center for Endodontic Research, Shahid Beheshti University of Medical Sciences, Tehran, Iran and the Department of Endodontics, Prosthodontics, and Operative Dentistry, School of Dentistry, University of Maryland, Baltimore, Maryland; †Department of Oral Biology, School of Dentistry, University of Washington, Seattle, Washington; and ‡Department of Endodontics, Dental School, Tehran University of Medical Sciences, Tehran, Iran. Address requests for reprints to Dr Negar Homayounfar, Graduate Student, Department of Oral Biology, School of Dentistry, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-7132. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.06.025

1428 Nosrat et al. JOE — Volume 38, Number 10, October 2012 Case Report/Clinical Techniques probable etiologies based on available case reports/series and animal Challenging Histologic Outcomes of Animal studies. These problems are as follows. Studies A few histologic and immmunohistologic studies have been Discoloration performed on the outcome of regenerative endodontic treatments in Discoloration of the tooth after regenerative endodontic treatments, dogs’ teeth (27, 30–33). Histologic evidence of hard-tissue deposition as revealed by Kim et al (25), is a problem mostly related to the use of on the root canal walls (43.9%), apical closure (54.9%), and formation minocycline in the triple antibiotic paste. They demonstrated that the of vital tissue in root canal space (29.3%) was demonstrated by Thibo- main reason for tooth discoloration after treatment was the contact of deau et al (33) in a dog model. They also evaluated the effect of blood minocycline in the triple antibiotic paste with coronal dentinal walls clot and a soluble collagen scaffold on the outcome. Histologic outcome during treatment procedure. A recent study suggested sealing dentinal of the treatment was not different in the presence or absence of blood walls of the access cavity by using dentin bonding agent and composite clot inside the root canal space. In addition, presence of soluble resin before placement of triple antibiotic paste inside the canal (26). collagen scaffold did not improve the results. A histologic study by da On the other hand, Kim et al examined the performance of this prevention Silva et al (27) revealed that the generated tissue inside the root canal technique for tooth discoloration. In this study teeth treated with dentin space after regenerative endodontic treatment was basically ingrowth of bonding were evaluated with naked eye and then with colorimeter. In the periodontal connective tissue instead of pulpal connective tissue. In eye assessment teeth did not have any change in color, but in the color- another study, histologic evaluation of the tissues produced inside the imeter assessment they had. They concluded that using dentin bonding root canal after regenerative endodontic treatment of dogs’ immature agents before placement of the triple antibiotic paste might not completely necrotic teeth revealed 3 types of tissues: cementum-like tissue that prevent tooth discoloration. A practical way to prevent discoloration is was responsible for increase in root length and thickness, bone-like replacing minocycline with an antibiotic that does not stain teeth. Thibo- tissue and periodontal ligament (PDL)–like tissue inside the canal deau and Trope (14) reported a successful regenerative endodontic space (30). There was only one case with partially survived pulp tissue treatment of a maxillary central incisor by using cefaclor instead of min- in which the presence of odontoblasts lining was seen. Formation of ocycline in the antibiotic mixture. Some efforts have been made to omit cemental bridges in different levels inside the canal was also demon- intracanal medication from the treatment procedure. Shin et al (16) pre- strated, which might be related to hard-tissue induction potential of sented a successful single-visit technique of regenerative endodontic MTA (30). In an effort to improve the root thickening and lengthening treatment. The technique used in this study consisted of irrigation of of treated immature necrotic teeth, Yamauchi et al (31) designed and the coronal portion of the root canal space with NaOCl and chlorhexidine evaluated a tissue engineering protocol including use of insoluble gluconate and then MTA placement without bleeding induction. A recent collagen sponge as a scaffold and ethylenediaminetetraacetic acid animal study on regenerative endodontic treatment introduced a new (EDTA) 17% as a demineralizing agent that could expose dentin matrix technique for root canal disinfection (27). They used NaOCl 2.5% and and possibly promote differentiation of mesenchymal cells and forma- apical negative pressure produced by EndoVac (Discus Dental, Culver tion of mineralized tissues. Outcomes revealed that the use of City, CA) system without antibiotic medication. Histologic evidence of cross-linked collagen significantly increased formation of mineralized successful treatment in this study demonstrated that the introduced tissues, and use of EDTA 17% significantly increased attachment of method of root canal disinfection is a promising protocol for immature newly formed mineralized tissues to the dentinal canal walls. Two forms necrotic teeth, and the use of triple antibiotic paste may not be necessary. of hard tissue were detected: dentin-associated mineralized tissues Both studies (16, 27) introduced novel methods to shorten the treatment (DAMT) that were adhered to or detached from dentinal walls, devoid period and also prevent tooth discoloration by omitting the intracanal of vasculature and cells; and bony islands (BI) that were in the inner medication process. lumen independent of dentinal walls and contained many embedded Although the main reason for discoloration after regenerative blood vessels, cells, and bone marrow–like tissues. In a separate study, endodontic treatments is minocycline (25), several studies revealed further histologic assessments and immunohistochemical analyses that gray MTA (26) and white MTA (15) can cause discoloration after were performed on DAMT and BI by the same group (32). Outcomes treatment. Discoloration after treatment of teeth that were treated with showed that DAMT was clearly different from dentin and bone and, to calcium hydroxide might be related to presence of MTA in cervical some extent, from cementum. Although lack of vasculature and immu- portion of the root canal space (28) (2 of 20 cases). A recent report nostaining patterns in DAMT resembled cementum, the organization on in anterior teeth revealed that presence of white and maturation of collagen fibers were significantly different (32). MTA in the crown can cause considerable discoloration (29). Immunoreactivity of dentin sialoprotein (DSP) and bone sialoprotein (BSP) in BI was similar to alveolar bone. No odontoblastic cell layer, dentin-like structure, and pulp-like tissue were detected (32). All in Treatment Period all, histologic findings of animal studies show that the tissue formed The required time for disinfection of the root canal space with inside the canal is not pulp and therefore does not function like pulp triple antibiotic paste or calcium hydroxide and increased number tissue. It means that this treatment procedure does not result in pulp of clinical sessions (compared with one-visit MTA apical barrier tech- regeneration in dogs’ teeth. However, the histologic outcomes of treat- nique) are other drawbacks of regenerative endodontic treatment. ment of human necrotic immature teeth might differ from that of dogs’ Elimination of antibiotic paste and bleeding induction should be teeth. Although the presence of stem cells has been demonstrated in done in the absence of clinical signs of inflammation (including apical papilla of human teeth (12) and in the blood clot formed inside pain, swelling, drainage from root canal, and presence of a sinus the root canal space after disinfection with triple antibiotic paste (34), tract) (18). The time spent on antibiotic disinfection in clinical none of the aforementioned studies have characterized these cells in studies done from 2004 varies between one (21) to eleven (14) dog’s apical papilla or in the blood clot formed inside the canals. On weeks. However, MTA apical barrier technique also should be done the other hand, if the tissue formed in immature human teeth after in more than one session in the presence of clinical signs of inflam- regenerative endodontic treatment is the pulp tissue, it should function mation (5). as a normal pulp. That means in cases with poor root development after

JOE — Volume 38, Number 10, October 2012 Regenerative Endodontic Treatments of Necrotic Immature Teeth 1429 Case Report/Clinical Techniques treatment there might be no vital tissue (35) or at least a different vital TABLE 1. Summary of Dental History of Reported Cases with Successful tissue inside the root canal space. However, positive responses to pulp Regenerative Endodontic Treatment and Continued Root Development from vitality tests including cold test (18, 24, 26) and electrical pulp test 2004 (EPT) (21, 36) in cases with continued root development after Dental regenerative endodontic treatments are indicative of re-innervation of Study Tooth no. history Duration the tissue inside the canal with nociceptors. These findings might be Banchs and 29 Swelling 1 month indicative of presence of pulp or pulp-like tissue inside the root canal Trope (18) space after regenerative treatment in cases with continued root develop- Chueh and 29 4 days ment. Histologic examination of the tissue formed inside the root canal Huang (22) space of one case with continued root development and positive Chueh and 20 Toothache 5 days Huang (22) response to EPT at follow-ups revealed presence of a pulp-like tissue Cotti et al (17) 8 Traumatic impact 1 month in this case (37). Reynolds 20 Pain and swelling 3 months et al (26) Shin et al (16) 29 Pain 3 days Poor Root Development Jung et al (52) 29 Swelling 2 months Jung et al (52) 29 Slight discomfort 1 month Ideal root development pattern in immature teeth includes Jung et al (52) 20 Throbbing pain 10 days increase in root length, increase in root wall thickness, and formation Jung et al (52) 20 Pain 3 months of the root apex. In some studies, the outcome of regenerative Jung et al (52) 20 Swelling 1 week endodontic treatments of necrotic immature teeth was lower than ideal, Jung et al (52) 29 Swelling 1 week Jung et al (52) 4 Pain 3 months including absence of increase in root length (15, 23), absence of Petrino et al (15) 8 Avulsion 6 months increase in root wall thickness (15, 28), or lack of formation Petrino et al (15) 9 Avulsion 6 months of tooth apex (28). Formation of a hard-tissue barrier inside the canal Torabinejad and 4 Accidental extraction 1 month between the coronal MTA plug and the root apex (28) is another Turman (36) Cehreli et al (39) 8 Extrusive luxation 1 week reported unfavorable outcome. A recent study revealed that root devel- Cehreli et al (39) 9 Extrusive luxation 1 week opment potential of immature necrotic teeth is related to the vitality of Hertwig epithelial root sheath (28). Therefore, there might be a corre- lation between dental history and quality of root development; the which the dental history of the patients did not show the duration of longer the duration of pulp necrosis, the lower the quality of root devel- chief complaint or the approximate time of the pulp necrosis before opment after regenerative endodontic treatments. Such an association treatment (14, 19, 21, 23–25, 28). This subject deserves further could be found in studies that reported decreased or no root develop- studies to determine case selection criteria based on patient’s dental ment. In a case series study by Petrino et al (15), a case of 2 traumatized history. necrotic immature maxillary central incisors (case 1) had a history of A retrospective evaluation of radiographic outcomes discovered impact trauma 6 years before initial visit. One year after treatment, that regenerative endodontic treatment with triple antibiotic dressing the apex of the left central incisor was blunt and closed, and the root increased root wall thickness significantly more than either calcium length did not increase. The right central incisor did not show any hydroxide or formocresol (38). In addition, this study revealed that sign of root development. Lenzi and Trope (35) reported regenerative in cases disinfected with calcium hydroxide, the radiographic location endodontic treatment in 2 traumatized necrotic immature maxillary of calcium hydroxide inside the root canal space influenced the root central incisors. The patient had a history of impact trauma 2.5 months development. When calcium hydroxide was radiographically limited earlier. On the basis of the severity of immaturity, they found that left to coronal half of the root canal space, the increase in root wall thick- central incisor had been traumatized long before the right one. ness was greater than when it was placed beyond coronal half. Twenty-one months after treatment, the left central incisor did not indi- cate any sign of root development, and just a radiopaque hard-tissue barrier formed at the apex. Meanwhile, the right one showed signs of Insufficient Bleeding successful treatment and full root development. The authors discussed Some authors have reported failure to induce bleeding (21, 23, the possibility that longstanding infection might destroy the cells capable 24). A recent study revealed that mesenchymal stem cells are of pulp regeneration. However, on the basis of successful outcomes of delivered into root canal space after bleeding induction in human regenerative endodontic treatments in cases with long-lasting apical teeth, a phenomenon that did not happen in the absence of blood periodontitis, they concluded that this might not be the reason. We clot inside the disinfected root canal space (34). In addition, it is reviewed the available history of all reported cases of successful regen- assumed that the blood clot formed inside the root canal space after erative endodontic treatment with continued root development done disinfection contains platelet-derived growth factors and serves as from 2004 to establish a probable relationship between dental history a protein-rich scaffold (13). An animal study demonstrated that root and the quality of root development (Table 1). This information shows canals that had a blood clot formation inside them after disinfection that history of pulp necrosis in cases with successful treatment is no had better radiographic outcome compared with those without blood longer than 6 months (mean, 47.8 days), which is much shorter clot (33). To facilitate bleeding after root canal disinfection, using local than 6 years. Therefore, there might be a relationship between the anesthetics without vasoconstrictors is recommended (15). However, duration of pulp necrosis and outcome of treatment. However, this lack of bleeding or insufficient bleeding after using plain local anes- information shows the time between initiation of patient’s symptoms thetics is reported (15, 23, 24), which has been shown to be related and treatment or the time between traumatic injury and treatment. to poor root development in some cases (23). On the other hand, There is a possibility that pulp necrosis occurs before initiation of several cases with sufficient bleeding and lack of root development patient’s symptoms (in cases with pain or swelling) or after traumatic have been reported (15, 35). One study assumed that there was a impacts (Table 1). In addition, there were several reports of successful possibility that blood clot broke down and left the root canal regenerative endodontic treatment with continued root development in space without scaffold into which the new vital tissue could

1430 Nosrat et al. JOE — Volume 38, Number 10, October 2012 Case Report/Clinical Techniques grow (35). In addition, there are several reports of successful regener- mentioned studies. In addition, Cehreli et al (24) followed their cases ative endodontic treatment and continued root development without for 9–10 months. The complete root canal calcification/obliteration has bleeding induction (16, 19, 22). A recent case report suggested use occurred at least within an average of 16 months after initial treatment in of platelet-rich plasma instead of blood clot inside the root canal space aforementioned studies. Therefore, the follow-up period of the study by (36). Interestingly, the histologic evaluation of the soft tissue produced Cehreli et al (24) might not be enough for a conclusion in this regard. inside the canal revealed presence of pulp-like tissue (37). Therefore, On the other hand, there is no report of complete root canal calcifica- this subject is controversial and should be studied more. tion/obliteration in cases disinfected by triple antibiotic paste. Although complete root canal calcification/obliteration is not mentioned as Root Canal Calcification/Obliteration a failure in cases that have undergone regenerative endodontic treat- Root canal calcification/obliteration is another problem after ment, it can cause serious challenges in case the involved tooth needs regenerative endodontic treatment of necrotic immature teeth that is re- root canal therapy. ported in cases disinfected by calcium hydroxide (19, 22, 28). In a case There are other issues that should be addressed. Randomized clin- series study by Chen et al (28) complete root canal calcification/oblit- ical trials comparing the long-term success of this new treatment with eration happened in 4 of 20 cases within an average follow-up time of 16 traditional ones, especially MTA apical plug techniques, have not months. Although it is stated that the maximum interval of calcium been performed. In addition, the criteria for case selection and criteria hydroxide therapy was 4 weeks in this study, there is not any information of success/failure have not been determined. Besides, there is a study about the duration of calcium hydroxide therapy in each case. A retro- reporting the continuation of symptoms after disinfection procedure spective study by Chueh et al (19) on 23 immature necrotic teeth treated with NaOCl 5.25% and triple antibiotic dressing (2 patients out of with calcium hydroxide revealed complete root canal obliteration in 2 12) that caused changes in treatment planning (21). cases within 17 and 59 months after initial treatment. The duration of calcium hydroxide therapy was 9 and 6 months, respectively. In addi- Case Report tion, other 21 teeth showed partial obliteration of the root canal space A healthy 14-year-old female patient was referred to the Endodontic in comparison with adjacent normal teeth. In a study by Chueh and Department of the Dental School, Tehran University of Medical Sciences Huang (22) on 4 cases of regenerative endodontic treatment, complete in 2005. As stated by her legal guardians, the patient’s chief complaints root canal calcification occurred in one case within 34 months (18.5 were repeated swelling and pain in the anterior maxilla within past few months of calcium hydroxide therapy), and severe narrowing of the months before initial visit and crown fracture of the anterior maxillary root canal space occurred in another case within 5 years. However, teeth. They reported a history of impact trauma to the anterior maxillary there were 3 studies by Cotti et al (17) (1 central incisor), Cehreli teeth 6 years before initial visit. Clinical examinations revealed extensive et al (24) (6 molars), and Cehreli et al (39) (2 central incisors) in caries of tooth #8, complicated crown fracture of tooth #8, and uncom- which calcium hydroxide was used as intracanal medicament, and no plicated fracture of tooth #9. Both teeth showed normal mobility. Cold root canal calcification/obliteration was reported. The duration of test by using Endo-Frost cold spray (Roeko; Coltene Whaledent, Lange- calcium hydroxide therapy in these 3 studies was 2, 3, and 3 weeks, nau, Germany) did not elicit any response in maxillary central incisors, respectively, which was much shorter than the same period in afore- whereas maxillary lateral incisors responded normally to the test. Both

Figure 1. (A) Preoperative periapical radiograph of teeth #8 and #9. Note periapical radiolucent lesions and immaturity of roots. (B) After regenerative endodontic treatment and permanent coronal restoration. (C) 6-year follow-up. The periapical lesions healed, and root apices formed without increase in length and thickness of the roots. Note the mesial coronal caries in tooth #9. (D) Immediately after root canal therapy. Note several sealer pathways at apex of tooth #9. (E) After casting dowel core and full crown restoration of both teeth.

JOE — Volume 38, Number 10, October 2012 Regenerative Endodontic Treatments of Necrotic Immature Teeth 1431 Case Report/Clinical Techniques maxillary central incisors were sensitive to percussion and palpation. a sterile amalgam carrier, approximately 3 mm of MTA was placed in There was no palpable periapical swelling on the maxillary central inci- the coronal third of the canals. MTA was gently adapted to the dentinal sors at the time of examination. Both maxillary central incisors were walls with a moistened cotton pellet. The access cavities were filled with immature with visible periapical radiolucent lesions in the radiographs saline, a moistened cotton pellet was placed inside the access cavities, (Figs. 1A and 2A). Considering the history of impact trauma and outcome and both teeth were temporarily restored. A week later the patient of the clinical and radiographic examinations, the concluding diagnosis was referred for permanent restoration of the teeth (Figs. 1B and 2B). for both central incisors was pulp necrosis with symptomatic periradic- The patient was recalled yearly for clinical and radiographic ular periodontitis. Taking into consideration that both teeth were imma- follow-ups. At follow-up sessions both maxillary central incisors were ture and necrotic, the treatment of choice was regenerative endodontic asymptomatic and functional. No recurrence of swelling was reported treatment. After explaining the treatment procedure, risks, and benefits by the patient. The teeth were not sensitive to percussion and palpation. to the patient’s legal guardians, a written consent was obtained. The response to the cold test was negative in all follow-up sessions. In After local anesthesia with 3% plain mepivacaine (Septodont, radiographic examinations the radiolucent lesions healed, and the Cedex, France) and rubber dam isolation, caries in tooth #8 was apices formed. However, there was no increase in the length and thick- removed, and access cavities on teeth #8 and #9 were prepared by using ness of the roots (Fig. 1C). a diamond-coated fissure bur (Diatech, Heerbrugg, Switzerland) and Six years after initial treatment the patient complained about the a high-speed handpiece. Each root canal was passively irrigated with appearance of her maxillary central incisors. Clinical examinations 20 mL NaOCl 5.25% without instrumentation. Canals were gently dried revealed moderate discoloration of both teeth and deep mesial caries with paper points (Ariadent, Tehran, Iran). Equal powdered propor- in maxillary left central incisor (Figs. 1C and 2C and D). In consulta- tions of metronidazole (ParsDaru, Tehran, Iran), ciprofloxacin tion with Department of Prosthodontics, because of the amount of re- (AminDaru, Tehran, Iran), and minocycline (Razak, Tehran, Iran) maining tooth structure, severity of discoloration, patient demands and were mixed with saline, the creamy paste was placed inside the canals expectations, and to get the ideal esthetic and better prognosis, full by using counterclockwise motion of a size 50 K-file (Dentsply Maillefer, crown restoration for both teeth was suggested. Therefore, the patient Tulsa, OK), and the teeth were restored temporarily with Cavite was scheduled for root canal therapy of both central incisors. The (Asia Chemi Teb Co, Tehran, Iran). Four weeks later, the patient was same clinician performed the root canal therapy. After local anesthesia asymptomatic, and none of the maxillary central incisors were sensitive and rubber dam isolation, restorative materials and caries were to percussion and palpation. Under rubber dam isolation and local removed, and access cavities were prepared by using a diamond- anesthesia with 3% plain mepivacaine, the temporary restorations coated bur and high-speed handpiece. The MTA barriers were hard were removed, and the antibiotic paste was eliminated by using copious and intact. They were removed by using a Cavitron ultrasonic scaler irrigation with NaOCl 5.25%. A sterile size 40 K-file was overextended (Dentsply International, York, PA) with copious water irrigation. beyond the root length several times in both teeth to irritate periradicular Both root canals were empty, and no vital tissue or bleeding was tissues and initiate bleeding. The blood could be seen on the apical part seen. Root canal preparation was performed by using hand instrumen- of the file, but not inside the canals. After 10 minutes, MTA powder (Pro- tation with K-files sizes 45, 50, 55, and 60, with copious NaOCl 1.25% Root tooth-colored MTA; Dentsply Tulsa Dental, Tulsa, OK) and distilled irrigation between instruments. The canals were dried with paper water were mixed according to manufacturer’s instructions. By using points. The apical thirds of the root canals were obturated with

Figure 2. (A) Preoperative photograph of the central maxillary incisors of a 14-year-old girl. The patient had a history of impact trauma. Note the fracture of the incisal edges in both teeth and the caries lesion in the right central maxillary incisor. (B) View after revascularization and permanent coronal restoration. (C) Facial view of both teeth 6 years after initial visit. Note the mesial caries in the left central maxillary incisor and the discoloration of both teeth. (D) Palatal view 6 years after initial visit. Note the discoloration.

1432 Nosrat et al. JOE — Volume 38, Number 10, October 2012 Case Report/Clinical Techniques warm vertical compaction of size 70 gutta-percha (Ariadent, Tehran, 1 month of triple antibiotic dressing) renders the root canal space Iran) and sealer (Pulp Canal Sealer, Kerr, MI) by using preheated sterile or has a long-lasting effect suppressing the regrowth of bacteria pluggers (M-series; Dentsply Maillefer). The radiograph after obtura- over time. The follow-up time in that study was 21 months. The pre- tion revealed several pathways of sealer extrusion in the apical area of sented case showed that empty root canal spaces remained bacteria the left maxillary central incisor (Fig. 1D). After root canal therapy free for 6 years, which confirms the conclusion of Lenzi and Trope. both teeth were temporarily restored, and the patient was referred Tight coronal seal is an important component of successful regen- to the prosthodontist for coronal restorations. Finally, both teeth erative endodontic treatment (13, 18). Most of the studies have used were restored with casting dowel core and full crown (Fig. 1E). a double seal consisting of MTA and a permanent resin restoration over the blood clot (15, 18, 26), similar to what was done in the presented case. The calcium ion released from MTA reacts with Discussion environmental phosphorus. The reaction leads to formation of A recent clinical study on the responses of necrotic immature hydroxyapatite crystals on the surface of MTA (50) and MTA-dentin permanent teeth to regenerative endodontic treatment revealed that interface (51). It is hypothesized that this bioactive reaction is respon- there would be 5 types of root development pattern based on sible for MTA’s sealing ability and biocompatibility (50, 51). Sealing survival of Hertwig epithelial root sheath (28). As stated in this ability of MTA against bacterial leakage is well-documented (6). The study, type 2 is ‘‘no significant continuation of root development presented case shows that MTA might maintain its sealing ability even with the root apex becoming blunt and closed’’. In the presented after the seal of the coronal permanent restoration is lost. However, case, after treatment, root apices of both central incisors formed presence or absence of leakage around MTA barriers could not be without any significant increase in the root length and root wall shown by clinical or radiographic examinations. thickness. Although the disinfection method used in this study was The importance of presence of blood clot inside the root canal different, this finding was almost comparable with type 2 develop- space as a protein-rich scaffold that contains platelet-derived growth- mental pattern described by Chen et al (28) (Fig. 1). However, factors and mesenchymal stem cells has been shown before (33, all developmental patterns described in that study showed signs 34). In addition, it has been demonstrated that insufficient bleeding of root wall thickening, which was not seen in the presented might be related to poor root development after regenerative case. Therefore, the radiographic findings of this case were similar endodontic treatment (23). Therefore, the absence of vital tissue inside to findings of an apexification treatment, the same as that reported the root canal spaces of both central incisors and lack of root develop- by Lenzi and Trope (35). In the present case, passage of a long time ment in this case might be partly related to insufficient bleeding and lack (6 years) without any treatment after traumatic impact might be of scaffold inside the root canals. As stated in previous studies, bleeding related to damaged Hertwig epithelial root sheath and, subsequently, induction is part of the treatment procedure and should be encouraged decreased root development potential. in cases without bleeding (15). Therefore, the presented case should be Root canal disinfection in regenerative endodontic treatments is considered as a deviation of the accepted technique. However, absence a challenge. Although in the traditional endodontic treatments, lowering of blood inside the canal space was confirmed by naked eye in the pre- of the bacterial loads and prevention of bacterial access to the periapical sented case. Using magnification in such cases helps the clinician to tissues might be conducive to healing, in pulp regeneration a higher evaluate the situation more accurately. level of disinfection is required (40). Bacterial cells are smaller in diam- A recent case report showed that the tissue formed inside eter compared with the pulpal end of dentinal tubules and therefore can the root canal space of human teeth after regenerative endodontic treat- penetrate deep into the dentinal tubules of infected teeth (41). More- ment is a pulp-like tissue (37). However, the information about the over, in the younger teeth bacteria penetrate through more dentinal hard tissue produced on dentinal walls and the cells responsible for tubules and advance deeper in comparison with older ones (42). hard-tissue production is still lacking. An animal study revealed that Although NaOCl is an effective irrigant to reduce bacterial loads, it a cementum-like tissue was deposited on the root canal dentinal walls cannot render the infected root canal space bacteria free (43), which after regenerative endodontic treatment. This tissue was irregular and would be an ideal condition for pulp regeneration. Two in vitro studies was assumed to be responsible for root development (30). In the by Hoshino et al (44) and Sato et al (45) revealed that the mixture of present case, the radiographic view of several sealer extrusion pathways ciprofloxacin, metronidazole, and minocycline was effective against in the apical region of the left maxillary central incisor might indicate endodontic pathogens and was able to deeply disinfect the infected that the hard tissue formed in the apical region is porous and irregular. dentinal tubules. Animal studies by Windley et al (46) and Cohenca In addition, although the treatment procedure was the same for both et al (47) revealed that irrigation with NaOCl followed by triple antibiotic teeth, radiographic view of the apex formed in the right central incisor paste dressing renders 70%–78% of the canals culture negative. On the was more uniform, and there were not any sealer extrusion pathways. other hand, culturing bacteria after antibiotic dressing is a problematic A case series study on regenerative endodontic treatment revealed issue. Bacteria may remain viable but uncultivable, and also it is difficult that a mean follow-up period of 8 months for evaluation of osseous to inactivate antibiotics in the culture medium (48). Meanwhile, several healing and 16 months for evaluation of root development are necessary successful cases have been reported that used the similar disinfection (19). There are several studies on this new treatment that have followed regimen (14, 23), the same as we used in the presented case. The their cases more than 16 months (18, 22, 26), but also there are lots of effect of disinfectants on the root canal dentinal walls is another issue studies with shorter follow-up periods (15, 24, 25). Except for one that should be addressed. Ring et al (49) demonstrated that irrigation study in which calcium hydroxide was used for root canal with NaOCl or chlorhexidine can interfere with the attachment of dental disinfection (22), none of the prospective clinical studies followed pulp stem cells to dentinal walls. the cases beyond the time of completion of the root development, Lenzi and Trope (35) demonstrated that healing of the periapical and there is no information about what happened after completion of lesion and production of hard-tissue barrier in the apex after regener- apical closure. The presented case is unique in this regard. ative endodontic treatment can be seen even in the absence of vital tissue The present review and poor outcomes of the reported case show inside the root canal space. They concluded that disinfection protocol in the importance of determining criteria for case selection in regenerative regenerative endodontic treatments (NaOCl irrigation followed by endodontic treatments. The ideal goal of this new treatment is pulp

JOE — Volume 38, Number 10, October 2012 Regenerative Endodontic Treatments of Necrotic Immature Teeth 1433 Case Report/Clinical Techniques regeneration inside the root canal space and, consequently, root devel- 24. Cehreli ZC, Isbitiren B, Sara S, Erbas G. Regenerative endodontic treatment (revas- opment. Healing of the periradicular disease has occurred in all cases cularization) of immature necrotic molars medicated with calcium hydroxide: with complete treatment, which shows the predictability of the disinfec- a case series. J Endod 2011;37:1327–30. 25. Kim J, Kim Y, Shin S, Park J, Jung I. Tooth discoloration of immature permanent tion protocols. Randomized clinical trials that compare long-term incisor associated with triple antibiotic therapy: a case report. J Endod 2010;36: outcomes of this new approach with the traditional ones, specifically 1086–91. MTA apical barrier technique, are recommended. 26. Reynolds K, Johnson J, Cohenca N. Pulp revascularization of necrotic bilateral bicus- pids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J 2009;42:84–92. 27. da Silva L, Nelson-Filho P, da Silva R, et al. Revascularization and periapical repair Acknowledgments after endodontic treatment using apical negative pressure irrigation versus conven- The authors thank Foad Iranmanesh, DDS, MS and Hossein tional irrigation plus triantibiotic intracanal dressing in dogs’ teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:779–87. Ghasemkhani, MS, PhD for their help and technical support and 28. Chen MY, Chen KL, Chen CA, Tayebaty F, Rosenberg PA, Lin LM. Responses of imma- Mrs Mona Ansari for managing the patient and follow-ups. ture permanent teeth with infected necrotic pulp tissue and apical periodontitis/ The authors deny any conflicts of interest related to this study. abscess to revascularization procedures. Int Endod J 2012;45:294–305. 29. Belobrov I, Parashos P. Treatment of tooth discoloration after the use of white mineral trioxide aggregate. J Endod 2011;37:1017–20. 30. Wang X, Thibodeau B, Trope M, Lin L, Huang G. Histologic characterization of re- References generated tissues in canal space after the revitalization/revascularization procedure 1. Waterhouse P, Whitworth J, Camp J, Fuks A. Pediatric endodontics: endodontic of immature dog teeth with apical periodontitis. J Endod 2010;36:56–63. treatment for the primary and young permanent dentition. In: Hargreaves K, 31. Yamauchi N, Yamauchi S, Nagaoka H, et al. Tissue engineering strategies for imma- Cohen S, eds. Pathways of the pulp. 10th ed. St Louis: Mosby Elsevier; 2011:808–57. ture teeth with apical periodontitis. J Endod 2011;37:390–7. 2. Rafter M. Apexification: a review. Dent Traumatol 2005;21:1–8. 32. Yamauchi N, Nagaoka H, Yamauchi S, Teixeira FB, Miguez P, Yamauchi M. Immu- 3. Sheehy E, Roberts G. 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