Ⅰ. INTRODUCTION

Regenerative endodontic treatment (RET) refers to procedure in which bleeding from apical tissues is evoked to induce migration of mesenchymal stem cells to the canal space. The blood clot serves as a scaffold and provides growth factors, which overall contribute to regeneration of pulp/dentin complex. (1, 2) This technique of revascularization was first reported in the work of Ostby in 1961 (3), in which evoked bleeding of immature permanent teeth led to ingression of vascular tissue in the canal space. Since then, numerous approaches have been developed to revascularize necrotic teeth as alternatives to conventional root canal treatment or apexification, and they have demonstrated positive results in clinical practices (4-7).

In addition to resolving apical periodontitis, RET also allows for continued root formation by increasing thickness or length of the root, and inducing apical closure. Therefore, it enhances mechanical strengths of the immature tooth and makes it more resistant to fracture (8). Moreover, RET contributes to restoring neurovascular system of the root canals and further, may revitalize the tooth. Innate immunity of root canals is obtained by recovery of vascular system which serves as primary defense against microbial invasion and reduces likelihood of reinfections. Furthermore, restoration of nerve and sensory function provides warning system for the tooth, in the event of tissue damages (9, 10).

RET has mainly been performed in immature permanent teeth due to larger apical sizes, which facilitate stem cells to migrate into the canal space. Stem cells from apical papilla (SCAPs), known for their potential to survive throughout pulp necrosis, also serve as a source of pulp and dentin regeneration in immature teeth (11, 12). Mature permanent teeth on the other hand, have constricted apical sizes and complicated canal anatomy that poses challenge to disinfection (9, 13). Moreover, age-related decline in stem cell proliferation and migration attribute to less regenerative potential in adult patients (14), which overall may reduce success rate of RET in mature teeth.

In recent years, however, RET has been suggested as a viable treatment option for mature teeth, as demonstrated by clinical cases reporting the healing of apical periodontitis through RET (9, 15-17). In particular, histologic study by Arslan et al. (18) revealed that mixture of connective tissue involving fibrous, osseous and vascular tissues were found in mature teeth that underwent RET. It demonstrated the feasibility of RET to be performed in mature teeth with fully developed apex, presenting histologic evidence of tissue revascularization. The present case report describes RET of previously treated mature permanent tooth, with a 3 year follow-up result showing complete resolution of a large periapical lesion.

Ⅱ. Case Report

A 34-year-old female patient visited the department of conservative dentistry of Seoul National University Dental Hospital, with discomfort in her maxillary lateral incisor (#22). The patient had trauma history of #22 and had received endodontic treatment 15 years ago. However, due to recurred swelling and pain of the tooth, she received nonsurgical endodontic re-treatment 6 months ago at a local dental clinic, but was notified that the prognosis would be poor and extraction may be necessary. At the time of the visit to our department, she had no pain but complained of intermittent swelling of gingiva around #22. She had no contributory medical history.

Upon visual inspection, she had no significant signs of pathosis extra-orally or intra-orally, without any swelling or lymphadenopathy. The tooth had no response to percussion and bite test, and probing depths were within normal limits of 3-4mm. However, the tooth was sensitive to palpation and showed class I mobility. Moreover, periapical radiograph examinations showed overextended gutta percha protruding approximately 5mm beyond apical foramen of #22 and periapical radiolucency of 10 X 8mm in diameter, encompassing entire apical root of #22 and the overextended gutta percha (Fig. 1A). Axial and sagittal view of cone-beam computed tomographic (CBCT) imaging revealed that periapical lesion of tooth #22 had invaded the palatal cortical bone, resulting in palatal plate perforation (Fig. 1B and C). Based on radiographic and clinical examinations, tooth #22 was diagnosed as “previously treated” and “symptomatic apical periodontitis.”

Procedure of regenerative endodontic treatment and possibility of surgical endodontic retreatment were explained to the patient and informed consent was obtained. Without the administration of local anesthesia, #22 was isolated with rubber dam, and all subsequent steps were performed under a surgical microscope (Carl Zeiss Meditac Inc, Dublin, CA). Access cavity was opened and old gutta percha was removed with barbed broach. Periapical radiograph was taken to confirm the removal of old filling material but it was identified that segment of gutta percha measuring about 5mm, extending beyond the root apex of #22 was remaining (Fig. 2A). To avoid damaging the dentinal structures of root apex, further attempts to remove the remaining gutta percha were not made. After working length of 21mm was determined by radiograph, canal was copiously irrigated with 20mL of 2.5% sodium hypochlorite, 10mL of sterile saline, 20mL of 17% EDTA and dried with paper points. Canal bleeding was induced by passing #20 K-file approximately 3mm beyond the apex, and it was verified under surgical microscope that the bleeding was filled up to 3mm beneath cemento-enamel junction (CEJ). Blood clot formed after 15 minutes and paste of calcium hydroxide (CleaniCal; Maruchi, Wonju, Korea) was applied above the blood clot. The tooth was temporized with cotton pellet and Caviton (GC, Tokyo, Japan) (Fig. 2B).

At 3-week follow-up (2^nd treatment visit, Table 1), with the patient reporting no symptoms and clinical examinations of #22 within normal limits, longer-term follow-up was planned. After isolation of #22 with rubber dam, Caviton was replaced with resin-modified glass ionomer cement (RMGI, Fuji II LC; GC, Tokyo, Japan) in order to enhance strength and sealing ability of the temporary restoration. After 3 months (3^rd treatment visit), the patient had no discomfort and tooth #22 was asymptomatic. Radiographic examinations revealed reduced radiolucency (Fig. 2C) and further follow-ups were scheduled without any intervention.

At sequential follow-ups at 6 months (4^th treatment visit) and 9 months (5^th treatment visit) after initial treatment, periapical radiographs showed evident resolution of periapical radiolucency (Fig. 2D and E). Therefore, at 5^th treatment visit RMGI temporary restoration was removed and canal medicament of calcium hydroxide was removed with 30mL of sterile saline. Bleeding from canal was observed when canal was dried with paper points. Blood clot formation was identified under surgical microscope and mineral trioxide aggregate (MTA; Dentsply Tulsa Dental, Tulsa, OK) was placed over the blood clot up to 3mm beneath CEJ of tooth #22 (Fig. 2F). The tooth was temporized with cotton pellet and Caviton, and was restored with RMGI and composite resin (Filtek Z250; 3M ESPE) at subsequent visits (6^th treatment visit).

At 18 months (7^th treatment visit) and 24 months (8^th treatment visit) follow-ups after initial treatment, the tooth remained asymptomatic and radiographic examinations indicated substantial recovery of the periapical lesion, with the exception of apical periodontal space widening (Fig. 3A and B). However, the tooth did not respond to either thermal or electric pulp test (EPT). During the 9^th radiographic evaluations which was 36 months after the initial treatment, complete healing of periapical radiolucency was observed with intact lamina dura and periodontal ligament (PDL) space around apex of tooth #22 (Fig. 3C). Negative response to the vitality test remained. Table 1 presents summary of dates, treatments and radiographic views of each visit of the patient.

Ⅲ. Discussion

Upon the patient’s initial visit to our department, several treatment options were considered. The CBCT images of #22 revealed palatal plate perforation, posing an impediment to a surgical approach. Given that apical surgeries are typically conducted through a buccal approach, additional bone reduction on the buccal cortical bone, in conjunction with the pre-existing palatal bone perforation, would potentially cause further harm to the patient. Moreover, since the patient had already undergone conventional endodontic retreatment for tooth #22, 6 months before visiting our department, it was necessary to prioritize considering an alternative approach over another conventional retreatment. Relatively large apical size of #22 observed in the preoperative periapical radiographs allowed for the consideration of RET procedure. Considering that typical mesio- distal size of maxillary lateral incisor apical foramen is 0.3mm (19), an apical size measurement of approximately 0.5mm appeared to be a feasible case for RET. Furthermore, the presence of gutta percha extending 5mm beyond the apex of #22 introduced additional risks to the compaction process during canal obturation. When filling the canal to its full length, there existed a potential for remaining gutta percha to be pushed further beyond the apex, into the alveolar bone. Hence, risks and benefits of RET procedure were evaluated and treatment plan was established with the patient’s consent.

RET has been primarily performed in immature teeth due to their higher likelihood for pulp regeneration (15). SCAP, defined as group of mesenchymal cells located in the apical papilla of immature teeth, is considered as a main source for primary odontoblasts and hence principal supplier for pulp regeneration (20, 21). In fully developed teeth, the absence of SCAP is replaced by dental pulp stem cells (DPSCs), alveolar bone stem cells or periodontal ligament (PDL) stem cells, which reside outside the root apex (22, 23). The concept of cell homing is employed to describe recruitment and differentiation of progenitor cells to desired sites of the tissue to differentiate into odontoblasts or pulp fibroblasts. Because cell homing does not require cell transplantation, it is also referred to as the cell-free approach, and is prompted by exogenous signaling molecules or endogenous growth factors. Therefore, in the case of RET in mature teeth, apical bleeding is mandatory for both migration of stem cells from outside the root apex and the release of the biologic cues (24-26). In the current case, apical bleeding was evoked twice; first before application of calcium hydroxide and then before application of MTA. At 9-month follow up and 2-year follow up after application of calcium hydroxide and MTA over blood clot, respectively, the periapical lesion gradually decreased. This suggests that innate immunity induced by revascularization prevented further infection of the root canals. It is noteworthy that, while induction of bleeding is omitted in certain instances of RET for immature teeth (27, 28), it is mandatory in mature teeth.

Apical diameter is another consideration when performing RET in mature teeth with closed apex. Traditionally it was considered that open apex with wider apical diameter allows for growth factors and stem cells to be delivered and activated through apical foramen (24, 29). However, recent studies have presented contradictory results suggesting that apical foramen sizes smaller than 1mm do not hinder the success of RET. Histologic study in in vivo canine model by Laureys et al. (30) demonstrated that apical sizes smaller than 1mm, particularly the size of 0.32mm, did not impede revascularization and ingrowth of vital tissue after autotransplantation, indicating that the size of apical foramen may not be a key element for the regeneration of pulp tissue. A review study by Fang et al. (31) also reported that apical diameters less than 1mm exhibited clinical success, with the specific range between 0.5mm and 1mm achieving the highest success rate. In the present case, the radiographic measurement indicated an apical foramen width of approximately 0.5mm, and the successful outcome of RET aligns with the findings of the aforementioned studies, suggesting that apical diameter may not be a critical factor. However, the limitation to this study lies in the negative response of #22 to the vitality test. Although this might be attributed to MTA restoration occupying large coronal portion of the canal, the uncertainty regarding whether actual revitalization occurred still remains.

Conventional RET protocol involves application of calcium silicate cement such as mineral trioxide aggregate (MTA) as a capping material over the blood clot. This is due to its bioactivity of releasing growth factors from dentin matrix to induce odontogenesis of mesenchymal cells and mineralization (10, 32, 33). However, as a downside of its great sealing ability, calcium silicate is difficult to remove once hardened. In the present case, calcium hydroxide was applied as a capping material on the top of the blood clot for 9 months due to the possibility of an unfavorable outcome. In the event of undesirable consequences, calcium hydroxide can be easily removed and conventional endodontic retreatment can be performed. Advantages of calcium hydroxide also involve providing an antimicrobial environment during the migration of mesenchymal stem cells without damaging the cells. According to in vitro study by Ruparel et al (34), calcium hydroxide did not damage human mesenchymal stem cells within the therapeutic concentration range of 0.01mg/mL to 100mg/mL. Case report by Paryni & Kim (9) also reported a reduction in apical lesion when applying collagen membrane dusted with ciprofloxacin powder during RET procedure. The authors inferred that low concentrations of ciprofloxacin had prevented migrated stem cells from being contaminated by residual bacteria in the root canal. Moreover, calcium hydroxide has been demonstrated to solubilize and release growth factors and bioactive molecules embedded in dentin matrix, and hence contribute to dentin regeneration (35). Overall, calcium hydroxide provides a beneficial environment for pulp-dentin regeneration through revascularization, and this is exhibited in the present case by the significant reduction in apical lesion size during the long-term application of calcium hydroxide over the blood clot.

Conducting RET on previously treated teeth is more challenging than on teeth with pulp necrosis due to the presence of different microbiota residing in the canal. While primary endodontic infections consist of gram-negative anaerobes, persistent endodontic infections mainly involve gram-positive facultative anaerobes that are more resistant to conventional disinfectants (e.g. sodium hypochlorite) (36-38). Moreover, RET procedures require more stringent aseptic environment compared to traditional root canal treatment because RET involves tissue engineering technique which is susceptible to bacterial infection (39). In vivo study by Verma et al. demonstrated that the presence of residual bacteria is correlated with intracanal inflammation and therefore, is a crucial factor for determining the success of pulp regeneration (40). Combined, the level of disinfection required in RET for previously treated teeth is higher than that needed for conventional root canal therapy or RET of necrotic pulps. However, the conventional chemical irrigants such as sodium hypochlorite and EDTA are often incapable of eradicating microorganisms residing in dentinal tubules or biofilms, and may fail to achieve advanced disinfection levels (41). In the present case, not only were further disinfections of the root canals achieved through the long-term application of calcium hydroxide, but also through the recovery of natural immunity induced by revascularization. This endowed the tooth with additional defensive capabilities against remaining microorganisms, making it less susceptible to reinfections.

In the current case, overextended gutta percha protruding about 5mm beyond the root apex could not be completely removed. Advantages and disadvantages of removing the segment of extruded gutta percha were considered, and it was decided to leave the remaining gutta percha to minimize damage to the apical root dentin within the limitations of non-surgical endodontic treatment. While overfilling of gutta percha may elicit unfavorable outcome of root canal treatment due to foreign body reaction (42, 43), there also have been reports demonstrating that the filling material itself is not the primary cause of the poor prognosis. The study by Halse and Molven (44) revealed that infection, rather than apical overfilling per se, is a key determinant of the failure of endodontic treatment. Research by Friedman (45) also emphasized on the adverse effect of overinstrumentation, which may push the infected dentinal chips and canal debris into periapical tissue. Meanwhile, study by Sjogren et al. (46) exhibited that particle sizes of gutta percha impacted the healing of periapical lesions, with solid piece inducing mild reaction while fine particles or dissolved gutta percha evoked an intense tissue response. In the present case, thorough disinfection within the canal and subsequent innate immunity achieved through revascularization may have contributed to periapical healing, unhindered by extruded solid gutta percha. Particularly at a 3-year follow up, the radiographic examinations show complete resolution of radiolucency around extruded gutta percha, which have become embedded in the alveolar bone. Also given that the patient displayed no symptoms, an additional surgical approach is not a current consideration.

In conclusion, although RET has been mainly performed in immature teeth in the past, there have been instances of studies performing RET in mature permanent teeth with successful outcomes, in recent years. In the present case, a previously treated mature permanent tooth was successfully treated with RET, employing long-term calcium hydroxide application over the induced blood clot. It is inferred that calcium hydroxide created an antimicrobial environment during stem cell migration and proliferation without causing harm to the cells. Moreover, the restoration of innate immunity through revascularization might have endowed additional defensive capacities against microorganisms, preventing the occurrence of reinfections. Therefore, this modified RET method may be a potentially viable treatment option for previously treated mature permanent teeth with apical periodontitis. However, further clinical studies with larger number of cases are recommended to verify its effect.