Regenerative Endodontic Treatment of a Maxillary Mature Premolar

Regenerative endodontic treatment was performed on a mature maxillary premolar diagnosed as chronic pulpitis. The root canals were chemomechanically prepared and placed intracanal medicaments at the first appointment. Then 2 weeks later, a blood clot was created in the canals, over which mineral trioxide aggregate was placed. At 6-month follow-up, cementum-like tissue seemed to be formed in the root canal along with nearly recovered pulp vitality. At 12-month recall, the radiographic results revealed evidence of root wall thickening. At 30-month recall, no periapical lesion was found. This case report indicates that regenerative endodontic treatment for the mature premolar is feasible. More cases are needed for further validation.


Introduction
Regenerative endodontics is the use of biologically based procedures designed to replace damaged structures, including dentin and root structures, as well as cells of the pulp-dentin complex [1]. It shows a broad prospect in both tissue engineering and clinical application, which was adopted as a procedure code by the American Dental Association in January 2011 [2].
Traditionally, for pulpitis or apical periodontitis, root canal treatment (RCT) is the most common treatment method. However, RCT results in complete removal of pulp tissues, which means loss of a warning system, immunoresponse and formation of dentin as active defense mechanisms against invading toxins and bacteria, and cease of root development in the cases of young patients. Moreover, in some special cases, the absence of an apical constriction makes RCT problematic. Apexi cation also has some disadvantages such as variability of treatment time, patient compliance, and increased risk of tooth fracture, which may do not promote the continued development of the root and just "closed" the open apex by a calci c barrier. In contrast, the goal of regenerative endodontics is to induce biologic replacement of dental tissues and regenerate a functional and healthy pulp-dentin complex. So, regenerative endodontic treatment may be a good alternative to conventional endodontic therapies, especially for immature permanent teeth.
To date, more and more case reports and case series showed the feasibility of regenerative endodontic treatment [3][4][5]; however, most of these cases were performed on immature teeth. Recently, cases on mature teeth with closed apex were occasionally reported [6,7]. In the present report, we described the treatment of a mature tooth with pulpitis by using regenerative endodontic therapy, and the follow-up period lasted for 30 months.

Case Report
A 15-year-old girl in good health presented to the Department of Endodontics, Wuhan University Hospital of Stomatology, in November, 2014, with a chief complaint of cold water pain on her left upper posterior tooth for the last 5 days. ree months earlier, she had caries lled in the same region. Intraoral examination revealed that tooth #13 had an existing distal occlusal composite restoration. Tooth #13 showed sensitivity to pulp test and no sensitivity to palpation and percussion. Radiographic examination showed distal a lling shadow close to the pulp and a fully developed root and closed apex ( Figure 1). On the basis of the results of the clinical and radiographic examinations, the diagnosis for tooth #13 was chronic pulpitis.
An informed consent was obtained from the patient and her parent for regenerative endodontic treatment. An access cavity preparation was performed after local anesthesia with 2% lidocaine and rubber dam isolation. e canal was irrigated copiously with 5.25% sodium hypochlorite and 17% EDTA. e canal was partially dried with sterile paper points and then dusted with a mixture powder of cipro oxacin and metronidazole.
e tooth was then temporized with zinc oxide eugenol cement.
At 2-week follow-up, the patient was asymptomatic. e tooth was not sensitive to percussion and palpation. After local anesthesia by using 2% lidocaine and rubber dam isolation, the temporary restoration was removed. e antibiotic mixture was completely removed with 5.25% sodium hypochlorite. e apical foramen was enlarged up to 0.6 mm with a size #60 hand le (K-le). Seventeen percent EDTA was used as a nal irrigating agent and left in the canal for about 1 minute. en, the canal was dried with sterile paper points. A size #25 K-le was introduced into the root canal to irritate the periapical tissue and to create some bleeding into the root canal. e mineral trioxide aggregate (MTA) was placed approximately 2 mm below the cementoenamel junction. e tooth was restored with glass ionomer cement (Figure 2). At 3-month recall, the patient was asymptomatic. e tooth was not sensitive to percussion or palpation and not responsive to pulp testing. e radiograph showed nearly a normal periodontal ligament space (Figure 3). e tooth was restored with composite resin after removal of the glass ionomer cement.
At 6-month follow-up, tooth #13 regained pulp sensibility and responded positively to the electric pulp tester (EPT) and Endo-Ice at a similar extent as the control tooth.
e radiographic results showed narrowing of the canal space with radiopaque shadows (Figure 4).
At 12-month recall, there was no pain on percussion and palpation. Tooth #13 was normally responsive to Endo-Ice and EPT. e radiographic results revealed evidence of root wall thickening ( Figure 5).
At 30-month recall, tooth #13 still showed normal pulp vitality compared to the control tooth, and no periapical lesion was found on the X-ray lm ( Figure 6). e recovery of pulp vitality at di erent follow-up time points was summarized in Table 1.

Discussion
Age of thirteen to fteen years is the period of a premolar maturation, during which the apex is gradually closed. In this case, the patient was 15 years old. e X-ray showed that the apex was fully developed. Furthermore, during the regenerative endodontic treatment, a #15 K-le was used to probe the apical foramen and an obvious sense of friction was found, so we judged the tooth in this case belongs to mature permanent tooth. Apical bleeding was induced into the root canal, leading to a suitable environment for pulp regeneration with an enrichment of host endogenous stem cells and growth factors in a bioactive sca old. e newgrown tissue was not a pulp tissue revealed by the calci ed shadows in the root canal. Instead, it might be a newly generated cementum-like tissue termed herein "intracanal cementum" as reported in relevant literature [8].
e concept of regenerating pulpal tissue was promulgated by the classic studies of Nygaard-Ostby in 1961.  His early research revealed tissue repair ( broblasts, collagen, and sparse vascularity) without histologic evidence of regeneration of the pulp-dentin complex, while a newly mineralized tissue appeared to be cementum was evident on some dentinal walls. e second case series 10 years later showed histologic evidence of an ingrowth of vascularized brous connective tissue in the most of the teeth. Wang et al. [8] analyzed the histologic characterization of regenerated tissues in the canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. It is found that the canal dentinal walls were thickened by the apposition of a newly generated cementum-like tissue which was similar to cellular cementum. One of the cases showed partial survival of pulp tissue juxtaposed withbrous connective tissue that formed intracanal cementum on canal dentin walls. Bone or bone-like tissue, the so-called intracanal bone, and connective tissue similar to periodontal ligament were also present in the canal space surrounding the intracanal cementum and/or intracanal bone. In the    present case, the intracanal cementum may be able to explain the imaging manifestations well, including the radiopaque shadows and root wall thickening. But further studies are still needed to prove whether the recovery of pulp vitality means there is regeneration of nerve bers and whether there are blood vessels, broblast, newly formed dentin, etc.
It is believed that stem cells play an important role in histogenesis as well as pulp regeneration. To our knowledge, at least ve types of postnatal mesenchymal stem cells can di erentiate into odontoblast-like cells based upon the studies of recent years, including dental pulp stem cells (DPSCs), stem cells of human exfoliated deciduous teeth (SHED), dental follicle progenitor cells (DFPCs), stem cells of the apical papilla (SCAP), and bone marrow-derived mesenchymal stem cells (BMMSCs). Promisingly, studies in vivo have shown that transplantation of stem cells can yield ectopic dental pulp-like tissues in tooth slices or fragments. However, no clinical trials of stem cell transplantation have been reported to date. It has been adopted by the American Dental Association (ADA) to induce apical bleeding into the root canal in immature permanent teeth with necrotic pulps which have been extirpated in January 2011 [9]. e evoked bleeding during endodontic regenerative procedures conducted on immature teeth reveals a massive in ux of mesenchymal stem cells into the root canal space. In contrast to cell-based approaches of stem cell transplantation and/or tissue engineering, this "induced bleeding" technique was named "cell-free approach" [10], which was applied in our present case as well.
Sca old is thought of as an indispensable element for pulp/dentin regeneration. In this report, sterile blood clot induced into the root canal also acted as the 3-dimensional sca old [11] for tissue ingrowth which supplied potent chemoattractants and growth factors simultaneously to induce cell migration, proliferation, and di erentiation. erefore, no other sca old materials or growth factors were used in this case while considering noninvasion and avoiding rejection. But they may be the substitution when ideal blood clot is absent. For instance, after dressing with the antibiotic mixture to resist in ammatory reaction, or if the root apex of the upper posterior tooth is adjacent to maxillary sinus, it may be more di cult to induce enough blood.
Clinical regenerative endodontics has been focused on immature teeth because they have a greater chance of pulp tissue regeneration. It is believed that immature teeth with open apices allow more stem cells to migrate into root canals, and more stem cells found near immature root apices have been shown to possess a great regeneration potential in pulp regeneration [12]. Moreover, there are narrower apical pathways for cell migration in mature teeth, and su cient disinfection is more challenging than that in immature teeth.
In conclusion, the present case demonstrates the resolution of clinical signs and symptoms with cementum-like tissue formed in the root canal and pulp vitality nearly recovered in the mature permanent premolar with pulpitis after regenerative endodontic treatment. Current regenerative procedures successfully produce root development, but still no evidence is there to reestablish a real pulp tissue. Higher level of evidence with large-scale investigations and randomized controlled clinical trials is still needed. Anyway, it might be possible that regenerative endodontic treatment could be widely used on mature teeth in the future and become an alternative to conventional endodontic therapies.

Conflicts of Interest
e authors declare that they have no con icts of interest.