Iwaya et al (1) showed that a human immature permanent tooth with necrotic pulp

Histologic Observation of a Human Immature Permanent Tooth with Irreversible Pulpitis after Revascularization/Regeneration Procedure Emi Shimizu, DDS, PhD,* George Jong, DDS,* Nicola Partridge, PhD, Paul A. Rosenberg, DDS,* and Louis M. Lin, BDS, DMD, PhD* Abstract Introduction: Histological studies of immature human permanent necrotic teeth with or without apical periodontitis after revascularization have not been reported. This case report describes the histological findings of tissue formed in the canal space of an immature permanent tooth #9 with irreversible pulpitis without apical periodontitis after revascularization. Methods: An immature human permanent tooth #9 was fractured 3.5 weeks after revascularization and could not be retained. The tooth was extracted and prepared for routine histological and immunohistochemical evaluation in order to examine the nature of tissue formed in the root canal following the revascularization procedure. Results: At 3.5 weeks after revascularization, more than one half of the canal was filled with loose connective tissue similar to the pulp tissue. A layer of flattened odontoblast-like cells lined along the predentin. Layers of epithelial-like cells, similar to the Hertwig s epithelial root sheath, surrounded the root apex. No hard tissue was formed in the canal. Conclusions: Based on the histological findings in the present case, regeneration of pulp-like tissue is possible after revascularization. In this case, both the apical papilla and the Hertwig s epithelial root sheath survived in an immature permanent tooth despite irreversible pulpitis but without apical periodontitis. (J Endod 2012;38:1293 1297) Key Words Immature permanent tooth, irreversible pulpitis, pulplike tissue regeneration, revascularization From the Departments of *Endodontics and Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, New York. Address requests for reprints to Dr Louis M. Lin, Department of Endodontics, New York University College of Dentistry, 345 East 24th Street, New York, NY 10010. E-mail address: lml7@nyu.edu 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.06.017 Iwaya et al (1) showed that a human immature permanent tooth with necrotic pulp and apical periodontitis/abscess after a revascularization procedure could induce increased thickening of the canal walls and continued root development. Since then, many similar cases have been reported (2 5). Radiographically, in some cases, revascularized human immature permanent teeth appear to show continued development as evidenced by the deposition of hard tissue on the canal walls and continued root development. Therefore, the revascularization of human immature permanent teeth with necrotic pulp and apical periodontitis/abscess has been considered to be a regenerative process (2 6). Regeneration is defined as the replacement of damaged tissue by the same parenchymal cells (7). However, regeneration is a histologic observation and cannot be determined radiographically. The nature of the tissue formed in the canal space in human revascularized immature permanent teeth with apical periodontitis is speculative because no histologic studies are available. Currently, the available animal studies of immature teeth with pulp necrosis and apical periodontitis after revascularization procedures show that the tissues formed in the canal space are cementoid or osteoid tissue and periodontal ligament like tissue (8 11). However, no studies have investigated the nature of the tissue present in immature teeth with irreversible pulpitis with normal periapical tissues after a revascularization/regeneration procedure in animals or humans. The purpose of this case report is to describe the histologic observation of a human immature permanent tooth clinically diagnosed as having irreversible pulpitis with normal periapical tissues after a revascularization/regeneration procedure. To our knowledge, this is the first histologic observation of a human revascularized immature permanent tooth with irreversible pulpitis. Materials and Methods A 10-year-old boy was referred from the Postgraduate Pediatric Clinic at New York University College of Dentistry to the Postgraduate Endodontic Clinic for the treatment of tooth #9. The child had a traumatic injury to his maxilla, which caused an uncomplicated crown fracture of tooth #8 and a complicated crown fracture with pulp exposure of tooth #9. The coronal one half of the crown of tooth #9 was horizontally fractured. According to the patient s mother, the general dentist removed part of the pulp, placed medication inside tooth #9, and advised the mother to bring the child to New York University College of Dentistry for further treatment. The patient and his mother visited the Postgraduate Pediatric Clinic approximately 1 month after treatment by the general dentist. Pulp sensibility tests with Endo-Ice (Coltene/Whaledent Inc, Cuyahoga, OH), heated gutta-percha, and electric current with the Vitality Scanner (SybronEndo, Glendora, CA) of teeth #8, #9, and #10 were conducted in the Postgraduate Endodontic Clinic. Teeth #8 and #10 responded to heated gutta-percha, Endo-Ice, and electric current within normal limits and were not sensitive to palpation or percussion. The crown of tooth #9 had an Intermediate Restorative Material (Dentsply Internation, Milford, DE) restoration. It was asymptomatic and responded erratically to pulp sensibility tests because the patient was apprehensive and perhaps also because of the attempt at pulp therapy performed by the general dentist. The tooth had a large canal JOE Volume 38, Number 9, September 2012 Histologic Observation of an Immature Permanent Tooth 1293

Figure 1. (A) A preoperative radiograph of tooth #9. (B) A radiograph of the fractured tooth 3.5 weeks after revascularization. (C) A photograph of the extracted tooth. Note a small mass of soft tissue attached to the root apex (arrow). M, mineral trioxide aggregate plug. space and an open apex surrounded by a well-circumscribed radiolucent area (Fig. 1A). The provisional clinical diagnosis of the pulpperiapical tissue complex of tooth #9 was pulp necrosis with normal periapical tissues. A cotton pellet was found in the canal after tooth #9 was accessed after adequate local anesthesia. Bleeding was noted near the midroot under a Zeiss surgical microscope (Carl Zeiss Meditac Inc, Dublin, CA). It appeared that a deep pulpotomy or a partial pulpectomy might have been previously performed on tooth #9. Consequently, the clinical diagnosis of irreversible pulpitis instead of pulp necrosis with normal periapical tissues was confirmed. The patient s dentist could not be contacted to confirm the prior treatment. It was not known if a deep pulpotomy or partial pulpectomy was performed under the rubber dam or the pulp had become infected after deep pulpotomy or partial pulpectomy because the crown was badly fractured. Therefore, a revascularization/regenerative procedure instead of apexogenesis was selected for tooth #9 in an attempt to promote possible pulp tissue regeneration, increased thickening of the canal walls, and continued root development by Hertwig s epithelial root sheath (HERS) and apical papilla. The revascularization/ regenerative procedure was performed according to the protocol suggested in our previous study with slight modifications (12). Briefly, at the first visit, the working length was determined, and the canal was minimally instrumented with hand K-files (Dentsply Mailefer, Ballaigues, Switzerland) and gently irrigated with copious amounts of 5.25% sodium hypochlorite solution (Sultan Healthcare, Hackensack, NJ) with an irrigation syringe penetrating to the apical portion of the canal. Calcium hydroxide (Henry Schein, Melville, NY) mixed with saline solution was used as an interappointment intracanal medication and carried into the canal with files to apical one third of the canal. At the second visit 2 weeks later, the tooth was asymptomatic. Under a surgical microscope, K-files were used to induce bleeding into the canal by irritating the periapical tissues. A thick mixture of ProRoot mineral trioxide aggregate (MTA) (Dentsply Tulsa Dental, Tulsa, OK) and saline solution was used as a coronal seal in the revascularized tooth. The MTA paste was placed into the coronal canal approximately 5 mm below the access opening against induced bleeding, and an adequate access cavity was provided for a composite resin restoration. The access cavity was restored with light-cured composite resin (Amelogen Plus; Ultradent, South Jordan, UT). Three and a half weeks after completion of the revascularization/ regeneration procedure, the lingual aspect of the crown fractured below the alveolar crest bone (Fig. 1) and the tooth could not be retained. No pulp sensitivity tests were performed. The tooth was extracted and processed for histologic and immunohistochemical evaluation. The crown and MTA plug were removed from the tooth to ensure adequate penetration of fixative into the canal. The tooth was immediately fixed in 4% formaldehyde for a week and decalcified in 10% EDTA (ph = 7.5) for 4 weeks at 4 C. The specimen was then soaked in 10% sucrose for 2 hours, in 20% sucrose for 6 hours, and in 30% sucrose overnight and embedded in optimal cutting temperature compound. Frozen series sections of approximately 10 mm thickness were cut along the long axis of the tooth and dried overnight. Hematoxylin-Eosin Staining The dried sections were stained with 0.1% Mayer s hematoxylin solution for 10 minutes. They were then rinsed in cool running double-distilled water for 5 minutes, dipped in 0.5 eosin 12 times, dipped in distilled water, and dehydrated in ascending concentrations of ethanol. The sections were dipped in xylene several times, mounted on slides, and covered with a coverslip with Cytoseal (Thermo Fisher Scientific, Waltham, MA) and examined under a light microscope. Immunohistochemical Staining To evaluate the localization of mesenchymal stem cells, STRO-1 (R and D System, Minneapolis, MN) was used. Immunohistochemical staining was performed using the avidin-biotin complex staining system (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer s instructions. The sections were washed in phosphate-buffered saline (PBS) and immersed in methanol containing 1% hydrogen peroxide to block endogenous peroxidase activity. The sections were incubated with 2% blocking serum and then with anti STRO-1 antibodies or normal immunoglobulin G as a negative control at 4 C overnight and washed in PBS. The sections were incubated with biotin-labeled anti immunoglobulin G and washed in PBS. Staining was completed by 10 minutes of incubation with 3,3 -diaminobenzidine (Santa Cruz Biotechnology, Santa Cruz, CA). Results At 3.5 weeks after the revascularization/regeneration procedure of the immature permanent tooth with clinically diagnosed irreversible pulpitis, a loose connective tissue with few collagen fibers filled the canal space up to the coronal MTA plug (Fig. 2A). The tissue in the canal and periapical tissues were devoid of inflammatory cells. The majority of cells in the canal space and in the periapical area were spindle-shaped young fibroblasts or mesenchymal cells. There were more blood vessels and cellular components in the canal than that at the apical area (Fig. 2B and C). No nerve-like fibers running alongside the blood vessels were observed. The tissue in the canal space appeared to be an extension of the periapical tissue (Fig. 2A). There was a layer of flattened odontoblast-like cells polarized along the predentin in the apical canal (Fig. 2B). No hard tissue was seen in the canal space and on the canal walls. Layers of epithelial-like cells, similar to HERS, surrounded the root apex (Fig. 2D). Strol-1 positive cells (solid arrows) were observed in the loose connective tissue near the apical foramen (Fig. 3A and B) and in the epithelial-like HERS surrounding the root apex. Compared with the mature dental pulp, the loose connective tissue in the canal 1294 Shimizu et al. JOE Volume 38, Number 9, September 2012

Figure 2. (A) Histology of the section of extracted revascularized tooth #9. A loose connective tissue with few collagen fibers has filled the canal space up to the coronal MTA plug (hematoxylin-eosin, original magnification 200). The MTA plug was removed before histologic tissue processing. (B) High magnification of the square in A (the apical root canal). Flattened odontoblast-like cells lined along the predentin (solid arrows). Many blood vessels filled with red blood cells (open arrows). No mature nerve-like bundles along the blood vessels are observed. Most cells are spindle shaped. (C) High magnification of the rectangle in A (the apical foramen). There are fewer blood vessels (arrow) and cellular components at the apical foramen than that in the canal. (D) High magnification of the square in C (part of the root apex). Layers of epithelial-like HERS (arrow) surrounding the root apex. Spaces in the tissue are artifacts caused by histologic preparation. was similar to an immature pulp tissue consisting of numerous spindleshaped young fibroblasts or mesenchymal cells, many blood vessels, few collagen fibers, and no mature nerve-like tissue. Discussion It is reasonable to ask why apexogenesis was not attempted in this case, which was initially diagnosed as an irreversible pulpitis. Irreversible pulpitis can be caused by trauma, chemical irritation, or bacterial infection. In this case, bacterial contamination was the major concern as described previously. If apexogenesis was selected, it was not known how much of the infected pulp tissue would have to be removed from the canal. Importantly, apexogenesis in the present case can only encourage root maturation. It cannot promote pulp tissue regeneration or ingrowth of vital tissue into the coronal canal space because of a calcified tissue barrier formed in the canal induced by calcium hydroxide or Figure 3. (A) The immunohistochemistry of the section of extracted revascularized tooth #9 (immunohistochemical staining, original magnification 200). (B) High magnification of square in A. Strol-1 positive cells in the pulp-like tissue near the apical foramen (arrows) and in the epithelial-like HERS surrounding the root apex (not shown in high magnification). JOE Volume 38, Number 9, September 2012 Histologic Observation of an Immature Permanent Tooth 1295

mineral trioxide aggregate in apexogenesis. A revascularization/regeneration procedure of immature permanent teeth with irreversible pulpitis involving the pulp tissue in the apical portion of the root canal might have the potential of pulp tissue regeneration into the coronal canal space, thickening of the canal walls, and continued root development because of the survival of HERS and the apical papilla. In addition, if apexogenesis fails and apical periodontitis develops, the apical papilla will likely be destroyed, and pulp regeneration will not occur as in animal studies (8 11). Based on the present case study, the tissue in the canal space appeared to be an extension from the periapical tissue after the revascularization/regeneration procedure of the immature permanent tooth with clinically diagnosed irreversible pulpitis. Three and a half weeks after the revascularization/regeneration procedure, loose connective tissue filled the canal space up to the coronal MTA plug. The tissue in the apical canal space was cell rich and well vascularized. This tissue is similar to cell-rich, well-vascularized connective tissue in the canal space described by Skoglund and Tronstad (13) in replanted and autotransplanted immature teeth of dogs at 30 days of histologic observations. It is also similar to cell-rich, well-vascularized connective tissue reported by Claus et al (14) in autotransplanted immature teeth after removal of the original pulp tissue in beagle dogs at 4 weeks of histologic observations. The loose connective tissue near the apical foramen contained fewer blood vessels and cell components than the tissue in the apical canal space. This tissue is similar to the apical papilla described by Sonoyama et al (15). The majority of the cells in the periapical tissue and loose connective tissue in the canal were spindle-shaped young fibroblasts or mesenchymal cells. A layer of flattened cells similar to root odontoblasts were polarized along the predentin in the apical canal. It is not known if these odontoblast-like cells were preexisting primary odontoblasts or newly differentiated odontoblasts from the apical papilla after revascularization/regeneration procedure of the immature permanent tooth with irreversible pulpitis. There were few collagen fibers. No nerve-like fibers running alongside the blood vessels as in mature dental pulp were observed (16). This may indicate that the loose connective tissue in the canal is a newly developed immature pulp-like tissue. No hard tissue was seen in the canal and on the canal walls. Skoglund and Tronstad (13) and Claus et al (14) observed hard-tissue formation in the canal at 30 days and 4 weeks, respectively, after autotransplantation of dog s immature teeth. However, Zhao et al (17) observed initial hard-tissue formation at 7 days after replantation of rat s immature teeth. Layers of epithelial-like cells, similar to HERS, surrounded the root apex, which would regulate continued root maturation (16). Strol-1 positive stem cells were observed in the loose connective tissue near the apical foramen. They might be from the apical papilla because stem cells from the developing apical papilla have greater numbers of STRO-1 positive cells than stem cells from the mature dental pulp (15). Mesenchymal stem cells from the apical papilla are capable of differentiating into odontoblasts under the influence of appropriate microenvironment cues (15). There are 2 possible scenarios that could explain the formation of the pulp-like loose connective tissue in the canal after the revascularization/regeneration procedure in the present case. First, the pulp-like tissue might be caused by the proliferation of the remaining apical pulp tissue after the revascularization procedures. However, it is not known if the residual apical pulp stump in the present case is capable of regenerating the pulp tissue to fill the coronal canal space after the revascularization/regeneration procedure. Sodium hypochlorite irrigation and intracanal calcium hydroxide medication might destroy part of the residual vital apical pulp tissue (18, 19) but not the apical papilla. Nevertheless, infection is the primary concern and cause of tissue destruction. It has to be controlled for wound healing (regeneration or repair) to occur. Second, the pulp-like tissue could be caused by the proliferation and differentiation of the apical papilla into the canal space. It has been shown that stem cells from the apical papilla have more potential than stem cells from the dental pulp to differentiate into odontoblast-like cells upon receiving appropriate inductive signals (15, 20, 21). Stem cells from the apical papilla are derived from a developing tissue that may represent a population of early stem/progenitor cells that may have a superior cell source for tissue regeneration (20). Therefore, the pulp-like tissue in the present case might be derived from the apical papilla. Nevertheless, the relationship between the apical papilla and the dental pulp needs to be characterized. If the revascularized tooth in the present case was not fractured, thickening of the canal walls by deposition of dentin and continued root development could occur because of the survival of HERS and the apical papilla and presence of odontoblast-like cells along the predentin (15, 22). Conclusion Based on histologic observation of the present case, regeneration of the pulp-like tissue is possible after a revascularization/regeneration procedure because both HERS and the apical papilla survived in an immature permanent tooth clinically diagnosed as having irreversible pulpitis. The ideal cases for pulp regeneration in revascularization/ regeneration procedures are likely to be immature permanent teeth with irreversible pulpitis involving the canal pulp without radiographic evidence of apical periodontitis or traumatized immature permanent teeth with a necrotic pulp without radiographic evidence of apical periodontitis. Apexogenesis is more suitable for irreversible pulpitis involving only the coronal pulp. Infection and/or inflammation hinder the potential of tissue regeneration and stem cell function (23). Accordingly, infection/inflammation must be controlled for wound healing (regeneration or repair) to occur (23). Acknowledgments The authors deny any conflicts of interest related to this study. References 1. Iwaya SI, Ikawa M, Kubota M. Revascularization of an immature permanent tooth with apical periodontitis and sinus tract. Dent Traumatol 2001;17:185 7. 2. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod 2004;30:196 200. 3. Chueh LH, Huang GT. Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a paradigm shift. J Endod 2006;32:1205 13. 4. Ding RY, Cheung GS, Chen J, Yin XZ, Wang QQ, Zhang CF. Pulp revascularization of immature teeth with apical periodontitis: a clinical study. J Endod 2009;35: 745 9. 5. Jung IY, Lee SJ, Hargreaves KM. Biologically based treatment of immature permanent teeth with pulpal necrosis: a case series. J Endod 2008;34:876 87. 6. Chueh LH, Ho YC, Kuo TC, Lai WH, Chen YH, Chiang CP. Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod 2009;35:160 4. 7. Kumar V, Abbas AK, Fausto N. Robbins and Cotran Pathologic Basis of Disease, 8th ed. Philadelphia: Saunders; 2009. 8. Thibodeau B, Trope M. Pulp revascularization of a necrotic infected immature permanent tooth: case report and review of the literature. Pediatr Dent 2007;29: 47 50. 9. Wang X, Thibodeau B, Trope M, Lin LM, Huang GT. Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod 2010;36: 56 63. 10. Yamauchi N, Yamauchi S, Nagaoka H, et al. Tissue engineering strategies for immature teeth with apical periodontitis. J Endod 2011;37:390 7. 11. Bezerra da Silva LA, Nelson-Filho P, da Silva RA, et al. Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs teeth 1296 Shimizu et al. JOE Volume 38, Number 9, September 2012

with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109:779 87. 12. Chen MY, Chen KL, Chen CA, Tayebaty F, Rosenberg PA, Lin LM. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J 2012;45: 294 305. 13. Skoglund A, Tronstad L. Pulpal changes in replanted and autotransplanted immature teeth of dogs. J Endod 1981;7:309 16. 14. Claus I, Laureys W, Cornelissen R, Dermaut LR. Histologic analysis of pulpal revascularization of autotransplanted immature teeth after removal of the original pulp tissue. Am J Orthod Dentofacial Orthop 2004;125:93 9. 15. Sonoyama W, Liu Y, Yamaza T, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 2008;34:166 71. 16. Nanci A. Ten Cate s Oral Biology. 7th ed. St Louis, MO: Mosby; 2007. 17. Zhao C, Hosoya A, Kurita H, et al. Immunohistochemical study of hard tissue formation in the rat pulp cavity after tooth replantation. Arch Oral Biol 2007;52:945 53. 18. Rosenfeld EF, James GA, Burch BS. Vital pulp tissue response to sodium hypochlorite. J Endod 1978;4:140 6. 19. Gordon TM, Damato D, Christner P. Solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue. J Endod 1981;7:466 9. 20. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissue vs. those from other sources: their biology and role in regenertive medicine. J Dent Res 2009;88:792 806. 21. Tziafas D, Kodonas K. Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. J Endod 2010;36:781 9. 22. Sonoyama W, Seo BM, Yamaza T, Shi S. Human Hertwig s epithelial root sheath cells play crucial roles in cementum formation. J Dent Res 2007;86:594 9. 23. Thomas MV, Puleo DA. Infection, inflammation, and bone regeneration: a paradoxical relationship. J Dent Res 2011;90:1052 61. JOE Volume 38, Number 9, September 2012 Histologic Observation of an Immature Permanent Tooth 1297