- Email: [email protected]
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex: An In Vivo Investigation
Regenerative Endodontics
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex: An In Vivo Investigation Mahmoud Torabinejad, DMD, MSD, PhD,* Arin Alexander, DMD, MSD, MBA,† Seyed Aliakbar Vahdati, DDS, MSD,‡ Anupama Grandhi, BDS, DDS,§ David Baylink, MD, FACB,§ and Shahrokh Shabahang, DDS, MS, PhDk Abstract Introduction: Current pulp revascularization procedures in teeth with necrotic pulps and open apices have produced histologic evidence of connective tissue growth, cementum, and bone within the root canals of experimental animals. This study aims to investigate the effect of maintaining uninflamed residual apical pulp tissue on the histologic outcome of pulp-dentin complex regeneration after a revascularization procedure in immature ferret cuspid teeth. Methods: Twenty-eight cuspid teeth from 7 young male ferrets were used in this experiment. Seven teeth were reserved to serve as positive control samples without any treatment. In another 7 teeth, the pulp was completely extirpated (negative control), whereas the pulp of the remaining 14 teeth were removed to either 1–2 mm short of the apex (7 samples) or 2–4 mm short of the apex (7 samples). Blood clots were covered with mineral trioxide aggregate at the cementoenamel junction level of each tooth. Three months later, block sections were removed for histologic evaluations, and the data were statistically analyzed with the chi-square test (P < .05). Results: All teeth with complete pulp extirpation showed the presence of bone inside the root canal. In contrast, the root canals for most teeth with pulp amputation 1–4 mm from the radiographic apex were filled with normal pulp, which extended coronally to the mineral trioxide aggregate, where hard tissue bridges had formed. Conclusions: Based on these results, we concluded that regeneration of the pulpdentin complex is possible when the apical 1–4 mm of the apical pulp remains intact in immature teeth. (J Endod 2018;44:1796–1801)
N
Key Words
All experimental procedures were performed in accordance with protocols approved by the Loma Linda University Animal Research Committee. A total of 28 immature cuspid teeth (14) in seven 97- to 101-day-old male ferrets were used in this
Bone remodeling/regeneration, cementum, dentinogenesis, endodontics, odontogenesis, pulp biology
umerous case reports Significance and case series as The results of this animal study show that complete well as a few prospective regeneration of the pulp-dentin complex is and retrospective cohort possible when 1–4 mm of normal pulp remains in studies and randomized the apical segment of the root canal system. controlled trials have been published showing radiographic illustration of the continuation of root development and the resolution of periapical lesions in teeth with necrotic pulp and open apices after treatment with “revascularization/regeneration” procedures (1). Except for a few human case reports, the outcomes for most of these reports were evaluated clinically and radiographically (1). Radiographic resolution of periapical lesions, root-end closure, and canal wall thickening in teeth with necrotic pulp and open apices give an indication that a normal, functional pulp can be regenerated. Several animal studies (2–8) and 3 human case reports (9–11) have examined the nature of tissues present in the root canals of teeth treated with regenerative endodontic procedures and have reported the presence of connective tissue, cementum, and bone in the root canals of the experimental teeth instead of pulp tissue. Only 2 human case reports have shown regeneration of pulplike tissue after performing regenerative endodontic procedures (12, 13). Because previous investigations have not determined the effect of inflammation and infection on the capability of an immature tooth to regenerate the pulp-dentin complex, an animal model is needed to determine the effect of these factors on the capacity of an immature tooth to regenerate the pulp-dentin complex at various stages of pulpal and periapical pathosis. The first phase of these investigations is to examine histologically the capacity of a tooth for true pulp-dentin regeneration under ideal conditions in immature teeth without any pulpal and/or periapical pathosis. The development of this model has the potential for future studies leading to the determination of factors affecting pulp-dentin regeneration. The aim of this study was to investigate the effect of residual uninflamed apical pulp tissue on the histologic outcome of pulp-dentin complex regeneration in immature ferret cuspid teeth.
Materials and Methods
From the *Torabinejad Institute of Surgical Education and Research Venues, Irvine, California; †Private practice, Los Angeles, California; ‡Private practice, Newport Beach, California; §Loma Linda University, Loma Linda, California; and kPrivate practice, Redlands, California. Address requests for reprints to Dr. Mahmoud Torabinejad, 9950 Irvine Center Drive, Irvine, CA 92618. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.09.005
1796
Torabinejad et al.
JOE — Volume 44, Number 12, December 2018
Regenerative Endodontics experiment. The teeth were randomly assigned to the following groups: group 1, positive control (7 teeth); group 2, complete pulp extirpation (7 teeth); group 3, pulp amputation to 1–2 mm short of the radiographic apex (7 teeth); and group 4, pulp amputation 2–4 mm from the radiographic apex (7 teeth). Preoperative cone-beam computed tomographic (CBCT) images were initially obtained to examine the condition of the apex and the absence of any preexisting pathosis in the cuspid teeth. These initial radiographic examinations also served as a guide for the levels of pulp amputation in groups 3 and 4 and were also a baseline for the comparison of root length, thickness, and apical closure before and after completion of the experiment. All procedures were conducted under a clean protocol with the use of sterile materials and equipment. After obtaining general anesthesia, each experimental tooth was decoronated with a sterile diamond bur approximately 3 mm above the gingival crest to expose the dental pulp. Seven teeth in group 1 served as positive control samples and did not receive any treatment. This was done to examine normal growth in length, dentin thickness, and apical closure of teeth in ferrets for comparison reasons with teeth in the experimental groups. The pulp tissue in 7 teeth in group 2 was completely extirpated with sterile barbed broaches (negative control). This procedure resulted in the filling of the canal with fresh blood from the periapical tissues and the formation of a blood clot below the cementoenamel junction. No root irrigation was used after the removal of the pulps in the group. The root canals of the remaining 14 teeth were cleaned with very large files (100–120 file sizes) to approximately either 1–2 mm from the apex (7 samples, group 3) or 2–4 mm short of the apex (7 samples, group 4) based on the measurements gathered from the preoperative CBCT images obtained before the experiment. Similar to teeth in group 2, this procedure also resulted in the production of fresh blood from the residual apical pulp tissues and the formation of a blood clot below the cementoenamel junction. No overinstrumentation beyond the apical foramen was performed in any of these specimens to induce bleeding from periapical tissues. No root irrigation was used after the removal of the pulps in these groups. Gray mineral trioxide aggregate (MTA) (Dentsply Tulsa Dental, Tulsa, OK) was gently placed on the clotted blood in all the experimental teeth, and the coronal access was sealed with amalgam. After 3 months, postoperative CBCT images were obtained, and all animals were sacrificed followed by perfusion. After the completion of perfusion, block sections containing the maxilla and the mandible were removed and placed into bottles containing 10% buffered formalin solutions for 2 weeks. Block sections were decalcified in 10% EDTA (Sigma-Aldrich, St Louis, MO) and were then embedded in paraffin (Thermo Fisher Scientific, Fair Lawn, NJ). Step serial sections of 5-mm thickness were
prepared and stained using hematoxylin-eosin. Samples were evaluated microscopically by a pathologist who was not aware of the nature of treatments performed in the experiment. Data were statistically analyzed with the chi-square test (P < .05) to evaluate the radiographic preoperative diameter of the apical opening of roots (mm), postoperative incidence of closure (yes) or nonclosure (no) of the apical opening, incidence of presence (yes) or absence (no) of normal pulp tissue, incidence of presence (yes) or absence (no) of intracanal and periapical inflammation, and incidence of presence (yes) or absence (no) of normal root canal wall thickening in the experimental teeth compared with the control samples.
Results The radiographic and histologic findings are summarized in Table 1. Radiographic examinations of the cuspid teeth using preoperative CBCT images showed the presence of an average apical foramen diameter of 0.56 mm. Similar examinations for the same teeth using postoperative CBCT imaging showed complete closure of the apices at the end of the experiment (Figs. 1–5). In the positive control teeth, normal tooth anatomy was noted (Fig. 1C–F). The root canals in this group were filled with normalpulp tissue. The canal walls were thick, and the apical region showed physiologic narrowing with apical deltas. The periradicular tissues were normal without any inflammation. In the teeth with full pulp extirpation (negative control), the canal walls were generally thin, and there was no evidence of physiologic apical narrowing or root canal wall thickening compared with the control samples. Instead of normal pulp tissue, there was a bony ingrowth in the canal space of these teeth that extended to the level of the MTA, where a hard tissue bridge had formed (Fig. 2C–F). No normal pulp was observed in any of the specimens in this group. There was no intracanal or periapical inflammation in these samples. In 6 of 7 teeth with pulp amputation 1–2 mm from the radiographic apex, the canal walls appeared normal, and there was evidence of a physiologic apical narrowing like those observed in the positive control samples (Fig. 3C–F). The canals of these teeth were filled with normal pulp tissue that extended to the level of the MTA, where a hard tissue bridge had formed. There was no evidence of apical bony ingrowth or inflammation in the canal space or periapical inflammation in 6 of 7 teeth in this group. In 1 sample in this group, there was connective tissue containing numerous blood vessels and fibroblasts plus bony ingrowth extending to the coronal third of the root (Fig. 4A–F). There was no inflammation in the canal space or in the periapical tissues in any of the teeth in this group. In all the teeth with pulp amputation 2–4 mm from the radiographic apex, the canal walls appeared normal, and there was evidence of a physiologic apical narrowing like those observed in the positive
TABLE 1. Radiographic Preoperative Diameter of the Apical Opening of Roots (mm), Postoperative Incidence of Closure (Yes) or Nonclosure (No) of the Apical Opening, Incidence of Presence (Yes) or Absence (No) of Normal Pulp Tissue, Incidence of Presence (Yes) or Absence (No) of Intracanal and Periapical Inflammation, and Incidence of Presence (Yes) or Absence (No) of Normal Root Canal Wall Thickening
Group
Preoperative average diameter of apical opening (mm)
Positive control Negative control 1–2 mm pulp left 2–4 mm pulp left
0.47 0.59 0.33 0.76
JOE — Volume 44, Number 12, December 2018
Presence (yes) or absence (no) of Presence (yes) or Postoperative intracanal and/or absence (no) of closure (yes) or periapical nonclosure (no) of normal pulp tissue inflammatory cells in the canal the apical opening Yes = 7, no = 0 Yes = 7, no = 0 Yes = 7, no = 0 Yes = 7, no = 0
Yes = 7, no = 0 Yes = 0, no = 7 Yes = 6, no = 1 Yes = 7, no = 0
Yes = 0, no = 7 Yes = 0, no = 7 Yes = 0, no = 7 Yes = 0, no = 7
Presence (yes) or absence (no) of normal root canal Apex wall thickening closure Yes = 7, no = 0 Yes = 0, no = 7 Yes = 6, no = 1 Yes = 7, no = 0
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex
Yes = 7 Yes = 7 Yes = 7 Yes = 7
1797
Regenerative Endodontics
Figure 1. (A) A preoperative CBCT scan of a maxillary cuspid, (B) a postoperative CBCT scan of the same tooth 3 months later, (C) a histologic section of the same tooth at the end of the experiment showing normal pulp and periradicular tissues (original magnification 1.1), (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
control samples (Fig. 5C–F). The canals of these teeth were filled with normal pulp tissue that extended to the level of the MTA, where a hard tissue bridge had formed. There was no apical bony ingrowth or inflammation in the canal space or periapical inflammation in any tooth in this group. No statistical differences were computed for comparison of the radiographic preoperative diameter of the apical opening of roots, postoperative incidence of closure or nonclosure of the apical opening, and incidence of the presence or absence of intracanal and/or periapical inflammation in the experimental teeth compared with the control samples. Statistically significant differences in responses were observed for the presence of normal pulp tissue (c23 = 23.8, P < .001) and root canal wall thickening (c23 = 23.8, df = 3, P < .001) in negative control teeth with total pulp extirpation and those with 1–2 mm residual pulp tissue, 2–4 mm residual pulp tissue, and the positive control samples. Statistically significant differences in responses were also found for the presence of normal pulp tissue (c23 = 23.8, P < .001) and root canal
wall thickening (c23 = 23.8, P < .001) in experimental teeth with 1– 2 mm residual pulp tissue and 2–4 mm residual pulp tissue as well as the positive controls. In addition, there was a statistically significant difference in the response to treatment comparing groups 2 (complete pulp extirpation), 3 (1–2 mm residual pulp tissue), and 4 (2–4 mm residual pulp tissue) (c22 = 17.365, P < .001). However, there was no statistically significant difference in outcome regarding the presence of normal pulp tissue and root canal wall thickening between group 3 (1–2 mm residual pulp tissue) and group 4 (2–4 mm residual pulp tissue) (c21 = 1.077, P = .299).
Discussion To determine the nature of tissues present in teeth that have had regenerative endodontics, it is necessary to remove the tissues within the root canals or to extract the whole tooth, thus leading to obvious ethical concerns in patients. Alternatively, such an approach can be
Figure 2. (A) A preoperative CBCT scan of a maxillary cuspid, (B) a postoperative CBCT scan of the same tooth 3 months later showing signs of intracanal calcification, (C) a histologic section of the same tooth with full pulp extirpation showing thin canal walls with no evidence of physiologic apical narrowing and a bony ingrowth into the canal space (original magnification 1.6), (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
1798
Torabinejad et al.
JOE — Volume 44, Number 12, December 2018
Regenerative Endodontics
Figure 3. (A) A preoperative CBCT scan of a mandibular cuspid; (B) a postoperative CBCT scan of the same tooth 3 months later showing complete closure of the apex and coronal bridge formation under MTA and no signs of intracanal calcification; (C) a histologic section of the same tooth with pulp amputation 1–2 mm from the radiographic apex showing thick root canal walls with a physiologic apical narrowing with apical deltas, normal pulp tissue containing fibroblasts, and blood vessels that extend to the level of the MTA, where a hard tissue bridge had formed(original magnification 2); (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
evaluated by performing animal experiments (14). A ferret has been suggested as a suitable animal model to perform endodontic studies (14, 15). The results of this experiment showed that true pulp regeneration after revascularization occurs when 1–4 mm of normal apical pulp tissue remains in immature teeth. The elements needed for pulp-dentin complex regeneration are 1. 2. 3. 4. 5.
A disinfected root canal space An adequate coronal seal An appropriate scaffold Stem cells with dental pulp progenitor capabilities Signaling molecules (16)
The use of various disinfecting agents and restorative materials in the access cavities appears to be effective in creating and maintaining disinfection before performing regenerative endodontics (17). A blood clot appears to be an inexpensive and adequate scaffold for growth factors necessary to promote cell growth and differentiation (17). A substantial number of mesenchymal stem cells are present in the apical tissues in mature and immature teeth (18). Growth factors and signaling molecules are present in the tissues surrounding the dental pulp and can stimulate cellular differentiation and proliferation (17). In the present study, the teeth in the positive control group had all the elements required for pulp-dentin complex formation. As expected, every tooth in this group had a normal pulp surrounded by thick dentin and normal apical closure with natural narrowing of the root canal space. The histologic features of the teeth in the negative control group with full pulp extirpation were dissimilar to those found in the positive JOE — Volume 44, Number 12, December 2018
control group and like those reported previously in experimental animals (2–8) and human teeth (9–11) treated with revascularization procedures. The findings in these teeth show that true regeneration of the pulp-dentin complex has not occurred (19). The tissue response of ferrets in this experiment might be different from what might be expected in humans. However, our findings corroborate those reported previously in experimental animals and human teeth treated with revascularization procedures (2–11). Because we found no inflammatory cells in any of the samples examined in this study, we did not quantify the types or number of inflammatory cells. The presence of thin root canal walls and ingrowth of bone into the root canal space and the absence of physiologic closure of the apical foramen in these teeth indicate the absence of ideal conditions for pulp-dentin complex regeneration. The total mechanical removal of the dental pulp was accomplished using barbed broaches. Because of the immaturity of teeth and the presence of large pulps, total removal of the pulp could be easily observed by the operators, and there was no need to instrument the apical foramen with standard instruments. This action might have eliminated the barrier that normally separates osteogenic cells from the surface of dentin (ie, calcium phosphate–containing tissues). It is well established that calcium phosphate is osteoconductive (20). The mechanism to explain osteoconductivity has not been established, but it seems likely that osteogenic cells respond to calcium ions that are present at the calcium phosphate surface of dentin through mechanisms including the L calcium channel and a calcium-sensing receptor, both of which are known to promote osteogenesis (21, 22). Accordingly, we propose this as a possible mechanism that accounts for the observed bone formation in the canal in teeth with total pulp extirpation.
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex
1799
Regenerative Endodontics
Figure 4. (A) A preoperative CBCT scan of a mandibular cuspid, (B) a postoperative CBCT scan of the same tooth 3 months later showing complete closure of the apex and coronal bridge formation under MTA and no signs of intracanal calcification, (C) a histologic section of the same tooth with pulp amputation 1–2 mm from the radiographic apex showing thin canal walls with no evidence of physiologic apical narrowing and connective tissue containing numerous blood vessels and fibroblasts plus bony ingrowth extending to the coronal third of the root (original magnification 1), (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
Miller et al (23) have recently reported that MTA promotes differentiation of stem cells in the apical papilla into a mineralizing phenotype with greater osteoblastic marker expression. Previous in vivo studies did not promote bone formation when MTA was used as a pulp capping or root-end filling material (24–27). Therefore, bone ingrowth in these teeth with immature apices may have resulted from elimination of the barrier that normally separates osteogenic cells from the surface of dentin and may not be related to differentiation of stem cells in response to MTA (20). Our results indicate that the method of pulp extirpation or necrosis may have a significant effect on the ability of an immature tooth to regenerate the pulp-dentin complex. The histologic findings in most teeth (13/14 samples) with pulp removal either 1–2 mm from the radiographic apex or 2–4 mm short
of the radiographic apex were like those found in the positive control group and those reported previously in the revascularization of replanted and transplanted immature teeth of experimental animals (28–30); traumatically avulsed teeth in humans after revascularization (31–33); a case report of an immature permanent incisor tooth with irreversible pulpitis and without apical periodontitis treated with revascularization procedures (12); and another case report of a permanent premolar tooth that was accidently extracted, replanted, had necrotic pulp, and possibly intact dental papilla when it was treated later with regenerative endodontics (13). The observed results might also be caused by the methods used for the removal of pulpal tissue despite using large files and leaving some peripheral pulp tissue intact, thus giving the final healed tissue the traditional pulpal histologic architecture.
Figure 5. (A) A preoperative CBCT scan of a maxillary cuspid; (B) a postoperative CBCT scan of the same tooth 3 months later showing complete closure of the apex, coronal bridge formation under MTA, and no signs of intracanal calcification; (C) a histologic section of the same tooth with pulp amputation 2–4 mm from the radiographic apex showing thick root canal walls with a physiologic apical narrowing with apical deltas, normal pulp tissue containing fibroblasts, and blood vessels that extend to the level of the MTA, where a hard tissue bridge had formed (original magnification 1.6), (D) a higher magnification of the coronal segment of the tooth (original magnification 4); (E) a higher magnification of the middle portion of the tooth (original magnification 4); and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
1800
Torabinejad et al.
JOE — Volume 44, Number 12, December 2018
Regenerative Endodontics The presence of thickened root canal walls and ingrowth of normal pulp tissue with occasional calcification in the root canal space and physiologic closure of the apical foramen in these teeth indicate the presence of stem cells from apical papilla with dental pulp progenitor abilities and/ or signaling molecules. It is well established that dental pulp contains stem cells with properties capable of generating new dental pulp (34). These findings show the ability of the pulp to regrow into the root canals of immature teeth. The histologic findings in 1 of 7 samples with pulp removal to 1 mm from the radiographic apex were like those of teeth with a total pulp extirpation, and it may be caused by damage of the apical papilla during pulp removal. Similar effects might happen after pulp necrosis and periapical lesion formation in teeth with necrotic pulp and open apices that undergo pulp regeneration procedures. Unlike the presence of bonelike material in the root canals of teeth with full pulp extirpation, most samples of teeth with pulp removal either 1–2 mm to the radiographic apex or 2–4 mm short of the radiographic apex had no intracanal bone formation. Based on these results, it appears that when some residual pulp tissues were left in the apical portion of the canal, in effect, it provided a barrier against osteogenic cells, precluding bone formation in the canal and allowing dentin deposition by the newly formed pulp. Bleeding from the residual pulp may have produced platelet-rich plasma (PRP) above the residual dental pulp. PRP contains mitogens for various cell types (35). Accordingly, we propose that the PRP may have acted on the stem cells in the residual pulp to allow regeneration of normal pulp tissue that filled the entire root canal space. Based on the results of this animal model, it appears that regeneration of the pulp-dentin complex is possible when 1–4 mm of pulp remains in the apical segment of immature teeth. This mechanistic approach provides a potential foundation for future vital pulp therapy and pulp regenerative procedures. Future studies are needed to investigate the potential of residual inflamed pulp on the regeneration of the pulp-dentin complex in immature and mature teeth.
Acknowledgments The authors thank Dr Kenneth Abramovitch from the Department of Radiology, School of Dentistry, Loma Linda University, Loma Linda, CA, for his assistance with taking and interpreting the cone-beam computed tomographic images of this study, Dr Udochukwu Oyoyo from the Department of Dental Educational Services, School of Dentistry, Loma Linda University for his assistance with statistical analysis of data of this study, and Mr John Hough from the Department of Pathology, School of Medicine, Loma Linda University for his histologic technical assistance. The authors deny any conflicts of interest related to this study.
References 1. Torabinejad M, Nosrat A, Verma P, Udochukwu O. Regenerative endodontic treatment or mineral trioxide aggregate apical plug in teeth with necrotic pulps and open apices: a systematic review and meta-analysis. J Endod 2017;43:1806–20. 2. 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 with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:779–87. 3. Torabinejad M, Faras H, Corr R, et al. Histological examinations of teeth treated with two scaffolds: a pilot animal investigation. J Endod 2014;40:515–20. 4. Torabinejad M, Milan M, Shabahang S, et al. Histologic examination of teeth with necrotic pulps and periapical lesions treated with 2 scaffolds: an animal investigation. J Endod 2015;41:846–52. 5. Scarparo RK, Dondoni L, Bottcher DE, et al. Response to intracanal medication in immature teeth with pulp necrosis: an experimental model in rat molars. J Endod 2011;37:1069–73. 6. Wang X, Thibodeau B, Trope M, et al. 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.
JOE — Volume 44, Number 12, December 2018
7. Yamauchi N, Yamauchi S, Nagaoka H, et al. Tissue engineering strategies for immature teeth with apical periodontitis. J Endod 2011;37:390–7. 8. Zuong XY, Yang YP, Chen WX, et al. [Pulp revascularization of immature anterior teeth with apical periodontitis]. Hua Xi Kou Qiang Yi Xue Za Zhi 2010;28: 672–4. 9. Martin G, Ricucci D, Gibbs JL, Lin LM. Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod 2013;39:138–44. 10. Nosrat A, Kolahdouzan A, Hosseini A, et al. Histologic outcomes of uninfected human immature teeth treated with regenerative endodontics: 2 case reports. J Endod 2015;41:1725–9. 11. Shimizu E, Ricucci D, Albert J, et al. Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. J Endod 2013;39:1078–83. 12. Shimizu E, Jong G, Partridge N, et al. Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure. J Endod 2012;38:1293–7. 13. Torabinejad M, Faras H. A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma. J Endod 2012;38:864–8. 14. Torabinejad M, Corr R, Buhrley M, et al. An animal model to study regenerative endodontics. J Endod 2011;37:197–202. 15. Fouad A, Walton RE, Rittman BR. Healing of induced periapical lesions in ferret canines. J Endod 1993;19:123–9. 16. Hargreaves KM, Geisler T, Henry M, Wang Y. Regeneration potential of the young permanent tooth: what does the future hold? Pediatr Dent 2008;30:253–60. 17. Wigler R, Kaufman AY, Lin S, et al. Revascularization: a treatment for permanent teeth with necrotic pulp and incomplete root development. J Endod 2013;39:319–26. 18. Lovelace TW, Henry MA, Hargreaves KM, Diogenes A. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 2011;37:133–8. 19. Kumar V, Abbas AK, Fausto N, Aster J. In: Robbins and Cotran Pathologic Basis of Disease, 8th ed. Philadelphia: Saunders; 2009. 20. Ko C-L, Chen W-C, Chen J-C, et al. Properties of osteoconductive biomaterials: calcium phosphate cement with different ratios of platelet-rich plasma as identifiers. Mater Sci Eng C Mater Biol Appl 2013;33:3537–44. 21. Wen L, Wang Y, Wang H, et al. L type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Biochem Biophys Res Commun 2012;424:439–45. 22. Goltzman D, Hendy GN. The calcium-sensing receptor in bone—mechanistic and therapeutic insights. Nat Rev Endocrinol 2015;11:298–307. 23. Miller AA, Takimoto K, Wealleans J, Diogenes A. Effect of 3 bioceramic materials on stem cells of the apical papilla proliferation and differentiation using a dentin disk model. J Endod 2018;44:599–603. 24. Andelin WE, Shabahang S, Wright K, Torabinejad M. Identification of hard tissue after experimental pulp capping using dentin sialoprotein (DSP) as a marker. J Endod 2003;29:646–50. 25. Pitt Ford TR, Torabinejad M, Abedi HR, et al. Using mineral trioxide aggregate as a pulp-capping material. J Am Dent Assoc 1996;127:1491–4. 26. Pitt Ford TR, Torabinejad M, McKendry DJ, et al. Use of mineral trioxide aggregate for repair of furcal perforations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:756–63. 27. Torabinejad M, Pitt Ford TR, McKendry DJ, et al. Histologic assessment of mineral trioxide aggregate as a root-end filling in monkeys. J Endod 1997;23:225–8. 28. 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. 29. Cvek M, Cleaton-Jones P, Austin J, et al. Pulp revascularization in reimplanted immature monkey incisors–predictability and the effect of antibiotic systemic prophylaxis. Endod Dent Traumatol 1990;6:157–69. 30. Skoglund A, Tronstad L. Pulpal changes in replanted and autotransplanted immature teeth of dogs. J Endod 1981;7:309–16. 31. Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM. Replantation of 400 avulsed permanent incisors. 2. Factors related to pulpal healing. Endod Dent Traumatol 1995;11:59–68. 32. Johnson WT, Goodrich JL, James GA. Replantation of avulsed teeth with immature root development. Oral Surg Oral Med Oral Pathol 1985;60:420–7. 33. Kling M, Cvek M, Mejare I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 1986;2: 83–9. 34. Potdar PD, Jethmalani YD. Human dental pulp stem cells: applications in future regenerative medicine. World J Stem Cells 2015;7:839–51. 35. Gruber R, Varga F, Fischer MB, Watzek G. Platelets stimulate proliferation of bone cells: involvement of platelet derived growth factor, microparticles and membranes. Clin Oral Implants Res 2002;13:529–35.
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex
1801
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex: An In Vivo Investigation Mahmoud Torabinejad, DMD, MSD, PhD,* Arin Alexander, DMD, MSD, MBA,† Seyed Aliakbar Vahdati, DDS, MSD,‡ Anupama Grandhi, BDS, DDS,§ David Baylink, MD, FACB,§ and Shahrokh Shabahang, DDS, MS, PhDk Abstract Introduction: Current pulp revascularization procedures in teeth with necrotic pulps and open apices have produced histologic evidence of connective tissue growth, cementum, and bone within the root canals of experimental animals. This study aims to investigate the effect of maintaining uninflamed residual apical pulp tissue on the histologic outcome of pulp-dentin complex regeneration after a revascularization procedure in immature ferret cuspid teeth. Methods: Twenty-eight cuspid teeth from 7 young male ferrets were used in this experiment. Seven teeth were reserved to serve as positive control samples without any treatment. In another 7 teeth, the pulp was completely extirpated (negative control), whereas the pulp of the remaining 14 teeth were removed to either 1–2 mm short of the apex (7 samples) or 2–4 mm short of the apex (7 samples). Blood clots were covered with mineral trioxide aggregate at the cementoenamel junction level of each tooth. Three months later, block sections were removed for histologic evaluations, and the data were statistically analyzed with the chi-square test (P < .05). Results: All teeth with complete pulp extirpation showed the presence of bone inside the root canal. In contrast, the root canals for most teeth with pulp amputation 1–4 mm from the radiographic apex were filled with normal pulp, which extended coronally to the mineral trioxide aggregate, where hard tissue bridges had formed. Conclusions: Based on these results, we concluded that regeneration of the pulpdentin complex is possible when the apical 1–4 mm of the apical pulp remains intact in immature teeth. (J Endod 2018;44:1796–1801)
N
Key Words
All experimental procedures were performed in accordance with protocols approved by the Loma Linda University Animal Research Committee. A total of 28 immature cuspid teeth (14) in seven 97- to 101-day-old male ferrets were used in this
Bone remodeling/regeneration, cementum, dentinogenesis, endodontics, odontogenesis, pulp biology
umerous case reports Significance and case series as The results of this animal study show that complete well as a few prospective regeneration of the pulp-dentin complex is and retrospective cohort possible when 1–4 mm of normal pulp remains in studies and randomized the apical segment of the root canal system. controlled trials have been published showing radiographic illustration of the continuation of root development and the resolution of periapical lesions in teeth with necrotic pulp and open apices after treatment with “revascularization/regeneration” procedures (1). Except for a few human case reports, the outcomes for most of these reports were evaluated clinically and radiographically (1). Radiographic resolution of periapical lesions, root-end closure, and canal wall thickening in teeth with necrotic pulp and open apices give an indication that a normal, functional pulp can be regenerated. Several animal studies (2–8) and 3 human case reports (9–11) have examined the nature of tissues present in the root canals of teeth treated with regenerative endodontic procedures and have reported the presence of connective tissue, cementum, and bone in the root canals of the experimental teeth instead of pulp tissue. Only 2 human case reports have shown regeneration of pulplike tissue after performing regenerative endodontic procedures (12, 13). Because previous investigations have not determined the effect of inflammation and infection on the capability of an immature tooth to regenerate the pulp-dentin complex, an animal model is needed to determine the effect of these factors on the capacity of an immature tooth to regenerate the pulp-dentin complex at various stages of pulpal and periapical pathosis. The first phase of these investigations is to examine histologically the capacity of a tooth for true pulp-dentin regeneration under ideal conditions in immature teeth without any pulpal and/or periapical pathosis. The development of this model has the potential for future studies leading to the determination of factors affecting pulp-dentin regeneration. The aim of this study was to investigate the effect of residual uninflamed apical pulp tissue on the histologic outcome of pulp-dentin complex regeneration in immature ferret cuspid teeth.
Materials and Methods
From the *Torabinejad Institute of Surgical Education and Research Venues, Irvine, California; †Private practice, Los Angeles, California; ‡Private practice, Newport Beach, California; §Loma Linda University, Loma Linda, California; and kPrivate practice, Redlands, California. Address requests for reprints to Dr. Mahmoud Torabinejad, 9950 Irvine Center Drive, Irvine, CA 92618. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.09.005
1796
Torabinejad et al.
JOE — Volume 44, Number 12, December 2018
Regenerative Endodontics experiment. The teeth were randomly assigned to the following groups: group 1, positive control (7 teeth); group 2, complete pulp extirpation (7 teeth); group 3, pulp amputation to 1–2 mm short of the radiographic apex (7 teeth); and group 4, pulp amputation 2–4 mm from the radiographic apex (7 teeth). Preoperative cone-beam computed tomographic (CBCT) images were initially obtained to examine the condition of the apex and the absence of any preexisting pathosis in the cuspid teeth. These initial radiographic examinations also served as a guide for the levels of pulp amputation in groups 3 and 4 and were also a baseline for the comparison of root length, thickness, and apical closure before and after completion of the experiment. All procedures were conducted under a clean protocol with the use of sterile materials and equipment. After obtaining general anesthesia, each experimental tooth was decoronated with a sterile diamond bur approximately 3 mm above the gingival crest to expose the dental pulp. Seven teeth in group 1 served as positive control samples and did not receive any treatment. This was done to examine normal growth in length, dentin thickness, and apical closure of teeth in ferrets for comparison reasons with teeth in the experimental groups. The pulp tissue in 7 teeth in group 2 was completely extirpated with sterile barbed broaches (negative control). This procedure resulted in the filling of the canal with fresh blood from the periapical tissues and the formation of a blood clot below the cementoenamel junction. No root irrigation was used after the removal of the pulps in the group. The root canals of the remaining 14 teeth were cleaned with very large files (100–120 file sizes) to approximately either 1–2 mm from the apex (7 samples, group 3) or 2–4 mm short of the apex (7 samples, group 4) based on the measurements gathered from the preoperative CBCT images obtained before the experiment. Similar to teeth in group 2, this procedure also resulted in the production of fresh blood from the residual apical pulp tissues and the formation of a blood clot below the cementoenamel junction. No overinstrumentation beyond the apical foramen was performed in any of these specimens to induce bleeding from periapical tissues. No root irrigation was used after the removal of the pulps in these groups. Gray mineral trioxide aggregate (MTA) (Dentsply Tulsa Dental, Tulsa, OK) was gently placed on the clotted blood in all the experimental teeth, and the coronal access was sealed with amalgam. After 3 months, postoperative CBCT images were obtained, and all animals were sacrificed followed by perfusion. After the completion of perfusion, block sections containing the maxilla and the mandible were removed and placed into bottles containing 10% buffered formalin solutions for 2 weeks. Block sections were decalcified in 10% EDTA (Sigma-Aldrich, St Louis, MO) and were then embedded in paraffin (Thermo Fisher Scientific, Fair Lawn, NJ). Step serial sections of 5-mm thickness were
prepared and stained using hematoxylin-eosin. Samples were evaluated microscopically by a pathologist who was not aware of the nature of treatments performed in the experiment. Data were statistically analyzed with the chi-square test (P < .05) to evaluate the radiographic preoperative diameter of the apical opening of roots (mm), postoperative incidence of closure (yes) or nonclosure (no) of the apical opening, incidence of presence (yes) or absence (no) of normal pulp tissue, incidence of presence (yes) or absence (no) of intracanal and periapical inflammation, and incidence of presence (yes) or absence (no) of normal root canal wall thickening in the experimental teeth compared with the control samples.
Results The radiographic and histologic findings are summarized in Table 1. Radiographic examinations of the cuspid teeth using preoperative CBCT images showed the presence of an average apical foramen diameter of 0.56 mm. Similar examinations for the same teeth using postoperative CBCT imaging showed complete closure of the apices at the end of the experiment (Figs. 1–5). In the positive control teeth, normal tooth anatomy was noted (Fig. 1C–F). The root canals in this group were filled with normalpulp tissue. The canal walls were thick, and the apical region showed physiologic narrowing with apical deltas. The periradicular tissues were normal without any inflammation. In the teeth with full pulp extirpation (negative control), the canal walls were generally thin, and there was no evidence of physiologic apical narrowing or root canal wall thickening compared with the control samples. Instead of normal pulp tissue, there was a bony ingrowth in the canal space of these teeth that extended to the level of the MTA, where a hard tissue bridge had formed (Fig. 2C–F). No normal pulp was observed in any of the specimens in this group. There was no intracanal or periapical inflammation in these samples. In 6 of 7 teeth with pulp amputation 1–2 mm from the radiographic apex, the canal walls appeared normal, and there was evidence of a physiologic apical narrowing like those observed in the positive control samples (Fig. 3C–F). The canals of these teeth were filled with normal pulp tissue that extended to the level of the MTA, where a hard tissue bridge had formed. There was no evidence of apical bony ingrowth or inflammation in the canal space or periapical inflammation in 6 of 7 teeth in this group. In 1 sample in this group, there was connective tissue containing numerous blood vessels and fibroblasts plus bony ingrowth extending to the coronal third of the root (Fig. 4A–F). There was no inflammation in the canal space or in the periapical tissues in any of the teeth in this group. In all the teeth with pulp amputation 2–4 mm from the radiographic apex, the canal walls appeared normal, and there was evidence of a physiologic apical narrowing like those observed in the positive
TABLE 1. Radiographic Preoperative Diameter of the Apical Opening of Roots (mm), Postoperative Incidence of Closure (Yes) or Nonclosure (No) of the Apical Opening, Incidence of Presence (Yes) or Absence (No) of Normal Pulp Tissue, Incidence of Presence (Yes) or Absence (No) of Intracanal and Periapical Inflammation, and Incidence of Presence (Yes) or Absence (No) of Normal Root Canal Wall Thickening
Group
Preoperative average diameter of apical opening (mm)
Positive control Negative control 1–2 mm pulp left 2–4 mm pulp left
0.47 0.59 0.33 0.76
JOE — Volume 44, Number 12, December 2018
Presence (yes) or absence (no) of Presence (yes) or Postoperative intracanal and/or absence (no) of closure (yes) or periapical nonclosure (no) of normal pulp tissue inflammatory cells in the canal the apical opening Yes = 7, no = 0 Yes = 7, no = 0 Yes = 7, no = 0 Yes = 7, no = 0
Yes = 7, no = 0 Yes = 0, no = 7 Yes = 6, no = 1 Yes = 7, no = 0
Yes = 0, no = 7 Yes = 0, no = 7 Yes = 0, no = 7 Yes = 0, no = 7
Presence (yes) or absence (no) of normal root canal Apex wall thickening closure Yes = 7, no = 0 Yes = 0, no = 7 Yes = 6, no = 1 Yes = 7, no = 0
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex
Yes = 7 Yes = 7 Yes = 7 Yes = 7
1797
Regenerative Endodontics
Figure 1. (A) A preoperative CBCT scan of a maxillary cuspid, (B) a postoperative CBCT scan of the same tooth 3 months later, (C) a histologic section of the same tooth at the end of the experiment showing normal pulp and periradicular tissues (original magnification 1.1), (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
control samples (Fig. 5C–F). The canals of these teeth were filled with normal pulp tissue that extended to the level of the MTA, where a hard tissue bridge had formed. There was no apical bony ingrowth or inflammation in the canal space or periapical inflammation in any tooth in this group. No statistical differences were computed for comparison of the radiographic preoperative diameter of the apical opening of roots, postoperative incidence of closure or nonclosure of the apical opening, and incidence of the presence or absence of intracanal and/or periapical inflammation in the experimental teeth compared with the control samples. Statistically significant differences in responses were observed for the presence of normal pulp tissue (c23 = 23.8, P < .001) and root canal wall thickening (c23 = 23.8, df = 3, P < .001) in negative control teeth with total pulp extirpation and those with 1–2 mm residual pulp tissue, 2–4 mm residual pulp tissue, and the positive control samples. Statistically significant differences in responses were also found for the presence of normal pulp tissue (c23 = 23.8, P < .001) and root canal
wall thickening (c23 = 23.8, P < .001) in experimental teeth with 1– 2 mm residual pulp tissue and 2–4 mm residual pulp tissue as well as the positive controls. In addition, there was a statistically significant difference in the response to treatment comparing groups 2 (complete pulp extirpation), 3 (1–2 mm residual pulp tissue), and 4 (2–4 mm residual pulp tissue) (c22 = 17.365, P < .001). However, there was no statistically significant difference in outcome regarding the presence of normal pulp tissue and root canal wall thickening between group 3 (1–2 mm residual pulp tissue) and group 4 (2–4 mm residual pulp tissue) (c21 = 1.077, P = .299).
Discussion To determine the nature of tissues present in teeth that have had regenerative endodontics, it is necessary to remove the tissues within the root canals or to extract the whole tooth, thus leading to obvious ethical concerns in patients. Alternatively, such an approach can be
Figure 2. (A) A preoperative CBCT scan of a maxillary cuspid, (B) a postoperative CBCT scan of the same tooth 3 months later showing signs of intracanal calcification, (C) a histologic section of the same tooth with full pulp extirpation showing thin canal walls with no evidence of physiologic apical narrowing and a bony ingrowth into the canal space (original magnification 1.6), (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
1798
Torabinejad et al.
JOE — Volume 44, Number 12, December 2018
Regenerative Endodontics
Figure 3. (A) A preoperative CBCT scan of a mandibular cuspid; (B) a postoperative CBCT scan of the same tooth 3 months later showing complete closure of the apex and coronal bridge formation under MTA and no signs of intracanal calcification; (C) a histologic section of the same tooth with pulp amputation 1–2 mm from the radiographic apex showing thick root canal walls with a physiologic apical narrowing with apical deltas, normal pulp tissue containing fibroblasts, and blood vessels that extend to the level of the MTA, where a hard tissue bridge had formed(original magnification 2); (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
evaluated by performing animal experiments (14). A ferret has been suggested as a suitable animal model to perform endodontic studies (14, 15). The results of this experiment showed that true pulp regeneration after revascularization occurs when 1–4 mm of normal apical pulp tissue remains in immature teeth. The elements needed for pulp-dentin complex regeneration are 1. 2. 3. 4. 5.
A disinfected root canal space An adequate coronal seal An appropriate scaffold Stem cells with dental pulp progenitor capabilities Signaling molecules (16)
The use of various disinfecting agents and restorative materials in the access cavities appears to be effective in creating and maintaining disinfection before performing regenerative endodontics (17). A blood clot appears to be an inexpensive and adequate scaffold for growth factors necessary to promote cell growth and differentiation (17). A substantial number of mesenchymal stem cells are present in the apical tissues in mature and immature teeth (18). Growth factors and signaling molecules are present in the tissues surrounding the dental pulp and can stimulate cellular differentiation and proliferation (17). In the present study, the teeth in the positive control group had all the elements required for pulp-dentin complex formation. As expected, every tooth in this group had a normal pulp surrounded by thick dentin and normal apical closure with natural narrowing of the root canal space. The histologic features of the teeth in the negative control group with full pulp extirpation were dissimilar to those found in the positive JOE — Volume 44, Number 12, December 2018
control group and like those reported previously in experimental animals (2–8) and human teeth (9–11) treated with revascularization procedures. The findings in these teeth show that true regeneration of the pulp-dentin complex has not occurred (19). The tissue response of ferrets in this experiment might be different from what might be expected in humans. However, our findings corroborate those reported previously in experimental animals and human teeth treated with revascularization procedures (2–11). Because we found no inflammatory cells in any of the samples examined in this study, we did not quantify the types or number of inflammatory cells. The presence of thin root canal walls and ingrowth of bone into the root canal space and the absence of physiologic closure of the apical foramen in these teeth indicate the absence of ideal conditions for pulp-dentin complex regeneration. The total mechanical removal of the dental pulp was accomplished using barbed broaches. Because of the immaturity of teeth and the presence of large pulps, total removal of the pulp could be easily observed by the operators, and there was no need to instrument the apical foramen with standard instruments. This action might have eliminated the barrier that normally separates osteogenic cells from the surface of dentin (ie, calcium phosphate–containing tissues). It is well established that calcium phosphate is osteoconductive (20). The mechanism to explain osteoconductivity has not been established, but it seems likely that osteogenic cells respond to calcium ions that are present at the calcium phosphate surface of dentin through mechanisms including the L calcium channel and a calcium-sensing receptor, both of which are known to promote osteogenesis (21, 22). Accordingly, we propose this as a possible mechanism that accounts for the observed bone formation in the canal in teeth with total pulp extirpation.
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex
1799
Regenerative Endodontics
Figure 4. (A) A preoperative CBCT scan of a mandibular cuspid, (B) a postoperative CBCT scan of the same tooth 3 months later showing complete closure of the apex and coronal bridge formation under MTA and no signs of intracanal calcification, (C) a histologic section of the same tooth with pulp amputation 1–2 mm from the radiographic apex showing thin canal walls with no evidence of physiologic apical narrowing and connective tissue containing numerous blood vessels and fibroblasts plus bony ingrowth extending to the coronal third of the root (original magnification 1), (D) a higher magnification of the coronal segment of the tooth (original magnification 4), (E) a higher magnification of the middle portion of the tooth (original magnification 4), and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
Miller et al (23) have recently reported that MTA promotes differentiation of stem cells in the apical papilla into a mineralizing phenotype with greater osteoblastic marker expression. Previous in vivo studies did not promote bone formation when MTA was used as a pulp capping or root-end filling material (24–27). Therefore, bone ingrowth in these teeth with immature apices may have resulted from elimination of the barrier that normally separates osteogenic cells from the surface of dentin and may not be related to differentiation of stem cells in response to MTA (20). Our results indicate that the method of pulp extirpation or necrosis may have a significant effect on the ability of an immature tooth to regenerate the pulp-dentin complex. The histologic findings in most teeth (13/14 samples) with pulp removal either 1–2 mm from the radiographic apex or 2–4 mm short
of the radiographic apex were like those found in the positive control group and those reported previously in the revascularization of replanted and transplanted immature teeth of experimental animals (28–30); traumatically avulsed teeth in humans after revascularization (31–33); a case report of an immature permanent incisor tooth with irreversible pulpitis and without apical periodontitis treated with revascularization procedures (12); and another case report of a permanent premolar tooth that was accidently extracted, replanted, had necrotic pulp, and possibly intact dental papilla when it was treated later with regenerative endodontics (13). The observed results might also be caused by the methods used for the removal of pulpal tissue despite using large files and leaving some peripheral pulp tissue intact, thus giving the final healed tissue the traditional pulpal histologic architecture.
Figure 5. (A) A preoperative CBCT scan of a maxillary cuspid; (B) a postoperative CBCT scan of the same tooth 3 months later showing complete closure of the apex, coronal bridge formation under MTA, and no signs of intracanal calcification; (C) a histologic section of the same tooth with pulp amputation 2–4 mm from the radiographic apex showing thick root canal walls with a physiologic apical narrowing with apical deltas, normal pulp tissue containing fibroblasts, and blood vessels that extend to the level of the MTA, where a hard tissue bridge had formed (original magnification 1.6), (D) a higher magnification of the coronal segment of the tooth (original magnification 4); (E) a higher magnification of the middle portion of the tooth (original magnification 4); and (F) a higher magnification of the apical portion of the tooth (original magnification 4).
1800
Torabinejad et al.
JOE — Volume 44, Number 12, December 2018
Regenerative Endodontics The presence of thickened root canal walls and ingrowth of normal pulp tissue with occasional calcification in the root canal space and physiologic closure of the apical foramen in these teeth indicate the presence of stem cells from apical papilla with dental pulp progenitor abilities and/ or signaling molecules. It is well established that dental pulp contains stem cells with properties capable of generating new dental pulp (34). These findings show the ability of the pulp to regrow into the root canals of immature teeth. The histologic findings in 1 of 7 samples with pulp removal to 1 mm from the radiographic apex were like those of teeth with a total pulp extirpation, and it may be caused by damage of the apical papilla during pulp removal. Similar effects might happen after pulp necrosis and periapical lesion formation in teeth with necrotic pulp and open apices that undergo pulp regeneration procedures. Unlike the presence of bonelike material in the root canals of teeth with full pulp extirpation, most samples of teeth with pulp removal either 1–2 mm to the radiographic apex or 2–4 mm short of the radiographic apex had no intracanal bone formation. Based on these results, it appears that when some residual pulp tissues were left in the apical portion of the canal, in effect, it provided a barrier against osteogenic cells, precluding bone formation in the canal and allowing dentin deposition by the newly formed pulp. Bleeding from the residual pulp may have produced platelet-rich plasma (PRP) above the residual dental pulp. PRP contains mitogens for various cell types (35). Accordingly, we propose that the PRP may have acted on the stem cells in the residual pulp to allow regeneration of normal pulp tissue that filled the entire root canal space. Based on the results of this animal model, it appears that regeneration of the pulp-dentin complex is possible when 1–4 mm of pulp remains in the apical segment of immature teeth. This mechanistic approach provides a potential foundation for future vital pulp therapy and pulp regenerative procedures. Future studies are needed to investigate the potential of residual inflamed pulp on the regeneration of the pulp-dentin complex in immature and mature teeth.
Acknowledgments The authors thank Dr Kenneth Abramovitch from the Department of Radiology, School of Dentistry, Loma Linda University, Loma Linda, CA, for his assistance with taking and interpreting the cone-beam computed tomographic images of this study, Dr Udochukwu Oyoyo from the Department of Dental Educational Services, School of Dentistry, Loma Linda University for his assistance with statistical analysis of data of this study, and Mr John Hough from the Department of Pathology, School of Medicine, Loma Linda University for his histologic technical assistance. The authors deny any conflicts of interest related to this study.
References 1. Torabinejad M, Nosrat A, Verma P, Udochukwu O. Regenerative endodontic treatment or mineral trioxide aggregate apical plug in teeth with necrotic pulps and open apices: a systematic review and meta-analysis. J Endod 2017;43:1806–20. 2. 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 with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:779–87. 3. Torabinejad M, Faras H, Corr R, et al. Histological examinations of teeth treated with two scaffolds: a pilot animal investigation. J Endod 2014;40:515–20. 4. Torabinejad M, Milan M, Shabahang S, et al. Histologic examination of teeth with necrotic pulps and periapical lesions treated with 2 scaffolds: an animal investigation. J Endod 2015;41:846–52. 5. Scarparo RK, Dondoni L, Bottcher DE, et al. Response to intracanal medication in immature teeth with pulp necrosis: an experimental model in rat molars. J Endod 2011;37:1069–73. 6. Wang X, Thibodeau B, Trope M, et al. 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.
JOE — Volume 44, Number 12, December 2018
7. Yamauchi N, Yamauchi S, Nagaoka H, et al. Tissue engineering strategies for immature teeth with apical periodontitis. J Endod 2011;37:390–7. 8. Zuong XY, Yang YP, Chen WX, et al. [Pulp revascularization of immature anterior teeth with apical periodontitis]. Hua Xi Kou Qiang Yi Xue Za Zhi 2010;28: 672–4. 9. Martin G, Ricucci D, Gibbs JL, Lin LM. Histological findings of revascularized/revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod 2013;39:138–44. 10. Nosrat A, Kolahdouzan A, Hosseini A, et al. Histologic outcomes of uninfected human immature teeth treated with regenerative endodontics: 2 case reports. J Endod 2015;41:1725–9. 11. Shimizu E, Ricucci D, Albert J, et al. Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. J Endod 2013;39:1078–83. 12. Shimizu E, Jong G, Partridge N, et al. Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure. J Endod 2012;38:1293–7. 13. Torabinejad M, Faras H. A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma. J Endod 2012;38:864–8. 14. Torabinejad M, Corr R, Buhrley M, et al. An animal model to study regenerative endodontics. J Endod 2011;37:197–202. 15. Fouad A, Walton RE, Rittman BR. Healing of induced periapical lesions in ferret canines. J Endod 1993;19:123–9. 16. Hargreaves KM, Geisler T, Henry M, Wang Y. Regeneration potential of the young permanent tooth: what does the future hold? Pediatr Dent 2008;30:253–60. 17. Wigler R, Kaufman AY, Lin S, et al. Revascularization: a treatment for permanent teeth with necrotic pulp and incomplete root development. J Endod 2013;39:319–26. 18. Lovelace TW, Henry MA, Hargreaves KM, Diogenes A. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 2011;37:133–8. 19. Kumar V, Abbas AK, Fausto N, Aster J. In: Robbins and Cotran Pathologic Basis of Disease, 8th ed. Philadelphia: Saunders; 2009. 20. Ko C-L, Chen W-C, Chen J-C, et al. Properties of osteoconductive biomaterials: calcium phosphate cement with different ratios of platelet-rich plasma as identifiers. Mater Sci Eng C Mater Biol Appl 2013;33:3537–44. 21. Wen L, Wang Y, Wang H, et al. L type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Biochem Biophys Res Commun 2012;424:439–45. 22. Goltzman D, Hendy GN. The calcium-sensing receptor in bone—mechanistic and therapeutic insights. Nat Rev Endocrinol 2015;11:298–307. 23. Miller AA, Takimoto K, Wealleans J, Diogenes A. Effect of 3 bioceramic materials on stem cells of the apical papilla proliferation and differentiation using a dentin disk model. J Endod 2018;44:599–603. 24. Andelin WE, Shabahang S, Wright K, Torabinejad M. Identification of hard tissue after experimental pulp capping using dentin sialoprotein (DSP) as a marker. J Endod 2003;29:646–50. 25. Pitt Ford TR, Torabinejad M, Abedi HR, et al. Using mineral trioxide aggregate as a pulp-capping material. J Am Dent Assoc 1996;127:1491–4. 26. Pitt Ford TR, Torabinejad M, McKendry DJ, et al. Use of mineral trioxide aggregate for repair of furcal perforations. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:756–63. 27. Torabinejad M, Pitt Ford TR, McKendry DJ, et al. Histologic assessment of mineral trioxide aggregate as a root-end filling in monkeys. J Endod 1997;23:225–8. 28. 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. 29. Cvek M, Cleaton-Jones P, Austin J, et al. Pulp revascularization in reimplanted immature monkey incisors–predictability and the effect of antibiotic systemic prophylaxis. Endod Dent Traumatol 1990;6:157–69. 30. Skoglund A, Tronstad L. Pulpal changes in replanted and autotransplanted immature teeth of dogs. J Endod 1981;7:309–16. 31. Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM. Replantation of 400 avulsed permanent incisors. 2. Factors related to pulpal healing. Endod Dent Traumatol 1995;11:59–68. 32. Johnson WT, Goodrich JL, James GA. Replantation of avulsed teeth with immature root development. Oral Surg Oral Med Oral Pathol 1985;60:420–7. 33. Kling M, Cvek M, Mejare I. Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod Dent Traumatol 1986;2: 83–9. 34. Potdar PD, Jethmalani YD. Human dental pulp stem cells: applications in future regenerative medicine. World J Stem Cells 2015;7:839–51. 35. Gruber R, Varga F, Fischer MB, Watzek G. Platelets stimulate proliferation of bone cells: involvement of platelet derived growth factor, microparticles and membranes. Clin Oral Implants Res 2002;13:529–35.
Effect of Residual Dental Pulp Tissue on Regeneration of Dentin-pulp Complex
1801