- Email: [email protected]
Regenerative Endodontics: Regeneration or Repair?
Pulp Regeneration—Translational Opportunities
Regenerative Endodontics: Regeneration or Repair? Stephane R.J. Simon, DDS, PhD,*†‡§ Phillip L. Tomson, PhD,§ and Ariane Berdal, PhD*‡ Abstract Recent advances in biotechnology and translational research have made it possible to provide treatment modalities that protect the vital pulp, allow manipulation of reactionary and reparative dentinogenesis, and, more recently, permit revascularization of an infected root canal space. These approaches are referred to as regenerative procedures. The method currently used to determine the origin of the tissue secreted during the repair/ regeneration process is largely based on the identification of cellular markers (usually proteins) left by cells that were responsible for this tissue production. The presence of these proteins in conjunction with other indicators of cellular behavior (especially biomineralization) and analysis of the structure of the newly generated tissue allow conclusions to be made of how it was formed. Thus far, it has not been possible to truly establish the biological mechanism controlling tertiary dentinogenesis. This article considers current therapeutic techniques to treat the dentin-pulp complex and contextualize them in terms of reparative and regenerative processes. Although it may be considered a semantic argument rather than a biological one, the definitions of regeneration and repair are explored to clarify our position in this era of regenerative endodontics. (J Endod 2014;40:S70–S75)
Key Words Dentin bridge, endodontics, healing, regeneration, repair
From the *Department of Oral Biology, School of Dentistry, University of Paris Diderot, Paris, France; †H^opital de la Pitie Saleptriere, Paris, France; ‡UMRS INSERM 1138 TEAM 5, Paris, France; and §Oral Biology, School of Dentistry, University of Birmingham, United Kingdom. This paper is based on a presentation from the International Association for Dental Research (IADR) Pulp Biology and Regeneration Group Satellite Meeting, which was held March 24–26, 2013 in San Francisco, California. Address requests for reprints to Dr Stephane R.J. Simon, UMRS 1138, Team 5, Centre de Recherche des Cordeliers, 18-20 Rue de l’ecole de medicine, 75006 Paris, France. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.024
S70
Simon et al.
S
ignificant progress in the field of prevention and treatment of pulpal and periradicular disease has led to an increasing amount of research into the role of the dentin-pulp complex and its ability to repair itself and regenerate mineralized tissue. For many years, research laboratories have investigated the pulp healing process with far reaching aims of enhancing its inherent regenerative capacity to completely regenerate this unique tissue. Recent advances in biotechnology and translational research have made it possible to provide treatment modalities that protect the vital pulp, allow manipulation of reactionary and reparative dentinogenesis, and, more recently, permit revascularization of an infected root canal space. Although the volume of the mature pulp is very small (less than 100 mL), it is conceivable that regeneration of such a small tissue should be relatively easy. Unfortunately, this is not the case. The dental pulp is a complex specialized connective tissue that is enclosed in a mineralized shell and has a limited blood supply; these are only a few of the many obstacles faced by the clinicians and researchers attempting to design new therapeutic strategies for its regeneration. Regenerative endodontics should be considered as 2 entities. One is dentin-pulp complex regeneration (which could also be called dentin/odontoblast complex regeneration). This relates to preservation of pulp vitality and pulp capping. The second is dental pulp regeneration. This relates to regeneration of a vital tissue into an empty but infected root canal space.
Dentin-Pulp Complex Regeneration Pulp capping and dentin bridge formation induction have been used for more than 70 years, with the first experimental studies published by Zander (1) in 1939. If the right environmental conditions persist, clinical results can be encouraging and tend to motivate the clinician to maintain pulp vitality as long as possible because of the advantages it can bring in terms of prolonging the life of the tooth. Nevertheless, high-quality robust clinical trials are lacking (2), and published findings are contradictory, not allowing for clear guidance for this clinical technique. A recent systematic review demonstrated success rates (pulp vitality maintained) at 3 years of 72.9% for pulp capping and 99.4% for partial pulpotomy (3). However, in a recent randomized clinical trial, Bjorndal et al (4) showed success rates to be much lower, 31.8% for pulp capping and 34.5% for partial pulpotomy. Clinically, the aim of such treatment is to keep the pulp vital and maintain its homeostatic functions, thus avoiding pulpectomy or extraction of the tooth. Success of the treatment is assessed by the symptoms reported by the patients and through the use of relatively rudimentary investigations such as thermal tests, electrical tests, tenderness to percussion or palpation, and radiographic assessment. It has been well-established that clinical signs and symptoms do not correlate to the histologic status of the tooth (5). Biological research methods allow for more sophisticated assessment and enable the researcher to observe and analyze the histologic structure, cell behavior, and immunologic/inflammation status of the tissue concerned. Such techniques allow the pulpal responses to be assessed with greater accuracy than in clinical studies. To study the physiological and reparative processes of the pulp, in vitro experiments with immortalized cells or primary cell cultures can be used. The limitations of these studies must be acknowledged; they can only mimic biological processes such as mineral production and cannot prove conclusively the type of mineralized tissue formed. Frequently these experiments are supported with gene expression data to establish the likely gene regulation that induced this mineral production. Until phenotypic markers associated with dentin production are shown,
JOE — Volume 40, Number 4S, April 2014
Pulp Regeneration—Translational Opportunities
Figure 1. (A and B) Endodontic treatment by revascularization on tooth #8 of a 16-year-old girl. Note formation of a mineralized barrier distant from coronal filling material (mineral trioxide aggregate) (arrow) (C). At 18-month recall, the bone healing is complete, and the mineral barrier is still present (D). Nevertheless, no root lengthening or apexogenesis is noticeable.
the results are often viewed with skepticism to whether the mineral tissue formed is from odontoblastic origin.
Regeneration/Repair and Remodeling Bone is constantly being remodeled, and newly generated tissue is replaced within a few months by new bone. The turnover of bone means that gradually the newly secreted tissue will merge with other tissue laid down at different times, and new and old tissue becomes homogenous. Some exceptions remain, for example as in the case of pseudarthrosis. This new non-resident tissue would be known as reparative tissue and be considered distinctly different from that of tissue that has formed through the process of regeneration. Remodeling can also be at the origin of destruction of regenerated tissue (partial or complete), if this one is not biologically identical to the original one (6). JOE — Volume 40, Number 4S, April 2014
Remodeling of dentin does not occur, and newly formed tissue will never be replaced. Histologically, tertiary dentin can appear to be similar to secondary dentin; however, it is never truly the same and does not form a continuum with preexisting dentin. From a semantic point of view, it may be pertinent to consider the union of pulp and dentin differently and think of the tissue as the dentinodontoblastic complex rather than the dentin-pulp complex. Dentin is uniquely penetrated by odontoblast processes that form an intimate and cohesive union with the underlying odontoblastic palisade. This layer could be regarded as a membrane that separates itself from the pulp underneath by the acellular layer of Weil. A breakage in this odontoblastic membrane due to caries, trauma, or iatrogenic damage results in exposure of the pulp tissue itself, leaving it unprotected and vulnerable. The method currently used to determine the origin of the tissue secreted by the processes of repair/regeneration is generally based on cellular markers (usually proteins) left by cells that were responsible
Regeneration or Repair?
S71
Pulp Regeneration—Translational Opportunities
Figure 2. Revascularization treatment on tooth #25 of a 12-year-old boy. (A) Preoperative x-ray, (B) postoperative, and (C and D) 6- and 12-month recall, respectively. Mineralization of the apical part of the canal is visible, almost obliterating this part of the canal.
for this tissue production. The presence of these proteins in conjunction with other indicators of cellular behavior (especially biomineralization) and analysis of the structure of the newly generated tissue allow conclusions to be made of how it was formed. Nevertheless, because of the lack of true specific molecular markers of newly secreted tissues, a new generation of cells is nominated with the suffix ‘‘-like’’ (osteoblast-like, odontoblast-like). This makes it possible to distinguish between normal tissue and altered tissue, suggesting that it is formed from reparative processes rather than regenerative ones. In biology, it is conventional to consider that the mineralized tissue secreted by dental cells is dentin. There are few in vitro and in vivo experiments that seek to characterize the type of mineral produced during synthesis of tertiary dentin. In a previous experiment, we showed by x-ray analysis that the crystal structure of reparative dentin formed after pulp capping was close to that of orthodentin but was different in terms of protein levels (7). There is a lack of understanding about the precise nature of mineralized tissue to the extent that it is not entirely clear what the exact difference is between S72
Simon et al.
dentin and bone, for example. Such knowledge would make it possible to describe the precise nature of secreted tissue and qualify the healing process as regenerative or reparative. Under specific physiopathologic conditions it is not only odontoblasts that can secrete dentin. Other pulp cells are also able to produce mineralized tissue. It is possible for pulp stones and other intrapulpal mineral tissue accumulations to form in the presence of chronic inflammatory processes (8, 9). Clinically it is possible to make a morphologic distinction between orthodentin and pulp mineralization, but in vitro, it is much more difficult to distinguish the pathologic tissue from true dentin. In vitro, under appropriate experimental conditions it is possible to demonstrate the production of mineralized tissue but impossible to assess the precise the nature of this tissue. A better knowledge and understanding of the ultrastructure of the mineral produced in these different physiopathologic situations would allow us to differentiate and determine in every in vitro situation the true origin of the mineralized tissue, for instance, whether it was from JOE — Volume 40, Number 4S, April 2014
Pulp Regeneration—Translational Opportunities are usually aimed at investigating a reparative process rather than a true process of regeneration (progenitor migration/recruitment/differentiation). The mechanism for material-induced dentin bridge formation is unknown. No published data decisively demonstrate that the dentin secreted in contact with a bioactive material is related to a true process of odontoblastic differentiation. It has long been suggested that pulp capping agents initially induce tissue irritation. It may be that this initial inflammatory reaction, brought about by contact of the biomaterial with the dental pulp, induces this reparative process and mineral formation (11). Because of the lack of certainty about the structure and ultrastructure of the mineralized tissue produced from pulp capping, it would be wise to regard this therapeutically induced wound healing as a repair of the dentin-pulp complex rather than a regeneration of the dentinodontoblastic complex.
Dental Pulp Regeneration or Root Canal Revascularization?
Figure 3. (A–C) Cone-beam computed tomography of the same tooth in Figure 2. Note the obliteration of the canal in its apical third and the absence of root thickening in the middle third.
odontoblastic phenotype cells or induced by an inflammatory process. In other tissues/organs such as the urinary system (10), it has been demonstrated that there is a correlation between the ultrastructure or chemical composition of the mineral and the etiology of its secretion. Whereas the synthesis of dentin-like tissue by odontoblast-like cells can be considered as a regenerative process, an ectopic biomineralization process would be considered more as a reparative one. The experimental methods used in in vitro and in vivo experiments JOE — Volume 40, Number 4S, April 2014
The therapeutic strategies discussed so far for inducing wound healing of the dental pulp tissue are based on limiting tissue degeneration and enabling the rest of the pulp tissue to remain vital. It may not be possible to preserve the pulp if there is severe pulp damage or the pulp has become severely inflamed or necrotic. Under such conditions, the clinician has to perform a pulpectomy, disinfect the whole canal system, and provide a root filling to prevent any recontamination by bacteria. Although current root canal techniques provide reliable outcomes, it appears that de novo synthesis of pulp or connective tissue inside the root canal system itself might be a better approach for endodontic treatment in the future. Treatment of an empty canal with a regenerative strategy provides a true challenge. It is a hostile environment in which to regenerate a complex tissue. Further research is needed to understand the basic cellular processes involved in engineering this tissue including the type of scaffold, the source and subsequent recruitment of stem cells, and the correct signaling molecules to induce the molecular responses required for tissue development, maturation, and neovascularization. First attempts to carry out root canal revascularization were made in the 1960s (12). The main objective of this treatment strategy is to regenerate de novo dental pulp tissue. One of the biggest issues identified by this early research was that the only possible local sources of viable cells could arise by inducing bleeding into the root canal space. This meant that these cells were derived from circulating cells, cementum, periodontal ligament, or alveolar bone and therefore not of pulpal origin. It is interesting that although first described in the 1960s, it was not until 2001 that the concept of root canal revascularization reemerged, and research on this topic became more popular. This has generated debate about whether this tissue is produced by repair or regenerative processes (13). It has been proposed that stem cells from the apical papilla could be introduced into the canal by disorganization of the apical papilla tissue with an endodontic file and carried into the canal space by the forming blood clot. Despite the publication of a significant number of case reports and case series, little is known about the processes involved in this therapeutic approach. Most case reports/case series show examples of revascularization of the pulp space where there is a preexisting lesion of endodontic origin. Vitality tests are very subjective; however, radiographic evidence of healing is more objective, and successful outcomes can be demonstrated. It has been shown that where incomplete apexogenesis has occurred because of pulp necrosis, this therapeutic intervention can result in increased root-end dentin thickness and reduction in volume of
Regeneration or Repair?
S73
Pulp Regeneration—Translational Opportunities root canal space. These observations prove regeneration of a dental pulp-like tissue inside the root canal, with peripheral cells showing dentinogenetic capability. This treatment is not always a success; some case reports have described instances where teeth have had to be extracted, and subsequent histologic analysis has been performed. The first histologic observations of tooth tissue that had been regenerated by using the revascularization technique were based on a dog model (14). The authors clearly show that inside the root canal space, dentinal walls were covered with a layer of cementum, a neo-ligament, and an osteoid structure. More recently, the histologic analysis of teeth treated by simple revascularization (15) or by filling with platelet-rich plasma (16) shows that a mineralized layer was deposited on the radicular walls. This newly formed tissue appeared to be of periodontal origin rather than pulpal origin and did not represent tissue that was formed by the process of dentinogenesis. In this case, the recruited progenitors migrating from the apical papilla or from the surrounding periradicular tissues would have differentiated into cells of periodontal origin. Radiographic analysis of these cases may have been deceptive thus far and may have led us to think that the mineralized tissue was dentin rather than cementum, as was shown by this histologic analysis. If this evidence is corroborated and it becomes established, the technique of induced apexogenesis by using revascularization will not be considered a regenerative process. Nevertheless, similar treatments with apexification are described in the literature (17, 18). Although an apical closure with a periodontal structure was not intended, it is still yet to be seen whether this treatment technique will result in successful long-term outcome. We have recently conducted a case series study with 22 treatments that used the revascularization technique. All the treatments used a consistent protocol based on that recommended by the American Association of Endodontics (19). Standard periapical intraoral radiographs were taken at 3, 6, 12, 18, and 24 months after operation, with conebeam computed tomography exams at 12 months for the 12 most recent cases. Clinical recall duration varies from 10–32 months (data unpublished). Our preliminary results show radiographic healing of periapical tissues with preexisting apical pathology. Other findings of interest include the presence of a mineralized tissue barrier close to the coronal filling material. This barrier was apparent on radiographic examination in most cases at 3 months after treatment (Fig. 1). At the time of publication, all 22 cases show no root lengthening or thickening of the dentinal walls. It does appear that an apical barrier forms in the majority of the cases, with canal obliteration in some patients (Figs. 2 and 3). Although this study is still in progress and is not complete, preliminary results raise doubt over the ability of this technique to induce regenerative processes within the pulp canal space. The tissue formed could only be considered reparative because it is non-resident. To determine whether this treatment modality could be considered a success, it is important to review what the objective is. If it is to induce healing of the periapical tissue, stimulate bone regeneration, and render the patient free from any signs or symptoms, then it would be termed a success. However, if the objective is to regenerate a pulp tissue ad integrum, this treatment would be deemed a failure. In short, it would be a clinical success but a biological failure. The aim of endodontic treatment in an infected tooth is first to disinfect the root canal system and second to prevent reinfection over time. Both of these are necessary for successful bone healing and regeneration of the periradicular tissues. Use of a biological tissue to fill the root canal space would avoid the disadvantages of a synthetic material, namely the potential loss of seal, toxicity, etc. Furthermore, biological tissue has the huge advantage of being immunocompetent and thus being able to defend itself from bacteria as normal pulp tissue would. S74
Simon et al.
Revascularization is an interesting technique that allows the pulp space to be filled with vital tissue. This tissue is different from the tissue that was initially present in the canal and will never be modified or reformed into what was the resident tissue. Among the case reports published, only one case shows the presence of a dental pulp inside a treated canal; it shows a true palisade of odontoblasts and a wellorganized dental pulp tissue. This difference with this case compared with most of those published about revascularization is the tooth had pulpitis and not necrosis. In such a case, the odontoblastic layer is mostly preserved, and the treatment consists of disorganizing remaining pulp tissue without completely destroying it. Although the dentinodontoblastic complex is highly specialized and difficult to regenerate, with resident cells still present, it was possible for it to reorganize, regenerate, and finally preserve itself. Generating a vital tissue in a vacant root canal space raises a further question of semantics. The precise term regeneration implies that pulp tissue has formed in the space and returned to normal homeostatic function. If this is the case, then none of our present therapeutic strategies should be considered regenerative because they cannot fulfill these demands. They should be regarded simply as reparative therapeutic strategies; however, if the demand is for generation of a vital biological tissue in this vacant space, then the technique known as revascularization could be regarded as a regenerative technique. Clinically, although these strategies have their place, clear indications and contraindications have not yet been determined. Beyond the semantics of the definitions, many questions remain about how a vital tissue can form in a vacant biological space. The presence of stem cells in a revascularized canal has been clearly shown (20), suggesting that the recruitment of stem cells from the apical papilla and their subsequent migration play a critical role in the formation of this new tissue; however, the origin of these cells remains unclear. At this point in time on the basis of these assumptions, it is suggested that the clinical indications of revascularization treatment should be confined to immature teeth. If progenitor cells could be recruited from a niche other than the apical papilla, the indications of treatment could be extended to the mature teeth. If progenitor cell niches lie within developed periapical tissues, then their recruitment into the root canal could be plausible. If this is the case, then treatment by using such a technique on mature teeth may be possible.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Zander H. Reaction of the pulp to calcium hydroxide. J Dent Res 1939;18:373–9. 2. Miyashita H, Worthington HV, Qualtrough A, Plasschaert A. Pulp management for caries in adults: maintaining pulp vitality. Cochrane Database Syst Rev 2007;(2): CD004484. 3. Aguilar P, Linsuwanont P. Vital pulp therapy in vital permanent teeth with cariously exposed pulp: a systematic review. J Endod 2011;37:581–7. 4. Bjorndal L, Reit C, Bruun G, et al. Treatment of deep caries lesions in adults: randomized clinical trials comparing stepwise vs direct complete excavation, and direct pulp capping vs partial pulpotomy. Eur J Oral Sci 2010;118:290–7. 5. Dummer PM, Hicks R, Huws D. Clinical signs and symptoms in pulp disease. Int Endod J 1980;13:27–35. 6. Leucht P, Kim J-B, Amasha R, et al. Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration. Development 2008;135: 2845–54. 7. Simon S, Cooper P, Smith A, et al. Evaluation of a new laboratory model for pulp healing: preliminary study. Int Endod J 2008;41:781–90. 8. Sundell JR, Stanley HR, White CL. The relationship of coronal pulp stone formation to experimental operative procedures. Oral Surg Oral Med Oral Pathol 1968;25: 579–89.
JOE — Volume 40, Number 4S, April 2014
Pulp Regeneration—Translational Opportunities 9. Goga R, Chandler NP, Oginni AO. Pulp stones: a review. Int Endod J 2008;41: 457–68. 10. Dessombz A, Meria P, Bazin D, Daudon M. Prostatic stones: evidence of a specific chemistry related to infection and presence of bacterial imprints. PLoS One 2012;7: e51691. 11. Cooper PR, McLachlan JL, Simon S, et al. Mediators of inflammation and regeneration. Adv Dent Res 2011;23:290–5. 12. Ostby BN. The role of the blood clot in endodontic therapy: an experimental histologic study. Acta Odontol Scand 1961;19:324–53. 13. Trope M. Letters to editor: reply. J Endod 2008;34:511. 14. Thibodeau B, Teixeira F, Yamauchi M, et al. Pulp revascularization of immature dog teeth with apical periodontitis. J Endod 2007;33:680–9. 15. 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;39:1–6.
JOE — Volume 40, Number 4S, April 2014
16. 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 2012;38:1–7. 17. Simon S, Rilliard F, Berdal A, Machtou P. The use of mineral trioxide aggregate in one-visit apexification treatment: a prospective study. Int Endod J 2007; 40:186–97. 18. Nosrat A, Li KL, Vir K, et al. Is pulp regeneration necessary for root maturation? J Endod 2013;39:1291–5. 19. Regenerative endodontics: AAE. Available at: http://www.aae.org/ publications-and-research/research/regenerative-database.aspx. Accessed November 14, 2011. 20. 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.
Regeneration or Repair?
S75
Regenerative Endodontics: Regeneration or Repair? Stephane R.J. Simon, DDS, PhD,*†‡§ Phillip L. Tomson, PhD,§ and Ariane Berdal, PhD*‡ Abstract Recent advances in biotechnology and translational research have made it possible to provide treatment modalities that protect the vital pulp, allow manipulation of reactionary and reparative dentinogenesis, and, more recently, permit revascularization of an infected root canal space. These approaches are referred to as regenerative procedures. The method currently used to determine the origin of the tissue secreted during the repair/ regeneration process is largely based on the identification of cellular markers (usually proteins) left by cells that were responsible for this tissue production. The presence of these proteins in conjunction with other indicators of cellular behavior (especially biomineralization) and analysis of the structure of the newly generated tissue allow conclusions to be made of how it was formed. Thus far, it has not been possible to truly establish the biological mechanism controlling tertiary dentinogenesis. This article considers current therapeutic techniques to treat the dentin-pulp complex and contextualize them in terms of reparative and regenerative processes. Although it may be considered a semantic argument rather than a biological one, the definitions of regeneration and repair are explored to clarify our position in this era of regenerative endodontics. (J Endod 2014;40:S70–S75)
Key Words Dentin bridge, endodontics, healing, regeneration, repair
From the *Department of Oral Biology, School of Dentistry, University of Paris Diderot, Paris, France; †H^opital de la Pitie Saleptriere, Paris, France; ‡UMRS INSERM 1138 TEAM 5, Paris, France; and §Oral Biology, School of Dentistry, University of Birmingham, United Kingdom. This paper is based on a presentation from the International Association for Dental Research (IADR) Pulp Biology and Regeneration Group Satellite Meeting, which was held March 24–26, 2013 in San Francisco, California. Address requests for reprints to Dr Stephane R.J. Simon, UMRS 1138, Team 5, Centre de Recherche des Cordeliers, 18-20 Rue de l’ecole de medicine, 75006 Paris, France. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.024
S70
Simon et al.
S
ignificant progress in the field of prevention and treatment of pulpal and periradicular disease has led to an increasing amount of research into the role of the dentin-pulp complex and its ability to repair itself and regenerate mineralized tissue. For many years, research laboratories have investigated the pulp healing process with far reaching aims of enhancing its inherent regenerative capacity to completely regenerate this unique tissue. Recent advances in biotechnology and translational research have made it possible to provide treatment modalities that protect the vital pulp, allow manipulation of reactionary and reparative dentinogenesis, and, more recently, permit revascularization of an infected root canal space. Although the volume of the mature pulp is very small (less than 100 mL), it is conceivable that regeneration of such a small tissue should be relatively easy. Unfortunately, this is not the case. The dental pulp is a complex specialized connective tissue that is enclosed in a mineralized shell and has a limited blood supply; these are only a few of the many obstacles faced by the clinicians and researchers attempting to design new therapeutic strategies for its regeneration. Regenerative endodontics should be considered as 2 entities. One is dentin-pulp complex regeneration (which could also be called dentin/odontoblast complex regeneration). This relates to preservation of pulp vitality and pulp capping. The second is dental pulp regeneration. This relates to regeneration of a vital tissue into an empty but infected root canal space.
Dentin-Pulp Complex Regeneration Pulp capping and dentin bridge formation induction have been used for more than 70 years, with the first experimental studies published by Zander (1) in 1939. If the right environmental conditions persist, clinical results can be encouraging and tend to motivate the clinician to maintain pulp vitality as long as possible because of the advantages it can bring in terms of prolonging the life of the tooth. Nevertheless, high-quality robust clinical trials are lacking (2), and published findings are contradictory, not allowing for clear guidance for this clinical technique. A recent systematic review demonstrated success rates (pulp vitality maintained) at 3 years of 72.9% for pulp capping and 99.4% for partial pulpotomy (3). However, in a recent randomized clinical trial, Bjorndal et al (4) showed success rates to be much lower, 31.8% for pulp capping and 34.5% for partial pulpotomy. Clinically, the aim of such treatment is to keep the pulp vital and maintain its homeostatic functions, thus avoiding pulpectomy or extraction of the tooth. Success of the treatment is assessed by the symptoms reported by the patients and through the use of relatively rudimentary investigations such as thermal tests, electrical tests, tenderness to percussion or palpation, and radiographic assessment. It has been well-established that clinical signs and symptoms do not correlate to the histologic status of the tooth (5). Biological research methods allow for more sophisticated assessment and enable the researcher to observe and analyze the histologic structure, cell behavior, and immunologic/inflammation status of the tissue concerned. Such techniques allow the pulpal responses to be assessed with greater accuracy than in clinical studies. To study the physiological and reparative processes of the pulp, in vitro experiments with immortalized cells or primary cell cultures can be used. The limitations of these studies must be acknowledged; they can only mimic biological processes such as mineral production and cannot prove conclusively the type of mineralized tissue formed. Frequently these experiments are supported with gene expression data to establish the likely gene regulation that induced this mineral production. Until phenotypic markers associated with dentin production are shown,
JOE — Volume 40, Number 4S, April 2014
Pulp Regeneration—Translational Opportunities
Figure 1. (A and B) Endodontic treatment by revascularization on tooth #8 of a 16-year-old girl. Note formation of a mineralized barrier distant from coronal filling material (mineral trioxide aggregate) (arrow) (C). At 18-month recall, the bone healing is complete, and the mineral barrier is still present (D). Nevertheless, no root lengthening or apexogenesis is noticeable.
the results are often viewed with skepticism to whether the mineral tissue formed is from odontoblastic origin.
Regeneration/Repair and Remodeling Bone is constantly being remodeled, and newly generated tissue is replaced within a few months by new bone. The turnover of bone means that gradually the newly secreted tissue will merge with other tissue laid down at different times, and new and old tissue becomes homogenous. Some exceptions remain, for example as in the case of pseudarthrosis. This new non-resident tissue would be known as reparative tissue and be considered distinctly different from that of tissue that has formed through the process of regeneration. Remodeling can also be at the origin of destruction of regenerated tissue (partial or complete), if this one is not biologically identical to the original one (6). JOE — Volume 40, Number 4S, April 2014
Remodeling of dentin does not occur, and newly formed tissue will never be replaced. Histologically, tertiary dentin can appear to be similar to secondary dentin; however, it is never truly the same and does not form a continuum with preexisting dentin. From a semantic point of view, it may be pertinent to consider the union of pulp and dentin differently and think of the tissue as the dentinodontoblastic complex rather than the dentin-pulp complex. Dentin is uniquely penetrated by odontoblast processes that form an intimate and cohesive union with the underlying odontoblastic palisade. This layer could be regarded as a membrane that separates itself from the pulp underneath by the acellular layer of Weil. A breakage in this odontoblastic membrane due to caries, trauma, or iatrogenic damage results in exposure of the pulp tissue itself, leaving it unprotected and vulnerable. The method currently used to determine the origin of the tissue secreted by the processes of repair/regeneration is generally based on cellular markers (usually proteins) left by cells that were responsible
Regeneration or Repair?
S71
Pulp Regeneration—Translational Opportunities
Figure 2. Revascularization treatment on tooth #25 of a 12-year-old boy. (A) Preoperative x-ray, (B) postoperative, and (C and D) 6- and 12-month recall, respectively. Mineralization of the apical part of the canal is visible, almost obliterating this part of the canal.
for this tissue production. The presence of these proteins in conjunction with other indicators of cellular behavior (especially biomineralization) and analysis of the structure of the newly generated tissue allow conclusions to be made of how it was formed. Nevertheless, because of the lack of true specific molecular markers of newly secreted tissues, a new generation of cells is nominated with the suffix ‘‘-like’’ (osteoblast-like, odontoblast-like). This makes it possible to distinguish between normal tissue and altered tissue, suggesting that it is formed from reparative processes rather than regenerative ones. In biology, it is conventional to consider that the mineralized tissue secreted by dental cells is dentin. There are few in vitro and in vivo experiments that seek to characterize the type of mineral produced during synthesis of tertiary dentin. In a previous experiment, we showed by x-ray analysis that the crystal structure of reparative dentin formed after pulp capping was close to that of orthodentin but was different in terms of protein levels (7). There is a lack of understanding about the precise nature of mineralized tissue to the extent that it is not entirely clear what the exact difference is between S72
Simon et al.
dentin and bone, for example. Such knowledge would make it possible to describe the precise nature of secreted tissue and qualify the healing process as regenerative or reparative. Under specific physiopathologic conditions it is not only odontoblasts that can secrete dentin. Other pulp cells are also able to produce mineralized tissue. It is possible for pulp stones and other intrapulpal mineral tissue accumulations to form in the presence of chronic inflammatory processes (8, 9). Clinically it is possible to make a morphologic distinction between orthodentin and pulp mineralization, but in vitro, it is much more difficult to distinguish the pathologic tissue from true dentin. In vitro, under appropriate experimental conditions it is possible to demonstrate the production of mineralized tissue but impossible to assess the precise the nature of this tissue. A better knowledge and understanding of the ultrastructure of the mineral produced in these different physiopathologic situations would allow us to differentiate and determine in every in vitro situation the true origin of the mineralized tissue, for instance, whether it was from JOE — Volume 40, Number 4S, April 2014
Pulp Regeneration—Translational Opportunities are usually aimed at investigating a reparative process rather than a true process of regeneration (progenitor migration/recruitment/differentiation). The mechanism for material-induced dentin bridge formation is unknown. No published data decisively demonstrate that the dentin secreted in contact with a bioactive material is related to a true process of odontoblastic differentiation. It has long been suggested that pulp capping agents initially induce tissue irritation. It may be that this initial inflammatory reaction, brought about by contact of the biomaterial with the dental pulp, induces this reparative process and mineral formation (11). Because of the lack of certainty about the structure and ultrastructure of the mineralized tissue produced from pulp capping, it would be wise to regard this therapeutically induced wound healing as a repair of the dentin-pulp complex rather than a regeneration of the dentinodontoblastic complex.
Dental Pulp Regeneration or Root Canal Revascularization?
Figure 3. (A–C) Cone-beam computed tomography of the same tooth in Figure 2. Note the obliteration of the canal in its apical third and the absence of root thickening in the middle third.
odontoblastic phenotype cells or induced by an inflammatory process. In other tissues/organs such as the urinary system (10), it has been demonstrated that there is a correlation between the ultrastructure or chemical composition of the mineral and the etiology of its secretion. Whereas the synthesis of dentin-like tissue by odontoblast-like cells can be considered as a regenerative process, an ectopic biomineralization process would be considered more as a reparative one. The experimental methods used in in vitro and in vivo experiments JOE — Volume 40, Number 4S, April 2014
The therapeutic strategies discussed so far for inducing wound healing of the dental pulp tissue are based on limiting tissue degeneration and enabling the rest of the pulp tissue to remain vital. It may not be possible to preserve the pulp if there is severe pulp damage or the pulp has become severely inflamed or necrotic. Under such conditions, the clinician has to perform a pulpectomy, disinfect the whole canal system, and provide a root filling to prevent any recontamination by bacteria. Although current root canal techniques provide reliable outcomes, it appears that de novo synthesis of pulp or connective tissue inside the root canal system itself might be a better approach for endodontic treatment in the future. Treatment of an empty canal with a regenerative strategy provides a true challenge. It is a hostile environment in which to regenerate a complex tissue. Further research is needed to understand the basic cellular processes involved in engineering this tissue including the type of scaffold, the source and subsequent recruitment of stem cells, and the correct signaling molecules to induce the molecular responses required for tissue development, maturation, and neovascularization. First attempts to carry out root canal revascularization were made in the 1960s (12). The main objective of this treatment strategy is to regenerate de novo dental pulp tissue. One of the biggest issues identified by this early research was that the only possible local sources of viable cells could arise by inducing bleeding into the root canal space. This meant that these cells were derived from circulating cells, cementum, periodontal ligament, or alveolar bone and therefore not of pulpal origin. It is interesting that although first described in the 1960s, it was not until 2001 that the concept of root canal revascularization reemerged, and research on this topic became more popular. This has generated debate about whether this tissue is produced by repair or regenerative processes (13). It has been proposed that stem cells from the apical papilla could be introduced into the canal by disorganization of the apical papilla tissue with an endodontic file and carried into the canal space by the forming blood clot. Despite the publication of a significant number of case reports and case series, little is known about the processes involved in this therapeutic approach. Most case reports/case series show examples of revascularization of the pulp space where there is a preexisting lesion of endodontic origin. Vitality tests are very subjective; however, radiographic evidence of healing is more objective, and successful outcomes can be demonstrated. It has been shown that where incomplete apexogenesis has occurred because of pulp necrosis, this therapeutic intervention can result in increased root-end dentin thickness and reduction in volume of
Regeneration or Repair?
S73
Pulp Regeneration—Translational Opportunities root canal space. These observations prove regeneration of a dental pulp-like tissue inside the root canal, with peripheral cells showing dentinogenetic capability. This treatment is not always a success; some case reports have described instances where teeth have had to be extracted, and subsequent histologic analysis has been performed. The first histologic observations of tooth tissue that had been regenerated by using the revascularization technique were based on a dog model (14). The authors clearly show that inside the root canal space, dentinal walls were covered with a layer of cementum, a neo-ligament, and an osteoid structure. More recently, the histologic analysis of teeth treated by simple revascularization (15) or by filling with platelet-rich plasma (16) shows that a mineralized layer was deposited on the radicular walls. This newly formed tissue appeared to be of periodontal origin rather than pulpal origin and did not represent tissue that was formed by the process of dentinogenesis. In this case, the recruited progenitors migrating from the apical papilla or from the surrounding periradicular tissues would have differentiated into cells of periodontal origin. Radiographic analysis of these cases may have been deceptive thus far and may have led us to think that the mineralized tissue was dentin rather than cementum, as was shown by this histologic analysis. If this evidence is corroborated and it becomes established, the technique of induced apexogenesis by using revascularization will not be considered a regenerative process. Nevertheless, similar treatments with apexification are described in the literature (17, 18). Although an apical closure with a periodontal structure was not intended, it is still yet to be seen whether this treatment technique will result in successful long-term outcome. We have recently conducted a case series study with 22 treatments that used the revascularization technique. All the treatments used a consistent protocol based on that recommended by the American Association of Endodontics (19). Standard periapical intraoral radiographs were taken at 3, 6, 12, 18, and 24 months after operation, with conebeam computed tomography exams at 12 months for the 12 most recent cases. Clinical recall duration varies from 10–32 months (data unpublished). Our preliminary results show radiographic healing of periapical tissues with preexisting apical pathology. Other findings of interest include the presence of a mineralized tissue barrier close to the coronal filling material. This barrier was apparent on radiographic examination in most cases at 3 months after treatment (Fig. 1). At the time of publication, all 22 cases show no root lengthening or thickening of the dentinal walls. It does appear that an apical barrier forms in the majority of the cases, with canal obliteration in some patients (Figs. 2 and 3). Although this study is still in progress and is not complete, preliminary results raise doubt over the ability of this technique to induce regenerative processes within the pulp canal space. The tissue formed could only be considered reparative because it is non-resident. To determine whether this treatment modality could be considered a success, it is important to review what the objective is. If it is to induce healing of the periapical tissue, stimulate bone regeneration, and render the patient free from any signs or symptoms, then it would be termed a success. However, if the objective is to regenerate a pulp tissue ad integrum, this treatment would be deemed a failure. In short, it would be a clinical success but a biological failure. The aim of endodontic treatment in an infected tooth is first to disinfect the root canal system and second to prevent reinfection over time. Both of these are necessary for successful bone healing and regeneration of the periradicular tissues. Use of a biological tissue to fill the root canal space would avoid the disadvantages of a synthetic material, namely the potential loss of seal, toxicity, etc. Furthermore, biological tissue has the huge advantage of being immunocompetent and thus being able to defend itself from bacteria as normal pulp tissue would. S74
Simon et al.
Revascularization is an interesting technique that allows the pulp space to be filled with vital tissue. This tissue is different from the tissue that was initially present in the canal and will never be modified or reformed into what was the resident tissue. Among the case reports published, only one case shows the presence of a dental pulp inside a treated canal; it shows a true palisade of odontoblasts and a wellorganized dental pulp tissue. This difference with this case compared with most of those published about revascularization is the tooth had pulpitis and not necrosis. In such a case, the odontoblastic layer is mostly preserved, and the treatment consists of disorganizing remaining pulp tissue without completely destroying it. Although the dentinodontoblastic complex is highly specialized and difficult to regenerate, with resident cells still present, it was possible for it to reorganize, regenerate, and finally preserve itself. Generating a vital tissue in a vacant root canal space raises a further question of semantics. The precise term regeneration implies that pulp tissue has formed in the space and returned to normal homeostatic function. If this is the case, then none of our present therapeutic strategies should be considered regenerative because they cannot fulfill these demands. They should be regarded simply as reparative therapeutic strategies; however, if the demand is for generation of a vital biological tissue in this vacant space, then the technique known as revascularization could be regarded as a regenerative technique. Clinically, although these strategies have their place, clear indications and contraindications have not yet been determined. Beyond the semantics of the definitions, many questions remain about how a vital tissue can form in a vacant biological space. The presence of stem cells in a revascularized canal has been clearly shown (20), suggesting that the recruitment of stem cells from the apical papilla and their subsequent migration play a critical role in the formation of this new tissue; however, the origin of these cells remains unclear. At this point in time on the basis of these assumptions, it is suggested that the clinical indications of revascularization treatment should be confined to immature teeth. If progenitor cells could be recruited from a niche other than the apical papilla, the indications of treatment could be extended to the mature teeth. If progenitor cell niches lie within developed periapical tissues, then their recruitment into the root canal could be plausible. If this is the case, then treatment by using such a technique on mature teeth may be possible.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Zander H. Reaction of the pulp to calcium hydroxide. J Dent Res 1939;18:373–9. 2. Miyashita H, Worthington HV, Qualtrough A, Plasschaert A. Pulp management for caries in adults: maintaining pulp vitality. Cochrane Database Syst Rev 2007;(2): CD004484. 3. Aguilar P, Linsuwanont P. Vital pulp therapy in vital permanent teeth with cariously exposed pulp: a systematic review. J Endod 2011;37:581–7. 4. Bjorndal L, Reit C, Bruun G, et al. Treatment of deep caries lesions in adults: randomized clinical trials comparing stepwise vs direct complete excavation, and direct pulp capping vs partial pulpotomy. Eur J Oral Sci 2010;118:290–7. 5. Dummer PM, Hicks R, Huws D. Clinical signs and symptoms in pulp disease. Int Endod J 1980;13:27–35. 6. Leucht P, Kim J-B, Amasha R, et al. Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration. Development 2008;135: 2845–54. 7. Simon S, Cooper P, Smith A, et al. Evaluation of a new laboratory model for pulp healing: preliminary study. Int Endod J 2008;41:781–90. 8. Sundell JR, Stanley HR, White CL. The relationship of coronal pulp stone formation to experimental operative procedures. Oral Surg Oral Med Oral Pathol 1968;25: 579–89.
JOE — Volume 40, Number 4S, April 2014
Pulp Regeneration—Translational Opportunities 9. Goga R, Chandler NP, Oginni AO. Pulp stones: a review. Int Endod J 2008;41: 457–68. 10. Dessombz A, Meria P, Bazin D, Daudon M. Prostatic stones: evidence of a specific chemistry related to infection and presence of bacterial imprints. PLoS One 2012;7: e51691. 11. Cooper PR, McLachlan JL, Simon S, et al. Mediators of inflammation and regeneration. Adv Dent Res 2011;23:290–5. 12. Ostby BN. The role of the blood clot in endodontic therapy: an experimental histologic study. Acta Odontol Scand 1961;19:324–53. 13. Trope M. Letters to editor: reply. J Endod 2008;34:511. 14. Thibodeau B, Teixeira F, Yamauchi M, et al. Pulp revascularization of immature dog teeth with apical periodontitis. J Endod 2007;33:680–9. 15. 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;39:1–6.
JOE — Volume 40, Number 4S, April 2014
16. 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 2012;38:1–7. 17. Simon S, Rilliard F, Berdal A, Machtou P. The use of mineral trioxide aggregate in one-visit apexification treatment: a prospective study. Int Endod J 2007; 40:186–97. 18. Nosrat A, Li KL, Vir K, et al. Is pulp regeneration necessary for root maturation? J Endod 2013;39:1291–5. 19. Regenerative endodontics: AAE. Available at: http://www.aae.org/ publications-and-research/research/regenerative-database.aspx. Accessed November 14, 2011. 20. 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.
Regeneration or Repair?
S75