Pulp Regeneration—Translational Opportunities

Regenerative Endodontics: Regeneration or Repair?St�ephane R.J. Simon, DDS, PhD,*†‡§ Phillip L. Tomson, PhD,§ and Ariane Berdal, PhD*‡

Abstract

Recent advances in biotechnology and translationalresearch have made it possible to provide treatment mo-dalities that protect the vital pulp, allow manipulation ofreactionary and reparative dentinogenesis, and, morerecently, permit revascularization of an infected rootcanal space. These approaches are referred to as regen-erative procedures. The method currently used to deter-mine the origin of the tissue secreted during the repair/regeneration process is largely based on the identifica-tion of cellular markers (usually proteins) left by cellsthat were responsible for this tissue production. Thepresence of these proteins in conjunction with otherindicators of cellular behavior (especially biomineraliza-tion) and analysis of the structure of the newly gener-ated tissue allow conclusions to be made of how itwas formed. Thus far, it has not been possible to trulyestablish the biological mechanism controlling tertiarydentinogenesis. This article considers current therapeu-tic techniques to treat the dentin-pulp complex andcontextualize them in terms of reparative and regenera-tive processes. Although it may be considered asemantic argument rather than a biological one, thedefinitions of regeneration and repair are explored toclarify our position in this era of regenerative endodon-tics. (J Endod 2014;40:S70–S75)

Key WordsDentin bridge, endodontics, healing, regeneration,repair

From the *Department of Oral Biology, School of Dentistry,University of Paris Diderot, Paris, France; †Hopital de la Piti�eSal�eptri�ere, Paris, France; ‡UMRS INSERM 1138 TEAM 5, Paris,France; and §Oral Biology, School of Dentistry, University of Bir-mingham, United Kingdom.

This paper is based on a presentation from the InternationalAssociation for Dental Research (IADR) Pulp Biology andRegeneration Group Satellite Meeting, which was held March24–26, 2013 in San Francisco, California.

Address requests for reprints to Dr St�ephane R.J. Simon,UMRS 1138, Team 5, Centre de Recherche des Cordeliers,18-20 Rue de l’�ecole de medicine, 75006 Paris, France. E-mailaddress: stephane.simon@univ-paris-diderot.fr0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2014.01.024

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Significant progress in the field of prevention and treatment of pulpal and perira-dicular 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 processwith far reaching aims of enhancing its inherent regenerative capacity to completelyregenerate this unique tissue. Recent advances in biotechnology and translationalresearch have made it possible to provide treatment modalities that protect the vitalpulp, allow manipulation of reactionary and reparative dentinogenesis, and, morerecently, permit revascularization of an infected root canal space. Although the vol-ume of the mature pulp is very small (less than 100 mL), it is conceivable that regen-eration of such a small tissue should be relatively easy. Unfortunately, this is not thecase. The dental pulp is a complex specialized connective tissue that is enclosed in amineralized shell and has a limited blood supply; these are only a few of the manyobstacles faced by the clinicians and researchers attempting to design new therapeu-tic strategies for its regeneration.

Regenerative endodontics should be considered as 2 entities. One is dentin-pulpcomplex regeneration (which could also be called dentin/odontoblast complex regen-eration). This relates to preservation of pulp vitality and pulp capping. The second isdental pulp regeneration. This relates to regeneration of a vital tissue into an emptybut infected root canal space.

Dentin-Pulp Complex RegenerationPulp 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 canbe encouraging and tend to motivate the clinician to maintain pulp vitality aslong as possible because of the advantages it can bring in terms of prolongingthe life of the tooth. Nevertheless, high-quality robust clinical trials are lacking(2), and published findings are contradictory, not allowing for clear guidancefor 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% forpartial pulpotomy (3). However, in a recent randomized clinical trial, Bjorndalet al (4) showed success rates to be much lower, 31.8% for pulp capping and34.5% for partial pulpotomy.

Clinically, the aim of such treatment is to keep the pulp vital and maintain itshomeostatic functions, thus avoiding pulpectomy or extraction of the tooth. Successof the treatment is assessed by the symptoms reported by the patients and throughthe use of relatively rudimentary investigations such as thermal tests, electrical tests,tenderness to percussion or palpation, and radiographic assessment. It has beenwell-established that clinical signs and symptoms do not correlate to the histologicstatus of the tooth (5). Biological research methods allow for more sophisticatedassessment and enable the researcher to observe and analyze the histologic struc-ture, cell behavior, and immunologic/inflammation status of the tissue concerned.Such techniques allow the pulpal responses to be assessed with greater accuracythan in clinical studies. To study the physiological and reparative processes of thepulp, in vitro experiments with immortalized cells or primary cell cultures canbe used. The limitations of these studies must be acknowledged; they can only mimicbiological processes such as mineral production and cannot prove conclusively thetype of mineralized tissue formed. Frequently these experiments are supported withgene expression data to establish the likely gene regulation that induced this mineralproduction. Until phenotypic markers associated with dentin production are shown,

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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 fromcoronal 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.

Pulp Regeneration—Translational Opportunities

the results are often viewed with skepticism to whether the mineraltissue formed is from odontoblastic origin.

Regeneration/Repair and RemodelingBone is constantly being remodeled, and newly generated tis-

sue is replaced within a few months by new bone. The turnover ofbone means that gradually the newly secreted tissue will merge withother tissue laid down at different times, and new and old tissuebecomes homogenous. Some exceptions remain, for example asin the case of pseudarthrosis. This new non-resident tissue wouldbe known as reparative tissue and be considered distinctly differentfrom that of tissue that has formed through the process of regen-eration. Remodeling can also be at the origin of destruction of re-generated tissue (partial or complete), if this one is not biologicallyidentical to the original one (6).

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Remodeling of dentin does not occur, and newly formed tissue willnever be replaced. Histologically, tertiary dentin can appear to besimilar to secondary dentin; however, it is never truly the same anddoes not form a continuum with preexisting dentin.

From a semantic point of view, it may be pertinent to consider theunion of pulp and dentin differently and think of the tissue as the dentin-odontoblastic complex rather than the dentin-pulp complex. Dentin isuniquely penetrated by odontoblast processes that form an intimateand cohesive unionwith the underlying odontoblastic palisade. This layercould be regarded as a membrane that separates itself from the pulp un-derneath by the acellular layer of Weil. A breakage in this odontoblasticmembrane due to caries, trauma, or iatrogenic damage results in expo-sure of the pulp tissue itself, leaving it unprotected and vulnerable.

The method currently used to determine the origin of the tissuesecreted by the processes of repair/regeneration is generally basedon cellularmarkers (usually proteins) left by cells that were responsible

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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.

Pulp Regeneration—Translational Opportunities

for this tissue production. The presence of these proteins in conjunctionwith other indicators of cellular behavior (especially biomineralization)and analysis of the structure of the newly generated tissue allow conclu-sions to bemade of how it was formed. Nevertheless, because of the lackof true specific molecular markers of newly secreted tissues, a new gen-eration of cells is nominated with the suffix ‘‘-like’’ (osteoblast-like,odontoblast-like). This makes it possible to distinguish between normaltissue and altered tissue, suggesting that it is formed from reparativeprocesses rather than regenerative ones. In biology, it is conventionalto consider that the mineralized tissue secreted by dental cells is dentin.There are few in vitro and in vivo experiments that seek to characterizethe type of mineral produced during synthesis of tertiary dentin. In aprevious experiment, we showed by x-ray analysis that the crystal struc-ture of reparative dentin formed after pulp capping was close to that oforthodentin but was different in terms of protein levels (7). There is alack of understanding about the precise nature of mineralized tissue tothe extent that it is not entirely clear what the exact difference is between

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dentin and bone, for example. Such knowledge would make it possibleto describe the precise nature of secreted tissue and qualify the healingprocess as regenerative or reparative.

Under specific physiopathologic conditions it is not only odonto-blasts that can secrete dentin. Other pulp cells are also able to producemineralized tissue. It is possible for pulp stones and other intrapulpalmineral tissue accumulations to form in the presence of chronic inflam-matory processes (8, 9). Clinically it is possible to make a morphologicdistinction between orthodentin and pulpmineralization, but in vitro, itis much more difficult to distinguish the pathologic tissue from truedentin. In vitro, under appropriate experimental conditions it ispossible to demonstrate the production of mineralized tissue butimpossible to assess the precise the nature of this tissue. A betterknowledge and understanding of the ultrastructure of the mineralproduced in these different physiopathologic situations would allowus to differentiate and determine in every in vitro situation the trueorigin of the mineralized tissue, for instance, whether it was from

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Figure 3. (A–C) Cone-beam computed tomography of the same tooth inFigure 2. Note the obliteration of the canal in its apical third and the absenceof root thickening in the middle third.

Pulp Regeneration—Translational Opportunities

odontoblastic phenotype cells or induced by an inflammatory process.In other tissues/organs such as the urinary system (10), it has beendemonstrated that there is a correlation between the ultrastructure orchemical composition of the mineral and the etiology of its secretion.

Whereas the synthesis of dentin-like tissue by odontoblast-likecells can be considered as a regenerative process, an ectopic biomin-eralization process would be considered more as a reparative one.The experimental methods used in in vitro and in vivo experiments

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are usually aimed at investigating a reparative process rather than atrue process of regeneration (progenitor migration/recruitment/differ-entiation).

The mechanism for material-induced dentin bridge formation isunknown. No published data decisively demonstrate that the dentinsecreted in contact with a bioactive material is related to a true processof odontoblastic differentiation. It has long been suggested that pulpcapping agents initially induce tissue irritation. It may be that this initialinflammatory reaction, brought about by contact of the biomaterial withthe dental pulp, induces this reparative process and mineral formation(11). Because of the lack of certainty about the structure and ultrastruc-ture of the mineralized tissue produced from pulp capping, it would bewise to regard this therapeutically induced wound healing as a repair ofthe dentin-pulp complex rather than a regeneration of the dentin-odontoblastic complex.

Dental Pulp Regeneration or Root CanalRevascularization?

The therapeutic strategies discussed so far for inducing woundhealing of the dental pulp tissue are based on limiting tissue degener-ation and enabling the rest of the pulp tissue to remain vital. It maynot be possible to preserve the pulp if there is severe pulp damage orthe pulp has become severely inflamed or necrotic. Under such condi-tions, the clinician has to perform a pulpectomy, disinfect the whole ca-nal system, and provide a root filling to prevent any recontamination bybacteria. Although current root canal techniques provide reliable out-comes, it appears that de novo synthesis of pulp or connective tissueinside the root canal system itself might be a better approach for end-odontic treatment in the future.

Treatment of an empty canal with a regenerative strategy provides atrue challenge. It is a hostile environment in which to regenerate a com-plex tissue. Further research is needed to understand the basic cellularprocesses involved in engineering this tissue including the type of scaf-fold, the source and subsequent recruitment of stem cells, and the cor-rect signaling molecules to induce the molecular responses requiredfor tissue development, maturation, and neovascularization.

First attempts to carry out root canal revascularization were madein the 1960s (12). The main objective of this treatment strategy is toregenerate de novo dental pulp tissue. One of the biggest issues iden-tified by this early research was that the only possible local sourcesof 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 notof pulpal origin.

It is interesting that although first described in the 1960s, it wasnot until 2001 that the concept of root canal revascularization ree-merged, and research on this topic became more popular. This hasgenerated debate about whether this tissue is produced by repair orregenerative processes (13). It has been proposed that stem cellsfrom the apical papilla could be introduced into the canal by disorga-nization of the apical papilla tissue with an endodontic file and carriedinto the canal space by the forming blood clot. Despite the publicationof a significant number of case reports and case series, little is knownabout the processes involved in this therapeutic approach. Most casereports/case series show examples of revascularization of the pulpspace where there is a preexisting lesion of endodontic origin. Vitalitytests are very subjective; however, radiographic evidence of healing ismore objective, and successful outcomes can be demonstrated. It hasbeen shown that where incomplete apexogenesis has occurredbecause of pulp necrosis, this therapeutic intervention can result inincreased root-end dentin thickness and reduction in volume of

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Pulp Regeneration—Translational Opportunities

root canal space. These observations prove regeneration of a dentalpulp-like tissue inside the root canal, with peripheral cells showingdentinogenetic capability. This treatment is not always a success;some case reports have described instances where teeth have had tobe extracted, and subsequent histologic analysis has been performed.The first histologic observations of tooth tissue that had been regener-ated by using the revascularization technique were based on a dogmodel (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 teethtreated by simple revascularization (15) or by filling with platelet-richplasma (16) shows that a mineralized layer was deposited on theradicular walls. This newly formed tissue appeared to be of peri-odontal origin rather than pulpal origin and did not represent tissuethat was formed by the process of dentinogenesis. In this case, the re-cruited progenitors migrating from the apical papilla or from the sur-rounding periradicular tissues would have differentiated into cells ofperiodontal origin. Radiographic analysis of these cases may havebeen deceptive thus far and may have led us to think that the miner-alized tissue was dentin rather than cementum, as was shown by thishistologic analysis. If this evidence is corroborated and it becomesestablished, the technique of induced apexogenesis by using revascu-larization 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 structurewas not intended, it is still yet to be seen whether this treatmenttechnique will result in successful long-term outcome.

We have recently conducted a case series study with 22 treatmentsthat used the revascularization technique. All the treatments used aconsistent protocol based on that recommended by the American Asso-ciation of Endodontics (19). Standard periapical intraoral radiographswere taken at 3, 6, 12, 18, and 24 months after operation, with cone-beam computed tomography exams at 12months for the 12most recentcases. Clinical recall duration varies from 10–32 months (data unpub-lished). Our preliminary results show radiographic healing of periapi-cal tissues with preexisting apical pathology. Other findings of interestinclude the presence of a mineralized tissue barrier close to the coronalfilling material. This barrier was apparent on radiographic examinationin most cases at 3 months after treatment (Fig. 1). At the time of pub-lication, all 22 cases show no root lengthening or thickening of thedentinal walls. It does appear that an apical barrier forms in themajorityof the cases, with canal obliteration in some patients (Figs. 2 and 3).Although this study is still in progress and is not complete, preliminaryresults raise doubt over the ability of this technique to induce regener-ative processes within the pulp canal space. The tissue formed couldonly be considered reparative because it is non-resident.

To determine whether this treatment modality could be consid-ered a success, it is important to review what the objective is. If it isto induce healing of the periapical tissue, stimulate bone regeneration,and render the patient free from any signs or symptoms, then it would betermed a success. However, if the objective is to regenerate a pulp tissuead integrum, this treatment would be deemed a failure. In short, itwould be a clinical success but a biological failure.

The aim of endodontic treatment in an infected tooth is first todisinfect the root canal system and second to prevent reinfection overtime. Both of these are necessary for successful bone healing andregeneration of the periradicular tissues. Use of a biological tissueto fill the root canal space would avoid the disadvantages of a syn-thetic material, namely the potential loss of seal, toxicity, etc. Further-more, biological tissue has the huge advantage of beingimmunocompetent and thus being able to defend itself from bacteriaas normal pulp tissue would.

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Revascularization is an interesting technique that allows the pulpspace to be filled with vital tissue. This tissue is different from the tissuethat was initially present in the canal and will never be modified or re-formed into what was the resident tissue. Among the case reports pub-lished, only one case shows the presence of a dental pulp inside atreated canal; it shows a true palisade of odontoblasts and a well-organized dental pulp tissue. This difference with this case comparedwith most of those published about revascularization is the tooth hadpulpitis and not necrosis. In such a case, the odontoblastic layer ismostly preserved, and the treatment consists of disorganizing remainingpulp tissue without completely destroying it. Although the dentin-odontoblastic 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 afurther question of semantics. The precise term regeneration impliesthat pulp tissue has formed in the space and returned to normal homeo-static function. If this is the case, then none of our present therapeuticstrategies should be considered regenerative because they cannot fulfillthese demands. They should be regarded simply as reparative therapeu-tic strategies; however, if the demand is for generation of a vital biolog-ical tissue in this vacant space, then the technique known asrevascularization could be regarded as a regenerative technique.

Clinically, although these strategies have their place, clear indica-tions and contraindications have not yet been determined. Beyond thesemantics of the definitions, many questions remain about how a vitaltissue can form in a vacant biological space. The presence of stem cellsin a revascularized canal has been clearly shown (20), suggesting thatthe recruitment of stem cells from the apical papilla and their subse-quent migration play a critical role in the formation of this new tissue;however, the origin of these cells remains unclear. At this point in timeon the basis of these assumptions, it is suggested that the clinical indi-cations of revascularization treatment should be confined to immatureteeth. If progenitor cells could be recruited from a niche other than theapical papilla, the indications of treatment could be extended to themature teeth. If progenitor cell niches lie within developed periapicaltissues, then their recruitment into the root canal could be plausible.If this is the case, then treatment by using such a technique on matureteeth may be possible.

AcknowledgmentsThe authors deny any conflicts of interest related to this study.

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