etp12039

Pulp regeneration in previouslyinfected root canal spaceASHRAF F. FOUAD & ALI NOSRAT

Contemporary pulp revascularization procedures have been successful in achieving endodontic infection controland promoting the root mineralization process. However, there is still no agreement on the methods to producepredictable clinical outcomes or optimal disinfection protocols. Furthermore, there is no evidence for actual pulpregeneration in humans or in in situ animal models. This review will outline the various factors involved inundertaking this procedure, with a special emphasis on the antimicrobial strategies employed and the availableinformation on their efficacy. Areas in which information is lacking and research is needed will also be presented.

Received 8 January 2013; accepted 1 May 2013.

Pathogenesis of endodonticinfections in immature teeth

It is generally accepted that full maturation of the apexof permanent teeth occurs about 3 years followingeruption. For most teeth, this period takes placeduring childhood and adolescence, a time duringwhich the tooth may be subjected to a number ofconditions that may result in pulpal degeneration andinfection of the root canal space. These conditionsinclude carious lesions, traumatic injuries, and thedevelopmental anomalies that result in pulp exposure.Carious lesions that expose the pulp in teeth with an

immature apex typically occur in the first and secondmolars. First molars in particular erupt at an age whenchildren are not capable of exercising adequate oralhygiene on their own. Caries is more prevalent andmore extensive in children from low income families;these patients usually do not have good oral hygieneand/or they have poor dietary habits conduciveto dental caries (1,2). The vital pulp associatedwith carious exposures occasionally becomes mildlysymptomatic, leading to presentation for treatment.In these cases, a vital pulp therapy procedure such aspulp capping (3) or pulpotomy (46) may allow forthe normal apexogenesis of the tooth. However,frequently the pulp becomes necrotic, and the patientpresents with symptomatic or asymptomatic apical

disease. In teeth with an immature apex, it isrecognized that an apical radiolucency may beassociated with the dental follicle and not necessarilywith apical periodontitis. That is why apicalperiodontitis in cases with immature teeth is onlyconfirmed if the teeth have mechanical allodynia, areassociated with swelling or a sinus tract, or havestunted maturation given the age of the patient andthe status of neighboring teeth (Fig. 1).Traumatic injuries may affect children of any

socioeconomic background, typically affect anteriorteeth, and have a variety of clinical presentations.These injuries may result in crown or root fracture,tooth avulsion, or other luxation injuries. Olderstudies have shown that dental pulp revascularizationcan occur spontaneously or can be inducedtherapeutically when the pulp space is relativelyuncontaminated (79).Amongst the most prevalent congenital tooth

anomalies that may devitalize the dental pulp at anearly age is dens evaginatus, which usually results inpulp exposure following attrition from normal toothfunction (1012). These cases usually result inasymptomatic pulp necrosis, development of periapicallesions, and formation of sinus tracts. Dens invaginatusmay also develop a similar pulpal pathosis. The latter isusually characterized with more complex root canalanatomy, and may have necrosis and an infectious

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Endodontic Topics 2013, 28, 2437All rights reserved

2013 John Wiley & Sons A/S.Published by John Wiley & Sons Ltd

ENDODONTIC TOPICS1601-1538

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process in an additional canal or dens space, sparingthe main dental pulp of the tooth.

Success of spontaneous or inducedrevascularization following traumaThe response of the dental pulp to traumatic injuries inpermanent teeth with immature apices has beendescribed in many studies. An examination of thisin the literature is revealing with respect to theconditions that can lead to spontaneous or inducedpulp revascularization, and the interplay between thebiological response and the progress of infection inthese cases.In a study of replanted avulsed teeth with an

immature apex, it was shown that teeth with an apicalradiographic width of less than 1 mm had no chance ofrevascularization (13). Those with a wider apicaldiameter had a probability of revascularization thatwas on average only about 18%. The probability forrevascularization was significantly increased if theextra-alveolar time was less than 45 min (39% forshorter periods vs. 11% for longer periods; p < 0.05).In teeth with successful revascularization, two patternsof mineralization were observed: one suggestive ofcontinued normal maturation of the tooth in terms of

root length and dentin thickness, and another in whichradiopaque material separated from the canal wall butcontinuous with apical bone developed in teeth thathad arrested length (13). Interestingly, the use ofsystemic antibiotics post-operatively did not have aneffect on the success of revascularization in this study.The reduction in success with long extra-alveolarperiods may be related to the death of cellularelements that are critical in the healing process, butmay also be related to microbial contamination andgrowth. In fact, with short extraoral storage time, thelong-term response has long been known to involvepulp canal obliteration, with or without elongation ofthe root (Fig. 2) (14,15). Occasionally, bone andlamina dura are seen to develop in the pulp space(Fig. 3) (16).A series of seminal papers on the subject (1518)

with a sample size of 94 teeth and the long-termoutcomes have been published. For teeth with rootdevelopment at stages 25 according to Moorreeset al. (19), where each stage represents one-quarter ofthe root length development up to complete apicalformation, but without any apical constriction,replantation of teeth resulted in spontaneousrevascularization in 34% of cases (18). There was atrend for cases with more root development to

Fig. 1. (A) Mandibular molar with a carious exposure in an 8-year-old child. Apical radiolucency may represent apicalinfection or remnants of the tooth follicle. Negative response to pulp testing and mechanical allodynia confirmed theformer. (B) Left central incisor with a history of trauma and a sinus tract with gutta percha tracing. The tooth wasresponsive to cold. Maturation of neighboring tooth and a test cavity confirmed the diagnosis of pulp necrosis andchronic apical abscess.

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result in less revascularization (p = 0.08). Most ofthese cases resulted in pulp canal obliteration (29 of 32teeth), which was first detected 6 months post-operatively (18). Again, when immediate implantation(within 5 min) was compared with delayedimplantation, pulpal revascularization was significantlymore prevalent (p = 0.02) in the former. Amultivariate logistic regression analysis showed thatthe most important predictor of pulp healing or

revascularization in this study was pulp length, whereshorter root/pulp length was more conducive torevascularization. These findings corroborated theearlier findings by Kling et al. (13). In a subsequentanalysis of root length (15), the authors showed thatcontinued root development occurred significantlymore often when pulp revascularization occurred, butthat there were several cases in which normal rootdevelopment occurred when the pulp was necrotic

Fig. 2. (A) Maxillary right central incisor replanted after 10 min extraoral dry storage time in a 7-year-old girl.(B) Same tooth as in A, 10 years and 8 months after trauma. Apical obliteration and arrested root developmentcompared with tooth #9 is seen. Clinically normal percussion sound was registered with this tooth. Reproduced withpermission from Andersson & Bodin, 1990 (14).

Fig. 3. Arrested root formation and ingrowth of pulpal bone and PDL, after replantation of the right central incisorat stage 3 of root development. Reproduced with permission from Andreasen et al., 1995 (16).

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(15). They concluded that the Hertwigs epithelialroot sheath (HERS) may function separately from thepulp, citing earlier reports of cases where immatureteeth were lost completely but new root formation wasevident.Induced revascularization was shown to occur in

immature teeth with luxation injuries (7,8,20). Inthese cases, the luxation injury was thought to be anegative predictor of pulp survival, and scaffolds thatinvolved calcium phosphate mixed with variousformulations of collagen were used to allow pulp tore-grow into the pulp space and continue normal rootdevelopment. The underlying principle in many ofthese older studies was that pulp revascularization isimportant for continued root development, but thatthis cannot take place in the presence of an infection.This principle had been presented in earlier work inwhich experimentation with revascularization inhuman teeth with various pulpal diagnoses led to theseconclusions (9,21,22).Collectively, these findings show that despite

severance of the blood supply and possible planktonicbacterial contamination during luxation injuries,including avulsion of immature teeth, host responsesare able to eliminate microbial factors and promotehealing with maturation of the tooth in many of thesecases. In general, the prognosis of pulp survival isusually much better following traumatic injuries ofimmature teeth than in teeth with a closed apex and

thus emergency management combined with carefulmonitoring is usually advocated to assess the eventualpulpal responses.In cases where pulp necrosis and apical periodontitis

develop, apexification with calcium hydroxide (ormore recently mineral trioxide aggregate, MTA) hasbeen advocated in order to control the infection andstimulate a mineralized barrier at the end of the root(Fig. 4). Calcium hydroxide apexification may lead toweakening of the root due to the direct effect oflong-term calcium hydroxide on dentin (23,24), aneffect that was not seen with MTA (25). Therefore,long-term treatment with calcium hydroxide showed a2877% chance of cervical root fracture depending onthe degree of preoperative root development (26).Apical MTA plugs have a reasonable prognosis thatranges from 8191% according to several cohortstudies (2730). However, the root structure is stillrelatively weak and can easily fracture compared tofully mature roots.

Root canal disinfection strategieswith regenerative techniquesIn cases of teeth with necrotic pulp and an immatureapex, the root canal is usually large in size, permittingeasier permeation of antimicrobial irrigants into theentire root canal space. In fact, the concern is usuallyhow to prevent relatively toxic antimicrobials from

Fig. 4. 8-year-old first presented for treatment with signs and symptoms of pulpal necrosis and apical periodontitis.(A) Calcium hydroxide placed during emergency visit several weeks before resulted in calcific barrier short of the apexof the right central incisor. (B) MTA apexification performed. (C) Back-filled with gutta percha and sealer. Courtesyof Dr. Afua Mireku.


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gaining access to periapical tissues, which may containthe very stem cells and vasculature that are necessaryfor regenerating tissue. In this regard, the use of theEndo-Vac may be a good strategy to avoid periapicalextrusion of irrigants such as sodium hypochlorite(NaOCl) (31). Another concern is further weakeningof the root canal walls if traditional instrumentationis performed. This represents a significant challengeto traditional root canal debridement because thebacterial biofilm on the canal wall needs to bemechanically disrupted in order to allow antimicrobialagents to permeate the biofilm and kill bacterial cells.In addition, the bacteria may have gained access tothe dentinal tubules, and their presence there mayinterfere with the attachment of the regenerating pulptissue. In fact, it has been shown that bacteria mayinfect more dentinal tubules and to a deeper level inteeth from young compared with older individuals(32). Additionally, root canal anatomical areas thatcannot be adequately disinfected using traditionaltechniques such as isthmuses, fins, lateral canals, andapical arborizations may pose a significant problemsince bacterial reservoirs may populate them andpresent a problem for the regenerating tissues.Strategies such as photodynamic disinfection (33) orultrasonic irrigation (34,35) have been proposed toadequately clean these areas in mature teethundergoing routine treatment. However, the safety ofthese techniques with respect to the apical papilla andits stem cell contents has not yet been investigated.The most common antimicrobial irrigant for teeth

with a mature apex is sodium hypochlorite. In clinicaltrials, it has been shown that half- or full-strengthsodium hypochlorite reduces the incidence ofcultivable bacteria by 4060% (36,37). However, theseconcentrations of sodium hypochlorite have beenshown to prevent stem cell attachment to the dentinsurface (38), to abrogate odontoblastic differentiationmediated by growth factors from root dentin (39),and are toxic to stem cells of the apical papilla(SCAP) (40). This last study also showed that 2%chlorhexidine was the most toxic irrigant of all of thosestudied on SCAP. Therefore, it is clear that whendisinfecting the root canal space for the purpose oftissue revitalization, revascularization, or regeneration,a different paradigm should be considered, namely onethat also takes into account the vitality of the apicaltissues from which the new tissue will arise, and thereservoir of important growth factors in the dentin. It

is worth noting that in some of the successfulrevascularization case reports that have beenpublished, full-strength sodium hypochlorite has beenused for irrigation, at least for the first visit (10,11).

Intracanal antibioticsAntibiotics in the form of the poly-antibiotic pastecontaining penicillin, bacitracin, streptomycin, andsodium caprylate had been placed topically in rootcanals during endodontic treatment a long time ago(41). However, in the 1960s, the use of antibiotics inthe root canals of teeth undergoing endodontictreatment was abandoned because it was seen thatteeth without antibiotics healed at a similar healingrate, and because there were concerns about thedevelopment of superinfections or patient sensitizationto penicillin specifically through the root canal route(42).A series of studies were undertaken in Japan in the

1990s to determine the optimal antibiotic to use inroot canals. The antibacterial effects of a mixture ofciprofloxacin, metronidazole, and a third antibiotic(100 mg/mL amoxicillin, cefaclor, cefroxadin,fosfomycin, or rokitamycin) against endodonticpathogens were tested by culturing methods (43).Bacteria were obtained from carious lesions andinfected root canal spaces. The study showed that all ofthe aforementioned combinations of the antibioticsinhibited the growth of bacteria in the culture plates.This group investigated the antibacterial effects of thecombination of ciprofloxacin, metronidazole, andcefaclor on carious lesions of extracted teeth and rootcanal spaces of extracted teeth with endodonticinfections and showed that no bacteria were recovered.This study concluded that carious lesions andendodontic infections can be sterilized in vitro byusing a combination of antibiotics. Another in vitrostudy was performed on the effects of a mixture ofciprofloxacin, metronidazole, and minocycline (tripleantibiotic paste) with or without rifampicin (25 mg/mL) on endodontic bacteria obtained from root canalwalls of extracted teeth with endodontic infections,carious lesions, pulp tissues extirpated due to cariousexposure and irreversible pulpitis, or periapical lesions(44). Bacterial samples were cultured in anaerobicchambers in the presence of antibiotic mixtures andshowed no growth in any of the samples. There were

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no differences between triple antibiotic paste (TAP)with rifampicin and without rifampicin.The same group then tested the effects of TAP

(0.5 mg) on the bacteria in the deep layers of rootcanal dentin (45). They performed various proceduresin their study and tested the effectiveness of theantibiotic mixtures by using culture methods. Theprocedures showed that TAP diffuses throughout theentire dentin thickness and effectively disinfects deeplayers of the root canal dentin.The effectiveness of the TAP was evaluated in an

animal model of apical periodontitis (46). In thisstudy, periapical lesions were induced in the immatureteeth of dogs. Root canals were irrigated with 1.25%NaOCl and then each canal received a TAP dressingfor 2 weeks. The outcomes of the study revealed that,although only 10% of the cases had no cultivablebacteria after NaOCl irrigation, the TAP increasedthis percentage to 70%. In this study, the authorsperformed spiking of negative culture plates withPorphyromonas gingivalis (strain A7436, which isknown to be sensitive to the three antibiotics) toensure the absence of antibiotics in these negativecultures.In a similar study, another group investigated the

antibacterial efficacy of the TAP in immature infectedteeth in dogs (47). They showed that 78% of the rootcanals did not have cultivable bacteria after NaOClirrigation followed by 2 weeks of TAP dressing.Studies on pulp revascularization in extracted and

re-implanted immature teeth in monkeys haverevealed that topical doxycycline applicationsignificantly increased the frequency of completepulp revascularization, decreased the frequency ofmicroorganisms in the pulp lumen, and decreased thefrequencies of ankylosis and inflammatory rootresorption (48). However, systemic administration ofdoxycycline failed to produce any significant benefitin the same model (49).While all of these studies (in vitro and animal

studies) have shown acceptable results for root canaland dentin disinfection with TAP, recent research hasshown that culturing bacteria after antibiotic dressingis a challenging issue (50). Bacteria may remain viablebut uncultivable. Also, it is difficult to inactivateantibiotics in the culture medium. The animal studiesshowed the efficacy of TAP compared with just theuse of sodium hypochlorite. However, subsequenthistological analysis of these specimens showed

revascularization with bone, cementum, and inflamedconnective tissue rather than pulp and dentin(31,51,52).It is worth noting here that antibiotics have been

explored for use in combination with other agents asmedicaments to disinfect root canals topically in manyother conventional treatment situations. Examplesof these include Ledermix Paste (Haupt PharmaGmbH, Wolfratshausen, Germany) (which combines asteroid, triamcinolone acetonide, with the antibioticdemeclocycline calcium) as well as the combination ofcalcium hydroxide with clindamycin (53), or withtetracycline or erythromycin (54). However, it wasrecently shown that calcium hydroxide formulationsreduce the antibiotic content of Ledermix andOdontopaste (a similar product but with clindamycinas the antibiotic) due to the chemical interaction of theantibiotics and calcium hydroxide (55). A slow-releaseclindamycin fiber has also been investigated in the rootcanal environment and found to be efficacious againstFusobacterium nucleatum (56).

Antibiotics in clinicalrevascularization casesIn the first report of successful revascularization of animmature mandibular premolar with infected pulp,a double antibiotic paste (metronidazole andciprofloxacin) was used as an intracanal medicament(57). 30-month follow-up of the case showed healingof the periapical disease and development of the root.In another case report, the same outcome wasobtained with TAP (10). These authors treated animmature mandibular premolar with necrotic pulp andchronic abscess by using the TAP dressing. Thetechnique introduced by Banchs & Trope includedirrigation with 6% NaOCl and final irrigation withchlorhexidine in the first visit, followed by TAPdressing for 4 weeks. At the second visit, they inducedbleeding inside the root canal space by irritating theperiapical tissues and then sealed the blood clot with alayer of MTA followed by a permanent coronalrestoration. Another report from the same institutionshowed successful pulp revascularization in animmature maxillary central incisor with infected pulpby using a mixture of ciprofloxacin, metronidazole,and cefaclor (modified TAP) as intracanal medicamentfor 11 weeks (58). Following these reports, severalcase reports/series studies have been published to


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investigate technical modifications to the originalmethod introduced by Banchs & Trope (10). One ofthese reports showed that pulp revascularization canoccur without bleeding induction (59). Another caseseries recommended the use of anesthetic solutionswithout epinephrine at the second visit and the use ofa collagen barrier beneath MTA in the blood clot(60). One other report showed successful pulprevascularization in infected permanent mandibularmolars (61). The authors showed that when there isnot enough bleeding from small canals in molar teeth,the larger canal is used as a source of blood for theothers. Finally, the use of platelet-rich plasma wasintroduced in another report (62). In none of thereports published did the investigators determine theantibiotic susceptibility of the bacteria in the root canalbefore making their final selection of which antibioticor type of scaffold material to use.As described in a review introducing this general

area of research (63), the main goal of thistreatment modality is dentinpulp regeneration thatconsequently results in root development. Therefore,the outcome of the treatment should be evaluated atthree levels:(i) Clinical evidence of periapical healing and a

positive response to pulp vitality tests. Thisincludes the absence of sensitivity to percussionor palpation, and the absence of sinus tracts orswelling. This finding has been documented byall of the clinical reports (10,57,58,6062). Apositive response to pulp vitality tests has beendocumented in a few studies. A positive responseto a cold test (10,64) and an electrical pulp test(EPT) (62,65) is indicative of re-innervation ofthe root canal space regardless of the type oftissue generated inside the root canal.

(ii) Radiographic evidence of periapical healing androot development. This includes completeosseous healing of the periapical lesion, anincrease in root length, an increase in root wallthickness, and the formation of a radiographicapex. Complete osseous healing of the periapicallesion has been documented by clinical reports(10,57,58,6062). This finding in conjunctionwith clinical evidence of periapical health isindicative of the absence of inflammation inperiapical tissues due to effective eliminationof endodontic infection. However, these areindirect evidence of effective disinfection. Some

of the clinical studies have shown unfavorableroot development regardless of favorableinfection control by a TAP dressing. Theseunfavorable outcomes include no rootdevelopment (60,66) or apical closurewithout anincrease in root length or root wall thickness(67). When considering the variables studiedfor revascularization of traumatized teethmentioned previously, it may be speculatedthat incomplete bacterial elimination or osseousproliferation into the pulp space may beresponsible for these variations in clinicalresponses.

(iii) Histological evidence of dentinpulpregeneration inside the root canal space. Asnoted before, several animal studies have beendone to evaluate this aspect of the healing.Histological studies have been performed onimmature teeth with infected pulp space in dogs(31,51,52). These studies followed the protocolintroduced in the report by Banchs & Trope(10) and also evaluated technical modificationsincluded using Endo-Vac instead of TAPdressing (31) or doing revascularization withoutbleeding induction (51,52). Although all ofthese animal studies documented osseoushealing of the periapical lesions and rootdevelopment following treatment, none ofthem showed regeneration of the dentinpulpcomplex in the root canals. The findingsincluded ingrowth of periodontal connectivetissues (31) into the root canal space thatcontained bone-like tissues (52) and theformation of cementum or cementum-liketissues (52) on the dentinal walls. In an attemptto improve the outcome of the treatment,another study was conducted at the same school:regenerative endodontic treatment in immatureinfected dog teeth was attempted by addingtissue engineering strategies to the originaltreatment protocol (68). The authors usedcross-linked collagen as a scaffold and ethylene-diamine-tetraacetic acid (EDTA) to exposedentin protein matrix, as it had been shown tobe conducive to dental pulp stem cell (DPSC)proliferation (69). Although they showed thatthe use of collagen matrix and exposure of thedentin matrix to EDTA caused a significantincrease in the formation of mineralized tissues,

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immunohistological evaluation of the tissuesrevealed that they did not regenerate the dentinpulp complex (70). The findings revealed theformation of cementum-like tissues on thedentinal walls and bony islands inside the rootcanals.

Histological evidence of the tissues formed insidethe root canal space in human teeth is still very limited.However, the available case reports show thatfollowing disinfection with the techniques describedpreviously, with or without the use of platelet-richplasma as a scaffold material, regeneration of the pulpdentin does not occur, but rather connective tissuewith cementum-like mineralization similar to theanimal findings appears to occur in humans as well(7173).

Biological studies on TAPThe response of rat subcutaneous tissue to TAP within90 days was recently reported (74). There was amoderate inflammation response to the paste at 15days, which was reduced to mild at 30 days. Theauthors concluded that TAP is biocompatible and willnot interfere with the process of healing. SCAP cellswere exposed to different dilutions of TAP, modifiedTAP, or a double antibiotic paste (ciprofloxacinand metronidazole) (75). They showed thatconcentrations more than 16 mg/mL weredetrimental to SCAP survival for all antibioticmixtures. Considering that for antibiotic pastes a1,000 mg/mL solution is needed to create a pastyslurry in a clinical situation, the current clinicalprocedures might pose a risk to the survival of SCAPcells during TAP dressing and consequently to theoutcome of dentinpulp regeneration.

Clinical outcomes of the treatmentprovided by revascularizationA retrospective study was performed to compare theradiographic outcomes of regenerative endodontictreatment with those of MTA apexification andformocresol or non-surgical root canal treatment (76).This study showed that regenerative treatments usingTAP or calcium hydroxide as an intracanal dressingproduced significantly greater increanses in rootlengths compared to controls, but that TAP was

significantly better than the other groups in terms ofroot wall thickness. More recently, a retrospectivestudy on clinical and radiographical outcomes ofregenerative endodontic treatment compared to MTAapexification and calcium hydroxide apexificationshowed a greater increase in root length and root wallthickness of the teeth treated with regenerativeprocedures (77). In addition, this study demonstrateda higher survival rate for teeth treated withregenerative procedures (100%) comparing to MTAapexification (95%, which was not statisticallysignificant) and calcium hydroxide apexification(77.2%, which was statistically significant).Altogether, the published reports clearly

demonstrate that, despite the differences in antibioticcombinations used, the important clinical outcome isthe control of infection and promotion of tissuegrowth into the canal space. This is significant giventhe fact that some side-effects may preclude the use ofone or more antibiotics, and this may not necessarilyaffect the overall control of infection. The outcomestudies also show some advantages to the use ofantibiotics over the use of calcium hydroxide. It wasnoted in one study that calcium hydroxide had pooroutcomes if it was placed to the apex of the tooth(76,78). The authors of this review have observedanecdotally that in some of these dens evaginatus casesin mandibular premolars, despite negative pulpresponses and the presence of an apical radiolucencyand a sinus tract, there was some bleeding internally,and the patient had some sensitivity in the mid-canalarea, necessitating additional anesthesia. Clearly, theseinfectious processes in children, which are presentat the same time as the proliferating apical papilla,may involve a more complex pathological situationcompared with traditional endodontic infections, onethat may just respond to the coronal placement ofcalcium hydroxide (78).

Contemporary regenerativeprotocols from adisinfection perspectiveAlthough the literature shows that the currentregenerative protocols do not regenerate the dentinpulp complex, recent studies revealed the importanceof regenerative approaches in increasing the survivalrate of immature teeth with necrotic pulp and theimpact that this has on the long-term oral health of the


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patients. Therefore, the protocol for regenerativeendodontic treatment should be examined carefully,and the best available evidence should consistently beapplied to maximize successful outcomes.A recent report on the revascularization of two

maxillary central incisors with necrotic pulps showedthat the criteria for predictable revascularization arestill lacking (66). Although previous studies (13) hadshown that the wider the apical foramen and theshorter the tooth, the more likely that therevascularization procedure will succeed, this reportshowed the opposite results.A recent review of the dental history of all of the

cases of regenerative endodontic treatment reportedfrom 2004 to 2012 showed that the longer the periodof pulp necrosis and periapical disease beforetreatment, the higher the chance of obtainingunfavorable root development after treatment (67).This may be due to the maturation of the bacterialbiofilm as described above, which results in moredifficult elimination by conventional protocols. Thatreview also listed the unfavorable outcomes of currentregenerative protocols, including discoloration, rootcanal calcification/obliteration, insufficient bleeding,and poor root development.Irrigation with sodium hypochlorite has always been

part of the disinfection protocol in regenerativeendodontic treatments in animal studies (52,68)and human reports (58,60,61,64). Differentconcentrations of NaOCl including 1.25% (58), 2.5%(78), 5.25% (10,60,61), and 6% (64,79) have beenshown to be associated with a successful clinicaloutcome. As we noted before, however, in vitro andsome animal studies have shown a number ofunfavorable effects of higher concentrations of NaOClon regenerative procedures. These data prove the needfor further studies aimed at finding a more appropriateand biocompatible irrigation protocol.Discoloration after treatment is a serious drawback

related to the use of TAP. As illustrated by Kim et al.(80), the main culprit is minocycline in TAP. Reynoldset al. (64) introduced the technique of sealing thedentinal walls of the access cavity with flowablecomposite resin before introducing TAP into thecanal. However, it was still shown that using dentinbonding before antibiotic dressing can reduce thechange in darkness of the tooth but cannot prevent it(80). A recent study on the discoloration potential ofseveral endodontic materials revealed that TAP was

associated with the highest amount of discoloration(81).In addition to discoloration, recent findings on the

toxicity of high concentrations of TAP and several ofits alternatives (75) show the necessity of studies onthe apical diffusion of antibiotics placed into the rootcanal environment. Using more effective antibioticsand investigating methods that allow for the slowrelease of antibiotic dressings in order to lower theconcentration should also be the subject of futurestudies. As well, the disinfection protocols need to beevaluated by both culture and molecular methods,which are complementary to one another and providea more comprehensive view of the infectious process(82).Since the main goal of regenerative endodontic

procedures is dentinpulp regeneration, the bestcriteria to assess the treatment is the histologicaloutcome. However, this method is not practical inhuman teeth. Osseous healing of the radiographiclesion and the absence of clinical signs of periapicaldiseases are indirectly indicative of efficient disinfectionprotocols.Moreover, a positive response to pulp vitalitytests is only indicative of re-innervation of tissues insidethe root canal space. The re-establishment of pulpvitality was shown in a case report of luxated humanimmature teeth using laser Doppler flowmetry (LDF),and was much faster than thermal responsiveness (83).Confirmation at this level shows the revitalization of thepulp tissue, but still does not reveal the nature of thetissue formed.

Analysis of endodonticmicrobial burdenData on the microbiota of immature teeth withendodontic infections, or endodontic infections inchildren categorically is very sparse. It is generallyaccepted in any infectious disease that accuratecharacterization of the microbial pathogens is criticalfor identifying the appropriate therapy. This isespecially true for therapies that involve antibiotics.Not only is it important to determine the rightantibiotics for the pathogens present, it is alsoimportant to continue this analysis given that bacteriain root canals contain a lot of antibiotic resistancegenes (82) and continuously evolve through plasmidexchange and other mutations (84).

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The use of specific antimicrobial therapies todisinfect root canals in preparation for the developingnew tissue has other advantages. Among these isallowing the antimicrobial agent to have a lastingeffect, which may give the growing tissue anopportunity to establish its own immune responses.This is essential for the regenerated tissue to be able toeffectively manage any residual infection, as well ascombat any new infection from a coronal, tubular,or apical source. At the same time, non-specificantimicrobial therapies may give a preferentialadvantage to virulent microorganisms to allow them toproliferate, thus having an antagonistic effect (85).The advent of sensitive molecular tools allows for the

rapid and deep coverage analysis of any microbiome ofinterest (86).Using these technologies has transformedthe study of endodontic microbiology from one thatdeals with a few detectable microorganisms to one thatstudies all of the microflora present, their sources, andtheir patterns of migration to form various signs ofthe disease (87,88). In the future, this analysis couldexpand to rapidly identify virulence determinants,antibiotic resistance genes, or vaccine targets that maybe of therapeutic value.

Antimicrobial efficacy and tissueengineering strategiesIt is generally accepted that tissue regenerationrequires the interaction of stem cells and growthfactors in a bioactive scaffold called a tissueengineering triad (89). For a scaffold, contemporaryprotocols use either a blood clot (63) or platelet-richplasma (PRP) (62). Growth factors are released fromthe root canal dentinal walls following EDTAirrigation (90). SCAP has been accepted as a sourceof stem cells for dentinpulp revascularization/regeneration. In this regard, SCAP was shown to bethe primary source of odontoblasts responsible forroot dentin production and root development (91),and human stem cell markers were significantly higherin the blood clot produced inside the root canalcompared to peripheral blood (92). However, the lackof dentinpulp regeneration in animal (52,68) andhuman (71) studies following current regenerativeprotocols shows that the process of disinfectionmay not be complete, and/or the process of celldifferentiation inside the root canal space is notpredictable. Introducing a known dental pulp stem cell

source to the current protocol should be the subject offuture studies. Also, the effect of growth factors on thedifferentiation of stem cells inside the root canal needsto be further studied.Regenerative techniques in an infected space, where

the bacteria cannot be totally eliminated, will alwayshave to go hand-in-hand with antimicrobial strategiesuntil the regenerated soft tissue has sufficientvascularity and immune response to spontaneouslycontinue its defense mechanisms. Removing theantimicrobial in order to start the regenerative processmay be the missing link in the regeneration equation,as bacteria may proliferate and negatively influence theprocess, leading to the success of only some but notthe desired tissues. In this regard, guided pulpregeneration should be studied, and adequate animalmodels must be developed for sufficient research inthis area.One potential strategy that may allow this

combination of tissue engineering and antimicrobialprotocols is to incorporate a slow-release antimicrobialinto the scaffolds that are used to deliver stem cells andallow the growth of the developing pulp tissue. Thisantimicrobial must be released from the degradingscaffolds in concentrations that are high enough toeffect the disinfection and abrogate bacterial growth,but at the same time not interfere with the viability anddifferentiation potential of the stem cells. Recently,there have been several attempts to investigate theseapproaches in various systems (9395).Finally, clinicians and the research community must

come to an agreement as to what is a clinicallyacceptable outcome. For example, is a tooth where theinfection has completely resolved but that hasundergone pulp canal obliteration with mineralizedtissue, which is not necessarily dentin, a clinicallysuccessful outcome? Answers to these and othersimilar questions will determine where theantimicrobial strategies evolve.

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