Alternative to Avoid Tooth Discoloration after Regenerative Endodontic Procedure: A Systematic Review

triple antibiotic paste (TAP metronidazole, ciprofloxacin, and minocycline) and grey mineral trioxide aggregate (GMTA) could avoid tooth discoloration in teeth submitted to Regenerative Endodontic Procedure (REP). It was also investigated if dental bleaching is able to reverse the color of darkened teeth due to REP. The search was conducted in four databases (Medline via PubMed, Scopus, ISI Web of Science and BVS Virtual health library), following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. The retrieved papers were uploaded in the software EndNoteTM and two reviewers independently selected the studies and extracted the data. Only studies in humans (case reports, case series, clinical trials) were included in the review. From 1,122 potentially eligible studies, 83 were selected for full-text analysis, and 38 were included in the review. The included studies were mainly case reports (76.3 %). The studies described a total of 189 teeth submitted to REP. From these, about 54% of teeth presented some degree of discoloration. Most teeth presenting color alteration were treated with TAP, especially when combined with GMTA. Only three studies performed dental bleaching to restore the color of teeth and neither bleaching technique was able to restore the original color of the crowns. The use of alternative materials to TAP and GMTA, such as double antibiotic paste or Ca(OH)2 pastes and white mineral trioxide aggregate or BiodentineTM, reduces the occurrence of tooth discoloration. Alternative to Avoid Tooth Discoloration after Regenerative Endodontic Procedure: A Systematic Review


Introduction
Regenerative endodontic procedure (REP) has been proposed as a treatment option for necrotic immature permanent teeth (NIPT) in detriment to apexogenesis with calcium hydroxide (CaOH 2 ) and apexification with trioxide mineral aggregate (MTA) (1). REP is based on the formation of blood clot into the root canal space creating a natural scaffold that serves as anchorage site for cells from apical papilla and periodontal tissues. Additionally, blood supplies growth factors that act along with those from dentin matrix, contributing to the formation of a new tissue into the pulp cavity and to the resumption of root development (1,2). Therefore, REP seems to provide an increase in root lengthening and thickening, as well as a remission of the signs and symptoms of apical periodontitis (2).
In the Clinical Considerations for a Regenerative Procedure, the American Association of Endodontists (AAE) (3) recommends for root canal disinfection the use of CaOH 2 or triple antibiotic paste (TAP -metronidazole, ciprofloxacin and minocycline), for a short period (3). Probably, the presence of minocycline, an antibiotic of tetracycline family, in TAP composition plays an important role in tooth discoloration (4,5). Minocycline binds to the calcium ions present in the dentin by means of chelation, being incorporated into the dentin matrix and inducing a change in the color of the dental structure (4). To avoid crown discoloration, it was proposed to seal the coronal dentinal tubules with bonding agent prior to the use of TAP as intracanal medication (6) as well as the replacement or eliminating minocycline from TAP composition. The alternatives to minocycline report in the literature are performed mainly with cefaclor (7), amoxicillin (8) or clindamicine (9). Some studies have adopted only the elimination of minocycline of conventional TAP, so called Double antibiotic paste (DAP -metronidazole, ciprofloxacin) (10,11).
Moreover, the blood clotted inside of the root canal must be protected in its cervical portion by a sealing material, conventionally the MTA (2). However, MTA also can lead to a change in the tooth color due to presence of minerals as bismuth oxide, its radiopacifier component (12). Although the AAE recommends the use of glass ionomer cement (GIC) to avoid the discoloration in areas where esthetic is primordial (3), several studies have performed the seal with white MTA (WMTA) due to less amount of metallic ions (7,13,14), which could reduce tooth crown discoloration.
Therefore, although satisfactory biological results are achieved after REP (1,2,15), several case reports have indicated color change, especially in those cases treated with TAP (16)(17)(18). A recent literature review (19) has also shown that TAP is the main reason to tooth discoloration in REP, although did not directly compare the possible reduction of tooth discoloration when alternatives materials (to TAP and gray MTA) are employed. Thus, it suggest that the use of pastes alternative to TAP and the replacement of grey MTA (GMTA) could decrease the percentage of tooth discoloration (5,20,21). Moreover, in cases of discoloration, dental bleaching can be performed aiming to minimize esthetic damages. However, the ability of dental bleaching in restore the original color of teeth did not was aim of systematic reviews. Similarly, little is investigated to ability of alternatives materials to TAP and GMTA to avoid the tooth discoloration in REP. Thus, the aim of this study was to review the literature systematically and evaluate whether alternatives to conventional TAP and GMTA could avoid tooth discoloration in teeth submitted to revascularization and investigate if dental bleaching is able to reverse the color of discolored teeth due to REP.

Material and Methods
This systematic review was carried out according to the guidelines of Cochrane Handbook for Systematic Reviews of Interventions, following the four-phase flow diagram of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (22). This report is based on the PRISMA Statement. The question of research was adapted from the PICO framework; Population -human immature permanent teeth; Intervention and Comparison -use of TAP and GMTA in regenerative endodontic procedures vs. alternative materials; Outcome -tooth discoloration.
A structured research was carried in Pubmed/Medline, Scopus, ISI Web of Science and BVS -Virtual health library. Date limit was set up to March 2017, with no language restriction. The search and the combinations of syntaxes are described in Table 1. The references of all eligible papers were also hand-searched. A wide search strategy was used to avoid missing information. Duplicated studies were removed by using EndNote TM X7 software (Thomson Reuters, Rochester, New York, NY, USA). The inclusion criteria were in vivo original studies (prospective, retrospective, clinical cases, case series, clinical trials) in which regenerative procedures were performed in immature permanent teeth, and which color maintenance or the tooth discoloration, as an outcome or not, were described. Studies were included independently of their follow-up intervals. The exclusion criteria were: reviews, in vitro studies, studies in animals, or studies that did not report the color maintenance or tooth discoloration.
Two independent reviewers (L. A. C. and C. G. S.) initially screened the titles of all identified studies. If the title indicated possible inclusion, the abstract was carefully appraised, and the manuscripts considered eligible for the review (or in case of doubt) were selected for full-text reading. Discrepancies were resolved by discussion with a third reviewer (L. G. P.) Standardized outline was used for data extraction of the studies that met the inclusion criteria. Due to the high heterogeneity of included studies, evaluation of risk of bias and meta-analysis could not be performed. Consequently, the characteristics of studies were summarized descriptively.

Studies selection
The flowchart of the systematic review is shown in Figure 1. A total of 1,122 records were identified and 843 remained after duplicated studies removal. After title and abstract evaluation, 83 papers were selected for full-text assessment. From these, 38 studies met the inclusion criteria of the present study. Forty-five studies were excluded after full-text reading once they did not report the color maintenance or the tooth discoloration , or since the tooth discoloration described had occurred prior to revascularization procedure (63)(64)(65)(66)(67)(68).

Synthesis of Results
Considering the 189 teeth evaluated, 54% (n=102) presented some degree of discoloration. Tooth color assessment was performed at the end of REP, therefore the evaluated teeth had already suffered the possible effects of intracanal medication as well as cervical sealing material. Thus, the results of the present review are described according the association of these factors: (1) TAP/GMTA, (2) TAP/alternative materials (AM) to GMTA (WMTA, Biodentine, and CEM), (3) AM to TAP/GMTA, and (4) AM to TAP/ AM to GMTA.
In three studies dental bleaching was performed in 16 teeth (4,9,83) to restore the natural dental color. Internal bleaching technique applying sodium perborate as whitening agent was employed in two studies (2 teeth). The third study, associated 10% hydrogen peroxide to the sodium perborate (83). In studies in which only sodium perborate was used, authors described an improvement in the color of the teeth, but teeth do not return to the original shade. Satisfactory results were reported for 11 of the 14 teeth treated with sodium perborate associated with hydrogen peroxide.

Discussion
The regeneration of NIPT is a recent approach presenting promissory results (1,2). However, the risk of tooth discoloration, observed in several cases (10,16,18,69,87,89,92,94), can compromise esthetic results and even the quality of life of the individuals (96). In the present study, the literature was systematically reviewed to assess whether alternative materials to conventional TAP and GMTA could avoid tooth discoloration after pulp revascularization. The included studies showed that there is great potential for tooth discoloration after REP. More than 50% of the revascularized teeth presented some color alteration, mainly when TAP was used as intracanal medication and GMTA as cervical sealing material.
TAP is the most used intracanal medication in revascularization (2), presenting biocompatibility (97) and adequate antimicrobial capacity, being able to perform root canal system disinfection (98)(99)(100). According to the American Association of Endodontics (AAE) antibiotic pastes should be used at concentrations ranging from 0.1 mg/ mL to 1 mg/mL, which promote low cytotoxicity to stem cells from apical papilla (3), maintaining its disinfection ability (101). However, even nowadays, authors have still employed pastes at different concentrations higher than that recommended. A recent case series study reported the use of 1g/mL on detriment to recommended due to clinical difficulties of applying pastes in low concentrations (72). Hence, crown discoloration has been more frequently reported after the use of 1g/mL TAP in root canal treatment. Some studies reports a rapid tooth discoloration after TAP application (89,93). The main reason for tooth staining (69), has been advocated to minocycline, a tetracycline derivate, present in TAP composition (102). Discoloration induced by minocycline normally occurs in the first 24 hours after its use, promoting a bluish-grey staining, clinically perceivable (11). Usually, this alteration is more evident in tooth crown, and in the cervical third of the root (102). Therefore, modifications to the original TAP (98) have been proposed, with the aim at avoiding discoloration, by the use of cefaclor (6,7,70,72,73), amoxicillin (8,74) and clindamicine instead of minocycline (9) Nevertheless, Figure 2. Tooth discoloration according with materials used to regenerative endodontic treatment. some degree of discoloration has been still observed after the use these alternative medications. Dabbagh et al. (7) associating WMTA to TAP/cefaclor observed discoloration in only one of the 18 revascularized teeth. An in vitro study showed that TAP with minocycline promoted higher discoloration than TAP with amoxicillin, doxycycline, or cefaclor (102). It was also observed that TAP induced a severe color change in the first day of the assessment, while the use of amoxicillin showed similar discoloration only after one week. On the other hand, double antibiotic paste (DAB -metronidazole and ciprofloxacin) and Ca(OH) 2 did not produced color alterations (102). Another in vitro study showed similar discolorations induced by these pastes, while the most esthetic outcomes were found when alternatives to minocycline were used (5).
A reduced number of cases reporting discoloration were observed when Ca(OH) 2 was applied as intracanal medication, however, from 20 teeth revascularized with Ca(OH) 2 six presented some color change (14,69,(76)(77)(78)(79)(80)(81). This may be explained by the fact that the materials used in the cervical plug can also promote discoloration, especially GMTA (80,81). Although in some cases is difficult to define if the color change was promote by antibiotic paste.
GMTA has been extensively used as cervical sealing material, being applied above the blood clot in resvacularized teeth (2). However, GMTA presents high levels of iron, magnesium, aluminum, as well as bismuth oxideas a radiopacifier, which can oxidize promoting tooth discoloration (12). Staining due to MTA can occur immediately after the first day of application and increase over the time (21). Aiming to avoid tooth discoloration, WMTA was developed, with significant reduction of minerals in its composition. Despite this, in vitro studies have shown that WMTA also could promote some degree of discoloration (20,21).
Only one study included in this review used Biodentine TM as cervical plug. As a result, all teeth treated showed minimal discoloration (72). In these cases, the color alteration probably can be attributed to the biochemical interaction between Biodentine TM and platelet-rich fibrin (PRF), mixed to the blood clot, since Biodentine presents zirconium oxide as radiopacifier instead of bismuth oxide (20,21). Biodentine TM was recently developed tricalcium silicatebased material, which presents high biocompatibility and mechanical resistance (103). Recently, in order to avoid the negative effect of the Portland cement-based materials on tooth color, AAE (3) recommends the use of glass ionomer cement in the cervical sealing, especially for the anterior teeth, in which esthetics assumes an important role. No study included in this review used GIC for this purpose.
Another reported way to minimize the risks of tooth discoloration is sealing dentinal tubules of the pulp chamber with adhesive agent prior to the use of TAP (3). However, more studies are necessary to confirm the role of adhesive agents in prevent color alterations following REP. In the present review, three studies performed this preventive technique (6,70,72). Bezgin et al. (6) applying adhesive agent previously intracanal medication with TAP/ cefaclor, followed by WMTA as cervical sealing material. No discoloration was observed in both two evaluated teeth. However, in a study performed in 20 cases of revascularization, the application of an adhesive agent before TAP/cefaclor, did not prevent discoloration in 12 teeth. Nevertheless, the staining was attributed to the GMTA, used as cervical sealing material (70). Finally, Bakhtiar et al. (72) also sealed coronal dentinal tubules prior to treat root canal with TAP/cefaclor; however, as cervical sealing material Biodentine TM was used rather MTA. Discoloration occurred in all the four revascularized teeth. Authors attributed the staining to the interaction between Biodentine and PRF. Confirming the results of those studies, an in vitro (4) one observed that the use of the adhesive (self-etching, 2-component adhesive dentin bonding system) reduced, but did not prevent staining. The samples became yellow, reproducing the color of minocycline. The explanation for this was probably due the use of a clear bonding agent, without the ability of completely blocking the yellowish color of the antibiotic. Besides, it is important highlight that the REP per se can promote some degree of discoloration. An recent in vitro study that evaluate by spectrophotometry the change color in teeth submitted to REP observed a low discoloration in the control group -no antibiotic pastes treatment (5).
Aiming restore tooth color, some studies reported perform dental bleaching. The crowns were treated by the internal bleaching technique with sodium perborate or sodium perborate associated to hydrogen peroxide (4,9,83). However, neither technique was able to restore the original color of the crowns. These results did not corroborate with a study that used spectrophotometer, before and after the use of materials commonly applied in REP, to investigate the efficacy of dental bleaching on the color of bovine teeth. The results showed that only one application of 10% carbamide peroxide, inside the pulp chamber and on the outer surface of the crown, was enough to make the color of the teeth similar to the original one (5).
In all the studies included in this review, the visual method of analysis was used to evaluate the color change. However, this method results in a subjective evaluation, which can be affected by factors such as the experience and the chromatic perception of the evaluator, the illumination of the environment (104). In order to avoid biases resulting from the evaluation of the human eye, spectrophotometers should be used instead. Spectrophtometer devices determine the color of the tooth from numerical values that represent light and hue, being considered reliable methods for color reading (105). However, these devices are not yet widely used in the clinical routine.
It is important to highlight that reports of tooth discoloration was found predominantly in cases treated with TAP and GMTA. Although the high amount of studies found at the initial search, many studies did not fulfill inclusion criteria of this review. Therefore, the present results should be interpreted with caution. Since the main outcome assessed in included studies was the survival of the teeth, dental color was mostly reported as secondary data. Therefore, tooth discoloration might had been underestimate, being reported only in those cases in which a major color change was noticed. Moreover, the lonely effect of the different materials involved in REP on tooth discoloration is a very hard task, since intracanal medication and cervical sealing are applied sequentially, and both have potential to induce tooth color alteration. The conduction of randomized clinical trials evaluating tooth color on each phase of regenerative endodontics is highly recommended to better understand the role of each material in tooth discoloration.
Alternatives to conventional Triple Antibiotic Pastes and Gray MTA avoid tooth discoloration in teeth submitted to revascularization. The association of alternatives to Triple Antibiotic Paste and alternatives to Gray MTA resulted in minor discoloration. Dental bleaching is able to reverse partially the color of discolored teeth due to REP. Minocycline seems to be the main responsible for discoloration induced by TAP.