Efficiency of Teeth Bleaching after Regenerative Endodontic Treatment: A Systematic Review

The aim of this review is to evaluate of effectiveness of bleaching procedures used to treat discolored teeth subsequent to regenerative endodontic procedures (REPs) based on the review of in vitro and in vivo studies. This literature review was carried out according to the PRISMA guidelines. Four databases (PubMed, Scopus, the Cochrane Library, and Web of Science databases) were searched electronically, until 30 January 2020 without a year limit. The quality of studies was assessed using a modified methodological index for non-randomized studies. After analyzing 1405 studies, 6 in vitro and 9 in vivo studies were eligible for this review. In in vitro studies, effectiveness of bleaching was assessed in teeth discolored by antibiotic pastes, blood, and barrier materials in various combinations. In all analyzed studies, bleaching was effective in teeth discolored by antibiotic pastes as well as by blood and barrier materials. Of 26 treated teeth in the in vivo studies, 17 teeth were bleached successfully. In six cases, there was improvement of the shade. In three cases, bleaching was not sufficient. Bleaching material, techniques, and times differed between studies. Whitening of discolored teeth after REPs is achievable. However, to establish precise guidelines, further long-term clinical studies should be performed.


Introduction
Regenerative endodontic procedures (REPs) are a recently expanding field in endodontics. They are "biologically based procedures designed to physiologically replace damaged tooth structures" [1]. Regeneration of damaged dentin and root structures, as well as the pulp-dentin complex, are fundamental goals of these procedures [1]. REPs are increasingly applied in immature permanent teeth with pulpal necrosis (with or without apical periodontitis) as an alternative treatment option to apexification. Unlike traditional apexification, physiological root development and maturation represent the greatest advantages of this method [2]. However, some studies report the occurrence of tooth discoloration subsequent to REPs [3,4], which is an unfavorable outcome of these procedures [5,6]. The potential causes of observed discoloration are respectively: intracanal medicaments, distribution of blood products, and compositions of barrier materials used in REPs.
Teeth discoloration may negatively impact the quality of life in young patients and their families [7], especially if the problem concerns anterior teeth. To minimize the risk of discoloration, placing of triple antibiotic paste (TAP) containing minocycline (TAPM)

Search Strategy and Study Selection
Four databases (PubMed, Scopus, the Cochrane Library, and Web of Science databases) were searched electronically by two independent reviewers (I.F and T.F) for publications involving bleaching of teeth discolored after REPs. Publications were searched without a year limit. The last search was conducted on 30 January 2020. The search phrases are presented in Table 1. After removing duplicates all titles and abstracts were examined.

Quality Assessment
The quality of in vitro and in vivo studies was assessed using a modified methodological index for non-randomized studies (MINORS) [19]. This index includes twelve items: the first eight items pertain to non-comparative studies and an additional four concern comparative studies. Search results of in vivo studies including case series and case reports. The risk of bias assessment tool to evaluate the methodological quality of case reports was not developed [20]. To show an overview and standardize different types of studies including case reports and case series a modified MINORS scale was used analogous to that of Benetti et al. [21]. The modified MINORS scale items were as follows: clear aim, clear REPs protocol, clear bleaching protocol, prospective collection of data, justification of sample size, follow-up period appropriate to the aim of the study, endpoints appropriate to the aim of the study, blinded analysis, an adequate control group, contemporary groups, baseline equivalence of groups, and adequate statistical analyses. The items were scored: 0, not reported; 1, reported but inadequate; and 2, reported and adequate. All twelve items were used to assess in vitro studies, of which the first eight items were used to evaluate the in vivo studies. The ideal score for comparative studies is 24 and for non-comparative studies 16 [19]. The classification of quality of the in vivo studies was made on according to that of Elkhadem et al. [20] into poor (0-5), fair (6-10), and good (11)(12)(13)(14)(15)(16) and analogously a similar classification was used for in vitro studies: poor (0-8), fair (9)(10)(11)(12)(13)(14)(15)(16), and good (17)(18)(19)(20)(21)(22)(23)(24). The results of each item, total score, and study quality are presented in Tables 2 and 3 for the in vitro and in vivo studies, respectively.

Results
After analyzing 1405 studies retrieved from the query of all databases, six in vitro and nine in vivo studies were qualified on using the PRISMA criteria ( Figure 1). Two of the included studies were searched from additional sources [11,30]. Articles excluded were studies in which the procedure did not correspond to REPs in in vitro studies [33][34][35]; studies where bleaching was performed after apexification [36] or partial pulpotomy [37] in in vivo studies; studies lacking information about bleaching agent and technique [38,39]; and review articles [4,40].
Bleaching material was placed inside [16,[22][23][24][25][26] or inside and outside [25] dental crowns. The authors evaluated color changes using spectrophotometric measurements [16,[22][23][24][25][26] which allows to obtain the CIELAB color coordinates for a quantitative evaluation of color changes and based on the VITA Classical A1-D4 ® Shade Guide, a standard and reference system used worldwide in tooth shade determination [25]. In studies in which antibiotic pastes were used, a tooth stained with TAPM bleached more than a tooth stained with TAP with doxycycline (TAPD) and amoxicillin (TAPA) using both 35% hydrogen peroxide and sodium perborate [16,26]. In studies in which TAP with cefaclor (TAPC), blood and barrier material such as Biodentine (Septodont, Lancasted, PA, USA), MM-MTA, (Micro Mega, Besancon Cedex, France), and ProRoot MTA (Dentsply, Tulsa, OK, USA) were used and bleaching was performed with 35% hydrogen peroxide, the group treated with Biodentine bleached significantly more than in other groups [23]. Information extracted from qualified in vitro studies including number and teeth type, intracanal medicament or/and coronal barrier, discoloration, bleaching material and technique, the measurement method, and time as well as bleaching effect are listed in Table 4. Figure 2 presents delta E values from studies, which included its value among the results.

Results of In Vivo Studies
Nine in vivo studies related to bleaching after REPs were identified in this review, seven of which were case reports [9,11,[27][28][29][30][31] and two case series [17,32]. Of the 26 treated teeth, 23 were anterior teeth [9,11,17,27,28,30,32] and three involved premolar teeth [29,31]. Bleaching was performed in teeth in which discoloration was caused by intracanal pastes In the studies discolored teeth were bleached with different bleaching agents: sodium perborate [9,11,27,28,31], hydrogen peroxide [29], and carbamide peroxide [32]. Two authors used the combination of sodium perborate and hydrogen peroxide [17,30]. Sodium perborate was the most frequent bleaching agent used in the in vivo studies. Whitening improved the color of the discolored teeth [11,[28][29][30] both without [32] and with patient satisfaction [27,31]. However, teeth did not always return to their original shade [9]. Of all the analyzed studies, three cases did not bleach sufficiently [17]. Table 5 contains details from in vivo studies including patient ages, tooth type, injury, preoperative diagnosis, medicaments applied in REPs, discoloration, bleaching material and methods, as well as bleaching outcome.
A statistical analysis could not be performed because of different parameters used by reviewed studies.

Discussion
This review investigated the efficiency of bleaching tooth discoloration after REPs. All qualified studies were divided into two groups, in vitro and in vivo studies, and were discussed separately.

Analysis of In Vitro Studies
Most of the analyzed studies evaluated the whitening effect of tooth discoloration caused by disinfectant pastes [16,22,24,26] because root canal disinfection is the most important and initial stage in the revascularization procedure in which discoloration could be observed. In the in vitro studies included in this analysis where TAPM was used, coronal discoloration was observed more often than when other disinfectant pastes were applied [16,25,26]. A tooth stained by TAPM whitened more compared to teeth discolored by TAPD, TAPA, TAPC, or DAP [26]. Yasa et al. [16] found that the whitening effect was greater when teeth were discolored by TAPM and TAPC compared to groups with TAPD and TAPA. However, the number of teeth in the groups was smaller (n = 5) than in the study by Fundaoglu Küçükekenci et al. [26] and there was no information on the concentration of TAPC used [16].
Effective bleaching was achieved using 35% hydrogen peroxide [16,26], sodium perborate [16,22,24], and 37% carbamide peroxide [25]. The whitening effect of 35% hydrogen peroxide was greater than that of sodium perborate [16]. The bleaching effect of 35% hydrogen peroxide exceeded the perceptibility threshold from the 4th day of the evaluation and increased over time [26]. The highest bleaching effect was noticed on the 12th day [26], but another study showed no statistically significant difference (P = 0.175) between the 8 th and 12th day of measurements [16]. The number of teeth in the test groups differed between studies [16,26]. Nd-YAG laser irradiation on a 35% hydrogen peroxide increased the efficacy of internal bleaching, but there was no significant difference between the walking bleach technique and the thermo/photo bleaching technique (P = 0.19) [26]. Akbulut et al. [23] assessed the effectiveness of whitening teeth in which discoloration was induced respectively by TAPC, blood, and barrier materials, such as Biodentine, MM-MTA, or ProRoot MTA. In this study, 35% hydrogen peroxide was applied over the coronal barrier. The group with teeth discolored by Biodentine was bleached significantly more than with MM-MTA and ProRoot MTA, while no statistically significant difference was observed between specimens in groups treated with MM-MTA and ProRoot MTA [23]. The differences could be related to the composition of barrier materials such as calcium silicate cements. Biodentine contains zirconium oxide as a radiopacifying component, while ProRoot MTA and MM-MTA contained bismuth oxide, which is associated with tooth discoloration. Calcium silicate cements with zirconium oxide exhibited less discoloration [41]. This may be attributed to it being a finer sized particle [42] and, therefore, its effect on calcium silicate material diffusion into dentin tubules [43]. Moreover, Biodentine has also a highly specific surface area [42], which possibly increases the effectiveness of the bleaching agent [23]. However, overoxidation of bismuth oxide contained in ProRoot MTA and MM-MTA could result in discoloration [41] and thus, may lead to a reduction in whitening efficiency.
Sodium perborate is effective as a whitening agent and improves discoloration caused by TAPM [22,24]. Kirchhoff et al. [22] studied the ability of sodium perborate to bleach stained teeth with open and closed apices. The results indicated that the group with open apices bleach was similar to groups with closed apices, which is an interesting result, especially since younger teeth have potentially wider dentinal tubules [44]. Iriboz et al. [24] observed that there were no significant differences in bleaching on stained teeth with TAPM and minocycline paste when sodium perborate was used without or with activation by heat from a hand instrument, using an ultrasonic instrument for 30 s at a frequency at 29 kHz or 60 s at a frequency at 28 kHz. Increasing the temperature of bleaching agent with a heated hand instrument has been historically described [45], although excessive heating may damage the dental pulp in vital tooth bleaching [46] and increases the risk of external cervical resorption in non-vital tooth bleaching [47]. Currently in clinical procedures bleaching agents' activation by heat from a hand instrument is not performed. Santos et al. [25] bleached specimens using 37% carbamide peroxide gel. Bleaching agent was placed two times with 1-week interval inside and outside the dental crown for 45 min. In the same group no difference was found between first and second bleaching. It may suggest that only one session of bleaching is effective to improve color shade.

Analysis of In Vivo Studies
Regenerative endodontic procedures involved all treated teeth, of which only one was completed in a single visit [31]. In analyzed studies, a change in color was noticed some time after the disinfectant paste was placed or post-treatment. In two of the included studies, grey and blue-greyish discoloration was observed six weeks after placing TAPM [9,27]. TAPM was most frequently used as an intracanal disinfectant [9,17,27,28,30]. However, other materials were also used (e.g., TAPK, Odontopaste, TAPA, DAP, CH, and 2% chlorhexidine gel). Parthiban et al. [30] noticed mild discoloration 28 days after TAPM placement and blue-greyish discoloration three months post-treatment. In other cases, posttreatment discoloration time occurrence varied between studies. It is worth mentioning that not all studies contained information about the color of the discoloration.
The research methodology differed between studies. Bleaching was performed after one week [9], 39 weeks [27], 3 months [30], 39 months [11], and 58 months [31]. Some authors [17,28,29] did not include information about the post-treatment time before starting whitening in their studies. Bleaching was carried out using different techniques: internal, external, or both of these techniques.
Internal bleaching in which the bleaching material was placed into the pulp chamber was similar to the walking bleach technique carried out for non-vital teeth. Although vital pulp-like tissue is formed in root canal following REPs, the coronal pulp space is empty. This allows using the non-vital teeth bleaching technique to treat discolored teeth after REPs because cervical sealing material protects the new vital pulp-like tissue from the bleaching agent [25].
Bleaching techniques were carried out in asymptomatic patients immediately after the regeneration procedure had been performed in the teeth in which the hard tissue bridge did not manage to form [9] or after some time from bleaching when continued lateral wall thickening [11], periapical healing and maturation of the root apex [31] was observed. Kim et al. [9] performed bleaching one week post-treatment. The barrier material (MTA) was left in canal and the bleaching agent was placed over the cervical sealant with glass ionomer cement (GI). Eight months after bleaching the radiograph showed evidence of continuation of apical closure. Despite the only barriers separating the newly formed pulp-like tissue were MTA and GI, respectively, revascularization procedures were successful, which was a very meaningful result, especially since bleaching materials may damage the pulp tissue and cause pulp inflammation [21]. However, it is worth noticing that Tsujimoto et al. [48] using a scanning electron microscope observed changes in surface structure of MTA after application of hydrogen peroxide as a bleaching agent. Discovered structural alterations described as e.g., globular structures, woodpecker holes and creases were dependent on hydrogen peroxide concentration and may predispose to microleakage occurrence. Although the findings suggest that MTA is an insufficient barrier against tooth bleaching, an in vitro study of 2-mm intra-orifice barriers of GMTA, WMTA and GI showed similar coronal leakage in all tested materials [49]. The studies with a fluid transport model [49] and protein leakage test [50] showed that cervical sealing material significantly reduces leakage even when bleaching agent is in use. Therefore, seeing the fact that sealing materials are highly required as they reduce the possibility of resorption further investigation is essential. The formation of the dentin bridge underneath the barrier material could act as an additional biological seal. The formation of the dentin bridge underneath the barrier material could act as an additional biological seal. Additionally, studies that analyzed the thickness of the dentin bridge formation after direct pulp capping showed that the mean thicknesses of the hard-tissue dentin bridge depended on the material used [51].
Unfortunately, there are risks associated with internal bleaching of non-vital teeth, such as weakening of the physical properties of dental hard tissues, penetration of the bleaching agent in the dentinal tubules, dental fracture during treatment, and the most serious, external cervical root resorption [52]. No information on the above-mentioned complications was included in the in vivo studies analyzed. To avoid cervical root resorption in the treatment of non-vital tooth discoloration, a cervical barrier should be placed to prevent diffusion of the bleaching agent throughout the dentinal tubules [53]. Similar to this technique, the authors in the analyzed studies also applied cervical sealing materials such as GI [9,11,30,32], Cawit W (3M, St Paul, MN, USA) [31], or Coltosol (Coltene Whaledent, Mahwah, NJ, USA) [28] which was placed in cases when barrier material (e.g., MTA, WMTA, PC) was removed [31] or left [9,11,28,32] in the canal. Some authors did not report on the cervical sealing material [17,27,29].
Among the widely used bleaching materials such as carbamide peroxide, hydrogen peroxide, and sodium perborate, the last one was mainly used. Sodium perborate has been classified as carcinogenic, mutagenic, or toxic to reproduction (CMR substances) and its use is prohibited in Europe (Cosmetics Regulation 1223/2009) [54]. In analyzed studies, sodium perborate was mixed with hydrogen peroxide [17,30], distilled water [9,28,31], sterile water [11], or saline [27] and was placed in the pulp chamber. McTigue et al. [17] also applied a cotton pellet saturated with Superoxol (Sultan Healthcare, Hackensack, NJ, USA) for 3 min. The duration of whitening time varied from one to three weeks. The bleaching agent was placed in the pulp chamber once [11,17,27,31], twice [28], and three times [9] depending on the study. Only in one study was calcium hydroxide placed in the access cavity after bleaching for one week [30] and in one study was the evaluation of color changes made using a digital spectrophotometer [28].
Antov et al. [32] showed three cases in which 10% carbamide peroxide was used in the bleaching procedure. In two cases, two weeks of internal/external bleaching was performed using vacuum formed bleaching trays with a reservoir over the labial surface of the bleached teeth. Before bleaching reduction of the barrier material and cervical sealing with GI were performed. Bleaching showed a satisfactory final result in the first case and minimal improvement of shade in the second case. In the third case, because of the lack of radiographic evidence of Portland cement and calcific barriers, and therefore, the risk of damage to revitalized tissue upon reduction of cement, two weeks of external bleaching was performed. Shade was improved but the patient was not fully satisfied with the result, which was why a direct composite veneer was provided.
From 26 treated teeth 17 teeth were bleached successfully [11,17,28,29,32]. In six cases, there was an improvement of the shade [9,27,[30][31][32] of which, in one case presented by Antov et al. [32], the effect was minimal and, in a case presented by Kim et al. [9], the tooth did not return to its original shade. In three cases bleaching was not sufficient [17].
The clinicians who perform REPs should be aware of the high-risk of post-treatment discoloration [32] and all procedures ought to be performed with minimal risk of potential discoloration. If the discoloration appears, bleaching should be considered ultimately to improve shade or in the transitional period before more invasive procedures were planned such as porcelain veneers or crowns.
There are no exact guidelines for dental bleaching of discolored teeth after REPs. There are no randomized trials on this topic. However, based on analyzed studies a summary of the current bleaching procedure was presented below.
Three different bleaching techniques were used in the analyzed studies: internal, internal-external, and external bleaching technique, of which internal bleaching was the most frequently used method in an asymptomatic patient [9,11,27,30,31]. It is worth mentioning that internal bleaching technique is commonly used in non-vital teeth and analyzed studies provide insufficient information about follow-up and long-term observations after this method was applied in terms of its effect on the outcome of teeth regeneration procedure. The barrier material was usually left in the canal [9,11,28,32], which may affect the additional seal and minimize the risk of damage to dentin bridge or revitalized pulp.
Cervical sealing with GI cement was frequently applied to the barrier material [9,11,32]. The bleaching agent was placed inside the dental crown analogously to a non-vital tooth bleaching technique [9,11,17,[27][28][29][30][31]. As a bleaching agent, three different substances were used: hydrogen peroxide, sodium perborate, and carbamide peroxide. It is impossible to conclude which bleaching agent should be recommended owing to high variability in the investigated studies-different bleaching agents, its concentration, time, and technique of application, but also inconsistent outcome evaluation. The problem with evaluation is especially seen in in vivo studies, where a quantitative method was used only once and qualitative methods were not standardized, because such a standardization does not exist. Difference in local legal permissibility is also a major issue in terms of guidelines preparation e.g., the use of sodium perborate is not legal in the Europe. Calcium hydroxide was placed in the pulp cavity after bleaching [30] only in one case. The access cavity was restored with composite [9,27,29,30].
Taking the above into consideration, a standardized protocol for the bleaching procedure and assessment should be introduced to obtain the most reliable results.

Conclusions
This systematic review indicated that whitening of discolored teeth after REPs is achievable. The internal method was the dominant one, but due to alternative method usage shortfall, it is hard to make a comprehensive comparison. Similar to difference in bleaching agent usage observed in analyzed studies, there is a wide difference in bleaching duration. Therefore, it is not possible to make a suitable conclusion. For the creation of precise guidelines that would define the appropriate bleaching technique, material, and duration in discolored teeth after REPs, further studies are required.