Different techniques for the treatment of teeth with an incomplete root development and pulp necrosis: a systematic review

129 Graduada em Odontologia, Universidade Luterana do Brasil, Cachoeira do Sul, RS, Brasil. Graduando em Odontologia, Universidade Luterana do Brasil, Cachoeira do Sul, RS, Brasil. Doutora em Ciências Odontológicas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil. Doutora em Ciências Odontológicas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil. Professora do Departamento de Endodontia, Universidade Luterana do Brasil, Cachoeira do Sul, RS, Brasil.

with the pulp regeneration technique, using cohort studies, and non-randomized and randomized clinical trials. Methods: The methodology was based on electronic research in the following databases: PubMed, MEDLINE, Google Scholar, SciELO, and LILACS. In addition, a manual search was carried out using the references that were listed in the articles found. Results: A total of 403 potentially eligible studies were found, with seven being included in the inclusion criteria of this systematic review. The seven studies involved a total of 312 teeth. The minimum time of a follow-up period was 12 months. The irrigation solution most used was sodium hypochlorite, for both of the apexification and revascularization techniques. The medication commonly chosen in the apexification groups was Ca(OH) 2 , with antibiotic triple paste in the revascularization groups. The clinical rate of success in the groups treated with revascularization varied from 76% to 100%, while in the groups treated with apexification, it ranged from 68% to 100%. Only two studies reached a success rate equal to 100%. Conclusions: Variable levels of evidence were observed in relation to the treatments. However, it was confirmed that revascularization is an excellent option since its outcomes produced a greater gain of thickness and root length, besides developing a decrease in the apical foramen.

Introduction
The regenerative endodontic protocol is an emerging alternative for the management of necrotic teeth that display an incomplete root development. Traditionally, dental elements that present apical periodontitis, thin and parallel walls, and an open apex, are treated by apexification 1 . Apexification can be conducted in one or multiple visits. This conventional method consists of successive exchanges of intracanal calcium hydroxide (Ca(OH) 2 ), in order to promote an apical calcific barrier 2 . However, this technique presents some limitations, specifically because it is a long-term therapy. This may compromise the patient's adherence to the treatment, which invariably lasts for more than 6 months, and it does not show any gain in dentin thickness, implying a fragility of the tooth. In addition, the prolonged use of Ca(OH) 2 may promote a weakening of the dentin 3,4 . Another apexification protocol is based on the use of of mineral trioxide aggregate (MTA), in order to make an artificial apical barrier that allows for root canal filling in the same session 5 . Although the clinical success of both variants of apexification is evident, these two protocols do not confer a dentin thickness gain, or a complete root development, resulting in a fragile and fracture susceptible tooth 6,7 .
Recently, an alternative therapy to apexification has been used in clinical practice. This technique is called regenerative endodontic therapy and it corroborates the viability of root formation and dentin thickness gain; consequently, there is a reinforcement of the root structure 8 . The initial concept of regenerative endodontics was proposed by Nygaard-Østby in the 1960s 9 . However, it was in the year 2000, based on two case reports, that regenerative endodontic activity aroused attention for its advantages. Thus began its introduction into clinical practice 6,10 . Banchs and Trope 7 suggested a protocol that recommended the disinfection with copious irrigation and a combination of three antibiotics as intracanal medication. After 26 days, over-instrumentation induce bleeding into the canal and a double seal of the coronal access should be carried out. The procedure produces a blood clot, which must be controlled at the cemento-enamel junction level.
The two aforementioned alternatives for the treatment of teeth suffering from pulp necrosis and an incomplete root development have demonstrated reliable clinical outcomes 1,7 . Regenerative endodontics is one of the most exciting advancements in dentistry nowadays, and it is constantly the subject of recent scientific research aiming to find the best treatment protocol. Although there are clinical considerations with emerging scientific evidence for the regenerative procedure 11 , a broader discussion about each stage of the therapy should be carried out in order to consolidate an effective and unquestionable protocol for regenerative endodontics. Therefore, the main objective of this systematic review was to assess the best available evidence and to collect appropriate data, in order to evaluate the effectiveness of the techniques of apexification with Ca(OH) 2 or MTA, and of a pulp regeneration with a blood clot or injectable scaffolds, from cohort studies and clinical trials. In addition, a critical analysis of the available information will be made and the success rate of the techniques evaluated.

Research question
The research questions that guided the following literature review were: "Does the revascularization technique, with a blood clot only, a blood clot and matrix, a blood clot plus a scaffold impregnated with a basic growth factor of fibroblasts, and a blood clot plus an absorbable collagen barrier, promote the best outcomes regarding periapical healing, an apical foramen closure, and an increased thickness in root length, with clinical success, if compared to the technique of apexification with Ca(OH) 2 or MTA, in human teeth with an incomplete root development and pulp necrosis?" and "Does revascularization demonstrate a higher survival rate in immature permanent teeth that exhibit necrotic pulp and an incomplete root development when compared to the apexification technique?".
These research questions were constructed according to the population, the intervention protocol, the comparison protocol, and the outcome (Population: human teeth with an incomplete root development and pulp necrosis; Intervention: pulp regeneration; Control: apexification with Ca(OH) 2 or MTA; Outcome: apical closure or root maturation, clinical and radiographic outcomes, clinical success rate and survival, an increase in length percentage and root thickness; Type of study: retrospective cohort, non-randomized and randomized clinical trial.

Strategy for the identification and the selection of studies
The electronic databases that were used were PubMed, MEDLINE, Google Scholar, SciELO, and LILACS. The search strategy is described in Appendix 1 and the words used were identified from the keywords that were present in the articles that dealt with the subject, as well as the terms that were obtained from MeSH (Medical Subject Headings of the United States National Library of Medicine). In addition, a manual search was also performed on the references cited by the selected studies from the databases. The search and the selection of articles were carried out by two independent researchers (C.B. and C.J.), without any language restriction, in the period of May 2020. After the electronic search, the titles and the abstracts were evaluated according to the inclusion and exclusion criteria. The reading of the articles in full was carried out from those papers that met the inclusion criteria. Alternatively, in the cases of studies with an insufficient amount of data in the title or in the abstract to establish a decision, a search was carried out in order to minimize the possibility of disregarding relevant research. The articles selected by both of the researchers were compared and when discrepancies were found, they were discussed. When necessary, a consensus was obtained from a third evaluator (M.C.B.).

Inclusion criteria
Retrospective cohort study, non-randomized and randomized clinical trial in human permanent teeth that displayed an incomplete root development and pulpal necrosis, subsequently comparing pulp revascularization techniques with a blood clot, a blood clot plus a scaffold that was impregnated with a basic growth factor of fibroblasts, and a blood clot plus an absorbable collagen barrier, when using the Ca(OH) 2 or MTA root apexification technique.

Exclusion criteria
• Literature reviews, case reports, dissertations, theses, textbook chapters, and annals of congress; • Deciduous teeth; • Permanent human teeth with a complete root formation; • Teeth without pulp necrosis; • Apexification with other materials that were different from MTA and/or Ca(OH) 2 .

Data synthesis
The following information was analyzed and extracted from the studies: authors, year of publication, patient's age, inclusion criteria, irrigation protocol, the intracanal medication used and the time it remained intracanal, treatment protocol (apexification with Ca(OH) 2 or MTA and revascularization), follow-up period, sealing used, radiographic outcomes (changes in root length, canal wall thickness, apical diameter closure, plus other outcomes), and clinical outcomes.

Selection of studies
The research strategy found a total of 403 articles in the electronic databases, of which 204 articles were duplicates and they were excluded. After reading the titles and the abstracts, 186 studies were excluded, as they did not meet the inclusion criteria. With a complete reading of the texts, five articles were excluded because they also did not meet the inclusion criteria of this study. In the end, seven articles were included for this review, according to Figure 1. Board 1 presents the characterization of the articles that were included in this review.

Type of study
Most of the studies included in this systematic review were of a retrospective cohort (n = 5) 12,14,15,17,21 . Meanwhile, the study by Lin et al. 19 was a randomized clinical trial and the study by Nagy et al. 20 was a non-randomized clinical trial.

Type of teeth
The seven studies involved a total of 312 teeth. The teeth most commonly treated were premolars (n = 139) 15,17,19 , followed by central incisors (n = 99) 17,19,20 . Some studies did not report on the specific teeth covered, with a total of 74 unspecified teeth 12,14,21 .

Age
In the studies analyzed, the age of the patients ranged from 06 to 46 years, with three studies not reporting on the age of the individuals 14,15,17 .

Follow-up time
There was great variability in the follow-up periods between the studies included in this review. However, most of the studies had a minimum period of 12 months, except for the study by Nagy et al. 20 , which was followed up in four moments, or that is, at 3, 6, 9, and 12 months. Jeeruphan et al. 17 had an average follow-up time of between 21.15 and 30.47 months. The maximum follow-up time was 96 months, according to the study by Silujjai and Linsuwanont 21 . One article did not report on the follow-up period 14 .

Disinfection protocol
As for the root canal disinfection protocol for the revascularization technique, sodium hypochlorite (NaOCl) was used in concentrations ranging from 1.5 to 2.6%, except for in the study by Alobaid et al. 12 who did not report on the concentrations of NaOCl, chlorhexidine, and/or ethylenediaminetetraacetic acid (EDTA). The study by Silujjai and Linsuwanont 21 used 17% EDTA for the NaOCl sequence, while the study by Lin et al. 19 used 17% EDTA plus 0.9% physiological saline solution. In the study by Nagy et al. 20 , for both of the revascularizations, they used 10 ml of 2.6% NaOCl, followed by a sterile saline solution. The study by Bose et al. 14 only used NaOCl, not to mention the concentration.
As for the groups in which the apexification technique was chosen, four studies did not present or record the irrigating solution that was used for the disinfection procedure 12,14,15,17 . While the studies by Lin et al. 19 and Silujjai and Linsuwanont 21 used NaOCl of between 1.5% and 2.5%, and the study by Lin et al. 19 used NaOCl with 0.9% physiological saline solution, followed by 20 mL of 17% EDTA. The study by Nagy et al. 20 used 10 ml of 2.6% NaOCl, followed by sterile saline for the apexification group with MTA.

Intracanal medication
Six studies 12,14,17,[19][20][21] used an antibiotic triple paste as an intracanal medication in their revascularization groups. However, there was a variation between them regarding the composition of the medication. The study by Bose et al. 14 did not report which drugs constituted the paste. All the remaining studies used two drugs in common, which were ciprofloxacin and metronidazole, with only the third element varying. The studies by Alobaid et al. 12 , Jeeruphan et al 17 and Silujjai and Linsuwanont 21 chose minocycline, while Lin et al. 19 chose clindamycin hydrochloride and, finally, Nagy et al. 20 used doxycycline. Chen and Chen 15 chose to use Ca(OH) 2 in the revascularization group, not reporting their choice in the apexification group.The medication commonly chosen in the apexification groups was Ca(OH) 2 12,15,17,19-21 .

Time of intracanal medication
The time of medication varied from 3 to 6 weeks for the revascularization groups 17,19,20 , while four studies did not report on the time of medication 12,14,15,21 . In the group that chose for the apexification technique, three studies varied the time between 1, 3, and 4 weeks, respectively 17,19,20 , and four articles did not describe the time of medication that they adopted 12,14,15,21 .

Revascularization technique
In the revascularization technique, all of the studies chose to induce the formation of a blood clot. However, the study by Bose et al. 14 did not inform on the technique of revascularization.

Radiographic outcomes
According to the study by Chen and Chen 15 , there were no differences between the two techniques regarding the periapical index scores (PAI). This periapical index quantifies the apical lesions in radiographic images. In addition, the stages of root development did not show a statistically significant difference after both of the treatments. On the other hand, Alobaid et al. 12 concluded that there was a difference between the groups regarding the root width (1.4% ± 3.2% for apexification versus 10.2% ± 4.0% for revascularization). In this study, a total of four cases (27%) were observed out of a total of 17 cases in the revascularization group, versus one out of a total of 15 in the apexification group; thus, there was a change of 20% or more out of any of the radiographic outcomes (width, length, and the area of the apical foramen). However, none of the radiographic outcomes showed a statistically significant improvement between the groups. In addition, it was also observed that among the 17 patients who were submitted to revascularization, three patients had a pulp canal obliteration (17.6%).
The studies by Alobaid et al. 12 and Lin et al. 19 also showed that in their radiographic outcomes, a greater root length and thickness was achieved in the group that used the revascularization technique when in comparison to the apexification group. Silujjai and Linsuwanont 21 demonstrated that the revascularization technique provided significantly greater percentage changes in root width when compared to the apexification technique with MTA. Likewise, Jeeruphan et al. 17 also demonstrated that the revascularization protocol significantly improved the root width (28.2%) when compared to the apexification protocol with MTA (0.00%) and with Ca(OH) 2 (1.52%). These authors also observed that the revascularization protocol significantly improved the root length (14.9%) when compared to the teeth treated with MTA (6.1%) or Ca(OH) 2 (0.4%) in the apexification protocol. Between the apexification groups, there were no statistically significant differences. For Bose et al. 14 , the outcomes showed that regenerative endodontic treatment with antibiotic triple paste and with Ca(OH) 2 for the root canal disinfection produced significant improvements in the root length when compared to the apexification groups with MTA and formocresol.
The triple antibiotic paste also produced larger differences in the root wall thickness than did the Ca(OH) 2 or formocresol groups, and the Ca(OH) 2 inserted position influenced the radiographic outcomes. The study by Nagy et al. 20 was the only study in all of the groups to quantify the bone density, showing significant improvements after 12 months of follow-up. After a monitoring period of 18 months, most of the cases presented radiographic evidence of periapical healing. The revascularization groups with a blood clot that were associated with an injectable scaffolding showed a progressive increase in the length and the width of the root, but with a decrease in the diameter of the apical foramen. However, the use of an injectable scaffolding was not essential for the root repair.

Clinical success
One study did not report on the clinical success rate 14 . The study by Chen and Chen 15 reported that both treatments exhibited considerable success rates, presenting no statistically significant differences. For the studies that quantitatively measured the success rate, there was an approximation between the findings 12,17,20,21 . The success rate in the groups that were treated with the revascularization technique ranged from 76% to 100%, while in the groups that were treated with apexification, it varied between 68% and 100%. Only the studies by Lin et al. 19 and Nagy et al. 20 achieved a success rate equal to 100%.
Few studies reported on any adverse events. The study by Alobaid et al. 12 observed a higher incidence in the revascularization group (42%) when compared to the apexification group (11%). According to the study by Chen and Chen 15 , the main complication of apexification was a tooth fracture (14.3%). In three cases, two had cervical fractures and one presented an apical fracture. The mean time to the occurrence of any complication was 1.7 years after the treatment. The study by Lin et al. 19 reported that after one year of follow-up, all of the teeth were asymptomatic, and the revascularization and apexification groups achieved comparable outcomes when in relation to the resolution of the symptoms. In addition, the authors observed that the etiology of necrosis had an impact on the outcome of revascularization and that the cases of "dens evaginatus" had a better prognosis than did the cases of trauma. They also observed other complications, such as discoloration in the revascularization group.

Survival rate
The studies by Bose et al. 14 , Chen and Chen 15 , Nagy et al. 20 , and Silujjai and Linsuwanont 21 , did not report on the survival rates. The study by Lin et al. 19 reported a 100% survival rate in both of the treatment groups, while the study by Jeeruphan et al. 17 presented a rate of 100%, but only in the revascularization group. The MTA and Ca(OH) 2 apexification groups had 95% and 77% survival rates, respectively. However, the study by Alobaid et al. 12 presented a survival rate of 100% in the apexification group, while in the revascularization group, the survival rate was equal to 95%.

Coronal sealing
The study by Bose et al. 14 did not report on the method used for coronal sealing in the groups that adopted the revascularization technique, or in the groups that adopted the technique of apexification. The study by Jeeruphan et al. 17 , which was subdivided into three groups, that is, the revascularization group, the apexification group with MTA, and the apexification group with Ca(OH) 2 , reported only on sealing in the apexification group with MTA, in which they used glass ionomer cement (GIC) and composite resin.
The studies of Chen and Chen 15 and Lin et al. 19 also adopted GIC when it was associated with a composite resin, but only in the revascularization groups. This was while in the apexification groups, Lin et al. 19 adopted only a composite resin, whilst Chen and Chen 15 used GIC and composite resin for the apexification group with MTA and gutta-percha for the apexification group with Ca(OH) 2 . Alobaid et al. 12 and Nagy et al. 20 used MTA when associated with composite resin for the sealing in the revascularization groups, with only a composite resin in the apexification groups. Finally, the study by Silujjai and Linsuwanont 21 used MTA and composite resin for sealing in the revascularization group, and only MTA in the apexification group.

Discussion
Permanent teeth with necrotic pulp and with an incomplete root formation are traditionally treated using the method of apexification with MTA or with Ca(OH) 2 . Both variations of the technique, despite demonstrating substantial clinical outcomes, also present disadvantages such as: reduction of root resistance mediated by calcium hydroxide, little gain in root thickness and length, teeth more susceptible to fracture, and the requirement for excellent patient compliance due to the need for several visits scheduled for many months 17 . In this sense, regenerative endodontic procedures are an alternative to conventional treatment with scientific evidence that shows satisfactory clinical outcomes from the continuation of the rhizogenesis process and gain in dentin thickness 22 . However, this technique imposes clinical challenges and its success is related to predictors of prognosis, which must be taken into account [23][24][25][26] .
Considering the studies included in this literature review, there was great variability in the patients' age, which ranged from 06 to 46 years old 12,[19][20][21] . All of these studies showed satisfactory clinical outcomes. However, the study by Silujjai and Linsuwanont 21 was the one with the lowest success rate. It is believed that this can be explained by the age group of patients included in the study (up to 46 years old). This line of reasoning is supported by the results of the studies by Estefan et al. 27 and Cai et al. 28 , which demonstrated that the closer the tooth age to the eruption time, the greater the potential of dental pulp regeneration. In addition, regeneration procedures on teeth with larger apical diameters demonstrate greater increase in thickness, length and apical narrowing of the root 27,29 . Therefore, the size of the radiographic apical opening of the pre-treatment can be considered as a factor that contributes to the success of revascularization, with the ideal opening being greater than 1.1 mm 30 . Of the seven articles that were inserted in the systematic review, only one added apical foraminal openings larger than 1 mm in diameter as inclusion criteria 19 .
As for the protocol for root canal disinfection in the cases of revascularization technique, the included studies that mentioned concentration used NaOCl as irrigant, which ranged from 1.5 to 2.6% 15,17,[19][20][21] . Alobaid et al. 12 used NaOCl and chlorhexidine as an irrigating solution. However, they did not mention the concentrations, nor did they report whether there was a difference in the results depending on the irrigant of choice.
The disinfection of the root canal system is essential for tissue regeneration and for the procedure to be successful 31,32 , so there is a need for decontamination with antimicrobial irrigators 33,34 and the use of intracanal medication 35 . According to Hoshino et al. 32 , antibiotic triple paste, which is composed of minocycline, metronidazole, and ciprofloxacin, has proved efficiency in eliminating the endodontic pathogens both in vitro and in vivo. Considering the included studies, Alobaid et al. 12 , Jeeruphan et al 17 and Silujjai and Linsuwanont 21 used the combination of these three antibiotics. Lin et al. 19 chose clindamycin hydrochloride, and Nagy et al. 20 used doxycycline instead of minocycline. However, all studies obtained considerable success rates, regardless of the combination chosen. Among the studies that mentioned the time of use of the intracanal medication, it varied from 3 to 6 weeks in the revascularization groups 17,19,20 . This period of intracanal medication seems to be adequate for clinical success, since Ghabraei et al. 36 demonstrated that it takes only 7 days to eliminate Enterococcus faecalis from dentinal tubules in the apical half of the root canal up to 40 microns (μ) depth.
An empty and disinfected intracanal environment does not allow for the growth of regenerated tissues, thus it requires a scaffold to support the undifferentiated cells 37, 38 . Several authors have suggested the induction of a blood clot, which would act as a support for the migration of the pluripotent cells. This would result in a biological framework, rich in growth factors, allowing for favorable support, in order to provide the appropriate conditions for the differentiation and the growth of tissue into the pulp space 7,[39][40][41] . Other methods that serve as a framework have been used and have been suggested in the literature, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) [42][43][44] . PRF is a non-thrombosed autologous fibrin mesh that serves as a reservoir for the slow release of the growth factors over a period of 7 to 14 days. PRP, on the other hand, exhibits a sudden release of the growth factors in approximately 7 to 14 hours 45 . However, there is still no consensus among authors on the best technique. According to Duggal et al. 42 , no additional benefit was found when compared to the use of a blood clot. Naggy et al. 20 consolidated the findings of Duggal et al. 42 when stating that the use of an artificial hydrogel scaffold, or a scaffold impregnated with the basic fibroblast growth factor, was not essential for the repair.
The compatibility of the material of choice for the coronal plug is vital to allow for the survival of the stem cells and for the regeneration of new tissues, which are sensitive to the conditions of the intracanal environment 46 . The MTA is considered the recommended material for regenerative procedures 47 . Of the articles included in this review that mentioned the material used, MTA was the material of choice 12,15,[19][20][21] . Although all have obtained considerable results, current scientific evidence demonstrates that Biodentine® is also an excellent alternative for this purpose. According to Aly et al. 48 , in a study without significant difference in the results, Biodentine® obtained a higher clinical success rate and provided an increase in root length higher than with MTA, suggesting a greater chance of maintaining the dental element. In this context, Biodentine® should be considered for a regenerative therapy.
The proservation period and the survival rate are extremely important to determine the success of revascularization. The success rate in the groups treated with revascularization ranged from 76% to 100% 12,17,20,21 . Silujjai and Linsuwanont 21 had the lowest success rate (76.46%) and observed cases of failure up to three years after the completion of treatment with revascularization. This demonstrates that supervision shorter than this period can compromise the success rate. This can also explain the high success rate (100%) observed by Lin et al. 19 and Nagy et al. 20 , whose follow-up period was 12 and 18 months, respectively. Regarding the survival rate, only three studies mentioned it 12,17,19 . In all studies, the survival rate was around 100% both in revascularization and apecification. This indicates that an adequate disinfection is probably the most important factor in the clinical success and in the survival of the dental elements submitted to these procedures, corroborating with the findings of Lui et al. 49 , which show the importance of eliminating persistent microorganisms, due to their ability to remain viable in dentinal tubules, hindering regeneration and repair processes. Failure in apecification is usually caused by tooth fracture, which results in tooth extraction 21 .
As already mentioned, the primary purpose of the revascularization technique is root maturation. However, in some cases, even if a complete root development is not achieved, success can be considered when there is a resolution of pain, infection, and periapical pathology, in addition to demonstrating the long-term retention of the dental element 50 . Chen and Chen 15 observed that in the revascularization technique, even with the presence of a persistent periapical lesion, continuous root development occurred. Silujjai and Linsuwanont 21 corroborated this finding since they observed two cases of revascularization that were classified as a failure, in which the periapical lesions increased. However, there was an extension of the root length and width. Other authors have also demonstrated considerably Different techniques for the treatment of teeth with an incomplete root... greater percentage changes in root length and width when compared with the apexification group with MTA or Ca(OH) 2 , ensuring superiority in the revascularization technique when considering root development 14,21,51,52 . Alobaid et al. 12 observed an increase in the length and width of the root process through the technique of revascularization. However, this was without statistically significant differences with the apexification technique, probably due to the small sampling.
Although the revascularization procedure has numerous advantages, there are also unwanted effects resulting from the technique. Alobaid et al. 12 evidenced in their results that there were three cases of intracanal calcified barrier formation and two cases of obliteration. Chen and Chen 15 also demonstrated that 17.6% of the cases that were treated with the revascularization technique, presented pulp canal obliteration. Lin et al. 19 reported on intracanal calcification, external resorption, and coronary discoloration, as being adverse effects, most of which became evident in a 6-month period.

Conclusion
Based on the data listed in this literature review, pulp revascularization proves to be a technique with high predictability and clinical success. Thus, the regenerative endodontic procedure can be considered as the treatment of choice for teeth with incomplete rhizogenesis and pulp necrosis. In addition, in case of failure of the revascularization procedure, which usually occurs due to the persistence of microbials in the root canal system, the apecification technique may still be the treatment of choice, thus increasing the survival rate of the dental element.