Regenerative endodontic treatment performed with platelet-rich plasma presents better periapical healing than only induced blot clot: a systematic review

This study comprehensively reviewed two different treatments for regenerative endodontic: platelet-rich plasma and induced blot clot, in regarding to periapical healing. Two investigators performed a systematic review. MEDLINE/PubMed, Cochrane Library and Scopus supplied relevant data from studies published until December 2020 to answer the PICO question. Primary outcome was periapical healing. Eight randomized clinical trials fulfilled eligibility criteria. Primary outcome indicated that platelet-rich plasma results in similar or better periapical healing compared to blot clot group. The reported failures were related to blot clot group due to incomplete radiographic parameters, pain and reinfection; however, few cases of unsuccess were reported to platelet-rich plasma group. Only two studies observed better results to blot clot group in relation to increase of radiographic area and partial pulp canal obliteration. This review showed that procedures using platelet-rich plasma were successful in treating permanent teeth with root development.


Introduction
Immature permanent teeth have been characterized, histologically, by no formation of the apical dentin coated with cementum (Leonardo, 2005). Radiographically, it is not possible to see the root closure -root development does not reach Nolla stage 10, the foraminal opening may have a larger diameter than the diameter of the canal in the middle and cervical third and the root walls are thin and fragile (Leonardo, 2005). Thus, the conventional mechanical preparation becomes unfeasible to be performed, needing other therapeutic alternatives, making this treatment a challenge for endodontic practice (Leonardo, 2005).
Currently, regenerative endodontics has been shown to be effective in the treatment of immature permanent teeth. Such therapy may be defined as a biologically based procedure designed to replace damaged structures, including dentin, root structure and pulp tooth complex cells (Murray et al., 2007). The most well-known and successful approach to regenerative therapy in endodontics is revascularization (Murray et al., 2007;Shah et al., 2008). This procedure is performed to restore the vitality of a dental element, to stimulate the apical closure, to increase the thickening of fragile dentinal walls and to lengthen the underdeveloped root (Jadhav et al., 2012).
The traditional method of regenerative endodontic treatment for immature permanent teeth is conducted under local anesthesia without vasoconstrictor, access to the root canal and abundant irrigation with sodium hypochlorite (Metlerska et al., 2019). Due to the reduced dentin thickness, the canal is not subjected to any type of instrumentation, but disinfection with chemical solution (Bezgin et al., 2015). To achieve the disinfection process, the root canal is filled with a tri-antibiotic paste for an average period of 3 weeks (Bezgin et al., 2014). In the second session, the tri-antibiotic paste is removed and a manual file or a needle is used to evoke bleeding and a clot to form in the root canal (Jadhav et al., 2012). The root canal is sealed with a synthetic material (for example MTA) and radiographic monitoring is carried out periodically (Metlerska et al., 2019).
Recent research on new proposals for endodontic regenerative treatments has shown interesting results when using platelet concentrate, called platelet-rich plasma (PRP) (Jadhav et al., 2012;Bezgin et al., 2015;Narang et al., 2015;Alagl et al., 2017;Shivashankar et al., 2017;Rizk et al., 2019;Ulusoy et al., 2019;ElSheshtawy et al., 2020). PRP contains growth factors, stimulates collagen production, recruits other cells to the injury site, produces anti-inflammatory agents, initiates vascular growth, induces cell differentiation and improves the healing of soft and hard tissue wounds (Sachdeva et al., 2015). For the use of PRP in revascularization, the patient's venous blood is collected, centrifuged and blood free of erythrocytes and leukocytes is inserted into the root canal up to 3 mm from the cement-enamel junction (Dohan et al., 2006). As in traditional regenerative treatment, the root canal is sealed with a synthetic material and radiographic monitoring is carried out periodically (Metlerska et al., 2019).
In 2019, Metlerska and colleagues conducted a systematic review with studies in humans and the answer to the PICO question "Is the use of platelet concentrates effective in regenerative endodontics?" was those procedures using autologous Research, Society andDevelopment, v. 10, n. 13, e147101321219, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21219 platelet concentrates contribute to the success of treating immature permanent teeth, especially when there are problems with bleeding, but the level of evidence is weak. Furthermore, they have observed that autologous platelet concentrates can lead to development of the root and protect the tooth from extraction. However, in 2019 and 2020 new randomized clinical trials have been published driving the need of a new systematic review (Rizk et al., 2019;Ulusoy et al., 2019;ElSheshtawy et al., 2020). Therefore, the purpose of this systematic review was to assess the outcomes of periapical healing after regenerative endodontic treatment with induced blot clot and with PRP. Null hypothesis was that both platelet-rich plasma and induced blot clot present similar periapical healing in regenerative endodontic treatment.

Protocol and registration
This article was designed according to the Cochrane criteria (Cochrane Handbook for Systematic Reviews of Interventions, version 5.1.0) (Higgins and Green, 2011;Koche et al., 2011;Ludke and Andre, 2013;Estrela, 2018;Pereira et al., 2018;Yin, 2015) for elaborating a systematic review and meta-analysis and adopted the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement (Liberati et al., 2009). The study was registered at the International Prospective Register of Systematic Reviews (PROSPERO -233444).

Eligibility criteria
The inclusion criteria were: (1) randomized controlled trials, (2) studies that evaluated periapical healing in patients who were submitted to regenerative endodontic treatment, (3) studies that used platelet-rich plasma compared to induced blot cot in regenerative endodontic treatment, and (4) studies published in English language with available abstracts. Exclusion criteria included any articles that failed to meet the inclusion criteria and studies that evaluated platelet-rich fibrin.
A specific clinical question was structured according to the PICO approach: The addressed focus question was: does regenerative endodontic treatment performed with platelet-rich plasma presents better results than only induced blot clot? In this process, (P) represents patients submitted to regenerative endodontic treatment that were treated with (I) platelet-rich plasma, (C) compared to induced blot clot; and, (O) the periapical healing as outcome.

Information Sources
An electronic search in the PubMed/MEDLINE, Cochrane Library and Scopus databases was conducted until August 2020. Furthermore, a manual search was conducted to identify gray literature (http://www.opengrey.eu) and registered trials not yet published until 2020 from the following journals: Journal of Endodontics, International Endodontic Journal, Journal of Dental Research, Journal of Dentistry and Clinical Oral Investigations.

Study Selection
Two researchers (M.F.K. and L.S.M.) independently selected the studies according to their titles and abstracts and categorized them as included or excluded. Any disagreements were settled through discussion and consensus with a third researcher (C.M.M). After that both investigators read the articles selected for inclusion and a manual search was performed on the reference list.

Data collection process and data items
Subsequently, the full-text of the obtained articles was analyzed. The analysis of these selected articles was used to answer the PICO questions. The researchers (M.F.K and L.S.M) collected relevant information from the articles, including author, year, study type, number of patient and teeth sample size, gender, age average, methods for diagnosis, pulp diagnosis, anesthesia, the use of rubber dam, the working length, the instrumentation technique and irrigating protocol, the use of intracanal medication protocol, the scaffolds for platelet-rich plasma, the platelet-rich plasma group initial sample size and the drop-out, the scaffolds for induced blot clot, the induced blot clot plasma group initial sample size and the drop-out, the final restoration, the follow up protocol, the periapical healing and cases of unsuccess, the complete apical closure, the increase of radiographic root area, the discoloration, the presence of partial pulp canal obliteration, positive vitality testing, wall thickening and root length. Then, a third researcher (C.M.M.) checked all of the collected information. A fourth researcher (V.E.S.B.) settled any disagreement between the investigators through discussion until a consensus was obtained. Duplicate subject publications within separate unique studies were not reported twice.

Risk of Bias
The risk of bias assessment in the included studies was evaluated using the Cochrane Collaboration's Tool for Assessing Risk of Bias in Randomized Trials (Higgins and Green, 2011). The assessment criteria are a domain-based evaluation in which critical assessments are made separately for different domains: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other bias (Higgins and Green, 2011). For each domain the risk of bias was graded as high, low or unclear based on criteria described in the Cochrane Handbook for Systematic reviews of Interventions 5.1.0 (Table 1) (Bezgin et al., 2015).

Study selection
A total of 220 articles were retrieved, of which only 8 fulfilled eligibility criteria (inter-reader agreement, Kappa = 1 for PubMed/Medline and Kappa = 1 for Cochrane Library) ( Figure 1). All the studies selected were randomized clinical trials and compared the outcomes of periapical healing after regenerative endodontic treatment using blot clot and PRP (Jadhav et al., 2012;Bezgin et al., 2015;Narang et al., 2015;Alagl et al., 2017;Shivashankar et al., 2017;Ulusoy et al., 2019;Rizk et al., 2019;ElSheshtawy et al., 2020). The risk of bias assessment of the included studies is described in Table 1. Extracted data are summarized in Table 2 and Table 3 12 density and the decrease of apical diameter. Pulp vitality was evaluated thru electric pulp, cold and heat stimulation tests. Ulusoy et al., 2020 Clinical and radiographical evaluation at 3, 6, 9, 12, 18, 24 months. Radiographical scores: 0 -Failure, persistence of clinical and radiographic findings; 1 -resolution of disease, absence of swelling, drainage, and pain along with radiographic evidence of osseous healing; no evidence of increased root dimensions; 2: score 1 + radiographic root development and a positive response to cold testing and electric pulp testing. Pulp vitality was evaluated thru electric pulp and cold stimulation tests.

Study characteristics
A total of 204 patients, regardless of gender, with an average age of 14.1 years had 244 teeth treated endodontically thru regenerative. From this, 104 teeth were treated with PRP and 106 teeth were treated with blot clot.
All cases treated were diagnosed with pulp necrosis with the presence or absence of periapical lesion. The methods or diagnosis used were clinical examination, periapical radiograph and pulp vitality test (electric pulp and cold stimulation) and only in one research CBCT was used (ElSheshtawy et al., 2020).
Four studies performed minimal mechanical instrumentation and the canals were not instrumented in the remained studies. Sodium hypochlorite (NaOCl) was the irrigant of choice in all 8 articles, with concentrations ranging from 1.25 to 5.25%. Bezgin et al. (2015) and Alagl et al. (2017) associated the irrigation with chlorhexidine. Rizk et al. (2019), Ulusoy et al. (2020) and ElSheshtaw et al. (2020) used EDTA to remove the smear layer after the use of tri antibiotic paste.
For achieve the scaffolds for PRP, all researchers followed the protocol of Dohan et al. in 2006. And the blot clot was obtained through evoke bleeding from periapices and only ElSheshtaw et al. (2020) placed a collagen over the blot clot.
The follow up was completed mostly evaluating the periapical healing and apical closure. It was observed that the periapical healing was similar or superior for PRP group compared to blot clot group. The reported failures were related to blot clot group due to incomplete radiographic parameters, pain and reinfection; however, few cases of unsuccess was reported to PRP group. Two studies (Ulusoy et al., 2019;Narang et al., 2015) observed better results to blot clot group, ElSheshtaw et al. 2020 observed similar results to both groups and most research points to a better result with the use of blot clot.
Some studies observed better results to blot clot group in relation to increase of radiographic area (Bezgin et al., 2015;Ulusoy et al., 2019) and partial pulp canal obliteration10. However, studies that evaluated discoloration pointed to better results to PRP Group (ElSheshtawy et al., 2020;Rizk et al., 2019). On the other hand, controversial results are observed or aspects positive vitality test, wall thickening and root length.

Discussion
The findings of the present systematic review deny the null hypothesis since the performance of the regenerative treatment with PRP was better when compared to regeneration with the stimulation of the blood clot. The main results show that periapical health with complete root formation (apical closure) was greater in most studies with the induction of PRP in the regenerative process than blood clot (Jadhav et al., 2012;Bezgin et al., 2015;Narang et al., 2015;Alagl et al., 2017;Shivashankar et al., 2017;ElSheshtawy et al., 2020). Furthermore, some studies have shown that the teeth treated with a blood clot had a lower success rate (Bezgin et al., 2015;Shivashankar et al., 2017;Ulusoy et al., 2019;ElSheshtawy et al., 2020). Not all studies have shown results regarding pulp vitality, however, in spite of those who evaluated after regenerative process, most demonstrated that the use of PRP was clinically better (Bezgin et al., 2015;Shivashankar et al., 2017;Rizk et al., 2019).
Treatment of immature permanent teeth is a challenge for clinicians and its success can be analyzed thru thickening and lengthening of the root wall and closure of the apical foramen. Osteoblasts/odontoblasts, adipocytes, and neuronal-like cells provided by periodontal ligament, with or without scaffold of PRP will develop the root and the apex. Adequate length with a sufficient thickness of the root canal leads to a greater resistance to masticatory forces, thus preventing possible fractures.
However, great disinfection is primordial in the process (Jadhav et al., 2012). Sodium hypochlorite (NaOCl) is most commonly used. NaOCl presents the characteristic of dissolve organic matter and in high concentrations, the substance may denature growth factors, besides affects stem cell attachment, survival, and the differentiation. The American Association of Endodontists (AAE) recommend using a 1.5% concentration of NaOCl in regenerative treatments. Nonetheless, it was observed that in cases treated in the present research different concentrations were used, ranging from 1.25 to 5.25%. Chlorhexidine was also used as complement to disinfection; however, its use is discouraged due to its cytotoxic to stem cells from human apical papilla.
In all cases, a tri antibiotic paste was used. Several combinations of antibiotics have been shown to be able to decontaminate the channels, but the mixture of metronidazole, monocycline and ciprofloxacin was the most used in the studies of our review (Jadhav et al., 2012;Bezgin et al., 2015;Alagl et al., 2017;Shivashankar et al., 2017;Ulusoy et al., 2019;Rizk et al., 2019). The main disadvantage in using this paste is the discoloration, which -for aesthetic reasons -can put in check the maintenance and longevity of the tooth. Kim et al. (2010) reported that there was coronary discoloration after the use of monocycline in the antibiotic paste introduced into the root canal during the regenerative process. In order to prevent, Dabbagh et al. (2012) proposed the replacement of monocycline by cefaclor, obtaining favorable clinical results, both from the periapical repair process and from coronary discoloration. In the present systematic review, only one study used cefaclor instead of monocycline, and they found results that corroborates with previous published research (Bezgin et al., 2015).
Discoloration was also evaluated comparing the treatment performed. Regenerative endodontics using the blood clot was better in terms of increasing the radiographic area and partial obliteration of the canal (Bezgin et al., 2015;Ulusoy et al., 2019). On the other hand, the group that used PRP had advantages related to coronary discoloration and periapical healing (Bezgin et al., 2015;Rizk et al., 2019;ElSheshtawy et al., 2020).
The blood clot is a traditional method for regenerative treatment. The blood clot acts as a scaffold, and the growth factors inside recruit stem cells, most likely from periapical papilla. An ideal blood clot is necessary to achieve the success; however, incomplete bleeding inhibits suitable clot formation and sometimes it happens. The use of anesthetics with vasoconstrictor impairs the formation of clots, so some studies guide the use of these substances free of vasoconstrictor (Jadhav et al., 2012;ElSheshtawy et al., 2020).
All studies of this systematic review predicted the use of rubber dam to avoid bacterial contamination in the canal, thus improving the prognosis of the treatment. In addition, all studies carried out exclusively chemical or mechanical minimally invasive preparation for disinfecting the root canal with sodium hypochlorite, avoiding conventional mechanical preparation, thus preventing wear and weakening of the dentinal wall (Jadhav et al., 2012;Bezgin et al., 2015;Narang et al., 2015;Alagl et al., 2017;Shivashankar et al., 2017;Ulusoy et al., 2019;Rizk et al., 2019;ElSheshtawy et al., 2020).
The role of scaffolding materials is to fix, proliferate, migrate, and organize the spatial population of cells required for structural and functional term replacement of the target tissue. The latest research concerns the use of platelet concentrates and the most popular platelet concentrates are plateletrich plasma (PRP). The use of PRP has been suggested as an adequate matrix for the stimulation of the endodontic regenerative process, due to the presence of alpha granules in the platelets, which are considered reservoirs of growth factors and pro inflammatory cells (Narang et al., 2015;Adel Ragab et al., 2019;ElSheshtawy et al., 2020). In addition, PRP acts as a reservoir of biochemical assets that are released slowly and continuously over a period of 7 to 14 days, increasing the proliferation of various types of cells, in addition to stimulating cell differentiation and angiogenesis (Adel Ragab et al., 2019).

Conclusion
This review showed that procedures using PRP were successful in treating permanent teeth with root development.
According to good properties, the promising results of the treatment of endodontic revascularization are understood using a matrix with PRP, in order to maintain the integrity and periapical healing, in addition to the longevity of the element in the oral cavity. However, it is necessary future researches regarding to randomized clinical trials to consolidate the therapy.