Alexandre Henrique dos Reis-Prado 1 | Sabrina de Castro Oliveira1 | Juliana Goto 2 | Gerluza Aparecida Borges Silva 3 | Luciano Tavares Angelo Cintra 2 | Ricardo Alves de Mesquita 4 | Raphael Escorsim Szawka 5 | Antônio Paulino Ribeiro-Sobrinho 1 | Francine Benetti 1 1 Restorative Dentistry, School of Dentistry, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
2 Restorative Dentistry, School of Dentistry, São Paulo State University (Unesp), Araçatuba, Brazil
3 Morphology, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
4 Oral Pathology and Surgery, School of Dentistry, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil 5 Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil Correspondence Francine Benetti, Department of Restorative Dentistry, Universidade Federal de Minas Gerais (UFMG), School of Dentistry, R. Prof. Moacir Gomes de Freitas, 688 – Pampulha, CEP. 31270-901, Belo Horizonte, MG, Brazil. Email: francine-benetti@ufmg.br Funding information Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Grant/Award Number: 88887.489995/2020-00
Abstract
Aim: To analyse the influence of ethylenediaminetetraacetic acid (EDTA) on the repair process in immature rat molars after a regenerative endodontic procedure (REP).
Methodology: The lower first molars of 12 4-week-old Wistar rats underwent pulpectomy in the mesial root and were divided into the following groups: sodium hypochlorite (NaOCl; n = 6) – the mesial canals were irrigated with 2.5% NaOCl for 5 min, and NaOCl-EDTA (n = 6) – the canals were irrigated with 2.5% NaOCl, followed by 17% EDTA for 5 min each. After evoking bleeding using a size 10 K-file, the cavities were sealed. Three molars on the untreated side were randomly used as control (control-15d; n = 3), and three molars from the other three rats untreated were used as immediate control (n = 3). After 15days (NaOCl, NaOCl-EDTA and control15d groups) or immediately (control-immediate), the animals were euthanized, and the teeth were subjected to histologic evaluation of tissue regeneration and presence of collagen fibres. Mann–Whitney U-test was used (p<.05).
Results: The experimental groups had newly formed cementum-like tissue and increased root length and thickness. Half of the specimens in NaOCl-EDTA group showed apical foramen closure, whilst the NaOCl group had partial apical closure. The experimental groups showed inflammatory infiltrate extending mainly to the medium third of the root canal. These parameters were similar between experimental groups (p>.05). Newly formed connective tissue in the pulp space was significantly higher in the NaOCl-EDTA group than in NaOCl group (p<.05). Regarding the collagen fibres, the NaOCl-EDTA group had more collagen fibres in the root tip, but there was no significant difference compared to NaOCl group, and both groups showed greater amount of immature fibres in this area; in the centre of the apical third of root canal, there was equivalence between mature and immature fibres from both groups (p>.05).
Conclusions: Ethylenediaminetetraacetic acid irrigation improved newly formed intracanal connective tissue after REP in immature molars of rats; however, EDTA did not influence cementum-like tissue formation, apical closure, inflammatory infiltrate and maturation of collagen fibres.
KEYWORDS : dental pulp, ethylenediaminetetraacetic acid, guided tissue regeneration, histologic outcomes, regenerative endodontics, root canal irrigants
INTRODUCTION Caries, traumatic dental injuries and dental anomalies may damage the pulp-dentine complex of immature permanent teeth, resulting in pulp necrosis and incomplete root development (Palma et al., 2017; Scarparo et al., 2011). In these cases, conventional endodontic treatment often presents challenges, since there is a major risk of root fractures and overflow of filling materials, due to the presence of thin dentinal walls and open apices (Shah et al., 2008; Thibodeau et al., 2007). In addition, the presence of thin remaining dentinal walls and reduced length due to disrupted root maturation leaves the tooth even more susceptible to fracture (Bracks et al., 2019). Therefore, new materials and approaches have been proposed for the treatment of immature teeth with pulp necrosis. Regenerative endodontic procedures (REPs) are biologically based treatments that have emerged, and it may help to restore the physiologically functional dentition by regenerating tissue in the canal space of immature necrotic teeth (Galler et al., 2016; Kim et al., 2018; Shamszadeh et al., 2019). These procedures are based on the pillars of tissue engineering that contributes to its long-term success, including stem cells, 3-dimensional scaffolds, signalling molecules and a bacteria-free environment (Bracks et al., 2019; Conde et al., 2016; Gomes-Filho et al., 2013; Zhang et al., 2014). Clinical studies using REP have demonstrated promising results with high survival and success rates ranging between 95%–100% (Alobaid et al., 2014; Arslan et al., 2019; Jeeruphan et al., 2012), and increased root length and thickness, with apical closure (Shah et al., 2008; Torabinejad & Faras, 2012). Despite those results, there is no standard irrigation protocol available in REP (Aksel et al., 2020; Aksel & Serper, 2014; Shamszadeh et al., 2019). Current clinical protocols of the European Society of Endodontology (Galler et al., 2016) and the American Association of Endodontics (2018) have proposed the use of 17% ethylenediaminetetraacetic acid (EDTA) after low concentrations of sodium hypochlorite (NaOCl). The use of EDTA is important to minimize the cytotoxicity of NaOCl and to enhance the release of bioactive molecules from the dentine (Chae et al., 2018; Kim et al., 2018). In addition, conditioning dentine with EDTA removes the smear layer, which may expose growth factors entrapped in the dentine matrix (Bracks et al., 2019; Conde et al., 2016; Gonçalves et al., 2016; Graham et al., 2006; Pang et al., 2014). The expression of those molecules from conditioned dentine could potentially modulate cellular activity in periapical tissues (Gonçalves et al., 2016; Taweewattanapaisan et al., 2019), playing a crucial role in the neoformation of intracanal tissues (Bracks et al., 2019). Thus, the additional irrigation with EDTA has been fully recommended in order to optimize the conditions of cell differentiation, tissue formation and regeneration (Conde et al., 2016; Pang et al., 2014). Nevertheless, EDTA may be capable of promoting some negative impact on cell viability (Aksel et al., 2020), cell migration (Deniz Sungur et al., 2019) and blood clot formation (Taweewattanapaisan et al., 2019) compared with other solutions, which might affect tissue regeneration. There have been a few histological analyses evaluating the tissue reaction involved in the repair process and regeneration after the use of NaOCl followed by EDTA irrigation as clinical recommendations for REP. Additionally, an in vivo collagenous content evaluation as a relevant component for the tissue repair and mineralization in immature teeth has not yet been investigated. The present study aimed to histologically evaluate the influence of the use of EDTA on the repair process of immature molars of rats that underwent a REP. The null hypothesis tested was that there are no differences in the histological findings after REP with or without EDTA. MATERIALS AND METHODS Animals A total of fifteen 4-week-old male Wistar albino rats (weighing approximately 80 g) were used. The sample size was calculated using data of periapical inflammation in rat molar submitted to REP of the two experimental groups of a previous study (Scarparo et al., 2014). The calculation was performed using Statistical Package for SigmaPlot (version 12.0; Systat Software Inc.) software program and t-test sample size. Considering 95% power and alpha-type error level of 0.05, the sample size was of five molars per experimental group. Considering the potential of animal mortality, one more animal was added in each group, resulting in six rats per group, similar to previous studies (Bracks et al., 2019; Scarparo et al., 2014). The animals were housed in a temperature-controlled environment (22–24°C) with a 12-h light–dark cycle and ad libitum access to water and feed. The study was approved by the local Animal Research Ethics Committee (CEUA 81/2020) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Pulp revascularization procedure The animals were anaesthetized via intramuscular (IM) injection using ketamine 10% (80 mg/kg; Ketamina Agener 10%, União Química Farmacêutica Nacional S/A) and xylazine 2% (15 mg/kg; Xilazin, Syntec do Brasil LTDA). The lower left or right first immature molars of 12 animals were randomly separated in the experimental groups NaOCl and NaOCl-EDTA based on the operative procedures. Lower untreated molars from these animals were randomly selected as control (Control-15 d, n = 3). Additionally, lower left or right immature molars of other three rats did not receive any intervention, and they were used as control-immediate (n = 3). These controls were included for histologic reference of natural embryogenesis. The teeth were isolated with gingival barrier (Top dam, FGM, Joinville, SC, Brazil), and special dental clamps designed for rat molars and made of stainless steel alloys (Figure S1). Then, endodontic access in mesial root canal was performed with a sterile long neck (LN) round bur (diameter of 0.06 mm; Dentsply Maillefer), in low speed coupled to an electric motor (Beltec) and under constant irrigation with saline. Operative procedures were performed using an operating microscope (Alliance) with 24× magnification. After exposing the dental pulps, concomitantly pulpectomy was performed using size 10 K-files (Dentsply Sirona). The canals were irrigated with sterile saline solution and dried with sterile paper points. To define the working length of the mesial root canals, the jaws of one animal of same age from those used in this experiment were removed during a pilot study. After endodontic access, a radiograph was taken with size 10 K-file introduced in mesial canal of the first molars on both sides, and a root canal length of 4 mm was registered. Afterwards, the irrigation protocols were performed according to the experimental groups (NaOCl and NaOCl-EDTA), using 29 ga NaviTip™ tips (length of 17 mm; Ultradent). For the NaOCl group, the mesial canals were carefully and gently irrigated with 0.1 ml/min 2.5% NaOCl for 5 min, and in the NaOCl-EDTA group, the mesial canals were carefully and gently irrigated with 2.5% NaOCl followed by 17% EDTA (0.1 ml/min) for 5 min each. Subsequently, the canals were irrigated with saline solution and dried with sterile absorbent paper points. Intracanal bleeding was induced by introducing a 10 K-file at 0.5 mm beyond the apical foramen, using a counterclockwise push-and-pull motion until the root canal was filled with blood from the periapical tissues (Bracks et al., 2019). PBS CIMMO HP (CIMMO), a mineral oxide-based cement (Santiago et al., 2021), was placed for coronal barrier, and the cavities were sealed with resin-modified glass ionomer cement lightcured (GC America Inc.). Sample preparation and histological analysis At the 4-week period (Control-immediate group), or 15 days after the pulp revascularization procedure (experimental groups and Control-15 d), the rats were euthanized with an overdose of anaesthetic solution (Thiopentax; Cristália-Produtos Químicos Farmacêuticos LTDA). The right and left jaws were separated, dissected, fixed in a solution of 4% buffered formaldehyde for 24 h and then decalcified in 10% EDTA solution for 45 days. The specimens were processed and embedded in paraffin. Serial histological sections (5 μm) were cut in the mesiosagittal plane and were selected from the point where the mesial root of the first molar was seen in all its longitudinal extension. Then, the histological sections were stained with haematoxylin-eosin (HE), Masson's trichrome (MT) and picrosirius red (PSR) (Cintra et al., 2017). The first slide obtained with histological sections was selected for HE staining and each the next two for MT or PSR analysis. The analyses were performed under light microscopy (400× magnification; DM4000 B; Leica Microsystems) by a single calibrated and blinded operator to the groups. Sections stained with HE were used for histopathological analysis. The intracanal and periapical tissues present in each group were analysed and scored according to the histopathological parameters involving mineralized tissues formed on root canal (Palma et al., 2017; Zhang et al., 2014), newly formed cementum-like tissue (Gomes-Filho et al., 2013), apical closed histologic (Palma et al., 2017), extension of the inflammatory infiltrate (Gomes-Filho et al., 2013) and presence of newly connective tissue. These rated parameters were described in Table 1. Sections stained by MT were used for detection of collagen, which is stained blue, differentiating it from other structures. The collagen fibre maturation was analysed in the sections stained by PSR under polarized light microscopy. The images were obtained of the apical tip of the root, and the centre of the apical third of the root canal (400× magnification; Leica QWin V3, Leica Microsystems), allowing the selection of corresponding colours for each type of collagen fibre. After colour selection, the program automatically calculated the marked area of each collagen fibre type inside the pulp chamber. Greenish-yellow fibres were classified as immature and thin, whilst yellowish-red fibres were considered mature and thick (Benetti et al., 2020; Cintra et al., 2017; Terayama et al., 2020) Statistical analysis Statistical Package for SigmaPlot (version 12.0, Systat Software Inc.) software program was used for the statistical analysis. The data were analysed using the Mann– Whitney U-test. Statistical significance was set at p<.05 for all analyses
RESULTS
Histologic findings
The effects of EDTA on immature molars of rats after REP were determined by comparing the same parameters between experimental groups. Representative images of the evaluated histopathological parameters are presented in Figure 1, and the results are summarized in Table 1. After 15days of REP, most specimens of NaOCl-EDTA group showed a concomitant increase of root length and thickness indicated by a deposition of cellular mineralized tissue compared to NaOCl group, where an increase in the root canal wall thickness was mostly observed. All teeth in the NaOCl and NaOCl-EDTA groups displayed cementum tissue formation, where many cementocyte-like cells were present in the newly layer of mineralized tissue formed on the root canal wall. Histologic evaluation showed progressive apical closure in NaOCl-EDTA group, where half of the specimens exhibited complete closure of the root apex whilst NaOCl without EDTA showed partial apical closure in one tooth.
Regarding the extension of inflammatory infiltrate, both experimental groups showed a variable inflammatory response to treatment with the presence of polymorphonuclear cells, extending mainly to the medium third of the root canal (Figure 1). These four evaluated parameters did not differ histologically between experimental groups (p>.05). Regarding the newly formed connective tissue into the pulp canal space, a partial ingrowth of connective tissue occurred significantly in most specimens of NaOCl-EDTA group, extending mainly from the apical to the medium third of teeth (p<.05). The tissue was characterized by a continuous layer of connective tissue of the periodontal ligament, with fibroblasts and blood vessels. Additionally, the presence of some islands of cellular cementum in the lumen of the root canal space was also noted in the NaOCl-EDTA group. On the other hand, newly formed connective tissue areas in the root canal space were predominantly absent in the NaOCl group
Presence and maturation of collagen fibres
Representative images of the MT and PSR staining are displayed in Figure 2, and the results are shown in Figure 2 and Table 2. Although NaOCl-EDTA group exhibited more collagen fibres in the root tip region, there was no significant difference compared with the NaOCl group, where both groups showed greater amount of immature fibres in this area. Regarding the centre area of the apical third, equivalent values between mature and immature fibres from both evaluated groups were found, without significant differences.
DISCUSSION
In the current study, a comparative histologic evaluation of the effect of EDTA on the tissue regenerative potential of immature molars of rats that underwent a REP with or without the use of EDTA was performed. It was found that the use of NaOCl-EDTA group promoted mineralized tissue deposition along the root canal walls, cementum tissue formation, apical closure and inflammatory response in most specimens similarly to NaOCl group. On the other hand, the former demonstrated a higher newly formed connective tissue within the pulp canal space than NaOCl without EDTA, as confirmed by the H & E analysis. A greater number of immature fibres were demonstrated in the root tip region, whilst similar values of mature and immature fibres were observed in the centre area of the apical third in the experimental groups. There were no significant differences between the groups at the evaluated areas. Thus, the null hypothesis of the study was partially accepted. An investigation of the steps of regenerative therapy, such as scaffold formation and irrigation, is necessary to establish promisor clinical protocols for REP (Bracks et al., 2019; Peters, 2014). Besides showing high repair potential, bleeding induction in immature teeth is necessary to produce a support in-growth of new tissue, including the delivery of stem cells and growth factors during regeneration process (Gomes-Filho et al., 2013; Jung et al., 2019). Additionally, adequate disinfection before inducing bleeding in the root canal space is important for the success of REP (Verma et al., 2017). Ethylenediaminetetraacetic acid is widely used as the final rinse in the protocols for REP, due to its ability to remove intracanal dressing and smear layer generated by instrumentation (Chae et al., 2018), in addition to expose and release the dentine matrix (Scarparo et al., 2011; Wang et al., 2010). Therefore, irrigation with 17% EDTA has also been incorporated after NaOCl in REP (Galler 2016), probably due to its high expression of proteins during calcium quenching associated with the stem cell differentiation, which might positively influence tissue neoformation (Bracks et al., 2019; Widbiller et al., 2017). Similarly to the present study, the formation of connective and cementum-like tissues was previously described in the regenerated tissues using other irrigating protocols in animal models submitted to REP (Gomes-Filho et al., 2013; Thibodeau et al., 2007; Yamauchi et al., 2011). These histological findings may be associated with the ingrowth of periodontal ligament stem cells into the canal space. Nevertheless, no pulp-like tissue with odontoblastlike cell layer was observed in these investigations, similarly to our study. Conversely, Ishizaka et al. (2012) also performed an in vivo study, which demonstrated the presence of pulp-like tissue regeneration when pulp, adipose and bone marrow-derived CD31(−) cells were deposited in the root canal by using a collagen scaffold as a stem cell homing with stromal cell-derived factor 1. Nevertheless, the study of Ishizaka et al. (2012) did not perform dentine conditioning, which do not simulate a clinical environment. There are still uncertainties about the histological improvements of irrigation with EDTA that need to be explored in REP. By using a rat model with immature teeth, we histologically assessed this point in the present report. Groups that did not receive any treatment were used as the immediate and 15-day period controls. The histological sections of the experimental groups were associated with continued root development, mineralized tissue formation, apical closure, inflammatory reaction, newly formed connective tissue and areas of collagen deposition in immature rat teeth after 15days of REP. H & E throughout evaluation revealed a continued root development, including increased root length and thickness, in most specimens irrigated with EDTA. Furthermore, a layer of cementum-like tissue deposited on the root canal walls of all samples and complete or partial apical closure was noticed. Interestingly, the NaOCl-EDTA showed complete closure in half of the samples, one of the main histologic findings of this study. These results might be related with the presence of calcium ions in the blood clot that play a crucial role in its osteoinductive properties (Taweewattanapaisan et al., 2019; Yamauchi et al., 2011). In addition, the ability of EDTA to expose signalling molecules following dentine demineralization, such as transforming growth factor (TGF)-β, that might regulate cellular activity (Bracks et al., 2019; Chae et al., 2018; Gonçalves et al., 2016; Kucukkaya Eren et al., 2021) might have enhanced biological apical closure in this group. A considerable inflammatory infiltration composed by polymorphonuclear cells, extending mainly to the medium third of the canal, was observed in the NaOCl and NaOCl-EDTA groups. In accordance with these findings, a previous histologic evaluation of immature dog teeth irrigated with 1.25% NaOCl during REP showed a significant number of inflammatory cells adjacent (and not in it) to the newly formed intracanal tissue (Wang et al., 2010). The liberation of bioactive molecules following dentine conditioning is involved in the recruitment of various immune-inflammatory cells (Galler et al., 2011). Similarly to Wang et al. (2010), the presence of inflammatory cells was not capable of interfering the cementumlike tissue neoformation in the current study, and it may even accelerate hard tissue deposition by providing factors to stimulate stem cell differentiation into cementoblasts (Wang et al., 2010). Thus, the presence of inflammatory cells in the treated area may be related to tissue repair. Moreover, Bracks et al. (2019) reported a higher interleukin (IL)-1 mRNA expression in the mice teeth submitted to irrigation to EDTA followed by blood clot formation at 7 and 14days of analysis. IL-1 is a pro-inflammatory cytokine that activates endothelial cells to participate in the immune-inflammatory response and also shows proangiogenic effects (Bracks et al., 2019). Histologic reports using immature human and animal teeth have shown the presence of fibrous tissue, and intracanal islands of cementum-like or bone-like tissues, but not as pulp-like tissue (Shimizu et al., 2013; Wang et al., 2010; Yamauchi et al., 2011). These histologic findings were similar to the regenerated tissue observed in the pulp space of teeth with mature roots, in which a vascular and fibrous connective tissue with areas of hard tissue deposition and inflammation was reported (Arslan et al., 2019; Gomes-Filho et al., 2013). Regenerated tissues may have characteristics of the tissue from which stem cells originate (Jung et al., 2019). This investigation found ectopic areas of cellular cementum-like tissue in the newly formed tissue in the lumen of the canal space in the NaOCl-EDTA, probably originated from stem cells derived from the periodontal ligament and alveolar bone. Regarding the newly generated tissue onto the pulp canal space, a significant partial ingrowth of connective tissue with fibroblasts and blood vessels that reached the medium third of the teeth irrigated with NaOCl followed by EDTA was identified in comparison with those without EDTA irrigation. These results may be associated with the ability of EDTA to release several growth factors entrapped in dentine, thereby promoting cell migration and differentiation. However, further long-term evaluations are necessary to assess the histologic characteristics of the new tissue formed into the root canal, in addition to the presence of growth factors and other bioactive molecules in immature teeth irrigated with EDTA in comparison to non-EDTA irrigation. The extracellular matrix of dental the pulp is composed with approximately 34% of collagen fibres, which represents one of the most important classes of extracellular macromolecules of the dental pulp (Cintra et al., 2017). Although type I collagen is the most predominant component in the mineralized tissues, a great distribution of collagen type I and III, or mature and immature fibres has been reported in dental pulp, respectively (Cintra et al., 2017; Garcia et al., 2003). These proteins participate in the process of tissue organization and mineralization of the tooth. In the present study, a visualization of the collagen was executed by using MT and PSR techniques. Areas of collagen were discovered especially in the apical area of the treated groups. Although there were no statistical differences between the experimental groups for total area of collagen fibres, there was histological evidence of more collagen fibres in the tip region of NaOClEDTA group, probably indicating faster repair. Concomitantly, a specific analysis for immature and mature collagen fibres detection was performed using polarized light microscopy. A greenish-yellow colour suggests that collagen is poorly packed, whereas a yellowish-red colour suggests better maturity and thick fibre organization (Benetti et al., 2020; Cintra et al., 2017). Immature fibres were predominant in the root tip region and may indicate a loose connective immature tissue-like, compatible with the animal's age. The increased number of collagen fibres in the tip region of NaOCl-EDTA may have been associated with the presence of ectopic mineralization areas onto the pulp space in this group. On the other hand, similar amount of mature and immature collagen fibres was observed in the centre region of the apical third from both experimental groups. These results disagree with the study by Gomes-Filho et al. (2013), who showed dense collagen fibres in the newly formed tissue inside the root canals. However, the analysis period of this previous study was 3 months after the REP, in addition to using the dog model. To our knowledge, there are no other studies that evaluated the maturation of collagen fibres in the newly formed tissue after pulp revascularization. These data are interesting, as greater collagen maturation can be related to older pulp tissue. Residual bacteria demonstrate a negative effect on the outcomes of REP, and it is associated with persistent periapical radiolucency, in addition to showing a reduction in newly formed mineralized tissue (Verma et al., 2017). Nevertheless, the negative effects of residual contamination on newly formed tissues were excluded from this study, so that any variable could be discarded, as well as performed in other investigations (Alexander et al., 2020; Bracks et al., 2019; Nosrat et al., 2019). The use of a noninfected model rather than induced periapical lesion is a reasonable alternative to focus on examining the histological outcomes of tissue engineering strategies for REP (Alexander et al., 2020; Nosrat et al., 2019), such as the ability of final irrigation with EDTA to favour tissue repair. However, it is important now to assess the influence of EDTA on the revascularization of a tooth with necrotic pulp. As a possible limitation, a human model would be favourable over animal models, to get even closer to the clinical reality, despite the great physiological, biological and anatomical similarity of rat molars with human teeth (Cintra et al., 2016; Dammaschke, 2010). This study is important because it shows that despite the existing contradiction about the effects of EDTA on pulp regeneration (Aksel et al., 2020; Deniz Sungur et al., 2019; Taweewattanapaisan et al., 2019), it favoured the neoformation of connective and mineralized tissue, strengthening the root structure, even if they are not original tissues of the pulp-dentine complex. Still, this study was carried out only within the period of 15days. Perhaps over a longer period, both groups had complete apical closure. However, the present results might indicate acceleration in the repair process by the use of EDTA. CONCLUSION
The use of EDTA in REPs improved newly formed intracanal connective tissue in immature molars of rats; however, EDTA did not influence cementum-like tissue formation, apical closure, inflammatory infiltrate, and maturation of collagen fibres. AUTHOR CONTRIBUTIONS
AH dos Reis-Prado: Conceptualization; Data Curation; Formal Analysis; Investigation; Funding Acquisition; Visualization; Writing-original draft. SC Oliveira: Formal Analysis; Investigation; Visualization; Writing-original draft. J Goto: Data Curation; Investigation; Writing-original draft. GAB Silva: Methodology; Project Administration; Resources. LTA Cintra: Formal Analysis; Resources; Writing – Review & Editing. RA Mesquita: Formal Analysis; Funding Acquisition; Writing – Review & Editing. RE Szawka: Project Administration; Resources; Supervision. AP Ribeiro-Sobrinho: Conceptualization; Project administration; Resources; Supervision; Writing – Review & Editing. F Benetti: Conceptualization; Data Curation; Formal Analysis; Investigation; Project Administration; Resources; Supervision; Funding Acquisition; Writing – review & editing. All authors collectively proofread the final version and approved it for publication. ACKNOWLEDGEMENTS
This research was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – 88887.489995/2020-00.
CONFLICT OF INTEREST
The authors have stated explicitly that there are no conflicts of interest in connection with this article.
DATA AVAILABILITY STATEMENT
Data available on request from the authors ORCID Alexandre Henrique dos Reis-Prado: https://orcid.org/0000-0002-5866-7137
Sabrina de Castro Oliveira : https://orcid.org/0000-0003-2659-8322
Juliana Goto : https://orcid.org/0000-0002-6040-3560
Luciano Tavares Angelo Cintrac: https://orcid.org/0000-0003-2348-7846
Ricardo Alves de Mesquita : https://orcid.org/0000-0002-2639-3469
Raphael Escorsim Szawka : https://orcid.org/0000-0002-2639-3469
Antônio Paulino Ribeiro-Sobrinho : https://orcid.org/0000-0002-3598-7592
Francine Benetti : https://orcid.org/0000-0002-5459-353X REFERENCES
Aksel, H., Albanyan, H., Bosaid, F. & Azim, A.A. (2020) Dentin conditioning protocol for regenerative endodontic procedures. Journal of Endodontics, 46, 1099–1104
Aksel, H. & Serper, A. (2014) Recent considerations in regenerative endodontic treatment approaches. Journal of Dental Sciences, 9, 207–213.
Alexander, A., Torabinejad, M., Vahdati, S.A., Nosrat, A., Verma, P., Grandhi, A. et al. (2020) Regenerative endodontic treatment in immature noninfected ferret teeth using blood clot or SynOss putty as scaffolds. Journal of Endodontics, 46, 209–215.
Alobaid, A.S., Cortes, L.M., Lo, J., Nguyen, T.T., Albert, J., AbuMelha, A.S. et al. (2014) Radiographic and clinical outcomes of the treatment of immature permanent teeth by revascularization or apexification: a pilot retrospective cohort study. Journal of Endodontics, 40, 1063–1070.
American Association of Endodontists. (2018) AAE clinical consideration for a regenerative procedure. Chicago: American Association of Endodontists. Revised 4-1-18.
Arslan, H., Şahin, Y., Topçuoğlu, H.S. & Gündoğdu, B. (2019) Histologic evaluation of regenerated tissues in the pulp spaces of teeth with mature roots at the time of the regenerative endodontic procedures. Journal of Endodontics, 45, 1384–1389.
Benetti, F., Bueno, C.R.E., Reis-Prado, A.H.D., Souza, M.T., Goto, J., J.M.P., Camargo et al. (2020) Biocompatibility, biomineralization, and maturation of collagen by RTR®, bioglass and DM bone® materials. Brazilian Dental Journal, 31, 477–484.
Bracks, I.V., Espaladori, M.C., Barros, P., de Brito, L.C.N., Vieira, L.Q. & Ribeiro Sobrinho, A.P. (2019) Effect of ethylenediaminetetraacetic acid irrigation on immune-inflammatory response in teeth submitted to regenerative endodontic therapy. International Endodontic Journal, 52, 1457–1465.
Chae, Y., Yang, M. & Kim, J. (2018) Release of TGF-B1 into root canals with various final irrigants in regenerative endodontics: an in vitro analysis. International Endodontic Journal, 12, 1389–1397.
Cintra, L.T., Benetti, F., Ferreira, L.L., Rahal, V., Ervolino, E., Jacinto Rde, C. et al. (2016) Evaluation of an experimental rat model for comparative studies of bleaching agents. Journal of Applied Oral Science, 24, 95–104.
Cintra, L.T.A., Ferreira, L.L., Benetti, F., Gastélum, A.A., GomesFilho, J.E., Ervolino, E. et al. (2017) The effect of dental bleaching on pulpal tissue response in a diabetic animal model. International Endodontic Journal, 50, 790–798.
Conde, M.C., Chisini, L.A., Demarco, F.F., Nör, J.E., Casagrande, L. & Tarquinio, S.B. (2016) Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. International Endodontic Journal, 49, 543–550.
Dammaschke, T. (2010) Rat molar teeth as a study model for direct pulp capping research in dentistry. Laboratory Animals, 44, 1–6.
Deniz Sungur, D., Aksel, H., Ozturk, S., Yılmaz, Z. & Ulubayram, K. (2019) Effect of dentine conditioning with phytic acid or etidronic acid on growth factor release, dental pulp stem cell migration and viability. International Endodontic Journal, 52, 838–846.
Galler, K.M. (2016) Clinical procedures for revitalization: current knowledge and considerations. International Endodontic Journal, 49, 926–936.
Galler, K.M., D'Souza, R.N., Federlin, M., Cavender, A.C., Hartgerink, J.D., Hecker, S. et al. (2011) Dentin conditioning codetermines cell fate in regenerative endodontics. Journal of Endodontics, 37, 1536–1541.
Galler, K.M., Krastl, G., Simon, S., van Gorp, G., Meschi, N., Vahedi, B. et al. (2016) European Society of Endodontology position statement: revitalization procedures. International Endodontic Journal, 49, 717–723.
Garcia, J.M.Q., Martins, M.D., Jaeger, R.G. & Marques, M.M. (2003) Immunolocalization of bone extracellular matrix proteins (type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells. International Endodontic Journal, 36, 404–410.
Gomes-Filho, J.E., Duarte, P.C., Ervolino, E., Mogami Bomfim, S.R., Xavier Abimussi, C.J., da Mota Silva Santos, L. et al. (2013) Histologic characterization of engineered tissues in the canal space of closed-apex teeth with apical periodontitis. Journal of Endodontics, 39, 1549–1556.
Gonçalves, L.F., Fernandes, A.F., Cosme-Silva, L., Colombo, F.A., Martins, N.S., Oliveira, T.M. et al. (2016) Effect of EDTA on TGF-β1 released from the dentin matrix and its influence on dental pulp stem cell migration. Brazilian Oral Research, 30, e131.
Graham, L., Cooper, P.R., Cassidy, N., Nor, J.E., Sloan, A.J. & Smith, A.J. (2006) The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials, 27, 2865–2873.
Ishizaka, R., Iohara, K., Murakami, M., Fukuta, O. & Nakashima, M. (2012) Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone marrow and adipose tissue. Biomaterials, 33, 2109–2118.
Jeeruphan, T., Jantarat, J., Yanpiset, K., Suwannapan, L., Khewsawai, P. & Hargreaves, K.M. (2012) Mahidol study 1: comparison of radiographic and survival outcomes of immature teeth treated with either regenerative endododontic or apexification methods: a retrospective study. Journal of Endodontics, 38, 1330–1336.
Jung, C., Kim, S., Sun, T., Cho, Y.-B. & Song, M. (2019) Pulp-dentin regeneration: current approaches and challenges. Journal of Tissue Engineering, 10, 1–13. Kim, S.G.,
Kim, S.G., Malek, M., Sigurdsson, A., Lin, L.M. & Kahler, B. (2018) Regenerative endodontics: a comprehensive review. International Endodontic Journal, 51, 1367–1388.
Kucukkaya Eren, S., Zırh, E.B., Zeybek, N.D., Örs, S.A., Aksel, H. & Parashos, P. (2021) Effect of benzalkonium chloride addition to EDTA on attachment and proliferation of dental pulp stem cells on dentin and on transforming growth factor-β1 release. Odontology, 109, 313–320.
Nosrat, A., Kolahdouzan, A., Khatibi, A.H., Verma, P., Jamshidi, D., Nevins, A.J. et al. (2019) Clinical, radiographic, and histologic outcome of regenerative endodontic treatment in human teeth using a novel collagen-hydroxyapatite scaffold. Journal of Endodontics, 45, 136–143.
Palma, P.J., Ramos, J.C., Martins, J.B., Diogenes, A., Figueiredo, M.H., Ferreira, P. et al. (2017) Histologic evaluation of regenerative endodontic procedures with the use of chitosan scaffolds in immature dog teeth with apical periodontitis. Journal of Endodontics, 43, 1279–1287.
Pang, N.-S., Lee, S.J., Kim, E., Shin, D.M., Cho, S.W., Park, W. et al. (2014) Effect of EDTA on attachment and differentiation of dental pulp stem cells. Journal of Endodontics, 40, 811–817.
Peters, O.A. (2014) Translational opportunities in stem cell-based endodontic therapy: where are we and what are we missing? Journal of Endodontics, 40, S82–S85.
Santiago, M.C., Gomes-Cornélio, A.L., de Oliveira, L.A., TanomaruFilho, M. & Salles, L.P. (2021) Calcium silicate-based cements cause environmental stiffness and show diverse potential to induce osteogenesis in human osteoblastic cells. Scientific Reports, 11, 16784.
Scarparo, R.K., Dondoni, L., Böttcher, D.E., Grecca, F.S., Figueiredo, J.A.P., Kantarci, A. et al. (2014) Intracanal delivery of Resolvin E1 controls inflammation in necrotic immature rat teeth. Journal of Endodontics, 40, 678–682.
Scarparo, R.K., Dondoni, L., Böttcher, D.E., Grecca, F.S., Rockenbach, M.I. & Batista, E.L., Jr. (2011) Response to intracanal medication in immature teeth with pulp necrosis: an experimental model in rat molars. Journal of Endodontics, 37, 1069–1073.
Shah, N., Logani, A., Bhaskar, U. & Aggarwal, V. (2008) Efficacy of revascularization to induce apexification/apexogensis in infected, nonvital, immature teeth: a pilot clinical study. Journal of Endodontics, 34, 919–925.
Shamszadeh, S., Asgary, S. & Nosrat, A. (2019) Regenerative endodontics: a Scientometric and bibliometric analysis. Journal of Endodontics, 45, 272–280.
Shimizu, E., Ricucci, D., Albert, J., Alobaid, A.S., Gibbs, J.L., Huang, G.T.J. et al. (2013) Clinical, radiographic, and histological observation of a human immature permanent tooth with chronic apical abscess after revitalization treatment. Journal of Endodontics, 39, 1078–1083.
Taweewattanapaisan, P., Jantarat, J., Ounjai, P. & Janebodin, K. (2019) The effects of EDTA on blood clot in regenerative endodontic procedures. Journal of Endodontics, 45, 281–286.
Terayama, A.M., Benetti, F., de Araújo Lopes, J.M., Barbosa, J.G., Silva, I.J.P., Sivieri-Araújo, G. et al. (2020) Influence of low-level laser therapy on inflammation, collagen fiber maturation, and tertiary dentin deposition in the pulp of bleached teeth. Clinical Oral Investigations, 24, 3911–3921.
Thibodeau, B., Teixeira, F., Yamauchi, M., Caplan, D.J. & Trope, M. (2007) Pulp revascularization of immature dog teeth with apical periodontitis. Journal of Endodontics, 33, 680–689.
Torabinejad, M. & Faras, H. (2012) A clinical and histological report of a tooth with an open apex treated with regenerative endodontics using platelet-rich plasma. Journal of Endodontics, 38, 864–868.
Verma, P., Nosrat, A., Kim, J.R., Price, J.B., Wang, P., Bair, E. et al. (2017) Effect of residual bacteria on the outcome of pulp regeneration in vivo. Journal of Dental Research, 96, 100–106.
Wang, X., Thibodeau, B., Trope, M., Lin, L.M. & Huang, G.T. (2010) Histologic characterization of regenerated tissues in canal space after the revitalization/revascularization procedure of immature dog teeth with apical periodontitis. Journal of Endodontics, 36, 56–63.
Widbiller, M., Eidt, A., Hiller, K.-A., Buchalla, W., Schmalz, G. & Galler, K.M. (2017) Ultrasonic activation of irrigants increases growth factor release from human dentine. Clinical Oral Investigations, 21, 879–888.
Yamauchi, N., Yamauchi, S., Nagaoka, H., Duggan, D., Zhong, S., Lee, S.M. et al. (2011) Tissue engineering strategies for immature teeth with apical periodontitis. Journal of Endodontics, 37, 390–397.
Zhang, D.D., Chen, X., Bao, Z.F., Chen, M., Ding, Z.J. & Zhong, M. (2014) Histologic comparison between platelet-rich plasma and blood clot in regenerative endodontic treatment: an animal study. Journal of Endodontics, 40, 1388–1393
SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.
How to cite this article: dos Reis-Prado, A.H., Oliveira, S.d.C., Goto, J., Silva, G.A.B., Cintra, L.T.A., de Mesquita, R.A. et al. (2022) Influence of ethylenediaminetetraacetic acid irrigation on the regenerative endodontic procedure in an immature rat molar model. International Endodontic Journal, 00, 1–11. Available from: https://doi.org/10.1111/iej.13846
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