EDTA enhances cementum-like tissue formation, TGF-β1 & VEGF in rat molars during regenerative endodontics

To analyse the influence of ethylenediaminetetraacetic acid (EDTA) on the repair process in immature rat molars after a regenerative endodontic procedure (REP). 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 5min, and NaOCl-EDTA (n=6) - the canals were irrigated with 2.5% NaOCl, followed by 17% EDTA for 5min each. After evoking bleeding using a size 10K-file, the cavities were sealed. Three molars on the untreated side were randomly used as control (control-15 d; n=3), and three molars from the other three rats untreated were used as immediate control (n=3). After 15 days (NaOCl, NaOCl-EDTA and control-15 d 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). 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). 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.

Regenerative endodontic cell-homing procedures are frequently performed on injured immature teeth diagnosed with pulp necrosis and/or apical periodontitis. The representative histological finding after those procedures is cementum-like tissues filling in the root canal but details of the healing process remain unknown. We investigated that healing process histologically using a mouse in situ model. Regenerative endodontic procedures were experimentally performed on noninfected maxillary first molars of 6-week-old male C57BL/6 mice, after which the healing process was investigated using histology and immunohistochemistry. Immediately after the regenerative endodontic cell-homing procedures, blood clots were seen in the root canals that disappeared over time. On day 7, the blot clot in the root canal was replaced by granulation tissue. From day 14 onward, cementum-like tissues were filled in the root canals, while the amount of fibrous tissue was reduced. Immunohistochemically, positive reactions for periostin were seen in the fibrous tissue in the root canal, the apex, and periodontal ligament cells. On the other hand, positive reactions for nestin were not detected in the root canal. CD31-positive cells with a luminal structure were also observed in the fibrous tissue around the apex and around the newly formed cementum-like tissues in the root canal. Thus, in this study, we have established an in situ mouse model of regenerative endodontic procedures. The results of this study suggest that periodontal ligament cells and vascular endothelial cells grow into the root canals from the apex, replace the blood clots, and participate in the formation of cementum-like tissues with angiogenesis during the healing process of regenerative endodontic procedures.

ABSTRACTPreserving the natural dentition in its normal form and function is one of the major goals of endodontic treatment. Re-establishing the lost vitality and development of root maturation in immature permanent teeth with pulp necrosis/apical periodontitis is quiet challenging clinically. The very basis of regenerative endodontics relies on the concept of tissue engineering using stem cells, biomimetic scaffold, and bioactive growth factors to regenerate the pulp tissue damaged by microbial infection, injury, or developmental defects. In clinical endodontics, this approach is referred to as a “paradigm shift.” Even though repair instead of true regeneration is achieved with current protocols, it is hoped that further research in the area of stem-cell-based tissue engineering will allow for true regeneration and improved treatment outcomes. The aim of this review is to discuss about the various aspects of regenerative endodontics, current clinical protocols, and the future of pulp regeneration techniques.

This study systematically reviews the literature on protocols used in regenerative endodontic procedures, focusing on the relationship between irrigants, intracanal medicaments, and the release of dentin-derived growth factors, which form one of the foundational pillars of regenerative endodontics. Systematic searches were conducted in PubMed, Web of Science, Scopus, Google Scholar, and gray literature for English studies (2015-2024). The included studies investigated growth factor release associated with irrigants or intracanal medicaments in human dentin. QUIN tool assessed methodological quality. Of the three-thousand-seven-hundred-and-seventy-four screened, seventeen studies were included in the review. Ethylenediaminetetraacetic acid (EDTA), either alone or combined with sodium hypochlorite (NaOCl) or chlorhexidine, and 10% citric acid were the most effective in releasing TGF-β1 and BMPs (BMP2 and BMP7). Although this systematic review demonstrates methodological heterogeneity among studies, there was a tendency for increased levels of TGF-β1 and BMP with the use of EDTA (10 or 17%) associated or not with NaOCl or chlorhexidine, while the release of VEGF and FGF2 was minimal or undetectable. Regarding the intracanal medicaments, the use of calcium hydroxide paste (Ca(OH)₂) paste demonstrated an increase in TGF-β1 release. However, this result should be interpreted with caution due to the limited number of available studies.

The effect of EDTA, EGTA, EDTAC, and tetracycline-HCl with and without subsequent NaOCl treatment on the microhardness of root canal dentin

Objectives: The present study aims to evaluate the effects of different concentrations of ethylenediamine tetraacetic acid (EDTA) on smear layer removal in primary teeth by using scanning electron microscopy (SEM). Materials and Methods: The present study was performed with 28 extracted upper primary incisors assigned into four main groups (n=7) as 5%, 10% and 17% EDTA, and 1% sodium hypochlorite (NaOCl). The root canal surfaces (coronal, middle and apical) were scanned by SEM and scores of smear layer removal were recorded and compared after the root canal irrigation procedures. The results were analyzed using Kruskal–Wallis, Friedman and Dunn tests. Results: In all the root portions, although EDTA groups removed the smear layer more effectively than NaOCl, a statistically significant difference was observed between 17% EDTA and 1% NaOCl only in middle third (p &amp;lt;0.05). Also, smear layer was removed more effectively in coronal than apical in most of the groups (10% EDTA, 17% EDTA and 1% NaOCl) (p &amp;lt;0.05). Erosive defects were mostly seen in 17% EDTA and less than 10% EDTA group. These findings were not detected in 5% EDTA and 1% NaOCl group. Conclusions: It is possible to recommend the use of 5% EDTA irrigation solution in root canal treatment of primary teeth due to its similar smear layer removal efficacy with NaOCl and high concentration EDTA groups, low erosive potential and low concentration for periapical safety.

Introduction:The aim of this study was to verify the effect of alternating 2.5% sodium hypochlorite (NaOCl) and 17% ethylenediaminetetraacetic acid (EDTA) on the smear layer removal from root canal surfaces. Methods and Materials:A total of 15 single-rooted human teeth, instrumented with ProTaper files, were randomly distributed in 3 groups. In group 1 (n=7) the canals were irrigated with 1 mL of 2.5% NaOCl between files and final irrigation was done with 1 mL of 2,5% NaOCl, followe by 1 mL of 17% EDTA, for a perio of 15 sec with new irrigtion of 1 mL of 2,5% NaOCl at each change of files. In group 3 (control group) (n=1), saline solution was used. All samples were cleaved into two sections, metalized and analyzed under scanning electron microscopy (SEM). The presence or absence of smear layer in the cervical, middle and apical thirds, with scores varying from 1 to 3, respectively were evaluated. The data were submitted to nonparametric Mann-Whitney U test. The level of significance was set at 0.05. Results:It was observed that there was a greater discrepancy between groups with respect to the apical third. In the other areas there was a greater similarity between the scores attributed to the groups. There was a statistically significant difference between the groups only in the apical third, when group 1 presented the higher median (P<0.05). Conclusion:The alternating use of EDTA during instrumentation with NaOCl was the most effective irrigation method to remove the apical smear layer. Both forms of irrigation were effective on removal of the smear layer in the coronal and middle thirds of the canals.

To evaluate the influence of an experimental solution of cobalt-doped F18 bioactive glass (F18Co) on tissue repair following regenerative endodontic procedure (REP) in rat molars. The F18Co solution was prepared at a ratio of 1:5 F18Co powder to distilled water. The right or left upper first molars of 12 Wistar rats were used, where the pulps were exposed, removed, and irrigated with 2.5% sodium hypochlorite (NaOCl), followed by 17% ethylenediaminetetraacetic acid (EDTA) (5 min each). Subsequently, the molars were divided into two groups (n = 6): REP-SS and REP-F18Co, where they received a final irrigation (5 min) with saline solution (SS) or F18Co solution, respectively. Then, intracanal bleeding was induced, and the tooth was sealed. Untreated molars were used as controls (n = 3). At 21 days, the rats were euthanized, and the specimens were processed for analysis of mineralized tissue and soft tissue formation inside the root canal using haematoxylin-eosin. The presence and maturation of collagen were evaluated by Masson's trichrome and picrosirius red staining. Immunolabelling analyses of proliferating cell nuclear antigen (PCNA) and osteocalcin (OCN) were performed. The data were submitted to the Mann-Whitney U-test (p < .05). There was a similar formation of mineralized tissue in thickness and length in REP-SS and REP-F18Co groups (p > .05). Regarding the presence of newly formed soft tissue, most specimens of the REP-F18Co had tissue formation up to the cervical third of the canal, whilst the REP-SS specimens showed formation up to the middle third (p < .05), and there was higher maturation of collagen in REP-F18Co (p < .05). The number of PCNA-positive cells found in the apical third of the root canal was significantly higher in the F18Co group, as well as the OCN immunolabelling, which was severe in most specimens of REP-F18Co, and low in most specimens of REP-SS. The final irrigation with F18Co bioactive glass solution in REP did not influence mineralized tissue formation but induced soft tissue formation inside the root canals, with higher collagen maturation, and an increase in PCNA-positive cells and OCN immunolabelling.

Evaluation of low-level diode laser irradiation and various irrigant solutions on the biological response of stem cells from exfoliated deciduous teeth

The effects of ethylenediaminetetraacetic acid (EDTA) on regenerative endodontic procedures (REPs) are controversial, because, despite releasing growth factors from dentine, some studies show negative effects on cell behaviour. The aim of the study was to investigate the influence of the use of EDTA in REP on the growth factors' release, cell behaviour and tissue regeneration. A systematic search was conducted (PubMed/Medline, Scopus, Cochrane Library, Web of Science, Embase, OpenGrey and reference lists) up to February 2021. Only in vivo and in vitro studies evaluating the effects of EDTA on the biological factors of dentine, pulp/periapical tissues and cell behaviour were eligible. Studies without a control group or available full text were excluded. The growth factors' release was the primary outcome. Risk of bias in the in vitro and in vivo studies was performed according to Joanna Briggs Institute's Checklist and SYRCLE's RoB tool, respectively. Of the 1848 articles retrieved, 36 were selected. Amongst these, 32 were in vitro, three animal studies and one with both models. The EDTA concentrations ranged from 3% to 15%, at different times. Regarding growth factors' release (17 studies), 15 studies found significant transforming growth factor (TGF)-β release after dentine conditioning with EDTA, and most found no influence on vascular endothelial growth factor release. Regarding cell behaviour (26 studies), eight studies showed no influence of EDTA-treated dentine on cell viability, whereas, five, nine and six studies showed higher cell migration, adhesion and differentiation respectively. No influence of EDTA conditioning was observed in animal studies. In vitro studies had a low risk of bias, whereas animal studies had high risk of bias. Meta-analysis was unfeasible. This review found that EDTA increased TGF-β release and improved cell activity. However, well-designed histological analyses using immature teeth models are needed. High-quality in vitro evidence suggests that EDTA-treated dentine positively influences TGF-β release, cell migration, attachment and differentiation; further research to evaluate its influence on tissue regeneration is necessary due to low methodological quality of the animal studies.

Objective: To evaluate the outcome of post space disinfection using Er,Cr:YSGG laser (ECYL), riboflavin photosensitizer (RFP), and photon-induced photoacoustic streaming (PIPS) along with ethylene diamine tetra acetic acid (EDTA) as final irrigant on the bond values of glass fiber post (GFP) to the canal wall. Materials and methods: Forty human mandibular premolars were subjected to root canal treatment after decoronation till the cementoenamel junction. Post space was prepared using a Gates-Glidden drill leaving 4 mm of gutta perch at the apical third of the root section. Random allocation of samples was performed into four groups based on the post space disinfection (n = 10 each): Group 1: 1% RFP and 17% EDTA, group 2: 5.25% sodium hypochlorite (NaOCl) and 17% EDTA (control), group 3: PIPS and 17% EDTA, and group 4: ECYL and 17% EDTA. GFP was luted using Rely X Unicem. A universal testing machine and stereomicroscope were used to assess the push-out bond strength (PBS) and failure mode, respectively. Analysis of variance determined the PBS amid different experimental groups at three root levels. Multiple group comparison using means of tested groups was identified using post hoc Tukey. Results: The coronal third of group 2 (5.25% NaOCl +17% EDTA) demonstrated the highest PBS (8.83 ± 0.11 MPa). However, the lowest bond integrity (4.15 ± 0.52 MPa) was displayed in an apical third of group 1 (RFP +17% EDTA). The intergroup comparison revealed that 5.25% NaOCl +17% EDTA at all three levels demonstrated comparable outcomes of PBS with PIPS +17% EDTA and ECYL +17% EDTA specimens (p > 0.05). However, riboflavin +17% EDTA demonstrated the lowest bond strength (p < 0.05). Conclusions: Er,Cr: YSGG and PIPS in combination with 17% EDTA demonstrated comparable outcomes of bond score with those of conventional gold standard irrigation regime 5.25% NaOCl.

Periodontal regeneration has been a relentless goal of the periodontist. Perhaps the oldest and most frequently attempted type of regeneration has involved chemical modification of the root surface. Varying results from histological and clinical studies have created controversy about the clinical effectiveness of root surface decalcification. This systematic review assesses the efficacy of root surface biomodification through the use of citric acid, tetracycline, or ethylenediaminetetraacetic acid (EDTA) in patients with chronic periodontitis. Does the use of chemical root declacification result in effective periodontal regeneration and improved clinical outcomes in patients with chronic periodontitis? The Cochrane Database of Systematic Reviews; Database of Abstracts of Reviews of Effectiveness; Cochrane Central Register of Controlled Trials; American College of Physicians Journal Club, evidence-based MEDLINE journals; and National Center for Biotechnology Information PubMed Journals, as well as Dogpile, Google, and Copernic search engines were screened. Hand searches were performed on the Journal of Clinical Periodontology, Journal of Periodontology, Journal of Periodontal Research, and Periodontology 2000. Searches were performed for relevant clinical trials published through September 25, 2002. Histological and clinical studies evaluating the effects of citric acid, tetracycline, or EDTA on root surfaces of patients with chronic periodontitis were considered for inclusion. Studies evaluating extracellular matrix proteins (e.g., fibronectin), enamel matrix proteins (e.g., amelogenins), or other proteins or growth factors applied to the root surface were not included. Primary outcome measures included changes in connective tissue attachment, cementogenesis, clinical attachment levels, probing depths, and gingival recession. Secondary outcome measures included changes in bone level, gingival inflammation, and plaque levels. Results for continuous outcome measures for primary variables (clinical attachment levels, probing depths, and recession) were expressed as mean differences or standardized mean differences. Clinical attachment levels and reduction in probing depth were evaluated using meta-analysis. All papers were rated according to methodological strength of evidence. 1. Thirty-four studies incorporating a total patient population of 575 were analyzed: 26 for citric acid, 5 for tetracycline, and 3 for EDTA treatment. 2. Four of 8 human histological studies reported regeneration with the use of citric acid. Only 1 of 18 clinical studies reported attachment gain. 3. Of the 5 studies examined using tetracycline, 1 histological study and 1 clinical study reported attachment gain. 4. No regeneration was reported in the 3 studies evaluating the use of EDTA. 5. Meta-analysis performed on 28 clinical trials did not show any significant effects of acid root treatment on attachment level gains or probing depth. 1. Evidence to date suggests that the use of citric acid, tetracycline, or EDTA to modify the root surface provides no benefit of clinical significance to regeneration in patients with chronic periodontitis. 2. The best method for ascertaining the clinical efficacy of acid-treated root regeneration would be to conduct a randomized clinical trial with sufficient statistical power that is supported by quantitative histological evaluation. 3. The majority of the studies that evaluated the regenerative potential of root surface modifiers were observational in nature; therefore, the value of conclusions reached in this manuscript must be carefully considered.

Could calcium hypochlorite and grape seed extract keep the mechanical properties of root dentin and fracture resistance of weakened roots?

Recent studies have shown that bone morphogenetic protein-2 (BMP-2) stimulates mineralization and osteoclast differentiation. Osteoclastic resorption by BMP-2 application may play an important role in the regulation of new cementum-like tissue formation on the dentin surfaces. Therefore, this study aimed to examine the effect of BMP-2 application on dentin resorption and cementum-like tissue formation at the dentin surfaces. Seventy-two flat dentin blocks were prepared from rat roots and treated with 24% EDTA. Each block was assigned to group 0, group 100, or group 400, and immersed correspondingly in 0, 100, or 400 microg/ml BMP-2. The dentin blocks were then implanted into palatal connective tissue of rats, and specimens were prepared 2, 4 and 8 wk after surgery for histologic and histomorphometric analyses. BMP-2 caused a dose-dependent increase in dentin resorption by osteoclastic cells. New cementum-like tissue was randomly formed on parts of the nonresorbed and resorbed dentin surfaces in groups 100 and 400. Dentin resorption in groups 100 and 400 was significantly greater than group 0 (p < 0.01). However, at 8 wk, new cementum-like tissue formed in 41.8% of group 100, as compared with 16.2% of group 400 (p < 0.05). Dentin resorption was stimulated by a high dose of BMP-2, and cementum-like tissue was induced by a low dose of BMP-2, effectively suggesting that BMP-2 application, at an appropriate dose, to a dentin surface may enhance periodontal regeneration.

AIM: To assess the combined use of tetracycline (TTC) and ethylenediaminetetraacetic acid (EDTA) on clot formation, considering that EDTA may neutralize TTC acidity. METHODS: Planed human tooth roots were treated with saline solution, EDTA, TTC and their combination (EDTA followed by TTC and TTC before EDTA). Fresh human blood was applied on the conditioned surfaces to check clot adhesion and stabilization. A previously calibrated (kappa = 0.93) and blinded examiner scored scanning electron micrographs of the samples. Statistical analyses were performed using one-way ANOVA and Tukey's test. RESULTS: Application of TTC before EDTA presented the best results with the highest number of cells adhered to the root surface (p=0.046). Use of EDTA alone and EDTA before TTC disturbed clot stabilization when compared to control group (p<0.01). CONCLUSIONS: The use of TTC before EDTA seems to be able to keep blood cells viable to establish an organized clot and could be used by clinicians together with the conventional mechanical root scaling and planing.