Effect of EDTA Activation on Blood Clot Structure in Regenerative Endodontics: A Scanning Electron Microscopy Study.

INTRODUCTION: EDTA plays a crucial role in regenerative endodontic therapy (RET) because of its significant biological effects. However, EDTA is also recognized as the preferred anticoagulant for hematologic tests. Thus, this study aimed to assess the influence of different EDTA activation techniques on the morphology of blood clots after conditioning the root canal dentin. METHODS: Forty extracted human teeth were prepared to simulate immature teeth and divided into the following 5 groups: (1) saline solution (negative control), (2) EDTA 17% + saline solution (CNI), (3) CNI + ultrasonic activation, (4) CNI + Easy clean activation, and (5) CNI + XP-endo Finisher activation. After irrigation, the roots were cleaved, and the root canals were filled with human blood to clot formation. The morphology and density of erythrocytes, platelets, and the fibrin network were observed using a scanning electron microscope. The fibrin network density was classified using a 4-point scale. Data were analyzed using the Friedman test and the Kruskal-Wallis test with Bonferroni adjustment (α = 5%). RESULTS: All groups exhibited consistent blood clot morphology characterized by a high density of erythrocytes, platelets, and white blood cells throughout the entire length of the root canal. The negative control group showed statistically significant high scores of fibrin density compared with the CNI group in all root thirds (P < .05). However, there was no statistical difference in the scores for the fibrin network density between the groups irrigated with EDTA with and without activation (P > .05). CONCLUSIONS: EDTA may impair the fibrin network formation compared with the saline group. However, EDTA activation did not significantly change the effects on the blood clot in contact with the conditioned intraradicular dentin.

Stem cells of the apical papilla regulate trigeminal neurite outgrowth and targeting through a BDNF-dependent mechanism.

Regenerative endodontic procedures have become a valuable alternative for the treatment of immature teeth with pulp necrosis. In addition to resolution of periradicular pathosis and promotion of continued root development, positive vitality testing has been observed in some regenerative clinical cases. Importantly, the positive response to electric stimulation of the regenerated tissue requires targeting of periradicular axons into the previously empty root canal space. However, the mechanism by which this process occurs is largely unknown. Since stem cells of the apical papilla (SCAP) have been proposed to populate the root canal following regenerative endodontic procedures, we hypothesized that SCAP regulate neurite outgrowth and axonal targeting. To test this hypothesis, we established primary co-cultures of human SCAP and rat trigeminal neurons, and performed neurite outgrowth assays using ELISA and confocal microscopy to determine the effect of increasing concentration of SCAP on the total neurite outgrowth and axonal targeting. In addition, we evaluated whether SCAP evoked axonal targeting in vivo using a matrigel subcutaneous implant assay. Data were analyzed by ANOVA with Bonferroni's post hoc test, and significance was set at p<0.05. The results demonstrated that SCAP release a soluble factor that regulates neurite outgrowth from cultured trigeminal neurons. Next, we demonstrated that this effect is completely abolished by pretreatment with a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by antibodies to other neurotrophins. Further, SCAP release BDNF in a concentration-dependent manner as detected by ELISA, and trigger directed axonal targeting both in vitro and in vivo as demonstrated by microfluidic and matrigel implant experiments, respectively. Collectively, these results suggest that SCAP may be responsible for the chemical signal driving axons to target regenerated tissue via a BDNF-dependent mechanism.

Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation.

INTRODUCTION: Intracanal disinfection is a crucial step in regenerative endodontic procedures. Most published cases suggest the use of sodium hypochlorite (NaOCl) as the primary irrigant. However, the effect of clinically used concentrations of NaOCl on the survival and differentiation of stem cells is largely unknown. In this study, we tested the effect of various concentrations of NaOCl on the stem cells of the apical papilla (SCAPs) survival and dentin sialophosphoprotein (DSPP) expression. METHODS: Standardized root canals were created in extracted human teeth and irrigated with NaOCl (0.5%, 1.5%, 3%, or 6%) followed by 17% EDTA or sterile saline. SCAPs in a hyaluronic acid-based scaffold were seeded into the canals and cultured for 7 days. Next, viable cells were quantified using a luminescence assay, and DSPP expression was evaluated using quantitative real-time polymerase chain reaction. RESULTS: There was a significant reduction in survival and DSPP expression in the group treated with 6% NaOCl compared with the untreated control group. Comparable survival was observed in the groups treated with the lower concentrations of NaOCl, but greater DSPP expression was observed in the 1.5% NaOCl group. In addition, 17% EDTA resulted in increased survival and DSPP expression partially reversing the deleterious effects of NaOCl. CONCLUSIONS: Collectively, the results suggest that dentin conditioning with high concentrations of NaOCl has a profound negative effect on the survival and differentiation of SCAPs. However, this effect can be prevented with the use of 1.5% NaOCl followed by 17% EDTA. The inclusion of this irrigation regimen might be beneficial in regenerative endodontic procedures.