The objective of the present in vitro study was to evaluate the influence of endodontic irrigation systems on the removal of smear layer and ion diffusion of calcium hydroxide through dentinal tubules in root external resorption models.
Materials and methods
Forty single-rooted teeth were divided into four groups (G1, G2, G3, and G4) according to the irrigation regimens: G1, saline solution; G2, 10% sodium hypochlorite with ultrasonic irrigation; G3, 14% EDTA with ultrasonic irrigation; G4, 14% EDTA with ultrasonic irrigation → 10% sodium hypochlorite with ultrasonic irrigation. Ultrasonic irrigation lasted 45 seconds and all the roots were irrigated with 2 mL of saline solution. In experiment 1, the samples were observed on the root canal wall using scanning electron microscope and the percentage of opened dentinal tubules (POD) was calculated. Experiment 2 evaluated the diffusion of calcium ions through dentinal tubules.
In the middle region of root canals, the mean POD values were 0.11, 5.02, 82.17 and 96.72 in G1, G2, G3, and G4, respectively. In the apical region of root canals, the mean POD values were 0.06, 0.43, 4.41, and 12.70 in G1, G2, G3, and G4, respectively. The diffusion of calcium ions in G4 was significantly high in all groups.
This study demonstrated that a combination of EDTA and sodium hypochlorite with ultrasonic irrigation was most effective in removing the smear layer and ion diffusion from the root canal to the surrounding media.
In daily clinical practice, we often see patients with oral injury. Oral injury is characterized by such symptoms as crown fracture, root fracture, damaged periodontal tissue, tooth luxation, and mandibular alveolar fracture. In the case of poor prognosis, it causes root resorption. Various studies on treatment for external root resorption reported the effects of calcium hydroxide (CH): induction of hard tissue formation , reduction of inflammatory cells , and pH changes . Mineral trioxide aggregate (MTA) forms calcium hydrate with water. The calcium hydrate from MTA prevents root resorption by increasing calcium ion concentration through the dentinal tubules . The amount of calcium ions in the dentinal tubules is important to prevent external root resorption . The amount of calcium ions in the dentinal tubules is affected by the root canal treatment materials and the percentage of opened dentinal tubules. Additionally, the root canal enlargement may obstruct the dentinal tubules by making a smear layer on the root canal wall . Removing the smear layer effectively by chemical irrigation invites calcium ion diffusion on the external surface of the root. It aids successful the root canal treatment. Previous studies examined various root canal irrigation systems: EDTA , EDTA and sodium hypochlorite , phosphoric acid , and EDTA and citric acid . Recently, the use of sodium hypochlorite as the main endodontic irrigant was strongly recommended . However, many opinions exist on effective irrigation systems: irrigant solution , vehicle , and using ultrasonic activation . Various studies also examined ion diffusion of calcium hydrate in dentinal tubules ; however, the method of measuring calcium ions is inconsistent. The objective of the present in vitro study was to evaluate the influence of endodontic irrigation systems associated with the removal of the smear layer and ion diffusion of CH through the dentinal tubules in root external resorption models.
Materials and methods
This study was approved by the Research Ethics Committee of Hokkaido University Graduate School of Dental Medicine (Number 2012-1).
Forty human permanent teeth with a single root and single root canal were used in this study. They had been extracted for severe periodontal disease or trauma of the teeth at Hokkaido University Hospital. Teeth were stored in saline solution at 4 °C before the experiment.
Endodontic preparation of root canals
The crowns were cut off at cement-enamel junction using a diamond bur (smooth cut AR2, GC, Tokyo, Japan) attached to a turbine-powered hand-piece with water. The apical foramen of each root was enlarged with a no. 15 size K-file (Dentsply Maillefer, Baillnages, Switzerland), and the working length was determined 1 mm from the apex. The root canals were enlarged sequentially using K-files from #15 to #80, and rinsed with 2.5 mL saline during each enlargement.
Root canal irrigation
The root canal was irrigated with different substances through a 23-gauge needle attached to a syringe (NIPRO, Osaka, Japan). The irrigants used in this experiment were 10% sodium hypochlorite (Neo Dental Chemical Products, Tokyo, Japan), 14% EDTA (Showa Yakuhin Kako, Tokyo, Japan), and saline solution (Otsuka Pharmaceutical, Tokyo, Japan). The specimens were divided into four groups subjected to the following irrigation regimens: G1, saline solution; G2, 10% sodium hypochlorite with ultrasonic irrigation; G3, 14% EDTA with ultrasonic irrigation; G4, 14% EDTA with ultrasonic irrigation→10% sodium hypochlorite with ultrasonic irrigation ( Fig. 1 ). Ultrasonic irrigation was conducted using an endodontic tip (ST49A-0.8tip; Osada, Tokyo, Japan, U-file size #15; Mokuda Dental, Hyogo, Japan) attached to an ultrasonic generator (ENAC10W; Osada, Tokyo, Japan) for 15 s, during which the tip was located 2 mm above the apical foramen. These procedures were repeated three times; the total volume of the irrigant was 0.5 mL. The saline solution of all groups was also used for the final irrigation.
Observation of root canal walls
For experiment 1, twenty teeth were divided into four groups (N = 5) according to the root canal irrigation methods (G1-G4). After root canal irrigation, they were cut parallel to the tooth axis and fixed in glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.3) for 24 h. The specimens were then dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, 95%, 100%), dried in the critical point, and the surfaces were sputter coated with Pt-Pd. The irrigated root canal walls of specimens were observed by scanning electron microscopy (SEM; Hitachi S-4000, Hitachi, Tokyo, Japan) at an accelerated voltage of 10 kV. SEM photographs were taken at 12 sites in the middle region and six sites in the apical region per tooth. The sites were randomly selected so that each site was not repeated. The middle and apical regions of the canals were scanned to evaluate the amount of smear layer. The percentage of opened dentinal tubules (POD) at the middle and apical regions of canals was calculated by using an SEM photograph taken at x1,500 magnifications. Each photograph measured 120 mm × 150 mm. POD was defined as follows:
Evaluation of the calcium ion diffusion
For experiment 2, we evaluated the diffusion of calcium ions through dentinal tubules. Twenty teeth were divided into four groups according to root canal irrigation (N = 5). After root canal irrigation, they were filled with CH paste (CH powder, Kanto Chemical Co., Inc., Tokyo, Japan; saline solution = 1:1). The root canal orifice and foramen of teeth were blocked by quick self-curing resin (Unifast II, GC, Tokyo, Japan). The external root resorption (2 mm × 3 mm × 1 mm, Fig. 2 ) was then simulated by creating two resorptive defects on the outer root surface using a 1.4-mm diamond round bur (smooth cut D4014, GC, Tokyo, Japan) with water. The surfaces of resorptive defects were irrigated by 14% EDTA and 10% sodium hypochlorite with ultrasonic irrigation to remove the smear layer. After that, the root surface was covered with nail varnish except for external defects. The specimens were stored in deionized water at 37 °C. The concentration of calcium ions in the water were then measured with ICP emission spectrometer (ICP-AES, P-4010, Hitachi, Tokyo, Japan) at 1, 2, 3, and 4 weeks after.
Statistical analyses were conducted using SPSS software (IBM, Tokyo, Japan). The POD values were statistically analyzed using one-way factorial analysis of variance (ANOVA) and Tukey’s test. The concentrations of the calcium were compared using two-way repeated measures ANOVA at a 5% significance level.
Observation of root canal wall
Middle region of root canals
In G1, the dentinal tubule was not visible because it was covered by the smear layer ( Fig. 3 a). In G2, some dentinal tubules were visible, but almost all were covered by the smear layer ( Fig. 3 b). In G3, almost all dentinal tubules were open, though some were not clearly so ( Fig. 3 c). In G4, almost all of the dentinal tubules were clearly open. The mean POD values were 0.11, 5.02, 82.17 and 96.72 in G1, G2, G3 and G4, respectively ( Table 1 ). Significant differences were observed in all groups (P < .05).