A comparison between two negative pressure irrigation techniques in simulated immature tooth: an ex vivo study.

OBJECTIVES: This ex vivo study evaluated the irrigation efficacy of a new apical negative pressure system (ANP) in canals with simulated immature teeth, by comparing it to EndoVac (EV) system in terms of smear layer (SL) removal and irrigation extrusion. MATERIALS AND METHODS: Three millimetres of the root end of 40 single canalled lower incisors were resected and decoronated to standardize root canal length. After instrumentation, the specimens were embedded in warm normal saline agar coloured with 1 % acid red and randomly divided into four groups; one control group and three experimental groups. Except in the control group where distilled water was used as irrigant using positive pressure irrigation needle, the canals were irrigated with 6 % NaOCl and 17 % EDTA using the intracanal negative pressure needle (iNP) system, the EV system or 27G open-ended needle under positive pressure (PP). NaOCl extrusion was determined by observing a discolouration of the agar surrounding the root. The SL was evaluated by observing scanning electron microscope images based on a four-level scoring system. RESULTS: Two specimens with irrigant extrusion were observed in the iNP group, which was significantly different (logistic regression, p < 0.05) to EV and PP. There were no significant differences (Kruskall-Wallis test, p > 0.05) among the experimental groups in terms of SL removal, but all were significantly different to the control group. CONCLUSIONS: Irrigation with the iNP could be a viable alternative to EV as an apical negative pressure irrigation technique especially while treating immature teeth. CLINICAL RELEVANCE: ANP in canal cleanliness is recommended to be utilized in treating immature teeth where periapical tissues should be saved and stimulated. The iNP system might have the potential to avoid irrigant extrusion while cleaning the canal till the apical end.

Performance of NiTi endodontic instrument under different temperatures.

The purpose of this study was to test nickel titanium (NiTi) instrument performance under different surrounding temperatures. Twenty-four superelastic NiTi instruments with a conical shape comprising a 0.30-mm-diameter tip and 0.06 taper were equally divided into 3 groups according to the temperature employed. Using a specially designed cyclic fatigue testing apparatus, each instrument was deflected to give a curvature 10 mm in radius and a 30° angle. This position was kept as the instrument was immersed in a continuous flow of water under a temperature of 10, 37, or 50 °C for 20 s to calculate the deflecting load (DL). In the same position, the instrument was then allowed to rotate at 300 rpm to fracture, and the working time was converted to the number of cycles to fracture (NCF). The statistical significance was set at p = 0.05. The mean DL (in N) and NCF (in cycles) of the groups at 10, 37, and 50 °C were 10.16 ± 1.36 and 135.50 ± 31.48, 13.50 ± 0.92 and 89.20 ± 16.44, and 14.70 ± 1.21 and 65.50 ± 15.90, respectively. The group at 10 °C had significantly the lowest DL that favorably resulted in the highest NCF. Within the limitations of this study, the surrounding temperature influences the cyclic fatigue resistance and DL of the superelastic NiTi instruments. Lower temperatures are found to favorably decrease the DL and extend the lifetime of the superelastic NiTi instrument. Further NiTi instrument failure studies should be performed under simulated body temperature.

Periostin is a negative regulator of mineralization in the dental pulp tissue.

The dental pulp tissue is encased in hard tissue and surrounded by hard tissue-forming cells, but remains in a non-mineralized state itself, suggesting the presence of regulatory mechanisms precluding pulp mineralization. This study aimed to reveal the regulatory function of periostin (Postn), which is essential for osteoblast differentiation, for odontoblast differentiation/mineralization. We evaluated the effects of Postn overexpression and RNAi-mediated suppression in mouse dental papilla cells (MDPs) on the expression of odontoblastic markers and Notch signaling molecules, and on the formation of mineralized nodules. Localization of Postn in the dental pulp tissue of normal and cavity-prepared molars was observed immunohistologically. Enforced overexpression of Postn in MDPs induced down-regulation of odontoblastic markers and in vitro mineralization. Conversely, silencing of Postn mRNA in MDPs induced up-regulation of odontoblastic markers and ALP activity. Up- and down-regulation of Postn caused increased and decreased expression, respectively, of Notch signaling molecules. Postn expression was minimal in normal dental pulp, but was rapidly and globally increased in the whole pulp tissue of molar teeth at 1 day after cavity preparation, decreasing thereafter. These results indicate that Postn may be a negative regulator of odontoblast differentiation/mineralization, and that may exert its actions via Notch signals.

Joint haemorrhage partly accelerated immobilization-induced synovial adhesions and capsular shortening in rats.

PURPOSE: To elucidate the effects of intra-articular haemorrhage on the joint capsule of immobilized knees in rats. METHODS: The unilateral knee joints were immobilized using a plastic plate and screws. Sham operated rats had only screws inserted. A single injection of fresh autologous blood was given postoperatively into the knee joints of the immobilized blood injection (Im-B) and the Sham blood injection (Sm-B) groups. Normal saline was administered for the immobilized-saline injection (Im-S) group. Sagittal sections were prepared from the medial midcondylar region of the knee and assessed with histological, histomorphometric, and immunohistochemical methods. The range of motion (ROM) was measured, and the mechanical property of the capsule was assessed by scanning acoustic microscope. RESULTS: Absorption of the injected blood was delayed and made severe adhesions in the Im-B group. The length of the synovial membrane in the Im-B group was significantly shorter than that of the other groups. The ROM was significantly restricted in the Im-B group compared with the other groups. The elasticity of the posterior capsule in the Im-B group was significantly lower than that in the Sm-B group. Iron deposition was observed in the Im-B and Sm-B groups. Strong immunoreactivities of CD68, TGF-ß1, and α-SMA were observed in the adhesion area of the Im-B group. Joint immobilization with blood injection caused severe capsular adhesion and limited range of motion. Immunostaining related to fibrosis increased with joint haemorrhage. CONCLUSION: Intra-articular haemorrhage with joint immobilization might be an accelerated risk factor for joint contracture. It is likely that leaving a haematoma inside an immobilized joint should be avoided.

Joint immobilization induced hypoxic and inflammatory conditions in rat knee joints.

The purpose of this study was to examine the hypoxic and inflammatory conditions after immobilization in the joint capsule of rat knees. The unilateral knee joints of adult male rats were immobilized with an internal fixator (Im group) for 1 day, 3 days, and 1, 2, 4, 8, and 16 weeks. Sham-operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). The number of cells and blood vessels in the capsule were histologically examined. The hypoxic condition in the capsule was histologically examined with a Hypoxyprobe™-1. The gene expressions related to the hypoxic (hypoxia inducible factor-1α, vascular endothelial growth factor, and fibroblast growth factor 2) and inflammatory conditions [interleukin-6 (IL-6), IL-1α, IL-1ß, tumor necrosis factor-α, and tumor necrosis factor-ß] were evaluated by quantitative reverse transcription polymerase chain reaction. The number of cells was unchanged at 1 day in the two groups; however, the number significantly increased at 3 days in the Im group. The number of blood vessels in the Im group gradually decreased. Strong immunostaining of Hypoxyprobe™-1 around the blood vessels was observed in the Im group. The gene expressions of hypoxia inducible factor-1α and fibroblast growth factor 2 were significantly higher in the Im group compared with those in the control group. The gene expressions of IL-6, IL-1α, IL-1ß, and tumor necrosis factor-ß were significantly higher in the Im group compared with those in the control group. These data indicated that joint immobilization induced hypoxic and inflammatory conditions in the joint capsule, which might be an initiating factor for joint contracture.

Remobilization does not restore immobilization-induced adhesion of capsule and restricted joint motion in rat knee joints.

Joint immobilization, which is used in orthopaedic treatments and observed in bedridden people, usually causes restricted joint motion. Decreased joint motion diminishes activities of daily living and increases burden of nursing-care. The purpose of this study was to clarify the reversibility of immobilization-induced capsular changes and restricted joint motion in rat knee joints. The unilateral knee joints of adult male rats were immobilized with an internal fixator for 1, 2, 4, 8, and 16 weeks as a model of immobilization after surgery or disuse of the joint. After the fixation devices were removed, the rats were allowed to move freely for 16 weeks. Sham-operated rats were used as controls. Sagittal sections at medial midcondylar regions were made and assessed with histological, histomorphometric, and immunohistochemical methods. Joint motion was measured using a custom-made device under x-ray control after removal of the periarticular muscles. In the 1/16-week and 2/16-week immobilization-remobilization (Im-Rm) groups, cord-like structures connecting the superior and inferior portions of the posterior capsule (partial adhesion) were observed without restricted joint motion. In the 4/16-, 8/16-, and 16/16-week Im-Rm groups, global adhesion of the posterior capsule and restricted joint motion were observed. The restricted joint motion was not completely restored after incision of the posterior capsule. These data indicate that immobilization alone causes irreversible capsular changes and arthrogenic restricted joint motion. Besides the joint capsule, other arthrogenic factors such as ligaments might influence the restricted joint motion. Prolonged immobilization over 4 weeks should be avoided to prevent irreversible joint contracture.

Comparison of articular cartilage images assessed by high-frequency ultrasound microscope and scanning acoustic microscope.

PURPOSE: The purpose of this study was to compare images of a newly developed high-frequency ultrasound imaging system (HFUIS) and scanning acoustic microscope (SAM) and to calculate their Pearson product moment correlations with a view to applying HFUIS for clinical use. METHODS: Cylindrical cartilage-bone complexes from adult male Sprague-Dawley rats were obtained. The specimens were immersed in normal saline and scanned by HFUIS. Intensity by HFUIS was normalised by reflection from a steel plate at the same distance. After the scanning, specimens were fixed with paraformaldehyde, decalcified and embedded in paraffin. Thinly sliced tissues were prepared for SAM evaluation. After the scanning, three layers of articular cartilage (superficial, middle and deep) were independently evaluated and their relationships calculated. RESULTS: The superficial and deep layers indicated high relative intensity, whereas the middle layer showed nonhomogeneous relative intensity by HFUIS. A high relative intensity by HFUIS and high sound speed area by SAM had strong correlations (Pearson product moment correlation, superficial layer 0.704, middle layer 0.731). CONCLUSIONS: HFUIS produced high-resolution images of the articular cartilage and its intensity was strongly correlated with sound speed by SAM.

Reversibility of immobilization-induced articular cartilage degeneration after remobilization in rat knee joints.

Joint immobilization is commonly used for the treatment of joint injuries, but it causes articular cartilage degeneration. The purpose of this study was to clarify the reversibility of immobilization-induced articular cartilage degeneration using rat knee joints. Immobilization of rat knee joints induces atrophic changes in the non-contact area, loss of chondrocytes in the contact area, and hypertrophy of chondrocytes in the transitional area of the articular cartilage. The unilateral knee joints of adult male rats were rigidly immobilized at 150° of flexion with a plate and screws for 1, 2, and 4 weeks. After the experimental periods, the fixation devices were removed and the rats moved freely for 16 weeks. Sham-operated rats were used as a control. Sagittal sections at medial midcondylar regions of the knee were assessed with histological and histomorphometric methods. Mechanical properties were assessed by measuring the sound speed with scanning acoustic microscope. Reduction of cartilage proteoglycan in the non-contact area was almost reversible, but hypertrophy of chondrocytes in the transitional area and loss of chondrocytes in the contact area were irreversible even at 1-week immobilization-remobilization group. Sound speed of the articular cartilage in the contact area was not restored. These results indicate that atrophic changes through decreased mechanical stress in the non-contact area were reversible, but loss of chondrocytes and hypertrophy of chondrocytes in the contact and transitional areas through increased mechanical stress by rigid immobilization were irreversible after remobilization. Clinicians should be aware that even a short-term rigid immobilization could cause irreversible articular cartilage damage.

Clinical management of dens invaginatus in a maxillary lateral incisor with the aid of cone-beam computed tomography--a case report.

This report describes non-surgical endodontic treatment of Oehlers' type III dens invaginatus in a maxillary lateral incisor with the aid of postobturation cone-beam computed tomography (CBCT). The endodontic treatment was initiated with the aid of a surgical operating microscope, and two canals, one of which represented the invagination, were instrumented, irrigated under passive ultrasonic activation and obturated with the lateral condensation technique. As postobturation periapical radiographs suggested the presence of untereated and/or unfilled areas in the root canal and invagination, CBCT was taken to assess the possibility of further treatment. The CBCT scans demonstrated inaccessible and unfilled canal and invagination areas because of complex internal morphology characterized by (i) C- or ring-shaped cross-sectional canal configuration with constrictions at different points in different root levels and (ii) a prominent intraradicular cavity that was communicated with the enamel-lined invagination and opened into the apical periodontium. Thus, it was judged that further endodontic treatment was not feasible. A 14-month follow-up revealed a satisfactory clinical and radiographic outcome, suggesting that the chemomechanical debridement may have sufficed to induce periapical healing. CBCT greatly helped the decision of avoiding further intervention that could have been difficult to negotiate.

Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj.

Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

Differentiation of odontoblasts is negatively regulated by MEPE via its C-terminal fragment.

Matrix extracellular phosphoglycoprotein (MEPE) is an extracellular matrix protein that is mainly expressed in mineralizing tissues, including the dental pulp. The purposes of this study were to clarify the localization of MEPE in the tooth germ and to investigate the roles of MEPE in the differentiation of odontoblasts. The immunohistochemical staining in the tooth germ of the upper first molars of male Wistar rats (postnatal day 3) revealed that MEPE was mainly localized in odontoblasts during dentinogenesis. Stable MEPE-overexpressing and MEPE-knockdown cell lines, which were established in odontoblast-lineage cells (OLCs), showed lower and higher differentiation capabilities, respectively. Eukaryotic proteins of the N-terminal fragment of MEPE produced in HEK cells had no effect on the differentiation of OLCs, whereas the C-terminal fragment containing an RGD sequence inhibited their differentiation. These results indicated that the C-terminal fragment of MEPE containing an RGD sequence, cleaved in odontoblasts, appeared to be the active form of MEPE, which may play important roles in dentinogenesis and pulpal homeostasis by keeping the odontoblasts in immature condition.

Expression of collagen types I and II on articular cartilage in a rat knee contracture model.

The purpose of our study was to clarify the expression patterns of collagen types I and II on articular cartilage after immobilization in a rat knee contracture model in 3 specific areas (noncontact area, transitional area, contact area). The unilateral knee joints of adult male rats were rigidly immobilized at 150 degrees of flexion using screws and a rigid plastic plate. Sham-operated animals had holes drilled in the femur and the tibia and screws inserted but were not plated. The expression patterns of collagen types I and II in each area were evaluated by in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative real-time polymerase chain reaction (qPCR). The expression of collagen type II in the noncontact area was decreased by ISH but appeared unchanged when examined by IHC. In the transitional and contact areas, the expression of collagen type II was initially shown to have decreased and then increased at the hypertrophic chondrocytes by ISH but appeared decreased by IHC. Quantitative PCR revealed the decreased expression of type II collagen in the contact area. Immunostaining of collagen type I was increased at the noncontact area and transitional areas. Alterations of collagen types I and II expression may also affect the degeneration of articular cartilage after immobilization and the changes were different in the three areas.

Distribution of type A and B synoviocytes in the adhesive and shortened synovial membrane during immobilization of the knee joint in rats.

Joint immobilization is commonly used for the treatment of joint injuries and diseases, but it also causes unfavorable outcomes such as joint contracture. The purpose of this study was to examine the morphological changes of the synovial membrane that is suspected as a cause of joint contracture, and localization of type A (macrophage-like) and type B (fibroblast-like) synoviocytes in the capsule after joint immobilization. Male Sprague-Dawley rats were used in this study. Unilateral knee joints were rigidly immobilized at 150 degrees of flexion with internal fixators for 3 days, 1, 2, 4, 8, and 16 weeks (7 rats/each immobilized group), while 42 rats were sham-operated. Sagittal sections of 5 mum were prepared from the medial midcondylar region of the knee joints and assessed with histological, histomorphometric, and immunohistochemical methods. Adhesions were observed both in the anterior and posterior synovial membranes in the immobilized group after 2 weeks. In the adhesion area, the cells were mainly composed of type A synoviocytes that were positive for CD68 and type B synoviocytes positive for prolyl 4-hydroxylase subunit beta. The length of synovial membrane in the immobilized group was significantly shorter than that in the control group after 2 and 4 weeks. After 8 weeks, the adhesion area in the immobilized group became fibrous and hypocellular. The staining intensity of hyaluronic acid-binding protein was increased after 16 weeks. Adhesion and shortening of the synovial membrane and the structural changes of the adhesion area may contribute to the development of joint contracture.

Increased expression of metalloproteinase-8 and -13 on articular cartilage in a rat immobilized knee model.

Joint immobilization is commonly used for the treatment of joint injuries and diseases, but it also causes cartilage degeneration. Damage to the fibrillar meshwork of type II collagen in the articular cartilage is a critical event for cartilage degeneration. Collagenases such as matrix metalloproteinase (MMP)-8 and MMP-13 have been considered the main enzymes responsible for the degradation of type II collagen. However, the mechanism of the articular cartilage degeneration after immobilization has not been revealed. The purpose of this study was to examine changes of the expression patterns of MMP-8 and MMP-13 after rigid immobilization of the knee joint. The unilateral knee joints of adult male rats were rigidly immobilized at 150 degrees of flexion using an internal fixator. Histological sections from the medial midcondylar region of the knee were obtained and evaluated in 3 specific areas (non-contact, transitional, and contact areas). The expression of MMP-8 and MMP-13 was evaluated by in situ hybridization. Total RNA was extracted from the articular cartilage in the contact area, and expression levels of MMP-8 and MMP-13 mRNAs were measured by quantitative real-time polymerase chain reaction. Localization of MMP-13 expression was also examined by immunohistochemistry. The expression of MMP-8 mRNA was decreased by 1 week after immobilization. After 4-week immobilization, hypertrophic differentiated chondrocytes were observed in the transitional and contact areas, and the expression of MMP-8 and MMP-13 mRNAs was increased in the chondrocytes. Rigid immobilization is associated with the increased expression of MMP-8 and MMP-13 in the hypertrophic differentiated chondrocytes. These two collagenases may play an important role in the articular cartilage degeneration after joint immobilization.

Root canal medicaments.

The ultimate goals of endodontic treatment are complete removal of bacteria, their byproducts and pulpal remnants from infected root canals and the complete seal of disinfected root canals. Intracanal medicaments have been thought an essential step in killing the bacteria in root canals; however, in modern endodontics, shaping and cleaning may be assuming greater importance than intracanal medicaments as a means of disinfecting root canals. Until recently, formocresol and its relatives were frequently used as intracanal medicaments, but it was pointed out that such bactericidal chemicals dressed in the canal distributed to the whole body from the root apex and so might induce various harmful effects including allergies. Furthermore, as these medicaments are potent carcinogenic agents, there is no indication for these chemicals in modern endodontic treatment. Today, biocompatibility and stability are essential properties for intracanal medicaments. The more modern meaning of intracanal dressing is for a blockade against coronal leakage from the gap between filling materials and cavity wall. Calcium hydroxide has been determined as suitable for use as an intracanal medicament as it is stable for long periods, harmless to the body, and bactericidal in a limited area. It also induces hard tissue formation and is effective for stopping inflammatory exudates. Single-visit endodontics, where intracanal medicaments are not used, is generally not now contraindicated and various reports have shown that the clinical outcomes between single- and multiple- visit endodontics are similar. There is no reason to counsel against single-visit endodontics: however, if multiple-visit endodontics is chosen, calcium hydroxide is recommended to be used as an intracanal medicament.

Analysis of factors related to needle-stick and sharps injuries at a dental specialty university hospital and possible prevention methods.

Needle-stick and sharps injuries (NSIs) can happen even when dental health care workers (DHCWs) act in compliance with standard precautions to prevent transmitting blood-borne infections. The objective of this study was to investigate causes of NSIs that had occurred at a dental specialty university hospital during the past 12 academic years. A total of 215 NSIs were reported during the investigation period, and NSIs ascribed to female DHCWs (n = 148; 68.8%) were significantly (P < 0.05) more common than those ascribed to male DHCWs (n = 67; 31.2%). One hundred twenty-six NSIs (58.6%) were caused by DHCWs with little experience (P < 0.05), and 37 of those (17.2% of the total) were ascribed to undergraduate students during clinical training (P < 0.05). The NSIs occurred both during treatment (n = 119; 55.3%) and during cleaning up used devices (n = 89; 41.4%). The NSIs at the dental hospital occurred with a probability of 0.004% of total therapeutic opportunities. Prevention of NSIs should be the responsibility of dental students and DHCWs, and should be a part of education about infection control to prevent the nosocomial transmission of blood-borne pathogens.

Irrigation effectiveness of continuous ultrasonic irrigation system: An ex vivo study.

This study evaluated the irrigation extrusion and smear layer (SL) removal of continuous ultrasonic irrigation (CUI). Forty root canals were shaped, embedded in warm sterile saline agar colored with 1% acid red, and distributed into four groups; three groups had the final irrigation sequence of sodium hypochlorite and EDTA solutions performed by using syringe irrigation (SI), EndoVac (EV), and CUI, respectively. Group 4 had the canals ultrasonically irrigated with saline (CUIS). Irrigant extrusion was verified by a color change of the agar. The SL was evaluated under scanning electron microscope. Irrigant extrusion was observed in two and four teeth in the CUI and SI, respectively, whilst none had irrigant extruded in the EV. In term of SL, the CUI was comparable to EV at 1 and 3 mm but different to SI at 1 mm. Although CUI might clean the root canal system effectively, it was unable to avoid irrigant extrusion.

Kinetic study of immunohistochemical colocalization of antigen-presenting cells and nerve fibers in rat periapical lesions.

Immune and nervous systems play key roles in periapical pathosis; however, their spatial associations, which may be a prerequisite for paracrine interactions in the progression of periapical lesions, remain to be examined. In this study we examined the distribution and frequency of spatial associations between major histocompatibility complex class II molecule-expressing antigen-presenting cells (APCs) and protein gene product 9.5-immunoreactive nerve fibers in experimentally induced rat periapical lesions using double-immunofluorescence staining and confocal laser scanning microscopy. When active lesion expansion started, macrophage-like APCs frequently associated with nerve fibers around the apex. When the lesions were starting to stabilize, however, close associations between APCs with dendritic morphology and nerve fibers were found mostly in the periphery of lesions. CD86+ mature dendritic cells were also observed in this area. These findings suggest that functional interactions between APCs and nerve fibers may play some roles in the development of self-defense reactions in periapical lesions.

Evaluation of the efficiency of a new file removal system in comparison with two conventional systems.

A novel file-removal system (FRS) was designed to address weak points of conventional file-removal methods. The purpose of this study was to compare file-removal time and dentin removal rates among the FRS, the Masserann kit (Micro-Mega, Besancon, France), and an ultrasonic file-removal method. Ninety extracted mandibular incisors with separated nickel titanium files were divided into 3 groups of 30 teeth each. Groups 1, 2, and 3 had file-removal attempts made by using the Masserann kit, a CPR-7 titanium ultrasonic tip (Obtura-Spartan Corp., Fenton, MO), and the FRS, respectively. Each group had three operators removing the separated files. Pre-/postoperative digital radiographs were downloaded into image analyzing software that calculated the amount of dentin removed. The FRS needed less time and had less dentin loss than the others (p<0.05). There were statistical differences between the experienced operator and less experienced operators regarding the file-removal time and the dentin removal rates (p<0.05).

Sealing ability of new adhesive root canal filling materials measured by new dye penetration method.

The sealing ability of new adhesive root canal filling materials was evaluated using a new dye penetration method. Twenty-eight single-rooted mandibular premolars were randomly divided into four groups of seven teeth each and filled by lateral condensation using one of these combinations: Resilon point with Epiphany sealer (RE); gutta-percha point with Sealapex sealer (GS); gutta-percha point with dentin activator and Superbond sealer (GDS); or gutta-percha point with Accel primer, dentin activator, and Superbond sealer (GADS). Amount of 0.06% methylene blue dye solution (MB) that leaked from the coronal portion to the apical area was measured with a spectrophotometer at one, four, eight, 15, and 30 days in an accumulative manner. The total amount of leaked MB on day 30 was significantly higher for GDS than the other combinations (p < 0.05).