Conservative management of double teeth in molar teeth with pulp or periapical disease: a report of five cases and literature review.

BACKGROUND: Double teeth are usually the result of an abnormality in the developing tooth germ. Double teeth can occur in either the primary or permanent dentition, with the majority of cases concerning permanent teeth reported in the anterior teeth and less frequently in the molar teeth. CASE PRESENTATION: This report illustrates five cases of double teeth in molars with pulp and periapical disease, including one case of geminated teeth and four cases of fused teeth. Radiographic findings revealed the presence of extra teeth on the buccal aspect of the molar in five cases, with or without communication between the two root canal systems. Root canal treatment was performed by using CBCT and a dental operating microscope. The treatment outcome was good in all five cases. CONCLUSION: The diagnosis and treatment of double teeth requires special attention. The root canal system should be carefully explored to obtain a full understanding of the anatomy, allowing it to be fully cleaned and obturated. Proper anatomical structure analysis prior to treatment facilitates the development of an appropriate treatment plan, thereby increasing the likelihood of successful treatment both aesthetically and functionally.

Rhodamine-based chemosensor for Sn2+ detection and its application in nanofibrous film and bioimaging.

A simple rhodamine-based compound CK was designed and synthesized as a fluorescent chemosensor for Sn2+ based on Sn2+-mediated cyclization. The optical investigation indicated that the probe could quantitatively detect Sn2+ in a concentration range of 10-30 µM, with a detection limit of 118 nM. Moreover, probe CK, with low cytotoxicity, was successfully applied for imaging of Sn2+ in HeLa cells and mice, exhibiting excellent biocompatibility and cell membrane permeability. For on-site monitoring, CK-hybridized polymethyl methacrylate (PMMA) nanofibers were prepared by electrospinning and successfully employed for the visual detection of Sn2+ in actual samples. All the results demonstrated that the chemosensor could be a promising tool for the detection of Sn2+ in vitro and in vivo.

Overexpression of programmed cell death ligand 1 reduces LPS-induced inflammatory cytokine upregulation and enhances osteo/odontogenic-differentiation of human dental pulp stem cells via upregulation of CCCTC-binding factor.

OBJECTIVE: The aim of this study was to explore the effect and mechanism of programmed cell death ligand 1 (PD-L1) in promoting the proliferation and osteo/odontogenic-differentiation of human dental pulp stem cells (hDPSCs) by mediating CCCTC-binding factor (CTCF) expression. DESIGN: The interaction between PD-L1 and CTCF was verified through co-immunoprecipitation. hDPSCs transfected with PD-L1 overexpression and CTCF knockdown vectors were treated with lipopolysaccharide or an osteogenic-inducing medium. Inflammatory cytokines and osteo/odontogenic-differentiation related genes were measured. Osteo/odontogenic-differentiation of hDPSCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining. RESULTS: Overexpression of PD-L1 inhibited LPS-induced pro-inflammatory cytokine upregulation, cell proliferation, ALP activity, and calcium deposition in hDPSCs and elevated the expression of osteo/odontogenic-differentiation related genes; however, such expression patterns could be reversed by CTCF knockdown. Co-immunoprecipitation results confirmed the binding of PD-L1 to CTCF, indicating that PD-L1 overexpression in hDPSCs increases CTCF expression, thus inhibiting the inflammatory response and increasing osteo/odontogenic-differentiation of hDPSCs. CONCLUSION: PD-L1 overexpression in hDPSCs enhances the proliferation and osteo/odontogenic-differentiation of hDPSCs and inhibit the inflammatory response by upregulating CTCF expression.

The complexing of cationic copolymer MPC30-DEA70 with TGF-ß1 antisense oligodeoxynucleotide and transfection into cardiomyocytes in vitro.

Although gene therapy is an attractive option for the treatment of cardiovascular diseases, the ideal gene delivery systems are still under investigation and must meet the following criteria: safety, adequate gene transfer efficiency, and stable expression of the transgene for a duration appropriate for treating the disease. In this study, we developed a cationic phosphorylcholine-containing diblock copolymer, namely MPC30-DEA70, as carrier systems to deliver a chemically synthesized transforming growth factor-beta 1(TGF-ß1) antisense oligonucleotide (AS-ODN) into cardiomyocytes (CMs) to observe the cell transfection efficiency of MPC30-DEA70 and the inhibition effect on the expression of TGF-ß1. MPC30-DEA70/TGF-ß1 AS-ODN complexes were formed through complexation between copolymer MPC30-DEA70 (N) and AS-ODN (P) at different N/P ratios and were characterized by DNA electrophoresis. Notably, the cytotoxicity and cell growth inhibition assay showed that the MPC30-DEA70 had low cytotoxicity to CMs within the effective transfection dosage range (<20 µL/mL). CLSM/TEM images displayed that most of the AS-ODN molecules engulfed by cells were located around the cell nuclei, and a few entered into the cell nuclei without harming the organelles in the cell. Transfection studies from CMs indicated a steady increase of transfection efficiency with increasing N/P ratios. The expression levels of TGF-ß1 mRNA and protein in CMs were significantly inhibited at high N/P ratios. This study shows that MPC30-DEA70 can function as an effective transgenic vector into CMs and that TGF-ß1 AS-ODN delivered by MPC30-DEA70 can silence the expression of the TGF-ß1 gene efficiently and specifically and thereafter antagonize TGF-ß1-mediated biological function in cardiomyocytes.

In vitroapplication of drug-loaded hydrogel combined with 3D-printed porous scaffolds.

Titanium mesh and three-dimensional titanium alloy scaffolds play a key role in addressing oral and maxillofacial bone defects, which can provide a specific environment and structure for bone growth and development. The two main causes of implant surgery failure are aseptic loosening and bacterial-induced implant-associated infections. To make bone defect implants effective for a long time, the ideal scaffold should take into account the two functions of osseointegration and anti-infection. Therefore, on the basis of the low-elastic-modulus Ti-10Ta-2Nb-2Zr (TTNZ) alloys developed by the research group in the early stage, this study intends to combine the vancomycin-loaded hydrogel with the 3D-printed through-hole porous titanium alloy scaffold to endow 3D-printed TTNZ scaffolds with antibacterial properties. The antibacterial properties of the complex were investigated by the zone of inhibition test and the adhesion/free antibacterial test. The effects of the composite system on osseointegration were investigated from the aspects of cell adhesion, cell proliferation and osteogenesis-related gene expression. The results showed that loading 2.5 wt.% and 5 wt.% vancomycin did not affect the structure of chitosan-hyaluronic acid hydrogel. The properties of the hydrogels were examined by scanning electron microscopy, Fourier-transform infrared, degradation experimentin vitroand vancomycin release experimentin vitro. When combined with porous scaffolds, the drug-loaded hydrogels exhibited slower drug release rates and longer release times. In addition,in vitrostudies found that the TTNZ scaffolds loaded with 5 wt.% vancomycin had a certain effect on the expression of osteogenesis-related genes in cells, but the antibacterial effect was the best. The porous scaffolds loaded with 2.5 wt.% vancomycin hydrogel TTNZ scaffolds did not inhibit cell proliferation, adhesion, alkaline phosphatase activity, and osteogenesis-related gene ex-pression, but the antibacterial effect on free bacteria was not as good as that of TTNZ scaffolds loaded with 5 wt.% vancomycin. This study, complementing the advantages of the two and controlling the local release rate of vancomycin, provides a new idea for future 3D printing of titanium alloy stents for anti-infection.

Augmented Reality Research of Measuring X-Ray Dental Film Alveolar Bone Based on Computer Image Analysis System.

The important application of computer imaging technology in the medical field is a necessary auxiliary method for clinical diagnosis and treatment. At present, many people are affected by various factors and have various problems caused by the dental cellular bone. Traditional treatment methods are complex and long, which can cause damage to body tissues. Based on this problem, this paper takes the augmented reality measurement of X-ray dental film as the research object. Based on the in-depth measurement algorithm of the computer image analysis system, two three-dimensional reconstruction methods based on the center of gravity and the matching of the front and side positions are proposed. These two methods only need two X-rays of the front and side of the dental film, the three-dimensional parameters are obtained through calculation and analysis of each spine in the X-ray film, and these parameters are used to fit the dental alveolar bone model. The experimental results prove that the computer-based image analysis system has a great effect on the measurement of X-ray dental film alveolar bone. The positive correlation coefficient reaches 0.87. Compared with the cerebral infarction caused by other methods, the proportion of people with dental film alveolar bone injury is about 15%; after treatment, the functional recovery rate reaches more than 80%. Studies have found that there is a great difference in the age of the population that needs to be treated for dental slices and alveolar bone. The grade of patients is generally under 20 and over 60. This shows that the measurement of X-ray dental film alveolar bone based on computer image analysis system can play an important role in protecting people's oral health.

The kinetics and release behaviour of curcumin loaded pH-responsive PLGA/chitosan fibers with antitumor activity against HT-29 cells.

The bioavailability and clinical effect of curcumin (Cur) are greatly restricted due to its physicochemical instability and high hydrophobicity. To overcome the disadvantages, the nanofibers of poly(lactide-glycolide)/chitosan loaded with Cur (PLGA/CS/Cur) was developed here by electrospinning technique for controlled Cur delivery. The incorporated Cur was well-dispersed and maintained crystalline form in PLGA/CS fiber matrix by hydrogen bonding. The incorporation of Cur had no obvious influence on the fiber size and morphology but exerted impacts on thermal stability. At pH 7.4, the release followed Fickian diffusion mechanism; while at pH 2.0, the release followed the coexistence of diffusion and erosion mechanisms. In addition, the amount of Cur released at pH 2.0 was much higher than that at pH 7.4. As a result, the nanofibers demonstrated higher anticancer activity at acidic environment. Therefore, the PLGA/CS/Cur nanofibers may be served as a potential pH responsive vehicle for the controlled drug delivery.

pH-responsive antibacterial film based polyvinyl alcohol/poly (acrylic acid) incorporated with aminoethyl-phloretin and application to pork preservation.

This study demonstrates a pH-responsive antibacterial film based on polyvinyl alcohol/poly (acrylic acid) incorporated with aminoethyl-phloretin (PVA/PAA-AEP) for intelligent food packaging. The thermal, mechanical, barrier and light transmittance properties of PVA/PAA are enhanced by PAA presence of ≤6%. The interactions between PVA and PAA were hydrogen and ester bonds. The pH-responsive characteristic is dependent on the protonation/deprotonation tendency of the carboxylic groups on PAA in acidic/alkaline environment. The PVA/PAA3 is selected for the incorporation of AEP and its pH-responsive swelling follows Ritger-Peppas and Schott second-order models. The AEP is hydrogen bonded with the matrix of PVA/PAA3 and the release of AEP is pH-responsive and a rate-limiting step following the First-order model. With pH decrease, the predominant release control was gradually changing from polymer relaxation to Fick diffusion. The PVA/PAA3-AEP films demonstrate AEP content dependent antioxidant and antimicrobial activities. Furthermore, the antibacterial efficiency against Listeria monocytogenes and Staphylococcus aureus is significantly better than Escherichia coli. The target film PVA/PAA3-AEP3 can effectively prolong the shelf-life of pork (TVB-N < 25 mg/100 g) by 4 days at 25 °C, suggesting its great potential in intelligent food packaging.

Kidney targeted delivery of asiatic acid using a FITC labeled renal tubular-targeting peptide modified PLGA-PEG system.

Chronic kidney disease (CKD) is one of the leading public health problems worldwide and finally progresses to end-stage renal disease. The therapeutic options of CKD are very limited. Thus, development of drug delivery systems specific-targeting to kidney may offer more options. Here we developed an efficient kidney-targeted drug delivery system using a FITC labeled renal tubular-targeting peptide modified PLGA-PEG nanoparticles and investigated the intrarenal distribution and cell-type binding. We found that the modified nanoparticles with an approximate diameter of 200 nm exhibited the highest binding capacity with HK-2 cells and fluorescence and immunohistochemical analysis showed they mainly localized in renal proximal tubules by passing through the basolateral side. Furthermore, these kidney-specific nanoparticles could significantly enhance the therapeutic effects of asiatic acid, an insoluble triterpenoid compound as drug delivery carriers. In conclusion, these results suggest the potential of the peptide modified PLGA-PEG nanoparticles as kidneytargeted drug delivery system to proximal tubular cells in treatment of CKD.

Colorimetric film based on polyvinyl alcohol/okra mucilage polysaccharide incorporated with rose anthocyanins for shrimp freshness monitoring.

In this work, a colorimetric film was designed for shrimp freshness monitoring by incorporating rose anthocyanins (RAs) in polyvinyl alcohol/okra mucilage polysaccharide (PVA/OMP) composite film. The presence of OMP changed the film-forming solution from Newtonian to non-Newtonian fluid. The addition of OMP and RAs decreased the crystalline of PVA due to the hydrogen bonds among RAs, OMP and PVA. An appropriate content of RAs and OMP could improve the film mechanical and barrier properties. The colorimetric film showed distinguishable color changes at pH 2-12 and was high sensitive to volatile ammonia. The target film of PVA/OMP-RAs could effectively monitor shrimp freshness in real time and the color changes were easily distinguished by naked eye, suggesting its potential in intelligent packaging for freshness monitoring of aquatic products and meat foods.

Temperature Responsive Shape-Memory Scaffolds with Circumferentially Aligned Nanofibers for Guiding Smooth Muscle Cell Behavior.

Structural simulation of the smooth muscle layer plays an important role in tissue engineering of blood vessels for the replacement of damaged arteries. However, it is difficult to construct small-diameter tubular scaffolds to homogenously locate and align smooth muscle cells (SMCs). In this work, novel temperature responsive shape-memory scaffolds are designed for SMC culturing. The scaffolds are composed of an outer layer of poly(lactide-glycolide-trimethylene carbonate) (PLGATMC) for programming the deformation from planar to small-diameter tubular shape and an inner layer of aligned nanofibrous membrane of poly(lactide-glycolide)/chitosan (PLGA/CS) to regulate cell adhesion, proliferation, and morphology. The SMC behaviors and functions are dependent on the PLGA/CS ratios of membranes, and the scaffold with PLGA/CS 7:3 membrane exhibits the most suitable ability to regulate SMC behavior. The PLGA/CS@PLGATMC scaffold can be deformed into a temporary planar at 20 °C for convenient seeding and attachment of SMCs and then immediately self-rolled into 3D tube at 37 °C. The proposed strategy offers a practical approach for the development of small-diameter vascular scaffolds from 2D planar into 3D tubular shape by self-rolling.

Antibacterial activity and cytotoxicity of novel silkworm-like nisin@PEGylated MoS2.

Recently, molybdenum disulfide functionalized with poly-ethylene glycol (PEGylated MoS2) has been widely used as a new drug delivery vehicle in biomedical field. However, the weak antibacterial activity of PEGylated MoS2 limits its application as an antibacterial agent. In this work, a novel silkworm-like conjugate of nisin loaded PEGylated MoS2 (nisin@PEGylated MoS2) was developed for antibacterial application. The morphology and structure of PEGylated MoS2 were strongly dependent on the Mo/S molar ratio of precursors during the solvothermal process. The silkworm-like skeleton was well kept after loading with nisin. A high level of reactive oxygen species (ROS) induced by the conjugate was an important cause of bacteria death. Due to the different structure of cell membranes, the sharp edges could more easily puncture into Escherichia coli (E. coli) as compared with Staphylococcus aureus (S. aureus) and produced more intracellular ROS, which improved the antibacterial activity of nisin against E. coli. As a result, nisin@PEGylated MoS2 displayed the antibacterial activity against both gram-positive and gram-negative bacteria. Furthermore, the toxicity of the conjugate was very low. Therefore, the target conjugate of nisin@PEGylated MoS2 may have great potential application as an antibacterial agent.

Graphene Oxide Immobilized PLGA-polydopamine Nanofibrous Scaffolds for Growth Inhibition of Colon Cancer Cells.

Graphene oxide (GO)/poly (lactide-co-glycolic acid) (PLGA) scaffolds have promising applications in the biomedical field. However, greater attention is focused on the incorporated system and its applications in normal cells. In this work, a novel GO immobilized PLGA nanofibrous scaffold assisted by polydopamine (PLGA-PDA-GO) is developed for growth inhibition of HT-29 colon cancer cells. The interactions between GO and PDA are attributed to a π-π conjugate interaction and electrostatic attraction. In addition to the enhancement of thermal stability and mechanical strength, the surface roughness, hydrophilicity, and electro-activity of the scaffolds are significantly improved by immobilization of GO. The scaffolds show good inhibition of HT-29, and immobilized GO is observed to be in contact with but not internalized in HT-29 cells. The cytotoxicity mechanism of scaffolds toward HT-29 is attributed to intracellular activated reactive oxygen species that result from the physical interaction of the sharp GO edges and electrical signals of π-π stacking between PDA and GO.

Ethylene-vinyl Alcohol Copolymer-Montmorillonite Multilayer Barrier Film Coated with Mulberry Anthocyanin for Freshness Monitoring.

Colorimetric films incorporated with anthocyanins as an indicator for freshness monitoring have aroused growing interest recently. However, the application of the films is limited by the easily oxidizable nature of anthocyanins. In this work, we developed a novel colorimetric film with a barrier by coating mulberry anthocyanin (MBA) on the internal side of an ethylene-vinyl alcohol copolymer-montmorillonite (EVOH-MMT) multilayer film. A facile layer-by-layer (LBL) assembly was employed under a parallel electric field to build the EVOH-MMT multilayer structure, in which the exfoliated MMT nanosheets were well-oriented and assembled on the EVOH matrix to form a tightly stacked layer between two EVOH layers. The interlayer of MMT significantly enhanced the barrier and mechanical properties of the films (below 40 layers). The interactions between EVOH and MMT and between EVOH and MBA were confirmed to be intermolecular hydrogen bonds. The colorimetric response of (EVOH-MMT)40-MBA3 to volatile ammonia and pH was sensitive, and the color change could be easily distinguished by the naked eye. The successful application of (EVOH-MMT)40-MBA3 to shrimp-freshness monitoring confirms its high potential for the freshness monitoring of packaged food.

Antibacterial [2-(Methacryloyloxy) ethyl] Trimethylammonium Chloride Functionalized Reduced Graphene Oxide/Poly(ethylene-co-vinyl alcohol) Multilayer Barrier Film for Food Packaging.

The objective of present work was to construct antibacterial [2-(methacryloyloxy) ethyl] trimethylammonium chloride functionalized reduced graphene oxide/poly(ethylene-co-vinyl alcohol) (MTAC-rGO/EVOH) multilayer barrier films by using layer-by-layer assembly under a parallel electric field. Besides barrier and mechanical properties, the antibacterial activities of the film and cytotoxicity of MTAC-rGO nanosheets were extensively investigated. The functionalization of rGO was achieved by grafting MTAC onto a graphene framework through C (sp3)-C bonds. The assembly of MTAC-rGO on the EVOH matrix not only significantly improved film mechanical strength, but also endowed the targeting film with outstanding moisture barrier even under a relative humidity of 99% (e.g., 0.019 g m-2 s-1 atm-1 for (MTAC-rGO/EVOH)20) besides good oxygen barrier (e.g., 0.07 cm3 m-2 d-1 atm-1 for (MTAC-rGO/EVOH)20). Among the testing films, MTAC-rGO/EVOH film had the best antibacterial activity, and the activity against S. aureus was better than E. coli. Meanwhile, the cytotoxicity of MTAC-rGO nanosheets was very low. Results suggest that MTAC-rGO/EVOH film may have great potential in food active packaging.

[Effect of root canal wall moisture and filling techniques on the sealability of iRoot sp].

PURPOSE: To study the effect of root canal wall moisture and filling techniques on the sealability of iRoot sp. METHODS: Sixty-two undamaged extracted human single-rooted teeth with fully developed apex were selected and prepared by crown-down technique. Two teeth were selected randomly to observe dentin tubules, opening, the residual teeth were randomly assigned to 3 groups: group A (wet), group B (slightly moist),group C(dry).The roots were further divided into 4 subgroups, group a: iRoot sp sealer without a core material (bulk-fill); group b: iRoot sp sealer with single cone obturation techniques; group c: iRoot sp sealer with heated gutta-purcha vertical pressure; group d: iRoot sp sealer with cold gutta-percha lateral compression technique. Glucose microleakage were measured in each group by glucose oxidase method. The differences in distribution of each group were analyzed with SPSS 19.0 software package. RESULTS: Group A and group B always showed the maximum and minimum amount of glucose penetration in the whole experimental process, and the difference was statistically significant at 28d (P<0.05). Under the same degree of moisture of root canal wall, glucose leakage of subgroup Aa was significantly higher than that of subgroup Ab and Ac at 15 d; and significantly higher than Ab, Ac, Ad at 21 d and 28 d(P<0.05). In group B and C, the subgroups a, b, c, d had no significant difference from each other during the experimental process(P>0.05). CONCLUSIONS: iRoot sp sealer has the best sealing effect when root canals were slightly moist. When the root canal wall is completely dry or slightly moist, the sealability of iRoot sp bulk-fill is similar to that of iRoot sp sealer with gutta-percha filling technique.

[The effect of different gutta-percha master cones on first penetration depth of spreader and apical microleakage].

PURPOSE: This study was to evaluate the effect of different master cones on apical microleakage through comparing first penetration depth of spreader and the leakage concentration of glucose in the curved root canal of the extracted posterior teeth filled with different gutta-percha master cones. METHODS: Sixty-six extracted roots of human posterior teeth with a single, curved canal were collected and divided into five experimental groups of 11 roots in each group (Group A:master cone 0.06 tapered, 25#; Group B: 0.06 tapered, 20#; Group C: 0.04 tapered, 30#; Group D: 0.04 tapered, 25#; Group E: 0.02 tapered, 25#) using cold lateral condensation as positive control group(group F, AH-PLUS sealer) of 5 roots and negative control (group G, integrated teeth) of 6 roots. The first penetration depth of spreader and the concentration of glucose in apical reservoir at 1, 2, 4, 7, 10, 15, 20 and 30 days were measured, respectively. The data was analysed with ANOVA and LSD test using SPSS 16.0 software package. RESULTS: The value of working length minus the first penetration depth of spreader in group D was significantly smaller than that in group A and group C(P<0.05),and that in group E was significantly smaller than that in group A ,B,C(P<0.05), but no significant difference(P>0.05) in other groups. The concentration of leakage glucose in group F was significantly higher than that in the other six groups(P<0.05),and there were no significant difference in the five experimental groups (P>0.05),which had no significant difference from the negative control group G(P>0.05). CONCLUSIONS: Choosing smaller tapered master cone could get better apical filling in curved canals, but it has no superiority in apical sealing in short time.

[Experimental study of preparing curved root canals with different instrument].

OBJECTIVE: To compare the shaping ability and the influence on apical foramen among hand ProTaper, stainless steel K-files and rotary ProTaper in preparing different curved root canal. METHODS: Forty simulated resin root canal blocks were randomly divided into four groups and prepared by hand ProTaper, stainless steel K-file and rotary ProTaper, respectively. Of them, 12 blocks in group A, B, C consist of six 200 curved root canals and six 30 degrees curved root canals each group. The curvature of the other 4 blocks in group D was less than 5 degrees. Taking photos of the models to the root canal orthotopically and apical foramen using digital camera before and after instrumentation. Finally, the transportation of root canal and the size of apical foramen were analyzed using special image software Auto-CAD. RESULTS: The transportation of center in group B was the highest than that in group A and group C (P<0.05). In some portions of root canal, the transportation of center in group C was higher than that in group A. The size of the apical foramen in group B was significantly bigger than the other groups and the size of the apical foramen in 30 degrees root canal was significantly bigger than that in 20 degrees root canal after instrumentation (P<0.05). There was no significantly different between group A and group C, though the size of apical foramen in group C was bigger than that in group A at the same curvature, and that in 30 degrees root canal was bigger than in 20 degrees root canal (P>0.05). CONCLUSION: Both of the two instruments engender root canal transportation, and curvature is the main reason of transportation. Comparing with stainless steel K-files, NiTi files can maintain the shape of the root canal and apical foramen well.

[The effect of smear layer on apical microleakage].

PURPOSE: To evaluate the effect of smear layer on apical microleakage in extracted root-filled teeth by SEM and glucose penetration model. METHODS: 51 straight single-rooted human teeth were randomly divided into 5 groups, instrumented and irrigated differently by groups. 11 teeth in group A were irrigated with 17% EDTA and 1% NaClO,11 teeth in group B with 1% tetracycline hydrochloride and 1% NaClO,11 teeth in group C with 10% citric acid and 1% NaClO, 9 teeth in group D with 1% NaClO only and 9 teeth in group E with 0.9% NaCl. One tooth in each group was observed under scanning electron microscopy. The other teeth were obturated using lateral condensation technique. Leakage along the root filling was measured by the concentration of glucose in apical reservoir at 1, 2, 4, 7, 10, 15, 20 and 30 days respectively with glucose oxidase method. The data were analyzed with repeated measure ANOVA and SNK-q test using SPSS 16.0 software package. RESULTS: No smear layer on the root canal wall was observed in group A, B and C, the leakage was lower than that in group D and E (P<0.05). The smear layer occurred in quantity in group D, E and their leakage increased. The microleakage was higher in group E than that in group D (P<0.05). CONCLUSION: Removal of smear layer on the root canal wall could reduce the root apical microleakage.