Markus Haapasalo

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Dynamics of killing biofilm bacteria (2013)

No abstract available.

Master's Student Supervision (2010 - 2018)
Dynamic dissolution and inhibition of biofilm development by endodontic disinfecting agents (2018)

Many disinfecting solutions have been developed to disrupt the biofilm and to kill biofilm bacteria. However, there is little data so far showing dynamic dissolution on biofilm. The present study aims to establish a standardized model that makes it possible to evaluate the dynamic dissolution of biofilm and inhibition of growth of multispecies biofilm by endodontic irrigation solutions.Biofilm was grown from plaque bacteria on collagen coated hydroxyapatite (HA) disks in brain-heart infusion broth for 3 days or 3 weeks under anaerobic conditions. Biofilms were stained by the LIVE/DEAD viability stain and subjected to sterile water, 2% sodium hypochlorite (NaOCl), 6% NaOCl, or 2% chlorhexidine (CHX) for 32 minutes. Dynamic change in fluorescence from each biofilm sample after treatment was analyzed using a live-cell imaging confocal laser scanning microscopy (LC-CLSM). Biovolume and proportion of dead bacteria were calculated. The biofilm structures after treatments were visualized by scanning electron microscopy (SEM). The treated biofilms on HA disks were collected and cultured on blood agar plates for the colony forming unit (CFU) test. Another set of sterile HA disks were immersed in 2%, 0.2% or 0.02% CHX solutions for 3 minutes. Plaque biofilm growth on these disks was monitored by LC-CLSM for 12 hours.For 3-day-old biofilms, 2% and 6% NaOCl reduced the biovolume by 63% and 94% in 32 minutes respectively. For 3-week-old biofilms, 75% and 86% of the biofilm was dissolved by 2% and 6% NaOCl respectively in 32 minutes. Six percent NaOCl was the most effective in dissolving and killing bacteria followed by 2% NaOCl and CHX. CFU results indicated difference in bacterial reduction between biofilm and planktonic culturing after disinfection. SEM showed biofilm bacteria disruption after CHX and NaOCl treatments. The use of 2% CHX and sterile water did not result in biofilm dissolution. However, prior exposure of HA disks to 2% and 0.2% CHX for 3 minutes prevented biofilm from growing on HA disk surfaces for at least 12 hours.In conclusion, NaOCl dissolved biofilm effectively, more with higher concentration and longer time of exposure. CHX does not dissolve biofilm but inhibits biofilm growth on HA surface.

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Removal of calcifications from distal canals of mandibular molars with the gentlewave™ system (2018)

Objective: Radiographic examination of teeth often reveals the presence of calcified structures in pulp chambers. Many studies have also reported the presence of calcifications throughout the root canal system. These can complicate root canal therapy by obliterating the pulp chamber or blocking root canals. Many studies have proposed techniques to remove calcifications using instrumentation. GentleWave is a novel system for non-instrumentational cleaning of root canals, however, its ability to remove calcifications has not been established. The aim of our study was to determine the efficiency of the GentleWave system in removing calcifications from the root canal system.Methods: Mandibular molars were accessed, and all canals identified. Patency of the distal canals was ensured with a size 10 K-File and the pulp chambers were irrigated with NaOCl. Micro-CT images were obtained to evaluate the initial volume of calcification in the distal canals. Teeth were then treated with the GentleWave system as recommended by the manufacturer and a second set of micro-CT images were obtained to assess for changes in the calcification volume. The Mann-Whitney test was used to compare calcification volumes before and after GentleWave treatment. The significance level was set at P ≤ .05.Results: All distal canals showed calcifications prior to GentleWave treatment. In teeth treated with the GentleWave system, distal canals showed statistically significant reduction in calcification volume when compared to the initial volume of calcification. Mean reduction in volume was 87.6%, with overall values ranging from 60-100%. Root canal volume prior to and after GentleWave treatment did not show any significant change. Conclusions: The GentleWave system showed significant reduction in root canal calcification after treatment. Almost half (42%) of the distal canals showed complete elimination of calcification after GentleWave treatment. Acknowledgements: Supported by the Canadian Academy of Endodontics. Sonendo provided the GentleWave unit for the cleaning of the teeth.

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A three dimensional analysis of the effect of irrigant flow velocity on endodontic biofilms (2017)

Introduction: Irrigation of the root canal system is an essential part of disinfection during root canal treatment. Some irrigants have the ability to dissolve or detach the biofilm from the dentin surface as well as kill residual bacteria. Several chemical and physical parameters affect the effectiveness of the irrigation process.Aim: The primary aim of this study is to investigate the effect of irrigation flow velocity on the biovolume of the biofilm and antimicrobial effect against biofilm microbes. Materials and methods: Hydroxyapatite discs coated with type I collagen were used as the biofilm substrate. Mixed supra- and sub-gingival plaque was collected from a single donor and suspended in Brain Heart Infusion broth (BHI). The discs were placed in 24-well tissue culture plates containing BHI-plaque suspension. They were then incubated under anaerobic conditions for three weeks. BHI medium was replaced with a fresh solution once a week. After three weeks of biofilm growth, the discs were placed in a CDC Biofilm reactor placed on a magnetic stir plate. The speed settings used were 60 rpm and 200 rpm both for 30 and 60 seconds. 0.1 % Sodium hypochlorite solutions and sterilized spring water were utilized for the experiment. Control samples were not subjected to any treatment. After treatment, all discs were stained with a viability stain and assessed under a confocal laser scanning microscope. The total biofilm volume and percentage of dead bacteria were calculated using the Bioimage L software.Results: Sodium hypochlorite NaOCl at 200 rpm was significantly more effective than control groups in reducing biofilm volume and killing biofilm bacteria. At slower flow velocity (60 rpm), there was no difference when compared to the negative control groups not subjected to any treatment. Multivariate analysis results revealed that irrigant type and flow velocity had a significant effect on reducing biofilm volume and killing biofilm bacteria. Exposure time influenced the biofilm volume only (p
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Evaluation of the effectiveness of two irrigation systems in reducing bacterial load in root canals in vitro by quantitative real-time polymerase chain reaction (2017)

Introduction: Irrigation is regarded as having a key role in one of the main goals of endodontic treatment, the killing and removing bacteria from the infected root canal. Different methods and techniques have been used to deliver irrigating solutions and to facilitate the effectiveness of irrigation. The purpose of this in vitro study was, by using quantitative real-time PCR to determine the effectiveness of two irrigation and cleaning systems in removing multispecies oral biofilms from root canals. Methods: Fifteen extracted human molars were instrumented to size #15/.02 and then cleaned with the GentleWave System (GW; Sonendo Inc, Laguna Hills, CA). The teeth were autoclaved to provide the same sterile baseline. The molars were filled with mixed plaque suspended in brain-heart infusion broth and centrifuged to help the bacteria spread all over the root canal system. After two weeks of incubation, samples were divided randomly into two treatment groups and instrumented into size #15/.04 (GW) and #35/.04 (PiezoFlow group) under needle irrigation with sterile water. The teeth were then cleaned either with GentleWave System or ProUltra PiezoFlow Active Ultrasonic System (Dentsply Tulsa, Tulsa, OK) using 3% NaOCl, 8% EDTA and sterile water as irrigants. Root canals were sampled with paper points before and after instrumentation (S1, S2) and after GW or PF cleaning (S3). Quantitative real-time PCR was performed, the presence of microorganism in the samples was determined by using universal bacterial, a genus specific and species-specific primers. Statistical analysis was performed using the Mann-Whitney U test with the significance level set at P
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Antimicrobial efficacy of different calcium hydroxide containing preparations against biofilms at different stages of biofilm development (2016)

Objective: To quantify and assess the antibacterial effect of different medicaments on young and aged biofilms, and to modify the medicaments in order to increase their antibacterial effect.Hypotheses: Microbes in aged biofilms grown from a mixture of oral bacteria are more resistant to the antimicrobial activity of calcium hydroxide than microbes in young biofilms. Biofilms are less resistant to calcium hydroxide combined with other antimicrobial agents than to pure calcium hydroxide.Methodology: Collagen coated hydroxyapatite disks were immersed in plaque suspension solution and incubated for one and three weeks to grow young and aged biofilms, respectively. The tested medicaments were calcium hydroxide, iodine potassium iodide, cetrimide, and the following combinations: iodine potassium iodide + cetrimide, calcium hydroxide, calcium hydroxide + iodine potassium iodide, calcium hydroxide + cetrimide, calcium hydroxide + iodine potassium iodide + cetrimide. After exposure to the medicaments for one day, one week, and two weeks, biofilms on disks were stained with a LIVE/DEAD viability stain and imaged using confocal laser scanning microscopy. The three-dimensional reconstructions of the images were done and proportions of green and red fluorescence were measured and statistically analyzed.Results: Aged biofilms were thicker than the young biofilms. All tested medicaments showed reduced antibacterial activity on the aged biofilms compared to young biofilms. Combining iodine potassium iodide to cetrimide had an additive effect and mixed with calcium hydroxide showed stronger antibacterial effect than calcium hydroxide alone.Conclusions: Aged biofilms are more resistant to antibacterial agents than young biofilms. Combining iodine potassium iodide and cetrimide to calcium hydroxide resulted in an antibacterial effect that was stronger than using calcium hydroxide alone.

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Quality of root filling by three different techniques in teeth after minimal root canal preparation and the GentleWave cleaning (2016)

Objective: This study aimed to examine the quality of root filling in canals obturated using three different filling techniques after minimal root canal preparation and GentleWave (GW) cleaning.Methods: Root canals of 30 mandibular molars were instrumented to size 20/.04 taper using hand K-files, and a Vortex Blue NiTi rotary file. Sodium hypochlorite (NaOCl) 6% was used in-between each instrument insertion and the canals were finally cleaned using a 7.5 min irrigation by the GW. The samples were scanned with micro-CT using a voxel size of 30 µm after instrumentation (pre-GW), post-GW, and after obturation. The samples were randomly allocated into three groups (n = 10) with regard to the root filling method: (1) single-cone with AH Plus sealer (SC/AH+), (2) single-cone with GuttaFlow sealer (SC/GF), and (3) GuttaCore with AH Plus sealer (GC/AH+). The reconstructed 3D images were analyzed for the volumetric percentages of debris, filling materials, gaps, and voids at three canal levels. Data was analyzed statistically using Kruskal-Wallis and Mann-Whitney U tests. P values
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Potential for potassium recovery as K-struvite (2015)

Crystallization of NH₄-struvite (MgNH₄PO4:6H₂O) pellets has proven to be a successful method of recovering nitrogen and phosphorus from wastewaters. Thus far, little work has been done on potassium recovery since it is not considered a water pollutant, nor do we face potassium shortages. However, potassium is an essential plant macronutrient and we are seeing worldwide imbalances in nutrient and fertilizer use, as well as a need for a slow release potassium fertilizer. Development of a full complement NPK fertilizer with NH₄-struvite and K-struvite components may have great potential. Given this, research into potassium recovery through crystallization of K-struvite (MgKPO₄: 6H₂O) is relevant and complements previous work done with NH₄-struvite.The goals of this research were to develop fundamental understanding of K-struvite formation as the first step to recovering potassium, and eventually produce a full complement NPK slow-release fertilizer from wastewaters. This required the determination of new solubility product values for K-struvite at different temperatures, followed by bench-scale experiments to assess K-struvite synthesis under various solution conditions. A model to simulate each batch experiment and to predict optimal supersaturation conditions for K-struvite precipitation was developed using PHREEQC, aqueous equilibrium modelling software. Finally, initial experiments in the UBC fluidized bed reactor (UBC-FBR) were undertaken to assess the pelletization potential of K-struvite.New solubility product values for K-struvite indicate that it is less soluble than previously reported, and the values determined at 10, 25 and 35°C fit the Van’t Hoff model. Optimal Mg:K:P molar ratio for synthesis of pure K-struvite was found to be approximately 3:50:1 in a wastewater matrix with pH 8, P-PO₄ concentration of 8 mM and a Mg:P ratio of 3:1. These concentrations were used in the UBC-FBR to assess the pelletization potential of K-struvite. These initial reactor runs were inconclusive due to an inability to stabilize the reactor without seeding. It would be recommended to seed the reactor during start-up in order to be able to compare process performance with the NH₄-struvite crystallization process in the UBC-FBR.This study showed that formation of pure K-struvite is possible given the right supersaturation conditions in solution, requiring high potassium concentrations.

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Effect of previous angular deformation on flexural fatigue resistance of controlled memory nickel-titanium endodontic instruments (2014)

Objective: To evaluate the effect of torsional stress preloading angle on fatigue resistance of Typhoon (TYP) CM instruments. Methodology: TYP NiTi 25/.04, TYP NiTi 40/.04, TYP CM 25/.04 and TYP CM 40/.04 were rotated until fracture to obtain the mean angular deflection according to the +-ISO 3630-1 standard. Files were pre-torqued to 25, 50, and 75% of their elastic limit and then subjected to cyclic loading in a three-point binding device until fracture. The fatigue life was recorded for each file.The fracture surface of each fragment was examined with a scanning electron microscope. Results: The angle of rotation at fracture of TYP CM was significantly higher than that of TYP instruments (P
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Influence of Previous Angular Deformation on Cyclic Fatigue Resistance of K3XF Instruments (2014)

Objective: To evaluate the effect of preloading various degrees of the maximum distortion angle on the cyclic fatigue resistance of post-machining heat-treated nickel-titanium (NiTi) instruments.Methodology: New K3XF and K3 NiTi instruments (size 25/.04 taper)( n = 15) were tested to obtain the mean number of cycles to failure (Nf) using a 3-point bending apparatus. Torque and distortion angles at failure were determined according to the ISO 3630-1 standard. New files were then pre-cycled to four conditions (0%, 25%, 50%, and 75% of the angular deflection) and fatigue resistance tests were performed. After torsional preloading, the total number of revolutions to failure (Nf) was measured for each file. The fracture surface of each fragment was examined with a scanning electron microscope. The crack-initiation sites and the percentage of dimple area of the whole fracture cross-sectional area were recorded.Results: The fatigue resistance of K3XF instruments was two times higher than that of K3 instruments (P
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Effect of fatigue on torsional failure of nickel-titanium controlled memory instruments (2013)

AbstractIntroduction: This study was undertaken to understand how fatigue affects the torsional properties of both traditional nickel-titanium (NiTi) and nickel-titanium controlled memory (CM) files. Methods: Typhoon (TYP) 25.04 and 40.04 rotary files, in both NiTi and CM, were tested using a three-point bending apparatus to obtain the mean number of cycles to failure (mNCF). New files were precycled to four conditions (i.e., 0%, 25%, 50%, and 75% of the mNCF) before torsional resistance tests were performed. Each file was exposed to torsional stress until failure, and at that point the torque and distortion angles were measured. The fracture surface of each fragment was examined with a scanning electron microscope. Results: The TYP CM files yielded a seven-fold improvement in mNCF over the TYP NiTi files (P ≤ 0.05). In both file sizes there was no difference in torque between the CM files and the conventional Ni-Ti files (P > 0.05). The torque of the size 40.04 files was significantly higher than the torque of the size 25/.04 files (P ≤ 0.05). In the 40.04 TYP files group, the 75% precycling group had a significantly lower torque to failure than the no precycling group (P ≤ 0.05). The CM files of both sizes had significantly higher distortion angles than the Ni-Ti files of both sizes (P ≤ 0.05). The 40.04 TYP CM files that were not precycled showed a significantly higher distortion angle than the precycled groups. The fractured files in the precycling groups showed the typical pattern of torsional failure. Conclusions: Cyclic fatigue has an effect on torsional fracture resistance of TYP and TYP CM files on size 40.04. TYP CM files displayed a similar torque value to TYP files but rotated a greater angle before fracture in both preloading and no preloading groups.

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The effect of agitation on the penetration depth of sodium hypochlorite into dentinal tubules. (2013)

Objective: The aim of this study was to compare the difference in irrigant penetration into dentinal tubules of 6% NaOCl using the EndoActivator®, ProUltra® PiezoFlow™ and EndoVac® and to compare them with the standard side-vented ProRinse® needle.Methods: Sixty extracted anterior teeth with single canals were accessed conventionally, the pulp tissue removed and canal patency verified using minimal instrumentation. Crystal Violet dye was placed in the canals for 5 days followed by instrumentation of the canals to standard shape with ProTaper rotary files to size F4 using 1ml of 6% NaOCl used between each file. The teeth were divided into four groups and each agitation system was used with 6% NaOCl as per manufacturers recommendations. Each tooth was mounted in acrylic and cut into 1 mm thick section perpendicular to the long axis of the tooth using the Isomet® Linear Precision Saw (Censico International Pvt. Ltd.) The sections were analyzed with a Nikon® Eclipse® Microscope at 40x magnification and NaOCl penetration was measured with the NIS Elements™ Software (Nikon Corporation).Results: The maximum penetration depth for the ProRinse® side-vented needle, EndoActivator and EndoVac irrigation methods occurred in the coronal third of the canal. However, the maximum penetration depth for the ProUltra® PiezoFlow™ Ultrasonic System occurred in the middle third. With regard to NaOCl Penetration area, the coronal and middle thirds showed better area penetration than the apical third in all irrigation groups.Conclusions: The irrigation methods may affect the highest penetration depth of NaOCl into dentinal tubules at different areas of the root canal position.

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Evaluation of ultrasonic irrigation systems for debris and smear removal in root canals : a scanning electron microscope study (2012)

Objective: The aim of this study was to compare the efficacy of continuous flow ultrasonic irrigation systems to conventional syringe irrigation in removing debris and smear layer in straight and curved root canals. Methods: Twenty-four maxillary recently extracted anterior teeth of curvature less than 10 degrees and 24 mesial roots of mandibular molars with a curvature between 15-30 degrees were instrumented to size 40, 0.04 taper and 35, 0.04 taper, respectively, using 3% sodium hypochlorite. The teeth were divided into three experimental groups according to the final irrigation technique: conventional syringe irrigation with a 30 gauge side vented needle, the PiezoFlow Ultrasonic irrigation system, and the VPro StreamClean Ultrasonic irrigation system. In all experimental groups, 15 mL of 3% sodium hypochlorite was used after instrumentation. Both ultrasonic systems were set at a flow rate of 15 mL/min and used for 1 minute at the ultrasonic power setting recommended by the manufacturer. This was followed by 3 mL of 17% EDTA for 2 minutes and 2 mL of sterile water. The teeth were sectioned and prepared for scanning electron microscope observation to assess the presence of debris and smear layer at the apical level (1, 3, 5 mm) with 200x and 1000x magnification, respectively. The debris was graded using Adobe Photoshop CS5 and two calibrated observers using a five-score scale graded the smear. All grading was blinded. The debris data was analyzed using one-way analysis of variance with Dunett’s test and the smear layer scores were analyzed using Kruskal Wallis. Results: Concerning debris removal, no significant differences among groups were detected, however, the PiezoFlow Ultrasonic system approached significance at the 1 and 3 mm levels in the straight canals. The PiezoFlow Ultrasonic system resulted in significantly more smear layer removal at the 1 mm level in the straight canals compared to conventional syringe irrigation. Conclusion. The final irrigation techniques were unable to completely remove debris or smear layer from the apical 5 mm of the straight and curved canals, however, the PiezoFlow removed significantly more smear layer at the 1 mm level in straight canals.

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Novel ex vivo biofilm model : a comparative study of root canal disinfection (2012)

Bacteria, organized as biofilms within the root canal, can cause apical periodontitis (AP). It has been shown that microorganisms harbouring in the anatomical irregularities of the root canal system such as fins, grooves, or isthmuses after treatment, decrease the outcome prognosis of endodontic therapy. Therefore, adequate removal of these microorganisms is essential for the prevention and treatment of AP. While difficulties in disinfection have been recognized, limited data are available to directly assess the effectiveness of bacteria removal by treatment. Thus, the aim of this study is to develop a standardized ex vivo biofilm model, closely resembling the in vivo clinical situation, to quantify and compare the efficacy of hand, rotary nickel-titanium (NiTi) and self-adjusting file (SAF) instrumentation in the removal of biofilm bacteria. Thirty-six extracted single-rooted human teeth with an ovoid cross-section canal were selected. Each tooth was split longitudinally and a 0.2 mm wide by 0.3 mm deep groove was placed in the apical 2 to 5 mm of the canal. After growing mixed bacteria biofilm inside the canal under anaerobic condition, the split halves were reassembled in a custom block, creating apical vapour lock. Teeth were randomly divided into 3 treatment groups (n = 10 per group) using: (1) hand stainless steel (SS) K-file; (2) ProFile NiTi rotary instrumentation; and (3) SAF. Irrigation consisted of 10 ml 3% sodium hypochlorite and 4 ml 17% ethylenediaminetetraacetic acid. Six teeth received no treatment. Areas of the canal inside and outside the groove were examined using a scanning electron microscope. Within the groove, a smaller area remained occupied by bacteria after the use of SAF than ProFile and SS K-file (3.25%; 19.25%; 26.98%) (P .05).Although all techniques equally removed bacteria outside the groove, SAF significantly reduced more bacteria from within the apical groove. No technique was able to completely remove the bacteria. The biofilm model represents a potentially useful tool for future study of root canal disinfection.

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The effect of flow rate, needle design, and needle placement on apical pressure and irrigant exchange in an in vitro tooth model (2012)

Heavy irrigation forces in a root canal can lead to irrigant extrusion accidents, causingnegative sequelae for patients. There are no guidelines for a safe and optimal irrigation flowrate and little data on the apical pressures generated during irrigation. This study aimed tomeasure the pressure generated during positive and negative pressure irrigation at theperiapex of an in vitro tooth model using a novel method of measurement, investigating theeffect of flow rate, depth of needle placement, and needle design. Apical pressure wascorrelated with extent of dye clearance from the end of a needle tip in a similar model.A mandibular molar was prepared and placed into a chamber coupled to a pressuretransducer. Irrigation was performed using a digital peristaltic pump using flow rates from1-15 ml/min with irrigation needles of different size and design. A similar plastic root canalmodel filled with dye was used to measure the extent of dye clearance beyond the needle tipusing the same irrigation conditions.Positive pressure irrigation revealed a flow rate dependent and depth of needle placementdependent increase in apical pressure (P
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A survey of the irrigation protocols used by dentists in British Columbia, Canada (2011)

The goal of endodontic treatment is to prevent and eliminate infection. This is achieved by mechanical and chemical cleaning of the root canal. Chemical cleaning is a process in which irrigants are introduced in the root canal to eradicate bacteria and flush out debris. A study was conducted to assess the type, concentration of sodium hypochlorite (NaOCl), volume, sequence and method of delivery system of different irrigants used by general practitioners (GP) and endodontists (ENDO) in treating teeth with vital (VP) or non-vital pulp (NVP). We hypothesized that there are no significant differences between the groups regarding the method of irrigation in root canal treatment. Methods: 1) A total of 68 samples of bleach were collected from GP offices in Vancouver. Using titration, the concentration of hypochlorite in these bleach samples was calculated. 2) A questionnaire was sent to 150 GP and 42 ENDO registered with the College of Dental Surgeons of British Columbia. Results: The overall response rate to our questionnaire was 70.3%. Irrigants used in treating teeth with VP were NaOCl: ENDO 93.4%, GP 89.9% (p=0.445), EDTA: ENDO 72.9%, GP 35.1% (p
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Quantitative analysis of the effect of irrigation sequences on root canal wall dentin erosion (2011)

Objectives: The purpose of the present study was to examine the effect on root canal wall dentin and compare the level of erosion caused by different irrigation sequences. Material and methods: Dentin specimens of the middle third of the root of extracted teeth with one root canal were instrumented and randomly divided into five groups. Each group was subjected to 17% ethylenediaminetetraacetic acid (EDTA), 17% ethylene-glycoltetraacetic acid (EGTA), or 10% citric acid (CA) and 5.25% sodium hypochlorite (NaOCl) varying the time of irrigation and the order of the irrigants. The blocks were prepared for and examined by scanning electron microscopy. Digital images at a magnification of 2000× were taken at randomized areas on the root canal dentin surface, and the area of tubule openings was measured by a semi-automatic method using image analysis software image-Pro Discovery 5.0. Results: Erosion of peritubular and intertubular dentin was detected when EDTA, EGTA or CA were used as the initial rinse (even for 30 s ), followed by 5.25% NaOCl. The area of dentin tubule opening increased markedly when compared to the sequences where NaOCl was used first, before the chelators or CA (P
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