Abstract
Objectives
The objective of the present in vitro study was to evaluate the canal wall smear layer removal capacity and mineral content distribution of root canal dentine after irrigation with QMix, 7% maleic acid (MA) and 17% ethylenediaminetetraacetic acid(EDTA).
Methods
Forty single-rooted teeth were subjected to root canal instrumentation and divided into four groups: [1] 7% MA + 2.5% sodium hypochlorite (NaOCl), [2] 17% EDTA+ 2.5% NaOCl, [3] QMix + 2.5% NaOCl and [4] 0.9% saline (negative control). After irrigation, the teeth were examined byscanning electron microscopy (SEM) to determine the presence or absence of smear layer. Formineral content assessment, 40 root-halves were divided into four groups and treated with 7% MA, QMix, 17% EDTA and saline. Mineral content was evaluated using SEM-energy dispersive X-ray analysis.
Results
There was no significant difference between QMix, MA and EDTA in removal of smear layer from coronal and middle third of the canal spaces. In the apical third, MA performed better. Calcium was decreased more with QMix with no difference between MA and EDTA. Phosphorous was reduced more with MA and QMix than EDTA with no difference between MA and QMix. Similar result was observed with magnesium level. Carbon was reduced more with EDTA with no difference between QMix and MA. Oxygen was reduced significantly more with MA with no difference between QMix and EDTA.
Conclusion
7% MA had superior smear layer removal ability compared with QMix and 17% EDTA. Calcium level was decreased more with QMix while phosphorus level was decreased more with 7% MA and QMix respectively.
Clinical significance
The present study highlights the effect of newer chelating agentson smear layer removal and decalcification of root canal dentine, which is required for disinfection of the root canal space and maintenance of the structural integrity of the teeth.
1
Introduction
Success of root canal treatment of teeth with pulpal necrosis and apical periodontitis depends on complete eradication of microorganisms fromthe infected canals. Cleaning and shaping ofthe root canal system are considered the key requirements in eliminating microorganisms. Hand and rotary instrumentation of the root canal system produces an irregular, granular andtenacious layer covering the canal wall surfaces known as the smear layer . It consists of inorganic dentine debris, pulp tissues, severed odontoblastic processes, necrotic debris, microorganisms and their metabolic products . Studies have demonstrated that removal of smear layer improves the fluid-tight seal offilled root canals, eliminates bacteria within the dentinal tubules and facilitates the penetration of intracanal medicaments, irrigating agents and sealers into the dentinal tubules . It has also been demonstrated thatsmear layer removal increases the bond strength ofresin sealers to root canal dentine , resulting in a significantly better apical seal .
To date, no irrigating solution is capable of removing both the organic and inorganic components of the smear layer. Hence, a number of studies suggested the use of a combination of irrigants . Combined application of ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite (NaOCl) is frequently recommended for effective removal ofthe smear layer from the root canal system . Seven percent maleic acid (MA) is a chelating agent that has been found to possess better smear layer removal capability when compared to 17% EDTA . It is also less cytotoxic when compared to17% EDTA . QMix ® 2in1 (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA; referred to as QMix thereafter) is a root canal irrigant which contains EDTA, chlorhexidine, a detergent and water. It has shown to removethe smear layer as effectively as 17% EDTA . QMix is also aseffective antimicrobial agent against Enterococcus faecalis biofilms when compared to BioPure ® MTAD ® (mixture of doxycycline, citric acid, and Tween 80; Dentsply Tulsa Dental Specialties) and chlorhexidine gluconate . Calcium (Ca 2+ ) and phosphorus (PO 4 3− )in carbonated apatite are the major inorganic components of dental hard tissues. Alteration in the Ca 2+ /PO 4 3− ratio changes the permeability, microhardness and solubility of root canal dentine and may also adversely affect the sealing ability of resin-based cements and sealers to root canal dentine .
Comparative studies evaluating the smear layer removal and demineralizing effect of QMixand MA on root canal dentine are lacking. Hence, the objectives of the present in vitro study were to evaluate the efficacy of smear layer removal from canal walls, and to determine mineral content distribution of root canal dentine after treatment with QMix, 7% MA and 17% EDTA solutions.The null hypothesistested were: (1) there are no differences in the ability of QMix, 7% MA or 17% EDTA to remove canal wall smear layer when these solutions are used as final root canal irrigants, and (2) there is no difference in the mineral content of root canal dentine after the use of QMix, 7% MA or 17% EDTA as final irrigants for root canal debridement.
2
Materials and methods
2.1
Smear layer evaluation
2.1.1
Specimen preparation
Ethical clearance for the use of human teeth for the experiments performed in the present study was obtained from the institutional review board (IEC 166/2015) of Manipal University, India. Forty extracted human maxillary anterior teeth with straight root and single canal were selected. All the teeth were radiographed to verify the presence of a single canal with closed apex, and the absence of intra radicular resorption or root canal filling. Superficial soft tissues were removed with a curette and the teeth were stored in 0.2% sodium azide (Millipore Sigma, St. Louis, MO, USA) at 4 °C until use. The teeth were decoronated to standardise the root length to 15 mm longand the specimenswere randomly divided into three experimental groups and a control group (n = 10). Working length was established by inserting a size 10 K file (Mani Inc., Tochigi Ken, Japan) into each root canal until it was just visible at the apical foramen (observed using magnifying loupes) and by subtracting 1 mm from the recorded length. Chemomechanical preparation was performed using ProTaper ® nickel titanium rotary instruments (Dentsply Maillefer, Ballaigues, Switzerland) and the canals wereinstrumented to size F3. Irrigation was performed with 2 mL of 2.5% of NaOCl (KMC Pharmacy, Manipal, India) after each instrument change.
2.2
Irrigation techniques
The final irrigation sequences were: (I) QMix group: 5 mL of 2.5% NaOCl for 1 min followed by 5 mL of QMix for 1 min; (II) MA group: 5 mL of 2.5% NaOCl for 1 min followed by 5 mL of 7% MA for 1 min; (III) EDTA group: 5 mL of 17% EDTA for 1 min; (IV) Negative control: 5 mL of 2.5% NaOCl for 1 min followed by 5 mL of 0.9% saline for 1 min. All the irrigating solutions were introduced into the canal using 29 gauge stainless steel needles (NaviTip ® Tips, Ultradent Products Inc., South Jordan, UT, USA). The needle tip was inserted to 1 mm of the working length in each canal. After the use of the aforementioned irrigation protocols, each root canal was irrigated with 5 mL of deionised water to remove any precipitate that might have been formed. The canals were then dried with sterile paper points (Dentsply, Maillefer, Ballaigues, Switzerland). Longitudinal grooves werethen prepared on the buccal and lingual surfaces of each root using a diamond disc (Horico Dental, Berlin, Germany) at slow-speed without perforating the canal. The roots were then split into two halves using a chisel and stored in deionised water at 37 °C for scanning electron microscope (SEM) examination.
2.3
Scanning electron microscopy
The specimens were dehydrated using ascending grades of ethanol (25%, 50%, 75% and 100%), mounted on metal stubs, coated with gold/palladium using an ion-sputtering machine and examined with aJSM-6010 SEM (JEOL, Tokyo, Japan) for the presence or absence of the smear layer. Images were taken to observe the canal wall surface morphology at 500× magnification and 20 kV alongthe coronal (10–12 mm from apex), middle (6–7 mm from apex) and apical (1–2 mm from apex) thirds of each specimen. These areas were evaluated by two independent evaluators who were blind to the group designations. The images were scored according to the criteria reported by Torabinejad et al. : 1 = no smear layer (no smear layer on the surface of the canalwall; all dentinal tubules clean and open); 2 = moderate smear layer (no smear layer on the surface of the canal wall; tubules contain debris); and 3 = heavy smear layer (smear layer covering the canal wall and tubules).
2.4
Evaluation of mineral content
Twenty human maxillary central incisors extracted for periodontal reasons were selected and prepared using the previously described criteria, and stored in 0.2% sodium azide at 4 °C until use. The teeth were decoronated at the cementoenamel junction and the apical portions of the teeth were removed using a high speed diamond disc (Horico Dental, Germany) under water cooling. The length of each root was standardised to 10 mm long. The roots were then split longitudinally to obtain 40 root-halves; pulpal tissues present were removed with a fine brush. All specimens were dehydrated using Drierite for 14 days and weighed on a precision balance (AT 261, Mettler, Greifensee, Switzerland) so that each specimen weighed 0.20 g. Weight equality among the specimens was achieved by reducing the weight whenever necessary with silicon carbide abrasive paper from the cementum of the specimens. Thereafter, each specimen was covered with two consecutive layers of nail varnish, leaving the root canal surface exposed. The specimens were randomly divided into 4 groups (n = 10):
- (I)
QMix group: treated with QMix for 1 min;
- (II)
MA group: treated with 7% MA for 1 min;
- (III)
EDTA group: treated with 17% EDTA for 1 min; and
- (IV)
Negative control: treated with deionised water for 1 min.
In each group, the specimens were immersed in a magnetic stirrer bath containing 10 mL of the respective experimental solution. After one min, all the specimens were removed from the stirrer bath, thoroughly rinsed with deionised water and dried using absorbent lint-free paper.The specimens were then mounted on metal stubs, sputtered with gold/palladiumand prepared for mineral content analysis. The levels of calcium (Ca), phosphorus (P), magnesium (Mg), carbon (C) and oxygen (O) in the canal wall dentine surface of each specimen were measured using SEM-energy dispersive X-ray spectrometry (EDS; JEOL, Japan) with a minimum detectable range of 1%. Each specimen was irradiated at the centre and at two other equidistant areas, at a voltage of 20 kV for 60 s. Changes in the mineral levels were recorded and differences among the groups were statistically analysed.
2.5
Statistical analyses
For smear layer evaluation, the inter-examiner’s reliability was rated using coefficient of Kappa test. The mean was used to summarize the smear scores of the photomicrographs taken at each level of each specimen to account for the clustered nature of the data. The Cochran–Mantel–Haenszel (CMH) test was then used to test for significant differences amongst treatment groups separately at each canal level. If a significant difference amongst treatment groups was found, the CMH method was used to test comparisons among the treatment groups, adjusting for the effect of canal level. For mineral content analysis, comparison was performed non-parametrically using Kruskal-Wallis analysis of variance. Post-hoc pairwise comparisons were performed using the Dunn’stest. For all analyses, the significance level was preset at α = 0.05.
2
Materials and methods
2.1
Smear layer evaluation
2.1.1
Specimen preparation
Ethical clearance for the use of human teeth for the experiments performed in the present study was obtained from the institutional review board (IEC 166/2015) of Manipal University, India. Forty extracted human maxillary anterior teeth with straight root and single canal were selected. All the teeth were radiographed to verify the presence of a single canal with closed apex, and the absence of intra radicular resorption or root canal filling. Superficial soft tissues were removed with a curette and the teeth were stored in 0.2% sodium azide (Millipore Sigma, St. Louis, MO, USA) at 4 °C until use. The teeth were decoronated to standardise the root length to 15 mm longand the specimenswere randomly divided into three experimental groups and a control group (n = 10). Working length was established by inserting a size 10 K file (Mani Inc., Tochigi Ken, Japan) into each root canal until it was just visible at the apical foramen (observed using magnifying loupes) and by subtracting 1 mm from the recorded length. Chemomechanical preparation was performed using ProTaper ® nickel titanium rotary instruments (Dentsply Maillefer, Ballaigues, Switzerland) and the canals wereinstrumented to size F3. Irrigation was performed with 2 mL of 2.5% of NaOCl (KMC Pharmacy, Manipal, India) after each instrument change.
2.2
Irrigation techniques
The final irrigation sequences were: (I) QMix group: 5 mL of 2.5% NaOCl for 1 min followed by 5 mL of QMix for 1 min; (II) MA group: 5 mL of 2.5% NaOCl for 1 min followed by 5 mL of 7% MA for 1 min; (III) EDTA group: 5 mL of 17% EDTA for 1 min; (IV) Negative control: 5 mL of 2.5% NaOCl for 1 min followed by 5 mL of 0.9% saline for 1 min. All the irrigating solutions were introduced into the canal using 29 gauge stainless steel needles (NaviTip ® Tips, Ultradent Products Inc., South Jordan, UT, USA). The needle tip was inserted to 1 mm of the working length in each canal. After the use of the aforementioned irrigation protocols, each root canal was irrigated with 5 mL of deionised water to remove any precipitate that might have been formed. The canals were then dried with sterile paper points (Dentsply, Maillefer, Ballaigues, Switzerland). Longitudinal grooves werethen prepared on the buccal and lingual surfaces of each root using a diamond disc (Horico Dental, Berlin, Germany) at slow-speed without perforating the canal. The roots were then split into two halves using a chisel and stored in deionised water at 37 °C for scanning electron microscope (SEM) examination.
2.3
Scanning electron microscopy
The specimens were dehydrated using ascending grades of ethanol (25%, 50%, 75% and 100%), mounted on metal stubs, coated with gold/palladium using an ion-sputtering machine and examined with aJSM-6010 SEM (JEOL, Tokyo, Japan) for the presence or absence of the smear layer. Images were taken to observe the canal wall surface morphology at 500× magnification and 20 kV alongthe coronal (10–12 mm from apex), middle (6–7 mm from apex) and apical (1–2 mm from apex) thirds of each specimen. These areas were evaluated by two independent evaluators who were blind to the group designations. The images were scored according to the criteria reported by Torabinejad et al. : 1 = no smear layer (no smear layer on the surface of the canalwall; all dentinal tubules clean and open); 2 = moderate smear layer (no smear layer on the surface of the canal wall; tubules contain debris); and 3 = heavy smear layer (smear layer covering the canal wall and tubules).
2.4
Evaluation of mineral content
Twenty human maxillary central incisors extracted for periodontal reasons were selected and prepared using the previously described criteria, and stored in 0.2% sodium azide at 4 °C until use. The teeth were decoronated at the cementoenamel junction and the apical portions of the teeth were removed using a high speed diamond disc (Horico Dental, Germany) under water cooling. The length of each root was standardised to 10 mm long. The roots were then split longitudinally to obtain 40 root-halves; pulpal tissues present were removed with a fine brush. All specimens were dehydrated using Drierite for 14 days and weighed on a precision balance (AT 261, Mettler, Greifensee, Switzerland) so that each specimen weighed 0.20 g. Weight equality among the specimens was achieved by reducing the weight whenever necessary with silicon carbide abrasive paper from the cementum of the specimens. Thereafter, each specimen was covered with two consecutive layers of nail varnish, leaving the root canal surface exposed. The specimens were randomly divided into 4 groups (n = 10):
- (I)
QMix group: treated with QMix for 1 min;
- (II)
MA group: treated with 7% MA for 1 min;
- (III)
EDTA group: treated with 17% EDTA for 1 min; and
- (IV)
Negative control: treated with deionised water for 1 min.
In each group, the specimens were immersed in a magnetic stirrer bath containing 10 mL of the respective experimental solution. After one min, all the specimens were removed from the stirrer bath, thoroughly rinsed with deionised water and dried using absorbent lint-free paper.The specimens were then mounted on metal stubs, sputtered with gold/palladiumand prepared for mineral content analysis. The levels of calcium (Ca), phosphorus (P), magnesium (Mg), carbon (C) and oxygen (O) in the canal wall dentine surface of each specimen were measured using SEM-energy dispersive X-ray spectrometry (EDS; JEOL, Japan) with a minimum detectable range of 1%. Each specimen was irradiated at the centre and at two other equidistant areas, at a voltage of 20 kV for 60 s. Changes in the mineral levels were recorded and differences among the groups were statistically analysed.
2.5
Statistical analyses
For smear layer evaluation, the inter-examiner’s reliability was rated using coefficient of Kappa test. The mean was used to summarize the smear scores of the photomicrographs taken at each level of each specimen to account for the clustered nature of the data. The Cochran–Mantel–Haenszel (CMH) test was then used to test for significant differences amongst treatment groups separately at each canal level. If a significant difference amongst treatment groups was found, the CMH method was used to test comparisons among the treatment groups, adjusting for the effect of canal level. For mineral content analysis, comparison was performed non-parametrically using Kruskal-Wallis analysis of variance. Post-hoc pairwise comparisons were performed using the Dunn’stest. For all analyses, the significance level was preset at α = 0.05.
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