Evaluation of the effect of cetrimide with edta on Microhardness and surface changes in root dentin – An invitro study

INTRODUCTION

The success of endodontic therapy depends on the technique, the quality of instrumentation, irrigation, disinfection and three dimensional obturation of the root canal system. Endodontic instrumentation produces a smear layer and plugs of organic and inorganic particles of calcified tissue. The smear layer contains additional organic elements such as pulp tissue debris, odontoblastic processes, micro-organisms and blood cells in the dentinal tubules1. The smear layer increases microflora and the inorganic toxins by increasing the potential for bacterial survival and reproduction.²

The smear layer has a negative impact on the root canal adhesion because it will act as a physical barrier between the filling material and canal walls, undermining sealer penetration and formation of intratubular tags.³ Whether to remove the smear layer or not, had been a matter of issue for many years. Arecent systematic review and meta-analysis of leakage studies had showed that the smearlayer removal improves the fluid tight seal of the root canal system.⁴ The canals are irrigated with a solution capable of disinfecting them anddissolving the organic matter during instrumentation.⁵

The current methods to remove the smear layer involve the use of achelating agent, in combination with other irrigants. NygardOstby in 1957 introduced chelating agents in endodontics for the preparation ofnarrow and calcified root canals. Ethylene Di-amine Tetra-acetic acid (EDTA) was the first chelator used in dentistry. Ostby showed that EDTA chelated metallic ions at neutral pH,allowing the instrumentation of difficult canals.6 EDTA is capable of softening the rootcanal dentin, dissolving the smear layer and increasing the dentin permeability.⁷ 17% EDTA(pH of 5−9) is the most commonly used chelating agent for endodontic irrigation.⁵

The efficacy of irrigating solutions can be improved by the addition of surface active agents.

These chemicals help in reducing the surface tension and increasing the wettability ofsolutions.Improved bactericidal effect of irrigants with the addition of surface active agents is related to the weakening of the cohesive forces in extracellular polymeric substance and bacterial membranes.Surface active agents are also known as surface modifiers or surfactants. EDTA solution acts only through direct contact with the substrate. Thus the addition of surfactants to the EDTA results in increased cleaning efficacy and improved ability to penetrate the dentin.⁷

Cetrimide and sodium lauryl ether sulfate have been generally used for this purpose. Addition of a surfactant to the EDTA results in effective smear layer removal compared to EDTA solution alone, producing clean dentinal surface as well as open dentinal tubules⁸. Cetrimide increases the capacity of EDTA to eradicate E.faecalis biofilms without affecting their decalcifying ability.⁹

The structural properties of dentin, such as microhardness, permeability and solubility may change after the use of chemical irrigants.⁵ In relation to root dentin, the chelating agent reacts with the calcium ions in the hydroxy apatite crystals.¹⁰ This process can cause changes in the microstructure of the dentin and also in Calcium-Phosphorus ratio (CaP ratio) of the dentin surface. Alterations in the Ca-P ratio in turn lead to the changes in permeability and solubility characteristics of the dentin.The changes in the mechanical, chemical and physical properties of dentin structure also affect its adhesiveness to the obturating materials.¹

EDTA demineralizes the inorganic components of dentin by chelating calcium ions, resulting in decreased microhardness of the dentin. Microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues.¹¹ CruzFilhoet al¹². Reported that the action of 17% EDTA in decreasing dentin microhardness can be observed within the first minute after application of this chelator. As the contact time with the solution increases the dentin microhardness decreases.

In addition to the decrease in the microhardness, irrigation solutions alter the surface texture of the root dentin. Application of EDTA to root dentin has shown eroded appearance and enlarged tubular orifice diameters.¹³ Surface roughness produced is directly proportional to the concentration of EDTA.⁵

The purpose of this in vitro study was to evaluate the effect of different concentrations of cetrimide with or without EDTA solution on the microhardness and surface changes of root dentin.

MATERIALS AND METHODS

One hundred and twenty extracted human mandibular premolar teeth were collected from the Department of Oral and Maxillofacial Surgery, D.A.P.M.R.V Dental College, Bengaluru. The premolar teeth that were extracted for orthodontic and periodontal reasons were used in this study. Only single rooted mandibular premolar teeth, with mature root apex, without anatomic variations, caries, cracks and restorations were selected. The teeth were cleaned of surface debris and calculus with ultrasonic scalers.

Access cavity was prepared using low speed micromotor and access opening bur. The length was recorded by placing #10 K-file just seen through the apical foramen. The working length was determined by reducing 1mm from this recorded length. The cleaning and shaping of root canals was done till # 35 size K- file upto the working length. Recapitulation was done during instrumentation. One Shape rotary system was used with crown down technique. The speed and torque were adjusted according to manufacturer specifications. During instrumentation, normal saline was used for root canal irrigation after each file use.

Teeth were decoronated at the cementoenamel junction using low speed diamond disc. The roots were sectioned longitudinally into 2 parts using low speed micromotor with diamond disc under water coolant. The root segments were horizontally embedded in autopolymerizing resin with their dentine surfaces exposed. The dentine surfaces of mounted specimens were grounded smooth with increasingly fine emery papers under distilled water and finally were polished using 0.1μm alumina suspension on felt cloth.

Preparation of test solutions:

5% EDTA solution was prepared by mixing 5 gm of 100% EDTA powder in 100ml of distilled water. 0.25% cetrimide and 0.50% cetrimide solutions were prepared by adding 0.25 gm and 0.50 gm of 100% cetrimide powder to 100ml of distilled water respectively. Magnetic stirrer was used to mix the contents in distilled water.

Classification of the experimental groups:

One hundred and twenty mandibular premolar roots were sectioned longitudinally to obtain two hundred and forty specimens. These specimens were randomly divided into following 6 groups (n=40) according to the irrigating solution used for treatment.

Group 1: The specimens were treated with 5% EDTA for 1 minute.

Group 2: The specimens were treated with 5%EDTA + 0.25% Cetrimide for 1 minute.

Group 3: The specimens were treated with 5%EDTA + 0.50% Cetrimide for 1 minute.

Group 4: The specimens were treated with 0.25% Cetrimide for 1 minute.

Group 5: The specimens were treated with 0.50% Cetrimide for 1minute.

Group 6: The specimens were treated with distilled water for 1 minute (control).

Every group was divided into two subgroups containing 20 specimens each. Group 1a, 2a, 3a, 4a, 5a and 6a specimens were employed for microhardness testing. Group 1b, 2b, 3b, 4b, 5b and 6b specimens were used to evaluate the surface changes of root dentin under stereomicroscope.

Surface treatment:

Immediately after baseline measurements of microhardness, each specimen of the first subgroup was immersed in 50ml of irrigating solution according to the test group for 1 minute. At the end of active treatment period, the specimens were rinsed with distilled water and blotted dry. Post-treatment microhardness values were obtained.

After the initial stereomicroscope evaluation, the specimens of second subgroup were treated by immersion in test solution as described above. Then specimens were rinsed with distilled water and blotted dry. Surface changes were evaluated under stereomicroscope.

Microhardness testing:

In this study dentin microhardness was determined using Future Tech microhardness indenter installed in the Department of Materials Science Engineering, Indian Institute of Science, Bangalore. The specimens were mounted on stage of Vicker’smicrohardness tester. 50-g load and a10-second dwell time was used. Three separate indentations were made parallel to the edge of the root lumen at the mid-root level. Indentations were made at the depth of 100μm from the pulp–dentin interface. The lengths of the 2 diagonals were used to calculate the vicker’smicrohardness number (VHN). The representative hardness values were calculated by taking the average of the 3 indentations. Microhardness values were obtained before and after treatment with test solutions. 

Evaluation of surface changes:

In this study surface changes were evaluated by stereomicroscope in the Department of Oral and Maxillofacial Pathology, D.A.P.M.R.V. Dental College, Bengaluru. Specimens were viewed at 4X magnification. Images were obtained before and after the surface treatment using a digital camera. Two observers evaluated the images to eliminate the bias.

The scorings were given as:

0- Significant erosion not seen.

1- Significant erosionseen.

Statistical analysis:

The means of Vickers hardness values of the specimens, before and after treatment with the test solutions, were calculated. The mean microhardness values of the specimens before and after the treatment with test irrigants were represented in the Table -1.

Comparison of the mean VHN of six groups was done using ANOVA followed by post hoc Tukey’s test. Pre- and post- treatment hardness values were compared using student paired t test. The surface roughness data was subjected to statistical analysis using Chi square test.

RESULTS

The data obtained by microhardness tests and surface roughness evaluation was statistically analyzed.Student paired t test was done to compare the mean microhardness of the specimens within each group before and after immersion in test irrigants. The results of student paired t test were given in Table-4 and represented as bar diagram in Graph-1. All the groups significantly reduced the microhardness of dentin (p<0.001) except for the distilled water. The greatest reduction in dentin microhardness was observed with Group-3.

Statistical analysis of the data obtained from erosion scores was given in TABLE-5. Chi square test was performed to compare surface erosion seen among in the specimens (p<0.001). 55% of the Group -1 specimens showed significant erosion.

DISCUSSION

Thorough cleaning and shaping of root canals is a pre-requisite for the endodontic obturation.

This can be achieved by proper instrumentation, debridement and disinfection of the root canal. Use of chelating agents is required to remove the smear layer created during instrumentation, thereby allowing medicaments to penetrate the dentinal tubules.⁷ Various chelating agents such as EDTA, citric acid, MTAD, Tetraclean, CDTA, EGTA, QMix, PAA have been used for smear layer removal during endodontic therapy^1,2,6,26.

17% EDTA is the commonly used chelating agent for endodontic irrigation. Studies have shown significant microhardness reduction^5,10,14,35 and excessive erosion^5,10 with this concentration of EDTA. In a study by Akcay and Sen,⁷ 5% EDTA showed significant microhardness reduction.⁴⁴ Thus 5% EDTA was used in this study.

Surfactants are wetting agents added to the irrigants in order to reduce the surface tension of the irrigating solutions. Cetrimide is commonly added to EDTA to improve its clinical performance.⁷ Different concentrations of cetrimide were used in combination with EDTA. Use of 0.2% cetrimide incombination with EDTA exhibited enhanced antimicrobial activity against E.faecalis.^9,22 As cetrimide was proposed to control crystallization process, to find out its effect with orwithout EDTA association and also in increased concentrations, 0.25% and 0.50% cetrimide solutions were used in the present study.

Microhardness can provide an indirect evidence of the mineral loss or gain in the dental hard tissues. Craig et al. had suggested that the microhardness of dentine may vary considerably within teeth.⁴⁶ Comparison of dentine hardness values before and after the irrigation treatment was made within the same root sample in order to minimize the effect of the structural variations of different teeth and to establish a reasonable baseline for evaluation.

EDTA caused aggressive canal wall erosion and decline in dentine flexural strength after prolonged use of NaOCl as an initial rinse.²¹ The evaluation of the effects of EDTA and cetrimide on the microhardness and surface changes would be influenced by the use of NaOCl, hence normal saline was used as an irrigant in this study.

A study on antimicrobial activity of irrigants had concluded that the combination of EDTA and cetrimide could eradicate E.faecalis biofilm after 1 minute of contact time.²² The demineralizing action of chelating agents results in increased exposure of organic content. This organic matrix of dentine acts as a limiting factor for further dissolution of inorganic content, thus reducing the decalcifying action of chelating solutions over time.³⁹ Thus in this study immersion time for the specimens in test solutions was limited to 1 minute.

In this study, Vickers microhradness tester was employed to determine the dentin microhardness. The indentations for the microhardness testing were made 100μm from the pulp-dentin interface.All the groups significantly reduced microhardness except for distilled water (p<0.001)indicating that these irrigants interfere with the chemical composition of dentin structure²⁸. 5% EDTA with 0.50% cetrimide resulted greater reduction in microhardness whereas 5% EDTA alone and its combination with 0.25% cetrimide had similar values⁴³. In a study by Akcay and Sen⁷ similar to our study, 5% EDTA combined with 0.50% cetrimide showed maximum reduction in dentin microhardness. In contrast, a study by Poggioet al39 concluded that cetrimideaddition did not improve decalcifying capability of EDTA.In present study, increased concentration of cetrimide might be the cause for greater reduction in microhardnessdue to facilitation of deeper penetration of irrigants¹⁹. Both EDTA and its combination with 0.50% cetrimide reduced dentin microhardness significantly than 0.25% and 0.50% concentrations of plain cetrimide. Thiscan be attributed to the strong chelating property of EDTA.

Cetrimide is a cationic surfactant exhibiting potent antimicrobial action on biofilm components with no decalcifying effect on the root dentin³⁹. This could be the reason for lesser reduction in microhardness with plain cetrimide than in association with EDTA. ⁵%EDTA and its combination with 0.25% cetrimide showed similar mean difference values for microhardness reduction. At lower concentrations of cetrimide addition microhardness reductionmight be largely due to chelating action of EDTA. Validating this result EDTA showedreduction in microhardness both in the presence and absence of surfactant in a study by Aslantaset al³⁶. In contrary, a previous study had showed that EDTA reduced microhardness more than EDTAC [EDTA and cetrimide]³⁵.

Association of EDTA with 0.50% cetrimide showed greater reduction in microhardness. This can be explained by the fact that cetrimide addition can elongate the hydroxyapatite nanorods. The length – diameter ratio of hydroxyapatite nanorods decreases when the cetrimide content increases⁷. In addition to the reduction in microhardness, the chelating agents cause erosion of the canal wall. Surface changes of dentin and enamel are of concern as they affect the interaction of these tissues with materials used for obturation and coronal restoration¹⁴.

Two investigators scored the erosion in a blind manner similar to the study conducted by Saghiri et al¹⁹.Significant erosion on the root dentin surface was observed under stereomicroscope with 5% EDTA. 55% of the Group -1 (5% EDTA) specimens showed dentin surface erosion. The erosive effect of EDTA has been established by a number of studies validating our result in this regard^5,10. In a study by Tartari et al³¹ only the irrigation regimens that used chelating agents were capable of increasing the roughness of canal dentin.

Saghiri et al. had tried to establish the relation between erosion and microhardness of root Dentin¹⁹. They found that erosion was not the main factor in decreasing the microhardness. Irrigant with greater depth of penetration caused greater reduction in microhardness.

In our study, 5% EDTA in association with 0.50% cetrimide showed greater reduction in microhardness whereas significant erosion was observed with 5% EDTA alone. This could be due to increased wettability by cetrimide addition resulted in greater microhardness reduction unrelated to its lower erosive effect compared to plain EDTA.

CONCLUSION

Within the limitation of the study it was concluded that:

• All the tested irrigating solutions reduced the microhardness of root dentin except distilled water

.• Addition of 0.50% cetrimide to EDTA caused maximum reduction in microhardness.

• EDTA increased the surface roughness ofroot dentin irrespective of cetrimide association.

• Cetrimide addition did not increase the surface erosion as observed understereomicroscope.