Retrievabilty of calcium hydroxide intracanal medicament with Chitosan from root canals: An in vitro CBCT volumetric analysis.

AIM: This study compared the amount of aqueous-based and oil-based calcium hydroxide [Ca(OH)2 ] remaining in the canal, after removal with two different chelators 17% EDTA and 0.2% Chitosan in combination with ultrasonic agitation.
MATERIALS AND METHODS: Cleaning and shaping of root canals of 28 mandibular premolar was done and canals were filled either with Metapex or Ca(OH)2 mixed with distilled water. Volumetric analysis was performed utilizing cone beam-computed tomography (CBCT) after 7 days of incubation. Ca(OH)2 was removed using either 17% EDTA or 0.2% Chitosan in combination with ultrasonic agitation. Volumetric analysis was repeated and percentage difference was calculated and statistically analysed using Kruskal-Wallis and Mann-Whitney U test.
RESULTS: Both the chelators failed to remove aqueous-based as well as oil-based Ca(OH)2 completely from the root canal. Aqueous-based Ca(OH)2 was easier to be removed than oil-based Ca(OH)2. 0.2% Chitosan was significantly more effective for removal of oil-based Ca(OH)2 (P < 0.01) while both 17% EDTA and 0.2% Chitosan were equally effective in removing aqueous-based Ca(OH)2 .
CONCLUSION: Combination of 0.2% Chitosan and ultrasonic agitation results in lower amount of Ca(OH)2 remnants than 17% EDTA irrespective of type of vehicle present in the mix.

INTRODUCTION

Bacteria have been implicated in the pathogenesis and progression of pulp and periapical diseases.[1] It can exist even after chemo-mechanical preparation within root canal itself or within other related regions such as the dentinal tubules, accessory canals, fins etc.[2] Ca(OH)2 is widely used as a root canal disinfectant during root canal treatment due to its alkaline pH.[34] Its antibacterial efficacy is also influenced by the type of vehicle used for mixing.[5]

Before obturating the canal, complete removal of Ca(OH)2 is mandatory as both in vitro and clinical case have shown that residual Ca(OH)2 may have negative influence on success of root canal treatment.[6789] Thus, complete and predictable removal of Ca(OH)2 before obturation is critical and several methods viz., ultrasonics, sonic, canal brush, etc. and irrigating agents including NaOCl, EDTA, their combination, maleic acid, etc. have been used to accomplish this task.[10111213]

Many studies conducted to assess the efficiency of techniques and agents for removal of Ca(OH)2 found, removal difficult and incomplete.[1415] Lambrianidis et al.[11] concluded that 45% surface remained covered with Ca(OH)2 when NaOCl and EDTA were used. They associated difficulty in retrieval with the type of vehicle used for mixing Ca(OH)2. Type of vehicle also affects calcium release and diffusion and consequently pH.[1617] Kenee et al.[14] found rotary instrumentation and ultrasonics better than hand instrumentation for removal of Ca(OH)2. Passive ultrasonic irrigation is accomplished with a small file placed at the center of a previously shaped root canal and activated to produce acoustic streaming that transfers its energy to the irrigant inside the canal.[18]

Chitosan, a natural polysaccharide prepared by the deacetylation of chitin, is endowed with properties of biocompatibility, biodegradability, bio-adhesion, and atoxicity. Its abundance in nature and low production costs has made it ecologically interesting for various applications in the areas of medicine and pharmaceuticals. In dentistry it has been used as a barrier membrane for periodontal therapy and as oral mucosal delivery agent for chlorhexidine. In addition it has high chelating capacity for different metal ions in acidic conditions. In a study conducted by Silva et al.[19] chitosan has effectively removed smear layer from the root canals after instrumentation. However, the effect of chitosan as an endodontic irrigant for the removal of Ca(OH)2 -based intracanal medicaments has not yet been established.

Different methods have been included for measuring residual Ca(OH)2 viz., direct visualization, digital microscopy, scanning electron microscopy, and spiral computed tomography.

The aim of this in vitro study was to assess the efficiency of two calcium chelators, 17% EDTA solution and 0.2% Chitosan in combination with ultrasonic agitation, in the removal of Ca(OH)2 when mixed with two different vehicles. The two formulations used in the study were the commercially available paste in silicone oil and freshly mixed Ca(OH)2 powder in distilled water. The volume of Ca(OH)2 removed was analyzed with cone beam computed tomography (CBCT).

MATERIALS AND METHODS

Twenty-eight extracted single canaled mandibular premolars free of fractures, cracks, or any other defects were selected. Accesses were prepared and the root canals were subjected to chemo-mechanical preparation with the crown down technique using ProTaper files (Densply-Mailiefer, Ballaigues, Switzerland) till F4 and 2 ml of 5.25% NaOCl was used as an irrigant after each instrument and 5ml of 17% EDTA for final flush. Canals were dried with paper points (Densply-Mailiefer, Ballaigues, Switzerland).

Two formulations of Ca(OH)2 with different vehicles were selected. Metapex (Meta Dental Corp. Ltd., Elmburst, NY), a commercially available product is composed Ca(OH)2, silicone oil, and iodoform. The chemically pure (95%) Ca(OH)2 powder (Merck India Ltd., Mumbai, India) was mixed with distilled water in 1:1 ratio and bismuth trioxide was added in the ratio of 1:8 by weight for radiopacity.[20]

The teeth were divided into two groups of 14 teeth each.

Group 1: Metapex was injected into the root canal until the material extruded through the apex.

Group 2: Ca(OH)2 mixed with distilled water was placed into the canals with lentulo spiral until the material extruded through the apex.

Teeth were held in wet sponge during placement of Ca(OH)2 formulations. Excess material was wiped off with moist cotton. The access cavities were temporarily sealed with a cotton pellet and Cavit and were stored at 37°C and 100% relative humidity for 7 days. Subsequently the teeth were mounted in a modeling wax for the purpose of CBCT.

After CBCT imaging, the volume of the filled material in each tooth were estimated in coronal section using OnDemand 3D software (Cybermed inc. Korea). The teeth in each group were held in wet sponge and further randomly divided into three subgroups on the basis of irrigant used for removal. 30-G endodontic needle was used for irrigation at 2 mm from the working length. During irrigation the needle was used in an up-down motion. Ultrasonic agitation was performed with a Satelac ultrasonic endodontic tip K15 Sonofile (Dentsply, Tulsa, OK) in endomode of ultrasonic unit (Woodpecker Dte-D5 ultrasonic scaler, China).

The teeth in each group were further randomly divided into three subgroups on the basis of irrigant used for removal.

Group 1E (n = 6): Metapex retrieved with 1 ml of 17% EDTA + ultrasonic agitation for 1 min + final rinse with 1 ml of distilled water.

Group 1 D (n = 2): Metapex retrieved with 1 ml of distilled water + ultrasonic agitation for 1 min + final rinse with 1 ml of distilled water.

Group 1C (n = 6): Metapex retrieved with 1 ml of 0.2% Chitosan solution + ultrasonic agitation for 1 min + final rinse with 1 ml of distilled water.

Group 2E (n = 6): Ca(OH)2 retrieved with 1 ml of 17% EDTA+ ultrasonic agitation for 1 min + final rinse with 1 ml of distilled water.

Group 2D (n = 2): Ca(OH)2 retrieved with 1 ml distilled water + ultrasonic agitation for 1 min + final rinse with 1 ml of distilled water.

Group 2C (n = 6): Ca(OH)2 retrieved with 1 ml of 0.2% Chitosan solution + ultrasonic agitation for 1 min + final rinse with 1 ml of distilled water.

For the preparation of 0.2% Chitosan solution, 0.2 g of Chitosan (Acros organics, 90% degree of deacetylation) was diluted with 100 ml of 1% acetic acid and the mixture was stirred for 2 h using a magnetic stirrer. The pH of the solution was measured using a digital pH meter and was adjusted to 3.2.[19]

A second CBCT was done and the volume of remaining material in each tooth was estimated as before.

Outcome assessment

The calculation of Ca(OH)2 volume in each specimen was performed using OnDemand 3D software (Cybermed Inc., Korea). Each dataset was also segmented using a uniform grayscale threshold (OnDemand 3D software) to visualize and quantify the volume of residual Ca(OH)2 material. Volumes of Ca(OH)2 were expressed as cubic mm. The removal efficiency was calculated as [(a-b) 100/a], where a was the volume of material packed in the root canal and b was the volume remaining after retrieval. The data was statistically analyzed using independent “t” test and Mann-Whitney U test.

RESULTS

The mean percentage of oil-based and aqueous Ca(OH)2 removed by 0.2% Chitosan was more (82.97 ± 8.73 and 96.45 ± 4.73) than for 17% EDTA ( 60.44 ± 9.69 and 91.15 ± 7.59) followed by distilled water (38.50 ± 6.95 and 75.93 ± 3.49). Both the chelators (17% EDTA and 0.2% Chitosan) and distilled water removed the aqueous-based significantly better (P < 0.05) than oil-based Ca(OH)2. Both 17% EDTA and 0.2% Chitosan were significantly more effective than distilled water for removal of Ca(OH)2 irrespective of vehicle used (P < 0.05) except for 17% EDTA for oil-based group. Both 17% EDTA and 0.2% Chitosan (P > 0.05) were equally good in removal of aqueous-based Ca(OH)2 but for oil-based Ca(OH)2 0.2% Chitosan was better than 17% EDTA (P < 0.05).

DISCUSSION

Intracanal medicaments have been recommended with goals of eliminating surviving bacteria in the root canal, prevent bacterial proliferation between appointments and act as a physiochemical barrier, preventing root canal re-infection and nutrient supply to the remaining bacteria.[21] Ca(OH)2 was chosen for the study as it is most frequently used as intracanal medicament. The vehicle used for mixing Ca(OH)2 is highly responsible for the velocity of ionic dissociation;[22] however, its influence on difficulty on removal is debatable.[101123] Therefore two different types of vehicles were included in the study.

Removal of Ca(OH)2 is accomplished through several irrigants including saline, NaOCl, several chelating agents e.g., EDTA, citric acid, malic acid, EDTA-T and combination of NaOCl and EDTA. 0.2% Chitosan had shown similar chelation activity as 15% of EDTA and 10% citric acid.[19] Hence Chitosan was tested in the present study. Silva et al.[19] concluded in their study that 0.2% Chitosan provided best results in reference to smear layer removal when compared with different concentrations of Chitosan. To the best of our knowledge its role as Ca(OH)2 removing agent has not been established, therefore 0.2% concentration of Chitosan was included in the study.

In addition, several techniques are also utilized viz., hand file, rotary file, canal brush, irrigation syringe, Endovac, sonics and passive ultrasonics for removal of Ca(OH)2 and passive ultrasonics has been found superior.[142425] Therefore, passive ultrasonics was used in the study. Different studies utilized different methods for assessing Ca(OH)2 remnants after removal of Ca(OH)2 from root canal. One such technique requires longitudinal sectioning of the specimen and digital imaging to measure the surface area covered by the Ca(OH)2,[81114] few authors also used scanning electron microscopy for evaluation.[2627] The disadvantage of these techniques was that it analyses the surface area covered with Ca(OH)2 not the volume. In addition these require sectioning of tooth which may cause loss of material and may influence the result. Nandani et al.,[10] Ballal et al.[28] and Wiseman et al.[25] used computed tomography. In the present study CBCT was used for the calculation of remaining Ca(OH)2. This technique not only enables volumetric analysis but also no sectioning is required and therefore no loss of material, thus more accurate.

The results of the present study demonstrate that none of the chelating agent tested were able to remove the Ca(OH)2 completely irrespective to the type of vehicle used. This is in accordance to the Nandini et al.,[10] Kenee et al.,[14] Kontakiotis et al.[29] and in contrast to De Faria et al.[23] Results also demonstrated that chelators helped in removal of aqueous-based and oil-based Ca(OH)2 when compared with distilled water irrespective to the vehicle used for mixing; however, the difference was insignificant for 17% EDTA when oil-based Ca(OH)2 was removed (P > 0.05). The reason may be that both 17% EDTA and distilled water failed to penetrate oil thus were ineffective in removal of Ca(OH)2. Majority of the Ca(OH)2 was found to be retained in apical third region which substantiate the findings of Nandini et al.[10]

Both 17% EDTA and 0.2% Chitosan removed aqueous-based Ca(OH)2 more effectively than oil-based Ca(OH)2. Silicone oil, which was the oily vehicle present, might have resisted its dissolution and removal from the root canal by tested chelators. However, 0.2% Chitosan performed better than 17% EDTA solution in removal of oil-based Ca(OH)2. This result is in accordance to previous studies,[1028] which found 10% citric acid more effective than 17% EDTA for removal of oil-based Ca(OH)2 (P < 0.05). This could be because of improved penetration of 0.2% Chitosan into the silicone oil and causing chelation than 17% EDTA that chelates calcium ion in water. Silva et al. demonstrated in their study that the chelation effect of Chitosan is due to its own properties rather than because of 1% acetic acid in which it is prepared.[19]

CONCLUSIONS

While none of the chelator in combination with ultrasonics was able to completely remove the Ca(OH)2, within limitations of this study it can be concluded vehicle used to prepare Ca(OH)2 influences its retrieval. Combination of ultrasonic agitation with chelator results in cleaner canal for both aqueous-based as well as oil-based Ca(OH)2. Aqueous-based Ca(OH)2 was found easy to be removed than oil-based Ca(OH)2. 0.2% Chitosan in combination with ultrasonics performed better than 17% EDTA in removal of oil-based Ca(OH)2.