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Year : 2014  |  Volume : 2  |  Issue : 1  |  Page : 13-19

In vitro evaluation of sealing ability and antimicrobial activity of hydraulic temporary sealing materials

1 Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India
2 Department of Dental Materials; Manipal College of Dental Sciences, Manipal University, Mangalore, Karnataka, India
3 Manipal College of Dental Sciences; Department of Oral Pathology, Manipal University, Mangalore, Karnataka, India
4 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India

Date of Web Publication20-Mar-2014

Correspondence Address:
Prashanthi Sampath Madhyastha
Manipal College of Dental Sciences, Manipal University, Mangalore - 575 001, Karnataka
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Source of Support: ICMR-STS 2012 Research proposal no.: 2012- 00733, Conflict of Interest: None

DOI: 10.4103/2321-4619.129008

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Context: A good seal ability and antimicrobial action is a desired feature of an effective temporary sealing material. Aims: To compare the sealing ability and antimicrobial activity of three temporary sealing materials: Caviton, MD Temp and IRM Materials and Methods: In the present in vitro study, sealing ability (dye penetration method using 2% methylene blue) was measured with class I cavities on human premolars restored using test materials. The antimicrobial activity (agar diffusion test) of the materials was evaluated against Streptococcus mutans (MTCC 497 and clinical isolate) and Candida albicans (ATCC 60193 and clinical isolate). Statistical Analysis Used: For sealing ability, data was statistically analyzed using Chi-square test at a significance level of 5% using Statistical Package for Social Sciences (SPSS) 15.0. Antimicrobial activity was evaluated by comparing the mean diameter of the inhibition zones formed around the respective wells. Results: IRM produced best marginal sealing (Fisher's exact test = 38.361 and P < 0.001) and was also associated with higher antimicrobial activity in comparison to Caviton and MD Temp. The inferior properties of MD temp can be attributed to thermal instability demonstrated by MD Temp leading to an inadequate seal, and also failed to produce a zone of inhibition. IRM proved effective and superior to Caviton and MD Temp in both these aspects. Conclusions: The success of an endodontic treatment depends on the effective seal achieved following debridement. This study stresses the need for an adequate marginal seal along with satisfactory antibacterial potential for a temporary sealing material.

Keywords: Agar diffusion test, antimicrobial potential, dye penetration test, hydraulic filling materials, microleakage, sealing ability

How to cite this article:
Gidwani K, Madhyastha PS, Gidwani K, Srikant N, Suman E, Kotian R. In vitro evaluation of sealing ability and antimicrobial activity of hydraulic temporary sealing materials. J Res Dent 2014;2:13-9

How to cite this URL:
Gidwani K, Madhyastha PS, Gidwani K, Srikant N, Suman E, Kotian R. In vitro evaluation of sealing ability and antimicrobial activity of hydraulic temporary sealing materials. J Res Dent [serial online] 2014 [cited 2020 Jul 2];2:13-9. Available from: http://www.jresdent.org/text.asp?2014/2/1/13/129008

  Introduction Top

Complete sealing of endodontic access openings between appointments and after completion of therapy is an essential element in achieving endodontic success. [1] A provisional restorative material plays a pivotal role in sealing a root canal and keeping the root canals sterile; thus preventing contamination of food debris, oral fluids, and microbes, which can result in postoperative failure.

In today's endodontic treatment hydraulic temporary sealing materials are gaining popularity because of their manipulative convenience and easy handling characteristics, along with its good sealing ability. [2] These materials are based on zinc sulfate and zinc oxide; they set upon contact with saliva in the oral cavity. During setting, the materials begin to chemically react and adhere to dentin as they undergo linear hygroscopic expansion like plaster, thereby resulting in good sealing ability.

Coronal microleakage can considerably affect the prognosis of endodontic treatment. An inadequate coronal seal will allow biologic contamination and penetration of saliva, nutrients, chemicals, and importantly microorganisms and their by-products. As a result endodontic failure may occur; coronal seal therefore can be regarded as important as the apical seal. [3]

Another important factor of restoration failure is the microflora around and beneath restorations. The presence of microorganisms in the oral cavity left after treatment or their invasion through a microcrack that can sometimes occur between the tooth and its filling can result in secondary caries. [4] Mutans streptococci adhere to the oral hard tissues and take a major part in initiation of caries, thus creating an inducive environment and attend in the proceeding demineralization of caries lesion. [5],[6] The establishment and maintenance of oral microbiota is related not only to interbacterial coaggregations, but also to interactions of these bacteria with yeasts like Candida albicans. [7] The adhesion of C. albicans to dental restorative materials in the human oral cavity may promote the occurrence of oral candidosis. [8]

Thus, the successful outcome of an endodontic treatment will depend on an effective seal from recontamination of associated microorganisms. A good seal ability and antimicrobial action is a desired feature of an effective temporary sealing materials used for dental fillings. Therefore, the study aims to evaluate and compare the marginal seal and antimicrobial action of hydraulic temporary sealing materials: Caviton and MD Temp with IRM, using dye penetration method and antimicrobial potential against Streptococcus mutans and C. albicans using agar diffusion test.

  Materials and Methods Top

The in vitro sealing ability and antimicrobial activity of the following temporary sealing materials will be evaluated using dye penetration method and agar diffusion test, respectively: Caviton (GC Corporation Tokyo, Japan) and MD Temp (MetaBiomed Co Ltd, Korea) in comparison with IRM (Dentsply International Inc, York, PA). The composition of the individual materials used in this study is given in [Table 1].
Table 1: Materials used in the study

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Marginal sealing ability

Forty-two freshly extracted intact caries free human premolar (extracted during orthodontic treatment) stored in 10% formalin solutions were selected under approval of the Institutional Ethics Committee of Manipal College of Dental Sciences, Mangalore. All teeth were examined at ×10 magnification and those with microcracks were excluded. The teeth were cleaned of soft tissue and debris before use, rinsed overnight in running water, and then immersed in deionized water for 24 h.

Class I endodontic access cavities with standardized outline were prepared using a hand piece under water cooling. The coronal access to the pulp chamber was done using cylindrical diamond and carbide burs. The burs were changed after 10 preparations. The pulp cavity and the root canals were rinsed with 5.25% NaOCl solution in order to remove debris. Root canals were dried through aspiration and using cotton pellets, and their entrance were filled with gutta-percha. To standardize the cavity depth, a periodontal probe was used to assure the existence of at least 4 mm between the cavity outline and the entrance of the root canals. [9]

The teeth were randomly assigned into five groups; three experimental (n = 10 each) and 2 control groups (n = 6 each). All materials were manipulated according to the manufacturers' specifications. IRM were prepared in 6 g mL -1 powder/liquid ratio, and inserted and adapted to the cavity walls with a dental spatula. Caviton and MD Temp, the cavity were left slightly moist and were incrementally introduced into to access opening from the bottom up with the use of a plastic instrument allowed to set in contact with a moist cotton pellet. Every effort was made to ensure that the filling materials were carefully pressed against the cavity walls. The negative controls had cavity prepared and teeth coated by two coats of nail varnish. Positive controls were not filled with any temporary restorative material, only a small dry cotton pellet was placed on the floor of the chamber. The root apices were sealed with self-cured resin (DPI) and teeth were covered with two coats of nail polish, except the restorations and a 1 mm area surrounding them.

After storage in saline for 7 days at 37°C, the teeth were submitted to 500 thermal cycles between 5 ± 5 and 55 ± 5°C, with 30 s dwell time and 3 s interval time. The teeth were then immersed in 2% methylene blue solution at neutral pH (pH = 7.0) in an incubator at 37°C and 100% humidity for 7 days. They were removed from the dye solution, washed under tap water, and air dried. Sectioning were performed mesiodistally to the long axis of the tooth using a diamond disc and the depth of dye penetration in mm, were assessed using compound microscope at ×4 magnification under reflected light using an ocular micrometer. [10] Data will be statistically analyzed using Chi-square test at a significance level of 5% using Statistical Package for Social Sciences (SPSS) 15.0.

Antibacterial activity

Selection of indicator organisms was either from clinical isolates or test strains. Oral rinse method and swab culture method was used for isolation of S. mutans and Candida species, respectively from oral cavity of healthy individuals in the presence of clinicians after obtaining informed consent according to Institutional Ethics Committee regulations. In the oral rinse method, healthy individuals were asked to rinse the mouth for 60 s with 10 ml of sterile normal saline and the saliva rinse was collected in a sterile container and processed within 1-2 h after the sampling onto mitis salivarius agar (Himedia Laboratories Pvt Ltd, Mumbai) (with bacitracin) plates for S. mutans were incubated aerobically at 37°C, 5-10% CO 2 for 48-72 h. In the swab culture method, the swabs were collected from the oral vestibule and cultured on sabourand dextrose agar with chloramphenicol. The plates were incubated at 37°C for 48-72 h (the swab culture method was later adopted for Candida because of the insufficient growth of the organism with the oral rinse method).

The readily available strain of S. mutans (gram positive coccus) (MTCC 497) and C. albicans (fungi) (ATCC 60193) were chosen as test strain for the study. The inoculum for the test organism will be prepared as follows:

For Candida: Before the experiment, 100 ml pure culture of Candida was prepared in sabourand dextrose broth (SDB) (Himedia Laboratories Pvt Ltd, Mumbai). Cells were harvested by centrifugation (750 g, 20 min) washed three times in sterile physiological saline, to a 0.5 McFarland which corresponds to a cell concentration of 1.5 ± 0.3 × 10 8 cells/ml.

For Streptococcus: After frozen (-60°C) precultures were established, the bacteria were exposed on an agar plate and incubated at 37°C for 48 h. Single colonies were cultivated in sterile mitis salivaris agar (MSA) (Himedia Laboratories Pvt Ltd, Mumbai), supplemented with bacitracin for 16 h at 37°C. The day before the experiment 1 ml of each bacterial suspension were inoculated with 250 ml of the brain heart agar infusion and incubated for 12 h at 37°C. The bacterial suspension were centrifuged for 5 min at 2,000 rpm at 18°C and washed twice with physiological saline. The turbidity was adjusted to 0.5 McFarland.

Three uniform perforations, 5 mm diameter were made at equidistant points in agar (SDB for Candida and tryptic soy broth (TSB) for Streptococcus) by means of sterile loop, to receive the testing materials. Lawn culture of the organism was done and the sealers under study were handled according to the manufactures instructions and were placed into the three wells immediately after mixing. After the sealers setting, the agar plates were inoculated at 37°C for 24 h to allow the microorganisms to grow and reagents to diffuse through the culture medium. After incubation, the mean diameter of the inhibition zones formed around the wells containing the sealers was measured. All the assays were done in triplicates, under aseptic conditions and the results were average of three records. [11] Data were then statistically analyzed.

  Results Top

Analysis of microleakage

The microleakage values of restoration with IRM, Caviton, and MDTemp were compared with each other and with the positive and negative controls using Chi-square test. Microleakage values showed significant differences (P < 0.001) amongst the materials tested [Table 2] and [Figure 1] compared with the control indicating the presence of microleakage in all test materials.
Table 2: Fisher's exact test comparing the microleakage of three materials in relation to controls

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Figure 1: Microleakage: Materials vs control

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To evaluate the differences in levels of microleakage between the materials, Fisher's exact test was performed among the three [Table 3], [Figure 2]. Out of the three materials tested, it was found that IRM showed least microleakage (60.6% with upper third) and was found better compared to Caviton (33.3%) and MD Temp (6.1%) [Figure 3]. Both the groups of Caviton and MD Temp presented microleakage to the deeper levels of the restoration after thermal cycling. Interestingly, microleakage till the base of the restorations was seen only in cases restored with MD Temp (4/60).
Table 3: Fisher's exact test comparing the three materials for the amount of microleakage

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Figure 2: Microleakage within materials

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Figure 3: Evaluation of sealing ability. (a) Sealed root apices. (b) Stained specimens. (c) Specimens showing leakage; Caviton, MD Temp, and IRM (left to right)

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Analysis of antimicrobial activity

In the present study, zone of inhibition was seen only in IRM samples. The antimicrobial activity was higher towards Candida species with a mean of 14.32 and 18.41 mm, respectively for ATCC and oral swab smear culture. S. mutans MTCC and oral rinse culture had inhibition zone of 14.03 and 13.94 mm, respectively [Figure 4]. MD Temp and Caviton did not yield any zones of inhibition in our study, indicating no antimicrobial activity.
Figure 4: Culture sensitivity of Streptococcus mutans and Candida albicans. (a) S. mutans oral rinse. (b) S. mutans MTCC. (c) C. albicans oral rinse

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  Discussion Top

A good antimicrobial activity and marginal seal between tooth and temporary endodontic restorative material is required to minimize contamination of root canal systems during and after endodontic therapy. Temporary filling materials, which prevent the entry of saliva and microorganisms, should be used. [1],[9],[12],[13]

Evaluation of sealing ability

Assessing microleakage with methylene blue dye penetration is one of the most common methods utilized. [14],[15],[16] This dye has high solubility in water, it can move by simple diffusion and it is not absorbed by the hydroxyapatite crystals of dentin. 0.5% basic fuchsine is another dye used; [10] however, marginal difference is present in the size, thus sensitivity remains the same.

Thermal cycling attempted to account for temperature variations that occur in vivo. [17] The present study used thermal cycling procedures to simulate intraoral conditions. Thermal cycling frequently increases microleakage within the margins of restorative material. The temperature range between 5 ± 5°C and 55 ± 5°C that was used in this study corresponds to the extremes of temperature that could be experienced in the oral environment. [16]

The literature is replete with both in vitro and in vivo studies evaluating the sealing ability of endodontic temporary restorative materials in intact teeth. Also the excellent seal of IRM and Caviton has been demonstrated by several researchers. To the best of our knowledge this study was the first experiment that addressed the microleakage of MD Temp.

IRM is zinc oxide-eugenol (ZnOE) cement reinforced with polymethyl methacrylate. This reinforcement provides the restoration with improved compressive strength, abrasion resistance, and hardness. [12],[18] IRM was assessed and compared to other temporary restorative materials in a number of studies both in vivo and in vitro with conflicting findings. The results obtained in the present study are comparable with those observed by Jacquot et al., [19] who found good sealing for this material. Conflicting the finding, leakage of IRM increased when subjected to thermal stress, which was attributed to its dimensional instability as observed by Anderson et al.[18] Some of the studies provided semiquantitative results where dye penetration was assessed in one longitudinal section, which limits three-dimensional penetration to a two-dimensional section. [20],[21] The majority of in vivo and in vitro studies employing bacteria demonstrated almost equal or better seal with IRM (or ZnOE) than with Cavit. [22] Pai et al., [23] found that dye leakage at the interface between an amalgam restoration and IRM, Caviton and a double seal of Caviton and IRM temporary restorations was less than the leakage between the temporary materials and tooth cavity walls. In the present study, IRM showed better sealability (60.6% with upper third) with least microleakage than compared to Caviton (33.3%) and MD Temp (6.1%).

Caviton and MD Temp are the two hydraulic temporary sealing material used in the present study. The hydraulic temporary sealing material does not have adhesiveness with cavity, but when a putty-like paste is filled in the cavity, the paste reacts with water such as saliva in an oral cavity and is set. Therefore, the hydraulic temporary sealing materials are gaining popularity since it does not need mixing/heating, and has good operativity. [2],[24] Further, the hydraulic temporary sealing material has good sealing property because it expands at a time of setting.

However, since the hydraulic temporary sealing material utilizes a setting mechanism due to the reaction of calcium sulfate in a composition with the water in the oral cavity, there are problems that a setting time is long and the initial setting time is particularly slow. A dental hydraulic temporary sealing material composition consists of calcium sulfate, a vinyl acetate resin, an inorganic filler, alcohols having a boiling point of 110°C or more, and a nonionic surfactant; whereby water on a cavity can adhere, without being repelled, to a paste surface due to hydrophilic effect between an organic solvent having proper hydrophilicity and the nonionic surfactant, the water can permeate and diffuse rapidly and stably through the inside of the paste by the hydrophilic effect so as to set faster than conventional composition. Furthermore, since the hydrophilic effect is effective for adhesiveness with cavity, filling property in an oral cavity becomes extremely good. As a result, the sealing property can be improved. Therefore, the composition is critical for the formability of material and any changes in composition deteriorate the properties including strength and sealing characteristics. [25]

Caviton is a premixed temporary restorative material that contains zinc oxide, plaster of Paris, and vinyl acetate. Caviton possess high coefficient of thermal expansion than compared to ZnOE based materials, which permits more expansion for the material to adapt more tightly to dentin walls; thus providing good seal under different conditions, including thermal cycling. A recent study compared Cavit and Caviton with Fermin (a zinc sulfate cement) and Canseal (a noneugenol cement requiring mixing) in a leakage study using methylene blue. [9] The best seal was provided by Fermin followed by Caviton, Cavit, and Canseal. The study indicated that thermal cycling procedures influenced seal more than load cycling. Lee et al., compared the sealing properties of IRM, Cavit, and Caviton. According to their report, Caviton provided the best seal, whereby dye penetration was within the dentinoenamel junction and that expansion by water absorption during setting caused the material to adhere closely to the cavity wall. [20]

However, the result of the present study does not concur with the consensus with the finding in other literature. In the groups where thermal cycling was applied, both the groups of Caviton and MD Temp were found to have been severely affected resulting in gap formation between filling material and tooth structure with subsequent leakage of the dye into the entirety of pulp chamber. The sealing ability of Caviton was intermediate between IRM and MD Temp and MD Temp was the worst. A similar finding was noted in previous studies as observed by Lee et al., [20] and Pai et al., [23] In the present study, the temporary sealing materials used were also hygroscopic. Therefore, they might have permitted dye penetration not only between dentin and the materials, but also directly into the materials. [2],[9] The discrepant results can be related to it manipulative variables like variations in volume resulting from contraction of the material, different methodologies applied, thickness of the material and its condensation into the cavity, and slow setting process that could partially explain poor sealing results with these materials. Also, the sealing property of hydraulic temporary sealing materials did not seem to be improved by the setting phenomenon, but by non-hydrophobic components remaining at an unset or set area that blocked water penetration. The materials required only surface setting to withstand mechanical stresses such as mastication force, and this was sufficient to prevent deformation or destruction in the oral cavity. However, the instability demonstrated by MD Temp when subjected to thermal cycling raises questions as to its ability to provide a tight seal and this suggests that its use as a temporary endodontic filling material requires reevaluation.

Further studies are needed for considering other factors such as masticatory factors, different immersion periods, that would affect microleakage. Additional studies may be needed to verify the quality of the seal provided by these materials for prolonged periods.

Evaluation of antimicrobial activity

Antimicrobial properties of dental materials were studied by many researchers and by different methods of testing. The most useful and popular one is the agar diffusion test also known as the disk diffusion test. [26],[27]

In the present study, IRM showed effective antibacterial activity against S. mutans and C. albicans; and was significantly better than the other groups. However, no antimicrobial activity was found with Caviton and MD Temp. IRM is a power liquid system which harnesses the antimicrobial properties of both ZnOE as well as eugenol. The high rate of generation of reactive oxygen molecules by ZnO has been indicated in the antibmicrobial activity. [28] Eugenol is known to increase the membrane permeability of the bacterial cell wall and disrupts the cytoplasmic membrane causing bacterial death. [29] The greater antibacterial activity shown by IRM might also be attributed to a softer mix which undergoes hydrolysis and subsequent better release of nascent oxygen and eugenol which may prevent bacterial colonization if leakage takes place as explained by Chandler and Heling. [30] Antibacterial action of zinc is attributed to its inhibitory actions on the cell glycolysis, transmembrane proton translocation, and acid tolerance. Daugela et al., in their study have shown bacteriostatic activity of S. mutans in ZnOE based cements. [31]

Similar components of Caviton and MD Temp can justify the lack of antimicrobial activity. Although ionization of these molecules releases hydroxyl ions and thereby increasing the pH of the medium, it requires time for its effective action against microorganisms. Also, due to the setting of calcium sulfate, one of the ingredients in the composition of the hydraulic filling material, it can result in a porous structure facilitating microbial growth. The lower percentage release of available zinc in these forms may also attribute to the lesser antibacterial activity.

Only Deveaux et al., [32] showed that Cavit was superior to IRM in preventing Streptococcus sanguis penetration. The authors related this finding to the presence of a growth-inhibiting factor (probably zinc ion) present in Cavit. However, in a pilot study with unpublished data, the authors found that IRM also demonstrated antibacterial effects as tested by agar diffusion tests.

Extrapolating the results, the instability demonstrated by MD Temp when subjected to thermal cycling and antibacterial potential raises questions as to its ability to provide a tight seal and this suggests that its use as a temporary endodontic filling material requires reevaluation.

  Conclusion Top

IRM produced best marginal sealing and was also associated with antimicrobial activity in comparison with all other materials tested. This study also showed that thermal cycling procedures seemed to offset the sealing ability of temporary filling material. To improve the sealing performance effectively, it is necessary to ensure the presence of nonhydrophobic components within the temporary sealing materials to block surplus water penetration. Thus, the choice of temporary filling material must be based on the sealing ability and if possible even on the antimicrobial potential. ZnOE cements present good sealing ability, resistance, and satisfactory antibacterial activity, coupled with it being economical.

  References Top

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2.Ogura Y, Katsuumi I. Setting properties and sealing ability of hydraulic temporary sealing materials. Dent Matl J 2008;27:730-5.  Back to cited text no. 2
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7.Jenkinson HF, Lala HC, Shepherd MG. Coaggregation of Streptococcus sanguisand other streptococci with Candida albicans. Infect Immun 1990;58:1429-36.  Back to cited text no. 7
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17.Hansen SR, Montgomery S. Effect of restoration thickness on the sealing ability of Term. J Endod 1993;19:448-52.  Back to cited text no. 17
18.Tuggle ST, Anderson RW, Pantera EA Jr, Neaverth EJ. A dye penetration study of retrofilling materials. J Endod 1989;15:122-4.  Back to cited text no. 18
19.Jacquot BM, Panighi MM, Steinmetz P, G'Sell C. Microleakage of Cavit, Cavit W, Cavit G and IRM by impedance spectroscopy. Int Endod J 1996;29:256-61.  Back to cited text no. 19
20.Lee YC, Yang SF, Hwang YF, Chueh LH, Chung KH. Microleakage of endodontic temporary restorative materials. J Endod 1993;19:516-20.  Back to cited text no. 20
21.Mayer T, Eickholz P. Microleakage of temporary restorations after thermocycling and mechanical loading. J Endod 1997;23:320-2.  Back to cited text no. 21
22.Barthel CR, Strobach A, Briedigkeit H, Göbel UB, Roulet JF. Leakage in roots coronally sealed with different temporary fillings. J Endod 1999;25:731-4.  Back to cited text no. 22
23.Pai SF, Yang SF, Sue WL, Chueh LH, Rivera EM. Microleakage between endodontic temporary restorative materials placed at different times. J Endod 1999;25:453-6.  Back to cited text no. 23
24.Balto H. An assessment of microbial coronal leakage of temporary filling materials in endodontically treated teeth. J Endod 2002;28:762-4.  Back to cited text no. 24
25.Available from: http://www.freepatentsonline.com/y2011/0244431.html. [2011 March 30].  Back to cited text no. 25
26.Cobankara FK, Altinöz HC, Ergani O, Kav K, Belli S. In vitro antibacterial activities of root canal sealers by using by using two different methods. J Endod 2004;30:57-60.  Back to cited text no. 26
27.Leonardo MR, da Silva LA, Tanomaru Filho M, Bonifácio KC, Ito IY. In vitro evaluation of antimicrobial activity of sealers and pastes used in endodontics. J Endod 2000;26:391-4.  Back to cited text no. 27
28.Padmavathy N, Vijayaraghavan R. Enhanced bioactivity of Zno nanoparticles-An antimicrobial study. Sci Technol Adv Mater 2008;9:1-7.  Back to cited text no. 28
29.Devi KP, Nisha SA, Sakthivel R, Pandian SK. Eugenol (an essential oil of Clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol 2010;130:107-15.  Back to cited text no. 29
30.Chandler NP, Heling I. Efficacy of three cavity liners in eliminating bacteria from infected dentinal tubules. Quintessence Int 1995;26:655-9.  Back to cited text no. 30
31.Daugela P, Oziuanas R, Zekonis G. Antibacterial potential of contemporary dental luting cements. Stomatologija 2008;10:16-21.  Back to cited text no. 31
32.Deveaux E, Hildelbert P, Neut C, Boniface B, Romond C. Bacterial microleakage of Cavit, IRM, and TERM. Oral Surg Oral Med Oral Pathol 1992;74:634-43.  Back to cited text no. 32


  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3]


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