ENDODONTOLOGY Volume: 25 Issue 2 December 2013 Original Research Antimicrobial activity of gutta-percha points containing root canal medications against E.faecalis and Candida albicans in simulated root canals - An in vitro study Balaram Naik #* Sheetal Shetty #** Mahantesh Yeli #* ABSTRACT Objective : The aim of the study was to evaluate the antimicrobial activity of Calcium hydroxide and Chlorhexidine (Activ points) releasing gutta-percha points to Enterococcus faecalis and Candida albicans in simulated root canals using microbiological method. Methadology : Sixteen sterile capillary tubes acted as simulated root canals. Eight sterile simulated root canals were filled with 20µl of E. faecalis suspension and divided into 4 groups with 2 samples each and were filled with Chlorhexidine points (Activ points), Calcium Hydroxide points, Conventional gutta-percha points, without guttapercha points (Control). The procedure was repeated with pure culture of Candida albicans (MTCC 3017). The samples were incubated for 37 0 and colony count was calculated with samples recovered after 10 mins and 5 hours after incubation. Results : Statistically significant difference in the antimicrobial efficacy of CHX gutta percha, Ca(OH) 2 gutta percha, normal gutta percha and no gutta percha against E. faecalis and candida albicans both at 10 minutes and 5 hours since the p value was <0.05. Conclusion: Within the limitation of this invitro study it can be concluded that there is significant difference in the antimicrobial activity of Calcium hydroxide points and Chlorhexidine impregnated gutta percha points (Activ points) against Enterococcus faecalis and Candida albicans Key words : Chlorhexidine points, Calcium Hydroxide points, Enterococcus faecalis, Candida albicans, colony forming units. Introduction Micro-organisms are the main etiologic factors in pulpal and periapical disease. The root canal infection is polymicrobial usually dominated by anaerobic bacteria. 1 The aim of root canal treatment is the elimination of microbes and thereby the infection from the root canal system. 2 Biomechanical preparation effectively reduce microbiota; these procedures do not completely eradicate bacteria in the lateral and accessory root canals, isthmi, culde- sac and apical deltas. 3 Enterococcus faecalis and Candida albicans, can survive in the root canals even after thorough mechanical instrumentation and irrigation procedures. 4 Enterococci are Gram positive facultative anaerobic cocci that can be seen singly, in pairs or in short chains. 5 They are the most # Dept of Conservative Dentistry and Endodontics, * SDM College of Dental Sciences and Hospital, Dharwad, ** Faculty of Dentistry, Melaka Manipal Medical College, Dental Wing, Manipal 8
ANTIMICROBIAL ACTIVITY OF GUTTA-PERCHA POINTS CONTAINING ROOT CANAL MEDICATIONS AGAINST E.FAECALIS AND CANDIDA ALBICANS IN SIMULATED ROOT CANALS - AN IN VITRO STUDY commonly isolated bacteria from root canals of teeth with endodontic treatment failure. Fungi have also been observed in the pulp space and periapical areas, where they have often been associated with persistent infections. The majority of the recovered yeasts were Candida, with C. albicans being the most widespread. 6 Therefore, an antimicrobial intracanal medicament is needed to get rid of the surviving resistant microorganisms. 4 Studies have been made to develop gutta percha points with antimicrobial efficacy, to function as inter-appointment intracanal dressing by incorporating calcium hydroxide, chlorhexidine and tetracycline. 7 Calcium hydroxide [Ca(OH) 2 ] has been extensively used an ideal root canal dressing. The antimicrobial property of Ca(OH) 2 is due to its high ph. 8 Chlorhexidine, a cationic bisguanide is a broad- spectrum antimicrobial agent. It has an ability to absorb onto dentine and onto the microorganism cell wall, causing leakage of the intracellular component. 9 The placement and removal of medicament from the root canal is time consuming and cumbersome procedure. These limitations focused the development of gutta percha points impregnated with calcium hydroxide and chlorhexidine, which could be placed and removed easily from the root canal. 7 The purpose of the study was to evaluate the antimicrobial activity of Calcium hydroxide and Chlorhexidine (Activ points) releasing gutta-percha points to Enterococcus faecalis and Candida albicans in simulated root canals using microbiological method. Materials and Methods Small glass capillary tubes of 1mm in diameter were used. The narrow end of the capillaries were sealed over a Bunsen burner to create small test tubes, into which gutta- percha cones were placed. The length of these tubes were approximately 28mm. The tubes acted as simulated root canals. These tubes were then placed in a glass beaker and sterilized in an autoclave at 121 0 C and 15 lbs pressure for 20 minutes. Subsequent to sterilization all the small test tubes were transported and manipulated under aseptic conditions using sterile instruments and equipments. All the microbial procedures were performed in the Biosafety Cabinet (Level 2 A 2). Pure culture of E. faecalis (ATCC 29212) grown in Brain Heart Infusion broth (BHI) for 24 hours and 37 0 C was used. A suspension of E. faecalis was prepared in BHI, which had optical density adjusted to approximately 1 10 8 cfu/ml by comparing its turbidity to a Mc Farland 0.5 standard. Procedure The beaker containing the sterile simulated root canals were opened under laminar air flow in the Biosafety Cabinet. Eight sterile simulated root canals were filled with 20µl of E. faecalis suspension each using micropipettes. Two simulated root canals per group were filled with different gutta- percha points: Group 1 (n=2) Simulated root canals were filled with Chlorhexidine points (Activ points) Group 2 (n=2) Simulated root canals were filled with Calcium Hydroxide points Group 3 (n=2) Simulated root canals were filled with Conventional gutta-percha points 9
BALARAM NAIK, SHEETAL SHETTY, MAHANTESH YELI Group 4 (n=2) Simulated root canals were left without gutta-percha points (Control) All guttapercha points used were straight out of the box and care was taken to avoid their contamination. Each of the simulated root canals were placed inside a sterile screw capped tube and were incubated at 37 0 C. From one simulated root canal per group the bacterial suspension was recovered immediately, which resulted in a contact time of the bacteria to the point of approximately 10 min. The other simulated root canals was incubated at 37 0 C and the suspension was recovered after 5 hours. To recover the bacterial suspension, the closed end of the glass capillaries was removed and the glass capillaries was washed with 1 ml of 0.9 % sterile saline solution each. The gutta percha points was also washed with 1 ml of 0.9 % sterile saline solution each to recover as many bacteria as possible. The resulting 2 ml per test tube was collected in sterile screw capped test tube and then vortexed for 30 seconds. A dilution sequence was completed up to 10-3 and 10-4. 100ml of these last two dilutions were plated on BHI agar plates using L rods for lawn cultures. All plates were read after 24 hours of incubation at 37 0 C. The number of Colony Forming Unit was calculated. This experimental procedure was repeated 10 times, so that each group consisted of a total of 20 samples; 10 samples recovered after 10 min and the other 10 samples recovered after 5 hours of incubation. The above procedure was also carried out using pure culture of Candida albicans (MTCC 3017) grown on Sabouraud dextrose agar. The recovered bacterial suspension underwent dilution. A dilution sequence was completed up to 10-3 and 10-4. 100µl of these last two dilutions were plated on Sabouraud dextrose agar plates using L rods for lawn cultures. All plates were read after 24 hours of incubation at 37 0 C. The number of Colony Forming Unit was calculated. This experimental procedure was also repeated 10 times. The statistical analysis were performed using Kruskal Wallis ANOVA, Mann-Whitney test and by Wilcoxon Signed Ranks test. The level of significance was set at p <0.05. Results M3 is the dilution upto 10-3 and M4 is the dilution upto 10-4. The statistical analysis were performed using Kruskal Wallis ANOVA, Mann- Whitney test and by Wilcoxon Signed Ranks test. Discussion Complex microbial flora consisting of cocci, rods, spirochetes, filaments and sometimes fungi are present in root canals of infected teeth. 10 The main objective of endodontic therapy is to eradicate microbes from the root canal system and prevention of reinfection. 10 E faecalis and C albicans are the most resistant microorganisms and are possible cause for the failure of root canal therapy. 11 E. faecalis is a Gram-positive, catalase negative, fermentative and non-sporing facultative anaerobic coccus. Its cells are ovoid with diameter ranging from 0.5 to 1µm. Surface colonies on blood agar are creamy white, circular, smooth, and entire. They are able to survive in hyper- osmotic conditions, at temperatures ranging from 10 0 C to 45 0 C and a ph over 9.6. Generally enterococci are facultative anaerobes, but some species are strict aerobes. Growth on bile esculin is a useful trait to identify enterococci. 12 10
ANTIMICROBIAL ACTIVITY OF GUTTA-PERCHA POINTS CONTAINING ROOT CANAL MEDICATIONS AGAINST E.FAECALIS AND CANDIDA ALBICANS IN SIMULATED ROOT CANALS - AN IN VITRO STUDY Table No 1: Summary statistics by groups with mean number of colonies of E. faecalis organism. Groups 10 minutes 5 hours M3 M4 M3 M4 1.CHX GP 10.5 3.62 6.4 2.75 7.7 3.68 2.3 1.82 2. Ca(OH) 2 GP 22.8 7.54 12.5 3.37 18.6 2.40 11.1 2.76 3.Normal GP 31.9 6.67 15.5 4.81 32.4 6.94 21.2 4.13 4.No GP 55.7 14.26 32.5 11.78 86.6 21.85 48.4 12.03 p- value of Kruskal Wallis ANOVA.001*.000*.000*.000* p- value of 1,2 (.000)* 1,2 (.001)* 1,2 (.000)* 1,2 (.000)* Mann-Whitney 1,3 (.000)* 1,3 (.000)* 1,3 (.000)* 1,3 (.000)* Test 1,4 (.000)* 1,4 (.000)* 1,4 (.000)* 1,4 (.000)* 2,3 (.005)* 2,3 (.190) 2,3 (.000)* 2,3 (.000)* 3,4 (.000)* 3,4 (.000)* 3,4 (.000)* 3,4 (.000)* 2,4 (.000)* 2,4 (.000)* 2,4 (.000)* 2,4 (.000)* The p- value obtained from Kruskal Wallis ANOVA shows that there was statistically significant difference in the antimicrobial efficacy of CHX gutta percha, Ca(OH) 2 gutta percha, normal gutta percha and no gutta percha against E. faecalis at M3 and M4 dilution, both at 10 minutes and 5 hours since the p value was <0.05. Table No 2: Pair wise intragroup comparison of four groups with mean number of colonies in M3 dilution of E. faecalis organism by Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval. Time Points CHS Ca(OH)2 Normal GP No GP 10 minutes 10.50 3.629 22.80 7.540 31.90 6.674 55.70 14.268 5 hours 18.60 3.688 7.70 2.406 32.40 6.947 86.60 21.854 P value Wilcoxon.084.024*.317.005* Signed Ranks Test The p- value obtained from Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval shows that there was statistically significant difference in the antimicrobial efficacy of CHX gutta percha, Ca(OH) 2 gutta percha and no gutta percha against E. feacalis at M3 dilution, since the p value was <0.05; however for normal gutta percha since the p value was 0.317 hence it was not statistically significant. 11
BALARAM NAIK, SHEETAL SHETTY, MAHANTESH YELI Table No 3: Pair wise intragroup comparison of four groups with mean number of colonies in M4 dilution of E. faecalis organism by Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval. Time Points CHS Ca(OH)2 Normal GP No GP 10 minutes 6.40 2.757 12.50 3.375 15.50 4.813 32.50 11.787 5 hours 2.30 1.829 11.10 2.767 21.20 4.131 48.40 12.039 P value Wilcoxon.007*.206.017*.031* Signed Ranks Test The p- value obtained from Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval shows that there was statistically significant difference in the antimicrobial efficacy of CHX gutta percha, normal gutta percha and no gutta percha against E. feacalis at M4 dilution, since the p value was <0.05; however for Ca(OH) 2 gutta percha the p value was 0.206, hence it was not statistically significant. Table No 4: Summary statistics by groups with mean number of colonies of C. albicans organism. Groups 10 minutes 5 hours M3 M4 M3 M4 1.CHX GP 17.5 6.83 9.8 5.26 6.4 3.47 1.9 2.23 2. Ca(OH) 2 GP 23.4 8.01 11.8 5.0 16.8 4.91 8.8 3.55 3.Normal GP 30.5 7.50 16.9 4.81 50.6 8.08 26.9 11.83 4.No GP 50.1 7.37 32.0 9.41 93.8 19.22 50.1 12.99 p- value of Kruskal Wallis ANOVA.103.000*.001*.000* p- value of 1,2 (.105) 1,2 (.353) 1,2 (.000)* 1,2 (.000)* Mann-Whitney 1,3 (.001)* 1,3 (.005)* 1,3 (.000)* 1,3 (.000)* Test 1,4 (.000)* 1,4 (.000)* 1,4 (.000)* 1,4 (.000)* 2,3 (.105)* 2,3 (.052) 2,3 (.000)* 2,3 (.000)* 3,4 (.000)* 3,4 (.000)* 3,4 (.000)* 3,4 (.001)* 2,4 (.000)* 2,4 (.000)* 2,4 (.000)* 2,4 (.000)* The p- value obtained from Kruskal Wallis ANOVA shows that there was statistically significant difference in the antimicrobial efficacy of CHX gutta percha, Ca(OH) 2 gutta percha, normal gutta percha and no gutta percha against C.albicans at M3 and M4 dilution, both at 10 minutes and 5 hours since the p value was <0.05; however for M3 dilution of C.albicans at 10 minutes the p value was 0.103 which shows that it was not statistically significant. 12
ANTIMICROBIAL ACTIVITY OF GUTTA-PERCHA POINTS CONTAINING ROOT CANAL MEDICATIONS AGAINST E.FAECALIS AND CANDIDA ALBICANS IN SIMULATED ROOT CANALS - AN IN VITRO STUDY Table No 5: Pair wise intragroup comparison of four groups with mean number of colonies in M3 dilution of C. albicans organism by Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval. Time Points CHS Ca(OH)2 Normal GP No GP 10 minutes 17.50 6.835 23.40 8.017 30.50 7.502 50.10 7.370 5 hours 6.40 3.471 16.80 4.917 50.60 8.086 93.80 19.228 P value Wilcoxon.012*.009*.005*.005* Signed Ranks Test The p- value obtained from Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval shows that there was statistically significant difference in the antimicrobial efficacy of CHX gutta percha, Ca(OH) 2 gutta percha, normal gutta percha and no gutta percha against C.albicans at M3 dilution, since the p value was <0.05 Table No 6: Pair wise intragroup comparison of four groups with mean number of colonies in M4 dilution of C. albicans organism by Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval. Time Points CHS Ca(OH)2 Normal GP No GP 10 minutes 9.80 5.26 11.80 5.0 16.90 4.81 32.00 9.41 5 hours 1.90 2.23 8.80 3.5 26.90 11.83 50.10 12.99 P value Wilcoxon.012*.106.026*.013* Signed Ranks Test The p- value obtained from Wilcoxon Signed Ranks Test at 10 minutes and 5 hours time interval shows that there was statistically significant difference in the antimicrobial efficacy of CHX gutta percha, normal gutta percha and no gutta percha against C.albicans at M4 dilution, since the p value was <0.05; however for Ca(OH) 2 gutta percha the p value was 0.106, hence it was not statistically significant. Enterococci possess several virulence factors that assist adherence to host cells and extracellular matrix and assist in tissue invasion. These factors include: (1) aggregation substance (2) enterococcal surface proteins such as, (3) gelatinase, (4) a cytolysin toxin, (5) extracellular superoxide production, (6) capsular polysaccharides and (7) antibiotic resistance determinant. 12,13 Another important feature for the pathogenesis of E. faecalis is its adhesion and infiltration into dentine. E. faecalis has a intrinsic capacity to resist a wide ph range which represents a problem for clinical antibacterial control. 13 The persistence of E. faecalis in the canal long after root filling causing failure is due to its ability to to adapt to fluctuating levels of nutrient supply. 14 Fungi form a small part of the oral microbiota. Candida species form the largest proportion of the fungal microbiota. Candida albicans is most commonly found in the oral cavity of both healthy 13
BALARAM NAIK, SHEETAL SHETTY, MAHANTESH YELI and medically compromised individuals. 15 Fungi has been more frequently isolated in obturated teeth in which the treatment has failed of which C albicans was the most common. 15 There are various factors that are responsible for the virulent nature of candida species that may play a role in disease causation. They have the capability to adapt to a variety of environmental conditions. They can also attach to types I and IV collagen. 15 C albicans has the ability to form biofilms on different surfaces which facilitates establishment in a given site and protection against potential hazards. 15 Candida species can coaggregate with certain oral bacteria which play a important role in the colonization of oral mucosal and hard tissues. 15 C albicans produces hydrolytic enzymes that cause damage to the periradicular tissues. These include secreted aspartyl proteinase, collagenase, aminopeptidases, glucosaminidases, acid and alkaline phosphatases, hyaluronidase and chondroitin sulfatase. 15 A number of antimicrobial agents have been tested for their ability to eliminate E. faecalis and C. albicans from the root canal system. These encompass both irrigants, such as sodium hypochlorite, hydrogen peroxide, chlorhexidine digluconate and iodine compounds, as well as interappointment dressings, such as calcium hydroxide, chlorhexidine gluconate, camphorated phenol and mixed antibiotic steroid combinations. 12 Ca(OH) 2 is bactericidal and buffers the remaining tissue debris in the root canal system. Estrela et al claimed that Ca(OH) 2 inhibits bacterial enzymes by means of hydroxyl ions of the bacteria s cytoplasmic membrane, generating the antibacterial effect. Ca(OH) 2 has a mineralizing effect due to the activation of alkaline phosphatase,. For calcium hydroxide to act sucessfully as an intracanal dressing, it should occupy all the pulp space and diffuse into areas inaccessible to instruments. Its high ph (around 12.5) has a damaging effect on cell membranes and protein structure. 16 Chlorhexidine (CHX) is a broad-spectrum antimicrobial agent that has been used as an effective intracanal medicament. 17 It is a synthetic cationic bis-guanide which consists of two symmetric 4- cholorophenyl rings and two biguanide groups, connected by a central hexamethylene chain. Its effectiveness is because of altering the microbial cells osmotic equilibrium due to the interaction of the positive charge of the molecule and the negatively charged phosphate groups on microbial cell walls. CHX molecule penetrates into the bacteria due to increase in permeability of the cell wall. The most common water soluble preparation, CHX gluconate which at physiologic ph, dissociates and releases the positively charged CHX component. At low concentration (0.2%), CHX is bacteriostatic, specifically potassium and phosphorous, will leak out of the cell. On the other hand, at higher concentration (2%), CHX is bactericidal as cytoplasmic contents precipitates, which results in cell death. 18 Different approaches have been used to test the effectiveness of antimicrobial agents in the laboratory. Agar diffusion tests are relatively easy, but testing of calcium hydroxide is difficult, because of the composition of agar plates which act as an effective buffer for the released hydroxyl ions. In 14
ANTIMICROBIAL ACTIVITY OF GUTTA-PERCHA POINTS CONTAINING ROOT CANAL MEDICATIONS AGAINST E.FAECALIS AND CANDIDA ALBICANS IN SIMULATED ROOT CANALS - AN IN VITRO STUDY another test model (Haapasalo & Ørstavik 1987), the canals of root sections of bovine teeth are inoculated with bacteria and allowed to grow into the dentinal tubules (Ørstavik & Haapasalo 1990). Intracanal medicaments can be placed and the growth of the bacteria quantified within ground dentine close to the root canal. 19 In previous investigation using natural teeth some difficulties were observed due to the inability to recover the bacteria that grow in dentinal tubules when they were used for a full quantitative model of an infected root canal (Bruß 1998). Thus, a simple root canal model, consisting of glass capillaries, which simulates the root canals were chosen. 26 This model is relatively simple to use and can serve as a screening method when assessing new materials to serve as intracanal medicaments. 19 E. faecalis and C. albicans are most commonly found in cases of persistent root canal infections. 6 Thus, they are the important microorganism in endodontics (Portenier et al. 2003), and were therefore chosen for the present study. 6,19 In the present study a new formulation of intracanal medicament that is calcium hydroxide and chlorhexidine impregnated gutta percha points (Activ points) were used. These gutta-percha points contain substances with antimicrobial activity that is released when they come in contact with moisture (Distler & Petschelt 1997). 19 The advantages of these materials are that there is no mixing procedure, easy to apply and remove, leaves no residue, and the root canals could be filled from the apex to coronal area. 20 The present study used Brain heart infusion agar and Sabouraud s dextrose agar as the culture media, since these media are easily available and commonly used media for Enterococcus faecalis and Candida albicans. Our study demonstrated that there was significant difference in the antimicrobial activity of Calcium hydroxide points and Chlorhexidine impregnated gutta percha points (Activ points) against Enterococcus faecalis and Candida albicans. At an exposure time of 10 minutes, the antibacterial efficacy of chlorhexidine impregnated gutta percha points (Activ points) was significantly greater than that of Calcium hydroxide points against Enterococcus faecalis and Candida albicans. Even after five hours, the antibacterial activity of chlorhexidine impregnated gutta percha points (Activ points) remained greater than Calcium hydroxide points. Various results have been reported for intracanal medicament antimicrobial effectiveness. For chlorhexidine impregnated gutta percha points (Activ points), the microbial counts were very less both for Enterococcus faecalis and Candida albicans. This may be due to the rapid release of CHX out of the points. These findings are in agreement with previous studies, showing the effectivity of CHX as a possible alternative to calcium hydroxide as intracanal medicament in cases, where E. faecalis is suspected (Gomes et al. 2003, Menezes et al. 2003, Siren et al. 2004, Oztan et al. 2006). 1,3,19,21,22 Other research have also shown that Chlorhexidine was effective against C. albicans. 18,23 For Calcium hydroxide points, the microbial counts were higher for both Enterococcus faecalis 15
BALARAM NAIK, SHEETAL SHETTY, MAHANTESH YELI Colonies of Enterococcus faecalis on BHI agar plate Colonies of Candida albicans on Sabouraud s Dextrose agar plate 16
ANTIMICROBIAL ACTIVITY OF GUTTA-PERCHA POINTS CONTAINING ROOT CANAL MEDICATIONS AGAINST E.FAECALIS AND CANDIDA ALBICANS IN SIMULATED ROOT CANALS - AN IN VITRO STUDY and Candida albicans. The calcium hydroxide released from the points restricted microbial growth to only a limited extent. 19 E. faecalis is resistant to calcium hydroxide. 3,24 and studies have also reported the failure of Ca(OH) 2 to eliminate E. faecalis effectively. 1,16 Similarly Bystrom et al. 1985 reported C. albicans were highly resistant to calcium hydroxide in vitro. 13,18,23,25,26 Conventional gutta percha did reveal some limited antimicrobial activity, which can be attributed to their content of zinc oxide (Moorer & Genet 1982, Weiger et al. 1993). 19 In another study (Barthel et al. 2002), activ points were less effective than chlorhexidine gel in disinfecting root canals infected by human oral flora. 19 Other studies have shown that Ca(OH) 2 or CHX impregnated gutta-percha points did not inhibit bacterial growth of E. faecalis completely. 6 Chlorhexidine diacetate containing Activ Points did not exhibit sufficient antimicrobial or anticandidal activity. These differences may have been caused by different amount of bacterial suspension used per medicated point and the different number of bacteria per ml. 19 Endodontic infections are primarily polymicrobial. The medicament that is effective against single microbe in vitro may not necessarily be effective against the same microbe in vivo since the root canal system contains multiple microorganisms. Future research needs to be conducted to evaluate the antimicrobial efficacy of medicated gutta-percha points in clinical conditions. References : 1. Siren EK, Haapasalo MPP, Waltimo TMT, Orstavik D. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodide on Enterococcus faecalis. Eur J Oral Sci 2004; 112: 326 331. 2. Lui JN, Sae- Lim V, Song PK, Chen NN. In vitro antrimicrobial effect of chlorhexidine- impregnated gutta percha points on Enterococcus faecalis. Int Endod J 2004; 37: 105-113. 3. Delgado RJR, Gasparoto TH, Sipert CR, Pinheiro CR, Moraes IG, Garcia RB, Bramante CM, Campanelli AP, Bernardineli N. Antimicrobial effects of Calcium Hydroxide and Chlorhexidine on Enterococcus faecalis. J Endod 2010; 36: 1389-1393. 4. Turk BT, Sen BH, Ozturk T. In vitro antimicrobial activity of calcium hydroxide mixed with different vehicles against Enterococcus faecalis and Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: 297-301. 5. Stuart CH, Scott AS. Enterococcus faecalis : its role in root canal treatment failure and current concepts in retreatment. J Endod 2006; 32(2): 93-98. 6. Oztan MD, Kiyan M, Gerceker D. Antimicrobial effect, in vitro, of gutta- percha points containing root canal medications against yeast and Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102: 410-416. 7. Vijay R, Makam S, Shashikala K. Evaluation of antimicrobial efficacy of chlorhexidine gutta percha and calcium hydroxide gutta percha against Enterococcus faecalis- an invitro study. Streamdent 2010; 1(3): 209-213. 8. Vianna ME, Gomes BPFA, Sena NT, Zaia AA, Ferraz CCR, Filho FJS. In vitro evaluation of the susceptibility of endodontic pathogens to calcium hydroxide combined with different vehicles. Braz Dent J 2005; 16(3):175-180. 9. Gomes BPFA, Souza SFC, Ferraz CCR, Teixeira FB, Zaia AA, Valdrighi L, Souza- Filho FJ. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003; 36: 267-275. 10. Sen BH, Piskin B, Demirci T. Observation of bacteria and fungi in infected root canals and dentinal tubules by SEM. Endod Dent Traumatol 1995; 11: 6-9. 11.Ercan E, Dalli M, Dulgergil CT. In vitro assessment of the effectiveness of chlorhexidine gel and calcium hydroxide paste with chlorhexidine against Enterococcus faecalis and Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102: 27-31. 12. Portenier I, Uomos MT, Waltimo, Haapasalo M. Enterococcus faecalis the root canal survivor and star in post treatment disease. Endod Topics 2003; 6: 135 159. 17
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