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An in vitro evaluation of antifungal activity of bioaggregate


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  • 1. An in vitro evaluation of antifungal activity of bioaggregate Abdullah Dohaithem, BDS,a Abdulrahman Al-Nasser, BDS,b Abdulhakim Al-Badah, BS,c Saad Al-Nazhan, BDS, MSD,d and Nassr Al-Maflehi, BSc, MSc-ISS,e Jeddah and Riyadh, Saudi Arabia KING FAHD ARMED FORCES HOSPITAL, MINISTRY OF HEALTH, AND KING SAUD UNIVERSITYObjective. The aim of this study was to evaluate, in vitro, the antifungal effect of bioaggregate (BA) against Candidaalbicans by using the direct contact method.Study design. BA was tested freshly mixed and after 24-hour set on C. albicans. The tested BA was incubated with C.albicans in plastic tissue clusters for 1 hour, 24 hours, and 3 and 5 days. Aliquots of 0.1 mL were taken from eachwell at the end of the incubation periods and transfered to tubes containing 5 mL fresh Sabouraud broth. All tubeswere vortexed and then incubated at 37°C and observed for the subsequent 5 days. Growth of the fungi was observeddaily by the presence of turbidity in the tubes. The results were statistically analyzed by using Kaplan-Meier test.Results. The freshly mixed and set BA had no antifungal effect at 1 and 24 hours of contact. Both mixes demonstratedcomplete fungicidal activity after 24 hours’ contact. Statistical analysis showed a highly significant difference betweenthe negative and positive control groups (P Ͻ .001) and a significant difference between the freshly mixed and 24-hour set BA groups (P Ͻ .001) at 24 hours.Conclusions. BA (freshly mixed and 24-hour set) was effective against C. albicans after 24 hours. (Oral Surg Oral MedOral Pathol Oral Radiol Endod 2011;112:e27-e30)Bioaggregate (BA), laboratory-synthesized water- evaluate, in vitro, the antifungal activity of BA by usingbased cement, was recently developed aiming for the the direct contact method.improvement of some properties of the well studiedmineral trioxide aggregate (MTA) cement. It has the MATERIAL AND METHODSsame indications for use as MTA, including vital pulp The effect of the antifungal activity of the BA (In-therapy, perforation repair, retrograde filling, and novative Biocaramix, Vancouver, Canada) was evalu-apexification, which has proved to be the gold standard ated (freshly prepared and after 24-hour set) againstof all materials used in surgical and nonsurgical end- Candida albicans.odontic treatment. In addition, BA has been reported to Stock cultures of clinically isolated C. albicansdisplay in vitro compatibility similar to MTA1,2 as well (strain no. 66027) provided by the Microbiology Lab-as antimicrobial activity against Enterococcus faeca- oratory of King Khalid University Hospital (King Saudlis.3 The antifungal properties of MTA have been eval- University, Riyadh, Saudi Arabia) were maintained inuated by several investigators.4-8 They all reported Sabouraud agar plate. A suspension was prepared bygood fungicidal effect against Candida albicans. How- transfering 3 colonies from the Sabouraud agar plate byever, the antifungal activity of BA was not yet been using a sterile 4-mm diameter platinum loop to 10 mmreported. Therefore, the aim of the present study was to Sabouraud dextrose broth in a sterilized 10 mL screw- capped test tube, followed by incubation for 2 days at 37°C. Six such test tubes were prepared.Supported by the Research Center of the Dental College, King SaudUniversity, Riyadh, Saudi Arabia.a Endodontic Saudi Board Resident, King Fahd Armed Forces Hos- Experiment procedurepital. The experiment was performed in plastic tissue cul-b Endodontic Saudi Board Resident, Dental Department, Ministry of ture clusters (Costar Corning, Corning, NY) containingHealth. 24 wells each with an inner diameter of 16 mm. A totalc Microbiology Laboratory, College of Dentistry, King Saud Univer-sity. of 40 wells were used and divided into 2 experimentald Division of Endodontics, Department of Restorative Dental Science, groups (freshly mixed BA and 24-hour set BA) andKing Saud University. control groups (positive and negative) of 10 wells each.e Department of Preventive Dental Sciences, King Saud University. For the BA groups, 1 pack of BA (1 g) was carefullyReceived for publication Jan 18, 2011; returned for revision Mar 10, mixed at the bottom of each culture well according to2011; accepted for publication Mar 25, 2011.1079-2104/$ - see front matter the manufacturer’s instructions. The 24-hour set BA© 2011 Mosby, Inc. All rights reserved. group was placed in the incubator at 37°C for 24 hoursdoi:10.1016/j.tripleo.2011.03.037 after mixing. Afterward, 2 mL Candida suspension e27
  • 2. OOOOEe28 Dohaithem et al. October 2011Table I. Evaluation of the effect of the direct contact of tested materials on cultured Candida albicans Contact (culturing) timeTube Neg. control Pos. control BA fresh mix BA (24-hr set) 1 1h 1d 3d 5d 1h 1d 3d 5d 1h 1d 3d 5d 1h 1d 3d 5d 2 — — — — * * * * * * — — * — — — 3 — — — — * * * * * * — — * — — — 4 — — — — * * * * * * — — * — — — 5 — — — — * * * * * * — — * — — — 6 — — — — * * * * * * — — * — — — 7 — — — — * * * * * * — — * — — — 8 — — — — * * * * * * — — * — — — 9 — — — — * * * * * * — — * — — — 10 — — — — * * * * * * — — * — — —BA, Bioaggregate; *, presence of growth; —, absence of growth (n ϭ 10 per test).solution was placed into the wells containing the Table II. Means and medians for survival timefreshly mixed BA as well as the 24-hour set BA. In Meanaaddition, 1 mL Sabouraud broth media was mixed with 95% Confidence1 mL of Candida suspension in a culture well. This intervalserved as positive control. For the negative control test, Lower Upper2 mL Sabouraud broth was placed in culture well. The Bioaggregate Estimate SE bound boundculture-cluster plates were then incubated at 37°C and CϪ 0.000 0.000 0.000 0.000evaluated after 1 hour and 1, 3, and 5 days. At the end Cϩ 120.000 0.000 120.000 120.000of each incubation period, aliquots of 0.1 mL were BA fresh mix 72.000 0.000 72.000 72.000taken from each well without mixing the content of the BA 24-h set 24.000 0.000 24.000 24.000 Overall 54.000 7.380 39.536 68.464well and transfered to tubes containing 5 mL freshSabouraud broth. All tubes were vortexed and then CϪ, Negative control; Cϩ, positive control; BA, bioaggregate. aincubated at 37°C and observed for the subsequent 5 Estimate is limited to the largest survival time if it is censored.days. Growth of the fungi was observed daily by the pres-ence of turbidity in the tubes. The presence of turbiditywas determined, and the purity of the culture was Table III. Overall comparisonschecked by morphology of colonies onto Sabouraud ␹2 Df Sig.agar. The results were statistically analyzed using Ka-plan-Meier test at the level of significance ␣ ϭ .05. Log rank (Mantel-Cox) 62.086 3 .000 Breslow (generalized Wilcoxon) 53.886 3 .000 Tarone-Ware 57.746 3 .000RESULTS Test of equality of survival distributions for the different levels of The results are summarized in Tables I-III. bioaggregate.Control No fungal growth was shown in the negative controlsamples during the period of examinations, whereas the 24-hour set BApositive control samples demonstrated entirely fungal Fungal growth was observed during 1-hour andgrowth. 1-day incubation of C. albicans with the set BA. When the incubation period increased to 3 and 5 days, noFreshly mixed BA fungal growth was observed. Fungal growth was observed during 1-hour and Statistical analysis showed a highly significant dif-1-day incubation of C. albicans with the freshly pre- ference between the negative and positive controlpared BA. Increasing the exposure time to 3 and 5 days groups (P ϭ .000) and between the freshly mixed andof C. albicans to the freshly prepared BA, however, 24-hour set BA groups (P ϭ .000) after 24 hours’resulted in complete inhibition of growth. observations.
  • 3. OOOOEVolume 112, Number 4 Dohaithem et al. e29DISCUSSION stances into the growth media of MTA and BA is The method used in this study has an advantageous primarily responsible for killing C. allowing direct contact between fungi and the mate- Tantalum oxide is the major difference betweenrial in solution.9 In addition, it minimizes possible MTA and BA. A significant amount of tantalum oxideconfounding factors in the experiment.3 Such advan- is present in the BA material. It has been used astages explained the rationale of its methodologic choice sutures, plates, and membranes in orthopedics becausein a previous study.4 The effectiveness of this method of its inertness.23,24 A strong inhibition zone whenwas confirmed by the observation of the positive con- osteoblasts were grown has been reported by Steine-trol samples. mann, whereas fibroblasts proliferated well on the tan- A great deal of scientific evidence indicates that talum disk.25 The antimicrobial activity of tantalummicroorganisms involved in intraradicular or extrara- oxide was reported by Pratt and Smith.26 Thus, thedicular infections are the major causative agents of presence of tantalum oxide could play a role in theendodontic therapy failure, including fungi.10-13 Can- antifungal effectiveness of the BA material.dida albicans has been reported to be the most com- In a previous study, we found that MTA (freshlymonly isolated fungal species. Siqueira and Sen re- mixed and 24-hour set) was effective in killing C.ported that C. albicans is able to colonize root canal albicans after 24 hours of contact.4 In the present study,walls and penetrate into dentinal tubules.14 Grossman15 BA displayed in vitro antifungal activity similar toreported that the presence of Candida organisms in white MTA.infected root canals causes a real problem in endodontic In conclusion, BA (freshly mixed and 24-hour set)treatment. He insisted in eliminating these organisms displayed an in vitro effect on the tested C. albicansfor better prognosis. Candida albicans was found to be after 24 hours of contact.more resistant than E. faecalis or Bacillus species whenevaluating the antimicrobial effects of citric acid and The authors thank Dr. Khalid Idriss his support andsodium hypochlorite.16 In addition, Sen et al.17 reported critique.that the antifungal properties of 0.12% chlorhexidine,1% NaOCl, and 5% NaOCl was affected by the pres- REFERENCESence of smear layer. They found C. albicans to be more 1. De-Deus G, Canabarro A, Alves G, Linbares A, Senne MI, Granjeiro JM. Optimal cytocompatibility of a bioceramic nano-resistant in the presence than in the absence of smear particulate cement in primary human mesenchymal cells. Jlayer. Furthermore, C. albicans cells were reported to Endod 2009; highly resistant to calcium hydroxide.18 Both MTA 2. Yuan Z, Peng B, Jiang H, Bian Z, Yan P. Effect of bioaggregateand BA produce calcium hydroxide by a hydration on mineral associated gene expression in osteoblast cells. J Endod 2010;36:1145-8.reaction. Siqueira et al.19 reported that even in a harsh 3. Zhang H, Pappen FG, Haapasalo M. Dentin enhances the anti-calcium hydroxide environment, it took 1 week to bacterial effect of mineral trioxide aggregate and bioaggregate. Jtotally eliminate C. albicans and 2 days’ exposure to Endod 2009;35:221-4.disinfect most of a specimen. A mixture of 2% chlo- 4. Al-Nazhan S, Al-Judai A. Evaluation of antifungal activity ofrhexidine and calcium hydroxide was found to be a mineral trioxide aggregate. J Endod 2003;29:826-7. 5. Al-Hezaimi K, Al-Hamdan K, Naghshbandi J, Oglesby S, Simonvery effective antifungal agent against C. albicans.20 JH, Rotstein I, et al. Effect of white-colored mineral trioxideThe involvement of fungi in cases of persistent and aggregate in different concentrations on Candida albicans insecondary infections associated with recalcitrant peri- vitro. J Endod 2005;31:684-6.radicular lesions require the use of intracanal medica- 6. Al-Hezaimi K, Naghshbandi J, Oglesby S, Simon JH, Rotstein I.ment and repaired filling material with antifungal ac- Comparison of antifungal activity of white-colored and gray- colored mineral trioxide aggregate (MTA) at similar concentra-tivity. tions against Candida albicans. J Endod 2006;32:365-7. The present study has demonstrated that BA, both 7. Mohammadi Z, Modaresi J, Yazdizadeh M. Evaluation of thefreshly mixed and 24-hour set, is effective in killing C. antifungal effects of mineral trioxide aggregate materials. Austalbicans at 3 and 5 days’ observation. The material did Endod J 2006;32:120-2.not show antifungal activity at 1 hour and 24 hours’ 8. Mohammadi Z, Khademi AA, Ezoddini-Ardakani F. In vitro evaluation of antifungal effects of mineral trioxide aggregate andobservation. A clear explanation of such delayed anti- Portland cement on Candida albicans. Iran J Endod 2006;1:fungal activity is still unknown. An earlier study dem- 137-40.onstrated that pH of BA peaked at 24 hours, which 9. Baron E, Peterson L Finegold S. Bailey and Scott’s diagnosticcould explain the delayed activity.3 In addition, C. microbiology. 9th ed. St. Louis: Mosby; 1994.albicans has been reported to be more resistant to high 10. Siqueira JF Jr. Aetiology of root canal treatment failure: why well-treated teeth can fail. Int Endod J 2001;34:1-10.pH in vitro than other persisting microorganisms, such 11. Nair PN, Sjögren U, Krey G, Kahnberg KE, Sundqvist G. Intra-as E. faecalis.18,21,22 Zhang et al.3 and Al-Nazhan and radicular bacteria and fungi in root-filled, asymptomatic humanAl-Judai4 reported that the release of diffusible sub- teeth with therapy-resistant periapical lesions: a long-term light