Year : 2008 | Volume
: 19 | Issue : 2 | Page : 112--115
The antimicrobial and antifungal efficacy of tetracycline-integrated gutta-percha
Emre Bodrumlu1, Tayfun Alacam2, Mustafa Semiz3,
1 Department of Operative Dentistry and Endodontics, Faculty of Dentistry, Ondokuz Mayis University, Samsun, Turkey
2 Department of Operative Dentistry and Endodontics, Faculty of Dentistry, Gazi University, Ankara, Turkey
3 Statistics Faculty, Selšuk University, Konya, Turkey
Department of Operative Dentistry and Endodontics, Faculty of Dentistry, Ondokuz Mayis University, Samsun
The purpose of this study was in vitro evaluation of the antimicrobial and antifungal efficacy of commercially available gutta-percha containing tetracycline on some potential endodontic pathogens. The test microorganisms were Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Porphyromonas endodontalis, and Candida albicans . Tetracycline-integrated gutta-percha (TGP) cones, tetracycline disc, and conventional gutta-percha cones of the same size were placed on the inoculated plates. The plates were incubated at 37░C aerobically or anaerobically. Growth inhibition zones on each plate were inspected at 24, 48, and 72 h. Tetracycline disc and TGP cones inhibited all the tested bacterial strains, however the greatest antimicrobial effect was seen on S. aureus. Tetracycline disc and TGP seemed less effective on E. faecalis and P. aeruginosa. However, all tested treatments were unable to affect C. albicans . Based on the results of this study, it seems that TGP offers an antimicrobial advantage over conventional gutta-percha.
|How to cite this article:|
Bodrumlu E, Alacam T, Semiz M. The antimicrobial and antifungal efficacy of tetracycline-integrated gutta-percha.Indian J Dent Res 2008;19:112-115
|How to cite this URL:|
Bodrumlu E, Alacam T, Semiz M. The antimicrobial and antifungal efficacy of tetracycline-integrated gutta-percha. Indian J Dent Res [serial online] 2008 [cited 2020 Feb 19 ];19:112-115
Available from: http://www.ijdr.in/text.asp?2008/19/2/112/40464
The major goal of endodontic treatment is to eliminate bacteria from the root canal and prevent reinfection. Biomechanical instrumentation, irrigation, intracanal medication, and root canal filling are the crucial processes for reducing the population of microorganisms in the root canal system.  However, removing all microorganisms in the canal before obturation has proven to be difficult, , and the choice of endodontic materials that have high antimicrobial efficacy can help in decreasing or avoiding growth of the microorganisms that remain.
Numerous studies ,,, have been performed to assess the antimicrobial efficacy of various endodontic materials. Gutta-percha has been in use for over a century to fill root canals, and it remains the material of choice. The antibacterial properties of gutta-percha cones are very limited  and is attributed to the zinc oxide component.  Recently, a new root-canal filling material, tetracycline-integrated gutta-percha (TGP), has been developed as an alternative to gutta-percha for root canal obturation.
TGP contains 20% gutta-percha, 57% zinc oxide, 10% tetracycline, 10% barium sulfate, and 3% beeswax. The developers (Medidenta International Inc, New York, USA) claim that the tetracycline in these gutta-percha cones remains inert until it comes in contact with tissue fluids, when it gets activated and becomes available to inhibit any bacteria that remain in the root canal or those that enter the canal via leakage. Melker et al .  detected some antibacterial properties of TGP, using different microorganisms. To date, little is known about the antibacterial properties of TGP. However, its antifungal activity has not been reported yet.
The purpose of this study was to evaluate the in vitro efficacy of a commercially available gutta-percha containing tetracycline in inhibiting the growth of some potential endodontic pathogens over different time periods.
Materials and Methods
The antimicrobial and antifungal efficacy of TGP was investigated using the disc diffusion method for different time periods.
The following microbial strains from the American Type Culture Collection in the Microbiology Laboratory of Refik Saydam National Hygiene Center were used for the study:
Enterococcus faecalis (ATCC 29212)
Pseudomonas aeruginosa (ATCC 27853)
Staphylococcus aureus (ATCC 95106)
Porphyromonas endodontalis (ATCC 35406)
Candida albicans (ATCC 10231)
These strains, except for P. endodontalis , were propagated in 5 ml of brain heart infusion broth (Difco Laboratories, Detroit, MI, USA). P. endodontalis was cultured on plates of brain heart infusion broth (Difco), supplemented with hemin (5 mg/l) and menadione (5 mg/l). P. endodontalis was incubated anaerobically for 48 h at 37░C. The other microorganisms were incubated aerobically for 24 h at 37░C.
Each broth culture suspension of bacteria and C. albicans was adjusted to no. 1 McFarland Standard (approximately 3 Î 10 8 cells/ml). One hundred microliter aliquots of each microbial suspension (except of P. endodontalis ) were dispersed on the surface of Mueller-Hinton agar medium (Merck, Germany) until the surface was covered. The inoculated plates were dried for 15 min at 37░C. Aliquots of the suspension containing P. endodontalis (100 μl) were dispersed on petri dishes containing anaerobic basal agar medium (Oxoid, England).
In accordance with the recommendations of the developers, before the test, TGP cones were soaked by complete immersion in 2 ml of sterile water in a test tube for 1 h for the release of free tetracyline. The conventional gutta-percha cones were soaked in 2 ml of sterile water for 1 h. After the soaking procedure, the wet TGP and conventional gutta-percha cones were dried with sterile paper. Afterwards, the cones were transferred to inoculated agar plates as described below.
A tetracycline Bio-disc« (Tetracycline TE-30 mcg, Gokhan Laboratory Company, Izmir, Turkey) and four TGP (no. 35 size) and four conventional gutta-percha cones (Diadent, ML.029, Korea) (no. 35 size) were aseptically transferred into three sections of each previously inoculated plate. Positive and negative controls, namely the inoculated plates and the uninnoculated plates, respectively, were maintained for the same time and under the same laboratory conditions.
Subsequently, the agar plates with the facultative anaerobic and aerobic bacteria were placed in an incubator and incubated aerobically for 24, 48, and 72 h at 37░C. Anaerobic conditions were achieved using the AnaeroGen atmosphere generation system (Oxoid Anaerogen System AN025A, Hampshire, England) with an anaerobic jar (Oxoid AnaeroJar AG025A, Unipath Ltd, Hampshire, England). The plate with P. endodontalis was incubated anerobically for 24, 48, and 72 h at 37░C.
After incubation, the diameters of the zones of inhibition were measured in millimeters from the sides of the cones and discs. For each strain, experiments were performed in triplicate and the average value was determined.
Statistical analyses using the Kruskal-Wallis test to compare k-independent samples used. The statistical tests and graphics were done using SPSS statistical software.
Uniform growth was evident on all control plates. Inhibition zones of tested materials for different time periods are given in [Table 1],[Table 2]. Samples from the triplicate trials yielded consistent results. Conventional gutta-percha cones did not exhibit any antimicrobial or antifungal effect on any of the tested microorganisms for all time periods.
Tetracycline disc and TGP cones inhibited the growth of all bacterial strains over a 24 h period. Regardless of time period or the pathogen strain, TGP was statistically more effective than conventional gutta-percha as shown in [Table 1] ( P 0.05). However, tetracycline discs and TGP cones had the lowest inhibition zones with E. faecalis (11.33 mm and 9.67 mm for tetracycline discs and TGP, respectively) and the second lowest inhibition zones with P. aeruginosa (12.67 mm and 11.33 mm, respectively) in a 24-h period. The effects of tetracycline disc and TGP on all bacteria seem to decrease over 48 and 72 h as seen at [Table 2] ( P 0.05). Additionally, no antifungal activity was detected in all three time periods.
Most cases of endodontic failure are thought to be due to continuing infection of the root canal system, which results in a chronic periapical lesion after endodontic treatment.  The antimicrobial efficacy of root canal filling material and sealer may help to eliminate residual microorganisms. Hence, the root filling materials should have an antibacterial effect to avoid the growth of microbial attachments.
The bacteria and fungi selected for this study are species which have been most commonly found in infected root canals. ,, The TGP cones were tested against five species of microorganisms, but the infected root canal usually contains many different species of pathogens. Also, the root canal contains necrotic and/or viable tissues and tissue fluids, which may reduce the antimicrobial activity of TGP. In addition, the antibacterial efficacy of TGP cones differed according to the strain of pathogen tested. This could be interpreted as selective potency and may lead to a quantitative or qualitative shift in the composition of the endodontic microflora. The maximum antibacterial efficacy was seen in the first 24 h, probably due to a higher degree of tetracycline release from the gutta-percha points. The ineffectiveness over the longer term may be due to decreased release of tetracycline from the points or it may have been because of the interaction of the medicament with the agar medium.
The tetracycline discs were used to evaluate the differences in the antimicrobial efficacies of the tetracycline discs and TGP, which has 10% tetracycline. The efficacy of tetracycline discs was found to be better than that of TGP. This discrepancy could be ascribed to the differences in tetracycline concentrations.
The P. endodontalis strains are sensitive to tetracyclines, penicillin, and clindamycin.  The results of this study were consistent with the study of Haapasalo et al .  It appeared that TGP cones exhibited a moderate antibacterial efficacy  on P. endodontalis for all observation periods.
E. faecalis was chosen as a test microorganism primarily because it is among the few facultative organisms associated with persistent apical periodontitis  and because previous experimental studies have used this microorganism. ,, Furthermore, several authors have reported the difficulty in eliminating enterococci, which are resistant to calcium hydroxide and iodine medication in root canal treatment. , Based on the results of this investigation, it appears that tetracycline disc and TGP cones have a mild antimicrobial effect  on the tested bacteria. However, the effectiveness of these cones on E. faecalis decreases over time. Melker et al .  detected antibacterial properties for TGP against Actinomyces israelii, Actinomyces naeslundi, E. faecalis , and Fusobacterium nucleatum . However, the authors' results with regard to E. faecalis supported the study of Melker et al . 
It is known that tetracycline has no effect against yeast.  However, C. albicans was chosen as a test organism in this study for assessing the antifungal efficacy of the other components of TGP because it has been found in infected root canals and in periradicular tissue as well.  TGP points had no antifungal efficacy against C. albicans in this study. The findings of this study support the study by MacNeill et al . 
In the present study, conventional gutta-percha exhibited no antimicrobial efficacy against the tested microorganisms. The findings of this study do not support Attin et al .  and Moorer and Genet.  The discrepancies between the studies may be due to differences in methodology, such as the inoculum density, agar viscosity, and type of agar medium.
The diffusion test used in this study is one of the most frequently used methods for assessment of the antimicrobial efficacy of endodontic materials. ,,,,, The environment in the dentinal tubule is certainly dissimilar to in vitro conditions, such as that in the agar plate. Therefore, the size of the zones of inhibition of bacterial growth does not necessarily reflect the strength of the antimicrobial agent. Solubility and diffusibility can influence the antimicrobial efficacy of the material.  Al-Khatib et al .  claimed that the antimicrobial activity of a material may change, depending on the type of the microorganisms used in a test and the penetrating ability of the tested material in agar plates. However, several limitations of this test should be noted. Standardization of affectional factors (i.e., inoculation density, incubation temperature of plates, and selection of agar medium  ) allows one to exclude numerous variables existing in vivo. Nevertheless, consistent and reproducible results may be obtained if most of these varibles are carefully controlled. The disc diffusion method permits such direct comparisons between the test materials. 
The antimicrobial characteristics of TGP may offer an advantage over conventional gutta-percha. However, the antimicrobial activity of the various components of sealers plays a more important role than the root canal filling materials in the success of endodontic treatment. Although the antimicrobial activity of TGP may help in reducing the bacterial population in the root canal system, and not merely in altering the species composition, TGP should not be used as a root canal filling material in patients hypersensitive to tetracycline. Furthermore, Szep et al .  stated that medicated gutta-percha materials caused cytotoxic reactions in varying degrees. Hence, the biocompatibility of TGP should be investigated before its clinical use.
There is probably no absolute way of determining the effectiveness of any endodontic materials via in vitro studies. Even considering that in vitro studies are accomplished with isolated bacteria, whereas endodontic infections generally involve complex microbial interactions that can interfere in the action of different antibacterial agents, this study allowed an objective quantitative evaluation of the action of a newly developed material. This study suggests that TGP cones could be recommended in terms of its antimicrobial efficacy.
The authors thank microbiologist S. Bodrumlu, Refik Saydam Hygiene Center, Ankara, Turkey, for the microbiologic evaluation.
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