ANTIBACTERIAL ACTIVITY OF TWO MINERAL TRIOXIDE AGGREGATE MATERIALS IN VITRO EVALUATION

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1 ANNALES ACADEMIAE MEDICAE STETINENSIS ROCZNIKI POMORSKIEJ AKADEMII MEDYCZNEJ W SZCZECINIE 2008, 54, 1, ELŻBIETA ŁUCZAJ-CEPOWICZ, MAŁGORZATA PAWIŃSKA 1, GRAŻYNA MARCZUK-KOLADA, KATARZYNA LESZCZYŃSKA 2, DANUTA WASZKIEL ANTIBACTERIAL ACTIVITY OF TWO MINERAL TRIOXIDE AGGREGATE MATERIALS IN VITRO EVALUATION ANTYBAKTERYJNE DZIAŁANIE DWÓCH PREPARATÓW MINERAL TRIOXIDE AGGREGATE OCENA IN VITRO Zakład Stomatologii Dziecięcej Akademii Medycznej w Białymstoku ul. Waszyngtona 15a, Białystok Kierownik: dr hab. n. med. Danuta Waszkiel 1 Zakład Stomatologii Zachowawczej Akademii Medycznej w Białymstoku ul. M. Skłodowskiej-Curie 24a, Białystok Kierownik: prof. dr hab. Wanda Stokowska 2 Zakład Diagnostyki Mikrobiologicznej Akademii Medycznej w Białymstoku ul. Waszyngtona 15a, Białystok Kierownik: dr hab. n. med. Elżbieta Tryniszewska Streszczenie Wstęp: Dowiedziono, że usunięcie zębiny próchnicowej z ubytku, oceniane według kryteriów klinicznych, nie eliminuje z niego całkowicie bakterii. Z tego powodu, w profilaktyce próchnicy i zapaleń miazgi, konieczne jest zastosowanie materiałów o dobrych właściwościach antybakteryjnych. Cel pracy: Określenie działania antybakteryjnego dwóch białych preparatów Mineral Trioxidate Aggregate (MTA) przeciwko bakteriom z rodzaju. Materiał i metoda: Badano preparaty MTA: ProRoot White (Dentsply, Tulsa Dental, USA) oraz MTA White Angelus (Angelus, Brazylia). Do oceny wykorzystano wzorcowe szczepy bakterii z rodzaju : mutans, sangui i salivarius. Efekt antybakteryjny określono metodą dyfuzji agarowej. Zawiesinę bakteryjną rozprowadzano wymazówką na podłoże tryptozowo-sojowe (TSA), w którym wycięto korkoborem otwory o średnicy 7 mm i głębokości 5 mm, po 4 otwory dla każdego materiału. Zagłębienia wypełniono szczelnie świeżo przygotowanymi preparatami MTA. Wyniki odczytywano mierząc strefy zahamowania wzrostu bakterii po 1, 2, 3 i 8 dobach. Wyniki: Wszystkie próbki ocenianych materiałów hamowały wzrost bakterii z rodzaju. Efekt antybakteryjny MTA utrzymywał się do końca badania (8 doba). ProRoot White wykazywał nieznacznie większe strefy zahamowania wzrostu salivarius i sanguis w porównaniu z MTA White Angelus. Średnie strefy zahamowania wzrostu mutans dla obu ocenianych preparatów okazały się takie same. Wniosek: Oba preparaty MTA wykazują dobre działanie przeciwbakteryjne w stosunku do wzorcowych szczepów mutans, sanguis i salivarius. H a s ł a: mineral trioxide aggregate działanie antybakteryjne paciorkowce. Summary Introduction: It has been documented that total removal of carious dentin, assessed according to clinical criteria, does not complete eradication of bacteria. For that reason in prevention of caries and pulp inflammations, application of the materials having high antibacterial properties seems to be indispensable. Aim: Determination of antibacterial activities of two white Mineral Trioxide Aggregate (MTA) preparations against bacteria of the genus.

2 148 ELŻBIETA ŁUCZAJ-CEPOWICZ, MAŁGORZATA PAWIŃSKA, GRAŻYNA MARCZUK-KOLADA et al. Material and methods: Antibacterial activities of the materials were determined by diffusion method on a solid tryptic-soy medium (Tryptic Soy Agar, Oxoid). Wells were prepared on the medium with a puncher of 7 mm diameter and 5 mm depth (four for each material). The bottom was poured with 10 microlitres of liquid TSA for sealing. Bacterial suspension was inoculated with cotton swabs onto the medium surface and then the wells were filled with the materials prepared ex tempore. The plates were left in room temperature for 30 minutes and incubated at 37 C for 24 h. Results were read by measuring the inhibition zones on day 1, 2, 3 and 8 of the culture. Results: All the study samples demonstrated inhibition of bacteria of the genus. The antibacterial effect of MTA was maintained till the end of the experiment (day 8). ProRoot White showed slightly larger inhibition zone against salivarius and sanguis as compared to MTA white Angelus. The inhibition zones against mutans appeared the same for both test materials. Conclusion: The two MTA preparations assessed in the current study show good antibacterial action against the standard strains mutans, sanguis and salivarius. K e y w o r d s: mineral trioxide aggregate antibacterial effect streptococci. Introduction Microorganisms play a pivotal role in the initiation and progression of dental caries or such complications as pulpitis and periapical pathology [1, 2, 3]. In deep carious lesions, bacterial toxins penetrate dentinal tubules, irritating the pulp and causing its inflammatory reaction. Thus, cavity preparation aims not only at the removal of pathological tissues, but is also expected to eliminate microorganisms. It has been documented that total removal of carious dentin, assessed according to clinical criteria, does not complete eradication of bacteria. Microbiological examinations of clinically healthy dentine collected from the cavities prepared by both traditional and manual method have revealed the presence of bacteria of the genus ( mutans and salivarius) and Lactobacillus [4, 5, 6]. It has been found in long-term observations, that these bacterial species are responsible for the initiation and progression of dental caries and its complications [1, 2, 3]. Therefore, apart from appropriate methods of carious dentin removal or precise clinical evaluation of dentin, also application of the materials having high antibacterial properties seems to be indispensable. This is particularly important once pulp exposure has occurred during dental procedure. For many years, calcium hydroxide-based sealers have been used in the biological treatment of the pulp, with high efficacy reaching 80% [1, 2, 7]. This is, on one hand, the result of good odontotropic properties of calcium hydroxide, leading to reparative tertiary dentin formation. On the other hand, high ph of ready-made preparations creates unfavourable environmental conditions for microorganisms, that have remained in the cavity, thus inhibiting their growth. However, calcium hydroxide-based preparations show a few unfavourable properties. Their poor adhesion may induce microleakage, they may dissolve in tissue fluid and in the presence of other dental materials, and may thus degrade under a filling. At the site where calcium hydroxide gets in contact with vital pulp, a necrotic focus is formed with an extensive inflammatory zone in the tissue [1, 2, 7]. A new material Mineral Trioxide Aggregate (MTA) has been introduced into dentistry in recent years. It is a powder containing hydrophylic molecules of oxides of calcium, silicon, bismuth, aluminum, iron, sulphur, phosphorus, magnesium, lithium and trace amounts of insoluble calcium oxide, potassium sulphate, sodium sulphate and crystalline silica [8, 9, 10, 11, 12]. Water-bound MTA forms a gel (ph = 12) [7, 13, 14]. Crystalline calcium oxides having amorphous structure are produced. The bound material has the advantages of high adhesion to tissues, slight microleakage, good mechanical strength, histobiocompatibility and excellent dentinogenic, cementogenic and osteogenic properties [7, 15, 16, 17, 18, 19, 20, 21]. Mineral Trioxidate Aggregate is not only indicated for biological treatment of the pulp (direct capping, pulpotomy), but also for apexificaton, root canal retrofilling after apical root resection, for sealing of root perforations, and as a filling material for dental tissue and alveolar bone lesions caused by resorption [2, 9, 18, 19, 20]. The study objective was the in vitro assessment of antibacterial activity of two Mineral Trioxide Aggregate preparations against bacteria. Material and methods Two MTA preparations, namely ProRoot White (Dentsply, Tulsa Detal, USA) and MTA White (Angelus, Brasil) were assessed in the study. The materials were manipulated directly before analysis in aseptic conditions (sterile glass plate and spatula). ProRoot White was prepared in the ratio of 1 g powder to 0.33 g sterile distilled water, whereas MTA White in the ratio of 1 measure (according to the manufacturer s instructions) to 1 drop of sterile distilled water. Antibacterial properties were evaluated by agar diffusion method. The following standard streptococcal strains were used: mutans ATCC 35668, salivarius ATCC and sanguis ATCC The strains were seeded on a Columbia agar medium supplemented with 5% sheep blood (Emapol). They were subsequently incubated for 18 h at 37 C. Next, a suspension was prepared in 0.85% NaCl solution (0.5 in McFarland s scale). The antibacterial activities of the materials were determined by diffusion method on a solid tryptic-soy medium

3 ANTIBACTERIAL ACTIVITY OF TWO MINERAL TRIOXIDE AGGREGATE MATERIALS IN VITRO EVALUATION 149 (TSA Tryptic Soy Agar, Oxoid). Wells were prepared on the medium with a puncher of 7 mm diameter and 5 mm depth (four for each material). The bottom was poured with 10 microliters of liquid TSA for sealing. Bacterial suspension was inoculated with cotton swabs onto the medium surface and then the wells were filled with the materials prepared ex tempore. The plates were left in room temperature for 30 minutes and incubated at 37 C for 24 h. Results were read by measuring growth inhibition zones on day 1, 2, 3 and 8 of the culture. The zones were measured in mm, including the sample diameter. In order to control the growth of standard strains, the bacterial strains were additionally cultured on TSA medium. Results Antibacterial activities of the materials are presented in Table 1. Both MTA preparations were shown to inhibit the growth of mutans, with identical inhibition zones. Antibacterial properties were maintained throughout the experiment (till day 8). ProRoot White had a better inhibitory effect on sanguis already on day 1 (13.5 mm) and the zones were maintained till day 8. MTA White Angelus showed a slightly weaker inhibitory action against sanguis (10 mm on day 1), but like in the case of ProRoot White, the inhibitory properties were maintained till the end of the experiment. The two materials did not differ much in their activity against salivarius. For ProRoot White the inhibition zone against this strain was 14.5 mm on day 1 (the largest inhibition zone in the study), being 10 mm on day 1 for MTA White Angelus. It seems to be important that both materials maintained good antibacterial properties in 1, 2, 3 and 8 day of experiment. No statistically significant differences were noted in the inhibition zones between day 1 and day 8 for all the bacterial species examined. Discussion The current study was performed for a few reasons. Firstly, antibacterial properties of dental materials are essential, especially in the case of those materials which get in a direct contact with the pulp, periodontium or alveolar bone. Secondly, we expected the MTA preparations used in the study, due to their composition, may have an antibacterial properties. The two MTA products of different manufacturers, namely ProRoot White (several years on the market) and MTA White Angelus (a novel MTA material) were assessed. Considering their similar composition, we could expect alike inhibitory activities against bacterial growth. The agar diffusion method, used by most researchers who evaluated antibacterial properties of solid dental materials, was applied in the experiment [6, 22, 23], thus some investigators used another methods [3]. Two methods used to read the inhibition zone have been described. Most authors determine its diameter including the material sample, and so did we in the current study (sample diameter 7 mm) [6]. More seldom the inhibition zone is presented excluding the sample diameter [23]. The study used three standard streptococcal strains as these species are known to play a major role in the initiation and progression of secondary caries and pulpitis. Literature reports describe beneficial antibacterial properties of Pro- Root against Enerococcus faecalis, Actinomyces odontolyticus, Fusobacteruim nucleatum, sanguis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus epidermidis, Bacillus subtilis and mutans [3, 15, 22, 23]. Statistically more significant antibacterial properties of ProRoot material against anaerobes than aerobes have been noted [22]. We manipulated the materials according to the manufacturer s instructions, i.e. using sterile distilled water in an appropriate ratio. Stowe and co-authors checked how the Badane preparaty Assessed preparations ProRoot White MTA White Angelus Bacteria strain Szczep bakterii mutans sanguis salivarius mutans sanguis salivarius T a b l e 1. Mean growth inhibition zones of bacteria in mm T a b e l a 1. Średnie sfery zahamowania wzrostu bakterii w mm of samples Liczba próbek I day / I doba II day / II doba III day / III doba VIII day / VIII doba

4 150 ELŻBIETA ŁUCZAJ-CEPOWICZ, MAŁGORZATA PAWIŃSKA, GRAŻYNA MARCZUK-KOLADA et al. change of a solvent from distilled water to 0.12% chlorhexidine gluconate affected the bacterial activity of MTA. They found ready-made MTA with chlorhexidine to show better antibacterial properties as compared to the traditionally prepared material [22]. However, the researchers did not assess whether chlorhexidine used as a solvent affected other MTA properties [22]. The authors revealed that Escherichia coli was the most susceptible microorganism. ProRoot had the weakest antibacterial action against sanguis, which was also confirmed by our study [3, 22]. In another experiment, Estrela et al. tested ProRoot Regular (grey MTA) [15]. They compared its inhibitory effect against Staphylococcus areus, Enterococcus faecalis, Pseudomonas aeruginosa, Bacillus subtilis with other materials: Portland cement, calcium hydroxide, Sealapex and Dycal. They showed that Portland and MTA well inhibited the bacterial growth, although inhibition zones were smaller than in the case of calcium hydroxide [15]. In our study, both test materials showed an antibacterial effect against the standard streptococci used. The inhibition zones were not large, although the antibacterial action was prolonged till day 8. Such an extended antibacterial activity of MTA materials has been confirmed by literature data and is of great significance from the clinical point of view. Conclusion Confirmed by our study beneficial properties of Pro- Root White and MTA White Angelus may have a major role in the biological pulp treatment, due to prevention of secondary caries and pulpitis. References 1. Jańczuk Z.: Leczenie endodontyczne. PZWL, Warszawa 1995, Barańska-Gachowska M.: Endodoncja wieku rozwojowego i dojrzałego. 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Endod. 2003, 29, Aminoshariae A., Hartwell G.R., Moon P.C.: Placement of mineral trioxide aggregate using two different techniques. J. Endod. 2003, 29, Zarzecka J., Gończowski K.: Zastosowanie materiału MTA (Mineral Trioxide Aggregate Dentsply, Tulsa Dental, USA) w zabiegach z zakresu mikrochirurgii endodontycznej przegląd piśmiennictwa. Por. Stom. 2003, 1, Lee Y.L., Lee B.S., Lin F.H., Lin A.Y., Lan W.H., Lin C.P.: Effects of physiological environments on the hydration behavior of mineral trioxide aggregate. Biomaterials, 2004, 25, Santos A.D., Moraes J.C.S., Araujo E.B., Yukimitu K., Filho Filio.V.: Physico-chemical properties of MTA and a novel experimental cement. Int. Endod. J. 2005, 38, Camilleri J., Montesin F.E., Brady K., Sweeney R., Curtis R.V., Pitt Ford T.R.: The consitution of mineral trioxide aggregate. Dent. Mater. 2005, 21, Fridland M., Rosado R.: Mineral trioxide aggregate (MTA) solubility and porosity with different water-to-powder ratios. J. 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Oral Sci. 2006, 14, 5, Komentarz Praca porusza niezwykle ciekawy problem właściwości przeciwbakteryjnych preparatów stosowanych m.in. w leczeniu biologicznym miazgi zęba. Autorzy ocenili w warunkach in vitro przeciwbakteryjne działanie dwu preparatów MTA przeciwko paciorkowcom, a więc bakteriom odpowiedzialnym za chorobę próchnicową. Badania z wykorzystaniem metody dysfuzji agarowej wykazały, że oba oceniane preparaty posiadają dobre właściwości przeciwbakteryjne. Należy zaznaczyć, że preparaty Mineral Trioxide Aggregate charakteryzują się również bardzo dobrym przyleganiem brzeżnym, są odporne na czynniki chemiczne (np. nie rozpuszczają się w płynie kanaliko-

5 ANTIBACTERIAL ACTIVITY OF TWO MINERAL TRIOXIDE AGGREGATE MATERIALS IN VITRO EVALUATION 151 wym) oraz posiadają dużą wytrzymałość mechaniczną. Niestety, cech tych pozbawione są nietwardniejące preparaty wodorotlekowo-wapniowe, które przez wiele lat były stosowane jako materiał z wyboru w leczeniu biologicznym miazgi. Tym można tłumaczyć m.in. niższy odsetek pozytywnych wyników leczenia obnażonej miazgi obserwowany w przypadku stosowania preparatów wodorotlenkowo-wapniowych, aniżeli preparatów na bazie tlenków wapnia, krzemu, bizmutu, glinu, żelaza, siarki, fosforu, magnezu i litu. dr hab. n. med. Mariusz Lipski