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Dental Tribune Middle East & Africa No. 2, 2017

Dental Tribune Middle East & Africa Edition | 2/2017 ◊Page 18 GENERAL DENTISTRY 19 action against anaerobic infections, and minocycline’s action against an- aerobic and aerobic infections (Sato et al., 1996; Takushige et al., 2004). Propolis is a natural resinous prod- uct of complex chemical composi- tion and varying color. It is produced by the Apis mellifera bee from resins and plant exudates that the bees collect from the plants around the hive, which they mix with wax and salivary excretions. More than 160 components have been identi- fied in propolis, 50% of them being phenolic, and it is to these that its pharmacological action has been at- tributed (Bankova, 2000; Bankova, 2005). This natural product has at- tracted much interest for endodon- tic treatment due to its antimicrobial action against many pathogenic mi- croorganisms (Santos et al., 2003), and so has become a new root canal disinfectant that is compatible with periapical tissues. Its antibacterial action can be explained by the com- bination of polyphenols, bioflavo- noids such as vitamin P, terpenes, aromatic acids, and esters (Kayaoglu et al., 2011). At present, NaOCL is the most widely used irrigant agent due to its wide antibacterial spectrum and its ca- pacity to dissolve organic remains and necrotic tissue. It can be used at different concentrations that range from 0.5-5%. At low concentrations (0.5-1%) it dissolves mainly necrotic tissue, while at higher concentra- tions its dissolving capability and its antibacterial properties increase, but also its tissue-toxic effects (Nav- arro-Escobar et al., 2010). It can also penetrate into dentinal tubules to a depth of up to 130 µm; but bacterial infection can persist at depths of ap- proximately 300 µm and occasion- ally up to 1.000 µm reaching the cementum-dentin junction (Berutti et al., 1997; Peters et al., 2001). In this context, the penetration ca- pacity of laser therapies can reduce the rate of endodontic failure. For this reason, new therapeutic strate- gies such as photodynamic therapy (PDT) are under investigation in endodontics because of their bacte- ricidal action in both primary and permanent dentition (Pinheiro et al., 2009). PDT is a new antimicrobial strategy that involves the combina- tion of a nontoxic photosensitizer (PS) and a light source (Demidova & Hamblin, 2004). The excited PS reacts with molecular oxygen to pro- duce highly reactive oxygen species, which induces injury and death of microorganisms (Wainwright, 1998). It has been established that the PS, which has a cationic charge, can rap- idly bind to and penetrate the bacte- rial cells and so shows a high degree of selectivity for killing microorgan- isms rather than host cells (Maisch et al., 2005). In the same way, ozone (triatomic molecule, consisting of three oxygen atoms) also offers a high penetration capability into the dentinal tubules and so is coming into wider use in endodontic treatment as a disin- fectant against E. faecalis (Noites et al., 2014). Ozone damages the bacte- rial cell membranes by ozonolysis and oxidates intracellular proteins leading to loss of organelle function (Sawadaishi et al., 1985). This action is selective to microbial cells and does not affect human body cells as the latter have good antioxidative ability (A et al., 2013). For endodontic treat- ment, three main forms of applica- tion are used: ozonated water, ozo- nated olive oil, and oxygen/ozone gas (Saini, 2011). There are now clini- cal devices that generate ozone by simulating lightning via an electrical discharge field based on (cold spark) corona discharge. Ozone production by corona discharge uses an energy source, anode, cathode and a suit- able dielectric spacer. Oxygen-rich air passes between the electrodes by means of a dielectric spacer, creat- ing an electric field (corona), which causes ozone formation. An hour later, 140 teeth (20 were used as a negative control group) were each inoculated with 0.1 mL E. faecalis (at a concentration of 3 x 108 cell/mL), which were then incubated at 37º C for 48 hours in vertical posi- tion (Figure 1). The aim of this study was to evalu- ate the antibacterial effect of 2.5% NaOCL, PDT, 2% CHX, TAM, propolis and ozone against E. faecalis in ex- perimentally infected root canals of extracted human teeth in vitro. Materials And Methods Preparation of the teeth The model used was a modification of the one previously described by Haapassalo and Orstavik (Haapas- salo & Orstavik, 1987). One hundred and sixty extracted, intact, adult, human, single-rooted, mature teeth with a single canal were collected and stored in sterile 0.5% NaOCL for 2-4 weeks. Calculus and stains were removed from the root surface using an ultrasonic scaler (Cavitron, Dent- sply Ltd, Ballaigues, Switzerland) (Estrela et al., 2003). The external surface of each tooth was cleaned with 10% povidone-iodine. After 5 min, the disinfectant was removed from the surface with isopropyl alco- hol (Kranz et al., 2011), and the tooth decoronated with a rotary diamond saw (Komet, Barcelona, Spain) at 700 rmp under water-cooling to facilitate access to the root (Cheng et al., 2012). Instrumentation of the root canals began with a Number 10 Hedström K file (Dentsply Maillefer, Ballaigues, Switzerland) used to calculate the working length (1 mm from the root apex). Afterwards, instrumentation of the canal system continued with Hedström K files numbers 15, 20, 25 or 30 depending on the width of the canal. Coronal thirds were prepared with Gates Glidden burs numbers 4, 3, and 2 (Dentsply Maillefer, Bal- laigues, Switzerland). Lastly, the apical third was instrumented with Wave One® rotary files (Dentsply Maillefer, Ballaigues, Switzerland), to the working length. With the use of each file or bur, the area was irrigated with saline and when instrumenta- tion was completed canals were ir- rigated with 17% EDTA (Dentaflux®, Madrid, Spain) for 1 minute to elimi- nate dentinal smear and then with 2.5% NaOCL (Tecnoquim S.L., Murcia, Spain). Lastly, the root canals were dried with number 25 paper points (Dentsply Maillefer, Ballaigues, Swit- zerland). The tooth apices underwent retro- grade sealing with resin-modified glass ionomer (Vitrebond®, 3M Espe, Madrid, Spain). The teeth were stored in demethylated ethanol 70% (Tec- noquim S.L., Murcia, Spain) for 24 hours and then autoclave processed at 121º C for 15 minutes. Afterwards, the teeth were placed in vertical posi- tion on 96-well microtitration plates (Biolab® S.L, Madrid, Spain) filled with sterile brain heart infusion broth (BHI) (Merck, Madrid, Spain) so that the roots were covered (Seal et al., 2002). The samples were placed in an incubator (Selecta®, Madrid, Spain) at 37º C for 1 hour. Cultivation of Enterococcous faecalis and root canals inocu- lation E. faecalis strain ATCC 2912 (Ameri- can Cell Culture Collection) was cultivated in its pure form in 1 mL BHI (Merck, Madrid, Spain) until it reached an optical turbidity of 1 Mc- Farland standard, with a concentra- tion of 3 x 108 cel/mL (quantified by spectrophotometry with a wave length of 600 nm and absorbance of 0.137 nm), and incubated at 37º C for 1 hour. Testing procedures After 48 hours incubation in vertical position, the teeth were randomly divided into eight experimental groups (20 teeth per group) accord- ing to post-instrumentation proce- dure. Randomization was performed using an online service (www.rand- omization.com). The eight groups were: Group 1 (2.5% NaOCl), Group 2 (PDT), Group 3 (2% CHX), Group 4 (TAM), Group 5 (propolis), Group 6 (ozone), Group 7 (positive control) and Group 8 (negative control). Group 1: the canals were filled with 2.5% NaOCL (Tecnoquim S.L., Murcia, Spain) for 5 minutes, removed with sterile paper points, and irrigated with normal 85% saline solution (Seal et al., 2002). Group 2: the root canals infected with E. faecalis were subjected to PDT. This was performed with a set- up for PDT (Helbo® Photodynamic Systems GmbH & KG, Walldorf, Germany), including a hand-held diode laser (Helbo® Theralite Laser 3D Pocket Probe, Helbo® Photody- namic Systems GmbH & KG, Wall- dorf, Germany) with a wavelength of 660 nm and a power density of 100 mW. To perform the treatment, 100 µl of methylene blue solution (the dye phenothiazine chloride, 3,7- bis (dimethylamino)-phenothiazin- 5-ium chloride [Helbo® Blue Pho- tosensitizer, Helbo® Photodynamic Systems GmbH & KG, Walldorf, Ger- many]) at a concentration of 0.01% (mass per volume) were applied to the root canal. It was left in situ for 3 minutes. Subsequently, the root canal was exposed to laser light for 60 seconds, following the manufac- turer’s instructions. Group 3: root canals received topical applications of 3 mL of 0.2% CHX glu- conate (Bohmclorh®, Laboratories Bohm, Madrid, Spain) for 5 minutes, removed with sterile paper points, and irrigated with normal 85% saline solution (Schafer & Boosman, 2005). Group 4: root canals received tri-an- tibiotic mixture (TAM) (0.5 g of cip- rofloxacin plus 0.5 g of minocycline plus 0.5 g of metronidazole; Clari- ent Life Sciences, Barcelona, Spain) mixed with a liquid consisting of macrogol and propilenglicol (1:1) at a proportion of 2:1 to produce a paste. The TAM was placed into the canals with paper points for 5 minutes, re- moved with sterile paper points, and irrigated with normal 85% saline so- lution (Madhubala et al., 2011). Group 5: in this group the teeth were treated with applications of 3 mL of 11% propolis. For the pre preparation of 11% alcoholic extract of propolis, a commercially available 33% alco- holic extract of propolis was diluted to make an 11% concentration using warm saline in 2:1 concentration (Liproline®, Novadiet S.A, Burgos, Spain). The propolis was placed into the canal for 5 minutes, removed with sterile paper points, and irrigat- ed with normal 85% saline solution (Shingare & Chaugule, 2011). Group 6: disinfection of the root canals was performed with applica- tions of ozone by means of corona- inducing dielectric barrier discharge (OzoneDTA®, Apoza, Taiwan, China). Following the manufacturer’s in- structions, the barrier discharge co- rona was applied using a probe with a power setting of 5-8 W for 90 sec- onds in a single application, supply- ing an O3 concentration of 1000 µg/ mL (Figure 1). Control groups consisted of infec- tion but no treatment (Group 7 posi- tive control), and no bacterium in- oculation (Group 8 negative control). Bacteriological evaluation Following all treatments, canals were filled with normal 85% saline solu- tion and samples were taken by the sequential use of three paper points (Dentsply Maillefer, Ballaigues, Switzerland) placed to the working length. Paper points were trans- ferred to Eppendorf tubes contain- ing 0.1 mL of normal 85% saline so- lution and centrifuged for 5 minutes at 10,000 rpm in a centrifuge for Ep- pendorf tubes (Beckmann Coulter® S.A., Madrid, Spain). Afterwards, the paper points were carefully removed and the superna- tant was eliminated from the Eppen- dorf tubes. Ten serial dilutions were placed (100 µl) in triplicate on the surface of Sabouraud Dextrose Agar (Scharlau® S.L., Barcelona, Spain) and incubated at 37ºC for 48 hours. The number of colonies was counted at the appropriate dilution and the number of colony forming units (CFU)/mL was calculated (Seal et al., 2002; Souza et al., 2010). SEM analysis Five teeth in each group were ana- lyzed by SEM. The teeth were fixed in a buffered formalin solution for 1 week, dehydrated by immersion in ethanol solutions of increasing con- centrations (70%, 95% and 100%), with two solution changes every 30 minutes. Longitudinal grooves were carefully made along the entire length of each root with a metallic water-refrigerated disk (Komet, Bar- celona, Spain) and, using a surgical chisel, a buccolingual split was cre- ated along the long axis to expose the entire extent of the root canal (Figure 2). Before analysis, the teeth were sub- mitted to metallographic prepara- tion in a scanning electron micro- scope (Oxford Instruments INCA 300 EDX System, Abingdon, Oxford- shire, United Kingdom). Initially, the specimens were analyzed by naviga- tion under different magnifications. For the comparative analysis be- tween groups, two SEM micrographs were obtained from each third. The root canal was measured and the central part of each middle third was evaluated. SEM images were obtained at 1,600 and 5,000 magni- fications. The images were then ana- lyzed to identify the presence or ab- sence of contamination and debris on the root canal surfaces (Estrela et al., 2012) using MIP-4® histomorpho- metry software (Digital Image, Barce- lona, Spain), calculating the percent- age of area with contamination and debris in relation to the total area under study. Statistical analysis Data were analyzed using the SPSS version 20.0 statistical pack- age (SPSS® Inc., Chicago, IL, USA). A descriptive study was made of each variable. ANOVA and Tukey’s test were applied to quantitative variables, in each case determining whether the variances were homo- geneous. Statistical significance was accepted for p<0.05. Results The E. Faecalis count in root canals, calculated as CFU/mL log10, was zero in the negative control group. The positive control group showed the highest number of colony-forming units per milliliter 6.11 ± 0.85 log10 CFU/mL, with statistically significant differences in comparison with all six treatment groups (p≤0.05). The ozone treatment group showed the lowest E. faecalis count (3.62 ± 0.92 log10 CFU/mL), followed by the PDT group (3.63 ± 1.61 log10 CFU/mL), which obtained similar values to the TAP group (3.71 ± 0.56 log10 CFU/mL) and the 2.5% NaOCL group (3.73 ± 1.95 log10 CFU/mL). The propolis group obtained higher values than the 2.5% NaOCL and 2% CHX groups (Table 1). Comparing the percentage of area with contamination and debris in relation to the total area under study (coronal + middle + apical) obtained by each group, the highest percent- age was obtained by the positive control group (25.22 ± 5.62 %) (Table 2) (Figures 3A and 3B), while the 2.5% NaOCL group showed the lowest percentage of area contaminated by bacteria and debris (20.75 ± 3.08 %) (Figure 3C and 3D), followed by the ozone group (21.71 ± 1.49 %) (Figures 4A and 4B) which obtained similar results to the PDT group (22.22 ± 6.82 %) (Figures 5C and 5D). But no statis- tically significant differences were found between any of the groups. Discussion In the present study, inoculation by E. faecalis was performed at a con- centration of 3 x 108 cell/mL, the same concentration as used by other researchers including Estrela et al., and Kranz et al., (Estrela et al., 2012; Kranz et al., 2011), and slightly higher than that used by Fimple et al., (2.5 x 108 cel/mL) (Fimple et al., 2008). On the basis of the results obtained in the positive control group, this was clearly an effective concentration for infecting root canals for in vitro ex- perimental purposes. In the field of endodontics, NaOCL shows broad-spectrum antimicro- bial activity against difficult-to-erad- icate microorganisms and biofilms of species such as Enterococcus, Ac- tinomyces and Candida (Heling et al., 2001). Consensus has not yet been reached as to the ideal concentra- tion for the treatment of endodontic infections (Estrela et al., 2012) and the concentrations used vary from 0.5-5% (Baumgartner et al., 1992), although the most widely used con- centration is 2.5% (Navarro-Escobar et al., 2010). This concentration achieves an adequate bactericidal effect while keeping its high tissue toxicity to a minimum, which could otherwise cause haemolysis, ulcers, neutrophil migration, and the de- struction of endothelial cells and fi- broblasts (Gerhardt et al., 2004). The toxicity is mainly due to the fact that NaOCL is a non-specific oxidizing agent that favors the rapid oxidation of proteins and the destruction of membrane lipid cells (Fuentes et al., 2009). In the present study, 5-min- ute applications of 2.5% NaOCL were seen to reduce the E. faecalis count in tooth canals with statistically signifi- cant difference compared with the positive control group. Numerous authors (Zou et al., 2010; Stojicic et al., 2001) claim that the antibacterial effect of NaOCL does not depend ex- clusively on its concentration but on a range of other variables including exposure time. In this way, 5-minute exposure to NaOCL at 2 or 2.5% has the same bactericidal effect as 2 min- utes exposure at 5.25%, 10 minutes at 2.5% or 30 minutes at 1.5% (Gerhardt et al., 2004; Radcliffe et al., 2004). Nevertheless, 2.5% reduces the risk of tissue damage. The use of CHX as an endodontic irrigant is based on its substantiv- ity and its long-lasting antimicro- bial effect that derives from its ad- hesion to hydroxyapatite (Evanov et al., 2004). Its antimicrobial action ÿPage 20

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