S. Haenni, P. R. Schmidlin, B. Mueller, B. Sener & M. Zehnder
Division of Endodontology, Department of Preventive Dentistry, Cariology, and Periodontology, Center for Dental Medicine, University of Zurich, Zurich, Switzerland.
Swiss Federal Institute for Environmental Science and Technology (EAWAG), Kastanienbaum, Switzerland.
Swiss Federal Institute of Technology (ETH), Limnological Research Center, Kastanienbaum, Switzerland.

Introduction.
Teeth with periapical lesions of endodontic origin may be divided into two main groups: teeth with necrotic pulps (primary endodontic infections) and teeth with failed root canal treatments. In cases of primary endodontic infections, the flora consists mostly of anaerobes (Sundqvist 1976). In such cases, a calcium hydroxide (Ca(OH)2) dressing for at least 1 week has been demonstrated to kill bacteria more thoroughly than all other common intracanal medications (Bystrom et al. 1985), and it has been recommended for the treatment of apical periodontitis in teeth with necrotic pulps. However, in cases of failed endodontic treatment, the intracanal flora is different, and facultative anaerobes may predominate (Molander et al.1998). In addition, yeasts have been associated with endodontic failures (Waltimo et al. 1997). Interestingly, Ca(OH)2 has been shown to be inefficient in the killing of both facultative anaerobes and yeasts, whilst other medications or irrigating solutions have been shown to be more effective in the killing of these microbiota in vitro (Crstavik & Haapasalo1990,Waltimo et al.1999). It has therefore been suggested that Ca(OH)2 powder should be mixed with iodine potassium iodide (IPI), chlorhexidine (CHx) or sodium hypochlorite (NaOCl) in order to obtain a wide-spectrum antimicrobial preparation with a long-lasting effect (Waltimo et al. 1999). However, the effects of these irrigation solutions on Ca(OH)2 and vice-versa have not been studied in detail.
When used as an intracanal dressing, Ca(OH)2 mixed with water or saline to a paste-like consistency results in a radially diminishing pH rise in dentine over time, until a steady state is reached (Tronstad et al.1980). Alkalis have a pronounced destructive effect on cell membranes and protein structure (Gordon et al. 1985), and antimicrobial properties of Ca(OH)2 are directly related to pH (Bystrom et al. 1985, Evans et al. 2002). Therefore, the purpose of this study was to evaluate the effect of mixing Ca(OH)2 powder with IPI, CHx digluconate and NaOCl solutions on the ability of Ca(OH)2 to raise the pH at the root surface in vitro. In addition, the antimicrobial potential of these solutions and mixtures was assayed with an agar diffusion test against Enterococus faecalis and Candida albicans.

Materials and methods.

Selection and preparation of teeth.
Eighty single-rooted human canines and premolars of similar length and diameter from the Department’s collection of extracted teeth were used. After collection, they were stored in 0.1% thymol solution. Crowns were amputated at the level of the cemento-enamel junction. Root canals were instrumented with ProTaper1 files (Dentsply Maillefer, Ballaigues, Switzerland) under copious irrigation with a1% NaOCl solution. The apices were intentionally over instrumented witha size 40Flexofi le (Maillefer) to facilitate sealing with glass ionomer cement (see below). After instrumentation, the root canals were flushed repeatedly with17% EDTA solution for 5 min to remove the smear layer. In the middle third of the buccal aspect of each root, a standardized well 0.75 mm deep, 3 mm long and 1.5 mm in diameter was prepared with a 1.5-mm diameter diamond round bur. To assess the remaining dentine thickness under the measuring well, digital radiographs were taken of the mesio-distal aspect of the teeth (Digora1, Soredex, Helsinki, Finland). After calibration, the dentine thickness was measured using the Digora1 software (Soredex). Before the experiments, teeth were washed in deionized water at room temperature for12 h.

Intracanal dressings.
The 80 experimental teeth were randomly divided into four different test groups and four corresponding control groups of 10 teeth each. The teeth were placed into individual vials containing unbuffered isotonic saline solution. After air-drying the teeth and sealing the apices with glass ionomer cement (Ketac ESPE, Seefeld, Germany) and nail varnish, intracanal medications were applied. Prepared root canals were filled with: Group 1, Ca(OH)2 (Merck, Darmstadt, Germany) mixed with unbuffered isotonic saline; Group 2, unbuffered isotonic saline; Group 3, 0.5% (w/v) CHx digluconate Ca(OH)2 paste; Group 4, 0.5% CHx; Group 5, 1% (w/v) NaOCl Ca(OH)2 paste; Group 6, 1% NaOCl; Group 7, 5% (w/v) IPI (I2 ÑŽ KI) Ca(OH)2 paste and Group 8,5%IPI.According to clinical standards, all pastes were mixed to a creamy consistency on a glass slab using Ca(OH)2 powder and the respective solutions (1 :1.5, w/v). Pastes were applied with a lentulo spiral (Maillefer), solutions with a syringe and a 26-gauge needle. Subsequently, access cavities were sealed in the same manner as the apices. Teeth were processed in batches containing one sample of each test (mixtures) and control (solutions only) group. To further avoid bias, the investigator taking the pH measurements was not aware of the content of the roots.

pH measurements.
Measurements of root surface pH were performed immediately after filling and then after 24 h, 3 days, 1, 2, 3 and 5 weeks postfill. For measuring, teeth were removed from their vials, cooled to room temperature in a 100% humid environment and dried with compressed air (GEPE-air, Image Trade, Safenwil, Switzerland). Two microlitres of unbuffered isotonic saline solution was placed into the standardized well. After an equilibration time of 5 min, pH was measured with a calibrated microelectrode (MI 415-2, Microelectrodes Inc., Bedford, NH, USA). Subsequently, teeth were returned to their vials filled with fresh saline and incubated at 378C.
In addition, the pH of 1:1.5 (w/v) mixtures of Ca(OH)2 with the irrigating solutions was measured with a semiconductor pH electrode (Sanwa Tsusho Co., Tokyo, Japan) and compared to the pH of the solutions (Table 1).

Table 1. pH values of irrigants/Ca(OH)2 medications and pH values measured at the root surface of teeth dressed with these in vitro at different points in time.

To assess the influence of removing the cementum layer on the experimental tooth surface (measuring well) on hydroxide ion (OH-) penetration, teeth were stained with a pH indicator (thymol blue, Merck). Thymol blue changes its colour from yellow to blue at a pH range of 8.0-9.2. Experimental and control teeth that had been dressed for 5 weeks were unsealed, and the root canals cleaned with saline and interproximal brushes. Teeth were then placed in eppendorf tubes containing 1mL of the indicator solution. Subsequently, specimens were centrifuged at 4000 g for 30 min, and longitudinally or horizontally sectioned through the centres of the measuring wells using a slow-speed diamond-coated rotary disc (Isometh, Buehler, Prufmaschinen AG, Zurich, Switzerland).The disc was cooled with kerosene to avoid influencing pH scores, and finally the cut surfaces were photographed.

Agar diffusion test.
Facultative bacteria (E. faecalis ATCC 29212) and yeasts (C. albicansOMZ110) were maintained separately influid universal medium (FUM) (Gmur & Guggenheim 1983). The latter microbiota were chosen because they are the facultatives and yeasts most frequently isolated from root canals of failed treatments (Waltimo et al.1997, Dahle. n et al. 2000). The optical density of FUM sample aliquots was measured at a wavelength of 550 nm in a spectrophotometer (U 2000, Hitachi, Boehringer-Mannheim, Rotkreuz, Switzerland) and adjusted by dilution with FUMto1.0. In Petri plates,25 mL of Columbia Blood Agar (Becton Dickinson Microbiology Systems, Sparks, MD,USA)was inoculatedwith5 mL of the broth containing the microorganisms. After the agar had set, round cavities, 5 mm in diameter, were punched out. These cavities were filled with the solutions (40 mL) and combinations (40 mL of solution mixed with 20 mg Ca(OH)2 powder). After 24 h incubation at 378C in ambient air, diameters of zones of inhibition were measured with a caliper. All experiments were performed in triplicate.
To test the influence of high OH- ion concentration on the antimicrobial effectiveness of irrigants, 5% IPI and 0.5% CHx solutions were adjusted to pH 12 by titration with a 1% sodium hydroxide (NaOH) solution (pH 14), and subjected to the agar diffusion test described above.

Statistical analysis.
As pH values are logarithmic, nonparametric statistics were applied to compare pH values at the root surface between groups: Kruskal-Wallis one-way analysis of variance followed by Mann-Whitney U-test for individual comparisons. Data obtained in the agar diffusion tests and dentine thickness values were compared using one-way analysis of variance (anova) followed by an unpaired t-test. Bonferroni adjustments were applied for multiple posthoc testing. Levels for rejection of null hypotheses were set at P < 0.05.