Results.

pH measurements.
High pH values in the range of11.7-12.4 were measured in suspensions of Ca(OH)2 powder with irrigating solutions, irrespective of the pH of the irrigating solution alone (Table 1). In the extracted single-rooted teeth used for the OH-ion diffusion assay, the average dentine thickness between the root canal space and the floor of the measuring well was1.8 _0.3 mm. No significant difference in dentine thickness was found between groups (anova, P = 0.76). Removing the cementum layer at the root surface to obtain measuring well did not influence OH- ion penetration (Fig. 1). Significant pH differences at the root surface between test and control groups were first noted after 2 weeks (Table 1; Kruskal-Wallis test, P < 0.05). These differences became more pronounced at weeks 3 and 5 (P < 0.005). Significant differences were found from week 2-5 (Mann-Whitney U-test, P < 0.05) when comparing pH values of the IPI/Ca(OH)2 group and the saline/Ca(OH)2 group with pH values obtained with their corresponding irrigation solutions. It took 3 weeks for the CHx/Ca(OH)2 group and 5 weeks for the NaOCl/Ca(OH)2 group to produce significant pH differences compared to controls (P < 0.05). There was a tendency of the NaOCl/Ca(OH)2 medication to release less OH- ions through dentine than the other Ca(OH)2/ irrigant mixtures. However, no significant differences between pH values measured with different Ca(OH)2 medications were noted at any time (P > 0.05). This statement was also true for pH differences between the four control groups.

Figure 1. Teeth dressed for 5 weeks with a calciumhydroxide (Ca(OH)2) medication (panels B and C) or saline (panel A) were perfused with thymol blue (original magnification 30x). The indicator changes its colour from yellow to blue at pH 8.0^9.2. Note that the removal of cementum in the measuring well apparently did not influence the penetration of hydroxide (OH-) ions through dentine (panels B and C).

Agar diffusion test.
On plates incubated with E. faecalis or C. albicans, zones of inhibition did not differ significantly between the four Ca(OH)2 medications (Table 2; anova, P > 0.05). Significantly larger areas of inhibition were obtained with CHx alone than with the same amount of CHx mixed with Ca(OH)2. Inhibit on areas with I2/I_ as well as with OCl- were significantly smaller than respective zones obtained with the corresponding mixtures with Ca(OH)2 (P < 0.05).
When the pH of the 0.5% CHx solution was increased to 12 by titration with NaOH, the agent precipitated. However, the solution was still as effective against C. albicans as the original at pH 6 (P > 0.05). In contrast, a 5% IPI solution made alkaline with NaOH turned clear at pH 10.9 and did no longer inhibit the growth of C. albicans.

Table 2. Diameters (mm) of the zones of inhibition against the test organisms (agar diffusion).

Discussion.
The antimicrobial effectiveness of Ca(OH)2 is based on its ability to release OH- ions (Proell1949). Because Ca(OH)2 is a strong base, weak acids in a Ca(OH)2 suspension do not influence chemical or antimicrobial properties of the latter. This was corroborated by the present study.
In this in vitro study, similar pH values were measured at the root surface as in an in vivo investigation in monkeys (Tronstad et al. 1980). In the absence of fluid movement in the nonvital tooth, OH- ions are transported through dentine tubuli by molecular diffusion (Nerwich et al.1993). Thus, the development of the pH in a defined solution volume outside the tooth can be calculated using Fick’s first law of diffusion (Crank1975). Starting with a pH of 12 at the Ca(OH)2-dentine interface, a diffusion length of 1.8 mm and an average liquid space of 0.5% in root dentine (Pashley 1991), a calculated pH of 9.6 would be expected after 5 min in a volume of 2 mL when all chemical interactions are neglected. However, the median pH value at the surface of roots filled with Ca(OH)2 pastes measured after 3 weeks was 8.7. Thus, the reported buffering capacity of dentine for alkalis (Wang&Hume1988) was confirmed in this study.This buffering capacity may also explain why NaOCl alone, at a similar pH as the Ca(OH)2 pastes, did not change the root pH in our experiments. Ca(OH)2 has low solubility in water. In the moist environment of the root canal system, undissolved Ca(OH)2 in a paste-like suspension will steadily dissolve, resulting in a sustained pH effect, which was not observed with the solutions alone. This is in agreement with a reported in vivo finding: a saturated solution of Ca(OH)2, pH 12,wasunable to eliminate E. faecalis fromthe root canal of a cat canine, whilst a paste-like aqueous suspension killed all cultivable bacteria (Stevens & Grossman1983).
As stated above, Ca(OH)2 effectiveness is entirely pH related (Bystrom et al.1985, Evans et al. 2002).Although direct clinical evidence is still elusive, it may therefore be stated that bacteria and yeasts with a high tolerance for basic pH levels, such as Enterococci and Candida species, may not effectively be killed by conventional Ca(OH)2 suspensions. In particular, Enterococci are hardy opportunistic invaders, able to survive as a mono infection in a sparse environment (Fabricius et al. 1982). Moreover, Enterococci readily invade dentinal tubules by division (Crstavik & Haapasalo1990). Therefore, facultative anaerobes and yeasts may find an ecological niche in dentinal tubules of root-treated teeth. In endodontic retreatments, when facultatives predominate, there appears to be a need for improved interappointment root canal dressings. Under the conditions of this study, no increase of antimicrobial efficacy was noted in suspensions of irrigating solutions with Ca(OH)2 compared to a conventional Ca(OH)2 medication. Our results are in accordance with a recently published study (Estrela et al. 2001), which could not demonstrate an additive antibacterial effect when mixing Ca(OH)2 powder with CHx, but showed that Ca(OH)2 does not lose its antibacterial properties in such a mixture. In the original article on mixtures of root canal disinfectants (Waltimo et al. 1999), irrigating solutions were saturated with Ca(OH)2 powder. In those mixtures, a saturated Ca(OH)2 solution alone was less effective against C. albicans than NaOCl, IPI or CHx solutions saturated with Ca(OH)2 powder in vitro. However, as stated above, a saturated Ca(OH)2 solution is ineffective as a root canal dressing in vivo (Stevens & Grossman 1983). Therefore, it may be more relevant to use Ca(OH)2/irrigant mixtures with a paste-like consistency for comparative in vitro studies.
An agar diffusion method was chosen in the current study to assess the antimicrobial effectiveness of irrigants and their mixtures with Ca(OH)2. It may be argued that, with the known inhibitory action of dentine on root canal medicaments (Haapasalo et al. 2000), a dentine block model may have been more appropriate. However, the primary goal of the present investigation was to study the direct interactions of irrigants and Ca(OH)2, and an agar diffusion test appeared suitable for this purpose.
In CHx/Ca(OH)2 mixtures, we detected an inhibition of antimicrobial activity of CHx, most likely caused by the high pH and the alkaline buffering capacity of the suspension. The reason for the reduced efficacy may be the deprotonation of the biguanide at pH >10 and hence a markedly reduced solubility and altered interaction with bacterial surfaces due to the change in the charge of the molecule (Jones et al.2000). Ina 0.5%CHx digluconate solution inwhichthepHwasadjustedto12byNaOH titration, the antimicrobial effect was maintained. An explanation for that phenomenon may be seen in the lower alkaline buffering capacity of a solution compared to a Ca(OH)2 suspension, resulting in a pH drop with increasing distance from the OH- source. Dilution and the buffering effect of the agar may have allowed the CHx to become reprotonated and regain its bactericidal activity. IPI appears to be ineffective in killing microbiota at pH levels higher than11. I2 at high pH disproportionates to IO_ and I_ and, since recombination is kinetically slow, looses its antimicrobial activity (Lengyel et al. 1993). In addition, the allergenic potential of iodine may be a contraindication for its long-term use as a root canal dressing.
Thus, the most promising Ca(OH)2 powder/irrigant combination might be the Ca(OH)2/NaOCl paste. Hypochlorite is chemically stable at high pH. Although apparently not more effective in an agar diffusion test, such a combination may have better tissue-dissolving properties than conventional Ca(OH)2/saline paste (Hasselgren et al.1988). Further research is necessary to confirm that hypothesis.

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