calcium hydroxide [60, 79, 83, 90, 91]. E. faecalis ability to resist high pH values seems to be related to a functioning proton pump, which drives protons into the cell to acidify the cytoplasm [91]. E. faecalis and Candida species are commonly found in root canal-treated teeth with posttreatment disease [49, 92–97].

Inactivation of Bacterial Virulence

Factors

Structural components of the bacterial cell are important virulence factors that stimulate and modulate the inflammatory response and induce indirect damage to host tissues. The main examples are lipopolysaccharides (LPS, a.k.a., endotoxins) and the lipoteichoic acid (LTA), components of the cell wall of gram-negative and gram-positive bacteria, respectively.

Lipid A is the portion of LPS that has been regarded as the main responsible factor for the biological effects of this molecule [98, 99]. In vitro studies demonstrated that calcium hydroxide can inactivate LPS by acting primarily on the lipid A portion, inducing the alkaline hydrolysis of ester bonds with consequent release of free hydroxy fatty acids with no or reduced toxic and pro-inflammatory effects [100–106]. However, this inactivating effect has been observed in vitro under optimal contact between LPS and calcium hydroxide. It is highly unlikely that hydroxyl ions released from calcium hydroxide can reach LPS molecules present in areas distant from the main canal in magnitude sufficient to inactivate these molecules. A clinical study revealed that the levels of LPS were reduced but still relatively high in the canal after chemomechanical preparation, and these levels were virtually unaltered after intracanal medication with calcium hydroxide, CHX, or a combination of both [107].

LTA is a polymer of glycerol phosphate linked to fatty acids. It has been demonstrated that calcium hydroxide can detoxify LTA and attenuate its pro-inflammatory ability [108]. These inactivating effects are supposed to be related to deacylation of LTA induced under high alkaline conditions. Deacylated LTA does not stimulate

Toll-like receptor 2, the host molecule responsible for recognition of and response to LTA, and the consequent release of pro-inflammatory cytokines [108]. There are no clinical studies reporting on the effects of calcium hydroxide medication on LTA intracanal levels.

Thus far, it remains to be determined whether these inactivating effects of calcium hydroxide on LPS and LTA can be consistently observed in vivo and, if so, what is the actual relevance for the long-term treatment outcome. After all, there is no clear indication that LPS or LTA molecules, in the absence of living bacteria, can induce or maintain periradicular inflammation beyond a certain point in time. Moreover, it is important to point out that virulence factors other than LPS and LTA can also be involved in the pathogenesis of apical periodontitis, usually in a mixture of factors released from multispecies biofilms [109]. This scenario makes the analysis of the effects against specific factors like LPS or LTA somewhat simplistic.

Combination with Biologically Active Vehicles

In an attempt to sidestep the limitations of calcium hydroxide pastes in inert vehicles (e.g., distilled water, saline, glycerin), association of this substance with other antibacterial medicaments, such as CPMC or CHX, has been evaluated [68, 83, 110, 111].

Paste in CPMC

In vitro studies have demonstrated that calcium hydroxide paste in CPMC has a broader antimicrobial spectrum (eliminating microorganisms that are resistant to calcium hydroxide) and a larger radius of antimicrobial action (eliminating microorganisms located in regions more distant from the vicinity where the paste was applied) kills microorganisms faster and is less affected by serum and necrotic tissue than mixtures of calcium hydroxide with inert vehicles [59, 68, 72, 83, 84, 112–119]. The larger radius of action may be a result of the low surface tension of CPMC and/or its high solubility in lipids. Glycerin has

been added to the paste to dilute CPMC and facilitate both handling and further removal of the paste from the canal. Although CPMC exhibits high toxicity when used alone, satisfactory biocompatibility results have been observed in animal studies [120, 121]. Clinical studies evaluating the incidence of postoperative pain [122], antibacterial activity [37, 40], and treatment outcome [123] have demonstrated optimal results when using an antibacterial protocol for treatment that includes a 7-day interappointment medication with calcium hydroxide/CPMC/glycerin paste.

Paste in CHX

In vitro studies investigating the antimicrobial effectiveness of the combination calcium hydroxide and CHX have shown conflicting results. Some studies demonstrated that the antimicrobial effects of calcium hydroxide are significantly increased when adding CHX in a paste [110, 124–126], while others have shown no significant increase in activity [112, 127]. However, the antibacterial efficacy of CHX may be significantly reduced after mixing with calcium hydroxide [112, 126, 127].

Although some clinical studies have shown no advantage in using calcium hydroxide combined with CHX [71, 107], others have reported good results for this association [38, 128–130]. Zerella et al. [128] reported that intracanal dressing with a mixture of 2 % CHX and calcium hydroxide was at least as effective as calcium hydroxide in an inert vehicle in the disinfection of root canal-treated teeth with apical periodontitis. In a clinical study evaluating the antibacterial effectiveness of a treatment protocol against primary infections, Siqueira et al. [38] used 0.12 % CHX as the irrigant during chemomechanical preparation and found an incidence of positive cultures of 54 %. Further intracanal medication with calcium hydroxide paste in 0.12 % CHX significantly decreased the number of positive cultures to 8 %. Paiva et al. [129] used several sensitive molecular biology techniques to evaluate the clinical antibacterial effects of chemomechanical preparation using NiTi rotary instrumentation and NaOCl irrigation (S2), a final rinse with

CHX (S3), and then 1-week interappointment medication with calcium hydroxide/CHX paste (S4). Treatment procedures promoted a decrease in microbial diversity and significantly reduced the incidence of positive results and the bacterial counts. In general, each subsequent treatment step improved disinfection. In S2, 64 % of samples were still positive for the presence of bacteria, decreasing to 43 % in S3 and then to 14 % in S4. The number of positive results was significantly lower for S4 when compared with S2, and the same was true for bacterial counting analysis. The authors concluded that supplementary steps consisting of a final rinse with CHX followed by calcium hydroxide/CHX interappointment medication promoted further decrease of the bacterial bioburden to levels significantly below those achieved by the chemomechanical procedures alone. Oliveira et al. [130] demonstrated that intracanal medication with calcium hydroxide/CHX paste had significant supplementary effects in eliminating endotoxins from infected canals and/or neutralizing their cytotoxic effects.

CHX remains stable at pH 5–8 and, as the pH increases, ionization decreases. Association of calcium hydroxide with CHX maintains a high pH value, which is similar to calcium hydroxide paste using water as vehicle [110, 128]. CHX antimicrobial activity is influenced by pH conditions, with the optimal range of 5.5–7, and at high pH values, it precipitates and may be unavailable as an antimicrobial agent [128]. Despite the expected high loss of CHX when mixed with calcium hydroxide, the combined resulting antimicrobial effect may still be of clinical significance, as demonstrated by the studies discussed above [38, 128–130]. This combination presents significant antibacterial effects, which may be related to small residues of active CHX still present in the paste, even though the effects of the high pH of the paste cannot be disregarded.

Table 16.2 summarizes several clinical studies investigating the percentage of cases that remained positive for the presence of detectable bacteria after using different treatment protocols.