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Chlorhexidine in Endodontics

Abstracts

Chemical auxiliary substances (CAS) are essential for a successful disinfection and cleanness of the root canals, being used during the instrumentation and if necessary, as antimicrobial intracanal medicaments. Different CAS have been proposed and used, among which sodium hypochlorite (NaOCl), chlorhexidine (CHX), 17% EDTA, citric acid, MTAD and 37% phosphoric acid solution. CHX has been used in Endodontics as an irrigating substance or intracanal medicament, as it possesses a wide range of antimicrobial activity, substantivity (residual antimicrobial activity), lower cytotoxicity than NaOCl whilst demonstrating efficient clinical performance, lubricating properties, rheological action (present in the gel presentation, keeping the debris in suspension); it inhibits metalloproteinase, is chemically stable, does not stain cloths, it is odorless, water soluble, among other properties. CHX has been recommended as an alternative to NaOCl, especially in cases of open apex, root resorption, foramen enlargement and root perforation, due to its biocompatibility, or in cases of allergy related to bleaching solutions. The aim of this paper is to review CHX's general use in the medical field and in dentistry; its chemical structure, presentation form and storage; mechanism of action; antimicrobial activity including substantivity, effects on biofilms and endotoxins, effects on coronal and apical microbial microleakage; tissue dissolution ability; interaction with endodontic irrigants; effects on dentin bonding, metalloproteinases and collagen fibrils; its use as intracanal medicament and diffusion into the dentinal tubules; its use as disinfectant agent of obturation cones; other uses in the endodontic therapy; and possible adverse effects, cytotoxicity and genotoxicity.

chlorhexidine; Endodontics; irrigants; intracanal medicaments; antimicrobial activity


Resumo

Substâncias químicas auxiliares (SQA) são essenciais para o processo de limpeza e desinfecção dos canais radiculares, sendo utilizadas durante a instrumentação dos canais radiculares e, se necessário, como medicamentos intracanais. Diferentes SQA têm sido propostas e utilizadas, entre elas: hipoclorito de sódio (NaOCl), clorexidina (CHX), EDTA 17%, ácido cítrico, MTAD e solução de ácido fosfórico a 37%. CHX tem sido usada na endodontia como SQA ou medicação intracanal. CHX possui uma ampla gama de atividade antimicrobiana; substantividade (atividade antimicrobiana residual); menor citotoxicidade que NaOCl, demonstrando desempenho clínico eficiente; propriedades de lubrificação; ação reológica (presente na apresentação gel, mantendo os detritos em suspensão); inibe metaloproteinases; é quimicamente estável; não mancha tecidos; é inodora; solúvel em água; entre outras propriedades. CHX tem sido recomendada como uma alternativa ao NaOCl, especialmente em casos de ápice aberto, reabsorção radicular, perfuração radicular e durante a ampliação foraminal, devido à sua biocompatibilidade, ou em casos de alergia ao NaOCl. O objetivo deste trabalho é fazer uma revisão do uso da clorexidina na medicina e na odontologia; sua estrutura química; forma de apresentação e armazenamento; mecanismo de ação, atividade antimicrobiana, incluindo, substantividade, efeitos sobre biofilmes e endotoxinas; efeito sobre infiltração microbiana coronal e apical; capacidade de dissolução do tecido; interação com os irrigantes; efeitos sobre a união à dentina, metaloproteinases e fibrilas de colágeno; a sua utilização como medicamento intracanal e difusão nos túbulos dentinários; a sua utilização como agente desinfetante de cones de obturação; seus outros usos na terapia endodôntica, possíveis efeitos adversos, citotoxicidade e genotoxicidade.


Introduction

Complete debridement and disinfection of the pulpal space are considered to be essential for predictable longterm success in endodontic treatment. Residual pulpal tissue, bacteria and dentin debris may persist in the irregularities of root canal systems, even after meticulous mechanical preparation (1,2). Therefore, several irrigating substances have been recommended for use in combination with canal preparation, including sodium hypochlorite (NaOCl), chlorhexidine gluconate (also called chlorhexidine digluconate or just chlorhexidine - CHX), 17% EDTA, citric acid, MTAD and 37% phosphoric acid solution. However, if these substances remain in the root canal, they might affect the penetration of the resin sealer in dentin and its polymerization. They might also degenerate dentin if they have a negative effect on the collagen fibers (3,4). It has long been recognized that the antibacterial effects of chemomechanical procedures can be enhanced by the subsequent placement of an antimicrobial intracanal medication, particularly in those cases of exudation, haemorrhage, perforation, root resorption, trauma or incomplete root formation (5-7). Nevertheless, the efficacy of both procedures also depends on the vulnerability of the involved microbial species present in the root canal system. Moreover, in order to avoid re-infection of the cleansed space, not only the placement of a host-compatible root canal filling but also of a permanent coronal restoration must be performed (2).

CHX can be applied clinically as antimicrobial agent during all phases of the root canal preparation, including the disinfection of the operatory field; during the enlargement of the canals orifices; removal of necrotic tissues before performing the root canal length determination; in the chemomechanical preparation prior to the foraminal patency and enlargement; as an intracanal medicament alone or combined with other substances (i.e. calcium hydroxide - CH); in the disinfection of obturation cones; for modeling the main gutta-percha cone; in the removal of gutta-percha cones during retreatment; in the disinfection of prosthetic space; among others. Therefore, the objective of this review was to report relevant aspects of chlorhexidine in Endodontics.

General Use

According to Tomás et al. (8), back in 1947, a complex study to synthesize new antimalarial agents led to the development of the polybiguanides (9). These compounds showed significant antimicrobial potential, particularly compound 10,040, a cationic detergent later called chlorhexidine (10). The first salt derived from compound 10,040 that reached the market was chlorhexidine gluconate. It was registered in 1954 by the Imperial Chemical Industries Co. Ltd. of Macclesfield (United Kingdom) under the brand name Hibitane®, the first internationally accepted antiseptic for cleaning skin, wounds and mucous membranes because of its strong affinity to such areas, with high level of antibacterial activity and low mammalian toxicity (11). In 1957, only 3 years after coming into the market, the broad antimicrobial spectrum of CHX led to an extension of its indications to include not only skin disinfection, but also use in the fields of ophthalmology, urology, gynecology and otorhinolaryngology. Although CHX started being used to control bacterial plaque in 1959, it use became widespread in dentistry only in the 1970s after the publication of the studies by Löe and Schiött (8,12,13).

CHX is currently considered the gold standard of oral antiseptics and is, along with fluoride, the most extensively researched preventive agent in dentistry (14). In addition to its effects on dental plaque and gingivitis, CHX is effective in the prevention and treatment of caries (15,16); secondary infections to oral surgical procedures, and in the maintenance of the health of peri-implant tissues. CHX reduces the bacterial load of aerosols and reduces bacteremia after dental manipulation. It is also employed in the treatment of recurrent aphthous and denture-related stomatitis. CHX is particularly indicated in certain population groups, such as individuals with orthodontic appliances, disabled people, and immunologically compromised patients (8,17). It also retains its activity in the presence of blood, wounds and burns (11). Soaking dentures in CHX has also been shown to be effective in reducing colonization with Candida species (11). It has been used in Endodontics as an irrigating substance (2,6,14,18-25) or intracanal medicament alone or in combination with CH (5,6,19,26-30), among other uses.

Chemical Structure

The structural formula of CHX consists of two symmetric 4-chlorophenyl rings and two biguanide groups connected by a central hexamethylene chain (11), as illustrated in the image below:

Presentation Form

CHX is an almost colorless to pale straw-colored substance or slightly opalescent, odorless or almost odorless substance. The 20% (w/w) CHX salt is the most commonly used. Solutions prepared from all salts have an extremely bitter taste that must be masked in formulations intended for oral use (11). There are a variety of products containing CHX used in dentistry, medicine, veterinary and food, namely Periogard, Perioxidin, Plac Out, Corsodyl, Chlorohex, Descutan, Hibiscrub, Hibitane, Savacol, among others. The most commonly used concentrations in commercially available CHX mouth rinses are 0.12 and 0.20%. The 2% concentration, used in Endodontics, can be prepared by pharmacies, under prescription (23,24,30,31).

For endodontic purposes, CHX can be used in a liquid or in a gel presentation. CHX gel consists of a gel base (1% natrosol, a hydroxyethylcellulose, pH 6-9) and chlorhexidine gluconate (23,31), in a optimal pH range of 5.5 to 7.0 Natrosol gel is a biocompatible carbon polymer (32) that is a water-soluble substance, and therefore can be easily removed from the root canal with a final flush of distilled water (23,31).

Some studies have shown that the antimicrobial activity of CHX liquid is equal or superior to that of CHX gel when the direct contact was used as a methodology (2,24,33). In other studies, using the agar diffusion test, 2% CHX gel was superior to 2% CHX liquid (34).

Ferraz et al. (23,34) showed that 2% CHX gel has several advantages over 2% CHX solution, in spite of having similar antimicrobial, substantivity and biocompatibility properties. The CHX gel lubricates the root canal walls, which reduces the friction between the file and the dentin surface, facilitating the instrumentation and decreasing the risks of instrument breakage inside the canal. In addition, by facilitating instrumentation, CHX gel improves the elimination of organic tissues, which compensates for its incapacity to dissolve them (23,31). Another advantage of CHX gel is the reduction of smear layer formation (23), which does not occur with the liquid form. CHX gel maintains almost all the dentinal tubules open because its viscosity keeps the debris in suspension (rheological action), reducing smear layer formation. Furthermore, the gel formulation may keep the “active principle” of CHX in contact with the microorganisms for a longer time, inhibiting their growth (24).

It is important to point out that during chemomecanical preparation, before using each file, 1 mL of CHX gel is introduced in the root canal with a syringe (24-gauge needle or smaller) and immediately after each instrumentation, 5 mL of distilled water is used to irrigate the canal. Before the use of EDTA or other chemical substance, a final flush with 10 mL of distilled water is recommended in order remove traces of CHX inside the root canals.

Storage

A shelf life of at least 1 year can be expected, provided that packaging is adequate, in a dark, refrigerated bottle (11,20). Regarding the gel formulation, it may keep its pH and satisfactory antimicrobial activity for approximately 10 months after the fabrication date. Color alteration has been found in samples 1 year after the fabrication date (unpublished data), probably due to the presence of breakdown products resulting from prolonged shelf life or exposure to high temperatures.

Mechanism of Action

CHX is a strong base and it is more stable in the form of its salts. The salts originally employed were acetate and hydrochlorite, both of which suffer from relatively poor water solubility and were largely replaced by the digluconate in 1957 (35), which is a highly water soluble salt. Aqueous solutions of CHX are more stable within the pH range from 5 to 8. The antimicrobial activity of CHX is pH-dependent, being the optimum range from 5.5 to 7.0, within which is the pH of body surfaces and tissues (11). It readily dissociates at the physiological pH, releasing the positively charged CH component.

The bactericidal effect of the drug is due to the cationic molecule binding to extra-microbial complexes and negatively charged microbial cell walls, thereby altering the osmotic equilibrium of the cells. At low concentrations, low molecular weight substances will leak out, specifically potassium and phosphorous, resulting in a bacteriostatic effect. At higher concentrations, CHX has a bactericidal effect due to precipitation and/or coagulation of the cytoplasm of bacterial cells, probably caused by protein cross-linking, resulting in cell death (14,36), and leaving cell debris in the root canals (37), which can be removed with a vigorous irrigation with distilled water.

Antimicrobial Activity

Regarding the spectrum of activity, CHX is bactericidal and effective against Gram-positive and Gram-negative bacteria, facultative and strict anaerobes (2,14,19,20,23,24,27,38-40), yeasts and fungi, particularly Candida albicans (24,33,34,41). It is active against some viruses (respiratory viruses, herpes, cytomegalovirus, HIV) and inactive against bacterial spores at room temperature (42-44). It also retains its activity in the presence of blood (11) and organic matters (45).

In the liquid presentation, CHX kills microorganisms in 30 s or less, while in the gel formulation it takes from 22 s (2% CHX gel) to 2 h (0.2% CHX gel) (24).

Several in vitro works using a broth dilution test have shown that 2.0% CHX (in both presentation forms) and 5.25% NaOCl have similar antimicrobial performance against all tested microorganisms (2,21,24,44), while others have shown the superiority of 2% CHX gel or liquid over 5.25% NaOCl (34) using the agar diffusion method.

Clinical investigations have also been performed to compare the antimicrobial activity of CHX and NaOCl, and reported that these two substances had comparable effects in eliminating bacteria (18,25).

Vianna et al. (37), in a clinical study, evaluated the degree of microbial reduction after chemomechanical preparation of human root canals containing necrotic pulp tissue when using two endodontic irrigating reagents, 5.25% NaOCl or 2% CHX gel. Assessment of the bacterial load was accomplished by use of real-time quantitative-polymerase chain reaction (RTQ-PCR) directed against the small subunit ribosomal DNA using the SYBRGreen and TaqMan formats. The bacterial load was reduced substantially in both groups (over 96%). The bacterial reduction in the NaOCl-group (SYBRGreen 99.99%; TaqMan: 99.63%) was significantly greater (p<0.01) than in the CHX-group (SYBRGreen 96.62%; TaqMan: 96.60%), probably due to the differences between the mechanisms of action of NaOCl and CHX.

Substantivity

The effectiveness of CHX stems from its capacity to absorb to negatively charged surfaces in the mouth (e.g. tooth, mucosa, pellicle, restorative materials), being slowly released from these retention sites and therefore maintaining prolonged antimicrobial activity for several hours (11). This process is known as substantivity, and only CHX and tetracycline have this property so far (46).

Regarding its substantivity, it has been found that the use of CHX as root canal irrigating substance prevented microbial activity from 48 h (22), 7 days (in the liquid and gel formulation) (39), 21 days (47), 4 weeks (46), up to 12 weeks (48).

It seems that the antimicrobial substantivity is related to the CHX molecules available to interact with the dentin (49). Furthermore, the outstanding substantivity of CHX to dentin (evaluated at an interval from 0.5 h to 8 weeks) and its reported effect on the inhibition of dentinal proteases may explain why CHX can prolong the durability of resin-dentin bonds (50), particularly in the presence of collagen (51).

CHX and Biofilms

A biofilm can be defined as communities of microorganisms attached to a surface, embedded in an extracellular matrix of polysaccharides. Within these microcolonies, bacteria have developed into organized communities with functional heterogeneity (52). It constitutes a protected mode of growth that allows survival in a hostile environment. Bacteria in such an environment differ greatly in phenotype when compared with their planktonic counterparts, and are far less susceptible to antimicrobial killing (33,52). It has been reported that microorganisms grown in biofilms could be 2-fold to 1000-fold more resistant than the corresponding planktonic form of the same organisms (49).

Several studies using a single-species biofilm model (33,53) and apical dentin biofilm (54) have reported that higher concentration of NaOCl (varying from 2.25% to 6%) and CHX solution (2%) were effective against the tested microorganisms. The mechanical agitation improved the antimicrobial properties of the chemical substances, favoring the agents in liquid presentation, especially 5.25% NaOCl and 2% CHX (33). Although CHX is effective against bacterial biofilms, NaOCl is the only irrigation solution with the capacity of disrupting biofilms (49).

Endotoxin Reduction

Lipopolysaccharide (LPS, endotoxin), an outer membrane component of gram negative bacteria predominantly involved in root canal infection is an important mediator in the pathogenesis of apical periodontitis and enhancing the sensation of pain in endodontic infections (55).

Concerning CHX detoxifying activity, Buck et al. (56) reported little or no efficacy on inactivating the biologically active portion of the endotoxin lipid A. Furthermore, in vitro studies (57-59) inoculating Escherichia coli LPS in root canals showed the low effectiveness of CHX in reducing LPS after chemomechanical preparation. However, Signoretti et al. (60) showed that CHX improved CH properties of reducing the endotoxin content in root canals in vitro.

Vianna et al. (6) evaluated clinically the effect of root canal procedures on endotoxins and endodontic pathogens. The canals were instrumented with K-files, 1 mm from the radiographic apex, irrigated with 2% CHX and medicated with either CH, CH plus CHX or 2% CHX gel alone for 7 days. After chemomechanical preparation a mean endotoxin reduction of 44.4% was found, with a mean reduction of bacteria (CFU) of 99.96%. After 7 days of intracanal medicament, endotoxin concentration decreased by only 1.4%. No significant difference was found among different intracanal medicaments. The authors concluded that relatively high values of endotoxin were still present in the root canal after chemomechanical preparation although the majority of bacteria were eliminated. No improvement was achieved by 7 days of intracanal medicament.

Gomes et al. (61), in a clinical study in primarily infected root canals, obtained a higher percentage of endotoxin reduction in the 2.5% NaOCl–group (57.98%), when compared with the 2% CHX-gel-group (47.12%) (p<0.05), using hand K-files for apical preparation 1 mm from the radiographic apex. This result supports the fact that 2.5% NaOCl and 2% CHX have no detoxifying effect on endotoxins. Therefore, it might be argued that the removal of more than 47% of the LPS content from the infected root canals is related to the mechanical action of the instruments in dentin walls accomplished by the flow and backflow of the irrigants.

Gomes et al. (62) in a clinical study comparing the endotoxin levels found in primary and secondary endodontic infections reported that teeth with primary endodontic infections had higher contents of endotoxins and a more complex gram-negative bacterial community than teeth with secondary infections.

Endo et al. (63), in a clinical study with secondarily infected root canals with post-treatment apical periodontitis, used hand K-files for apical preparation, and 2% CHX gel for root canal irrigation. They found that higher levels of endotoxin were related to larger size of radiolucent area. Chemomechanical preparation was more effective in reducing bacteria (99.61%) than endotoxin (60.6%).

CHX and Coronal Microleakage

Canals medicated with CHX alone or in combination with CH retard the entrance of microorganisms through the coronal portion of the tooth into the root canal system, due to its wide antimicrobial activity and substantivity (64). Such a finding is interesting, especially if the coronal restoration becomes defective or if it is lost. However, even though a temporary seal delays the entrance of saliva and microorganisms into the canal system, it does not prevent microleakage, juying efforts to incorporate dentin-bonding agents and resin for coronal seal (64).

Regarding coronal microleakage during the intracoronal bleaching (65,66), it was found that CHX used as a vehicle for sodium perborate enhanced its antimicrobial activity and did not affect adversely dentin microhardness (67).

CHX and Apical Fluid Penetration

Canals irrigated or medicated with CHX do not affect negatively the ability of root fillings to prevent fluid penetration into the root canal system through the apical foramen (49,68-70).

Tissue Dissolution Capacity

As far as the use of an auxiliary chemical substance in Endodontics is concerned, several studies have been performed in the search for a substance with major desirable properties for root canal irrigation, which includes the capacity to dissolve organic tissues (49). The tissue dissolution capacity of a substance depends mainly on three factors: the frequency of shaking, the amount of organic matter in relation to the amount of irrigant in the canal system and the surface area of tissue that is available for contact with the irrigant (71).

Chlorhexidine gluconate has been recommended as a root canal irrigant (2) because of its broad spectrum antimicrobial action, substantivity and low toxicity (21). However, CHX's incapacity of tissue dissolution has been pointed out as its major disadvantage. Some attempts have been made to evaluate the activity of CHX to dissolve organic matter, demonstrating that both preparations of this substance, aqueous solution or gel, were not able to dissolve pulp tissues (68,72). Bleeding in case of vital pulp will stop only with the complete removal of the pulp tissue by a full instrumentation of the root canal within its whole extension. Therefore, when CHX is used as an irrigant, emphasis should be given to full canal instrumentation in order to remove all pulp tissue rests, as CHX does not promote a superficial necrosis.

On the other hand, Ferraz et al. (23) showed that 2% CHX gel produced the cleanest dentin wall surfaces when compared with other irrigants, including NaOCl. Due to its viscosity and rheological action, which keeps the debris in suspension, the gel seems to compensate for CHX's inability to dissolve pulp tissue, by promoting a better mechanical cleansing of the root canal and removing dentin debris and remaining tissues. The mechanical properties of the gel seem to be the main factor for this difference because the same chemical agent in the liquid form showed lower cleaning efficiency, although presenting similar antimicrobial activity (2,24).

Another important fact to be pointed out is that due to the complexity of the root canal system, even irrigation with 5.25% NaOCl does not remove all debris and organic tissues. On the other hand, dentin and organic tissues that get in contact with CHX during irrigation maintain a prolonged antimicrobial activity, as CHX is slowly released from these retention sites (11,73). Furthermore, if CHX is extruded through the apex, it does not induce pain to the patients.

Interaction with Endodontic Irrigants

Due to its wide spectrum antimicrobial activity and its inability of dissolving organic tissues, an irrigation regimen has been proposed, in which NaOCl would be used throughout instrumentation, followed by EDTA, and CHX would be used as a final irrigant (74).

The combination of NaOCl and CHX has been advocated to enhance their antimicrobial properties, and the advantage of using a final rinse with CHX would be the prolonged antimicrobial activity due to the CHX substantivity (75).

Kuruvilla and Kamath (76) reported that the antimicrobial effect of 2.5% NaOCl and 0.2% CHX used in combination was better than that of either component. However, Vianna and Gomes (75) found that the association of NaOCl and CHX did not improve the antimicrobial activity of CHX alone.

Apart from the antimicrobial aspect, the association of NaOCl with CHX leads to the formation of an orange-brown precipitate, resulting in a chemical smear layer that covers the dentinal tubules and may interfere with the seal of the root filling (31,77). In addition, this precipitate changes the color of the tooth (40,78,79) and is cytotoxic (80).

Heling and Chandler (81) investigated NaOCl and CHX, with and without EDTA, when used in combination as endodontic irrigants against Enterococcus faecalis, and verified that combining EDTA with NaOCl or CHX was more effective than using EDTA alone. However, CHX combined with EDTA also leads to the formation of precipitates, resulting in a chemical smear layer that covers the dentinal tubules.

Prado et al. (4) used electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) analyses to investigate the byproducts formed with the combinations between the most commonly used endodontic irrigants. Regarding the CHX combinations, 2% CHX gel and solution immediately produced an orange-brown precipitate when combined with 1%, 2.5% and 5.25% NaOCl solutions, and an orange-white precipitate, when combined with 0.16% NaOCl. In combination with EDTA, CHX produced a white-milky precipitate, related to the acid-base reactions. When combined with saline and ethanol, a salt precipitation was produced. No precipitate was observed when CHX was used together with distilled water, citric acid or phosphoric acid.

Regarding the orange-brown precipitate, it occurs due the presence of NaOCl, an oxidizing agent causing chlorination of the guanidino nitrogens of the CHX (4). Basrani et al. (82) detected the presence of para-chloroaniline (PCA) in this precipitate. On the other hand, Thomas and Sem (83), Nowicki and Sem (84) and Prado et al. (4) failed to detect it using different methodologies. PCA has been found to be mutagenic in microorganisms (14,85) and cytotoxic (80). Some concern over possible carcinogenicity has also been expressed (82).

Thus, after chemomechanical preparation with NaOCl, the use of CHX as a final irrigant or as an intracanal medicament would require the removal of NaOCl from the canal (77). Do Prado et al. (77) found that with regard to the use of NaOCl with CHX, 10 mL of distilled water in association or not with 17% EDTA and 10% citric acid was not enough to inhibit the formation of the chemical smear layer. In the cases where one wants to associate these substances, the protocol using phosphoric acid did not induce formation of chemical smear layer.

In summary, it is important to remove all traces of the substances used inside the root canals in order to avoid interactions between them.

CHX and Dentin Bonding

Coronal leakage has been extensively demonstrated as a negative contributor to the prognosis of endodontic treatments. Clinical trials have shown that apical periodontal health depends both on the effectiveness of coronal restorations and on the quality of the endodontic therapy (86,87).

Prevention of coronal leakage has usually been accomplished by using temporary restorative materials. However, these products are originally intended for temporary use and therefore have a finite lifetime. Thus, the immediate sealing of endodontically treated teeth using restorative materials has been considered as a powerful resource in preventing early coronal leakage (88,89). Among non-temporary restorative materials, dentin adhesives have been advocated for use in the pulp chamber in an attempt to work as a durable barrier against microleakage (89).

Bonding to pulp chamber dentin is affected differently by endodontic chemical irrigants. NaOCl have been extensively used in endodontic therapy to provide gross debridement, disinfection, lubrication and dissolution of tissues. Nevertheless, this powerful antimicrobial agent (90) has been shown to jeopardize the polymerization of bonding resins due to its oxidizing action on dentin substrate (91). It is hypothesized that NaOCl might lead to the oxidation of some component of the dentin matrix (91), perhaps demineralized collagen, forming protein-derived radicals (92). These radicals would compete with the propagating vinyl free-radicals generated by the light-activation of resin adhesives, resulting in premature chain termination and incomplete polymerization (91). For this reason, bonding to oxidized dentin has shown to be significantly weak (91,93-97). Furthermore, reductions in the mechanical properties of dentin, such as its elastic modulus, flexural strength and microhardness, have been reported after irrigation of root canals with 5% NaOCl (98-100), which can also contribute to decrease the micromechanical interaction between adhesive resins and NaOCl-treated dentin.

It has been shown that CHX application prior to acid-etching has no adverse effects on immediate composite-adhesive bonds in coronal dentin (101-103), pulp chamber dentin (104), enamel (102,105), or with resin-reinforced glass-ionomer cements (106).

Erdemir et al. (97) reported that endodontic irrigation with CHX solution significantly increased bond strength to root dentin. These authors suggested that adsorption of CHX by dentin may favor the resin infiltration into dentinal tubules, which supposedly explain the high bond strength values obtained. However, the reliability of such concept remains unclear and needs to be tested.

Santos et al. (104) considered that as a non-oxidizing agent, 2% CHX in water solution or in a gel base did not interfere with the interaction of a self-etching adhesive system to pulp chamber dentin. An exception to this tendency was observed when the CHX gel is combined with EDTA. Despite the gel base used with CHX is a water-soluble carbon polymer, which showed to be easily removed from the root canal (31), an occasional presence of residual CHX gel on dentin could react with EDTA, forming products that affect resin infiltration and/or resin polymerization, providing bond strength values slightly lower than those observed for the control group. Therefore, all efforts should be taken to remove traces of the chemical substances inside the canal through intermediate flushes with inert solutions.

De Assis et al. (107) observed that a final flush with CHX favor the wettability of AH Plus and Real Seal SE sealers on dentin surface. Additionally, Hashem et al. (108) verified that the bond strength of ActiV GP was improved by using 2% CHX in the final irrigation after 17% EDTA. Prado et al. (4) found that the irrigation protocols influenced the bond strength of the resin sealers to dentin. In the gutta-percha/AH Plus groups, the bond strength was higher when NaOCl was combined with phosphoric acid or the CHX with EDTA. In Resilon/Real Seal SE groups, the protocol combining CHX with phosphoric acid showed better results. The use of CHX as a final irrigant did not affect negatively the bond strength.

The in vitro and in vivo application of 2% CHX in cavities after acid etching and before hybridization with adhesive monomers prevents the loss of bond strength with time (109) and preserves the integrity of the hybrid layer (110). In radicular dentin, the use of CHX as an endodontic irrigant may also inhibit the bacteria-related activation of metalloproteinases (111).

CHX, Metalloproteinases and Collagen Fibrils

Matrix metalloproteinases (MMPs) are members of an enzyme family that require a zinc ion in their active site for catalytic activity. MMPs are critical for maintaining tissue allostasis. MMPs are active at neutral pH and can therefore catalyze the normal turnover of extracellular matrix (ECM) macromolecules such as the interstitial and basement membrane collagens, proteoglycans such as aggrecan, decorin, biglycan, fibromodulin and versican as well as accessory ECM proteins such as fibronectin (112).

MMPs are present in sound coronal and radicular dentin and play a role in collagen network degradation in bonded restorations. Collagen is dentin's main organic component, and it has the important function of acting as a matrix for the deposition of apatite crystals (113). It also plays an important role in the bonding between dentin and adhesive systems. During bonding procedures, resin monomers infiltrate demineralized dentin, thus forming a structure named hybrid layer (114,115). The reduction of the bond strength seen between adhesive systems and dentin walls may occur because of the removal of collagen fibrils from the dentin surface by sodium hypochlorite and may impede the formation of a consistent hybrid layer (93). The decrease in bond strength values mentioned in many studies may be caused by collagen degradation and also by structural disorganization of reminiscent fibrils (116).

In a previous study, 2% CHX gel, whether combined or not with 17% EDTA, did not promote alterations in the morphological structure of dentin organic matrix. It is an auxiliary chemical substance that does not interfere with the collagen present in the organic matrix of root dentin, and maintains the quality of the dentin substrate for posterior obturation or restoration of the tooth with resin-based materials (116).

CHX has also shown the capacity to preserve the durability of the hybrid layer and bond strength in vitro and in vivo (109,110), probably to do its effectiveness as a MMP inhibitor (117), resulting in lower degradation of hybrid layer and sub-hybrid layer collagen fibrils. It is a remarkable property because one reason for losing of resin-dentin bonds integrity with time is the degradation of denuded collagen fibrils exposed in incompletely infiltrated hybrid layers (118). This degradation is attributed to an endogenous proteolytic mechanism involving the activity of MMPs present in dentin (119).

CHX as Intracanal Medicament

CH is one of the most versatile medicaments in dentistry, especially for use as an intracanal medicament in vital and non-vital teeth (7). It is believed to have many of the properties of an ideal root canal medicament, mainly due to its alkaline pH (120). It is bactericidal and neutralizes the remaining tissue debris in the root canal system (121). CH also promotes an alkalinizing osteogenic environment on the surrounding tissues through the continuous release of OH- ions (120). Moreover, CH mediates the neutralization of lipopolysaccharides (122), helping in the cleansing the root canal (123). However, CH cannot be considered as a universal intracanal medicament, since it is not equally effective against all bacteria found in the root canal (30). Indeed, several studies (124-126) have reported difficulty in eliminating enterococci effectively, as they tolerate high pH values, varying from 9 to 11 (9).

CHX has been used in endodontics and proposed as both an irrigant and an intracanal medicament. It is active against a wide range of microorganisms, such as Gram-positive and Gram-negative bacteria (including Enterococcus faecalis), yeasts and fungi. One of the mechanisms that can explain its efficacy is based on the interaction between the positive charge of the molecule and the negatively charged phosphate groups on the bacterial cell wall, which allows the CHX molecule to penetrate into the bacteria with toxic effects (29). Therefore, its antimicrobial activity is not related to its pH (between 5.5 to 7).

The antimicrobial activity of CHX has also been tested for its use as an intracanal medicament alone (5-7,19,26-30,127-130) or in combination with other substances (5-7,30,127-129,131-133).

When used as an intracanal medicament, CHX is more effective than CH against E. faecalis infection in dentinal tubules (5,127,129,134). In fact, the antimicrobial activity of CHX is reduced when combined with other substances, including CH, CH plus zinc oxide, among others (5,7,30,129,135). However, CHX alone does not act as a physical barrier and does not present radiopacity. The use of CHX gel as intracanal medicament is recommended for a short period of time (3-5 days), particularly in those cases where the canals were fully instrumented but could not be root-filled due to the lack of time. It is also recommended in cases of exudation (unpublished data), as it retains its antimicrobial activity in the presence of blood and other organic matters (11,45). CHX gel is delivered into the canals with a syringe (e.g.: 24-gauge needle), being easily introduced and removed from the root canals.

On the other hand, the antimicrobial activity of CH increases with the combination with CHX (5,6,7,30,64,127-129,131-133). Such combination aims to increase the antimicrobial properties of CH, while maintaining its biological characteristics, mechanical properties, action as a physical barrier (30). It has been reported that the antimicrobial effect of this association is not due to the CHX molecule, but to the action of different byproducts generated by CHX fragmentation. Such byproducts exhibit both antioxidant and pro-oxidant properties, and have a high pH (136). No traces of PCA have been found in the combination of CHX with CH, due to the immediate degradation of CHX (137), even though this mixture liberated reactive oxygen species (ROS) at all time points.

Studies have also shown that CH pastes added with CHX gel, alone or with ZnO, have greater antimicrobial activity than those prepared with distilled water or saline (5,7,30,129,138).

The main advantages of this association are: a) higher antimicrobial than that of CH alone (5,30,129); b) pH around 13 (5,7,30,64), which is greater than that of CHX alone (pH 5.5-7.0) and could help in the control of the inflammatory internal- and external- root resorption (29,139); c) substantivity due to the presence of CHX (30,129); d) physical and chemical barrier better than that of 2% CHX gel alone, preventing root canal re-infection and interrupting the nutrient supply to the remaining bacteria (30); e) the contact angle of CH combined with CHX is lower than that observed when CH is combined with water, increasing the wettability of the medicament, which may explain the increase in the antimicrobial activity of its association with CHX (138); f) CHX improves CH properties of reducing the endotoxin content in root canals in vitro (60); g) diffusion through the dentinal tubules (129); h) radiopacity (129).

The paste consistency should be similar to the toothpaste and its radiopacity is similar to that of the root dentin.

To act only as a physical barrier, this medicament can be used for a short period of time. It was observed that to achieve its best antimicrobial activity, it should stay for a period of 15 to 30 days inside the root canal, without being changed. The immediate antimicrobial action of the paste in the first 7 days seems to be related to the antimicrobial effect of CHX. This effect remains stable up to 14 days. The best action is observed within 30 days, with the diffusion of hydroxyl ions through the dentinal tubules (unpublished data). The use of a temporary sealing with composite ensures an effective seal, preventing contamination of the root canal and solubilization of the medicament by oral fluids, especially in periods longer than 7 days.

Diffusion into the Dentinal Tubules

It has been shown that 2% CHX containing medicaments is able to diffuse into the dentin tubular structure and reach the outer root surface, exerting antimicrobial action. Therefore, the root canal could be considered as a reservoir for the release of intracanal medicament to the whole dentin and to the external root surface (129). The antimicrobial effects of the tested medicaments could be ranked from strongest to weakest as follows: 2% CHX, CH + 2%CHX, CH + 2%CHX +ZnO, CH + sterile saline.

Disinfection of Obturation Cones (Gutta-Percha and Resilon Cones)

The efficacy of NaOCl and CHX as auxiliary chemical substances and their action as disinfectant agents of guttapercha cones do not involve additional costs to clinicians, since these substances are commonly used in endodontic therapy. CHX has the ability to kill vegetative forms within short periods of time. However, this agent is not able to eliminate some spores, as does NaOCl (43,44).

As a strong oxidizing agent, 5.25% NaOCl is able to cause local changes in surface roughness of gutta-percha cones (140) observed by atomic force microscopy (AFM) (141,142). Moreover, formation of crystals on the surface of guttapercha cones has been ideied after a rapid sterilization with 2.5% and 5.25% NaOCl (43,143) showing that the final rinse with distilled water is essential, especially when NaOCl is used for cone disinfection (143). Other studies showed that 2% CHX did not change gutta-percha cone properties after exposure for up to 30 min, suggesting that this substance is less harmful to the structure of guttapercha (141,143). It was also found that 5.25% NaOCl and 2% CHX did not produce any changes on Resilon surface (142). Resilon cones exposed to CHX gel presented some residual antibacterial action. The clinical importance of CHX release in endodontic cones might be related to its immediate antimicrobial effect inside the root canal, during the obturation time (140).

CHX and NaOCl lead to an increase in the surface free energy (wettability) of the gutta-percha cones and Resilon surfaces, thereby interfering positively with the adhesion mechanism. This change can be due to chemical modifications on the surface of these materials caused by the action of these solutions. Comparing the two solutions, CHX was a better disinfectant compared with NaOCl, that is, presented high values of surface free energy. Cones disinfected with CHX presented smaller contact angles than NaOCl, favoring the interaction between the solid surface (cone) and the liquid, in this case, the sealer (144).

Other Uses in the Endodontic Therapy

Before 1990's, CHX gluconate was used in Endodontics as an irrigating solution, but always in a liquid form. One of the first reports of its use in Endodontics dates back to 1964 (145), demonstrating its effectiveness in enhancing radicular dentin permeability. Kennedy et al. (146), in 1967, recommended the use of 14.6% EDTA and 0.005% Hibitane, as separate solutions or combined, for irrigation of vital and non-vital teeth. They reported that these solutions not only reduce the number of microorganisms in the root canals, but also have the advantage of being well tolerated by soft tissue or wounds, provided the contact is not prolonged. However, according to them, chlorinated soda solution should never be used with Hibitane, as it forms a brown precipitate that stains the teeth. CHX in a gel presentation was evaluated by Siqueira and de Uzeda (27) as an intracanal medicament, demonstrating good performance. In 2001, Ferraz et al. (23) proposed the use of CHX gel as endodontic irrigant.

CHX can be used during all phases of the root canal treatment, including in the disinfection of the operatory field (125), due to its antimicrobial and substantivity properties. CHX has been recommended as an alternative to NaOCl, especially in cases of open apex, root resorption, foramen enlargement and root perforation, due to its biocompatibility, or in cases of allergy related to bleaching solutions (24).

Clinical investigations have been performed using 2% CHX gel for root canal preparation in the full extension of the root canal, with foraminal patency and enlargement, followed by root canal filling in the same visit. The results showed that approximately 93% of the patients did report postoperative pain (unpublished data). With foramen enlargement, the risk of irrigant extrusion through the apex increases, favoring the use of CHX, for being less irritating to the periapical tissues than NaOCl and not inducing pain. Irrigation with 17% EDTA for a better smear layer removal is recommended after instrumentation of the root canals with CHX, which should be previously removed with distilled water.

CHX gel can also be used for modeling gutta-percha cones, which improves their adaptation to the apical dentin wall (unpublished data).

The use of CHX gel during retreatment has also been investigated. In vitro, groups that used CHX gel with manual or rotary instrumentation showed smaller debris extrusion as well as the cleanest root canal walls than the ones where solvents were used (unpublished data). A clinical investigation of retreatment cases has reported that chemomechanical preparation with 2% CHX gel was more effective in reducing bacteria (99.61%) than endotoxin (60.6%) (63).

Adverse Effects

No adverse effects have been published regarding CHX use as irrigant or intracanal medicament. However, the direct effect in an in vitro test on human stem cells of apical papilla showed lack of viable cells after its use (147). The in vitro cytotoxic effect of the CHX on human osteoblastic cells seems to be dose dependent (148). There is a consensus that all irrigating substances when applied direct to cells would impact to a certain degree on cell viability (149). On the other hand, animal studies have shown that 2.0% CHX did not induce intense inflammatory response when injected into the peritoneal cavity of mice (58) or in root canals of dogs, when used as intracanal medicament (59).

CHX adverse effects are usually more related to its topical or oral application. The use of CHX dental gel derices and mouthwashes has been associated with reversible discoloration of the tongue, teeth, and silicate or composite restorations (11,42). Removal of the brownish discoloration can be done with abrasive pastes or instruments (14). However, it should not be used concomitantly with derices, as CHX interacts with detergents and fluoride in toothpaste. The CHX products should be used 30 min after brushing.

Transient taste disturbances and a burning sensation of the tongue may occur on initial use. Oral desquamation and occasional parotid glad swelling have been reported with the use of mouthwash (11). The incidence of skin irritation and hypersensitivity is low when CHX is applied at its recommended concentrations. Strong solutions may cause irritation of the conjunctive and other sensitive tissues, such as brain, meninges and middle ear (11,42). Syringes and needles that have been immersed in CHX solutions should be thoroughly rinsed with sterile water or saline before use. A recent study demonstrated that immediate hypersensitivity to CHX has increased in the United Kingdom (150), therefore it is important to investigate previous allergy to CHX during the history taking and prior its clinical application or prescription.

Cytotoxicity and Genotoxicity

Cytotoxicity is the degree to which an agent has specific destructive action on certain cells, while genotoxicity is related to the potential damage of certain substances to the DNA, which is not proof of their dangerousness to humans, but does render them potentially mutagenic or carcinogenic.

Results of a previous study have shown that bactericidal concentrations of CHX diacetate were lethal to canine embryonic fibroblasts in vitro, whereas non-lethal concentrations allowed significant bacterial survival (151). Moreover, higher concentration of CHX induces necrosis and lower concentration is associated with apoptosis (152).

CHX is cytotoxic in cell culture with different cell lines and its cytotoxicity is not cell type specific (153). CHX showed cytotoxic effects in human gingival fibroblasts (154), human periodontal ligament cells (155), human alveolar bone cells (156), human osteoblastic cells (157).

Gianelli et al. (153) investigated the in vitro cytotoxicity of CHX on osteoblastic, endothelial and fibroblastic cell lines. They reported that CHX affected cell viability in a dose and time-dependent manners, particularly in osteoblasts. Its toxic effect consisted in the induction of apoptotic and autophagic/necrotic cell deaths and involved disturbance of mitochondrial function, intracellular Ca2+ increase and oxidative stress. These findings agree with those of Li et al. (152), who studied the cytotoxicity of CHX in RAW264.7 murine macrophage cells. The genotoxicity of CHX in RAW264.7 cells had shown DNA damage in a dose-dependent manner.

However, Ribeiro et al. (158) evaluated the genotoxicity of formocresol, paramonochlorophenol, CH and CHX at final concentration ranging from 0.01% to 1% against Chinese hamster ovary cells. Results showed that none of the mentioned agents contributed to DNA damage.

The mechanisms of the cytotoxicity of CHX are still unclear and it is important to understand that the cytotoxic effects of CHX on cell culture are directly dependent on the exposure dose, frequency and duration, and also depend on the composition of the exposure medium (159).

It has been reported that PCA, an industrial chemical, is found in CHX products as a trace contaminant (11). PCA has been shown to be mutagenic in microorganisms (85). However, no evidence of carcinogenicity was found in rats after 2 years of up to 40 mg/kg/day CHX plus 0.6 mg/kg/day p-chloroaniline (11). No detrimental effects were caused by CHX application in man over a 2-year period was found (20). Therefore, the human safety experience with CHX supports its suitability for long-term oral use. However, the development of tooth staining, in a topical or oral application, imposes a practical cosmetic limitation to such use (160). Although sensitivity to CHX is rare, it should be kept in mind during CHX application (49).

In conclusion: 1) CHX is effective in the control of dental plaque and gingivitis, in the prevention and treatment of caries, and in the maintenance of implants; after dental manipulation, in the treatment of recurrent aphthous and denture-related stomatitis. It is particularly effective in individuals with orthodontic appliances, disabled people, and immunologically compromised patients. In Endodontics, it is used as an irrigating substance, intracanal medicament, among others. 2) Its structural formula consists of two symmetric 4-chlorophenyl rings and two biguanide groups connected by a central hexamethylene chain. 3) For endodontic purposes, CHX can be used in a liquid or gel presentation. The concentration most frequently used is 2%. 4) A shelf-life of at least 1 year can be expected, provided that the packaging is adequate and in the dark bottle. 5) The bactericidal effect of the drug is due to the cationic molecule binding to extra-microbial complexes and negatively charged microbial cell walls, thereby altering the cell's osmotic equilibrium. 6) CHX is bactericidal and effective against Gram-positive, Gram-negative, facultative and strict anaerobes, yeasts and fungi, particularly Candida albicans. It is active against some viruses (respiratory viruses, herpes, cytomegalovirus, HIV) and inactive against bacterial spores at room temperature. 7) CHX shows substantivity up to 12 weeks. 8) Although CHX is effective against bacterial biofilms, NaOCl is the only irrigation solution with the capacity of disrupting biofilms. 9) 2% CHX have no detoxifying effect on endotoxins, but it improves CH properties of reducing the endotoxin content in root canals in vitro. 10) Canals medicated with CHX alone or in combination with CH delay the entrance of microorganisms through the coronal portion of the tooth into the root canal system. Coronal microleakage is also delayed when CHX is used as a vehicle for sodium perborate during the intracoronal bleaching. 11) Canals irrigated or medicated with CHX do not affect adversely the ability of root fillings to prevent fluid penetration into the root canal system through the apical foramen. 12) CHX does not dissolve organic tissues. 13) CHX in contact with NaOCl, EDTA, saline and ethanol forms precipitate. However, no precipitate was observed when CHX was combined with citric acid, phosphoric acid or distilled water. It is important to remove all traces of the substances used inside the root canals through intermediate flushes with distilled water in order to avoid interaction between them. 14) The in vitro and in vivo application of 2% CHX in cavities after acid etching and before hybridization with adhesive monomers prevents the loss of bond strength with time and preserves the integrity of the hybrid layer. Irrigation with CHX increases the bond strength to root dentin. 15) CHX does not interfere with the collagen present in the organic matrix of root dentin and inhibits MMPs. 16) CHX increases the antimicrobial activity of CH. 17) 2% CHX containing medicaments is able to diffuse into the dentin and reach the outer surface, exerting antimicrobial action. 18) CHX is effective in disinfecting gutta-percha and Resilon cones, although it does not eliminate bacterial spores. 2% CHX does not change the properties of gutta-percha and Resilon cones. CHX and NaOCl lead to an increase in the surface free energy (wettability) of the gutta-percha cones and Resilon surfaces. 19) CHX can be used during all phases of the root canal preparation, including the disinfection of the operative field, during the enlargement of the canals orifices and removal of necrotic tissues before root canal length determination; in the chemomechanical preparation: alternating its use with an irrigation with an inert solution (i.e. distilled water, sterile saline); prior to the foraminal patency and enlargement; as intracanal medicament alone or combined with other substances (i.e. CH); in the disinfection of gutta-percha cones; for modeling the main gutta-percha cone; in the removal of gutta-percha cones during retreatment; in the disinfection of prosthetic space; among others. If it extrudes through the apex, during instrumentation and foramen enlargement, it does not induce pain, for being less irritating to the periapical tissues than NaOCl. CHX has been recommended as an alternative to NaOCl, especially in cases of open apex, root resorption, foramen enlargement and root perforation or in cases of allergy related to bleaching solutions. 20) No adverse effects have been published regarding CHX use as an irrigant or intracanal medicaments. CHX adverse effects are usually related to its topical or oral application, including reversible discoloration of the tongue, teeth, and silicate or composite restorations, transient taste disturbances and a burning sensation of the tongue. The incidence of skin irritation and hypersensitivity is low and the biocompatibility is acceptable.

We would like to thank Ariane C. S. Martinho, Giselle P. C. Abi-Rached and Thais M. Duque for their assistance. We would like also to thank the Brazilian agencies FAPESP, CNPq and CAPES.

References

  • 1
    Abou-Rass M, Piccinino MV. The effectiveness of 4 clinical irrigation methods on the removal of root-canal debris. Oral Surg Oral Med Oral Pathol 1982;54:323-328.
  • 2
    Gomes BP, Ferraz CC, Vianna ME, Berber VB, Teixeira FB, Souza FJ. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and chlorhexidine gluconate in the elimination of Enterococcus faecalis. Int Endod J 2001;34:424-428.
  • 3
    Rocha AW, de Andrade CD, Leitune VC, Collares FM, Samuel SM, Grecca FS, et al. Influence of endodontic irrigants on resin sealer bond strength to radicular dentin. Bull Tokyo Dent Coll 2012;53:1-7.
  • 4
    Prado M, Simão RA, Gomes BP. Impact of different irrigation protocols on resin sealer bond strength to dentin. J Endod 2013 - in press.
  • 5
    Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267-275.
  • 6
    Vianna ME, Horz HP, Conrads G, Zaia AA, Souza FJ, Gomes BP. Effect of root canal procedures on endotoxins and endodontic pathogens. Oral Microbiol Immun 2007;22:411-418.
  • 7
    de Souza-Filho FJ, Soares A de J, Vianna ME, Zaia AA, Ferraz CC, Gomes BP. Antimicrobial effect and pH of chlorhexidine gel and calcium hydroxide alone and associated with other materials. Braz Dent J 2008;19:28-33.
  • 8
    Tomás I, Rubido S, Donos N. In situ antimicrobial activity of chlorhexidine in the oral cavity. Formatex 2011;530-541.
  • 9
    Lang NP, Brecx MC. Chlorhexidine digluconate: an agent for chemical plaque control and prevention of gingival inflammation. J Periodontal Res 1986;21:74-89.
  • 10
    Davies GE, Francis J, Martin AR, Rose FL, Swain G. 1:6-di-4′-chlorophenyldiguanidohexane (“Hibitane”). Laboratory investigation of a new antibacterial agent of high potency. Br J Pharmacol Chemother 1954;9:192–196.
  • 11
    Denton G.W. 1991. Chlorhexidine. In: Disinfection, Sterilization and preservation. Block SS (Editor). 4th ed. Philadelphia: Lea & Febiger, 1991; p.274-289.
  • 12
    Loe H, Schiott CR, Karring G, Karring T. Two years oral use of chlorhexidine in man. I. General design and clinical effects. J Periodontal Res 1976;11:135-144.
  • 13
    Loe H, Schiott CR. The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Periodontal Res 1970;5:79-83.
  • 14
    Fardal O, Turnbull RS. A review of the literature on use of chlorhexidine in dentistry. J Am Dent Assoc 1986;112:863-869.
  • 15
    Epstein JB, McBride BC, Stevenson-Moore P, Merilees H, Spinelli J. The efficacy of chlorhexidine gel in reduction of Streptococcus mutans and Lactobacillus species in patients treated with radiation therapy. Oral Surg Oral Med Oral Pathol 1991;71:172-178.
  • 16
    Clark DC, Morgan J, MacEntee MI. Effects of a 1% chlorhexidine gel on the cariogenic bacteria in high-risk elders: a pilot study. Spec Care Dentist 1991;11:101-103.
  • 17
    Enrile de Rojas FJ, Alemany AS, Burguera AC, Dios PD. Aplicaciones clínicas adicionales de colutorios antisépticos. Periodoncia 2006;16:95-104.
  • 18
    Siqueira JF Jr, Rocas IN, Paiva SS, Guimaraes-Pinto T, Magalhaes KM, Lima KC. Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:122-130.
  • 19
    Delany GM, Patterson SS, Miller CH, Newton CW. The effect of chlorhexidine gluconate irrigation on the root canal flora of freshly extracted necrotic teeth. Oral Surg Oral Med Oral Pathol 1982;53:518-523.
  • 20
    Greenstein G, Berman C, Jaffin R. Chlorhexidine. An adjunct to periodontal therapy. J Periodontol 1986;57:370-377.
  • 21
    Jeansonne MJ, White RR. A comparison of 2.0% chlorhexidine gluconate and 5.25% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20:276-278.
  • 22
    Leonardo MR, Tanomaru Filho M, Silva LA, Nelson Filho P, Bonifacio KC, Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. J Endod 1999;25:167-171.
  • 23
    Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. In vitro assessment of the antimicrobial action and the mechanical ability of chlorhexidine gel as an endodontic irrigant. J Endod 2001;27:452-455.
  • 24
    Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza Filho FJ. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79-84.
  • 25
    Ercan E, Ozekinci T, Atakul F, Gül K. Antibacterial activity of 2% chlorhexidine gluconate and 5.25% sodium hypochlorite in infected root canal: in vivo study. J Endod 2004;30:84-87.
  • 26
    Heling I, Steinberg D, Kenig S, Gavrilovich I, Sela MN, Friedman M. Efficacy of a sustained-release device containing chlorhexidine and Ca(OH)2 in preventing secondary infection of dentinal tubules. Int Endod J 1992;25:20-24.
  • 27
    Siqueira JF Jr, de Uzeda M. Intracanal medicaments: Evaluation of the antibacterial effects of chlorhexidine metronidazole, and calcium hydroxide associated with three vehicles. J Endod 1997;23:167-169.
  • 28
    Barbosa CA, Goncalves RB, Siqueira JF Jr, de Uzeda M. Evaluation of the antibacterial activities of calcium hydroxide, chlorhexidine, and camphorated paramonochlorophenol as intracanal medicament. A clinical and laboratory study. J Endod 1997;23:297-300.
  • 29
    Lindskog S, Pierce AM, Blomlöf L. Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space. Endod Dent Traumatol 1998;14:186-190.
  • 30
    Gomes BP, Vianna ME, Sena NT, Zaia AA, Ferraz CC, de Souza Filho FJ. In vitro evaluation of the antimicrobial activity of calcium hydroxide combined with chlorhexidine gel used as intracanal medicament. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:544-550.
  • 31
    Vivacqua-Gomes N, Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. Influence of irrigants on the coronal microleakage of laterally condensed gutta-percha root fillings. Int Endod J 2002;35:791-795.
  • 32
    Miyamoto T, Takahashi S, Ito H, Inagaki H, Noishiki Y. Tissue biocompatibility of cellulose and its derivatives. J Biomed Mater Res 1989;23:125-133.
  • 33
    Sena NT, Gomes BP, Vianna ME, Berber VB, Zaia AA, Ferraz CC, et al. In vitro antimicrobial activity of sodium hypochlorite and chlorhexidine against selected single-species biofilms. Int Endod J 2006;39:878-885.
  • 34
    Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, Souza-Filho FJ. Comparative study of the antimicrobial efficacy of chlorhexidine gel, chlorhexidine solution and sodium hypochlorite as endodontic irrigants. Braz Dent J 2007;18:294-298.
  • 35
    Foulkes DM. Some toxicological observations on chlorhexidine. J Periodontal Res Suppl 1973;12:55-60.
  • 36
    Greenstein G, Jaffin RA, Hilsen KL, Berman CL. Repair of anterior gingival deformity with durapatite. A case report. J Periodontol 1985;56:200-203.
  • 37
    Vianna ME, Horz HP, Gomes BP, Conrads G. In vivo evaluation of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue. Int Endod J 2006;39:484-492.
  • 38
    Oncag O, Hosgor M, Hilmioglu S, Zekioglu O, Eronat C, Burhanoglu D. Comparison of antibacterial and toxic effects of various root canal irrigants. Int Endod J 2003;36:423-432.
  • 39
    Dametto FR, Ferraz CC, Gomes BP, Zaia AA, Teixeira FB, de Souza-Filho FJ. In vitro assessment of the immediate and prolonged antimicrobial action of chlorhexidine gel as an endodontic irrigant against Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:768-772.
  • 40
    Basrani B, Lemonie C. Chlorhexidine gluconate. Aust Endod J 2005;31:48-52.
  • 41
    Ferguson JW, Hatton JF, Gillespie MJ. Effectiveness of intracanal irrigants and medications against the yeast Candida albicans. J Endod 2002;28:68-71.
  • 42
    Westcott WW, Martindale W. Martindale, The Extra Pharmacopoeia. Reynolds JEF (Editor). 29th ed London. The Pharmaceutical Press, 1989.
  • 43
    Siqueira JF Jr, da Silva CH, Cerqueira M das D, Lopes HP, de Uzeda M. Effectiveness of four chemical solutions in eliminating Bacillus subtilis spores on gutta-percha cones. Endod Dent Traumatol 1998;14:124-126.
  • 44
    Gomes BP, Vianna ME, Matsumoto CU, Rossi V de P, Zaia AA, Ferraz CC, et al. Disinfection of gutta-percha cones with chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:512-517.
  • 45
    Gelinas P, Goulet J. Neutralization of the activity of eight disinfectants by organic matter. J Appl Bacteriol 1983;54:243-247.
  • 46
    Khademi AA, Mohammadi Z, Havaee A. Evaluation of the antibacterial substantivity of several intra-canal agents. Aust Endod J 2006;32:112-115.
  • 47
    Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315-317.
  • 48
    Rosenthal S, Spangberg L, Safavi K. Chlorhexidine substantivity in root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488-492.
  • 49
    Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J 2009;42:288-302.
  • 50
    Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A, et al. Substantivity of chlorhexidine to human dentin. Dent Mater 2010;26:779-785.
  • 51
    Baca P, Junco P, Arias-Moliz MT, Castillo F, Rodriguez-Archilla A, Ferrer-Luque CM. Antimicrobial substantivity over time of chlorhexidine and cetrimide. J Endod 2012;38:927-930.
  • 52
    Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;21:1318-1322.
  • 53
    Spratt DA, Pratten J, Wilson M, Gulabivala K. An in vitro evaluation of the antimicrobial efficacy of irrigants on biofilms of root canal isolates. Int Endod J 2001;34:300-307.
  • 54
    Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to irrigant solutions on apical dentin biofilms in vitro. J Endod.2006;32:434-437.
  • 55
    Martinho FC, Chiesa WM, Zaia AA, Ferraz CC, Almeida JF, Souza-Filho FJ, et al. Comparison of endotoxin levels in previous studies on primary endodontic infections. J Endod 2011;37:163-167.
  • 56
    Buck RA, Cai J, Eleazer PD, Staat RH, Hurst HE. Detoxification of endotoxin by endodontic irrigants and calcium hydroxide. J Endod 2001;27:325-327.
  • 57
    de Oliveira LD, Jorge AO, Carvalho CA, Koga-Ito CY, Valera MC. In vitro effects of endodontic irrigants on endotoxins in root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:135-142.
  • 58
    Tanomaru JM, Leonardo MR, Tanomaru Filho M, Bonetti Filho I, Silva LA. Effect of different irrigation solutions and calcium hydroxide on bacterial LPS. Int Endod J 2003;36:733-739.
  • 59
    Silva LA, Leonardo MR, Assed S, Tanomaru Filho M. Histological study of the effect of some irrigating solutions on bacterial endotoxin in dogs. Braz Dent J 2004;15:109-114.
  • 60
    Signoretti FGC, Gomes BP, Montagner F, Tosello FB, Jacinto RC. Influence of 2% chlorhexidine gel on calcium hydroxide ionic dissociation and its ability of reducing endotoxin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:653-658.
  • 61
    Gomes BP, Martinho FC, Vianna ME. Comparison of 2.5% sodium hypochlorite and 2% chlorhexidine gel on oral bacterial lipopolysaccharide reduction from primarily infected root canals. J Endod 2009;35:1350-1353.
  • 62
    Gomes BP, Endo MS, Martinho FC. Comparison of endotoxin levels found in primary and secondary endodontic infections. J Endod 2012;38:1082-1086.
  • 63
    Endo MS, Martinho FC, Zaia AA, Ferraz CC, Almeida JF, Gomes BP. Quaication of cultivable bacteria and endotoxin in post-treatment apical periodontitis before and after chemo-mechanical preparation. Eur J Clin Microbiol Infect Dis 2012;31:2575-2583.
  • 64
    Gomes BP, Sato E, Ferraz CC, Teixeira FB, Zaia AA, Souza-Filho FJ. Evaluation of time required for recontamination of coronally sealed canals medicated with calcium hydroxide and chlorhexidine. Int Endod J 2003;36:604-609.
  • 65
    Oliveira DP, Gomes BP, Zaia AA, Souza-Filho FJ, Ferraz CC. In vitro assessment of a gel base containing 2% chlorhexidine as a sodium perborate's vehicle for intracoronal bleaching of discolored teeth. J Endod 2006;32:672-674.
  • 66
    Oliveira DP, Gomes BP, Zaia AA, Souza-Filho FJ, Ferraz CC. Ex vivo antimicrobial activity of several bleaching agents used during the walking bleach technique. Int Endod J 2008;41:1054-1058.
  • 67
    de Oliveira DP, Teixeira EC, Ferraz CC, Teixeira FB. Effect of intracoronal bleaching agents on dentin microhardness. J Endod 2007;33:460-462.
  • 68
    Marley JT, Ferguson DB, Hartwell GR. Effects of chlorhexidine gluconate as an endodontic irrigant on the apical seal: short-term results. J Endod 2001;27:775-778.
  • 69
    Ferguson DB, Marley JT, Hartwell GR. The effect of chlorhexidine gluconate as an endodontic irrigant on the apical seal: long-term results. J Endod 2003;29:91-94.
  • 70
    Wuerch RM, Apicella MJ, Mines P, Yancich PJ, Pashley DH. Effect of 2% chlorhexidine gel as an intracanal medication on the apical seal of the root-canal system. J Endod 2004;30:788-791.
  • 71
    Mohammadi Z. Sodium hypochlorite in endodontics: an update review. Int Dent J 2008;58: 329-341.
  • 72
    Okino LA, Siqueira EL, Santos M, Bombana AC, Figueiredo JA. Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. Int Endod J 2004;37:38-41.
  • 73
    Parsons GJ, Patterson SS, Miller CH, Katz S, Kafrawy AH, Newton CW. Uptake and release of chlorhexidine by bovine pulp and dentin specimens and their subsequent acquisition of antibacterial properties. Oral Surg Oral Med Oral Pathol 1980;49:455-459.
  • 74
    Zehnder M. Root canal irrigants. J Endod 2006;32:389-398.
  • 75
    Vianna ME, Gomes BPFA. Efficacy of sodium hypochlorite combined with chlorhexidine against Enterococcus faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:585-589.
  • 76
    Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24:472-476.
  • 77
    Do Prado M, Simao RA, Gomes BP. Evaluation of different irrigation protocols concerning the formation of chemical smear layer. Microsc Res Tech 2013;76:196-200.
  • 78
    Akisue E, Tomita VS, Gavini G, de Figueiredo JAP. Effect of the combination of sodium hypochlorite and chlorhexidine on dentinal permeability and scanning electron microscopy precipitate observation. J Endod 2010;36:847-850.
  • 79
    Bui TB, Baumgartner JC, Mitchell JC. Evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin. J Endod 2008;34:181-185.
  • 80
    Burkhardt-Holm P, Oulmi Y, Schroeder A, Storch V, Braunbeck T. Toxicity of 4-chloroaniline in early life stages of zebrafish (Danio rerio): II. Cytopathology and regeneration of liver and gills after prolonged exposure to waterborne 4-chloroaniline. Arch Environ Contam Toxicol 1999;37:85-102.
  • 81
    Heling I, Chandler NP. Antimicrobial effect of irrigant combinations within dentinal tubules. Int Endod J 1998;31:8-14.
  • 82
    Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966-969.
  • 83
    Thomas JE, Sem DS. An in vitro spectroscopic analysis to determine whether para-chloroaniline is produced from mixing sodium hypochlorite and chlorhexidine. J Endod 2010;36:315-317.
  • 84
    Nowicki JB, Sem DS. An in vitro spectroscopic analysis to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite and chlorhexidine. J Endod 2011;37:983-988.
  • 85
    Prasad I. Mutagenic effects of the herbicide 3′,4′-dichloropropionanilide and its degradation products. Can J Microbiol 1970;16:369-372.
  • 86
    Ray HA, Trope M. Periapical status of endodontically treated teeth in relation to the technical quality of the root filling and the coronal restoration. Int Endod J 1995;28:12-18.
  • 87
    Hommez GM, Coppens CR, De Moor RJ. Periapical health related to the quality of coronal restorations and root fillings. Int Endod J 2002;35:680-689.
  • 88
    Uranga A, Blum JY, Esber S, Parahy E, Prado C. A comparative study of four coronal obturation materials in endodontic treatment. J Endod 1999;25:178-180.
  • 89
    Belli S, Orucoglu H, Yildirim C, Eskitascioglu G. The effect of fiber placement or flowable resin lining on microleakage in Class II adhesive restorations. J Adhes Dent 2007;9:175-181.
  • 90
    Spangberg L, Engstrom B, Langeland K. Biologic effects of dental materials. 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg Oral Med Oral Pathol 1973;36:856-871.
  • 91
    Morris MD, Lee KW, Agee KA, Bouillaguet S, Pashley DH. Effects of sodium hypochlorite and RC-prep on bond strengths of resin cement to endodontic surfaces. J Endod 2001;27:753-757.
  • 92
    Hawkins CL, Davies MJ. Hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation. Biochem J 1999;1;340:539-548.
  • 93
    Nikaido T, Takano Y, Sasafuchi Y, Burrow MF, Tagami J. Bond strengths to endodontically-treated teeth. Am J Dent 1999;12:177-180.
  • 94
    Ishizuka T, Kataoka H, Yoshioka T, Suda H, Iwasaki N, Takahashi H, et al. Effect of NaClO treatment on bonding to root canal dentin using a new evaluation method. Dent Mater J 2001;20:24-33.
  • 95
    Ari H, Yasar E, Belli S. Effects of NaOCl on bond strengths of resin cements to root canal dentin. J Endod 2003;29:248-251.
  • 96
    Ozturk B, Ozer F. Effect of NaOCl on bond strengths of bonding agents to pulp chamber lateral walls. J Endod 2004;30:362-365.
  • 97
    Erdemir A, Ari H, Gungunes H, Belli S. Effect of medications for root canal treatment on bonding to root canal dentin. J Endod 2004;30:113-116.
  • 98
    Grigoratos D, Knowles J, Ng YL, Gulabivala K. Effect of exposing dentine to sodium hypochlorite and calcium hydroxide on its flexural strength and elastic modulus. Int Endod J 2001;34:113-119.
  • 99
    Sim TP, Knowles JC, Ng YL, Shelton J, Gulabivala K. Effect of sodium hypochlorite on mechanical properties of dentine and tooth surface strain. Int Endod J 200;34:120-132.
  • 100
    Saleh AA, Ettman WM. Effect of endodontic irrigation solutions on microhardness of root canal dentine. J Dent 1999;27:43-46.
  • 101
    Perdigão J, Denehy GE, Swift EJ, Jr. Effects of chlorhexidine on dentin surfaces and shear bond strengths. Am J Dent 1994;7:81-84.
  • 102
    el-Housseiny AA, Jamjoum H. The effect of caries detector dyes and a cavity cleansing agent on composite resin bonding to enamel and dentin. J Clin Pediatr Dent 2000;25:57-63.
  • 103
    de Castro FL, de Andrade MF, Duarte Junior SL, Vaz LG, Ahid FJ. Effect of 2% chlorhexidine on microtensile bond strength of composite to dentin. J Adhes Dent 2003;5:129-138.
  • 104
    Santos JN, Carrilho MR, De Goes MF, Zaia AA, Gomes BP, Souza-Filho FJ, et al. Effect of chemical irrigants on the bond strength of a self-etching adhesive to pulp chamber dentin. J Endod 2006;32:1088-1090.
  • 105
    Filler SJ, Lazarchik DA, Givan DA, Retief DH, Heaven TJ. Shear bond strengths of composite to chlorhexidine-treated enamel. Am J Dent 1994;7:85-88.
  • 106
    Cunningham MP, Meiers JC. The effect of dentin disinfectants on shear bond strength of resin-modified glass-ionomer materials. Quintessence Int 1997;28:545-551.
  • 107
    de Assis DF, Prado M, Simao RA. Evaluation of the interaction between endodontic sealers and dentin treated with different irrigant solutions. J Endod 2011;37:1550-1552.
  • 108
    Hashem AA, Ghoneim AG, Lutfy RA, Fouda MY. The effect of different irrigating solutions on bond strength of two root canal-filling systems. J Endod 2009;35:537-540.
  • 109
    Carrilho MR, Carvalho RM, de Goes MF, di Hipolito V, Geraldeli S, Tay FR, et al. Chlorhexidine preserves dentin bond in vitro. J Dent Res 2007;86:90-94.
  • 110
    Hebling J, Pashley DH, Tjaderhane L, Tay FR. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. J Dent Res 2005;84:741-746.
  • 111
    Itoh T, Nakamura H, Kishi J, Hayakawa T. The activation of matrix metalloproteinases by a whole-cell extract from Prevotella nigrescens. J Endod 2009;35:55-59.
  • 112
    Malemud CJ. Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci 2006;11:1696-1701.
  • 113
    Butler WT. Dentin extracellular matrix and dentinogenesis. Oper Dent 1992;5:18-23.
  • 114
    Nakabayashi N, Kojima K, Masuhara E. The promotion of adhesion by the infiltration of monomers into tooth substrates. J Biomed Mater Res 1982;16:265-273.
  • 115
    Nakabayashi N. The hybrid layer: a resin-dentin composite. Proc Finn Dent Soc 1992;88 Suppl1:321-329.
  • 116
    Moreira DM, Almeida JF, Ferraz CC, Gomes BP, Line SR, Zaia AA. Structural analysis of bovine root dentin after use of different endodontics auxiliary chemical substances. J Endod 2009;35:1023-1027.
  • 117
    Gendron R, Grenier D, Sorsa T, Mayrand D. Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clin Diagn Lab Immunol 1999;6:437-439.
  • 118
    Wang Y, Spencer P. Hybridization efficiency of the adhesive/dentin interface in wet bonding. J Dent Res 2003;82:141–145
  • 119
    Santos J, Carrilho M, Tervahartiala T, Sorsa T, Breschi L, Mazzoni A, et al. Determination of matrix metalloproteinases in human radicular dentin. J Endod 2009;35:686-689.
  • 120
    Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1981;7:17-21.
  • 121
    Torneck CD, Moe H, Howley TP. The effect of calcium hydroxide on porcine pulp fibroblasts in vitro. J Endod 1983;9:131-136.
  • 122
    Safavi KE, Nichols FC. Alteration of biological properties of bacterial lipopolysaccharide by calcium hydroxide treatment. J Endod 1994;20:127-129.
  • 123
    Hasselgren G, Olsson B, Cvek M. Effects of calcium hydroxide and sodium hypochlorite on the dissolution of necrotic porcine muscle tissue. J Endod 1988;14:125-127.
  • 124
    Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1-7.
  • 125
    Gomes BP, Lilley JD, Drucker DB. Variations in the susceptibility of components of the endodontic microflora to biomechanical procedures. Int Endod J 1996;29:235-241.
  • 126
    Pinheiro ET, Gomes BP, Ferraz CC, Teixeira FB, Zaia AA, Souza Filho FJ. Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. Oral Microbiol Immunol 2003;18:100-103.
  • 127
    Basrani B, Tjaderhane L, Santos JM, Pascon E, Grad H, Lawrence HP, et al. Efficacy of chlorhexidine- and calcium hydroxide-containing medicaments against Enterococcus faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:618-624.
  • 128
    Schäfer E, Bössmann K. Antimicrobial efficacy of chlorhexidine and two calcium hydroxide formulations against Enterococcus faecalis. J Endod 2005;31:53-56.
  • 129
    Gomes BP, Montagner F, Berber VB, Zaia AA, Ferraz CC, de Almeida JF, et al. Antimicrobial action of intracanal medicaments on the external root surface. J Dent 2009;37:76-81.
  • 130
    Lenet BJ, Komorowski R, Wu XY, Huang J, Grad H, Lawrence HP, et al. Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles. J Endod 2000;26:652-655.
  • 131
    Siren EK, Haapasalo MPP, Waltimo TMT, Orstavik D. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodide on Enterococcus faecalis. Eur J Oral Sci 2004;112:326-231.
  • 132
    Zerella JA, Fouad AF, Spangberg LS. Effectiveness of a calcium hydroxide and chlorhexidine digluconate mixture as disinfectant during retreatment of failed endodontic cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:756-761.
  • 133
    Podbielski A, Spahr A, Haller B. Additive antimicrobial activity of calcium hydroxide and chlorhexidine on common endodontic bacterial pathogens. J Endod 2003;29:340-345.
  • 134
    Almyroudi A, Mackenzie D, McHugh S, Saunders WP. The effectiveness of various disinfectants used as endodontic intracanal medications: An in vitro study. J Endod 2002;28:163-167.
  • 135
    Cury JA, Rocha EP, Koo H, Francisco SB, Del Bel Cury AA. Effect of saccharin on antibacterial activity of chlorhexidine gel. Braz Dent J 2000;11:29-34.
  • 136
    Yeung SY, Huang CS, Chan CP, Lin CP, Lin HN, Lee PH, et al. Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. Int Endod J 2007;40:837-844.
  • 137
    Barbin LE, Saquy PC, Guedes DF, Sousa-Neto MD, Estrela C, Pecora JD. Determination of para-chloroaniline and reactive oxygen species in chlorhexidine and chlorhexidine associated with calcium hydroxide. J Endod 2008;34:1508-1514.
  • 138
    Basrani B, Ghanem A, Tjaderhane L. Physical and chemical properties of chlorhexidine and calcium hydroxide-containing medications. J Endod 2004;30:413-417.
  • 139
    Beltes PG, Pissiotis E, Koulaouzidou E, Kortsaris AH. In vitro release of hydroxyl ions from six types of calcium hydroxide nonsetting pastes. J Endod 1997;23:413-415.
  • 140
    Gomes BP, Berber VB, Montagner F, Sena NT, Zaia AA, Ferraz CC, et al. Residual effects and surface alterations in disinfected gutta-percha and Resilon cones. J Endod 2007;33:948-951.
  • 141
    Valois CRA, Silva LP, Azevedo RB. Effects of 2% chlorhexidine and 5.25% sodium hypochlorite on gutta-percha cones studied by atomic force microscopy. Int Endod J 2005;38:425–429.
  • 142
    Prado M, Gusman H, Gomes BP, Simao RA. Effect of disinfectant solutions on gutta-percha and resilon cones. Microsc Res Tech 2012;75:791-795.
  • 143
    Prado M, Gusman H, Gomes BPFA, Simao RA. The importance of final rinse after disinfection of gutta-percha and Resilon cones. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:e21-e24.
  • 144
    Prado M, de Assis DF, Gomes BP, Simão RA. Effect of disinfectant solutions on the surface free energy and wettability of filling material. J Endod 2011;37:980-982.
  • 145
    - Kennedy GDC, McLundie AC, Day RM. Calcium hydroxide, its role in a simplified endodontic technique. Dent Mag Oral Top 1967;84:51-57.
  • 146
    Atkinson AM, Hampson EL. Sterilization of root canals. Br Dent J 1964;116:526–532.
  • 147
    Trevino EG, Patwardhan AN, Henry MA, Perry G, Dybdal-Hargreaves N, Hargreaves KM, et al. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod 2011;37:1109-1115.
  • 148
    Lee TH, Hu CC, Lee SS, Chou MY, Chang YC. Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels. Int Endod J 2010;43:430-435.
  • 149
    Navarro-Escobar E, González-Rodríguez MP, Ferrer-Luque CM. Cytotoxic effects of two acid solutions and 2.5% sodium hypochlorite used in endodontic therapy. Med Oral Patol Oral Cir Bucal 2010;15:e90-e94.
  • 150
    Nakonechna A, Dore P, Dixon T, Khan S, Deacock S, Holding S, et al. Immediate hypersensitivity to chlorhexidine is increasingly recognised in the United Kingdom. Allergol Immunopathol (Epub ahead of print. DOI: 10.1016/j.aller.2012.08.001).
  • 151
    Sanchez IR, Nusbaum KE, Swaim SF, Hale AS, Henderson RA, McGuire JA. Chlorhexidine diacetate and povidone-iodine cytotoxicity to canine embryonic fibroblasts and Staphylococcus aureus. Vet Surg 1988;17:182-185.
  • 152
    Li YC, Kuan YH, Lee SS, Huang FM, Chang YC. Cytotoxicity and genotoxicity of chlorhexidine on macrophages in vitro. Environ Toxicol 2012 Apr 4. doi: 10.1002/tox.21771. [Epub ahead of print. DOI: 10.1002/tox.21771].
  • 153
    Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A. Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. Toxicol In vitro 2008;22:308-317.
  • 154
    Pucher JJ, Daniel JC. The effects of chlorhexidine digluconate on human fibroblasts in vitro. J Periodontol 1992;63:526-532.
  • 155
    Chang YC, Huang FM, Tai KW, Chou MY. The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:446-450.
  • 156
    Cabral CT, Fernandes MH. In vitro comparison of chlorhexidine and povidone-iodine on the long-term proliferation and functional activity of human alveolar bone cells. Clin Oral Investig 2007;11:155-164.
  • 157
    Lee TH, Hu CC, Lee SS, Chou MY, Chang YC. Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels. Int Endod J 2010;43:430-435.
  • 158
    Ribeiro DA, Scolastici C, de Almeida PLA, Marques PLA, Marques MEA, Salvadori MF. Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:637-640.
  • 159
    Babich H, Wurzburger BJ, Rubin YL, Sinensky MC, Blau L. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995;11:79-88.
  • 160
    Rushton A. Safety of Hibitane. II. Human experience. J Clin Periodontol 1977;4:73-79.

Publication Dates

  • Publication in this collection
    Mar-Apr 2013

History

  • Received
    15 Feb 2013
  • Accepted
    2 Apr 2013
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