chx 2009

REVIEW ARTICLE

Antimicrobial substantivity of root canal irrigants andmedicaments: A reviewaej_164 131..139Zahed Mohammadi, DMD, MSD1 and Paul V. Abbott, BDSc, MDS, FRACDS(Endo)2

1 Shahid Sadoughi Dental School, Department of Endodontics, Yazd, Iran

2 School of Dentistry, The University of Western Australia, Perth, Australia

Keywordsendodontics, irrigants, medicaments,

substantivity.

CorrespondenceDr Zahed Mohammadi, Department of

Endodontics, Shahid Sadoughi Dental School,

Imam Avenue, Fazaye Sabz Cross Road, Yazd,

Iran. Email: [email protected]

doi:10.1111/j.1747-4477.2009.00164.x

Abstract

Disinfection of the root canal system is one of the primary aims of root canaltreatment. This can be achieved through the use of various antimicrobialagents in the form of irrigants and medicaments. These agents are only used forrelatively short periods of time ranging from minutes (for irrigants) up to daysor several weeks (for medicaments) and therefore their long-term antimicro-bial effects rely on whether or not the particular agent has any properties ofsubstantivity. If irrigants and medicaments had effective long-term residualantimicrobial activity that could prevent re-infection of the root canal system,then the long-term outcomes of endodontic treatment might be improved.Only a small number of studies have investigated the short-term substantivityof commonly used antimicrobial agents and the results show substantivity ofchlorhexidine lasts for up to 12 weeks and tetracycline for up to 4 weeks.However, it is not known whether the substantivity of these agents will last forlonger periods of time as this has not been investigated.

Introduction

The essential role of micro-organisms in the developmentand perpetuation of pulp and periapical diseases has beenclearly demonstrated in many animal model and humanstudies (13). Elimination of micro-organisms frominfected root canal systems (RCSs) is a complicated task.Numerous measures have been described to reduce thenumber of micro-organisms from the RCS, including theuse of various mechanical instrumentation techniques,irrigation regimes and intracanal medicaments. There isno definitive evidence in the literature to show thatmechanical instrumentation alone will predictably resultin bacteria-free RCSs, which is not surprising given thecomplex anatomy of the RCS (4). On the contrary, thereis both in vitro and clinical evidence that mechanicalinstrumentation leaves significant portions of the rootcanal walls untouched (5). Hence, complete eliminationof bacteria from the RCS by instrumentation alone isunlikely to be achieved (6). It is assumed, but not dem-onstrated, that any pulp tissue left in the RCS can serve asa source of nutrient for bacteria but this is likely to be

only for a very short time as any such remaining pulptissue is likely to necrose and be digested by the bacteriawithin 12 months, depending on whether the canal isopen to the oral environment or not (7). Furthermore,tissue remnants may impede the antimicrobial effects ofroot canal irrigants and medicaments. Therefore, someform of chemical irrigation and disinfection is necessaryto remove tissue and other debris from the RCS and tokill any remaining micro-organisms. Chemical treat-ment of the RCS can be arbitrarily divided into severalphases, namely irrigants, rinses and inter-appointmentmedicaments.

Importance of substantivity

Following cleaning of the RCS, after root canal filling andwhile placing the final coronal restoration, there areseveral situations which can lead to the root canal spacebecoming infected again. Of significance are the periodsof time when the tooth is only temporarily restored andtimes when rubber dam is not used during the finalcoronal restoration procedures. Calcium sulphate-based

Aust Endod J 2009; 35: 131139

131 2009 The AuthorsJournal compilation 2009 Australian Society of Endodontology

and zinc oxide-eugenol-based materials are commonlyused for temporary restorations of endodontically treatedteeth. However, there are many studies using dye, radio-isotopes and bacteria, which have demonstrated thatthese restorations provide a relatively poor seal, especiallyagainst moisture penetration (812). Thus, micro-organisms may penetrate into the RCS via any defects inthe coronal aspect of the tooth and the temporary resto-rations. Furthermore, re-infection of the RCS can occurbecause of re-growth of any residual micro-organismsthat have survived the endodontic treatment procedures(6,13). The most popular irrigant currently used in endo-dontics is sodium hypochlorite (NaOCl), which possessesmany desirable properties including the ability to dissolvenecrotic tissue as well as having potent antimicrobialaction (14). However, it has not been reported to haveany residual antimicrobial activity (15).

Calcium hydroxide [Ca(OH)2] is an intracanal medica-ment that is commonly used because of its ability topredictably disinfect the RCS. However, after mechani-cally removing Ca(OH)2, there does not appear to be anyresidual antimicrobial effect (16,17). A treatment proto-col worth considering for preventing re-infection of theRCS and thus improving the outcome of endodontictreatment is to use irrigants and medicaments withresidual antimicrobial activity. Substantivity is the pro-longed association between a material and a substrate, anassociation that can be greater or more prolonged thanwould be expected from a simple deposition mechanism(18).

Chlorhexidine

Chlorhexidine (CHX) consists of two symmetric4-cholorophenyl rings and two biguanide groups con-nected by a central hexamethylene chain (19). CHX is ahydrophobic and lipophilic molecule which dissociates insolutions to form positively charged ion that interacts withphospholipids and lipopolysaccharides on the cell mem-brane of bacteria and then enters the cell through sometype of active or passive transport mechanism (20). Itsefficacy is due to the interaction of the positive charge ofthe molecule and the negatively charged phosphategroups on the microbial cell walls (20), thereby alteringthe cells osmotic equilibrium. This increases the perme-ability of the cell wall, which allows the CHX molecule topenetrate into the bacterial cell. CHX is a base and is stableas a salt. The most common oral preparation, CHX glucon-ate, is water-soluble and, at physiologic pH, it readilydissociates and releases the positively charged CHX com-ponent (20). At low concentration (such as 0.2%), lowmolecular weight substances specifically potassium andphosphorous will leak out. On the other hand, at higher

concentrations (e.g. 2%), CHX is bactericidal and precipi-tation of cytoplasmic contents occurs which results in celldeath (20). It has a wide antimicrobial spectrum and it iseffective against both Gram-positive and Gram-negativebacteria as well as yeasts, but mycobacteria, bacterialspores and most viruses are resistant to CHX (14).

The beneficial effect of CHX is a result of its anti-bacterial, substantive properties and its ability to inhibitadherence of certain bacteria (20). CHX has muchgreater activity against Gram-positive than Gram-negative organisms. The least susceptible of the Gram-negative micro-organisms include strains of Proteus,followed by Pseudomonas, Enterobacter, Actinobacter andKlebsiella (20).

Chlorhexidine possesses adequate antimicrobial pro-perties to enable it to be used as an antimicrobial endo-dontic irrigant. Gomes et al. (21) have compared the invitro antimicrobial activity against endodontic pathogensof three concentrations (0.2%, 1% and 2%) of two formsof CHX (gel and liquid) and five concentrations of NaOCl(0.5%, 1%, 2.5%, 4% and 5.25%). All irrigants wereeffective in killing Enterococcus faecalis, but at differenttimes. CHX in the liquid form at all concentrations tested(0.2%, 1% and 2%) and NaOCI (5.25%) were the mosteffective irrigants. However, the time required by 0.2%CHX liquid and 2% CHX gel to promote negative cultureswas only 30 s and 1 min respectively. Even though alltested irrigants possessed antibacterial activity, the timerequired to eliminate E. faecalis depended on the concen-tration and type of irrigant used. On the other hand,Siqueira et al. (22) found that 4% NaOCl was statisticallysignificantly better than 0.2% and 2% CHX against fourblack-pigmented Gram-negative antimicrobial aerobesand four facultative anaerobes. For the first time, Ferrazet al. (23) introduced the 2% CHX gel as an endodonticirrigant. They investigated both the ability of CHX gel todisinfect root canals contaminated in vitro with E. faecalisas well as its cleaning ability compared with commonlyused irrigants, such as NaOCl and CHX liquid. The resultsindicated that the CHX gel produced a cleaner root canalsurface and had an antimicrobial ability comparable withthat obtained with other solutions tested. It was con-cluded that CHX gel had the potential for use as anendodontic irrigant. Sena et al. (24) investigated the anti-microbial activity of 2.5% and 5.25% NaOCl and 2.0%CHX gel and liquid as endodontic-irrigating substancesagainst selected single-species biofilms. Findings showedthat mechanical agitation improved the antimicrobialproperties of the chemical substances tested using abiofilm model, favouring the agents in liquid presenta-tion, especially 5.25% NaOCl and 2% CHX.

In an in vivo antimicrobial study, Zamany (25) exam-ined whether adding a 2% CHX rinse to their conven-

Substantivity of Irrigants and Medicaments Z. Mohammadi and P. V. Abbott


tional treatment protocol increased the effectiveness ofdisinfection of the RCS. They reported that cultivablebacteria were retrieved at the conclusion of the firstappointment in only one of the CHX cases, whereas thecontrol group had seven out of 12 cases showing growthof micro-organisms. This difference was statistically sig-nificant. Recently, Siqueira et al. (26) compared the effec-tiveness of 2.5% NaOCl and 0.12% CHX as irrigants inreducing the cultivable bacteria in infected root canals ofteeth with apical periodontitis. They found that bothsolutions were comparable in removing bacteria frominfected root canals and suggested that both solutionscould be used as irrigants. In another study, Siqueira et al.(27) evaluated the effectiveness of four intracanal medi-cations in disinfecting the root dentine in bovine teeththat had been infected with Candida albicans. Infecteddentine cylinders were exposed to four different medica-tions namely, calcium hydroxide/glycerin, calciumhydroxide/0.12% CHX, calcium hydroxide/camphoratedparamonochlorophenol/glycerin and 0.12% CHX/zincoxide. They reported that the specimens exposed topastes containing either calcium hydroxide/camphoratedparamonochlorophenol/glycerin or CHX/zinc oxide werecompletely disinfected after 1 h of exposure whereas thecalcium hydroxide/glycerin paste required 7 days ofexposure and calcium hydroxide mixed with CHX wasineffective in disinfecting dentine even after 1 week.

Combination of CHX and Ca(OH)2 as anintracanal medicament

As mentioned above, CHX is a cationic biguanide. CHXsoptimal antimicrobial activity is achieved within a pHrange of 5.57.0 (20). Therefore, adding Ca(OH)2 to CHX(i.e. creating an alkaline pH) will precipitate CHX mol-ecules and decrease its effectiveness (20).

When used as an intracanal medicament, CHX has beenreported to be more effective than Ca(OH)2 ineliminating E. faecalis from inside dentinal tubules (20).In a study by Almyroudi et al. (28), all of the CHXformulations used, including a CHX/Ca(OH)2 50:50 mix,were efficient in eliminating E. faecalis from the dentinaltubules with a 1% CHX gel working slightly better than theother preparations. These findings were corroborated byGomes et al. (29) in bovine dentine and Schafer and Boss-mann (30) in human dentine where 2% CHX gel hadgreater activity against E. faecalis, followed by the CHX/Ca(OH)2 mixture and then Ca(OH)2 used alone. In anin vitro study using human teeth, Ercan et al. (31) showed2% CHX gel was the most effective agent against E. faecalisinside dentinal tubules, followed by a Ca(OH)2/2% CHXmix, while Ca(OH)2 alone was totally ineffective, evenafter 30 days. The 2% CHX gel was also significantly more

effective than the Ca(OH)2/2% CHX mix against C.albicans after 7 days, although there was no significantdifference after 15 and 30 days. Ca(OH)2 alone was com-pletely ineffective against C. albicans. In another in vivostudy using primary teeth, a 1% CHX gluconate gel, bothwith and without Ca(OH)2, was more effective against E.faecalis than Ca(OH)2 alone over a 48-h time period (32).Schafer and Bossmann (30) reported that 2% CHX glu-conate was significantly more effective against E. faecalisthan Ca(OH)2 used alone, or a mixture of the two. This wasconfirmed by Lin et al. (33) although in a study by Evanset al. (34) using bovine dentine, 2% CHX with Ca(OH)2was shown to be more effective than Ca(OH)2 in water. Inan animal study, Lindskog et al. (35) reported that teethmedicated with CHX for 4 weeks had reduced inflamma-tory reactions in the periodontium (both apically andmarginally) and less root resorption. In an in vitro study,Gomes et al. (36) investigated the time required for recon-tamination of the RCS of teeth with and without coronalrestorations medicated with either calcium hydroxide, 2%CHX gel or with a combination of both. The canals withouta coronal restoration, but medicated with CHX, showedrecontamination after an average time of 3.7 days; thegroup with Ca(OH)2 after 1.8 days and the group withCHX + Ca(OH)2 after 2.6 days. The canals medicated withCHX and restored with intermediate restorative material(IRM) showed recontamination within 13.5 days; thegroup with Ca(OH)2 + IRM after 17.2 days and the groupwith CHX + Ca(OH)2 + IRM after 11.9 days. The groupwith no medication, but restored with IRM, showedrecontamination after an average time of 8.7 days. Therewere statistically significant differences between thegroups. All groups without a coronal restoration wererecontaminated significantly more quickly than thoserestored with IRM, except those teeth that had a restora-tion but no medicament. The groups with intracanal medi-cation and a coronal restoration were not significantlydifferent from each other.

Substantivity of CHX

Chlorhexidine has a unique feature in that dentinemedicated with it acquires antimicrobial substantivity.The positively charged ions of CHX can adsorb ontodentine and prevent microbial colonization on thedentine surface for some time beyond the actual period ofmedication (20).

Antimicrobial substantivity of CHX has been assessed inseveral periodontal and endodontic studies. In an in vivoperiodontal study, Stabholz et al. (37) evaluated the sub-stantivity of the human root surface after in situ subgin-gival irrigation with tetracycline HCL and CHX. Theyfound that the substantivity of 50 mg mL-1 tetracycline

Z. Mohammadi and P. V. Abbott Substantivity of Irrigants and Medicaments


was significantly greater than CHX over 12 days andgreater than saline over 16 days. In an in vitro study,White et al. (38) evaluated the antimicrobial substantivityof a 2% CHX solution as an endodontic irrigant and theyreported that substantivity lasted 72 h.

In an in vivo study to evaluate the substantivity of 2%CHX solution, Leonardo et al. (39) found that CHX pre-vents microbial activity with residual effects in the RCSfor up to 48 h. However, other studies have shown thatthe substantivity of CHX can last for longer periods oftime. Khademi et al. (40) found that a 5-min applicationof a 2% CHX solution induced substantivity for up to4 weeks while Rosenthal et al. (41) reported that a10-min application of a 2% CHX solution resulted in CHXbeing retained in the root canal dentine in antimicrobiallyeffective amounts for up to 12 weeks. Dametto et al. (42)found that 2% CHX gel and liquid were more effectivethan 5.25% NaOCl in keeping low colony-forming unit(CFU) of E. faecalis for 7 days after the biomechanicalpreparation.

Antimicrobial substantivity depends on the number ofCHX molecules available to interact with the dentine.Therefore, medicating the canal with a more concen-trated CHX preparation should result in increased resis-tance to microbial colonization. Recently, antibacterialsubstantivity of three concentrations of CHX solution(4%, 2% and 0.2%) after 5 min has been evaluated.Results revealed a direct relationship between the con-centration of CHX and its substantivity (43). In contrast,Komorowski et al. (44) reported that 5-min application ofCHX did not induce substantivity at all and they recom-mended that the dentine should be treated with CHX for7 days.

Tetracyclines

Tetracyclines, including tetracycline-HCl, minocycline,demeclocycline (demethylchlortetracycline, Ledermycin)and doxycycline, are a group of broad-spectrum anti-biotics that are effective against a wide range of micro-organisms (45). Tetracyclines are bacteriostatic in nature(46). This property may be advantageous because, in theabsence of bacterial cell lysis, antigenic by-products suchas endotoxin are not released (45). Tetracyclines alsohave many unique properties other than their antimicro-bial effect such as the inhibition of mammalian collage-nases which prevents tissue breakdown (47,48) and theinhibition of clastic cells (4850) which results in anti-resorptive activity (51).

Inflammatory diseases such as periodontitis include apathological excess of tissue collagenases which may beblocked by tetracyclines, leading to enhanced formationof collagen and bone (47).

In periodontics, tetracyclines are used to remove thesmear layer (i.e. dentine conditioning) and surface con-taminants such as endotoxin. Surface demineralisationwidens the orifices of the dentinal tubules and exposesthe cementum collagen matrix which stimulates fibro-blast attachment and growth (45). In endodontics, tet-racyclines have been used to remove the smear layerfrom instrumented root canal walls (47,52), for irriga-tion of retrograde cavities during periapical surgical pro-cedures (53), and as an intracanal medicament (54).Abbott et al. (5557) have shown that when Ledermixpaste is used as a medicament, the demeclocyclinecomponent is released from the compound and diffusesthrough the dentine over time. This diffusion of deme-clocycline was measured for up to 12 weeks followinginsertion into the canal in an in vitro model. A concen-tration gradient formed across the dentine (55) andsmall quantities of the drug also diffused through thecementum to reach the peri-radicular tissues (56). Theconcentration of demeclocycline in the dentine immedi-ately adjacent to the root canal wall is high enough toinhibit most of the commonly reported endodontic bac-teria during the first few days of use but the concentra-tion drops over time and by about 1 week afterinsertion, it is too low to be reliable as an antimicrobialagent. In the dentine adjacent to the cementum, theconcentration is too low to be reliable (55). The diffusionof the demeclocycline was significantly slower thanthat of the corticosteroid component of Ledermix paste,triamcinolone (55,57). The diffusion of demeclocyclinecould also be slowed down significantly by combiningthe Ledermix paste with a calcium hydroxide paste (57).These slower rates of diffusion were a result of thebinding of the tetracycline to the dentine and to thecalcium component of the calcium hydroxide (57). Thisbinding was not permanent as the drug was able to bereleased from the tooth into the bathing solution inwhich the teeth were stored (5557).

Barkhordar et al. (47) evaluated the effect of doxycy-cline hydrochloride on the smear layer on instrumentedcanal walls. Their findings showed that doxycycline HCleliminated smear layer in a concentration-dependentmanner. Results showed that 100 mg mL-1 doxycyclineHCl was more effective than lower concentrations inremoving the smear layer. In another investigation,Haznedaroglu and Ersev (52) used scanning electronmicroscopy to assess the effect of tetracycline hydrochlo-ride as an endodontic irrigant to remove smear layer.Their results revealed that tetracycline HCl was as effec-tive as citric acid in removing the smear layer. Barkhordarand Russell (53) evaluated the effect of doxycycline onthe apical seal of the retrograde filling materials. Theirfindings revealed that teeth that with retrograde IRM or



amalgam fillings placed following doxycycline irrigationhad significantly less dye penetration.

Substantivity of tetracyclines

Tetracyclines readily attach to dentine and are subse-quently released without losing their antibacterial activity(45). This property creates a reservoir of active antibac-terial agent, which is then released from the dentinesurface in a slow and sustained manner. In periodontics,several studies have been conducted on the antibacterialsubstantivity of tetracyclines. In an in vivo study, Stabholzet al. (37) compared the antibacterial substantivity oftwo concentrations of tetracycline HCl (50 mg mL-1,10 mg mL-1) and 0.12% CHX. Their findings showed thatboth concentrations of tetracycline demonstrated residualantibacterial activity and the antibacterial substantivity ofthe three solutions in descending order was: 50 mg mL-1

tetracycline >10 mg mL-1 tetracycline >0.12% CHX.In endodontics, only one study has been reported

regarding the antibacterial substantivity of tetracyclines.This was an in vitro study by Khademi et al. (40) whocompared the antibacterial substantivity of 2% CHX,100 mg mL-1 doxycycline HCL and 2.6% NaOCl inbovine root dentine over five experimental periods of 0,7, 14, 21 and 28 days. Their findings revealed that after7 days, the NaOCl and doxycycline HCl groups showedthe lowest and the highest number of CFU, respectively.However, after the other experimental periods, the CHXgroup showed the lowest number of CFU count.

BioPure MTAD

BioPure (Dentsply, Tulsa Dental, Tulsa, OK, USA) other-wise known as MTAD, is a relatively new root canalirrigant which was introduced by Torabinejad et al. in2003 (45). This solution is a mixture of 3% doxycycline,4.25% citric acid and a detergent (0.5% Polysorbate 80)(58). Several studies have evaluated the effectiveness ofMTAD for disinfection of root canals. Torabinejad et al.have showed that MTAD is able to remove the smearlayer (45) and is effective against E. faecalis (5961).

Shabahang et al. (60) cleaned and shaped root canalsof extracted human teeth and exposed them to humansaliva. They then compared the antibacterial efficacy of acombination of 1.3% NaOCl as a root canal irrigant andMTAD as a final rinse with that of 5.25% NaOCl. Theirfindings showed that the use of MTAD was more effectivethan 5.25% NaOCl alone in disinfecting root canals.However, Tay et al. (62) found that when MTAD is appliedto 1.3% NaOCl-irrigated dentine, its antimicrobial sub-stantivity is reduced. They attributed this phenomenon tothe oxidation of MTAD by NaOCl in a manner similar tothe peroxidation of tetracycline by reactive oxygen

species. In another study, Shabahang and Torabinejad(61) compared the antibacterial effects of MTAD withthose of NaOCl and ethylene diamine tetraacetic acid(EDTA) by using standard in vitro microbiological tech-niques and they reported that MTAD was significantlymore effective against E. faecalis. Kho and Baumgartner(63) compared the antimicrobial efficacy against E. faecalisof 1.3% NaOCl/BioPure MTAD with that of the combinedalternate use of 5.25% NaOCl and 15% EDTA for rootcanal irrigation. Bacterial samples taken early in the canalcleaning process revealed growth in none of the 20samples with 5.25% NaOCl/15% EDTA irrigation and ineight of 20 samples with 1.3% NaOCl/BioPure MTADirrigation. Further samples taken after additional canalenlargement revealed growth in none of 20 sampleswhen 5.25% NaOCl/15% EDTA were used but therewas growth in 10 of the 20 samples when 1.3% NaOCl/BioPure MTAD was used. This investigation showedconsistent disinfection of infected root canals whena combination of 5.25% NaOCl/15% EDTA was used.However, the combination of 1.3% NaOCl/BioPureMTAD left nearly 50% of the canals contaminated withE. faecalis. Krause et al. (64) compared the antimicrobialeffect against E. faecalis of MTAD, two of its components(doxycycline and citric acid), and sodium hypochlorite intwo in vitro models using two different methods. In thetooth model, NaOCl and doxycycline were more effectivethan the control in killing E. faecalis at shallow bur depthsinto dentine, but at deeper bur depths, the NaOCl wassuperior. In the agar diffusion model, NaOCl producedless inhibition of bacteria than MTAD or doxycycline.

Substantivity of MTAD

As stated earlier, tetracyclines (including doxycycline)readily attach to dentine and are subsequently releasedwithout losing their antibacterial activity. The presence ofdoxycycline in MTAD suggests that MTAD may havesome substantive antimicrobial action. To date, there areonly two studies on the substantivity of MTAD. In anin vitro study, Mohammadi and Yazdizadeh (65) evaluatedthe substantivity of NaOCl, CHX and MTAD using abovine dentine tube model. Dentine chips were removedfrom the canals with sequential sterile low-speed roundburs with increasing diameters of ISO sizes: 025, 027,029, 031 and 033 at experimental times of 0, 7, 14, 21and 28 days following irrigation with the test solution. Inthe first culture, the NaOCl group and the CHX groupshowed the lowest and highest number of CFU countrespectively. In each group, the number of CFU countincreased significantly by time lapse. The authors con-cluded that the substantivity of MTAD was significantlygreater than CHX and NaOCl.



In another study, Mohammadi (66) assessed the sub-stantivity of three concentrations (100%, 10% and 1%)of MTAD using a bovine dentine tube model. Dentinechips were removed from the canals with sequentialsterile low-speed round burs with increasing diameters ofISO sizes: 025, 027, 029, 031 and 033 at experimentaltimes of 0, 7, 14, 21 and 28 days. Results showed that inthe first culture, the MTAD 100% group and the MTAD1% group showed the lowest and highest number of CFUcount respectively. In each group, the number of CFUcount increased significantly by time lapse (P < 0.05). Inconclusion, the substantivity of 100% MTAD was signifi-cantly greater than the two other concentrations ofMTAD.

Discussion

Chlorhexidine is a synthetic cationic biguanide, whichhas a wide range of activity against both Gram-positiveand Gram-negative bacteria as well as fungi, especially C.albicans. It can be used as both liquid and gel forms (67).In most investigations, the recommended concentrationof CHX for endodontic applications was 2% (2025).Only a small number of studies have investigated theshort-term substantivity of commonly used antimicrobialagents and the results show substantivity of CHX lasts forup to 12 weeks (41). Antimicrobial substantivity dependson the number of CHX molecules available to interactwith the dentine. Therefore, medicating the canal with amore concentrated CHX preparation should result inincreased resistance to microbial colonization. There aretwo different viewpoints regarding the treatment timewith CHX to induce substantivity. Some studies showedthat dentine treatment for 510 min induces substantiv-ity (4142). On the other hand, Lin et al. (68) attributedthe substantivity of CHX to absorption of the medicationto dentine during the first hour of application and theystated that the antimicrobial capability of CHX onlyincreases with time after the saturation point had beenreached by the end of the first hour of application.

It is not known whether the substantivity of CHX willlast for longer than 12 weeks as this has not been inves-tigated. Further research is required to determine thelong-term substantivity of these agents and any otheragents used as endodontic antimicrobial irrigants ormedicaments. Further research is required to determinethe long-term substantivity of these agents and anyothers used as endodontic antimicrobial irrigants ormedicaments.

Haenni et al. (69) demonstrated that the alkalinity ofthe calcium hydroxide when mixed with CHX remainedunchanged. Despite its excellent antimicrobial activityand substantivity, CHX should not be advocated as a

standard irrigation solution because it is less effective onGram-negative than Gram-positive bacteria and it isunable to dissolve pulpal tissue remnants. However, itmay be very useful for a final rinse solution (45).

Tetracyclines are a group of broad-spectrum bacterio-static antibiotics that are effective against a wide range ofmicro-organisms (46). Bacteriostaticity may be an advan-tage for tetracyclines because, in the absence of bacterialcell lysis, antigenic by-products such as endotoxin are notreleased (46). Studies on the substantivity of tetracyclinesare very limited. Substantivity of doxycycline has beendemonstrated for up to 4 weeks (40). Furthermore, it hasbeen shown that, such as CHX, substantivity of doxycy-cline has a direct relationship to its concentration (70).Adding a detergent (Tween 80) to doxycycline in MTADhas increased its penetration depth into dentinal tubulesby decreasing its surface tension (46). Substantivity ofMTAD has been revealed for up to 4 weeks in human(71) and bovine (66) root dentine blocks. Furthermore,its concentration-dependent substantivity has beenshown (66).

The root canal milieu is a complex mixture of a varietyof organic and inorganic compounds. Hydroxyapatite, themain component of dentine, is the major representativeof inorganic components present. In addition, inflamma-tory exudate, entering the apical root canal in purulentinfections, is rich in proteins such as albumin. The relativeimportance of the various organic and inorganic com-pounds in the inactivation of root canal disinfectants hasbeen studied restrictively (69). Haapasalo et al. (72) intro-duced a new dentine powder model for studying theinhibitory effect of dentine on various root canal irrigantsand medicaments. They reported that 0.05% CHX acetatekilled greater than 99.9% of E. faecalis cells within 1 hwhen dentine was not present. Addition of dentine (18%w/v) totally prevented killing of the bacteria during thefirst hour. However, after 24 h all bacteria were killed inboth groups. They also found that pre-incubation withdentine slightly weakened the long-term effect (substan-tivity) of CHX. Portenier et al. (73) found that CHX(0.05%) was strongly inhibited by bovine serumalbumin (BSA) and slowed down by dentine. However,hydroxyapatite (HA) had little or no inhibitory effect onCHX. A reduction of dentine from 28 mg 150 mL-1 to onetenth (2.8 mg 150 mL-1) was followed by a similar reduc-tion of the inhibition of the antibacterial activity of CHX.One major mechanism for survival of E. faecalis in the rootcanal filled with Ca(OH)2 may be the buffering effect ofdentine against the pH rise. Inorganic HA had little or noinhibitory activity against CHX as compared with dentine,whereas BSA was the strongest inhibitor of CHX, withmore than 10% of E. faecalis cells still viable after 24 h ofincubation with the medicament. This indicates that



periapical inflammatory exudate entering the root canalmay be a greater threat to the activity of CHX than thedentine walls. In another study Portenier et al. (74)revealed that dentine matrix and heat-killed microbialcells were strong inhibitors of CHX, whereas dentine pre-treated by citric acid or EDTA showed only slight inhibi-tion. Dentine and skin collagen showed some inhibitionat 1 h but not after 24 h. Another study showed thatdentine and BSA caused a marked delay in the killing ofE. faecalis by CHX and MTAD (75). The inhibitory effect ofBSA on the antibacterial activity of CHX has been con-firmed by Sassone et al. (76). Taken together, it seems thatdentine, dentine components (HA and collagen), killedmicro-organisms and inflammatory exudate in the RCSreduce or inhibit the antibacterial activity of medicamentsand irrigants.

Conclusions

1. CHX has a wide range of activity against both Gram-positive and Gram-negative bacteria.2. CHX is an effective antifungal agent especially againstC. albicans.3. CHX has antibacterial substantivity for up to 12 weeks.4. Dentine, dentine components (HA and collagen),killed micro-organisms and inflammatory exudate in theRCS may reduce or inhibit the antibacterial activity ofCHX and MTAD.5. It seems that that the mixture of CHX and Ca(OH)2 isless effective than CHX alone.6. Substantivity of MTAD and doxycycline has beenshown for up to 4 weeks.7. Because CHX has favourable antimicrobial activityand substantivity it should be used as a final rinse inendodontic treatment protocols.8. Owing to the greater activity against Gram-positivethan Gram-negative organisms, application of CHX asintracanal medicament is preferred in retreatment casescompared with initial endodontic infections.

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