Aust Endod J 2009; 35: 131139

R E V I E W A RT I C L E

Antimicrobial substantivity of root canal irrigants and

medicaments: A review
aej_164

131..139

Zahed 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

Keywords
endodontics, irrigants, medicaments,
substantivity.
Correspondence
Dr Zahed Mohammadi, Department of
Endodontics, Shahid Sadoughi Dental School,
Imam Avenue, Fazaye Sabz Cross Road, Yazd,
Iran. Email: mohammadi_zahed@yahoo.com
doi:10.1111/j.1747-4477.2009.00164.x

Abstract
Disinfection of the root canal system is one of the primary aims of root canal
treatment. This can be achieved through the use of various antimicrobial
agents in the form of irrigants and medicaments. These agents are only used for
relatively short periods of time ranging from minutes (for irrigants) up to days
or several weeks (for medicaments) and therefore their long-term antimicrobial effects rely on whether or not the particular agent has any properties of
substantivity. If irrigants and medicaments had effective long-term residual
antimicrobial 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 substantivity
of commonly used antimicrobial agents and the results show substantivity of
chlorhexidine 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 for
longer periods of time as this has not been investigated.

Introduction
The essential role of micro-organisms in the development
and perpetuation of pulp and periapical diseases has been
clearly demonstrated in many animal model and human
studies (13). Elimination of micro-organisms from
infected root canal systems (RCSs) is a complicated task.
Numerous measures have been described to reduce the
number of micro-organisms from the RCS, including the
use of various mechanical instrumentation techniques,
irrigation regimes and intracanal medicaments. There is
no definitive evidence in the literature to show that
mechanical instrumentation alone will predictably result
in bacteria-free RCSs, which is not surprising given the
complex anatomy of the RCS (4). On the contrary, there
is both in vitro and clinical evidence that mechanical
instrumentation leaves significant portions of the root
canal walls untouched (5). Hence, complete elimination
of bacteria from the RCS by instrumentation alone is
unlikely to be achieved (6). It is assumed, but not demonstrated, that any pulp tissue left in the RCS can serve as
a source of nutrient for bacteria but this is likely to be

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only for a very short time as any such remaining pulp

tissue is likely to necrose and be digested by the bacteria
within 12 months, depending on whether the canal is
open to the oral environment or not (7). Furthermore,
tissue remnants may impede the antimicrobial effects of
root canal irrigants and medicaments. Therefore, some
form of chemical irrigation and disinfection is necessary
to remove tissue and other debris from the RCS and to
kill any remaining micro-organisms. Chemical treatment of the RCS can be arbitrarily divided into several
phases, namely irrigants, rinses and inter-appointment
medicaments.

Importance of substantivity
Following cleaning of the RCS, after root canal filling and
while placing the final coronal restoration, there are
several situations which can lead to the root canal space
becoming infected again. Of significance are the periods
of time when the tooth is only temporarily restored and
times when rubber dam is not used during the final
coronal restoration procedures. Calcium sulphate-based
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and zinc oxide-eugenol-based materials are commonly

used for temporary restorations of endodontically treated
teeth. However, there are many studies using dye, radioisotopes and bacteria, which have demonstrated that
these restorations provide a relatively poor seal, especially
against moisture penetration (812). Thus, microorganisms may penetrate into the RCS via any defects in
the coronal aspect of the tooth and the temporary restorations. Furthermore, re-infection of the RCS can occur
because of re-growth of any residual micro-organisms
that have survived the endodontic treatment procedures
(6,13). The most popular irrigant currently used in endodontics is sodium hypochlorite (NaOCl), which possesses
many desirable properties including the ability to dissolve
necrotic tissue as well as having potent antimicrobial
action (14). However, it has not been reported to have
any residual antimicrobial activity (15).
Calcium hydroxide [Ca(OH)2] is an intracanal medicament that is commonly used because of its ability to
predictably disinfect the RCS. However, after mechanically removing Ca(OH)2, there does not appear to be any
residual antimicrobial effect (16,17). A treatment protocol worth considering for preventing re-infection of the
RCS and thus improving the outcome of endodontic
treatment is to use irrigants and medicaments with
residual antimicrobial activity. Substantivity is the prolonged association between a material and a substrate, an
association that can be greater or more prolonged than
would be expected from a simple deposition mechanism
(18).

Chlorhexidine
Chlorhexidine (CHX) consists of two symmetric
4-cholorophenyl rings and two biguanide groups connected by a central hexamethylene chain (19). CHX is a
hydrophobic and lipophilic molecule which dissociates in
solutions to form positively charged ion that interacts with
phospholipids and lipopolysaccharides on the cell membrane of bacteria and then enters the cell through some
type of active or passive transport mechanism (20). Its
efficacy is due to the interaction of the positive charge of
the molecule and the negatively charged phosphate
groups on the microbial cell walls (20), thereby altering
the cells osmotic equilibrium. This increases the permeability of the cell wall, which allows the CHX molecule to
penetrate into the bacterial cell. CHX is a base and is stable
as a salt. The most common oral preparation, CHX gluconate, is water-soluble and, at physiologic pH, it readily
dissociates and releases the positively charged CHX component (20). At low concentration (such as 0.2%), low
molecular weight substances specifically potassium and
phosphorous will leak out. On the other hand, at higher
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concentrations (e.g. 2%), CHX is bactericidal and precipitation of cytoplasmic contents occurs which results in cell
death (20). It has a wide antimicrobial spectrum and it is
effective against both Gram-positive and Gram-negative
bacteria as well as yeasts, but mycobacteria, bacterial
spores and most viruses are resistant to CHX (14).
The beneficial effect of CHX is a result of its antibacterial, substantive properties and its ability to inhibit
adherence of certain bacteria (20). CHX has much
greater activity against Gram-positive than Gramnegative organisms. The least susceptible of the Gramnegative micro-organisms include strains of Proteus,
followed by Pseudomonas, Enterobacter, Actinobacter and
Klebsiella (20).
Chlorhexidine possesses adequate antimicrobial properties to enable it to be used as an antimicrobial endodontic irrigant. Gomes et al. (21) have compared the in
vitro antimicrobial activity against endodontic pathogens
of three concentrations (0.2%, 1% and 2%) of two forms
of CHX (gel and liquid) and five concentrations of NaOCl
(0.5%, 1%, 2.5%, 4% and 5.25%). All irrigants were
effective in killing Enterococcus faecalis, but at different
times. CHX in the liquid form at all concentrations tested
(0.2%, 1% and 2%) and NaOCI (5.25%) were the most
effective irrigants. However, the time required by 0.2%
CHX liquid and 2% CHX gel to promote negative cultures
was only 30 s and 1 min respectively. Even though all
tested irrigants possessed antibacterial activity, the time
required to eliminate E. faecalis depended on the concentration and type of irrigant used. On the other hand,
Siqueira et al. (22) found that 4% NaOCl was statistically
significantly better than 0.2% and 2% CHX against four
black-pigmented Gram-negative antimicrobial aerobes
and four facultative anaerobes. For the first time, Ferraz
et al. (23) introduced the 2% CHX gel as an endodontic
irrigant. They investigated both the ability of CHX gel to
disinfect root canals contaminated in vitro with E. faecalis
as well as its cleaning ability compared with commonly
used irrigants, such as NaOCl and CHX liquid. The results
indicated that the CHX gel produced a cleaner root canal
surface and had an antimicrobial ability comparable with
that obtained with other solutions tested. It was concluded that CHX gel had the potential for use as an
endodontic irrigant. Sena et al. (24) investigated the antimicrobial activity of 2.5% and 5.25% NaOCl and 2.0%
CHX gel and liquid as endodontic-irrigating substances
against selected single-species biofilms. Findings showed
that mechanical agitation improved the antimicrobial
properties of the chemical substances tested using a
biofilm model, favouring the agents in liquid presentation, especially 5.25% NaOCl and 2% CHX.
In an in vivo antimicrobial study, Zamany (25) examined whether adding a 2% CHX rinse to their conven-

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Z. Mohammadi and P. V. Abbott

tional treatment protocol increased the effectiveness of

disinfection of the RCS. They reported that cultivable
bacteria were retrieved at the conclusion of the first
appointment in only one of the CHX cases, whereas the
control group had seven out of 12 cases showing growth
of micro-organisms. This difference was statistically significant. Recently, Siqueira et al. (26) compared the effectiveness of 2.5% NaOCl and 0.12% CHX as irrigants in
reducing the cultivable bacteria in infected root canals of
teeth with apical periodontitis. They found that both
solutions were comparable in removing bacteria from
infected root canals and suggested that both solutions
could be used as irrigants. In another study, Siqueira et al.
(27) evaluated the effectiveness of four intracanal medications in disinfecting the root dentine in bovine teeth
that had been infected with Candida albicans. Infected
dentine cylinders were exposed to four different medications namely, calcium hydroxide/glycerin, calcium
hydroxide/0.12% CHX, calcium hydroxide/camphorated
paramonochlorophenol/glycerin and 0.12% CHX/zinc
oxide. They reported that the specimens exposed to
pastes containing either calcium hydroxide/camphorated
paramonochlorophenol/glycerin or CHX/zinc oxide were
completely disinfected after 1 h of exposure whereas the
calcium hydroxide/glycerin paste required 7 days of
exposure and calcium hydroxide mixed with CHX was
ineffective in disinfecting dentine even after 1 week.

Combination of CHX and Ca(OH)2 as an

intracanal medicament
As mentioned above, CHX is a cationic biguanide. CHXs
optimal antimicrobial activity is achieved within a pH
range of 5.57.0 (20). Therefore, adding Ca(OH)2 to CHX
(i.e. creating an alkaline pH) will precipitate CHX molecules and decrease its effectiveness (20).
When used as an intracanal medicament, CHX has been
reported to be more effective than Ca(OH)2 in
eliminating E. faecalis from inside dentinal tubules (20).
In a study by Almyroudi et al. (28), all of the CHX
formulations used, including a CHX/Ca(OH)2 50:50 mix,
were efficient in eliminating E. faecalis from the dentinal
tubules with a 1% CHX gel working slightly better than the
other preparations. These findings were corroborated by
Gomes et al. (29) in bovine dentine and Schafer and Bossmann (30) in human dentine where 2% CHX gel had
greater activity against E. faecalis, followed by the CHX/
Ca(OH)2 mixture and then Ca(OH)2 used alone. In an
in vitro study using human teeth, Ercan et al. (31) showed
2% CHX gel was the most effective agent against E. faecalis
inside dentinal tubules, followed by a Ca(OH)2/2% CHX
mix, while Ca(OH)2 alone was totally ineffective, even
after 30 days. The 2% CHX gel was also significantly more

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Substantivity of Irrigants and Medicaments

effective than the Ca(OH)2/2% CHX mix against C.

albicans after 7 days, although there was no significant
difference after 15 and 30 days. Ca(OH)2 alone was completely ineffective against C. albicans. In another in vivo
study using primary teeth, a 1% CHX gluconate gel, both
with 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 gluconate was significantly more effective against E. faecalis
than Ca(OH)2 used alone, or a mixture of the two. This was
confirmed by Lin et al. (33) although in a study by Evans
et al. (34) using bovine dentine, 2% CHX with Ca(OH)2
was shown to be more effective than Ca(OH)2 in water. In
an animal study, Lindskog et al. (35) reported that teeth
medicated with CHX for 4 weeks had reduced inflammatory reactions in the periodontium (both apically and
marginally) and less root resorption. In an in vitro study,
Gomes et al. (36) investigated the time required for recontamination of the RCS of teeth with and without coronal
restorations medicated with either calcium hydroxide, 2%
CHX gel or with a combination of both. The canals without
a coronal restoration, but medicated with CHX, showed
recontamination after an average time of 3.7 days; the
group with Ca(OH)2 after 1.8 days and the group with
CHX + Ca(OH)2 after 2.6 days. The canals medicated with
CHX and restored with intermediate restorative material
(IRM) showed recontamination within 13.5 days; the
group with Ca(OH)2 + IRM after 17.2 days and the group
with CHX + Ca(OH)2 + IRM after 11.9 days. The group
with no medication, but restored with IRM, showed
recontamination after an average time of 8.7 days. There
were statistically significant differences between the
groups. All groups without a coronal restoration were
recontaminated significantly more quickly than those
restored with IRM, except those teeth that had a restoration but no medicament. The groups with intracanal medication and a coronal restoration were not significantly
different from each other.

Substantivity of CHX
Chlorhexidine has a unique feature in that dentine
medicated with it acquires antimicrobial substantivity.
The positively charged ions of CHX can adsorb onto
dentine and prevent microbial colonization on the
dentine surface for some time beyond the actual period of
medication (20).
Antimicrobial substantivity of CHX has been assessed in
several periodontal and endodontic studies. In an in vivo
periodontal study, Stabholz et al. (37) evaluated the substantivity of the human root surface after in situ subgingival irrigation with tetracycline HCL and CHX. They
found that the substantivity of 50 mg mL-1 tetracycline
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was significantly greater than CHX over 12 days and

greater than saline over 16 days. In an in vitro study,
White et al. (38) evaluated the antimicrobial substantivity
of a 2% CHX solution as an endodontic irrigant and they
reported 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 prevents microbial activity with residual effects in the RCS
for up to 48 h. However, other studies have shown that
the substantivity of CHX can last for longer periods of
time. Khademi et al. (40) found that a 5-min application
of a 2% CHX solution induced substantivity for up to
4 weeks while Rosenthal et al. (41) reported that a
10-min application of a 2% CHX solution resulted in CHX
being retained in the root canal dentine in antimicrobially
effective amounts for up to 12 weeks. Dametto et al. (42)
found that 2% CHX gel and liquid were more effective
than 5.25% NaOCl in keeping low colony-forming unit
(CFU) of E. faecalis for 7 days after the biomechanical
preparation.
Antimicrobial substantivity depends on the number of
CHX molecules available to interact with the dentine.
Therefore, medicating the canal with a more concentrated CHX preparation should result in increased resistance to microbial colonization. Recently, antibacterial
substantivity of three concentrations of CHX solution
(4%, 2% and 0.2%) after 5 min has been evaluated.
Results revealed a direct relationship between the concentration of CHX and its substantivity (43). In contrast,
Komorowski et al. (44) reported that 5-min application of
CHX did not induce substantivity at all and they recommended that the dentine should be treated with CHX for
7 days.

Tetracyclines
Tetracyclines, including tetracycline-HCl, minocycline,
demeclocycline (demethylchlortetracycline, Ledermycin)
and doxycycline, are a group of broad-spectrum antibiotics that are effective against a wide range of microorganisms (45). Tetracyclines are bacteriostatic in nature
(46). This property may be advantageous because, in the
absence of bacterial cell lysis, antigenic by-products such
as endotoxin are not released (45). Tetracyclines also
have many unique properties other than their antimicrobial effect such as the inhibition of mammalian collagenases which prevents tissue breakdown (47,48) and the
inhibition of clastic cells (4850) which results in antiresorptive activity (51).
Inflammatory diseases such as periodontitis include a
pathological excess of tissue collagenases which may be
blocked by tetracyclines, leading to enhanced formation
of collagen and bone (47).
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In periodontics, tetracyclines are used to remove the

smear layer (i.e. dentine conditioning) and surface contaminants such as endotoxin. Surface demineralisation
widens the orifices of the dentinal tubules and exposes
the cementum collagen matrix which stimulates fibroblast attachment and growth (45). In endodontics, tetracyclines have been used to remove the smear layer
from instrumented root canal walls (47,52), for irrigation of retrograde cavities during periapical surgical procedures (53), and as an intracanal medicament (54).
Abbott et al. (5557) have shown that when Ledermix
paste is used as a medicament, the demeclocycline
component is released from the compound and diffuses
through the dentine over time. This diffusion of demeclocycline was measured for up to 12 weeks following
insertion into the canal in an in vitro model. A concentration gradient formed across the dentine (55) and
small quantities of the drug also diffused through the
cementum to reach the peri-radicular tissues (56). The
concentration of demeclocycline in the dentine immediately adjacent to the root canal wall is high enough to
inhibit most of the commonly reported endodontic bacteria during the first few days of use but the concentration drops over time and by about 1 week after
insertion, it is too low to be reliable as an antimicrobial
agent. In the dentine adjacent to the cementum, the
concentration is too low to be reliable (55). The diffusion
of the demeclocycline was significantly slower than
that of the corticosteroid component of Ledermix paste,
triamcinolone (55,57). The diffusion of demeclocycline
could also be slowed down significantly by combining
the Ledermix paste with a calcium hydroxide paste (57).
These slower rates of diffusion were a result of the
binding of the tetracycline to the dentine and to the
calcium component of the calcium hydroxide (57). This
binding was not permanent as the drug was able to be
released from the tooth into the bathing solution in
which the teeth were stored (5557).
Barkhordar et al. (47) evaluated the effect of doxycycline hydrochloride on the smear layer on instrumented
canal walls. Their findings showed that doxycycline HCl
eliminated smear layer in a concentration-dependent
manner. Results showed that 100 mg mL-1 doxycycline
HCl was more effective than lower concentrations in
removing the smear layer. In another investigation,
Haznedaroglu and Ersev (52) used scanning electron
microscopy to assess the effect of tetracycline hydrochloride as an endodontic irrigant to remove smear layer.
Their results revealed that tetracycline HCl was as effective as citric acid in removing the smear layer. Barkhordar
and Russell (53) evaluated the effect of doxycycline on
the apical seal of the retrograde filling materials. Their
findings revealed that teeth that with retrograde IRM or

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Z. Mohammadi and P. V. Abbott

amalgam fillings placed following doxycycline irrigation

had significantly less dye penetration.

Substantivity of tetracyclines
Tetracyclines readily attach to dentine and are subsequently released without losing their antibacterial activity
(45). This property creates a reservoir of active antibacterial agent, which is then released from the dentine
surface in a slow and sustained manner. In periodontics,
several studies have been conducted on the antibacterial
substantivity of tetracyclines. In an in vivo study, Stabholz
et al. (37) compared the antibacterial substantivity of
two concentrations of tetracycline HCl (50 mg mL-1,
10 mg mL-1) and 0.12% CHX. Their findings showed that
both concentrations of tetracycline demonstrated residual
antibacterial activity and the antibacterial substantivity of
the 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) who
compared the antibacterial substantivity of 2% CHX,
100 mg mL-1 doxycycline HCL and 2.6% NaOCl in
bovine root dentine over five experimental periods of 0,
7, 14, 21 and 28 days. Their findings revealed that after
7 days, the NaOCl and doxycycline HCl groups showed
the lowest and the highest number of CFU, respectively.
However, after the other experimental periods, the CHX
group showed the lowest number of CFU count.

BioPure MTAD
BioPure (Dentsply, Tulsa Dental, Tulsa, OK, USA) otherwise known as MTAD, is a relatively new root canal
irrigant which was introduced by Torabinejad et al. in
2003 (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 of
MTAD for disinfection of root canals. Torabinejad et al.
have showed that MTAD is able to remove the smear
layer (45) and is effective against E. faecalis (5961).
Shabahang et al. (60) cleaned and shaped root canals
of extracted human teeth and exposed them to human
saliva. They then compared the antibacterial efficacy of a
combination of 1.3% NaOCl as a root canal irrigant and
MTAD as a final rinse with that of 5.25% NaOCl. Their
findings showed that the use of MTAD was more effective
than 5.25% NaOCl alone in disinfecting root canals.
However, Tay et al. (62) found that when MTAD is applied
to 1.3% NaOCl-irrigated dentine, its antimicrobial substantivity is reduced. They attributed this phenomenon to
the oxidation of MTAD by NaOCl in a manner similar to
the peroxidation of tetracycline by reactive oxygen

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species. In another study, Shabahang and Torabinejad

(61) compared the antibacterial effects of MTAD with
those of NaOCl and ethylene diamine tetraacetic acid
(EDTA) by using standard in vitro microbiological techniques and they reported that MTAD was significantly
more effective against E. faecalis. Kho and Baumgartner
(63) compared the antimicrobial efficacy against E. faecalis
of 1.3% NaOCl/BioPure MTAD with that of the combined
alternate use of 5.25% NaOCl and 15% EDTA for root
canal irrigation. Bacterial samples taken early in the canal
cleaning process revealed growth in none of the 20
samples with 5.25% NaOCl/15% EDTA irrigation and in
eight of 20 samples with 1.3% NaOCl/BioPure MTAD
irrigation. Further samples taken after additional canal
enlargement revealed growth in none of 20 samples
when 5.25% NaOCl/15% EDTA were used but there
was growth in 10 of the 20 samples when 1.3% NaOCl/
BioPure MTAD was used. This investigation showed
consistent disinfection of infected root canals when
a combination of 5.25% NaOCl/15% EDTA was used.
However, the combination of 1.3% NaOCl/BioPure
MTAD left nearly 50% of the canals contaminated with
E. faecalis. Krause et al. (64) compared the antimicrobial
effect against E. faecalis of MTAD, two of its components
(doxycycline and citric acid), and sodium hypochlorite in
two in vitro models using two different methods. In the
tooth model, NaOCl and doxycycline were more effective
than the control in killing E. faecalis at shallow bur depths
into dentine, but at deeper bur depths, the NaOCl was
superior. In the agar diffusion model, NaOCl produced
less inhibition of bacteria than MTAD or doxycycline.

Substantivity of MTAD
As stated earlier, tetracyclines (including doxycycline)
readily attach to dentine and are subsequently released
without losing their antibacterial activity. The presence of
doxycycline in MTAD suggests that MTAD may have
some substantive antimicrobial action. To date, there are
only two studies on the substantivity of MTAD. In an
in vitro study, Mohammadi and Yazdizadeh (65) evaluated
the substantivity of NaOCl, CHX and MTAD using a
bovine dentine tube model. Dentine chips were removed
from the canals with sequential sterile low-speed round
burs with increasing diameters of ISO sizes: 025, 027,
029, 031 and 033 at experimental times of 0, 7, 14, 21
and 28 days following irrigation with the test solution. In
the first culture, the NaOCl group and the CHX group
showed the lowest and highest number of CFU count
respectively. In each group, the number of CFU count
increased significantly by time lapse. The authors concluded that the substantivity of MTAD was significantly
greater than CHX and NaOCl.
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In another study, Mohammadi (66) assessed the substantivity of three concentrations (100%, 10% and 1%)
of MTAD using a bovine dentine tube model. Dentine
chips were removed from the canals with sequential
sterile low-speed round burs with increasing diameters of
ISO sizes: 025, 027, 029, 031 and 033 at experimental
times of 0, 7, 14, 21 and 28 days. Results showed that in
the first culture, the MTAD 100% group and the MTAD
1% group showed the lowest and highest number of CFU
count respectively. In each group, the number of CFU
count increased significantly by time lapse (P < 0.05). In
conclusion, the substantivity of 100% MTAD was significantly greater than the two other concentrations of
MTAD.

Discussion
Chlorhexidine is a synthetic cationic biguanide, which
has a wide range of activity against both Gram-positive
and 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 concentration
of CHX for endodontic applications was 2% (2025).
Only a small number of studies have investigated the
short-term substantivity of commonly used antimicrobial
agents and the results show substantivity of CHX lasts for
up to 12 weeks (41). Antimicrobial substantivity depends
on the number of CHX molecules available to interact
with the dentine. Therefore, medicating the canal with a
more concentrated CHX preparation should result in
increased resistance to microbial colonization. There are
two different viewpoints regarding the treatment time
with CHX to induce substantivity. Some studies showed
that dentine treatment for 510 min induces substantivity (4142). On the other hand, Lin et al. (68) attributed
the substantivity of CHX to absorption of the medication
to dentine during the first hour of application and they
stated that the antimicrobial capability of CHX only
increases with time after the saturation point had been
reached by the end of the first hour of application.
It is not known whether the substantivity of CHX will
last for longer than 12 weeks as this has not been investigated. Further research is required to determine the
long-term substantivity of these agents and any other
agents used as endodontic antimicrobial irrigants or
medicaments. Further research is required to determine
the long-term substantivity of these agents and any
others used as endodontic antimicrobial irrigants or
medicaments.
Haenni et al. (69) demonstrated that the alkalinity of
the calcium hydroxide when mixed with CHX remained
unchanged. Despite its excellent antimicrobial activity
and substantivity, CHX should not be advocated as a
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standard irrigation solution because it is less effective on

Gram-negative than Gram-positive bacteria and it is
unable to dissolve pulpal tissue remnants. However, it
may be very useful for a final rinse solution (45).
Tetracyclines are a group of broad-spectrum bacteriostatic antibiotics that are effective against a wide range of
micro-organisms (46). Bacteriostaticity may be an advantage for tetracyclines because, in the absence of bacterial
cell lysis, antigenic by-products such as endotoxin are not
released (46). Studies on the substantivity of tetracyclines
are very limited. Substantivity of doxycycline has been
demonstrated for up to 4 weeks (40). Furthermore, it has
been shown that, such as CHX, substantivity of doxycycline has a direct relationship to its concentration (70).
Adding a detergent (Tween 80) to doxycycline in MTAD
has increased its penetration depth into dentinal tubules
by decreasing its surface tension (46). Substantivity of
MTAD has been revealed for up to 4 weeks in human
(71) and bovine (66) root dentine blocks. Furthermore,
its concentration-dependent substantivity has been
shown (66).
The root canal milieu is a complex mixture of a variety
of organic and inorganic compounds. Hydroxyapatite, the
main component of dentine, is the major representative
of inorganic components present. In addition, inflammatory exudate, entering the apical root canal in purulent
infections, is rich in proteins such as albumin. The relative
importance of the various organic and inorganic compounds in the inactivation of root canal disinfectants has
been studied restrictively (69). Haapasalo et al. (72) introduced a new dentine powder model for studying the
inhibitory effect of dentine on various root canal irrigants
and medicaments. They reported that 0.05% CHX acetate
killed greater than 99.9% of E. faecalis cells within 1 h
when dentine was not present. Addition of dentine (18%
w/v) totally prevented killing of the bacteria during the
first hour. However, after 24 h all bacteria were killed in
both groups. They also found that pre-incubation with
dentine slightly weakened the long-term effect (substantivity) of CHX. Portenier et al. (73) found that CHX
(0.05%) was strongly inhibited by bovine serum
albumin (BSA) and slowed down by dentine. However,
hydroxyapatite (HA) had little or no inhibitory effect on
CHX. A reduction of dentine from 28 mg 150 mL-1 to one
tenth (2.8 mg 150 mL-1) was followed by a similar reduction of the inhibition of the antibacterial activity of CHX.
One major mechanism for survival of E. faecalis in the root
canal filled with Ca(OH)2 may be the buffering effect of
dentine against the pH rise. Inorganic HA had little or no
inhibitory activity against CHX as compared with dentine,
whereas BSA was the strongest inhibitor of CHX, with
more than 10% of E. faecalis cells still viable after 24 h of
incubation with the medicament. This indicates that

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Journal compilation 2009 Australian Society of Endodontology

Z. Mohammadi and P. V. Abbott

periapical inflammatory exudate entering the root canal

may be a greater threat to the activity of CHX than the
dentine walls. In another study Portenier et al. (74)
revealed that dentine matrix and heat-killed microbial
cells were strong inhibitors of CHX, whereas dentine pretreated by citric acid or EDTA showed only slight inhibition. Dentine and skin collagen showed some inhibition
at 1 h but not after 24 h. Another study showed that
dentine and BSA caused a marked delay in the killing of
E. faecalis by CHX and MTAD (75). The inhibitory effect of
BSA on the antibacterial activity of CHX has been confirmed by Sassone et al. (76). Taken together, it seems that
dentine, dentine components (HA and collagen), killed
micro-organisms and inflammatory exudate in the RCS
reduce or inhibit the antibacterial activity of medicaments
and irrigants.

Conclusions
1. CHX has a wide range of activity against both Grampositive and Gram-negative bacteria.
2. CHX is an effective antifungal agent especially against
C. 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 the
RCS may reduce or inhibit the antibacterial activity of
CHX and MTAD.
5. It seems that that the mixture of CHX and Ca(OH)2 is
less effective than CHX alone.
6. Substantivity of MTAD and doxycycline has been
shown for up to 4 weeks.
7. Because CHX has favourable antimicrobial activity
and substantivity it should be used as a final rinse in
endodontic treatment protocols.
8. Owing to the greater activity against Gram-positive
than Gram-negative organisms, application of CHX as
intracanal medicament is preferred in retreatment cases
compared with initial endodontic infections.

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