IEJ366.
fm Page 184 Friday, March 9, 2001 10:01 AM
Inactivation of root canal medicaments by dentine,
Blackwell Science, Ltd
hydroxylapatite and bovine serum albumin
I. Portenier1, H. Haapasalo1, A. Rye1, T. Waltimo2, D. Ørstavik2 & M. Haapasalo1
1
Department of Endodontics, Dental Faculty, University of Oslo, Oslo; and 2Scandinavian Institute of Dental Material Research,
Haslum, Norway
Abstract
Portenier I, Haapasalo H, Rye A, Waltimo T,
Ørstavik D, Haapasalo M. Inactivation of root canal
medicaments by dentine, hydroxylapatite and bovine serum
albumin. International Endodontic Journal, 34, 184–188, 2001.
Aim This study examined and compared the inhibition
of the antibacterial effect of saturated calcium hydroxide
solution, chlorhexidine acetate and iodine potassium
iodide by dentine, hydroxylapatite and bovine serum
albumin.
Methodology Enterococcus faecalis strain A197A
prepared to a suspension of 3 × 108 cells per ml in 0.5%
peptone water was used. Fifty µL of saturated calcium
hydroxide solution, 0.05% chlorhexidine acetate or
0.2/0.4% iodine potassium iodide were incubated at
37 °C with 28 mg dentine powder (DP), hydroxylapatite
(HA) or bovine serum albumin (BSA) in 50 µL water for
1 h before adding 50 µL of the bacterial suspension.
Samples for bacterial culturing were taken from the
suspension 1 and 24 h after adding the bacteria. In further
experiments, the amount of dentine was stepwise reduced
from 28 mg 150 µL−1 to 2.8 mg 150 µL−1.
Results Calcium hydroxide was totally inactivated by
the presence of 28 mg of DP, HA or BSA. Chlorhexidine
Introduction
Elimination of the microbial infection in root canals
is essential in the treatment of apical periodontitis.
Chemomechanical preparation alone is not enough to
predictably eradicate all bacteria, and a small portion of
the flora survives (Byström & Sundqvist 1981, Gomes
et al. 1996). Therefore, local disinfecting agents are
Correspondence: Isabelle Portenier, Department of Endodontics,
Dental Faculty, PO Box 1109, Blindern, N-0317 Oslo, Norway
(fax: + 47 2285 2344; e-mail: isaport@odont.uio.no).
184 International Endodontic Journal, 34, 184–188, 2001
(0.05%) was strongly inhibited by BSA and slowed
down by dentine. However, HA had little or no inhibit-
ory effect on chlorhexidine. The antibacterial effect of
0.2/0.4% iodine potassium iodide on E. faecalis was
totally inhibited by dentine (28 mg), but was practically
unaffected by HA or BSA. A stepwise reduction of
dentine from 28 mg 150 µL−1 to 2.8 mg 150 µL−1 was
followed by a similar reduction of the inhibition of the
antibacterial activity of chlorhexidine. Iodine potassium
iodide was not inhibited at all with dentine amounts less
than 28 mg. However, the effect of saturated calcium
hydroxide solution was totally eliminated by dentine, in
all four concentrations.
Conclusion Inhibition by dentine of the anti-
bacterial activity of calcium hydroxide, chlorhexidine and
iodine potassium iodide occurs by different mechanisms.
Different components of dentine may be responsible
for the inhibition of these three medicaments. Calcium
hydroxide was particularly sensitive to inhibition by
both inorganic and organic compounds.
Keywords: bovine serum albumin, calcium hydroxide,
chlorhexidine, dentine, Enterococcus faecalis, hydro-
xylapatite, inactivation, iodine potassium iodide.
Received 19 January 2000; accepted 27 March 2000.
applied in the canal after preparation and left for
1–3 weeks to eliminate residual microbes. In primary
apical periodontitis, calcium hydroxide has proved to
be a reliable and effective means of canal disinfection
(Byström et al. 1985). However, several studies have
shown that 10 – 20% of primary lesions and 20 –50%
of the retreatment cases do not heal after root canal
treatment (Sjögren et al. 1990). Molander et al. (1998)
showed that Enterococcus faecalis was present in some
50% of the culture positive retreatment cases, and
Peciuliene et al. (1999) found E. faecalis in 71% of
culture positive teeth undergoing retreatment. In both
© 2001 Blackwell Science Ltd
IEJ366.fm Page 185 Friday, March 9, 2001 10:01 AM
studies, E. faecalis was often isolated in pure culture.
Earlier in vitro and in vivo studies have established that
E. faecalis tolerates high alkalinity well. E. faecalis may
survive in the root canal after calcium hydroxide treat-
ment (Sirén et al. 1997, Molander et al. 1998) and it is
possible that medicaments other than calcium hydroxide
may be more effective in the elimination of this species
from the root canal (Byström et al. 1985, Haapasalo &
Ørstavik 1987, Gomes et al. 1996, Molander et al. 1998).
Yeasts, which are more frequently found in retreatment
than in primary cases, are even more resistant than
E. faecalis to calcium hydroxide (Waltimo et al. 1997,
1999).
Calcium hydroxide has, however, remained a popular
local disinfectant in endodontics. Yet calcium hydroxide
may be less optimal for the treatment of previously
root-filled teeth with apical periodontitis, whilst other
medicaments may have greater potential (Molander et al.
1998). One of the assumed benefits of calcium hydroxide
is its long-lasting effect in the root canal. It is a commonly
held opinion that other medicaments may rapidly lose
their activity in the root canal environment. However, a
review of the literature does not give a clear picture of
inactivation of various medicaments, including calcium
hydroxide, in the root canal. The number of such studies
is surprisingly low (Engström 1958, Messer & Chen
1984). Studies by Möller (1966) indicated that the time
iodine zinc iodide and calcium hydroxide remain active
in the root canal is limited to a few days. One reason for
the lack of such studies may be the obvious difficulties
in designing experiments that will give reliable and
comparable data. Recently, Haapasalo et al. (1999) intro-
duced a new dentine powder model for studying the
inhibitory effect of dentine on various root canal medic-
aments. The results of the study showed that dentine
had the potential to inhibit all medicaments tested,
calcium hydroxide, chlorhexidine, sodium hypochlorite
and iodine potassium iodide, depending on the concen-
tration and time of contact.
The root canal milieu is a complex mixture of a variety
of organic and inorganic compounds. Hydroxylapatite,
the main component of dentine, is the major repres-
entative 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 is not known. In the present study, the
inhibition by dentine, hydroxylapatite, and bovine serum
albumin of the antibacterial activity of three commonly
used disinfectants in root canal treatment was studied.
© 2001 Blackwell Science Ltd
Portenier et al. Inactivation of medicaments
Materials and methods
Medicaments
The medicaments tested were (i) saturated calcium
hydroxide solution (Merck, Darmstadt, Germany)
(ii) 0.05% chlorhexidine acetate in water, obtained on
prescription from a pharmacist as a 0.5% stock solution
and (iii) 0.2/0.4% iodine potassium iodide solution,
obtained on prescription from a pharmacist as a 2/4%
stock solution. Sterile water was used as a negative control.
Test organism
A culture of E. faecalis A197A, isolated from a persist-
ent apical periodontitis (Sirén et al. 1997) and grown
overnight on TSA plates (Tryptic-soy-agar, Difco, MI,
USA), was checked for purity, suspended in 0.5%
peptone water (Bacto peptone, Difco) and adjusted
spectrophotometrically to give a cell density of 3 × 108
colony forming units (cfu) per mL for the stock solution.
Inhibitors
The substances tested for the inhibition of the anti-
bacterial activity of the medicaments were (i) dentine
powder (ii) hydroxylapatite (Bio-Rad Laboratories,
Richmond, CA, USA) and bovine serum albumin (Sigma
Chemical Co., St. Louis, MO, USA). All compounds were
used in powder form. Of each powder, 28 mg was
suspended in 50 µL water. The dentine powder was
prepared by crushing roots from human third molars
as previously described (Haapasalo et al. 1999), giving
a particle size of 0.2–20 µm.
Inhibition of the antibacterial effect of the medicaments
by the potential inhibitors
Fifty µL of the different inhibitor suspensions were thor-
oughly mixed and incubated with 50 µL medicament
in sealed test tubes at 37 °C for 1 h before adding 50 µL
of the bacterial suspension, giving a total volume of
150 µL. One control group consisted of 50 µL sterile
water instead of the inhibitor and the other control
group consisted of 50 µL sterile water instead of the
medicament. The suspensions were carefully mixed
and incubated at 37 °C in air. Ten-µL samples were taken
1 and 24 h after adding the bacteria. Serial 10-fold dilu-
tions were made of the samples, each dilution was care-
fully mixed, and droplets of 20 µL were cultured on TSA
plates for 24 h at 37 °C. The plates were inspected for
International Endodontic Journal, 34, 184–188, 2001 185
IEJ366.fm Page 186 Friday, March 9, 2001 10:01 AM

Inactivation of medicaments Portenier et al.

growth under a stereo-microscope and bacterial

colonies were counted. All experiments were completed
in triplicate.
In the second part of the experiment only dentine
powder was used. Here, the liquid volumes were increased
stepwise so that the concentration of the medicament,
as well as the bacterial cfu/mL, remained constant,
whereas the dentine powder concentration decreased
from 18.6 mg 100 µL−1 (28 mg 150 µL−1) to 1.86 mg
100 µL−1. The purpose of these additional experiments
was to assess the sensitivity of the selected medicaments Figure 1 Killing of E. faecalis A197A by saturated calcium
to inhibition by dentine. The experiments were performed hydroxide solution within 1 h and 24 h in the presence of
as above including the one-hour preincubation before dentine powder, hydroxylapatite (HA), or bovine serum
adding the bacteria, sampling at 1 h, serial 10-fold dilu- albumin (BSA). Sterile water was used as a control, replacing
either the disinfectant or the inhibitor in this and all
tions and culturing, and colony counting. As above, all
subsequent experiments.
experiments were done in triplicate.

Results
The antimicrobial effect of the three medicaments on
E. faecalis was variably inhibited by dentine, hydroxylapatite
and bovine serum albumin. Whilst water, used as a
control, showed no inhibition of calcium hydroxide that
reduced E. faecalis to 7% in 1 h and below detection limit
in 24 h, all three test inhibitors completely abolished the
antibacterial effect of calcium hydroxide (Fig. 1).
Hydroxylapatite showed only little inhibition of
chlorhexidine acetate (Fig. 2). On the other hand, the Figure 2 Killing of E. faecalis A197A by 0.05% chlorhexidine
inhibitory effect of dentine on chlorhexidine was marked acetate within 1 and 24 h in the presence of dentine powder,
1 h after adding the bacteria, and the number of viable hydroxylapatite, or bovine serum albumin.
cells was unchanged as compared to start and over 103
times more than without any inhibition of chlor-
hexidine (Fig. 2). However, after 24 h of incubation,
bacterial counts were reduced below the detection limit
in the presence of both dentine and HA. When BSA was
incubated, together with chlorhexidine and bacteria,
much of the antibacterial effect of chlorhexidine was
lost and even after 24 h of incubation more than 20% of
E. faecalis cells were viable.
Iodine potassium iodide lost all its activity against
E. faecalis when incubated with dentine (Fig. 3). However,
neither HA nor BSA alone showed inhibitory activity on
iodine potassium iodide: after 1 h less than 0.1% of cells Figure 3 Killing of E. faecalis A197A by 0.2/0.4% iodine
survived, and after 24 h no viable cells could be found potassium iodide within 1 and 24 h in the presence of dentine
(Fig. 3). powder, hydroxylapatite, or bovine serum albumin.
Control experiments, in which water replaced either
the disinfectants or the inhibitors, showed that water
had no antibacterial activity against E. faecalis or inhibit- hydroxide solution, as no change in bacterial counts
ory activity against the disinfectants. was detected (Fig. 4). With 0.05% chlorhexidine acetate
The reduction of the amount of dentine to one tenth the decrease in dentine concentration was followed by a
did not reduce its inhibitory effect on saturated calcium stepwise reduction of the inhibition (Fig. 4). With iodine

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IEJ366.fm Page 187 Friday, March 9, 2001 10:01 AM
Figure 4 Killing of E. faecalis A197A by saturated calcium
hydroxide, 0.05% chlorhexidine acetate or 0.2/0.4% iodine
potassium iodide within 1 h in the presence of declining
amount of dentine powder.
potassium iodide only the maximum (18.6 mg 100 µL−1)
concentration of dentine inhibited its antibacterial
activity (Fig. 4), whereas all lower amounts of dentine
failed to inhibit iodine.
Discussion
The results of the present study showed clearly that
different compounds were responsible for the inhibition
of the antimicrobial activity of the three medicaments
(Figs 1– 3). Surprisingly, calcium hydroxide was sensit-
ive to the inhibitory effect of all three materials tested.
The inhibition of calcium hydroxide by dentine and by
the other compounds is, of course, dependent on their
quantitative relationships. The supplemental experi-
ments in the present study showed that reducing the
amount of dentine to one-tenth of that used initially did
not reduce the inhibition. Calcium hydroxide is applied
in the canal as a paste. When the ions diffuse from the
paste into dentine, their concentrations will be equal to
or lower than in a saturated solution. Our earlier experi-
ments, measuring the effect of packed calcium hydrox-
ide in the main canal to the pH of surrounding dentine
have indicated that the effect may be limited (Sirén et al.
unpublished). Taken together, the present results and
those of earlier studies indicate that E. faecalis may
survive in the root canal filled with calcium hydroxide
because of the buffering effect of dentine against the pH
rise by alkaline materials (Wang & Hume 1988). One of
the key virulence factors of E. faecalis is that it is eco-
logically extremely tolerant. Calcium hydroxide paste,
due to its physical characteristics, effectively blocks
nutrients entering the root canal, which is detrimental
to most other bacteria. However, E. faecalis has been
shown to be able to survive even long periods with
nutritional difficulties.
© 2001 Blackwell Science Ltd
Portenier et al. Inactivation of medicaments
Inhibition experiments with chlorhexidine acetate
showed a quite different pattern. Inorganic hydroxylap-
atite had little or no inhibitory activity as compared to
dentine, whereas BSA was the strongest inhibitor of
chlorhexidine, with more than 10% of E. faecalis cells
still viable after 24 h of incubation with the medicament
(Fig. 2). The results may point to periapical inflammat-
ory exudate entering the root canal as a greater threat
to the activity of chlorhexidine than the dentine walls.
Although dentine seemed to slow down the effect of
chlorhexidine as measured at 1 h, after 24 h the bac-
teria were eliminated. This corroborates our previous
results (Haapasalo et al. 1999) and is consistent with
the concept of substantivity of chlorhexidine on dentine
surfaces as expressed by prolonged antibacterial activ-
ity of dentine after pretreatment with chlorhexidine
( Jung et al. 1995, Cuccia et al. 1995). An explanation
for these findings may be that binding of chlorhexidine
to dentine modifies but does not abolish its antibacterial
activity against E. faecalis. When dentine concentration
was reduced in three steps to one-tenth of the original
concentration, the inhibition of chlorhexidine was gradu-
ally decreased. Within the chosen concentration range,
0.05% chlorhexidine acetate was not nearly as sensitive
to inhibition by dentine as saturated calcium hydroxide
solution.
Iodine potassium iodide also showed a unique pattern
of inhibition by the three compounds. Dentine powder
(18.6 mg 100 µL−1) totally eliminated the antibacterial
effect of 0.2/0.4% iodine potassium iodide, which is in
agreement with our previous study (Haapasalo et al.
2000). However, the major component of dentine,
hydroxylapatite did not affect the antibacterial effect of
IKI, nor did BSA (Fig. 3). It is generally known that
blood rapidly inactivates the antibacterial activity of
iodine compounds. Further experiments are needed to
clarify the key compounds required for the inactivation
of iodine potassium iodide in the root canal. When the
dentine concentration was gradually reduced, it rapidly
lost its inhibitory effect on 0.2/0.4% iodine potassium
iodide, and all bacteria were killed in 1 h already at
13.1 mg 100 µL−1.
The results showed little variation within the parallel
settings. The dentine powder model has documented
a variable, but sometimes strong inhibitory effect by
dentine on root canal medicaments. The present experi-
ments with separate compounds available in the infected
root canal point to particular roles of the different or-
ganic and inorganic components. The resulting informa-
tion, highlighting the modes of inhibitory activity, aids
in the selection and usage of medicaments either alone
International Endodontic Journal, 34, 184–188, 2001 187
IEJ366.fm Page 188 Friday, March 9, 2001 10:01 AM
Inactivation of medicaments Portenier et al.
or in combination. Furthermore, it creates a basis for
the development of targeted treatment by locally used
medicaments in different types of endodontic infections.
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