JOURNAL OF ENDODONTICS
Copyright 2004 by The American Association of Endodontists
BASIC RESEARCHTECHNOLOGY
Preliminary Evaluation of Bioactive Glass S53P4 as
an Endodontic Medication In Vitro
Matthias Zehnder, DMD, Eva Sderling, PhD, Jukka Salonen, DDS, PhD, and
Tuomas Waltimo, DDS, PhD
Calcium hydroxide is the gold standard end-
odontic medication, but it may fail to eliminate
certain facultative bacteria and yeasts found in
root canal systems. In this study, standardized
bovine dentin blocks infected with Enterococcus
faecalis were treated with an aqueous calcium
hydroxide or bioactive glass S53P4 (BAG) pow-
der suspension. While calcium hydroxide was in-
effective, the BAG suspension eliminated the in-
fection in the sampled dentin layers after 5 days.
In a direct exposure test, preincubation with hu-
man dentin boosted the BAG-killing efficacy
against E. faecalis ATCC29212, Candida albicans
CCUG19915, Pseudomonas aeruginosa
ATCC9027, Streptococcus sanguis ATCC10556,
and S. mutans ATCC25175. BAG placed in the
root canals of extracted teeth did not alter root-
dentin pH. Consequently, the conditions under
which the microbiota were killed were not pH-
mediated. Further studies are ongoing to help
clarify the antimicrobial BAG effect in the pres-
ence of dentin.
All endodontic infections are not of a similar nature. Mixed cul-
tures of Gram-negative anaerobes predominate primary endodontic
infections (1). In contrast, the flora in teeth with failed root canal
treatments may consist mainly of facultative bacteria. Enterococ-
cus faecalis is isolated from roughly 50% to 70% of culture-
positive retreatment cases (2, 3).
In primary endodontic infections, an aqueous calcium hydroxide
Ca(OH)2 suspension is advocated over other common intracanal
medications because of its ability to render the root canal system
free of cultivable bacteria (4). However, calcium hydroxide may
not be the medication of choice for endodontic retreatments.
E. faecalis can not be eliminated by calcium hydroxide from
dentinal tubules (57) because of its high tolerance for an alkaline
220
Printed in U.S.A.
VOL. 30, NO. 4, APRIL 2004
environment (4), which is because of the proton pump in this
species (8). The high buffering capacity of dentin (9) causes root
dentin pH levels to drop with increasing distance from the hydrox-
ide ion source, i.e. the calcium hydroxide medication (10).
One major advantage of a calcium hydroxide medication is its
ability to remain active for extended time periods in the root canal
system (11). Calcium hydroxide has low solubility in water. In the
root canal environment, undissolved Ca(OH)2 in a paste-like aque-
ous suspension will steadily dissolve, resulting in a sustained pH
effect (10, 11). Recent approaches to mix calcium hydroxide with
irrigating solutions to obtain a better effect against facultative
bacteria and yeasts (12) may be hampered by the limited long-term
action of irrigants (13).
This pilot study was designed to test the efficacy of a slow-
release antimicrobial agent, bioactive glass S53P4 (BAG) in an
aqueous suspension (14), to kill E. faecalis in dentinal tubules.
Based on the findings of this preliminary test, the efficacy of BAG
was assayed against different microorganisms in the presence of
human dentin in suspension. Finally, because the antimicrobial
effect of bioactive glass has been attributed to its ability to create
an alkaline environment (15), pH changes in root dentin were
evaluated in extracted teeth treated with a bioactive glass or cal-
cium hydroxide suspension.
MATERIALS AND METHODS
Materials
The bioactive glass (BAG) powder used in this study was S53P4
(AbminDent 1, Lot AD1-99/04, Abmin Technologies Ltd, Turku,
Finland, U.S. Patent 6,190,643 and 6,342,207). It is composed of
53% SiO2 (w/w), 23% Na2O, 20% CaO, and 4% P2O5, and was
prepared from reagent grade Na2CO3, CaHPO4 2H2O, CaCO3
(Merck, Darmstadt, Germany) and Belgian sand as described else-
where (14). The glass powder was sieved to a particle size of 45
m, with mean diameter of approximately 20 m. Human dentin
powder with an average particle size of 110 m was prepared from
desiccated extracted third molar roots, ground in a ball grinder for
2 min (MM 200, Retsch, Haan GmbH, Germany), as previously
described (16). Particle sizes were determined in aqueous suspen-
Vol. 30, No. 4, April 2004
sions using laser diffraction analysis (CILAS 1064, Marcoussis,
France). In bovine dentin blocks approximately 4-mm high and
6-mm wide, a standardized central pulpal lumen diameter of 3.1
mm was prepared by widening the original pulp space with an ISO
031 bur as previously described (17). For the pH measurements,
single-rooted teeth of similar shape were selected from the depart-
ments collection of extracted teeth. The protocol of this study did
not in any way influence the treatment plan of any patient, nor did
it alter the premortal treatment or the slaughtering process of the
calves from which the teeth for the dentin block assay were
collected. Informed consent was obtained from all patients before
the extractions, which were performed according to each subjects
treatment plan. Therefore, this study was conducted in accordance
with the ethical guidelines for medical research of the Canton of
Zrich, Switzerland and the declaration of Helsinki.
Dentin Block Assay
Thirty-nine standardized bovine dentin blocks were used for this
assay: 36 were used for the disinfection experiments, and 3 blocks
were used to verify bacterial penetration before disinfection. Or-
ganic and inorganic debris, including smear layer, were removed
from the dentin blocks by ultrasonic treatment in 17% EDTA for
10 min. The specimens were then sterilized by autoclaving (121C,
15 min) in 50-ml tryptic soy broth (TSB, Oxoid, Hampshire,
England). This procedure allows optimal penetration of the nutri-
ent broth into the dentinal tubules, as shown in pilot experiments.
Subsequently, the TSB containing the dentin blocks was seeded
with E. faecalis ATCC 29212 by transferring five colonies of
overnight culture of this microorganism on tryptic soy agar (TSA,
Oxoid). Blocks were kept in TSB at 37C for 2 weeks during which
the broth was regularly changed at 2- to 3-day intervals. Strict
asepsis was maintained to avoid contamination, and the purity of
the cultures was checked regularly based on colony morphology
and cellular appearance of subcultures on TSA. Penetration of the
dentinal tubules by the bacteria was verified in three randomly
selected blocks by scanning electron microscopy (6).
The infected dentin specimens were taken from their vials and
washed in sterile saline to remove any remnants of the incubation
broth. Blocks were then medicated with a 1:1.5 (wt/vol) mixture of
Ca(OH)2 powder (Merck, Darmstadt, Germany) with unbuffered
isotonic saline solution (0.9% NaCl), a 1.5:1 (wt/vol) mixture of
bioactive glass with saline, or saline alone (negative controls). The
mixing ratios were chosen for the suspensions to have a paste-like
consistency, which easily can be applied. Medications were placed
in the canal lumina and on the top and bottom surfaces of the
blocks. Subsequently, the blocks sealed with paraffin and incu-
bated at 37C in sterile airtight containers. Bacteriologic samples
were taken after 24 h (calcium hydroxide, BAG), 3 days (BAG),
and 5 days (calcium hydroxide, BAG, and negative controls).
Before sampling, medications were carefully rinsed from the dentin
blocks using sterile saline solution. Dentin samples were then har-
vested by drilling with round burs, ranging in size from ISO 033 to
040 (i.e. 450 m into the dentin) through the canal lumen. The dentin
powder was collected in separate test tubes containing 2 ml of TSB
and incubated for up to 3 days to detect bacterial growth. Purity of the
growth was controlled by subculturing 100 l from each test tube on
TSA, followed by examination of colony morphology and cellular
appearance. These experiments were repeated five times each.
Bioactive Glass Disinfects Dentin 221
Killing Efficacy of BAG in the Presence of Dentin Powder
To evaluate antimicrobial activity of BAG in the presence of
human dentin, a modification of a previously described protocol
was used (16). Briefly, two freshly prepared suspensions, sterile
BAG in sterile water (1:1.5 wt/vol) and sterile dentin powder in
sterile water (1:2 wt/vol) were thoroughly mixed together (1:1
vol/vol) and incubated at 37C for 24 h. E. faecalis ATCC29212
and reference strains of other common root canal isolates: Pseudo-
monas aeruginosa ATCC9027, Streptococcus sanguis
ATCC10556, S. mutans ATCC25175, and Candida albicans
CCUG19915 were chosen for this experiment. Spectrophotometri-
cally standardized suspensions of these microorganisms were pre-
pared in 0.9% NaCl (approximately 2 107 colony-forming units
(CFU)/ml for bacteria and 2 106 CFU/ml for yeasts). These were
mixed (1:1 vol/vol) with the BAG or the BAG dentin suspension
described earlier, or with sterile 0.9% NaCl and incubated at 37C
for 100 min. Ten-fold serial dilutions down to 105 were made in
saline, and aliquots of 20 l were cultivated from each dilution on
TSA (Oxoid) and incubated at 37C for 24 h. All experiments were
repeated twice. Colonies were counted and CFUmean SD /ml
were calculated. To equalize the differences between the species,
the results are presented as percentages of log10 values, NaCl-
control representing 100%.
pH Measurements
For pH measurements, 24, single-rooted, human teeth of similar
shape were randomly divided into three groups of eight teeth each.
Teeth were instrumented and prepared as described (18). A stan-
dardized well, 0.75-mm deep, 3-mm long, and 1.5-mm wide, was
prepared in the middle third of the buccal aspect of each root, with
a 1.5-mm diameter diamond round bur. The teeth were placed into
individual vials containing unbuffered isotonic saline solution.
After air-drying the teeth and sealing the apices with glass ionomer
cement (Ketac, ESPE, Seefeld, Germany) and nail varnish, the
prepared root canals were filled with Ca(OH)2 mixed with 0.9%
NaCl at a 1:1.5 ratio (wt/vol), 0.9% NaCl as a control, or BAG
mixed with 0.9% NaCl (1.5:1, wt/vol). Pastes were applied with a
lentulo spiral (Maillefer, Ballaigues, Switzerland) and down-
packed under visual control using Schilder Pluggers (Henry
Schein, Konstanz, Germany). Measurements of pH were per-
formed immediately after filling and then after 1 day, 2 days, 1
week, 2 weeks, and 3 weeks after filling. This procedure was
performed as follows: teeth were removed from their vials, cooled
to room temperature in a 100% humid atmosphere, and carefully
dried with compressed air (GEPE-air, Image Trade, Safenwil,
Switzerland). Two microliters of unbuffered, isotonic, saline so-
lution was placed into the standardized well. After an equilibration
time of 5 min, pH was measured using a calibrated microelectrode
(MI 415-2, Microelectrodes Inc., Bedford, NH). Subsequently, the
teeth were returned to their vials filled with fresh saline and
incubated further at 37C.
Nonparametric statistics were used to compare pH scores at
the root surface between groups: Kruskal-Wallis one-way analy-
sis of variance followed by Mann-Whitney U test for individual
comparisons. Bonferroni adjustment was applied for multiple
post-hoc testing. Levels for rejection of null hypotheses were set at
p 0.05.
222 Zehnder et al. Journal of Endodontics

TABLE 1. Dentin disinfection of E. faecalis-infected calf tooth blocks

24 h Ca(OH)2
Depth (m)
Block 1 Block 2 Block 3 Block 4 Block 5 Block 6
100
200
300
450

24 h BAG
Block 7 Block 8 Block 9 Block 10 Block 11 Block 12
100
200
300
450

3 day BAG
Block 13 Block 14 Block 15 Block 16 Block 17 Block 18
100
200
300
450

5 day Ca(OH)2
Block 19 Block 20 Block 21 Block 22 Block 23 Block 24
100
200
300
450

5 day BAG
Block 25 Block 26 Block 27 Block 28 Block 29 Block 30
100
200
300
450
The samples were incubated in TSB at 37C up to 5 days. Dentin was sampled with ISO burs of increasing size: no growth, sparse growth, moderate growth, vigorous
growth.

FIG 1. Survival SD of different microorganisms in suspensions of bioactive glass S53P4, bioactive glass preincubated with dentin for 24 h,
and saline control (representing 100%). Incubation at 37C for 100 min.

RESULTS
Dentin blocks infected with E. faecalis for 2 weeks and then
dressed with saline solution for 5 days (negative controls) showed
vigorous growth in all sampled dentin layers. The calcium hydroxide
treatment had only limited effectiveness against E. faecalis in the
tested dentin layers both after 1 day and after 5 days (Table 1).
Similarly, little efficacy against E. faecalis was observed with a
bioactive glass S53P4 dressing for 1 day. After 3 days of incubation,
one of six blocks dressed with BAG was disinfected in all sampled
dentin layers; the other five blocks remained contaminated. After 5
days, however, the BAG medication disinfected all of the sampled
dentin layers in all of the six test blocks completely.
On direct exposure in aqueous suspension for 100 min, BAG
had no effect against E. faecalis, S. sanguis, or S. mutans under
the conditions of the current study (Fig. 1). Some killing effect
of BAG was observed on C. albicans and P. aeruginosa. When
BAG was preincubated with dentin, its antimicrobial action was
boosted.
In the BAG dentin samples, the percent log10 CFU value of
E. faecalis compared with control was 60 3%, which equals
a reduction of more than 99% in absolute CFU counts. With the
Vol. 30, No. 4, April 2004
FIG 2. pH values measured in standardized wells in root surfaces of extracted teeth dressed with a calcium hydroxide suspension, a bioactive
glass S53P4 suspension, or saline, plotted as a function of time.
other tested microorganisms, no viable bacteria were detected in
dentin BAG suspensions.
When root canals were filled with a BAG suspension, no in-
crease above control pH levels was observed in the outer root
dentin during the observation period of 3 weeks (Fig. 2). In
contrast, median pH levels in the calcium hydroxide group were
significantly (p 0.05) increased compared with controls after 14
(median, 8.9; range, 7.59.8 versus median, 7.7; range, 7.57.9)
days and 21 days (median, 8.8; range, 7.8 9.9 versus median, 7.6;
range, 7.57.8).
DISCUSSION
Antimicrobial efficacy of bioactive glass S53P4 against E. fae-
calis in dentinal tubules was better than that observed with
Ca(OH)2 in the current study. The dentin block assay is commonly
used to test endodontic antiseptic irrigants and medications. An
aqueous calcium hydroxide suspension, the gold standard end-
odontic medication, was used as a control. As previously shown
(6), survival of the alkali-resistant E. faecalis in dentinal tubules
was only marginally affected by the presence of Ca(OH)2 in the
canal lumen. This may be explained by the buffering effect of
dentin (9). In contrast, the aqueous bioactive glass S53P4 suspen-
sion had a substantial late-onset effect against E. faecalis in den-
tinal tubules, similar to the one observed with Ca(OH)2 on anaer-
obic bacteria in the root canal system (19). Therefore, like calcium
hydroxide, BAG seems not to be an immediately effective bacte-
ricidal agent such as iodine potassium iodide, sodium hypochlorite,
or chlorhexidine (6), but rather a slowly acting, antimicrobial
material with a long-lasting effect. This feature may be of impor-
tance for an intracanal medication.
All common endodontic medications, including calcium hy-
droxide, and antiseptic irrigants tested in the dentin powder model
are inhibited when preincubated with dentin (16, 20, 21). Surpris-
ingly, under the conditions of the present study, BAG effectiveness
on direct exposure for 100 min against several oral microorganisms
associated with endodontic failures or dental caries was increased
by preincubation with dentin powder. BAG alone had little or no
effect on the bacteria.
The BAG S53P4 suspensions used in this study had a pH of 11
because of the release of sodium oxide from the glass surface on
wetting. The antimicrobial action of BAG has been attributed to its
ability to increase the pH in bacterial suspensions (15). However,
unlike Ca(OH)2, BAG has a low alkaline buffering capacity. Be-
cause of the high buffering capacity of dentin, the results obtained
with BAG dentin suspensions may not be explained by a simple
Bioactive Glass Disinfects Dentin 223
pH effect (9). This was substantiated by the pH measurements on
the exposed root dentin surfaces. In addition, in the direct exposure
test, pH values in the BAG dentin suspensions were not higher
than in the BAG suspensions alone. Currently, the mechanism for
the increased killing efficacy of BAG in the presence of dentin is
speculative. In the presence of excess Ca(II) and P(V) in a BAG
suspension, Ca/P precipitates were observed not only on glass
surfaces but also up to 2-mm away from the material surface in
vitro (22). Dentin may serve as a source of Ca and P ions, which
allow the BAG to mineralize the bacteria, i.e. form Ca/P pre-
cipitates on their surface, thereby destroying their cellular integrity.
However, further research is necessary to confirm or disprove this
theory. In this context, it should be mentioned that the BAG to
dentin ratio, absolute number of bacteria, growth phase of the
microorganisms, incubation time, and other conditions affect the
killing efficacy of BAG dentin (data not shown).
In conclusion, it may be stated that the current results are new,
surprising, and definitely worth exploring further. If the BAG
effect can be observed in vivo and its mechanism revealed, new
approaches to treat endodontic and periodontal infections and/or
dental caries may evolve. However, it must be reiterated that the
results were obtained in an ideal environment in vitro. It is,
therefore, premature to draw any conclusions regarding the action
of BAG in situ. Blood, tissue remnants, occluded dentinal tubules,
and many other confounding factors may affect the effectiveness of
BAG in the root canal system.
The authors thank Beatrice Sener and Pasi Juntunen for technical assis-
tance, and Prof. Markus Haapasalo and Dr. Fred Barbakow for their construc-
tive criticism.
Dr. Zehnder is affiliated with the Division of Endodontics, Department of
Preventive Dentistry, Cariology, and Periodontology, University of Zrich Cen-
ter for Dental Medicine, Zrich, Switzerland. Drs. Sderling and Waltimo are
affiliated with the Institute of Dentistry, University of Turku, Turku, Finland. Dr.
Salonen is affiliated with the Turku Centre for Biomaterials, Turku, Finland. Dr.
Waltimo is affiliated with the Department of Preventive Dentistry and Oral
Microbiology; University of Basel Center for Dental Medicine, Basel,
Switzerland.
Address requests for reprints to Dr. Matthias Zehnder, Division of End-
odontics, Department of Preventive Dentistry, Cariology, and Periodontology,
University of Zrich Center for Dental Medicine, Plattenstrasse 11, CH 8028
Zrich, Switzerland. E-mail zehnder@zzmk.unizh.ch.
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