Journal

of
Dentistry
Journal of Dentistry 27 (1999) 313–316

Short communication

Effect of sterilisation methods on the structural integrity of artificial

enamel caries for intra-oral cariogenicity tests
B.T. Amaecha*, S.M. Higham, W.M. Edgar
Cariology Research Group, Department of Clinical Dental Sciences, School of Dentistry, The University of Liverpool, Liverpool L69 3BX, UK
Received 20 February 1998; received in revised form 30 April 1998

Abstract
Enamel blocks bearing artificial caries are used in intra-oral appliances for cariogenicity tests. These blocks are often sterilised to prevent
the possibility of cross-infection via this route. This study therefore aimed to determine the effect of sterilisation methods on the structural
integrity of artificial enamel caries used for intra-oral cariogenicity tests.
Four experimental groups were devised. Ten bovine incisors were used in each group. Artificial caries was produced in each tooth which
was subsequently cut into two halves. One half of each tooth was reserved as control while the other was sterilised. The four groups were
subjected to respective sterilisation methods: gamma irradiation (艑25 KGy), steam autoclaving (121⬚C for 15 min), sodium hypochlorite
(12% w/v for 24 h) and povidone–iodine (7.5% w/v for 24 h). The control and sterilised specimens in each group were examined for
microbial growth after incubation in nutrient broth for up to 7 days at 37⬚C under aerobic and anaerobic conditions. Mineral loss and lesion
depth were quantified from microradiographs of sections from control and sterilised specimens using transverse microradiography. Data
were analysed statistically by paired Student‘s t-test.
Microbial growth was observed only in control specimens. Gamma irradiation and NaOCL caused cream discolouration and bleaching of
the enamel surface, respectively. Autoclaving, sodium hypochlorite and povidone–iodine resulted in further demineralisation of the
lesions.The four sterilisation methods were all effective sterilants for artificial caries. However, gamma irradiation appears the most
acceptable method considering the more adverse effects of the other methods with regards to cariogenicity tests. 䉷 1999 Elsevier Science
Ltd. All rights reserved.
Keywords: Artificial caries; Sterilisation; Gamma irradiation; Autoclave; Sodium hypochlorite; Povidone–iodine

1. Introduction the most efficient and popular means of sterilisation in medi-

In intra-oral cariogenicity tests, artificial caries are
produced in vitro in teeth of human or animal origin.
These are subsequently used in the oral cavity of a human
volunteer via intra-oral appliances for cariogenicity experi-
ments. Salivary microorganisms can survive for extended
time periods at room temperature [1], hence the potential
exists for the transfer of viruses, bacteria and spores present
in these materials to the volunteers. As a preventive
measure, these teeth are being sterilised before they are
used in intra-oral appliances. The means of sterilisation, in
addition to being effective, must not affect the structural
integrity of the artificially-produced lesion.
Gamma irradiation from a cobalt-60 source, the typical
method used for the sterilisation of hospital supplies [2], is
lethal to all forms of microbial life [3]. Steam autoclaving is
* Corresponding author. Tel.: +44 0151 706 5288; Fax: +44 0151 706
5809; e-mail: b.t.amaechi@liv.ac.uk
0300-5712/99/$ - see front matter 䉷 1999 Elsevier Science Ltd. All rights reserved.
PII: S0300-571 2(98)00064-5
cal and dental fields, and its effectiveness has been demon-
strated [4]. Many workers have demonstrated the
bactericidal [5], viricidal [6] and fungicidal [7] properties
of sodium hypochlorite. Povidone–iodine has been shown
to be effective against most bacteria [8], fungi [7], viruses
[6] as well as spores [3]. The effect of using these methods
of sterilisation on artificial enamel caries used in intra-oral
appliances has not been investigated.
The aim of this study, therefore, was to determine the
effect of the above sterilisation methods on the structural
integrity of artificial enamel caries (white spot lesions) to be
used for intra-oral cariogenicity tests (ICT).
2. Materials and methods
Forty freshly extracted bovine permanent incisors were
polished with wet pumice to remove organic contaminants.
Four experimental groups were devised and 10 teeth were
314 B.T. Amaecha et al. / Journal of Dentistry 27 (1999) 313–316
Fig. 1. Effect of sterilisation methods on the structural integrity of artificial
caries in bovine permanent enamel as measured by (A) mean mineral loss
and (B) mean lesion depth in specimens in each sterilisation group. (n ˆ
10). (a) Gamma irradiation, (b) Steam autoclaving, (c) Sodium hypochlor-
ite, (d) povidone–iodine. (*) Difference statistically significant.
used in each group. Each tooth was coated with two layers
of acid resistant nail varnish except for an exposed enamel
window (6 × 2 mm) on the labial surface of the tooth.
Caries-like lesions were created by demineralisation of the
samples in acidic buffer solutions containing 2.2 mM
KH2PO4, 50 mM acetic acid, 2.2 mM CaCl2 and 0.5 ppm
fluoride at a pH of 4.5 [9]. Following a 3-day exposure
period, nail varnish was removed with acetone, and each
tooth was then cut vertically into two portions using a
water-cooled diamond wafering blade (Buehler, Warwick,
UK). One portion from each tooth was reserved as a control
while the other was subjected to the following sterilisation
methods with respect to their group.
Samples in group 1 were immersed in 20 ml of sterile de-
ionised distilled water in a volumetric container and were
sterilised with gamma irradiation from a cobalt-60 source
with a particle energies of 0.315 Mev and irradiation expo-
sure of 3.4 Gy/min at a source to specimen distance of
100 cm and field size of 15 × 15 cm for 5 days resulting
in a total dose of approximately 25 KGy.
Samples in group 2 were packaged in an autoclave paper
sealed with autoclave tape, and were subjected to a steam
autoclaving (Series 32, model DS38, Rodwell, Hornchurch,
Essex, England) at 121⬚C for 5 min followed by 10 min air
drying at subatmospheric pressure.
All samples in group 3 were immersed in 200 ml of
sodium hypochlorite (pH 12.5) with 12% w/v available
chlorine (BDH, Poole, England), in a closed glass container
for 24 h at room temperature (approximately 20⬚C). Follow-
ing sterilisation, the samples were aseptically passed
through four bottles (each containing 400 ml) of sterile
de-ionised distilled water with thorough shaking to remove
residual sodium hypochlorite.
Samples in group 4 were immersed in 200 ml of aqueous
povidone–iodine (Betadine, Seton, Oldham, England) solu-
tion (pH 5.3) at concentration of 7.5% w/v in a closed glass
container for 24 h at room temperature. Following sterilisa-
tion, povidone–iodine was removed through three
processes. The samples were first thoroughly shaken in
400 ml of sterile de-ionised distilled water. This procedure
left some traces of iodine stains on the samples. The stains
were removed by immersion of the samples in 200 ml of
freshly prepared 0.1 N sodium thiosulphate solution (BDH,
Poole, England) for 15 min at room temperature. Samples
were finally passed through four bottles (each containing
400 ml) of sterile de-ionised distilled water to remove the
sodium thiosulphate. Complete removal of iodine was
confirmed by dropping one of the samples into a 1% starch
solution.
Following sterilisation, each specimen (both control and
sterilised) in every group was placed in a bottle containing
20 ml of brain heart infusion broth (Amersham, Bury,
England; Batch no. 007548) and vortexed vigorously.
Each specimen was again transferred aseptically to a second
bottle also containing 20 ml of brain heart infusion broth.
The first set of broth bottles were labelled accordingly and
incubated anaerobically (with 5% CO2, 5% H2 and 90% N2)
at 37⬚C. The second set of broth bottles containing the speci-
mens were incubated aerobically in 4% CO2 at 37⬚C. All
media under both aerobic and anaerobic conditions were
examined for evidence of growth (turbidity) for up to
7 days. Aseptic procedures were strictly followed. Ten-
fold dilutions of the broth cultures showing growth were
made in sterile saline down to 10 ⫺6 dilutions. 100 ml of
each dilution were plated out on to 5% (v/v) horse blood
agar plates (Amersham, Bury, England; Batch no. 006297)
 B.T. Amaecha et al. / Journal of Dentistry 27 (1999) 313–316
in duplicate. Plates from the first set of broth bottles were
again incubated anaerobically and examined for growth for
up to 7 days, while those from the second set of broth bottles
were incubated aerobically for up to 48 h. All plates were
incubated at 37⬚C, and the presence of viable microorgan-
isms was verified by the growth of colonies on agar plates.
After incubation, two sections were cut from each sample
using a water-cooled diamond saw (Well, Walter Ebner, Le
Locle, Germany) and polished to give planoparallel speci-
mens of 80 mm thick using a diamond disc. Each section
was imbibed with water and examined under polarised light
using a Nikon, Optiphot light microscope (Nikon, Tokyo,
Japan) at a magnification of × 450. Following this, micro-
radiographs of the sections were produced as described in
Amaechi et al. [10] Examination of the microradiographs
was carried out using a Leica DMRB microscope (Leica,
Wetzlar, Germany), and the integrated mineral loss (vol%
mm) and lesion depth (mm) were quantified as described by
de Josselin de Jong et al. [11] The data from image analysis
were examined statistically using paired Student‘s t-tests,
with a level of significance pre-chosen at 0.05.
3. Results
Gamma irradiation changed the colour of the enamel and
the caries lesions to a cream colour. There was an obvious
bleaching of the enamel and the lesions by sodium hypo-
chlorite. No observable macroscopic change on the lesion
surface was observed following autoclaving or povidone–
iodine treatment. Microbial growth was observed only in
control samples. Under the polarised light microscope, arti-
ficial caries created in all samples were observed to have
sound surface layers and subsurface lesions, and there was
no observable change in the outline and structure of these
lesions following sterilisation. Comparing the control (non-
sterilised) with the sterilised samples, mineral loss was
significantly greater in sterilised specimens following auto-
claving ( p ⬍ 0:05) or NaOCl treatment ( p ⬍ 0:01).
Although not significant, numerical values of mineral loss
were lower in sterilised specimens in the gamma group and
greater also in sterilised specimens in povidone–iodine
group (Fig. 1A). Lesion depth in sterilised specimens was
greater in the autoclave group and lower in the other three
groups when compared with the control (Fig. 1B) the dif-
ference being significant only with the autoclave group
( p ⬍ 0:05).
4. Discussion
Although the four sterilisation methods were all effective
for sterilisation of enamel artificial caries, some of them
may not be acceptable for this purpose considering their
effects on the lesion which may adversely affect the result
of the ICT. Some of these effects were not only observed in
this study but have also been reported by other workers.
315
A significant softening of bovine enamel following
sterilisation by steam autoclaving was reported by Chandler
[12] who observed that the changes in microhardness
recorded was comparable to those produced by some experi-
mental cariogenic substrates. This may be responsible for
the observed significant increase in mineral loss and lesion
depth in the present study. This effect would invalidate the
result of any cariogenicity test in which the samples were
sterilised by autoclaving.
The observation of an increase in mineral loss following
sterilisation of the artificial caries with sodium hypochlorite
may be attributed to the dissolution of some mineral follow-
ing the treatment of calcified tissue with sodium hypochlor-
ite [13]. Treatment of enamel with sodium hypochlorite is
also known to cause removal of organic materials by depro-
teinisation [14] which may result in a decrease in lesion
depth because of tissue contraction, hence the slight
decrease in lesion depth observed in this study. The removal
of organic components has been shown to cause an increase
in permeability of the enamel [15] resulting in an improved
uptake of calcium into the subsurface layer of a carious
lesion [14] with a consequent improvement in remineralisa-
tion [16]. With this effect, the result of a remineralisation
test in which the test artificial caries was sterilised by
sodium hypochlorite may lack comparability with other
tests using alternative sterilisation methods. With the low
pH (5.3) of povidone–iodine this chemical would be
capable of dissolving minerals from enamel/carious lesions,
and this may explain the increase in mineral loss observed
with povidone–iodine in the present study. The possibility
exists for penetration and retention of povidone–iodine or
sodium hypochlorite inside the subsurface layer of the arti-
ficial caries lesion which would inevitably prevent the
growth of bacterial plaque on the sterilised enamel block
in in situ caries models[17] used in de/remineralisation
studies. This is presently under investigation.
Irradiation may have caused denaturation of the organic
component of the tooth which presented as cream disco-
louration of the enamel/artificial caries. The decrease in
mineral loss and lesion depth in artificial caries following
gamma irradiation may also be attributed to the effect of the
irradiation on the organic component of the enamel. Jansma
et al. [18] reported a decrease in the organic and water
content of the enamel and hence tissue contraction, follow-
ing X-ray irradiation. This may also be applicable to gamma
irradiation. It is suspected that these effects were dose-
dependent and the high dose of irradiation (25 KGy) used
in the present study, which is a typical dose for sterilisation
of hospital supplies [2], may be responsible for the present
observations. This is currently under investigation. As the
observed decrease in mineral loss and lesion depth in this
study is not significant, and considering the adverse effect of
the other methods, gamma irradiation appears the most
acceptable means of sterilisation of enamel samples for
use in intra-oral appliances for cariogenicity tests.
It can be concluded that the use of steam autoclaving,
316 B.T. Amaecha et al. / Journal of Dentistry 27 (1999) 313–316
sodium hypochlorite or povidone–iodine as a means of
sterilisation of enamel blocks with artificial caries for
intra-oral appliances may not be acceptable considering
the drawbacks associated with their application. Gamma
irradiation may be acceptable to the structural integrity of
the enamel blocks provided all the samples are sterilised by
the same irradiation dose. Further investigations are under
way to determine the effects of these sterilisation methods
on the growth of bacterial plaque on the enamel blocks in in
situ caries models.
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