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-

cal and dental fields, and its effectiveness has been demon-
In intra-oral cariogenicity tests, artificial caries are strated [4]. Many workers have demonstrated the
produced in vitro in teeth of human or animal origin. bactericidal [5], viricidal [6] and fungicidal [7] properties
These are subsequently used in the oral cavity of a human of sodium hypochlorite. Povidone–iodine has been shown
volunteer via intra-oral appliances for cariogenicity experi- to be effective against most bacteria [8], fungi [7], viruses
ments. Salivary microorganisms can survive for extended [6] as well as spores [3]. The effect of using these methods
time periods at room temperature [1], hence the potential of sterilisation on artificial enamel caries used in intra-oral
exists for the transfer of viruses, bacteria and spores present appliances has not been investigated.
in these materials to the volunteers. As a preventive The aim of this study, therefore, was to determine the
measure, these teeth are being sterilised before they are effect of the above sterilisation methods on the structural
used in intra-oral appliances. The means of sterilisation, in integrity of artificial enamel caries (white spot lesions) to be
addition to being effective, must not affect the structural used for intra-oral cariogenicity tests (ICT).
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
2. Materials and methods
lethal to all forms of microbial life [3]. Steam autoclaving is
Forty freshly extracted bovine permanent incisors were
* Corresponding author. Tel.: +44 0151 706 5288; Fax: +44 0151 706 polished with wet pumice to remove organic contaminants.
5809; e-mail: b.t.amaechi@liv.ac.uk Four experimental groups were devised and 10 teeth were
0300-5712/99/$ - see front matter 䉷 1999 Elsevier Science Ltd. All rights reserved.
PII: S0300-571 2(98)00064-5
314 B.T. Amaecha et al. / Journal of Dentistry 27 (1999) 313–316

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
Fig. 1. Effect of sterilisation methods on the structural integrity of artificial
caries in bovine permanent enamel as measured by (A) mean mineral loss 20 ml of brain heart infusion broth (Amersham, Bury,
and (B) mean lesion depth in specimens in each sterilisation group. (n ˆ England; Batch no. 007548) and vortexed vigorously.
10). (a) Gamma irradiation, (b) Steam autoclaving, (c) Sodium hypochlor- Each specimen was again transferred aseptically to a second
ite, (d) povidone–iodine. (*) Difference statistically significant. bottle also containing 20 ml of brain heart infusion broth.
The first set of broth bottles were labelled accordingly and
used in each group. Each tooth was coated with two layers incubated anaerobically (with 5% CO2, 5% H2 and 90% N2)
of acid resistant nail varnish except for an exposed enamel at 37⬚C. The second set of broth bottles containing the speci-
window (6 × 2 mm) on the labial surface of the tooth. mens were incubated aerobically in 4% CO2 at 37⬚C. All
Caries-like lesions were created by demineralisation of the media under both aerobic and anaerobic conditions were
samples in acidic buffer solutions containing 2.2 mM examined for evidence of growth (turbidity) for up to
KH2PO4, 50 mM acetic acid, 2.2 mM CaCl2 and 0.5 ppm 7 days. Aseptic procedures were strictly followed. Ten-
fluoride at a pH of 4.5 [9]. Following a 3-day exposure fold dilutions of the broth cultures showing growth were
period, nail varnish was removed with acetone, and each made in sterile saline down to 10 ⫺6 dilutions. 100 ml of
tooth was then cut vertically into two portions using a each dilution were plated out on to 5% (v/v) horse blood
water-cooled diamond wafering blade (Buehler, Warwick, agar plates (Amersham, Bury, England; Batch no. 006297)
 B.T. Amaecha et al. / Journal of Dentistry 27 (1999) 313–316 315

in duplicate. Plates from the first set of broth bottles were A significant softening of bovine enamel following
again incubated anaerobically and examined for growth for sterilisation by steam autoclaving was reported by Chandler
up to 7 days, while those from the second set of broth bottles [12] who observed that the changes in microhardness
were incubated aerobically for up to 48 h. All plates were recorded was comparable to those produced by some experi-
incubated at 37⬚C, and the presence of viable microorgan- mental cariogenic substrates. This may be responsible for
isms was verified by the growth of colonies on agar plates. the observed significant increase in mineral loss and lesion
After incubation, two sections were cut from each sample depth in the present study. This effect would invalidate the
using a water-cooled diamond saw (Well, Walter Ebner, Le result of any cariogenicity test in which the samples were
Locle, Germany) and polished to give planoparallel speci- sterilised by autoclaving.
mens of 80 mm thick using a diamond disc. Each section The observation of an increase in mineral loss following
was imbibed with water and examined under polarised light sterilisation of the artificial caries with sodium hypochlorite
using a Nikon, Optiphot light microscope (Nikon, Tokyo, may be attributed to the dissolution of some mineral follow-
Japan) at a magnification of × 450. Following this, micro- ing the treatment of calcified tissue with sodium hypochlor-
radiographs of the sections were produced as described in ite [13]. Treatment of enamel with sodium hypochlorite is
Amaechi et al. [10] Examination of the microradiographs also known to cause removal of organic materials by depro-
was carried out using a Leica DMRB microscope (Leica, teinisation [14] which may result in a decrease in lesion
Wetzlar, Germany), and the integrated mineral loss (vol% depth because of tissue contraction, hence the slight
mm) and lesion depth (mm) were quantified as described by decrease in lesion depth observed in this study. The removal
de Josselin de Jong et al. [11] The data from image analysis of organic components has been shown to cause an increase
were examined statistically using paired Student‘s t-tests, in permeability of the enamel [15] resulting in an improved
with a level of significance pre-chosen at 0.05. 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
3. Results test in which the test artificial caries was sterilised by
sodium hypochlorite may lack comparability with other
Gamma irradiation changed the colour of the enamel and
tests using alternative sterilisation methods. With the low
the caries lesions to a cream colour. There was an obvious
pH (5.3) of povidone–iodine this chemical would be
bleaching of the enamel and the lesions by sodium hypo-
capable of dissolving minerals from enamel/carious lesions,
chlorite. No observable macroscopic change on the lesion
and this may explain the increase in mineral loss observed
surface was observed following autoclaving or povidone–
with povidone–iodine in the present study. The possibility
iodine treatment. Microbial growth was observed only in
exists for penetration and retention of povidone–iodine or
control samples. Under the polarised light microscope, arti-
sodium hypochlorite inside the subsurface layer of the arti-
ficial caries created in all samples were observed to have
ficial caries lesion which would inevitably prevent the
sound surface layers and subsurface lesions, and there was
growth of bacterial plaque on the sterilised enamel block
no observable change in the outline and structure of these
in in situ caries models[17] used in de/remineralisation
lesions following sterilisation. Comparing the control (non-
studies. This is presently under investigation.
sterilised) with the sterilised samples, mineral loss was
Irradiation may have caused denaturation of the organic
significantly greater in sterilised specimens following auto-
component of the tooth which presented as cream disco-
claving ( p ⬍ 0:05) or NaOCl treatment ( p ⬍ 0:01).
louration of the enamel/artificial caries. The decrease in
Although not significant, numerical values of mineral loss
mineral loss and lesion depth in artificial caries following
were lower in sterilised specimens in the gamma group and
gamma irradiation may also be attributed to the effect of the
greater also in sterilised specimens in povidone–iodine
irradiation on the organic component of the enamel. Jansma
group (Fig. 1A). Lesion depth in sterilised specimens was
et al. [18] reported a decrease in the organic and water
greater in the autoclave group and lower in the other three
content of the enamel and hence tissue contraction, follow-
groups when compared with the control (Fig. 1B) the dif-
ing X-ray irradiation. This may also be applicable to gamma
ference being significant only with the autoclave group
irradiation. It is suspected that these effects were dose-
( p ⬍ 0:05).
dependent and the high dose of irradiation (25 KGy) used
in the present study, which is a typical dose for sterilisation
4. Discussion of hospital supplies [2], may be responsible for the present
observations. This is currently under investigation. As the
Although the four sterilisation methods were all effective observed decrease in mineral loss and lesion depth in this
for sterilisation of enamel artificial caries, some of them study is not significant, and considering the adverse effect of
may not be acceptable for this purpose considering their the other methods, gamma irradiation appears the most
effects on the lesion which may adversely affect the result acceptable means of sterilisation of enamel samples for
of the ICT. Some of these effects were not only observed in use in intra-oral appliances for cariogenicity tests.
this study but have also been reported by other workers. It can be concluded that the use of steam autoclaving,
316 B.T. Amaecha et al. / Journal of Dentistry 27 (1999) 313–316

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