IN VIVO CARIES M O D E L S -
MECHANISMS FOR CARIES INITIATION AND
ARRESTMENT
A. THYLSTRUP
C. BRUUN
L. HOLMEN
Department of Cariology and Endodontics
School of Dentistry, Health Science Faculty
University of Copenhagen
20 N0rre Alle
DK-2000 Copenhagen N, Denmark
Adv Dent Res 8(2): 144-157, July, 1994
Abstract—The effects of intra-oral mechanical forces on
caries initiation, progression, and arrestment are evaluated by
examination of different in vivo caries models. The models are
grouped in four categories: (1) a population study, (2) short-
term clinical trials, (3) clinical experiments, and (4) controlled
clinical observations. Taken together, these in vivo studies
convincingly demonstrate that partial or total elimination of
the intra-oral mechanical forces operating during mastication
or toothbrushing leads to evolution of cariogenic plaque,
resulting in localized carious enamel dissolution. In addition,
they show that re-exposure to the partly or totally eliminated
mechanical forces not only arrests further lesion progression,
but also results in partial lesion regression. The data from in
vivo caries studies also show that the clinical and structural
changes associated with lesion arrestment or partial regression
are not related to any salivary repair mechanism, but are solely
the result of mechanical removal of the cariogenic biomass
which is physically interrelated with the eroded surface of the
active, dull-whitish enamel lesion. No indications of superficial
mineral deposition or "blocking" of the external intercry stalline
spaces are seen in the surface layer of lesions arrested in vivo.
For this reason, the conventional usage of the terminology
'remineralization' is considered absolutely misleading when
used to describe the mechanisms responsible for the arrest of
lesion progression in vivo.
This manuscript was presented at a Symposium entitled
"Mechanisms and Agents in Preventive Dentistry", held
October 28-November 1, 1992, in Chester, England, under
the auspices of the Council of Europe Research Group on
Surface and Colloid Phenomena.
Printing of the color plate is supported by Colgate-Palmolive, Denmark.
144
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C
aries research has for many years been almost
synonymous with fluoride research, and relatively little
attention has been devoted to the pathology of dental
caries compared with the multitude of laboratory studies
on fluorides during the past 20 years. It is therefore not
surprising that current discussions on the anti-caries mechanisms
of fluoride are based almost entirely on the wealth of clinical
trial data and in vitro studies. There is no doubt that the many
different approaches that mimic carious dissolution in the
laboratory have provided a great deal of insight concerning the
physico-chemical processes operating during enamel
demineralization, which eventually leads to formation of a
visible lesion, often simply referred to as the "white spot"
lesion. Considering, however, past and ongoing intensive
attempts to unravel the mystery of dental caries, it is surprising
that this apparently very important stage in the disease process
is considered as simply "a white spot". According to the
literature, this "white spot" lesion undergoes several changes
in the clinic or in vivo as well as in the laboratory. It may thus
be transformed into a "brown spot lesion", a remineralized
lesion, an arrested lesion, and occasionally it even disappears
completely as a "caries reversal".
The explanatory models proposed on the basis of laboratory
experiments are, for obvious reasons, focusing on the few
parameters which have been strictly controlled in the
experiments. The essential processes appear to be dissolution
and re-precipitation of the mineral phase of enamel, often
described as demineralization and remineralization processes.
Extrapolation of the simplistic laboratory explanatory models
to the complicated in vivo situation requires at least, however,
that the terminology of the essential issues cover the same
processes or phenomena. Most important is, of course, whether
the widely used, but vaguely defined, stage during caries
development, designated the "white spot" lesion, actually
covers a well-defined concept among the users of the
terminology. It is equally important to consider whether the
intra-oral mechanisms leading to initiation of the "white spot"
lesion and to its progression, arrestment, or "reversal" are
similar in the laboratory and in vivo.
By a review of in vivo caries models, this paper will attempt
to clarify and discuss essential concepts and mechanisms
involved in vivo in caries initiation, progression, and arrestment.
The studies are described in four categories: a population
study, short-term clinical trials, clinical experiments, and
controlled clinical observations. The final part combines the
experiences arising from these studies and discusses definitions
and terminology on the basis of an updated view of the "white
spot" lesion.
VOL. 8(2)
A POPULATION STUDY
In 1966, Backer Dirks published his classic work on
"Posteruptive Changes in Dental Enamel". He examined about
90 boys and girls yearly at ages 7 to 15 years. The results were
reported in three categories: fissures and pits, proximal surfaces,
and free smooth surfaces. The results concerning the readily
visible buccal surfaces are of particular interest, because a
more detailed examination procedure was used than that
performed on the other surfaces. The free smooth surfaces
were first cleaned with a toothbrush, and then the surfaces were
scraped with the side of an explorer. Each surface was dried
with compressed air for about 3 s. The surfaces were classified
in three categories: "sound", "caries white" (if the surface
showed a white opaque lesion with or without a partial loss of
surface gloss), and "carious cavity" (if there was a break in the
continuity of the enamel perceptible with the explorer). The
Table summarizes the fate of the same buccal surface of
maxillary first molars from age 8 to age 15. Only 9 of the 72
opaque spots progressed to a cavity stage. More than half of the
surfaces with white opaque spot lesions were diagnosed as
sound at age 15. The change of a surface with a "white spot"
lesion to a surface clinically not different from sound took
place at all ages—from 8 to 9, lOto 11,12 to 13,and 14to 15—
and it occurred, respectively, in 11, 10, 12, and 4 surfaces (=
37). In the discussion part, it is stated that white spot lesions in
buccal surfaces develop soon after eruption, since 48% were
seen within half a year after eruption and 84% within 172 years
after eruption. Backer Dirks explains this phenomenon by
quoting Hardwick (1965): "when a tooth is erupting, temporarily
there is an area of high acidity near the gingival margin."
The disappearance of the white opaque lesions in the buccal
surfaces was considered to take place by either remineralization
or surface abrasion, or both. Backer Dirks discusses the
possibility that recrystallization may explain the observed
changes, but he also adds that the further eruption of the first
molar after the age of 8 signifies better usage, and thus also
better natural cleaning by mastication, leading to non-
progression and disappearance of buccal smooth surface lesions.
The marked environmental changes taking place from year 7
to year 15 in the gingival level of the buccal surface of
maxillary first molars are illustrated in Fig. 1. During this
period, there is gradual physiologic peeling off of the gingival
attachment from the surface of the tooth and continuing exposure
of the clinical crown. Also during this period, the second
maxillary molar erupts leading to a further re-position of the
gingival attachment of the distal first molar part. Thus, the
physiologic passive exposure of the crown leads to a change in
local conditions for plaque accumulation. It is therefore possible
to conclude from this study that favorable conditions for
accumulation along the gingival margin of erupting first
maxillary molars lead to early development of "white spot"
lesions. Further eruption leads to changes in the local
environment which favor mechanical removal or suppression
of cariogenic plaque, causing either arrestment of further
lesion progression or complete disappearance of lesions.
Another approach to study the impact of oral mechanical
forces on caries development is to withdraw toothbrushing in
controlled periods.
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IN VIVO CARIES MODELS 145
TABLE
DISTRIBUTION OF BUCCAL SURFACES OF
MAXILLARY FIRST PERMANENT MOLARS IN
THREE CATEGORIES OF DIAGNOSIS AT AGE 8 AND
AGE 15 OF THE SAME SURFACES
Diagnosis Age Total
8 15
Sound 74 111
37
White spot 15
lesion 41
26
\
Caries with
cavitation 32
k
19
184
From Backer Dirks (1966)
SHORT-TERM CLINICAL TRIALS
On the basis of previously conducted studies on an experimental
model for examination of initiation of gingivitis in humans,
von der Fehr et al (1970) developed a similar model for
experimental human caries studies. Twelve male dental students
volunteered for the experiments. During a pre-experimental
period, sound gingival conditions were established. The tooth
surfaces were carefully cleaned and thoroughly dried and
given a score: (0) surface appears intact; (1) limited greyish
tinge, with and without accentuated perikymata; (2) perikymata
well-accentuated, in some areas forming confluent greyish-
white spots; and (3) pronounced white decalcification. The
recording was made by means of a binocular dissection
microscope fitted with two spotlights (xl6). All participants
then refrained from oral hygiene procedures during 23 days.
Six participants were assigned to a sucrose group, which
performed 9 daily mouthrinses with 10 mL of a watery solution
of sucrose. The two-minute rinses were carried out between
meals. At the end of the "no hygiene" period, the teeth were
carefully cleaned and polished, and re-examined for caries.
Oral hygiene was re-instituted, and the participants performed
daily mouthrinses with a 0.2% NaF solution. After one month,
the teeth were cleaned and polished, and caries examination
was performed. After another month with oral hygiene and F
rinses, the final caries examination was performed after the
teeth were cleaned and polished, and the experiment was
terminated.
At the end of the "no hygiene" period, the mean Caries
Index increased in both groups, but the index of the sucrose
group was considerably higher than that of the control group
(Fig. 2). The mean Caries Index scores varied within the
dentition, however, at the start as well as at the end of the "no
hygiene" period, where lower first premolars showed the
greatest absolute caries increment, closely followed by the
upper canines, and the lateral and central incisors. At the end
146
Figs, la (top) and b (bottom)—(la) Erupting maxillary first
permanent molar with the buccal surface portion partially
covered by gingival tissue. Along this margin, Backer Dirks
(1966) observed early signs of caries due to favorable
conditions for plaque accumulation, (lb) In an individual at
age 15, physiologic passive exposure of the crown and the
eruption of the second permanent molar have altered local
conditions for plaque accumulation, leading to better
natural cleaning by mastication.
of the two periods with resumed oral hygiene and F, the mean
index returned to pre-experimental levels.
The study showed that omission of daily mechanical removal
or disturbance of bacterial accumulations on teeth leads to
formation of cariogenic plaque, causing development of early
signs of bucco-gingival enamel demineralization, and that
daily addition of sucrose rinses between meals further
accelerates caries progression. The following period with daily
mechanical plaque control in addition to three professionally
performed tooth cleanings and F rinses showed not only an end
of caries progression, but also that the clinical signs of enamel
dissolution returned to normal levels. In principle, identical
results were obtained in a study in which a similar experimental
design was used (Loe et al., 1972).
In a preliminary re-examination of the experimental caries
method proposed by von der Fehr et al. (1970), Jenkins et al.
(1973) were unable to confirm the need for frequent sucrose
rinses in inducing caries-like changes in enamel. Controls not
rinsing with sucrose showed an equally large rise in Caries
Index scores, suggesting that, with increased plaque
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THYISTRUP ET AL. ADV Dm RES Mr 1994
Caries
Index
1.0
Sucrose
0.8
0.6
Pre-experimental No Hygiene Hygiene • F " Hygiene • F "
0.4 -II-
4
Months
Fig. 2—Mean Caries Index scores for bucco-gingival areas
throughout the experiment (excluding molars and
mandibular incisors). Data from von der Fehr et al., 1970.
accumulation, dietary carbohydrates alone sufficed to produce
a maximal change. Geddes etal. (1978) therefore repeated the
experiment with minor technical modifications and used a 14-
day experimental period, which gave adequate change in
Caries Index (Edgar etal., 1978). The results of the new study
confirmed the original findings of von der Fehr et al. (1970),
since mean Caries Index scores rose during the period without
dental hygiene, and the rise was highest in the group where 9
daily sucrose rinses were performed. They also reported that
mean Caries Index values returned to pre-experimental levels
during a month with resumed oral hygiene.
The experimental caries studies showed that withdrawal of
oral hygiene caused development of caries with intra- and
inter-individual variations in lesion progress. Frequent rinsing
with sucrose during the experimental period with undisturbed
plaque accumulation seemed to increase the caries progression
rate, but still with large individual variations. The deposition of
plaque along the gingival margin is clinically visible in less
than 24 h without toothbrushing (Loe, 1970). After this initial
establishment, plaque rapidly accumulates in the coronal
direction until, after approximately one week without
toothcleaning, the thickness and clinical extension of the
plaque on different teeth and tooth surfaces have reached their
maxima. Whereas no major differences in the thickness of the
gingivally located plaque occur, its occlusal and incisal
extension may vary in different groups of teeth as well as on the
various surfaces, presumably reflecting individual masticatory
patterns (Loe, 1970). While friction through mastication has an
effect on incisal and occlusal growth of plaque (Carvalho etal.,
1989, 1991; Ekstrand et al, 1993), examination of plaque
development (Loe et al., 1965) as well as experimental studies
(Wilcox and Everett, 1963; Lindhe and Wicen, 1969) indicate
that cervical tooth areas and the gingival margin are not
subjected to physical stress from food particles in the modern
diet. Since signs of caries in the studies also occurred along the
gingival margin, it can be concluded that visible signs of caries
develop where bacterial deposits have been protected against
oral mechanical disturbance for the longest period of time
Vol.. 812)
Fig. 3—Local
protection against
oral mechanical
forces established on
buccal premolar
surface by means of
an orthodontic band.
From Thylstrup et al.,
1987. Reprinted with
permission from
Munksgaard.
fThylstrup, 1990). The meticulous professional toothcleaning
before caries re-examinations, in conjunction with resumed
oral hygiene procedures, resulted not only in arrestment of
further lesion progression, but also in regression of the
superficial enamel lesions to an extent where they were not
readily recognized in the clinic.
We have now considered the long-term and the short-term
effects of intra-oral mechanical forces' impact on plaque
accumulation, caries initiation, progression, and arrestment. In
the next part, we will look at the effect of total local elimination
of oral mechanical forces.
CLINICAL EXPERIMENTS
The first attempt to establish total elimination of oral mechanical
forces on surfaces of teeth was made by Nygaard 0stby et al.
(1957), who used a gold plate retained to the tooth by two
pinledges. In this way they were able to produce opaque spots
during periods of 4 to 6 weeks. Von der Fehr (1965) used the
same method to examine histological features of enamel caries.
The caries-inducing periods varied from a few weeks to several
months. Macroscopically, von der Fehr (1965) observed loss
of enamel translucency corresponding to the area which was
protected against oral mechanical forces by the gold plate. The
changes ranged from slight accentuation of the perikymata to
distinct white spots. Microradiographic examination revealed
an x-ray-dense surface zone overlying zones with low x-ray
absorption ("inner spots") running in parallel with the outer
surface.
In areas where the surface enamel was abraded prior to the
experiment, a radiopaque surface layer was observed, but was
thinner and often with a lower density than in the control areas.
In order to study changes in the oral environment, after
removal of the gold plate, "white spot" lesions were established
in vivo as previously described. Wedge-shaped grindings were
made so that the effect of surface layer removal could be
examined. The teeth remained in the mouth for from 2 to 57
days without the protection of the gold plate. Visual examination
disclosed that lesions either disappeared or decreased in severity
when re-exposed to the oral environment. In abraded areas,
there was no tendency to surface layer formation. Von der Fehr
also states that "Contrary to what was expected, the lesions
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IN VIVO CARIES MODELS 147
(n e -n 0 ) X10 4
30
20
l\
10
Q
N30 60 90 120 180 240 ym
-10
-20
\
— One week
Control
-30
Air
Fig. 4—Imbibition graphs of a one-week lesion. The graphs
indicate total birefringence in air plotted from the surface.
Data from Holmen et al., 1985a.
tended to remineralize throughout as demonstrated by the faint
and diffuse appearance of the demineralized areas. In some
teeth, highly mineralized layers appeared in the deeper parts of
the lesions, but they did not seem to develop from the abraded
surface and inward."
Hals and Simonsen (1972) modified the technique by
means of a pre-formed orthodontic band. Two metal posts 0.3
or 0.5 mm thick were welded to the inner surface of the band
so that a space between the buccal tooth surface and the band
would be secured (Fig. 3). They applied this technique in
studies of in vivo caries around amalgam fillings. The outer
lesion type that developed had the same histological features,
including a surface zone in polarized light, as previously
described in natural "white spot" lesions (Silverstone, 1973).
0gaard et al. (1983) used the same technique to examine
topical fluoride interaction with in vivo demineralized enamel
(for review, see 0gaard, 1985, 0gaard and R0lla, 1992). It is
surprising, however, that the lesions produced by 0gaard et al.
(1986,1988) and by Arends etal. (1987) have been repeatedly
characterized as a surface softening, i.e., lesions without a
surface layer. Their observations on the nature of the lesions,
which clinically resemble typical "white spot" lesions (0gaard
and R0lla, 1992), are based solely on densitometric tracings on
the basis of microradiograms. However, von der Fehr (1965,
1967), also using the microradiographic technique, always
identified a radiodense surface layer, and the discrepancy
between von der Fehr's findings and those of 0gaard et al.
(1986, 1988) is therefore presumably a result of the
densitometric tracing, which is inaccurate in depicting the
mineral content of the outermost surface layer due to
instrumental limitations (Buskes et al., 1985; Arends and
Christoffersen, 1986). This assumption receives support by the
SEM micrographs illustrating the surface of the typical lesion
(Figs. 4-6 in Arends et al., 1987), because the appearance is
very close to that of normal enamel, not as would be expected
from the densitometric tracings, dominated by severe signs of
outer surface dissolution.
In order to study the effect of complete elimination of
148
physical disturbance in relation to time under identical
environmental conditions, Holmen et al. (1985a,b) examined
4 premolars in each of three individuals after 1, 2, 3, and 4
weeks with local protection against mechanical disturbance by
means of orthodontic bands, as described by Hals and Simonsen
(1972) and 0gaard et al. (1983). The children were not exposed
to any fluoride regimen during the experiment, and they used
Fig. 5a (top)—SEM micrograph of a one-week lesion. Note
dissolution of the external micro-surface after one week with
undisturbed plaque. Figs. 5b (bottom left) shows crystals of
the unprotected part of the buccal surface. (5c) (bottom right)
shows partial dissolution of external crystals after one week
with undisturbed plaque. From Holmen et al., 1985b.
Reprinted with permission from Karger.
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THYLSTKUF ET A L ADV Dm RES Mr 1994
a non-fluoride dentifrice for oral hygiene. The enamel reactions
were examined macroscopically, in polarized light, and in the
SEM. Examination of those parts of the buccal surfaces which
were exposed to oral removal forces revealed no changes at the
macroscopic and microscopic levels. In the SEM, the
unprotected surface part displayed various degrees of surface
wear. Detailed examination disclosed the contours of individual
Figs. 6 and 7—SEM micrographs of a four-week lesion. The
external erosion is more marked with loss of larger parts of
perikymata overlappings (Fig. 6). Erosion of perikymata
overlapping has exposed underlying rod and interrod
enamel at different stages of dissolution. From Holmen et
al., 1985b. Reprinted with permission from Karger.
VOL. 8(2)
crystals, or aggregates of crystals, in the outer microsurface,
with various degrees of intercrystalline spaces (Fig. 5b). No
enamel changes were seen macroscopically, even following
prolonged air-drying, after one week with complete elimination
of mechanical disturbance. After 2, 3, and 4 weeks without
mechanical disturbance, the enamel gradually became more
opaque. After 4 weeks, the surface had lost its normal luster
and appeared dull-whitish. At the light-microscopic level, it
was possible to visualize mineral loss even after one week in
terms of a translucent zone. With increasing time of protection,
the enamel reactions gradually adopted the structural features
of the classic enamel lesion, with an inner translucent zone, a
dark zone, the body of the lesion, and a surface zone (Silverstone,
1973). Quantitative imbibition studies (Holmen et al, 1985a)
showed that the outermost surface enamel from the very
beginning, i.e., after one week, was always less porous than the
underlying enamel, indicating a reduced tendency to dissolution
of enamel tissue near the surface (Fig. 4).
In the SEM, the most important observation was that direct
dissolution of the external micro-surface also occurred from
the very beginning of the experimental period (Holmen et al,
1985b). This was predominantly seen as partial dissolution of
individual crystals after one week with protection against
mechanical forces (Figs. 5a and 5c). Complete dissolution of
external crystals became more prominent in relation to length
of the experiment. Thus, the four-week specimens showed the
most extensive signs of direct surface dissolution, since parts
of the outer micro-surface were missing (Figs. 6 and 7). Even
though variations in the dissolution process, as observed at low
levels of magnifications, were obviously related to the structural
configuration of the surface, it was noticeable that examination
at the crystal level indicated a consistent reaction, which was
independent of the original surface micro-anatomy (Holmen et
al, 1985b).
In another experiment, Holmen et al. (1987a,b) examined
the effect of re-exposing active enamel lesions to the oral
environment. Active enamel lesions were developed as
previously described during a period of 4 weeks. The orthodontic
bands were removed from 4 premolars in each of four children
undergoing orthodontic treatment. One premolar in each
individual was extracted and served as control, whereas the
remaining teeth were extracted after 1, 2, or 3 weeks. The
children were not exposed to any fluoride regimen during the
entire experimental period. The teeth were not professionally
cleaned after removal of the bands, but the children were told
to maintain oral hygiene as usual. All experimental teeth were
photographed in situ before being banded and after being
debanded, and immediately before extraction. The enamel
reactions were examined in polarized light and in the SEM.
After 4 weeks with local protection, all teeth showed
evidence of active enamel caries, partly covered by remnants
of bacterial deposits. During the following three weeks, a
gradual reduction in the whitish lesion areas was noted. These
clinical signs of lesion regression were already evident after
one week of exposure to the oral environment. Polarized light
observations indicated a gradual smoothing out of perikymata
overlappings with increasing time of exposure to oral
mechanical forces. This phenomenon was associated with a
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IN VIVO CARIES MODELS 149
(ne-n0) x 1(
40
30
Central lesion area
20
10
0 A 'C^ i
) 90 120 180 240 M™
-10
-20
-30 "\^——____^^ Control
-40
Three weeks after
removal of protection
Fig. 8—Imbibition graphs of active control lesion and
arrested lesion three weeks after removal of protection. The
graphs indicate total birefringence in water along a
traverse through the central part of the lesions. Data from
Holmen et al., 1987a.
reduction in surface-layer thickness. Lesion depth tended to
diminish, particularly corresponding to lesion border areas,
which accordingly became macroscopically translucent.
Examination in quinoline showed a gradual widening of the
dark zones. Quantitative imbibition studies showed a reduction
in tissue porosity in the deeper parts of the lesions soon after
re-exposure to the oral environment (Fig. 8). Remnants of
microbial deposits in surface irregularities and along
perikymata overlappings were seen in the SEM. After removal
of organic material, the control teeth showed typical features
of active enamel lesions with surface erosion and enlarged
intercrystalline pathways (Fig. 9). Following one week of
exposure to oral mechanical forces, the surface was dominated
by multiple micro-scratches in the eroded outer surface enamel
(Fig. 10). Two weeks of exposure resulted in micro-wear,
which was seen as removal of parts of the porous external
surface (Fig. 11). The surfaces of the three-week specimens
were characterized by classic wear striation in different
directions (Fig. 12). At the crystal level of examination,
removal of the outermost, loosely bound crystals was seen
after one week (Fig. 10). This tendency was more pronounced
after 2 or 3 weeks (Figs. 11 and 12). The crystals of the "new"
surface appeared tightly packed, and individual crystals
frequently seemed to have merged together in groups. However,
a fine network of intercrystalline pathways could still be
discerned (Fig. 12).
On the basis of these two experiments, it can be concluded
that local elimination of oral mechanical disturbance promoted
progressive lesion formation, and re-exposure to naturally
occurring mechanical disturbance, including oral hygiene
procedures, not only arrested further lesion progress, but also
resulted in partial regression of lesions. The gradual
enhancement of micro-wear in relation to time supports the
concept that mechanical removal of the cariogenic biomass
has been responsible for the observed arrestment (Holmen and
Thylstrup, 1986).
150 THYLSTRUP ET AL. Anv DM Rus JULY 1994

Figs. 9 to 12—SEM micrographs. Overview (left) and high-magnification detail (right). (Fig. 9) (top left) Control showing
typical features of active enamel lesion with partial and complete dissolution of outermost crystals. (Fig. 10) (bottom left)
After one week with exposure to the oral environment, multiple micro-scratches can be seen in the outermost partly dissolved
crystal layer. Loosely bound crystals have been worn away (right). (Fig. 11) (top right) Micro-wear after two weeks of
exposure. Parts of the porous external microsurface have been removed by wear. (Fig. 12) (bottom right) After three weeks,
the surface appears smoother, with classic wear striation patterns owing to more complete removal of the eroded micro-
surface (left). The complete removal of loosely bound and partly dissolved crystals has exposed tightly packed crystals
separated by a distinct network of intercrystalline spaces. From Holmen et al., 1987b. Reprinted with permission from
Scandinavian University Press.

In order to examine the effect of regular disturbance/
removal of dental plaque, Holmen et al. (1988) used the
above-described model in a sample of 15 children undergoing
orthodontic treatment. Two homologous premolars were
banded for 5 weeks. One tooth in each pair served as control
and had the band cemented for the entire test period of 5
weeks. The other band was removed weekly, and the buccal
surface cleaned, either by being careful pumiced with a non-
fluoride toothpaste, or by simple being cleaned with a cotton
pellet. As with the previous studies, no fluoride of any kind
was added during the entire test period. The teeth were
examined macroscopically, in polarized light, and in the SEM.
The enamel changes in the control teeth ranged from
slightly accentuated perikymata overlappings to pronounced
white opaque lesions. In contrast, all the experimental teeth
appeared clinically sound. Examination of control lesions in
polarized light showed classic subsurface lesions at different
stages of progress, while no subsurface dissolution seemed to
have taken place in any of the experimental teeth, regardless of
cleaning procedure. Quantitative imbibition studies revealed,
however, a slight increase in tissue porosity in the outer 5-10
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u.m of the surface in the teeth cleaned weekly (Fig. 13). The
graphs document the remarkable effect of weekly plaque
control in two individuals with rapid caries progression. SEM
examination of the controls showed signs of active carious
dissolution as previously described, while the pumiced surfaces
were dominated by a general smoothing out of surface
irregularities, in addition to several micro-scratches. The cotton-
pellet-cleaned surfaces appeared very similar but were less
dominated by micro-wear. However, high-resolution
examination of the areas cleaned weekly disclosed initial
dissolution of the outermost surface, regardless of cleaning
procedure. The importance of intra-oral mechanical forces for
caries initiation and progression was convincingly demonstrated
in this study. Thus, complete elimination caused development
of caries in all individuals, without the exclusion of the
complex interplay of other individual factors, indicated by the
variation noted in the rate of lesion progress (Fig. 14). In spite
of this, mechanical suppression of bacterial activities without
additional use of fluoride overrules all other factors, since no
visible indications of caries were noted in any of the experimental
teeth.
VOL. 8(2) IN VIVO CARIES MODELS 151

CONTROLLED CLINICAL OBSERVATIONS

Caries is a frequently occurring side-effect to orthodontic 40
treatment (Wisth and Nord, 1977; Lundstrom and Hamp,
30
1980; Gorelick et al.9 1982; Mizrahi, 1983; Artun and Pumiced
20
Brobakken, 1986). The lesions developed in conjunction with
10
the direct bonding technique are typically seen in the gingival Distance from surface

enamel part (Gorelick et al., 1982; Artun and Brobakken, 0

180 240 /urn
1986). Artun and Thylstrup (1986, 1989) examined such -10
lesions after debonding in six children who had received -20
Control
routine orthodontic treatment for about two years. These -30
children were examined at time of debonding, and after 1,2,3, -~~ Experimental
-40
4, 8, and 12 weeks, and 3 years. Detailed examinations of the -50
demineralized areas, adjacent to the bonded brackets, were
carried out on the maxillary incisors. The examinations included:
(i) clinical examinations of plaque distribution, extension and
surface texture of the lesions, and clinical estimation of enamel
opacity; (2) color slides at each appointment, before and after
removal of microbial deposits, with the second slide being (n e -n 0 ) *
40
made after the teeth were air-dried for 20 s; and (3) SEM
examination of replicas made at each appointment, after being 30
Cleaned with cotton-pellet
pumiced and washed with a solution of 5% v/v hypochlorite for
30 s. Positive replicas were made and prepared for the SEM. At
20
10
A
Distance from surface
the time of debonding, a furrow was made with a sharp hand- 0
3/ 0\
instrument in the bonding area as a reference for SEM
examinations. At the time of debonding, heavy accumulations
-10 J ^60 90 120 180 240 ,um

of dental plaque were observed in all areas gingival to the
brackets. After routine cleaning, the exposed gingival region
of the labial tooth surface showed the characteristic chalky and
white appearance of active enamel caries. The border between
the lesion and the sound enamel, which had been covered by
the bonding material, appeared very distinct (Fig. 15a). During
the observation period, the surface texture of the lesions
gradually changed from being chalky and soft at the time of
debonding to appearing shiny. Gentle probing also disclosed
that the lesions on the surface gradually resumed the hardness
of the adjacent sound enamel. These changes took place from
2 to 8 weeks after the debonding. Concomitant with these
surface alterations, the marked white lesion appearance at
the time of the debonding changed to a more diffuse opacity
(Fig. 15b).
After 3 years, only remnants of opaque enamel could be
found. Small surface micro-cavities, which developed during
the first weeks of observation, were seen in some cases. In
these micro-cavities, normal enamel translucency occurred
relatively rapidly. In the SEM, there was a marked step
between the active lesion surface and the adjacent sound
enamel (Fig. 16a). After three months, the difference in levels
between the lesion surface and the sound surface became more
marked, indicating that the wear of the "soft" lesion surface
had been somewhat larger than that of the sound enamel (Fig.
16b). After 3 years, the furrow in the bonding area could not be
discerned, but the step between the lesion and the bonding area
was still distinct. The three-year observations generally showed
marked wear, and the surface micro-cavities were either leveled
out or barely discernible.
From this study, it can be concluded that long periods with
undisturbed plaque, due to orthodontic appliances, result in
-20
^ ^ - ^ _ _ _ _ _ Control
-30 Experimental
-40
-50J
Fig. 13—Imbibition graphs showing distribution of tissue
porosity in control teeth and their respective experimentally
cleaned teeth by pumice (top) or with a cotton pellet
(bottom). The graphs indicate total birefringence in
individuals with a relatively rapid caries progression. Data
from Holmen et al., 1988.
more marked caries dissolution than in the previous short-term
studies. The direct dissolution of the outer surface therefore
became much more evident, since it resulted in a visible
difference in surface levels of the lesion area and the sound
enamel. The clinical impression of a less whitish arrested
lesion was therefore predominantly a result of the wearing
away and polishing of the partly dissolved "chalky" surface of
the active lesion. This phenomenon also explained the clinical
impression of resumed surface hardness. This study again
demonstrated that removal of cariogenic plaque resulted in
arrestment of further lesion progression, and that the clinical
impression of lesion regression is related to intra-oral wear and
tear, including oral hygiene procedures.
DISCUSSION
Taken together, these in vivo studies convincingly demonstrate
that partial or total elimination of the intra-oral mechanical
forces leads to evolution of cariogenic plaque, resulting in
carious enamel dissolution. In addition, they show that re-

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152
Caries signs
Caries progression
in teeth with
undisturbed plaque
Visible
Micro-
Caries progression
scopical in teeth with
weekly plaque removal
i Weeks
Fig. 14—Complete elimination of mechanical forces results
in cariogenic plaque evolution and caries initiation in all
individuals, whereas the variations in rate of caries
progression are determined by the complex interplay of
other external and internal factors. Mechanical suppression
of bacterial activities, performed weekly, overruled all other
factors, since no indications of caries were noted in the
experimental teeth. Modified from Thylstrup (1988a).
exposure to the partly or totally eliminated mechanical forces
not only arrests further progression, but also results in partial
lesion regression. In all studies, the localized loss of normal
enamel translucency, which clinically is visualized as white
opacities or "white spots", was used as an indication of carious
dissolution.
Some of the studies also used different techniques such as
polarized light, microradiography, or SEM for estimations of
mineral loss in the enamel tissue. In the first part of this
discussion, we will therefore consider the nature of the natural
"white spot" lesion on the basis of recent work, but with all due
regard to previous studies, in order to arrive at a common
agreement on the phenomenon and the terminology we are
using.
The term "white spot" lesion originates from the observation
that extracted, macroscopically sound teeth often have small
opaque white regions positioned at the cervical margin of the
interdental facet (Silverstone, 1973). Itis therefore only natural
that the interproximal "white spot" lesion has been regarded
largely as being synonymous with the onset of dental caries
(Thylstrup et al., 1983). For this reason, the interproximal
caries lesion has been in the focus of attention for caries
research to such an extent that the conically shaped interproximal
lesion is considered identical to natural caries as opposed to,
for example, lesions with advancing fronts, maintaining a
direction parallel to the enamel surface (Silverstone, 1973).
While the conically shaped lesion is characteristic of
interproximal caries, due to the specific environmental
conditions offered for growth of microbial communities in this
part of the dentition (Thylstrup and Qvist, 1987; Bj0rndal,
1991), it is not necessarily more "natural" than lesions paralleling
the outer surface. When oral bacteria are offered total protection
against oral removal forces, they produce lesions which, in
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THYLSTRUP ET AL. ADV DENT RES JULY 1994
polarized light, are indistinguishable from the classic
interproximal lesions, but the advancing fronts at any stage of
lesion progression are parallel to the outer surface (Holmen et
al., 1985a, 1987a, 1988), in keeping with microradiographic
observations (von der Fehr, 1965, 1967). For this reason,
results obtained by examination of lesions, produced under
controlled conditions in vivo with respect to the oral mechanical
forces, can be extrapolated to all types of naturally occurring
enamel caries.
In principle, the interproximal "white spot" lesion may be
considered as a relatively late stage in the caries process
(Thylstrup et al, 1983). The tissue alterations taking place
before the lesion becomes macroscopically visible have, in
controlled studies, been shown to be characterized by direct
dissolution of the external microsurface (Holmen et al., 1985b;
Thylstrup et al, 1990), in accordance with observations on
erupting teeth (Thylstrup and Fredebo, 1982; Thylstrup et al,
1983; Holmen and Thylstrup, 1984). The corresponding light
microscopic changes were identified in polarized light as a
translucent zone, when examined in quinoline (Holmen et al,
1985a), as also reported by Darling (1958), who saw similar
changes in the contact point region of macroscopically sound
teeth. Quantitative imbibition studies revealed that the outermost
enamel was more porous than unaffected enamel and that the
enamel immediately beneath the surface was more porous than
that on the surface (Fig. 4). In other words, from the very
beginning there appeared to be a tendency toward subsurface
dissolution (Holmen et al, 1985a). As lesion progression
continued, the external surface dissolution became more evident,
the thickness of the surface layer increased, and the subsurface
demineralization was growing, as visualized in Fig. 17. Signs
of direct dissolution of the outer surfaces of enamel subsurface
lesions have been described by Thylstrup and Fejerskov (1981),
Haikel et al. (1983), and Frank (1990). The direct surface
dissolution therefore explains why examinations in the TEM
of ultrathin sections cut through the interface of the overlying
microbial biomass and the eroded surface enamel give the
impression of a superficial invasion of bacteria into the enamel
(Frank and Brendel, 1966; Johnson, 1967; Frank, 1990). At
more advanced stages of caries, the direct surface erosion
became so evident that the surface can be clinically described
as chalky with a dull-whitish appearance, and with a distinct
level between the active lesion surface and the adjacent
unaffected enamel (Artun and Thylstrup, 1986, 1989). It is
therefore more correct to use the phrase "the surface zone", as
was also done by Silverstone (1973), instead of the common
description "the relatively unaffected or intact surface layer",
because the external enamel part participates in the dynamics
of lesion development in the same manner as in the other three
zones of enamel lesions. To put it differently, the surface zone
is not an inert structural feature overlying the body of the
lesion, both because of structural changes including significant
mineral loss, and because of changes in the chemical
composition continously taking place during gradual lesion
development (Weatherell et al, 1977).
In light of the prevailing concept of the relatively unaffected
surface layer, it is no wonder that the entry of caries attack
through the surface has been the subject of controversial
VOL. 8(2)
opinions. Light microscopic examinations gave rise to the
theory that caries enters the enamel along the stria of Retzius
(Darling, 1958). Ultrastructural studies could not confirm this
hypothesis, but suggested that the structural configurations at
the rod level represented major potential pathways for diffusion
(Frank and Brendel, 1966; Johnson, 1967; Mortimer and
Tranter, 1971; Scott et al, 1974; Simmelink et al, 1974;
Theunsefa/., 1982;Haikelefa/., 1983;Frank, 1990). Moreover,
Arends and Christoffersen (1986) assumed that the external
solution maintains contact with the internal liquid phase through
peculiar small "holes" in the enamel surface, being about 0.1-
1.0 urn.
However, some workers using the TEM noted that the
carious destruction may not be dependent on the structural
detail of the tissue as previously believed (Johnson, 1967), and
that carious invasion is able to follow narrow lanes, such as
Figs. 15a (top) and b (bottom)—Clinical features
immediately after removal of orthodontic appliances and
cleaning (top) and after three months (bottom). Note the
characteristic white and chalky surface appearance of the
active lesion. The eroded surface layer has been worn away
and polished, giving the shiny and hard surface of the
arrested lesion (bottom).
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IN VIVO CARIES MODELS 153
intercrystalline spaces, in addition to larger pathways (Frank
and Brendel, 1966). Enamel is a micro-porous solid composed
of crystals, and because the caries lesion is the result of acids
reacting with individual crystals, it is reasonable to consider
the intercrystalline spaces as being the most important pathways
forthe diffusion of ions into and out of the enamel, particularly
at initial stages of lesion formation (Holmen et al., 1985b).
Several reviews have considered a variety of explanatory
models proposed for the relative preservation of the surface
zone during subsurface demineralization (Silverstone, 1973;
Thylstrupefa/., 1983; Arends and Christoffersen, 1986;Frank,
1990). It is interesting, however, that while much attention has
been paid to the "preservation" of the surface zone, none of the
proposed mechanistic explanatory models has considered the
ongoing external surface erosion, which is clinically expressed
by loss of surface luster or, in more advanced lesions, as a dull-
Figs. 16a (top) and b (bottom)—SEM micrographs of
replicas of the active lesion (top) and after three months
(bottom). Note the distinct step between the eroded surface
of the active lesion and the adjacent sound enamel, open
arrows (top). After three months, the furrow (arrows) has
almost disappeared, and the step between the sound enamel
and the arrested lesion surface is slightly enhanced
(bottom). From Thylstrup, 1988b.

154
Active lesions Arrested lesions
B v\\\\\\\\\\\\\\\\\\\\\V
Fig. 17—Schematic drawing of the initiation (A) and
gradual progression of an enamel caries lesion (B to D).
Caries initiation begins beneath undisturbed plaque with
direct dissolution of the external micro-surface (A). Further
progression leads to increased surface dissolution and
preferential subsurface dissolution (B to D). Arrows
indicate structural changes in corresponding stages of
lesion arrestment. At each stage, the mechanical removal of
the cariogenic plaque, which is physically closely
interrelated with the eroded surface layer, leads to surface
wear and polishing. The "new" surface of the arrested
lesion is therefore hard and shiny, in contrast to the chalky
and "soft" surface of the active lesion. Porosity in the
interior part of the subsurface lesion is reduced, due to
gradual return to normal pH, promoting reprecipitation of
minerals from the internal enamel fluids.
whitish, chalky surface (Fig. 15a).
The opaque or white appearance of the enamel lesion is
conventionally assumed to be a result of the internal increase
in enamel porosity (Silverstone, 1973). When air-dried, lesions
in the clinic become more visible due to replacement of water
with air in the intercry stalline spaces (Thylstrup and Fejerskov,
1994). Thylstrup and Fejerskov (1994) compared the clinical
estimation of mineral loss with the principles behind the use of
polarized light in conjunction with different imbibition media
for estimating enamel porosity in enamel sections.
In light of the SEM examinations by Holmen etal (1985b,
1987b) and Artun and Thylstrup (1986, 1989), it is more
appropriate, however, to relate the white appearance of the
active enamel lesion with its characteristic chalky surface
luster to two phenomena: The first one is the previously
discussed internal increase in enamel porosity due to subsurface
demineralization. The second phenomenon is caused by direct
surface erosion. The enamel loses its shiny appearance, because
the irregular surface generated by the erosion gives rise to a
diffuse back-scattering of the light. On this basis, it is possible
to explain the clinical changes taking place when active lesions
have been turned into inactive or arrested lesions (Fig. 17). The
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ADV DENT RES JULY 1994
THYLSTRUP ET AL.
examination of results from the in vivo caries models provided
evidence that lesion arrestment was due to mechanical removal
of the cariogenic plaque. The mechanical removal of the
microbial biomass, which is physically interrelated with the
eroded enamel surface, also causes removal or wearing away
of the partly dissolved enamel interface. The mechanical wear
and polishing thus explain the marked reduction in clinical
"whiteness" and the resumed surface luster of arrested lesions.
The simultaneous exposure of the tightly packed crystals
comprising the deeper parts of the surface zone explains the
clinical impression of resumed surface hardness.
In addition, the internal porosity is slightly reduced (Fig. 8)
due to the removal of the acid-producing plaque (Holmen etal.,
1987a). The complete end of acid production at the surface
results in a gradual return to neutral pH in the inner lesion part,
promoting an outward diffusion of protons. The reduced
porosity in the inner lesion part, as also observed by von der
Fehr (1965), is therefore the result of a gradual return of enamel
fluids to supersaturation, causing a shift in equilibrium and re-
precipitation of minerals in the sites of demineralization. The
return to normal pH in the interior lesion is presumably a long
process which may last several weeks (Holmen etal, 1987a).
Arrested lesions have commonly been described as
"remineralized" lesions, because it has been believed that the
clinical observations of regression, glossy appearance, and
surface hardness are the results of salivary repair (Gr0n, 1973;
ten Cate, 1983,1992). This concept has been widely accepted,
because human saliva is supersaturated with calcium and
phosphate salts that form dental enamel (Gr0n, 1973). However,
precipitation of calcium phosphate salts directly onto dental
enamel is rarely observed in spite of the supersaturation. It is
now realized that salivary proline-rich phosphoproteins and
other inhibitors, such as statherin, inhibit crystal growth and
spontaneous precipitation of calcium phosphate salts from
supersaturated solutions (Hay, 1984; Hay etal, 1984). These
inhibitors, which also form essential components of the enamel
pellicle, are responsible for maintenance of the supersaturation
of calcium phosphate salts in saliva without precipitations on
the dental hard tissues. Accordingly, the function of the
inhibitors makes it unlikely that carious enamel is restored
significantly in vivo by salivary repair mechanisms. Similar
observations have been made after enamel surface etchings,
since the apparent repair has been the result of masking by
salivary proteins instead of mineral deposition (Lenz and
Miihlemann, 1963; Miihlemann et al, 1964; Meurmann and
Asikainen, 1976;GarberoglioandCozzani, 1979). It is therefore
possible to conclude that the enamel surface defects will not be
refilled in vivo, either at the ultrastructural level (Thylstrup et
al, 1983) or at the macroscopic level (Gr0n, 1973; Artun and
Thylstrup, 1986,1989). This also explains why surface defects
at any level of examination in fluorosed teeth do not show signs
of repair (Thylstrup and Fejerskov, 1979; Thylstrup, 1983;
Thylstrup et al, 1990), and that the gradual disappearance of
the larger defects are explained by post-eruptive functional
wear (Thylstrup, 1983).
In short, the data from in vivo caries studies show that the
clinical and structural changes associated with lesion arrestment
or partial regression are not related to any salivary repair
VOL. 8(2)
mechanism but are solely the result of mechanical removal of
the cariogenic biomass which is physically interrelated with the
eroded surface of the active lesion. Obviously, this does not
preclude subtle alterations at the crystal level, but our SEM
studies did not reveal obliteration or "blocking" (ten Cate,
1992) of either the external intercrystalline spaces or the
surface layer, as also indicated by polarized light observations
(Holmen etal., 1987a,b).
The results obtained by Backer Dirks (1966) in his classic
study have been used in several review papers and textbooks as
an indication of "remineralization" due to some vaguely defined
salivary repair mechanism. In addition, examination of more
recent literature makes it clear that the word "remineralization"
is widely used to describe completely different phenomena
caused by acid reactions, including fluoride interactions with
the dental hard tissues.
Communication depends to a large extent on a mutual
agreement with regard to the meaning of the words. Scientific
communication is thus based on an elaborate and specialized
terminology in order to ensure that we are dealing with the same
phenomena. There are therefore several reasons for a profound
re-evaluation of the current uniform and inaccurate usage of the
word "remineralization" in scientific communications. The
need to avoid unnecessary confusion becomes particularly
evident in relation to the clinical management of caries, because
"remineralization" in terms of superficial mineral redeposition
or "blocking" is an illusion which does not lead to lesion
arrestment. The complete end of lesion progression in vivo
requires mechanical removal of the bacterial origin of the
disease, the cariogenic plaque, which, in effect, is equivalent to
the withdrawal of enamel specimens from acids in the laboratory
model systems in order to arrest further demineralization.
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