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C
A. THYLSTRUP aries research has for many years been almost
C. BRUUN synonymous with fluoride research, and relatively little
L. HOLMEN attention has been devoted to the pathology of dental
Department of Cariology and Endodontics caries compared with the multitude of laboratory studies
School of Dentistry, Health Science Faculty on fluorides during the past 20 years. It is therefore not
University of Copenhagen surprising that current discussions on the anti-caries mechanisms
20 N0rre Alle of fluoride are based almost entirely on the wealth of clinical
DK-2000 Copenhagen N, Denmark trial data and in vitro studies. There is no doubt that the many
different approaches that mimic carious dissolution in the
Adv Dent Res 8(2): 144-157, July, 1994 laboratory have provided a great deal of insight concerning the
physico-chemical processes operating during enamel
demineralization, which eventually leads to formation of a
Abstract—The effects of intra-oral mechanical forces on visible lesion, often simply referred to as the "white spot"
caries initiation, progression, and arrestment are evaluated by lesion. Considering, however, past and ongoing intensive
examination of different in vivo caries models. The models are attempts to unravel the mystery of dental caries, it is surprising
grouped in four categories: (1) a population study, (2) short- that this apparently very important stage in the disease process
term clinical trials, (3) clinical experiments, and (4) controlled is considered as simply "a white spot". According to the
clinical observations. Taken together, these in vivo studies literature, this "white spot" lesion undergoes several changes
convincingly demonstrate that partial or total elimination of in the clinic or in vivo as well as in the laboratory. It may thus
the intra-oral mechanical forces operating during mastication be transformed into a "brown spot lesion", a remineralized
or toothbrushing leads to evolution of cariogenic plaque, lesion, an arrested lesion, and occasionally it even disappears
resulting in localized carious enamel dissolution. In addition, completely as a "caries reversal".
they show that re-exposure to the partly or totally eliminated The explanatory models proposed on the basis of laboratory
mechanical forces not only arrests further lesion progression, experiments are, for obvious reasons, focusing on the few
but also results in partial lesion regression. The data from in parameters which have been strictly controlled in the
vivo caries studies also show that the clinical and structural experiments. The essential processes appear to be dissolution
changes associated with lesion arrestment or partial regression and re-precipitation of the mineral phase of enamel, often
are not related to any salivary repair mechanism, but are solely described as demineralization and remineralization processes.
the result of mechanical removal of the cariogenic biomass Extrapolation of the simplistic laboratory explanatory models
which is physically interrelated with the eroded surface of the to the complicated in vivo situation requires at least, however,
active, dull-whitish enamel lesion. No indications of superficial that the terminology of the essential issues cover the same
mineral deposition or "blocking" of the external intercry stalline processes or phenomena. Most important is, of course, whether
spaces are seen in the surface layer of lesions arrested in vivo. the widely used, but vaguely defined, stage during caries
For this reason, the conventional usage of the terminology development, designated the "white spot" lesion, actually
'remineralization' is considered absolutely misleading when covers a well-defined concept among the users of the
used to describe the mechanisms responsible for the arrest of terminology. It is equally important to consider whether the
lesion progression in vivo. 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
This manuscript was presented at a Symposium entitled involved in vivo in caries initiation, progression, and arrestment.
"Mechanisms and Agents in Preventive Dentistry", held The studies are described in four categories: a population
October 28-November 1, 1992, in Chester, England, under study, short-term clinical trials, clinical experiments, and
the auspices of the Council of Europe Research Group on controlled clinical observations. The final part combines the
Surface and Colloid Phenomena. experiences arising from these studies and discusses definitions
Printing of the color plate is supported by Colgate-Palmolive, Denmark. and terminology on the basis of an updated view of the "white
spot" lesion.
144
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VOL. 8(2) IN VIVO CARIES MODELS 145
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146 THYISTRUP ET AL. ADV Dm RES Mr 1994
Caries
Index
1.0
Sucrose
0.8
0.6
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Vol.. 812) IN VIVO CARIES MODELS 147
Fig. 3—Local
protection against (n e -n 0 ) X10 4
oral mechanical 30
forces established on 20
buccal premolar l\
10
surface by means of
an orthodontic band. Q
N30 60 90 120 180 240 ym
From Thylstrup et al., -10
1987. Reprinted with
permission from -20
\
— One week
Munksgaard. Control
-30
Air
physical disturbance in relation to time under identical a non-fluoride dentifrice for oral hygiene. The enamel reactions
environmental conditions, Holmen et al. (1985a,b) examined were examined macroscopically, in polarized light, and in the
4 premolars in each of three individuals after 1, 2, 3, and 4 SEM. Examination of those parts of the buccal surfaces which
weeks with local protection against mechanical disturbance by were exposed to oral removal forces revealed no changes at the
means of orthodontic bands, as described by Hals and Simonsen macroscopic and microscopic levels. In the SEM, the
(1972) and 0gaard et al. (1983). The children were not exposed unprotected surface part displayed various degrees of surface
to any fluoride regimen during the experiment, and they used wear. Detailed examination disclosed the contours of individual
Fig. 5a (top)—SEM micrograph of a one-week lesion. Note Figs. 6 and 7—SEM micrographs of a four-week lesion. The
dissolution of the external micro-surface after one week with external erosion is more marked with loss of larger parts of
undisturbed plaque. Figs. 5b (bottom left) shows crystals of perikymata overlappings (Fig. 6). Erosion of perikymata
the unprotected part of the buccal surface. (5c) (bottom right) overlapping has exposed underlying rod and interrod
shows partial dissolution of external crystals after one week enamel at different stages of dissolution. From Holmen et
with undisturbed plaque. From Holmen et al., 1985b. al., 1985b. Reprinted with permission from Karger.
Reprinted with permission from Karger.
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VOL. 8(2) IN VIVO CARIES MODELS 149
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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/ u.m of the surface in the teeth cleaned weekly (Fig. 13). The
removal of dental plaque, Holmen et al. (1988) used the graphs document the remarkable effect of weekly plaque
above-described model in a sample of 15 children undergoing control in two individuals with rapid caries progression. SEM
orthodontic treatment. Two homologous premolars were examination of the controls showed signs of active carious
banded for 5 weeks. One tooth in each pair served as control dissolution as previously described, while the pumiced surfaces
and had the band cemented for the entire test period of 5 were dominated by a general smoothing out of surface
weeks. The other band was removed weekly, and the buccal irregularities, in addition to several micro-scratches. The cotton-
surface cleaned, either by being careful pumiced with a non- pellet-cleaned surfaces appeared very similar but were less
fluoride toothpaste, or by simple being cleaned with a cotton dominated by micro-wear. However, high-resolution
pellet. As with the previous studies, no fluoride of any kind examination of the areas cleaned weekly disclosed initial
was added during the entire test period. The teeth were dissolution of the outermost surface, regardless of cleaning
examined macroscopically, in polarized light, and in the SEM. procedure. The importance of intra-oral mechanical forces for
The enamel changes in the control teeth ranged from caries initiation and progression was convincingly demonstrated
slightly accentuated perikymata overlappings to pronounced in this study. Thus, complete elimination caused development
white opaque lesions. In contrast, all the experimental teeth of caries in all individuals, without the exclusion of the
appeared clinically sound. Examination of control lesions in complex interplay of other individual factors, indicated by the
polarized light showed classic subsurface lesions at different variation noted in the rate of lesion progress (Fig. 14). In spite
stages of progress, while no subsurface dissolution seemed to of this, mechanical suppression of bacterial activities without
have taken place in any of the experimental teeth, regardless of additional use of fluoride overrules all other factors, since no
cleaning procedure. Quantitative imbibition studies revealed, visible indications of caries were noted in any of the experimental
however, a slight increase in tissue porosity in the outer 5-10 teeth.
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VOL. 8(2) IN VIVO CARIES MODELS 151
-20
^ ^ - ^ _ _ _ _ _ Control
of dental plaque were observed in all areas gingival to the
-30 Experimental
brackets. After routine cleaning, the exposed gingival region
-40
of the labial tooth surface showed the characteristic chalky and -50J
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 Fig. 13—Imbibition graphs showing distribution of tissue
gradually changed from being chalky and soft at the time of porosity in control teeth and their respective experimentally
debonding to appearing shiny. Gentle probing also disclosed cleaned teeth by pumice (top) or with a cotton pellet
that the lesions on the surface gradually resumed the hardness (bottom). The graphs indicate total birefringence in
of the adjacent sound enamel. These changes took place from individuals with a relatively rapid caries progression. Data
2 to 8 weeks after the debonding. Concomitant with these from Holmen et al., 1988.
surface alterations, the marked white lesion appearance at
the time of the debonding changed to a more diffuse opacity more marked caries dissolution than in the previous short-term
(Fig. 15b). studies. The direct dissolution of the outer surface therefore
After 3 years, only remnants of opaque enamel could be became much more evident, since it resulted in a visible
found. Small surface micro-cavities, which developed during difference in surface levels of the lesion area and the sound
the first weeks of observation, were seen in some cases. In enamel. The clinical impression of a less whitish arrested
these micro-cavities, normal enamel translucency occurred lesion was therefore predominantly a result of the wearing
relatively rapidly. In the SEM, there was a marked step away and polishing of the partly dissolved "chalky" surface of
between the active lesion surface and the adjacent sound the active lesion. This phenomenon also explained the clinical
enamel (Fig. 16a). After three months, the difference in levels impression of resumed surface hardness. This study again
between the lesion surface and the sound surface became more demonstrated that removal of cariogenic plaque resulted in
marked, indicating that the wear of the "soft" lesion surface arrestment of further lesion progression, and that the clinical
had been somewhat larger than that of the sound enamel (Fig. impression of lesion regression is related to intra-oral wear and
16b). After 3 years, the furrow in the bonding area could not be tear, including oral hygiene procedures.
discerned, but the step between the lesion and the bonding area
was still distinct. The three-year observations generally showed DISCUSSION
marked wear, and the surface micro-cavities were either leveled Taken together, these in vivo studies convincingly demonstrate
out or barely discernible. that partial or total elimination of the intra-oral mechanical
From this study, it can be concluded that long periods with forces leads to evolution of cariogenic plaque, resulting in
undisturbed plaque, due to orthodontic appliances, result in carious enamel dissolution. In addition, they show that re-
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152 THYLSTRUP ET AL. ADV DENT RES JULY 1994
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VOL. 8(2) IN VIVO CARIES MODELS 153
opinions. Light microscopic examinations gave rise to the intercrystalline spaces, in addition to larger pathways (Frank
theory that caries enters the enamel along the stria of Retzius and Brendel, 1966). Enamel is a micro-porous solid composed
(Darling, 1958). Ultrastructural studies could not confirm this of crystals, and because the caries lesion is the result of acids
hypothesis, but suggested that the structural configurations at reacting with individual crystals, it is reasonable to consider
the rod level represented major potential pathways for diffusion the intercrystalline spaces as being the most important pathways
(Frank and Brendel, 1966; Johnson, 1967; Mortimer and forthe diffusion of ions into and out of the enamel, particularly
Tranter, 1971; Scott et al, 1974; Simmelink et al, 1974; at initial stages of lesion formation (Holmen et al., 1985b).
Theunsefa/., 1982;Haikelefa/., 1983;Frank, 1990). Moreover, Several reviews have considered a variety of explanatory
Arends and Christoffersen (1986) assumed that the external models proposed for the relative preservation of the surface
solution maintains contact with the internal liquid phase through zone during subsurface demineralization (Silverstone, 1973;
peculiar small "holes" in the enamel surface, being about 0.1- Thylstrupefa/., 1983; Arends and Christoffersen, 1986;Frank,
1.0 urn. 1990). It is interesting, however, that while much attention has
However, some workers using the TEM noted that the been paid to the "preservation" of the surface zone, none of the
carious destruction may not be dependent on the structural proposed mechanistic explanatory models has considered the
detail of the tissue as previously believed (Johnson, 1967), and ongoing external surface erosion, which is clinically expressed
that carious invasion is able to follow narrow lanes, such as by loss of surface luster or, in more advanced lesions, as a dull-
Figs. 15a (top) and b (bottom)—Clinical features Figs. 16a (top) and b (bottom)—SEM micrographs of
immediately after removal of orthodontic appliances and replicas of the active lesion (top) and after three months
cleaning (top) and after three months (bottom). Note the (bottom). Note the distinct step between the eroded surface
characteristic white and chalky surface appearance of the of the active lesion and the adjacent sound enamel, open
active lesion. The eroded surface layer has been worn away arrows (top). After three months, the furrow (arrows) has
and polished, giving the shiny and hard surface of the almost disappeared, and the step between the sound enamel
arrested lesion (bottom). and the arrested lesion surface is slightly enhanced
(bottom). From Thylstrup, 1988b.
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ADV DENT RES JULY 1994
154 THYLSTRUP ET AL.
Active lesions Arrested lesions 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
B v\\\\\\\\\\\\\\\\\\\\\V
"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
Fig. 17—Schematic drawing of the initiation (A) and fluids to supersaturation, causing a shift in equilibrium and re-
gradual progression of an enamel caries lesion (B to D). precipitation of minerals in the sites of demineralization. The
Caries initiation begins beneath undisturbed plaque with return to normal pH in the interior lesion is presumably a long
direct dissolution of the external micro-surface (A). Further process which may last several weeks (Holmen etal, 1987a).
progression leads to increased surface dissolution and Arrested lesions have commonly been described as
preferential subsurface dissolution (B to D). Arrows "remineralized" lesions, because it has been believed that the
indicate structural changes in corresponding stages of clinical observations of regression, glossy appearance, and
lesion arrestment. At each stage, the mechanical removal of surface hardness are the results of salivary repair (Gr0n, 1973;
the cariogenic plaque, which is physically closely ten Cate, 1983,1992). This concept has been widely accepted,
interrelated with the eroded surface layer, leads to surface because human saliva is supersaturated with calcium and
wear and polishing. The "new" surface of the arrested phosphate salts that form dental enamel (Gr0n, 1973). However,
lesion is therefore hard and shiny, in contrast to the chalky precipitation of calcium phosphate salts directly onto dental
and "soft" surface of the active lesion. Porosity in the enamel is rarely observed in spite of the supersaturation. It is
interior part of the subsurface lesion is reduced, due to now realized that salivary proline-rich phosphoproteins and
gradual return to normal pH, promoting reprecipitation of other inhibitors, such as statherin, inhibit crystal growth and
minerals from the internal enamel fluids. spontaneous precipitation of calcium phosphate salts from
supersaturated solutions (Hay, 1984; Hay etal, 1984). These
inhibitors, which also form essential components of the enamel
whitish, chalky surface (Fig. 15a). pellicle, are responsible for maintenance of the supersaturation
The opaque or white appearance of the enamel lesion is of calcium phosphate salts in saliva without precipitations on
conventionally assumed to be a result of the internal increase the dental hard tissues. Accordingly, the function of the
in enamel porosity (Silverstone, 1973). When air-dried, lesions inhibitors makes it unlikely that carious enamel is restored
in the clinic become more visible due to replacement of water significantly in vivo by salivary repair mechanisms. Similar
with air in the intercry stalline spaces (Thylstrup and Fejerskov, observations have been made after enamel surface etchings,
1994). Thylstrup and Fejerskov (1994) compared the clinical since the apparent repair has been the result of masking by
estimation of mineral loss with the principles behind the use of salivary proteins instead of mineral deposition (Lenz and
polarized light in conjunction with different imbibition media Miihlemann, 1963; Miihlemann et al, 1964; Meurmann and
for estimating enamel porosity in enamel sections. Asikainen, 1976;GarberoglioandCozzani, 1979). It is therefore
In light of the SEM examinations by Holmen etal (1985b, possible to conclude that the enamel surface defects will not be
1987b) and Artun and Thylstrup (1986, 1989), it is more refilled in vivo, either at the ultrastructural level (Thylstrup et
appropriate, however, to relate the white appearance of the al, 1983) or at the macroscopic level (Gr0n, 1973; Artun and
active enamel lesion with its characteristic chalky surface Thylstrup, 1986,1989). This also explains why surface defects
luster to two phenomena: The first one is the previously at any level of examination in fluorosed teeth do not show signs
discussed internal increase in enamel porosity due to subsurface of repair (Thylstrup and Fejerskov, 1979; Thylstrup, 1983;
demineralization. The second phenomenon is caused by direct Thylstrup et al, 1990), and that the gradual disappearance of
surface erosion. The enamel loses its shiny appearance, because the larger defects are explained by post-eruptive functional
the irregular surface generated by the erosion gives rise to a wear (Thylstrup, 1983).
diffuse back-scattering of the light. On this basis, it is possible In short, the data from in vivo caries studies show that the
to explain the clinical changes taking place when active lesions clinical and structural changes associated with lesion arrestment
have been turned into inactive or arrested lesions (Fig. 17). The or partial regression are not related to any salivary repair
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VOL. 8(2) IN VIVO CARIES MODELS 155
mechanism but are solely the result of mechanical removal of and caries on occlusal surfaces of first permanent molars in
the cariogenic biomass which is physically interrelated with the relation to stage of eruption. J Dent Res 68:773-779.
eroded surface of the active lesion. Obviously, this does not Carvalho JC, Ekstrand KR, Thylstrup A (1991). Results of 1
preclude subtle alterations at the crystal level, but our SEM year of non-operative occlusal caries treatment of erupting
studies did not reveal obliteration or "blocking" (ten Cate, permanent first molars. Community Dent Oral Epidemiol
1992) of either the external intercrystalline spaces or the 19:23-28.
surface layer, as also indicated by polarized light observations Darling AI (1958). Studies on the early lesion of enamel caries
(Holmen etal., 1987a,b). with transmitted light, polarized light and microradiography.
The results obtained by Backer Dirks (1966) in his classic Its nature, mode of spread, points of entry and its relation to
study have been used in several review papers and textbooks as enamel structure. BrDent J 105:119-135.
an indication of "remineralization" due to some vaguely defined Edgar WM, Rugg-Gunn AJ, Jenkins GN, Geddes DAM (1978).
salivary repair mechanism. In addition, examination of more Photographic and direct visual recording of experimental
recent literature makes it clear that the word "remineralization" caries-like changes in human enamel. Arch OralBiol 23:667-
is widely used to describe completely different phenomena 673.
caused by acid reactions, including fluoride interactions with Ekstrand KR, Nielsen LA, Carvalho JC, Thylstrup A (1993).
the dental hard tissues. Dental plaque and caries on permanent first molar occlusal
Communication depends to a large extent on a mutual surfaces in relation to sagittal occlusion. ScandJ Dent Res
agreement with regard to the meaning of the words. Scientific 101:9-15.
communication is thus based on an elaborate and specialized Frank RM (1990). Structural events in the caries process in
terminology in order to ensure that we are dealing with the same enamel, cementum, and dentin. J Dent Res 69(Spec Iss):559-
phenomena. There are therefore several reasons for a profound 566.
re-evaluation of the current uniform and inaccurate usage of the Frank RM, Brendel A (1966). Ultrastructure of the approximal
word "remineralization" in scientific communications. The dental plaque and the underlying normal and carious enamel.
need to avoid unnecessary confusion becomes particularly Arch OralBiol 11:883-912.
evident in relation to the clinical management of caries, because Garberoglio R, Cozzani G (1979). In vivo effect of oral
"remineralization" in terms of superficial mineral redeposition environment on etched enamel: a scanning electron
or "blocking" is an illusion which does not lead to lesion microscope study. J Dent Res 58:1859-1865.
arrestment. The complete end of lesion progression in vivo Geddes DAM, Cooke JA, Edgar WM, Jenkins GN (1978). The
requires mechanical removal of the bacterial origin of the effect of frequent sucrose mouthrinsing on the induction in
disease, the cariogenic plaque, which, in effect, is equivalent to vivo of caries-like changes in human dental enamel. Arch
the withdrawal of enamel specimens from acids in the laboratory OralBiol 23:663-665.
model systems in order to arrest further demineralization. Gorelick L, Geiger A, Gwinnett AJ (1982). Incidence of white
spot formation after bonding and banding. Am J Orthod
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