Australian Dental Journal 2008; 53: 281285

SYMPOSIUM REPORT
doi: 10.1111/j.1834-7819.2008.00063.x

Remineralization of deep enamel dentine caries lesions

JM ten Cate*
*Department of Cariology Endodontology Pedodontology, Academic Center for Dentistry Amsterdam (ACTA), Amsterdam, The Netherlands.

ABSTRACT
Enamel remineralization is generally studied in superficial (up to 100 lm) lesions, but in vivo caries lesions may be tenfold
deeper. This article addresses the question whether deep lesions, and extending into dentine, can be remineralized under
optimal conditions and if this process is influenced by agents affecting calcium phosphate precipitation and dissolution.
Lesions through enamel into dentine were first formed in thin sections and then continuously remineralized for periods up to
200 days. With longitudinal assessment by transversal microradiography it was showed that remineralization throughout
the depth of the lesion and into the dentine was possible, although this process is very slow. Fluoride and bisphosphonate
treatments affected mainly the deposition in the outer enamel. Although it was assumed that this would affect the diffusion
of ions to deeper layers, the treatments had no impact on remineralization in the inner enamel or dentinal parts of the
lesions. These findings are discussed with relevant theoretical considerations, and in their possible clinical implications.
Key words: Remineralization, dentine, minimal intervention dentistry, fluoride, bisphosphonate.
Abbreviations and acronyms: DEJ = dentino-enamel junction; GIC = glass ionomer cements; KHN = Knoop Hardness Numbers; MID =
minimal invasive dentistry; QLF = quantitative light fluorescence.
(Accepted for publication 19 May 2008.)

comprehensive packages of non-invasive care. New

INTRODUCTION
The past decades have led to major changes in
restorative and preventive dentistry. Various investiga-
tors reported that early caries lesions can be remineral-
ized from saliva. This process was studied in detail in
laboratory experiments and in clinical trials.1,2 Adhesive
dentistry has resulted in new non-metallic filling mate-
rials, such as composites based on acrylate resin and
glass ionomer cements (GIC) based on an acid base
precipitation mechanism. Restorations made with
adhesive materials no longer require a large cavity
preparation, but merely the removal of the tissue
affected by caries. Moreover, the notion became gener-
ally accepted that restorative intervention is generally
the beginning of a long sequence of re-restorations,
often leading to crowns and implants, irrespective of
how well the first filling was prepared.
The concept of minimal intervention or minimal
invasive dentistry (MID) has combined these three
major (paradigm) shifts in operative dentistry and
this philosophy is currently accepted worldwide. MID
now has its own materials, congresses and research
organization.3
In caries prevention, most protocols are still built
around fluoride, although improving oral hygiene, sugar
substitutes and antimicrobials are also part of the more
2008 Australian Dental Association
methodologies of caries diagnosis have been developed,
using the early changes of fluorescence induced by caries
in the tissue detected by Quantitative Light Fluorescence
(QLF),4 or with chromophores produced by bacteria (in
the Diagnodent device)5 as indicators.
Remineralization of enamel and dentine is studied
from two perspectives. First of all, it is the process of
mineral deposition from saliva or plaque fluid filling up
small enamel or dentine defects formed during the
demineralization episodes resulting from acid attack on
the tooth. The relative magnitudes of demineralization
and remineralization determine whether a tooth surface
remains sound or a caries lesion develops. This lesion
may then increase in severity eventually resulting in
a (deep) cavity. (For more details about this caries
equilibrium, see Featherstone.2) Alternatively, reminer-
alization is studied and described as the repair of
established lesions. Such lesions have developed over a
long period but may be filled in with calcium phosphates
when external conditions favour mineral deposition.
This type of remineralization may either be complete
and partial; when the mineral precipitating in the lesion
is less soluble than the original tissue, this remineraliza-
tion will help in preventing or limiting future tissue loss.
Remineralization of superficial enamel lesions is
well documented in hundreds of studies completed at
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JM ten Cate
numerous laboratories in the last century. Studies
on the basic mechanism of remineralization and on
methods to stimulate this process have led to the
conclusion that the caries preventive effect of fluoride is
beyond any doubt. This is partly attributed to the
enhancing effect of fluoride on calcium phosphate
precipitation, hence remineralization.6
A topic that has received limited attention is whether
there is a point of no return beyond which remineral-
ization can or does no longer occur. Koulourides (one
of the pioneers in remineralization research), stated that
if caries has weakened the tooth structure to below a
hardness of 150 Knoop Hardness Numbers (KHN),
remineralization could no longer be achieved.7,8 Con-
ceptually this was seen as the point where the mineral
structure is destroyed to the extent that reprecipitation
of mineral on remaining hydroxyapatite crystallites was
no longer possible. In caries diagnosis using X-rays, the
extent of caries is graded at various depth levels in the
enamel and dentine, realizing that loss of tissue has
occurred also considerably beyond the depth identified
on the X-ray picture. The current consensus is that
caries beyond the dentino-enamel junction (DEJ)
should be treated with restorations, and lesions up to
that point should receive extra preventive care. How-
ever, it was never studied whether deep lesions,
extending into dentine, can be remineralized if such
lesions are subjected to a continuous remineralization
scheme. Obviously, such lesions should be protected
from mechanical damage. Ideally, a study with this
objective should be carried out in vivo or using an in
situ model. However, given the fact that remineraliza-
tion is a very slow process, it seemed technically
impossible to complete such a study with volunteer
subjects or with patients, within an acceptable time
period.
This article reiterates previous results from in vitro
remineralization experiments of deep lesions, extending
into dentine.9 The aim was to explore whether such
lesions can still be remineralized and how this could
be affected by treatments that would stimulate or inhibit
calcium phosphate precipitation. These findings are then
discussed from a theoretical and clinical perspective.
Experimental design and results
The experiment was performed in groups of 100 lm
thick sections cut from ground bovine incisors. Sections
were fully embedded in Araldite, and after setting of the
resin the outer 200 lm of the enamel was cut with a
diamond coated wire sectioning machine. This was
done to remove surface enamel and to reduce the
enamel thickness to the DEJ. Lesions through enamel
and into dentine were formed during 1015 days
in individual solutions containing 1.5 mM CaCl2,
0.9 mM KH2PO4 and 50 mM acetate buffer (pH 4.8),
282
with the addition of 0.1 ppm KF to prevent surface
loss.10,11 Next the lesions were immersed in solutions
supersaturated and stochiometric to hydroxyxapatite
(HAP), so as to achieve remineralization (1.5 mM
CaCl2, 0.9 mM KH2PO4 20 mM HEPES buffer pH 7.0
and 130 mM KCl). All steps in this process were
monitored and recorded by taking microradiographs, as
was the remineralization phase by taking microradio-
graphs weekly during 200 days. Profiles were scanned
at fixed positions of the specimens. Five independent
specimens were run at each condition.
Treatments tested were control (no additional treat-
ments), weekly five-minute fluoride rinses with 1000
ppmF, addition of 1 ppmF to the remineralizing
solution, and a five-minute single treatment with the
calcium phosphate precipitation inhibitor bisphospho-
nate (as 2 mMethaneHydroxy-BisPhosphonate).
The primary findings of these experiments are given
in Fig 1. To average out variation between specimens in
enamel thickness and overall lesion depth, data are
expressed as percentage repair in four zones: outer
enamel, inner enamel, outer dentine, inner dentine. The
relative remineralization parameter shows that remin-
eralization in dentine (panels C and D) may be up to 80
per cent after 200 days, while remineralization in inner
enamel (panel B) plateaus at around 40 per cent. Only
in the outer enamel (panel A) is the remineralization
significantly affected by the various treatments, thus
resulting in relative remineralization values between 40
and 100 per cent. (For full experimental details and
results, see ten Cate.9)
Theoretical considerations
Whether remineralization occurs in enamel-dentine
lesions extending beyond the DEJ depends on several
factors. First, the concentration of mineral ions at the
site of precipitation should exceed supersaturation to
hydroxyapatite. This requires that not all calcium and
phosphate ions entering through the lesion pores have
already been precipitated in the layers closer to the
surface. Secondly, the site of precipitation requires
nuclei for precipitation, considering that substantially
larger degrees of supersaturation are needed for
de novo precipitation of apatite or precipitation onto
remaining organic matrix than on fragments of enamel
or dentine apatite crystals. Detailed electron micro-
scopic analysis of crystallites in various zones of the
lesion confirmed that remineralization occurs by
growth of existing crystals to dimensions larger than
the original crystallites.12
Rate control
Considering the theory of crystallization kinetics,
a precipitation at greater depth requires that the
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 Remineralization of deep caries lesions

(a) 120 (c)

100

80

60
Relative remineralization (%)

40

20

0
(b) (d)

100

80

60

40

20

0
0 50 100 150 200 0 50 100 150 200

Time (days)
Fig 1. Average relative remineralization, with time of remineralization, for the five experimental groups in the four zones of interest: a: outer
enamel, b: inner enamel, c: outer dentine, and d: inner dentine (n = 5 per Group). Markers indicate the five experimental groups: control (r),
weekly 1000 ppm fluoride treatments (m), continuous presence of 1 ppm fluoride (s), single MHBP treatment (h), and combination
treatment: single MHDP treatment at start followed at 56 days by weekly 1000 ppm treatments (n). Relative remineralization is defined as the
percentage of mineral deposited at a given depth to fill in the mineral removed during lesion formation. (Reprinted with permission from JM ten
Cate; J Dent Res 2001;80:14071411.)

precipitation reaction is slow compared to mass transfer
of ions, thus allowing diffusion to supply ions throughout
the lesion. If such a condition is met, the mineral ion
concentrations are uniform throughout the lesion.
Little data are available to confirm this assumption.
With Arrhenius plots (temperature dependence of
reactions), we previously determined that the activation
energies of remineralization of subsurface lesions versus
surface softened enamel were significantly different. We
then concluded that diffusion processes were rate
limiting in the lesion remineralization (judging from
the lower activation energies), while surface softened
lesion remineralization was controlled by surface
reactions.13 The current findings seem to contradict
these early observations.
Demineralization
More potentially relevant data are available on enamel
demineralization, showing that its rate is determined by
diffusion processes, although differences in mechanism
were noted between demineralization in vitro and
in vivo.14,15 Recently, we developed an artificial groove
model, in thin sections, to simulate demineralization in
fissures. In this model we periodically monitored mineral
loss with microradiography and pH and calcium
2008 Australian Dental Association
concentrations throughout the depth of the grooves with
microelectrodes. The observed gradients in pH and
calcium activities pointed to diffusion inhibition for
demineralization even in the 250 lm wide grooves (ten
Cate and Buijs, unpublished data). Making this compar-
ison it should be noted that precipitation rates are
probably 10 times slower than dissolution rates at
relevant super- and undersaturation conditions.
Enamel-dentine continuum
Even if enamel and dentine form a continuum within the
tooth, their respective apatite crystallites differ in size
and composition, reflected in differences in solubility. In
an aqueous environment this would eventually lead to
the complete dissolution of the dentine crystallites in
favour of the enamel crystallites, a process known as
Ostwald ripening in crystal chemistry. Findings in
accordance with this principle were observed when
enamel and dentine (lesions) were de- and remineralized,
respectively, when placed in juxtaposition.16
Matrix
For in vivo remineralization, additional mechanisms
could play a role as indicated by in situ studies
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JM ten Cate

completed by van Strijp and colleagues.17 In a compar- (a) enamel dentine

ison of various fluoride toothpastes they observed full
remineralization of dentinal lesions in many of the
participating subjects, and small changes in the contra-
laterally placed enamel lesions. This observation hints
that remineralization conditions for dentine lesions may
be favourable compared to enamel lesions, although the mineral

full explanation of this finding is yet lacking. One may concentration (IP)

formulate the hypothesis that the demineralized organic

(b) enamel dentine
matrix of dentine may constitute a scaffold to enhance
remineralization.
Furthermore, non-collagenous components of this
matrix (SIBLINGs, osteocalcin, proteoglycans).18,19
may interact directly with crystal formation and crystal
growth during dentine remineralization. In the context mineral
of the current experiment, this all could suggest that
concentration (IP)
remineralization of dentine proceeds faster than for
enamel and would create a concentration sink beyond (c) enamel dentine
the DEJ.

Modelling
Many of the abovementioned findings need further
study. However, given the duration of remineraliza- mineral

tion studies of deep lesions, it seems worthwhile to concentration (IP)

consider in silico experiments; computer simulations
of remineralization using model parameters that can (d) enamel dentine

be determined individually or are available in the

literature (rates of apatite precipitation, dissolution,
diffusion constants, etc.). Obviously, as with any
simulation model, this approach helps to identify or
illustrate the relative importance of the individual
steps in a complex process, in this case diffusion,
precipitation, tortuosity, etc. It is beyond the scope of
this presentation to describe the numerical approach in
detail or give all the equations for the separate steps in
the process (for details see ten Cate).20 Examples of
such computational exercises are included as illustra-
tion in Fig 2.
Enhancing remineralization of deep lesions
mineral
concentration (IP)
Fig 2. Computer simulation of diffusion precipitation processes in
pore in enamel lesion extending into dentine. Two dimensional
diffusion was modeled using Ficks law of mass transfer, and
precipitation using the thermodynamic laws of dissolution and
precipitation (for details see ten Cate).20 Various conditions were
modeled: a, b: fast precipitation resulting in concentration gradient
in pore and preferential deposition in outer enamel c: slow
precipitation resulting in homogeneous concentration and precipita-
tion throughout the depth of the pore d: preferential deposition of
mineral in dentine, resulting in concentration sink towards dentine.

Fluoride
Traditionally, the focus in the development of agents
aimed to enhance remineralization has been on fluoride.
The presence of low levels of fluoride increases the
degree of supersaturation with respect to fluoridated
hydroxyapatite. This thermodynamic property is the
rationale for the enhancement of remineralization
by fluoride. This, however, could lead to excessive
remineralization of the surface layer of lesions and
consequently lesion arrestment.21 In the described
experiments (Fig 1) the tested agents (fluoride and
bisphosphonates) were found to give rise to diverging
results in the outer enamel, but these treatments did not
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significantly affect precipitation of mineral in the inner
enamel and dentine. Fluoride treatments, whether as
1000 ppm topical treatments or continuously present at
1 ppm, were both beneficial for repair of deep lesions,
at least in the outer enamel.
Calcium
After analysing comprehensive literature data on
in situ remineralization, saliva and plaque mineral
ion compositions and saliva flow dynamics, we have
proposed that, in addition to fluoride, calcium may be
rate limiting in remineralization.22 Since then many
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new products, including toothpastes and chewing
gums, have been formulated with the aim to supply
calcium ions to the oral cavity.23,24 Recently, clinical
studies and in situ trials have confirmed the potential of
this remineralization approach.25,26 There seems scope
to also study such products with the aim to enhance
deep rather than superficial lesions.
Clinical aspects
Obviously, conditions in the mouth are very different
from the ideal remineralization conditions used in the
experiments described in this article. An important
in vivo challenge to the remineralization of deep lesions
are the periods of low pH in the plaque which worsen
the degree of mineralization rather than improving it.
Moreover, the teeth are subject to mechanical forces
which could break the surface of the lesion and create a
retention site for bacteria. Once the surface is irrepa-
rably damaged, clearly the chance for a non-invasive
repair of the lesion is lost.
CONCLUSIONS
The experiments described in this study show that
remineralization of lesions extending into the dentine is
possible. This process takes a considerably long time,
which clinically is unacceptable. Nevertheless, studies
to further our knowledge in this field would contribute
to the area of minimal intervention dentistry.
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Address for correspondence:
Professor JM ten Cate
University of Amsterdam and Free University
Amsterdam
Academic Center for Dentistry Amsterdam (ACTA)
Royal Netherlands Academy of Arts and Sciences
Louwesweg 1
1066EA Amsterdam
The Netherlands
Email: j.t.cate@acta.nl
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