Eur J Oral Sci 2002; 110: 392–395 Copyright  Eur J Oral Sci 2002

Printed in UK. All rights reserved European Journal of

Oral Sciences
ISSN 0909-8836

Short communication
R. P. Shellis1, A. S. Hallsworth2,
Organic material and the optical J. Kirkham2, C. Robinson2
1
Division of Restorative Dentistry, Department
properties of the dark zone in caries of Oral and Dental Science, University of
Bristol, Bristol, UK, 2Department of Oral
Biology, University of Leeds Dental Institute,
lesions of enamel Leeds, UK

Shellis RP, Hallsworth AS, Kirkham J, Robinson C: Organic material and the optical
properties of the dark zone in caries lesions of enamel. Eur J Oral Sci. 2002; 110: 392–395.
 Eur J Oral Sci, 2002
Dr R. P. Shellis, Division of Restorative
Sections of uncavitated natural caries lesions of human enamel were extracted with Dentistry, University of Bristol Dental School,
various solvents and examined by polarizing microscopy. After lipid extraction, the Lower Maudlin Street, Bristol BS1 2LY, UK
dark zone enlarged by 19–162% and its birefringence increased, while after protein
Telefax: +44–117–9284778
extraction it shrank by 37–92% and became less birefringent. It is concluded that E-mail: r.p.shellis@bris.ac.uk
occlusion of submicroscopic pores by organic material largely accounts for the optical
properties of the dark zone. The results are not consistent with occlusion of pores by Key words: dental enamel; caries; protein; lipid;
reprecipitation of mineral. On the basis of previous work, organic material in this polarizing microscopy
location could influence demineralization and remineralization. Accepted for publication July 2002

The dark zone lies close to the inner boundary of early
natural caries lesions of enamel. In sections immersed in
organic liquids of high refractive index (e.g. quinoline), it
is pseudo-isotropic or positively birefringent in polarized
light, unlike the overlying body of the lesion and the
underlying translucent zone, which are both negatively
birefringent (1). The optical properties of the dark zone
are believed to be due to submicroscopic pores with
molecular-sieve properties (2). Although the pores can be
dehydrated by a solvent such as quinoline, which can
dissolve a limited amount of water (3), the pores or their
entrances are too narrow to permit entrance of the sol-
vent itself. The pores thus remain filled with water
vapour and acquire a low refractive index. This in turn
generates positive form birefringence which compensates
the negative birefringence of the enamel mineral and
causes the net birefringence of the dark zone to be zero
(pseudo-isotropy) or positive. There is some evidence
that pores in sound and carious enamel are occluded by
organic material (4, 5) and the aim of this study was to
determine whether this could contribute to the optical
properties of the dark zone.
Material and methods
Eight extracted human teeth, stored in 70% ethanol,
showing uncavitated white- or brown-spot approximal
lesions were sectioned vertically through the lesions
(Microslice 2 saw; Malvern Instruments, Malvern, UK).
The sections were lapped to about 80 lm thickness
with 1200 grade silicon carbide in water. There were six
white-spot lesions (W1–W5; W1a and 1b on the same tooth)
and three brown-spot lesions (B1–B3).
In a first experiment (experiment 1), sections were
exposed to a sequence of treatments: (a) Soxhlet extraction
in a 1 : 1 mixture of chloroform and methanol (Ch-Me)
(24 h); (b) phosphate buffer (0.1 mol l)1, pH 7.0, room
temperature, 24 h); (c) urea solution (6 mol l)1, pH 7.0,
room temperature, 24 h); (d) Soxhlet extraction in anhy-
drous ethylenediamine (ED) (48 h). The sections were wa-
shed in ethanol after treatment (a), in water after treatments
(b) and (c) and washed by Soxhlet extraction with 100%
ethanol for 4 h after treatment (d). Before the experiment,
and after each treatment, sections were blotted dry, im-
mersed in iso-propanol for 24 h, then in quinoline for 24 h
and finally photographed on colour positive film.
The effects of treatments were quantified by the changes
in the area (AIP) occupied by the dark zone. In lesions W3,
W5, and B1–B3 the combined areas of small isotropic or
positively birefringent areas superficial to the dark zone
were added to AIP because it was often impossible to assign
them unequivocally to the dark zone or the body of the
lesion. Using a Nikon 6CT2 profile projector (magnification
·210), the outlines of all isotropic/positive regions were
traced on to acetate sheet, taking the junction of the iso-
tropic fringe with the negatively birefringent enamel as the
boundary. The enamel surface and other ÔlandmarksÕ were
traced to enable tracings of large lesions, which required
more than one photograph, to be montaged. Areas were
measured using a graphics tablet and ScionImage software
(Scion Corporation, Frederick, MD, USA: www.scion-
corp.com). Where outlines were not complete – where the
 Organic material in enamel caries 393

Fig. 1. Dark zone in white-spot lesion W2. (A) untreated; (B) Following chloroformlmethanol extraction; (C) Following subsequent
ethylene diamine extraction. Bar, 0.5 mm.

dark zone merged with isotropic inner enamel or where part

of a section was lost after one of the treatments – meas-
urement was restricted to the area common to all photo-
graphs of that section.
In a second experiment (experiment 2), sections of lesions
W1, W3, W4, and W5 were recorded and quantified as above,
before and after dehydration for 48 h in 100% methanol,
either at room temperature or by Soxhlet extraction.
Part of a sample of powdered human enamel, prepared as
described previously (6), was placed in a sintered-glass
thimble, Soxhlet-extracted for 48 h with ED, then with
ethanol for 24 h and air-dried. Samples of the original and
extracted enamel were dissolved in HCl and analysed for
calcium, magnesium, inorganic phosphorus and carbonate
as described previously (6).

Results and discussion

In experiment 1, because of repeated handling, sections
of lesions W5 and W1a were lost after buffer and ED
treatment, respectively. The sections of lesions W4 and
B3 were incomplete after ED treatment. In experiment 2,
the section of lesion W1a was incomplete after Soxhlet
extraction with methanol.
All intact lesions showed dark zones, each with a weakly
positively birefringent central region, grading into an
isotropic fringe (Figs 1A and 2A). After Ch-Me extrac-
tion, AIP increased (Figs 1B and 2B, Table 1). In addition,
the central region of positive polarization colour enlarged
and became brighter, clearer and much more clearly de-
marcated from the isotropic fringe. After phosphate buf-
fer treatment, the dark zone in one area of lesion W1b
showed a small increase in area but otherwise neither this
treatment nor urea extraction produced any change. The
ED extraction had the opposite effects to Ch-Me. First,
AIP decreased to less than in the untreated sections (Ta-
ble 1) and isotropic/positive areas within the body of le-
sions W3 and B1-B3 disappeared (Figs 1C and 2C).
Second, all residual areas were isotropic and showed no
positive birefringence. After dehydration with either hot
or cold methanol, AIP increased, but to a much smaller
Fig. 2. Brown-spot lesion B3, showing dark zone and isotropic/
extent than after Ch-Me treatment (Table 1). The ED positive regions, B, in body of the lesion. (A) Untreated; (B)
extraction did not alter enamel composition (Table 2). following chloroform/methanol extraction; (C) Following
We aimed to extract first lipid and then protein, to subsequent ethylenediamine extraction. P, area of positive
study the effects in sequence. Phosphate buffer at birefringence. Bar, 0.5 mm.
394 Shellis et al.

Table 1
Changes in area (AIP) of dark zones and other positive or isotropic regions within lesions, relative to the area in the untreated sections

% Change after dehydration in 100% methanol

% Change after % Change after
Lesion no. lipid removal ethylenediamine extraction Hot Cold

W1a +29 –* +3.7, +10.1 –

W1b +19 )62 – +9.9
W2 +100 )75 – –
W3 +162 )37 – +4.5
W4 +72 )84– +25.6 )10.9
W5 +87 –* )2.9 –
B1 +22 )92 – –
B2 +88 )63 – –
B3 +93 )67– – –

*Section lost after ethylenediamine extraction.

–Part of section lost after ethylenediamine extraction. Measurement relates to corresponding region of section before treatment.

1 mol l)1 desorbs many proteins from apatite (7), while with the most soluble fraction of the tissue (8). If ED
urea solubilizes proteins by disrupting hydrogen bond- does not solubilize mineral, then the greatly reduced AIP
ing. Both treatments leave the protein molecules intact, and the loss of positive birefringence after ED treatment
whereas hot ED causes degradation of protein. Our must be due to removal of protein that was occluding
results show that organic material is a major factor in the pores. Thus, the molecular-sieve effect responsible for the
extent and optical properties of the dark zone. More- dark zone is not due to small pore size. This is consistent
over, isotropic/positive patches in the body of the lesion with electron microscopy observations (9, 10) showing
responded to extraction of organic material in the same that even the smallest intracrystalline pores created by
way as the dark zone, suggesting that their optical early carious demineralization of enamel are sufficiently
properties have the same structural basis. large (1–10 nm diameter) to accept quinoline molecules
Because of its small molecular size, methanol replaces (mean molecular radius about 0.26 nm). The almost
water in pores inaccessible to other solvents (2). How- complete lack of effect of phosphate buffer or urea could
ever, the relatively small effect of 100% methanol shows be due to the protein in the dark zone being too firmly
that the effect of Ch-Me extraction is mostly due to lipid bound to the mineral or too insoluble to be extracted, or
extraction and not to efficient dehydration. The marked to enamel pores being too small to allow significant
expansion and increased positive birefringence of the diffusion of intact protein molecules under the treatment
dark zone seen after this treatment suggests that the zone conditions.
contains small pores which, in intact sections, do not The organic material responsible for the isotropic/
generate enough form birefringence to compensate the positive regions could be the enamel matrix, which
negative birefringence of the mineral. This could be due comprises approximately equal parts of lipid and protein
to the pores containing lipid instead of water or to (11). However, it is known that exogenous protein is
occlusion of pore entrances by lipid, which prevents taken up by lesions (12, 13) and albumin has been
removal of water. Lipid extraction would leave residual identified in natural lesions (14).
protein, occluding the pores or their entrances, which Our results are inconsistent with the concept (15) that
would allow transfer of water but prevent ingress of the pores in the dark zone arise by occlusion of larger
quinoline. pores, created earlier in the caries process, by reprecipi-
Anhydrous ED treatment caused no change in the tation of mineral, since removal of organic material
composition of enamel mineral. In particular, there was would have no effect if that were the case. The repre-
no loss of magnesium or carbonate, which are associated cipitation hypothesis is also inconsistent with the lower
mineral content of the dark zone compared with the
translucent zone (8) and with ultrastructural observation
Table 2 of increased numbers of intra- and inter-crystalline pores
Composition of powdered enamel before and after ethylenedi- in the dark zone (9, 16).
amine extraction Lesion formation is strongly influenced by both pro-
teins (17) and lipids (18) and it has been suggested that
Ethylenediamine-
albumin, which appears to be significantly more abun-
Untreated enamel extracted enamel
dant in white-spot enamel than sound enamel (14) and is
Calcium (% w/w) 35.9 ± 0.6 35.2 ± 0.4 an inhibitor of apatite crystal growth (19), could interfere
Phosphorus (% w/w) 17.5 ± 0.5 17.0 ± 0.7 with lesion remineralization (14). It is possible that the
Magnesium (% w/w) 0.257 ± 0.007 0.237 ± 0.009 occlusion of pores by organic material which we have
Carbonate (% w/w) 3.89 ± 0.33 3.97 ± 0.27 demonstrated, not only in the dark zone, close to the
Ca/P (mol mol)1) 1.59 ± 0.05 1.60 ± 0.02
advancing front of the lesion, but also in the body of the
Values are means of triplicate determinations ± SD lesion in some lesions, might influence demineralization

and remineralization. We therefore believe that further
work on the interaction of organic material with the
caries process would be valuable.
Acknowledgements – We thank Mr A R Lee for technical
assistance. R.P.S. is grateful to Glaxo SmithKline PLC for
financial support.
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