Clinical Oral Investigations (2022) 26:2597–2605

https://doi.org/10.1007/s00784-021-04229-4

ORIGINAL ARTICLE

ICCMS™ root caries lesions stages and their underlying depth

towards the pulp: an in vitro study with histologic evaluation
Kim R. Ekstrand1 · Thais Cordeschi2 · Ninoska Abreu‑Placeres1,3

Received: 12 August 2020 / Accepted: 13 October 2021 / Published online: 20 October 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
Objective To examine the relationship between the ICCMS™ (International Caries Classification and Managing System)
features of root caries lesions and the underlying depth of the lesion towards the pulp. In order to control for bias, the study
followed the tailored document for risk of bias assessment (RoB-tool) recently published. A pilot study showed that the
outline of the pulp was much clearer on horizontal compared to vertical sections through the lesions (p = 0.03) and that the
histological stereomicroscopical (SM) assessed lesion depth towards the pulp was not influenced by the cutting direction
(p = 0.155).
Material and methods A sample of extracted permanent molar teeth (n = 100) were classified independently by two of the
authors according to ICCMS™ as no sign of root caries lesion 0 = sound; 1 = initial lesion (non-cavitated); 2 = moderate
lesion (cavity depth ≤ 2 mm) and 3 = extensive lesion (cavity depth > 2 mm). After horizontal sectioning (HS) through the
lesion, the depth of the underlying lesion was SM assessed independently by two of the authors as 0 = no lesion; 1 = lesion
in outer 1/3; 2 = middle 1/3; and 3 = inner 1/3 of the dentine towards the pulp.
Results Intra- and inter-reproducibility (weighted kappa values ≥ 0.83); the accuracy (Spearman’s rho-values) = 0.94 and
0.95; and specificity/sensitivities/AUC values (three different thresholds) were ≥ 0.91, ≥ 0.93, and ≥ 0.96, respectively.
Conclusion Under the umbrella of the RoB-tool, the validity in terms of the reproducibility and accuracy of the ICCMS™
root caries scoring system was high.
Clinical relevance By means of the ICCMS™ root caries scoring system, the underlying lesion depth can be estimated, which
must be considered when managing the lesion.

Keywords Dental caries · Root caries · Demineralization · Caries detection

Introduction occurs, and many elderly people are medically compromised

Root caries lesions are already a problem and maybe an
increasing problem in the future, as many of our popula-
tions maintain their teeth throughout life, gingival retraction
* Kim R. Ekstrand
kek@sund.ku.dk
1
Faculty of Health and Medical Sciences, Section
of Cariology and Endodontics, University of Copenhagen,
Copenhagen, Denmark
2
Department of Pediatric Dentistry, University of Sao Paulo,
Sao Paulo, Brazil
3
Biomaterials and Dentistry Research Center (CIBO‑UNIBE),
Academic Research Department, Universidad
Iberoamericana, Santo Domingo, Dominican Republic
[1].
In order to assist in the detection of caries lesions, a
number of scoring systems staging root caries lesions have
been devised [2–6]. Banting [7] pointed to the fact that the
reliability of the visual-tactile diagnosis of primary root
caries lesions is not strong. The ICDAS (International Car-
ies Detection and Assessment System) root caries scoring
system includes 3 stages: code 0 = no caries, code 1 = car-
ies lesion, but no cavitation (0.5-mm loss of anatomical
structure is accepted), and code 2 = caries lesion and cavita-
tion ≥ 0.5 mm [5]. Mendieta Facetti [8] mapped the associa-
tion between the ICDAS root caries system and the demin-
eralization depth of the lesions and found that code 2 lesions
were significantly deeper than code 1 lesions. As with coro-
nal caries lesions, correct detection and assessment of root

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Vol.:(0123456789)
2598
caries lesions are crucial in order to provide an accepted
diagnosis and to manage the lesions accordingly [9].
The aim of the present in vitro study was to demonstrate
the relationship between the visual features of the root caries
lesions, classified with the successor to the ICDAS scoring
system the ICCMS™ systems (International Caries Clas-
sification and Managing System) [5, 6], and the depth of
the underlying lesion towards the pulp, where the latter was
assessed histologically.
Materials and methods
Recently, a caries research group has based on the STARD
recommendation—recommendations and checklists for
Standard Reporting of detection and diagnostic studies—
e.g., within medicine [10], developed a tailored risk of bias
assessment tool for caries diagnostic studies (RoB-tool)
[11]. This initiative arises due to the need to diminish bias
in future caries studies by allowing better comparability
between original studies and higher quality of systematic
reviews. It was decided to structure the present study, tak-
ing into account the listed 16 indicators for low versus high
risk of bias suggested in the RoB-tool (see supplementary
material). This called for a pilot study in order to find the
best way of cutting through the lesions (vertically or hori-
zontally) and to devise a precise way of measuring the depth
of the lesion on the section faces using a stereomicroscope
supplemented by a software system.
Ethical considerations
In the present in vitro study, both in the pilot and in the main
study, we used extracted teeth. As the human teeth were
extracted for therapeutic reasons and, collected from anony-
mous donors, as leftover biological material, no notification
to the Danish National Ethical Committee is required.
Pilot, training, and calibration
The pilot study was conducted by the first author and initi-
ated before the main study. A total of 21 extracted permanent
molar (n = 16) and premolar teeth (n = 5), stored in 0.1%
thymol water, each with one or more located root caries
lesions were selected. Photos (× 5, Fig. 1) of the lesions were
made by a stereomicroscope (SM) (Zeizz Stereo Discovery
V.8 – lens 1.0, Oberkochen, Germany) with the aid of a
software program (DeltaPix, Smørum, Denmark). All 21
selected lesions were then hemi-sectioned in a vertical direc-
tion through the center of the lesion with a saw (EXAKT,
Advanced Technologies, GmbH, Norderstedt, Germany) and
photos were made of the two-section faces using the SM.
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Clinical Oral Investigations (2022) 26:2597–2605
This was followed by sectioning through the lesions in a
horizontal direction on both vertical sections. SM analyses
showed that in 5 cases the pulp could not be identified on
any of the vertical cut sections, while the pulp always could
be noted on the horizontally cut sections (sign test; p = 0.03).
Both pairs of the vertical cut section faces of the remaining
16 teeth were examined in the SM and the section show-
ing the pulp most clear was chosen for further examination.
The depth of the lesion towards the pulp on these sections
was measured in the following way (Fig. 1a): a line was
made from the outer surface of the lesion to the deepest
part of the lesion to the pulp, following the directions of
the dentine tubules. Still in the microscope, the line was
divided into 1/3 using the software program measuring the
distance on the lines in micrometer. An X was made illus-
trating the depth of the lesion and each lesion was assessed
if the lesion extended into the outer 1/3 (initial), middle 1/3
(moderate), or inner 1/3 (extensive) of the distance from
the surface towards the pulp. A color change from brown-
ish/yellowish to grayish discoloration was used to estimate
carious dentine from non-carious dentine as suggested in
the literature [12–15].
Concerning the horizontal counterpart to the vertically
cut section faces examined above, a line following a “90°”
direction from the surface through the deepest part of the
lesion towards the pulp was made (Fig. 1b). The same pro-
cedures to divide the distance from the surface to the pulp
through the lesion into 1/3 as described above were used for
the horizontally cut sections. The first author repeated all the
measurements concerning the depth of the lesions 29 days
after the first assessment.
Concerning lesion depth on the 16 cases where the pulp
could be seen on both section faces (both the vertical and
the horizontally), in 14 cases (88%), the lesion depth was
assessed similar deep whether cut vertically or horizon-
tally (ordinal logistic regression, adjusted for cluster effect;
n = 32; association coefficient =  − 0.278; chi-square = 2.02;
p = 0.155; CI 95% − 0.66 to 0.10). There was no difference
between first and second assessments. Based on the results,
it was decided, in the main study, to cut the lesions hori-
zontally and assess the lesion depth towards the pulp as
described above.
The first author then trained the two other authors in the
following: ICCMS™ concerning the root caries part [5, 6]
and in the Ekstrand, Ricketts, and Kidd (ERK) caries histo-
logical assessment system [13, 14]. This session took about
4 h and consisted of power points presentations and examin-
ing extracted teeth.
The ICCMS™ system divides the severity of the root
caries lesion into 4 stages, sound, initial, moderate, and
extensive. The initial lesions are non-cavitated, while mod-
erate (≤ 2 mm) and extensive root caries lesions (> 2 mm)
are cavitated lesions. In the present study, the depth of the
Clinical Oral Investigations (2022) 26:2597–2605 2599

Fig. 1  Extracted tooth with a

root caries lesion on the approx-
imal surface (× 4). (1a) shows
one of the section faces in the
vertical hemi-sectioned tooth
through the root caries lesion
(× 6). A line in the direction of
the dentine tubule is made from
the surface through the deepest
part of the lesion and to the pulp
(red line). This line is divided
into thirds. Thus, the deepest
part of the lesion (arrow) in
this case is into the inner third.
In addition, a horizontal cut
is made through the deepest
part of the lesion (1a arrow).
The apical part of this is seen
in (1b) (× 6). A straight line is
made from the surface to the
pulp through the lesion and
divided into thirds. Also, on the
horizontal cut section face, the
lesion extends (white arrow)
into the inner third of the lesion

cavitation was measured by a perio-probe (Model 8-520B,
Parainen, Finland). The probe was used to identify any
breakdown due to caries. The deepest part of the cavity was
explored by the perio-probe. The perio-probe was held in a
“90°” direction from the bottom of the cavity and the nearest
non-cavitated part of the tooth was used as a reference point
for assessing if the depth was ≤ 2 mm or above 2 mm. If in
doubt, we scored low.
After the powerpoint presentation, showing numerous
examples of root caries lesions, 10 permanent molar teeth,
where 8 had root caries lesions, were examined and dis-
cussed. After the theoretical session, the 10 extracted teeth
were sectioned, horizontally, through the lesions or at the
sound part of the roots and the histological reactions (demin-
eralization versus hypermineralized dentine) were examined
using a microscope (× 5) in relation to where the pulp was
located. The first and second authors measured the lesion
depth on the section face as described above. The second
author then examined twice an additional 20 teeth with
root caries lesions in order to get familiar with both scoring
systems to be used in the main study. Perfect agreement for
the second author (C.T.) was for the ICCMS™ 17 out of
20 (85%) and for the histological assessment, 17 out of 19
(89%), then one tooth broke during the cutting procedure.
Sample size estimation
Our calculation for the sample size was based on the fol-
lowing consideration: a correlation coefficient of 0.33,
power (1—β) = 0.9 and α = 0.05, giving a sample size of
92 teeth [16]. A total of 100 posterior teeth were therefore
to be selected as we counted that 10% of the teeth would be
destroyed due to the preparation technique used in the study.
Visual acuity and clinical/scientifical experience
of the examiners
The visual acuity of the authors was tested using Snellen
Chart online (https://​www.​provi​su.​ch/​images/​PDF/​Snell​
encha​r t_​en.​pdf) and the results were for all 20/20 when

13
2600
using glasses. All authors are dentists, K.E. is Ph.D. and
experienced with performing dentistry on grown-ups, and
T.C. and N.A. are both pediatric dentists and Ph.D. students.
Inclusion and exclusions criteria
One criterion for inclusion in the main study was that the
selected teeth should all be permanent molar teeth, another
was that no roots should be broken, so it could be controlled,
that the root apexes were completed [11]. Teeth with resto-
rations located close to the examination site were excluded.
The teeth used in the pilot study were not included in the
main study.
The authors selected the 100 permanent molar teeth from
a tooth bank containing thousands of extracted teeth, located
at the Department of Odontology, University of Copenha-
gen, Denmark The teeth were kept in 0.1% thymol solution
until selected for the study.
After selection, the teeth were cleaned with a dry tooth-
brush and rinsed in running water for 2 h before the initial
examination to select a particular area (sound) or lesion if
there was more than one lesion on the root. This part of the
procedure was done by the last author. The teeth were placed
Fig. 2  This shows a tooth with a moderate root caries lesion (encir-
cled) located at the enamel-cement border on an approximal surface
(× 6). (2a) (× 5) is the related section face through the lesion after sec-
tioning. (2b) (× 5) the deepest part of the lesion is marked with an X.
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Clinical Oral Investigations (2022) 26:2597–2605
in silicone with the surface of interest facing upwards. The
teeth were then numbered and photographed in position in
a stereomicroscope × 6 (Zeizz Stereo Discovery V.8 – lens
1.0, Fig. 2).
Visual detection
Initially, the last author mapped the part of the root that
was of interest (approximal or smooth root surface, Fig. 2)
and the location of the lesion/sound area from the enamel-
cement border (whole mm, Fig. 2).
Then, the first and second authors (K.E. and T.C.) inde-
pendently, by the naked eye, but with optimal light condi-
tions and with a perio-probe with a scale from 0–10 mm
visually assessed the areas of interest. Afterwards, the area
of interest was classified according to Pitts et al. [5], as
0 = sound (no signs of root caries disease at the evaluated
surface); 1 = initial lesion (root caries, but not cavitated);
2 = moderate lesion (root caries lesion, where the depth of
the cavity was ≤ 2 mm, Fig. 2); and 3 = extensive lesion (root
caries lesion, where the cavitation is deeper than 2 mm). In
case of uncertainty whether the lesion should be classified
as moderate or extensive, it was decided to score low. After
(2c) (× 5) lines are placed SX and SP through the deepest part of the
lesion (X). Thus, a fraction can be calculated as SX/SP and expressed
in percentage
Clinical Oral Investigations (2022) 26:2597–2605
10–12 days, the same examiners scored all lesions again,
using the same visualization classification. All examinations
took place in the same area of a modern laboratory. The tem-
perature was about 21 °C. It is important to underline that
both examiners examined the teeth in a shuffled allocation of
the order of teeth prepared by the technical assistant. When
teeth were not under examination, they were kept in 0.1%
thymol water. Just before the teeth were under investigation,
they were placed in tap water for at least 5 min.
Cutting and histological evaluation
The teeth were sectioned by the same saw as in the pilot
study into two parts, in the horizontal direction just in the
middle of the lesion. To reduce the risk of error, a thin line,
with a pen with a tip of 0.5 mm, was drawn on the examined
surface indicating where the section should be done (Fig. 2).
This was done by the last author. The cutting procedure was
done with the help of a technical assistant. At this point,
3 teeth were excluded from the sample, because rupture
occurred during the sectioning.
The section surfaces were then examined with a magni-
fying lens (× 4) (Hama GmbH & Co KG, Monheim, Ger-
many) by the first author (K.E.), who then decided, which
of the two-section surfaces showed the deepest part of the
lesion. Photographs were taken (technical assistant) of the
selected section surfaces (Zeizz Stereo Discovery V.8 – lens
1.6) (Fig. 2a).
Microscopic photography and measurement
of lesion depth
Using the SM and the added software, both examiners inde-
pendently marked the deepest part of the lesion with an X
on the chosen section face (Fig. 2b). The distance from the
outer surface to the X (SX) was measured in micrometer.
Similarly, the distance from the outer surface (S) through
the deepest part of the lesion towards the pulp (P) was meas-
ured (Fig. 2c). A fraction SX/SP was calculated from each
specimen and converted to the following scoring: 0 = (0%);
1 = (1 to 33%); 2 = (34 to 66%); and 3 = (67 to 100%). All
measurements were repeated between 14 to 23 days after the
first examination by the two examiners.
Statistical analysis
Chi-square tests were performed to see if the severity of the
lesion was associated with the site on the roots (approximal
or at smooth surfaces) or location of the lesion with respect
to the enamel-cement border (0–1 mm, versus > 1 mm).
Yate’s correction was used in the calculation. The intra-
reproducibility using weighted kappa values (quadratic
weights) were expressed from the following variables: the
2601
first visual examination versus the second visual examina-
tion; the first microscopic assessment of the depth of the
lesion versus the second microscopic assessment. The inter-
reproducibilities were measured using the first set of obser-
vations for both examiners.
The accuracies between the naked eye feature of the
lesion and the histological depth of the lesion assessed
microscopically were assessed by Spearman’s rho. The first
assessments were used for the calculations. Finally, also
using the first assessments, the sensitivity, specificity, and
AUC values (area under the ROC curves) were calculated
from three different thresholds.
All calculations were performed using the Statistical
Package for the Social Sciences (SPSS). These data analyses
were performed by independent statisticians.
Results
As 3 teeth broke during the preparation technique, the final
sample size was 97 permanent molar teeth. A total of 60% of
the sites to be analyzed were on roots on approximal surfaces
while 40% of the sites were on roots on buccal or lingual/
palatal surfaces. The sections were done at the enamel-
cement border or 1 mm below in 78% of the cases, while in
22% the cuts were done 2–4 mm below the enamel-cement
border. The severity of the lesions was not related to the site
of the tooth (chi-square = 5.61; df = 3; p = 0.13) or location
on the root in terms of the location related to the enamel-
cement border (chi-square = 2.453; df = 3; p = 0.48).
As shown in Table 1, each clinical score (sound, initial,
moderate, or extensive) represented between 18 and 30% of
the full sample of scores, both at the first and second assess-
ments. There were fewer moderate lesions compared to the
other stages. The intra-reproducibility in terms of weighted
kappa was for K.E. = 0.93 (p < 0.001) and for C.T. = 0.96
(p < 0.001). The inter-reproducibility was 0.83 (p < 0.001).
Table 2 shows the association between the first and
second histological assessments for both examiners. The
achieved intra-kappa values were 0.91 (K.E.) and 0.93
(C.T.). The inter-kappa value was 0.85.
The correlation between the first clinical assessment
and the first histological assessment was 0.94 (p < 0.0001)
(K.E.) and 0.95 (p < 0.0001) (C.T.). The raw data is shown
in Table 3. Internal analyses disclosed for both examiners
that about 1/3 of the extensive lesions extended into the pulp
(histological assessment).
Table 4a–c present raw data, so the accuracy between
the clinical scoring system and the histological scoring
system could be expressed by the terms sensitivity, spec-
ificity, and AUC. In Table 4a the threshold is between
sound, and any caries lesion and the specificity/sensitiv-
ity/AUC values were 0.91/0.93/0.96 (SE = 0.02, p < 0.001)
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2602 Clinical Oral Investigations (2022) 26:2597–2605

Table 1  Shows raw data in absolute and percentage at the first and second clinical assessment for both examiners (K.E. and T.C.)
Observer K.E. and T.C
Score at the first assessment
Score at the sec- Sound Initial Moderate Extensive Total
ond assessment
K.E T.C K.E T.C K.E T.C K.E T.C K.E T.C

Sound 25 23 1 5 0 0 0 0 26/27% 28/29%

Initial 1 2 27 20 1 1 0 0 29/30% 24/25%
Moderate 0 0 1 1 17 20 0 2 18/18% 23/24%
Extensive 0 0 0 0 0 0 24 22 24/25% 22/23%
26/27% 25/26% 29/30% 26/27% 18/18% 21/21% 24/24% 24/25% 97 97

Table 2  This shows raw data of the first and second microscopic assessment for both examiners
Microscopic assessment 1
Sound Initial Moderate Extensive Total
K.E T.C K.E T.C K.E T.C K.E T.C K.E T.C

Microscopic Sound 18 22 0 0 0 0 0 0 18/19% 22/23%

assessment 2 Initial 2 0 27 38 3 0 0 0 32/33% 38/39%
Moderate 0 0 5 0 19 15 0 0 24/25% 15/15%
Extensive 0 0 0 0 0 0 23 22 23/24% 22/23%
Total 20/21% 22/23% 32/33% 38/39% 22/23% 15/15% 23/24% 22/23% 97 97

Table 3  This shows raw data of the first clinical assessment and first microscopic assessment for both examiners
Microscopic assessment 1
Sound Initial Moderate Extensive Total
K.E T.C K.E T.C K.E T.C K.E T.C K.E T.C

Clinical Sound 20 22 6 3 0 0 0 0 26/28% 25/26%

assess- Initial 0 0 26 36 3 0 0 0 29/30% 36/37%
ment 1
Moderate 0 0 0 0 16 9 2 2 18/19% 11/11%
Extensive 0 0 0 0 3 5 21 20 24/25% 25/26%
Total 20/22% 22/23% 32/31% 39/40% 22/23% 14/14% 23/23% 22/23% 97 97

(K.E.) and 1.0/0.96/0.98 (SE = 0.01, p < 0.001) (C.T.). In
Table 4b, the threshold is between sound/initial caries
and moderate/extensive caries and the specificity/sensi-
tivity were 1.0/0.91/0.98 (SE = 0.01, p < 0.001) (K.E.)
and 1.0/1.0/1.0 (SE = 0.0, p < 0.001) (C.T.). Finally, in
Table 4c, the threshold was between sound/initial/mod-
erate versus extensive. The specificity/sensitivity values
were 0.97/0.91/0.97 (SE = 0.02, p < 0.001) (K.E.) and
0.93/0.91/0.96 (SE = 0.02), p < 0.001) (C.T.).
In the supplementary material, we have devised a sheet
showing the relationship between representative stages of
root caries lesion in their clinical appearance and histo-
logical presentation.
Discussion
The present study was dependent on a clear view of the
pulp for the histological assessment of lesion depth and the
first author had experienced that the pulp far from always
was visible or the periphery of the pulp was not possible to
locate, when cutting the teeth vertically through the lesions.

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Clinical Oral Investigations (2022) 26:2597–2605 2603

Table 4  Raw data to express

specificity and sensitivity a Test*
values using three different Histological scores** S I+M+E Total
thresholds: a, sound (S) versus K.E T.C K.E T.C K.E T.C
any staged caries lesion (initial S 20 22 6 0 26 22
(I) + moderate (M) + extensive
(E); b, sound (S) plus initial (I) I+M+E 0 3 71 72 71 75
staged lesions versus moderate Total 20 25 77 72 97 97
(M) and extensive (E) staged b Test
lesions; and c, sound (S) plus Histological scores S+I M+E Total
initial (I) and moderate (M)
staged lesions versus extensive K.E T.C K.E T.C K.E T.C
(E) staged lesions *test = visual S+I 52 61 3 0 55 61
score by K.E. or T.C., M+E 0 0 42 36 42 36
**histological score by K.E. or Total 52 61 45 36 97 97
by T.C
c Test
Histological scores S+I+M E Total
K.E T.C K.E T.C K.E T.C
S+I+M 71 70 2 2 73 72
E 3 5 21 20 24 25
Total 74 75 23 22 97 97

S, sound; I, initial; M, moderate; E, extensive

A pilot study is needed, therefore, to be conducted address-
ing (a) if the pulp was visible more often, if the cutting was
performed horizontally compared to vertically through the
root caries lesion, (b) to develop a precise method for assess-
ing the depth of the lesion on the section faces using SM
with the aid of a software program, irrespectively of the
cutting direction, and (c) to examine if the lesion depth was
influenced by the cutting directly through the lesion (hori-
zontally compared to vertically). The authors were aware
from the literature that the dentine tubules do not follow a
straight path towards the pulp, rather they extend to a more
apically positioned location when they reach the pulp area.
Therefore, a horizontal cut through the lesion might not
show the real depth of the lesion. The pilot study disclosed
that in about 25% (5 out of 21), we could not localize the
pulp on the vertically cut sections, while the pulp was clearly
visible when the cut was done horizontally (p = 0.03). Our
pilot study also showed that it was possible to express the
depth of the lesion as a fraction of the lesion depth com-
pared to the distance from the surface to the pulp. Finally,
the pilot study documented that there were no differences
in lesion depth whether we used a vertical or a horizontal
cutting procedure (p = 0.155). For these reasons, we chose
to use a horizontal cutting procedure through the lesions in
the main study.
The RoB-tool [11] advocates for those teeth used in the
pilot study are not a part of the sample used in the main
study. Furthermore, the sample of teeth consists of perma-
nent molar teeth, if possible. Both recommendations were
followed in the present study (signaling questions 10 and 2,
respectively, see supplementary material).
The RoB-tool also advocates for sample size estimations
(signaling question 4, see supplementary material). Prior to
this study, we did not know the correlation between the two
parameters investigated and thus based on statistical advice
agreed to set the correlation coefficient corresponding to a
weak level (a correlation coefficient of 0.33). The sample
size calculation estimated that around 100 teeth were needed
to achieve reasonable power in the study. This included
teeth that had to be potentially excluded due to the prepara-
tion technique. This large number of teeth also reduced the
chance of the examiners to remember the scorings.
As previously described in epidemiological studies, e.g.,
by Fejerskov et al. [2], root caries develops on both smooth
and approximal surfaces. Thus, we included all surfaces in
this study. The fraction in this study was 60% for lesions on
approximal surfaces and 40% on smooth surfaces.
The first author is highly experienced in the ICDAS and
ICCMS™ including how to stage both coronal and root
caries lesions and to perform histological validation [3, 5,
17–19]. The training of the co-authors was very thorough
involving both theoretical education as well as practical
exercises. The visual acuity for each of the 3 authors was
adequate, when they used their glasses. The root caries
lesions were staged by the ICCMS™ scoring system oper-
ating with 4 scores. The difference between the scores is that
initial lesions are non-cavitated lesions, while the difference
between moderate and extensive lesions is whether the depth
of the cavity was less/equal or above 2 mm measured by a
perio-probe. Thus, a rather easy scoring system to be used
under the in vitro conditions, a statement supported by the
high reproducibility achieved both during training (perfect

13
2604
agreement was 85% for examiner 2), and for the main study,
kappa values were ≥ 0.83 for both first and second examiner
(Table 1). It is tempting to suggest that observer bias was
under control.
Taking the mean between K.E. and T.C. about 28% of the
sample did not have any root caries lesions (sound surfaces),
approximately 27% of the sample had initial staged caries
lesions, 21% of the sample had moderate staged lesions, and
24% had extensively staged lesions. Thus, spectrum bias
(signaling question 3, see supplementary material) seemed
also to be under control.
Hypermineralized dentine on a section surface becomes
more grayish in color compared to sound dentine. Demin-
eralized dentine also changes color to a more brownish/yel-
lowish compared to sound dentine. Thus, the border between
the brownish/yellowish and the grayish dentine was used to
assess the depth of the lesion [13, 20]. This assessment was
done by means of the microscope (× 5); eventually, very high
values of reproducibilities were obtained, both under the
training (perfect agreement 89% for the second examiner)
and in the main study (weighted kappa values ≥ 0.83).
Using section faces to assess the depth of the lesion cre-
ates a problem, which counts for all 4 histological meth-
ods described below. Under examination, the examiner can
actually see if the lesion is cavitated or not. The blinding
issue between the two tests, which in the RoB-tool is called
the index test (ICCMS™) and the reference test (the histo-
logically assessed depth of the lesion) is then strictly not
separated, which was also the case for the two examiners in
the present study (signaling questions 8 and 9, see supple-
mentary material). As long as dentists who are familiar with
caries pathogenesis do the histological readings on section
faces, blinding between the index and the reference tests is
not possible to perform. Maybe machine learning can help
with this bias-creating situation in the future. The problem
is perhaps not so big in the present study, as we eventu-
ally classified the depth of the lesion as initial, moderate, or
extensively deep. As each stage has a distance, e.g., 2 mm,
an influence from knowing that there is a lesion and, whether
it is cavitated or not, might not change, that too many lesions
will be classified as moderate instead of extensive and so on.
The response options for signaling questions 8 and 9 were
therefore probably low risk of bias.
Bias due to attritions was not a problem in the present
study, as only 3% of the teeth were excluded for the reason
that the histological assessment could not be done (signaling
question 14, see supplementary material).
The type of the validation method influences the validity
of the study [21]. When it comes to the registration of the
depth of caries lesions, on section faces, thus histologically,
in general, 4 methods have been used: stereomicroscopical
assessments based on changes in color; stereomicroscopi-
cal assessment, but where the section faces are colored,
13
Clinical Oral Investigations (2022) 26:2597–2605
e.g., by rhodamine; microradiography; and microhardness
measurements. However, the overall statement is that none
of the methods can show the real lesion depth [21, 22], not
even the more recent microradiographic methods [23]. Thus,
due to the fact that the stereomicroscopical method still is
acknowledged as the most popular gold standard for assess-
ing the lesion depth on section faces [21, 22] and that the
main author has great experiences in that method [13, 14,
18, 19], it was logical to use that method.
The data shows a strong and a significant relationship
between the clinical feature of a root caries lesion and the
demineralization depth of the same lesion, seen in relation
to the location of the pulp (see the sheet in supplementary
material). Even under three different thresholds, the speci-
ficity and sensitivity values were each ≥ 0.91, and combined
the sum was always ≥ 1.84. Furthermore, the corresponding
AUC values were ≥ 0.96.
Ekstrand et al. [3, 24] tested a scoring system for assess-
ing activity, using the following clinical parameters: texture
of the dentine (whether the dentine is soft, leathery, or hard
to gentle probing); contour of the lesion (whether the lesion
was in a cavitated or non-cavitated stage); location of the
lesion (whether the lesion was in a plaque stagnation area or
not); and if the lesion was yellowish or brown. Combining
the assessments presented in the present study (ICCMS™
and histology using 1/3) with the activity assessments just
mentioned, the following diagnosis can be established for
individual root caries lesions: initial active/arrested; mod-
erate active/arrested; or extensive active/arrested. Thus, the
same characteristics can be used for root caries lesions as
that for coronal lesions [5, 6]. The active lesions require
caries care management [9].
In conclusion, the present study shows a strong relation-
ship between the external feature of the root caries lesion
characterized by the ICCMS™ scoring system and the actual
demineralization depth of the lesion. Both the ICCMS™
scoring system and the histological scoring system used are
precise, and due to the structuring of the present study, fol-
lowing by large the recommendation of the RoB-tool, it is
tempting to state that the risk of bias related to the overall
results of the study is low (ten response options to the signal-
ing questions were graded as low risk of bias and 5 probably
low risk of bias, the last one of the 16 signaling question
could not be assessed as that was about patient selection, see
supplementary material).
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00784-0​ 21-0​ 4229-4.
Acknowledgements We are thankful for the help received from
Associate Professor Azam Bakhshandeh, University of Copenhagen,
concerning the use of the microscope for assessing lesion depth, for
the help from technical assistant Camilla Gündüz who to care of the
material and cut the teeth and for the statistical advice from Associate
Clinical Oral Investigations (2022) 26:2597–2605
Professor Luis Garrido, Pontificia Universidad Catolica Madre y Maes-
tra, Santo Domingo, Dominican Republic, and from Professor Fausto
Mendes, University of Sao Paulo, Brazil.
Author contribution K.E. discussed the idea with T.C. and N.A.P. K.E.
trained T.C. in the techniques. All were involved in the selection of
teeth. K.E. and T.C. did the final microscopic and histological assess-
ments. K.E., T.C., and N.A.P. drafted the paper and N.A.P. did the work
with tables, figures, and references. All authors critically reviewed the
papers before the submission.
Funding This research was conducted at the University of Copenha-
gen and only the participation of the second author (T.C.) was pos-
sible thanks to the funding of the Foundation for Research Support
(FAPESP—Sao Paulo City) and the Research Internship Abroad
(BEPE—process number 2018/ 08066–4).
Declarations
Ethical approval This study does not contain any human participants
or animals. Thus, ethical approval was not necessary.
Informed consent For this type of study, formal consent is not required
in Denmark.
Conflict of interest The authors declare no competing interests.
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