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Odontology
https://doi.org/10.1007/s10266-022-00692-x
ORIGINAL ARTICLE
Repeatability ofdental shade bydigital spectrophotometry incurrent,
former, andnever smokers
GianlucaConte1,2· SebastianoAntonioPacino2,3· SalvatoreUrso3· RosaliaEmma4· EugenioPedullà1,5·
FabioCibella6· MartinaStefanini7· GiovanniZucchelli7· RiccardoPolosa3,5,8
Received: 6 August 2021 / Accepted: 24 January 2022
© The Author(s) 2022
Abstract
Cigarette smoking contributes to poor oral health and dental discoloration. Therefore, stopping smoking may translate into
measurable amelioration of dental shade indices. We compared dental shade parameters by digital spectrophotometry among
current, former, and never smokers and verified their repeatability at 7 and 30days. Dental shade parameters (CIE L*a*b*
and corresponding whiteness index for dentistry-WID) were measured in current, former, and never smokers with a digital
spectrophotometer (Vita Easyshade V) on three separate study visits: at baseline (day 0), at day 7, and day 30. Dental shade
parameters were analyzed in 18 current, 18 former, and 20 never smokers. The repeatability of shade parameters was consist-
ent in current, former, and never smokers. L*, a*, b*, and WID show significant short and long-term repeatability (p < 0.0001,
by regression analyses). The mean (± SD) WID score of 13.42 (± 4.9) in current smokers was significantly lower compared
to the WID score of 20.38 (± 5.3) in never smokers (p = 0.001). No significant differences were observed between current
and former smokers and between former smokers and former smokers. Dental shade measurements by digital spectropho-
tometry were highly reproducible and showed that teeth whiteness of current smokers is substantially inferior compared
to never smokers. Objective discrimination of dental shade can be a valuable regulatory science endpoint for investigating
oral hygiene and dental aesthetics of consumer care products, smoking cessation medications, and tar-free tobacco products
(e-cigarettes, heated tobacco products, oral nicotine products) for cigarette substitution.
Clinical trial registration: the study was not registered in ClinicalTrials.gov considering that it is a pilot study, parts of a
larger project with ID: NCT04649645
Keywords Smoking· Smoking cessation· Dental shade· Digital spectrophotometer· CIE L*a*b*· Whiteness index for
dentistry· Reproducibility
Introduction
Cigarette smoking is known to contribute to poor oral
health and tooth discoloration [1, 2]. The particulate mat-
ter of cigarette combustion (known as “tar”) contains
pigments that can stain and discolor human tissue, includ-
ing skin, fingernails, and teeth [3–5]. The extent of smoke-
related discoloration of the teeth may depend on the inten-
sity and duration of exposure to cigarette smoke [6, 7].
Of note, intrinsic characteristics of the tooth may also
* Riccardo Polosa
polosa@unict.it
1 Department ofGeneral Surgery andMedical-Surgical
Specialties, University ofCatania, Catania, Italy
2 Addendo Srl, Dental Clinic, Catania, Italy
3 ECLAT Srl, Spin-off oftheUniversity ofCatania, Catania,
Italy
4 Department ofBiomedical andBiotechnological Sciences,
University ofCatania, Catania, Italy
5 Center ofExcellence fortheAcceleration ofHArm
Reduction (CoEHAR), University ofCatania, Catania, Italy
6 Research Council ofItaly, Institute ofBiomedicine
andMolecular Immunology, Palermo, Italy
7 Department ofBiomedical andNeuromotor Sciences,
University ofBologna, Bologna, Italy
8 Department ofClinical andExperimental Medicine,
University ofCatania, Catania, Italy
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contribute; for example, rough/irregular enamel surfaces
can facilitate tar adhesion [8].
Teeth color and appearance vary widely [9]; human
eye perception, lighting conditions, tooth translucency
and opacity, and individual differences in tooth color
can contribute to this variability. Therefore, teeth’ visual
color determination varies significantly, leading to inac-
curate evaluation [10, 11], and tooth shade guides have
been introduced to minimize variability. Dentists evaluate
teeth color and assess how much whiter teeth can get after
using various interventions (e.g., toothbrush, mouthwash
or whitening solutions) using tooth shade guides. How-
ever, studies with tooth shades guide have shown poor
inter-observer correspondence [12, 13] with only 25–35%
accuracy for human visual determination of teeth shades
[14, 15]. Human shade evaluation cannot be considered for
high-quality analysis and research applications, and more
precise and reproducible techniques are required.
Innovative technologies for color measurement have
been recently introduced to allow more accurate meas-
urements of dental shade. Digital spectrophotometers are
considered the most reliable, practical and versatile color
matching system [16], with much greater accuracy of den-
tal shade matching than human eye observation [17].
The*a*b* color space principle, developed in 1976 by
the Commission International de l’Eclairage (CIE), is used
in most studies on tooth color determinations with digi-
tal spectrophotometers [18]. L* expresses the intensity of
lightness reflected by an object, whereas a* and b* express
the definedchromaticity measure of green/red (for a*) and
blue/yellow (for b*)–the so-called “forbidden colors” of
human vision. The whiteness index for dentistry (WID)
has been validated to show that it can accurately capture
visual whiteness differences using the CIE L*a*b* color
space [19]. Moreover, clinical studies have demonstrated
that WID outperformed previous similar indices when
assessing the effectiveness of different bleaching proce-
dures on natural teeth [20, 21]. Avisually perceivable dif-
ference in whiteness between twoteeth occurs when WID
difference is > 2.90 WID units [22].
To the best of our knowledge, changes in the dental
shade in smokers who quit smoking have never been
reported. Our research hypothesis is that stopping smoking
can translate into measurable amelioration of dental shade
indices. However, smoking-induced dentaldiscoloration
may be permanent, with limited whiteness restoration after
quitting smoking. If this is true, former smokers’ dental
shade values should not be much different from current
smokers. Moreover, more information is needed to vali-
date within subjects repeatability of digital spectropho-
tometry in current and former smokers. This is mandatory
to increase confidence in the value of this technology for
future clinical research.
The objective of the study is to: (a) verify the short
(7days) and long term (30 days) repeatability of dental
shade indices obtained by digital spectrophotometry; and
(b) compare these measurements between current, former,
and never smokers. CIE L*a*b* color space parameters and
corresponding WID values were considered for the analyses.
Methods
Study population
The study population consists of three study groups iden-
tified among a pool of subjects who attended a smoking
cessation clinic (CPCT, Centro per la Prevenzione e Cura
del Tabagismo of the University of Catania) in the previous
2years or contacted among hospital staff.
Study group 1 consisted of current smokers, defined as
smokers of > 10 cigarettes per day with an exhaled carbon
monoxide (eCO) level of ≥ 7ppm.
Study group 2 consisted of former smokers, defined
as quitters of at least 12months and who were still absti-
nent when contacted for enrollment, with an eCO level
of < 7ppm.
Study group 3 consisted of never smokers, defined as
having never smoked or who reported having smoked less
than 100 cigarettes in their lifetime [23]. Their eCO had to
be < 7ppm to exclude subjects passively exposed to cigarette
smoke or environmental sources of carbon monoxide.
Current, former, and never smokers had to satisfy the fol-
lowing inclusion criteria:
1. Healthy adult subjects (age 18–50 yrs)
2. Presence of at least ten natural anterior teeth (cuspid to
cuspid, lower and upper jaw), with no composite restora-
tions and no prosthetics or crown
Furthermore, they had to satisfy the following exclusion
criteria:
1. Any conditions that could interfere with dental shade
measurements, including:
1. Regular daily use of mouth rinse containing essen-
tial oil (EO), cetylpyridinium chloride (CPC), or
chlorhexidine (CHX) for at least the preceding seven
days before screening visit
2. Subjects wearing fixed or removable orthodontic
appliances or prostheses (limited to the 12 natural
anterior teeth)
2. Significant exposure to passive smoking (excludes cur-
rent smokers)
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3. Had undergone professional dental cleaning within six
months before screening
4. Pregnancy
The study was conducted according to the Principles of
Good Clinical Practice (GCP) and Declaration of Helsinki.
The local Ethics Committee reviewed and approved the
study.
Study design
This is an observational study to compare digital spectro-
photometric parameters for dental shade evaluation by Vita
Easyshade V among three study populations (current, for-
mer, and never smokers) and to assess their repeatability.
The study consists of a total of four visits: screening visit,
baseline visit at day 0 (Visit 1), short-term follow-up visit at
day 7 (± 1days) (Visit 2), and a long-term follow-up visit at
day 30 (± 3days) (Visit 3) (Fig.1). Subjects were asked to:
1. Not to change their habitual oral hygiene (toothbrush-
ing, mouth washing, interdental flossing) pattern for the
whole duration of the study
2. Avoid scaling and polishing procedures for the entire
period of the study
3. Not to daily use mouth rinse for the whole of the course
of the study
4. Not to smoke for at least 2h before each study visit
5. Not to toothbrush for at least 2h before each study visit
6. Not to eat and drink (except water) for at least 2h before
each study visit
Study visits
Screening visit
Potential participants attended a screening visit to (1)
receive information about the rationale and objectives of
the research; (2) verify eligibility criteria by reviewing
their inclusion and exclusion criteria; (3) assess smoking
status and oral hygiene habit (i.e., frequency of toothbrush-
ing, type of toothpaste, etc.); and (4) record general socio-
demographic characteristics (i.e., sex, age, and occupation).
All eligible subjects were invited to participate in Baseline
Visit (Visit 1).
Baseline visit (visit 1)
Carried out within ten days of the Screening Visit. Subjects
were asked to go over a patient information sheet and sign
a consent form. After re-checking inclusion/exclusion crite-
ria and reviewing study restrictions, eCO measurement and
dental shade assessment were carried out, and baseline data
were recorded. Subjects were instructed not to change their
habitual oral hygiene pattern and invited to attend the next
study visit (Visit 2).
Fig. 1 Study Design
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Day‑7 visit (visit 2)
Carried out 7 (± 1) days after Visit 1. Eligibility criteria
were verified again. Dental shade assessment was repeated
for short-term repeatability. Subjects were instructed not
to change their habitual oral hygiene pattern and invited to
attend the next study visit (Visit 3).
Day‑30 visit (visit3)
Carried out 30 (± 3) days after Visit 1. After re-checking
eligibility criteria, a dental shade assessment was repeated
for long-term repeatability.
Exhaled carbon monoxide measurement
The smoking status was objectively verified by measuring
exhaled carbon monoxide (eCO) levels (eCO > 7ppm indi-
cating smoking status) with a portable CO monitor (Micro
CO; Micro Medical Ltd, UK). Subjects were asked not to
smoke cigarettes for at least 2h before eCO measurements.
Subjects were invited to exhale slowly into a disposable
mouthpiece attached to the eCO monitor per the manufac-
turer's recommendations. The value of eCO readings was
noted.
Dental shade assessment
Before dental shade assessment, participants were asked to
rinse their mouths with water. They were subjected to gentle
flushing and drying by triple syringe to remove any food
debris.
All measurements were performed in the same examina-
tion room, under the exact ambient illumination condition,
and by the same operator (GC). The digital spectrophotom-
eter (Vita Easyshade V) was calibrated and used according
to the manufacturer’s instructions.
Subjects were instructed to open their mouths with the
tongue away from the anterior teeth.
The shade was measured at the central tooth area of the
vestibular surface in “base shade determination” mode,
with the measuring tip staying at 90° on the tooth surface to
achieve an accurate measurement. Subjects were asked to
withhold breathing for a few seconds during measurements,
to avoid fogging of the measuring tip as this could cause
inaccurate shade readings.
CIE L*a*b* color parameters were measured for the ves-
tibular aspect of each anterior tooth (cuspid to cuspid, upper
and lower jaw).
The coordinate L* measures lightness, ranging from 0
(black) to 100 (white); the a* and b* coordinates express
chromaticity measures of green/red and blue/yellow, respec-
tively. For each subject, total CIE L*a*b* scores were
obtained by adding the individual values of each tested
anterior tooth and dividing by the number of teeth examined.
Whiteness index fordentistry (WID)
The whiteness index for dentistry (WID), which is based on
CIE L*a*b* coordinates, was calculated as the following
equation [18]:
Each subject’s total WID score was obtained by adding
the value of each tested anterior tooth and dividing by the
number of teeth examined. High WID values indicate whiter
teeth, while low WID values indicate discolored or less
white teeth. Differences in the WID index were evaluated in
consideration of the whiteness 50:50% acceptability thresh-
old (WAT = 2.90 ΔWID units) [22]; someone can visually
appreciate a clear variation in whiteness between two teeth
if the ΔWID difference is > 2.90 units.
Data analysis
Short-term repeatability of CIE L*a*b* and WID scores
was evaluated by linear regression analysis of measurements
obtained at V1 and at V2 for each study group. Long-term
repeatability was assessed by linear regression analysis of
measurements at V1 and at V3. Scatter plots of linear regres-
sion analyses summarized repeatability results. Moreover,
“Bland and Altman” plots were created to illustrate the level
of agreement between V1 vs. V2 and V1 vs. V3 for each
study group. A 1-tailed sample t test was performed to assess
the mean difference between two measurements from zero.
Normality was assessed using Shapiro–Wilk’s normality
test. Counts and percentages summarized categorical data;
continuously distributed data, with symmetrical distribution,
were summarized using the mean (standard deviation; SD);
continuously distributed data, with skewed distribution, was
summarized using the median (interquartile range; IQR).
Clinical data comparisons among the groups were carried
out by Chi-square test for categorical data and Kruskal–Wal-
lis test for continuously skewed data. Comparison of L* was
performed using the Kruskal–Wallis test followed by Wil-
coxon pairwise test with Bonferroni’s adjustment for mul-
tiple comparisons. Comparisons of a*, b*, and WID scores
were performed by one-way ANOVA followed byTukey's
adjustment for multiple comparisons. Moreover, multi-way
ANOVA was performed to assess the interaction of age, gen-
der, daily toothbrushing frequency, weekly mouth-washing
frequency, and weekly dental flossing frequency on WID
score among the study groups.
All analyses were considered significant with a P
value < 0.05. R version 3.4.3 (2017-11-30) was utilized for
data analysis and generation of graphs.
WID =0.511 L ∗ −− 2.324 a ∗ −− 1.100 b ∗
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Results
Study participants
We screened a total of 70 subjects: five failed eligibil-
ity screening and four failed to attend their baseline
visit. Therefore, 61 subjects were enrolled in this study,
of which 5 participants were excluded from statistical
analysis after failing to attend their short- or long-term
follow-up visit.
Complete analysis of dental shade assessment was
carried out in 56 subjects (21 F; mean ± SD age of
34.73 ± 11.5years) 18 current, 18 former, and 20 never
smokers (Table1).
Dental shade repeatability innever smokers
Linear regression analyses were performed to assess short
term (7days) and long term (30days) repeatability of
dental shade parameters for never smokers. Analyses are
summarized in Table2.
Short‑term repeatability
For L*, a significant regression was observed between
study visits (R2 = 0.9287; p = 0.0001). No subjects had
the L* difference between V1 and V2 outside the 95%
confidence interval, and the mean of differences between
L* at V2 and V1 was not different from zero (p = 0.080).
Also, for the parameters a* and b* we observed significant
regression between study visits (R2 = 0.9146, 0.0001 for a*;
R2 = 0.8690, p = 0.0001 for b*). Only two subjects had the *
difference between V1 and V2 outside the 95% confidence
interval. Moreover, the mean differences between a* at V2
and V1 were not different from zero (p = 0.890). Only one
subject had the b* difference between V1 and V2 outside
the 95% confidence interval. Moreover, the mean differ-
ences between b* at V2 and V1 were not different from zero
(p = 0.233).
For WID scores, a significant regression was observed
between study visits (R2 = 0.9293; p = 0.0001) (Table 2,
Fig.2A). Only one subject had the WID difference between
V1 and V2 outside the 95% confidence interval (Fig.2B).
Moreover, the mean differences between WID at V2 and V1
were not different from zero (p = 0.884).
Table 1 Clinical characteristic
of study groups
Data are presented as median (IQR), n/N (%)
CO carbon monoxide
Current smokers Former smokers Never smokers p value
Subjects n 18 18 20
Age 31 (26–37) 32 (25–43) 34 (29.5–37) 0.871
Female 4/17 (23.5%) 7/17 (41.2%) 10/19 (52.6%) 0.202
Exhaled CO 15 (11–14.9) 3 (3–4) 3 (2–4) < 0.0001
n. Cigarette/day 15 (10–15) // // NA
Pack/years 9.6 4.7 // NA
Year non-smoking // 2 (1.5–9) // NA
Toothbrushing frequency/daily 2 (1.5–3) 2 (2–3) 2 (2–2.25) 0.454
Mouth washing frequency/daily 0 (0–0) 0 (0–0) 0 (0–2) 0.008
Dental flossing frequency/weekly 0 (0–1.5) 1 (0–1.5) 1 (0–1.5) 0.791
Table 2 Dental shade parameters repeatability analysis in Never Smokers
Parameters 7days (short term) repeatability 30days (long term) repeatability
Regression analysis
V2-V1 R value (p
value)
Mean of the difference V2-V1 being
different from zero? YES/NO (p value)
Regression analysis
V3-V1 R-value (p
value)
Mean of the difference V3-V1 being
different from zero? YES/NO (p
value)
L* 0.9287 (p = 0.0001) NO (p = 0.080) 0.8673 (p = 0.0001) NO (p = 0.169)
a* 0.9146 (p = 0.0001) NO (p = 0.890) 0.8644 (p = 0.0001) NO (p = 0.443)
b* 0.8690 (p = 0.0001) NO (p = 0.233) 0.8633 (p = 0.0001) YES (p = 0.004)
WID 0.9293 (p = 0.0001) NO (p = 0.884) 0.8672 (p = 0.0001) NO (p = 0.338)
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Long‑term repeatability
For L*, a significant regression was observed between study
visits (R2 = 0.8673; p = 0.0001). Only one subject had the L*
difference between V1 and V3 outside the 95% confidence
interval. The mean differences between L* at V3 and V1 were
not different from zero (p = 0.169). Also, for the parameters
a* and b* we observed significant regression between study
visits (R2 = 0.8644, p = 0.0001 for a*; R2 = 0.8633, p = 0.0001
for b*). Only one subject had the * difference between V1
and V3 outside the 95% confidence interval. Moreover, the
mean differences between a* at V3 and V1 were not different
from zero (p = 0.443). Only one subject had the b* differ-
ence between V1 and V3 outside the 95% confidence interval.
However, the mean differences between b* at V3and V1 were
significantly different from zero (p = 0.004).
For WID scores, a significant regression was observed
between study visits (R2 = 0.8672; p = 0.0001) (Table2,
Fig.3A). Only one subject had the WID difference between
V1 and V3 outside the 95% confidence interval (Fig.3B).
Moreover, the mean differences between WID at V3 and V1
were not different from zero (p = 0.338).
Dental Shade Repeatability inCurrent
Smokers
Linear regression analyses were performed to assess short
term (7days) and long term (30days) repeatability of dental
shade parameters for current smokers. Analyses are sum-
marized in Table3.
Short‑term repeatability
For L*, a significant regression was observed between
study visits (R2 = 0.9495; p = 0.0001). Only one subject
had the L* difference between V1 and V2 outside the 95%
confidence interval, and the mean of differences between
L* at V2 and V1 was not different from zero (p = 0.110).
Also, for the parameters a* and b* we observed significant
regression between study visits (R2 = 0.9603, 0.0001 for
a*; R2 = 0.8573, p = 0.0001 for b*). One subject had the *
difference between V1 and V2 outside the 95% confidence
interval. Moreover, the mean differences between a* at
V2 and V1 were not different from zero (p = 0.660). One
subject had the b* difference between V1 and V2 outside
the 95% confidence interval. Moreover, the mean differ-
ences between b* at V2 and V1 were not different from
zero (p = 0.446).
For WID scores, a significant regression was observed
between study visits (R2 = 0.9076; p = 0.0001) (Table 3,
Fig.4A). Only one subject had the WID difference between
V1 and V2 outside the 95% confidence interval (Fig.4B).
Moreover, the mean differences between WID at V2 and V1
were not different from zero (p = 0.773).
Long‑term repeatability
For L*, a significant regression was observed between study
visits (R2 = 0.9038; p = 0.0001).
No subjects had the L* difference between V1 and
V3 outside the 95% confidence interval. Moreover, L*
the mean differences between L* at V3 and V1 were not
Fig. 2 Short-term repeatability (V2 vs. V1) of WID scores in Never
Smokers. A shows the scatter plot of regression analysis between
visit 2 (V2) and visit 1 (V1) for WID scores. B shows the difference
between the measurements taken at V1 and V2 concerning the mean
in each subject in the Bland Altman plot
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different from zero (p = 0.712). Also, for the parameters
a* and b* we observed significant regression between
study visits (R2 = 0.9455, p = 0.0001 for a*; R2 = 0.8033,
p = 0.0001 for b*). One subject had the * difference
between V1 and V3 outside the 95% confidence interval.
Moreover, the mean differences between a* at V3 and V1
were not different from zero (p = 0.394). Two subjects
had the b* difference between V1 and V3 outside the
95% confidence interval, and the mean of the differences
between b* at V3 and V1 was significantly different from
zero (p = 0.012).
For WID scores, a significant regression was observed
between study visits (R2 = 0.8292; p = 0.0001) (Table 3,
Fig.5A). No subjects had the WID difference between V1
and V3 outside the 95% confidence interval (Fig.5B). More-
over, the mean differences between WID at V3 and V1 was
not different from zero (p = 0.424).
Dental shade repeatability informer
smokers
Linear regression analyses were performed to assess short-
and long-term repeatability of dental shade parameters for
former smokers. Analyses are summarized in Table4.
Short‑term repeatability
For L*, a significant regression was observed between study
visits (R2 = 0.9256; p = 0.0001).
No subjects had the L* difference between V1 and
V2 outside the 95% confidence interval, and the mean of
differences between L* at V2 and V1 was not different
from zero (p = 0.927). Also for the parameters a* and b*
we observed significant regression between study visits
(R2 = 0.9846, 0.0001 for a*; R2 = 0.9646, p = 0.0001 for
b*). No subjects had the * difference between V1 and V2
Fig. 3 Long-term repeatability (V3 vs. V1) of WID scores in Never
Smokers. A shows the scatter plot of regression analysis between
visit 3 (V3) and visit 1 (V1) for WID scores. B shows the difference
between the measurements taken at V1 and V3 concerning the mean
in each subject in the Bland Altman plot
Table 3 Dental shade parameters repeatability analysis in Current Smokers
Parameters 7days (short term) repeatability 30days (long term) repeatability
Regression analysis
V2-V1 R value (p
value)
Mean of the difference V2-V1 being
different from zero? YES/NO (p value)
Regression analysis
V3-V1 R-value (p
value)
Mean of the difference V3-V1 being
different from zero? YES/NO (p
value)
L* 0.9495 (p = 0.0001) NO (p = 1) 0.9038 (p = 0.0001) NO (p = 0.712)
a* 0.9603 (p = 0.0001) NO (p = 0.6) 0.9455 (p = 0.0001) NO (p = 0.394)
b* 0.8573 (p = 0.0001) NO (p = 0.446) 0.8033 (p = 0.0001) YES (p = 0.012)
WID 0.9076 (p = 0.0001) NO (p = 0.773) 0.8292 (p = 0.0001) NO (p = 0.4)
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outside the 95% confidence interval, and the mean of dif-
ferences between a* at V2 and V1 was not different from
zero (p = 0.278). Only one subject had the b* difference
between V1 and V2 outside the 95% confidence interval.
However, the mean differences between b* at V2 and V1
were significantly different from zero (p = 0.012).
For WID scores, a significant regression was observed
between study visits (R2 = 0.9855; p = 0.0001) (Table4,
Fig. 6A). Only one subject had the WID difference
between V1 and V2 outside the 95% confidence interval
(Fig.6B). However, L* the mean differences between
L* at V2 and V1 were significantly different from zero
(p = 0.038).
Fig. 4 Short-term repeatability (V2 vs. V1) of WID scores in Cur-
rent Smokers. A shows the scatter plot of regression analysis between
visit 2 (V2) and visit 1 (V1) for WID scores. B shows the difference
between the measurements taken at V1 and V2 concerning the mean
in each subject in the Bland Altman plot
Fig. 5 Long-term repeatability (V3 vs. V1) of WID scores in Cur-
rent Smokers. A shows the scatter plot of regression analysis between
visit 3 (V3) and visit 1 (V1) for WID scores. B shows the difference
between the measurements taken at V1 and V3 concerning the mean
in each subject in the Bland Altman plot
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Long‑term repeatability
For L*, a significant regression was observed between
study visits (R2 = 0.9215; p = 0.0001).
No subjects had the L* difference between V1 and V3
outside the 95% confidence interval. Moreover, the mean
differences between L* at V3 and V1 were not different
from zero (p = 0.548). Also, for the parameters a* and b*
we observed significant regression between study visits
(R2 = 0.9692, p = 0.0001 for a*; R2 = 0.9103, p = 0.0001
for b*).
Only one subject had the * difference between V1 and
V3 outside the 95% confidence interval. Moreover, the
mean differences between a* at V3 and V1 were not dif-
ferent from zero (p = 0.921). No subjects had the b* dif-
ference between V1 and V3 outside the 95% confidence
interval, and the mean of differences between b* at V3 and
V1 was not different from zero (p = 0.102).
For WID scores, a significant regression was observed
between study visits (R2 = 0.9492; p = 0.0001) (Table4,
Fig.7A). No subjects had the WID difference between
V1 and V3 outside the 95% confidence interval (Fig.7B).
Moreover, the mean differences between WID at V3 and
V1 were not different from zero (p = 0.408).
Summary ofrepeatability analysis
In all study groups (current, former, and never smokers),
dental shade parameters L*, a*, b*, and WID showed sig-
nificant short- and long-term repeatability. Validation of
the repeatability of digital spectrophotometry can increase
confidence in the value of this technology for future clini-
cal research.
Table 4 Dental shade parameters repeatability analysis in Former Smokers
Parameters 7days (short term) repeatability 30days (long term) repeatability
Regression analysis
V2-V1 R value (p
value)
Mean of the difference V2-V1 being
different from zero? YES/NO (p value)
Regression analysis
V3-V1 R-value (p
value)
Mean of the difference V3-V1 being
different from zero? YES/NO (p
value)
L* 0.9256 (p = 0.0001) NO (p = 0.927) 0.9215 (p = 0.0001) NO (p = 0.548)
a* 0.9846 (p = 0.0001) NO (p = 0.278) 0.9692 (p = 0.0001) NO (p = 0.921)
b* 0.9646 (p = 0.0001) YES (p = 0.012) 0.9103 (p = 0.0001) NO (p = 0.102)
WID 0.9855 (p = 0.0001) YES (p = 0.018) 0.9492 (p = 0.0001) NO (p = 0.408)
Fig. 6 Short-term repeatability (V2 vs. V1) of WID scores in For-
mer Smokers. A shows the scatter plot of regression analysis between
visit 2 (V2) and visit 1 (V1) for WID scores. B shows the difference
between the measurements taken at V1 and V2 concerning the mean
in each subject in the Bland Altman plot
Odontology
1 3
Comparison betweencurrent, former,
andnever smokers
Median (IQR) and Mean (± SD) values of L*, a*, b*, and
WID scores for the three study groups are summarized in
Table5. The L*values were similar, and no significant differ-
ence was observed among the three study groups (p = 0.475).
Moreover, no significant difference in the L* score was
observed when cross-comparisons were performed (p val-
ues > 0.05). This means that tooth lightness (L*) seems not
to be affected by smoking habits. Significant differences
were observed between study groups for both a* (p = 0.016)
and b* (p < 0.001), meaning that smoking cigarettes could
alter tooth color, increasing yellowness (b*) and redness (a*)
perception and causing tooth discoloration.
A significant difference was observed for WID scores
among the study groups (p = 0.002) (Fig.8). The mean
(± SD) WID score of 13.42 (± 4.9) in current smokers was
significantly lower compared to the WID score of 20.38
(± 5.3) in never smokers (p = 0.001), indicating discolored
or much darker teeth. No significant differences in WID
scores were observed between current smokers and former
smokers (p = 0.153) and between never smokers and for-
mer smokers (p = 0.167).
Assessment ofinteraction eects onWID
score
A statistically significant simple main effect of age for
WID score (p = 0.042). No other meaningful interactions
were observed for gender, daily toothbrushing frequency,
weekly mouth-washing frequency, and weekly dental floss-
ing frequency on WID score among the study groups.
Fig. 7 Long-term repeatability (V3 vs. V1) of WID scores in For-
mer Smokers. A shows the scatter plot of regression analysis between
visit 3 (V3) and visit 1 (V1) for WID scores. B shows the difference
between the measurements taken at V1 and V3 concerning the mean
in each subject in the Bland Altman plot
Table 5 Comparison of L*,
a*, b*, and WID scores among
Current, Former, and Never
smokers
Data are presented as median (interquartile range) or mean ± standard deviation (SD). The overall p values
were calculated by Kruskal–Wallis Test* or one-way ANOVA§
L* a* b* WID
Median (IQR) Mean ± SD Mean ± SD Mean ± SD
Current smokers 79.69 (73.9–81.5) 0.57 ± 0.8 22.71 ± 2.8 13.42 ± 4.9
Former smokers 79.78 (77.9–83.3) 0.26 ± 0.99 20.87 ± 2.9 16.99 ± 6.2
Never smokers 80.15 (77.6–83.1) −0.22 ± 0.6 18.92 ± 3 20.38 ± 5.3
Overall P value P = 0.475* P = 0.02§P = 0.001§P = 0.002§
Odontology
1 3
Discussion
Avoiding cigarette smoke toxicants may translate into meas-
urable amelioration of teeth appearance. Information about
changes in dental shade indexes in smokers who quit smok-
ing is not available. This study is the first to investigate the
impact of smoking and smoking abstinence on teeth appear-
ance by comparing digital spectrophotometry measurements
of dental shade in current, former, and never smokers. Den-
tal shade measurements showed that the teeth whiteness of
current smokers is substantially inferior compared to never
smokers.
Former and current smokers had lower WID values (indi-
cating discolored or less white teeth), with a statistically
significant difference being observed only between current
and never smokers, but not between current and former
smokers. Therefore, dental discolorations caused by smok-
ing may be persistent, with only negligible improvement
after stopping smoking. An alternative explanation for the
lack of significant difference between current and former
smokers is that the relatively short (2years on average) dura-
tion of smoking abstinence in our sample of former smokers
was not long enough to allow a complete appreciable rever-
sal of dental discoloration. Moreover, it is possible that the
observed trend could have become statistically significant
with a larger sample size.
The reported difference of teeth whiteness between cur-
rent smokers and never smokers were not only statistically
significant but also of practical/clinical relevance because
it largely exceeded the 2.90 ΔWID units’ threshold, above
which a visual difference in whiteness between two teeth
starts to be apparent [22]. The observation that teeth white-
ness of current smokers is substantially inferior compared
to never smokers was not unexpected; the conclusion is
consistent with what we know about pigments in the par-
ticulate matter of cigarette smoke and its staining of human
tissues [3–5]. Our clinical findings are compatible with
recent experimental work with human premolars extracted
for orthodontic reasons [6] and bovine enamel blocks [7],
showing that cigarette smoke induces substantial dental
discoloration.
A few factors and limitations need to be considered
when interpreting these study findings. First, the study
populations consisted of relatively young subjects, and
their dental shade measurements may not be representa-
tive of the general population. This is particularly impor-
tant, considering that age was the only factor causing a
statistically significant interaction with WID scores. Con-
sequently, additional studies with more representative
age groups are needed to confirm our findings. Second,
dental shade measurements were performed only on the
vestibular aspect of each anterior tooth (cuspid to cuspid,
upper and lower jaw). However, it is unlikely that study
findings would have changed significantly by extending
measurements to all existing natural teeth (including the
lingual/palatal portion). Third, COVID-19 restrictions had
Fig. 8 Individual WID scores
among Current, Former, and
Never smokers. The diamond
points with a black horizontal
bar illustrate the means of each
study group. High WID values
indicate whiter teeth, with low
WID values showing discolored
or much darker teeth
Odontology
1 3
only a minimal impact on the study conduct because it
was carried out between the first and second wave of the
pandemic in Italy (July 2020–October 2020) when clear
dental settings guidelines were already set in place and
most restrictions to hospital access were lifted.
Last but not least, this is a proof-of-concept pilot study,
which has been conducted to validate the repeatability of a
new methodology and increase researchers’ confidence in
the value of this measurement for clinical research, includ-
ing our ongoing large multicentre randomized controlled
trial [24]. No previous data for WID values could be used
for power calculation. However, based on experimental
studies (6, 7) the extent of smoke-related discoloration of
the teeth is so considerable that a small sample size can
be adequate. This has been confirmed by the data of the
current study; when comparing dental shade parameters of
18 current smokers with those of 20 never smokers, mean
WID scores resulted significantly different (13.42 for current
smokers vs. 20.38 for never smokers; p = 0.001). Therefore,
a sample of 18–20 subjects is adequate to detect significant
and aesthetically relevant differences (WID difference was
well above 2.9 units—the visually perceivable whiteness
difference between two teeth). However, it is possible that
the absence of significant differences with former smokers
(i.e., current smokers vs. former smokers; never smokers vs.
former smokers) could be due to a small sample size and that
a much larger sample could have detected significant differ-
ences. When considering within-subject repeatability, the
narrow confidence intervals indicate very low measurement
variability suggesting that a small group of 18–20 subjects
can be informative.
This study also confirms the good reproducibility of den-
tal shade indices measured by VITA Easyshade V, both short
term and long term; our findings are in agreement with those
obtained with the same digital spectrophotometer in several
studies [25–27].
This is the first study to investigate dental shade repeat-
ability in current, former, and never smokers. Study find-
ings show significant short- and long-term repeatability in
all study groups for all dental shade indices (except L*). In
particular, a high level of repeatability was found for WID
score with significant regression between study visits (both
short and long term) in all study groups.
The issue of test variability is significant when investigat-
ing dental outcomes in clinical trials of subjects with signifi-
cant exposure to tobacco smoke. We believe that the good
reproducibility of dental shade measurements in this study
was due to (1) optimization of environmental conditions
with standardization of ambient light; (2) careful consid-
eration of factors that could significantly affect study meas-
urements (e.g., asking participants not to alter the habitual
pattern of their oral hygiene practices, avoiding fogging of
the measuring tip during measurements); (3) well-trained
operators performing the test correctly and accurately, and
(4) using an accurate and reliable digital spectrophotometer.
Dental shade measurements by digital spectrophotometry
were highly reproducible and showed that teeth whiteness of
current smokers is substantially inferior compared to never
smokers. These findings may have important implications
for smoking cessation, especially for those smokers for
which bad breath and teeth appearance (due to tooth dis-
coloration and “tar”/tobacco stains) are often perceived as
a significant problem [28–30]. Moreover, objective, repro-
ducible discrimination of dental shade measurements will
increase confidence in their value for a range of applica-
tions, including clinical and regulatory research applied to
combustion-free tobacco products (e.g., e-cigarettes, heated
tobacco products, oral tobacco/nicotine products), smoking
cessation medications, and consumer care product for oral
hygiene and dental aesthetics.
Acknowledgements The authors expressed their thanks to all study
participants and Mike Coughlan for the professional editing of this
manuscript.
Author contributions All authors contributed to the study’s concep-
tion and design. GC, FC, RE performed material preparation, data
collection, and analysis. RP, GC and EP wrote the first draft of the
manuscript, which was subsequently reviewed by MS and GZ. SAP and
SU overlooked study’s logistic and administration. All authors read and
approved the final manuscript.
Funding ECLAT Srl. a spin-off of the University of Catania, sponsored
this investigator-initiated study through a grant from the Foundation for
a Smoke-Free World, a US nonprofit 501(c)(3) private foundation with
a mission to end smoking in this generation. The contents, selection,
and presentation of facts, as well as any opinions expressed herein, are
the sole responsibility of the authors and under no circumstances shall
be regarded as reflecting the positions of the Foundation for a Smoke-
Free World, Inc. ECLAT Srl. is a research-based company from the
University of Catania that delivers solutions to global health problems
with particular emphasis on harm minimization and technological inno-
vation. Smile Study (COE1-05).
Declarations
Conflict of interest RP is full tenured professor of Internal Medicine
at the University of Catania (Italy) and Medical Director of the In-
stitute for Internal Medicine and Clinical Immunology at the same
University. In relation to his recent work in the area of respiratory
diseases, clinical immunology, and tobacco control, RP has received
lecture fees and research funding from Pfizer, GlaxoSmithKline, CV
Therapeutics, NeuroSearch A/S, Sandoz, MSD, Boehringer Ingelheim,
Novartis, Duska Therapeutics, and Forest Laboratories. Lecture fees
from a number of European EC industry and trade associations (in-
cluding FIVAPE in France and FIESEL in Italy) were directly donated
to vaper advocacy no-profit organizations. RP has also received grants
from European Commission initiatives (U-BIOPRED and AIRPROM)
and from the Integral Rheumatology and Immunology Specialists Net-
work (IRIS) initiative. He has also served as a consultant for Pfizer,
Global Health Alliance for treatment of tobacco dependence, CV
Therapeutics, Boehringer Ingelheim, Novartis, Duska Therapeutics,
ECITA (Electronic Cigarette Industry Trade Association, in the UK),
Odontology
1 3
Arbi Group Srl., Health Diplomats, and Sermo Inc. RP has served on
the Medical and Scientific Advisory Board of Cordex Pharma, Inc.,
CV Therapeutics, Duska Therapeutics Inc, Pfizer, and PharmaCielo.
RP is also founder of the Center for Tobacco prevention and treatment
(CPCT) at the University of Catania and of the Center of Excellence
for the acceleration of HArm Reduction (CoEHAR) at the same Uni-
versity, which has received support from Foundation for a Smoke-Free
World to conduct 8 independent investigator-initiated research projects
on harm reduction. RP currently involved in a patent application con-
cerning an app tracker for smoking behavior developed for ECLAT Srl.
RP is also currently involved in the following pro bono activities: sci-
entific advisor for LIAF, Lega Italiana Anti Fumo (Italian acronym for
Italian Anti-Smoking League), the Consumer Advocates for Smoke-
free Alternatives (CASAA) and the International Network of Nicotine
Consumers Organizations (INNCO); Chair of the European Technical
Committee for standardization on “Requirements and test methods for
emissions of electronic cigarettes” (CEN/TC). The other authors de-
clare that they have no conflict of interest.
Ethical approval This study was conducted following the Declara-
tion of Helsinki. The details of the IRB/oversight body that provided
approval or exemption for the research described are given below:
Comitato Etico Catania 2 n. 73/2020/CECT2.
Informed consent Informed consent was obtained from all individual
participants included in the study.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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