ARTICLE
Auteur(s) : Emi
Nishijima Sakanashi1, Mitsuaki
Matsumura1, Katsuko Kikuchi2, Masaomi
Ikeda3, Hiroyuki Miura1
1Fixed Prosthodontics, Department of Restorative
Sciences, Division of Oral Health Sciences, Graduate School,
Tokyo Medical and Dental University, 1-5-45, Yushima,
Bunkyo-Ku, 113-8549, Tokyo, Japan
2Department of Dermatology, Tohoku University
Graduate School of Medicine, 1-1, Seiryou-chou, Aoba-Ku,
980-8574, Sendai, Japan
3School of Dental Technologists, Faculty
of Dentistry, Tokyo Medical and Dental University,
1-5-45, Yushima, Bunkyo-Ku, 113-8549, Tokyo, Japan
accepté le 9 Juin 2010
Traditionally, different types of alloys have been used in
restorative dentistry. The common criterion for all these materials
is their permanent existence in the oral cavity for a prolonged
period of time and this exposure may sensitize patients. The
clinical manifestations of contact allergy to dental alloy are not
uniform. Diseases such as pustulosis palmaris et plantaris, lichen
planus, systemic or palmoplantar eczema, symptoms like glossodynia,
cheilitis related with ions released from these metals are well
documented [1-6]. Furthermore, the Japanese Ministry of Health and
Welfare reported in 1997 that allergy affects approximately 30% of
the population in Japan and recently, the frequency of dental metal
allergy has risen significantly [7-9].
Patch testing is the primary tool to diagnose allergens causing
allergic contact dermatitis. On the other hand, this method is
strongly dependent on the experience of the observer, and
distinguishing doubtful-positive from positive patch test (PT)
reactions for different reagents remains difficult [10-14].
Reflectance confocal laser microscopy (RCLM) has been used to image
human skin non-invasively, providing a virtual window into tissues
in vivo without staining processes or destruction of the skin
[15-18]. More recently, diagnostic criteria with RCLM were
investigated to characterize features of allergic contact
dermatitis. These criteria include vesicle formation, inflammatory
infiltrate, spongiosis and exocytosis [19-22]. However, skin
reactions to different metal allergens and changes in epidermal
thickness are still not well understood [23, 24]. The aim of this
study was to compare suprabasal epidermal thickness, using RCLM,
with clinical grading of PT from two major allergen metals i.e.,
nickel (Ni) and cobalt (Co) in Japan.
Materials and methods
Subjects
Eight healthy volunteers (3 males and 5 females, mean age
30 years), and eleven patients (1 male and 10 females,
mean age 47 years) with suspected contact allergy to dental
metals and confirmed presence of dental metal restoration from the
Dental Allergy Clinic of Tokyo Medical and Dental University were
recruited for this study (table 1).
We received approval from the Dental Research Ethics Committee
of Tokyo Medical and Dental University and informed consent was
obtained from all contributors.
Table 1 Characteristics of patients
|
Patient
|
Age/ Sex
|
Symptoms & systemic diseases
|
Years of symptoms & systemic diseases
|
Dental metal restorations
|
Mean years in use dental metal restoration
|
|
1
|
36/F
|
CD (jewellery)
|
20
|
Au-Ag-Pd alloy
|
10
|
|
2
|
67/F
|
CD (jewellery, leather)
|
6
|
Au-Ag-Pd alloy
|
20
|
|
|
HF
|
5
|
|
|
|
3
|
28/F
|
eczema (face, neck, head) HF, sinusitis
|
1 month 8
|
Au-Ag-Pd alloy
|
8
|
|
4
|
38/F
|
CD (jewellery)
|
15
|
Amalgam
|
22
|
|
|
stomatitis
|
2
|
Au-Ag-Pd alloy
|
10
|
|
5
|
67/F
|
CD (jewellery)
|
17
|
Au-Ag-Pd alloy
|
40
|
|
|
adenoiditis
|
50
|
Co-Cr alloy
|
20
|
|
|
loxoprofen allergy
|
7
|
|
|
|
6
|
58/F
|
stomatitis
|
1
|
Au-Ag-Pd alloy
|
25
|
|
7
|
68/F
|
oral lichen planus on buccal surface adjacent
to the metal restoration
|
2
|
Au-Ag-Pd alloy
|
40
|
|
8
|
40/M
|
pustulosis palmaris et plantaris
|
8 months
|
Au-Ag-Pd alloy
|
20
|
|
9
|
66/F
|
CD (jewellery)
|
26
|
Au-Ag-Pd alloy
|
41
|
|
10
|
51/F
|
CD (jewellery)
|
10
|
Au-Ag-Pd alloy
|
20
|
|
|
|
|
Ni-Cr alloy
|
30
|
|
11
|
44/F
|
cheilitis facial erythema
|
2
|
Au-Ag-Pd alloy
|
20
|
|
|
|
|
Co-Cr alloy
|
2
|
Exposure to reagents
The upper back skin was exposed to aqueous solutions of 5% nickel
sulfate (Ni) and 2% cobalt chloride (Co) (Torii Pharmaceutical Co.
Ltd, Tokyo, Japan) using patch tester TORII® (9-mm
diameter, Torii Pharmaceutical Co. Ltd, Tokyo, Japan). The testers
were removed by the subject after 48 h on day 2 (D2).
Evaluation
Clinical evaluation
Skin readings were performed 20 min after removal of the
testers on D2, D3, and, D7 according to the clinical scoring
criteria recommended by the International Contact Dermatitis
Research Group [21] (table 2).
Positive reactions on D3 or D7 were judged as allergic contact
sensitivity. Skin reaction only on D2 and faint erythema or absence
of reaction on D3 and/or D7 was judged as doubtful-positive
reaction. In addition, clinical photographs of skin reactions were
taken under standardized conditions with a digital camera (Nikon
Coolpix 950, Nikon Corp, Tokyo, Japan).
Table 2 Criteria for clinical scoring
of allergic contact dermatitis reactions
|
Score
|
Allergic contact dermatitis
|
|
0
|
Negative
|
|
0.5
|
Barely erythema
|
|
1
|
Erythema, infiltration, possibly papules
|
|
2
|
Erythema, infiltration, papules, vesicles
|
|
3
|
Intensive erythema, bullous reaction or erosion
|
RCLM evaluation
A commercially available RCLM (Vivascope 1500 Plus, Lucid Inc,
Henrietta, NY) was used to produce horizontal (surface) images of
skin sites with X, Y, and vertical (in depth) images with Z plane
micrometer screws. This device use a diode laser at 830 nm
with a power less than 16 mW at tissue level. The X30
water-immersion lens of numerical aperture 0.9 was applied to the
skin. It was immobilized with a tissue ring and template fixture
device to provide standardized mechanical contact with the RCLM.
Details of the instrument are given elsewhere [16, 17]. In each of
the skin sites analyzed, a systematic 4 mm2 X-Y
mapping was performed and 5 images were captured in Z plane
per 1μm in depth, beginning at the stratum corneum and going
through the epidermis and into the upper reticular dermis. The
suprabasal epidermis (from the surface of the stratum corneum with
presence of corneocytes to the bottom of the cells in the uppermost
portion of the stratum basale) were used to calculate thicknesses
before (D0) and after testers were removed (D2, D3, D7) (figure 1, A panel).
Statistical analysis
Suprabasal epidermal thickness of healthy volunteers and patients
before PT was analyzed using the Student t-test. Changes of
suprabasal epidermal thickness at the time points of skin readings
were analyzed using one-way ANOVA and Tukey HSD test [25, 26].
Furthermore, in patients, the paired t-test was used on the same
subjects to analyze differences in suprabasal epidermal thickness
between Ni and Co. Clinical scores were performed with the
Mann-Whitney U test and Regression analyses to determine the
correlations between suprabasal epidermal thickness and elapsed
period of days [27]. All analyses were conducted using SPSS
Ver.12.0J for Windows. For global significance, P less than 0.05
and for Bonferroni, P less than 0.016 was considered statistically
significant.
Results
Clinical evaluation
Healthy volunteers
All subjects were found to be clinically negative to Ni
and Co. However, punctante erythema and hemorrhagic around the
hair follicles openings, presenting small dots, irregularly
distributed within the test area were observed on D2 and D3 for Co
on two of 8 healthy volunteers (figures 2, A and
B panel).
Patients
From 11 subjects three were found to be doubtful-positive PT
to Co, two were positive PT to Co and four were positive PT to Ni.
(P < 0.05 and P < 0.016) (figures 2, C and
D panel; tables 3, 4).
Table 3 Severity of clinical scoring in patch
test for Ni and Co
|
Patient
|
Clinical scores
|
|
Ni
|
Co
|
|
1
|
2
|
0
|
|
2
|
2
|
0
|
|
3
|
1
|
0
|
|
4
|
1
|
0
|
|
5
|
0
|
0
|
|
6
|
0
|
0.5
|
|
7
|
0
|
0
|
|
8
|
0
|
1
|
|
9
|
0
|
0.5
|
|
10
|
0
|
1
|
|
11
|
0
|
0.5
|
Table 4 Percentage distribution of clinical scores
and average of suprabasal epidermal thickness from D2,
D3, D7 among Ni and Co in patients
|
Reagent
|
Patch test result
|
Clinical Scores median (25th/75th)
|
Average on suprabasal epidermal thickness from D2, D3, D7 mean
(SD) μm
|
|
Ni
|
positive (n = 4) negative (n = 7)
|
1 (1/2) a* 0 (0/0) a*
|
37.25 (8.14) a* 25.33 (5.92) a*
|
|
Co
|
positive (n = 2) doubtful positive (n =3) negative (n = 6)
|
1 (1/1) b1* 0.5 (0.5/0.5) b2* 0 (0/0) b1* b2*
|
30.83 (6.7) b1* 30 (4.97) b2* 24.06 (2.38) b1* b2*
|
RCLM evaluation
Suprabasal epidermal thickness did not show a significant
difference between healthy volunteers and patients on D0 (P >
0.05) (figure 1
B panel) [18].
Healthy volunteers
All subjects showed an increasing tendency of suprabasal epidermal
thickness on D2, decreasing consequently until normal parameters on
D7 (figure 3).
Statistical analysis showed no significant differences among time
points of assessment for Ni and Co, nor between the reagents (P
> 0.05).
Patients
The increasing suprabasal epidermal thickness on Ni and Co was
greater in patients than healthy volunteers (t = – 5.942, P
< 0.0001). We found statistical differences between D0 and D2,
D0 and D3, D0 and D7 for Ni and Co respectively (P < 0.05).
Between the reagents, statistical significance showed on D2 and D7
(t = 2.094, P = 0.049) (t = 0.692, P = 0.0497). For each PT result,
the average suprabasal epidermal thicknesses from D2, D3, and D7;
Ni-positive and Ni-negative, Co-positive and Co-negative,
Co-doubtful-positive and Co-negative were statistically significant
(P < 0.05 and P < 0.016) (figure 4, table 4).
Regression analyses of suprabasal epidermal thickness in
positive reactions to Ni vs. elapsed period by days were
significantly strong positive (R2 = 0.6191, P < 0.0003), and
negative Ni reactions showed a significantly moderate positive
correlation (R2 = 0.3119, P < 0.0071) (figures 5A, B).
For Co, regression analysis of suprabasal epidermal thickness
vs. elapsed period by days showed moderate positive significance in
positive Co reactions (R2 = 0.5686, P = 0.0322), strong positive
significant correlation in negative Co reactions (R2 = 0.6993, P
< 0.009) and not significant correlation in doubtful-positive Co
reactions (P = 0.0535) (figures 5C, D,
E).
Discussion
PT is said to be the most conventional evaluation method for
allergic contact dermatitis [28]. The instrument used for clinical
assessment is a combination of vision and feel in the form of
palpation with the examiner's finger, and this measurement is a
totally subjective method based on the clinician's knowledge and
experience.
Interestingly, previous reports of PT readings have shown the
disagreement on scoring among examiners under the same conditions
[29]. The RCLM represents a useful measurement for determining an
individual's skin hypersensitivity [30, 31].
In our study, we evaluated the efficiency of the RCLM in grading
the severity of allergic skin reactions to Ni and Co and determined
the correlation with routine visual PT results. Patients showed a
significant increase in suprabasal epidermal thickness compared
with healthy volunteers. This indicated that the differences
observed on the skin response could be explained by the occlusive
effect and the metal reagents of the patch tester acting as
mechanical stimuli with the additional response of specific T-cell
involvement in patients [32].
The Ni-positive group showed an overall increase in suprabasal
epidermal thickness, intercellular edema, acanthosis and great
vesicle formation. This result might be due to a stronger
proliferative response of keratinocytes and T-cell involvement in
the pathogenesis of metal hypersensitivity than that to Co [19, 33,
34]. The Co-positive group showed an increase of the suprabasal
epidermal thickness mainly on D2 and D3, and both positive groups
were well correlated with respective clinical aspects of PT scoring
in our study.
In Co-doubtful positive group, the RCLM images showed two
different aspects.
- – The presence of superficial disruption in the stratum
corneum on two of 3 patients. However, we did not observe RCLM
morphologic features common to allergic contact dermatitis [24, 29,
35].
- – The increase of suprabasal epidermal thickness in the
remaining one of the 3 patients, which could be recognized as
positive in RCLM, the visual assessment by PT displayed doubtful
positive. The facial erythema and cheilitis of this patient were
improved after the removal of a cobalt-chrome metal frame-removable
denture with no additional treatment.
Moreover, we found that Co showed more doubtful-positive PT
reactions than Ni (Ni-positive: 21.44%, Ni doubtful-positive:
5.15%, Co-positive: 10.72%, Co doubtful-positive: 7.01%,
(χ2 = 10.527, Haberman's test, p < 0.002) from
485 patients (aged between 27 and 82 years, mean age
48 years) who visited our clinic from 2006 to 2007 (data not
shown) [36]. This result supported previous studies that the
characteristics of allergic reactions in Co showed weaker signs
compared to those of the Ni group [23, 24, 37, 38].
Although we assessed only two specific allergens, Ni and Co, the
morphology of PT reactions can vary with different metals. For
instance, palladium and gold have a tendency to appear as a skin
reaction 5 days after removing the reagent [39]. Therefore,
the observations of epidermal thickness and morphological changes
by the RCLM should each be performed after the visual
assessment.
One of the main limitations of the RCLM technique is the
fundamental inability to image deep objects in the dermis. In
addition, acanthosis due to intercellular edema, vesicle formation
and keratinocyte proliferation can also restrict the visualization.
In some other studies different concentrations of allergen were
used, different types of vehicles and other types of patch testers,
for example T.R.U.E. test (Thin-layer Rapid Use Epicutaneous Test,
Allerderm Lab, Phoenix, Ariz.) [40, 41]. Since different approaches
and materials could lead to variations in the results, the
selection criteria of the tests should be considered and structured
to obtain more accurate results of the reaction processes of the
allergic contact dermatitis.
Co and Ni are components of many types of alloy that can be
found in daily life. However, in dental treatment, it is possible
to avoid such allergens by proper selection of materials or by
using metal free restorations [42, 43].
In conclusion, our study demonstrated the potential of the RCLM
to assist in more accurate interpretation of test results between
allergic, doubtful and irritant reaction on each allergen, rather
than by using visual assessment alone.
Disclosure
Acknowledgements: we would like to express gratitude to Mr.
Hoshiaki Sawata to gives us insightful comments and great help
during preparation of this paper. Financial support: none. Conflict
of interest: none.
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