Sample
Sample size calculation for this single-blind randomized control clinical trial was based on the formula by Brown and Prescott (2006).
n= 2×(z1-a/2 + zβ )2×σ2×(1+(m-1)×ρ)/(m×Δ2)
= 2×(z1-a/2 + zβ )2×(σ2/ Δ2)×(1+(m-1)×ρ)/m
In the present study two factors were included, namely “GROUP” and
“INTERVENTION” with three and two levels respectively. Therefore, there were 2×3=6 independent groups of patients. The main outcome variable was color difference ΔΕ and it was calculated 6 times (as the difference of 6 follow up TIMEPOINTS from the baseline measurements using the color parameters L*, a* and b*). Each tooth was considered a unit with minimum significant difference between mean ΔΕ values of at least 0.9 SD of ΔΕ with probability at least 80%, and the alpha error probability was set to 0.05. Accordingly, the number of patients in each of the 6 groups was:
n= 2×(1.964+0.842)2×(1.1)2×(1+5×0.5)/6 = 11.1
Rounding up to an even number for randomization purposes, 12 was the number of subjects in each of the six groups, overall N=72 subjects; 432 teeth;
2 repeated measurements per tooth, and 6048 captures in total. Under the hypothesis of a 30% attrition rate, the number of patients in each of the 6 groups was calculated equal to 16.
Randomization of the participants into two groups (BLEACHING and PLACEBO) was performed by the statistician and was communicated to the author of this thesis for each patient on the first day of intervention. The BLEACHING-
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PLACEBO randomization was performed with http://www.randomizer.org. The results for the three levels of INTERVENTION were:
Patients 3, 9, 12, 13, 16, 17, 18, 19, 20, 21, 24, 26, 27, 29, 30, 32 were allocated to the combination BLEACHING-CONTROL and the rest to the PLACEBO- CONTROL.
Patients 2, 3, 5, 6, 7, 8, 10, 12, 13, 15, 17, 21, 23, 25, 31, 32 were allocated to the combination BLEACHING-DEBONDED and the rest to the PLACEBO- DEBONDED.
Patients 1, 2, 6, 11, 12, 15, 16, 17, 18, 22, 23, 25, 26, 27, 28, 32 were allocated to the combination BLEACHING-RETENTION and the rest to the PLACEBO- RETENTION.
All patients in the three groups that were invited to enroll in this study voluntarily consented to participate and were informed accordingly to the ethical principles for medical research involving human subjects. The Ethical Committee of the Faculty of Dentistry, Aristotle University of Thessaloniki reviewed and approved the protocol and assured that all procedures detailed, were contacted in accordance with the guidelines of good clinical practice (protocol number 220-16/11/2010). Each volunteer who participated in the study was provided with and signed a written informed consent.
The first group (DEBONDED) included 24 consecutively selected orthodontic patients, 14 females and 10 males with a mean age 24.8±13.4 years, who received comprehensive fixed appliances orthodontic treatment at the Postgraduate Orthodontic Clinic of the Aristotle University of Thessaloniki and were debonded by the author. The bonding protocol was identical for each subject and according to the manufacturer’s instructions. Enamel surface was etched with 37% phosphoric acid for 15sec, rinsed with distilled water for
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10sec, air-dried for 10 sec at a 5 cm distance and coated with Transbond XT®
primer (3M/Unitek, Monrovia, CA, USA). Each bracket (Clarity Series or Victory Series; 3M/Unitek, Monrovia, CA, USA) received a layer of Transbond XT®
adhesive resin (3M/Unitek, Monrovia, CA, USA) on its base and is positioned and pressed on the buccal surface of each tooth. Excess adhesive was removed and the material was light cured (Ortholux XT® lamp, 3M/Unitek, Monrovia, CA, USA) for 10sec on each side. For this group, in order for a subject to be included in the study, the following inclusion criteria had to be met: (1) comprehensive fixed appliances orthodontic treatment of at least twelve months; (2) non-fixed retention regimen; (3) removal of orthodontic appliances (debonding) performed by the author; (4) full permanent dentition (third molars were not required to be present) and completed root formation of the investigated teeth; (5) devoid of dental caries, prosthetic restorations, enamel attrition, enamel microfractures, decalcifications, and anatomical/morphological deviations in the measured teeth; (6) no reported usage of chlorexidine or fluoride mouthrinse except from the fluoride toothpaste; (7) absence of dietary habits or medication intake that cause tooth discoloration; and (8) non-smokers. These patients were further randomized into two groups: GROUP BLEACHING received a 38% bleaching agent (Opalescence Xtra Boost 38%, Ultradent Products, South Jordan, UT, USA) according to manufacturer’s instructions, while GROUP PLACEBO was treated with application of placebo agent (saline solution).
The second group (RETENTION) included 24 consecutively treated orthodontic patients in retention during the start of this investigation, 13 females and 11 males, with a mean age 21.9±5.9 years. These patients had already received comprehensive orthodontic treatment with fixed appliances at the Graduate Orthodontic Clinic of the Aristotle University of Thessaloniki. Appliances had to be removed within a period of 12-18 months. For the second group the
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inclusion criteria were: (1) comprehensive treatment of at least twelve months; (2) non-fixed retention regimen; (3) absence of dental caries, prosthetic restorations, decalcifications, anatomical/morphological deviations, severe rotations or inclinations in the measured teeth; (4) no reported usage of chlorexidine or fluoride mouthrinse except from the fluoride toothpaste; (5) absence of dietary habits or medication intake that cause tooth discoloration;
and (6) non-smokers. The same BLEACHING/PLACEBO regimen was also adopted for this group.
The third group (CONTROL) consisted of 24 arbitrarily selected (simple random sampling) fourth-year and fifth-year undergraduate dental students at the Aristotle University of Thessaloniki, 14 females and 10 males with a mean age 21.9±0.7 years without a fixed appliances orthodontic treatment history. The same BLEACHING/PLACEBO regimen was also adopted for this group. For the third group the inclusion criteria were: (1) no previous fixed appliances orthodontic treatment; (2) absence of dental caries, prosthetic restorations, decalcifications, anatomical/morphological deviations, severe rotations or inclinations in the measured teeth; (3) no reported usage of chlorexidine or fluoride mouthrinse except from the fluoride toothpaste; (4) absence of dietary habits or medication intake that cause tooth discoloration; and (5) non- smokers. A flowchart diagram of the study indicating patient enrolment, allocation, follow-up and data analysis of the study is presented in Figure 1.
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Figure 1.
Flowchart diagram of the study
Volunteers assessed for eligibility criteria n=51, GROUP 1 (DEBONDED) n=67, GROUP 2 (RETENTION) n=40, GROUP 3 (CONTROL)
GROUP 1 (DEBONDED) Not meeting inclusion criteria n=18 Declined to participate n=1 GROUP 2 (RETENTION) Not meeting inclusion criteria n=32 Declined to participate n=3 GROUP 3 (CONTROL) Not meeting inclusion criteria n=8 Excluded
(n=96)
Allocated to DEBONDED (n=32)
Received allocated intervention (n=30) Did not receive allocated intervention
(dental work, trauma) (n=2)
Allocated to RETENTION (n=32)
Received allocated intervention (n=29) Did not receive allocated intervention
(retreatment, smoking, tea dye) (n=3)
Allocated to CONTROL (n=32)
Received allocated intervention (n=30) Did not receive allocated intervention
(food dye discoloration) (n=2)
Lost to follow up (n=3) Discontinued intervention (dental work, relocation) (n=3)
Lost to follow up (n=1) Discontinued intervention (dental work) (n=1)
Lost to follow up (n=4) Discontinued intervention (relocation) (n=2)
Analyzed (n=24) Excluded from analysis (poor image
quality due to tongue posture) (n=1)
Analyzed (n=24) Analyzed (n=24) Excluded from analysis (for grouping purposes) (n=4) Intervention
Bleaching (n=15)
Intervention Placebo (n=15)
Intervention Bleaching (n=14)
Intervention Placebo (n=15)
Intervention Bleaching (n=15)
Intervention Placebo (n=15)
Randomization Randomization Randomization
34 Data Collection
Teeth included in the study were the upper central incisors (teeth 11 and 21), upper lateral incisors (teeth 12 and 22) and upper canines (teeth 13 and 23).
For the first group on the day of appliance removal, brackets were debonded and residual adhesive material attached to the enamel surface was removed with a 12-fluted carbide finishing bur (No 7803, S.S. White, Lakewood, NJ, USA) in a high-speed handpiece (above 200,000 rpm) using water as a coolant. The underlying enamel was finished with graded medium, fine and extra fine Sof- Lex disks (3M, ESPE, St. Paul, USA) operated at 10,000 rpm with adequate air cooling. Prior to data collection all patients enrolled in the study received final dental polishing of all teeth surfaces using a rubber cup and Zircate prophy paste (L.D. Caulk Division, Dentsply International Inc., Milford, DE, USA).
Appliance and adhesive removal was performed by the author. The debonding/cleaning protocol adopted was described in another study (Zarrinnia et al., 1995). The above-mentioned cleaning protocol was also used in the second group to ensure absence of residual resin material. Identical toothbrushes and dentifrices were provided to all patients who also received oral hygiene instructions and were recalled in 7-14days following appliances’
removal. For all patients, data collection initiated one day before the bleaching/placebo intervention with the first spectrophotometric color measurement (T1). The following day patients received the second spectrophotometric measurement (T2) prior to application of the BLEACHING or placebo agents. Twenty-thirty minutes following the application of the BLEACHING or placebo agents a third measurement was performed (T3). For all groups in addition to the spectrophotometric recordings that were performed at the completion of BLEACHING, other measurements were made after 7 (T4), 14 (T5), 21 (T6), and 90 days (T7). All patients were instructed not to drink coffee or other staining beverages for one hour prior to each measurement.
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Also, 30 minutes before the measuring process, all teeth were thoroughly rinsed with water.
Methods
In the present study, one method was employed to assess tooth color. A reflectance spectrophotometer, SpecroShadeTM Micro (MHT, Zürich, Switzerland; software version 2.20) was used, with a LED light source that transforms into monochromatic light (λ = 400 – 720 nm) by means of a grating.
This light is split so that teeth can be illuminated simultaneously from two sides at a 45° angle with the use of an intra-oral camera. The reflected light is directed at 0° on both the system’s detector areas (640 x 480 pixels, 8-bit depth). One detector area has a color CCD chip responsible for the generation of the colored video image. A CCD detector area records the spectrophotometric data. During a measuring process, light originating from the monochromator of the device emits in 20-nm intervals on a tooth so that 17 photometric spectra of each object is recorded. To finalize a measuring process, the monochromator is brought into a position where no light is emitted. A reflection spectrum is recorded due only to surrounding ambient light. This ambient spectrum subtracts from an object’s total spectrum to obtain the object’s spectrum that originates only from reflection of the standard illumination. Polarization filters are used to eliminate surface gloss.
Spectrophotometric images consisted of 300,000 pixels. CIE-LAB L* [lightness ranging from 0 (black) to 100 (white)], a* (red/green), b* (yellow/blue) color parameters were calculated for each object (CIE, Commission Internationale de l’Eclairage, 2004). The coordinate L* is a measure of lightness similar to
‘value’ and the readings a* and b*, when combined, provide the same information as ‘hue’ and ‘chroma’ in the Munsell system (Munsell, 1961).
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A standardized protocol of tooth preparation and in vivo spectrophotometric evaluation was adopted for all patients. Measurements were performed in the same examination room with standardized lighting conditions (Karamouzos et al., 2007). All spectrophotometric images were recorded by the same operator at seven time intervals. SpectroShade Micro was calibrated according to manufacturer’s instructions and a sterile mouthpiece was placed on the optic hand-piece for each patient (Figure 2). The optic hand-piece was then positioned at 90-degree angle to the target tooth, flush against the gingival border and image was visually checked for signs of tongue, lips, opposing teeth. Patient was asked not to breath directly onto the mouthpiece causing fogging and inaccurate shade reading. Four spectrophotometric images were recorded for each tooth; 4 consecutive recordings by positioning, removing and repositioning the optic handpiece at a 30sec interval, then the same procedure was repeated for each tooth. The acquired data was further compared for quality control through the synchronization tool of SpecroShade Micro. This software can overlap one image onto another by aligning the colorimetric data-points of two images and produce an overall comparison in ΔΕ units (Figure 3). When two images were compared the ΔΕ had to be below 1.00 (Figure 4). The CIE color parameters (L*, a*, b*) were also measured and averaged for each tooth. The resultant ΔΕ between the timepoints was calculated according to the following equation:
ΔΕ = [(L*i – L*ii)2 + (a*i – a*ii)2 + (b*i – b*ii)2]1/2
where i and ii represent the color measurements made at two different timepoints (Kuehni and Marcus, 1979; Seghi et al., 1989).
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Figure 3. The synchronization tool of SpectroShade™ Micro software.
Figure 2. Calibration was performed by placing the optic handpiece on the white and a green ceramic tile before each image capture.
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Figure 4. The best two image captures were selected per measurement and color parameters L*, a* and b were calculated; when the two images were compared the ΔΕ had to be below 1.00.
Statistical Analysis and Method’s Error
Intra-rater agreement was estimated with the Intra-class Correlation Coefficient based for two repeated measurements of the parameters L*, a*
and b* on 24 patients. Method’s error was calculated using the repeatability coefficient set by the British Standards Institution and given by the formula 2 × SD difference, where SD difference is the standard deviation of the difference between the repeated measurements. Intra-rater agreement and method’s error were recalculated for three types of teeth, 13, 11 and 22. Regarding parameters L*, a* and b*, the data at baseline were analyzed with analysis of variance model in order to study differences between groups due to random allocation of the patients. In case where differences were observed between groups at baseline, comparisons between follow up and baseline was done with a linear mixed model (algorithm MIXED IBM Statistics SPSS 21.0 [2,3,4]).
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The effects of GROUP, INTERVENTION, TIME and the interactions between them where then analyzed with another linear mixed model where the baseline measurement was used as a covariate in order to adjust the estimated means for baseline differences. If there were no differences at baseline a linear mixed model was used that also included the baseline level in the factor TIME. Regarding parameter ΔΕ the analysis was performed with a linear mixed model on the color differences between follow up TIMEPOINTS and baseline measurements including the main effects and the interactions of the factors GROUP, INTERVENTION and TIME. Further, the analysis of each of the foregoing parameters was rerun with a series of linear mixed models that included the confounders “DURATION OF TREATMENT”, “ANGLE CLASSIFICATION”, “ORTHODONTIC TREATMENT PLAN“, and “MONTHS IN RETENTION”, in order to study any effects of them in the results. The choice of the appropriate residual variance-covariance matrix in the linear mixed models used in the analysis was based on the Schwarz's Bayesian Criterion (BIC) while in addition the assumptions of residual normality and homoscedasticity were verified with the Kolmogorov Smirnov Statistic and the scatterplot between predicted values and residuals, respectively. The analysis was performed with the statistical software IBM SPSS Statistics 21.0 and the statistical significance for all tests was set at p<0.05.
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