The use of qlf to quantify in vitro whitening in a product testing model

ABSTRACT BACKGROUND Professional and consumer interest in whitening products continues to increase against a background of both increased oral health awareness and demand for cosmetic

procedures. In the current legal climate, few dentists are providing 'in-office' whitening treatments, and thus many patients turn to home-use products. The most common of these

are the whitening toothpastes. Researchers are keen to quantify the effectiveness of such products through clinically relevant trials. AIM Previous studies examining whitening products have

employed a variety of stained substrates to monitor stain removal. This study aimed to quantify the removal of stain from human enamel using a new device, quantitative light-induced

fluorescence (QLF). The experimental design follows that of a product-testing model. MATERIALS AND METHOD A total of 11 previously extracted molar teeth were coated with transparent nail

varnish leaving an exposed window of enamel. The sound, exposed enamel was subject to a staining regime of human saliva, chlorhexidine and tea. Each of the eleven teeth was subjected to

serial exposures of a positive control (Bocasan), a negative control (water) and a test product (Yotuel toothpaste). Following each two-minute exposure QLF images of the teeth were taken (a

total of 5 applications). Following completion of one test solution, the teeth were cleaned, re-stained and the procedure repeated with the next solution. QLF images were stored on a PC and

analysed by a blinded single examiner. The ΔQ value at 5% threshold was reported. ANOVA and paired t-tests were used to analyse the data. RESULTS The study confirmed the ability of QLF to

longitudinally quantify stain reduction from human enamel. The reliability of the technique in relation to positive and negative test controls was proven. The positive control had a

significantly (α=0.05) higher stain removal efficacy than water (p=0.023) and Yotuel (p=0.046). Yotuel was more effective than water (p=0.023). CONCLUSION The research community, the

practicing clinician and the consumer all require sound product evaluation data. The use of human enamel specimens may offer more relevant clinical data. QLF has been designed as an _in

vivo_ device. Further development of the technique should permit _in vivo_ clinical whitening trials. You have full access to this article via your institution. Download PDF SIMILAR CONTENT

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IN-OFFICE PEROXIDE-FREE TOOTH-WHITENING GELS: BLEACHING EFFECTIVENESS, ENAMEL SURFACE ALTERATIONS, AND CELL VIABILITY Article Open access 22 June 2020 MAIN With the measurable improvement in

the nation's oral health, many individuals are looking to enhance their appearance through 'whiter' teeth, rather than simply seeking restorative treatments.1,2,3 The use of

professionally applied bleaching products has come under judicial scrutiny in the UK, and with no clear message to practitioners, many do not offer such procedures. Patients must therefore

use 'at-home' products to achieve tooth colour change, typically through the removal of extrinsic stain. The majority of these products are marketed as tooth-whitening dentifrices.

The causes of extrinsic tooth staining are complex and have been covered in the literature recently.4 The method of action of these pastes is believed to be a combination of mechanical and

chemical effects.5 Research has assessed both of these modalities, with the current study concentrating on the chemical properties.6 In order to properly assess these toothpastes a number of

studies have been performed. One of the most comprehensive was by Sharif _et al_.5 The techniques used in Sharif's paper were developed by Addy and involve the use of stained Perspex

with spectrophotometer analysis.7 The strengths of such a technique include the ability to control the application of the stain, the exposure of the test product to the specimen and also to

longitudinally monitor any reduction in stain. However, it would be ideal to retain these experimental strengths while using a more authentic stained substrate, human enamel. Using a new

technology for the detection of early carious lesions, the current study describes such a technique. QUANTITATIVE LIGHT-INDUCED FLUORESCENCE (QLF) The detection of very early (and hence

reversible) carious lesions by optical methods has been a major research focus of cariologists.8 One of the most promising devices is the QLF (Inspektor Research Systems BV, NL).9,10 The

underlying principle of the QLF technique is that teeth will auto-fluorescence under certain light conditions, and that demineralised enamel will fluoresce less than sound enamel. When

examined under the QLF, demineralised lesions appear as dark areas. With analytical software, the loss of fluorescence characteristic of demineralised areas can be compared with the

unaffected fluorescence of the sound enamel and a quantifiable value, ΔQ, reported. Developed from early experimentation with lasers, the QLF device has been the subject of numerous

validation articles.11,12 The QLF handpiece is shown in Figure 1. QLF AND STAINING Amaechi has described the potential for QLF to quantify stain removal.13 The stain produced on the teeth

appears on the QLF image in a similar manner to demineralised lesions. The analytical software is able to compare the stained enamel to adjacent unstained enamel in the same way as

demineralised and sound enamel is compared. This study furthers the initial work by incorporating it into a product-testing model. By employing this mechanism a longitudinal and quantifiable

analysis of stain reduction on human enamel specimens is possible. MATERIALS AND METHODS A total of 11 previously extracted human mandibular third molars were selected. Buccal surfaces were

devoid of intrinsic stain, enamel cracks or fractures, caries or other enamel defects. The teeth were gently pumiced and their buccal surfaces gently abraded with wet-and-dry paper. Clear

nail varnish (MaxFactor, Proctor and Gamble, Ltd., UK) was applied to the buccal surfaces leaving an exposed window, mean area 13 mm2. The teeth were then mounted into the black plastic caps

of 50 ml bottles with dental compound (Kerr Dental, UK). The caps containing the teeth were attached to a series of 50 ml bottles (Jencons PLC, UK) containing 30 ml of a)human saliva,

b)chlorhexidine mouthwash and c)tea. The specimens were gently agitated in an orbital incubator at 37°C for a total of two minutes in each solution. The teeth were subjected to two staining

cycles. Following staining, the nail varnish was removed leaving a stained area surrounded by periphery of un-stained enamel (see Figure 2). The QLF camera was mounted in a fixed position

over a laboratory jack (see Figure 3). Each cap was placed onto the jack's platform and baseline QLF images taken. Fine focussing of the QLF image was obtained by the adjustment of the

jack in vertical relation to the QLF camera. Markings on the platform and plastic caps enabled reproducible placement of the teeth on each occasion. Black plastic caps reduced internal

reflection of the QLF light source, providing a sharp clear image. Three test preparations were used. Bocasan (Oral B, UK) as a positive control, distilled water as a negative control and

Yotuel whitening toothpaste (Yotuel Classic, Biocosmetics, Spain) as a test product. The test preparations were prepared as per Sharif's method; Bocasan, 1 sachet dissolved in 15 ml of

water and Yotuel as a slurry (3 g in 15 ml of water). Each specimen was gently agitated in the solution for 2 minutes at 37°C. Following each application, the specimen was gently washed and

5 QLF images taken. This was repeated for 5 applications (10 minutes total exposure). An example of the image series obtained is shown in Figure 4. Following completion, the teeth were

cleaned, re-stained and the procedure repeated with the next test preparation. Immediately following cleaning, baseline QLF images were taken to ensure the integrity of the enamel. These

images were compared with the baselines for previous staining cycles to ensure no demineralisation or other optical event had occurred. QLF images were stored on a PC and were analysed by a

single, blinded examiner using QLF V2.00 (Inspektor Research Systems BV, NL). The QLF analysis method is illustrated in Figure 5. Following capture of the image, an analysis

'patch' was placed around the area of interest (AOI), in this case the stained area. It was essential that the borders of the patch fell on sound, or unstained, enamel. The image

was then analysed by the software and the reconstructed stained area shown. The reconstruction was checked to ensure that it mimicked the original AOI morphology, and if inconsistencies were

noted, the analysis was repeated. Mean ΔQ values were reported and the % stain removed calculated. ANOVA followed by paired t-tests was used to analyse significant differences between the

test preparations. RESULTS Mean baseline (unstained) ΔQ values for each test preparation were 1.76 (±2.90) (Bocasan), 3.29 (±5.16) (water) and 1.72 (±2.79) (Yotuel). Mean baseline (stained)

ΔQ values for each test preparation were 240.80 (±119.67) (Bocasan), 235.12 (±110.97) (water) and 326.35 (±134.39) (Yotuel). The % decrease in stain following each exposure to test

preparation is shown in Figure 6. Bocasan had significantly better extrinsic stain removal than water (p=0.005) and Yotuel (p=0.046). Yotuel had significantly better extrinsic stain removal

than water (p=0.023). Alpha was pre-set at 0.05. DISCUSSION AND CONCLUSIONS Many novel techniques have been suggested for the quantification of stain removal from enamel specimens. McCaslin

_et al_ used digitised photographs and NIH Image (National Institutes of Health, US) to detect whitening via 256 shades of grey.14 However, studies that examined the presence of white spots

adjacent to orthodontic brackets (a broadly similar technique) found that photographs were sensitive to flash reflections, angulation and magnification factors that must be tightly

controlled.15 Amaechi used the ShadeEye device (Shofu, USA) to longitudinally monitor extrinsic stain removal from enamel.16 However the ShadeEye device does not permit the user to visualize

the tooth image and provides quantitative data via printout only. The reliability and operational definition of this device for stain removal has not been determined and further research is

required to assess its use in product testing models. Other groups have used a combination of optical (colorimeter), photographic (35 mm slides, subsequently digitised) and shade guide

techniques to monitor _in vivo_ changes.17 The comparison of such techniques in terms of reliability and validity (i.e. what exactly are they measuring?) has yet to be carried out. The QLF

images, and their subsequent analysis demonstrate the ability of the technique to quantifiably monitor the longitudinal decrease in stain following application of test products with

appropriate positive and negative controls. The described methodology represents a more authentic stained substrate in the form of human enamel. The study has shown that the Yotuel product

is able to remove artificial extrinsic stain from teeth. The QLF analysis software enables visualisation of stain reduction, in combination with a quantitative assessment of the action of

the test preparations. There are several advantages of the QLF technique over other optical methods. As QLF uses the auto-fluorescence of the teeth to produce an image, a flash is not

required. This reduces image variability between exposures and increases the reliability of the technique. By storing the captured digital images QLF permits the blinded assessment of the

data and, of particular importance in product testing trials, the ability for other interested parties to examine the raw data obtained during trials. The same examiner, using the same

images, can repeat QLF analyses and thus repeat testing and verification is possible. Analysed images are stored with their unanalysed counter-parts and may easily be archived. A technique

such as a colorimeter or the ShadeEye does not permit such archiving or blind testing. The QLF device was primarily designed as an _in vivo_ device to detect and quantify early enamel

demineralisation. Its application to stain measurement has been demonstrated. The _in vivo_ capability of the device will allow future clinical trials on whitening products to be conducted

out of the laboratory and thus increase the validity of product testing. Further research to incorporate the more generalised staining of teeth _in situ_ into the methodology is required.

The difficulty arises from the need for QLF to compare stained tissue to unstained. The use of a generic stain-free mask over the acquired _in vivo_ images may resolve this problem. Methods

such as photographic assessment and the ShadeEye do not suffer this problem, as they do not require un-stained enamel to conduct a comparison. As such the QLF technique requires subjective

input from the examiner at the image analysis stage (the placement of the analysis patch, see Figure 5). Such subjectivity could introduce variability to the assessment of stain reduction. A

study performed looking at the reliability and effect of subjectivity on QLF analyses found that both intra- and inter-examiner reproducibility was high when assessing _in vitro_ caries

lesions.18 The assessment of stain reduction is simpler, and it is reasonable to extrapolate these results to the reliability of stain assessment. Reliability can also be increased by using

multiple examiners or repeated measures by the same examiner.18 QLF presents an expeditious, user-friendly, economical and resilient method of assessing stain reduction. The data produced

can be archived and the ability to blind examiners and conduct repeated analysis of the images strengthens studies employing QLF. Its ability to longitudinally monitor stain reduction from

enamel specimens _in vitro_ has been proven and its use in product testing demonstrated. The future could present the general practitioner with a method of quantifying, and therefore

documenting, the effectiveness of any whitening procedure. Such a technique would satisfy medico-legal and patients' requirements for proof of efficacy. REFERENCES * Pine C M, Pitts B

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Inter-examiner reliability of QLF analyses. _Caries Res_ 2001; 35: 269. Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Doctoral Student, Department of

Clinical Dental Sciences, The University of Liverpool, The Edwards Building, Daulby Street, Liverpool, L69 3GN I A Pretty * Emeritus Professor, Department of Clinical Dental Sciences, The

University of Liverpool, The Edwards Building, Daulby Street, Liverpool, L69 3GN W M Edgar * Senior Lecturer, Department of Clinical Dental Sciences, The University of Liverpool, The Edwards

Building, Daulby Street, Liverpool, L69 3GN S M Higham Authors * I A Pretty View author publications You can also search for this author inPubMed Google Scholar * W M Edgar View author

publications You can also search for this author inPubMed Google Scholar * S M Higham View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING

AUTHOR Correspondence to I A Pretty. ADDITIONAL INFORMATION _R_ _efereed_ _ P_ _aper_ RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Pretty, I., Edgar,

W. & Higham, S. The use of QLF to quantify _in vitro_ whitening in a product testing model. _Br Dent J_ 191, 566–569 (2001). https://doi.org/10.1038/sj.bdj.4801235 Download citation *

Received: 21 February 2001 * Accepted: 03 September 2001 * Published: 24 November 2001 * Issue Date: 24 November 2001 * DOI: https://doi.org/10.1038/sj.bdj.4801235 SHARE THIS ARTICLE Anyone

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