Oral Health Group
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Evaluation of Plaque Removal Efficacy of a Novel Dye-Containing Toothpaste: A Clinical Trial


June 30, 2017
by Benjamin Z. Belavsky, DDS; Carla A. Evans, DDS, DMSc; Maria Grace Viana, MSc; Christine D. Wu, MS, PhD

Background
Ineffective dental plaque removal has been shown to cause demineralization, caries, and periodontal disease. 1,2 This results in physical and cosmetic damage to both oral soft and hard tissues in the form of gingivitis, white spot lesions, enamel discoloration, the need for restorations, and potentially tooth loss. Prevalence of tooth decay and periodontal disease is high despite many patients’ claims of following recommended oral hygiene protocols. In fact, the prevalence of caries in adults is 92% and periodontal disease is 8.52%. 3

Poor oral hygiene skill and low dental IQ negatively influence plaque removal efficacy and the ability to evaluate accurately one’s oral status. Increasing education and technique instruction is one way to address the problem, but another option is to improve oral hygiene products to enhance plaque removal efficiency. This is particularly important in populations of low socioeconomic status and the elderly that generally have less access to regular professional oral care. 4 Adding a visible dye to toothpaste to disclose remaining plaque after tooth brushing has the potential to enhance patients’ awareness and encourage them to be more thorough when performing oral hygiene.

Plaque determination and analysis has previously been performed in dental clinics using various manual indices such as those developed by Ramfjord, Silness and Loe, Turesky, and Elliott. 5 However, according to Pretty et al., “traditional plaque indices are problematic due to their integral nature and their failure to detect small, but potentially clinically relevant changes in plaque area”. 6 These procedures are also time consuming, more subjective, and more invasive to patients. The digital plaque imaging analysis (DPIA) method utilizes photography and computer software to accelerate data collection, operator consistency, reproducibility of results, the ability to store data for later use and analysis. Patient comfort is also an important factor. 7,8,9 Dental plaque can be stained with a disclosing agent, e.g. fluorescein disodium salt (D&C No. 8), a well-documented method for intraoral plaque disclosure. 7,8,9 Long wave UV light (405nm), commonly used in medical and scientific applications has been used to excite the fluorescein on plaque, gingiva, and enamel with significant photographic color separation for quantitative analysis. 7 Its efficacy, safety, and reliability have been tested extensively. 9,10,11,12

The objective of this study was to compare the efficacy of plaque removal between dye-containing fluoride toothpaste versus a placebo using the DPIA method. The dye in the experimental toothpaste is an FDA-registered organic food colorant that adheres to plaque and stains it green to provide visual indication of its location. We would determine whether the presence of a visual indicating dye, coupled with instructions on how to use the toothpaste containing it, would cause a difference in plaque removal efficacy. We hypothesize that brushing with an indicating dye-containing toothpaste, along with proper instruction, results in higher plaque removal than using a traditional non-dye containing toothpaste.

Methods
A. Design and Treatment Protocol
Preliminary studies were performed to establish optimal methodology and study protocol for accurate and reproducible data collection for the proposed study.

Thirty-nine participants were recruited in this randomized, controlled clinical study. Participants were recruited with flyers and a classified ad and were selected to represent the general population as best as possible. The study protocol was approved by the Institutional Review Board. The screening questionnaire was administered to interested participants in person or via phone. See Figure 1 for protocol flowchart.

Fig. 1 
Recruitment and subsequent steps taken during data collection appointments.

Inclusion Criteria: Participants must be 18 years of age or older, in good general health (self-assessment), and must have all 12 anterior teeth present (canine to canine).

Exclusion Criteria: Participants should not be pregnant or nursing, not be a dental student/faculty member, not have taken antibiotics within two weeks prior to testing, not have dry mouth symptoms or significant food allergies, not have dental restorations or caries (canine to canine in both arches), and not have had anterior dental work or prophylaxis performed within 30 days prior to testing.

Qualified participants were randomly assigned to one of the two groups – control and experimental. All participants were required to make two visits to the university for this study. Participants in the control group only used the control toothpaste during visits, whereas participants in the experimental group used the control toothpaste at one visit and the experimental (PlaqueHD) at the other. PlaqueHD is an FDA-registered toothpaste which contains an FDA-registered seed-extract dye (Annato (Bixo orellana)), plus FD&C Blue No. 1, which gives the toothpaste a green color that adheres to intraoral plaque. Both toothpastes had similar chemical compositions, apart from the presence of a plaque-indicating dye in the experimental toothpaste.

Participants were instructed to refrain from brushing, flossing, or using other oral hygiene aids the evening prior to and morning of the visit. At the first visit, informed consent was administered to confirm that participants understood the risks and benefits and had an opportunity to ask questions. Participants were then asked to brush their teeth with the control toothpaste using a provided manual toothbrush (Henry Schein Inc., Melville, NY) for one minute in front of a mirror. They were then asked to complete the following procedure for plaque disclosing: rinse for 10 seconds with 25mL of phosphate buffer, rinse for one minute with 5.0 mL of 1240-ppm fluorescein (FD&C yellow No.8) in phosphate buffer, rinse three times for 10 seconds with 25mL of phosphate buffer. 7 The phosphate buffer consisted of 3.62g monosodium phosphate and 0.349g disodium phosphate in 2L of water at a pH of 5.5. Participants expectorated the solution after each rinse.

After rinsing, a digital frontal intraoral photograph of the participant’s upper and lower teeth was taken. Images were taken using a Canon Rebel Xi camera (Canon, Melville, NY) with a Tamron 90 mm fixed focal lens (Tamron, Commack, NY) and a mounted black-light emitting flash (Digi-Slave L-Ring Ultra II UV, SR Electronics, Dallas, TX). Images were oriented to be as perpendicular to the incisors as possible, and centered on the maxillary midline such that the anterior teeth were in focus. Participants held cheek retractors during image capture to increase the visibility of their teeth and gingiva. The room was darkened prior to image capture and images were then saved to a desktop computer.

At the second visit, participants in the control group brushed with the control toothpaste again while the treatment group brushed with the experimental toothpaste (PlaqueHD). The brushing, disclosing, and image-capture procedures were identical to those that took place during the first appointment, however, specific instructions were given on how to use the experimental product, i.e. “brush for one minute and concentrate on removing any green stains.”

B. Data Analysis and Statistics
Using computer software, the twelve anterior teeth in each digital photo were masked (Fig. 2) by the operator to define the area of analysis. The ratio of (plaque pixels)/(plaque + teeth pixels) x 100% was calculated to give an overall patient plaque coverage score. Statistical analysis assessed results of the plaque reduction. 7

Using IBM SPSS Statistics for Windows, Version 22.0 Armonk, NY: IBM Corp., statistics were performed to test the mean of plaque reduction in the control and experimental groups.

Fig. 2
Image masking for data analysis.

Results
Preliminary data from the pilot study, obtained without specific hygiene instructions, showed no mean significant differences in plaque reduction between the control and experimental toothpaste, (p-value>0.05) (data not shown).

Subsequently, 39 subjects were recruited and divided into control (20 subjects) and experimental (19 subjects) groups. Six subjects dropped out due to scheduling conflicts leaving 16 subjects in the control group and 17 in the experimental group. All remaining participants completed two visits; no complaints or adverse effects were reported after using the experimental toothpaste.

The second set of data, obtained after instructions about removing visible stains were given, showed there was no statistically significant mean differences between the control group and experimental group at the baseline initial appointment when using the control dentifrice, t(37) = (0.737), p-value=0.466. And no statistically significant differences in mean plaque reduction between the first and second visits for the control group [t(15) = (-1.377), p-value=0.189] (Fig. 3).

Fig. 3
Comparison of Mean Plaque Coverage (%) of the Control Group between appointments.

The data showed statistically significant mean plaque reduction between the initial baseline appointment and the second appointment for the experimental group using PlaqueHD, [t(16) = (2.718), p-value=0.015] (Fig. 4). The data in Figure 5 show that participants using the experimental toothpaste had more than six times as much plaque elimination than those using the control toothpaste (8.3% mean change versus 51.3% mean change), with the mean change in the control group not deemed of statistically significant value (p=0.189).

Fig. 4
Comparison of Mean Plaque Coverage (%) of the Control Group between appointments.

Fig. 5
Comparison of percentage of plaque remaining, as well as mean change, at both visits.

The data also showed a statistically significant mean difference between the control group and the experimental group after the second appointment, (t(31) = (2.241), p-value=0.032).

The data analysis is reported using parametric Student paired samples t-test and independent t-test. The Shapiro-Wilk Normality test results indicated that the raw data are not distributed on a normal curve, therefore corresponding non-parametric tests were run, as well. Similar results were found with parametric and non-parametric tests, so parametric data were reported. Statistical significance was set at 0.05.

Discussion
Self-performed mechanical plaque removal in adults is frequently ineffective13 and Schäfer et al. were able to demonstrate that providing patients with a means to assess their hygiene habits at home can significantly improve plaque removal efficacy. 14 It seems apparent that by simplifying oral hygiene patients are likely to have improved homecare.

Patients and providers have long been aware of various plaque indicating products, but their use outside of dental offices is typically limited. Disclosing tablets and solutions are commonly used in dental offices, however, to our knowledge, no product that incorporates a plaque-indicating dye and toothpaste was previously available on the market for direct-to-consumer use.

Toothpaste combined with a plaque-disclosing agent is also more likely to work for patients than using multiple products concurrently to achieve the same result. One might speculate that this type of toothpaste may reinforce improved hygiene habits by showing patients where they are deficient in removing plaque (Fig. 6). Choo et al explained that without such reinforcement, patients are likely to revert to old, possibly ineffectual, habits. This is especially important in children, where establishing an easy, visual method of performing good oral hygiene is important for a lifetime of reduced oral disease. 15 In Renz et al.’s review, several studies were cited demonstrating that, for adults, effective plaque removal is based not only on the ability to remove plaque but an understanding of how to do so.16 By combining proper oral hygiene instructions and a toothpaste that discloses remaining plaque, adults should be more effective at removing dental plaque.

Fig. 6
Clinical photographs and predicted outcomes of orthodontic and non-orthodontic patients using PlaqueHD.

In our preliminary study, we discovered that there was no significant difference between a control toothpaste and PlaqueHD when there were no oral hygiene instructions. Our experimental study showed that participants given instructions on how to use a plaque-disclosing toothpaste demonstrated a significant improvement in plaque removal efficacy. Our results demonstrate that when subjects used the control toothpaste at both visits, there was no significant difference in plaque on their teeth – this is because they had no signal to perform hygiene any differently than they normally did and maintained their same oral-hygiene habits. Those patients using PlaqueHD as instructed were able to demonstrate significantly better mechanical plaque removal because they were able to visualize remaining plaque and then remove it. The long-term use of such a product is therefore likely to promote better hygiene habits, and possibly reduce oral diseases.

Further studies with increased subject sample size are warranted. Studies focused on patients with fixed orthodontic appliances would also be beneficial because these patients are typically at higher risk for plaque-mediated disease. 8

Conclusions
This study demonstrates that using a toothpaste with plaque-indicating dye, combined with instructions on how to best use such a product, significantly increases plaque removal efficacy. OH

List of abbreviations
DPIA = Digital Plaque Imaging Analysis
OPRS = Office for the Protection of Research Subjects
SD = Standard Deviation
UV = Ultraviolet

Oral Health thanks Dr. Belavsky for submitting this article.

Acknowledgements
We would also like to thank TJA Health, LLC, for their donation of single-dose samples of PlaqueHD for use in this study.

References
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About the Author
Dr. Ben Belavsky is a practicing Orthodontist in the Northern suburbs of Chicago, where he resides with his wife. He also dedicates two days each week teaching the Orthodontic residents and the dental students at the University of Illinois at Chichago College of Dentistry. Outside of orthodontics, his interests are primarily food-oriented, and he will spend as much time cooking as he does treatment planning. bbelavsky@gmail.com