INTRODUCTION

Orthodontic appliances create obstacles to oral hygiene, resulting in increased plaque accumulation,¹ inadequate oral hygiene,² and orthodontic-induced gingivitis.^3,4 Residual plaque in orthodontic subjects is often found in challenging-to-reach areas (near the gum line, around brackets, and beneath wires).¹

A paucity of literature exists regarding proteolytic hydrolysis in dental oral hygiene. Due to the innate proteolytic activity of enzymes, proteins are hydrolyzed, yielding peptides and amino acids. Enzymes have the potential to lyse adhesive bonds between bacteria and the dental pellicle, delaying bacteria from colonizing on tooth surfaces.⁵ In vivo enzyme studies have demonstrated reductions in plaque and gingivitis.⁶

Bromelain, a naturally occurring cysteine protease derived from pineapple stalks, is used in food and medical industries and listed on the US Food and Drug Administration's “Generally Recognized As Safe” list.⁷ An in vitro bromelain study demonstrated antibacterial effects on oral pathogens.⁸ Bromelain was found to prevent biofilm formation by interfering with bacteria-bacteria adhesion and or adhesion to the enamel surface.⁹ Clinical trials with toothpaste containing bromelain demonstrated improved plaque and gingivitis scores¹⁰ and extrinsic enamel stain removal capabilities.¹¹

The aim of this study was to investigate the impact of a bromelain pre-rinse adjunct on oral plaque removal in orthodontic subjects. The null hypothesis was that there would be no statistically significant difference in visual plaque scores with or without the use of a proteolytic enzyme rinse aid.

MATERIALS AND METHODS

This randomized, single site, crossover, double-blind clinical trial was approved by the Indiana University-Purdue University Institutional Review Board (IRB protocol number: 1802369383). Forty-six (46) subjects were recruited from the Indiana University School of Dentistry, Department of Orthodontics and Oral Facial Genetics clinic. Subjects recruited for the study were existing patients in active treatment, who had fixed metal twin bracket appliances placed prior to enrollment. No attempt was made to standardize patients with one particular bracket type or slot size. Recruited subjects had both 0.018” and 0.022” brackets manufactured by three companies: 3M, American, and ORMCO. Additionally, the subjects recruited were at various stages of active orthodontic treatment. All required treatment consents were obtained from the subjects and or their legal guardians prior to participation in the study. Investigators reviewed subject medical histories to ensure all subjects possessed good health and were fit for participation. Oral soft and hard tissue exams were performed at study visits to ensure subject safety. Subjects were asked to abstain from oral hygiene procedures the morning of and day prior to study visits. A washout period between interventions (1 week ± 2 days) ensured adequate baseline plaque levels were present. At the subsequent visit, subjects performed the same protocol, but received the converse test rinse. Data acquisition took place over 1 month. An overview of the clinical protocol is illustrated in Figure 1.

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Inclusion criteria were: male or female; 10 to25 years of age; willing to consent to treatment; able to follow instructions; and undergoing active orthodontic treatment with fixed metal twin orthodontic appliances. Exclusion criteria were: unwilling/unable to follow study instructions; documented/suspected pineapple allergy; proteolytic enzymes allergy; food dye allergy; and or smoker.

One gram of the enzyme (bromelain) or control (powdered sugar) was weighed on a digital scale (Uniweigh, Shanghai, China), placed into an individual 20-mL vial, and sealed with a plastic cap to prevent contamination and or spillage. Vials were wrapped with paper to conceal the contents. After vial preparation was complete, a neutral individual worked with the biostatistician to code the vials in preparation for the randomized stratification process. After the preparation and coding process was complete, the investigators were blinded and unaware of the vial content or sequence of vial distribution. The bromelain enzyme was obtained in bulk from Ultra Bio-logics Inc. (Chateauguay, Quebec, Canada). The amount of enzyme used in this study was in accordance with the manufacturer's recommendation for dietary oral consumption. Bromelain is stable at room temperature in dry powder form but active in liquid form. The dry powders were reconstituted chairside with 15 mL of warm pineapple juice just prior to administration. A 5” disposable swizzle straw (AmerCareRoyal, Oakville, Ontario, Canada) was used to stir the solution. Canned pineapple juice (Dole Food Company, Westlake Village, CA) was used since the canning manufacturing process destroys the active form of bromelain, while maintaining an optimal enzymatic pH for reconstituting the powder. This ensured a standard quantity of enzyme for all subjects. Bromelain's optimal activity is 55°C, so a temperature-controlled hot plate warmed an electric kettle of pineapple juice to 50°C.

All protocol steps were timed (Figure 1) with a digital timer (Sunbeam Products Inc., Boca Raton, FL) for consistency. The same brand of C-shaped cheek retractors, size L (EZGO, Ontario, Canada) and digital camera (Pentax K-50 digital camera, Ricoh, Tokyo, Japan) with Lester Dine 105-mm F/2.8 macro lens (Dine Corp., Palm Beach Gardens, FL) and 52-mm Eitar SER-VII UV filter (Eitar, Japan) were used for all subjects. The same camera settings (f-stop: 32, shutter speed: 1/60, ISO: 200) were used for all images. The camera was mounted on a tripod and photos were captured under the same clinical conditions. All subjects used the same brand of pre-pasted, individually wrapped, soft bristle, toothbrush: Vivid Premium Quality Toothbrush (Pearson Dental Supply Co, Sylmar, CA), Waterpik: Aquarius Professional Designer Series (Waterpik Inc., Fort Collins, CO), and Two Tone chewable disclosing tablets (TePe USA, Anaheim, CA). Subjects received a new toothbrush and disclosing tablet at each visit.

Intraoral photographs were acquired of every subject at each treatment step to document plaque scores. The photographic series included one frontal and two buccal images of the dentition (Figure 2). One investigator (JR) reviewed all photographs immediately after acquisition to ensure acceptable diagnostic quality.

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The clinical procedure began with subjects chewing and swishing a disclosing tablet in their mouth for 30 seconds. They were then asked to expectorate prior to baseline image acquisition. Thereafter, subjects received the designated vial and were instructed to place the entire solution in their mouth and swish for 2 minutes. They expectorated the solution after the allotted time and had post-rinse photographs taken. Subjects then brushed with a provided toothbrush and were instructed to “brush your teeth as you normally would.” After the attainment of post-brushing photographs, the subjects used the Waterpik and were instructed to “use this device (Waterpik) to the best of your ability to clean your teeth.” The waterjet reservoir (600 mL) was filled with tap water at room temperature and a standard waterjet tip was used. Subjects were allowed to use a mirror, which was covered in pink tinted wrap (The Michaels Companies, Inc., Irving, TX) to obscure visualization of the disclosing agent on their teeth.

A single examiner from the Indiana University School of Dentistry, Department of Dental Hygiene assessed the subjects' degree of plaque retention via digital photographs. The examiner's reliability was calibrated by scoring 10 randomly selected images, which were re-scored after a 2-week washout period. Adequate reliability was assessed using intraclass correlation coefficients and a threshold of 0.8 was set for the study. Once the reliability threshold was reached, the examiner scored each photograph in the study. The coding scheme used to label the photographs did not allow the examiner to link a photograph with a specific subject, intervention, or stage. Each photograph was independently scored using the modified Orthodontic Plaque Index visual scale¹² (Figure 3). Plaque scores were assigned for the maxillary and mandibular incisors and a total score was obtained by summing the values of the eight teeth.

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RESULTS

Forty-three subjects (23 female and 20 male) completed the study and were included in the statistical analysis. Mean subject age was 15.8 (SD = 2.6) and ranged from 11 to 22 years. A total of 53% (23 of 43) of the study subjects were female. Three consented subjects dropped out of the study. One subject elected not to participate after providing consent to participate but before the first data collection appointment. The other two subjects violated the established protocol (brushed their teeth on the morning of the second data collection appointment) and their complete data was excluded from statistical analysis. No subjects presented with or reported any discomfort during this study.

No significant differences were found between enzyme and placebo at baseline (P = .406) or post-rinse (P = .151) (Table 1). Baseline and post-rinse stages were not significantly different for enzyme (P = .055) or placebo (P = .947). The changes from baseline to post-rinse (P = .190), post-brushing (P = .764), and post-Waterpik (P = .882) were not significantly different between interventions. Enzyme application resulted in significantly lower composite plaque scores than placebo post-brushing (P = .012) and post-Waterpik (P = .005).

Table 1. Paired t-Test Assessing Average Sum Plaque Scores

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The plaque scores for all other stages were significantly different from each other for both interventions (P < .001): baseline and post-rinse > post-brushing > post-Waterpik. Stage impacted plaque score (P < .001), as there was a significant reduction of plaque in both arms of the study post-brushing and post-Waterpik. Subject gender (P = .449) and age (P = .220) did not significantly impact plaque scores (Table 2).

Table 2. ANOVA Comparisons of Different Effects on Plaque Scores^a

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DISCUSSION

No significant differences were noted for plaque scores at baseline or post-rinse for either arm of intervention, which indicated there was no bias in data toward test rinse, either before or after rinsing, with or without enzyme. The bromelain pre-rinse alone did not significantly increase plaque removal.

The data demonstrate that, even under controlled settings, subject plaque removal was inadequate (only 64% plaque removal with manual tooth brushing alone). Similarly, van der Weijden and Slot found that manual tooth brushing removed an average of 42% of dental plaque.¹³ Likewise, Atassi and Awartani found that 30% of orthodontic subjects had poor oral hygiene, with average plaque scores around 65%.¹⁴

Enzyme use alone did not yield a significant reduction in composite plaque scores. However, the data demonstrated a trend that suggests proteolytic enzymes could be a potentially beneficial adjunctive agent when combined with traditional forms of oral hygiene. Study results indicated a small benefit by including a proteolytic enzyme in the cleansing routine of orthodontic subjects. This was hypothesized to occur through a number of possible actions including bromelain enzymatically detaching plaque from the dental pellicle, the enzyme dissolving the protein components of the biomatrix of dental plaque, or both. It is likely that hydrolysis interfered with adhesion of the dental pellicle to the tooth surface, since the dental pellicle is made up of glycoproteins¹⁵ and the enzyme acted to cleave protein bonds. In vivo and in vitro studies in the literature demonstrated that protease limits oral biofilm formation by digesting fimbriae and inhibiting biofilm formation.¹⁶ Enzymes hydrolyze extracellular glucans, which thereby inhibit bacteria from adhering and forming plaques.¹⁷

Waterpik use after manual brushing significantly decreased residual plaque an average of 42%. The total combined effect of manual brushing and Waterpik use resulted in a 79% reduction of plaque. These findings were similar to other studies that found that use of pulsating water jet resulted in biofilm reduction¹⁸ and decreased plaque,^4,19 more so than manual brushing alone.^19,20 With the addition of all three modalities, some subjects in the current study were able to completely remove all visual plaque.

The data demonstrated that gender and age did not affect dental plaque scores. This contradicts work by Kudirkaite et al., who found that older adolescent subjects with fixed orthodontic appliances had better oral hygiene.²¹ Unlike the current findings, Broadbent et al. determined that 15-year-old individuals had the highest average plaque scores.²² Work by Atassi and Awartani observed no impact of gender on oral hygiene, which was similar to the results of the current study.¹⁴ However, the current findings contrasted with those of Mei et al.,²³ who found that female adults had the least amount of dental biofilm.

The current study had a few acknowledged limitations. The sample size was slightly smaller than desired due to subject dropout. Additionally, a longer enzyme exposure time may have demonstrated effects that were more significant; however, such a change would have resulted in a protocol that was less clinically applicable. Additionally, the subjects recruited into the study had fixed metal twin brackets manufactured by different companies and with different slot sizes. The subtle variations in bracket size and configuration also could have served as a minor confounding factor for the current study.

Although this study did not find that a bromelain pre-rinse was independently effective in decreasing dental plaque, further studies with a larger sample size and additional standardization steps could demonstrate its utility as an oral hygiene adjunct. Additionally, the use of bacterial co-aggregation to evaluate the effectiveness of the proteolytic enzyme pre-rinse opposed to visual plaque evaluation may provide results that are more conclusive.

CONCLUSIONS

• Use of an oral bromelain pre-rinse alone did not increase dental plaque removal.

• Manual tooth brushing and Waterpik demonstrated significant plaque reduction.

• Further clinical studies are warranted to investigate the impact of enzyme pre-rinses on dental plaque removal and oral hygiene in orthodontic subjects.