Clinical trial NCT04512482
Conditions: Dental Plaque Dental Caries
Keywords: proteolytic enzyme plaque removal oral hygiene orthodontic treatment
Sponsor: Indiana University
Start date: 2018-06-14; Completion date: 2019-03-04
Enrollment: 46 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
This study aims to investigate the impact of a bromelain pre-rinse adjunct on oral plaque removal in orthodontic subjects. The study's null hypothesis states that there is no statistically significant difference in visual plaque scores among orthodontic subjects with or without proteolytic enzyme rinse aid.

Detailed description:
Bromelain, a naturally occurring cysteine protease, from pineapple stalks, is used in food and medical industries and listed on the FDA's "Generally Recognized As Safe" (GRAS) list. In vitro bromelain study demonstrated antibacterial effect on oral pathogens. Bromelain was found to prevent biofilm formation by interfering with bacteria-bacteria adhesion and/or adhesion to enamel surface. Clinical trials with toothpaste containing bromelain demonstrated improved plaque and gingivitis scores and extrinsic enamel stain removal capabilities. The impact of bromelain application prior to traditional mechanisms of plaque removal have not been clearly evaluated.

Arms: arm 1: Subjects were asked to chew a disclosing tablet, swish, and expectorate. Intraoral photographs were obtained. They then received a vial containing 1 gram of bromelain that was reconstituted with 15ml of pineapple juice at 50 degrees F. Subjects swished the solution in their mouth for 2 minutes and then expectorated. Intraoral photographs were obtained. Subjects then received a toothbrush and were asked to brush their teeth for 2 minutes. Intraoral photographs were obtained. The subjects were then given a Waterpik and instructed to use the appliances to clean their teeth and brackets to the best of their ability, for 2 minutes. Intraoral photographs were obtained. After a 1 week washout period, they received the placebo rinse (powdered sugar), following the same protocol and series of intraoral photographs. arm 2: Subjects were asked to chew a disclosing tablet, swish, and expectorate. Intraoral photographs were obtained. They then received a vial containing 1 gram of powdered sugar that was reconstituted with 15ml of pineapple juice at 50 degrees F. Subjects swished the solution in their mouth for 2 minutes and then expectorated. Intraoral photographs were obtained. Subjects then received a toothbrush and were asked to brush their teeth for 2 minutes. Intraoral photographs were obtained. The subjects were then given a Waterpik and instructed to use the appliances to clean their teeth and brackets to the best of their ability, for 2 minutes. Intraoral photographs were obtained. After a 1 week washout period, they received the bromelain rinse, following the same protocol and series of intraoral photographs.
Interventions: intervention 1: Bromelain intervention 2: Powdered sugar
Intervention descriptions: intervention 1: The subjects were randomized to either receive the bromelain pre-rinse or the powdered sugar placebo pre-rinse. Subjects then had a 5-9 day washout period and was then exposed to the other intervention. intervention 2: The subjects were randomized to either receive the bromelain pre-rinse or the powdered sugar placebo pre-rinse. Subjects then had a 5-9 day washout period and was then exposed to the other intervention.

Protocol documents:
Prot_SAP_001.pdf: 
OFFICIAL TITLE OF STUDY: A VISUAL EVALUATION OF ORAL PLAQUE REMOVAL UTILIZING AN ADJUNCT 
ENZYME PRE-RINSE IN ORTHODONTIC SUBJECTS  
 
NCT NUMBER: N/A  
 
DATE: MAY 29, 2018 


To:     
Kelton  Stewart
DENTISTRY-ORTHO & ORAL FACIAL
     
Frank  Lippert
CARIOLOGY/OPERATIVE&DENTAL PUBLIC HEALTH
Jennifer  Rose
UNIVERSITY LEVEL
 
From:
Chair - IRB-04
Human Subjects Office
Office of Research Compliance – Indiana University
Date:
May 29, 2018
RE:     
NOTICE OF APPROVAL - NEW STUDY
Protocol Title:
A visual comparison of oral plaque removal in the orthodontic patient utilizing an oral rinse
with and without proteolytic enzyme prior to dental brushing and subsequent pulsating water
jet cleansing
Study #:
1802369383
Funding Agency/Sponsor:
DENTISTRY-ORTHO & ORAL FACIAL
Review Level:
Response
Study Approval Date: May 29, 2018
Study Expiration Date: May 14, 2019
The Indiana University Institutional Review Board (IRB) IRB00000219 | IRB-04 recently took action on the above-referenced protocol. The IRB has
subsequently verified that the investigator’s response to the review satisfies the conditions for approval. In compliance with (as applicable) 21 C.F.R. §
56.109 (e) and 46 C.F.R. § 46.109 (d), this letter serves as written notification of the IRB’s determination.
The Board made the following determination(s), as applicable:
. Plan for soliciting the assent of the children and/or the parent/guardian permission is appropriate.
. Child Category 3: greater than minimal risk and no prospect of direct benefit to individual child-subject, but likely to yield
generalizable knowledge about the child-subject's disorder or condition.
Approval of this study is based on your agreement to abide by the policies and procedures of the Indiana University Human Research Protection Program
and does not replace any other approvals that may be required. Relevant policies and procedures governing Human Subject Research can be found at:
http://researchcompliance.iu.edu/hso/hs_guidance.html.
As a reminder, IRB approval is required prior to implementing any changes or amendments in the protocol, regardless of how minor, except to eliminate
immediate hazards to subjects. No changes to the informed consent document may be made without prior IRB approval.
If you submitted and/or are required to provide participants with an informed consent document, a copy of the most recently approved stamped
document is enclosed and  must be used to enroll participants.


The study expiration date is noted above. Failure to receive notification from the Human Subjects Office will not relieve you of your
responsibility to ensure compliance with Federal Regulations regarding annual review [as applicable, 21 C.F.R. § 56.109(f) and 45 C.F.R. §
46.109(e)].
You should retain a copy of this letter and all associated approved study documents for your records. Please refer to the assigned study number and exact
study title in future correspondence with our office. Additional information is available on our website at at http://researchcompliance.iu.edu/hso/.
If your source of funding changes, you must submit an amendment to update your study documents immediately via an amendment.
If you have any questions or require further information, please contact the Human Subjects Office via email at irb@iu.edu or via phone at
(317)274-8289.
The study expiration date is noted above. Failure to receive notification from the Human Subjects Office will not relieve you of your
responsibility to ensure compliance with Federal Regulations regarding annual review [as applicable, 21 C.F.R. § 56.109(f) and 45 C.F.R. §
46.109(e)].
You should retain a copy of this letter and all associated approved study documents for your records. Please refer to the assigned study number and exact
study title in future correspondence with our office. Additional information is available on our website at at http://researchcompliance.iu.edu/hso/.
If your source of funding changes, you must submit an amendment to update your study documents immediately via an amendment.
If you have any questions or require further information, please contact the Human Subjects Office via email at irb@iu.edu or via phone at
(317)274-8289.


1 
 
Indiana University School of Dentistry 
Graduate Student Research Proposal Abstract Form 
(For review by IUSD IRB Committee) 
 
Project Title:  A visual comparison of oral plaque removal in the orthodontic 
patient utilizing an oral rinse with and without proteolytic enzyme prior to 
dental brushing and subsequent pulsating water jet cleansing 
 
 
Name                                                
Signature* 
Principal Investigator: Kelton T. Stewart, DDS 
MS 
 
Co-Investigator: Jennifer Rose, DDS 
 
Co-Investigator: Frank Lippert, MSc, PhD 
 
Co-Investigator:  Ahmed Ghoneima, BDS, 
MSc, PhD, MSD 
 
Research Committee:  Lisa Maxwell, LDH, BS, 
MSM  
 
Biostatistician:  George Eckert, MS 
 
Department/Program:    Department of 
Orthodontics and Oral Facial Genetics 
 
Cost of Project:   
$3500 
Dates of Project:  
 April 2018-May 2018 
Use of Human Subjects:   
Yes 
Use of Animals:   
No 
Use of Biohazards or rDNA:   
No 
Reviewed by Sue Kelly (IRB):   
 
Reviewed by Sue Kelly (Good Clinical 
Practice): 
 
 
*Signature indicates that the proposal has been read and approved. 
 
 
 
 
 


2 
 
 
 
Enzyme Research Protocol 
 
 
Study Number: 
 
Title: A Visual Comparison of Oral Plaque Removal in the Orthodontic Patient 
Utilizing an Oral Rinse with and without Proteolytic Enzyme Prior to Dental Brushing 
and Subsequent Pulsating Waterjet Cleansing 
 
Phase: Not Applicable 
 
Funding:         WaterPik, Inc. (Providing free WaterPik devices only) 
 
           1730 East Prospect Road. 
 
            Fort Collins, Colorado 80553 
 
 
Principal Investigator:            Kelton T. Stewart, DDS MS 
 
 
 
 
Department of Orthodontics 
 
 
 
 
Indiana University School of Dentistry 
 
 
 
 
1121 West Michigan St. 
 
 
 
 
Indianapolis, Indiana 46202  
Phone: (317) 278-1087  
Fax: (317) 278-1438  
 
 
 
 
 
E-mail: keltstew@iu.edu 
 
Co-Investigator: 
 
Jennifer Rose, DDS 
 
 
 
 
Department of Orthodontics 
 
 
 
 
Indiana University School of Dentistry 
 
 
 
 
1121 West Michigan St. 
 
 
 
 
Indianapolis, IN 46202 
 
 
 
 
Phone: (785) 760-6244 
 
 
 
 
E-mail: jschaetz@iupui.edu 
 
 
 
 
 
 
Co-Investigator: 
 
Frank Lippert, MSc, PhD  
 
 
 
 
Oral Health Research Institute 
 
 
 
 
415 Lansing St. 
 
 
 
 
Indianapolis, IN 46202 
 
 
 
 
Phone: (317) 274-3983 
 
 
 
 
Fax: (317) 274-5425 
 
 
 
 
E-mail: flippert@iupui.edu  
 
Co-Investigator: 
        
Ahmed Ghoneima, BDS, MSc, PhD, MSD 
 
 
 
 
Director, Orthodontic Clinic 
 
 
 
 
Director, MSD Program 
 
 
 
 
Department of Orthodontics 
 
 
 
 
Indiana University School of Dentistry 
 
 
 
 
1121 West Michigan Street, DS 249 


3 
 
 
 
 
 
Indianapolis, Indiana 46202  
Phone: (317) 278-1653 
Fax: (317) 278-1438  
 
 
 
 
E-mail: aghoneim@iu.edu 
 
Committee Member:  
Lisa Maxwell, LDH, BS, MSM  
Dental Hygiene Program Director 
Indiana University School of Dentistry 
1121 West Michigan Street, S408 
Indianapolis, Indiana, 46202 
Phone: (317) 274-2611 
E-mail: lmax@iu.edu 
 
Statistician: 
 
 
George Eckert, MAS 
 
 
 
 
Department of Biostatistics 
 
 
 
 
Indiana University School of Medicine 
 
 
 
 
410 W. 10th St. 
 
 
 
 
Indianapolis, IN 46202 
 
 
 
 
Phone: (317) 274-2884 
 
 
 
 
Fax: (317) 274-2678 
 
 
 
 
E-mail: geckert@iu.edu 
 
IRB Advisor:  
 
Sue Kelly 
 
 
 
 
Director, Clinical Research Core  
Indiana University Oral Health Research Institute 
415 Lansing Street 
 
 
 
 
Indianapolis, IN 46202 
 
 
 
 
Phone: (317) 274-3954  
E-mail: sakelly@iu.edu 
 
 
 
 
Fax: (317) 274-5425 
 
IRB: 
 
 
 
IU Institutional Review Board 
 
 
 
 
Human Subjects Office 
 
 
 
 
Office of Research Administration, Room 3315 
 
 
 
 
980 Indiana Ave. 
 
 
 
 
Indianapolis, IN 46202 
 
 
 
 
Phone: (317) 274-8289 
 
 
 
 
Fax: (317) 274-5932 
 
 
 
 
E-mail: resrisk@iupui.edu  
FWA #: 00003544, exp IRB# Indiana University- 
Purdue University at Indianapolis (IUPUI): 
 
Version: 
 
 
  April 5th, 2018 
 
 
 
 
 
 
 
 


4 
 
 
 
 
 
 
STATEMENT OF COMPLIANCE 
 
Refer to: 
http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.htm#46 
http://www.fda.gov/cder/guidance/959fnl.pdf 
http:// grants.nih.gov/grants/guide/notice-files/NOT-OD-01-061.html 
http://www.cancer.gov/clinicaltrials/learning/page3 
 
The study will be carried out in accordance with good clinical practice (GCP) as required by the 
following: 
 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
 
ICH E6; 62 Federal Register 25691 (1997) 
 
Department of Orthodontics, Research Review Committee 
 
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed Human Subjects Protection Training. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


5 
 
 
 
 
 
 
SIGNATURE PAGE 
 
The signature below constitutes the approval of this protocol and attachments, and provides the necessary assurances 
that this trial will be conducted according to all stipulations of the protocol, including all statements regarding 
confidentiality, and in accordance to local legal and regulatory requirements and applicable US federal regulations and 
IHC guidelines. 
 
 
 
Signed: _________________________________Date:    _____________________ 
 
 
Name: Kelton T. Stewart, DDS MS 
 
 
Title: Principal Investigator 
 
                           
 
 
 
Signed: _______________________________Date: _______________________ 
 
 
Name: Jennifer Rose, DDS 
 
 
Title: Student-investigator 
 
 
 
 
 
Signed: _________________________________Date: ________________________ 
 
 
Name: Frank Lippert, MSc, PhD 
 
 
Title: Co-investigator 
 
 
 
 
 
Signed: _________________________________Date: ________________________ 
 
 
Name: Ahmed Ghoneima, BDS, MSc, PhD, MSD 
 
 
Title: Co-investigator 
 
 
 
 
 
 
 
 
 
 
 


6 
 
 
 
 
 
Table of Contents 
 
 
1. 
STUDY OBJECTIVES AND RATIONALE 
  1.1. Primary Objective………………………………………………………….. …....... 8 
  1.2. Rationale…………………………………………………………………………… 8-10 
  1.3. Potential Risks and Benefits………………………………………………...……... 10-11 
  1.4.    Adequacy of Protection against Risks…………………………………………….. 11 
  1.5. Known Potential Benefits………………………………………………….………. 11 
 
2. 
STUDY DESIGN …………………………………………………………………. 11-12 
  2.1. Study schedule ………………………………………………………………..…… 12-13 
 
3. 
STUDY POPULATION …………………………………………………………... 12 
  3.1. Source and Number of Subjects …………………………………………………… 13 
  3.2. Subject-Selection Criteria ……………………………………………….………… 13 
  3.3.    Subject Recruitment………………………………………………………………....13-14 
 
4. 
TEST TREATMENTS ……………………………………………………………..14 
  4.1 
Administration of Study Investigational Product…………………………...…....... 14 
  4.2 
Blinding/Unblinding Procedures ……………………………………………..…… 14-15 
  4.3 
Product Labeling ……………………………………………………………..……. 15 
  4.4 
Treatment Compliance………………………………………………………...…… 15 
  4.5 
Randomization Procedures …………………………………………………….….. 15 
 
5. 
STUDY METHODOLOGY ……………………………………………….……… 15 
  5.1. Preparation of Investigational Products……………………………………….…… 15-16 
  5.2. Clinical Procedures …………………………………………………………...…… 16-17 
  5.3. Informed Consent Process………………………………………………………......17 
  5.4 
Oral Photography Process ………………………………………………………..... 17 
 
6. 
EFFICACY MEASUREMENTS AND EVALUATIONS ……………………….. 17 
  6.1. Training of Examiner……………………………………………………………….17 
  6.2. Dental Plaque Evaluation and Data Management………………………..……….. 17-18 
  6.3. Retention of Photographs…………………………………………………………... 18 
 
7. 
ASSESSMENT OF SAFETY…………………………………………………….. 18 
  7.1. Study Safety Management…………………………………………………………. 18 
  7.2. Medical/Dental Monitor…………………………………………………………… 18-19 
  7.3.  Specification of Safety Parameters………………………………………………… 19 
  7.4. Methods for Assessing Safety Parameters…………………………………………. 19 
  7.5. Safety Oversight Plan…………………………………………………………….... 19 
 
8. 
DATA ANALYSIS………………………………………………………………… 19 
  8.1. Sample Size Determination…………………………………………………………19 
  8.2. Statistical Analysis………………………………………………………………... 19-20 
 


7 
 
9. 
OBLIGATION OF THE INVESTIGATOR………………………………………. 20 
  9.1 
Institutional Review………………………………………………………………... 20 
  9.2 
Subject Assent …………………………………………………………………..…. 20 
  9.3 
Subject Consent ………………………………………………………………...…. 20 
  9.4 
Data Collection …………………………………………………………………… 20-21 
  9.5 
Adherence to Protocol………………………………………………………………21 
  9.6 
Records Retention………………………………………………………………….. 21 
  9.7 
Investigator’s Final Report……………………………………………………...…. 21 
 
10.     APPENDIX……………………………………………………………………...… 22 
 
11. 
BUDGET………………………………………………………………...…............ 23 
 
12. 
REFERENCES ……………………………………………………………………..24-25 
 
 


8 
 
1.  
 
STUDY OBJECTIVE AND RATIONALE 
 
 
1.1 
 
Objective 
 
The objective of this study is to determine if residual dental plaque following brushing can be 
mitigated by pre-rinse with broad spectrum oral proteolytic enzyme rinse and subsequent oral 
pulsating waterjet appliance, enhancing the oral hygiene of patients receiving orthodontic 
treatment. 
 
 
1.2. 
 
RATIONALE 
 
Since the advent of modern orthodontic treatment, maintaining optimal oral hygiene has been 
troublesome for both the patient and the clinician. Fixed orthodontic appliances in the oral cavity 
are a physical barrier which can interfere with optimal oral hygiene. The tooth surface 
beneath the wire and adjacent to the orthodontic bracket are challenging areas for the 
patient to properly clean. The placement of this hardware creates obstructions and 
obstacles to the abrasive function of the toothbrush bristles. Poor removal of plaque 
adjacent to the brackets often leaves a visible halo or shadow effect on the tooth surface 
when the orthodontic hardware is removed. This discoloration halo (white spot lesion) is due 
to acidic demineralization of the tooth surface beneath the adherent dental plaque. The tooth 
surface beneath the bracket is protected from plaque development and resultant acidic 
leaching of dental minerals. Upon bracket removal, the discrepancy in tooth mineralization 
between these adjacent areas is now exposed creating the unaesthetic white spots. 
Unfortunately, many orthodontic patients to some degree, develop these unaesthetic white 
spots on their teeth. 
 
The purpose of this research study is to determine if utilizing a proteolytic oral pre-rinse can 
hydrolyze the glycoprotein bonds which adheres the dental plaque to the dental pellicle (tooth 
surface in vivo).  It is proposed here that the proteolytic enzyme may loosen the bond enough to 
allow for dislodgement by standard brushing, and further reduction in oral plaque with subsequent 
water jet propulsion.  
 
The question to be answered is thus: in an adolescent patient with orthodontic appliances and poor 
oral hygiene, can the water jet more easily reach those areas protected from the bristles of the 
toothbrush (created by the orthodontic hardware) and dislodge the enzymatically loosened 
plaque? If so, an enzyme pre-rinse may have a place in orthodontic hygiene 
procedures.  
 
The findings of this study may lead to future research studies investigating whether the addition of 
proteolytic enzymes can also enhance the overall oral hygiene in the general population. Possibly 
removing plaque between teeth and other areas where brushing alone can’t reach. 
 
It is well known that upon initiation of fixed orthodontic appliance therapy there is a 
significant increase in dental plaque accumulation, in which patient education and oral 
hygiene instruction alone has proven insufficient to overcome(1). Poor oral hygiene during 
orthodontic treatment results in a high prevalence of white spot lesions on anterior tooth 
surfaces surrounding brackets. Martignon et al showed 96% of patients had one or more white 
spot lesions following orthodontic treatment (2). 


9 
 
 
The problem appears to be that the orthodontic wire and bracket impede the toothbrush 
bristles from close contact with an otherwise naturally smooth tooth surface. This hardware 
interferes with the mechanical contact and abrasive function of the toothbrush bristles to 
dislodge adherent dental plaque from the tooth surface. Residual plaque following brushing in 
orthodontic patients has been found along the gum line, around brackets and beneath wires(3). 
 
The orthodontic wires make flossing an arduous task, which is why many patients give up and 
never adequately remove dental plaque from orthodontic hardware and dental contact points. 
Numerous studies have correlated fixed orthodontic treatment with less than optimal oral hygiene (4). 
Orthodontic induced gingivitis(5) (7) typically develops within the first few months of fixed 
appliance placement(6). Following the placement of the orthodontic fixed appliance, the resultant 
change in oral hygiene creates an environment favorable to Streptococcus mutans(8) and an 
increase in gram negative bacteria which are associated with periodontal disease(9). Improved 
oral hygiene would likely prevent many adverse sequelae commonly associated with 
orthodontic treatment (2). 
 
Previous studies have used pre-rinsing agents as an oral hygiene adjunct and demonstrated 
mixed results. Boyd found Hydrogen peroxide-sodium fluoride rinse is more effective than 
sodium fluoride rinse alone in preventing gingivitis and decalcification in adolescent 
orthodontic patients(10). Miranda, conducted a trial using water, Listerine, or Plax, as pre-
rinse agents and concluded that pre-rinsing in children demonstrated no difference in plaque 
removal (11). However, Tufekci showed the addition of Listerine rinse to an oral hygiene 
protocol significantly reduced plaque (more than just brushing and flossing alone in 
orthodontic patients). He concluded that the addition of Listerine was helpful for fixed 
orthodontic patients to reduce their risk of white spots development and gingivitis (12).  
Pontier’s study concluded there was no difference between pre-rinse (Plax TM versus 
placebo) for plaque removal in orthodontic patients(13). 
 
Water irrigation (pulsating water jet therapy), another adjunct to oral hygiene, has 
demonstrated a significant difference in biofilm (14) and plaque removal - especially in 
difficult to reach dental plaque retention areas, such as around the orthodontic brackets and 
wires (6). The plaque removal was also increased utilizing a specialized orthodontic water jet 
tip as compared to a standard water jet tip (15). 
 
Surprisingly little has been investigated concerning proteolytic hydrolysis (to loosen the 
dental biofilm) influence on oral hygiene (16). No published studies could be located 
(PubMed) that involve use of proteases as a plaque removal aid in the orthodontic patient. 
Literature search revealed a study of Krillase (enzyme obtained from ocean krill) included in a 
chewing gum to reduce gingivitis and plaque acumination (17). Published articles utilizing the 
plant enzyme papain in orthodontics include: orthodontic bonding and sheer bond strength 
study (18) and the effect of different concentrations of papain gel have on orthodontic bracket 
adhesion study (19). 
 
Papain and Bromelain are naturally occurring serine proteases. Papain is derived from the 
latex from green papaya fruit (Carica papaya) while bromelain is obtained from pineapple 
stalks (Ananas comosus). Because of their innate proteolytic activity, papain and bromelain 
have been used extensively in the food and medical industries. Both enzymes are included on 
the FDA’s “Generally Recognized as Safe” (GRAS) list. Both bromelain and papain are used 
commercially for tenderizing meat and producing protein hydrolysates (20). These proteolytic 
enzymes have a broad range of substrate specificity, which easily and efficiently hydrolyze 


10 
 
most soluble proteins, yielding peptides and amino acids byproducts. 
 
Other uses of these enzymes are in toothpaste (papain/bromelain combination as the active 
ingredient) for extrinsic enamel stain removal (21, 22)(23). These enzymes have also been 
used for wound debridement for of damaged collagen and necrotic tissue in humans (24). 
Finally, health food enthusiasts claim dietary supplementation with papain and bromelain may 
be useful in alleviating joint pain and inflammation and supplementing natural digestive 
proteases.  
 
1.3. 
 
Potential Risks and Benefits  
 
1.3.1.  
Potential Risks 
 
 
 
 
 
Product Risks 
 
Irritation of the oral cavity can occur, (also known as stomatitis or angular chelitis), and has 
occurred among those individuals who eat extraordinary amounts of fresh pineapple. The use of an 
active protease in an oral rinse may cause stomatitis or angular chelitis and possibly irritate an 
undetected oral ulcer and cause the subject discomfort. 
 
Adverse reactions 
 
Hypersensitivity/allergic reactions have been reported in individuals who have supplemented their 
diet with bromelain and are allergic to pineapple. Nausea, vomiting, and diarrhea were reported in 
such instances following ingestion.  
 
Contraindications 
 
 
 
Use of bromelain is contraindicated in those who are hypersensitive or allergic to pineapple or 
proteases. Gastrointestinal or oral ulcers, use of anticoagulant/blood thinner medication, antibiotics 
or anticipating surgery would also be contraindications to ingestion of active proteases. 
 
Precautions 
 
Bromelain has a long history of being used in the food industry and no serious side effects or 
deaths have been reported. Nevertheless, certain precautions will be taken in using this natural 
protease. 
 
Rinses will be expectorated. Only a negligible amount of enzyme may be ingested unless the test 
subject inadvertently swallows the solution. Even in this scenario, this should not be a problem 
since quantities in greater amounts are frequently ingested by individuals wishing to supplement 
their natural pancreatic digestive capabilities.  
 
Interactions 
 
Since bromelain is an active serine protease, it is susceptible to inactivation or denaturation by 
excessive heat, chemicals or ions. Therefore, the oral rinse test solutions will not have alcohol or 
fluorine compounded within the solution tested.  
 
 
Over dosage 
 


11 
 
Bromelain has been shown to be safe when ingested in less than 130,000 FCC papain units per day 
(approximately 1.5 g of enzyme preparation). Symptoms of over dosage using bromelain as an oral 
rinse would include development of oral ulcers, stomatitis or angular chelitis. This would produce 
mild discomfort for a few days while the oral mucosa regenerates. 
 
Additional Risks 
 
Additional potential risk to the subjects include cross contamination. This risk will be managed by 
the use of experienced IU dental personnel proctoring the test subjects, whom will follow strict 
infection control guidelines outlined by the IU School of Dentistry. This includes handwashing 
between subjects, use of single use nitrile gloves, and facemasks (for researchers) during the 
intraoral exam. Vials and cheek retractors will be single use. 
 
Loss of confidentiality of private information collected is another risk. To counter this risk, all data 
collected on paper will be stored in a locked area and limited to study personnel. Electronic data 
will be stored in an encrypted, password protected computer file with access limited to study 
personnel. All records will be identified by study number only. A master list will be maintained 
which will match the subject to their study number and this list will be stored in a secured area. 
 
1.4.     Adequacy of Protection against Risks 
 
To limit the potential risks of ingestion of the active ingredients, persons with the following 
conditions will be excluded from participation in the study:  
1. gastrointestinal or oral ulcer 
2. taking an anticoagulant/blood thinner medication or antibiotics  
3. anticipating surgery in the next six months  
4. allergic to proteolytic enzymes, pineapples or disclosing agent 
5. pregnant, breastfeeding or intending to become pregnant in the next six months 
 
1.5. 
Known Potential Benefits 
 
There is no known benefit for the subject.  
  
2. 
STUDY DESIGN 
 
This will be a single site, cross-over, double-blind study with up to 55 subjects enrolled in the 
study. Since each person varies with technique and diligence in brushing their teeth, each 
subject will serve as their own control in this cross-over study. There will be at least a week 
time interval between the two arms of the study (length of wash out phase: one week). The study 
will be completed over the course of two weeks. Each step in the protocol will be timed to 
reduce inter- and intra-subject variability. Toothbrushes used in the study will be uniform, a 
new tooth brush will be used at each arm of the study. All oral hygiene aids will be of the 
same brand to eliminate variability (eg: all toothbrushes will be of the same brand, all 
disclosing tablets of the same brand, etc). For standardization and optimal photography, the same 
type of cheek retractor and digital camera will be used for all subjects. 
 
Hypothesis 
 
Null: There is no statistically significant difference in visual plaque scores with or without use 
of a proteolytic enzyme rinse aid. 
 


12 
 
Alternative: There is a statistically significant difference in visual plaque scores with use of 
proteolytic enzyme rinse aid. 
Product Description 
 
This study will use bromelain as the proteolytic enzyme in the oral rinse. Bromelain is an enzyme 
naturally found in high concentration in the stem of pineapples. This proteolytic, protein-
digesting enzyme breaks down (hydrolyzes) proteins by reducing large complex proteins into 
smaller peptides or individual amino acids (25). For this reason, bromelain is commonly used 
as a digestive aid, (in pill form) or (in powder form) as a meat tenderizer.  
 
2.1. 
Study Schedule 
 
In both arms of the study, the steps will be exactly the same to isolate the effect of the enzyme. 
Data (digital photograph) will be obtained after each oral hygiene step to prevent confounding 
variables. 
 
First Visit 
 
a. Describe study and obtain informed consent, if not previously consented by phone(see 
attachment).  
b. An oral soft tissue exam will be completed (see section 7.4). 
c. Randomize subject and select a subject kit (test box)   Patient writes name on outside of 
box and on WaterPik box.  The patient name on outside of box allows easy retrieval for 
second visit. 
d. Stain oral plaque with two-tone plaque disclosing tablet (individually packaged disclosing 
tab). Instructions for test subject using oral disclosing agent: “chew tablet completely, then 
swish around in your mouth and between your teeth. I will time you for 30 seconds and tell 
you when to spit it out in the sink.” Rinse with 15ml of tap water to remove unbound stain. 
e. First photograph (base line plaque before oral hygiene procedures) obtained with unique 
identifying number (see 9.4). 
f. Subject chooses one of the test vials from their box and the serial number on the vial is 
recorded (placebo rinse or enzymatic rinse). (The second vial will be used on the second 
testing visit.) 
g. The student investigator will reconstitute oral rinse powder in vial with 15 mL warm 
(50°C) pineapple juice from coffeepot on hotplate. Stir into solution with disposable 
swizzle stick. 
h. Hand the vial with the dissolved oral rinse solution to the subject. The subject will then 
place the entire contents at once into their mouth and swish it between their teeth and side 
to side for two minutes (timed).  At the end of two minutes the subject expectorates 
contents of mouth into sink.  
i. Second frontal photograph obtained (post-rinse). (This photo will include a different 
random identifying number unique for subject, stage and visit). 
j. The subject will then be asked to brush their teeth with a new toothbrush (individually 
packaged and pre-pasted from manufacturer). The only instructions in regards to 
brushing: “brush your teeth as you normally do.” Subject brushing time will be 
standardized (two minutes) and a digital alarm clock will keep time. The subject will 
expectorate contents of mouth at the end of two minutes.   
k. Third frontal photograph again will be obtained including another identifying number 
specific to this stage of the experiment and visit.  
l. The subject will then be asked to use a water jet cleansing device (WaterPik). This will also 


13 
 
be timed (two minutes). The instructions given to the subject: “Use this Waterpik to clean 
around the brackets and under the wires as best you can”. All Waterpik devices will be set 
at the same medium pulsating pressure. 
m. Fourth frontal photograph for this visit obtained (including another random serial 
identifying number unique for subject, stage and visit). 
n. Subject   receives $25 Visa gift card and dismissed. 
o. Subject (or guardian) is phoned later that day & questioned if they have any concerns or 
side effects. Subject & Guardian are reminded of phone number contact of student 
researcher. 
 
Second Visit (1week later) 
 
 
a. Each subject will repeat the procedure as described above on the subsequent visit (a-n) 
with the second (vial) oral rinse treatment (placebo or enzyme).  
 
3. 
STUDY POPULATION  
 
 
3.1. 
Source and Number of Subjects 
 
55 adolescent and young adult subjects (10-25 years, male or female). Subjects will be in active 
fixed (edgewise) orthodontic appliances at the Indiana University School Dentistry. 
 
3.2.        Subject-Selection Criteria  
 
Inclusion Criteria  
Individuals may qualify to participate in the study if they meet all the following requirements: 
• Subject is willing to consent 
• male or female  
• age range from 10-25 years  
• willing to follow study instructions 
• in active orthodontic treatment with fixed orthodontic appliances 
• patient at Indiana University School of Dentistry Orthodontic Clinic 
 
Exclusion Criteria 
Individuals will be excluded from participation if they possess any of the following conditions: 
• pregnancy, breast feeding or intending to become pregnant in the next six month period  
• latex allergy  
• not willing/able to follow study instructions  
• pineapple  allergy 
• proteolytic enzymes allergy 
• food dye allergies (to assure no issues with using disclosing solution) 
• smoker 
• has a gastrointestinal or oral ulcer 
• is taking an anticoagulant/blood thinner medication or antibiotics  
• is anticipating surgery in the next six months  
• person with dental training (dental students, hygiene students, faculty, & assistants) 
  
3.3  
Subject Recruitment 
 


14 
 
Potential subjects will be recruited from patients at the Indiana University School of Dentistry 
Orthodontic Department. Recruitment methods will include the following: 
 
1) An IRB approved flyer will be hung in the waiting room and various locations within the 
clinic.   
2) The student investigator will call patients who are her patients in the clinic to determine 
interest. 
3) All residents and faculty will assist in distributing an IRB approved flyer that contains a 
section with contact information for the subject to complete if they are interested in 
learning more about the study. Potential subjects will be able to place completed interest 
forms with their contact phone number in a secured drop box in the clinic for the student 
investigator to access. The student investigator will then call the potential subjects to 
discuss the study. 
 
In all cases, the student investigator will use an IRB approved telephone script to review the study 
with the potential subjects. If the potential subject is interested in participating, and if time allows, 
the consent and assent will be sent to the potential subject by mail so they will have the 
opportunity to review the information before their appointment. Consenting and assenting, as 
applicable, will occur in the clinic prior to any study procedures being performed. Consenting may 
also occur by phone, using the consent documents sent by mail. Test subjects will attend two 
testing visits approximately one week apart. These visits will be outside from their normal 
orthodontic visits. 
 
Subjects will be compensated for their time in the study by gift of the testing Waterpik 
appliance they used and $50 ($25 per visit) for participating in the study. Payment will be in 
the form of Visa gift cards. 
 
4. 
TEST TREATMENTS 
 
4.1.      Administration of Study Investigational Product 
 
The components of each treatment will be formulated prior to treatment and placed in a plastic vial. 
Each of the vials for the oral rinse treatments will be labeled with a unique set of numbers for subject 
and stage of treatment (only decoded by use of master list). 
 
4.2. 
Blinding/Unblinding Procedures 
 
To eliminate potential human bias, photos will be graded independently by one examiner from 
Indiana University School of Dentistry with dental clinical experience. The examiner will be 
blinded with regard to the stage and visit of the oral hygiene procedures. The identification on the 
photos will not allow the examiner to know which photos came from the same subject, which 
series they are evaluating (baseline staining, post-rinse, post-brush, post-Waterpik) and which 
treatment.  
 
Each photo will be independently reviewed and scored assessing the degree of plaque staining 
from one to five. An example photograph of each degree (1-5) will be given to the examiner 
for standardization.  
 
The researchers and subjects will be blinded to knowing which vial has the proteolytic enzyme 
treatment. The subsequent visit the subject will receive the second vial. The study records will be 
maintained by this committee member (DR. KS).  


15 
 
 
Each photograph will be labeled by only a serial number and can only identified through use of a 
master sheet. Each subject will have eight randomly assigned serial numbers corresponding to each 
testing step and visit (two different study visits with four photographs each). 
 
4.3     Photo Labeling  
 
A photographic sequence identifier (A-D) will be added to the patient’s random subject ID to 
properly log and sequence the obtained photographs. The following codes will be utilized in the 
study: 
A: After use of the disclosing tablet 
B: After 2 minute rinse with provided solution  
C: After brushing 
D: After the use of the WaterPik  
 
4.4. 
Treatment Compliance 
 
 
It is expected most subjects enrolling in the study will complete both treatment visits. Since the 
treatments are to be administered to the subject and supervised, compliance with the protocol is 
expected. 
 
4.5. 
Randomization Procedures  
 
Subjects will be stratified randomly by sex and age groups.  Subjects will be allocated random sets 
of serial numbers kept on a master list which will be retained in a secured location. The faculty 
member grading the photographs will be given the 440 photographs completely in random order, 
randomized by the primary investigator. 
 
5. 
STUDY METHODOLOGY 
 
5.1. 
Preparation of Investigational Products 
 
The dry components of each rinse will be formulated prior to study and placed in a plastic vial 
with lid.  The statistician will generate a second randomization list to identify which vial (A or B) 
the patient will receive at the first and second visits, respectively.  
 
Prior to beginning the study, the dry powdered components for each treatment rinse will be 
weighed and placed into clean 20 mL vials, (for both the enzymatic and placebo rinse) and sealed 
with plastic caps. This will prevent contamination or spillage. All vials will be prepared at one 
time in a clean room prior to the study. 
 
Selected committee members will measure out one gram of enzyme and one gram of placebo 
(powdered sugar) for each subject. Both the placebo and the proteolytic rinse compounds will be 
weighed, formulated and placed into darkened 20 mL vials with a plastic top. Each vial will then 
be assigned the appropriate code (Product A or Product B) by the co-investigator K.S. Only this 
investigator (K.S.) will know the identity of the vial contents and will share this information with 
the statistician at the time of statistical analysis. The formulated compounds placed in the clean 
vials will be stored in a locked secured location.  
 
Manufacture of Bromelain Rinse: 
 


16 
 
The bromelain enzyme will be obtained (donated) in bulk powder form from Ultra Bio-logics Inc. 
(Chateauguay, Quebec, Canada). The Ultra Bio-logic bulk bromelain concentrated supplement is 
standardized (by manufacturer) to 2400 Gelatin Digesting Units (GDU)/gram. The strength of an 
enzyme is quantified in GDU/gram units. GDU/gram tells us how much protein a gram of 
bromelain can digest. The proteolytic rinse will contain one gram of bromelain brought into 
solution with 15ml of pineapple juice. One gram of bromelain is within the recommended daily 
dosage for oral consumption by the manufacturer. 
 
Bromelain is stable at room temperature for more than a year in its dry form. It is active when 
reconstituted with warm water. (In this study- warm pineapple juice will be substituted for warm 
water). Since bromelain is active in the liquid form, the rinse will be constituted with canned 
pineapple juice just prior to administration while ensuring complete dissolution. Bromelain’s 
enzymatic activity is destroyed by the pasteurization process during canning. Thus, the canned 
pineapple juice has the correct pH for optimal activity but does not contribute any enzymatic 
activity to the test solution. 
 
 Bromelain’s optimal activity is at 55°C. This is approximately the temperature of very warm 
coffee. To achieve optimal results a temperature controlled hot plate will be used to keep a pot of 
warm pineapple juice at 50°C available to mix with the powdered mixture just prior to 
administration.  
 
Manufacture of Placebo Rinse: 
 
The placebo rinse will consist of powdered sugar and pineapple juice.  Powdered sugar has the 
same color and consistency as the bromelain powder.  This placebo will not be discernible once 
weighed and placed in sealed vials. Canned pineapple juice contains no active bromelain, which is 
destroyed in the pasteurization process. 
 
The components of the two study rinses are as follows: 
 
TREATMENT A: Placebo 
 
TREATMENT B: Proteolytic Rinse 
Pineapple juice (15 mL) 
Pineapple juice (15 mL) 
Powdered sugar (1.0 gm) 
Bromelain powder (1.0 g) 
 
5.2. 
Clinical Procedures  
 
All study materials needed for each test subject will be contained in a new cardboard box.  Each box 
will contain: 
• Vial of enzyme powder with attached set of assigned random numbers 
• Vial of placebo powder with attached set of assigned random numbers 
• 10 disposable cheek retractors 
• Two disclosing tablets for staining 
• Waterpik device 
• Two toothbrushes (pre-pasted and individually sealed by manufacturer) 
 
After explaining the experiment in obtaining consent (either by phone or in person), the subject will 
be randomized and given their assigned study box. The subject will then put their name on the 
outside of the box and on the box of the Waterpik. The name on the outside of the box will allow for 
retrieval for the second visit. The student researcher will then record the first serial number with the 
name of the test subject. This will be entered on the master list. The master list will then have been 


17 
 
pre-prepared and grouped into 55 patient sets. Each set of random numbers for each patient will be 
divided and assigned with four numbers attached to each test vial. At the completion of the first visit, 
the cardboard box containing the Waterpik and the remaining materials will be sealed with adhesive 
tape and secured in a locked room. When the patient returns for their next testing visit, that subjects’ 
box will be retrieved and the second vial labeled with the subject’s second set of numbers will then 
be used.  The test procedure will be repeated as with the first visit as described in 2.1. 
 
5.3. 
Informed Consent Process 
 
Informed consent will be obtained from subjects and two parents (if the subject is a minor <18 yrs 
and if 2nd parent is reasonably available within the CFR regulations) prior to participation in study 
either in person or by phone (see 10.2). Assent will be obtained from all children under age 18. For 
children 14 to 17, the adult consent will be used as the assent form.  
 
5.4. 
Oral Photography Process  
 
Standardization of photographs: all photographs will be taken from the same angle (frontal), to 
include only the teeth and lips of the subject, with a single use, hands-free cheek retractor. Subject 
will be partially open so that the lower dentition is fully visible for photography.  Each photograph 
will be at the same magnification (see 10.4). The adhesive label with the corresponding serial 
number will be placed on the subject’s chin and will be included in the photograph. 
 
6. 
EFFICACY MEASUREMENTS AND EVALUATIONS 
 
6.1      Training & Calibration of Examiner: 
 
One examiner from the Department of Periodontics and Allied Dental Programs or Cariology, 
Operative Dentistry and Dental Public Health, will be recruited to assess the plaque index of the 
digital photographs. An example of five oral photographs with varying degrees of stained adherent 
dental plaque will be graded according to the Orthodontic Plaque Index and used as a reference in 
grading the study photographs. The examiner will independently grade the residual plaque in each 
photograph.  
 
The examiner will first be calibrated by scoring 10 randomly selected images and re-score the 
same images after a one week washout period. Once reliability testing of the examiner is at 0.8 or 
higher as measured by the intra-class correlation coefficient, the examiner will proceed to judge 
each photograph of the study.  
 
6.2. 
Dental Plaque Evaluation and Data Management 
 
Teeth will be disclosed and the facial tooth surfaces will be graded for visual plaque.  
Incisors (#s: 7-10 and 23-26) will be visually evaluated on digital photographs and assigned a 
score (0-4) according to the orthodontic plaque index (see figure 1 below). Examiner will be given 
a visual scale of comparison, which corresponds with the Orthodontic Plaque Index (OPI) for 
diagnosing dental plaque around orthodontic brackets. Each tooth will each receive an individual 
score, in addition all tooth scores will be added together to calculate a composite score for upper 
incisor and lower incisor segments.  All recorded plaque scores will be recorded in an excel 
document on a password protected laptop computer by the grader. 
 
To eliminate potential human bias, a single examiner from the Indiana University School of 
Dentistry with clinical dental experience, will judge each photograph. This examiner will be 


18 
 
blinded with regard to the stage of the oral hygiene procedure as described above.  
 
Data/digital photographs will be viewed on the same computer and will be backed up daily onto a 
secure box.iu.edu health account, which will only be accessible to researchers and by two step 
authentications and passphrase. Research laptop will remain in secure location and locked 
whenever not in use. 
 
Plaque scoring: 
 
 
Figure 1: Orthodontic Plaque index scoring system(Beberhold).  
 
6.3. 
Retention of Photographs 
 
Digital photographs and any study material will be maintained for seven years following the 
completion of this study, as per Indiana law. Paper records will be stored in locked cabinets that only 
study personnel can access. Electronic records will be stored in encrypted, password protected files 
only accessible by study personnel. 
 
7. 
ASSESSMENT OF SAFETY 
 
7.1. 
Study Safety Management 
 


19 
 
The PI will be notified immediately if a serious adverse event occurs. An adverse event will be 
considered any unfavorable and unintended sign, symptom or disease associated with the use of the 
bromelain (investigational product) or the conduct of the study.  
 
All adverse events will be reported to the IRB during annual review.  
All serious adverse events will be reported immediately to the IRB. Serious adverse events include:  
any event resulting in death, is life-threatening, requires hospitalization, results in 
disability/permanent damage, congenital anomaly/birth defect, or requires intervention to prevent 
permanent impairment or damage, and/or other medical events(31). 
7.2. 
Medical/Dental Monitor 
 
The principal investigator and student investigator will be responsible for overseeing the conduct of 
the study. 
 
7.3.  
Specification of Safety Parameters 
 
The temperature of the pineapple juice (to reconstitute the solutions) will be monitored to make 
sure it is 50°C plus or -1°C. The formulated compounds placed in the clean vials will be stored in a 
locked secured location.  
 
7.4. 
Methods for Assessing Safety Parameters 
 
Each subject will be given the student investigator’s phone number to call if they should experience 
any discomfort or unusual symptoms. On the second visit each subject will be questioned as to 
whether they experienced any discomfort or symptoms from the treatment at the first visit and an 
oral soft tissue exam will be completed.  
 
7.5. 
Safety Oversight Plan 
 
All subjects will receive a follow- up phone call the night of the study and reminded of the contact 
phone number of the student investigator in case of emergency or adverse side-effects. Subject will 
be instructed to make contact, should they experience any discomfort or unusual symptoms.  
This is part of the study’s safety plan to assure the subjects are not reacting negatively to the 
product. 
 
On the second visit each subject will be questioned as to whether they experienced any discomfort 
or symptoms from the treatment (symptoms of over dosage using bromelain as an oral rinse may 
include development of oral ulcers, stomatitis or angular chelitis) at the first visit & an oral soft 
tissue exam will be completed. 
 
If a subject experiences unusual side effects or appears to be experiencing an allergic reaction the 
subject will immediately be excluded from the study and will seek medical care if indicated. 
 
8. 
DATA ANALYSIS  
8.1.      Sample Size Determination 
 
55 subjects will be enrolled to account for dropout. Standard deviation of the average plaque scores 
is assumed to be 1, with a correlation of 0.5 between the two study arms. With a minimum completed 


20 
 
test sample size of 45 subjects, the study will have 90% power to detect a difference of 0.5 between 
the average plaque scores with and without enzyme after waterjet, assuming a two-sided paired t-
tests conducted at a two-sided 5% significance level.  
 
8.2. 
Statistical Analysis 
 
Intra-examiner repeatability of the individual surface scores will be evaluated using weighted kappa 
statistics and repeatability of the subject-level index scores will be evaluated using intra-class 
correlation coefficients (ICCs) and Bland-Altman plots. Subject-level plaque scores will be 
summarized by treatment (with or without enzyme) and stage (baseline, post-rinse, post-brushing, 
post-waterjet). The effects of the treatment will be evaluated using a repeated measures ANOVA 
suitable for a crossover study. The ANOVA will include fixed effects for treatment, stage, and their 
interaction, will treat stage as a repeated factor within subject for each study arm, and will include a 
factor for study arm. Age and sex groups will be included as covariates due to the stratified 
randomization. The ANOVA will also include an effect for treatment sequence to evaluate the model 
for a carry-over effect. If the treatment sequence effect is significant, the full analysis may need to 
be interpreted cautiously or the analysis may need to be limited to using only the results from the 
first study arm. A 5% significance level will be used for all tests. Statistical analyses will be 
performed using SAS version 9.4 (SAS Institute, Inc., Cary, NC).  
 
9. 
OBLIGATION OF THE INVESTIGATOR 
 
9.1 
Institutional Review 
 
The proposal and protocol will be reviewed within the School of Dentistry. After it is approved by 
the School of Dentistry the proposal will be submitted to the Institutional Review Board for their 
approval. 
 
9.2 
Subject Assent 
 
Informed assent will be obtained from subjects (who are minors) prior to participation in study.  
 
9.3 
Subject Consent 
 
Informed consent will be obtained from subjects (and both parents of minors unless, per CFR 
regulations, only one parent signature is needed) prior to participation in study.  
 
9.4 
Data Collection and Measurements  
 
Digital photography will make up the data for this study. Digital photography will be recorded at 
each step, for each subject-for evaluation of facial surfaces of anterior incisors (upper and 
lower). An intraoral dental plaque disclosing agent will be used to stain the oral plaque present, 
which will be documented via digital photography. 
This study involves two separate visits. At each visit there will be a total of four photographs 
obtained. Each time the subject’s teeth are photographed it will involve a frontal photograph to 
include only the facial surfaces of the teeth of each subject. Lingual surfaces of teeth will not be 
taken into account as the focus of this study relates to oral hygiene procedures around fixed 
appliances- all fixed appliances in this study will be on facial surfaces. 


21 
 
Each subject will have eight unique serial numbers assigned to them to differentiate the four different 
photograph at the two separate visits. A hands free cheek retractor will be utilized for all 
photographs. For consistency, fixed appliances including orthodontic wires will remain in place 
throughout the study. 
The same type of camera (Canon Rebel EOS with 100mm macro lens) and camera settings (f-stop: 
32, shutter speed: 1/60, ISO: 200) will be used for each subject at every data collection. 
Photographs will be taken at the same magnification and focus. The student investigator will 
review all photos immediately following to ensure that they are of diagnostic quality.  
 
Diagnostic quality of data (digital photos of dentition) will be defined as non-blurry digital photos 
of dentition of upper and lower incisors with orthodontic fixed appliances in place. The dentition 
visualized will be the same in each subject and for both study visits.  Facial surfaces of upper and 
lower incisors will be clearly and completely visualized in the digital photos.  No lips will obstruct 
the tooth in the photo.  Orthodontic appliance will remain on the teeth which are photographed and 
plaque surrounding the bracket and wire will be graded according to visual plaque index. 
 
Data/photo collection will be standardized so that all photos are taken at the same step in the 
hygiene process/step of protocol (in this way, a photo is taken after each oral hygiene procedure to 
evaluate any change in amount of dental plaque) (See Flow Chart in Appendix, page 22).  
 
 
1) Baseline plaque photograph following chewing/spitting of disclosing tablet.  
2) Post-treatment rinse (enzyme or placebo) photograph. 
3) Post- brushing photograph.  
4) Post-Waterpik photograph. 
 
Each photograph will be independently reviewed using the same computer and scored assessing 
the degree of plaque according to the Orthodontic Plaque index method by a single examiner. 
 
9.5 
Adherence to Protocol 
 
Strict adherence to the protocol is essential to eliminate variables which would invalidate the study 
design. Subjects unwilling or unable to follow the protocol will be eliminated from the study. 
 
9.6 
Records Retention 
 
A digital copy of all the photographs and scoring will be retained by the principal investigator for 
no less than seven years, as per Indiana law. 
 
9.7 
Investigator’s Final Report 
 
The data and scoring from each subject will be compared with respect to each arm of the study. A 
report will be written with the results and statistical analysis of the study results. A paper will be 
written and submitted for publication with the summation of the findings. 
 
APPENDIX 


22 
 
PROCEDURE FLOWCHART 
 
EXPLAIN STUDY, ANSWER QUESTIONS 
AND OBTAIN WRITTEN CONSENT
STAIN ORAL PLAQUE (30 sec.), THEN 
PHOTO #1
GIVE SUBJECT TEST RINSE (2 MIN.) 
THEN PHOTO #2
SUBJECT BRUSHES TEETH (2 MIN.), 
THEN PHOTO #3
SUBJECT USES WATERPIK (2 MIN.), 
THEN PHOTO #4


23 
 
Indiana University School of Dentistry 
Student Research Proposal 
Budget Worksheet 
Name: Jennifer Rose, DDS 
Title of Proposal: A visual comparison of oral plaque removal in the orthodontic subject utilizing an 
oral rinse with and without proteolytic enzyme prior to dental brushing and subsequent pulsating 
water jet cleansing 
Anticipated Start Date: April 2018 
$300 Requested from Research Committee 
$3200 supplied “in kind” from Other Sources 
 
Budget.  
Description: 
Amount ($): 
Source: 
Materials: 
 
SIM card 
$40 
IU research committee 
Funds  
 
Toothbrushes (“Ready Brush” 
disposable, individually wrapped 
& pre-pasted) 
$100 
Disclosing tablets (Two tone 
chewable by Plaqsearch) 
$30 
Vials 
$100 
Recruiting flyers 
$50 
Pineapple juice 
$10 
Powdered Bromelain  
$50 
Poster fabrication 
$50 
Donated by IUSD 
 
Waterpik®s (55) 
$1925 
Donated by Waterpik 
 
Digital Camera: (Canon Rebel 
EOS ) 100mm macro lens 
 
Borrowed from IUSD 
Orthodontic Department 
 
Cheek retractors 
$100 
Will be purchased from 
Amazon & disposed of 
after one use 
Panelist/Subject Pay: 
$50 gift card 
$2750 
Grant from Delta Dental 
Biostatistics: 
Statistical analysis 
$3200 
IU research committee 
 
 
 
 
 
 
 
 
 
 
 
 
 


24 
 
 
12. 
REFERENCES…………………………………………………………………… 
 
1. 
Hagg U, Kaveewatcharanont P, Samaranayake YH, Samaranayake LP. The effect of fixed 
orthodontic appliances on the oral carriage of Candida species and Enterobacteriaceae. European journal 
of orthodontics. 2004;26(6):623-9. 
2. 
Martignon S, Ekstrand KR, Lemos MI, Lozano MP, Higuera C. Plaque, caries level and oral 
hygiene habits in young patients receiving orthodontic treatment. Community dental health. 
2010;27(3):133-8. 
3. 
Klukowska M, Bader A, Erbe C, Bellamy P, White DJ, Anastasia MK, et al. Plaque levels of 
patients with fixed orthodontic appliances measured by digital plaque image analysis. American journal of 
orthodontics and dentofacial orthopedics : official publication of the American Association of 
Orthodontists, its constituent societies, and the American Board of Orthodontics. 2011;139(5):e463-70. 
4. 
Wites M, Panuszka J, Dyras M. [Evaluation of oral and orthodontic appliance hygiene in 
orthodontically treated patients]. Przeglad lekarski. 2003;60 Suppl 6:126-8. 
5. 
Ristic M, Vlahovic Svabic M, Sasic M, Zelic O. Clinical and microbiological effects of fixed 
orthodontic appliances on periodontal tissues in adolescents. Orthodontics & craniofacial research. 
2007;10(4):187-95. 
6. 
Attarzadeh F. Water irrigating devices for the orthodontic patient. International journal of 
orthodontics. 1990;28(1-2):17-22. 
7. 
Zurfluh MA, van Waes HJ, Filippi A. The influence of fixed orthodontic appliances on halitosis. 
Schweizer Monatsschrift fur Zahnmedizin = Revue mensuelle suisse d'odonto-stomatologie = Rivista 
mensile svizzera di odontologia e stomatologia. 2013;123(12):1064-75. 
8. 
Attin R, Thon C, Schlagenhauf U, Werner C, Wiegand A, Hannig C, et al. Recolonization of 
mutans steptococci on teeth with orthodontic appliances after antimicrobial therapy. European journal of 
orthodontics. 2005;27(5):489-93. 
9. 
Rego RO, Oliveira CA, dos Santos-Pinto A, Jordan SF, Zambon JJ, Cirelli JA, et al. Clinical and 
microbiological studies of children and adolescents receiving orthodontic treatment. American journal of 
dentistry. 2010;23(6):317-23. 
10. 
Boyd R. Two-Year Longitudinal Study of a Peroxide-Fluoride Rinse on the Calcification in 
Adolescent Orthodontic Patients. Journal of Clinical Dentistry  1992;3(3): 83-7. 
11. 
Miranda Rda S, Marques RA, Dummel C, Soares FZ, Oliveira MD, Rocha Rde O. The influence 
of prebrushing mouthwashes on plaque removal in children. Pediatric dentistry. 2014;36(3):211-5. 
12. 
Tufekci E, Casagrande ZA, Lindauer SJ, Fowler CE, Williams KT. Effectiveness of an essential 
oil mouthrinse in improving oral health in orthodontic patients. The Angle orthodontist. 2008;78(2):294-8. 
13. 
Pontier JP, Pine C, Jackson DL, DiDonato AK, Close J, Moore PA. Efficacy of a prebrushing rinse 
for orthodontic patients. Clinical preventive dentistry. 1990;12(3):12-7. 
14. 
Kato K, Tamura K, Nakagaki H. Quantitative evaluation of the oral biofilm-removing capacity of a 
dental water jet using an electron-probe microanalyzer. Archives of oral biology. 2012;57(1):30-5. 
15. 
Gorur A, Lyle DM, Schaudinn C, Costerton JW. Biofilm removal with a dental water jet. 
Compendium of continuing education in dentistry (Jamesburg, NJ : 1995). 2009;30 Spec No 1:1-6. 
16. 
Daep CA, Novak EA, Lamont RJ, Demuth DR. Selective substitution of amino acids limits 
proteolytic cleavage and improves the bioactivity of an anti-biofilm peptide that targets the periodontal 
pathogen, Porphyromonas gingivalis. Peptides. 2010;31(12):2173-8. 
17. 
Hellgren K. Assessment of Krillase chewing gum for the reduction of gingivitis and dental plaque. 
The Journal of clinical dentistry. 2009;20(3):99-102. 
18. 
Agarwal RM, Yeluri R, Singh C, Munshi AK. Enamel Deproteinization using Papacarie and 10% 
Papain Gel on Shear Bond Strength of Orthodontic Brackets Before and After Acid Etching. The Journal 
of clinical pediatric dentistry. 2015;39(4):348-57. 
19. 
Pithon MM, Ferraz CS, Oliveira GD, Dos Santos AM. Effect of different concentrations of papain 


25 
 
gel on orthodontic bracket bonding. Progress in orthodontics. 2013;14:22. 
20. 
: Enzyme Development Corporation;  [Available from: 
http://www.enzymedevelopment.com/applications/tenderizer/. 
21. 
Chakravarthy P, Acharya S. Efficacy of extrinsic stain removal by novel dentifrice containing 
papain and bromelain extracts. Journal of young pharmacists : JYP. 2012;4(4):245-9. 
22. 
Kalyana P, Shashidhar A, Meghashyam B, Sreevidya KR, Sweta S. Stain removal efficacy of a 
novel dentifrice containing papain and Bromelain extracts--an in vitro study. International journal of 
dental hygiene. 2011;9(3):229-33. 
23. 
Patil PA, Ankola AV, Hebbal MI, Patil AC. Comparison of effectiveness of abrasive and 
enzymatic action of whitening toothpastes in removal of extrinsic stains - a clinical trial. International 
journal of dental hygiene. 2015;13(1):25-9. 
24. 
Goldman M, Kronman JH. A preliminary report on a chemomechanical means of removing caries. 
Journal of the American Dental Association (1939). 1976;93(6):1149-53. 
25. 
Group DE. The Health Benefits of Bromelain: Global Healing Center;  [Available from: 
https://www.globalhealingcenter.com/natural-health/bromelain/. 
27. 
Keukenmeester RS SD, Rosema NAM, Van der Weijden GA. . Determination of a Comfortable 
Volume of Mouthwash for Rinsing. . International Journal of Dental Hygiene. 2012;10:169-74. 
28. 
Al-Anezi SA, Harradine NW. Quantifying plaque during orthodontic treatment. The Angle 
orthodontist. 2012;82(4):748-53. 
29. 
Thienpont V, Dermaut LR, Maele GV. Comparative study of 2 electric and 2 manual toothbrushes 
in patients with fixed orthodontic appliances. American Journal of Orthodontics and Dentofacial 
Orthopedics. 2001;120(4):353-60. 
30. 
Clerehugh V, Williams P, Shaw WC, Worthington HV, Warren P. A practice-based randomised 
controlled trial of the efficacy of an electric and a manual toothbrush on gingival health in patients with 
fixed orthodontic appliances. Journal of Dentistry. 1998;26(8):633-9. 
31. 
FDA. What is a Serious Adverse Event? 
32. 
Arici S, Alkan A, Arici N. Comparison of different toothbrushing protocols in poor-toothbrushing 
orthodontic patients. European journal of orthodontics. 2007;29(5):488-92. 
 
Beberhold, Katrin & Sachse-Kulp, Anika & Schwestka-Polly, Rainer & Hornecker, Else & Ziebolz, Dirk. 
(2012). The Orthodontic Plaque Index: An oral hygiene index for patients with multibracket appliances.. 
Orthodontics : the art and practice of dentofacial enhancement. 13. 94-9.