Induced Gingivitis & Intra-oral Imaging
Phase: NA
Conditions: Plaque Induced Gingivitis
Sponsor: University of Washington

 
 
__________________________________________________________________________ 
 
Induced Gingivitis & Intra-oral Imaging 
Protocol Number  CRO-2018-06-EXG-JG 
 
ClinicalTrials.gov ID: NCT03750955 
____________________________________________________________________ 
 
 
 
 
 
Protocol Date: July 18, 2018 
 
Sponsored by: 
 
Colgate-Palmolive Technology Center 
909 River Road 
Piscataway, NJ  08855 
 
Protocol Approval  
 
Signature Page 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dr. Maria Ryan  
 
 
 
 
 
 
Date 
Vice President and Chief Dental Officer 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
Dr. LaTonya Kilpatrick-Liverman  
 
 
 
 
Date 
Word Wide Director – Oral Care Clinical Research  
 
 
 
 
 
 
 
 
 
 
 

Protocol Number  CRO-2018-06-EXG-JG 
Confidential 
 
 
 
 
 
 
 
Induced Gingivitis & Intra-oral Imaging  
Protocol Number:   CRO-2018-06-EXG-JG 
 
 
Sponsored by: 
 
Dental Clinical Research 
Colgate-Palmolive Technology Center 
909 River Road 
Piscataway, NJ  08855 
 
 
Protocol Approval 
 
 
PRINCIPAL INVESTIGATOR APPROVAL AND AGREEMENT 
 
I, the undersigned, have reviewed this protocol and I agree to conduct this protocol in accordance with 
the Guidelines for Good Clinical Practice, the ethical principles set forth in the Declaration of Helsinki, 
and with the U.S. Code of Federal Regulations governing the protection of human subjects (21 CFR 50) 
and the institutional review boards (21 CFR 56). 
 
 
 
 
 
 
____________________________________________ 
_________________ 
Richard Darveau, PhD 
Date 
Principal Investigator 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Number  CRO-2018-06-EXG-JG 
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STUDY AIM: 
To evaluate the ability of an intra-oral imaging device to assess the level of gingival inflammation 
present and the process of resolution using a localized experimentally induced gingivitis model. 
 
BACKGROUND AND SIGNIFICANCE: 
This project is significant for public health because it aims to validate a non-invasive imagining 
examination for determining gingival (gum) inflammation. Given that the prevalence of gingivitis in the 
US population is greater than 50%, and that gingivitis can progress to periodontitis and tooth loss if left 
untreated, the proposed non-invasive imaging method can be an invaluable asset in clinicians' quivers 
for the early prevention of this prevalent disease.  
  
STUDY DESIGN 
This study is a modification of classical experimental gingivitis model. The study will prospectively enroll 
subjects and a maxillary sextant to an intervention, while the other sextant will serve as control in a split-
mouth design of localized experimental gingivitis, utilizing localized stent-induced biofilm overgrowth 
model (SIBO). The intervention will consist of cessation of oral hygiene versus an active control of oral 
hygiene with a fluoridated toothpaste that will be provided by the investigators. The intervention, i.e. oral 
hygiene cessation will manipulate the participants’ oral environment by leading to reversible 
inflammation of the gingival tissues.  This is a well established protocol that was first introduced in 1965 
(Loe’s experimental gingivitis) and is safe, reliable and widely utilized in periodontal clinical trials. The 
purpose of this study is to evaluate the effects of oral hygiene cessation in the gingival tissues by an 
innovative non-invasive microimaging technique, i.e. OCT. 
 
The study will include the following phases – 1) Hygiene phase of 2 weeks; 2) Gingivitis induction phase 
with stent lasting up to 2 weeks; and 3) Resolution phase of 2 weeks. This pilot study will enroll up to 15 
generally healthy adults aged 18-35 years that are self-referred or referred to the UW School of 
Dentistry. Recruitment will be completed in 3 waves. The study will include an oral health history review, 
probing depths, plaque & gingival evaluations, biospecimens collection and imaging of the buccal 
gingiva of teeth #5 - 12. 
 
RECRUITMENT 
All study participants will be patients of record at the UW School of Dentistry, or referred. All study 
participants will undergo informed consent process, prior to enrollment in the study. Categorization of 
participants and sites will be based on the clinical assessments. Other than the study-related oral health 
history recorded, there is no intention to obtain information from the participants’ other dental or medical 
records. The first participant will be assigned a number, and all the following subjects will be assigned 
consecutive numbers by accession. 
 
Inclusion criteria: 
 
Aged 18-35 years 
 
In good general health, ASA I 
 
No clinical signs of gingival inflammation at >90% sites observed 
 
Probing Depth(PD) < 3.0 mm 
 
Attachment Loss (AL) = 0 mm 
 
Gingival health at baseline visit (Day 0): Gingival Index (GI) ≤ 0.5, Bleeding on probing (BOP)(-) 
 
Non-smokers 
 
Fluent in English 
 
 

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Exclusion criteria: 
 
Medical condition which requires premedication prior to dental treatments/visits 
 
Subjects unable or unwilling to undergo informed consent process. 
 
Subjects currently using anti-gingivitis oral rinses (washout period of 1 week) 
 
History of periodontal disease 
 
History of systemic inflammatory or immune conditions 
 
Use of antibiotic or anti-inflammatory drugs within 30 days of enrollment 
 
Pregnant or breastfeeding at time of screening 
 
Concurrent orthodontic treatment 
 
Untreated carious lesions and/or inadequate restorations, implants, crowns on maxillary 
non-molar teeth 
 
Participation in any other clinical study or test panel within 1 week prior to enrollment into this 
study 
 
Use of tobacco products 
 
Subjects who must receive dental treatment during the study dates 
 
Orthodontic bands, appliances, or crowns and bridges, or removable partial dentures affecting 
the non-molar maxillary teeth 
 
Immune compromised individuals (HIV, AIDS, immuno suppressive drug therapy) 
 
METHODS 
Induced Gingivitis Model 
In each participant, one maxillary quadrant will be assigned as test quadrant (experimental gingivitis) 
and contralateral quadrant as control quadrant. Assignment of quadrants will be random using a 
computer program for randomization.  
 
At the initial visit all participants will undergo the informed consent process, with thorough discussion of 
study participation details. Full clinical assessments, biospecimen collections and imaging will be 
performed and maxillary impression will be taken for acrylic stent. Maxillary acrylic stents will be 
fabricated to cover teeth on test quadrant extending at least 4-5 mm apical to the marginal gingiva both 
buccal and palatal and will be relieved around interproximal areas to prevent plaque disturbance. An 
oral prophylaxis will be administered and thorough oral hygiene instructions will be given. Participants 
will return 14 days after this initial visit and will have baseline measurements and biospecimen 
collections taken. These measurements include an abbreviated health assessment with a periodontal 
probe to measure pocket depths, gingival and plaque index and Imaging of the buccal gingival on eight 
maxillary teeth (#’s 5-12), and collection of crevicular fluid and plaque samples37. 
 
At the baseline visit the acrylic stent will 
be given to participant with detailed 
instructions for use. Participants will be 
given instructions not to brush teeth on 
test quadrant (under the provided stent) 
and not to use any other measure of oral 
hygiene (flossing, interdental aids, xylitol 
gum, and mouth rinses). Participants will 
be provided with Colgate Cavity 
Protection Toothpaste and a soft bristle 
toothbrush to use during their entire 
participation in this study. In order to 
prevent accidental brushing of the 
experimental sites, subjects will be 
asked to wear the maxillary stent during 
regular brushing. Participants will be 
UW - Colgate Imaging Study 
Test Site - GI 
Control Site – No GI 
Transition 
Zone 
UW Periodontics 2018
UW Periodontics 2018

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rechecked at Day 2, 4, 7 & 14 and a full assessment of their plaque and gingival status will be carried 
out, specimens collected, and imaging performed each time using the same criteria as at the baseline 
examination. The course of the "no-brushing" part of the experiment will last up to 14 days to allow all 
the participants to develop gingivitis. Following clinical assessment and biospecimen collections at day 
14, a thorough prophylaxis will be administered.  
 
Participants will be given detailed instructions in oral hygiene methods using a manual toothbrush and 
Colgate Cavity protection toothpaste. Subjects will also usedental floss beginning the same day and 
continuing once in the morning and once at night for the duration of the study. Assessment of plaque 
and gingival condition along with imaging will continue during the entire reversal phase, Day 21 and Day 
28. At a point where the Gingival Index and Plaque Index scores approach zero (approximately day 28), 
all measurements and biospecimen samples will be taken, along with imaging and that visit will be 
recorded as end-of-study. 
 
 
 
Medical imaging device 
The medical imaging device is a home-built multi-functional OCT system (MI-OCT), which is also 
illustrated in Figure1. The functionality of the system includes: 
 
Anatomical structure imaging (OCT) 
 
Functional angiography (OCTA) 
 
Molecular imaging: Red and green autofluorescence imaging (MI) 
The components of the system include: 
 
A laser light source (NIR, 1300nm peak, 100nm bandwidth, peak power 3.5mW) 
 
Optical components that transmit OCT light beam into biological tissue, including: optical fiber 
and optics table, galvanometer mirror scanners, and several other optics components (lenses, 
dichroic mirrors, prism, etc.) 
 
CMOS camera for molecular imaging (MI) 
 
An excitation light source in violet-near UVA wavelength (405±5 nm) 
 
A computer and monitor for real-time imaging display, data processing, and data analysis. 
The home-built MI-OCT device include OCT module, which is similar to commercially FDA approved 
OCT devices, and was also approved by the IRB as ‘Non-Significant’ risk for skin imaging (IRB No. 
41841) and for periodontal imaging (IRB No. 2827). The difference between MI-OCT and the 
aforementioned units are: 
 
Violet-near UVA excitation light source (LED ring, 405±5 nm wavelength). CMOS 3-channel 
red-green-blue camera with added optics (optical filter, lenses, etc.) 
 
Wiring and cable harness, providing power to the LED ring and CMOS camera 
Although the wavelength is within the visible spectrum and the LED ring is operated at lower power level 
(25mW, radiating over a surface of 10×10cm2, which yields 0.25 mW/cm2), participants will be given 
UVA-protective goggles to reduce the risk of exposure to possible residual UVA emissions from the 
LED. The length of exposure will also be limited to maximum 15 minutes in the MI mode (2 minutes for 
each scanning sites, total 6 scan sites) 

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Fig 1a.  
 
 
 
Fig. 1b  
 
Fig. 1c 
Figure 1 a) MI-OCT probe which is attached to a stable frame, b) MI-OCT in operation, showing visible 
violet excitation light when operated in autofluorescence mode, c) components of the stable frame 
 
Imaging procedure 
Participants will be instructed to wear a dental retractor to expose the maxillary incisors, canines, and 1st 
bicuspids prior to the scan. The scanning protocols for each scan-site (6 in total) are as followed: 
 
Positioning: participants will be asked to rest their chins on the chin-rest of the stable frame. 
They will also be asked to bite on the bite bar (Figure 1c) during the whole scanning procedure. 
Operator will re-position the scanning probe for each scan-site. This step takes approximately 1 
to 3 minutes for each scan-site. 
 
Real-time autofluorescence imaging and registration: participants will be exposed to 405nm 
excitation light source for approximately 2 minutes; autofluorescence images are captured and 
addition registration (re-positioning of the probe) is performed when needed. This step takes 
approximately 2-3 minutes 
 
OCT scan: participants will be asked to stay particularly still in this step. The scan will be 
performed within a window of less than 1 minute. 
 
 
OUTCOMES 
In the present study, we will be examining the sensitivity, specificity, and practicality of a new 
microimaging technique for assessing experimental gingivitis, as compared with standard health-related 
biomedical outcomes (i.e. gingival index and gingival crevicular fluid volume).  
 
Abbreviated periodontal health assessment 
Clinical data will be documented based on probing depth (PD), visible plaque index (VPI), gingival index 
(GI) and bleeding on probing (BOP) 38.  All clinical measurement will be conducted using a manual 
periodontal probe. BOP will be recorded within 20 seconds of probing. The following four maxillary teeth 
will be used in each test and control quadrant: first premolar – central incisor. For each tooth, all 
measurements and biospecimen collection will be taken on mesiobuccal and mesiopalatal surfaces. 
Staggering of sites (mesiobuccal & mesiodistal) will be performed for plaque collection.  
 
Visible Plaque Index 
The surfaces of the study teeth will be scored and given either a “0” no plaque or “1” plaque 
assessment: 
0 = No plaque 
1 = Visible Plaque 
 
 

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Loe and Silness Gingival Index (GI) 
The surfaces of the study teeth will be evaluated using light air and a periodontal probe. A score is given 
based on the following scale: 
0 = Normal gingiva 
1 = Mild inflammation, slight change in color, slight edema, no bleeding on probing 
2 = Moderate inflammation, moderate glazing, redness, bleeding on probing 
3 = Severe inflammation, marked redness and hypertrophy, ulceration, tendency to spontaneous 
bleeding 
 
Gingival Crevicular Fluid (GCF) collection 
Prior to GCF collection the plaque index will be recorded. The sites to be sampled will be isolated with 
cotton rolls and gently air-dried. GCF samples will be collected with sterile paper strips (Periopaper 
strips, Oraflow Inc.) that will be inserted into the gingival crevice until mild resistance is felt and left in 
place for 30 seconds. 
 
Amount of GCF samples collected will be quantified with a Periotron 6000 (Oraflow, Inc.) and will be 
expressed in microliter.39 GCF will be collected from the mesiobuccal and mesiopalatal surfaces of 
teeth #’s 5- 12. Paper strips visibly contaminated with saliva and blood will be excluded from the study. 
All samples will be collected in micro centrifuge tubes. They will be transported on ice and stored within 
24 hours at -80oC until processing. 
 
Plaque collection 
Sub-gingival plaque will be collected using sterile paper points inserted in the gingival sulcus for 30s 
from the mesiobuccal and mesiodistalsurfaces of teeth #’s5- 12. Plaque samples will be placed in micro 
centrifuge tubes then transported on ice to -80oC freezer in lab. 
 
Analysis of GCF chemokines  
GCF samples will undergo assay of 3 angiogenesis chemokines using commercially available kits. The 
following list of chemokine will be analyzed: angiopoietin-1, vascular endothelial growth factor, and 
hypoxia-inducible factor 1 alpha. The expression of these chemokines have all been shown to 
contribute to angiogenesis and possibly homeostasis in gingival tissue. Commercially available ELISA 
kits will determine the concentration of Myeloperoxidase (MPO). 
 
Analysis of microbial composition 
Plaque samples will be stored in the Periodontics Repository for future studies. The samples will not be 
analyzed as part of this study. 
 
Imaging 
1) Autofluorescence imaging 
Autofluorescence images are captured and analyzed for two purposes: 
a. 
Plaque (biofilm) detection (red autofluorescence)  
b. 
Enamel absorption and anomalies (green autofluorescence) 
The images are registered in each subsequent visit. This registration step allows observation on the 
progression of a) plaque and b) tissues response over the whole study period. 
 
2) OCT and OCTA 
Data is captured for each visit and is analyzed off-line. The following features are extracted and 
monitored: 
a. 
The rete-ridges structure (OCT) in free gingiva. During inflammation, the rete-ridges 
layer becomes thinner and is replaced by connective tissues. 
b. 
The depth of the free gingiva (OCT+OCTA), which is defined by a line joining: i) the 

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surface of the gingiva, ii) projected perpendicular to the gingival sulcus line, and iii) passing 
through the point where connective blood vessels in free/attached gingiva disappear 
c. 
The vasculature (OCTA) in the free gingiva.  
d. 
Possible changes in other morphology (reserved for exploratory parameters). 
 
Recovery and Termination from the Study 
All subjects will be evaluated weekly during induction phase. In the case that participants develop GI 
score 3 in 25% or more of their experimental teeth, they will be immediately moved to the resolution 
phase. Otherwise the participants will finish the full induction phase and then be moved into resolution 
phase. All subjects will be followed until gingivitis condition is completely resolved, beyond the two 
weeks period if necessary. If any participants have persistent gingivitis that does not resolve, they will be 
referred to the Graduate Periodontics Clinic at the University of Washington School of Dentistry. 
 
Tooth Stent Construction 
 
An alginate impression will be taken of one maxillary quadrant of each subject for the 
construction of an individual toothshield stent for use during the trial period.  The quadrant will be 
randomly selected. A working model cast will be prepared immediately from the impression using dental 
stone and labeled with the subject's ID.  After curing for an hour, the cast will be removed from the 
impression mold, trimmed to the desired shape and size, then dried at room temperature overnight. 
 
The toothshield stents will be constructed from 3-mm-thick plastic mouthguard material to 
include only the teeth and gingival margin and to eliminate contact with the cervical margin of each 
tooth, thereby reducing the risk of plaque being disturbed during insertion or removal of the shield.  
Elimination of cervical contact is accomplished by blocking out around the gingival margin and proximal 
surfaces using a spacer made from 1-mm-thick mouthguard material.  Dipping the cast in water, 
allowing it to drain, and then adapting the 1-mm-thick material by using a vacuum former.  After cooling, 
the material is scribed with a scalpel to a height one-third of the clinical crowns, following the contour of 
the gingivae; removed from the cast; and trimmed with scissors to ~2 mm above the gingival margin.  
The 1-mm-thick spacer is placed on the model; coated with silicone spray; then the 3-mm-thick material 
is vacuum-adapted over the model.  When cooled, the mouthguard material is removed from the model 
and the spacer peeled out.  The 3-mm material is trimmed vertically on the buccal side to a length just 
short of the vestibule and frenum attachments, and on the lingual side to 2 mm above the gingival 
margin.  It is trimmed mesially to the midline, and distally behind the first premolar.  
 
 A label with the subject's ID and the date of preparation is affixed to the buccal surface, and then 1-
mm-thick mouthguard material is vacuum-adapted over the toothshield and trimmed to the original 
contours.  The shields are prepared prior to the prophylaxis, chemically disinfected, and packed in 
individual plastic bags with each subject's name. 
 
Statistical Analysis Plan 
Multiple sites within each subject will be evaluated using a Gingival Bleeding Index at baseline and 
subsequent timepoints during clinical exams. The gingivitis bleeding scores will be averaged to provide 
a mean score per subject for each exam period.  Product means and standard deviations will be 
calculated for each time period.  Analysis of Variance techniques (possibly including Analysis of 
Covariance using the baseline scores as the covariant) will be used to compare the difference between 
the two test treatments using the 95% confidence level (p ≤ 0.05).   
 
Based upon our initial study the selection of 15 participants is expected to identify statistically significant 
differences in clinical parameters, GCF chemokines level, MPO expression and microbial composition 
over time among the subjects of the study. Participants will be enrolled in cohorts of 5 and we have 
planned for 3 waves to reach the final sample size N=15. 

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REPORT OF ADVERSE REACTIONS 
 
Subjects will be told of any possible adverse reactions or side effects from lack of oral hygiene and stent 
use, i.e. oral irritation.  At the all clinic visits/examinations each participant will be asked if they have 
experienced any discomfort or oral irritation.  In addition, all subjects will be examined for signs of 
epithelial desquamation (sloughing) and discoloration of the teeth and/or tongue. The investigators will 
record any and all adverse reactions.  If the evidence indicates that the adverse reaction may be due to 
lack of oral hygiene, the subject will be instructed to resume oral hygiene and appropriate treatment will 
be provided.  If an adverse event occurs it is expected to be mild and abate (stop) when oral hygiene is 
resumed.  In the event of an adverse experience, emergency or other problems or questions regarding 
participation in this study subjects will be advised to contact the following investigator(s): 
 
Richard Darveau, MS, PhD  
Office: 206-543-5043 
 
In the event of a medical emergency, subjects will be instructed to contact their physician. 
 
 
Definitions 
Adverse Events (AEs) and Serious Adverse Events (SAEs) are defined by the ICH Guideline for 
Good Clinical Practice (ICH GCP) as follows: 
 
Adverse Event:  Any untoward medical occurrence in a patient or clinical investigations subject 
administered a pharmaceutical product and which does not necessarily have to have a causal 
relationship with this treatment.  An adverse event (AE) can therefore be any unfavorable and 
unintended sign (including abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a medicinal product, whether or not considered related to the 
medicinal product. 
 
AEs include any clinically significant deterioration of a subject’s medical status, after enrolled and 
signing an Informed Consent Form.  The AE may involve any organs or systems and can be 
represented by the new onset or the deterioration of a disease, a syndrome, a symptom, a physical 
sign, as well as by findings and results of instrumental examinations and laboratory tests.  Any medically 
relevant and untoward change from baseline, including frequency or pattern changes for a fluctuating 
condition (e.g., migraine), occurring after the first administration of study medication is an adverse 
event.  All such occurrences must be recorded and reported accordingly, whether they appear causally 
related to the study medication, or not. 
 
Serious Adverse Event: Any adverse event occurring at any dose that results in any of the 
following outcomes: 
 
Death 
 
Life threatening adverse event 
 
Inpatient hospitalization, or prolongation of existing hospitalization 
 
Persistent or significant disability/incapacity 
 
Congenital anomaly/birth defect 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse experience when, based upon appropriate 
medical judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
 
Examples of such important medical events include allergic bronchospasm requiring intensive treatment 
in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient 
hospitalization, or the development of drug dependency or drug abuse. 

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IMPORTANT NOTES: The concepts of Adverse Event / Experience1 (AE) and Serious Adverse Event / 
Experience (SAE) represent regulatory instruments used to evaluate and monitor the safety of clinical 
trial subjects.  Therefore, these terms only apply in light of their regulatory definition.  The term 
“serious”, in a regulatory sense, does not necessarily mean “severe”.  All adverse events (serious and 
non-serious) reported during a study will be taken into account when analyzing the study data and 
establishing the safety profile of the investigation, and will be included in the final study report.  The SAE 
concept is primarily used to identify, during the conduct of the trial, those adverse events that may 
require an expedited reporting procedure to regulatory authorities. 
 
Death: The outcome of death requires that the AE that resulted in death be reported as an SAE.  Death, 
in and of itself, is not an AE; it is only an outcome.  The cause of death is the AE; therefore, the 
investigator should make every effort to obtain and document the cause of death for all subjects who die 
during the study.  If, despite all efforts, the cause of death remains unknown, the AE it should be 
documented as “unspecified fatal event”. 
 
Life-threatening Adverse Event:  Any adverse event that places the patient or subject, in the view of 
the investigator, at immediate risk of death from the reaction as it occurred (i.e., it does not include a 
reaction that had it occurred in a more severe form, might have caused death). 
 
Hospitalization:  It should be noted that hospitalization, in and of itself, does not represent an 
SAE.  It is the adverse event leading to the subject’s hospitalization that becomes “serious” 
when it requires inpatient care.  Consequently, SAE should not be reported in case of pre-
planned hospitalizations for pre-existing conditions that did not worsen during the study. 
 
Disability: A substantial disruption of a person’s ability to conduct normal life functions. 
 
Documenting and Reporting Adverse Events 
 
General Procedures for All Adverse Events 
All clinical complaints, symptoms, or signs that meet the adverse event definition will be recorded on the 
Adverse Reaction Form using a recognized medical term or diagnosis that accurately reflects the event. 
 Source documentation should be maintained that allows for clear identification of each adverse event 
and the following parameters required for the form: 
 
AE description 
 
Date of onset 
 
Date of resolution 
 
Outcome 
 
Severity 
 
Seriousness 
 
Relationship to study drug (causality) 
 
Actions taken 
 
Adverse events will be assessed by the investigator or designee for severity, relationship to the study 
intervention, possible etiologies, and whether the event meets the criteria as a serious adverse event 
and therefore requires immediate notification of the sponsor. 
 
For data collection purposes, the outcome of all adverse events recorded on the Adverse Reaction 
Form will be designated as of the completion of the final evaluation or examination.  However, the 
investigator is responsible for following all adverse events until resolution or until no longer of clinical 
concern, and providing these data to the sponsor. 
                     
 
 

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Reporting Procedures for Serious Adverse Events 
Any adverse event that is serious or potentially serious requires additional detailed reports and follow-
up.  A serious adverse event must be reported via telephone to the sponsor’s representative 
immediately (within 24 hours) so as to facilitate discussion and implementation of necessary follow-up 
measures, and to enable the sponsor to submit necessary reports to regulatory authorities and other 
investigators.  Following the initial telephone notification, the investigator must complete and submit a 
Serious Adverse Event Report Form to the sponsor within five calendar days.  Serious Adverse Event  
Report Forms will be provided to the investigator upon initiation of the study.  Once the sponsor reviews 
the Serious Adverse Event Report Form, additional information may be requested from the investigator 
to allow appropriate medical evaluation and determine the regulatory reporting requirements. 
The investigator is responsible for following all adverse events, especially those deemed  “serious”, until 
resolution or until the event is no longer of clinical concern, and for providing these data to the sponsor 
in an agreed-upon format.  The investigator is also responsible for reporting all serious adverse events 
to the Institutional Review Board (IRB) overseeing the conduct of the study at the respective study 
center, according to the rules and procedures established by the IRB. 
 
COMPENSATION 
Participants in this study will receive up to $500 in gift cards for their participation. A $50 gift card will be 
given at the end of each visit, and subjects who attended all study visits will receive an additional $100 
gift card at the final visit (28-day visit). Payment will be prorated for visits attended only. No therapeutic 
or other benefit will be received by participating in the study.   
 
QUALITY ASSURANCE/CONDUCT OF THE STUDY 
This clinical research study will be conducted in compliance with this protocol and U.S. Federal 
Regulations governing informed consent (21 CFR 50), Institutional Review Board (21 CFR 56), 
applicable regulations governing Investigator conduct (21 CFR 312) and/or any local regulatory agency 
(where applicable). 
 
It is the responsibility of the Investigator to ensure that all subject data are collected and reported 
according to the study protocol. Subject records will indicate subject and examination information such 
as visit dates, examiners, etc. that is unique to this study and the subjects. Proper documentation of all 
adverse events and final resolutions will be maintained. Case report forms will be used for recording all 
clinical data. All CRFs used in this study will be provided by the study sponsor.  The Investigator will be 
responsible for maintaining original consent forms, case report forms (CRFs), and other source 
documentation. 
 
Data Management Responsibilities 
Data collection is the responsibility of the clinical research site staff under the direction of the Principal 
Investigator. The Investigator will keep a copy of every document (clinical and laboratory) related to the 
research study. 
 
Data generated at the clinical site will be transferred to the sponsor by the Co-Investigator via mail or 
electronic email for statistical analyzes. The principal Investigator and study manager are responsible 
for the review and interpretation of the analyzed data.  
 
Subject Termination/Procedures 
A genuine effort will be made to determine the reason(s) why a subject fails to return for the necessary 
visit(s) or is dropped from the study. Subjects could be dropped from the study if any of the following 
occur: 
1. Subject fails to substantially comply with the protocol requirements. 

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2. Subject fails to report for a scheduled examination. 
3. Subject is treated with medications during the course of the study, which may interfere with 
the parameters of the study. 
4. Subject develops an adverse reaction.  The study Investigator will immediately notify the 
study monitor and information will be recorded on Adverse Reaction Form. 
5. Subject receives emergency dental or medical treatment, which may interfere with the 
parameters of the study. 
6. Sponsor elects to terminate the study. 
7. Subject elects to terminate participation in the study. 
 
Removal of Subjects From Study 
Subjects will be dropped from the study if they receive emergency dental treatment, which in the opinion 
of the monitor could influence the parameters of the study. Any subject treated with an antibiotic during 
the study will be dropped from the study. Either the investigator or the sponsor may terminate the study 
at any time for well documented reasons, provided written notice is submitted at a reasonable time in 
advance of the intended termination. 
 
Pregnancy  
No pregnant women will intentionally be enrolled in this study. All female subjects being considered for 
this study will be asked about their pregnancy status.  All enrolled female subjects will be required to 
report to the clinical/research investigator if they become pregnant during the course of the 6 week 
study. In the event a women enrolled in this clinical research study becomes pregnant at any time 
during this study, participation in this study will be terminated upon the clinical staff’s notification of the 
event. The subject’s medical records used in this study will be updated to reflect the pregnancy and 
there will be follow- up contact until the end of the pregnancy to record the outcome in the clinical file.  
 
 
ETHICAL & REGULATORY REQUIREMENTS/HUMAN SUBJECT PROTECTION  
 
Protocol Approval, Study Monitoring and Compliance 
Prior to initiation of the study, the Investigator will obtain approval from Product Safety and Regulatory 
for the study protocol, the informed consent document, study instructions, and any forms of advertising 
in compliance with regulations. The reviewers will also review any change(s) in the protocol before the 
change is initiated.  
 
Adherence to Protocol/Amendment(s) 
The Investigator will be required to adhere to the final protocol. Any changes to the protocol, except 
those necessary to eliminate apparent hazards, will require prior approval by the local reviewers through 
the submission of a protocol amendment.  These changes to the protocol must be implemented only 
through formal written protocol amendments and only upon joint approval by the sponsor and 
investigator.  If a protocol amendment requires changes to the informed consent form, the revised 
consent form must also be approved by any local board. Departures from eligibility requirements may 
be allowed on a case-by-case basis by the medical monitor or other authorized sponsor representative. 
 Such departures must be medically and scientifically justified, must be pre-authorized, and must be 
documented in the case report form and tracked as official eligibility waivers. 
 
Institutional Review Board 
Prior to initiation of the study, the Investigator will obtain Institutional Review Board approval for the 
study protocol, the informed consent document, study instructions, and any forms of advertising in 
compliance with regulations. The IRB will review the investigation at least once a year and will review 
any significant change in the protocol before the change is initiated. The Investigator will maintain all 

Protocol Number  CRO-2018-06-EXG-JG 
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original correspondence. IRB approval for this study will be obtained from: 
 
Human Subjects Division  
University of Washington Box 359470 
Seattle, Washington 98195 
Telephone:  (206) 543-0098 
 
Advertising 
No newspaper, radio, or television advertising will be used for recruitment purposes. A flyer will be 
created to recruit participants. The flyer will be approved by an Institutional Review Board prior to 
posting or dissemination. 
 
Informed Consent Process 
Written informed consent will be obtained from all subjects prior to their enrollment into the study. The 
purpose and description of the study in lay language, possible adverse reactions, risks and benefits of 
participation and the subject’s right to withdraw without prejudice at any time must be explained to each 
subject in the presence of a witness.  The subject must read, understand and sign the informed consent 
form provided. The informed consent form and any other written information for subjects should meet 
local requirements of language and interpretation (i.e., non-English speaking subjects must be 
presented with informed consent forms in a language that they can understand). The consent form will 
comply with all applicable regulations governing protection of the participating subjects in the study, and 
include basic elements specified in the U. S. Code of Federal Regulations, 21 CFR 50.25(a) and 50.27 
and ICH-GCPs, Chapter 4, subpart 4.8. The Informed Consent form will be reviewed and approved by 
the clinical site’s IRB.  
 
Each subject will be given unlimited time to read the consent form and ask questions. Subjects who 
agree to participate will be asked to sign and date an IRB-approved informed consent form. A copy of 
the signed and dated consent form will be given to each subject prior to their participation in the study.  
The original signed and dated informed consent document will be retained by the Investigator. All 
informed consent forms will be documented in a log by date and subject ID; the log will be kept as 
source documentation.  All study procedures must be explained in non-technical terms.  Study 
personnel will assure that participants are clearly informed regarding their roles and obligations to 
protect vulnerable subjects and ensure they are not under coercion or undue influence.  Subjects have 
the right to withdraw consent at any time.  
 
Confidentiality 
All records of subject participation in this study are confidential and these records are available only to 
the Investigator, supervising dentist, sponsoring company and possibly the Food and Drug 
Administration of the United States.  The results of this study may be published in a scientific journal or 
a government public clinical database.  If any publication occurs, subjects will not be identified.   
 
New Findings 
Subjects will be informed of any significant new findings related to study products or procedures when 
they become known during the course of this clinical research study. Such information may affect the 
subject’s decision to continue participation in the study. 
 
Publication 
All manuscripts or presentations based upon this study, including press statements and internal public 
notices and memoranda must be submitted to the sponsor for review and approval prior to release for 
publication or presentation.  This review period will be up to 60 days in duration.  Review of an abstract 
may be expedited in some circumstances. 

Protocol Number  CRO-2018-06-EXG-JG 
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ADMINISTRATIVE ASPECTS 
 
Curriculum Vitae 
The investigator will complete the FDA form 1572 and provide the sponsor with copies of his/her 
curriculum vitae and those of all sub-investigators listed on the form, at sponsor’s request. 
 
Data collection in the case report form 
All study data will be recorded in the case report form supplied by the sponsor.  All entries will be written 
clearly in black ink.  Only the principal investigator, sub-investigators, or study coordinators may make 
entries in the case report forms.  If erroneous data are entered on the case report forms, corrections to 
the data must be made by crossing out the incorrect entry with a single line (such that the initial entry 
remains legible) and entering the correction.  All corrections on a case report form will be initialed and 
dated by the investigator, sub-investigator, or study coordinator making the correction. 
 
Documentation of Consent and storage of study documents  
All informed consent forms will be documented in a log by date and subject ID; the log will be kept as 
source documentation.  Informed consent forms will be stored in a secure locked room designated for 
research charts storage. 
 
Study Management 
 
Under the direct supervision of the Principal Investigator, certain duties may be delegated during the 
course of the study. These responsibilities will be documented on the Transfer of Responsibilities form 
maintained in the Investigator’s clinical file for the study. 
 
Study monitoring 
Appropriate phone calls or visits will be made by the monitor to the investigative site in order to review 
the protocol and to ascertain that the facility is adequate for satisfactory conduct of the study, as well as 
to discuss the obligations of both the sponsor and the investigator. 
 
The investigator will permit a representative of the sponsor or the sponsor's designate and the FDA, if 
requested, to inspect all case report forms and corresponding portions of the study subjects' original 
office, hospital and/or clinic medical records at regular intervals throughout the study. These inspections 
are for the purpose of assessing the progress of the study, verifying adherence to the protocol, 
determining the completeness and exactness of the data being entered on the case report forms.  
During on-site visits, case report forms will be examined by the study monitor(s) and the data verified by 
comparison with corresponding source data (such as clinic, hospital and/or office records).  Due to the 
geographic distance of the clinical site from the sponsor, phone conferences or teleconferences may be 
used to complete the monitoring requirement. 
 
Final Report 
Following the completion of the study, the Investigator shall prepare a final study report. The final report 
will include a general description of the conduct of the study including protocol deviations, subject 
withdrawals, discussion of any adverse events, safety and efficacy data, laboratory data, and statistical 
analysis of the data if available. This report will be approved and signed by the Principal Investigator. 
 
Record Retention and Access to Source Data/Documents 
Source documents must be kept for at least six (6) years after terminating the study. The Investigator 
will maintain all study documentation for all subjects entered into the study in a secure area ensuring the 
confidentiality of the information collected. Securing records includes placing written forms in locked file 
cabinets and/or sealed and labeled storage boxes in a locked room 

Protocol Number  CRO-2018-06-EXG-JG 
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References 
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15 Nussbaum G, Shapira L. How has neutrophil research improved our understanding of periodontal  
pathogenesis? J Clin Periodontol. 2011;38 Suppl 11:49-59. Epub 2011/02/26. doi: 10.1111/j.1600- 
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21 Borregaard N. Neutrophils, from marrow to microbes. Immunity. 2010;33(5):657-70. Epub  
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22 Sadik CD, Kim ND, Luster AD. Neutrophils cascading their way to inflammation. Trends in  
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27 Teles R, Sakellari D, Teles F, Konstantinidis A, Kent R, Socransky S, Haffajee A. 2010   
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nine acute phase proteins in healthy human plasma, saliva and urine. 2012 Journal of Proteomics  
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29 Loe H, Jensen SB, Theilade E. Experimental gingivitis in man. Journal of Periodontology. 1965; 
36(3):177-87. 
 
30 Braun TM, Giannobile WV, Kinney JS, Morelli T, Oh M, , Ramseier CA, Sugai JV,. Crevicular Fluid 
Biomarkers and Periodontal Disease Progression. J Clinical Periodontol. 2014; 41(2):113-120. 
 
31 Aspiras, M, Barros S, Offenbacher S, Mauriello S, Mendoza L, Moss K, Preisser J. Changes in  
Gingival Crevicular Fluid Inflammatory Mediator Levels during the Induction and Resolution of  
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32 Leishman SJ, Seymour GJ, Ford PJ. Local and Systemic Inflammatory Responses to Experimentally 
Induced Gingivitis. Disease markers. 2013; 35(5):543-549. 
 
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peri- implant mucositis compared with experimental gingivitis in humans. Clinical Oral Implants  
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assessment of 3 different instruments. Journal of Periodontal Research. 1986; 21(1):39-44.

Protocol Number  CRO-2018-06-EXG-JG 
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Appendix A: draft flyer 
 
 
 
 
 
 
 
 
 
 
How do unhealthy gums become healthy 
again? 
 
Researchers at the University of Washington want to 
learn more about the mechanisms in gingivitis. 
 
Sign Up and Help Us Learn! 
You may participate in this study if you: 
 
• 
Are between the ages of 18 and 35 
• 
Have healthy gums and overall are  
 
generally healthy 
• 
Are a non-smoker 
 
What happens in the research? 
 
• 
You will have a dentist examine your gums  
• 
If qualified, you will be asked to attend several  
dental visits 
• 
Participants can receive up to $500 in Gift Cards 
• 
Contact us to learn more details! 
 
 
 
 
 
 
 

Protocol Number  CRO-2018-06-EXG-JG 
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APPENDIX B 
 
Protocol Title 
Induced Gingivitis and Intra-oral Imaging 
 
Study Duration 
Approximately 42 days total 
14 day “no brushing” period 
Pre-experimental 
(Hygiene) phase  
 (Day -14 to 0) 
Visit at Day -14 
At Day -14 visit: 
 
Screening (may be done at separate visit) 
 
Informed Consent  
 
The following measurements will be taken on six surfaces of all teeth 
o 
Plaque Evaluation 
o 
Gingival Index Evaluation 
o 
Probe pocket depths  
o 
Attachment Level  
o 
Bleeding on Probing 
 
The following Biospecimen will be taken from mesiobuccal and mesiopalatal 
surfaces of teeth #’s 5- 12, 
o 
Gingival Crevicular Fluid 
o 
Plaque sample 
 
Prophylaxis (scaling and polishing) 
 
Oral hygiene instructions  
 
Impression for maxillary stent 
 
Baseline  
Visit at Day 0 
 
 
The following measurements will be taken from mesiobuccal and mesiopalatal 
surfaces of teeth #’s5- 12 
o Plaque Evaluation 
o Gingival Index Evaluation 
o Probe pocket depths  
o Attachment Level  
o Bleeding on Probing 
o Gingival Crevicular Fluid 
o Plaque sample 
 
Stent will be delivered 
 
Imaging buccal gingival teeth #’s 5 - 12 
 
Begin oral hygiene abstinence  

Protocol Number  CRO-2018-06-EXG-JG 
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Gingivitis induction 
phase (14 Days) 
Visit at Day 2 
Visit at Day 4 
Visit at Day 7 
Visit at Day 14 
 
 
Assessments over 14 days (4 visits) 
 
The following measurements will be taken from mesiobuccal and mesiopalatal 
surfaces of teeth #’s5- 12 
o Plaque Evaluation 
o Gingival Index Evaluation 
o Probe pocket depths  
o Attachment Level  
o Bleeding on Probing 
o Gingival Crevicular Fluid 
o Plaque sample 
 
Imaging buccal gingival teeth #’s 5 - 12 
 
At Day 14 after data collection: 
o Prophylaxis (scaling and polishing) 
o Reinstitution of regular oral hygiene  
Resolution Phase (14 
Days) 
Visit at Day 21 
Final Visit at Day 28  
  
 
 
 
The following measurements will be taken from mesiobuccal and mesiopalatal 
surfaces of teeth #’s5- 12 
o Plaque Evaluation 
o Gingival Index Evaluation 
o Probe pocket depths  
o Attachment Level  
o Bleeding on Probing 
o Gingival Crevicular Fluid 
o Plaque sample 
 
Imaging buccal gingival teeth #’s 5 - 12