Download Appendix A Patient Inclusion and Exclusion Criteria Participants

Appendix A
Patient Inclusion and Exclusion Criteria
Participants aged 18 to 40 years with ≥ 20 permanent teeth were considered candidates.
Other inclusion criteria consisted of; 1) probing pocket depth (PD) of  4mm at six sites per
tooth on all teeth; 2) mean clinical attachment level (CAL) of  2mm on all teeth; 3)
bleeding on probing (BOP) score of > 30% at the screening visit and a BOP score of  10%
at the baseline visit (or re-baseline visit 2 weeks thereafter); and 3) compliance with all
study requirements.
Participants were excluded if they; 1) had a history of alcoholism or drug abuse; 2) had
medical conditions that may affect the study outcome such as neurologic, immune, or
psychiatric disorders; 3) were pregnant or breast-feeding; 4) previous known
hypersensitivity reactions or oral allergies to any ingredient in the study dentifrice; 5) were
current smokers, smokers who quit < 1 one year ago, or had a pack-year history of ≥ 10
(pack-years were calculated by multiplying the number of years smoked by the average
number of cigarette packs smoked per day); 6) had a positive urine cotinine analysis (>200
ng/mL); 7) had antibiotic therapy within 3 months of the baseline visit or needed antibiotic
therapy for prophylaxis; 8) took medications known to affect periodontal status; 9) were
new oral contraceptive users within 3 months of the baseline visit, or users who were
planning on starting oral contraceptives or use of contraceptive injection (Depo-Provera,
Pharmacia & Upjohn Company, Division of Pfizer, New York, NY) during the study; 10) used
antiseptics within 30 days prior to the baseline visit; 11) had current orthodontic or
periodontal treatment; and 12) had untreated carious lesions or defective restorations
which could exacerbate during a period of oral hygiene abstinence.
Appendix B
Description of stent fabrication:
A uniform layer of silicone material was used to block out the free gingival margin area to
provide sufficient space for a standardized amount of dentifrice to accumulate, minimize
ejection of material, and prevent disruption of the plaque and gingival tissues. The stent
was fabricated using a uniform thermoplastic material, which was vacuum fitted to the
models and extended over one half of the mandibular quadrant 1mm short of the mucosal
folds.
Appendix C
Participant instructions for using the stent:
To ensure thorough understanding of the instruction, participants were shown a power
point presentation, had a practice-session with an examiner, and were provided a take
home instruction sheet. Participants received the same type of toothbrush and were
instructed to refrain from using any home oral health care products (mouth rinses,
dentifrice, interproximal aids, etc…) other than those distributed as part of the study
protocol or from using dental floss in their mandibular arch.
Appendix D
Supplemental information about salivary biomarker array procedures:
Upon receipt, the array kits were stored at -20°C. Prior to each assay, whole saliva samples
were thawed and centrifuged for 15 minutes at ~10-12,000 g at 40C to remove insoluble
material and obtain a cell-free supernatant. Each third slide contained known cytokine
standards for making serial dilutions with sample diluents serving as the negative control
and the remaining slides contained experimental whole saliva samples. The detection
antibody was bound to the antigens within each well. Cyanine 3 equivalent dye-conjugated
streptavidin was pipetted into each well, which bound to the detection antibody associated
with immune complexes. Slides were incubated, covered with aluminum foil, and
fluorescence intensity was detected using a laser scanner (Axon Gene Pix, RayBiotech, Inc,
Norcross, GA). The signals of the experimental samples were compared to the standard
curve for each of the cytokines to determine the concentrations of each cytokine within the
samples. Data were extracted and analyzed using microarray analysis software
(Quantibody® Q-Analyzer software, RayBiotech, Inc, Norcross, GA).
Appendix E
Supplemental information about checkerboard DNA-DNA hybridization techniques:
Samples were lysed and DNA was pipetted on a nylon membrane using a miniblotter
apparatus (MiniSlot, Immunetics, Boston, MA). After fixation of DNA to the membrane, the
membrane was placed in another blotting apparatus (Miniblotter 45, Immunetics).
Digoxigenin-labeled whole chromosomal DNA probes and hybridization buffer were
injected into individual lanes of the miniblotter. After hybridization, membranes were
washed twice at low stringency and then twice at high stringency. Hybrids were detected
using anti-digoxigenin antibody conjugated with alkaline phosphatase, followed by
detection using a chemifluorescence substrate (AttoPhos, GE Healthcare, Arlington Heights,
IL) . The intensity of the fluorescence signals was read with computer-linked fluorimager
(Storm Fluorimager, GE Healthcare, Sunnyvale, CA). Signals were compared to standards
on the same membrane to generate absolute counts.
Appendix F
Supplemental GCF Analysis information:
20μl of extraction solution (24.5mL Phosphate Buffered Saline [pH 7.4], 125ml
phenylmethylsulfonylfluoride (PMSF;Sigma Chemical, St. Louis, MO), 200mM in MeOH250
ml Aprotinin (Sigma Chemical , St. Louis, MO), 1 mg/ml in water 83.5 ml of 30% Human
Serum Albumin (Sigma Chemical, St. Louis, MO)) was pipetted onto the cellulose portion of
each of strips. Individual strips were centrifuged at 2000 rpm at 4° C for 5 minutes. This
process was repeated 4 additional times to yield a total of 100ml final volume per tube. The
GCF samples were quantitatively analyzed for specific biomarker concentrations using a
Quantibody Human Cytokine Array and software (RayBiotech, Inc, Norcross, GA).
Appendix G
Statistical Analyses
This was an exploratory, pilot study analyzing oral fluid biomarkers in an experimental
gingivitis study model design focusing on the anti-microbial effects of triclosan when used
as a locally applied agent without the use of mechanical tooth brushing.
Group differences in demographic characteristics were assessed using independent
samples t-tests for continuous characteristics and Fisher's Exact Test for categorical
characteristics. The longitudinal patterns of mean PI and GI were modeled using
generalized estimating equations (GEE) to account for the correlation of values measured
on the same participant; robust standard errors were used to assess the statistical
significance of changes over time within each group as well as for the statistical significance
of differences between the groups at each time point. The same modeling approach was
used for the longitudinal patterns of GCF (both weekly and hourly) and salivary biomarkers
(weekly only). The areas under the hourly longitudinal curves of the GCF biomarkers were
also computed and compared between the groups using GEE. Mean counts of bacterial
species were stratified by dentifrice treatment and pie charts were created demonstrating
proportions of each bacterial complex in relation to their total DNA over the study period.
The Wilcoxon Signed Rank test was used to examine differences from Day 0 to Day 14 in
both test and control groups for each pathogen, and the Wilcoxon Rank Sum test was used
to examine if the Day 0 to Day 14 changes differed significantly between the test and
control groups.
Logistic regression was used to model the probability of GI susceptibility as a function of
combinations of GCF biomarkers and microbial species. Participants were stratified into
two groups based on clinical manifestations of gingivitis; ‘high’ responders were classified
as having a GI score of >1.5 during the course of the study whereas ‘low’ responders
consisted of participants who had GI scores of ≤1.5 (Lee et al., 2012, Trombelli, 2004). A
limited number of GCF biomarkers and microbial species were investigated, including; IL-
1β, IL-6, IL-8, MMP-8, A. naeslundii, A. oris, C. ochracea, F. nucleatum ss polymorphum, and F.
periodonticum. The abilities of these models to correctly identify GI progression were
summarized graphically in receiver operating characteristic (ROC) curves, and numerically
with areas under the ROC curves (AUC) and the odds ratio corresponding to a doubling
of IL-1β. AUCs were computed non-parametrically.
Inter- and intra-examiner calibration data for CAL and PD were analyzed using correlation
coefficient tests in SPSS.