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CLINICAL EFFECTIVENESS OF NEXT SCIENCE ORAL RINSE IN CONTROLLING

PLAQUE AND GINGIVAL INFLAMMATION. A DOUBLE-BLINDED RANDOMIZED

CONTROLLED TRIAL

By

BIANCA NEWMAN

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2019

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© 2019 Bianca Newman

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To my amazing family whose love and support made this possible

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ACKNOWLEDGMENTS

I want to thank my love, Daniel, and my parents for their unconditional love and unwavering

support throughout this journey. I will forever be eternally grateful to my co-residents Claire

Rosebrough, Fred Silva and Wagner Duarte for their encouragement, motivation, and unyielding

support since day one. I thank Dr. Ikramuddin Aukhil for his great mentorship and guidance

through all phases of my project. I would also like to thank Dr. Claire Rosebrough, Dr. Ana Dias

Ribeiro, Janice Braddy, Renita Jenkins, Gabriela Marchegiani, and Aaliyah Stateson for their

help with the clinical portions of my study. I must thank Dr. Ann Progulske-Fox, Dr. Gary

Wang, and Next ScienceTM and their labs for their support and resources. Additionally, I would

like to thank Dr. Rodrigo Neiva, Dr. Jia Chang and Dr. Kesavalu Naidu Lakshmyya for being a

part of my committee and helping me complete this thesis. Lastly, I would like to express my ap-

preciation and gratitude to the faculty and staff of UF Department of Periodontology, especially

Dr. Rodrigo Neiva, for their support and guidance through my periodontal education.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ........................................................................................................... 4

LIST OF TABLES ……………………………………………………………………..…….… 7

LIST OF FIGURES.…………………………………………………………………….……….8

ABBREVIATIONS…………………………………………………….………………………. 9

ABSTRACT ............................................................................................................................... 10

CHAPTER

1 INTRODUCTION ……………………………………………………….…….………..… 12

Biofilm Formation…………………………….……………………………….….….……. 13

Supragingival Plaque………………………….……………………………….….….……. 15

Plaque-induced Gingivitis…………………….…………………….………….….….……. 15

Plaque Index/Gingival Index………………….………………………………..…….……. 17 Treatment of Gingivitis……………………………………………………………..…….…21

Oral Antimicrobials……..…………………….…………………….………….……….…. 22

Study Aim…………………………………………………………………………………...22

2 MATERIALS AND METHODS ……………………………….………………………… 23

Study Design........................................................................................................................ 23

Baseline, Visit 1 ……………………………………………………………………….. 24

Visit 2: Dental Prophylaxis, Oral Hygiene Instruction and Product Dispensation.…..…24

Visit 3: Week 6…………………………………………………………………….……25

Visit 4: Week 12…………………………………………………………………….…..26

Plaque Samples…………………………………………………………………….……26

Inclusion Criteria……………………..…………….…………………………….….….27

Exclusion Criteria………………………………………………………………….……27

Continuance Criteria……………………………………………………………….……28

Identity of Investigational Product(s)…………………………………..………….……29

Product Usage……..……………………………………………………………….……29

Blinding, Labeling and Shipping……………….………………………………….……30

Method of Assigning Subjects to Treatment Groups……………..………………..……31

Determination of Sample Size……………………….…………………………….……31

Safety Observation and/or Measurements………………………………………………31

Oral Examination……………………………………………………………….……….31

Efficacy Variable(s).…………………………………………………………….………32

Hypothesis………..………………………………………………………….….……….32

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Statistics and Analytical Plans………………………………………….…….…………33

Monitoring……………………………………………………………………..………..34

Confidentiality…….…………………………………………………….………………34

Institutional Review…………………………………………………….…….…………34

Potential Risks………………………………………………………….……………….35

Potential Benefits….………………………………………………….…………………35

Obligation of the Investigator…………..…………………………….…………………35

Record Retention….………………………………………………….…………….……36

Study Medication Dispensing, Storage, and Accounting…………….…………….……36

Subject Consent……………………………………………………….…………………36

3 RESULTS ..............................................................................................................................38

4 DISCUSSION ........................................................................................................................44

REFERENCE LIST …………………………………………………………..…………………48

BIOGRAPHICAL SKETCH ………………………….………………………..……………….51

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LIST OF TABLES

Table page

1-1 Study schedule by procedure type and visit.……………………………………….……….23

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LIST OF FIGURES

Figure page

1-1 Scoring criteria for the Turesky plaque index……………………………………………….17

1-2 Diagram of the Turesky plaque index……………………………………………….………18

1-3 Scoring criteria for the Loe and Silness gingival index…………….…………………….….19

1-4 Diagram of the Loe and Silness gingival index………………….…………………………..20

3-1 Comparison of treatment group plaque scores with control plaque scores…........................39

3-2 Confidence and power values for treatment vs. control group ..............................................40

3-3 T-test of plaque values for the Next Scince group at each time point………………………40

3-4 Confidence and Power Values for Next Science treated time points......................................40

3-5 Comparing the gingivitis scores of Next Science to Control at various time points…….….41

3-6 T-test of gingivitis values for the Next Science group at each time points.…………………41

3-7 Confidence and Power Values for Next Science treated at T12………….…………………..41

3-8 T-test for bleeding score Next Science to Control at various time points………….….….…42

3-9 T-test for probing depths Next Science to Control at various time points……………..……42

3-10 Clinical photograph of an adverse effect………..………………………………………….43

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LIST OF ABBREVIATIONS

AAP American Academy of Periodontology

AE Adverse Effects

BOP Bleeding on probing

CPC Cetylpyridinium chloride

CT Connective tissue

GCF Gingival Crevicular Fluid

GI Gingival Index

ICF Informed Consent Form

JE Junctional Epithelium

MGI Modified Gingival Index

MPI Modified Plaque Index

NSPH Non-specific Plaque Hypothesis

OTU Operational Taxonomic Unit

PD Pocket Depth

PDL Periodontal Ligament

PI Plaque Index

PMN Polymorphonuclear neutrophils

SPH Specific Plaque Hypothesis

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Abstract of Thesis Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Master of Science

CLINICAL EFFECTIVENESS OF NEXT SCIENCE ORAL RINSE IN CONTROLLING

PLAQUE AND GINGIVAL INFLAMMATION. A DOUBLE-BLINDED RANDOMIZED

CONTROLLED TRIAL

By

Bianca Newman

May 2019

Chair: Ikramuddin Aukhil

Major: Dental Sciences – Periodontology

Periodontal disease is a multifactorial inflammatory infection. Its microbiological origin

arising from supragingival and subgingival dental plaque and the susceptibility of the host re-

sponse contributes to the destruction of the underlying tissues. Plaque is a bacterial biofilm that

colonizes and adheres to the tooth surface1. The biofilm causes an inflammatory reaction within

the sulcus creating a microbial anaerobic environment2. Non-surgical periodontal therapy has

been proven to be a very effective and integral part of treating periodontal disease for decades.

Through mechanic debridement and reinforcement of oral hygiene instruction, the primary goal

is to reduce the bacterial pathogens and its by-products and ultimately restore and maintain peri-

odontal health. The use of antimicrobial therapy as an adjunctive treatment can aid in reducing

the bacterial loads and improve the clinical signs of oral diseases.

Methods: 118 adult patients with gingivitis were recruited for the study. Baseline measurements

were taken for gingival index3, plaque index4 and probing depth. Supragingival plaque samples

were collected from selected teeth and surfaces. A professional prophylaxis was completed at the

second visit, and mouth rinse bottles were distributed. A random selection was performed so

that half of the patients received the treatment mouth rinse and half of the patients received the

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carrier rinse control. Patients were given standardized toothbrushes and toothpaste and in-

structed to use no additional oral hygiene aids. Rinsing was performed twice a day for 1 minute.

Clinical measurements and plaque sampling were repeated at 6 and 12 weeks.

Results: Clinical data showed a significant decrease in plaque score among the treatment group

(p=0.003). No significant difference was noted for bleeding scores and probing depths. The gi-

ingival index scores showed both groups were statistically equivalent at various time points

when using the t-test analysis. When comparing the time points within the test group, the product

yielded statistically significant improvement (p=0.02) compared to baseline measurements. The

microbial results will be discussed by Dr. Claire Rosebrough.

Conclusions: The Next ScienceTM mouth rinse had a positive effect on plaque reduction and

some gingival improvements by the end of the 12-week period. No significant difference was

seen in the probing depth or bleeding measurements, but both groups did have improvement

from baseline. This is consistent with the fact that both groups were treated with a prophylaxis.

Further investigations are needed to evaluate the long-term effects of the product.

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CHAPTER 1

INTRODUCTION

The oral cavity of an average healthy individual contains hundreds of different bacterial

species, many of which form biofilms that are resistant to mechanical stress2. This environment

of bacterial colonization occurs immediately after brushing the teeth and eventually leads to the

production of a mature biofilm that adheres to the tooth surface. Socransky and Haffajee used the

DNA probes and DNA-DNA hybridization methods to categorize bacterial subgroups in healthy

and diseased populations and found five major bacterial complexes. Of those five, they found

that one complex was related to clinical measurements often seen in periodontal disease. This

complex was termed the “red complex” and consisted of P. gingivalis, B. forsythus (now T. for-

sythia), T. denticola5. The presence of these microorganisms within the sulcus can cause an in-

flammatory reaction and promote the onset of periodontal diseases such as gingivitis and perio-

dontitis. Through longitudinal studies, these microorganisms are known to increase the risk of

disease when harboring in pockets and saliva6. In addition to mechanical debridement, it is sug-

gested that oral rinse solutions can facilitate in controlling the amount of bacterial present and

thereby delaying the process of bacterial biofilm formation. Next ScienceTM has developed an

oral mouth rinse aiming to delay the onset of this process by reducing the bacteria count on the

tooth surface and thereby reducing the risk of gingivitis. This oral rinse has been tested in a pre-

vious in-vitro study comparing its effectiveness to ListerineⓇ and PerioguardⓇ. Their results re-

vealed superior efficacy to Listerine against both subgingival and supragingival bacterial biofilm

formation5. Clinical studies showed significant reduction in plaque scores after 12 weeks of us-

ing the rinse but did not show improvements in the gingival index.

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Biofilm Formation

Early studies investigating the microbiology of disease showed certain trends in microor-

ganism between healthy and inflamed gingiva. Loe H. et al. (1965) wrote a classic paper in

which 12 periodontally healthy patients discontinued oral hygiene and the sequence of changes

in microbiota and gingiva were recorded. The results showed that gingivitis developed in all sub-

jects between 10-21 days7. The process of the bacterial colonization was categorized into three

phases. Initially the gingiva contained cocci and short rods. The second phase began within 2-4

days and showed slender rods, filamentous forms, and cocci still present in large numbers. The

final phase occurred at 6-10 days and found predominantly vibrios and spirochetes. When nor-

mal oral hygiene was re-introduced, the microbiota returned to cocci and short rods7.

The bacterial mass, termed dental plaque, is a structurally complex and organized biofilm

that is formed through a cascade of events. It can produce a variety of irritants, such as endotox-

ins, acids, and antigens, capable of destroying the supporting tissues over time8. In 1975, Lo-

esche described two possible hypotheses to explain the clinical effects of plaque on periodontal

disease: the specific plaque hypothesis (SPH) and the non-specific plaque hypothesis (NSPH).

SPH states that only certain specific pathogens can cause disease9. NSPH states that any accumu-

lation of microorganisms present supra-gingivally or sub-gingivally would produce irritants lead-

ing to an inflammatory response9. That inflammation eventually leads to the destruction of the

periodontium if not treated appropriately. Theilade and Kornman (1986) reported two papers to

further investigate these two hypotheses. They stated that in chronic periodontitis, the gingival

sulcus is colonized by a complex system of over 200 microbial species that contribute to disease

onset and progression. No single species appears in active sites that are not commonly present at

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inactive regions, but the shift of the microbial flora from gram-positive to gram-negative species

can produce the pathogenic potential for periodontal destruction9.

The growth and developmental patterns of bacterial plaque has been studied extensively

using light and electron microscopy and bacterial culture on enamel and dentin surfaces as well

as artificial surfaces such as metal and acrylic materials9. The plaque formation process begins

with the formation of a conditioning film, called the acquired pellicle, on the tooth surface im-

mediately after cleaning of a solid surface in the mouth. Primary colonization is dominated by

facultative anaerobic Gram-negative cocci (mostly Streptococcus sanguis) as they adsorb onto

the pellicle-coated surface. This film also contains salivary glycoproteins and antibodies that al-

ter the charge and free energy of the surface which increases the efficiency of bacterial adhe-

sion8. Through different attachment molecules such as fimbriae and extracellular polymeric sub-

stances, the film becomes a layer of molecules a bacterium can adhere to upon contact and coag-

gregate (Gram-positive facultative cocci and rods). At this time, the surface receptors on the de-

posited Gram-positive cocci and rods allow subsequent adherence of Gram-negative organisms

with poor ability to attache directly to the pellicle8. The bacterial mass will begin to increase and

multiply due to the continued adhesion of new bacteria (Fusobacterium nucleatum and

Prevotella intermedia). As the mass grows, an oxygen gradient is created allowing anaerobic

bacteria to thrive at the base of the growing biofilm8. As plaque becomes more mature, more

Gram-negative strictly anaerobic bacteria (Porphyromonas gingivalis and Capnocytophaga sp.)

colonize and contribute to an increased pathogenicity of the biofilm.

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Supragingival Plaque

The microbial biofilm formed on the tooth surface may occur at the gingival margin, su-

pragingivally, and/or below the gingival margin, subgingivally. Supragingival plaque has been

thoroughly examined in a number of studies for decades. In studies looking at the composition

and structure of plaque, samples are required in which the deposits of plaque are kept in their

original relation to the surface where they had developed8. This was done by light and electron

microscopy in order to gain information on the bacteria’s internal structure8. Originally devel-

oped in 1911 by Dr. G.V. Black, this method of plaque collection was reintroduced during the

1950s using artificial surfaces, such as plastic materials, for the collection of plaque8. This re-

search provided the tools needed to achieve the resolution to identify the substructure of plaque

and the specific bacterial composition of plaque. Supragingival plaque is often present on the

tooth surface but if not removed over a period of time, the flora will migrate subgingivally, af-

fecting the underlying gingival tissues. The inflammatory response from the host and the toxic

bacterial products will eventually affect the alveolar bone supporting the tooth.

Plaque-Induced Gingivitis

To understand the pathogenic changes that occur within the sulcus of a tooth, it is im-

portant to understand the anatomy and clinical appearance of the gingiva in its healthy state. The

periodontium anatomy consists of the oral surface of the gingiva consisting of keratinized oral

epithelium which becomes sulcular epithelium as it reaches the inner lining of the sulcus. The

apical portion of the sulcus is the junctional epithelium that is attached to the tooth by hemi-

desmosomes. Supporting the oral and junctional epithelia is the attachment network of connec-

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tive tissue consisting primarily of collagen fibers8. Clinically “healthy” gingiva consistently fea-

tures a small infiltrate of inflammatory cells that are within the junctional epithelium (JE) and

connective tissue (CT) attachment10. This response is a reaction to continuous presence of bacte-

ria and bacterial by-products. The host’s defense system may be able to protect the host from the

onset of disease depending on each individual’s susceptibility. Page and Schroeder (1976)

staged the pathogenesis of gingivitis into four distinct phases: initial, early, established, and ad-

vanced lesions. The initial lesion occurs within the first 2-4 days of bacterial invasion and in-

volves the junctional epithelium and the most coronal portion of the connective tissue attach-

ment. During this phase, the vessels subadjacent to the JE start to dilate resulting in tissue edema,

loss of perivascular collagen, and an increase in gingival crevicular fluid (GCF). Polymorphonu-

clear neutrophils (PMNs) cell migration to the affected sites are facilitated with macrophages and

T-lymphocytes. Beginning around days 4-7 and lasting through day 14 after initial plaque accu-

mulation, the site develops into the early lesion phase. The vessels in the dentogingival plexus

remain dilated, and the capillary beds opening. At this stage of gingivitis, the lymphocytes and

PMNs are the predominant infiltrates with very few plasma cells present. The basal cells also

begin to proliferate within the JE along the root surface. As the exposure to the bacterial plaque

continues to further enhances the inflammatory reaction of the gingival tissues, the site enters the

established lesion phase. The specific timing of this lesion depends on the host’s susceptibility

but will usually occur at 2-3weeks. As defined by Page and Schroeder (1976), this lesion has an

increased number of leukocytes and is predominantly comprised of plasma cell infiltrates. The

proliferation, apical migration, and lateral extension of the JE occurs with further loss of collagen

resulting in collagen-depleted spaces extending deeper into the tissues. This region can now oc-

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cupy available infiltration of leukocytes. Two types of established lesion appear to exist:one re-

mains stable and does not progress for months or even years11,12, while the other becomes more

active and progressed to the destructive advanced lesion. As the pocket deepens, the biofilm con-

tinues its apical migration and flourishes in the anaerobic environment eventually causing loss of

connective tissue attachment and alveolar bone. This phase is often referred to as the condition,

periodontitis.

Plaque Index/Gingival Index

Figure 1-1. Scoring criteria described by Turesky et al.28 in 1970. This index was used to score

plaque after the use of a disclosing solution at the baseline, week 6 and week 12 visits.

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Figure 1-2. Diagram of the plaque index proposed by Turesky et al.4 in 1970. This index was

used to score plaque after the use of a disclosing solution at the baseline, week 6 and week 12

visits.

To quantify the plaque for each patient, we used the modified Quigely Hein Index4 which

was modified by Turesky et al. in 1970. This plaque index is scored from 0 to 5 and was evalu-

ated on the buccal and palatal/lingual surfaces of each tooth included in the study (exclusion cri-

teria included teeth with crowns, teeth with restorations extending to the gingival one-third of

tooth crown or teeth with orthodontic appliances). Plaque was disclosed with a pink disclosing

solution which was applied to each tooth surface using a cotton tip applicator. The disclosing so-

lution was thoroughly rinsed with water and plaque scores were recorded. The scoring was com-

pleted using the following values:

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0: No visible plaque

1: Isolated flecks of plaque at the gingival margin

2: Thin continuous band of plaque at the gingival margin (≤1mm)

3: Band of plaque at the gingival margin that is wider than 1mm but covering less than one-third

of tooth crown

4: Plaque covering at least one-third but less than two-thirds of tooth crown

5: Plaque covering two-thirds or more of the crown of the tooth

Figure 1-3. Scoring criteria for the gingival index, proposed by Loe and Silness3 in 1963. This

index was used to grade gingival health at the baseline, week 6 and week 12 visits.

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Figure 1-4. A pictorial description of the gingival index, proposed by Loe and Silness3 in 1963.

This index was used to grade gingival health at the baseline, week 6 and week 12 visits.

To evaluate each patient’s gingival health, the gingival index3 proposed by Loe and Sil-

ness in 1963 was used. The probe is gently inserted ≤1mm below the gingival margin and run

mesiodistally under the gingival margin on both the buccal and lingual. Gingival inflammation is

quantified on a scale of 0 to 3 with the following criteria:

0: Absence of inflammation with no bleeding

1: Mild inflammation, slight color gingival color change, no bleeding

2: Moderate inflammation, redness, edema and bleeding after probe insertion

3: Severe inflammation, marked redness, gingival ulceration, spontaneous bleeding after probe

insertion

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Treatment of Gingivitis

The main goal of periodontal therapy is the preservation of the natural dentition by

achieving and maintaining a healthy functional periodontium. This can be accomplished through

the elimination of bacterial plaque through therapy. Non-surgical therapy is the first approach

when attempting to treat periodontal disease. This consists of mechanical removal of the su-

pragingival and subgingival bacterial pathogens present, patient motivation for home care, and

oral hygiene instructions. By reducing the periodontal pathogens or reducing to below the “criti-

cal mass”13, an equilibrium can be established between the remaining normal flora.

The disruption of the colonized microbial plaque build-up is necessary in order to reduce

and eliminate the pathogens and their byproducts and ultimately restore periodontal health. Sonic

and ultrasonic instrumentation can be utilized during debridement. They can be air driven and

have vibrations to help break up the plaque or calculus deposits. Two types of ultrasonic units

include magnetostrictive (elliptic vibration) and piezoelectric (linear vibration). Ultrasonic ther-

apy is usually followed by manual instrumentation which includes instruments such as: curettes,

scalers, files and hoes. Scalers have pointed tips and are utilized for supragingival plaque and

calculus removal while curettes have rounded toes and are utilized for subgingival use. Although

mechanical removal of the periodontal pathogens is necessary part of therapy, patient motivation

and oral hygiene instruction are also very important to maintain health8.

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Oral Antimicrobials

Another adjunctive form of periodontal therapy involved the use of locally delivered anti-

microbials. The rationale for their use is based on the following: pathogens are difficult to reach

(deep vertical defects, furcations, etc.) in addition to adverse reactions and poor patient compli-

ance that may occur with systemic antibiotic administration. Examples of locally delivered de-

vices include PerioChip (chlorhexidine gluconate), Atridox (Doxycycline), Arestin (Minocy-

cline), and Elyzol (Metronidazole gel).

PerioChip is a product known for its ability to inhibit plaque formation is due to its bind-

ing to anionic acid groups on salivary glycoproteins, which reduce the pellicle formation and

plaque colonizations and by binding to salivary bacteria and interfering with adsorption to

teeth14. The efficacy of PerioChip was used as an adjunct to scaling and root planing in two dou-

ble-blind, randomized, placebo-controlled multicenter clinical trials. After 9 months, significant

reductions in probing depths and clinical attachment levels were seen in the Chlorhexidine group

compared to the mechanical control group15.

The advantages of a local delivery device include prolonged drug levels within therapeu-

tic range, minimal systemic adverse reactions and improved in patient compliance. Treating peri-

odontal disease with adjunct antimicrobials can enhance the results in sites than may not respond

well to conventional therapy alone.

Study Aim

In the present study, Next ScienceTM aims to further investigate the clinical and microbi-

ological effects of the oral rinse compared to a placebo rinse on patients with gingivitis.

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CHAPTER 2

MATERIALS AND METHODS

Study Design

In this randomized, controlled and double-blind plaque and gingivitis prevention study,

132 subjects were seen at the Baseline Visit in order to enroll approximately 100 healthy adult

volunteers with gingivitis. All subjects received a full-mouth dental prophylaxis and were ran-

domized to either the control or the test group. The effects of the oral rinse on biofilm formation

were monitored by plaque index (PI) scores, gingivitis index (GI) and composition of oral micro-

bial flora through supragingival plaque sampling. Gingival inflammation and bleeding were as-

sessed clinically after 6 and 12 weeks of product use.

Table 1-1. Study Schedule by Procedure Type and Visit

Procedure

Baseline

Visit 1

Oral Prophy-

laxis

Visit 2/Product

Dispensation

Week 6

Visit 3

Week 12

Visit 4

Informed Consent X

Medical History Review X

Demographics X

Inclusion/Exclusion Criteria X

Oral Examination X X X X

Gingivitis Examination/Scoring X X X

Plaque Collection/Scoring X X X

Dental Prophylaxis X

Product Distribution X X

General Comments X X X X

Adverse Effects X X X

Subject Accountability X

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Visit 1: Baseline

Prior to this visit subjects were asked not to perform any oral hygiene the morning of the

visit. They were also asked not to use medicated lozenges, breath mints, toothpicks, eat, drink

(except water), smoke and/or chew gum the morning of the study visit. Subjects were asked to

read and sign an informed consent and given a signed copy. Demographic information and en-

trance criteria were assessed. Eligibility of the participants were confirmed. If subject did not

qualify, he/she was exited from the study without compensation. Ineligible subjects’ personal

information was reviewed and retained as site source documentation. If subject met all inclusion

and none of the exclusion criteria, a periodontal charting for pocket depths (PD), plaque scores

as well as gingival index (GI) and bleeding scores were recorded. Plaque samples were collected

from the following four sites- mesial surface of maxillary first molar on left side, distal surface

of maxillary first premolar on right side, mesial surface of mandibular second molar on left side

and distal surface of mandibular lateral incisor on right side. These samples were collected from

the supragingival tooth surface with a sterile curette. All four plaque samples were placed in the

same tube. One tube of plaque was collected per patient per visit. If any of these four teeth are

missing, the next tooth mesial to it was used to collect the plaque sample. General comments, if

applicable, were recorded. Subjects meeting the selection criteria was scheduled for the second

visit.

Visit 2: Dental Prophylaxis, Oral Hygiene Instruction and Product Dispensation

Subjects received a whole-mouth dental prophylaxis by the dental hygienist. For logisti-

cal reasons, the Dental Prophylaxis visit occurred within 2 weeks of the Baseline Visit. Follow-

ing dental prophylaxis, an oral examination was conducted. This visit included instructions on

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oral hygiene, dispensation of toothbrush/toothpaste and the oral rinse product. The experimental

oral rinse product and the placebo provided by the manufacturer was fully coded in prepacked

bottles with the code numbers. Subjects were instructed to brush their teeth and rinse their mouth

with the dispensed product twice daily (AM/PM) for 1 minute. A sheet of paper with pre-marked

dates was provided to each subject for them to record the time of the day they rinsed and dura-

tion. Subjects were asked not to perform any oral hygiene the morning of their next visit. They

were also asked not to use medicated lozenges, breath mints, toothpicks, eat, drink (except wa-

ter), smoke and/or chew gum the morning of the next study visit.

Visit 3: Week 6

Subjects were asked if they had performed any oral hygiene the morning of the visit.

They were also asked if they had used medicated lozenges, breath mints, toothpicks, eaten, had

anything to drink (other than water), smoked and/or chewed gum the morning of the study visit.

The paper sheet with recording of their rinsing habits were collected. An oral examination was

conducted, an assessment of PI and GI scores occurred with periodontal charting for PDs, plaque

scores as well as GI and bleeding scores. Dental plaque samples were collected from the same

sites as the prior appointment. Any untoward experiences and findings were recorded (example-

change in taste sensation, any ulcers, blisters, etc.). New paper sheet to document their daily use

of oral rinse were provided. Subjects were asked not to perform any oral hygiene the morning of

their next visit. They were also asked not to use medicated lozenges, breath mints, toothpicks,

eat, drink (except water), smoke and/or chew gum the morning of the next study visit.

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Visit 4: Week 12

Subjects were asked if they had performed any oral hygiene the morning of the visit.

They were also asked if they had used medicated lozenges, breath mints, toothpicks, eaten, had

anything to drink (other than water), smoked and/or chewed gum the morning of the study visit.

The paper sheet with recording of their rinsing habits were collected. Full mouth periodontal

charting to include plaque and bleeding scores as well as GI were recorded and dental plaque

samples were collected as before (from the same sites). Any untoward experiences and findings

were recorded (example- change in taste sensation, any ulcers, blisters, etc.). Patients were in-

formed of any dental treatment needs and general comments and Adverse Effects, if applicable,

were recorded. A subject accountability form was completed and subjects were dismissed from

the study. A subject accountability form was also be completed for subjects who drop out of the

study prior to its completion.

Plaque Samples

Plaque samples were immediately placed on ice after collection from each patient. Fol-

lowing each clinical session, samples were taken to Dr. Gary Wang’s lab at the University of

Florida College of Medicine. Samples were frozen until time of analysis.

Plaque samples were treated with a solution to lyse bacterial cells followed by PCR am-

plification. Bacteria in the plaque samples were sequenced using 16s rRNA. Variable regions

within rRNA V1-V3 regions were used to identify the bacterial strains. Next, mapping was used

to attach the name of each organism to its sequence. The total number of sequences found for

each organism provided data regarding the size of that bacterial population.

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Inclusion Criteria

In order to be included in the study, each subject must

• be between the ages of 18 and 60;

• provide written informed consent prior to participation and be given a signed copy of the in-

formed consent form (ICF);

• be in good general health as determined by the investigator/designee based on a review of the

Medical history/update for participation in the study;

• have at least 20 gradable teeth;

• have 10 or more bleeding sites at Baseline.

Exclusion Criteria

Subjects are excluded from study participation where there is evidence of

• known allergies or sensitivity to oral rinse products, especially those containing cetylpyri-

dinium chloride (CPC), cinnamaldehyde, sodium hydroxide or potassium dihydrogen phos-

phate;

• severe periodontal disease, as characterized by purulent exudate, generalized mobility, and/or

severe recession;

• active treatment for periodontitis;

• bleeding disorder or using a blood thinner;

• orthodontic appliances;

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• diabetes;

• antibiotic use within three months of the Baseline, Visit 1;

• pregnancy (If you are a female of child-bearing age you will be asked at each visit if you are

pregnant);

• any diseases or conditions that could be expected to interfere with the subject safely complet-

ing the study.

Continuance Criteria

Subjects may be excluded from the study or the analysis due to

• use of antibiotics any time during the study;

• use of any non-study oral hygiene products during the study;

• a non-study dental prophylaxis or other elective dentistry during the study.

In addition, subjects would be discontinued from the trial at subject’s request, or if the investiga-

tor determined that their well-being or safety was at risk or for non-compliance or study viola-

tions. The reason for discontinuation would be documented in the research chart for any subject

that was withdrawn from the trial

29

Identity of Investigational Product(s)

• Test: Next ScienceTM OTC Oral Rinse with Essential Oils

(Supplier Sigma Aldrich - Cetylpyridinium chloride monohydrate, meets USP testing

specifications, Cinnamaldehyde certified food grade product, Puriss sodium hydroxide, meets

analytical specification of Ph. Eur., BP, NF, E524, 98-100.5%, Puriss Potassium phosphate mon-

obasic, anhydrous, ACS reagent, ISO, Ph. Eur., 99.5-100.5%., Menthol, racemic, >.98%, Euca-

lyptol, Methyl Salicylate, Thymol, Ethanol.)

(Supplier B. Braun - Sterile Water for Irrigation USP)

• Control: OTC Oral Rinse Control

(Supplier Sigma Aldrich – Cinnamaldehyde, certified food grade product

(Supplier B. Braun – Sterile Water for Irrigation USP)

Product Usage

Subjects were instructed to brush thoroughly with the toothbrush and toothpaste provided

and rinse with water after brushing. Each were instructed to rinse with 20 mL (volume was

measured using a dose cup provided) of oral rinse for 1 minute (rinsing was timed using the

timer provided). Subjects were instructed to brush and rinse twice daily and to use the product in

place of normal oral hygiene for the duration of the study. Flossing was not permitted for the du-

ration of the study.

30

Blinding, Labeling and Shipping Plan

The identity of the experimental oral rinse and its placebo was not disclosed to the clini-

cal investigators. Test and control kit boxes each contained a toothbrush, a toothpaste, dose cups

and a timer. The kit boxes were labeled with a unique kit number representing the test product or

placebo known only to the manufacturer. The kit box labels contained the study number, emer-

gency phone number, distributor name/address, appropriate caution statements, content statement

and other information as required by internal regulations and clinical SOPs. Kit box content

statement will be worded to maintain the study blind.

The site will be provided with the code breaker report in a sealed envelope. The sealed

code breaker report contains documents that list the kit box number or treatment code. If the

study blind needs to be broken, an individual subject’s investigational product may be ascer-

tained by opening the sealed code breaker report, locating the subject’s kit box number and treat-

ment code and matching the identity. The sealed code breaker report will be opened only if a

clinically serious AE occurs or management of the subject requires knowledge of the identity of

the investigational product. The investigator will immediately inform the Sponsor that the code

will be broken and record the date, time and reason for breaking the code in writing. After the

study is complete and the study database has been finalized and locked, the site will return the

code breaker report to the Sponsor using the self- addressed stamped envelope provided by the

sponsor.

31

Method of Assigning Subjects to Treatment Groups

The sponsor will provide adequate supplies of the oral rinse solution containing either the

experimental or placebo compounds. The investigators will be unaware of the identity of the bot-

tles. Enough oral rinse will be provided to last 12 weeks.

Determination of Sample Size

Assuming the dropout rate does not exceed 10%, finishing with a total of 90 subjects, 45

subjects per treatment group, should provide at least 80% power in 1-sided testing (alpha=5%) to

detect a difference between the Test oral rinse and the placebo groups. This assumes that the ef-

fect size (treatment difference divided by within subject standard deviation) is at least 0.528.

Safety Observations and/or Measurements

Safety will be assessed by the absence of irreversible side effects. Whole body adverse

events will be collected only if related to product use.

Oral Examination

Assessment of the oral soft tissue is conducted via a visual examination of the oral cavity

and perioral area utilizing a standard dental light, dental mirror, and gauze. The structures exam-

ined included the gingiva (free and attached), hard and soft palate, oropharynx/uvula, buccal mu-

cosa, tongue, floor of the mouth, labial mucosa, mucobuccal/mucoliabial folds, lips, and perioral

area. Assessment of the oral hard tissues will be conducted via a visual examination of the denti-

tion and restorations utilizing a standard dental light, dental mirror, and air syringe. All abnor-

mal findings will be recorded and categorized by their location: hard tissue findings will be cate-

32

gorized as “other”. An AE will be recorded if a new abnormal finding is noted after product dis-

pensing or any previously noted abnormal finding increases in severity during the treatment pe-

riod.

Efficacy Variable(s)

Plaque and bleeding scores as well as gingival index will be indicative of improvements

in biofilm accumulation and consequent gingival inflammation. At baseline, Visits 3 and 4 we

will be assessing the plaque and bleeding scores as well as gingival index of participants. Finally,

plaque samples collected from four selected sites at baseline, immediately, at 6 and 12 weeks af-

ter oral prophylaxis will be analyzed by PCR-based HOMIM (Human Oral Microbe Identifica-

tion MicroArray). This HOMIM analysis is an accurate qualitative and quantitative assay for

analysis of oral microbial ecology at various stages of this study (baseline, 6 and 12 weeks after

the prophylaxis). Collectively, clinical parameters (plaque/bleeding scores/gingival index), sali-

vary diagnostics (microbial flora and proteomics) and plaque analysis by HOMIM will provide

definite data on the effectiveness of the experimental oral rinse in reducing biofilm formation

and its harmful effects (inflammation) over the placebo.

Hypotheses

The following hypotheses will be tested at 6 and 12 weeks:

Primary Hypotheses:

Null The mean plaque, gingival and bleeding scores for the Test Rinse group are

lower than the mean for the Placebo Rinse group.

33

Alternative The mean plaque, gingival and bleeding scores for the Test Rinse group

are the same as the mean for the Placebo Rinse group.

Null The mean number and composition of the plaque microorganisms implied

in gingival inflammation for the Test Rinse group are lower and more

leaning towards a health-associated flora than the mean for the Placebo

Rinse group.

Alternative The mean number and composition of the plaque microorganisms implied

in gingival inflammation for the Test Rinse group are the same as the

mean number and composition of the plaque microorganisms implied in

gingival inflammation for the Placebo Rinse group.

Statistical and Analytical Plans

Whole-mouth average plaque, gingival and bleeding scores were calculated for each sub-

ject separately at Baseline, week 6 and week 12, by summing the gradable site-level plaque and

bleeding scores and dividing by the number of gradable sites. Analysis of covariance will be

used to model the adjusted treatment group means in whole-mouth average scores with the Base-

line score used as the covariate. The number and types of periodontal disease-associated plaque

microorganisms were analyzed in the similar way. If the model assumptions were not satisfied,

the data was mathematically transformed prior to analysis or nonparametric methods applied.

Additional analyses were performed to better understand the data. All treatment comparisons

were 1-sided with the significance level set to 5%.

34

Monitoring

During the course of the trial, the clinical site was monitored by Next ScienceTM to ensure

compliance with the protocol, regulations and guidelines, adequacy of the equipment and facili-

ties, and satisfactory data collection. The study was conducted in accordance with the ethical

principles of the Declaration of Helsinki and consistent with Good Clinical Practice and applica-

ble laws and regulations. Harmonization’s Good Clinical Practice Consolidated Guidelines.

[ICH-GCP’s, as published by the FDA on 9 May 1997, Federal Register, Volume 62, Number 90

pages 25691 – 25709]).

Confidentiality

All subjects were given information regarding the research site’s privacy policy and

signed the HIPAA authorization prior to participation in the study. Subjects were not individu-

ally identified in any publications that result from this study. Individual patient’s medical infor-

mation obtained as a result of this study was considered confidential and was not disclosed to

parties other than those noted below. With the subject and/or guardian’s permission, medical in-

formation was shared with the subject’s personal physician or other medical personnel responsi-

ble for their welfare. Data generated by this study was made available for inspection upon re-

quest by representatives of the U.S. FDA, local and national health authorities, the Sponsor or its

representatives, and by the IRB.

Institutional Review

Prior to study initiation, the investigator obtained institutional review and approval of

both the Protocol and the consent form, in compliance with the US Code of Federal Regulations.

35

Title 21, Part 56 or the ICH-GCP’s Consolidated Guidelines. Chapter 3. The investigator main-

tained any original authorization letter(s) and forwards copies to Next ScienceTM. IRB approval

letters included the study title, study number, the address of the IRB, date of request, and the sig-

nature of the IRB chairperson/designate. Additionally, the letter acknowledged that both the

Protocol and consent form have been approved by the IRB, with notification of any changes re-

quired. The study did not begin until Principal Investigator had received written confirmation of

IRB approval. The investigator notified the IRB when the study was completed.

Potential Risks

There were no known systemic risks with the utilization of The Next ScienceTM mouth

rinse when used in accordance to their indication and instructions for use. The products were not

be used in subjects known to have allergic reaction to one or more ingredients.

Potential Benefits

Management and control of plaques, gingivitis and bleeding is a key component of oral

health. Participant was presume to benefit from the oral prophylactic treatment and in addition

benefit from improved oral health from the potential reduction in plaques, gingivitis and bleed-

ing.

Obligation of the Investigator

Following completion of the study, the investigator submited a final report within 30 days

to Next ScienceTM describing the conduct of the study, deviations from planned conduct, early

withdrawals, and subject accountability, adverse events, and other information on study conduct

necessary for full interpretation of collected data.

36

Records Retention

The investigator retained the subject identification codes, informed consent documenta-

tion, clinical materials, inventory, CRF’s, medical records and other source data for a minimum

of 2 years after the last regulatory approval has been received or the discontinuation of the study.

The investigator received written authorization from the Sponsor before destroying any study

document. The investigator made the records available for inspection and copying upon the re-

quest of an authorized employee of a government authority or the Sponsor, at reasonable times.

In the event the investigator retires, relocates, or for any other reason withdraws from the respon-

sibility for maintaining records for the period of time required, custody of the records were trans-

ferred to another person who accepted responsibility for the records. Notice of such a transfer

was given in writing to the Sponsor.

Study Medication Dispensing, Storage and Accounting

Study products were stored in a secure area, under environmental condition as required

by label instructions or as described in the Protocol, and dispensed only under the authorization

of the investigator. The storage condition was properly documented. Both the receipt and dis-

pensation of all test products (used and unused) were documented using forms provided by Next

ScienceTM or suitable forms provide by the site provided the site had an existing SOP for the de-

struction of clinical materials and prior written approval from Next ScienceTM.

Subject Consent

The Investigator obtained written informed consent for each subject prior to that subject’s

participation in the study, per the US Code of Federal Regulations, Title 21, Parts 50.25 and

50.27 and ICH-GCPs. Chapter 4, subpart4, 8. Subjects, or their legal guardian, were required to

37

read, sign and date an IRB approved consent form with the investigator also maintaining a signed

and dated copy. The subject or legal guardian were given a copy of the consent form. All study

procedures were explained in non-technical terms.

38

CHAPTER 3

RESULTS

A total of 80 subjects completed this study from the original 132 patients (ages 18-60

years old) screened for eligibility. Fourteen patients did not qualify based on the inclusion and

exclusion criteria. A total of 38 patients dropped out due to either adverse effects of the oral rinse

or inability to complete the duration of the study. By the end of the trial period (12 weeks), 39

patients had received the Next ScienceTM oral rinse and 41 patients received the control placebo.

Clinical Results

After using t-test analysis, the plaque index, gingival index, and bleeding, there were no

statistically significant findings between the control and test groups for gingivitis scores from

baseline (T0) to 12 weeks (T12). There was however, statistically significant improvement in

plaque between the treatment and placebo group from baseline to 6 weeks (T6) and baseline to 12

weeks. Table 3-1 displays that both groups were equivalent at T0 for plaque score, however at 6

weeks and 12 weeks, the Next ScienceTM oral rinse group yielded a statistically superior result at

p-values of 0.01 for T6 and 0.003 for T12. The treatment groups also had statistically significant

improvements in gingival index and bleeding from baseline to 12 weeks.

39

Plaque Scores

Time Control Next Science

(weeks) Plaque Score St. Dev. # Plaque Score St. Dev. # t-test

0 2.58 0.43 59 2.61 0.45 59 0.735

6 2.55 0.43 42 2.32 0.38 40 0.011

12 2.61 0.38 41 2.34 0.40 39 0.003

Figure 3-1. Comparing the plaque score of the Next Science TM to Control at various time points.

To determine the power of these results, the power analysis was performed using the dif-

ferences between these results and standard deviation. Equation used to calculate power: Power

for mean 1 = mean 2 + difference α = 0.1. The calculations were performed in MiniTab, resulting

in the power at T0 being 0.123986, T6 being 0.858213, and T12 power was 0.911091. Table 3-2

represents the confidence and power values for Next ScienceTM vs. Control and revealed the Next

ScienceTM product to be superior to the Control product at all three phases of the trial. Lastly,

when comparing the plaque values of the test group at different time points using the t-test analy-

sis, the results showed that the product yielded statistically significant improvements at T6 and

T12 compared to T0. The p-values at T6 was 0.001 and T12 0.003 (Table 3-3). To determine the

power of these results, the same power analysis was conducted revealing the power at T6 was

0.947558 and T12 was 0.904763. Table 3-4 shows the confidence and power values for the Next

ScienceTM test group treated at specific time points and found statistically significant improve-

ments.

40

Time Next Science Vs. Control

(weeks) Confidence/Power

0 90/10

6 90/85

12 90/90

Figure 3-2. Confidence and Power Values for Next ScienceTM vs. Control

Time Plaque Score St. Dev. # t-test to T0

0 2.61 0.45 59

6 2.32 0.38 40 0.001

12 2.34 0.40 39 0.003

Figure 3-3. T-test of plaque values for the Next ScienceTM group at each time point

Time To Versus Time

(weeks)

Confi-

dence/Power

6 90/95

12 90/90

Figure 3-4. Confidence and Power Values for Next ScienceTM treated time points

Gingivitis Scores

Table 3-5 displays that both groups were equivalent for the gingivitis score at T0, T6, and

T12, revealing that there was no statistically significant difference between the test group and

control group. The treatment group did, however, have statistically significant improvements (p-

value of 0.02) in gingivitis scoring over time (Table 3-6). To determine the power of these re-

sults, the same power analysis was conducted revealing the power at T12 was 0.373711. Table 3-

7 shows the confidence and power values for the Next ScienceTM test group comparing T0 to T12

41

showing that the power of this test was low. This low power indicates that more samples would

be required to demonstrate a 90/80 or higher confidence and power.

Time Control Next Science

(weeks)

Gingivitis

Score St. Dev. #

Gingivitis

Score St. Dev. # t-test

0 1.06 0.37 59 1.00 0.29 59 0.294

6 0.97 0.41 42 1.02 0.26 40 0.471

12 0.85 0.37 42 0.87 0.42 37 0.890

Figure 3-5. Comparing the gingivitis score Next Science TM to Control at various time points.

Time Gingivitis Score St. Dev. # t-test to To

0 1.00 0.29 59

6 1.02 0.26 40 0.69

12 0.87 0.42 37 0.02

Figure 3-6. T-test of gingivitis values for the Next ScienceTM group at each time point

Time To Versus Time

(weeks) Confidence/Power

12 90/37

Figure 3-7. Confidence and Power Values for Next ScienceTM treated at T12

42

Bleeding Scores and Probing Depths

When comparing the control and test group for the bleeding scores (Table 3-8) at various

time points using the t-test, the two populations were statistically equivalent at all time points in-

dicating no statistical improvement. Both groups showed slight improvement over time. The re-

sults of probing depths (Table 3-9) showed the two groups were statistically equivalent at all

time points of the study.

Time Control Next Science

(weeks) Bleeding St.

Dev. # Bleeding St. Dev. # t-test

0 1.10 0.72 59 0.99 0.62 59 0.38

6 0.81 0.71 41 0.81 0.61 40 1.00

12 0.76 0.58 40 0.71 0.51 39 0.68

Figure 3-8. T-test of bleeding score Next Science TM to Control at various time points

Time Control Next Science

(weeks) Pocket Depth St. Dev. # Pocket Depth St. Dev. # t-test

0 2.5 0.8 9728 2.5 0.8 9688 0.060

6 2.4 0.8 6899 2.4 0.7 6464 0.442

12 2.3 0.8 6732 2.3 0.8 6448 0.111

Figure 3-9. T-test of probing depths Next Science TM to Control at various time points

The microbial analysis will be discussed in a separate paper by Dr. Rosebrough.

43

Adverse Effects

Throughout the duration of the 12-week trial period, there were some adverse effects pa-

tients within the control group had experienced and for some, was the reason for discontinuing

the study. Signs of these effects included small, white round granules on the attached gingival

and mucosal regions (Figure 3-1). Other complaints included painful sensation of the gingiva

and/or tongue, ulcers, loss of taste, staining, sloughing of the gingiva, burning sensation of the

tongue, and irritation of the gingiva, all of which returned to normal upon discontinued use of

oral rinse. Upon discontinuation of the placebo oral rinse, the signs and symptoms disappeared

and returned to normal. A total of 20 subjects dropped the study due to these findings. 18 sub-

jects dropped out of the study due to lost of follow up.

Figure 3-10. Clinical intra-oral photograph of adverse effects of the placebo oral rinse.

44

CHAPTER 4

DISCUSSION

Through the course of the study, gingival index scores improved for all patients. There

was no significant difference between patients in the control versus treatment group, but this im-

provement may be attributed to the prophylaxis that each patient received after the baseline visit.

The amount of dental visits of each patient prior to this study was widely variable and many pa-

tients did not have any professional cleanings in many years. For this reason, the prophylaxis

may have been a contributing factor to this study’s clinical results.

The benefits of dental prophylaxis for improvement in gingivitis are widely researched

over the years. In 1986, Loe et al. investigated experimental gingivitis in 12 healthy patients with

healthy gingiva by withdrawing of all active effects directed towards oral hygiene and studied

the sequence of changes in the microbial flora. The same gingival index (Loe and Silness 1963)

and plaque index (Silness and Loe 1964) used in the present study was used to evaluate their

clinical findings, and they found that after 21 days of abstained oral hygiene practice, evidence

of gingivitis developed. Once oral hygiene was re-established, the condition of their gingival tis-

sue returned to their state prior to the start of the study.

Furthermore, home oral hygiene may have improved because the patients were aware that

they were participating in a dental study and they knew that we would be evaluating their plaque

scores. No calibration was completed for oral hygiene habits aside from the distribution of stand-

ardized toothbrushes and toothpaste for usage for the duration of the study.

Clinically, this study found a significant difference between the treatment group and the

placebo rinse group in plaque reduction at week 12. The test group had a significantly lower

45

plaque index (p-value: 0.003) compared to the baseline measurements, which may be partially

due to the patients heightened awareness of participating in a dental study. This change in behav-

ior called the Hawthorne effect often occurs when a subject of a study is aware of being ob-

served. There was no calibration of the subjects’ practice of oral hygiene which may have an in-

fluence on the effectiveness of their final results. Standardized toothbrushes and toothpastes were

given to every patient to normalize their hygiene tools used and to minimize possible chemical

adverse reactions they may have for the duration of the study.

This study revealed that adjunct mouth rinse can be beneficial to plaque control and the

clinical signs of inflammation. Al-Kamel et al. (2019) conducted a study comparing N-acetyl

cysteine to Chlorhexidine mouth rinses to prevent and treat experimental gingivitis. They ran-

domly assigned 60 subjects to three groups: N-acetyl cysteine; Chlorhexidine, and placebo. After

professional prophylaxis was conducted at baseline, the subjects were instructed to stop oral hy-

giene practice and begin rinsing twice a day. They did this to assess each mouth rinses effective-

ness to prevention of inflammation. At 21 days, a subgroup was selected and assigned to either

test mouth rinse groups to assess its effectiveness to treatment. They found that all groups accu-

mulated plaque and developed some degree of gingivitis, with the placebo group accumulating

the most. The N-acetyl cysteine group demonstrated slight preventative properties at 14 and 21

days. Similar to the present study, the chlorhexidine group showed reduction in plaque and in-

flammation, while the N-acetyl cysteine group resulted in insignificant reductions. These find-

ings reveal that mouth rinses can have a beneficial effect on plaque accumulation27.

Chlorhexidine as an adjunct to periodontal therapy can often have adverse effects to the

gingival tissues and taste buds. Greenstein et al. (1986) found staining in approximately 50% of

the sample within several days28.

46

In the periodontal classic literature, there are two opposing theories on the origin of

plaque: The non-specific plaque hypothesis and the specific plaque hypothesis. The non-specific

plaque hypothesis states that the quantity of plaque is responsible for the development and pro-

gression of periodontal disease. Under this theory, the reduction in the critical mass of plaque

caused by the Next Science TM mouth rinse would have improved the clinical measurements in-

cluding bleeding, inflammation, and redness in the test group. In this study, no significant differ-

ence was noted in gingival index between the treatment and control groups at week 12. This is

more consistent with the specific plaque index and shows that decreasing the critical mass of

plaque does not correlate with improvement in disease indicators.

The specific plaque hypothesis, proposed by Loesche (1976), states that the development

of periodontal breakdown arises from specific types of bacteria present in the plaque biofilm.

The accumulation of these pathogenic microbia produce harmful virulence factors, giving them

the increased ability to activate the host immune response. Treatment for the specific plaque hy-

pothesis is aimed to reduce or eliminate of these specific microorganisms18.

One limitation of the study is the individual components of the mouth rinse are already

proven to be effective against plaque and gingivitis. Cetylpyridinium chloride (CPC) is a cationic

quaternary ammonium compound with antiseptic and surfactant properties. It has an antimicro-

bial effect through the disruption of bacterial cell membranes resulting in an alteration of cellular

metabolism, and eventually cell death23.

The essential oils in the Next ScienceTM rinse are also used in Listerine®, a known effec-

tive mouth rinse against plaque and gingivitis. These oils include menthol, eucalyptol, methyl sa-

licylate and thymol. Essential oils have antibacterial activity via alteration of the bacterial cell

wall. They are known to significantly reduce plaque and gingival bleeding24.

47

To our knowledge, this is the first clinical trial that investigates the clinical and microbio-

logical effects of a rinse that combines Cetylpyridinium chloride (CPC) and essential oils, but

numerous studies report on the effects of a CPC rinse vs. placebo rinse.

In 2005, Mankodi et al. completed a study that evaluated the effects of a 0.07% CPC

rinse (Crest® PRO-HEALTH™) vs. a placebo rinse25. This was a 6-month double-blind RCT

with 119 patients that completed the study. Patients rinsed with 20mL of their assigned rinse for

1 minute, twice a day following brushing. Clinical measurements were completed at baseline,

three months and six months and included the modified gingival index, modified bleeding index,

and the modified Quigley-Hein plaque index. Mankodi reported that at the end of the six months,

the CPC rinse patients showed 15.4% less gingival inflammation, 33.3% less gingival bleeding,

and 15.8% less plaque when compared to the placebo rinse patients.

The evidence clearly shows that CPC and essential oil rinses are independently effective

in reducing plaque and gingivitis. Future research for the Next ScienceTM rinse should evaluate

whether the Next ScienceTM combination of these ingredients is more effective than either of the

ingredients alone.

48

REFERENCE LIST

1. Seneviratne, C. J., Zhang, C. F., & Samaranayake, L. P. (2011). Dental plaque biofilm in

oral health and disease. Chinese Journal of Dental Research, 14, 87–94.

2. Avila M, Ojcius DM, and Yilmaz Ozlem. The Oral Microbiota: Living with a Permanent

Guest. DNA and Cell Biology 2009; volume 28, number 8: 1-7

3. Löe, H. The Gingival Index, the Plaque Index and the Retention Index Systems. Journal

of Periodontology 1967. 38(6): 610-616

4. Turesky S, Gilmore ND, Glickman I. Reduced plaque formation by the chloromethyl an-

alogue of Victamine C. J Periodontol. 1970; 41:41-3.

5. Socransky SS, Haffajee AD et al. Microbial complexes in subgingival plaque. J Clin Periodontol 1998; 25: 134-144

6. Umeda M, Chen C, et al. Risk indicators for harboring periodontal pathogens. J Perio-

dontol 1998; 69: 1111-1118

7. Loe, H., Theilade, E. & Jensen, S.B. Experimental gingivitis in man. J Periodontol 1965;

36: 177-187

8. Lindhe J: Clinical Peridontology and Implant Dentistry. Ed. 5. 2008.

9. Theilade E. The non-specific theory theory in microbial etiology of inflammatory perio-

dontal disease. J Clin Periodontol 1986; 13(10): 905

10. Page, R.C. & Schroeder, H.E. Pathogenesis of inflammatory periodontal disease. A sum-

mary of current work. Laboratory Investigation, 1976; 33, 235-249.

11. Lindhe, J., Hamp, S.E. & Loe, H. Plaque-induced periodontal disease in beagle dogs.

Scandinavian Journal of Dental Research, 1975; 10, 243-255.

12. Page, R.C., Simpson, D.M. & Ammons, W.F. Host tissue response in chronic inflamma-

tory periodontal disease. IV. The periodontal and dental status of a group of aged great

apes. Journal of Periodontology, 1975; 46, 144-155.

13. Cobb, C. M. Non-surgical mechanical. Ann periodontol 1996; 1, 443–490

14. Greenstein, G., C. Berman, et al. Chlorhexidine. An adjunct to periodontal therapy. Jour-

nal of Periodontology, 1986; 57 (6); 370-377.

49

15. Jeffcoat, M. K., K. S. Bray, et al. (1998). "Adjunctive use of a subgingival controlled-re-

lease chlorhexidine chip reduces probing depth and improves attachment level compared

with scaling and root planing alone." J Periodontol 69(9): 989-997.

16. Socransky SS, Haffajee AD, Cugini MA et al. Microbial complexes in subgingival

plaque. J Clin Periodontol 1998; 25 (2): 134–144.

17. JoAnn R. Gurenlian. The role of dental plaque biofilm in oral health. J Dental Hygiene

2007 Oct. 81 (5)

18. Loesche WJ. Chemotherapy of dental plaque infections. Oral Sci Rev. 1976;9: 65-107.

19. Wunderlich RC, Singleton M, O’Brien WJ, Caffesse RG. Subgingival penetration of ap-

plied solutions. Int J Periodontics Restorative Dent 1984: 6: 65-71

20. Pitcher GR, Newman HN, Strahan JD. Access to subgingival plaque by disclosing agents

using mouthrinsing and direct irrigation. J Clin Periodontol 1980; 7: 300–308.

21. Waerhaug J. Effect of toothbrushing on subgingival plaque formation. J Periodontol

1981; 52: 30-34.

22. Eakle WS, Ford C, Boyle RL. Depth of penetration in periodontal pockets with oral irri-

gation. J Clin Periodontol 1986: 13: 39-44

23. Asadoorian, J., Williams, K. Cetylpyridinium Chloride Mouth rinse on Gingivitis and

Plaque. Journal of Dental Hygiene 2008 Oct; 82(5)

24. Albert-Kiszely A, Pjetursson BE, Salvi GE, Witt J, Hamilton A, Persson GR, Lang NP.

Comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse

on dental plaque and gingivitis - a 6-month randomized controlled clinical trial. J Clin

Periodontol. 2007 Aug; 34(8):658-67

25. Mankodi S, Bauroth K, Witt JJ, Bsoul S, He T, Gibb R, Dunavent J, Hamilton A. A 6-

month clinical trial to study the effects of a cetylpyridinium chloride mouth rinse on gin-

givitis and plaque. Am J Dent 2005;18 (special issue):9A-14A.

26. Fei Teng, Tao He, Shi Huang, Cun-Pei Bo, Zhen Li, Jin-Lan Chang, Ji-Quan Liu, Duane

Charbonneau, Jian Xu, Rui Li, Jun-Qi Ling. Cetylpyridinium chloride mouth rinses alle-

viate experimental gingivitis by inhibiting dental plaque maturation. Intl J Oral Science

2016. 8: 182-190

27. Al-Kamel A, Al-Hajj WA, Halboub E, Abduirab S, Al-Tahami K, Al-Hebshi NN. N-Ac-

etyl cysteine versus Chlorhexidine mouthwashes in prevention and treatment of experi-

mental gingivitis: a randomized, triple-blind placebo-controlled clinical trial. Clin Oral

Investig. 2019 Jan; DOI: http://dx.doi.org/10.1007/s00784-019-02813-3

50

28. Greenstein, G., C. Berman, et al. Chlorhexidine. An adjunct to periodontal therapy. J

Perio. 1986. 57 (6): 370-377.

51

BIOGRAPHICAL SKETCH

Bianca Newman was born in El Paso, Texas. She completed her undergraduate degree with

Bachelor of Science in Exercise Science at Florida State University in Spring 2012. Her dental

degree was completed at Meharry Medical College and graduated May of 2016 with Doctor of

Dental Surgery. She then received her Master of Science from the University of Florida in spring

of 2019 and anticipates graduating in May 2019 with a certificate in Periodontics. Following

graduation, she plans to practice clinical periodontology in Orlando, Florida.