microbiological effectiveness of next science oral …
TRANSCRIPT
MICROBIOLOGICAL EFFECTIVENESS OF NEXT SCIENCE ORAL RINSE IN
CONTROLLING PLAQUES AND GINGIVAL INFLAMMATION. A DOUBLE-BLINDED
RANDOMIZED CONTROLLED TRIAL
By
CLAIRE ROSEBROUGH
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 Claire Rosebrough
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ACKNOWLEDGMENTS
First, I would like to thank our clinical staff. Janice Braddy and Renita Jenkins worked
with us to contact and schedule patients and to help with patient visits. They always made sure to
accommodate everyone and patients looked forward to seeing them. They were so patient and
such a joy to be around. Thank you to Gabriela Marchegiani and Aaliya Stateson for your hard
work during the patient visits. You were both very helpful and efficient. You made the visits go
smoothly but kept them fun at the same time.
I would like to thank Dr. Ikramuddin Aukhil for being my research project mentor and
for being available to help with the writing of this thesis. You have been a great resource
throughout this project as well as throughout my entire residency program.
Thank you to my program director Dr. Rodrigo Neiva and to our other committee
member, Dr. Jia Chang. You have both been supportive in the clinic and in teaching us the
intricacies of periodontal treatment!
Thank you to Dr. Bianca Newman and Dr. Ana Dias Ribeiro who worked alongside me
on this clinical project. You both made the best team!
Finally, thank you to Dr. Gary Wang and Dr. Ann Progulske-Fox for your help
processing and analyzing the microbiological data. Thank you for taking the time to provide us
with the data but also to explain it.
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TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ............................................................................................................ .3
LIST OF TABLES ..........................................................................................................................5
LIST OF FIGURES ........................................................................................................................6
LIST OF ABBREVIATIONS…………………………………………….……………………….7
ABSTRACT ....................................................................................................................................8
CHAPTER
1 INTRODUCTION ............................................................................................................10
1.1 Biofilm Formation…………….…………...……………….…….……. …………11
1.2 Supragingival Plaque…..……………...…………………….…….……. ………...13
1.3 Plaque-induced Gingivitis…………..………………...…….…….………………. 14
1.4 Plaque Index/Gingival Index…...…...…………………………….……………… 16
1.5 Treatment of Gingivitis…………………………..……..………..……………...…16
1.6 Oral Antimicrobials……..………………………….……...………….…….……. 17
1.7 Study Aim…………………………………….………………….…...……………18
2 MATERIALS AND METHODS ..................................................................................... 21
2.1 Study Design ............................................................................................................21
2.2 Baseline, Visit 1 .......................................................................................................21
2.3 Visit 2: Dental Prophylaxis, Oral Hygiene Instruction and Product Dispensation...22
2.4 Visit 3: Week 6……...………………………………………………………..……22
2.5 Visit 4: Week 12…...…………………………………………………………..…..23
3 RESULTS .........................................................................................................................34
4 DISCUSSION ...................................................................................................................46
REFERENCE LIST ......................................................................................................................53
BIOGRAPHICAL SKETCH ........................................................................................................55
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LIST OF TABLES
Table page
2-1 Study schedule by procedure type and visit.…………………………………….……….33
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LIST OF FIGURES
Figure page
1-1 Scoring criteria for the Turesky plaque index…………………………………………....19
1-2 Diagram of the Turesky plaque index……………………………………………………19
1-3 Scoring criteria for the Loe and Silness gingival index…...……………………………..20
1-4 Diagram of the Loe and Silness gingival index...………………………………………..20
3-1 ANOVA analysis for alpha diversity comparisons............................................................38
3-2 Unifrac analysis showing microbiological distributions at baseline………………......…39
3-3 Unifrac analysis reporting microbiological distributions at week 12……...…………….39
3-4 Unifrac analysis of microbiological data for baseline and week 12 in control…………..40
3-5 Unifrac analysis of microbiological data for baseline and week 12 in treatment...…...…40
3-6 Permanova analysis of weighted unifrac data……………………………………………41
3-7 Permanova analysis of unweighted unifrac data…………………………………………41
3-8 LDA scores of bacterial species in control and treatment at baseline……...……………42
3-9 LDA scores of bacterial species in control and treatment at week 12…………...……....43
3-10 LDA scores comparing control baseline and control week 12 bacteria……...…………..44
3-11 LDA scores comparing treatment baseline and treatment week 12 bacteria…………….45
4-1 Socransky color-coded bacterial complexes……………………………….......………...47
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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
MPI Modified Plaque Index
MGI Modified Gingival 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
MICROBIOLOGICAL EFFECTIVENESS OF NEXT SCIENCE ORAL RINSE IN
CONTROLLING PLAQUES AND GINGIVAL INFLAMMATION. A DOUBLE-BLINDED
RANDOMIZED CONTROLLED TRIAL
By
Claire Rosebrough
May 2019
Chair: Ikramuddin Aukhil
Major: Dental Sciences – Periodontics
Introduction
Periodontal disease is a multifactorial disease influenced by factors including smoking,
diabetes, and oral hygiene, but the initiation of disease must be preceded by bacteria present in
dental plaque. Plaque is a bacterial biofilm that colonizes and adheres to the tooth surface1. The
plaque biofilm initiates an inflammatory reaction through release of endotoxins as well as
through initiation of the host immune response 2. Therapies available for the treatment of
periodontal disease include non-surgical periodontal therapy such as scaling and root planing or
surgical therapy such as osseous resective surgery. Both of these treatment modalities are
proven to be effective in the treatment of periodontal disease and with professional treatment
combined with good home care and regular maintenance visits, periodontal health may be
achieved and maintained. Although disease progression can be slowed or stopped, the best
treatment for periodontal disease is prevention. The use of antimicrobial therapies as adjunctive
treatments are commonly employed to maintain gingival health or to supplement the treatments
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previously described. This study evaluates a novel mouth rinse and the rinse’s effects on plaque
biofilm accumulation, gingival health, and the microbiologic changes that are occurring within
the supragingival plaque. The current study will discuss the micobiologic effects of the mouth
rinse. An additional thesis will be submitted by Dr. Bianca Newman to review the clinical results
on plaque accumulation and gingival changes.
Materials and Methods
80 adult patients with gingivitis were recruited and completed 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 carrier rinse control. Patients were given standardized toothbrushes and toothpaste
and instructed to use no additional oral hygiene aids. Rinsing was performed twice a day for 1
minute. Clinical measurements were repeated following 6 and 12 weeks of mouth rinse use.
Supragingival plaque samples were collected following 12 weeks of mouth rinse use.
Results
A shift was noted in the microbiota community in the treatment group. No change was
noted in the control. It is unknown whether this shift produced a positive or negative change, but
it was apparent that the treatment rinse had an effect on the microbiological community
structure.
Conclusion
Microbiologic data suggests that the Next ScienceTM rinse has an effect on the microbiota
community structure but the implication of this shift is unknown.
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CHAPTER 1
INTRODUCTION
The oral cavity is home to hundreds of bacterial species. The bacteria present in a healthy
patient are very different from the bacterial species found in a patient effected by periodontal
disease. Some of these species exist within a commensal relationship and other species cause
harm to the host. The more virulent bacterial species are capable of biofilm formation. A biofilm
provides protection for the bacteria against mechanical stress as well as antimicrobial agents2.
Biofilm formation occurs quickly but biofilm maturation requires time. Early colonizer bacteria
which are bacteria present at health, initiate the biofilm adherence to the tooth surface. Once
these early colonizing bacteria are present, a bridging bacteria, Fusobacterium nucleatum, can
attach to the tooth surface. Once Fusobacterium nucleatum is present, the more virulent bacteria
may attach to F. nucleatum and join the biofilm5. Oral hygiene efforts are aimed at removal of
bacteria and prevention of biofilm formation, but initial bacterial colonization begins within
seconds of oral hygiene efforts. This initial colonization eventually matures into a well-organized
biofilm that is firmly adherent to the tooth surface if the biofilm is not regularly disturbed.
Socransky and Haffajee sampled plaque from healthy and diseased patients. They used
DNA probes to categorize bacterial subgroups on a spectrum from healthy to disease-causing
bacteria. They proposed five major bacterial complexes. Of those five complexes, they found
that one complex was particularly virulent and related to periodontal breakdown. This complex
was termed the “red complex”. The red complex consists of P. gingivalis, B. forsythus (now T.
forsythia), and T. denticola5. The presence of red complex bacteria within the sulcus can cause
an inflammatory reaction and promote the onset of periodontal diseases, first presenting as
gingivitis and then progressing to periodontitis.
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Furthermore, longitudinal studies have proven that the presence of these particular “red-
complex” microorganisms is related to disease initiation and progression when present in
periodontal pockets and saliva6. After biofilm disruption and removal, it is possible that oral
rinses with antimicrobial effects will provide additional benefits in controlling the quantity and
quality of bacterial present. These benefits may delay the process of bacterial biofilm formation.
Next ScienceTM developed an oral mouth rinse that is intended to delay the onset of the
periodontal disease process by reducing plaque accumulation and slowing the development of
gingivitis. The oral rinse has been tested in a previous in-vitro study. In this study, its action
against the previously discussed red complex periodontal pathogens was compared to the effects
of ListerineⓇ and PerioguardⓇ. The results revealed superior efficacy to Listerine in reducing the
number of red complex bacteria in-vitro. This study will evaluate the microbiologic changes that
occur after use of the Next ScienceTM oral mouth rinse in-vivo through analysis of supragingival
plaque samples.
1.1 Biofilm Formation
Plaque, or biofilm, formation begins with the formation of a conditioning film called the
acquired pellicle7. The acquired pellicle forms on the tooth surface immediately after oral
hygiene efforts are completed. This pellicle is composed of glycoproteins from saliva. The
acquired pellicle film alters the charge and free energy of the surface and increase the efficiency
of bacterial adhesion8. After formation of the acquired pellicle, primary bacteria colonization is
completed by predominately Gram-positive aerobic cocci, which adsorb onto the pellicle-coated
enamel surface. The film grows and forms a layer of molecules that new Gram-positive
facultative cocci and rods can adhere to and use for co-aggregation. At this time, the surface
receptors on the Gram-positive cocci and rods allow subsequent adherence of Gram-negative
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organisms that were unable to adhere directly to the initial pellicle8. The bacterial mass increases
and multiplies with the continued adhesion of new bacteria. As the biofilm grows, an oxygen
gradient is created which allows anaerobic bacteria to thrive at the base of the biofilm8. As
plaque becomes more mature, more strictly anaerobic Gram-negative red complex bacteria
colonize and contribute to an increased pathogenicity of the biofilm.
The first comprehensive study to look at the effect of poor oral hygiene on plaque
accumulation, gingival status and microbiology was presented by Loe et al. in 19659. Loe began
the study with 12 periodontally healthy patients who were instructed to fully discontinue oral
hygiene, including brushing, flossing or the use of mouth rinses, for twenty-one days. Plaque
samples were collected at various time points and changes in the appearance and texture of the
gingiva were recorded. The results showed that gingivitis developed in all subjects between 10-
21 days. The process of the bacterial colonization was categorized into three phases. In the initial
phase, cocci and short bacterial rods predominated. The second phase began within 2-4 days.
During this phase, filamentous bacteria were observed but cocci and rods continued to be
present. The final phase occurred at 6-10 days. During this phase, predominantly spirochetes
were observed. After the twenty-one day period of oral hygiene abstinence concluded, patients
were given a professional cleaning and encouraged to resume normal oral hygiene. When oral
hygiene was resumed, the microbiota returned to the initial phase with predominately cocci and
short rods. All signs of gingival inflammation subsided within twenty-one days for all patients.
This study showed that changes in the microbial structure were correlated to clinically observed
changes. The study also revealed that gingival inflammation is reversible.
Dental plaque is a structurally complex and organized biofilm that is formed through a
series of events. Bacteria present in plaque can produce a variety of irritants, including
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endotoxins, enzymes, toxins, acids, and antigens. These irritants are capable of destroying the
supporting periodontal tissues when present over an extended period of time8. In 1975, Loesche
described two hypotheses to explain the clinical effects of bacterial plaque on periodontal
disease. These two theories were termed the specific plaque hypothesis (SPH) and the non-
specific plaque hypothesis (NSPH). The specific plaque hypothesis states that only certain
specific pathogens can cause disease due to their virulence factors or their ability to initiate a
response from the host immune system7. The non-specific plaque hypothesis states that the
supra-gingival and sub-gingival accumulation of plaque will lead to an inflammatory response,
regardless of the quality of bacteria present7. In this hypothesis, it is the quantity of bacteria that
determines the disease response. Inflammation triggered by plaque will eventually lead to the
destruction of the periodontium, if left untreated. Theilade and Kornman continued the
discussion on these differing hypotheses in 1986. They wrote two papers which 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. Theilade argued that no single species is
responsible for the development of periodontal disease, but the overall shift of the microbial flora
from Gram-positive to Gram-negative species can produce the pathogenic potential for
periodontal destruction7. Loesche supported the opposing hypothesis and argued that some
bacteria are present in health and will not initiate or exacerbate disease. He stated that there are
certain bacteria that must be present to tip the scales between health and disease.
1.2 Supragingival Plaque
Biofilm formation can occur at any location on the tooth surface including at the gingival
margin, supragingivally (above the gingival margin) or subgingivally (below the gingival
margin). As early as 1911, G.V. Black used light and electron microscopy to view supragingival
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plaque while it was still attached to the enamel surface. In the 1950s, studies looked at the plaque
structure by allowing a biofilm to grow in vitro, on artificial surfaces such as plastic material8.
The study of supragingival plaque and its structure led clinicians to see that supragingival plaque
will migrate subgingivally, if left undisturbed for a long enough period of time. Once plaque
migrates subgingivally, it affects the gingival tissues and eventually the alveolar bone. These
effects result from the toxic bacterial products as well as the host response that is initiated in
response to the bacteria.
1.3 Plaque-Induced Gingivitis
To understand the pathogenic changes that occur within the sulcus of a tooth, it is
important to understand the anatomy and clinical appearance of the gingiva in its healthy state.
The periodontium consists of the oral surface of the gingiva. The oral gingival surface is
composed 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 and is
attached to the tooth by hemidesmosomes. Supporting the oral and junctional epithelia is the
attachment network of connective tissue which is primarily composed of collagen fibers8. Even
clinically “healthy” gingiva has a small infiltrate of inflammatory cells that are within the
junctional epithelium (JE) and connective tissue (CT) attachment10, but this inflammatory
infiltrate grows as gingivitis develops. This inflammatory response is a reaction to continuous
presence of bacteria and bacterial by-products. The host’s defense system may be able to protect
the host from the onset of disease with this constant inflammatory infiltrate, but this depends on
each individual’s susceptibility. If the host cannot control the bacterial growth, gingivitis will
develop.
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Page and Schroeder (1976) described the initiation of gingivitis into four distinct phases:
initial, early, established, and advanced lesions. The initial lesion occurs within the first 2-4 days
of bacterial invasion and involves the junctional epithelium and the most coronal portion of the
connective tissue attachment. During this phase, the vessels sub-adjacent to the junctional
epithelium dilate, resulting in tissue edema, loss of perivascular collagen, and an increase in
gingival crevicular fluid (GCF). Polymorphonuclear neutrophils (PMNs) migrate to the affected
sites. Later the PMNs are accompanied by macrophages and T-lymphocytes. The phase of the
early lesion begins at 4-7 days and continues through day 14. During the early phase, the vessels
in the dentogingival plexus remain dilated, and the capillary beds open. At this stage of
gingivitis, the lymphocytes and PMNs are the predominant infiltrates with very few plasma cells
present. The basal cells begin to proliferate within the JE and along the root surface. As exposure
to bacterial plaque continues to enhance the inflammatory reaction of the gingival tissues, the
site enters the established lesion phase. The specific timing of the established lesion depends on
the host’s susceptibility but usually occurs at 2-3weeks. As defined by Page and Schroeder
(1976), the established lesion has an increased number of leukocytes and plasma cell infiltrates.
The proliferation, apical migration, and lateral extension of the JE occurs with further loss of
collagen. This results in collagen-depleted spaces, which extend deeper into the tissues. At this
point, the lesion may remain in the established phase for an indefinite period of time or it may
progress to become an advanced lesion11,12. The advanced lesion is more active and leads to
destruction of the periodontium. As the advanced phase progresses, pocket depth increases and
the biofilm migrates apically. Several subgingival bacteria flourish in the anaerobic environment
and cause loss of connective tissue attachment and alveolar bone resorption. This progression
leads the advanced phase to become periodontal disease.
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1.4 Plaque Index/Gingival Index
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
evaluated on the buccal and palatal/lingual surfaces of each tooth included in the study
(exclusion criteria 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 solution was thoroughly rinsed with water and plaque scores were recorded. Plaque
disclosing solutions contain a dye which reacts with dental plaque and stains the plaque pink so
that it can be easily visualized. The dye is easily removed, along with the stained plaque, after
brushing or a dental cleaning is completed. The scoring was completed using the values in Figure
1-1 and according to the pictorial diagram presented in Figure 1-2.
To evaluate each patient’s gingival health, the gingival index3 proposed by Loe and
Silness 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 criteria listed in Figure 1-3 and as described in the picture
in Figure 1-4.
1.5 Treatment of Gingivitis
The main goal of periodontal therapy is the preservation of the natural dentition through
the maintenance of a healthy and functional periodontium. This is accomplished through regular
elimination of bacterial plaque. If a healthy periodontium is not maintained and periodontal
disease develops, non-surgical therapy is the first approach in the treatment of disease. Non-
surgical therapy, or scaling and root planing (SRP), consists of mechanical removal of the
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supragingival and subgingival bacterial plaque, removal of hardened deposits such as calculus,
patient oral hygiene instructions, and a 3-month maintenance recall schedule. By reducing the
periodontal pathogens below the “critical mass”13, an environment is created that predominately
harbors “healthy” or early colonizing Gram-positive aerobic bacteria.
Biofilm disruption is the first step in reducing and eliminating the pathogens and their
byproducts. Sonic and ultrasonic instrumentation are often the first tools used during
debridement. These instruments may be air driven or electric powered and they utilize a
vibration motion to break up the plaque or calculus deposits. There are two types of ultrasonic
units that are commonly used in periodontal therapy. The first is a magnetostrictive unit which
uses an elliptic vibration. The second is a piezoelectric unit which uses a linear vibration.
Ultrasonic therapy is followed by hand instrumentation with instruments including curettes and
scalers. Scalers have pointed tips and are utilized for supragingival plaque and calculus removal.
Curettes have rounded toes, making them gentler and more indicated for subgingival use.
Following mechanical removal of supragingival and subgingival microbes, patient motivation,
oral hygiene instructions and a regular maintenance schedule are very important factors in
maintaining periodontal health8.
1.6 Oral Antimicrobials
In addition to the initial non-surgical periodontal therapy, locally delivered antimicrobials
may be used as supplemental treatment. The rationale for antimicrobial use is that bacterial
pathogens are difficult to reach in deep periodontal pockets or furcation defects. Irrigation with
antimicrobials may access bacteria that were not reached with the ultrasonic instrument or
curettes. One reason that local antimicrobials are preferred to systemic medications is that
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patients often report adverse reactions and poor compliance to systemic antibiotic administration.
Furthermore, the overuse of systemic antibiotics leads to the development of antibiotic
resistance. Examples of locally delivered devices include PerioChip which has an active
ingredient of chlorhexidine gluconate, Atridox which has active ingredient Doxycycline, Arestin
with active ingredient Minocycline, and Elyzol with active ingredient Metronidazole gel.
PerioChip is used because chlorhexidine has the ability to inhibit plaque formation due to
its anionic acid groups which bind salivary glycoproteins. This slows the formation of the
acquired pellicle as well as subsequent bacterial colonization. Chlorhexidine also binds to
salivary bacteria and prevents bacterial adsorption to the enamel14. The efficacy of PerioChip
was tested in two double-blind, randomized, placebo-controlled multicenter clinical trials.
PerioChip was used as an adjunctive therapy to scaling and root planing in the treatment group.
After 9 months, significant reductions in probing depths and clinical attachment levels were seen
in the chlorhexidine group compared to the mechanical debridement alone control group15.
The advantages of a local delivery device include prolonged drug levels within the
therapeutic range, minimal systemic adverse reactions and improved in patient compliance.
Treating periodontal disease with adjunct antimicrobials can enhance the results in sites than
may not respond well to conventional therapy alone.
1.7 Study Aim
The aim of the present study is to investigate the clinical and microbiological effects of
the Next ScienceTM oral rinse when compared to a placebo rinse on patients with gingivitis.
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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.
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.
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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.
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.
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CHAPTER 2
MATERIALS AND METHODS
2.1 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
randomized 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 microbial flora through supragingival plaque sampling. Gingival inflammation and bleeding
were assessed clinically after 6 and 12 weeks of product use. The events that were completed at
each visit are summarized in Table 2-1.
2.2 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
entrance 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
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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.
2.3 Visit 2: Dental Prophylaxis, Oral Hygiene Instruction and Product Dispensation
Subjects received a whole-mouth dental prophylaxis by the dental hygienist. For
logistical reasons, the Dental Prophylaxis visit occurred within 2 weeks of the Baseline Visit.
Following dental prophylaxis, an oral examination was conducted. This visit included
instructions on 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 30 seconds. 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 duration. 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.
2.4 Visit 3: Week 6 Clinical Measurements
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
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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.
2.5 Visit 4: Week 12 Clinical Measurements and Plaque Sampling
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
informed 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 were collected with curettes, placed in 0.2mL Eppendorf tubes, and
immediately placed on ice after collection from each patient. Following each clinical session,
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samples were taken to Dr. Gary Wang’s laboratory at the University of Florida College of
Medicine. Samples were frozen until time of analysis.
Plaque samples were treated to lyse bacterial cells followed by PCR amplification.
Bacterial DNA 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 align
the name of each organism to its sequence. The total number of sequences found for each
organism indicates the abundance of that bacteria in the population.
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
informed 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 the following:
• Known allergies or sensitivity to oral rinse products, especially those containing
cetylpyridinium chloride (CPC), cinnamaldehyde, sodium hydroxide or potassium dihydrogen
phosphate.
• Severe periodontal disease, as characterized by purulent exudate, generalized mobility, and/or
severe recession.
• Active treatment for periodontitis.
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• Bleeding disorder or using a blood thinner
• Braces
• Diabetes
• Antibiotic use within three months of the Baseline, Visit 1.
• Pregnancy (Females of child-bearing age will be asked at each visit if they are pregnant.)
• Any diseases or conditions that could be expected to interfere with the subject safely
completing 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
investigator determined that their well-being or safety was at risk or for non-compliance or study
violations. The reason for discontinuation would be documented in the research chart for any
subject that was withdrawn from the trial
Investigational Product:
• 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
monobasic, anhydrous, ACS reagent, ISO, Ph. Eur., 99.5-100.5%., Menthol, racemic, >.98%,
Eucalyptol, Methyl Salicylate, Thymol, Ethanol.)
(Supplier B. Braun - Sterile Water for Irrigation USP)
• Control: OTC Oral Rinse Control
26
(Supplier Sigma Aldrich – Cinnamaldehyde, certified food grade product
(Supplier B. Braun – Sterile Water for Irrigation USP)
Subjects were instructed to brush thoroughly with the standardized toothbrush and
Colgate 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 30 seconds
(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 duration of the study.
The identity of the experimental oral rinse and its placebo was not disclosed to the
clinical 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, emergency 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
ascertained by opening the sealed code breaker report, locating the subject’s kit box number and
treatment 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
27
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.
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
bottles. Enough oral rinse will be provided to last 12 weeks.
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
effect size (treatment difference divided by within subject standard deviation) is at least 0.528.
Safety will be assessed by the absence of irreversible side effects. Whole body adverse
events will be collected only if related to product use.
Assessment of the oral soft tissue was conducted via a visual examination of the oral
cavity and perioral area utilizing a standard dental light, dental mirror, and gauze. The structures
examined included the gingiva (free and attached), hard and soft palate, oropharynx/uvula,
buccal mucosa, tongue, floor of the mouth, labial mucosa, mucobuccal/mucolabial folds, lips,
and perioral area. Assessment of the oral hard tissues was conducted via a visual examination of
the dentition and restorations utilizing a standard dental light, dental mirror, and air syringe. All
abnormal findings were recorded and categorized by their location and hard tissue findings will
be categorized as “other”. An AE was recorded if a new abnormal finding is noted after product
28
dispensing or any previously noted abnormal finding increases in severity during the treatment
period.
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
after oral prophylaxis will be analyzed by PCR-based HOMIM (Human Oral Microbe
Identification 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), salivary 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.
The following hypotheses was 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.
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
29
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.
Whole-mouth average plaque, gingival and bleeding scores were calculated for each
subject 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
Baseline 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%.
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
facilities, 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
applicable 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]).
30
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
individually identified in any publications that result from this study. Individual patient’s
medical information 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 information was shared with the subject’s personal physician or other medical personnel
responsible for their welfare. Data generated by this study was made available for inspection
upon request by representatives of the U.S. FDA, local and national health authorities, the
Sponsor or its representatives, and by the IRB.
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.
Title 21, Part 56 or the ICH-GCP’s Consolidated Guidelines. Chapter 3. The investigator
maintained 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 signature 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 required. 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.
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.
31
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
bleeding.
Following completion of the study, the investigator submitted 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.
The investigator retained the subject identification codes, informed consent
documentation, 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 request 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 responsibility for maintaining records for the period of time required,
custody of the records were transferred to another person who accepted responsibility for the
records. Notice of such a transfer was given in writing to the Sponsor.
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
dispensation of all test products (used and unused) were documented using forms provided by
32
Next ScienceTM or suitable forms provide by the site provided the site had an existing SOP for
the destruction of clinical materials and prior written approval from Next ScienceTM.
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
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.
33
Table 2-1. Study Schedule by Procedure Type and Visit (Note: Visit 1 and 2 may be combined)
PROCEDURE
Baseline
Visit 1
Oral Prophylaxis
Visit 2/ Product
Dispensation
Week 6
Visit 3
Month 3
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
AEs X X X
Subject Accountability X
34
CHAPTER 3
RESULTS
A total of 80 subjects completed this study from the original 132 patients with ages 18
years old to 60 years old were screened for eligibility. Fourteen patient 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 12-
week trial period, 39 patients had received the Next ScienceTM oral rinse and 41 patients received
the control placebo.
Plaque samples were treated with a solution to lyse bacterial cells followed by PCR
amplification. 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.
A two-way repeated measures ANOVA analysis showed that there was no significant
difference when comparing the time, treatment and treatment to 6 week or 12 week visits. This
shows that the mouth rinse does not have a significant effect on alpha diversity.
Unifrac analysis was completed to compare bacterial communities between the samples.
The 3D charts acquired via this analysis showed that the control and treatment groups had
similar distributions of bacteria at the baseline visit (Figure 3-3). When comparing the control
and treatment groups at the week 12 visit, the chart showed that the control bacterial community
distribution was very similar to the baseline for both the control rinse and the treatment mouth
rinse. For the treatment group, a definite shift to the left was noted (Figure 3-4).
35
When comparing the baseline and week 12 data for the control group alone, the data
points representing each bacterial sample had very similar distribution. They were all grouped
within the same general area (Figure 3-5).
When comparing the baseline and week 12 data for the treatment mouth rinse group, the
data points for the baseline bacterial samples were in the same area as the previously seen control
group baseline and week 12 values. The week 12 data points for the treatment mouth rinse were
shifted to the left (Figure 3-6).
To determine whether this shift in the week 12 treatment mouth rinse bacterial
community was significant compared to the baseline treatment group data and the week 12
control rinse data, a permanova analysis was conducted.
The permanova analysis of the weighted unifrac results (Table 3-8) showed that bacterial
community differences between patients in the control group at baseline and the treatment group
at baseline were not significant p-value: 0.263). The control group at baseline compared to the
control samples at 12 weeks bacterial communities had a small but significant change (p-value:
0.035). The greatest significance was observed when comparing the control group at baseline to
the treatment mouth rinse group at week 12 (p-value: 0.001). The next greatest significance was
seen when comparing the control group at week 12 to the treatment group at week 12 (p-value:
0.002). There was no significant difference when comparing the treatment group at baseline to
the treatment group at week 12 (p-value: 0.058).
The permanova analysis of the unweighted unifrac results (Table 3-9) showed that the
only significant difference was when comparing the baseline control data with the week 12
mouth rinse data (p-value 0.012). The unweighted unifrac data does not take into account the
abundance of each bacterial species present.
36
Linear discriminant analysis effect size (LEfSe) was completed to provide data on sample
species richness. The diversity of the most abundant bacteria was only slightly different between
the treatment and control groups at baseline (Table 3-8), but there was a much larger difference
between the two groups at week 12. This can be seen by the greater number of bars associated
with various bacterial species on the week 12 graph (Figure 3-8). These are the bacterial species
that are abundant in the treatment group, indicated by the green bars, but not in the control group
as well as the species abundant in the control group, indicated by the red bars, but not in the
treatment group.
LEfSe analysis also shows the difference in baseline control and week 12 control
bacterial species abundance (Figure 3-9). Figure 3-10 shows the difference in baseline treatment
and week 12 treatment bacterial species abundance. There are many more bars on the treatment
graph which indicates that the treatment group bacteria had a greater divergence over the 12-
week period than the control group bacteria.
Overall, the mouth rinse had the following effects on enriching some bacteria when
compared to the week 12 control patients and in depleting other bacteria when compared to the
week 12 control patients:
Organisms Enriched in Mouthwash Week 12:
Actinomyces_sp. oral taxon 169_oral_taxon_16
Actinomyces_sp. oral taxon 175_oral_taxon_175
Actinomyces_sp. oral taxon 525_oral_taxon_525
Campylobacter_gracilis_oral_taxon_623
Clostridiales[F-2][G-2]_sp. oral taxon 085_oral_taxon_85
Fusobacterium_nucleatum subsp. animalis_oral_taxon_420
Parvimonas_sp. oral taxon 110_oral_taxon_110
Peptostreptococcaceae[11][G-4]_sp. oral taxon 369_oral_taxon_369
Rothia_mucilaginosa_oral_taxon_681
Streptococcus_intermedius_oral_taxon_644
Streptococcus_sp. oral taxon 066_oral_taxon_66
Treponema_socranskii_oral_taxon_769
37
Treponema_sp. oral taxon 231_oral_taxon_231
Organisms Depleted in Mouthwash Week 12:
Neisseria_sp. oral taxon 014_oral_taxon_14
Cardiobacterium_valvarum_oral_taxon_540
Leptotrichia_sp. oral taxon 225_oral_taxon_225
Actinomyces_sp. oral taxon 177_oral_taxon_177
Bergeyella_sp. oral taxon 322_oral_taxon_322
Neisseria_subflava_oral_taxon_476
Capnocytophaga_granulosa_oral_taxon_325
Corynebacterium_durum_oral_taxon_595
Aggregatibacter_sp. oral taxon 513_oral_taxon_513
Fusobacterium_periodonticum_oral_taxon_201
Aggregatibacter_sp. oral taxon 458_oral_taxon_458
Lautropia_mirabilis_oral_taxon_22
Neisseria_flavescens_oral_taxon_610
Leptotrichia_hofstadii_oral_taxon_224
Terrahaemophilus_aromaticivorans_oral_taxon_826
Porphyromonas_sp. oral taxon 279_oral_taxon_279
Fusobacterium_nucleatum subsp. polymorphum_oral_taxon_202
Corynebacterium_matruchotii_oral_taxon_666
38
Figure 3-1. Two-way repeated measures ANOVA analysis for Alpha Diversity Comparisons.
39
Figure 3-2. Unifrac analysis showing microbiological distributions for baseline data plots for
the control mouth rinse and treatment mouth rinse groups.
Figure 3-3. Unifrac analysis showing microbiological distributions for week 12 bacterial data
plots for the control mouth rinse and treatment mouth rinse groups.
40
Figure 3-4. Unifrac analysis showing microbiological distributions between baseline and week
12 data for the control mouth rinse group.
Figure 3-5. Unifrac analysis showing microbiological distributions between baseline and week
12 data for the treatment mouth rinse group.
41
Figure 3-6. Permanova analysis of weighted unifrac data. C1 and C4 indicate the control
values at visit 1 and visit 4. M1 and M4 indicate the mouth rinse values at visit 1 and visit 4.
Figure 3-7. Permanova analysis of unweighted unifrac data.
42
Figure 3-8. Linear Discriminant Analysis (LDA) scores showing the difference in abundant
bacterial species in the control group at baseline vs. the treatment group at baseline.
43
Figure 3-9. LDA scores (effect size) showing the difference in abundant bacterial species in
the control group at baseline vs. the treatment group at week 12.
44
Figure 3-10. LDA scores (effect size) showing the difference in abundant bacterial species in
the control group at baseline vs. the control group at week 12.
45
Figure 3-11. LDA scores (effect size) showing the difference in abundant bacterial species in
the treatment group at baseline vs. the treatment group at week 12
46
CHAPTER 4
DISCUSSION
Over the course of the study, gingival index scores improved for all patients (See Dr.
Bianca Newman’s Thesis). There was no significant difference between patients in the control
versus treatment group. The reason for this improvement in both the treatment and control group
may be attributed to the prophylaxis that each patient received after the baseline visit. The
frequency of dental visits prior to this study was widely variable and many patients had not had a
professional cleaning in many years. The benefits of dental prophylaxis for improvement in
gingivitis are widely accepted. 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 standardized toothbrushes and toothpaste for usage for the duration of
the study.
Clinically, there was a significant difference between the treatment group and the placebo
rinse group in plaque reduction at week 12. The mouth rinse treatment group had a significantly
lower plaque index (p-value: 0.003). Initial plaque colonization consists primarily of gram
positive bacteria. If allowed to accumulate and mature, plaque composition will shift to a
predominantly gram-negative microbiota16. This shift in bacteria results in more orange and red
complex bacteria which, in combination with the host immune response, are responsible for the
destruction of periodontal disease. Plaque maturation results in a biofilm formation that is highly
organized and possesses increased resistance to saliva, gingival crevicular fluid, antibiotics and
antiseptics17.
47
Figure 4-1. Bacteria placed into color-coded complexes according to pathogenicity.
The red complex is the most virulent in initiation and progression of periodontal disease5.
In the periodontal literature, there are two opposing theories on plaque: The specific
plaque hypothesis and the non-specific plaque hypothesis. The nonspecific plaque hypothesis
states that the quantity of plaque is responsible for the development of periodontal disease and
disease progression rather than the quality of the plaque, or specific types of bacteria present
within the plaque9. If treatment is dictated by the assumption that this theory is correct, then
decreasing the critical mass of plaque will prevent or improve disease. The specific plaque
hypothesis was proposed by Loesche in 1976. This alternate hypothesis states that only certain
types of bacteria within the plaque can initiate and exacerbate periodontal break down. Increased
proportions of these pathogenic bacteria cause disease because these bacteria produce more
harmful virulence factors and have an increased ability to activate the host immune response.
Under the principles of the specific plaque hypothesis, treatment must be aimed at reduction or
elimination of these specific microorganisms18.
48
If the nonspecific plaque hypothesis were true, the reduction in the critical mass of plaque
that resulted from the use of the treatment mouth rinse would initiate a significant reduction in
the periodontal measurement parameters such as bleeding, gingival swelling and gingival redness
for the treatment group patients. In this study, no significant difference 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.
A question that may arise regarding the study design is why supragingival plaque samples
were collected instead of subgingival plaque samples. The rationale behind the decision to
collect supragingival plaque samples was multifactorial. First, gingivitis patients harbor less
complex subgingival periodontal biofilm with fewer red complex bacteria. This study excluded
patients with periodontal disease and only included patients with active gingivitis. Furthermore,
Wunderlich showed in 1984 that mouth rinses only penetrate an average of 0.2mm below the
gingival margin19. If at home use of the mouth rinse is not physically reaching the subgingival
environment, it makes more sense to sample the supragingival plaque where the rinse is in direct
contact. Even in a clinical setting, irrigation of a pocket (PD≥5mm) with an irrigating syringe
penetrates an average of 1.8mm subgingivally20. When looking at other oral hygiene aids,
toothbrushes have been shown to penetrate an average of 0.9mm subgingivally21. Waterpik® at a
high-pressure setting may deliver solution to an average of half way between the free gingival
margin and the most coronal connective tissue attachment22. These may be an adequate depths
for our gingivitis patients, but this study is testing the results of at-home use of the mouth rinse
without any adjunctive oral hygiene aids. Future studies may be warranted on the use of the Next
ScienceTM mouth rinse in combination with irrigating syringes or Waterpik® devices.
49
In the microbiological data for the Next ScienceTM rinse, the Unifrac analysis showed a
distinct shift of the bacterial community for the mouth rinse group. The Permanova analysis
reported that this shift was significant when comparing the control baseline bacterial community
with the mouth rinse week 12 bacterial community (p-value: 0.001) and when comparing the
control week 12 bacterial community with the mouth rinse week 12 bacterial community (p-
value: 0.002). An unexpected result was that this analysis showed no significant difference
between the baseline mouth rinse bacterial communities and the week 12 mouth rinse bacterial
communities. The p-value was 0.058 which was not significant but very close to significance.
Another result of interest was that there was a small but significant change (p-value:
0.035) in the control baseline compared to the control data at week 12. This may be attributed to
the dental prophylaxis that all patients received or to the changes in oral hygiene that resulted
because patients were prohibited from flossing during the 12-week study period.
The Permanova results show that there was a distinct shift in the microbiological
community that is attributed to the use of the treatment mouth rinse, but the data does not tell us
whether this shift was positive or negative. It only states that the mouth rinse caused a change.
We can infer that this was a positive change when we combine the data with the clinical results,
namely the decrease in plaque index for the treatment group.
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
antimicrobial effect through the disruption of bacterial cell membranes resulting in an alteration
of cellular metabolism, and eventually cell death23.
50
The essential oils in the Next ScienceTM rinse are also used in Listerine®, a known
effective mouth rinse against plaque and gingivitis. These oils include menthol, eucalyptol,
methyl salicylate and thymol. Essential oils have antibacterial activity via alteration of the
bacterial cell wall. They are known to significantly reduce plaque and gingival bleeding24.
To our knowledge, this is the first study that looks at the clinical and microbiological
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 or an essential oil rinse vs. placebo
rinse. There are even studies that compare the effects of a CPC rinse against an essential oils
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
30 seconds, 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.
In 2016, Teng et al.26 conducted another study that evaluated the effects of a 0.07% CPC
rinse (Crest® PRO-HEALTH™) vs. a placebo water rinse. This was a double-blind randomized
controlled trial of 91 patients that completed the study. All patients started the study with
twenty-one days of rigorous oral hygiene methods, including a dental prophylaxis. Following
this initial period to ensure gingival health, patients completed another twenty-one day period
51
with no oral hygiene, aside from the use of their assigned rinse. Patients could not brush or floss
but they did use 20mL of the rinse, twice daily for thirty seconds each rinse.
Supragingival plaque samples were collected at baseline and 21 days. Clinical
measurements including BOP and modified gingival index were recorded at baseline, 7 days, 14
days and 21 days. Results showed that the CPC rinse inhibited the growth of 17 gingivitis
associated bacteria (Porphyromonas, Peptostreptococcus, Prevotella, Peptococcus,
Selenomonas, Solobacterium, SR1, Tannerella, TM7 genus, Lachnospiraceae, Atopobium,
Gemella, Megasphaera, Mogibacterium, Moraxella, Oribacterium and Shuttleworthia) and that
the CPC rinse maintained the original bacteria of the healthy plaque. A Shannon index showed
that control rinse had significant increase in genus richness, but the treatment group had no
significant change in α-diversity.
Clinically, there was significantly greater BOP and gingival index scores in the control
group.
One limitation of this study is that it could not be fully double-blinded when the placebo
patients were rinsing with water. A water rinse would be too obvious to the control patients that
they are not part of the treatment group.
A study that compared CPC with an essential oil mouth rinse was reported by Albert-
Kiszely et al. in 200724. This was a six month double-blind randomized controlled trial with 127
patients completing the study. Patients rinsed twice a day with 20mL of their assigned rinse for
30 seconds following brushing. The two mouth rinse groups were a 0.07% CPC rinse (Crest®
PRO-HEALTH™ Rinse) and an essential oil rinse (Listerine®). Clinical measurements and
subgingival plaque samples were collected at 3 months and 6 months. Clinical measurements
included the gingival index (Loe and Silness 1963) and plaque index (Silness and Loe 1964).
52
No significant difference was observed between rinses for gingival status, gingival
bleeding or plaque accumulation. Both groups improved from baseline. This improvement may
be attributed to the effectiveness of both mouth rinses, to the fact that each patient received a
professional prophylaxis after baseline readings, or to a combination of these. Microbiological
data showed that the community profiles were similar for both rinse groups.
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.
53
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BIOGRAPHICAL SKETCH
Claire Rosebrough graduated from the University of Florida with her Bachelor of Science
degree in spring of 2012. Her major was biochemistry with a minor in business administration.
Claire began dental school at the University of Florida in the fall of 2012 and completed her
Doctor of Dental Medicine degree in spring of 2016. She began a three-year residency program
to obtain a Master of Science degree and a certificate in periodontics in the fall of 2016. She
graduated from the University of Florida with a Master of Science degree and a certificate in
periodontics in spring of 2019.