Resveratrol Effect on Oral Inflammatory Load in Chronic Periodontitis: A Pilot Study

by

Faryn Berger BSc, DDS

A thesis submitted in conformity with the requirements for the degree of Master of Science Periodontology

Faculty of Dentistry University of Toronto

© Copyright by Faryn Berger 2018

ii

Resveratrol Effect on Oral Inflammatory Load in Chronic

Periodontitis: A Pilot Study

Faryn Berger BSc, DDS

Master of Science Periodontology

Faculty of Dentistry University of Toronto

2018

Abstract

Chronic periodontitis is an inflammatory disease that diminishes tooth-supporting structures and

is dependent on neutrophil recruitment and oxidative stress. An anti-oxidant, resveratrol (3, 4’, 5-

trihydroxystilbene) blocks neutrophil recruitment and oxidative bursts. Thus, resveratrol can help

lessen oral inflammatory load (OIL), thereby reducing clinical manifestations of periodontitis. It

was hypothesized that OIL will be further reduced in periodontitis patients treated with SRP plus

resveratrol, compared to SRP alone. A randomized triple-blind clinical trial was conducted. By

measuring clinical parameters, oral neutrophil numbers, and neutrophil phenotypes, it was

demonstrated that, while not statistically significant, resveratrol treatment further reduced OIL.

Importantly, patients treated with resveratrol demonstrated earlier responses to treatment

compared to those on placebo. This pilot study demonstrated the potential benefits of resveratrol,

as an adjunctive treatment to SRP. Further research should be conducted with larger cohorts to

examine the benefits of resveratrol in patients with periodontal diseases, particularly those with

co-morbidities.

iii

Acknowledgments

Firstly, I would like to thank my mentor and supervisor for this thesis project, Dr. Howard

Tenenbaum. His encouragement of my interest in the field of periodontology greatly contributed

to my decision to apply for the Master of Science Program at the University of Toronto. Not only

did he provide me with the opportunity to shadow his work at Mount Sinai Hospital, but his

guidance has also been invaluable over the past 5 years. I would also like to thank my committee

members, Dr. Michael Goldberg and Dr. Michael Glogauer. Their knowledge and assistance has

been a true help in the completion of this Master of Science Thesis.

The members of Dr. Michael Glogauer’s Lab also require acknowledgement for helping with the

processing of samples and providing laboratory assistance and guidance. Noah Fine, Morvarid

Oveisi, Nimali Wellappuli, Chunxiang Sun and Oriyah Barzilay, your technical expertise and

willingness to teach me the needed laboratory skills was greatly appreciated.

Thank you to the Alpha Omega Dental Fraternity for helping to financially support this research

project. Your gracious assistance allowed this endeavour to proceed. The Graduate Periodontal

Department has also been instrumental in the success of this project. Dr. Lai and the clinical

staff’s dedication to the treatment of patients and donation of their time, requires

acknowledgement and recognition.

Lastly, I would like to thank my family for allowing me the opportunity to pursue my education,

while constantly providing the love and encouragement needed to excel in my chosen field.

Their unwavering support has been invaluable and will forever be greatly cherished.

iv

Table of Contents

Acknowledgments.................................................................................................................. iii

TableofContents ................................................................................................................... iv

ListofTables ..........................................................................................................................vi

ListofFigures ........................................................................................................................ vii

Abbreviations ...................................................................................................................... viii

Chapter1LiteratureReview....................................................................................................1

1.1 ChronicPeriodontitis.............................................................................................................. 1

1.1.1 Prevalence............................................................................................................................... 1

1.1.2 Pathogenesis ........................................................................................................................... 2

1.1.3 RiskFactors ............................................................................................................................. 5

1.2 PolymorphonuclearCells........................................................................................................ 6

1.2.1 PMNFormation....................................................................................................................... 6

1.2.2 PMNsandPeriodontalDisease............................................................................................... 6

1.2.3 SmokingandPMNs ................................................................................................................. 8

1.2.4 OralInflammatoryLoad ........................................................................................................ 10

1.2.5 PMNPhenotyping ................................................................................................................. 11

1.3 TreatmentApproachesforChronicPeriodontitis.................................................................. 13

1.3.1 ScalingandRootPlaning....................................................................................................... 13

1.3.2 AntibioticsandPeriodontalTreatment ................................................................................ 14

1.3.3 HostModulation ................................................................................................................... 15

1.4 Resveratrol........................................................................................................................... 16

1.4.1 Biosynthesis .......................................................................................................................... 16

1.4.2 TherapeuticBenefits ............................................................................................................. 17

1.4.3 ResveratrolandPeriodontalDisease .................................................................................... 19

1.4.4 Smoking,Resveratrol,andPeriodontalDisease ................................................................... 20

1.4.5 ResveratrolandPeriodontalDiseaseinHumans .................................................................. 21

Chapter2StatementoftheProblem.....................................................................................22

v

Chapter3MaterialsandMethods .........................................................................................23

3.1SubjectRecruitment................................................................................................................. 23

3.2RandomizationandBlinding .................................................................................................... 24

3.3ResveratrolAdministration ...................................................................................................... 24

3.4TrialProtocol ........................................................................................................................... 24

3.5OralPMNCollectionandQuantification................................................................................... 25

3.6BloodPMNCollectionandQuantification ................................................................................ 26

3.7MulticolourFlowCytometry .................................................................................................... 26

3.8GingivalCrevicularFluid ........................................................................................................... 27

3.9StatisticalAnalysis ................................................................................................................... 27

Chapter4Results ..................................................................................................................28

4.1PatientDemographics .............................................................................................................. 28

4.2BleedingonProbing ................................................................................................................. 29

4.3ProbingDepth.......................................................................................................................... 29

4.4OralPMNQuantificationandOIL ............................................................................................. 30

4.5PMNPhenotype....................................................................................................................... 30

Chapter5Discussion .............................................................................................................32

Chapter6ConclusionandFutureDirections ..........................................................................38

FiguresandTables.................................................................................................................40

ContributionstotheThesisandManuscript ..........................................................................57

References ............................................................................................................................58

vi

List of Tables

Table 1: Inclusion and Exclusion Criteria

Table 2: Trial Design

Table 3: CD Antibody Markers for Flow Cytometry Master Mix

Table 4: Demographic and Periodontal Characteristics per Treatment Group

Table 5: Percent change in proportion of sites with BOP

Table 6: Percent change in proportion of sites with PD >6mm

Table 7: Percent change in proportion of sites with PD 4-5mm

Table 8: Percent change in proportion of sites with PD 1-3mm

Table 9: Percent change in oPMNs

vii

List of Figures

Figure 1: Flow of Participants through the clinical trial

Figure 2: Percent change in proportion of sites exhibiting BOP in resveratrol treated patients

compared to placebo

Figure 3: Percent change in proportion of sites exhibiting PD >6mm in resveratrol treated

patients compared to placebo

Figure 4: Percent change in proportion of sites exhibiting PD 4-5mm in resveratrol treated

patients compared to placebo

Figure 5: Percent change in proportion of sites exhibiting PD 1-3mm in resveratrol treated

patients compared to placebo

Figure 6: Percent change in oPMNs in resveratrol treated patients compared to placebo

Figure 7: CD marker expression of oPMNs in resveratrol treated patients compared to placebo

viii

Abbreviations

1O2 - Singlet Oxygen

AhR – Aryl Hydrocarbon Receptor

AHSG - Alpha-2-Heremans Schmid glycoprotein

AO – Antioxidant

BaP - Benzo[a]pyrene

BOP – Bleeding on Probing

CAL – Clinical Attachment Level

CD – Cluster of Differentiation

CP – Chronic Periodontitis

CRP - C-Reactive Protein

CXCR - CXC Chemokine Receptors

DMBA - Dimethylbenz[a]anthracene

DNA- Deoxyribonucleic Acid

ELISA - Enzyme-linked Immunosorbent Assay

FACS - Fluorescence-Activated Cell Sorting

ƒMLP - f-Met-Leu-Phe

FSC – Forward Scatter

GCF – Gingival Crevicular Fluid

G-CSF - Granulocyte Colony-Stimulating Factor

ix

H2O2 - Hydrogen Peroxide

HClO - Hypochlorous Acid

HTS – High-Throughput Screening

ICAM-1 – Intercellular Adhesion Molecule 1

IL – Interleukin

LPS – Lipopolysaccharide

NAD – Nicotinamide Adenine Dinucleotide

NADPH – Nicotinamide Adenine Dinucleotide Phosphate

NET – Neutrophil Extracellular Trap

NF-κB - Nuclear Factor Kappa-B

NSAIDs - Non-Steroidal Anti-Inflammatory Drugs

Nrf2 - Nuclear Factor Erythroid 2-related Factor

MFI - Mean Fluorescence Intensities

MMP – Matrix Metalloproteinases

MPO – Myeloperoxidase

MSE – Melingo Seed Extract

O3 - Ozone

OH- - Hydroxy Radicals

OH.- - Superoxide Anion

OIL – Oral Inflammatory Load

x

oPMN – Oral Polymorphonuclear Cells or Oral Neutrophils

PAH – Polycyclic Aromatic Hydrocarbons

PBS – Phosphate-Buffered Saline

PD – Probing Depth

PDL – Periodontal Ligament

PFA – Paraformaldehyde

PI3K - Phophatidylinositol 3-Kinase

PMN – Polymorphonuclear Cells or Neutrophils

RANK - Receptor Activator of Nuclear Factor κB

RANKL - Receptor Activator of Nuclear Factor κB Ligand

ROS – Reactive Oxygen Species

SSC – Side scatter

SOD – Superoxide Dismutase

SRP – Scaling and Root Planing

TNF – Tumor Necrosis Factor

WBC – White Blood Cell

1

Chapter 1 Literature Review

1.1 Chronic Periodontitis 1.1.1 Prevalence

Periodontal disease is a highly prevalent inflammatory disease of the periodontium, involving the

hard and soft connective and integumentary tissues surrounding the teeth, and affects up to 70%

of the US population [1]. Chronic periodontitis (CP), the most common form of periodontal

disease, is characterized by progressive destruction of tooth-supporting structures. This is the

result of altered host-biofilm interactions in the gingival crevice and unresolved inflammation [2,

3]. Approximately, 8% of individuals demonstrate a severe form of the disease, characterized by

extensive destruction of the periodontal apparatus [4]. This can ultimately lead to a hopeless

prognosis for the dentition, resulting in tooth loss, as well as significant associated morbidity

ranging from occlusal discomfort to pain and swelling. These sequelae have a substantial

economic impact in that patients suffering from chronic periodontitis miss work and spend

significant funds in an attempt to maintain their dentition [5]. Predicting what sites in the mouth

that will experience periodontal breakdown has proven to be a difficult task. Paradoxically, even

teeth that have had severe attachment loss in the past might not necessarily experience increased

destruction in the future [6]. Periodontal probing has been used in periodontal therapy as a

diagnostic tool for identification and measurement of periodontal lesions, called periodontal

pockets. Use of measurements obtained by periodontal probing has stood as the ‘gold standard’

for determining the severity of disease, but cannot be used to identify sites of active disease, at

least with a single measurement. In relation to this it is known that periodontitis is not active on

a continual basis but progresses in bursts, with intervening periods of quiescence [7]. Yet the

periodontal examination, with heavy reliance upon measurements obtained by way of

periodontal probing, has remained fairly unchanged since the 1960s. This produces clinical data

including probing depths (PDs) (as measures of periodontal pocket depths; the deepest pockets

being the most ‘severe’), along with additional clinical parameters, such as the clinical

attachment level (CAL) of hard and soft connective tissues about any one or any group of teeth

to arrive at a diagnosis [8]. However, it should be emphasized that these measures merely

indicate the amount of destruction of the periodontal tissues that has already occurred as a result

2

of the progression of periodontitis. These clinical data cannot be used either to predict future

disease progression or just as importantly overall prognosis (i.e. potential to response to

treatment). In spite of this, a study conducted by Claffey et al. in 1990 demonstrated that an

increase in PD, when combined with the presence of bleeding on probing (BOP) in greater than

75% of the sites, corresponded with a diagnostic predictability relating to disease progression of

82%-87% [9]. Therefore, although deep PDs alone fail to show a high specificity and sensitivity

in detecting the presence of active periodontal disease, when combined with additional clinical

parameters, and considered over a longitudinal period of time, periodontal probing can be more

useful in identifying areas that might require additional treatment [9, 10]. Furthermore as alluded

to above, periodontitis is not a continually progressive condition. Rather, CP is characterized by

asynchronous random “bursts” of disease activity, where single or multiple sites will

demonstrate active progression of attachment loss often reflected by increasing probing depth

measurements, followed by a period of remission [10]. As a result, active periodontitis might be

occurring in relatively few sites at any given time, even in individuals who demonstrate more

severe forms of periodontal disease (i.e. severe loss of periodontal structures).

1.1.2 Pathogenesis

Inflammation is a physiologic response to the presence of injury or invasion by bacterial species.

This process attempts to heal, repair, and regenerate lost or damaged tissues. The initial response

to the presence of noxious stimuli is acute inflammation and is predominated by the activation of

the ‘innate immune response’. Innate immunity is the first line of defence against pathogens and

includes non-specific physiologic activities aimed at protecting the host. This response usually

includes the accumulation of transudate fluid, followed by an influx of a cell-rich inflammatory

infiltrate [11]. The innate immune response is of short duration; however, failure to resolve the

microbial insult or other inflammation-inducing stimuli can lead to the development of a chronic

inflammatory state, where the adaptive immune system predominates [12]. The presence of

specific bacteria, such as Porphyromonas gingivalis, Tannerella Forsythia, and Treponema

denticola has been implicated as a primary microbial etiological factor that plays an important

role in triggering periodontitis, as well as being associated with the more advanced forms of this

condition. However, it is also known that the mere presence of these bacterial species does not

3

always result in the development of severe periodontal attachment loss [13]. More important than

the strict presence of specific bacterial species insofar as disease induction and propagation is

concerned is the fact that after some time, these and other microorganisms can mature into a

structurally organized multi-species community of microbes called a biofilm [14]. The species

within biofilms are not distributed randomly, but rather are spatially organized to allow for

enhanced tolerance of environmental factors and increased growth [15]. These predominately

gram-positive communities have the ability to colonize gingival tissues and exist symbiotically

within the host. This provides protection by competitively inhibiting the growth of pathogenic

bacteria, most of which are thought to be Gram-negative anaerobes [16]. However, an

environmental change, such as the development of an inflammatory response to the increased

microbial load, as well as increased virulence caused by ongoing growth and maturation of a

biofilm near the gingival tissues, can favour the shift to a more dysbiotic biofilm (i.e. symbiotic

towards the support of so-called periodontal pathogenic bacteria). This, along with factors related

to developing inflammation, will then promote the proliferation of gram-negative anaerobic

bacteria such as P. gingivalis. The virulence factors of these periodontal pathogens allow them

to invade the gingival sulcus and evade some host defenses. This initiates the host immuno-

inflammatory response and ultimately, if untreated, periodontal disease ensues [17]. In light of

these concepts it can be suggested that periodontal pathogenic bacteria and the participation of

other oral microbes are necessary, but not sufficient to cause and propagate periodontal tissue

loss associated with periodontitis.

In relation to this, it is understood that in response to the presence of subgingival periodontal

pathogens, the host immune system responds in a coordinated effort to protect the periodontium

and re-establish homeostasis. Thus, in order to prevent invasion and infection by these or any

other oral organisms, a ‘generic’ inflammatory response occurs [12]. During the initial phase of

development of a periodontal lesion, bacterial products induce the release of pro-inflammatory

mediators (e.g. cytokines). It is believed that this increased release of pro-inflammatory

cytokines is dependent on nuclear factor kappa-B transcription (NF-κB) [18]. This process is

activated by the presence of lipopolysaccharide (LPS), a harmful endotoxin found in the cell

walls of gram-negative bacteria [19]. The pro-inflammatory mediators released include cytokines

and interleukins, which signal the recruitment of an inflammatory infiltrate [20]. The study of

specific cytokines, such as interleukins (IL) and tumor necrosis factors (TNF) has helped to

4

increase the understanding of the underlying host response to the presence of periodontal

pathogens that ultimately results in destruction of the periodontium.

In trying to understand the pathophysiological reactions that cause periodontitis, animal models

for periodontitis have been developed. These include the use of ligature-induced periodontitis in

rodent and non-human primate models that mimic periodontitis seen in humans [21]. In using

these model systems it has been possible to demonstrate that the presence of periodontal

inflammation leads to increased circulation of pro-inflammatory cytokines, such as TNF-α, IL-

1β, IL-6, and IL-8 [22]. The increase in these mediators causes vasodilation and chemical

gradients that facilitate the migration of polymorphonuclear neutrophils (PMNs) from the

vasculature to the site of injury. As the periodontal lesion becomes more advanced, lymphocytes

and plasma cells have been shown to accumulate and predominate in the gingival tissues [23]. As

well, cytokines have an impact on connective tissue metabolism and bone turnover [24]. The

release of IL-1β can increase expression of receptor activator of nuclear factor κB ligand

(RANKL) [25]. Expressed by osteoblasts, fibroblasts, and activated T cells, RANKL binds

directly to receptor activator of nuclear factor κB (RANK) on the surface of pre-osteoclasts. This

results in the differentiation, activation, and survival of osteoclasts, which are responsible for

bone resorption and have a major role in the pathogenesis of CP [26]. As well, IL-1, IL-6, and

TNF-α increase PMN-mediated release of matrix metalloproteinases (MMPs). MMPs are a

group of enzymes that are responsible for the degradation of the extracellular matrix [27]. This

includes collagen and other proteins that could regulate the inflammatory process, such as Alpha-

2-Heremans Schmid glycoprotein (AHSG), a serum glycoprotein that can inhibit ectopic arterial

calcification, as well as osteodifferentiation [28]. Thus, increased MMP release has negative

consequences for the periodontium and manifests as direct MMP-mediated destruction of tissues

within the periodontium, while also possibly indirectly regulating inflammation through

activation of inflammatory mediators [29, 30]. This ultimately leads to further loss of the tooth

supporting apparatus, including the periodontal ligament and alveolar bone [31].

5

1.1.3 Risk Factors

A vast range of factors related to host susceptibility to periodontal disease has been described,

which cannot be attributed to bacteria or their byproducts alone [23]. These concepts fit with the

notion that microbial pathogens, as stated above, are necessary but not sufficient to cause

periodontitis. As a result, while some patients exposed to copious volumes of etiologic factors,

including a heavy microbial load and the presence of periodontal pathogenic bacteria, exhibit a

robust amount of periodontal destruction; others, with a similar degree of exposure to the same

levels of etiologic factors, actually have an unexpectedly limited loss of the clinical attachment

apparatus [32]. This conundrum has spurred much biomedical research in this area over the past

40 years in an attempt to understand the multi-factorial nature of periodontal disease and to help

explain the different presentations of this condition as noted above. This includes the attempt to

identify systemic, genetic, and behavioural factors that are correlated with periodontal disease

[33].

While age, race, sex, socio-economic status, and diabetes have all been identified as important

risk factors for periodontal disease, cigarette smoking has been deemed to be one of the more

important epigenetic influences on the loss of the periodontal attachment apparatus [34].

Cigarette smoking has been demonstrated to be a strong predictor of periodontal disease and this

relationship is dose dependent [35]. This has been demonstrated by the fact that individuals who

smoke fewer than 9 cigarettes per day have an odds ratio of 2.8 for developing periodontal

disease, while this increases to an odds ratio of 5.9 when the daily consumption of tobacco

exceeds 31 cigarettes [36]. As a result, smokers tend to demonstrate deeper PDs [37], more CAL

[38], increased bone loss [39] and fewer teeth [40]. Not only do patients who currently smoke

have more severe periodontitis compared to their non-smoking counterparts, but they also do not

respond as well to periodontal treatment. Smokers have been noted to achieve only 50-75% of

the expected reduction in PD following non-surgical or surgical periodontal therapy [41].

Alternatively, individuals who were former smokers tend to demonstrate improved responses to

treatment, compared to smokers [42]. Therefore, the acquired risk to the periodontium from

cigarette smoke appears to be reversible with proper smoking cessation [43]. This said, it still has

to be recognized that while smoking is one of several risk factors that have been identified, it is

still believed that a significant proportion of disease susceptibility is due to genetic variations in

the host immuno-inflammatory response [44].

6

1.2 Polymorphonuclear Cells 1.2.1 PMN Formation

PMNs represent 50-70% of the white blood cells (WBCs) in human blood, and thus are the most

abundant leukocytes in the human body [45]. PMN production occurs in the bone marrow from

the differentiation of myeloid lineage progenitor cells [46]. This tightly regulated process is

mediated by granulocyte colony-stimulating factor (G-CSF) and expression of CXC chemokine

receptors (CXCR) [46, 47]. In healthy individuals, a steady state of granulopoiesis results in the

release of mature PMNs into the bloodstream, where they have a short lifespan with limited

functional activity [48]. The presence of increased quantities of circulating PMNs is often

considered a sign of acute bacterial infection. This is because in response to acute inflammation

and the presence of pro-inflammatory cytokines, the bone marrow significantly increases the

release of PMNs into the blood [49]. In order to replenish the bloodstream’s PMN population as

quickly as possible, PMN maturation time within the bone marrow is actually reduced. This

leads to an increase in the amount of immature PMNs in the blood stream [50].

1.2.2 PMNs and Periodontal Disease

As discussed above, it is clear that PMNs are important for protection against systemic infection

by putative invading organisms. Along the same lines, PMNs are also the primary cell type

mobilized into the gingival crevice in response to increased oral microbial load (i.e. the biofilm

referred to above) and at least initially, this response is critically important for the maintenance

of periodontal health [51]. In order to leave the vasculature, PMNs follow a stepwise process

with the initial step referred to as diapedesis, defined as the rolling, adhesion, polarization and

transmigration of the PMNs between the endothelial cells of capillaries to reach the periodontal

interstitium [52, 53]. PMNs can then migrate through the tissue following a chemo-attractant

gradient of cytokines produced by pathogenic bacteria, as well as other host cells in the

periodontium, in order to accumulate at the site of the initial bacterial insult. To facilitate this

movement through the tissues, PMNs release MMPs that are capable of degrading the extra-

cellular matrix, thereby allowing easier migration through the blood vessels and into the nearby

tissues of the periodontium [54].

7

As the most abundant leukocytes in blood, the accumulation of PMNs plays a major role in

mediating the innate immune response. This process is also modulated by a variety of

mechanisms. This includes increased production of pro-inflammatory cytokines to allow for

additional increases in the accumulation of cells representing the inflammatory infiltrate [55].

Furthermore, in the early stages of inflammation, in response to the presence of excessive

microbial load, PMNs facilitate the elimination of bacteria within the gingival sulcus. By

utilizing recognition receptors that allow the innate immune system to be able to distinguish

pathogens from host cells, PMNs are able to engulf microbes through a process termed

phagocytosis [12]. Once bacteria have been ingested by PMNs, bacterial degradation can occur,

thereby setting the stage for re-establishment of gingival health [56]. This is accomplished

through the use of lysosomes containing various proteolytic enzymes [57]. These degradation

enzymes include myeloperoxidase (MPO) and nicotinamide adenine dinucloetide phosphate

(NADPH) oxidase. NADPH oxidase neutralizes and helps degrade engulfed material by

transporting electrons into the bacteria-containing phagosomes [58]. This creates a respiratory

burst that results in the formation of superoxide anions (OH.-) [59, 60]. These superoxide anions

can form hydrogen peroxide (H2O2) that when combined with MPO produces hypochlorous acid

(HClO) [61]. Alternatively, H2O2 can combine with superoxide to generate reactive oxygen

species (ROS). These ROS include hydroxy radicals (OH-), singlet oxygen (1O2), and ozone

(O3) [62]. Superoxide dismutase (SOD) is protein and an anti-oxidant enzyme that cells can then

utilize to inhibit the negative impact of oxidative stress. Thus bacterial ingestion can be

completed, while preventing damage to the mitochondria [63]. Another mechanism employed by

PMNs to kill invading pathogens is the creation of extracellular traps known as neutrophil

extracellular traps (NETs). NETs consist of extracellularly released proteolytic proteins in

combination with strands of deoxyribonucleic acid (DNA) that are capable of recognizing,

trapping, and destroying noxious bacteria [64] This process is also mediated by the presence of

ROS, with the suppression of ROS formation having been shown to reduce overall production of

NETs [65]. The release of NETs by PMNs is a terminal event for PMNs, termed NETosis, and is

an alternative to programmed cell death (apoptosis) [66]. Both phagocytosis and NETosis play

an essential role in mediating the innate immune response, and when these protective

mechanisms are in a homeostatic state, they help to reduce levels of pathogens and also to

minimize damage to the tissues.

8

As suggested, as long as the protective mechanisms related to the activity of the innate immune

system are in homeostasis, the net effect of activity of this system is that of protection. However,

in some instances, there is dysregulation of the innate immune response (upregulation generally)

that can actually provoke tissue destruction, a hallmark of active periodontitis [56]. Chronic

inflammation, such as what exists in active periodontal disease, can cause changes in the

oxidative state - the net balance between ROS and antioxidants (AO). An imbalance favouring

excessive ROS production leads to the development of so-called ‘oxidative stress’. Increased

ROS production by oral neutrophils (oPMNs) has been recognized as a central pathobiological

process and risk factor for mediation of the destruction of periodontal tissues [67]. ROS are

known to contribute to periodontal tissue destruction through various mechanisms, including

induction of osteoclastogenesis and osteoclast recruitment, which directly leads to bone

resorption and net loss of bone [68]. This process is mediated by nuclear factor erythroid 2-

related factor (Nrf2), which in response to oxidative stress increases the expression of AO.

However, in patients with CP, reduced production of endogenous AO has been demonstrated,

and this then represents the loss of an important mechanism for protection of tissues against the

damaging effects of higher than normal levels of ROS [69]. As well, failure of the body to

achieve appropriate PMN clearance can prevent the resolution of the inflammatory response, and

thereby, also lead to an increase in the destruction of periodontal tissues [70].

1.2.3 Smoking and PMNs

As discussed previously, it is well known that smoking represents a very significant epigenetic

risk factor for not only the development of periodontitis, but also influences its severity and

reduces treatment response. Various studies have aimed to elucidate the underlying mechanism

that underpins the relationship between smoking and loss of periodontal attachment. While

studies of the microbiome have demonstrated some variations in plaque composition as well as

alterations in vasculature, the ability of smoking to perturb the immuno-inflammatory response

appears to be of even greater importance [71, 72]. Although smoking increases the amount of

circulating PMNs found in the blood stream, a similar elevation in the quantity of PMNs within

the oral cavity is not seen [73, 74]. Therefore, it has been determined that smoking inhibits

transmigration and chemotaxis of PMNs through the periodontal tissues [71]. In this regard,

9

smoking could then interfere with early responses to increased microbial load and result in

progression of biofilm-induced disease. As well, this might represent inhibition of the protection

that could be afforded by a fully functioning innate immune system.

Having said this, it is also known that hyperactivity of the innate immune system contributes to

the advancement and severity of periodontitis. For example, PMNs express the aryl hydrocarbon

receptor (AhR), a cytosolic transcription factor [75, 76]. AhRs are activated by polycyclic

aromatic hydrocarbons (PAHs), such as, 7,12-dimethylbenz[a]anthracene (DMBA), and

benzo[a]pyrene (BaP), which are found in high concentrations in cigarette smoke [77]. It is well

known that if these receptors are over-activated, there is a resulting increase in transcription of

AhR-responsive genes that regulate production of mediators of inflammation in both human

periodontal ligament (PDL) cells and PMNs. This contributes to inflammation and inflammation-

associated destruction of the periodontal tissues due to inappropriate turnover and apoptosis of

cells within the PDL [78]. As well, smoking can cause dysregulation of several endogenous

factors that can have negative consequences. For instance, in smokers, intercellular adhesion

molecule 1 (ICAM-1), a soluble circulating immuno-modulator, has an increased concentration

[79]. ICAM-1 has the ability to bind to β2-integrin complexes expressed on PMNs and thus,

stimulates increased elastase and MMP release [80, 81]. This manifests as an increase in collagen

degradation and tissue destruction within the periodontium. Another process that may be affected

by cigarette smoking is the ability of PMNs to eliminate periodontal pathogens through the

respiratory burst [82]. Research has shown that smoking may enhance the release of ROS, such

as superoxide [83, 84, 85]. While release of superoxides is important insofar as bacterial kill is

concerned, it is also known that when ROS production is dysregulated and thus very high, the

resulting oxidative stress damages local cells and tissues [86]. In fact, this issue is not restricted

solely to the oral cavity, since smokers and individuals diagnosed with various inflammatory

mediated chronic conditions, such as diabetes mellitus, are also known to have high levels of

oxidative stress at the systemic level [87].

Clearly, cigarette smoking has a large negative impact on periodontal health and these effects are

mediated through more than one pathway [71]. Abstinence from smoking is very important, and

is the ultimate goal in improving smokers’ overall health, as well as reducing the severity,

prevalence, and poor response to therapy seen in these patients. However this goal must be

pondered realistically. In this regard, even the best studies focused on smoking cessation

10

therapies (pharmacological, behavioural and both) have produced results that are at most

nominal and in many cases short-lived [88]. Research in this area has shown quit rates of only

2% to 12% [89] and even with those quit rates, the follow-up periods have generally been very

conservative, usually 6 to 24 months [85, 90]. Thus, smoking cessation has at most only modest

effects, and even in the absence of smoking it mustn’t be forgotten that oxidative stress plays an

important role in the progression of periodontitis. Therefore, there is a need to identify ways to

modulate these underlying mechanisms and focus on approaches that could produce

improvements in overall inflammation, and therefore oxidative stress. This should minimize the

damage to tooth supporting structures in all patients and particularly in those who smoke or have

only recently quit smoking given the long half-life of PAHs [77].

1.2.4 Oral Inflammatory Load

Not only are PMNs the most prevalent WBC in the blood; but, they are also extremely prevalent

within the oral cavity. PMNs recruited to the periodontal tissues eventually accumulate in the

gingival sulcus, and gingival crevicular fluid (GCF) [91, 92]. While the presence of oPMNs

within the gingival sulcus is considered to be physiologic, previous research has demonstrated

that the amount of oPMNs present is elevated in patients with periodontitis when compared to

patients with healthy gingiva [93]. The concept of measuring oPMN levels as a method for

measuring disease severity and response to periodontal therapy was first proposed in 1978 [94].

This methodology is based on the fact that unlike plasma cells, PMNs that are present in the

gingival sulcus are continuously washed into the saliva through GCF flow. However, only in

more recent research has the development of a standardized protocol for collection,

quantification, and classification of standardized quantities of oPMNs in health and disease been

examined [95]. The development of a rapid, noninvasive oral rinse method to collect oPMNs,

has previously demonstrated a correlation between the quantity of oPMNs that are present and

the severity of periodontal disease [96]. This led to the concept of oral inflammatory load (OIL),

which is defined as the inflammatory burden from oral inflammatory disease in the mouth and

the body [95]. This measure is quantifiable and has been demonstrated to increase as the severity

of a patient’s periodontal disease increases. This methodology has also been demonstrated to

have high sensitivity (0.83) and positive predictive value (0.94) making it a reliable assessment

11

of active periodontal disease [96]. As well, while probing depth measurements quantify past

destruction, as has been mentioned above, these measurements are not necessarily diagnostic for

the identification of sites that are experiencing or will experience active disease and attachment

loss [9]. By combining clinical parameters (e.g. PDs), with the quantification of OIL, clinicians

may gain additional insight as to the relationship between disease progression and prognosis.

However, since sources of inflammation in the mouth may not be from the periodontal

attachment apparatus exclusively, and can occur in response to the presence of noxious stimuli

affecting other oral tissues, the importance of correlating these measurements with clinical

periodontal measures must be emphasized. Full examination of all oral tissues must therefore be

completed to rule out the presence of other intraoral sources of inflammation such as traumatic

lesions or mucocutaneous diseases that may influence OIL.

1.2.5 PMN Phenotyping

There has been a recent paradigm shift in the understanding of the PMN population of cells.

Researchers now appreciate that there is no ‘single’ PMN phenotype, but rather that there is a

heterogeneity of PMN phenotypes, and in particular a phenotypic spectrum of oPMNs that have

been identified in a number of physiological and pathological conditions [97, 98]. Most recently,

several oral phenotypes have been identified and have been shown to be associated with various

degrees of inflammation. It has been demonstrated that while PMNs are recruited in greater

numbers as part of the pro-inflammatory response, which is associated with ongoing periodontal

breakdown (i.e. active periodontitis), these oPMNs are also found to be more active [99]. As

well, an intermediary state of para-inflammation also exists, which is associated with healthy

gingiva. This so-called ‘para-inflammatory’ state occurs when the body is responding to low-

grade noxious stimuli, but no overt signs of inflammation are clinically present [100]. The PMNs

present in this state are not activated to their full potential. The ability to differentiate amongst

the different phenotypes of PMNs is based on heterogeneic expression of combinations of cluster

of differentiation (CD) markers on the cell surfaces of various PMN sub-populations [101]. CD

markers are formed in granules within PMNs and transported to the cell surface. By fusing with

the plasma membrane, there is a net increase in the number of cell surface receptors, which

12

primes the PMN for increased activity that relates to the individual functions of the CD proteins

thus expressed [102].

To identify various PMN phenotypes, scientists have employed the concept of utilizing specific

antibodies that adhere to certain surface proteins [103]. However, since individual CD markers

can be expressed on a multitude of various cell types, conjunctive use of several CD markers can

be utilized to systematically identify specific cell populations; each with its own unique

assortment of proteins both in presence and amount. This has led to the development of flow

cytometric assessment of PMNs by way of immunophenotyping, to identify specific cell

phenotypes from a heterogeneous PMN population [104, 105]. In the past, there was limited

consistency in regard to the panel of CD markers used to isolate PMN populations. However,

previous research conducted at the University of Toronto, utilized high-throughput screening

(HTS) flow cytometry to identify CD markers consistently expressed on the PMN surface. By

using this approach it is now possible to sort cells using CD11b, CD16 and CD66b markers so

that populations of mixed white blood cells can be purified to the point where 99% of the cells

are shown to be PMNs, regardless of disease state and whether the sample was obtained from

oral rinses or peripheral blood [101]. This panel of CD markers can be utilized to identify and

isolate mature PMNs from patient samples. As well, the gating strategy previously described by

Fine et al. will be utilized to identify the para- and pro-inflammatory PMN phenotypes by

focusing on differential expression of CD markers found on the cell surfaces as alluded to above.

This utilizes gating with CD18+ve to initially isolate PMNs and remove debris from the sample.

Further gating using forward scatter (FSC) and side scatter (SSC) to select for specific cell sizes

and granularity respectively has been shown to further select for PMN populations [106]. As

well, it has previously been shown that oPMNs in patients with CP have increased expression of

CD11b, CD18, CD55, CD63, CD64, and CD66. The functionality of these CD markers can be

grouped into three categories. CD63, CD64, and CD66 are markers of activation/degranulation

[107, 108, 109], while, CD11b and CD18 are adhesion receptors, and CD55 is a complement

inhibitor [110, 111]. Accordingly, the researchers identified PMNs with this CD marker

expression profile as being in a pro-inflammatory state. These PMNs are believed to be more

active and thus more responsible for the deleterious consequences observed in the periodontium

when periodontitis is present. This contrasts with oPMNs isolated from healthy subjects, which

13

display different arrays of CD markers (as well as lower expression of CD markers in general)

and have been classified as being para-inflammatory [106].

1.3 Treatment Approaches for Chronic Periodontitis 1.3.1 Scaling and Root Planing

Scaling and root planing (SRP) is considered the primary initial treatment for patients

experiencing CP. This form of non-surgical therapy focuses on mechanical removal of microbial

biofilms and the calcified products they produce (i.e. calculus) from the surface of the teeth. As

well, it is thought that by use of SRP it is possible to remove surface cemental layers that are

likely impregnated with bacterial LPS and other noxious elements. This reduces the bacterial

load and pro-inflammatory modulators present and thus restores homeostasis in the periodontium

[112]. This is accomplished by reducing the load of etiologic factors below the disease threshold,

such that a patient’s immune system can re-establish a healthy state where inflammation and

tissue destruction is no longer favoured.

Complete elimination of bacterial debris through scaling and root planing alone may be

impossible, since the deeper the initial PD, the more difficult it is to access the root surface for

proper debridement [113]. In fact, Caffesse et al. noted that in sites with PD greater than 5mm,

only 32% of calculus was removed [114]. However, the simple disruption of a mature biofilm

may help to shift a dysbiotic environment to one where homeostasis is supported, and healing

can occur despite the presence of residual etiological factors. In fact, some research has

suggested that there is no magnitude of initial PD where non-surgical SRP is no longer effective

in improving tissue health. Therefore, the impact of SRP on improving periodontal health is quite

large [115]. When comparing SRP to surgical intervention, it was noted over time that both

treatment modalities can result in a reduction in PD and a gain in CALs [116]. As a result, before

any surgical treatment is considered, SRP should be undertaken. As well, since surgery

inherently carries added risks and possible co-morbidities for patients, new adjunctive treatments

are being sought. This would hopefully allow further improvements in periodontal health not

only from SRP alone, but also from newer and hopefully, non-invasive adjunctive treatment

approaches. The best outcomes (absent regeneration) would be to stop progression of disease,

14

and from the perspective of the patient, to reduce the need for periodontal flap surgery. It is also

noteworthy that the beneficial effects of SRP are so profound, that it is often difficult to

determine whether or not a novel adjunctive approach to management of periodontitis can be

shown to produce both statistically and clinically significant improvements versus SRP alone.

This fact makes it more difficult to determine the effectiveness of adjunctive therapies when

done in concert with SRP; a challenging problem.

1.3.2 Antibiotics and Periodontal Treatment

Since periodontal disease is initiated by a microbial etiology, the adjunctive use of antibiotics in

the treatment of CP has been extensively researched. The utilization of antibiotics is aimed at

eliminating residual bacteria that is not removed by SRP alone, because of the ability of

microbes to invade the gingival tissues or due to some areas being inaccessible to

instrumentation [117]. Antibiotics that have demonstrated a slight, but statistically significant

clinical benefit in treating CP include amoxicillin, clindamycin, metronidazole, and tetracyclines

[118, 119, 120, 121]. However, judicious use of antibiotics must be employed due to the

potential adverse side effects. These include but are not limited to, gastrointestinal

complications, anaphylaxis and antibiotic resistance [122, 123]. As a result, while antibiotics

have been shown to provide some clinical benefit in the treatment of CP, the potential risks

necessitate a conservative approach. Since patients usually respond well to SRP alone, antibiotic

usage is typically reserved for cases that are severe, aggressive, or do not respond to

conventional treatment modalities [124, 125]. It is generally recommended that antibiotics only

be utilized in combination with SRP for specific patients (i.e. medically compromised) and when

appropriate, following sensitivity testing, in an attempt to minimize potential negative

consequences [117].

15

1.3.3 Host Modulation

As noted in section 1.3.2, treatment with antibiotics can lead to important improvements in

periodontal health. However, antimicrobial treatment does not always address all of the clinical

problems, particularly in patients with refractory periodontal diseases [126]. In addition, as

mentioned above, there are growing issues pertaining to the stewardship of antimicrobial use that

make it more challenging to support the constant use and re-use of antimicrobials when treating

chronic periodontal diseases [123]. Accordingly, numerous studies have focused on the

pathophysiological nature of periodontitis in order to allow for the development of adjunctive

therapies that can improve upon overall treatment outcomes and do not require the use of

antibiotics [127, 128, 129]. When used either alone or combined with SRP and/or surgery, the

goal is to modify the underlying mechanisms of disease (i.e. the host response), as opposed to

merely focusing on the microbial elements of periodontitis. With regard to this investigation the

concept of ‘host modulation therapy’ will be a major focus. Although it’s recognized that

microbial colonization plays a critical role in the pathogenesis of periodontitis, it must be

reiterated that microbial colonization does not operate in a vacuum insofar as the development

and progression of periodontitis is concerned. In this regard, and as has been made clear above,

the host response to oral microbial colonization on and under gingival tissues, plays a key and

critically important role in the overall disease process [130]. Therefore, host modulation therapy

as an approach to treatment has been studied and developed in order to modify patients’

immuno-inflammatory responses to the presence of specific bacterial challenges [131]. By

reducing pro-inflammatory pathways or promoting inherent processes that assist the body’s

natural inflammation resolution mechanisms, host modulation therapy should reduce the

sequelae of dysregulated or upregulated immune responses seen in patients with CP [132].

Several adjunctive treatments have been suggested to help improve the clinical response in

conjunction with SRP. These include, statin medications, non-steroidal anti-inflammatory drugs

(NSAIDs), and bisphosphonates to name a few [133, 134]. However, all these treatment

modalities possess potentially serious side effects if used systemically, while single time local

application would likely only have transient effects. Potential complications include, but are not

limited to, anaphylaxis, gastric bleeding, and increased cardiovascular risks [135]. As a result,

there has been the need to discover additional remedies that can provide therapeutic benefit by

regulating the dysbiotic host-mediated response to the presence of bacterial biofilms; hopefully,

with fewer reported side effects than other agents.

16

1.4 Resveratrol 1.4.1 Biosynthesis

First isolated in 1939, resveratrol (3, 4’, 5-trihydroxystilbene), a phytoalexin, is a defense

mechanism produced by plants in response to the presence of noxious stimuli [136]. A natural

compound, resveratrol is synthesized from the condensation of one molecule of p-coumaroyl-

COA (4-coumaroyl-COA) and 3-malonyl-CoA by the enzyme stilbene synthase [137]. This

enzyme has been identified in various plant species, including grapevine and as a result,

resveratrol can be found in high concentration in grape skins and thus red wine [138].

Resveratrol has two geometric isomers depending on the configuration of its two phenolic rings.

Existing in either a cis- or trans- form, the trans- configuration is considered to be more

biologically active [139]. Despite its early identification and use as a natural source of

antioxidants, resveratrol was not studied extensively until after 1992, when an article related to

the “the French Paradox”, with a focus on resveratrol, was published. This paper suggested that

wine consumption, and thus, resveratrol intake in the French population was the underlying

reason why consumption of high levels of saturated fat and cigarette smoking did not lead to

higher rates of coronary artery disease in that population [140]. However, resveratrol

concentration in red wine is relatively low, with red wine containing 0.361-1.972 mg/L [141].

Therefore, while the amount of resveratrol present in red wine varies greatly depending on the

type of grape and source region, the average 150mL glass of red wine contains a limited amount

of resveratrol [142]. As a result, in order to ingest a therapeutic daily dose of pure resveratrol

(considered to be about 500 mg), individuals would have to consume over 250 litres of red wine.

This undoubtedly would have significant detrimental health effects. However, it should also be

considered that there are many other similar molecules within red wine and it can be speculated

that these molecules could, in concert, actually have much greater effects than any one molecule

used on its own [143]. Indeed, there is some evidence that more highly purified extracts made

from grape stems and canes can be a valuable source of a variety of oligostilbenoids that, in

combination with resveratrol, have significant antioxidant capacity and might be superior to

resveratrol alone [144].

From a therapeutic point of view then, it is necessary to use higher levels of resveratrol than

might be found in food, including wine, in order to achieve the desired biological effects. In

order to increase intake of resveratrol, numerous commercial and nutritional products have been

17

developed to supplement resveratrol ingestion, either alone or in combination with other

antioxidants, vitamins and minerals [136]. Approved by Health Canada as a natural food

supplement and as an antioxidant, consumption of up to 500mg per day of resveratrol has been

found to be safe for adult human ingestion with few side effects [145]. It has been shown in

various studies that resveratrol has good absorption by passive diffusion in the intestines.

Exhibiting a relatively high first pass effect, limited amounts of resveratrol reach systemic

circulation before being conjugated in the liver into glucuronides and sulfates and eliminated

from the body [146]. However, if ingested at quantities greater than 500mg a day, resveratrol can

induce abdominal pain, nausea, or diarrhea [147, 148]. These symptoms are more commonly

observed at higher doses of resveratrol (2.5 – 5 grams per day), which is higher than maximum

recommended daily dose in the Health Canada monograph [147]. Furthermore, these side effects

relate mainly to resveratrol that has been purified from natural plant sources, most commonly

polygonum cuspidatum (knotweed) due to the presence of a contaminant known as emodin [149].

These potential side effects can be minimized through the biosynthesis of resveratrol in modified

yeast species, such as Saccharomyces cerevisiae, since production of resveratrol by this

methodology does not result in the formation of emodin as a byproduct. While various

resveratrol products vary in absorption, elimination and bioavailability, resveratrol generally

reaches peak activity at 12 hours and its effect is limited following a 48-hour period [150]. Thus

daily administration is commonly recommended to maintain resveratrol’s therapeutic benefits.

1.4.2 Therapeutic Benefits

Since the 1990s, extensive research has been conducted to examine the wide range of biological

properties of resveratrol. Believed to provide benefits through its anti-oxidant, anti-inflammatory

and Sirt1 signal-activating abilities, it has not been possible yet to identify a single mechanism to

explain the potential benefits of resveratrol [136]. However, resveratrol’s ability to target a

variety of biological systems may be the underlying reason for its large potential benefit in

numerous ailments. Resveratrol’s therapeutic and preventive properties have been demonstrated

in animal models for various conditions, including diabetes, cardiovascular disease, osteoporosis,

rheumatoid arthritis and in experimental periodontitis [151, 152, 153]. These promising results

18

have encouraged researchers to investigate resveratrol’s pharmacodynamics further to allow for

use of this natural compound in the treatment of human subjects.

One proposed mechanism to explain these potential health benefits has been that resveratrol has

been shown to minimize the negative effects of ROS. Exposure of PMNs to TNF-α and bacterial

LPS can prime these cells to increase ROS production when exposed to the bacterial peptide f-

Met-Leu-Phe (ƒMLP) [154]. Through inhibition of phophatidylinositol 3-kinase (PI3K), a

mediator in the activation of NADPH, and likely by inactivating the AhR, resveratrol reduces the

response to the presence of ƒMLP, and thus minimizes the respiratory burst [155, 156]. Not only

has resveratrol been shown to inhibit ROS production by PMNs, but it likely also acts by

inducing the upregulation of Nrf2 transcription factor and increasing SOD activity [157]. This

effect is also probably mediated initially through the AhR and through this mechanism,

mitochondrial damage from oxidative stress could be prevented [158]. In addition to its ability to

interfere with production of ROS and to up-regulate endogenous antioxidants, the resveratrol

molecule is itself an antioxidant and can act as a scavenger of superoxide, hydroxyl radicals, and

peroxynitrite on its own [146]. Therefore, there would be an exaggerated antioxidant effect of

resveratrol, and more health benefits, compared to other biologically available antioxidants, such

as vitamin C [159].

Significant research has also been conducted on the effects of resveratrol on bone homeostasis.

Through activation of estrogen receptors, studies in estrogen deficient rats have demonstrated

that resveratrol ingestion can activate osteoblast formation and prevent bone loss [152, 160].

This process is potentiated through the activation of Sirt1, a nicotinamide adenine dinucleotide

(NAD) dependent protein deacetylase. Sirt1 removes an acetyl group from acetyl lysine in both

non-histone and histone substrates. This increases the activation of mesenchymal stem cells and

promotes differentiation to osteoblasts. Binding of resveratrol to Sirt1 causes a conformational

change that increases its enzymatic activity and thus, stimulates increased osteoblast formation

[161]. It has also been shown that in order for RANKL to induce osteoclastogenesis, a supply of

ROS is required. Given the fact that resveratrol can significantly downregulate ROS levels, it

stands to reason that resveratrol would therefore, also inhibit RANKL mediated

osteoclastogenesis and stimulation of osteoclast apoptosis [162]. Thereby, the number of active

osteoclasts present in the periodontium would be reduced and this would interfere with bone loss

as seen in periodontitis. Resveratrol’s potential ability to stimulate osteoblastic activity, while

19

inhibiting the deleterious effects of osteoclasts, is also thought to be the underlying reason that

health benefits have been appreciated in vivo following intake of resveratrol [163, 164]. Other

findings in research studies utilizing resveratrol have shown resveratrol’s ability to down

regulate inflammatory induced biomarkers. These include pro-inflammatory cytokines, such as

IL-1β, IL-6, TNF-α, IFN-γ and IL-17, as well as C-reactive protein (CRP), and triglycerides

[165]. Up regulation of inflammation-reduced biomarkers, such as IL-10, an anti-inflammatory

cytokine has also been shown [136]. Since periodontal disease is an immuno-inflammatory

response to the presence of bacterial biofilms, the potential for host modulation with resveratrol

to negate the negative consequences of this process is of particular interest. It is noteworthy that

these effects occur independent of the deleterious effects of smoking (and hence oxidative stress)

on the periodontium; but, as will be discussed below, they figure significantly in smokers.

1.4.3 Resveratrol and Periodontal Disease

The expectation of positive treatment results is related to the notion that chronic periodontitis is a

disease dependent on PMN recruitment and oxidative stress. As noted previously, resveratrol

causes a decrease in oxidative burst capacity of PMNs, thereby reducing their ability to produce

ROS, while also neutralizing ROS that have already been produced. ROS cause various degrees

of tissue damage and are also required for RANKL-mediated induction of osteoclast

differentiation, meaning that resveratrol-induced reduction in ROS levels or activity would

correspondingly reduce osteoclast formation and the bone loss seen in periodontitis [158]. Other

stimulatory effects on osteo-differentiation would help to promote formation of bone; another

activity that could protect periodontal bone tissues [166]. Resveratrol also reduces the levels of

pro-inflammatory cytokines produced by leukocytes. These activities essentially prevent hyper-

activated PMN-mediated dysregulation of periodontal inflammation and therefore, should also

help to prevent the subsequent destruction of periodontal tissues [145]. These qualities of

resveratrol should therefore, reduce OIL, which would be reflected also by clinical

improvements in patients with CP. In support of the concepts alluded to above, positive

treatment results in humans with periodontitis can be anticipated if they are treated with

resveratrol based on positive preliminary results in animal studies and human trials. A study

conducted by Casati et al. (2013) demonstrated resveratrol’s inhibitory effect on bone

20

destruction during ligature-induced periodontitis in rats. These findings suggest that resveratrol

may be an effective therapeutic option for treatment of CP [165]. This was shown further in a

study conducted by Tamaki et al. (2014) examining the benefits of resveratrol in a ligature

induced rat model of periodontitis. Rats with ligature-induced periodontitis that were taking

resveratrol had higher Sirt1 expression levels and a reduction in AMPK phosphorylation [146].

Since Sirt1 has been shown to be a potent regulator in human PDL cells, activation of Sirt1 by

resveratrol can lead to increased osteoblast differentiation and prevention of periodontal bone

loss [167]. The NF-κB pathway and thus downstream phosphorylation, which is typically

elevated in periodontitis, was also reduced in rats that had ingested resveratrol [146]. This

process may provide an additional explanation for resveratrol’s ability to prevent periodontal

disease. As well, resveratrol contributes to the up regulation of antioxidant defenses through

activation of the Nrf2 pathway. This can lead to increased levels of SOD and a reduction in

inducible nitrous oxide synthase (iNOS) expression [146]. These findings, along with a reduction

in the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, within the

gingival tissues, explain resveratrol’s protective effects in relation to the progression of

periodontitis [136]. In addition to studies demonstrating that resveratrol can be used to prevent

the development of periodontitis and the associated tissue destruction, our laboratory has also

demonstrated that a resveratrol-rich extract made from melinjo seed (melinjo seed extract; MSE),

can actually reverse loss of periodontal tissues in established experimental periodontitis lesions

in mice. This occurred during resveratrol treatment even without removal of etiological factors

such as, plaque and biofilm-coated silk ligatures tied around the test teeth that induce and

maintain experimental periodontitis [168].

1.4.4 Smoking, Resveratrol, and Periodontal Disease

As discussed above, it is known that smokers have generally increased levels of oxidative stress

and therefore, these individuals have increased risk for the development of periodontitis. As

well, if they do develop periodontitis it is more severe than in non-smokers, and treatment in

smokers tends to be more difficult [37]. As alluded to previously, cigarette smoke contains

products of combustion known as PAHs [169]. These bind to and activate their cognate

receptor, the AhR, which leads to stimulation of ROS production, elevation in MMP production

21

and increased expression of pro-inflammatory cytokines [170, 171]. Notably, resveratrol is an

antagonist of the AhR and can therefore, block the deleterious effects of aryl hydrocarbons by

inhibiting their ability to stimulate the AhR. This quality likely explains the ability of resveratrol

to inhibit the formation of ROS by PMNs [162]. In addition, smoke related aryl hydrocarbons

inhibit osteoblast cell differentiation; but resveratrol, through its interactions with the AhR,

mitigates these deleterious effects [166]. It has been shown that this may be due to resveratrol’s

ability to block RANKL production [172]. As a result, smokers, who represent a population that

typically doesn’t gain all the potential benefits of periodontal therapy, may experience additional

resolution of disease from the adjunctive use of resveratrol in combination with routine

periodontal therapy [173]. Through the reduction of smoke-induced inhibition of osteoblasts,

direct inhibition of neutrophil ROS production, and decreased oxidative stress, osteoblastic bone

formation would be increased while osteoclast mediated destruction of bone would be inhibited

[174]. This would have an overall effect of reducing the overall pattern of inflammation and

restoring periodontal health.

1.4.5 Resveratrol and Periodontal Disease in Humans

To date, very limited data exist examining the potential benefits of resveratrol in humans in

regard to periodontal disease. In a recent open label human trial, participants who were smokers,

had generalized severe chronic periodontitis and had responded poorly to treatment were treated

with systemic resveratrol (500mg/d). Routine three-month maintenance therapy was continued

(SRP) and at six months, marked improvement in BOP levels and pocket depth reduction was

observed [175]. While these results were promising, a more systematic randomized control trial

was needed to examine empirically the potential therapeutic benefits of resveratrol in curtailing

the progression of periodontal disease and promoting resolution of periodontal inflammation.

22

Chapter 2 Statement of the Problem

Chronic periodontitis is a widespread condition affecting a large proportion of the North

American population. This condition is initiated by an immuno-inflammatory response, primarily

involving PMNs, to the presence of complex bacterial biofilms. Periodontal disease has been

demonstrated to be a multi-factorial condition that is influenced by various local, systemic,

genetic, epigenetic and behavioural risk factors. If left untreated, CP can lead to significant

destruction of the tooth supporting apparatus and ultimately, tooth loss. This can lead to

functional, behavioural and esthetic morbidities for patients, which can include pain and in some

cases life threatening infections. As such, numerous methods have been examined that can help

with host modulation, affecting a patient’s inflammatory response in the presence of

periodontitis inducing bacteria. One such substance that has been considered is resveratrol.

Produced by plants in response to noxious stimuli, resveratrol, a natural phytoalexin, is a

compound approved by Health Canada as a source of anti-oxidants. Research over the past

several years have highlighted the potential therapeutic benefits of resveratrol in the treatment of

a variety of inflammatory mediated conditions, such as cardiovascular disease, diabetes, and

cancer. One of the proposed methods of action for resveratrol is the reduction of ROS. This is

achieved by minimizing the oxidative burst capacity of PMNs and upregulating SOD to quench

any residual ROS in the tissues. Since periodontal disease is a condition that is dependent on

PMN recruitment and oxidative stress, it is believed that resveratrol can be a useful adjunctive

tool in the treatment of CP. As a result, the goal of this pilot study was to examine the

therapeutic effect of systemically administrated resveratrol on the OIL and clinical parameters in

patients diagnosed with chronic periodontitis. In this proposed clinical trial, it was hypothesized

that in patients with chronic periodontitis, OIL will be further reduced following resveratrol

treatment adjunctive to the accepted non-surgical periodontal therapy, SRP, when compared to

placebo treatment with SRP. This will manifest as an improvement in clinical parameters of

periodontal health, and a reduction of total OIL. As well, through the use of flow cytometry to

achieve appropriate immunophenotyping, a decrease in the proportion of pro-inflammatory

PMNs, and an increase in the amount of para-inflammatory PMN subpopulations was expected.

23

Chapter 3 Materials and Methods

3.1 Subject Recruitment

This triple-blind, randomized, controlled, clinical trial received approval by the University of

Toronto’s Research Ethics Board (Protocol # 32114). Potential participants with severe forms of

chronic periodontitis were recruited from the University of Toronto’s Faculty of Dentistry

through the Graduate Periodontal Department. Potential participants received an initial

comprehensive periodontal examination by the principle investigator. Clinical parameters that

are indicators of inflammation and tissue breakdown were measured at six sites per tooth, using a

periodontal probe. These included: 1) PD, 2) BOP and 3) CAL. Patients who satisfied the

inclusion criteria, and did not meet any of the predetermined exclusion criteria were invited to

take part in the study (Table 1). In order to be eligible for participation in the trial, participants

needed to be over 18 years of age and have active moderate to severe chronic periodontitis,

defined as having at least eight teeth with probing depths greater than 5mm with BOP. Patients

were excluded if their medical history revealed: 1) a history of systemic or mucocutaneous

disease, 2) current pregnancy or breast-feeding, 3) use of chronic anti-inflammatory or antibiotic

medications in the past 3 months or 4) current blood thinner or Vitamin E intake that may

promote gingival bleeding. Patients with short bowel syndrome or a history of high output

ostomies (typically ileostomies), who are more liable to dehydration and diarrhea, were also

excluded. This is related to the fact that these potential adverse effects can occur during

treatment with resveratrol that has been purified from plant sources. This exclusion was required

by the Research Ethics Board for these reasons. However, it is noteworthy and important to

point out that the resveratrol used in this study, being biosynthesized from yeast, did not contain

any of the biproduct emodin, and so in reality there was no reason to expect this side effect to

occur [149]. The primary investigator then presented the study and obtained informed consent.

24

3.2 Randomization and Blinding

Participants who consented to being part of the clinical trial were randomly allocated to one of

two treatment groups: 1) Resveratrol + SRP or 2) Placebo + SRP. The randomization was

performed with the use of a computer generated random number list. This study was

characterized as triple blind since the participants, the principle clinical examiner, and the

individuals who assisted in processing of patient samples were not informed of the treatment

group to which the patients had been assigned. The master patient list was in the sole possession

of an independent clinician, who was responsible for providing monthly quantities of either

resveratrol or placebo tablets to participants in this investigation.

3.3 Resveratrol Administration

Currently there are no generally accepted therapeutic dose ranges for resveratrol. However,

based on the literature noted previously, 500mg per day of resveratrol has been used

overwhelmingly in human trials and this dose was therefore, selected for this study. Purified

trans-resveratrol (Fluxome A/S Evolva) manufactured using a modified strain of S. cerevisiae

was acquired. Both resveratrol and placebo (microcrystalline cellulose) were compounded into

identical non-transparent capsules (Haber’s Compounding Pharmacy, 1584 Bathurst Street,

Toronto, ON). Resveratrol and placebo capsules were prescribed for six months and participants

were instructed to take 1 capsule by mouth, once per day. Bags containing only monthly amounts

of tablets were provided to each participant to prevent misplacement. Patients were encouraged

to return all unconsumed tablets in an attempt to monitor patient compliance throughout the trial.

3.4 Trial Protocol

Participants were asked to attend 10 clinical visits over the course of the 6 month clinical trial.

Patients were initially seen at 2-week intervals for the first 2 months, then monthly from months

3 through 6. Oral rinse samples were collected at all re-evaluation visits (visits 2-10) to be used

for assessment of OIL (See Section 3.5 below). Peripheral blood was collected at baseline (visit

2), four weeks (visit 4), three months (visit 7) and at six months (visit 10). All samples were

25

collected at the beginning of each appointment, prior to any instrumentation, in order to avoid

bleeding that might interfere with the results. All participants received SRP treatment, under

local anesthetic as needed, at baseline and at 3 months. Ultrasonic and hand instruments, such as

curettes and scalers, were used to debride all tooth surfaces in a single session. The trial was

completed at visit 10 during which final SRP therapy was performed. Individualized oral hygiene

instruction was provided at each trial visit and patients were provided with oral hygiene

armamentarium as needed. A more detailed description of the trial design can be found in Table

2. Upon completion of the study, participants were provided with the option to either continue

their periodontal care at the Graduate Periodontal Clinic or go back to their primary dental

provider for continuity of care.

3.5 Oral PMN Collection and Quantification

At each visit, participants were asked to rinse with 3ml of saline water for 30 seconds. Rinse

samples were collected in 3-minute intervals until 30mL total of oral rinse sample had been

acquired. Samples were kept on ice until quantification and preparation for flow cytometry could

be completed. Oral rinse samples were fixed with 3.3ml of 16% paraformaldehyde (PFA), mixed

well, and allowed to fix on ice for 15 minutes. The sample was then split into two 50ml falcon

tubes, diluted with phosphate buffered saline (PBS), and centrifuged for 10 minutes at 2500 rpm.

The supernatant was then aspirated from both tubes, and the combined pellets re-suspended in

10mls of PBS. To remove bacteria, debris, and epithelial cells from the sample, sequential nylon

mesh filtration was performed through two 40µm and two 11µm filters, with each filter being

rinsed with an additional 5ml of PBS to increase the PMN yield. The samples were then

centrifuged again for 10 minutes at 2500 rpm and the supernatant aspirated. All the isolated cells

were then resuspended in 1ml of cold PBS and a Coulter counter (Beckman Coulter, Brea, CA)

was used to obtain a cell count of the PMNs present in 20ml of sample. The remaining sample

was then once again centrifuged for 5 minutes at 2500 rpm. The remaining pellet was re-

suspended in FACS buffer at a concentration of 0.5 million cells per 34.75µl and set aside for

labeling in preparation for multicolour flow cytometry.

26

3.6 Blood PMN Collection and Quantification

Whole blood was collected from participants by way of antecubital fossa venipuncture into

vacuum tubes containing 0.1vol of sodium citrate anticoagulant (Sigma-Aldrich). Samples were

kept on ice until peripheral blood PMN isolation could be completed (within 3 hours). Blood

PMNs were isolated using a one-step PMN isolation solution, as described previously in the

literature [176]. Briefly, 0.5mls of whole blood, which is equivalent to approximately 8 million

WBCs, was added to 55.5µl of 16% PFA. This was fixed for 15 minutes on ice, diluted with PBS

and centrifuged for 5 minutes at 2500 rpm. The supernatant was aspirated and the pellet

suspended in 1-2mls BD pharm lyse for 5 minutes at 4°C. The centrifugation, aspiration and

lysis were then repeated. A blood PMN count was then obtained using a Coulter counter

(Beckman Coulter, Brea, CA). In preparation for multicolour flow cytometry, a concentration of

0.5 million cells per 34.75µl of FACS buffer was formulated and set aside for labeling.

3.7 Multicolour Flow Cytometry

Following isolation, previously prepared peripheral blood PMNs and oPMN samples were used

for analysis of CD marker expression. Samples were initially blocked with 1µl rat serum and 2µl

of mouse IgG. Samples were then stained with 24.5µl of an antibody panel (Table 3) to allow for

appropriate staining and recognition of various CD markers. The staining was completed by

incubating the samples at 4°C for 30 minutes without any exposure to ambient light. The samples

were then washed 3 times to remove any excess stain and re-suspended in 400µl of FACS buffer.

Flow cytometry was then completed to examine PMN phenotype according to previously

described protocols [67, 106]. An LSR Fortessa (BD Biosciences) flow cytometer with channel

voltages that had been manually calibrated using rainbow beads was then used to acquire at least

2x104 gated events. Fluorescently tagged isotype control antibodies were used for each CD

marker in order to subtract auto-fluorescent signals from the mean fluorescence intensities

(MFIs). Gating was then used to eliminate doublets and examine the cells of interest. Flow

cytometry data were analyzed using FlowJo (vX) software and MFI of the various PMN CD

markers was compared between treatment groups. As well, samples were stained in duplicate on

the same day by different technicians to confirm the reproducibility of the results.

27

3.8 Gingival Crevicular Fluid

At each appointment, GCF was collected from each patient at the four deepest periodontal

pocket sites, as determined during the initial exam. Two designated Periopaper® strips were

inserted into the pocket until mild resistance was felt. Strips were kept in the crevice for several

seconds and analyzed, as previously described in the literature, using a Periotron® micro-

moisture meter [177]. Strips were frozen until future enzyme-linked immunosorbent assay

(ELISA) assessments could be performed to examine the presence of MMPs, as well as other

molecules of interest, including inflammatory cytokines. This analysis will be done as part of a

future study and will not be reported as part of this clinical trial.

3.9 Statistical Analysis

Descriptive statistics were analyzed for demographic characteristics. Continuous variables were

demonstrated using mean, medians and standard deviations while categorical factors were

reported in percentages and frequency counts. A one-way ANOVA was performed. A Fisher’s

Exact Test was also done to compare responders with non-responders, as defined as at least a

33% reduction in OIL, between the two treatment groups. All data was analyzed using the

Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL). A p-value of < 0.05 was

used to indicate statistical significance.

28

Chapter 4 Results

4.1 Patient Demographics

Seventeen patients were enrolled in the study from March 2016 to October 2017. Several

patients who qualified to participate in the study declined participation due to the significant time

commitment required as outlined by the clinical trial protocol. Eight of the enrolled patients were

assigned to the SRP plus resveratrol group, while nine were in the SRP plus placebo group.

There were no significant differences found between the 2 groups with regard to age, number of

teeth present, severity of initial periodontal disease, and pre-treatment OIL (Table 4). However,

it was noted that there were significantly more women and fewer smokers in the placebo cohort

compared to the resveratrol group (p<0.05). Although there was no significant difference noted

between the two treatment groups in regard to initial severity in periodontal disease or number of

teeth, to account for some level of inter-patient variability, it was decided that all changes in PD,

BOP and OIL were to be reported as a percent change from each patient’s individual baseline.

To prevent single sites with deeper PDs from masking overall treatment results, PDs were also

examined by looking at proportion of sites that had PDs of 1-3mm, 4-5mm and >6mm

respectively, as opposed to calculating mean probing depths.

Throughout the clinical trial, patients were asked to report any adverse signs or symptoms

associated with the ongoing treatment. While one patient reported generalized fatigue and

another noted darker urine, these were attributed to increasing stress and dehydration

respectively. No other severe side effects were reported by any of the enrolled participants.

All nine participants enrolled in the placebo group completed the clinical trial. However, only

five of the patients taking resveratrol completed the entire clinical trial, since three were lost to

follow up for a dropout rate of 37.5% (Figure 1). The reason for loss to follow up was unknown

for two of the participants since they could not be reached using the contact information

provided, while a third reported a conflicting work schedule that prevented him from attending

further appointments.

29

4.2 Bleeding on Probing

Regardless of treatment group, at 6 months, all patients who completed the clinical trial

demonstrated an overall reduction in frequency of sites with BOP (p<0.05). Further in this

regard, it was found prior to treatment that 70% (± 19%) of sites bled on probing in the

resveratrol treated group, compared to 65% (± 23%) in the placebo group, suggesting similar

inflammation profiles from the perspective of bleeding. By two weeks there were still no

statistically significant differences in bleeding when both groups were compared (Table 5). At 4

weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months and 6 months, the resveratrol group

showed a greater percent reduction in the proportion of bleeding sites, but this was only

statistically significant (p<0.05) at 4 weeks from baseline (Figure 2).

4.3 Probing Depth

Comparing to each patient’s individual baseline, both treatment groups demonstrated a percent

decrease in the proportion of sites that had PDs greater than 6mm (p<0.05). There was a slight

increase in the percent change in proportion of sites that were greater than 6mm in the resveratrol

group at 2 weeks. However, by the end of the 6 month trial, the SRP plus resveratrol treated

participants exhibited a greater percent reduction in the proportion of sites with PDs greater than

6mm compared to the group that received SRP with placebo (Figure 3). However, given the ‘n’,

this was a trend only. It was also noted that there was no statistically significant difference, no

matter the time-point, between the two treatment groups in regards to percent change in

proportion of sites exhibiting PDs 6mm or greater (Table 6). A similar trend was also appreciated

when the examiners looked at the percent change in the proportion of sites that probed 4 to 5mm.

Once again, overall the participants in the resveratrol cohort had a greater reduction in percent

change by the end of 6 months (Figure 4); however, no statistically significant difference was

found (Table 7). For percent change in proportion of sites exhibiting PDs ranging from 1 to

3mm, as expected, the opposite trend was observed in that both groups experienced an increased

percent change following 6 months of treatment. The placebo group was noted to have increased

percent change in proportion of sites with PDs of 1 to 3mm at 4 weeks, 8 weeks, and 3 months.

However, the resveratrol group had greater percent change at 2 weeks, 6 weeks, 4 months, 5

30

months, and 6 months (Figure 5). Once again, statistical analysis demonstrated no statistically

significant differences between the treatment groups (Table 8).

4.4 Oral PMN Quantification and OIL

oPMN counts were also examined to determine OIL. Interestingly, the resveratrol group

experienced an initial increase in oPMN counts compared to the placebo cohort. This trend was

seen at 2 weeks, 4 weeks, 6 weeks, 8 weeks and 3 months. However, differences between the

two groups were only statistically significant at 2 weeks (Table 9). By the end of the 6-month

trial, the resveratrol group demonstrated a greater percent reduction in oPMNs, but this finding

was not statistically significant (Figure 6). Based on previous research on OIL, a 33% reduction

from baseline in oPMNs was demonstrated to be a benchmark with good specificity and

sensitivity that can be used to classify patients as being responders or non-responders to

treatment [96]. As such, 5 individuals out of a total of 9 (56%) patients in the placebo group and

4 participants out of 5 (80%) in the resveratrol treated cohort were deemed to be responders to

treatment based on OIL reduction. While it is evident that a higher proportion of those in the

resveratrol group were responders (80% vs. 56%), a Fisher’s Exact Test showed no statistical

significance between the two groups (p=0.238).

4.5 PMN Phenotype

Utilizing multi-colour flow cytometry, CD marker expression was analyzed on oral and blood

PMNs in an attempt to identify various PMN phenotypes at baseline, 1 month, 3 months and 6

months. There were no significant differences between treatment groups in the expression of

oPMN CD markers CD63, CD66, CD64, CD55, CD16, CD11b and CD18 throughout the trial

period (Figure 7). As well, examining all patients combined, there was no significant difference

in expression of any of the CD markers at any time point compared to baseline. However, it was

noted that overall CD63 and CD66 expression trended lower at the 3-month time point. This also

corresponded to an overall increase in SSC percentage. This may indicate an increase in para-

inflammatory PMNs at 3-months; however, this trend was not statistically significant. The CD

31

marker expression and SSC percentage returned to approximately baseline levels by the 6-month

time point. CD marker expression was also compared between blood and oral PMNs collected

from patients. After the removal of 2 patient outliers from the data analysis, as expected, CD66,

CD64, CD63, CD55, CD18, CD14, and CD11b, had reduced expression on blood PMNs when

compared to oPMNs (p<0.05). CD16 was the only CD marker that had increased expression on

blood PMNs. This is consistent with previous studies examining PMN phenotypes in blood and

oral samples and demonstrates that circulating PMNs are in an inactive state. These PMNs

become primed through a marked change in CD maker expression and are activated for function

only once they leave the vasculature and enter the interstitium.

32

Chapter 5 Discussion

This pilot study aimed to examine the potential therapeutic benefits of resveratrol in conjunction

with SRP in the treatment of periodontal disease in human subjects. As would be expected,

significant improvements in clinical parameters, OIL, and oPMN yields, compared to baseline

for both groups were shown. Thus, all patients received benefit from this clinical trial and it was

reinforced that SRP alone positively influences the periodontium. Indeed, this might be an

important factor when attempting to evaluate adjunctive treatments for periodontitis when SRP is

also being used. While trends towards additional benefit were noted in the resveratrol group

when examining BOP, PDs, oPMN counts and OIL, most of the findings were deemed to not be

statistically significant. Yet these were consistent trends. More importantly, it was noted that

patients who received resveratrol demonstrated an earlier treatment response than those taking

placebo. This was demonstrated at 2 weeks when there appeared to be earlier recruitment of

PMN cells, which are required to mount an initial defense and at 4-weeks with patients on

resveratrol demonstrating significant reductions in BOP compared to those on placebo. As might

be anticipated though, over time these differences were reduced because the effects of SRP are

so profound that ultimately both treatment groups experienced the same clinical benefit [115]. As

such, in order to better appreciate the therapeutic benefits of resveratrol, future studies might be

designed to reduce the use of SRP.

It is also axiomatic that as a consequence of the small sample size obtained for this investigation,

the study was underpowered and statistical significance could not be achieved. This was despite

longer term trends towards superior treatment responses noted in resveratrol-treated patients.

Based on previous research examining other adjunctive periodontal treatments, calculations were

performed prior to initiating the study to determine the sample size needed to provide a power of

80% in detecting intergroup differences in probing depths of 1.0mm at a significance level of 5%

[178]. To achieve sufficient statistical power, 16 patients per group were recommended.

However, due to the potential withdrawal of patients or poor treatment adherence over the course

of the study (approximately 20%), 20 patients per group should have been enrolled for a total of

40 participants. Increasing the overall ‘n’ would have not only resulted in greater statistical

power, but would have also allowed examiners to stratify patients further into responders and

33

non-responders. Given the differences observed in this clinical trial, it would seem that there

would then have been significantly higher numbers of ‘responders’ in the resveratrol-treated

group, as compared to the placebo-treated group.

Several factors were identified to explain why a greater sample size was not achieved in this

clinical trial. Firstly, due to substantial time commitment of 10 appointments required for

participation in the study, numerous patients were not interested in taking part following the

informed consent process and in some cases, even if they did participate initially, they dropped

out later for this reason. Another factor contributing to the smaller sample size obtained related

to the strict inclusion and exclusion criteria. Hence, even patients who had severe forms of

periodontal disease did not qualify to participate due to underlying systemic conditions, such as

hypertension and diabetes, which like periodontal disease, are modulated by the immuno-

inflammatory response. While excluded from this study, patients with these systemic conditions

may also gain significant benefit from adjunctive resveratrol treatment due to the various ways

that this compound can modulate the inflammatory response. Previous research has shown that

simply non-surgical periodontal therapy can result in improvement in the control of these

systemic conditions, such as glycemic control in diabetics [179]. Even a slight reduction in

overall inflammatory load, through the delivery of periodontal treatment, can manifest as

significant reductions in health care costs associated with emergency visits and continual care for

these chronic conditions [180, 181]. If such an impact can be achieved with SRP alone, then

further improvement with adjunctive resveratrol may not only help to improve clinical outcomes

in these patients, but it may manifest as even greater reductions in complications and subsequent

health care costs in the treatment of these systemic conditions. Therefore, further research is

indicated to examine the potential therapeutic benefits of resveratrol in patients with various

chronic conditions, such as cerebral vascular disease, coronary artery disease, and type II

diabetes.

A single examiner was responsible for all data collection at patient appointments and preparation

of samples for quantification and multi-color flow cytometry. It was estimated that 35 hours of

clinical and laboratory time was dedicated for each individual patient enrolled in this pilot study.

As well, due to limited clinic availability and the need to process samples shortly after collection

to maintain the viability of the cells, only a few participants could be enrolled in the study at any

given time. This also prevented the recruitment of patients in greater numbers. Another reason

34

that this study was deemed to be underpowered was due to loss to follow up. A significantly high

proportion of participants dropped out of the resveratrol group compared to the placebo treated

cohort (37.5% vs. 0%). However, the cause for the withdrawal from the study was not reported

by any of the participants to be due to the negative side effects of treatment, and no worrisome

sequelae were noted by any of the patients. Thus resveratrol ingestion at 500mg per day does not

appear to pose any negative health effects.

It should be highlighted that a percent reduction in the proportion of sites with deep PDs and

BOP, as well as a decrease in OIL was noted in the resveratrol group, despite this cohort having

a significantly greater number of smokers. Yet as discussed above, it is known that cigarette

smoking is a known risk factor for periodontal disease and that smokers tend to not respond as

well to periodontal therapy compared to non-smokers. Despite this and possibly because

resveratrol is also an AhR antagonist, the potential of resveratrol to negate the deleterious effects

of smoking on the periodontium cannot be dismissed, even with this small population. As well,

while at baseline there were no statistically significant differences between the treatment groups,

it was appreciated that individually, patients enrolled in the study presented with a range in the

severity of their periodontal disease. As such, the inclusion of several patients who presented

with more moderate periodontitis and who would have been expected to respond well to SRP

alone might have skewed the outcomes, thus preventing examiners from appreciating the full

benefit of resveratrol to reduce OIL. In fact it is probably that more treatment effects from

resveratrol could have been observed in patients with more severe disease; particularly when

mean data were analyzed for both study populations.

It should also be noted that the examiners in this pilot study observed these positive results while

being unable to confirm resveratrol absorption in the treatment group. While participants were

asked to take provided tabs daily, patient compliance was difficult to assess and relied on patient

self-reporting. Oral delivery in the form of a pill is limited by patient compliance and while tests

exist to measure urinary levels of resveratrol, these were not available for use during this study.

As well, access to hospital-based hematological testing was limited in the Faculty of Dentistry

setting. As a result, examination of other known markers of inflammation (such as HS-CRP),

which have been shown to be down regulated in the presence of resveratrol and could have acted

as an indirect assessment of compliance and resveratrol absorption, and even as an indirect

indication of benefits for systemic health, was not possible [165]. Future research should be

35

considered to assess the impact of topical treatments with resveratrol, which could provide a

much more potent, albeit localized antioxidant effect. Inasmuch as resveratrol is non-polar, there

is every reason to expect that it will be absorbed when applied topically and previous in vitro

studies have demonstrated resvertrol’s ability to act locally [182, 183]. Resveratrol can then be

delivered directly to the sites of interest; however, preliminary research would be needed to

determine ideal concentrations to induce an effect. If a different formulation of resveratrol were

successful, this would eliminate the concerns over patient compliance and dosing limitations of

oral tabs.

It is unclear why, on average, resveratrol patients had an increase in oPMN counts compared to

placebo treated individuals following the first administration of SRP, but we surmise that in fact

this is reflective of early recruitment of PMNs required for the initial defense of the host. As

well, the examiners noted that baseline oPMN quantification for certain enrolled patients in the

resveratrol cohort were unexpectedly low at baseline, even in the presence of severe periodontal

disease. As a result, oPMN quantification at subsequent visits was consistently found to be

elevated when compared to these patient’s baseline values. This finding may be due to sampling

error that occurred at the outset of the trial and possibly during the course of the clinical trial.

Coincidentally, the patients with lower than expected initial OIL were both in the resveratrol

group and both dropped out of the clinical trial at 4 weeks and 3 months respectively. As such,

the observation of these outliers with increased oPMN relative to baseline at the beginning weeks

of the trial were eliminated in the final calculations at the 6-month time point. It is also

noteworthy that tissue trauma induced during the delivery of SRP might also account for an

initial increase in oPMN quantification. However, if the elevated response of oPMNs was as a

result of inadvertent injury to the gingival tissues, then an elevation in oPMNs would be

expected to be limited to only a few weeks after SRP and would have occurred in not just the

resveratrol group, and should in fact have been shown in both treatment groups. This theory is

also refuted by the fact that this increase in oPMNs was not appreciated in either treatment group

following the SRP delivered at 3 months. Another possible mechanistic explanation for this trend

is the fact that one of the mechanisms of action for resveratrol is through the reduction of ROS

generation and an increase in ROS scavenging. As a result, ROS-mediated apoptosis or NETosis

may be reduced within PMNs in the resveratrol treated cohort; therefore, actually manifesting as

an initial increase in oPMN levels [184]. Regardless of the mechanism though, these findings

36

still support the notion that resveratrol treatment seems to induce an earlier response to treatment

as compared to placebo. After the 3-month time point, this phenomenon was no longer noted

possibly due to decreased PMN recruitment and the PMNs present in the tissues finally

undergoing natural degeneration and clearance.

This study focused primarily on PMNs due to the availability of a non-invasive collection

technique that has been shown previously to correlate with OIL [96]. However, PMNs are not

the only immune cell recruited to sites of periodontitis and constitute just one cellular component

of the complex immuno-inflammatory response [23]. It should be noted, that reliable,

conservative, non-invasive techniques have not yet been developed to to allow for simple

collection of the other cell types from the gingival tissues. Once such collection methods have

been developed, further research can be conducted to allow clinicians to gain a better

understanding of the interplay of these various cells types in the resolution of periodontitis using

the approaches described here.

Even though a positive treatment response was seen in all patients regardless of treatment group

(as shown by reductions in PD, BOP, and OIL) no definite shift in PMN phenotypes within the

oral samples was noted. Therefore, while the majority of patients experienced some level of

reduction in inflammation, this did not appear to correlate with significant shifts in oPMN

functionality as defined by CD phenotyping. As a result, by the end of the study, the treatment

rendered may only have been able to reduce PMN recruitment, but not overall activity. It is

uncertain based on these findings if this was merely due to the fact that full disease resolution

was not achieved in the patients enrolled in the study due to the severe nature of their CP. To

date, no study has shown a shift in PMN phenotype from disease to health in a single CP patient.

Since patients in this study were only treated with SRP, complete disease resolution may not

have been achieved. Thereby, a shift to a PMN phenotype profile that is typically seen in health

with more para-inflammatory over pro-inflammatory PMNs may not have occurred. However, it

was noted that when looking at patients overall, CD66 and CD63, both markers of activation

were reduced at the 3 month time point. This was correlated with an increase in the proportion of

SSC high PMNs, indicative of oPMNs with reduced degranulation [106]. Therefore, a slight shift

towards a more para-inflammatory, as opposed to a more pro-inflammatory population may have

occurred at the 3-month time point only. However, since this finding was not statistically

significant and not noted at 6 months, then a definitive shift in PMN phenotype cannot be

37

established. Alternatively, these findings may indicate that a more pro-inflammatory phenotype

PMN population may be something inherent in individuals prone to periodontal disease; indeed

they are probably predisposed, genetically, to expression of a pro-inflammatory PMN phenotype.

As such, these patients may not experience a normalization of their PMN phenotype despite

treatment. This would emphasize further the importance of regular treatment to prevent the

deleterious consequences of this chronic condition. In this regard, it might not be possible to alter

phenotypic changes in PMN cells, but at the least it should be possible to obtund their more

damaging activities, possibly through the use of adjunctive resveratrol. Therefore, further

research is needed to fully understand the potential applications and therapeutic benefits of

resveratrol in the treatment of periodontal disease.

38

Chapter 6 Conclusion and Future Directions

Chronic periodontitis is a widespread condition affecting a large proportion of the North

American population. This condition is initiated by an immuno-inflammatory response to the

presence of bacterial microbes arranged in biofilms. If left untreated, periodontal disease can

lead to significant destruction of the tooth supporting apparatus and ultimately, tooth loss. As

such, numerous methods have been proposed that can help with host modulation. One such

substance that has been considered is resveratrol. Based on the outcome of this pilot study, it was

concluded that resveratrol might provide an additional benefit in the treatment of periodontal

disease when combined with SRP delivered at 3-month intervals. This conclusion was based on a

greater improvement in clinical parameters, such as PD and BOP, as well as in a reduction in

OIL based on oPMN quantification. Through its ability to modulate the immuno-inflammatory

response seen in CP, resveratrol may help in the re-establishment of homeostasis in the

periodontal tissues and negate damage of tooth supporting structures.

No side effects were noted from resveratrol treatment, further showing that resveratrol is safe for

human ingestion at quantities of 500mg per day. Therefore, clinicians can recommend resveratrol

intake to patients, particularly those with a more severe amount of destruction to the

periodontium or those with known risk factors that are at a higher likelihood of disease

development, since potential benefits outweigh non-existent risks. This is in contrast to many

host modulation therapies that are currently being used such as COX-2 inhibitors, and

antimicrobials that have well documented negative sequelae, especially when taken for long

periods of time.

By improving clinical and patient-based outcome measures, adjunctive use of resveratrol in the

treatment of periodontal disease, if demonstrated conclusively to be effective, might also help to

reduce surgical need. Although surgical need, as an outcome measure, was not assessed as part

of the study, this parameter could be studied in future studies with larger samples sizes. Although

this is not necessarily a routine clinical measure, it is perhaps one of the most important

outcomes for patients, and has been examined by other researchers in the past [185]. Studies

evaluating this patient outcome should be conducted in the future to further emphasize

resveratrol’s potential therapeutic benefit. As well, while this study included only systemically

39

healthy patients, further investigation should be undertaken to determine if resveratrol could be

beneficial in treating periodontal disease in patients with confounding inflammatory mediated

ailments, such as cardiovascular disease, and diabetes mellitus.

Finally, this pilot study has made significant advancement on demonstrating the potential

benefits of resveratrol use in the treatment of CP. However, the need to repeat this study with a

larger sample size, possibly as a multi-centre trial, to validate these preliminary findings cannot

be over-emphasized. As well, future studies may be able to compound resveratrol into a rinse,

gel, or paste that can be applied either at home or in a dental office. This may allow for increased

concentration of resveratrol, delivered directly to inflamed gingival tissues, and help to minimize

the reliance on patient compliance in taking a capsule on a daily basis. While patients enrolled in

this clinical trial received the standard of care for initial periodontal therapy, for many patients

with severe forms of periodontal disease, non-surgical treatment is commonly insufficient to

achieve full disease resolution in the form of a healthy and maintainable periodontium. As such,

further research where the end point is complete disease resolution should be conducted to see if

a shift in PMN phenotype could be achieved. With complete resolution of the inflammation

present, a change from a more pro-inflammatory PMN population to an increasing para-

inflammatory PMN phenotype, as demonstrated by a difference in CD marker expression, may

be better appreciated. However, the examiners recognize that a more pro-inflammatory PMN

phenotype may be a genetic variant that makes individuals more prone to severe forms of

periodontitis. As a result, further research on the possible genetic determinants of PMN

phenotypes is needed, in addition to focusing on treatment methods that can modulate PMN

function.

40

Figures and Tables

Table 1: Inclusion and Exclusion Criteria

Inclusion Exclusion

• Over 18 years old

• With active moderate and/or severe chronic periodontitis

• Willing to provide informed consent and follow the complete trial design

• Patients who are non-smokers, smokers or past smokers

• Patients which will be suggested for antibiotic therapy as part of their periodontal treatment

• Systemic disease (e.g. blood disorders, immunosuppressive disorders, cancer).

• Patients taking, on a long term basis, anti-inflammatory or antibiotic medications (defined as 30 days or more in the past 3 months)

• Patients in need of pre-medication for dental treatment

• Pregnant and breast feeding women

• Patients with chronic aphthous ulcers, tonsillitis, or mucocutaneous disease (e.g. erosive lichen planus)

• Patients with Short Bowel Syndrome and high-output ostomies

• Patients taking blood thinners and/or large amount of Vitamin E (>30U/day)

41

Table 2: Trial Design

Visit Content

1 Full periodontal exam, recruitment and informed consent process, random assigning of

participants to a study group

2 Scaling and root planing, oral rinse sample & blood collection

3 2 weeks re-evaluation, oral rinse sample collection

4 4 weeks re-evaluation, oral rinse sample & blood sample collection

5 6 weeks re-evaluation, oral rinse sample collection

6 8 weeks re-evaluation, oral rinse sample collection

7 12 weeks re-evaluation, scaling and root planing, oral rinse sample & blood collection

8 16 weeks re-evaluation, oral rinse sample collection

9 20 weeks re-evaluation, oral rinse sample collection

10 24 weeks re-evaluation, scaling and root planing, oral rinse sample & blood sample

collection

42

Table 3: CD Antibody Markers for Flow Cytometry Master Mix

CD Marker Protein - Function Flour Voltage

Channel

Isotype µ l

1 CD66 Ceacam – Adhesion,

degranulation

APC R1 - 660 IgG2a 1.25

2 CD16 Fc-γRIII – Low affinity Fc-

receptor

AF700 R2 - 730 IgG1 1

3 CD11b αM-integrin – Adhesion,

complement receptor

APC-Cy7 R3 - 780 IgG1 1.25

4 CD18 β2-integrin – Adhesion,

complement receptor

BV421 V1 - 450 IgG1 1.25

5 CD55 DAF – Complement

inhibitor

FITC B1 - 530 IgG2a 7.5

6 CD63 Granulophysin – Degranulation

PerCP/Cy5.5 B4 - 695 IgG1 1.25

7 CD64 Fc-γRI – High affinity Fc-receptor

PE Y1 - 585 IgG1 8

8 CD14 Macrophages PE-Cy7 Y5 - 780 IgG1 2.5

24µ l

43

Table 4: Demographic and Periodontal Characteristics per Treatment Group

Characteristic* Resveratrol (n=8)

Placebo (n=9)

Intergroup p-value

Age (years) 48.8 ± 8.2

52 ± 11.7

0.252

Female (%) 1 (12.5%) 7 (77.8%) 0.012

Smoking 0.044

Never 2 7 -

Current smoker 6 2 -

Pack Years 10.6 ± 12.7 4.9 ± 13.2 0.987

Number of teeth 26.6 ± 2.7 26.1 ± 3.1 0.689

% Sites with BOP 70 ± 19 65 ± 23 0.583

% Sites with PD >6mm 14 ± 14 16 ± 14 0.776

Oral Neutrophil Count (x106)**

4.1 ± 3.6

9.2 ± 11.1

0.137

*Mean ± STDev **Neutrophil yield was per 20ml of saliva

44

Figure 1: Flow of Participants through the clinical trial

Chronic Periodontitis Patients n=17

SRP + Resveratrol n=8

Completed study n=5

Lost to follow up

n=3

SRP + Placebo n=9

Completed study n=9

Lost to follow up

n=0

45

Table 5: Percent change in proportion of sites with BOP

Resveratrol Placebo Intergroup p-value

2 weeks* -40 ± 16 -55 ± 35 0.326

4 weeks* -59 ± 22 -33 ± 25 0.045

6 weeks* -51 ± 12 -43 ± 32 0.561

8 weeks* -52 ± 28 -35 ± 22 0.226

3 months* -41 ± 16 -30 ± 25 0.376

4 months* -53 ± 11 -38 ± 23 0.197

5 months* -56 ± 9 -45 ± 21 0.272

6 months* -57 ± 8 -45 ± 22 0.267

*Mean ± STDev

46

Figure 2: Percent change in proportion of sites exhibiting BOP in resveratrol treated patients compared to placebo

-70%

-60%

-50%

-40%

-30%

-20%

-10%

0%

placebo

resveratrol

47

Table 6: Percent change in proportion of sites with PD >6mm

Resveratrol Placebo Intergroup p-value

2 weeks* -15 ± 107 -43 ± 35 0.144

4 weeks* -33 ± 65 -56 ± 27 0.336

6 weeks* -27 ± 34 -56 ± 37 0.129

8 weeks* -50 ± 50 -46 ± 36 0.872

3 months* -17 ± 38 -46 ± 32 0.139

4 months* -65 ± 24 -65 ± 24 0.999

5 months* -80 ± 23 -64 ± 30 0.340

6 months* -77 ± 24 -68 ± 27 0.521

*Mean ± STDev

48

Figure 3: Percent change in proportion of sites exhibiting PD >6mm in resveratrol treated patients compared to placebo

-100%

-80%

-60%

-40%

-20%

0%

20%

40%

60%

placebo

resveratrol

49

Table 7: Percent change in proportion of sites with PD 4-5mm

Resveratrol Placebo Intergroup p-value

2 weeks* -17 ± 13 -41 ± 48 0.216

4 weeks* -43 ± 28 -23 ± 15 0.090

6 weeks* -18 ± 41 -36 ± 33 0.333

8 weeks* -39 ± 33 -23 ± 24 0.286

3 months* -26 ± 20 -19 ± 26 0.503

4 months* -37 ± 34 -24 ± 24 0.400

5 months* -39 ± 35 -34 ± 27 0.771

6 months* -38 ± 30 -23 ± 26 0.336

*Mean ± STDev

50

Figure 4: Percent change in proportion of sites exhibiting PD 4-5mm in resveratrol treated patients compared to placebo

-70%

-60%

-50%

-40%

-30%

-20%

-10%

0%

10%

placebo

resveratrol

51

Table 8: Percent change in proportion of sites with PD 1-3mm

Resveratrol Placebo Intergroup p-value

2 weeks* 24 ± 15 -9 ± 83 0.317

4 weeks* 32 ± 64 42 ± 42 0.690

6 weeks* 40 ± 15 29 ± 61 0.636

8 weeks* 17 ± 61 38 ± 36 0.404

3 months* 32 ± 17 39 ± 43 0.716

4 months* 77 ± 74 48 ± 56 0.429

5 months* 87 ± 85 57 ± 62 0.466

6 months* 83 ± 79 53 ± 81 0.512

*Mean ± STDev

52

Figure 5: Percent change in proportion of sites exhibiting PD 1-3mm in resveratrol treated patients compared to placebo

-60%

-40%

-20%

0%

20%

40%

60%

80%

100%

120%

140%

placebo

resveratrol

53

Table 9: Percent change in oPMNs

Resveratrol Placebo Intergroup p-value

2 weeks* 130 ± 157 -23 ± 43 0.014

4 weeks* 200 ± 350 -22 ± 47 0.078

6 weeks* 109 ± 183 -26 ± 50 0.051

8 weeks* 125 ± 246 -4 ± 72 0.156

3 months* 172 ± 297 20 ± 114 0.180

4 months* -4 ± 69 4 ± 84 0.865

5 months* -23 ± 50 -8 ± 90 0.736

6 months* -50 ± 37 -14 ± 97 0.458

*Mean ± STDev

54

Figure 6: Percent change in oPMNs in resveratrol treated patients compared to placebo

-100%

-50%

0%

50%

100%

150%

200%

250%

300%

placebo

resveratrol

55

Figure 7: CD marker expression of oPMNs in resveratrol treated patients compared to placebo

Degranulation/Activation

0

5000

10000

15000

20000

25000

30000

D0 1month 3month 6month

gMFI

CD63

0

5000

10000

15000

20000

25000

D0 1month 3month 6month

gMFI

CD66

0

200

400

600

800

1000

1200

1400

1600

1800

D0 1month 3month 6month

gMFI

CD64

placebo resveratrol

0

5

10

15

20

25

30

35

40

45

D0 1month 3month 6month

gMFI

SSCHighPercent

56

Complement Immunoregulation

Adhesion

0

1000

2000

3000

4000

5000

6000

7000

D0 1month 3month 6month

gMFI

CD55

placebo resveratrol

0

1000

2000

3000

4000

5000

6000

7000

D0 1month 3month 6monthgM

FI

CD16

0

1000

2000

3000

4000

5000

6000

D0 1month 3month 6month

gMFI

CD11b

placebo resveratrol

0

2000

4000

6000

8000

10000

12000

D0 1month 3month 6month

gMFI

CD18

57

Contributions to the Thesis and Manuscript

Faryn Berger is solely responsible for the content of this thesis. Faryn Berger designed the

experiment, performed the data analysis, interpreted the data and prepared the manuscript.

Faryn Berger was the sole examiner and was the only clinician who recruited, treated and

examined all participating patients. All patient data collection and collection of patient samples

was also conducted by Faryn Berger.

Noah Fine contributed to the project by helping to process patient samples, performing the flow

cytometry portion of the experiments and assisting with data interpretation.

Morvarid Oveisi, Nimali Wellappuli, Chunxiang Sun and Oriyah Barzilay also contributed by

assisting with the processing of patient samples in preparation for flow cytometry.

58

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