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T2D OSTEOCLASTS ARE RESISTANT TO LPS-INDUCED DEACTIVATION

By

DOUGLAS IAN STORCH

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2012

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© 2012 Douglas Ian Storch

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To my grandparents who gave me everything and asked only for love in return, my parents to whom I am eternally grateful, and my beautiful wife Melissa, whose love and

patience is unequalled

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ACKNOWLEDGMENTS

I would like to thank the faculty members of the University of Florida, College of

Dentistry who have had contributed not only to my education but to hundreds of other

student dentists that represent the future of our field. In particular, I would like to thank

Shannon Wallet, Rodrigo Neiva, Theofilos Koutouzis, Peter Harrison, and Ikramuddin

Aukhil for their guidance in this endeavor. Lastly, I would like to acknowledge Luciana

Shaddox, Shannon Holliday, and Joseph Riley for their contributions to my education in

this thesis.

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TABLE OF CONTENTS page

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

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

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

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 11

CHAPTER

1 INTRODUCTION .................................................................................................... 13

2 BACKGROUND ...................................................................................................... 15

The Periodontium in Health .................................................................................... 15

The Periodontium in Disease .................................................................................. 16

Progression of Periodontitis .................................................................................... 17

Chronic Periodontitis ........................................................................................ 19

Aggressive Periodontitis ................................................................................... 20

Etiology ............................................................................................................. 20

Diabetes .................................................................................................................. 21

Classification and Diagnosis ............................................................................. 21

Complications of Diabetes ................................................................................ 23

Effects of Diabetes on the Periodontium .......................................................... 24

Bone ....................................................................................................................... 25

Intramembranous and Endochondral Ossification ............................................ 26

Osteoblast Differentiation and Function ........................................................... 26

Osteoclast Differentiation and Function ............................................................ 26

Bone Metabolism in Periodontitis ............................................................................ 29

Bone Metabolism in Diabetes ................................................................................. 30

3 MATERIALS AND METHODS ................................................................................ 32

Participant Population ............................................................................................. 32

Monocyte Isolation .................................................................................................. 33

Osteoclast Differentiation ........................................................................................ 33

TRAP Staining ........................................................................................................ 34

Osteoclast Stimulation ............................................................................................ 34

Scanning Electron Microscopy ................................................................................ 34

Collagen Telopeptide ELISA ................................................................................... 35

Cathepsin K ELISA ................................................................................................. 36

Soluble Mediator Analysis ....................................................................................... 36

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Statistical Analysis .................................................................................................. 36

4 RESULTS ............................................................................................................... 37

Clinical Characteristics of Participant Population .................................................... 37

Differentiation Potential ........................................................................................... 37

Bone Resorption ..................................................................................................... 38

Extracellular Milieu .................................................................................................. 39

5 DISCUSSION ......................................................................................................... 47

LIST OF REFERENCES ............................................................................................... 53

BIOGRAPHICAL SKETCH ............................................................................................ 63

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LIST OF TABLES

page 4-1 Clinical characteristics of participant population ................................................. 42

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LIST OF FIGURES

page

4-1 Elevated glucose and HBA1c levels in T2D participants. ................................... 43

4-2 T2D does not alter differentiation potential of osteoclasts. ................................. 44

4-3 T2D Osteoclasts are not deactivated by LPS.. ................................................... 45

4-4 Osteoclasts retain precursor functions ............................................................... 46

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LIST OF ABBREVIATIONS

AgP Aggressive periodontitis

CEJ Cemento-enamel junction

CP Chronic periodontitis

ELISA Enzyme-linked Immunosorbent Assay

GAP Generalized aggressive periodontitis

GCF Gingival crevicular fluid

GDM Gestational Diabetes Mellitus

HbA1c Glycated hemoglobin

IFN-γ Interferon gamma

IL-10 Interleukin 10

IL1-β Interleukin 1 - beta

IL-6 Interleukin 6

IP-10 Interferon gamma-induced protein

LAP Localized aggressive periodontitis

LPS Lipopolysaccharide, endotoxin

MCP-1 Monocyte chemoattractant protein-1

M-CSF Macrophage colony stimulating factor

MIP-1α Macrophage inflammatory protein 1α

ND Non-Diabetic Participant

OPG Osteoprotegrin

PDL Periodontal ligament

RANK Receptor activator of nuclear factor kappa-B

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RANK-L Receptor activator of nuclear factor kappa-B ligand

T1D Type 1 Diabetes Mellitus

T2D Type 2 Diabetes Mellitus

TNF-α Tumor necrosis factor - alpha

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

T2D OSTEOCLASTS ARE RESISTANT TO LPS-INDUCED DEACTIVATION

By

Douglas Ian Storch

May 2012

Chair: Shannon Wallet Major: Dental Sciences

Periodontal disease is experienced by 4-40% of the US population. Those patients

that also suffer from Type 2 Diabetes encounter greater alveolar bone resorption for a

variety of reasons. Many studies suggest this results from altered bone and collagen

metabolism in addition to altered host response to a bacterial infection. The osteoclast

is the only cell in the body with the unique ability to resorb bone. Its actions are

controlled by osteoblasts, the cell responsible for producing new bone. However, in

Type 2 Diabetes, the mechanisms of action in response to infection are not clearly

understood. This study was proposed to clarify the role of osteoclasts in increased

alveolar bone loss in Type 2 Diabetics with periodontal disease.

To accomplish this, peripheral blood samples were taken from persons with Type

2 Diabetes (T2D) and Diabetes-free individuals (ND). Monocytes were isolated and

stimulated to become osteoclasts. Once osteoclastogenesis was confirmed, the

samples were placed on bone slices and stimulated further with and without the addition

of bacterial endotoxin (LPS). The samples were evaluated for collagen release, markers

of osteoclasts activity, and markers of inflammation. Our results show T2D osteoclasts

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were not deactivated by LPS and produced a more pro-inflammatory cytokines and

chemokines than ND samples.

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CHAPTER 1 INTRODUCTION

Periodontal diseases are a number of chronic inflammatory diseases that affect

the supporting structures of the teeth. Periodontal disease is experienced by 4-40% of

the US population1-3. Many factors contribute to or a associated with periodontal

disease, including diabetes mellitus. Although the initiation of periodontal disease

requires a bacterial infection, the severity can be greatly affected by the ensuing host

response.

Those patients that also suffer from Type 2 Diabetes encounter greater alveolar

bone resorption for a variety of reasons. Many studies suggest this results from altered

bone and collagen metabolism in addition to altered host response to a bacterial

infection. The osteoclast is the only cell in the body with the unique ability to resorb

bone. Its actions are controlled by osteoblasts, the cell responsible for producing new

bone. Osteoclasts can be influenced by many factors including glucose concentration,

AGEs, bacterial components, and inflammatory mediators, all of which are present in

the oral cavity in patients with diabetes. Therefore, it is plausible an altered milieu alters

the function of osteoclasts, resulting in increased alveolar bone loss. This study was

proposed to clarify the osteoclast’s role in increased alveolar bone loss in T2D

participants with periodontal disease.

We hypothesize that osteoclasts from T2D samples are capable of resorbing more

bone than normal controls intrinsically. Also, these samples will show an augmented

response to infectious stimuli. Therefore the aim of this study was to evaluate the

differentiation capacity of osteoclasts, bone resorbing ability and the extracellular milieu

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in relation to bone resorption. Our aims were evaluated through light microscopy and

ELISA analysis.

To accomplish this peripheral blood samples were taken Type 2 Diabetics (T2D)

and Non-Diabetics (ND). Monocytes were isolated and stimulated to become

osteoclasts. Once osteoclastogenesis was confirmed, the samples were placed on bone

slices and stimulated further with and without the addition of bacterial endotoxin (LPS).

The samples were evaluated for collagen release, markers of osteoclasts activity, and

markers of inflammation. Our results show T2D osteoclasts were not deactivated by

LPS and produced a more pro-inflammatory cytokines and chemokines than ND

samples.

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CHAPTER 2 BACKGROUND

The Periodontium in Health

The periodontium - comprised of the gingiva, the periodontal ligament, root

cementum, and the alveolar bone proper – is responsible for anchoring the tooth to

bone and is also known as the “attachment apparatus”. Gingiva is a mucosa that

surrounds the teeth and covers the alveolar process. Cementum is found on the tooth

root surface and serves as one insertion of the periodontal ligament (PDL), a fibrous

connective tissue. Alveolar bone proper – also known as “bundle bone” lines the tooth

socket and is the other attachment of the periodontal ligament fibers4.

The gingiva microscopically shows two main tissue types in its composition: an

epithelial layer and an underlying connective tissue layer called the lamina propria. The

epithelial layer differs depending on its position in relation to the tooth. Oral epithelium

faces the oral cavity and is the most heavily keratinized. Oral sulcular epithelium faces,

but is not attached to, the tooth and shows a decreased keratinization. The junctional

epithelium attaches to the tooth surface via hemidesmosomes5 immediately coronal to

the connective tissue attachment which proceeds apically along the tooth root surface.

In health, the tissue attachment to the tooth creates a small crevice between the tooth

and the gingiva called the sulcus5.

The PDL attaches to the root cementum just apical to the junctional epithelial

attachment. It is a highly vascularized tissue which joins the root cementum to the

socket walls via Sharpey’s fibers. The PDL also contains nerve fibers, fibroblasts,

osteoblasts, osteoclasts and epithelial cells5.

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Cementum, which covers the roots of teeth is a specialized tissue containing both

hydroxyapatite and collagen, allowing attachment of the PDL4. Deposition of cementum

continues throughout life, resulting in increased thickness and mineralization over time.

Cementum shows different composition in relation to area of the root surface. Acellular

cementum, on the coronal two-thirds of the root surface, contains mainly Sharpey’s

fibers – an essential component to the attachment apparatus. Cellular cementum exists

in the apical one-third of the root and in furcations4.

The alveolar process is a bony extension of both the maxilla and the mandible and

provides the housing for all the teeth. The wall of each socket is lined by a cortical layer

of bone called bundle bone. Cancellous bone is found below the cortical bone. Bone is

a dynamic structure characterized by its mineralized organic matrix of collagenous and

non-collagenous proteins. Bone remodels over time in response to functional demands

as a part of its role in protecting vital organs and serving as a reservoir for minerals

which contribute homeostasis of the body.

The Periodontium in Disease

Periodontal disease refers to number of inflammatory conditions that affect the

supporting structures of the teeth. Depending on the measurement definitions and the

populations studied, periodontal disease may affect 4-40% of the adult population in the

United States1-3. Gingivitis always precedes periodontitis and is widely prevalent in the

United States. Periodontitis is a cumulative condition, initiated by bacteria but

perpetuated by individual host factors6. Susceptibility to periodontitis is enhanced by the

interaction between acquired, environmental and genetic factors which modify the host

response toward putative pathogens7.

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Bacteria, the initiating factor in periodontitis, colonize the sulcus and surrounding

areas in a complex structure called a biofilm. Bacterial products of the biofilm, such as

lipopolysacchride (LPS), are recognized by the gingiva by a family of pattern recognition

receptors called Toll-like receptors8, 9. Activation of this pathway leads to an

inflammatory response called gingivitis, or inflammation of the gingiva. Regular

mechanical removal of all bacterial deposits from the tooth-tissue interface eliminates

signs of inflammation is the primary prerequisite to disease prevention10. When not

removed, the biofilm proliferates and invades the deeper structures of the attachment

apparatus causing destruction of its underlying components. Clinically, the sulcus

enlarges due to inflammation. As the periodontium is destroyed, the junctional epithelial

attachment migrates apically, exposing the root surface to a diseased environment and

creating a periodontal pocket.

In the presence of meticulous oral hygiene, patients can achieve clinically healthy

gingiva. In this state, the gingiva is keratinized and continuous with the junctional

epithelium, which is attached to the root surfaces. Immediately adjacent to this

attachment is the dentogingival plexus of venules which contributes the nutrients and

defenses to the epithelium. The continual presence of bacteria in the gingival sulcus

perpetuates a transudate called gingival crevicular fluid (GCF) containing mainly

neutrophils, plasma proteins, lymphocytes and macrophages.

Progression of Periodontitis

It is important to note that sites with clinically healthy gingiva appear to deal with

the constant microbial challenge without progressing to gingivitis due to several factors

such as an intact barrier provided the junctional epithelium, positive GCF flow which

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mechanically eliminates planktonic bacteria, and perpetual presence of antibodies,

complement, neutrophils and macrophages.

The transitions from clinically healthy gingiva to gingivitis and further to

periodontitis are accompanied by specific clinical and histologic features classically

separated into four phases: initial, early, established, and advanced lesions11. The four

stages represent a continuum on the transition from health to disease and are

sometimes not easily distinguished and are subject to extensive subject and site

variability.

The initial lesion, representative of an accumulation of the biofilm for

approximately 24 hours, shows dilation of the dentogingival plexus which is

accompanied by increased blood flow and permeability of the microvascular beds. This

equates to an appreciable increase in fluid flow into the tissue and GCF flow into the

sulcus.

The early lesion forms following one week of plaque accumulation. Again, marked

fluid exudate translates into increased lymphocyte migration ad neutrophil infiltration. In

this stage, fibroblasts and collagen degenerate to provide space needed for infiltrating

fluid and inflammatory cells. Few plasma B-cells are seen in this stage. In an attempt to

wall off the increasing infection, the junctional epithelium proliferates in an apical

direction. It is during this stage that signs of inflammation are clinically detectable such

as edema and erythema.

After an undetermined time period, the established lesion develops. This lesion,

known as gingivitis, shows more obvious inflammatory changes including leukocyte

infiltrate, plasma cell influx and collagen loss. The junctional epithelium proliferates to

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become the pocket epithelium, which is poorly adherent to teeth and ulcerated. The

lesion may continue in this state or progress to a more destructive stage depending on

host susceptibility

The final stage of the disease process, the advanced lesion, represents chronic

periodontitis. The apical growth of the junctional epithelium in response to increased

microbial deposits creates a deeper pocket, more hospitable to anaerobic bacteria. In

addition to all the features of the established lesion, the advanced lesion includes

alveolar bone loss, severe collagen fiber damage, and migration of the junctional

epithelial attachment apical to the cemento-enamel junction. Plasma cells dominate the

inflammatory response. The lesion continues apically and laterally into the connective

tissue, destroying alveolar bone in its wake.

Chronic Periodontitis

Chronic periodontitis does not represent continual tissue destruction, but

progresses through periods of acute exacerbation resulting in clinical attachment loss.

Progression of attachment loss has been correlated to presence of certain Gram-

negative, anaerobic bacteria including Porphyromonas gingivalis, Prevotella intermedia,

Bacteroides forsythus, Aggregatibacter (formerly Actinobacillus)

actinomycetemcomitans and Treponemadenticola12.

Diagnosis of chronic periodontitis describes both severity and extent of disease.

Severity of chronic periodontitis is designated by slight, moderate, and severe clinical

attachment loss and corresponds to 1-2mm, 3-4mm, and >5mm respectively. Clinical

attachment level is measured with a periodontal probe and is the distance from a fixed

reference point, the cemento-enamel junction (CEJ), to the base of the probable

crevice13. The disease is considered localized if up to 30% of the teeth are affected and

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generalized when >30% of the teeth are involved14. Clinicians usually combine all the

clinical findings into a global statement by assigning a diagnosis for the entire patient,

but can be phrased many different ways depending on the desired level of detail so long

as description aids in communication of disease status12.

Aggressive Periodontitis

Aggressive periodontitis (AgP) is a rapidly progressing disease form characterized

by rapid attachment loss and bone destruction, a non-contributory medical history with a

tendency toward familial aggregation. Secondary characteristics of AgP include

inconsistent amount of microbial deposits in relation to severity of tissue destruction,

elevated proportions of Aggregatibacter (formerly Actinobacillus)

actinomycetemcomitans and/or Porphyromonas gingivalis, phagocyte abnormalities,

hyper-responsive macrophage phenotype, elevated production of prostaglandin E2

(PGE2) and interleukin-1β, and self-limiting progression of disease12, 15. The pattern of

the disease is also diagnostic for aggressive periodontitis and helps distinguish between

localized and generalized forms. Localized aggressive periodontitis (LAP) is defined by

presentation of interproximal attachment loss on at least two permanent teeth, one of

which is a first molar, but involving no more than two teeth other than first molars and

incisors. Generalized aggressive periodontitis (GAP) is delineated by generalized

interproximal attachment loss occurring on at least three permanent teeth other than the

first molars and incisors, showing pronounced episodic destruction of the periodontium.

Etiology

Though bacteria are necessary to initiate the periodontal lesion, its presence is not

always accompanied by the clinical signs or symptoms of the disease. Rather, the

disease development is dependent on a variety of factors. Aside from causal factors,

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risk factors are an aspect of personal behavior or life-style, an environmental exposure,

or an inborn or inherited characteristic which, based on epidemiologic evidence, can be

associated with disease related conditions. Such exposure may be associated with an

increased probability of disease occurrence without necessarily being a causal factor.

Systemic factors modify periodontitis mainly through their effects on the immune

and inflammatory systems. Although the role of systemic diseases modifying the

progress of periodontal disease is complex, it has become consensus that several

conditions contribute to increased prevalence, incidence or severity of periodontitis.

Diabetes

Diabetes, the most common endocrine disorder, is a group of metabolic diseases

representing disorders that affect metabolism of carbohydrates, lipids, and proteins. The

predominant feature of diabetes – hyperglycemia - results from insulin production,

action, or a combination of the two. Insulin is a hormone responsible for regulating

carbohydrate and fat metabolism by liver, muscle and adipose tissue to take up glucose

from the blood and store it as glycogen. The deficiency in insulin action is most

commonly due to either inadequate insulin secretion by β-cells of the pancreas or a

failure to respond (insulin resistance) by the target tissue cells. Those who suffer from

this syndrome experience damage, dysfunction and failure of many organs including

heart, kidneys, eyes, nerves and vasculature. The damage directly results from chronic

hyperglycemia.

Classification and Diagnosis

Diabetes classification is based on pathophysiology of hyperglycemia. Type 1

diabetes (T1D) results from pancreatic β-cell destruction usually leading to insulin

secretion deficiency. Also known as insulin-dependent diabetes (IDDM) or juvenile

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onset diabetes, this disease form represents approximately 5-10% of patients. Its

causes are unknown but several components appear to contribute. Cellular-mediated

autoimmune destruction of the β-cells has been linked to islet cell autoantibodies,

autoantibodies to insulin, autoantibodies to GAD (GAD65), and autoantibodies to the

tyrosine phosphatases IA-2 and IA-2β.Also, the disease has strong human leukocyte

antigen (HLA) associations, with HLA-DR/DQ alleles found to be either predisposing or

protective. The lack of insulin production in these patients means the use of insulin

supplementation is necessary to survive. In its absence, patients will develop a deadly,

acute condition called ketoacidosis, in which the body cannot regulate the byproduct of

fatty acid and protein degradation, ketones.

Type 2 Diabetes (T2D), previously referred to as non-insulin-dependent diabetes

(NIDDM) or adult-onset diabetes, accounts for 90-95% of diabetics. Autoimmune

destruction of β-cells is not seen in T2D. These individuals develop insulin deficiency

due to secretory dysfunction or cellular resistance in the tissue. More specifically, target

cells of insulin lose responsiveness to the insulin hormone. In fact, while T2D patients

have normal or elevated levels of insulin, blood glucose levels remain high meaning

insulin secretion is functioning well. Thus, these patients rarely experience ketoacidosis.

But, hyperglycemia may be undiagnosed for years until symptoms are severe enough to

arise Initially, T2D patients show no signs of β-cell destruction. However, after years of

exogenous insulin supplementation, the pancreas may lose its ability to produce insulin

due to β-cell death.

Another common form of diabetes, gestational diabetes mellitus (GDM), occurs in

approximately 4% of pregnancies in the US, with an onset usually in the third trimester

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of pregnancy. Although mothers often return to normoglycemic state after childbirth, a

history of GDM significantly increases the risk of future T2D development. To meet

normal demands of pregnancy, insulin secretion must meet 1.5-2.5 times

normoglycemic conditions. Women with limited beta-cell reserve may be incapable of

compensating for this increase, resulting in hyperglycemia.

Symptoms of diabetes include polyuria, polydipsia, polyphagia, fatigue, weight loss

and blurred vision among others. Diagnosis of diabetes requires positive laboratory

values confirmed on two subsequent days from the following tests: plasma glucose

concentration >200mg/dL (>11.1 mmol/L) any time of day without regard to the previous

meal; fasting plasma glucose (no caloric intake for >8 hours) >126 mg/dL (>7 mmol/L);

or 2-hour post-load glucose >200 mg/dL (>11.1 mmol/L) in an oral glucose tolerance

test using 75g anhydrous glucose16.

As a result of higher average blood glucose over time, non-enzymatic binding of

glucose to hemoglobin yields a highly stable molecule, glycohemoglobin. This binding

lasts the lifespan of the erythrocyte and its estimation provides an accurate

determination of the average glucose levels over the preceding 1-3 months. Known as

HbA1C, glycated hemoglobin levels correlate well with the development of diabetic

complications17. The recommended HbA1c target value for being a well-controlled

diabetic is <7.0% (normal is <6%)18. However, a recent study showed only 36% of

people with T2D achieve this target19.

Complications of Diabetes

Complications of diabetes can be acute or chronic. Acute complications result

from an overabundance or an extreme lack of insulin. Diabetic ketoacidosis, an acute

complication resulting from a lack of insulin causing the body to metabolize fatty acids

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resulting in highly acidic ketone bodies in the blood, is the leading cause of diabetes

related fatalities20. Hyperglycemia can lead to a metabolic abnormality causing extreme

dehydration, lethargy, and coma requiring hospitalization18. Hypoglycemia, a far more

common and but equally lethal condition, results from insulin excess causing an unsafe

drop in blood glucose levels leading to confusion, anxiety, dizziness, seizures and even

loss of consciousness18.

Chronic complications can divided into macro- and microvascular complications.

Macrovascular complications include cardiovascular disease. Microvascular

complications include nephropathy, retinopathy and neuropathy18.

Effects of Diabetes on the Periodontium

Periodontal disease has been called the sixth complication of diabetes21. Diabetes

has been described as a risk factor for gingivitis and periodontitis across all age groups,

with the level of glycemic control an important determinant in the relationship22, 23.

Diabetes has been associated with an increased incidence of periodontitis compared to

non-diabetics, showing an ability to increase initiation of alveolar bone loss in

diabetics24. In addition, well-controlled diabetics have a decreased risk of disease

progression and respond better to periodontal therapy. Poor glycemic control yields a

higher prevalence and severity of gingival inflammation in both T1D and T2D subsets of

children, adolescents and adults25-28. Longitudinal studies confirm these observations29-

31.

Though increased inflammation is responsible for a more destructive periodontal

condition, its source is not related to differences in the microbiota between diabetics and

non-diabetics because none have been shown to exist32, 33. Instead, this suggests that

the chronic biofilm-induced wound produces an altered host response in diabetics.

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Diabetes results in changes in the function of innate immune cells such as

neutrophils, monocytes and macrophages.34 Adherence, chemotaxis and phagocytosis

are impaired in neutrophils which may inhibit bacterial killing in the periodontal pocket35-

37. In addition, neutrophil activation and priming may result in increased tissue

damage38, 39. In response to periodontopathogens, the upregulated

monocyte/macrophage cell line produces a prolonged response as well as increased

pro-inflammatory cytokines and mediators, namely IL1-β and TNF-α40-42. This

increased inflammatory response is evident in the GCF, more so in poorly-controlled

diabetics43.

Progressive periodontal bone loss in diabetics is also related to changes that alter

the balance between resorption and deposition phases of bone metabolism. Changes

such as impaired osseous healing, inhibition of osteoblastic proliferation and function,

and decreased collagen function and deposition are results of hyperglycemia that

accelerate tissue destruction44.

Bone

Bone is a dynamic structure characterized by its mineralized organic matrix of

collagenous and non-collagenous proteins. Bone remodels over time in response to

functional demands as a part of its role in protecting vital organs and serving as a

reservoir for minerals which contribute homeostasis of the body. The remodeling

process is a complex continuum of resorption and deposition in which bone is degraded

by osteoclasts and produced by osteoblasts in the form of the bone multi-cellular units

(BMU). Osteoblasts and osteoclasts communicate through a variety of stimulatory and

inhibitory pathways in order to meet these demands.

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Intramembranous and Endochondral Ossification

When a fracture occurs, homeostasis is disrupted. Clot formation and localized

ischemia signal an inflammatory cascade. Osteoclasts are signaled to help removed

damaged bone. At a distance from the fracture but in contact with existing blood supply,

osteoblasts directly deposit new bone in a random structure (woven bone) which is

remodeled by osteoblasts and osteoclasts, recreating the original lamellar structure of

bone45. At the fracture and its immediate vicinity, endochondral ossification occurs

where the blood supply is disrupted. Fibrous tissue, initially formed by fibroblasts, is

mineralized by chondrocytes resulting in a cartilaginous region around the fracture. This

is then removed by osteoclasts and finally replaced by woven bone laid down by

osteoblasts.

Osteoblast Differentiation and Function

Bone metabolism is driven by a variety of signals. The environment produced by

fracture, inflammation or infection provides a stimulus for the chemoattraction,

migration, proliferation, and differentiation of mesenchymal cells into osteoblasts.

Osteoblasts originate from mesenchymal progenitor cell after stimulation by bone

morphogenic proteins (BMP) and are stimulated to proliferate by platelet-derived growth

factors (PDGF), insulin-like growth factors (IGF), and fibroblast growth factors (FGF) –

all members of the transforming growth factor-β family of signaling proteins. Osteoblasts

are responsible for producing a variety of proteins, including type 1 collagen that forms

mineralizable bone matrix.

Osteoclast Differentiation and Function

Resorption of bone is carried out by osteoclasts. Osteoclasts share a cell lineage

with macrophages and dendritic cells, the hematopoietic stem cell. When stimulated by

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two factors produced by osteoblasts, macrophage colony stimulating factor (M-CSF)

and receptor activator of NFκB ligand (RANK-L) , these monocytes commit to

osteoclastogenesis46. The receptor for M-CSF is c-fms on osteoclasts precursors and

activates signaling through MAP kinases and ERKs during early differentiation47. RANK-

L binds to its receptor on the osteoclasts precursor, RANK, and induces differentiation

into mature osteoclasts via NFκB, c-Fos, phospholipase Cγ (PLCγ) and nuclear factor of

activated T cells c1 (NFATc1) pathways48. Upon further stimulation with RANK-L, fusion

of pre-osteoclasts yields multi-nucleated mature osteoclasts.

Bone degradation occurs upon osteoclasts binding to the bone via a series of actin

ring-containing podosomes, as well as integrins, creating a sealed, isolated

microenvironment to facilitate resorption49. Secretion of HCL produced by a vacuolar

H+-ATPase (V-ATPase) dissolves the mineral portion of the bone49. This activates

cathepsin K, an enzyme necessary to cleave type 1 collagen. Products of this process

are endocytosed by the osteoclast released at the opposite membrane of the cell49.

The cycle ends when osteoblasts produce osteoprotegrin which binds to RANK,

preventing further stimulation of the osteoclast.

It is important to note that both bone-regulating cells share precursors with

immune cells, both of which begin in the bone marrow. The bone marrow, in this regard,

is considered a “loosely compartmentalized lymphoid organ” in which immune cells and

bone cells interact intensely50. Many of the soluble mediators of immune cells, including

cytokines, chemokines, and growth factors, regulate the activities of osteoblasts and

osteoclasts, producing a wide variety of physiologic and pathologic effects.

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The RANKL-RANK-OPG signaling axis plays a central role in osteoimmunology.

Once stimulated by MCSF, pre-osteoclasts begin to express RANK, which shares high

homology with CD40. RANKL is the osteoclast differentiation factor, but can be inhibited

by its decoy, osteoprotegrin (OPG)51, 52. Increase in the RANKL/OPG ratio shifts bone

homeostasis into bone degradation pathway, leading to bone loss. RANKL is expressed

mainly by osteoblasts in response to many signals including 1,25-dihydroxyvitamin D3,

prostaglandin E2 (PGE2), and parathyroid hormone53. T-cells express soluble RANKL

which can also activate osteoclastogenesis and may play a significant role in

inflammatory regulation through the adaptive immune system48, 54.

Inflammatory cytokines that are produced by macrophages such as IL-1, TNF and

IL-6, promote osteoclastogenesis and are considered osteolytic cytokines on the basis

of their bone resorbing effects53, 55.

Interleukin-6 (IL-6) is a multipotent cytokine with a wide variety of activities. It has

been shown to regulate development of mature osteoclasts56 as well as directly

stimulate the production of RANK-L and OPG57. IL-6 is produced by a variety of cells

including T cells, monocytes, epithelial cells, fibroblasts, osteoblasts/stromal cells,

synovial cells, various cancer cells, and also mature osteoclasts58-60.

Interleukin-10 (IL-10) is known as an anti-inflammatory cytokine partly due to its

inhibitory function on osteoclastogenesis and osteoblastogenesis. IL-10 causes

decreased NFATc1 expression, the key transcription factor in osteoclastogenesis61.

Also IL-10 directly inhibits RANK-L production while enhancing OPG production

resulting in decreased osteoclastogenesis62.

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Several chemokines contribute to osteolclastogenesis. Monocyte chemoattractant

protein-1 (MCP-1, CCL2) is highly expressed in osteoblasts in inflamed sites and

enhances osteoclasts formation63-65. Macrophage inflammatory protein-1α (MIP-1α,

CCL3) directly stimulates osteoclastogenesis through its receptors66, 67. It may stimulate

motility but suppress osteoclasts resorptive activity68.

Another chemokine, IP-10, is secreted by several cell types, including monocytes,

endothelial cells and fibroblasts, in response to IFN-γ. IP-10 may serve as a

chemoattractant for inflammatory cells such as monocytes/macrophages and T cells

and is involved in bone erosion69.

Bone Metabolism in Periodontitis

The pathogenesis of periodontitis depends upon the immune and inflammatory

response to bacterial colonization of the gingival sulcus7, 70. An initial activation of the

innate immune system is amplified in periodontitis, resulting in an expansion of the

inflammatory response approximating the alveolar bone71-73. The response is modified

by a variety of osteoimmune cells abundant in the periodontium such as osteoblasts,

fibroblasts, PDL cells and T and B lymphocytes, all showing capability of RANKL

expression through a variety of cellular mediators74, 75. In addition, the production of

bacterial endotoxin, lipopolysaccrhride (LPS) can also stimulate RANKL production

through toll-like receptor pathways8. As mentioned previously, RANKL is one of the

master regulators of bone metabolism, along with its receptor (RANK) and antagonist

(OPG). Increase in the RANKL/OPG ratio leads to alveolar bone loss. It has also been

shown that an increase in the RANKL/OPG ratio may be associated with increasing

severity of periodontitis76-79. It is clear that bacterial presence in the gingival sulcus of a

30

susceptible host can lead to an exacerbated inflammatory response and, thus, alveolar

bone loss through the RANK/OPG pathway.

While the vast majority of studies have demonstrated that LPS may directly

stimulate osteoclastogenesis, a more recent study showed this direct stimulation only

occurs in the presence of RANKL. Without the support of RANKL, LPS instead inhibits

osteoclastogenesis80.

Bone Metabolism in Diabetes

In diabetes, the state of bone is altered, leading to increased fracture risk, delayed

healing, and potentially non-union, regardless of greater bone mineral density. 81-84 A

vast amount of research has shown how diabetes alters bone in a variety of subject

types, mainly for T1D. Osteopenia, and even osteoporosis, is found in both humans and

animal models, showing thinner bone cortex, mineral content and diminished

mineralization and bone formation rate85-88. In addition, reduced blood flow due to poor

microvasculature contributes to poor bone quality89.

Cell-specific changes have also been found. Decreased osteoblasts number and

activity was observed in T1D rodents, leading to decreased osteoid in new bone

formation and diminished collagen production90-92 Both chondrocytes and mesenchymal

stromal cells display down-regulation, diminished proliferation, and decreased

differentiation45. Osteoclasts in T1D humans and rats have shown both increased

numbers and function87, 88, 93. In T2D, poorer bone quality was observed, displaying

increased risk of fracture84. Decreased osteoblasts and thus osteoid surface was seen

in a T2D rat model94.

As stated previously progressive periodontal bone loss in diabetics may be related

to changes in bone metabolism resulting from altered cellular functions44. Naturally,

31

focusing on the cell responsible for bone degradation will elucidate its role in increased

alveolar bone loss among T2D persons. Our overall hypothesis is that osteoclasts

inherently experience altered differentiation and function resulting in increased alveolar

bone resorption. Furthermore, we hypothesize this phenomenon will be greater when

stimulated additionally with LPS.

32

CHAPTER 3 MATERIALS AND METHODS

Participant Population

The Institutional Review Board (IRB) for protection of human subjects at the

University of Florida approved this protocol. All data and samples were obtained under

informed consent. Participants were recruited from the University of Florida, College of

Dentistry undergoing comprehensive dental care. Participants for the experimental

group were selected based upon the following inclusion criteria: subjects aged 13-75

years old, patients diagnosed with Type 1 Diabetes (T1D) or Type 2 Diabetes (T2D)

without diabetic ketoacidosis, currently under the care of a physician, non-smoker.

Participants in the control group (normoglycemic) were selected upon the following

inclusion criteria: subjects aged 13-75 years old, patients who have never been

diagnosed with Type 1 Diabetes (T1D) or Type 2 Diabetes (T2D), non-smoker.

Participants were excluded from the study based on the following criteria: diabetic

complications such as proliferative retinopathy, macrovascular diseases or kidney

and/or liver failure, clinically significant neurological, hepatic, renal, gastrointestinal,

hematologic, dermatologic, metabolic, autoimmune or immune deficiency diseases that,

in the opinion of the principal investigator and/or study physician, would affect the safety

or compliance or that could influence the course of either periodontal disease or diabetic

care and glucose control, history of Sjogren’s syndrome, parotid or submandibular gland

disease, received and immunosuppressive, antibiotic or glucocorticoid therapy over the

last 6 months; current smokers; oral tobacco use, bisphosphonate drug use.

Venous blood samples (30mL) were collected from all participants. Prior to

monocyte purification, blood glucose levels and glycated hemoglobin (HbA1C) were

33

measured. Blood glucose levels were measured with the Ascensia Contour blood

glucose meter (Bayer). Glycated hemoglobin was measured using an AC1Now meter

(Bayer).

Monocyte Isolation

Peripheral blood monocytes were isolated from whole blood using Ficoll-Plaque

and centrifugation. Briefly, whole blood samples were divided equally into 50mL conical

tubes containing 10mL of a salt solution [1 part 1.0g/L Anhydrous D-glucose, 0.0074g/L

CaCl2x2H2O, 0.1992g/L MgCl2x6H2O, 0.4026g/L KCL, 17.565g/L Tris, ConcHClpH7.6 :

9 parts0.14M/L NaCl]. Diluted blood samples were overlayed onto 15mL of Ficoll-

Plaque (General Electric) and centrifuged at400xg for 40 minutes. Following

centrifugation, the interface was removed and washed three times with a salt solution.

The resulting cell pellet was resuspended in α-MEM complete media (Sigma Aldrich)

and cells counted using a hemocytometer.

Osteoclast Differentiation

Purified peripheral blood monocytes were seeded in a T25 flask at a concentration

of 1.5x106 cells/mL supplemented with 10mL α-MEM complete media (Sigma Aldrich)

and 10ng/mL recombinant human M-CSF [rhM-CSF] (Peprotech) and allowed to culture

for 14 days at 37C and 5% CO2 with media refreshed every 3 days. After which, non-

adherent cells were removed and adherent cells seeded at 5.9x105 cells/mL in 24-well

plates on either glass coverslips (Fisher) or 1 cm2 bovine bone slices cut with an Isomet

Low Speed Saw (Buehler). All cultures were supplemented with 10ng/mL rhM-CSF and

50ng/mL recombinant human soluble RANK-L [rhsRANK-L] (Peprotech) and allowed to

culture for 12d with complete media containing rhsRANK-L and rhM-CSF refreshed

every 3d.

34

TRAP Staining

After 12d of differentiation, cells plated on glass coverslips were fixed with 2%

paraformaldehyde/PBS (Fisher) for 15mins. Cells were washed 2 times with PBS and

permeablized for 10mins in 0.5% Triton X-100/PBS (Fisher). Cells were washed and

probed for leukocyte acid phosphatase (TRAP) [1:1:1:2:4 Fast Garnet GBC Base

Solution:Sodium Nitrite Solution:Napthol AS-BI Phosphate Solution:Tartrate

Solution:Acetate Solution] (Sigma Aldrich) for 1 hr in dark at 37C. Cells were washed

with dH2O and glass coverslips mounted on glass slides with MOWIOL 4-88 solution

(Calbiochem). TRAP positive cells [purple in color] were counted according to number

of nuclei present: pre-osteoclasts [1 nucleus], multinucleated osteoclasts [2-10 nuclei],

and giant osteoclasts [11+ nuclei] using light microscopy at 40x magnification.

Osteoclast Stimulation

After 12d of differentiation, media was refreshed with α-MEM complete media

supplemented with 10ng/mL rhM-CSF and 50ng/mL rhsRANK-L. Cells were allowed to

resorb bone for 72hrs in the presence or absence of 1ug/mL Escherichia coli LPS

(Sigma). Cultures were permeablized with 1% Triton X-100 for 10mins. Supernatants

were stored at -80C until cathepsin K ELISA, collagen type I telopeptide ELISA, MMP

ELISA and soluble mediator analysis were performed. Bone was made devoid of cells

with 10% sodium hypochlorite/PBS for 10mins after which they were washed with PBS

and stored in Trump’s fixative (Fisher) at 4C until scanning electron microscopy [SEM]

could be performed.

Scanning Electron Microscopy

Bone slices were sputter-coated with gold and visualized with S-4000 FE-SEM

scanning electron microscope (Hitachi). Three random scanning electron micrographs

35

(8-bit grayscale) of bone slices were acquired at 40x magnification with a 2048x1594

resolution and pits were defined as “scalloped” areas with clearly identifiable borders.

Identical procedures were applied to every image from all experimental groups utilizing

NIH ImageJ software to quantify the surface area resorbed. Prominent repeating

elements in the frequency domain were identified and removed after which the inverse

fast Fourier transformation was applied yielding the original image with reduced saw

marks. The CLAHE algorithm 95 was used to increase contrast (block size: 256,

histogram bins: 255, maximum slope: 8), and a Gaussian blur (σ: 8 pixels) was applied

to the result. The rolling ball algorithm96 was applied (radius: 100 pixels) to achieve

background intensity equalization. A threshold value (77) was used to convert the result

to a 1-bit image (0: normal bone, 1: region of resorption) used for quantitative analysis.

Images which contained prominent artifacts spanning 5% or more of the total area were

not included for analysis.

Collagen Telopeptide ELISA

Collagen carboxy-terminal telopeptides were detected using an ELISA according

to manufacturer instructions (Immunodiagnostic Systems). Supernatants were pre-

incubated with both a biotin conjugated anti-telopeptide and a horseradish-peroxidase

[HRP] conjugated anti-telopeptide and incubated 2h in an ELISA plate coated with

streptavidin [SAV]. Following five washes, a tetramethylbenzidine [TMB] substrate was

used to develop for 1hr followed by quenching with H2SO4. Colorimetric reactions were

detected using a Benchmark Microplate Reader spectrophotometer (Bio-Rad) set at a

dual wavelength reading of 450nm with a reference of 655nm. Microplate Manager

Software (Bio-Rad) and a standard curve were used to determine nM concentrations.

36

Cathepsin K ELISA

Active cathepsin K was detected using an ELISA according to manufacturer

instructions (Alpco). Supernatants pre-incubated with HRP-conjugated anti-cathepsin K

were added to an ELISA plate pre-coated with polyclonal sheep anti-cathepsin K and

allowed to incubate overnight. Following five washes, a TMB substrate was used to

develop for 30mins followed by quenching with STOP solution. Colorimetric reactions

were detected using a Benchmark Microplate Reader spectrophotometer (Bio-Rad) set

at a dual wavelength reading of 450nm with a reference of 655nm. Microplate

Manager Software (Bio-Rad) and a standard curve were used to determine pM/L

concentrations of active cathespin K.

Soluble Mediator Analysis

Cytokines and chemokines from resorption supernatants were detected and

quantified using a human14-cyto/chemokine multiplex (Millipore). Supernatant and

antibody-coated beads were allowed to incubate overnight at 4C in a 96-well primed

plate. Following three washes, biotinylated detection antibodies were allowed to

incubate for 1h, after which SAV-phycoerythrin [PE] was allowed to incubate for 30mins.

All incubations occurred while gently shaking. Following three washes, beads were

resuspended in sheath fluid and reactivity acquired using a Luminex 200 IS system with

Xponent software (Millipore). Milliplex analyst software (Viagene), 5-parameter logistics

and a standard curve were used to determine pg/mL concentrations.

Statistical Analysis

One-way ANOVA with Bonferroni’s multiple comparisons as well as one-tailed

unpaired t-tests with Welch’s correction were used to analyze and determine statistical

significance (p<0.05) as appropriate.

37

CHAPTER 4 RESULTS

Clinical Characteristics of Participant Population

The characteristics of the experimental and control groups are detailed in Table 4-

1. The control group (normoglycemic participants) consisted of 7 males and 8 females,

age 28.1 + 5.57. The experimental group (T2D participants) consisted of 14 males and

14 females, age 54.2 +16.87.

In order to determine whether the glycemic status effects osteoclast differentiation

or function, peripheral venous blood was drawn on the participant population and blood

glucose (mg/dL) and glycated hemoglobin (HbA1c) was determined on each participant

sample. (Figure 4-1) Although T2D participants show elevated non-fasting glucose

(NFG) and glycated hemoglobin (HbA1C) in comparison to control groups indicating

some degree of insulin resistance the reported levels are also indicative of well

controlled diabetics.

Differentiation Potential

Since T2D patients with periodontal disease experience excessive alveolar bone

resorption we sought to clarify whether increased destruction was due to higher

numbers of osteoclasts in the infection site or an enhanced ability of osteoclasts to

become stimulated and resorb more bone. First we sought to determine the

differentiation potential of osteoclasts from T2D and ND participants under

normoglycemic culture conditions. Peripheral monocytes were isolated from venous

blood samples and differentiated into osteoclasts in the presence of M-CSF and RANK-

L for 26 days. After TRAP staining, samples were evaluated under magnification to

determine presence of multi-nucleated osteoclasts (2-10 nuclei) and giant osteoclasts

38

(11+ nuclei). (Figure 4-2 A, B) Results show no significant differences in differentiation

of monocytes into either multi-nucleated cells or giant osteoclasts, indicating that well-

controlled T2D does not alter the differentiation potential of osteoclast precursors

(Figure 4-2 C,D).

Bone Resorption

Although differentiation may not be altered, it is possible that mature osteoclasts of

T2D participants have an enhanced ability to be stimulated to resorb bone. Bone

resorption occurs as cathepsin K and MMP-9 degrade collagen fibers, the products of

which are released into the extracellular milieu. In order to determine the bone

resportive potential of T2D, osteoclasts, cultures were differentiated on bovine bone

slices and stimulated with rhsRANKL (Figure 4-3). As a measure of bone resorption,

collagen (Figure 4–3 A) release in the culture supernatant was measured. No significant

differences in the amount of collagen in supernatants were observed. As an additional

measure of osteoclast function the levels of cathepsin K and MMP9 were also evaluated

in the supernatants (Figure 4-3 B,C). Again, no significant difference was observed in

the levels of cathepsin K (Figure 4-3 B), although OCs derived from T2D participants

produced significantly higher levels of MMP9 (Figure 4-3 C).

Bacterial components can act as the inflammatory stimulus which activates bone

resorption. However, in the absence of supporting cells, osteoclasts can respond to

bacterial components by halting resorption. Thus, samples were also stimulated with

LPS in the presence of RANKL in order to simulate the affect of bacterial infection would

have on osteoclast function. Following stimulation with RANKL+LPS the amount of

collagen, cathepsin K and MMP-9 were significantly greater in T2D samples (Figure 4-

4). In addition ND osteoclasts cultures had significantly lower amounts of, collagen,

39

cathepsin K and MMP-9 in the presence of LPS than in its absence (Figure 4-4)

Together these data indicate that T2D osteoclasts did not stop resorption with LPS and

remained active at levels similar to RANK-L-only stimulated cultures. Thus, the failure to

deactivate in the presence of LPS suggests an inherent defect in T2D osteoclasts since

this occurs in a normoglycemic environment and not in the hyperglycemic state

Extracellular Milieu

One possible explanation for this LPS induced deactivation in ND osteoclasts is

the need to recruit other immune cells via cyto/chemokine secretion instead of resorbing

bone. Pro-inflammatory cytokines such as IL-6 activate immune cells including T and B

lymphocytes while chemokines such as MCP-1, IP-10, MIP-1a, and MIP-1b recruit other

immune cells, such as monocytes, lymphocytes, and neutrophils. We tested our

hypothesis of retained precursor functions in ND osteoclasts by analyzing

cyto/chemokine release in cell culture supernatants. In order to determine the

extracellular milieu of the osteoclast response to bacterial infection, participant samples

were stimulated by RANKL or RANKL+LPS (Figure 4-5). Concentrations of IL-6 were

significantly higher in T2D cultures after stimulation with RANKL and RANKL+LPS,

respectively, compared to controls. Also, RANKL+LPS stimulation resulted in

significantly higher concentrations of IL-6 compared to RANKL stimulation alone in both

groups (Figure 4-5 A). Concentrations of MCP-1 were significantly higher in T2D

samples after stimulation by RANKL+LPS compared to controls. Also, RANKL+LPS

stimulation resulted in significantly higher concentrations of MCP-1 compared to RANKL

stimulation alone in both groups (Figure 4-5 B). Concentrations of IL-10 were

significantly higher in samples stimulated by RANK+LPS compared to RANKL alone in

both ND and T2D groups. Concentrations of IL-10 were significantly higher in ND

40

samples after stimulation by RANKL+LPS, compared to T2D (Figure 4-5 C). IL-10

concentrations were significantly higher after stimulation by RANKL+LPS compared to

controls and T2D samples stimulated by RANKL alone (Figure 4-5 D). Concentrations

of MIP-1α were significantly greater in T2D samples in both RANKL and RANKL+LPS

when compared to the control groups, respectively (Figure 4-5 E). Concentrations of

MIP-1β were significantly higher in T2D samples after stimulation by both RANKL and

RANKL+LPS, respectively, compared to controls. Also, RANKL+LPS stimulation

resulted in significantly higher concentrations of MIP-1β compared to RANKL

stimulation alone in both groups (Figure 4-5 F). In summary, baseline MCP-1, IP-10,

and IL-10 were similar in T2D and ND cohorts (Figure 4–4 B, D). IL-6, MIP-1α, and

MIP-1β were increased with RANK-L stimulation in T2D compared to ND controls

suggesting higher baseline levels of inflammatory capability in T2D (Figure 4-4

A,E,F). As expected, with LPS stimulation, IL-6, MCP-1, IL-10, and MIP-1βincreased in

ND controls, with IP-10 and MIP-1α showing slight increases. However, this increase

was significantly higher in T2D cohorts for IL-6, MCP-1, IP-10, MIP-1α, and MIP-1β.

This suggests T2D osteoclasts have an increased ability to recruit and activate other

immune cells, including osteoclasts precursors. IL-10 decrease in LPS-stimulated T2D

was opposite to that seen in ND samples (Figure 4-5 C), suggesting defective

regulatory responses to inflammation and reduced ability to moderate osteoclasts

maturation. Overall, the data indicate a switch of ND osteoclasts to that of an immune

cell recruiter that halts resorption in the presence of LPS. On the other hand T2D

osteoclasts retain both functions and perpetuate even more inflammation that can lead

41

to increased osteoclastogenesis. This could cause a feedback loop of uncontrolled

inflammation causing excessive bone loss during infections like periodontal disease.

42

Table 4-1. Clinical characteristics of participant population

Variable Diabetics (n=28) Non-Diabetics (n=15)

M F M F

Gender 14 14 7 8

Smoker 0 0 0 0

Age (years) 56.4 + 15.09 52 + 18.77 28.4 + 2.12 27.9 + 1.41

Combined Age (years) 54.2 + 16.87 28.1 + 5.57

White/Non-white 13/1 12/2 6/2 4/3

43

Figure 4-1.Elevated glucose and HBA1c levels in T2D participants. Venous blood

samples (30mL) were collected from all participants after which A) blood glucose levels and B) Glycated hemoglobin (HbA1C) were measured. A) Blood glucose levels were measured with the Ascensia Contour blood glucose meter. B) Glycated hemoglobin was measured using an AC1Now meter. *p value<0.05 one-way ANOVA with Bonferroni’s multiple comparisons.

44

Figure 4-2. T2D does not alter differentiation potential of osteoclasts. 14d following M-

CSF induced adherence enrichment of purified peripheral blood monocytes, 5.9x105 adherent cells/mL were seeded onto glass coverslips and supplemented with 10ng/mL rhM-CSF and 50ng/mL recombinant human soluble RANK-L [rhsRANK-L]. Cultures were continued for an additional 12d with media and supplements refreshed every 3d. After which cells were permeablized with 0.5% Triton X-100/PBS, probed for leukocyte acid phosphatase (TRAP) activity [1:1:1:2:4 Fast Garnet GBC Base Solution:Sodium Nitrite Solution:Napthol AS-BI Phosphate Solution:Tartrate Solution:Acetate Solution] and mounted on glass slides with MOWIOL 4-88 solution. A) Representative picture of osteoclast cultures at 20x and 40x magnification. TRAP positive cells [purple in color] B) TRAP positive cells [purple in color] were counted according to number of nuclei present: multinucleated osteoclasts [2-10 nuclei] and giant osteoclasts [11+ nuclei] using light microscopy at 40x magnification.

45

Figure 4-3. T2D Osteoclasts are not deactivated by LPS. After 12d of differentiation, cultures supplemented with 10ng/mL rhM-CSF and 50ng/mL rhsRANK were allowed to resorb bone for 72hrs in the presence or absence of 1ug/mL Escherichia coli LPS. After which cultures were permeablized with 1% Triton X-100 and A) collagen carboxy-terminal telopeptides, B) active cathepsin K and C) active MMP-9 were detected using ELISA according to the manufactures’ instructions. Microplate Manager Software and a standard curve were used to determine A) nM, B) pM/L or C) ng/mL.*p value<0.05 one-way ANOVA with Bonferroni’s multiple comparisons.

46

Figure 4-4. Osteoclasts retain precursor functions. After 12d of differentiation, cultures supplemented with 10ng/mL rhM-CSF and 50ng/mL rhsRANK were allowed to resorb bone for 72hrs in the presence or absence of 1ug/mL Escherichia coli LPS. After which cultures were permeablized with 1% Triton X-100 and A) IL6, B) MCP1, C) IL10, D) IP10, E) MIP1α, and F) MIP1β were detected and quantified using a human 14-cyto/chemokine multiplex. Milliplex analyst software, 5-parameter logistics and a standard curve were used to determine pg/mL concentrations. *p value<0.05 one-way ANOVA with Bonferroni’s multiple comparisons.

47

CHAPTER 5 DISCUSSION

The results of our study have the potential to explain some key factors in the

pathogenesis of one complication of T2D, namely periodontal disease. A hallmark of

periodontal disease is destruction of alveolar bone. Loss of alveolar bone can be

attributed to multiple mechanisms of altered bone remodeling. Data generated here

may help explain increased or accelerated alveolar bone loss in T2D patients with

periodontal disease. Bone remodeling is regulated by osteoblasts and osteoclasts which

lay down and break down bone respectively. Much interest has been given to the role of

the osteoclasts in periodontal patients due to its unique role in bone turnover as it is the

only cell known to degrade bone. Importantly, the target of many drugs aimed at

regulating bone remodeling is osteoclasts. Thus an aberration in osteoclast abundance

or function would likely have a profound effect on bone homeostasis and

responsiveness to treatments.

The literature suggests decreased osteoclast numbers or function can be

attributed to lowered number of osteoblasts, yielding less bone to be degraded in

models other than periodontal disease97. Thus one could propose that a more direct

relationship is also likely with increased numbers of osteoclasts resulting in increased

bone resorption. Both scenarios have been shown in the literature. For instance,

Verhaeghe et al. showed decreased osteoblast and osteoclast numbers were

associated with decreased bone mass and bone metabolism in a T1D animal model in

tibia and vertebrae as measured by decreased osteoid surface, mineral apposition rate

and plasma osteocalcin levels98. Also, Hamada et al. showed decreased osteoblast

number and function as well as decreased number of osteoclasts in T1D mice were

48

associated with suppressed bone resorption90. On the other hand Pacios (2011)

showed an increase in osteoclast numbers and activity in a T2D rat model resulted in an

augmented inflammatory response99. Furthermore this study showed treatment with a

TNF inhibitor lowered osteoclast numbers and inflammation99.

Mechanisms of increased alveolar bone resorption in T2D were tested in our

study. We demonstrated thatT2D does not alter the differentiation potential of peripheral

blood monocytes. In addition the resulting osteoclasts resorbed similar levels of bone as

those isolated from diabetes-free individuals. It is important to note that in our study, we

did not evaluate the contribution of glycemic control in T2D on the monocytes’ ability to

differentiate into osteoclasts nor its contribution to osteoclast function, as all T2D

participants in this study were under glycemic control. Unpublished data from our

laboratory using mouse models of T2D does suggest that hyperglycemia can increase

the number and size of osteoclasts differentiated from bone marrow. Thus these

findings provide explanation for data in studies of the contribution of T2D and glycemic

control to periodontal disease severity and/or responses to treatment where the

contributions of osteoclast-specific and hyperglycemia-specific mechanisms have not

been delineated. For example, cross-sectional and longitudinal studies concur that T1D

or T2D patients with poor glycemic control present with poorer periodontal attachment

levels than subjects with good glycemic control100, 101. This dose-dependent response is

also shown in response to periodontal therapy. Tervonen et al. showed increased

periodontal breakdown was observed more frequently in subjects with poor metabolic

control with or without increased diabetic complications receiving treatment for existing

periodontal disease102.

49

In this study, we have also demonstrated an absence of LPS-induced deactivation

in T2D derived osteoclasts from participants under glycemic control. Importantly, our

murine models of T2D corroborate these findings (unpublished data). These data are

the first of their kind to demonstrate an osteoclast-specific mechanism of altered bone

remodeling in T2D. This data is significant in the field of periodontal disease in that LPS

and other bacterial components are a key contributing factor to the initiation and

progression of disease. In addition, this finding corroborates evidence found in many

studies showing increased alveolar bone loss in chronic periodontitis patients with T2D

in comparison to non-diabetic patients31, 103-105.

LPS, a cell wall component of gram negative bacteria, has been found to be highly

immunogenic and induces the production of pro-inflammatory cytokines by various

immune cells such as macrophages and dendritic cells. Osteoclasts and their

precursors, which share the same lineage as macrophages and dendritic cells, express

many innate immune receptors and thus can respond to bacterial components106-108. In

a co-culture of osteoclasts and osteoblasts, LPS, the ligand for TLR4, augments bone

resorption107. However, when supporting cells such as osteoblasts or other immune

cells are absent, LPS inhibits bone resorption in osteoclast pure cultures, although the

exact mechanism(s) is not known80. Interestingly, we found that additional stimulation of

T2D cultures with LPS caused these osteoclasts cultures to produce significantly higher

pro-inflammatory levels compared to those from diabetes-free participants. This finding

is in agreement with several studies citing increased inflammatory response in T2D

patients with chronic periodontitis105. But, production of inflammatory mediators is not a

normal function seen in osteoclasts. It is, however, seen in the monocyte/macrophage

50

cell line. Hyper-responsiveness of this lineage in T2D and T1D has been shown in other

studies40-42. This finding could be explained by one of two scenarios. First, the

inflammatory mediators could have been produced by the undifferentiated monocytes,

or T2D osteoclasts may retain some of the functions of their monocyte precursors.

Our data also demonstrates that while diabetes-free derived osteoclasts resorb

less bone in the presence of high amounts of LPS, they do produce pro-inflammatory

cytokines and chemokines. This suggests that LPS shunts osteoclast precursors to that

of an immune cell phenotype to help fight infection rather than towards mobilization to

resorb bone. Here we demonstrated that in addition to being more resorptive in

response to LPS, T2D-derived osteoclasts were also more inflammatory in nature as

indicative of increased soluble mediator secretion. Thus, LPS in the context of T2D is a

double edged sword perpetuating a pro-osteoclastic environment through multiple

mechanisms. Thus, one can postulate that increased presence of these bacterial

components in combination with a predisposition for enhanced osteoclast-LPS

responsiveness can lead to exacerbated bone resorption. The distinction between of the

role (or lack thereof) of hyperglycemia in osteoclast differentiation and function is a very

important finding which may explain why glycemic control in T2D directly affects the

responsiveness to conventional periodontal treatment such as SRP. One can imagine

that in a patient under glycemic control, removal of the sub-gingival plaque (containing

the offending osteoclast stimulator – LPS) would allow for resolution of osteoclast

function. On the other hand in the hyperglycemic patient, removal of the LPS would not

counteract the effect of hyperglycemia on increased osteoclast number and function.

Together these data also suggests that increased alveolar bone loss in T2D is not alone

51

due to a result of greater numbers of osteoclasts, but rather in combination with an

intrinsic alteration in osteoclast responsiveness. Thus, this data is very important to

consider when evaluating osteoclasts-specific mediated treatments for alterations in

bone remodeling such as periodontal disease.

These findings together support the literature suggesting diabetics experience

greater bone loss due to an enhanced inflammatory response. Augmented inflammatory

response may explain why some studies suggest inflammation lasts longer in T2D

subjects. Naguib et al. showed aT2D animal model in which P. gingivalis infection

prolonged the expression of TNF-α, MCP-1 and MIP-2 compared to controls40.

Recently, Pacios et al. showed this prolonged inflammation was normalized by TNF-α

inhibition. Also, TNF-α inhibition allowed new bone and osteoid formation along with

increased numbers of osteoblasts99. The results of our study support the argument that

enhanced inflammatory response may result in longer periods of periodontal

destruction. But rather than resulting from an inhibition of expression of critical factors

needed to stimulate bone formation99, inducing expression of critical factors responsible

for stimulating bone resorption. Another potential implication of these findings is the

potential absence of a negative feedback mechanism in the bone resorptive pathway. If

osteoclasts are producing inflammatory mediators, this will cause osteoblasts to

produce more RANKL, resulting in increased osteoclastogenesis. This coupled with a

failure of LPS to inhibit osteoclast activation, result in augmented bone resorption.

Compounding this issue is the LPS-induced recruitment of more osteoclast precursors

through soluble mediator expression providing more differentiation and resorptive

capacity at the lesion. Thus defining the pathways in which LPS fails to inhibit osteoclast

52

activation is imperative for the development of appropriate osteoclasts-specific

mechanisms for T2D patients.

The results of this study are also important to implant dentistry. Although poor

osseointegration has been shown on implants in patients with poorly controlled

diabetes, successful dental implant integration and high implant survival rates can be

accomplished in subjects with good metabolic control109-112. However, implants are not

free from risk of infection in the adjacent supporting tissue113. Although the

pathogenesis of peri-implant diseases are similar to periodontal diseases, several

important differences exist114. Peri-implant infections tend to be greater in size and

intensity of inflammatory infiltrate and can eventually progress into the bone marrow115,

116.Considering the evidence presented in this study as well as others, poorly- controlled

diabetics could be at a greater risk for peri-implant diseases risk due to poor bone

healing, disabled osteoclasts deactivation, as well as enhanced inflammatory response.

This study was a very important step in elucidating the mechanisms of increased

alveolar bone resorption in T2D patients with chronic periodontal disease. It is apparent

that osteoclasts are not deactivated by LPS and, in fact, display a pro-inflammatory

phenotype akin to other cells with which they share a common precursor, including

monocytes, macrophages and dendritic cells. This pro-inflammatory phenotype could be

responsible for an augmented inflammatory response.

53

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BIOGRAPHICAL SKETCH

Dr. Douglas Ian Storch received his Bachelor of Science in zoology from

University of Florida in spring 2003. He then attended the University of Florida College

of Dentistry where he received his Doctor of Dental Medicine (DMD) degree in the

spring of 2007. He then completed a General Practice Residency at Harvard School of

Dental Medicine, followed by one year of private practice in Peabody, MA. Douglas

returned to the University of Florida to complete a post-doctoral residency in

periodontics. After graduation, Douglas will begin practicing periodontics in Northeast

Florida.