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T2d Osteoclasts Are Resistant to LPS-Induced Deactivation

Permanent Link: http://ufdc.ufl.edu/UFE0044167/00001

Material Information

Title: T2d Osteoclasts Are Resistant to LPS-Induced Deactivation
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Storch, Douglas I
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diabetes -- lps -- osteoclast -- storch -- t2d
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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 were not deactivated by LPS and produced a more pro-inflammatory cytokines and chemokines than ND samples.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Douglas I Storch.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Wallet, Shannon.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044167:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044167/00001

Material Information

Title: T2d Osteoclasts Are Resistant to LPS-Induced Deactivation
Physical Description: 1 online resource (63 p.)
Language: english
Creator: Storch, Douglas I
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diabetes -- lps -- osteoclast -- storch -- t2d
Dentistry -- Dissertations, Academic -- UF
Genre: Dental Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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 were not deactivated by LPS and produced a more pro-inflammatory cytokines and chemokines than ND samples.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Douglas I Storch.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Wallet, Shannon.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044167:00001


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1 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 O F SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Douglas Ian Storch

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3 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 patie nce is unequalled

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4 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 Lucia na Shaddox, Shannon Holliday, and Joseph Ri ley for their contributions to my education in this thesis.

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5 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 Pe riodontium ................................ .......................... 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 Parti cipant 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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6 Statistical Analysis ................................ ................................ ................................ .. 36 4 RESULTS ................................ ................................ ................................ ............... 37 Clinical Characteristics of Participant Population ................................ .................... 37 Differentiation Potential ................................ ................................ ........................... 37 Bone Resorption ................................ ................................ ................................ ..... 38 Extracellular Mil ieu ................................ ................................ ................................ .. 39 5 DISCUSSION ................................ ................................ ................................ ......... 47 LIST OF REFERENCES ................................ ................................ ............................... 53 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 63

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7 LIST OF TABLES page 4 1 Clinical characteristics of participant population ................................ ................. 42

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8 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 Osteocl asts retain precursor functions ................................ ............................... 46

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9 LIST OF ABBREVIATION S 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 Mell itus HbA1c Glycated hemoglobin IFN Interferon gamma IL 10 Interleukin 10 IL1 Interleukin 1 beta IL 6 Interleukin 6 IP 10 Interferon gamma induced protein LAP Localized aggressive periodontitis LPS Lipopolysaccharide, endotoxin MCP 1 Monocyt e chemoattractant protein 1 M CSF Macrophage colony stimulating factor MIP ND Non Diabetic Participant OPG Osteoprotegrin PDL Periodontal ligament RANK Receptor activator of nuclear factor kappa B

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10 RANK L Receptor activator of nuclear factor kappa B ligand T1D Type 1 Diabetes Mellitus T2D Type 2 Diabetes Mellitus TNF Tumor necrosis factor alpha

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11 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 osteocla st 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 Diabet es (T2D) and Diabet es 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 sam ples were evaluated for collagen release, markers of osteoclasts activity, and markers of inflammation. Our results show T2D osteoclasts

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12 were not deactivated by LPS and produced a more pro inflammatory cytokines and chemokines than ND samples.

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13 CHAPTER 1 I NTRODUCTION 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 population 1 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 cel l 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 alter s the func tion of osteoclasts, resulting in increased alveolar bone loss This study was T2D participants with periodontal disease. We hypothesize that osteoclasts from T2D sa mples 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 reso rbing ability and the extracellular milieu

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14 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 rel ease, 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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15 CHAPTER 2 BACKGROUND The Periodontium in Healt h The periodontium comprised of the gingiva, the periodontal ligament, root cementum, and the alveolar bone proper is responsible for anchoring the tooth to surrounds the te eth 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 socket and is th e other attachment of the periodontal ligament fibers 4 The gingiva microscopically shows two main tissue types in its composition: an epithelial layer an d 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, b ut is not attached to, the tooth and shows a decreased keratinization. The junctional epithelium attaches to the tooth surface via hemidesmosomes 5 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 c revice between the tooth and the gingiva called the sulcus 5 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 osteoblasts osteoclasts and epithelial cells 5

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16 Cementum, which covers the roots of teeth is a specialized tissue containing both hydroxyapatite and collagen, allowing attachment of the PDL 4 Deposition of cementum continues thro ughout 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 fibers a n essential component to the attachment apparatus. Cellular cementum exists in the apical one third of the root and in furcations 4 The alveolar process i s 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 structu re 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 contrib ute 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 d isease may affect 4 40% of the adult population in the United States 1 3 Gingivitis always precedes periodonti tis and is widely prevalent in the United States. Periodontitis is a cumulative condition, initiated by bacteria but perpetuated by individual host factors 6 Susceptibility to periodontitis is enhanced by the interaction between acquired, environmental and genetic factors which modify the host response toward putative pathogens 7

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17 Bacteria, the initiating factor in periodontitis, colonize the sulcus and surround ing 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 receptors 8 9 Activation of this pathway leads to an inflammatory response called gingi vitis, or inflammation of the gingiva. Regular mechanical removal o f all bacterial deposits from the tooth tissue interface eliminates signs of inflammation is the primary prerequisite to disease prevention 10 When not removed, the biofilm prol iferates 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 a pically, 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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18 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 lesions 11 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 lesi on, 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 i s 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 le ukocyte infiltrate, plasma cell influx and collagen loss. The junctional epithelium proliferates to

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19 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 hospit able 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. P lasma 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 attachmen t loss has been correlated to presence of certain Gram negative anaerobic bacteria including Porphyromonas gingivalis, Prevotella intermedia, Bac teroides forsythus, Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans and Treponemadenticol a 12 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 distan ce from a fixed reference point the cemento enamel junction (CEJ) to the base of the probable crevice 13 The disease is considered localized if up to 30% of the teeth are affected and

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20 generalized when >30% of the teeth are involved 14 Clinicians usually combine all the clinical findings into a global statemen t 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 status 12 Aggressive Periodontitis Aggressive periodontitis (AgP) is a rapidly progressing disease form characterized by rapid attac hment 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 prop ortions of Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans and/or Porphyromonas gingivali s phagocyte abnormalities, hyper responsive macrophage phenotype, elevated production of prostaglandin E 2 ( PGE 2 ) and interleukin limiting pr ogression of disease 12 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 attac h ment 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 episo dic 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 varie ty of factors. Aside from causal factors,

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21 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 cond itions. 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 r ole 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 diso rder, 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 secr 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 base d on pathophysiology of hypergly cemia. Type 1 diabetes (T1D) results from pa n cell destruction usually leading to insulin secreti on deficiency. Also known as insulin dependent diabetes (IDDM) or juvenile

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22 onset diabetes, this disease form represents approximately 5 10% of patients. Its causes are unknown but several components appear to contribute. Cellular mediated autoimmune destru 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 Also, the disease has strong human leukocyte antigen (HLA) associatio ns, 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 ca lled ketoacidosis, in which the body cannot regulate the byproduct of fatty acid and protein degradation, ketones. Type 2 D iabetes (T2D), previously referred to as non insulin dependent diabetes (NIDDM) or adult onset diabetes, accounts for 90 95% of diab etics. Autoimmune 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 hormo ne. 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 cell destruction. However, after years of exogenous insulin supplementation, the pancreas may lose its ability to produce insulin cell death. Another common for m 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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23 of pregnancy. Although mothers often return to normoglycemic state after childbirth, a history of GDM sign ificantly 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, resul ting 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 conce ntration >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 h our post load glucose >200 mg/dL (>11.1 mmol/L) in an oral glu cose tolerance test using 75g anhydrous glucose 16 As a result of higher average blood glucose over time, non enzymatic binding of glucose to hemoglobin yields a highly stable molecul e, 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 dev elopment of diabetic complications 17 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 target 19 Complications of D iabetes Complications of d iabetes 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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24 resulting in highly acidic ketone bodies in the blood, is the leading cause of diabetes related fatalities 20 Hyperglycemia can lead to a metabolic abnormality causing extreme dehydration, lethargy, and coma requirin g hospitalization 18 Hypoglycemia, a far more common and but equally lethal condition, results from insulin excess causin g an unsafe drop in blood glucose levels leading to confusion, anxiety, dizziness, seizures and even loss of consciousness 1 8 Chronic complications can divided into macro and microvascular complications. Macrovascular complications include cardiovascular disease. Microvascular complications include nephropathy, retinopathy and neuropathy 18 Effects of Diabetes on the Periodontium Periodontal disease has been called the sixth complication of diabetes 21 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 relationship 22 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 diabetics 24 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 subse ts of children, adolescents and adults 25 28 Longitudinal studies confirm these observations 29 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 exist 32 33 Instead, this suggests that the chronic biofilm induced wound produces an alter ed host response in diabetics.

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25 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 ki lling in the periodont al pocket 35 37 In addition, neutrophil activation and priming ma y result in increased tissue damage 38 39 In response to periodontopathogens, the upregulated monocyte/macrophage cell line produces a prolong ed response as well as increased pro inflammatory cytokines and mediators, namely IL1 and TNF 40 42 This increased inflammatory response is evident in the GCF, more so in poorly controlled diabetics 43 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 i mpaired osseous healing, inhibition of osteoblastic proliferation and function, and decreased collagen function and deposition are results of hyperglycemia that accelerate tissue destruction 44 Bone Bone is a dynamic structure characterized by its mineralized organic matrix of collagenous and non collage nous proteins. Bone remodels over time in response to functional demands as a part of its role in protecting vital organs and serv ing as a reservoir for minerals which contribute homeostasis of the body. The remodeling process is a complex continuum of res orption 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 mee t these demands.

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26 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 fro m 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 bone 4 5 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 prov ides 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 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. Oste oclasts share a cell lineage with macrophages and dendritic cells, the hematopoietic stem cell. When stimulated by

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27 two factors produced by osteoblasts, macrophage colony stimulating factor (M CSF) and receptor activator of NF B ligand (RANK L) these mono cytes commit to osteoclastogenesis 46 The receptor for M CSF is c fms on osteoclasts precursors and activates signaling through MAP kinases and ERKs during ear ly differentiation 47 RANK L binds to its receptor on the osteoclasts precursor, RANK, and induces differentiation into mature osteoclasts via NF B, c Fos, phospholipase C (PLC ) and nuclear factor of activated T cells c1 (NFATc1) pathways 48 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 resorption 49 Secretion of HCL produced by a vacuolar H+ ATPase (V ATPase) dissolves the mineral portion of the bone 49 This activates cathepsin K, an enzyme necessary to cleave type 1 collage n. Products of this process are endocytosed by the osteoclast released at the opposite membrane of the cell 49 The cycle ends when ost eoblasts produce osteoprotegrin which binds to RANK, preventing further stimulation of the osteoclast. It is impo rtant 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, bone cells inter act intensel y 50 Many of the soluble mediators of immune c ells, 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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28 The RANKL RANK OPG signaling axis plays a central role in osteoimmunology. O nce 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 D 3 prostaglandin E 2 (PGE 2 ), and parathyroid hormone 53 T cells express soluble RANKL which can also activate osteoclastogenesis and may play a signi ficant role in inflammatory regulation through the adaptive immune system 48 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 ba sis of their bone resorbing effects 53 55 Interleukin 6 (IL 6) is a multipotent cytokine with a wide variety of activities. It has been shown to regulate development of mature osteoclasts 56 as well as directly stimulate the production of RANK L and OPG 57 IL 6 is prod uced by a variety of cells including T cells, monocytes, epithelial cells, fibroblasts, osteoblasts/stromal cells, synovial cells, various cancer cells, and also mature osteoclasts 58 60 Interleukin 10 (IL 10) is known as an anti inflammatory cytokine partly due to its inhibitory function o n osteoclastogenesis and osteoblastogenesis. IL 10 causes decreased NFATc1 expression, the key transcription factor in osteoclastogenesis 61 Also IL 10 directly inhibits RANK L production while enhancing OPG production resulting in decreased osteoclastogenesis 62

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29 Several chemokines contribute to osteolclastogenesis. Monocyte chemoattractant protein 1 (MCP 1, CCL2) is highly expressed in osteoblasts in inflamed sites and enhances osteoclasts forma tion 63 65 Macrophage infla mmatory protein 1 (MIP 1 CCL3) directly stimulates osteoclastogenesis through its receptors 66 67 It may stimulate motility but suppress osteoclasts resorptive activity 68 Another chemokine, IP 10 is secreted by several cell types, including monocytes, endothelial cells and fibroblasts, in response to IFN 10 may serve as a chemoattractant for inflammatory cells such as monocytes/macrophages and T cells and is involved in bone erosion 69 Bone Metabolism in Periodontitis The pathogenesis of periodontitis depends upon the immune and inflammatory response to bacterial colonization of the gingival sulcus 7 70 An initial activation of the innate immune system is amplified in periodontitis, resulting in an expansion of the inflammatory response approximating the alveolar bone 71 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 mediators 74 75 In addition, the production of bacterial endotoxin, lipopolysaccrhride (LPS) can also stim ulate RANKL production through toll like receptor pathways 8 As mentioned previously RANKL is one of the master regulators of bone metabolism, alon g 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 periodontitis 76 79 It is clear that bacterial presence in the gingi val sulcus of a

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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 r ecent study showed this direct stimulation only occurs in the presence of RANKL. Without the support of RANKL, LPS instead inhibits osteoclastogenesis 80 Bone Metabo lism 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 rate 85 88 In addition, reduced blood flow due to poor microvasculature contributes to poor bone quality 89 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 an d diminished collagen production 90 92 Both chondrocytes and mesenchymal stromal cells display down regulation, diminished proliferation, and decreased differentia tion 45 Osteoclasts in T1D humans and rats have shown both increased numbers and function 87 88 93 In T2D, poorer bone quality was observed, displaying increased risk of fracture 84 Decreased osteoblasts and thus osteoid surface was seen in a T2D rat model 94 As stated previously progressive periodontal bone loss in diabetics may be related to changes in bone metabolism resulting from altered cellular functions 44 Naturally,

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31 focusing on the cell responsible for bone degradation will elucidate its role in increased alveolar bone loss among T2D persons. O ur 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.

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32 CHAPTE R 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 recru ited 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 ol d, 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 di seases 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 phy sician, would affect the safety or compliance or that could influence the course of either periodontal disease or diabetic 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 (HbA 1C) were

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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 CaCl 2 x2H 2 O, 0.1992g/L MgCl 2 x6H 2 O, 0.4026g/L KCL, 17.565g/L Tris, C oncHClpH7.6 : 9 parts0.14M/L NaCl]. Diluted blood samples were overlayed onto 15mL of Ficoll Plaque (General Electric) and centrifuged at 400 xg for 40 minutes Following centrifugation the interface was removed and washed three times with a salt solution. MEM complete media (Sigma Aldrich) and cells counted using a hemocytometer. Osteoclast Differentiation Purified peripheral blood monocytes were seeded in a T 2 5 flask at a concentration of 1.5x10 6 cells/mL sup MEM complete media (Sigma Aldrich) and 10ng/mL recombinant human M CSF [rh M CSF] ( Peprotech ) an d allowed to culture for 14 days at 37C and 5% CO 2 with media refreshed every 3 days After which, non adherent cells were removed and adh erent cells seeded at 5.9x10 5 cells/mL in 24 well plates on either glass coverslips (Fisher) or 1 cm 2 bovine bone slices cut with an Isomet Low Speed Saw (Buehler). All cultures were supplemented with 10ng/mL r h M CSF and 50ng/mL recombinant human soluble R ANK L [rh sRANK L] ( Peprotech ) and allowed to culture for 12d wi th complete media containing rhsRANK L and rhM CSF refreshed every 3d.

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34 TRAP Staining After 12 d of differentiation, cells plated on glass coverslips were fixed with 2% paraf ormaldehyde/PBS (Fis her) for 15 mins. 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 Soluti on] (Sigma Aldrich) for 1 hr in dark at 37C. Cells were washed with dH 2 O and glass coverslips mounted on glass slides with MOWIOL 4 88 solution (Calbiochem). TRAP positive cells [purple in color] w ere 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 12 d of differentiation m MEM complete media supplemented with 10ng/mL r h M CSF and 50ng/mL r h sRANK 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 t hey were washed with PBS 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 r andom scanning electron micrographs

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35 (8 bit grayscale) of bone slices were acquired at 40x magnification with a 2048x1594 resolution s with clearly identifiable borders. Identical proce dures 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 trans formation 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 ( : 8 pixels) was applied to the result. The rolling ball algorithm 96 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) u sed 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 ma nufacturer 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 t etramethylbenzidine [TMB] substrate was used to develop for 1hr followed by quenching with H 2 SO 4 Colorimetric reactions were detected using a Benchmark Microplate Reader spectrophotometer (Bio Rad) set at a dual waveleng th reading of 450nm with a reference of 655nm. Microplate Manager Software (Bio Rad) and a standard curve were used to determine nM concentrations.

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36 Cathepsin K ELISA Active cathepsin K was detected using an ELISA according to manufacturer instructions (Alp co). 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 f ollowed 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). Supernata nt 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 30mi ns. 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 cu rve were used to determine pg/mL concentrations. Statistical Analysis One tailed unpaired t cal significance (p<0.05) as appropriate.

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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 o f 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 + 16.8 7 In order to determine whether the glycemic status effects osteoclast differentiation or function, peripheral venous b lood was drawn on the participant population and blood glucose (mg/dL) and glycated hemoglobin (HbA1c) was determined o n eac h participant sample. (Figure 4 1) Although T2D participants show elevated non fasting glucose (NF G) 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 re sorption 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 pot ential 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

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38 (11+ nuclei). (Fig ure 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 os teoclast precursors (Figure 4 2 C,D). Bone Resorption Although differentiation may not be altered, it is possible that mature oste oclasts 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 (Fig ure 4 3). As a measure of b one 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 eva luate d in the supernatants (Figure 4 3 B,C). Again, no significant difference was observe d in the levels of cathepsin K (Fig ure 4 3 B), although OCs derived from T2D participants produced significantly higher levels of MMP9 (Fig ure 4 3 C). Bacterial components can act as the inflammatory stimulus which activates bone resorption. However, in t he 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 func tion. Following stimulation with RANKL+LPS the amount of collagen, cathepsin K and MMP 9 were significantly greater in T2D samples (Fig ure 4 4). In addition ND osteoclasts cultures had significantly lower amounts of, collagen,

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39 cathepsin K and MMP 9 in the presence of LPS than in its abse nce (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 recrui t 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 respon se to bacterial infection, participant samples were stimul ated 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 (Fig ure 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 (Fig ure 4 5 B). Concentrations of IL 10 were significantly higher in samples stimulated by RANK+LPS compared to RANKL a lone in both ND and T2D groups. Concentrations of IL 10 were significantly higher in ND

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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 contro ls and T2D samples stimulated by RANKL alone (Fig ure 4 5 D). Concentrations of MIP 1 were significantly greater in T2D samples in both RANKL and RANKL+LPS when compared to the co ntrol groups, respectively (Figure 4 5 E). Concentrations of MIP 1 were significantly higher in T2D samples after stimulation by both RANKL and RANKL+LPS, resp ectively, compared to controls. Also, RANKL+LPS stimulation resulted in significantly higher concentrations of MIP 1 compared to RANKL stimul ation alone in both groups (Figure 4 5 F). In summary, b aseline MCP 1, IP 10, and IL 10 were similar in T2D and N D cohort s (Figure 4 4 B D ). IL 6, MIP 1 and MIP 1 were increased with RANK L stimulation in T2D compared to ND controls suggesting higher baseline levels of infl ammatory capability in T2D (Figure 4 4 A,E,F ). As expected, with LPS stimulation, IL 6, M CP 1, IL 10, and MIP 1 increased in ND controls, with IP 10 and MIP 1 showing slight increases. However, this increase was significantly higher in T2D cohorts for IL 6, MCP 1, IP 10, MIP 1 and MIP 1 This suggests T2D osteoclasts have an increased abi lity to recruit and activate other immune cells, including osteoclasts precursors. IL 10 decrease in LPS stimulated T2D was opposite to th at seen in ND samples (Figure 4 5 C), suggesting defective regulatory responses to inflammation and reduced ability t o 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 inf lammation that can lead

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41 to increased osteoclastogenesis. This could cause a feedback loop of uncontrolled inflammation causing excessive bone loss during infections like periodontal disease.

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42 Table 4 1. Clinical characteristics of participant population V ariable Diabetics (n=28) Non Diabet i c s (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

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43 Figur e 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) G lycated hem oglobin (HbA1C) were measured. A) Blood glucose levels were measured with the Ascensi a Contour blood glucose meter. B) Glycated hemoglobin was measured using an AC1Now meter. *p value<0.05 one comparisons.

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44 Figure 4 2. T2D does not alter differentiation potential of osteoclasts. 14d foll owing M CSF induced adherence enrichment of purified peripheral blood mono cytes, 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 conti nued 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 slid es with MOWIOL 4 88 solution. A) Representative picture of osteoclast cultures at 20x and 40x magnification. TRAP po sitive cells [purple in color] B) TRAP positi ve 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.

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45 Figure 4 3. T2D Osteoclasts are not deactivated by L PS. 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 an d A) collagen carboxy terminal telopeptides, B) active cathepsin K and C) active MMP 9 were detected using ELISA according to the d curve were used to determine A) nM, B) pM/L or C) ng /mL .*p value<0.05 one

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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 permea blized with 1% Triton X 100 and A) IL6, B) MCP1, C) IL10, D) IP10, quantified using a human 14 cyto/chemokine multiplex. Milliplex analyst software, 5 parameter logistics and a standard cu rve were used to determine pg/mL concentrations. *p value<0.05 one multiple comparisons.

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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 bo ne. 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 oste o blasts and osteoclasts which lay down an d 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 T he literature suggest s decreased osteoclast numbers or function can be at tributed to lowered number of osteoblasts, yielding less bone to be degraded in models other than periodontal disease 97 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 i nstance, 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 pla sma osteocalcin levels 98 Also, Hamada et al showe d decreased osteoblast number and function as well as decreased number of osteoclasts in T1D mice were

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48 associated with suppressed bone resorption 90 On the other h and Pacios ( 2011 ) showed an increase in osteoclast numbers and activity in a T2D rat model resulted in an augmented inflammatory response 99 Furthermore this study showed treatment with a TNF inhibitor lowered osteoclast numbers and inflammation 99 Mechanisms of increased alv eolar 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 stud y 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 t his 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 finding s 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, c ross 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 control 100 101 This dose dependent response is also shown in response to periodontal therapy. Tervonen et al showed i ncreased periodontal breakdown was observed more frequently in su bjects with poor metabolic control with or without increased diabetic complications receiving treatment for existing periodontal disease 102

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49 In this study, we have also demonstrated an absence of LPS induced deactivation in T2D derived ost eoclasts 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 T his data is significant in the field of periodontal disease in that LPS and other bacterial components are a key contributing factor to the i n it ia tion and progression of disease. In addition, t his finding corroborates evidence found in many studies sho wing increased alveolar bone loss in chronic periodontitis patients with T2D in comparison to non diabetic patients 31 103 105 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. Osteoclast s and their precursors, which share the same lineage as macrophages and dendritic cells, express many innate immune receptors and thus can respond to bacterial components 106 108 In a co culture of osteoclasts and osteoblasts, LPS, the ligand for TLR4, augments bone resorption 1 07 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 known 80 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 periodontitis 105 But, production of inflammatory mediators is not a normal function seen in osteocla sts. It is, however, seen in the monocyte/macrophage

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50 cell line. Hyper responsiveness of this lineage in T2D and T1D has been shown in other studies 40 42 This fi nding 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 functio ns of their monocyte precursors. Our data also demonstrates th at w hile diabetes free derived osteoclast s 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 f ight 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 se cretion. 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 co ntrol 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 al low 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 al veolar bone loss in T2D is not alone

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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 MCP 1 and MIP 2 compared to controls 40 Recently, Pacios et al showed this prolonged inflammation was normalized by TNF inhibition. Also, TNF inhibition allowed new bone and osteoid formation along with increased numbers of osteoblasts 99 The results o f our study support the argument that enhanced inflammatory response may result in longer periods of periodontal destruction B ut rather than resulting from a n inhibition of expression of critical factors needed to stimulate bone formation 99 inducing expression of critical factors responsible for stimulating bone resorption. Another potential implication of these findings is the potential abse nce 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 recruit ment of more osteoclast precursors through soluble mediator expression providing more differentiation and resorptive capacity at the lesion Thus defining the pathways in which LPS fail s to inhibit osteoclast

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52 activation is imperative for the develo pment of appropriate osteoclasts specific mechanism s 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 control 109 112 However, i mplants are not free from risk of infection in the adjacent supporting tissue 113 Although the pathogenesis of peri implant diseases are similar to periodontal diseases, several important differences exist 114 Peri implant infections tend to be g reater in size and intensity of inflammatory infiltrate and can eventually progress into the bone marrow 115 116 Considering the evidence presented in this study as well as others, poorly controlled d iabetics 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 stud y was a very important step in elucidat ing 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 i nflammatory 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

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53 LIST OF REFERENCES 1. Alb andar JM, Brunelle JA, Kingman A. Destructive periodontal disease in adults 30 years of age and older in the United States, 1988 1994. J Periodontol 1999;70(1):13 29. 2. Borrell LN, Burt BA, Taylor GW. Prevalence and trends in periodontitis in the USA: the [corrected] NHANES, 1988 to 2000. J Dent Res 2005;84(10):924 30. 3. Borrell LN, Crawford ND. Social disparities in periodontitis among United States adults 1999 2004. Community Dent Oral Epidemiol 2008;36(5):383 91. 4. Nanci A, Bosshardt DD. Structure of periodontal tissues in health and disease. Periodontol 2000 2006;40:11 28. 5. Schroeder HE, Listgarten MA. The gingival tissues: the architecture of periodontal protection. Periodontol 2000 1997;13:91 120. 6. Kinane DF. Causation and pathogenesis of period ontal disease. Periodontol 2000 2001;25:8 20. 7. Page RC, Kornman KS. The pathogenesis of human periodontitis: an introduction. Periodontol 2000 1997;14:9 11. 8. Mahanonda R, Pichyangkul S. Toll like receptors and their role in periodontal health and disea se. Periodontol 2000 2007;43:41 55. 9. Takeda K, Akira S. Toll like receptors in innate immunity. Int Immunol 2005;17(1):1 14. 10. Loe H, Theilade E, Jensen SB. Experimental Gingivitis in Man. J Periodontol 1965;36:177 87. 11. Page RC, Schroeder HE. Pathog enesis of inflammatory periodontal disease. A summary of current work. Lab Invest 1976;34(3):235 49. 12. Armitage GC. Periodontal diagnoses and classification of periodontal diseases. Periodontol 2000 2004;34:9 21. 13. Armitage GC. Clinical evaluation of p eriodontal diseases. Periodontol 2000 1995;7:39 53. 14. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4(1):1 6. 15. Flemmig TF. Periodontitis. Ann Periodontol 1999;4(1):32 8.

PAGE 54

54 16. Diagnosis and classification of diabetes mellitus. Diabetes Care 2011;34 Suppl 1:S62 9. 17. Davidson MB, Schriger DL, Peters AL, Lorber B. Glycosylated hemoglobin as a diagnostic test for type 2 diabetes mellitus. JAMA 2000;283(5):606 7. 18. Mealey BL, Ocampo GL. D iabetes mellitus and periodontal disease. Periodontol 2000 2007;44:127 53. 19. Koro CE, Bowlin SJ, Bourgeois N, Fedder DO. Glycemic control from 1988 to 2000 among U.S. adults diagnosed with type 2 diabetes: a preliminary report. Diabetes Care 2004;27(1):1 7 20. 20. Kitabchi AE, Nyenwe EA. Hyperglycemic crises in diabetes mellitus: diabetic ketoacidosis and hyperglycemic hyperosmolar state. Endocrinol Metab Clin North Am 2006;35(4):725 51, viii. 21. Loe H. Periodontal disease. The sixth complication of diabe tes mellitus. Diabetes Care 1993;16(1):329 34. 22. Papapanou PN. Periodontal diseases: epidemiology. Ann Periodontol 1996;1(1):1 36. 23. Lalla E, Cheng B, Lal S, Tucker S, Greenberg E, Goland R, et al. Periodontal changes in children and adolescents with d iabetes: a case control study. Diabetes Care 2006;29(2):295 9. 24. Nelson RG, Shlossman M, Budding LM, Pettitt DJ, Saad MF, Genco RJ, et al. Periodontal disease and NIDDM in Pima Indians. Diabetes Care 1990;13(8):836 40. 25. Campus G, Salem A, Uzzau S, Bal doni E, Tonolo G. Diabetes and periodontal disease: a case control study. J Periodontol 2005;76(3):418 25. 26. Ervasti T, Knuuttila M, Pohjamo L, Haukipuro K. Relation between control of diabetes and gingival bleeding. J Periodontol 1985;56(3):154 7. 27. G usberti FA, Syed SA, Bacon G, Grossman N, Loesche WJ. Puberty gingivitis in insulin dependent diabetic children. I. Cross sectional observations. J Periodontol 1983;54(12):714 20. 28. Karjalainen KM, Knuuttila ML. The onset of diabetes and poor metabolic c ontrol increases gingival bleeding in children and adolescents with insulin dependent diabetes mellitus. J Clin Periodontol 1996;23(12):1060 7. 29. Seppala B, Seppala M, Ainamo J. A longitudinal study on insulin dependent diabetes mellitus and periodontal disease. J Clin Periodontol 1993;20(3):161 5.

PAGE 55

55 30. Taylor GW, Burt BA, Becker MP, Genco RJ, Shlossman M. Glycemic control and alveolar bone loss progression in type 2 diabetes. Ann Periodontol 1998;3(1):30 9. 31. Tervonen T, Oliver RC. Long term control of diabetes mellitus and periodontitis. J Clin Periodontol 1993;20(6):431 5. 32. Sastrowijoto SH, Hillemans P, van Steenbergen TJ, Abraham Inpijn L, de Graaff J. Periodontal condition and microbiology of healthy and diseased periodontal pockets in type 1 diab etes mellitus patients. J Clin Periodontol 1989;16(5):316 22. 33. Zambon JJ, Reynolds H, Fisher JG, Shlossman M, Dunford R, Genco RJ. Microbiological and immunological studies of adult periodontitis in patients with noninsulin dependent diabetes mellitus. J Periodontol 1988;59(1):23 31. 34. Mealey B. Diabetes and periodontal diseases. J Periodontol 1999;70(8):935 49. 35. Manouchehr Pour M, Spagnuolo PJ, Rodman HM, Bissada NF. Impaired neutrophil chemotaxis in diabetic patients with severe periodontitis. J D ent Res 1981;60(3):729 30. 36. McMullen JA, Van Dyke TE, Horoszewicz HU, Genco RJ. Neutrophil chemotaxis in individuals with advanced periodontal disease and a genetic predisposition to diabetes mellitus. J Periodontol 1981;52(4):167 73. 37. Park S, Rich J Hanses F, Lee JC. Defects in innate immunity predispose C57BL/6J Leprdb/Leprdb mice to infection by Staphylococcus aureus. Infect Immun 2009;77(3):1008 14. 38. Ayilavarapu S, Kantarci A, Fredman G, Turkoglu O, Omori K, Liu H, et al. Diabetes induced oxid ative stress is mediated by Ca2+ independent phospholipase A2 in neutrophils. J Immunol 2010;184(3):1507 15. 39. Karima M, Kantarci A, Ohira T, Hasturk H, Jones VL, Nam BH, et al. Enhanced superoxide release and elevated protein kinase C activity in neutro phils from diabetic patients: association with periodontitis. J Leukoc Biol 2005;78(4):862 70. 40. Naguib G, Al Mashat H, Desta T, Graves DT. Diabetes prolongs the inflammatory response to a bacterial stimulus through cytokine dysregulation. J Invest Derma tol 2004;123(1):87 92. 41. Salvi GE, Collins JG, Yalda B, Arnold RR, Lang NP, Offenbacher S. Monocytic TNF alpha secretion patterns in IDDM patients with periodontal diseases. J Clin Periodontol 1997;24(1):8 16.

PAGE 56

56 42. Salvi GE, Yalda B, Collins JG, Jones BH, Smith FW, Arnold RR, et al. Inflammatory mediator response as a potential risk marker for periodontal diseases in insulin dependent diabetes mellitus patients. J Periodontol 1997;68(2):127 35. 43. Engebretson SP, Hey Hadavi J, Ehrhardt FJ, Hsu D, Celenti RS, Grbic JT, et al. Gingival crevicular fluid levels of interleukin 1beta and glycemic control in patients with chronic periodontitis and type 2 diabetes. J Periodontol 2004;75(9):1203 8. 44. Mealey BL, Oates TW. Diabetes mellitus and periodontal diseases J Periodontol 2006;77(8):1289 303. 45. Blakytny R, Spraul M, Jude EB. Review: The diabetic bone: a cellular and molecular perspective. Int J Low Extrem Wounds 2011;10(1):16 32. 46. Boyce BF, Yao Z, Xing L. Osteoclasts have multiple roles in bone in addit ion to bone resorption. Crit Rev Eukaryot Gene Expr 2009;19(3):171 80. 47. Fukuchi M, Fukai Y, Masuda N, Miyazaki T, Nakajima M, Sohda M, et al. High level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esoph ageal squamous cell carcinoma. Cancer Res 2002;62(24):7162 5. 48. Takayanagi H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat Rev Immunol 2007;7(4):292 304. 49. Teitelbaum SL. Bone resorption by osteoclasts. Scie nce 2000;289(5484):1504 8. 50. Lee SH, Kim TS, Choi Y, Lorenzo J. Osteoimmunology: cytokines and the skeletal system. BMB Rep 2008;41(7):495 510. 51. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, et al. Osteoprotegerin: a novel secreted pr otein involved in the regulation of bone density. Cell 1997;89(2):309 19. 52. Tsuda E, Goto M, Mochizuki S, Yano K, Kobayashi F, Morinaga T, et al. Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis. Biochem Biophys Res Commun 1997;234(1):137 42. 53. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999; 20(3):345 57. 54. Takayanagi H. Mechanistic insight into osteoclast differentiation in osteoimmunology. J Mol Med (Berl) 2005;83(3):170 9.

PAGE 57

57 55. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423(6937):337 42. 56. Mano lagas SC, Jilka RL. Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med 1995;332(5):305 11. 57. Palmqvist P, Persson E, Conaway HH, Lerner UH. IL 6, leukemia inhibitory factor, and oncostati n M stimulate bone resorption and regulate the expression of receptor activator of NF kappa B ligand, osteoprotegerin, and receptor activator of NF kappa B in mouse calvariae. J Immunol 2002;169(6):3353 62. 58. Blanchard F, Duplomb L, Baud'huin M, Brounais B. The dual role of IL 6 type cytokines on bone remodeling and bone tumors. Cytokine Growth Factor Rev 2009;20(1):19 28. 59. Heymann D, Rousselle AV. gp130 Cytokine family and bone cells. Cytokine 2000;12(10):1455 68. 60. Li H, Hong S, Qian J, Zheng Y, Ya ng J, Yi Q. Cross talk between the bone and immune systems: osteoclasts function as antigen presenting cells and activate CD4+ and CD8+ T cells. Blood 2010;116(2):210 7. 61. Evans KE, Fox SW. Interleukin 10 inhibits osteoclastogenesis by reducing NFATc1 ex pression and preventing its translocation to the nucleus. BMC Cell Biol 2007;8:4. 62. Liu D, Yao S, Wise GE. Effect of interleukin 10 on gene expression of osteoclastogenic regulatory molecules in the rat dental follicle. Eur J Oral Sci 2006;114(1):42 9. 6 3. Rahimi P, Wang CY, Stashenko P, Lee SK, Lorenzo JA, Graves DT. Monocyte chemoattractant protein 1 expression and monocyte recruitment in osseous inflammation in the mouse. Endocrinology 1995;136(6):2752 9. 64. Kim MS, Day CJ, Morrison NA. MCP 1 is induc ed by receptor activator of nuclear factor {kappa}B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony stimulating factor suppression of osteoclast formation. J Biol Chem 2005;280(16):16163 9. 65. Li X, Qin L, Bergenstock M Bevelock LM, Novack DV, Partridge NC. Parathyroid hormone stimulates osteoblastic expression of MCP 1 to recruit and increase the fusion of pre/osteoclasts. J Biol Chem 2007;282(45):33098 106. 66. Kukita T, Nomiyama H, Ohmoto Y, Kukita A, Shuto T, Hotoke buchi T, et al. Macrophage inflammatory protein 1 alpha (LD78) expressed in human bone marrow: its role in regulation of hematopoiesis and osteoclast recruitment. Lab Invest 1997;76(3):399 406.

PAGE 58

58 67. Watanabe T, Kukita T, Kukita A, Wada N, Toh K, Nagata K, e t al. Direct stimulation of osteoclastogenesis by MIP 1alpha: evidence obtained from studies using RAW264 cell clone highly responsive to RANKL. J Endocrinol 2004;180(1):193 201. 68. Fuller K, Owens JM, Chambers TJ. Macrophage inflammatory protein 1 alpha and IL 8 stimulate the motility but suppress the resorption of isolated rat osteoclasts. J Immunol 1995;154(11):6065 72. 69. Kwak HB, Ha H, Kim HN, Lee JH, Kim HS, Lee S, et al. Reciprocal cross talk between RANKL and interferon gamma inducible protein 10 is responsible for bone erosive experimental arthritis. Arthritis Rheum 2008;58(5):1332 42. 70. Offenbacher S. Periodontal diseases: pathogenesis. Ann Periodontol 1996;1(1):821 78. 71. Graves DT, Cochran D. The contribution of interleukin 1 and tumor necro sis factor to periodontal tissue destruction. J Periodontol 2003;74(3):391 401. 72. Assuma R, Oates T, Cochran D, Amar S, Graves DT. IL 1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J Immunol 1998;160( 1):403 9. 73. Graves DT, Delima AJ, Assuma R, Amar S, Oates T, Cochran D. Interleukin 1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis. J Periodontol 1998;69(12):1419 25. 74. Cochran DL. Inflammation and bone loss in periodontal disease. J Periodontol 2008;79(8 Suppl):1569 76. 75. Belibasakis GN, Bostanci N, Hashim A, Johansson A, Aduse Opoku J, Curtis MA, et al. Regulation of RANKL and O PG gene expression in human gingival fibroblasts and periodontal ligament cells by Porphyromonas gingivalis: a putative role of the Arg gingipains. Microb Pathog 2007;43(1):46 53. 76. Bostanci N, Ilgenli T, Emingil G, Afacan B, Han B, Toz H, et al. Gingiva l crevicular fluid levels of RANKL and OPG in periodontal diseases: implications of their relative ratio. J Clin Periodontol 2007;34(5):370 6. 77. Bostanci N, Ilgenli T, Emingil G, Afacan B, Han B, Toz H, et al. Differential expression of receptor activato r of nuclear factor kappaB ligand and osteoprotegerin mRNA in periodontal diseases. J Periodontal Res 2007;42(4):287 93. 78. Liu D, Xu JK, Figliomeni L, Huang L, Pavlos NJ, Rogers M, et al. Expression of RANKL and OPG mRNA in periodontal disease: possible involvement in bone destruction. Int J Mol Med 2003;11(1):17 21.

PAGE 59

59 79. Lu HK, Chen YL, Chang HC, Li CL, Kuo MY. Identification of the osteoprotegerin/receptor activator of nuclear factor kappa B ligand system in gingival crevicular fluid and tissue of patien ts with chronic periodontitis. J Periodontal Res 2006;41(4):354 60. 80. Liu J, Wang S, Zhang P, Said Al Naief N, Michalek SM, Feng X. Molecular mechanism of the bifunctional role of lipopolysaccharide in osteoclastogenesis. J Biol Chem 2009;284(18):12512 2 3. 81. Beam HA, Parsons JR, Lin SS. The effects of blood glucose control upon fracture healing in the BB Wistar rat with diabetes mellitus. J Orthop Res 2002;20(6):1210 6. 82. Kayal RA, Alblowi J, McKenzie E, Krothapalli N, Silkman L, Gerstenfeld L, et al. Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment. Bone 2009;44(2):357 63. 83. Loder RT. The influence of diabetes mellitus on the healing of closed fractures. Clin Orthop Relat Res 1988(232):2 10 6. 84. Khazai NB, Beck GR, Jr., Umpierrez GE. Diabetes and fractures: an overshadowed association. Curr Opin Endocrinol Diabetes Obes 2009;16(6):435 45. 85. Bain S, Ramamurthy NS, Impeduglia T, Scolman S, Golub LM, Rubin C. Tetracycline prevents cancell ous bone loss and maintains near normal rates of bone formation in streptozotocin diabetic rats. Bone 1997;21(2):147 53. 86. Herrero S, Calvo OM, Garcia Moreno C, Martin E, San Roman JI, Martin M, et al. Low bone density with normal bone turnover in ovarie ctomized and streptozotocin induced diabetic rats. Calcif Tissue Int 1998;62(3):260 5. 87. Mathiassen B, Nielsen S, Johansen JS, Hartwell D, Ditzel J, Rodbro P, et al. Long term bone loss in insulin dependent diabetic patients with microvascular complicati ons. J Diabet Complications 1990;4(4):145 9. 88. Suzuki K, Miyakoshi N, Tsuchida T, Kasukawa Y, Sato K, Itoi E. Effects of combined treatment of insulin and human parathyroid hormone(1 34) on cancellous bone mass and structure in streptozotocin induced dia betic rats. Bone 2003;33(1):108 14. 89. Oikawa A, Siragusa M, Quaini F, Mangialardi G, Katare RG, Caporali A, et al. Diabetes mellitus induces bone marrow microangiopathy. Arterioscler Thromb Vasc Biol 2010;30(3):498 508.

PAGE 60

60 90. Hamada Y, Fujii H, Kitazawa R, Yodoi J, Kitazawa S, Fukagawa M. Thioredoxin 1 overexpression in transgenic mice attenuates streptozotocin induced diabetic osteopenia: a novel role of oxidative stress and therapeutic implications. Bone 2009;44(5):936 41. 91. Verhaeghe J, Suiker AM, Viss er WJ, Van Herck E, Van Bree R, Bouillon R. The effects of systemic insulin, insulin like growth factor I and growth hormone on bone growth and turnover in spontaneously diabetic BB rats. J Endocrinol 1992;134(3):485 92. 92. Spanheimer RG, Umpierrez GE, St umpf V. Decreased collagen production in diabetic rats. Diabetes 1988;37(4):371 6. 93. Bjorgaas M, Haug E, Johnsen HJ. The urinary excretion of deoxypyridinium cross links is higher in diabetic than in nondiabetic adolescents. Calcif Tissue Int 1999;65(2): 121 4. 94. Fujii H, Hamada Y, Fukagawa M. Bone formation in spontaneously diabetic Torii newly established model of non obese type 2 diabetes rats. Bone 2008;42(2):372 9. 95. Karel Z. Contrast limited adaptive histogram equalization. Graphics gems IV: Acad emic Press Professional, Inc.; 1994. p. 474 85. 96. Sternberg SR. Biomedical Image Processing. Computer 1983;16(1):22 34. 97. Karsdal MA, Martin TJ, Bollerslev J, Christiansen C, Henriksen K. Are nonresorbing osteoclasts sources of bone anabolic activity? J Bone Miner Res 2007;22(4):487 94. 98. Verhaeghe J, Suiker AM, Nyomba BL, Visser WJ, Einhorn TA, Dequeker J, et al. Bone mineral homeostasis in spontaneously diabetic BB rats. II. Impaired bone turnover and decreased osteocalcin synthesis. Endocrinology 1 989;124(2):573 82. 99. Pacios S, Kang J, Galicia J, Gluck K, Patel H, Ovaydi Mandel A, et al. Diabetes aggravates periodontitis by limiting repair through enhanced inflammation. FASEB J 2011. 100. Tsai C, Hayes C, Taylor GW. Glycemic control of type 2 diab etes and severe periodontal disease in the US adult population. Community Dent Oral Epidemiol 2002;30(3):182 92. 101. Taylor GW, Burt BA, Becker MP, Genco RJ, Shlossman M, Knowler WC, et al. Non insulin dependent diabetes mellitus and alveolar bone loss pr ogression over 2 years. J Periodontol 1998;69(1):76 83.

PAGE 61

61 102. Tervonen T, Karjalainen K. Periodontal disease related to diabetic status. A pilot study of the response to periodontal therapy in type 1 diabetes. J Clin Periodontol 1997;24(7):505 10. 103. Emri ch LJ, Shlossman M, Genco RJ. Periodontal disease in non insulin dependent diabetes mellitus. J Periodontol 1991;62(2):123 31. 104. Shlossman M, Knowler WC, Pettitt DJ, Genco RJ. Type 2 diabetes mellitus and periodontal disease. J Am Dent Assoc 1990;121(4) :532 6. 105. Cutler CW, Machen RL, Jotwani R, Iacopino AM. Heightened gingival inflammation and attachment loss in type 2 diabetics with hyperlipidemia. J Periodontol 1999;70(11):1313 21. 106. Dumitrescu AL, Abd El Aleem S, Morales Aza B, Donaldson LF. A m odel of periodontitis in the rat: effect of lipopolysaccharide on bone resorption, osteoclast activity, and local peptidergic innervation. J Clin Periodontol 2004;31(8):596 603. 107. Jiang J, Zuo J, Hurst IR, Holliday LS. The synergistic effect of peptidog lycan and lipopolysaccaride on osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96(6):738 43. 108. Matsuo K, Irie N. Osteoclast osteoblast communication. Arch Biochem Biophys 2008;473(2):201 9. 109. Javed F, Romanos GE. Impact of diabetes mellitus and glycemic control on the osseointegration of dental implants: a systematic literature review. J Periodontol 2009;80(11):1719 30. 110. Kopman JA, Kim DM, Rahman SS, Arandia JA, Karimbux NY, Fiorellini JP. Modulating the effects of diab etes on osseointegration with aminoguanidine and doxycycline. J Periodontol 2005;76(4):614 20. 111. McCracken MS, Aponte Wesson R, Chavali R, Lemons JE. Bone associated with implants in diabetic and insulin treated rats. Clin Oral Implants Res 2006;17(5):4 95 500. 112. Nevins ML, Karimbux NY, Weber HP, Giannobile WV, Fiorellini JP. Wound healing around endosseous implants in experimental diabetes. Int J Oral Maxillofac Implants 1998;13(5):620 9. 113. Zitzmann NU, Berglundh T. Definition and prevalence of per i implant diseases. J Clin Periodontol 2008;35(8 Suppl):286 91. 114. Lang NP, Berglundh T. Periimplant diseases: where are we now? -Consensus of the Seventh European Workshop on Periodontology. J Clin Periodontol 2011;38 Suppl 11:178 81.

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62 115. Berglundh T, Zitzmann NU, Donati M. Are peri implantitis lesions different from periodontitis lesions? J Clin Periodontol 2011;38 Suppl 11:188 202. 116. Lang NP, Bosshardt DD, Lulic M. Do mucositis lesions around implants differ from gingivitis lesions around teeth? J Clin Periodontol 2011;38 Suppl 11:182 7.

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63 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 h is 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 comple te a post doctoral residency in p eriodontics. After graduation, Douglas will begin practicing p eriodontics in North east Florida.