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Augmented Osteoclast Function in Type II Diabetes Mellitus

Permanent Link: http://ufdc.ufl.edu/UFE0044264/00001

Material Information

Title: Augmented Osteoclast Function in Type II Diabetes Mellitus
Physical Description: 1 online resource (53 p.)
Language: english
Creator: Britten, Todd Michael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diabetes -- osteoclast -- periodontitis
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: Altered osteoclast function in diabetes mellitus might account for exaggerated alveolar bone destruction in periodontitis. The aim of this study was observe differences in osteoclast differentiation and function, as well as soluble inflammatory mediator secretion in a murine model of type II diabetes (T2D). Eleven type II diabetic mice and eight normoglycemic mice were included in the test and control groups, respectively. Bone marrow-derived osteoclast (BMOC) precursor cells were isolated from murine subjects and induced to differentiate into osteoclasts with recombinant murine soluble receptor activator of nuclear factor kappa- B ligand (rmsRANK-L) and recombinant murine macrophage colony stimulating factor (rmM-CSF). Osteoclasts were plated on bovine bone slices and allowed to degrade bone under normal conditions and under induction from lipopolysaccharide (LPS) derived from Escherichia coli. Bone degradation was measured with collagen telopeptide and cathepsin K enzyme-linked immunosorbant assays (ELISAs). Bone slices were observed under SEM magnification to identify bone resorption. Soluble mediator analysis was used to detect pro-inflammatory cytokine activity. Diabetic mice produced more multinucleated and giant cell osteoclasts than the normoglycemic controls. The diabetic osteoclasts secreted greater levels of cathepsin K and produced more collagen via bone resorption than the control group. They also showed increased resistance to LPS-induced deactivation. Production of pro- inflammatory and pro-osteoclastic cytokines and chemokines was significantly greater in dbdb mice. This study indicated that osteoclast production and function is significantly altered in diabetic subjects with marked hyperglycemia and may account for augmented patterns of periodontal bone loss in diabetes.
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 Todd Michael Britten.
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: UFE0044264:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044264/00001

Material Information

Title: Augmented Osteoclast Function in Type II Diabetes Mellitus
Physical Description: 1 online resource (53 p.)
Language: english
Creator: Britten, Todd Michael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diabetes -- osteoclast -- periodontitis
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: Altered osteoclast function in diabetes mellitus might account for exaggerated alveolar bone destruction in periodontitis. The aim of this study was observe differences in osteoclast differentiation and function, as well as soluble inflammatory mediator secretion in a murine model of type II diabetes (T2D). Eleven type II diabetic mice and eight normoglycemic mice were included in the test and control groups, respectively. Bone marrow-derived osteoclast (BMOC) precursor cells were isolated from murine subjects and induced to differentiate into osteoclasts with recombinant murine soluble receptor activator of nuclear factor kappa- B ligand (rmsRANK-L) and recombinant murine macrophage colony stimulating factor (rmM-CSF). Osteoclasts were plated on bovine bone slices and allowed to degrade bone under normal conditions and under induction from lipopolysaccharide (LPS) derived from Escherichia coli. Bone degradation was measured with collagen telopeptide and cathepsin K enzyme-linked immunosorbant assays (ELISAs). Bone slices were observed under SEM magnification to identify bone resorption. Soluble mediator analysis was used to detect pro-inflammatory cytokine activity. Diabetic mice produced more multinucleated and giant cell osteoclasts than the normoglycemic controls. The diabetic osteoclasts secreted greater levels of cathepsin K and produced more collagen via bone resorption than the control group. They also showed increased resistance to LPS-induced deactivation. Production of pro- inflammatory and pro-osteoclastic cytokines and chemokines was significantly greater in dbdb mice. This study indicated that osteoclast production and function is significantly altered in diabetic subjects with marked hyperglycemia and may account for augmented patterns of periodontal bone loss in diabetes.
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 Todd Michael Britten.
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: UFE0044264:00001


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1 AUGMENTED OSTEOCLAST FUNCTION IN TYPE II DIABETES MELLITUS By TODD MICHAEL BRITTEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTE R OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Todd Michael Britten

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3 To my wife Mary Ellen and newborn son Daniel

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4 ACKNOWLEDGMENTS I would like to thank my family for their unwavering support for me throughout my long academic career. I would also like to thank the faculty members of the University of Florida Department of Periodontics for their commitment to my education and to our profession. I would especially like to thank my wife for her amazing patience, understanding, and suppo rt for me throughout all of the long days and nights that I have dedicated towards bettering myself as a dentist.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 LIST OF ABBREVIATIONS ................................ ................................ ............................. 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 BACKGRO UND ................................ ................................ ................................ ...... 13 The Periodontium ................................ ................................ ................................ ... 13 Periodontal Disease ................................ ................................ ................................ 13 Etiology of Periodontal Disease ................................ ................................ ........ 14 Bacterial colonization ................................ ................................ ................. 14 Host response to bacterial challenge ................................ ......................... 16 Mechanisms of Alveolar Bone Destruction in Periodontitis ................................ ..... 17 Physiologic Bone Remodeling ................................ ................................ .......... 17 Osteoblasts ................................ ................................ ................................ ...... 17 Osteoclasts ................................ ................................ ................................ ....... 17 Regulation and development of osteoclasts ................................ ............... 18 Osteoclast resorption of bone ................................ ................................ .... 18 Osteoimmunology ................................ ................................ ............................ 19 Innate immunity ................................ ................................ .......................... 19 T cell regulation ................................ ................................ .......................... 20 LPS induced osteoclast deactivation ................................ ......................... 20 Mechanisms of increased bone resorption in inflammation and periodontitis ................................ ................................ ............................ 21 Diabetes Mellitus ................................ ................................ ................................ .... 21 Physiological Action of Insulin and Glucagon ................................ ................... 22 Classification of Diabetes ................................ ................................ ................. 22 Diagnostic Criteria for Diabetes ................................ ................................ ........ 23 Clinical Presentati on of Diabetes ................................ ................................ ...... 24 Complications of Diabetes ................................ ................................ ................ 25 Periodontal Effects of Diabetes ................................ ................................ ........ 25 Bone Metabolism in Di abetes ................................ ................................ ........... 26 Mouse Model for Type 2 Diabetes ................................ ................................ .......... 27 3 MATERIALS AND METHODS ................................ ................................ ................ 29 Mouse Models ................................ ................................ ................................ ........ 29

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6 Osteoclast Differentiation ................................ ................................ ........................ 29 Tartrate resistant Acid Phosphatase Staining ................................ ......................... 30 Osteoclast Stimulation ................................ ................................ ............................ 31 Scanning Electron Microscopy ................................ ................................ ................ 31 Collagen Telopeptide Enzyme linked Immunosorbant Assay ................................ 32 Cathepsin K ELISA ................................ ................................ ................................ 32 Soluble Mediator Analysis ................................ ................................ ....................... 33 4 RESULTS ................................ ................................ ................................ ............... 37 Diabetic Mice Had Higher Plasma Glucose Concentrations ................................ ... 37 More Osteoclasts Derived From T2D Bone Marrow ................................ ............... 37 T2D derived OCs Secrete Mor e Cathepsin K and Degrade More Collagen ........... 37 T2D derived Osteoclasts Are Less Responsive to LPS induced Deactivation ........ 38 T2D Cultures Secrete Elevated Levels of Pro Inflammatory/Pro Osteoclastic Mediators ................................ ................................ ................................ ............. 38 5 DISCUSSION ................................ ................................ ................................ ......... 44 LIST OF REFERENCES ................................ ................................ ............................... 47 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 53

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7 LIST OF FIGURES page 3 1. Example of a db/db mouse. ................................ ................................ .................. 34 3 2. Example of TRAP staining ................................ ................................ .................... 35 3 3. Cathepsin K ELISA. ................................ ................................ .............................. 36 4 1. Differentiation of osteoclast precursor cells ................................ ........................... 39 4 2. Diabetic mice displayed more bone re sorption ................................ ..................... 40 4 3. Soluble mediator analysis, non LPS induced ................................ ........................ 41 4 4. Change in bone degradation after LPS induction. ................................ ................. 42 4 5. LPS induced changes in soluble mediators ................................ .......................... 43

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8 LIST OF ABBREVIATION S B6 Normoglycemic mouse of a C57BL/6 background BMOC Bone marrow derived osteoclast CEJ C ement o enamel junction CP C hronic periodontitis D B / DB Diabetic mouse of a C57BL/KSJ background ELISA Enzyme linked i mmunosorbent a ssay GCF Gingival crevicular fluid G CSF Granulocyte colony stimulating factor H B A1 C Glycated hemoglobin IFN Interferon gamma IL 10 Int erleukin 10 IL1 Interleukin 1 beta IL 6 Interleukin 6 IL 8 In terleukin 8 IP 10 Interferon gamma induced protein KC Keratinocyte chemoattractant LPS Lipopolysaccharide MCP 1 Monocyte chemoattractant protein 1 M CSF Macrophage colony stimulating factor MIP 1 ND Non Diabetic OC Osteoclast OPG Osteoprotegrin PDL Periodontal ligament

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9 RANK Receptor activator of nuclear factor kappa B RANK L Receptor activator of nuclear factor kappa B ligand RANTES R eg ulated upon activation, normal T cell expressed, and secreted rmM CSF R ecombinant murine macrophage colony stimulating factor RMS RANK L R ecombinant murine soluble r eceptor activator of nuclear factor kappa B ligand T1D Type 1 Diabetes T2D Type 2 Diabet es TLR 4 Toll like receptor 4 complex TNF Tumor necrosis factor alpha

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10 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 AU GMENTED OSTEOCLAST FUNCTION IN TYPE II DIABETES MELLITUS By Todd Michael Britten May 2012 Chair: Shannon Wallet Major: Dental Sciences Periodontal disease and diabetes mellitus are two common conditions in the United States that appear to have a synergi stically destructive effect on patients if present together. As periodontal disease progresses, patients experience irreversible destruction of the alveolar bone supporting the teeth. Osteoclasts (OCs) being the lone cell capable of resorbing bone, may have potentially altered function in diabetes. This may account for the increased alveolar bone resorption seen in diabetic patients with periodontitis. The aim of this study was observe differences in osteoclast differentiation and function, as well as soluble inflammatory mediator secretion in a murine model of type II diabetes (T2D). Eleven type II diabetic mice and eight normoglycemic mice were included in the test and control groups, respectively. Bone marrow derived osteoclast (BMOC) precursor cell s were isolated fro m murine subjects and induced with recombinant murine soluble r eceptor activator of nuclear factor kappa B ligand (rms RANK L) and recombinant murine macrophage colony stimulating factor (rmM CSF) to differentiate into osteoclasts Osteo clasts were plated on bovine bone slices and allowed to degrade bone under normal conditions and under induction from lipopolysaccharide (LPS)

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11 derived from Escherichia coli. B one degradation was measured with collagen telopeptide and cathepsin K enzyme li nked immunosorbant assays (ELISAs). Bone slices were observed under scanning electron microscope ( SEM ) to identify bone resorption. Soluble mediator analysis was used to detect pro inflammatory cytokine activity. Our results show that d iabetic mice prod uced more multinucleated and giant cell osteoclasts than the normoglycemic controls. Additionally, t he diabetic osteoclasts secreted greater levels of cathepsin K and produced more collagen via bone resorption than the control group. They also showed inc reased resistance t o LPS induced deactivation. This study indicated that osteoclast production and function is significantly altered in diabetic mice with marked hyperglycemia and may account for augmented patterns of periodontal bone loss in diabetes.

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12 CHAPTER 1 INTRODUCTION The periodontium represents a unique surface in the body in which soft tissue joins hard tissue that is exposed to an external environment. It is c onstantly populated by bacterial and viral pathogens that form a complex interaction with the host immune system. I f left untreated, this multispecies challenge can lead to progressive destruction of the tissues that sup port the teeth and eventually severe loss of alveolar bone support leads to tooth loss. It is therefore the goal of pe riodontal therapy to maintain the periodontal tissues in a state of health, comfort, and function with minimal tooth loss. Diabetes mellitus, like periodontal disease, involves chronic inflammation and is just as complex. Elevated levels of plasma glucose concentrations are a hallmark of the disease and can have many local and systemic complications. One complication is its exacerbative effect on periodontal disease and periodontal wound healing. Diabetic patients commonly display an increase in alveolar bone destruction via decreased osteoblast production, hyperexpression of pro inflammatory cytokines and chemokines, and increased collagen degradation As osteoclasts are the sole cell capable of bone resorption, i t is therefore possible that OC s have al tered function in diabetes, which may also account for the increased periodontal destruction. We hypothesize tha t osteoclasts derived from a type 2 diabetic mouse will have altered differentiation and/or function when compared to normoglycemic controls. A dditionally, they may have an altered response to bacterial endotoxin. Therefore, the aim of this study was to use a murine model of T2D to compare osteoclast differentiation and function in a hyperglycemic environment.

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13 CHAPTER 2 BACKGROUND The Periodont ium The periodontium refers to the tissues that invest and support the teeth including the gingiva, alveolar mucosa, cementum, periodontal ligament (PDL) and alveolar and supporting bone. In a state of health, connective tissue and junctional epithelium form the attachment apparatus to the tooth, coronal to the alveolar bone crest. The gingival sulcus is a shallow fissure surrounding the tooth lined by enamel, sulcular epithelium, and possibly cementum or dentin. Also in health, a state of equilibrium e xists between the commensal bacterial species acquired defense mechanisms. Alterations in any number of factors in the equilibrium of the periodontium can lead to disease and tissue destruction. Periodontal Disease Periodontal diseases encompass a large number of afflictions of the periodontal tissues, including gingivitis and periodontitis. Gingivitis is defined as inflammation of the gingiva. Typical clinical presentation includes erythema, edema and bl eeding of the gingiva. It is reversible, however, and health can be reestablished with the proper control of the plaque and bacterial biofilm. Periodontitis is differentiated from gingivitis in that irreversible destruction of alveolar bone and soft tissu e attachment to the teeth occurs as inflammation extends from the gingiva into the adjacent bone and ligament The understanding of this disease has evolved tremendously over the past three centuries

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14 surgical removal of excessive amounts of bone and gingiva with the aim to remove infection. Periodontitis is now understood to be a complex, chron ic process that involve s a host response to a polymicrobial invasion of the gingival sulcus. As the disease progresses so do host defense factors change. Initial disease is accompanied by neutrophil and lymphocyte mi gration to the site of microbial insul t. Later, as the diseased lesion becomes established, macrophage and plasma cell influx and collagen destruction can be observed. Finally, in advanced or chronic disease, d estruction of alveolar bone, connective tissue and PDL occurs as the lesion progre sses apically. Etiology of Periodontal Disease Bacterial c olonization A large number of bacterial species populate the human oral cavity in both health and diseas e. 1 The teeth are unique however, in that they provide a non shedding surface for bacterial colonization and proliferation. The bacterial biofilm forms readily in the gingival sulcus It remains there in a protected state unless physically disrupted Thus, daily disruption of the subgingival biofilm via tooth brushing, flossing or use of other interdental devices, as well as routine professional debridement are necessary to maintain this biofilm in a non pathogenic state. Bacterial colonization o f the periodontal tissues occurs in steps that eventually lead to the formation of a mature pathogenic biofilm, a dynamic colony of microorganisms that adheres to a surface. These steps include: 1) the binding of macromolecules to form a pellicle, a layer of molecules a bacterium can adhere to, 2) bacterial adherence to the pellicle, followed by 3) increase in the bacterial mass as more bacteria bind and multiply, 4) as the mass grows and oxygen gradient is created allowing anaerobic bacteria to thrive at the base of the biofilm.

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15 Deep in the gingival crevice nutrients from dietary sources and saliva are scarce and thus bacteria resort to destruction of host tissues for nutrition to survive. These bacteria have proteolytic enzymes that enable them to obtai n nutrients from the blood and gingival tissues around them. This can in part explain the destructive nature of periodontal disease. It is well documented that periodontal disease is not related to one specific bacterial species but rather a complex shif t towards polymicrobial pathogenesis. The subgingival microflora transitions from health, when gram positive cocci and short rods predominate, to disease, in which gram negative bacteria, vibrios and spirochetes predominate as the subgingival biofilm matu res. 2 Socransky and Haffajee identified several bacterial species that are most commonly found in periodontitis. This includes Porphyromonas gingivalis Treponema denticola and Tan n erella forsythia. 1 Other common periodontal pathogens i nclude Prevotella intermedia Fusobacterium Nucleatum and Aggregati bacter actinomycetemcomitans. P. gingivalis, a non motile, gram negative anaerobic rod, is particularly well studied for its many host virulence factors. Virulence factors are molecules or mechanisms that allow the bacteria to uniquely adapt to the gingival crevice while often harming the host. These include the use of two types of adhesion mechanisms, fi m briae and hemag g lutanin. P. gingivalis has the ability to cleave host defense pro teins such as collagen, to dysregulate host immune function, and to invad e neighboring epithelial cells. 3 P. gingivalis also contains a lipopolysachharide (LPS) macromolecule within its cell wall that elicits an immune response from the host that is

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16 designed to eli minate the intruder. 4 Lipid A, also known as endotoxin, is a portion of the LPS that provokes this immune response that, when present in the bloodstream can lead to septic shock. When bound to the Toll like receptor 4 complex (TLR 4) it triggers the release of multiple pro inflammatory cytokines, including tumo r necrosis factor alpha (TNF ) interleukin 1 beta (IL ), interleukin 6 (IL 6) and interleukin 8 (IL 8) However, the LPS found in oral gram negative bacteria such as P. gingivalis elicits a far weaker immune respon se than say E. coli which may in part account for its ability to evade host immune cells from elimin ating the pathogen. 4 Host response to bacterial c hallenge It is now understood that despite the presence of pathogenic bacterial species and absence of professional debridement, huma n host response to the polymicrobial c hallenge varies significantly and can lead to advanced disease in some patients and essentially no progression of disease in others. 5 In fact, recognition that the h ost immune response significantly contributes to tissue destruction and alveolar bone destruction, which are hallmarks of the disease, has been a key concept in the last three decades. Extensive evidence supports this hypothesis. For example, gingival cr evicular fluid (GCF) from diseased sites contains concentrations of cytokines and inflammatory mediators that are significantly higher than those found in the GCF from healthy sites 6 In certain knockout mice, removal of certain molecules responsible for neutrophil transit to the site of infection leads to advanced periodontal disease, highlighting the importance of proper neutrophil funct ion in health. 7 Other studies suggest that an overly aggressive immune response leads to accentuated bone resorpt ion. 8 9 In f act, Dayan and colleagues demonstrated that even in the absence of bacteria periodontal bone

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17 loss can occur during the production of excessive levels of host inflammat ory mediators. 9 Mechanisms of Alv eolar Bone Destruction in Periodontitis Physiologic Bone Remodeling Bone resorption and apposition are coupled, and in a healthy adult, are in a constant state of balance. As osteoclasts resorb an area of bone, osteoblasts are signa led to come in and rep lace it. The resorptive phase occurs over a 3 4 week period, while the formative phase occurs over a 3 4 month period. 10 The reversal phase is a period in between the resorptive and formative phases in which cells that appear morphologically inactive line the lacunae. The net result is a turnover of nearly 10% of the skeleton in a 1 year period. 11 Osteoblasts Osteoblasts (OB) are the cells respo nsible for bone formation and are derived from mesenchymal cells. Precursor cells differentiate into preosteoblastic cells via bone morphogenic proteins (BMPs), where after it matures into an osteoblast. The osteoblast subsequently becomes an osteocyte b y becoming trapped within a bony matrix, then dies or becomes a lining cell. 10 Osteoclasts Osteoclasts (OCs) are derived from progenitor cells of the hematopoetic lineage. They gain access to the sites of action via the blood supply. They are multinuclear cells, meaning they have more than 1 nuclei (typically more than 3), and can become giant osteoclasts, having 11 or more nuclei. Osteoclasts can be identified via their expression of an osteoclastic enzyme called tartrate resistant acid p hosphatase (TRAP), which stains purple.

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18 The structure of the OC includes a clear zone and a ruffled border, which allows it to bind to bone and create a seal with the aid of integrins. This sealed zone under the ruffled border ensures the resorptive pr ocess is localized under the OC. A proton pump regulates the hydrogen ion concentration and pH within its microenvironment. Regulation and d evelopment of osteoclasts Several cells are involved in formation of osteoclasts, including osteoblasts or bone m arrow stromal cells and lymphocytes. There are 2 major molecules considered essential to OC formation (osteoclastogenesis): macrophage colony stimulating factor (M CSF) and receptor activator of nulear factor kappa B ligand (RANKL). M CSF is a cytokine released from osteoblasts as a result of endocrine stimulation from parathyroid hormone. 12 It binds to receptors on osteoclast precursor cells (OPC) and induces differentiation into OCs. RANKL is a membrane bound protein found on stromal cells, OBs, or lymphocytes and directly bind s to its receptor, RAN K, to induce differentiation. into an active osteoclast in the presence of M CSF and in the absence of granulocyte and macrophage colony stimulating factor (GM CSF). 12 Osteoprotegrin (OPG), which is also produced by OBs, binds to RANK and competitively inhibits RANKL from binding, effectively inhibiting osteoclastogenesis. Osteoclast resorption of bone Bone is composed of approximately 70% mineral content and 30% organic content, the majority of which is type I colla gen. 11 Bone resorption begins with the osteoclast binding to the bony matrix via F actin forming an actin ring along the ruffled border, creating a seal. 13 The proton pump then produces hydrochloric acid (HCL) to dissolve the hydroxyapatite laden mineralized content. Meanwhile, the organic

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19 component of bone is degra ded by a number of enzymes released by the OC, but the primary enzyme is cathepsin K. Degraded collagen fragments are then transported to the extracellular matrix through vesicles. Specific cross linked collagen fragments, called collagen c terminal telop eptides, can be identified using immunoassays and are good markers for identifying active bone resorption. The size of the OC and number of nuclei may or may not be relevant to its resorptive capacity. Piper and colleagues observed a positive correlation between the number of OC nuclei and the volume of the resorptive pit that it made. However, Lees et al. observed no difference in resorption per nuclei when comparing small osteoclasts (2 5 nuclei) to large ones (10+ nuclei). 14 15 It is more likely that the size of the cell as a whole is more relevant to its resorptive capacity than the number of nuclei. Osteoim munology Innate immunity The innate immune system is a first line of defense against invading pathogens. It involves recognition of pathogens via pattern recognition receptors such as toll like receptors (TLRs), which are expressed in macrophages, dendri tic cells, B cells, and certain types of T cells. Once a pathogen is recognized TLRs initiate signaling pathways that include pro inflammatory cytokines to help promote elimination of the invading organism. The activation of TLRs in committed osteoclast precursors, mature osteoclasts and osteoblasts resu lts in increased os teoclastogenesis and is the most likely mechanism by which pathogen induced bone loss occurs. 12 Interestingly, Kollet et al showed that stress induced by bacterial LPS resulted in the appearance of many osteoclasts to the endosteum a nd mobilization of many osteoclast progenitor cells from the bone marrow to circulation. 16

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20 It is important to recognize that under normal physiologic conditions, certain cytokines will have pro inflammatory and pro osteoclastic bone resorptive effects. These include interleukin 1 (IL 1), IL 6, IL 11, IL 15, a nd TNF have similar inhibitory effects on bone resorption and include IL 4, IL 10, IL 12, and interferon gamma (IF 17 IL 1, in particular, is a potent pro inflammatory bone resorptive agent commonly identified in periodontal disease. In fact, humans with genetic polymorphisms of this cytokine that in increased levels upon bacterial challenge exhibit greater severity of periodontal disease. 18 Certain chemokines can also contribute to osteoclastogenesis. Macrophage inflammatory protein ) is expressed in bone and bone marrow cells and directly induces osteoclastogenesis through its receptors. M onocyte chemoattractant protein 1 (MCP 1) is another chemokine that is expressed by OBs and induce OC fo rmation. Other pro inflammatory chemokines include regulated upon activation normal T cell ex pressed and secreted (RANTES) and interferon inducible protein 10 (IP 10). I P 10 may serve as a chemoattractant for inflammatory cells such as monocytes/macropha ges and T cells and is involved in bone resorption. 19 T cell regulation While the osteo blast is the primary expressor of RANKL, activated T cells can also directly express RANKL. Additionally, they can indirectly induce RANKL expression in OBs via cytokines, resulting in increased osteoclastogenesis. 12 LPS induced osteoclast deactivation Most studies support the notion of LPS inducing os teoclastogenesis. 20 22 However, two recent studies have suggested that bacterial LPS actually inhibits

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21 osteoclastic precursor cells from differentiating into mature osteoclasts. 23 24 It remains controversial as to which mechanism is true. Mechanisms of increased bone resorption in inflammation and periodontitis It is apparent that chronic periodontitis results from the hosts inflammatory destruction of its own tissue in response to a chronic bacterial infection of the periodontium. The bone resorption/apposition proc ess, normally in equilibrium, is pushed towards resorption and results in irreversible destruction of alveolar bone. As the only cell capable of bone resorption, osteoclasts are involved in the pathogenesis of bone resorption in periodontitis. Certain pro inflammatory cytokines may play a key role in increased inflammatory bone resorption. For instance, while Preiss et al showed that IL increased at periodontitis sites Hou and colleagues demonstrated reduced levels of IL herapy. 25 26 Other inflammatory mediators and host derived enzymes that have been found in higher concentratio ns at periodontitis sites include IL 6 prostaglandin E 2 (PGE 2 ), and collagenases. 27 31 Diabetes Mellitus Diabetes mellitus is a group of metabolic diseases t hat represent disorders affecting the metabolism of lipids, proteins, and carbohydrates. The underlying common feature of all forms of diabetes is an elevation in blood glucose levels. Hyperglycemia results from either an inability to produce insulin, de ficiency in insulin effect to the target tissues, or both. Chronic hyperglycemia leads to damage to and potential failure of multiple vital organs, including the kidneys, eyes, heart, nerves, and vascular system.

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22 Physiological Action of Insulin and Glucag on Plasma glucose is regulated ove r a normal range of 55 165 mg/dL during the course of 24 h ours Insulin is primarily responsible for glucose regulation and homeostasis and has major effects on muscle, adipose tissue and the liver. The insulin receptor is a protein that consists subunits that must be able to autophosphorylate and phosphorylate intracellular substrates in order to mediate the complex cellular responses to insulin. ion, whereby it suppresses hepatic glucose output by stimulating glycogen synthesis, a storage mechanism for plasma glucose. It also inhibits glycogenolysis and gluconeogenesis, effectively decreasing plasma glucose concentrations. Insulin is an anabolic hormone that promotes lipid synthesis and suppresses lipid degradation. Target tissues for glucose use include the brain, liver, intestines, and muscle. Glucagon is also an important mediator of glucose homeostasis, whereby inhibition of glugagon secreti on causes a profound reduction in endogenous glucose production and a reduction in plasma glucose concentrations. As a consequence of diabetes, hyperinsulemia inhibits glucagon production, thus suppressing hepatic g lucose production. 32 Classification of Diabetes Classification of diabetes is based on the pathophysiology of hyperglycemia. Type 1 diabetes (T1D), which accounts for 5 10% of all diabetic cases, resu lts from an autoimmune destruction of pancreatic beta cells and results in absolute insulin insufficiency. It is usually diagnosed early in life with peak incidence at 10 14 years of

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23 age. Patients with Type 1 diabetes typically require daily exogenous in sulin supplementation t o sustain life. 32 Type 2 diabetes (T2D), previously known as non insulin dependent diabetes, occurs as the normal target tissues for insuli n action develop resistance to insulin. Type 2 diabetic patients often have altered insulin production as well. Early in the disease insulin production is often increased due to insufficient effect at target tissues, resulting in increased insulin produc dysfunction, which later leads to deficient insulin production and a relative insulin deficiency. At this point exogenous insulin supplementation is often required. However, complete destruction of some ability to produce insulin. 33 Most patients with T2D are obese and have an excess of body fat i n the abdominal region. Adipose tissue plays a predominant role in insulin resistance. Elevated circulating levels of free fatty acids inhibit glucose uptake, glycogen synthesi s, and glycoly s is. 34 This also increases hepatic glucose production. The r isk of dev eloping T2D increases with age, obesity, previous history of gestational diabetes, and lack of physical activity. 32 Diagnostic Criteria for Diabetes The level of blood glucose used to diagnose diabetes is based on the level of glucose above which microvascular complications have been shown to increase. As an example, the risk for developing a retinopathy increases when the fasting plasma glucose con centration exc eeds 108 116 mg/dL when the 2 h postprandial (after meal consumption) level rise s above 185 mg/dL and when the hemoglobin A1c level is greater tha n 5.9 6.0%. 32

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24 There are currently three diagnostic criteria for diagnosing diabetes, and each positive laboratory value must be re tested and confirmed on a separate day. These 200 mg/dl at any point in the day without r egard to last meal along with symptoms of diabetes (polyuria, polydipsia and unexp lained weight loss ) (2) fasting plasma glucose lev and (3) 2 h post l during an oral gluco se tolerance test. 35 The HbA1c test measures the amount of glycated hemoglobin in the bloodstream. Glycated hemoglobin forms in erythrocytes as a product of a reaction between he moglobin and glucose, and remains for the lifespan of the eryt hrocyte, approximately 123 days. 36 HbA 1c values rise with elevated levels of plasma glucose concentrations and are accurate measures of glycemic control over a 1 3 month period. However, due to lack of standardization of the a ssay performed for testing it is not included as a diagnostic crite ria for diabetes. 32 A normal value for HbA1c is <6.0% and the recommended target values for di abetics is <7.0%. Clinical Presentation of Diabetes Type 1 diabetes generally presents with an abrupt onset. Due to the altered osmolarity caused by chronic hyperglycemia, patients will have frequent urination (polyuria), followed by frequent thirst (po lydispia), hunger (polyphagia) and also unexplained weight loss. During periods of extreme hyperglycemia, patients can experience ketoacidosis, causing nausea, vomiting, and various levels of alte red consciousness. 32 Type 2 diabetic patients can typically be asymptomatic or experience polydispia and polyuria. They can also present with acute skin infections such as candidiasis.

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25 Type 2 patients are typically obes e and their symptoms can go unnoticed for many years until diagnosis, at which point significant systemic complications may have already occurred. Complications of Diabetes In periods of hyperglycemia the plasma glucose conce n trations can exceed the c apacity of the kidneys to recapture glucose. Glucose excretion in the urine can lead to severe dehydration. If insulin production is deficient as in T1D, ketone formation in the liver can lead to ketoacidosis, a potentially life threatening condition. V ascular complications as a result of narrowing of vascular lumens are commonly seen as well and can lead to complications of the brain, heart, and limbs N ephropathies and retinopathies of th e kidneys and eyes are also common. Lastly, nerve involvement re sults in diabe tic neuropathy, which commonly a ffects the peripheral limbs and can result in amputation. 32 Periodontal Effects of Diabetes Diabetes has significan t effects on the periodontium and renders treatment of periodontal disease in a diabetic patient challenging. Cianciola et al observed that the prevalence of gingivitis in T1D children was hi gher than age matched non diabet ics with similar plaque levels. 37 Biologic mechanisms of complica tions: Several mechanisms have been proposed to explain the increased prevalence and severity o f periodontitis in diabetic patien ts. First, i mpaired neutrophil chemotaxis has been demonstrated in diabetics with periodontal disease. 38 Other authors have shown a hyperresponsive production of pro inflammatory cytokines in response to bacterial LPS, such as TNF 39 40 Further, t he chronic state of hyperglycemia of the diabetic patient leads to the formation

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26 of advanced glycated end products (AGEs) via the non enzymatic glycation of proteins and lipids. The accumulation of these long lived proteins also leads to a chronic hyper inflammatory state involving cytokines and free radicals. It is known t hat high plasma glucose concentration leads to high glucose concentration in the gingival crevicular fluid and this impairs the wound healing capacity of fibroblasts in the periodontium, which are responsible for maintaining tissue health. Additionally, F rantzis et al. observed increased thickness of gingival capillaries in diabetics, which potentially limits oxygen and nutrient diffusion and limit the defense capacity of the periodontal tissues. 41 Finally, impaired collagen production and degradation in diabetics may alter the response to microbial insult. 42 43 Glycemic control: Literature has consistently shown that in regards to common periodontal parameters and/or response to periodontal therapy, diabetics with good glycemic control are similar to non diabetics, while poorly controlled diabetics respond less favorably. Cutler and colleagues showed that poorly controlled diabetics showed the greatest amount of gingival inflammation. 44 Other studies suggest that w ith similar plaque conditions, adult subjects with long term poorly controlled diabetes lost more approximal attachment and bone than the subjects with better metabolic control 45 A longitudinal study of diabetic Pima Indians observed that poor glycemic control of type 2 fold increased risk of progressive bone loss com pared to the non diabetic controls. Well controlled diabetic subjects (HbA1c <9%) had no significant increase in this risk 46 Bone Metabolism in Diabetes Hyperg lycemia also has marked effects on bone metabolism. This includes inhibition of osteoblastic cell proliferation and collagen production. 47 Overall, diabetics

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27 di splay poorer bone quality with decreased mineral content and mineralization rate. A lso, osteoclasts show increased numbers and function, while mesencymal stromal cells and osteoblasts are down regulated or diminished. Additionally, microvasculature leads to poor blood flow and diminished healing. 48 51 Mouse Model for Type 2 Diabetes In period ontal disease, human studies have traditionally been limited by the d if ficulty in establishing relationsh ips between periodontal breakdown and causative factors. Additionally, there are ethical limitations of inducing disease in an otherwise healthy individual. Thus, animal models are often utilized in periodontal research. Along with testing caus e and effect relationships, animal models can be utilized for tests of principle to establish advanced therapeutics. However, no perfect animal model exists, as periodontal disease involves complex host factors together with compl ex pathogen mechanisms and interactions. The selection of a n animal model should therefore be optimized to the goals of the study and should not necessarily reflect all aspects of periodontal disease. 52 Rodent models have several useful features for investigating the underlying mechanisms involved in inflammatory diseases The existence of rodent strains with targeted genetic deletions allows investigators to iso late factors that may play a role in overlying diseases, such as diabetes. The db/db murine model is a useful animal model for diabetes. The db/db mouse has a point mutation i n the leptin receptor gene that provides a deficiency in the leptin receptor. This receptor is responsible for the regulation of the leptin hormone, which controls food intake and energy expenditure. Knockouts for this receptor gene lead to an increase in appetite and consequently obesity and rapid development of type 2 di abetes. The mice develop significant o besity

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28 at 6 weeks of age and are hyperglycemic a nd hyperinsulinemic at fasting. 53 The aim of this study is to use a murine model of T2D to test for alterations in osteoclast differentiation and function in a bacterial LPS induced and non induced state.

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29 CHAPTER 3 MATERIALS AND METHOD S Mouse Models The test group for this study included d b /db (type 2 diabetic) mice on a C5 7BL/KSJ background. The control mice consisted of C57BL/6 (B6 normoglycemic) mice, all purchased from the Jackson Laboratory (Bar Harbor, Maine). Experiments were initiated when the mice were 10 11 w ee k s old and diabet ic for approximately 20 d ays The degree of hyperglycemia was similar among mice in the diabetic group typically having plasma glucose concentrations of 400 450 mg per dL. Glyc ated hemoglobin levels were 10% 15% for db/db mice and under 5% for normoglycemi c littermates. All mice were maintain ed in a specific pathogen free (SPF) environment at the breeding facilities of the University of Florida. Blood glucose levels were measured at time of sacrifice with the Ascensia Contour Blood Glucose Meter (B ayer). Bone marrow and pancreata were harvested fro m fe male mice aged 10 12 weeks of age. All experimental procedures were approved and conducted in accordance with the guidelines of the University of Florida Institutional Animal Care and Use Committee (IACUC) Osteoclast Differentiation The isolation and differentiation of murine osteoclasts first involved harvesting bone marrow from the specimens. Upon euthanasia, f emora and tibiae were surgically isolated, excess tissue removed, and marrow expelled from bones using a syringe with MEM complete media (Sigma Aldrich) [ 10% fetal bovine serum (Mediatech), 1% L glutamine (Thermo Scientific), 1% penicillin/streptomycin/amphotericin B (Fisher)]. In

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30 order to induce differentiation into osteoclasts, c ells were seeded in T75 flasks at a concentration of 1.5 x 10 6 cells/mL supplemented with 5 ng/mL recombinant murine macrophage colony stimulating factor (rmM CSF) (Peprotech Rocky Hill, NJ ) Cells were allowed to cul ture for 24 h at 37 o C in a 5% CO 2 incubator. Non adher ent cells were removed and 5.9 x 10 5 cells/mL of adherent cells were seeded in 24 well plates on either UpCell tissue culture plastic (Nunc Rochester, NY ), glass coverslips (Fisher Scientific, Waltham, MA ), or 1 cm 2 bovine bone slices cut with an Isomet L ow Speed Saw (Buehler Lake Bluff, IL ). All cultures were supplemented with 10 ng/mL rmM CSF and 50 ng/mL recombinant murine soluble r eceptor activator of nuclear factor kappa B ligand (rms RANK L) (Peprotech) and allowed to culture for 6 days with complete media refreshed every 3 d ays T artrate resistant Acid Phosphatase Staining After 6 d ays of differentiation, cells u nderwent a staining process for t artrate resistant acid phosphatase (TRAP) in order to determine the quantity and variety of cells that wer e obtained. Cells plated on glass coverslips were fixed with 2% paraformaldehyde/PBS (Fisher Scientific ) for 15 min. Cells were washed 2 times with PBS and permeablized for 10 min ute s in 0.5% Tr iton X 100/PBS (Fisher Scientific ). The cells were then was hed and probed for TRAP [1:1:1:2:4 Fast Garnet GBC Base Solution:Sodium Nitrite Solution:Napthol AS BI Phosphate Solution:Tartrate Solution:Acetate Solution] (Sigma Aldrich St. Louis, MO ) for 1 h ou r in the dark at 37 o C. The c ells were washed with d e ioni zed H 2 O and covered with glass coverslips mounted on glass slides with MOWIOL 4 88 solution (Cal biochem San Diego, CA ). The TRAP positive cells (purple in color) were counted according to number of n uclei present: pre

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31 osteoclasts (1 nucleus), multinuclea ted osteoclasts (2 10 nuclei), and giant osteoclasts (11+ nuclei) using light microscopy at 40x magnification. Osteoclast Stimulation Osteocla st function was examined under normal conditions and under lipopolysaccharide (LPS) stimulation To do this medi MEM complete media supplemented with 10 ng/mL rmM CSF and 50 ng/mL rmsRANK L. Cells were allowed to resorb bovine bone for 72 h in the presence or absence of 1ug/mL Escherichia coli LPS (Sigma Aldrich ). Cultures were permeab a lized w ith 1% Triton X 100 for 10 min Supernatants were stored at 80 o C until cathepsin K ELISA, collagen type I telopeptide ELISA, an d Luminex cyto/chemokine analyse s were performed. Bone was made devoid of cells with 10% sodium hypochlorite/PBS for 10 min af Scientific ) at 4 o C unti l SEM could be performed. Scanning Electron Microscopy SEM was utilized to identify the bone reso rption process on bovine slices As each osteoclast resorb s bone it leaves pits that are visible under SEM, which can then be counted. Bone slices were sputter coated with gold and visualized with S 4000 FE SEM scanning electron microscope (Hitachi Santa Clara, CA ). Three random scanning electron micrographs ( 8 bit grayscale) of bone slices were acquired at 40x magnification with a 2048 x identifiable borders. Identical procedures were applied to every image from all experimental groups ut ilizing NIH ImageJ software to quantify the surface area resorbed. Prominent repeating elements in the frequency domain were identified and removed after which the inverse fast Fourier transformation was applied yielding the

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32 original image with reduced sa w marks. The CLAHE algorithm was used to increas e contrast (block size: 256, histogram bins: 255, maximum slope: 8), and a Gaussian blur orithm was applied (radius: 100 pixels) to achieve background intensity equa lization. A threshold value wa s used to convert the result to a 1 bit image (0: normal bone, 1: region of resorption) used for quantitative analysis. Images which contained prominent artifacts spanning 5% or more of the total area were not included for data analysis. Collagen Telope ptide E nzyme linked Immunosorban t Assay The collagen telopeptide ELISA measures an end point of bone resorption and was therefore used to determine bone resorption activity q uantitatively from each sample Collagen carboxy terminal telopeptides were detec ted using an ELISA according to manufacturer instructions (Immunodiagnostic Systems Scottsdale, AZ ). Supernatants were p re incubated with both a biotin conjugated anti telopeptid e and a horseradish peroxidase (HRP) conjugated anti telopeptide and incubat ed 2 h in an ELISA plate coated with streptavidin (SAV) Following five washes, a tetramethylbenzidine (TMB) substrate was used to develop for 1 hr followed by quenching with H 2 SO 4 Colorimetric reactions were then detected using a Benchmark Microplate Reader spectrophotometer (Bio Rad Hercules, CA ) set at a dual wavelength reading of 450 nm with a reference of 655 nm to quantify the results Microplate Manager Software (Bio Rad) and a standard curve were used to determine nM concentrations. Cathepsin K ELISA Cathepsin K is a protease enzyme expressed by osteoclasts with the ability to catabolize elastin, gelatin, and collagen, allowing it to break down bone and cartilage.

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33 Active cathepsin K was thus targeted quantitatively in all samples and was dete cted using an ELISA according to manufacturer instructions (Alpco Salem, NH ). 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 30 min ute s followed by quenching with STOP solution (Cell Signaling Technology, Danvers, MA) Colorimetric reactions were detected using a Benchmark Microplate Reader spectrophotometer (Bio Rad) set at a dual wavelength reading of 450 nm with a reference of 655 nm. Microplate Manager Software (Bio Rad) and a standard curve were used to determine pM/L c onc entrations of active cathepsin K Soluble Mediator Analysis Cytokines and chemokines f rom resorption supernatants were detected and quantified in order to observe any possible differences in signaling pathways between the type 2 diabe tic and normoglycemic mice This was done using a mouse 22 cyto/chemokine multiplex (Millipore Billerica, MA ). Supernatant and antibody coated beads were allowed to incubate overnight at 4 o C in a 96 well primed plate. Following three washes, biotinylated detection antibodies were allowed to incubate for 1 h, after which SAV phycoerythrin (PE) was allowed to incubate for 30 min. 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 (Viag ene Tech, Carlisle, MA ), 5 parameter logistics and a standard curve were used to determine pg/ml concentrations.

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34 Figure 3 1. Example of a db/db mouse Deficient leptin receptors rendered the mice obese and development of T2D ensued. Mice were sacrif iced at 10 11 weeks old.

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35 3 2. Example of TRAP staining. TRAP staining revealed A) Pre osteoclasts (1 nucleus), B) Multi nucleated osteoclasts (2 10 nuclei), and C) Giant osteoclasts (11+ nuclei)

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36 3 3 Cathepsin K ELISA ELISA was performed acco rding to manufacturer instructions (Alpco). Colorimetric readings were then taken using a Benchmark Microplate Reader spectrophotometer (Bio Rad) set at a dual wavelength reading of 450 nm with a reference of 655 nm

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37 CHAPTER 4 RESULTS Diabetic Mice Had H igher Plasma Glucose Concentrations Eleven db/db mice were included in the test group and 8 B6 mice were included in the control group. In order to identify hyperglycemic status in the experimental and control groups, we measured blood glucose concentrati ons just before animal sacrifice. Db/db mice exhibited significantly higher (p <0.05) plasma glucose concentrations compared to the c ontrol B6 mice (Figure 4 1) More Osteoclasts Derived From T2D Bone Marrow In order to induce differentiation of the bone marrow isolates into osteoclasts, cells were supplemented with rmM CSF and RANK L for 6 days. TRAP staining and subsequent cell counting under microscopic examination revealed significantly higher counts of differentiated multinucleated osteoclasts in the db/db group compared to the B6 group (Figure 4 1) Db/db mice also showed significantly more formation of giant osteoclasts than the B6 controls T2D derived OCs Secrete More Cathepsin K and Degrade More Collagen In order to determine if osteoclasts fro m the T2D mice secrete more cathepsin K and degrade more collagen than the normoglycemic mice, we plated the cells onto bone slices and allowed them to resorb bone under induction and non induction from E. coli LPS. Figure 4 2 reveals that o steoclasts der ived from the T2D group secreted more cathepsin K than those derived from the B6 mice The difference was statistically significant (p <0.05) Diabetic mice also degraded significantly more collagen than the controls

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38 T2D derived Osteoclasts Are Less Res ponsive to LPS induced D eactivation Under direct stimulation from LPS, we observed a decrease in cathepsin K expression in normoglycemic mice, but no reduction in expression from db/db osteoclasts (figure 4 4) This difference in reduction was statistical ly significant (p <0.05) Additionally, significantly more reduction in collagen production from osteoclastic bone resorption was seen in the test group compared to the B6 group, which had no reduction in degradation. T2D Cu ltures Secrete Elevated Levels o f Pro Inflammatory/Pro Osteoclastic Mediators In order to observe differences in secretion levels of pro inflammatory and pro osteoclastic mediators soluble mediator analysis was performed via Luminex technology. Significantly higher levels of several pro inflammatory and pro osteoclastic mediators were produced by the db/db mice (p <0.05) (figure 4 3) These include G CSF, IL 6, IP 10, TNF Under stimulatory conditions with E. coli LPS, we observed significantly higher se cretory levels of CSF, IL 1, IP 10 and MIP (p <0.05) (figure 4 5)

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39 4 1 Differentiation of osteoclast precursor cells. Db/db mice produced more A) multinucleated osteoclasts, B) giant osteocl asts, and C) had higher plasma glucose concentrations before sacrifice. *p < 0.05

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40 4 2 Diabetic mice displayed more bone resorption. A) Bone degradation in db/db group produced significantly more collagen. B) Db/db mice secreted more cathepsin K en zyme. C) Plasma glucose levels for db/db mice were well above the 250 mg/dL threshold. *p < 0.05

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41 4 3 Soluble media tor analysis, non LPS induced. Db/db mice secreted more A) G CSF, B) IL 6, D) IP 10, E) TNF and G) MIP No significant differences in secretory levels of H) KC, and I) MCP 1. *p < 0.05

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42 4 4. Change in bone degradation after LPS induction. Production of A) Collagen and, B) Cathepsin K decreased in the B6 control group in response to E. coli LPS, while the db/db group did not respond.

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43 4 5. LPS induced changes in soluble mediators. Db/db subjects continued to secrete hig h levels of A) G CSF B) 1L 1. E) B6 mice secreted higher levels of IP 10 when compared to the db/db group. No difference was seen in levels of G) KC, H) RANTES, and I) TNF

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44 CHAPTER 5 DISCUSSION In the present study, leptin receptor de ficient mice developed overt hyperglycemia and subsequent type II diabetes mellitus. When bone marrow derived osteoclast (BMOC) precursor cells were allowed to differenti ate under induction from RANK L, a significantly higher volume of osteoclasts were pro duced when compared to normoglycemic mice. We believe that the increased differentiation of osteoclasts accounts for the significantly elevated production of the cathepsin K enzyme, which is then responsible for the increased rate of bone degradation into collagen, as observed in the present data. One possible explanation for increased BMOC differentiation is that the db/db osteoclastic precursor cells are potentially more sensitive to RANK L and thus under the same levels of available RANK L, more db/db d erived cells differentiate into osteoclasts. In periodontal disease, destruction of the periodontal attachment apparatus and alveolar bone commonly occurs on a site specific basis. Socransky and colleagues thesized that periodontal destruction occurs rapidly at some points in time with intermittent periods of relative latency. 54 The established periodontal lesion involves a complex interaction of inflammatory cells, includi ng plasma cells, polymorphonuclear cells (PMNs), macrophages, and dendritic cells, to name a few. 55 If these served as alternative sources for low level secretion of RANK L and diabetic osteoclastic precursors wer e indeed more sensitive to RANK L, they would potentially be induced to form more osteoclasts in the site of active inflammation. Increased presence of osteoclasts would then shift the homeostatic

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45 regulation of bone metabolism towards greater destruction and loss of supporting bone. The combination of RANK L sensitive osteoclast precursors and low level secretion of RANK L from the periodontal lesion would explain the site specificity of bone loss to the per iodontium. In this study, diabetic mice secreted elevated levels of pro inflammatory chemokines and cytokines. Additionally, when compared to the B6 control mice, they secreted more soluble mediators that promote osteoclastogenesis. This is consistent w ith previous literature in which diabetic patients exhibit hyper expression of pro inflammatory soluble mediators. 40 56 57 A recent study by Liu et al. indicate d that LPS induction deactivates osteoclastic bone resorption. 58 In contrast db/db osteoclasts in the present study did not show an LPS induced deactivation. The OCs effectively overcame the tolerance to LPS and continued to resorb significantly mo re bone than th e control mice, while also secreting higher levels of pro inflammatory and pro osteoclastogenetic cytokines and chemokines. This vicious cycle of increased bone resorption may in fact account for the increased rate of alveolar destruction seen in diabetic s with periodontal disease. The question exists as to whether increased expression of osteoclasts in the db/db mice is a result of their genetic background or of their severe hyperglycemic state. If the latter is true, it would account for the fact that periodontitis is more manageable and less severe in well controlled diabetics. W ell controlled diabetics respond as well to periodontal therapy as normoglycemic patients do. 59 60 Furthur studies are needed to determine if db/db mice produce similar amounts of osteoclasts in less e xtreme

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46 hyperglycemic conditions. This would involve testing the same db/db m ice against a control group before the db/db mice develop overt hyperglycemia.

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47 LIST OF REFERENCES 1. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL, Jr. Microbial complexes in subgingival plaque. Journal of clinical periodontolo gy 1998;25(2):134 44. 2. Loe H, Theilade E, Jensen SB. Experimental Gingivitis in Man. The Journal of periodontology 1965;36:177 87. 3. Lamont RJ, Chan A, Belton CM, Izutsu KT, Vasel D, Weinberg A. Porphyromonas gingivalis invasion of gingival epithelial cells. Infection and immunity 1995;63(10):3878 85. 4. Jain S, Darveau RP. Contribution of Porphyromonas gingivalis lipopolysaccharide to periodontitis. Periodontology 2000 2010;54(1):53 70. 5. Loe H, Anerud A, Boysen H, Morrison E. Natural history of pe riodontal disease in man. Rapid, moderate and no loss of attachment in Sri Lankan laborers 14 to 46 years of age. Journal of clinical periodontology 1986;13(5):431 45. 6. Champagne CM, Buchanan W, Reddy MS, Preisser JS, Beck JD, Offenbacher S. Potential f or gingival crevice fluid measures as predictors of risk for periodontal diseases. Periodontology 2000 2003;31:167 80. 7. Niederman R, Westernoff T, Lee C, Mark LL, Kawashima N, Ullman Culler M, et al. Infection mediated early onset periodontal disease in P/E selectin deficient mice. Journal of clinical periodontology 2001;28(6):569 75. 8. 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. Journal of immu nology 1998;160(1):403 9. 9. Dayan S, Stashenko P, Niederman R, Kupper TS. Oral epithelial overexpression of IL 1alpha causes periodontal disease. Journal of dental research 2004;83(10):786 90. 10. Manolagas SC. Birth and death of bone cells: basic regul atory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocrine reviews 2000;21(2):115 37. 11. McCauley LK, Nohutcu RM. Mediators of periodontal osseous destruction and remodeling: principles and implications for diagnosis and therapy. Journal of periodontology 2002;73(11):1377 91. 12. Bar Shavit Z. The osteoclast: a multinucleated, hematopoietic origin, bone resorbing osteoimmune cell. Journal of cellular biochemistry 2007;102(5):1130 9.

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48 13. Vaananen K. Mechanism of osteoc last mediated bone resorption -rationale for the design of new therapeutics. Advanced drug delivery reviews 2005;57(7):959 71. 14. Lees RL, Sabharwal VK, Heersche JN. Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclas ts cultured on collagen hydroxyapatite films. Bone 2001;28(2):187 94. 15. Piper K, Boyde A, Jones SJ. The relationship between the number of nuclei of an osteoclast and its resorptive capability in vitro. Anatomy and embryology 1992;186(4):291 9. 16. Kol let O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, et al. Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nature medicine 2006;12(6):657 64. 17. Walsh MC, Kim N, Kadono Y, Rho J, Lee SY, Lor enzo J, et al. Osteoimmunology: interplay between the immune system and bone metabolism. Annual review of immunology 2006;24:33 63. 18. Kornman KS, di Giovine FS. Genetic variations in cytokine expression: a risk factor for severity of adult periodontitis Annals of periodontology / the American Academy of Periodontology 1998;3(1):327 38. 19. Lee SH, Kim TS, Choi Y, Lorenzo J. Osteoimmunology: cytokines and the skeletal system. BMB reports 2008;41(7):495 510. 20. Henderson B, Nair SP. Hard labour: bacter ial infection of the skeleton. Trends in microbiology 2003;11(12):570 7. 21. Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B. Bacterially induced bone destruction: mechanisms and misconceptions. Infection and immunity 1996;64(7):2371 80. 22. Nagasawa T, Kiji M, Yashiro R, Hormdee D, Lu H, Kunze M, et al. Roles of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin in periodontal health and disease. Periodontology 2000 2007;43:65 84. 23. Zou W, Bar Shavit Z. Dual modulation of osteoclast differentiation by lipopolysaccharide. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2002;17(7):1211 8. 24. Takami M, Kim N, Rho J, Choi Y. Stimulation by toll l ike receptors inhibits osteoclast differentiation. Journal of immunology 2002;169(3):1516 23. 25. Preiss DS, Meyle J. Interleukin 1 beta concentration of gingival crevicular fluid. Journal of periodontology 1994;65(5):423 8.

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49 26. Hou LT, Liu CM, Rossomando EF. Crevicular interleukin 1 beta in moderate and severe periodontitis patients and the effect of phase I periodontal treatment. Journal of clinical periodontology 1995;22(2):162 7. 27. Geivelis M, Turner DW, Pederson ED, Lamberts BL. Measurements of int erleukin 6 in gingival crevicular fluid from adults with destructive periodontal disease. Journal of periodontology 1993;64(10):980 3. 28. Guillot JL, Pollock SM, Johnson RB. Gingival interleukin 6 concentration following phase I therapy. Journal of perio dontology 1995;66(8):667 72. 29. Offenbacher S, Odle BM, Braswell LD, Johnson HG, Hall CM, McClure H, et al. Changes in cyclooxygenase metabolites in experimental periodontitis in Macaca mulatta. Journal of periodontal research 1989;24(1):63 74. 30. Golu b LM, Siegel K, Ramamurthy NS, Mandel ID. Some characteristics of collagenase activity in gingival crevicular fluid and its relationship to gingival diseases in humans. Journal of dental research 1976;55(6):1049 57. 31. Villela B, Cogen RB, Bartolucci AA, Birkedal Hansen H. Collagenolytic activity in crevicular fluid from patients with chronic adult periodontitis, localized juvenile periodontitis and gingivitis, and from healthy control subjects. Journal of periodontal research 1987;22(5):381 9. 32. Meale y BL, Ocampo GL. Diabetes mellitus and periodontal disease. Periodontology 2000 2007;44:127 53. 33. Rhodes CJ. Type 2 diabetes a matter of beta cell life and death? Science 2005;307(5708):380 4. 34. Bergman RN, Ader M. Free fatty acids and pathogenesis o f type 2 diabetes mellitus. Trends in endocrinology and metabolism: TEM 2000;11(9):351 6. 35. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mell itus provisional report of a WHO consultation. Diabetic medicine : a journal of the British Diabetic Association 1998;15(7):539 53. 36. Virtue MA, Furne JK, Nuttall FQ, Levitt MD. Relationship between GHb concentration and erythrocyte survival determined from breath carbon monoxide concentration. Diabetes care 2004;27(4):931 5. 37. Cianciola LJ, Park BH, Bruck E, Mosovich L, Genco RJ. Prevalence of periodontal disease in insulin dependent diabetes mellitus (juvenile diabetes). Journal of the American Dent al Association 1982;104(5):653 60.

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50 38. Manouchehr Pour M, Spagnuolo PJ, Rodman HM, Bissada NF. Impaired neutrophil chemotaxis in diabetic patients with severe periodontitis. Journal of dental research 1981;60(3):729 30. 39. Engebretson SP, Hey Hadavi J, E hrhardt 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. Journal of periodontology 2004;75(9):1203 8. 40. Salvi GE, Collins JG, Y alda B, Arnold RR, Lang NP, Offenbacher S. Monocytic TNF alpha secretion patterns in IDDM patients with periodontal diseases. Journal of clinical periodontology 1997;24(1):8 16. 41. Frantzis TG, Reeve CM, Brown AL, Jr. The ultrastructure of capillary base ment membranes in the attached gingiva of diabetic and nondiabetic patients with periodontal disease. Journal of periodontology 1971;42(7):406 11. 42. Willershausen Zonnchen B, Lemmen C, Hamm G. Influence of high glucose concentrations on glycosaminoglyca n and collagen synthesis in cultured human gingival fibroblasts. Journal of clinical periodontology 1991;18(3):190 5. 43. Sorsa T, Ingman T, Suomalainen K, Halinen S, Saari H, Konttinen YT, et al. Cellular source and tetracycline inhibition of gingival cr evicular fluid collagenase of patients with labile diabetes mellitus. Journal of clinical periodontology 1992;19(2):146 9. 44. Cutler CW, Machen RL, Jotwani R, Iacopino AM. Heightened gingival inflammation and attachment loss in type 2 diabetics with hype rlipidemia. Journal of periodontology 1999;70(11):1313 21. 45. Safkan Seppala B, Ainamo J. Periodontal conditions in insulin dependent diabetes mellitus. Journal of clinical periodontology 1992;19(1):24 9. 46. Taylor GW, Burt BA, Becker MP, Genco RJ, Shl ossman M. Glycemic control and alveolar bone loss progression in type 2 diabetes. Annals of periodontology / the American Academy of Periodontology 1998;3(1):30 9. 47. Mealey BL, Oates TW. Diabetes mellitus and periodontal diseases. Journal of periodontol ogy 2006;77(8):1289 303. 48. Oikawa A, Siragusa M, Quaini F, Mangialardi G, Katare RG, Caporali A, et al. Diabetes mellitus induces bone marrow microangiopathy. Arteriosclerosis, thrombosis, and vascular biology 2010;30(3):498 508.

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53 BIOGRAPHICAL SKETCH Dr. Todd Britten grew up in Tampa, Florida where he attended Jesuit High School. He has an older brother, Matthew, and an identical twin brother, Nick, who is also a dentist and practices in Tampa with their father, Leonard Britten. Todd is married to his wife of nearly 4 years, Mary Ellen who he met while attending the Un iversity of Florida. They had their first c hild, Daniel, in March of 2012. Upon graduation, Tod d and Mary Ellen moved to the Tampa area where Todd now practices clinical periodontics He plans to continue his contribution to the profession through clin ical instruction and lecturing. He is an avid traveler, golfer, and enjoys movies, music, and football.