EFFECT OF NON SURGICAL PERIODONTAL THERAPY ON SERUM LEVELS OF IL-6 AND TNF- α IN CHRONIC

EFFECT OF NON SURGICAL PERIODONTAL THERAPY ON SERUM LEVELS OF IL-6 AND TNF- α IN CHRONIC

PERIODONTITIS PATIENTS WITH AND WITHOUT HYPOTHYROIDISM

DISSERTATION

Submitted to The Tamil Nadu Dr. M.G.R Medical University in partial fulfilment of the requirement for the degree of

MASTER OF DENTAL SURGERY

BRANCH II PERIODONTOLOGY

2013 - 2016

CERTIFICATE

This is to certify that the dissertation titled “Effect of Non Surgical Periodontal Therapy on serum levels of IL-6 and TNF-α in Chronic Periodontitis patients with and without Hypothyroidism” is a bonafide record of the work done by Dr. R. S. Indhuja, Post graduate student during the period 2013-2016 under our guidance and supervision. The dissertation is submitted to THE TAMIL NADU DR.M.G.R MEDICAL UNIVERSITY, CHENNAI, in partial fulfilment of the requirement for the award of the Degree of MASTER OF DENTAL SURGERY IN PERIODONTOLOGY, BRANCH II. It has not been submitted (partial or full) for the award of any other degree or diploma.

GUIDE CO GUIDE

Dr. Arun Sadasivan, MDS Dr. Elizabeth Koshi, MDS

Professor Professor & HOD

Department of Periodontics Department of Periodontics SreeMookambika Institute of SreeMookambika Institute of

Dental Sciences, Dental Sciences,

Kulasekharam. Kulasekharam.

SCIENCES, KULASEKHARAM

ENDORSEMENT BY THE PRINCIPAL / HEAD OF THE INSTITUTION

This is to certify that the dissertation entitled “Effect of Non Surgical Periodontal Therapy on serum levels of IL-6 and TNF-α in Chronic Periodontitis patients with and without Hypothyroidism” is a bonafide research work done by Dr. R.S. Indhuja under the guidance of Dr. Arun Sadasivan, M.D.S, Professor, Department of Periodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam.

Dr. Elizabeth Koshi, MDS, PRINCIPAL

Sree Mookambika Instituteof Dental Sciences, V.P.M Hospital Complex,

Padanilam, Kulasekharam,

Kanyakumari District, Tamil Nadu - 629161

I bow in gratitude to the Almighty God, for showering upon me His blessings that gave me the courage to venture out this thesis.

My foremost thanks to my guide, Dr. Arun Sadasivan MDS, Professor for his invaluable guidance, constant encouragement, immense patience and everlasting inspiration in every step of my research work.

I am ever grateful to my co-guide, Dr. Elizabeth Koshi MDS, Principal, Professor & Head Department of Periodontics, Sree Mookambika Institute of Dental Sciences for her valuable insight, suggestions and meticulous supervisionwhich shaped this work.

I submit my bouquet of thanks to Dr. C.K. Velayuthan Nair MBBS, MS., Chairman, Dr. Rema V Nair MBBS, MD, DGO., Director, Dr. Vinu Gopinath MS, Mch.

and Dr. R.V. Mookambika MD, DM., Trustees Sree Mookambika Institute of Dental Sciences for the sanction of resources and facilitating the conduct of my project.

I would like to acknowledge Dr. Anu Fernz MDS, Dr. Nikhil Das MDS, Dr. Shilpa Jayakumar MDS in the Department of Periodontics, for their practical advice and constant encouragement.

I owe a huge debt of gratitude to Dr. Radhakrishnan Nair, Scientist E at Rajiv Gandhi Institute of Biotechnology for his invaluable help in allowing utilization of lab resources and his technical expertise.

statistical analysis involved in this study.

I gratefully acknowledge my batchmate Dr. Steffi Vijayakumar and my fellow Post graduates Dr. Sheethal V. Menon, Dr. Vishnu J.S, Dr. Shamna, Dr. Anju, Dr. Blessing and Dr. Anina for their motivation and encouraging words.

With deep sense of gratitude, I remember the love, support and encouragement I received from my family and friends, I thank them with all my heart for being with me throughout my ups and downs.

Sl. No Index Page No

1. List of Abbreviations i-ii

2. List of Tables iii

3. List of Graphs iv

4. List of color plates v

5. List of Appendices vi

6. Abstract vii-viii

7. Introduction 1-4

8. Aims and objectives 5

9. Review of literature 6-29

10. Materials and Methods 30-37

11. Results and Observations 38-46

12. Discussion 47-51

13. Summary and conclusion 52

14. Bibliography ix-xvii

15. Appendices

i ALP ^- Alkaline Phosphatase

BUN ^- Blood Urea Nitrogen

cAMP ^- Cyclic Adenosine Monophosphate

CD 16 ^- Cluster of Differentiation 16 CD 56 ^- Cluster of Differentiation 56 CD 40 ^- Cluster of Differentiation 40 CRP ^- C Reactive Protein

CPITN ^- Community Periodontal Index of Treatment Needs ELISA ^- Enzyme Linked Immunosorbent Assay

ESR ^- Erythrocyte Sedimentation Rate GCF ^- Gingival Crevicular Fluid

hs-CRP ^- High sensitivity C reactive Protein HOMA ^- The Homeostasis Model Assessment HDL-C ^- High Density Lipoprotein cholesterol hBMSCS ^- Human Bone marrow stromal cells

IL-1β ^- Interleukin 1 beta

IL-6 ^- Interleukin 6 IL-10 ^- Interleukin 10 IL-8 ^- Interleukin 8

IFN - - Interferon gamma

LDL ^- Low Density Lipoprotein MMP ^- Matrix metalloproteinase mRNA ^- Messenger Ribonucleic Acid

ii PGE2 ^- Prostaglandin E2

RANK ^- Receptor Activator of Nuclear factor kappa B RANKL ^- Receptor Activator Nuclear factor kappa B Ligand SCH ^- Subclinical hypothyroidism

SGOT ^- Serum glutamic oxaloacetic transaminase SGPT ^- Serum glutamic pyruvic transaminase T3 ^- Triiodothyronine

T4 ^- Tetraiodothyronine

TSH ^- Thyroid Stimulating Hormone TNF-α ^- Tumour necrosis factor alpha TRAP ^- Tartrate Resistant Acid Phosphatase

iii

Table No Title

Table 1 Demographic characteristics of the study population

Table 2 Comparison of mean Periodontal and Biochemical parameters values within the study group

Table 3 Comparison of mean Periodontal and Biochemical parameters within the control group

Table 4 Multiple comparisons of Plaque Index between the groups Table 5 Multiple comparisons of Gingival Index between the groups Table 6 Multiple comparisons of Probing pocket depth between the groups Table 7 Multiple comparisons of Clinical attachment level between the groups Table 8 Multiple comparisons of TNF-α between the groups

Table 9 Multiple comparisons of IL-6 between the groups Table 10 Comparison of Thyroid function Test in study group

iv

Graph No Title

Graph 1 Age distribution in the study population among the groups Graph 2 Multiple comparisons of Plaque Index between the groups Graph 3 Multiple comparisons of Gingival Index between the groups Graph 4 Multiple comparisons of Probing pocket depth between the groups Graph 5 Multiple comparisons of Clinical attachment level between the groups Graph 6 Multiple comparisons of TNF-α between the groups

Graph 7 Multiple comparisons of IL-6 between the groups

v

Color plate No. Title of color plate

CP-1 Armamentarium for sample collection CP-2 Armamentarium for scaling and root planing CP-3 Blood collection

CP-4 Blood sample transferred to clot activator tube CP-5 Blood sample kept for 30 min

CP-6 Blood sample placed in Kemi C48 table top centrifuge CP-7 Supernatant serum is formed

CP-8 Serum transferred to cryotube CP-9 Serum in cryotube

CP-10 Liquid nitrogen container CP-11 Human IL-6 ELISA Kit CP-12 Human TNF-α ELISA Kit CP-13 Collected samples

CP-14 Vortex mixture

CP-15 Samples in the microplate well

CP-16 Covered with a plastic plate cover and incubated CP-17 ELISA wash procedure

CP-18 Addition of Streptavidin-HRP solution CP-19 Color reaction

CP-20 100 µl of H₂ SO₄ added

CP-21 Stop reagent incorporated into all wells CP-22 Absorbance value of each well was read

vi

Appendix No Title of Appendix

Appendix - 1 Permission letter from Rajiv Gandhi Centre for Biotechnology Appendix - 2 Certificate from Institutional Research Committee

Appendix - 3 Certificate from Institutional Human Ethics Committee Appendix - 4 Patient Information sheet

- English - Malayalam - Tamil

Appendix - 5 Patient Consent Form - English - Malayalam - Tamil Appendix - 6 Case record form

ABSTRACT

vii Background

IL-6 and TNF-α has been implicated in the pathogenesis of both periodontal disease and hypothyroidism. IL-6 and TNF-α are two major pro-inflammatory cytokines that are locally produced in different tissues in thyroid dysfunction. This enters systemic circulation and stimulate resident cells of the periodontium to produce matrix metalloproteinases, leading to alveolar bone destruction.

Aim of the study

To evaluate the effect of non-surgical periodontal therapy on serum levels of IL-6 and TNF-α in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing.

Objectives

1. To find out the effect of non-surgical periodontal therapy on clinical parameters like plaque index, gingival index, recession, probing pocket depth and clinical attachment level in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing.

2. To evaluate the serum levels of IL-6 and TNF-α in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing.

Materials and methods

This was an comparative interventional study including a total of 40 subjects (20 systemically healthy subjects with chronic periodontitis and 20 subjects with chronic periodontitis with hypothyroidism). Clinical parameters (Plaque index, Gingival index, Probing pocket depth and Clinical attachment level) were recorded

viii at baseline and 4 weeks after non surgical periodontal therapy. Non surgical periodontal therapy included oral hygiene instructions and full mouth scaling and root planing. 5 ml of venous blood sample was collected from each subject at baseline and 4 weeks after non surgical periodontal therapy. Serum IL-6 and TNF-α were analysed by ELISA sandwich method. T_3, T₄ and TSH hormone levels were evaluated at baseline and 4 weeks after non surgical periodontal therapy.

Results

The periodontal parameters like plaque index, gingival index, probing pocket depth and clinical attachment level and biochemical parameters like IL-6 and TNF-α in both the groups showed pronounced reduction at the end of four weeks following non surgical periodontal therapy from its baseline values. However, simultaneous comparison of baseline and post treatment periodontal parameters and serum levels of IL-6 and TNF-α between the two groups was not statistically significant.

Conclusion

Serum level of IL-6 and TNF-α were reduced following non surgical periodontal therapy in systemically healthy subjects with chronic periodontitis and chronic periodontitis patients with hypothyroidism. This study provide evidence that non surgical periodontal therapy contribute to reduction in serum level of IL-6 and TNF-α in these patients. Larger controlled trials are needed to confirm these findings.

INTRODUCTION

Page 1 Periodontitis is defined as an inflammatory disease of the supporting tissues of the teeth caused by specific microorganism or groups of specific microorganism, resulting in progressive destruction of periodontal ligament and alveolar bone with pocket formation, recession or both.¹ Initiation and progression of periodontitis are dependent on the presence of virulent microorganisms capable of causing disease.

Although bacteria are initiating agents in periodontitis, host response to the pathogenic infection is critical to disease progression.²

The primary etiologic factor in periodontal disease is the accumulation of bacteria in the gingival sulcus.³ Monocytes, macrophages and other cells responds to the dental plaque microorganisms by secreting a number of chemokines and inflammatory cytokines especially TNF–α, PGE2, interleukins (IL-1β and IL-6).⁴ Production of numerous proinflammatory cytokines is amplified by several bacterial served virulence factors, there by leading to the destruction of soft tissues and bone.⁵

Thyroid is the principal endocrine gland that regulates body functions, growth and metabolism. Among all the endocrine disorders, thyroid diseases are the most common glandular disorder. Various studies have estimated that around 42 million people suffer from thyroid diseases in India with a significantly higher prevalence in women.^6,7

Thyroid gland is a bilobular structure composed of a right and a left lobe connected by an isthmus. The gland produces two important hormones, triiodothyronine or T3 and thyroxine or T4, from a basic protein thyroglobulin, which is present in the thyroid follicles. Iodine is also essential for the formation of these hormones. Thyroid hormones production is controlled by feedback mechanisms and

Page 2 a deficiency of T3 or T4 can adversely affect growth and development of the infant and will decrease metabolic functions in adults. An excess or over production can lead to serious and life threatening complications if not detected and treated on time.⁸

Hypothyroidism is a condition where in thyroid gland function and production of the thyroid hormones is reduced. The causes of hypothyroidism can range from severe iron deficiency anemia, insufficient stimulation of the gland, inflammatory (Common Hashimoto’s thyroiditis), radioactive iodine, surgery, pregnancy (Post partum thyroiditis) and use of pharmacological agents such as lithium and amiodarone.⁹

The clinical features of hypothyroidism include anemia, cardiomegaly, intolerance to cold, constipation, dry and brittle hair, elevation in aspartate transaminase, alanine transaminase and lactate dehydrogenase, lethargy, weight gain, tachycardia, seizures, reduction in cardiac output, reduced respiratory rate, myxedema, parasthesia.¹⁰ The oral manifestations of hypothroidsm are, in childhood hypothryoidism known as cretinism is characterized by thickening of lips, large protruding tongue (macroglossia), malocclusions and delayed eruption of teeth.

Thickening of the lips and macroglossia is due to increased accumulation of subcutaneous mucopolysaccharides i.e. glycosaminoglycans due to decrease in the degradation of these substances. The long term effects of severe hypothyroidism on craniofacial growth and dental development have also included impaction of the mandibular second molars. This seems to be caused by a dissociation of ramus growth and failure of normal resorption of the internal aspect of the ramus, resulting in insufficient space for proper eruption of these teeth.¹¹ The other oral findings in

Page 3 hypothyroidism include dysgeusia, poor periodontal health, delayed wound healing, enamel hypoplasia, anterior open bite.⁸ Cytokines are important host response to infections, tissue degradation and repair. Therefore, cytokines play a crucial role in the maintenance of tissue homeostasis. Various periodontopathic bacteria possess different cytokines released in inflamed periodontium, TNF- α and IL-6 seems to be an important candidate as a causative mediator of periodontal tissue destruction.¹²

IL–6 is considered as a pleiotropic cytokine which like IL-1β and TNF–α appears early in the immune response development in periodontitis. Activated by other cytokines (IL-1β & TNF -α ) it produces a wide spectrum of cells activated T–

cells, B–cells, macrophages, dendritic cells and the so called non – immune cells (keratinocytes, fibroblasts and endothelial cells). It signals the proteins of the acute phase response (C-reactive protein) in hepatocytes and the localized immune response in periodontal disease. It regulates the proliferation and differentiation of B cells, the differentiations of dendritic cells, stimulates bone resorption and osteoclast development. Taylor believes that IL-6 has the power to overturn the course of the disease (localized bone turn over).¹³

TNF-α can be considered as an essential mediator of the immune response in periodontitis as it is produced directly by a number of cells and generally activates the immune response through the secondary mediator molecules induces chemokines, adhesion molecules and prostaglandin E2. TNF-α elevates phagocytic and neutrophil activity, induces the secretion of matrix metalloproteinases, stimulates the differentiation of osteoclasts and causes apoptosis in fibroblasts. In plaque induced inflammation the levels of TNF-α are increased.^13,14

Page 4 Alveolar bone resorption is the most important clinical parameter used to assess the severity of periodontal disease, therefore variations of T₃ and T₄ blood levels may be considered a modulating factor in chronic periodontitis. Thyroid hormones play an important role in bone resorption by influencing OPG and RANKL mechanism.¹⁵ This is accompanied by local production of IL-6 and other bone regulating factors.^16,17 Thyroid dysfunction has a negative effect on IL-6 and TNF-α which play an important role in osteoclast differentiation and function, without any influence from RANKL- RANK interactions .¹⁸

IL-6 and TNF-α are two major pro-inflammatory cytokines that are locally produced in different tissues in different pathological situations, including thyroid dysfunction.¹⁹ It is thought that cytokines stimulate resident cells of the periodontium to produce metalloproteinases, molecules that mediate connective tissue destruction and induce the differentiation and activation of osteoclasts, leading to alveolar bone destruction.²⁰ Till date, there are only limited data concerning the influence of scaling and root planing in chronic periodontitis patients with hypothyroidism. Hence an attempt has been made in this study to evaluate and compare the serum level of IL- 6 and TNF- α in chronic periodontitis patients with and without hypothyroidism before and after non surgical periodontal therapy.

Serum is preferred to plasma as it has been shown that different anticoagulants may affect absolute cytokine levels differently.²¹

AIMS & OBJECTIVES

Page 5 Aim of the study:

To evaluate the effect of non-surgical periodontal therapy on serum levels of IL-6 and TNF-α in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing.

Objectives:

1. To find out the effect of non-surgical periodontal therapy on clinical parameters like plaque score, gingival score, recession, probing pocket depth and clinical attachment level in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing.

2. To evaluate the serum levels of IL-6 and TNF-α in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing.

REVIEW OF

LITERATURE

Page 6 The association between periodontal diseases and systemic disease such as thyroid dysfunction had received importance in periodontal literature.²² The concept that oral diseases and systemic diseases influence each other goes back to the history of focal infection. In 1891, Miller published his theory regarding focal infection in which he indicated that microorganisms and their products are able to access parts of the body that are adjacent to or distant from the mouth.²³ The proponents of this concept assume that microorganisms present in dental plaque and their metabolic products may enter the bloodstream, thereby causing many systemic diseases and sometimes resulting in degenerative conditions. In recent years, the concept of focal infection has changed and now mostly relies on the correlation between chronic periodontitis and systemic diseases. Offenbacher introduced the concept of

‘Periodontal Medicine’ as a discipline focusing on validating this relationship and its biological plausibility using human and animal studies.²⁴

The thyroid gland is a bilobular structure that lies on either side of the trachea. Thyroid activity is controlled by the hypothalamic-pituitary-thyroid axis.

Thyroid-releasing hormone secreted by the hypothalamus, induces the secretion of TSH by the anterior pituitary, which in turn stimulates thyroid hormone synthesis and secretion by the thyroid gland.²⁵ Thyroid dysfunction is the second most common glandular disorder of the endocrine system (after diabetes mellitus) and is increasing, predominantly among women.²⁶ Hypothyroidism is defined by a decrease in thyroid hormone production and thyroid gland function. Hypothyroidism can occur as a congenital or acquired condition.²⁷

Page 7 The hypothyroidism can be divided into 3 main categories:

• Primary hypothyroidism due to permanent loss or atrophy of thyroid tissue

• Goitrous hypothyroidism (hypothyroidism with compensatory thyroid enlargement due to impairment of hormone synthesis) and

• Secondary hypothyroidism due to insufficient stimulation of a normal gland (hypothalamic or pituitary disease or defects in the TSH molecule).⁸

Thyroid hormones have an important role in controlling bone resorption through their action on the OPG and RANKL mechanism¹⁵ and on bone regulating factors such as IL-6.²⁸ Since changes in bone are prominent features of periodontal disease, alternations of thyroid hormone levels may be suggested to be a modulating factor in periodontal disease, as other systemic conditions such as smoking and diabetes.²⁹ The cytokines that were produced due to thyroid dysfunction might have been the initiators of an amplified inflammatory cascade. In combination with endotoxins produced by germs in dental plaque this might lead to higher local inflammatory mediator concentrations, including cytokines and prostaglandins, higher concentrations of MMP and of other proteinases with destructive effects on bone and conjunctive tissue, in the end leading to osteoporosis and periodontal breakdown.³⁰ Feitosa et al conducted a study to evaluate histologically in rats, the influence of thyroid hormones on the rate of periodontal bone loss, resulting from ligature placement and on the quality of the tooth-supporting alveolar bone. The results showed that decreased serum levels of thyroid hormones may enhance periodontitis- related bone loss, as a function of an increased number of resorbing cells.³¹

Page 8 IL-6 and TNF-α:

Cytokines are small polypeptides with a wide spectrum of inflammatory, hemopoietic, metabolic and immuno modulatory properties. They are produced by a variety of cells, including the macrophage monocyte system dendritic cell, lymphocytes, neutrophils, endothelial cells and fibroblasts. Cytokines are a large group of small protein with certain features of biological activity, whose main function is to regulate host response in periodontitis. The group includes interleukins, interferons and TNF (tumor neorosis factor) family.³²

IL-6 is a 26 KD protein released by fibroblasts, T lymphocytes, endothelial cells and monocytes.³³ It is pleiotropic cytokine produced by a wide spectrum of cells activated T-cells, B-cells, macrophages, dendritic cells and the so called non – immune cells (keratinocytes, fibroblasts and endothelial cells). It is active in the immune system, the cardiovascular system, nervous system, and hematopoiesis. It signals the proteins of the acute phase response (C-reactive protein) in hepatocytes and the localized immune response (Periodontal disease) it regulates the proliferation and differentiation of B cells, the differentiation of dendritic cells, stimulates bone resorption and osteoclast development.^34,35,36

Page 9 Elevated level of IL-6 in blood or biological fluid have been reported in association with immune pathologies, such as in tissue injury, infection and some inflammatory disease.³⁷ Osteoblasts and bone marrow stromal cells produce bone active cytokines such as IL-6 which are critical for osteoclast formation and bone resorption.³⁸

TNF-α is a multifunctional cytokine produced by macrophages and other cells during inflammatory responses.³⁹ TNF-α is predominantly produced by activated macrophages. TNF- α has a strong potential for increasing boneresorption and is involved in the degradation of connective tissue by stimulating PGE2 and collagenase respectively.⁴⁰ In plaque induced inflammation (such as the one associated with chronic periodontitis) the levels of TNF-α are increased.¹² Cells expressing CD4 secrete TNF α, while CD8+ve cells secrete little or no TNF-α. Besides the direct effect on the pathogenesis of periodontal disease, TNF-α

IL-6 producing cells and biological activities of IL-6.³³

Page 10 upregulates the production of other classic proinflammatory innate immune cytokines such as IL-1β and IL-6.⁴¹

IL-6 and TNF-α in hypothyroidism:

Ahmet et al. 1999⁴² conducted a study to evaluate the serum levels of cytokines IL-6 and TNF-α to examine their relationship in patients with hyperthyroidism, hypothyroidism and with normal thyroid functions. The study was designed to evaluate IL-6 and TNF-α levels and to examine their relationship in 27 patients with hyperthyroidism, 23 with hypothyroidism and 18 with normal thyroid functions. All of control subject and patient groups were not taking any medication known to affect thyroid metabolism. Serum T3, T4, TSH levels were determined by automated chemiluminescence assay. Serum IL-6 and TNF-α were measured by an enzyme-linked immunosorbent assay. Mean serum levels of IL-6 and TNF-α in patient with untreated hyperthyroidism were higher than those of control group.

However, these values were lower in hypothyroidism than in the controls. After treatment, both of the patients groups had IL-6 and TNF-α value in accordance with those of controls and these values were decreased in hyperthyroid patients, but increased in hypothyroid ones. There were significant correlations between before and after treatment serum IL-6 and TNF-α values in hyperthyroid patients and between before and after treatment serum IL-6 and TNF-α in hypothyroid patient. There were significant correlations between serum IL-6 and TNF-α in control subjects and it was concluded that serum IL-6 and TNF-α are increased in patients with hyperthyroidism, but decreased in patients with hypothyroidism subjects, and become normal by successful surgical treatment or ten or more months after medical treatment.

Page 11 Kim et al. 1999⁴³ conducted a study to examine the effects of thyroid hormone on basal IL-1 stimulated IL-6 and IL-11 production in primary cultured human bone marrow stromal cells (hBMSCS). In this study, they studied the effects of 3,5,3-tri-iodothyronine (T₃) on basal and interleukin-1 (IL-1)-stimulated IL-6/IL- 11 production in primary cultured human bone marrow stromal cells. T₃ at 10^-12 – 10^-8M concentration significantly increased basal IL-6 production in a dose- dependent manner. It also had an additive effect on IL-1-stimulated IL-6 production, but failed to elicit a detectable effect on basal or IL-1-stimulated IL-11 production.

Treatment with 17β-estradiol (10^-8 M) did not affect the action of T3 on IL-6/IL-11 production. These results suggest that thyroid hormone may stimulate bone resorption by increasing basal and IL-1 induced IL-6 production from osteoblast- lineage cells, and these effects are independent of estrogen status.

Sekeroglu MR et al. 2006¹⁹conducted a study to assess serum IL-6 and TNF-α levels, as well as bone turn over markers in hyperthyroidism and hypothyroidism. A total of 20 female patients with hyperthyroidism, 15 with subclinical hyperthyroidism, 16 with hypothyroidism, and 15 with subclinical hypothyroidism constituted the patient groups. In all, 15 age-matched healthy female volunteers were recruited as controls. When compared with controls, serum TNF-α levels showed no significant difference in any of the patient groups. In the groups with hyperthyroidism and subclinical hyperthyroidism, IL-6 levels were significantly higher compared with control group values. Hyperthyroid patients showed higher levels of ALP and osteocalcin, and a higher urinary deoxypyridinoline/creatinine ratio, compared with controls. In subclinical hyperthyroidism, only ALP was found to be higher compared with control values.

Page 12 No significant correlations were made in any group between serum IL-6 or TNF-α level and bone turnover markers. In the group with hyperthyroidism and subclinical hyperthyroidism IL -6 levels were significantly higher compared with control group values. Results suggested that serum IL-6 level and markers of bone turnover rate seem to be increased in hyperthyroidism.

Tokuda et al. 1998¹⁷ conducted a study to investigate the effect of T3 on IL - 6 synthesis induced by several agonists in osteoblast like MC3T3 – E1 cells. T3, which by itself had little effect on IL-6 synthesis, significantly reduced the IL-6 synthesis induced by PGE1 in a dose-dependent manner in the range between 10 pM and 10 nM. T3 also reduced PGE1-induced activation of protein kinase A. T3

inhibited the IL-6 synthesis induced by cholera toxin, an activator of Gs, or forskolin, which directly activates adenylate cyclase. However T3 did not affect (Bu)2cAMP-induced IL-6 synthesis. In addition, T3 reduced PGF2a-induced IL-6 synthesis dose dependently in the range between 10 pM and 10 nM. T3 also inhibited IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C. On the other hand, T3 markedly enhanced IL-1 induced IL-6 synthesis. This enhancement by T₃ was potentiated in protein kinase C down-regulated cells. T₃ hardly affected the protein kinase C activation induced by PGF2a (Prostaglandin F2α) or IL-1. These results strongly suggest that T3 modulates IL-6 synthesis at two points in osteoblasts as follows; one is exerted at the point between adenylate cyclase and protein kinase A, and the other is at a point downstream from protein kinase C activation.

Adriana Monea et al. 2014³⁰ conducted a study to determine if thyroid dysfunction can induce periodontal disease by comparing salivary and serum levels

Page 13 of IL -6 and TNF-α in patients with thyroid dysfunction without clinical periodontal changes to healthy controls. Venous blood and unstimulated saliva were assessed for TNF-α and IL-6 levels, using ELISA-sandwich methods. Mean values of serum TNF-α concentration for patients with hypothyroidism were 4 times higher than in subjects with hyperthyroidism and almost 15 times higher than in controls, but there was no significant difference between hypo- and hyperthyroid subjects. Mean values of salivary TNF-α were significantly higher in hyperthyroid subjects than in controls, also in hypothyroid subjects than in controls, but there was no significant difference between hypo- and hyperthyroid subjects. Serum IL-6 had apparently much higher levels in study groups (99.39±54.41pg/ml hyperthyroid and 68.69±45.94pg/ml in hypothyroid) compared with controls (11.23±2.14pg/ml), but the differences were not statistically significant. Mean values of salivary IL-6 were significantly higher both in hyperthyroid and hypothyroid subjects than in controls, and there was an extremely significant difference between hypo- and hyperthyroid subjects. This study concluded that serum and salivary levels of TNF-α and IL-6 represent a well individualized biologic indicator for appreciating the development of periodontitis in subjects with thyroid dysfunction. Hyperthyroidism can induce more important periodontal destructions than hypothyroidism.

Taddei et al. 2006⁴⁴ conducted a study to assess whether low grade systemic inflammation might contribute to the pathogenesis of endothelial dysfunction in patients with subclinical hypothyroidism and autoimmune thyroiditis. In sHT and healthy subjects, the forearm blood flow (strain-gauge plethysmography) response to intrabrachial acetylcholine (Ach) with and without local vascular COX inhibition by intrabrachial indomethacin or nitric oxide synthase blockade by N-mono methyl

Page 14 arginine (L-NMMA) or the antioxidant vitamin C were studied. The protocol was repeated 2 hours after systemic nonselective COX inhibition (100 mg indomethacin) or selective COX-2 blockade (200 mg celecoxib) oral administrations. The results showed that in controls, vasodilation to Ach was blunted by L-NMMA and unchanged by vitamin C. In contrast, in sHT, the response to Ach, reduced in comparison with controls, was resistant to L-NMMA and normalized by vitamin C.

In these patients, systemic but not local indomethacin normalized vasodilation to Ach and the inhibition of L-NMMA on Ach. Similar results were obtained with celecoxib. When retested after indomethacin administration, vitamin C no longer succeeded in improving vasodilation to Ach in sHT patients and concluded that subclinical hypothyroid patients showed higher C reactive protein and IL-6 values.

In sHT patient low grade chronic inflammation cause endothelial dysfunction and impaired nitric oxide availability by a COX-2 dependent pathway leading to increased production of oxidative stress.

Turemen et al. 2011⁴⁵ conducted a study to investigate the presence of the well known preceding clinical situations of atherosclerosis like endothelial dysfunction and inflammation in SCH. Endothelial dysfunction was measured by examining brachial artery responses to endothelium-dependent (flow mediated dilation, FMD) and endothelium-independent stimuli (sublingual nitroglycerin (NTG)). Serum TNF-α, IL-6, and hs-CRP were measured. The estimate of insulin resistance by HOMA score was calculated with the formula: fasting serum insulin (µIU/ mL) x fasting plasma glucose (µM/L)/22.5. There were no significant differences in age, body mass index, waist circumference, HOMA scores. There was a statistically significant difference in endothelium-dependent (FMD) and

Page 15 endothelium-independent vascular responses (NTG) between the patients with subclinical hypothyroidism and the normal healthy controls. The groups were well matched for baseline brachial artery diameter. The TSH and LDL, IL-6, TNF-α and hs-CRP levels in the patient group were significantly higher than those in control group. A positive correlation was found only between endothelium-dependent vasodilation and TNF-alpha, hs-CRP and IL-6, TSH, total cholesterol, LDL and triglycerides. Endothelium-independent vascular response was not correlated with any of the metabolic or hormonal parameters. Neither of the groups were insulin resistant and there was not any difference either in fasting insulin or in glucose levels. This study found endothelial dysfunction in subclinical hypothyroidism group. Endothelium-dependent (FMD) and endothelium-independent vascular responses (NTG) were lower in patient group. This study also suggested that there is endothelial dysfunction and low grade chronic inflammation in SCH was due to autoimmune thyroiditis. There are several contributing factors which can cause endothelial dysfunction in SCH such as changes in lipid profile, hyperhomocysteinemia. According to this results low grade chronic inflammation may be one of these factors.

Marchiori et al. 2015⁴⁶ conducted a study to assess inflammatory biomarkers and oxidative stress in patients with primary hypothyroidism at baseline and after 6 and 12 months of levothyroxine replacement therapy (LRT). The following parameters were measured before and at 6 and 12 months of levothyroxine treatment with an average dose of 1.5 to 1.7 µg/kg/day: TSH, free thyroxine (fT4), hs-CRP, IL-1, IL-6, IL-10, INF-γ, TNF-α, thiobarbituric acid- reactive substances (TBARS), activity of aminolevulinic acid dehydratase (δ-ALA-

Page 16 D), nonprotein and total thiol (NP-SH and T-SH) groups, total cholesterol (TC), HDL-C, LDL-C and TG. Generalized estimating equation (GEE) modeling was used to analyze the effects of LRT (at pre-treatment, 6 months and 12 months) on those variables. The hypothyroidism status (i.e., overt or subclinical hypothyroidism) was included as a confounder in all analyses. An additional GEE post hoc analysis was made to compare time points. The findings suggested that there was a significant decrease in TSH over time. There were significant changes in interleukin levels over time, with a significant increase in IL-10 and significant decrease in IL-1, IL-6, IFN- and TNF-α. In conclusion, this study observed a significant reduction of pro- inflammatory cytokines and an increase of one anti-inflammatory cytokine in hypothyroid patients using levothyroxine. In these patients, a decrease in low-grade chronic inflammation may have clinical relevance due to the known connection between chronic inflammation, atherosclerosis and cardiovascular events.

Siddiqi et al. 1998⁴⁷ conducted a study to investigate whether the T₃-induced increase in bone resorption could be due to the regulation of cytokine production by human osteoblasts. They examined the effects of T3 on medium cytokine concentrations and cytokine gene expression in cultured hOb-like cell lines, primary cultures of hOb (human osteoblasts) and hBMS. The effects of T3 (1, 10, 100 nM) and IL-1α (100 U/ml) as the positive control were examined on cytokine protein release and mRNA levels in cultured hOb cell lines (MG63, SaOs-2), primary hOb and human bone marrow stromal (hBMS) cells. T3 increased IL-6 and IL-8 mRNA levels as well as IL-6 and IL-8 protein release into the culture media from MG63 and hBMS cells in a time- and dose-dependent manner. The maximal effect on protein release in hBMS cells occurred at 24 hours with a dose of T3 10 nM (IL-6

Page 17 5·5&1·1-fold above controls; IL-8 3·7&0·5-fold above controls). At the same time, mRNA levels in hBMS cells were increased 6·2 & 0·8-fold for IL-6 and 5·7&0·8- fold for IL-8. Similar results were obtained in MG63 cells but no response was seen in SaOs-2 (hOb -like cell lines) or hOb cells despite measurable basal production.

Nor was there detectable regulation of IL-1α, IL-3, IL-11, IL-4 or granulocyte macrophage-colony stimulating factor by T3 in any cell type and concluded that T3

increases IL-6 and IL-8 by MG63 and hBMS cells, suggesting that IL-6 and IL-8 may be T₃ regulated genes in osteoblasts.

Gaurav Gupta et al. 2015⁴⁸ conducted a study to investigate the relationship between subclinical hypothyroidism and inflammatory markers (ESR, CRP and IL- 6) TSH, fT4& T3 were estimated by enzyme linked Immunosorbent assay (ELISA) for diagnosis of subclinical hypothyroidism. Total cholesterol, triglycerides, and HDL-C were estimated by spectrophotometric method. LDL-C was calculated by Friedewald formula. Inflammatory markers (ESR, C-reactive protein & Interleukin 6) were also estimated by ELISA. In this study the level of TSH Mean ± SD and T3

Mean±SD were significantly higher in subclinical hypothyroidism. Serum concentration of fT₄ was not significantly different between the groups. Total cholesterol, triglycerides, and LDL-C were significantly higher in patients group.

While the level of HDL-C was significantly lower in SCH patients compared to euthyroid group. TSH level was positively correlated with inflammatory markers in subclinical hypothyroidism, which were significantly different in subclinical hypothyroidism and concluded that SCH patients have increased inflammatory markers along with dyslipidemia and due to that future risk of further development of cardiovascular disorder can occur.

Page 18 IL-6 and TNF-α in chronic periodontitis

The primary etiologic factor in periodontal disease is the accumulation of bacteria in the gingival sulcus. Bacteria and their products accumulate in the gingival sulcus and mediate connective tissue destruction through the ability of the antigens from their cell walls to stimulate pro-inflammatory cytokines by circulating mononuclear cells production of numerous pro-inflammatory cytokines is amplified by several bacteria derived virulence factors, there by leading to the destruction of soft tissues and bone.⁴⁹

The cytokines such as TNF-α appears to play a central role in the progressive migration of an inflammatory front towards the alveolar bone. This suggests that the production of cytokines at deeper levels within the gingival connective tissue leads to an inflammatory cascade in this area. Once a “Critical level” of proinflammatory cytokine TNF-α production is reached, a physiologic response becomes a pathologic response. If the inflammatory front occurs predominantly in the area of attachment to cementum, the result will be loss of attachment. If it occurs near the alveolar crest, the result would be a loss of bone.

If the inflammatory front has not progressed far from the epithelium, the resulting lesion will be restricted to gingivitis.⁵⁰

Nyugen et al. 1991⁵¹ conducted a study to evaluate the effect of IL-1β on bone resorption and formation in adult rats in vivo. Resorption was assessed by serum and urinary calcium levels, osteoclast number, and active resorption surface.

Bone formation was determined by measurement of serum osteocalcin levels, and by quantitation of bone apposition rate using tetracycline labeling. A modest dose of IL-1β (1 microgram/kg) was found to stimulate transient increases in serum

Page 19 calcium, and a persistent elevation of urinary calcium excretion. IL-1β treatment also resulted in decreases in serum iron levels and in the albumin/globulin ratio, well-established in vivo effects of IL-1. SGOT, SGPT, BUN, creatinine, and total protein were unaffected, indicating that IL-1β treatment did not compromise kidney or liver function, and that animals were systemically healthy. This was further evidenced by normal weight gain in IL-1β treated animals. Low doses (50 micrograms/kg) of synthetic human parathyroid hormone (PTH 1-34) also stimulated resorption, as shown by a sustained increase in serum calcium without increased urinary excretion. Both IL-1β and parathyroid hormone (PTH) stimulated similar increases in osteoclast number (N.Oc) and active resorption surface [Oc.S(%)] and concluded that application of cytokines like IL-1β and TNF-α in vivo not only stimulates bone resorption but limits bone formation by inhibiting the coupling process. The net bone loss observed in periodontitis is due to the uncoupling of bone resorption and bone formation.

Page 20 Bone loss in periodontitis is due to uncoupling of bone resorption and formation⁵¹

Ikezawa et al. 2005⁵² conducted a study to investigate the levels of TNF-α, soluble TNF receptor type 1 and 2 in gingival crevicular fluid (GCF) and serum of healthy subjects and chronic periodontitis patients GCF was collected from probing pocket depth (PPD) < or =3 mm sites of healthy subjects, PPD < or =3, 4-6 and > or

=7 mm sites of chronic periodontitis patients. The levels of TNF-α, soluble TNF receptor type 1 and 2 in the serum and GCF were quantified by enzyme-linked

Page 21 immunosorbant assay. The results showed no significant difference in serum TNF-α levels between healthy and chronic periodontitis patients, where as GCF TNF-α levels estimated in healthy periodontium were 0.16 pg/site and 0.24 pg/site in chronic periodontitis and concluded that imbalance between soluble TNF receptor type 1 and 2 levels in GCF could be related to chronic periodontitis severity.

Gorska et al. 2003⁵³ conducted a study to assess the relation between clinical parameters and concentrations of the key (IL-1β, TNF-α, IL-2, IFN-, IL-4, IL-10) cytokines, important in the initiation and progression of periodontal diseases, within inflamed gingival tissues and serum samples from patients with severe chronic periodontitis. Clinical examinations including probing depth, clinical attachment loss, plaque index, and bleeding index were performed before periodontal treatment. Gingival tissue biopsies were collected from one active site of each patient and from healthy individuals, and blood samples were withdrawn on the day of tissue biopsy. The concentrations of cytokines were determined by an enzyme-linked immunosorbent assay, and the relationship between their profiles in situ and in circulation with clinical parameters was analysed. The concentrations of IL-1β, TNF-α, IL-2, IFN- were, on average, significantly higher in serum samples and gingival tissue biopsies from periodontitis patients than in healthy controls. On the contrary, the levels of IL-4 and IL-10 in both kinds of samples obtained from patients and controls were generally low or even undetectable, and remained, on average, on the same level. However, the frequency of IL-4 and IL-10 was much higher in healthy gingival tissues. These results indicated that high variability of cytokine concentrations and low frequency of their detection in serum samples from periodontitis patients make these determinations useless for the detection of disease

Page 22 presence and/or its severity. In contrast, high absolute levels of IL-1β, TNF-α, IL-2 and IFN- and, especially their high ratios to IL-4 and IL-10 found in inflamed tissue biopsies, were closely associated with periodontal disease severity.

Prabhu et al. 1996⁵⁴ conducted a study to investigate the simultaneous presence of cytokines produced by Th1, Th2, and inflammatory cells which could be involved in periodontitis. This study investigated the simultaneous presence of cytokines produced by Th1,Th2, and inflammatory cells which could be involved in periodontitis. This study also compared the expression of these cytokine mRNAs in healthy and diseased tissues. No significant differences were detected at the protein or mRNA levels of the cytokines in the systemic circulation of patients and controls.

The surface markers CD16 and CD56 were expressed on significantly fewer peripheral mononuclear cells of patients when compared to controls. Gamma delta T cells(γδ+T cells) were found in half of the diseased tissues, but in none of the healthy tissues of either patients or controls. Finally, significant differences were observed between healthy and inflamed gingival tissues in the cytokine mRNA profile. Expression of IL-6 and IFN-δ mRNA was significantly higher in diseased tissues compared to healthy tissues in patients.

Dongari-Bagtzoglou et al. 1998⁵⁵ conducted a study to test the hypothesis that gingival fibroblasts from diseased sites contribute to pathogenesis by possessing a secretory phenotype characterized by an exuberant secretion of inflammatory mediators and cytokines. This study showed that fibroblasts from periodontal lesions produce in vitro greater amounts of IL-6 and IL-8 constitutively than healthy controls. When fibroblasts were stimulated with a panel of endogenous or exogenous response modifiers, the magnitude of cytokine and mediator stimulation

Page 23 above constitutive levels did not differ between health and disease. A strong positive correlation was identified between IL-6 or IL-8 constitutive secretion levels in vitro and the in situ expression of these cytokines within the connective tissues from where these cells originated, indicating that the in vitro phenotype mirrors their invivo function. Furthermore, this study presented evidence which indicates that increased cytokine secretion by fibroblasts in disease is due to an elevated proportion of subpopulations with higher cytokine secretory capacity. Finally, this study demonstrated that cultures from diseased sites are composed of cells with higher levels of constitutive CD40 expression, which may contribute to the increased IL-6 and IL-8 secretory phenotype.

Loos et al. 2000⁵⁶ conducted a study to assess CRP and IL-6 levels in periodontitis patients. Consecutive adult patients with periodontitis and healthy controls, all without any other medical disorder, were recruited and peripheral blood samples were collected. The results showed that patients with generalized periodontitis and localized periodontitis had higher median CRP levels than controls. 52% of generalized periodontitis patients and 36% of the localized periodontitis patients were sero- positive for IL-6, compared to 26% of controls. Plasma IL-6 levels were higher in periodontitis patients than in controls. Leukocytes were also elevated in generalized periodontitis compared to localized periodontitis and controls; this finding was primarily explained by higher numbers of neutrophils in periodontitis.

IL-6 and CRP correlated with each other, and both CRP and IL-6 levels correlated with neutrophils and finally concluded that periodontitis results in higher systemic levels of CRP, IL-6, and neutrophils. These elevated inflammatory factors may

Page 24 increase inflammatory activity in atherosclerotic lesions, potentially increasing the risk for cardiac or cerebrovascular events.

Min Ki Noh et al. 2013⁵⁷ conducted a study to quantify IL-6, IL-8 and TNF-α levels in the human gingival tissues of patients with periodontitis and to assess the correlation of these three cytokines with each other. In this study, human gingival tissues from 19 patients with periodontitis were collected. The tissues were homogenized, centrifuged and the protein in the supernatant was quantified. Enzyme linked immunosorbent assay (ELISA) was used in the measurement of the IL-6, IL-8 and TNF-α levels. The mean levels of IL-8 were higher than those of the other two cytokines. In each sample, the level of TNF-α expression was consistently high, with little difference between the results, which contrasted with the fluctuations in IL-6 and IL-8 levels. The expression of the two ILs (IL-6 and IL-8) showed a positive correlation, whereas TNF-α levels were not correlated with IL-6 or IL-8 levels. These results suggested that IL-6, IL-8 and TNF-α may be relevant in the pathophysiology of periodontitis, and the measurement of these cytokines may be beneficial in the identification of patients with periodontitis.

Rashmi Heralgi et al. 2011⁵⁸ conducted a study to assess the relationship between the levels of TNF-α in the gingival crevicular fluid and periodontal disease severity. The subjects included in the study were categorized into following groups:

I-Healthy, II-Gingivitis, III- Periodontitis based on Community Periodontal Index (CPI) scores. GCF collected from each individual was subjected to biochemical analysis to detect TNF-α level by using commercially available Enzyme Linked Immunosorbent Assay (ELISA) kit. This study demonstrated that the periodontitis group had a higher mean TNF-α levels when compared to the gingivitis group and

Page 25 healthy group. The gingivitis group had a higher mean TNF-α level when compared to the healthy group. The mean difference in the TNF-α levels between the groups was found to be statistically significant. TNF-α level in GCF progressively increase from periodontal health to periodontal disease. Therefore, from the results of this study it can be concluded that TNF-α plays a key role in the progression of periodontal disease and also provides site specific information on changes in TNF-α levels serving as a strong clinical marker of disease activity.

D Aiuto et al. 2004⁵⁹ conducted a study to assess whether the degree of response to periodontal therapy was associated with changes in serological markers of systemic inflammation. Systemically healthy subjects with severe generalized periodontitis participated in a prospective six-month blind intervention trial.

Periodontal parameters and inflammatory markers [CRP and IL-6] were evaluated prior to and 2 and 6 months after delivery of standard non-surgical periodontal therapy. Six months after treatment, significant reductions in serum IL-6 and CRP were observed. Decreases in inflammatory markers were significant in subjects with above average clinical response to periodontal therapy after correction for possible confounders. Periodontitis may add to the systemic inflammatory burden of affected individuals. This study demonstrated that IL-6 levels were significantly reduced for 6 months after periodontal treatment of patients with periodontal disease.

Goutoudi P et al. 2012⁶⁰ conducted a study to examine the GCF levels of IL-6 and IL-8 in periodontal sites with varying degrees of destruction and inflammation of periodontal patients prior to and following surgical and/or nonsurgical periodontal therapy GCF samples were obtained from non-diseased and diseased sites of 12 periodontal patients prior to as well as at 6, 16, and 32 weeks

Page 26 following non-surgical and surgical periodontal therapy. IL-6 and IL-8 levels were determined by ELISA. The results showed that periodontal treatment improved all clinical parameters. Both treatment modalities resulted in similar IL-6 as well as IL- 8 levels. Mean IL-6 and IL-8 concentrations were significantly higher in non- diseased compared to diseased sites and increased significantly following treatment in diseased sites. Mean total amounts of IL-6 and IL-8 (TAIL-6, TAIL-8) did not differ significantly between diseased and non-diseased sites, while following therapy TAIL-8 levels decreased significantly. The data suggested that periodontal therapy reduced the levels of IL-8 in GCF. However, a strong relationship between IL-6, IL-8 amounts in GCF and periodontal destruction and inflammation was not found.

Association between Hypothyroidism and Chronic Periodontitis

Venkatesh Babu et al. 2016⁶¹ conducted a study to assess and compare the oral health status of children suffering from thyroid disorders and healthy children.

The study group consisted of 100 children with thyroid dysfunction (hypothyroidism/ hyperthyroidism), while the control group consisted of 100 healthy children. Gingival index, plaque index, DMFT (Decayed missing filled teeth Index for permanent teeth) & Dmft index (Decayed missing filled teeth Index for primary teeth) and modified developmental defects of enamel (DDE) index were recorded and data were analyzed statistically. The results showed that plaque and gingival scores were significantly higher in the thyroid group compared to the control group.

DMFT and dmft scores were higher in the thyroid group than the control group but the difference in score was not statistically significant. Statistically significant DDE score was found in the thyroid group. Apart from increased susceptibility to caries and poor periodontal health condition, children with thyroid disorders were also

Page 27 found to have other oral manifestations such as macroglossia, open bite, and change in eruption pattern and concluded that thyroid dysfunction (both hypothyroidism and hyperthyroidism) has impact on the oral health status. Children with thyroid disorders showed high prevalence of dental caries and periodontal disease compared to the control group.

Hanau KJ et al. 2012⁶² conducted a study to estimate the periodontal health status and periodontal treatment need among Iraqi females with thyroid dysfunction.

Adult Iraqi females allocated into three comparable groups: group A (patients with hypothyroidism), group B (patients with hyperthyroidism) and group C (control group).

CPITN, CAL & TN indices were recorded and the data were analyzed statistically using SPSS program. Results showed that generally, healthy women found to have better periodontal health and less clinical attachment loss compared to those with thyroid dysfunction but these results was not significant for all CPITN scores. On the other hand all population sample were found to need periodontal treatment in different levels and it is concluded that thyroid dysfunction in its both types (hypothyroidism &

hyperthyroidism) has its impact on periodontal health status.

Feitosa et al. 2008³¹ conducted a study to evaluate histologically in rats, the influence of thyroid hormones on the rate of periodontal bone loss resulting from ligature placement and on the quality of tooth-supporting alveolar bone. Thirty-six male wistar rats were randomly assigned to the following groups: healthy, hypothyroidism and hyperthyroidism. Once alterations were confirmed by total serum levels of T₃ and T4, ligatures were randomly placed around one of the first mandibular molars. Thirty days later, the animals were killed and specimens routinely processed for serial decalcified sections. The parameters assessed were periodontitis-related bone loss,

Page 28 quality of tooth-supporting alveolar bone and the number of cells positive for TRAP, a marker of bone resorption. The results showed that, at the ligated sites, intergroup analysis revealed that hypothyroidism group showed significantly increased bone loss resulting from ligature-induced periodontitis and the number of TRAP-positive cells on the linear surface of bone crest. In addition, no significant differences were detected regarding the quality of the bone or the number of TRAP-positive cells in the area of the interradicular bone for ligated teeth among the groups. It may be concluded that decreased serum levels of thyroid hormones may enhance periodontitis-related bone loss, as a function of an increased number of resorbing cells, whereas the tooth- supporting alveolar bone seems to be less sensitive to alterations in hormone levels.

Patil BS et al. 2011⁶³ reported that the strongest type of causal relationship is the association of systemic and periodontal disease. Hashimotos thyroiditis has also been considered as one of the causes of periodontal disease. Hashimoto's thyroiditis (chronic autoimmune thyroiditis) is the most common cause of hypothyroidism in iodine-sufficient areas of the world, leading to reduced caliber and a greater number and tortuosity of gingival capillary loops in the interdental region, observed in Hypothyroid cases. The altered gingival microcirculation compromises the first line of defense, with increased PGE2 and cytokines leading to periodontitis.

Chandana et al. 2011²⁶ reported that management of patient with a thyroid dysfunction, as well as the patient taking medications for it, requires proper risk management before considering dental treatment by the dentist. Patients with long standing hypothyroidism may have increased subcutaneous mucopolysaccharides due to decrease in the degradation of these substances. The presence of excess subcutaneous mucopolysaccharides may decrease the ability of small blood vessels to constrict when

Page 29 cut and may result in increased bleeding from infiltrated tissues, including mucosa and skin. Local pressure for an extended time will probably control the bleeding from the small vessels adequately. Patient with hypothyroidism may have delayed wound healing due to decreased metabolic activity in fibroblasts. Delayed wound healing may be associated with an increased risk for infection because of the longer exposure of the unhealed tissue to pathogenic organisms. Hypothyroid patients are not considered to be immunocompromised. Patients who have hypothyroidism are susceptible to cardiovascular disease from arteriosclerosis and elevated LDL. Before treating such patients, consult with their primary care providers who can provide information on their cardiovascular statuses. Patients who have atrial fibrillation can be on anticoagulation therapy and might require antibiotic prophylaxis before invasive procedures, depending on the severity of the arrhythmia. If valvular pathology is present, the need for antibiotic prophylaxis must be assessed. Patients who have hypothyroidism are sensitive to central nervous system depressants and barbiturates, so these medications should be used sparingly. It has been found that recent exposure to a surgical antiseptic that includes iodine (such as Povidone) can increase the risk of thyroiditis or hypothyroidism.

Patients with underlying thyroid antibodies and a tendency toward autoimmunity appear to be at more risk. Drug interactions of l-thyroxine include increased metabolism due to phenytoin, rifampicin and carbamazepine, as well as impaired absorption with iron sulfate, sucralfate and aluminum hydroxide. When l-thyroxine is used, it increases the effects of warfarin sodium and, because of its gluconeogenic effects; the use of oral hypoglycemic agents must be increased. Concomitant use of tricyclic antidepressants elevates l-thyroxine levels. Appropriate coagulation tests should be available when the patient is taking an oral anticoagulant and thyroid hormone replacement therapy.

MATERIALS &

METHODS

Page 30 Study setting:

The subjects for this study were selected from the Outpatients of Department of Periodontics, Sree Mookambika Institute of Dental Sciences, Out patients of Department of General medicine and General Surgery of Sree Mookambika Institute of Medical Sciences, (SMIMS) Kulasekharam.

Study design:

This is a comparative interventional study for evaluating the levels of serum IL-6 and TNF-α in chronic periodontitis patients with and without hypothyroidism before and after scaling and root planing procedure.

Study duration:

3 months from January 2015 to March 2016.

Informed consent and Ethical considerations

The study protocol was approved by Ethical committee of Sree Mookambika Institute of Medical Sciences, Kulasekharam (Ref No: SMIMS/IHEC/2015/A/07).

The Clinical trial registration number is REF/2016/01/010418.

Study protocol was explained to patient. Information on the nature and potential benefit of their participation in the study was also explained. Informed consent was obtained from all subjects after screening.

Sample size selection:

The Number of participants required in each group, n, is given by:

n = ^{

ഀ

మ }^మ

∆^మ

where ݖ ቀ1 −ቁ and ݖሺ1 −ߚሻ represent percentage points of the normal distribution of the significance level and power, respectively.

Page 31

∆ represents the standardized difference. (i.e. the treatment difference divided by its standard deviation) in one of the outcome parameters i.e. TNF-α.

The selected patients were assigned into 2 groups each group consist of 20 female patients.

Group 1: 20 Systemically healthy subjects with chronic periodontitis Group 2: 20 subjects with chronic periodontitis and hypothyroidism Inclusion Criteria:

Patients with chronic periodontitis will be diagnosed according to the Criteria of AAP 1999. ⁶⁴

• Attachment loss of > 5mm at more than 30% of the site

• Patients with bleeding on probing

• Patients with > 20 functional teeth

• Female patients between age group 18-60 years.

Exclusion criteria

• Patients with any systemic condition except hypothyroidism

• Smokers and former smokers

• Alcoholics and former alcoholics

• Obese, pregnant and lactating woman

• Subjects over 60 years of age

• Patients presenting any kind of inflammatory disease within last 3 months

• Patients who had received professional periodontal treatment during the 6 month period prior to the study.