Review of Literature

BEFORE AND AFTER PHASE I PERIODONTAL THERAPY IN AGGRESSIVE PERIODONTITIS

A Dissertation submitted in partial fulfillment of the requirements

for the degree of

MASTER OF DENTAL SURGERY

BRANCH – II PERIODONTOLOGY

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY CHENNAI – 600 032

2017 - 2020

I thank God for providing this wonderful opportunity in my life and I pray for his continuous blessings in my life.

It is my privilege to extend my sincere gratitude and heartfelt thanks to Dr. K. MALATHI, MDS., HOD, Professor, Department of Periodontology, Tamil Nadu

Government Dental College and Hospital, Chennai – 600003 for her valuable guidance and sincere support throughout my post-graduation.

My sincere thanks, gratitude and heartfelt thanks to my guide Dr. JAISHREE TUKARAM KSHIRSAGAR MDS., Professor, Department of

Periodontology, Tamil Nadu Government Dental College and Hospital, Chennai – 600003 for her valuable guidance, support and encouragement throughout my post graduate course and to bring this dissertation to a successful completion.

I express my gratitude to Dr. P. BHUVANESWARI, MDS., Professor, Department of Periodontology, Tamil Nadu Government Dental College and Hospital, Chennai – 600 003 for her valuable guidance and continuous encouragement throughout the dissertation preparation.

I wish to express my gratitude to Dr.G. VIMALA, MDS., Principal, Tamil Nadu Government Dental College and Hospital, Chennai – 600003 for her invaluable guidance in completing this dissertation.

My sincere thanks to Dr.B. SARAVANAN, MDS., PhD., former Principal, Tamil Nadu Government Dental College and Hospital, Chennai – 600003 for his kind

permission and encouragement.

Dr. A. MUTHUKUMARASWAMY, MDS., Associate Professor, Department of Periodontology, Tamil Nadu Government Dental College and Hospital, Chennai – 600003 for his valuable guidance, constant support and sharing his clinical knowledge and continuous encouragement throughout my PG course.

I am grateful to Dr. VIDHYAALBERT, MDS., Associate Professor, Dr. A.J. ANAND, MDS., Dr. R. KARTHIKEYAN, MDS., Dr. P.R. GANESH, MDS.,

Dr. D. JAYANTHI, MDS., Dr. M. SHABBIR AHAMED, MDS., Dr. SIVSANKAR P, MDS., Senior Assistant Professors, Department of Periodontology, Tamil Nadu Government Dental College and Hospital, Chennai – 600003, for helping me with my dissertation and during my study period.

I am also extremely grateful to Dr. KALAISELVI P, Assistant Professor,

Department of Medical Biochemistry, University of Madras, Taramani Campus, Chennai -600 113 for granting me permission to conduct the study in their department and

Mr. PUGAZHENDHI,M.S.C., Ph.D for his assistance and co-operation throughout the study.

I thank my batchmates Dr.V.VALARMATHY., Dr.V.DASARATHAN., Dr.M.NOUFA., Dr.G.D.RAMKUMAR., Dr.R.THANMANAM., for their constant support, help and motivation throught the postgraduation period.

I would also like to express my gratitude to my seniors especially Dr.S.RUBINE M.D.S., Dr.M.BALAMURUGAN M.D.S., Dr.S.AISHWARYA M.D.S., Dr.M.AADHIRAI M.D.S.,Dr.S.RAJSUNDAR M.D.S., Dr.HEMALATHA M.D.S., Dr.A. JAREEN MDS., who have stood by me always and have been a constant source of encouragement for me during this period.

Dr.T.GOWRI., Dr.GOWRIDEVI., Dr.LISHAMOLTHOMAS., Dr.ARIVUKARASU for their co-operation and help whenever I needed.

I thank Dr. CYRIL BENEDICT, MDS, for helping me with the statistical work of my dissertation.

A special mention of thanks to all my patients for their very kind co-operation throughout my study.

I also would like to mention a special thanks my daughter ASHIKAA for her constant love and support throughout my post-graduation period.

I also would like to thank my husband AKILAN for his constant love, support and motivation throughout my post- graduation.

I also would like to thank my parents, SUNDARI and RENGA RAJAN for their love, constant support in taking care of my daughter throughout my post- graduation period.

I also would like to thank my brothers, SUNDARARAJAN and KARTHIK RAJAN for their love and encouragement to pursue my post-graduation.

I also would like to thank my sisters in law, KIRUTHIKA and RUBIYA, nephew, JAITHREN for their love and support in my endeavours.

I also would like to thank my father in law and mother in law, MUTHURATHINAM and THIRUVALAR SELVI for their support and blessings throughout my post-graduation period.

ABSTRACT

BACKGROUND: Leptin, an adipocytokine with pro-inflammatory properties plays an important role in regulation of immunologic and inflammatory responses of the body. The role of non-surgical periodontal therapy (NSPT) in altering the leptin concentrations in serum of Aggressive periodontitis patients is yet to be ascertained.

AIM: The aim of the present study is to assess and evaluate the concentration of leptin in serum before and after phase I periodontal therapy in aggressive periodontitis patients.

MATERIALS AND METHOD: Level of serum leptin was determined in healthy subjects (control group, n=30) and patients with aggressive periodontitis (study group, n=30) and compared with clinical parameters like gingival bleeding index, plaque index, probing pocket depth and clinical attachment level before and after phase-I therapy.

RESULTS: Clinical parameters and leptin level were increased in the serum of patients with aggressive periodontitis before phase I therapy compared to healthy subjects.

Statistically significant (p<0.01) reduction in clinical parameters and serum leptin level were seen after Phase-I therapy.

CONCLUSION: These results suggest that periodontal inflammation may up-regulate serum leptin level and increase clinical parameter values in patients with

aggressive periodontitis and phase I therapy was able to restore the serum leptin levels and clinical parameters.

KEYWORDS: Serum Leptin, Aggressiveperiodontitis, Phase I therapy

S.NO TITLE PAGE NO

1 INTRODUCTION 1

2 AIM AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 21

5 PHOTOGRAPHS 33

6 STATISTICAL ANALYSIS 39

7 RESULTS 40

8 DISCUSSION 54

9 SUMMARY AND CONCLUSION 60

10 BIBLIOGRAPHY 61

11 ANNEXURES 69

S.NO TITLE PAGE NO

1 GROUP I PATIENT BEFORE THERAPY 33

2 GROUP II PATIENT BEFORE THERAPY 33

3 ORTHOPANTAMOGRAPH (OPG) OF THE GROUP II PATIENT

33

4 GROUP II PATIENT 3 MONTHS AFTER THERAPY 34

5 ARMAMENTARIUM FOR BLOOD COLLECTION 34

6 COLLECTION OF VENOUS BLOOD SAMPLE 35

7 ARMAMENTARIUM FOR PHASE I THERAPY 35

8 ELISA PLATE WITH WELLS 36

9 PIPETTES 37

10 COOLING CENTRIFUFE 37

11 STORAGE REFRIGERATOR FOR SERUM SAMPLES 38

12 ELISA READER 38

S.NO TITLE PAGE NO

1 GROUP I (CONTROL GROUP) HEALTHY SUBJECTS AT BASELINE

43

2 GROUP II (STUDY GROUP) AGGRESSIVE PERIODONTITIS AT BASELINE

44

3 GROUP II (STUDY GROUP) AGGRESSIVE

PERIODONTITIS AT 3 MONTHS AFTER THERAPY

45

4 COMPARISON OF PLAQUE SCORES 46

5 COMPARISON OF GINGIVAL BLEEDING INDEX 46

6 COMPARISON OF PROBING POCKET DEPTH 46

7 COMPARISON OF CLINICAL ATTACHMENT LEVEL 47

8 COMPARISON OF SERUM LEPTIN LEVEL 47

9 PAIRED T TEST FOR COMPARISON OF BASELINE AND 3 MONTHS POST-OP VALUES OF GROUP II

(AGGRESSIVE PERIODONTITIS)

48

10 INDEPENDENT T TEST FOR COMPARISON OF THE BASELINE VALUES OF GROUP I AND GROUP II

48

S.NO TITLE PAGE NO

1 PATHOGENESIS OF PERIODONTITIS 5

2 COMPARISON OF GINGIVAL BLEEDING INDEX BETWEEN GROUP I AND GROUP II AT BASELINE

49

3 COMPARISON OF PROBING POCKET DEPTH BETWEEN GROUP I AND GROUP II AT BASELINE

49

4 COMPARISON OF CLINICAL ATTACHMENT LEVEL

BETWEEN GROUP I AND GROUP II AT BASELINE

50

5 COMPARISON OF PLAQUE INDEX BETWEEN GROUP I AND GROUP II AT BASELINE

50

6 COMPARISON OF SERUM LEPTIN LEVEL BETWEEN

GROUP I AND GROUP II AT BASELINE

51

7 COMPARISON OF GINGIVAL BLEEDING INDEX FOR GROUP II AT BASELINE AND 3 MONTHS

51

8 COMPARISON OF PROBING POCKET DEPTH FOR

GROUP II AT BASELINE AND 3 MONTHS

52

9 COMPARISON OF CLINICAL ATTACHMENT LEVEL FOR GROUP II AT BASELINE AND MONTHS

52

10 COMPARISON OF PLAQUE INDEX FOR GROUP II AT BASELINE AND 3 MONTHS

53

11 COMPARISON OF SERUM LEPTIN FOR GROUP II AT BASELINE AND 3 MONTHS

53

AP Aggressive Periodontitis

LEP Leptin

kDa Kilodalton

GCF Gingival Crevicular Fluid

BOP Bleeding on Probing

PI Plaque Index

GBI Gingival Bleeding Index

PPD Probing Pocket Depth

CAL Clinical Attachment Level

SRP Scaling and Root Planning

NSPT Nonsurgical Periodontal Therapy

CEJ Cemento – Enamel Junction

FPLC Fast Protein Liquid Chromatography

mRNA Messenger –RNA

IL-1 Interleukin-1

IL-6 Interleukin-6

IL-11 Interleukin-11

IL-12 Interleukin-12

LIF Leukaemia Inhibitory Factor

ELISA Enzyme-Linked Immunosorbent Assay

STAT3 Signal Transducer and Activator Of Transcription 3

PD Probing Depth

OD Optical Density

APR Acute Phase Reactant Protein

LPS Lipopolysaccharide

BMI Body Mass Index

VEGF Vascular Endothelial Growth Factor

VCAM-1 Vascular Cell Adhesion Molecule -1

Introduction

1 INTRODUCTION

The periodontal disease is a chronic inflammatory process that affects the tooth supporting tissues and occurs as result of interaction between the periodontopathic bacteria and the host immune system. It can be broadly divided into gingivitis (which is a reversible form that isn't accompanied by attachment loss) and periodontitis (which is an irreversible form and results in attachment loss) .¹

Aggressive periodontitis was defined as a complex disease exhibiting microbial alterations and cellular dysfunction that differentiates the underlying molecular mechanism of its pathogenesis from chronic periodontal disease .²

It has been indicated that systemic factors play a greater role in etiology of aggressive periodontitis which in turn exert greater effects on systemic conditions.³ Higher serum levels of inflammatory mediators, such as interleukin (IL)-17, tumor necrosis factor- α (TNF-α) and C-reactive protein (CRP), have been higher in Aggressive Periodontitis patients, even if whose clinical parameters were less severe.^4,5

It's been recently found that a hormone-like protein called leptin, initially known for its effects on body weight regulation, body metabolism and reproduction, may also be part of some inflammatory diseases via its direct impact on the innate and adaptive immune responses.⁷

obesity genewas found at Rockefeller University in 1994 which encodes Leptin.⁸ It has a molecular weight of nearly 16 kilodaltons.

2 Leptin, a polypeptide hormone, has been classified as a cytokine as it shares structural and functional similarities with members of the long chain helical cytokines: IL- 6, IL-11, IL-12, leukemia inhibitory factor, granulocyte-colony–stimulating factor, and oncostatin M.^9,10 Thus, leptin should be classified as a cytokine. Circulating leptin in humans is mainly secreted from adipose tissue.¹¹ It has been suggested that leptin orchestrates the host response to inflammatory and infectious stimuli as it stimulates the immune system by enhancing cytokine production and phagocytosis by macrophages.

In recent years, clinical studies indicated an association between leptin and periodontal infection. Serum leptin concentration was reported to be significantly elevated in periodontitis patients compared with periodontal healthy subjects and was positively correlated with periodontal parameters including probing depth and clinical attachment loss and could be effectively reduced by periodontal treatment .^12,13,14These results suggested that the systemic leptin level could be greatly influenced by the periodontal conditions, and is a potential mediator between periodontitis and systemic health.

This study will help to estimate leptin level in aggressive periodontitis patients before and after phase 1 periodontal therapy.

Aim and objectives

3 AIM AND OBJECTIVES

AIM

The purpose of this study is to assess and evaluate the concentration of leptin in serum before and after phase I periodontal therapy in aggressive periodontitis patients.

OBJECTIVES

1. To measure serum leptin concentration in healthy subjects (control group)

2. To measure serum leptin concentration in aggressive periodontitis patients before phase I therapy (study group)

3. To measure serum leptin concentration in aggressive periodontitis patients after phase I therapy (study group)

4. To compare and evaluate the serum leptin concentration in healthy subjects (control group) and in aggressive periodontitis before &after phase I therapy (study group).

Review of Literature

4 REVIEW OF LITERATURE

Periodontitis is an inflammatory disease initiated by microorganisms, which results in inflammatory breakdown of tooth-supporting hard and soft tissues. The presence of periodontopathogens alone is inadequate for progression and severity of disease. The onset, progression and severity of periodontal disease are related to interaction between periodontal microorganisms and the host immune response.

Aggressive periodontitis is the disease of periodontium occurring in otherwise healthy adolescent, which is characterized by rapid loss of alveolar bone about more than one tooth of the permanent dentition. The amount of destruction is not commensurate with the amount of local irritants.

To arrive at periodontal diagnosis, the dentist must rely upon factors such as (a)presence and absence of signs and symptoms, including pain and amount of observable plaque and calculus, (b)patients medical and dental history, (c) presence or absence of clinical signs of inflammation like bleeding on probing, (d) probing depths and extent and pattern of clinical attachment and bone loss. These parameters provide a measure of past destruction and are of limited use in early diagnosis frodge et al (2008).¹⁵ Because of increasing prevalence and associated comorbidities, screening and diagnostic modalities for the early identification of periodontitis, its initiation and progression, as well as objective measures for response to therapy, are being sought Mealey et al (2006)^.16

A myriad of biomarkers in serum/plasma, saliva and gingival crevicular fluid (GCF) have been evaluated in periodontitis patients. These markers have the potential to provide an insight much beyond the classical and radiographic findings of the disease process.

5 The definition of biomarkers as established by the National Institute of Health (NIH) is as follows: biomarkers are the biological, biochemical, anthropometric, physiological, etc. characteristics, which are objectively measurable, capable of identifying physiological or pathological processes, or a pharmacological response or a therapeutic intervention .¹⁷ There are different types of biomarkers; the ideal biomarker must be specific, sensitive, predictive, rapid, economical, noninvasive, and stable in vivo and in vitro. Additionally, it must have enough preclinical and clinical relevance to modify decisions regarding the pathological process in which it is applied.

Figure1: Schematic diagram to illustrate the pathogenesis of periodontitis.

The dental plaque biofilm is complex, dynamic, and variable; it is subject to quantitative and qualitative ecological shifts in response to changes in the local environment (e.g., pH changes), changes in localized immune regulation, and extrinsic factors such as smoking. Bacteria in dental plaque signal the local tissue cells and immune

6 cells via intrinsic and secreted microbe-associated molecular patterns (MAMPs ) such as lipopolysaccharide (LPS )and specific antigens (e.g.,fimbrial proteins).The healthy periodontium is maintained by an effective innate response to commensal (nonpathogenic) microflora in dental plaque which is restricted to the gingival/plaque margin and in which neutrophils play a pivotal role regulated by low levels of cytokines such as IL-1𝛽 and IL-8. An ecological shift in dental plaque towards a more pathogenic microflora dominated by species such as Porphyromonas gingivalis, Treponemadenticola, and Tannerellaforsythia leads to an enhanced immune response through enhanced stimulation of cytokine responses from a wide range of periodontal and immune cells. The development of periodontitis is driven by an exaggerated activation of intrinsic periodontal cells, a heightened primary and thereafter secondary cytokine response leading to activation of innate effector responses and in particular recruitment and activation of neutrophils (in response to elevated IL-1𝛽 and IL-8) and osteoclasts (in response to RANKL).Enhanced local activity of neutrophils in the periodontium is reflected by increased levels of MMP-8 (neutrophil collagenase),MMP-9(neutrophil gelatinase), and 𝛽- glucuronidase among other enzymes. Activated macrophages and T-and B- lymphocytes may also contribute to the cytokine milieu through secretion of TNF-𝛼, IL-12, IL-17, and IL-18 and the balance of the proinflammatory cytokines with immunosuppressive mediators such as IL-10 and TGF-𝛽 may be an important determinant of disease progression. Persistence of this proinflammatory response, coupled with aberrant resolution, leads to tissue destruction characteristic of periodontitis which involves loss of the soft connective tissues of the periodontium (lamina propria of the gingiva and the periodontal ligament) and alveolar bone which eventually leads to compromised tooth

7 function. The presentation and progress of periodontitis are influenced by a number of secondary factors such as age, smoking, coexisting metabolic disorders (e.g., diabetes, obesity), and genetic susceptibility.

Inflammation has evolved as a protective response to an injury, is a primordial response that eliminates or neutralizes foreign organisms or materials in general; the innate inflammatory response starts in minutes and, if all is well, resolves in a matter of hours. In contrast, chronic inflammation persists for weeks, months, or even years.¹⁸ The inflammatory response that occurs in periodontal disease is mediated mainly by B and T lymphocytes, neutrophils, and monocytes/macrophages. These are activated to produce inflammatory mediators, including cytokines and chemokines.¹⁹ Several pro-inflammatory cytokines including interleukins like IL-1, IL-6, IL-12, IL-17, IL-18, and IL-21; tumor necrosis factor alpha (TNFα); and interferon (IFN-γ) have been demonstrated to be involved in the pathogenesis of periodontitis .²⁰

Balkwill et al (1989)²¹ found that cytokines interact in a network firstly by inducing each other, secondly, trans modulating cell surface receptors and thirdly, by a synergistic, additive or antagonistic interaction on cell function.

Yamazaki et al (1997) in a study concluded that the cytokines affect various cells and play important roles in various inflammatory diseases such as periodontitis²¹ Growth factors, hormones, and chemokines are the other immunoregulatory molecules that regulate the development and progression of periodontal diseases.

Leptin, a 16-KDa hormone identified and cloned in 1994, is synthesized and secreted specifically from white adipose cells. In minor quantities by placenta, gastric

8 epithelium, T cells, osteoblasts and intralobular ducts of the major salivary glands. Leptin, a 167- amino acid polypeptide with a putative 21-amino acid signal sequence giving rise to a 146-aminoacid secreted polypeptide, acts through its specific receptors that exhibit an almost universal distribution.²³ Leptin receptors exist in at least 6 isoforms. The so-called long form, OB-Rd, with one transmembrane domain and a long intracellular carboxy terminal tail, is thought to be the most important for transmitting the leptin signals in cells^23,24,25. The OB-Ra, OB-Rc, OB-Rd and OB-Rf are prematurely terminated receptor proteins with short intracellular tails, and they are presumed to facilitate the transport of leptin across the blood-brain barrier. The OB-Re lacks the transmembrane domain and, therefore, it may function as a soluble receptor to bind and inactivate circulating leptin²⁶ Leptin receptors are found in a variety of tissues, but OB-Rb is located predominantly in hypothalamus. Leptin is lipostatic hormone that is released from adipose tissue and regulates the amount of body fat by reducing food intake and increasing energy expenditure through its action on the hypothalaus.^27,28However, specific receptors for leptin have been found ubiquitously in the body (e.g., thyroid gland, adrenal glands, lung, placenta, kidney, liver, and endothelial cells .^29,30,31This suggests a peripheral role for Leptin Interestingly, a specific leptin receptor was recently identified in the gastric mucosa

32 and in other parts of the gastrointestinal tract .³³

Leptin is produced, stored, and secreted by the salivary glands and is expressed in oral mucosa. Using FPLC analysis, free immunoreactive leptin could clearly be identified in human saliva. The identity of leptin was confirmed by Western blot presenting the 16- KDa band of leptin. Cross-reactions of the leptin antibody with other salivary components

9 appear to be unlikely because of the strict linearity of leptin values measured in serial dilutions.³⁴

Leptin has a variety of important central and peripheral actions to regulate energy balance and metabolism, fertility, and bone metabolism that are mediated by specific cell surface leptin receptors ^.35,36 Importantly, leptin may also exert actions related to cardiovascular homeostasis that are potentially atherogenic, thrombotic, and angiogenic^.

37,38,39 Leptin has peripheral actions to stimulate vascular inflammation, oxidative stress, and vascular smooth muscle hypertrophy that may contribute to pathogenesis of type 2 diabetes mellitus, hypertension, atherosclerosis, and coronary heart disease ^.40 Leptin bears structural homology to long chain helical family of cytokines - TNF-α, IL-6, IL- 11, IL-12, oncostatin M and leukemia inhibitory factor (LIF) and shows cytokine like expression during inflammation. Leptin, besides being a satiety signal for the central nervous system and to be related to insulin and glucose metabolism, may also play an important role in regulating vascular tone because of the widespread distribution of functional receptors in the vascular cells. Evidence shows that leptin acts as a proinflammatory stimulus, including IL-6, IL-1, or lipopolysaccharide, regulating leptin mRNA expression and circulating leptin levels⁴¹. inflammation, including periodontitis, indicates that leptin is part of the immune response and host defense mechanisms. Furthermore, leptin is involved in at least two different bone-controlling mechanisms: it has a direct stimulatory effect on bone growth through osteoblast proliferation and differentiation, and it prolongs the life span of human primary osteoblasts by inhibiting apoptosis ⁴², or it has an indirect suppressive effect on bone through the hypothalamus.⁴³ Leptin seems to mediate not only periodontal local inflammation but also bone-controlling mechanisms.

10 Several large population-based studies have suggested that there were strong positive associations between high leptin levels and cardiovascular complications, including hypertension, diabetes, coronary heart disease and stroke, in different populations.44,45,46,47 Clinical studies also provide evidence for this association in various patient populations, including patients with type 2 diabetes and those with hypertension.

48,49 However, some studies have shown evidence against leptin as a prospective risk marker for CVD.^50,51 An in vivo experiment reported that exogenous leptin given during early reperfusion in an isolated mouse heart model reduced infarct size. One recent study showed that adding leptin restored heart function by increasing STAT3 activation during myocardial infarction in ob⁄ob mice .⁵² Furthermore, a community-based Framingham Heart Study showed that leptin concentrations were inversely associated with left ventricular (LV) mass, LV wall thickness and left atrial size, which shows that leptin has a cardioprotective influence on LV remodeling. Collectively, these data report para-doxical results of the leptin-related effects on the cardiovascular system; that is, excessive exogenous leptin and a leptin deficiency often lead to cardiovascular dysfunction.

Zimmermann GS et al (2004), concluded in his study that Obesity is associated with oral diseases particularly periodontitis and recent studies have strongly suggested that leptin could be one of the potential mechanisms that links obesity to periodontitis since higher serum levels of leptin are seen in these individuals.^53,54

There is a drop-in leptin levels in GCF in periodontitis and the raise in serum leptin can be because periodontitis causes increase in systemic inflammatory markers including CRP, IL 1, IL 6, IL 8, and TNF α.

11 Johnson RB (2001) et al, in his study said that the reduced GCF leptin levels found in periodontitis could be because of (1) Expansion of the vascular network caused by vascular endothelial growth factor during gingival inflammation might increase the net rate of leptin depletion from the gingival tissues. (2) Inflammation may cause cytopathic changes on endothelial cells with increasing expression of leptin receptors, which might allow more leptin–leptin receptor complex formation in the gingival tissue, resulting in decreased detectable GCF (3) Transition of T lymphocytes to B lymphocytes, as it is known that one source of OB is T lymphocytes. (4) the high biomechanical forces in reduced periodontium due to periodontitis could inhibit and impact local leptin synthesis by PDL cells. (5) Periodontopathic bacteria and inflammation can cause a significant down regulation of leptin and its receptors in PDL cells.55,56,57,58,59

Carlson et al (1999)⁶⁰ suggested that higher gingival crevicular fluid (GCF) leptin levels in healthy sites of patients with periodontitis may play a protective role in periodontal pocket. The cytopathic changes during inflammation in endothelial cells may affect leptin receptor expression on these cells. Greater leptin receptor expression might allow more leptin-leptin receptor complex formation in the gingival tissue resulted in decreased detectable GCF leptin levels, and they attributed this variation to possible mechanisms and individual host response against inflammation. They could not explain the protective mechanism in the absence of adipose tissue in gingiva. However, Soderberg et al 1999⁶¹ reported strongly associated higher plasma leptin levels and risk of acute myocardial infarction (AMI), showing a positive association between plasma leptin concentration attributable to inflammatory condition.

12 Johnson &Serio et al 2001⁶² and Bozkurt et al 2006⁶³ reported that leptin is present within healthy and marginally inflamed gingiva and decreases in concentration as probing depth increases. When leptin concentrations decreased, vascular endothelial growth factor concentrations increased, suggesting that leptin could be released from gingiva coincident to vascular expansion.

Bozkurt et al 2006⁶³ reported higher leptin GCF levels in healthy sites of periodontitis patients, and they suggested that leptin may play a protective role in periodontal disease.

Several reports have indicated an association between leptin levels and periodontitis. The studies conducted by Wallace et al & Yamagishi et al (2001)^64,65 and more recently Karthikeyan & Pradeep et al¹² also have considered elevated serum leptin concentration >10,000 pg/mL as a risk factor for cardiovascular disease.

NON-SURGICAL PERIODONTAL THERAPY(NSPT):

Nonsurgical periodontal therapy (NSPT) is the cornerstone of periodontal therapy and the first recommended approach to the control of periodontal infections. It is also known as “Cause-related therapy, Drisko et al (2001)⁶⁷ “Phase I therapy or Etiotrophic phase,” or “Initial therapy.” It is defined as “plaque removal, plaque control, supragingival and subgingival scaling and root planing (SRP), and adjunctive use of chemical agents.”

Although NSPT has evolved over the years, it is still considered to be the “gold standard”

to which other treatment methods are compared (Ehizele et al 2013)68,69

The term periodontal debridement was suggested by Smart et al. to describe the light overlapping strokes used for instrumenting the tooth with a sonic or ultrasonic scaler

13 Subsequently, the term has been used more broadly to describe the gentle but thorough instrumentation (by power driven and hand scaling) aiming at the removal of plaque, endotoxin and calculus but not cementum ^70,71,72.

The primary objective of (SRP) is to regain gingival health by completely removing elements that are responsible for the gingival inflammation (i.e., plaque, calculus, and endotoxins) in the oral environment. Both hand instruments and ultrasonic instruments are capable of dramatically reducing the numbers of subgingival microorganisms. The outcome of any treatment method is determined by complete and adequate access to pocket areas, the time devoted by the operator to the procedure, and the thoroughness of the procedure. Ower et al (2013)⁷³

McNabb et al ⁷⁴ reported that supragingival plaque control by professional tooth cleaning induced significant changes in the composition of subgingival microflora, including a decrease of Porphyromonas gingivalis and spirochetes.

Haffajee et al ⁷⁵ reported reduction in gingival redness, bleeding reduction of 40 bacterial species including Aggregatibacter actinomycetemcomitans, P. gingivalis, Prevotella intermedia, and Treponema denticola and mean gain in attachment level.

EFFECTS OF NONSURGICAL THERAPY ON THE PERIODONTAL TISSUES:

1. Changes in gingival inflammation

Several investigators have demonstrated higher probability of periodontal breakdown and the range of reduction of the occurrence of bleeding after probing after the 1^st month was 6–64%, 12–80% at 3 months posttreatment, 12–87% at 6 months and 37–

87% at 12 months after completion of the nonsurgical periodontal treatment.

14 Cercek et al (1983)⁷⁶ reported that patients maintaining oral hygiene showed approximate 25% decrease in bleeding tendency, 0.5 mm probing depth (PD) reduction, 0.7 mm of recession and no gain of clinical attachment.

It was concluded that supragingival plaque control can help resolve signs of inflammation associated with gingivitis but does not predictably alter the bacterial composition in pockets <5 mm. Therefore, subgingival debridement is necessary in addition to personal oral hygiene to achieve periodontal health

2. Changes in probing pocket depth and clinical attachment level

For nonmoral sites, with initial PD between 1 and 3 mm, the amount of gingival recession was approximately 1 mm, and with moderately deep (4–6 mm) or deep (7 mm or more) PD at baseline, the gingival recession were 1.2 mm and 1.9 mm, respectively. There was significantly less gingival recession at the molar furcation sites than nonmolar sites.

No significant changes were seen when hand instruments or ultrasonic instruments were used.

Proye et al (1982)⁷⁷ reported reduction in recession after 1 week and a gain of clinical attachment by 3 weeks after a single episode of SRP.

It was concluded the magnitude of recession was related to initial PDs and inflammatory status of the tissues. The most gingival shrinkage occurred interproximally.

The greatest gain of clinical attachment occurred at sites which initially had deep pockets.

Conversely, at shallow sites, hand instrumentation usually caused a small amount of attachment loss.

15 3. Reduced efficacy in molar furcation defects

Nordland et al (1987)⁷⁸ reported similar pocket reduction and gain of clinical attachment in molar or nonmolar sites, but there was a tendency for recurrence in furcation defects within a year. In addition, the reduction of anaerobes was only 2-fold at furcation defects, whereas there was a 100-fold reduction at other sites.

4. Changes in alveolar bone structures

Renvert and Egelberg et al (1981)⁷⁹ reported that probing bone levels increased by 0.6 mm after surgery and there was virtually no bone fill after root planing.

Isidor et al (1985)⁸⁰ found that surgery resulted in 0.5 mm coronal growth of bone in angular defects and no changes following root planing. Isidor et al observed no changes in the bone height with horizontal bone loss when treated with NSPT.

Hardy et al (1982)⁸¹, concluded that intraosseous defects treated with NSPT showed an increase in bone probing levels of 0.2 mm at 6 months, 0.3 mm at 12 months, and 0.5 mm at 24 months after therapy. After 3 years, the gain in bone probing levels after NSPT was gradually lost, most probably due to the absence of any additional professional subgingival instrumentation during the 5-year follow-up in these studies.

STUDIES SHOWING THE EFFECT OF NONSURGICAL PERIODONTAL THERAPY ON PERIODONTITIS:

From 1980 to 1986 a group of seven studies were done to show the effect of SRP on moderately advanced and advanced periodontitis as follows:

A study done by Anita Badersten, Rolf Nilveus and Jan Egelberg et al ⁸¹ in 1981 evaluated the effect of NSPT on moderately advanced periodontitis with hand instrument and ultrasonic instrumentation and showed no difference at the time of treatment but more

16 reduction in pocket depth and more attachment gain for surfaces with 6–7.5 mm initial depth than for surfaces with 4–5.5 mm initial depth was seen.

In 1984, they reported marked improvement of gingival conditions not only in periodontal pockets of moderate depth but also in pockets up to 12 mm deep by non- surgical therapy.

In another study in Cercek et al (1983)⁷⁷, they compared the effects of single versus repeated instrumentation and demonstrated no difference in results could and suggest that recurrence of disease due to subgingival recolonization by microorganisms during healing phase may not be a major problem.

Badersten et al (1985) ⁸² demonstrated that deep periodontal pockets may be successfully treated by plaque control with one episode of instrumentation and operator variability may be limited.

Continuing the study in 1985 they showed that probing and attachment loss in sites with nonresponsive to initial periodontal therapy follows a gradual, linear course, or approximately a linear pattern for the vast majority of treated sites

In 1985, they suggested that the majority of sites with attachment loss were found amongst initially shallow or moderately deep a lesion which indicates that the attachment loss is due to trauma associated with therapy rather than loss as a result of a continuing inflammatory disease process.

Badersten et al (1985)⁸³ showed that sites with probing attachment loss were more frequent for sites with high scores for plaque, bleeding, residual PD, and suppuration than in sites with low scores.

17 Non-surgical periodontal therapy has been proved to be effective in controlling the disease in most patients Claffey & Egelberg et al (1995).⁸⁴ Furthermore, most of the stabilized periodontal conditions can be maintained throughout the time if the patients are committed to have supportive periodontal care Renvert & Persson et al (2004).⁸⁵

With a moderate level of certainty, Christopher et al (20150 found approximately a 0.5-millimeter average improvement in CAL with SRP. Combinations of SRP with assorted adjuncts resulted in a range of average CAL improvements between 0.2 and 0.6 mm over SRP alone. They judged the following 4 adjunctive therapies as beneficial with a moderate level of certainty: systemic sub antimicrobial-dose doxycycline, systemic antimicrobials, chlorhexidine chips, and photodynamic therapy with a diode laser. There was a low level of certainty in the benefits of the other included adjunctive therapies. ⁸⁶

MICHIGAN STUDY

Michigan study I was done by Ramfjord et al⁸⁷ in 1973 found greater gain in clinical attachment level by NSPT with no difference in clinical attachment level in surgical and nonsurgical treatment after 5 years.

Michigan study II was done in 1975 by Ramfjord et al⁸⁸ found no difference after 1 year in surgical and nonsurgical therapy. However, after 5 years, pocket depth ≥7 mm treated with Modified Widman flap had greater attachment gain than NSPT.

Michigan study III was done by Morrison, Ramfjord, Hill et al in 1980 found greater reduction in PD by surgery when compared to NSPT and when pocket depth was 4–6 mm there was less loss (greater gain) with NSPT and PD ≥7 mm showed no difference by surgical and nonsurgical therapy. After 5 years, PD reduction was greater by surgery

18 when pocket depth was 4–6 mm before surgery. However, when pocket depth was ≥7 mm there was no difference in PD reduction by surgical and NSPT.

SWEDISH STUDY

Sweden study I was done by Rosling et al in 1976⁸⁹ found no difference in PD reduction by flap surgery with osseous and without osseous resection.

Sweden study II was done by Lindhe et al in 1982⁹⁰ found greater PD reduction and more attachment level gain with surgical therapy than nonsurgical therapy and no difference in surgical and NSPT with pocket depth ≥4 mm.

Sweden study III was done by Lindhe and Nyman et al in 1985⁹¹ found no difference in surgical and NSPT in PD reduction and attachment level gain.

Sweden study IV and V was done by Westfelt et al in 1985⁹² found more attachment gain in pocket ≥7 mm with surgery and after 1 year no difference was noticed with surgical and NSPT in PD reduction and attachment level gain.

MINNESOTA STUDY

This study was done by Pihlstrom et al in 1981⁹³ found greater reduction in PD with surgical therapy as compared to nonsurgical therapy. When initial PD was 1–3, no difference was found in surgical and NSPT. After 5 years, no difference in surgical and NSPT was found with PD 1–6 mm and with PD ≥7 mm greater reduction in probing was found by surgical therapy. In case of attachment level gain after 1 year and 5 years duration, pocket with 4–6 mm there was greater gain in attachment in NSPT and pocket with <7 mm there was no difference in surgical and nonsurgical therapy.

19 DENMARK STUDY

The Denmark study was done by Isidor F, Karring et al in 1984⁹⁴ found that there was greater PD reduction after 1-year duration, but there was no difference after 5 years. In case of attachment level gain, there was greater attachment gain by NSPT but after 5 years there was no difference.

LOMA LINDA STUDY

This study was done by Durwin et al in 1985⁹⁵ found greater gain in attachment level by surgical therapy after 1 year. However, after ≥5 years duration there was no difference in surgical and NSPT.

NEBRASKA STUDY

The Nebraska study was done by Kalkwarf et al in 1988⁹⁶ found greater PD reduction by surgical procedures after 1 year. However, in case of attachment level gain, there was more gain with NSPT when PD was 5–6 mm after 1 year and with PD ≥ 7 mm there was no difference in surgical and NSPT.

However, conventional mechanical debridement procedures do not remove all periodontopathic bacteria from the subgingival environment, especially those in inaccessible areas such as furcations, grooves, concavities, and deep pockets. It appears that the effects of mechanical therapy might be augmented using antimicrobial agents which further suppress the

20 remaining pathogens⁹⁷ A recent advance which is receiving much attention is the application of lasers, photodynamic therapy (PDT), and hyperbaric oxygen therapy (HBOT) in NSPT.

Materials and Methods

21 MATERIALS AND METHOD

STUDY POPULATION:

The study population needs to be selected from the outpatient section of the Department of Periodontology, Tamil Nadu Government Dental College and Hospital, Chennai.

SAMPLE SIZE: 60

INCLUSION CRITERIA:

• Patients with age group between 30-60years

• Gender – Either sex

• Systemically healthy individuals

• Probing pocket depth: 5-8mm.

• Periodontitis involving more than 30% of the sites assessed

• Patients who will not use chemotherapeutic mouth rinse and oral irrigation devices during the study period.

• Patients who have not undergone any type of regenerative periodontal therapy over a period of six months prior to the initial examination.

• Patients with established willingness and ability to perform adequate oral hygiene.

EXCLUSION CRITERIA:

• Patients having received any periodontal therapy in the preceding 6 months.

• Chronic periodontitis patients.

• Patients with underlying systemic diseases.

22

• Medically compromised patients such as uncontrolled diabetes, immunosuppression, bleeding disorders, cancer, stroke and severe osteoporosis

• Obese individuals

• Patients with habits of smoking and alcoholism, tobacco and pan chewing

• Pregnant and lactating women.

• Patients who received medications like antibiotics, anti-inflammatory, steroids during the past 4 to 6 months

• Patients under bisphosphonates.

SUBJECTS:

A total of 60 patients need to be selected for the study (30 cases and 30 controls).

Group I – Control group (30 subjects).

Healthy patients without gingivitis & periodontitis Group II – Study group (30 subjects)

2A – Aggressive periodontitis patients before phase-I periodontal therapy.

2B – Aggressive periodontitis patients after phase-I periodontal therapy.

STUDY DESIGN:

The study is of randomized controlled trial type. The study participants will be recruited prospectively in this study.

Sex: Either sex

Ethical clearances were obtained from the Institutional Ethical Committee and ethical principles were meticulously followed throughout the study. After explaining the study protocol, informed consent was obtained from all the selected subjects. A thorough medical and dental history of the subjects was taken. All the subjects

23 underwent full-mouth periodontal probing and charting and clinical and laboratory evaluation.

METHOD OF COLLECTING DATA:

ARMAMENTARIUM:

For clinical examination 1. Mouth mirror 2. William’s Probe 3. Curved Explorer 4. Tweezer

5. Cotton rolls

6. Sterilized disposable gloves

7. Disposable face masks and head cap For collection of blood sample 1. Sterile cotton

2. Surgical spirit

3. Disposable syringe with 22gauge needle 4. Sterile EDTA coated test tubes

5. Tourniquet.

24 For Phase I therapy

1. Mouth mirror

2. William’s Probe, Explorer 3. Hu-Friedy Gracey Currettes 4. Ultrasonic scaling unit 5. Kidney Tray

6. Cotton Rolls&Gauze 7. Disposable Gloves 8. Disposable Facemask 9. Disposable Head cap

10. Disposable syringe 24 gauge needle 11. Local Anaesthetic solution

12. 0.9 % Normal saline 13. Patient apron

CLINICAL PARAMETERS ASSESSMENT The following clinical parameters were evaluated for all the subjects:

1. Plaque index – Silness and Loe 1964

2. Gingival bleeding index – Ainamo and Bay 1975

3. Probing pocket depth in mm (PPD) – Carranza 10th ed 4. Clinical attachment level in mm (CAL) – Carranza 10th ed

25 Plaque Index (Silness and Loe 1964)

All teeth were examined at 4 sites each (disto-facial, facial, mesio-facial, lingual / palatal) and were scored as follows:

Scoring Criteria:

Score 0: No plaque in the gingival area.

Score 1: A film of plaque adhering to the free gingival margin and adjacent area of the tooth. The plaque is recognized only by running a probe across the tooth surface.

Score 2: Moderate accumulation of plaque within the gingival pocket and on the gingival margin and / or adjacent tooth surface that can be seen by the naked eye.

Score 3: Abundance of soft deposits within the gingival pocket and / or on the gingival margin and adjacent tooth surface.

Calculation:

Plaque index per tooth = Total score/4

Plaque index per individual ═ Total P I per tooth / Total number of teeth examined Interpretation:

Score 0 – Excellent oral hygiene 0.1 to 0.9 – Good oral hygiene 1.0 to 1.9 – Fair oral hygiene 2.0 to 3.0 - Poor oral hygiene

Gingival Bleeding Index (Ainamo & Bay 1975)

Starting distobuccally, the probe was gently inserted into the sulcus and run to the buccal and mesial surfaces of every tooth at an angle of about 45º. This was repeated for all the teeth present. Similarly probing was carried out at palatal/lingual sites. The total number of bleeding sites per tooth was thus recorded for every tooth except the third molar.

26 Scoring Criteria:

Positive score (1) - Presence of bleeding within 10 seconds Negative score (0) - Absence of bleeding

% of bleeding sites = Total number of positive score x 100 Total number of surfaces of all teeth Probing Pocket Depth (PPD)

Probing Pocket Depths were measured from the gingival margin to the base of the pocket in millimeters using William’s Periodontal Probe. The probe was walked within the gingival sulcus along the circumference of the tooth. Keeping the probe parallel to the long axis of the selected tooth, six measurements were made per tooth (Mesiobuccal, Distobuccal, Midbuccal, Mesiolingual, Distolingual and Midlingual).

Clinical Attachment Level (CAL)

Clinical Attachment Level was measured from the Cemento – Enamel Junction (CEJ) to the base of the pocket using William ‘s Periodontal Probe.

When the gingival margin was located on the anatomic crown, the level of the attachment was determined by subtracting from the probing depth, the distance from the gingival margin to the CEJ. If both were the same, the loss of attachment was calculated to be zero.

When the gingival margin coincided with the CEJ, the loss of attachment was calculated as equalling the probing depth.

When the gingival margin was located apical to the CEJ, the loss of attachment was greater than the probing depth and therefore the distance between the CEJ and the gingival margin were added to the PD.

27 Three measurements were made on the buccal aspect and three on the lingual aspect of each tooth – total of six sites per tooth (Mesiobuccal, Midbuccal, Distobuccal, Mesiolingual, Midlingual, and Distolingual).

RADIOGRAPHIC PARAMETER:

• Radiographs were taken to assess the bone loss.

ROUTINE BLOOD INVESTIGATION WAS DONE:

• Hemoglobin%

• Bleeding time

• Clotting time

• Total leukocyte count

• Differential leukocyte count

• Random sugar

ESTIMATION OF SERUM LEPTIN

METHOD OF COLLECTION OF BLOOD SAMPLE:

Venous blood was drawn from the participants selected for the study. The subjects were informed, and consent was taken. They were made to tighten a fist so that vein was more palpable, and antecubital vein was selected for venipuncture. A tourniquet was applied about 1-2 inches above the antecubital fossa. After cleansing the puncture site with 10% isopropanol solution, blood was withdrawn using a syringe with 24- gauge needle. Tourniquet was released as the blood flow began. After drawing 3 ml of blood, sterile cotton ball was placed on the puncture site and needle was withdrawn.

28 The subjects were instructed to apply mild finger pressure on the site for few minutes to avoid oozing out of blood.

Quantitative determination of leptin in patient's serum was done by enzyme-linked immunosorbent assay (ELISA) method.

PRINCIPLE OF THE ASSAY

The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to assay the level of Human Leptin（LEP）in samples. Add Leptin（LEP）

to monoclonal antibody Enzyme well which is pre-coated with Human Leptin（LEP）monoclonal antibody, incubation; then, add Leptin (LEP）antibodies labeled with biotin, and combined with Streptavidin-HRP to form immune complex;

then carry out incubation and washing again to remove the uncombined enzyme. Then add Chromogen Solution A, B, the color of the liquid changes into the blue, and at the effect of acid, the color finally becomes yellow. The chroma of color and the concentration of the Human Substance Leptin（LEP）of sample were positively correlated.

29 Washing method:

Manually washing method: shake away the remain liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside down strongly.

Inject at least 0.35ml after-dilution washing solution into the well, and marinate 1~2 minutes. Repeat this process according to your requirements.

Automatic washing method: if there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance.

Precision:

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Human LEP were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Human LEP were tested on 3 different plates, 8 replicates in each plate.

CV (%) = SD/meanX100 Intra-Assay: CV<10%

Inter-Assay: CV<12%

LEPTIN DETERMINATION PROCEDURE:

1. Standard dilution: （this test kit will supply one original Standard reagent, please dilute it by yourself according to the instruction. ）

2. The quantity of the plates depends on the quantities of to be tested samples and the standards. It is suggested to duplicate each standard and blank well. Every sample

30 shall be made according to your required quantity, and try to use the duplicated well as possible.

3. Inject samples: ① Blank well: don’t add samples and LEP-antibody labeled with biotin, Streptavidin-HRP, only Chromogen solution A and B, and stop solution are allowed; other operations are the same.② Standard wells: add standard 50μl, Streptavidin-HRP 50μl (since the standard already has combined biotin antibody, it is not necessary to add the antibody);③ To be test wells: add sample 40μl, and then add both LEP-antibody 10μl and Streptavidin-HRP 50μl. Then seal the sealing memberance, and gently shaking, incubated 60 minutes at 37 ℃.

4. Confection: dilute 30 times the 30×washing concentrate with distilled water as standby.

5. Washing: remove the memberance carefully, and drain the liquid, shake away the remaining water.

6. Add chromogen solution A50μl, then chromogen solution B 50μl to each well.

Gently mixed, incubate for 10 min at 37℃ away from light.

7. Stop: Add Stop Solution 50μl into each well to stop the reaction (the blue changes into yellow immediately).

8. Final measurement: Take blank well as zero, measure the optical density (OD) under 450nm wavelength which should be carried out within 15min after adding the stop solution.

9. According to standards’ concentration and the corresponding OD values, calculate out the standard curve linear regression equation, and then apply the OD values of the

31 sample on the regression equation to calculate the corresponding sample’s concentration. It is acceptable to use kinds of software to make calculations.

SUMMARY:

Preparing reagents, samples and standards

Add prepared samples and standards, antibodies labeled with enzyme, reacting 60 minutes at 37 ℃

Plate washed five times, adding Chromogen solution A, B, reacting 10 minutes at 37 ℃

Add stop solution

measure the OD value within 10min

Calculation

32 CALCULATION:

Take the standard density as the horizontal, the OD value for the vertical, draw the standard curve on graph paper, find out the corresponding density according to the sample OD value by the Sample curve (the result is the sample density) or calculate the straight-line regression equation of the standard curve with the standard density and the OD value, with the sample OD value in the equation, calculate the sample density.

Photographs

33 PHOTOGRAPHS

Photograph 1: Group I Patient (Control group)

Photograph 2: Group 2 patient (study group) at baseline

Photograph 3: Orthopantomograph (OPG) of the Group II patient

34 Photograph 4: Group 2 patient (study group) at 3months after phase 1 therapy

Photograph 5: Armamentarium for blood collection

35 Photograph 6: Collection of venous blood sample

Photograph 7: Armamentarium for phase I therapy

36 Photograph 8: ELISA plate with wells

37 Photograph 9: Pipettes

Photograph 10: Cooling centrifuge

38 Photograph 11: Storage refrigerator for serum samples

Photograph 12: ELISA Reader

Statistical Analysis

39 STATISTICAL ANALYSIS

In the present study, 60 subjects were selected, of which 30 were categorized as Group I which consisted of healthy subjects, 30 were categorized as Group II which consisted of patients with generalized aggressive periodontitis. The mean average of age in group I is 35.57±6.26, group II is 26.87±4.59. The clinical parameters assessed were plaque index, gingival bleeding index, pocket probing depth, clinical attachment level and laboratory parameters assessed were serum leptin level at baseline in group I, at baseline and 3 months after phase I therapy in group II.

Statistical analysis was done by IBM SPSS (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.)

Statistical Tests used were:

1. Paired t test 2. Unpaired t test

PAIRED t TEST

It is applied to paired data of independent observations from one sample only when each individual gives a pair of observations

UNPAIRED t TEST

This test is applied to unpaired data of independent observations made on individuals of two different or separate groups or samples drawn from two populations, to test if the difference between the means is real or it can be attributed to sampling variability.

Results

40 RESULTS

The present study was done to assess and compare the serum leptin levels in patients with periodontal disease & periodontally healthy individuals. 60 peripheral blood samples were collected from the 2 groups – Group I (30 periodontally healthy individuals) and Group II (30 patients with periodontal disease). Leptin levels in serum were assessed using ELISA technique. Absorbance was measured at 450nm as primary wavelength as reference wave length in terms of pg/ml, and the results were obtained.

1. PLAQUE INDEX Intragroup comparison

Group I: The mean plaque index score at baseline was 0.690±0.104.

Group II: The mean plaque index score at baseline was 2.183±0.253 and at 3 months was 0.711±0.176. The mean difference in plaque score from baseline to 3 months was 1.47

±0.32which was found to be statistically significant (p<0.001).

Intergroup comparison

Mean difference between group I and group II at baseline was 1.49±0.05 which was statistically significant (p<0.001).

2. GINGIVAL BLEEDING INDEX Intragroup comparison

Group I: The mean gingival bleeding index score at baseline was 10.035±1.740

Group II: The mean gingival bleeding index score at baseline was 82.01±6.48 and at 3 months was 17.14±5.20. The mean difference in gingival bleeding index score from

baseline to 3 months was 64.86 ±8.39which was statistically significant (p<0.001).

41 Intergroup comparison

Mean difference between group I and group II at baseline was 71.9±1.22 which was statistically significant (p<0.001).

3. PROBING POCKET DEPTH Intragroup comparison

Group I: The mean PPD at baseline was 2.074±0.23.

Group II: The mean PPD at baseline was 5.726±1.65 and at 3 months was 3.75±1.11. The mean difference in PPD from baseline to 3 months was 1.97±0.66 which was statistically significant (p<0.001).

Intergroup comparison

Mean difference between group I and group II at baseline was 3.6±0.31 which was also statistically significant (p<0.001).

4. CLINICAL ATTACHMENT LEVEL Intragroup comparison

Group I: The mean CAL at baseline was 0.

Group II: The mean CAL at baseline was 5.622±1.63 and at 3 months was 3.734±117. The mean difference in CAL from baseline to 3 months was 1.88±0.65 which was statistically significant (p<0.001).

Intergroup comparison

Mean difference between group I and group II at baseline was 5.6±0.3 which was statistically significant (p<0.001).

42 5. SERUM LEPTIN

Intragroup comparison

Group I: The mean serum leptin at baseline was 8178.20±407.41

Group II: The mean serum leptin at baseline was 16333.1±2159.9 and at 3 months was 9154.80±768.86. The mean difference in serum leptin baseline to 3 months was 7178.3±2138.12. which was statistically significant (p=0.032).

Intergroup comparison

Mean difference between group I and group II at baseline was 8154.9±3943.57 which was statistically significant (p=0.032).

43 TABLE 1: GROUP I (CONTROL GROUP) - HEALTHY SUBJECTS

AT BASELINE

S. No AGE SEX GBI% PPD (mm)

CAL (mm)

PI% SL (pg/ml0

1 45 M 10.44 2.43 0 0.68 8138

2 30 M 10.44 2.43 0 0.68 8138

3 25 F 8.78 2.13 0 0.74 7804

4 32 F 7.3 2.2 0 0.54 8846

5 35 F 10.22 2.08 0 0.54 7863

6 40 M 8.78 2.13 0 0.74 7804

7 27 M 10.24 2.24 0 0.54 8846

8 22 F 10.24 2.08 0 0.82 7964

9 42 F 8.69 1.64 0 0.76 8218

10 32 F 9.66 2.5 0 0.82 7778

11 46 F 8.78 1.82 0 0.62 8348

12 33 M 11.46 1.56 0 0.86 7652

13 37 M 8.36 2.04 0 0.58 8136

14 38 M 14.62 2.36 0 0.54 8168

15 41 F 10.68 1.68 0 0.74 7808

16 31 F 9.92 1.84 0 0.64 8843

17 36 M 10.44 2.28 0 0.7 8198

18 34 F 12.42 1.68 0 0.82 8450

19 44 F 8.78 1.74 0 0.68 9034

20 30 F 14.72 1.9 0 0.88 7748

21 33 M 8.52 2.44 0 0.86 7894

22 34 F 9.72 1.92 0 0.64 7932

23 39 F 10.88 2.22 0 0.81 8380

24 30 M 12.56 2.3 0 0.68 7922

25 46 F 7.88 2.44 0 0.66 7656

26 42 M 8.78 2.03 0 0.67 7840

27 31 F 9.92 1.84 0 0.64 7940

28 42 F 9.62 2.63 0 0.68 8842

29 38 M 9.52 1.88 0 0.58 8604

30 32 M 8.67 1.78 0 0.56 8552

44 TABLE 2: GROUP II (STUDY GROUP) - AGGRESSIVE PERIODONTITIS AT

BASELINE

S.No AGE SEX GBI% PPD (mm)

CAL (mm)

PI% SL (pg/ml)

1 22 F 77.67 7.46 7.32 1.88 12887

2 30 M 78.75 6.21 6.92 2.52 13013

3 36 F 81.45 7.53 7.47 1.92 13013

4 28 F 74.84 7.38 7.42 1.96 12424

5 26 F 75.26 6.94 6.83 1.72 12628

6 23 F 79.52 6.99 6.82 2.33 12376

7 37 M 83.63 7.08 7.02 2.31 12115

8 25 F 80.29 7.92 7.88 1.79 11709

9 22 F 77.69 7.46 7.32 1.88 12239

10 21 M 75.52 6.71 6.83 2.57 12112

11 26 M 81.41 6.53 6.61 2.46 12909

12 19 M 70.17 7.27 7.15 2.41 13490

13 24 M 83.96 6.93 7.02 1.84 12446

14 32 M 85.2 4.50 4.50 2.04 12274

15 24 M 65.7 4.63 4.50 2.14 13189

16 30 F 91.6 3.5 3.61 2.24 12951

17 28 M 83.2 4.45 4.40 2.00 12780

18 28 M 89.5 4.08 4.92 2.41 11876

19 20 F 90.5 2.91 3.01 1.96 12294

20 30 M 86.8 4.52 4.48 2.38 12598

21 30 F 75.4 4.40 4.30 2.46 130621

22 29 M 88.4 4.50 4.50 2.32 13856

23 29 M 85.7 3.80 3.90 2.34 11835

24 25 M 89.5 2.81 3.02 1.93 12276

25 26 M 84.8 3.52 3.56 2.36 12244

26 27 F 87.51 6.85 6.64 2.39 11724

27 18 F 87.51 6.85 6.64 2.39 11804

28 30 M 87.6 3.26 3.48 2.16 10264

29 30 F 73.63 7.25 7.21 1.79 11582

30 31 M 87.6 3.21 3.48 2.16 12464