EXPRESSION OF MATRIX METALLOPROTEINASE-9 (GELATINASE-B) IN

GINGIVAL TISSUES OF CHRONIC PERIODONTITIS PATIENTS WITH AND

WITHOUT DIABETES MELLITUS: AN IMMUNOHISTOCHEMICAL STUDY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfillment for the degree of

MASTER OF DENTAL SURGERY

BRANCH – II

PERIODONTOLOGY

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

CHENNAI – 600032

2017 – 2020
 Acknowledgement

ACKNOWLEDGEMENT

First of all, I want to thank MY GOD for providing me this opportunity to

complete my dissertation work successfully.

I would like to express my gratitude to my guide Dr. H. Esther Nalini, M.D.S.,

Professor & Head, Department of Periodontology, K.S.R Institute of Dental Science and

Research, Tiruchengode, who supported and motivated me throughout the study period.

Without her support it’s not possible to finish my dissertation work successfully.

My grateful sincere thanks to Dr. P. Arun Kumar Prasad, M.D.S., Professor,

Department of Periodontology, K.S.R Institute of Dental Science and Research,

Tiruchengode, who constantly motivated and encouraged me a lot to improve in

academics as well as in clinical side and helped to complete my dissertation.

I would like to express my sincere thanks to Dr. R. Renuka Devi, M.D.S.,

Professor, Department of Periodontology, K.S.R Institute of Dental Science and

Research, Tiruchengode, for her moral support and guidance.

I would also like to thank the staff members, Department of Periodontology,

K.S.R Institute of Dental Science and Research, Tiruchengode, Dr. Tamilselvi, Dr.

Kokila Priya and Dr. Bharathi, Dr. Nithya for their guidance.

I would like to thank the Principal of our Institute, Dr. G.S. Kumar, M.D.S., for

providing me all the facilities needed for my dissertation.

 Acknowledgement

I would also like to express my grateful thanks to my batchmates, Dr. Visalakshi,

Dr. Ramesh, Dr. Lakshana and all my juniors especially Dr. Sonika who helped me a

lot in all aspects throughout my study period.

It gives me a great pleasure to thank my parents and my brother for their

continuous moral support in all situations. Without them, my postgraduation is

impossible.

A special thanks to all the patients who participated in the study. This

dissertation would not have been possible without their support and co-operation

Finally, I take this opportunity to thank all the people who helped me directly or

indirectly to complete my dissertation work.

 Contents

CONTENTS

S.NO TITLE PAGE NO

1 INTRODUCTION 1-2

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4-23

4 MATERIALS AND METHODS 24-47

5 STATISTICAL ANALYSIS 48

6 RESULTS 49-60

7 DISCUSSION 61-65

8 SUMMARY AND CONCLUSION 66

9 BIBLIOGRAPHY 67-71

10 ANNEXURES 72-78
 Contents

LIST OF FIGURES

FIGURE NO CONTENTS PAGE NO

1 Domain structures of MMPs 7

2 Structure of MMP-9 8

3 Diabetes leading to periodontitis 11

4 Periodontitis leading to diabetes 12

5 Effect of periodontal treatment on diabetes 12

6 Armamentarium for tissue collection 36

7 Armamentarium for IHC analysis 36

8 Tissue collection during crown lengthening 37

9 Tissue collection during gingivectomy 38

10 Tissue collection during extraction 39

11 Preparation of wax block and slides for IHC analysis 40-42

12 Compound microscope 43
 Contents

13 Negative expression of MMP-9 44

14 Mild expression of MMP-9 45

15 Moderate expression of MMP-9 46

16 Severe expression of MMP-9 47

 Contents

LIST OF TABLES

TABLE NO CONTENTS PAGE NO

1 Descriptive statistics of all parameters 51

2 Comparison of MMP-9 expression between the 52

groups

3 Post hoc pairwise comparison of MMP-9 53

expression between the groups

4 Post hoc pairwise comparison of periodontal 53

clinical parameters between the groups

5 Distribution of age between the groups 55

6 Gender wise distribution between the groups 56

Comparison of Random blood glucose between the

7 57
groups

8 Comparison of MMP-9 expression based on gender 58

Correlation of MMP-9 expression with PD and

9 CAL 59
 Contents

LIST OF GRAPHS

GRAPH NO CONTENTS PAGE NO

1 Comparison of MMP-9 expression scores among groups 52

2a Comparison of PI, GI scores among the three groups 54

2b Comparison of PD and CAL among experimental 54

groups

3 Comparison of mean age between the groups 55

4 Comparison of Gender distribution between the groups 56

5 Distribution of random blood glucose between the 57

groups

6 Comparison of MMP-9 expression based on gender 58

7a Correlation of MMP-9 expression with Probing depth 59

7b Correlation of MMP-9 expression with Clinical 60

Attachment level
 Abbreviations

ABBREVIATIONS

AGE Advanced Glycated End Products

AP Aggressive Periodontitis

APES 3- Aminopropyltriethoxysilane

CP Chronic Periodontitis

CAL Clinical attachment level

DM Diabetes Mellitus

DAB Diaminobenzidine chromogen

DM Diabetes Mellitus

DPX Dibutylpthalate xylene mountant

ELISA Enzyme linked immunosorbent assay

ECM Extracellular matrix

GCF Gingival crevicular fluid

GI Gingival index

HDL High Density Lipoprotein

HRP Horse Radish peroxidase

hs-CRP High sensitive C Reactive Protein

IL-6 Interleukin -6
 Abbreviations

IL-1ß Interleukin-1ß

IHC Immunohistochemistry

ICAM-1 Intercellular Adhesion Molecule-1

LDL Low Density Lipoprotein

LDD Local Drug Delivery

LPS Lipopolysaccharide

m-RNA Messenger Ribonucleic Acid

MMP Matrix metalloproteinases

MPO Myeloperoxidase

PLGF Placental growth factor

PlI Plaque Index

PPD Probing Pocket Depth

PMN Polymorphonuclear Neutrophil

RAGE Receptor of Advanced Glycated End Products

RT-PCR Real Time Polymerase Chain Reaction

SDD Sub antimicrobial Dose Doxycycline

SRP Scaling and root planing

T1DM Type 1 diabetes mellitus

T2DM Type 2 diabetes mellitus

 Abbreviations

TBS Tris buffer saline

TGF-β Transforming growth factor beta

TNF-α Tumor Necrosis Factor alpha

TIMP Tissue Inhibitor of Matrix metalloproteinase

VEGF Vascular endothelial growth factor

VCAM-1 Vascular cell Adhesion Molecule 1

 Introduction

INTRODUCTION

Periodontitis is a chronic inflammatory disease which leads to destruction of

the periodontal tissues and alveolar bone. Among the various host factors which are

involved in destruction of periodontal tissues, one of the most important factor is

matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are a large family of calcium-dependent

zinc containing endopeptidases which are responsible for tissue remodeling and

degradation of ECM including collagens, elastins, gelatin and proteoglycans.1 They

are divided into six groups, one among them consists of gelatinases MMP-2

(gelatinase A) and MMP-9 (gelatinase B). MMP-8, MMP-13 and MMP-9 are the

main proteases involved in periodontal tissue destruction. Among these MMP-8 and

MMP-9 are the most abundant MMPs which reflect the periodontal disease severity,

progression and treatment response. 2

Several previous studies showed that the levels of MMP-9 is higher in gingival

biopsies and gingival crevicular fluid of patients with chronic periodontitis. And also

their levels have significantly decreased after periodontal therapy which confirms

their role in periodontal tissue destruction.

Diabetes mellitus is a complex metabolic disorder which is characterised by

chronic hyperglycaemia. Chronic hyperglycaemia impairs the structure and functions

of collagen which in turn alters the integrity of periodontal tissues. According to the

American Diabetes Association, periodontitis is the sixth complication of diabetes

mellitus. Hence diabetes mellitus can be considered as a risk factor for periodontitis.3

An epidemiological study by Tsai et al showed that patients with poor glycaemic

control were at 2.9 times increased risk for periodontitis.4 MMP-9 is a permissive

1
 Introduction

factor for insulin degradation in patients with DM. Evidence shows that the

expression of MMP-9 is increased in chronic periodontitis with DM when compared

to chronic periodontitis patients without DM. Hence MMP-9 can be considered as a

biomarker in the diagnosis of periodontal disease.5

In most of the studies, the MMP-9 levels and their association with diabetes

mellitus were detected using ELISA, gelatin zymography, western blot and

polymerase chain reaction methods in gingival tissues. Very few studies has been

done on immunohistochemical analysis (IHC) of MMP-9 in chronic periodontitis

patients with and without diabetes mellitus.

Immunohistochemistry (IHC) is a diagnostic technique which is used for the

identification of tissue constituents such as antigens by antigen-antibody reactions.

And the antibody binding site is identified by direct labelling of primary antibody or

by secondary antibody. IHC analysis is done by using two methods, which includes

fluorophore-labeled and enzyme-labeled antibodies for the detection of specific

protein molecules in the tissues. In our present study, the enzyme- labeled method has

been used.

It requires tissue biopsies and their sections that are incubated with

appropriate antibodies. The color of the antigen-antibody site is determined by the

chromogen used auch as diaminobenzidine (brown) or aminoethylcarbazole (red) and

the site is visualized under microscope.6

Thus, aim of our present study is to evaluate the immunohistochemical

expression of MMP-9 (Gelatinase-B) in gingival tissues of chronic periodontitis

patients with and without diabetes mellitus.

2
 Aims and Objectives

AIMS AND OBJECTIVES

To evaluate and to compare the immunohistochemical expression of MMP-9 in gingival

tissues of patients with gingivitis, systemically healthy patients with chronic periodontitis

and diabetic patients with chronic periodontitis.

3
 Review of Literature

REVIEW OF LITERATURE

Periodontitis is a chronic inflammatory disease, which often results in the

destruction of periodontal tissues and alveolar bone due to the release of

proinflammatory mediators. Even though, microorganisms (anaerobic bacteria) plays

an initial role in the periodontal disease, the progression and development of disease

are influenced by various factors which includes pro-inflammatory cytokines, growth

factors, reactive oxygen species, matrix metalloproteinases (MMPs) and their

inhibitors (TIMPs).1

Role of MMPs in periodontitis:

Matrixmetalloproteinases also called as matrixins, are a family of metal (zinc)

dependent endopeptidases responsible for the degradation of ECM components. 7

Gross and Lapier found that some diffusible proteinases are capable of degrading

fibrillar collagen in tadpole tail and lead to the discovery of MMP-1 in the year1962.

Since then several families and subclasses of MMPs has been identified. MMPs are

numbered according to the order of their discovery.8 MMPs are composed of 25

members, and they are grouped into six groups according to their substrate

specificity.7

Collagenases

MMP-1 (collagenase-1, interstitial collagenase), MMP-8 (collagenase-2, neutrophil

collagenase), and MMP-13 (collagenase-3).

Gelatinases

MMP-2 (gelatinase A, 72kDa gelatinase), MMP-9 (gelatinase B, 92kDa gelatinase).

4
 Review of Literature

Stromelysins

MMP-3 (stromelysin-1), MMP-10 (stromelysin-2), MMP-11 (stromelysin-3), and

MMP-12 (metalloelastase).

Matrilysins

MMP-7 (matrilysin, PUMP-1) and MMP-26 (matrilysin-2).

MT-MMPs (membrane type)

MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP), MMP-17

(MT4-MMP), MMP-24 (MT5MMP), and MMP-25 (MT6-MMP).

Other MMPs

MMP-18, MMP-19, MMP-20 (enamelysin), MMP-21, MMP23, MMP-27, and MMP-

28 (epilysin).7

MMPs are involved in both physiological and pathological roles.

Physiological roles such as tissue development, remodeling, in wound healing, and

cell to cell communication. Pathological role which includes periodontal disease

progression and also in other oral diseases such as dental caries, apical periodontitis

and oral cancer.9 In case of periodontitis, periodontal pathogens in the dental plaque

are responsible for stimulating the host cells to produce MMPs in an increased

amount, which in turn leads to the destruction of collagen fibers. Since collagen is a

major component extrcellular matrix (ECM) in the periodontium, it determines the

pathogenesis of periodontal dissease (Konopka et al). 1

Activities of MMPs are controlled by endogenous inhibitors known as TIMPs

(tissue inhibitors of matrix metalloproteinases). They are classified into four types

TIMP-1, 2, 3 and 4. Any imbalance between MMPs and TIMPs leads to the increased

5
 Review of Literature

degradation of ECM.1 MMPs are found in the periodontal tissues as pro-forms, active

forms, complexed species, fragmented species and cell bound MMPs.

Among various MMPs, MMP-8 and MMP-9 are the most abundant MMPs

which have been detected in their different enzymatic forms in gingival tissue, GCF

and saliva reflecting the severity and progression of periodontal disease. 2

MMP-9 also known as gelatinase B of molecular weight 92kDa is an

important member of MMPs family (Uitto et al. 2003) and they are highly expressed

during periodontal disease. MMP-9 was cloned from HT1080 fibrosarcoma cell line.8

MMP-9 is located on the human chromosome 20q11.2-q13.1. MMP-9 digest the

denatured collagens and gelatins. Several studies showed that that the levels of MMP-

9 were significantly high in periodontitis patients when compared to healthy controls

(Pozo et al. 2005). And also it has been found that genetic variation which affects the

expression of MMP-9 influences the susceptibility and severity of periodontitis. The

promoter region of MMP-9 gene harbours a functional C-to-T single nucleotide

polymorphism (SNP) at position -1562 (rs3918242) which affects the expression and

activity of MMP-9 (Blankenberg et al 2003).10

6
 Review of Literature

Structure of MMP-9:

Most of the MMPs exhibit the following domains, they are

• Catalytic domain

• Amino terminal propeptide

• Signal peptide

• Hemopexin like domain important for substrate specificity11

FIGURE 1: DOMAIN STRUCTURES OF MMPs

7
 Review of Literature

FIGURE 2: STRUCTURE OF MMP-9

S, signal peptide; Pro, propeptide; Cat, catalytic domain; Zn, active-site zinc; Hpx,

hemopexin domain; Fn, fibronectin domain.

MMP-9 has three repeats of a type II fibronectin domain which are inserted into the

catalytic domain that binds to gelatin, collagens, and laminin.10

Substrates for MMP-9:

Types I, IV, V, VII, X, XI, XIV, XVII, gelatin, elastin, fibronectin, laminin, aggrecan,

vitronectin, decorin, plasminogen, proTNF-α , α1antichymotrypsin, a2M, a1PId.8

PMNs has been considered as the important source of MMP-9 in periodontitis.

Tumor necrosis factor-α and Interleukin (IL)-1β are proinflammatory cytokines both

stimulates MMP-9 production in keratinocytes of gingival mucosa. The bacterial

lipopolysaccharide (LPS) is also a very potent inducer of MMP-9. TGF-β1 up

regulates the activity of MMP-9 in fibroblasts. TGF-β induces the production of

MMP-9in peripheral blood monocytes, and also in keratinocytes of gingival mucosa.

Though decreased level of MMP-13 has been detected in periodontal soft

tissue destruction, it induces pro MMP-9 activation in the gingival tissues of

periodontitis patients. So, MMP-13 along with the MMP-9 leads to alveolar bone

resorption and periodontal tissue breakdown. 2

8
 Review of Literature

Diabetes mellitus and Periodontitis – Two way relationship:

Diabetes mellitus is a metabolic disease which is characterised by

hyperglycaemia resulting from a defect in insulin secretion, a defect in its action, or a

combination of both. It has been considered that periodontitis affects the pathogenesis

of certain systemic diseases, which lead to the development of “periodontal

medicine”.12

Classification of diabetes mellitus:

 Type 1 DM (insulin dependent) results from autoimmune destruction of beta

cells of islets of pancreas which in turn leads to decreased insulin secretion. It

commonly affects children and adolescents.

 Type 2 DM (non-insulin dependent) which results from insulin resistance and

commonly occurs in elder individuals. It is the most common type of

diabetes.4

It has been found that periodontitis has become the sixth complication of DM.

Inflammation is the common characteristic for both DM and periodontitis. Both type

1 and type 2 DM are associated with increased levels of inflammatory markers such

as IL-1β, TNF-α, IL-6 and MMPs. The two way relationship between diabetes

mellitus and periodontitis which means diabetes can lead to periodontitis and

periodontitis which in turn can lead to DM.12

Diabetes mellitus as a risk factor for Periodontitis:

In case of DM, inflammation is increased which leads to both macrovascular

and microvascular complications, hyperglycaemic state activates the pathways of

inflammation that leads to the production of inflammatory markers such as IL-1beta,

TNF-alpha, and MMPs resulting in Periodontitis.13 Salvi et al showed that the TNF-

9
 Review of Literature

alpha was upregulated in individuals with diabetes as a response to gram negative

periodontal pathogens in monocytes of blood when compared to non-diabetic

patients. Diabetes alters the functions of various immune cells (neutrophils,

macrophages and monocytes) that leads to impairment in chemotaxis and

phagocytosis which in turn favours the environment for periodontal pathogens.14

In chronic hyperglycaemic state numerous protein molecules undergoes a

process called nonezymatic glycosylation that also leads to the formation of AGEs

(advanced glycation end products). Even at normal glucose levels, the production of

AGEs occurs, whereas in hyperglycaemia the production is excessive. AGEs affects

the endothelial cells or phagocytic cells and they bind to their receptors (RAGEs) in

these cells. Accumulation of AGEs in the gingival tissues increases the vascular

permeability, favours the growth of gram negative bacteria which results in

periodontitis.12

Diabetes and MMP-9:

Hyperglycaemic state enhances the activity and expression of several MMPs

such as MMP-8 and MMP-9 in specific cells which occurs mainly through the

production of increased advanced glycation end products (AGEs) in diabetes

mellitus.12 Due to the increased expression of these MMPs, the equilibrium state

maintained between MMPs and TIMPs are altered which in turn leads to the

destruction of collagen fibers.

Also the collagen is cross linked by AGEs, which makes the collagen less

soluble the reparative process is affected. 3 Several studies proved that hyperglycaemic

state induces the expression of MMP-9 in monocyte-derived macrophages and also

increased gelatinolytic activity of MMP-9 has been found in blood samples and aortic

10
 Review of Literature

specimens of diabetic rats. Hence the levels of MMP-9 were found to be higher in

patients with DM.13

FIGURE 3: DIABETES LEADING TO PERIODONTITIS

Periodontitis as a risk factor for diabetes mellitus:

Subgingival plaque is an important factor in the pathogenesis of

periodontal disease. Periodontal pathogens especially gram negative bacteria in the

subgingival plaque expel their contents into the gingival crevicular space. And it

results in the increased secretion of proinflammatory mediators which in turn leads to

increased insulin resistance.15 So periodontitis acts as an accelerator in elevating the

blood glucose levels. Saremi et al investigated the mortality effect of periodontitis on

diabetic patients. The death rate was 8.5 times higher in patients with severe

periodontitis.14 Periodontal treatment eliminates the bacteria and reduces the

inflammation, thereby it results in improved glycemic control.

11
 Review of Literature

FIGURE 4: PERIODONTITIS LEADING TO DIABETES

FIGURE 5: EFFECT OF PERIODONTAL TREATMENT ON DIABETES

Since the expression of MMP-9 is increased in both the periodontitis and DM, MMP-

9 can be used as a biomarker to detect the severity and progression of DM and

periodontal disease.

12
 Materials and Methods

MATERIALS AND METHODS

Patient selection:

A total of 45 subjects were selected from the outpatient ward, reported to the

department of periodontics, K.S.R. Institute of Dental Science and Research,

Tiruchengode. Informed consent was obtained from the subjects fulfilling the

following inclusion criteria and the study protocol was approved by the ethical

committee.

Study design:

45 subjects were divided into 3 groups.

Group A- 15 Gingivitis

Group B- 15 Systemically healthy patients with chronic periodontitis

Group C- 15 Chronic periodontitis patients with diabetes

INCLUSION CRITERIA

Group A (Gingivitis)

Probing depth ≤ 3 mm

No clinical attachment loss

Age : 20-60 years

Group B (Chronic Periodontitis without diabetes)

Probing Depth (PD) ≥ 5mm

Clinical Attachment Level (CAL) ≥ 1mm

Age : 20-60 years

24
 Materials and Methods

GROUP C (Chronic periodontitis with diabetes)

Probing depth (PD) ≥ 5mm

Clinical Attachment Level (CAL) ≥ 1mm

Age : 20-60 years

Fasting plasma glucose level of ≥126 mg/dl

Post prandial glucose level of ≥140 mg/dl

EXCLUSION CRITERIA

 Pregnancy subjects and lactating women

 Smokers

 Patients who underwent periodontal treatment within a duration of 6 months

 Use of systemic antibiotics within the last 6 months

 Patients with systemic diseases ( Except diabetes in group C )

Following clinical parameters were taken:

1. Plaque Index - Loe’s modification (1967)

2. Gingival Index - Loe’s modification (1967)

3. Probing pocket depth (PPD)

4. Clinical attachment level (CAL)

25
 Materials and Methods

PLAQUE INDEX (PI)

The Plaque Index was described by Silness J and Loe H in 1964 and modified

by Loe H in 1967. The plaque was assessed using a mouth mirror and dental explorer

after air drying the teeth to assess plaque on the different areas namely mesiofacial,

facial, distofacial and lingual surfaces.

Instruments used:

 Mouth mirror

 Dental explorer

The teeth were air dried and examined visually. When no plaque was visible, an

explorer was used on the surface. The explorer was passed across the surface in the

cervical third and near the entrance to the gingival sulcus. The following scores were

given.

26
 Materials and Methods

Scores for Plaque Index

Score Criteria

0 No plaque

A film of plaque adhering to the free gingival margin and adjacent area of the

tooth. The plaque may be seen only by running a probe, across the tooth

1 surface.

Moderate accumulation of soft deposits within the gingival pocket, on the

gingival margin and/or adjacent tooth surface, which can be seen by the naked

2 eye.

Abundance of soft matter within the gingival pocket and /or on the gingival

3 margin and adjacent tooth surface.

Pl score for the individual:

The indices for each of the teeth are added and then divided by the total number of

teeth examined. The scores range from 0 to 3.

27
 Materials and Methods

Interpretation:

Excellent ‘0’

Good 0.1 – 0.9

Fair 1.0 – 1.9

Poor 2.0 – 3.0

GINGIVAL INDEX (GI)

The Gingival Index (GI) was developed by Loe H and Silness J in 1963,

solely for the purpose of assessing the severity of gingivitis and its location in four

possible areas by examining only the qualitative changes (i.e., severity of the lesion)

of the gingival soft tissue. In 1967, Loe detailed the sequence of examination to

include entire teeth instead of six teeth.

Instruments used:

 Mouth mirror

 Periodontal probe.

The tissues surrounding each tooth were divided into four gingival scoring units:

distofacial papilla, facial margin, mesio-facial papilla and the entire lingual gingival

margin. The teeth and gingival should be dried lightly with a blast of air and /or

cotton rolls. Each of the 4 gingival units were assessed and following scores were

given.

28
 Materials and Methods

Scores for Gingival Index

Score Criteria

0 Absence of inflammation/normal gingiva.

1 Mild inflammation, slight change in color, slight edema; no bleeding on

probing.

2 Moderate inflammation; moderate glazing, redness, edema and hypertrophy,

bleeding on probing.

3 Severe inflammation; marked redness and hypertrophy, ulceration, tendency to

spontaneous bleeding.

GI score for the individual:

The indices for each of the teeth are added and then divided by the total number of

teeth examined. The scores range from 0 to 3.

Gingival score interpretation

Gingival scores Condition

0.1 – 1.0 Mild Gingivitis

1.1 – 2.0 Moderate Gingivitis

2.1 – 3.0 Severe Gingivitis

29
 Materials and Methods

PROBING POCKET DEPTH (PPD) AND CLINICAL ATTACHMENT LEVEL

(CAL):

Probing pocket depth was measured as the distance between the free gingival

margin and the base of the pocket and clinical attachment level was measured as the

distance between the cementoenamel junction and the base of the pocket using

periodontal probe.

EXTRACTION OF GINGIVAL TISSUES:

Gingival tissues were collected during extraction, crown lengthening, gingivectomy

and gingivoplasty procedures, periodontal flap surgery and stored in 10% formalin.

Expression of MMP-9 in the gingival tissues were determined using

immunohistochemical analysis.

EQUIPMENTS AND MATERIALS USED IN THE STUDY:

The following equipments and materials were used for the immunohistochemical

staining.

1. APES (3-Aminopropyltriethoxysilane) pre-coated slides

2. Normal glass sides

3. Microtome

4. Xylene

5. 100%, 90%, 70%, and 50% alcohol

6. Distilled water

7. Citrate buffer (pH: 6)

30
 Materials and Methods

8. Pressure cooker

9. Humidifying chamber

10. Micropipettes with plastic disposable pipette tips

11. Tris phosphate wash buffer (pH: 7.4)

12. Peroxide block

13. Power block

14. Primary antibody – MMP-9

15. Universal secondary kit - 3% hydrogen peroxide block

- Secondary antibody

- Streptavidin conjugated with horse radish peroxidase

- Diluting buffer

- Diaminobenzidinechromogen (DAB)

16. Dibutylpthalate xylene (DPX) Mountant

17. Cover slip

18. Slide holding box

19. Compound microscope

31
 Materials and Methods

Antigen retrieval (Sodium citrate) buffer (10 mmol sodium citrate, pH 6.0)

• Tri-sodium citrate (dihydrate) -2.94g

• Distilled water-1000ml

• 1mol hydrochloric acid

All the above ingredients were mixed to dissolve. The pH was adjusted to 6.0 with 1

mol hydrochloric acid.

Wash buffer - Tris buffer saline (TBS, pH 7.4)

• Tris - 0.605gms

• Sodium chloride - 8gms

• 1 mol Hydrochloric acid - 4.4ml

All the above ingredients were mixed to dissolve. The pH was adjusted to 7.4 with 1

mol Hydrochloric acid.

Preparation of tissues for IHC staining

Sectioning:

For immunohistochemistry, each section of about 3 to 4 μm thickness was prepared

by using a semiautomatic microtome. The sections were placed on APES pre-coated

slides. The slides were preserved in a slide holding box until they were stained.

32
 Materials and Methods

Deparaffinization:

The sections were deparaffinized by heating on the slide warmer at 60°C for one and

half hours. The sections were then dewaxed in 2 changes of xylene, each lasting 15

minutes.

Rehydration:

The sections were rehydrated in decreasing grades of alcohol (100%, 90%, 70%, and

50%), 5 minutes each and kept under water for 10 minutes.

Antigen Retrieval:

1.5 liters of Sodium citrate buffer (pH 6.0) was added into the pressure cooker and

brought to boil (without securing the lid).

Once the buffer started boiling the slide racks were placed into the hot buffer and the

lid was sealed.

The pressure cooker was allowed to reach full pressure, then left for about 20 minutes

The pressure cooker was cooled until all the pressure was released.

IHC STAINING PROCEDURE:

All the reagents stored in the refrigerator were brought to room temperature prior to

immunostaining. All the incubations were performed at room temperature using a

humidifying chamber unless otherwise mentioned. The tissue sections were not

allowed to dry at any point during the staining procedure.

33
 Materials and Methods

Step 1: Endogenous peroxidase blocking:

Sections were covered with peroxidase block for 10 minutes to prevent nonspecific

binding of secondary antibody. The slides were then rinsed twice with wash buffer for

5 minutes.

Step 2: Power Block:

Antibodies may attach nonspecifically to highly charged sites. To prevent this, power

block was used to avoid cross reactions and to reduce nonspecific binding. The

sections were covered with power block solution for 10 minutes.

Step 3: Incubation with primary antibody:

Excess buffer was tapped off and the sections were incubated with VEGF Antibody

(Sigma Aldrich) at 1:10 dilution at 4°C.

Step 4: Super enhancer application

The sections were covered with super enhancer for 20 minutes and washed with tris

buffer for 5 minutes and excess was wiped out using blotting paper.

Step 5: Secondary antibody application:

The slides were washed and treated with secondary antibody tagged with horseradish

peroxidase enzyme (HRP) for 30 min.

Step 6: Substrate chromogen application:

The slides were then washed with phosphate buffer saline and immunostaining was

developed by treatment with freshly prepared DAB (3-diaminobenzidene tetra

34
 Materials and Methods

hydrochloride) solution for 5 minutes following which they were washed in distilled

water to remove excess chromogen.

Step 7: Counter stain:

The slides were immersed in Mayer’s hematoxylin for 30 seconds, and bluing was

done using distilled water.

Step 8: Dehydration and cleaning:

The sections were dehydrated in 100% alcohol, followed by clearing in xylene.

Step 9: Mounting:

The sections were mounted using dibutylpthalate xylene (DPX), a non-aqueous

permanent mounting medium.

A red–brown cytoplasmic staining was considered as positive for antibody staining.

The staining intensity for antibodies in inflammatory cells was regarded as

 intense (+++)

 moderate (++)

 mild (+) and

 negative (-)35

35
 Materials and Methods

FIGURE 6: ARMAMENTARIUM FOR TISSUE COLLECTION

FIGURE 7: ARMAMENTARIUM FOR IMMUNOHISTOCHEMICAL

ANALYSIS

36
 Materials and Methods

FIGURE 8: TISSUE COLLECTION DURING CROWN LENGTHENING

A. Pre-op B. Incision made using scalpel

C. Gingival tissue collected D. Post-op

37
 Materials and Methods

FIGURE 9: TISSUE COLLECTION DURING GINGIVECTOMY

38
 Materials and Methods

FIGURE 10: TISSUE COLLECTION DURING EXTRACTION

39
 Materials and Methods

FIGURE 11: PREPARATION OF WAX BLOCK AND SLIDES FOR

IMMUNOHISTOCHEMICAL ANALYSIS

Tissue processing Wax block made

Tissue sectioning done using microtome

40
 Materials and Methods

Slides heated using hot plate

Primary antibody

41
 Materials and Methods

Secondary antibody kit

The slides are prepared for IHC analysis in the following order

42
 Materials and Methods

FIGURE 12: SLIDES VIEWED BY COMPOUND MICROSCOPE

43
 Materials and Methods

FIGURE 13: NEGATIVE EXPRESSION OF MMP-9

(10X magnification)

44
 Materials and Methods

FIGURE 14: MILD EXPRESSION OF MMP-9

(10X magnification)

10X magnification

45
 Materials and Methods

FIGURE 15: MODERTE EXPRESSION OF MMP-9

(10X magnification)

46
 Materials and Methods

FIGURE 16: SEVERE EXPRESSION OF MMP-9

(10X magnification)

47
 Statistical Analysis

STATISTICAL ANALYSIS

All analysis were done using SPSS version 16(SPSS Inc., Chicago, IL).

Descriptive statistics was performed. The results were presented as mean, Standard

deviation, median and IQR for continuous data and as frequencies percentages for

categorical data. The clinical, demographic, IHC variables were compared between

three groups. Based on the distribution of the data, Comparisons of the continuous

data between the groups were performed by One way ANOVA with post hoc Tukey’s

HSD test and Kruskal-Wallis test with series of post hoc mann whitney U test

appropriately. Qualitative differences between the groups were analyzed by the Chi-

square test. Spearman correlation analysis was used to detect the linear correlations

between variables. Two-sided p-values < 0.05 were considered to indicate statistical

significance.

48
 Results

RESULTS

Table 1 shows the descriptive statistics of Demographic, clinical parameters

and expression of MMP-9 among the three groups. The variables are age in years,

MMP-9 expression, plaque index, gingival index, probing depth and clinical

attachment level.

Table 2 and graph 1 shows the comparative analysis of MMP-9 expression

between the groups. Kruskal Wallis test has been used to compare the mean values.

The mean values of the subjects in each group A, B, C were 0.20 ± 0.41, 2.20 ± 0.77,

2.60 ± 0.51 respectively. Compared to group A, significantly higher expression of

MMP-9 were found in groups B and C (P<0.05). No significant difference was found

between the groups B and C.

Table 3 shows the pairwise comparison of MMP-9 expression between the

groups. Mann whitney U test was used for comparison. When compared to group A,

significant difference was found in group B and C. But no significant difference was

found between group B and C.

Table 4 and graphs 2a and 2b shows the pairwise comparison of periodontal

clinical parameters of gingival index, plaque index, probing depth and clinical

attachment level between the three groups. Non parametric post hoc series of mann

whitney U test was used for comparison. Statistically significant difference was found

in groups B and C when compared to group A (P<0.05).

Table 5 and graph 3 shows the distribution of age between the three groups.

One way ANOVA test was used to compare the mean values. The mean values in

each group A, B and C were 24.13, 48.50 and 50.47 respectively. The mean age

distribution between group B and C were almost similar.

49
 Results

Table 6 and graph 4 shows the distribution of gender (males and females)

among the groups. Chi square test was used to compare the values. The gender

distribution in group A was 66.7% of (females) & 33.3% of males, in group B was

66.7% of (females) & 33.3% of (males), in group C was 53.3% of (females) & 46.7%

of (males) respectively. No significant difference was found between the groups.

Table 7 and graph 5 shows the distribution of random blood glucose (RBG)

levels in gingivitis subjects, periodontitis patients with and without diabetes. The

mean values were 92.80 ± 8.58, 108.13 ± 6.76,176.40 ± 26.07 respectively and

statistically significant higher RBG values were found in group C compared to group

A & B(P<0.05).

Table 8 and graph 6 shows the gender wise comparison of MMP-9

expression. Mann whitney U test was used to compare the mean values. The mean

values were 1.82 ± 1.13 in males and 1.57 ± 1.26 in females. No statistically

significant difference was found between males and females.

Table 9 and graph 7a & 7b shows the correlation coefficient (r value) of

MMP-9 expression with the clinical parameters PD and CAL. Spearman correlation

analysis was used to analyze the relationship between these variables. Positive correlation

has been found between MMP-9 expression and PD, CAL.

50
 Results

Table 1: Descriptive statistics of Demographic, clinical parameters and antibody

immunostaining of MMP-9 among gingivitis and periodontitis patients with and
without T2DM

Parameters Groups Mean Std. Minimum Maximum Median IQR

Deviation
Age (yrs) GROUP A
24.13 5.13 16.00 32.00 24.00 9.00
GROUP B
48.80 8.07 36.00 60.00 49.00 13.00
GROUP C
50.47 6.32 40.00 59.00 52.00 10.00
MMP-9 expression GROUP A
0.20 0.41 0.00 1.00 0 0
GROUP B
2.20 0.77 1.00 3.00 2.0 1
GROUP C
2.60 0.51 2.00 3.00 3.0 1
Plaque index GROUP A
0.31 0.08 0.21 0.46 0.31 0.11
GROUP B
1.65 0.06 1.50 1.70 1.70 0.10
GROUP C
2.26 0.30 1.70 2.70 2.20 0.30
Gingival index GROUP A
0.27 0.08 0.20 0.43 0.23 0.12
GROUP B
1.65 0.06 1.50 1.70 1.70 0.10
GROUP C
1.91 0.20 1.70 2.20 1.80 0.40
Probing depth (mm) GROUP A
1.39 0.27 1.01 1.95 1.33 0.41
GROUP B
4.22 0.26 3.65 4.52 4.30 0.39
GROUP C
4.20 0.24 3.82 4.64 4.22 0.40
Clinical attachment level GROUP A
0.00 0.00 0.00 0.00 0.00 0.00
(mm)
GROUP B
5.90 0.52 5.09 6.60 5.93 0.90
GROUP C
5.83 0.44 4.80 6.42 5.94 0.72
Group A - Clinically healthy (controls); Group B - Generalized chronic periodontitis; Group C - Generalized Chronic Periodontitis with
T2DM.

51
 Results

Table 2: Comparison of MMP-9 expression between gingivitis and periodontitis

patients with and without T2DM

Antibody Groups Mean ±Std. Median ± P value

Immunostaining Deviation IQR
MMP-9 expression * GROUP A 0.20± 0.41 0±0

0.001*
GROUP B 2.20± 0.77 2±1

GROUP C 2.60± 0.51 3±1

* Non parametric Statistical comparison among the three groups (Kruskalwallis test). * shows (p < 0.05)

Graph: 1 Comparison of MMP-9 expression scores among groups

52
 Results

Table 3: Post hoc pairwise comparison of MMP-9 expression between the groups

MMP-9 expression
P value
Group A Group B 0.001*
Group C 0.001*

Group B Group C 0.147

Series of mannwhitney U test * shows (p < 0.05)

Table 4 : Post hoc pairwise comparison of periodontal clinical parameters between

the groups

PI* GI* PD† CAL†

Group A Group 0.001* 0.001* 0.001* 0.001*

B

Group 0.001* 0.001* 0.001* 0.001*

C

Group B Group 0.001* 0.001* 0.983 0.890

C

*shows (p<0.05)
* Non parametric Post hoc Series of mannwhitney U test ; †Tukey,s HSD post hoc test

53
 Results

Graph2a: Comparison of PI, GI scores among the three groups

2.5
2.2
2.0
1.8
1.7 1.7

1.5

1.0

0.5 0.3 0.2

0.0
GROUP A GROUP B GROUP C

PI GI

Graph 2b: Comparision of PD and CAL among experimental groups

8.0

7.0
5.9 5.8
6.0

5.0
4.2 4.2
4.0

3.0

2.0 1.4
1.0
0.0
0.0
GROUP A GROUP B GROUP C

Probing depth Clinical attachment loss

54
 Results

Table 5: Distribution of age between the groups

Mean Std. Median IQR P value

Deviation
Group A – Gingivitis 24.13 5.13 24.00 9

Group B -Generalized 48.80 8.07 49.00 13

chronic periodontitis 0.001*

Group C -Generalized 50.47 6.32 52.00 10th

chronic periodontitis
(Diabetes)
One way ANOVA test; shows *(p<0.05)

Graph 3: Comparision of mean age between the groups

60.0

48.8 50.5
50.0

40.0
GROUP A
30.0
24.1 GROUP B

20.0 GROUP C

10.0

0.0
GROUP A GROUP B GROUP C

55
 Results

Table 6: Gender wise distribution between the groups

Group A – Group B – Group C – P value

Gingivitis Generalized chronic Generalized chronic
periodontitis periodontitis
(Diabetes)
Female 10 (66.7%) 10 (66.7%) 8 (53.3%) 0.685

Male 5 (33.3%) 5 (33.3%) 7 (46.7%)

Chi square Test; Not significant

Graph 4: Comparision of Gender distribution between the groups

70.0% 66.7% 66.7%

60.0%
53.3%
50.0% 46.7%

40.0% 33.3% 33.3%

30.0%

20.0%

10.0%

0.0%
GROUP A GROUP B GROUP C

Female Male

56
 Results

Table 7: Comparison of Random blood glucose between the groups

Mean Std. Median IQR P value

Deviation
Group A – Gingivitis 92.80 8.58 98.00 14.00
Group B -
Generalized chronic 0.001*
108.13 6.76 110.00 6.00
periodontitis
Group C -
Generalized chronic
176.40 26.07 193.00 41.00
periodontitis
(Diabetes)
One way ANOVA test; shows *(p<0.05)

Graph 5: Distribution of random blood glucose between the groups

200.00
176.40
180.00
160.00
140.00
120.00 108.13
GROUP A
100.00 92.80
GROUP B
80.00
GROUP C
60.00
40.00
20.00
0.00
GROUP A GROUP B GROUP C

57
 Results

Table 8: Comparision of MMP-9 expression based on gender

Sex Mean Std. Median

Deviation

Male 1.82 1.13 2.00 0.534

Female 1.57 1.26 2.00

mannwhitney U test ; Not significant

Graph 6: Comparision of MMP-9 expression based on gender

2.00 2.00
2.00
1.80
1.60
1.40
1.20 Male
1.00 Female
0.80
0.60
0.40
0.20
0.00
Male Female

58
 Results

Table 9: Correlation of MMP-9 expression with PD and CAL

variables MMP-9 expression

r value p value
PD 0.676 0.001**

CAL 0.671 0.001**

** Correlation is significant at the 0.01 level (2-tailed).Spearman correlation test

Graph 7a: Correlation of MMP-9 expression with Probing depth

Positive Correlation between MMP- 9 with probing depth

(r=+0.676)
5

4
PD (mm)

2 PD
Linear (PD)
1

0
0 1 2 3
MMP-9

59
 Results

Graph 7b: Correlation of MMP-9 expression with Clinical Attachment Level

Positive Correlation between MMP- 9 with CAL

(r=+0.671)
7
6
5
CAL(mm)

4
3 CAL
2 Linear (CAL)
1
0
0 1 2 3
MMP-9

60
 Discussion

DISCUSSION

Chronic periodontitis is an infectious disease resulting in inflammation of the

supporting tissues of the teeth, with attachment loss and bone loss. Dental plaque

composed of gram negative bacteria (Porphyromonas gingivalis, Tannerella forsythia,

Aggregatibacter actinomycetemcomitans) is considered to be the primary etiological

agent in the development of periodontal disease.

Diabetes mellitus is a metabolic disorder which causes chronic

hyperglycaemia. Inflammation is the common characteristic of both DM and

periodontal disease. Neutrophils as the first line of defense migrates to the site of

inflammation and they store MMP-9 into their secondary secretory granules.

Whenever inflammation occurs they readily undergo degranulation and release MMP-

9.19 The virulence factors of periodontal pathogens and proinflammatory cytokines

stimulates the degranulation of neutrophils.2

In case of DM, there will be accumulation of anomalous glycoproteins that

leads to the increased production of advanced glycation end products and also

neutrophil function impairment, which in turn results in an increased release of

proinflammatory mediators.13,14 Both DM and periodontitis, leads to the production of

proinflammatory cytokines like IL-1β, IL-6, TNF-α including MMPs (MMP-8 and

MMP-9).12 These MMPs are mainly regulated by these proinflammatory cytokines

and growth factors.27 So the bidirectional relationship between DM and periodontitis

has been established. And periodontitis has been considered as the sixth complication

of DM. Hence, hyperglycaemic individuals with uncontrolled blood glucose level are

highly susceptible to periodontal disease.

61
 Discussion

Matrix metalloproteinases (MMPs) are the enzymes responsible for the

degradation of ECM compoments (mainly collagen). Since the major component of

periodontal ECM is type I collagen, special attention has been paid to collagenases

(MMP-8, -13) and gelatinases (MMP-2, -9) in the progression of periodontal disease.

Collagenases degrade type I fibrillar collagen into N and C terminal fragments of

denatured collagen which results in the formation of gelatin. Gelatinases (MMP-2 and

MMP-9) contains gelatin ligation domain and it further degrades the type I denatured

collagen leads to periodontal tissue destruction. So, MMP-13 activates pro-MMP-9

which in turn activates pro-MMP-2.2, 34

Several studies demonstrated that MMP-9 (Gelatinase-B) plays an important

role in both DM and periodontal disease. It has been found that elevated blood

glucose level stimulates the production of MMP-9.5 Diabetes affects the balance

between MMPs and their inhibitors (TIMPs), which results in delayed wound healing

and occurrence of deep periodontal pockets. 24 Compromised periodontium in the

diabetes directly reflects the levels of MMP-9 that leads to the massive degradation of

collagen. And also it has been found that the gingival tissues of diabetic patients were

infiltrated with neutrophils and increased collagen diffusion which is directly

proportional to the levels of MMP-9.5

Very few studies were done on immunohistochemical analysis of MMP-9

expression in periodontitis patients with and without diabetes. So, the aim of our

present study is to evaluate the immunohistochemical expression of MMP-9

(Gelatinase-B) in gingival tissues of chronic periodontitis patients with and without

diabetes mellitus.

62
 Discussion

Clinical parameters which includes plaque index, gingival index, probing

depth and clinical attachment level and random blood glucose were evaluated and the

total of 45 subjects were divided into 3 groups. Group A - patients with gingivitis,

Group B – systemically healthy patients with chronic periodontitis, Group C –

chronic periodontitis patients with diabetes. Gingival tissues were collected from the

patients during extraction, crown lengthening, gingivectomy and periodontal flap

surgery. They were stored in 10% formalin. Gingival tissues were then processed and

the expression of MMP-9 in the samples were evaluated by immunohistochemical

analysis as negative, mild expression, moderate expression, severe expression.

Expression scores were evaluated under 10X magnification. Moderate to severe

expression of MMP-9 has been found to be associated with periodontitis and type 2

diabetes mellitus. In the present study basal cells of epithelium and connective tissue

cells (fibroblasts) showed immunostaining of MMP-9.

Statistical analysis was done. The results showed that the expression of MMP-

9 were significantly higher in group B and C when compared to group A. But no

significant difference in expression of MMP-9 was found between the groups B and

C. The mean values of the groups A, B, C were 0.20 ± 0.41, 2.20 ± 0.77, 2.60 ± 0.51

respectively.

Also positive correlation was found between MMP-9 expression and probing

depth, clinical attachment level. Comparative analysis of MMP-9 expression on

gender was also done, but no significant difference was found between the males and

females. In both the groups B and C, MMP-9 levels were found to be higher and

hence the results shows the bidirectional relationship between periodontitis and

diabetes mellitus. Studies showed that the levels of MMP-9 were significantly higher

in aggressive periodontitis patients when compared to healthy subjects.33 In these

63
 Discussion

patients, systemic antibiotics of tetracycline has been used to reduce the levels of

matrix metalloproteinases.14

The present study results are consistent with the following studies conducted by

several authors.

Rai et al (2008) and Makela et al (1994) conducted a study to compare the

levels of MMP-9 in patients with gingivitis and periodontitis by evaluating gingival

tissue samples and gingival crevicular fluid respectively. They found that the MMP-9

levels were elevated significantly in periodontitis patients when compared to

gingivitis.15, 26

Ozdemir et al (2012) conducted an animal study in wistar rats with chronic

periodontitis and diabetes and evaluated the expression of MMP-9 in the gingival

tissues by immunohistochemical analysis. The results showed that the expression

levels were high in the diabetes + periodontitis group compared to diabetes control

group.31

Kumar et al (2006) compared the expression of MMP-9 in the gingival tissues

of chronic periodontitis patients with and without DM and healthy controls. Samples

were analysed using gelatin zymography, and found that the expression of MMP-9

was significantly higher in CP patients with DM than without DM and healthy

controls.5

Goncalves et al (2008) also compared the expression of MMP-9 in healthy and

inflammed gingival samples by zymography and western blotting. They found that

the activity of MMP-9 was higher in inflamed tissue samples compared to healthy

controls.23

Marcaccini et al in the year 2009 and 2010 analysed the levels of MMP-9 in

chronic periodontitis patients in plasma and gingival crevicular fluid respectively and

64
 Discussion

also compared their levels before and after the non-surgical periodontal therapy. At

baseline, the levels of MMP-9 were high, but after non-surgical periodontal therapy

their levels were found to be decreased.19, 29

The limitations of the study includes the following:

 HbA1c which is a reliable test for the diagnosis of DM was not done in

diabetic patients.

 Also the density of inflammation was not evaluated (inflammatory cells were

not counted)

 Comparison of MMP-9 levels were not done before and after the periodontal

therapy.

Hence, only very few studies has been conducted using immunohistochemical

analysis in gingival biopsies, further randomized controlled trials and longitudinal

studies has to be done to evaluate the exact role of MMP-9 in the pathogenesis of

periodontal disease and diabetes in order to establish the relationship that exists

between periodontitis and diabetes mellitus.

65
 Summary and Conclusion

SUMMARY AND CONCLUSION

From the results of our present study, it can be concluded that

 Significantly higher immunohistochemical expression of MMP-9 was found

to be associated with systemically healthy chronic periodontitis patients and

chronic periodontitis patients with type 2 diabetes mellitus when compared to

gingivitis patients.

 Also, positive correlation was observed between MMP-9 expression and PD,

CAL.

Therefore, both chronic periodontitis and diabetes are found to be associated

with the elevated levels of MMP-9 in gingival tissues and bidirectional relationship

between CP and DM was observed. Since subgingival plaque plays an important role

in both the diseases, it should be eliminated at the correct time. And non-surgical

periodontal therapy seems to reduce the levels of MMP-9. It was found that the

patients with poor glycaemic control shows higher levels of MMP-9 than patients

with controlled diabetes, so it is important to maintain the blood glucose levels.

Considering within the limitations of the study, future large scale

epidemiological studies in the form of randomised controlled trials has to be done, to

establish the pathogenesis and treatment protocol in patients with chronic

periodontitis and diabetes to avoid further progression of periodontal disease.

66
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9 and-13 in gingival tissue of patients with type 1 diabetes and periodontitis.

Rom J Morphol Embryol. 2014 Jan 1;55(3 Suppl):1137-41.

35. Watanabe K, Petro BJ, Shlimon AE, Unterman TG. Effect of periodontitis on

insulin resistance and the onset of type 2 diabetes mellitus in Zucker diabetic

fatty rats. J Periodontol. 2008 Jul;79(7):1208-16.

71
 Annexures

ANNEXURE 1

INFORMATION SHEET

We are conducting a study on “EXPRESSION OF MATRIX

METALLOPROTEINASE-9 (GELATINASE-B) IN GINGIVAL TISSUES OF

CHRONIC PERIODONTITIS PATIENTS WITH AND WITHOUT DIABETES

MELLITUS: AN IMMUNOHISTOCHEMICAL STUDY”

The identity of the patients participating in the research will be kept confidential

throughout the study. In the event of any publication or presentation resulting from the

research, no personally identifiable information will be shared.

Taking part in the study is voluntary. You are free to decide whether to participate

in the study or to withdraw at any time; your decision will not result in any loss of

benefits to which you are otherwise entitled.

The results of the special study may be intimated to you at the end of the study

period or during the study if anything is found abnormal which may aid in the

management or treatment.

Name of the patient Signature / Thumb impression

Name of the investigator Signature

Date

72
 Annexures

ANNEXURE 2

INFORMED CONSENT FORM

EXPRESSION OF MATRIX METALLOPROTEINASE-9 (GELATINASE-B) IN

GINGIVAL TISSUES OF CHRONIC PERIODONTITIS PATIENTS WITH AND

WITHOUT DIABETES MELLITUS: AN IMMUNOHISTOCHEMICAL STUDY

K.S.R. INSTITUTE OF DENTAL SCIENCE AND RESEARCH

DEPARTMENT OF PERIODONTICS

I ..................................hereby declare that I have read this consent and clearly

understood the procedures, possible discomforts, as well as possible benefits of the

study. I whole heartedly give permission for the above mentioned institution /

individual / organization / hospital to disclose my records to the individual /

organization listed above.

SIGNATURE: ………………………………… DATE: ………………………..

I have explained the procedure involved in the study and answered all the questions

asked by the patient.

SIGNATURE:................................................... DATE: ....................................

73
 Annexures

ஒஒஒஒஒஒஒ ஒஒஒஒஒஒஒஒஒஒஒ

--------------------------------------------------------------------- ஆஆஆஆ ஆஆஆஆ ஆஆஆஆஆஆஆஆஆ

ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆ
ஆஆஆஆஆஆஆஆ . ஆஆஆஆஆஆ ஆஆஆஆ ஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆஆஆ
ஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆ ஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ
ஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆஆஆஆஆ.

-------------------------------------------
ஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆ ----------------------------
---

ஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆ ஆஆஆ

ஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆஆஆஆஆஆஆ

------------------------------------------
ஆஆஆஆஆஆஆஆஆஆஆஆ ஆஆஆஆஆஆஆஆஆ ஆஆஆஆ ---------------------
--------

74
 Annexures

ANNEXURE 3

EXPRESSION OF MATRIX METALLOPROTEINASE-9 (GELATINASE-B) IN

GINGIVAL TISSUES OF CHRONIC PERIODONTITIS PATIENTS WITH AND

WITHOUT DIABETES MELLITUS: AN IMMUNOHISTOCHEMICAL STUDY

PROFORMA

S. No :

Name: O.P.no:

Age/Gender: Date:

Occupation:

Address:

Contact No:

Chief complaints:

Past Medical history:

Past Dental history:

Laboratory investigations:

75
 Annexures

PERIODONTAL EXAMINATION

Date:

PLAQUE INDEX – MODIFIED BY LOE (1967)

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

PI score:

GINGIVAL INDEX – MODIFIED BY LOE(1967)

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

GI score:

76
 Annexures

PROBING POCKET DEPTH (in mm)

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

Mean-

CLINICAL ATTACHMENT LEVEL (in mm)

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

Mean-

77
 Annexures

GINGIVAL BIOPSY:

PLAQUE INDEX MEAN SCORE:

GINGIVAL INDEX MEAN SCORE:

PROBING DEPTH MEAN SCORE:

CAL MEAN SCORE:

SIGNATURE OF THE GUIDE

78
RESEARCH METHODOLOGY AND BIOSTATISTICS CERTIFICATE
RESEARCH METHODOLOGY AND BIOSTATISTICS CERTIFICATE
PLAGIARISM REPORT