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Immunohistochemical Study of Matrix

Metalloproteinase-1 (MMP-1) Expression in

Oral Submucous Fibrosis

Dissertation submitted to

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

In partial fulfillment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH IV

ORAL AND MAXILLOFACIAL PATHOLOGYMarch 2007

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ACKNOWLEDGEMENT

I extend my sincere thanks to Dr. K. Ranganathan, MDS, MS (Ohio), Professor and

Head, Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital,

for his valuable guidance, support, encouragement and all help throughout my post graduate

curriculum. I thank him for the time spent on going through my work, giving ideas and

correcting all the minute mistakes without which this thesis could not be presented in the

present form.

My heartful gratitude to Dr. T. R. Saraswathi, MDS, M.Sc (Lon), Professor, for her

constant encouragement and valuable advices throughout my post graduate course.

I extend my sincere thanks to Dr. M. Uma Devi, Professor, Department of Oral and

Maxillofacial Pathology, Ragas Dental College and Hospital, for her guidance, support and

help throughout my post graduate curriculum, and various advices in completion of this

work.

I earnestly thank Dr. Elizabeth Joshua, Associate Professor, Department of Oral and

Maxillofacial Pathology, Ragas Dental College and Hospital for her constant

encouragement and support throughout my study.

I thank Dr. S. Nalin Kumar, Reader and Dr. S. Balasundaram & Dr. Sandhya,

Lecturers, Department of Oral and Maxillofacial Pathology, Ragas Dental College and

Hospital, who helped me with their guidance throughout my post graduate course.

Sincere thanks to the Principal, Dr. S. Ramachandran, Ragas Dental College and

Hospital for his permission to use the facilities of the institution.

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I extend my sincere thanks to Research Assistant Mrs. Kavitha, Bio-statistician Mrs.

Hemalatha, Lab Technician Mr. Rajan, Ragas Dental College and Hospital, for all the

patience shown and the constant help they rendered in completion of this study.

I wish to thank Computer Assistant Mrs. Rupa, Ragas Dental College and Hospital,

for her valuable help.

I acknowledge gratefully all my batch mates Dr. Antony, Dr. Jayanthi, Dr. Raghu,

Dr. Siva and Dr. Vidya, all my seniors & juniors, for their constant help and support

throughout my postgraduate life & my friends Dr. Ghazan and Dr. Vidya KM for being there

when I needed them.

I would like to dedicate my work to my parents Sri. Brajesh Mishra & Dr. Manju

Mishra, for their sacrifices, abundant support, faith, understanding and love & my

Grandmother for her prayers and blessings. I would like to remember my beloved Late

Grandfather whose teachings and ideologies have always been my source of inspiration.

Last but not the least, I thank God Almighty, the Supreme Being without whose will

nothing would have been possible.

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CONTENTS

S. No. INDEX PAGE No.

1. INTRODUCTION 1

2. AIMS AND OBJECTIVES 4

3. MATERIALS AND METHODS 5

4. STATISTICAL ANALYSIS 15

5. REVIEW OF LITERATURE 16

6. RESULTS 50

7. TABLES AND GRAPHS 55

8. PHOTOMICROGRAPHS 65

9. DISCUSSION 71

10. SUMMARY AND CONCLUSION 76

11 BIBLIOGRAPHY 77

12. ANNEXURE 92

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Introduction

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Oral submucous fibrosis (OSF) is a form of pathological fibrosis affecting the oral

mucosa and contiguous areas of the upper aerodigestive tract, having a relentless course of

progression 63. The condition was aptly described by Sirsat and Pindborg as “an insidious

chronic disease affecting any part of the oral cavity and sometimes pharynx. Although

occasionally preceded by and/or associated with vesicle formation, it is always associated

with juxtaepithelial inflammatory reaction followed by fibroelastic changes in the lamina

propria, with epithelial atrophy leading to stiffness of the oral mucosa causing trismus and

difficulty in eating” 51.

WHO definition for an oral precancerous condition- “a generalized pathological state

of the oral mucosa associated with a significantly increased risk of cancer”, accords well with

the characteristics of OSF 59.

OSF predominantly involves the oral cavity. Buccal mucosa, retromolar area and the

soft palate are the most commonly involved sites. Other sites include floor of the mouth,

oropharynx, and tongue. The mucosa in the involved site becomes pale followed by

progressive stiffness.

It occurs predominantly in the Indian subcontinent and South Asian countries where

this habit is more prevalent. The frequency of the population affected by OSF ranges from

35% to 100% 37.

There is compelling evidence to implicate the habitual chewing of arecanut with the

development of OSF. Arecoline, an active alkaloid found in betel nuts, stimulates fibroblasts

to increase production of collagen by as much as 150%. Flavanoids, catechin, and tannin in

arecanut cause collagen fibers to cross-link, making them less susceptible to collagenase 37.

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Since collagens are the major structural components of connective tissues, including

oral submucosa, the composition of collagen within each tissue needs to be precisely

regulated to maintain tissue integrity13. Collagenase among other enzymes plays an important

role in this process. Collagenase is a type of matrix metalloproteinase (MMP).

MMPs are a family of neutral proteases that are produced by variety of cells during

both physiologic conditions like normal development, wound healing and wide variety of

pathological processes 40.

Currently 26 human MMPs have been identified and these enzymes have been

classified according to their substrate specificity and structural similarities. They are

produced by several cell types including fibroblasts, macrophages, neutrophils, synovial cells

and some epithelial cells. Their secretion is induced by certain stimuli, including growth

factors, cytokines and physical stress 31.

Collagenase, a member of the MMP family, is secreted as a latent precursor

(procollagenase) and is activated by chemicals, such as free radicals produced during the

oxidative burst of leukocytes and proteinases 31. Collagenase is the principal human enzyme

that cleaves native fibrillar collagen 13. Collagenase-1 (MMP-1) is produced by a wide

variety of normal cells, eg. stromal fibroblasts, macrophages, endothelial cells and epithelial

cells, as well as by numerous tumors, suggesting a broad-base role for this collagenase in

biology 6.

MMP expression in various tissue types has been examined through

immunohistochemical analysis and insitu hybridization26. Collagenase activity has been

found to be lower in OSF than in the normal oral mucosa. This implies that OSF may be a

collagen-metabolic disorder resulting from alkaloid exposure and individual variation in

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collagen metabolism13. This study was carried out to evaluate the expression of MMP-1 in

OSF and in normal mucosa.

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Aims & Objectives

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AIM

1. To evaluate the expression of Matrix Metalloproteinase (MMP) -1 in different

histological grades of oral submucous fibrosis (OSF) by immunohistochemistry in

paraffin embedded tissues.

2. To evaluate the expression of MMP-1 in normal buccal mucosal tissues by

immunohistochemistry in paraffin embedded tissues.

3. To compare the expression of MMP-1 in OSF and normal buccal mucosa.

HYPOTHESIS

Matrix Metalloproteinase-1 (MMP-1) expression is decreased in oral submucous

fibrosis (OSF).

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Materials & Methods

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Study setting

The study was conducted in the Department of Oral and Maxillofacial Pathology,

Ragas Dental College and Hospital, Chennai, to study the expression of MMP-1 in OSF by

immunohistochemistry in paraffin embedded tissues.

Study subject

The study comprised of 2 groups:

Group 1- (CASES)

o 30 consecutive patients of OSF, diagnosed clinically and confirmed

histopathologically, were selected.

o Inclusion Criteria-

Clinical:

x Habit of chewing arecanut in some form.

x The presence of fibrous bands in the labial and/ or buccal

mucosa.

x Loss of elasticity of the buccal/ labial mucosa.

x Restriction of mouth opening.

Histopathological:

x Juxtaepithelial hyalinization.

x Submucosal dense and vascular collagenous connective tissue.

x Epithelial atrophy

x Variable number of chronic inflammatory cells 63.

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Group 2- (CONTROLS)

o 10 patients who had clinically normal buccal mucosa, reporting to the out

patient Department of Oral and Maxillofacial Surgery for removal of impacted

third molar constituted the control group.

o Inclusion Criteria-

They had no habit of smoking, alcohol consumption or chewing

arecanut.

They were apparently healthy with no systemic disorders.

They were not on any medications.

Methodology

1. A detailed case history including age, sex, occupation, past medical & dental history,

history of habits, drugs and trauma were recorded.

2. General examination and intra oral examination was done.

3. Biopsy was done in both cases and controls.

a. Group 1- The site for biopsy was the region of maximum palpable fibrotic

bands in the buccal mucosa.

b. Group 2 - Normal buccal mucosa adjacent to the site of extraction of impacted

3rd molar was taken for biopsy.

c. Incisional biopsy of sufficient width and depth to ensure inclusion of

connective tissue was taken from buccal mucosa.

4. The tissue taken was immediately transferred to 10 % buffered formalin.

5. After adequate fixation, tissues were embedded in paraffin.

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6. From the paraffin embedded blocks 5 micron thick, sections were cut and used for

routine hematoxylin and eosin (H & E) staining and immunohistochemical (IHC)

staining.

HEMATOXYLIN & EOSIN STAINING

Reagents

x Harry’s hematoxylin

x 1% acid alcohol

x Eosin

Procedure

x The slides were dewaxed in xylene and hydrated through graded alcohol to water.

x The sections on the slides were flooded with Harry’s hematoxylin for 5 minutes.

x The slides were washed in running tap water for 5 minutes.

x The slides were differentiated in 1% acid alcohol for 5 minutes.

x The slides were washed well in running tap water for 5 minutes.

x The tissue sections on the slides were then stained in Eosin for 30 seconds.

x The slides were washed in running tap water for 1 minute.

x The slides were then dehydrated through alcohol, cleared, mounted and viewed under

light microscope.

IMMUNOHISTOCHEMISTRY (IHC)

Armamentarium

x Microtome

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x Autoclave

x Hot air oven

x Slide warmer

x Couplin jars

x Measuring jar

x Weighing machine

x APES coated slides

x Slide carrier

x Aluminium foil

x Micro-pipettes

x Toothed forceps

x Electronic timer

x Beakers

x Rectangular steel tray with glass rods

x Sterile gauze

x Cover-slips

x Light microscope

Reagents used

1. conc. HCl

2. Laxbro soln

3. APES ( 3 amino propyl tri ethoxy silane)

4. Acetone

5. Tri-sodium citrate buffer

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6. Tri-phosphate buffer saline (TBS)

7. 3% H2O2

8. Deionized distilled water

9. Haematoxylin

10. Absolute alcohol

11. Xylene

Antibodies used

1. Primary antibody

Anti-MMP-1 Mouse Monoclonal Antibody, Clone: 41-1E5; Isotype: IgG2a

(Calbiochem – Merck International EMD Biosciences, Cat. No. IM35L)

2. Secondary antibody

IHC Select Immunoperoxidase Secondary Detection System.

(Chemicon International, Cat. No. DAB150)

IHC Procedure

Pretreatment of the slides

x The slides were first washed in tap water for few minutes

x The slides were then soaked in detergent solution for 1 hour

x After 1 hour, each slide was brushed individually using the detergent solution and

were transferred to distilled water.

x The slides were washed in two changes of distilled water.

x The slides were washed in autoclaved distilled water.

x The slides were immersed in 1 N HCL (100 ml HCl in 900 ml distilled water)

overnight.

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x The following day slides were taken out of acid and washed in two changes of

autoclaved distilled water.

x All the slides were then transferred to slide trays, wrapped in aluminium foil and

baked in hot air oven for 4 hours at 180 degrees centigrade.

APES (3 Amino propyl tri ethoxy silane) coating

Slides first dipped in couplin jar containing acetone for 2 minutes

Dipped in APES for 5 minutes

Dipped in two changes of distilled water for 2 minutes each

Slides left to dry

Preparation of paraffin sections

After the slides were dry, tissue section of 5 micron thickness were made in a rotary

manual microtome. The ribbons of tissue section were transferred onto the APES coated slide

from the tissue float both such that two tissue bits come on to the slide with a gap in between.

One of the tissue sections was labeled positive (P) and the other negative (N).

Procedure

The slides with tissue sections were treated with three changes of xylene to

remove paraffin wax. They were put in descending grades of alcohol and then rehydrated

with water. Slides were then treated with 5% hydrogen peroxide for 20 minutes to quench

endogenous peroxidase activity of cells that would otherwise result in non – specific staining.

The slides were then put in two changes of distilled water. Then the slides were transferred to

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citrate buffer and autoclaved for antigen retrieval at 15 lbs pressure for 15 minutes. The

slides were then washed in two changes of distilled water. Then the slides were dipped in 3

changes of Tri-phosphate buffer saline (TBS) for 5 minutes each. The slides were then wiped

carefully without touching the tissue section with gauze to remove excess TBS. Circles were

drawn around the tissues, so that the antibodies added later on do not spread and are

restricted to the circle. Blocking reagent was added to both the specimens and incubated for 5

minutes. The sections taken out were washed in three changes of cold TBS for 5 minutes in

each. Then the slides were wiped carefully without touching the tissue section to remove

excess TBS. The primary antibody, Anti-MMP-1 Mouse Monoclonal Antibody, 1:40 dilution

was added to P tissue on the slide and then to the N TBS was added. The slides were

incubated for 1 hour. The sections taken out were washed in three changes of cold TBS for 5

minutes in each to remove the excess antibody. Then the slides were wiped carefully without

touching the tissue section to remove excess TBS. Then a drop of biotnylated link from

secondary antibody kit, was added on both the sections and the slides were incubated for 20

minutes. Later slides were washed in three changes of cold TBS for 5 minutes in each. The

slides were wiped carefully without touching the tissue section to remove excess TBS. Then

a drop of Streptavidin- Horse Radish Peroxidase (HRP) from the secondary antibody kit was

added on both the sections and the slides were incubated for 10 minutes. The sections were

washed in 3 changes of cold TBS for 5 minutes in each. Then the slides were wiped carefully

without touching the tissue section to remove excess TBS. Then a drop of freshly prepared

DAB (3’ – Diaminobenzidine Tetra Hydrochloride – a substrate chromogen) was added on

both sections. Slides were then washed in running distilled water to remove excess DAB and

counter stained with hematoxylin. The slides were placed in a tray with tap water for bluing.

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Then the slides were transferred to 70% alcohol, 100% alcohol and two changes of xylene.

The tissue sections were mounted with DPX. The slides were then observed under the

microscope. Throughout the procedure care was taken not to dry the tissues.

Positive Control

Breast carcinoma specimen tissues were fixed, processed, embedded, sectioned and

stained in same manner and used as positive control. One positive control tissue slide was

included for each batch of staining.

Observations

The mounted slides were viewed under the light microscope. The positively stained

MMP-1 cells take up brown color within the cytoplasm. The intensity was evaluated and

graded using the staining intensity of the breast carcinoma tissues (positive control) as:

¾ (-) negative

¾ (+) mild

¾ (++) moderate

¾ (+++) intense staining

(Photomicrograph 1, 2)

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IHC PROCEDURE FLOW CHART

APES coated slides with 2 paraffin embedded tissues

Placed in xylene thrice (5 minutes each)

Placed in 100% isopropanol (5minutes)

Placed in 70% isopropanol (5minutes)

Washed in distilled water thrice (5 minutes each)

Placed in 3% hydrogen peroxide (20 minutes)

Washed in distilled water thrice (5 minutes each)

Kept in citrate buffer at pH 6 and autoclaved at 210C at 121psi for Antigen retrieval

and bench cooled for 40 minutes

Washed in TBS thrice (5 minutes each)

Blocking reagent added to both specimens and incubated at room temperature in an enclosed

hydrated container (5 minutes)

Washed in TBS thrice (5 minutes each)

Primary antibody added to study specimen and incubated in an enclosed hydrated container

(1 hour)

Washed in TBS thrice (5 minutes each)

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Secondary antibody added and incubated in an enclosed hydrated container

(20 minutes)

Washed in TBS thrice (5 minutes each)

Streptavidin HRP added and incubated in an enclosed hydrated container

(10 minutes)

Washed in TBS thrice (5 minutes each)

DAB added and incubated in an enclosed hydrated container (5 minutes)

Washed in TBS thrice (5 minutes each)

Stained with haematoxylin (1 minute)

Washed in tap water

Placed in ammonia (1 minute)

Washed in tap water

Placed in 70% isopropanol (5 minute)

Placed in 100% isopropanol (5 minute)

Placed in xylene twice (5 minutes each)

Slides were mounted using DPX

Slides were observed under the LM and graded

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Statistical Analysis

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Data entry was performed and analysed with the aid of Statistical Package for Social

Sciences (SPSS 10.0.5 version). Descriptive analysis was presented for all the variables. Chi-

square test of association was used to find out the difference in expression of MMP-1

between oral submucous fibrosis (OSF), normal buccal mucosa and among different

histological grading of OSF. A p value < 0.05 was considered significant.

Kappa analysis was performed for measurement of agreement with a second

evaluator to assess inter-observer variation.

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Review of Literature

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ORAL SUBMUCOUS FIBROSIS

Oral submucous fibrosis (OSF) is a chronic, progressive, scarring disease that

predominantly affects the people of South-East Asian origin. It is considered to be a

premalignant stage of oral cancer, affecting predominantly Indians and other Asians 33.

HISTORY

Sushruta (400 BC)69 the ancient Indian medical text-describes a condition termed

“VIDARI” under mouth and throat diseases as progressive narrowing of mouth,

depigmentation of oral mucosa and pain on taking food.” All these are characteristics of

OSF.

Schwartz J (1952)68 while examining five Indian women from Kenya ascribed the

descriptive term “atrophia idiopathica mucosae oris” to this chronic progressive scarring

disease.

Joshi SG (1953)28 an otolaryngolygist from Bombay redesignated the condition as

“oral submucous fibrosis”, implying predominantly its histological nature.

Lal D (1953)34 observed and named the condition as “diffuse oral submucous

fibrosis”.

Rao RV (1954)65 examined and termed the condition as “Idiopathic palatal fibrosis”.

Su IP (1954)76 was the one who called the condition as “Idiopathic scleroderma of

the mouth”.

Behl PN (1957)4 suggested to name the condition as “Sclerosing stomatitis”.

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GEOGRAPHIC DISTRIBUTION

Schwartz J (1952)68 first described the condition among five East African women of

Indian origin.

Lal D and Joshi SG (1953)34 were the first to report a case of oral submucous from

India.

Su IP (1954)76 was the first one to report oral submucous fibrosis among non-Indians

from Taiwan.

Pindborg JJ and Sirsat SM (1966)55 stated that it occurs predominantly in India,

Southeast Asia, the South Pacific Islands and South Africa.

Laskaris G, Bovopoulou O and Nicolis G (1981)35 reported a case of OSF in a 67

year old Greek female.

Canniff JJ (1986)8 were the first to report 27 cases of oral submucous fibrosis from

UK.

Rajendran R (2003)59 reported about a survey of Indian villages and the prevalence

of OSF as 0.2% in Gujarat, 0.4% in Kerala, 0.4% in Andhra Pradesh and 0.07% in Bihar.

Ariyawardana A, Athukorala ADS and Arulanandam A (2006)2 reported the

cases of OSF from Sri Lanka.

ETIOLOGY

Etiological agents proposed in the pathogenesis of OSF are as following:

Arecanut

Arecanut is the endosperm of the fruit of the Areca catechu tree.

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Lal D (1953)3 4 stated that all cases without exception gave history of chewing

“supari” (Hindi word for betel nut).

Paissat DK (1981)51 observed that in the three new cases in his study, all chewed

betelnut. He suggested betelnut as a common denominator in the occurrence of severe

changes in oral mucosa both in India and Papua New Guinea.

Sinor PN, Gupta PC, Murti PR et al (1990)73 confirmed arecanut as the most

important etiologic factor in OSF.

Rajendran R, Radhakrishnan NS, Kartha CC et al (1993)60 stated that chewing

arecanut can be an important etiologic factor of OSF.

Maher R, Lee AJ, Warnakulasuriya KAAS et al (1994)39 inferred that habit of

chewing betel nut is the most important etiologic factor in pathogenesis of OSF.

Murti P R (1995)47 calculated the incidence of OSF from a 10-yr prospective study

and results underscored the usual role of arecanut chewing in OSF.

Chang YC, Tai KW, Li CK et al (1999)9 stated that arecoline is the main alkaloid of

arecanut, which may inhibit fibroblast proliferation and collagen synthesis and thereby cause

OSF.

Rajendran R (2003)5 9 stated that there is compelling evidence to implicate the

habitual chewing of arecanut with development of OSF.

Ranganathan K, Devi MU, Joshua E et al (2004)63 conducted a case-control study

and confirmed a strong association between arecanut use and OSF.

Chilli consumption

Sirsat SM and Khanolkar VR (1962)6 8 studied 85 palatal biopsies from OSF

patients and hypothesized chilli as an etiological agent. They reported that “capsaicin”, one

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of the ingredients of chilli, produced mild changes in the connective tissue in wistar rats

similar to oral submucous fibrosis.

Laskaris G (1981)35 conducted an experimental study on wistar rats using capsaicin

and inferred that capsaicin was capable of evoking a limited connective tissue response in an

unimpaired animal system.

Chiu CJ, Chang ML, Chiang CP et al (2002)13 conducted a case control study betel

quid chewers with OSF and betel quid chewers without OSF, and found no association

between OSF and chilli\ hot pepper consumption.

Nutritional deficiency

Pindborg JJ, Chawla TN, Srivastav AN et al (1965)5 4 reported clinical and

pathological changes of OSF in vitamin B and protein deficient animals.

Ramanathan K (1981)57 summarized the evidence for OSF being a mucosal change

secondary to chronic iron and\or vitamin B complex deficiency.

Genetic susceptibility

Canniff JP, Harvey W and Harris M (1986)8 stated that the patients have a genetic

predisposition which renders their oral mucosa susceptible to chronic inflammatory changes

if they chew betel nut.

CLINICAL FEATURES

Paissat DK (1981)51 observed the most initial symptoms as burning sensation in

mouth while eating spicy food followed by vesicle formation, ulceration or recurrent

stomatitis with excessive salivation, defective gustatory sensation and dryness of mouth.

Later there is difficulty in mouth opening, inability to whistle and difficulty in swallowing.

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Canniff JP, Harvey W and Harris M (1986)8 reported the major features of OSF as:

x Burning sensation with hot or spicy food

x Progressive inability to open the mouth

x Sudden onset of inflammation

x Oral ulcerations

x Dry mouth

x Difficulty in mastication, speech and swallowing

x Shrinkage and deformity of uvula

x Depapillation of tongue.

Pindborg JJ (1989)37 clinically divided OSF into 3 stages and the physical findings

vary accordingly, as follows:

x Stage 1: Stomatitis includes erythematous mucosa, vesicles, mucosal ulcers,

melanotic mucosal pigmentation, and mucosal petechia.

x Stage 2: Fibrosis occurs in ruptured vesicles and ulcers when they heal, which is the

hallmark of this stage.

o Early lesions demonstrate blanching of the oral mucosa.

o Older lesions include vertical and circular palpable fibrous bands in the buccal

mucosa and around the mouth opening or lips, resulting in a mottled marble

like appearance of the mucosa because of the vertical, thick, fibrous bands

running in a blanching mucosa. Specific findings include the following:

Reduction in the mouth opening (trismus)

Stiff and small tongue

Blanched and leathery floor of the mouth

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Fibrotic and depigmented gingiva

Rubbery soft palate with decreased mobility

Blanched and atrophic tonsils.

Shrunken budlike uvula.

Sunken cheeks, not commensurate with age or nutritional status.

x Stage 3: Sequelae of OSF are as follows:

o Leukoplakia is precancerous and is found in more than 25% of individuals

with OSF.

o Speech and hearing deficits may occur because of involvement of the tongue

and the eustachian tubes.

Khanna JN and Andrade NN (1995)30 developed a group classification system for the

surgical management of OSF:

¾ Group I: Very early cases.

Common symptom is burning sensation in the mouth.

Acute ulceration and recurrent stomatitis

Not associated with mouth opening limitation.

x Histology:

Fine fibrillar collagen network interspersed with marked edema.

Blood vessels dilated and congested.

Large aggregate of plump, young fibroblasts present with abundant

cytoplasm.

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Inflammatory cells mainly consists of polymorphonuclear leukocytes with few

eosinophils.

Epithelium normal and nonkeratinised.

¾ Group II: Early cases.

Buccal mucosa appears mottled and marble-like.

Widespread sheets of fibrosis palpable.

Patients with an interincisal distance of 26-35 mm.

x Histology:

Juxtaepithelial hyalinization is present.

Collagen present as thickened but separate bundles.

Blood vessels are dilated and congested.

Young fibroblasts seen in moderate number.

Inflammatory cells mainly consists of polymorphonuclear leukocytes with few

eosinophils and occasional plasma cells.

Flattening or shortening of epithelial rete pegs evident with varying degree of

keratinization.

¾ Group III: Moderately advanced cases.

Trismus evident, with an interincisal distance of 15-25 mm.

Buccal mucosa appears pale and firmly attached to underlying tissues.

Atrophy of vermilion border.

Vertical fibrous bands palpable at the soft palate, pterygomandibular raphe

and anterior faucial pillars.

x Histology:

Juxtaepithelial hyalinization is present.

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Thickened collagen bundles faintly discernible, separated by very slight,

residual edema.

Blood vessels, mostly constricted.

Mature fibroblasts with scanty cytoplasm and spindle-shaped nuclei.

Inflammatory exudate consists mainly of lymphocytes and plasma cells.

Epithelium markedly atrophic with loss of rete pegs.

Muscle fibers are seen interspersed with thickened and dense collagen fibers.

¾ Group IVA: Advanced cases.

Trismus is severe with interincisal distance of less than 15 mm.

The fauces is thickened, shortened and firm to palpation.

Uvula is shrunken and appears as small, fibrous bud.

Tongue movements are limited.

On palpation of lips, circular band is felt around entire mouth.

¾ Group IVB: Advanced cases with premalignant and malignant changes.

Leukoplakia or squamous cell carcinoma can be seen.

o Histology:

Collagen hyalinized as a smooth sheet.

Extensive fibrosis obliterated the mucosal blood vessels and eliminated the

melanocytes.

Fibroblasts were markedly absent within the hyalinized zones.

Total loss of epithelial rete pegs.

Mild to moderate atypia present.

Extensive degeneration of muscle fibers is evident.

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Lai DR, Chen HR and Huang YL et al (1995)33 classified OSF on the basis of mouth

opening as:

¾ Group A: Mouth opening greater than 35mm

¾ Group B: Mouth opening between 30-35mm

¾ Group C: Mouth opening between 20-30mm

¾ Group D: Mouth opening less than 20mm

Ranganathan K et al (2001)6 4 conducted a baseline study on the mouth opening

parameters of normal patients and divided the OSF patients as-

¾ Group I: Only symptoms. No restricted mouth opening.

¾ Group II: Limited mouth opening. 20mm and above.

¾ Group III: Mouth opening less than 20 mm.

¾ Group IV: OSF advanced with limited mouth opening.

Precancerous or cancerous changes seen throughout the mucosa.

Rajendran R (2003)59 reported the clinical features as follows:

¾ Early OSF: Burning sensation in the mouth. Blisters especially on the palatal

ulceration or recurrent generalized inflammation of oral mucosa, excessive salivation,

defective gustatory sensation and dryness of the mouth.

¾ Advanced OSF: Blanched and slightly opaque mucosa, fibrous bands in buccal

mucosa running in vertical direction. Palate and the faucial pillars are the areas first

involved. Gradual impairment of tongue movement occurs with difficulty in mouth

opening.

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PATHOPHYSIOLOGY

The pathogenesis of the disease is not well established, but is believed to have a

multifactorial cause. A number of factors trigger the disease process by causing a

juxtaepithelial inflammatory reaction in the oral mucosa29.

Canniff JP and Harvey W (1986)8 found that arecoline, an active alkaloid found in

betel nuts, stimulates fibroblasts to increase production of collagen by 150%.

Harvey W, Scutt A, Meghji S et al (1986)22 indicated that fibroblasts metabolize

arecoline to arecaidine, which may enhance fibroblast proliferation and thus increase

collagen. They proposed that flavanoid, catechin and tannin in betel nuts cause collagen

fibers to cross-link, making them less susceptible to collagenase. They found that OSF

remains active even after cessation of the chewing habit, suggesting that components of the

arecanut initiate OSF and then affect gene expression in the fibroblasts, which then produce

greater amounts of normal collagen.

Sheih TY and Yang JF (1992)7 2 conducted a study to examine the relationship

between the fibrosis and collagenase activity. Collagenase activity was determined by using

soluble 14C-glycine-labeled collagen as a substrate in a solution incubated for 30 hours at 35

degrees C. The results showed that the collagenase activity of OSF was much lower than that

of normal oral mucosa. They therefore suggested decreased secretion of collagenase as a

possible etiology for OSF.

Kuo MYP (1995)32 mentioned the production of collagen with more stable structure

(collagen type 1 trimer) as the reason for OSF.

Trivedy C (1999)79 suggested that upregulation of lysyl oxide may be an important

factor in the pathogenesis of OSF.

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Tsai CC (1999)60 reported deficiency in collagen phagocytosis by OSF fibroblasts as

the possible cause of OSF.

Chiu CJ, Chang ML, Chiang CP et al (2002)13 reported that collagen fibers form a

three-dimensional scaffolding by combining cross-linked collagen molecules with other

extracellular matrix components. The terminal regions of each collagen molecule consist of

terminal peptides, which contain the sites of intra- and intermolecular cross-links. These

areas are resistant to attack by collagenases but can be attacked by a number of other serine

and cysteine proteinases. Collagenase activity has been found to be lower in OSF than in the

normal oral mucosa. This evidence implies that OSF may be considered a collagen-metabolic

disorder resulting from alkaloid exposure and individual variation in collagen metabolism.

Shieh DH, Chiang LC, Lee CH et al (2003)7 1 mentioned that increased mRNA

expression of TIMP-1 in buccal mucosa fibroblast by arecoline and safrole has a possible

role in the pathogenesis for oral submucous fibrosis.

Rajendran R (2003)5 9 associated genetic susceptibility with OSF because raised

frequencies of HLA-A10, B7 and DR3 were found in OSF patients compared to the normal

subjects.

Rajalalitha P and Vali S (2005)57 reviewed the autoregulatory pathway of TGF-ȕ,

which acts as the main trigger for both increased collagen production and decrease

degradation pathways, causing collagen deposition in the oral tissue, leading to fibrosis.

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HISTOPATHOLOGICAL FEATURES

Pindborg JJ and Sirsat SM (1966)55 reported juxta-epithelial chronic inflammatory

cell infiltration, strong fibroblastic response in lamina propria and thickened collagen bundles

in the connective tissue. Secondary to connective tissue changes there is an atrophic

epithelium, intercellular edema, basal cell hyperplasia, keratinisation and epithelial dysplasia.

They gave a histological grading for OSF:

o Very early stage: Finely fibrillar collagen dispersed with marked edema.

Plump young fibroblasts containing abundant cytoplasm. Blood vessels are

dilated and congested. Inflammatory cells, mainly polymorphonuclear

leukocytes with occasional eosinophils are found.

o Early stage: Juxtaepithelial area shows early hyalinization. Collagen still in

separate thick bundles. Moderate number of plump young fibroblasts is

present. Dilated and congested blood vessels are present. Inflammatory cells

are primarily lymphocytes, eosinophils and occasional plasma cells are

present.

o Moderately Advanced stage: Collagen is moderately hyalinised. Thickened

collagen bundles are separated by slight residual edema. Fibroblastic response

is less marked. Blood vessels are either normal or compressed. Inflammatory

exudate consists of lymphocytes and plasma cells.

o Advanced stage: Collagen is completely hyalinised. Smooth sheets with no

separate bundles of collagen are seen. Edema is absent. Hyalinised area is

devoid of fibroblasts. Blood vessels are completely obliterated or narrowed.

Inflammatory cells found are lymphocytes and plasma cells.

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Canniff JP, Harvey W and Harris M (1986)8 analyzed 44 biopsies and reported

an atrophic epithelium with mild atypia, flattened epidermal\dermal junction and an

accumulation of collagen beneath the basement membrane.

Van Wyk CW, Seedat HA and Phillips VM (1990)85 examined the ultrastructural

features of the collagen in the moderate and advanced examples of OSF and found no

obvious abnormality of the collagen fibrils. They concluded that although there is an

excessive increase of collagen, especially Type I in OSF, the fibrils are still morphologically

normal.

Rajendran R, Radhakrishnan NS and Kartha CC (1993)6 0 observed thin

epithelium with ortho or parakeratinisation, with mild atypia. Rete ridges were blunt or

absent. Well defined basal membrane zone, with widened lamina propria with juxtaepithelial

fibrosis was seen. Connective tissue with chronic inflammatory cells was present. Small

arteries and fibrous tissue thick walled blood vessels were seen. Subepithelial fibrosis

extended deeper down into the muscle bundles. Extensive fatty infiltration was seen.

Chiu CJ , Chang ML, Chiang CP et a l (2002)13 stated that the main

histopathological characteristic of OSF is the deposition of collagen in the oral submucosa. It

has been found that alkaloid exposure of buccal mucosal fibroblasts may result in the

accumulation of collagen.

Rajendran R, Sunil, Twinkle SP et al (2004)6 0 reported a reduction in overall

thickness of the epithelium due to a decline in the number of mean cellular layers rather than

due to a diminution of the size of the cells in case of OSF.

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Utsunomiya H, Tilakratne WM, Oshiro K et al (2005)84 histologically divided OSF

as:

o Early stage: Large number of lymphocytes in subepithelial zone along with

myxoedematous changes.

o Intermediate stage: Granulation changes close to the muscle layer and hyalinization

appears in subepithelial zone where blood vessels are compressed by fibrous bundles.

Reduced inflammatory cells in the subepithelial layer.

o Advanced stage: Inflammatory cell infiltrate hardly seen. Number of blood vessels

dramatically small in subepithelial zone. Marked fibrosis areas with hyaline changes

extending from subepithelial to superficial muscle layers. Atrophic, degenerative

changes start in muscle fibers.

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MATRIX METALLOPROTEINASES

Birkedal-Hansen H (1987)86 stated that the matrix metalloproteinases (MMPs) are a

family of enzymes secreted by resident and inflammatory cells that are collectively capable

of degrading most or all of the constituent macromolecules of the extracellular matrix.

HISTORY

Gross J and Lapiere CM (1962)19 produced a diffusible collagenolytic factor

operating at neutral pH and physiologic temperature on undernatured collagen, in Rana

Catesbiana tadpole’s tail fin tissues. Cultivation of bullfrog tadpole tissues on thermally

reconstituted neutral calf and guinea pig skin collagen gels resulted in degradation of the

substrate to dialyzable collagen peptides. They proposed that the experimental system used

by them could be developed into a quantitative assay for collagenolytic activity in small

tissue fragments.

Gross J and Nagai Y (1965)20 incubated thin strips of sterile living tadpole tail fin or

back skin on filter paper discs floating in amphibian Tyrode solution at 37°C for several

days. They observed that relatively large amount of collagenolytic enzyme appears in the

culture media. It had pH optimum 6-8, loses activity at 50-60°C after 10minutes and is

inhibited by ethylene diamine tetra acetic acid (EDTA) and cysteine. This enzyme, isolated

from tadpole tissue culture medium, cleaved native collagen molecules into two fragments

(one three-quarters and the other one-quarter the length of the molecule) at neutral pH and

physiologic temperatures without disrupting the helical structure of either of the two parts.

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Lazarus GS, Brown RS and Daniels JR (1968)3 5 suggested a mechanism for

collagen degradation mediated by human granulocytic leukocytes. A specific collagenase,

which is extractable from human granulocytes, was partially purified chromatography. This

collagenolytic enzyme was operative at physiological pH and was inhibited by ethylene

diamine tetra acetic acid (EDTA), cysteine and reduced glutathione but not by human serum.

The enzyme cleaved the collagen molecule into two specific products, without loss of helical

conformation. Experiments with crude extracts from granulocytes suggested that the specific

products of granulocyte collagenase activity were then degraded by other proteases present in

the human granulocyte.

Harper E, Bloch KJ and Gross J (1971)21 identified the latent form of tadpole

collagenase, a zymogen which could be activated to its active form by an unknown factor.

Werb Z and Burleigh MC (1974)88 found that the latent collagenase in rabbit

fibroblast can be activated by organomercurials. Extracts of rabbit skin and synovium in

tissue culture secreted a specific collagenase into their culture media. Primary cultures of

fibroblast-like cells, which were obtained from these tissues and maintained in culture for up

to 14 subculture passages, also secreted high activities of a specific collagenase into serum-

free culture medium. Secretion of enzyme activity from the cell monolayer was at constant

rate for over 100 hours and continued for up to 8 days in serum-free culture medium. The

enzymic activity released was proportional to the number of cells in the monolayer. The

fibroblast collagenase was maximally active between pH 7 and 8. At 24°C the collagenase

decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved

into three-quarters and one-quarter length fragments as demonstrated by electron microscopy

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of segment-long-spacing crystallites and by polyacrylamide-gel electrophoresis of the

denatured products.

Werb Z, Banda MJ and Jones PA (1980)87 investigated the neutral and lysosomal

enzymes of mouse macrophages and indicated that the macrophages at inflammatory sites

contain and secrete proteolytic enzymes that could degrade the extracellular matrix.

Chin JR, Murphy G and Werb Z (1985)11 identified stromelysin, a connective

tissue degrading enzyme in parallel to collagenase.

Okada Y, Nagase H, Harris ED (1986)4 9 proposed the nomenclature Matrix

Metalloproteinases with the abbreviation MMP.

Murphy G, Cockett MI, Stephens PE et al (1987)46 identified metalloproteinase,

Stromelysin which can activate pro-collagenase.

Hasty KA, Stricklin GP, Hibbs MS et al (1987)23 compared the immunologic cross-

reactivity of human neutrophil and skin fibroblast collagenases, utilizing polyclonal antisera

prepared to purify enzymes. Polyclonal antisera from rabbits immunized with neutrophil

collagenase recognized fibroblast collagenase, as well as the neutrophil enzyme, when

analyzed by immunoblot techniques. The cross-reactive epitopes constituted a major

proportion of the antibody population, as shown by competitive inhibition of enzyme-linked

immunosorbent assay; 50% of the antibody to neutrophil collagenase was inhibited by skin

collagenase. Paradoxically, antisera to fibroblast collagenase failed to recognize the

neutrophil enzyme, either by immunoblot techniques or competitive inhibition enzyme-

linked immunosorbent assay, an observation which supports the notion that there are unique

immunodominant epitopes. The cross-reactivity with skin fibroblast collagenase shown by

the neutrophil antibody suggested a conservation of epitopes between collagenases of

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different cellular origins. They opined that presence of epitopes unique for each enzyme

could lead to a feasible approach for investigating the differential contribution of various cell

types to collagenolytic activity in inflamed tissues.

Muller D, Quantin B, Gesnel MC et al (1988)45 reported that the collagenase gene

family in humans consists of at least four members who have several common structural

features, including an N terminal, a C terminal with hemopexin like domain, and a Zn

binding site.

William GSS (1996)70 identified a new member of MMP inhibitor family, tissue

inhibitor of MMPs (TIMP) -2.

Woessner FJ (1991)90 gave the recommended nomenclature and glossary of MMPs,

depending on their substrate specificity and broadly classified them as collagenase, gelatinase

and stromelysin. He proposed the name “Matrixin” for this family of proteases at the First

International Symposium on Matrix Metalloproteinases held at Destin, Florida, USA in Sept

1989. He listed the following as the chief characteristics of MMPs:

1. The catalytic mechanism depends on zinc at the active center.

2. The proteinases are secreted in zymogen form.

3. The zymogens can be activated by proteinases or by organomercurials.

4. The cDNA sequences all show homology to that of collagenase.

5. The enzymes cleave one or more components of the extracellular matrix.

6. Activity is inhibited by tissue inhibitor of metalloproteinases (TIMPs).

Pavloff N, Staskus PW, Kishnani NS et al (1992)52 identified the third member of

TIMP family from chick embryo fibroblast.

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Sorsa T and Salo T (1994)75 showed that MMP-2 and MMP-9 were secreted by

stromal fibroblast during human mucosal wound healing.

Massova I, Kotra LP, Fridman R et al (1998)42 suggest that MMPs may be even

more ancient than previously thought, and could be traced before the emergence of

vertebrates from invertebrates; since the discovery of a similar metal-containing enzyme

from Bacteroides fragilis, which has been around for more than 3.5 billion years. They

reviewed that a total of 66 MMPs belonging to 23 distinct sub-groups have been sequenced

to date, of which 17 have been sequenced from humans, three from plants, three from

nematode Caenorhabditis elegans, and one each from Drosophila fly, sea urchin, hydra and

green algae. It is not clear how many MMPs exist in nature; many more are yet to be

discovered and sequenced.

Overall CM, King AE, Sam DK et al (1999)50 identified the TIMP-2 binding site on

hemopexin carboxyl domain of MMP-2.

Rao VH, Singh RK, Finnell RH et al (1999)66 listed the following as the

distinguishing characteristics of MMPs:

1. They are proteinases that degrade at least one component of the extracellular matrix.

2. They contain a zinc ion and are inhibited by chelating agents.

3. They are secreted in a latent form, requiring activation for proteolytic activity.

4. They are inhibited by TIMPs.

5. They share common amino acid sequences as revealed by molecular cloning.

Sorsa T, Salo T, Teronen O et al (2001)75 did “in vitro” studies on rat teeth and

showed that MMP inhibition significantly down-regulated dentinal caries lesion progression.

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Chau I, Rigg A and Cunningham D (2003)10 reported that 20 MMPs have been

identified and these endopeptidases enzyme function at physiological temperature and pH,

and are either secreted or are membrane bound.

Mandal M, Mandal A, Das S et al (2003)40 reported that MMPs comprise of a large

family of endopeptidases, that share common structural and functional elements but are

products of a different gene.

Mantyla P, Kinane DF, Tikanoja S et al (2003)41 developed a sensitive, specific,

rapid and practical immunological chair-side dip-stick test for MMP-8 in gingival crevicular

fluid (GCF) and peri-implant sulcular fluid. The MMP-8 test stick is based on the

immunochromatography principle that uses two monoclonal antibodies specific for different

epitopes of MMP-8. One is immobilized into nitrocellulose membrane to form a catching

zone and other onto blue latex particles. This test can be performed by the dentist without

specific equipments and measures the GCF MMP-8 level in 5minutes. It differentiates

healthy and gingivitis sites from periodontitis sites.

Uitto VJ, Overall CM and McCulloch C (2003)83 opined that MMPs are often up-

regulated in groups forming activation cascades both in inflammatory and malignant

diseases.

Sorsa T, Tjaderhane L and Salo T (2004)75 reviewed the presence, activity,

function and regulation of MMPs in healthy and diseased oral tissues. They suggested the

role of collagenases, especially MMP-8 in periodontitis and peri-implantitis, as example for

unwanted tissue destruction related to increased presence and activity of MMPs at the site of

destruction. Evidences were also brought forward for involvement of MMP in other diseases

as dental caries and oral cancer.

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Ylipalosaari M, Thomas GJ, Nystrom M et al (2005)92 using beta6-transfected and

control OSCC cells demonstrated that alphavbeta6 integrin down-regulates MMP-13

expression at both mRNA and protein level. Although expressing less MMP-13, beta6-

transfected cells have similar collagenolytic activity as control cells and invade at similar

levels through type I collagen. Growth of the tumour cells in organotypic culture and

confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6

expression. They suggested that alphavbeta6 down-regulates MMP-13 expression in oral

squamous cell carcinoma (OSCC) cells and that MMP-13 is not essential for the degradation

of type I collagen by OSCC cells.

STRUCTURE (Annexure 1-3)

MMPs are spherical strucWXUHV�FRPSRVHG�PDLQO\�RI�WZLVWHG�ILYH�VWUDQGHG�ȕ�VKHHWV�DQG�

WKUHH�Į�KHOLFHV��ZLWK�WZR�ELQGLQJ�VLWHV�HDFK�IRU�FDOFLXP�DQG�]LQF��7KH�VWUXFWXUDO�]LQF�IRUPV�D�

topknot for the sphere and is attached via three histidines and one asparagine to the catalytic

domain.

Mostly all the MMPs share a basic structure. This consists of-

1. a signal peptide \ prepeptide domain

2. a amino terminal propeptide domain that has to be cleaved to ensure enzyme

activity

3. a catalytic domain

4. a hinge \ linker domain

5. a C-terminal \ hemopexin domain

6. a trans membrane domain

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COPY

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1. Signal peptide domain-

It is rich in hydrophobic amino acids and directs MMP synthesis inside the cell. It is

removed before MMPs are secreted.

2. Propeptide domain-

The propeptide has a characteristic egg-like shape and contains a conserved sequence