Post on 29-Jan-2021
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ACKNOWLEDGEMENT It gives me great pleasure to express my sincere-most tribute and heart-felt appreciation towards the
following people for their invaluable help and support
DDrr.. AAHHMMEEDD MMUUJJIIBB BB..RR.. PPRROOFFEESSSSOORR AANNDD HHEEAADD,, DDeeppaarrttmmeenntt ooff OOrraall PPaatthhoollooggyy && MMiiccrroobbiioollooggyy BBaappuujjii DDeennttaall CCoolllleeggee aanndd HHoossppiittaall,, DDaavvaannggeerree..
DDrr.. KK.. SSAADDAASSHHIIVVAA SSHHEETTTTYY PPRRIINNCCIIPPAALL,, BBaappuujjii DDeennttaall CCoolllleeggee aanndd HHoossppiittaall,, DDaavvaannggeerree.. DDrr.. VVIIKKRRAAMM SS.. AAMMBBEERRKKAARR AASSSSIISSTTAANNTT PPRROOFFEESSSSOORR,, DDeeppaarrttmmeenntt ooff OOrraall PPaatthhoollooggyy && MMiiccrroobbiioollooggyy BBaappuujjii DDeennttaall CCoolllleeggee aanndd HHoossppiittaall,, DDaavvaannggeerree..
DDrr.. LL.. AASSHHOOKK,, PPrrooffeessssoorr && HHeeaadd,, DDeepptt.. ooff OOrraall MMeeddiicciinnee aanndd RRaaddiioollooggyy BBaappuujjii DDeennttaall CCoolllleeggee aanndd HHoossppiittaall,, DDaavvaannggeerree..
DDrr.. KK.. KKIIRRAANN KKUUMMAARR DDeepptt.. ooff OOrraall PPaatthhoollooggyy && MMiiccrroobbiioollooggyy,, SS..DD..MM.. DDeennttaall CCoolllleeggee,, DDhhaarrwwaadd..
SSppeecciiaall tthhaannkkss ttoo DDrr.. SSHHUUBBHHAADDAA SS.. BBAADDVVEE
MMYY CCOOLLLLEEAAGGUUEESS DDrr.. SSYYEEDD MMUUKKIITTHH DDrr.. RRAAHHUULL,, DDrr.. SSHHEELLLLYY DDrr..NNIIDDHHII,,DDrr..KKIIRRAANN DDrr..RRAASSHHMMII,, DDrr..NNEEEETTHHUU && DDrr.. PPOOOORRNNIIMMAA,, DDrr..PPRRAADDEEEEPP,, DDrr.. SS..MMAANNII DDrr.. GGOOKKUULL,, DDrr.. AASSHHWWIINN && DDHHRRIITTIIMMAANN MMrr.. PPRRAAKKAASSHH MM..KK.. SSEENNIIOORR LLAABB TTEECCHHNNIICCIIAANN.. MMrrss.. SSHHEERRII TTHHOOMMAASS SSrrii.. NNEEEELLAAPPPPAA,, SSmmtt.. VVIIJJAAYYAAMMMMAA DDeeppaarrttmmeenntt ooff OOrraall PPaatthhoollooggyy && MMiiccrroobbiioollooggyy MMrr.. DD..KK.. SSAANNGGAAMM BBIIOO--SSTTAATTIISSTTIICCIIAANN,,
MMrr.. SSUURREENNDDRRAA SSUURRYYAAWWAANNSSHHII DDYYNNAA CCOOMMPPUUTTEERRSS,, DDAAVVAANNGGEERREE..
And my FAMILY that stood by me to accomplish this venture in countless ways.
Dr. Diwakar Gajendra Mallappa
LIST OF ABBREVIATIONS USED
BMP1 Bone morphogenetic protein 1 COL Collagen EGF Epidermal Growth Factor ELISA Enzyme linked Immunosorbent Assay FGF Fibroblast Growth Factor H&E Haematoxylin and Eosin HLA Human Leukocyte Antigen HP Histopathological grade IFN γ Interferon Gamma IGF Insulin like Growth Factor IL Interleukin LOX Lysyl oxidase MHC Major Histocompatibility Complex mm Millimeter MMP Matrix Metalloproteinase MO Mouth opening mRNA Messenger Ribonucleic Acid MTS Masson trichrome stain OSF Oral Submucous Fibrosis PAI Plasminogen activator inhibitor PCP Pro-collagen C- proteinase PDGF Platelet Derived Growth Factor PGE2 Prostaglandin E2 PNP Pro-collagen N- proteinase TGF Transforming Growth Factor TIMP Tissue inhibitor of matrix metalloproteinase TNF Tumor Necrosis Factor V & G Van-Gieson Stain
VI
ABSTRACT
Background: Oral submucous fibrosis is a common premalignant condition of the mouth
with areca nut chewing habit in Asian subcontinent. It is characterized by restricted
mouth opening, tongue protrusion and cheek flexibility. There are few reports to correlate
the clinical stage with histopathological grade and muscle alterations in Oral submucous
fibrosis. The present study was undertaken to describe the extent of fibrosis and to
correlate it with the mouth opening in Oral submucous fibrosis patients and also to see
the muscular changes.
Method: A total of 50 cases were selected for this study which were clinically diagnosed as
oral submucous fibrosis in the Department of Oral Medicine and Radiology and confirmed
by Histopathology in Department of Oral Pathology, Bapuji Dental College & Hospital.
The histopathological fibrosis and muscle degeneration was compared with clinical mouth
opening.
Results: In Group I, 4(16%), 3(12%) and 18(72%) were in grade I, II, III, respectively
and in Group II, 4(16%),5(20%),16(64%)were in grade I,II, III respectively. In Group I,
18 (72%) patients showed muscle degenerative changes and in 7(28%) it was absent. In
group II, 16 (64%) patients showed muscle degenerative changes and in 9 (36%) it was
absent.
Conclusion: The statistical analysis showed that there is no correlation between stage of
mouth opening and grading of fibrosis as indicated by the insignificant `p' value. This
implies that the cause of reduction of MO in OSF is multifactorial and is determined by
factors like site, extent of fibrosis, regional anatomic variation and tone of the muscle, the
neuromuscular coordination, anatomical and physiological integrity of the underlying
oral musculature.
Keywords: Oral submucous fibrosis; muscle alteration; fibrosis in OSF.
VII
CONTENTS
Page No.
1. Introduction 01
2. Objectives 04
3. Review of Literature 05
4. Methodology 37
5. Results and Observations 46
6. Discussion 57
7. Conclusion 63
8. Summary 64
9. Bibliography 67
10. Annexures 75
VIII
LIST OF TABLES
Sl. No Tables
Page No.
1 Distribution of study subjects according to age, gender and mouth opening 46
2 Correlation between the clinical mouth opening and fibrosis on histopathology 47
3 Showing correlation between mouth opening and muscle degeneration 48
4 Histopathological grade and mean mouth opening 48
5 Histopathological features 49
IX
LIST OF FIGURES
Sl. No Figures
Page No
1 Soft tissue microtome 44
2 Reagents used for H & E 44
3 Reagents used for V & G 44
4 Reagents used for MTS 45
5 DMRB Leica research microscope with detachable Sony camera 45
6 Photograph showing measurements of mouth opening 52
7. Photograph showing blanching of right buccal mucosa 52
8 Photograph showing blanching of lower labial mucosa 52
9. Photomicrograph showing Grade I of OSF (H&E) 53
10 Photomicrograph showing Grade I of OSF (V&G) 53
11 Photomicrograph showing Grade I of OSF (MTS) 53
12 Photomicrograph showing Grade II of OSF (H&E) 54
13 Photomicrograph showing Grade II of OSF (V&G) 54
14 Photomicrograph showing Grade II of OSF (MTS) 54
15 Photomicrograph showing Grade III of OSF (H&E) 55
16 Photomicrograph showing Grade III of OSF (V&G) 55
17 Photomicrograph showing Grade III of OSF (MTS) 55
18 Photomicrograph showing juxta epithelial hyalinization (V&G) 56
19 Photomicrograph showing juxta epithelial hyalinization (MTS) 56
20 Photomicrograph showing muscle atrophy (MTS) 56
X
LIST OF GRAPHS
Sl.No Figures Page No.
1. Histogram illustrating correlation between the clinical mouth opening and the histopathological grades. 50
2. Histogram illustrating correlation between the mean mouth opening and fibrosis 50
3. Histogram illustrating correlation between mouth opening and muscle degeneration 51
XI
IInntrtroodduuccttiioonn
Introduction
INTRODUCTION
The journey of a thousand miles begins with a single step. Disease – has no
boundaries. In fact, there is nothing so insatiably ‘international’ as disease. The
undeletable dark clouds commemorating the advent of disease has pondered the sanity of
mankind since time immemorial. However, with the glorious dawn of the ‘modern
civilization’ - an era of hope; man has sought a million ways to combat the phenomenon
of disease. The various intensified and sophisticated studies have only related to the
discovery of unbelieving variations in the aura of the disease process. The recognition of
less vigilant or low risk diseases to severe life-threatening ones. Adverse habits
symbolizing addictions of any kind, merely represent man’s unbounded weakness and his
search for secondary whims and fancies to hide his inert weakness and a form of
psychological push to make them move on with life, if not face it then forget about it.
Such adverse habits may follow the ladders of culture, nature, fantasy or mere addiction;
which forms a platform or a magnet of risk for disease to show its synchronous presence.
Oral Submucous Fibrosis (OSF) can be cited as one significant encounter to be put
forward as an appropriate example. Dental and medical practitioners throughout the globe
come across a wide spectrum of oral mucosal lesions in their everyday clinical practice.
OSF – one such entity; which is predominantly seen in the geographical locations
covering entire South-east Asia and the Indian subcontinent, is undoubtedly a habit-
related ‘woe’ in the truest sense.
Dental and Medical practitioners often encounter a wide spectrum of oral mucosal
lesions in their day to day clinical practice. These mucosal lesions vary in nature from
1
Introduction
simple to life threatening ones. Many of these lesions including oral submucous fibrosis
are caused by betel nut and tobacco habits. It is estimated that 47% of Indians aged 15
years and above use tobacco and betel nut in one form or the other. However, the etiology
of oral submucous fibrosis is also thought to be of autoimmune nature similar to
scleroderma.
Oral submucous fibrosis is of concern as it is more prevalent in India affecting
young adults addicted to the above mentioned habits. Although there are regional
variations in the type of areca nut products used in India, the betel quid has been the most
popular and prevalent habit in ancient Indian culture. But in 1980, both areca quid
products such as Pan Masala and Gutkha were introduced in Indian market as
commercial preparations. Since then there has been an increase in the use of Pan Masala
and Gutkha in the younger age groups, which had lead to increased incidence of OSF.
Recently, it has been documented that the habit of chewing Gutkha had gained
considerable popularity among the young men. The rapidly increasing prevalence of this
habit can be judged from the reports that the Indian market for Pan masala and Guthka is
worth 25 billion (US$500 million).1
Oral submucous fibrosis was first described in the Indian medical literature by
Schwartz J. in 1952, as "Atropica idopathica Mucosae Oris.2
Other terminologies which were used by various authors are diffused oral
submucous fibrosis, idiopathic scleroderma of the mouth, idiopathic palatal fibrosis and
sclerosing stomatitis of the mouth". Joshi 1953, from Bombay described the disease for
the first time in India and coined the term Oral submucous fibrosis. This particular
disease progresses with an insidious onset. It predominantly occurs among Indians and
2
Introduction
people of Indian origin living out side India mainly because of habits. Occasionally
occurs in other Asians and sporadically in Europeans. In India the prevalence and
incidence rates are higher in south India, where the incidence of oral cancer is also high.
The possible pre-cancerous nature of submucous fibrosis was mentioned by
Paymaster. In 1956, he observed the development of squamous cell carcinoma in one
third of cases of submucous fibrosis. 2
Physicians in their daily practice often encounter these mucosal fibrotic bands,
leading to mucosal rigidity of varied intensity, which is due to the juxta-epithelial fibrosis
along with atrophy of overlying epithelium and accumulation of hyalinized collagen
beneath the basement membrane.
Oral submucous fibrosis is a common premalignant condition of the mouth with
areca nut chewing habit in Asian subcontinent. It is characterized by restricted mouth
opening, tongue protrusion and cheek flexibility. There are few reports to correlate the
clinical stage to histopathological grade and muscle alterations in Oral submucous
fibrosis. The present study was undertaken to describe the extent of fibrosis and to
correlate this with the mouth opening in Oral submucous fibrosis patient and also to see
the muscle changes. Although the histopathological grading & muscle alterations can be
studied under H& E staining itself, additional techniques of special staining were used in
the present study to check whether these techniques are beneficial in developing an
accurate method for grading and to check the extent of muscular alterations.
3
OObbjjeecctitivveess
Objectives
OOBBJJEECCTTIIVVEESS
1. To study the association between mouth opening and fibrosis.
2. To study degree of alterations involving muscle fibers as disease progresses.
4
RReevviieeww oof f LLititeerraattuurree
CONTENTS
Page No.
1) Historical review 05 2) Definition 06 3) Epidemiology 07 4) Etiology 09 5) Pathogenesis 10 6) Clinical features 22 7) Histopathological features 26 8) OSF and malignant transformation 32 9) Special stains 34
Review of Literature
HISTORICAL REVIEW In ancient medicine Sushrutha described a condition, “Vidari” under mouth and
throat diseases. He noted progressive narrowing of mouth, depigmentation of oral
mucosa, and pain on taking food. These features precisely fit in with the symptomatology
of oral submucous fibrosis.3
Schwartz, for the first time reported a case of “Atrophia idiopathica tropica
mucosae oris” occurring in Indians migrated to East Africa. He had seen five such cases
in ten years, all of them were Indian women in East Africa and described blanching and
stiffness of the oral mucosa, difficulty in mouth opening and inability to tolerate spicy
food.4
This condition was first described in India by Lal and Joshi in 1953. 5
The fibrotic process extending to the lateral wall of pharynx via the faucial pillars
down to the pyriform fossa was described by Rao.6
The first histological four consecutive grades of the OSF was described by
Pindborg JJ, Sirsat SM.2
5
Review of Literature
DEFINITION
Submucous fibrosis is an “insidious, chronic disease affecting any part of oral
cavity and sometimes the pharynx. Although occasionally preceded by and / or associated
with vesicle formation, it is always associated with a juxta-epithelial inflammatory
reaction followed by a fibro-elastic change of the lamina propria, with epithelial atrophy,
leading to stiffness of the oral mucosa and causing trismus and inability to eat”.2
6
Review of Literature
EPIDEMIOLOGY
The geographical distribution of OSF shows confinement to tropical areas
primarily in the Indian subcontinent. There were isolated reports from other countries
where expatriates of Indian origin have settled and contracted the disease. OSF can occur
at any decade, but the majority of patients are between 20 and 40 years of age.7
The disease occurs almost exclusively among Indians, Pakistanis and Burmese,
but sporadic cases have been observed in China, Nepal, Thailand and South Vietnam.
Occasional cases involving Europeans have also been recorded.8
An epidemiological assessment of the prevalence of OSF among Indian villagers,
based on baseline data, recorded a prevalence of 0.2% in Gujarat, 0.4% in Kerala, 0.04%
in Andhra Pradesh and 0.07% in Bihar. The prevalence among 1, 01,761 villagers in the
state of Maharastra was 0.3%. The general female preponderance may be related to
factors like oral habits, deficiency states of iron and vitamin B-complex among many
others prevalent in Indian women.
This condition affects approximately 0.5% (5-million people) of the population in
the Indian subcontinent and following migration from this region, is now a health care
problem in many parts of the world, including the United Kingdom9
The yearly incidence of OSF per 1, 00,000 persons per year was found to be 9 for
males and 20 for females over a 10-year period in the Ernakulam District of Kerala,
South India. 10
7
Review of Literature
The prevalence rate in India varies from 0.2 – 0.5%, with a higher percentage
being found in southern areas of the country. Reports of the sex ratio vary; however, the
majority demonstrates a female predominance. The largest percentages of cases occur
between 20-40 years of age.11
In a population based study in Pakistan, it was reported that the incidence of OSF
in women was almost twice as high as in men.12
Recent epidemiological data indicates that, the number of cases of OSF has raised
rapidly in India from an estimated 2, 50,000 cases in 1980 to 2 million cases in 1993. The
reasons for the rapid increase of the disease are reported to be due to an upsurge in the
popularity of commercially manufactured areca nut preparations (pan masala) in India
and an increased uptake of this habit by young people, due to easy access and marketing
strategies13
A hospital-based cross-sectional study on various habit patterns associated with
OSF was performed in Nagpur over a 5-year period. A total of 1000 OSF cases from 266
418 out patients comprised the study sample. The male-to-female ratio of OSF was 4.9:1.
Occurrence of OSF was at a significant younger age group (
Review of Literature
ETIOLOGY
A number of epidemiological surveys, case series reports, large sized cross
sectional surveys, case control studies, cohort and intervention studies provide over
whelming evidence that arecanut is the main etiological factor for OSF. Most convincing
evidence is derived from case control studies that estimate the odds ratio for arecanut use
among OSF cases and a definite causation of the disease. It has been showed that daily
use appears to be more important than the duration of the habit. 12
A recent case control study has shown that the risk of developing OSF was almost
double for the subjects below 21years of age compared with that for the 21-40 years age
group; The younger group developed features of OSF in 3.5 years whilst the older group
took 6.5 years from the start of the habit. 13
In a recent study, a clear dose dependent relationship was observed for both
frequency and duration of chewing areca nut (without tobacco) in the development of
OSF.11
The severity and the time taken for development of the disease may also vary
according to the preparation of arecanut consumed. The commercially freeze dried
products such as pan masala, gutkha and mawa (areca and lime) have high concentrates
of arecanut per chew and appears to cause OSF more rapidly than by self prepared
conventional betel quid which contained smaller amounts of arecanut.14
9
Review of Literature
PATHOGENESIS
The role of the constituents of areca nut in the pathogenesis of OSF has been
studied in detail over the last two decades. It is apparent that the fibrosis and
hyalinization of subepithelial tissues account for most of the clinical features encountered
in this condition. The substantial amount of research on elucidating the etiology and
pathogenesis appear to have been focused on the changes in the extracellular matrix.
Various studies have hypothesized that the increased collagen synthesis and or reduced
collagen degradation as possible mechanisms in the development of the disease. And
there are numerous biological pathways involved in the above processes and is likely that
the normal regulatory mechanisms are either down regulated or up regulated at different
stages of the disease.14
Studies on the pathogenesis of oral submucous fibrosis have suggested that the
occurrence may be due to:
1) Areca alkaloids causing fibroblast proliferation and increased collagen synthesis
An in-vitro study conducted on human fibroblasts using areca extracts or
chemically purified arecholine showed that among the chemical constituents, alkaloids
from areca nut are most important biologically. Four alkaloids which have been
conclusively identified are arecholine, arecaidine, guvacine, guvacholine, of which
arecholine is the main agent. The study supports the theory of fibroblastic proliferation
and increased collagen formation that is also demonstrable histologically in human OSF
tissues. Hydrolysis of arecholine produces arecaidine that has pronounced effects on
fibroblasts. Arecholine in high doses is cytotoxic and cells show detachment from the
10
Review of Literature
culture surface. There was a concentration dependent stimulation of collagen synthesis
when fibroblasts were exposed to both arecholine and arecaidine. And the stimulation
was greater with arecaidine. In addition, it was evident that the correlation between the
hydrolysis rates of different esters and the extent to which they stimulate collagen
synthesis, this suggests that hydrolysis of arecholine into arecaidine is necessary before
fibroblast stimulation can occur.15
This view was further supported by the finding that, addition of slaked lime to
areca nut in pan facilitates hydrolysis of arecholine to arecaidine making this agent
available in the oral environment.16
In the study conducted to examine the effects of arecholine on both normal and
OSF fibroblasts in culture revealed an elevated rate of collagen synthesis by OSF
fibroblasts compared with normal fibroblasts. Although the reason for this elevation is
not clear, the authors have proposed that it could reflect the clonal selection of a cell
population in the altered tissues under the influence of local factors such as interleukin-1
from inflammatory cells.17
2) Stabilization of collagen structure by tannins and catachins polyphenols –
In a study conducted on effects of betel nut polyphenols on collagen synthesis
indicated that one of the mechanisms that can lead to increased fibrosis is by reduced
degradation of collagen, forming a more stable collagen structure. Treatment of
reconstituted collagen fibrils with crude extracts of areca nut increased their resistance to
both human and bacterial collagenases in a concentration dependent manner. The study
says that the same mechanism may operate in patients with OSF causing fibrosis below
the damaged oral epithelium.18
11
Review of Literature
This evidence was also supported by another study which showed that the ability
of large quantities of tannins present in areca nut reduced collagen degradation by
inhibiting collagenases and proposed the basis for fibrosis as the combined effect of
tannin and arecholine by reducing degradation and increased production of collagen
respectively.19
Collagenase activity was measured with soluble glycine labeled collagen as a
substrate and showed reduced activity in fibroblasts from OSF compared with controls.
Further, they confirmed that the cleavage pattern of the collagen is similar to that of
typical mammalian collagen.20
Another study using cell culture methodology showed that there was a 1.5 fold
increase in collagen production in OSF fibroblasts with a ratio of type I to type III
collagens similar to normal fibroblast.21
It was postulated that the reason for high level of collagen production in OSF is
that these fibroblasts are a subset with increased potential for proliferation among the
heterogenic fibroblasts.22
However, this may not be the only reason for increased collagen as fibroblast
strains isolated from OSF failed to show the same phenomena. In a study done on the
action of mammalian collagenases, excess α 1 chain relative to α 2 was seen suggesting
an alteration of collagen molecules during the progression of the disease. Although the
biological function of this trimer is not known, it is regarded as more resistant to
degradation than the normal collagen molecule.23
12
Review of Literature
3) Copper in areca nut and fibrosis:
Study on the copper content in arecanut products and OSF has showed that the
copper content in areca nut is high and the level of copper in saliva is raised in areca
chewers.2.
It has also been showed that the oral mucosa of areca nut chewers have
significantly raised levels of copper when compared with the control subjects.24
The association between copper and OSF has been linked on the basis that excess
copper is found in tissues of other fibrotic disorders – Wilson’s disease, Indian childhood
cirrhosis and primary biliary cirrhosis. The enzyme lysyl oxidase is found to be
upregulated in OSF.25
Lysyl oxidase is a copper dependent enzyme and plays a key role in collagen
synthesis and its cross linkage. The possible role of copper as a mediator of fibrosis is
supported by the demonstration of upregulation of lysyl oxidase enzyme in OSF
biopsies26.
Copper added at various concentrations in vitro has also been shown to increase
proliferation of fibroblasts in culture27.
The fibroblasts in OSF have not only increased lysyl oxidase activities but also
specific characteristics. This was evident with the reported cell doubling time of 3.2 days
for OSF and 3.6 days for normal fibroblasts.28
However another study based on ultrasound investigations of visceral organs in
OSF patients reported that there was no evidence of fibrotic changes elsewhere. Fecal
copper was also normal suggesting that the copper levels were within the tolerance
levels29.
13
Review of Literature
As oral mucosa is directly exposed to the copper challenge in chewers its effect
may be well local. These different growth characteristics may either be due to the direct
effects of ingredients of areca nut or secondary to inflammatory factors mediated by
arecanut such as IL-1, TGF-β, IGF, and EGF30.
4) Upregulation of COX -2 –
It is known that OSF is associated with inflammatory changes in atleast some
stages of the disease. Prostaglandin is one of the main inflammatory mediators and its
production is controlled by various enzymes such as COX. Biopsies from buccal mucosa
of OSF cases and from controls were stained for COX 2 by immunohistochemistry and
revealed that there was increased expression of the enzyme in moderate fibrosis and this
disappeared in advanced fibrosis. This finding is compatible with the histology of the
disease as there is lack of inflammation in the advanced disease.31
Similar data have been reported in another study quoting 1.4 -3.4 fold increase of
PGE2 production and 1.1-1.7 fold increase of PGE 1α when gingival keratinocytes were
exposed to areca nut extracts.32
5) Fibrogenic cytokines -
Changes in cytokine secretion in OSF have been investigated. Endothelial and
TGFβ1 estimated by radioimmunoassay and ELISA respectively were increased in OSF
fibroblasts compared to fibroblasts of normal individuals. Therefore it has been
postulated that external stimuli such as areca nut may induce the development of the
disease by increased levels of cytokines in the lamina propria.33
Another study compared spontaneous and stimulated production of cytokines by
peripheral mono nuclear cells from OSF patients with those of genetically related control
14
Review of Literature
subjects. They were able to demonstrate increased levels of proinflammatory cytokines
and reduced antifibrotic IFN -γ in patients with the disease.29
These observations may suggest that disease process in OSF may be altered
version of wound healing as the recent findings show the expression of various
extracellular molecules are similar to those seen in maturation of a granulation tissue21.
6) Genetic polymorphisms predisposing to OSF –
Polymorphisms of the genes coding for TNF-α has been reported as significant
risk factor of OSF. A study of 809 patients with OSF has revealed that the high
production of allele TNF-2 to be significantly lower compared to an areca chewing
control group.34
In another study a possible relationship existed between the MHC class I chain
related gene A and OSF. They showed that phenotype frequency of allele A6 of MHC
class I chain related gene A in test subjects was significantly higher than the controls.35
In some genotypes of cytotoxic T lymphocytes associated antigen 4, a negative
regulator of T lymphocyte activation seems to have susceptibility for various
autoimmune diseases. Interestingly, G allele at position 49 of exon 1 was found to be
significantly associated with OSF compared with controls36.
7) Inhibition of collagen phagocytosis -
Degradation of collagen by fibroblasts through phagocytosis is an important
pathway of physiological remodeling of the extracellular matrix in connective tissue. As
OSF shows a gross imbalance in extracellular matrix remodeling, this putative
mechanism was investigated in vitro. Fibroblasts from OSF patients and controls were
15
Review of Literature
incubated with collagen beads and found that the proportion of phagocytic cells to be
35% and 75% respectively.37
There was a dose dependent enhancement of phagocytic cells when the cultures
were treated with corticosteroids. In another study reduced collagen phagocytosis by
fibroblasts was inversely dose dependent to the levels of arecholine, safrole and
nicotine.38
8) Stabilization of extracellular matrix –
Increased and continuous deposition of extracellular matrix may take place as a
result of disruption of the equilibrium between MMPs and tissue inhibitors of MMPs.
When normal control fibroblasts and fibroblasts of OSF patients were subjected to
arecholine and arecaidine in culture, OSF fibroblasts produced more tissue inhibitors
MMPs protein than normal fibroblasts. mRNA expression of tissue inhibitors MMPs in
OSF fibroblasts was also higher.39
Another recent study reports that the main gelatinolytic proteins secreted by
buccal fibroblasts (MMP 2 and MMP 9) are found in minimal amounts in diseased
tissues. The study further showed that arecholine reduced the MMP 2 secretion and
increased the tissue inhibitor MMP 1 levels resulting in increased deposition of collagen
in the extracellular matrix.40
Although the main pathologic change present in OSF appears to markedly
increased production of extracellular matrix, there is little information on the actual
remodeling of connective tissue with the progression of the disease. In a study conducted
to investigate the remodeling of extracellular matrix in OSF and the patterns of
expression of several molecules in various phases of the disease, it was apparent that the
16
Review of Literature
expression of tenascin disappeared when the lesion advanced from early to intermediate
phase. Heparan sulphate proteoglycans (perlecan), fibronectin, type III collagen and
elastin appeared in the early and intermediate phases but there was complete replacement
by collagen type I when the lesion progressed to an advanced phase. The pattern of
expression of most of these molecules followed a similar pattern to the organization of
granulation tissue.41
9) OSF as an autoimmune disorder –
Autoimmunity as an etiological factor for OSF has been examined. The reasons
for investigating an autoimmune basis included, slight female predilection and
occurrence in the middle age as reported in some of the studies. The presence of varying
autoantibodies at varying antibody titers has been reported in several studies suggesting
the possibility of an autoimmune basis to the disease. The first report on this concept
came in 1986 showing 65% of the sample being positive for atleast one of the
autoantibodies.36
Few studies reported on HLA typing in OSF patients. The frequencies of HLA
A10, DR 3 and DR 7 proved to be significantly different compared with an ethnically,
regionally and age-matched control group.42
Another study using polymerase chain reaction has shown a significant increase
in frequencies of HLA A2, DRB 1-11. The association of HLA and OSF does not appear
consistently as one study showed that there was no demonstrable specific pattern of HLA
antigen frequencies in chewers with or without the disease.43
17
Review of Literature
10) Collagen related genes – molecular aspects of OSF
Collagen related genes play an important role in the homeostasis of collagen in
the body. As OSF is a disease with dysregulation of collagen metabolism, it is important
to identify the enzymes and various other molecules that may contribute to genetic
modulation during the progression of the disease which includes different type of
enzymes such as collagenases and lysyl oxidase together with cytokines namely the
TGFβ. There is evidence to suggest that the collagen related genes are altered due to
ingredients in the quid. The genes COL1A2, COL3A1, COL6A1, COL6A3, and
COL7A1 have been identified as definite TGFβ targets and induced in fibroblasts at early
stages of the disease.
A study was done to compare the association of OSF and polymorphisms of six
collagen related genes. They found that genotypes associated with highest OSF risk for
collagen 1A1, collagen 1A2, collagenase-1, TGFß 1, lysyl oxidase and cystatin C were
found in low exposure group.44
A case series analysis of 30 cases of OSF was carried out for bFGF expression
using immunohistochemistry. The contribution of bFGF in disease progression and
consequent stromal changes with increase in the severity of disease was studied. They
found bFGF immunoreactivity was found to be increased in fibroblasts and in endothelial
cells in early OSF cases, while expression of bFGF in stroma increased notably in
advanced fibrosis.45
18
Review of Literature
Molecular pathogenesis of OSF 44
Collagen production pathway
Collagen Production
TGF-ß
PNP Procollagen gene activation
BMPI/PCP
procollagen ProLOX LOX
Collagen (Soluble form)
Collagen (Insoluble form)
Copper in areca nut
LOX
Flavonoids in areca nut
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Collagen degradation pathway44
TGF-ß
Activation of Plasminogen activator gene(PAI)
Activation of TIMP gene
PAI
Inhibits activated collagenase
TIMPs
Plasminogen Plasmin
in collagenase activity
Procollagenase Collagenase
Flavonoids in areca nut
in collagen degradation
20
Review of Literature
Overall effect of activated TGF –ß pathway44
Increase in collagen production
Decrease in collagen degradation
Increase in collagen (Insoluble form)
Fibrosis
Oral submucous fibrosis
21
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CLINICAL FEATURES
The most common initial symptoms of OSF are burning sensation of the oral
mucosa aggrevated by spicy food (42%), followed by either hypersalivation or dryness of
the mouth (25%). The most common and initial clinical sign as well as a regular feature
of OSF is blanching i.e. marble like appearance of the oral mucosa.
Localized blanching is caused by the impairment of the local vascularity. The
disease often starts as a blanched area and palpable fibrous bands develop over time.
Blanching may be localized, diffuse or reticular. The period between initiation of the
habit and the development of OSF may range from few months to several decades.
In diffuse blanching a greater part of the oral mucosa is involved. Blanching may
be asymptomatic or accompanied by a burning sensation of the oral mucosa, and salivary
changes.
Reticular blanching consists of blanched areas with intervening, clinically normal
mucosa, giving it a lace like appearance. Over a period of time, one type of blanching
may change into another.46
The prodromal symptoms include burning sensation in the mouth when
consuming spicy food, appearance of blisters especially in the palate, ulceration or
recurrent generalized inflammation of the oral mucosa, excessive salivation, defective
gustatory sensation and dryness of the mouth.38
Study on the early clinical forms of OSF has revealed that there are periods of
exacerbations manifested by the appearance of small vesicles in the cheek and palate. The
22
Review of Literature
intervals between such exacerbations vary from three months to one year. Focal vascular
dilatations manifest clinically as petechiae in the early stages of the disease.47
The reason for the focal vascular dilations may be as a part of response due to
hypersensitivity of the mucosa towards some external irritant like chilli or arecanut
Petechiae were observed in about 22% of OSF cases, mostly on the tongue followed by
the labial and buccal mucosa with no signs of blood dyscrasias or systemic disorders.32
As the disease progresses, the oral mucosa become blanched and slightly opaque,
and white fibrous bands appear. The buccal mucosa becomes first affected at an early
stage although it was thought, palate and faucial pillars are the areas involved first.40
The oral mucosa is involved symmetrically and the fibrous bands in the buccal
mucosa run in a vertical direction. The density of the fibrous deposit varies from a
slightly whitish area on the soft palate causing no symptoms to dense fibrosis causing
fixation and shortening or even the deviation of the uvula and soft palate. The fibrous
tissue in the faucial pillars varies from a slight submucosal accumulation in both pillars to
a dense fibrosis extending deep into the pillars with strangulation of the tonsils.38
The patients with OSF can be classified into 4 stages as per increasing intensity of
the trismus 1) Very early stage (2) Early stage (3) Moderately advanced stage and (4)
Advanced stage
Very early stage:
The patient complains of burning sensation in the mouth or ulcerations without
difficulty in opening the mouth.
23
Review of Literature
Early stage:
Prodromal symptoms - This includes the burning sensation in the mouth when
consuming spicy food, appearance of blisters especially on the palate, ulcerations or
recurrent generalized inflammation of the oral mucosa, excess salivation and defective
gustatory sensation. There are periods of exacerbations, manifested by the appearance of
small vesicles in the cheek and palate. Focal vascular dilatations manifest clinically as
petechiae in the early stages of the disease.
Moderately advanced stage:
The trismus is marked to such an extent that patients cannot open his / her mouth
more than 2 fingers wide. Patient therefore experiences difficulty in mastication.
Advanced stage
Patient is under nourished, anemic and has a marked degree of trismus and/or
other symptoms as mentioned above.15
Clinically it can also be classified into six grades:
Grade –1 : Only blanching of oral mucosa
Grade – II : Burning sensation, dryness of mouth, vesicles and ulcers.
Grade – III : Grade –II + Restricted mouth opening.
Grade – IV : Grade – III + Palpable fibrotic bands without involvement of tongue.
Grade – V : Grade – IV + Tongue involvement.
Grade – VI : OSF with histologically proven cancer.48
24
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As the disease progresses, the oral mucosa become blanched and slightly opaque
and white fibrous bands appear. The buccal mucosa and lips may be affected at an early
stage, although it was thought that the palate and the facial pillars are the areas involved
first. The fibrous bands in buccal mucosa run in a vertical direction. Sometimes the
fibrosis spreads to the pharynx and down to the piriform fossae. There is impairment of
tongue movement in patients with advanced OSF with significant atrophy of the tongue
papillae. With progressing fibrosis, there is difficulty in opening mouth, inability to
whistle or blow out a candle and difficulty in swallowing.41
The most outstanding feature and the best reliable sign of OSF is the presence of
palpable fibrous bands in the oral mucosa. Furthermore, the patients may suffer from a
burning sensation aggravated by spicy foods, dryness of the mucosa or hypersalivation.47
The fibrosis also leads to difficulty in mastication, speech and swallowing, pain in
the throat and ears and a relative loss of the auditory acuity due to stenosis of the opening
of the Eustachian tube. In early cases, the fibrous tissue is seen arching from the anterior
pillars into the soft palate as a delicate reticulum of interlacing white strands that later
become confluent. The floor of the mouth becomes pale and thickened, the tongue
reduced in size and mobility and bands of encircling collagen distal to the lips.36, 49
In advanced cases, the jaws may be inseparable and the totally inelastic mucosa is
forced against the buccal aspects of the teeth where sharp edges or restorations may cause
ulceration that becomes secondarily infected. If the fibrosis extends into the esophagus,
the patients may experience progressive dysphagia and reduced esophageal mobility may
be confirmed with a Barium swallow.36
25
Review of Literature
HISTOPATHOLOGIC FINDINGS
The epithelial changes in the different stages of OSF are predominantly
hyperplasia (early) and atrophy (advanced), associated with an increased tendency for
keratinizing metaplasia. The epithelial atrophy is the marked epithelial change in
advanced OSF, which contrasts with the predominantly hyperplastic epithelium of early
OSF. Lesions involving the palate showed predominantly orthokeratosis and those of the
buccal mucosa, parakeratosis. The high mitotic count in parakeratotic epithelium, which
is more common with OSF and the association with parakeratotic leukoplakia and
atrophic epithelial changes predisposes OSF to malignancy.
SUBEPITHELIAL CHANGES :
On the basis of the histopathological appearance of stained (H&E) sections, OSF
can be grouped into four clearly definable stages : very early, early, moderately advanced
and advanced. These stages are based not only on the amount and nature of the
subepithelial collagen, but also on the following criteria taken together:
• Presence or absence of edema,
• Physical state of the mucosal collagen,
• Overall fibroblastic response (number of cells and age of individual cells),
• State of the blood vessels,
• Predominant cell type in the inflammatory exudates.
A vascular response due to inflammation, apart from the connective tissue repair
process, has been very commonly found in OSF. Normal, dilated and constricted blood
vessels have been seen often in combination, in the same section. The apparent
26
Review of Literature
narrowing of the smaller vessels appears first in the upper mucosa and spreads gradually
to the larger, deeper vessels. Persistent dilation has also been seen in many moderately
advanced and advanced biopsies. A rise in mast cells occurs in the earlier stages of the
tissue reaction but in advanced stages, the counts are fewer in number.
The inflammatory cells seen are mainly lymphocytes and plasma cells. The
connective tissue in advanced stages is characterized by the submucosal deposition of
extremely dense and avascular collagenous tissues with variable numbers of chronic
inflammatory cells. Epithelial dysplasia without carcinoma is found in 10-15 percent of
cases submitted for biopsy and carcinoma is found in at least 5 percent of sampled cases.
The excessive fibrosis in the mucosa seems to be the primary pathology in OSF. The
atrophic changes in the epithelium are secondary.9
EPITHELIAL CHANGES:
In a study on epithelial changes in 34 biopsies of OSF patients, it was noticed that
in 33 biopsies the epithelium was not only reduced in thickness but the reteridges had
completely disappeared. Of 33 biopsies 15 had unkeratinized surface, 18 had undergone
keratinizing metaplasia, and 10 of these were orthokeratotic, 2 hyperorthokeratotic, 5
parakeratotic and one hyperparakeratotic. In 4 biopsies, signet cells like degeneration was
observed mainly occurring in the spinous layer. Liquefaction of the basal cell layer, inter
cellular edema and subepithelial vesicle were also seen.17
In a histomorphological study of OSF, it was noticed that hyperpalsia or atypical
epithelial hyperplasia was present in 102 of 104 cases with only one case showing an
atrophic epithelium and keratinizing metaplasia in all cases, and an increase mitotic
activity in advanced cases.20
27
Review of Literature
In more than 90% of OSF cases, the oral epithelium in the clinically affected
areas was markedly atrophic. The reteridges were completely lost. The atrophic
epithelium also exhibited;
- Intercellular edema in 18% of cases
- Signet cells in 13% of cases
- Epithelial atypia (focal dysplasia) in 7%2.
In a histologic section Lemmer J and Shear M noted that the epithelium was
extremely thick and there was ballooning of the superficial cells.50
In histological evaluation of the oral epithelial changes in 30 Indians with oral
cancer and submucous fibrosis, oral biopsies were taken from 30 patients either in the
remote areas or in the vicinity of cancer. They observed epithelial atypia in 11.5% in
areas remote from cancer and 71.4% in the vicinity of the cancer, along with
keratinization in varying degree.51
Studies on epithelial changes in 38 biopsies of OSF showed that there was normal
thickness of epithelium in 16 cases (42.1%), atrophic epithelium in 13 (34.2%) and
hyperplasia in 9 (23.7%). Non-keratinized epithelium was noticed in 16 (42.1%) whereas
the other 22 (57.9%) cases showed keratinization of different types either separately or
combined. Hyperorthokeratosis was noticed in 11 cases.19
In a study of 36 cases of OSF, atrophy of the epithelium was a major feature .52
On analysis of epithelial changes in OSF, out of 48 cases, 30 were parakeratosis
(62.50%) and 12 cases keratosis (25%). In 6 cases both keratosis as well as parakeratosis
(12.50%) were present. 45 cases showed atrophy of epithelium (93.75%),while 3 cases
showed nearly normal epithelium (6.25%), 46 cases showed flattened rete-ridges
28
Review of Literature
(95.83%) while 2 cases showed nearly normal epithelium (6.25%), 46 cases showed
flattening of rete-ridges (95.83%) while 2 cases showed rete-ridges having maximum
width. Granular cell layer was indistinct in 41 cases (85.4%). 2 cases showed intercellular
edema (4.16%), 6 showed nuclear hyperchromatism (12.5%), 4 showed irregular
stratification (8.33%) and 12 showed cellular pleomorphism (25%). Intercellular bridges
were conspicuous in all cases (100%). Change in nucleo-cytoplasmic ratio and individual
cell keratinization was seen in 3 cases (6.25%) and signet cells were seen in 2 cases
(4.16%). Increased in density of the cells were seen in 4 (8.32%) and loss of polarity was
observed in 2 cases (4.16%). Basal cell layer showed increase in density in 11 cases
(22.09%) and mitosis was increased in 3 cases (6.25%) out of which one had showed
atypical mitosis (2.08%). Basement membrane was intact in all the cases (100%).53
In a histological examination of 30 OSF cases showed these changes in the
epithelium.
- 26 (87%) of the biopsies showed an atrophic epithelium.
- 8 (27%) were reported as having flattened basement membrane.
- 10 (33%) had non-keratinized or poorly keratinized epithelium. In the remaining
20 (67%) cases, the epithelium had undergone keratinizing metaplasia i.e. 13
(43%) were parakeratinized and 7 (23%) hyperorthokeratinized.
- 8 (27%) cases had mild atypia within the epithelium.
- 2(7%) cases showed mild to moderate changes and one (3%) had marked atypia.47
In a report of 8 cases, they observed atrophy of the epithelium in 6, and one
specimen exhibited the so called signet cell-like degeneration.54
29
Review of Literature
In a hematological and histological study in 113 cases of OSF, the epithelium
showed hyperkeratinisation in 95.75%, epithelial atrophy in 38.29% and atypia in only
4.4% of cases.55
In hospital-based case –control study of 185 OSF patients, atrophy of the
epithelium was observed.13
A study on rats, areca nut-treated oral epithelium showed progressive changes in
epithelial thickness leading to atrophy, increased cellularity of fibroblasts, fibrosis of
connective tissue, focal infiltration of inflammatory cells and muscle atrophy. On killing
of rats after 600 days of treatment, the scores on cellularity, inflammation and muscle
atrophy were significantly different to the control group (P ¼ 0.03).56
CONNECTIVE TISSUE CHANGES:
Rao A.B.N observed that the submucosa showed increased dense collagen which
varied from normal to abnormal and showed fragmentation6
In a histological sections of OSF, it was observed that dense bands of collagen in
the lamina propria and submucosa. These infiltrated between, surrounded, and sometimes
replaced striated muscle bundles. Perivascular fibrosis was a prominent feature.50
A study of 53 patients with OSF, an amorphous change in the connective tissue
commensing downward from the epithelial basement membrane was evident. In
advanced stage of OSF, the connective tissue appeared hyalinized without discernible
collagen bundles.57
30
Review of Literature
In a study of 36 cases of OSF, subepithelial hyalinization was observed just below
the basement membrane with marked fibrosis and varying amount of round cell
infiltration.52
Shiau Y.Y and Kwan H.W in their study of 35 patients observed hyalinization of
collagen fibres in the connective tissue layer. Collagen fibres were seen as a smooth sheet
without discernible separated bundles. Dense collagen filled almost the entire submucosal
area.58
In a study of oxytalan fibres in OSF, fibres were found to be greater in early
stages and lesser in late stages. The other changes in the submucosa were impairment of
vascularity and inflammatory cellular infiltrate which increased with the progression of
the disease.59
A study on OSF reported that the lamina propria exhibited hyalinization and
condensation of collagen bundles and was paucicellular with some capillaries. The deeper
connective tissue had a patchy lymphocytic infiltrate that involved muscle fibres,
neurovascular bundles and accessory salivary gland.36
In a study of 11 cases of OSF, all cases exhibited a similar connective tissue
pattern. Adjacent to the basement membrane there was a thin zone relatively sparse
individual collagen fibrils and loosely arranged group of fibrils running parallel to the
epithelial-connective tissue junction. Deeper in the lamina propria the collagen consisted
of thick dense bundles arranged in a regular interwoven pattern.24
In a case report, it was observed that the lamina propria contains a patchy
distribution of chronic inflammatory cell infiltrate concentration in some regions and also
distributed in a band along the subepithelial region. Melanin pigment – containing
31
Review of Literature
macrophages were conspicuous in the superficial lamia propria. The most striking feature
of the connective tissue is the presence of dense collagen bundles, randomly orientated
and extending into the underlying striated muscles.60
In a hematological and histological study of 113 cases of OSF, it was observed
that collagen deposition of varying degrees and chronic inflammatory cell infiltrate
consisting of lymphocytes, monocytes, plasma cells and occasional macrophages. And
hyalinization of collagen bundles was also present.55
The thickening of the basement membrane with marked reduction in the
vascularity in the connective tissue was inversely propotional to increase density of
collagen which appeared hyalinized.61
A hospital based case control study on habits and OSF over a three year period in
185 patients observed that a number of cases showed submucosal dense and avascular
collagenous connective tissue and variable number of chronic inflammatory cells.13
ORAL SUBMUCOUS FIBROSIS AND ORAL CANCER
Various authors in past have suggested oral submucous fibrosis as a precancerous
condition.
Summarized criteria to support the precancerous nature of this disease are:
1. Higher prevalence of leukoplakia among submucous fibrosis patients.
2. High frequency of epithelial dysplasia.
3. Concurrent findings of submucous fibrosis in oral cancer patients.
4. Histological diagnosis of oral cancer without clinical suspicion among
submucous fibrosis cases.
5. Higher incidence of oral cancer among patients with submucous fibrosis.22
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In a study malignant potential of submucous fibrosis in 89 patients at the time of
first examination oral cancer was found in 9 patients (10%) and for the rest of the cases
the rate of malignant transformation was 4.5%.22
In an observation of the malignant transformation rate in oral submucous fibrosis
over a 17 year period analyses showed that oral submucous fibrosis possesses a high
degree of malignant potential. Within 2 years of observation the malignant transformation
rose from 4.5% to 7.6%.5
In a study, to investigate the relationship between hypoxia inducible factor-1α and
epithelial dysplasia using immunohistochemistry and RT-PCR.it was found that hypoxia
inducible factor-1α is upregulated at both protein and mRNA levels in OSF and the
correlation with epithelial dysplasia is stastically significant. it was concluded that
hypoxia inducible factor-1α may play a role in malignant transformation of
OSF.further,over expression of hypoxia inducible factor-1α may contribute to the
progression of fibrosis.62
Malignant Transformation
The precancerous nature of OSF was first described by Paymaster in 1956, when
he observed slow growing squamous cell carcinoma in one third of the patients with the
disease. This was confirmed by various groups and put forward five criteria to prove that
the disease is precancerous. They included, high occurrence of OSF in oral cancer
patients, higher incidence of squamous cell carcinoma in patients with OSF, histological
diagnosis of cancer without any clinical suspicion in OSF, high frequency of epithelial
dysplasia and higher prevalence of leukoplakia among OSF cases.2
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Most of the earlier studies have focused on the prevalence of epithelial dysplasia
in OSF. It has been so far the most reliable indicator for predicting potential malignant
transformation of an oral precancerous condition though new markers are emerging.63
According to the current awareness of the disease and some refined criteria for
grading dysplasia, it is reasonable to assume that the prevalence of dysplasia is more
towards the midway of the reported range. Malignant transformation rate of OSF was
found to be in the range of 7-13%.64
Recently, the carcinogenicity of areca nut without tobacco has been identified.
The strong association of arecanut with OSF, its dose dependent effects and the
confirmation of OSF as a potentially malignant disease leading to oral cancer provided
further evidence for this assertion.65
A review article on the pathogenesis of OSF hypothesized that dense fibrosis and
less vascularity of the corium, in the presence of an altered cytokine activity creates a
unique environment for carcinogens from both tobacco and areca nut to act on the
epithelium. The authors have assumed that carcinogens from areca nut accumulate over a
long period of time either on or immediately below the epithelium allowing the
carcinogens to act for a longer duration before it diffuses into deeper tissues. Less
vascularity may deny the quick absorption of carcinogens into the systemic circulation14.
A study was conducted over a one year period which included 58 patients of OSF.
On observation 15 (25.86%) patients showed mild dysplasia, 3 (5.17%) moderate and 2
(3.45%) severe.66
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SPECIAL STAINS
The fibrosis and muscle involvement in OSF has been studied previously using
special stains.
The study was conducted by Ragas Dental College & Hospital, Chennai, where
incisional biopsy sections of 30 Oral submucous fibrosis patients were stained with
Masson Trichrome stain, to study fibrosis involving muscle. They have chosen Masson's
trichrome stain as this offers a simultaneous contrast of color to the collagen fibers along
with muscle fiber and muscle bundle. Where collagen stained blue while the muscle took
a brilliant red color. This colour contrast facilitated a better visual discrimination between
muscle and collagen. The technique is also simple and easily reproducible.67
The study was conducted on 53 oral submucous fibrosis patients. This included
oral biopsies of 53 Oral submucous fibrosis patients among 50,915 rural villagers in the
Indian states of Kerala, Gujarat, Bihar, and Andhra Pradesh. Histological sections
revealed a definite alteration in the tinctorial quality of connective tissues when stained
by the Rinehart and Van Gieson method. The connective tissue in oral submucous
fibrosis patient tends to be amorphous and non bundular, in contrast to that seen in the
normal controls.57
Studies on the areca nut-treated oral epithelium showed progressive changes in
epithelial thickness leading to atrophy, increased cellularity of fibroblasts, fibrosis of
connective tissue, focal infiltration of inflammatory cells and muscle atrophy. There
study provides further evidence that areca nut contributes to the development of OSF in
treated animals. In there studies histological sections were stained with haematoxylin and
eosin, van Gieson for collagen fibres and Masson’s trichrome for the young collagen.
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Tissue sections were examined by light microscopy and scored independently and blindly
for epithelial atrophy, connective tissue fibrosis, cellularity (population of fibroblasts)
and vascularity (population of blood vessels), hyalinization of the connective tissue
interface, inflammation and muscle atrophy.56
A case series analysis of 30 cases of OSF was carried out for bFGF expression
using immunohistochemistry. The contribution of bFGF in disease progression and
consequent stromal changes with increase in the severity of disease was studied. The
connective tissue changes in these cases were corroborated using aldehyde fuschin and
verhoeffs haemotoxylin special stain.45
Muscle involvement in OSF has been studied previously using special stains. SC
Gupta and Hamner JC and Mehta FS have used Van Gieson as a special stain for
collagen.68
36
MMeetthhooddoollooggyy
Methodology
METHODOLOGY
A number of studies have been reported in the literature on clinical and
histological changes in oral mucosa in patients with oral submucous fibrosis. The fibrosis
tends to present itself clinically in the form of palpable fibrous bands running vertically in
the buccal mucosa. Similar bands are seen affecting the labial mucosa running
circumorally. Hence the present study was aimed at evaluating correlation between
mouth opening clinically and fibrosis histologically & also to see muscle changes.
A total of 50 cases were selected for this study which were clinically diagnosed as
Oral submucous fibrosis in the department of Oral Medicine and Radiology and
confirmed by histopathology in Department of Oral Pathology. Cases with the complaint
of difficulty in opening the mouth with associated blanching of oral mucosa and palpable
fibrous bands were included for this study. Distance between the inter-incisal edges in
millimeters was measured using divider for recording the mouth opening. Patients were
divided into two groups (according to the mouth opening.) based on first two parameters
of functional staging given by S.M. Haider et al criteria.69
Group I – mouth opening more than 20 mm
Group II – mouth opening between 11-19 mm.
Prior to biopsy the hematological and biochemical investigations were done in
Clinical laboratory of the Oral Pathology department. Further the incisional biopsy was
performed on buccal mucosa for the selected cases in the department of Oral Medicine
and Radiology. The biopsy tissues of both the groups were fixed in 10% neutral buffered
formalin & further processed and embedded in paraffin wax. Further five micrometer
37
Methodology
thick sections were made by using semi automatic “Leica RM 2155 Soft Tissue
Microtome” (Fig.1). The obtained sections were stained by Hematoxylin & Eosin stains
(Fig.2), Van-Gieson (Fig.3), Masson Trichrome stain (Fig.4)70 and observed under
Binocular compound microscope (Leica Biomed, Germany) and LEICA DMRB
Research Microscope attached with detachable Sony camera for photomicrographs.
(Fig.5)
Equipments and Materials utilized for the preparation of histopathological specimens for
the study are as follows:
1. Paraffin wax blocks of the selected subjects for the study.
2. Sectioning
- Rotary Microtome for making 5 μm thick sections (Leica RM 2155)
- Hot water bath
- Microscopic slides 75 mm x 25 mm, 1.45 mm thick (BLUE STAR,
Mumbai).
- Mayer’s egg albumin (Equal parts of egg white and glycerin with a pinch
of thymol).
- Hot air oven
3. Staining
- Staining trays
- Glass trough with lids
- Measuring jars
- Coupling jars
- Pipettes
38
Methodology
- Electronic balance (Adair Dutt Company)
- Amber colored bottles.
4. Ehrlich’s Haematoxylin Stain:
Haematoxylin – 2 gm (Merck Chemicals Ltd),
Absolute alcohol – 100 ml (S.D. Fine Chemicals, Mumbai)
Glycerin – 100 ml (Movika Pharmaceuticals, Mumbai)
Glacial acetic acid – 10 ml (S.D. Fine Chemicals, Mumbai).
Distilled water – 100 ml.
5. Eosin stain
Eosin – 0.5 gms (Glaxo Laboratories, Mumbai)
Absolute alcohol –50 ml.
6. Celestine Blue
Celestine Blue B - 2.5gm (Loba Chemicals, Mumbai),
Ferric ammonium sulphate - 25gm
Glycerin – 70ml (Movika Pharmaceuticals, Mumbai)
Distilled water – 500 ml.
7. Van – Gieson stain
Saturated aqueous picric acid solution – 50ml,
(Picric acid - Thomas baker chemical limited, Mumbai)
1% aqueous acid fuschin – 9.0ml (S.D. Fine Chemicals, Mumbai).
Distilled water-50ml,
8. Masson Trichrome Stain
Solution A
39
Methodology
Acid fuschin – 0.5g (S.D. Fine Chemicals, Mumbai).
Glacial acetic acid – 0.5g (S.D. Fine Chemicals, Mumbai).
Distilled water – 100ml
Solution B
Phosphomolybdic acid – 1.0g (Loba Chemicals, Mumbai),
Distilled water – 100ml
Solution C
Light Green – 2.0g (Loba Chemicals, Mumbai),
Glacial acetic acid – 2.5ml (S.D. Fine Chemicals, Mumbai).
Distilled water – 100ml
9. Cleaning and mounting.
Absolute alcohol (S.D fine chemical Ltd, Mumbai),
Xylene (Qualigens Fine Chemicals, Mumbai).
Microscopic slide cover glass - 22 x 25 mm, 0.16 mm thick (BLUESTAR,
Mumbai).
Disterene polysterene xylene (DPX) (NICE CHEMICALS, Cochin).
10. Microscopy
Binocular compound microscope (Leica Biomed, Germany)
Leica DMRB Research Microscope
METHOD
1. Staining Procedure: Haematoxylin and Eosin70
1. The sections were deparaffinized in hot air oven and two changes of xylene.
2. Hydrated through descending grades of alcohol.
40
Methodology
3. Brought to running water for 5 minutes
4. Stained with Ehrlich’s Hematoxylin solution for 5 minutes
5. Brought to running water for 5 minutes
6. Differentiation was done by one dip in 1% acid alcohol.
7. Washed in water.
8. Stained with Eosin for 1 minutes.
9. Dehydrated in ascending grades of alcohol.
10. Cleared in xylene.
11. Mounted in DPX
2. Staining Procedure Van- Gieson stain.70
1. The sections were deparaffinized in hot air oven and two changes of xylene.
2. Hydrated through descending grades of alcohol.
3. Brought to running water for 5 minutes
4. Stained with Celestine blue for 5minutes.
5. Stained with Ehrlich's haematoxylin for 5 minutes
6. Washed in tap water
7. Differentiation was done by one dip in 1% acid alcohol.
8. Washed in tap water for 5 minutes.
9. Stained with Van-Gieson’s stain for 5minutes.
10. Blotted dry.
11. Dehydrated through ascending grades of alcohols.
12. Cleared in xylene
13. Mounted in DPX.
41
Methodology
3. Staining Procedure Masson Trichrome stain70
1. The sections were deparaffinized in hot air Oven and two changes of xylene.
2. Hydrated through descending grades of alcohol.
3. Brought to running water for 5 minutes
4. Stained with Celestine blue for 5minutes.
5. Stained with Ehrlich's haematoxylin for 5 minutes
6. Washed in tap water
7. Then the sections were differentiated in 1% acid alcohol -one dip.
8. Treat with 1% acid fuschin in 1% acetic acid for 5 minutes.
9. Washed in tap water.
10. Treat with aqueous phosphomolybdic acid for 5 minutes.
11. Treat with 1% light green in 1% acetic acid for 2-3 minutes.
12. Rinse in acetic acid
13. Dehydrated through ascending grades of alcohols.
14. Cleared in xylene
15. Mounted in DPX.
The sections were studied under 5X, 10X and 40X magnifications employing
Binocular compound microscope (Leica Biomed, Germany) and Leica DMRB Research
Microscope and the histological grading: was done according to the Rooban T et al a
criterion where fibrosis was graded as follows
Stage 1: Fibrosis limiting to lamina propria alone.
Stage 2: Fibrosis involving superficial region of muscle bundle.
42
Methodology
Stage 3: Fibrosis involving deeper regions of muscle bundle.
Stage 4: Muscle bundle replaced by fibrosis.67
Statistical Analysis:
Results are expressed as mean ± standard deviation and range values. From
continuous data and number and percentages for categorical data, one way ANOVA was
used for multiple group comparisons and ‘t’ test for two group comparisons.
Chi-square test was used for analysis categorical data.
A ‘p’ value of 0.05 or less was considered as statistically significant.
43
Methodology
44
Methodology
45
RReessuu tlltss && OObbsseerrvvaatitioonnss
Results & Observations
RESULTS AND OBSERVATIONS
A total of 50 cases of OSF were included in the present study and the cases were
divided into two groups –
Group I : Includes patients having maximum mouth opening of more than 20 mm
Group II : Includes patients having mouth opening between 11-19 mm.
Group I : Included 23 (92%) males and 2 (8%) females, between age range of 18-48
years (mean of 28.5 ± 6.5), having mouth opening of 21-40 mm with (mean of 28.7 ±
4.8).
Group II: Included 24 (96%) males and 1 (4%) female in the age range of 18-35 years
(mean of 24.0 ± 4.0), having mouth opening of 12-19 mm with (mean of 16.9 ± 2.4).
TABLE – I: Distribution of study subjects according to age, gender and mouth opening
Group I
(MO > 20 mm) (N=25)
Group II (MO =11-19
mm) (N=25)
t-Value p-Level
Mean ± SD 28.5 ± 6.5 24.0 ± 4.0 Age (Years) Range 18 – 48 18 – 35
2.99
Results & Observations
Group I.: 4 (16%) patients, 3 (12%) patients and 18 (72%) patients exhibited Grade I –
Fibrosis limited to lamina propria, Grade II – Fibrosis superficial to muscle bundle.
Grade III – fibrosis in deeper region of muscle bundles respectively.
Group II: 4 (16%) patients, 5 (20%) patients and 16 (64%) patients with Grade I, II and
III respectively, on stastical analysis shows insignificant ‘p’ value (p>0.05).
TABLE – II: Showing correlation between the clinical mouth opening and fibrosis
on histology
Fibrosis Sl. No. Mouth Opening L–LP
(Grade I) S –MB
(Grade II) D-MB
(Grade III)
I Group I 25 Cases
(MO>20 mm)
4 (16.0%)
3 (12.0%)
18 (72%)
II Group II 25 Cases
(MO = 11-19 mm)
4 (16.0%)
5 (20%)
16 (64.0%)
X
2 = 0.62 P = 0.73 NS (Not Significant) p>0.05 MO : Mouth opening
L – LP: Fibrosis limiting to lamina propria
S – MB: Fibrosis superficial to muscle bundle
D – MB: Fibrosis deep within muscle bundle.
On histologic sections in Group I, muscle degeneration was evident in 18(72%)
patients and in 7(28%) patients no such changes was observed. In Group II, 16 (64%)
patients showed muscle degeneration while 9 (36%) patients didn’t show any change.
Over all, 34 (68%) patients showed muscle degeneration irrespective of mouth opening
while 16 (32%) patients showed no changes (p=0.54, p>0.05 not significant). (Table III)
47
Results & Observations
TABLE – III: Showing correlation between mouth opening and muscle
degeneration
Muscle Degeneration Sl. No. MO Groups No. of Cases Present
N (%) Absent N (%)
1 Group I (>20 mm) 25 18
(72%) 7
(28%)
2. Group II (11 – 19 mm) 25 16
(64%) 9
(36%) X2 = 0.37 p = 0.54, NS (Not significant) p>0.05
Table IV shows correlation between histopathological grading with mean
clinical mouth opening where, 8 cases of grade I, exhibited mean mouth opening of 22.9
± 6.6mm, 8 case of grade II had a mean mouth opening of 22.0 ± 6.7mm, while in 34
cases of grade stage III, a mean mouth opening of 23.0 ± 7.0mm was noted. Though
68% of subjects where categorized into grade III as per histopathologic section, the
ANOVA test fail to demonstrate a significant reduction of mouth opening.
TABLE – IV: Histopathological grade and mean mouth opening
Sl.No. HP Grades No. of Cases Mouth Opening Mean ± SD 1 I 8 22.9 ± 6.6
2 II 8 22.0 ± 6.7
3 III 34 23.0 ± 7.0 ANOVA: F = 0.06, p = 0.94, (Not Significant) On stastical analysis of the histopathologic features all the patients of grade I, II,
III, exhibited 100% keratinized stratified squamous epithelium. Hyalinization was
evident in 5 patients, 7 patients & 16 patients of grade I, II, III, respectively, while
48
Results & Observations
muscle degeneration was demonstrated in 100% of patients in grade III as against to 0%
in grade I and II patients.
TABLE – V: Histopathological features
Histopathological grading Sl.No. Histopathological features
1 (n=8) 2 (n=8) 3 (n=34)
1. Epithelium Keratinization 8 8 34
Limiting to LP 8 - -
Superficial to MB - 8 - 2. Fibrosis
Deep to MB - - 34
Present 5 7 16 3. Hyalinization
Absent 3 1 18
Present - - 34 (100%) 4. Muscle Degeneration Absent 8 8 -
In the present study the association between clinical staging of mouth opening and
histological grading of fibrosis did not show any statistical significance.
49
Results & Observations
Graph 1: Histogram illustrating correlation between the clinical mouth opening and
the histopathological grade
4 3
18
4 5
16
0
5
10
15
20
No.
of c
ases
Group I (MO>20 mm) Group II (MO 11-19 mm)
HP Grade in relation to mouth opening
Grade I Grade II Grade III
Graph 2: Histogram illustrating correlation between the mean mouth openings and fibrosis
Mouth opening in relation to HP grading
23.022.022.9
0
5
10
15
20
25
30
35
Grade I Grade II Grade III
Mea
n M
O (m
m)
50
Results & Observations
Graph 3: Histogram illustrating correlation between mouth opening groups and muscle degeneration
72
28
64
36
01020304050607080
% o
f cas
es
Group I (MO>20 mm) Group II (MO 11-19 mm)
Mouth opening groups
Muscle Degeneration
PresentAbsent
51
Results & Observations
52
Results & Observations
53
Results & Observations
54
Results & Observations
55
Results & Observations
56
DDiissccuussssiioonn
Discussion
DISCUSSION
Oral submucous fibrosis “an insidious chronic disease affecting any part of the
oral cavity and sometimes the pharynx. Although occasionally preceded by and / or
associated with vesicle formation, it is always associated with a juxta-epithelial
inflammatory reaction followed by a fibroelastic change of the lamina propria, with
epithelial atrophy leading to stiffness of the oral mucosa and causing trismus and inability
to eat.”2
Oral submucous fibrosis is a common premalignant condition of the mouth with
areca nut chewing habit in Asian subcontinent. It is characterized by restricted mouth
opening, tongue protrusion and cheek flexibility. Reports on muscle changes in Oral
submucous fibrosis using light microscope are few. The present study was undertaken to
describe the extent of fibrosis and to correlate this with the mouth opening in Oral
submucous fibrosis patient and also to see muscle changes.
The study was conducted by Ragas Dental College & Hospital, Chennai, where
incisional biopsy sections of 30 Oral submucous fibrosis patients were stained with
Masson Trichrome stain, to study fibrosis involving muscle. Wide spectrum of changes
were observed, starting from fibrosis that was evident only subepithelially without
extending into the muscle bundles, to the point, wherein only few remnants of muscle
fibers were seen and missing muscle bundles being replaced by fibrous tissue.67
A study was conducted by Eastman Dental College, England. They investigated
muscle changes ultrastructurally in two groups of patients having Oral submucous
fibrosis.
i) Patients with no evidence of restricted mouth opening.
57
Discussion
ii) Patients with restricted mouth opening.
A electron microscopic examination showed that the majority of muscle fibers
taken from first group appeared normal with only occasional muscle fiber showing
accumulation of homogenous material and compression of sacromere closest to this
material. In contrast, tissue from second group showed severe changes and necrosis of
muscle fibers in a higher proportion. The necrotic muscle fiber exhibited complete loss of
plasma membrane in which the outline was maintained by an intact basal lamina. It is
suggested from this study that restricted mouth opening in Oral submucous fibrosis might
depend not only on the subepithelial fibrosis but also on the extent of muscle
degeneration.71
A study was conducted on 53 oral submucous fibrosis patients. This included oral
biopsies of 53 Oral submucous fibrosis patients among 50,915 rural villagers in the
Indian states of Kerala, Gujarat, Bihar, and Andhra Pradesh. Histological sections
revealed a definite alteration in the tinctorial quality of connective tissues when stained
by the Rinehart and Van Gieson method. The connective tissue in oral submucous
fibrosis patient tends to be amorphous and non bundular, in contrast to that seen in the
normal controls. However in this study the correlation between mouth opening and
fibrosis was not done.57
A literature survey shows wide variation in age & sex distribution of OSF. Few
epidemiological surveys in India have shown a female predominance in the occurrence of
this entity. Study conducted in India by Sinor showed a male predominance.72 An
Hospital-Based Cross-Sectional Study of 1000 cases from central India showed male-to-
female ratio of 4.9:11. In our study out of 50 cases Group I consist of 23(92%) males,
58
Discussion
2(8%) females & Group II consist of 24(96%) male & 1(4%) females, so over all out of
50 cases 47(94%) males & 3(6%) females which show a male predominance.
The study population in Group I, on an average consists of age range between 18-
48 years (mean of 28.5 ± 6.5) and Group II on an average consist of age range between
18-35 years (mean of 24.0 ± 4.0). In over all study average age of occurrence of OSF was
18-35. Only one case was reported at age of 48. This observation was different from that
of Pindborg who reported maximum number of cases in 40-49 years in their study.
Although there are regional variations in the type of areca nut products used in
India, the betel quid was the most popular and prevalent habit in ancient Indian culture.
But in 1980, both areca quid products such as Pan Masala and Gutkha were introduced in
Indian market as commercial preparations. Since then there has been an increase in the
use of Pan Masala and Gutkha in the younger age groups, which had lead to increased
incidence of OSF.
Recently, it has been documented that the habit of chewing Gutkha had gained
considerable popularity among the younger men. The rapidly increasing prevalence of
this habit can be judged from the reports that the Indian market for Pan masala and
Guthka is worth Rupees 25 billion (US$500 million).1
In a study of 800 normal patients in south India conducted by Ranganathan et al
it is reported that average size of mouth opening was 47.5% & 44.6% in males & female.
Our study included two Groups based on interincisal distance with Group I having more
than 20mm (average of 21-40mm), mean of 28.7±4.8 and Group II less than 20mm
(average 12-19), mean of 16.9±2.4. It is well documented that in OSF there is progressive
59
Discussion
inability to open the mouth and tongue movement get restricted depending on severity
of the disease process.
OSF is a disease of altered collagen metabolism. The lesion is characterized by
increased collagen fiber formation in the initial stage followed by formation of dense
collagen fibre bundles and different degrees of hyalinization. This alters the flexibility of
the mucosal tissue leading to restriction in the ability to open the mouth.
The cross sectional study was conducted by University Hospital, Pakistan, on 325
patients with oral submucous fibrosis in Karachi during January 1992 to October 1994.
They concluded that the bands were formed initially in the fauces followed by buccal and
labial areas this is accompanied by an increase in the severity of disease as measured by
restriction in the ability to open the mouth.69
In our studies we have taken two groups of patients depending on clinical mouth
opening & histological grading into four grades based on by Rooban et alcriteria, In
Group I, we have - 4(16%), 3(12%) and 18(72%) in grade I, II, III, and in Group II -
4(16%),5(20%),16(64%) in grade I,II, III respectively. We didn’t find any association
between clinical staging and histopathological grading.
Along with fibrosis hyalinization was noted in 22(44%) patients & this feature
was absent in 28(56%) patients irrespective of mouth opening. This doesn’t have any
effect on mouth opening.
Over all, amongst 50 patients, we found 34 patients in grade III, 8 in grade II, and
8 in grade I. In our study, biopsy site was buccal mucosa as it’s difficult for a surgeon to
take biopsy more posteriorly in OSF patients, however it was soon evident that the
60
Discussion
severity of fibrosis is more intensitified in posterior areas of oral cavity compared to
midbuccal mucosa and anterior buccal mucosa. Hence this could very well be the reason
for a patient showing prominently reduced mouth opening yet with a histologically
grade I. This standpoint is however similar to the findings obtained by Rooban et al &
Kiran kumar et al.67,73
The site and extent of the fibrosis and its role in causation of trismus are
determined by several factors including the anatomical and physiological integrity of the
underlying musculature.74
Muscle involvement in OSF has been studied previously using special stains. SC
Gupta and Hamner JC and Mehta FS have used Van Gieson, a special stain for
collagen.68
There is also study by Rooban et al where Masson’s trichrome stain was used
which offers a simultaneous contrast color to the collagen fibers along with muscle fiber
and muscle bundle. This colour contrast facilitated a better visual discrimination between
muscle and collagen. The technique is also simple and easily reproducible. In the present
study we have used Van Gieson, a special stain for collagen & Masson trichrome for
better discrimination between muscle & collagen.
Rajendran et al studied the histopathology of OSF from buccal mucosa using light
and electron microscope. They described fibroblasts, bundles of collagen, mast cells,
macrophages and subepithelial fibrosis in the lamina propria. They also observed that the
fibrosis in some cases was extending deep into muscle bundle under light microscope.
They observed focal myofibrillar lysis, hypercontraction of myofibres and extensive fatty
infiltration between muscle bundles under electron microscope.75
61
Discussion
Gupta SC et al studied histopathologically the palatal muscle of OSF patients and
reported degenerative changes in muscle bundle. 20.8% of their cases exhibited
degenerative changes either as a loss of cross st