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com

Review Article
Universal Journal of Pharmacy ISSN 2320-303X
Take Research to New Heights

DIAGNOSTIC APPROACHES FOR ORAL SUBMUCOUS FIBROSIS

Ramachandran Sudarshan*1, Sree Vijayabala G2, Dinesh Raj KS3
1
Department of Oral Medicine and Radiology, Best Dental Science College, Madurai.
2
Assistant Professor, Faculty of Dentistry, ESI hospital, KK nagar, Chennai.
3
Sustainability Expert,Accenture, Chennai

Received 21-02-2013; Revised 10-03-2013; Accepted 06-04-2013

ABSTRACT
Oral submucous fibrosis is a potentially malignant disorder with increased rate of malignant transformation. It is due to
well known etiology areca nut. Recognition of this condition is so clear-cut that it is considered as a spot diagnosis. But
determining its malignant transformation requires histopathology. Histopathological investigation is considered to be
gold standard in diagnosing oral submucous fibrosis. Still many investigators researched on other modalities for simpler
and non invasive technique. This review portrays the different modalities of investigation tried in oral submucous
fibrosis.
Keywords: Oral submucous fibrosis (OSMF), Potentially malignant disorder, Collagen, Fibroblast

INTRODUCTION opening and causing difficulty in mastication, speech,

Oral submucous fibrosis (OSMF) is a high risk
precancerous condition characterized by changes in the
connective tissue fibers of the lamina propria and
deeper parts leading to stiffness of the mucosa and
restricted mouth opening. The strongest risk factor for
OSMF is the chewing of betel quid containing areca nut.
OSMF is seen most frequently in communities resident
in the Indian sub-continent and has a reported
incidence of between 0.2–1.2 percent of the urban
population attending dental clinics1.
The symptoms and signs of OSMF are due to
inflammation and, primarily, fibrosis. The most
common initial symptoms and signs are a burning
sensation, dry mouth, blanching of oral mucosa and
ulceration. The burning sensation usually occurs while
chewing spicy food. Blanching of the oral mucosa is
caused by impairment of local vascularity because of
increasing fibrosis and results in a marble-like
appearance. Blanching may be localized, diffuse or
reticular. In the more advanced stage of the disease,
the essential feature is a fibrous band restricting mouth
*Corresponding author:
Dr. SUDARSHAN.R,
Senior Lecturer
Department of Oral Medicine and Radiology
Best Dental Science College, Madurai
Mobile :9444498736, Email: Sudharshanram@yahoo.co.in
swallowing and maintaining oral hygiene2.
Histopathology findings are the mainstay of diagnosis at
present. The principal features of OSMF are less
vascularized collagenous submucosa with a range of
atrophy in the neighboring striated muscle fibers, mild
to moderate chronic inflammation, and epithelial
changes consisting of atrophy and a variable degree of
dysplasia. The important pathological feature of OSMF
is submucosal accumulation of collagen leading to
epithelial atrophy. It has been discovered that
exposure of buccal mucosal fibroblasts to alkaloids may
cause aggregation of collagen3. But several other
invasive and noninvasive analysis for OSMF was
researched with or without histopathology as gold
standard. This review illustrates the other diagnostic
modalities tried in OSMF.
Collagen in OSMF
OSMF fibroblasts synthesized larger amount of collagen
when compared to normal fibroblasts and they have
higher pro collagen mRNA levels; they produce type I
collagen trimer, which is resistant to degradation and
cytokines from the inflammatory cells also stimulate
the collagen synthesis4. Most of the collagen fibers
appeared to be parallel to the epithelium and the
reason for unidirectional alignment of clinical fibrous
bands could be due to chronic stimulation of oral
mucosa by the irritants leading to change in the
orientation of collagen fiber bundles, which might

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Ramachandran et al. UJP 2013, 02 (02): Page 37-41
result in scar formation similar to that of wound
healing, where the collagen fibers are oriented parallel
to the epidermis5.
Blood chemistry and hematological variations
Significant haematological abnormalities have been
reported in OSMF.
It was also reported that OSMF was associated with
eosinophilia. The basis for this is the reported finding
of mast cells in submucous layers. Mast cells regulate
the release of histamine which explains the occasional
complaint of itching sensation in the oral mucosa in
these patients6. Biopsy specimen stained with Toluidine
blue stains the mast cell granules metachromatically
due to its reaction with sulphated
mucopolysaccharides. Interleukin-1 from the mast cells
could cause increased fibroblastic response and mast
cell derived tryptase causes increased production of
type-I collagen and fibronectin thereby attributing to
the increased fibrosis7.
In a study of 113 cases of OSMF, the absolute
eosinophil count for these cases was elevated and
hemoglobin level of less than 12 gm% was found in 24
cases and 12 cases had hemoglobin level of less than 10
gm%6.
A significant increase in total protein level due to
increase in the globulin fraction and other plasma
protein in advanced stage of OSMF was observed.
Serum iron level was significantly lowered in advanced
stage of OSMF. The levels of ascorbic acid was reduced
in the early stage, moderately advanced stage and
decreased to a greater extent in advanced stage of
OSMF. Collagen levels were increased significantly in
early stage, moderately advanced stage and highly
significant in advanced stage8.
Several studies on OSMF indicated that there was
decreased red blood cell count9, hemoglobin
percentage9, iron levels10 and increased ESR9,
eosinophil count9, gamma globulins9, copper levels10
and Circulating immune complex11.
Serum levels of total and lipid bound sialic acid were
significantly elevated in patients with OSMF and there
was a positive correlation between serum levels of the
markers and the extent of malignant disease (TNM
Clinical staging) as well as histopathological grades12.
A study in which Tissue malonaldehyde levels were
significantly higher as the grading progressed but tissue
levels in grade 3 oral submucous fibrosis were lower.
This decrease in tissue malonaldehyde could possibly
be associated to collagen cross linking occurring during
the advanced stages of oral submucous fibrosis13.
Cytogenetics
Chromosomal instability has long been associated with
the neoplastic process and the quantitative assay of
sister chromatid exchange (SCE) provides an easy, rapid
and sensitive method for studying chromosome / DNA
instability and its subsequent repair processes14.
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A study of 60 patients in which among the controls, the
mean SCE value was 5.12±SD. Mean value in OSMF
patients was 8.23±SD. This increase may be attributed
to the genotoxic effect of the constituents of betel
quid. The role of areca nut alkaloids in this regard may
be significant. But 3 OSMF patients showed
exceptionally high values of SCE (more than 10.00)15.
AgNOR
Silver-binding nucleolar organizer region proteins (Ag
NORs) comprise a simple and reproducible cytological
test indicative of the proliferative status of cells
particularly of epithelial and haematopoitic origin. It
was found that the pooled mean Ag NOR count in
clinically advanced OSMF was higher than in moderately
advanced cases14.
A study of 20 patients with OSMF in which Ag NORs
count was done and the mean Ag NORs count in normal
epithelium was 3.62±SD. OSMF patients showed mean
AgNORs count of 7.21±SD. Ag NORs count in OSMF and
the oral cancer patients were significantly higher than
normal patients. But 3 OSMF patients showed
exceptionally high values of Ag NORs count of (more
than 8.0)15.
Immunological studies
An immunological study conducted in which 32 OSMF
patients were studied for circulating T, B and null cell
populations were quantified. The absolute null count
was significantly increased, while the absolute T
lymphocytes were significantly decreased. The B cell
populations were within normal limits16.
Spinous layer being a sub compartment of surface
epithelium of oral mucosa plays a major role in
investigating oral submucous fibrosis. The epithelium
was segmented using anisotropic diffusion and Otsu’s
thresholding. Wavelet based multi-resolution technique
is applied to extract 12 textural features from spinous
layer. Bayesian and SVM based classification shows an
overall accuracy of 93.33% and 96.66% respectively
which are comparable emphasizing that texture of
spinous layer plays an important role in oral cancer
diagnosis17.
The malignant transformation with OSMF is often
associated with changes at the genetic level that in
turn is reflected by the altered expression of proteins
related to cell cycle, proliferation, and apoptosis. The
p53, Ki67, and bax profiles of OSMF were altered18.
Cell kinetic studies
When fibroblasts are exposed to the alkaloids of areca
nut their proliferation rate increased; a finding in
contrast to the results of others. The second group
found a dose-dependent inhibition of growth by
alkaloids. These studies prompt one to propose that the
connective tissue changes in OSMF is probably not due
to increased fibroblast proliferation. On the other
hand, OSMF fibroblasts can play a role in over-
production of collagen19.
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Ramachandran et al. UJP 2013, 02 (02): Page 37-41
The UK group in 1987 found that collagen formation by
fibroblasts increases when exposed to nut extracts and
alkaloids, a finding which has been questioned by Jeng
et al, who found the opposite. Thus it would seem that
OSMF fibroblasts have the capability to produce
collagen in excess, but what triggers it, is
controversial20.
A study in 1997 demonstrated the copper content in
areca nut to be relatively high and that it is released in
the mouth with chewing. Other reasons proposed for
excessive accumulation of' collagen are decreased
collagenase activity in OSMF mucosa and reduced
phagocytosis by OSMF researchers. In both instances
accumulation of collagen is enhanced21.
Analysis of OSMF grades morphometrically depicted
mean blood vessel area and the mean vessel diameter
showed a marked increase in grade II and a marker
decrease in grade IV and the grade III, collagen
thickness (mum) increased according to increasing
grade while density of endothelial cells decreases22.
Myofibroblasts are contractile cells expressing α-
smooth muscle actin (α-SMA) and are considered
primary producers of extracellular matrix after injury.
Their accumulation has been established as a marker of
progressive fibrosis in organs like lungs, liver, kidney
and skin. It was found that there was a progressive
increase in myofibroblasts from early to advanced
stages23.
Light based system
The fluorescence emission spectra represented reduced
intensity in OSMF patients can be attributed to the
generalized distortion of fluorescence emission by the
presence of collagen. Collagen causes distortion of
fluorescence emitted by other fluorophores such as
tryptophan, NADH, FAD, and hemoglobin24.
Polarized microscopic, examination revealed, there
was a gradual decrease in the green-greenish yellow
color of the fibers and a shift to orange red-red color
with increase in severity of the disease. Thereby, it
appeared that the tight packing of collagen fibers in
OSMF progressively increased as the disease progressed
from early to advanced stages25.In the OSMF mucosa,
the epithelium (EP) thickness becomes smaller and the
standard deviation (SD) of A-mode scan intensity in the
laminar propria (LP) layer also becomes smaller based
on the noninvasive clinical scanning of a swept-source
optical coherence tomography (OCT) system26.
Ultrasonography
A Study to evaluate OSMF by clinical and
histopathological examination, and compare the results
with Color Doppler and spectral Doppler showed
decreased vascularity and Peak systolic velocity (PSV)
in lesional area27.
Precancerous nature and malignant transformation
The precancerous nature of OSMF was first described
by Paymaster in 1956 when he observed slow growing
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quamous cell carcinoma (SCC) in one third of the
patients with the disease28.
An archival series of oral biopsies from Karachi,
Pakistan in 1998 consisted of 21 cases of OSMF and 27
cases of SCC, of which 6 had arisen from OSMF, were
used to examine the aberrations in the structure and
expression of the p53 tumor suppressor gene. Mutation
in p53 or loss of heterozygosity (deletion of a band)
were seen in 13/21 cases of OSMF and 15/27 cases of
SCC29.
A study in 2003 was taken up (i) to determine the
expression of aberrant p53 in Oral Sub Mucous Fibrosis
(OSMF) and Oral SCC patients. (ii)To study correlation if
any relation exist between p53 expression and degree
of dysplasia in OSMF and SCC patients and (iii)To study
correlation if any between p53 expression and habits in
OSMF and SCC patients. Results of the study reveals 18
cases of OSMF and 26 cases of SCC were positive for
p53 protein. Only 4 cases of SCC showed (++) grade and
the rest all had (+) grade. Out of 75 patients, 65 had
the habit of smoking and chewing, 4 patients history of
habit was not known. Among patients with habits (65),
40 specimens were +ve for p53 stain and 2 out of 6
without history of habit, 2 out of 4 unknown history of
habit took up p53 stain30.
A study in 2010 in which Polymerase chain reaction-
restriction fragment length polymorphism (PCR-RFLP)
analysis was used to determine the FAS and FASL
polymorphisms in 294 oral SCC, 53 OSMF, and 84 oral
leukoplakia (OL) patients, as well as in 333 healthy
controls. A two to four fold difference in the risks of
betel quid chewing, alcohol consumption, and smoking
on oral SCC development were observed between
participants with different FAS polymorphisms. FAS
polymorphisms were significantly correlated with the
malignant potential of OSMF. It was found that FAS A (-
1377)-G (-670) haplotype may play a role in the
malignant potential of OSMF31.
CONCLUSION
Even though many modalities are tried in OSMF still
histopathology is considered as gold standard. Many
more investigations are required so that it can be
simple and noninvasive for the Physician and patient to
tolerate. Further this condition has likely malignant
transformation so an early diagnosis is essential and
more imperative for patient to quit the habit.
REFERENCES
1. Pundir S, Saxena S, Aggrawal P. Oral submucous
fibrosis a disease with malignant potential -
Report of two Cases. J Clin Exp Dent., 2010;
2(4): 215-8.
2. Hasan S, Sherwani O, Ahmed S, Khan MA. Oral
submucous fibrosis turning into malignancy- A
39
Ramachandran et al. UJP 2013, 02 (02): Page 37-41
case report and review of literature. J Orofac
Sci., 2011; 3(2): 30-36.
3. Yazdanpanah MJ, Banihashemi M, Pezeshkpoor
F, Famili S, LayeghP , Katebi M, Hamidi H. Oral
submucous fibrosis in a young patient. Acta
Dermatoven APA., 2009; 18(4): 176-178.
4. Kuo MYP, Chen HM, Hahn LJ, Hsieh CC, Chiang
CP. Collagen Biosynthesisin Human Oral
Submucous Fibrosis Fibroblast Cultures J Dent
Res., 1995 ; 74: 1783-8.
5. Smitha B, Donoghue M. Clinical and
histopathological evaluation of collagen fiber
orientation in patients with oral submucous
fibrosis. J Oral Maxillofac Pathol., 2011; 15(2):
154-60.
6. Shah N, Kumar R, Singh MK. Hematological and
Histological studies in Oral Submucous Fibrosis.
JIDA., 1993; 64: 383-8.
7. Ankle MR, Kale AD, Nayak R. Mast cells are
increased in leukoplakia, oral submucous
fibrosis, oral lichen planus and oral squamous
cell carcinoma. J Oral Maxillofac Pathol., 2007;
11: 18-22
8. Anuradha CD, Devi CSS. Serum protein, ascorbic
acid and iron and tissue collagen in oral
submucous fibrosis- a preliminary study. Indian
J Med Res., 1993; 98: 147-51.
9. Desa JV. Submucous fibrosis of the palate and
cheek. 6TH International Congress of
Otolaryngology, Washington, D.C. 1957.
10. Trivedy CR, Warnakulasuriya KAAS, Peters CJ,
Hazarey VK, Johnson KW. Raised tissue copper
levels in oral submucous fibrosis. J Oral Path
Med., 2000; 29: 241-8.
11. Rai S, Mody RN. Serum circulating immune
complexes as prognostic indicators in
premalignant and malignant lesions of oral
cavity during and following radiotherapy. J
Cancer Res Ther., 2012; 8 Suppl 1:S116-22.
12. Sawhney H, Kumar CA. Correlation of serum
biomarkers (TSA & LSA) and epithelial dysplasia
in early diagnosis of oral precancer and oral
cancer. Cancer Biomark., 2011-2012; 10(1): 43-
9.
13. Shetty SR, Babu SG, Kumari S, Rao V, Vijay R,
Karikal A. Malondialdehyde levels in oral sub
mucous fibrosis: a clinicopathological and
biochemical study. N Am J Med Sci., 2012; 4(3):
125-8.
14. Rajendran R. Oral submucous fibrosis. J Oral
Maxillofac Pathol 2003;7:1-4
15. Ankathil R, Anil S, Vijaykumar T, Bhattathiri
VN, Madhavan VNJ, Nair KM. High risk
predictive cytogenetic markers in oral
premalignant conditions. Proceedings of 3rd
International congress on oral cancer (3A).
Universal Journal of Pharmacy, 02(02), Mar-Apr 2013
www.ujponline.com
16. Phatak AG. Lymphocyte subpopulations B, T
and Null in oral submucous fibrosis. Ind J
Otolaryngol., 1979; 31: 72-5.
17. Patra R, Chakraborty C, Chatterjee J. Textural
Analysis of Spinous Layer for Grading Oral
Submucous Fibrosis International Journal of
Computer Applications., 2012; 48(22): 33-37.
18. Ranganathan K, Kavitha R. Proliferation and
apoptosis markers in oral submucous fibrosis. J
Oral Maxillofac Pathol., 2011; 15(2): 148-53.
19. Van Wyk CW, Olivier A, Hoal-van Helden EG,
Grobler-Rabie AF. Growth of oral and skin
fibroblasts from patients with oral submucous
fibrosis. J Oral Pathol Med., 1995; 24: 349-53.
20. Scutt A, Meghji S, Canniff JP, Harvey W.
Stabilization of collagen by betel nut
polyphenols as a mechanism in oral submucous
fibrosis. Experientia., 1987; 43: 391-3.
21. Trivedy C, Baldwin D, Warnakulasuriya S,
Johnson N, Peters T. Copper content in areca
catechu (betel nut) products and oral
submucous fibrosis. The Lancet., 1997; 349:
1447.
22. Singh M, Chaudhary AK, Pandya S, Debnath S,
Singh M, Singh PA et al. Morphometric analysis
in potentially malignant head and neck lesions:
oral submucous fibrosis. Asian Pac J Cancer
Prev., 2010; 11(1):257-60.
23. Angadi PV, Kale AD, Hallikerimath S. Evaluation
of myofibroblasts in oral submucous fibrosis:
correlation with disease severity. J Oral Pathol
Med., 2011; 40(3): 208-13.
24. Ponnam SR, Chandrasekhar T, Ramani P, Anuja.
Autofluorescence spectroscopy of betel quid
chewers and oral submucous fibrosis: A pilot
study. J Oral Maxillofac Pathol., 2012; 16: 4-9
25. Ceena DE, Bastian TS, Ashok L, Annigeri RG.
Comparative study of clinicofunctional staging
of oral submucous fibrosis with qualitative
analysis of collagen fibers under polarizing
microscopy. Indian J Dent Res., 2009; 20(3):
271-6.
26. Lee CK, Tsai MT, Lee HC, Chen HM, Chiang CP,
Wang YM et al. Diagnosis of oral submucous
fibrosis with optical coherence tomography. J
Biomed Opt., 2009; 14(5): 054008.
27. Manjunath K, Rajaram PC, Saraswathi TR,
Sivapathasundharam B, Sabarinath B,
Koteeswaran D, Krithika C. Evaluation of oral
submucous fibrosis using ultrasonographic
technique: a new diagnostic tool. Indian J Dent
Res., 2011; 22(4): 530-6.
28. Paymaster JC. Cancer of the buccal mucosa; A
clinical study of 650 patients. Cancer., 1956; 9:
431-5.
40
Ramachandran et al. UJP 2013, 02 (02): Page 37-41 www.ujponline.com

29. Trivedy C, Warnakulasuriya KAAS, Tavassoli M, immunohistochemistry. Indian J Dent Res.,

Steingrimsdottir H, Penhallow J, Maher R. p53
aberrations in oral submucous fibrosis and oral
squamous cell carcinoma detected by
immunocytochemistry and PCR-SSCP. J Oral
Pathol Med., 1998; 27: 72–7.
30. Bathi RJ, Prabhat. p53 aberrations in oral sub
mucous fibrosis and oral cancer detected by
2003; 14: 214-9.
31. Wang LH, Ting SC, Chen CH, Tsai CC, Lung O,
Liu TC et al. Polymorphisms in the apoptosis-
associated genes FAS and FASL and risk of oral
cancer and malignant potential of oral
premalignant lesions in Taiwanese population.
J Oral Pathol Med., 2010; 39: 155-61.

Source of support: Nil, Conflict of interest: None Declared

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