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Year : 2005  |  Volume : 16  |  Issue : 4  |  Page : 131-4
A light microscopic study of fibrosis involving muscle in oral submucous fibrosis

Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, Chennai 600119, India

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Presence of two canals with type 11 pattern in mandibular canine is around 15%. Even though the incidence rate is high, multiple angled diagnostic radiographs are not regularly taken. This article highlights the importance of multiple-angled radiographs and indicates that the dentist should have a mind set to look out for extra canals in all root canal cases.

Keywords: Type II canal pattern, multiple angled radiographs, second canals

How to cite this article:
Rooban T, Saraswathi T R, Al Zainab FH, Devi U, Eligabeth J, Ranganathan K. A light microscopic study of fibrosis involving muscle in oral submucous fibrosis. Indian J Dent Res 2005;16:131

How to cite this URL:
Rooban T, Saraswathi T R, Al Zainab FH, Devi U, Eligabeth J, Ranganathan K. A light microscopic study of fibrosis involving muscle in oral submucous fibrosis. Indian J Dent Res [serial online] 2005 [cited 2023 Oct 4];16:131. Available from:

   Introduction Top

Oral submucous fibrosis (OSF) is a chronic insidious disease, affecting any part of the oral cavity and sometimes pharynx Although occasionally preceded by and/ or associated with vesicle formation, it is always associated with a juxta-epithelial inflammatory reaction followed by a fibroblastic change of the lamina propria, with epithelial atrophy leading to stiffness of the oral mucosa and causing trismus and inability to eat [1]. Epidemiological studies revealed the premalignant potential of OSE Although the etiology of OSE is not completely understood, there is a close association with the habit of chewing arecanut[2].

Various mechanisms were suggested for the etiopathogenesis of OSE. These include stimulation of fibroblast proliferation and collagen synthesis by arecanut alkaloids (Harvey W et al , 1986), clonal selection of fibroblasts with a high amolmt of collagen production during the long term exposure to arecanut ingredients (Meghji S et al , 1987), stabilization of collagen structure by catechin and tannins from arecanut (Scutt A et al , 1987), decreased secretion of collagenase (Shieh TY et al , 1992), production of stable collagen (type I trimer) by OSF fibroblasts (Kuo MYP et al , 1995), increase in collagen cross linking by up-regulation of lysyl oxidase (Ma RH et al , 1995), deficiency in collagen phagocytosis (Tsai CC et al , 1999) and effect of fibrogenic cytokines secreted by activated macrophages and T lymphocytes on fibroblasts (Hague ME et al , 2000) [3].

OSE has been graded histologically into four stages, depending on fibroblasticresponse, hyalinization and inflammation [1]. Binnie and Cawson have reported a homogenous collagenous subepithelial zone along with degeneration of muscle fibers [4]. Oliver AJ et al reported the presence of dense collagen bundles that were randomly oriented and extended into the underlying striated muscles [5]. The electron microscopic features of muscle fibers of OSF patients revealed partial to complete loss of plasma membrane, filled with large pools of homogenous material and often surrounded with edematous fluid [6].

Though there are many histological studies of OST reporting fibrosis and hyalinization in the sub-epithelium, there is a paucity of information related to the association of fibrosis and muscle in this condition. The present work has been undertaken to describe the nature and extent of fibrosis within muscle and to correlate this with the mouth opening (MO) in OSE patients.

   Materials and method Top

The study population was patients attending Ragas Dental College and Hospital, Chennai, India who were clinically and histologically diagnosed as OSF. The clinical criteria stipulated by Ranganathan et al was followed [7].

The mouth opening (interincisal distance of maxillary and mandibular incisors at maximum possible mouth opening) was measured and graded as follows: grade 1 (> 40rmn), grade 2 (20-39mm) and grade 3 (<19 mm). Incisional biopsy from right buccal mucosa opposite to premolar-molar junction was taken and tissues were processed for routine haemotoxylin and eosin staining and Masson's Trichrome staining for histopathological study.

The fibrosis was graded as

Stage 1

Fibrosis limiting to laminapropriaalone

Stage 2

Fibrosis involving superficial region of muscle bundle

Stage 3

Fibrosis involving deeper regions of MB

Stage 4

MB replaced by fibrosis.

Hyalinization was graded as present (+) or absent(-). Data were entered and analyzed using Statistical Package for Social Servicesa, Version 10.0.5. Pearson's Chi square test was done to find the association between grade of MO and the stage of fibrosis. `p' value of less than 0.05 was considered as statistically significant.

   Results Top

According to MO, there were 26.67% (8 cases) in grade 1 OSF, 66.67% (20 cases) in grade 2 OSF and 6.67% (2 cases) in grade 3 OSE. In 23.34% (7 cases) of OSF cases, belonged to stage-1 fibrosis was evident only in sub­epithelial zone, not involving the muscle. In these cases the MB was arranged as fascicles, covered by a thin layer of fibrous connective tissue both in cross and longitudinal sections [Figure - 1]. In 40% (12 cases) of OSF cases, fibrosis was seen extending into the superficial regions of MB (stage-2) [Figure - 2] while in 10% (3 cases) of all OSF cases had involvement of deeper regions of MB (stage-3) including neurovascular bundles [Figure - 3].Thin collagen fibers were present in-between muscle fiber (MF) and ME showed broken sarcolemma [Figure - 4]. In 26.67% (8 cases) of all OSF cases (stager 4), only a few remnants of ME were seen and the missing MB area was replaced by fibrous tissue [Figure - 5].

Of the OSF patients in stage-1 fibrosis 42.9% of cases belonged to grade 1 OSF and 42.9% to grade 2 OSE. 83.3% of cases in stage-2 fibrosis belonged to grade 2 OSF whereas 66.7% of stage-3 fibrosis and 62.5% of stage-4 fibrosis OSF cases belonged to grade 2 OSF [Table 1].

Along with fibrosis, 33.34% of cases exhibited hyalinization in all stages with varying proportion [Table 1]. Hyalinization was also seen extending into the MB zone associated with muscle atrophy [Figure - 6].

The correlation between clinical grading of MO and histological staging of fibrosis did not show any statistical significance.

   Discussion Top

In OSF, the oral mucosa appears pale and blanched. Fibrous bands appear earlier in the course of disease, often symmetrically, in a vertical direction usually in the retromolar region and adjacent buccal mucosa. The clinical appearance varies from a localized pale white area to wide blanched region depending on the fibrosis. 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 [8]. Hence the role of fibrosis in muscle warrants investigation. The muscle could be damaged primarily and repaired by fibrosis or progression of fibrosis inducing secondary muscle changes.

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 [9], [10]. Though there are more specific and sensitive special stains like Phospho 'Iungstic Acid Hematoxylin (PTAH) to observe changes in muscle fibers, we have chosen Masson's Trichrome stain as this offered a simultaneous contrast color to the collagen fibers along with ME and MB. The collagen was stained blue while the muscle took a brilliant red color [11]. This colour contrast facilitated a better visual discrimination between muscle and collagen. The technique is also simple and easily reproducible.

Normal striated muscles are aligned as fascicles, each of which is invested by a thin fibrous connective sheath known as perimysium. Within this sheath nerves, arteries, arterioles and vein travel. Each muscle fiber normally appears to be invested by endomysium, a mesenchymal matrix made of collagen, elastic and reticulin fibers to provide support to the vasculature of the fascicles.

Under normal situations, the injured muscle initiates the repair of myofibres by the proliferation of satellite cells present in endo and perimysium. These are reserve pool of stem cells that can generate differentiated myocyte after injury. Inflammatory cells appear soon, becoming a source for pro-inflammatory cytokines. The satellite cells are activated by growth factors. These cells proliferate into myocytes and fuse to form multinucleated myofibres [12]. The same inflammatory mediators also stimulate the fibroblasts to proliferate and help in the healing process. Successful functional repair of injured muscle is characterized by ME regeneration while impairment denotes that it is replaced by fibrosis.

The role of the cytoxicity and genotoxicity of arecanut products, trace elements and cytokines in the causation of fibrosis has been well documented in the literature [13]. The effect of other additive materials including catechu, lime and/ or tobacco is not known. The role of free oxygen radicals could not be ignored. Arecanut chewing causes vasoconstriction of blood vessels, which may lead to ischaemia [14]. The tissue edema resulting secondarily to the inflammatory changes also promotes fibroblast activity and collagen production, by the action of soluble mediators of inflammation. It has been reported that the over-expression of transforming growth factor B-1 (TGF­Bl) in various injured tissues is the major cause of fibrosis in animals and humans [15]. Hsu I-IJ et al and Haque et al , studied the role of TGF - B in OSF and reported that the levels of TGF- B in OSF are higher [16],[17],[18]. The general increase in pro-inflammatory cytokines and growth factors along with reduced production of interferon may play an important role in the fibrosis observed in this condition [17],[18].

Rajendran et al studied the histopathology of OST from buccal mucosa using light and electron microscope. They described fibroblasts, bindles 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 MB under light microscope. They observed focal myofibrillar lysis, hypercontraction of myofibres and extensive fatty infiltration between muscle bindles under electron microscope [19]. Gupta SC et al studied histopathologically the palatal muscle of OSF patients and reported degenerative changes in ME. 20.8% of their cases exhibited degenerative changes either as loss of cross striations or edema of ME surrounded by inflammatory cells or as atrophy [9].Our study using light microscopy is consistent with the findings of Rajendran et al and Gupta SC et al . Though fibrosis inside the muscle was reported earlier, our study highlights the effect of fibrosis on the ME.

In 23.34% of OSF cases where fibrosis was evident sub­epithelially but not extended in to the muscle, we consider that the muscle is spared from the direct noxious effect of the arecanut products. In these cases, MB was well enclosed in fascicles by a thin layer of fibrous connective tissue in cross and longitudinal sections, indicating that there is no demonstratable damage to the muscle.

In some OSF cases, we observed fibrosis extending in to the superficial (40%) and deeper regions (10%) without visible changes in MB. In the other OSF cases, it was observed that there were thin collagen fibers present in ­between ME that showed broken sarcolemma Along with this, fibrosis with hyalinization was also seen extending into the MB zone resulting in atrophy of the muscle whereas in 26.67% other cases only a few remnants of ME were seen and the missing MB area was replaced by fibrous tissue.

Progression of fibrosis as asequelae of the disease process in and around the muscle as well in the region of neurovascular bundles could cause functional and nutritional impairment. Due to the compression of NIB along with neuromuscular bundles by thick collagen fibers, the myofibres suffers damage. Compartmenta­lization of muscle tissues by dense collagen may cause myoischemia. The fibrosis makes the muscle more susceptible for repeated injury upon function. The role of such repeated muscle trauma and the consequent micro­hemorrhages could not be underestimated.

There is a poor statistical correlation between grade of MO and staging of fibrosis as indicated by the insignificant `p' value. This implies that the cause of reduction of MO in OSF is multifactorial and determined by factors like site and extent of fibrosis, regional anatomic variation, tone of the muscle, the neuromuscular coordination and the anatomical and physiological integrity of the underlying oral musculature. The observation of grade 3 OSF (MO) seen in 2 cases, exhibiting stage-1 and 2 of fibrosis implies the possibility of biopsy being taken in the more anterior region whereas severe fibrosis would have been present in the posterior region. Cases exhibiting stage-3 and stage-4 fibrosis occurring in cases belonging to grade 1 MO supports the above view. Hyalinization was observed in 100% of grade 3 OSF, 30% (6 cases) of grade 2 OSF and 25 % of grade 3 OSF cases. This did not have any influence on the grade of MO.

   Conclusion Top

The light microscopic study of OSF revealed varying degrees of alterations involving the muscle fibers as the disease progresses. We consider that the damage to the ME appear more as consequences of fibrosis rather than by a direct injury to the muscle by the arecanut products. The factors other than fibrosis that restrict MO in OSF cases need to be explored.

   References Top

1.Pindborg JJ and Sirsat SM (1966): Oral submucous fibrosis oral Surg Oral Med Oral Path, 22:764-79.  Back to cited text no. 1    
2.Pindorg JJ (1989): Oral submucous fibrosis: a review, Annals of the Academy of Medicine Singapore, 18:603-07.  Back to cited text no. 2    
3.Rajendran R (2003): Oral Submucous Fibrosis, JOMFP, 7[1]:1-4.  Back to cited text no. 3    
4.Binnie WA and Cawson RA (1972)_ Anew ultra structural finding in Oral Submucous Fibrosis, BrfDennato1,86:286.  Back to cited text no. 4    
5.Oliver AJ and Radden BG (1992): Oral Submucous Fibrosis. Case report and review of literature, Aust Dent J, 37[1]:31-34.  Back to cited text no. 5    
6.El-Labban NG and Canniff JP (1985): Ultra structural finding in Oral Submucous Fibrosis, J Oral Pathol, 14: 709-17.  Back to cited text no. 6    
7.Ranganathan K, UmaDevi M, Elizabeth Joshua, Kiran Kumar K, Saraswathi TR (2004): Oral Submucous Fibrosis: a case- control study in Chennai, South India, J Oral Pathol Med, 33: 274-77.  Back to cited text no. 7    
8.Rajendran R (1994): Oral Submucous Fibrosis, etiology, pathogenesis and future research, Bulletin of World health Organization, 72 [6]: 985-96.  Back to cited text no. 8    
9.Gupta SC, Sanjay Khanna, Mangal Singh, Singh PA (2000): Histological changes to palatal and paratubal muscle in oral submucous fibrosis, The Journal of Laryngology and Otology, 114: 947-950.  Back to cited text no. 9    
10.Hamner JC 3rd, MehtaFS, Pindborg ff. Daftary DK (1971): Altered staining reaction of connective tissue in 53 submucous fibrosis patients, JDentRes, 50 [2]:388-92.  Back to cited text no. 10    
11.Bancroft JD and Alan Stevens (1996): Theory and Practice of Histological Techniques (fourth Edition), Churchill Livingston, Pearsons Professional Limited, Hong Kong, Page 129.   Back to cited text no. 11    
12.Vinay Kumar, Abul K Abbas, Nelson Fausto, (Eds) (2004): Robbins and Cotran - Pathologic basis of disease (7th Ed), Saunders, Elseiver Inc, Pennsylvania, page 94.  Back to cited text no. 12    
13.ChangYC, Tai KW, Chang MII, Chou LS, Chou MY (1998): Cytotoxic and non-genotoxic effects of arecoline on human buccal fibroblasts in vitro, J Oral Pathol Med. 27[2]:68-71.   Back to cited text no. 13    
14.Boucher BJ, Mannan N (2002): Metabolic effects of the consumption of areca catechu, Addiction Biology, 7:103-10.  Back to cited text no. 14    
15.Vinay Kumar, Abul K Abbas, Nelson Fausto, (Eds) (2004): Robbins and Cotran - Pathologic basis of disease (7th Ed), Saunders, Elseiver Inc, Pennsylvania, page 96-97.  Back to cited text no. 15    
16.Hsu HJ, Chang KL, Yang YR. Stitch TY (2001): The effects of arecoline on the release of cytokines using cultured peripheral blood mononuclear cells from patients with oral mucous diseases, Kaohsiung J Med Set, 17[4]: 175-82. (Abstract)  Back to cited text no. 16    
17.Haque ME, Harris M, Meghji S, Barrett AW (1998): Imnnrnolocalization of cytokmes and growth factors in oral submucous fibrosis, Cytokine,10[9]: 713-9. (Abstract)  Back to cited text no. 17    
18.Haque MF, Meghji S, Khitab U, Harris M (2000): Oral submucous Fibrosis patients have altered levels of cytokine production, J Oral Pathol Med 29[3]:123-8.  Back to cited text no. 18    
19.Rajendran R, Radhakrishnan NS, Kartha CC (1993): Light and Electron microscopic studies on Oral Submucous fibrosis, JIDA, 64[5]: 157-­162.  Back to cited text no. 19    

Correspondence Address:
T Rooban
Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, Chennai 600119
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.29909

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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]

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