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ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 3  |  Page : 181-184

Oral submucous fibrosis: Clinical and histopathological correlation of collagen fibers using Masson's trichrome and Van Gieson stains


1 General Practitioner, Duhita Multispeciality Dental Clinic, Vijayawada, Andhra Pradesh, India
2 Department of Oral Pathology, SIBAR Institute of Dental Sciences, Tekkallapadu, Guntur, Andhra Pradesh, India
3 General Practiner, Tirupati, Andhra Pradesh, India

Date of Web Publication17-Sep-2018

Correspondence Address:
Dr. Kiran K Kattappagari
Department of Oral Pathology, SIBAR Institute of Dental Sciences, Tekkallapadu, Guntur, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_78_17

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  Abstract 


Background: Oral sub mucous fibrosis (OSMF) is chronic insidious diseases of the oral cavity, pharynx and larynx with unknown etiology. It is characterized by mucosal rigidity. The proposed etiological factor for causing OSMF areca nut. Areca nut and arecoline cause fibroblast proliferation and collagen production.
Aim and Objectives: Correlate the clinical and histopathological grading using Masson's trichrome and Van Gieson's stain in OSMF.
Materials and Methods: Thirty patients with clinically proven cases of OSMF and grouped according to their mouth opening. Incisional biopsy was done. The sections were stained with hematoxylin and eosin for histopathological conformation. Selected cases were sectioned and stained with Masson's trichrome and Van Gieson special stain and graded histopathologically. Data was analyzed using SPSS 20.0 version. Pearson's correlation coefficient and Chi-square test were done to find out the association between variables. Student's t-test was used to find clinical and histopathological correlation.
Results: The mean age of OSMF patients was 20–40 years and when compared with gender males were affected more than females. The stains between Masson's trichrome with Van Gieson's stain showed significant correlation, but when compared between clinical and histopathological grading there was no statistically significant.
Conclusion: Clinical staging and histopathological grading in OSMF there is no correlation when using special stains such as Masson's trichrome and Van Gieson's stain.

Keywords: Clinical, histopathological, massan trichrome stain, oral sub mucous fibrosis, Van Gieson's stain


How to cite this article:
Kanneganti S, Kattappagari KK, Tanuja K, Chandra K L, Poosarla C, Baddam VR. Oral submucous fibrosis: Clinical and histopathological correlation of collagen fibers using Masson's trichrome and Van Gieson stains. J NTR Univ Health Sci 2018;7:181-4

How to cite this URL:
Kanneganti S, Kattappagari KK, Tanuja K, Chandra K L, Poosarla C, Baddam VR. Oral submucous fibrosis: Clinical and histopathological correlation of collagen fibers using Masson's trichrome and Van Gieson stains. J NTR Univ Health Sci [serial online] 2018 [cited 2018 Dec 18];7:181-4. Available from: http://www.jdrntruhs.org/text.asp?2018/7/3/181/241292




  Introduction Top


Diseases of different types affect the human beings. However, some of them show predilection to certain races. One among them is oral sub mucous fibrosis (OSMF) which predominantly occurs in Indians and South East Asians. OSMF is “an insidious, chronic disease affecting any part of the oral cavity, sometimes pharynx, and esophagus. It is characterized by mucosal rigidity of varying intensity due to the fibroblastic transformation of the juxta epithelial layer, resulting in a progressive inability to open the mouth”[1] it was first reported by Schwartz (1952) it was Sushruta, an ancient Indian medical treatise who described a condition called VIDARI. It is a disease of mouth and throat in which progressive narrowing of mouth opening, depigmentation of oral mucosa, and pain on taking food were seen.[2] Various mechanisms were suggested for the etiopathogenesis of OSMF. These include stimulation of fibroblast proliferation and collagen synthesis by areca nut alkaloids, clonal selection of fibroblasts with high amount of collagen production and stabilization of collagen structure by catechins and tannins from areca nut.[3]

The amount of fibrosis, hyalinization, and muscle degeneration can be studied using stains starting from routine hematoxylin and eosin to special stains such as Masson's trichrome, Van Gieson, Mallory's hematoxylin, and other stains.[4] Though OSMF has been graded clinically and histologically there is paucity of information related to association between clinical mouth opening and extent of fibrosis.[5] The present study was undertaken to correlate clinical staging and histopathological grading and qualitatively assess which stain among hematoxylin and eosin, Masson trichrome, and Van Gieson are superior for staining collagen under light microscope.


  Materials and Methods Top


Thirty patients with OSMF cases were included for this study. All the subjects were patients attending the department of Oral and Maxillofacial Pathology, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh during the period of 2012 to 2014. The clinical criteria for diagnosis of OSMF were the presence of burning sensation while eating spicy food, presence of fibrous bands and loss of elasticity of buccal mucosa, labial mucosa, and inability to open the mouth.[6] Difficulty in opening the mouth due to reasons like inflammation, temporomandibular joint abnormalities was excluded from the study. Selected clinical diagnosis was confirmed by incisional biopsy. Each of the OSMF showed an atrophic epithelium juxta-epithelial hyalinization with dense bundles of collagen fibers and variable chronic inflammatory cell infiltration. The clinical stages of OSMF grouped into three groups based on mouth opening according to Ranganathan K et al., clinical staging as follows:

  • Stage 1: Mouth opening >45 mm
  • Stage 2: Restricted mouth opening 20–44 mm
  • Stage 3: Mouth opening <20 mm.[7]


For all the subjects of OSMF Hb% was estimated to rule out anemia and method used was Sahli's colorimetric method and late sent for incisional biopsy. The tissues were processed for paraffin embedding. Three sections of 4 μm thickness were prepared using semiautomatic microtome (Leica RM 2255, Leica Biosystem rotary microtome, Walldorf, Germany) for hematoxylin and eosin, Masson's trichrome, and Van Gieson staining. The staining procedure was standardized according to Bancroft et al. 2002.[8]

The following histological classification is followed according to Kiran Kumar et al. 2007.

  • Grade I: Loose/dense fibrous connective tissue
  • Grade II: Loose or dense with partial hyalinization
  • Grade III: Complete hyalinization of the fibrous connective tissue with muscle degeneration [9]


Statistical analysis

Data analysis and database management were done using SPSS version 20.0. Pearson's correlation coefficient and Chi-square test were done to find out the association between variables. Student t- test was utilized to find out the mean of parameters.


  Results Top


Out of 30 cases of OSMF taken for the study majority, i.e., 27 (90%) were male and 3 (10%) were female [Table 1], [Graph 1]. More than 60% of the subjects were less than 26 years of age group. The association between histopathological grading with clinical staging, maximum cases of OSMF, i.e., 15 are in clinical stage 2 When histopathological grade I followed by nine cases of OSMF are in stage 2 with histopathological grade II. There is no significant correlation between clinical staging and histopathological grading when observed with hematoxylin and eosin stain. When the association between clinical staging and histopathological grading is observed with Masson's trichrome majority of cases fall under clinical stage 2 and histopathological grade II. There is no significant correlation in association with clinical staging and histopathological grading.
Table 1: Distribution of samples by gender

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The association between clinical staging and histopathological grading there was correlation is observed with Van Gieson's staining 24 (80%) subjects are in clinical stage 2 followed by 3 (10%) stage 1 and 3. 6) Association between clinical staging and histopathological grading in relation with Van Gieson's showed no significant correlation with clinical staging and histological grading. When the association between histological grading with all three stains (hematoxylin and eosin, Masson's trichrome, and Van Gieson's) Van Gieson's shows better histological features when compared to hematoxylin and eosin. When compared with histopathological grading with Masson's trichrome and Van Gieson's the observations showed statistically significant with P ≤ 0.032. When correlation between clinical staging with histopathological grading using all three stains, the histopathological grading showed stastically significant with P ≤ 0.028 only in Masson's trichrome and Van Gieson's stain [Table 2].
Table 2: Correlation between clinical staging, histological grading H and E, histological grading MT, and histological grading VG by spearman's rank correlation coefficient method

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  Discussion Top


OSMF, a crippling disease of the oral mucosa has evoked the interest of dental professionals in different parts of the world.[10] Its occurrence in various parts of India has been reported as case reports, review, and original research. The disease shows idiosyncrasy in that, it is confined to a particular geographic area.[11] The etiology of OSMF is still perplexing. OSMF is a disease of altered collagen metabolism. The lesion is characterized by increased collagen fiber formation in initial stages, followed by formation of dense collagen fiber bundles and different degrees of hyalinization. Alteration in the collagen metabolism changes the flexibility of mucosal tissue, leading to restricted mouth opening.[3] According to Sinor et al. in 1990 and Kiran Kumar K et al., in 2003 have done a case control study in OSMF and observed male predominance with wide variation in age and sex distribution of OSMF in Indian population. [9,12] In our study we observed that younger age group (26 years) was involved with OSMF. There was one study by Kiran Kumar et al., in 2007 done to compare between clinical and histopathological correlation, they observed that there is no significant correlation between clinical staging and histopathological grading. This could be because fibrous bands formed retro molar area to anterior and site of biopsy should be specific.[9] In the current study, 80% of the patients showed clinical stage 2, of them only 43% of the cases showed histopathological grade II. The remaining 17% of the cases showed histopathological grade III. Statistically no significant correlation was observed between clinical staging and histopathological grading. These findings were similar to the study done by above study. James E Hamner et al. in 1971 used Van Gieson stain for identification of collagen fibers in OSMF. They observed that juxta-epithelial connective tissue band was amorphous, hyalinized, non-bundler, and stained faint grayish pink, rather than customary deep red.[13] In this current study, 78% of the histopathological sections stained with Van Gieson's, showed the faint grayish pink color rather than the customary deep red. This could be because of collagen metabolism disorder which is characterized by increased collagen production and decreased collagen degradation which leads to hyalinization. These hyalinized areas showed faint grayish pink color. The other reason could be because of Bouin's fixative.

There was one more study done by Rooban T et al. in 2005, the study was conducted to demonstrate collagen fibers using Masson's Trichrome stain. They observed both collagen fibers and muscle fibers simultaneously; using this stain. The collagen was stained blue while the muscle took brilliant red color. This color contrast facilitated a visual discrimination between muscle and collagen. This technique is very simple and easily reproducible.[14] In the current study, majority of the cases were in clinical stage 2 (75%) and histopathological grade II (84%). The collagen fibers were observed dark blue in color whereas muscle fibers were stained bright red. These findings are similar to a study done by Rooban et al., 2005[14] Karan Rajpal et al., 2013 done a study to compare the efficacy of Masson's Trichrome stain and Van Gieson's to stain collagen fibers in OSMF. They have observed that Masson's Trichrome will give better collagen stain when compared with Van Gieson's and other stains such as hematoxylin and eosin.[15] Contrary to the above study, this study showed collagen fibers and muscle fibers were stained superior with Van Gieson's, compared to Masson's Trichrome. In our study, Van Gieson's showed better collagen staining property when compared with Masson's Trichrome. This could be because of the property of collagen and its structural alterations.


  Conclusion Top


This study is designed to compare the association of hematoxylin and eosin, Masson's Trichrome, and Van Gieson stains with clinic staging and histopathological grading in OSMF patients and also to know which stain is better to visualize under light microscope. Hematoxylin and eosin stain was widely used for histopathological examination. This could be because of simplicity and ability to demonstrate the collagen fibers. Other stains we have used are Masson's Trichrome and Van Gieson stain to demonstrate the collagen stain intensity. Van Gieson stain showed the amorphous changes in the connective tissues in a striking fashion.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Pindborg JJ, Sirsat SM. Oral Submucous fibrosis. Oral Surg 1966;22:764-79.  Back to cited text no. 1
    
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Constantine VS, Mowry RW Selective staining of human dermal collagen II. The use of picrosirious red F3BA with polarizing microscopy J Invest Derm 1968;50:419-23.  Back to cited text no. 5
    
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Neville BW, Damm DD, Allen CM, Bouquot JE, Allen CM, Chi AC. Oral and Maxillofacial Pathology. 2nd edition. St Louis: Elsevier Saunders; 2002. p. 349-50.  Back to cited text no. 6
    
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Ranganathan K, Uma Devi M, Joshua Elizabeth, Arun Bharadwaj, Rooban T, Viswanathan R. Mouth opening, cheek fl exibility and tongue protrusion parameters of 800 normal patients in Chennai, SouthIndia a base line study to enable assessment of alteration in Oral sub mucous fibrosis. J Indian Dent Assoc 2001:72;78-80.  Back to cited text no. 7
    
8.
John D Bancroft, Marilyn Gamble. Theory and Practice of Histological techniques. 5th edition. Churchill Living Stone; 2002. p. 153.  Back to cited text no. 8
    
9.
Kiran Kumar K, Saraswathi TR, Ranganathan K, Uma Devi M, Elizabeth J. Oral Sub mucous fibrosis: A clinic-histopathological study in Chennai. Indian J Dent Res 2007;18:106-11.  Back to cited text no. 9
    
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Rao AB. Idiopathic palatal fibrosis. Brit J Surg 1962;6:23-5.  Back to cited text no. 10
    
11.
Mohd Akbar. Oral submucous fi brosis-Clinical study. J India Dent Assoc 1976;48:365-74.  Back to cited text no. 11
    
12.
Sinor PN, Gupta PC, Murthi PR, Bhonsle RP, Daftary DK, Mehta FS, et al. A case control study of oral submucous fibrosis with special reference to the etiologic role of areca nut. J Oral Pathol Med 1990;19:182-7.  Back to cited text no. 12
    
13.
Hamner JE 3rd, Mehta FS, Pindborg JJ, Daftary DK. Altered staining reaction of connective tissues in 53 submucous fibrosis patients. J Dent Res 1971;50:388-92.  Back to cited text no. 13
    
14.
Rooban T, Saraswathi TR, Al Zainab FH, Devi U, Eliqabeth J, Ranganathan K. A light microscopic study of fibrosis involving muscle in oral sub mucous fibrosis. Indian J Dent Res 2005;16:131-4.  Back to cited text no. 14
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15.
Rajpal K, Grover N, Senguptha S, Sing S, Sing N, Singh P, et al. Qualitative Comparison of Various Stains to Assess the Tinctorial Properties of Collagen in Oral Submucous Fibrosis (OSMF). J Orofacial Health Sci 2013;4:76-8.  Back to cited text no. 15
    



 
 
    Tables

  [Table 1], [Table 2]



 

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