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ORIGINAL ARTICLE
Year : 2012  |  Volume : 1  |  Issue : 2  |  Page : 99-105

Quantitative and qualitative analysis of collagen in oral submucous fibrosis


Department of Oral & Maxillofacial Pathology, Panineeya Mahavidyalaya Institute of Dental Sciences & Research Centre, Dilshukhnagar, Hyderabad, Andhra Pradesh, India

Date of Web Publication11-Jul-2012

Correspondence Address:
Gannepalli Ashalata
Professor, Department of Oral & Maxillofacial Pathology, Panineeya Mahavidyalaya Institute of Dental Sciences & Research Centre, Road No.5, Kamala Nagar, Dilshukhnagar, Hyderabad, Andhra Pradesh - 500060
India
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DOI: 10.4103/2277-8632.98350

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  Abstract 

Background: Oral submucous fibrosis (OSMF) is considered as a potentially malignant disease, which is associated with betel quid and pan masala chewing. It is thought to be due to the defective collagen synthesis and degradation. The amount of collagen in the diseased varies with the functional stages and histopathological grades of the disease.
Aims: The aim of the study was to assess the severity of the disease in oral submucous fibrosis, by correlating the functional staging with the histopathological grading and analyze the collagen distribution in different grades of OSMF using picrosirius red stain under polarizing microscope and also to assess the quantitative changes in collagen with respect to various histopathological grades of OSMF using spectrophotometry.
Materials and Methods: The study conducted on 40 subjects, which included 30 subjects with different functional and histopathological grades of oral submucous fibrosis (OSMF), and 10 were in control group. Functional staging and histopathological grading was done based on the definite criteria. A Histopathological and quantitative analysis of collagen was conducted using picrosirius red stain, polarizing microscope and spectrophotometer, respectively. The results were analyzed using chi-square test, and the significance was tabulated.
Results: The functional staging and the histopathological grading did not show statistical correlation, but as the histopathological grade of the disease increased; there was a shift in the polarizing color from yellow-green to orange-red. In the colorimetric estimation, the optical densities of the sample decreased as with increased grades of OSMF.
Conclusion: In the present study, we observed that the histopathological grading of OSMF is not correlating with the functional staging of OSMF. Therefore, it mandates the proper correlation between the functional staging and the histopathological grading before the commencement of the treatment to render better outcome of the treatment.

Keywords: Collagen, oral submucous fibrosis, polarizing microscope, picrosirius red, spectrophotometry


How to cite this article:
Ashalata G, Baghirath PV, Krishna AB, Kumar PU, Tom A. Quantitative and qualitative analysis of collagen in oral submucous fibrosis. J NTR Univ Health Sci 2012;1:99-105

How to cite this URL:
Ashalata G, Baghirath PV, Krishna AB, Kumar PU, Tom A. Quantitative and qualitative analysis of collagen in oral submucous fibrosis. J NTR Univ Health Sci [serial online] 2012 [cited 2014 Oct 1];1:99-105. Available from: http://www.jdrntruhs.org/text.asp?2012/1/2/99/98350


  Introduction Top


In 1952, Schwartz described 5 Indian women at Kenya with a condition of the oral mucosa including the palate and pillars of the fauces, which he called "atrophia idiopathica." Later, it was termed oral submucous fibrosis (OSMF). Other names for this condition are "diffuse oral submucous fibrosis, idiopathic scleroderma of the mouth, idiopathic palatal fibrosis, sclerosing stomatisis, and juxta-epithelial fibrosis." [1]

The number of cases of OSMF has raised rapidly in India from an estimated 250,000 cases in 1980 to 2 million cases in 1993 with an overall prevalence rate of 0.2% to 0.5% and prevalence by gender varying from 0.2 to 2.3% in males and 1.2 to 4.57% in females. [2],[3] The age of occurrence for OSMF is ranging between 20 and 40 years of age. [4] It has been suggested that areca nut chewing, ingestion of chilies, genetic susceptibility, nutritional deficiencies, altered salivary constituents, autoimmunity and collagen disorders may be involved in the pathogenesis of this condition. [1],[5] The chewing of betel quid (BQ) and pan masala has been recognized as the one of the most important risk factors for OSMF as per the recent epidemiological data. [5]

The possible precancerous nature of OSMF was first mentioned by Paymaster who described among patients in Bombay, the development of slow growing squamous cell carcinoma in a third of the patients with OSMF. [4] It is now considered as a potentially malignant disorder with an overall transformation rate of 7.6% over a period of 17 years, among the Indian population. [6]

Various histopathological, immunological, and electron microscopic studies have shown that the pathogenesis of OSMF is associated with the increase in the deposition of extracellular matrix (ECM) by increasing the matrix proteins like collagen and decreasing its degradation by various inhibitory mechanisms. [5] The clinical and histopathological grades of OSMF varies with the amount of collagen present in the ECM, major studies have been focused on the qualitative assessment of the collagen fibers in OSMF. Hence, in the present study, we made an attempt to correlate between the functional staging and histopathological grading and different histopathological grades with respect to the qualitative and quantitative analysis of collagen.


  Materials and Methods Top


The study group comprised of 30 patients with clinical signs and symptoms of OSMF and 10 subjects in the control group who were devoid of any symptoms and any other systemic disorders. An incisional biopsy was performed after obtaining the written consent from all subjects. OSMF cases were clinically categorized into subjects in the study group were grouped functionally based on the criteria by Kiran et al as given below [Figure 1]: [7]
Figure 1: Showing reduced mouth opening (Stage 3 OSMF)

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Stage 1: Mouth opening ≥45 mm

Stage 2: Restricted mouth opening 20-44 mm

Stage 3: Mouth opening ≤20 mm

The hematoxylin and eosin (H and E) stained sections were viewed under the light microscope for the histopathologic grading based on the criteria, which were modified from the original criteria given by Pindborg and Sirsat as given below: [8]

Grade 1: Early hyalinization of juxtaepithelial area, plump young fibroblasts, dilated or congested blood vessels, and presence of inflammatory cells mostly mononuclear lymphocytes, eosinophils, and occasional plasma cells.

Grade 2: Moderately hyalinized collagen, less marked fibroblastic response, presence of mostly fibrocytes, constricted blood vessels, and inflammatory exudate consist of mostly lymphocytes and plasma cells with occasional eosinophils.

Grade 3: Complete hyalinization of collagen with depletion of fibroblasts in those areas, blood vessels are completely obliterated or narrowed, and inflammatory cells are lymphocytes and plasma cells [Figure 2].
Figure 2: Image showing complete hyalinization of the extracellular matrix and loss of rete ridges

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Procedure for polarizing microscopy

The paraffin-embedded tissues were cut into 5 mm thick sections and were stained using Picrosirius red stain. The stained sections were examined under a polarizing microscope to observe the polarizing colors of the collagen fibers in the connective tissue. On considering the variable thickness of normal mucosal collagen fibers as 1-12 mm, collagen fibers with thickness less than or equal to 5 mm were considered as thin fibers and those with more than 5 mm, was considered as thick fibers. The connective tissue was divided into upper half, immediately below the basal lamina and lower half, immediately above the sub mucosa, and a total of 5 randomly selected high power (100 ×) fields were examined in each half, and the mean was considered as the number of fibers pertaining to that area. Only well discernible and distinct fibers were selected in each field. The same procedure was adopted for the control group also.

The thickness and color of the collagen fibers were measured and analyzed using the image analysis software Sigma scan pro 5.

Colorimetric quantification of collagen

The paraffin-embedded sections of 10 μm thicknesses were placed on the glass slide, deparaffinized and stained with 0.1% picrosirius red saturated with picric acid for 30 min in the dark, at ambient temperature. After staining, the sections were rinsed several times with distilled water, and with the help of shaving blade, sections were agglomerated to an assay tube which contained 1 ml of solution of 0.1% of NaOH in absolute methanol (1:1, v:v) to wash out the bound dye from the sections. The absorbency of the washed tissue was read in a spectrophotometer at 540 nm. This absorbency refers to the collagen content in the specimens. As the collagen content of the sample increases, the absorbency decreases.

Statistical analysis

Statistical analysis was done using Medi calc software, Chi square test was done to compare the significance between various groups at the level of significance 0.5.


  Results Top


Total study group was comprised of 30 subjects of OSMF with different functional stages and histopathological grades of OSMF and 10 in the control group. The age group in the study group was ranging from 18 to 37 years with the mean age of 27.13 years and for the control group, 22 to 28 years with the mean age of 26.4 years.

Functional staging of the study group was done based on the mouth opening. Stage 1 comprised of 2 cases, stage 2 comprised of 18 cases, and stage 3 comprised of 10 cases [Table 1]. The experimental group was divided into 3 groups based on the histopathological grading based on the defined criteria, grade 1 comprised of 10 cases, grade 2 comprised of 10 cases, and grade 3 comprised of 10 cases [Table 2].
Table 1: Showing total number of cases with respect to the functional stages

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Table 2: Showing total number of cases with respect to the histopathological grading

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Comparison between the functional staging and the histopathological grading did not show any statistical significance (P = 0.0663) [Table 3].
Table 3: Showing comparison between functional staging and the histopathological grading

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Polarizing microscopic evaluation

The study of the tissue sections in the upper half of the connective tissue revealed that the number of thin fibers with green-grey color decreased in number and the orange-red color increased as the grade of the disease progressed and the number of thick fibers with green-grey color decreased and the orange-red color increased as the grade of the disease advances, and the result obtained was significant (P < 0.001) [Figure 3], [Figure 4], [Figure 5] and [Figure 6], [Table 4] and [Table 5].
Figure 3: Polarizing photomicrograph showing the upper half of the connective tissue from the normal buccal mucosa

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Figure 4: Polarizing photomicrograph showing the upper half of the connective tissue in grade 1 OSMF

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Figure 5: Polarizing photomicrograph showing the upper half of the connective tissue in grade 2 OSMF

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Figure 6: Polarizing photomicrograph showing the upper half of the connective tissue in grade 3 OSMF

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Table 4: Showing average number of thin fibers in the upper half of the connective tissue

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Table 5: Showing the average number of the thick fibers in the upper half of the connective tissue

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In the lower half, the thin fibers showed the reduction of green-grey color and a considerable increase in the number of orange-red fibers as with the progress of disease severity. Also, for the thick fibers, the number of green-grey fibers reduced and the orange-red fibers increased as the grade of disease progressed, and the obtained result showed high significance (P < 0.001) [Figure 7], [Figure 8], [Figure 9] and [Figure 10], [Table 6] and [Table 7].
Figure 7: Polarizing photomicrograph showing the lower half of the connective tissue in normal buccal mucosa

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Figure 8: Polarizing photomicrograph showing the lower half of the connective tissue in grade 1 OSMF

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Figure 9: Polarizing photomicrograph showing the lower half of the connective tissue in grade 2 OSMF

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Figure 10: Polarizing photomicrograph showing the lower half of the connective tissue in grade 3 OSMF

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Table 6: Showing the average number of the thin fibers in the lower half of the connective tissue

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Table 7: Showing the average number of the thick fibers in the lower half of the connective tissue

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Colorimetric analysis

The colorimetric evaluation of collagen revealed a progressive reduction in the optical densities as the disease grade increased. The result obtained was statistically significant (P < 0.001) [Table 8], [Graph 1].
Table 8: Showing the comparison between the histopathological grading and the derived optical densities from spectrophotometry

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  Discussion Top


The cause of increased collagen deposition in the connective tissue in OSMF is directly attributed to the effect of areca nut (betel nut) component of the betel quid, which cause an abnormal increase in the deposition of collagen. [9],[10] The alkaloids and the flavonoids present in the areca nut have shown to exert a stimulatory effect on the buccal mucosal fibroblasts and thus resulting in the accumulation of collagen. This collagen accumulation is directly attributed to the increased collagen cross linking, which in-turn result in reduced collagen degradation along with the reduced collagenase activity. [5]

Recent studies on pathogenesis of OSMF revealed that the arecaidine, a product formed due to the hydrolysis of arecoline, along with copper results in fibroblast stimulation. Along with the increased fibroblast production, the tannins which is another constituent of the areca nut leads to the stabilization of the collagen structure. [2]

Many of the previous studies conducted OSMF to correlate between the clinical staging and the histopathological grading did not show any significant correlation. [7],[8],[11],[12] As the major criteria used in our clinical practice in establishing the treatment protocol for the OSMF patients is the functional staging, but in the present study, the correlation between the functional staging and histopathological grading was not significant, especially between stage 2 and grade 2. It could be due to the fact that the initial fibrosis beginning at the fauces, followed by buccal and labial areas. [13] The preferred biopsy site in our study was the buccal mucosa and due to the inaccessibility of the surgeon to the posterior most areas near the fauces, which could shift most of the stage 1 cases to stage 2, or it could be due to the fact that physiological range of mouth opening varies in different individuals which in turn affect the rate of decreased mouth opening due to the disease. Hence, we correlated histopathological grading with the advanced aids like polarizing microscopy and spectrophotometry, which estimates collagen qualitatively and quantitatively.

The upper and the lower half of the connective tissue revealed that the number of thick and thin orange red fibers increased as the grade of the disease progressed. The possible reason for this finding could be due to the increased collagen cross linking which in-turn result in reduced collagen degradation along with the reduced collagenase activity as the disease progressed. [5] In the advanced stages of the disease, the collagen fibers appeared to be highly hyalinized and the delineation between the fibers was difficult. This tight packing of the collagen fibers could be the other reason for appearance of orange-red color. [11]

When comparing with the upper half of the connective tissue, there was a statistically significant increase in the number of thick fibers in lower half in the study group. It could be due to the commencement of the initial fibrosis in the lower sub mucosa compared to the upper and also may be due to the presence of more healthy and thick collagen fibers and bundles in the submucosa. [14],[15]

There was a progressive reduction in the optical densities in the colorimetric evaluation; the possible reason for this could be the increase in the amount of collagen as the grade of the disease progress and which results in the increased intake of the stain. The increased amount of the stain in the fluid, which is read in the spectrophotometer, results in increased absorbance of the photons which in turn results in the reduction of the optical densities. [16],[17]

In conclusion, the present study revealed that the current criteria used in the clinics for the treatment i.e., functional staging, is showing no correlation with the histopathological grading. We could establish a positive correlation with the histopathological grading to the quantity and quality of collagen present in different grades of OSMF. This fact, therefore, necessitate the need of proper histopathological grading of the disease before commencing the treatment, which could result in better outcome for the treatment and prevent further malignant transformation.

 
  References Top

1.Rajendran R. Oral submucous fibrosis: Etiology, pathogenesis, and future research. Bull World Health Organ 1994;72:985-96.  Back to cited text no. 1
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2.Tilakaratne WM, Kinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncol 2006;42:561-8.  Back to cited text no. 2
    
3.Reddy V, Wanjari PV, Banda NR, Reddy P. Oral submucous fibrosis: Correlation of clinical grading to various habit factors. International Journal of Dental Clinics 2011;3:21-24.  Back to cited text no. 3
    
4.Pindborg JJ. Oral submucous fibrosis as a precancerous condition. J Dent Res. 196645 (Suppl 3):546-53.  Back to cited text no. 4
    
5.Rajalalitha P, Vali S. Molecular pathogenesis of oral submucous fibrosis - a collagen metabolic disorder. J Oral Pathol Med 2005;34:321- 8.  Back to cited text no. 5
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6.Murti PR, Bhonsle RB, Pindborg JJ, Daftary DK, Gupta PC, Mehta FS. Malignant transformation rate in oral submucous fibrosis over a 17- year period. Community Dent Oral Epidemiol 1985;13:340-1.  Back to cited text no. 6
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7.Kiran KK, Saraswathi TR, Ranganathan K, Uma Devi M, Joshua E. Oral submucous fibrosis: A clinic-histopathological study in Chennai. Indian J Dent Res 2007;18:106-11.  Back to cited text no. 7
    
8.Shruthi P, Ajay KC, Mamta S, Mangal S, Ravi M. Correlation of histopathological diagnosis with habits and clinical findings in oral submucous fibrosis. Head Neck Oncol 2009;1-10.  Back to cited text no. 8
    
9.Canniff JP, Harvey W. The aetiology of oral submucous fibrosis: The stimulation of collagen synthesis by extracts of areca nut. Int J Oral Surg 1981;10:163-7.  Back to cited text no. 9
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10.Harvey W, Scutt A, Meghji S, Canniff JP. Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids. Arch Oral Biol 1986;31:45-9.  Back to cited text no. 10
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11.Ceena DE, Bastian TS, Ashok L, Rajeswari GA. Comparative study of clinicofunctional staging of oral submucous fibrosis with qualitative analysis of collagen fibres under polarizing microscopy. Indian J Dent Res 2009;20:271-6.  Back to cited text no. 11
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12.Saurabh G, Junaid A, Mohit PS, Prashant N. Oral submucous fibrosis: A clinico-histopathological comparative study in population of Southern Rajasthan. J Carcinogene Mutagene 2010;1.  Back to cited text no. 12
    
13.Haider SM, Merchant AT, Fikree FF, Rahbar MH. Clinical and functional staging of oral submucous fibrosis. Br J Oral Maxillofac Surg 2000;38:12-5.  Back to cited text no. 13
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14.Anna PJ, Rajendran R. Submucosa precedes lamina propria in initiating fibrosis in oral submucous fibrosis -evidence based on collagen histochemistry. Oral Maxillofac Pathol 2010;1.  Back to cited text no. 14
    
15.Van Wyk CW, Seedat HA, Philips VM. Collagen in submucous fibrosis: An electron-microscopic study. J Oral Pathol Med 1990;19:182-9.  Back to cited text no. 15
    
16.Rodrigues Jr AJ, Rodrigues CJ, da Cunha AC, Jin Y. Quantitative analysis of collagen and elastic fibres in the transversalis fascia in direct and indirect inguinal hernia. Rev Hosp Clin Fac Med Sao Paulo 2002;57:265-70.  Back to cited text no. 16
    
17.Finkelstein I, Trope GE, Basu PK, Hasany SM, Hunter WS. Quantitative analysis of collagen content and aminoacids in trabecular meshwork. Br J Ophthalmol 1990;74:280-2.  Back to cited text no. 17
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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