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ORIGINAL RESEARCH Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 3  |  Page : 271-276
Comparative study of clinicofunctional staging of oral submucous fibrosis with qualitative analysis of collagen fibers under polarizing microscopy

1 Department of Oral Medicine and Radiology, Manipal College of Dental Sciences, Mangalore, India
2 Department of Oral Pathology and Microbiology, Sardar Patel Institute of Dental and Medical Sciences, Lucknow, India
3 Department of Oral Medicine and Radiology, Bapuji Dental College and Hospital, Davangere, India
4 Department of Oral Medicine and Radiology, College of Dental Sciences, Davangere, India

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Date of Submission20-Feb-2008
Date of Decision31-Mar-2008
Date of Acceptance20-Sep-2008
Date of Web Publication30-Oct-2009


Objective : The aim of the study was to assess the severity of the disease in oral submucous fibrosis (OSF), correlate the clinical, functional staging with histopathological staging, and analyze collagen distribution in different stages of OSF using the picrosirius red stain under polarizing microscopy.
Materials and Methods : The study included randomly incorporated 50 subjects, of whom 40 were patients with OSF, and 10 were in the control group. Clinical, functional staging in OSF cases was done depending upon definite criteria. A histopathological study was conducted using the hematoxylin and eosin stain and picrosirius red stain. Collagen fibers were analyzed for thickness and polarizing colors. Furthermore, clinical, functional, and histopathological stages were compared.
Statistical Analysis : Descriptive data which included mean, SD, and percentages were calculated for each group. Categorical data were analyzed by the chi-square test. Multiple group comparisons were made by one-way ANOVA followed by Student's t-test for pairwise comparisons. For all tests, a P-value of 0.05 or less was considered for statistical significance.
Results : As the severity of the disease increased, clinically, there was definite progression in subjective and objective symptoms. 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 OSF progressively increased as the disease progressed from early to advanced stages. We observed that the comparison of functional staging with histopathological staging was a more reliable indicator of the severity of the disease.
Conclusion : In the present study, we observed that mouth opening was restricted with advancing stages of OSF. The investigation also points to the importance of assessing the
cases of OSF, especially with regard to functional and histological staging in planning the treatment.

Keywords: Collagen thickness, oral submucous fibrosis, picrosirius red stain, polarizing microscopy

How to cite this article:
Ceena DE, Bastian T S, 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;20:271-6

How to cite this URL:
Ceena DE, Bastian T S, 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 [serial online] 2009 [cited 2017 Feb 19];20:271-6. Available from:
In ancient medicine, Shrusrutha described the condition similar to oral submucous fibrosis (OSF) as "Vidari" under mouth and throat diseases. [1] Joshi described the disease for the first time in India and coined the term oral submucous fibrosis. [2]

Clinically, patients suffer from gradual, progressive alterations in oral mucosa resulting in compromised mouth opening to various degrees. The treatment of OSF has been a major concern and more cases are being reported due to causative deleterious habits.

In OSF, connective tissue changes are characterized by a deposition of dense collagen fibers. Even though collagen could be stained with variety of dyes, for histological studies, the picrosirius red stain was found to be superior to other stains. It stained finer collagen fibers more intensely and increased their birefringence spectacularly. [3] It was observed that Sirius red stains are more stable and do not fade easily when compared to other stains. [4]

Picrosirius red staining followed by polarizing microscopy can selectively demonstrate collagen. The differences in polarization colors are caused by fiber thickness as well as packing of collagen. [5] The purpose of the present study was to evaluate collagen fibers in OSF, and assess the severity, which in turn may help to assess treatment and prognosis of OSF.

   Materials and Methods Top

The study comprised 40 patients with OSF, with 10 subjects in the control group, judged by established clinicopathologic parameters. Patients who were using areca nut and areca nut-containing products, tobacco mixed with areca nut in different forms, and who were clinically exhibiting the signs and symptoms of OSF were included in the present study. The control group consisted of tissue sections obtained from normal oral mucosa, from patients who reported for minor oral surgical procedures. Controls were selected after confirming that they were not having any deleterious habits like smoking and chewing tobacco. For the study conducted, ethical protocols were adhered to and written consent was obtained to include them in this study. The patient's personal history was recorded with chewing habits, frequency, duration of chewing, and the brand of tobacco used. The questionnaire provided to the patients included factors like the site of keeping the quid, time duration, and whether he/she swallows it or spits it out. Patients were also asked whether he/she had burning sensation to spicy food. Symptoms like burning sensation, restricted mouth opening, difficulty in swallowing and speech, and their duration were noted in every case. Furthermore, fibrous bands were palpated and their location was recorded. Routine hematological work-up was done in the experimental group and it comprised total leukocyte count, differential count, hemoglobin values, clotting and bleeding times, and the platelet count.

Clinical and functional staging of OSF was done according to Haider et al. [5] However, a slight modification of the criteria was made since early manifestations of OSF in patients were in the buccal mucosa rather than the faucial pillars. Hence, stage I comprised presence of buccal bands only. According to Haider et al., [6] patients exhibited early changes in the faucial bands since they were in the habit of chewing and swallowing the areca nut, unlike patients in India who spat out the chewed deleterious products [Figure 1],[Figure 2],[Figure 3].

Clinical staging was performed based on the presence of palpable fibrous bands in various sites: stage I - buccal bands only; stage II - faucial and buccal bands only; stage III - faucial, buccal, and labial bands.

The histopathological staging of OSF was done according to Pindborg et al. [7] Hematoxylin and eosin sections were used for histological staging of all the cases. Furthermore, they were categorized into four different stages depending on the severity of the disease. After the histological staging, picrosirius red-stained sections were further studied.

Polarizing microscopy

The picrosirius red-stained sections were examined using polarizing microscopy, and the thickness of collagen fibers and polarizing colors of the collagen fibers in the connective tissue were assessed [Figure 4],[Figure 5],[Figure 6]. Considering the variable thickness of normal mucosal collagen fibers as 1-12 mm, the collagen fibers with a thickness 5 mm and less were considered as thin fibers and those with a thickness above 5 mm were considered as thick fibers. Polarization colors were determined separately for thin and thick fibers and recorded. A total of five high- power (100Χ) fields were selected from the sections. Twenty five fibers, which were well discernible and distinct in each section, were randomly selected from the subepithelial region and deeper regions in the connective tissue for evaluation. The same procedure was adopted for the control group. The results thus obtained by the histopathological grading of OSF were compared with respect to the various parameters.

The thickness of collagen fibers were measured by using a Leica research microscope (Leica DMRB; Germany) under polarized light and the color of the fibers in varying thickness was assessed.

Statistical analysis

Categorical data were analyzed by the chi-square test. Multiple group comparisons were made by one-way ANOVA followed by Student's t-test for pairwise comparisons. For all the tests, a P-value of 0.05 or less was considered for statistical significance.

   Results Top

The study included a total of 50 subjects, which included 40 patients with OSF and 10 in the control group. The 40 experimental subjects were in the age range of 18-48 with a mean age of 26 ± 6.3 years. The control group comprised 10 healthy individuals in the age group of 24-42 of whom 8 were males. Of the 40 subjects in the experimental group, 2 were females and 38 were males. Thus, it showed a male predominance. It was noted that the majority of subjects in the present study chewed only gutkha. It was also observed that all 40 subjects had a burning sensation, and only 4 subjects had difficulty in speech and 25 had difficulty in opening the mouth.

Clinical staging was done for all the subjects. Of the 40 OSF patients, 6 had fibrotic buccal bands, 24 had buccal and faucial bands, 10 had buccal, faucial, and labial bands and they were staged into stage I, stage II, and stage III, respectively [Table 1].

Functional staging was assessed depending on the patient's ability to open the mouth. The experimental subjects were divided into three groups depending on the degree of mouth opening. There were 29 patients in stage A, 9 in stage B, and 2 in stage C [Table 2].

The experimental subjects were divided into four stages, depending upon the histological features. The staging was done as per the criteria laid down by Pindborg et al. [7] The distribution of the subjects based on histopathological criteria are presented in [Table 3].

Fiber thickness was measured in all cases of OSF and was compared with that of normal tissue collagen. Statistical analysis using one-way ANOVA showed decrease in mean thickness of thin fibers with increase in severity of the disease (P = 0.29), and a significant increase in thickness of thick fibers with increase in severity of the disease (P<0.001).

Analysis of the difference between various groupsusing student's 't'-test showed the difference among thin fiber thickness to be not significant. Whereas when thick fibers were taken into consideration, the difference between the normal and stage I did not reveal any significance; however, when compared to stage II and III, it showed a highly significant difference [Table 4],[Figure 7]. In advanced stages, due to the tight packing of the collagen fibers, the individual fiber thickness could not be assessed.

Polarizing colors of collagen fibers in 40 cases of OSF patients were compared with those of 10 cases of the control group. We observed that as the stages advanced, the polarization color of thick fibers significantly showed orange red-red color and there was a considerable decrease in the greenish yellow color. This is due to the fact that the overall number of thin fibers diminished markedly as the stages of disease progressed. In advanced stages, as the collagen fibers appeared hyalinized and tightly packed, delineation into thick and thin fibers was not observed. However, the tight packing of collagen fibers resulted in orange red-red color throughout the sections [Table 5],[Figure 4],[Figure 5],[Figure 6],[Figure 8],[Figure 9].

Analysis of clinical and histopathological staging, showed no significant correlation between the two staging systems (P > 0.05) [Table 6],[Figure 10]. However, when functional staging was compared with histopathological staging it was highly significant (P < .01) [Table 6].

   Discussion Top

Studies on collagen in OSF has been a major focus in research as arecoline, the main alkaloid constituent of the areca nut, has been reported to induce excessive collagen synthesis. Studies have shown that arecoline induces collagen formation. The present study was aimed to study the collagen fibers in OSF using the picrosirius red stain and polarization microscopy. The fiber thickness as well as polarizing colors of collagen fibers in normal mucosa and various stages of the disease were studied.

In the present study, all the 40 subjects exhibited the presence of one or more of the clinical characteristics like palpable bands, blanching of the mucosa, and restricted mouth opening. The cases selected were clinically and functionally graded by modifying the criteria laid down by Haider et al., [6] and staging of the disease was done histopathologically as per the criteria laid down by Pindborg et al. [7]

Once the disease progresses to early stages from very early stage and if the patient continues with the areca nut chewing habits, the fibrotic changes in the connective tissue accelerate in severity. So the very early stage could be considered as the appropriate stage for secondary prevention of the disease. The remarkable differences in the various stages reveal the fact that in moderately advanced stages, the collagen is tightly packed and the thickness of collagen fibers increases considerably when compared to the very early stages. This observation was statistically highly significant (P < 0.01).

Furthermore, the collagen in the advanced stages appeared as tightly packed bundles, where the ability to identify the individual fibers was practically impossible. This is probably due to an ever-increasing thickness of the fibers as the disease progresses. However, the polarizing color appeared as predominantly red in the advanced stages.

In the present study, there is a trend showing increase in collagen fiber thickness as the severity of the disease increases. However, the subunits of collagen, the fibrils which aggregate to form the fibers, have been described as thinner by few authors. [8] Many authors have the opinion that though dense fibers are observed in OSF, the periodicity of collagen interlinking is not altered and that the collagen formed in OSF is morphologically normal.

Despite the considerable amounts of work done on collagen, not many methods have been proposed to demonstrate this protein in tissue sections. However, the picrosirius red stain is preferred for the demonstration of collagen and has provided excellent results consistently. This is due to the fact that in polarized microscopy, the parallel relationship between dye and the collagen results in an enhanced birefringence. Even the very thin fibrils undetectable in normal microscopy become visible by this method.

However, some authors have reported that various picric acid techniques employed to demonstrate collagen may provide different qualitative results. This can create some confusion even though factors like pH of the stain, concentration of the stain, and duration of the stain may not be directly attributed to variation in results. These noticeably different results apply even when the above- mentioned parameters were varied.

In the present study, we found that, when picrosirius red staining was done on formalin-fixed tissues which were fixed for more than "a few days," picrosirius red stain demonstrated a decreased staining particularly of fine collagen fibers in such tissues.

So it is advisable not to use this staining technique on tissues preserved in formalin for many number of days. Further studies should focus on collagen in OSF in detail, as it appears that the basic defect lies in collagen metabolism.

   Conclusion Top

A comparison of clinical, functional, and histopathological stages of OSF was statistically analyzed. It was found that the comparison of clinical and histopathological staging did not provide any valuable information as to the severity of the disease. However, the comparison of the functional staging with histopathological staging was more reliable in providing the severity of the disease. And this observation stems from the present study of collagen fibers using polarization microscopy. The study in particular revealed that the collagen fiber thickness gradually increased as the severity of the disease increased, and the polarizing color gradually shifted from greenish yellow to orange red and to red as the severity of the disease increased. This reveals that in OSF, the basic defect lies in the collagen synthesis and degradation.

In future, studies should also compare OSF cases in different demographic features of ethnicity, geographic localization, and socioeconomic status backed with epidemiological and experimental evidences. Research must also be aimed at the ultrastructural features of collagen fibers in various stages of OSF and their correlation to clinical and functional staging. The uncertainty and flaws in the OSF management protocol can be dealt with, only if research is focused to characterize the pathogenesis in spite of numerous difficulties encountered.

   References Top

1.Gupta SC, Yadav YC. "MISI" an etiologic factor in oral submucous fibrosis. Indian J Otolaryngol 1978;30:5-6.  Back to cited text no. 1      
2.Shah N, Ranikumar, Singh MK. Hematological and histological studies in oral submucous fibrosis. J Indian Dent Assoc 1993;64:383-8.  Back to cited text no. 2      
3.Constantine VS, Mowry RW. Selective staining of human dermal collagen. The use of picrosirius red F3BA with polarization microscopy. J Invest Dermatol 1968;50:419-23.  Back to cited text no. 3      
4.Sweat F, Puchtler H, Rosenthal IS. Sirius red F3BA as a stain for connective tissue. Arch Pathol 1964;78:69-72.  Back to cited text no. 4      
5.Junqueira LCU, Bignolas G, Brentani RR. A simple and sensitive method for the quantitative estimation of collagen. Anal Biochem 1979;94:96-9.  Back to cited text no. 5      
6.Haider SM, Merchant AT, Fikree FF, Rahbar MH. Clinical and functional staging of OSF. Br J Oral Maxillofac Surg 2000;38:12-5.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Pindborg JJ, Odont, Sirsat SM. Oral submucous fibrosis. Oral Surg Oral Med Oral Pathol 1966;22:764-9.  Back to cited text no. 7      
8.Binnie WH, Cawson RA. A new ultrastructural finding in oral submucous fibrosis. Br J Dermatol 1972;86:286-90.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]  

Correspondence Address:
Denny E Ceena
Department of Oral Medicine and Radiology, Manipal College of Dental Sciences, Mangalore
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.57356

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

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

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