| Abstract|| |
Background: Oral submucous fibrosis (OSMF), a well-recognized oral potentially malignant disorder, results due to increased collagen production and reduced collagen degradation. Aims and Objectives: To qualitatively compare the staining properties of collagen in OSMF using two special stains based on their birefringent property using polarizing microscopy. The study also assessed the distribution and orientation of collagen fibers in different grades of OSMF. Materials and Methods: A total of 73 subjects with different clinical and histopathological staging of OSMF comprised the study population. Histopathological examination was done using hematoxylin and eosin stain, Van Gieson and picrosirius red. Collagen fibers were analyzed for polarization colors, distribution, and orientation. Results: Picrosirius red stained both thick and thin collagen fibers. Irrespective of the histopathological grades reddish orange and yellowish orange were the most predominant colors. Parallel arrangement of fibers was observed when stained with Van Gieson but picrosirius red stained sections showed a majority of parallel type I fibers with perpendicular type III fibers which increased with advancement in the histopathological grade. Yellowish orange and greenish yellow fibers were predominant in the lamina propria, while reddish orange fibers were predominant in the submucosa. Conclusion: Picrosirius red was found to be a better stain. Histopathological grading and polarization colors showed no association with each other. Collagen fibers were more thickly and tightly packed in the submucosa indicating that the process of fibrosis began there. The increase in perpendicular type III fibers with advancing histopathological grades suggested their role in fibrosis.
Keywords: Birefringence, collagen, oral submucous fibrosis, picrosirius red, special stains, Van Gieson
|How to cite this article:|
Nishat R, Kumar H. Collagen fibers in oral submucous fibrosis - A polarizing microscopy study using two special stains. Indian J Pathol Microbiol 2019;62:537-43
|How to cite this URL:|
Nishat R, Kumar H. Collagen fibers in oral submucous fibrosis - A polarizing microscopy study using two special stains. Indian J Pathol Microbiol [serial online] 2019 [cited 2020 May 30];62:537-43. Available from: http://www.ijpmonline.org/text.asp?2019/62/4/537/269066
| Introduction|| |
Oral submucous fibrosis (OSMF) is a chronic irreversible debilitating disease associated with juxta epithelial inflammatory reaction followed by a fibro elastic change of lamina propria with epithelial atrophy leading to stiffness of oral mucosa, causing trismus, and inability to eat. This well-recognized oral potentially malignant disorder, most commonly seen in the Indian subcontinent has mainly been attributed to the use of areca nut and its products. With the increased use of these preparations, prevalence of OSMF is on the rise among the younger age group and majority of the cases occur between 20 and 30 years of age., According to a study the overall prevalence of oral potentially malignant disorders in the Indian subcontinent was 13.7% in which OSMF (8.06%) was the most common entity.
The study of collagen has been the mainstay of investigative histological procedures in understanding the pathogenesis of OSMF. Traditionally, stains such as Van Gieson and various forms of trichromes have been used to detect collagen fibers in tissues which rely on differential binding by tissue components. These stains can be used under both light and polarizing microscopy and can differentiate between thick and thin fibers under polarization. Picrosirius red dyes are also being widely used due to their specific reactivity toward most of the collagen subtypes.,, Moreover, combining picrosirius red staining with the use of polarizing microscopy technique enhances the specificity for collagen and also increases the sensitivity and resolution manifold.
There appears to be no study in the literature on comparison of collagen fibers in OSMF using Van Gieson and picrosirius red stains using polarizing microscopy. Taking all this into consideration, this study was conducted to qualitatively compare the above two stains to assess the staining properties of collagen in OSMF using polarizing microscopy.
| Materials and Methods|| |
A total of 73 patients who were clinically diagnosed with OSMF and agreed for a biopsy formed the study group. Informed consent was obtained from all patients and approval from the Institute Ethics Committee was taken before pursuing the research project (Ref no.: KIMS/KIIT/IEC/024/2014).
Patients with history of using areca nut in different forms, patients with restricted mouth opening, palpable fibrous bands, and history of burning sensation on consumption of spicy food, patients with clinically diagnosed and histopathologically confirmed OSMF formed the study population. Patients with restricted mouth opening due to any other cause other than OSMF and cases histopathologically not diagnosed to be OSMF were excluded from the study. Ethical committee approval has been obtained on 12.12.2014.
Clinical assessment and sample collection
A detailed case history was recorded for patients clinically diagnosed with OSMF. Patients were examined and the relevant data entered into the clinical data collection Performa [Figure 1]. Clinical staging of severity of fibrosis was carried out based on the classification given by Lai, in which the OSMF population was divided based on the interincisal distance as:
|Figure 1: Intraoral photographs showing blanching of (a) labial mucosa, (b) buccal mucosa, and (c) palate which are characteristic features of OSMF|
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- Group A - mouth opening greater than 35 mm
- Group B - mouth opening between 30 and 35 mm
- Group C - mouth opening between 20 and 30 mm
- Group D - mouth opening less than 20 mm
- After performing the routine hematological investigations, incisional biopsy was done and sent for histopathological evaluation where the specimens were duly processed and stained.
Staining of samples using routine and special stains
Staining techniques used has been elaborated in [Figure 2].,
|Figure 2: Protocols for (a) Hematoxylin and Eosin staining, (b) Van Gieson staining, and (c) picrosirius red staining|
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Van Gieson staining
To prepare the stain, 9 ml of 1% aqueous acid fuchsin was dissolved in 50 ml of saturated aqueous picric acid solution, and 50 ml of distilled water was added to this mix.
Picrosirius red staining technique
Picrosirius red solution was prepared by dissolving 0.5 g of Sirius Red F3B in 500 ml of saturated picric acid solution. 5 ml of glacial acetic acid was added to 1 liter of distilled water to prepare acidified water. Weigerts hematoxylin was prepared by adding equal quantities of solution A and B. Solution A comprised of Delafield hematoxylin solution and isopropyl alcohol, while solution B was composed of ferric chloride hexahydrate, concentrated hydrochloric acid and distilled water.
The Hematoxylin and Eosin stained sections were examined under light microscopy and cases were categorized depending on connective tissue changes into very early, early, moderately advanced and advanced stages according to Sirsat and Pindborg [Figure 3]. After histopathological grading, picrosirius red and Van Gieson staining was carried out and the stained sections were examined using polarizing microscope.
|Figure 3: Photomicrographs showing sections of (a) early stage, (b) moderately advanced stage, and (c) advanced stage of OSMF [Hematoxylin and eosin staining, x10]|
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Under a magnification of 10X, the overall predominant polarization color was assessed and recorded. Polarization colors and orientation of fibers in the lamina propria and submucosa were evaluated separately. The efficacy of stain was determined based on the staining properties and birefringence. Under light microscopy, these stained sections were also used to evaluate infiltration of muscles by collagen fiber bundles.
The data recorded in the process were analyzed using appropriate statistical tools and techniques using Statistical Package for the Social Sciences (SPSS) (version 16.0).
| Results|| |
A total of 73 subjects forming the study group were in the age range of 18--61 years with a mean age of 31.75 ± 9.85 years. The model age group was 26--35 years having the highest frequency of 33 cases. The maximum number of subjects had a mouth opening ranging between 20 and 30 mm and hence belonged to Stage C of clinical staging. The histopathological grading was done as per the criteria laid down by Pindborg and Sirsat. Not a single case was reported to be in very early stage, 15 (20.5%) were diagnosed to be in the early stage, 51 (69.9%) in moderately advanced stage, and 7 (9.6%) in the advanced stage.
Association of clinical staging with histopathological grading was studied using chi square test of association. The chi square test did not indicate any significant association of clinical staging with histopathological grading (P = 0.567) implying that histopathological grades were more or less uniformly distributed within the clinical stages. 97.2% of the cases showed invasion into the muscles.
Van Gieson and picrosirius red stains were evaluated under polarizing microscope at a magnification of 10X for staining properties, overall polarization colors, and orientation of collagen fiber bundles. Van Gieson showed an overall reddish polarization color, while picrosirius red showed a wide range of birefringence colors including greenish yellow yellowish orange and reddish orange birefringence [Figure 4] and [Figure 5]. Hence, based on the staining properties and polarization colors obtained, picrosirius red was found to be a better stain than Van Gieson for staining collagen in OSMF.
|Figure 4: Photomicrograph showing polarization colors in (a) early stage, (b) moderately advanced stage, and (c) advanced stage of OSMF [Van Gieson staining, x10]|
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|Figure 5: Photomicrograph showing polarization colors in (a) early stage, (b) moderately advanced stage, and (c) advanced stage of OSMF [picrosirius red staining, x10]|
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In the early stage, out of 15 cases, 2 (13.3%) showed an overall greenish yellow polarization color, 7 (46.7%) showed a predominant yellowish orange color and 6 (40%) showed reddish orange color. In the moderately advanced stage, out of 51 cases, 6 (11.8%) showed a predominant greenish yellow color, 26 (51%) showed yellowish orange color, 17 (33.3%) showed a reddish orange color, 1 showed equal distribution of greenish yellow and reddish orange color, while 1 case showed equal proportion of all the three colors, i.e., greenish yellow, yellowish orange, and reddish orange. In the advanced stage, 1 case showed a predominant greenish yellow color and 3 cases each showed yellowish orange and reddish orange birefringence [Table 1]. Irrespective of the histopathological grades, yellowish orange and reddish orange were the most predominant colors, which indicated no association between histopathological grading and polarization colors. Chi square test of association did not reveal any significant association.
|Table 1: Association of histopathological grading with polarization colors of picrosirius red stain|
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89% of the Van Gieson stained sections showed a parallel arrangement of collagen fiber bundles in relation to the epithelium. Of the 73 cases, 15 (20.5%) showed a parallel arrangement of collagen fiber bundles in relation to the epithelium, 54 (74%) showed parallel arrangement of collagen fiber bundles with presence of greenish yellow perpendicular fibers, 3 (4.1%) did not show a parallel arrangement and 1 showed a nonparallel arrangement with presence of greenish yellow perpendicular fibers [Table 2]. It was seen that with increasing histopathological grade, there was an increase in parallel collagen fiber bundles with greenish yellow perpendicular fibers - 66.7% in early, 74.5% in moderately advanced, and 85.7% in advanced stage.
On comparison of distribution of collagen fibers in lamina propria and submucosa, a trend was observed. Reddish orange birefringence was seen in 35.6% cases in the lamina propria which increased up to 54.3% in the submucosa. On the contrary, yellowish orange birefringence was seen in 49.3% of the cases in the lamina propria which declined to 37.1% in the submucosa. Similarly, greenish yellow birefringence was seen in 13.7% of the cases in lamina propria which reduced to 7.1% in the submucosa. Hence, it can be suggested that yellowish orange and greenish yellow fibers were predominant in the lamina propria, while reddish orange fibers were predominant in the submucosa [Figure 6].
|Figure 6: Bar diagram showing comparison of distribution of collagen fibers in lamina propria and submucosa using picrosirius red stain|
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| Discussion|| |
Collagen in OSMF remains the focus for various research projects as the disease occurs mainly due to its increased synthesis and reduced degradation. Collagen being a birefringent material has the ability of double refraction. This anisotropic property of collagen is best exploited using a polarization microscope which allows researchers to obtain information regarding color absorption, structure, composition, light refraction, and other properties. Moreover, this technique enhances the sensitivity and quality of image contrast manifold.
According to a search in English language literature, no studies were found comparing the tinctorial properties of Van Gieson and picrosirius red stains under polarizing microscopy. Thus, the present study was undertaken to study the polarization colors, orientation and distribution of collagen fibers in the various stages of OSMF using the two special stains and polarization microscopy. Muscle invasion by collagen fibers was also assessed using special stains under light microscopy.
Association of clinical staging with histopathological grading did not indicate any significant association (P = 0.567). Thus, based on our findings we conclude that clinical staging and histopathological grading may be termed independent of each other. This finding was similar to the results obtained by Ashalata et al., Rooban et al., and Kumar et al. Factors like site and extent of fibrosis, regional anatomical variation, muscle tone, neuromuscular coordination, and the physioanatomical integrity of the underlying oral musculature could determine the degree of trismus.,, Buccal mucosa was the preferred site of biopsy, and the inaccessibility of the surgeon to the posterior most areas could shift most of the stage I cases to stage II as the process of fibrosis begins from the fauces and proceeds anteriorly., However, Ceena et al. and Modak et al. reported that with the advancement of histopathological grade, fibrosis, and thickness of collagen increased which clinically resulted in trismus.,
Based on the staining properties and polarization colors, picrosirius red stain was found to be a better stain as compared to Van Gieson. Van Gieson stain does not stain thin collagen fibers and hence may result in underestimation of the degree of fibrosis. Moreover, the stain has the demerit of fading with time. On the contrary, picrosirius red stains collagen fibers more intensely and increases the birefringence dramatically. Increased birefringence is attributed to the parallel relationship between dye and collagen molecules. Fiber thickness, packing of collagen, and the distinct pattern of physical aggregation exhibited by collagen are the factors responsible for the differences in polarization colors., In addition to this, picric acid in the stain prevents indiscriminate staining of noncollagenous structures by sirius red, hence providing better results.
According to our study, irrespective of the histopathological grades, yellowish orange and reddish orange were the most predominant colors, which indicated no association between histopathological grading and polarization colors. Similar results were obtained by Kamath et al.
Various polarizing microscopy studies on collagen using picrosirius red stain have concluded that greenish yellow to yellow birefringence is given by type III collagen, while type I collagen exhibits a red to reddish yellow color. Type I collagen corresponds to the thick fibers and type III to thin fibers. Implying these findings to our study, type I collagen was more predominant in all the cases irrespective of histopathological grades. The results obtained in our study were not in accordance with those obtained by Ashalata et al., Ceena et al., Parveen et al., Velidandla et al., Modak et al., and Radhika et al. who observed a shift from yellow green to orange red color with advancement in the histopathological grade.,,,, According to them, this shift in color was attributed to factors like fiber thickness, packing of collagen, and the distinct pattern of physical aggregation exhibited by collagen. In the early stages of OSMF, collagen fibers are comparatively thinner which gives a greenish yellow birefringence but with the advancement in histopathological grade, collagen fibers tend to aggregate thus forming thicker bundles which gives a reddish orange birefringence.,,
In our study, a parallel arrangement of type I collagen fibers was noted in majority of the cases irrespective of histopathological grades with both Van Gieson and picrosirius red stains. Several other authors like Smitha et al., Parveen et al., and Patel et al. also found similar parallel orientation of collagen fibers to the epithelium.,, In addition, picrosirius red staining also showed a perpendicular arrangement of type III collagen fibers which increased with the advancement of histopathological grade, which may be indicative of the fact that these fibers are responsible for the increasing amount of fibrosis. This finding is unique to our study. Parallel type I and perpendicular type III fibers result in a mesh-like formation which is histopathologically seen as increased amount of fibrosis. Thus, intervention by local administration of antitype III collagen may inhibit the progression of fibrosis.
According to our findings, yellowish orange and greenish yellow fibers were predominant in the lamina propria, while reddish orange fibers were predominant in the submucosa suggesting that collagen fibers were more thicker and tightly packed in the submucosa than the lamina propria. This may suggest that fibrosis in OSMF begins in the submucosa and proceeds into the lamina propria which may explain xerostomia and trismus experienced clinically by the patients. Structures like muscles, salivary gland are present in the submucosa whose early involvement in fibrosis may act as a barrier for nutrient diffusion into these structures, thereby resulting in their degenerative changes, thus resulting in functional impairment. This finding was in accordance with the results obtained by Joseph and Rajendran.
In our study of 71 cases showing presence of muscles, 69 (97.2%) showed invasion into the muscles, while only 2 (2.8%) did not. This may be attributed to the fact that the initiation of fibrosis in OSMF occurs from the submucosal layer (as mentioned above). Since, muscles are present in the submucosa, they tend to get affected at an early stage.
| Conclusion|| |
Picrosirius red stain was a better stain as compared to Van Gieson due to its stability and its ability to stain both thick and thin collagen fibers. Most of the cases showed a parallel arrangement of collagen fibers when stained with Van Gieson but picrosirius red stained sections showed a majority of parallel type I collagen fibers with perpendicular type III fibers which increased with advancement in the histopathological grade, suggesting the role of type III fibers in increased fibrosis. Collagen fibers were more thickly and tightly packed in the submucosa as compared to the lamina propria, thus indicating that the process of fibrosis begins in the submucosa.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
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Senior Resident, Department of Dentistry, Nalanda Medical College and Hospital, Patna - 7, Bihar
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]