|Year : 2013 | Volume
| Issue : 2 | Page : 123-127
Image analyses of collagen types and thickness in oral sub mucous fibrosis stained with picrosirius red under polarizing microscope
Venkatesh V Kamath, Krishnanand Satelur, Y Komali, Shruthi S Krishnamurthy
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Bengaluru, Karnataka, India
|Date of Web Publication||3-Jan-2014|
Venkatesh V Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Munnekolala, Marathahalli, Bengaluru - 560 037, Karnataka
Source of Support: None, Conflict of Interest: None
Context: Oral submucous fibrosis (OSF) is a potentially malignant oral disorder leading to increased fibrosis in the sub-epithelial layer. The collagen in the condition has been a subject of intense scrutiny in an attempt to understand the pathogenesis of the disease. Aim: The present study aims to quantify and qualify the collagen fibers in different histological grades of OSF using picrosirius red stain under the polarizing microscope. The quantification of the fibrosis was carried out using image analysis software and the fibers were graded according to staining hue and intensity into their respective subtypes. Comparison was done with normal mucosa, scar/keloid tissue samples. Materials and Methods: The present study included OSF (n = 50) of differing histological grades, keloid/scar (n = 4) and normal mucosa (n = 6) as control cases. Histological assessment was performed on hematoxylin and eosin stained sections. Picrosirius red stained slides were observed under a polarizing microscope for assessment of collagen subtypes. Quantification of collagen was done under polarizing microscope and image parameters were analyzed using ProReg® Capture Pro 2.8.8 (Lawrence and Mayo India Pvt Ltd, 2011) image analysis software. Results: The epithelial thickness in OSF, scar and keloid is less than that of normal mucosa and progressive decrease in the epithelial thickness is seen in the successive stages of OSF. The fibrosis increases with increasing grades of OSF, was higher in scar and keloid and was highly statistically significant. Type I collagen was more predominant in all stages of OSF, in normal oral mucosa and scar/keloid tissue samples as compared with type III. Though quantitative analysis of the collagen types I and III is possible, with picrosirius red qualitative analysis is an arduous task. The specificity of detection of collagen subtypes was acceptable with the picrosirius red stain, but the sensitivity left a lot to be desired.
Keywords: Collagen, image analysis, oral submucous fibrosis, picrosirius red, polarizing microscopy
|How to cite this article:|
Kamath VV, Satelur K, Komali Y, Krishnamurthy SS. Image analyses of collagen types and thickness in oral sub mucous fibrosis stained with picrosirius red under polarizing microscope. J Orofac Sci 2013;5:123-7
|How to cite this URL:|
Kamath VV, Satelur K, Komali Y, Krishnamurthy SS. Image analyses of collagen types and thickness in oral sub mucous fibrosis stained with picrosirius red under polarizing microscope. J Orofac Sci [serial online] 2013 [cited 2020 Feb 23];5:123-7. Available from: http://www.jofs.in/text.asp?2013/5/2/123/124258
| Introduction|| |
Oral submucous fibrosis (OSF) has been described as an "insidious, chronic disease affecting any part of the oral cavity and sometimes the pharynx. Although occasionally preceded by and/or associated with vesicle formation, it is always associated with a juxta-epithelial inflammatory reaction followed by a fibro-elastic change of the lamina propria, with epithelial atrophy, leading to stiffness of the oral mucosa and causing trismus and inability to eat." 
Almost 0.5% (5 million) population in Indian subcontinent is affected with OSF. It is alarmingly increasing in South-East Asian countries especially among the young group due to the increasing popularity of chewing areca nut, which has been etiologically implicated in the pathogenesis of the disease. 
The overproduction of collagen fibers, a hallmark of this disorder, has been attributed to various components of the areca nut mixture consumed by the individual. Arecoline, a major component, is known to up-regulate intake of the enzyme, lysyl-oxidase, which increases the cross linkage of collagen fibers, causes stimulation of the production and hinders the degradation of collagen. Thus, OSF is considered a collagen metabolic disorder. 
The study of collagen has been the mainstay of investigative histological procedures in understanding the pathogenesis of the lesion. Conventional stains for collagen like Van Gieson and trichrome stains lack selectivity and thus are not ideal for assessing collagen subtypes. 
Combination of sirius red and picric acid was first considered to be a special stain for connective tissue in 1964, especially for differentiating the subtypes of collagen. It works on the principle that sulphonic group of sirius red reacts with the basic groups in collagen molecules. 126 sirius dye molecules binds to purified collagen types I, II and III. The enhanced birefringence of collagen is due to the attachment of the elongated dye molecules parallel to the long axis of the collagen. The orientation of the fibers to polars and collagen birefringence provides brightness to the collagen. Thickness, density of packing and spatial arrangement determines the polarization birefringence of the collagen. ,, Picrosirius red stain thus helps in better understanding of the collagen function and pathology.
The purpose of this study was to quantify and qualify subtypes of collagen in OSF, keloid and scar as compared with tissue samples of normal oral mucosa.
| Materials and Methods|| |
The present study included 50 patients with OSF of various histological grading, four cases of keloid and scar tissue and six cases of normal oral mucosa were used as the control group. The histological grading of the lesion was performed according to Pindborg and Sirsat  criteria.
Picrosirius red staining protocol (from Coleman, 2011): 
- Dewax sections in xylene and hydrate through graded ethanols to water.
- Stain for 10 min in a solution of 1% picric acid with 0.1% fast green.
- Wash 3 times in 1% acetic acid.
- Stain for 20 min (or up to 1 h) in a solution of 0.2% sirius red F3B with 0.2% picric acid.
- Wash in 1% acetic acid.
- Air-dry at room temperature (several hours or overnight).
- Add distrene, plasticizer, xylene mountant and coverslip.
Picrosirius red stained sections were observed under polarizing microscope and analyzed with ProReg® Capture Pro2.8.8 (2011) image analysis software. The total collagen content was determined by the grid of visible (non-zero) pixels in submucosa depending on the red, reddish-yellow, yellowish-green and green. Readings of each case of OSF, scar, keloid and normal was tabulated.
| Results|| |
The tabulated readings were statistically analyzed by one-way analysis of variance test. Pearson's correlation test was employed for comparison between various parameters in the different groups.
The mean value of epithelial thickness in OSF in various stages was 0.17 μ, 0.16 μ and 0.14 μ, less than that of the normal mucosa (0.23 μ). There was a progressive decrease in the epithelial thickness with the increasing stages of OSF. The mean of the epithelial thickness of scar and keloid was same as that of the OSF in stage 2 (0.16 μ) [Table 1].
|Table 1: Mean values for epithelial thickness and total fibrosis with statistical analyses (one-way ANOVA and Pearson's correlation)|
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The mean value of fibrosis in OSF in various stages was 0.27 μ (Grade 1), 0.28 μ (Grade 2) and 0.40 μ (Grade 3) when compared with normal mucosa (0.15 μ). Higher mean of 0.77 μ was seen in scar/keloid tissue samples. The results were found to be statistically significant (P < 0.05) [Table 1].
Pearson's correlation between the epithelial thickness and fibrosis in the different groups showed statistical significance (P < 0.05); there was increasing fibrosis with decreasing thickness of the epithelium [Table 1] and [Figure 1].
|Figure 1: Graph showing the relationship of epithelial thickness to total content of fibers in all the tissue samples|
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Collagen subtypes were identified based on the difference in hues. Type I collagen exhibited a red to reddish-yellow coloration compared with type III which exhibited a yellowish-green to green coloration.
The mean area of sub-epithelial tissue occupied by type I collagen fibers in Grade 1, Grade 2 and Grade 3 OSF was 0.36 μ, 0.36 μ and 0.37 μ respectively, which was almost equal to that of normal mucosa (0.36 μ), whereas in scar/keloid it was 0.56 μ [Table 2].
|Table 2: Comparison of types I and III collagen fibers in OSF, scar, keloid tissue and in normal oral mucosa with statistical analysis (one-way ANOVA)|
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The mean of type III collagen fibers in Grade 1, Grade 2 and Grade 3 OSF was 0.20 μ, 0.33 μ and 0.19 μ respectively. This was more than in normal mucosa (mean 0.15 μ) and in scar/keloid (means 0.16 μ) [Table 2].
A comparative correlation between types I and III fibers in all tissue samples examined revealed the type I fibers to be less dense (occupying lesser area) than the type III fibers in all stages of OSF. The relation between the two groups of fibers in scar/keloid and normal oral mucosal samples showed an inverse result with type I fibers appearing to be more denser than type III. This reversal of relationship could be considered pathognomic and was found to be statistically significant [Table 2] and [Figure 2].
|Figure 2: Graph showing the relationship between distribution of types I and III fibers in all tissue sample.|
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[Figure 3], [Figure 4] show the staining reactions of tissue samples stained with hematoxylin and eosin (H and E), picrosirius red and picrosirius red stained sections observed under polarizing microscope.
| Discussion|| |
Ever since the identification and establishment of OSF as a definitive oral disorder, the collagen deposited as a part of the pathologic process has been a subject of intense scrutiny. At the light microscopic level identification of collagen with standard H and E stains is at best a visual exercise with no specificity and sensitivity. The localization and detection of collagen subtypes is not possible and differentiation from other connective tissue components is superficial and gross. The adoption of special stains thus becomes a necessity in an attempt to localize tissue collagen and identify its subtypes. The affinity of the picrosirius red stain to the components of collagen impart an almost immunological specificity during staining resulting in a bright color of collagen ranging from red, yellow-green to green that helps in easy visualization. In addition the picrosirius red stained sections when observed under a polarizing microscope show birefringence due to the anisotrophic properties of collagen. , The polarization birefringence colors appear to be a measure of the thickness of the collagen fibers, the density of their packing and spatial arrangement.  In the past, it was thought that the polarization colors were indicative of the collagen subtype. This is now known to be untrue and specific collagen types (numbering more than 20) can only be determined using immunohistochemical techniques with specific antibodies. 
In the present study, epithelial thickness decreased predictably with increasing grades of OSF. The inverse relationship between the epithelial thickness and fibrosis is well-known and is explained by factors like lack of vascularity in the fibrotic submucosa depriving the overlying epithelium of nutritional components and the constant pressure from the increasing fibers.
The density of fibers (fibrosis) was higher in scar/keloid tissue as compared with OSF and proportionately more than that in normal oral mucosa. The mean value of type III fiber content of the submucosa was more in stage II OSF than in stage III. This may be explained based on the compaction (removal of extracellular matrix substance) during progressive maturation of the fibers.
The reversal of fiber type content was an interesting observation in our study. The predominance of type III as compared with type I in OSF was supported statistically and could be considered pathognomic. The reversal could probably explain the pattern of deposition of fibers, lack of action of collagenase in remodeling and the wide affliction of various parts of the oral mucosa. In scar/keloid tissue interestingly, though the density of collagen fibers measured as a whole was more that OSF there was no change in the normal ratios of types I and III varieties. The fact that scar/keloid tissues tend to rather more localized and form as a result of direct injury to the site may be a plausible explanation.
Very few studies in the literature were available which had an assessment of collagen fibers using picrosirius red stain and polarized microscopic analyses. None had an image analyses quantitative assessment as was done in our study. In a study by Ceena et al.  analyzed 40 cases of OSF using picrosirius red stain and polarized microscopy. The authors found an increase in red (type I) fibers compared with green (type III) fibers in increasing grades of severity of the lesion. Smitha and Donoghue  analyzed 33 cases of OSF stained with picrosirius red observed under the polarizing microscope. They reported fibers as being unidirectional and parallel to the epithelium. van Wyk et al. (1998)  carried out an EM study coupled with picrosirius red staining. The authors reported that type I collagen fibers formed the bulk of the sub-epithelial collagen while type III was localized around blood vessels, epithelial-mesenchymal junction, salivary glands and muscle fibers. Both types of fibers were said to be morphologically normal.
Picrosirius red stain is said to be collagen specific and highlights types I and III collagen, though the sensitivity is much less than standard immunological techniques in usage today. Types I and III, which are specifically highlighted gave a red and green color respectively in the birefringent pattern and thus delineation was easy. Though picrosirius red and polarization highlighted collagen content of the submucosa, the overlap, mode of distribution and spread in the connective tissue make efforts to quantify collagen very difficult in vivo. The use of computerized image analysis software gives an objective basis to the subjective data. Predictably type I collagen shows increased predominance when compared with type III in the normal mucosa. In the present study type I was more than type III in all grades of OSF as well as in scar and keloid. We observed certain significant points in the study. The staining of collagen using picrosirius stain, though much better than H and E, did not delineate fibers sharply, there was considerable overlap, masking and disordered fiber orientation. The observation of red and green color was not always clear, sharp and delineating, shades of red and green were admixed with bright red and bright green. This was especially seen in normal mucosa slides, where no delineation of fibers subtype was visible. Literature supports this contention that fiber composition may not be localized to single subtype may also be composed of collagen of types other than types I and III. Thus, polarization colors being the measure of the collagen content is known to be untrue.  The literature recommends thick sections of 7-10 μ when assessing collagen in the tissue using picrosirius red and polarizer; we found an increase in overlap and masking when thicker sections were used. It is our opinion that an accurate assessment of collagen subtype in tissues is not possible with picrosirius red even when the technique is enhanced by use of polarizer.
| Conclusion|| |
Based on the above study it can be concluded that picrosirus red stain with the use of a polarizer enhances the identification of collagen subtypes in OSF, scar and keloid. Use of computerized image analysis software convert's subjective conversion into objective. The increase in the density and total content of fibers is conclusively demonstrated by the staining technique. There are significant limitations and accurate mapping of collagen in OSF cannot be done solely on the basis of this technique. Advanced techniques of using immunohistochemistry and collagen gene identification are probably more specific and sensitive in this regard.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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