|Year : 2021 | Volume
| Issue : 1 | Page : 47-53
Evaluation of Collagen Birefringence in Oral Reactive Lesions Using Picrosirius Red Stain Under Polarized Light Microscopy: An Observational Microscopic Study
A. P. Shirona, Usha Hegde, H. S. Sreeshyla
Department of Oral Pathology and Microbiology, JSS Dental College & Hospital, JSS AHER, Mysuru, Karnataka, India
|Date of Submission||15-Apr-2021|
|Date of Acceptance||14-Jun-2021|
|Date of Web Publication||06-Aug-2021|
Dr. Usha Hegde
Department of Oral Pathology and Microbiology, JSS Dental College & Hospital, JSS AHER, Mysuru 570015, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Oral reactive lesions are relatively common. Collagen is the basic component of such overgrowths that could be fibrous or inflammatory. Hence, understanding the type, nature, and distribution of collagen fibers can aid in our knowledge and better management of these lesions. Materials and Methods: Ten paraffin blocks of each of the following lesions − giant-cell granuloma (L1), inflammatory fibrous hyperplasia (L2), pyogenic granuloma (L3), fibroma (L4), and peripheral ossifying fibroma (L5) − were retrieved from the archives. They were analyzed for their collagen birefringence property under polarized microscopy after picrosirius red stain. The demographic details of all the cases were recorded and evaluated. Results: In our study, giant-cell granulomas were commonly observed between third and seventh decades of life, inflammatory fibrous hyperplasias between second and fifth decades of life, yogenic granulomas in sixth decade, fibromas in fifth and sixth decades, and peripheral ossifying fibromas in second and fourth decade of life. All the lesions were predominant in females and commonly observed on gingiva. L5 lesions showed more areas of green birefringence followed by L1, L2, and L3 groups of lesions showed more of red birefringence than other lesions. Mixed birefringence of orange-red and green-yellow was almost same in all the lesions. All these findings were significant statistically. Conclusion: A plausible conclusion that the lesions with mature red fibers have better prognosis than the lesions with immature green fibers, in reactive oral lesions could be drawn.
Keywords: Fibroma, inflammatory fibrous hyperplasia, oral reactive lesions, peripheral giant-cell granuloma, peripheral ossifying fibroma, picrosirius red stain, polarized microscopy, pyogenic granuloma
|How to cite this article:|
Shirona AP, Hegde U, Sreeshyla HS. Evaluation of Collagen Birefringence in Oral Reactive Lesions Using Picrosirius Red Stain Under Polarized Light Microscopy: An Observational Microscopic Study. J Orofac Sci 2021;13:47-53
|How to cite this URL:|
Shirona AP, Hegde U, Sreeshyla HS. Evaluation of Collagen Birefringence in Oral Reactive Lesions Using Picrosirius Red Stain Under Polarized Light Microscopy: An Observational Microscopic Study. J Orofac Sci [serial online] 2021 [cited 2021 Sep 29];13:47-53. Available from: https://www.jofs.in/text.asp?2021/13/1/47/323360
| Introduction|| |
The collagen fibers form an integral component of the connective tissue stroma in both health and disease conditions. Different collagen types exhibit different structural characteristics. The amount, distribution, and structural organization of fibrillar collagen are all important factors of the tissues. Studying the basic structure and the altered types of the fibers has helped in understanding the pathogenesis and in predicting the course of the disease in certain pathologic conditions.
Specific orientation and alignment of fibers play an important role in progression and metastasis of cancer. Collagen has been helpful as a biomarker in wound healing, aging, and also in other pathologies such as fibrosis, atherosclerosis, and diabetes. Collagen has been shown to be of value in human tooth germ papillae, skin lesions, odontogenic tumors, odontogenic cysts, and in various carcinomas.,,,,,,,
Polarized light microscopy has an anisotrophic material that has the ability to change or acquire different properties in different directions. Birefringence is one such property and most studies have used this property on a picrosirius red-stained section to study the birefringent collagen fibers. Birefringence is an optical property of a material having a refractive index that depends on the polarization and propagation of direction of light. Different objects differ in their intensity of brightness. The intensity of collagen birefringence depends on fibers which are more hydrated, perfectly aligned, cross-linking between the fibers, aging, thickness, expiry date of reagents, mounting media, and artifacts.
Collagen molecules are made up of basic amino acids and react with acidic dyes. Picrosirius polarizing red special stain is one such acidic dye used to study the individual and different collagen molecules. By staining with picrosirius red, procollagen, intermediate, and pathologic collagen fibers, which are not tightly packed, can be differentiated based on the birefringent properties.
Reactive lesions in the oral cavity are commonly encountered. However, not much is known regarding the type and distribution of collagen fibers in oral reactive lesions. Understanding the type, nature and distribution of collagen fibers in these seemingly common oral lesions will aid in furthering our knowledge and better management of these lesions. Therefore, the following oral reactive lesions − inflammatory fibrous hyperplasia, oral pyogenic granuloma, fibroma, peripheral ossifying fibroma, and peripheral giant-cell granuloma − were studied under polarized microscopy after picrosirus red staining for their collagen profile.
| Materials and Methods|| |
Ethical approval for this study (JSSDCH IEC Research protocol No:29/2018) was obtained from the Institutional Ethical Committee, JSS Dental College and Hospital, Mysuru on 5th November 2018. Five different reactive oral lesions were included in the study to analyze the collagen birefringence under polarized microscopy after picrosirius red staining. The samples were obtained from the archives of department of oral pathology and microbiology. Demographic details such as age (20–30, 31–40, 41–50, 51–60, and above 70), sex (female and male), and site (buccal mucosa [BM], gingiva [GI], palate, tongue, vestibule) were collected. Ten tissue blocks each for five lesions (L) were included in the study. The lesions included were giant-cell granuloma (L1), inflammatory fibrous hyperplasia (L2), pyogenic granuloma (L3), fibroma (L4), and peripheral ossifying fibroma (L5). A total of 50 blocks were collected and all the demographic details recorded. Two sections of each lesion were performed, one for hematoxylin and eosin stain (H&E stain) and one for picrosirius red stain.
The H&E-stained slides of each tissue of lesions were observed under light microscope. The diagnosis of the lesions was ascertained and the lesions were grouped into the five categories considered. The staining procedure for picrosirius red stain was performed on the paraffin sections of the tissues, as per the Sirius red staining kit, AMD labs. Statistical data were analyzed by qualitative data as frequencies and percentages. Chi-squared test was used to assess the collagen birefringence in different oral reactive lesions using picrosirius red stain under polarized light microscope. Statistical significance was fixed at 0.05. Statistical analysis was performed using SPSS 22.
| Results|| |
The demographic details of all the lesions were analyzed and grouped into different age categories and gender [Table 1]. It was reported that there was a male predilection in age groups 20–30, 31–40, and 61–70 years and female predilection for the other groups. However, these results were not statistically significant (P = 0.09).
|Table 1 The percentage of males and females included in the study in different age groups|
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L1 was commonly observed between third and seventh decades of life, L2 among second to fifth decades of life, and L3 commonly in sixth decade and least commonly between third and fourth decades of life. L4 was predominant among fifth and sixth decades and least observed in fourth decade of life. L5 was reported commonly in second and fourth decades, but least in fifth decade of life [Table 2].
The percentage of occurrence of each lesion was analyzed among males and females. It was reported that L2 and L3 cases were more in males, whereas L1, L4, and L5 cases were higher in females [Table 3]. However, the results were not statistically significant (P = 0.44).
|Table 3 The percentage of distribution of different reactive lesions among males and females|
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The various lesions in the current study were observed on BM, GI, palate (P), tongue (T), and vestibule (V). The percentage of different lesions occurring at different sites was analyzed [Table 4]. It was reported that GI was the most common site for occurrence for all the lesions, followed by BM and palate. However, these results were not statistically significant (P = 0.18).
|Table 4 The number and percentage distribution of the lesions at different sites in the oral cavity|
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The nature of the birefringent property of collagen fibers was analyzed. In picrosirius red-stained sections, the fibers stained green, green-yellow, orange-red, or red. The green-colored fibers [Figure 1] were immature, green-yellow and orange-red fibers [Figure 2] and [Figure 3], respectively] were identified as mixed intermediate and the red fibers [Figure 4] as mature fibers. Based on the distribution and amount of these colors, they were graded as 1 (few areas), 2 (moderate areas), and 3 (most areas), and the percentage amount of distribution of the fibers in all the lesions was recorded [Table 5]. In picrosirius red stain, the amount of mature fibers in descending order was L2 = L3 > L4 > L1 = L5. The findings were statistically significant (P = 0.04).
|Figure 1 Predominantly green color of collagen fibers under 10× magnification of polarized microscope using picrosirius red stain.|
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|Figure 2 Predominantly green-yellow color of collagen fibers under 10× magnification of polarized microscope using picrosirius red stain.|
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|Figure 3 Predominantly orange-red color of collagen fibers under 10× magnification of polarized microscope using picrosirius red stain.|
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|Figure 4 Predominantly red color of collagen fibers under 10× magnification of polarized microscope using picrosirius red stain.|
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|Table 5 Percentage of amount of different fibers − mature, mixed, and immature − based on the picrosirius red-stained sections under polarized microscopy|
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Mixed, intermediate fibers were equally distributed in L1, L2, and L3 groups, followed by L4 and least in L5. These findings were also statistically significant (P = 0.04).
Immature green fibers were high in L5 > L1 > L4 > L2 = L3. The statistical analysis was significant (P = 0.04).
| Discussion|| |
The extracellular matrix is a three-dimensional network of extracellular macromolecules, such as collagen, enzymes, and glycoproteins. It provides structural and biochemical support to the surrounding cells. Collagen fibers provide tensile strength to the tissues and are the most abundant structural proteins in the matrix. Collagen is made up of amino acids, the building blocks of proteins that give structural support to the extracellular matrix. It is rigid and resists stretching.
All the collagen is made up of three polypeptide alpha chains, coiled around to form the triple helix. Variations among the collagen assembly of the polypeptide chain, length of the triple helix, interruptions, termination of the helical ends, and the spatial arrangements further divide the collagen into several types. There are 28 different types of collagen and type I (90%) and type IV constitute the majority in human beings.
Maturation of collagen fibers occur physiologically by cross-linking of the fibers. When the immature collagen fibers attach with the divalent cross-links, they will be enzymatically converted to mature collagen fibers with stable trivalent cross-links. These mature collagen fibers are exposed to nonenzymatic cross-linking known as glycation or Maillard reaction with the formation of advanced glycation end products. Pathologic process of maturation of collagen fibers is formed by over deposition of unaltered collagen due to overstimulation and overproduction of normal fibroblasts with reduced or absent collagenase enzyme production.
Collagen molecules are rich in basic amino acids and react strongly with acidic dyes. Different stains used to study collagen fibers are Trichrome, Mallory, Masson, and Van Gieson. These stains help in identifying the collagen fibers but do not distinguish the variations in the maturity of the fibers. The maturity of the fibers varies depending on the different structural composition and molecular aggregation of the fibers. The staining of these fibers hence also depends on the three-dimensional organization of fibers, the altered birefringence property and thus their visual appearance.
Picrosirius red (F3BA) stain is a simple and sensitive method to identify collagen fibers. It is a strong, anionic dye consisting of six sulfonate groups associated with cationic collagen fibers. It gives natural birefringence under the polarized light. This evaluates the qualitative and quantitative properties of collagen alterations in degenerative pathologies and inherited or acquired diseases. The effect of the dye picrosirius stain will be visible for a minimum of 15 months at pH of 2.8. However, longer the fixation time in 10% formalin, the picrosirius red stain fades by blocking hydrogen bond between the sulfonic groups. It has longer shelf life when compared with Van Geison stain.
Normal collagen molecules are arranged parallel to each other. Collagen molecules are rich in basic amino acids. Hence, they readily react with acidic sulfonic dyes. Sirius red stain is an elongated dye which reacts with the collagen molecule and aligns parallel to the long axis of the collagen molecule. This enhances the birefringence property of the collagen molecules based on its orientation.
The birefringence of the collagen fibers varies from red, orange-red, green-yellow to green. The variations in the colors of the fibers are based on their birefringence property associated with the maturity, thickness, and orientation of the fibers. The red to orange-red to green-yellow to green color shows the descending levels of maturity of the fibers, with red-colored fibers being highly mature to green ones being immature fibers. Further based on the type of collagen, red colored fibers represent type I collagen and green-colored fibers the type III collagen., The thick tightly packed collagen fibers appear red colored and thin loosely arranged collagen appears green colored.
It has been noted that the variation in maturity of collagen fibers has an implication on the prognosis of certain lesions. Reactive oral lesions are quite common in the oral cavity and have collagen as an important component in their histopathology finding. However, very little is known about the distribution of various maturation patterns of collagen fibers and its implication in oral reactive lesions.
Many studies have suggested that giant-cell granulomas (L1) are commonly observed between fourth and sixth decade of life.,, Our study showed a slightly wider age range and presented between third and seventh decade of life. Inflammatory fibrous hyperplasias (L2) are observed commonly among older age groups caused by ill-fitting denture. But our study showed these lesions to occur between second and fifth decades of life. Pyogenic granuloma (L3) is reported to occur between second and third decades of life., In the present study, it was commonly observed in sixth decade and least commonly observed among third to fourth decades of life. Fibromas (L4) are reported to occur in any age group., In the present series, it was common in fifth to sixth decade, followed by fourth decade of life. Peripheral ossifying fibromas (L5) are noted commonly in second to third decade with similar findings in the present study.,
When the clinical data were examined, literature says that giant-cell granuloma, inflammatory fibrous hyperplasia, pyogenic granuloma, fibroma, and peripheral ossifying fibroma have predilection for occurrence in female gender.,,,,,,,,, Although similar results were evident in the present analysis of our archives, it was not statistically significant.
The most common site of occurrence for giant-cell granuloma is GI and BM and GI for inflammatory fibrous hyperplasias.,,, Our study showed similar findings of GI being the common site for both these lesions (60% in giant-cell granuloma and 70% in inflammatory fibrous hyperplasia). Pyogenic granuloma is reported commonly on GI, lips, tongue, BM, and hard plate.,, Our study showed its presentation on GI (90%) followed by BM (10%). Fibromas occur commonly on BM, but our cases showed a slight predilection for GI (50%) over BM (40%)., Peripheral ossifying fibromas are reported usually on maxillary GI and our findings were concurrent with it.,
Evaluation of the collagen fibers in inflammatory fibrous hyperplasia was performed using picrosirius red stain under polarizing microscopy. The study showed the collagen fibers to have red-colored birefringence, implying mature collagen fiber. Our results were similar as we noticed red birefringence to be predominant in these lesions and it was statistically significant.
A study using picrosirius red stain and polarized microscopy was carried out to compare peripheral ossifying fibroma, central ossifying fibroma and fibrous dysplasia. It revealed more areas of green immature fibers in peripheral ossifying fibroma than the other lesions. Our results also showed similar findings with green immature birefringence of collagen fibers predominating significantly in peripheral ossifying fibroma.
Literature search did not reveal any studies of peripheral giant-cell granulomas or pyogenic granulomas or fibromas under polarized microscopy after picrosirus red staining. Our study showed more of immature fibers (green birefringence) in peripheral giant-cell granulomas, more mature fibers (red birefringence) in pyogenic granulomas, and mixed mature and immature collagen fibers (both green and red birefringence) in fibromas.
The literature search for recurrence rate of the above peripheral lesions was carried out. Peripheral giant-cell granuloma is a reactive, proliferative, extra-osseous lesion. The recurrence rate has been reported to vary from 5% to 70.6%., Inflammatory fibrous hyperplasia is caused by irritating factors, because of the poor maintenance of oral hygiene, a recurrence rate of 8.9% has been reported., The relapse of fibromas depends on the removal of the etiologic factors. About 8% to 20% relapse of the cases has been reported when it is incompletely excised. Recurrence rate of pyogenic granuloma varies from 5.8% to 15.8%. About 20% of peripheral ossifying fibromas involves periodontium and periodontal ligament. Hence, complete surgical excision has to be performed with strict follow up to eliminate the relapse of the cases.
Reports of the recurrences of these reactive lesions based on the birefringence properties of the collagen fibers under polarized microscope after picrosirius red staining have not been established. In our current study also, we were unable to relate these findings, as ours was a retrospective study with samples collected from the hospital archives and the follow-up details of the patients could not be established. Hence, a comparison of the findings from the present study was carried out with the recurrence rates of the similar lesions reported in literature.
In our study, peripheral ossifying fibromas and peripheral giant-cell granulomas showed more of immature green birefringent fibers and inflammatory fibrous hyperplasia and pyogenic granulomas showed predominant thick mature red fibers. In fibromas, both immature and mature fibers were distributed similarly. Mixed type (orange-red and green-yellow) were almost same in all the lesions in the current study. All these findings were significant statistically.
The progression and recurrences of the oral reactive lesions are dependent on many factors such as the causatives agents, removal of the causative agents, location of the lesion, gender, completeness of the surgical removal, and hormonal influences. Despite all these factors, when the recurrence rates and the types of collagen fibers are correlated, significant conclusions could be drawn from the present study. Giant-cell granulomas followed by peripheral ossifying fibromas and fibromas showed highest recurrence rates. In both giant-cell granulomas and peripheral ossifying fibromas, the predominant fibers were the immature green fibers. In fibromas, both immature and mature fibers were seen almost equally. The less recurrent inflammatory fibrous hyperplasia and pyogenic granulomas showed red mature fibers. Thus a tentative conclusion that mature red fibers have better prognosis than the immature green fibers, in reactive oral lesions could be arrived at. However, larger samples with prospective cases and regular follow up of cases are necessary to establish these findings.
| Conclusion|| |
From the literature, the available recurrent rates for lesions in consideration were correlated with the type of collagen birefringence in our cases. A plausible conclusion that the lesions with mature red fibers have better prognosis than the lesions with immature green fibers, in reactive oral lesions could be drawn. However, larger samples with prospective cases and regular follow up are necessary to establish these findings. As the current study samples were obtained from the archives of the department, the details of the follow up of the cases could not be obtained.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]