|Year : 2015 | Volume
| Issue : 2 | Page : 119-124
Study of polarization colors in the connective tissue wall of odontogenic cysts using picrosirius red stain
Anusha Shetty, Avinash Tamgadge, Sudhir Bhalerao, Treville Periera, Sandhya Tamgadge, Swati Gotmare
Department of Oral and Maxillofacial Pathology and Microbiology, Padmashree Dr. D Y Patil Dental College and Hospital, Navi Mumbai, Maharashtra, India
|Date of Web Publication||17-Nov-2015|
Dr. Sandhya Tamgadge
Department of Oral and Maxillofacial Pathology and Microbiology, Padmashree Dr. D Y Patil Dental College and Hospital, Sector 7, Nerul, Navi Mumbai - 400 706, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Lesions of odontogenic origin comprise the heterogeneous group ranging from hamartomatous proliferations, cysts to benign and malignant tumors. Interplay between the epithelium and connective tissue can be assumed to play a significant role in the pathogenesis of odontogenic cysts. Aims and Objectives: A study was taken up to show the role of picrosirius red (PSR) stain to demonstrate the fibers and also to assess the difference in the nature of the fibers (different color patterns) and to find out the role of it, if any in the pathogenesis and biological behavior of the commonly occurring odontogenic cysts. Materials and Methods: Collagen fibers of 30 cases of odontogenic cysts (10 radicular cysts, 10 odontogenic keratocysts (OKC's), and 10 dentigerous cysts) were studied by staining the sections with PSR stain and examining them under bright field and polarizing microscope. Results: Sixty-seven percentage of the thin collagen fibers and 55% of the thick fibers in radicular cyst showed green-yellow birefringence. Fifty-seven percentage of the thin collagen fibers and 15% of the thick fibers in OKC showed green-yellow birefringence. Eighty-two percentage of the thin collagen fibers and 66% of the thick fibers in dentigerous cysts showed green-yellow birefringence. Rest of the fibers showed orange-red birefringence. Statistical analysis with one-way ANOVA was significant with a P < 0.01 only for thick fibers. Moreover, comparison of polarization colors of thick fibers of odontogenic cysts with duration of the lesion gave statistically significant results. Conclusion: The observations in the present study with respect to color profiles of the collagen fibers in the three commonly occurring odontogenic cysts possibly explain the biological behavior of the lesions. The predominant orange-red birefringence in OKC's in comparison to radicular and dentigerous cysts suggests that OKC's exhibit well organized and tightly packed fibers. This may possibly explain the reason for the poorer prognosis of OKC's. It is suggested that though the epithelium plays an important role in the pathogenesis of these lesions, even stroma is likely to play an equally important role in the pathogenesis and biological behavior.
Keywords: Collagen fibers, dentigerous cyst, odontogenic keratocyst, picrosirius stain, polarization colors, polarized microscopy, radicular cyst
|How to cite this article:|
Shetty A, Tamgadge A, Bhalerao S, Periera T, Tamgadge S, Gotmare S. Study of polarization colors in the connective tissue wall of odontogenic cysts using picrosirius red stain. J Orofac Sci 2015;7:119-24
|How to cite this URL:|
Shetty A, Tamgadge A, Bhalerao S, Periera T, Tamgadge S, Gotmare S. Study of polarization colors in the connective tissue wall of odontogenic cysts using picrosirius red stain. J Orofac Sci [serial online] 2015 [cited 2019 Jul 20];7:119-24. Available from: http://www.jofs.in/text.asp?2015/7/2/119/169771
| Introduction|| |
Lesions of odontogenic origin comprise the heterogeneous group ranging from hamartomatous proliferations, cysts to benign and malignant tumors.
Despite the fact that proliferation of epithelial cells is an indispensable ingredient for cyst formation, connective tissue may be regarded as a functional part of the cyst and not just a structural support. Interplay between the epithelium and connective tissue can be assumed to play a significant role in the pathogenesis of odontogenic cysts.
Collagen, the most abundant protein in the body, which constitutes 34% of the total extracellular matrix (ECM) proteins plays a vital role in maintaining the functional integrity of tissues including the odontogenic apparatus and was thought that it might also play a role in pathogenesis and expansion of odontogenic cyst.  In pathological conditions, collagen can show variations in the way the individual fibrils are organized into fibers. Picrosirius red (PSR) staining in combination with polarization microscopy has been used to visualize and study collagen in paraffin-embedded tissue sections.
Keeping this in view, a study was taken up to show the role of PSR stain to demonstrate the fibers and also to assess the difference in the nature of the fibers (different color patterns) and to find out the role of it, if any in the pathogenesis and biological behavior of the commonly occurring odontogenic cysts i.e., radicular cysts, odontogenic keratocysts (OKC's), and dentigerous cysts. We have also related age, gender, site, and duration of the lesion to polarization colors of odontogenic cysts to determine if these attributes play a role in the biologic behavior of commonly occurring odontogenic cysts.
| Materials and Methods|| |
Thirty cases of odontogenic cysts were retrieved from the archives; among which 10 cases were radicular cysts, 10 cases were OKC's, and 10 cases were dentigerous cysts.
Two paraffin-embedded sections were obtained for each case. The diagnosis of each case was confirmed by examining one section stained with hematoxylin and eosin using bright field microscopy. The second section was stained using PSR. , The PSR-stained sections were examined under bright field and polarizing microscope. All images were then clicked under oil immersion (×100), and fiber thickness was measured. A total of 4 high-power (×100) fields were selected from the sections, and a total of 20 fibers from each section were measured for thin (0.8 lm or less) and thick fibers (1.2-2.4 m) by image analysis software version of Leica Microsystems (CMS GmbH Ernst-Leitz-Street 17-37, 35578 Wetzlar, Germany) under polarized light. The polarizing colors and thickness of the collagen fibers were determined by two independent observers to minimize the subjectivity. Further statistical analysis was done to derive the results.
Polarization colors were divided into two groups: Green-yellow and orange-red. Categorical data were analyzed by Chi-square test multiple group comparisons were made by one-way ANOVA. For all the tests, a P-value of 0.05 or less was considered for statistical significance.
| Results|| |
Collagen fibers appeared uniformly dark pink under bright field microscope [Figure 1] and birefringence colors under polarized light microscopy.
|Figure 1: Odontogenic keratocyst (picrosirius red stain, ×10) under bright field microscopy|
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Sixty-seven percentage of the thin collagen fibers and 55% of the thick fibers in radicular cyst showed green-yellow birefringence. Fifty-seven percentage of the thin collagen fibers and 15% of the thick fibers in OKC showed green-yellow birefringence. Seventy-two percentage of the thin collagen fibers and 66% of the thick fibers in dentigerous cysts showed green-yellow birefringence. Rest of the fibers showed orange-red birefringence [Table 1] and [Table 2].
|Table 1: Comparison of polarization colors of thin collagen fibers between radicular cyst, OKC, and dentigerous cyst|
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|Table 2: Comparison of polarization colours of thick collagen fibers between radicular cyst, OKC and dentigerous cyst|
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Polarization colors of thin and thick fibers of the radicular cyst, OKC, and dentigerous cyst were compared. Statistical analysis with one-way ANOVA was significant with a P < 0.01 only for thick fibers.
The 30 experimental cases of odontogenic cysts were in the age range of 8-56 years with a mean age of 26.4 ± 11.11 years. Patients with the age group ranging between 1 and 20 years showed predominance of thick orange-red fibers (37.78% green to yellow and 62.22% orange to red), when compared with patients with the age group ranging between 21 and 40 years (47.37% green to yellow and 52.63% orange to red) and 41-60 years (50% green to yellow and 40% orange to red). Statistical analysis with Pearson's Chi-square test was significant with P < 0.05. Comparison of polarization colors of thin fibers of odontogenic cysts with the age of the subject did not give significant results.
Out of 30 subjects, 19 (63.33%) were males, and 11 (36.67%) were females. Comparison of polarization colors of thin and thick fibers of odontogenic cysts with the gender of the subject gave nonsignificant results.
Out of 30 subjects with odontogenic cysts, 11 lesions occurred in the maxilla, and 19 lesions occurred in the mandible. Lesions occurring in mandible showed a predominance of thick orange-red fibers (43.16% green to yellow and 56.84% orange to red), when compared with maxilla (49.09% green to yellow and 50.91% orange to red). Statistical analysis with Pearson's Chi-square test was significant with P < 0.05.
Out of 30 subjects with odontogenic cysts, 22 subjects gave a history of duration ranging between 1 and 30 days, 3 subjects gave a history of duration ranging between 30 and 60 days, and 5 subjects gave a history of the duration of above 60 days. Comparison of polarization colors of thick fibers of odontogenic cysts with duration of the lesion gave statistically significant results whereas that of thin fibers were not significant [Table 3] and [Table 4].
|Table 3: Comparison of polarization colours of thin fibers of odontogenic cysts with duration of the lesion|
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|Table 4: Comparison of polarization colours of thick fibers of odontogenic cysts with duration of the lesion|
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| Discussion|| |
Tissues that contribute to odontogenesis undergo stages of differentiation with the period. Any of the tissues participating in this process may be involved in the development of myriad pathologies either in the form of cysts, hamartomas, or tumors collectively often referred as odontogenic cysts and odontogenic tumors. 
Epithelial-mesenchymal interactions are essential in maintaining homeostatic equilibrium in adult tissues with the stromal cells maintaining control over cell size, function, and response to wounds and other pathologic conditions through modification of the ECM. , The ECM performs a very important role in growth, regulation, tissue differentiation, and organization.  In the developing tooth, morphogenesis and cell differentiation are controlled by epithelial-mesenchymal interaction. Development of odontogenic tumors and cysts is also dependent on these interactions. 
Earlier, extensive research works were done on the epithelial component of the odontogenic cyst to explain its role in the expansion of these cysts without the involvement of its nonepithelial component. It was Vedtofte et al., who showed the importance of stromal component from a study on the transplanted keratocyst epithelium in nude mice, which led the authors to believe that the differentiation of the cystic epithelium is not independent of the stroma, and suggested that the stromal component has a role to play in the biological behavior by establishing ectomesenchymal interaction. ,
Collagen is an anisotropic structure, which exhibits the phenomenon of birefringence, which can be selectively visualized using polarized light microscopy. Weak birefringence in biological specimens is enhanced by the addition of dyes, or impregnating metals in an orderly linear arrangement. Thus, polarized light has the ability to enhance histological assessment of tissue and can provide additional insight into the composition and structure of collagen. Following this line of reasoning, the present study was carried out. 
In a study of various collagen staining techniques, Constantine and Mowry found that none was specific for collagen. They found the picric acid methods to be the most selective, particularly when acid fuchsin or sirius red F3BA were used.  It is a strong anionic dye, which stains collagen via its sulfonic acid group. Sirius red is an elongated dye. It attaches to the collagen fiber in such a way that their long axes are parallel to the long axis of each collagen molecule. This results in enlarged birefringence of the collagen.
The enhancement of birefringence promoted by the picrosirius polarization method is, therefore, specific for collagenous structures composed of aggregates of oriented molecules. 
It was found in our study, that when stained with PSR, structures other than collagen did not exhibit any birefringence proving that this histochemical stain is selective for collagen fibers. This is supported by other studies by Constantine and Mowry and Mahajan et al.,
In the present study, the radicular cysts exhibited a slight predominance of greenish-yellow birefringence [Figure 2] whereas OKC's showed a slight predominance of greenish-yellow birefringence among thin fibers, and significant predominance of orange-red fibers among thick fibers [Figure 3]. These findings were in conjunction with studies by Aggarwal and Saxena (2011), who noted a predominance of orange-red birefringence in odontogenic keratocysts, dentigerous cysts, and ameloblastomas when compared to radicular cysts, which showed a predominance of green-greenish yellow fibers. 
|Figure 2: Picrosirius red stained section showing predominantly green-yellow color of collagen fibers in radicular cyst (×10) under polarized microscopy|
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|Figure 3: Picrosirius red stained section showing predominantly orange-red color of collagen fibers in odontogenic keratocyst (×10) under polarized microscopy|
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The present study contrasted the studies by Hirshberg et al. and Mahajan et al. (2013), who found that OKC's in comparison with dentigerous cysts and radicular cysts exhibited a predominant greenish-yellow birefringence of both thick and thin fibers, which were suggestive of procollagen, intermediate or pathologic collagen. ,
The slight predominance of green-yellow polarization colors in radicular cyst can be explained by the fact that the radicular cyst is an inflammatory cyst, and the inflammatory cells could affect the packing of collagen fibers. Teronan et al. held the view that macrophages and neutrophils have a common feature of elaboration of a tissue collagenase, which is capable of causing hydrolytic breakdown of peptide bonds in the helical region of collagen. Collagenolytic activity is least in normal and mildly inflamed tissue, and greatest in severely inflamed hyperplastic tissue. Thus, inflammatory cells could affect the arrangement of collagen. ,
Recently, it has been proposed that inflammation is necessary to trigger the initiation of fibrosis; chronic inflammation leads to tissue destruction, ongoing wound healing responses, and eventually fibrosis. This could explain the presence of orange-red fibers which were also present in significant amount in radicular cysts. 
Longstanding nature of the lesion might be the cause of closely packed collagen fibers and the presence of predominantly orange-red polarization colors of collagen fibers in the connective tissue wall of OKC's.
In a study by Aggarwal and Saxena, the predominant color of collagen fibers in the stroma of odontogenic tumors was also found to be orangish-red. Similar collagen characterization of OKC's and odontogenic tumors provides further evidence regarding the comparable nature and biological behavior of these lesions. Hence, the treatment modality of these OKC's may be compared to that of odontogenic tumors. 
The probable explanation of exhibition of green-yellow birefringence color in dentigerous cyst [Figure 4] may be due to the fact that they arise due to the expansion of the dental follicle. This finding is comparable with the study conducted by Abrahão et al., who analyzed collagen fibers in tooth germ papillae, which showed a predominance of delicate green fibers. They also found a predominance of green fibers in the connective tissue of dental follicles. 
|Figure 4: Picrosirius red stained section showing predominantly green-yellow color of collagen fibers in dentigerous cyst (×10) under polarized microscopy|
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The effect of duration on the odontogenic cysts were assessed. Thin fibers did not show a significant change in the polarization colors of collagen fibers. Thick fibers, however, showed a statistically significant increase in orange-red birefringence colors as the duration of the lesion increased. This result is comparable to the study on irritation fibroma by Dayan et al., who noted an increase in the number of yellowish-orange and orange fibers, and a decrease in blue-green and green fibers as the lesion matured.
This is because as the duration of the lesions increase the amount of collagen fiber, which exhibit yellowish-orange and orange polarization colors indicating tighter packing and better alignment of the microfibrils similar to normal mature collagen. 
| Conclusion|| |
Based on these observations from the present study, it was seen that PSR stain is an important tool in a rapid and easy demonstration of collagen fibers, and also provide a powerful complement to immunohistochemistry, in situ hybridization and other diagnostic technologies.
The observations in the present study with respect to color profiles of the collagen fibers in the three commonly occurring odontogenic cysts, possibly explain the biological behavior of the lesions. The predominant orange-red birefringence in OKC's in comparison to radicular and dentigerous cysts suggests that OKC's exhibit well organized and tightly packed fibers. This may possibly explain the reason for the poorer prognosis of OKC's.
Therefore, it is suggested that though the epithelium plays an important role in the pathogenesis of these lesions, even stroma is likely to play an equally important role in the pathogenesis and biological behavior.
However, further investigations on the biochemical and molecular studies are required on a larger sample to know the major role of collagen fibers in the pathogenesis of odontogenic cysts, and also the influence of mesenchyme in the behavior of the odontogenic cysts.
Extensive studies with large samples of such lesions may shed light on their biologic behavior especially the aggressive ones thus helping precise treatment planning and patient well-being.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]
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