 Abstract | | |
Introduction: Collagen forms an integral part of connective tissue and maintains its structural integrity. It has natural birefringence which is attributed to the arrangement of its fibers and is enhanced by special stains such as picrosirius red through polarizing microscopy. The polarization colors differ according to the fiber thickness and pattern of arrangement which in turn related to aggressiveness. Hence, the present study was conducted to evaluate collagen fibers in keratocystic odontogenic tumor (KCOT) and ameloblastoma using polarizing microscopy. Aim: This study aims to compare and correlate different types and patterns of collagen fibers in KCOT and ameloblastoma using picrosirius red stain under polarizing microscopy to delineate their aggressiveness. Materials and Methods: The color, thickness, and orientation of collagen fibers in the KCOTs (n = 15) and ameloblastomas (n = 15) were studied histochemically by staining the sections with picrosirius red and examined under polarizing microscope using image analyzer software. Results: When collagen fiber bundles in KCOT and ameloblastoma were compared, significant difference was noted between yellowish-orange collagen fiber bundles, but no significant difference was observed between greenish-yellow and orange-red collagen bundles. With respect to orientation and organization, the results are statistically significant (P < 0.05). Conclusion: The connective tissue stroma of KCOT could be regarded not just as a structural support but as a functional part of the lesion. In KCOT, the thin, parallel, and loosely arranged greenish-yellow collagen fibers may be attributed to its high recurrence rate and biological aggressiveness.
Keywords: Ameloblastoma, keratocystic odontogenic tumor, polarizing microscopy
How to cite this article:
Peddapelli K, Rao V, Kumar MP, Sravya T, Rakesh D. Analysis of collagen fibers in keratocystic odontogenic tumor and ameloblastoma: A polarizing microscopic study. Indian J Dent Res 2019;30:731-5 |
How to cite this URL:
Peddapelli K, Rao V, Kumar MP, Sravya T, Rakesh D. Analysis of collagen fibers in keratocystic odontogenic tumor and ameloblastoma: A polarizing microscopic study. Indian J Dent Res [serial online] 2019 [cited 2023 Mar 22];30:731-5. Available from: https://www.ijdr.in/text.asp?2019/30/5/731/273437 |
| Introduction | |  |
Odontogenic keratocyst was first described by Philipsen in 1956[1],[2],[3],[4] and was categorized as a cyst.[5] Toller in 1967 suggested it as a benign cystic neoplasm rather than a simple odontogenic cyst owing to its neoplastic behavior clinically.[1],[4] The propensity for recurrence and the aggressive behavior clinically and histologically has necessitated the reclassification of these lesions by the WHO working group on odontogenic tumors as keratocystic odontogenic tumors (KCOTs) to address their neoplastic nature.[1],[2],[6] According to WHO 2005, KCOT is defined as “benign uni- or multi-cystic intraosseous tumor of odontogenic origin.”[1] However, the reclassification has not yet been universally accepted.[1],[2],[4]
Among all the odontogenic tumors, ameloblastoma is more aggressive clinically. The epithelium of the above two lesions has been investigated extensively regarding their role in the proliferative and aggressive behavior. However, extensive studies need to be done to evaluate the role of the connective tissue wall in their behavior.
The reciprocal interactions between epithelium and mesenchyme are thought to play a crucial role in normal odontogenesis. Thus, the mesenchymal influence may play an important role in the maintenance of epithelial expression.[7]
Collagen, the major component of the fibrous capsule, plays a vital role in maintaining the structural integrity and in determining the tissue function.[2],[8],[9] In pathological setting, collagen can show variations in the way individual fibrils are organized into fibers and in terms of diameter and cross-sectional profile.[7],[10] Hence, the study of the organization of collagen fibers may be helpful in understanding the behavior of the lesion.
Picrosirius red staining in combination with polarization microscopy has been used to study the individual collagen fibers and to determine their content in the specific tissue.[3],[9],[10],[11] Collagen molecules, rich in basic amino acids which are disposed in a parallel orientation, can strongly react with the acidic dyes. Sirius red is an elongated dye molecule which reacts with collagen and promotes an enhancement of its normal birefringence. The birefringence is mainly because of the parallel arrangement of dye molecules along the long axis of each collagen molecule.[7] Both fiber thickness and packing of collagen can cause differences in polarization colors.[7],[12],[13] This method can serve as a procedure to differentiate procollagens, intermediates, and pathological collagen fibers.[2],[7],[10],[12],[13] Hence, the present study was undertaken to analyze the nature of collagen fibers in KCOT and ameloblastoma.
| Materials and Methods | | |
After obtaining the Ethical Committee clearance from our college and hospital, 15 cases of formalin-fixed paraffin-embedded tissue blocks each of KCOT (n = 15) and ameloblastomas (n = 15) which were histopathologically confirmed were retrieved. Sections of 3-μm thickness were obtained and stained using picrosirius red stain (Sigma-Aldrich). The sections were then examined under polarizing microscope for birefringence of collagen fibers and were recorded. With the application of image analyzer software (Image-Pro Insight 8.0 version, Olympus), thickness of collagen fiber bundles is measured. In each section, two separate high-power fields were examined. The connective tissue showed polarization colors varying from greenish-yellow to yellow-orange to orange-red. The color of the collagen fibers was noted by two independent observers to eliminate the interobserver bias. Collagen fiber orientation in relation to the epithelial component was also observed. The analysis was performed at ×100 and ×400 magnifications. Each case was classified according to the fiber orientation pattern as not parallel and parallel. The organization of collagen fibers was also evaluated in areas near and distant from the epithelial component. Each case was classified as (1) loose bundles of collagen fibers (loosely arranged and interwoven in all directions) and (2) dense (well-defined organization with orderly organized collagen fibers forming collagen lamellae). Statistical analysis was done using SPSS version 14 (Statistical Package for Social Sciences, Chicago, Illinois, USA) software and findings obtained were subjected to Student's t-test and Chi-square test.
| Results | | |
In the present study, the mean distribution values of greenish-yellow, yellowish-orange, and orange-red collagen fiber bundles in 15 cases each of KCOT and ameloblastoma are shown in [Table 1]. In KCOT, greenish-yellow fibers dominated in the areas near the epithelium whereas yellowish-orange and orange-red fibers dominated in the areas away from the epithelium [Figure 1]. In case of ameloblastoma, greenish-yellow fibers dominated in both the areas [Figure 2]. There was a significant difference between yellowish-orange collagen fiber bundles, but no significant difference was observed between greenish-yellow and orange-red collagen bundles in KCOT and ameloblastoma [Figure 3]. With respect to orientation, it was revealed that 13/15 (86.67%) cases of KCOT and 7/15 (46.67%) cases of ameloblastoma showed parallel arrangement of collagen fibers in the areas near the epithelium [Table 2]. In relation to the organization, 13 (86.67%) cases of KCOT and 10 (66.67%) cases of ameloblastoma showed loosely arranged collagen fibers in areas near the epithelium, whereas 9 (60%) cases in KCOT and 5 (33.33%) cases of ameloblastoma showed densely arranged collagen fibers in the areas away from the epithelium [Table 3]. This different pattern of collagen orientation and packing was found to be statistically significant (P < 0.05). | Table 1: Comparison of distribution of collagen fiber bundles in keratocystic odontogenic tumor and ameloblastoma
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 | Figure 1: Keratocystic odontogenic tumor revealing dominated greenish-yellow fibers in the areas near the epithelium whereas yellowish-orange and orange-red fibers in the areas away from the epithelium
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 | Figure 2: Ameloblastoma revealing dominated greenish-yellow fibers in the areas near the epithelium and away from the epithelium
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 | Figure 3: The distribution of collagen fiber bundles in keratocystic odontogenic tumor and ameloblastoma
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 | Table 2: Comparison of orientation of collagen fibers near the epithelium and away from the epithelium in keratocystic odontogenic tumor and ameloblastoma
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 | Table 3: Comparison of organization/packing of collagen fibers near the epithelium and away from the epithelium in keratocystic odontogenic tumor and ameloblastoma
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| Discussion | | |
Numerous studies have been done to classify the odontogenic lesions based on the clinicopathological criteria. With the development of new technologies in diagnostics such as immunohistochemistry, special stains, molecular biology, and genetics, as well as the clinical and epidemiological follow-up, some of the lesions are reclassified according to their biological behavior.[14] Odontogenic keratocyst is one such lesion which is highly controversial with respect to its histopathologic features and clinical behavior. Earlier, it was thought to be a cyst; currently, it is considered as a benign locally aggressive neoplasm and has been renamed as KCOT.[1],[2],[7]
KCOT is always a subject of controversy since its first description by Philipsen in 1956. Although the WHO has suggested term KCOT, there is no universal agreement regarding it.[2] The present study focussed on this controversy and analyzed the collagen fibers in KCOT and compared to ameloblastoma to elucidate the biological behavior of KCOT.
Interplay between the epithelium and connective tissue plays a significant role in the pathogenesis of odontogenic lesions.[9],[15] According to Vedtofte et al., the biologic behavior is dependent on both the epithelium and connective tissue.[8],[16],[17] The epithelium of these lesions has been investigated extensively with regard to their role in the proliferative and aggressive behavior of the lesions. However, the role of the connective tissue wall in their behavior has not been studied as extensively.[9] Collagen forms an integral part of the connective tissue stroma and plays a vital role in maintaining the structural integrity and in determining tissue function.[2] Collagen has natural birefringence which is attributed to the arrangement of its fibers and is enhanced by special stains such as Van Gieson, Masson's trichrome, and picrosirius red. Van Gieson and trichrome stains may not be ideal for detection of collagen fibers as both these methods fail to reveal thin collagen fibers, a disadvantage which can lead to a underestimation of collagen content. This perplexing issue made Puchtler and his colleagues. seek a better method and they found that sirius red F3BA (color index 35780) dissolved in a saturated picric acid solution (picrosirius red) consistently stained thin collagen fibers, did not fade, and was appropriate for use with polarized light microscopy.[9]
Collagen fibril formation is complex and depends on numerous secondary or posttranslational modifications. Defects in these modifications are associated with a number of diseases.[7] Moreover, evidence exists that collagen fibers undergo a rapid maturation in fibrotic processes where the intensity of their birefringence increases and at the same time, their polarization color changes. Newly formed collagen fibers appear in green polarization color. Later, in a more mature stage, they become yellow, orange, or red under crossed polaroids. As collagen matures, there is change in proteoglycan content of the fibers causing dehydration of fibers resulting in increase in diameter of collagen fibers and intensity of birefringence. Hence, the change in polarizing colors.[9],[15]
Aparna V et al. in their study using picrosirius red staining followed by polarizing microscopy, selectively demonstrated collagen with an observable difference in the polarizing colors caused by fiber thickness as well as by the packing of the collagen.[13] According to Junqueira et al.,[18] Szendröi et al.,[19] and Dayan et al.,[20] green to greenish-yellow fibers represent immature, thin, loosely arranged fibers which uptake less stain and show weak birefringence. Yellowish-orange fibers represent intermediate fibers in the process of maturation of collagen. Orange-red fibers represent thick, mature, and tightly packed fibers which uptake more stain and show strong birefringence.[18],[19],[20] The tightly packed and presumably, better-aligned collagen molecules had polarization colors of longer wavelengths,[3],[10] and poorly packed had polarization colors of shorter wavelengths.[3] Sharf et al. in 1997 conducted nuclear resonance studies on physical aggregation of collagen and suggested that orange to red polarization color is due to well-packed fibers and green to greenish-yellow is due to poorly packed fibers.[21] Thus, examination of collagen fibers in picrosirius red-stained sections by polarizing microscopy can serve as a procedure for differentiating procollagens, intermediates, and other nontightly packed collagen fibers from normal tightly packed fibers.
According to Zhang et al., the stroma of KCOT could be regarded not just as a structural support of the cyst wall but as playing a part in the neoplastic behavior of the cyst.[7]
In the present study, the amount of greenish-yellow fibers in KCOT were almost approximating with that of ameloblastoma. Hence, it can be conferred that KCOT has an aggressive behavior clinically and may can be regarded as tumor instead of a cyst. However, in areas away from the epithelium, KCOT has increased amount of densely packed yellowish-orange fibers, whereas ameloblastoma showed loosely packed greenish-yellow fibers suggesting that the aggressiveness of KCOT is less when compared to ameloblastoma. Moreover, the parallel arrangement of collagen in KCOT reflects the detachable nature of epithelium affecting the complete surgical removal and becoming the cause for recurrence. This loose and parallel pattern of collagen arrangement observed in KCOT may be useful to facilitate the separation of the epithelial lining from the underlying connective tissue wall, the presence of satellite cysts, and the invasive potential of the lesion, which are important characteristics of this tumor.[22]
In the present study, greenish-yellow fibers in KCOT are due to collagen that is loosely packed and may be composed of procollagen, intermediates, or other pathologic collagen that is not tightly packed. The normal collagen because of its stable helical structure is resistant to degradation. However, these parallel, loosely packed, and denatured collagens are more susceptible to degradation by collagenases and proteinases reflecting their aggressive nature.[18],[23]
In this study, we have tried to reassess the aggressive nature of the individual lesions based on histopathology alone by studying the polarization color collagen fibers in KCOT and ameloblastoma to assess the biological behavior of these lesions or to reflect their aggressiveness. A further study evaluating larger sample size with inclusion of all radiological types of KCOT and histological types of ameloblastoma may help in arriving at the above conclusions more accurately.
| Conclusion | | |
Connective tissue stroma has been suggested to play an important role in the pathogenesis of KCOT. The connective tissue stroma of KCOT could be regarded not just as a structural support but as a functional part of the lesion. In KCOT, the thin, parallel, and loosely arranged greenish-yellow collagen fibers may be attributed to its high recurrence rate and biological aggressiveness.
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Conflicts of interest
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Correspondence Address:
Dr. Taneeru Sravya
Department of Oral Pathology and Microbiology, Mamata Dental College, Giriprasad Nagar, Khammam - 507 002, Telangana
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijdr.IJDR_77_16
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3] |
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