Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
HOME | ABOUT US | EDITORIAL BOARD | AHEAD OF PRINT | CURRENT ISSUE | ARCHIVES | INSTRUCTIONS | SUBSCRIBE | ADVERTISE | CONTACT
Indian Journal of Dental Research   Login   |  Users online: 849

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         

 


 
Table of Contents   
ORIGINAL RESEARCH  
Year : 2019  |  Volume : 30  |  Issue : 5  |  Page : 731-735
Analysis of collagen fibers in keratocystic odontogenic tumor and ameloblastoma: A polarizing microscopic study


Department of Oral Pathology and Microbiology, Mamata Dental College, Khammam, Telangana, India

Click here for correspondence address and email

Date of Submission05-Feb-2016
Date of Acceptance07-Jul-2017
Date of Web Publication18-Dec-2019
 

   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 2020 Aug 5];30:731-5. Available from: http://www.ijdr.in/text.asp?2019/30/5/731/273437



   Introduction Top


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 Top


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 Top


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

Click here to view
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

Click here to view
Figure 2: Ameloblastoma revealing dominated greenish-yellow fibers in the areas near the epithelium and away from the epithelium

Click here to view
Figure 3: The distribution of collagen fiber bundles in keratocystic odontogenic tumor and ameloblastoma

Click here to view
Table 2: Comparison of orientation of collagen fibers near the epithelium and away from the epithelium in keratocystic odontogenic tumor and ameloblastoma

Click here to view
Table 3: Comparison of organization/packing of collagen fibers near the epithelium and away from the epithelium in keratocystic odontogenic tumor and ameloblastoma

Click here to view



   Discussion Top


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 Top


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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Ba K, Li X, Wang H, Liu Y, Zheng G, Yang Z, et al. Correlation between imaging features and epithelial cell proliferation in keratocystic odontogenic tumour. Dentomaxillofac Radiol 2010;39:368-74.  Back to cited text no. 1
    
2.
Singh HP, Shetty S, Patil P, Sethi N, Singh A, Raghunandan B. Correlation between collagen fibers and radiographic patterns of keratocystic odontogenic tumour. J Clin Diagn Res 2014;8:ZC16-8.  Back to cited text no. 2
    
3.
Kaijkar MS, Joshi PS, Chougule M. A comparative study of influence of inflammation and collagen deposition in the cystic wall of infected and non-infected odontogenic keratocyst using picrosirius red and polarizing microscopy. Indian J Oral Sci 2014;5:83-7.  Back to cited text no. 3
  [Full text]  
4.
Madras J, Lapointe H. Keratocystic odontogenic tumour: Reclassification of the odontogenic keratocyst from cyst to tumour. J Can Dent Assoc 2008;74:165-165h.  Back to cited text no. 4
    
5.
Adyanthaya S, Shetty P. The effect of inflammation on the epithelial lining and polarization colors of collagen fibres in odontogenic keratocysts: A retrospective histopathological study. J Int Oral Health 2011;3:15-21.  Back to cited text no. 5
    
6.
Nagraja A, Anigol PS, Kamath VV, Setlur KP. Keratocystic odontogenic tumour of the maxilla: Report of a rare case and review of literature. World J Dent 2012;3:100-8.  Back to cited text no. 6
    
7.
Zhang JY, Dong Q, Li TJ. Differences in collagen fibres in the capsule walls of parakeratinized and orthokeratinized odontogenic cysts. Int J Oral Maxillofac Surg 2011;40:1296-300.  Back to cited text no. 7
    
8.
Aggarwal P, Saxena S. Stromal differences in odontogenic cysts of a common histopathogenesis but with different biological behavior: A study with picrosirius red and polarizing microscopy. Indian J Cancer 2011;48:211-5.  Back to cited text no. 8
[PUBMED]  [Full text]  
9.
Singh HP, Shetty DC, Wadhwan V, Aggarwal P. A quantitative and qualitative comparative analysis of collagen fibers to determine the role of connective tissue stroma on biological behavior of odontogenic cysts: A histochemical study. Natl J Maxillofac Surg 2012;3:15-20.  Back to cited text no. 9
[PUBMED]  [Full text]  
10.
Rabau MY, Dayan D. Polarization microscopy of picrosirius red stained sections: A useful method for qualitative evaluation of intestinal wall collagen. Histol Histopathol 1994;9:525-8.  Back to cited text no. 10
    
11.
Junqueira LC, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 1979;11:447-55.  Back to cited text no. 11
    
12.
Hirshberg A, Sherman S, Buchner A, Dayan D. Collagen fibres in the wall of odontogenic keratocysts: A study with picrosirius red and polarizing microscopy. J Oral Pathol Med 1999;28:410-2.  Back to cited text no. 12
    
13.
Aparna V, Charu S. Evaluation of collagen indifferent grades of oral squamous cell carcinoma by using the picrosirius red stain - A histochemical study. J Clin Diagn Res 2010;4:3444-9.  Back to cited text no. 13
    
14.
Shear M. The aggressive nature of the odontogenic keratocyst: Is it a benign cystic neoplasm? Part 1. Clinical and early experimental evidence of aggressive behaviour. Oral Oncol 2002;38:219-26.  Back to cited text no. 14
    
15.
Vij R, Vij H, Rao NN. Evaluation of collagen in connective tissue walls of odontogenic cysts – A histochemical study. J Oral Pathol Med 2011;40:257-62.  Back to cited text no. 15
    
16.
Vered M, Shohat I, Buchner A, Dayan D. Myofibroblasts in stroma of odontogenic cysts and tumors can contribute to variations in the biological behavior of lesions. Oral Oncol 2005;41:1028-33.  Back to cited text no. 16
    
17.
Vedtofte P, Holmstrup P, Dabelsteen E. Human odontogenic keratocyst transplants in nude mice. Scand J Dent Res 1982;90:306-14.  Back to cited text no. 17
    
18.
Junqueira LC, Montes GS, Sanchez EM. The influence of tissue section thickness on the study of collagen by the Picrosirius-polarization method. Histochemistry 1982;74:153-6.  Back to cited text no. 18
    
19.
Szendröi M, Vajta G, Kovács L, Schaff Z, Lapis K. Polarization colours of collagen fibres: A sign of collagen production activity in fibrotic processes. Acta Morphol Hung 1984;32:47-55.  Back to cited text no. 19
    
20.
Dayan D, Waner T, Tal H, Nyska A. Polarization microscopy of picrosirius red-stained collagen from oxodipine-induced hyperplastic gingiva of beagle dogs. Int J Exp Pathol 1993;74:225-8.  Back to cited text no. 20
    
21.
Sharf Y, Knubovets T, Dayan D, Hirshberg A, Akselrod S, Navon G. The source of NMR-detected motional anisotropy of water in blood vessel walls. Biophys J 1997;73:1198-204.  Back to cited text no. 21
    
22.
Hirshberg A, Lib M, Kozlovsky A, Kaplan I. The influence of inflammation on the polarization colors of collagen fibers in the wall of odontogenic keratocyst. Oral Oncol 2007;43:278-82.  Back to cited text no. 22
    
23.
Hand AR, Tencate AR. Cytoskeleton, junction and fibroblasts. In: Tencate AR, editor. Oral Histology. 5th ed. Singapore: Harcourt Asia Pvt, Ltd.; 1999. p. 61-78.  Back to cited text no. 23
    

Top
Correspondence Address:
Dr. Taneeru Sravya
Department of Oral Pathology and Microbiology, Mamata Dental College, Giriprasad Nagar, Khammam - 507 002, Telangana
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_77_16

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed298    
    Printed2    
    Emailed0    
    PDF Downloaded33    
    Comments [Add]    

Recommend this journal