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ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 3  |  Page : 206-210

Evaluation of biological behavior of odontogenic keratocyst and orthokeratinized odontogenic cyst using picrosirius red stain: A clinicopathological retrospective study


Department of Oral Pathology and Microbiology, SVS Institute of Dental Sciences, Mahabubnagar, Telangana, India

Date of Submission18-Jun-2019
Date of Acceptance20-Jun-2019
Date of Web Publication17-Oct-2019

Correspondence Address:
Dr. Suvarna Manthapuri
Department of Oral Pathology and Microbiology SVS Institute of Dental Sciences, Mahabubnagar, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_69_19

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  Abstract 


Background: Odontogenic keratocyst (OKC) and orthokeratinized odontogenic cyst (OOC) show considerable difference in biological behavior, and the study of the organization of collagen fibers may be helpful in understanding the nature of these variants.
Aims: The purpose of this study is to determine the clinicopathological features and to analyze the birefringence of collagen fibers in OKC and OOC using picrosirius red stain to delineate its exact role in biological behavior.
Materials and Methods: Clinical data were obtained from the case records of 71 patients, and for the analysis of birefringence, paraffin-embedded blocks of 10 cases each of OKC and OOC were retrieved from the archives of our department during the period of 2010–2018. Tissue sections were obtained and stained with picrosirius red stain to determine the birefringence of collagen fibers in both the variants. Statistical analysis was done by unpaired t-test.
Results: Out of 71 cases, 61 were OKC and 10 were OOC. The clinicopathological data revealed that the most common affected age group was third decade with male predilection and the most common site noted was posterior mandible, and considerable difference in birefringence was noted in the collagen fibers of OKC and OOC.
Conclusion: The study results analyzing the clinicopathological parameters of OKC and OOC could act as a viable record for the distribution of odontogenic lesions in the sample population. Significant differences in birefringence were demonstrated in OKC and OOC, suggesting that different collagen fibers may exist in the two lesions.

Keywords: Birefringence, odontogenic keratocyst, orthokeratinized odontogenic cyst


How to cite this article:
Manthapuri S, Sanjeevareddygari S, Mantha H, Oruganti RV, Reddy S, Vamshi V R. Evaluation of biological behavior of odontogenic keratocyst and orthokeratinized odontogenic cyst using picrosirius red stain: A clinicopathological retrospective study. J NTR Univ Health Sci 2019;8:206-10

How to cite this URL:
Manthapuri S, Sanjeevareddygari S, Mantha H, Oruganti RV, Reddy S, Vamshi V R. Evaluation of biological behavior of odontogenic keratocyst and orthokeratinized odontogenic cyst using picrosirius red stain: A clinicopathological retrospective study. J NTR Univ Health Sci [serial online] 2019 [cited 2019 Nov 15];8:206-10. Available from: http://www.jdrntruhs.org/text.asp?2019/8/3/206/269491




  Introduction Top


Odontogenic keratocyst (OKC) and orthokeratinized odontogenic cyst (OOC) are two histopathological subtypes which show keratinized cystic epithelium with variation in incidence, distributional pattern, and their biological behavior.[1],[2],[3]

Several studies have indicated that OOC showed less proliferative activity compared with OKC. The mesenchymal influence of the fibrous capsules may play an important role in the epithelial expression, and because the major component of the stroma comprises collagen fibers, the study of the organization of collagen fibers may be helpful in understanding the behavior of the lesion.[4],[5],[6]

Information regarding demographic profile of these lesions in different population is very limited, especially in the south Indian population. Thus, this study is conducted to determine the distribution, clinical aspects, and also to compare and correlate the birefringence of collagen fibers in OKC and OOC using picrosirius red stain to delineate its exact role in biological behavior.


  Materials and Methods Top


For the analysis of clinical data, 71 cases were obtained from the case records of the patients among which 61 were diagnosed as OKC and 10 were diagnosed as OOC. For the analysis of birefringence of collagen fibers, 20 cases were selected of which 10 were OKC and 10 were OOC. Formalin-fixed paraffin-embedded tissue samples of both the variants were retrieved from the archives of the Department of Oral and Maxillofacial Pathology, SVS Institute of Dental Sciences, Mahabubnagar, Telangana, during the period of 2010–2018.

The demographic data included age, site, gender, association with impacted tooth, and radiological features. For visualization of the collagen fibers, 4-μm-thick sections were obtained and stained with picrosirius red. In brief, after deparaffinization in xylene and ethanol, the sections were hydrated in distilled water, followed by incubation in sirius red (0.1% in saturated picric acid) for 1 hour at room temperature and rinsed with distilled water. The sections were examined by polarizing microscopy, and image analysis was done using image analysis software ProgResCapturePro v2.8.8. For each case, birefringent colors were noticed from five microscopic fields under 10× magnification. Both the clinical features and birefringence were analyzed.


  Results Top


The clinical features of 71 cases were reviewed, of which 61 cases were OKC and 10 cases were OOC. The most common affected age group was 21–30 years (43.6%) followed by 31–40 years (25.3%) as shown in [Graph 1]. Males (63.28%) were most commonly affected compared with females (36.6%) as depicted in [Graph 2]. The most common site noted was posterior mandible (73.2%) followed by maxilla (12.6%). Multiple OKCs were noticed in 7% of the cases and 2.8% showed involvement of maxillary sinus as shown in [Graph 3]. Out of 71 cases, 23 cases showed the presence of impacted tooth. On radiographic examination, 68% showed unilocular radiolucency and 32% showed multilocular radiolucency.



On examining the tissue sections under the polarizing microscope, the collagen fibers in the connective tissue wall of OKC exhibited predominantly greenish yellow birefringence [Figure 1] followed by yellow orange and orange red, whereas collagen fibers in OOC showed predominantly orange red [Figure 2] birefringence followed by yellow orange. The statistical analysis with unpaired t-test showed statistically significant P value (<0.0001) for the parameter birefringence as depicted in [Table 1].
Figure 1: High power magnification showing predominantly greenish yellow birefringence in OKC using polarising microscope

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Figure 2: High power magnification showing Predominantly yellow orange birefringence in OOC under polarising microscope

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Table 1: Statistical Analysis Of Birefringence

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  Discussion Top


OKC is a unique cyst with respect to its aggressive behavior, enlargement, and recurrence. The role of the cystic capsule in OKC represents a greater part in its behavior, and even then, very little consideration has been given to it till recently. The biologic behavior of OKC is dependent on both the epithelium and underlying stroma which was suggested by Vedtofte et al. when they successfully demonstrated that transplanted keratocyst epithelium in nude mice retained its typical histological appearance only when supported by its own stroma.[7] Studies have shown that OOC presents a completely different biologic behavior compared with OKC.[2] Hence, a clinicopathological retrospective study and analysis of birefringence of collagen fibers were carried out.

The clinical features obtained in our study were coinciding with most of the studies. In this study, the most common age group affected is the third decade, with male predilection and the most common site involved is posterior mandible. These results are coinciding with the studies carried out by Myoung et al.,[8] Ramachandra et al.,[9] and Brannon.[10]

The association with impacted tooth is found to be 32% in this study and 27% in studies conducted by Sánchez-Burgos et al.[11] and Mortazavi and Baharvand,[12] which indicates a geographical variation in different sample populations and discrepancies in the size of the sample.

Radiographically, 68% of cases showed unilocular radiolucency and 32% showed multilocular radiolucency which is almost similar to a study conducted by Sánchez-Burgos et al.[11]

Many studies have focused on the proliferative activity of the epithelial component in odontogenic lesions, but the role of mesenchyme still remains unrevealed. Stromal changes in these odontogenic lesions can be depicted by picrosirius red stain and this stain imparts birefringence to collagen fibers specifically.[13]

The color exhibited by these fibers depends on their alignment, size, packing, molecular organization, ground substances, and water content.[14] The different types of collagen show different colors and intensities of birefringence. Collagen type I presents an intense birefringence with yellow to red color, collagen type II shows a variable color according to the tissue, whereas collagen type III displays a weak birefringence of green color.[15],[16]

In the pathological conditions, collagen shows variations in the organization of fibers. The birefringent colors range from shades of green, yellow, to orange red in various lesions. Normally, green to greenish yellow corresponds to poorly packed fibers, whereas orange red represents well-packed fibers.[14]

In this study, a predominance of yellowish red color, which originates from well-packed fibers, has been found in OOC. The major polarization color of OKC is greenish yellow, which differs significantly from OOC, suggesting that the collagen found in these lesions is loosely packed and might be composed of precollagens, intermediates, or pathologic collagens rather than the normal tightly packed fibers seen in OOC. These results are in accordance with the study conducted by Zhang et al.[5]

All the other polarization studies carried out by different authors for comparison between odontogenic cysts and tumours showed predominantly greenish yellow birefringence for more aggressive tumours and yellow or orange red birefringence for less aggressive cysts. The greenish yellow birefringence observed in OKC was in agreement with studies conducted by Singh et al.[17] and Hirshberg et al.[18]

A study conducted by Raj et al.[19] for the evaluation of the nature of collagen fibers in cysts and tumors, KCOT and ameloblastomas showed a predominantly greenish yellow birefringence which indicates that the behavior of OKC is similar to that of aggressive odontogenic tumors such as ameloblastoma.

To the best of our knowledge, there are only few studies conducted to evaluate the difference in polarization of collagen fibers exclusively in OKC and OOC.

Owing to different polarization colors, the composition of the mesenchymal component of OKC appeared to differ from that of OOC. It has been demonstrated that the lining epithelium of OOC has less proliferative and self-renewal potential compared with that of OKC, which appeared to reflect the contrasting differences in the biological behavior of these two lesions. It is interesting to speculate that the differences in collagen fibers within the fibrous capsules of OKC and OOC may also be related to their behavioral differences.[20]


  Conclusion Top


The study results analyzing the clinicopathological parameters of OKC and OOC could act as a viable record for the distribution of these odontogenic lesions in the sample population.

Significant differences in polarization color were demonstrated in the subepithelial zones between OKC and OOC, suggesting that different collagen fibers may exist in the two lesions and their role in pathogenesis requires further attention. More studies with large sample size are to be carried out to corroborate our view which in turn may be helpful to predict the nature of these lesions in terms of biological behavior, prognosis, and treatment modalities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Shear M. The aggressive nature of the odontogenic keratocyst: Is it a benign cystic neoplasm? Part 2. Proliferation and genetic studies. Oral Oncol 2004;38:323-31.  Back to cited text no. 1
    
2.
Dandena VK, Thimmaiah SY, Kiresur MA, Hunsigi P, Roy S, Rashmi M. A comparative study of odontogenic keratocyst and orthokeratinized odontogenic cyst using Ki67 and α smooth muscle actin. Journal of oral and maxillofacial pathology. 2017;21:458.  Back to cited text no. 2
    
3.
Kramer IR, Pindborg JJ, Shear M. The WHO histological typing of odontogenic tumours. A commentary on the second edition. Cancer 1992;70:2988-94.  Back to cited text no. 3
    
4.
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. 4
    
5.
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. 5
    
6.
Montes GS, Junqueira LC. The use of the Picrosirius-polarization method for the study of the biopathology of collagen. Mem Inst Oswaldo Cruz 1991;86:1-1.  Back to cited text no. 6
    
7.
Vedtofte P, Holmstrup P, Dabelsteen E. Human odontogenic keratocyst transplant in nude mice. Scand J Dent Res 1982;90:306-14.  Back to cited text no. 7
    
8.
Myoung H, Hong SP, Hong SD, Lee JI, Lim CY, Choung PH, et al. Odontogenic keratocyst: Review of 256 cases for recurrence and clinicopathologic parameters. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:328-33.  Back to cited text no. 8
    
9.
Ramachandra P, Maligi P, Raghuveer HP. A cumulative analysis of odontogenic cysts from major dental institutions of Bangalore city: A study of 252 cases. J Oral Maxillofac Pathol 2011;15:1.  Back to cited text no. 9
[PUBMED]  [Full text]  
10.
Brannon RB. The odontogenic keratocyst: A clinicopathologic study of 312 cases. Part I. Clinical features. Oral Surg Oral Med Oral Pathol 1976;42:54-72.  Back to cited text no. 10
    
11.
Sánchez-Burgos R, González-Martín-Moro J, Pérez-Fernández E, Burgueño-García M. Clinical, radiological and therapeutic features of keratocystic odontogenic tumours: A study over a decade. J Clin Exp Dent 2014;6:e259.  Back to cited text no. 11
    
12.
Mortazavi H, Baharvand M. Jaw lesions associated with impacted tooth: A radiographic diagnostic guide. Imaging Sci Dent 2016;46:147-57.  Back to cited text no. 12
    
13.
Moure SP, Carrard VC, Lauxen IS, Manso PP, Oliveira MG, Martins MD, et al. Collagen and elastic fibers in odontogenic entities: Analysis using light and confocal laser microscopic methods. Open Dent J 2011;5:116.  Back to cited text no. 13
    
14.
Constantine VS, Mowry RW. Selective staining of human dermal collagen: II. The use of picrosirius red F3BA with polarization microscopy. J Invest Dermatol 1968;50:419-23.  Back to cited text no. 14
    
15.
Eyden B, Tzaphlidou M. Structural variations of collagen in normal and pathological tissues: Role of electron microscopy. Micron 2001;32:287-300.  Back to cited text no. 15
    
16.
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. 16
    
17.
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 behaviour of odontogenic cysts: A histochemical study. Natl J Maxillofac Surg 2012;3:15-20.  Back to cited text no. 17
[PUBMED]  [Full text]  
18.
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. 18
    
19.
Raj Y, Sekhar MS, Shylaja S, Bhavani SN, Ramanand OV, Patha S, et al. Evaluation of the nature of collagen fibers in KCOT, dentigerous cyst and ameloblastoma using picrosirius red stain – A comparative study. J Clin Diagn Res 2015;9:ZC01.  Back to cited text no. 19
    
20.
Hirshberg A, Lib M, Kozlovsky A, Kaplan I. The influence of inflammation on the polarization colours of collagen fibres in the wall of odontogenic keratocyst. Oral Oncol 2007;43:278-82.  Back to cited text no. 20
    


    Figures

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