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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 52  |  Issue : 3  |  Page : 408-412
 

Stromal characterization and comparison of odontogenic cysts and odontogenic tumors using picrosirius red stain and polarizing microscopy: A retrospective and histochemical study


1 Department of Oral Pathology and Microbiology, Seema Dental College and Hospital, Rishikesh, Uttarakhand State, India
2 Department of Oral Pathology and Microbiology, KLEVK's Institute of Dental Sciences, KLE University, Belgaum, Karnataka, India
3 DKLEVK's Institute of Dental Sciences, KLE University, Belgaum, Karnataka, India

Date of Web Publication18-Feb-2016

Correspondence Address:
P B Jahagirdar
Department of Oral Pathology and Microbiology, Seema Dental College and Hospital, Rishikesh, Uttarakhand State
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.176742

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 » Abstract 

Introduction: Odontogenic lesions represent a range of conditions, the features of which probably depend on the stage of induction towards tooth formation reached prior to neoplastic or hamartomatous proliferation. It has been also suggested that inductive changes may allow progression from one type of odontogenic tumor to another. The epithelium also 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. So, this study was performed to investigate, compare, and correlate different types of collagen fibers in odontogenic cysts and odontogenic tumors. Materials And Methods: Thirty each pre-diagnosed odontogenic cysts and tumors were histochemically analyzed using a special stain (Picrosirius red stain) and polarizing microscopy. Results: Seven cases (99%) of inflammatory cysts exhibited predominantly greenish-yellow birefringence indicating procollagen, intermediate, or pathologic collagen fibers suggestive of loosely packed collagen fibers. Predominant yellowish-orange birefringence exhibited by 21 cases (99%) of developmental cysts was comparable to the yellowish-orange and orangish-red to red birefringence exhibited by odontogenic tumors suggesting tightly packed fibers. Conclusions: The Picrosirius red stain in conjunction with polarizing microscopy serves as a specific and sensitive tool in characterizing collagen fibers in odontogenic cysts and odontogenic tumor.


Keywords: Birefringence, odontogenic cysts, odontogenic tumors, picrosirius red stain, polarizing microscopy, procollagen


How to cite this article:
Jahagirdar P B, Kale A D, Hallikerimath S. Stromal characterization and comparison of odontogenic cysts and odontogenic tumors using picrosirius red stain and polarizing microscopy: A retrospective and histochemical study. Indian J Cancer 2015;52:408-12

How to cite this URL:
Jahagirdar P B, Kale A D, Hallikerimath S. Stromal characterization and comparison of odontogenic cysts and odontogenic tumors using picrosirius red stain and polarizing microscopy: A retrospective and histochemical study. Indian J Cancer [serial online] 2015 [cited 2019 Aug 19];52:408-12. Available from: http://www.indianjcancer.com/text.asp?2015/52/3/408/176742



 » Introduction Top


No organ in the body is immune to disease. The oral cavity bears no exception to this.[1] Many attempts to understand pathogenesis and biological behavior of various processes have been made which has helped to render best health care measures to mankind. The lesions affecting the jaws and oral cavity could either be developmental, inflammatory, cystic, or neoplastic in origin.

Odontogenic lesions derived from the odontogenic tissues constitute an unusually diverse group of oral lesions.[2] Despite, the general prevalence of these odontogenic lesions, their multiformality reflects a complexity in understanding its pathogenesis and biological behavior. Distinct tissue interactions have been known to occur both during odontogenesis (physiological) and disease process (pathological). Tissues that contribute to odontogenesis undergo stages of differentiation with the period of time. Thus, 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.[3]

The complexity of the induction effects within the odontogenic apparatus and the involvement of both ectodermal and mesodermal tissues are probably responsible for bewildering variety of forms, which the odontogenic lesions exhibit and because of this presentation, odontogenic lesions are more commonly studied, discussed, and debated on.[3]

A sequence of inductive interactions between the epithelium of the enamel organ and the mesenchymal connective tissue of dental sac, which is embryologically derived from the neural crest, occurs during odontogenesis. Pindborg et al. indicated that odontogenic lesions represent a range of conditions, the features of which probably depend on the stage of induction towards tooth formation reached prior to neoplastic or hamartomatous proliferation. Furthermore, it has been suggested that inductive changes may allow progression from one type of odontogenic tumor to another.[4]

The stroma of the odontogenic lesions consists of fibrous collagenous matrix with proliferating fibroblasts, exhibiting predominantly types I, III, and type IV collagen fibers along with oxytalan, elastin, and reticulin fibers. Inflammatory component is seen, if secondarily infected. The lesions also show good vascularization. Along with these changes evidence of enameloid, dentinoid, metaplastic changes, degenerative changes (fibro-myxoid, hyalinization), dystrophic calcifications are seen in many lesions such as odontogenic keratocyst, calcifying epithelial odontogenic cysts, adenomatoid odontogenic tumor, ameloblastoma, and ameloblastic fibroma as a result of epithelial-mesenchymal interactions.[5]

It can be conjectured from various studies that odontogenic lesions show a varying degree of inductive changes in the connective tissue and that stroma plays an important role along with the epithelium in exhibiting their biological behavior from the origin to their progression.[6]

The epithelium also 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. The evidence of characteristic extracellular matrix proteins, presence of enameloid, dentinoid, granulo-filamentous material, eosinophilic substances (ameloblastomas, ameloblastic fibro-odontomas), cementum and calcifications (calcifying epithelial odontogenic tumor), and the presence of dystrophic bone (ameloblastomas, odontogenic keratocysts) support and prove the epithelial-mesenchymal interactions. It is also believed that extracellular matrix proteins are integral components of the stromal connective tissue, which plays an important role in histomorphological features and biological behavior of the lesions.[6]

To understand the biological behavior of these lesions, studies on the stromal changes have been employed by various techniques ranging from histochemistry, immunohistochemistry, electron microscopy, and polymerase chain reaction.[7] Various special stains have been employed to study stroma selectively and these include Masson's trichome, Van Geison's and Picrosirius red stains. Picrosirius red stain, an anionic dye that selectively reacts and stains collagen fibers is been of specific importance in differentiating different types of collagen fibers in various oral conditions including odontogenic cysts and odontogenic tumors.[8] Birefringence is a phenomena exhibited by anisotropic objects which can be selectively visualized by polarized light microscopy. Quartz and collagen exhibit positive birefringence.[9]

Examination of collagen fibers stained with Picrosirius red stain in conjunction with polarizing microscopy serves as a selective method to differentiate procollagen, intermediate, and other forms of collagen fibers.[10],[11]

This study was aimed to evaluate the packing nature of collagen fibers in the odontogenic cysts and odontogenic tumors with an attempt to understand sensitivity and specificity of the stain by determining the polarization colors exhibited by the collagen fibers with picrosirius red stain under a polarizing microscope and also to understand biologic behavior of these aggressive lesions by means of comparing the birefringence exhibited between the cysts and tumors, thus, predicting treatment plan and prognosis.


 » Material and Methods Top


The retrospective study comprised of 30 previously diagnosed cases, each of odontogenic cysts and odontogenic tumors respectively, which were retrieved from the archives of Department of Oral Pathology and Microbiology, K.L.E.S's Institute of Dental Sciences, Belgaum and Government Dental College, Nagpur.

Two formalin-fixed, paraffin-embedded tissue sections of 4 μm thickness were obtained. Relevant clinical information about the patients was obtained from archives. One section of each odontogenic cysts and odontogenic tumors was stained with routine Hematoxylin and Eosin stain and other section was stained using Picrosirius red stain.

Picrosirius red staining method:

  • Picrosirius red stain (Sigma-Aldrich Company Ltd)
  • Sirius red F3B (C.I. 35782): 0.5 g
  • Saturated aqueous solution of picric acid: 500 ml
  • Add a little solid picric acid to ensure saturation.


Procedure

The method is most frequently used on paraffin sections (fixed at least 24 hours in neutral buffered formalin).

  • Dewaxed sections were treated with Weigert's hematoxylin A and B solutions (10-15 minutes) for achieving nuclear staining, attained by mixing both the solutions simultaneously just before staining and then the slides were washed for 10 min in running tap water
  • Sections were later stained with Picrosirius red for 1hour (this gives near-equilibrium staining, which does not increase with increasing time. Shorter times should not be used even if the colors look appreciable)
  • Dehydration by two changes of 100% absolute alcohol and simultaneous mounting in a resinous medium was done.[11]


The tissue sections were observed under 4× and 10× magnifications using Leica BX 200 Trinocular Research Microscope (Leica microsystems Gmbh, Germany) by three observers and interpretation of the sections was done. The interpretation of the sections was done based on the birefringence exhibited by collagen fibers as observed by Sharf et al.[12] and Junqueira et al.[13] who suggested that polarization colors are dependent on the physical aggregation of the collagen fibers and are also based on the packing nature of the collagen fibers respectively. The interpretation of the color profiles exhibited by the collagen fibers ranged from green to greenish-yellow and orange to orangish-red.


 » Results Top


In the present study, 30 each case of odontogenic cysts and odontogenic tumors respectively were studied and the type and nature of the odontogenic lesions, site, sex predilections were recorded as tabulated in [Table 1].
Table 1: The total number of cases, sex predilection and location of odontogenic lesions

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The picrosirius red stained sections were reviewed independently by three senior oral pathologists under polarizing microscope. The color intensities were observed ranging from green to greenish-yellow and orange to orangish-red to red.

Data were read independently by three observers to eliminate inter-observer bias and were found to be in consensus [Table 2] and [Table 3]. The statistical analysis was done using the Chi-square test and probability P ≤ 0.05 was considered to be significant.
Table 2: Comparison of the predominant polarization colors exhibited by collagen fibers in odontogenic cysts and odontogenic tumor

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Table 3: Comparison of the predominant polarization colors exhibited by collagen fibers in developmental cysts and inflammatory cysts

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 » Discussion Top


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.[3]

The complexity of the induction effects within the odontogenic apparatus and the involvement of both ectodermal and mesodermal tissues are probably responsible for bewildering variety of forms in which the odontogenic lesions exhibit.[6]

Studies by Hirshberg et al.[6] have focused on the importance of the epithelial-mesenchymal interactions in odontogenic lesions and that stroma plays an important role in the inductive changes.

Collagen, the most abundant protein in the body, is responsible for 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. To understand the biological behavior of these lesions, studies on the stromal changes have been employed by various methods, which include histopathology, immunohistochemistry, electron microscopy, etc., The most common and routinely used method for studying the stromal characterization is histopathology.[14]

Many histochemical stains have been used to demonstrate collagen fibres like Van Gieson, Masson's trichrome, Weigert's Resorcin Fushsin, modified Movat's stain, Goldner's Trichrome method, Wilder Modification of Bielschowsky's method, etc.[15],[16],[17]

The dye sirius red was first introduced in a microtechnique as a substitute for Acid Fuchsin in Van Geison's staining method. The enhanced birefringency produced by the stain under polarizing light helps in differentiating the different types of collagen fibers such as procollagen, intermediate collagen, mature collagen, and pathologic collagen fibers. It is observed that sirius red enhances the normal birefringency of collagen fibers in tissue sections.[14]

The use of polarizing microscopy in conjunction with picrosirius red stain is not only responsible for its specificity, but also for increasing considerably the method's sensitivity and resolution.[18] Thus, this study was carried out to evaluate the staining intensity of collagen fibers by picrosirius red stain in odontogenic cysts and odontogenic tumors.

It was observed in the present study that 22 (73.33%) developmental cysts which included odontogenic keratocysts, dentigerous cysts, and calcifying epithelial odontogenic cysts exhibited predominant yellowish-orange birefringence suggesting well-organized and thickly packed collagen fibers [Figure 1]a. The present findings were in conjunction with the studies by Sharf et al.[12] who in their studies on Nuclear Magnetic Resonance (NMR) in motional anisotropy of water in blood vessel walls suggested that color profile is related to the physical aggregation of the collagen and that yellowish-orange and orangish-red originates from well-packed fibers.
Figure 1: (a) Photomicrograph showing odontogenic developmental odontogenic cysts and predominant yellowish-orange birefringence exhibited by collagen fibers. (b) Photomicrograph showing radicular cyst and predominant greenish-yellow birefringence exhibited by collagen fibers

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In the present study, eight (26.66%) cases of odontogenic cysts were inflammatory cysts which included radicular cysts and seven (99%) cases exhibited predominantly greenish-yellow birefringence suggesting weakly organized or loosely packed collagen fibers [Figure 1]b. The present findings were in conjunction with the studies by Sharf et al.,[12] who suggested that green to greenish-yellow originates from loosely packed fibers. The findings in the present study were also comparable to the studies of L.C.U Junqueira et al.,[10] who suggested that collagen type III forms thin fibers composed of loosely packed thin fibrils and thus displays a weak birefringence of a greenish color.

The present study contrasted the studies done by Hirshberg et al.,[6] who studied collagen fibers in odontogenic keratocyts, dentigerous cysts, and radicular cysts in which they suggested that thin fibers with green to greenish-yellow birefringence and that thick fibers exhibited orangish-red birefringence which were suggestive of normal fibers. It was found that, odontogenic keratocysts in comparison with dentigerous cysts and radicular cysts exhibited predominant greenish-yellow birefringence of both thick and thin fibers, which were suggestive of procollagen, intermediate or pathologic collagen.

The inflammatory cysts in the present study exhibited predominant greenish-yellow birefringence. The probable explanation for such exhibition of the collagen fibers may be due to local destruction of the tissue caused due to the release of cytokines, lymphokines, and endotoxins by the inflammatory cells and also due to the collagenase activity.[19] These agents probably degrade collagenous matrix leading to repair and regeneration of new matrix. 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.[20],[21],[22] But in the developmental cysts because of their longstanding duration may cause degeneration of the stroma in the form of hyalinization and may probably explain such an exhibition by the collagen fibers. These regions also exhibited either a change in phenotype or thickness of overlying epithelium.[23]

The statistical analysis in the present study suggested that the results were significant with a 'P' value being 0.0005, in comparison to the polarization colors between the developmental and inflammatory cysts [Table 4].
Table 4: Comparison of the percentage of predominant yellowish-orange birefringence exhibited by collagen fibers in odontogenic cysts and odontogenic tumors and comparison of the percentage of predominant orangish-red color exhibited by collagen fibers in odontogenic cysts and odontogenic tumors

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In the present study, 25 (80%) cases of odontogenic tumors were of epithelial origin and 5 (16.66%) cases were of mesenchymal origin, respectively. The predominant color exhibited was orangish-red which was suggestive of highly organized and tightly packed mature collagen fibers. Seven cases were adenomatoid odontogenic tumors (AOT), which showed predominant yellowish-orange birefringence [Figure 2]a, [Figure 2]b and [Figure 2]c. The findings were in conjunction with the studies done by Nyska et al.[24] who suggested that human desmoplastic ameloblastomas also exhibited predominant yellowish-orange to orangish-red suggesting tightly packed fibers. The findings in the present study can also be compared to the findings of L.C.U Junqueira et al.[13] that type I collagen fibers forms thick fibers, composed of closely packed thick fibrils and consequently present an intense birefringence with yellow to red.[18]
Figure 2: (a,b and c) Photomicrographs showing odontogenic tumors and predominant orangish-red to red birefringence exhibited by collagen fibers

Click here to view


The observations in the present study with respect to color profiles of the collagen fibers in both odontogenic cysts and odontogenic tumors possibly explain the biological behavior of these lesions [Figure 3]. The predominant yellowish-orange birefringence in the developmental cysts in comparison to the inflammatory cysts suggests that developmental cysts exhibit well-organized and tightly packed collagen fibers. This may possibly explain the reason for the poorer prognosis of the developmental cysts. It can also be suggested that the color predominance exhibited by developmental cysts were comparable to stromal reactions of that of the odontogenic tumors and thus this may possibly explain the clinical and histological aggressive nature of these cysts. Seven cases of AOT's in the present study predominantly exhibited yellowish-orange birefringence in comparison to predominant orangish-red to red birefringence of other tumors probably suggesting less mature collagen fibers than the fibers in the other tumors. This is the probable reason why adenomatoid odontogenic tumors are less aggressive than other tumors in their biological behavior.
Figure 3: Graph showing comparison of the predominant polarization colors exhibited by collagen fibers in odontogenic cysts and odontogenic tumors

Click here to view



 » Conclusion Top


The study elicits that picrosirius red stain in conjunction with polarizing microscopy serves as a specific and sensitive tool in characterizing collagen fibers in various odontogenic lesions.

  • It can be used as an adjunct for routine staining procedures, where the advanced diagnostic methods are not available
  • Extensive studies with large samples of such lesions would shed light on their biologic behavior by means of stromal characterization
  • Furthermore, markers for epithelium and mesenchyme can be used to know the exact nature of epithelial and mesenchymal interactions.


 
 » References Top

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2.
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Becker J, Reichart PA, Schuppan D, Philipsen HP. Ectomesenchyme of ameloblastic fibroma reveals a characteristic distribution of extracellular matrix protein. J Oral Pathol Med 1992;21:156-9.  Back to cited text no. 4
    
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Sharf Y, Knubovets T, Dayan D, Hirshberg A, Akselrod S, Navon G. The source of NMR detected motional anisotropy of water in blood vessel water. Biophys J 1997;73:1198-204.  Back to cited text no. 12
    
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Constantine VS, Mowry RW. Selective staining of human dermal collage II. The use of picrosirius red F3BA with polarization microscopy. J Invest Dermatol 1968;50:419-23.  Back to cited text no. 14
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21.
Teronen O, Salo T, Konttinen YT, Rifkin B, Vernillo A, Ramamurthy NS, et al. Identification and characterization of gelatinases/type IV collagenases in jaw cysts. J Oral Pathol Med 1995;24:78-84.  Back to cited text no. 21
    
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23.
Nyska A, Dayan D. Ameloblastic fibroma in a young cat. J Oral Pathol Med 1995;24:233-6.  Back to cited text no. 23
    
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Vij R, Vij H, Rao NN. Evaluation of collagen in connective tissue walls of odontogenic cysts-A histochemical study. J Oral Pathol Med2011;40:257-62.  Back to cited text no. 24
    


    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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