Indian Journal of Cancer Medknow Publications on behalf of Indian Cancer Society ISSN: 0019-509X EISSN: 1998-4774 Vol. 48, Num. 2, 2011, pp. 211-215

Indian Journal of Cancer, Vol. 48, No. 2, April-June, 2011, pp. 211-215

Original Article

Stromal differences in odontogenic cysts of a common histopathogenesis but with different biological behavior: A study with picrosirius red and polarizing microscopy

P Aggarwal, S Saxena

Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut, India

Correspondence Address:P Aggarwal Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut India drpooja_path@yahoo.co.in

Code Number: cn11051

DOI: 10.4103/0019-509X.82897

Abstract

Keywords: Collagen fibers, odontogenic cysts, picrosirius red, polarizing microscopy

Introduction

Odontogenic cysts are usually classified into three main groups, namely, dental/radicular cysts, dentigerous cysts and odontogenic keratocysts. The proposed histogenetic differences and the clinically more aggressive behavior of odontogenic keratocysts compared with other cyst types has prompted studies aimed at characterizing possible differences between their fluid aspirates and epithelial linings. Little research has been done with respect to their connective tissue walls. ^[1]

Collagen plays a vital role in maintaining structural integrity and in determining tissue function. Therefore, methods to detect, quantify and analyze collagen are valuable. ^[2]

Picrosirius red stain, when used with polarized microscopy, not only characterizes collagenous material specifically and reliably, but also shows very clearly the orientation of collagen fibers. ^[2],[3]

The picrosirius-polarization methodology has occasionally been used in dental and gingival research to demonstrate pathological changes in collagen fibers, such as skin lesions, hyperplastic gingiva and odontogenic tumors. ^{[4],[5],[6],[7]} Very few studies have been done in odontogenic cysts using this stain.

This study was undertaken to detect and compare the pattern of collagen fibers in odontogenic cysts and also to find out if this methodology could be used to predict the aggressive nature of odontogenic cysts by comparing with the odontogenic tumors.

Materials and Methods

Thirty-nine cases of odontogenic cysts [11 odontogenic keratocysts (group 1), 14 dentigerous cysts (group 2), 14 radicular cysts (group 3)] and 10 cases of ameloblastoma (group 4) were retrieved from the files of the Oral Pathology and Microbiology Department. The diagnosis of the cysts and tumors was based on radiographic evidence, clinical description and histological features (H and E stained section).

Sections of 4 μm thickness were prepared, and after deparaffinization and hydration in distilled water, sections were incubated in 0.1% Sirius red F3B (C.I. 35780) in saturated picric acid solution for 1 hour at room temperature. This was followed by rinsing with distilled water, staining with Weigert′s hematoxylin, differentiation in 1% acid alcohol, alkalinization with tap water, dehydration and mounting with Dibutylphathalate xylene (DPX).

To evaluate the birefringence pattern of collagen fibers, all the sections were examined under 4× magnification using Olympus polarized microscope. In odontogenic cysts, predominant colors of collagen fibers in connective tissue in relation to the epithelium were evaluated. For odontogenic tumors, the predominant color around islands was considered.

Data were read independently by three observers to eliminate inter-observer bias and were found to be comparable. The results were then tabulated and subjected to statistical analysis using Z test for proportion.

Results

In the present study, the predominant color of collagen fibers in stroma of odontogenic keratocysts, dentigerous cysts and odontogenic tumors was found to be orangish red [Figure - 1], [Figure - 2], [Figure - 3], whereas in radicular cyst it was found to be green [Figure - 4]. Whereas calculated Z value for all colours between Group 1 and group 4 [Table - 1] and group 1 and group 2 [Table - 2] was found to be nonsignificant.

Using Z test for proportions, at 5% and 1% level of significance, the birefringence pattern of collagen fibers between different groups was compared. On comparing, the calculated Z values for greenish yellow color between group 2 and group 4 [Table - 3], for green and orangish red between group 3 and group 4 [Table - 4], for green and orangish red between group 1 and group 3 [Table - 5], for green, greenish yellow and orangish red between group 2 and group 3 [Table - 6] were found to be statically significant (P < 0.05).

Discussion

Epithelial-mesenchymal interactions are essential in maintaining homeostatic equilibrium in adult tissue, with the stromal cells maintaining control over cell size, function and response to wounds and other pathologic conditions through modification of the extracellular matrix (ECM). ^[8]

The ECM performs a very important role in growth regulation, tissue differentiation and organization. ^[9],[10]

Collagen is a major organic component that constitutes nearly 34% of the total ECM proteins. Type I and type III collagens are the most predominant types. ^{[11],[12],[13]}

It was observed that the collagens displayed different interference colors and intensities of birefringence, when studied by the picrosirius-polarization method, ranging from green to greenish yellow to yellow to orangish red, and red. ^[3],[14]

This can be explained by the widely known fact that these different interstitial collagens display distinct patterns of physical aggregation. ^[14] Collagen type I forms thick fibers composed of closely packed thick fibrils and consequently presents an intense birefringence with yellow to red color. Collagen type III (reticular fibers) forms thin fibers composed of loosely packed thin fibrils and thus displays a weak birefringence of a greenish color. ^{[5],[7],[8],[9]}

Recent studies have focused on the importance of the epithelial-mesenchymal interactions in odontogenic cysts and have demonstrated that the expansion involves degradation of bone matrix and cell attachment to the ECM components. ^{[15],[16],[17]}

Vedtofte et al, demonstrated that transplanted keratocyst epithelium in nude mice retained its typical histological appearance only when supported by its own stroma. Thus, it can be suggested that the biological behavior of odontogenic keratocyst is dependent not only on the epithelium but also on the underlying stroma. ^[18]

The capsule essentially is composed of collagen fibers consisting of both thin and thick fibers arranged in bundles. The nature of arrangement in odontogenic cysts is little understood.

Hirsberg et al, used picrosirius red to note the differences in collagen in odontogenic cysts. ^[19]

In the present study, the predominant color of collagen fibers in stroma of odontogenic keratocysts and dentigerous cysts was found to be orangish red. When comparison between these two groups was done, there was no significant difference statistically (P > 0.05). The predominant color in radicular cysts in 10 out of 14 cases (71.43%) was green and the other 4 cases showed greenish yellow birefringence.

In our study, green to greenish yellow color of collagen fibers in radicular cysts suggests that the collagen found in these lesions is loosely packed and might be composed of procollagens, intermediate or pathologic collagens rather than the normal tightly packed fibers seen in dentigerous cysts and odontogenic keratocysts. This could be explained by the fact that the radicular cyst is an inflammatory cyst and the inflammatory cells could affect the packing of collagen fibers. ^[8]

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. ^{[15],[16],[17]}

In the present study, the predominant color of collagen fibers around the tumor islands in odontogenic tumors was orangish red. When the comparison of color of collagen fibers of odontogenic tumors with that of odontogenic keratocysts and dentigerous cysts was done, there was no significant difference statistically (P > 0.05). When odontogenic tumors were compared to radicular cysts, there was a statistically significant difference between these two groups (P < 0.05).

The present study shows that connective tissue stroma of odontogenic keratocysts, dentigerous cysts and odontogenic tumors predominantly exhibited orangish red collagen fibers, which may suggest that they belong to the same group, i.e., are developmental in origin, and their longstanding nature might be the cause of closely packed collagen fibers.

Zegarelle concluded from his investigations of induced ameloblastoma in mice that odontogenic cysts are stages in the development of ameloblastoma. ^[20]

Spouge postulated a "Unitarian concept" that relates the histology and clinical behavior of odontogenic tumors to the embryonic properties and functions and to the degree of differentiation of their appropriate parent tissue. This theory brings ameloblastoma and odontogenic cysts under the same category, which may help to clarify their relationship. ^[21]

It can be concluded from our study that similarity between dentigerous cysts, odontogenic keratocysts and odontogenic tumors may indicate that these lesions have a common histogenesis with a broad spectrum of biological behavior.

Conclusions

Similar collagen characterization of dentigerous cysts, odontogenic keratocysts and odontogenic tumors provides further evidence regarding the comparable nature and biological behavior of these lesions. Hence, the treatment modality of these developmental cysts may be compared to that of odontogenic tumors.

On the other hand, the dissimilar pattern of collagen arrangement in radicular cysts suggests a different biological behavior, thus warranting a more conservative treatment and a positive role of inflammation on the polarization color characteristic of collagen fibers.

From the present study, we can suggest that the connective tissue stroma of odontogenic cysts may be regarded as a functional part of the cyst and not just a structural support.

References

Copyright 2011 - Indian Journal of Cancer

The following images related to this document are available:

Photo images