The study material included a total of 20 histopathologically diagnosed cases of DC, KCOT, UA and SMA. Five formalin-fixed, paraffin-embedded tissue blocks of each of DC, KCOT, UA and SMA were retrieved from the archives of Department of Oral and Maxillofacial Pathology, SVS Institute of Dental Sciences, Mahabubnagar, Telangana.

Sections of 4-μm thickness were taken and floated on to the slides treated with Mayer’s egg albumen (slide adhesive) and incubated at 47°C on a slide warmer for ensuring adhesion of the sections to the slides. Following deparaffinization, and later hydration in distilled water; the sections were stained with Harris’s haematoxylin, differentiated in 1% HCl. The sections were then subjected to alkalization with tap water, followed by incubation in 0.1% (w/v) Sirius red F3B (Sigma-Aldrich) in saturated Picric acid solution for 1 hour at room temperature. The sections were rinsed in acidified water {5 ml acetic acid (glacial) to 1 liter of water (tap or distilled)}, followed by dehydration and then mounted with D.P.X. The sections were then examined under bright field and polarizing microscope. On examination under polarizing microscope, the collagen fibers exhibited birefringence. We stained another section with routine haematoxylin and eosin, to facilitate the comparision of similar sections with those stained with picrosirius red stain.

The analysis was performed under 10X magnification independently by two observers and the average value of their observations was taken. Areas of polarization colours were observed in five fields and predominant colour was noted for each case. The findings of the study were subjected to chi-square test for statistical analysis.

It was observed that 80% of KCOT, 80% of UA and 60% of SMA cases showed a predominant greenish-yellow birefringence whereas a yellow-red birefringence was observed in 80% of dentigerous cyst cases [Table/Fig-1,2,3,4 and 5]. [Table/Fig-6] shows staining *lesion cross-tabulation which depicts the percent of staining within the lesion and also overall staining. Of 20 cases studied, it was seen that most of the cases (60%; 12/20) showed a greenish-yellow birefringence (positive staining) and remaining 40% (8/20) of the cases showed a yellowish-red birefringence (negative staining) [Table/Fig-6].

[Table/Fig-1]:

KCOT (H&E) (40X); KCOT showing predominant greenish-yellow collagen fibers (arrow) (40X)

[Table/Fig-2]:

Unicystic ameloblastoma (H&E) (40X); UA showing predominant greenish-yellow collagen fibers (arrow) (40X)

[Table/Fig-3]:

SMA (H&E) (40X); SMA showing predominant greenish-yellow collagen fibers (arrow) (40X)

[Table/Fig-4]:

Dentigerous cyst (H&E) (40X); DC showing predominant yellowish-red collagen fibers (arrow) (40X)

[Table/Fig-5]:

Positive represents greenish-yellow birefringence and negative represents predominant yellowish-red birefringence

The greenish yellow polarization colour which denotes young and immature collagen increased with increasing aggressive nature of the lesions (KCOT, UA and SMA). However, the results were not statistically significant (p-value obtained = 0.172; p-value < 0.05 was considered significant) [Table/Fig-7].

During odontogenesis, morphogenesis and cell differentiation are controlled by epithelial–mesenchymal interactions [15,16]. Similarly, development of odontogenic cysts and tumours is also dependent on such interactions [17,18]. Majority of the studies have focused on the evaluation of the proliferative activity in the epithelium of the odontogenic lesions [19–21], with fewer studies focusing on the role of epithelial-mesenchymal interactions in the progression of these odontogenic entities [4,5,7–14].

Tumour stroma has a very important role to play in the progression of a neoplasm. The tumour stroma acts as a physical barrier to ward off the host immunological reactions. It also plays a vital role in supplying the essential nutrients to the tumour via blood and also serves to remove waste products [22]. Nevertheless, the amount of stroma differs from one tumour to another; some tumours have abundant stroma while others have minimal stroma.

Collagen is the major constituent of the extracellular matrix and there is an alteration in the nature and structure of collagen in various pathological conditions. Various studies done using polarizing microscopy confirm the above statement and suggest that these differences may influence their biologic behaviour. These studies observed that young and immature fibers imparted greenish-yellow birefringence and collagen fibers which were mature and densely packed showed a yellowish-red birefringence [4,7,9–13].

In the present study, collagen which forms the chief constituent of the connective tissue was evaluated, to note if there was any difference in the nature and character of collagen fibers in DC, KCOT, and SMA and to know if there was any correlation between the difference in the nature of collagen and the clinical behaviour of these lesions. It was found that the collagen in KCOT, UA, and SMA imparted a predominant greenish-yellow birefringence and collagen present in the stroma of DC imparted a predominant yellowish-red birefringence [Table/Fig-1,2,3 and 4].

In our study, 4 out 5 cases (80%) of DC showed a predominant yellowish-red birefringence which was in accordance with HP Singh et al., [4]. This yellowish-red birefringence of collagen fibers seen in DC could possibly attribute to the mature, densely packed collagen. As the collagen fibers mature, there is change in proteoglycan content of fibers causing dehydration of fibers resulting in increase in diameter of collagen fibers and intensity of birefringence. Hence, the change in polarizing colour of the fibers from greenish-yellow to yellowish-red is seen [8].

Further, a predominant greenish-yellow birefringence was observed in most of the cases of KCOT (80%), UA (80%) which was in agreement with HP Singh et al., VN Nayak et al., and JY Zhang et al., [4,9,10]. The greenish-yellow birefringence seen in KCOT, UA and SMA can be attributed to the young and immature collagen fibers. HP Singh et al., evaluated different types of collagen fibers in KCOT using picrosirius red stain under polarizing microscopy and correlated the results with different radiographic patterns of KCOT to elucidate its biological behaviour [11]. They found significantly more greenish yellow collagen fibers in multilocular KCOT as compared to its unilocular variant, indicating a more aggressive behaviour of the former lesion.

Kajikar MS et al., evaluated the role of inflammation in the connective tissue wall of infected and non-infected odontogenic keratocyst [12]. They observed that cases of OKC with no or mild inflammation had majority of poorly packed fibers with green and yellow birefringence and moderate to severely inflamed cases had well-packed and thick fibers showing orange to red birefringence. Thus, indicating that inflammation influences polarization colour and packing of collagen fibers in the connective tissue wall of infected and non-infected OKC. However, in our study, we did not take into account the role of inflammation in cysts and tumours. Hirshberg suggested that the greenish-yellow collagen fibers seen in these lesions represent procollagen, intermediate or pathological collagen [13]. This assumption can also justify the aggressive behaviour of these lesions.

Not many studies have evaluated the nature of collagen fibers in SMA. It was found in our study that 60% of the cases of SMA showed greenish-yellow birefringence whereas 40% showed a predominant yellowish-red birefringence, which were in contrast to study done by P Aggarwal et al., where a predominant orangish-red birefringence of the collagen fibers around the tumour islands was observed. It was suggested by them that due to the long-standing nature of the lesion more matured and densely packed collagen fibers were seen in SMA [14].

Eighty percent cases of KCOT showed predominant greenish-yellow birefringence as compared to 60% cases of SMA. This was not in correlation with the biological behaviour of these lesions as SMA is found to be more aggressive than KCOT [23]. However, this disparity could be due to the small sample size of our pilot study.

In the present study a predominant yellowish-red birefringence was observed in majority of cases of DC whereas a primary greenish-yellow birefringence was observed in most of the cases of KCOT, UA and SMA. However, the results were not statistically significant. Therefore, we opine that the quality of collagen fibers can have an impact on the biological behaviour of the lesion; thus, substantiating that connective tissue plays a vital role in the progression of odontogenic cysts and tumours. Owing to the small sample size, the results were not statistically significant. Hence, a study with a larger sample size will be favorable to corroborate our view.