|Year : 2014 | Volume
| Issue : 4 | Page : 243-248
Evaluation of mast cells and eosinophils in odontogenic cysts: A histochemical approach
Madhusudan Astekar1, Junaid Ahmed1, Rashmi Metgud1, Khushboo Phull1, GV Sowmya2
1 Department of Oral and Maxillofacial Pathology, Pacific Dental College and Hospital, PAHER University, Debari, Udaipur, Rajasthan, India
2 Senior Lecturer, Department of Oral Medicine and Radiology, Institute of Dental Sciences, Bareilly, Uttar Pradesh, India
|Date of Web Publication||10-Dec-2014|
Ph.D. Scholar, Department of Oral and Maxillofacial Pathology, PAHER University, Pacific Dental College and Hospital, Debari, Udaipur - 313 024, Rajasthan
Source of Support: None, Conflict of Interest: None
Background: Odontogenic cysts and tumors are most common benign destructive lesions in the oral cavity. These cystic lesions with its central fluid, reservoir of non-physiological composition, is in itself likely to provoke an inflammatory response in the surrounding host tissues, which may result in its formation and enlargement.
Aims and Objectives: 1. To evaluate the number of mast cells and eosinophils in radicular cyst, keratocystic odontogenic tumor and dentigerous cyst and to intercompare it with the degree of inflammation. 2. To evaluate and intercompare the staining quality of toluidine blue and thionin for mast cells identification and Carbol Chromotrope and Congo red for eosinophils evaluation.
Study Design: The study comprised of total 47 cases of which 20 were of radicular cyst, 15 were keratocystic odontogenic tumor and 12 were dentigerous cyst. Sections of 4μm thick were cut and stained with gold standard hematoxylin and eosin staining procedure for routine diagnosis. The amount of inflammation in each cyst was graded. They were further stained with toluidine blue and thionin for mast cells identification and Carbol Chromotrope and Congo red for eosinophils identification. The statistical analysis used was one way ANOVA (Analysis of Variance) and Pearson's Correlation Coefficient.
Results: The mean number of cell count per high power field of mast cells was higher than the eosinophils. On correlating with degree of inflammation the results came out to be statistically significant. The staining quality for mast cells was equally good with toluidine blue and thionin, whereas Carbol Chromotrope had a significantly better staining quality than Congo red in case of eosinophils.
Conclusion: The higher number of mast cells detected in odontogenic cysts could contribute in the release of inflammatory mediators involved in the pathogenesis of odontogenic cysts formation and enlargement. The histochemistry using various stains has proved to be promising in identification of mast cells and eosinophils.
Keywords: Basophils, coloring agents, dentigerous cyst, eosinophils, keratocystic odontogenic tumor, mast cells, odontogenic cyst, radicular cyst, special stains, toluidine blue
|How to cite this article:|
Astekar M, Ahmed J, Metgud R, Phull K, Sowmya G V. Evaluation of mast cells and eosinophils in odontogenic cysts: A histochemical approach. J NTR Univ Health Sci 2014;3:243-8
|How to cite this URL:|
Astekar M, Ahmed J, Metgud R, Phull K, Sowmya G V. Evaluation of mast cells and eosinophils in odontogenic cysts: A histochemical approach. J NTR Univ Health Sci [serial online] 2014 [cited 2020 Mar 28];3:243-8. Available from: http://www.jdrntruhs.org/text.asp?2014/3/4/243/146610
| Introduction|| |
Cysts of the jaws are probably the most common destructive bone lesions in the human maxillofacial skeleton.  Odontogenic cysts are derived from the epithelium which is associated with the dental apparatus. They are either of developmental origin or may result from inflammation.  The most common odontogenic cysts are radicular cysts followed by keratocystic odontogenic tumor and dentigerous cysts.  Despite this, our knowledge of their pathogenesis is at best rudimentary. A cystic lesion with its central fluid, reservoir of non-physiological composition, is in itself likely to provoke an inflammatory response in the surrounding host tissues. Thus, at the time of microscopic examination of a cyst, some degree of inflammatory change is present in its wall. 
Cystic expansion is also influenced by a number of factors like mural growth, hydrostatic enlargement and bone resorbing factor. The hydrostatic pressure of the luminal fluid is important in cyst enlargement and mast cell activity might contribute to this by increasing the osmotic pressure of the fluid.  From the time of their discovery by Paul Ehrlich, tissue mast cells have been of interest because of their characteristic granules. These are rich in Histamine, Heparin, Zinc, Cationic protein and various active enzymes.  On degranulation of the mast cell, dissociation of heparin proteoglycan may provide a mechanism for activation of the granule associated proteases.  A variety of hydrolytic enzymes are released on degranulation of mast cells and these enzymes would be expected to degrade components of the connective tissue capsule of odontogenic cysts. , Because of poor lymphatic drainage in the cyst wall, these released components would then largely diffuse into the luminal fluid where they would be expected to contribute to the osmotic pressure therein. ,
Mast cells have also been suggested to promote collagenolytic activity, which in view of the reported collagenase activity in odontogenic cysts, may be pertinent to their pathogenesis. Mast cells liberate Eosinophils chemo-attractant factor and histamine, which attract eosinophils in tissue. Both cells can stimulate the production of prostaglandins, important in bone resorption which may aid in cyst growth. 
Mast cells are present in mucosal and connective tissue environments. They possess cytoplasmic granules that stain metachromatically under ordinary conditions. , Metachromasia may be defined as the staining of tissue or tissue components such that the color of the tissue bound dye complex differs significantly from the color of the original dye complex to give a marked contrast color. Under Metachromasia Toluidine Blue and Thionin Stain the tissue components purple-red, while Carbol Chromotrope and Congo red give red color to the Eosinophilic granules and stain the nuclei blue. These special stains are wonderful; they allow us to see which we cannot through routine hematoxylin and eosin staining and thus in turn help to identify the cells specifically. 
Hence the present study was carried out to assess the amount of mast cells and eosinophils in odontogenic cystic lining and to inter-compare the staining quality of special stains. Later its correlation with the degree of inflammation was also evaluated.
| Materials and Methods|| |
The study comprised of total 47 cases of which 20 were of radicular cyst, 15 keratocystic odontogenic tumors and 12 dentigerous cysts. Formalin fixed paraffin embedded tissue blocks of these odontogenic cysts were retrieved from the archives of the department of oral and maxillofacial pathology. All the cases were diagnosed on the basis of clinical and histopathological correlation. The serial sections of 4-5 μm thick were used for staining with hematoxylin and eosin and special stains.
Special stains like toluidine blue and thionin were used for mast cells identification and Carbol Chromotrope and Congo red were used for eosinophils identification. Under the preparation section, (a) for mast cell staining  0.5% toluidine blue solution was prepared and sections stained for 1 hr,  and 1.3% aqueous thionin solution was prepared and sections stained for 30 min. (b) For eosinophils staining  0.5% Carbol Chromotrope solution was prepared, sections were first stained with Delafield's Hematoxylin for 3 min and then counterstained with Carbol Chromotrope for one hour  and 1% Congo red solution was prepared, sections stained for 30 min and counterstained with hematoxylin. All the sections were then washed in tap water and dehydrated through ascending grades of alcohol, cleared and mounted in DPX. Under low power view high density areas were selected in tissue section and cells counted in randomly chosen ten high power fields.
The degree of inflammation present in the cyst wall was scored as per the criteria given by Priyanka D and Smith G , as no inflammation (zero), mild (+), moderate (++) and severe inflammation (+++).
The mean and Standard Deviation (SD) values for each cyst group were determined using one way ANOVA (Analysis of Variance). The correlation of mast cells and eosinophils was then carried out with degree of inflammation using Pearson's correlation coefficient. All the statistical analysis was carried out using Statistical Package for Social Sciences (SPSS) software, version 14.
| Results|| |
On evaluation, the mean and standard deviation values of mast cells using toluidine blue and thionin stain and eosinophils using Congo red and Carbol Chromotrope stain were 4.38 ± 0.60, 4.17 ± 0.62, 1.26 ± 0.11, 1.61 ± 0.15 in radicular cyst, 3.57 ± 0.19, 3.49 ± 0.22, 0.98 ± 0.17, 1.29 ± 0.11 in keratocystic odontogenic tumor and 3.16 ± 0.18, 3.04 ± 0.11, 0.68 ± 0.08, 1.22 ± 0.08 in dentigerous cyst respectively [Table 1].
|Table 1: The Mean and SD Value of Mast Cells and Eosinophils In 3 Cysts Groups|
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Assessment of inflammation based on mild (+), moderate (++) and severe (+++) grading was noted in 5, 10 and 5 cases among radicular cyst, in 7, 6 and 2 cases of keratocystic odontogenic tumor, and in 6, 5 and 1 case of dentigerous cyst respectively [Graph 1].
On intercomparison of staining quality for mast cell between toluidine blue and thionin, a statistically non-significant result was obtained (P > 0.05). Whereas on comparing the staining quality for eosinophils identification between Carbol Chromotrope with Congo red, the results showed a statistical significant difference (P < 0.05) [Table 2]. This clearly indicates that the staining intensity of Carbol Chromotrope was superior to Congo red in differentiating eosinophils [Figure 1].
|Figure 1: The toluidine blue and thionin staining for mast cells at 40× (a and c) and 100× magnifi cation (b and d) respectively. Similarly Carbol Chromotrope and Congo red staining for eosinophils at 40× (e and g) and 100×magnification (f and h) respectively|
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|Table 2: The Correlation of Stains Used In Identification of Mast Cells and Eosinophils In Different Odontogenic Cysts|
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The mean and standard deviation values for both mast cells in toluidine blue and thionin and wofor eosinophils in Carbol Chromotrope and Congo red were 3.49 ± 0.16, 3.41 ± 0.40, 1.39 ± 0.15, 0.97 ± 0.21 in mild inflammation, 4.0 ± 0.75, 3.89 ± 0.69, 1.46 ± 0.23, 1.10 ± 0.27 in moderate inflammation and 4.2 ± 0.65, 4.04 ± 0.63, 1.6 ± 0.22, 1.18 ± 0.13 in sever inflammation respectively. Upon intercomparison the results revealed a significant positive correlation with the degree of inflammation (P < 0.05) [Table 3].
|Table 3: The Correlation of Mast Cells and Eosinophils With Inflammation Grading|
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| Discussion|| |
Odontogenic cysts are characterized by an expansile growth resulting in smooth, usually unilocular cavity containing fluid or semi-fluid material lined by an epithelium and supported by fibrous connective tissue capsule.  These cysts are derived from the epithelium associated with the development of the dental apparatus which are either of developmental origin or may result from inflammation. However, the cysts of developmental origin may show inflammatory changes secondary to infection. 
Mast cells contribute to a broad spectrum of physiologic, immunologic and pathologic processes of inflammation.  Activated mast cell synthesizes vasoactive and chemotactic mediators namely platelet derived growth factor, pro-inflammatory cytokines like Interleukins 3, 6 and TNF-α which increases the vascular permeability. On degranulation, mast cells release heparin and hydrolytic enzymes which facilitate the breakdown of glycosaminoglycan's and proteoglycan's.  The Hyaluronic acid content degrade the capsular extra-cellular matrix components thereby facilitating their passage into the fluid; raising the osmolality and thereby increasing the internal hydrostatic pressure resulting in cyst enlargement. , Mast cells also liberate eosinophils chemo-attractant factor and histamine, which attract eosinophils into the tissue. Both mast cells and eosinophils have been implicated in stimulating the production of prostaglandins, important in bone resorption. ,
In the present study special stains like toluidine blue and thionin were used for mast cells and Carbol Chromotrope and Congo red were used for eosinophils identification and quantification in radicular cyst, keratocystic odontogenic tumor and dentigerous cyst. Assessment of inflammation in the cyst wall was also quantified and intercompared.
In the present study, maximum infiltration of mast cells was distinguished in radicular cyst in comparison with other two odontogenic cysts. According to Goldsmith et al.  the presence of mast cells was in similar numbers in keratocystic odontogenic tumor, radicular cyst and dentigerous cyst, implying the similar content of heparin in the capsules from these cysts. Whereas, results of the study conducted by Chatterjee S  showed significant higher concentration in keratocystic odontogenic tumor than dentigerous and radicular cyst, suggesting increased breakdown of capsular matrix in keratocystic odontogenic tumor. Keratocystic odontogenic tumor epithelium has been shown to be non-keratinized at places, which causes a transport of breakdown products into the cystic lumen and consequently can determine an elevated osmolality of the cystic fluid, which partly explains the greater aggressiveness of keratocystic odontogenic tumor comparing to other odontogenic cyst. In 2010 a study accomplished by Debta P  showed that number of mast cells was more in radicular cyst in comparison keratocystic odontogenic tumor followed by dentigerous cyst. This may be associated with rather more inflammation in the connective tissue stroma of radicular cyst.
Numerous special stains have come up which clearly demarcate mast cells from other inflammatory cells like eosinophils and basophils.  Mast cells can be distinguished from other connective tissue cells by the presence of cytoplasmic granules and their larger size.  Toluidine blue special stain with its property of metachromasia is the most widely and routinely used stain for mast cells identification.  In the present study, the staining intensity of toluidine blue for mast cell was equally efficient when compared with thionin; which were in accordance with the study performed by Priyanka et al.  On comparing the economic value, the commercial cost of thionin comes out to be much cheaper as compared to toluidine blue stain. Hence thionin may be used as an alternate to toluidine blue for staining of mast cells in routine practice. In contrast to the present study Joseph et al.  compared two thiazide stains, namely toluidine blue and thionin and noted that toluidine blue staining showed more intact mast cells as compared to thionin. Schwartz et al.,  and Teronen et al.,  observed that toluidine blue, does not stain degranulated mast cells (phantom cells) and suggested that this could lead to variations in the number of mast cells observed in the different zones. 
Bischoff et al.,  Rodini et al.  and Santos Netto et al.  observed a reduction in the number of mast cells count in toluidine blue stained sections when compared with immunohistochemical staining using monoclonal antibodies against the human mast cell proteases, tryptase and chymase. The obvious reason for this discrepancy was that only few of the activated mast cells were detectable in toluidine blue stained sections whereas maximal degranulated and resting mast cells were stained immunohistochemically. Goldsmith et al.,  used a combination of avidin-biotin staining procedure with 0.5% toluidine blue counterstaining and observed very distinct black cells with a well-defined granular appearance on a negative background distinguishing the cells which only took up the toluidine blue counterstain. This was beneficial in detecting the more immature mast cells with their less dense staining.
In the present study, Carbol Chromotrope staining method to demonstrate tissue eosinophil revealed better results than Congo red staining, showing red colored eosinophil granules in pale background of the cells with distinct outline. This was in accordance with the study performed by Priyanka et al. 
In the present study, the infiltration of mast cells and eosinophils revealed significant positive correlation to degree of inflammation. Goldsmith et al.,  also showed a similar finding indicating maximum association rather more inflammation in radicular cyst than dentigerous and keratocystic odontogenic tumor. It was noted that generally, mast cells were not found within areas of dense inflammatory infiltrate but rather more adjacent to them. According to the study performed by Priyanka et al.,  mast cells and eosinophils appeared to be a feature common to both keratinizing and non-keratinizing cysts and their infiltration did not necessarily correlate with the degree of inflammation.
Mast cells and eosinophils stimulate the production of prostaglandins, Interleukin-1α, tryptase and other collagenases aiding in bone resorption, which is a feature in cyst enlargement. Hence, it is very likely that reduction of intracystic pressure is a key factor in controlling the enlargement of cyst. It may also be postulated that reducing the concentration of inflammatory mediators by irrigation of the cyst lumen could reduce the epithelial proliferation and reverse bone resorption, leading to shrinkage of the cyst cavity. ,
A significant raise in the number of mast cells and eosinophils may play a vital role in the pathogenesis of cyst formation and enlargement. The influence of mast cell antagonist on treatment of cystic lesions needs to be evaluated. Further studies with larger sample size are required to examine these possibilities.
| Acknowledgment|| |
We would like to thank all the faculty members in the department of oral and maxillofacial pathology for their general support.
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[Table 1], [Table 2], [Table 3]