Shylaja S .Mast Cells In Odontogenic

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SHYLAJA S.MAST CELLS IN ODONTOGENIC CYSTS. Journal of Clinical and Diagnostic Research [serial online] 2010 April [cited: 2010 April 5]; 4:2226-2236.

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Journal of Clinical and Diagnostic Research. 2010 April ;(4):2226-2236 Shylaja S .Mast Cells In Odontogenic Cysts

ORIGINAL ARTICLE

Mast Cells in Odontogenic Cysts

SHYLAJA S

ABSTRACT

Background: are probably the most common destructive in the human maxillofacial skeleton. Odontogenic cysts are derived from the epithelium which is associated with the development of the dental apparatus and can be either developmental or inflammatory in origin. The most common odontogenic cysts are radicular cysts, dentigerous cysts and odontogenic . However, the cysts of developmental origin may show inflammatory changes secondary to . Mast cell degranulation plays an important role in the inflammatory response and it is speculated that alteration in their number and distribution could contribute to the pathogenesis of odontogenic cysts. So, an attempt was made to evaluate the significance and distribution of mast cells in radicular , odontogenic and using toluidine blue staining. Materials and Methods: This retrospective study was undertaken by retrieving the records and the paraffin blocks of 40 confirmed cases of odontogenic cysts, out of which 19 were Radicular cysts, 12 were odontogenic keratocysts and 9 were dentigerous cysts. Sections of 5µm thickness were prepared and stained with haematoxylin and eosin, as well as with toluidine blue. The toluidine blue stained mast cells were then counted under a high power microscopic field (40X) for each specimen in three different zones and the mean value obtained. The mean number of mast cells was then compared between different zones by using the Relative Deviate ‘Z’ test. Results: In cases of radicular cyst, the highest mean number of mast cells per high power field was seen in the age group of 10-19 years. A statistically significant difference (p<0.05) amongst the distribution of mast cells was noted between the sub-epithelial and the deep zones. In cases of , the highest mean number of mast cells per high power field was seen in the age group of 20-29 years. A statistically significant difference (p<0.01) amongst the distribution of mast cells was noted between the sub- epithelial and the deep zones, as well as between the sub-epithelial and the intermediate zones. In cases of dentigerous cyst, the highest mean number of mast cells per high power field was seen in the age group of 10-19 years. A statistically significant difference (p<0.05) amongst the distribution of mast cells was noted between the sub-epithelial and the deep zones.

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Conclusion: In the present study, the maximum number of mast cells were noted in the sub-epithelial zone as compared to other zones. The number of mast cells were seen to decrease with age and there was no gender predilection. However, further studies using immunohistochemical techniques may help in the better understanding of the pathogenesis of these cysts. Key Words : Mast cells, Odontogenic cysts, Radicular Cyst, Odontogenic Keratocyst, Dentigerous cyst, Toluidine blue.

______Department of Oral & Maxillofacial Mast cells have been the object of SVS Institute of Dental Sciences investigation for almost a century, but Mahabubnagar,Andhra-Pradesh (INDIA). Corresponding Author: still remains an enigma in terms of their Dr. Sanjeevareddigari Shylaja function in situ. They have often MIG-276, 4th road, KPHB Colony, puzzled investigators from the time that Kukatpally, Hyderabad-500072(INDIA) they were first identified and were Phone No. - 09441023999 named by Ehrlich in 1887 [4]. Email id: [email protected]

Introduction Mast cells are present in mucosal and connective tissue environments. They Cysts of the jaws are probably the most possess cytoplasmic granules that stain common destructive bone lesions in the meta-chromatically under ordinary human maxillofacial skeleton. The head conditions [5]. Degranulations of mast and neck region and the jaw in cells play an important role in the particular, collectively comprise one of initiation of the inflammatory response. the most common sites for the This action is significant in the occurrence of cysts [1]. pathogenesis of different lesions like Odontogenic cysts are derived from the , early periodontal epithelium which is associated with the diseases, ulcerative colitis, pulmonary development of the dental apparatus. fibrosis, inflammatory bowel, systemic They are either of developmental origin mastocytosis and odontogenic cysts [6]. or may result from . The most common odontogenic cysts are Mast cells are recognized in non- radicular cysts, odontogenic keratocysts keratinizing and keratinizing and dentigerous cysts [2]. odontogenic cysts. Alteration in their Cystic expansion is influenced by a number and distribution could number of factors like mural growth, contribute to the pathogenesis of hydrostatic enlargement and bone odontogenic cysts [3]. The special stains resorbing factor. The hydrostatic used for mast cell staining are toluidine pressure of the luminal fluid is blue, azur A, bismark brown, thionin important in cyst enlargement and mast and alcian blue [7]. cell activity might contribute to this by increasing the osmotic pressure of the Mast cells might play a role in the fluid [3]. pathogenesis of odontogenic cysts. So,

an attempt was made to evaluate the significance and distribution of mast

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cells in radicular cysts, odontogenic taken. The mean number of mast cells keratocysts and dentigerous cysts by were compared between the different using toluidine blue staining. zones by using the Relative Deviate ‘Z’ test. Aims and Objectives To study the presence and the number of Results and Observations mast cells in radicular cysts, Out of the total 40 cases, 30 were males odontogenic keratocysts and dentigerous and 10 were females. cysts. To evaluate the distribution of mast cells in the subepithelial, Age intermediate and deep zones. To In radicular cysts, the age ranged from 7 correlate the number of mast cells and to 48 years. The maximum number of their distribution with age, sex and the cases were in the age group of 10 to 19 different zones which were considered. years (8 cases). In odontogenic Materials and Methods keratocysts, the age ranged from 15 to This retrospective study was undertaken 58 years. The maximum number of by retrieving the records and the cases were in the age group of 20 to 29 paraffin blocks of confirmed cases of years (10 cases). In dentigerous cysts, odontogenic cysts. The paraffin blocks the age ranged from 11 to 45 years. The were sectioned and stained with maximum number of cases were in the haematoxylin and eosin and toluidine age group of 10 to 19 years (4 cases) blue. A total of 40 cases were included [Table / Fig 1]. in this study, out of which 19 were radicular cysts, 12 were odontogenic Sex keratocysts and 9 were dentigerous Out of 19 cases of radicular cysts, 12 cysts. (63.2%) were males and 7 (36.8%) were females, with a male to female ratio of Mast cell count was done in sections 1.7:1. Out of 12 cases of odontogenic which were stained with toluidine blue. keratocysts, 10 (83.3%) were males and Four areas were randomly selected 2 (16.7%) were females, with a male to under a high power field (40x) for each female ratio of 5:1. Out of 9 cases of specimen. The area encompassed by one dentigerous cysts, 8 (88.9%) were males high power field (HPF) was taken as one and 1 (11.1%) was a female, with a male microscopic field (MF). In each area, to female ratio of 8:1 [Table / Fig 1]. three zones were considered. The subepithelial zone was the area just (Table / Fig 1) Age and Sex-Wise below the epithelium and the next two Distribution of Mast Cells in Odontogenic consecutive microscopic fields were the Cysts intermediate and the deep zones.

Statistical Analysis The number of mast cells per microscopic field for individual cyst was calculated at the three zones and the means (±S.D) for each type of cyst were

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Mast Cell Distribution Sex Wise Distribution of Mast Age Wise Distribution of Mast Cells Cells In all the three cysts, statistically In radicular cysts, the mean number of significant differences was not noted in mast cells per HPF was high in the age the distribution of mast cells between group of 10 to 19 years [Table / Fig 2] males and females [Table/ Fig 3] (Table (Table 2). In odontogenic keratocysts, 5), (Table 6), (Table 7). the mean number of mast cells per HPF was high in the age group of 20 to 29 years [Table/Fig 2] (Table 3). In (Table/Fig 3) Table 5: Sex Wise dentigerous cysts, the mean number of Distribution of Mast Cells in Radicular mast cells per HPF was high in the age Cyst group of 10 to 19 years [Table / Fig 2] (Table 4).

(Table/Fig 2) Table2: Age Wise Distribution Of Mast Cells In Radicular (Table/Fig 3) Table 6: Sex Wise Cyst Distribution of Mast Cells in Odontogenic Keratocyst

(Table/Fig 3) Table 7: Sex Wise (Table/Fig 2) Table 3: Age Wise Distribution of Mast Cells in Dentigerous Distribution Of Mast Cells In Cysts Odontogenic Keratocyst

Zone Wise Distribution of Mast Cells In radicular cysts, the mean number of (Table/Fig 2) Table 4: Age Wise mast cells per HPF was high in the Distribution Of Mast Cells In Dentigerous Cysts subepithelial zone as compared to the intermediate and deep zones. A statistically significant difference (P<0.05) was noted between the subepithelial and the intermediate zones [Table/Fig 4](Table 8).

In odontogenic keratocysts, the mean number of mast cells per HPF was high in the subepithelial zone as compared to

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the intermediate and the deep zones. A Statistically significant differences was statistically significant difference not noted in the distribution of the mast (P<0.01) was noted between the cells between the cysts when the zones subepithelial and the intermediate zones are compared [Table/Fig 4] (Table and between the subepithelial and the 11),[Table/Fig 5] (Fig12), (Fig13), (Fig deep zones [Table/Fig 4](Table 9). 14), (Fig15), (Fig16), (Fig 17).

In dentigerous cysts, the mean number (Table/Fig 4) Table 11: Comparison of of mast cells per HPF was high in the Mast Cells between Odontogenic Cysts subepithelial zone as compared to the intermediate and the deep zones. A statistically significant difference (P<0.05) was noted between the OKC – Odontogenic Keratocyst; DC – subepithelial and the deep zones Dentigerous Cyst; [Table/Fig 4](Table10). RC – Radicular Cyst

(Table/Fig 4) Table 8: Zone Wise Distribution Of Mast Cells In Radicular Cysts

SEZ – Subepithelial Zone; IZ – Intermediate Zone; DZ – Deep Zone.

(Table/Fig 4) Table 9: Zone Wise Distribution of Mast Cells in Odontogenic Keratocysts (Table/Fig 5) Fig 12: Photomicrograph Of H & E Stained Section Of Radicular Cyst (10x).

(Table/Fig 4) Table 10: Zone Wise Distribution of Mast Cells in Dentigerous Cysts

(Table/Fig 5) Fig 13: Photomicrograph Comparison of Mast Cells Of Toluidine Blue Stained Section Of between Odontogenic Cysts Radicular Cyst Showing Mast Cells (10x).

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(Table/Fig 5) Fig 14: Photomicrograph (Table/Fig 5) Fig 17: Photomicrograph Of H & E Stained Section Of Of Toluidine Blue Stained Section Of Odontogenic Keratocyst (40x). Dentigerous Cyst Showing Mast Cells (10x).

Discussion Mast cells are the normal members of the cell population in fibrillar connective tissue. For three quarters of a century, mast cells were biological curiosities. Few people knew anything about them, their contents and functions, though occasionally, few investigators made (Table/Fig 5) Fig15: Photomicrograph some studies without any conclusive Of Toluidine Blue Stained Section Of results. In recent years, mast cells have Odontogenic Keratocyst Showing Mast received increasing attention and a Cells (40x). number of investigations and reviews have appeared in the literature. With the advent of new techniques, various attempts have been made to study the response of these cells in physiological and different pathological conditions [8].

The mast cells in Hand E stained sections showed predominantly abundant basophilic granules and with toluidine blue, the mast cells stained (Table/Fig 5) Fig 16: Photomicrograph Of H & E Stained Section Of Dentigerous red-purple (metachromatic staining) and Cyst (20x). the rest of the section stained blue. Mast cells can be confused with basophils. The functional and the biochemical characteristics of both the cells are much identical. A distinction between mast cells and basophils can be made upon

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morphology, natural history, and by the Matsson L ( 1993 ) (12) and Sjolin K.E expression of cell surface structures. (1946 ) [13] showed that in man, there Other cells that have been confused with was a decrease in the number of dermal mast cells include immature eosinophils mast cells with increasing age from or eosinophilic melanocytes and childhood to adults. Wolf J.E et al. macrophages [14]. (1973 ) [14] noted a decrease in the number of mast cells with age in Mast cells might play a role in the germfree rats in the connective tissue of pathogenesis of odontogenic cysts [9]. mandibular alveolar mucosa and bone They contribute to cyst enlargement by marrow. This decrease in the number of increasing the osmotic pressure of the mast cells with age could be due to fluid at least in three ways 1) by direct increasing degranulation which is noted release of heparin into the luminal fluid, in older age [14]. Abdel et al. ( 1976 ) 2) by release of hydrolytic enzymes [15] reported an increase in the human which could degrade capsular dermal mast cells from 5 to 15 years of extracellular matrix components, age, followed by a gradual decrease. thereby facilitating their passage into the fluid and by the action of histamine on Matsson L. ( 1993 ) [12] noted a smooth muscle contraction and vascular decrease in the number of mast cells permeability, thus encouraging the from juvenile to adult rats in the tongue, transudation of serum proteins [10]. buccal mucosa and gingival mucosa. Degranulation of mast cells has been The high number of mast cells in implicated in the generation of various oral sites of juvenile rats prostaglandins, which would promote suggested that the of bone resorption and remodeling to growing animals have a higher readiness accommodate the growing cyst [11]. for reaction, where mast cells take an Because of their possible importance in active part than that of adults. the pathogenesis of odontogenic cysts, an attempt was made to evaluate the It is a pure conjecture, whether the presence and distribution of mast cells reduced number of mast cells with in the connective tissue and to correlate increasing age in all the three this data with age, sex, and the different odontogenic cysts is a result of the zones considered. normal aging process or whether it is related to the pathogenesis of these When the age groups were compared in cysts. So, further studies have to be all the three cysts, the mean number of carried out to know whether it is related mast cells per HPF showed progressive to age or whether some other factors are decrease with age. responsible. Mast cell count varies between species. Therefore, studies of Although there are no studies possible age variation in the mast cell mentioning the correlation between mast population in the oral mucosa of man cell count and age in odontogenic cysts, are needed. in general, it was noted that mast cell count decreases in the connective tissue In the present study, no statistically with age, as noted by some authors. significant difference was noted in the

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mast cell count between the sexes in all Smith G. et al. ( 1988 ) [3], in their the three odontogenic cysts. Coleman histochemical studies on E.J (1974 ) [16] noted no statistical glycosaminoglycans of odontogenic difference in the mast cell count cysts, observed a subepithelial band of between males and females in hamster alcian blue staining and this appeared to gingiva. be due to the presence of heparin, which could have been released by the In the present study, the number of mast degranulation of mast cells beneath the cells was more in the subepithelial zone epithelium to produce this staining as compared to the intermediate and pattern. deep zones in all the three odontogenic cysts. The difference in the distribution In the present study, a statistically of mast cells between the subepithelial significant difference was not noted and deep zones was statistically between other zones like the significant (P<0.05) in all the three subepithelial and the intermediate zones, odontogenic cysts. In odontogenic and between the intermediate and the keratocysts, a statistically significant deep zones, except for odontogenic difference (P<0.01) was noted in the keratocysts which showed a statistically subepithelial and the intermediate zones. significant difference (P<0.01) between This is compatible with that the reports the subepithelial and the intermediate of Smith G. et al. (1989 ) [9] who also zones. This is in contrast to the reports noted a statistically significant by Smith G. et al. ( 1989 ) [9] who noted difference (P< 0.05) between the a significant difference (P<0.05) subepithelial and the deep zones of all between the above mentioned zones. the three odontogenic cysts and also between the subepithelial and the Smith G. et al. ( 1989 ) [9] also noted a intermediate zones in case of statistically significant difference odontogenic keratocysts. (P<0.03) in the subepithelial zones of radicular cysts and odontogenic The subepithelial collection of mast keratocysts, which was not observed in cells in odontogenic cysts could be the present study. attributed to the chemotactic stimulus to mast cells, attracting them to the These differences could be attributed to epithelial lining or luminal fluid the specificity of the avidian-peroxidase contents. The nature of such stimulus is staining as compared to the toluidine unclear, but the secretory matrix blue staining. They found that the avidin proteins of the normal odontogenic peroxidase stain was specific for mast epithelium have been reported to be cells and the differentiation of the mast chemotactic to mast cells. Although cells from other tissue elements was odontogenic cysts are not known to good, because there was no staining on secrete enamel matrix proteins, the other cells of the inflammatory infiltrate epithelial lining stains positively for or the connective extracellular matrix. and has been shown to share Toluidine blue was not found to be common antigenic determinants with effective due to poor metachromasia, enamel matrix proteins [9].

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probably arising from acid in toluidine blue stained sections and by decalcification of the tissue. the immunohistochemical staining of mast cells by using monoclonal Several reasons may account for the antibodies against the human mast cell difficulty to compare the data of proteases, tryptase and chymase. They different studies, like size of the sample, found a decreased number of mast cells type of tissue examined, the method of in toulidine blue stained sections. The fixation, the type of staining used, the obvious reason for this discrepancy is morphological criteria of the mast cells that only a few of the activated mast considered by the investigator, the cells are detectable in toluidine blue method used for counting the cells, the stained sections. Maximal degranulated number of cells examined per biopsy, or and resting mast cells can be stained maybe the investigated tissue area was immunohistochemically. too small to reduce the possible error caused by the inhomogeneity of cellular Joseph S. et al. (2003) in their study distribution [17], [18]. comparing toluidine blue and thionin noted that toluidine blue showed more Schwartz J. and Dibble M. ( 1975 ) (19) intact mast cells as compared to and Teronen O. et al. ( 1996 ) [20] thionine. Hence, Carnoy’s fixative observed that toluidine blue, the toluidine blue is a better stain as routinely used special stain for mast compared to thionin [21]. cells, does not stain degranulated mast cells (phantom cells). This might lead to Before any definitive conclusion can be variations in the number of mast cells reached, additional research must be observed in the different zones. conducted at histochemical levels. Such research should make use of specific Degranulated mast cells can be histochemical labeling techniques so detected by immunohistochemical that the products of expelled granules staining by using monoclonal antihuman may be identified in the connective tryptase antibodies and Western blotting tissue and so that the decrease in the by using specific antihuman mast cell mast cell counts may be correlated to the tryptase antiserum [20]. increased concentration of the degranulation products. Teronen O (1996) [20] noted that the density of intact mast cells decreased Standardization of the conventional outwards from the cyst lumen. methods, strict requirement for mast cell Degranulated mast cells were highest at identification, immunohistochemical the periphery of the cysts, at their border staining of mast cells using antibodies with , indicating higher activity of raised against granule proteases, and mast cells in this area. This was Western blotting with specific observed using immunohistochemical antihuman mast cell tryptase on jaw cyst staining of mast cell tryptase. extract samples may throw more light on the role of mast cells in the Bischoff S.C.et al. (1996) [17] observed pathogenesis of cysts. a difference in the number of mast cells

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Mast cell plays a contributory role in the [6] Weedon D et al. The skin London: pathogenesis of odontogenic cysts Churchill Livingstone., 1992, 1005pp. [7] Strobel S. et al. Human intestinal which are often treated by surgical mucosal mast cells: evaluation of enucleation. In extreme cases where fixation and staining techniques. J Clin surgical intervention is not possible, one Pathol 1981; 34:851-58. might speculate the role of the [8] Galli SJ. New concepts about the mast inhibitors of mast cells or that of mast cell. N. Eng. J. Med 1993; 328:257-65. [9] Smith G, Smith A.J. and Basu M.K. Mast cell tryptase. In future, studies which cells in human odontogenic cysts. J examine the influence of mast cell Oral Pathol Med 1989; 18:272-78. antagonists on cyst pathogenesis may [10] Pepys J. and Edwards AM. The mast completely unravel the role of mast cells its role in health and disease. cells. Great Britian: Bath Pitman Press 1979, 9-19. [11] Mckenna MJ. and Frame B. The mast Conclusion cell and bone. Clin Orthoped Res 1985; In the present study, more number of 20:226-33. mast cells was noted in the subepithelial [12] Matsson L. Presence of mast cells in various oral mucosal sites in juvenile zone as compared to the intermediate and adult rats. Scand J. Dent Res 1993; and the deep zones. The number of mast 101:292-98. cells decreased with age. No gender [13] Sjolin KE. Quantitative estimation of dependence on mast cell count was mast cells in the dermal connective of noted. haemophilic patients. Acta Pathol Microbial Scand 1964; 61:537-42. [14] Wolf JE. et al. Mast cells and Basophils Further studies using in the mandibular alveolar connective immunohistochemical techniques of tissue and bone marrow of germfree mast cells and the influence of mast cell Albian rats.J Dent Res 1973; 52:1092- 96. antagonists on cyst pathogenesis may [15] Abdel- Aal et al. Quantitative and help unravel the role of mast cells in the qualitative changes in mast cells in the pathogenesis of odontogenic cysts. skin of normal Egyptians. Acta Derm Venerol (Stockh) 1976; 56:435- 39.

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[21] Joseph S, Das S, Chand R, Roopa R, and Rat Mesentery Using Carnoy’s Fixative. Thomas I M. Comparison of Toluidine J Anat. Soc. India 2003;52(2):166-67. Blue Vs Thionin For Mast Cells In

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