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ORIGINAL ARTICLE
Year : 2009  |  Volume : 27  |  Issue : 4  |  Page : 235-241
 

Quantitative estimation of AgNORs in inflammatory gingival overgrowth in pediatric patients and its correlation with the dental plaque status


Department of Paedodontia, Dr. R Ahmed Dental College & Hospital, Kolkata, India

Date of Web Publication14-Nov-2009

Correspondence Address:
S Mukhopadhyay
18-1C Diamond City, 68, Jessore Road, P.O:- Nager Bazar, Kolkata - 700 055, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.57659

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   Abstract 

Background and Objectives: Nucleolar organizer Regions (NORs) are situated within the nucleolus of a cell. The proteins are selectively stained by the silver colloid technique that is known as the AgNOR technique. AgNOR stain can be visualized as a black dot under the optical microscope. The present study aimed to evaluate the cases for quantitative estimation of AgNORs in the epithelial cells in various grades of gingival overgrowth to that of normal gingival tissues. Materials and Methods: Only preadolescent and adolescent groups aged up to 14 years were selected. Twenty normal and 31 disease cases of gingival overgrowth were selected. The tissue sections were stained by the hematoxylin and eosin (HandE) technique for the routine histological evaluation, while the AgNOR counts were performed through the improved one-step method of Ploton et al. Results: HandE staining revealed five different types of gingival overgrowth. The plaque index (PI), gingival index (GI), and AgNOR count were not significantly (P >0.05) higher than that of control cases in pyogenic granuloma, puberty gingivitis, and in drug-induced gingival overgrowth cases. In gingival fibromatosis cases, for comparison of different indices t-tests were done. The PI when compared with AgNOR count was found significant at 5% level and 0.1% level for mixed and permanent dentition, respectively. The GI when compared with AgNOR count was found significant at 1% level and 0.1% level in mixed and permanent dentitions, respectively.


Keywords: AgNOR staining, gingival overgrowth, gingival index, plaque index


How to cite this article:
Mukhopadhyay S. Quantitative estimation of AgNORs in inflammatory gingival overgrowth in pediatric patients and its correlation with the dental plaque status. J Indian Soc Pedod Prev Dent 2009;27:235-41

How to cite this URL:
Mukhopadhyay S. Quantitative estimation of AgNORs in inflammatory gingival overgrowth in pediatric patients and its correlation with the dental plaque status. J Indian Soc Pedod Prev Dent [serial online] 2009 [cited 2019 Jul 19];27:235-41. Available from: http://www.jisppd.com/text.asp?2009/27/4/235/57659



   Introduction Top


The most common form of gingival disease in children is chronic nonspecific gingivitis. An increase in size of the gingiva is a common feature of gingival disease. Accepted current terminology for this condition is gingival enlargement and gingival overgrowth. These are strictly clinical descriptive terms. [1] Gingival overgrowth represents an overt, exuberant response to various local and systemic conditions. Local factors like plaque and calculus may cause gingival enlargement. Hormonal changes during puberty or pregnancy and intake of certain drugs like phenytoin, cyclosporine, and calcium channel blockers can result in gingival overgrowth.

The gingival overgrowth in terms of inflammation or other disorders can be studied with specific cell proliferation markers. In the present study, quantitative analysis of silver stained nucleolar organizer regions (NOR) was considered. NORs are situated within the nucleolus of a cell that are of central importance in the transcription of DNA to ribosomal RNA and hence in the ultimate assembly of proteins. [2],[3],[4] NORs are located, one each on the short arms of the five acrocentric chromosomes namely 13, 14, 15, 21, and22 as secondary constrictions close to the centromere. [5] NORs are stained with silver nitrate under proper conditions and the structures visualized are termed as AgNORs. [5],[6] The number of NOR-bearing chromosomes are seen in the karyotype. [7] This technique is very useful in assessing various pathological conditions. The present study aimed to evaluate the cases for quantitative estimation of AgNORs in the epithelial cells in various grades of gingival overgrowth to that of normal gingival tissues.


   Materials and Methods Top


A total of 31 patients who had gingival overgrowth were selected from the department of Pedodontia, Dr. R Ahmed Dental College and Hospital, Kolkata, as a study material. For control study, 20 volunteers (10 having mixed dentition and 10 from permanent dentition) of normal healthy gingiva were chosen. All subjects belonging to the study and control groups were between the age group of 7-14 years. An informed written consent was taken from patient's parents. Ethical committee clearance was obtained from concerned authority.

The complete clinical and oral examinations were done and recorded in a preformed case recording sheet designed for the study. The papillary, marginal, and attached (PMA) gingival index was utilized to study gingival status following the Massler and Schour method. [8]

A plaque-disclosing solution was used for evaluation of dental plaque. Dentition status was recorded according to FDI classification. Incisional biopsy samples were taken from the most representative area of the diseased tissue. The biopsy specimens were then fixed in 10% buffered formalin and subsequently processed to prepare paraffin blocks.

Two separate sets of sections of 4 μ thickness were obtained from each block. One set of sections was stained by the hematoxylin and eosin (HandE) technique for routine histological evaluation and confirmation of diagnosis.

However, the other set of sections were stained for localization and quantification of AgNORs in the lining epithelium of the enlarged gingival specimen by the improved one-step method of Ploton et al. (1986) and were carefully recorded. [9] AgNOR counts were recorded as black dots [Figure 1].

Preparatory steps for AgNOR staining are:

Solution A

A 50% silver nitrate solution

Solution B

Gelatin - 2 g

Pure formic acid - 1 cc

De-ionized water - 99 cc

A working solution was prepared by mixing two parts of solution A with one part of solution B. The sections were dewaxed in xylene for 5 minutes and rehydrated by passing through descending grades of alcohol. The slides were stained in small batches of 10 slides and kept in the dark for 45 minutes at room temperature. Following removal from staining, the slides were thoroughly washed in running deionised water for 5 minutes. Dehydration of the stained slides was done by passing through ascending grades of alcohol. Finally, the slides were cleared in xylene and mounted in DPX.

Results of staining

Background - pale yellow and clear

Nucleus - yellow and / or brown

AgNOR sites - intra-nucleolar black dots

Microscopic evaluation of the stained sections:

The counting of AgNORs in 100 cells involving the full thickness of gingival lining was performed and the findings were carefully recorded. The counting was repeated in all the sections to eliminate any related error in primary observation [Figure 2] and [Figure 3]. All the HandE stained sections were carefully viewed under the light microscope using a magnification of 10Χ and 40Χ in reference to the light microscopic diagnostic parameters of gingival overgrowth. The findings were viewed immediately following staining under the light microscope using 100Χ oil immersion lens and cedarwood oil [Figure 4] and [Figure 5].


   Results Top


The present study was conducted on20 normal subjects and 31 disease cases [Table 1] and [Table 2]. Plaque index (PI), Gingival index (GI) and AgNOR count were recorded in normal and disease cases [Table 3],[Table 4],[Table 5],[Table 6],[Table 7],[Table 8]. Comparison of PI, GI and AgNOR counts were made [Table 9],[Table 10],[Table 11],[Table 12].

In [Table 4], PI, GI, and AgNOR count in mixed and permanent dentition category of pyogenic granuloma were not statistically significant (P >0.05) in comparison to normal cases.

From [Table 5], PI, GI, and AgNOR count in puberty gingivitis in both mixed and permanent dentitions were not significantly higher (P >0.05) than that of the control group.

In [Table 6], the differences here were all significant due to comparatively lower magnitude of standard deviation values.

In [Table 7], the averages due to PI and AgNOR or between GI and AgNOR did not differ significantly (P >0.05) for mixed as well as for permanent dentition category.

In [Table 9], to compare the average indices, ' t' tests were performed. None of the averages were seen to differ significantly (P >0.05) with one another. The tests were done for mixed dentition and permanent dentition separately, and also for mixed and permanent dentition combined.

In [Table 11], here, 't ' tests were performed to compare the average indices (PI and GI) and AgNOR count separately for mixed dentition and permanent dentition, and for mixed and permanent dentition combined. In all cases, the difference between any two averages (as shown in the table above) did not reach the significance level (P >0.05). Only for mixed dentition and permanent dentition, combined average due to GI and that of AgNOR count, the difference between the averages just touches the significant level (P =0.05).

In [Table 11], the differences were all significant due to comparatively lower magnitude of standard deviation values.

In [Table 12], here, the averages due to PI and AgNOR and between GI and AgNOR did not differ significantly (P >0.05) from mixed dentition, permanent dentition, and also mixed and permanent dentition combined.


   Discussion Top


The increase in the size of the gingiva is a common feature of gingival disease. Local factors like plaque and calculus are commonly associated with gingival enlargement. Plaque is a highly selective entity resulting from a sequential colonization of microorganisms on the surface of the tooth. The plaque is the main causative agent of gingivitis. Lang et al., Loesche, Matsson et al. were of the view that plaque deposition had a important role in gingival inflammatory changes. [10],[11],[12] During puberty gingival enlargement and gum bleeding are common findings in girls. Estrogen and progesterone do increase the permeability of gingival blood vessels. [13] Sutcliffe and Biswas et al. also found gingival overgrowth during puberty. [14],[15] The present study noted similar findings though gingival inflammation was not significantly higher (P > 0.05) than that of control groups.

Gingival inflammation along with enlargement associated with plaque deposition and/or tendency to bleed with slightest provocation is a feature of pyogenic granuloma. The present study noted change in the PI and GI in pyogenic granuloma. However, the values were not significant statistically (P >0.05).

Gingival overgrowth is a common side effect of some anticonvulsant drugs, immunosuppressants and calcium channel blockers. McLaughlin and others noted clinically significant gingival overgrowth. Plaque scores and gingival inflammation were greater in patients taking phenytoin in comparison to non-drug taking groups. [16] Saymour and Smith suggested that the overgrowth of gingiva would depend on the interaction between dental plaque and drug therapy. [17] The present study also noted gingival overgrowth in boys and girls who took anticonvulsant and immunosuppressant drugs. The GI, PI, and gingival inflammation were significantly higher (P <0.01) than that of control groups.

Gingival fibromatosis is a relatively uncommon oral disease characterized by the enlargement of gingiva. It may be seen as an isolated finding or as part of a syndrome. [18] The proliferation rate of fibroblasts is significantly higher in gingival fibromatosis than that of fibroblasts of normal gingiva. [18]

Nucleolar organizer regions (NORs) are situated within the nucleolus of a cell. NORs are associated with certain non-histone proteins which are essential for RNA synthesis. The proteins are selectively stained by the silver colloid technique that is known as the AgNOR technique. AgNOR stained can be visualized as a black dot under the optical microscope. [19]

Hematoxylin and eosin (H and E) staining of the section is necessary for diagnostic purpose following clinical evaluation of cases. In the present study, 51 cases were stained with HandE stain. Out of these 51 cases, 20 were normal samples and 31 were disease cases.

Normal gingival epithelium was used as a standard. Coletta and others found the mean AgNOR dots of normal gingiva to be 1.64 ± 1.10. In this study, the AgNOR counts of normal gingiva of the mixed dentition group were 0.89 ± 0.02 and 1.23 ± 0.28 in the permanent dentition group.

Coletta et al. found that the mean AgNOR dot in gingival fibromatosis cases was 2.26 ± 1.69. [18] In the present study, the mean AgNOR count for gingival fibromatosis cases was 2.66 ± 0.08 and 3.04 ± 0.09, respectively for mixed and permanent dentition cases.

The mean AgNOR counts for drug-induced gingival overgrowth in mixed and permanent dentition cases in this study were 1.94± 0.91 and 2.01± 0.24, respectively.

In the present study, only one case of fibroma was included. TheGI, PI, and AgNOR count were 2.14, 1.89, and 2.61, respectively.

The mean AgNOR counts of pyogenic granuloma cases recorded in our study was 2.14± 0.46 and 2.18 ± 0.30 in mixed and permanent dentitions, respectively.

In the present study, the mean AgNOR count in puberty gingivitis was 2.30 ± 0.04 and 1.92 ± 0.51 in mixed and permanent dentitions, respectively.

Present study noted changes of AgNOR counts (from control cases) in puberty gingivitis, pyogenic granuloma, gingival fibromatosis and fibroma non-significantly (p<0.05) and significantly (p<0.01) in drug induced gingival overgrowth cases. As no previous study was conducted on gingival overgrowth of pediatric age group hence the present study cannot be corroborated with any other previous study.


   Conclusion Top


From the present study, it was seen that the PI, GI and AgNOR count in mixed and permanent dentition cases were not significantly (P >0.05) higher than normal in pyogenic granuloma, puberty gingivitis, and in drug-induced gingival overgrowth patients.

In gingival fibromatosis cases, for comparison of different indices t- tests were done. The plaque index (PI) when compared with AgNOR count was found significant at 5% level in mixed dentition and 0.1% level at permanent dentition. The GI when compared to AgNOR count was found significant at 1% level in mixed dentition and 0.1% level in permanent dentition.

Thus, the present study indicated that the AgNOR counts were not of diagnostic significance and could not be used to distinguish between various gingival overgrowths.

However, further in depth studies are required to understand the biologic behavior and prognostic aspects of different gingival enlargements.

 
   References Top

1.Carranza FA, Hogan EL. Gingival enlargement. In: Newman MG, Takei HH, Carranza FA, eds. Carranza's clinical periodontology 9 th ed. Philadelphia. WBSaunders Company, 2003. p. 279-96.  Back to cited text no. 1      
2.Howat AJ, Giri DD, Cotton DW, Slater DN. Nucleolar Organizer Regions in spitz nevi and malignant melanomas. Cancer 1989;63:474-8.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Schwint AE, Folco A, Morales A, Cabrini RL, Itoiz ME. AgNOR mark epithelial foci undergoing malignant transformation in hamster cheek pouch carcinogenesis. J Oral Pathol Med 1996;25:20-4.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]  
4.Uno T, Hashimoto S, Shimono M. A study of the proliferative activity of long junctional epithelium using AgNOR staining. J Periodontal Res 1998;33:298-309.   Back to cited text no. 4  [PUBMED]  [FULLTEXT]  
5.Vuhahula EA, Nikai H, Ogawa I, Miyauchi M, Takata T, Ito H, et al. Correlation between argyrophilic nucleolar organizer region (AgNOR) counts and histologic grades with respect to biologic behavior of salivary adenoid cystic carcinoma. J Oral Pathol Med 1995;24:437-42.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.Crocker J. Nucleolar organizer regions (NORs) in neoplasms. In: McGee JD, Issacson PG, et al, eds. Oxford text book of pathology vol 1. Oxford: Oxford university press; 1992. p. 586-90.  Back to cited text no. 6      
7.Landini G. Nucleolar organizing regions (NORs) in pleomorphic adenomas of the salivery glands. J Oral Pathol Med 1990;19:257-60.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
8.Schour I, Massler M. Survey of gingival disease using the PMA index. J Dent Res 1948;27:733-4.  Back to cited text no. 8      
9.Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ. Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level. Histochem J 1986;18:5-14.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]  
10.Lang NP, Cumming BR, Lφe H. Tooth brushing frequency as it relates to plaque development and gingival health. J Periodontol 1973;44:396-405.  Back to cited text no. 10      
11.Loesche WJ. Clinical and microbiological aspects of chemotherapeutic agents used according to the specific plaque hypothesis. J Dent Res 1979;58:2404-14.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]  
12.Matsson L, Goldberg P. Gingival inflammatory reaction in children at different ages. J Clin Periodontol 1985;12:98-103.  Back to cited text no. 12  [PUBMED]    
13.Guyton AC. Female physiology before pregnancy and female hormone. In: Guyton AC, editor. Text book of medical Physiology.8 th ed. Bangalore: Prism books pvt ltd; 1991. p. 899-914.  Back to cited text no. 13      
14.Sutcliffe P. A longitudinal study of gingivitis and puberty. J Periodontal Res 1972;7:52-8.  Back to cited text no. 14  [PUBMED]    
15.Biswas S, Duperon DF, Chebib FS. Study of periodontal disease in children and young adolescents. J Periodontal Res 1977;12:250-64.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]  
16.McLaughlin WS, Ball DE, Seymour RA, Kamali F, White K. The pharmacokinetics of phenytoin in gingival crevicular fluid and plasma in relation to gingival overgrowth. J Clin Periodontol 1995; 22:942-8.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]  
17.Seymour RA, Smith DG. The effect of a plaque control programme on the incidence and severity of cyclosporine induced gingival changes. J Clin Periodontol 1991;18:107-10.  Back to cited text no. 17  [PUBMED]    
18.Coletta RD, Almeida OP, Graner E, Page RC, Bozzo L. Differential proliferation of fibroblasts cultured from hereditary gingival fibromatosis and normal gingival. J Periodontal Res 1998;33:469-75.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]  
19.Crocker J, Boldy DA, Egan MJ. How should we count AgNORs? Proposals for a standardized approach. J Pathol 1989;158:185-8.  Back to cited text no. 19      


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]


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