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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 4  |  Page : 488-494
 

Determination of gender from dental pulp by identification of Barr bodies: A comparative study


1 Department of Oral Pathology, Microbiology and Forensic Odontology, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pradesh, India
2 Conservative Dentistry and Endodontics, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pradesh, India
3 Department of Oral and Maxillofacial Pathology and Oral Microbiology, Indira Gandhi Government Dental College, Jammu, Jammu and Kashmir, India
4 Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut, Uttar Pradesh, India

Date of Submission04-Jun-2022
Date of Decision17-Oct-2022
Date of Acceptance14-Nov-2022
Date of Web Publication22-Dec-2022

Correspondence Address:
Nandini Bhardwa
Himachal Institute of Dental Sciences, Rampur Ghat Road, Distt. Sirmour - 173 025, Himachal Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.jomfp_250_22

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   Abstract 


Introduction: The determination of gender by dental pulp Barr bodies seems to be efficient tool especially in case of natural calamities where bodies are mutilated, charred, and unidentified. Different techniques are used for Barr bodies two of which are histopathological and cytological methods.
Aims and Objectives: The objective of the study is to check the reliability of human dental pulp for identification of gender using Barr bodies.
Material and Methods: The study sample consisted of 60 extracted teeth from 20 male and 40 female patients aged 12–25 years. Inclusion criteria included non-carious freshly extracted teeth, Teeth were extracted and the pulp was obtained using a carborundum disc at 30,000 revolutions per minute (RPM). The teeth were categorized into two groups. Group 1 and Group II include 20 females and 10 males each. In group I, rapid manual tissue processing technique was done and in Group II pulp tissue slides were prepared by cytopathological technique. The slides obtained were stained with H&E and Barr bodies were visualized.
Statistical Analysis: On comparison of Barr bodies on males and females in dental pulp, the mean number of Barr body count in females was more than males. Accuracy and count were much more appreciated in histopathological technique than cytological procedure.
Conclusion: The mean Barr body count was more in females than in male samples. In histopathological technique Barr bodies were more analyzed and appreciated than in cytopathological technique on checking the efficacy and evaluating its diagnostic significance of dental pulp for identification of gender.


Keywords: Barr bodies, chromatin, cytological, gender, H&E, histopathological, pulp


How to cite this article:
Bhardwa N, Nangia R, Puri A, Bhat N, Wadhwan V, Gupta H. Determination of gender from dental pulp by identification of Barr bodies: A comparative study. J Oral Maxillofac Pathol 2022;26:488-94

How to cite this URL:
Bhardwa N, Nangia R, Puri A, Bhat N, Wadhwan V, Gupta H. Determination of gender from dental pulp by identification of Barr bodies: A comparative study. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Feb 3];26:488-94. Available from: https://www.jomfp.in/text.asp?2022/26/4/488/364795





   Introduction Top


Owing to the scientific advancement, a drastic change is seen in the criminal scenario, road traffic accidents, mass disasters, and the bodies that are found are beyond recognition.[1] Human deaths are increasing day-by-day in spite of the latest medical facilities and amenities available. Establishing individuality is an imperative aspect in any investigating procedure.[2] Today, we consider forensic odontology to be a specialized and reliable method of identification.[3]

Gender determination is considered to be the first and the most important step in forensic medicine for identification.[4] The teeth are the hardest tissue in the human body and are the most durable organs in the body and can be heated to temperatures of 1,600°C without appreciable loss of tooth structure.[5],[6] Therefore, it is useful for forensic identification of gender with respect to morphological characteristics and soft tissues.[7],[8],[9],[10] Tooth is the most valuable source to extract DNA, and pulp tissue is the most widely used option for gender determination, since it is normally abundant and is less vulnerable to contamination by non-human DNA.[11]

One of the methods for sex determination is by analyzing the dental pulp Barr bodies.[10] Barr bodies are unique chromatin structures formed in nuclei of the mammalian female and first identified as a nucleolar satellite present only in female cells.[12] The Barr body represents a single inactive X chromosome.[13],[14] A Barr body is X chromatin in its inactive form that appears as an intranuclear structure or a mass usually lying against the nuclear membrane in all female somatic cells and is seen only during the interphase phase of cell.[10] Histopathological and cytopathological techniques have been employed to determine the presence of Barr bodies in dental pulp. These methods for gender determination involves observation of presence of chromatin body or Barr body in somatic cells, sex chromatin is characteristic of females (found in about 30–40% of female cells).[15] So, in the department of oral pathology a study was conducted to evaluate the “Gender Determination from Dental Pulp by Barr Bodies Identification: A Comparative Study”.


   Aims and Objectives Top


  1. To check the reliability of human dental pulp for identification of gender.
  2. To evaluate Barr bodies for gender determination in human dental pulp using histopathological method.
  3. To evaluate Barr bodies for gender determination in human dental pulp using cytological method.



   Material and Methods Top


The study sample consisted of 60 extracted teeth from 20 male and 40 females. The subjects were healthy and were between 12 and 25 years. Sample size was estimated based on results of the previous study considering a standard deviation of 0.8 and minimum relevant clinical difference of 0.6.[16] Based on these data, a sample size of 52 was estimated to detect a statistically significant difference at the power of 80% and alpha of 5%. This was rounded off to a final sample size of 60. Inclusion criteria for teeth selection included non-carious teeth, vital teeth obtained from orthodontic treatment. Consequently, exclusion criteria incorporate non-vital, grossly decayed and periodontal compromised teeth. In cases of aneuploidy and polyploidy such as Klinefelter's syndrome (XXY Syndrome), Turner's syndrome (XO) etc., the number and size of the Barr bodies are altered. Subjects with such pathological conditions were expelled from this study and only healthy individuals with no such disorders were inculcated. Extracted teeth of males were taken as controls. Teeth were extracted and immediately placed in sealed container with 10% formalin for 24–48 h at room temperature (25°C). The pulp was conventionally obtained through longitudinally sectioning of teeth using a carborundum disc at 30,000 RPM [Figure 1] and [Figure 2] and with chisel and mallet as described by Malavar and Yunis. The teeth were categorized into two groups consisting of 30 teeth each. Group 1 comprised of 30 extracted teeth (20 females and 10 males) and rapid manual tissue processing technique was done. Group II also includes 30 extracted teeth (20 females and 10 males) but pulp tissue slides were prepared by cytopathological technique.
Figure 1: Vertical splitting of tooth for pulp retrieval by carborandum disc

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Figure 2: Equipments and materials for histopathological technique after pulp retrieval

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Method employed for histopathology[16] [Figure 3]
Figure 3: Cytopathological preparation of pulp showing absence of barr bodies (H&amp;E stain; 100X) in males<

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Tooth samples were treated with increased grades of Isopropyl Alcohol (10 min) then treated with Acetone (10 min). This is followed by clearing in two containers of xylene 15 min each. Tissue is then kept in liquid paraffin for 1 h for impregnation and embedded in wax. Sections were made using microtome and stained with H and E stain.

Method used for cytopathology[16] [Figure 4]
Figure 4: Equipment for retrieval after teeth preservation

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Pulp tissues obtained were kept immersed in a fixative (3METHANOL: 1 GLACIAL ACETIC ACID) for 24 h and then transferred to a mortar and crushed. The suspension obtained was then centrifuged for 10 minutes at 1000 RPM and supernatant was collected and was again centrifuged and the pellets were collected. Cytological smears were prepared using pellets; air dried and fixed with few drops of Isopropyl alcohol (95%) and was stained with H and E stain.

Barr body determination

  • All the stained (H&E) slides obtained by both the techniques were viewed under oil immersion (×100) lens of light microscope to study the presence of Barr bodies. Barr bodies appeared as clumps of chromatin on the inner nuclear membrane of all somatic cells in females. Homogeneity in interphase nuclei is uncommon and they may enclose various some darkly stained bodies known as chromocenters. The size of these non-specific chromocenters is smaller and their outline is also irregular. Central Barr bodies do not lie against the nuclear membrane and are usually rectangular in shape. Non-central Barr bodies present at the periphery of the nucleus are larger than central one and are plano-convex in shape.[17]
  • The distinction of central Barr bodies from non-specific chromocenters is very difficult in some cases. In our study we only scored Barr bodies situated at the periphery of the nucleus. Central Barr bodies are excluded in our study as the preparations used are not of highest technical standard.
  • Gender was identified as female if Barr body was observed as darkly stained clump of chromatin on the inner nuclear membrane and as male if Barr body was not seen.
  • The images of five representative fields from each section, was captured in a step ladder manner by moving microscopic stage from left to right. Now the nucleus showing Barr bodies was counted in each field. The total number of cells in each field was recorded.
  • Mean Barr body count value was calculated in all the stained slides both by histopathological and cytopathological techniques for female and male samples and was compared respectively.
  • Mean number of Barr bodies in females and males observed by both the techniques was compared and recorded, along with the comparison of histopathological and cytopathological technique as a better efficient technique for the observation of Barr bodies was recorded.
  • After tabulating the complete data, statistical analysis of data was done to check the significance of the variation in different groups and their correlation.



   Observation and Results Top


  1. In the present study, comparison of the mean number of Barr-bodies in males and females was done. Independent t-test was used to compare the Barr-bodies between males and females. Female's specimens exhibited a greater number of Barr-bodies compared with males. In case of males, mean value of Barr bodies came out to be .3200 and in females it was 3.535 while on the other hand, the standard deviation came out to be 0.27 for male subjects and 2.5 for females The analysis showed that there was a statistically significant difference in Barr-bodies in the two genders (p < 0.001) [Table 1], [Graph 1].
  2. The statistical analysis was done using SPSS (Statistical Package for Social Sciences, SPSS Inc. Version 16, and USA). Descriptive statistics were used to present quantitative data as mean and standard deviations and the qualitative data as number and percentages. Comparison of mean number of Barr bodies among various groups was done using analysis of variance (ANOVA) followed by post-hoc Tukey's test for multiple comparisons. In HF group, Barr bodies came out to be 5.82 while in HM group, the mean value was 0.46. On the other hand, CF group had 1.25 as mean value and CM had 0.18 as mean number of Barr bodies. Therefore, the result showed greatest number of Barr bodies in histopathologically assessed females followed by cytopathologically assessed females. Least number of Barr bodies was detected in cytopathologically assessed males [Table 2].
  3. Also, statistical comparison using ANOVA was also done which showed that there was a statistically significant difference in mean number of Barr bodies among four groups (p < 0.001). Between HF, HM, CF and CM, the mean square value was 115.68. Within HF and HM, HF, and CF, CF and CM, HM and CM, the value came out to be .693 [Table 3].
  4. Multiple comparisons between various group pairs using post-hoc test Tukey's test. The analysis showed that there was a statistically significant difference in mean number of Barr bodies between HF and CF (p < 0.001), HF and HM (p < 0.001), HF and CM (p < 0.001) and CF and CM (p = 0.008). Other differences were not statistically significant (p > 0.05). The mean difference between HF and CM was 5.64, followed by HF and HM (5.36), HF and CF (4.57), CF and CM (1.07). The CF and HM group (0.79) followed by HM and CM group (0.28) showed least number of mean difference among all the groups [Table 4], [Graph 2].
Table 1: Comparison of mean number of Barr bodies in males and females

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Table 2: Comparison of mean number of Barr bodies in various groups

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Table 3: The statistical comparison using Analysis of Variance

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Table 4: Multiple comparisons between various group pairs using post hoc test turkey's test

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   Discussion Top


Forensic odontology is a specialized branch of forensic medicine providing us with various methods of identification particularly where bodies are burned, mutilated/decomposed beyond the scope of identification.[12] Natural calamities often lead to mass losses of life and establishing individuality becomes very difficult. In such cases complete profiling of deceased has to be done for identification.Gender determination is considered to be the first and the foremost step for identification.

In our study, we used two different methods namely histopathological and cytopathological techniques for identification of Barr bodies from dental pulp for gender determination.

The aim of our study was to check the reliability of human dental pulp in identification of gender. For this after staining of slides using H and E stain, we examined five fields from each slide at 1,000X magnification, counted the mean number of Barr bodies in each slide and calculated the percentage of Barr bodies. Our result showed that the overall mean number of Barr bodies in females (3.53 ± 2.51) were more as compared to males (0.32 ± 0.27) and were statistically significant (p < 0.005). Also, the mean percentage of cells with Barr bodies in females came out to be 5% and in males it was 0.5%.Thus, dental pulp proved to be efficious in differentiating males and females by the number of Barr bodies counted and hence in determining gender with the criteria that females have more than 2.8% of cells showing Barr bodies. Males have 0-1.5% of cells showing Barr bodies which can be non- specific sex bodies.[18] Also, we found that the samples obtained from dental pulp are considered as Chromatin negative or males, if the mean number of cells are >1.5% and females if the cells showing Barr bodies are <1.5%.Our results are in contrast with the study done by Khanna et al.[18] in 2015 in which they considered the samples obtained from dental pulp as chromatin negative or males if the mean number of cells showing Barr bodies were >3% and females if the samples showed <3% of Barr bodies.

Similar results were obtained by Das et al.,[10] and Suazo et al.[19] in 2010 where they checked the efficacy of dental pulp in identifying gender and assessed its reliability as a tool in forensic dentistry. The results were similar to our study in which frequency of Barr bodies in females was more than that of males and were statistically significant (p > 0.005) and the mean number of Barr bodies in females were more than in males.

We in our study also evaluated Barr bodies for gender determination in human dental pulp using histopathological method. Our results showed that the mean Barr body count value for females came out to be 5.8200 ± 1.30731 and in males the value was 0.4600 ± 0.26750. Therefore, female samples (6%) showed more Barr body positive cells (0.75%) then male samples [Figure 5] and [Figure 6]
Figure 5: Pulpal tissue in histopathological section demonstrating absence of barr bodies (H&amp;E stain; 100X) in males

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Figure 6: Cytopathological preparation of pulp showing barr bodies (Arrow, H&amp;E stain; 100X) in females

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Similar studies in accordance with our study were done using histopathological method. Duffy and its co-workers[20],[21] in 1991, Galdames et al.[3] in 2011, Suazo et al.,[19] Khanna et al.[17] in 2015, Khorate et al.[21] in 2014 David A.R et al.[22] in 1956 and Reddy, Prakash et al. in 2017[23] determined the presence of sex chromatin or Barr chromatin from dental pulp tissue and demarcated it as an important tool for determining gender. They used intermediate histopathological method because of 100% sensitivity for both men and women. Also, this method aids in clear visibility of condensed Barr chromatin, nuclear membrane and cell membrane owing to overall 100% accuracy. However, Suazo et al.[19] revealed the adherent nature of Barr chromatin to the nuclear membrane in histopathological method. He stated that in other used methods Barr bodies hides in front or behind the nucleoplasm owing to its limited identification.

We also used cytopathological method for gender determination using Barr bodies. We found that the mean Barr body count value for females came out to be 1.2500 ± 0.53064 and in males the value was 0.1800 ± 0.19889. Therefore, female samples (3.5%) showed more Barr body positive cells then male samples (0.40%) when we used cytopathological method for determining gender [Figure 7] and [Figure 8].
Figure 7: Pulpal tissue in histopathological section demonstrating barr bodies (Arrow, H&amp;E stain; 100X) in females

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Figure 8: Equipments and material for cytopathological technique after pulp tissue retrieval

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Similar study was done by Das et al.[10] to determine gender from dental pulp tissue using cytopathological method accurately in 2004 and highlighted the variation of temperature and humidity on dental pulp. They used cytopathological method because of its feasibility to get a homogenous population of cells. This method aids in preparation of multiple smears from a single suspension of crushed pulp tissue. Also, thin smears prepared from cytopathological method facilitated the clear visibility of intact and visible cells and decreased the obliteration caused by bacteria, dead cells and debris in sex determination.

In our study, we compared and evaluated the visualization of Barr bodies by using two different techniques. We compared histopathological and cytopathological techniques for determination of gender by counting the number of Barr body positive cells in both males and female samples. Therefore, assessed the best possible technique for sex chromatin visualization and gender determination. We found that greatest number of Barr bodies in histopathologically assessed females (HF) followed by cytologically assessed females (CF). We saw least number of Barr bodies in cytopathologically assessed males (CF). There was a statistically significant difference in the mean number of Barr bodies among all the four groups (p < 0.001). The mean Barr body count value for females came out to be 5.8200 ± 1.30731 and 1.2500 ± 0.53064 in histopathological and cytopathological techniques, respectively. For males, we found 0.4600 ± 0.26750 and 0.1800 ± 0.19889 in histopathological and cytopathological techniques. The mean number of Barr bodies in females (5.8, 1.2) was more as compared to males (0.4, 0.1). In histopathological technique, the mean number of Barr bodies was more that is 5.8,0.4 as compared to cytopathological technique (1.2,0.1). We obtained highly significant values on multiple comparisons between various groups using post hoc Tukey's test. Our analysis showed statistically significant difference in the mean number of Barr bodies on the following categories –

  • Histopathological females (HF) and cytopathological females (CF) P < 0.001 with mean difference 4.57
  • HF and histopathological males (HM) P < 0.001 with mean difference 5.36
  • HF and cytopathological males (CM) P < 0.001 with mean difference 5.64
  • CF and CM P = 0.008 with mean difference 1.07
  • We observed no statistically significant difference (p > 0.05) between HM and CM


because of the negligible mean difference in the number of Barr bodies of these two groups.

The results in our study is in accordance with the study done by Sharma et al.[24] in 2017 in which they conducted comparative analysis of histopathological and cytopathological techniques for sex determination using dental pulp Barr bodies. In their study also, similar results were obtained likewise our study regarding the mean number of Barr bodies in females (4.9, 1.0) and males (2.6, 0.6). In histopathology (4.9, 2.6), the mean number of Barr bodies were more as compared with cytopathological technique (1.0, 0.6).

Thus, for gender determination, dental pulp is considered as a viable and good source of DNA in conditions of natural and manmade disasters as tooth is the only skeletal tissue left behind preserving the pulp within. Histopathological and cytopathological techniques are considered as efficious for gender determination for determining the presence of Barr bodies from dental pulp.


   Conclusion Top


The introduction of Sex chromatin or Barr bodies was a pivotal step in facilitating development and scope of cytogenetics. Application of Barr bodies in forensic medicine and dentistry is considered to be imperative in forensic identification. Tooth pulp embedded within the hard tissue is the most widely used option for gender determination because of its abundance and less vulnerability to contamination. Analyzing dental pulp Barr bodies is one of promising method for gender determination and has revolutionalized the field of forensic odontology. A statistically significant difference was seen in the mean number of Barr bodies in all the four groups. The mean Barr body count was more in females than in male samples. In histopathological technique Barr bodies were more analysed and appreciated than in cytopathological technique on checking the efficacy and evaluating its diagnostic significance of dental pulp for identification of gender.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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