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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 8  |  Issue : 2  |  Page : 73-80

Age estimation using tooth cementum annulations method by different types of microscope: A comparative study


1 Private Practitioner, Panineeya Institute of Dental Science and Hospital, Hyderabad, Telangana, India
2 Department of Oral Pathology, Panineeya Institute of Dental Science and Hospital, Hyderabad, Telangana, India

Date of Web Publication18-Dec-2018

Correspondence Address:
H L Geetha
91/E 5-5-149, 5th Road Hills Colony, Vanastlialipuram, Hyderabad, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijohs.ijohs_56_18

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  Abstract 


Introduction: Age is an essential factor in establishing the identity of a person. Age estimation through dental parameters can be of valuable assistance in human identification as teeth can survive in most conditions encountered at death and during decomposition. Tooth cementum annulations are one of the many methods for determination of an individual's age. Incremental lines in cementum are counted in this method.
Objective: The aim of this study was to examine the correlation between chronologic age and estimated age and to find the most accurate method of calculating cemental annulations using different types of microscopes (light microscope, phase contrast microscope, polarized, and stereomicroscope).
Methodology: The extracted teeth were cleaned and polished. Block was prepared by embedding two third of each tooth root horizontally in self-cure acrylic. Using a hard tissue microtome (SP 1600) longitudinal sections of 100 μm thickness were made. The sections were cleaned in alcohol and were mounted on a glass slide. These sections were observed under different types of microscopes (light microscope, phase contrast microscope, polarized, and stereomicroscope) to count cemental annulations.
Results: The relationship between chronological age and estimated age using light microscope, phase contrast and polarized microscope showed stronger correlation while stereo microscope showed a weaker correlation.
Conclusion: The use of polarized microscope increases the accuracy and thus, can predict the near estimate of the age for all the age groups.

Keywords: Age estimation, cemental annulations, light microscope, phase contrast microscope, polarized microscope, stereomicroscope


How to cite this article:
Geetha H L, Baghisath VP, Vinay HB, Sudheer B, Kumar VJ, Gayathri C H. Age estimation using tooth cementum annulations method by different types of microscope: A comparative study. Int J Oral Health Sci 2018;8:73-80

How to cite this URL:
Geetha H L, Baghisath VP, Vinay HB, Sudheer B, Kumar VJ, Gayathri C H. Age estimation using tooth cementum annulations method by different types of microscope: A comparative study. Int J Oral Health Sci [serial online] 2018 [cited 2019 May 20];8:73-80. Available from: http://www.ijohsjournal.org/text.asp?2018/8/2/73/247815




  Introduction Top


Age is one of the essential factors, which play an important role in every aspect of life. The age estimation through dental parameters can be of valuable assistance in human identification. It may also help in other situations such as: determining the legal liability of teenagers and adults of unknown age, assist adoption processes, release retirement funds for adults of unknown age as well as support research in archeology and paleodemography.[1]

Estimating age from teeth is generally reliable as they are naturally preserved long after all other tissues have disintegrated.[2] Teeth can survive in most conditions encountered at death and during decomposition, even when the body is exposed to extreme forces and/or temperatures.[3] This resistance has made teeth useful indicators for age estimation.[4]

Dental age estimation methods are either based on a well-ordered cascade of changes that occur during the formation and eruption of teeth or they rely on a continuous process that alter and diminish the quality of dental tissues even when individual growth is completed.[5] Tooth cementum annulations (TCA) is a microscopic method for the determination of an individual's age based on the analysis of acellular extrinsic fiber cementum.[6]

The distance from one cemental line to the next represents a yearly incremental deposition of cementum in many mammals, and counting of these lines has been used routinely for estimation of the age of animals. Incremental lines in cementum have also been observed in sections of human teeth, and there are a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, cut from formalin-fixed human dental roots (either paraffin-embedded or frozen sections), can be stained using several techniques. The other methods include fluorescence, confocal laser scanning, interference contrast, and scanning electron microscopy.[7]


  Methodology Top


A prospective study was done to estimate age by using cemental annulations with different types of Microscopes in the Department of Oral Pathology and Microbiology, Panineeya Mahavidyalaya Institute of Dental Sciences, Hyderabad (Telangana).

After the approval of the study from the Institutional Ethical Review Board, with the written consent of the subjects, a total of 100 single-rooted extracted teeth were collected from the patients who were visiting the Department of Oral and Maxillofacial Surgery. Criteria for inclusions were normal sound teeth without any developmental anomalies of diseases such as caries or trauma from the age group of 20–70 years. After extraction of the tooth, name, and sex of the patient, outpatient number, date of birth and date of extraction of a tooth (to obtain the chronological age of the patient), were noted and were grouped into five: Group 1: 21–30 years, Group 2: 31–40 years, Group 3: 41–50 years, Group 4: 51–60 years, and Group 5: 60–70 years. The age of the patients according to the demographic data is referred as the chronological age while the age calculated using the TCA method is referred to as the estimated age.

The extracted teeth were cleaned with pumice slurry to remove the debris left on the tooth and polishing was done using a brush under slow speed rotator handpiece. Block was prepared by embedding two third of each tooth root horizontally in self-cure acrylic. Using a hard tissue microtome (SP 1600) fitted with the diamond coated blade rotating at a speed of 3000 rpm using tap water as a coolant; longitudinal sections of 100 μm thickness were made. The sections were cleaned in alcohol and were mounted on a glass slide, on which the coverslip was placed using DPX mountant [Figure 1]. These sections were observed under different types of microscopes to count cemental annulations. The types of microscopes included light microscope, phase contrast microscope, polarized, and stereomicroscope [Figure 2], [Figure 3], [Figure 4], [Figure 5].
Figure 1: Resin-mounted teeth for the preparation of ground sections

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Figure 2: Light microscope

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Figure 3: Phase-contrast microscope

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Figure 4: Polarized microscope

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Figure 5: Stereomicroscope

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The mounted sections were examined under the ×10 objective lens of the microscope, and middle one-third of the roots were selected for the count of cemental annulations. Images of the areas selected were captured using Camera two to three images per se ctions were taken, and the best photographic slide was used for counting annulations [Figure 6], [Figure 7], [Figure 8]. The same process was repeated for all the microscopes. The images were stored in JPEG format and later analyzed using computer software. The images were then uploaded into the software, and the following measurements were noted using the measuring tool of Adobe Photoshop (Adobe Systems Incorporated, San Jose, California, United States) [Figure 9].
Figure 6: Tooth cementum annulation using light microscope

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Figure 7: Tooth cementum annulation using phase-contrast microscope

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Figure 8: Tooth cementum annulation using polarized microscope

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Figure 9: Analyzing the image (tooth cemental annulations) using Image Analysis software

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The distance between the cementodentinal junction to the surface was considered as D1, and the distance between the two prominent cemental lines was considered as D2. The chronological age was calculated by using the formula:

Estimated age = Eruption age + Total cemental width

The distance between two incremental lines.

EA = Eruption age + D1/D2

Mean of chronologic age and estimated age was calculated. To know the relationship between chronologic age and estimated age; chronologic age and number of cemental annulations, Pearson's correlation (r) and coefficient of determination (r2) was employed. P ≤ 0.05 was considered for statistical significance. Statistical analysis was performed with SPSS version 20 (IBM, Armonk, NY, USA) and Microsoft Excel 2010 (Microsoft Corporation, Redmond, Washington).


  Results Top


Of 100 Samples, 57 were male and 43 were female. The age and gender distribution among the five study groups has been summarized in [Table 1].
Table 1: Age and gender distribution among the study groups

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The mean age with standard deviation (SD) for the age group of 20–70 years is about 48.26 ± 11.31. The estimated age with light microscope is about 47.01 ± 11.08, with phase contrast about 49.47 ± 9.64, with polarizing microscope 47.84 ± 10.82 and with stereo microscope 58.22 ± 22.91. Stronger ‘r’ value of about 0.86 was shown with a polarized microscope, and about 0.76 of “r” value was shown by light microscope. A significant coefficient of correlation of about 0.000 was found with all the microscopes except stereomicroscope. Polarized microscope showed less Standard Error of Estimate (SEE)of about 5.7 years and for light microscope about 7.34 years [Table 2] and [Graph 1].
Table 2: Mean chronological and estimated age for all study groups

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Mean and SDs of Chronological age of group I (21–30 years) is 25.73 ± 2.61 and the mean and SD values of the estimated using different microscopes are as follows: with light microscope is 27.36 ± 3.26, with phase contrast is 44.09 ± 12.25, polarized microscope is about 27.00 ± 2.49, and with stereo microscope, it was about 59.36 ± 28.35. The mean and SDs of chronological age of Group II (31–40 years) is 37.22 ± 3.42, and the mean and SD values of the estimated using different microscopes are as follows, with light microscope is 39.00 ± 5.09, with phase contrast is 44.44 ± 5.89, polarized microscope is 38.89 ± 4.56, and with stereo microscope it is 54.11 ± 15.42 [Table 3] and [Graph 2].
Table 3: Chronological age and estimated age using different microscopes for different age groups

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The mean and SDs of chronological age of Group III (41–50 years) is 46.06 ± 2.94, and the mean and SD values of the estimated using different microscopes are as follows, with light microscope is 46.19 ± 4.67, with phase contrast is 46.86 ± 8.25, polarized microscope is 46.58 ± 3.42, and with stereo microscope it is 60.28 ± 27.39. The mean and SDs of chronological age of Group IV (51–60 years) is 55.74 ± 2.68, and the mean and SD values of the estimated using different microscopes are as follows: with light microscope is 53.91 ± 9.10, with phase contrast is 54.43 ± 8.20, polarized microscope is about 55.63 ± 5.46, and with stereo microscope, it was about 58.54 ± 20.26. The mean and SDs of Chronological age of Group V (61–70 years) is 66.06 ± 4.12 and the mean and SD values of the estimated using different microscopes are as follows, with light microscope is 55.44 ± 11.8, with phase contrast is 56.11 ± 11.2, polarized microscope is 57.00 ± 13.7 and with stereo microscope it is 51.44 ± 11.64.

The relationship between chronological age and estimated age using light microscope is as follows, Group II (31–40 years) showed stronger “r” (Pearson's correlation) value of 0.875, with significant P = 0.002 with variance (R2) of 76% and SEE of 1.77, followed by Group I (21–30 years) and Group III (41–50 years). Least Pearson's correlation, R2 value was shown among Group IV (51–60 years) and Group V (61–70 years) with nonsignificant P value [Table 4] and [Graph 3].
Table 4: Correlation between chronological age and estimated age using light microscope

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The relationship between chronological age and estimated age using phase contrast microscope is as follows: Group I (21–30 years) showed stronger “r” (Pearson's correlation) value of 0.866, with statistically significant P = 0.001 with variance (R2) of 75% and SEE of 1.77, followed by Group IV (51–60 years) and Group III (41–50 years). Least Pearson's correlation, R2 value, and non-significant “P” value were shown among Group IV (51–60 years) [Table 5] and [Graph 4] and Group V (61–70 years) with a non-significant P value.
Table 5: Correlation between chronological age and estimated age using phase-contrast microscope

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The relationship between chronological age and estimated age using polarized microscope is as follows, Group I (21–30 years) showed stronger “r” (Pearson's correlation) value of 0.877, with significant P = 0.000 with Variance (R2) of 76% and SEE of 1.32, followed by Group IV (51–60 years) and Group III (41–50 years). Least Pearson's correlation, R2 value, and nonsignificant “P” value were shown among Group IV (51–60 years) and Group V (61–70 years) with nonsignificant P value [Table 6] and [Graph 5].
Table 6: Correlation between chronological age and estimated age using polarizing microscope

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The relationship between Chronological age and estimated age using stereomicroscope which showed a weaker correlation with lesser R2 value and “P” values which are not significant among all age groups [Table 7].
Table 7: Correlation between chronological age and estimated age using stereomicroscope

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Correlation between chronological age and number of cemental annulations for males and females

As shown in [Table 8] and [Table 9], the correlation between the chronological age and the number of cemental annulations using different microscopes for males and females. Among males, polarized microscope showed a higher r value (Pearson's correlation) and R2 = 0.829 and 68%, followed by light microscope with r and R2 = 0.672 and 45%. Among females, the polarized microscope showed a higher r value (Pearson's correlation) and R2 = 0.871 and 75%, followed by light microscope with r and R2 = 0.819 and 67%. The correlation is statistically significant (<0.01) for both males and females.
Table 8: Correlation between chronological age and number of cemental annulations using different microscopes in males

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Table 9: Correlation between chronological age and number of cemental annulations using different microscopes in females

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  Discussion Top


In the present study, we had taken single-rooted teeth for our analysis. Jankauskas et al.[8] and Kaur et al.[9] typically used incisors, canines, premolars, and molars to count cementum layers. Dias et al.,[1] Avadhani et al.[10] and Aggarwal et al.[4] also studied all the maxillary and mandibular teeth. Condon et al.[11] and Renz et al.[12] in their studies indicated that premolars are a more reliable age indicator. Lipsinic et al.[13] chose to use maxillary bicuspids; others such as Solheim stated that mandibular second bicuspids and central incisors have the best correlation for annulations count. Radovic[6] in his study examined samples consisting of only permanent premolars from the upper and lower jaws.

There has been no consensus in the sectioning method used. Many authors prefer sections to be longitudinal, whereas others prefer cross-sections. Both methods are seen to have their own advantages and limitations. In the present study, 100 longitudinal ground sections were prepared for age estimation, which gave an opportunity to count lines in both the cellular and acellular cementum on the same tooth. Aggarwal et al.[4] and Joshi et al.[14] in their studies also used longitudinal ground sections similar to our study. Avadhani et al.[10] studied 25 teeth, half of which were sectioned longitudinally and the other half group were cross-sectioned.

In the present study, we used the middle third region of the root for counting cementum annulations. In the mid-root region of a tooth, the cementum present is usually acellular, undisturbed and even in growth such that annulations can be counted easily without any hindrance. Lieberman and Meadow[15] in their study stated that annulations of acellular cementum are more easily microscopically resolved because in this region cementum is less compressed than the cementum near the cementoenamel junction and contains lesser cellular cementum than the root apex. According to Huffman and Antoine,[16] the apical region of root proved to be the best area to observe and count the cementum layers. The more rapidly growing cellular cementum found at the apex of root showed the clearest layers, whereas the slower and thinner acellular cementum layers found in the middle and cervical regions were difficult to observe.

In our study, the extracted teeth were examined under four microscopes (light, phase contrast, polarized, and stereomicroscope) and it showed that the Polarized microscope is better among all microscopes to assess the cemental annulations. This is contrary to the study conducted by Kaur et al.[9] in which age estimation was done using light, polarized, and phase contrast microscopy. It was seen that cementum annulations were more clearly visible under the phase contrast microscope as compared to the light microscope and polarizing microscope. The polarized microscope was preferred over the light microscope as it showed better discernability of the annulations. Aggarwal et al.[4] also found similar results in their study. Joshi et al.[14] and Pundir et al.[17] in their study concluded that among the methods of counting incremental lines by various types of microscopy, phase contrast microscopy was more reliable for age estimation than polarizing microscope.

The relationship between chronological age and estimated age using polarized microscope, light microscope, and phase contrast microscope showed stronger “r” (Pearson's correlation) value of 0.877, 0.875, and 0.866, respectively. Stereo microscope showed a weaker correlation. In their study, Kaur et al.,[9] the correlation of ages estimated by light, polarized, and phase contrast microscopy with the actual age of subjects gave correlation values (r) as 0.347, 0.557 and 0.989 respectively. Kasetty et al.[18] and Lipsinic et al.[13] in their studies observed a positive correlation between estimated age and known age as r = 0.42 and 0.51, respectively. However, in a study by Stein et al.,[19] a much higher correlation between predicted age and known age was seen (r = 0.93). Wittwer-Backofen et al.,[20] Solheim[21] and Kvaal and Solheim[7] derived correlation of cementum annulations and age as r = 0.98, 0.63 and 0.84 in their respective studies.

However, there were limitations of the present study, which necessitates further studies considering these limitations. The method of counting cementum annulations is subjective which makes it prone to interobserver error; clinical data for knowing the actual age were collected from patients which may not be reliable; age of eruption of a tooth may show minor variations genetically from individual to individual; the field of focus for a specimen may vary when different microscopes are used which may induce errors in the results.


  Conclusion Top


TCA method for age estimation is a valuable aid for forensic identification. The use of polarized microscope increases the accuracy and thus, can predict the near estimate of the age for all the age groups followed by light microscope, with a reliability of 74%. However, considering the limitations, further research aiming at involving larger sample, including not only age and gender but also other parameters such as race, culture, the effect of parafunctional habits, diet, periodontal regression, enamel hypoplasia, should be carried out to validate the results of the present study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Dias PE, Beaini TL, Melani RF. Age estimation from dental cementum incremental lines and periodontal disease. J Forensic Odontostomatol 2010;28:13-21.  Back to cited text no. 1
    
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Lipsinic FE, Paunovich E, Houston GD, Robison SF. Correlation of age and incremental lines in the cementum of human teeth. J Forensic Sci 1986;31:982-9.  Back to cited text no. 13
    
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Joshi PS, Chougule MS, Agrawal GP. Comparison of polarizing and phase contrast microscopy for estimation of age based on cemental annulations. Indian J Forensic Odontol 2010;3:87-95.  Back to cited text no. 14
    
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Lieberman DE, Meadow RH. The biology of cementum increments (with an archaeological application). Mammal Rev 1992;22:57-77.  Back to cited text no. 15
    
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Huffman M, Antoine D. Analysis of cementum layers in archaeological material. Dent Anthropol 2010;23:67-73.  Back to cited text no. 16
    
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Pundir S, Saxena S, Aggrawal P. Estimation of age based on tooth cementum annulations using three different microscopic methods. J Forensic Dent Sci 2009;1:82-7.  Back to cited text no. 17
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Stein TJ, Corcoran JF. Pararadicular cementum deposition as a criterion for age estimation in human beings. Oral Surg Oral Med Oral Pathol 1994;77:266-70.  Back to cited text no. 19
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

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



 

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