Year : 2008 | Volume
: 19 | Issue : 4 | Page : 326--330
Incremental lines in root cementum of human teeth: An approach to their role in age estimation using polarizing microscopy
Pooja Aggarwal, Susmita Saxena, Puja Bansal
Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut, UP, India
Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut, UP
Age estimation is an important factor in the identification of an individual in forensic science. The hard tissues of the human dentition are able to resist decay and degradation long after other tissues are lost. This resistance has made teeth useful indicators for age calculation. Recent research indicates that tooth cementum annulations (TCA) may be used more reliably than any other morphological or histological traits of the adult skeleton for age estimation. The purpose of this study was to examine the correlation between age and the number of incremental lines in human dental cementum and to ascertain the best method of studying cementum with respect to different forms of microscopy. Thirty nonrestorable teeth were extracted from 20 people, and longitudinal ground section of each tooth was prepared. Photomicrographs of the area at the junction of apical and middle third of the root under light and polarized microscope were taken. The cementum was composed of multiple light and dark bands that were counted on the photomicrograph with the help of image analysis software and added to the average eruption time of individual tooth. The predicted age of the individual was thus obtained. Results showed a significant correlation between the predicted age and actual chronological age of the individual. These data indicate that quantitation of cementum annuli is a moderately reliable means for age estimation in humans and polarizing microscopy is a better means of visualizing incremental lines of cementum compared to light microscopy.
|How to cite this article:|
Aggarwal P, Saxena S, Bansal P. Incremental lines in root cementum of human teeth: An approach to their role in age estimation using polarizing microscopy.Indian J Dent Res 2008;19:326-330
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Aggarwal P, Saxena S, Bansal P. Incremental lines in root cementum of human teeth: An approach to their role in age estimation using polarizing microscopy. Indian J Dent Res [serial online] 2008 [cited 2023 Feb 8 ];19:326-330
Available from: https://www.ijdr.in/text.asp?2008/19/4/326/44536
Age determination plays an important role in forensic medicine, not only in identification of bodies but also in connection with crimes. When the subjects have undergone changes so extensive that external characteristics yield no information, the teeth are often the only means of identification. 
The hard tissues of the human dentition are able to resist decay and degradation long after other tissues are lost. This resistance has made teeth useful indicators for assessing variation in diet, expression of metabolic diseases, and calculation of age at the time of death. 
Gradual structural changes in teeth throughout the life are the basis for age estimation. The enamel, dentin, and cementum that comprise teeth have been used to estimate the chronologic age of unidentified individuals. Because of its position, cementum has not been used to the extent of enamel and dentin. However, the counting of cemental annulations may offer a more accurate method for age estimation in human beings. 
Zander and Hürzeler  stated that cementum is potentially a better age-estimating tissue due to its unique location in the alveolar process. It has been hypothesized that these incremental lines in the tooth cementum can be used as a more reliable age marker than any other morphological or histological traits in the human skeleton. This hypothesis is based on the biological factors of the tooth cementum annulations (TCA) formation known so far.
The purpose of this study was to examine the correlation between age and the number of incremental lines in human dental cementum and to ascertain the best method of studying cementum with respect to different forms of microscopy.
Materials and Methods
This study was carried out in the Department of Oral Pathology and Microbiology, Subharti Dental College, Meerut.
The study sample consisted of 30 teeth. They included seven maxillary canines, six mandibular canines, four mandibular central incisors, two maxillary lateral incisors, six mandibular premolars, and five mandibular molars.
The age of the individuals (when the tooth was extracted) ranged from 13-69 years. Teeth were extracted because of periodontal disease, caries, orthodontic, and prosthetic reasons. Teeth with periapical pathologies were not included in the study. The presence of periodontal disease, however, was not the criteria for exclusion because we were interested to see the possible impact of periodontal pathologies on TCA.
Among the 30 teeth in the sample, 19 originated from multiple extractions in seven individuals. These multiple extractions provided the basis for establishing intraindividual correlations in TCA.
Longitudinal ground section of each tooth was prepared and examined under light microscope and polarized microscope [Figure 1] and [Figure 2].
The measurements and counting were made without the age being known by two observers, to check for interobserver variation.
In each section, the area at the junction of apical and middle third of root [Figure 3] and the area where the lines were easiest to count, disregarding whether the cementum was cellular or acellular, were selected for counting. These areas were photographed and images were transmitted from the microscope to a computer monitor, and counting was done with the help of image analysis software [Figure 4].
First, the width of the cementum from the dentin-cemental junction to the surface of the cementum was measured in an area where the lines seemed to run approximately parallel. Then the measurement was made of the width occupied by the two adjacent incremental lines [Figure 5], which were most easily recognizable, and the number of incremental lines in the total cementum width was calculated.
Number of incremental lines (n) = X/ Y
Where, X is the total width of cementum (from DEJ to cementum surface) and Y the width of cementum between the two incremental lines.
By adding average age of eruption in years for each tooth as presented in Gray's Anatomy  in the counted number of incremental lines, the chronological age of the individual was obtained.
E = n + t
Where,Estimated age = number of incremental lines (n) + eruption age of tooth (t)
Calculated and the actual chronological ages of individuals are shown in [Table 1], which represents the data of 30 specimens for ages between13-69 years. This indicates a fairly strong positive correlation between the two variables of estimated age and actual age.
Cementum is the calcified tissue that surrounds the root portion of dentin and forms the attachment site for the periodontal fibers that links the tooth to the alveolar bone. In cementum formation, hypermineralized layers of extracellular matrix alternate with less-mineralized layers. The first layer of acellular cementum is produced before the tooth erupts, and further layers are added during and after eruption. 
A biological explanation for these alternating layers was given by Lieberman  and Schroder.  They suggested that the dark lines are stop phases of mineralization during continual growth of the fibroblasts, leading to a change in mineral crystal orientation. This pattern is visible under the microscope as a series of alternating light and dark lines or bands. The dark lines have been referred to as incremental lines and the cementum between each two lines as incremental bands.
In our study, we found that these lines were more clearly visible under polarized microscope than light microscope [Table 2]. Under light microscope, cemental annulations could not be found in each examined tooth and their visibility differed across specimens.
Along the axis of the tooth root there are two zones of different cementum types: the acellular cementum that grows close to the cervical part of the root, and the cellular cementum that mainly covers the apical part of the root. In the present study, we focused on the acellular cementum, predominantly seen in the middle third of the root. 
It was shown that each pair of lines corresponds to one year of life and constitutes a biological record that can be used to estimate the age of the individual. ,
Variations in cementogenesis that change the appearance of lines may be induced by different factors, including biomechanical forces, nutrition, hormonal cycle, or ecological conditions such as temperature, ultraviolet light, humidity, altitude, or pollution. 
The appearance of cementum lines, observed in more than 50 different mammalian species all over the world, has been said to reflect the natural metabolic rhythm of seasonal changes. Seasonal rhythms in cementum annulations, as observed in the alternating dark and light bands, can be explained by the metabolism of the parathyroid hormone, which in connection with vitamin D regulates the resorption of calcium. 
Thus, both hormones and vitamins may interact to produce a circannual rhythm by a complex mechanism of environmental and physiochemical synchronizers. Many questions remain regarding the mechanisms of TCA and its influencing factors, particularly concerning the interpretation of seasonal increments. ,,
The first use of cementum in human age estimation began with measurements of width of the total cementum layer, rather than the number of incremental lines. 
Stott et al.,  in 1982 first used TCA, as an age estimation method in humans. Further technical improvements  led to the suggestion that the TCA is superior to other tooth-based methods of age estimation in the adult skeleton.
Initially, the TCA method was applied to freshly extracted teeth, but Grosskopf  showed that the method was also applicable to historical skeletons and cremations. This was confirmed by others and extended to forensic cases. These findings add further support to the idea that the number of incremental lines is a stable property, even under circumstances where other characteristics of the lines (e.g., width and degree of mineralization) have been altered by environmental or physiological perturbations. It was on the basis of these kinds of results that the TCA method was recently recommended as a reliable technique for age estimation in adults using skeletal materials. 
However, some problems remain regarding the full application of the TCA method. For example, the small samples used in previous studies limited the establishment of statistical parameters needed for practical paleodemographic and forensic applications and the question of whether dental disease, particularly periodontal disease, has an impact on TCA. 
One of the more contentious discussion associated with TCA is related to the effect of periodontal disease. As the cementum annulations are interrelated with the Sharpey fibers, it is plausible that with increasing periodontal decline, the Sharpey fibers may lose their functional significance and decay owing to the reducing alveolar bone. An arrest of the cementum annulation process might occur. In our sample, however, we did not observe any correlation between the deviation of TCA age from true age and degree of periodontal decline. From our study, we conclude that the accuracy of the TCA age estimation is independent of periodontal disease, a finding that supports the results of Grosskopf et al.,  rather than those of Kragerer and Grup. 
To analyze reliability by tooth type, we had to split our sample into small subgroups. However, as no sex differences in TCA estimation error were observed, different tooth types were not analyzed separately based on the sex of the individual. The results of our study do not suggest the selection of a specific tooth type as a favorable tooth type for age estimation by cementum annulations. All teeth in our sample from the maxilla as well as from the mandible provided results of comparable reliability.
The sectioning method to be used has also not been a topic of agreement. Many authors prefer the sections to be longitudinal, whereas others prefer cross sections. Both methods seem to have advantages and limitations. Although the longitudinal sections allow viewing the whole root surface such as advocated by Klevezal and Kleinenberg,  others such as Stott et al.,  prefer a cross section that allows a series of observations. The study of cemental incremental lines in longitudinal sections as in the present study gave the opportunity to count the lines in both the cellular and acellular cementum on the same tooth.
Annulation counts in younger age group (et al.,  study and the present study may be due to a decreased apposition of cementum in individuals over 60 years. Solheim  showed that cementum apposition diminishes by one-thirds after the age of 60 years.
Our results suggest that there is no significant influence of sex, age, periodontal disease, or tooth type on the estimation quality of the TCA method. This suggests that the accuracy and repeatability of the method is not dependent on tooth type or location, and this method can be applied to general populations regardless of systemic or periodontal health.
For a successful application in historical populations, the influence of living conditions that severely affect the calcium metabolism in the human body still has to be tested, including severe malnutrition or specific diseases. So, further studies are needed to show whether the same accuracy can be achieved in a historical known age collection exposed to different environmental conditions than in a modern population.
Finally, we can conclude from the current study that the application of TCA age estimation improves individual age estimation and it may serve as a valuable aid for forensic identification.
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