ORIGINAL RESEARCH 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 Received : 23-01-08 Review completed : 19-05-08 Accepted : 22-05-08 PubMed ID : 19075436 ABSTRACT 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 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. Key words: Age estimation, cementum, incremental lines Age determination plays an important role in forensic medicine, not only in identiþcation 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 identiþcation. [1] 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. [2] 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 unidentiþed individuals. Because of its position, cementum has not been used to the extent of enamel and dentin. However, the counting of cemental Correspondence: Dr. Pooja Aggarwal, E-mail: drpooja_path@yahoo.co.in annulations may offer a more accurate method for age estimation in human beings. [3] Zander and Hürzeler [4] 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. 326 326 CMYK
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 Þve 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 and polarized [Figures 1 and 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 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 [5] 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) RESULTS 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. DISCUSSION Cementum is the calciþed tissue that surrounds the root portion of dentin and forms the attachment site for the periodontal Þbers that links the tooth to the alveolar bone. In cementum formation, hypermineralized layers of extracellular matrix alternate with less-mineralized layers. The Þrst layer of acellular cementum is produced before the tooth erupts, and further layers are added during and after eruption. [6] A biological explanation for these alternating layers was given by Lieberman [7] and Schroder. [8] They suggested that the dark lines are stop phases of mineralization during continual growth of the Þbroblasts, leading to a change in mineral crystal orientation. This pattern is visible under the 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 than light [Table 2]. Under light, 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. [5] It was shown that each pair of lines corresponds to one year of life and constitutes a biological record that can be used [7, 9] 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. [6] The appearance of cementum lines, observed in more than 50 different mammalian species all over the world, has been said to reßect the natural metabolic rhythm of seasonal changes. Seasonal rhythms in cementum annulations, as observed in 327 CMYK 327
Figure 1: The cementum incremental lines under light Figure 2: The cementum incremental lines under polarized Figure 3: The area selected for counting (middle third of root cementum) Figure 4: Research with photomicrographic attachment and computer setup with image analysis software with vitamin D regulates the resorption of calcium. [6] 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 inßuencing factors, particularly concerning the interpretation of seasonal increments. [7,10,11] The Þrst use of cementum in human age estimation began with measurements of width of the total cementum layer, rather than the number of incremental lines. [12] Figure 5: Measured total cementum width (L1) and one incremental band width (L2) using image analysis software the alternating dark and light bands, can be explained by the metabolism of the parathyroid hormone, which in connection Stott et al., [10] in 1982 Þrst used TCA, as an age estimation method in humans. Further technical improvements [13] 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 [14] showed that the method was also 328 328 CMYK
applicable to historical skeletons and cremations. This was conþrmed by others and extended to forensic cases. These Þndings 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. [6] 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. [6] 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 Þbers, it is plausible that with increasing periodontal decline, the Sharpey Þbers may lose their functional signiþcance 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 Þnding that supports the results of Grosskopf et al., [14] rather than those of Kragerer and Grup. [9] 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 speciþc 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, [15] others such as Stott et al., [11] 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. Table 1: Counts of cemental annulations with eruption time estimates compared with actual age of individuals (in years) Specimen Tooth number No. of annulations Eruption range (years) Estimated age (years) Actual age (years) Mean age number error (years) 1 45 31 11 42 40 2 2 21 19 7 26 28 2 3 41* 19 7 26 27 1 4 25* 40 10 50 65 10** 5 45 20 11 31 29 2 6 42* 36 7 43 45 2 7 22 25 8 33 30 3 8 23 42 11 53 56 3 9 15 45 10 55 58 3 10 44 43 9 52 55 3 11 45 42 10 52 54 2 12 24 32 9 41 43 2 13 34 45 10 55 67 12** 14 33 46 11 57 60 3 15 44* 50 9 59 60 1 16 11* 53 7 60 69 9** 17 14 32 9 41 43 2 18 24 18 10 28 30 2 19 15 48 10 58 59 1 20 24 25 9 34 36 2 21 33 40 11 51 52 1 22 35* 41 10 51 52 1 23 24 45 9 54 56 2 24 11* 41 7 48 50 2 25 32* 49 8 57 59 2 26 24 32 10 42 42 0 27 15 30 10 40 42 2 28 13 42 11 53 55 2 29 21 50 7 57 58 2 30 31* 48 7 55 57 2 **Mean age error (> ±2) (sample of actual age > 60 years), *Teeth with periodontal disease 329 CMYK 329
Table 2: Number of annulation counted by light and polarized Specimen Tooth No. of annulations No. of annulations number counted by light counted by polarized 1 45 28 31 2 21 19 19 3 41 17 19 4 25 32 40 5 45 17 20 6 42 29 36 7 22 25 25 8 23 40 42 9 15 14 45 10 44 35 43 11 45 35 42 12 24 27 32 13 34 32 45 14 33 41 46 15 44 43 50 16 11 51 53 17 14 20 32 18 24 14 18 19 15 40 48 20 24 19 25 21 33 39 40 22 35 40 41 23 24 45 45 24 11 41 41 25 32 47 49 26 24 26 32 27 15 24 30 28 13 40 42 29 21 48 50 30 31 39 48 Annulation counts in younger age group (<55 years) appear to be very close to the actual age. Whereas, annulation for individuals above 55 years of age showed an increased tendency to be unrepresentative. Inaccurate age estimation was obtained in three samples, all of which had actual age more than 60 years. The lower correlation in older persons in the Lipsinic et al., [16] study and the present study may be due to a decreased apposition of cementum in individuals over 60 years. Solheim [17] showed that cementum apposition diminishes by one-thirds after the age of 60 years. CONCLUSION Our results suggest that there is no signiþcant inßuence 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 speciþc 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 identiþcation. REFERENCES 1. Stein TJ, Corcoran JF, Arbor A, Mich. Pararadicular cementum deposition as a criterion for age estimation in human beings. Oral Surg Oral Med Oral Pathol 1994;77:266-70. 2. Stein TJ. Anatomy of root apex and histological changes with age. Oral Surg Oral Med Oral Pathol 1990;69:266-70. 3. Tencate A. The estimation of age of skeletal remains from color of roots of teeth. J Can Dent Assoc 1977;2:83-6. 4. Zander HA, Hürzeler B. Continuous cementum apposition. J Dent Res 1958;37:1035-44. th 5. Gray s anatomy. In: Warwick R, Williams PL, editors. 35 ed. W B Saunders Co; 1973. p. 1184-5. 6. Backofen UW, Gampe J, Vaupel JW. Tooth cementum annulation for age estimation: Results from a large known-age validation study. Am J Phys Anthropol 2004;123:119-29. 7. Lieberman DE. The biological basis for seasonal increments in dental cementum and their applications to archaeological research. J Archaeol Sci 1994;21:525-39. 8. Schröder HE. Orale strukturbildung: Etwicklungsge-schichte, struktur und function normaler hartgewebe und weichgewebe der mundhöhle und des kiefergelenkes. Berlin: Thieme Verlag; 2000. 9. Kagerer P, Grupe G. Age at deathdiagnosis and determination of life history parameters by incremental lines in human dental cementum as an identification aid. Forensic Sci Int 2001;118:75-82. 10. Stott GG, Sis RF, Levy BM. Cementum annulation as an age criterion in forensic dentistry. J Dent Res 1982;61:814-7. 11. Cipriano A. Cold stress in captive great apes recorded in incremental lines of dental cementum. Folia Primatol (basel) 2001;73:21-31. 12. Gustafson G. Age determinations on teeth. J Am Dent Assoc 1950;41: 45-54. 13. Naylor JW, Miller WG, Stokes GN, Stott GG. Cemental annulation enhancement: A technique for age determination in man. Am J Phys Anthropol 1985;68:197-200. 14. Grosskopf B. Incremental lines in prehistoric cremated teeth. Z Morpho Anthro 1989;77:309-11. 15. Klevezal GA, Kleinenberg SE. Age determination of mammals from annual layers in teeth and bones. Springfield, Virginia: US Department of Commerce; 1967. 16. Lipsinic FE. Paunovich E, Houston GD, Robison SF. Corrlation of age and incremental lines in the cementum of human teeth. J Forensic Sci 1986;31:982-9. 17. Solheim T. Dental cementum apposition as an indicator of age. Scand J Dent Res 1990;98:510-9. 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: 326-30. Source of Support: Nil, Conflict of Interest: None declared. 330 330 CMYK