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International Journal Of Scientific Research And Education ||Volume||2||Issue|| 3 ||Pages|354-367||2014|| ISSN (e): 2321-7545 Website: http://ijsae.in “Teeth” –The focus of research in forensic age diagnostics Dr. Nagalaxmi V1, Dr. Naga Jyothi M2, Dr. Kotya Naik M,3 Dr.Srikanth kodangal4 1 Professor & Head, Sri Sai College Of Dental Surgery, Vikarabad, Andhra Pradesh, India. Post Graduate Student, Sri Sai College Of Dental Surgery, Vikarabad, Andhra Pradesh, India. 3 Post Graduate Student, Sri Sai College Of Dental Surgery, Vikarabad, Andhra Pradesh, India. 4 Senior lecturer, Sri Sai College of Dental Surgery, Vikarabad, Andhra Pradesh, India. 2 Email: [email protected] Abstract: Forensic dentistry is the specialized branch of forensic medicine uses dental evidence for criminal, civil, legal proceedings, establishing the identity of unknown and missing individuals along with enormous applications. In forensic dentistry various craniofacial structures are subjected to investigation to arrive at conclusive data pertaining to age estimation. Age estimation using the dental evidence is an important area of research in this field, where teeth serve as valuable tool in this specialized era of forensic age diagnostics as they are least affected and destructed by the post-mortem changes and it also extends its applications to other allied areas of forensic specialty like Anthropology, Palaeodontology, and Palaeoanthropology. This review assembles various methods in forensic dental age estimation under one roof. Keywords: Age estimation, Forensic odontology, Teeth, Bureau of Legal Dentistry. 1.Introduction: Forensic dentistry is a specialized branch of forensic medicine and may be described as that part of odontology which, in the interests of justice, deals with the handling and examination of dental evidence, from which a proper evaluation and presentation of dental findings can be made. (Cameron and Sims, 1974 and Keiser-Nielson, 1980). Forensic odontology (forensic dentistry) involves the correct collection, management, interpretation, evaluation and presentation of dental evidence for criminal or civil legal proceedings: a combination of various aspects of the dental, scientific and legal professions. The bones and teeth of the craniofacial region constitute an invaluable aid in the identification of an unknown individual thus considered as the essential Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 354 parameters by the forensic Odontologists. Forensic dentistry was relatively in a state of infancy in its research and advancements until the 1960s when the period of renaissance awoked by the first formal constitutional program in forensic dentistry held in the United States at the Armed Forces Institute of Pathology. Since then the field of “forensic odontology” has ramified its branches to such an extent that the teeth and its supporting bone alone were able to render a plenty of reliable information not only to the dental profession, but also to law enforcement agencies and other forensic groups [1]. Saunders, a dentist, was the first to publish information regarding dental implications in age assessment by presenting a pamphlet entitled ‘‘Teeth A Test of Age’’ to the English parliament in 1837. While quoting the results from his study on 1000 children, he pointed out the value of dentition in age estimation [2]. Dentistry has much to offer law enforcement in the detection and solution of crime or in civil proceedings. Forensic identifications by their nature are multidisciplinary team efforts of law enforcement officials, forensic pathologists, forensic odontologists, forensic anthropologists, serologists, criminalists involving a combination of positive identification methodologies as well as presumptive or exclusionary methodologies. Forensic dental fieldwork requires an interdisciplinary knowledge of dental science. Most often the role of the forensic odontologist is to establish a person’s identity. Teeth, with their physiologic variations, pathoses and the ability to withstand extreme conditions of post mortem changes comprising of decomposition, carbonization and fragmentation, are capable of producing information that remains throughout life and beyond. Forensic odontology has its applications in (1) diagnostic and therapeutic examination and evaluation of injuries to jaws, teeth, and oral soft tissues, (2) the identification of individuals, especially casualties in criminal investigations and / or mass disasters, and (3) identification, examination, and evaluation of bite marks in sexual assaults, child abuse cases, and in personal defense situations. The odontological identification and examination of a decedent is based on a systematic comparison of the pre- and postmortem dental characteristics of the individual based on the dental record and supporting radiographs (apical films, pantomographs and medical skull films). A variety of techniques can be used to narrow the search and are based on presumptive identification data which are often sorted with computer assistance. The challenge for the forensic dentist is to acquire the pertinent information, organize and summarize it into a conclusive data leading to an accurate diagnosis for the identification of the deceased. Forensic Odontologists are confronted with the problem of determining the age of unknown bodies and also living persons. Attempts to use teeth as an indicator of age estimation originate from England. Despite the alleged use of the eruption of second molars by the ancient Romans to evaluate readiness for military service (Muller,1990), age estimation (sometimes known as age evaluation, age determination, age diagnostics or age assessment) in living individuals is a relatively recent area of applied research within the forensic sciences. Its value and importance as an assessment tool has risen exponentially as the needs for an informed Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 355 opinion on the age of an individual have assumed increasing importance for the assessment of both criminal culpability and legal/social categorization. There are many areas in which the evaluation of age in the living has become relevant but the most prevalent concern issues pertaining to refugee and asylum seekers, criminals and their victims, human trafficking and child pornography. Age estimation is a sub-discipline of forensic sciences and is an important part of identification process when information regarding deceased is unavailable. 2.Methods of age estimation: Forensic biological age estimation using dental traits utilizes the following methods; BIOLOGICAL AGE ESTIMATION Morphological Genetic Histological Radiological Biochemical 2(A). Morphological Method: It is worth to speculate the different characteristics of dentition that are taken in consideration for the quantitative and qualitative biologic age estimation by means of either histological or biochemical or genetic methods [3]. Appearance of tooth buds Initial finding of calcification Alterations in chemical composition and discoloration of teeth Degree of completion of unerupted tooth Gingival recession & root resorption Deposition of secondary dentin & root dentin transparency Dental parameters Formation of enamel & neonatal line Clinical eruption Degree of completion of roots of erupted teeth & resorption of deciduous teeth Cemental apposition Attrition of the clinical crown Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 356 The chronological age of an individual gives the estimate of actual age but this article replaces the term with biological age as it deals with the age estimation methods of deceased and unidentified in the context of forensic dentistry. The biological age determination is done at three stages of development; 1) Age estimation in prenatal, neonatal & early postnatal period. 2) Children & adolescents 3) Adults Age estimation in children includes the sequential phases of growth and developmental changes in primary and permanent dentition sometimes influenced by the geographic distribution, environmental influences, hormonal, and nutritional factors where as in adults in it includes the regressive alterations of morphology and histological composition of teeth. Age estimation in children employs atlas and scoring system utilizing the radiographic method of assessment. Various stages of tooth mineralization are studied radiographically and correlated with the standard charts where the approximate age is determined. The morphological and radiological methods of age estimation go hand-in-hand in the forensic dental age estimation, probably radiological method can be considered as advanced front of the morphological method. Henceforth, this is the reason this article reviews both the methods simultaneously for comprehensive understanding of the elaborative methods. The point scoring method of age estimation having the scores being calculated statistically to derive a regression formula for the subsequent forensic research studies also plays a vital role these days for the accurate age estimation. The atlas method proposed by Schour and Massler [4] shows 20 chronological stages of tooth development spanning from 4th month of post natal life to 21 years of adulthood depicting the appearance of tooth germ, early spurts of tooth mineralization, eruption sequence of deciduous and permanent dentition [5]. These charts are periodically revised and serve the useful estimate of the individual’s age. The drawback of this method is it lacks specificity as it is not designed separate for males and females [6]. Moores, Fanning, Hunt studied the developmental and mineralization stages of teeth in 14 steps including the third molar Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 357 development and the mean age estimate for each stage of the permanent teeth is charted out separately for both the sexes and specifically for each tooth[7]. They used panoramic radiographs in estimating the stages. Delmirjian, Goldstein, Tanner [8] method enumerates the stages of teeth development in 8 phases scored in the order of A – H for the left mandibular teeth from incisors to second molars and allotted a particular maturity score statistically where the procedure involves the summing up of all the scores to derive total maturity score that is compared with the standard values in the tables and graphs plotted or substituting the standard regression formula for both boys and girls. A- Is the initiation of calcification taking the shape of inverted cones. B- Is the fusion of the mineralized cusps. C- Is the coronal half with the evidence of pulp chamber is formed. D- Is the crown formation is completed till the CEJ junction with the initial evidence of root formation. E- Is the initial evidence of root bifurcation in multi-rooted teeth. F- Is the open apical ends in the form of a funnel. G- Is the parallelism of the root canal walls with partially open apical end. H- Is the complete closure of the apical end of the root canal with uniform width of the periodontal membrane around the root and apex. Formula (males) - (0.000055 ×S3) - (0.0095 × S2) + (0.647 × S) – 8.4583. Formula (females) – (0.0000615 ×S3) - (0.0106 × S2) + (0.6997×S) – 9.3178. Third molar development is not considered which also shows variations in age estimation and the presence of all mandibular teeth is important [9]. Many studies used orthopantomograms as an aid in this method. Nolla gave ten stages of development for both the maxillary and mandibular teeth including third molar for both girls and boys as tables where the values are correlated to get actual age [10]. The earliest age estimation method for adults using retrogressive changes in the permanent dentition is the actual scientific method can be done with or without sectioning of teeth which holds its advantage and is the well known and familiar and widely accepted with reliable results comparatively. The following are the 6 regressive changes along with the established statistical regression formulae to compute the results. 1) Attrition of the occlusal surfaces (A). 2) Periodontal recession.(P) 3) Secondary dentin formation.(S) Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 358 4) Cemental apposition(C) 5) Root resorption(R) 6) Root dentin transparency.(T) Each stage is assigned a score point from 0 to 3 as A0-3, P0-3, S0-3, C0-3, R0-3, T0-3.This method can be determined both destructively by histological ground sections and also from radiographs. The Gustafson’s regression equation AGE = 11.43 + 4.56X (X is the total score) [11]. Error is ± 3.6years. Maples and Rice modified the Gustafson’s equation as AGE = 13.45 + 4.26X [12]. Johanson formula using the same above criteria is given as, AGE = 11.02 + (5.14×A) + (2.3×S) + (4.14×P) + (3.71×C) + (5.57×R) + (8.98×T) [13]. Dalitz’s regression equation using Gustafson’s criteria includes, AGE = 8.691 + 5.146A + 5.338P + 1.866S + 8.411T [14]. Root resorption and cemental apposition is not taken into the study and limits only to the anterior teeth but not the bicuspids and molars where they are the only teeth remaining in the deceased. Later Bang and Ramm still simplified the above 6 criteria and considered only single tooth criteria that is the length of the apical root dentin transparency in mm for a given tooth where they considered incisors and cuspids but not the bicuspids and molars as the latter couldn’t give accurate readings. The distal, mesial and palatal root surfaces are studied, the intact and sectioned teeth are taken into account. They proposed two separate equations along with the based on the apical translucent length less than or equal to 9mm and one with greater than 9mm.The accuracy of the readings increased when the above criteria are used. Length (X) ≤ 9mm the regression equation for age estimation is as follows, AGE = B0 + (B1 × X) + (B2 × X2). Length (X) ≥ 9mm the regression formula is AGE = B0 + (B1× X). B0, B1, B2 values are obtained from the tables given by Bang and Ramm [15]. All the above methods can be classified under combined destructive and non-destructive methods of age estimation as they employ both histological sections and radiological interpretations. 2(B).Radiological method: Various modalities of imaging ranging from conventional to advanced can be utilized in the age estimation with the association of good image analysis and interpretation for accurate measurements. Kvaal et al and Solheim [16] elaborated the radiological methods of age estimation using periapical radiographs and orthopantomograms both conventional and digital versions and also volume rendering CT and CBCT imaging. The basis for this is the gradual reduction in the pulp volume due to secondary dentin deposition with age and can be performed on both living and deceased individuals without the need for tooth preparations. Only six teeth are studied and the maxillary first premolars and molars are excluded. (11/21, 12/22, 15/25, 32/42, 33/43, 34/44) are used with the following parameters for Pulp-Tooth Ratios; Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 359 1) Pulp/Root length(P) 2) Pulp/Tooth length(R) 3) Tooth/Root length(T) 4) Pulp/Root width at CEJ (A) 5) Pulp/Root width at midpoint between A and C (B). 6) Pulp/Root width at mid-root length(C). Mean value of all ratios excluding T is designated as (M), Mean value of width ratios B and C as (W) and Mean value of length ratios P and R as (L). The regression formula as per this method is, AGE = 129.8 – (316.4 × M) - 6.8 × (W – L) [17]. The above method using periapical radiographs and orthopantomograms likely to have inherent image distortions giving fluctuations in the above values, allows only 2-dimensional method of studying toot-pulp ratios .Using computed tomography (CT) 3-D reconstruction images from 3-d remodeling software for all the 4 canines, the tooth – pulp volumes and tooth surfaces can be measured accurately with an average error of 8.3 to 12.86 years [18].This method again can be used in living and deceased as well which in turn can be used in forensic dentistry and anthropology. All the four canines must be present which a limitation is but canines are the last ones to get deteriorated in the process of environmental changes after death as per literature. The ratio of pulp volume (PV) and tooth volume (TV) is calculated for each canine say 13,23,33,43 and Spearman's rank correlation coefficient is used to evaluate the correlation between age and the PV/TV ratio×100 [19]. Right maxillary canine (13): (PV13/TV13) ×100 was coded as X13 Left maxillary canine (23): (PV23/TV23)×100 was coded as X23 Left mandibular canine (33): (PV33/TV33) ×100 was coded as X33 Right mandibular canine (43): (PV43/TV43) ×100 was coded as X43 Maxillary canines (13 and 23): [(PV13/TV13+PV23/TV23)/2] ×100 was coded as Xmax Mandibular canines (33 and 43) :[( PV33/TV33+PV43/TV43)/2] ×100 was coded as Xmand Four canines (13, 23, 33 and 43):[(PV13/TV13+PV23/TV23+ PV33/TV33+PV43/TV43)/4]×100 was coded as X4c. The regression equations for age estimation is as follows, 13 (right maxillary canine) AGE = 74.426– 41.853X13+ 7.767X213. 23 (left maxillary canine) AGE = 74.252– 41.146X23 + 7.351X223. 33 (left mandibular canine) AGE = 71.632- 45.82X33 +10.021X233. 43 (right mandibular canine) AGE = 70.933 – 43.843X43 + 9.122X243. Maxillary Canines AGE = 73.460– 40.051Xmax+ 7.099 X2max Mandibular Canines AGE = 71.211– 45.195Xmand+ 9.670X2mand. 4 Canines AGE = 73.193- 43.445X4c +8.528 X24c. Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 360 One of the radiological method using orthopantomograms (digital) more appropriately evaluates the open apices of the teeth particularly seven left mandibular teeth at various stages of open apices. The number of teeth with root development completed with apical ends completely closed was calculated (N0). For the teeth with incomplete root development, which is with open apices, the distance between inner sides of the open apex was measured (A). For the teeth with two roots, the sum of the distances between inner sides of two open apices was evaluated. To nullify the magnification, the measurement of open apex or apices (if multirooted) was divided by the tooth length (L) for each tooth and these normalized measurements of seven teeth were used forage estimation. The dental maturity was calculated as the sum of normalized open apices (S) and the numbers of teeth with root development complete (N0). The values are substituted in the following regression formula for age estimation [20]. AGE = 8.971 + 0.375g + 1.631×5 + 0. 674 N0 - 1.034s – 0.176s. N0. (Where g is a variable equal to 1 for boys, and 0 for girls). A new Indian formula from the Cameriere’s European formula [21] using the above criteria with slight modification to fit to Indian children in age estimation is as follows, AGE = 9.402 - 0.879 C + 0.663N0-0.711s-0.106s N0. (Where C is a dummy variable, 0 for north and central India, and 1 for south India). Other methods are based on measuring Coronal pulp cavity index radiographically and assessing third molar mineralization. 2(C).Histological method: Dental tissues show wide variations which are genetically determined and depict many changes with age which forms the basis in forensic age diagnostics. Enamel attrition shows different patterns and it is used as one of the scoring criteria in Gustafson’s method which can be done both histologically and radiologically. The regular time dependency of some dental features of the tooth crown, examined by histological techniques, allow the assessment of development to estimate chronological age. Age is estimated from enamel by counting the prism cross striations which are bundles of hydroxyapatite crystallites that later transform into mature enamel. These periodic structures, visible in transmitted light microscopy of ground sections or fractured enamel, are known as prism cross striations. Another dental feature visible in crown enamel is the incremental lines of Retzius which run from the dentinoenamel junction (DEJ) in occlusal direction which represents the zones of hypo- mineralization during amelogenesis. The neonatal line, which constitutes a borderline between pre and postnatal formed enamel (and dentin), is frequently used for age at death estimation. This feature, combined with the total number of cross-striations, gives an estimate in days for the age at death. Dentin, contrary to enamel, is continuously deposited which we call as secondary or reparative dentin as a normal physiological process with the increase in age. Secondary dentin formation is initiated subsequent to Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 361 dentinogenesis (Costa 1986). There is only little difference between primary and secondary dentin, which can sometimes be detected in stained preparations, via micro-radiography or in polarizing microscopy. This consequently results in decreased pulp volume which is again studied in combination with radiographs proposed by Kvaal et al. Dentin sclerosis (root transparency) is another feature of the pulpo-dentinal complex which was first described by Tomes in 1861, which shows variations with age. Root dentin sclerosis results in reduction of the diameter of dentinal tubules. The refraction index of the intratubular substance becomes the same as that of peritubular dentine. This process leads to a milk-glass like consistency of the dentin, which starts in the late adolescence at the apex of the tooth root and progresses towards the dentino - enamel junction (DEJ), where this histological variant is the part of the Gustafson’s criteria of age estimation. The most accurate histological method of age estimation is by counting the tooth cemental annulations from the histological ground sections under microscopy with stained or unstained sections. It is proved that the absolute count can be done with a mean error of ± 5 years using advanced microscopic techniques like polarizing microscopy, confocal laser scanning microscopy, scanning electron microscopy .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 Alternate light and dark pairs are counted as 1 pair of cemental line. The biological explanation for the alternating layers was given by Liberman and Schroeder [22] who suggested that dark lines are the stop phases of mineralization during the continuous growth of fibroblasts, leading to change in mineral crystal orientation. The biological age of each tooth is determined by the formula E = N + T. (where E is the estimated age, N is the number of incremental lines and T is the eruption age of each tooth) [23]. They are also useful in the fields of Archaeology, Anthropology, and Palaeodontology in determining the age at death estimation. 2(D).Biochemical method: The various changes in the chemical composition of the dental hard tissues are the basis of the biochemical method of age estimation. Aspartic acid racemization is considered to be one of the advanced, reliable, accurate and complex of the biochemical methods. Assessment of age using aspartic acid racemization methodology was first described in 1975 by Helfman and Bada [24]. During the course of aging, L-forms of amino acids are transformed by racemization to the D-forms. Thus, the extent of racemization of amino acids may be used to estimate the age of various tissues. Of all stable amino acids, aspartic acid has one of the fastest racemization rates and is therefore the amino acid most commonly used for age estimation. Rates of change of L-form amino acids to D-forms are influenced by environmental factors, such as temperature, humidity, pH, etc. Because of the continuous formation and removal or degradation of amino acids, tissues with low metabolic rates provide better age estimates than those with high metabolic rates. The only limitation here is the tooth requires sectioning mechanically. Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 362 Among the different types of teeth examined, the rate of aspartic acid racemization (k) is the highest in the second molar and decreases in the following order: first molar > second premolar > central incisor > first premolar > lateral incisor > canine[25].This method gives more accurate values when performed in dentin tissues compared to tooth enamel. All these age estimation methods use gas chromatography (GC). As an alternative to GC, very few estimations used high performance liquid chromatography (HPLC) coupled with ultraviolet detection [26,27] and with fluorescence detection[28].Quantification of Deoxypyridinoline crosslinks[29] and the evidence of presence of gelatinase A in human dentin SDS – PAGE electrophoresis and Zymography[30]. Radiocarbon analysis of tooth is relatively a new technique using Accelerator Mass spectroscopy where Radiocarbon dating of dental enamel with Carbon-14 tells us the date of birth of identified and unidentified individuals [31,32]. 2(E).Genetic methods: Since DNA can be recovered from tooth pulp as well as dentin for forensic analysis by RFLP (Restriction Fragment Length Polymorphisms) and PCR (Polymerase Chain Reactions), continuous research is ongoing in this front [33]. Telomere shortening based on dental pulp DNA is anew and useful approach to estimate age of the subject at the time of death [34]. Terminal Restriction Fragment (TRF) is taken as telomere length in actual age estimation. Mitochondrial DNA obtained by cryogenic grinding of tooth dentin is the source of genetic material for age estimation at death. The only disadvantage of this method is it may not be economically feasible and the material used from tooth is available for the process in trace amounts and also requires sectioning and chemical and mechanical manipulations of tooth material. 3.Conclusion: The advent of sophisticated and well equipped clinical and biochemical laboratories made many developments in the fields of medicine and dentistry. Forensic age estimation of unidentified human bodies and human remains for the purpose of identification has been a traditional feature of forensic discipline. They are useful in Anthropological, Palaeodontological, Palaeoanthropological and forensic investigation as biomarkers of aging as they are preserved for a long time against post-mortem changes. In rare cases, it may be necessary to determine the age of the living persons particularly in cases when individual is either unwilling or unable to reveal his identity. Though there are many method proposed by many researchers the accuracy relies on the combination methods of age estimation thus helps in avoiding inaccuracies in the estimation. It is advisable to have regression formulae for each individual tooth instead of having a single equation for all teeth. With the inception of Dental age calculating software it is possible to automate the dental age calculations allowing the repeatability and reproducibility ensuring the reliable results. This software programme enabled the forensic odontologists to feed the calculated dental parameters in the Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 363 programme to retrieve the correct data. Tremendous research and experiments are underway in the field of forensics to refine the conventional methods for easy and convenient applications in the coming future. Inherent to this is the establishment of BOLD (Bureau Of Legal Dentistry) which is a forensic odontology laboratory at the University of British Columbia, A centre of excellence aims to act as a resource for odontologists and other forensic scientists who deal with teeth ,bones, saliva, DNA, and dental records. Experts provide assistance with Investigations, Identifications, Analysis and Testimony. Conflicts Of Interest : NIL References 1. L.Luntz L, “History of forensic dentistry”, Dent Clin North Am, vol. 21, no. 1, pp. 7-17, 1977. 2. E.Saunders, “The Teeth A Test of Age’ considered with the reference to the factory children, addressed to the members of both Houses of Parliament. London: Renshaw”, 1837. 3. I A.Pretty, “The use of dental aging technique in Forensic Odontological practice”, J Foren Sci, vol. 48, pp.1127-1132, 2003. 4. I .Schour and M. Massler, “Development of human dentition”, J Am Dent Assoc, vol. 20, pp. 379-427, 1941. 5. L. Ciapparelli, “ The chronology of dental development and age assessment. In: Clark DH, (Eds). Practical forensic odontology”, Oxford: Wright Butterworth-Heinemann Ltd, pages:22–42, 1992. 6. F.R. Karjodkar , “ Role of dental radiology in forensic odontology”, Text book of dental and maxillofacial radiology, (2ndedt). New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, pages: 929–963, 2009. 7. C.F.A Moores, A.Fanning , E.E.Hunt, “Age variation of formation stages for ten permanent teeth”, J Dent Res, vol.42, pp. 1490-1502, 1963. 8. A. Delmirjian, H.Goldstein , J.M.Tanner, “ A new system of dental age assessment”, Hum Biol, vol.45, pp. 211-227, 1973. 9. A. Delmirjian and H.Goldstein, “ New systems for dental maturity based on seven and four teeth”, Ann Hum Biol, vol.3, pp. 411-421, 1976. 10. C.M.Nolla, “ The development of permanent teeth”, J Dent Child, vol.27, pp.254-266, 1960. 11. G.Gustafson, “ Age determination on teeth”, J Am Dent Assoc,vol.41, pp. 45-54, 1950. 12. 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T.Benesova , A.Honzatko , A.Pilin , J.Votruba , M.Flieger, “ A modified HPLC method for the determination of aspartic acid racemization in collagen from human dentin and its comparison with gas chromatography (GC)”, J Sep Sci, vol.27, pp.330-334, 2004. Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 365 29. S.Martin-de lasHeras , A.Valenzuela , E.Villanueva , “ Deoxypyridinoline crosslinks in human dentin and estimation of age”, Int J Legal Med, vol.112, pp. 222-6, 1999. 30. S.Martin-de lasHeras , A.Valenzuela , C.M.Overall , “ Gelatinase A in human dentin as a new biochemical marker for age estimation”, J Forensic Sci, vol.45, no.4, pp.807-811, 2000. 31. G.T.Cook , E.Dunbar , S.M.Black , S.Xu , “ A preliminary assessment of age at death determination using the nuclear weapons testing 14C activity of dentine and enamel”, Radiocarbon, vol.48, pp.305-310, 2006. 32. K.L.Spalding , B.A.Buchholz , L.E.Bergman , H.Druid , J.Frisen , “ Forensics: age written in teeth by nuclear tests”, Nature, vol.437, pp.333-334, 2005. 33. H.Pfeiffer , J.Huhne , B.Seitz , B.Brinkmann , “ Influence of soil storage and exposure period on DNA recovery from teeth”, Int J Legal Med, vol.112, pp.142-144, 1999. 34. T.Takasaki , A.Tsuji ,N. Ikeda, M.Ohishi , “ Age estimation in dental pulp DNA based on human telomere shortening”, Int J Legal Med, vol.117, pp.232-234, 2003. Dr. Nagalaxmi V et al. IJSRE Volume 1 Issue 3 March 2014 Page 366