DENTAL AGE ESTIMATION.pptx

Methods for dental age estimation

1. DENTAL AGE ESTIMATION Submitted by :- Dr. Yakita Singh ‹#›

2. CONTENTS -INTRODUCTION -VARIOUS METHODS VISUAL HISTOMORPHOLOGICAL RADIOLOGICAL BIOCHEMICAL GENETIC AND EPIGENETIC METHODS -REFERENCES ‹#›

3. INTRODUCTION Age systems include formal age classes of individuals of similar numerical age, age grades or developmental stages based on social and biological development, and relative ages of individuals. Body development is not completely associated with biological and chronological age. Hence, different parameters such as dental age, bone age, mental age, and other factors such as menarche, voice change, height, and weight are considered as proxy indicator for biological age and body development. Dental development is more reliable as an indicator of biological maturity in children as it is less affected by nutritional and endocrine status. The first known attempts that used teeth as an indicator of age originated from England. In the early 19th century, because of economic depression due to the industrial revolution, juvenile work and criminality were serious social problems. Edwin 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. ‹#›

4. VARIOUS METHODS FOR DENTAL AGE ESTIMATION : VISUAL METHOD HISTOLOGICAL RADIOGRAPHIC BIOCHEMICAL GENETIC AND EPIGENETIC METHODS ‹#›

5. VISUAL METHODS DENTAL ATTRITION SEQUENCE OF ERUPTION OF TEETH COLOR OF TEETH - Naked eye visual method - Spectrometric method ‹#›

6. DENTAL ATTRITION SEQUENCE OF ERUPTION OF TEETH ( Clinical measurement of tooth wear: Tooth wear indices ) (Wheeler's dental anatomy, physiology, and occlusion-9th edition) ‹#›

7. COLOR OF TEETH NAKED EYE VISUAL METHOD A popular system for visual determination of color is the Munsell color system, the parameters of which are represented in three dimensions. Value (lightness) is determined first by the selection of a tab that most nearly corresponds with the lightness or darkness of the color. Value ranges from white (10/) to black (0/). Chroma is determined next with tabs that are close to the measured value but are of increasing saturation of color. Chroma ranges from achromatic or gray (/0) to a highly saturated color (/18). Hue is determined last by matching with color tabs of the “value” and “chroma” already determined. Hue is measured on a scale from 2.5 to 10 in increments of 2.5 for each of the 10 color families (red, R; yellow-red, YR; yellow, Y; green-yellow, GY; green, G; blue-green, BG; blue, B; purple-blue, PB; purple, P; red-purple, RP). SPECTROMETRIC METHOD Martin-de las Heras in 2002 proposed spectroradiometry as an objective method of dentin color measurements for estimation of age. It produced an associated error of ±13.7 years. They found that dentinal colors white, cream, and yellow were associated with the age group 12–37 years, while dark yellow and brown were associated with the age group 55–64 years . Spectrophotometric dental colour measurement to assess age in living adults ‹#›

8. HISTOLOGICAL/MORPHOLOGICAL METHODS 1- Combination of different regressive alterations of teeth Gustafson’s method (1950) Dalitz method (1962) Bang and Ramm method (1970) Johanson method (1971) Maples method (1979) Kashyap and Koteshwar method (1990) Lamendin method (1992) Solheim method (1993) 2- Dentinal translucency—as a sole indicator 3- Secondary dentin deposition—as a sole indicator 4- Cementum—as a sole indicator Thickness of cementum Annulations or rings of cementum 5- Fluorescence from dentin and cementum 6- Microscopic measurements by scanning electron microscope (SEM) ‹#›

9. 1- GUSTAFSON’S METHOD OF AGE ESTIMATION (1950) Gustafson (1950) and Thoma (1944) described the age changes occurring in the dental tissues and noted six changes related to age. They are: Attrition of the incisal or occlusal surfaces due to mastication (A) Periodontitis (P) Secondary dentin (S) Cementum apposition (C) Root resorption (R) Transparency of the root (T) Gustafson suggested the last two changes. In the method proposed, each sign was ranked and allotted 0, 1, 2, 3 points. The point values of each age-change are added according to the following formula: Aₙ+ Pₙ + Sₙ + Cₙ + Rₙ + Tₙ = points . The exact equation calculated was: y = 11.43 + 4.56x , where, y = age and x = points according to the formula above. The error of estimation as calculated by Gustafson (1950) was ±3.6 years. Limitations: It cannot be used in living individuals. Multiple evaluations make it a time-consuming method. Periodontal ligament assessment is difficult in decomposed bodies. One regression line was given to all teeth irrespective of their eruption time and morphological differences. COMBINATION OF DIFFERENT REGRESSIVE ALTERATIONS OF TEETH ‹#›

10. Gösta Gustafson, D. Odont Malmö,Age Determinations on Teeth,The Journal of the American Dental Association,Volume 41, Issue 1,1950,Pages 45-54,ISSN 0002-8177, https://doi.org/10.14219/jada.archive.1950.0132 . (https://www.sciencedirect.com/science/article/pii/S0002817750110057) ‹#›

11. 2- DALITZ METHOD (1962) Dalitz improvised Gustafson's method with a 5-point system scoring from 0 to 4. Root resorption and secondary cementum criteria were discarded. The remaining four criteria for the 12 anterior teeth corresponded well with the age. This method excludes the use of posterior teeth. Use of upto four out of the 12 anterior teeth from one individual was recommended by Dalitz. The standard deviation in age estimated by this method is ±6 years. E =8.691 + 5.146A +5.338P + 1.866S + 8.411T Limitations : It does not include premolars and molars which are more likely to be sustained in case of severe trauma or mass disasters. 3- BANG AND RAMM METHOD (1970) They found that the root dentine appears to become transparent during the third decade starting at the tip of the root and advancing coronally with age. This variation is due to decrease diameter of dentinal tubules caused by increased intratubular calcification. As age increases dentinal translucency increases towards crown. Traditionally it is measured with help of vernier caliper.A new digital method was also described in 2014 to measure the dentinal translucency. Advantage : Good results are obtained by measuring intact roots only. Disadvantage : Irregular junction of translucent and non translucent zones makes it difficult to measure the length . ‹#›

12. 4- JOHANSON METHOD (1971) He suggested seven grades (0, 0.5, 1, 1.5, 2, 2.5, and 3) for the same six criteria. A tooth section thickness of 0.25 mm was recommended for better evaluation of root transparency, and the following multiple regression formula was suggested: Age = 11.02 + (5.14 × A) + (2.3 × S) + (4.14 × P) + (3.71 × C) + (5.57 × R) + (8.98 × T) Age changes were differentiated into seven different stages (A0 -A3 ) and evaluated for the same six criteria, mentioned earlier, attrition (A), secondary dentine formation (S), periodontal attachment loss (P), cement apposition (C), root resorption (R), and apical translucency (T). 5- MAPLES METHOD (1979) In this method the use of only two criteria of the total six Gustafson was recommended i.e. secondary dentine formation and root transparency, in order to make the method more simple and accurate. New formula was designed to estimate age in adults which is as follows: Age = + 13.45 4.26X. ‹#›

13. 6- KASHYAP AND KOTESWARA RAO METHOD (1990) They omitted periodontitis and root resorption from Gustafson’s method and calculated the index values of various parameters undergoing regressive changes. Age= (A)+ (D)+ (T)+ (CE)/ 4 Their modified method gave an error of 土1.59 years and Spearman coefficient value (r) of 0.998 . 7- LAMENDIN METHOD (1992) Lamendin et al. proposed a technique to estimate age at death for adults by analyzing single-rooted teeth. They expressed age as a function of two factors: translucency of the tooth root and periodontitis (gingival regression). By using a simple formulae ( A = 0.18 x P + 0.42 x T + 25.53 , where A = Age in years, P = Periodontitis height x 100/root height, and T = Transparency height x 100/root height), Lamendin et al. were able to estimate age at death with a mean error of +/- 10 years on their working sample and +/- 8.4 years. 8- SOLHELM METHOD (1993) Solheim used five of the changes that Gustafson recommended (attrition, secondary dentin, periodontitis, cementum apposition, and root transparency) and added another three new changes that showed a significant correlation in different types of teeth. The three new age-related changes were surface roughness, color of teeth, and gender of individual. They eliminated the use of root resorption as it was found to be negatively correlated with age, periodontal status was excluded due to evaluation difficulties after soft-tissue decomposition, and varying influences of dietary and occlusal conditions led to elimination of enamel attrition. This exclusion enabled this method to be applied on teeth with broken crowns, populations of contemporary and prehistoric eras, and teeth with damaged cementum. ‹#›

14. Phulari, R.G.S., Dave, E.J. Evolution of dental age estimation methods in adults over the years from occlusal wear to more sophisticated recent techniques. Egypt J Forensic Sci 11, 36 (2021). https://doi.org/10.1186/s41935-021-00250-6 ‹#›

15. DENTINAL TRANSLUCENCY Bang and Ramm (1970) concluded that accurate results were obtained from teeth sections until 75 years of age. Lamendin (1992) applied two parameters—dentin transparency and periodontal height and observed a mean error of ±8.4 years. Kamann (1998) evaluated the width of dentinal tubules on cryosections and found that the diameter of dentinal tubules decreased from 3–4 to 2 μm with increasing age. Acharya (2010) attempted to quantify translucency using digital aids where the analog signal is converted to digital and subsequent image processing is done using customized software programs. They concluded that the digital method could better estimate age with a mean error of ±5 years . SECONDARY DENTIN DEPOSITION The continuous formation of secondary dentin is thought to be a biological response and an indicator of aging. Bodecker (1925) was the first one to show the correlation between deposition of secondary dentin and chronological age. Gustafson (1950) considered it as one of the parameters. ‹#›

16. CEMENTUM Age estimation based on cementum can be done in two ways: cementum apposition and cementum annulations. Zander and Hurzeler (1958) discovered a linear relationship between the growth of cementum and chronological age for the first time. They concluded that the cemental thickness tripled over the study age of 11 to 76 years. The determination of the age of the tree is carried out by counting the annulations of the bark of a tree. Similarly, the annulations in cementum were thought to be an ideal parameter for dental age estimation. FLUORESCENCE FROM DENTIN AND CEMENTUM Kvaal and Solheim (1989) evaluated the fluorescence of dentin and cementum in human premolars and showed that fluorescence from cementum was stronger than that of dentin. Also, teeth from human remains gave stronger intensity of fluorescence than teeth from living patients, but it is not well accepted as they used it as an arbitrary scale . Extensive research of parameters by numerous modifications concluded that dentin translucency reproduced the best correlation while periodontal attachment gave the least correlation . ‹#›

17. MICROMETRIC MEASUREMENTS BY SCANNING ELECTRON MICROSCOPE The diameter of dentinal tubules reduces with age due to the deposition of peritubular dentin, which is a mineralized deposit formed centripetally in the dentinal tubules. Kosa et al. (1900) first used scanning electron microscopy (SEM) to observe aging changes as shrinkage of the pulp tissue and presence of a predentin layer microscopically. They determined weight concentrations of calcium and phosphorus and further calculated Ca/P weight ratio with the help of electron probe microanalysis in the dentin of single-rooted teeth and obtained a higher correlation with age (r=0.9712). Kvaal et al. (1994) investigated the amount of peritubular dentin and extent of obliteration of tubules and correlated with age. Kedici et al. (2000) used a SEM micrometric scaler to obtain 20 measurements of different variables in incisor teeth. Their results showed a better correlation with gender differences . Histological methods although accurate, need an extraction, and sectioning of teeth. Hence, it is an invasive procedure, practically difficult in living beings yet the most preferred method in deceased. ‹#›

18. RADIOGRAPHIC METHODS Radiographic assessment of age is a simple, non-invasive and reproducible method that can be employed both on living and unknown dead. The radiological age determination is based on assessment of various features as follows: Jaw bones prenatally Appearance of tooth germs Earliest detectable trace of mineralization or beginning of mineralization Early mineralization in various deciduous teeth during intrauterine life Degree of crown completion Eruption of the crown into the oral cavity Degree of root completion of erupted or unerupted teeth. Degree of resorption of deciduous teeth Measurement of open apices in teeth Volume of pulp chamber and root canals/formation of physiological secondary dentine Tooth-to-pulp ratio Third molar development and topography ‹#›

19. Age estimation is grouped into three phases: 1- Prenatal, neonatal and postnatal 2- Children and adolescents 3- Adults Radiographic methods : 1- Pulp-tooth dimension ratio Kvaal: based on length Cameriere: based on area 2- Tooth coronal pulp cavity index—Ikeda method 3- Age calculation using X-ray micro-focus computed tomographical scanning of teeth 4- Age estimation using mental foramen and mandibular canal ‹#›

20. AGE ESTIMATION IN PRENATAL,NEONATAL AND POSTNATAL Radiographically, the mineralization of deciduous incisors starts at the 16th week of intrauterine life. Before the mineralization of tooth germs starts, the tooth germs may be visible as radiolucent areas on the radiograph; the subsequent radiographs of the mandible will depict the deciduous teeth in various stages of mineralization as per the prenatal age of the fetus. The method employed is : Stages by Kraus and Jordan (1965) They studied the early mineralization in various deciduous teeth as well as the permanent first molar. The development is described in 10 stages, denoted by Roman numerals from I to X; the IXth stage includes three stages and the Xth stage includes five stages. Panchbhai AS. Dental radiographic indicators, a key to age estimation. Dentomaxillofac Radiol . 2011;40(4):199-212. doi:10.1259/dmfr/19478385 ‹#›

21. Panchbhai AS. Dental radiographic indicators, a key to age estimation. Dentomaxillofac Radiol . 2011;40(4):199-212. doi:10.1259/dmfr/19478385 ‹#›

22. AGE ESTIMATION IN CHILDREN AND ADOLESCENT Dental age estimation in children and adolescents is based on the time of emergence of the tooth in the oral cavity and the tooth calcification. The radiographic analysis of developing dentition, especially when there is no clinical evidence available (2.5-6 years) as well as the clinical tooth emergence in various phases will help in age determination. Methods applied for age determination in children and adolescents are : 1- Schour and Massler method (1941) They studied the development of deciduous and permanent teeth, describing 21 chronological steps from 4 months to 21 years of age and published the numerical development charts. DISADVANTAGE : These charts do not have separate surveys for males and females. Panchbhai AS. Dental radiographic indicators, a key to age estimation. Dentomaxillofac Radiol . 2011;40(4):199-212. doi:10.1259/dmfr/19478385 ‹#›

23. 2- Nolla’s method (1960) Nolla evaluated the mineralization of permanent dentition in 10 stages. After every tooth is assigned a reading, a total is made of the maxillary and mandibular teeth and then the total is compared with the table given by Nolla. Advantages : It can be applied to an individual with or without the third molar and girls and boys are dealt separately. 3- Moorees, Fanning and Hunt method (1963) In this method, the dental development was studied in the 14 stages of mineralization for developing single and multirooted tooth. Permanent teeth and the mean age for the corresponding stage was determined. The earliest age in the survey was 6 months and the data also include the development of the third mandibular molar. Panchbhai AS. Dental radiographic indicators, a key to age estimation. Dentomaxillofac Radiol . 2011;40(4):199-212. doi:10.1259/dmfr/19478385 ‹#›

24. 4- THE LONDON ATLAS OF HUMAN TOOTH DEVELOPMENT AND ERUPTION ‹#› Alqahtani, Sakher & Hector, Mark & Liversidge, Helen. (2010). The London Atlas of Human Tooth Development and Eruption. American Journal of Physical Anthropology. 142. 481 - 490. 10.1002/ajpa.21258. Held at the Royal College of Surgeons of England, and the Natural History Museum, London UK. A comprehensive evidence based atlas to estimate age using both tooth development and alveolar eruption for individuals between 28 weeks in utero to 23 years. It shows a sequence of diagrams representing a continuum of developmental ages without gaps or overlaps.

25. 5- DEMIRJIAN, GOLDSTEIN, AND TANNER METHOD (1973) Demirjian, Goldstein and Tanner rated seven mandibular permanent teeth in the order of second molar (M2), first molar (M1), second premolar (PM2), first premolar (PM1), canine (C), lateral incisors (I2) and central incisor (I1) and determined eight stages (A to H) of tooth mineralization together with stage zero for non-appearance as follows: If there is no sign of calcification, the rating 0 is given; the crypt formation is not taken into consideration. Stage description: (A) In both uniradicular and multi radicular teeth, a beginning of calcification is seen at the superior level of the crypt in the form of an inverted cone or cones. There is no fusion of these calcification points; (B) Fusion of calcified points forms one or more cusps which unite to give a regularly outlined occlusal surface, or mineralized cusps are united so the mature coronal morphology is well defined; (C) Crown half-formed, pulp chamber is evident, dentinal deposition is occurring; (D) The crown formation is completed down to the cemento-enamel junction, pulp chamber has a trapezoidal form and beginning of root formation is seen; (E) Initial formation of the radicular bifurcation is seen, the root length is still less than the crown height; (F) The apex ends in a funnel shape; the root length is equal to or greater than the crown height; (G) The walls of the root canal are now parallel and its apical end is still partially open; and (H) The apical end of the root canal is completely closed; the periodontal membrane has a uniform width around the root and the apex. ‹#›

26. The stages are the indicators of dental maturity of each tooth. The differences in the dental development between males and females are not usually apparent until the age of 5 years. The maturity scores (S) for all the teeth are added and the total maturity score may be converted directly into a dental age as per the standard table given or they are substituted in regression formula. Girls and boys have separate formulas. In females, the formula is And in males,the formula is In this method, missing teeth from one side can be replaced by those from the other side. If the first molar is absent, the central incisor can be substituted for it as their developmental age coincides. DRAWBACK : The survey does not include the developing third molar and the mandibular teeth need to be present for the survey to be applicable. ‹#›

27. Jain V, Kapoor P, Miglani R. Demirjian approach of dental age estimation: Abridged for operator ease. J Forensic Dent Sci . 2016;8(3):177. doi:10.4103/0975-1475.195103 ‹#›

28. 6- Age estimation using open apices (Cameriere method) In this study the seven left permanent mandibular teeth were valued. The number of teeth with root development completed with apical ends completely closed was calculated (N。) . For the teeth with incomplete root development, that 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 for age estimation. The dental maturity was calculated as the sum of normalized open apices (s) The numbers of teeth with root development complete (N0) . The values are substituted in the following regression formula for age estimation. Where g is a variable equal to 1 for boys and 0 for girls. ‹#›

29. AGE ESTIMATION IN ADULTS Clinically, the development of permanent dentition completes with the eruption of the third molar at the age of 17–21 years, after which the radiographic age estimation becomes difficult. The two methods commonly followed are the assessment of volume of teeth and the development of the third molar. 1. Volume assessment of teeth: pulp-to-tooth ratio method by Kvaal coronal pulp cavity index 2. Development of third molar: Harris and Nortje method Van Heerden system ‹#›

30. VOLUME ASSESSMENT OF TEETH The age estimation in adults can be achieved by radiological determination of the reduction in size of the pulp cavity resulting from a secondary dentine deposition, which is proportional to the age of the individual. 1- Method by Kvaal et al In this method, pulp-to-tooth ratio were calculated for six mandibular and maxillary teeth, such as maxillary central and lateral incisors; maxillary second premolars; mandibular lateral incisor; mandibular canine; and the first premolar. Using intraoral periapical radiographs, pulp-root length (R) , pulp-tooth length (P) , tooth-root length (T) , pulp-root width at cemento-enamel junction (A) , pulp-root width at mid-root level (C) and pulp-root width at midpoint between levels C and A (B) for all six teeth were measured . Finally, mean value of all ratios excluding T (M) , mean value of width ratio B and C (W) and mean value of length ratio P and R (L) were substituted in the given formula : Jain V, Kapoor P, Miglani R. Demirjian approach of dental age estimation: Abridged for operator ease. J Forensic Dent Sci . 2016;8(3):177. doi:10.4103/0975-1475.195103 ‹#›

31. 2- The coronal pulp cavity index Only mandibular premolars and molars were considered, as the mandibular teeth are more visible than the maxillary ones. Panoramic radiography is used to measure the length (mm) of the tooth crown (coronal length, [ CL ]) and the length (mm) of the coronal pulp cavity (coronal pulp cavity height or length [ CPCH ]). The tooth-coronal index ( TCI ) is computed for each tooth and regressed on the real age of the sample using the formula: ‹#› Jain V, Kapoor P, Miglani R. Demirjian approach of dental age estimation: Abridged for operator ease. J Forensic Dent Sci . 2016;8(3):177. doi:10.4103/0975-1475.195103

32. DEVELOPMENT OF THIRD MOLAR The radiographic age estimation becomes problematic after 17 years of age as eruption of permanent dentition completes by that age with the eruption of the third molar. Later, the development of the third molar may be taken as a guide to determine the age of the individual. 1- Harris and Nortje method Harris and Nortje have given five stages of third molar root development with corresponding mean ages and mean length: Stage 1 (cleft rapidly enlarging—one-third root formed, 15.8 ± 1.4 years, 5.3 ± 2.1 mm); Stage 2 (half root formed, 17.2 ± 1.2 years, 8.6 ± 1.5 mm); Stage 3 (two-third root formed, 17.8 ± 1.2 years, 12.9 ± 1.2 mm); Stage 4 (diverging root canal walls, 18.5 ± 1.1 years, 15.4 ± 1.9 mm); Stage 5 (converging root canal walls, 19.2 ± 1.2 years, 16.1 ± 2.1 mm Jain V, Kapoor P, Miglani R. Demirjian approach of dental age estimation: Abridged for operator ease. J Forensic Dent Sci . 2016;8(3):177. doi:10.4103/0975-1475.195103 ‹#›

33. 2- Van Heerden system Van Heerden assessed the development of the mesial root of the third molar to determine the age. He described the development of the mesial root in five stages using panoramic radiographs . The males and females were surveyed separately and no significant differences were found between them. Jain V, Kapoor P, Miglani R. Demirjian approach of dental age estimation: Abridged for operator ease. J Forensic Dent Sci . 2016;8(3):177. doi:10.4103/0975-1475.195103 ‹#›

34. PULP-TOOTH DIMENSION RATIO Kvaal and Solheim ( 1994) The method used length and width of the tooth and dental pulp on IOPA radiographs. A standard error of ±9.5 years was obtained by using the following equation: Age=129.8−(316.4 x M) (6.8 x [W−L]) Parameters used were as follows: Pulp-root length–(R) Pulp-tooth length–(P) Tooth-root length–(T) Pulp-root width at the cement-enamel junction (CEJ)–(A) Pulp-root width at mid-root level–(C) Pulp-root width at the midpoint between levels A and C–(B) Mean value of all ratios excluding T–(M) Mean value of width ratio B and C–(W) Mean value of length ratio P and R–(L) Salemi, F., Farhadian, M., Askari Sabzkouhi, B. et al. Age estimation by pulp to tooth area ratio in canine teeth using cone-beam computed tomography. Egypt J Forensic Sci 10, 2 (2020). https://doi.org/10.1186/s41935-019-0176-9 ‹#›

35. TOOTH-CORONAL PULP CAVITY INDEX Ikeda et al.(1985) developed a new index, called the tooth-coronal index or tooth coronal pulp cavity index (TCI), which is based on two linear measurements on dental radiographs of extracted human teeth, crown height (CH), and coronal pulp cavity height. TCI=(CPCH X 100)/CL {CPCH= coronal pulp cavity height or length, CL= coronal length} X-RAY MICROFOCUS SCANNING Calculation is based on the ratio of pulp volume and tooth volume. Limitations : Expensive equipment and time-consuming procedure. Gotmare SS, Shah T, Periera T, et al. The coronal pulp cavity index: A forensic tool for age determination in adults. Dent Res J (Isfahan) . 2019;16(3):160-165. Aboshi, Hirofumi et al. “Age estimation using microfocus X-ray computed tomography of lower premolars.” Forensic science international 200 1-3 (2010): 35-40 . ‹#›

36. MENTAL FORAMEN AND MANDIBULAR CANAL Bhardwaj et al. ( 2014) used digital panoramic radiographs for assessing gonial angle, antegonial angle, mental foramen, mandibular canal, and mandibular foramen for age estimation and found mandibular canal and mandibular foramen as highly significant parameters. Recently, technological advancements have made the application of artificial intelligence (AI) possible in the forensic world. One such study by Kim et al.(2021) considered only first molars from OPGs. They obtained an accuracy of 89.05 to 90.27% indicating excellent outcomes. The AI utilized a convolutional neural network (CNN) that focused on tooth pulp, alveolar bone level, and interdental space depending on age and location of tooth . Dosi T, Vahanwala S, Gupta D. Assessment of the Effect of Dimensions of the Mandibular Ramus and Mental Foramen on Age and Gender Using Digital Panoramic Radiographs: A Retrospective Study. Contemp Clin Dent . 2018;9(3):343-348. doi:10.4103/ccd.ccd_26_18 ‹#›

37. Phulari, R.G.S., Dave, E.J. Evolution of dental age estimation methods in adults over the years from occlusal wear to more sophisticated recent techniques. Egypt J Forensic Sci 11, 36 (2021). https://doi.org/10.1186/s41935-021-00250-6 ‹#›

38. BIOCHEMICAL METHODS In the natural aging process, several molecular changes occur most commonly in the long-living proteins and hard tissues like the teeth and bone. Aspartic acid racemization, collagen cross links,lead accumulation, advanced glycation-end products, enamel uptake of radioactive carbon 14 and mitochondrial DNA (mtDNA) mutations are some of the most established methods of biochemical age estimation. Among the above methods, the racemization of aspartic acid can be considered as the most precise method. Advantage : The sample can be collected from tissues (teeth) protected from various environmental and nutritional factors. If all the confounding factors are stable, the utilization of advanced glycation-end products can also be considered valuable. ‹#›

39. 1- ASPARTIC ACID RACEMIZATION (AAR) In most living organisms, optically active amino acids initially consist of only L-forms, which partially get converted into D-forms until an equilibrium is obtained. Under this equilibrium, the D/L ratio of aspartic acid is 1.0. This conversion is known as racemization, which causes alterations in the conformation of metabolically stable proteins, thereby inducing changes in their biochemical activities . It is dependent on temperature, pH, and humidity. WHY ASPARTIC ACID? Among the amino acids, aspartic acid has the fastest rate of racemization, followed by alanine, glutamic acid, isoleucine, and leucine. Therefore, aspartic acid is most commonly used for age estimation. The sample can be collected from tissues most protected from environmental and nutritional factors (teeth). The chemical instability of asparaginyl and aspartyl residues in proteins may result in modifications that increase the D-aspartate residue with age . In humans, the presence of long-living proteins is observed in hard tissues of the teeth, bone (type I collagen, telopeptides, osteocalcin), sclera of the eye (elastin), lung parenchyma (elastin), arterial wall (elastin), and intervertebral disc, articular cartilage (proteoglycans), brain (tubulin, synapsin, proteoglycans, myelin base protein, white matter, β amyloid protein, tar protein), ocular lens (αA-crystallin), cartilage, membrane proteins of erythrocytes, and skin. ‹#›

40. Even after all other soft tissues have degenerated, hard tissues like the teeth and bone are well preserved. The teeth, in particular, are frequently conserved even when most of the bones have been destroyed or mutilated. In 1975, Helfman et al first used the aspartic acid racemization method in dentin to assess age. Researchers found a strong correlation (0.96–0.98) between chronological age and aspartic acid racemization levels, with a standard estimation error of 2.95–4.84 years. A correlation between the age of dentin and the extent of aspartic acid racemization was identified to be approximately 0.96 with a standard error of 5.69 years . The aspartic acid racemization is also observed in deciduous teeth and a correlation between chronological age and rate of racemization has been identified ( r =0.824–0.98), proving it to be applicable in deciduous teeth. The extent of post mortem preservation on aspartic acid racemization in the dentin of healthy, impacted, and carious teeth were studied and noted that the teeth can be preserved for up to 10 years, showing a negligible effect on estimated values with an error of 4 years . ‹#›

41. High power liquid chromatography (HPLC) and gas chromatography (GC) are generally used to analyze the racemic mixture. In the various available HPLCs, ion-exchange chromatography (IEC) is usually preferred. The gas chromatographic method is considered as the most sensitive method. Procedure Extraction of acid , which results in two parts: the acid-soluble and the insoluble acid fraction. The soluble acid fraction mainly consists of collagen, and the insoluble fraction consists of non-collagenous proteins. The insoluble acid fraction undergoes constant remodeling. The rate of racemization is rapid in the non-collagenous proteins. The D-aspartate accumulates with age predominantly in the non-collagenous proteins. Such non-collagenous proteins like osteocalcin are also found in bone, and the extent of aspartic acid racemization using osteocalcin is the measure of the aging of these proteins, thereby measuring an individual’s age. The correlation between the rate of racemization and age varies with the type of bone used, the highest being in the sternum and the lowest in the pelvic and sacral bone. The alveolar bone, a metabolically more active bone, shows an increased ratio of racemization with age. The rate is significantly higher in males than in females. The alveolar bone cannot be used in edentulous individuals. ‹#›

42. ‹#› Wochna, K., Bonikowski, R., Śmigielski, J. et al. Aspartic acid racemization of root dentin used for dental age estimation in a Polish population sample. Forensic Sci Med Pathol 14, 285–294 (2018). https://doi.org/10.1007/s12024-018-9984-8

43. Non-dental tissues like the yellow ligament of the spine, and sclera, contain long-living proteins like elastin that accumulate D-aspartate residues, making it a suitable testing sample with results closer to the actual age and less time consuming. However, it is not reliable for corpses under the influence of high temperatures. FACTORS AFFECTING ASPARTIC ACID RACEMIZATION The presence of caries in teeth has been shown to influence the rate of racemization . Deviations up to 20.39 years were noted in carious teeth. It is assumed that caries induced protein degradation, which thereby generates small fragments of lower steric hints, leading to faster accumulation of D-aspartic acid. With the increase in temperature, the extent of aspartic acid racemization increases significantly ( r =0.913; p <0.01) with heating time, and the stability rates of dentin at different temperatures (22–25°C, 4°C, and −30°C) showed no significant changes after 1 year with an error range of 5 years . The position of teeth and the time taken for dentin formation also influence the rate of racemization and is highest in the first molars in the middle-aged population and second molars in elderly individuals . No differences in racemization rates were noted between the jaws . Pillalamarri, M., Manyam, R., Pasupuleti, S. et al. Biochemical analyses for dental age estimation: a review. Egypt J Forensic Sci 12, 2 (2022). https://doi.org/10.1186/s41935-021-00260-4 ‹#›

44. 2- COLLAGEN CROSSLINKS The crosslink patterns are characteristic and are formed by two divalent crosslinks of dehydro-hydroxylysinonorleucine and dehydro-dihydroxylysinonorleucine. The bone and dentin collagen contain two non-reducible crosslinks of hydroxypyridinium, namely pyridinoline and deoxypyridinoline. The calcified tissues of bone and teeth show a significant peak in lysyl hydroxypyridinium residues. Pyridinoline is a non-reducible crosslink, which is the main maturation product of reducible crosslinks prominent in bone and dentin. It is abundant in adult cartilage at one residue per collagen molecule. Almost all reducible crosslinks in cartilage collagen seem to progress rapidly to hydroxypyridinum crosslinks. Deoxypyridinoline has been identified as a minor component in the adult dentin residues. It is observed that the hydroxypyridinium crosslinks increased with age. Analysis of these crosslinks is studied using enzyme immunoassay and chromatography. ‹#›

45. 3- ADVANCED GLYCATION-END PRODUCTS (AGEs) Louis–Camille Maillard( 1912) discovered that when amino acids are heated in the presence of reducing sugars, they turn brown. This biological process results in the formation of advanced glycation-end products, known as Maillard reaction. Maillard reaction can be described in three stages: early, intermediate, and late stages . In the early stage , reducing sugars like glucose, fructose, mannose, and galactose react with different molecules like proteins, nucleic acids, and lipids to form a stable ketoamide called Amadori compound. In the intermediate stage , this Amadori compound further degrades into various carbonyl compounds like glyoxal, 3-deoxyglucosone, and methylglyoxal. The final stage involves reactions between the carbonyl compound with amino acids, leading to complex rearrangements, cleavage, and covalent binding reactions of the Amadori products .This Amadori arrangement is irreversible and results in the formation of stable adducts and protein crosslinks called advanced glycation products. (Formation of advanced glycation-end products) Pillalamarri, M., Manyam, R., Pasupuleti, S. et al. Biochemical analyses for dental age estimation: a review. Egypt J Forensic Sci 12, 2 (2022). https://doi.org/10.1186/s41935-021-00260-4 ‹#›

46. Various end products including pentosidine , fructoselysine , and carboxymethyl lysine (CML) have been analyzed. These AGEs accumulate in long-lived proteins and easily bind to collagen and act as a crosslink between the collagen fibrils in collagen-rich tissues like the crystalline lens ,articular cartilage ,aorta ,rib cartilage ,skin collagen, intervertebral disc, and dentin. Along with nutrients, dentin also receives AGEs from the blood vessels. They form cross links with collagen fibers and induce various mechanical and morphological changes in dentin, along with brownish discoloration. By analyzing the degradation of dentin collagen by carboxylic protease, the characteristic Maillard fluorescence can be noticed. An early Maillard reaction product, called furosine, has been examined in healthy and carious dentin . Pentosidine levels in root dentin (including healthy, diabetic, stored, and heated root dentin) can be quantified and used for age estimation. Heated and carious teeth show high levels of pentosidine. It is assumed that the glycation-based changes stabilize the affected region and protect it from degradation. In a recently conducted study on archaeological samples, results have shown that the age estimation by measuring the pentosidine levels has proven to be more accurate in comparison to D-Asp . The advanced glycation-end products can be analyzed using fluorescence spectroscopy, mechanical indentation analysis, immunohistochemical staining, and immune-electron microscopy , HPLC , gas chromatography-mass spectroscopy (GC-MS), and enzyme-linked immunosorbent assay (ELISA) . ‹#›

47. 4- LEAD ACCUMULATION About 90% of lead is accumulated in the bones. The teeth, blood, and other soft tissues also contain considerable amount of lead which progressively increases with age. However, the blood lead levels are instantaneous, which reflect an immediate lead exposure . In the bone, it is removed over time due to its remodeling, but in the teeth, once deposited, it cannot be removed as there is no turnover of apatite. Therefore, the teeth are the most suitable material for studying total past lead exposure. Lead levels in the teeth can also be utilized to analyze an individual’s age due to its progressive accumulation. In the teeth, dentin has been identified as the leading site for lead accumulation as compared with enamel . The lead is separated using anion exchange chromatography and measured using mass spectrophotometry, and atomic absorption spectrophotometry. It is essential to note that the atmospheric lead levels influence the amount accumulated, making it population and region-specific. It may therefore be inapplicable as a generalized method for age estimation. Although if region-specific lead levels can be determined and correlated with age, this method can be employed. ‹#›

48. 5- MITOCHONDRIAL DNA MUTATIONS Mitochondrial DNA is present near the inner membrane of the mitochondria and is influenced by the presence of free radicals which accumulate with age. As stated by Harman in the theory of aging, the production of free radicals increases with age. Several studies have found a relation between mtDNA mutations and aging in tissues like the brain, skeletal muscle, and heart . A semiquantitative PCR conducted on dentin and pulp of third molars demonstrated a decrease in the quantity of mtDNA with age. A strong linear negative correlation has been seen between the amplification of mtDNA and dentin age using real-time PCR in third molars . The mtDNA damage is measured using real-time PCR. DRAWBACKS : It is time-consuming, expensive, and technique-sensitive. ‹#›

49. 6- ENAMEL UPTAKE OF RADIOACTIVE CARBON 14 Carbon dating which is conventionally used by archaeologists to measure the age of geological matter found its application in dental age estimation also. Spalding et al. (2005) showed the levels of radioactive C14 isotope levels and radioactive CO2 which is formed by the reaction of C14 with oxygen have drastically increased after the nuclear tests. This radioactive CO2 which has a radioactive signature is uptaken by plants. It is deposited in human dental enamel while consuming these plants. By calculating these levels of deposited C14 in enamel, the date of birth can be calculated. Initial reports claim the accuracy of estimated age by the mean error of ±1.5 years . Alkass et al. ( 2009) proposed a combined method where both aspartic acid racemization and radioactive carbon uptake could be used. The age at death determined by racemization and the date of birth from C14 levels provide the closest estimation of age with a difference of 1–1.5 years. ‹#›

50. GENETIC AND EPIGENETIC METHODS Telomeres at the end of the chromosomes shorten with each cell division, limiting the proliferation of human cells and inducing senescence, differentiation, or cell death. Telomere shortening may be an effective method to estimate age. Takasaki et al. (2003) determined the terminal restriction fragment (TRF) length as telomere length in dental pulp DNA and determined the age. A variety of T cell receptor (TCR) molecules is generated when each immature T lymphocyte undergoes somatic rearrangement. This process leads to the elimination of DNA sequences of TCR and circulation of TCR into “Signal joint TCR excision circles (sjTRECs)”. LIMITATIONS : recent, expensive, and technique-sensitive and yield variable results which are not reliable. Recently, following genetics, even epigenetic modifications have been tried out such as DNA methylation. Bekaert et al. (2015) carried out DNA methylation on both blood and teeth DNA. Guillani et al. ( 2016) studied DNA methylation in DNA from three tissues- dentin, pulp, and cementum. This technique is highly technique-sensitive and requires sophisticated and expensive equipment, so is not quite common in routine setup. ‹#›

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54. THANK YOU ‹#›