Archives of Oral Biology 87 (2018) 7–14

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Review

When forensic odontology met biochemistry: Multidisciplinary approach in T

forensic human identification
⁎
Joe Adserias-Garrigaa, Christian Thomasb, Douglas H. Ubelakerb, Sara C. Zapicob,c,
a
University of Girona, Emili Grahit 77, Girona, Cataluña, Spain
b
Smithsonian Institution, NMNH, MRC112, Anthropology Department, 10th and Constitution Ave, NW, PO Box 37012, 20560, Washington, DC, United States
c
Florida International University, Department of Chemistry and Biochemistry and International Forensic Research Institute, 11200 SW 8 St. CP323, Miami, FL, 33199,
United States

A R T I C L E I N F O A B S T R A C T

Keywords: When human remains are found, the priority of the investigation is to ascertain the identity of the deceased. A
Forensic odontology positive identification is a key factor in providing closure for the family of the deceased; it is also required to
Biochemistry issue the death certificate and therefore, to settle legal affairs. Moreover, it is difficult for any forensic in-
Biological profile vestigation involving human remains to be solved without the determination of an identity. Therefore, personal
Geographical origin
identification is necessary for social, legal and forensic reasons.
Personal identification
In the last thirty years forensic odontology has experienced an important transformation, from primarily
involving occasional dental identification into a broader role, contributing to the determination of the biological
profile. In the same way, “DNA fingerprinting” has evolved not only in terms of improving its technology, but
also in its application beyond the “classical”: helping with the estimation of sex, age and ancestry. As these two
forensic disciplines have developed independently, their pathways have crossed several times through human
identification operations, especially the ones that require a multidisciplinary approach. Thus, the aim of this
review is to describe the contributions of both forensic odontology and molecular biology/biochemistry to
human identification, demonstrating how a multidisciplinary approach can lead to a better and more efficient
identification.

1. Introduction presumptive identification occurs when there are several consistencies

When human remains are found, the first priority of the investiga-
tion is to ascertain the identity of the deceased; indeed, any forensic
investigation involving human remains would be very difficult to solve
without this information. Several methods and techniques from diverse
fields, depending on the remains available, can be applied to human
identification. The first step in the identification process is to build up a
biological profile, which is a general description of the individual’s
ancestry, sex, age-at-death and stature.
This information is the post-mortem data. The ante-mortem data is
any information concerning the individual (provided by the missing
person’s family or relatives) that could be used for identification.
Comparisons between ante-mortem and post-mortem data can lead to a
positive identification, presumptive identification or an exclusion.
A positive identification is scientifically proven, and is usually
achieved through fingerprinting, dental data or DNA. In contrast,
between ante-mortem and post-mortem data, but no single factor alone
justifies the identification (Thompson & Black, 2006). A presumptive
identification may be based on personal effects, scars, tattoos, con-
textual evidence, testimony recognition or facial approximation. When
the ante-mortem and post-mortem data are not consistent without ex-
planation, that leads to an exclusion.
Dental identification is extremely useful when attempting to achieve
a positive identification or exclusion, either in ordinary cases of iden-
tification or in disaster victim identification (DVI) scenarios, where
forensic odontology offers an expeditious and scientific method of
comparative identification. The field of forensic odontology has ex-
perienced a significant change in the last thirty years, from first in-
volving forensic odontologists only occasionally in identification cases,
to them playing a key role in the identification process (Senn & Stimson
2010). Nowadays, most medical examiner/coroner offices, as well as
most police departments around the world, have forensic odontology

⁎
Corresponding author at: Current address: Florida International University, Department of Chemistry and Biochemistry and International Forensic Research Institute, 11200 SW 8 St.
CP323, Miami, FL, 33199, United States.
E-mail addresses: mjadserias@hotmail.com (J. Adserias-Garriga), crf.thomas@gmail.com (C. Thomas), ubelaked@si.edu (D.H. Ubelaker),
scasadoz@fiu.edu, casado-zapicos@si.edu, saiczapico@gmail.com (S. C. Zapico).

https://doi.org/10.1016/j.archoralbio.2017.12.001
Received 8 May 2017; Received in revised form 30 November 2017; Accepted 3 December 2017
0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
J. Adserias-Garriga et al.
consultants that are routinely involved in cases of dental identification,
age estimation from dental structures, and patterned injuries that may
have been created by teeth.
As new technologies advance, new techniques are emerging, and
forensic odontology is incorporating these advances into research to be
subsequently applied to casework. Recent technological developments
are creating new opportunities to perform robust and validated scien-
tific measurements. These technological advances have the potential to
strongly increase the speed and efficacy of the criminal justice process.
However, such benefits are only realized when quality assurance and
control can be guaranteed, so findings can be used as forensic evidence
in court (Kloosterman et al., 2015).
In current practice, DNA molecular analysis is an extremely useful
tool in forensic investigations. DNA profiling is based on the short
tandem repeats (STR) and aids in human identification from biological
samples. In the last decade, because of the advances in the field of
biochemistry, new biomarkers have been studied and proposed for use
in forensic identification (Dumache, Ciocan, Muresan, & Enache, 2016).
Likewise, the current trend is to apply biochemical methodologies to
determine the biological profile: sex, age and ancestry (Cloos &
Fledelius, 2000; Murakami et al., 2000; Witas, Tomczyk,
Jedrychowska-Danska, Chaubey, & Ploszaj, 2013). This article aims to
review the methods and techniques that can be applied to teeth and oral
structures to identify the deceased, from the reconstruction of a biolo-
gical profile to the ante-mortem and post-mortem comparison of dental
data. While some of these techniques can also be applied to the living,
there are certain techniques that are applicable only to the dead. Re-
viewed here are forensic odontology methods as well as biochemical
techniques applied to dental structures.
2. Forensic odontology and biochemical methods applied to
biological profile reconstruction
In human identification cases, a biological profile must be re-
constructed from identifiers in bones and teeth. Forensic anthropology
offers a great number of biological profile identifiers to estimate an-
cestry, sex, age, stature and, in certain cases, pathology. Forensic
odontologists in particular, through examination of teeth and oral
structures, can provide information for several characteristics of the
individual such as age, ancestry, geographical origin, sex, occupation,
habits and past or present pathology (Berman, Bush, et al., 2013), such
that dental and maxillofacial structures can help in the reconstruction
of the biological profile of the unknown. Additionally, molecular
methodologies have been developed and applied to ancestry estimation
such as mitochondrial DNA and single nucleotide polymorphisms
(SNPs) (Witas et al., 2013). In the same line of research, biochemical
parameters have been applied to age assessment such as aspartic acid
racemization (Cloos & Fledelius, 2000), mitochondrial DNA mutations
(Zapico & Ubelaker, 2016), epigenetics (Bekaert, Kamalandua, Zapico,
Van De Voorde, & Decorte, 2015), collagen crosslinks (Martin-De Las
Heras, Valenzuela, & Villanueva, 1999), advanced glycation end (AGEs)
products (Baynes, 2001) or telomere shortening (Tsuji, Ishiko,
Takasaki, & Ikeda, 2002). Even though age estimation is a part of the
biological profile, this topic will be discussed separately due to the great
contribution of forensic odontology and biochemistry to that identifier.
2.1. Estimation of ancestry
There are some dental traits that can be used as indicators of a
higher probability of certain ancestral groups. Traits that are illustrative
of a Caucasian origin are Carabelli’s cusp on the first maxillary molars,
a bi-lobulated chin or deep canine fossae. Negroid ancestry indicators
consist of multicusped premolars, maxillary midline diastema and
pronounced prognathism. Mongoloid ancestry indicators include
shovel-shaped incisors, buccal pits and incisor rotations (Berman, Bush,
et al., 2013). Even though these dental characteristics can help in
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ancestry estimation, the final conclusion on ancestry estimation should
be carried out by anthropological assessment. Molecular methodologies
can also be applied to estimate ancestry, such as mitochondrial DNA
profiling and SNPs. However, there are few studies that apply these
methodologies to teeth, as researchers mainly use blood. The studies
developed in teeth were mostly archaeological, applying mtDNA (Witas
et al., 2013) or comparing ancient specimens with current populations
(Goncalves et al., 2010).
2.2. Geographical origin
The type of dental restorations present, quality of treatment, and
materials used may indicate a country or region where the dental
treatment was completed. Silver or gold color metal crowns on anterior
teeth are very frequent in Central and South America; full cast metal
crowns with acrylic facings on anterior teeth are usually found in
Eastern Europe. Some dental conditions may offer information about
the geographical origin of the remains. For instance, dental fluorosis
can be indicative of Texas, New Mexico, rural United States, China,
Africa or India (Berman, Bush, et al., 2013). Also, dental modifications
in the present time are practiced in certain parts of South Africa
(Friedling & Morris, 2005; Hollowell & Childers, 2007).
Isotope analysis can be used to determine geographic origin, like
13
C. This is a stable isotope that constitutes about 1.1% of all carbon.
Plants can discriminate between 12C and 13C, creating differences in the
levels of these isotopes among types of plants. Based on the fixation of
CO2 during photosynthesis, it is possible to distinguish between C4
plants (like corn and sugar cane), which contain higher amounts of 13C
than C3 plants (like potato, wheat and sugar beet), since it diffuses out
through the stomatal pores into the ambient air (Kubasek, Urban, &
Santrucek, 2013). C4 plants tend to grow in hotter or drier climates
than C3 plants. Animals, including humans, with diets based mainly on
C4 plants will incorporate more 13C than those that have C3 plant based
diets, thus differentiating geographical origin. Another stable isotope
that shows geographic variation is 18O. The incorporation of this iso-
tope in animal tissues is correlated to the levels in drinking water, and
these levels vary with latitude due to differences in the evaporation and
condensation propensity between 16O and 18O (Chesson, Podlesak,
Thompson, Cerling, & Ehleringer, 2008). Studies analyzing these iso-
topes in dental enamel point out their usefulness towards providing
information about the geographical origin of an individual (Alkass,
Buchholz, Druid, & Spalding, 2011; Alkass et al., 2013).
2.3. Sex assessment
Although there are several scientific studies on morphological sex
dimorphism of teeth (Kapila, Nagesh, Iyengar, & Mehkri, 2011;
Schwartz & Dean, 2005; Pettenati-Soubayroux, Signoli, & Dutour, 2002;
Silva et al., 2016), its use in diagnosis requires further scientific vali-
dation (Berman, Bush, et al., 2013). Sex assessment in forensic case-
work should be carried out by anthropological study or molecular
analysis. The determination of sex in skeletal remains is made more
challenging if these remains are found fragmented or commingled.
Moreover, from an anthropological perspective, the determination of
sex in skeletal remains of children and preadolescents is difficult owing
to the lack of development of sex characteristics (Potsch, Meyer,
Rothschild, Schneider, & Rittner, 1992; Murakami et al., 2000). Mole-
cular methods have been used to combat these complications. Among
these methods, the amplification of the Y chromosome-specific alphoid
centromeric repeat sequence (DYZ3) by polymerase chain reaction
(PCR) reported in 1989 by Witt and Erickson (Akane et al., 1991;
Horiuchi, Morisaki, Fujii, & Miwa, 1988; Fukushima, Hasekura, &
Nagai, 1988; Kobayashi, Nakauchi, Nakahori, Nakagome, &
Matsuzawa, 1988; Tyler, Kirby, Wood, Vernon, & Ferris, 1986), which
can detect X chromosome-specific alphoid centromeric repeat sequence
(DXZ1) as well as DYZ3, is considered to provide accurate
J. Adserias-Garriga et al.
determination. This method was also studied in teeth (Murakami et al.,
2000), producing good results both in pulp and hard tissues under some
conditions, although sometimes the pulp tissues failed to provide a sex
determination, leaving only hard tissues to use for results. A study from
our group (Zapico & Ubelaker, 2013a, 2013b) analyzed the efficiency of
sex determination in dentin and pulp tissues, but in this case based on
the amplification of amelogenin genes. These genes are located in the X
and Y chromosomes in humans and express different intron sequences.
The amelogenin gene on the X chromosome is 106 based-pairs in
length, whereas the same gene on the Y chromosome has 112 base pairs
(Bansal, Shetty, Bindal, & Pathak, 2012). Thus, females have two
identical AMEL genes but males do not (Sivagami, Rao, & Varshney,
2000), which can be used for sex determination. Our study pointed out
the possibility of sex determination from pulp and minimal amounts of
dentin, although the efficiency of DNA isolation depends more on the
type of tooth analyzed, with molars providing the best results.
3. Age estimation
A basic step in biological profile reconstruction is to estimate the
age-at-death of the unknown. Age estimation can be inferred by a
variety of different approaches, the selection of the methods and
techniques to be used to estimate age depends on the conditions of the
remains as well as their applicability to the age category of the in-
dividual.
3.1. Dental age estimation
Dental age estimation can be assessed by their development, erup-
tion and post-formation changes. As teeth are highly mineralized and
usually well preserved, they are especially useful in age estimation. In
addition, they are minimally affected by environmental and nutritional
insult compared with other skeletal age indicators (Cunha et al., 2009;
Elamin & Liversidge, 2013; Garn, Lewis, & Kerewsky, 1965). Thus, age
estimation based on dental development and eruption shows more ac-
curacy than other anthropological methods based on development and
growth.
3.2. Fetal dental age estimation
Dentition is an excellent age indicator applicable from the time of
intrauterine development, due to embryonic tooth development which
begins early in fetal development. The degree of morphological enamel
mineralization is easily viewed through X-rays (Lewis & Senn, 2013).
Histological analysis on incremental growth lines within the enamel,
the Retzius striae (Copenhaver et al., 1978) can also be performed.
When a systemic disturbance occurs, the enamel mineralization
process is interrupted and the currently developing striae will appear
darker. The physiologic stress suffered at birth will be reflected in the
enamel mineralization process, by the appearance of the darkest and
largest incremental growth line in the deciduous teeth, which is called
the neonatal line. (Bath-Balogh & Fehrenbach, 2006). The presence of a
neonatal line may indicate that the child lived after birth, but it must be
taken into account that the neonatal line takes some hours to days to
form after birth.
3.3. Child dental age estimation
Dental age estimation in children is based on tooth development
and eruption. There are basically two methodological categories for
child age assessment: the atlas style and the incremental staging system.
Both methodologies rely on radiographic examination. Schour and
Massler (1941), Ubelaker (1989), and The London Atlas by AlQahtani
et al. (2010) are the most frequently used atlases in odontology and
anthropology, and are applicable in both archaeological and forensic
settings (Schour & Massler,1941; Ubelaker, 1989; AlQahtani, Hector, &
9
Archives of Oral Biology 87 (2018) 7–14
Liersidge, 2010). Atlases can be used from weeks in utero to maturity.
While no sex differences are reflected in the atlases, AlQahtani’s com-
puter application permits male, female and unknown data to be en-
tered; even though there is no statistical significance between sexes, the
results obtained when entering male or female data are slightly dif-
ferent.
Special attention must be paid to the concept of eruption, because
while Ubelaker defines eruption as the point in time that the tooth
emerges through the gingival tissue, AlQahtani defines eruption as
emergence through the alveolar bone (Lewis & Senn, 2013). Regarding
the staging system, the Moorrees system (Moorrees et al., 1963a,
1963b) and Demirjian system (Demirjian, Goldstein, & Tanner, 1973;
Demirjian & Goldstein, 1976) are the most often used in forensic
odontology. The Moorrees system provides sex specific graphs for de-
velopmental staging of deciduous and permanent maxillary and man-
dibular teeth, including incisors, canines, premolars and molars
(Moorrees et al., 1963a, 1963b). The Demirjian system consists of
scoring the mandibular left quadrant excluding the third molar in eight
stages (A to H) (Demirjian & Goldstein, 1976; Demirjian et al., 1973).
3.4. Adolescent age estimation
As the skeletal development process progresses, fewer age indicators
based on development remain useful for age assessment. Skeletal ele-
ments can provide age estimation in adolescents and early adulthood
like long bone development, maturation of the hand and wrist, fusion of
the spheno-occipital/basilar synchondrosis, iliac crest and vertebral
ring and fusion of the clavicle sternal end (Cunha et al., 2009). Re-
garding dental age estimation in that age range, the only remaining
tooth undergoing growth and formation by the age of 14 is the third
molar (Lewis & Senn, 2013). Even though the third molar is the most
developmentally variable tooth, it is the most reliable biological in-
dicator during adolescence and early adulthood (Harris, Mincer,
Anderson, & Senn, 2010; Lewis & Senn 2013).
Mincer developed a scoring system in 1993 applying Demirjian’s
eight developmental stages on third molars, labeled A to H. This
method is widely used to assess the legal majority of age, thus when
stage “H” is achieved there is a high probability that the individual is at
least eighteen years old (Lewis & Senn, 2013).
UT-Age is a computer application that simplifies and expedites age
assessment based on the Mincer method (Lewis, Senn, & Alder, 2001;
Lewis, Senn, & Silvaggi, 2008). This computer tool allows the selection
of ancestry based on different population specific studies (Arany, Iino,
& Yoshioka, 2004; Blankenship, Mincer, Anderson, Woods, & Burton,
2007; Kasper, Austin, Kvanli, Rios, & Senn, 2009; Mincer, Harris, &
Berryman, 1993).
The combination of dental and skeletal indicators results in in-
creased accuracy of the estimation. Schmeling et al. (2004) proposed
age estimations combining third molar, hand and wrist bones and cla-
vicle development (Schmeling, Olze, Reisinger, & Geserick, 2004).
Cameriere, Ferrante and Cingolani (2004) established that the pulp-
tooth area ratio of the second molar and the stage of development of the
third molar in combination resulted in better assessment of whether an
individual was eighteen or older than either method alone (Cameriere
et al., 2004; Harris et al., 2010).
3.5. Adult age estimation
When growth and development achieve their conclusion, skeletal
and dental age assessment can only be based on degenerative changes
of the skeleton. Gustafson (1950) used six tooth post-formation changes
(attrition, periodontosis, secondary dentin, cementum apposition, root
resorption, and root translucency) scored between 0 and 3 (Gustafson,
1950). This study assumes that all variables are equally effective in age
assessment, that staging occurs equally among the six variables, and
that statistical variables are independent (Harris et al., 2010).
J. Adserias-Garriga et al.
A modification of this methodology was carried out by Johanson
(1971), grading microscopically the same post-formation changes
(Johanson, 1971).The order of age assessment variables from best
correlation of age to least is root transparency, secondary dentin, at-
trition, gingival recession, and cementum apposition (Maples & Rice,
1979). Maples (1978) generated different regression formulas con-
sidering secondary dentin apposition and root translucency (Maples,
1978). Lamendin et al. (1992) considered root transparency and peri-
odontal recession for age estimation (Lamendin et al., 1992), whilst
Prince and Ubelaker (2002) modified Lamendin’s method taking into
account sex and ancestry (Prince & Ubelaker, 2002). Kvaal and Solheim
(1994) developed a method to estimate age by evaluating progressive
pulp size changes due to secondary dentin apposition. Even though
tooth wear is traditionally used to estimate age in ancient remains
(Brothwell, 1989), it is of little use in contemporary populations since
in recent times attrition is so moderate even in old age. There several
external factors that must be considered because they can affect age
estimation such as orthodontic treatment, root canal therapy, dental
restorations, trauma and occlusion. Therefore, techniques that rely on
the evaluation of non-restored teeth in normal occlusion and utilize
root translucency and secondary dentin formation as variables in the
age assessment tend to be more accurate (Lewis & Senn, 2013).
3.6. Age estimation based on biochemical parameters from dental samples
New methodologies for age estimation are based on the natural
process of aging, leading to alterations of tissues and organs on different
biochemical levels (Zapico & Ubelaker, 2013a, 2013b). In this line of
research, some of these new biochemical techniques have been applied
to teeth to determine this parameter.
3.7. Aspartic acid racemization
Aspartic acid racemization seems to be the most accurate technique
among all the new biochemical techniques. In fact, from its discovery in
1975 (Helfman & Bada, 1975), several studies have pointed out its
precision and its wide application among tissues. This technique is
based on a natural process, which converts optically active compounds
into a racemic mixture. In living systems, L-amino acids are commonly
found due to the stereochemical specificity of enzymes, which utilize
only the L-enantiomers. Racemization converts this L-form into D-form,
which alters the conformation of the metabolically stable proteins, in-
ducing changes in their biological or chemical activities (Masters et al.,
1977). These alterations may contribute to the progressive changes
associated with the aging process (Helfman, Bada, & Shou, 1977). In
particular, aspartic acid has one of the fastest racemization rates,
making it the ideal amino acid for use in forensic studies (Cloos &
Fledelius, 2000).
Thus, the forensic studies of the application of this technique to age-
at-death estimation in dentin (Helfman & Bada, 1976; Ohtani, 1995a;
Ritz, Schutz, & Schwarzer, 1990) and cementum (Ohtani, 1995c) found
a positive correlation between aspartic acid racemization and age.
Dentin was the best tissue for estimation of chronological age, based on
accuracy, simplicity and time required, since it suffers less con-
tamination than other dental tissues. With respect to the specific
technique for measurement, gas chromatography (GC) seems to be the
chosen method. However, this methodology is not exempt of draw-
backs, the degree of racemization is dependent of the type of tooth, and
different values were observed when labial and lingual portions of one
tooth were compared (Ritz et al., 1990, 1993). It is good practice to
analyze the “entire dentin of central longitudinal sections” and stan-
dardize sampling (Ohtani & Yamamoto, 1991a, 1991b, 1992). This
methodology is not reliable for corpses that have been exposed to
higher temperatures (Ritz, Schutz, & Peper, 1993, 1990) since racemi-
zation is a first-order chemical reaction influenced by temperature and
other factors (Arany & Ohtani, 2010). In spite of these drawbacks,
10
Archives of Oral Biology 87 (2018) 7–14
several studies have demonstrated the precision of this method in the
estimation of age in human corpses with low errors (around +3 years)
(Ogino, Ogino, & Nagy, 1985; Ohtani & Yamamoto, 2010; Ohtani,
1995b).
3.8. Lead
Lead is one of the most significant pollutants in the environment
(Al-Qattan & Elfawal, 2010). Its exposure is measured as tooth lead
concentration since dentin is the main site for lead deposition
(Steenhout & Pourtois, 1981). Thus, some authors analyzed the re-
lationship of lead accumulation to age. The results have been con-
troversial, in deciduous teeth, some studies found a linear increase with
age (Altshuller, Halak, Landing, & Kehoe, 1962), while others only
found this in one kind of tooth, like the canine (Mackie, Stephens, &
Townshend, 1977) or negative regression of the lower molar (Pinchin,
Newham, & Thompson, 1978). Other authors did not find any re-
lationship (Habercam, Keil, Reigart, & Croft, 1974; Holtzman et al.,
1968). In contrast, in permanent teeth several authors found a positive
correlation between lead levels and age (Al-Qattan & Elfawal, 2010;
Bercovitz & Laufer, 1991; Steenhout & Pourtois, 1981; Strehlow &
Kneip, 1969). In fact, with forensic purposes, Al-Qattan and Elfawal (Al-
Qattan & Elfawal, 2010) found a significant correlation between age
and dentin lead levels in a Kuwaiti population, with a difference be-
tween real age and calculated age 1.3 + 4.8 years. However, the pro-
posed formula is only applicable to the Kuwaiti population. Further
research is needed in other populations and environments.
3.9. Collagen crosslinks
The collagenous matrices of dentin and other skeletal connective
tissues are stabilized by covalent crosslinks between collagen molecules
(Eyre, 1987; Mechanic, Gallop, & Tanzer, 1971). These crosslinks are
formed through the intermolecular reactions of aldehyde residues made
on the protein monomers of lysyl oxidase (Piez, 1968). However, they
disappear as connective tissues mature (Eyre, Dickson, & Van Ness,
1988; Walters & Eyre, 1983), it is thought by further spontaneous re-
actions within the collagen polymer to form mature non-reducible
compounds (Bailey & Shimokomaki, 1971; Robins, Shimokomaki, &
Bailey, 1973). Deoxypyridinoline (DPD), a component of non-reducible
crosslinks, was analyzed in permanent molars from patients between 15
and 73 years old with forensic purposes (Martin-De Las Heras et al.,
1999) using an enzyme immunoassay method. They found an increase
in the DPD ratio in relation to the age, however, the estimated error of
this technique is +14.9 years at a 65% level of confidence.
3.10. Chemical composition
The mechanical characteristics of dentin decay with age. In fact, the
aging process produces a gradual formation of non-carious transparent
dentin, starting from the apex of the root and sometimes extending into
the coronal dentin (Micheletti Cremasco, 1998; Vasiliadis, Darling, &
Levers, 1983a; Vasiliadis, Darling, & Levers, 1983b). The transparency
of dentin is due to the mineralization of the dentin substance around the
dentinal tubules (peritubular dentin) and to the gradual reduction of
the dentinal tubules (Amprino & Engstrom, 1952). These tubules be-
come thinner with increasing age and their number is also reduced. It is
likely that these age-related changes are accompanied by changes in
chemical composition (Kosa, Antal, & Farkas, 1990). Thus, Raman
spectrum was used to analyze this composition, establishing a partial
correlation coefficient between predictors and age, providing mainly a
high value with a mean error of 5 years (Tramini, Bonnet, Sabatier, &
Maury, 2001).
Utilizing this technique in combination with UV resonance
(UVRRS), an increase was found in the amide I peak height in dehy-
drated and demineralized dentin, because of the increased interaction
J. Adserias-Garriga et al.
between collagen fibrils caused by stretching and intrafibrillar move-
ment (Ager et al., 2006).
3.11. Advanced Glycation Endproducts (AGEs)
The Maillard reaction is a complex series of reactions between re-
ducing sugars and amino groups on proteins, which lead to browning,
fluorescence, and cross-linking of proteins. AGEs products, formed
during the later stages of these reactions, accumulate in long-lived
tissue proteins, implicated in the development of complications in aging
and associated diseases (Baynes, 2001). Based on this browning reac-
tion and color changes, these compounds have been used to attempt to
make age estimations on hard dental tissues, like dentin and enamel
(Brudevold et al., 1957; De, 1950; Solheim, 1993), which become more
yellow with age. Ten Cate reached the same conclusions, finding an
increase in yellow roots with age (Ten Cate, Thompson, Dickinson, &
Hunter, 1977). However, Lackovic and Wood (Lackovic & Wood, 2000),
found that mesial surfaces of the roots were less yellow than the other
three surfaces, although they still found a positive increase in the per-
centage of the measured color with age. Lately, Martin de las Heras
et al. (Martin-De Las Heras et al., 2003) analyzed color changes with the
spectroradiometry technique, finding a correlation with age, although
the error was 13.7 years and was affected by the postmortem interval.
3.12. Telomere shortening
Chromosomes in eukaryotes are protected from degradation and
abnormal recombination by specialized end structures termed telo-
meres, simple repeating sequences of six bases in humans (Moyzis et al.,
1988). These structures are replicated by telomerase, a specific reverse
transcriptase that maintains the length of chromosomes. However, be-
cause of the requirement for an RNA primer, DNA polymerases cannot
replicate the extreme 3 ́ end of a parental DNA strand and, in the ab-
sence of compensatory mechanisms, telomeres shorten with each cell
division (Counter et al., 1992), and thus with aging. Taking this into
account, and using Southern blot non-radiactive analysis, telomere
length has been analyzed in pulp DNA from 100 healthy molars, finding
a high inverse correlation between telomere lengths and aging
(Takasaki, Tsuji, Ikeda, & Ohishi, 2003). This technique was applied to
forensic cases, although the error was between 7.52-10 years between
estimated and actual age (Tsuji et al., 2002). The cause of death and the
postmortem period are probable key factors.
3.13. Mitochondrial DNA
According to the aging theory presented by Harman (Harman,
1960), the production of free radicals rises with age, and plays a key
role in the degenerative processes of senescence as the origin of cellular
molecule damage. Particularly, mitochondrial DNA (mtDNA) is located
near the inner membrane of the mitochondria where the breakdown of
enzymes produces an abundance of free radicals causing heteroplasmic
mutations. These mutations induce decay in mitochondrial respiratory
function, which induces more mutations in mtDNA (Beckman & Ames,
1998a, 1998b; Harman, 1972; Wei & Lee, 2002). These mutations seem
to accumulate with age, so they could help to improve the estimation of
age-at-death. Although this approach has been analyzed in different
tissues, with a good correlation of mitochondrial mutations to age,
there are only two studies that have investigated this methodology in
teeth. Particularly, Mornstad et al. (Mornstad, Pfeiffer, Yoon, & Teivens,
1999) demonstrated a decrease in the amount of mtDNA in dentin with
age, analyzing the hypervariable region 2 (HV2) of the mtDNA in third
molars. A recent study from our group (Zapico & Ubelaker, 2016), also
developed in third molars, studied the efficiency of amplification of
HV2 through Real-Time PCR in dentin and pulp, finding a negative
strong linear correlation between mtDNA amplification and age in
dentin. However, no correlation was found in pulp, likely due to the
11
Archives of Oral Biology 87 (2018) 7–14
placement of the majority of the mitochondria in dentin. In this study,
two different populations were used finding alterations in mtDNA da-
mage between them, which suggests an ancestry component.
3.14. Epigenetic techniques
More recently, epigenetic research points to a new and growing field
for age-at-death estimation. Different studies indicate that global DNA
methylation levels decrease during aging (Fraga, 2009), even though
specific local CpG sites can either become hypo- or hyper-methylated
with age (Florath, Butterbach, Muller, Bewerunge-Hudler, & Brenner,
2014). CpG sites located in CpG islands in particular become hy-
permethylated, unlike the hypomethylated CpGs, which are usually
located outside these islands (Johansson, Enroth, & Gyllensten, 2013).
Using that knowledge, several groups have identified numerous CpG
sites that are significantly correlated with age, inferring this parameter
with linear models using a select set of DNA methylation markers in
single or multiple tissues (Johansson et al., 2013). Despite these studies
in different tissues, only a recent paper (Bekaert et al., 2015) explored
the application of this methodology in teeth. Methylation levels from
three age-associated genes (PDE4C, ELOVL2 and EDARADD), were
analyzed in dentin samples and quadratic was performed, with r2 0.74
and a mean absolute deviation (MAD) of 4.86 years. This preliminary
study in teeth opens the door to a new line of research towards age-at-
death estimation purposes.
One of the relatively new approaches is the use of radiocarbon
analysis to determine the year of birth regardless of the individual’s age
at death (Spalding, Bhardwaj, Buchholz, Druid, & Frisen, 2005;
Spalding, Buchholz, Bergman, Druid, & Frisen, 2005). Classically,
radiocarbon analysis (14C) has been used for dating archaeological
material, however it has been adapted for use in modern forensic cases
based on the radioactive decay of 14C in biological material (Libby,
Berger, Mead, Alexander, & Ross, 1964). This methodology is called
bomb-pulse 14C and it takes advantage of the substantial increase in
global 14C levels caused by above-ground nuclear test bomb detona-
tions 1955–1963 (Nydal & Lovseth, 1965; Spalding, Bhardwaj, et al.,
2005). Repeated measurements of 14C in the atmosphere and in bio-
logical products of known age has over time resulted in reference values
to which analytical results can be compared to offer an estimate of the
age (Spalding, Bhardwaj, et al., 2005). In fact, this analysis has been
improved by the application of accelerated mass spectrometry tech-
nology (AMS). This methodology counts all Ce 14 atoms and offers
more precise results using smaller samples than previously available
standard procedures (Ubelaker, 2014). Although different tissues have
been tested with this technique, bones and teeth seem to be most fre-
quently used. Spalding et al. (Spalding, Buchholz, et al., 2005) de-
scribed this analysis in dental enamel to assess the birth date of in-
dividuals if crown formation occurred during the bomb-pulse era.
Dental enamel does not remodel and preserves the radiocarbon values
at the time of formation. Lately, the same author presented additional
details on this approach (Buchholz & Spalding, 2010). In 2010, Alkass
et al. (Alkass et al., 2010) used dental enamel and radiocarbon analysis
to estimate the date of birth and combined the data with aspartic acid
racemization to determine the age-at-death. One year later, Ubelaker
and Parra (Ubelaker & Parra, 2011) analyzed radiocarbon levels in
dental enamel, femoral cortical bone and trabecular bone from the
vertebral bodies of four individuals (age-at-death between 16 and 56
years) from Peru. They found that dental enamel data were more ac-
curate with the known birth dates in consideration of the timing of
dental formation and the bomb curve values in the southern hemi-
sphere. In contrast, the radiocarbon values of the trabecular bone were
closer to the atmospheric values at the date of death than were those of
cortical bone. Several studies pointed out the accuracy of this technique
for estimation of date of birth with an error around 1.8 + 1.3 years
(Alkass et al., 2011; Alkass et al., 2013).
J. Adserias-Garriga et al.
4. Personal identification
Personal identification of the deceased is necessary for social, legal
and forensic reasons. A positive identification is a key factor for the
grieving process, because it can help in understanding and acceptance
of the loss, contributing to closure. In addition, the death certificate,
issued when a positive identification is established, is required to settle
legal affairs such as debt payment, life insurances, remarriage or child
custody. Moreover, criminal investigation is difficult to carry out
without the victim’s identification.
Personal identification involves the comparison of post-mortem
data and ante-mortem data. The process of identification and the spe-
cific method used depends on the circumstances of the case.
Nevertheless, the process of identification must always be accurate and
based on scientific principles (Herschaft, Alder, Ord, Rawson, & Smith,
2007).
4.1. Comparative identification by dental structures
Comparative identification by dental structures involves the post-
mortem dental examination of human remains, charting, taking radio-
graphic documentation of the specimens, performing analysis and ap-
plying the techniques that forensic odontologists have in their arsenal.
This post-mortem data and its scientific interpretation is then compared
to the ante-mortem data of missing persons to match with the remains.
The dentist of the deceased usually provides ante-mortem records.
Dental charts, radiographs, dental models, prosthetic and orthodontic
appliances or dental splints can be used for comparisons with the post-
mortem data. Smiling pictures (which have not been digitally altered),
where teeth are shown, can be also of great help to achieve an identity.
Finally, a forensic report will be written regarding the findings and the
conclusion of the comparison.
The American Board of Forensic Odontology established standards
and guidelines for dental identifications in 1994. They set forth that a
dental comparison and subsequent identification can lead to one of the
following four conclusions. The first is positive identification, which is
obtained when the ante-mortem and post-mortem data match in suffi-
cient detail to determine that they are from the same individual and
there are no irreconcilable discrepancies. The second, possible identi-
fication, is defined as the circumstances when the ante-mortem and
post-mortem data has consistent features, but, due to the quality of the
data, it is not possible to establish a positive dental identification. The
third possible outcome is insufficient evidence, which occurs when the
available information is insufficient to form the basis for a conclusion.
And finally, exclusion is reached when the ante-mortem and post-
mortem data are clearly inconsistent (ABFO, 2013; Berman, Bush, et al.,
2013; Herschaft et al., 2007). Computer technology advances have been
incorporated into the forensic identification process. DVI scenarios are
especially impacted by this need, where robust software programs
capable of handling ante-mortem and postmortem record management
are essential.
Computer Assisted Post Mortem Identification (CAPMI), developed
in late 1980s was the first software for dental identification. CAPMI was
used in the Alfred P. Murrah Federal Building bombing in Oklahoma
City, Oklahoma in 1995 and in the Crash of TWA Flight 800 off the
coast of Long Island, New York in 1996 (Berman, Chrz, et al., 2013).
WinID was developed by Dr. James McGivney as a free, computer-
assisted dental identification application. WinID has been widely used
in USA in instances such as the World Trade Center terrorist attack or
hurricanes Katrina and Ike (Berman, Chrz, et al., 2013). DVI System
International by PlassData Software is a global mass disaster program,
which includes a dental section. DVI System International is used
worldwide in international disasters following INTERPOL guidelines,
such as the Thai Tsunami in 2005, the Black Saturday bushfires in
Australia in 2009 or the German Wings plane crash in French Alps in
April 2015 Plass Data, 2016 (http://www.plassdata.com).
12
Archives of Oral Biology 87 (2018) 7–14
UVIS (Unified Victim Identification System) was developed for the
City of New York following the September 11th attack. UVIS Dental
Identification Module (UDIM) is the dental section of UVIS developed
by Dr. Kenneth Aschheim, forensic odontologist of the NYC Office of the
Chief Medical Examiner (NYC-OCME). This user-friendly program
permits unlimited image importation, partial jaw fragment manage-
ment, and linking and joining of specimens, all of which is very helpful
in cases that involve body fragmentation (Berman, Chrz, et al., 2013;
UVIS, 2009). UVIS/UDIM is used in DVI scenarios as well as in daily
casework at NYC-OCME, offering great advantages of personnel
training and identification speed.
OdontoSearch is a computer program developed by Dr. Bradley
Adams that provides an objective means of assessing the frequency of
occurrence of a certain dental pattern. The program works by com-
paring an individual's pattern of missing, filled, and unrestored teeth to
a large, representative sample of the U.S. population. Currently the
OdontoSearch 3.0 database includes dental records of 57,980 adults
(http://odontosearch.com). Unlike the other computer-assisted systems
mentioned above, OdontoSearch does not compare ante-mortem and
post- mortem dental records, but rather provides a scientific assessment
of the frequency of certain dental pattern in the general population.
4.2. DNA profiling from dental structures
As previously pointed out, teeth are the most durable structures in
the human body, and they can survive long after soft and skeletal tis-
sues have been destroyed owing to their inert and mineralized struc-
tures (Zapico & Ubelaker, 2013a, 2013b). Thus, in cases involving
skeletal remains, teeth are the main source used for DNA profiling.
Their role in this has been extensively reviewed (Sakari, Jimson,
Masthan, & Jacobina, 2015; Pajnic, 2016). However, it is important to
consider that in cases of burnt remains, sometimes even teeth are not
useful. A recent study from our group (Adserias, Ubelaker, & Zapico,
2016) analyzed the macroscopic changes and efficiency of DNA pro-
filing at different temperatures and times, pointing out that the highest
temperature at which it is still possible to get a full DNA profile is
around 300 °C. Also, our study demonstrated that some STRs are more
prone to degradation than others, depending on where they are placed
in the chromosome. With its basis in the housekeeping gene amplifi-
cation efficiency, this study suggests looking for other identification
markers like the oldest RFLPs for this type of case.
5. Conclusions
The first step in the identification process is to build up a biological
profile of the unknown, followed by a comparison between the ante-
mortem data collected and the post-mortem data obtained from the
study of the remains. Fingerprinting, dental record comparison and
DNA matching are the scientific methods to use to achieve a positive
identification.
The examination of teeth and oral structures can provide several
identified characteristics of the individual contributing to the re-
construction of biological profile. In comparative identification, for-
ensic odontology offers an expeditious method of identification based
on dental traits, treatment and pathology. DNA profiling has been tra-
ditionally used for human identification, but recent advances in bio-
chemistry are applying new methodologies to determine the biological
profile.
Teeth are excellent samples for biochemical analysis and simulta-
neously the field of forensic odontology is evolving, introducing new
technologies. These two facts lead to a grand partnership between
forensic odontology and biochemistry, contributing to the advancement
of forensic science.
J. Adserias-Garriga et al.
References
American Board of Forensic Odontology (ABFO) (2013). Diplomates reference manual.
http://www.abfo.org.
Adserias, J., Ubelaker, D. H., & Zapico, S. C. (2016). Evaluation of macroscopic changes
and the efficiency of DNA profiling from burnt teeth. Science and Justice, 56, 437–442.
Ager, J. W., 3rd, Nalla, R. K., Balooch, G., Kim, G., Pugach, M., Habelitz, S., ... Ritchie, R.
O. (2006). On the increasing fragility of human teeth with age: A deep-UV resonance
Raman study. Journal of Bone and Mineral Research, 21, 1879–1887.
Akane, A., Shiono, H., Matsubara, K., Nakahori, Y., Seki, S., Nagafuchi, S., ... Nakagome,
Y. (1991). Sex identification of forensic specimens by polymerase chain reaction
(PCR): two alternative methods. Forensic Science International, 49, 81–88.
Al-Qattan, S. I., & Elfawal, M. A. (2010). Significance of teeth lead accumulation in age
estimation. Journal of Forensic and Legal Medicine, 17, 325–328.
AlQahtani, S. J., Hector, M. P., & Liersidge, H. M. (2010). Brief communication: The
London atlas of human tooth development and eruption. American Journal of Physical
Anthropology, 142, 481–490.
Alkass, K., Buchholz, B. A., Ohtani, S., Yamamoto, T., Druid, H., & Spalding, K. L. (2010).
Age estimation in forensic sciences: Application of combined aspartic acid racemi-
zation and radiocarbon analysis. Molecular & Cellular Proteomics, 9, 1022–1030.
Alkass, K., Buchholz, B. A., Druid, H., & Spalding, K. L. (2011). Analysis of 14C and 13C in
teeth provides precise birth dating and clues to geographical origin. Forensic Science
International, 209, 34–41.
Alkass, K., Saitoh, H., Buchholz, B. A., Bernard, S., Holmlund, G., Senn, D. R., ... Druid, H.
(2013). Analysis of radiocarbon, stable isotopes and DNA in teeth to facilitate iden-
tification of unknown decedents. PloS One, 8, e69597.
Altshuller, L. F., Halak, D. B., Landing, B. H., & Kehoe, R. A. (1962). Deciduous teeth as an
index of body burden of lead. The Journal of Pediatrics, 60, 224–229.
Amprino, R., & Engstrom, A. (1952). Studies on x ray absorption and diffraction of bone
tissue. Acta Anatomica, 15, 1–22.
Arany, S., & Ohtani, S. (2010). Age estimation by racemization method in teeth:
Application of aspartic acid, glutamate, and alanine. Journal of Forensic Sciences, 55,
701–705.
Arany, S., Iino, M., & Yoshioka, N. (2004). Radiographic survey of third molar develop-
ment in relation to chronological age among Japanese juveniles. Journal of Forensic
Sciences, 49, 534–538.
Bailey, A. J., & Shimokomaki, M. S. (1971). Age related changes in the reducible cross-
links of collagen. FEBS Letters, 16, 86–88.
Bansal, A. K., Shetty, D. C., Bindal, R., & Pathak, A. (2012). Amelogenin: A novel protein
with diverse applications in genetic and molecular profiling. Journal of Oral and
Maxillofacial Pathology, 16, 395–399.
Bath-Balogh, M., & Fehrenbach, M. J. (2006). EnamelDental embryology, histology, and
anatomy (2nd ed.). St. Louis, MO: Elsevier Saunders179–189.
Baynes, J. W. (2001). The role of AGEs in aging: Causation or correlation. Experimental
Gerontology, 36, 1527–1537.
Beckman, K. B., & Ames, B. N. (1998a). The free radical theory of aging matures.
Physiological Reviews, 78, 547–581.
Beckman, K. B., & Ames, B. N. (1998b). Mitochondrial aging: Open questions. Annals of
the New York Academy of Sciences, 854, 118–127.
Bekaert, B., Kamalandua, A., Zapico, S. C., Van De Voorde, W., & Decorte, R. (2015).
Improved age determination of blood and teeth samples using a selected set of DNA
methylation markers. Epigenetics, 1–9.
Bercovitz, K., & Laufer, D. (1991). Age and gender influence on lead accumulation in root
dentine of human permanent teeth. Archives of Oral Biology, 36, 671–673.
Berman, G. M., Bush, M. A., Bush, P. J., Freeman, A. J., Loomis, P. W., & Miller, R. G.
(2013). Dental identification. In D. R. Senn, & R. A. Weems (Eds.). Manual of forensic
odontology (pp. 81–87). (5th ed.). Boca Raton: CRC Press.
Berman, G. M., Chrz, B., Nawrocki, L. A., Hermsen, K. P., Miller, R. G., & Weems, R. A.
(2013). Disaster victim identification. In D. R. Senn, & R. A. Weems (Eds.). Manual of
forensic odontology (pp. 186–191). (5th ed.). Boca Raton: CRC Press.
Blankenship, J. A., Mincer, H. H., Anderson, K. M., Woods, M. A., & Burton, E. L. (2007).
Third molar development in the estimation of chronologic age in American Blacks as
compared with Whites. Journal of Forensic Sciences, 52(2), 428–433.
Brothwell, D. R. (1989). The relationship of tooth wear to aging. In M. Y. Isçan (Ed.). Age
markers in human skeleton (pp. 303–316). Springfield: CC Thomas.
Brudevold, F., Hein, J. W., Bonner, J. F., Nevin, R. B., Bibry, B. G., & Hodge, H. C. (1957).
Reaction of tooth surfaces with one ppm of fluoride as sodium fluoride. Journal of
Dental Research, 36, 771–779.
Buchholz, B. A., & Spalding, K. L. (2010). Year of birth determination using radiocarbon
dating of dental enamel. Surface and Interface Analysis, 42, 398–401.
Cameriere, R., Ferrante, L., & Cingolani, M. (2004). Precision and reliability of pulp/tooth
area ration (RA) of second molar as indicator of adult age. Journal of Forensic Sciences,
49, 1319–1323.
Chesson, L. A., Podlesak, D. W., Thompson, A. H., Cerling, T. E., & Ehleringer, J. R.
(2008). Variation of hydrogen, carbon, nitrogen, and oxygen stable isotope ratios in
an American diet: Fast food meals. Journal of Agricultural and Food Chemistry, 56,
4084–4091.
Cloos, P. A., & Fledelius, C. (2000). Collagen fragments in urine derived from bone re-
sorption are highly racemized and isomerized: A biological clock of protein aging
with clinical potential. The Biochemical Journal, 345(Pt 3), 473–480.
Copenhaver, W. M., Kelly, D. E., & Wood, R. L. (1978). The digestive systemBailey’s textbook
of histology (17th ed.). Baltimore, MD: Williams & Wilkins455–551.
Counter, C. M., Avilion, A. A., Lefeuvre, C. E., Stewart, N. G., Greider, C. W., Harley, C. B.,
& Bacchetti, S. (1992). Telomere shortening associated with chromosome instability
is arrested in immortal cells which express telomerase activity. The EMBO Journal, 11,
1921–1929.
Cunha, E., Baccino, E., Martrille, L., Ramsthaler, F., Prieto, J., Schuliar, Y., ... Cattaneo, C.
(2009). The problem of aging human remains and living individuals: A review.
Forensic Science International, 193(1-3), 1–13.
De, J. T. (1950). Senescence of the dentition. Paradentologie, 4, 83–98.
13
Archives of Oral Biology 87 (2018) 7–14
Demirjian, A., & Goldstein, H. (1976). New systems for dental maturity based on seven
and four teeth. Annals of Human Biology, 3, 411–421.
Demirjian, A., Goldstein, H., & Tanner, J. M. (1973). A new system of dental age as-
sessment. Human Biology, 45, 211–227.
Dumache, R., Ciocan, V., Muresan, C., & Enache, A. (2016). Molecular DNA analysis in
forensic identification. Clinical Laboratory, 62(1-2), 245–248.
Elamin, F., & Liversidge, H. M. (2013). Malnutrition has no effect on the timing of human
tooth formation. PLoS ONE, 8, e72274.
Eyre, D. R., Dickson, I. R., & Van Ness, K. (1988). Collagen cross-linking in human bone
and articular cartilage: Age-related changes in the content of mature hydro-
xypyridinium residues. The Biochemical Journal, 252, 495–500.
Eyre, D. (1987). Collagen cross-linking amino acids. Methods in Enzymology, 144,
115–139.
Florath, I., Butterbach, K., Muller, H., Bewerunge-Hudler, M., & Brenner, H. (2014).
Cross-sectional and longitudinal changes in DNA methylation with age: An epi-
genome-wide analysis revealing over 60 novel age-associated CpG sites. Human
Molecular Genetics, 23, 1186–1201.
Fraga, M. F. (2009). Genetic and epigenetic regulation of aging. Current Opinion in
Immunology, 21, 446–453.
Friedling, L. J., & Morris, A. G. (2005). The frequency of culturally derived dental
modification practices on the Cape Flats in the Western Cape. SADJ, 60(3), 97–102.
Fukushima, H., Hasekura, H., & Nagai, K. (1988). Identification of male bloodstains by
dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA)
probe. Journal of Forensic Sciences, 33, 621–627.
Garn, S. M., Lewis, A. B., & Kerewsky, R. S. (1965). Genetic, nutritional and maturational
correlates of dental development. Journal of Dental Research, 44, 228–243.
Goncalves, V. F., Parra, F. C., Goncalves-Dornelas, H., Rodrigues-Carvalho, C., Silva, H. P.,
& Pena, S. (2010). Recovering mitochondrial DNA lineages of extinct Amerindian
nations in extant homopatric Brazilian populations. Investigative Genetics, 1, 13.
Gustafson, G. (1950). Age determination on teeth. Journal of American Dental Association,
41, 45–54.
Habercam, J. W., Keil, J. E., Reigart, J. R., & Croft, H. W. (1974). Lead content of human
blood, hair, and deciduous teeth: Correlation with environmental factors and growth.
Journal of Dental Research, 53, 1160–1163.
Harman, D. (1960). The free radical theory of aging: The effect of age on serum mer-
captan levels. Journal of Gerontology, 15, 38–40.
Harman, D. (1972). The biologic clock: The mitochondria? Journal of the American
Geriatrics Society, 20, 145–147.
Harris, E. F., Mincer, H. H., Anderson, K. M., & Senn, D. R. (2010). Age estimation from
oral and dentalstructures. In D. R. Senn, & P. G. Stimson (Eds.). Forensic dentistry (pp.
263–291). (2nd ed.). Boca Raton: CRC Press.
Helfman, P. M., & Bada, J. L. (1975). Aspartic acid racemization in tooth enamel from
living humans. Proceedings of the National Academy of Sciences of the United States of
America, 72, 2891–2894.
Helfman, P. M., & Bada, J. L. (1976). Aspartic acid racemisation in dentine as a measure
of aging. Nature, 262, 279–281.
Helfman, P. M., Bada, J. L., & Shou, M. Y. (1977). Considerations on the role of aspartic
acid racemization in the aging process. Gerontology, 23, 419–425.
Herschaft, E. E., Alder, M. A., Ord, D. K., Rawson, R. D., & Smith, E. S. (2007). Manual of
forensic odontology: American society of forensic odontology (4th ed.). Boca Raton: CRC
Press [pp. 7].
Hollowell, W. H., & Childers, N. K. (2007). A new threat to adolescent oral health: The
grill. Paediatric Dentistry. 29(4), 320–322.
Holtzman, R. B., Lucas, H. F., Jr., & Ilcewicz, F. H. (1968). The concentration of lead in
human bone ANL-7615 . Anl. 43–49.
Horiuchi, N., Morisaki, T., Fujii, H., & Miwa, S. (1988). A simple human sex determi-
nation method using biotin-labeled probes. Nihon hoigaku zasshi= The Japanese
Journal of Legal Medicine, 42, 351–353.
Johanson, G. (1971). Age determinations from human teeth: A critical evaluation with
special consideration of changes after fourteen years of age. Odontologisk Revy, 22,
1–126.
Johansson, A., Enroth, S., & Gyllensten, U. (2013). Continuous aging of the human DNA
methylome throughout the human lifespan. PloS One, 8, e67378.
Kapila, R., Nagesh, K. S., Iyengar, A. R., & Mehkri, S. (2011). Sexual dimorphism in
human mandibular canines: A radiomorphometric study in south indian population.
Journal of Dental Research, Dental Clinics and Dental Prospects, 5(2).
Kasper, K. A., Austin, D., Kvanli, A. H., Rios, T. R., & Senn, D. R. (2009). Reliability of
third molar development for age estimation in a Texas Hispanic population: A
comparison study. Journal of Forensic Sciences, 54(3), 651–657.
Kloosterman, A., Mapes, A., Geradts, Z., van Eijk, E., Koper, C., van den Berg, J., ... van
Asten, A. (2015). The interface between forensic science and technology: How
technology could cause a paradigm shift in the role of forensic institutes in the
criminal justice system. Philosophical Tranactions of the Royal Society of London, Series
B, Biological Sciences, 370, 20140264.
Kobayashi, R., Nakauchi, H., Nakahori, Y., Nakagome, Y., & Matsuzawa, S. (1988). Sex
identification in fresh blood and dried bloodstains by a nonisotopic deoxyribonucleic
acid (DNA) analyzing technique. Journal of Forensic Sciences, 33, 613–620.
Kosa, F., Antal, A., & Farkas, I. (1990). Electron probe microanalysis of human teeth for
the determination of individual age. Medicine, Science, and the Law, 30, 109–114.
Kubasek, J., Urban, O., & Santrucek, J. (2013). C4 plants use fluctuating light less effi-
ciently than do C3 plants: A study of growth, photosynthesis and carbon isotope
discrimination. Physiologia Plantarum, 149(4), 528–539.
Kvaal, S. I., & Solheim, T. (1994). A non-destructive dental method for age estimation.
Journal of Forensic Odontostomatology, 12, 6–11.
Lackovic, K. P., & Wood, R. E. (2000). Tooth root color as a measure of chronological age.
The Journal of Forensic Odonto-Stomatology, 18, 37–45.
Lamendin, H., Baccino, E., Humbert, J. F., Tavernier, J. C., Nossintchouk, R. M., & Zerilli,
A. (1992). A simple technique for age estimation in adult corpses: The two criteria
dental method. Journal of Forensic Sciences, 37(5), 1373–1379.
Lewis, J. M., & Senn, D. R. (2013). Dental age estimation. In D. R. Senn, & R. A. Weems
J. Adserias-Garriga et al.
(Eds.). Manual of forensic odontology (pp. 211–255). (5th ed.). Boca Raton: CRC Press.
Lewis, J. M., Senn, D. R., & Alder, M. E. (2001). Standardization, automation and data-
base creation for age determination reports. Paper presented at the annual american
academy of forensic sciences meeting.
Lewis, J. M., Senn, D. R., & Silvaggi, J. (2008). UT-Age 2008: an update. Paper presented at
the annual american academy of forensic sciences meeting.
Libby, W. F., Berger, R., Mead, J. F., Alexander, G. V., & Ross, J. F. (1964). Replacement
rates for human tissue from atmospheric radiocarbon. Science, 146, 1170–1172.
Mackie, A. C., Stephens, R., & Townshend, A. (1977). Tooth lead levels in Birmingham
children. Archives of Environmental Health, 32, 178–185.
Maples, W. R., & Rice, W. R. (1979). Some difficulties in the Gustafson dental age esti-
mations. Journal of Forensic Sciences, 24, 168–172.
Maples, W. R. (1978). An improved technique using dental histology for estimation of
adult age. Journal of Forensic Sciences, 23(4), 764–770.
Martin-De Las Heras, S., Valenzuela, A., & Villanueva, E. (1999). Deoxypyridinoline
crosslinks in human dentin and estimation of age. International Journal of Legal
Medicine, 112, 222–226.
Martin-De Las Heras, S., Valenzuela, A., Bellini, R., Salas, C., Rubino, M., & Garcia, J. A.
(2003). Objective measurement of dental color for age estimation by spectro-
radiometry. Forensic Science International, 132, 57–62.
Masters, P. M., Bada, J. L., & Zigler, J. S., Jr. (1977). Aspartic acid racemisation in the
human lens during aging and in cataract formation. Nature, 268, 71–73.
Mechanic, G., Gallop, P. M., & Tanzer, M. L. (1971). The nature of crosslinking in col-
lagens from mineralized tissues. Biochemical and Biophysical Research Communications,
45, 644–653.
Micheletti Cremasco, M. (1998). Dental histology: Study of aging processes in root den-
tine. Bollettino Della Societa Italiana Di Biologia Sperimentale, 74, 19–28.
Mincer, H. H., Harris, E. F., & Berryman, H. E. (1993). The A. B. F. O. study of third molar
devel- opment and its use as an estimator of chronological age. Journal of Forensic
Sciences, 38(2), 379–390.
Moorrees, C. F. A., Fanning, E. A., & Hunt, E. E., Jr. (1963a). Formation and resorption of
three deciduous teeth in children. American Journal of Physical Anthropology, 21,
205–213.
Moorrees, C. F. A., Fanning, E. A., & Hunt, E. E., Jr. (1963b). Age variation of formation
stages for ten permanent teeth. Journal of Dental Research, 42, 1490–1502.
Mornstad, H., Pfeiffer, H., Yoon, C., & Teivens, A. (1999). Demonstration and semi-
quantification of mtDNA from human dentine and its relation to age. International
Journal of Legal Medicine, 112, 98–100.
Moyzis, R. K., Buckingham, J. M., Cram, L. S., Dani, M., Deaven, L. L., Jones, M. D., ... Wu,
J. R. (1988). A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the
telomeres of human chromosomes. Proceedings of the National Academy of Sciences of
the United States of America, 85, 6622–6626.
Murakami, H., Yamamoto, Y., Yoshitome, K., Ono, T., Okamoto, O., Shigeta, Y., ... Ishizu,
H. (2000). Forensic study of sex determination using PCR on teeth samples. Acta
Medica Okayama, 54, 21–32.
Nydal, R., & Lovseth, K. (1965). Distribution of radiocarbon from nuclear tests. Nature,
206, 1029–1031.
Ogino, T., Ogino, H., & Nagy, B. (1985). Application of aspartic acid racemization to
forensic odontology: Post mortem designation of age at death. Forensic Science
International, 29, 259–267.
Ohtani, S., & Yamamoto, K. (1991a). Age estimation using the racemization of amino acid
in human dentin. Journal of Forensic Sciences, 36, 792–800.
Ohtani, S., & Yamamoto, K. (1991b). Estimation of ages from racemization of an amino
acid in teeth?assessment of errors under various experimental conditions. Nihon
hoigaku zasshi = The Japanese Journal of Legal Medicine, 45, 124–127.
Ohtani, S., & Yamamoto, K. (1992). Estimation of age from a tooth by means of race-
mization of an amino acid, especially aspartic acid–comparison of enamel and dentin.
Journal of Forensic Sciences, 37, 1061–1067.
Ohtani, S., & Yamamoto, T. (2010). Age estimation by amino acid racemization in human
teeth. Journal of Forensic Sciences, 55, 1630–1633.
Ohtani, S. (1995a). Estimation of age from dentin by using the racemization reaction of
aspartic acid. The American Journal of Forensic Medicine and Pathology, 16, 158–161.
Ohtani, S. (1995b). Estimation of age from the teeth of unidentified corpses using the
amino acid racemization method with reference to actual cases. The American Journal
of Forensic Medicine and Pathology, 16, 238–242.
Ohtani, S. (1995c). Studies on age estimation using racemization of aspartic acid in ce-
mentum. Journal of Forensic Sciences, 40, 805–807.
Pajnic, I. Z. (2016). Extraction of DNA from human skeletal material. Methods Molecular
Biology, 1420, 89–108.
Pettenati-Soubayroux, I., Signoli, M., & Dutour, O. (2002). Sexual dimorphism in teeth:
Discriminatory effectiveness of permanent lower canine size observed in a XVIIIth
century osteological series. Forensic Science International, 126(3), 227–232.
Piez, K. A. (1968). Cross-linking of collagen and elastin. Annual Review of Biochemistry, 37,
547–570.
Pinchin, M. J., Newham, J., & Thompson, R. P. (1978). Lead, copper, and cadmium in
teeth of normal and mentally retarded children. Clinica Chimica Acta; International
Journal of Clinical Chemistry, 85, 89–94.
Plass Data webside: http://www.plassdata.com.
Potsch, L., Meyer, U., Rothschild, S., Schneider, P. M., & Rittner, C. (1992). Application of
DNA techniques for identification using human dental pulp as a source of DNA.
International Journal of Legal Medicine, 105, 139–143.
Prince, D. A., & Ubelaker, D. H. (2002). Application of Lamendin’s adult aging technique
to a diverse skeletal sample. Journal of Forensic Sciences, 47(1), 107–116.
14
Archives of Oral Biology 87 (2018) 7–14
Ritz, S., Schutz, H. W., & Schwarzer, B. (1990). The extent of aspartic acid racemization in
dentin: A possible method for a more accurate determination of age at death?
Zeitschrift Fur Rechtsmedizin Journal of Legal Medicine, 103, 457–462.
Ritz, S., Schutz, H. W., & Peper, C. (1993). Postmortem estimation of age at death based
on aspartic acid racemization in dentin: Its applicability for root dentin. International
Journal of Legal Medicine, 105, 289–293.
Robins, S. P., Shimokomaki, M., & Bailey, A. J. (1973). The chemistry of the collagen
cross-links: Age-related changes in the reducible components of intact bovine col-
lagen fibres. The Biochemical Journal, 131, 771–780.
Sakari, S. L., Jimson, S., Masthan, K. M., & Jacobina, J. (2015). Role of DNA profiling in
forensic odontology. Journal of Pharmacy & Bioallied Sciences, 7, S138–141.
Schmeling, A., Olze, A., Reisinger, W., & Geserick, G. (2004). Forensic age diagnostics of
living people undergoing criminal proceedings. Forensic Science International, 144,
243–245.
Schour, I., & Massler, M. (1941). The development of the human dentition. Journal of the
American Dental Association, 28, 1153–1160.
Schwartz, G. T., & Dean, M. C. (2005). Sexual dimorphism in modern human permanent
teeth. American Journal of Physical Anthropology, 128(2), 312–317.
Senn, D. R., & Stimson, P. G. (2010). Future of forensic dentistry. In D. R. Senn, & P. G.
Stimson (Eds.). Forensic dentistry (pp. 405–406). (2nd ed.). Boca Raton: CRC Press.
Silva, A. M., Pereira, M. L., Gouveia, S., Tavares, J. N., Azevedo, A., & Caldas, I. M.
(2016). A new approach to sex estimation using the mandibular canine index. Med Sci
Law, 56(1), 7–12.
Sivagami, A. V., Rao, A. R., & Varshney, U. (2000). A simple and cost-effective method for
preparing DNA from the hard tooth tissue, and its use in polymerase chain reaction
amplification of amelogenin gene segment for sex determination in an Indian po-
pulation. Forensic Science International, 110, 107–115.
Solheim, T. (1993). A new method for dental age estimation in adults. Forensic Science
International, 59, 137–147.
Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., & Frisen, J. (2005).
Retrospective birth dating of cells in humans. Cell, 122, 133–143.
Spalding, K. L., Buchholz, B. A., Bergman, L. E., Druid, H., & Frisen, J. (2005). Forensics:
Age written in teeth by nuclear tests. Nature, 437, 333–334.
Steenhout, A., & Pourtois, M. (1981). Lead accumulation in teeth as a function of age with
different exposures. British Journal of Industrial Medicine, 38, 297–303.
Strehlow, C. D., & Kneip, T. J. (1969). The distribution of lead and zinc in the human
skeleton. American Industrial Hygiene Association Journal, 30, 372–378.
Takasaki, T., Tsuji, A., Ikeda, N., & Ohishi, M. (2003). Age estimation in dental pulp DNA
based on human telomere shortening. International Journal of Legal Medicine, 117,
232–234.
Ten Cate, A. R., Thompson, G. W., Dickinson, J. B., & Hunter, H. A. (1977). The esti-
mation of age of skeletal remains from the color of roots of teeth. Dental Journal, 43,
83–86.
Thompson, T., & Black, S. (2006). Forensic human identification: An introduction. CRC Press
[ISBN 9780849339547].
Tramini, P., Bonnet, B., Sabatier, R., & Maury, L. (2001). A method of age estimation
using Raman microspectrometry imaging of the human dentin. Forensic Science
International, 118, 1–9.
Tsuji, A., Ishiko, A., Takasaki, T., & Ikeda, N. (2002). Estimating age of humans based on
telomere shortening. Forensic Science International, 126, 197–199.
Tyler, M. G., Kirby, L. T., Wood, S., Vernon, S., & Ferris, J. A. (1986). Human blood stain
identification and sex determination in dried blood stains using recombinant DNA
techniques. Forensic Science International, 31, 267–272.
Unified Victim Identification System (UVIS) (2009). Unified victim identification system
(UVIS) guide. [www.nyc.gov/…/UVIS%20Information%20Guide_20090917.pdf].
Ubelaker, D. H., & Parra, R. C. (2011). Radiocarbon analysis of dental enamel and bone to
evaluate date of birth and death: Perspective from the southern hemisphere. Forensic
Science International, 208, 103–107.
Ubelaker, D. H. (1989). Human skeletal remains, excavation analysis, interpretation (2nd
ed.). Washington, DC: Taraxacum.
Ubelaker, D. H. (2014). Radiocarbon analysis of human remains: A review of forensic
applications. Journal Of Forensic Sciences, 59, 1466–1472.
Vasiliadis, L., Darling, A. I., & Levers, B. G. (1983a). The amount and distribution of
sclerotic human root dentine. Archives of Oral Biology, 28, 645–649.
Vasiliadis, L., Darling, A. I., & Levers, B. G. (1983b). The histology of sclerotic human root
dentine. Archives of Oral Biology, 28, 693–700.
Walters, C., & Eyre, D. R. (1983). Collagen crosslinks in human dentin: Increasing content
of hydroxypyridinium residues with age. Calcified Tissue International, 35, 401–405.
Wei, Y. H., & Lee, H. C. (2002). Oxidative stress, mitochondrial DNA mutation, and im-
pairment of antioxidant enzymes in aging. Experimental Biology and Medicine
(Maywood), 227, 671–682.
Witas, H. W., Tomczyk, J., Jedrychowska-Danska, K., Chaubey, G., & Ploszaj, T. (2013).
mtDNA from the early Bronze Age to the Roman period suggests a genetic link be-
tween the Indian subcontinent and Mesopotamian cradle of civilization. PloS One, 8,
e73682.
Zapico, S. C., & Ubelaker, D. H. (2013a). Applications of physiological bases of aging to
forensic sciences: Estimation of age-at-death. Ageing Research Rreviews, 12, 605–617.
Zapico, S. C., & Ubelaker, D. H. (2013b). Sex determination from dentin and pulp in a
medicolegal context. Journal of American Dental Association, 144, 1379–1385.
Zapico, S. C., & Ubelaker, D. H. (2016). Relationship between mitochondrial DNA mu-
tations and aging. Estimation of age-at-death. The Journals of Gerontology: Series A,
Biological Sciences and Medical Sciences, 71(4), 445–450.