Tanya M. Smith
Methods of dental histology as applied in archaeological science, including sample
preparation and analysis.
Because they are highly mineralised, teeth are one of the best preserved and most
commonly recovered elements in archaeological and fossil assemblages. They have
inspired more than a century of comparative studies of hominin tooth size and
shape (e.g., Bailey 2002; Brace et al. 1991; Dubois 1892; Hanihara 2008; Hanihara and
Ishida 2005; Hooijer 1948; Irish and Guatelli-Steinberg 2003; Keith 1913; Le Gros
Clark 1950; Weidenreich 1937; Wolpoff 1971; Wood et al. 1991). Additional valuable
information is recorded on outer surfaces and inner aspects of the dental hard
tissues (enamel, dentine, and cementum) that make up tooth crowns and roots,
providing a permanent record of growth. Initial study of dental histology, or
microscopic tooth structure, predates the ﬁelds of archaeology and evolutionary
biology by several centuries. The innovative microscopist Anthony Leeuwenhoeck
ﬁrst described the structure of enamel in the 1600s, noting that it was made of
longitudinal “pipes”(enamel prisms) that appeared as “globules”when viewed end-
on (Leeuwenhoeck 1677–1678). During the 1800s and early 1900s, microscopic
investigations revealed the tubular nature of dentine and the presence of successive
temporal lines in enamel and dentine (reviewed in Dean 1995; Smith 2006). By the
1940s, American and Japanese teams had experimentally demonstrated the pres-
ence of circadian structural features, as well as the neonatal (birth) line, which
allows one to relate developmental time to chronological (calendar) age in juvenile
dentitions. These incremental features form the basis of a growing area of
anthropological study that is illuminating aspects of human evolutionary develop-
mental biology, as well as the health and demography of past human populations.
Tooth development in humans begins before birth and continues throughout
adolescence. Like many biological systems, the formation of dental hard tissue is
characterised by a circadian rhythm, which manifests in the enamel and dentine
and remains unchanged in these tissues for millions of years after death. Enamel is
secreted by cells known as ameloblasts, which differentiate at the enamel-dentine
junction and migrate outward toward what becomes the surface of the crown. The
tracks left by these individual cells are known as enamel prisms. These prisms show
cross-striations that result from the circadian rhythm of enamel secretion
(Figure 8.1) (Bromage 1991; Smith 2006). The successive positions of the advancing
front of forming enamel are preserved as long-period incremental structures
termed Retzius lines, which contact the enamel surface and form circumferential
rings known as perikymata (illustrated below). Cross-striations and Retzius lines
are also frequently referred to as short- and long-period structures due to their
respective 24-hour and greater than 24-hour rhythms. An important relationship
exists between these types of internal lines, since the long-period line periodicity
(repeat interval or number of days between long-period lines) can be determined
only by counting cross-striations between Retzius lines. This value is believed to be
the same for all teeth in an individual’s dentition, although it may vary among
individuals (FitzGerald 1995; FitzGerald 1998). Modern human and fossil hominin
periodicities range from 6–12 days (Antoine et al. 2009; FitzGerald 1998; Smith
2008); tentative evidence suggests that fossil hominin ranges were once as wide as
5–13 days (Smith et al. 2015). Dentine is produced by cells known as odontoblasts
that generate dentine tubules, and shows daily incremental lines known as von
Ebner’s lines (equivalent to cross-striations), long-period structures known as
Andresen lines (equivalent to Retzius lines) and periradicular bands (equivalent to
perikymata) (reviewed in Dean 1995; Smith 2008; Smith and Reid 2009). Tooth
cementum shows annual incremental features known as cementum annulations
(e.g., Kay et al. 1984; Lieberman 1994; Wittwer-Backofen et al. 2004), but due to the
difﬁculty of accurately identifying a full series of these lines in adult teeth (Renz and
Radlanski 2006; Wittwer-Backofen et al. 2008), they are rarely used in archaeo-
Counts and measurements of incremental features have been used to determine
the timing of tooth formation, stress experienced during development, and the age
at death in juvenile fossil humans and apes (reviewed in Dean 2006; Hillson 2014;
Smith 2008). Archaeological applications, which are reviewed below, have mainly
dental histology 171
ﬁgure 8.1 Polarised light images of enamel and dentine microstructure in the Scladina Nean-
derthal upper ﬁrst molar. A. long-period Retzius lines in enamel (arrowed); B. overview of the
embedded block (prior to reconstruction) showing the position of higher magniﬁcation images in
A and D; C. Retzius lines (arrowed) and cross-striations in enamel (light and dark bands indicated by
brackets); D. Andresen lines (arrowed) and von Ebner’s lines in dentine (light and dark bands
indicated by brackets). (Adapted from Smith et al. 2014)
172 tan ya m. s mit h
focused on aspects of human developmental variation, determinations of age at
death and defects of enamel formation. These data may provide insight into
population-level developmental variation, mortality rates/demographic structure
and living conditions of prehistoric cultures, respectively. Dental microstructure
analyses also hold promise for the integration of temporal and chemical infor-
mation locked inside teeth (e.g., Austin et al. 2013; Humphrey et al. 2007; Humphrey
et al. 2008a,b; Richards et al. 2008; Sponheimer et al. 2006). In the subsequent
sections, common methods of histological preparation and analysis are presented
(Table 8.1), followed by a review of archaeological applications of dental histology
and a case study illustrating how these methods may be used to detail the dental
development, weaning process, and age at death of a juvenile Belgian Neanderthal.
2 methods of study
Impressions and Casts
Histological study typically requires careful preparation of high-resolution impres-
sions of external tooth surfaces, along with physical sectioning and generation of
thin (histological) sections. In both cases, high-resolution microscopy (with optical
magniﬁcation factors of 200–500X) is needed to image and quantify incremental
features on tooth surfaces or from histological sections. Recently, a new non-
destructive approach (virtual histology) has been developed using synchrotron
phase-contrast X-ray imaging (Figure 8.2; reviewed in Le Cabec et al. 2015; Smith
and Tafforeau 2008; Tafforeau and Smith 2008), which is currently possible at the
European Synchrotron Radiation Facility in Grenoble, France, the Swiss Light
Source in Villigen, Switzerland and Elettra in Trieste, Italy. In the following section,
aspects of conventional sample preparation are reviewed, followed by descriptions
and illustrations of the various imaging techniques commonly used to assess
microscopic tooth structure.
Initial analytical steps consist of macroscopic or stereoscopic photography,
followed by micro-computed tomography (micro-CT) of particularly valuable
samples. It is advisable to create a careful record prior to physical sectioning;
micro-CT provides a means of digitally archiving a sample in three dimensions,
which can be virtually sectioned in advance to guide physical sectioning, or
quantiﬁed volumetrically and physically reproduced with a 3D printer (e.g., Mac-
chiarelli et al. 2006; Olejniczak et al. 2007; Smith et al. 2007a; Smith and Tafforeau
dental histology 173
Table 8.1Methods for studying tooth growth and development.
Technique Application Exemplars
Replicate surface microstructure
(perikymata, hypoplasias from molds
Beynon 1987; Hillson
Thin Sectioning Prepare samples (~0.1mm thick) for light
Antoine et al. 2009;
Reid et al. 1998a
Image dental thin sections from a controlled
Stereomicroscopy Image surface microstructure with focal
Smith et al. 2007a
Same as stereomicroscopy, higher
Boyde et al. 1988;
Hillson and Bond
King et al. 2002
Image cut and polished internal surfaces
based on density differences and
Boyde and Jones 1983;
Witzel et al. 2008
Image subsurface microstructure to
approx. 0.1mm depth
Boyde and Martin 1987;
Illuminate experimental labels or
Bromage 1991; Dean
et al. 1993
Proﬁleometer Register variation in surface topography
at microscopic scale
Hillson and Jones 1989;
King et al. 2002
- Flat Plane
Image internal tissues based on density
Beynon et al. 1998;
Moorrees et al. 1963
Absorption Based Image density differences in 3D w/ near-
micron resolution, quantitative
Hayakawa et al. 2000;
Smith and Tafforeau
174 tanya m . smi th
2008; Smith et al. 2009; Tafforeau 2004). Original tooth crown surfaces are often
difﬁcult to image directly due to the transparency of enamel; light tends to reﬂect
poorly, complicating the resolution of ﬁne surface details (Hillson 1992a). Marks
et al. (1996) detail a method whereby tooth surfaces can be temporarily enhanced by
coating with ammonium chloride (performed within a fume hood). Alternatively,
tooth surfaces can be cleaned with ethanol or acetone, and molded with high-
resolution dental impression materials, such as Struers’(Struers Inc., Westlake,
Technique Application Exemplars
Image internal interfaces based on phase
Tafforeau et al. 2006;
Tafforeau and Smith
Image external surfaces with Phong’s
algorithm and lighting effects
Le Cabec et al. 2015
ﬁgure 8.2 The ﬁrst fossil hominin assessed with virtual phase-contrast X-ray imaging: the
Jebel Ihroud 3mandible. A. The North African juvenile fossil mandible showing the location of
the incisor tooth enamel (white box) sampled with the Grenoble synchrotron. B. Close up of
enamel fragment, with the area of interest (on right) shown in the white box. C. Synchrotron
image showing Retzius lines (white arrows) with 10 daily cross-striations between them (white
brackets). The scale bar is 0.2mm. (Adapted from Smith et al. 2007c)
dental histology 175
Ohio) Repliset or Coltène Whaledent’s (Coltène/Whaledent Inc., Cuyahoga Falls,
Ohio) light body systems (polyvinyl silicone). This facilitates production of epoxy
replicas (casts) for preserving a copy and for efﬁciently visualizing hypoplasias, ﬁne
external growth lines (perikymata), and microwear.
Impression materials can be administered via a handheld dispenser (or gun)
with disposable tips that mix the base and catalyst, or via a two-stage process
(termed the Beynon technique by Hillson [1992a] after oral biologist David
Beynon). In the latter case, a ﬁrst impression is made with a “coarse”impression
material (such as Coltène Whaledent’s soft body putty), which serves after
hardening as a template for a ﬁner impression made by lightly coating the tooth
with Coltène’s light body, and rapidly placing it inside the soft body impression to
polymerise. Guatelli-Steinberg and Mitchell (2003) note that an advantage of the
Repliset system over the use of Coltène is the stability of impressions (or molds)
under an electron beam, rendering them directly useful for scanning electron
microscopy. These authors also demonstrate that the Repliset impression material
has a slight advantage in resolution over the Coltène Whaledent light body
After dental impressions have hardened, replicas can be produced with the use of
slow curing low viscosity resin. Various resins (e.g., Epo-Tek 301 resin: Rose 1983;
Spurr resin: Beynon 1987; Araldite resin: Hillson 1992a) that have been employed
have similar properties, including stability under an electron beam. Replicas can be
coloured by the addition of commercially available resin or acrylic paint colouring
agents, or their surfaces can be coated with ammonium chloride, graphite, gold,
silver, or palladium to enhance the appearance of hypoplasias and incremental
features (reviewed in Beynon 1987; Hillson 1992a). Often an electron microscopy
sputter-coater is used to lay down a very thin coating of carbon or metal, which
reduces the transparency of epoxy casts, and facilitates either stereomicroscopy or
scanning electron microscopy (detailed below).
When permissible, additional developmental information can be obtained by thin
sectioning via cutting, grinding, and polishing, yielding a thin slice (approx. 0.1
mm) that allows transmission of light and visualisation of internal features. Arch-
aeological dental remains, especially the dentine and cementum, are particularly
vulnerable to diagenetic alteration (e.g., Bell et al. 1991). These tissues may appear
176 tan ya m. s mit h
very brittle or soft and chalk-like (Hillson 1996), and are liable to fragment when
force is applied. In order to protect samples during the sectioning process, teeth are
typically coated with successive layers of cyanoacrylate (super-glue) or embedded in
a slow-curing epoxy resin or methylmethacrylate polymer. Methylmethacrylate
(MMA) has been lauded for its ability to penetrate and ﬁll very small spaces within
samples (Boyde 1989), including dentine tubules that are approximately 0.001 mm
in diameter. It can be removed by soaking samples in a dichloromethane, which will
soften and eventually dissolve the MMA. However, epoxy resins may be preferable
for embedding since they are less toxic than MMA and do not require the use of a
Once coated or embedded, dental samples are slowly cut with a diamond-tipped
blade mounted on a peripheral or annular saw. The sample is then advanced
approximately 0.5to 1.0mm, and a second cut is made (Figure 8.3). This method
results in a “thick section”of approximately 0.2–0.7mm, which is bonded to a
microscope slide, slowly ground down to a ﬁnal thickness of approximately 0.1mm,
lapped or polished with a ﬁne-grain suspension solution (e.g., 1.0micron Alumina),
cleaned with an ultrasonicator, dehydrated in an alcohol series, cleared in xylene,
and covered with a cover slip and mounting medium (typically xylene-based, such
as DPX [Fluka Chemicals]). After the mount dries, the thin section is ready for
ﬁgure 8.3 Preparation of the initial thick section of the Scladina Neanderthal upper ﬁrst
molar. A. The embedded tooth mounted to the cutting arm of the Logitech APD1annular saw
prior to the initial cut. B. Sectioned tooth showing the “thick section”still attached to the distal
half of the embedded block. (Adapted from Smith et al. 2014)
dental histology 177
microscopic imaging. Additional descriptions of histological preparation proced-
ures may be found in Caropreso et al. (2000), Füsun et al. (2005), Hillson (1996),
Marks et al. (1996), and Reid et al. (1998a,b); and details of ultrathin section
preparation may be found in Gray and Opdyke (1962), Murphy and McNeil
(1964), and Neal and Murphy (1969). An illustrated description of the sectioning
process using Logitech equipment is also available in PDF format (by permission of
Logitech Ltd, Glasgow, Scotland, UK) at https://www.drtanyamsmith.com/wp-con
An added advantage of sectioning teeth is the potential to sample internal
surfaces for light or heavy isotopes, trace elements, enamel proteins, or preserved
DNA in concert with analyses of dental development (e.g., Lakonis Neanderthal
molar: Nielsen-Marsh et al. 2009; Richards et al. 2008; Smith et al. 2009; Scladina
Neanderthal molar: Austin et al. 2013; Smith et al. 2007a; Nielsen-Marsh et al. 2009).
Sectioned teeth are of great value for these complimentary approaches, since the
internal aspects of tooth crowns, particularly the enamel portion, are subject to less
diagenetic modiﬁcation than are external surfaces (Schoeninger et al. 2003).
Following sectioning, the tooth can be reconstructed using colour-matched dental
restorative materials (Figure 8.4; also see Smith et al. 2009:114,ﬁgure 4) or dental
sticky wax (e.g., Schwartz and Dean 2001:273,ﬁgure 1).
ﬁgure 8.4 Image of the Scladina Neanderthal ﬁrst molar before (left) and after (right)
sectioning. Note a small portion of the root was removed prior to sectioning for ancient
DNA analysis; this was not lost due to the physical sectioning process.
(Adapted from Smith et al. 2014)
178 tan ya m. s mit h
Incremental features in histological sections have traditionally been assessed with
polarised (transmitted) light microscopy. This technique is ideal for revealing the
birefringent (optically varied) properties of enamel and dentine, and it often
enhances microstructure clarity (Figure 8.1; also see Boyde 1989; Schmidt and Keil
1971). Modern image analysis systems include transmitted light microscopes (out-
ﬁtted with polarisers and analysers) coupled via high-resolution digital cameras to
workstations with image analysis software. Most commercially available software
packages include tools for performing linear measurements and manual counts of
incremental features. Unfortunately, it has not yet been possible to automate
incremental feature analysis because of subtle inhomogeneities of dental micro-
structure (which is also true for virtual histology).
Additional forms of incremental feature analysis include stereomicroscopy and
scanning electron microscopy (SEM), which are particularly effective for visualizing
features such as hypoplasias and perikymata on tooth surfaces (Figure 8.5). Scanning
electron microscopy is also useful for imaging microstructure in sectioned and
polished teeth (Figure 8.6) or on developing enamel surfaces (e.g., Boyde and Jones
1983; Boyde et al. 1988;Boyde1989). An advantage of SEM over traditional stereo-
scopic imaging is the improved depth of focus at high resolution in secondary electron
mode. Furthermore, back-scattered SEM yields density-dependent images of a single
plane, as opposed to the optical averaging of light microscopic imaging of 0.1mm
sections (roughly the thickness of 20 prisms stacked longitudinally). Another tech-
nique that has been applied to human and ape dental material is confocal microscopy
(Figure 8.7;alsoseeBoydeandMartin1987; Dean 2004), which is particularly valuable
for naturally fractured teeth that are nearly ﬂat, facilitating sub-surface microstructure
imaging. Confocal microscopy has been applied to the study of hominin enamel
secretion rates, periodicities, crown formation times (e.g., Lacruz et al. 2006;Lacruz
et al. 2008), and enamel-prism packing patterns (e.g., Boyde and Martin 1987).
3 archaeological applications
Crown Formation Time and Estimation of Age at Death
Bullion (1987) completed the ﬁrst doctoral dissertation on the study of incre-
mental enamel structures in an archaeological population, followed by
dental histology 179
ﬁgure 8.5 Stereomicroscopic imaging of dental material. Left –the Le Moustier palate on the
base of the Olympus SZX 9stereomicroscope. Right –image of a sputter-coated epoxy cast of a
premolar viewed with stereomicroscopy. Note the horizontal long-period perikymata encircling
the tooth crown. The scale bar is 2mm long. (Photo: Tanya M. Smith)
ﬁgure 8.6 Scanning electron micrograph of cut, etched and polished fossil ape enamel. Note
the relatively straight course of enamel prisms from the bottom right of the image toward the
tooth surface (beyond the top left of ﬁeld), and the ﬁne light and dark bands that represent daily
cross-striations spaced 4.1microns (0.0041 mm) apart on average. The scale bar is 0.1mm long.
(Adapted from Smith et al. 2004)
180 tan ya m. s mit h
FitzGerald (1995), Antoine (2000), and Thomas (2003), who detailed aspects of
incremental growth and the prevalence of developmental stress. Several other
studies have focused on the age of individuals at death, which may provide
insight into the mortality rates, demography, and living conditions of ancient
cultures. Archaeological applications of tooth histology include studies of the
British Spitalﬁelds collection (Antoine et al. 1999;Antoine2000;Antoineetal.
1990;),aswellasothermedievalBritishcollections(Boyde1963; Bullion 1987;
FitzGerald 1995;HudaandBowman1995), medieval French individuals (Reid
et al. 1998b), medieval Danish individuals (Reid and Ferrell 2006;Smith
et al. 2007b; Thomas 2003), imperial Roman children (FitzGerald et al. 1999;
FitzGerald and Saunders 2005; FitzGerald et al. 2006), ancient Greek infants
(FitzGerald and Hillson 2009) and prehistoric Native Americans (e.g., Goodman
et al. 1980;FitzGerald1995;Roseatal.1978;Simpson1999).
ﬁgure 8.7 Spinning-disk white light confocal images of Neanderthal enamel and dentine,
illustrating various incremental features and growth disturbances in a naturally fractured sample
(shown in the center of image). (Photos: Tanya M. Smith)
dental histology 181
These studies provide information on human developmental variation, which is
critical for consideration of individual dentitions or isolated teeth from living or
fossil hominins. Currently it appears that crown formation times do not differ
greatly between modern and recent archaeological European populations (e.g.,
Antoine 2000; Reid et al. 1998b; Smith et al. 2007b). Smith et al. (2007b) compared
molar crown formation times among three modern populations and medieval
Danish individuals, and found few differences between the modern and medieval
specimens. No differences were found between crown formation times in the
Danish and northern European samples. Less is known about histological develop-
ment in non-European archaeological populations. Given developmental variation
among modern northern European and southern African anterior and premolar
teeth (Reid and Dean 2006; Reid et al. 2008), it is likely that additional archaeo-
logical samples will demonstrate similar regional variation. In a study of the oldest-
known Homo sapiens juvenile from northern Africa, Smith et al. (2007c) reported
long crown formation times and dental eruption ages that were quite similar to
those of modern European children. Current evidence suggests that the slow-
growing modern developmental condition is unique to Homo sapiens; recent data
on a second fossil Homo sapiens individual and several juvenile Neanderthals
supports this conclusion (reviewed below and in Smith et al. 2010).
Permanent ﬁrst molars in apes and humans begin forming a few weeks before
birth, permanently recording the birth process as an accentuated line known as the
neonatal line (Rushton 1933; Schour 1936). Identiﬁcation of the neonatal line in ﬁrst
molars, or calculation of postnatal delay in other teeth, allows age at death to be
estimated in juveniles whose dental development is incomplete. This approach has
been applied most often to fossil hominins (reviewed in Smith 2008:218, Table 3,
also see Smith et al. 2015), and also to humans from recent archaeological contexts
in limited cases (e.g., Antoine 2000; Boyde 1963; Huda and Bowman 1995). FitzGer-
ald and Hillson (2009) examined the postnatal survivorship of Greek infants in a
cemetery population based on the presence and position of the neonatal line in
deciduous teeth (which begin forming several months before birth). Similarly,
Schwartz et al. (2010) analysed the presence or absence of the neonatal line in
50 deciduous teeth from Carthage in order to assess whether individuals were likely
to have been sacriﬁced. It appeared that roughly half the sample died before one to
two weeks after birth, leading the authors to conclude that other factors likely
contributed to the death of perinatal individuals in this particular cemetery.
Studies of age at death in the known-age Spitalﬁelds material have also been used
to demonstrate the high degree of accuracy (+ /2per cent) of histological methods
182 tanya m. s mi th
(Antoine 2000; Antoine et al. 2009;Stringeretal.1990). Although there are potential
applications of tooth histology for forensic identiﬁcation, very few published cases
exist (likely because pinpointing age at death is time-consuming and technically
demanding). Katzenberg et al. (2005) present a case study where the remains of an
unknown infant were assessed through dental histology, historical records and DNA
analysis. Histology yielded an age at death of 4.8–5.1months, supporting putative
historical identiﬁcation of a 5-month-old female (see also a similar study by Skinner
and Anderson 1991). Precise reconstruction of juvenile age at death represents one of
the most valuable applications of dental histology, particularly when compared to
other commonly employed anthropological aging methods.
The most frequent application of tooth histology in archaeology is the detection of
developmental stress. External manifestations of stress are known as hypoplasias on
tooth crowns (Berten 1895; reviewed in Hillson 2014; Hillson and Bond 1997;
Simpson 1999) and accentuated rings on tooth roots (Smith and Reid 2009).
Internal manifestations of developmental stress in enamel and dentine are termed
accentuated lines (or, less commonly, Wilson bands in enamel [see FitzGerald and
Saunders 2005] and lines of Owen in dentine [see Dean 1995]). Numerous studies
have reported on the frequency and timing of hypoplasias in archaeological popu-
lations and fossil hominins, a subject beyond the scope of this review (see references
in Goodman and Rose 1990; Guatelli-Steinberg 2004; Hillson 1992b; Hillson 1996;
Hillson 2014; Hillson and Bond 1997; Katzenberg et al. 1996; King et al. 2002;
Skinner 1996). Relatively less attention has been paid to the frequency or causation
of internal accentuated lines (but see references in FitzGerald and Saunders 2005;
Smith and Boesch 2015; Thomas 2003; Witzel et al. 2008;).
Unfortunately, both hypoplasias and accentuations arise from nonspeciﬁc
stresses experienced during development, limiting the utility of these features for
reconstructions of past environments and population health. Numerous studies
have posited relationships between these features and vitamin D deﬁciency (rick-
ets), hypothyroidism (hypocalcemia), acute dehydration, starvation, exanthematous
fevers, or other systematic disturbances, severe trauma, weaning, parturition, psy-
chological disorders, seasonal resource availability, disease cycles, or rainfall pat-
terns (e.g., Bowman 1991; Boyde 1970; Bracha 2004; Nikiforuk and Fraser 1979;
Schwartz et al. 2006; Simpson 1999; Skinner 1986; Skinner and Hopwood 2004;
dental histology 183
reviewed in Dirks et al. 2002; FitzGerald and Saunders 2005; Goodman and Rose
1991; Guatelli-Steinberg 2001; Guatelli-Steinberg and Benderlioglu 2006; Hillson
2014; Katzenberg et al. 1996; Smith and Boesch 2015). This area of study would
beneﬁt from additional systematic or experimental studies of defect expression
from a range of pre- and postnatal conditions in order to provide a framework
for interpreting the signiﬁcance of these features in archaeological and fossil
A substantial debate has ensued in the archaeological literature about the most
appropriate approach to determining the timing or age of hypoplasia formation
(reviewed in Goodman and Rose 1991; Goodman and Song 1999; Hillson and Bond
1997; Martin et al. 2008; Reid and Dean 2000; Ritzman et al. 2008). The standard
approach for much of the past century has been to divide the tooth crown into
equally spaced and equally timed divisions (assuming constant linear growth rates).
However, it is clear that primate teeth do not grow at a constant rate, as evidenced
by varying extension rates along the enamel-dentine junction and changing peri-
kymata spacing along the tooth surface. Furthermore, the ﬁrst-formed enamel in
the cuspal regions, which forms over several months to more than a year, is not
represented by perikymata on the tooth surface, complicating attempts to assign
ages to positions on the tooth surface without knowledge of internal development.
In contrast to decades of previous archaeological studies, Reid and Dean (2000)
have shown that no relationship exists between tooth crown height and the total
time of formation. They initially proposed a set of developmental standards from
115 northern European anterior teeth for approximate ages of crown surface deciles
from the cusp tip to the cervix (Reid and Dean 2000:138,ﬁgure 1). This was
followed by standards for southern African anterior teeth and molars from both
populations (Reid and Dean 2006). It is important to note that since anterior tooth
development varies among human populations (Reid and Dean 2006), histological
information will be most effectively applied to the populations from which the
standards are derived. Recent studies have explored differences in the timing of
defects using traditional models and histological information (Martin et al. 2008;
Ritzman et al. 2008), showing deviations from several months to more than a year
A number of studies have used patterns of enamel hypoplasias or accentuated
lines to match teeth forming at the same time (e.g., Boyde 1963; Gustafson 1955;
King et al. 2002; Schwartz et al. 2006; Smith et al. 2007a). This aspect of hard tissue
registry allows for more precise estimates of the timing of crown initiation and
completion than dissection or radiographic techniques and is also critical for
184 tan ya m. s mit h
estimates of age at death in older juvenile material (detailed in the case study
below). Kelley (2008) illustrates an interesting application of enamel hypoplasia
matching in a potential birth cohort of the (presumably) rare ape species Gripho-
pithecus from the Miocene locality of Paşalar (Turkey). Based on similar periky-
mata counts and the presence of an identical pattern of linear enamel hypoplasias
on nine incisors, he suggested that the individuals attributed to this taxon were all at
the same maturational stage, and that they experienced the same two stressful
episodes simultaneously. From this, he inferred that these animals were part of
the same cohort, that they experienced birth seasonality, and that they all died at the
same time (based on identical amounts of wear). This elegantly demonstrates how
incremental features can be used to reconstruct not only the “intimate history of the
individual,”as Gysi (1931) notes, but also the history of a group of individuals and
the environmental conditions they experienced.
4 case study: scladina juvenile neanderthal
This case study documents the determination of crown formation time in the
127,000-year old female juvenile Neanderthal from Scladina, Belgium (Toussaint
and Pirson 2006; Peyregne et al. 2019), in addition to identiﬁcation of developmen-
tal stress, leading to an estimate of the age at death (originally published in Smith
et al. 2007a) as well as the age at weaning (Austin et al. 2013). Following the
preparative methods detailed above (photography, micro-CT scanning, molding,
and casting), the upper ﬁrst molar of an associated dentition was cut and a
histological section prepared (Figures 8.1,8.3, and 8.4). This tooth was sectioned
to determine the long-period line periodicity and to register chronological and
developmental time via the neonatal line. Birth occurred approximately 13 days
after cusp initiation, and crown formation time was determined by tracking days of
growth along enamel prisms (according to the procedure described in Boyde [1963;
1990]) and adding this to the number of Retzius lines multiplied by their periodicity
(Figure 8.8). The formation time of this ﬁrst molar cusp is approximately 872 days,
and subtraction of the prenatal enamel yields an age at crown completion of 2.35
years. A record of postnatal developmental stress was also determined; a particu-
larly marked accentuated line formed at approximately 435 days of age, followed by
a second accentuated line at 875 days of age in the root dentine of both mesial cusps.
All associated teeth were molded and cast, allowing counts of long-period
incremental features on tooth crowns and roots, and micro-CT scanned for
dental histology 185
quantiﬁcation of the cuspal enamel thickness. Crown formation times were calcu-
lated from these variables and the long-period line periodicity of 8days from the
sectioned molar (Smith et al. 2007a; Smith et al. 2014). These data were compared
with incremental development in modern humans from northern England and
southern Africa, as well as a large sample of Neanderthals (Guatelli-Steinberg and
Reid 2008; Reid et al. 2008; Reid and Dean 2006; Smith et al. 2007b;), revealing
particularly short post-canine crown formation times in the Scladina individual.
This pattern is due to thin cuspal enamel and rapid rates of crown extension in
Neanderthals (Smith et al. 2007a; Smith et al. 2010), yielding teeth similar in size to
those of living humans that form more rapidly. Finally, a sequence of developmen-
tal stress was mapped across the dentition, which allowed registry of teeth forming
at the same time, and estimation of the age at death.
Developmental stress in the enamel and dentine of the ﬁrst molar at approxi-
mately 435 and 875 days of age was matched to hypoplasias on anterior teeth (Smith
et al. 2007a: 20,222,ﬁgure 1). A third developmental stress event was identiﬁed and
matched at 1,779 days of age in the developing lower third premolar and upper
canine, and subsequent developmental time was added to establish that the indi-
vidual was approximately 8years old at death (~2,939 days). This registry of stress
across the dentition allowed the establishment of crown initiation and completion
ages, yielding an overall developmental chronology for this young Neanderthal
(Smith et al. 2007a: 20,223,ﬁgure 2).
ﬁgure 8.8 Case study: Scladina Neanderthal dental development. The neonatal line is
indicated by the ﬁrst drawn line on the lower left (0), with subsequently calculated time
indicatedforaseriesofstresseventsas153 days, 227 days, 348 days, and 435 days postnatal
age. The stress event at 435 days (1.2years) was the most marked until later stressors at
875 and 1779 days of age (not shown). The scale bar is 1mm long. (Adapted from Smith et al.
2007a; Smith et al. 2014)
186 tan ya m. s mit h
When the Scladina juvenile is compared to modern humans, its tooth calciﬁcation
stages and eruption status appear to be advanced by several years over modern
juveniles at the same chronological age (Liversidge 2003; Smith 1991). For example,
the Scladina second molar had advanced beyond clinical (gingival) occlusion by
8years of age; second molar eruption occurs on average at 10–13 years of age in
global human populations. It is clear that from these data and other studies that age at
death in Neanderthals should not be assessed by comparison with modern human
standards, particularly those derived from European populations. Furthermore, this
individual has shed light on the on-going debate about Neanderthal life history,
illustrating a developmental pattern that appears to be intermediate between that of
fossil Homo sapiens and Homo erectus (Smith et al. 2007a). More recent studies
employing virtual histology have conﬁrmed this pattern of rapid dental development
in other Neanderthals (Smith et al. 2010), implying that the prolonged childhood and
slow developmental schedule of modern humans is, in fact, unique to our species.
Finally, the Scladina juvenile also represents the ﬁrst fossil hominin for which a
precise age at weaning has been reported (Austin et al. 2013). By integrating information
on the timing of ﬁrst molar incremental growth with patterns of the incorporation of
barium, a trace element enriched in breast milk, Austin et al. (2013) demonstrated
barium/calcium patterns in enamel that appear to coincide with periods of placental
nutrition, exclusive breastfeeding for 7months, and supplementation followed by an
abrupt cessation of nursing at 1.2years of age. While this fossil is particularly well
preserved, having yielded enamel proteins (Nielsen-Marsh et al. 2009)andDNA
(Peyrégne et al. 2019), biogenic elemental patterns and diagenetic indicators suggest
that tooth histology and elemental mapping can be employed to document the timing
of early life diet transitions in fossil enamel. A recent study has shown what appears to
be a more normative Neanderthal weaning age at 2.5years, as well as the seasons of
birth and weaning (Smith et al. 2018). Importantly, this approach may be extended to
test theories about changes in the timing of human weaning (reviewed in Reynard and
Tuross 2015;Smith2013; also see Beaumont et al. 2015).
Fossil samples were provided by Jean-Jacques Hublin, Shannon McPherron, Michel
Toussaint, Louis de Bonis, Almut Hoffmann, and Debbie Guatelli-Steinberg. Com-
ments on the text were kindly provided by JP Zermeno, Christina Warinner, Don
Reid, Karola Kirsanow, and Janet Fink. This work was funded by the Max Planck
Society and Harvard University.
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