Formation Times in Thermally Altered EnamelPatrick Mahoney Ph.D. and Justyna Miszkiewicz Ph.D.Human Osteology Research Laboratory, School of Anthropology and Conservation, University of Kent, Kent, UK
CHAPTER 20
Introduction
Estimating age at death is a fundamental step when creating a biological profile for a human juvenile skeleton recovered in a forensic context. Several macroscopic methods are available for this purpose, such as age estimation from long bone growth or the chronology of epiphyseal fusion (e.g., Hoppa, 1992; Schwartz, 1995). If teeth are still forming at the point of death, then the stage of enamel development can be compared with growth standards that have been created for permanent (e.g., Smith, 1991) and deciduous dentition (Mahoney, 2011, 2012). Alternatively, a developing tooth can be sectioned using histological methods and its formation time can be reconstructed from enamel incremental markings. From this, the duration of postnatal enamel growth can be calculated, which will equate to age at death (e.g., Boyde, 1963). This latter method is more time-consuming compared with the macroscopic methods, but it is accurate (Smith et al., 2006; Antoine et al., 2009).
Previous studies have gained insights into the colour, weight, fracture lines and histological processing (sectioning and staining) of tooth enamel exposed to heat (e.g., Carr et al., 1986; Yamamoto et al., 1990; Fairgrieve, 1994; Myers et al., 1999; Beach et al., 2008; Fereira et al., 2008; Hughes and White, 2009). However, incremental line preservation for age at death estimation purposes has only been evaluated in cremated and experimentally heated cementum (e.g., Grosskopf, 1989, 1990; Gocha and Schutkowski, 2013). No study has evaluated the effect of heat on enamel incremental markings, or whether formation times can be calculated from thermally altered tooth enamel. Given the accuracy of age at death estimates derived from these markings, it is important to understand if and how they are influenced by heat.
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Aim
This study assesses the visibility of enamel incremental markings in a sample of human permanent molars exposed to various temperatures for durations of either 20 min or 1 h. The aim is to determine at what temperature it is no longer possible to reconstruct molar enamel formation time.
Materials and Methods
Fourteen human permanent molars were extracted from either the mandible or maxilla of human skeletons dating to the British medieval period (Table 20.1). All skeletons were from the same cemetery. Teeth were either unworn or had slight wear at the tip of the enamel cusp.
Molars were exposed to heat using two methods. In the first method, a fire was constructed using hard woods and coal outside in a well-ventilated area. Four flat trays approximately 5 × 5 cm were constructed out of aluminium foil. These were placed at different locations in the fire. The fire was lit and allowed to burn for 10 min. Following this, two molar teeth were placed onto each tray using metal tongs for a duration of 20 min. The temperature at the base of each tray, directly under the teeth, was measured several times using a digital total range thermometer (Fisher Scientific Traceable Total Range Thermometer). The temperature reported is the maximum temperature each tray experienced during that 20-min period. Temperatures varied from a maximum of 250–515°C depending on the location of the tray in the fire.
In the second method, six molar teeth were heated in a bottom-loaded Carbolite Eurotherm high-temperature furnace oven in the School of Physical Sciences, University of Kent, United Kingdom. This allowed us to increase the duration and temperature to which the teeth were exposed. Two molars were heated to a temperature of 500°C for 1 h. Two more were heated to
Table 20.1 Temperature, sample size and duration of exposure.
600°C, and another two were heated to 700°C, for 1 h. Specimens were placed in a standard ceramic crucible (e.g., Beach et al., 2008). A large enough crucible was chosen to ensure that the samples were separated, and that any shattered enamel remained inside the vessel.
After the teeth were removed from the heat, they were allowed to cool to room temperature. Each tooth was photographed and placed into a plastic bag and labelled. Each tooth was sectioned using standard methods (e.g., Reid et al., 1998) in the Human Osteology Research Lab, School of Anthropology and Conservation, University of Kent, United Kingdom. The teeth were embedded in polyester resin to reduce the risk of splintering while sectioning. Using a diamond-wafering blade (Buehler IsoMet 4000 precision saw), buccal–lingual sections were taken through the mesial cusps, travelling through the outermost enamel cusp tip, the tip of the enamel–dentin junction (EDJ) and the most cervical extension of the enamel. When teeth were fractured, the same orientation was used during sectioning. Each section was mounted on a microscope slide, lapped down to 100–120 µm (Buehler EcoMet 300) using a graded series of grinding pads to reveal the incremental (and accentuated) lines, polished with a 0.3 mm aluminum oxide powder, placed in an ultrasonic bath to remove surface debris, dehydrated through a series of alcohol baths, cleared (Histoclear®) and mounted with a cover slip using a xylene-based mounting medium (DPX®).
Sections were examined under a high-powered microscope (Olympus BX51) using transmitted and polarised light. Images were captured using an Olympus DP25 digital camera linked to the microscope. For each section, the effect of heat on the enamel microscopic structures and the visibility of the incremental (and accentuated) markings were recorded. Following this, cusp formation times (cuspal + lateral formation time) were reconstructed (e.g., Mahoney, 2008) for three permanent first molars heated to 250°C, 370°C and 472°C, respectively, using incremental (cross-striations, Retzius lines) and accentuated markings (for another method see Dean, 2012). Cross-striations are lines in enamel that form every 24 h in humans and other primates (e.g., Schour and Poncher, 1937; Bromage, 1991). Retzius lines form between 6 and 12 days in permanent teeth (Smith, 2008). Accentuated markings only form sporadically, leaving a line in enamel at birth in the protoconid cusp of first molars (the neonatal line) and during subsequent periods of systemic stress (e.g., Boyde, 1989). Because enamel does not remodel, all of these lines are retained in teeth after death.
Cuspal formation times were calculated using a standard formula ([enamel thickness × correction factor]/daily enamel secretion rate [e.g., Mahoney, 2008]). Enamel thickness was measured from the tip of the dentin horn to the position of the first Retzius line at the cusp tip tooth surface. A correction factor of 1.05 was used because decussation was not marked in these teeth (Mahoney, 2008). Cuspal enamel was divided into three regions of equal thickness (inner, mid and outer), and divided by the mean daily enamel secretion rate (calculated from cross-striations) from each of those regions. The three formation times were then summed to give an overall cuspal formation time.
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Lateral enamel formation time was recorded by multiplying the number of Retzius lines by the periodicity. Where Retzius lines were indistinct, or not present, or in areas where only accentuated markings were present, enamel prism lengths divided by local mean daily enamel secretion rates were used to navigate through the enamel (see Mahoney et al., 2007: Figure 20.1). The time taken to form these prisms was included in the estimate of lateral enamel formation time.
Results
Results are summarised in Table 20.2. Teeth were darker on the side that faced the flame (Figure 20.1). When sectioned and viewed under the microscope, molars heated to a temperature of 250°C for 20 min had a few microfractures (Figure 20.2) (these microfractures are similar to those found on the outer aspects of burned bones and teeth; see Schmidt and Uhlig, 2012, for more on microfractures that occur on ectosurfaces). The microfractures followed the direction of the enamel prisms, running from the EDJ to the outer enamel surface. A dark carbon deposit was present in some of the fractures. The neonatal line and other accentuated markings, Retzius lines and daily cross-striations were visible. A cusp
M1 surface facing flame
M1 surface facing away from flame
Figure 20.1Notice the colour difference between those surfaces facing the heat versus those facing away.
Formation Times in Thermally Altered Enamel 359
formation time of 1052 days (2.88 years) was calculated for an M1 metaconid, which lies within the normal range of formation times for modern human first molars (e.g., Reid and Dean, 2006; Mahoney, 2008).
Teeth heated to a temperature of 370°C showed microfractures, and also two larger fractures in one cusp (Figure 20.3). Carbon deposits became more pronounced and darker. Incremental lines and accentuated markings were visible. A postnatal cusp formation time of 1091 days (2.98 years) was calculated for an M1 protoconid where the enamel was still intact. At a temperature of 472°C, large fractures led to complete enamel separation in an M1 protocone and M1 protoconid. Incremental and accentuated markings were still present and visible in both of these cusps. A cusp formation time of 905 days (2.48 years) was calculated for an M1 metaconid. Both formation times lie within the normal range for modern humans.
When exposed to a temperature of 515°C for 20 min, the enamel of one tooth shattered. In the other intact tooth carbon deposits were more pronounced and darker, which started to obscure the visibility of incremental lines that were adjacent to the EDJ. A large fracture produced complete enamel separation in one of the cusps. Formation time could not be calculated for the other cusp.
Teeth exposed to a temperature of 500°C for 1 h showed numerous microfractures and large fractures. One area of the enamel was completely detached from the cusp. Incremental markings were still visible in some areas, although carbon deposits obscured the markings in other areas. A formation time could not be calculated. When heated to a temperature of 600°C and 700°C for 1 h, either large areas of enamel detached from the cusp or the enamel
Table 20.2 Summary of histological changes with heat.
Temperature (Method)
Enamel Formation TimeHistology Markings
250°C (fire) Microfractures, dark carbon deposits Incremental and accentuated markings visible
Yes
370°C (fire) Microfractures, larger fractures, dark carbon deposits
Incremental and accentuated markings visible
Yes
472°C (fire) Microfractures, larger fractures, dark carbon deposits
shattered (Figure 20.4). When the fragments were viewed under the microscope (at 10× or 40×), carbon deposits obscured the prism structure and the incremental markings.
Discussion
Results support previous research that reported that enamel fractured at a temperature of 500°C or 600°C in human (Fereira et al., 2008) and nonhuman specimens (Yamamoto et al., 1990). In the present study, enamel started to peel away from the dentin at a temperature of 515°C and shattered at higher temperatures. Generally, the number of fractures in a tooth increased with temperature, which supports previously reported results (Muller et al., 1998).
Figure 20.2Microfractures in enamel heated to 250°C, and close-up showing daily cross-striations.
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Incremental markings were visible in some areas of enamel up to a temperature of 515°C. However, it was not possible to calculate formation times in this small sample of teeth beyond a temperature of 472°C. At the higher temperature, carbon deposits obscured large areas of the markings. Large fractures in the enamel also made it difficult to navigate though a cusp when reconstructing formation time. These temperatures are slightly lower compared with a study by Gocha and Schutkowski (2013), which examined the visibility of incremental
Figure 20.3Incremental and accentuated markings, histological structures and fractures in a first molar
protoconid enamel cusp heated to 370°C for 20 min and viewed under polarised light.
Figure 20.4Molar enamel heated to 600°C and 700°C.
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markings in cementum that has been exposed to heat and reported that age could be calculated up to a temperature of 600°C.
Conclusion
This study assessed the visibility of incremental markings in thermally altered human permanent molar enamel to determine at what temperature enamel formation times could no longer be reconstructed. It was found that cusp formation times could be calculated in molars heated to temperature of 472°C for 20 min. Formation times could not be calculated in teeth heated to higher temperatures and for longer periods of time, either because incremental markings became obscured by carbon deposits or because the enamel fractured.
Acknowledgements
We thank Christopher W. Schmidt for inviting us to contribute to this edition. Alan Chadwick and Marc Williams from the University of Kent provided technical assistance.
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