Diagnostic models to predict nuclear DNA and mitochondrial DNA recovery from incinerated teeth.

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).

Freeze-drying improves DNA yield from teeth.

The common method of preparing teeth prior to DNA extraction involves cleaning, decontamination, drying and pulverisation. Moisture in post-mortem teeth can promote bacterial growth and hydrolytic damage that could contribute to DNA degradation, whilst also possibly reducing the efficiency of sample pulverisation and DNA release. Here we compared DNA extraction from pig teeth, with- and without freeze-drying, to examine the impact of removing moisture on DNA yield. Quantitative real-time polymerase chain reaction (qPCR) was used to quantify an 83 bp mitochondrial DNA fragment and two nuclear DNA fragments of 82 bp and 150 bp. The comparative results showed that sample preparation with freeze-drying resulted in a higher DNA yield without compromising the DNA quality. This study highlights the advantage of incorporating a freeze-drying to improve the DNA yield and minimising the loss of DNA during sample preparation of teeth.

Comparison of anterior and posterior trabecular bone microstructure of human mandible using cone-beam CT and micro CT.

BACKGROUND: The aim of this study was to compare the trabecular bone microstructures of anterior and posterior edentulous regions of human mandible using cone-beam computed tomography (CBCT) and micro computed tomography (µCT). METHODS: Twenty volumes of interests consisting of six anterior and fourteen posterior edentulous regions were obtained from human mandibular cadavers. A CBCT system with a resolution of 80 µm (3D Accuitomo 170, J. Morita, Kyoto, Japan) and a µCT system with a resolution of 35 µm (SkyScan 1173, Kontich, Belgium) were used to scan the mandibles. Three structural parameters namely, trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analysed using CTAn software (v 1.11, SkyScan, Kontich, Belgium). For each system, the measurements obtained from anterior and posterior regions were tested using independent sample t-test. Subsequently, all measurements between systems were tested using paired t-test. RESULTS: In CBCT, all parameters of the anterior and posterior mandible showed no significant differences (p > 0.05). However, µCT showed a significant different of Tb.Th (p = 0.023) between anterior and posterior region. Regardless of regions, the measurements obtained using both imaging systems were significantly different (p ≤ 0.021) for Tb.Th and Tb.N. CONCLUSIONS: The current study demonstrated that only the variation of Tb.Th between anterior and posterior edentulous region of mandible can be detected using µCT. In addition, CBCT is less feasible than µCT in assessing trabecular bone microstructures at both regions.

The development of a tool to predict temperature-exposure of incinerated teeth using colourimetric and hydroxyapatite crystal size data.

This study presents a novel tool to predict temperature-exposure of incinerated pig teeth as a proxy for understanding impacts of fire on human teeth. Previous studies on the estimation of temperature-exposure of skeletal elements have been limited to that of heat-exposed bone. This predictive tool was developed using a multinomial regression model of colourimetric and hydroxyapatite crystal size variables using data obtained from unheated pig teeth and teeth incinerated at 300 °C, 600 °C, 800 °C and 1000 °C. An additional variable based on the observed appearance of the tooth was included in the tool. This enables the tooth to be classified as definitely burnt (600 °C-1000 °C) or uncertain (27 °C/300 °C). As a result, the model predicting the temperature-exposure of the incinerated teeth had an accuracy of 95%. This tool is a holistic, robust and reliable approach to estimate temperature of heat-exposed pig teeth, with high accuracy, and may act as a valuable proxy to estimate heat exposure for human teeth in forensic casework.

Integrating spectrophotometric and XRD analyses in the investigation of burned dental remains.

Heat alters colour and crystallinity of teeth by destruction of the organic content and inducing hydroxyapatite crystal growth. The colour and crystallite changes can be quantified using spectrophotometric and x-ray diffraction analyses, however these analyses are not commonly used in combination to evaluate burned dental remains. In this study, thirty-nine teeth were incinerated at 300-1000 °C for 15 and 30 min and then measured using a spectrophotometer and an x-ray diffractometer. Response variables used were lightness, L*, and chromaticity a* and b* and luminance (whiteness and yellowness) for colour, and crystal size for crystallinity. Statistical analysis to determine the attribution of these variables revealed yellowness and crystal size were significantly affected by temperature (p < 0.05), whilst duration of heat-exposure showed no significant effect. This study suggests the inclusion of both spectrophotometric and x-ray diffraction in investigating thermal-heated teeth is useful to accurately estimate the temperature teeth are exposed to.

A comparative study between xerographic, computer-assisted overlay generation and animated-superimposition methods in bite mark analyses.

This study was to compare the suitability and precision of xerographic and computer-assisted methods for bite mark investigations. Eleven subjects were asked to bite on their forearm and the bite marks were photographically recorded. Alginate impressions of the subjects' dentition were taken and their casts were made using dental stone. The overlays generated by xerographic method were obtained by photocopying the subjects' casts and the incisal edge outlines were then transferred on a transparent sheet. The bite mark images were imported into Adobe Photoshop® software and printed to life-size. The bite mark analyses using xerographically generated overlays were done by comparing an overlay to the corresponding printed bite mark images manually. In computer-assisted method, the subjects' casts were scanned into Adobe Photoshop®. The bite mark analyses using computer-assisted overlay generation were done by matching an overlay and the corresponding bite mark images digitally using Adobe Photoshop®. Another comparison method was superimposing the cast images with corresponding bite mark images employing the Adobe Photoshop® CS6 and GIF-Animator©. A score with a range of 0-3 was given during analysis to each precision-determining criterion and the score was increased with better matching. The Kruskal Wallis H test showed significant difference between the three sets of data (H=18.761, p<0.05). In conclusion, bite mark analysis using the computer-assisted animated-superimposition method was the most accurate, followed by the computer-assisted overlay generation and lastly the xerographic method. The superior precision contributed by digital method is discernible despite the human skin being a poor recording medium of bite marks.