Investigation of the Effects of Heat on Bone Tissues to Inform Forensic Analysis

Abstract

Following an incineration event bone is often the only surviving tissue, making the skeleton vital for obtaining a positive identification of the deceased. Discriminating features such as age, sex, height, and antemortem injuries can often be observed in skeletal remains and used as secondary identifiers in forensic examination. Understanding the effects of incineration on bone tissues and how this may impact skeletal markers is essential to improving standard methods of anthropological profiling for forensic identification. In addition to these secondary identifiers, bone provides a protective barrier for DNA which is highly individualising and hence is considered a primary scientific method of identification. Due to the structural degradation of bone, and the loss of organic material, obtaining a viable DNA sample from incinerated bone is problematic. There is a need for improved DNA extraction protocols for these incinerated samples, as well as methods of triaging multiple samples to facilitate an optimal outcome. The primary aim of this research was to study heat-induced changes in bone microstructure to inform forensic evidence recovery and analysis. This was achieved by employing a range of techniques including x-ray powder diffraction (XRPD), micro computed tomography (microCT), and scanning electron microscopy (SEM) with energy dispersive x-ray (EDX). As previous research has suggested heat-induced changes in bone mineral resemble long term diagenetic changes, it was hypothesised that ancient DNA extraction methods may be appropriate for modern incinerated samples. This theory was tested by comparing demineralisation techniques designed for ancient DNA extraction to simpler techniques used for fresh samples. The quantity of DNA recovered was compared between methods to assess the optimal method for extraction from burned samples. As bone crystallinity has an impact on both DNA preservation and the ability to extract DNA from mineralised tissue, XRPD was used to assess heat induced changes in hydroxyapatite crystal size and structure across a range of temperature intervals. SEM and EDX were then used to visually assess these changes and provide basic elemental analysis. In the final part of this project, MicroCT was used to create 3D reconstructions of incinerated bone samples to visually and quantitatively assess changes to bone porosity as a result of thermal insult. The application of ancient bone demineralisation techniques was not shown to increase DNA yield from incinerated bones, with a shorter demineralisation period and retention of EDTA supernatant producing optimal results. XRPD analysis provided a possible explanation for this, showing that heatinduced changes to bone hydroxyapatite were far more impactful than the diagenetic changes seen in ancient bone, which could not be distinguished from unburned modern samples. Similarly marked heat-induced changes in bone structure were observed using MicroCT, where bone porosity was greatly decreased at high temperatures. This research highlights the variable nature of heat induced changes in bone which could prove valuable in prioritising sampling based on the likelihood of obtaining identifying information such as DNA.