The Effect of Bacteria vs Environment on Bone Diagenesis

The aim of this research was to determine the origins of the microbes that drive bone diagenesis. Studying the microstructural changes skeletal tissue can undergo after death could lead to more accurate ways of establishing postmortem intervals when dealing with skeletonised remains. Previous research has allowed the development of methods to determine the post mortem interval (PMI) during the decomposition of the soft tissue, but this estimation becomes less accurate as the PMI increases. This study focused on the physical changes that occurred, from the macroscopic level of weathering and surface modifications, to the histological changes to the microstructure of the hard tissue. Proteomic analysis to chart changes in the bone proteome over time was also conducted. This research compared different tissue types and different deposition environments; three tissue types, defleshed bone, excised fleshed limbs, and whole rat; two deposition environments, buried in soil, exposed on the surface. Forty-six medium sized domestic rats (Rattus rattus) were used for this experiment, giving three repeats per condition plus control (day 0) analysis. This was a multi-analytical approach with a variety of techniques being implemented; soft tissue decomposition was recorded using the total body score (TBS) followed by the macroscopic analysis of bone surface weathering. Ultra-violet (UV) analysis was conducted alongside histological analysis using compound microscopy and digital microscopy. Further analysis of the bone was conducted using Confocal Laser Scanning Microscope (LSM). Bone proteomics were added as a result of collaboration with Dr Noemi Procopio at Northumbria University. Overall, this experiment produced statistically significant results for the increasing presence of diagenetic changes to the bone with increasing PMI. P values of less than 0.05 were obtained between samples at 4 weeks PMI and 28 weeks PMI for the histological analysis, Confocal LSM and proteomic analysis, which confirm the increasing destruction of bone integrity with increasing PMI. It was also found that the earliest signs of microscopic foci of destruction (MFD) occurred prior to the skeletonisation of the remains, agreeing with previous research. It was found that the deposition environment did not play the significant role in bone diagenesis that was hypothesised. The same was true for the presence of the gut microbiome, which did accelerate the initial diagenesis of the bone within the first 8 weeks. Diagenesis appeared to slow after the first 8 weeks, and all samples showed little comparable differences in diagenetic changes by 28 weeks. This research contributes to our understanding of bone diagenesis in forensic timescales. It gives us more information about the role of gut and soil bacteria in bone diagenesis and could aid the estimation of PMI in certain situations. However, more research is needed with longer timescales, and human subjects, to improve accuracy