Potential of mechanical and physicochemical analysis of human cortical bone for forensic age estimation

Age estimation remains one of the most challenging tasks for forensic practitioners when establishing the biological profile of unknown skeletonised remains. Morphological methods based on developmental markers of bones can provide accurate age estimates. Yet, these methods tend to be unreliable when the individuals are over 35 years of age as all the key developmental markers gradually disappear. Current methods are also highly population- and sex-specific, and individual differences must be considered when interpreting the results. Aspartic acid racemisation, radiocarbon dating and DNA-methylation have all shown to be accurate to only a margin of ± 5 years. A recent method using quantification of biomechanical properties in conjunction with bone microstructure for the mid-femur cortical bone has shown potential and a better accuracy. In the present research, analyses of samples from the 4th rib are detailed and a methodology for estimation of age of individuals is presented. The physical characteristics of ribs are less influenced by mechanical stress compared to weight bearing bones and ribs are relatively accessible from the thoracic cage during autopsy, which increases the applicability of any such rib-based method. This study exploits the changes in the biomechanical properties of bone tissue and matrix. These properties continue to change with age even after skeletal maturity and they are valuable for age estimation. The relationship of 28 variables at the macroscopic, microscopic and structural/compositional level were investigated in 113 ribs retrieved during autopsies. The experimental design comprehensively documents the changes in mineral size and composition, organic matrix quality and their combined effect on mechanical properties. The powder x-ray diffraction (XRD) method is applied to measure the crystal size and strain, and lattice parameters. Further, Fourier-transform infrared (FTIR) spectroscopy is used to investigate the changes in carbonate substitution and crystallinity. This examination allows a dependable evaluation of the increase in mineral to matrix ratio and therefore, the increase in collagen. Subsequent analysis of combined mineral and organic changes with age was done through thermal analysis. The physicochemical characterisation was compared to functional mechanical properties. The study showed that, in contrast to other bones, skeletal maturity for the rib cortical bone is reached between 40 and 50 years of age. The study revealed that with age, there is an increase in crystal size and mineral content that is combined with the decrease in organic matrix amount and quality. It was also possible to confirm the importance of standardisation of the procedures. It was found out that a simple defatting procedure based on consecutive baths of chloroform, methanol and ethanol affects the evaluation of crystal size, which in turn affects the age estimation when using the powder XRD method. The 28 parameters measured using standard biomechanical (nanoindentation and microindentation), physical (DSC/TGA, FTIR and XRD) and histomorphometry (porosity-ImageJ) procedures were used to obtain an age-at-death estimation for unknown skeletal remains. Stepwise regression was employed to develop relations that would produce the best 'estimates of age-at-death' vs real age of the cadavers. Seven relationships were proposed: the relationship of primary importance containing 13 parameters and resulting in an R2 = 0.873 with a mean absolute error of ± 4.47. The relationship that performs poorly utilised only five parameters and resulted in an R2 = 0.840, with an average error estimate of ± 4.96 years. To conclude, physicochemical and mechanical characterisation of bone cortical bone shows good potential in accurate age-at-death estimation. Furthermore, the multi-factorial approach allows the combination of different methods to reach the best performance. A further employment of this method may involve histomorphometry and macroscopic examination of the age related degeneration if the sternal rib end, still largely used in forensic anthropology.