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

Extraction and Untargeted Analysis of Metabolome from Undemineralised Cortical Bone Matrix

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) untargeted metabolomics has become the gold standard for the profiling of low-molecular-weight compounds. Recently, this discipline has raised great interest in forensic sciences, especially in the field of toxicology and for post-mortem interval estimation. The current study aims at evaluating three extraction protocols and two LC-MS/MS assays run in both positive and negative mode to identify the most suitable method to conduct post-mortem metabolomic profiling of bone tissue. A fragment of the anterior tibia of a 82 years-old male sampled from a Human Taphonomy Facility was powdered via freeze-milling. The powdered sub-samples were extracted in five replicates per protocol. Methods tested were (I) a biphasic chloroform-methanol-water protocol (II) a single phase methanol-water protocol and (III) a single phase methanol-acetonitrile-water protocol. LC-MS/MS analyses were carried out via High Performance Liquid Chromatography, either on hydrophilic interaction (HILIC) or on reversed-phase (C18) columns in both positive and negative ionisation modes, coupled with a Q-TOF mass spectrometer. Results suggest that the highest consistency between replicates and quality control samples was obtained with the single phase extractions (i.e., methanol-acetonitrile-water), whilst the ideal combination of instrumental set up HILIC chromatography in positive ionisation mode and of C18 chromatography in negative ionisation mode. For the purpose of forensic investigations, a combination of a single phase extraction and the two aforementioned chromatographic and mass spectrometry modes could represent an ideal set up for obtaining bone metabolomic profiles from taphonomically altered bones

Decomposition in an extreme cold environment and associated microbiome—prediction model implications for the postmortem interval estimation

Introduction: The accurate estimation of postmortem interval (PMI), the time between death and discovery of the body, is crucial in forensic science investigations as it impacts legal outcomes. PMI estimation in extremely cold environments becomes susceptible to errors and misinterpretations, especially with prolonged PMIs. This study addresses the lack of data on decomposition in extreme cold by providing the first overview of decomposition in such settings. Moreover, it proposes the first postmortem microbiome prediction model for PMI estimation in cold environments, applicable even when the visual decomposition is halted. Methods: The experiment was conducted on animal models in the second-coldest region in the United States, Grand Forks, North Dakota, and covered 23 weeks, including the winter months with temperatures as low as −39°C. Random Forest analysis models were developed to estimate the PMI based either uniquely on 16s rRNA gene microbial data derived from nasal swabs or based on both microbial data and measurable environmental parameters such as snow depth and outdoor temperatures, on a total of 393 samples. Results: Among the six developed models, the best performing one was the complex model based on both internal and external swabs. It achieved a Mean Absolute Error (MAE) of 1.36 weeks and an R2 value of 0.91. On the other hand, the worst performing model was the minimal one that relied solely on external swabs. It had an MAE of 2.89 weeks and an R2 of 0.73. Furthermore, among the six developed models, the commonly identified predictors across at least five out of six models included the following genera: Psychrobacter (ASV1925 and ASV1929), Carnobacterium (ASV2872) and Pseudomonas (ASV1863). Discussion: The outcome of this research provides the first microbial model able to predict PMI with an accuracy of 9.52 days over a six-month period of extreme winter conditions

Age estimation for two Mediterranean populations:rib histomorphometry applied to forensic identification and bone remodelling research

Numerous intrinsic and extrinsic factors influence bone remodelling rates and have shown to affect the accuracy of histological aging methods. The present study investigates the rib cortex from two Mediterranean skeletal collections exploring the development of population-specific standards for histomorphometric age-at-death estimation. Eighty-eight standard ribs from two samples, Cretans and Greek-Cypriots, were processed histologically. Thirteen raw and composite histomorphometric parameters were assessed and observer error tested. The correlation between age and the parameters and the differences between sex and population subsamples were explored through group comparisons and analysis of covariance. General linear models assessed through data fit indicators and cross-validation were generated from the total dataset, and by sex and population subsamples. Most of the histological variables showed a statistically significant correlation with age with some differences observed by sex and by sample. From the twelve models generated, the optimal model for the whole sample included osteon population density (OPD), osteon perimeter, and osteon circularity producing an error of 10.71 years. When sex and samples were separated, the best model selected included OPD and osteon perimeter producing an error of 8.07 years for Greek-Cypriots. This research demonstrates the feasibility of quantitative bone histology to estimate age, obtaining errors rates in accordance with macroscopic ageing techniques. Sex and sample population differences need further investigation and inter-population variation in remodelling rates is suggested. Moreover, this study contributes to the creation of population-specific standards for Cretans and Greek-Cypriots

The posterior portion of the ilium as a sex indicator:A validation study

Establishing a biological profile of skeletal remains is a key task of forensic anthropologists. Sex estimation is essential in forensic examination, as other elements of the biological profile, such as age at death or stature, are sex dependent. Visual assessment is considered low-cost and quick, therefore it is a commonly applied method of sex estimation. The most reliable results can be obtained with the analysis of the anterior part of the pelvis, however, these skeletal elements are fragile and prone to destruction. In contrary, the more robust posterior portion of the pelvis is often recovered. Several features of the posterior pelvis have been explored in the context of sexual dimorphism. The aim of the present study was to test three previously published methods of sex assessment based on the analysis of the inferior shape of the auricular surface (Novotný, 1975), the greater sciatic notch shape (Walker, 2005 (revised)) and overall morphology, apex morphology and inflection of the auricular surface (Luna et al., 2017). The sample consisted of 194 individuals of Greek origin from a documented modern collection. Four features of the auricular surface and shape of the greater sciatic notch were examined. Logistic regression analysis was applied to produce a sex discriminatory formula. The method proposed by Luna et al. (2017) failed to produce satisfactory results with overall accuracies of 36%, 50% and 53% for overall morphology, apex morphology and inflection respectively. Slightly better results (64%) were obtained with the inferior shape morphology (Novotný, 1975). However, the highest accuracy rate of 81% was noted for the greater sciatic notch shape (Walker, 2005 (revised)). The formula produced in this study allowed correct classification of 83.2% of the sample. This study illustrates that in spite of the presence of sexual dimorphism in the posterior portion of ilium, features of the auricular surface proposed as sex indicators by Novotný (1975) and Luna et al. (2017) should not be used for sex estimation purposes in the Greek population. The formula produced in this study and the greater sciatic notch shape should only be used as additional methods in cases where neither the cranium nor the anterior portion of the pelvis is present

Bone-mineral density: clinical significance, methods of quantification and forensic applications

Bone-mineral density (BMD) is a measure of the inorganic mineral content inbone, and is one of the more informative assessments of bone quality in both clinical studiesand forensic investigations. Several factors, such as age, sex, disease, genetics, and lifestyle,affect BMD measurements, and normative standards must be applied for specific groups andindividuals. One of the most common disorders associated with low BMD is osteoporosisand increased fracture risk, due to a decrease in bone strength and an increase in bonefragility. Medical conditions like diabetes or hyperthyroidism and other parameters like peakbone mass and postmenopausal estrogen deficiency also impact BMD. Single- and dual-energy photon absorptiometry, quantitative computet tomography, and magnetic resonanceimaging are some of the technological modalities for BMD quantification, and each presentsdistinct advantages and limitations, depending on the purpose of the analysis, the specificcharacteristics of the individual, the bone site under examination, and the equipment andtrained personnel available. Recently, BMD values were applied to forensic medicine in avariety of scenarios ranging from age and sex estimation to the assessment of malnutritionand the use offinite-element modelling. Despite technical and methodological inconsisten-cies reported in the literature on BMD readings, there is scope for expanding the use of thisvariable in forensic settings (4) (PDF) Bone-mineral density: clinical significance, methods of quantification and forensic applications. Available from: https://www.researchgate.net/publication/334676972_Bone-mineral_density_clinical_significance_methods_of_quantification_and_forensic_applications [accessed Aug 22 2019]

Insights into the Differential Preservation of Bone Proteomes in Inhumed and Entombed Cadavers from Italian Forensic Caseworks

Bone is a hard biological tissue and a precious reservoir of information in forensic investigations as it retains key biomolecules commonly used for identification purposes. Bone proteins have recently attracted significant interest for their potential in estimating post-mortem interval (PMI) and age at death (AAD). However, the preservation of such proteins is highly dependent on intrinsic and extrinsic factors that can hinder the potential application of molecular techniques to forensic sciences. The present study aims at investigating the effects that two commonly used types of burial practices (entombment and inhumation) have on bone protein survival. The sample consists of 14 exhumed individuals from cemeteries in Southern Italy with different AADs (29–85 years) and PMIs (1–37 years). LC-MS/MS analyses show that 16 proteins are better preserved under the entombed conditions and 4 proteins are better preserved under the inhumed conditions, whereas no clear differences are detected for post-translational protein modifications. Furthermore, several potential “stable” protein markers (i.e., proteins not affected by the burial environment) are identified for PMI and AAD estimation. Overall, these results show that the two burial environments play a role in the differential preservation of noncollagenous proteins, confirming the potential of LC-MS/MS-based proteomics in forensic sciences

Bone Diagenesis in Short Timescales: Insights from an Exploratory Proteomic Analysis

The evaluation of bone diagenetic phenomena in archaeological timescales has a long history; however, little is known about the origins of the microbes driving bone diagenesis, nor about the extent of bone diagenesis in short timeframes—such as in forensic contexts. Previously, the analysis of non-collagenous proteins (NCPs) through bottom-up proteomics revealed the presence of potential biomarkers useful in estimating the post-mortem interval (PMI). However, there is still a great need for enhancing the understanding of the diagenetic processes taking place in forensic timeframes, and to clarify whether proteomic analyses can help to develop better models for estimating PMI reliably. To address these knowledge gaps, we designed an experiment based on whole rat carcasses, defleshed long rat bones, and excised but still-fleshed rat limbs, which were either buried in soil or exposed on a clean plastic surface, left to decompose for 28 weeks, and retrieved at different time intervals. This study aimed to assess differences in bone protein relative abundances for the various deposition modalities and intervals. We further evaluated the effects that extrinsic factors, autolysis, and gut and soil bacteria had on bone diagenesis via bottom-up proteomics. Results showed six proteins whose abundance was significantly different between samples subjected to either microbial decomposition (gut or soil bacteria) or to environmental factors. In particular, muscle- and calcium-binding proteins were found to be more prone to degradation by bacterial attack, whereas plasma and bone marrow proteins were more susceptible to exposure to extrinsic agents. Our results suggest that both gut and soil bacteria play key roles in bone diagenesis and protein decay in relatively short timescales, and that bone proteomics is a proficient resource with which to identify microbially-driven versus extrinsically-driven diagenesis

Preliminary investigation of the effect of maceration procedures on bone metabolome and lipidome

The study of post-mortem changes is a crucial component of forensic investigation. Human forensic taphonomic facilities (HFTFs) are the only institutions allowing the design and execution of controlled human decomposition experiments. When bodies are skeletonised, bones are normally stored in skeletal collections and used for anthropological studies. However, HFTFs apply chemical and/or thermal treatments to the remains prior bone long-term storage. These treatments are believed to alter heavily the original biochemical and molecular signature of bone material. The present study aims to evaluate the effect of these procedures on the bone metabolome and lipidome by using an animal bone model. Three intact bovine tibiae were processed using three protocols routinely applied at HFTFs, and their three counterparts were used as non-treated controls. Bone powder samples were subjected to biphasic extraction and both metabolites and lipids were analysed via liquid chromatography tandem mass-spectrometry. Results showed severe reductions in the abundances of both metabolites and lipids, and the presence of contamination introduced by cleaning agents. Despite the preliminary nature of the study, we demonstrated that the biochemical profile of bone is heavily affected by the maceration procedures. Ideally, these treatments should be avoided, or replaced by minimally invasive procedures agreed across HFTFs

Bone histomorphometry of the clavicle in a forensic sample from Albania

Forensic assessment of skeletal material includes age estimation of unknown individuals. When dealing with extremely fragmented human remains that lack macro-features used in age estimation, histological assessment of the skeletal elements can be employed. Historically, microscopic methods for age assessment used by forensic anthropologists have been available since 1965. Several skeletal elements have been used for this purpose. Among them, the clavicle has garnered very little attention. The purpose of this study is to explore the validity of clavicular histomorphometry as an age marker in a modern Balkan sample. This study examined a modern clavicular autopsy sample from Albania. The sample consisted of 33 individuals of known age and cause of death. Data were collected for micro-anatomical features including osteon population density (OPD) and cortical area. Intra- and inter-observer errors were assessed through technical error of measurement (TEM) and R coefficient. A validation study was performed in order to test the accuracy of existing histological formulae. Regression analysis was run to developed age prediction models with the best models tested through cross-validation and the comparison between OPD for the Albanian sample and a European-American sample examined. Intra- and inter-observer error TEM results demonstrated values falling within the limits of acceptance. The existing histological methods did not perform accurately on the sample under study. Regression equations for Albanians produced age estimations deviating 8 and 11 years from known age. Cross-validation on the most accurate regression formula which includes OPD as a single variable demonstrated similar mean errors. Statistically significant differences were observed between the Albanian and the European-American population when the two samples were compared. The research presented is the fifth article published and the fifth population explored on clavicular microstructure. The potential of histology to estimate age on the Albanian population is shown here; however, population effect, diet and health status might be considered. Further inclusion of individuals will corroborate our preliminary findings