An overview of the heat-induced changes of the chemical composition of bone from fresh to calcined.

When bone is exposed to thermal stress, the chemical composition changes. This affects bone tissue regeneration after surgery, and these changes can also aid in reconstructing ante-, peri-, and post-mortem events in forensic investigations and past activities on cremation practices in archaeology. However, to date, no complete overview exists on the chemical composition of both fresh and thermally altered bone. Therefore, we aimed (i) to present the chemical composition of fresh bone and (ii) to present an overview of heat-induced chemical changes in bone under both reducing and oxidizing conditions. From the overview, it became clear that some chemical changes occur at a consistent temperature, independent of exposure duration, meaning there is a temperature threshold. However, the occurrence of other chemical changes appeared to be more inter-experimentally variable, and therefore, it is recommended to further investigate these changes.

Temperature-specific spectral shift of luminescing thermally altered human remains.

Human bone has shown to have luminescent properties that remain throughout the phases of cremation, with the exception of fully carbonized bone, when excited with a narrow band light source. During this research, an alternate light source (420-470nm, peak at 445nm) was used to visualize and investigate latent details relevant for forensic investigations of human remains recovered at fire scenes. As fire is a destructive force, it induces a vast variety of physical and chemical alterations to all components of the bone, making the subsequent analysis and interpretation of burned human remains challenging. A spectral shift in emission bandwidth, from green to red, was previously observed when the exposure temperature increased from 700 to 800 °C. This spectral shift was reproduced on a total of 10 human forearms, divided into 20 segments, by burning at 700 °C and 900 °C in an ashing furnace. The shift of emission bandwidth caused only by an increase in temperature was furthermore investigated by colorimetric analysis, proving the spectral shift to be significant. By easily quantifying the spectral shift, substantiation is provided for the use of this technique in practice to improve the interpretation of heat induced changes of bone.

Preliminary results of an investigation on postmortem variations in human skeletal mass of buried bones.

Extreme fragmentation can complicate the inventory of human skeletal remains. In such cases, skeletal mass can provide information regarding skeleton completeness and the minimum number of individuals. For that purpose, several references for skeletal mass can be used to establish comparisons and draw inferences regarding those parameters. However, little is known about the feasibility of establishing comparisons between inherently different materials, as is the case of curated reference skeletal collections and human remains recovered from forensic and archaeological settings. The objective of this paper was to investigate the effect of inhumation, weather and heat exposure on the skeletal mass of two different bone types. This was investigated on a sample of 30 human bone fragments (14 trabecular bones and 16 compact bones) that was experimentally buried for two years after being submitted to one of four different heat treatments (left unburned; 500 °C; 900 °C; 1000 °C). Bones were exhumed periodically to assess time-related mass variation. Skeletal mass varied substantially, decreasing and increasing in accordance to the interchanging dry and wet seasons. However, trends were not the same for the two bone types and the four temperature thresholds. The reason for this appears to be related to water absorption and to the differential heat-induced changes in bone microporosity, volume, and composition. Our results suggest that mass comparisons against published references should be performed only after the skeletal remains have been preemptively dried from exogenous water.

The circles of life: age at death estimation in burnt teeth through tooth cementum annulations.

Age at death estimation in burnt human remains is problematic due to the severe heat-induced modifications that may affect the skeleton after a burning event. The objective of this paper was to assess if cementochronology, which focuses on the cementum incremental lines, is a reliable method of age estimation in burnt remains. Besides the classical approach based on the counting of incremental lines, another approach based on the extrapolation of incremental lines taking into account the cement layer thickness and the incremental line thickness was investigated. A comparison of the performance of the two techniques was carried out on a sample of 60 identified monoradicular teeth that were recently extracted at dentist offices and then experimentally burnt at two maximum temperatures (400 and 900 °C). Micrographs of cross-sections of the roots were taken via an optical microscope with magnification of ×100, ×200 and ×400. Incremental line counting and measurements were carried out with the ImageJ software. Age estimation based on incremental line counting in burnt teeth had no significant correlation with chronological age (p = 0.244 to 0.914) and led to large absolute mean errors (19 to 21 years). In contrast, age estimation based on the extrapolation approach showed a significant correlation with known age (p = 0.449 to 0.484). In addition, the mean absolute error of the latter was much smaller (10 to 14 years). The reason behind this discrepancy is the heat-induced dimensional changes of incremental lines that affect their visibility and individualization thus complicating line counting. Our results indicated that incremental lines extrapolation is successful at solving this problem and that the resulting age estimation is much more reliable.

Rather yield than break: assessing the influence of human bone collagen content on heat-induced warping through vibrational spectroscopy.

Warping has been used to determine the pre-burning condition of human skeletal remains. In the literature, this modification has been associated more often with the burning of fleshed and green bones, but it also arises during the burning of dry bones. The objective of this paper was to assess if bone collagen content has a significant effect on the occurrence of warping in a sample of experimentally burned human bones. The presence of collagen was analyzed in two different samples through a vibrational spectroscopy technology-FTIR. One of them was composed of 40 archeological bones from the seventeenth to twentieth centuries AD. The other one was composed of bones from 14 skeletons belonging to the 21st century identified skeletal collection. The results confirmed that the amide I band assigned to the collagen was much more intense in bones presenting heat-induced warping. Nonetheless, although significant (p = 0.040), the collagen content was not as useful as other variables to the regression model we proposed for explaining warping. Factors such as the maximum temperature (p < 0.001) and burning time (p = 0.001) contributed more significantly. Results demonstrated that the mere preservation of collagen is not enough to explain warping. Burning dynamics seem to have an important role as well although we failed to clearly document its specificities. Other factors such as the asymmetric distribution of collagen and other components within bone, the gravity force, the shape of the bone, and the position in which it is burned may also play an important role on heat-induced changes and require further analysis.

The effects of exogenous substances on the color of heated bones.

OBJECTIVES: Burned bone coloration has been used for decades to help in the bioanthropological analysis of burned human bones. However, there is a variety of factors that can interfere with the coloration manifested by bones exposed to heat, resulting in colors that differ from the usual black to white gradient. In this study, we evaluated possible causes of unusual coloration changes and hues in burned bone. MATERIALS AND METHODS: For that purpose, defleshed fresh pig (Sus scrofa) ribs as well as fresh and dry human clavicles were burned at four different temperatures (500, 700, 900 and 1100°C) in contact with different materials (CaO, Zn, Fe, Cu, Mn, and polyester cloth). Observable color changes were assessed through naked eye observation and description, Munsell color charts, and reflectance spectrophotometry. Additionally, chemical changes in bone were assessed using Fourier-transform infrared spectroscopy in attenuated total reflectance (FTIR-ATR) and x-ray fluorescence (XRF). RESULTS: Our results showed that some materials did affect usual burned bone coloration (Fe, Mn, Cu, Zn) and correspondent FTIR-ATR and XRF spectra. As for other materials, although no effect on visual bone coloration was observed, they still affected FTIR-ATR and XRF spectra (CaO and cloth). DISCUSSION: This study can contribute to the anthropological analysis of burned human remains, providing some answers to what can cause unusual types of heat-induced colorations.

Reaching new heights: Testing the performance of metric approaches to estimate stature from burned skeletal remains.

Bone heat-induced changes complicate osteometric stature estimation of human remains from forensic settings. The validity of current methods has not been tested to a great extent. Our aim was to determine how precise are stature estimations obtained from three different approaches, namely by using (i) Rösing's method (Rösing 1977), (ii) a 10% shrinkage correction factor (Strzalko et al. 1972) and (iii) chemosteometry (Gonçalves et al. 2020). For this purpose, pre- and post-burned head measurements from the humerus, radius and femur were used. The sample comprised 46 human skeletons of known sex and age-at-death. These were experimentally burnt to maximum temperatures ranging from 700 to 1100°C (attained after 90-188 min) for other research purposes. Stature estimations were performed through measurements in both pre-burned and burned bones using the three approaches and based on the method of Olivier and Tissier (1975). Mean absolute differences and the relative technical errors of measurements (TEM%) between the pre-burned and the estimations were calculated. Absolute mean differences indicated that the chemosteometric approach allowed for potentially more precise stature estimations than the other two procedures. However, the TEM% was very low for all approaches (smaller or close to 1%), and stature estimations were thus well within the error margin reported by Olivier and Tissier (1975). Results suggest that the chemosteometric approach enables more accurate predictions of the actual heat-induced shrinkage of each bone thus rendering more precise stature estimations. Nonetheless, the other procedures also provided quite reliable estimations although they require confirmation that the bone is calcined.

Time burnt away: The impact of heat-induced changes on skeletal age-at-death diagnostic features.

This paper investigates the established notion that bone calcination has a major impact on age estimation while low-intensity burns have a mere negligible impact. Few systematic researches have been carried out so far about this topic so the true impact of heat-induced changes on diagnostic age features is mostly unknown. The agreement between pre-burning and post-burning observations of age features was investigated on 51 human skeletons (22 males and 29 females with ages-at-death ranging from 61 to 93 years old) subjected to experimental burns. These skeletons belong to the 21st Century Identified Skeletal Collection housed at the Laboratory of Forensic Anthropology of the Department of Life Sciences, University of Coimbra, Portugal. The Suchey-Brooks method based on the pubic symphysis and the method developed on the auricular surface by Buckberry and Chamberlain (2002) were scrutinized. The Suchey-Brooks method provided better agreement between pre- and post-burn observations than the method from Buckberry and Chamberlain (2002). However, it became clear that heat-induced changes affected both methods regardless of heat intensity since both calcined bones and bones burnt at lower intensities often showed less than perfect agreement. Therefore, this research demonstrates that the analysis of age-at-death can be impaired in burnt bones, even those not subjected to calcination, with clear impact for forensic and archaeological investigations.

Chemical trace XRF analysis to detect sharp force trauma in fresh and burned bone.

Forensic anthropologists may not always be able to differentiate heat-induced fractures from fractures with other aetiologies, namely sharp force traumas, with clear nefarious impact on medico-legal conclusions. The objective of this study was to experimentally investigate if blade chemical traces are transferred to defleshed bone tissue and if they remain there after a burning event. This was accomplished by prompting sharp force traumas in 20 macerated fresh pig ribs with five different instruments, namely a stainless steel knife, an artisanal knife and a ceramic kitchen knife, a small axe and a large axe. Another pig rib was used as control, not being subjected to any trauma. All instruments were probed by X-ray fluorescence (XRF) to establish the composition of each blade. Bone samples, both pre-burned and post-burned (at 500 °C, 700 °C, 900 °C and 1100 °C), were then probed by XRF. All sharp force instruments left detectable chemical traces on pre-burned bone, although not in all samples. Furthermore, traces were still detected after experimental burning in most cases. Potentially, XRF can provide relevant information about the aetiology of fractures in burned and unburned bones, although the effect of soft tissues and diagenesis must still be investigated.

The burning maze: The potential value of the human bony labyrinth in estimating sex of calcined remains.

Estimating sex from burnt human remains is a challenging task in bioanthropology, mainly due to their high level of alteration and fragmentation. Protected within the petrous part of the temporal bone, the bony labyrinth may be particularly valuable for assessing the sex of burnt remains. This prospective study aims at testing predictive models, already found reliable on unburnt bony labyrinths, to burnt specimens. Six discriminant functions were applied on six bony labyrinths of donated adult cadavers of known sex, before and after outdoor burning experiments. Comparisons between unburnt and burnt measurements were executed using Mann-Whitney U tests while shape and size differences induced by fire exposure were examined through a geometric morphometrics (GM) analysis. Predicted sex on unburnt bony labyrinths was consistent with known sex in five cases while a systematic misclassification for males was highlighted on burnt specimens. Higher values of shrinkage were found in males for two measurements included in the equations. GM analysis revealed significant differences in centroid size among males after calcination. Visualization of mean consensus of both female and male bony labyrinths evidenced a reduction in cochlear size and variations in the width and length of semicircular canals of burnt specimens. This exploratory study seems to confirm that designing sex estimation standards specifically for burnt bony labyrinth may be advisable. Understanding how the burning process could impact its morphology is highly recommended through further experiments on larger samples and in controlled environments.

Effects of heating on the physical properties of premixed calcium silicate-based root canal sealers.

The purpose of this study was to examine the effect of heating on the physical properties of four types of premixed calcium silicate-based root canal sealers. Endoseal MTA, Well-Root ST, EndoSequence BC Sealer, EndoSequence BC Sealer HiFlow, and AH Plus (epoxy resin root canal sealer) were heated at 100°C for 1 min, and changes in setting time, flow, and film thickness were evaluated in accordance with ISO 6876:2012 standards. In addition, pH measurement and scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analyses of the set materials were performed. All root canal sealers heated at 100°C showed significant decreases in setting time and flow, particularly Endoseal MTA. In addition, the film thickness of Endoseal MTA increased significantly after heating at 100°C. The pH and SEM/EDS results were not affected by heating. Heating calcium silicate-based root canal sealers accelerated setting time, decreased flow, and increased film thickness. However, the degree of these changes varied among the products. The present findings indicate that heat-induced changes in the physical properties of calcium silicate-based root canal sealers could adversely affect the quality of warm vertical condensation technique.

Assessment of skeletal changes after post-mortem exposure to fire as an indicator of decomposition stage.

Forensic anthropologists are tasked with interpreting the sequence of events from death to the discovery of a body. Burned bone often evokes questions as to the timing of burning events. The purpose of this study was to assess the progression of thermal damage on bones with advancement in decomposition. Twenty-five pigs in various stages of decomposition (fresh, early, advanced, early and late skeletonisation) were exposed to fire for 30 min. The scored heat-related features on bone included colour change (unaltered, charred, calcined), brown and heat borders, heat lines, delineation, greasy bone, joint shielding, predictable and minimal cracking, delamination and heat-induced fractures. Colour changes were scored according to a ranked percentage scale (0-3) and the remaining traits as absent or present (0/1). Kappa statistics was used to evaluate intra- and inter-observer error. Transition analysis was used to formulate probability mass functions [P(X=j|i)] to predict decomposition stage from the scored features of thermal destruction. Nine traits displayed potential to predict decomposition stage from burned remains. An increase in calcined and charred bone occurred synchronously with advancement of decomposition with subsequent decrease in unaltered surfaces. Greasy bone appeared more often in the early/fresh stages (fleshed bone). Heat borders, heat lines, delineation, joint shielding, predictable and minimal cracking are associated with advanced decomposition, when bone remains wet but lacks extensive soft tissue protection. Brown burn/borders, delamination and other heat-induced fractures are associated with early and late skeletonisation, showing that organic composition of bone and percentage of flesh present affect the manner in which it burns. No statistically significant difference was noted among observers for the majority of the traits, indicating that they can be scored reliably. Based on the data analysis, the pattern of heat-induced changes may assist in estimating decomposition stage from unknown, burned remains.

Volume analysis of heat-induced cracks in human molars: A preliminary study.

CONTEXT: Only a few methods have been published dealing with the visualization of heat-induced cracks inside bones and teeth. AIMS: As a novel approach this study used nondestructive X-ray microtomography (micro-CT) for volume analysis of heat-induced cracks to observe the reaction of human molars to various levels of thermal stress. MATERIALS AND METHODS: Eighteen clinically extracted third molars were rehydrated and burned under controlled temperatures (400, 650, and 800°C) using an electric furnace adjusted with a 25°C increase/min. The subsequent high-resolution scans (voxel-size 17.7 µm) were made with a compact micro-CT scanner (SkyScan 1174). In total, 14 scans were automatically segmented with Definiens XD Developer 1.2 and three-dimensional (3D) models were computed with Visage Imaging Amira 5.2.2. The results of the automated segmentation were analyzed with an analysis of variance (ANOVA) and uncorrected post hoc least significant difference (LSD) tests using Statistical Package for Social Sciences (SPSS) 17. A probability level of P < 0.05 was used as an index of statistical significance. RESULTS: A temperature-dependent increase of heat-induced cracks was observed between the three temperature groups (P < 0.05, ANOVA post hoc LSD). In addition, the distributions and shape of the heat-induced changes could be classified using the computed 3D models. CONCLUSION: The macroscopic heat-induced changes observed in this preliminary study correspond with previous observations of unrestored human teeth, yet the current observations also take into account the entire microscopic 3D expansions of heat-induced cracks within the dental hard tissues. Using the same experimental conditions proposed in the literature, this study confirms previous results, adds new observations, and offers new perspectives in the investigation of forensic evidence.

X-ray scattering evaluation of ultrastructural changes in human dental tissues with thermal treatment.

Micro- and ultrastructural analysis of burned skeletal remains is crucial for obtaining a reliable estimation of cremation temperature. Earlier studies mainly focused on heat-induced changes in bone tissue, while this study extends this research to human dental tissues using a novel quantitative analytical approach. Twelve tooth sections were burned at 400-900°C (30-min exposure, increments of 100°C). Subsequent combined small- and wide-angle X-ray scattering (SAXS/WAXS) experiments were performed at the Diamond Light Source synchrotron facility, where 28 scattering patterns were collected within each tooth section. In comparison with the control sample, an increase in mean crystal thickness was found in burned dentine (2.8-fold) and enamel (1.4-fold), however at a smaller rate than reported earlier for bone tissue (5-10.7-fold). The results provide a structural reference for traditional X-ray scattering methods and emphasize the need to investigate bone and dental tissues separately to obtain a reliable estimation of cremation temperature.

Weight references for burned human skeletal remains from Portuguese samples.

Weight is often one of the few recoverable data when analyzing human cremains but references are still rare, especially for European populations. Mean weights for skeletal remains were thus documented for Portuguese modern cremations of both recently deceased individuals and dry skeletons, and the effect of age, sex, and the intensity of combustion was investigated using both multivariate and univariate statistics. The cremains from fresh cadavers were significantly heavier than the ones from dry skeletons regardless of sex and age cohort (p < 0.001 to p = 0.003). As expected, males were heavier than females and age had a powerful effect in female skeletal weight. The effect of the intensity of combustion in cremains weight was unclear. These weight references may, in some cases, help estimating the minimum number of individuals, the completeness of the skeletal assemblage, and the sex of an unknown individual.

Application of the Clarifying Reagent Index as a Heat-Induced Parameter in Commercial Milk.

The absorbance at 340 nm of transparent modified milk solutions (designated as Clarifying Reagent index) from industrially processed samples as well as from milk-resembling systems was measured in order to test the validity of this index for determining the extent of previous heat treatment. A statistical study (n = 81) of the Clarifying Reagent index of different commercial milks was carried out. There was a significant difference (P < 0.001, Student's t test) between three groups of samples: (1) raw and pasteurized milk, (2) UHT-treated milk, and (3) in-bottle sterilized milk. The Clarifying Reagent index showed significant relationships (P < 0.001, Student's t test) with other heat-induced parameters such as hydroxymethylfurfural formation, loss of available lysine, and free fluorescent intermediary compounds formed by the Maillard reaction. Between 90 and 140°C, the Clarifying Reagent index of the clarified milk or milk-resembling systems followed zero-order kinetics as determined by nonlinear regression analysis.