Beyond metrics and morphology: the potential of FTIR-ATR and chemometrics to estimate age-at-death in human bone.

In forensic anthropology, the application of traditional methods for estimating the biological profile of human skeletal remains is often hampered by poor preservation and skeletal representativeness, compromising their reliability. Thus, the development of alternative methods to the morphometric analysis of bones to estimate the biological profile of human remains is paramount. The age of an individual can cause changes in bone morphology, mass and size, as well as in its chemical composition. In this sense, the main objective of this research was to evaluate if the contents of bone collagen (Am/P), carbonate type A (API), carbonate type B (BPI), the relation between the carbonate content (types A and B) to type B carbonate (C/C), carbonate-phosphate ratio (C/P) and crystallinity index (CI), spectroscopic indices obtained from relationships between infrared absorption band intensities (FTIR-ATR), can be used as age-at-death predictors. A sample of femora and humeri from the 21st Century Identified Skeleton Collection (N = 80, 44 females and 36 males) was employed. Results show that, with advancing age, women's femora have lower CI values, but BPI and C/P indices increase, and the deformation and disorder of the crystal lattice are probably affected by the integration of type B carbonate content of the femur. The ratios analysed, especially the CI and the BPI, show potential to estimate age-at-death in human skeletal remains, when sex is already known, thus helping to assess the biological profile when conventional methods cannot be applied.

Stochastic interdigitation as a toughening mechanism at the interface between tendon and bone.

Reattachment and healing of tendon to bone poses a persistent clinical challenge and often results in poor outcomes, in part because the mechanisms that imbue the uninjured tendon-to-bone attachment with toughness are not known. One feature of typical tendon-to-bone surgical repairs is direct attachment of tendon to smooth bone. The native tendon-to-bone attachment, however, presents a rough mineralized interface that might serve an important role in stress transfer between tendon and bone. In this study, we examined the effects of interfacial roughness and interdigital stochasticity on the strength and toughness of a bimaterial interface. Closed form linear approximations of the amplification of stresses at the rough interface were derived and applied in a two-dimensional unit-cell model. Results demonstrated that roughness may serve to increase the toughness of the tendon-to-bone insertion site at the expense of its strength. Results further suggested that the natural tendon-to-bone attachment presents roughness for which the gain in toughness outweighs the loss in strength. More generally, our results suggest a pathway for stochasticity to improve surgical reattachment strategies and structural engineering attachments.

Extraction and Physicochemical Characterization of Hydroxyapatites From Horse Humerus Bones of Different Ages (1, 3, 6, and 8 Years old) Calcined at Low Temperature.

The aim of this work is to investigate the changes in the physicochemical properties of hydroxyapatite (HAp) extracted from horse humerus bones of different ages (1, 3, 6, and 8 years) subjected to low temperature calcination (600°C). Thermal analysis revealed significant mass loss due to water, collagen, organic compounds, carbonates, and age-related magnesium out-diffusion. Higher fat content in older bones contributed to increased mass loss. Phosphorus content remained constant across age groups, while calcium and sodium showed age-related fluctuations. Magnesium levels decreased with age, emphasizing its importance for early bone development. The Ca/P ratio deviated from the stoichiometric values due to additional ions from biogenic sources. Infrared spectroscopy identified functional groups in carbonated HAp, with changes observed before and after calcination. The full width at half maximum (FWHM) of the 961 cm-1 band decreased with age, indicating improved crystalline quality. The molar absorption coefficients provided information on the changes in molecular concentration and emphasized the differences between the age groups. X-ray analysis revealed nanocrystalline HAp in all samples, with crystallite size increasing with age. Rietveld analysis showed that the lattice parameters were affected by the presence of organic material, but the lattice constants remained stable, confirming high crystallinity independent of age. TEM analysis confirmed nanocrystalline structures, with crystallite size increasing with age. SEM images showed the characteristic porosity of calcined HAp, with particle size correlating positively with age. Calcination at 600°C preserved the nanoscale properties and microcrystal formation. Raman spectroscopy confirmed the identity of HAp, with FWHM variations indicating age-related changes in crystalline quality. EHAp1 showed increased FWHM, indicating lower crystalline quality and increased trace element content.

Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth.

People with cystic fibrosis (CF) exhibit growth defects. That observation has been attributed, in part, to decreased insulin-like growth factor 1 (IGF1) levels, and the reduction has been blamed on malnutrition and pulmonary inflammation. However, patients with CF already have a reduced weight at birth, a manifestation not likely secondary to poor nutrition or inflammation. We found that, like humans, CF pigs were smaller than non-CF littermates and had lower IGF1 levels. To better understand the basis of IGF1 reduction, we studied newborn pigs and found low IGF1 levels within 12 h of birth. Moreover, humerus length and bone mineral content were decreased, consistent with less IGF1 activity in utero. These findings led us to test newborn humans with CF, and we found that they also had reduced IGF1 levels. Discovering lower IGF1 levels in newborn pigs and humans indicates that the decrease is not solely a consequence of malnutrition or pulmonary inflammation and that loss of cystic fibrosis transmembrane conductance regulator function has a more direct effect. Consistent with this hypothesis, we discovered reduced growth hormone release in organotypic pituitary slice cultures of newborn CF pigs. These findings may explain the long-standing observation that CF newborns are smaller than non-CF babies and why some patients with good clinical status fail to reach their growth potential. The results also suggest that measuring IGF1 levels might be of value as a biomarker to predict disease severity or the response to therapeutics. Finally, they raise the possibility that IGF1 supplementation beginning in infancy might be beneficial in CF.

Profiling of human burned bones: oxidising versus reducing conditions.

Complementary optical and neutron-based vibrational spectroscopy techniques (Infrared, Raman and inelastic neutron scattering) were applied to the study of human bones (femur and humerus) burned simultaneously under either aerobic or anaerobic conditions, in a wide range of temperatures (400 to 1000 °C). This is the first INS study of human skeletal remains heated in an oxygen-deprived atmosphere. Clear differences were observed between both types of samples, namely the absence of hydroxyapatite's OH vibrational bands in bone burned anaerobically (in unsealed containers), coupled to the presence of cyanamide (NCNH2) and portlandite (Ca(OH)2) in these reductive conditions. These results are expected to allow a better understanding of the heat effect on bone´s constituents in distinct environmental settings, thus contributing for an accurate characterisation of both forensic and archaeological human skeletal remains found in distinct scenarios regarding oxygen availability.

Nanocomposite Gel as Injectable Therapeutic Scaffold: Microstructural Aspects and Bioactive Properties.

The development of tissue scaffolds able to provide proper and accelerated regeneration of tissue is a main task of tissue engineering. We developed a nanocomposite gel that may be used as an injectable therapeutic scaffold. The nanocomposite gel is based on biocompatible gelling agents with embedded nanoparticles (iron oxide, silver, and hydroxyapatite) providing therapeutic properties. We have investigated the microstructure of the nanocomposite gel exposed to different substrates (porous materials and biological tissue). Here we show that the nanocomposite gel has the ability to self-reassemble mimicking the substrate morphology: exposition on porous mineral substrate caused reassembling of nanocomposite gel into 10× smaller scale structure; exposition to a section of humerus cortical bone decreased the microstructure scale more than twice (to ≤3 µm). The reassembling happens through a transitional layer which exists near the phase separation boundary. Our results impact the knowledge of gels explaining their abundance in biological organisms from the microstructural point of view. The results of our biological experiments showed that the nanocomposite gel may find diverse applications in the biomedical field.

First analysis of ancient burned human skeletal remains probed by neutron and optical vibrational spectroscopy.

Burned skeletal remains are abundant in archaeological and paleontological sites, the result of fire or of ancient funerary practices. In the burning process, the bone matrix suffers structural and dimensional changes that interfere with the reliability of available osteometric methods. Recent studies showed that these macroscopic changes are accompanied by microscopic variations are reflected in vibrational spectra. An innovative integrated approach to the study of archaeological combusted skeletal remains is reported here, where the application of complementary vibrational spectroscopic techniques-INS (inelastic neutron scattering), FTIR (Fourier transform infrared), and micro-Raman-enables access to the complete vibrational profile and constitutes the first application of neutron spectroscopy to ancient bones. Comparison with data from modern human bones that were subjected to controlled burning allowed identification of specific heating conditions. This pioneering study provides archaeologists and anthropologists with relevant information on past civilizations, including regarding funerary, burial, and cooking practices and environmental settings.

Bullet Caliber and Type Categorization from Gunshot Wounds in Sus Scrofa (Linnaeus) Long Bone.

Studies on ballistic trauma to the ribs and thorax, cranium, and long bones demonstrate the potential of obtaining a bullet caliber from an entrance wound. In order to validate prior research on caliber estimation in bone tissue and assess the viability of bullet type determination based on the macroscopic evidence at the entrance wound, thirty fleshed pork (Sus scrofa) shoulders (humeri) were shot with either lead or copper jacketed bullets in one of three calibers; 0.22, 9 mm, or 0.38. Overall, our findings are consistent with previous research indicating that calibers can be grouped into "small" and "large" categories. Bullet type, lead or copper jacket, can be ascertained based on cortical flaking and the analysis of materials deposited around the entry wound. The addition of this evidence holds value in cases where no firearm or ballistic evidence is recovered from a crime scene.

Analytical bone calcium and bone ash from mature laying hens correlates to bone mineral content calculated from quantitative computed tomography scans.

Laying hen skeletal health continues to be an industry priority. Bone ash and bone Ca quantification in laying hen long bones provides valuable information on skeletal health. Unfortunately, these measurements can only be accomplished by sacrificing hens, thus making longitudinal measurements on the same hen impossible. Quantitative computed tomography (QCT), used with a calcium hydroxyapatite phantom, has been used to determine bone density of wings and legs as well as live hens throughout the production cycle by scanning with a calcium hydroxyapatite phantom. QCT has also been used to scan live hens throughout the production cycle. The purpose of this study was to determine how QCT calculated bone mineral content (QCT BMC) corresponds to analytical bone Ca and bone ash. Wing and leg quarters from 72-wk-old W-36 hens were QCT scanned along with a QCT Phantom. After scanning, humeri, femurs, and tibias were cleaned, divided into epiphysis (E) and diaphysis (D), fat extracted, ashed, and digested under nitric acid, and Ca was determined by atomic absorption spectroscopy was used to determine E, D, and whole bone Ca. Four bones/type were used for E and D, while 6 bones/type were used for whole bone measurements. A second set of bones were prepared to determine correlation of BMC to bone ash. QCT scans were analyzed with Mimics software (Materialise NV, Leuven, Belgium) to calculate bone volume and density in Hounsfield units. Utilizing the QCT phantom and bone volume, BMC was calculated for E, D, and whole bone. Data were analyzed with regression analysis and Pearson correlation coefficients were determined. Analytical Ca was correlated to QCT BMC for E (R = 0.84, P < 0.01), D (R = 0.99, P < 0.01), and whole bone (R = 0.97, P < 0.01). Whole bone ash was highly correlated to QCT BMC for femur (N = 47, R = 0.97, P < 0.001), tibia (N = 50, R = 0.94, P < 0.001), and keel (N 50, R = 0.94, P < 0.001). Whole bone ash and QCT BMC values of femur and tibia were not different (P = 0.39 and 0.22 respectively). Based on this information, QCT could provide relative quantitative assessment of total bone mineral in live birds proving useful in long-term studies.

Evaluation of DNA Extraction Methods from Waterlogged Bones: A Pilot Study.

When deaths occur in water, soft tissue decomposes after a temperature-dependent period, making DNA identification dependent on bone. This study examined the effects of water on bone DNA purity and quantity, and determined the best of three extraction methods for isolating DNA. The organic phenol-chloroform method consistently extracted DNA approximating the accepted 260/280 purity value (~1.8); ChargeSwitch® gDNA Plant Kit and DNeasy Blood and Tissue Kit produced fair and unacceptable values, respectively. The purity value for humerus and rib samples was consistent across accumulated degree days (ADD). Significant differences in quantification among extraction methods and between bone types were identified. Ribs and ChargeSwitch® gDNA Plant Kit samples produced the lowest mean Ct values of the bone types and the extraction methods, respectively. Therefore, this study proposes that magnetic bead technology extraction methods and ribs be considered when processing bones that have been submerged in water for any length of time.

Evaluation of Intraosseous Fluid as an Alternative Biological Specimen in Postmortem Toxicology.

The postmortem redistribution phenomenon is an important factor in the interpretation of blood drug concentrations as a cause or factor in death. Intraosseous fluid (IOF) may serve as an alternative matrix for drug testing. Intraosseous fluid was collected from the left and right tibias and humerus of 29 decedents using the Arrow EZ-IO Intraosseous Vascular Access System. Standard autopsy specimens including blood were also collected at the same time during autopsy. Blood and IOF specimens were screened by immunoassay for opioids, fentanyl analogs, oxycodone, methadone, cocaine, methamphetamine, amphetamines, phencyclidine, tricyclic antidepressants, benzodiazepines and cannabinoids, using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Correlation between cardiac/central blood ELISA and IOF ELISA results was mostly 100% for drug targets. Further blood confirmation analysis was performed by gas chromatography mass spectrometry also showed comparable correlation to IOF screen results. There was no significant difference between the IOF sites or sides of the body. This novel study supports the use of IOF as an alternative postmortem specimen for toxicological investigations as a potentially less-compromised tissue in decomposed or traumatized bodies. Preliminary data is provided for the screening of common drugs of abuse in IOF that may show to be subject to alternative rates of postmortem redistribution than to that of other biological specimens in future studies that quantitate IOF drug concentrations.

Comparison of lead residues among avian bones.

To determine if significant differences exist in lead (Pb) accumulation in different bones, especially those most often used for bone-Pb studies in wildlife, we compared Pb concentrations in radius, ulna, humerus, femur, and tibia of Common Eider (Somateria mollissima); and radius/ulna (combined), femur, and tibia of American Woodcock (Scolopax minor). There were no significant differences in bone-Pb concentrations among woodcock bones over a wide range of Pb concentrations (3-311 microg/g). In eider, where bone-Pb concentrations were low (<10 microg/g), leg bones had significantly higher Pb concentrations (approximately 30-40%) than wing bones from the same individuals. The variation among individual birds was greater than the variation among different bones within a bird. Based on our findings, we conclude that one type of bone may be substituted for another in bone-Pb studies although the same bone type should be analyzed for all birds within a study, whenever possible.

An absorbed dose map of bone tissue treated with a radiopharmaceutical in vivo.

A beagle humerus treated with Ho-chelate radiopharmaceutical in vivo was examined by electron paramagnetic resonance (EPR) dosimetry. The bone was sectioned and the absorbed dose to each bone fragment was determined by additive re-irradiation of the bone tissue with calibrated doses of gamma radiation. The measured doses ranged from 4.3 Gy to 62 Gy. The highest doses were recorded in the predominately trabecular bone tissue and the lowest doses in the predominately cortical bone tissue. The mean absorbed dose for the entire bone was 17 Gy. The data from 50 bone fragments were combined to create an absorbed dose map of the interior bone surface.

Lead isotopes and lead shot ingestion in the globally threatened marbled teal (Marmaronetta angustirostris) and white-headed duck (Oxyura leucocephala).

Lead isotope ratios ((206)Pb/(207)Pb and (208)Pb/(207)Pb) and concentrations in the livers and bones of marbled teal and white-headed duck found dead or moribund were determined in order to establish the main lead source in these waterfowl species. Lead concentrations in bone (dry weight) and liver (wet weight) were found to be very high in many of the white-headed ducks (bone: geometric mean=88.9 ppm, maximum=419 ppm; liver: geometric mean=16.8 ppm, maximum=57.0 ppm). Some of the marbled teal had high lead levels in the bones but liver lead levels were all low (bone: geometric mean=6.13 ppm, maximum=112 ppm; liver: geometric mean=0.581 ppm, maximum=4.77 ppm). Ingested lead shot were found in 71% of the white-headed duck and 20% of the marbled teal. The (206)Pb/(207)Pb ratio in livers and bones of white-headed ducks and marbled teals showed no significant differences compared to the ratios obtained from lead shot. The (206)Pb/(207)Pb ratio in bones of marbled teal ducklings with the highest lead concentrations tended to resemble the ratios of lead shot, which supports our hypothesis that the lead was derived from the hens. We also found that the lead ratios of lead shot and lead ratios described for soils in the area overlapped, but also that the isotopic ratio (206)Pb/(207)Pb in lead shot used in Spain has a narrow range compared with those used in North America. The principal source of lead in many of these birds was, however, most likely lead shot, as supported by the similar isotopic ratios, high lead concentrations in tissues and evidence of ingested shot.

Evaluation of RF heating on humerus implant in phantoms during 1.5T MR imaging and comparisons with electromagnetic simulation.

PURPOSE: To evaluate the effect of radiofrequency (RF) heating on a metallic implant during magnetic resonance imaging (MRI), temperatures at several positions of an implant were measured, and results are compared with electromagnetic simulations using a finite element method. METHODS: A humerus nail implant made of stainless steel was embedded at various depths of tissue-equivalent gel-phantoms with loop (loop phantom) and partially cut loop (loop-cut phantom), and the phantoms were placed parallel to the static magnetic field of a 1.5T MRI device. Scans were conducted at maximum RF for 15 min, and temperatures were recorded with 2 RF-transparent fiberoptic sensors. Finally, electromagnetic-field analysis was performed. RESULTS: Temperatures increased at both ends of the implants at various depths, and temperature increase was suppressed with increasing depth. The maximum temperature rise was 12.3 degrees C at the tip of the implant and decreased for the loop-cut phantom. These tendencies resembled the results of electromagnetic simulations. CONCLUSION: RF heating was verified even in a nonmagnetizing metal implant in a case of excessive RF irradiation. Particularly, rapid temperature rise was observed at both ends of the implant having large curvatures. The difference in temperature increase by depth was found to reflect the skin-depth effect of RF intensity. Electromagnetic simulation was extremely useful for visualizing the eddy currents within the loop and loop-cut phantoms and for evaluating RF heating of a metallic implant for MRI safety.

Detection of toluene in a body buried for years with a fatal cardiac contusion.

This report aimed to present the postmortem finding of toluene in a homicide victim buried under the ground for six years. The bones of the skull and limbs were exposed, and the remainder of the subcutaneous tissues, brain and heart had formed into adipocere. There were numerous fractures in the skull and the anterior side of the ribs. A cardiac contusion extending into the cavity of the right ventricle was also observed. No other obvious injuries were identified on the body. The concentration of toluene in the bone marrow within the head of the humerus was 58.4 µg/g. The cause of death was suspected as heart rupture, possibly from a forceful impact or compression of the anterior chest under toluene intoxication. This report presents a rare case where toluene intake by a human was disclosed by autopsy even after several years of death.

Preliminary Study to Test the Feasibility of Sex Identification of Human (Homo sapiens) Bones Based on Differences in Elemental Profiles Determined by Handheld X-ray Fluorescence.

Sex assignment of human remains is a crucial step in forensic anthropological studies. The aim of this study was to examine elemental differences between male and female bones using X-ray fluorescence (XRF) and determine if elemental profiling could be used for sex discrimination. Cranium, humerus, and os coxae of 60 skeletons (30 male, 30 female) from the Chiang Mai University Skeletal Collection were scanned by XRF and differences in elemental profiles between male and female bones determined using discriminant analysis. In the cranium, three elements (S, Ca, Pb) were significantly higher in males and five elements (Si, Mn, Fe, Zn, Ag) plus light elements (atomic number lower than 12) were higher in females. In humerus and os coxae, nine elements were significantly higher in male and one element was higher in female samples. The accuracy rate for sex estimation was 60, 63, and 61 % for cranium, humerus, and os coxae, respectively, and 67 % when data for all three bones were combined. We conclude that there are sex differences in bone elemental profiles; however, the accuracy of XRF analyses for discriminating between male and female samples was low compared to standard morphometric and molecular methods. XRF could be used on small samples that cannot be sexed by traditional morphological methods, but more work is needed to increase the power of this technique for gender assignment.

Bone structural similarity score: a multiparametric tool to match properties of biomimetic bone substitutes with their target tissues.

BACKGROUND: One of the hardest tasks in developing or selecting grafts for bone substitution surgery or tissue engineering is to match the structural and mechanical properties of tissue at the recipient site, because of the large variability of tissue properties with anatomical site, sex, age and health conditions of the patient undergoing implantation. We investigated the feasibility of defining a quantitative bone structural similarity score based on differences in the structural properties of synthetic grafts and bone tissue. METHODS: Two biocompatible hydroxyapatite porous scaffolds with different nominal pore sizes were compared with trabecular bone tissues from equine humerus and femur. Images of samples' structures were acquired by high-resolution micro-computed tomography and analyzed to estimate porosity, pore size distribution and interconnectivity, specific surface area, connectivity density and degree of anisotropy. Young's modulus and stress at break were measured by compression tests. Structural similarity distances between sample pairs were defined based on scaled and weighted differences of the measured properties. Their feasibility was investigated for scoring structural similarity between considered scaffolds or bone tissues. RESULTS: Manhattan distances and Quadrance generally showed sound and consistent similarities between sample pairs, more clearly than simple statistical comparison and with discriminating capacity similar to image-based scores to assess progression of pathologies affecting bone structure. CONCLUSIONS: The results suggest that a quantitative and objective bone structural similarity score may be defined to help biomaterials scientists fabricate, and surgeons select, the graft or scaffold best mimicking the structure of a given bone tissue.

Rapid increase of carbonate in cortical bones of hens during laying period.

The incorporation of carbonate has been recognized as an evident change in bone mineral (bioapatite) during aging. Laying hens (Gushi layer) at 4 stages of age (8 hens each stage) were studied by Raman spectroscopy and X-ray radiography to investigate the mineralogical changes and bone density, respectively. Cortical bones of the humerus and femur show a rapid increase of carbonate (∼1.9 wt.%) from sexual maturity to the peak of hens' laying period, while the densities of the cortical bones are relatively stable. Before sexual maturity, the density of the cortical bones increases considerably during aging. However, after the peak of the laying period, only femoral density continues elevating. Carbonate contents in the cortical bones reach the maximum at the peak of the laying period. Two pathways (halted growth of density and Ca-release due to the CO3 incorporation) could both contribute to the intense Ca requirement for egg laying. Crystallization, however, has no significant changes during aging and the laying period. This study could shed light on the mechanism of mineral losses due to CO3 incorporation, and also shows the advantages of Raman spectroscopy in tracking mineral loss in poultry bone.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens.

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with beta-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed beta-D-glucuronic acid and N-acetyl beta-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.