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The Epigravettian human remains from Riparo Tagliente in northern Italy represent some of the earliest evidence of human occupation in the southern Alpine slopes after the Last Glacial Maximum. Genomic analyses of the 17,000-year-old Tagliente 2 mandible revealed the oldest presence of a genetic profile with affinities to the Near East in the Italian peninsula, which later became the most widespread hunter-gatherer ancestry across Europe. However, a comparable biomolecular characterization of the Tagliente 1 burial remains unavailable, preventing us from defining its biological relationships with Tagliente 2. Here, we apply paleogenomic, isotopic, and radiocarbon dating analyses on a femur fragment of Tagliente 1 and compare the reconstructed data with previously reported results from Tagliente 2. Despite their different isotopic signatures and non-overlapping radiocarbon dates, we reveal that the two human remains belong to the same male individual. We determine that the distinct isotopic values can be explained by different dietary practices during lifetime, whereas the non-overlapping radiocarbon dates can be caused by minimal radiocarbon contamination, possibly deriving from chemical treatments for conservation purposes. These findings highlight the importance of interdisciplinary biomolecular studies in offering new perspectives on the Palaeolithic fossil record and addressing long-standing bioarchaeological questions., (© 2024. The Author(s).)

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70-80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone. The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)-epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The volume of human carbon (δ13C) and nitrogen (δ15N) isotope data produced in archaeological research has increased markedly in recent years. However, knowledge of bone remodelling, its impact on isotope variation, and the temporal resolution of isotope data remains poorly understood. Varied remodelling rates mean different elements (e.g., femur and rib) produce different temporal signals but little research has examined intra-element variability. This study investigates human bone remodelling using osteon population density and the relationship with carbon and nitrogen isotope data at a high resolution, focusing on variation through femoral cross-sections, from periosteal to endosteal surfaces. Results demonstrate considerable differences in isotope values between cross-sectional segments of a single fragment, by up to 1.3‰ for carbon and 1.8‰ for nitrogen, illustrating the need for standardised sampling strategies. Remodelling also varies between bone sections, occurring predominantly within the endosteal portion, followed by the midcortical and periosteal. Therefore, the endosteal portion likely reflects a shorter period of life closer to the time of death, consistent with expectations. By contrast, the periosteal surface provides a longer average, though there were exceptions to this. Results revealed a weak negative correlation between osteon population density and δ15N or δ13C, confirming that remodelling has an effect on isotope values but is not the principal driver. However, a consistent elevation of δ15N and δ13C (0.5‰ average) was found between the endosteal and periosteal regions, which requires further investigation. These findings suggest that, with further research, there is potential for single bone fragments to reconstruct in-life dietary change and mobility, thus reducing destructive sampling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Gruchy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mitochondrial DNA (mtDNA) is of great value in forensics to procure information about a person when a next of kin, personal belongings, or other sources of nuclear DNA (nDNA) are unavailable, or nDNA is lacking in quality and quantity. The quality and reliability of the results depend greatly on ensuring optimal conditions for the given method, for instance, the optimal input of the copy number (CN) in next-generation sequencing (NGS) methods. The unavailability of commercial quantitative PCR (qPCR) methods to determine mtDNA CN creates the necessity to rely on recommendations to infer mtDNA CN from nDNA yield. Because nDNA yield varies between individuals, tissues, parts of the same tissue, and because mtDNA CN varies between tissues, such assumptions must be examined for a specific context, rather than be generalized. This study compares mtDNA CN calculated from nDNA yield and qPCR measured mtDNA CN. Seventy-five femurs from the Second World War victims were used as samples; they were cut below the greater trochanter, surface contaminants were removed by mechanical and chemical cleaning, samples were fully demineralized, and DNA was isolated. PowerQuant® Kit (Promega) was used to analyze DNA yield. An in-house method was used to determine mtDNA CN. Comparison of mtDNA CN from nDNA derived calculations and measured mtDNA CN highlighted vast differences. The results emphasize the need to perform qPCR to assess mtDNA CN before NGS analyses of aged bones' mitogenomes rather than estimating mtDNA CN from nDNA yield to ensure the quality and reliability of the results of NGS analysis., (© 2023. The Author(s).)

The purpose of this study is to investigate the effect of orthodontic anchor screws (OASs) inserted into the femur of growth-phase or mature rats using histological observation and bone structure analysis. The experimental animals are growth-phase (6-week-old) or mature (25-week-old) male Wistar rats. OAS was placed into the point one-third of the femoral length from the proximal end of the femur, and the response of the surrounding bone was observed and measured. The results showed at the OAS bone interface, in growth-phase rats, bone mineral density (BMD) was reduced and the running angle of collagen fiber bundles varied significantly. In mature rats, more osteoid was observed and biological apatite (BAp) crystals showed a different orientation. It was suggested that after the insertion of OASs, bone volume and quality are decreased, but after a sufficient healing period, a new bone micro/nano structure, different from the original structure, are reconstructed.

Bone samples are valuable for examining the cause of death and circumstance leading up to death when body fluids are not available for forensic toxicological analysis. Examined were heat-induced changes in methamphetamine and amphetamine concentrations in femurs removed from methamphetamine-injected mice to determine if the burned bones could be used for toxicology testing. The femurs were heated at 100°C, 300°C, or 500°C for 10 or 30 min. The tissue structure of the heated femurs was preserved at 100°C for 30 min but was destructed at higher temperatures. Methamphetamine and amphetamine were detected in femurs heated at 100°C for 10 min, 100°C for 30 min, and 300°C for 10 min (with methamphetamine and amphetamine concentrations ranging from 0.36 to 35 μg/g and 0.54 to 47 μg/g, respectively). Methamphetamine and amphetamine were detectable when heated above their decomposition temperature as a result of limited heat transfer do to protection provide by the femoral muscle. Thus, the bone could be a useful analytical sample in cases of burn-related deaths, where it is difficult to collect body fluids., (© 2023 American Academy of Forensic Sciences.)

The radiocarbon ( 14 C) analysis of skeletonised human remains can provide vital information regarding the time since death. This study analysed the 14 C content in both cortical and trabecular bone from the femur and rib of 18 human remains donated to the Australian Facility for Taphonomic Experimental Research. To determine the most appropriate bone to sample for 14 C analysis when estimating the time since death, differences in turnover time between the bone types, and the difference between environmental 14 C during the year of death (YOD) and the level of 14 C found within the bone (i.e., the lag time) were examined. Overall, the average lag time for all donors was 25 years, with the cortical bone from femur samples providing the largest lag time (39 years) and the trabecular bone from rib samples containing the lowest level of 14 C (most modern), with an average lag time of 13 years. The results of this study suggest that in cases of unidentified human remains, the analysis of trabecular bone from a rib may be preferred sample, as this bone contains the most recent 14 C, and can provide an estimate of both the time since death, and a broad approximation of YOD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In forensic anthropology, there is an inherent difficulty in estimating the post-mortem interval (PMI). This study aimed to assess whether there is a correlation between changes in the bone mineral component and the PMI estimation. Samples of femur and humerus from 80 identified individuals with known post-mortem interval were analyzed. Infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) was applied for this analysis, several indices having been obtained from the infrared bands most representative of the bone's relative contents in carbonate and phosphate. Specific relationships between sex/age and PMI were attained: for larger PMIs, there was an increase in the amount of B-type carbonate (BPI), A-type carbonates (API), and in the carbonate/phosphate (C/P) ratio, and a decrease of the crystallinity index (CI) and in the carbonate ratio (C/C). Two particular infrared indices (CI and C/C) were identified as the most suitable for post-mortem interval estimation, especially in females, controlling the effects of sex and age (in the statistical analysis)., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Freezing bone samples to preserve their biomolecular properties for various analyses at a later time is a common practice. Storage temperature and freeze-thaw cycles are well-known factors affecting degradation of molecules in the bone, whereas less is known about the form in which the tissue is most stable. In general, as little intervention as possible is advised before storage. In the case of DNA analyses, homogenization of the bone shortly before DNA extraction is recommended. Because recent research on the DNA yield from frozen bone fragments and frozen bone powder indicates better DNA preservation in the latter, the aim of the study presented here was to investigate and compare the chemical composition of both types of samples (fragments versus powder) using ATR-FTIR spectroscopy. Pairs of bone fragments and bone powder originating from the same femur of 57 individuals from a Second World War mass grave, stored in a freezer at - 20 °C for 10 years, were analyzed. Prior to analysis, the stored fragments were ground into powder, whereas the stored powder was analyzed without any further preparation. Spectroscopic analysis was performed using ATR-FTIR spectroscopy. The spectra obtained were processed and analyzed to determine and compare the chemical composition of both types of samples. The results show that frozen powdered samples have significantly better-preserved organic matter and lower concentrations of B-type carbonates, but higher concentrations of A-type carbonates and stoichiometric apatite. In addition, there are more differences in the samples with a low DNA degradation index and less in the samples with a high DNA degradation index. Because the results are inconsistent with the current understanding of bone preservation, additional research into optimal preparation and long-term storage of bone samples is necessary., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In cases involving identification of missing persons, mass disasters and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA profiling. Standard methods for extraction of DNA from such samples involve grinding of the bone and teeth samples. Here, we present an extremely efficient protocol for recovery of DNA from bone samples by a method of scrapping. The study was carried out on 25 samples and it was found that the quantity of DNA isolated by the scrapping method was up to 1.131 ng/µl with a success rate of 93% as compared to a much lower yield of 0.359 ng/µl DNA isolated with a success rate of 28% through the grinding method. The scrapping method of DNA extraction has been proven to be extremely useful in forensic examination of challenging samples that had multiple failures using the traditional grinding method., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest whatsoever and all the authors have read and approved the manuscript., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hydroxyapatite (HAp) powder was produced from chicken (femur and beak) and fishbone wastes and used as a green adsorbent to decrease Cd 2+ from aqueous media. The HAp powder was generated at 900 °C and characterized using physicochemical techniques. Chicken femur' HAp (16.72 m 2 /g) had a higher surface compared to chicken beak and fishbone ones. The solution pH was the most important parameter in removing Cd 2+ . The highest Cd 2+ removal was achieved at pH 6, temperature of 25 °C, contact time of 80 min, and adsorbent mass of 2 g/L. The Cd 2+ adsorption data fitted well with the quasi-second-order model in kinetics and the Freundlich model in isotherm. The highest adsorption capacity of Cd 2+ using HAp-chicken femur, HAp-fish bone, and HAp-chicken beak was determined 22.94 mg/g, 21.54 mg/g, and 21.45 mg/g, respectively. The Cd 2+ adsorption using HAp powder was a spontaneous and exothermic process and accidental collisions at the liquid-solid interface were reduced. The decrease of Cd 2+ adsorption efficiency was not significant after multiple recovery steps of the desired powders. In addition to Cd 2+ , other parameters of real wastewater (shipbuilding industry) were reduced by the proposed adsorbents. The utilization of hydroxyapatite powder is expected to be a cheap and eco-friendly method for eliminating metals such as Cd 2+ ., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The 0.2% strain offset approach is mostly used to calculate the yield stress and serves as an efficient method for cross-lab comparisons of measured material properties. However, it is difficult to accurately determine the yield of the bone. Especially when computational models require accurate material parameters, clarification of the yield point is needed. We tested 24 cortical specimens harvested from six bovine femora in three-point bending mode, and 11 bovine femoral cortical specimens in the tensile mode. The Young's modulus and yield stress for each specimen derived from the specimen-specific finite element (FE) optimization method was regarded as the most ideal constitutive parameter. Then, the strain offset optimization method was used to find the strain offset closest to the ideal yield stress for the 24 specimens. The results showed that the 0 strain offsets underestimated (- 25%) the yield stress in bending and tensile tests, while the 0.2% strain offsets overestimated the yield stress (+ 65%) in three-point bending tests. Instead, the yield stress determined by 0.007 and 0.05% strain offset for bending and tensile loading respectively, can effectively characterize the biomechanical responses of the bone, thereby helping to build an accurate FE model., (© 2021. Biomedical Engineering Society.)

Biogenic hydroxyapatite (BHAp) is a widely used material in the biomedical area due to its similarities with the bone tissue mineral phase. Several works have been spotlighted on the thermal behavior of bone. However, little research has focused on determining the influence of calcination temperature in the physicochemical and bioactive properties of BHAp. In this work, a study of the physicochemical properties' changes and bioactive response of BHAp produced from porcine femur bones using calcination temperatures between 900 to 1200 °C was conducted. The samples' structural, morphological, and compositional changes were determined using XRD, SEM, and FTIR techniques. XRD results identified three temperature ranges, in which there are structural changes in BHAp samples and the presence of additional phases. Moreover, FTIR results corroborated that B-type substitution is promoted by increasing the heat treatment temperature. Likewise, samples were immersed in a simulated biological fluid (SBF), following the methodology described by Kokubo and using ISO 23317:2014 standard, for 3 and 7 days. FTIR and SEM results determined that the highest reaction velocity was reached for samples above 1000 °C, due to intensity increasing of phosphate and carbonate bands and bone-like apatite morphologies, compared to other temperatures evaluated.

DNA testing of human bones is performed for identification when there is no remaining soft tissue, which often means the samples are old or environmentally compromised. Under these circumstances, it can be difficult to obtain a STR DNA profile. It is important to recover the highest quantity and quality of DNA for STR typing. This study compared the DNA recovery and STR profiles from five anatomical locations in five femora and five tibiae. These locations include the proximal metaphysis, proximal diaphysis, mid-diaphysis, distal diaphysis, and distal metaphysis. Twenty-five femur samples and 25 tibia samples were analyzed using the Qiagen Investigator Quantiplex Pro RGQ Kit for quantitating the extracted DNA and the Qiagen Investigator 24plex QS Kit for STR DNA typing. The highest DNA recovery of the five regions tested in both the femur and the tibia was from the midshaft diaphysis. The femur samples resulted in a significantly higher DNA recovery than the tibia samples as analyzed using a Kruskal-Wallis test (P = 0.002103). The midshaft diaphysis and distal diaphysis yielded the most complete STR DNA profiles in the femora, while the distal and proximal diaphysis yielded the most complete STR DNA profiles in the tibiae. There was no correlation between the amount of DNA recovered and the completeness of the STR DNA profile produced with low template extracts in this study., (© 2020 American Academy of Forensic Sciences.)

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D 2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D 2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Sphingosine kinase (SPHK) is the rate-limiting enzyme in S1P production and has 2 isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-). SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a 14% lower spinal bone mineral density (BMD; P < 0.01) and males a 22% lower BMD at the same site (P < 0.001). SPHK2-/- and control mice were subsequently treated either with daily parathyroid hormone [PTH](1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2-/-mice. The mean femoral bone volume to total volume fraction (BV/TV) increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (P < 0.01). In contrast, in the SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs13.3%, PTH vs vehicle, P = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2-/- mice (29.7% vs 23.1%, PTH vs vehicle, in controls, P < 0.05; 26.9% vs 19.5% PTH vs vehicle in SPHK2-/- mice, P = NS). The spine responded normally in the SPHK2-/- female mice. Interestingly, suppression of sclerostin was blunted in the SPHK2-/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The reduction in estrogen levels results in a decrease in bone density at menopause. Irisin is a myokine that modulates the benefits of exercise, which may include bone health. This study was planned to examine irisin's impact in preventing osteoporosis after ovariectomy. 4 groups of female albino rats (10 rats/group): control, sham-operated, ovariectomized (OVX-control), and OVX-irisin-treated. Serum levels of bone markers [osteocalcin (OC), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), calcium (Ca ++ ), phosphorus (P)], glucose, and insulin were being measured. Body mass index, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), dry and ash femur weight, and bone contents of Ca ++ and P were investigated. The femur was examined histopathologically. The OVX-control group showed an increase in serum levels of OC, BALP, TRAP, calcium, phosphorus, BMI, glucose, insulin, and HOMA-IR ( P < 0.05) and a reduction in dry and ash weight of the femur, the concentration of calcium and phosphorus content in bone ash ( P < 0.05). The OVX-irisin-treated group exhibited a decrease in serum levels of OC, BALP and TRAP, calcium, phosphorus, BMI, glucose, insulin, HOMA-IR ( P < 0.05), and a rise in dry and ash weight of the femur, the concentration of calcium and phosphorus in bone ash ( P < 0.05). Histological examination of the distal femur diaphysis of the OVX-irisin-treated group exhibited proper bone architecture and density compared with that of the OVX-control group. It is concluded that irisin treatment in the OVX rats safeguarded the regular bone architecture and normal levels of serum bone biomarkers. Irisin may be a possible novel target in the prohibition of postmenopausal osteoporosis., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Enas N. Morgan et al.)

High-intensity interval training (HIIT) is of scientific interest due its role in improving physical fitness, but the effects of HIIT on bone health need be carefully explored. Further, it is necessary to know whether HIIT effects on bone health are dependent on the physical activity levels. This may be experimentally tested since we have built a large cage (LC) that allows animals to move freely, promoting an increase of spontaneous physical activity (SPA) in comparison to a small cage (SC). Thus, we examined the effects of HIIT on biophysical, biomechanical and biochemical parameters of bone tissue of C57BL/6J mice living in cages of two different sizes: small (SC) or large (LC) cages with 1320 cm 2 and 4800 cm 2 floor space, respectively. Male mice were subdivided into two groups within each housing type: Control (C) and Trained (T). At the end of the interventions, all mice were euthanized to extract the femur bone for biophysical, biomechanical and biochemical analyses. Based a significant interaction from two-way ANOVA, trained mice kept in large cage (but not for trained mice housed in SC) exhibited a reduction of tenacity and displacement at failure in bone. This suggests that long-term HIIT program, in addition with a more active lifestyle correlates with exerts negative effects on the bone of healthy mice. A caution must also be raised about the excessive adoption of physical training, at least regarding bone tissue. On the other hand, increased calcium was found in femur of mice housed in LC. In line with this, LC-C mice were more active (i.e. SPA) than other groups. This implies that an active lifestyle without long-term high intensity physical training seems to play a role in promoting benefits to bone tissue. Our data provides new insights for treatment of osteo-health related disorders.

The YPRKDETGAERT peptide (PME-1) identified from the Mytilus edulis proteins has been shown to promote the proliferation and differentiation of osteoblasts and it has good bone-forming activity in vitro. Further, PME-1 has been shown to prevent osteoporosis in vivo. PME-1 can be absorbed through the gastrointestinal tract, and the passing rate in monolayer Caco-2 cells was 6.57%. PME-1 can also enter the blood circulation and the concentration of PME-1 in serum reached the maximum, 61.06 ± 26.32 ng mL-1, 20 min after feeding. The multifunctional in vivo imager was used to further determine the distribution of the 5-FITC-(Acp)-YPRKDETGAERT peptide (PME-1-FITC) 2 h after feeding the peptide, and the result confirmed the above results and showed that a part of PME-1-FITC can affect bone in vivo. Therefore, PME-1 not only was easily absorbed in the gastrointestinal tract, but also has the potential beneficial effect on preventing osteoporosis.

Recovering DNA from modern incinerated bones can be challenging and may require alteration of routine DNA extraction protocols. It has been postulated that incinerated bones share some similarities with ancient bones, including fragmented DNA, surface contamination and highly mineralised structure, all of which can inhibit the successful recovery of genetic material. For this reason, ancient DNA extraction protocols are often used for incinerated modern samples; however, their effectiveness is still somewhat unclear. Much of this uncertainty exists around the demineralisation step of extraction, specifically the length of incubation and retention or removal of supernatant. As obtaining human samples for forensic research can be challenging, porcine models (Sus scrofa domesticus) are often used as substitutes. This study developed real time PCR assays for porcine nuclear DNA in order to investigate the effects of modified demineralisation protocols on DNA yield from femurs exposed to either short (60 min) or prolonged (120 min) burning. Gradient PCR results indicated 56 °C was the ideal amplification temperature for targeted amplicons, with melt curve analysis showing short and long amplicons corresponded to 80.3 °C and 83 °C peaks respectively. Results of altered extraction protocol showed a trend towards higher DNA yields from longer demineralisation periods however this was not significant. By comparison, retaining supernatant post-demineralisation resulted in significantly greater DNA yields compared to discarding it (P < 0.009). Although DNA content yield decreased with burn duration, the demineralisation treatment variations appeared to have the same effect for all burn lengths. These results suggest that for incinerated modern bone retaining the supernatant following demineralisation can dramatically increase DNA yield., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bone mineral density (BMD) and lean body mass (LBM) not only have a considerable heritability each, but also are genetically correlated. However, common genetic determinants shared by both traits are largely unknown. In the present study, we performed a bivariate genome-wide association study (GWAS) meta-analysis of hip BMD and trunk lean mass (TLM) in 11,335 subjects from 6 samples, and performed replication in estimated heel BMD and TLM in 215,234 UK Biobank (UKB) participants. We identified 2 loci that nearly attained the genome-wide significance (GWS, p < 5.0 × 10 -8 ) level in the discovery GWAS meta-analysis and that were successfully replicated in the UKB sample: 11p15.2 (lead SNP rs12800228, discovery p = 2.88 × 10 -7 , replication p = 1.95 × 10 -4 ) and 18q21.32 (rs489693, discovery p = 1.67 × 10 -7 , replication p = 1.17 × 10 -3 ). The above 2 pleiotropic loci may play a pleiotropic role for hip BMD and TLM development. So our findings provide useful insights that further enhance our understanding of genetic interplay between BMD and LBM.

Solid-state nuclear magnetic resonance is a promising technique to probe bone mineralization and interaction of collagen protein in the native state. However, many of the developments are hampered due to the low sensitivity of the technique. In this article, we report solid-state nuclear magnetic resonance (NMR) experiments using the newly developed BioSolids CryoProbe™ to access its applicability for elucidating the atomic-level structural details of collagen protein in native state inside the bone. We report here approximately a fourfold sensitivity enhancement in the natural abundance 13 C spectrum compared with the room temperature conventional solid-state NMR probe. With the advantage of sensitivity enhancement, we have been able to perform natural abundance 15 N cross-polarization magic angle spinning (CPMAS) and two-dimensional (2D) 1 H- 13 C heteronuclear correlation (HETCOR) experiments of native collagen within a reasonable timeframe. Due to high sensitivity, 2D 1 H/ 13 C HETCOR experiments have helped in detecting several short and long-range interactions of native collagen assembly, thus significantly expanding the scope of the method to such challenging biomaterials., (© 2020 John Wiley & Sons, Ltd.)

As a local delivery carrier of bone metabolic proteins, we have previously reported hydroxyapatite/chondroitin sulfate composite microparticles (HAp/ChS) and their formulation method using zinc cations (Zn), and the in vitro release properties of proteins from the microparticles. Herein, we report the release properties of model antibodies such as immunoglobulin (IgG), human IgG (hIgG), and denosumab (Dmab) from HAp/ChS using this formulation method. Adding Zn in the formulation of IgG loaded with HAp/ChS microparticles enhanced the release of antibodies from HAp/ChS in phosphate buffer saline. In addition, the biological activity of Dmab released from HAp/ChS formulated with Zn was significantly higher than that without Zn. These results suggest a possible beneficial effect on the treatment for local bone diseases. The sclerostin monoclonal antibody (Sclmab) promotes fracture healing. We prepared HAp/ChS microparticles loaded with Sclmab and locally administered the microparticles into a drilled hole in the distal femoral bone of young rats. After three weeks, the area of the newly formed osteoid around the drilled hole where HAp/ChS loaded with Sclmab and Zn was locally administered was significantly higher than that observed in the control group (normal saline). Thus, HAp/ChS microparticles and the formulation method of monoclonal antibodies using Zn could be useful in the treatment of local bone diseases.

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 (NCNH 2 ) 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.

Microenvironments play a significant part in understanding the post-mortem interval in forensic taphonomy. Recently, the value of weathering factors in relation to obtaining a PMI has been investigated further. In this study, observations were made to calculate the length of time it takes for three different bone elements (femur, rib, and scapula) to bleach in a UK summer and winter. This research also investigated whether there were any physicochemical modifications to the bone caused by bleaching. Porcine femora, scapulae, and ribs were placed into open and shaded areas of an outdoor research facility located in Oxfordshire, UK, during summer (July-Sep) and winter months (Dec-Mar). The specimens were monitored at 3-week intervals using photography, and an observational scoring method was developed to quantify the extent of bleaching. As temperatures are typically much lower in the UK compared with warmer climates, a controlled indoor-simulated desert experiment was also undertaken to be used as a control. This allowed sun bleaching and changes to the bone chemistry to be monitored in a controlled, high-UV environment for comparison with the UK outdoor experiments. Fourier transform infrared spectroscopy (FTIR) was employed to analyze physicochemical modifications to both the mineral and organic components of the bone. The FTIR was used to calculate crystallinity index (CI), mineral to organic ratio, and the relative amount of carbonate concentrations. Weather data was collected and a positive correlation was found between both ultraviolet (UV) levels and accumulated degree days (ADD) when compared with observational bleaching scores. Bleaching (whitening) of the bone samples occurred in both seasons but at different rates, with the bleaching process occurring at a slower rate in winter. During summer, the initial bleaching process was evident at 6 weeks, and by 9 weeks, the bones were an off-white colour. During the winter period, whitening of the bone started at 9 weeks; however, only the scapula and rib samples displayed a similar off-white colour. This colouration was observed at 13 weeks rather than at 9 weeks. The desert simulation samples started bleaching in a similar pattern to the outdoor samples after 1 week but the bones did not fully bleach. The bone chemistry, based on physicochemical properties obtained from the FTIR, showed a significant statistical difference between the simulated desert and winter season when compared against a control sample. For the winter samples, the mineral to organic ratio was significantly higher than that in the control, suggesting a reduction in the proportion of organic. For the samples in the simulated desert environment, the crystallinity index was significantly higher than that in the control samples, suggesting an increase in crystallinity. The results of this experiment support the fact that it is possible to achieve bleaching in a UK environment and that the minimal time frame for this to occur differs in seasons.

Brillouin-Raman microspectroscopy is presented as an innovative label-free all-optical investigation approachable to characterize the chemical composition and the mechanical properties of human tissues at micrometric resolution. Brillouin maps unveil mechanical heterogeneities in a human femoral diaphysis, showing a ubiquitous co-existence of hard and soft components, even in the most compact sections. The novel correlative analysis of Brillouin and Raman maps shows that the relative intensity of Brillouin peaks is a good proxy for the fraction of mineralized fibers and that the stiffness (longitudinal elastic modulus) of the hard component is linearly dependent on the hydroxyapatite concentration. For the soft component, a gradient of composition is found, ranging from an abundance of proteins in the more compact, external, bone to abundance of lipids, carotenoids, and heme groups approaching the trabecular, inner, part of the diaphysis. This work unveils the strong potential of correlative mechano-chemical characterization of human tissues at a micrometric resolution for both fundamental and translational research.

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.

The imprints left by persistent DNA viruses in the tissues can testify to the changes driving virus evolution as well as provide clues on the provenance of modern and ancient humans. However, the history hidden in skeletal remains is practically unknown, as only parvovirus B19 and hepatitis B virus DNA have been detected in hard tissues so far. Here, we investigated the DNA prevalences of 38 viruses in femoral bone of recently deceased individuals. To this end, we used quantitative PCRs and a custom viral targeted enrichment followed by next-generation sequencing. The data was analyzed with a tailor-made bioinformatics pipeline. Our findings revealed bone to be a much richer source of persistent DNA viruses than earlier perceived, discovering ten additional ones, including several members of the herpes- and polyomavirus families, as well as human papillomavirus 31 and torque teno virus. Remarkably, many of the viruses found have oncogenic potential and/or may reactivate in the elderly and immunosuppressed individuals. Thus, their persistence warrants careful evaluation of their clinical significance and impact on bone biology. Our findings open new frontiers for the study of virus evolution from ancient relics as well as provide new tools for the investigation of human skeletal remains in forensic and archaeological contexts., (Copyright © 2020. Published by Elsevier B.V.)

Owing to selection for rapid growth and heavy muscle, bone deformities and injuries in broiler chickens are common and present a welfare concern. Recently, clinical quantitative computed tomography scans (QCTs) have been used for laying hens with significant, strong correlations between QCT-generated bone mineral content (BMC), bone ash, and analytical calcium. The objectives of this study were to determine if QCT-generated bone mineral density of tibias and femurs correlated to fat-free ash and if analytical Ca could be correlated to bone ash and digitally calculated density in immature broilers. Male broilers (Ross 708, n = 125) were raised from day 1 after hatching, and at 42 D, right leg quarters were collected from 50 randomly selected birds and frozen at -20°C until analysis. Leg quarters were scanned with muscle and feathers intact and arranged in rows on plexiglass, and each QCT included a solid Ca hydroxyapatite phantom. Tibias and femurs were removed from leg quarters after autoclaving, ashed, and weighed. Pearson's correlation analysis was conducted to understand the association between analytical bone ash and QCT BMC while paired t tests determined the amount of difference between QCT BMC and ash. Ash weight was strongly correlated to QCT BMC in both the femur (R = 0.86, P < 0.001) and the tibia (R = 0.91, P < 0.001). The average difference between the amount of actual ash weighed and BMC calculated from the QCT was 0.03 ± 0.22 g (P = 0.3) for the femur and 0.04 ± 0.22 g (P = 0.2) for the tibia. This study confirms that this technique can supply invaluable skeletal health information without sacrificing birds., (Copyright © 2020. Published by Elsevier Inc.)

Background: In this paper, mulberry leaf powder (MLP) and konjac glucomannan (KGM) flour were used as raw materials, and animal experiments were designed to evaluate the effects of a mixture of MLP and KGM on bone density. The femoral bone microstructure of mice and pathological changes were observed by using micro-computed tomography) and haematoxylin and eosin (HE) staining methods, respectively. A three-point bending test was used to determine the biomechanical properties of the femur., Results: Results indicated that the calcium content of MLP was high, reaching 16 148.5 mg kg -1 , and the total proportion of water-soluble calcium, calcium pectinate, and calcium carbonate accounted for about 60% of the total calcium content. Serum alkaline phosphatase (AKP) activity was significantly lower, and serum calcium content was significantly higher (P < 0.05), in the MLP + KGM group (KM) than in the low-calcium control group, whereas no significant difference (P > 0.05) was found for serum phosphorus content. KM had a longer femur length, a higher bone mineral density (BMD) (P > 0.05), and significantly greater femur diameter, dry weight, index and bone calcium content (P < 0.05). However, these parameters were not significantly different from those of the calcium carbonate control group (P > 0.05)., Conclusion: The results indicate that the MLP/KGM mixture can reduce the high rate of bone turnover and the corresponding loss of bone mass caused by calcium deficiency and is thus effective in enhancing bone density. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

We evaluated the influence of pine bark extract (PBE) on organs, the cytochrome-P450 (CYP) activities in liver and estrogenic effects in normal and ovariectomized (OVX) female mice. The PBE did not affect organ weights and liver-function indexes (activities of alkaline phosphatase, aspartate amino transferase, and alanine amino transferase) at doses; 0.04%, 0.4%, and 2.0% PBE in the diet, in normal and OVX female mice. In the OVX mice, CYP1A1 activity was significantly higher in the 0.4% and 2.0% PBE groups than in the OVX control group, and in the 0.4% and 2.0% PBE groups were significantly higher than in the 0.04% PBE group. CYP1A2 and 3A4 activities were significantly higher in the 2.0% PBE group than in all other groups. The PBE did not affect uterine weight and femoral bone mineral density at all PBE doses. These results showed that the dose of PBE at the recommended human intake, had no toxic and estrogenic effects in normal female and OVX mice, however, it may need attention to use the excess intake of PBE with some drugs in postmenopausal women., (© 2020 Institute of Food Technologists®.)

Age prediction can help identify skeletal remains by limiting the search range for a missing person. Although age prediction methods based on odontology and anthropology are frequently used in the forensic field, DNA methylation is particularly promising age-predictive biomarker. In this study, we generated genome-wide DNA methylation profiles of bone samples from 32 identified skeletal remains with an age at death ranging from 31 to 96 years. Only 12 provided more than 800 K quality-filtered CpG methylation values using Illumina's Infinium MethylationEPIC BeadChip array. Methylation ages of the bone samples calculated using a recently developed skin & blood clock composed of 391 CpG sites were found to be very similar to their actual ages (MAD = 6.4 years). However, the low success rate in methylation profiling of bone DNA samples may prevent researchers from applying the array to this type of samples. Therefore, we selected a set of CpG sites that would enable age prediction based on only a few CpG sites in bone DNA samples. Nineteen age-associated CpG marker candidates were selected from 720 K quality-filtered CpG values of 21 male skeletal remain samples. Because age signatures for blood, such as markers on the ELOVL2, FHL2, KLF14 and TRIM59 genes, had showed strong age associations in 12 bone samples, we further tested the age association of the 5 well-known markers in a blood-based model and the 13 out of 19 CpG markers from the array of 21 bone samples with an independent set of 30 skeletal remain samples using SNaPshot multiplex based on single nucleotide primer extension. Four CpG sites on TMEM51, TRIM59, ELOVL2, and EPHA6 genes showed moderate or weak correlations between methylation and age, which suggests further investigation of these markers to predict the age of bones., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Osteoporosis, a disease associated with ovarian hormone deficiency following menopause, is the most common cause of age-related bone loss. Although an optimal intake of Ca is vital - both bone accretion during growth and maintenance in adult life - a great percentage of the population consumes far below the recommended amounts of this mineral. On the other hand, there are evidences that fructans enhance not only Ca absorption, but bone calcium as well., Objective: In the knowledge that estrogen deficiency and insufficient Ca in the diet during postmenopause cause serious problems with resultant osteoporosis, the aim of this study is to assess the effects of a diet enriched in fructan-containing sources alone, or in the "strawberry matrix", on the structure of bone in OVX rats under calcium hypoalimentation., Material and Methods: Experimental animals were female Wistar rats, sham-operated or ovariectomized. The treatment with Ca-restricted diets also contained one of the sources of fructan (Jerusalem artichoke, yacon, Beneo Orafti Synergy1), in the amount providing 8% of fructans. Femur architecture of rats was assessed by tomography and Ca content by the AAS method., Results: Ovariectomy led to a significant decrease in femoral Ca content, total mineral content and bone density of rats. This study shows that a diet containing inulin-type fructan (especially as a component of strawberry product) improved bone quality (i.e. increase in Ca content in femur, total density in middle part of bone, as well as decrease of endosteal circumference) in OVX rats under calcium hypoalimentation., Conclusions: These findings suggest that a fructan-enriched diet could be potentially useful for postmenopausal osteoporosis. It is important to determine an optimal dietary level of fructan with the long-term goal of developing a dietary strategy in osteoporosis prevention.

Bones are often found in mass grave crime scene. To increase DNA identification success rates, a highly efficient DNA extraction method should be selected. Several DNA extraction methods for human bones have been published yet never been systematically compared, and some are time-consuming or complex. As such, a quick and highly efficient DNA extraction method was developed and compared with three published methods (Hi-Flow silica-based, total demineralization (TD) and PrepFiler BTA) using 70 fresh and 22 casework bones from different body parts. The highest median DNA concentrations were obtained from developed method (135.85 ng/μL and 0.224 ng/μL for fresh and casework bones, respectively). For residual PCR inhibitors, the threshold cycle (Ct) of the internal positive control (IPC) showed that developed method and PrepFiler BTA removed most PCR inhibitors. Similarly, 95.45% of casework STR profiles obtained using the developed protocol meet the standard requirements for Australian National Criminal Investigative DNA Database (NCIDD) entry, followed by 86.35% using TD, 81.82% using PrepFiler BTA, and 45.45% using Hi-Flow. Additionally, DNA extracts from seven different bones revealed that the 1 st distal phalange of the hand contained the highest DNA concentration of 338.43 ng/μL, which was three times higher than the tibia and femur. Our findings suggest that developed method was highly efficient for casework bone analysis. It significantly reduced the extraction processing time down to 4 h and is two to four times cheaper compared with other methods. In practice, both the extraction method and the bone sampling must be considered by a forensic DNA analyst to increase the chances of successful identification.

Rationale: Whole-bone proteomic analyses rely on lengthy sample preparation including demineralization and digestion to break bone down into peptides to recover using mass spectrometry. However, microwave-assisted acid hydrolysis, a technique used in proteomic analyses of other soft tissues and cells, will combine both demineralization and digestion and only take minutes., Methods: To test microwave-assisted hydrolysis on whole moose bone, we microwaved five concentrations of acetic and formic acids (15%, 12.5%, 10%, 7.5% and 5%) for three times (10, 20 and 30 min) at 140°C using an ETHOS UP high performance microwave digestion system. Peptides were injected and separated using Thermo BioBasic C18 columns and detected with an LTQ Orbitrap Velos mass spectrometer. We searched the raw data on PEAKS 8.5 against the white-tailed deer database., Results: Formic acid hydrolysis led to the most complete digestion, and therefore the highest number of peptide spectrum matches, more protein groups and better sequence coverage for collagenous proteins. However, for the formic acid samples there is a tradeoff with digestion completeness and a higher incidence of in vitro modifications (i.e. formylation) that are not induced using acetic acid. Acetic acid has greater cleavage specificity and higher sequence coverage for non-collagenous proteins., Conclusions: Depending on the goals of analysis, there are benefits and drawbacks to using both acetic acid and formic acid. Overall, microwave-assisted acid hydrolysis was successful in demineralizing and digesting bone fragments to considerably speed up the preparation for bottom-up proteomics analysis., (© 2019 John Wiley & Sons, Ltd.)

We investigated changes in oxidative biomarkers in brain regions such as brainstem, cerebellum, and cerebral cortex of 3-, 6-, 18-, 24-, and 30-month-old rats. We also assessed the effects of low-intensity exercise on these biomarkers in these regions of 6-, 18-, and 24-month-old rats that started exercise on a treadmill at 3, 15, and 21 months of age, respectively. Radiographic images of the femur were taken for all rats. A total of 25 rats (age: twelve 6-, ten 18-, ten 24-, and three 30-month-old rats) were used. Lipid hydroperoxide levels increased in cerebellum at 18 months. Total antioxidant activity exhibited lowest values in brainstem at 3 months. Superoxide dismutase activity did not exhibit significant changes during aging. Total thiol content exhibited lowest values in brain regions of 24- and 30-month-old rats. Exercise reduced total thiol content in brainstem at 6 months, but no change occurred in other regions and other ages. Femur increased its length and width and cortical thickness with advancing age. No change occurred in medullary width. Radiolucency increased and sclerosis was found in cortical and medullary bone with advancing age. Exercise reduced radiolucency and medullary sclerosis. Therefore, aging differentially changed oxidative biomarkers in different brain regions and radiographic measures of the femur. Low-intensity exercise only ameliorated some radiographic measurements of femur. Since the present study possessed limitations (small number of rats per group), a beneficial effect of regular low-intensity exercise on oxidative markers in brain cannot be ruled out.

Calcium contents of demineralised human cortical bone determined by titrimetric assay and atomic absorption spectrophotometry technique were verified by comparing to neutron activation analysis which has high recovery of more than 90%. Conversion factors determined from the comparison is necessary to correct the calcium content for each technique. Femurs from cadaveric donors were cut into cortical rings and demineralised in 0.5 M hydrochloric acid for varying immersion times. Initial calcium content in the cortical bone measured by titration was 4.57%, only 21% of the measurement by neutron activation analysis; while measured by atomic absorption spectrophotometer was 13.4%, only 61% of neutron activation analysis. By comparing more readings with the measurements by neutron activation analysis with 93% recovery, a conversion factor of 4.83 was verified and applied for the readings by titration and 1.45 for atomic absorption spectrophotometer in calculating the correct calcium contents. The residual calcium content started to reduce after the cortical bone was demineralised in hydrochloric acid for 8 h and reduced to 13% after 24 h. Using the linear relationship, the residual calcium content could be reduced to less than 8% after immersion in hydrochloric acid for 40 h. Atomic absorption spectrophotometry technique is the method of choice for calcium content determination as it is more reliable compared to titrimetric assay.

Laying hens suffer from osteoporosis during their laying period, which causes bone fragility and susceptibility to fracture. This study evaluated the changes of mechanical properties of their bones during the laying period (from 18 to 77 wk) by using nano-indentation, atomic force microscope, X-Ray diffraction, and Raman spectroscopy. Results indicated that the crystallite sizes of bioapatite in femur decreased significantly from 34.45 to 29.26 nm during aging from 18 to 49 wk. Then, the value increased to 37.79 nm at 77 wk. Despite the abundance in bone (usually >50 wt.%), bioapatite mineral content showed no continuous enhancement during aging. The fibrils demonstrated more regular and organized structure during the laying period. Meanwhile the elastic moduli (E) and hardness (H) of femur increased from 10.84 to 18.39 GPa and 43.79 to 97.21 Vickers respectively during this period. The changes in mechanical properties are hence tightly related to the structure of bone (composed of both collagen and mineral), rather than directly related to the mineralogical properties of bone bioapatite. This study addressed the importance of the interaction between collagen and bioapatite mineral during the laying period of hens by microscopic, physicochemical, and mechanical analysis., (© 2019 Poultry Science Association Inc.)

Hydroxyapatite (HAp) is a bioactive and vital material which has found many applications in the biomedical and clinical fields. This bio-ceramic powder can be synthesized via different bio-waste materials. In this study, the production of natural nanohydroxyapatite was produced through calcination of untreated turkey femur-bone waste powder at 850 °C followed by ball milling the powder. The obtained powder was characterized using X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis. The morphology, size, and elemental composition of obtained turkey hydroxyapatite (THA) particles were investigated by scanning electron microcopy (SEM), transmission electron microcopy (TEM), and energy dispersive spectroscopy (EDS) analysis, in which the average particle size of ball milled THA was found to be about 85 nm with a Ca/P ratio of 1.63. The powder was then cold pressed and later sintered at 850, 950, 1050, and 1150 °C to evaluate its mechanical properties in terms of compressive strength and hardness. The results revealed that the strength and hardness of the samples increased by increasing the sintering temperature up to 1150 °C. Finally, the maximum values of hardness and compressive strength of the sintered THA were obtained at 1150 °C (37.44 MPa and 3.2 GPa, respectively).

To study the effects of pre-lay dietary Ca and strain on Ca utilization and femur quality at 1st through to 50th egg, 30 Lohmann Brown (LB) and 30 Lohmann Selected Leghorn-Lite (LSL) pullets (14 weeks of age, woa) reared under same management regimen were used. Six pullets/strain were necropsied for baseline femur samples and the rest (24 pullets/strain) placed in individual cages (65 × 30 × 45 cm3) and fed 1% Ca developer diet for 2 wk. At 16 woa, all pullets were weighed and allocated within strains to pre-lay diets (2.5 vs. 4.0% Ca) effectively creating a 2 × 2 factorial arrangement. The pullets were offered pre-lay diets for 2 wk and switched to 4% Ca diet at 18 woa. The diets contained TiO2 to determine apparent retention (AR) of Ca. The age, BW, and feed intake (FI) at 1st, 25th, and 50th egg were recorded. Excreta samples were taken during pre-lay, at 1st and 25th egg and 4 hens per treatment were necropsied for femur samples at 1st, 25th, and 50th egg. There was no interaction (P > 0.05) between pre-lay Ca and strain on Ca intake, femur mineral density (FMD), mineral content (FMC), breaking strength (FBS), and ash (FA) content at 1st, 25th, and 50th egg. At 1st egg, pre-lay Ca interacted with strain on AR of Ca (P = 0.014) such that LB hens retained more Ca at 2.5 vs. 4.0% Ca or LSL at 2.5% Ca. Pre-lay Ca had no effect (P > 0.05) on FMD, FMC, FBS, and FA at 1st, 25th, and 50th egg lay. Compared with LB hens, LSL hens had higher FMD (0.30 vs. 0.19 g/cm2; P = 0.010) and FA (51.9 vs. 42.5%; P < 0.01) at 1st egg and FBS (259.4 vs. 173.8 N, P < 0.01) at 25th egg. In conclusion, except at 25th egg, pre-lay Ca and strain had independent effect on Ca utilization. Femur attributes to 1st egg suggested innate need for LSL birds to accumulate critical bone mass prior to first oviposition., (© 2019 Poultry Science Association Inc.)

Quick setting and poor injectability due to liquid-solid phase separation have limited the clinical use of brushite and monetite cements. The presence of certain ions in the cement during the setting reaction moderate the setting time and properties of the cement. This study reports the preparation of injectable bone cement by using biphasic calcium phosphate (BCP) extracted from femur lamb bone by calcination at 1450 °C. EDX analysis infers the presence of Mg and Na ions as trace elements in BCP. X-ray diffraction patterns of the prepared cement confirmed the formation of brushite (DCPD) along with monetite (DCPA) as a minor phase. DCPA phase diminished gradually with a decrease in powder to liquid ratio (PLR). Initial and final setting time of 5.3 ± 0.5 and 14.67 ± 0.5 min respectively are obtained and within the acceptable recommended range for orthopedic applications. Exceptional injectability of ≈90% is achieved for all prepared bone cement samples. A decrease in compressive strength was observed with increase in the liquid phase of the cement, which is attributed to the higher degree of porosity in the set cement. Immersion of bone cement in simulated body fluid (SBF) for up to 7 days resulted in the formation of apatite layer on the surface of cement with Ca/P ratio 1.71, which enhanced the compressive strength from 2.88 to 9.15 MPa. The results demonstrate that bone cement produced from BCP extracted from femur lamb bone can be considered as potential bone substitute for regeneration and repair of bone defects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background & Aims: Altering the lipid component in diets may affect the incidence of metabolic bone disease in patients dependent on parenteral nutrition. Consumption of polyunsaturated fatty acids (PUFA) can impact bone health by modulating calcium metabolism, prostaglandin synthesis, lipid oxidation, osteoblast formation, and osteoclastogenesis. The aim of this study was to evaluate the dietary effects of PUFA on murine bone health., Methods: Three-weeks-old male (n = 30) and female (n = 30) C57BL/6J mice were randomized into one of three dietary groups. The diets differed only in fat composition: soybean oil (SOY), rich in ω-6 PUFA; docosahexaenoic acid alone (DHA), an ω-3 PUFA; and DHA with arachidonic acid, an ω-6 PUFA, at a 20:1 ratio (DHA/ARA). After 9 weeks of dietary treatment, femurs were harvested for micro-computed tomographic analysis and mechanical testing via 3-point bending. Separate mice from each group were used solely for serial blood draws for measurement of biomarkers of bone formation and resorption., Results: At the microstructural level, although some parameters in cortical bone reached differences that were statistically significant in female mice, these were too small to be considered biologically relevant. Similarly, trabecular bone parameters in male mice were statistically different in some dietary groups, although the biological interpretation of such subtle changes translate into a lack of effect in favor of any of the experimental diets. No differences were noted at the mechanical level and in blood-based biomarkers of bone metabolism across dietary groups within gender., Conclusions: Subtle differences were noted at the bones' microstructural level, however these are likely the result of random effects that do not translate into changes that are biologically relevant. Similarly, differences were not seen at the mechanical level, nor were they reflected in blood-based biomarkers of bone metabolism. Altogether, dietary consumption of PUFA do not seem to affect bone structure or metabolism in a healthy model of growing mice., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Exposure to polychlorinated biphenyls (PCBs), which are persistent lipophilic environmental pollutants, has a variety of adverse effects on wildlife and human health, including bone mineralization, growth and mechanical strength. The present study evaluated the effects of lactational exposure to nondioxin-like PCB-155 and dioxin-like PCB-169, individually and in combination, on pubertal rat femur development and its biomechanics. After offspring delivery, Wistar rat mothers were divided into four groups, i.e., PCB-169, PCB-155, PCB-155+169 and control, and were administered PCBs intraperitoneally. Data on bone geometry, biomechanics and mineral composition were obtained by analysis of femurs from 42-day-old offspring by microCT scanning, three-point bending test and inductively coupled plasma mass spectrometry. Decreased somatic mass and femur size, i.e., mass, periosteal circumference and cross sectional area, were observed in the PCB-169 and PCB-155 groups. Additionally, lactational exposure to planar PCB-169 resulted in harder and more brittle bones containing higher amounts of minerals. Combined exposure to structurally and functionally different PCBs demonstrated only mild alterations in bone width and mineralization. To conclude, our results demonstrated that alterations, observed on postnatal day 42, were primarily induced by PCB-169, while toxicity from both of the individual congeners may have been reduced in the combined group., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: The stress fractures (SFs) are a common condition in athletes and military recruits, characterized by partial fracture caused by repetitive applications of stresses that are lower than the stress required to fracture the bone in a single loading. Fourier transform infrared (FTIR) spectroscopy gives information about the bone composition and also can determine the amount of a molecule. For this reason, the FTIR spectroscopy may be used as a tool for diagnosis of certain bone diseases related to the bone strength. In this research, we established the contributions of mineral and collagen properties to SF risk through FTIR spectroscopy, analyzing the biochemical profile differences between the healthy bone and the bone with an SF., Materials and Methods: Previous written informed consent was obtained, and samples of the hip with an SF ( n  = 11) and healthy bone from the femur with traumatic fracture ( n  = 5) were obtained and analyzed employing FTIR spectroscopy and its biochemical mapping function. Then, using FTIR spectra and the map, the collagen content and ratios corresponding to matrix maturity, mineralization, carbonate substitution, acid phosphate substitution, and crystallinity were calculated. Moreover, a histopathological analysis through Masson's staining was conducted., Results: The biochemical analysis showed that the bone with an SF presented a bone immaturity characterized by a higher content of collagen, lower matrix maturity, mineralization, carbonate and acid phosphate substitutions, and greater crystallinity compared to the healthy bone, being checked by the ratio analysis and biochemical mapping. Besides, Masson's stain showed a higher collagen content in the bone with an SF., Conclusions: The bone with an SF presented alterations in its biochemical composition, showing bone immaturity, which broadens the panorama of the condition to investigate future treatments or prophylactic techniques., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Monica Maribel Mata-Miranda et al.)

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-µm-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

Background: Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible., Methods: In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 μl saline, 100 μl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 μl hydrogel loaded with 1 μg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination., Results: Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples., Conclusions: In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation.

Objectives: The research explores whether the combined study of cortical bone histology, bone morphology, and dietary stable isotopes can expand insights into past human health and adaptations, particularly dietary sufficiency and life span., Materials and Methods: Midthoracic rib cortices from 54 South African Late Holocene adult skeletons (28 M, 24 F, two sex undetermined) are assessed by transmitted-light microscopy for cross-sectional area measurements, osteon area (On.Ar), osteon population density, and presence/absence of secondary osteon variants. Values for δ 13 C bone collagen , δ 15 N bone collagen , 14 C dates, Southwestern and Southern Cape geographic regions, body size measures, estimated ages-at-death from both morphological and histological methods are integrated into analyses, which include Spearman correlations, χ 2 tests and Kruskal-Wallis ANOVAs., Results: There is reduced On.Ar variability with higher δ 15 N (r = -.41, p = .005); rib %cortical area and δ 15 N are negatively correlated in the Southern Cape group (r = -.60, p = .03). Osteon variants are more common in older adults; histological ages at death are significantly older than those determined from gross morphology., Discussion: We found bone tissue relationships with measures of diet composition, but indicators of dietary adequacy remain elusive. Relationships of tissue quality and isotopes suggest that some Southern Cape adults lived long lives. Osteon variants are associated with age-at-death; some association with diet remains possible. Gross morphological methods appear to underestimate adult ages-at-death, at least among small-bodied adults., (© 2019 Wiley Periodicals, Inc.)