Decellularized ECM derived from normal bone involved in the viability and chemo-sensitivity in multiple myeloma cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy. The progression of MM is closely related to the bone microenvironment. Bone matrix proteins are remodeled and manipulated to govern cancer growth during the process of MM. However the role of normal bone extracellular matrix in MM is still unclear. In this study the decellularized extracellular matrix derived from normal SD rats' skulls (N-dECM) was prepared by decellularization technology. The CCK 8 assay and the dead-live cell kit assay were used to determine the viability of MM cells and the sensitivity to bortezomib. The Realtime PCR and Western blot assay were used to assay the mRNA and protein related to MM. Under the treatment of N-dECM, we found that the viability of MM cells was inhibited and the sensitivity of MM cells to bortezomib was increased. Additionally, the expression levels of APRIL and TACI, which participated in the progression of MM, were significantly decreased in MM cells. It suggested that N-dECM might inhibit the development of MM via APRIL-TACI axis, and our study may provide a novel and potential biomaterial for MM therapy.

Temporal, spatial and gender-based dietary differences in middle period San Pedro de Atacama, Chile: A model-based approach.

To explore the possible emergence and lived consequences of social inequality in the Atacama, we analyzed a large set (n = 288) of incredibly well preserved and contextualized human skeletons from the broad Middle Period (AD 500-1000) of the San Pedro de Atacama (Chile) oases. In this work, we explore model-based paleodietary reconstruction of the results of stable isotope analysis of human bone collagen and hydroxyapatite. The results of this modeling are used to explore local phenomena, the nature of the Middle Period, and the interaction between local situations and the larger world in which the oases were enmeshed by identifying the temporal, spatial, and biocultural correlates and dimensions of dietary difference. Our analyses revealed that: 1) over the 600-year period represented by our sample, there were significant changes in consumption patterns that may evince broad diachronic changes in the structure of Atacameño society, and 2) at/near 600 calAD, there was a possible episode of social discontinuity that manifested in significant changes in consumption practices. Additionally, while there were some differences in the level of internal dietary variability among the ayllus, once time was fully considered, none of the ayllus stood out for having a more (or less) clearly internally differentiated cuisine. Finally, sex does not appear to have been a particularly salient driver of observed dietary differences here. While we do not see any de facto evidence for complete dietary differentiation (as there is always overlap in consumption among individuals, ayllus, and time periods, and as isotopic analysis is not capable of pinpointing different foods items or preparations), there are broad aspects of dietary composition changing over time that are potentially linked to status, and foreignness. Ultimately, these stand as the clearest example of what has been termed "gastro-politics," potentially tied to the emergence of social inequality in the San Pedro oases.

The Analysis of Gap Junctional Intercellular Communication Among Osteocytes in Chick Calvariae by Fluorescence Recovery After Photobleaching.

This chapter describes the use of fluorescence recovery after photobleaching (FRAP) for analyzing gap junctional intercellular communication (GJIC) among osteocytes in chick calvariae by confocal laser scanning microscope.

Drugs in bone: Detectability of substances of toxicological interest in different states of preservation.

In forensic contexts of advanced decomposition, when conventional matrices are no longer available for toxicological analyses, finding alternative matrices is necessary. The skeleton, which is fundamental for anthropologists and geneticists, could be useful also for toxicological purposes. The present study aims to examine what kind of information toxicological analysis performed on bones (the cranium and the ribs) in different states of preservation could provide to the forensic practitioner. Thirty cadavers with known pharmacological history, subjected to forensic autopsy at the Institute of Legal Medicine of Milan, were selected. Rib and cranium samples were collected from each body and separated into two parts in order to create two different states of preservation: One was cleaned from soft tissues and analyzed as a well-preserved bone sample; the other was submitted to a long maceration process, simulating complete skeletonization. All specimens were then processed with accelerated solvent extraction and the eluates analyzed using Q-Exactive™ Orbitrap™ Mass Spectrometer. The analysis of blood and skeletal matrices showed positive results for the tested substances in 63% of cases, mainly benzodiazepines, antidepressants, and drug abuse. Significant Pearson correlations were observed between non-macerated vs. macerated bone samples: r = 0.79 for rib samples, r = 0.61 for cranium samples, and r = 0.69 for all bone samples. As a consequence, the positive results confirm the potential of the bone tissue as an alternative matrix in forensic toxicology, even in cases of extremely decomposed bodies. This study also highlighted important elements for reconstructing the biological profile in cases of forensic anthropological concern.

Variation in skull bone mineral density of ringed seals (Phoca hispida) from the Gulf of Bothnia and West Greenland between 1829 and 2019.

Bone is remodelled constantly through a balance of bone formation and resorption. This process can be affected by various factors such as hormones, vitamins, nutrients and environmental factors, which can create an imbalance resulting in systemic or local bone alteration. The aim of the present study was to analyse the changes in bone mineral density (BMD) over time in skulls of ringed seals (Pusa hispida) from the Baltic and Greenland using museum samples. Overall, 303 skulls (102 Male, 89 Female, 112 unknown) were used for bone investigations and were divided into three periods according to collection year: before 1958 (n = 167), between 1958 and 1989 (n = 40) and after 1994 up to 2019 (n = 96). All skulls were examined by dual-energy X-ray absorptiometry to obtain the BMD. Skull BMD of the Baltic seals was positively correlated with the historical polychlorinated biphenyls (PCB) contamination having potential effects on the constitution of bones. BMD fluctuated between the three study periods (LM: p-value < 0.001, F-value = 47.5) with the lowest BMD found between 1897 and 1957, in the Gulf of Bothnia, where the highest peak of contaminant concentration was in the second period. BMD levels increased with increasing PCB concentration (LM: p < 0.001). The Greenland population showed significant lower BMD levels in the pollution and post-pollution period than the Baltic population (LM: p < 0.001). It also revealed a higher BMD in males than in females (LM: p = 0.03). In conclusion, the variations between 1829 and 2019 in the Baltic Sea and Greenland may to a certain extent reflect normal fluctuations; however, this study revealed several factors affecting BMD, including sex and PCB levels.

First Discovery of the European Buffalo Bubalus murrensis (Artiodactyla, Bovidae) from the Pleistocene of the Russian Plain.

The skull of a buffalo from the Upper Pleistocene deposits in the Lukerino village near the city of Kolomna (Moscow Region) was found to belong to Bubalus murrensis (Berckhemer, 1927). In Western Europe, this species occurred in the Middle and Late Pleistocene during interglacials. In the Pleistocene of the East European Plain, the fossil Bubalus is discovered for the first time. According to the radiocarbon (14C) analysis of the collagen, the fossil dates to the Allerød interstadial (the age is ca. 12 800 years).

An investigation into structural behaviors of skulls chewing food in different occlusal relationships using FEM.

OBJECTIVES: This study aims to investigate the effect of different occlusal relationships on skull structural and mechanical behaviors through simulation of chewing food. METHODS: Finite element (FE) skull models of occlusion for Class I, end-on Class II, and full-cusp Class II were generated. End-on Class II and full-cusp Class II were chosen as mild and severe Class II occlusions, respectively. A simplified food bolus was introduced between the upper and lower dentition of the right molars. Chewing food was simulated in the skulls by moving the mandible. An experiment was conducted to measure strains at selective locations and compared them to the analytical results for validation. RESULTS: In the early stages of mandibular movement, masticatory forces predicted from the skull models without food were lower than the skull models with food but increased drastically after occluding teeth full enough. As a result, the relationship between masticatory force and mandible movement shows that there is no significant difference between the skull models with food and without food in the range of human masticatory force, approximately 250 N. In all the cases of skulls including a food bolus, stress was similarly propagated from the mandible to the maxilla and concentrated in the same regions, including the mandibular notch and alveolar bone around the lower molars. CONCLUSION: It is predicted that there is no significant difference of bite force-mandible movement relationships and stress distributions of skull and teeth, between end-on Class II and full-cusp Class II models. When simulating chewing activities on candy and carrot, it is also found that there is no difference of masticatory performance between Class II occlusions, from structural as well as mechanical perspectives.

Occurrence and vertical distribution of Ca, Cl, Cr, Fe, Mn, Mo, K, Rb, Sr, S, Sn, and Zn in the skull bones of alpine bullhead (Cottus poecilopus) in the West Carpathians.

The aim of this study was to examine the contents of calcium, chlorine, chromium, iron, manganese, molybdenum, potassium, rubidium, strontium, sulfur, tin, and zinc in the bones of the alpine bullhead (Cottus poecilopus) related to its vertical distribution in the mountain river Javorinka. This river is located on the northern side of the High Tatras, the West Carpathians. Sampling took place during 2017, 2018, and 2019. One hundred three individuals of bullheads were collected. Only deceased individuals were selected for sampling; there was no deliberate harm to any sampled individuals. Weight, body length, and head length and width were measured. The amounts of Ca, Rb, and Mo were significantly dependent on the altitude and the fish size. At higher altitudes, there are smaller fish that colonize more inaccessible waters with smaller habitat flow options and these smaller fish contain relatively more Ca, Rb, and Mo in their skulls than large fish. The presence of Mn, Sr, and Zn was related to the altitude. Concentrations of Zn and Sr increase with altitude in the alpine bullhead skulls. The amounts of S, Cl, K, Cr, Sn, and Mo did not differ among fish living at different elevations.

Place of origin of the sacrificial victims in the sacred Cenote, Chichén Itzá, Mexico.

OBJECTIVES: The ancient city of Chichén Itzá in the northern Yucatán of Mexico was one of the most important in the Maya area, but its origins and history are poorly understood. A major question concerns the origins of the peoples who founded and later expanded the ancient city. Hundreds of people were ritually executed and their bodies thrown into the waters of the Sacred Cenote at Chichén. MATERIALS AND METHODS: In this study, we use strontium and oxygen isotopes to study the place of origin of a large sample of these individuals. Isotopes are deposited in human tooth enamel. Enamel forms during the first years of life, remains largely unchanged long past death, and can provide a signature of the place of birth. If the isotope ratios in enamel are different from the place of death, the individual must have moved during his/her lifetime. RESULTS: Comparison of our results from the cenote with information on isotope ratios across the Maya region and elsewhere suggests that the individuals in the cenote came from a number of different parts of Mexico and possibly beyond. DISCUSSION: It is not known if all of the sacrificial victims resided in Chichén Itzá, but their suggested origins likely reflect patterns of population movement and social networks that existed between Chichén Itzá and both neighboring and distant regions. Various lines of evidence point to places in the Yucatán, along the Gulf Coast, Central America, or even in the Central Highlands of Mexico.

Getting Ahead: Extraction of DNA from Skeletonized Cranial Material and Teeth.

Between 1990 and 2018, the Defense POW/MIA Accounting Agency submitted 2177 cranial elements and 1565 teeth to the Armed Forces Medical Examiner System-Armed Forces DNA Identification Laboratory for DNA testing. In an effort to identify missing United States service members, materials were recovered from wartime losses inclusive of World War II, the Korean War, and Southeast Asia. Using four different DNA extraction protocols, DNA testing was performed using mitochondrial DNA Sanger sequencing, modified AmpFlSTR® Yfiler™, AmpFlSTR® MiniFiler™, PowerPlex® Fusion, or Next Generation Sequencing. This paper aims to provide optimal strategies for the DNA testing of skeletonized cranial materials. Cranial elements produced the most consistent results in Sanger sequencing using an organic purification; however, teeth were most successful for the same platform with an inorganic purification. The inverse is true for STR testing of cranial bones. Of the cranial elements, the temporal provided the most consistent results.

Cortex-wide neural interfacing via transparent polymer skulls.

Neural computations occurring simultaneously in multiple cerebral cortical regions are critical for mediating behaviors. Progress has been made in understanding how neural activity in specific cortical regions contributes to behavior. However, there is a lack of tools that allow simultaneous monitoring and perturbing neural activity from multiple cortical regions. We engineered 'See-Shells'-digitally designed, morphologically realistic, transparent polymer skulls that allow long-term (>300 days) optical access to 45 mm2 of the dorsal cerebral cortex in the mouse. We demonstrate the ability to perform mesoscopic imaging, as well as cellular and subcellular resolution two-photon imaging of neural structures up to 600 µm deep. See-Shells allow calcium imaging from multiple, non-contiguous regions across the cortex. Perforated See-Shells enable introducing penetrating neural probes to perturb or record neural activity simultaneously with whole cortex imaging. See-Shells are constructed using common desktop fabrication tools, providing a powerful tool for investigating brain structure and function.

Compound-specific radiocarbon dating and mitochondrial DNA analysis of the Pleistocene hominin from Salkhit Mongolia.

A skullcap found in the Salkhit Valley in northeast Mongolia is, to our knowledge, the only Pleistocene hominin fossil found in the country. It was initially described as an individual with possible archaic affinities, but its ancestry has been debated since the discovery. Here, we determine the age of the Salkhit skull by compound-specific radiocarbon dating of hydroxyproline to 34,950-33,900 Cal. BP (at 95% probability), placing the Salkhit individual in the Early Upper Paleolithic period. We reconstruct the complete mitochondrial genome (mtDNA) of the specimen. It falls within a group of modern human mtDNAs (haplogroup N) that is widespread in Eurasia today. The results now place the specimen into its proper chronometric and biological context and allow us to begin integrating it with other evidence for the human occupation of this region during the Paleolithic, as well as wider Pleistocene sequences across Eurasia.

The Protective Effect of α-Tocopherol on the Content of Selected Elements in the Calvaria for Exposed Hens to TCDD in the Early Embryonic Period.

This paper focuses on negative effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on element content in male chicken calvaria and α-tocopherol (vitamin E) ability to reduce its toxic potential on bone mineralization in offspring. In the experiment carried out once, a solution containing only DMSO, TCDD, TCDD + α-tocopherol, and exclusively α-tocopherol was administrated. Subsequently, on the 5th day after hatching, the mineral composition of the chicken calvaria was evaluated. The results obtained suggest that the use of α-tocopherol contributes to the maintenance of the concentration of calcium, magnesium, and manganese in the chicken calvaria treated with TCDD in the embryonic period. In turn, vitamin E increases the level of zinc. It has been found that α-tocopherol in chicken embryos has a protective effect against disturbance of level of chosen trace elements in the bones of offspring caused by the TCDD.

Combination of a Bioceramic Scaffold and Simvastatin Nanoparticles as a Synthetic Alternative to Autologous Bone Grafting.

The fragile nature of porous bioceramic substitutes cannot match the toughness of bone, which limits the use of these materials in clinical load-bearing applications. Statins can enhance bone healing, but it could show rhabdomyolysis/inflammatory response after overdosing. In this study, the drug-containing bone grafts were developed from poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles encapsulating simvastatin (SIM) (SIM-PP NPs) loaded within an appropriately mechanical bioceramic scaffold (BC). The combination bone graft provides dual functions of osteoconduction and osteoinduction. The mechanical properties of the bioceramic are enhanced mainly based on the admixture of a combustible reverse-negative thermoresponsive hydrogel (poly(N-isopropylacrylamide base). We showed that SIM-PP NPs can increase the activity of alkaline phosphatase and osteogenic differentiation of bone marrow stem cells. To verify the bone-healing efficacy of this drug-containing bone grafts, a nonunion radial endochondral ossification bone defect rabbit model (N = 3/group) and a nonunion calvarial intramembranous defect Sprague Dawley (SD) rat model (N = 5/group) were used. The results indicated that SIM-PP NPs combined with BC can improve the healing of nonunion bone defects of the radial bone and calvarial bone. Therefore, the BC containing SIM-PP NPs may be appropriate for clinical use as a synthetic alternative to autologous bone grafting that can overcome the problem of determining the clinical dosage of simvastatin drugs to promote bone healing.

Cell-based RNAi screening and high-content analysis in primary calvarian osteoblasts applied to identification of osteoblast differentiation regulators.

Osteoblasts are responsible for the maintenance of bone homeostasis. Deregulation of their differentiation is etiologically linked to several bone disorders, making this process an important target for therapeutic intervention. Systemic identification of osteoblast regulators has been hampered by the unavailability of physiologically relevant in vitro systems suitable for efficient RNAi and for differentiation read-outs compatible with fluorescent microscopy-based high-content analysis (HCA). Here, we report a new method for identification of osteoblast differentiation regulators by combining siRNA transfection in physiologically relevant cells with high-throughput screening (HTS). Primary mouse calvarial osteoblasts were seeded in 384-well format and reverse transfected with siRNAs and their cell number and differentiation was assayed by HCA. Automated image acquisition allowed high-throughput analyses and classification of single cell features. The physiological relevance, reproducibility, and sensitivity of the method were validated using known regulators of osteoblast differentiation. The application of HCA to siRNAs against expression of 320 genes led to the identification of five potential suppressors and 60 activators of early osteoblast differentiation. The described method and the associated analysis pipeline are not restricted to RNAi-based screening, but can be adapted to large-scale drug HTS or to small-scale targeted experiments, to identify new critical factors important for early osteoblastogenesis.

Microstructure and mechanical properties of different keratinous horns.

Animal horns play an important role during intraspecific combat. This work investigates the microstructure and mechanical properties of horns from four representative ruminant species: the bighorn sheep (Ovis canadensis), domestic sheep (Ovis aries), mountain goat (Oreamnos americanus) and pronghorn (Antilocapra americana), aiming to understand the relation between evolved microstructures and mechanical properties. Microstructural similarity is found where disc-shaped keratin cells attach edge-to-edge along the growth direction of the horn core (longitudinal direction) forming a lamella; multiple lamellae are layered face to face along the impact direction (radial direction, perpendicular to horn core growth direction), forming a wavy pattern surrounding a common feature, the tubules. Differences among species include the number and shape of the tubules, the orientation of aligned lamellae and the shape of keratin cells. Water absorption tests reveal that the pronghorn horn has the largest water-absorbing ability due to the presence of nanopores in the keratin cells. The loading direction (compressive and tensile) and level of hydration vary among the horns from different species. The differences in mechanical properties among species may relate to their different fighting behaviours: high stiffness and strength in mountain goat to support the forces during stabbing; high tensile strength in pronghorn for interlocked pulling; impact energy absorption properties in domestic and bighorn sheep to protect the skull during butting. These design rules based on evolutionary modifications among species can be applied in synthetic materials to meet different mechanical requirements.

A comparative analysis of the avian skull: Woodpeckers and chickens.

Woodpeckers peck at trees without any reported brain injury despite undergoing high impact loads. Amongst the adaptations allowing this is a highly functionalized impact-absorption system consisting of the head, beak, tongue and hyoid bone. This study aims to examine the anatomical structure, composition, and mechanical properties of the skull to determine its potential role in energy absorption and dissipation. An acorn woodpecker and a domestic chicken are compared through micro-computed tomography to analyze and compare two- and three-dimensional bone morphometry. Optical and scanning electron microscopy with energy dispersive X-ray spectroscopy are used to identify the structural and chemical components. Nanoindentation reveals mechanical properties along the transverse cross-section, normal to the direction of impact. Results show two different strategies: the skull bone of the woodpecker shows a relatively small but uniform level of closed porosity, a higher degree of mineralization, and a higher cortical to skull bone ratio. Conversely, the chicken skull bone shows a wide range of both open and closed porosity (volume fraction), a lower degree of mineralization, and a lower cortical to skull bone ratio. This structural difference affects the mechanical properties: the skull bones of woodpeckers are slightly stiffer than those of chickens. Furthermore, the Young's modulus of the woodpecker frontal bone is significantly higher than that of the parietal bone. These new findings may be useful to potential engineered design applications, as well as future work to understand how woodpeckers avoid brain injury.

Retrodeformation of fossil specimens based on 3D bilateral semi-landmarks: Implementation in the R package "Morpho".

Many fossil specimens exhibit deformations caused by taphonomic processes. Due to these deformations, even important specimens have to be excluded from morphometric analyses, impoverishing an already poor paleontological record. Techniques to retrodeform and virtually restore damaged (i.e. deformed) specimens are available, but these methods genenerally imply the use of a sparse set of bilateral landmarks, ignoring the fact that the distribution and amount of control points directly affects the result of the retrodeformation. We propose a method developed in the R environment and available in the R-package "Morpho" that, in addition to the landmark configurations, also allows using a set of semi-landmarks homogeneously distributed along curves and on surfaces. We evaluated the outcome of the retrodeformation, regarding the number of semi-landmarks used and its robustness against asymmetric noise, based on simulations using a virtually deformed gorilla cranium. Finally, we applied the method to a well-known Neanderthal cranium that exhibits signs of taphonomically induced asymmetry.