Ashing of bone: errors due to loss of CO2 and their correction.

INTRODUCTION: Ashing is widely used to determine weight fraction of water-free bone that is mineral, but no standard procedure exists and the range of techniques used spans a range of temperatures and times over which the amount of weight loss is variable. We show that variability is largely due to progressive loss of CO2 from CO3 2- ions in the apatite crystal lattice, beginning at 600 ℃, typically used for ashing. We test the effect of varying temperature, time, and weight of sample and develop a reliable method, using small samples. MATERIALS AND METHODS: Replicate samples of bovine cortical bone were tested at 500 ℃, 600 ℃, and 700 ℃ for times ranging up to 24 h. We also tested samples of multiple humans at what we concluded to be the optimal conditions. RESULTS: Varying conditions of ashing resulted in variations in apparent ash weight % by up to 7%. Samples between 5 and 20 mg heated to 600 ℃ for 1 h gave results agreeing with generally accepted values, but with much smaller variability. Ash wt% values for multiple human bone samples differed by up to 4.8%, but replicate data for individuals agree to ± 1 wt%. DISCUSSION: In conclusion, a satisfactory method is given for ash weight determination using small samples, and yielding highly reproducible data. If accepted widely, ash weight values between laboratories could be used to study variations due to diet, age, drug treatment, and disease.

X-ray diffraction and in situ pressurization of dentine apatite reveals nanocrystal modulus stiffening upon carbonate removal.

Bone-like materials comprise carbonated-hydroxyapatite nanocrystals (c-Ap) embedding a fibrillar collagen matrix. The mineral particles stiffen the nanocomposite by tight attachment to the protein fibrils creating a high strength and toughness material. The nanometer dimensions of c-Ap crystals make it very challenging to measure their mechanical properties. Mineral in bony tissues such as dentine contains 2~6 wt.% carbonate with possibly different elastic properties as compared with crystalline hydroxyapatite. Here we determine strain in biogenic apatite nanocrystals by directly measuring atomic deformation in pig dentine before and after removing carbonate. Transmission electron microscopy revealed the platy 3D morphology while atom probe tomography revealed carbon inside the calcium rich domains. High-energy X-ray diffraction in combination with in situ hydrostatic pressurization quantified reversible c-Ap deformations. Crystal strains differed between annealed and ashed (decarbonated) samples, following 1 or 10 h heating at 250 °C or 550 °C respectively. Measured bulk moduli (K) and a-/c-lattice deformation ratios (η) were used to generate synthetic Ksyn and ηsyn identifying the most likely elastic constants C33 and C13 for c-Ap. These were then used to calculate the nanoparticle elastic moduli. For ashed samples, we find an average E11=107 GPa and E33 =128 GPa corresponding to ~5% and ~17% stiffening of the a-/c-axes of the nanocrystals as compared with the biogenic nanocrystals in annealed samples. Ashed samples exhibit ~10% lower Poisson's ratios as compared with the 0.25~0.36 range of carbonated apatite. Carbonate in c-Ap may therefore serve for tuning local deformability within bony tissues. STATEMENT OF SIGNIFICANCE: Carbonated apatite nanoparticles, typical for bony tissues, stiffen the network of collagen fibrils. However, it is not known if the biogenic apatite mechanical (elastic) properties differ from those of geologic mineral counterparts. Indeed the tiny dimensions and variable carbonate composition may have strong effects on deformation resistance. The present study provides experimental measurements of the elastic constants which we use to estimate Young's moduli and Poisson's ratio values. Comparison between ashed and annealed dentine samples quantifies the properties of both carbonated and decarbonated apatite nanocrystals. The results reveal fundamental attributes of bony mineral and showcase the additive advantages of combining X-ray diffraction with in situ hydrostatic compression, backed by atom probe and transmission electron microscopy tomography.

Interfacial bonding between mineral platelets in bone and its effect on mechanical properties of bone.

Bone is a composite material consisting principally of apatite mineral, collagen fibrils, non-collagenous proteins, and other organic species. Recent electron microscopy studies have shown that the mineral in bone occurs as stacks of thin polycrystalline sheets ("mineral lamellae," MLs) which surround and lie between the collagen fibrils. We focus on the effect of the interface between these mineral lamellae on the mechanical properties of bone. Previous studies on bone treated with sodium hypochlorite (NaClO) to remove all organic material showed a greatly weakened mineral framework. Here, we treated femoral cortical bone with ethylenediamine (EDA), which only removes collagen, to study the effect of its removal on bone properties. We tested the degree of completion of the treatment by Raman spectroscopy and thermogravimetric analysis. When only collagen is removed, a continuous mineral structure remains and is less weakened than by NaClO treatment. Transmission electron microscopy study of finely ground particles of the EDA treated bone shows that stacks of MLs remain joined, whereas in NaClO treated bone, only isolated crystals are present. Thus, we infer that the MLs in bone are held together in stacks by an organic glue, which is destroyed by NaClO, but which survives the EDA treatment. We show that this glue may contribute to the stiffness, strength, and energy absorption of bone. Further studies are needed to discover the chemical nature of this glue. This study provides a starting point for such investigations.

Identification of collagen fibrils in cross sections of bone by electron energy loss spectroscopy (EELS).

Transmission electron microscopic (TEM) images of ion-milled bovid cortical bone cut approximately normal to the axes of fibrils show that mineral occurs in the form of plates surrounding and laying between circular or elliptical features about 50 nm in diameter. The classification of these features as either pores or collagen fibrils is highly debated. Electron energy loss spectroscopy (EELS) mapping of these features in ion milled sections shows that they are lacking significant amounts of mineral or collagen, although their appearance suggests that they are cross sections of collagen fibrils. However, analogous sections prepared using an ultramicrotome show that, while these circular features show reduced concentrations of calcium and phosphorus, some of them contain quantities of carbon and nitrogen in bonding states comparable to the composition of collagen. This work demonstrates that the observed circular features are sections of collagen fibrils, but that bombardment by argon ions during broad beam ion milling destroys the collagen and associated gap-zone mineral.

Ultrastructure of Bone: Hierarchical Features from Nanometer to Micrometer Scale Revealed in Focused Ion Beam Sections in the TEM.

The ultrastructure of bone has been widely debated, in part due to limitations in visualizing nanostructural features over relevant micrometer length scales. Here, we employ the high resolving power and compositional contrast of high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) to investigate new features in human bone with nanometer resolution over microscale areas. Using focused ion beam (FIB)-milled sections that span an area of 50 µm2, we have shown how most of the mineral of cortical human osteonal bone occurs in the form of long, thin polycrystalline plates (mineral lamellae, MLs) which are either flat or curved to wrap closely around collagen fibrils. Close to the collagen fibril (< 20 nm), the radius of curvature matches that of the fibril diameter, while at greater distances, MLs form arcs with much larger radii of curvature. In addition, stacks of closely packed planar (uncurved) MLs occur between fibrils. The curving of mineral lamellae both around and between the fibrils would contribute to the strength of bone. At a larger scale, rosette-like clusters of fibrils are noted for the first time, arranged in quasi-circular arrays that define tube-like structures in alternating osteonal lamellae. At the boundary between adjacent osteonal lamellae, the orientation of fibrils and surrounding mineral lamellae changes abruptly, resembling the "orthogonal" patterns identified by others (Reznikov et al. in Acta Biomater 10:3815-3826, 2014). These features spanning nanometer to micrometer scale have implications for our understanding of bone structure and mechanical integrity.

Mapping the origins of Imperial Roman workers (1st-4th century CE) at Vagnari, Southern Italy, using 87 Sr/86 Sr and δ18 O variability.

OBJECTIVES: We obtained the oxygen and strontium isotope composition of teeth from Roman period (1st to 4th century CE) inhabitants buried in the Vagnari cemetery (Southern Italy), and present the first strontium isotope variation map of the Italian peninsula using previously published data sets and new strontium data. We test the hypothesis that the Vagnari population was predominantly composed of local individuals, instead of migrants originating from abroad. MATERIALS AND METHODS: We analyzed the oxygen (18 O/16 O) and strontium (87 Sr/86 Sr) isotope composition of 43 teeth. We also report the 87 Sr/86 Sr composition of an additional 13 molars, 87 Sr/86 Sr values from fauna (n = 10), and soil (n = 5) samples local to the area around Vagnari. The 87 Sr/86 Sr variation map of Italy uses 87 Sr/86 Sr values obtained from previously published data sources from across Italy (n = 199). RESULTS: Converted tooth carbonate (δ18 ODW ) and 87 Sr/86 Sr data indicate that the majority of individuals buried at Vagnari were local to the region. ArcGIS bounded Inverse Distance Weighting (IDW) interpolation of the pan-Italian 87 Sr/86 Sr data set approximates the expected 87 Sr/86 Sr range of Italy's geological substratum, producing the first strontium map of the Italian peninsula. DISCUSSION: Results suggest that only 7% of individuals buried at Vagnari were born elsewhere and migrated to Vagnari, while the remaining individuals were either local to Vagnari (58%), or from the southern Italian peninsula (34%). Our results are consistent with the suggestion that Roman Imperial lower-class populations in southern Italy sustained their numbers through local reproduction measures, and not through large-scale immigration from outside the Italian peninsula.

The ultrastructure of bone as revealed in electron microscopy of ion-milled sections.

Mineral makes up more than half the volume of bone, but its spatial and structural relationship to collagen and other proteins is still a matter of debate. Due to the nanometer-size of bone crystals this matter can be resolved only with transmission electron microscope (TEM) images. Using sections cut with an ultramicrotome, previous investigators determined most mineral lies in the 40nm wide gap zone in collagen fibrils. Using less invasive sectioning methods (ion milling and focused ion beam [FIB]) reveals that most mineral is extrafibrillar, occurring in the form of mineral lamellae, polycrystalline plates 300nm or more long, packed around collagen fibrils in stacks of four or more lamellae <1nm apart. While Ca and P also occur in the gap zone, they do not appear to be in the form of well-crystallized apatite. This new model for bone ultrastructure resolves outstanding problems presented by the previous model.

Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals.

In a previous study we showed that most of the mineral in bone is present in the form of "mineral structures", 5-6nm-thick, elongated plates which surround and are oriented parallel to collagen fibrils. Using dark-field transmission electron microscopy, we viewed mineral structures in ion-milled sections of cortical human bone cut parallel to the collagen fibrils. Within the mineral structures we observe single crystals of apatite averaging 5.8±2.7nm in width and 28±19nm in length, their long axes oriented parallel to the fibril axis. Some appear to be composite, co-aligned crystals as thin as 2nm. From their similarity to TEM images of crystals liberated from deproteinated bone we infer that we are viewing sections through platy crystals of apatite that are assembled together to form the mineral structures.

Isotopic studies of the diet of the people of the coast of British Columbia.

In 1982, Chisholm et al. used δ(13) C data for human burials from shell midden sites widely distributed on the coast of British Columbia (BC) to show the extreme dependence of these individuals on high trophic level marine consumers, principally salmon and marine mammals. Here, we present previously unpublished analyses of δ(15) N for some of the same individuals as well as δ(13) C data for additional individuals. Nitrogen isotope data show that the diet was dominated by high trophic level marine fauna including carnivorous fish and marine mammals. Although most burials were found in shell middens, marine mollusks made up of only a minor component of diet. The data for δ(13) C demonstrate that terrestrial faunal foods are undetectable in the diet of the majority of individuals, and seldom constitute more than 10% of the dietary protein of individuals living on the coast although terrestrial fauna were widely available as a potential source of protein. This dietary pattern of exclusion of land-based animals from their diet persisted for almost 6,000 years along a wide expanse of coastline. In contrast, people from the BC interior (100 km or more from the coast) consumed a mixed diet of terrestrial and marine foods including spawning salmon.

Scanning transmission electron microscopic tomography of cortical bone using Z-contrast imaging.

Previously we presented (McNally et al., 2012) a model for the ultrastructure of bone showing that the mineral resides principally outside collagen fibrils in the form of 5 nm thick mineral structures hundreds of nanometers long oriented parallel to the fibrils. Here we use high-angle annular dark-field electron tomography in the scanning transmission electron microscope to confirm this model and further elucidate the composite structure. Views of a section cut parallel to the fibril axes show bundles of mineral structures extending parallel to the fibrils and encircling them. The mineral density inside the fibrils is too low to be visualized in these tomographic images. A section cut perpendicular to the fibril axes, shows quasi-circular walls composed of mineral structures, wrapping around apparently empty holes marking the sites of fibrils. These images confirm our original model that the majority of mineral in bone resides outside the collagen fibrils.

A model for the ultrastructure of bone based on electron microscopy of ion-milled sections.

The relationship between the mineral component of bone and associated collagen has been a matter of continued dispute. We use transmission electron microscopy (TEM) of cryogenically ion milled sections of fully-mineralized cortical bone to study the spatial and topological relationship between mineral and collagen. We observe that hydroxyapatite (HA) occurs largely as elongated plate-like structures which are external to and oriented parallel to the collagen fibrils. Dark field images suggest that the structures ("mineral structures") are polycrystalline. They are approximately 5 nm thick, 70 nm wide and several hundred nm long. Using energy-dispersive X-ray analysis we show that approximately 70% of the HA occurs as mineral structures external to the fibrils. The remainder is found constrained to the gap zones. Comparative studies of other species suggest that this structural motif is ubiquitous in all vertebrates.

Paleoclimate during Neandertal and anatomically modern human occupation at Amud and Qafzeh, Israel: the stable isotope data.

The δ(13)C(en) and δ(18)O(en) values of goat and gazelle enamel carbonate indicate that Neandertals at Amud Cave, Israel (53-70 ka) lived under different ecological conditions than did anatomically modern humans at Qafzeh Cave, Israel (approximately 92 ka). During the Last Glacial Period, Neandertals at Amud Cave lived under wetter conditions than those in the region today. Neither faunal species ate arid-adapted C(4) plants or drought-stressed C(3) plants. The variation in gazelle δ(18)O(en) values suggests multiple birth seasons, which today occur under wetter than normal conditions. The magnitude and pattern of intra-tooth variation in goat δ(18)O(en) values indicate that rain fell throughout the year unlike today. Anatomically modern humans encountered a Qafzeh Cave region that was more open and arid than Glacial Period Amud Cave, and more open than today's Upper Galilee region. Goat δ(13)C(en) values indicate feeding on varying amounts of C(4) plants throughout the year. The climate apparently ameliorated higher in the sequence; but habitats remained more open than at Amud Cave. Both gazelles and goats fed on C(3) plants in brushy habitats without any inclusion of C(4) plants. The magnitude of intra-tooth variation in goat δ(18)O(en) values, however, suggest that some rain fell throughout the year, and the relative representation of woodland dwelling species indicates the occurrence of woodlands in the region. Climate differences affecting the distribution of plants and animals appear to be the significant factor contributing to behavioral differences previously documented between Neandertals and anatomically modern humans in the region. Climate forcing probably affected the early appearances of anatomically modern humans, although not the disappearance of Neandertals from the Levant.

Possible paleohydrologic and paleoclimatic effects on hominin migration and occupation of the Levantine Middle Paleolithic.

This paper explores the impact of major glacial/interglacial paleohydrologic variations in the Middle-Paleolithic Levant on hominin migration and occupation. The climatic reconstruction is based primarily on the most straight-forward paleohydrologic records recently published. These terrestrial proxies convey direct paleoenvironmental signals of effective precipitation and aquifer recharge. The two main proxies are temporal changes of terminal lake levels in the Dead Sea basin and periods of deposition or non-deposition of speleothems. Other records, such as stable isotopes, if interpreted correctly, correspond well with these two direct proxies. All the records consistently indicate that the last two glacial periods in the central Levant were generally wet and cool, while the last two interglacials were dry and warm, so more water was available for the ecosystem and thus hominins during glacial periods than during interglacials. Some proxies indicate that the higher precipitation/evaporation ratio during glacial periods involved higher precipitation rather than only reduced evaporation. Beyond the general mean glacial/interglacial climate suggested here, variations occurred at all temporal scales throughout glacial or interglacial periods. In the Sahara-Negev arid barrier, moister conditions occurred during Marine Isotope Stage (MIS) 6a-5e, when Anatomically Modern Humans apparently migrated out of Africa. We suggest that this migration, as well as the later Neanderthal expansion from Southeast Europe or the Anatolian plateau into the Levant during early MIS 4, could be facilitated by the observed major climatic variations.

A new method for determination of postmortem interval: citrate content of bone.

Few accurate methods exist currently to determine the time since death (postmortem interval, PMI) of skeletonized human remains found at crime scenes. Citrate is present as a constituent of living human and animal cortical bone at very uniform initial concentration (2.0 ± 0.1 wt %). In skeletal remains found in open landscape settings (whether buried or not), the concentration of citrate remains constant for a period of about 4 weeks, after which it decreases linearly as a function of log(time). The upper limit of the dating range is about 100 years. The precision of determination decreases slightly with age. The rate of decrease appears to be independent of temperature or rainfall but drops to zero for storage temperature <0°C.

Isotopic and dental evidence for infant and young child feeding practices in an imperial Roman skeletal sample.

This study integrates isotopic, palaeopathological, and historical evidence to investigate infant and young child feeding practices in a Roman period (1st to 3rd centuries AD) skeletal sample from the Isola Sacra necropolis (Rome, Italy). Stable isotope analysis of bone collagen from 37 rib samples indicates that transitional feeding began by the end of the first year and weaning occurred by 2-2.5 years of age. Both delta(15)N and delta(13)C data clearly show the trophic level effect associated with breastfeeding. Childhood diet is investigated using dental pathology data in the deciduous dentitions of 78 individuals aged between 1 and 12 years. The presence of calculus, caries, and tooth wear in young children suggests that individuals were provided complementary foods and other items that impacted their dental health at an early age. The isotopic and dental data are generally consistent with the historical evidence from the Roman period with respect to the general timetable of weaning and the character of complementary foods. This is the first study to integrate isotopic and deciduous dental pathology data to explore infant and young child feeding practices in the Roman world.

Isotopic evidence for age-related immigration to imperial Rome.

Oxygen stable isotope ratios (delta(18)O) have been determined in carbonate in paired first and third molar teeth from individuals (N = 61) who lived in the town of Portus Romae ("Portus") and who were buried in the necropolis of Isola Sacra (First to Third centuries AD) near Rome, Italy. We compare these analyses with data for deciduous teeth of modern Roman children. Approximately one-third of the archaeological sample has first molar (M1) values outside the modern range, implying a large rate of population turnover at that time, consistent with historical data. Delta (18)O(ap) values suggest that a group within the sample migrated to the area before the third molar (M3) crown had completely formed (i.e., between 10 and 17.5 years of age). This is the first quantitative assessment of population mobility in Classical antiquity. This study demonstrates that migration was not limited to predominantly single adult males, as suggested by historical sources, but rather a complex phenomenon involving families. We hypothesize that migrants most likely came from higher elevations to the East and North of Rome. One individual with a higher delta(18)O value may have come (as a child) from an area isotopically similar to North Africa.

New dates for the Fontéchevade (Charente, France) Homo remains.

Homo I from the site of Fontéchevade, France, has long been an anomaly in the European fossil record. The specimen is a fragment of human frontal bone that lacks a supraorbital torus and appears to belong to an anatomically modern Homo sapiens. However, the level from which it was recovered in 1947 was dated on the basis of associated faunal and lithic material to the last interglacial or earlier. As a result, Homo I has been interpreted, among other things, as a representative of a pre-sapiens lineage in Europe. This paper reports on recent ESR and radiocarbon dates that indicate that the specimen almost certainly dates to oxygen isotope stage 3, which brings it in line with other evidence for the entry of modern Homo sapiens into Europe.

Isotopic evidence for age-related variation in diet from Isola Sacra, Italy.

This study examines collagen (N=105) and apatite (N=65) data from an Imperial Roman skeletal sample from the necropolis of Isola Sacra (Rome, Italy). This paper explores correlations between the isotopic composition of bone samples and the inferred age and sex of these individuals (aged 5--45+ years). The collagen of males, and older individuals in general, was significantly enriched in (15)N but not (13)C. Bone carbonate was somewhat depleted in (13)C in some older individuals, suggesting increased consumption of olive oil and possibly wine. Subadults (>5 years) in the sample appear to have consumed an almost exclusively terrestrial diet. This study demonstrates a clear trend in dietary patterns between adult age groups, as well as between adults and children within a population.

Chronometric dating in archaeology: a review.

Most archaeological dating methods are based on decay of a naturally occurring radioisotope. (14)C activity of fossil bones and charcoal decreases with age, but must be calibrated for past changes in atmospheric activity. Uranium absorbed by shells and stalagmites is used to date on a 10(5)-year scale by observing the decay of (234)U to (230) Th. Thermoluminescence, optical luminescence, and electron spin resonance detect trapped electronic charges generated by natural radioactivity in burned flint, beach sands, shells, and tooth enamel. Rate of racemization of amino acids in fossil shells is constant at constant T, and age can be tracked from an increase in the D/L ratio.