Focused ion beam-SEM 3D study of osteodentin in the teeth of the Atlantic wolfish Anarhichas lupus.

The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin. Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small 'pearls' which surround the denteonal canals, and characterize their structure.

Unique three-dimensional structure of a fish pharyngeal jaw subjected to unusually high mechanical loads.

We examine the structure of the bone of the pharyngeal jaws of a large fish, the black drum (Pogonias cromis), that uses its tooth-jaw complex to crush hard-shelled bivalve mollusks. During mastication huge compressive forces are concentrated in a tiny zone at the tooth-bone interface. We report on the structure of this bone, with emphasis on its contact with the teeth, at different hierarchical levels and in 3D. Micro-CT shows that the molariform teeth do not have roots and are supported by a circular narrow bony rim that surrounds the periphery of the tooth base. The lower pharyngeal jaw is highly porous, as seen by reflected light microscopy and secondary electron microscopy (SE-SEM). Porosity decreases close to the bone-tooth interface and back-scattered electron (BSE-SEM) microscopy shows a slight elevation in mineral density. Focused ion beam - scanning electron microscopy (FIB-SEM) in the serial surface view (SSV) mode reveals a most surprising organization at the nanoscale level: parallel arrays of mineralized collagen fibrils surrounding channels of ~100 nm diameter, both with their long axes oriented along the load direction. The channels are filled with organic matter. These fibril-channel arrays are surrounded by a highly disordered mineralized material. This unusual structure clearly functions efficiently under compression, but the precise way by which this unique arrangement achieves this function is unknown.

Relation between cross-sectional bone geometry and double zonal osteon frequency and morphology.

OBJECTIVES: While double-zonal osteons (DZ) are characterized by a hyper-mineralized ring inside their lamellae, recent findings suggest that this ring is also defined by a change in the collagen fibers' orientation. Collagen and minerals are essential components to the maintenance of adequate bone strength and their alteration can modify the mechanical properties of the bone tissue. Consequently, the aim of this study is to explore the effect of past loads, as estimated from cross-sectional geometric properties, on the formation of DZ osteons compared to type I (common) osteons. MATERIALS AND METHODS: The sample consists of paired humerus and femur midshaft sections (n = 23) of Eurocanadian settlers from the historical St. Matthew cemetery, Quebec City (1771-1860). Histomorphometric variables included in this study are osteon density for DZ and type I osteons (DZD; OPD), osteon area (DZOn.Ar; On. Ar), Haversian canal area (DZH.Ar; H.Ar), and the area within the hypermineralized ring (HR. Ar). Loading history is estimated from cross-sectional properties including the following variable: cortical and total area (CA, TA), maximum and minimum second moment of area (Imax , Imin ) and polar moment of area (J). RESULTS: When the humerus and femur of the same individuals are compared, the femur has a higher OPD, DZD, and relative DZD (DZD/OPD). DZ osteons have a smaller area and Haversian canal area compared to type I osteons. The area within the hypermineralized ring in DZ is higher than the Haversian canal area of the type I osteons. Correlations between the residual scores of the regression of histomorphometric variables and cross-sectional properties of the humerus on the femur were not significant. DISCUSSION: Based on the analysis of the entire cross-section, the lack of correlation between variations in cross-sectional properties and remodeling combined with the significant differences between humeri and femura suggests that the creation of DZ or type I osteons in the bone tissue might be due to a bone specific response, possibly related to differences in bone tissue age that needs to be further investigated. Definitive conclusion regarding biomechanical loads still seem to be premature as regional variations associated with mechanical properties remain to be explored.

Brief communication: Test of a method to identify double-zonal osteon in polarized light microscopy.

OBJECTIVES: Double-zonal osteons (DZ) have been of interest in paleopathological research because they might be linked to physiological pathology. DZ are thought to be evidence of arrested osteon formation with a brief but abrupt increase in mineralization of lamellae occurring during bone remodeling. Originally identified from microradiographs as hypermineralized rings, recent studies have identified DZ from linear polarized light microscopy (PLM). However, PLM does not guarantee the adequate detection of DZ since PLM captures bone birefringence and not hyper-mineralization. Scanning electron microscopy with backscatter electrons (BSE-SEM) allows observation of DZ by detecting differences in mineralization. The purpose of this study is to investigate whether DZ, as identified by BSE-SEM, can indeed be identified with PLM. MATERIALS AND METHODS: The sample consists of an archaeological collection of adult midshaft femurs (n = 30) from St. Matthew cemetery, Quebec City (1771-1860). DZ were identified and counted independently with PLM and BSE-SEM for the same sections. Results from both methods were compared. RESULTS: Chi-square test shows that there was no significant difference between the two methods (p = 0.404). No significant bias was found on Bland-Altman analysis and Cohen's kappa shows a substantial agreement between the two methods (Κ = 0.66). PLM shows a good accuracy (sensitivity 79%, specificity 99.4%) and reliability (Positive Predictive Value: 86.71%; Negative Predictive Value: 99.45%). DISCUSSION: These findings indicate that the two methods are interchangeable. PLM, using our proposed protocol, is reliable to accurately identify DZ. We discuss how PLM and BSE-SEM that measure different features of the bone tissue can converge on the identification of DZ.

Triazine-Based Graphitic Carbon Nitride Thin Film as a Homogeneous Interphase for Lithium Storage.

Triazine-based graphitic carbon nitride is a semiconductor material constituted of cross-linked triazine units, which differs from widely reported heptazine-based carbon nitrides. Its triazine-based structure gives rise to significantly different physical chemical properties from the latter. However, it is still a great challenge to experimentally synthesize this material. Here, we propose a synthesis strategy via vapor-metal interfacial condensation on a planar copper substrate to realize homogeneous growth of triazine-based graphitic carbon nitride films over large surfaces. The triazine-based motifs are clearly shown in transmission electron microscopy with high in-plane crystallinity. An AB-stacking arrangement of the layers is orientationlly parallel to the substrate surface. Eventually, the as-prepared films show dense electrochemical lithium deposition attributed to homogeneous charge transport within this thin film interphase, making it a promising solution for energy storage.

Initial Upper Palaeolithic material culture by 45,000 years ago at Shiyu in northern China.

The geographic expansion of Homo sapiens populations into southeastern Europe occurred by ∼47,000 years ago (∼47 ka), marked by Initial Upper Palaeolithic (IUP) technology. H. sapiens was present in western Siberia by ∼45 ka, and IUP industries indicate early entries by ∼50 ka in the Russian Altai and 46-45 ka in northern Mongolia. H. sapiens was in northeastern Asia by ∼40 ka, with a single IUP site in China dating to 43-41 ka. Here we describe an IUP assemblage from Shiyu in northern China, dating to ∼45 ka. Shiyu contains a stone tool assemblage produced by Levallois and Volumetric Blade Reduction methods, the long-distance transfer of obsidian from sources in China and the Russian Far East (800-1,000 km away), increased hunting skills denoted by the selective culling of adult equids and the recovery of tanged and hafted projectile points with evidence of impact fractures, and the presence of a worked bone tool and a shaped graphite disc. Shiyu exhibits a set of advanced cultural behaviours, and together with the recovery of a now-lost human cranial bone, the record supports an expansion of H. sapiens into eastern Asia by about 45 ka.

Logistics of Bone Mineralization in the Chick Embryo Studied by 3D Cryo FIB-SEM Imaging.

During skeletal development, bone growth and mineralization require transport of substantial amounts of calcium, while maintaining very low concentration. How an organism overcomes this major logistical challenge remains mostly unexplained. To shed some light on the dynamics of this process, cryogenic focused ion beam-scanning electron microscopy (cryo-FIB/SEM) is used to image forming bone tissue at day 13 of a chick embryo femur. Both cells and matrix in 3D are visualized and observed as calcium-rich intracellular vesicular structures. Counting the number of these vesicles per unit volume and measuring their calcium content based on the electron back-scattering signal, the intracellular velocity at which these vesicles need to travel to transport all the calcium required for the mineral deposited in one day within the collagenous tissue can be estimated. This velocity at 0.27 µm s-1 is estimated, which is too large for a diffusion process and rather suggests active transport through the cellular network. It is concluded that calcium logistics is hierarchical and based on several transport mechanisms: first through the vasculature using calcium-binding proteins and the blood flow, then active transport over tens of micrometers through the network of osteoblasts and osteocytes, and finally diffusive transport over the last one or two microns.

High resolution 3D structures of mineralized tissues in health and disease.

A thorough knowledge of the structures of healthy mineralized tissues, such as bone or cartilage, is key to understanding the pathological changes occurring during disease. Such knowledge enables the underlying mechanisms that are responsible for pathology to be pinpointed. One high-resolution 3D method in particular - focused ion beam-scanning electron microscopy (FIB-SEM) - has fundamentally changed our understanding of healthy vertebrate mineralized tissues. FIB-SEM can be used to study demineralized matrix, the hydrated components of tissue (including cells) using cryo-fixation and even untreated mineralized tissue. The latter requires minimal sample preparation, making it possible to study enough samples to carry out studies capable of detecting statistically significant differences - a pre-requisite for the study of pathological tissues. Here, we present an imaging and characterization strategy for tissue structures at different length scales, describe new insights obtained on healthy mineralized tissues using FIB-SEM, and suggest future research directions for both healthy and diseased mineralized tissues.

Focused ion beam-SEM 3D analysis of mineralized osteonal bone: lamellae and cement sheath structures.

The mineralized collagen fibril is the basic building block of bone, and hence is the key to understanding bone structure and function. Here we report imaging of mineralized pig bone samples in 3D using the focused ion beam-scanning electron microscope (FIB-SEM) under conditions that reveal the 67 nm D-banding of mineralized collagen fibrils. We show that in adult pig osteons, the lamellar bone comprises alternating layers with either collagen fibrils predominantly aligned in one direction, and layers in which fibrils are predominantly aligned in two directions. The cement sheath contains thin layers of both these motifs, but its dominant structural component comprises a very complex layer of fibrils predominantly aligned in three or more directions. The degree of mineralization of the cement sheath is comparable to that of the osteon interior. The extent of alignment (dispersion) of the collagen fibrils in the osteonal lamellar bone is significantly higher than in the cement sheath. Canaliculi within the cement sheath are mainly aligned parallel to the cement sheath boundary, whereas in the lamellar bone they are mainly aligned perpendicular to the lamellar boundaries. This study further characterizes the presence of two types of collagen fibril arrangements previously identified in demineralized lamellar bone from other species. The simple sample preparation procedure for mineralized bone and the lower risk of introducing artifacts opens the possibility of using FIB-SEM to study more samples, to obtain automatic quantitative information on collagen fibril organization and to evaluate the degrees of mineralization all in relatively large volumes of bone.

A Paleolithic bird figurine from the Lingjing site, Henan, China.

The recent identification of cave paintings dated to 42-40 ka BP in Borneo and Sulawesi highlights the antiquity of painted representations in this region. However, no instances of three-dimensional portable art, well attested in Europe since at least 40 ka BP, were documented thus far in East Asia prior to the Neolithic. Here, we report the discovery of an exceptionally well-preserved miniature carving of a standing bird from the site of Lingjing, Henan, China. Microscopic and microtomographic analyses of the figurine and the study of bone fragments from the same context reveal the object was made of bone blackened by heating and carefully carved with four techniques that left diagnostic traces on the entire surface of the object. Critical analysis of the site's research history and stratigraphy, the cultural remains associated with the figurine and those recovered from the other archeological layers, as well as twenty-eight radiometric ages obtained on associated archeological items, including one provided by a bone fragment worked with the same technique recorded on the object, suggest a Late Paleolithic origin for the carving, with a probable age estimated to 13,500 years old. The carving, which predates previously known comparable instances from this region by 8,500 years, demonstrates that three-dimensional avian representations were part of East Asian Late Pleistocene cultural repertoires and identifies technological and stylistic peculiarities distinguishing this newly discovered art tradition from previous and contemporary examples found in Western Europe and Siberia.

The rachitic tooth: The use of radiographs as a screening technique.

This study investigates morphological changes in pulp chambers of living and archaeological individuals with past vitamin D deficiency. Living individuals (n=29), four with detailed medical and dental records and three groups of archaeological individuals (n=25) were radiographed; selected individuals were further evaluated histologically for the presence of incremental interglobular dentin (IIGD), indicative of deficiency (28 living; 17 archaeological). Measurements of pulp horns/chambers from radiographs were conducted to quantify morphological observations. One group had clear skeletal evidence of rickets from St. Matthew, Quebec (n=1) and St. Jacques, France (n=4); a second group had slight skeletal indicators from Bastion des Ursulines, Quebec (n=6); and a third group lacked both skeletal and radiological evidence of deficiency from St. Antoine (n=6) and Pointe-aux-Trembles (n=4). Results showed archaeological individuals with clear and slight skeletal evidence of past deficiency displayed constricted or chair shaped pulp horns. Living individuals with deficiency exhibited similar pulp chamber morphology. Radiographic pulp horn/chamber measurements corroborated morphological findings and significant differences were found in pulp horn/chamber measurements between those with and without deficiency. Results suggest that radiograph assessment of teeth can be used as a screening technique to elucidate patterns of deficiency and select individuals for microCT or histological assessment.