Determining isotopic life history trajectories using bone density fractionation and stable isotope measurements: a new approach

A number of recent studies have attempted to trace diet at different stages of an individual's life by comparing isotope ratios of bone from different gross anatomical sites within the skeleton. In this study we develop this approach further by separating bone of differing mineral densities within one skeletal element, where each density fraction represents a different period of time. Isotope ratios are measured for these fractions. Each density fraction represents a period of bone formation and maturation, where younger (more recently formed) bone is less well-mineralized and therefore less dense than relatively older packets of bone. In an adult, bone is therefore able to partition approximately the last 15 years of life. Bone fractions were recovered by stepped ultracentrifugation in a series of organic solvents of increasing density, and then collagen was recovered by decalcification in dilute acid, and stable carbon isotope ratios ((13)C/(12)C) were measured. Bone density microstructure was checked for bacterial remodelling using backscattered electron imaging in a scanning electron microscope. Our results indicate that the bone density fractionation method is applicable to archaeological material, here extending to a maximum of 5,000 years BP, and that collagen can successfully be extracted from such fractions. The carbon isotope values for bone fractions of different densities patterned out as expected in one modern control bone and in specimens from five archaeological human skeletons, including three precolonial hunter-gatherers and two 18th/19th century individuals. The latter two are known (from previous assessments) to have undergone marked changes in diet during their lifetimes. Postmortem alteration was evident in some of the specimens examined. The bone density fractionation approach has allowed greater resolution of diet than has hitherto been possible and has provided access to the elusive last years and months of an individual's life.     

Cartilage,bone, and intermandibular connective tissue in the australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

The connective tissue that links the bones of the mandible in the Australian lungfish, Neoceratodus forsteri, has been described as an intermandibular cartilage, and as such has been considered important for phylogenetic analyses among lower vertebrates. However, light and electron microscopy of developing lungfish jaws demonstrates that the intermandibular tissue, like the connective tissue that links the bones of the upper jaw, contains fibroblasts and numerous bundles of collagen fibrils, extending from the trabeculae of the bones supporting the tooth plates. It differs significantly in structure and in staining reactions from the cartilage and the bone found in this species. In common with the cladistian Polypterus and with actinopterygians and some amphibians, lungfish have no intermandibular cartilage. The connective tissue linking the mandibular bones has no phylogenetic significance for systematic grouping of lungfish, as it is present in a range of different groups among lower vertebrates. J. Morphol. 274:1085–1089, 2013. © 2013 Wiley Periodicals, Inc.     

The mineralized osteocyte: a living fossil

We report here on an enigmatic and biologically mysterious event in which a single cell, the osteocyte, mineralizes in vivo and in this process the cell's organelles, cytoskeleton and membrane, are mineralized in a dying state. That the bony lacuna in which the lone osteocyte resides becomes infilled with mineral in vivo is not a new observation and was noted by early microscopists. This study has applied scanning and transmission electron microscopy to modern, archaeological, and fossil bone to investigate the mineral and organic structure and content of this cell. The results from this study revealed that within this mineral lies a visibly identifiable cell, which has an apoptotic-like morphology. The mechanisms by which this cell mineralizes are so intimate chemically that remnant cell organelles, membranes, cytoskeleton, and potentially nucleic bodies are morphologically identifiable. We have further identified mineralized osteocytes surviving in archaeological and fossil mammal bone up to 5 million years BP. The significance of our findings demonstrates that a single cell may itself mineralize in vivo via an unknown set of biochemical events. Importantly, the location and survival of extra cellular and cellular proteins, including nuclear and mitochondrial DNA in bone after death, has been an area of some speculation, and this unique fossil cell provides a preservation locus within human and mammalian bone, which might be fruitfully targeted in future biomolecular studies.     

Rethinking the nature of fibrolamellar bone: an integrative biological revision of sauropod plexiform bone formation

We present novel findings on sauropod bone histology that cast doubt on general palaeohistological concepts concerning the true nature of woven bone in primary cortical bone and its role in the rapid growth and giant body sizes of sauropod dinosaurs. By preparing and investigating longitudinal thin sections of sauropod long bones, of which transverse thin sections were published previously, we found that the amount of woven bone in the primary complex has been largely overestimated. Using comparative cellular and light‐extinction characteristics in the two section planes, we revealed that the majority of the bony lamina consists of longitudinally organized primary bone, whereas woven bone is usually represented only by a layer a few cells thin in the laminae. Previous arguments on sauropod biology, which have been based on the overestimated amount, misinterpreted formation process and misjudged role of woven bone in the plexiform bone formation of sauropod dinosaurs, are thereby rejected. To explain the observed pattern in fossil bones, we review the most recent advances in bone biology concerning bone formation processes at the cellular and tissue levels. Differentiation between static and dynamic osteogenesis (SO and DO) and the revealed characteristics of SO‐ versus DO‐derived bone tissues shed light on several questions raised by our palaeohistological results and permit identification of these bone tissues in fossils with high confidence. By presenting the methods generally used for investigating fossil bones, we show that the major cause of overestimation of the amount of woven bone in previous palaeohistological studies is the almost exclusive usage of transverse sections. In these sections, cells and crystallites of the longitudinally organized primary bone are cut transversely, thus cells appear rounded and crystallites remain dark under crossed plane polarizers, thereby giving the false impression of woven bone. In order to avoid further confusion in palaeohistological studies, we introduce new osteohistological terms as well as revise widely used but incorrect terminology. To infer the role of woven bone in the bone formation of fast‐growing tetrapods, we review some aspects of the interrelationships between the vascularity of bone tissues, basal metabolic rate, body size and growth rate. By putting our findings into the context of osteogenesis, we provide a new model for the diametrical limb bone growth of sauropods and present new implications for the evolution of fast growth in vertebrates. Since biomechanical studies of bone tissues suggest that predominant collagen fibre orientation (CFO) is controlled by endogenous, functional and perhaps phylogenetic factors, the relationship between CFO and bone growth rate as defined by Amprino's rule, which has been the basis for the biological interpretation of several osteohistological features, must be revised. Our findings draw attention to the urgent need for revising widely accepted basic concepts of palaeohistological studies, and for a more integrative approach to bone formation, biomechanics and bone microstructural features of extant and extinct vertebrates to infer life history traits of long extinct, iconic animals like dinosaurs.     

The cryomicrotomy of rat dental tissues: a technique for histological and immunohistochemical analysis

Summary We have applied a method developed for the cryomicrotomy of non-decalcified bone to the histological preparation of the tooth and related dental tissues. Cryosections of rat mandible have been cut on a heavy-duty freezing microtome. Both cellular and extracellular structure was well preserved and the sections of tooth and bone appeared to be suitable for optical and scanning electron microscopy and for immunohistochemical analysis. However, there was an overall strong non-specific binding of immunohistochemical reagents to enamel which was not evident in the other mineralized tissues of the mandible. This may relate to important differences in the nature of this tissue.     

Variations in cortical material properties throughout the human dentate mandible

Material properties and their variations in individual bone organs are important for understanding bone adaptation and quality at a tissue level, and are essential for accurate mechanical models. Yet material property variations have received little systematic study. Like all other material property studies in individual bone organs, studies of the human mandible are limited by a low number of both specimens and sampled regions. The aims of this study were to determine: 1) regional variability in mandibular material properties, 2) the effect of this variability on the modeling of mandibular function, and 3) the relationship of this variability to mandibular structure and function. We removed 31 samples on both facial and lingual cortices of 10 fresh adult dentate mandibles, measured cortical thickness and density, determined the directions of maximum stiffness with a pulse transmission ultrasonic technique, and calculated elastic properties from measured ultrasonic velocities. Results showed that each of these elastic properties in the dentate human mandible demonstrates unique regional variation. The direction of maximum stiffness was near parallel to the occlusal plane within the corpus. On the facial ramus, the direction of maximum stiffness was more vertically oriented. Several sites in the mandible did not show a consistent direction of maximum stiffness among specimens, although all specimens exhibited significant orthotropy. Mandibular cortical thickness varied significantly (P < 0.001) between sites, and decreased from 3.7 mm (SD = 0.9) anteriorly to 1.4 mm posteriorly (SD = 0.1). The cortical plate was also significantly thicker (P < 0.003) on the facial side than on the lingual side. Bone was 50-100% stiffer in the longitudinal direction (E(3), 20-30 GPa) than in the circumferential or tangential directions (E(2) or E(1); P < 0.001). The results suggest that material properties and directional variations have an important impact on mandibular mechanics. The accuracy of stresses calculated from strains and average material properties varies regionally, depending on variations in the direction of maximum stiffness and anisotropy. Stresses in some parts of the mandible can be more accurately calculated than in other regions. Limited evidence suggests that the orientations and anisotropies of cortical elastic properties correspond with features of cortical bone microstructure, although the relationship with functional stresses and strains is not clear.     

Analysis of arch‐like bones: The rodent mandible as a case study

Bone strength is determined by the mechanical properties of bone material, and the size and shape of the whole bone, i.e., its architecture. The mandible of vertebrates has been traditionally regarded as a beam oriented in relation to main masticatory loads, i.e., the longer dimension of its cross‐section being parallel to the load. Rodents follow this pattern but, in addition, their mandible possesses an intriguing arch‐like shape that is apparent when seen in the lateral view. Little attention was given to the structural capacity of this trait. The advantage of an arch is that it can withstand a greater load than a horizontal beam. The objective of this study was to model the rodent mandible like an arch to evaluate its structural strength. The bending moment in an arch‐like mandible was 15–25% lower with respect to a beam‐like mandible. Further, bending varies with mandible “slenderness” and incisor procumbency, a functionally relevant rodent trait. In the rodent Ctenomys talarum (Caviomorpha; Ctenomyidae), bone stress was substantially reduced when the mandible was modeled as an arch‐like structure as compared with a beam‐like structure, and safety factors were 15–34% higher. The shape of rodents' mandible might confer a functional advantage to high and repeatedly applied loads resulting from a unique feeding mode: gnawing. J. Morphol. 277:879–887, 2016. © 2016 Wiley Periodicals, Inc.     

Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)     

Morphometric and hormonal changes during the chimpanzee menstrual cycle

BACKGROUND: Sex steroids affect many peripheral tissue sites in female mammals. Receptors for these hormones have been found in skin, fat, and bone. In women, these tissues can show morphological changes during the menstrual cycle that may be directly related to steroid secretion. METHODS: The present study was done on chimpanzees to document morphometric markers associated with these tissues (anogenital swelling volume, skin fold thickness as indicator of subcutaneous fat, bony diameters of mandible, wrist, and elbow) and to compare them with cyclic patterns of estradiol, progesterone, testosterone, gonadotropins, and prolactin. RESULTS: Swelling volume changed significantly over the menstrual cycle. All other morphometric parameters showed variation without statistical significance. Skin folds were thickest during the luteal phase. Bony diameters displayed similar but less distinctive changes. Testosterone correlated positively with diameter sites, inversely with subcutaneous fat. No relationships with either estradiol or progesterone were found. We assume that subcutaneous fat and morphometric bone parameters exhibit cycle-dependent changes that may be caused by changes in steroid secretion.     

The dietary anthocyanin delphinidin prevents bone resorption by inhibiting Rankl-induced differentiation of osteoclasts in a medaka (Oryzias latipes) model of osteoporosis

The anthocyanin delphinidin is a natural compound found as water-soluble pigment in coloured fruits and berries. Anthocyanin-rich diets have been proposed to have bone protective effects in humans and mice, but the underlying mechanisms remain unclear. In this study, we used a medaka (Oryzias latipes) osteoporosis model to test the effects of delphinidin on bone cells in vivo. In this model, inducible transgenic expression of receptor-activator of NF-kβ ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, similar to the situation in human osteoporosis patients. Using live imaging in medaka bone reporter lines, we show that delphinidin significantly reduces the number of osteoclasts after Rankl induction and protects bone integrity in a dose-dependent manner. Our in vivo findings suggest that delphinidin primarily affects the de novo differentiation of macrophages into osteoclasts rather than the recruitment of macrophages to sites of bone resorption. For already existing osteoclasts, delphinidin treatment affected their morphology, leading to fewer protrusions and a more spherical shape. Apoptosis rates were not increased by delphinidin, suggesting that osteoclast numbers were reduced primarily by impaired differentiation from macrophage progenitors and reduced maintenance of pre-existing osteoclasts. Importantly, and in contrast to previously reported cell culture experiments, no effect of delphinidin on osteoblast differentiation and distribution was observed in medaka in vivo. Our study is the first report on the effects of delphinidin on bone cells in fish embryos, which are a unique model system for compound testing that is suitable for live imaging of bone cell behaviour in vivo.     

Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.     

Selective intra-lysosomal concentration of niobium in kidney and bone marrow cells: a microanalytical study

Niobium is used as an alloy in the industrial and biomedical fields. The concentration of the toxic element in organs of a number of animal species has been defined by using radioactive niobium (⁹⁵Nb). However, tissue lesions induced by niobium have only been studied at the light microscopy level. In this study, we used an electron probe X-ray analyzer equipped with a transmission electron microscope to define the localization of this element in kidney and bone marrow cells. Results demonstrated that niobium is located in the lysosome and that this element coprecipitates with phosphate. In kidney, lysosomes and precipitates are eliminated in the tubular lumen. In contrast, precipitates appear to be eliminated more slowly from the lysosomes of bone marrow macrophages. These processes therefore correspond to one of the mechanisms by which lysosomes eliminate certain toxic mineral elements and thus play a role in the more general process of the body's defenses.     

Bone remodelling in Neanderthal mandibles from the El Sidrón site (Asturias, Spain)

Skull morphology results from the bone remodelling mechanism that underlies the specific bone growth dynamics. Histological study of the bone surface from Neanderthal mandible specimens of El Sidrón (Spain) provides information about the distribution of the remodelling fields (bone remodelling patterns or BRP) indicative of the bone growth directions. In comparison with other primate species, BRP shows that Neanderthal mandibles from the El Sidrón (Spain) sample present a specific BRP. The interpretation of this map allows inferences concerning the growth directions that explain specific morphological traits of the Neanderthal mandible, such as its quadrangular shape and the posterior location of the mental foramen.     

Helicobacter pylori produces unique filaments upon host contact in vitro

Helicobacter pylori exists in 2 distinct morphological states, helicoid and coccoid. Both have been observed in in vitro culture and in gastric biopsies. We visualized H. pylori during AGS cell infections using immunofluorescence microscopy. Anti-H. pylori mouse serum as well as human serum from H. pylori-positive patients recognized long, thin bacterial filaments, which formed on helicoids and more frequently on coccoids. These filaments reached lengths of 59 microm and often connected bacteria. Periodate oxidation abolished antibody recognition, suggesting that carbohydrates compose a major antigenic component of the filaments. Similar to results obtained using immunofluorescence microscopy, scanning electron microscopy imaging revealed thin filamentous structures, which were absent on uninfected cells. Both coccoid conversion and filament development increased over the time course of infection with peak filament formation at 4 h. The number of visible filaments then decreased as bacteria clustered on the apical surface of AGS cells. Since the observed filaments were clearly distinct from previously described surface structures, including flagella and the cag type IV secretion system, our results demonstrate that these filaments represent a unique, previously unrecognized, organelle.     

Standard measurements, elasticity values and tensile strength behavior of the human mandible, a contribution to the biomechanics of the mandible--I

This paper is intended to help to characterize the physiological behavior of compact bone structure, so that realistic account can be taken of it when performing finite element (FE) analysis with the aim of solving biomechanical problems. In addition, further knowledge about the complex mechanical behavior of bone structure is provided. In accordance with anthropometric considerations, 13 mandibles have been measured, and a standard mandible defined for use in representative model calculations. In 4 mandibles (fully dentulous, partly dentulous, edentulous), orthotropic mechanical behavior of the fresh bone was determined experimentally. A comparison was also made with published data, as well as our own unpublished data obtained in other bone structures. Orthotropic behavior of the bone structure was confirmed. The material data of the individual bone structure vary considerably, but remain within a certain range. The proportion of the direction-dependent data is, however, almost constant. Our data correspond well with published data, although a direct comparison with the data of the mandible was not possible. A comparison of the data with those from the lower and upper extremities shows significant differences in rigidity, Young's modulus, and extension. A gradation can be found--starting with a maximum--that reveals the following order: mandible, upper extremity, lower extremity. In the case of extension, this order is reversed.     

Flexural and Creep Properties of Human Jaw Compact Bone for FEA Studies

The aim of this work was to improve the constitutive model of the human mandible and dentition system by taking into account the non-linear material properties of the structural boney matrix that forms the human jaw bone or mandible. Due to the specific structure of the jaw bone the time dependence of the mechanical properties also forms an important stage of the quantification process. The lack of specific experimental data of this type of material prevents the implementation of these properties into finite element simulations which results in poor quality modelling. Here an attempt was made to determine elastic and viscoelastic mechanical characteristics of the compact bone tissue forming the mandible. The elastic properties of compact bone were determined experimentally from 3 point bending tests and the viscoelastic properties were evaluated from creep tests in compression. A particular human jaw from this complex study was used to reconstruct a geometric model for further numerical experiments.     

Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes

Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson’s correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.     

Phenotypic characterization of craniofacial bone marrow stromal cells: unique properties of enhanced osteogenesis,cell recruitment,autophagy, and apoptosis resistance

Previous studies have shown that craniofacial bone marrow stromal cells (MSCs) have greater osteogenic potential than appendicular bone MSCs. However, detailed phenotypic characterization of MSCs from bone marrow in the different sites remains unclear. To investigate bone repair and regeneration of craniofacial MSCs and the regulatory mechanisms underlying their unique properties, we compared osteogenesis, cell recruitment, autophagy, and apoptosis resistance of MSCs from the mandible (M-MSCs) to those from tibia (T-MSCs) in vitro and in vivo. Compared with T-MSCs, M-MSCs formed more colonies, possessed stronger proliferation activity, exhibited higher expression of pluripotency genes such as Oct4 and Nanog, and held stronger osteogenic differentiation in osteogenic medium. Moreover, M-MSCs had greater autophagy and anti-apoptotic capacities than T-MSCs under hypoxia and serum deprivation conditions. M-MSCs were found to be more capable of recruiting more MSCs than T-MSCs. When these MSCs were transplanted into mandible critical-sized defects, more bone formed in the M-MSC-treated animals than in their T-MSC counterparts. Collectively, these findings reveal that MSCs have unique characteristics and bone-repairing properties from the mandible as compared with those from tibia, presumably by enhanced osteogenic potential, cell recruitment, autophagy and apoptosis resistance.     

Evaluation of bone remodeling around single dental implants of different lengths: a mechanobiological numerical simulation and validation using clinical data

Algorithmic models have been proposed to explain adaptive behavior of bone to loading; however, these models have not been applied to explain the biomechanics of short dental implants. Purpose of present study was to simulate bone remodeling around single implants of different lengths using mechanoregulatory tissue differentiation model derived from the Stanford theory, using finite elements analysis (FEA) and to validate the theoretical prediction with the clinical findings of crestal bone loss. Loading cycles were applied on 7-, 10-, or 13-mm-long dental implants to simulate daily mastication and bone remodeling was assessed by changes in the strain energy density of bone after a 3, 6, and 12 months of function. Moreover, clinical findings of marginal bone loss in 45 patients rehabilitated with same implant designs used in the simulation (n = 15) were computed to validate the theoretical results. FEA analysis showed that although the bone density values reduced over time in the cortical bone for all groups, bone remodeling was independent of implant length. Clinical data showed a similar pattern of bone resorption compared with the data generated from mathematical analyses, independent of implant length. The results of this study showed that the mechanoregulatory tissue model could be employed in monitoring the morphological changes in bone that is subjected to biomechanical loads. In addition, the implant length did not influence the bone remodeling around single dental implants during the first year of loading.