- Berlin, Berlin, Germany
ron shahar
The Hebrew University of Jerusalem, VETRINARY, Faculty Member
- Bone biomechanics, structure and functionedit
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Journal of Biomechanics, Volume 41, Issue null, Pages S47, July 2008, Authors:Meir Max Barak; Steve Weiner; Ron Shahar.
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In order to understand complex-hierarchical biomaterials such as bones and teeth, it is necessary to relate their structure and mechanical-properties. We have adapted electronic speckle pattern-correlation interferometry (ESPI) to make... more
In order to understand complex-hierarchical biomaterials such as bones and teeth, it is necessary to relate their structure and mechanical-properties. We have adapted electronic speckle pattern-correlation interferometry (ESPI) to make measurements of deformation of ...
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Synopsis Teeth are composed of the hardest tissues in the vertebrate body and have been studied extensively to infer diet in vertebrates. The morphology and structure of enamel is thought to reflect feeding ecology. Snakes have a... more
Synopsis Teeth are composed of the hardest tissues in the vertebrate body and have been studied extensively to infer diet in vertebrates. The morphology and structure of enamel is thought to reflect feeding ecology. Snakes have a diversified diet, some species feed on armored lizards, others on soft invertebrates. Yet, little is known about how tooth enamel, and specifically its thickness, is impacted by diet. In this study, we first describe the different patterns of enamel distribution and thickness in snakes. Then, we investigate the link between prey hardness and enamel thickness and morphology by comparing the dentary teeth of 63 species of snakes. We observed that the enamel is deposited asymmetrically at the antero-labial side of the tooth. Both enamel coverage and thickness vary a lot in snakes, from species with thin enamel, only at the tip of the tooth to a full facet covered with enamel. There variations are related with prey hardness: snakes feeding on hard prey have a t...
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1. The structure, composition, and shape of teeth have been related to dietary specialization in many vertebrate species, except snakes. Yet, snakes have diverse dietary habits that may impact the shape of their teeth. We hypothesize that... more
1. The structure, composition, and shape of teeth have been related to dietary specialization in many vertebrate species, except snakes. Yet, snakes have diverse dietary habits that may impact the shape of their teeth. We hypothesize that prey properties, such as hardness and shape, as well as feeding behavior, such as aquatic or arboreal predation, or holding vigorous prey, impose constraints on the evolution of tooth shape in snakes. 2. We compared the morphology of the dentary teeth of 63 species that cover the phylogenetic and dietary diversity of snakes, using 3D geometric morphometrics and linear measurements. 3. Our results show that prey hardness, foraging substrate and the main mechanical challenge are important drivers of tooth shape, size, and curvature. 4. Overall, long, slender, curved teeth with a thin layer of hard tissue are observed in species that need to maintain a grip on their prey. Short, stout, less curved teeth are associated with species that undergo high or...
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Teeth are one of the most studied hard tissues in vertebrates. Their structure, composition and shape are related to dietary specialization in many species. At first glance, snake teeth all look similar; conical, sharp, curved. Yet,... more
Teeth are one of the most studied hard tissues in vertebrates. Their structure, composition and shape are related to dietary specialization in many species. At first glance, snake teeth all look similar; conical, sharp, curved. Yet, snakes, like other vertebrates, have very diverse diets that may have affected their shape. We compared the morphology of the teeth of 63 species that cover both the phylogenetic and dietary diversity of snakes. We predicted that prey properties play a role in shaping snakes teeth along with their feeding behavior. Limblessness combined to the peculiar feeding behavior of snakes impose strong functional constraints on their teeth, especially during arboreal or aquatic feeding. Our results show that prey hardness, foraging substrate and the main feeding constraint are drivers of tooth shape, size, and curvature. We highlight two main morphotypes: long, slender, curved with a thin layer of hard tissue for snakes that need a good grip on their prey and shor...
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Dietary protein is necessary throughout all life stages. Adequate intake of protein during juvenile years is essential to enable appropriate synthesis of bone matrix and achieve the full peak bone mass (PBM). Due to socio-demographic... more
Dietary protein is necessary throughout all life stages. Adequate intake of protein during juvenile years is essential to enable appropriate synthesis of bone matrix and achieve the full peak bone mass (PBM). Due to socio-demographic changes, accompanied by environmental damage and ethical problems, a transition to the consumption of different and alternative protein sources in the human diet must occur. This transition requires the precise evaluation of protein quality. Here, we utilize a preclinical model of young rats during their post-natal developmental period to define the nutritive quality of a number of alternative protein sources (soy, spirulina, chickpea, and fly larvae) by their health impact on growth performance and skeletal development. We indicate that when restricted (10% of calories) not one of the tested alternative protein sources have succeeded in causing optimal growth, as compared to the referenced source, casein; yet fly larvae protein followed by chickpea flo...
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Bone is a fascinating biomaterial comprised mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, with water molecules residing at the interfaces between these phases. They are hierarchically organized with... more
Bone is a fascinating biomaterial comprised mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, with water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications to the mechanical properties of the particular bone. STATEMENT OF SIGNIFICANCE: Bone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.
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When describing the architecture and ultrastructure of animal skeletons, introductory biology, anatomy and histology textbooks typically focus on the few bone and cartilage types prevalent in humans. In reality, cartilage and bone are far... more
When describing the architecture and ultrastructure of animal skeletons, introductory biology, anatomy and histology textbooks typically focus on the few bone and cartilage types prevalent in humans. In reality, cartilage and bone are far more diverse in the animal kingdom, particularly within fishes, where cartilage and bone types exist that are characterized by features that are anomalous or even pathological in human skeletons. Here, we discuss the curious and complex architectures of fish bone and shark and ray cartilage, highlighting similarities and differences with their mammalian skeletal tissue counterparts. By synthesizing older anatomical literature with recent high-resolution structural and materials characterization work, we frame emerging pictures of form-function relationships in these tissues and of the evolution and true diversity of cartilage and bone.
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SummaryThe purpose of this study was to develop a reliable and repeatable radiographic protocol for the measurement of joint angles in the standing dog, and to use this protocol to determine all standing joint angles for dogs over a wide... more
SummaryThe purpose of this study was to develop a reliable and repeatable radiographic protocol for the measurement of joint angles in the standing dog, and to use this protocol to determine all standing joint angles for dogs over a wide range of body weights. The radiographic technique and the method of joint angle measurements were found to be highly repeatable, suggesting that the technique is reliable. Most joint angles did not vary between dogs of different weights. In those few instances where significant differences (p<0.05) were found, certain trends were followed and represent differences in conformation. This paper presents a complete description of the angles defining the position of the joints in a standing dog. This information is important for biomechanical studies, for clinical assessment of dogs, and for the design of surgical procedures such as arthrodesis.
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SummaryA wide variety of surgical techniques to stabilize the coxo-femoral joint have been described in the veterinary literature. In this paper twenty small animals (19 dogs and 1 cat) were treated successfully with open reduction and an... more
SummaryA wide variety of surgical techniques to stabilize the coxo-femoral joint have been described in the veterinary literature. In this paper twenty small animals (19 dogs and 1 cat) were treated successfully with open reduction and an extra-capsular suture taken between the greater trochanter of the femur and the origin of the rectus femoris muscle. The technique is simple, quick and safe, and avoids many of the potential complications associated with other methods.
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In order to examine the differences in bone properties between fast-growing and slow-growing broiler embryos and to understand the effects of genotype and egg size on these differences, fast- and slow-growing hens and males were... more
In order to examine the differences in bone properties between fast-growing and slow-growing broiler embryos and to understand the effects of genotype and egg size on these differences, fast- and slow-growing hens and males were reciprocally crossed to create 4 egg groups: FST (laid by fast-growing hens, inseminated by fast-growing males), H-FST (fast-growing hens and slow-growing males), H-SLW (slow-growing hens and fast-growing males), and SLW (slow-growing hens and slow-growing males). Embryos (n = 8) from these 4 groups were sacrificed and weighed, and both tibiae were harvested on embryonic d (E) 17, 19, and 21. Left tibiae were tested for their whole-bone mechanical properties using a micromechanical device. Cortical bone structure and bone mineral density (BMD) were examined by micro-computed tomography of the left tibiae. Bone mineralization was evaluated by measuring BMD and ash content, while the rate and location of mineralization were evaluated by fluorochrome labeling. ...
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Velocity and pressure fields for Stokes flow due to a force singularity (Stokeslet) of arbitrary orientation and at arbitrary location inside an infinite circular pipe are obtained. Two alternative expressions for the solution, one in... more
Velocity and pressure fields for Stokes flow due to a force singularity (Stokeslet) of arbitrary orientation and at arbitrary location inside an infinite circular pipe are obtained. Two alternative expressions for the solution, one in terms of a Fourier-Bessel type expansion, and the other as a doubly infinite series, are given. The latter is especially suitable for computational purposes as it is shown to be an exponentially decaying series. From the series it is found that all velocity components decay exponentially to zero up- or downstream away from the Stokeslet. This is also true for pressure fields of Stokeslets perpendicular to the axis of the pipe. A Stokeslet parallel to the axis of the pipe raises the pressure difference between − ∞ to + ∞ by a finite non-zero amount. Some numerical results for a Stokeslet parallel to the axis are given. Comparison of the results with flow in a two-dimensional channel is also discussed.
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The periodontal ligament (PDL), a soft tissue connecting the tooth and the bone, is essential for tooth movement, bone remodeling and force dissipation. A collagenous network that connects the tooth root surface to the alveolar jaw bone... more
The periodontal ligament (PDL), a soft tissue connecting the tooth and the bone, is essential for tooth movement, bone remodeling and force dissipation. A collagenous network that connects the tooth root surface to the alveolar jaw bone is one of the major components of the PDL. The organization of the collagenous component and how it changes under load is still poorly understood. Here using a state-of-the-art custom-made loading apparatus and a humidified environment inside a microCT, we visualize the PDL collagenous network of a fresh rat molar in 3D at 1 μm voxel size without any fixation or contrasting agents. We demonstrate that the PDL collagen network is organized in sheets. The spaces between sheets vary thus creating dense and sparse networks. Upon vertical loading, the sheets in both networks are stretched into well aligned arrays. The sparse network is located mainly in areas which undergo compressive loading as the tooth moves towards the bone, whereas the dense network functions mostly in tension as the tooth moves further from the bone. This new visualization method can be used to study other non-mineralized or partially mineralized tissues, and in particular those that are subjected to mechanical loads. The method will also be valuable for characterizing diseased tissues, as well as better understanding the phenotypic expressions of genetic mutants.
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Research Interests: Materials Science, Biomedical Engineering, Medicine, Anatomy, Interferometry, and 12 moreAnimals, Three Dimensional Imaging, Medical Physiology, Mechanical Stress, Incisor, Macaca Mulatta, Dental Stress Analysis, Dental Enamel, X Ray Microtomography, Structure Function, Biomechanical Phenomena, and Electronic Speckle Pattern Interferometry
The naked mole rat (NMR) is a small rodent with a remarkable array of properties, such as unique physiology, extremely long life-span and unusual social life. However, very little is known regarding its skeleton. The aim of this study was... more
The naked mole rat (NMR) is a small rodent with a remarkable array of properties, such as unique physiology, extremely long life-span and unusual social life. However, very little is known regarding its skeleton. The aim of this study was to describe the structure, composition and mechanical properties in an ontogenetic series of naked mole rat bones. Since common small rodents like mice and rats have an unusual structure of cortical bone, which includes a central region of non-lamellar (disordered) bone, mineralized cartilaginous islands and total lack of remodeling, this study could also determine if these features result from their short life-span. Sixty-one NMRs were included in the study and were divided into the following four age groups: 0-0.5 years old (n = 17), 0.5-3 years old (n = 25), 3-10 years old (n = 13), and >10 years (n = 6). Femora, vertebrae and mandibulae were examined using micro-CT, light microscopy, polarized light microscopy and scanning electron microscopy, thermogravimetric analysis was used to determine their dry ash content and their derived elastic modulus and hardness were determined using micro-indentation. Our findings show that NMR bones are similar in composition and mechanical properties to those of other small rodents. However, in contrast to other small rodents, the cortical bone of NMRs is entirely circumferential-lamellar and lacks mineralized cartilage islands. Furthermore, despite their long life-span, their bones did not show evidence of remodeling at any of the age groups, thus proving that lack of cortical remodeling in small rodents is not caused by their short life span.
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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... more
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), with porosity decreasing close to the bone-tooth interface, and back-scattered electron (BSE-SEM) microscopy showing 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.
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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... more
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. Understanding mineralized tissues, such as bone and cartilage, in health and disease requires a thorough knowledge of their structures. This Perspective proposes a new imaging and characterization strategy for mineralized tissue research that utilizes focused ion beam-scanning electron microscopy.
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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... more
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.
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ABSTRACT
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Ultra-processed foods have known negative implications for health; however, their effect on skeletal development has never been explored. Here, we show that young rats fed ultra-processed food rich in fat and sugar suffer from growth... more
Ultra-processed foods have known negative implications for health; however, their effect on skeletal development has never been explored. Here, we show that young rats fed ultra-processed food rich in fat and sugar suffer from growth retardation due to lesions in their tibial growth plates. The bone mineral density decreases significantly, and the structural parameters of the bone deteriorate, presenting a sieve-like appearance in the cortices and poor trabecular parameters in long bones and vertebrae. This results in inferior mechanical performance of the entire bone with a high fracture risk. RNA sequence analysis of the growth plates demonstrated an imbalance in extracellular matrix formation and degradation and impairment of proliferation, differentiation and mineralization processes. Our findings highlight, for the first time, the severe impact of consuming ultra-processed foods on the growing skeleton. This pathology extends far beyond that explained by the known metabolic eff...
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Bones are nanocomposites of protein, mineral and water that form mineralized collagen fibrils arranged in a variety of layered lamellae. Bone material has a long evolutionary record and specific bones attain shapes and microstructures... more
Bones are nanocomposites of protein, mineral and water that form mineralized collagen fibrils arranged in a variety of layered lamellae. Bone material has a long evolutionary record and specific bones attain shapes and microstructures that have well stood the test of time such that they can be considered optimized to match their function. Further, most bones typically contain entombed living cells, osteocytes responsible for adaptation, healing and biochemical signaling. The bones of pike fish (Esox lucius) are different because, as with other advanced teleost species, they evolved to eliminate osteocytes from the microstructure. This suggests that these cells are not needed because these bones are more damage resistant than mammalian bones. Here we explore details of this biologically-grown structure, using a combination of light and X-ray based characterization methods. We report the three-dimensional arrangement and composition of the heavily cyclically-loaded pivot of the cleith...
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With an exclusive diet of hard-shelled mollusks, the black drum fish (Pogonias Cromis) exhibits one of the highest bite forces among extant animals. To understand the structural basis for these integrated functional requirements, here we... more
With an exclusive diet of hard-shelled mollusks, the black drum fish (Pogonias Cromis) exhibits one of the highest bite forces among extant animals. To understand the structural basis for these integrated functional requirements, here we present the results of a systematic microstructural, chemical, crystallographic, and mechanical analysis of the black drum teeth. The teeth are based on a two-layered enameloid/dentin design. At the microscopic level of the outer enameloid region, Zn-doped hydroxyapatite/fluorapatite crystals and their preferential co-alignment of crystallographic c-axes along the biting direction contribute to the local stiffening, hardening, and toughening of the tooth surface (Er = 126.9 ± 16.3 GPa, H = 5.0 ± 1.4 GPa, and KIc = 1.12 MPa·m1/2). Our analysis also shows that this microstructure promotes local yielding instead of fracture when crushing mollusk shells. At the whole-tooth scale, the unique molar-like teeth, with smooth top surfaces, high density of dentin tubules, enlarged pulp chamber, and special dentin-bone interfacial connections, all contribute to the teeth functional requirements, including confinement of contact compressive stress in the stiff enameloid, enhanced energy absorption in the compliant and tough dentin, reduction of stress/strain concentration in the weakest bone, and controlled failure of the teeth under excessive loads. These results show that the multi-scale structure of black drum teeth is adapted to feed on mollusks.
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SummaryThe use of acrylic connecting bars in external fixators has become widespread in veterinary orthopaedics. One of the main advantages of an acrylic connecting bar is the ability to contour it into a curved shape. This allows the... more
SummaryThe use of acrylic connecting bars in external fixators has become widespread in veterinary orthopaedics. One of the main advantages of an acrylic connecting bar is the ability to contour it into a curved shape. This allows the surgeon to place the transcortical pins according to safety and convenience considerations, without being bound by the requirement of the standard stainless steel connecting bar, that all transcortical pins be in the same plane.The purpose of this study was to evaluate the stiffness of unilateral and bilateral medium-sized external fixator frames with different curvatures of acrylic connecting bars. Finite element analysis was used to model the various frames and obtain their stiffness under four types of load: Axial compression, four-point medio-lateral bending, fourpoint antero-posterior bending and torsion. The analysis also provided the maximal pin stresses occurring in each frame for each loading condition.Based on the results of this study, curva...
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<p>(a) A simplified loading graphic of the forces applied to the model. Lateral bending of the vertebral column during oscillatory swimming creates forces on the flat articular surfaces of adjacent centra, F Comp. on one side and F... more
<p>(a) A simplified loading graphic of the forces applied to the model. Lateral bending of the vertebral column during oscillatory swimming creates forces on the flat articular surfaces of adjacent centra, F Comp. on one side and F tens. on the opposite side. F Muscs acting on fish vertebrae are rather difficult to model because of the high complexity of the musculature as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000140#pbio.3000140.s002" target="_blank">S1 Fig</a> and are represented here in a simplified manner. (b) 3D representation of von Mises strains in the vertebra. (c) Strain distribution in 2 contralateral views of a loaded vertebra. Locations of several transverse sections are marked by white dashed lines and are shown in (d). (d) Strain distribution in the 3 numbered 2D transverse sections shown in (c). Peak strains occur primarily near the external surfaces of the VB. Strain magnitudes (no units) for all images are shown in the color bar on the right. F Comp., compression force; FEA, finite element analysis; F Musc, muscle force; F Tens., tension force; VB, vertebral bone.</p
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The severe impairment of bone development and quality was recently described as a new target for unbalanced ultra-processed food (UPF). Here, we describe nutritional approaches to repair this skeletal impairment in rats: supplementation... more
The severe impairment of bone development and quality was recently described as a new target for unbalanced ultra-processed food (UPF). Here, we describe nutritional approaches to repair this skeletal impairment in rats: supplementation with micro-nutrients and a rescue approach and switching the UPF to balanced nutrition during the growth period. The positive effect of supplementation with multi-vitamins and minerals on bone growth and quality was followed by the formation of mineral deposits on the rats’ kidneys and modifications in the expression of genes involved in inflammation and vitamin-D metabolism, demonstrating the cost of supplementation. Short and prolonged rescue improved trabecular parameters but incompletely improved the cortical parameters and the mechanical performance of the femur. Cortical porosity and cartilaginous lesions in the growth-plate were still detected one week after rescue and were reduced to normal levels 3 weeks after rescue. These findings highligh...
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<p>(a) Double fluorescent ISH, showing <i>SOST</i> (red) and <i>Col1a1</i> (green) in the spine and adjacent R of untrained (left) and trained (right) medaka (<i>n</i> = 3 for each group). In... more
<p>(a) Double fluorescent ISH, showing <i>SOST</i> (red) and <i>Col1a1</i> (green) in the spine and adjacent R of untrained (left) and trained (right) medaka (<i>n</i> = 3 for each group). In trained medaka, <i>SOST</i> expression appears down-regulated (as inferred from changes in staining intensity), while osteoblast recruitment is increased. (b) RT-qPCR results of <i>SOST</i> and <i>Col1a1</i> expression after 1 hour (<i>SOST</i>: swim trained <i>n</i> = 5, untrained <i>n</i> = 7 and <i>Col1a1</i>: swim trained <i>n</i> = 4, untrained <i>n</i> = 6) and 10 days of swim training of medaka (for both <i>SOST</i> and <i>Col1a1</i>: swim trained <i>n</i> = 5, untrained <i>n</i> = 4), (compared to levels in untrained medaka: gray, horizontal “control” line). Results were normalized to the rlp-7 housekeeping gene. Asterisks indicate a significant difference (<i>p</i> < 0.05) between trained and untrained (control) fish. Original raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000140#pbio.3000140.s010" target="_blank">S1 Data</a>. (c) Top: illustration of splice site blocking MO-binding site and primers used to detect morphant transcripts (E1, i1). Bottom: RT-PCR of medaka injected with standard control (C1, C2) and with <i>SOST</i> MO (MO1, MO2). Bands indicative of <i>SOST</i> splice blocking appeared in <i>SOST</i> MO-injected samples (black arrows) and not in the standard control samples. (d) Bone-formation dynamics marked by fluorochromes (red: alizarin red, injected at <i>t</i> = 0 weeks; green: calcein green, injected at <i>t</i> = 2.5 weeks) in vertebra of medaka injected with standard control MO (left) and <i>SOST</i> MO (right). Dashed, vertical white lines mark the border between vertebrae. NSs are cropped in fluorescence images in (d). <i>Col1a1</i>, collagen type I alpha 1; E1, exon-spanning forward primer; HS, hemal spine; ISH, in situ hybridization; i1, intron-spanning reverse primer; MO, vivo-morpholino; NS, neural spine; PCR, polymerase chain reaction; R, radial; RT-PCR, reverse transcription PCR; RT-qPCR, real-time quantitative PCR.</p
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<p>(a–d) An NS of medaka vertebra and adjacent fin R. (a) HE-stained section. The blue and yellow squares mark 2 regions in the dorsal fin R, shown in (b) and (c), respectively. (b–c) ISH showing <i>SOST</i> expression... more
<p>(a–d) An NS of medaka vertebra and adjacent fin R. (a) HE-stained section. The blue and yellow squares mark 2 regions in the dorsal fin R, shown in (b) and (c), respectively. (b–c) ISH showing <i>SOST</i> expression in the cartilaginous core of the dorsal fin R (white arrowheads) and surface osteoblasts of the fin R and NS (black arrowheads). (d) Double fluorescent ISH, showing both <i>Col1a1</i> (red) and <i>Col2a1</i> (green) expression in the <i>SOST</i>-positive osteoblasts (black arrowheads) of the NS and fin R and <i>Col2a1</i> in the <i>SOST</i>-positive chondrocytes (white arrowhead) in the cartilaginous core of the fin R. (e–h) The IVR in the caudal vertebral column of medaka, (e) HE-stained section. The blue and yellow squares mark the 2 regions of the intravertebral region shown in (f) and (g), respectively. (f–g) ISH showing <i>SOST</i> expression in chordoblasts (gray arrowheads), identified by their distinct morphology and location. (h) Double fluorescent ISH in the IVR, showing <i>Col1a1</i> expression (red) in osteoblasts (black arrowhead) in the external region of the IVR and <i>Col2a1</i> expression (green) in <i>SOST</i>-positive chordoblasts (gray arrowhead) in the internal region of the IVR (scale bar same as in e). Dashed, vertical white lines mark the border between vertebrae. Results for individual stains (e.g., DAPI, <i>SOST</i>, col1, col2) are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000140#pbio.3000140.s005" target="_blank">S4 Fig</a>. <i>Col1a1</i>, collagen type I alpha 1; <i>Col2a1</i>, collagen type II alpha 1; HE, hematoxylin–eosin; ISH, in situ hybridization; IVR, intervertebral region; J, joint; NS, neural spine; R, radial; VB, vertebral bone.</p
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<p>High-resolution tomography of caudal vertebrae of zebrafish (left) and medaka (right). O, colored in black in the zebrafish scan, show the ubiquity of cells residing in the bone material of zebrafish while being completely absent... more
<p>High-resolution tomography of caudal vertebrae of zebrafish (left) and medaka (right). O, colored in black in the zebrafish scan, show the ubiquity of cells residing in the bone material of zebrafish while being completely absent from medaka bone material. Note that the distal parts of the NS and HS were cropped in the original scans and are only drawn here for reference; therefore, these parts do not contain lacunae in the zebrafish rendering. Inset images show unsegmented tomography slices at a higher magnification. The vertebrae of both species are hourglass shaped along the cranio–caudal axis (see 3D representation in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000140#pbio.3000140.g003" target="_blank">Fig 3A</a>), with NS and HS extending caudally from the vertebral body. HS, hemal spine; NS, neural spine; O, osteocytic lacunae.</p