Bone is a complex hierarchically structured family of materials that includes a network of cells and their interconnected cell processes. New insights into the 3-D structure of various bone materials (mainly rat and human lamellar bone and minipig fibrolamellar bone) were obtained using a focused ion beam electron microscope and the serial surface view method. These studies revealed the presence of two different materials, the major material being the well-known ordered arrays of mineralized collagen fibrils and associated macromolecules, and the minor component being a relatively disordered material composed of individual collagen fibrils with no preferred orientation, with crystals inside and possibly between fibrils, and extensive ground mass. Significantly, the canaliculi and their cell processes are confined within the disordered material. Here we present a new hierarchical scheme for several bone tissue types that incorporates these two materials. The new scheme updates the hierarchical scheme presented by Weiner and Wagner (1998). We discuss the structures at different hierarchical levels with the aim of obtaining further insights into structure-function-related questions, as well as defining some remaining unanswered questions. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Fetal and postnatal bone development in humans is traditionally viewed as a process characterized by progressively increasing mineral density. Yet, a temporary decrease in mineral density has been described in the long bones of infants in the immediate postnatal period. The mechanism that underlies this phenomenon, as well as its causes and consequences, remain unclear. Using daily mu CT scans of murine femora and tibiae during perinatal development, we show that a temporary decrease in tissue mineral density (TMD) is evident in mice. By monitoring spatial and temporal structural changes during normal growth and in a mouse strain in which osteoclasts are non-functional (Src-null), we show that endosteal bone resorption is the main cause for the perinatal decrease in TMD. Mechanical testing revealed that this temporary decrease is correlated with reduced stiffness of the bones. We also show, by administration of a progestational agent to pregnant mice, that the decrease in TMD is not the result of parturition itself. This study provides a comprehensive View of perinatal long bone development in mice, and describes the process as well as the consequences of density fluctuation during this period. (C) 2012 Elsevier Inc. All rights reserved.

Bone includes cavities in various length scales, from nanoporosities occurring between the collagen fibrils and the mineral crystals all the way to macrocavities like the medullary cavity. In particular, bone is permeated by a vast number of channels (the lacunar-canalicular system), that reduce the stiffness and, more importantly, the strength of the bone that they permeate. These consequences are presumably a price worth paying for the ability of the lacunar-canalicular system to detect changes in the strain environment within the bone material and, when deleterious, to trigger processes like modeling or remodeling which 'rectify' it. Here we review the size and density of the various types of cavities in bone, and discuss their effect on the mechanical properties of cortical bone. 

In this respect the bones of advanced teleost fish species (probably the majority of all vertebrate species) are an unsolved conundrum because they lack bone cells (and therefore lacunae and canaliculi) in their skeleton. Yet, despite being acellular, some of these fish can undergo considerable remodeling in at least some parts of their skeleton. We address, but do not solve this mystery. (c) 2013 Elsevier Inc. All rights reserved.

The proinflammatory cytokine interleukin-1 (IL-1) signals through IL-1 receptor type I (IL-1RI) and induces osteoclastogenesis and bone resorption mainly during pathological conditions. Little is known about the effect of excess or absence of IL-1 signaling on the physiological development of the growth plate and bone. In this study, we examine growth plate morphology, bone structure, and mechanical properties as well as osteoclast number in IL-1RI knockout mice to evaluate the role of IL-1RI in the normal development of the growth plate and bone. We show for the first time that IL-1RI knockout mice have narrower growth plates due to a smaller hypertrophic zone, suggesting a role for this cytokine in hypertrophic differentiation, together with higher proteoglycan content. The bones of theses mice exhibit higher trabecular and cortical mass, increased mineral density, and superior mechanical properties. In addition, IL-1RI knockout mice have significantly reduced osteoclast numbers in the chondro-osseous junction, trabecular bone, and cortical bone. These results suggest that IL-1RI is involved in normal growth plate development and ECM homeostasis and that it is significant in the physiological process of bone modeling.

The objective of this study was to examine the effect of embryonic nutritional enrichment on the development and properties of broiler leg bones (tibia and femur) from the prenatal period until maturity. To accomplish the objective, 300 eggs were divided into 2 groups: a noninjected group (control) and a group injected in ovo with a solution containing minerals, vitamins, and carbohydrates (enriched). Tibia and femur from both legs were harvested from chicks on embryonic days 19 (E19) and 21 (E21) and d 3, 7, 14, 28, and 54 posthatch (n = 8). The bones were mechanically tested (stiffness, maximal load, and work to fracture) and scanned in a micro-computed tomography (mu CT) scanner to examine the structural properties of the cortical [cortical area, medullary area, cortical thickness, and maximal moment of inertia (I-max)] and trabecular (bone volume percent, trabecular thickness, and trabecular number) areas. To examine bone mineralization, bone mineral density (BMD) of the cortical area was obtained from the mu CT scans, and bones were analyzed for the ash and mineral content. The results showed improved mechanical properties of the enriched group between E19 and d 3 and on d 14 (P < 0.05). Differences in cortical morphology were noted between E19 and d 14 as the enriched group had greater medullary area on E19 (femur), reduced medullary area on E21 (both bones), greater femoral cortical area on d 3, and greater I-max of both bones on d 14 (P < 0.05). The major differences in bone trabecular architecture were that the enriched group had greater bone volume percent and trabecular thickness in the tibia on d 7 and the femur on d 28 (P < 0.05). The pattern of mineralization between E19 and d 54 showed improved mineralization in the enriched group on E19 whereas on d 3 and 7, the control group showed a mineralization advantage, and on d 28 and 54, the enriched group showed again greater mineralization (P < 0.05). In summary, this study demonstrated that in ovo enrichment affects multiple bone properties pre- and postnatally and showed that avian embryos are a good model for studying the effect of embryonic nutrition on natal and postnatal development. Most importantly, the enrichment led to improved mechanical properties until d 14 (roughly third of the lifespan of the bird), a big advantage for the young broiler. Additionally, the improved mineralization and trabecular architecture on d 28 and 54 indicate a potential long-term effect of altering embryonic nutrition.

One of the hallmarks of tetrapod bone is the presence of numerous cells (osteocytes) within the matrix. Osteocytes are
vital components of tetrapod bone, orchestrating the processes of bone building, reshaping and repairing (modeling and
remodeling), and probably also participating in calcium-phosphorus homeostasis via both the local process of
osteocytic osteolysis, and systemic effecton the kidneys. Given these critical roles of osteocytes, it is thought-provoking
that the entire skeleton of many fishes consists of bone material that does not contain osteocytes. This raises the
intriguing question of how the skeleton of these animals accomplishes the various essential functions attributed to
osteocytes in other vertebrates, and raises the possibility that in acellular bone some of these functions are either
accomplished by non-osteocytic routes or not necessary at all. In this review,we outline evidence for and against the fact
that primary functions normally ascribed to osteocytes, such as mechanosensation, regulation of osteoblast/clast
activity and mineral metabolism, also occur in fish bone devoid of these cells, and therefore must be carried out through
alternative and perhaps ancient pathways. To enable meaningful comparisons with mammalian bone, we suggest
thorough, phylogenetic examinations of regulatory pathways, studies of structure and mechanical properties and
surveys of the presence/absence of bone cells in fishes. Insights gained into the micro-/nanolevel structure and
architecture of fish bone, its mechanical properties and its physiology in health and disease will contribute to the
discipline of fish skeletal biology, but may also help answer questions of basic bone biology.

Lamellar bone is a major component of most mammalian skeletons. A prominent component of individual lamellae are parallel arrays of mineralized type I collagen fibrils, organized in a plywood like motif. Here we use a dual beam microscope and the serial surface view (SSV) method to investigate the three dimensional collagen organization of circumferential lamellar bone from rat tibiae after demineralization and osmium staining. Fast Fourier transform analysis is used to quantitatively identify the mean collagen array orientations and local collagen fibril dispersion. Based on collagen fibril array orientations and variations in fibril dispersion, we identify 3 distinct sub-lamellar structural motifs: a plywood-like fanning sub-lamella, a unidirectional sub-lamella and a disordered sub-lamella. We also show that the disordered sub-lamella is less mineralized than the other sub-lamellae. The hubs and junctions of the canalicular network, which connect radially oriented canaliculi, are intimately associated with the disordered sub-lamella. We also note considerable variations in the proportions of these 3 sub-lamellar structural elements among different lamellae. This new application of Serial Surface View opens the way to quantitatively compare lamellar bone from different sources, and to clarify the 3-dimensional structures of other bone types, as well as other biological structural materials. (C) 2012 Elsevier Inc. All rights reserved.

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 mu m 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. (C) 2012 Elsevier Inc. All rights reserved.

The laboratory rat is one of the most frequently-used animal models for studying bone biology and skeletal diseases. Here we show that a substantial portion of the cortical bone of mature rats is primary endochondral bone, consisting of a disorganized arrangement of mineralized collagen fibers. We characterize the structure and mechanical properties of the cortical bone of the rat. We show that the cortical bone consists of two architecturally distinct regions. One region, consisting of well-organized circumferential lamellae (CLB), is located in the endosteal and/or the periosteal regions while another, disorganized region, is located in the more central region of the cortex. Unexpectedly, we found that the disorganized region contains many islands of highly mineralized cartilage. 

Micro tomography showed different structural and compositional properties of the two primary structural elements; the CLB region has lower mineral density, lower porosity, larger but fewer blood vessels and fewer lacunae. However, no difference was found in the average lacunar volume. Additionally the mean indentation modulus of the CLB region was lower than that of the disorganized region. The islands of calcified cartilage were found to be extremely stiff, with an indentation modulus of 33.4 +/- 3.5 GPa. 

We conclude that though the cortical bone of rats is in part lamellar, its architecture is markedly different from that of the cortical bone of humans, a fact that must be borne in mind when using the rat as a model animal for studies of human bone biology and disease. (c) 2013 Elsevier Inc. All rights reserved.

Designed peptides may generate molecular scaffolds in the form of hydrogels to support tissue regeneration. We studied the effect of hydrogels comprising beta-sheet-forming peptides rich in aspartic amino acids and of tricalcium phosphate (beta-TCP)-loaded hydrogels on calcium adsorption and cell culture in vitro, and on bone regeneration in vivo. The hydrogels were found to act as efficient depots for calcium ions, and to induce osteoblast differentiation in vitro. In vivo studies on bone defect healing in rat distal femurs analyzed by microcomputerized tomography showed that the peptide hydrogel itself induced better bone regeneration in comparison to non-treated defects. A stronger regeneration capacity was obtained in bone defects treated with beta-TCP-loaded hydrogels, indicating that the peptide hydrogels and the mineral act synergistically to enhance bone regeneration. In vivo regeneration was found to be better with hydrogels loaded with porous beta-TCP than with hydrogels loaded with non-porous mineral. It is concluded that biocompatible and biodegradable matrices, rich in anionic moieties that efficiently adsorb calcium ions while supporting cellular osteogenic activity, may efficiently promote beta-TCP turnover into bone mineral. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The development of broilers is an extreme example of rapid growth, increasing in weight from 40 g at hatch to 2,000 g 5 to 6 wk later. Such rapid growth requires a correspondingly fast development of the skeleton.Bone development is a genetically programmed process that is modified by epigenetic factors, mainly muscle-induced stresses and strains. In this study, we describe the temporal changes in bone morphology and material properties during the prehatch period [embryonic day (E) 14, E17, E19, E21] and posthatch d 3 and 7. The bones were examined for their weight, length, ash content, mechanical properties, and cortical structure. We show that the cross-sectional shape of the tibia and femur changes during the examination period from circular to elliptical. Additionally, the changes in bone properties are time-dependent and nonuniform: from E14 to E17 and from d 3 to 7, fast bone growth was noted, with major increases in both mechanical properties (stiffness, ultimate load, and energy to fracture) and geometric properties (cross-sectional area and thickness, medullary area, and moment of inertia). On the other hand, during the last days of incubation, most mechanical and geometric properties remain unchanged or even decrease. The reasons for this finding may relate to the hatching process but also to mineral shortage during the last daysof incubation. This study leads to better understanding of bone development in ovo and posthatch in fast-growing broilers.

Maternal malnutrition during pregnancy was shown by numerous studies to result in the birth of offspring exhibiting altered bone characteristics, which are indicative of bone loss. We hypothesized that not only maternal malnutrition but also some developmental toxicants (teratogens) given at a dose inducing neither structural anomalies nor growth retardation can detrimentally affect skeletal health in adult offspring. To check this hypothesis, pregnant mice were exposed to a single injection of 5-aza-2-deoxycytidine (5-AZA) (a teratogen capable of inducing phocomelia of the hind limbs) at a sub-threshold teratogenic dose. Micro-computed tomography scanning revealed that femora of 5-month-old male offspring exposed in uterus to 5-AZA had trabecular microarchitecture indicative of bone loss. Furthermore, exposure to 5-AZA increased the susceptibility of offspring to postnatal chronic mild stress, which has been shown to induce bone loss in mice. While exploring possible mechanisms underlying this phenomenon, we observed that the expression of some microRNAs, which have been demonstrated as regulators of key osteo-blastogenic genes, was altered in hind limb buds of embryos exposed to 5-AZA. Furthermore, the expression of receptor activator of nuclear factor kappa B ligand (RANKL) in femoral stromal/osteoblastic cells of 5-month-old offspring of 5-AZA-treated females was found to be increased. Collectively, this study implies for the first time that single low-dose exposure to a teratogen can induce bone loss in adult offspring, possibly via alteration of embryonic microRNAs and RANKL expression.

The histological diversity of the skeletal tissues of fishes is impressive compared with that of other vertebrate groups, yet our understanding of the functional consequences of this diversity is limited. In particular, although it has been known since the mid-1800s that a large number of fish species possess acellular bones, the mechanical advantages and consequences of this structural characteristic - and therefore the nature of the evolution of this feature - remain unclear. Although several studies have examined the material properties of fish bone, these have used a variety of techniques and there have been no direct contrasts of acellular and cellular bone. We report on a comparison of the structural and mechanical properties of the ribs and opercula between two freshwater fish - the common carp Cyprinus carpio (a fish with cellular bone) and the tilapia Oreochromis aureus (a fish with acellular bone). We used light microscopy to show that the bones in both fish species exhibit poor blood supply and possess discrete tissue zones, with visible layering suggesting differences in the underlying collagen architecture. We performed identical micromechanical testing protocols on samples of the two bone types to determine the mechanical properties of the bone material of opercula and ribs. Our data support the consensus of literature values, indicating that Young's moduli of cellular and acellular bones are in the same range, and lower than Young's moduli of the bones of mammals and birds. Despite these similarities in mechanical properties between the bone tissues of the fish species tested here, cellular bone had significantly lower mineral content than acellular bone; furthermore, the percentage ash content and bone mineral density values (derived from micro-CT scans) show that the bone of these fishes is less mineralized than amniote bone. Although we cannot generalize from our data to the numerous remaining teleost species, the results presented here suggest that while cellular and acellular fish bone may perform similarly from a mechanical standpoint, there are previously unappreciated differences in the structure and composition of these bone types.

The mechanical properties of rat bone at micron length scales have been evaluated as a function of environmental conditions using an in situ atomic force microscope (AFM) setup while observing using scanning electron microscopy (SEM). Focused ion beam fabricated rat bone cantilever samples were tested in both low and high vacuum conditions in the SEM as well as wet in air using the AFM to measure their elastic modulus. The elastic modulus of rat bone at micron length scales is shown to be independent of the environmental testing conditions and indicates water is bound to bone material even under relatively high vacuum conditions. Our work therefore shows how in situ mechanical testing of bone while observing using high resolution SEM can provide results similar to testing wet in air. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.

Although fish species represent probably about half of all vertebrate species, the structure and material properties of fish skeletons are poorly understood. Since osteocytes (cells within the bone tissue) are believed to orchestrate the modeling and remodeling responses in mammalian bone, the unique lack of these cells in the bones of the majority of extant fishes (neoteleosts), raises several intriguing questions. In particular, how do their bones handle forces, especially those applied repeatedly and over long periods of time. This paper reviews the available information regarding the structure-function relationship of teleost bones namely their known structural features, mechanical properties and response to load with a particular focus on the acellular bone of neoteleosts. We present preliminary results of ongoing investigations in these areas and highlight topics (e.g. mechanisms of tissue fatigue, repair and resistance to high strain rates) we believe particularly ripe for and demanding further investigation.

Teeth sustain high loads over a lifetime and yet intact tooth failure is rare. The different structures of the tooth, jaw bone and the intervening soft periodontal ligament enable the tooth to endure repeated loading during mastication. Although mechanical and functional properties of the different components are thoroughly investigated, the manner in which the whole tooth functions under load is still enigmatic. A custom-made loading system inside a microCT scanner was used to directly visualize the root movements in relation to the jaw bone as the rat molar tooth was loaded. At low loads no contact was observed between the root surface and the bone, whereas at higher loads three specific contact areas between the root surface and the jaw bone were observed. These contact areas restrict tooth movement in the buccal-lingual direction, but enable the tooth to rock in a "seesaw" like manner in the distal-mesial direction. The contact areas appear to play a role in determining tooth motion and in turn define the manner in which the whole tooth moves when loaded. These observations are important for understanding basic structure-function relations of the tooth-PDL-bone system, and have direct implications for better understanding pathological and therapeutic processes in orthodontics, periodontics and jaw bone regeneration. (C) 2011 Elsevier Inc. All rights reserved.

The components of the tooth-periodontal ligament (PDL)-alveolar bone complex act in a synergistic manner to dissipate the loads incurred during mastication. The complex incorporates a diverse array of structural features for this purpose. These include the non-mineralized and hence soft PDL that absorbs much of the initial loads. The internal structure of the tooth also includes soft interphases that essentially surround the dentine core. These interphases, although stiffer than the PDL, still are more compliant than the dentine core, and are thus key components that allow the tooth itself to deform and hence help dissipate the compressive loads. There is also direct evidence that even under moderate compressive loads, when the tooth moves in the alveolar bone socket, this movement is guided by specific locations where the tooth comes into contact with the bone surface. The combination of all these responses to load is that each tooth type appears to move and deform in a specific manner when loaded. Much, however, still remains to be learned about these three-dimensional responses to load and the factors that control them. Such an understanding will have major implications for dentistry, that include a better understanding of phenomena such as abfraction, the manner in which tooth implants function even in the absence of a PDL-like tissue and the implications to bone remodelling of the movements imposed during orthodontic interventions. (C) 2012 Elsevier Ltd. All rights reserved.

Whereas detrimental effects of vitamin D deficiency are known over century, the effects of vitamin D receptor activation by 1,25(OH)2D3, the principal hormonal form of vitamin D, on the growing bone and its growth plate are less clear. Currently, 1,25(OH)2D3 is used in pediatric patients with chronic kidney disease and mineral and bone disorder (CKD-MBD) and is strongly associated with growth retardation. Here, we investigate the effect of 1,25(OH)2D3 treatment on bone development in normal young rats, unrelated to renal insufficiency. Young rats received daily i.p. injections of 1 µg/kg 1,25(OH)2D3 for one week, or intermittent 3 µg/kg 1,25(OH)2D3 for one month. Histological analysis revealed narrower tibial growth plates, predominantly in the hypertrophic zone of 1,25(OH)2D3-treated animals in both experimental protocols. This phenotype was supported by narrower distribution of aggrecan, collagens II and X mRNA, shown by in situ hybridization. Concomitant with altered chondrocyte maturation, 1,25(OH)2D3 increased chondrocyte proliferation and apoptosis in terminal hypertrophic cells. In vitro treatment of the chondrocytic cell line ATDC5 with 1,25(OH)2D3 lowered differentiation and increased proliferation dose and time-dependently. Micro-CT analysis of femurs from 1-week 1,25(OH)2D3-treated group revealed reduced cortical thickness, elevated cortical porosity, and higher trabecular number and thickness. 1-month administration resulted in a similar cortical phenotype but without effect on trabecular bone. Evaluation of fluorochrome binding with confocal microscopy revealed inhibiting effects of 1,25(OH)2D3 on intracortical bone formation. This study shows negative effects of 1,25(OH)2D3 on growth plate and bone which may contribute to the exacerbation of MBD in the CKD pediatric patients.

The blood supply of bone cells in compact bone is provided primarily by blood vessels located within Haversian canals forming the centre of osteons. Mid-diaphysial cross-sections of radii and third metacarpal bones from two horses and radii from two mature dogs were studied using reflective light microscopy to quantify the spatial ordering of canals and compared to a computational model. The distributions of canals were analyzed using: 1) the autocorrelation function (ACF), which describes the probability of finding two canals separated by a given distance and 2) the shortest distance distribution (SDD), which describes the probability that a site within bone is located at a given distance from the nearest canal. The order in the investigated horse radii, as characterized by the oscillations of the ACF, was found to be independent of the anatomical location although, in the metacarpal bone the order was higher in the lateral than in the cranial location. Among the dogs, marked differences were only found in the ACF. An analysis of the SDD demonstrates that ordering of canals minimizes the distance of osteocytes from a blood vessel. This suggests that the efficiency of the blood supply can be adapted through differences in the order of the Haversian canals. In our model, the ordering of canals is achieved via an exclusion zone around each canal, imposed upon newly formed osteons. Simulations demonstrate that differences in the observed order can be explained by either a larger size or a larger variability of this exclusion zone. Anat Rec, 294:1093-1102, 2011. (C) 2011 Wiley-Liss, Inc.

Objective: Skeletal abnormalities are one of the hallmarks of growth delay during gestation. The aim of this study was to determine changes induced by leptin in skeletal growth and development in a rat model of intrauterine growth retardation (IUGR) and to elucidate the possible underlying mechanisms. 

Methods: Intrauterine growth retardation was induced prepartum and the effects of leptin to mothers prenatally or to offspring postnatally were studied. Radii were harvested and tested mechanically and structurally. Tibias were evaluated for growth-plate morphometry. 

Results: On day 40 postpartum, total bone length and mineral density and tibial growth-plate width and numbers of cells within its zones of offspring treated with leptin were significantly greater than in the control group. 

Conclusion: Postnatal leptin administration in an IUGR model improves the structural properties and elongation rate of bone. These findings could pave the way to preventing some phenotypic presentations of IUGR. (C) 2011 Elsevier Inc. All rights reserved.