When Cortical Bone Matrix Properties Are Indiscernible between Elderly Men with and without Type 2 Diabetes, Fracture Resistance Follows Suit.

Type 2 diabetes mellitus (T2DM) is a metabolic disease affecting bone tissue and leading to increased fracture risk in men and women, independent of bone mineral density (BMD). Thus, bone material quality (i.e., properties that contribute to bone toughness but are not attributed to bone mass or quantity) is suggested to contribute to higher fracture risk in diabetic patients and has been shown to be altered. Fracture toughness properties are assumed to decline with aging and age-related disease, while toughness of human T2DM bone is mostly determined from compression testing of trabecular bone. In this case-control study, we determined fracture resistance in T2DM cortical bone tissue from male individuals in combination with a multiscale approach to assess bone material quality indices. All cortical bone samples stem from male nonosteoporotic individuals and show no significant differences in microstructure in both groups, control and T2DM. Bone material quality analyses reveal that both control and T2DM groups exhibit no significant differences in bone matrix composition assessed with Raman spectroscopy, in BMD distribution determined with quantitative back-scattered electron imaging, and in nanoscale local biomechanical properties assessed via nanoindentation. Finally, notched three-point bending tests revealed that the fracture resistance (measured from the total, elastic, and plastic J-integral) does not significantly differ in T2DM and control group, when both groups exhibit no significant differences in bone microstructure and material quality. This supports recent studies suggesting that not all T2DM patients are affected by a higher fracture risk but that individual risk profiles contribute to fracture susceptibility, which should spur further research on improving bone material quality assessment in vivo and identifying risk factors that increase bone fragility in T2DM. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dimorphic Mechanisms of Fragility in Diabetes Mellitus: the Role of Reduced Collagen Fibril Deformation.

Diabetes mellitus (DM) is an emerging metabolic disease, and the management of diabetic bone disease poses a serious challenge worldwide. Understanding the underlying mechanisms leading to high fracture risk in DM is hence of particular interest and urgently needed to allow for diagnosis and treatment optimization. In a case-control postmortem study, the whole 12th thoracic vertebra and cortical bone from the mid-diaphysis of the femur from male individuals with type 1 diabetes mellitus (T1DM) (n = 6; 61.3 ± 14.6 years), type 2 diabetes mellitus (T2DM) (n = 11; 74.3 ± 7.9 years), and nondiabetic controls (n = 18; 69.3 ± 11.5) were analyzed with clinical and ex situ imaging techniques to explore various bone quality indices. Cortical collagen fibril deformation was measured in a synchrotron setup to assess changes at the nanoscale during tensile testing until failure. In addition, matrix composition was analyzed including determination of cross-linking and non-crosslinking advanced glycation end-products like pentosidine and carboxymethyl-lysine. In T1DM, lower fibril deformation was accompanied by lower mineralization and more mature crystalline apatite. In T2DM, lower fibril deformation concurred with a lower elastic modulus and tendency to higher accumulation of non-crosslinking advanced glycation end-products. The observed lower collagen fibril deformation in diabetic bone may be linked to altered patterns mineral characteristics in T1DM and higher advanced glycation end-product accumulation in T2DM. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

3D Image Registration Marginally Improves the Precision of HR-pQCT Measurements Compared to Cross-Sectional-Area Registration in Adults With Osteogenesis Imperfecta.

Repositioning error in longitudinal high-resolution peripheral-quantitative computed tomography (HR-pQCT) imaging can lead to different bone volumes being assessed over time. To identify the same bone volumes at each time point, image registration is used. While cross-sectional area image registration corrects axial misalignment, 3D registration additionally corrects rotations. Other registration methods involving matched angle analysis (MA) or boundary transformations (3D-TB) can be used to limit interpolation error in 3D-registering micro-finite-element data. We investigated the effect of different image registration methods on short-term in vivo precision in adults with osteogenesis imperfecta, a collagen-related genetic disorder resulting in low bone mass, impaired quality, and increased fragility. The radii and tibiae of 29 participants were imaged twice on the same day with full repositioning. We compared the precision error of different image registration methods for density, microstructural, and micro-finite-element outcomes with data stratified based on anatomical site, motion status, and scanner generation. Regardless of the stratification, we found that image registration improved precision for total and trabecular bone mineral densities, trabecular and cortical bone mineral contents, area measurements, trabecular bone volume fraction, separation, and heterogeneity, as well as cortical thickness and perimeter. 3D registration marginally outperformed cross-sectional area registration for some outcomes, such as trabecular bone volume fraction and separation. Similarly, precision of micro-finite-element outcomes was improved after image registration, with 3D-TB and MA methods providing greatest improvements. Our regression model confirmed the beneficial effect of image registration on HR-pQCT precision errors, whereas motion had a detrimental effect on precision even after image registration. Collectively, our results indicate that 3D registration is recommended for longitudinal HR-pQCT imaging in adults with osteogenesis imperfecta. Since our precision errors are similar to those of healthy adults, these results can likely be extended to other populations, although future studies are needed to confirm this. © 2022 American Society for Bone and Mineral Research (ASBMR).

Potential Role of Perilacunar Remodeling in the Progression of Osteoporosis and Implications on Age-Related Decline in Fracture Resistance of Bone.

PURPOSE OF REVIEW: We took an interdisciplinary view to examine the potential contribution of perilacunar/canalicular remodeling to declines in bone fracture resistance related to age or progression of osteoporosis. RECENT FINDINGS: Perilacunar remodeling is most prominent as a result of lactation; recent advances further elucidate the molecular players involved and their effect on bone material properties. Of these, vitamin D and calcitonin could be active during aging or osteoporosis. Menopause-related hormonal changes or osteoporosis therapies affect bone material properties and mechanical behavior. However, investigations of lacunar size or osteocyte TRAP activity with age or osteoporosis do not provide clear evidence for or against perilacunar remodeling. While the occurrence and potential role of perilacunar remodeling in aging and osteoporosis progression are largely under-investigated, widespread changes in bone matrix composition in OVX models and following osteoporosis therapies imply osteocytic maintenance of bone matrix. Perilacunar remodeling-induced changes in bone porosity, bone matrix composition, and bone adaptation could have significant implications for bone fracture resistance.

Multisite longitudinal calibration of HR-pQCT scanners and precision in osteogenesis imperfecta.

BACKGROUND: For high-resolution peripheral quantitative computed tomography (HR-pQCT) to be used in longitudinal multi-center studies to assess disease and treatment effects, data must be aggregated across multiple timepoints and scanners. This requires an understanding of the factors contributing to scanner precision, and multi-scanner cross-calibration procedures, especially for clinical populations with severe phenotypes, like osteogenesis imperfecta (OI). METHODS: To address this, we first evaluated single- and multi-center short- and long-term precision errors of standard HR-pQCT parameters. Two imaging phantoms were circulated among 13 sites (7 XtremeCT and 6 XtremeCT2) and scanned in triplicate at 3 timepoints/site. Additionally, duplicate in vivo radial and tibial scans were acquired in 29 individuals with OI. Secondly, we investigated subject- and scanner-related factors that contribute to precision errors using regression analysis. Thirdly, we proposed a reference site selection criterion for multisite cross-calibration and demonstrated the external validity of phantom-based calibrations. RESULTS: Our results show excellent short-term single-site precision in both phantoms (CV % < 0.5%) and in density, microarchitecture and finite element parameters of OI participants (CV % = 0.75 to 1.2%). In vivo reproducibility significantly improved with (i) cross sectional area image registration versus no registration and (ii) scans with no motion artifacts. While reproducibility was similar across OI subtypes and anatomical sites, XtremeCT2 scanners achieved ~2.5% better precision than XtremeCT for trabecular parameters. Finally, we demonstrate that multisite longitudinal precision errors resulting from inconsistencies between scanners can be partially corrected through scanner cross-calibration. CONCLUSIONS: This study is the first to assess long-term reproducibility and cross-calibration in a study using first and second generation HR-pQCT scanners. The results presented in this context provide timely guidelines for future use of this powerful clinical imaging modality in multi-center longitudinal clinical trials.

Breaking new ground in mineralized tissue: Assessing tissue quality in clinical and laboratory studies.

Mineralized tissues, such as bone and teeth, have extraordinary mechanical properties of both strength and toughness. This mechanical behavior originates from deformation and fracture resistance mechanisms in their multi-scale structure. The term quality describes the matrix composition, multi-scale structure, remodeling dynamics, water content, and micro-damage accumulation in the tissue. Aging and disease result in changes in the tissue quality that may reduce strength and toughness and lead to elevated fracture risk. Therefore, the capability to measure the quality of mineralized tissues provides critical information on disease progression and mechanical integrity. Here, we provide an overview of clinical and laboratory-based techniques to assess the quality of mineralized tissues in health and disease. Current techniques used in clinical settings include radiography-based (radiographs, dual energy x-ray absorptiometry, EOS) and x-ray tomography-based methods (high resolution peripheral quantitative computed tomography, cone beam computed tomography). In the laboratory, tissue quality can be investigated in ex vivo samples with x-ray imaging (micro and nano-computed tomography, x-ray microscopy), electron microscopy (scanning/transmission electron imaging (SEM/STEM), backscattered scanning electron microscopy, Focused Ion Beam-SEM), light microscopy, spectroscopy (Raman spectroscopy and Fourier transform infrared spectroscopy) and assessment of mechanical behavior (mechanical testing, fracture mechanics and reference point indentation). It is important for clinicians and basic science researchers to be aware of the techniques available in different types of research. While x-ray imaging techniques translated to the clinic have provided exceptional advancements in patient care, the future challenge will be to incorporate high-resolution laboratory-based bone quality measurements into clinical settings to broaden the depth of information available to clinicians during diagnostics, treatment and management of mineralized tissue pathologies.

Bone adaptation: Safety factors and load predictability in shaping skeletal form.

Much is known about skeletal adaptation in relation to the mechanical functions that bones serve. This includes how bone adapts to mechanical loading during an individual's lifetime as well as over evolutionary time. Although controlled loading in animal models allows us to observe short-term bone adaptation (epigenetic mechanobiology), examining an assemblage of extant vertebrate bones or a group of fossils' bony structures can reveal the combined effects of long-term trends in loading history and the effects of natural selection. In this survey we examine adaptations that take place over both time scales and highlight a few of the extraordinary insights first published by John Currey. First, we provide a historical perspective on bone adaptation control mechanisms, followed by a discussion of safety factors in bone. We then summarize examples of structural- and material-level adaptations and mechanotransduction, and analyze the relationship between these structural- and material-level adaptations observed in situations where loading modes are either predictable or unpredictable. We argue that load predictability is a major consideration for bone adaptation broadly across an evolutionary timescale, but that its importance can also be seen during ontogenetic growth trajectories, which are subject to natural selection as well. Furthermore, we suggest that bones with highly predictable load patterns demonstrate more precise design with lower safety factors, while bones that experience less predictable loads or those that are less capable of repair and adaptation are designed with a higher safety factor. Finally, exposure to rare loading events with high potential costs of failure leads to design of structures with very high safety factor compared to everyday loading experience. Understanding bone adaptations at the structural and material levels, which take place over an individual's lifetime or over evolutionary time has numerous applications in translational and clinical research to understand and treat musculoskeletal diseases, as well as to permit the furthering of human extraterrestrial exploration in environments with altered gravity.

On the Origins of Fracture Toughness in Advanced Teleosts: How the Swordfish Sword's Bone Structure and Composition Allow for Slashing under Water to Kill or Stun Prey.

The osseous sword of a swordfish (Xiphias gladius) is specialized to incapacitate prey with stunning blows. Considering the sword's growth and maturation pattern, aging from the sword's base to the tip, while missing a mechanosensitive osteocytic network, an in-depth understanding of its mechanical properties and bone quality is lacking. Microstructural, compositional, and nanomechanical characteristics of the bone along the sword are investigated to reveal structural mechanisms accounting for its exceptional mechanical competence. The degree of mineralization, homogeneity, and particle size increase from the base toward the tip, reflecting aging along its length. Fracture experiments reveal that crack-growth toughness vastly decreases at the highly and homogeneously mineralized tip, suggesting the importance of aging effects. Initiation toughness, however, is unchanged suggesting that aging effects on this hierarchical level are counteracted by constant mineral/fibril interaction. In conclusion, the sword of the swordfish provides an excellent model reflecting base-to-tip-wise aging of bone, as indicated by increasing mineralization and decreasing crack-growth toughness toward the tip. The hierarchical, structural, and compositional changes along the sword reflect peculiar prerequisites needed for resisting high mechanical loads. Further studies on advanced teleosts bone tissue may help to unravel structure-function relationships of heavily loaded skeletons lacking mechanosensing cells.

Mechanical Competence and Bone Quality Develop During Skeletal Growth.

Bone fracture risk is influenced by bone quality, which encompasses bone's composition as well as its multiscale organization and architecture. Aging and disease deteriorate bone quality, leading to reduced mechanical properties and higher fracture incidence. Largely unexplored is how bone quality and mechanical competence progress during longitudinal bone growth. Human femoral cortical bone was acquired from fetal (n = 1), infantile (n = 3), and 2- to 14-year-old cases (n = 4) at the mid-diaphysis. Bone quality was assessed in terms of bone structure, osteocyte characteristics, mineralization, and collagen orientation. The mechanical properties were investigated by measuring tensile deformation at multiple length scales via synchrotron X-ray diffraction. We find dramatic differences in mechanical resistance with age. Specifically, cortical bone in 2- to 14-year-old cases exhibits a 160% greater stiffness and 83% higher strength than fetal/infantile cases. The higher mechanical resistance of the 2- to 14-year-old cases is associated with advantageous bone quality, specifically higher bone volume fraction, better micronscale organization (woven versus lamellar), and higher mean mineralization compared with fetal/infantile cases. Our study reveals that bone quality is superior after remodeling/modeling processes convert the primary woven bone structure to lamellar bone. In this cohort of female children, the microstructural differences at the femoral diaphysis were apparent between the 1- to 2-year-old cases. Indeed, the lamellar bone in 2- to 14-year-old cases had a superior structural organization (collagen and osteocyte characteristics) and composition for resisting deformation and fracture than fetal/infantile bone. Mechanistically, the changes in bone quality during longitudinal bone growth lead to higher fracture resistance because collagen fibrils are better aligned to resist tensile forces, while elevated mean mineralization reinforces the collagen scaffold. Thus, our results reveal inherent weaknesses of the fetal/infantile skeleton signifying its inferior bone quality. These results have implications for pediatric fracture risk, as bone produced at ossification centers during children's longitudinal bone growth could display similarly weak points. © 2019 American Society for Bone and Mineral Research.

Mechanically stimulated ATP release from murine bone cells is regulated by a balance of injury and repair.

Bone cells sense and actively adapt to physical perturbations to prevent critical damage. ATP release is among the earliest cellular responses to mechanical stimulation. Mechanical stimulation of a single murine osteoblast led to the release of 70 ± 24 amole ATP, which stimulated calcium responses in neighboring cells. Osteoblasts contained ATP-rich vesicles that were released upon mechanical stimulation. Surprisingly, interventions that promoted vesicular release reduced ATP release, while inhibitors of vesicular release potentiated ATP release. Searching for an alternative ATP release route, we found that mechanical stresses induced reversible cell membrane injury in vitro and in vivo. Ca2+/PLC/PKC-dependent vesicular exocytosis facilitated membrane repair, thereby minimizing cell injury and reducing ATP release. Priming cellular repair machinery prior to mechanical stimulation reduced subsequent membrane injury and ATP release, linking cellular mechanosensitivity to prior mechanical exposure. Thus, our findings position ATP release as an integrated readout of membrane injury and repair.

Sclerostin Neutralizing Antibody Treatment Enhances Bone Formation but Does Not Rescue Mechanically Induced Delayed Healing.

During bone healing, tissue formation processes are governed by mechanical strain. Sost/sclerostin, a key Wnt signaling inhibitor and mechano-sensitive pathway, is downregulated in response to mechanical loading. Sclerostin neutralizing antibody (SclAb) increases bone formation. Nevertheless, it remains unclear whether sclerostin inhibition can rescue bone healing in situations of mechanical instability, which otherwise delay healing. We investigated SclAb's influence on tissue formation in a mouse femoral osteotomy, stabilized with rigid or semirigid external fixation. The different fixations allowed different magnitudes of interfragmentary movement during weight bearing, thereby influencing healing outcome. SclAb or vehicle (veh) was administeredand bone healing was assessed at multiple time points up to day 21 postoperatively by in vivo micro-computed tomography, histomorphometry, biomechanical testing, immunohistochemistry, and gene expression. Our results show that SclAb treatment caused a greater bone volume than veh. However, SclAb could not overcome the characteristic delayed healing of semirigid fixation. Indeed, semirigid fixation resulted in delayed healing with a prolonged endochondral ossification phase characterized by increased cartilage, lower bone volume fraction, and less bony bridging across the osteotomy gap than rigid fixation. In a control setting, SclAb negatively affected later stages of healing under rigid fixation, evidenced by the high degree of endosteal bridging at 21 days in the rigid-SclAb group compared with rigid-veh, indicating delayed fracture callus remodeling and bone marrow reconstitution. Under rigid fixation, Sost and sclerostin expression at the gene and protein level, respectively, were increased in SclAb compared with veh-treated bones, suggesting a negative feedback mechanism. Our results suggest that SclAb could be used to enhance overall bone mass but should be carefully considered in bone healing. SclAb may help to increase bone formation early in the healing process but not during advanced stages of fracture callus remodeling and not to overcome delayed healing in semirigid fixation. © 2018 American Society for Bone and Mineral Research.

Inter-site variability of the osteocyte lacunar network in the cortical bone underpins fracture susceptibility of the superolateral femoral neck.

BACKGROUND: The osteocytic lacunar network is considered to be an integral player in the regulation of bone homeostasis, and reduction in osteocytes is associated with reduced bone strength. Here, we analyzed site-specific patterns in osteocyte characteristics and matrix composition in the cortical compartment of the femoral neck to reveal the structural basis of its fragility. METHODS: Cross-sections of the human femoral neck - one of the most common fracture sites - were acquired from 12 female cadavers (age 34-86 years) and analyzed with backscattered scanning electron microscopy and high-resolution micro-computed tomography (µ-CT). The 2D/3D density and size of the osteocyte lacunae as well as bone mineral density distribution (BMDD) were measured in two regions subject to different biomechanical loads in vivo: the inferomedial (medial) region (habitually highly loaded in compression) and the superolateral (lateral) region (lower habitual loading intensity). Using quantitative polarized light microscopy, collagen fiber orientation was quantified in these two regions, accordingly. RESULTS: In 2D measurements, the inferomedial region displayed lower mineralization heterogeneity, 19% higher osteocyte lacunar density (p = 0.005), but equal lacunar size compared to the superolateral region. 3D measurements confirmed a significantly higher osteocyte lacunar density in the inferomedial region (p = 0.015). Osteocyte lacunar density decreased in aged individuals, and inter-site differences were reduced. Site-specific osteocyte characteristics were not accompanied by changes in collagen fiber orientation. CONCLUSIONS: Since osteocyte characteristics may provide valuable insights into bone mechanical competence, the variations in osteocyte properties might reflect the increased fracture susceptibility of the superolateral neck.

In situ deformation of growth plate chondrocytes in stress-controlled static vs dynamic compression.

Longitudinal bone growth in children/adolescents occurs through endochondral ossification at growth plates and is influenced by mechanical loading, where increased compression decreases growth (i.e., Hueter-Volkmann Law). Past in vivo studies on static vs dynamic compression of growth plates indicate that factors modulating growth rate might lie at the cellular level. Here, in situ viscoelastic deformation of hypertrophic chondrocytes in growth plate explants undergoing stress-controlled static vs dynamic loading conditions was investigated. Growth plate explants from the proximal tibia of pre-pubertal rats were subjected to static vs dynamic stress-controlled mechanical tests. Stained hypertrophic chondrocytes were tracked before and after mechanical testing with a confocal microscope to derive volumetric, axial and lateral cellular strains. Axial strain in hypertrophic chondrocytes was similar for all groups, supporting the mean applied compressive stress's correlation with bone growth rate and hypertrophic chondrocyte height in past studies. However, static conditions resulted in significantly higher lateral (p<0.001) and volumetric cellular strains (p≤0.015) than dynamic conditions, presumably due to the growth plate's viscoelastic nature. Sustained compression in stress-controlled static loading results in continued time-dependent cellular deformation; conversely, dynamic groups have less volumetric strain because the cyclically varying stress limits time-dependent deformation. Furthermore, high frequency dynamic tests showed significantly lower volumetric strain (p=0.002) than low frequency conditions. Mechanical loading protocols could be translated into treatments to correct or halt progression of bone deformities in children/adolescents. Mimicking physiological stress-controlled dynamic conditions may have beneficial effects at the cellular level as dynamic tests are associated with limited lateral and volumetric cellular deformation.

The Formation of Calcified Nanospherites during Micropetrosis Represents a Unique Mineralization Mechanism in Aged Human Bone.

Osteocytes-the central regulators of bone remodeling-are enclosed in a network of microcavities (lacunae) and nanocanals (canaliculi) pervading the mineralized bone. In a hitherto obscure process related to aging and disease, local plugs in the lacuno-canalicular network disrupt cellular communication and impede bone homeostasis. By utilizing a suite of high-resolution imaging and physics-based techniques, it is shown here that the local plugs develop by accumulation and fusion of calcified nanospherites in lacunae and canaliculi (micropetrosis). Two distinctive nanospherites phenotypes are found to originate from different osteocytic elements. A substantial deviation in the spherites' composition in comparison to mineralized bone further suggests a mineralization process unlike regular bone mineralization. Clearly, mineralization of osteocyte lacunae qualifies as a strong marker for degrading bone material quality in skeletal aging. The understanding of micropetrosis may guide future therapeutics toward preserving osteocyte viability to maintain mechanical competence and fracture resistance of bone in elderly individuals.

The incorporation of fluoride and strontium in hydroxyapatite affects the composition, structure, and mechanical properties of human cortical bone.

Strontium ranelate and fluoride salts are therapeutic options to reduce fracture risk in osteoporosis. Incorporation of these elements in the physiological hydroxyapatite matrix of bone is accompanied by changes in bone remodeling, composition, and structure. However, a direct comparison of the effectiveness of strontium and fluoride treatment in human cortical bone with a focus on the resulting mechanical properties remains to be established. Study groups are composed of undecalcified specimens from healthy controls, treatment-naïve osteoporosis cases, and strontium ranelate or fluoride-treated osteoporosis cases. Concentrations of both elements were determined using instrumental neutron activation analysis (INAA). Backscattered electron imaging was carried out to investigate the calcium content and the cortical microstructure. In comparison to osteoporotic patients, fluoride and strontium-treated patients have a lower cortical porosity indicating an improvement in bone microstructure. Mechanical properties were assessed via reference point indentation as a measure of bone's resistance to deformation. The strontium-incorporation led to significantly lower total indentation distance values compared to osteoporotic cases; controls have the highest resistance to indentation. In conclusion, osteoporosis treatment with strontium and fluoride showed positive effects on the microstructure and the mechanical characteristics of bone in comparison to treatment-naïve osteoporotic bone. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 433-442, 2017.

Intrinsic mechanical behavior of femoral cortical bone in young, osteoporotic and bisphosphonate-treated individuals in low- and high energy fracture conditions.

Bisphosphonates are a common treatment to reduce osteoporotic fractures. This treatment induces osseous structural and compositional changes accompanied by positive effects on osteoblasts and osteocytes. Here, we test the hypothesis that restored osseous cell behavior, which resembles characteristics of younger, healthy cortical bone, leads to improved bone quality. Microarchitecture and mechanical properties of young, treatment-naïve osteoporosis, and bisphosphonate-treated cases were investigated in femoral cortices. Tissue strength was measured using three-point bending. Collagen fibril-level deformation was assessed in non-traumatic and traumatic fracture states using synchrotron small-angle x-ray scattering (SAXS) at low and high strain rates. The lower modulus, strength and fibril deformation measured at low strain rates reflects susceptibility for osteoporotic low-energy fragility fractures. Independent of age, disease and treatment status, SAXS revealed reduced fibril plasticity at high strain rates, characteristic of traumatic fracture. The significantly reduced mechanical integrity in osteoporosis may originate from porosity and alterations to the intra/extrafibrillar structure, while the fibril deformation under treatment indicates improved nano-scale characteristics. In conclusion, losses in strength and fibril deformation at low strain rates correlate with the occurrence of fragility fractures in osteoporosis, while improvements in structural and mechanical properties following bisphosphonate treatment may foster resistance to fracture during physiological strain rates.

Deterioration of teeth and alveolar bone loss due to chronic environmental high-level fluoride and low calcium exposure.

OBJECTIVES: Health risks due to chronic exposure to highly fluoridated groundwater could be underestimated because fluoride might not only influence the teeth in an aesthetic manner but also seems to led to dentoalveolar structure changes. Therefore, we studied the tooth and alveolar bone structures of Dorper sheep chronically exposed to very highly fluoridated and low calcium groundwater in the Kalahari Desert in comparison to controls consuming groundwater with low fluoride and normal calcium levels within the World Health Organization (WHO) recommended range. MATERIALS AND METHODS: Two flocks of Dorper ewes in Namibia were studied. Chemical analyses of water, blood and urine were performed. Mineralized tissue investigations included radiography, HR-pQCT analyses, histomorphometry, energy-dispersive X-ray spectroscopy and X-ray diffraction-analyses. RESULTS: Fluoride levels were significantly elevated in water, blood and urine samples in the Kalahari group compared to the low fluoride control samples. In addition to high fluoride, low calcium levels were detected in the Kalahari water. Tooth height and mandibular bone quality were significantly decreased in sheep, exposed to very high levels of fluoride and low levels of calcium in drinking water. Particularly, bone volume and cortical thickness of the mandibular bone were significantly reduced in these sheep. CONCLUSIONS: The current study suggests that chronic environmental fluoride exposure with levels above the recommended limits in combination with low calcium uptake can cause significant attrition of teeth and a significant impaired mandibular bone quality. CLINICAL RELEVANCE: In the presence of high fluoride and low calcium-associated dental changes, deterioration of the mandibular bone and a potential alveolar bone loss needs to be considered regardless whether other signs of systemic skeletal fluorosis are observed or not.

The fracture mechanics of human bone: influence of disease and treatment.

Aging and bone diseases are associated with increased fracture risk. It is therefore pertinent to seek an understanding of the origins of such disease-related deterioration in bone's mechanical properties. The mechanical integrity of bone derives from its hierarchical structure, which in healthy tissue is able to resist complex physiological loading patterns and tolerate damage. Indeed, the mechanisms through which bone derives its mechanical properties make fracture mechanics an ideal framework to study bone's mechanical resistance, where crack-growth resistance curves give a measure of the intrinsic resistance to the initiation of cracks and the extrinsic resistance to the growth of cracks. Recent research on healthy cortical bone has demonstrated how this hierarchical structure can develop intrinsic toughness at the collagen fibril scale mainly through sliding and sacrificial bonding mechanisms that promote plasticity. Furthermore, the bone-matrix structure develops extrinsic toughness at much larger micrometer length-scales, where the structural features are large enough to resist crack growth through crack-tip shielding mechanisms. Although healthy bone tissue can generally resist physiological loading environments, certain conditions such as aging and disease can significantly increase fracture risk. In simple terms, the reduced mechanical integrity originates from alterations to the hierarchical structure. Here, we review how human cortical bone resists fracture in healthy bone and how changes to the bone structure due to aging, osteoporosis, vitamin D deficiency and Paget's disease can affect the mechanical integrity of bone tissue.

Alendronate treatment alters bone tissues at multiple structural levels in healthy canine cortical bone.

Bisphosphonates are widely used to treat osteoporosis, but have been associated with atypical femoral fractures (AFFs) in the long term, which raises a critical health problem for the aging population. Several clinical studies have suggested that the occurrence of AFFs may be related to the bisphosphonate-induced changes of bone turnover, but large discrepancies in the results of these studies indicate that the salient mechanisms responsible for any loss in fracture resistance are still unclear. Here the role of bisphosphonates is examined in terms of the potential deterioration in fracture resistance resulting from both intrinsic (plasticity) and extrinsic (shielding) toughening mechanisms, which operate over a wide range of length-scales. Specifically, we compare the mechanical properties of two groups of humeri from healthy beagles, one control group comprising eight females (oral doses of saline vehicle, 1 mL/kg/day, 3 years) and one treated group comprising nine females (oral doses of alendronate used to treat osteoporosis, 0.2mg/kg/day, 3 years). Our data demonstrate treatment-specific reorganization of bone tissue identified at multiple length-scales mainly through advanced synchrotron x-ray experiments. We confirm that bisphosphonate treatments can increase non-enzymatic collagen cross-linking at molecular scales, which critically restricts plasticity associated with fibrillar sliding, and hence intrinsic toughening, at nanoscales. We also observe changes in the intracortical architecture of treated bone at microscales, with partial filling of the Haversian canals and reduction of osteon number. We hypothesize that the reduced plasticity associated with BP treatments may induce an increase in microcrack accumulation and growth under cyclic daily loadings, and potentially increase the susceptibility of cortical bone to atypical (fatigue-like) fractures.

Bone as a Structural Material.

As one of the most important natural materials, cortical bone is a composite material comprising assemblies of tropocollagen molecules and nanoscale hydroxyapatite mineral crystals, forming an extremely tough, yet lightweight, adaptive and multi-functional material. Bone has evolved to provide structural support to organisms, and therefore its mechanical properties are vital physiologically. Like many mineralized tissues, bone can resist deformation and fracture from the nature of its hierarchical structure, which spans molecular to macroscopic length-scales. In fact, bone derives its fracture resistance with a multitude of deformation and toughening mechanisms that are active at most of these dimensions. It is shown that bone's strength and ductility originate primarily at the scale of the nano to submicrometer structure of its mineralized collagen fibrils and fibers, whereas bone toughness is additionally generated at much larger, micro- to near-millimeter, scales from crack-tip shielding associated with interactions between the crack path and the microstructure. It is further shown how the effectiveness with which bone's structural features can resist fracture at small to large length-scales can become degraded by biological factors such as aging and disease, which affect such features as the collagen cross-linking environment, the homogeneity of mineralization, and the density of the osteonal structures.