Differences and relationships between the physical properties and the microscopic structure of human femoral,tibial and fibular cortical bone

Coefficients of correlation between certain physical properties and the histological components of the break area were calculated on an IBM 7090 computer for 56 femoral, 79 tibial and 37 fibular specimens of embalmed cortical bone of standardized size and shape. Strong positive correlations (0.01–0.02 significance level) were found between tensile strength and the percentage of interstitial lamellae in the break area; between hardness and the number of osteons/mm²; and between hardness and the percentage of osteons in the break area. Equally high negative correlations were found between tensile strength and percentage of osteons in the break area; between shearing strength and average area/osteon remnant; between elastic modulus and percentage of spaces in the break area; and an even higher correlation (0.001) between hardness and percentage of spaces in the break area. Negative correlations (at slightly more than 0.05 significance level) were found between shearing strength and modulus and average area/osteon. Osteons tend to reduce the tensile strength and elastic modulus of bone while interstitial lamellae tend to increase them. The probable reason is the relatively greater amount of cement lines, which are sites of weakness where failure can occur, in Haversian bone as compared with lamellar bone. The predominant orientation of collagen fibers and the amount and distribution of calcium may also be involved. These factors are now being investigated.     

Changes in Bone Remodeling Rate Influence the Degree of Mineralization of Bone

Mineralization of bone matrix implies two successive steps, a primary mineralization on the calcification front followed by a slow process of secondary mineralization progressively adding about 50-60% of the mineral content on bone matrix. Our model is that antiresorptive agents prolong the lifetime of the basic structural units (BSUs) by causing a marked reduction in the birth rate of basic multicellular units (BMUs), and increase the degree of mineralization of bone (DMB) by allowing a more complete secondary mineralization. Conversely, agents or events provoking an augmentation of the birthrate of BMUs and a decrease of the lifetime of BSUs lead to resorption of new BSUs before they have fully completed their secondary mineralization, leading to the presence of incompletely mineralized BSUs and a low mean DMB value, as measured by quantitative microradiography. Measurements of DMB (distribution and mean value) under circumstances with various remodeling activities favor our model. In postmenopausal osteoporotic women treated during 2 or 3 years with alendronate (10 mg/day), an increase of the mean DMB of approximately 7 to 10% was found, due to a marked reduction in the bone remodeling. In contrast, an activation of bone remodeling as in primary hyperparathyroidism lowered the mean DMB.     

Changes in bone remodeling rate influence the degree of mineralization of bone

Mineralization of bone matrix implies two successive steps, a primary mineralization on the calcification front followed by a slow process of secondary mineralization progressively adding about 50-60% of the mineral content on bone matrix. Our model is that antiresorptive agents prolong the lifetime of the basic structural units (BSUs) by causing a marked reduction in the birth rate of basic multicellular units (BMUs), and increase the degree of mineralization of bone (DMB) by allowing a more complete secondary mineralization. Conversely, agents or events provoking an augmentation of the birthrate of BMUs and a decrease of the lifetime of BSUs lead to resorption of new BSUs before they have fully completed their secondary mineralization, leading to the presence of incompletely mineralized BSUs and a low mean DMB value, as measured by quantitative microradiography. Measurements of DMB (distribution and mean value) under circumstances with various remodeling activities favor our model. In postmenopausal osteoporotic women treated during 2 or 3 years with alendronate (10 mg/day), an increase of the mean DMB of approximately 7 to 10% was found, due to a marked reduction in the bone remodeling. In contrast, an activation of bone remodeling as in primary hyperparathyroidism lowered the mean DMB.     

Mechanical implications of collagen fibre orientation in cortical bone of the equine radius

Mechanical test specimens were prepared from the cranial and caudal cortices of radii from eight horses. These were subjected to destructive tests in either tension or compression. The ultimate stress, elastic modulus and energy absorbed to failure were calculated in either mode of loading. Analysis was performed on the specimens following mechanical testing to determine their density, mineral content, mineral density distribution and histological type. A novel technique was applied to sections from each specimen to quantify the predominant collagen fibre orientation of the bone near the plane of fracture. The collagen map for each bone studied was in agreement with the previously observed pattern of longitudinal orientation in the cranial cortex and more oblique to transverse collagen in the caudal cortex. Bone from the cranial cortex had a significantly higher ultimate tensile stress (UTS) than that from the caudal cortex (160 MPa vs 104 MPa; P<0.001) though this trend was reversed in compression, the caudal cortex becoming relatively stronger (185 MPa vs 217 MPa; P<0.01). Bone from the cranial cortex was significantly suffer than that from the caudal cortex both in tension (22 GPa vs 15 GPa; P<0.001) and compression (19 GPa vs 15 GPa; P<0.01). Of all the histo-compositional variables studied, collagen fibre orientation was most closely correlated with mechanical properties, accounting for 71% of variation in ultimate tensile stress and 58% of variation in the elastic modulus. Mineral density and porosity were the only other variables to show any significant correlation with either UTS or elastic modulus. The variations in mechanical properties around the equine radius, which occur in close association with the different collagen fibre orientations, provide maximal safety factors in terms of ultimate stress, yet contribute to greater bending of the bone as it is loaded during locomotion, and thus lower safety factors through the higher strains this engenders.     

Evaluation of the mechanical properties of rat bone under simulated microgravity using nanoindentation

Exposure to microgravity causes a decrease in bone mass and altered bone geometry due to the lack of weight-bearing forces on the skeleton. The mechanical properties of bone are due not only to the structure and geometry, but also to the tissue properties of the bone material itself. To study the effects of microgravity on bone tissue, the mechanical properties of tail suspension rat femurs were investigated. Twelve Sprague–Dawley rats were randomly divided into two groups, tail suspension (TS) and control (CON). On days 0 and 14, the bone mineral density (BMD) of the femurs was determined by Dual Energy X-ray Absorptiometry. After 14 days, three-point bending was used to test the mechanical properties of the whole femur and nanoindentation was used to measure the mechanical properties of the bone materials. The BMD of femurs in TS was significantly lower than that in CON. In the three-point bending testing, the breaking load, stiffness and energy absorption all decreased significantly in the TS group. In the nanoindentation tests, there was no significant difference between TS and CON in elastic modulus (E), while hardness (H) was significantly decreased and E/H significantly increased in TS. Weightlessness affects the intrinsic mechanical properties of bone at the bone material level. It is necessary to investigate further the effect of microgravity on the collagen bone matrix. Nanoindentation is a relatively new technique that is useful for investigating the above changes induced by microgravity and for assessing the efficacy of intervention.     

Principal stiffness orientation and degree of anisotropy of human osteons based on nanoindentation in three distinct planes

Haversian systems or 'osteons' are cylindrical structures, formed by bone lamellae, that make up the major part of human cortical bone. Despite their discovery centuries ago in 1691 by Clopton Havers, their mechanical properties are still poorly understood. The objective of this study is a detailed identification of the anisotropic elastic properties of the secondary osteon in the lamella plane. Additionally, the principal material orientation with respect to the osteon is assessed. Therefore a new nanoindentation method was developed which allows the measurement of indentation data in three distinct planes on a single osteon. All investigated osteons appeared to be anisotropic with a preferred stiffness alignment along the axial direction with a small average helical winding around the osteon axis. The mean degree of anisotropy was 1.75±0.36 and the mean helix angle was 10.3°±0.8°. These findings oppose two well established views of compact bone microstructure: first, the generally clear axial stiffness orientation contradicts a regular 'twisted plywood' collagen fibril orientation pattern in lamellar bone that would lead to a more isotropic behavior. Second, the class of transverse osteons were not observed from the mechanical point of view.     

Different fibrillar architectures coexisting in Haversian bone

Samples of compact bone were deproteinated by heat treatment and analysed by SEM. This technique removes very effectively cells and vascular structures and brings into full view the mineralization front along the wall of the Havers canal. The present study was confined to samples of equine bone that are known to be subjected to different functional requirements. Bone subjected to high tensile stress exhibited collagen fibrils substantially aligned with the stress direction, and the vast majority of its osteons appeared made of almost parallel fibrils crossing at very narrow angles. On the contrary, bone subjected to prevalent compressive forces showed either an orthogonal alternation of collagen lamellae, or a multidirectional arrangement corresponding to the twisted plywood described by other authors. Our observations substantiate the classical concept of the osteon structure as well as the twisted plywood; they indicate that several osteon architectures coexist in the same specimen, and that the different structures have a different preferential distribution in different parts of the same bone; and suggest that the relative distribution of the osteon ultrastructure across the bone matrix is modulated by mechanical factors.     

The alignment of MC3T3-E1 osteoblasts on steps of slip traces introduced by dislocation motion

Bone tissue shows a highly anisotropic microstructure comprising biological apatite and collagen fibrils produced by the mutual activities of bone cells, which dominates its mechanical function. Accordingly, directional control of osteoblasts is crucial for forming anisotropic bone tissue. A new approach was proposed for controlling cell directionality by using crystallographic slip traces caused by dislocation glide. Dislocations were introduced into α-titanium single crystals by plastic deformation of (011ˉ0)[21ˉ1ˉ0] slip system, inducing a step-like structure with acute angles between the surface normal and the slip plane. Topographical properties of step patterning, including step interval and step height, could be controlled by varying the compressive plastic strain. The step geometry introduced by plastic deformation strongly influenced osteoblast elongation, and it aligned preferentially along slip traces. Ti substrates under 10% plastic strain with step height of approximately 300?nm and step interval of 10?μm induced osteoblast alignment most successfully. Actin stress fibers elongated parallel to slip traces, with polarized vinculin accumulation between steps.     

Biomechanical Design in Osteogenic Engineering

The bone is a naturally occurring composite system comprising collagen matrix and hydroxyapatites capable of generating sufficient strength and toughness to support mechanical loads and resist fracture,respectively.The material strength depends largely on the elastic properties,whereas the toughness depends on not only the elastic,but also the plastic properties.Thus,both elastic and plastic properties must be considered in the analysis of bone biomechanics and the design of osteogenic materials.The bone is capable of optimizing its elastic and plastic properties by integrating stiff hydroxyapatites and ductile collagen fibrils into a hierarchically ordered architecture,an effective mechanism to support the bone strength and toughness.Such a mechanism can be used as a model for designing osteogenic materials.     

哈弗式骨单元的微观结构模型

本文根据骨的微观结构,运用复合材料力学中的方法,构造也骨板的本构关系。建立了骨单元的微观结构模型,并给出该模型轴对称应变问题的解析解。根据该模型可知,密质骨的?观弹性系数的不仅与骨的孔隙率有关,而且还与骨组织中胶原和矿物持的含量以及胶原纤维的排列方向有关。     

Comparison of the structure and mechanical properties of bovine femur bone and antler of the North American elk (Cervus elaphus canadensis)

Antler and limb bone have a similar microstructure and chemical composition. Both are primarily composed of type I collagen and a mineral phase (carbonated apatite), arranged in osteons in compact (cortical bone) sections and a lamellar structure in the cancellous (spongy or trabecular bone) sections. The mineral content is lower in antler bone and it has a core of cancellous bone surrounded by compact bone running through the main beam and tines. The mineral content is higher in the compact compared with the cancellous bone, although there is no difference in ratios of the mineral elements with calcium. Mechanical tests (bend and compression) on longitudinal and transverse orientations of dry and rehydrated compact bone of North American elk (Cervus elaphus canadensis) antlers are compared with known data on other antlers as well as bovine femora. Both dry and rehydrated bones are highly anisotropic, with the bending and compressive strength and elastic modulus higher in the longitudinal than in the transverse direction. There is no significant difference between the bend strength and elastic modulus between dry and rehydrated samples tested in the transverse direction. The elastic modulus measured from the bending tests is compared with composite models. The elastic modulus and bend strengths are lower in the rehydrated condition, but the strain to failure and fracture toughness is much higher compared with dry samples. All antler bone mechanical properties are lower than that of bovine femora. The antler has a much higher fracture toughness compared with bovine femora, which correlates with their main function in intraspecific combat as a high impact resistant, energy absorbent material. A model of compression deformation is proposed, which is based on osteon sliding during shear.     

Effects of osteoporosis and nutrition supplements on structures and nanomechanical properties of bone tissue

In this study, the bone structures, nanomechanical properties and fracture behaviors in different groups of female C57BL/6 mice (control, sham operated, ovariectomized, casein supplemented, and fermented milk supplemented) were examined by micro-computed tomography, scanning and transmission electron microscopy, and nanoindentation. The control and sham operated mice showed dense bone structures with high cortical bone mineral densities of 544 mg/cm³ (average) and high hardness of 0.9–1.1 GPa; resistance to bone fracture was conferred by microcracking, crack deflections and ligament bridging attributed to aligned collagen fibers and densely packed hydroxyapatite crystals. Bone mineral density, hardness and fracture resistance in ovariectomized mice markedly dropped due to loose bone structure with randomly distributed collagens and hydroxyapatites. The acidic casein supplemented mice with blood acidosis exhibited poor mineral absorption and loose bone structure, whereas the neutralized casein or fermented milk supplemented mice were resistant to osteoporosis and had high bone mechanical properties.     

Evidence of altered matrix composition in iliac crest biopsies from patients with idiopathic juvenile osteoporosis

Purpose: Idiopathic juvenile osteoporosis (IJO) is a rare condition in children, characterized by bone pain and long bone and vertebral fractures. Previously, IJO bone was solely characterized by histomorphometry and quantitative computed tomography. The goal of this study is to describe IJO bone composition. Materials and methods: Fourier transform infrared imaging (FTIRI), a vibrational spectroscopic technique providing spatially resolved images of chemical composition, was used to determine whether iliac crest biopsies from children with IJO differed in composition from and age- and sex-matched controls, and, as a secondary analysis, whether IJO bone showed the same disease dependent change in composition as do iliac crest bone biopsies from women with post-menopausal osteoporosis (PMO). Wilcoxon rank tests and linear regressions were used to analyze FTIRI variables (mineral-to-matrix ratio, carbonate-to-phosphate ratio, crystallinity, acid phosphate substitution, collagen maturity) and their individual pixel distributions (heterogeneity). Results: Mineral-to-matrix ratio was comparable in IJO and age-matched controls. Contrastingly, collagen maturity (also known as collagen cross-link ratio) was higher in cortical and cancellous IJO bone compared with juvenile controls. Acid phosphate substitution was greater in IJO cancellous bone than in age-matched controls, suggesting IJO bone mineral is formed more recently, reflecting a slower mineralization process. This agrees with findings of increased heterogeneity for mineral-to-matrix and collagen maturity ratios in IJO cancellous bone. There were negative correlations between cancellous collagen maturity and previously reported histomorphometric bone formation markers. There were no correlations with indices of remodeling. Conclusions: IJO bone, similar to PMO bone, had elevated collagen maturity relative to its age-matched controls. This emphasizes the importance of the collagen matrix for bone health. IJO bone differed from PMO bone as IJO bone contains more recently formed mineral than age-matched controls but has a more mature matrix, whereas in PMO bone both mineral and matrix have older characteristics.     

Differences in osteonal micromorphology between tensile and compressive cortices of a bending skeletal system: Indications of potential strain-specific differences in bone microstructure

Background: It has been hypothesized that bone has the capacity to accommodate regional differences in tension and compression strain mode and/or magnitude by altering its osteonal microstructure. We examined a simple cantilevered bone to determine whether regional differences in particular strain-related features are reflected in the microstructural organization of compact bone. Methods & Results: The artiodactyl (e.g., sheep and deer) calcaneus has a predominant loading condition which is typified by prevailing compressive and tensile strains on opposite cortices, and variations in strain magnitudes across each of these cortices. Microscopic examination showed osteon density and cortical porosity differences between tension (caudal) and compression (cranial) cortices, averaging 11.4% more osteons in the compression cortex (P < 0.01) and 80.2% greater porosity in the tension cortex (P < 0.01). There is 43.5% more interstitial bone in the compression cortex (P < 0.01). Osteons in the compression cortex also have smaller areas in contrast to the larger area per osteon in the tension cortex. Although no definite transcortical gradient in osteonal density or cortical porosity is found, fractional area of interstitial bone is largest and osteon population density is lowest in the endocortical regions of both tension and compression cortices. The endocortical regions also have greater porosity than their corresponding middle and pericortical regions (P < 0.01). Conclusions: These osteonal microstructure and cortical porosity differences may be adaptations related to regional differences in strain mode and/or strain magnitude. This may be related to the disparity in mechanical properties of compact bone in tension vs. compression. These differences may reflect a capacity of bone to process local and regional strain-related information. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public dotmain in the United states of America

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Osteogenesis imperfecta: Ultrastructural and histological findings on examination of skin revealing novel insights into genotype-phenotype correlation

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity to fracture. Over 90% of patients with OI have a mutation in COL1A1/COL1A2, which shows an autosomal dominant pattern of inheritance. In-depth phenotyping and in particular, studies involving manifestations in the skin connective tissue have not previously been undertaken in OI. The aims of the study were to perform histological and ultrastructural examination of skin biopsies in a cohort of patients with OI; to identify common and distinguishing features in order to inform genotype-phenotype correlation; and to identify common and distinguishing features between the different subtypes of OI. As part of the RUDY (Rare Diseases in Bone, Joints and/or Blood Vessels) study, in collaboration with the NIHR Rare Diseases Translational Research Collaboration, we undertook a national study of skin biopsies in patients with OI. We studied the manifestations in the skin connective tissue and undertook in-depth clinical and molecular phenotyping of 16 patients with OI. We recruited 16 patients: analyses have shown that in type 1 collagen mutation positive patients (COL1A1/ COL1A2) (n-4/16) consistent findings included: variable collagen fibril diameter (CFD) and presence of collagen flowers. Histological examination in these patients showed an increase in elastic fibers that are frequently fragmented and clumped. These observations provide evidence that collagen flowers and CFD variability are consistent features in OI due to type 1 collagen defects and reinforce the need for accurate phenotyping in conjunction with genomic analyses.     

Derivation of elastic stiffness from site-matched mineral density and acoustic impedance maps

200 MHz acoustic impedance maps and site-matched synchrotron radiation micro computed tomography (SR-muCT) maps of tissue degree of mineralization of bone (DMB) were used to derive the elastic coefficient c(33) in cross sections of human cortical bone. To accomplish this goal, a model was developed to relate the DMB accessible with SR-muCT to mass density. The formulation incorporates the volume fractions and densities of the major bone tissue components (collagen, mineral and water), and accounts for tissue porosity. We found that the mass density can be well modelled by a second-order polynomial fit to DMB (R(2) = 0.999) and appears to be consistent with measurements of many different types of mineralized tissues. The derived elastic coefficient c(33) correlated more strongly with the acoustic impedance (R(2) = 0.996) than with mass density (R(2) = 0.310). This finding suggests that estimates of c(33) made from measurements of the acoustic impedance are more reliable than those made from density measurements. Mass density and elastic coefficient were in the range between 1.66 and 2.00 g cm(-3) and 14.8 and 75.4 GPa, respectively. Although SAM inspection is limited to the evaluation of carefully prepared sample surfaces, it provides a two-dimensional quantitative estimate of elastic tissue properties at the tissue level.     

Secondary osteon variants and remodeling in human bone

Histomorphometric analysis of human cortical bone has documented the occurrence of secondary osteon variants. These include drifting osteons which form tails as they move erratically through the cortex and Type II osteons which show partial resorption and redeposition within the cement line of the osteon. Little is known about the biological significance of these variants. Prior studies suggested correlations with age, biomechanics, diet, and mineral homeostasis. No study has yet tested for osteon variant associations with static measures of bone remodeling. In this study, thin sections (n = 112) of the posterior femur representing a late English Medieval adult human osteological collection, subdivided by age, sex, and socio-economic status, were examined to determine whether remodeling indicators reconstructed from osteon parameters (area, diameter, area ratios) and densities differed between categories of presence or absence of Type II and drifting osteon variants. Of the 112 sections, 33 presented with Type II osteons, and 38 had drifting osteons. Sporadic statistically significant results were identified. Haversian canal:osteon area ratio differed (p = 0.017) with Type II osteon presence, Type II osteons were more prevalent in males than females (p = 0.048), and drifting osteons were associated with smaller osteon (p = 0.049) and Haversian canal area (p = 0.05). These results may be explained through some biological (sex) and social (status) processes such as a period of physiological recovery (e.g., following lactation, malnutrition). However, the general lack of consistent relationships between osteon variants and remodeling indicators suggests they occur as a result of natural variation.     

密质骨各向异性性质的力学实验及细观分析

目的研究牛股骨密质骨的微观结构特征与其力学行为间的关系。方法对牛股骨密质骨纵向和横向试样分别进行压缩实验;对破坏后的纵向和横向试样进行断裂路径、断裂表面微结构特征观察;基于复合材料细观力学的理论,分析密质骨中骨单元方向对骨纵向和横向弹性模量及断裂极限强度的影响。结果纵向试样的压缩弹性模量和断裂极限强度均明显大于横向试样的压缩弹性模量和断裂极限强度;纵向试样的断裂路径与加载方向近似为0°,且比较平直,而横向试样的断裂路径与加载方向近似为45°,且比较曲折;纵向试样断面中存在许多与加载方向平行的条状结构,断面较为光滑,而横向试样断面存在许多圆弧形凹坑或凸起,断面较为粗糙。结论牛股骨密质骨具有各向异性的力学性质,其各向异性力学性质与骨单元方向密切相关。     

Morphometric analysis of osteonal architecture in bones from healthy young human male subjects using scanning electron microscopy

The shape and structure of bones is a topic that has been studied for a long time by morphologists and biologists with the goal of explaining the laws governing their development, aging and pathology. The osteonal architecture of tibial and femoral mid‐diaphyses was examined morphometrically with scanning electron microscopy in four healthy young male subjects. In transverse sections of the mid‐diaphysis, the total area of the anterior, posterior, lateral and medial cortex sectors was measured and analysed for osteonal parameters including osteon number and density, osteon total and bone area and vascular space area. Osteons were grouped into four classes including cutting heads (A), transversely cut osteons (B), longitudinally cut osteons (C) and sealed osteons (D). The morphometric parameters were compared between the inner (endosteal) and outer (periosteal) half of the cortex. Of 5927 examined osteons, 24.4% cutting heads, 71.1% transversely cut osteons, 2.3% longitudinally cut osteons and 2.2% sealed osteons were found. The interosteonic bone (measured as the area in a lamellar system that has lost contact with its own central canal) corresponded to 51.2% of the endosteal and 52.4% of the periosteal half‐cortex. The mean number of class A cutting heads and class B osteons was significantly higher in the periosteal than in the endosteal half‐cortex (P < 0.001 and P < 0.05, respectively), whereas there was no significant difference in density. The mean osteon total area, osteon bone area and vascular space area of both classes A and B were significantly higher (P < 0.001 for all three parameters) in the endosteal than in the periosteal half‐cortex. The significant differences between the two layers of the cortex suggest that the osteoclast activity is distributed throughout the whole cortical thickness, with more numerous excavations in the external layer, but larger resorption lacunae closer to the marrow canal. A randomly selected population of 109 intact class B osteons was examined at higher magnification (350×) to count osteocyte lacuna and to analyse their relationship with osteon size parameters. The distribution frequency of the mean number of osteocyte lacunae increased with the increment in the sub‐classes of osteon bone area, whereas the density did not show significant differences. The number of osteocyte lacunae had a direct correlation with the osteon bone area and the mean osteon wall thickness, as well as the mean number of lamellae. The osteocyte lacunae density showed an inverse relationship. These data suggest a biological regulation of osteoblast activity with a limit to the volume of matrix produced by each cell and proportionality with the number of available cells in the space of the cutting cone (total osteon area). The collected data can be useful as a set of control parameters in healthy human bone for studies on bone aging and metabolic bone diseases.     

Multiscale imaging of bone microdamage

Abstract

Bone is a structural and hierarchical composite that exhibits remarkable ability to sustain complex mechanical loading and resist fracture. Bone quality encompasses various attributes of bone matrix from the quality of its material components (type-I collagen, mineral and non-collagenous matrix proteins) and cancellous microarchitecture, to the nature and extent of bone microdamage. Microdamage, produced during loading, manifests in multiple forms across the scales of hierarchy in bone and functions to dissipate energy and avert fracture. Microdamage formation is a key determinant of bone quality, and through a range of biological and physical mechanisms, accumulates with age and disease. Accumulated microdamage in bone decreases bone strength and increases bone’s propensity to fracture. Thus, a thorough assessment of microdamage, across the hierarchical levels of bone, is crucial to better understand bone quality and bone fracture. This review article details multiple imaging modalities that have been used to study and characterize microdamage; from bulk staining techniques originally developed by Harold Frost to assess linear microcracks, to atomic force microscopy, a modality that revealed mechanistic insights into the formation diffuse damage at the ultrastructural level in bone. New automated techniques using imaging modalities, such as microcomputed tomography are also presented for a comprehensive overview.