Mathematical analysis of trabecular 'trajectories' in apparent trajectorial structures: the unfortunate historical emphasis on the human proximal femur

Wolff's "law" of the functional adaptation of bone is rooted in the trajectory hypothesis of cancellous bone architecture. Wolff often used the human proximal femur as an example of a trajectorial structure (i.e. arched trabecular patterns appear to be aligned along tension/compression stress trajectories). We examined two tenets of the trajectory hypothesis; namely, that the trabecular tracts from the tension- and compression-loaded sides of a bending environment will: (1) follow 'lines' (trajectories) of tension/compression stress that resemble an arch with its apex on a neutral axis, and (2) form orthogonal (90 degrees ) intersections. These predictions were analysed in proximal femora of chimpanzees and modern humans, and in calcanei of sheep and deer. Compared to complex loading of the human femoral neck, the chimpanzee femoral neck reputedly receives relatively simpler loading (i.e. temporally/spatially more consistent bending), and the artiodactyl calcaneus is even more simply loaded in bending. In order to directly consider Wolff's observations, measurements were also made on two-dimensional, cantilevered beams and curved beams, each with intersecting compression/tension stress trajectories. Results in the calcanei showed: (1) the same nonlinear equation best described the dorsal ("compression") and plantar ("tension") trabecular tracts, (2) these tracts could be exactly superimposed on the corresponding compression/tension stress trajectories of the cantilevered beams, and (3) trabecular tracts typically formed orthogonal intersections. In contrast, trabecular tracts in human and chimpanzee femoral necks were non-orthogonal (mean approximately 70 degrees ), with shapes differing from trabecular tracts in calcanei and stress trajectories in the beams. Although often being described by the same equations, the trajectories in the curved beams had lower r(2) values than calcaneal tracts. These results suggest that the trabecular patterns in the calcanei and stress trajectories in short beams are consistent with basic tenets of the trajectory hypothesis while those in human and chimpanzee femoral necks are not. Compared to calcanei, the more complexly loaded human and chimpanzee femoral necks probably receive more prevalent/predominant shear, which is best accommodated by non-orthogonal, asymmetric trabecular tracts. The asymmetrical trabecular patterns in the proximal femora may also reflect the different developmental 'fields' (trochanteric vs. neck/head) that formed these regions, of which there is no parallel in the calcanei.     

Application of homogenization theory to the study of trabecular bone mechanics.

It is generally accepted that the strength and stiffness of trabecular bone is strongly affected by trabecular microstructure. It has also been hypothesized that stress induced adaptation of trabecular bone is affected by trabecular tissue level stress and/or strain. At this time, however, there is no generally accepted (or easily accomplished) technique for predicting the effect of microstructure on trabecular bone apparent stiffness and strength or estimating tissue level stress or strain. In this paper, a recently developed mechanics theory specifically designed to analyze microstructured materials, called the homogenization theory, is presented and applied to analyze trabecular bone mechanics. Using the homogenization theory it is possible to perform microstructural and continuum analyses separately and then combine them in a systematic manner. Stiffness predictions from two different microstructural models of trabecular bone show reasonable agreement with experimental results, depending on metaphyseal region, (R2 greater than 0.5 for proximal humerus specimens, R2 less than 0.5 for distal femur and proximal tibia specimens). Estimates of both microstructural strain energy density (SED) and apparent SED show that there are large differences (up to 30 times) between apparent SED (as calculated by standard continuum finite element analyses) and the maximum microstructural or tissue SED. Furthermore, a strut and spherical void microstructure gave very different estimates of maximum tissue SED for the same bone volume fraction (BV/TV). The estimates from the spherical void microstructure are between 2 and 20 times greater than the strut microstructure at 10-20% BV/TV.     

A theoretical model to predict distribution of the fabric tensor and apparent density in cancellous bone

 The adaptation of cancellous bone to mechanical forces is well recognized. Theoretical models for predicting cancellous bone architecture have been developed and have mainly focused on the distribution of trabecular mass or the apparent density. The purpose of this study was to develop a theoretical model which can simultaneously predict the distribution of trabecular orthotropy/orientation, as represented by the fabric tensor, along with apparent density. Two sets of equations were derived under the assumption that cancellous bone is a biological self-optimizing material which tends to minimize strain energy. The first set of equations provide the relationship between the fabric tensor and stress tensor, and have been verified to be consistent with Wolff’s law of trabecular architecture, that is, the principal directions of the fabric tensor coincide with the principal stress trajectories. The second set of equations yield the apparent density from the stress tensor, which was shown to be identical to those obtained based on local optimization with strain energy density of true bone tissue as the objective function. These two sets of equations, together with elasticity field equations, provide a complete mathematical formulation for the adaptation of cancellous bone. Received: 25 February 1997/Revised version: 23 September 1997     

Three-dimensional micro-level computational study of Wolff's law via trabecular bone remodeling in the human proximal femur using design space topology optimization

The law of bone remodeling, commonly referred to as Wolff's Law, asserts that the internal trabecular bone adapts to external loadings, reorienting with the principal stress trajectories to maximize mechanical efficiency creating a naturally optimum structure. The goal of the current study was to utilize an advanced structural optimization algorithm, called design space optimization (DSO), to perform a micro-level three-dimensional finite element bone remodeling simulation on the human proximal femur and analyse the results to determine the validity of Wolff's hypothesis. DSO optimizes the layout of material by iteratively distributing it into the areas of highest loading, while simultaneously changing the design domain to increase computational efficiency. The result is a "fully stressed" structure with minimized compliance and increased stiffness. The large-scale computational simulation utilized a 175 μm mesh resolution and the routine daily loading activities of walking and stair climbing. The resulting anisotropic trabecular architecture was compared to both Wolff's trajectory hypothesis and natural femur samples from literature using a variety of visualization techniques, including radiography and computed tomography (CT). The results qualitatively revealed several anisotropic trabecular regions, that were comparable to the natural human femurs. Quantitatively, the various regional bone volume fractions from the computational results were consistent with quantitative CT analyses. The global strain energy proceeded to become more uniform during optimization; implying increased mechanical efficiency was achieved. The realistic simulated trabecular geometry suggests that the DSO method can accurately predict bone adaptation due to mechanical loading and that the proximal femur is an optimum structure as the Wolff hypothesized.     

Functional adaptation of cancellous bone in human proximal femur predicted by trabecular surface remodeling simulation toward uniform stress state

Two-dimensional simulation of trabecular surface remodeling was conducted for a human proximal femur to investigate the structural change of cancellous bone toward a uniform stress state. Considering that a local mechanical stimulus plays an important role in cellular activities in bone remodeling, local stress nonuniformity was assumed to drive trabecular structural change to seek a uniform stress state. A large-scale pixel-based finite element model was used to simulate structural changes of individual trabeculae over the entire bone. As a result, the initial structure of trabeculae changed from isotropic to anisotropic due to trabecular microstructural changes caused by surface remodeling according to the mechanical environment in the proximal femur. Under a single-loading condition, it was shown that the apparent structural property evaluated by fabric ellipses corresponded to the apparent stress state in cancellous bone. As is observed in the actual bone, a distributed trabecular structure was obtained under a multiple-loading condition. Through these studies, it was concluded that trabecular surface remodeling toward a local uniform stress state at the trabecular level could naturally bring about functional adaptation phenomenon at the apparent tissue level. The proposed simulation model would be capable of providing insight into the hierarchical mechanism of trabecular surface remodeling at the microstructural level up to the apparent tissue level.     

Trabecular bone remodeling around smooth and porous implants in an equine patellar model

The objective of this investigation was to examine the stress-morphology relationships for trabecular bone around implants with different surface characteristics. Stainless steel spheres with either a polished surface or a sintered-bead porous coating were implanted unilaterally into equine patellae and maintained for a 6 month period. Stereological methods were used to quantify the trabecular bone morphology and finite element analyses were performed to predict the trabecular bone stresses. In general, the remodeling response around the smooth implants was greater than that around those porous implants that exhibited bone ingrowth. In accordance with these differences, the finite element models predicted greater changes in the stresses adjacent to the smooth implants due to the nonlinear boundary conditions. However, it did not appear that the trajectorial theory, in its simplest form, was applicable to the remodeling induced by the implants. A linear relationship between the change in bone areal density and the change in von Mises effective stress provides support for the hypothesis that the architecture of trabecular bone corresponds to an optimal structure. The results also demonstrated that, under certain circumstances, small changes in the stress state may result in large changes in the principal material orientation.     

A theoretical framework for strain-related trabecular bone maintenance and adaptation

It is assumed that density and morphology of trabecular bone is partially controlled by mechanical forces. How these effects are expressed in the local metabolic functions of osteoclast resorption and osteoblast formation is not known. In order to investigate possible mechano-biological pathways for these mechanisms we have proposed a mathematical theory (Nature 405 (2000) 704). This theory is based on hypothetical osteocyte stimulation of osteoblast bone formation, as an effect of elevated strain in the bone matrix, and a role for microcracks and disuse in promoting osteoclast resorption. Applied in a 2-D Finite Element Analysis model, the theory explained the formation of trabecular patterns. In this article we present a 3-D FEA model based on the same theory and investigated its potential morphological predictability of metabolic reactions to mechanical loads. The computations simulated the development of trabecular morphological details during growth, relative to measurements in growing pigs, reasonably realistic. They confirmed that the proposed mechanisms also inherently lead to optimal stress transfer. Alternative loading directions produced new trabecular orientations. Reduction of load reduced trabecular thickness, connectivity and mass in the simulation, as is seen in disuse osteoporosis. Simulating the effects of estrogen deficiency through increased osteoclast resorption frequencies produced osteoporotic morphologies as well, as seen in post-menopausal osteoporosis. We conclude that the theory provides a suitable computational framework to investigate hypothetical relationships between bone loading and metabolic expressions.     

Trabecular eccentricity and bone adaptation

It is well established that bones functionally adapt by mechanisms that control tissue density, whole bone geometry, and trabecular orientation. In this study, we propose the existence of another such powerful mechanism, namely, trabecular eccentricity, i.e. non-central placement of trabecular bone within a cortical envelope. In the human femoral neck, trabecular eccentricity results in a thicker cortical shell on the inferior than superior aspect. In an overall context of expanding understanding of bone adaptation, the goal of this study was to demonstrate the biomechanical significance of, and provide a mechanistic explanation for, the relationship between trabecular eccentricity and stresses in the human femoral neck. Using composite beam theory, we showed that the biomechanical effects of eccentricity during a habitual loading situation were to increase the stress at the superior aspect of the neck and decrease the stress at the inferior aspect, resulting in an overall protective effect. Further, increasing eccentricity had a stress-reducing effect equivalent to that of increasing cortical thickness or increasing trabecular modulus. We conclude that an asymmetric placement of trabecular bone within a cortical bone envelope represents yet another mechanism by which whole bones can adapt to mechanical demands.     

Computer simulation of trabecular remodeling in human proximal femur using large-scale voxel FE models: Approach to understanding Wolff's law

Ever since Julius Wolff proposed the law of bone transformation in the 19th century, it has been widely known that the trabecular structure of cancellous bone adapts functionally to the loading environment. To understand the mechanism of Wolff's law, a three-dimensional (3D) computer simulation of trabecular structural changes due to surface remodeling was performed for a human proximal femur. A large-scale voxel finite element model was constructed to simulate the structural changes of individual trabeculae over the entire cancellous region. As a simple remodeling model that considers bone cellular activities regulated by the local mechanical environment, nonuniformity of local stress was assumed to drive the trabecular surface remodeling to seek a uniform stress state. Simulation results demonstrated that cell-scale (～10 μm) remodeling in response to mechanical stimulation created complex 3D trabecular structures of the entire bone-scale (～10 cm), as illustrated in the reference of Wolff. The bone remodeling reproduced the characteristic anisotropic structure in the coronal cross section and the isotropic structures in other cross sections. The principal values and axes of a structure characterized by fabric ellipsoids corresponded to those of the apparent stress of the structure. The proposed large-scale computer simulation indicates that in a complex mechanical environment of a hierarchical bone structure of over 10⁴ length scale (from ～10 μm to ～10 cm), a simple remodeling at the cellular/trabecular levels creates a highly complex and functional trabecular structure, as characterized by bone density and orientation.     

Finite element analysis of ramming in Ovis canadensis

The energy produced during the ramming of bighorn sheep (Ovis canadensis) would be expected to result in undesirable stresses in their frontal skull, which in turn would cause brain injury; yet, this animal seems to suffer no ill effects. In general, horn is made of an α-keratin sheath covering a bone. Despite volumes of data on the ramming behavior of Ovis canadensis, the extent to which structural components of horn and horn-associated structure or tissue absorb the impact energy generated by the ramming event is still unknown. This study investigates the hypothesis that there is a mechanical relationship present among the ramming event, the structural constituents of the horn, and the horn-associated structure. The three-dimensional complex structure of the bighorn sheep horn was successfully constructed and modeled using a computed tomography (CT) scan and finite element (FE) method, respectively. Three different three-dimensional quasi-static models, including a horn model with trabecular bone, a horn model with compact bone that instead of trabecular bone, and a horn model with trabecular bone as well as frontal sinuses, were studied. FE simulations were used to compare distributions of principal stress in the horn and the frontal sinuses and the strain energy under quasi-static loading conditions. It was noticed that strain energy due to elastic deformation of the complex structure of horn modeled with trabecular bone and with trabecular bone and frontal sinus was different. In addition, trabecular bone in the horn distributes the stresses over a larger volume, suggesting a mechanical link between the structural constituents and the ramming event. This phenomenon was elucidated through the principal stress distribution in the structure. This study will help designers in choosing appropriate material combinations for the successful design of protective structures against a similar impact.     

An experimental and computational investigation of the post-yield behaviour of trabecular bone during vertebral device subsidence

Interbody fusion device subsidence has been reported clinically. An enhanced understanding of the mechanical behaviour of the surrounding bone would allow for accurate predictions of vertebral subsidence. The multiaxial inelastic behaviour of trabecular bone is investigated at a microscale and macroscale level. The post-yield behaviour of trabecular bone under hydrostatic and confined compression is investigated using microcomputed tomography-derived microstructural models, elucidating a mechanism of pressure-dependent yielding at the macroscopic level. Specifically, microstructural trabecular simulations predict a distinctive yield point in the apparent stress–strain curve under uniaxial, confined and hydrostatic compression. Such distinctive apparent stress–strain behaviour results from localised stress concentrations and material yielding in the trabecular microstructure. This phenomenon is shown to be independent of the plasticity formulation employed at a trabecular level. The distinctive response can be accurately captured by a continuum model using a crushable foam plasticity formulation in which pressure-dependent yielding occurs. Vertebral device subsidence experiments are also performed, providing measurements of the trabecular plastic zone. It is demonstrated that a pressure-dependent plasticity formulation must be used for continuum level macroscale models of trabecular bone in order to replicate the experimental observations, further supporting the microscale investigations. Using a crushable foam plasticity formulation in the simulation of vertebral subsidence, it is shown that the predicted subsidence force and plastic zone size correspond closely with the experimental measurements. In contrast, the use of von Mises, Drucker–Prager and Hill plasticity formulations for continuum trabecular bone models lead to over prediction of the subsidence force and plastic zone.     

Relationships between loading history and femoral cancellous bone architecture

A theory relating bone maintenance to mechanical loading history has been applied to successfully predict the distribution of bone density and trabecular orientation in the adult proximal femur. The loading history was simulated by determining the stress fields in a two-dimensional finite element model exposed to various discrete loading cases and making assumptions about the relative number of loading cycles associated with each load case. The total stimulus to bone maintenance was then calculated by a linear superposition of the stimulus of each loading case. Based on the calculated total stimulus, the apparent density and material properties of each element were changed and the stress solutions were again determined. Using this iterative technique, the bone apparent density and orientation characteristics were predicted. The results indicate that the trabecular morphology of the femur can only be explained by considering the joint loadings from multiple directions. Contrary to the 'trajectorial theory' promoted by Wolff (The Law of Bone Remodelling, 1892), trabecular orientations predicted from our multiple-load analyses are not necessarily perpendicular and do not correspond to the principal stress directions of any one loading condition. Our predicted orientations correspond better to the drawing of bone trabecular morphology by von Meyer (Archs Anat. Physiol. wiss. Med. 34, 615-628, 1867) than to the classic drawing by Wolff and suggest that further study of the trajectorial theory is warranted.     

Static and dynamic finite element analyses of an idealized structural model of vertebral trabecular bone.

An idealized three-dimensional finite element model of a rodlike trabecular bone structure was developed to study its static and dynamic responses under compressive loading, considering the effects of bone marrow and apparent density. Static analysis of the model predicted hydraulic stiffening of trabecular bone due to the presence of bone marrow. The predicted power equation relating trabecular bone apparent elastic modulus to its apparent density was in good agreement with those of the reported experimental investigations. The ratio of the maximum stress in the trabecular bone tissue to its apparent stress had a high value, decreasing with increasing bone apparent density. Frequency analyses of the model predicted higher natural frequencies for the bone without marrow than those for the bone with marrow. Adding a mass relatively large compared to that of bone rendered a single-degree-of-freedom response. In this case, the resonant frequency was higher for the bone with marrow than that for the bone without marrow. The predicted vibrational measurement of apparent modulus was in good agreement with that of the static measurement, suggesting vibrational testing as a method for nondestructive measurement of trabecular bone elastic moduli.     

Length scale parameter of single trabecula in cancellous bone

Biomechanics and Modeling in Mechanobiology - In this paper, the material length scale parameter of the modified couple stress theory for trabecular bones is studied. For this reason, experimental...     

Altered architecture and cell populations affect bone marrow mechanobiology in the osteoporotic human femur

Age-related increases in trabecular bone porosity, as seen in osteoporosis, not only affect the strength and stiffness, but also potentially the mechanobiological response of bone. The mechanical interaction between trabecular bone and bone marrow is one source of mechanobiological signaling, as many cell populations in marrow are mechanosensitive. However, measuring the mechanics of this interaction is difficult, due to the length scales and geometric complexity of trabecular bone. In this study, a multi-scale computational scheme incorporating high-resolution, tissue-level, fluid–structure interaction simulations with discrete cell-level models was applied to characterize the potential effects of trabecular porosity and marrow composition on marrow mechanobiology in human femoral bone. First, four tissue-level models with different volume fractions (BV/TV) were subjected to cyclic compression to determine the continuum level shear stress in the marrow. The calculated stress was applied to three detailed models incorporating individual cells and having differing adipocyte fractions. At the tissue level, compression of the bone along its principal mechanical axis induced shear stress in the marrow ranging from 2.0 to 5.6 Pa, which increased with bone volume fraction and strain rate. The shear stress was amplified at the cell level, with over 90% of non-adipocyte cells experiencing higher shear stress than the applied tissue-level stress. The maximum shear stress decreased by 20% when the adipocyte volume fraction (AVF) increased from 30%, as seen in young healthy marrow, to 45 or 60% AVF typically found in osteoporotic patients. The results suggest that increasing AVF has similar effects on the mechanobiological signaling in bone marrow as decreased volume fraction.     

Trabecular Bone Structure Correlates with Hand Posture and Use in Hominoids

Bone is capable of adapting during life in response to stress. Therefore, variation in locomotor and manipulative behaviours across extant hominoids may be reflected in differences in trabecular bone structure. The hand is a promising region for trabecular analysis, as it is the direct contact between the individual and the environment and joint positions at peak loading vary amongst extant hominoids. Building upon traditional volume of interest-based analyses, we apply a whole-epiphysis analytical approach using high-resolution microtomographic scans of the hominoid third metacarpal to investigate whether trabecular structure reflects differences in hand posture and loading in knuckle-walking (Gorilla, Pan), suspensory (Pongo, Hylobates and Symphalangus) and manipulative (Homo) taxa. Additionally, a comparative phylogenetic method was used to analyse rates of evolutionary changes in trabecular parameters. Results demonstrate that trabecular bone volume distribution and regions of greatest stiffness (i.e., Young''s modulus) correspond with predicted loading of the hand in each behavioural category. In suspensory and manipulative taxa, regions of high bone volume and greatest stiffness are concentrated on the palmar or distopalmar regions of the metacarpal head, whereas knuckle-walking taxa show greater bone volume and stiffness throughout the head, and particularly in the dorsal region; patterns that correspond with the highest predicted joint reaction forces. Trabecular structure in knuckle-walking taxa is characterised by high bone volume fraction and a high degree of anisotropy in contrast to the suspensory brachiators. Humans, in which the hand is used primarily for manipulation, have a low bone volume fraction and a variable degree of anisotropy. Finally, when trabecular parameters are mapped onto a molecular-based phylogeny, we show that the rates of change in trabecular structure vary across the hominoid clade. Our results support a link between inferred behaviour and trabecular structure in extant hominoids that can be informative for reconstructing behaviour in fossil primates.     

Creep does not contribute to fatigue in bovine trabecular bone

In both cortical and trabecular bone loaded in fatigue, the stress-strain loops translate along the strain axis. Previous studies have suggested that this translation is the result of creep associated with the mean stress applied in the fatigue test. In this study, we measured the residual strrain (corresponding to the translation of the stress-strain loops) in fatigue tests on bovine trabecular bone and compared it to an upper bound estimate of the creep strain in each test. Our results indicate that the contribution of creep to the translation of the stress-strain loops is negligible in bovine trabecular bone. These results, combined with models for fatigue in lower density bone, suggest that that creep does not contribute to the fatigue of normal human bone. Creep may make a significant contribution to fatigue in low-density osteoporotic bone in which trabeculae have resorbed, reducing the connectivity of the trabecular structure.     

Trabecular bone adaptation with an orthotropic material model.

Most bone adaptation algorithms, that attempt to explain the connection between bone morphology and loads, assume that bone is effectively isotropic. An isotropic material model can explain the bone density distribution, but not the structure and pattern of trabecular bone, which clearly has a mechanical significance. In this paper, an orthotropic material model is utilized to predict the proximal femur trabecular structure. Two hypotheses are combined to determine the local orientation and material properties of each element in the model. First, it is suggested that trabecular directions, which correspond to the orthotropic material axes, are determined locally by the maximal principal stress directions due to the multiple load cases (MLC) the femur is subject to. The second hypothesis is that material properties in each material direction can be determined using directional stimuli, thus extending existing adaptation algorithms to include directionality. An algorithm is utilized, where each iteration comprises of two stages. First, material axes are rotated to the direction of the largest principal stress that occurs from a multiple load scheme applied to the proximal femur. Next, material properties are modified in each material direction, according to a directional stimulus. Results show that local material directions correspond with known trabecular patterns, reproducing all main groups of trabeculae very well. The local directional stiffnesses, degree of anisotropy and density distribution are shown to conform to real femur morphology.     

Hip bone trabecular architecture shows uniquely distinctive locomotor behaviour in South African australopithecines

Cancellous bone retains structural and behavioural properties which are time and strain-rate dependent. As the orientation of the trabeculae (trajectories) follows the direction of the principal strains imposed by daily loadings, habitual postural and locomotor behaviours are responsible for a variety of trabecular architectures and site-specific textural arrangements of the pelvic cancellous network. With respect to the great ape condition, the human trabecular pattern is characterized by a distinctive ilioischial bundle, an undivided sacropubic bundle, and a full diagonal crossing (approximately 100 degrees) over the acetabulum between the ilioischial and the sacropubic bundles. Advanced digital image processing (DIP) of hip bone radiographs has revealed that adolescent and adult South African australopithecines retained an incompletely developed human-like trabecular pattern associated with gait-related features that are unique among the extant primates.     

Incorporating tissue anisotropy and heterogeneity in finite element models of trabecular bone altered predicted local stress distributions

Trabecular bone is composed of organized mineralized collagen fibrils, which results in heterogeneous and anisotropic mechanical properties at the tissue level. Recently, biomechanical models computing stresses and strains in trabecular bone have indicated a significant effect of tissue heterogeneity on predicted stresses and strains. However, the effect of the tissue-level mechanical anisotropy on the trabecular bone biomechanical response is unknown. Here, a computational method was established to automatically impose physiologically relevant orientation inherent in trabecular bone tissue on a trabecular bone microscale finite element model. Spatially varying tissue-level anisotropic elastic properties were then applied according to the bone mineral density and the local tissue orientation. The model was used to test the hypothesis that anisotropy in both homogeneous and heterogeneous models alters the predicted distribution of stress invariants. Linear elastic finite element computations were performed on a 3 mm cube model isolated from a microcomputed tomography scan of human trabecular bone from the distal femur. Hydrostatic stress and von Mises equivalent stress were recorded at every element, and the distributions of these values were analyzed. Anisotropy reduced the range of hydrostatic stress in both tension and compression more strongly than the associated increase in von Mises equivalent stress. The effect of anisotropy was independent of the spatial redistribution high compressive stresses due to tissue elastic heterogeneity. Tissue anisotropy and heterogeneity are likely important mechanisms to protect bone from failure and should be included for stress analyses in trabecular bone.