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The skeleton accommodates changes in mechanical environments by increasing bone mass under increased loads and decreasing bone mass under disuse. However, little is known about the adaptive changes in micromechanical behavior of cancellous and cortical tissues resulting from loading or disuse. To address this issue, in vivo tibial loading and hindlimb unloading experiments were conducted on 16-week-old female C57BL/6J mice. Changes in bone mass and tissue-level strains in the metaphyseal cancellous and midshaft cortical bone of the tibiae, resulting from loading or unloading, were determined using microCT and finite element (FE) analysis, respectively. We found that loading- and unloading-induced changes in bone mass were more pronounced in the cancellous than cortical bone. Simulated FE-loading showed that a greater proportion of elements experienced relatively lower longitudinal strains following load-induced bone adaptation, while the opposite was true in the disuse model. While the magnitudes of maximum or minimum principal strains in the metaphyseal cancellous and midshaft cortical bone were not affected by loading, strains oriented with the long axis were reduced in the load-adapted tibia suggesting that loading-induced micromechanical benefits were aligned primarily in the loading direction. Regression analyses demonstrated that bone mass was a good predictor of bone tissue strains for the cortical bone but not for the cancellous bone, which has complex microarchitecture and spatially-variant strain environments. In summary, loading-induced micromechanical benefits for cancellous and cortical tissues are received primarily in the direction of force application and cancellous bone mass may not be related to the micromechanics of cancellous bone.

Microcracking in trabecular bone is responsible both for the mechanical degradation and remodeling of the trabecular bone tissue. Recent results on trabecular bone mechanics have demonstrated that bone tissue microarchitecture, tissue elastic heterogeneity and tissue-level mechanical anisotropy all should be considered to obtain detailed information on the mechanical stress state. The present study investigated the influence of tissue microarchitecture, tissue heterogeneity in elasticity and material separation properties and tissue-level anisotropy on the microcrack formation process. Microscale bone models were executed with the extended finite element method. It was demonstrated that anisotropy and heterogeneity of the bone tissue contribute significantly to bone tissue toughness and the resistance of trabecular bone to microcrack formation. The compressive strain to microcrack initiation was computed to increase by a factor of four from an assumed homogeneous isotropic tissue to an assumed anisotropic heterogenous tissue.

Bone tissue is composed at the nanoscale of apatite minerals, collagen molecules and water that form the mineralized collagen fibril (MCF). Water has a crucial role in bone biomineralization. We developed a 3D random walk model to investigate the water diffusion process within the MCF for three different scenarios, namely low, intermediate and high mineral volume fraction. The MCF geometric model is obtained after applying 6·106 translational and rotational perturbations to an ordered arrangement of mineral. Subsequently, we compute 300 random trajectories of water molecules within the MCF for each mineral volume fraction. Every trajectory is constituted of up to 500 k positions of the water particle. We determined the diffusion coefficient from the linear fit of the mean squared displacement of water molecules as a function of time. We investigate changes in the diffusivity values in relation to variation of bone mineral content. The analysis performed on the random walk data, for all mineralization conditions, leads to diffusion coefficients in good agreement with the diffusivity outcomes achieved from previous experimental studies. Thus, the 3D geometrical configuration adopted in this numerical study appears suitable for modelling the MCF with different volume fractions, from hypo- to hyper-mineralized conditions. We observed that low mineral content is associated with an increase of the water diffusion, while lower values of diffusivity are determined in hypermineralized conditions. In agreement with experimental data, our results highlight the influence of the structural alterations on the mass transport properties.

Biomaterial based reconstruction is still the most commonly employed method of small bone defect reconstruction. Bone tissue-engineered techniques are improving, and adjuncts such as vascularization technologies allow re-evaluation of traditional reconstructive methods for healingofcritical-sized bone defect. Slow infiltration rate of vasculogenesis after cell-seeded scaffold implantation limits the use of clinically relevant large-sized scaffolds. Hence, in vitro vascularization within the tissue-engineered bone before implantation is required to overcome the serious challenge of low cell survival rate after implantation which affects bone tissue regeneration and osseointegration. Mechanobiological interactions between cells and microvascular mechanics regulate biological processes regarding cell behavior. In addition, load-bearing scaffolds demand mechanical stability properties after vascularization to have adequate strength while implanted. With the advent of bioreactors, vascularization has been greatly improved by biomechanical regulation of stem cell differentiation through fluid-induced shear stress and synergizing osteogenic and angiogenic differentiation in multispecies coculture cells. The benefits of vascularization are clear: avoidance of mass transfer limitation and oxygen deprivation, a significant decrease in cell necrosis, and consequently bone development, regeneration and remodeling. Here, we discuss specific techniques to avoid pitfalls and optimize vascularization results of tissue-engineered bone. Cell source, scaffold modifications and bioreactor design, and technique specifics all play a critical role in this new, and rapidly growing method for bone defect reconstruction. Given the crucial importance of long-term survival of vascular network in physiological function of 3D engineered-bone constructs, greater knowledge of vascularization approaches may lead to the development of new strategies towards stabilization of formed vascular structure.

Bone quality as well as its quantity at the implant interface is responsible for determining stability of the implant system. The objective of this study is to examine the nanoindentation based elastic modulus (E) at different bone regions adjacent to titanium dental implants with guided bone regeneration (GBR) treated with DBM and BMP-2 during different post-implantation periods. Six adult male beagle dogs were used to create circumferential defects with buccal bone removal at each implantation site of mandibles. The implant systems were randomly assigned to only GBR (control), GBR with demineralized bone matrix (DBM), and GBR with DBM + recombinant human bone morphogenetic protein-2 (rhBMP-2) (BMP) groups. Three animals were sacrificed at each 4 and 8 weeks of post-implantation healing periods. Following buccolingual dissection, the E values were assessed at the defects (Defect), interfacial bone tissue adjacent to the implant (Interface), and pre-existing bone tissue away from the implant (Pre-existing). The E values of BMP group had significantly higher than control and DBM groups for interface and defect regions at 4 weeks of post-implantation period and for the defect region at 8 weeks (p 0.043). DBM group had higher E values than control group only for the defect region at 4 weeks (p 0.001). The current results indicate that treatment of rhBMP-2 with GBR accelerates bone tissue mineralization for longer healing period because the GBR likely facilitates a microenvironment to provide more metabolites with open space of the defect region surrounding the implant.

Osteoporosis is a major medical burden and its impact is expected to increase in our aging society. It is associated with low bone density and microstructural deterioration. Treatments are available, but the critical factor is to define individuals at risk from osteoporotic fractures. Computational simulations investigating not only changes in net bone tissue volume, but also changes in its microstructure where osteoporotic deterioration occur might help to better predict the risk of fractures. In this study, bone remodeling simulations with a mechanical feedback loop were used to predict microstructural changes due to osteoporosis and their impact on bone fragility from 50 to 80 years of age. Starting from homeostatic bone remodeling of a group of seven, mixed sex whole vertebrae, five mechanostat models mimicking different biological alterations associated with osteoporosis were developed, leading to imbalanced bone formation and resorption with a total net loss of bone tissue. A model with reduced bone formation rate and cell sensitivity led to the best match of morphometric indices compared to literature data and was chosen to predict postmenopausal osteoporotic bone loss in the whole group. Thirty years of osteoporotic bone loss were predicted with changes in morphometric indices in agreement with experimental measurements, and only showing major deviations in trabecular number and trabecular separation. In particular, although being optimized to match to the morphometric indices alone, the predicted bone loss revealed realistic changes on the organ level and on biomechanical competence. While the osteoporotic bone was able to maintain the mechanical stability to a great extent, higher fragility towards error loads was found for the osteoporotic bones.

Osteoporosis leads to bone fragility and represents a major health problem in our aging societies. Bone is a quasi-brittle hierarchical composite that exhibits damage with distinct crack morphologies in compression and tension when overloaded. A recent study reported the complex damage response of bovine compact bone under four different cyclic overloading experiments combining compression and tension. The aim of the present work is to propose a mechanistic model by which cracking bone accumulates residual strain and reduces elastic modulus in distinct compressive and tensile overloading modes. A simple rheological unit of bone with two types of cracks is formulated in the framework of continuum damage mechanics. A statistics of these rheological units is then assembled in parallel to compute the response of a macroscopic bone sample in which compressive and tensile cracks are opened, closed or propagated towards failure. The resulting constitutive model reproduces the key macroscopic features of bone tissue damage and delivers an excellent agreement with the four cyclic overloading experiments. The remarkable predictions of the model support the presence of (1) friction between the crack surfaces producing hystereses, (2) an incomplete closure of cracks leading to residual strains, (3) a bridging mechanism of collagen fibrils which failure reduces elastic modulus, and (4) two distinct classes of cracks where compressive cracks have a strong influence on tensile damage and tensile cracks have a limited impact on compressive damage. This work is expected to help improve our understanding of the bone damage mechanisms contributing to skeletal fragility and to foster a proper generalization of this damage behavior in 3D for computational analysis of bone and bone-implant systems.

Bone quantity, or density, has insufficient power to discriminate fracture risk in individuals. Additional measures of bone quality, such as microarchitectural characteristics and bone tissue properties, including the presence of damage, may improve the diagnosis of fracture risk. Microdamage and microarchitecture are two aspects of trabecular bone quality that are interdependent, with several microarchitectural changes strongly correlated to damage risk after compensating for bone density. This study aimed to delineate the effects of microarchitecture and estrogen depletion on microdamage susceptibility in trabecular bone using an ovariectomized sheep model to mimic post-menopausal osteoporosis. The propensity for microdamage formation in trabecular bone of the distal femur was studied using a sequence of compressive and torsional overloads. Ovariectomy had only minor effects on the microarchitecture at this anatomic site. Microdamage was correlated to bone volume fraction and structure model index (SMI), and ovariectomy increased the sensitivity to these parameters. The latter may be due to either increased resorption cavities acting as stress concentrations or to altered bone tissue properties. Pre-existing damage was also correlated to new damage formation. However, sequential loading primarily generated new cracks as opposed to propagating existing cracks, suggesting that pre-existing microdamage contributes to further damage of bone by shifting load bearing to previously undamaged trabeculae, which are subsequently damaged. The transition from plate-like to rod-like trabeculae, indicated by SMI, dictates this shift, and may be a hallmark of bone that is already predisposed to accruing greater levels of damage through compromised microarchitecture.

Glenoid loosening is still a main complication for shoulder arthroplasty. We hypothesize that cement and bone stresses potentially leading to fixation failure are related not only to glenohumeral conformity, fixation design or eccentric loading, but also to bone volume fraction, cortical thickness and degree of anisotropy in the glenoid. In this study, periprosthetic bone and cement stresses were computed with micro finite element models of the replaced glenoid depicting realistic bone microstructure. These models were used to quantify potential effects of bone microstructural parameters under loading conditions simulating different levels of glenohumeral conformity and eccentric loading simulating glenohumeral instability. Results show that peak cement stresses were achieved near the cement-bone interface in all loading schemes. Higher stresses within trabecular bone tissue and cement mantle were obtained within specimens of lower bone volume fraction and in regions of low anisotropy, increasing with decreasing glenohumeral conformity and reaching their maxima below the keeled design when the load is shifted superiorly. Our analyses confirm the combined influences of eccentric load shifts with reduced bone volume fraction and anisotropy on increasing periprosthetic stresses. They finally suggest that improving fixation of glenoid replacements must reduce internal cement and bone tissue stresses, in particular in glenoids of low bone density and heterogeneity.

The structure of a bone tissue is capable of adapting to mechanical loading through the process of bone remodeling, which is regulated by osteoblasts and osteoclasts. Fluid flow within trabecular porosity under cyclic loading is one of the factors stimulating the biological response of osteoblasts and osteoclasts. However, the relation between loading directions and interstitial fluid flow was seldom studied. In the present study, a finite element model based on micro-computed tomographic reconstructions is built by using a mouse femur. Results from the fluid-solid coupling numerical simulation indicate that the loading in different directions generates a distinct distribution of von Mises stress in the bone matrix and a fluid shear stress (FSS) in the bone marrow. The loading along the physiological direction leads to a more uniform distribution of solid stress and produces an FSS level beneficial to the biological response of osteoblasts and osteoclasts compared with those along the non-physiological direction. There was a minimum threshold line of wall FSS with a specific solid stress at the bone surface, suggesting that the wall FSS is mainly induced by the solid strain. These results may offer fundamental data in understanding the mechanical environment around osteoblasts and osteoclasts and the cellular and molecular mechanisms of mechanical loading-induced bone remodeling.

Mechanical analysis of animal long bones often makes use of beam theory to estimate tissue properties from bending tests. An earlier study (van Lenthe et al., 2008) found that beam theory leads to a considerable underestimation of the Young's modulus of mice femora. However, we hypothesized that beam theory might still be an accurate tool for the determination of bone strength from experimental data. The first goal was to test this hypothesis, along with checking if the underestimation of the tissue modulus is also valid for rat femurs. The second goal was to investigate if micro-FE and beam theory would yield similar increases in Young's moduli and yield stress during aging. Twelve rat femurs (12 and 16 weeks old) were scanned using micro-CT and subjected to a three-point bending test from which the bending stiffness and the yield force were obtained. The Young's modulus and yield stress then were calculated by regressing the experimental results with results obtained from beam theory and micro-FE analysis based on the micro-CT scans. It was found that bone strength calculated using beam theory overestimated that calculated from micro-FE by 8.0%. The Young's modulus did not significantly differ. When comparing age groups, similar increases in tissue modulus and yield strength were found for beam theory and micro-FE, with significant differences only for the micro-FE yield stress. We concluded that the use of beam theory to calculate bone yield strength from 3-point bending test results of rat femurs leads to its overprediction.

Lumbar spinal fusion is the most common approach for treating spinal disorders such as degeneration or instability. Although this procedure has been performed for many years, there are still important challenges that must be overcome and questions that need to be addressed regarding the high rates of non-union. The present finite element model study aimed to investigate the influence of different cage designs on the fusion process. An axisymmetric finite element model of a spinal segment with an interbody fusion cage was used. The fusion process was based on an existing mechano-regulation algorithm for tissue formation. With this model, the following principal concepts of cage design were investigated: (1) different cage geometries with constant compressive stiffness and (2) cage designs optimized to provide the ideal mechanical stimulus for bone formation, first at the beginning of fusion and then throughout the entire fusion process. The cage geometry substantially influenced the fusion outcome. A cage that created an optimized initial mechanical stimulus did not necessarily lead to accelerated fusion, but rather resulted in delayed fusion or non-union. In contrast, a cage made of a degradable material produced a significantly higher amount of bone and resulted in higher segmental stiffness. However, different compressive loads (250, 500 and 1000 N) substantially affected the amount of newly formed bone tissue. The results of the present study suggest that aiming for an optimal initial mechanical stimulus may be misleading because the initial mechanical environment is not preserved throughout the bone modeling process.

The biomechanics literature contains many well-understood mechanisms behind typical fracture types that have important roles in treatment planning. The recent association of “atypical” fractures with long-term use of drugs designed to prevent osteoporosis has renewed interest in the effects of agents on bone tissue-level quality. While this class of fracture was recognized prior to the introduction of the anti-resorptive bisphosphonate drugs and recently likened to stress fractures, the mechanism(s) that lead to atypical fractures have not been definitively identified. Thus, a causal relationship between these drugs and atypical fracture has not been established. Physicians, bioengineers and others interested in the biomechanics of bone are working to improve fracture-prevention diagnostics, and the design of treatments to avoid this serious side-effect in the future. This review examines the mechanisms behind the bone tissue damage that may produce the atypical fracture pattern observed increasingly with long-term bisphosphonate use. Our recent findings and those of others reviewed support that the mechanisms behind normal, healthy excavation and tunnel filling by bone remodeling units within cortical tissue strengthen mechanical integrity. The ability of cortical bone to resist the damage induced during cyclic loading may be altered by the reduced remodeling and increased tissue age resulting from long-term bisphosphonate treatment. Development of assessments for such potential fractures would restore confidence in pharmaceutical treatments that have the potential to spare millions in our aging population from the morbidity and death that often follow bone fracture.

There are two main types of fluid in bone tissue, blood and interstitial fluid. The chemical composition of these fluids varies with time and location in bone. Blood arrives through the arterial system containing oxygen and other nutrients and the blood components depart via the venous system containing less oxygen and reduced nutrition. Within the bone, as within other tissues, substances pass from the blood through the arterial walls into the interstitial fluid. The movement of the interstitial fluid carries these substances to the cells within the bone and, at the same time, carries off the waste materials from the cells. Bone tissue would not live without these fluid movements. The development of a model for poroelastic materials with hierarchical pore space architecture for the description of blood flow and interstitial fluid flow in living bone tissue is reviewed. The model is applied to the problem of determining the exchange of pore fluid between the vascular porosity and the lacunar-canalicular porosity in bone tissue due to cyclic mechanical loading and blood pressure. These results are basic to the understanding of interstitial flow in bone tissue that, in turn, is basic to understanding of nutrient transport from the vasculature to the bone cells buried in the bone tissue and to the process of mechanotransduction by these cells.

The study of short implants is relevant to the biomechanics of dental implants, and research on crown increase has implications for the daily clinic. The aim of this study was to analyze the biomechanical interactions of a singular implant-supported prosthesis of different crown heights under vertical and oblique force, using the 3-D finite element method. Six 3-D models were designed with Invesalius 3.0, Rhinoceros 3D 4.0, and Solidworks 2010 software. Each model was constructed with a mandibular segment of bone block, including an implant supporting a screwed metal-ceramic crown. The crown height was set at 10, 12.5, and 15 mm. The applied force was 200 N (axial) and 100 N (oblique). We performed an ANOVA statistical test and Tukey tests; p0.05 was considered statistically significant. The increase of crown height did not influence the stress distribution on screw prosthetic (p0.05) under axial load. However, crown heights of 12.5 and 15 mm caused statistically significant damage to the stress distribution of screws and to the cortical bone (p0.001) under oblique load. High crown to implant (C/I) ratio harmed microstrain distribution on bone tissue under axial and oblique loads (p0.001). Crown increase was a possible deleterious factor to the screws and to the different regions of bone tissue.

Surgical bone drilling is performed variously to correct bone fractures, install prosthetics, or for therapeutic treatment. The primary concern in bone drilling is to extract donor bone sections and create receiving holes without damaging the bone tissue either mechanically or thermally. We review current results from experimental and theoretical studies to investigate the parameters related to such effects. This leads to a comprehensive understanding of the mechanical and thermal aspects of bone drilling to reduce their unwanted complications. This review examines the important bone-drilling parameters of bone structure, drill-bit geometry, operating conditions, and material evacuation, and considers the current techniques used in bone drilling. We then analyze the associated mechanical and thermal effects and their contributions to bone-drilling performance. In this review, we identify a favorable range for each parameter to reduce unwanted complications due to mechanical or thermal effects.

Non-collagenous proteins are a vital component of bone matrix. Amongst them, osteocalcin (OC) and osteopontin (OPN) hold special significance due to their intimate interaction with the mineral and collagenous matrix in bone. Both proteins have been associated with microdamage and fracture, but their structural role in energy dissipation is unclear. This study used bone tissue from genetic deficient mice lacking OC and/or OPN and subjected them to a series of creep-fatigue-creep tests. To this end, whole tibiae were loaded in four-point bending to 70% stiffness loss which captured the three characteristic phases of fatigue associated with initiation, propagation, and coalescence of microdamage. Fatigue loading preceded and followed creep tests to determine creep and dampening parameters. Microdamage in the form of linear microcracks and diffuse damage were analyzed by histology. It was shown that OC and OPN were 'activated' following stiffness loss associated with fatigue damage where they facilitated creep and dampening parameters (i.e. increased energy dissipation). More specifically, post-fatigue creep rate and dampening were significantly greater in wild-types (WTs) than genetic deficient mice (p 0.05). These results were supported by microdamage analysis which showed significant increase in creep-associated diffuse damage formation in WTs compared to genetic deficient groups (p 0.05). Based on these findings, we propose that during local yield events, OC and OPN rely on ionic interactions of their charged side chains and on hydrogen bonding to dissipate energy in bone.

Trabecular bone is viscoelastic under dynamic loading. However, it is unclear how tissue viscoelasticity controls viscoelasticity at the apparent-level. In this study, viscoelasticity of cylindrical human trabecular bone samples (n = 11, male, age 18–78 years) from 11 proximal femurs were characterized using dynamic and stress-relaxation testing at the apparent-level and with creep nanoindentation at the tissue-level. In addition, bone tissue elasticity was determined using scanning acoustic microscope (SAM). Tissue composition and collagen crosslinks were assessed using Raman micro-spectroscopy and high performance liquid chromatography (HPLC), respectively. Values of material parameters were obtained from finite element (FE) models by optimizing tissue-level creep and apparent-level stress-relaxation to experimental nanoindentation and unconfined compression testing values, respectively, utilizing the second order Prony series to depict viscoelasticity. FE simulations showed that tissue-level equilibrium elastic modulus (Eeq) increased with increasing crystallinity (r = 0.730, p = .011) while at the apparent-level it increased with increasing hydroxylysyl pyridinoline content (r = 0.718, p = .019). In addition, the normalized shear modulus g1 (r = −0.780, p = .005) decreased with increasing collagen ratio (amide III/CH2) at the tissue-level, but increased (r = 0.696, p = .025) with increasing collagen ratio at the apparent-level. No significant relations were found between the measured or simulated viscoelastic parameters at the tissue- and apparent-levels nor were the parameters related to tissue elasticity determined with SAM. However, only Eeq, g2 and relaxation time τ1 from simulated viscoelastic values were statistically different between tissue- and apparent-levels (p < .01). These findings indicate that bone tissue viscoelasticity is affected by tissue composition but may not fully predict the macroscale viscoelasticity in human trabecular bone., final draft, peerReviewed

The increased risk for fracture with age is associated not only with reduced bone mass but also with impaired bone quality. At the microscale, bone quality is related to porosity, microstructural organization, accumulated microdamage and intrinsic material properties. However, the link between these characteristics and fracture behavior is still missing. Bone tissue has a complex structure and as age-related compositional and structural changes occur at all hierarchical length scales it is difficult to experimentally identify and discriminate the effect of each mechanism. The aim of this study was therefore to use computational models to analyze how microscale characteristics in terms of porosity, intrinsic toughness properties and microstructural organization affect the mechanical behavior of cortical bone. Tensile tests were simulated using realistic microstructural geometries based on microscopy images of human cortical bone. Crack propagation was modelled using the extended finite element method where cement lines surrounding osteons were modelled with an interface damage law to capture crack deflections along osteon boundaries. Both increased porosity and impaired material integrity resulted in straighter crack paths with cracks penetrating osteons, similar to what is seen experimentally for old cortical bone. However, only the latter predicted a more brittle failure behavior. Furthermore, the local porosity influenced the crack path more than the macroscopic porosity. In conclusion, age-related changes in cortical bone affect the crack path and the mechanical response. However, increased porosity alone was not driving damage in old bone, but instead impaired tissue integrity was required to capture brittle failure in aging bone.

High-resolution in vivo bone micro-architecture assessment, as possible now for the distal forearm, in combination with bone remodelling simulation algorithms could, eventually, predict patient-specific bone morphology changes. To simulate load-adaptive bone remodelling, however, physiological loading conditions must be defined. In this paper we test a previously developed algorithm to estimate such physiological loading conditions from the bone micro-architecture. The aims of this study were to investigate if realistic boundary forces and moments are predicted for the scanned distal radius section and how these predicted forces and moments should be distributed to the scanned section in order to obtain a load transfer similar to that in situ. Images at in vivo resolution were generated for the clinically measured section of nine distal radius cadaver bones, converted to micro-finite element models and used for load estimation. Models of the full distal radius were created to analyse tissue loading distributions of the sections in situ. It was found that predicted forces and moments at the boundaries of the scanned region varied considerably but, when translated to equivalent radiocarpal joint forces, agreed well with values reported in the literature. Bone tissue loading distribution was in best agreement with in situ distributions when loading was applied to an extra layer of material at both ends of the clinical scan region. The agreement of the predicted loading to previous studies and the wide range of predicted loading values indicate that subject-specific bone loading estimation is possible and necessary.

Cortical bone has a hierarchical structure, spanning from the macrostructure at several millimeters or whole bone level, the microstructure at several hundred micrometers level, to the nanostructure at hydroxyapatite (HAp) crystals and collagen fibrils levels. The aim of the study is to understand the relationship between the HAp crystal orientation and the elastic modulus and the relationship between the osteon area fraction and the deformation behavior of HAp crystals in cortical bone. In the experiments, five strip specimens (40×2×1 mm 3 ) aligned with the bone axis were taken from the cortical bone of a bovine femur. The degree of c -axis orientation of HAp crystals in the specimens was measured with the X-ray diffraction technique with the imaging plate. To measure the deformation behavior of HAp crystals in the specimens, tensile tests under X-ray irradiation were conducted. The specimens were cut at the X-ray measurement positions and osteon area fraction and porosity at the transverse cross-sections were observed. Further, the volume fraction of HAp of the specimens was measured. Results showed the degree of c -axis orientation of HAp crystals was positively correlated with the elastic modulus of the specimens ( r =0.94). The volume fraction of HAp and the porosity showed no statistical correlation with the elastic modulus and the tensile strength. The HAp crystal strain e H increased linearly with the bone tissue strain e . The average value of e H / e was 0.69±0.13 and there was no correlation between the osteon area fraction and e H / e ( r =−0.27, p =0.33). The results suggest that the degree of c -axis orientation of HAp crystals affects the elastic modulus and the magnitude of HAp crystal strain does not depend on the osteon area fraction.

International audience; The mechanical properties of cortical bone are determined by a combination bone tissue composition, and structure at several hierarchical length scales. In this study the spatial distribution of tissue level properties within a human femoral shaft has been investigated. Cylindrically shaped samples (diameter: 4.4 mm, N=56) were prepared from cortical regions along the entire length (20–85% of the total femur length), and around the periphery (anterior, medial, posterior and lateral quadrants). The samples were analyzed using scanning acoustic microscopy (SAM) at 50 MHz and synchrotron radiation micro computed tomography (SRμCT). For all samples the average cortical porosity (Ct.Po), tissue elastic coefficients (cij) and the average tissue degree of mineralization (DMB) were determined. The smallest coefficient of variation was observed for DMB (1.8%), followed by BV/TV (5.4%), cij (8.2–45.5%), and Ct.Po (47.5%). Different variations with respect to the anatomical position were found for DMB, Ct.Po and cij. These data address the anatomical variations in anisotropic elastic properties and link them to tissue mineralization and porosity, which are important input parameters for numerical multi-scale bone models.

Microdamage in bone tissue is typically studied using destructive, two-dimensional histological techniques. Contrast-enhanced micro-computed tomography (micro-CT) was recently demonstrated to enable non-destructive, three-dimensional (3-D) detection of microdamage in machined cortical and trabecular bone specimens in vitro. However, the accumulation of microdamage in whole bones is influenced by variations in the magnitude and mode of loading due to the complex whole bone morphology. Therefore, the objective of this study was to detect the presence, spatial location, and accumulation of fatigue microdamage in whole rat femora in vitro using micro-CT with a BaSO(4) contrast agent. Microdamage was detected and observed to accumulate at specific spatial locations within the cortex of femora loaded in cyclic three-point bending to a 5% or 10% reduction in secant modulus. The ratio of the segmented BaSO(4) stain volume (SV) to the total volume (TV) of cortical bone was adopted as a measure of damage. The amount of microdamage measured by micro-CT (SV/TV) was significantly greater for both loaded groups compared to the control group (p0.05), but the difference between loaded groups was not statistically significant. At least one distinct region of microdamage, as indicated by the segmented SV, was observed in 85% of loaded specimens. A specimen-specific finite element model confirmed elevated tensile principal strains localized in regions of tissue corresponding to the accumulated microdamage. These regions were not always located where one might expect a priori based upon Euler-Bernoulli beam theory, demonstrating the utility of contrast-enhanced micro-CT for non-destructive, 3-D detection of fatigue microdamage in whole bones in vitro.

During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes, in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage.

This study investigated whether changes in hardness of human trabecular bone are associated with osteoarthritis. Twenty femoral heads extracted from subjects without musculoskeletal diseases (subject age: 49-83 years) and twenty femoral heads extracted from osteoarthritic subjects (subject age: 42-85 years) were tested. Sixty indentations were performed along the main trabecular direction of each sample at a fixed relative distance. Two microstructures were found on the indenting locations: packs of parallel-lamellae (PL) and secondary osteons (SO). A 25gf load was applied for 15s and the Vickers Hardness (HV) was assessed. Trabecular tissue extracted from osteoarthritic subjects was found to be about 13% less hard compared to tissue extracted from non-pathologic subjects. However, tissue hardness was not significantly affected by gender or age. The SO was 10% less hard than the PL for both pathologic and non-pathologic tissues. A hardness of 34.1HV for PL and 30.8HV for SO was found for the non-pathologic tissue. For osteoarthritic tissue, the hardness was 30.2HV for PL and 27.1HV for SO. In the bone tissue extracted from osteoarthritic subjects the occurrence of indenting a SO (28%) was higher than that observed in the non-pathological tissue (15%). Osteoarthritis is associated with reduced tissue hardness and alterations in microstructure of the trabecular bone tissue. Gender does not significantly affect trabecular bone hardness either in non-pathological or osteoarthritic subjects. A similar conclusion can be drawn for age, although a larger donor sample size would be necessary to definitively exclude the existence of a slight effect.

The presence of the residual stresses in bone tissue has been noted and the authors have reported that there are residual stresses in bone tissue. The aim of our study is to measure the residual stress distribution in the cortical bone of the extremities of vertebrates and to describe the relationships with the osteon population density. The study used the rabbit limb bones (femur, tibia/fibula, humerus, and radius/ulna) and measured the residual stresses in the bone axial direction at anterior and posterior positions on the cortical surface. The osteons at the sections at the measurement positions were observed by microscopy. As a result, the average stresses at the hindlimb bones and the forelimb bones were 210 and 149 MPa, respectively. In the femur, humerus, and radius/ulna, the residual stresses at the anterior position were larger than those at the posterior position, while in the tibia, the stress at the posterior position was larger than that at the anterior position. Further, in the femur and humerus, the osteon population densities in the anterior positions were larger than those in the posterior positions. In the tibia, the osteon population density in the posterior position was larger than that in the anterior position. Therefore, tensile residual stresses were observed at every measurement position in the rabbit limb bones and the value of residual stress correlated with the osteon population density (r=0.55, P

The goal of this retrospective article is to place the body of my lab's multiscale mechanobiology work in context of top-down and bottom-up engineering of bone. We have used biosystems engineering, computational modeling and novel experimental approaches to understand bone physiology, in health and disease, and across time (in utero, postnatal growth, maturity, aging and death, as well as evolution) and length scales (a single bone like a femur, m; a sample of bone tissue, mm-cm; a cell and its local environment, μm; down to the length scale of the cell's own skeleton, the cytoskeleton, nm). First we introduce the concept of flow in bone and the three calibers of porosity through which fluid flows. Then we describe, in the context of organ-tissue, tissue-cell and cell-molecule length scales, both multiscale computational models and experimental methods to predict flow in bone and to understand the flow of fluid as a means to deliver chemical and mechanical cues in bone. Addressing a number of studies in the context of multiple length and time scales, the importance of appropriate boundary conditions, site specific material parameters, permeability measures and even micro-nanoanatomically correct geometries are discussed in context of model predictions and their value for understanding multiscale mechanobiology of bone. Insights from these multiscale computational modeling and experimental methods are providing us with a means to predict, engineer and manufacture bone tissue in the laboratory and in the human body.

Uniformity of tissue mineralisation is a strongly debated issue, due to its relation with bone mechanical behaviour. Bone mineral density (BMD) is measured in the clinical practice and is applied in computational application to derive material properties of bone tissue. However, BMD cannot identify if the variation in bone density is related to a modification of tissue mineral density (TMD), a change in bone volume or a combination of the two. This study was aimed to investigate whether TMD can be assumed as a constant in adult human bone (trabecular and cortical). A total number of 115 cylindrical bone specimens were collected. An inter-site analysis (96 specimens, 2 donors) was performed on cortical and trabecular specimens extracted from different anatomical sites. An intra-site study (19 specimens, 19 donors) was performed on specimens extracted from femoral heads. Bone volume fraction (BV/TV) was computed by means of a micro-computed tomography. Furthermore, ash density (ρ(ash)) was measured. TMD was computed as the ratio between ρ(ash) and BV/TV. It was found that the TMD of trabecular (1.24 ± 0.16 g/cm(3)) and cortical (1.19 ± 0.06 g/cm(3)) bone were not statistically different (p=0.31). Furthermore, the linear regression between ρ(ash) and BV/TV was statistically significant (r(2)=0.99, p0.001). Intra- and inter-site analyses demonstrated that the mineral distribution was independent of the extraction site. The present study suggests that TMD can be assumed reasonably constant in non-pathological adult bone tissue. Consequently, it is suggested that TMD can be managed as a constant in computational models, varying only BV in relation to clinical densitometric analysis.

Although bone-tissue stiffness is closely related to the degree to which bone has been mineralized, other determinants are yet to be identified. We, therefore, examined the extent to which the mineralization degree, collagen, and its cross-links are related to bone-tissue stiffness. A total of 50 cancellous and cortical bone samples were derived from the right mandibular condyles of five young and five adult female pigs. The degree of mineralization of bone (DMB) was assessed using micro-computed tomography. Using high-performance liquid chromatography, we quantified the collagen content and the number of cross-links per collagen molecule of two enzymatic cross-links: hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and one non-enzymatic cross-link: pentosidine (Pen). Nanoindentation was used to assess bone-tissue stiffness in three directions, and multiple linear regressions were used to calculate the correlation between collagen properties and bone-tissue stiffness, with the DMB as first predictor. Whereas the bone-tissue stiffness of cancellous bone did not differ between the three directions of nanoindentation, or between the two age groups, cortical bone-tissue stiffness was higher in the adult tissue. After correction for DMB, the cross-links studied did not increase the explained variance. In the young group, however, LP significantly improved the explained variance in bone-tissue stiffness. Approximately half of the variation in bone-tissue stiffness in cancellous and cortical bone was explained by the DMB and the LP cross-links and thus they cannot be considered the sole determinants of the bone-tissue stiffness. © 2011 Elsevier Ltd.

A number of successful results have been reported in bone tissue engineering, although the routine clinical practice has not been reached so far. One of the reasons is the poor understanding of the role of each scaffold design parameter in its functional performance, which yields an uncertain outcome of each clinical application. Specifically, the role of internal scaffold microarchitectural shape on the regeneration rate and distribution of newly formed bone is still unknown. This work is focused on the in-silico determination of the role of scaffold microstructural anisotropy in bone tissue regeneration. A multiscale approach of the problem is established distinguishing between macroscopic region domain (bone organ and scaffold) and microscopic domain (scaffold microstructure). Results show that, once the scaffold microstructure is properly interconnected and the porosity is sufficiently high, similar rates of bone regeneration are found. However, the main conclusion of the work is that initial scaffold microstructural anisotropy has important consequences since it determines the spatial distribution of the newly formed tissue.

Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of bone growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs. High resolution micro computed tomography (Micro-CT) was performed 10 and 35 weeks post MSC implantation, followed by micro finite element (Micro-FE) analysis. The results have shown that the regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2 to 1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post treatment. These results could be attributed to the fusion that occurred between in the ulna and radius bones. In conclusion, although MSCs induce bone formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair.

in Undetermined Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n=8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoindentation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference. The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9–40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9–10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols.

Until date, many devices have been developed for cutting human bones during orthopedic surgeries. However, bones are anisotropic material, and their machining characteristics depend on the tool feed direction. In this study, microcutting of the bovine cortical bone is performed and its structure observed under a microscope. Furthermore, the formation of cutting chips and measurement of the cutting force during bone machining are dynamically observed while considering the anisotropy of bone tissue. In particular, the fracture of secondary osteons and crack propagation in bones are observed and analyzed. The results indicate that when the cut depth exceeds 20mum and is greater than the interval of concentric lamellae, cracks are formed together with chips. A new method for bone machining is proposed. This method is based on the characteristics of crack propagation in bones and is expected to produce low mechanical stress and realize highly efficient and precise machining of living tissues such as bones.

In this work, a three-dimensional model for bone remodeling is presented, taking into account the hierarchical structure of bone. The process of bone tissue adaptation is mathematically described with respect to functional demands, both mechanical and biological, to obtain the bone apparent density distribution (at the macroscale) and the trabecular structure (at the microscale). At global scale bone is assumed as a continuum material characterized by equivalent (homogenized) mechanical properties. At local scale a periodic cellular material model approaches bone trabecular anisotropy as well as bone surface area density. For each scale there is a material distribution problem governed by density-based design variables which at the global level can be identified with bone relative density. In order to show the potential of the model, a three-dimensional example of the proximal femur illustrates the distribution of bone apparent density as well as microstructural designs characterizing both anisotropy and bone surface area density. The bone apparent density numerical results show a good agreement with Dual-energy X-ray Absorptiometry (DXA) exams. The material symmetry distributions obtained are comparable to real bone microstructures depending on the local stress field. Furthermore, the compact bone porosity is modeled giving a transversal isotropic behavior close to the experimental data. Since, some computed microstructures have no permeability one concludes that bone tissue arrangement is not a simple stiffness maximization issue but biological factors also play an important role.

The elastic behaviour of trabecular bone is a function not only of bone volume and architecture, but also of tissue material properties. Variation in tissue modulus can have a substantial effect on the biomechanical properties of trabecular bone. However, the nature of tissue property variation within a single trabecula is poorly understood. This study uses nanoindentation to determine the mechanical properties of bone tissue in individual trabeculae. Using an ovariectomised ovine model, the modulus and hardness distribution across trabeculae were measured. In both normal and ovariectomised bone, the modulus and hardness were found to increase towards the core of the trabeculae. Across the width of the trabeculae, the modulus was significantly less in the ovariectomised bone than in the control bone. However, in contrast to this hardness was found not to differ significantly between the two groups. This study provides valuable information on the variation of mechanical material properties in healthy and diseased trabecular bone tissue. The results of the current study will be useful in finite element modelling where more accurate values of trabecular bone modulus will enable the prediction of the macroscale behaviour of trabecular bone.

An experimental-numerical study was performed to investigate the relationships between computed tomography (CT)-density and ash density, and between ash density and apparent density for bone tissue, to evaluate their influence on the accuracy of subject-specific FE models of human bones. Sixty cylindrical bone specimens were examined. CT-densities were computed from CT images while apparent and ash densities were measured experimentally. The CT/ash-density and ash/apparent-density relationships were calculated. Finite element models of eight human femurs were generated considering these relationships to assess their effect on strain prediction accuracy. CT and ash density were linearly correlated (R(2)=0.997) over the whole density range but not equivalent (intercep t0, slope1). A constant ash/apparent-density ratio (0.598+/-0.004) was found for cortical bone. A lower ratio, with a larger dispersion, was found for trabecular bone (0.459+/-0.100), but it became less dispersed, and equal to that of cortical tissue, when testing smaller trabecular specimens (0.598+/-0.036). This suggests that an experimental error occurred in apparent-density measurements for large trabecular specimens and a constant ratio can be assumed valid for the whole density range. Introducing the obtained relationships in the FE modelling procedure improved strain prediction accuracy (R(2)=0.95, RMSE=7%). The results suggest that: (i) a correction of the densitometric calibration should be used when evaluating bone ash-density from clinical CT scans, to avoid ash-density underestimation and overestimation for low- and high-density bone tissue, respectively; (ii) the ash/apparent-density ratio can be assumed constant in human femurs and (iii) the correction improves significantly the model accuracy and should be considered in subject-specific bone modelling.

Experimental evidence demonstrates that collagen cross-linking in bone tissue significantly influences its deformation and failure behavior yet difficulties exist in determining the independent biomechanical effects of collagen cross-linking using in vitro and in vivo experiments. The aim of this study is to use a nano-scale composite material model of mineral and collagen to determine the independent roles of enzymatic and non-enzymatic cross-linking on the mechanical behavior of a mineralized collagen fibril. Stress-strain curves were obtained under tensile loading conditions without any collagen cross-links, with only enzymatic cross-links (modeled by cross-linking the end terminal position of each collagen domain), or with only non-enzymatic cross-links (modeled by random placement of cross-links within the collagen-collagen interfaces). Our results show enzymatic collagen cross-links have minimal effect on the predicted stress-strain curve and produce a ductile material that fails through debonding of the mineral-collagen interface. Conversely, non-enzymatic cross-links significantly alter the predicted stress-strain response by inhibiting collagen sliding. This inhibition leads to greater load transfer to the mineral, which minimally affects the predicted stress, increases modulus and decreases post-yield strain and toughness. As a consequence the toughness of bone that has more non-enzymatically mediated collagen cross-links will be drastically reduced.

Trabecular bone strength is marked not only by the onset of local yielding, but also by post-yield behavior. To study and predict trabecular bone elastic and yield properties, micro-finite element (micro-FE) models were successfully applied. However, trabecular bone strength predictions require micro-FE models incorporating post-yield behavior of trabecular bone tissue. Due to experimental difficulties, such data is currently not available. Here we used micro-FE modeling to determine failure behavior of trabecular bone tissue indirectly, by iteratively fitting FE simulation to experimental results. Failure parameters were fitted to an isotropic plasticity model based on Hill's yield function, using materially and geometrically nonlinear micro-FE models of seven bovine trabecular bone specimens. The predictive value of the averaged effective tissue properties was subsequently tested. The results showed that compression softening had to be included on the tissue level in order to accurately describe the apparent-level behavior of the bone specimens. A sensitivity study revealed that the simulated response was less sensitive to variations in the post-yield properties of the bone tissue than variations in the elastic and yield properties. Due to fitting of the tissue properties, apparent-level behavior could be accurately reproduced for each specimen separately. Predictions based on the averaged and fixed tissue properties were less accurate, due to inter-specimen variations in the tissue properties.

Microdamage is an important component of bone quality and affects bone remodeling. Improved techniques to assess microdamage without the need for histological sectioning would provide insight into the role of microdamage in trabecular bone strength by allowing the spatial distribution of damage within the trabecular microstructure to be measured. Nineteen cylindrical trabecular bone specimens were prepared and assigned to two groups. The specimens in group I were damaged to 3% compressive strain and labeled with BaSO(4). Group II was not loaded, but was labeled with BaSO(4). Micro-computed tomography (Micro-CT) images of the specimens were obtained at 10 microm resolution. The median intensity of the treated bone tissue was compared between groups. Thresholding was also used to measure the damaged area fraction in the micro-CT scans. The histologically measured damaged area fraction, the median CT intensity, and the micro-CT measured damaged area fraction were all higher in the loaded group than in the unloaded group, indicating that the micro-CT images could differentiate the damaged specimen group from the unloaded specimens. The histologically measured damaged area fraction was positively correlated with the micro-CT measured damaged area fraction and with the median CT intensity of the bone, indicating that the micro-CT images can detect microdamage in trabecular bone with sufficient accuracy to differentiate damage levels between samples. This technique provides a means to non-invasively assess the three-dimensional distribution of microdamage within trabecular bone test specimens and could be used to gain insight into the role of trabecular architecture in microdamage formation.

Stem cell-based gene therapy and tissue engineering have been shown to be an efficient method for the regeneration of critical-sized bone defects. Despite being an area of active research over the last decade, no knowledge of the intrinsic ultrastructural and nanomechanical properties of such bone tissue exists. In this study, we report the nanomechanical properties of engineered bone tissue derived from genetically modified mesenchymal stem cells (MSCs) overexpressing the rhBMP2 gene, grown in vivo in the thigh muscle of immunocompetent mice for 4 weeks, compared to femoral bone adjacent to the transplantation site. The two types of bone had similar mineral contents (61 and 65 wt% for engineered and femoral bone, respectively), overall microstructures showing lacunae and canaliculi (both measured by back-scattered electron microscopy), chemical compositions (measured by energy dispersive X-ray analysis), and nanoscale topographical morphologies (measured by tapping-mode atomic force microscopy imaging or TMAFM). Nanoindentation experiments revealed that the small length scale mechanical properties were statistically different with the femoral bone (indented parallel to the bone long axis) being stiffer and harder (apparent elastic modulus, E approximately 27.3+/-10.5 GPa and hardness, H approximately 1.0+/-0.7G Pa) than the genetically engineered bone (E approximately 19.8+/-5.6 GPa, H approximately 0.9+/-0.4G Pa). TMAFM imaging showed clear residual indents characteristic of viscoelastic plastic deformation for both types of bone. However, fine differences in the residual indent area (smaller for the engineered bone), pile up (smaller for the engineered bone), and fracture mechanisms (microcracks for the engineered bone) were observed with the genetically engineered bone behaving more brittle than the femoral control.

A parametric finite element model of an osteocyte lacuna was developed to predict the microstructural response of the lacuna to imposed macroscopic strains. The model is composed of an osteocyte lacuna, a region of perilacunar tissue, canaliculi, and the surrounding bone tissue. A total of 45 different simulations were modeled with varying canalicular diameters, perilacunar tissue material moduli, and perilacunar tissue thicknesses. Maximum strain increased with a decrease in perilacunar tissue modulus and decreased with an increase in perilacunar tissue modulus, regardless of the thickness of the perilacunar region. An increase in the predicted maximum strain was observed with an increase in canalicular diameter from 0.362 to 0.421 microm. In response to the macroscopic application of strain, canalicular diameters increased 0.8% to over 1.0% depending on the perilacunar tissue modulus. Strain magnification factors of over 3 were predicted. However, varying the size of the perilacunar tissue region had no effect on the predicted perilacunar tissue strain. These results indicate that the application of average macroscopic strains similar to strain levels measured in vivo can result in significantly greater perilacunar tissue strains and canaliculi deformations. A decrease in the perilacunar tissue modulus amplifies the perilacunar tissue strain and canaliculi deformation while an increase in the local perilacunar tissue modulus attenuates this effect.

Assessment of the mechanical properties of trabecular bone is of major biological and clinical importance for the investigation of bone diseases, fractures and their treatments. Finite element (FE) methods are getting increasingly popular for quantifying the elastic and failure properties of trabecular bone. In particular, voxel-based FE methods have been previously used to calculate the effective elastic properties of trabecular microstructures. However, in most studies, bone tissue moduli were assumed or back-calculated to match the apparent elastic moduli from experiments, which often lead to surprisingly low values when compared to nanoindentation results. In this study, voxel-based FE analysis of trabecular bone is combined with physical measures of volume fraction, micro-CT (microCT) reconstructions, uniaxial mechanical tests and specimen-specific nanoindentation tests for proper validation of the method. Cylindrical specimens of cancellous bone were extracted from human femurs and their volume fraction determined with Archimede's method. Uniaxial apparent modulus of the specimens was measured with an improved tension-compression testing protocol that minimizes boundary artefacts. Their microCT reconstructions were segmented to match the measured bone volume fraction and used to create full-size voxel models with 30-45 microm element size. For each specimen, linear isotropic elastic material properties were defined based on specific nanoindentation measurements of its embedded bone tissue. Linear FE analyses were finally performed to simulate the uniaxial mechanical tests. Additional parametric analyses were performed to evaluate the potential errors on the predicted apparent modulus arising from variations in segmentation threshold, tissue modulus, and the use of 125-mm(3) cubic sub-regions. The results demonstrate an excellent correspondence between experimental measures and FE predictions of uniaxial apparent modulus. In conclusion, the adopted voxel-based FE approach is found to be a robust method to predict the linear elastic properties of human cancellous bone, provided segmentation of the microCT reconstructions is carefully calibrated, tissue modulus is known a priori and the entire region of interest is included in the analysis.

The mechanical behavior of bone tissue's ultra- and micro- structure is fundamental to assessment of macroscopic bone mechanics. This paper explores the ultra-structural characteristics of human femoral tissue responsible for energy absorption of secondary osteons under mechanical loading. A novel mathematical interpretation of single osteon mechanics elucidates the behavior of the collagen-apatite interface. Fully calcified single osteon specimens were mechanically tested quasi-statically under cyclic torsional loading about their longitudinal axis. On each hysteretic diagram, all cycles after the initial monotonic cycle appear pinched and share two points. Stiffness degradation and pinching degradation were investigated on the torque versus deflection-angle-per-unit-length diagrams as the number of cycles increases, in relation to the appearance of osteons in cross-section under circularly polarized light microscopy. Material science's Bauschinger effect, originally defined for metals and later extended to structures reinforced with metal bars, is adapted to describe pinching. Material science's prying effect, defined as amplification of eccentric tensile load through lever action, is employed to explain pinching. The presence of the two points shared by all complete cycles is analyzed in terms of the mathematical fixed point theorem. The results allow formulation of the following conjectures: (1) the prying of carbonated apatite crystallites at the interface with the 40 nm long bands of non-calcified collagen fibrils causes pinching; (2) the prying effect increases with the increasing percentage of collagen-apatite elements that form a larger angle with the osteon axis; and (3) micro-cracks increase more in number than in length as the number of cycles increases.

Mechanical signalling plays a pivotal role in maintaining bone cell function and remodelling of the skeleton. Our previous work has highlighted the potential role of mechano-induction in tissue engineering applications. In particular, we have highlighted the potential for using magnetic particle techniques for tissue engineering applications. Previous studies have shown that manipulation of integrin attached magnetic particles leads to changes in intracellular calcium signalling within osteoblasts. However, due to the phenomenon of particle internalisation, previous studies have typically focused on short-term stimulation experiments performed within 1-2 h of particle attachment. For tissue engineering applications, bone tissue growth occurs over a period of 3-5 weeks. To date, no study has investigated the cellular responses elicited from osteoblasts over time following stimulation with internalised magnetic particles. Here, we demonstrate the long-term biocompatibility of 4.5 microm RGD-coated particles with osteoblasts up to 21 days in culture, and detail a time course of responses elicited from osteoblasts following mechanical stimulation with integrin attached magnetic particles (2h post attachment) and internalised particles (48h post attachment). Mechanical manipulation of both integrin attached and internalised particles were found to induce intracellular calcium signalling. It is concluded that magnetic particles offer a tool for applying controlled mechanical forces to osteoblasts, and can be used to stimulate intracellular calcium signalling over prolonged periods of time. Magnetic particle technology presents a potentially valuable tool for tissue engineering which permits the delivery of highly localised mechano-inductive forces directly to cells.

Cortical bone is a composite material composed of hydroxyapatite (HAp) and collagen. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the strain of HAp crystals. However, HAp crystals in bone tissue have been known to have the low degree of crystallization. Authors have proposed an X-ray diffraction method to measure the lattice strain of HAp crystals from the diffusive intensity profile due to low crystallinity. The precision of strain measurement was greatly improved by this method. In order to confirm the possibility of estimating the bone tissue strain with measurements of the strain of HAp crystals, this work investigates the relationship between bone tissue strain on a macroscopic scale and the lattice strain of HAp crystals on a microscopic scale. The X-ray diffraction experiments were performed under tensile loading. Strip bone specimens of 40×6×0.8 mm in size were cut from the cortical region of a shaft of bovine femur. A stepwise tensile load was applied in the longitudinal direction of the specimen. By detecting the diffracted X-ray beam transmitted through the specimen, the lattice strain was directly measured in the loading direction. As a result, the lattice strain of HAp crystals showed lower value than the bone tissue strain measured by a strain gage. The bone tissue strain was described with the mean lattice strain of the HAp crystals and the elastic modulus.

Cell-sheet techniques have been proven effective in various soft tissue engineering applications. In this experiment, we investigated the feasibility of bone tissue engineering using a hybrid of mesenchymal stem cell (MSC) sheets and PLGA meshes. Porcine MSCs were cultured to a thin layer of cell sheets via osteogenic induction. Tube-like long bones were constructed by wrapping the cell sheet on to PLGA meshes resulting in constructs which could be cultured in spinner flasks, prior to implantation in nude rats. Our results showed that the sheets were composed of viable cells and dense matrix with a thickness of about 80-120 microm, mineral deposition was also observed in the sheet. In vitro cultures demonstrated calcified cartilage-like tissue formation and most PLGA meshes were absorbed during the 8-week culture period. In vivo experiments revealed that dense mineralized tissue was formed in subcutaneous sites and the 8-week plants shared similar micro-CT characteristics with native bone. The neo tissue demonstrated histological markers for both bone and cartilage, indicating that the bone formation pathway in constructs was akin to endochondral ossification, with the residues of PLGA having an effect on the neo tissue organization and formation. These results indicate that cell-sheet approaches in combination with custom-shaped scaffolds have potential in producing bone tissue.

Bone adapts to mechanical stimuli. While in vivo mechanical loading has been shown to increase the density of cancellous bone, theory suggests that the relationship between tissue stress/strain and subsequent bone formation occurs at the scale of individual trabeculae. Here we examine bone formation one week following mechanical stimulus. Three bouts of cyclic loading (300 cycles/day on 3 consecutive days) were applied to caudal vertebrae of female rats (n=7). Bone formation was determined using three-dimensional images of fluorescent markers of bone formation (0.7×0.7×5.0μm(3)) and local tissue stress/strain was determined using high-resolution finite element models. Three days of mechanical stimuli resulted in an increase in mineralizing surface (loaded: 17.68±2.17%; control: 9.05±3.20%; mean±SD) and an increase in the volume of bone formed (loaded: 7.09±1.97%; control: 1.44±0.50%). The number of bone formation sites was greater in loaded animals (650.71±118.54) than pinned not loaded controls (310.71±91.55), a difference that was explained by the number of formation sites at regions with large local tissue strain energy density (SED). In addition, the probability of observing bone formation was greater at locations of the microstructure experiencing greater SED, but did not exceed 32%, consistent with prior work. Our findings demonstrate that bone formation in the week following a short term mechanical stimulus occurs near regions of bone tissue experiencing high tissue SED, although the ability of finite element models to predict the locations of bone formation remains modest and further improvements may require accounting for additional factors such as osteocyte distribution or fluid flow.

The role of bone water in the activity of this organ is essential in structuring apatite crystals, providing pathways for nutrients and waste involved in the metabolism of bone cells and participating in bone remodelling mechanotransduction. It is commonly accepted that bone presents three levels of porosity, namely the vasculature, the lacuno-canalicular system and the voids of the collagen-apatite matrix. Due to the observation of bound state of water at the latter level, the interstitial nanoscopic flow that may exist within these pores is classically neglected. The aim of this paper is to investigate the possibility to obtain a fluid flow at the nanoscale. That is why a molecular dynamics based analysis of a water-hydroxyapatite system is proposed to analyze the effect of water confinement on transport properties. The main result here is that free water can be observed inside hydroxyapatite pores of a few nanometers. This result would have strong implications in the multiscale treatment of the poromechanical behaviour of bone tissue. In particular, the mechanical properties of the bone matrix may be highly controlled by nanoscopic water diffusion and the classical idea that osteocytic activity is only regulated by bone fluid flow within the lacuno-canalicular system may be discussed again.