QCT-based computational bone strength assessment updated with MRI-derived 'hidden' microporosity.

Microdamage accumulated through sustained periods of cyclic loading or single overloading events contributes to bone fragility through a reduction in stiffness and strength. Monitoring microdamage in vivo remains unattainable by clinical imaging modalities. As such, there are no established computational methods for clinical fracture risk assessment that account for microdamage that exists in vivo at any specific timepoint. We propose a method that combines multiple clinical imaging modalities to identify an indicative surrogate, which we term 'hidden porosity', that incorporates pre-existing bone microdamage in vivo. To do so, we use the third metacarpal bone of the equine athlete as an exemplary model for fatigue induced microdamage, which coalesces in the subchondral bone. N = 10 metacarpals were scanned by clinical quantitative computed tomography (QCT) and magnetic resonance imaging (MRI). We used a patch-based similarity method to quantify the signal intensity of a fluid sensitive MRI sequence in bone regions where microdamage coalesces. The method generated MRI-derived pseudoCT images which were then used to determine a pre-existing damage (Dpex) variable to quantify the proposed surrogate and which we incorporate into a nonlinear constitutive model for bone tissue. The minimum, median, and maximum detected Dpex of 0.059, 0.209, and 0.353 reduced material stiffness by 5.9%, 20.9%, and 35.3% as well as yield stress by 5.9%, 20.3%, and 35.3%. Limb-specific voxel-based finite element meshes were equipped with the updated material model. Lateral and medial condyles of each metacarpal were loaded to simulate physiological joint loading during gallop. The degree of detected Dpex correlated with a relative reduction in both condylar stiffness (p = 0.001, R2 > 0.74) and strength (p < 0.001, R2 > 0.80). Our results illustrate the complementary value of looking beyond clinical CT, which neglects the inclusion of microdamage due to partial volume effects. As we use clinically available imaging techniques, our results may aid research beyond the equine model on fracture risk assessment in human diseases such as osteoarthritis, bone cancer, or osteoporosis.

In situ synchrotron radiation µCT indentation of cortical bone: Anisotropic crack propagation, local deformation, and fracture.

The development of treatment strategies for skeletal diseases relies on the understanding of bone mechanical properties in relation to its structure at different length scales. At the microscale, indention techniques can be used to evaluate the elastic, plastic, and fracture behaviour of bone tissue. Here, we combined in situ high-resolution SRµCT indentation testing and digital volume correlation to elucidate the anisotropic crack propagation, deformation, and fracture of ovine cortical bone under Berkovich and spherical tips. Independently of the indenter type we observed significant dependence of the crack development due to the anisotropy ahead of the tip, with lower strains and smaller crack systems developing in samples indented in the transverse material direction, where the fibrillar bone ultrastructure is largely aligned perpendicular to the indentation direction. Such alignment allows to accommodate the strain energy, inhibiting crack propagation. Higher tensile hoop strains generally correlated with regions that display significant cracking radial to the indenter, indicating a predominant Mode I fracture. This was confirmed by the three-dimensional analysis of crack opening displacements and stress intensity factors along the crack front obtained for the first time from full displacement fields in bone tissue. The X-ray beam significantly influenced the relaxation behaviour independent of the tip. Raman analyses did not show significant changes in specimen composition after irradiation compared to non-irradiated tissue, suggesting an embrittlement process that may be linked to damage of the non-fibrillar organic matrix. This study highlights the importance of three-dimensional investigation of bone deformation and fracture behaviour to explore the mechanisms of bone failure in relation to structural changes due to ageing or disease. STATEMENT OF SIGNIFICANCE: Characterising the three-dimensional deformation and fracture behaviour of bone remains essential to decipher the interplay between structure, function, and composition with the aim to improve fracture prevention strategies. The experimental methodology presented here, combining high-resolution imaging, indentation testing and digital volume correlation, allows us to quantify the local deformation, crack propagation, and fracture modes of cortical bone tissue. Our results highlight the anisotropic behaviour of osteonal bone and the complex crack propagation patterns and fracture modes initiating by the intricate stress states beneath the indenter tip. This is of wide interest not only for the understanding of bone fracture but also to understand other architectured (bio)structures providing an effective way to quantify their toughening mechanisms in relation to their main mechanical function.

Nonlinear micro finite element models based on digital volume correlation measurements predict early microdamage in newly formed bone.

Bone regeneration in critical-sized defects is a clinical challenge, with biomaterials under constant development aiming at enhancing the natural bone healing process. The delivery of bone morphogenetic proteins (BMPs) in appropriate carriers represents a promising strategy for bone defect treatment but optimisation of the spatial-temporal release is still needed for the regeneration of bone with biological, structural, and mechanical properties comparable to the native tissue. Nonlinear micro finite element (µFE) models can address some of these challenges by providing a tool able to predict the biomechanical strength and microdamage onset in newly formed bone when subjected to physiological or supraphysiological loads. Yet, these models need to be validated against experimental data. In this study, experimental local displacements in newly formed bone induced by osteoinductive biomaterials subjected to in situ X-ray computed tomography compression in the apparent elastic regime and measured using digital volume correlation (DVC) were used to validate µFE models. Displacement predictions from homogeneous linear µFE models were highly correlated to DVC-measured local displacements, while tissue heterogeneity capturing mineralisation differences showed negligible effects. Nonlinear µFE models improved the correlation and showed that tissue microdamage occurs at low apparent strains. Microdamage seemed to occur next to large cavities or in biomaterial-induced thin trabeculae, independent of the mineralisation. While localisation of plastic strain accumulation was similar, the amount of damage accumulated in these locations was slightly higher when including material heterogeneity. These results demonstrate the ability of the nonlinear µFE model to capture local microdamage in newly formed bone tissue and can be exploited to improve the current understanding of healing bone and mechanical competence. This will ultimately aid the development of BMPs delivery systems for bone defect treatment able to regenerate bone with optimal biological, mechanical, and structural properties.

Multiscale mechanical consequences of ocean acidification for cold-water corals.

Ocean acidification is a threat to deep-sea corals and could lead to dramatic and rapid loss of the reef framework habitat they build. Weakening of structurally critical parts of the coral reef framework can lead to physical habitat collapse on an ecosystem scale, reducing the potential for biodiversity support. The mechanism underpinning crumbling and collapse of corals can be described via a combination of laboratory-scale experiments and mathematical and computational models. We synthesise data from electron back-scatter diffraction, micro-computed tomography, and micromechanical experiments, supplemented by molecular dynamics and continuum micromechanics simulations to predict failure of coral structures under increasing porosity and dissolution. Results reveal remarkable mechanical properties of the building material of cold-water coral skeletons of 462 MPa compressive strength and 45-67 GPa stiffness. This is 10 times stronger than concrete, twice as strong as ultrahigh performance fibre reinforced concrete, or nacre. Contrary to what would be expected, CWCs retain the strength of their skeletal building material despite a loss of its stiffness even when synthesised under future oceanic conditions. As this is on the material length-scale, it is independent of increasing porosity from exposure to corrosive water or bioerosion. Our models then illustrate how small increases in porosity lead to significantly increased risk of crumbling coral habitat. This new understanding, combined with projections of how seawater chemistry will change over the coming decades, will help support future conservation and management efforts of these vulnerable marine ecosystems by identifying which ecosystems are at risk and when they will be at risk, allowing assessment of the impact upon associated biodiversity.

Deep learning approach to assess damage mechanics of bone tissue.

Machine learning methods have the potential to transform imaging techniques and analysis for healthcare applications with automation, making diagnostics and treatment more accurate and efficient, as well as to provide mechanistic insights into tissue deformation and fracture in physiological and pathological conditions. Here we report an exploratory investigation for the classification and prediction of mechanical states of cortical and trabecular bone tissue using convolutional neural networks (CNNs), residual neural networks (ResNet), and transfer learning applied to a novel dataset derived from high-resolution synchrotron-radiation micro-computed tomography (SR-microCT) images acquired in uniaxial continuous compression in situ. We present the systematic optimization of CNN architectures for classification of this dataset, visualization of class-defining features detected by the CNNs using gradient class activation maps (Grad-CAMs), comparison of CNN performance with ResNet and transfer learning models, and perhaps most critically, the challenges that arose from applying machine learning methods to an experimentally-derived dataset for the first time. With optimized CNN architectures, we obtained trained models that classified novel images between failed and pristine classes with over 98% accuracy for cortical bone and over 90% accuracy for trabecular bone. Harnessing a pre-trained ResNet with transfer learning, we further achieved over 98% accuracy on the cortical dataset, and 99% on the trabecular dataset. This demonstrates that powerful classifiers for high-resolution SR-microCT images can be developed even with few unique training samples and invites further development through the inclusion of more data and training methods to move towards novel, fundamental, and machine learning-driven insights into microstructural states and properties of bone.

Time-resolved in situ synchrotron-microCT: 4D deformation of bone and bone analogues using digital volume correlation.

Digital volume correlation (DVC) in combination with high-resolution micro-computed tomography (microCT) imaging and in situ mechanical testing is gaining popularity for quantifying 3D full-field strains in bone and biomaterials. However, traditional in situ time-lapsed (i.e., interrupted) mechanical testing cannot fully capture the dynamic strain mechanisms in viscoelastic biological materials. The aim of this study was to investigate the time-resolved deformation of bone structures and analogues via continuous in situ synchrotron-radiation microCT (SR-microCT) compression and DVC to gain a better insight into their structure-function relationships. Fast SR-microCT imaging enabled the deformation behaviour to be captured with high temporal and spatial resolution. Time-resolved DVC highlighted the relationship between local strains and damage initiation and progression in the different biostructures undergoing plastic deformation, bending and/or buckling of their main microstructural elements. The results showed that SR-microCT continuous mechanical testing complemented and enhanced the information obtained from time-lapsed testing, which may underestimate the 3D strain magnitudes as a result of the stress relaxation occurring in between steps before image acquisition in porous biomaterials. Altogether, the findings of this study highlight the importance of time-resolved in situ experiments to fully characterise the time-dependent mechanical behaviour of biological tissues and biomaterials and to further explore their micromechanics under physiologically relevant conditions. STATEMENT OF SIGNIFICANCE: Time-resolved synchrotron X-ray tomography in combination with in situ mechanical testing provided the first four-dimensional analysis of the mechanical deformation of bone and bone analogues. To unravel the interplay of damage initiation and progression with local deformation, digital volume correlation was used to map the local strain field while microstructural changes were tracked with high temporal and spatial resolution. The results highlighted the importance of fast imaging and time-resolved in situ experiments to capture the real deformation of complex porous materials to fully characterize the local strain-damage relationship. The findings are notably improving the understanding of time-dependent mechanical behaviour of bone tissue, with the potential to be extend to highly viscoelastic biomaterials and soft tissues.

Low-cycle full-field residual strains in cortical bone and their influence on tissue fracture evaluated via in situ stepwise and continuous X-ray computed tomography.

As a composite material, the mechanical properties of bone are highly dependent on its hierarchical organisation, thus, macroscopic mechanical properties are dictated by local phenomena, such as microdamage resulting from repetitive cyclic loading of daily activities. Such microdamage is associated with plastic deformation and appears as a gradual accumulation of residual strains. The aim of this study is to investigate local residual strains in cortical bone tissue following compressive cyclic loading, using in situ X-ray computed tomography (XCT) and digital volume correlation (DVC) to provide a deeper insight on the three-dimensional (3D) relationship between residual strain accumulation, cortical bone microstructure and failure patterns. Through a progressive in situ XCT loading-unloading scheme, localisation of local residual strains was observed in highly compressed regions. In addition, a multi-scale in situ XCT cyclic test highlighted the differences on residual strain distribution at the microscale and tissue level, where high strains were observed in regions with the thinnest vascular canals and predicted the failure location following overloading. Finally, through a continuous in situ XCT compression test of cycled specimens, the full-field strain evolution and failure pattern indicated the reduced ability of bone to plastically deform after damage accumulation due to high number of cyclic loads. Altogether, the novel experimental methods employed in this study, combining high-resolution in situ XCT mechanics and DVC, showed a great potential to investigate 3D full-field residual strain development under repetitive loading and its complex interaction with bone microstructure, microdamage and fracture.

Full-Field Strain Uncertainties and Residuals at the Cartilage-Bone Interface in Unstained Tissues Using Propagation-Based Phase-Contrast XCT and Digital Volume Correlation.

A deeper understanding of the cartilage-bone mechanics is fundamental to unravel onset and progression of osteoarthritis, enabling better diagnosis and treatment. The aim of this study is therefore to explore the capability of X-ray computed (XCT) phase-contrast imaging in a lab-based system to enable digital volume correlation (DVC) measurements of unstained cartilage-bone plugs from healthy adult bovines. DVC strain uncertainties were computed for both articular cartilage and mineralized tissue (calcified cartilage and subchondral bone) in the specimens at increasing propagation distances, ranging from absorption up to four times (4× such effective distance. In addition, a process of dehydration and rehydration was proposed to improve feature recognition in XCT of articular cartilage and mechanical properties of this tissue during the process were assessed via micromechanical probing (indentation), which was also used to determine the effect of long X-ray exposure. Finally, full-field strain from DVC was computed to quantify residual strain distribution at the cartilage-bone interface following unconfined compression test (ex situ). It was found that enhanced gray-scale feature recognition at the cartilage-bone interface was achieved using phase-contrast, resulting in reduced DVC strain uncertainties compared to absorption. Residual strains up to ~7000 µÎµ in the articular cartilage were transferred to subchondral bone via the calcified cartilage and micromechanics revealed the predominant effect of long phase-contrast X-ray exposure in reducing both stiffness and hardness of the articular cartilage. The results of this study will pave the way for further development and refinement of the techniques, improving XCT-based strain measurements in cartilage-bone and other soft-hard tissue interfaces.

Centre of Rotation of the Human Subtalar Joint Using Weight-Bearing Clinical Computed Tomography.

Accurate in vivo quantification of subtalar joint kinematics can provide important information for the clinical evaluation of subtalar joint function; the analysis of outcome of surgical procedures of the hindfoot; and the design of a replacement subtalar joint prosthesis. The objective of the current study was to explore the potential of full weight-bearing clinical computed tomography (CT) to evaluate the helical axis and centre of rotation of the subtalar joint during inversion and eversion motion. A subject specific methodology was proposed for the definition of the subtalar joint motion combining three-dimensional (3D) weight-bearing imaging at different joint positions with digital volume correlation (DVC). The computed subtalar joint helical axis parameters showed consistency across all healthy subjects and in line with previous data under simulated loads. A sphere fitting approach was introduced for the computation of subtalar joint centre of rotation, which allows to demonstrate that this centre of rotation is located in the middle facet of the subtalar joint. Some translation along the helical axis was also observed, reflecting the elasticity of the soft-tissue restraints. This study showed a novel technique for non-invasive quantitative analysis of bone-to-bone motion under full weight-bearing of the hindfoot. Identifying different joint kinematics in patients with ligamentous laxity and instability, or in the presence of stiffness and arthritis, could help clinicians to define optimal patient-specific treatments.

Exploratory Full-Field Strain Analysis of Regenerated Bone Tissue from Osteoinductive Biomaterials.

Biomaterials for bone regeneration are constantly under development, and their application in critical-sized defects represents a promising alternative to bone grafting techniques. However, the ability of all these materials to produce bone mechanically comparable with the native tissue remains unclear. This study aims to explore the full-field strain evolution in newly formed bone tissue produced in vivo by different osteoinductive strategies, including delivery systems for BMP-2 release. In situ high-resolution X-ray micro-computed tomography (microCT) and digital volume correlation (DVC) were used to qualitatively assess the micromechanics of regenerated bone tissue. Local strain in the tissue was evaluated in relation to the different bone morphometry and mineralization for specimens (n = 2 p/treatment) retrieved at a single time point (10 weeks in vivo). Results indicated a variety of load-transfer ability for the different treatments, highlighting the mechanical adaptation of bone structure in the early stages of bone healing. Although exploratory due to the limited sample size, the findings and analysis reported herein suggest how the combination of microCT and DVC can provide enhanced understanding of the micromechanics of newly formed bone produced in vivo, with the potential to inform further development of novel bone regeneration approaches.

Full-Field Strain Analysis of Bone-Biomaterial Systems Produced by the Implantation of Osteoregenerative Biomaterials in an Ovine Model.

Osteoregenerative biomaterials for the treatment of bone defects are under much development, with the aim of favoring osteointegration up to complete bone regeneration. A detailed investigation of bone-biomaterial integration is vital to understand and predict the ability of such materials to promote bone formation, preventing further bone damage and supporting load-bearing regions. This study aims to characterize the ex vivo micromechanics and microdamage evolution of bone-biomaterial systems at the tissue level, combining high-resolution synchrotron microcomputed tomography, in situ mechanics and digital volume correlation. Results showed that the main microfailure events were localized close to or within the newly formed bone tissue, in proximity to the bone-biomaterial interface. The apparent nominal compressive load applied to the composite structures resulted in a complex loading scenario, mainly due to the higher heterogeneity but also to the different biomaterial degradation mechanisms. The full-field strain distribution allowed characterization of microdamage initiation and progression. The findings reported in this study provide a deeper insight into bone-biomaterial integration and micromechanics in relation to the osteoregeneration achieved in vivo for a variety of biomaterials. This could ultimately be used to improve bone tissue regeneration strategies.

Preservation of Bone Tissue Integrity with Temperature Control for In Situ SR-MicroCT Experiments.

Digital volume correlation (DVC), combined with in situ synchrotron microcomputed tomography (SR-microCT) mechanics, allows for 3D full-field strain measurement in bone at the tissue level. However, long exposures to SR radiation are known to induce bone damage, and reliable experimental protocols able to preserve tissue properties are still lacking. This study aims to propose a proof-of-concept methodology to retain bone tissue integrity, based on residual strain determination using DVC, by decreasing the environmental temperature during in situ SR-microCT testing. Compact and trabecular bone specimens underwent five consecutive full tomographic data collections either at room temperature or 0 °C. Lowering the temperature seemed to reduce microdamage in trabecular bone but had minimal effect on compact bone. A consistent temperature gradient was measured at each exposure period, and its prolonged effect over time may induce localised collagen denaturation and subsequent damage. DVC provided useful information on irradiation-induced microcrack initiation and propagation. Future work is necessary to apply these findings to in situ SR-microCT mechanical tests, and to establish protocols aiming to minimise the SR irradiation-induced damage of bone.

Effect of SR-microCT radiation on the mechanical integrity of trabecular bone using in situ mechanical testing and digital volume correlation.

The use of synchrotron radiation micro-computed tomography (SR-microCT) is becoming increasingly popular for studying the relationship between microstructure and bone mechanics subjected to in situ mechanical testing. However, it is well known that the effect of SR X-ray radiation can considerably alter the mechanical properties of bone tissue. Digital volume correlation (DVC) has been extensively used to compute full-field strain distributions in bone specimens subjected to step-wise mechanical loading, but tissue damage from sequential SR-microCT scans has not been previously addressed. Therefore, the aim of this study is to examine the influence of SR irradiation-induced microdamage on the apparent elastic properties of trabecular bone using DVC applied to in situ SR-microCT tomograms obtained with different exposure times. Results showed how DVC was able to identify high local strain levels (> 10,000 µÎµ) corresponding to visible microcracks at high irradiation doses (~ 230 kGy), despite the apparent elastic properties remained unaltered. Microcracks were not detected and bone plasticity was preserved for low irradiation doses (~ 33 kGy), although image quality and consequently, DVC performance were reduced. DVC results suggested some local deterioration of tissue that might have resulted from mechanical strain concentration further enhanced by some level of local irradiation even for low accumulated dose.

3D Printing and Electrospinning of Composite Hydrogels for Cartilage and Bone Tissue Engineering.

Injuries of bone and cartilage constitute important health issues costing the National Health Service billions of pounds annually, in the UK only. Moreover, these damages can become cause of disability and loss of function for the patients with associated social costs and diminished quality of life. The biomechanical properties of these two tissues are massively different from each other and they are not uniform within the same tissue due to the specific anatomic location and function. In this perspective, tissue engineering (TE) has emerged as a promising approach to address the complexities associated with bone and cartilage regeneration. Tissue engineering aims at developing temporary three-dimensional multicomponent constructs to promote the natural healing process. Biomaterials, such as hydrogels, are currently extensively studied for their ability to reproduce both the ideal 3D extracellular environment for tissue growth and to have adequate mechanical properties for load bearing. This review will focus on the use of two manufacturing techniques, namely electrospinning and 3D printing, that present promise in the fabrication of complex composite gels for cartilage and bone tissue engineering applications.

A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part II, modeling.

Mucociliary clearance is one of the major lines of defense of the human respiratory system. The mucus layer coating the airways is constantly moved along and out of the lung by the activity of motile cilia, expelling at the same time particles trapped in it. The efficiency of the cilia motion can experimentally be assessed by measuring the velocity of micro-beads traveling through the fluid surrounding the cilia. Here we present a mathematical model of the fluid flow and of the micro-beads motion. The coordinated movement of the ciliated edge is represented as a continuous envelope imposing a periodic moving velocity boundary condition on the surrounding fluid. Vanishing velocity and vanishing shear stress boundary conditions are applied to the fluid at a finite distance above the ciliated edge. The flow field is expanded in powers of the amplitude of the individual cilium movement. It is found that the continuous component of the horizontal velocity at the ciliated edge generates a 2D fluid velocity field with a parabolic profile in the vertical direction, in agreement with the experimental measurements. Conversely, we show than this model can be used to extract microscopic properties of the cilia motion by extrapolating the micro-bead velocity measurement at the ciliated edge. Finally, we derive from these measurements a scalar index providing a direct assessment of the cilia beating efficiency. This index can easily be measured in patients without any modification of the current clinical procedures.