Romane Blanchard

Intravoxel bone micromechanics for microCT-based finite element simulations

While micro-FE simulations have become a standard tool in computational biomechanics, the choice of appropriate material properties is still a relevant topic, typically involving empirical grey value-to-elastic modulus relations. We here derive the voxel-specific volume fractions of mineral, collagen, and water, from tissue-independent bilinear relations between mineral and collagen content in extracellular bone tissue (J. Theor. Biol. 287: 115, 2011), and from the measured X-ray attenuation information quantified in terms of grey values. The aforementioned volume fractions enter a micromechanics representation of bone tissue, as to deliver voxel-specific stiffness tensors. In order to check the relevance of this strategy, we convert a micro Computer Tomograph of a mouse femur into a regular Finite Element mesh, apply forces related to the dead load of a standing mouse, and then compare simulation results based on voxel-specific heterogeneous elastic properties to results based on homogeneous elastic properties related to the spatial average over the solid bone matrix compartment, of the X-ray attenuation coefficients. The element-specific strain energy density illustrates that use of homogeneous elastic properties implies overestimation of the organ stiffness. Moreover, the simulation reveals large tensile normal stresses throughout the femur neck, which may explain the mouse femur necks trabecular morphology being quite different from the human case, where the femur neck bears compressive forces and bending moments.

A multiscale analytical approach for bone remodeling simulations : linking scales from collagen to trabeculae

Bone is a dynamic and hierarchical porous material whose spatial and temporal mechanical properties can vary considerably due to differences in its microstructure and due to remodeling. Hence, a multiscale analytical approach, which combines bone structural information at multiple scales to the remodeling cellular activities, could form an efficient, accurate and beneficial framework for the prognosis of changes in bone properties due to, e.g., bone diseases. In this study, an analytical formulation of bone remodeling integrated with multiscale micromechanical models is proposed to investigate the effects of structural changes at the nanometer level (collagen scale) on those at higher levels (tissue scale). Specific goals of this study are to derive a mechanical stimulus sensed by the osteocytes using a multiscale framework, to test the accuracy of the multiscale model for the prediction of bone density, and to demonstrate its multiscale capabilities by predicting changes in bone density due to changes occurring at the molecular level. At each different level, the bone composition was modeled as a two-phase material which made it possible to: (1) find a closed-form solution for the energy-based mechanical stimulus sensed by the osteocytes and (2) describe the anisotropic elastic properties at higher levels as a function of the stiffness of the elementary components (collagen, hydroxyapatite and water) at lower levels. The accuracy of the proposed multiscale model of bone remodeling was tested first by comparing the analytical bone volume fraction predictions to those obtained from the corresponding μFE-based computational model. Differences between analytical and numerical predictions were less than 1% while the computational time was drastically reduced, namely by a factor of 1 million. In a further analysis, the effects of changes in collagen and hydroxyapatite volume fractions on the bone remodeling process were simulated, and it was found that such changes considerably affect the bone density at the millimeter scale. In fact, smaller tissue density induces remodeling activities leading to finally higher overall bone density. The multiscale analytical model proposed in this study potentially provides an accurate and efficient tool for simulating patient-specific bone remodeling, which might be of importance in particular for the hip and spine, where an accurate assessment of bone micro-architecture is not possible.

Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair

Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called “Biopen”. To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10 seconds of exposure to 700 mW/cm2 of 365 nm UV-A, containing >90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering.

In situ handheld three-dimensional bioprinting for cartilage regeneration

Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ three‐dimensional (3D) printing is an exciting and innovative biofabrication technology that enables the surgeon to deliver tissue‐engineering techniques at the time and location of need. We have created a hand‐held 3D printing device (biopen) that allows the simultaneous coaxial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single‐session surgery. This pilot study assessed the ability of the biopen to repair a full‐thickness chondral defect and the early outcomes in cartilage regeneration, and compared these results with other treatments in a large animal model. A standardized critical‐sized full‐thickness chondral defect was created in the weight‐bearing surface of the lateral and medial condyles of both femurs of six sheep. Each defect was treated with one of the following treatments: (i) hand‐held in situ 3D printed bioscaffold using the biopen (HH group), (ii) preconstructed bench‐based printed bioscaffolds (BB group), (iii) microfractures (MF group) or (iv) untreated (control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bioprinting was performed in all animals without any intra‐ or postoperative complication. The HH biopen allowed early cartilage regeneration. The results of this study show that real‐time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage.

Estimation of anisotropic permeability in trabecular bone based on microCT imaging and pore-scale fluid dynamics simulations

In this paper, a comprehensive framework is proposed to estimate the anisotropic permeability matrix in trabecular bone specimens based on micro-computed tomography (microCT) imaging combined with pore-scale fluid dynamics simulations. Two essential steps in the proposed methodology are the selection of (i) a representative volume element (RVE) for calculation of trabecular bone permeability and (ii) a converged mesh for accurate calculation of pore fluid flow properties. Accurate estimates of trabecular bone porosities are obtained using a microCT image resolution of approximately 10 μm. We show that a trabecular bone RVE in the order of 2 × 2 × 2 mm3 is most suitable. Mesh convergence studies show that accurate fluid flow properties are obtained for a mesh size above 125,000 elements. Volume averaging of the pore-scale fluid flow properties allows calculation of the apparent permeability matrix of trabecular bone specimens. For the four specimens chosen, our numerical results show that the so obtained permeability coefficients are in excellent agreement with previously reported experimental data for both human and bovine trabecular bone samples. We also identified that bone samples taken from long bones generally exhibit a larger permeability in the longitudinal direction. The fact that all coefficients of the permeability matrix were different from zero indicates that bone samples are generally not harvested in the principal flow directions. The full permeability matrix was diagonalized by calculating the eigenvalues, while the eigenvectors showed how strongly the bone samples orientations deviated from the principal flow directions. Porosity values of the four bone specimens range from 0.83 to 0.86, with a low standard deviation of ± 0.016, principal permeability values range from 0.22 to 1.45 ⋅ 10 −8 m2, with a high standard deviation of ± 0.33. Also, the anisotropic ratio ranged from 0.27 to 0.83, with high standard deviation. These results indicate that while the four specimens are quite similar in terms of average porosity, large variability exists with respect to permeability and specimen anisotropy. The utilized computational approach compares well with semi-analytical models based on homogenization theory. This methodology can be applied in bone tissue engineering applications for generating accurate pore morphologies of bone replacement materials and to consistently select similar bone specimens in bone bioreactor studies.

Coupling multiscale X-ray physics and micromechanics for bone tissue composition and elasticity determination from micro-CT data, by example of femora from OVX and sham rats

ABSTRACT Due to its high resolution, micro-CT (Computed Tomograph) scanning is the key to assess bone quality of sham and OVX (ovariectomized) rats. Combination of basic X-ray physics, such as the energy- and chemistry-dependence of attenuation coefficients, with results from ashing tests on rat bones, delivers mineral, organic, and water volume fractions within the voxels. Additional use of a microelastic model for bone provides voxel-specific elastic properties. The new method delivers realistic bone mass densities, and reveals that OVX protocols may indeed induce some bone mass loss, while the average composition of the bone tissue remains largely unaltered.

The Application of Pulsed Electromagnetic Fields (PEMFs) for Bone Fracture Repair: Past and Perspective Findings

Bone fractures are one of the most commonly occurring injuries of the musculoskeletal system. A highly complex physiological process, fracture healing has been studied extensively. Data from in vivo, in vitro and clinical studies, have shown pulsed electromagnetic fields (PEMFs) to be highly influential in the fracture repair process. Whilst the underlying mechanisms acting to either inhibit or advance the physiological processes are yet to be defined conclusively, several non-invasive point of use devices have been developed for the clinical treatment of fractures. With the complexity of the repair process, involving many components acting at different time steps, it has been a challenge to determine which PEMF exposure parameters (i.e., frequency of field, intensity of field and dose) will produce the most optimal repair. In addition, the development of an evidence-backed device comes with challenges of its own, with many elements (including process of exposure, construct materials and tissue densities) being highly influential to the field exposed. The objective of this review is to provide a broad recount of the applications of PEMFs in bone fracture repair and to then demonstrate what is further required for enhanced therapeutic outcomes.

Modal analysis of nanoindentation data, confirming that reduced bone turnover may cause increased tissue mineralization/elasticity

It is widely believed that the activities of bone cells at the tissue scale not only govern the size of the vascular pore spaces (and hence, the amount of bone tissue available for actually carrying the loads), but also the characteristics of the extracellular bone matrix itself. In this context, increased mechanical stimulation (in mediolateral regions of human femora, as compared to anteroposterior regions) may lead to increased bone turnover, lower bone matrix mineralization, and therefore lower tissue modulus. On the other hand, resorption-only processes (in endosteal versus periosteal regions) may have the opposite effect. A modal analysis of nanoindentation data obtained on femurs from the Melbourne Femur Research Collection (MFRC) indeed confirms that bone is stiffer in endosteal regions compared to periosteal regions (E̅endost = 29.34 ± 0.75 GPa >E̅periost = 24.67 ± 1.63 GPa), most likely due to the aging-related increase in resorption modeling on endosteal surfaces resulting in trabecularization of cortical bone. The results also show that bone is stiffer along the anteroposterior direction compared the mediolateral direction (E̅anteropost = 28.89 ± 1.08 GPa >E̅mediolat = 26.03 ± 2.31 GPa), the former being aligned with the neutral bending axis of the femur and, thus, undergoing more resorption modeling and consequently being more mineralized.

Biofabrication of human articular cartilage: a path towards the development of a clinical treatment

Cartilage injuries cause pain and loss of function, and if severe may result in osteoarthritis (OA). 3D bioprinting is now a tangible option for the delivery of bioscaffolds capable of regenerating the deficient cartilage tissue. Our team has developed a handheld device, the Biopen, to allow in situ additive manufacturing during surgery. Given its ability to extrude in a core/shell manner, the Biopen can preserve cell viability during the biofabrication process, and it is currently the only biofabrication tool tested as a surgical instrument in a sheep model using homologous stem cells. As a necessary step toward the development of a clinically relevant protocol, we aimed to demonstrate that our handheld extrusion device can successfully be used for the biofabrication of human cartilage. Therefore, this study is a required step for the development of a surgical treatment in human patients. In this work we specifically used human adipose derived mesenchymal stem cells (hADSCs), harvested from the infra-patellar fat pad of donor patients affected by OA, to also prove that they can be utilized as the source of cells for the future clinical application. With the Biopen, we generated bioscaffolds made of hADSCs laden in gelatin methacrylate, hyaluronic acid methacrylate and cultured in the presence of chondrogenic stimuli for eight weeks in vitro. A comprehensive characterisation including gene and protein expression analyses, immunohistology, confocal microscopy, second harmonic generation, light sheet imaging, atomic force mycroscopy and mechanical unconfined compression demonstrated that our strategy resulted in human hyaline-like cartilage formation. Our in situ biofabrication approach represents an innovation with important implications for customizing cartilage repair in patients with cartilage injuries and OA.