Nanoindentation analysis of the micromechanical anisotropy in mouse cortical bone.

Studies investigating micromechanical properties in mouse cortical bone often solely focus on the mechanical behaviour along the long axis of the bone. Therefore, data on the anisotropy of mouse cortical bone is scarce. The aim of this study is the first-time evaluation of the anisotropy ratio between the longitudinal and transverse directions of reduced modulus and hardness in mouse femurs by using the nanoindentation technique. For this purpose, nine 22-week-old mice (C57BL/6) were sacrificed and all femurs extracted. A total of 648 indentations were performed with a Berkovich tip in the proximal (P), central (C) and distal (D) regions of the femoral shaft in the longitudinal and transverse directions. Higher values for reduced modulus are obtained for indentations in the longitudinal direction, with anisotropy ratios of 1.72 ± 0.40 (P), 1.75 ± 0.69 (C) and 1.34 ± 0.30 (D). Hardness is also higher in the longitudinal direction, with anisotropic ratios of 1.35 ± 0.27 (P), 1.35 ± 0.47 (C) and 1.17 ± 0.19 (D). We observed a significant anisotropy in the micromechanical properties of the mouse femur, but the correlation for reduced modulus and hardness between the two directions is low (r2 < 0.3) and not significant. Therefore, we highly recommend performing independent indentation testing in both the longitudinal and transverse directions when knowledge of the tissue mechanical behaviour along multiple directions is required.

3D Printing of Hierarchical Silk Fibroin Structures.

Like many other natural materials, silk is hierarchically structured from the amino acid level up to the cocoon or spider web macroscopic structures. Despite being used industrially in a number of applications, hierarchically structured silk fibroin objects with a similar degree of architectural control as in natural structures have not been produced yet due to limitations in fabrication processes. In a combined top-down and bottom-up approach, we exploit the freedom in macroscopic design offered by 3D printing and the template-guided assembly of ink building blocks at the meso- and nanolevel to fabricate hierarchical silk porous materials with unprecedented structural control. Pores with tunable sizes in the range 40-350 µm are generated by adding sacrificial organic microparticles as templates to a silk fibroin-based ink. Commercially available wax particles or monodisperse polycaprolactone made by microfluidics can be used as microparticle templates. Since closed pores are generated after template removal, an ultrasonication treatment can optionally be used to achieve open porosity. Such pore templating particles can be further modified with nanoparticles to create a hierarchical template that results in porous structures with a defined nanotopography on the pore walls. The hierarchically porous silk structures obtained with this processing technique can potentially be utilized in various application fields from structural materials to thermal insulation to tissue engineering scaffolds.

3D Printing of Emulsions and Foams into Hierarchical Porous Ceramics.

Bulk hierarchical porous ceramics with unprecedented strength-to-weight ratio and tunable pore sizes across three different length scales are printed by direct ink writing. Such an extrusion-based process relies on the formulation of inks in the form of particle-stabilized emulsions and foams that are sufficiently stable to resist coalescence during printing.

Hierarchically roughened microplatelets enhance the strength and ductility of nacre-inspired composites.

Rough interfaces featuring nanoscale asperities are known to play a major role in the mechanics of nacre. Transferring this concept to artificial bioinspired composites requires a detailed understanding about the effect of the surface topography of reinforcing elements on the mechanical performance of such materials. To gain further insights into the effect of asperity size, hierarchy and coverage on the mechanics of nacre-inspired composites, we decorate alumina microplatelets with silica nanoparticles of selected sizes and use the resulting roughened platelets as reinforcing elements (15vol%) in a commercial epoxy matrix. For a single layer of silica nanoparticles on the platelet surface, increased ultimate strain and toughness are obtained with a large roughening particle size of 250nm. On the contrary, strength and stiffness are enhanced by decreasing the size of asperities using 22nm silica particles. By combining particles of two different sizes (100nm and 22nm) in a hierarchical fashion, we are able to improve stiffness and strength of platelet-reinforced polymers while maintaining high ultimate strain and toughness. Our results indicate that carefully designed hierarchically roughened interfaces lead to a more homogeneous stress distribution within the polymer matrix between the stiff reinforcing elements. By enabling the deformation of a larger fraction of the polymer matrix, this design concept improves the mechanical response of bioinspired composites and can possibly also be exploited to enhance the performance of conventional fiber-reinforced polymers.

Orientation and size-dependent mechanical modulation within individual secondary osteons in cortical bone tissue.

Anisotropy is one of the most peculiar aspects of cortical bone mechanics; however, its anisotropic mechanical behaviour should be treated only with strict relationship to the length scale of investigation. In this study, we focus on quantifying the orientation and size dependence of the spatial mechanical modulation in individual secondary osteons of bovine cortical bone using nanoindentation. Tests were performed on the same osteonal structure in the axial (along the long bone axis) and transverse (normal to the long bone axis) directions along arrays going radially out from the Haversian canal at four different maximum depths on three secondary osteons. Results clearly show a periodic pattern of stiffness with spatial distance across the osteon. The effect of length scale on lamellar bone anisotropy and the critical length at which homogenization of the mechanical properties occurs were determined. Further, a laminate-composite-based analytical model was applied to the stiffness trends obtained at the highest spatial resolution to evaluate the elastic constants for a sub-layer of mineralized collagen fibrils within an osteonal lamella on the basis of the spatial arrangement of the fibrils. The hierarchical arrangement of lamellar bone is found to be a major determinant for modulation of mechanical properties and anisotropic mechanical behaviour of the tissue.

Adipose-derived stem cells and rabbit bone regeneration: histomorphometric, immunohistochemical and mechanical characterization.

BACKGROUND: In the last few years, several attempts have been made to treat large bone loss, including the use of tissue engineering with osteoinductive scaffolds and cells. This study highlights the role of mesenchymal stem cells from adipose tissue (ASCs; adipose-derived stem cells) in a rabbit bone regeneration model. METHODS: We compared the neoformed bone tissues achieved by treating critical tibial defects with either hydroxyapatite alone (HA, group I) or hydroxyapatite-autologous ASC constructs (ASCs-HA, group II), investigating their histomorphometric, immunohistochemical and biomechanical properties. RESULTS: After eight weeks of follow-up, we observed advanced maturation and a spatial distribution of new bone that was more homogeneous in the inner parts of the pores in group II, not just along the walls (as seen in group I). The new tissue expressed osteogenic markers, and biomechanical tests suggested that the newly formed bone in group II had a higher mineral content than that in group I. Although variability in differentiation was observed among the different cell populations in vitro, no differences in bone healing were observed in vivo; the variability seen in vitro was probably due to local microenvironment effects. CONCLUSIONS: Tibial defects treated with rabbit ASCs-HA showed an improved healing process when compared to the process that occurred when only the scaffold was used. We suggest that implanted ASCs ameliorate the bone reparative process either directly or by recruiting resident progenitor cells.

Predicting fatigue life of a PMMA based knee spacer using a multiaxial fatigue criterion.

PURPOSE: Experimental tests have played a major role in the assessment of fatigue endurance of orthopedic prostheses; however, cyclic tests on devices entail high costs. Here, a multiaxial fatigue criterion coupled with computational simulations and material properties measurements has been employed to predict fatigue life of the tibial component of a polymeric PMMA spacer. The ultimate aim is to obtain valid information on fatigue behavior avoiding fatigue tests on the device. METHODS: First, an accurate measurement of the static and fatigue properties of PMMA samples is performed. Then, numeric simulations of the fatigue behavior of the PMMA spacer reproducing the experimental test conditions according to ISO 14879-1 were carried out in order to calculate the stress field throughout the device. Finally, a Risk Index was calculated by using a proper multiaxial fatigue criterion for brittle materials (Kakuno-Kawada) for the assessment of the device fatigue behavior by predicting the F-N curves. RESULTS: The numeric results were validated by comparing the predictions against experimental data already published by our group. The multiaxial fatigue criterion was able to predict the most critical point on the spacer upper surface and the fatigue behavior of the device that nicely matched the experimental curves. CONCLUSIONS: This approach represents a valuable tool to investigate the mechanical reliability of implantable devices; nevertheless, the use of advanced and specific failure criteria coupled with accurate data of the device's material is mandatory to represent a real alternative to the experimental approach in fatigue life prediction.??Key words: Acrylic bone cement, Fatigue endurance, Finite element analyses, Knee spacer.

Role of damage mechanics in nanoindentation of lamellar bone at multiple sizes: experiments and numerical modeling.

The aim of this paper is to show that damage mechanisms can account for the response of lamellar bone to nanoindentation tests, with particular regards to the decrease of indentation stiffness with increasing penetration depth and to the loss of contact stiffness during the unloading phase of the test. For this purpose, indentation experiments on bovine cortical bone samples along axial and transverse directions have been carried out at five penetration depths from 50 to 450 nm; furthermore, a continuum damage model has been implemented into finite element analyses, which are able to simulate indentation experiments. Experiments along the axial direction have shown a decrease of about 20% of the indentation modulus with indentation depth; a similar trend was found along the transverse direction. All unloading branches of the force-displacement indentation curves exhibited relevant stiffness loss (curve concavity). The numerical model with damage was able to correctly predict the indentation stiffness and hardness at 300 nm penetration depth along both axial and transverse directions. Furthermore, stiffness loss during unloading was simulated with both qualitative and quantitative agreement with experiments. A final validation has been provided by simulating axial indentation experiments at the remaining penetration depths using the same set of constitutive parameters as those used to simulate the experiments at 300 nm depth. These results support the hypothesis that damage plays a relevant role in the mechanics of lamellar bone and should be taken into account when studying bone mechanical properties at multiple scales.

Nanoindentation testing and finite element simulations of cortical bone allowing for anisotropic elastic and inelastic mechanical response.

Anisotropy is one of the most peculiar aspects of cortical bone mechanical behaviour, and the numerical approach can be successfully used to investigate aspects of bone tissue mechanics that analytical methods solve in approximate way or do not cover. In this work, nanoindentation experimental tests and finite element simulations were employed to investigate the elastic-inelastic anisotropic mechanical properties of cortical bone. The model allows for anisotropic elastic and post-yield behaviour of the tissue. A tension-compression mismatch and direction-dependent yield stresses are allowed for. Indentation experiments along the axial and transverse directions were simulated with the purpose to predict the indentation moduli and hardnesses along multiple orientations. Results showed that the experimental transverse-to-axial ratio of indentation moduli, equal to 0.74, is predicted with a ∼3% discrepancy regardless the post-yield material behaviour; whereas, the transverse-to-axial hardness ratio, equal to 0.86, can be correctly simulated (discrepancy ∼6% w.r.t. the experimental results) only employing an anisotropic post-elastic constitutive model. Further, direct comparison between the experimental and simulated indentation tests evidenced a good agreement in the loading branch of the indentation curves and in the peak loads for a transverse-to-axial yield stress ratio comparable to the experimentally obtained transverse-to-axial hardness ratio. In perspective, the present work results strongly support the coupling between indentation experiments and FEM simulations to get a deeper knowledge of bone tissue mechanical behaviour at the microstructural level. The present model could be used to assess the effect of variations of constitutive parameters due to age, injury, and/or disease on bone mechanical performance in the context of indentation testing.

Mechanical properties of open-cell, self-expandable shape memory alloy carotid stents.

Mechanical properties of carotid stents are important for the selection of the implantable device. The efficacy of cell design is usually investigated on the basis of clinical follow-ups; however, for the specific carotid application the relative importance of the cell design on the stenting outcome is debated. Ideally, a carotid stent should have at the same time low bending stiffness (i.e., high flexibility) to correctly conform to the vessel curvatures in the stenotic region and high stiffness to sustain the vessel wall in the radial direction. In this study, experimental tests on six carotid open-cell design self-expandable stents were carried out to assess bending and radial stiffness. A four-point bending test was adopted to measure the bending stiffness. Radial stiffness was obtained by measuring the stent resistance to local compression in a V-shaped support. Geometrical features, such as the link positioning and their length, were examined as a possible determinant of the mechanical properties of the tested stents. The tested stents showed very different geometrical features and mechanical properties despite similar material and a common open-cell structure consisting in rings connected by roughly straight links. In particular, different strategies are evident regarding the link position. Three distinct behaviors were observed: stents with both low radial and bending stiffness, moderate bending and high radial stiffness, and high bending and moderate radial stiffness. In general, a large link length allows the stent to be more flexible under bending condition, whereas the mutual links positioning plays a key role in the radial stiffness of these devices. Furthermore, for a fixed strut layout, a correlation was found between the strut cross area, as well as metal-to-artery ratio and the resultant radial and bending stiffness.

Experimental evaluation of the biomechanical performances of a PMMA-based knee spacer.

Infection of knee prostheses is still one of the major concerns of the reliability over time of these implantable devices. The preferred treatment of this condition has turned out to be the use of a knee spacer in a two-stage reimplantation technique. The advantages of this technique associated with the use of a mobile spacer lies both in the possibility for the patient to move during the interim period, thus decreasing the risk of muscle contracture due to immobilisation, as well as in the ability to release antibiotics directly to the site of infection. The evaluation of the biomechanical properties of new mobile spacers preformed in three different sizes has been carried out subjecting the spacers to i) cyclic tests on a knee simulator for 500,000 walking cycles, ii) constraint tests in medio-lateral, antero-posterior and internal-external directions, iii) fatigue tests on the tibial tray. Particular attention was addressed to the evaluation of the mechanical resistance of the devices, to the quantity of wear debris produced during the tests and to the extent that such debris was influenced by the test parameters and geometrical dimensions of the spacers themselves. Results showed no sign of failure for any of the tested spacers, the constraint and fatigue behaviours were similar to those shown by a total knee prosthesis and the amount of debris turned out to be directly correlated to the size of the devices: in conclusion, the devices showed a good level of mechanical performance and, consequently, a sufficiently high degree of suitability for clinical use.