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Introduction: The in vivo tibial loading mouse model has been extensively used to evaluate bone adaptation in the tibia after mechanical loading treatment. However, there is a prevailing assumption that the load is applied axially to the tibia. The aim of this in silico study was to evaluate how much the apparent mechanical properties of the mouse tibia are affected by the loading direction, by using a validated micro-finite element (micro-FE) model of mice which have been ovariectomized and exposed to external mechanical loading over a two-week period. Methods: Longitudinal micro-computed tomography (micro-CT) images were taken of the tibiae of eleven ovariectomized mice at ages 18 and 20 weeks. Six of the mice underwent a mechanical loading treatment at age 19 weeks. Micro-FE models were generated, based on the segmented micro-CT images. Three models using unitary loads were linearly combined to simulate a range of loading directions, generated as a function of the angle from the inferior-superior axis (θ, 0°-30° range, 5° steps) and the angle from the anterior-posterior axis (ϕ, 0°: anterior axis, positive anticlockwise, 0°-355° range, 5° steps). The minimum principal strain was calculated and used to estimate the failure load, by linearly scaling the strain until 10% of the nodes reached the critical strain level of -14,420 µÎµ. The apparent bone stiffness was calculated as the ratio between the axial applied force and the average displacement along the longitudinal direction, for the loaded nodes. Results: The results demonstrated a high sensitivity of the mouse tibia to the loading direction across all groups and time points. Higher failure loads were found for several loading directions (θ = 10°, ϕ 205°-210°) than for the nominal axial case (θ = 0°, ϕ = 0°), highlighting adaptation of the bone for loading directions far from the nominal axial one. Conclusion: These results suggest that in studies which use mouse tibia, the loading direction can significantly impact the failure load. Thus, the magnitude and direction of the applied load should be well controlled during the experiments.

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Strain energy density (SED) is considered to be the primary remodelling stimulus influencing the process of bone growth into porous implants. A bone remodelling algorithm incorporating the concept of bone connectivity, that newly formed bone should only grow from existing bone, was developed to provide a more biologically realistic simulation of bone growth. Results showed that the new algorithm prevented the occurrence of unconnected mature bone within porous implants, an unrealistic phenomenon observed using conventional adaptive elasticity theories. The bone connectivity algorithm had minimal effect (0.67% difference) on the final bone density distribution for standard bending and torsional moment cases. For a porous implant model, both algorithms, with and without bone connectivity implementation, reached the same final stiffness, with a difference of less than 0.01%. The bone connectivity algorithm predicted a slower and more gradual bone remodelling process, requiring at least 50% additional time for full remodelling compared to the conventional adaptive elasticity algorithm, which should be accounted for in the planning of rehabilitation strategies. The developed modelling can be employed to improve porous implant designs to achieve better clinical outcomes.

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Combined treatment with PTH(1-34) and mechanical loading confers increased structural benefits to bone than monotherapies. However, it remains unclear how this longitudinal adaptation affects the bone mechanics. This study quantified the individual and combined longitudinal effects of PTH(1-34) and mechanical loading on the bone stiffness and strength evaluated in vivo with validated micro-finite element (microFE) models. C57BL/6 mice were ovariectomised at 14-week-old and treated either with injections of PTH(1-34), compressive tibia loading or both interventions concurrently. Right tibiae were in vivo microCT-scanned every 2 weeks from 14 until 24-week-old. MicroCT images were rigidly registered to reference tibia and the cortical organ level (whole bone) and tissue level (midshaft) morphometric properties and bone mineral content were quantified. MicroCT images were converted into voxel-based homogeneous, linear elastic microFE models to estimate the bone stiffness and strength. This approach allowed us for the first time to quantify the longitudinal changes in mechanical properties induced by combined treatments in a model of accelerated bone resorption. Both changes of stiffness and strength were higher with co-treatment than with individual therapies, consistent with increased benefits with the tibia bone mineral content and cortical area, properties strongly associated with the tibia mechanics. The longitudinal data shows that the two bone anabolics, both individually and combined, had persistent benefit on estimated mechanical properties, and that benefits (increased stiffness and strength) remained after treatment was withdrawn.

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Few studies have investigated the effect of a monosaturated diet high in ω-9 on osteoporosis. We hypothesized that omega-9 (ω-9) protects ovariectomized (OVX) mice from a decline in bone microarchitecture, tissue loss, and mechanical strength, thereby serving as a modifiable dietary intervention against osteoporotic deterioration. Female C57BL/6J mice were assigned to sham-ovariectomy, ovariectomy, or ovariectomy + estradiol treatment prior to switching their feed to a diet high in ω-9 for 12 weeks. Tibiae were evaluated using DMA, 3-point-bending, histomorphometry, and microCT. A significant decrease in lean mass (p = 0.05), tibial area (p = 0.009), and cross-sectional moment of inertia (p = 0.028) was measured in OVX mice compared to the control. A trend was seen where OVX bone displayed increased elastic modulus, ductility, storage modulus, and loss modulus, suggesting the ω-9 diet paradoxically increased both stiffness and viscosity. This implies beneficial alterations on the macro-structural, and micro-tissue level in OVX bone, potentially decreasing the fracture risk. Supporting this, no significant differences in ultimate, fracture, and yield stresses were measured. A diet high in ω-9 did not prevent microarchitectural deterioration, nevertheless, healthy tibial strength and resistance to fracture was maintained via mechanisms independent of bone structure/shape. Further investigation of ω-9 as a therapeutic in osteoporosis is warranted.

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The influence of diet on the development of osteoporosis is significant and not fully understood. This study investigated the effect of diets of varying lipid profiles and ω-3, ω-6 and ω-9 composition on the structural and mechanical properties of bone. The hypothesis studied was that a diet high in saturated fat would induce osteoporosis and produce an overall increased detrimental bony response when compared with a diet high in unsaturated ω-6, or ω-9. Male C57BL/6J mice were fed either a control diet, 50:50 mix (saturated:unsaturated) high in ω-9 (HFD50:50), a diet high in saturated fat (HSF) or a polyunsaturated fat diet high in ω-6 (PUFA) over an 8-week duration. Tibiae were retrieved and evaluated using DMA, 3-point-bending, histomorphometry, and microCT. Mice fed a HSF diet displayed key features characteristic of osteoporosis. The loss tangent was significantly increased in the HFD50:50 diet group compared with control (p = 0.016) and PUFA-fed animals (p = 0.049). HFD50:50-fed mice presented with an increased viscous component, longer tibiae, increased loss modulus (p = 0.009), and ultimate stress, smaller microcracks (p < 0.001), and increased trabecular width (p = 0.002) compared with control animals. A diet high in ω-9 resulted in an overall superior bone response and further analysis of its role in bone health is warranted.

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Interventions for bone diseases (e.g. osteoporosis) require testing in animal models before clinical translation and the mouse tibia is among the most common tested anatomical sites. In vivo micro-Computed Tomography (microCT) based measurements of the geometrical and densitometric properties are non-invasive and therefore constitute an important tool in preclinical studies. Moreover, validated micro-Finite Element (microFE) models can be used for predicting the bone mechanical properties non-invasively. However, considering that the image processing pipeline requires operator-dependant steps, the reproducibility of these measurements has to be assessed. The aim of this study was to evaluate the intra- and inter-operator reproducibility of several bone parameters measured from microCT images. Ten in vivo microCT images of the right tibia of five mice (at 18 and 22 weeks of age) were processed. One experienced operator (intra-operator analysis) and three different operators (inter-operator) aligned each image to a reference through a rigid registration and selected a volume of interest below the growth plate. From each image the following parameters were measured: total bone mineral content (BMC) and density (BMD), BMC in 40 subregions (ten longitudinal sections, four quadrants), microFE-based stiffness and failure load. Intra-operator reproducibility was acceptable for all parameters (precision error, PE < 3.71%), with lowest reproducibility for stiffness (3.06% at week 18, 3.71% at week 22). The inter-operator reproducibility was slightly lower (PE < 4.25%), although still acceptable for assessing the properties of most interventions. The lowest reproducibility was found for BMC in the lateral sector at the midshaft (PE = 4.25%). Densitometric parameters were more reproducible than most standard morphometric parameters calculated in the proximal trabecular bone. In conclusion, microCT and microFE models provide reproducible measurements for non-invasive assessment of the mouse tibia properties.

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Osteoporosis is one of the most common skeletal diseases, but current therapies are limited to generalized antiresorptive or anabolic interventions, which do not target regions that would benefit from improvements to skeletal health. To improve the evaluation of treatment plans, we used a spatio-temporal multiscale approach that combines longitudinal in vivo micro-computed tomography (micro-CT) and in silico subject-specific finite element modeling to quantitatively map bone adaptation changes due to disease and treatment at high resolution. Our findings show time and region-dependent modifications in bone remodelling following one and two sets of mechanical loading and/or pharmacological interventions. The multiscale results highlighted that the distal section was unaffected by mechanical loading alone but the proximal tibia had the greatest gain from positive interactions of combined therapies. Mechanical loading abated the catabolic effect of PTH, but the main benefit of combined treatments occurred from the additive interactions of the two therapies in periosteal apposition. These results provide detailed insight into the efficacy of combined treatments, facilitating the optimisation of dosage and treatment duration in preclinical mouse studies, and the development of novel interventions for skeletal diseases. STATEMENT OF SIGNIFICANCE: Combined mechanical loading and pharmacotherapy have the potential to slow osteoporosis-induced bone loss but current therapies do not target the regions in need of strengthening. We show for the first time spatial region-dependant interactions between PTH and mechanical loading treatment in OVX mouse tibiae, highlighting local regions in the tibia that benefitted from separate and combined treatments. Combined experimental-computational analysis also detailed the lasting period of each treatment per location in the tibia, the extent of positive (or negative) interactions of the combined therapies, and the impact of each treatment on the regulation of bone adaptation spatio-temporally. This approach can be used to create hypothesis about the interactions of different treatments to optimise the design of biomaterials and medical interventions.

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The in vivo mouse tibial loading model is used to evaluate the effectiveness of mechanical loading treatment against skeletal diseases. Although studies have correlated bone adaptation with the induced mechanical stimulus, predictions of bone remodeling remained poor, and the interaction between external and physiological loading in engendering bone changes have not been determined. The aim of this study was to determine the effect of passive mechanical loading on the strain distribution in the mouse tibia and its predictions of bone adaptation. Longitudinal micro-computed tomography (micro-CT) imaging was performed over 2 weeks of cyclic loading from weeks 18 to 22 of age, to quantify the shape change, remodeling, and changes in densitometric properties. Micro-CT based finite element analysis coupled with an optimization algorithm for bone remodeling was used to predict bone adaptation under physiological loads, nominal 12N axial load and combined nominal 12N axial load superimposed to the physiological load. The results showed that despite large differences in the strain energy density magnitudes and distributions across the tibial length, the overall accuracy of the model and the spatial match were similar for all evaluated loading conditions. Predictions of densitometric properties were most similar to the experimental data for combined loading, followed closely by physiological loading conditions, despite no significant difference between these two predicted groups. However, all predicted densitometric properties were significantly different for the 12N and the combined loading conditions. The results suggest that computational modeling of bone's adaptive response to passive mechanical loading should include the contribution of daily physiological load.

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Osteoporosis disrupts the healthy remodelling process in bone and affects its mechanical properties. Mechanical loading has been shown to be effective in stimulating bone formation to mitigate initial bone loss. However, no study has investigated the effects of repeated mechanical loading, with a pause of one week in between, in the mouse tibia with oestrogen deficiency. This study uses a combined experimental and computational approach, through longitudinal monitoring with micro-computed tomography, to evaluate the effects of loading on bone adaptation in the tibiae of ovariectomised (OVX) C57BL/6 mice from 14 to 22 weeks of age. Micro-FE models coupled with bone adaptation algorithms were used to estimate changes in local tissue strains due to OVX and mechanical loading, and to quantify the relationship between local strain and remodelling. The first in vivo mechanical loading increased apposition, by 50-150%, while resorption decreased by 50-60%. Both endosteal and periosteal resorption increased despite the second mechanical loading, and periosteal resorption was up to 70% higher than that after the first loading. This was found to correlate with an initial decrease in average strain energy density after the first loading, which was lower and more localised after the second loading. Predictions of bone adaptation showed that between 50 and 90% of the load-induced bone apposition is linearly strain driven at the organ-level, but resorption is more biologically driven at the local level. The results imply that a systematic increase in peak load or loading rate may be required to achieve a similar bone adaptation rate in specific regions of interests.

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Additive manufactured, porous bone implants have the potential to improve osseointegration and reduce failure rates of orthopaedic devices. Substantially porous implants are increasingly used in a number of orthopaedic applications. HA plasma spraying-a line of sight process-cannot coat the inner surfaces of substantially porous structures, whereas electrochemical deposition of calcium phosphate can fully coat the inner surfaces of porous implants for improved bioactivity, but the osseous response of different types of hydroxyapatite (HA) coatings with ionic substitutions has not been evaluated for implants in the same in vivo model. In this study, laser sintered Ti6Al4V implants with pore sizes of Ø 700 µm and Ø 1500 µm were electrochemically coated with HA, silicon-substituted HA (SiHA), and strontium-substituted HA (SrHA), and implanted in ovine femoral condylar defects. Implants were retrieved after 6 weeks and histological and histomorphometric evaluation were compared to electrochemically coated implants with uncoated and HA plasma sprayed controls. The HA, SiHA and SrHA coatings had Ca:P, Ca:(P+Si) and (Ca+Sr):P ratios of 1.53, 1.14 and 1.32 respectively. Electrochemically coated implants significantly promoted bone attachment to the implant surfaces of the inner pores and displayed improved osseointegration compared to uncoated scaffolds for both pore sizes (p<0.001), whereas bone ingrowth was restricted to the surface for HA plasma coated or uncoated implants. Electrochemically coated HA implants achieved the highest osseointegration, followed by SrHA coated implants, and both coatings exhibited significantly more bone growth than plasma sprayed groups (p≤0.01 for all 4 cases). SiHA had significantly more osseointegration when compared against the uncoated control, but no significant difference compared with other coatings. There was no significant difference in ingrowth or osseointegration between pore sizes, and the bone-implant-contact was significantly higher in the electrochemical HA than in SiHA or SrHA. These results suggest that osseointegration is insensitive to pore size, whereas surface modification through the presence of an osteoconductive coating plays an important role in improving osseointegration, which may be critically important for extensively porous implants.

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Understanding how bone adapts to mechanical stimuli is fundamental for optimising treatments against musculoskeletal diseases in preclinical studies, but the contribution of physiological loading to bone adaptation in mouse tibia has not been quantified so far. In this study, a novel mechanistic model to predict bone adaptation based on physiological loading was developed and its outputs were compared with longitudinal scans of the mouse tibia. Bone remodelling was driven by the mechanical stimuli estimated from micro-FEA models constructed from micro-CT scans of C57BL/6 female mice (N = 5) from weeks 14 and 20 of age, to predict bone changes in week 16 or 22. Parametric analysis was conducted to evaluate the sensitivity of the models to subject-specific or averaged parameters, parameters from week 14 or week 20, and to strain energy density (SED) or maximum principal strain (εmaxprinc). The results at week 20 showed no significant difference in bone densitometric properties between experimental and predicted images across the tibia for both stimuli, and 59% and 47% of the predicted voxels matched with the experimental sites in apposition and resorption, respectively. The model was able to reproduce regions of bone apposition in both periosteal and endosteal surfaces (70% and 40% for SED and εmaxprinc, respectively), but it under-predicted the experimental sites of resorption by over 85%. This study shows for the first time the potential of a subject-specific mechanoregulation algorithm to predict bone changes in a mouse model under physiological loading. Nevertheless, the weak predictions of resorption suggest that a combined stimulus or biological stimuli should be accounted for in the model.

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New porous implant designs made possible by additive manufacturing allow for increased osseointegration, potentially improving implant performance and longevity for patients that require massive bone implants. The aim of this study was to evaluate how implantation and the strain distribution in the implant affect the pattern of bone ingrowth and how changes in tissue density within the pores alter the stresses in implants. The hypothesis was that porous metal implants are susceptible to fatigue failure, and that this reduces as osteointegration occurs. A phenomenological, finite element analysis (FEA) bone remodelling model was used to predict partial bone formation for two porous (pore sizes of 700 µm and 1500 µm), laser sintered Ti6Al4V implants in an ovine condylar defect model, and was compared and verified against in vivo, histology results. The FEA models predicted partial bone formation within the porous implants, but over-estimated the amount of bone-surface area compared to histology results. The stress and strain in the implant and adjacent tissues were assessed before, during bone remodelling, and at equilibrium. Results showed that partial bone formation improves the stress distribution locally by reducing stress concentrations for both pore sizes, by at least 20%. This improves the long-term fatigue resistance for the larger pore implant, as excessively high stress is reduced to safer levels (86% of fatigue strength) as bone forms. The stress distribution only changed slightly in regions without bone growth. As the extent of bone formation into extensively porous bone implants depends on the level of stress shielding, the design of the implant and stiffness have significant influence on bone integration and need to be considered carefully to ensure the safety of implants with substantial porous regions. To our knowledge this is the first time that the effect of bone formation on stress distribution within a porous implant has been described and characterised.

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Bone loss caused by stress shielding of metallic implants is a concern, as it can potentially lead to long-term implant failure. Surface coating and reducing structural stiffness of implants are two ways to improve bone ingrowth and osteointegration. Additive manufacturing, through selective laser sintering (SLS) or electron beam melting (EBM) of metallic alloys, can produce porous implants with bone ingrowth regions that enhance osteointegration and improve clinical outcomes. Histology of porous Ti6Al4V plugs of two pore sizes with and without electrochemically deposited hydroxyapatite coating, implanted in ovine condyles, showed that bone formation did not penetrate deep into the porous structure, whilst significantly increased bone growth along coated pore surfaces (osteointegration) was observed. Finite Element simulations, combining new algorithms to model bone ingrowth and the effect of surface modification on osteoconduction, were verified with the histology results. The results showed stress shielding of porous implants made from conventional titanium alloy due to material stiffness and implant geometry, limiting ingrowth and osteointegration. Simulations for reduced implant material stiffness predicted increased bone ingrowth. For low modulus Titanium-tantalum alloy (Ti-70%Ta), reduced stress shielding and enhanced bone ingrowth into the porous implant was found, leading to improved mechanical interlock. Algorithms predicted osteoconductive coating to promote both osteointegration and bone ingrowth into the inner pores when they were coated. These new Finite Element algorithms show that using implant materials with lower elastic modulus, osteoconductive coatings or improved implant design could lead to increased bone remodelling that optimises tissue regeneration, fulfilling the potential of enhanced porosity and complex implant designs made possible by additive layer manufacturing techniques.

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Extracortical bone growth with osseointegration of bone onto the shaft of massive bone tumour implants is an important clinical outcome for long-term implant survival. A new computational algorithm combining geometrical shape changes and bone adaptation in 3D Finite Element simulations has been developed, using a soft tissue envelope mesh, a novel concept of osteoconnectivity, and bone remodelling theory. The effects of varying the initial tissue density, spatial influence function and time step were investigated. The methodology demonstrated good correspondence to radiological results for a segmental prosthesis.

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Radiological features alone do not allow the discrimination between accidental paediatric long bone fractures or those sustained by child abuse. Therefore, there is a clinical need to elucidate the mechanisms behind each fracture to provide a forensic biomechanical tool for the vulnerable child. Four-point bending and torsional loading tests were conducted at more than one strain rate for the first time on immature bone, using a specimen-specific alignment system, to characterise structural behaviour at para-physiological strain rates. The bones behaved linearly to the point of fracture in all cases and transverse, oblique, and spiral fracture patterns were consistently reproduced. The results showed that there was a significant difference in bending stiffness between transverse and oblique fractures in four-point bending. For torsional loading, spiral fractures were produced in all cases with a significant difference in the energy and obliquity to fracture. Multiple or comminuted fractures were seen only in bones that failed at a higher stress or torque for both loading types. This demonstrates the differentiation of fracture patterns at different strain rates for the first time for immature bones, which may be used to match the case history given of a child and the fracture produced.

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The choice of coordinate system and alignment of bone will affect the quantification of mechanical properties obtained during in-vitro biomechanical testing. Where these are used in predictive models, such as finite element analysis, the fidelic description of these properties is paramount. Currently in bending and torsional tests, bones are aligned on a pre-defined fixed span based on the reference system marked out. However, large inter-specimen differences have been reported. This suggests a need for the development of a specimen-specific alignment system for use in experimental work. Eleven ovine tibiae were used in this study and three-dimensional surface meshes were constructed from micro-Computed Tomography scan images. A novel, semi-automated algorithm was developed and applied to the surface meshes to align the whole bone based on its calculated principal directions. Thereafter, the code isolates the optimised location and length of each bone for experimental testing. This resulted in a lowering of the second moment of area about the chosen bending axis in the central region. More importantly, the optimisation method decreases the irregularity of the shape of the cross-sectional slices as the unbiased estimate of the population coefficient of variation of the second moment of area decreased from a range of (0.210-0.435) to (0.145-0.317) in the longitudinal direction, indicating a minimisation of the product moment, which causes eccentric loading. Thus, this methodology serves as an important pre-step to align the bone for mechanical tests or simulation work, is optimised for each specimen, ensures repeatability, and is general enough to be applied to any long bone.

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