Bone-inspired microarchitectures achieve enhanced fatigue life.

Microarchitectured materials achieve superior mechanical properties through geometry rather than composition. Although ultralightweight microarchitectured materials can have high stiffness and strength, application to durable devices will require sufficient service life under cyclic loading. Naturally occurring materials provide useful models for high-performance materials. Here, we show that in cancellous bone, a naturally occurring lightweight microarchitectured material, resistance to fatigue failure is sensitive to a microarchitectural trait that has negligible effects on stiffness and strength-the proportion of material oriented transverse to applied loads. Using models generated with additive manufacturing, we show that small increases in the thickness of elements oriented transverse to loading can increase fatigue life by 10 to 100 times, far exceeding what is expected from the associated change in density. Transversely oriented struts enhance resistance to fatigue by acting as sacrificial elements. We show that this mechanism is also present in synthetic microlattice structures, where fatigue life can be altered by 5 to 9 times with only negligible changes in density and stiffness. The effects of microstructure on fatigue life in cancellous bone and lattice structures are described empirically by normalizing stress in traditional stress vs. life (S-N) curves by √ψ, where ψ is the proportion of material oriented transverse to load. The mechanical performance of cancellous bone and microarchitectured materials is enhanced by aligning structural elements with expected loading; our findings demonstrate that this strategy comes at the cost of reduced fatigue life, with consequences to the use of microarchitectured materials in durable devices and to human health in the context of osteoporosis.

Customized mandibular reconstruction plates improve mechanical performance in a mandibular reconstruction model.

The purpose of this paper was to analyze the biomechanical performance of customized mandibular reconstruction plates with optimized strength. The best locations for increasing bar widths were determined with a sensitivity analysis. Standard and customized plates were mounted on mandible models and mechanically tested. Maximum stress in the plate could be reduced from 573 to 393 MPa (-31%) by increasing bar widths. The median fatigue limit was significantly greater (p < 0.001) for customized plates (650 ± 27 N) than for standard plates (475 ± 27 N). Increasing bar widths at case-specific locations was an effective strategy for increasing plate fatigue performance.

Strain energy density gradients in bone marrow predict osteoblast and osteoclast activity: a finite element study.

Huiskes et al. hypothesized that mechanical strains sensed by osteocytes residing in trabecular bone dictate the magnitude of load-induced bone formation. More recently, the mechanical environment in bone marrow has also been implicated in bone׳s response to mechanical stimulation. In this study, we hypothesize that trabecular load-induced bone formation can be predicted by mechanical signals derived from an integrative µFE model, incorporating a description of both the bone and marrow phase. Using the mouse tail loading model in combination with in vivo micro-computed tomography (µCT) we tracked load induced changes in the sixth caudal vertebrae of C57BL/6 mice to quantify the amount of newly mineralized and eroded bone volumes. To identify the mechanical signals responsible for adaptation, local morphometric changes were compared to micro-finite element (µFE) models of vertebrae prior to loading. The mechanical parameters calculated were strain energy density (SED) on trabeculae at bone forming and resorbing surfaces, SED in the marrow at the boundary between bone forming and resorbing surfaces, along with SED in the trabecular bone and marrow volumes. The gradients of each parameter were also calculated. Simple regression analysis showed mean SED gradients in the trabecular bone matrix to significantly correlate with newly mineralized and eroded bone volumes R(2)=0.57 and 0.41, respectively, p<0.001). Nevertheless, SED gradients in the marrow were shown to be the best predictor of osteoblastic and osteoclastic activity (R(2)=0.83 and 0.60, respectively, p<0.001). These data suggest that the mechanical environment of the bone marrow plays a significant role in determining osteoblast and osteoclast activity.

Bone adaptation to cyclic loading in murine caudal vertebrae is maintained with age and directly correlated to the local micromechanical environment.

The ability of the skeleton to adapt to mechanical stimuli (mechanosensitivity) has most often been investigated at the whole-bone level, but less is known about the local mechanoregulation of bone remodeling at the bone surface, especially in context of the aging skeleton. The aim of this study was to determine the local and global mechanosensitivity of the sixth caudal vertebra during cyclic loading (8 N, three times per week, for six weeks) in mice aged 15, 52, and 82 weeks at the start of loading. Bone adaptation was monitored with in vivo micro-computed tomography. Strain energy density (SED), assumed as the mechanical stimulus for bone adaptation, was determined with micro-finite element models. Mechanical loading had a beneficial effect on the bone microstructure and bone stiffness in all age groups. Mineralizing surface was on average 13% greater (p<0.05) in loaded than control groups in 15- and 82-week-old mice, but not for 52-week-old mice. SED at the start of loading correlated to the change in bone volume fraction in the following 6 weeks for loaded groups (r(2)=0.69-0.85) but not control groups. At the local level, SED was 14-20% greater (p<0.01) at sites of bone formation, and 15-20% lower (p<0.01) at sites of bone resorption compared to quiescent bone surfaces for all age groups, indicating SED was a stimulus for bone adaptation. Taken together, these results support that mechanosensitivity is maintained with age in caudal vertebrae of mice at a local and global level. Since age-related bone loss was not observed in caudal vertebrae, results from the current study might not be translatable to aged humans.

The effects of tensile-compressive loading mode and microarchitecture on microdamage in human vertebral cancellous bone.

The amount of microdamage in bone tissue impairs mechanical performance and may act as a stimulus for bone remodeling. Here we determine how loading mode (tension vs. compression) and microstructure (trabecular microarchitecture, local trabecular thickness, and presence of resorption cavities) influence the number and volume of microdamage sites generated in cancellous bone following a single overload. Twenty paired cylindrical specimens of human vertebral cancellous bone from 10 donors (47­78 years) were mechanically loaded to apparent yield in either compression or tension, and imaged in three dimensions for microarchitecture and microdamage (voxel size 0.7×0.7×5.0 µm3). We found that the overall proportion of damaged tissue was greater (p=0.01) for apparent tension loading (3.9±2.4%, mean±SD) than for apparent compression loading (1.9±1.3%). Individual microdamage sites generated in tension were larger in volume (p<0.001) but not more numerous (p=0.64) than sites in compression. For both loading modes, the proportion of damaged tissue varied more across donors than with bone volume fraction, traditional measures of microarchitecture (trabecular thickness, trabecular separation, etc.), apparent Young׳s modulus, or strength. Microdamage tended to occur in regions of greater trabecular thickness but not near observable resorption cavities. Taken together, these findings indicate that, regardless of loading mode, accumulation of microdamage in cancellous bone after monotonic loading to yield is influenced by donor characteristics other than traditional measures of microarchitecture, suggesting a possible role for tissue material properties.

The Clinical Biomechanics Award 2012 - presented by the European Society of Biomechanics: large scale simulations of trabecular bone adaptation to loading and treatment.

BACKGROUND: Microstructural simulations of bone remodeling are particularly relevant in the clinical management of osteoporosis. Before a model can be applied in the clinics, a validation against controlled in vivo data is crucial. Here we present a strain-adaptive feedback algorithm for the simulation of trabecular bone remodeling in response to loading and pharmaceutical treatment and report on the results of the large-scale validation against in vivo data. METHODS: The algorithm follows the mechanostat principle and incorporates mechanical feedback, based on the local strain-energy density. For the validation, simulations of bone remodeling and adaptation in 180 osteopenic mice were performed. Permutations of the conditions for early (20th week) and late (26th week) loading of 8N or 0N, and treatments with bisphosphonates, or parathyroid hormone were simulated. Static and dynamic morphometry and local remodeling sites from in vivo and in silico studies were compared. FINDINGS: For each study an individual set of model parameters was selected. Trabecular bone volume fraction was chosen as an indicator of the accuracy of the simulations. Overall errors for this parameter were 0.1-4.5%. Other morphometric indices were simulated with errors of less than 19%. Dynamic morphometry was more difficult to predict, which resulted in significant differences from the experimental data. INTERPRETATION: We validated a new algorithm for the simulation of bone remodeling in trabecular bone. The results indicate that the simulations accurately reflect the effects of treatment and loading seen in respective experimental data, and, following adaptation to human data, could be transferred into clinics.

Image interpolation allows accurate quantitative bone morphometry in registered micro-computed tomography scans.

Time-lapsed in vivo micro-computed tomography is a powerful tool to analyse longitudinal changes in the bone micro-architecture. Registration can overcome problems associated with spatial misalignment between scans; however, it requires image interpolation which might affect the outcome of a subsequent bone morphometric analysis. The impact of the interpolation error itself, though, has not been quantified to date. Therefore, the purpose of this ex vivo study was to elaborate the effect of different interpolator schemes [nearest neighbour, tri-linear and B-spline (BSP)] on bone morphometric indices. None of the interpolator schemes led to significant differences between interpolated and non-interpolated images, with the lowest interpolation error found for BSPs (1.4%). Furthermore, depending on the interpolator, the processing order of registration, Gaussian filtration and binarisation played a role. Independent from the interpolator, the present findings suggest that the evaluation of bone morphometry should be done with images registered using greyscale information.

Local mechanical stimuli regulate bone formation and resorption in mice at the tissue level.

Bone is able to react to changing mechanical demands by adapting its internal microstructure through bone forming and resorbing cells. This process is called bone modeling and remodeling. It is evident that changes in mechanical demands at the organ level must be interpreted at the tissue level where bone (re)modeling takes place. Although assumed for a long time, the relationship between the locations of bone formation and resorption and the local mechanical environment is still under debate. The lack of suitable imaging modalities for measuring bone formation and resorption in vivo has made it difficult to assess the mechanoregulation of bone three-dimensionally by experiment. Using in vivo micro-computed tomography and high resolution finite element analysis in living mice, we show that bone formation most likely occurs at sites of high local mechanical strain (p<0.0001) and resorption at sites of low local mechanical strain (p<0.0001). Furthermore, the probability of bone resorption decreases exponentially with increasing mechanical stimulus (R(2) = 0.99) whereas the probability of bone formation follows an exponential growth function to a maximum value (R(2) = 0.99). Moreover, resorption is more strictly controlled than formation in loaded animals, and ovariectomy increases the amount of non-targeted resorption. Our experimental assessment of mechanoregulation at the tissue level does not show any evidence of a lazy zone and suggests that around 80% of all (re)modeling can be linked to the mechanical micro-environment. These findings disclose how mechanical stimuli at the tissue level contribute to the regulation of bone adaptation at the organ level.

Mineralization kinetics in murine trabecular bone quantified by time-lapsed in vivo micro-computed tomography.

Trabecular bone is a highly dynamic tissue due to bone remodeling, mineralization and demineralization. The mineral content and its spatial heterogeneity are main contributors to bone quality. Using time-lapsed in vivo micro-computed tomography (micro-CT), it is now possible to resolve in three dimensions where bone gets resorbed and deposited over several weeks. In addition, the gray values in the micro-CT images contain quantitative information about the local tissue mineral density (TMD). The aim of this study was to measure how TMD increases with time after new bone formation and how this mineralization kinetics is influenced by mechanical stimulation. Our analysis of changes in TMD was based on an already reported experiment on 15-week-old female mice (C57BL/6), where in one group the sixth caudal vertebra was mechanically loaded with 8N, while in the control group no loading was applied. Comparison of two consecutive images allows the categorization of bone into newly formed, resorbed, and quiescent bone for different time points. Gray values of bone in these categories were compared layer-wise to minimize the effects of beam hardening artifacts. Quiescent bone in the control group was found to mineralize with a rate of 8 ± 1 mgHA/cm(3) per week, which is about half as fast as observed for newly formed bone. Mechanical loading increased the rate of mineral incorporation by 63% in quiescent bone. The week before bone resorption, demineralization could be observed with a drop of TMD by 36 ± 4 mgHA/cm(3) in the control and 34 ± 3 mgHA/cm(3) in the loaded group. In conclusion, this study shows how time-lapsed in vivo micro-CT can be used to assess changes in TMD of bone with high spatial and temporal resolution. This will allow a quantification of how bone diseases and pharmaceutical interventions influence not only microarchitecture of trabecular bone, but also its material quality.

Trabecular bone adapts to long-term cyclic loading by increasing stiffness and normalization of dynamic morphometric rates.

Bone has the ability to adapt to external loading conditions. Especially the beneficial effect of short-term cyclic loading has been investigated in a number of in vivo animal studies. The aim of this study was to assess the long-term effect (>10 weeks) of cyclic mechanical loading on the bone microstructure, bone stiffness, and bone remodeling rates. Mice were subjected to cyclic mechanical loading at the sixth caudal vertebra with 8N or 0N (control) three times per week for a total period of 14 weeks. Structural bone parameters were determined from in vivo micro-computed tomography (micro-CT) scans performed at week 0, 4, 6, 8, 10, 12, and 14. Mechanical parameters were derived from micro-finite element analysis. Dynamic bone morphometry was calculated using registration of serial micro-CT scans. Bone volume fraction and trabecular thickness increased significantly more for the loaded group than for the control group (p = 0.006 and p = 0.002 respectively). The trabecular bone microstructure adapted to the load of 8N in approximately ten weeks, indicated by the trabecular bone volume fraction, which increased from 16.7% at 0 weeks to 21.6% at week 10 and only showed little change afterwards (bone volume fraction of 21.5% at 14 weeks). Similarly bone stiffness - (at the start of the experiment 649N/mm) - reached 846N/mm at 10 weeks in the loaded group and was maintained to the end of the experiment (850N/mm). At 4 weeks the bone formation rate was 32% greater and the bone resorption rate 22% less for 8N compared to 0N. This difference was significantly reduced as the bone adapted to 8N, with 8N remodeling rates returning to the values of the 0N group at approximately 10 weeks. Together these data suggest that once bone has adapted to a new loading state, the remodeling rates reduce gradually while maintaining bone volume fraction and stiffness.

Strain-adaptive in silico modeling of bone adaptation--a computer simulation validated by in vivo micro-computed tomography data.

Computational models are an invaluable tool to test different mechanobiological theories and, if validated properly, for predicting changes in individuals over time. Concise validation of in silico models, however, has been a bottleneck in the past due to a lack of appropriate reference data. Here, we present a strain-adaptive in silico algorithm which is validated by means of experimental in vivo loading data as well as by an in vivo ovariectomy experiment in the mouse. The maximum prediction error following four weeks of loading resulted in 2.4% in bone volume fraction (BV/TV) and 8.4% in other bone structural parameters. Bone formation and resorption rate did not differ significantly between experiment and simulation. The spatial distribution of formation and resorption sites matched in 55.4% of the surface voxels. Bone loss was simulated with a maximum prediction error of 12.1% in BV/TV and other bone morphometric indices, including a saturation level after a few weeks. Dynamic rates were more difficult to be accurately predicted, showing evidence for significant differences between simulation and experiment (p<0.05). The spatial agreement still amounted to 47.6%. In conclusion, we propose a computational model which was validated by means of experimental in vivo data. The predictive value of an in silico model may become of major importance if the computational model should be applied in clinical settings to predict bone changes due to disease and test the efficacy of potential pharmacological interventions.

Microdamage caused by fatigue loading in human cancellous bone: relationship to reductions in bone biomechanical performance.

Vertebral fractures associated with osteoporosis are often the result of tissue damage accumulated over time. Microscopic tissue damage (microdamage) generated in vivo is believed to be a mechanically relevant aspect of bone quality that may contribute to fracture risk. Although the presence of microdamage in bone tissue has been documented, the relationship between loading, microdamage accumulation and mechanical failure is not well understood. The aim of the current study was to determine how microdamage accumulates in human vertebral cancellous bone subjected to cyclic fatigue loading. Cancellous bone cores (n = 32) from the third lumbar vertebra of 16 donors (10 male, 6 female, age 76 ± 8.8, mean ± SD) were subjected to compressive cyclic loading at σ/E0 = 0.0035 (where σ is stress and E0 is the initial Young's modulus). Cyclic loading was suspended before failure at one of seven different amounts of loading and specimens were stained for microdamage using lead uranyl acetate. Damage volume fraction (DV/BV) varied from 0.8 ± 0.5% (no loading) to 3.4 ± 2.1% (fatigue-loaded to complete failure) and was linearly related to the reductions in Young's modulus caused by fatigue loading (r(2) = 0.60, p<0.01). The relationship between reductions in Young's modulus and proportion of fatigue life was nonlinear and suggests that most microdamage generation occurs late in fatigue loading, during the tertiary phase. Our results indicate that human vertebral cancellous bone tissue with a DV/BV of 1.5% is expected to have, on average, a Young's modulus 31% lower than the same tissue without microdamage and is able to withstand 92% fewer cycles before failure than the same tissue without microdamage. Hence, even small amounts of microscopic tissue damage in human vertebral cancellous bone may have large effects on subsequent biomechanical performance.

Bone morphology allows estimation of loading history in a murine model of bone adaptation.

Bone adapts its morphology (density/micro- architecture) in response to the local loading conditions in such a way that a uniform tissue loading is achieved ('Wolff's law'). This paradigm has been used as a basis for bone remodeling simulations to predict the formation and adaptation of trabecular bone. However, in order to predict bone architectural changes in patients, the physiological external loading conditions, to which the bone was adapted, need to be determined. In the present study, we developed a novel bone loading estimation method to predict such external loading conditions by calculating the loading history that produces the most uniform bone tissue loading. We applied this method to murine caudal vertebrae of two groups that were in vivo loaded by either 0 or 8 N, respectively. Plausible load cases were sequentially applied to micro-finite element models of the mice vertebrae, and scaling factors were calculated for each load case to derive the most uniform tissue strain-energy density when all scaled load cases are applied simultaneously. The bone loading estimation method was able to predict the difference in loading history of the two groups and the correct load magnitude for the loaded group. This result suggests that the bone loading history can be estimated from its morphology and that such a method could be useful for predicting the loading history for bone remodeling studies or at sites where measurements are difficult, as in bone in vivo or fossil bones.

Advances in multimodality molecular imaging of bone structure and function.

The skeleton is important to the body as a source of minerals and blood cells and provides a structural framework for strength, mobility and the protection of organs. Bone diseases and disorders can have deteriorating effects on the skeleton, but the biological processes underlying anatomical changes in bone diseases occurring in vivo are not well understood, mostly due to the lack of appropriate analysis techniques. Therefore, there is ongoing research in the development of novel in vivo imaging techniques and molecular markers that might help to gain more knowledge of these pathological pathways in animal models and patients. This perspective provides an overview of the latest developments in molecular imaging applied to bone. It emphasizes that multimodality imaging, the combination of multiple imaging techniques encompassing different image modalities, enhances the interpretability of data, and is imperative for the understanding of the biological processes and the associated changes in bone structure and function relationships in vivo.

Longitudinal assessment of in vivo bone dynamics in a mouse tail model of postmenopausal osteoporosis.

Recently, it has been shown that transient bone biology can be observed in vivo using time-lapse micro-computed tomography (µCT) in the mouse tail bone. Nevertheless, in order for the mouse tail bone to be a model for human disease, the hallmarks of any disease must be mimicked. The aim of this study was to investigate whether postmenopausal osteoporosis could be modeled in caudal vertebrae of C57Bl/6 mice, considering static and dynamic bone morphometry as well as mechanical properties, and to describe temporal changes in bone remodeling rates. Twenty C57Bl/6 mice were ovariectomized (OVX, n = 11) or sham-operated (SHM, n = 9) and monitored with in vivo µCT on the day of surgery and every 2 weeks after, up to 12 weeks. There was a significant decrease in bone volume fraction for OVX (-35%) compared to SHM (+16%) in trabecular bone (P < 0.001). For OVX, high-turnover bone loss was observed, with the bone resorption rate exceeding the bone formation rate (P < 0.001). Furthermore there was a significant decrease in whole-bone stiffness for OVX (-16%) compared to SHM (+11%, P < 0.001). From these results we conclude that the mouse tail vertebra mimics postmenopausal bone loss with respect to these parameters and therefore might be a suitable model for postmenopausal osteoporosis. When evaluating temporal changes in remodeling rates, we found that OVX caused an immediate increase in bone resorption rate (P < 0.001) and a delayed increase in bone formation rate (P < 0.001). Monitoring transient bone biology is a promising method for future research.

Mouse tail vertebrae adapt to cyclic mechanical loading by increasing bone formation rate and decreasing bone resorption rate as shown by time-lapsed in vivo imaging of dynamic bone morphometry.

It is known that mechanical loading leads to an increase in bone mass through a positive shift in the balance between bone formation and bone resorption. How the remodeling sites change over time as an effect of loading remains, however, to be clarified. The purpose of this paper was to investigate how bone formation and resorption sites are modulated by mechanical loading over time by using a new imaging technique that extracts three dimensional formation and resorption parameters from time-lapsed in vivo micro-computed tomography images. To induce load adaptation, the sixth caudal vertebra of C57BL/6 mice was cyclically loaded through pins in the adjacent vertebrae at either 8 N or 0 N (control) three times a week for 5 min (3000 cycles) over a total of 4 weeks. The results showed that mechanical loading significantly increased trabecular bone volume fraction by 20% (p<0.001) and cortical area fraction by 6% (p<0.001). The bone formation rate was on average 23% greater (p<0.001) and the bone resorption rate was on average 25% smaller (p<0.001) for the 8 N group than for the 0 N group. The increase in bone formation rate for the 8 N group was mostly an effect of a significantly increased surface of bone formation sites (on average 16%, p<0.001), while the thickness of bone formation packages was less affected (on average 5% greater, p<0.05). At the same time the surface of bone resorption sites was significantly reduced (on average 15%, p<0.001), while the depth of resorption pits remained the same. For the 8 N group, the strength of the whole bone increased significantly by 24% (p<0.001) over the loading period, while the strain energy density in the trabecular bone decreased significantly by 24% (p<0.001). In conclusion, mouse tail vertebrae adapt to mechanical loading by increasing the surface of formation sites and decreasing the surface of resorption sites, leading to an overall increase in bone strength. This new imaging technique will provide opportunities to investigate in vivo bone remodeling in the context of disease and treatment options, with the added value that both bone formation and bone resorption parameters can be nondestructively calculated over time.

In vivo loading increases mechanical properties of scaffold by affecting bone formation and bone resorption rates.

A successful bone tissue engineering strategy entails producing bone-scaffold constructs with adequate mechanical properties. Apart from the mechanical properties of the scaffold itself, the forming bone inside the scaffold also adds to the strength of the construct. In this study, we investigated the role of in vivo cyclic loading on mechanical properties of a bone scaffold. We implanted PLA/ß-TCP scaffolds in the distal femur of six rats, applied external cyclic loading on the right leg, and kept the left leg as a control. We monitored bone formation at 7 time points over 35 weeks using time-lapsed micro-computed tomography (CT) imaging. The images were then used to construct micro-finite element models of bone-scaffold constructs, with which we estimated the stiffness for each sample at all time points. We found that loading increased the stiffness by 60% at 35 weeks. The increase of stiffness was correlated to an increase in bone volume fraction of 18% in the loaded scaffold compared to control scaffold. These changes in volume fraction and related stiffness in the bone scaffold are regulated by two independent processes, bone formation and bone resorption. Using time-lapsed micro-CT imaging and a newly-developed longitudinal image registration technique, we observed that mechanical stimulation increases the bone formation rate during 4-10 weeks, and decreases the bone resorption rate during 9-18 weeks post-operatively. For the first time, we report that in vivo cyclic loading increases mechanical properties of the scaffold by increasing the bone formation rate and decreasing the bone resorption rate.

In vivo validation of a computational bone adaptation model using open-loop control and time-lapsed micro-computed tomography.

Cyclic mechanical loading augments trabecular bone mass, mainly by increasing trabecular thickness. For this reason, we hypothesized that an in silico thickening algorithm using open-loop control would be sufficient to reliably predict the response of trabecular bone to cyclic mechanical loading. This would also mean that trabecular bone adaptation could be modeled as a system responding to an input signal at the onset of the process in a predefined manner, without feedback from the outputs. Here, time-lapsed in vivo micro-computed tomography scans of mice cyclically loaded at the sixth caudal vertebra were used to validate the in silico model. When comparing in silico and in vivo data sets after a period of four weeks, a maximum prediction error of 2.4% in bone volume fraction and 5.4% in other bone morphometric indices was calculated. Superimposition of sequentially acquired experimental images and simulated structures revealed that in silico simulations deposited thin and homogeneous layers of bone whilst the experiment was characterized by local areas of strong thickening, as well as considerable volumes of bone resorption. From the results, we concluded that the proposed computational algorithm predicted changes in bone volume fraction and global parameters of bone structure very well over a period of four weeks while it was unable to reproduce accurate spatial patterns of local bone formation and resorption. This study demonstrates the importance of validation of computational models through the use of experimental in vivo data, including the local comparison of simulated and experimental remodeling sites. It is assumed that the ability to accurately predict changes in bone micro-architecture will facilitate a deeper understanding of the cellular mechanisms underlying bone remodeling and adaptation due to mechanical loading.

In vivo micro-computed tomography allows direct three-dimensional quantification of both bone formation and bone resorption parameters using time-lapsed imaging.

Bone is a living tissue able to adapt its structure to external influences such as altered mechanical loading. This adaptation process is governed by two distinct cell types: bone-forming cells called osteoblasts and bone-resorbing cells called osteoclasts. It is therefore of particular interest to have quantitative access to the outcomes of bone formation and resorption separately. This article presents a non-invasive three-dimensional technique to directly extract bone formation and resorption parameters from time-lapsed in vivo micro-computed tomography scans. This includes parameters such as Mineralizing Surface (MS), Mineral Apposition Rate (MAR), and Bone Formation Rate (BFR), which were defined in accordance to the current nomenclature of dynamic histomorphometry. Due to the time-lapsed and non-destructive nature of in vivo micro-computed tomography, not only formation but also resorption can now be assessed quantitatively and time-dependent parameters Eroded Surface (ES) as well as newly defined indices Mineral Resorption Rate (MRR) and Bone Resorption Rate (BRR) are introduced. For validation purposes, dynamic formation parameters were compared to the traditional quantitative measures of dynamic histomorphometry, where MAR correlated with R = 0.68 and MS with R = 0.78 (p < 0.05). Reproducibility was assessed in 8 samples that were scanned 5 times and errors ranged from 0.9% (MRR) to 6.6% (BRR). Furthermore, the new parameters were applied to a murine in vivo loading model. A comparison of directly extracted parameters between formation and resorption within each animal revealed that in the control group, i.e., during normal remodeling, MAR was significantly lower than MRR (p < 0.01), whereas MS compared to ES was significantly higher (p < 0.0001). This implies that normal remodeling seems to take place by many small formation packets and few but large resorption volumes. After 4 weeks of mechanical loading, newly extracted trabecular BFR and MS were significantly higher (p < 0.01) in the loading compared to the control group. At the same time, ES was significantly decreased (p < 0.01). This indicates that modeling induced by mechanical loading takes place primarily by increased area, not width of formation packets. With these results, we conclude that the non-invasive direct technique is well suited to extract dynamic bone morphometry parameters and eventually gain more insight into the processes of bone adaptation not only for formation but also resorption.

Comparison of bone loss induced by ovariectomy and neurectomy in rats analyzed by in vivo micro-CT.

We hypothesized that osteoporosis due to estrogen deficiency progresses faster than due to disuse and that at the same amount of bone loss, disuse leads to less favorable bone structure and mechanical properties than estrogen deficiency. Adult rats were either ovariectomized (OVX) (n = 9) or neurectomized (NX) (n = 8). At week 0, 1, 2, 3, and 4, in vivo micro-CT scans were made of the proximal tibia. Segmented CT-scans at weeks 0 and 4 were used to build a 3D voxel-based micro finite element model (FEM). Displacement in the longitudinal direction was prescribed at the proximal end leading to a compression step of 1%. The severe reduction in metaphyseal bone volume fraction was not significantly different between OVX and NX. Epiphyseal bone loss was less severe in both groups, and BV/TV was significantly lower after NX. Trabecular separation and degree of anisotropy in the metaphysis and connectivity and trabecular number in the epiphysis were significantly more deteriorated after NX. FEM-derived stiffness decreased in both groups, but more after NX. Osteoporosis due to estrogen-deficiency progressed overall at a rate similar to osteoporosis due to disuse. At the same amount of induced bone loss, disuse led to more deteriorated bone structure and mechanical properties than estrogen deficiency.