Lars Mulder

Accuracy of microCT in the quantitative determination of the degree and distribution of mineralization in developing bone

PURPOSE To evaluate the accuracy and applicability of a commercially available microCT system for comparative measurements of the degree and distribution of mineralization of developing bone. MATERIAL AND METHODS Homogeneous K2HPO4 solutions with different concentrations (range 0-800 mg/cm3) were used to assess the accuracy of a microCT system equipped with a polychromatic X-ray source. Both high (45 kV) and low (70 kV) tube peak voltages were explored. The resulting attenuation was compared with calculated theoretical attenuation values to estimate the accuracy. As an example of its applicability, the method was used to assess changes in the degree of mineralization of various regions of the mandible from two pigs of different developmental age. RESULTS On average, the estimated error of the measured linear attenuation was 10% or less. Accuracy was dependent on the average mineral concentration, the size of the sample, and the energy of the X-ray beam. The accuracy of the microCT system appeared sufficient to distinguish regional differences in the degree of mineralization within and between specimens of developing mandibular bone. Furthermore, the resolution of the system allowed identification of different degrees of mineralization within trabeculae. CONCLUSION Accuracy of microCT with polychromatic radiation can be considered adequate for assessment of the degree of mineralization of developing bone. Therefore, this method provides a three-dimensional means by which to simultaneously investigate the bone structure as well as the degree of mineralization during development in a non-destructive manner and with high resolution.

Porosity of human mandibular condylar bone

Quantification of porosity and degree of mineralization of bone facilitates a better understanding of the possible effects of adaptive bone remodelling and the possible consequences for its mechanical properties. The present study set out first to give a three‐dimensional description of the cortical canalicular network in the human mandibular condyle, in order to obtain more information about the principal directions of stresses and strains during loading. Our second aim was to determine whether the amount of remodelling was larger in the trabecular bone than in cortical bone of the condyle and to establish whether the variation in the amount of remodelling was related to the surface area of the cortical canals and trabeculae. We hypothesized that there were differences in porosity and orientation of cortical canals between various cortical regions. In addition, as greater cortical and trabecular porosities are likely to coincide with a greater surface area of cortical canals and trabeculae available for osteoblastic and osteoclastic activity, we hypothesized that this surface area would be inversely proportional to the degree of mineralization of cortical and trabecular bone, respectively. Micro‐computed tomography was used to quantify porosity and mineralization in cortical and trabecular bone of ten human mandibular condyles. The cortical canals in the subchondral cortex of the condyle were orientated in the mediolateral direction, and in the anterior and posterior cortex in the superoinferior direction. Cortical porosity (average 3.5%) did not differ significantly between the cortical regions. It correlated significantly with the diameter and number of cortical canals, but not with cortical degree of mineralization. In trabecular bone (average porosity 79.3%) there was a significant negative correlation between surface area of the trabeculae and degree of mineralization; such a correlation was not found between the surface area of the cortical canals and the degree of mineralization of cortical bone. No relationship between trabecular and cortical porosity, nor between trabecular degree of mineralization and cortical degree of mineralization was found, suggesting that adaptive remodelling is independent and different between trabecular and cortical bone. We conclude (1) that the principal directions of stresses and strains are presumably directed mediolaterally in the subchondral cortex and superoinferiorly in the anterior and posterior cortex, (2) that the amount of remodelling is larger in the trabecular than in the cortical bone of the mandibular condyle; in trabecular bone variation in the amount of remodelling is related to the available surface area of the trabeculae.

Structural and Mechanical Properties of Mandibular Condylar Bone

The trabecular bone of the mandibular condyle is structurally anisotropic and heterogeneous. We hypothesized that its apparent elastic moduli are also anisotropic and heterogeneous, and depend on trabecular density and orientation. Eleven condyles were scanned with a micro-CT system. Volumes of interest were selected for the construction of finite element models. We simulated compressive and shear tests to determine the principal mechanical directions and the apparent elastic moduli. Compressive moduli were relatively large in directions acting in the sagittal plane, and small in the mediolateral direction. The degree of mechanical anisotropy ranged from 4.7 to 10.8. Shear moduli were largest in the sagittal plane and smallest in the transverse plane. The magnitudes of the moduli varied with the condylar region and were proportional to the bone volume fraction. Furthermore, principal mechanical direction correlated significantly with principal structural direction. It was concluded that variation in trabecular structure coincides with variation in apparent mechanical properties.

Mineral heterogeneity affects predictions of intratrabecular stress and strain

Knowledge of the influence of mineral variations (i.e., mineral heterogeneity) on biomechanical bone behavior at the trabecular level is limited. The aim of this study is to investigate how this material property affects the intratrabecular distributions of stress and strain in human adult trabecular bone. Two different sets of finite element (FE) models of trabecular samples were constructed; tissue stiffness was either scaled to the local degree of mineralization of bone as measured with microCT (heterogeneous) or tissue stiffness was assumed to be homogeneous. The influence of intratrabecular mineral heterogeneity was analyzed by comparing both models. Interesting effects were seen regarding intratrabecular stress and strain distributions. In the homogeneous model, the highest stresses were found at the surface with a significant decrease towards the core. Higher superficial stresses could indicate a higher predicted fracture risk in the trabeculae. In the heterogeneous model this pattern was different. A significant increase in stress with increasing distance from the trabecular surface was found followed by a significant decrease towards the core. This suggests trabecular bending during a compression. In both models a decrease in strain values from surface to core was predicted, which is consistent with trabecular bending. When mineral heterogeneity was taken into account, the predicted intratrabecular patterns of stress and strain are more consistent with the expected biomechanical behavior as based on mineral variations in trabeculae. Our findings indicate that mineral heterogeneity should not be neglected when performing biomechanical studies on topics such as the (long-term or dose dependent) effects of antiresorptive treatments.

Inhibitory regulation of osteoclast bone resorption by signal regulatory protein α

Osteoclasts mediate bone resorption, which is critical for bone development, maintenance, and repair. Proper control of osteoclast development and function is important and deregulation of these processes may lead to bone disease, such as osteoporosis. Previous studies have shown that the cytosolic protein tyrosine phosphatase SHP‐1 acts as a suppressor of osteoclast differentiation and function, but putative inhibitory receptors that mediate recruitment and activation of SHP‐1 in osteoclasts have remained unknown. In the present study, we identify the SHP‐1‐recruiting inhibitory immunoreceptor signal regulatory protein (SIRP) α as a negative regulator of osteoclast activity. SIRPα is expressed by osteoclasts, and osteoclasts from mice lacking the SIRPα cytoplasmic tail and signaling capacity display enhanced bone resorption in vitro. Consequently, SIRPα‐mutant mice have a significantly reduced cortical bone mass. Fur‐thermore, osteoclasts from SIRPα‐mutant mice show an enhanced formation of actin rings, known to be instrumental in bone resorption. SIRPα mutation did not significantly affect osteoclast formation, implying that the role of SIRPα was limited to the regulation of mature osteoclast function. This identifies SIRPα as a bona fide inhibitory receptor that regulates the bone‐resorption activity and supports a concept in which osteoclast function is balanced by the signaling activities of activating and inhibitory immunoreceptors.—Van Beek, E. M., de Vries, T. J., Mulder, L., Schoenmaker, T., Hoeben, K. A., Matozaki, T., Langenbach, G. E. J., Kraal, G., Everts, V., van den Berg, T. K. Inhibitory regulation of osteoclast bone resorption by signal regulatory protein α. FASEB J. 23, 4081‐4090 (2009). www.fasebj.org

Architecture and mineralization of developing cortical and trabecular bone of the mandible

Ossification of the presumptive trabecular bone in the mandibular condyle and the presumptive cortical bone in the mandibular corpus of the pig mandible was investigated during development, using micro-computed tomography (microCT). Three-dimensional architecture and mineralization characteristics were assessed from ten pigs of different developmental ages. In the condyle, increases in trabecular thickness and separation and a decrease in the trabecular number, led to an unchanged bone volume fraction. A conversion from rod-like into plate-like trabeculae was observed. Bone volume and trabecular thickness were always higher in the corpus, where an increase in bone volume fraction was caused by an increase in the trabecular thickness and a decrease in separation. A transition from a plate-like structure into a more compact structure took place. The average degree of mineralization in the condyle and the corpus increased with age. In the corpus, the degrees of mineralization were higher than in the condyle. The differences between the condyle and corpus and the changes with age could be explained by differences in the distribution of mineralization within the trabecular elements. Generally, the degrees of mineralization increased from the surface toward the centers of the trabecular elements, indicating growth of the trabecular elements by the surface apposition of new mineral.

Determination of vertebral and femoral trabecular morphology and stiffness using a flat-panel C-arm-based CT approach

The importance of assessing trabecular architecture together with bone mineral density to determine bone stiffness and fracture risk in osteoporosis has been well established. However, no imaging modalities are available to assess trabecular architecture at clinically relevant sites in the axial skeleton. Recently developed flat-panel CT devices, however, offer resolutions that are potentially good enough to resolve bone architecture at these sites. The goal of the present study was to investigate how accurate trabecular architecture and stiffness can be determined based on images from such a device (XperCT, Philips Healthcare). Ten cadaver human C3 vertebrae, twelve T12 vertebrae and 12 proximal femora were scanned with XperCT while mimicking in-vivo scanning conditions and compared to scans of the same bones with microCT. Standard segmentation and morphology quantification algorithms were applied as well as finite element (FE) simulation based on segmented and gray value images. Results showed that mean trabecular separation (Tb.Sp) and number (Tb.N) can be accurately determined at all sites. The accuracy of other parameters, however, depended on the site. For T12 no other structural parameters could be accurately quantified and no FE-results could be obtained from segmented images. When using gray-level images, however, accurate determination of cancellous bone stiffness was possible. For the C3 vertebrae and proximal femora, mean bone volume fraction (BV/TV), Tb.Sp, Tb.N, and anisotropy (C3 only) could be determined accurately. For Tb.Th, structure model index (SMI, femur only), and anisotropy good correlations were obtained but the values were not determined accurately. FE simulations based on segmented images were accurate for the C3 vertebrae, but severely underestimated bone stiffness for the femur. Here also, this was improved by using the gray value models. In conclusion, XperCT does provide a resolution that is good enough to determine trabecular architecture, but the signal to noise ratio is key to the accuracy of the morphology measurement. When the trabeculae are thick e.g. in the femur or the noise is low, e.g. cervical spine, architecture and stiffness could be determined accurately, but when the trabeculae are thin and the noise is high, e.g. thoracic spine, architecture could not be determined accurately and the connectivity was lost and hence no mechanical properties could be calculated directly.

Determination of the relationship between collagen cross-links and the bone–tissue stiffness in the porcine mandibular condyle

Although bone-tissue stiffness is closely related to the degree to which bone has been mineralized, other determinants are yet to be identified. We, therefore, examined the extent to which the mineralization degree, collagen, and its cross-links are related to bone-tissue stiffness. A total of 50 cancellous and cortical bone samples were derived from the right mandibular condyles of five young and five adult female pigs. The degree of mineralization of bone (DMB) was assessed using micro-computed tomography. Using high-performance liquid chromatography, we quantified the collagen content and the number of cross-links per collagen molecule of two enzymatic cross-links: hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and one non-enzymatic cross-link: pentosidine (Pen). Nanoindentation was used to assess bone-tissue stiffness in three directions, and multiple linear regressions were used to calculate the correlation between collagen properties and bone-tissue stiffness, with the DMB as first predictor. Whereas the bone-tissue stiffness of cancellous bone did not differ between the three directions of nanoindentation, or between the two age groups, cortical bone-tissue stiffness was higher in the adult tissue. After correction for DMB, the cross-links studied did not increase the explained variance. In the young group, however, LP significantly improved the explained variance in bone-tissue stiffness. Approximately half of the variation in bone-tissue stiffness in cancellous and cortical bone was explained by the DMB and the LP cross-links and thus they cannot be considered the sole determinants of the bone-tissue stiffness.

The Influence of Mineralization on Intratrabecular Stress and Strain Distribution in Developing Trabecular Bone

The load-transfer pathway in trabecular bone is largely determined by its architecture. However, the influence of variations in mineralization is not known. The goal of this study was to examine the influence of inhomogeneously distributed degrees of mineralization (DMB) on intratrabecular stresses and strains. Cubic mandibular condylar bone specimens from fetal and newborn pigs were used. Finite element models were constructed, in which the element tissue moduli were scaled to the local DMB. Disregarding the observed distribution of mineralization was associated with an overestimation of average equivalent strain and underestimation of von Mises equivalent stress. From the surface of trabecular elements towards their core the strain decreased irrespective of tissue stiffness distribution. This indicates that the trabecular elements were bent during the compression experiment. Inhomogeneously distributed tissue stiffness resulted in a low stress at the surface that increased towards the core. In contrast, disregarding this tissue stiffness distribution resulted in high stress at the surface which decreased towards the core. It was concluded that the increased DMB, together with concurring alterations in architecture, during development leads to a structure which is able to resist increasing loads without an increase in average deformation, which may lead to damage.

Higher number of pentosidine cross-links induced by ribose does not alter tissue stiffness of cancellous bone

The role of mature collagen cross-links, pentosidine (Pen) cross-links in particular, in the micromechanical properties of cancellous bone is unknown. The aim of this study was to examine nonenzymatic glycation effects on tissue stiffness of demineralized and non-demineralized cancellous bone. A total of 60 bone samples were derived from mandibular condyles of six pigs, and assigned to either control or experimental groups. Experimental handling included incubation in phosphate buffered saline alone or with 0.2M ribose at 37°C for 15 days and, in some of the samples, subsequent complete demineralization of the sample surface using 8% EDTA. Before and after experimental handling, bone microarchitecture and tissue mineral density were examined by means of microcomputed tomography. After experimental handling, the collagen content and the number of Pen, hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were estimated using HPLC, and tissue stiffness was assessed by means of nanoindentation. Ribose treatment caused an up to 300-fold increase in the number of Pen cross-links compared to nonribose-incubated controls, but did not affect the number of HP and LP cross-links. This increase in the number of Pen cross-links had no influence on tissue stiffness of both demineralized and nondemineralized bone samples. These findings suggest that Pen cross-links do not play a significant role in bone tissue stiffness.