Ex vivo culture platform for assessment of cartilage repair treatment strategies.

There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors specific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, matrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivo testing.

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.

The correlation between mineralization degree and bone tissue stiffness in the porcine mandibular condyle.

The aim of this study was to correlate the local tissue mineral density (TMD) with the bone tissue stiffness. It was hypothesized that these variables are positively correlated. Cancellous and cortical bone samples were derived from ten mandibular condyles taken from 5 young and 5 adult female pigs. The bone tissue stiffness was assessed in three directions using nanoindentation. At each of three tested sides 5 indents were made over the width of 5 single bone elements, resulting in a total number of 1500 indents. MicroCT was used to determine the local TMD at the indented sites. The TMD and the bone tissue stiffness were higher in bone from the adult animals than from the young ones, but did not differ between cancellous and cortical bone. In the adult group, both the TMD and the bone tissue stiffness were higher in the center than at the surface of the bone elements. The mean TMD, thus ignoring the local mineral distribution, had a coefficient of determination (R(2)) with the mean bone tissue stiffness of 0.55, p < 0.05, whereas the correlation between local bone tissue stiffness and the concomitant TMD appeared to be weak (R (2) 0.07, p < 0.001). It was concluded that the mineralization degree plays a larger role in bone tissue stiffness in cancellous than in cortical bone. Our data based on bone from the mandibular condyle suggest that the mineralization degree is not a decisive determinant of the local bone tissue stiffness.

Waterjet drilling in porcine bone: the effect of the nozzle diameter and bone architecture on the hole dimensions.

Using waterjets instead of rigid drill bits for bone drilling can be beneficial due to the absence of thermal damage and a consequent sharp cut. Additionally, waterjet technology allows the development of flexible instruments that facilitate maneuvering through complex joint spaces. Controlling the drilling depth is of utmost importance to ensure clinical safety, but is challenging given the local variations in structural properties of the bone. The goal of this study was to deduce a descriptive mathematical equation able to predict the hole depth and diameter based on the local structural properties of the bone at given waterjet diameters. 210 holes were drilled in porcine femora and tali with waterjet diameters (Dnozzle) of 0.3, 0.4, 0.5 and 0.6mm at a pressure of 700bar and a 5s jet time. Hole depths (Lhole), diameters (Dhole) and bone architectural properties were determined using microCT scans. The most important bone architectural property is the bone volume fraction (BV/TV), resulting in the significant predictive equations: Lhole=34.3 (⁎) Dnozzle(2)-17.6 (⁎) BV/TV+10.7 (R(2)=0.90, p<0.001), and hole Dhole=3.1(⁎) Dnozzle-0.45(⁎)BV/TV+0.54 (R(2)=0.58, p=0.02), with Lhole, Dhole and Dnozzle in mm. Drilling to a specific depth in bone tissue with a known BV/TV is possible, thereby contributing to the safe application of waterjet technology in orthopedic surgery.

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.

Age-related changes in collagen properties and mineralization in cancellous and cortical bone in the porcine mandibular condyle.

Collagen is an important constituent of bone, and it has been suggested that changes in collagen and mineral properties of bone are interrelated during growth. The aim of this study was to quantify age-related changes in collagen properties and the degree of mineralization of bone (DMB). The DMB in cancellous and cortical bone samples from the mandibular condyle of 35 female pigs aged 0-100 weeks was determined using micro-computed tomography. Subsequently, the amount of collagen and the number of pentosidine (Pen), hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were quantified by means of high-performance liquid chromatography. The amount of collagen increased with age in cancellous bone but remained unchanged in cortical bone. The number of Pen and LP cross-links decreased in both bone types. In contrast, the number of HP cross-links decreased only in cancellous bone. The sum of the number of HP and LP cross-links decreased with age in cancellous bone only. The DMB increased in cancellous and cortical bone. It was concluded that the largest changes in the number of mature collagen cross-links and the mineralization in porcine cancellous and cortical bone take place before the age of 40 weeks. The low number of mature cross-links after this age suggests that the bone turnover rate continues to be high and thereby prevents the development of mature cross-links.

Inhibitory regulation of osteoclast bone resorption by signal regulatory protein alpha.

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) alpha as a negative regulator of osteoclast activity. SIRPalpha is expressed by osteoclasts, and osteoclasts from mice lacking the SIRPalpha cytoplasmic tail and signaling capacity display enhanced bone resorption in vitro. Consequently, SIRPalpha-mutant mice have a significantly reduced cortical bone mass. Furthermore, osteoclasts from SIRPalpha-mutant mice show an enhanced formation of actin rings, known to be instrumental in bone resorption. SIRPalpha mutation did not significantly affect osteoclast formation, implying that the role of SIRPalpha was limited to the regulation of mature osteoclast function. This identifies SIRPalpha 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.

Development of the micro architecture and mineralization of the basilar part of the pig occipital bone.

In this study, the development of the architecture and the degree and distribution of mineralization in the basilar part of the pig occipital bone, one of the contact points between the spine and skull base, was investigated. Multiple regions of the basiocciput of pig specimens of different gestational ages were examined with three-dimensional microcomputed tomography (microCT). The cortex of the basilar part developed from a structure with a large intertrabecular separation into a more compact one, whereas its center maintained a trabecular structure. The cortex displayed a significant increase in bone volume fraction with age, together with an increase in trabecular thickness. In the center no change in bone volume fraction was observed, because of a combined decrease in trabecular number and increase in trabecular separation. During development the degree of mineralization was almost identical in both the center and the cortex and it tended to increase with age. This chance was, however, insignificant. The distribution of mineralization within the trabecular elements of both regions demonstrated that the cores of the elements were more highly mineralized than their surfaces. This difference in mineralization confirms the preexisting notion that trabecular elements grow in size due to surface apposition of new bone that initially is less mineralized.

Relationship between tissue stiffness and degree of mineralization of developing trabecular bone.

It is unknown how the degree of mineralization of bone in individual trabecular elements is related to the corresponding mechanical properties at the bone tissue level. Understanding this relationship is important for the comprehension of the mechanical behavior of bone at both the apparent and tissue level. The purpose of the present study was, therefore, to determine the tissue stiffness and degree of mineralization of the trabecular bone tissue and to establish a relationship between these two variables. A second goal was to assess the change in this relation during development. Mandibular condylar specimens of four fetal and four newborn pigs were used. The tissue stiffness was measured using nanoindentation. A pair of indents was made in the cores of 15 trabecular elements per specimen. Subsequently, the degree of mineralization of these locations was determined from microcomputed tomography. The mean tissue stiffness was 11.2 GPa (+/-0.5 GPa) in the fetal group and 12.0 GPa (+/-0.8 GPa) in the newborn group, which was not significantly different. The degree of mineralization of the fetal trabecular cores was 744 mg/cm3 (+/-28 mg/cm3). The one in the newborn bone measured 719 mg/cm(3) (+/-34 mg/cm3). Again, the difference was statistically insignificant. A significant relationship between tissue stiffness and degree of mineralization was obtained for fetal (R = 0.42, p < 0.001) and newborn (R = 0.72, p < 0.001) groups. It was concluded that woven bone tissue in fetal and newborn trabecular cores resembles adult trabecular bone in terms of tissue properties and is strongly correlated with degree of mineralization.

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.

Intratrabecular distribution of tissue stiffness and mineralization in developing trabecular bone.

The purpose of this study was to investigate the relation between bone tissue stiffness and degree of mineralization distribution and to examine possible changes during prenatal development. Understanding this may provide insight into adaptation processes and into deformation mechanisms of the bone microstructure. Mandibular condyles from four fetal and newborn pigs were used. Tissue stiffness was measured using nanoindentation, the degree of mineralization with microCT. Eight indents were made over the trabecular width of 15 trabeculae in each specimen, leading to a total of 960 indents. Subsequently, the degree of mineralization of these locations was determined. Intratrabecular variations in bone tissue stiffness and degree of mineralization showed a similar pattern; low at trabecular surfaces and higher in the cores. A strong correlation was found between the two variables, which remained unchanged during development. It was concluded that bone tissue in fetal and newborn trabecular cores resembles adult trabecular bone tissue properties and is distributed in a regular radial pattern in trabeculae. For the first time, it was shown that the intratrabecular tissue stiffness develops along the same path as the degree of mineralization. Knowledge regarding intratrabecular tissue stiffness and mineralization results in a better understanding of trabecular bone mechanical behavior on a structural and tissue level.

Age-related changes in microarchitecture and mineralization of cancellous bone in the porcine mandibular condyle.

The mandibular condyle is considered a good model for developing cancellous bone because of its rapid growth and high rate of remodeling. The aim of the present study was to analyze the simultaneous changes in microarchitecture and mineralization of cancellous bone during development in a three-dimensional fashion. Eight mandibular condyles of pigs aged 8 weeks prepartum to 108 weeks postpartum were scanned using microCT with an isotropic spatial resolution of 10 microm. The number of trabeculae decreased during development, whereas both the trabecular thickness and the distance between the trabeculae increased. The bone surface to volume ratio decreased during development, possibly limiting the amount of (re)modeling. Both the mean degree of mineralization and intratrabecular differences in mineralization between the surfaces and cores of trabecular elements increased during development. The trabecular surfaces were more highly mineralized in the older condyles compared to the younger ones. Together with the observed decrease in the relative size of trabecular surface, this finding suggests a decrease in (re)modeling activity during development. In accordance with the general growth and development of the pig, it was concluded that most developmental changes in cancellous bone occur until the age of 40 weeks postpartum.

Biomechanical consequences of developmental changes in trabecular architecture and mineralization of the pig mandibular condyle.

The purpose of the present study was to examine the changes in apparent mechanical properties of trabecular bone in the mandibular condyle during fetal development and to investigate the contributions of altering architecture, and degree and distribution of mineralization to this change. Three-dimensional, high-resolution micro-computed tomography (microCT) reconstructions were utilized to assess the altering architecture and mineralization during development. From the reconstructions, inhomogeneous finite element models were constructed, in which the tissue moduli were scaled to the local degree of mineralization of bone (DMB). In addition, homogeneous models were devised to study the separate influence of architectural and DMB changes on apparent mechanical properties. It was found that the bone structure became stiffer with age. Both the mechanical and structural anisotropies pointed to a rod-like structure that was predominantly oriented from anteroinferior to posterosuperior. Resistance against shear, also increasing with age, was highest in the sagittal plane. The reorganization of trabecular elements, which occurred without a change in bone volume fraction, contributed to the increase in apparent stiffness. The increase in DMB, however, contributed more dominantly. Incorporating the observed inhomogeneous distribution of mineralization decreased the apparent stiffness, but increased the mechanical anisotropy. This denotes that there might be a directional dependency of the DMB of trabecular elements, i.e. differently orientated trabecular elements might have different DMBs. In conclusion, the changes in DMB and its distribution are important to consider when studying mechanical properties during development and should be considered in other situations where differences in DMB are expected.

Regional differences in architecture and mineralization of developing mandibular bone.

The goal of this study was to investigate the mutual relationship between architecture and mineralization during early development of the pig mandible. These factors are considered to define the balance between the requirements for bone growth on the one hand and for load bearing on the other. Architecture and mineralization were examined using micro-CT, whereas the mineral composition was assessed spectrophotometrically in groups of fetal and newborn pigs. The development of the condyle coincided with a reorganization of bone elements without an increase in bone volume fraction, but with an increase in mineralization and a change in mineral composition. In the corpus, the bone volume fraction and mineralization increased simultaneously with a restructuring of the bone elements and a change in mineral composition. The growth of the condyle was reflected by regional differences in architecture and mineralization. The anterior and inferior regions were characterized by a more dense bone structure and a higher mineralization as compared to posterior and superior regions, respectively. In the corpus, growth was mainly indicated by differences between buccal and lingual plates as well as between anterior, middle, and posterior regions characterized by a more compact structure and higher mineralization in the lingual and middle regions. In conclusion, the architecture and mineralization in the condyle and corpus started to deviate early during development toward their destiny as trabecular and cortical bone, respectively. These results were compatible with those obtained with mineral composition analysis. Regional differences within condyle and corpus reflected known developmental growth directions.

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.

Architecture and mineralization of developing trabecular bone in the pig mandibular condyle.

Architecture and mineralization are important determinants of trabecular bone quality. To date, no quantitative information is available on changes in trabecular bone architecture and mineralization of newly formed bone during development. Three-dimensional architecture and mineralization of the trabecular bone in the mandibular condyle from six pigs of different developmental ages were investigated with micro-CT. Anteriorly in the condyle, a more advanced state of remodeling was observed than posteriorly, where more active growth takes place. Posteriorly, the bone volume fraction increased with age (r=0.87; P<0.05) by an increase of trabecular thickness (r=0.88; P<0.05), while the number of trabeculae declined (r=-0.86; P<0.05). Anteriorly, despite an increase in trabecular thickness (r=0.97; P<0.001), there was no change in bone volume fraction due to a simultaneous decline in trabecular number (r=-0.84; P<0.05) and increase in trabecular separation (r=0.95; P<0.01). Posteriorly, rods were remodeled into plates as expressed by the structure model index (r=-0.97; P<0.001), whereas anteriorly, a plate-like structure was already present in early stages. The trabecular structure had a clear orientation throughout the developmental process. The global degree of mineralization increased both anteriorly (r=0.86; P<0.05) and posteriorly (r=0.89; P<0.05). We suggest that the degree of mineralization does not depend on the bone volume, but on the thickness of the trabeculae as the mineralized centers of trabeculae were getting larger and more highly mineralized with age compared to their appositional layers. This indicates that besides apposition of new bone material on the surface of trabeculae, the mineralized tissue in their centers still changes and matures.