Andreas Roschger

Effect of sclerostin antibody treatment in a mouse model of severe osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually caused by mutations affecting collagen type I production in osteoblasts. Stimulation of bone formation through sclerostin antibody treatment (Sost-ab) has shown promising results in mouse models of relatively mild OI. We assessed the effect of once-weekly intravenous Sost-ab injections for 4weeks in male Col1a1(Jrt)/+mice, a model of severe dominant OI, starting either at 4weeks (growing mice) or at 20weeks (adult mice) of age. Sost-ab had no effect on weight or femur length. In OI mice, no significant treatment-associated differences in serum markers of bone formation (alkaline phosphatase activity, procollagen type I N-propeptide) or resorption (C-telopeptide of collagen type I) were found. Micro-CT analyses at the femur showed that Sost-ab treatment was associated with higher trabecular bone volume and higher cortical thickness in wild type mice at both ages and in growing OI mice, but not in adult OI mice. Three-point bending tests of the femur showed that in wild type but not in OI mice, Sost-ab was associated with higher ultimate load and work to failure. Quantitative backscattered electron imaging of the femur did not show any effect of Sost-ab on CaPeak (the most frequently occurring calcium concentration in the bone mineral density distribution), regardless of genotype, age or measurement location. Thus, Sost-ab had a larger effect in wild type than in Col1a1(Jrt)/+mice. Previous studies had found marked improvements of Sost-ab on bone mass and strength in an OI mouse model with a milder phenotype. Our data therefore suggest that Sost-ab is less effective in a more severely affected OI mouse model.

Sclerostin Deficiency Is Linked to Altered Bone Composition

High bone mass in animals and humans with sclerostin deficiency is associated with increased bone strength, which is not the case for all disorders with high bone mineral density, some of which are even associated with fragility fractures owing to unfavorable bone composition. In the current study we investigated whether alterations in bone composition may contribute to the bone strength characteristics associated with lack of sclerostin. We examined cortical bone of Sost‐knockout (KO) mice (n = 9, 16 weeks old) and sclerosteosis patients (young [4 to 14 years], n = 4 and adults [24 and 43 years], n = 2) by quantitative backscattered electron imaging and Raman microspectroscopy and compared it to bone from wild‐type mice and healthy subjects, respectively. In Sost‐KO mice endocortical bone exhibited altered bone composition, whereas subperiosteal bone was unchanged. When comparing endocortical bone tissue of identical tissue age as defined by sequential dual fluorochrome labeling the average bone matrix mineralization was reduced −1.9% (p < 0.0001, younger tissue age) and −1.5% (p < 0.05, older tissue age), and the relative proteoglycan content was significantly increased. Similarly, bone matrix mineralization density distribution was also shifted toward lower matrix mineralization in surgical samples of compact bone of sclerosteosis patients. This was associated with an increase in mineralization heterogeneity in the young population. In addition, and consistently, the relative proteoglycan content was increased. In conclusion, we observed decreased matrix mineralization and increased relative proteoglycan content in bone subcompartments of Sost‐KO mice—a finding that translated into sclerosteosis patients. We hypothesize that the altered bone composition contributes to the increased bone strength of patients with sclerostin deficiency.

Relationship between the v2PO4/amide III ratio assessed by Raman spectroscopy and the calcium content measured by quantitative backscattered electron microscopy in healthy human osteonal bone

Abstract. Raman microspectroscopy and quantitative backscattered electron imaging (qBEI) of bone are powerful tools to investigate bone material properties. Both methods provide information on the degree of bone matrix mineralization. However, a head-to-head comparison of these outcomes from identical bone areas has not been performed to date. In femoral midshaft cross sections of three women, 99 regions (20×20  μm2) were selected inside osteons and interstitial bone covering a wide range of matrix mineralization. As the focus of this study was only on regions undergoing secondary mineralization, zones exhibiting a distinct gradient in mineral content close to the mineralization front were excluded. The same regions were measured by both methods. We found a linear correlation (R2=0.75) between mineral/matrix as measured by Raman spectroscopy and the wt. %Mineral/(100-wt. %Mineral) as obtained by qBEI, in good agreement with theoretical estimations. The observed deviations of single values from the linear regression line were determined to reflect biological heterogeneities. The data of this study demonstrate the good correspondence between Raman and qBEI outcomes in describing tissue mineralization. The obtained correlation is likely sensitive to changes in bone tissue composition, providing an approach to detect potential deviations from normal bone.

Pediatric reference Raman data for material characteristics of iliac trabecular bone

Bone material characteristics are important contributors in the determination of bone strength. Raman spectroscopic analysis provides information on mineral/matrix ratio, mineral maturity/crystallinity, relative pyridinoline (Pyd) collagen cross-link content, relative proteoglycan content and relative lipid content. However, published reference data are available only for adults. The purpose of the present study was to establish reference data of Raman outcomes pertaining to bone quality in trabecular bone for children and young adults. To this end, tissue age defined Raman microspectroscopic analysis was performed on bone samples from 54 individuals between 1.5 and 23 years with no metabolic bone disease, which have been previously used to establish histomorphometric and bone mineralization density distribution reference values. Four distinct tissue ages, three well defined by the fluorescent double labels representing early stages of bone formation and tissue maturation (days 3, 12, 20 of tissue mineralization) and a fourth representing old mature tissue at the geometrical center of the trabeculae, were analyzed. In general, significant dependencies of the measured parameters on tissue age were found, while at any given tissue age, sex and subject age were not confounders. Specifically, mineral/matrix ratio, mineral maturity/crystallinity index and relative pyridinoline collagen cross-link content index increased by 485%, 20% and 14%, respectively between days 3 and 20. The relative proteoglycan content index was unchanged between days 3 and 20 but was elevated in the old tissue compared to young tissue by 121%. The relative lipid content decreased within days 3 to 20 by -22%. Thus, the method allows not only the monitoring of material characteristics at a specific tissue age but also the kinetics of tissue maturation as well. The established reference Raman database will serve as sensitive tool to diagnose disturbances in material characteristics of pediatric bone biopsy samples.

Reversible Deterioration in Hypophosphatasia Caused by Renal Failure With Bisphosphonate Treatment.

Hypophosphatasia is an inborn error of metabolism caused by mutations in the ALPL gene. It is characterized by low serum alkaline phosphatase (ALP) activity and defective mineralization of bone, but the phenotype varies greatly in severity depending on the degree of residual enzyme activity. We describe a man with compound heterozygous mutations in ALPL, but no previous bone disease, who suffered numerous disabling fractures after he developed progressive renal failure (for which he eventually needed dialysis treatment) and was prescribed alendronate treatment. A bone biopsy showed marked osteomalacia with low osteoblast numbers and greatly elevated pyrophosphate concentrations at mineralizing surfaces. In vitro testing showed that one mutation, T117H, produced an ALP protein with almost no enzyme activity; the second, G438S, produced a protein with normal activity, but its activity was inhibited by raising the media phosphate concentration, suggesting that phosphate retention (attributable to uremia) could have contributed to the phenotypic change, although a pathogenic effect of bisphosphonate treatment is also likely. Alendronate treatment was discontinued and, while a suitable kidney donor was sought, the patient was treated for 6 months with teriparatide, which significantly reduced the osteomalacia. Eighteen months after successful renal transplantation, the patient was free of symptoms and the scintigraphic bone lesions had resolved. A third bone biopsy showed marked hyperosteoidosis but with plentiful new bone formation and a normal bone formation rate. This case illustrates how pharmacological (bisphosphonate treatment) and physiologic (renal failure) changes in the “environment” can dramatically affect the phenotype of a genetic disorder.

Spatial heterogeneity in the canalicular density of the osteocyte network in human osteons

Osteocytes interconnect with each other forming an intricate cell network within the mineralized bone matrix. One important function of the osteocyte network is the mechano-regulation of bone remodeling, where a possible mechanism includes the fluid flow through the porosity housing the cell network - the osteocyte lacuno-canalicular network (OLCN). In our study the OLCN in human osteons was three-dimensionally imaged with the aim to obtain a quantitative description of the canalicular density and spatial variations of this quantity within osteons. The topology of the OLCN was determined by first staining the bone samples with rhodamine, then imaging the OLCN with confocal laser scanning microscopy and finally using image analysis to obtain a skeletonized version of the network for further analysis. In total 49 osteons were studied from the femoral cortical bone of four different middle-aged healthy women. The mean canalicular density given as length of the canaliculi in a unit volume was 0.074 ± 0.015 μm/μm3 (corresponding to 74 km/cm3). No correlation was found between the canalicular density and neither the size of the osteon nor the volume fraction occupied by osteocyte lacunae. Within osteons the canalicular density varied substantially with larger regions without any network. On average the canalicular density decreases when moving from the Haversian canal outwards towards the cement line. We hypothesize that a decrease in accessible canaliculi with tissue age as a result of micropetrosis can reduce the local mechanosensitivity of the bone. Systematic future studies on age- and disease-related changes on the topology of the OLCN have to demonstrate the diagnostic potential of the presented characterization method.

Mapping dynamical mechanical properties of osteonal bone by scanning acoustic microscopy in time-of-flight mode.

An important determinant of mechanical properties of bone is Youngs modulus and its variation in individual osteons of cortical bone tissue. Its mechanical behavior also depends on deformation rate owing to its visco- or poroelastic properties. We developed a method to measure dynamical mechanical properties of bulk bone tissue at osteonal level based on scanning acoustic microscopy (SAM) using time-of-flight (TOF) measurements in combination with quantitative backscattered electron imaging (qBEI). SAM-TOF yields local sound velocities and qBEI corresponding material densities together providing elastic properties. Osteons (n=55) were measured in three human femoral diaphyseal ground bone sections (∼ 30 µm in thickness). In addition, subchondral bone and mineralized articular cartilage were investigated. The mean mineral contents, the mean sound velocities, and the mean elastic modulus of the osteons ranged from 20 to 26 wt%, from 3,819 to 5,260 m/s, and from 21 to 44 GPa, respectively. There was a strong positive correlation between material density and sound velocity (Pearsons r=0.701; p<0.0001) of the osteons. Sound velocities between cartilage and bone was similar, though material density was higher in cartilage (+4.46%, p<0.0001). These results demonstrate the power of SAM-TOF to estimate dynamic mechanical properties of the bone materials at the osteonal level.

Increased zinc accumulation in mineralized osteosarcoma tissue measured by confocal synchrotron radiation micro X-ray fluorescence analysis

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X‐ray fluorescence analysis, we characterized the spatial distribution of Zn in high‐grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer‐Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer. Copyright

Altered matrix mineralization in a case of a sclerosing osteosarcoma.

Little is known about the tumor matrix mineralization of highly sclerotic osteosarcoma. We used quantitative backscattered electron imaging (qBEI) to determine the Bone mineralization density distribution (BMDD) of a highly sclerosing osteosarcoma of the proximal tibia as well as adjacent normal bone of a 10-year-old girl following chemotherapy according to the EURAMOS-1 protocol. Data were compared to recently published normative reference data for young individuals. Backscattered electron imaging of the tumor region revealed a dense accumulation of mineralized tumor bone matrix (up to 90% of the medullar space). The BMDD was shifted tremendously towards higher matrix mineralization (CaMean +18.5%, CaPeak +22.5%, CaHigh +100 fold) compared to normal bone. Additionally the BMDD became much wider, indicating a higher heterogeneity in mineralization (CaWidth +40%). In contrast to lamellar bone, which mineralizes via a mineralization front, the mineralization of the tumor matrix starts by randomly distributed spots of mineral clusters fusing together to a highly mineralized non-lamellar bone matrix. We also found an altered BMDD of the patients normal bone when compared with the reference BMDD of young individuals. In conclusion this high radiodensity region of the sclerosing sarcoma is not only due to the high amount of tumor matrix but also to its high mineralization density. Chemotherapy may lead to altered matrix mineralization of normal bone due to suppression of bone turnover. The mechanism of matrix mineralization in a sclerosing osteosarcoma warrants further studies.

Synchrotron radiation micro X-ray fluorescence spectroscopy of thin structures in bone samples: comparison of confocal and color X-ray camera setups

To find the ideal synchrotron radiation induced imaging method for the investigation of trace element distributions in bone tissue, experiments with a scanning confocal micro X-ray fluorescence system and a full-field color X-ray camera setup were performed.