Detection and imaging of gadolinium accumulation in human bone tissue by micro- and submicro-XRF.

Gadolinium-based contrast agents (GBCAs) are frequently used in patients undergoing magnetic resonance imaging. In GBCAs gadolinium (Gd) is present in a bound chelated form. Gadolinium is a rare-earth element, which is normally not present in human body. Though the blood elimination half-life of contrast agents is about 90 minutes, recent studies demonstrated that some tissues retain gadolinium, which might further pose a health threat due to toxic effects of free gadolinium. It is known that the bone tissue can serve as a gadolinium depot, but so far only bulk measurements were performed. Here we present a summary of experiments in which for the first time we mapped gadolinium in bone biopsy from a male patient with idiopathic osteoporosis (without indication of renal impairment), who received MRI 8 months prior to biopsy. In our studies performed by means of synchrotron radiation induced micro- and submicro-X-ray fluorescence spectroscopy (SR-XRF), gadolinium was detected in human cortical bone tissue. The distribution of gadolinium displays a specific accumulation pattern. Correlation of elemental maps obtained at ANKA synchrotron with qBEI images (quantitative backscattered electron imaging) allowed assignment of Gd structures to the histological bone structures. Follow-up beamtimes at ESRF and Diamond Light Source using submicro-SR-XRF allowed resolving thin Gd structures in cortical bone, as well as correlating them with calcium and zinc.

Melorheostotic Bone Lesions Caused by Somatic Mutations in MAP2K1 Have Deteriorated Microarchitecture and Periosteal Reaction.

Melorheostosis is a rare non-hereditary condition characterized by dense hyperostotic lesions with radiographic "dripping candle wax" appearance. Somatic activating mutations in MAP2K1 have recently been identified as a cause of melorheostosis. However, little is known about the development, composition, structure, and mechanical properties of the bone lesions. We performed a multi-method phenotype characterization of material properties in affected and unaffected bone biopsy samples from six melorheostosis patients with MAP2K1 mutations. On standard histology, lesions show a zone with intensively remodeled osteonal-like structure and prominent osteoid accumulation, covered by a shell formed through bone apposition, consisting of compact multi-layered lamellae oriented parallel to the periosteal surface and devoid of osteoid. Compared with unaffected bone, melorheostotic bone has lower average mineralization density measured by quantitative backscattered electron imaging (CaMean: -4.5%, p = 0.04). The lamellar portion of the lesion is even less mineralized, possibly because the newly deposited material has younger tissue age. Affected bone has higher porosity by micro-CT, due to increased tissue vascularity and elevated 2D-microporosity (osteocyte lacunar porosity: +39%, p = 0.01) determined on quantitative backscattered electron images. Furthermore, nano-indentation modulus characterizing material hardness and stiffness was strictly dependent on tissue mineralization (correlation with typical calcium concentration, CaPeak: r = 0.8984, p = 0.0150, and r = 0.9788, p = 0.0007, respectively) in both affected and unaffected bone, indicating that the surgical hardness of melorheostotic lesions results from their lamellar structure. The results suggest a model for pathophysiology of melorheostosis caused by somatic activating mutations in MAP2K1, in which the genetically induced gradual deterioration of bone microarchitecture triggers a periosteal reaction, similar to the process found to occur after bone infection or local trauma, and leads to an overall cortical outgrowth. The micromechanical properties of the lesions reflect their structural heterogeneity and correlate with local variations in mineral content, tissue age, and remodeling rates, in the same way as normal bone. © 2018 American Society for Bone and Mineral Research.

Confocal laser scanning microscopy-a powerful tool in bone research.

The confocal laser scanning microscope (CLSM) enables the collection of images picturing selected planes in depth of thick samples, thus giving 3D information while keeping the sample intact. In this article we give an overview of our CLSM applications in bone research: (i) the characterization of osteoblasts and osteoclasts properties in cell biology, (ii) the visualization of the three dimensional (3D) osteocyte lacunar canalicular network in undemineralized plastic-embedded bone samples, (iii) the observation of tetracycline labels in bone biopsy samples from patients in combination with information on the mineralization density from quantitative backscatter electron imaging, which enables the time course of mineral accumulation in newly formed bone to be followed, (iv) the precise measurement of the thickness of thin ground bone sections, a prerequisite for the mapping of local mechanical properties by scanning acoustic microscopy.

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 © 2016 The Authors. X-Ray Spectrometry published by John Wiley & Sons Ltd.

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.

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 patient's 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.