Increased strontium uptake in trabecular bone of ovariectomized calcium-deficient rats treated with strontium ranelate or strontium chloride

Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl2). Three-month-old ovariectomized rats were treated for 90 days with doses of 25 mg kg-1 d-1 and 150 mg kg-1 d-1 of SrR or SrCl2 at low (0.1% Ca) or normal (1.19% Ca) Ca diet. Vertebral bone tissue was analysed by confocal synchrotron-radiation-induced micro X-ray fluorescence and by backscattered electron imaging. Principal component analysis and k-means clustering of the acquired elemental maps of Ca and Sr revealed that the newly formed bone exhibited the highest Sr fractions and that low Ca diet increased the Sr uptake by a factor of three to four. Furthermore, Sr uptake in bone of the SrCl2-treated animals was generally lower compared with SrR. The study clearly shows that inadequate nutritional calcium intake significantly increases uptake of Sr in serum as well as in trabecular bone matrix. This indicates that nutritional calcium intake as well as serum Ca levels are important regulators of any Sr treatment

3D interrelationship between osteocyte network and forming mineral during human bone remodeling

During bone remodeling, osteoblasts are known to deposit unmineralized collagenous tissue (osteoid), which mineralizes after some time lag. Some of the osteoblasts differentiate into osteocytes, forming a cell network within the lacunocanalicular network (LCN) of bone. To get more insight into the potential role of osteocytes in the mineralization process of osteoid, sites of bone formation are three-dimensionally imaged in nine forming human osteons using focused ion beam-scanning electron microscopy (FIB-SEM). In agreement with previous observations, the mineral concentration is found to gradually increase from the central Haversian canal toward pre-existing mineralized bone. Most interestingly, a similar feature is discovered on a length scale more than 100-times smaller, whereby mineral concentration increases from the LCN, leaving around the canaliculi a zone virtually free of mineral, the size of which decreases with progressing mineralization. This suggests that the LCN controls mineral formation but not just by diffusion of mineralization precursors, which would lead to a continuous decrease of mineral concentration from the LCN. The observation is, however, compatible with the codiffusion and reaction of precursors and inhibitors from the LCN into the bone matrix

Phenotypic spectrum in osteogenesis imperfecta due to mutations in TMEM38B: unravelling a complex cellular defect.

Context: Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Objectives: Clinical and bone material phenotype description and osteoblast differentiation studies. Design and Setting: Natural history study in paediatric research centres. Patients: Eight patients with type XIV OI. Main Outcome Measures: Clinical examinations included: bone mineral density, radiographs, echocardiography and muscle biopsy. Bone biopsy samples (n=3) were analysed using histomorphometry, quantitative backscattered electron microscopy and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Results: The clinical phenotype of type XIV OI ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband L1-L4 bone density Z-score was reduced (median -3.3 [range -4.77 to +0.1; n=7]), and increased by +1.7 (1.17 to 3.0; n=3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased late and mineralization-related markers. Predominance of TRIC-B over TRIC-A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. Conclusions: OI type XIV has a bone histology, matrix mineralization and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities

Formation of Ultracracks in Methacrylate-Embedded Undecalcified Bone Samples by Exposure to Aqueous Solutions

Back-scattered electron (BSE) imaging allows the visualization and evaluation of mineralized bone structures down to the micrometer range. To produce undecalcified bone sections with adequate structural and surface integrity, bone specimens are usually resin-embedded, followed by cutting, grinding , and polishing procedures. In samples prepared this way, so-called ultracracks were detected as black clefts in the lamellar bone matrix by BSE-imaging at magnifications ranging from 1000x to 3000x. By charging phenomena in the secondary electron (SE) mode of the scanning electron microscope (SEM), these clefts can be proven to be open cracks in the sample surface, and thus, as being created after embedding. These ultracracks seem to be a swelling effect of the bone matrix when it is exposed to water on the sample surface, followed by shrinking during drying . They did not occur, when water-free preparation techniques, like micromilling, were used and all water contact with the sample surface was avoided. This observation using the BSE-technique in SEM, and the simple method of discrimination between cracks existing before embedding and cracks newly generated during or after embedding, seem important for ultrastructural investigations of mineralized bone tissue, particularly for the evaluation of microcracks after loading or for the study of bone-implant interfaces

Lifelong challenge of calcium homeostasis in male mice lacking TRPV5 leads to changes in bone and calcium metabolism

Trpv5 plays an important role in calcium (Ca2+) homeostasis, among others by mediating renal calcium reabsorption. Accordingly, Trpv5 deficiency strongly stresses Ca2+ homeostasis in order to maintain stable serum Ca2+. We addressed the impact of lifelong challenge of calcium homeostasis on the bone phenotype of these mice. Aging signifi

Postembedding iodine staining for contrast-enhanced 3D imaging of bone tissue using focused ion beam-scanning electron microscopy

For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high-resolution imaging techniques. In the case of bone, focused ion beam-scanning electron microscopy (FIB-SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin-embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB-SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization

A New Scanning Electron Microscopy Approach to the Quantification of Bone Mineral Distribution: Backscattered Electron Image Grey-Levels Correlated to Calcium Kα-Line Intensities

The introduction of backscattered electron (BSE) imaging in scanning electron microscopy (SEM) has led to new possibilities for the evaluation of mineral distributions in bone on a microscopic level. The different grey-levels seen in the BSE-images can be used as a measure for the local mineral content of bone. In order to calibrate these BSE-grey-levels (BSE-GL) and correlate them to mineral contents, various attempts, using reference samples with known weighted mean atomic number and/or using simulated bone tissues with known hydroxyapatite concentrations, have been made. In contrast, a new approach is presented here based on measurements of the X-ray intensities of the calcium Kα-line on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlation between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. The resulting mineral concentration histograms have a dynamic range from O to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The presented modifications of the BSE method strongly enhance its feasibility in the field of bone research and its application as a special diagnostic tool for bone diseases

Heterogeneity of the osteocyte lacuno-canalicular network architecture and material characteristics across different tissue types in healing bone

Various tissue types, including fibrous connective tissue, bone marrow, cartilage, woven and lamellar bone coexist in healing bone. Similar to all bone tissue type, healing bone contains a lacuno-canalicular network (LCN) housing osteocytes that are known to orchestrate bone remodeling in healthy bone by sensing mechanical strains and translating them into biochemical signals. The structure of the LCN is also hypothesized to influence mineralization processes. Hence, the aim of the present study was to visualize and correlate spatial variations in the LCN topology with mineral characteristics, within and at the interfaces of the different tissue types that comprise healing bone. We applied a correlative multi-method approach to visualize the LCN architecture and quantify mineral particle size and orientation within healing femoral bone in a mouse osteotomy model (26 weeks old C57BL/6 mice). This approach revealed structural differences across several length scales during endochondral ossification within the following regions: calcified cartilage, bony callus, cortical bone and the transition zone between the cortical region and callus that developed during 21 days after the osteotomy. In this transition zone, we observed a continuous convergence of mineral characteristics and osteocyte lacunae shape as well as discontinuities in the lacunae volume and LCN connectivity. The bony callus exhibits a 34% higher lacunae number density with 40% larger lacunar volume compared to cortical bone. The presented correlations between LCN architecture and mineral characteristics improves our understanding of how bone develops during healing and may indicate a contribution of osteocytes to bone (re)modeling

The contribution of the pericanalicular matrix to mineral content in human osteonal bone

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.The osteocyte lacunar-canalicular network (LCN) penetrates bone and houses the osteocytes and their processes. Despite its rather low volume fraction, the LCN represents an outstanding large surface that is possibly used by the osteocytes to interact with the surrounding mineralized bone matrix thereby contributing to mineral homeostasis. The aim of this study was to quantitatively describe such contributions by spatially correlating the local density of the LCN with the mineral content at the same location in micrometer-sized volume elements in human osteons. For this purpose, 65 osteons from the femur midshaft from healthy adults (n = 4) and children (n = 2) were structurally characterized with two different techniques. The 3D structure of the LCN in the osteons was imaged with confocal laser scanning microscopy after staining the bone samples with rhodamine. Subsequent image analysis provided the canalicular length density, i.e. the total length of the canaliculi per unit volume (μm/μm3). Quantitative information on the mineral content (wt%Ca) from the identical regions was obtained using quantitative backscattered electron imaging. As the LCN-porosity lowers the mineral content, a negative correlation between Ca content and network density was expected. Calculations predict a reduction of around −0.97 fmol Ca per μm of network. However, the experiment revealed for 62 out of 65 osteons a positive correlation resulting in an average additional Ca loading of +1.15 fmol per μm of canalicular network, i.e. an accumulation of mineral has occurred at dense network regions. We hypothesize that this accumulation happens in the close vicinity of canaliculi forming mineral reservoirs that can be utilized by osteocytes. Significant differences found between individuals indicate that the extent of mineral loading of the reservoir zone reflects an important parameter for mineral homeostasis.German Federal Ministry of Education and ResearchAUVA (Research Funds of the Austrian Workers Compensation Board, Austria)WGKK (Viennese sickness insurance funds, Austria)