Quantitative backscattered electron imaging of bone using a thermionic or a field emission electron source

Quantitative backscattered electron imaging is an established method to map mineral content distributions in bone and to determine the bone mineralization density distribution (BMDD). The method we applied was initially validated for a scanning electron microscope (SEM) equipped with a tungsten hairpin cathode (thermionic electron emission) under strongly defined settings of SEM parameters. For several reasons, it would be interesting to migrate the technique to a SEM with a field emission electron source (FE-SEM), which, however, would require to work with different SEM parameter settings as have been validated for DSM 962. The FE-SEM has a much better spatial resolution based on an electron source size in the order of several 100 nanometers, corresponding to an about 105 to 106 times smaller source area compared to thermionic sources. In the present work, we compare BMDD between these two types of instruments in order to further validate the methodology. We show that a transition to higher pixel resolution (1.76, 0.88, and 0.57 μm) results in shifts of the BMDD peak and BMDD width to higher values. Further the inter-device reproducibility of the mean calcium content shows a difference of up to 1 wt% Ca, while the technical variance of each device can be reduced to ±0.17 wt% Ca. Bearing in mind that shifts in calcium levels due to diseases, e.g., high turnover osteoporosis, are often in the range of 1 wt% Ca, both the bone samples of the patients as well as the control samples have to be measured on the same SEM device. Therefore, we also constructed new reference BMDD curves for adults to be used for FE-SEM data comparison

Novel PLS3 variants in X-linked osteoporosis: Exploring bone material properties

BACKGROUND: Idiopathic Juvenile Osteoporosis (IJO) refers to significantly lower than expected bone mass manifesting in childhood with no identifiable aetiology. IJO classically presents in early pubertal period with multiple fractures including metaphyseal and vertebral crush fractures, and low bone-mass. METHODS: Here we describe two patients and provide information on their clinical phenotype, genotype and bone material analysis in one of the patients. RESULTS: Patient 1: 40-year old adult male diagnosed with IJO in childhood who re-presented with a hip fracture as an adult. Genetic analysis identified a pathogenic PLS3 hemizygous variant, c.1765del in exon 16. Patient 2: 15-year old boy with multiple vertebral fractures and bone biopsy findings suggestive of IJO who also has a diagnosis of autism spectrum disorder. Genetic analysis identified a maternally inherited PLS3 pathogenic c.1295T>A variant in exon 12. Analyses of the transiliac bone sample revealed severe reduction of trabecular volume and bone turnover indices and elevated bone matrix mineralisation. DISCUSSION: We propose that genetic testing for PLS3 should be undertaken in patients presenting with a current or previous history of IJO as this has implications for genetic counselling and cascade screening. The extensive evaluation of the transiliac biopsy sample of Patient 2 revealed a novel bone phenotype. CONCLUSION: This report includes a review of IJO and genetic causes of osteoporosis, and suggests that existing cases of IJO should be screened for PLS3. Through analysis of bone material properties in Patient 2, we can conclude that PLS3 does have a role in bone mineralisation

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

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

Combination of Nanoindentation and Quantitative Backscattered Electron Imaging Revealed Altered Bone Material Properties Associated with Femoral Neck Fragility

Osteoporotic fragility fractures were hypothesized to be related to changes in bone material properties and not solely to reduction in bone mass. We studied cortical bone from the superior and inferior sectors of whole femoral neck sections from five female osteoporotic hip fracture cases (74–92 years) and five nonfractured controls (75–88 years). The typical calcium content (CaPeak) and the mineral particle thickness parameter (T) were mapped in large areas of the superior and inferior regions using quantitative backscattered electron imaging (qBEI) and scanning small-angle X-ray scattering, respectively. Additionally, indentation modulus (E) and hardness (H) (determined by nanoindentation) were compared at the local level to the mineral content (CaInd) at the indent positions (obtained from qBEI). CaPeak (−2.2%, P = 0.002), CaInd (−1.8%, P = 0.048), E (−5.6%, P = 0.040), and H (−6.0%, P = 0.016) were significantly lower for the superior compared to the inferior region. Interestingly, CaPeak as well as CaInd were also lower (−2.6%, P = 0.006, and –3.7%, P = 0.002, respectively) in fracture cases compared to controls, while E and H did not show any significant reduction. T values were in the normal range, independent of region (P = 0.181) or fracture status (P = 0.551). In conclusion, it appears that the observed femoral neck fragility is associated with a reduced mineral content, which was not accompanied by a reduction in stiffness and hardness of the bone material. This pilot study suggests that a stiffening process in the organic matrix component contributes to bone fragility independently of mineral content

Upregulation of MMP-13 and TIMP-1 expression in response to mechanical strain in MC3T3-E1 osteoblastic cells

<p>Abstract</p> <p>Background</p> <p>Mechanical strain plays a significant role in the regulation of bone matrix turnover, which is mediated in part by matrix metalloproteinase (MMP)-13 and tissue inhibitors of matrix metalloproteinase (TIMP)-1. However, little is known about the correlation between mechanical strain and osteoblastic cell activities, including extracellular matrix (ECM) metabolism. Herein, we determined the effect of different magnitudes of cyclic tensile strain (0%, 6%, 12%, and 18%) on MMP-13 and TIMP-1 mRNA and protein expression in MC3T3-E1 osteoblasts. Furthermore, we employed specific inhibitors to examine the role of distinct signal transduction pathways known to mediate cellular responses to mechanical strain.</p> <p>Results</p> <p>We identified a magnitude-dependent increase in MMP-13 and TIMP-1 mRNA and protein levels in response to mechanical strains corresponding to 6%, 12%, and 18% elongation. The strain-induced increases in MMP-13 and TIMP-1 mRNA expression were inhibited by PD098059 and cycloheximide, respectively.</p> <p>Conclusions</p> <p>Our results suggest a mechanism for the regulation of bone matrix metabolism mediated by the differential expression of MMP-13 and TIMP-1 in response to increasing magnitudes of mechanical strain.</p

Collagen packing and mineralization. An X-ray scattering investigation of turkey leg tendon

Several recent results are suggesting that the collagen packing in mineralized tissues is much less regular than in the case of other nonmineralizing collagen, e.g., rat tail tendon. To clarify this question we have investigated the molecular arrangement in mineralized and unmineralized turkey leg tendon as a model for the collagen of mineralized tissues. Using a combination of diffuse x-ray scattering and computer simulation, it could be shown quantitatively that, although the collagen fibril structure is periodic in the axial direction, it is similar to a two-dimensional fluid in the lateral plane. This has important consequences for the understanding of the mineralization process, which is also discussed