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We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.

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Traumatic heterotopic ossification (HO) is frequently observed in Service Members following combat-related trauma. Estimates suggest that ~65% of wounded warriors who suffer limb loss or major extremity trauma will experience some type of HO formation. The development of HO delays rehabilitation and can prevent the use of a prosthetic. To date there are limited data to suggest a standard mechanism for preventing HO. This may be due to inadequate animal models not producing a similar bone structure as human HO. We recently showed that traumatic HO growth is possible in an ovine model. Within that study, we demonstrated that 65% of sheep developed a human-relevant hybrid traumatic HO bone structure after being exposed to a combination of seven combat-relevant factors. Although HO formed, we did not determine which traumatic factor contributed most. Therefore, in this study, we performed individual and various combinations of surgical/traumatic factors to determine their individual contribution to HO growth. Outcomes showed that the presence of mature biofilm stimulated a large region of bone growth, while bone trauma resulted in a localized bone response as indicated by jagged bone at the linea aspera. However, it was not until the combinatory factors were included that an HO structure similar to that of humans formed more readily in 60% of the sheep. In conclusion, data suggested that traumatic HO growth can develop following various traumatic factors, but a combination of known instigators yields higher frequency size and consistency of ectopic bone.

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Metastasis of cancer to the spine impacts bone quality. This study aims to characterize vertebral microdamage secondary to metastatic disease considering the pattern of damage and its relationship to stress and strain under load. Osteolytic and mixed osteolytic/osteoblastic vertebral metastases were produced in athymic rats via HeLa cervical or canine Ace-1 prostate cancer cell inoculation, respectively. After 21 days, excised motion segments (T12-L2) were µCT scanned, stained with BaSO4 and re-imaged. T13-L2 motion segments were loaded in axial compression to induce microdamage, re-stained and re-imaged. L1 (loaded) and T12 (unloaded) vertebrae were fixed, sample blocks cut, polished and BSE imaged. µFE models were generated of all L1 vertebrae with displacement boundary conditions applied based on the loaded µCT images. µCT stereological analysis, BSE analysis and µFE derived von Mises stress and principal strains were quantitatively compared (ANOVA), spatial correlations determined and patterns of microdamage assessed qualitatively. BaSO4 identified microdamage was found to be spatially correlated with regions of high stress in µFEA. Load-induced microdamage was shown to be elevated in the presence of osteolytic and mixed metastatic disease, with diffuse, crossed hatched areas of microdamage present in addition to linear microdamage and microfractures in metastatic tissue, suggesting diminished bone quality.

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The Wnt signalling pathway is a critical regulator of bone mass and quality. Several heterozygous mutations in the LRP5 gene, a Wnt co-receptor, causing high bone mass (LRP5-HBM) have been described to date. The pathogenic mechanism is thought to be a gain-of-function caused by impaired inhibition of the canonical Wnt signalling pathway, thereby leading to increased bone formation. We report the cases of two affected family members, a 53-year-old mother and her 23-year-old daughter, with high bone mass (T-scores mother: lumbar spine 11.4, femoral neck 10.5; T-scores daughter: lumbar spine 5.4, femoral neck 8.7), increased calvarial thickness, and thickened cortices of the long bones but no history of fractures. Whereas the mother did not show any indications of the mutation, the daughter suffered from congenital hearing impairment resulting in cochlear implantation, recurrent facial palsy, and migraine. In addition, she had stenosis of the foramen magnum. In both individuals, we detected a novel heterozygous duplication of six basepairs in the LRP5 gene, resulting in an insertion of two amino acids, very likely associated with a gain-of-function. When the daughter had part of the occipital bone surgically removed, the bone sample was used for the visualization of bone lamellar structure and bone cells as well as the measurement of bone mineralization density distribution (BMDD). The bone sample revealed two distinctly different regions: an intra-cortical region with osteonal remodeling, typical osteonal lamellar orientation, associated with relatively higher heterogeneity of bone matrix mineralization, and another periosteal region devoid of bone remodeling, with parallel bone lamellae and lower heterogeneity of mineralization. In conclusion, we present data on bone tissue and material level from an LRP5-HBM patient with a novel mutation in the LRP5 gene. Our findings indicate normal morphology of osteoclasts and osteoblasts as well as normal mineralization in skull bone in LRP5-HBM.

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These experiments were designed to determine whether skin debris (desquamated epithelial cells and apparent skin oils) affects gunshot residue (GSR) particle detection on the sticky tape lift samples prepared for scanning electron microscopy (SEM). A dabbing experiment showed that GSR particles accumulate not only on the adhesive surface of the sampler, but also on the epithelial cell surfaces. Samplers were loaded with target GSR followed by dabbing 30 times on the back of a hand. Backscatter electron images were taken at 20 kV and for some at 30 kV of the same areas. The samplers were then treated with a sodium/calcium hypochlorite solution (bleach) to remove skin debris and again imaged in the SEM. Comparison of these images shows more GSR particles will likely be revealed at 30 kV than 20 kV and more particles revealed by the bleach treatment in an automated SEM system.

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Duchenne muscular dystrophy (DMD) is a genetic disorder causing progressive muscle weakness. To prolong independent ambulation, DMD patients are treated with glucocorticoids, which, in turn, can increase bone fragility. In a cohort with vertebral fractures, intravenous bisphosphonate (iv BP) therapy stabilized vertebrae and reduced back pain. To characterize the effects of glucocorticoid therapy and bisphosphonate treatment on bone tissue and material properties, paired transiliac biopsy samples (before and after on average 2.4 years of iv BP) from 9 boys with DMD were studied for histomorphometry and bone mineralization density distribution (BMDD) and compared to reference values. Before iv BP, the boys had low cancellous bone volume (BV/TV) and cortical thickness (Ct.Wi) (both on average 56% of the healthy average, p < 0.001 versus reference), and mineralizing surface (MS/BS) in the lower normal range (on average 74% of the healthy average). The average degree of mineralization of cancellous (Cn.CaMean) and cortical compartments (Ct.CaMean) was 21.48 (20.70, 21.90) wt% and 20.42 (19.32, 21.64) wt%, respectively (median [25th, 75th percentiles]), which was not different from reference. After iv BP, BV/TV and Ct.Wi were, on average, unchanged. However, at the individual patient level, BV/TV Z-scores increased in 2, remained unchanged in 4, and declined in 3 patients. Additionally, on average, MS/BS decreased (-85%, p < 0.001), Cn.CaMean (+2.7%) increased, whereas the heterogeneity of cancellous (Cn.CaWidth -19%) and cortical bone mineralization (Ct.CaWidth -8%, all p < 0.05) decreased versus baseline. The changes in bone mineralization are consistent with the antiresorptive action of iv BP. At the same time, our observations point to the need for novel therapies with less or absent bone turnover suppression, including the fact that bone turnover was low even before bisphosphonate therapy, that bone turnover declined further (as expected) with treatment, and that declines in trabecular bone volume were observed in some boys despite bisphosphonate therapy. © 2015 American Society for Bone and Mineral Research.

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UNLABELLED: Bone matrix mineralization based on quantitative backscatter electron imaging remained unchanged during the first year of menopause in paired transiliac biopsy samples from healthy women. This suggests that the reported early perimenopausal reductions in bone mineral density are caused by factors other than decreases in the degree of mineralization. INTRODUCTION: It is unknown whether perimenopausal loss of bone mass is associated with a drop in bone matrix mineralization. METHODS: For this purpose, we measured the bone mineralization density distribution (BMDD) by quantitative backscatter electron imaging (qBEI) in n = 17 paired transiliac bone biopsy samples at premenopausal baseline and 12 months after last menses (obtained at average ages of 49 ± 2 and 55 ± 2 years, respectively) in healthy women. For interpretation of BMDD outcomes, previously measured bone mineral density (BMD) and biochemical and histomorphometric markers of bone turnover were revisited for the present biopsy cohort. RESULTS: Menopause significantly decreased BMD at the lumbar spine (-4.5 %) and femoral neck (-3.8 %), increased the fasting urinary hydroxyproline/creatinine ratio (+60 %, all p < 0.01) and histomorphometric bone formation rate (+25 %, p < 0.05), but affected neither cancellous nor cortical BMDD variables (paired comparison p > 0.05). Mean calcium concentrations of cancellous (Cn.CaMean) and cortical bone (Ct.CaMean) were within normal range (p > 0.05 compared to established reference data). Ct.CaMean was significantly correlated with Cn.CaMean before (R = 0.81, p < 0.001) and after menopause (R = 0.80, p < 0.001) and to cortical porosity of mineralized tissue (Ct.Po.) after menopause (R = -0.57, p = 0.02). CONCLUSIONS: Surprisingly, the BMDD was found not affected by the changes in bone turnover rates in this cohort. This suggests that the substantial increase in bone formation rates took place shortly before the second biopsy, and the bone mineralization changes lag behind. We conclude that during the first year after the last menses, the degree of bone matrix mineralization is preserved and does not contribute to the observed reductions in BMD.

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Chronic obstructive pulmonary disease (COPD) is associated with low aBMD as measured by DXA and altered microstructure as assessed by bone histomorphometry and microcomputed tomography. Knowledge of bone matrix mineralization is lacking in COPD. Using quantitative backscatter electron imaging (qBEI), we assessed cancellous (Cn.) and cortical (Ct.) bone mineralization density distribution (BMDD) in 19 postmenopausal women (62.1 ± 7.3 years of age) with COPD. Eight had sustained fragility fractures, and 13 had received treatment with inhaled glucocorticoids. The BMDD outcomes from the patients were compared with healthy reference data and were correlated with previous clinical and histomorphometric findings. In general, the BMDD outcomes for the patients were not significantly different from the reference data. Neither the subgroups of with or without fragility fractures or of who did or did not receive inhaled glucocorticoid treatment, showed differences in BMDD. However, subgroup comparison according to severity revealed 10% decreased cancellous mineralization heterogeneity (Cn.CaWidth) for the most severely affected compared with less affected patients (p=0.042) and compared with healthy premenopausal controls (p=0.021). BMDD parameters were highly correlated with histomorphometric cancellous bone volume (BV/TV) and formation indices: mean degree of mineralization (Cn.CaMean) versus BV/TV (r=0.58, p=0.009), and Cn.CaMean and Ct.CaMean versus bone formation rate (BFR/BS) (r=-0.71, p<0.001). In particular, those with lower BV/TV (<50th percentile) had significantly lower Cn.CaMean (p=0.037) and higher Cn.CaLow (p=0.020) compared with those with higher (>50th percentile) BV/TV. The normality in most of the BMDD parameters and bone formation rates as well as the significant correlations between them suggests unaffected mineralization processes in COPD. Our findings also indicate no significant negative effect of treatment with inhaled glucocorticoids on the bone mineralization pattern. However, the observed concomitant occurrence of relatively lower bone volumes with lower bone matrix mineralization will both contribute to the reduced aBMD in some patients with COPD.

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