Quantitative Backscattered Electron Imaging Research Articles

Hypodynamia Alters Bone Quality and Trabecular Microarchitecture.

Disuse induces a rapid bone loss in humans and animals; hypodynamia/sedentarity is now recognized as a risk factor for osteoporosis. Hypodynamia also decreases bone mass but its effects are largely unknown and only few animal models have been described. Hypodynamic chicken is recognized as a suitable model of bone loss but the effects on the quality have not been fully explored. We have used ten chickens bred in a large enclosure (FREE group); ten others were confined in small cages with little space to move around (HYPO group). They were sacrificed at 53 days and femurs were evaluated by microcomputed tomography (microCT) and nanoindentation. Sections (4µm thick) were analyzed by Fourier Transform InfraRed Microspectroscopy (FTIR) to see the effects on mineralization and collagen and quantitative backscattered electron imaging (qBEI) to image the mineral of the bone matrix. Trabecular bone volume and microarchitecture were significantly altered in the HYPO group. FTIR showed a significant reduction of the mineral-to-matrix ratio in the HYPO group associated with an increase in the carbonate content and an increase in crystallinity (calculated as the area ratio of subbands located at 1020 and 1030cm-1) indicating a poor quality of the mineral. Collagen maturity (calculated as the area ratio of subbands located at 1660 and 1690cm-1) was significantly reduced in the HYPO group. Reduced biomechanical properties were observed at the tissue level. Confined chicken represents a new model for the study of hypodynamia because bone changes are not created by a surgical lesion or a traumatic method. Animals have a reduced bone mass and present with an altered bone matrix quality which is less mineralized and whose collagen contains less crosslinks than in control chicken.

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.

Bone histomorphometric changes in children with rheumatic disorders on chronic glucocorticoids.

BackgroundRheumatic diseases are associated with an increased fracture risk. The tissue level characteristics of the bone involvement in children have not been well elucidated. Our objectives were to describe the bone micro-architectural characteristics in children with rheumatic diseases on chronic glucocorticoids, and to determine associations between micro-architectural findings with clinical and radiological variables.MethodsChildren on chronic glucocorticoids for an underlying rheumatic disease were referred for evaluation of bone fragility given the presence of vertebral compression fractures. A trans-iliac bone biopsy was performed as part of the clinical assessment. Histomorphometric analysis and quantitative backscattered electron imaging (qBSE) of the biopsy samples were undertaken.ResultsData of 15 children (14.0 ± 3.2 years) with a duration of glucocorticoid exposure of 6.2 ± 4.1 years and average prednisone dose of 14.1 ± 6.2 mg/m2/day were assessed. Histomorphometric analyses demonstrated significant decrease in trabecular thickness (p = 0.01), osteoid thickness (p < 0.01), osteoblast surface (p = 0.02) and increase in trabecular separation (p = 0.04) compared to published age-matched normative data. Severity of the trabecular deficit was correlated to glucocorticoid dose, height and body mass index Z score, but not bone mineral density or measures of disease activity. Using qBSE to measure bone mineralization, the subjects were shown to have a heterogeneous and hypermineralized profile, with higher cumulative glucocorticoid dose being associated with greater mineralization (p < 0.01).ConclusionsIn children with rheumatic diseases presenting with vertebral fractures, there is evidence of abnormal bone matrix mineralization and impairments of bone micro-architecture that correlate to glucocorticoid dose.

Prevalent role of porosity and osteonal area over mineralization heterogeneity in the fracture toughness of human cortical bone

Changes in the distribution of bone mineralization occurring with aging, disease, or treatment have prompted concerns that alterations in mineralization heterogeneity may affect the fracture resistance of bone. Yet, so far, studies assessing bone from hip fracture cases and fracture-free women have not reached a consensus on how heterogeneity in tissue mineralization relates to skeletal fragility. Owing to the multifactorial nature of toughening mechanisms occurring in bone, we assessed the relative contribution of heterogeneity in mineralization to fracture resistance with respect to age, porosity, and area fraction of osteonal tissue. The latter parameters were extracted from quantitative backscattered electron imaging of human cortical bone sections following R-curve tests of single-edge notched beam specimens to determine fracture toughness properties. Microstructural heterogeneity was determined as the width of the mineral distribution (bulk) and as the sill of the variogram (local). In univariate analyses of measures from 62 human donors (21 to 101 years), local but not bulk heterogeneity as well as pore clustering negatively correlated with fracture toughness properties. With age as covariate, heterogeneity was a significant predictor of crack initiation, though local had a stronger negative contribution than bulk. When considering all potential covariates, age, cortical porosity and area fraction of osteons explained up to 50% of the variance in bone׳s crack initiation toughness. However, including heterogeneity in mineralization did not improve upon this prediction. The findings of the present work stress the necessity to account for porosity and microstructure when evaluating the potential of matrix-related features to affect skeletal fragility.

High fat-fed diabetic mice present with profound alterations of the osteocyte network

Diabetes mellitus is considered to be an independent risk factor for bone fragility fractures. Reductions in bone mass, observed only with type 1 diabetes mellitus, as well as modifications of bone microarchitectures and tissue material properties are landmarks of diabetes-related bone alterations. An interesting feature observed in type 2 diabetes mellitus (T2DM) is the augmented concentration in circulating sclerostin. This observation prompts us to hypothesize that modifications of osteocyte network and perilacunar mineralization occur in T2DM. As such, the aims of the present study were to ascertain by quantitative backscattered electron imaging, confocal microscopy and image analysis, modifications of perilacunar tissue mineral density, osteocyte morphology and osteocyte network topology in a mouse model of high fat-induced type 2 diabetes. As compared with lean control animals, diabetic mice exhibited a significant 48% decrease in perilacunar mineralization heterogeneity although mean perilacunar mineralization was unchanged. Furthermore, in diabetic animals, osteocyte volume was significantly augmented by 34% with no change in the overall number of dendrite processes. Finally, the network topology was profoundly modified in diabetic mice with increases in the mean node degree, mean node volume and hub numbers whilst the mean link length was reduced. Overall, it appeared that in diabetic animals, the dendritic network exhibited features of a scale-free network as opposed to the single-scale characteristic observed in lean controls. However, it is important to ascertain whether diabetic patients exhibit such modifications of the osteocyte network and whether anti-diabetic drugs could restore normal osteocyte and network parameters, thereby improving bone quality and protecting against fragility fractures.

Doses effects of zoledronic acid on mineral apatite and collagen quality of newly-formed bone in the rat's calvaria defect

Due to their inhibitory effects on resorption, bisphosphonates are widely used in the treatment of diseases associated to an extensive bone loss. Yet, little is known about bisphosphonates effects on newly-formed bone quality. In the present study, adult male Sprague-Dawley rats (n=80) with a bone defect calvaria area were used and short-term effects of zoledronic acid (ZA) were studied on the healing bone area. Three ZA treatments were tested by using either: 1°) a low single dose (120μgZA/kg, n=10; equivalent to human osteoporosis treatment), 2°) a low fractionated doses (20μgZA/kg daily for 6days either a total of 120μg/kg, n=15), and 3°) a high fractionated doses, (100μgZA/kg weekly for 6weeks, n=15; equivalent to 6months of human bone metastasis treatment). For each treatment, a control “vehicle” treatment was performed (with an identical number of rats). After ZA administration, the intrinsic bone material properties were evaluated by quantitative backscattered electron imaging (qBEI) and Raman microspectroscopy. Neither single nor fractionated low ZA doses modify the intrinsic bone material properties of the newly-formed bone compared to their respective control animals. On the opposite, the high ZA treatment resulted in a significant decrease of the crystallinity (−25%, P< 0.05) and of the hydroxyproline-to-proline ratio (−30%, P<0.05) in newly-formed bones. Moreover, with the high ZA treatment, the crystallinity was positively correlated with the hydroxyproline-to-proline ratio (ρ=0.78, P<0.0001). The present data highlight new properties for ZA on bone formation in a craniofacial defect model. As such, ZA at high doses disrupted the apatite crystal organization. In addition, we report here for the first time that high ZA doses decreased the hydroxyproline-to-proline ratio suggesting that ZA may affect the early collagen organization during the bone healing.

Effects of odanacatib on bone matrix mineralization in rhesus monkeys are similar to those of alendronate

Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K which is an important enzyme for the degradation of collagen I. Aim of the present work was the head-to-head comparison between the effects of ODN and alendronate (ALN) on bone mineralization density distribution (BMDD), based on quantitative backscattered electron imaging in relation to changes in histomorphometric mineralizing surface per bone surface (MS/BS) in 12–22years old ovariectomized rhesus monkeys. Trabecular and cortical BMDD derived parameters from vertebrae and proximal tibiae were compared among vehicle (VEH, n=8), odanacatib low dose (ODN-L, n=8), odanacatib high dose (ODN-H, n=8), and alendronate (ALN, n=6) treated animals. Additionally, data from an intact, non-treated group of animals are shown (INT, n=8). In trabecular bone from the vertebra and metaphyseal tibia, the BMDD of the ODN and ALN treatment groups was shifted toward higher mineralization densities (p<0.001) consistent with the significant reduction of MS/BS (p<0.05 in ODN-H and ALN) compared to VEH. Vertebral trabecular CaMean (average degree of mineralization) was significantly higher in ODN-L (+6.5%), ODN-H (+6.1%), and ALN (+6.7%, all p<0.001). Tibial osteonal cortical bone revealed also significantly increased CaMean for ODN-L (+1.4%, p<0.05), ODN-H (+2.2%, p<0.05), and ALN (+3.4%, p<0.001) versus VEH, while primary cortical bone (devoid of secondary osteons) did not show any significant differences between the study groups. The percentage of primary bone area in the tibial cross-sections (on average 45±12%) was also not significantly different between the study groups (p=0.232). No significant differences in any BMDD parameters of all studied skeletal sites between ODN and ALN treatment were found. Correlation analysis revealed that MS/BS was highly predictive for trabecular BMDD in vertebral bone. The higher MS/BS, the lower was CaMean. Our findings are consistent with the inhibition of bone resorption of ODN and ALN in trabecular and osteonal compartments.

Differential Effects of Teriparatide and Zoledronic Acid on Bone Mineralization Density Distribution at 6 and 24 Months in the SHOTZ Study

The Skeletal Histomorphometry in Patients on Teriparatide or Zoledronic Acid Therapy (SHOTZ) study assessed the progressive effects of teriparatide (TPTD) and zoledronic acid (ZOL) on bone remodeling and material properties in postmenopausal women with osteoporosis. Previously, we reported that biochemical and histomorphometric bone formation indices were significantly higher in patients receiving TPTD versus ZOL. Here we report bone mineralization density distribution (BMDD) results based on quantitative backscattered electron imaging (qBEI). The 12-month primary study was randomized and double blind until the month 6 biopsy, then open label. Patients (TPTD, n = 28; ZOL, n = 31) were then eligible to enter a 12-month open-label extension with their original treatment: TPTD 20 μg/d (subcutaneous injection) or ZOL 5 mg/yr (intravenous infusion). A second biopsy was collected from the contralateral side at month 24 (TPTD, n = 10; ZOL, n = 10). In cancellous bone, ZOL treatment was associated at 6 and 24 months with significantly higher average degree of mineralization (CaMEAN, +2.2%, p = 0.018; +3.9%, p = 0.009, respectively) and with lower percentage of low mineralized areas (CaLOW , -34.6%, p = 0.029; -33.7%, p = 0.025, respectively) and heterogeneity of mineralization CaWIDTH (-12.3%, p = 0.003; -9.9%, p = 0.012, respectively), indicating higher mineralization density and more homogeneous mineral content versus TPTD. Within the ZOL group, significant changes were found in all parameters from month 6 to 24, indicating a progressive increase in mineralization density. In sharp contrast, mineralization density did not increase over time with TPTD, reflecting ongoing deposition of new bone. Similar results were observed in cortical bone. In this study, TPTD stimulated new bone formation, producing a mineralized bone matrix that remained relatively heterogeneous with a stable mean mineral content. ZOL slowed bone turnover and prolonged secondary mineralization, producing a progressively more homogeneous and highly mineralized bone matrix. Although both TPTD and ZOL increase clinical measures of bone mineral density (BMD), this study shows that the underlying mechanisms of the BMD increases are fundamentally different. © 2016 American Society for Bone and Mineral Research.

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation viaTet1andTet2and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2,Col1a1,Bglap2,Sp7,Atf4, andAlpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of theTet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorptionin vivo Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis.

Rsk2, the Kinase Mutated in Coffin-Lowry Syndrome, Controls Cementum Formation

The ribosomal S6 kinase RSK2 is essential for osteoblast function, and inactivating mutations of RSK2 cause osteopenia in humans with Coffin-Lowry syndrome (CLS). Alveolar bone loss and premature tooth exfoliation are also consistently reported symptoms in CLS patients; however, the pathophysiologic mechanisms are unclear. Therefore, aiming to identify the functional relevance of Rsk2 for tooth development, we analyzed Rsk2-deficient mice. Here, we show that Rsk2 is a critical regulator of cementoblast function. Immunohistochemistry, histology, micro–computed tomography imaging, quantitative backscattered electron imaging, and in vitro assays revealed that Rsk2 is activated in cementoblasts and is necessary for proper acellular cementum formation. Cementum hypoplasia that is observed in Rsk2-deficient mice causes detachment and disorganization of the periodontal ligament and was associated with significant alveolar bone loss with age. Moreover, Rsk2-deficient mice display hypomineralization of cellular cementum with accumulation of nonmineralized cementoid. In agreement, treatment of the cementoblast cell line OCCM-30 with a Rsk inhibitor reduces formation of mineralization nodules and decreases the expression of cementum markers. Western blot analyses based on antibodies against Rsk1, Rsk2, and an activated form of the 2 kinases confirmed that Rsk2 is expressed and activated in differentiating OCCM-30 cells. To discriminate between periodontal bone loss and systemic bone loss, we additionally crossed Rsk2-deficient mice with transgenic mice overexpressing the osteoanabolic transcription factor Fra1. Fra1 overexpression clearly increases systemic bone volume in Rsk2-deficient mice but does not protect from alveolar bone loss. Our results indicate that cell autonomous cementum defects are causing early tooth loss in CLS patients. Moreover, we identify Rsk2 as a nonredundant regulator of cementum homeostasis, alveolar bone maintenance, and periodontal health, with all these features being independent of Rsk2 function in systemic bone formation.

Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice

Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix.

Bone matrix mineralization is preserved during early perimenopausal stage in healthy women: a paired biopsy study.

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. It is unknown whether perimenopausal loss of bone mass is associated with a drop in bone matrix mineralization. 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 12months after last menses (obtained at average ages of 49 ± 2 and 55 ± 2years, 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. 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). 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.

Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis

A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh−120/+) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh−120/+ mice. We also find a much larger fibril strain/tissue strain ratio in Crh−120/+ mice (~1.5) compared to the wild-type mice (~0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.

Adynamic Bone Decreases Bone Toughness During Aging by Affecting Mineral and Matrix.

Adynamic bone is the most frequent type of bone lesion in patients with chronic kidney disease; long-term use of antiresorptive therapy may also lead to the adynamic bone condition. The hallmark of adynamic bone is a loss of bone turnover, and a major clinical concern of adynamic bone is diminished bone quality and an increase in fracture risk. Our current study aims to investigate how bone quality changes with age in our previously established mouse model of adynamic bone. Young and old mice (4 months old and 16 months old, respectively) were used in this study. Col2.3Δtk (DTK) mice were treated with ganciclovir and pamidronate to create the adynamic bone condition. Bone quality was evaluated using established techniques including bone histomorphometry, microcomputed tomography, quantitative backscattered electron imaging, and biomechanical testing. Changes in mineral and matrix properties were examined by powder X-ray diffraction and Raman spectroscopy. Aging controls had a natural decline in bone formation and resorption with a corresponding deterioration in trabecular bone structure. Bone turnover was severely blunted at all ages in adynamic animals, which preserved trabecular bone loss normally associated with aging. However, the preservation of trabecular bone mass and structure in old adynamic mice did not rescue deterioration of bone mechanical properties. There was also a decrease in cortical bone toughness in old adynamic mice that was accompanied by a more mature collagen matrix and longer bone crystals. Little is known about the effects of metabolic bone disease on bone fracture resistance. We observed an age-related decrease in bone toughness that was worsened by the adynamic condition, and this decrease may be due to material level changes at the tissue level. Our mouse model may be useful in the investigation of the mechanisms involved in fractures occurring in elderly patients on antiresorptive therapy who have very low bone turnover.

Microstructure and wettability of root canal dentine and root canal filling materials after different chemical irrigation

The objective of this study was to determine the effects of various irrigation solutions on root canal dentine and gutta-percha surface properties. In addition, the effects of disinfectant chemicals on the wettability and surface morphological properties of the filling materials were evaluated. Ethylenediaminetetraacetic acid (EDTA), citric acid, and ozone were employed as irrigation solutions for dentine and gutta-percha treatment. Thereafter, the samples’ microstructure, degree of mineralization, and mechanical properties were assessed by means of quantitative backscattered electron imaging (qBEI) and reference point indentation (RPI). A contact angle analyzer was used to measure adhesion on the tested materials. Here, EDTA had the most significant affect on both the mechanical properties and the adhesive behavior of dentine. Citric acid did not affect dentine wettability, whereas the indentation properties and the mineralization were reduced. Similar effects were observed when ozone was used. The dentinal tubules were significantly widened in citric acid compared to the ozone group. EDTA causes considerable micromechanical surface alteration of dentine and gutta-percha, but represents the best option in clinical cases where a high adhesiveness of the filling materials is desired.

PTH(1-84) Administration in Hypoparathyroidism Transiently Reduces Bone Matrix Mineralization.

Patients with hypoparathyroidism have low circulating parathyroid (PTH) levels and higher cancellous bone volume and trabecular thickness. Treatment with PTH(1-84) was shown to increase abnormally low bone remodeling dynamics. In this work, we studied the effect of 1-year or 2-year PTH(1-84) treatment on cancellous and cortical bone mineralization density distribution (Cn.BMDD and Ct.BMDD) based on quantitative backscattered electron imaging (qBEI) in paired transiliac bone biopsy samples. The study cohort comprised 30 adult hypoparathyroid patients (14 treated for 1 year; 16 treated for 2 years). At baseline, Cn.BMDD was shifted to higher mineralization densities in both treatment groups (average degree of mineralization Cn.CaMean +3.9% and +2.7%, p < 0.001) compared to reference BMDD. After 1-year PTH(1-84), Cn.CaMean was significantly lower than that at baseline (-6.3%, p < 0.001), whereas in the 2-year PTH(1-84) group Cn.CaMean did not differ from baseline. Significant changes of Ct.BMDD were observed in the 1-year treatment group only. The change in histomorphometric bone formation (mineralizing surface) was predictive for Cn.BMDD outcomes in the 1-year PTH(1-84) group, but not in the 2-year PTH(1-84) group. Our findings suggest higher baseline bone matrix mineralization consistent with the decreased bone turnover in hypoparathyroidism. PTH(1-84) treatment caused differential effects dependent on treatment duration that were consistent with the histomorphometric bone formation outcomes. The greater increase in bone formation during the first year of treatment was associated with a decrease in bone matrix mineralization, suggesting that PTH(1-84) exposure to the hypoparathyroid skeleton has the greatest effects on BMDD early in treatment.

Subtle changes in bone mineralization density distribution in most severely affected patients with chronic obstructive pulmonary disease

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±7.3years 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.

Skeletal maturation substantially affects elastic tissue properties in the endosteal and periosteal regions of loaded mice tibiae

Although it is well known that the bone adapts to changes in the mechanical environment by forming and resorbing the bone matrix, little is known about the influence of mechanical loading on tissue material properties of the pre-existing and newly formed bone. In this study, we analyzed the newly formed and pre-existing tissue after two weeks of controlled in vivo axial compressive loading in tibia of young (10week-old) and adult (26week-old) female mice and compared to the control contralateral limb, by means of scanning acoustic microscopy. Additionally, we used quantitative backscattered electron imaging to determine the bone mineral density distribution within the newly formed and pre-existing bone of young mice. No significant differences were found in tissue stiffness or mineral density in the pre-existing bone tissue as a result of external loading. In the endosteal region, 10 and 26week loaded animals showed a 9% reduction in bone tissue stiffness compared to control animals. An increase of 200% in the mineral apposition rate in this region was observed in both age groups. In the periosteal region, the reduction in bone tissue stiffness and the increase in bone mineral apposition rate as a result of loading were two times higher in the 10 compared to the 26week old animals. These data suggest that, during growth and skeletal maturation, the response of bone to mechanical loading is a deposition of new bone matrix, where the tissue amount but not its mineral or elastic properties are influenced by animal age.

A polyadenylation site variant causes transcript-specific BMP1 deficiency and frequent fractures in children.

We had previously published the clinical characteristics of a bone fragility disorder in children that was characterized mainly by lower extremity fractures and a mineralization defect in bone tissue but not on the growth plate level. We have now performed whole-exome sequencing on four unrelated individuals with this phenotype. Three individuals were homozygous for a nucleotide change in BMP1, affecting the polyadenylation signal of the transcript that codes for the short isoform of BMP1 (BMP1-1) (c.*241T>C). In skin fibroblasts of these individuals, we found low levels of BMP1-1 transcript and protein. The fourth individual was compound heterozygous for the c.*241T>C variant in BMP1-1 and a variant in BMP1 exon 15 (c.2107G>C) that affected splicing in both BMP1-1 and the long isoform of BMP1 (BMP1-3). Both the homozygous 3'UTR variant and the compound heterozygous variants were associated with impaired procollagen type I C-propeptide cleavage, as the amount of free C-propeptide in the supernatant of skin fibroblasts was less than in controls. Peripheral quantitative computed tomography showed that all individuals had elevated volumetric cortical bone mineral density. Assessment of iliac bone samples by histomorphometry and quantitative backscattered electron imaging indicated that the onset of mineralization at bone formation sites was delayed, but that mineralized matrix was hypermineralized. These results show that isolated lack of BMP1-1 causes bone fragility in children.

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.