Andrea Berzlanovich

Trabecular bone microstructure and local gene expression in iliac crest biopsies of men with idiopathic osteoporosis

Male idiopathic osteoporosis (MIO) is a metabolic bone disease that is characterized by low bone mass, microstructural alterations, and increased fracture risk in otherwise healthy men. Although the detailed pathophysiology of MIO has yet to be clarified, evidence increasingly suggests an osteoblastic defect as the underlying cause. In this study we tested the hypothesis that the expression profile of certain osteoblastic or osteoblast‐related genes (ie, WNT10B, RUNX2, Osterix, Osteocalcin, SOST, RANKL, and OPG) is different in iliac crest biopsies of MIO patients when compared with healthy controls. Furthermore, we investigated the relation of local gene expression characteristics with histomorphometric, microstructural, and clinical features. Following written informed consent and diligent clinical patient characterization, iliac crest biopsies were performed in nine men. While RNA extraction, reverse‐transcription, and real‐time polymerase chain reactions (PCRs) were performed on one biopsy, a second biopsy of each patient was submitted for histomorphometry and micro–computed tomography (µCT). Age‐matched bone samples from forensic autopsies served as controls. MIO patients displayed significantly reduced WNT10B, RUNX2, RANKL, and SOST expression. Performing µCT for the first time in MIO biopsies, we found significant decreases in trabecular number and connectivity density. Trabecular separation was increased significantly, but trabecular thickness was similar in both groups. Histomorphometry revealed decreased BV/TV and osteoid volume and fewer osteoclasts in MIO. By providing evidence for reduced local WNT10B, RUNX2, and RANKL gene expression and histomorphometric low turnover, our data support the osteoblast dysfunction model discussed for MIO. Further, MIO seems to lead to a different microstructural pathology than age‐related bone loss.

Relationship between the v2PO4/amide III ratio assessed by Raman spectroscopy and the calcium content measured by quantitative backscattered electron microscopy in healthy human osteonal bone

Abstract. Raman microspectroscopy and quantitative backscattered electron imaging (qBEI) of bone are powerful tools to investigate bone material properties. Both methods provide information on the degree of bone matrix mineralization. However, a head-to-head comparison of these outcomes from identical bone areas has not been performed to date. In femoral midshaft cross sections of three women, 99 regions (20×20  μm2) were selected inside osteons and interstitial bone covering a wide range of matrix mineralization. As the focus of this study was only on regions undergoing secondary mineralization, zones exhibiting a distinct gradient in mineral content close to the mineralization front were excluded. The same regions were measured by both methods. We found a linear correlation (R2=0.75) between mineral/matrix as measured by Raman spectroscopy and the wt. %Mineral/(100-wt. %Mineral) as obtained by qBEI, in good agreement with theoretical estimations. The observed deviations of single values from the linear regression line were determined to reflect biological heterogeneities. The data of this study demonstrate the good correspondence between Raman and qBEI outcomes in describing tissue mineralization. The obtained correlation is likely sensitive to changes in bone tissue composition, providing an approach to detect potential deviations from normal bone.

Differential accumulation of lead and zinc in double-tidemarks of articular cartilage

INTRODUCTION Long-term exposure to increased lead (Pb) concentrations is associated with several chronic diseases. The divalent cation zinc (Zn) is essential for numerous enzymes. In a recent study we found remarkably elevated concentrations of Pb and Zn in the tidemark (TM), which is the mineralization front of human articular cartilage. OBJECTIVE Duplication or multiplication of TMs occurs with advancing age or degeneration. We hypothesized that trace elements accumulate in TMs as a function of time. Thus, in cases of double TMs, the deep (older) TM should contain higher Pb and Zn concentrations than the superficial (younger) TM. DESIGN Undecalcified tissue from articular cartilage and subchondral bone of femoral heads and patellae was examined by synchrotron radiation induced confocal micro X-ray fluorescence analysis and by quantitative backscattered electron imaging to determine the local distribution of Ca, Zn, and Pb in this tissue. RESULTS The evaluation of X-ray fluorescence intensities in double TMs revealed in average a 2.6-fold higher Pb level in the deep TM compared to the superficial TM while Zn concentrations were similar. Pb and Zn contents were significantly enhanced in the deep TM (Pb: 35-fold, Zn: five-fold) and in the superficial TM (Pb: 12-fold, Zn: five-fold) compared to the bone level. CONCLUSION For the first time a differential accumulation of Pb and Zn is documented in regions with double TMs revealing various timescales for the accumulation of these elements. Increased amounts of Pb are present in the TMs (up to the 62-fold of the bone level) featuring a potential source of internal Pb release if the TM region is destroyed.

The slow death of autopsies: a retrospective analysis of the autopsy prevalence rate in Austria from 1990 to 2009

Autopsies are still the gold standard by which treatmentmethods, clinical decision-making procedures and diag-nostic tools can be measured. They not only help toestablish the true cause of death, they also provide aninsight into how patients’ deaths can be prevented infuture.In Austria, where autopsy enjoyed its apotheosis in the1800s, the law permits non-forensic autopsies to be donewithout the consent of next of kin in cases in which there isa clear medical or scientific interest. Autopsy rate reportingmay vary between institutions, depending on whetherforensic, other outpatient cases, stillbirth cases, and casesreferred from outside the hospital are included; many of therates are reported from individual hospitals and appear inpublished studies.This study is unique in the literature due to the fact thatit is based on a huge data set containing individual infor-mation for every single person deceased in Austria between1990 and 2009. Our data provided by ‘‘Statistics Austria’’[1] include: status and kind of autopsies (medicolegal,clinico-pathological, others), sex, religion, family status,province, nationality, institution/place and date of death,age of deceased in years. The effect of the interestingvariables as well as their interaction terms on the autopsywas estimated by a logistic regression model. 95 % confi-dence intervals are given in brackets.The autopsy preva-lence rate for the 1.565.151 cases with no missing values inthe explanatory variables during the observation perioddecreased from 34.2 % (33.8, 34.5) in 1990 to 17.5 % in2009 (17.3, 17.8).Between 2004 and 2009 the frequency of clinico-path-ological autopsies decreased from 18.2 % (18.0, 18.5) to14.4 % (14.2, 14.6) of all deaths. The medicolegal autopsyprevalence rate remained stable on a low level at about1.5 %.All variables in the regression model turned out to besignificant (p\0.01). Table 1 presents odds ratios (OR)for all variables that do not include an interaction term with‘‘year of death’’.The probability of autopsy is highest among personswith unknown religious affiliation with an OR of 1.21compared to the reference group Roman Catholic. Incontrast Jews demonstrate the lowest autopsy prevalencerate with an OR of 0.28.Foreigners have a slightly higher probability of postmortem examinations with an OR of 1.04 compared toAustrians.The probability of autopsy is higher among singles,widowed and divorced people compared to married cou-ples. Divorced people display the highest OR with a valueof 1.42.Persons dying in a rest home are subject to a much lowerprobability for an autopsy with an OR of 0.03 compared topersons dying in a hospital. Moreover, all other locations(living address, transport, and other institutions/places ofdeath) express a lower probability than the location hospital.Persons dying between February and December have alower probability of autopsy than in the reference monthJanuary. The largest difference demonstrates Decemberwith an OR of 0.91.The weekday of death shows a quite small influence onthe autopsy probability. The largest difference to the

Assessment of chemical species of lead accumulated in tidemarks of human articular cartilage by X-ray absorption near-edge structure analysis.

Lead is a toxic trace element that shows a highly specific accumulation in the transition zone between calcified and non-calcified articular cartilage, the so-called ‘tidemark’. Excellent agreement has been found between XANES spectra of synthetic Pb-doped carbonated hydroxyapatite and spectra obtained in the tidemark region and trabecular bone of normal human samples, confirming that in both tissues Pb is incorporated into the hydroxyapatite crystal structure of bone. During this study the µ-XANES set-up at the SUL-X beamline at ANKA was tested and has proven to be well suited for speciation of lead in human mineralized tissue samples.

Spatial heterogeneity in the canalicular density of the osteocyte network in human osteons

Osteocytes interconnect with each other forming an intricate cell network within the mineralized bone matrix. One important function of the osteocyte network is the mechano-regulation of bone remodeling, where a possible mechanism includes the fluid flow through the porosity housing the cell network - the osteocyte lacuno-canalicular network (OLCN). In our study the OLCN in human osteons was three-dimensionally imaged with the aim to obtain a quantitative description of the canalicular density and spatial variations of this quantity within osteons. The topology of the OLCN was determined by first staining the bone samples with rhodamine, then imaging the OLCN with confocal laser scanning microscopy and finally using image analysis to obtain a skeletonized version of the network for further analysis. In total 49 osteons were studied from the femoral cortical bone of four different middle-aged healthy women. The mean canalicular density given as length of the canaliculi in a unit volume was 0.074 ± 0.015 μm/μm3 (corresponding to 74 km/cm3). No correlation was found between the canalicular density and neither the size of the osteon nor the volume fraction occupied by osteocyte lacunae. Within osteons the canalicular density varied substantially with larger regions without any network. On average the canalicular density decreases when moving from the Haversian canal outwards towards the cement line. We hypothesize that a decrease in accessible canaliculi with tissue age as a result of micropetrosis can reduce the local mechanosensitivity of the bone. Systematic future studies on age- and disease-related changes on the topology of the OLCN have to demonstrate the diagnostic potential of the presented characterization method.

Mapping dynamical mechanical properties of osteonal bone by scanning acoustic microscopy in time-of-flight mode.

An important determinant of mechanical properties of bone is Youngs modulus and its variation in individual osteons of cortical bone tissue. Its mechanical behavior also depends on deformation rate owing to its visco- or poroelastic properties. We developed a method to measure dynamical mechanical properties of bulk bone tissue at osteonal level based on scanning acoustic microscopy (SAM) using time-of-flight (TOF) measurements in combination with quantitative backscattered electron imaging (qBEI). SAM-TOF yields local sound velocities and qBEI corresponding material densities together providing elastic properties. Osteons (n=55) were measured in three human femoral diaphyseal ground bone sections (∼ 30 µm in thickness). In addition, subchondral bone and mineralized articular cartilage were investigated. The mean mineral contents, the mean sound velocities, and the mean elastic modulus of the osteons ranged from 20 to 26 wt%, from 3,819 to 5,260 m/s, and from 21 to 44 GPa, respectively. There was a strong positive correlation between material density and sound velocity (Pearsons r=0.701; p<0.0001) of the osteons. Sound velocities between cartilage and bone was similar, though material density was higher in cartilage (+4.46%, p<0.0001). These results demonstrate the power of SAM-TOF to estimate dynamic mechanical properties of the bone materials at the osteonal level.

Presence of pyrophosphate in bone from an atypical femoral fracture site: A case report

Long-term antiresorptives use has been linked to atypical subtrochanteric and diaphyseal femoral fractures (AFF), the pathogenesis of which is still unknown. In the present case report we present the results of analysis of bone chips from a 74-year old female patient that had been on alendronate, ibandronate and denosumab treatment, and who sustained an atypical femoral fracture, by histology, quantitative backscattered electron imaging, and Raman spectroscopic analysis. The results indicate ongoing osteoclastic resorption, but also several abnormalities: 1) an altered arrangement of osteons; 2) impaired mineralization; 3) the presence of pyrophosphate, which might contribute to the impaired mineralization evident in the present case. Taken together, these changes may contribute to the focally reduced bone strength of this patient.

Coalignment of osteocyte canaliculi and collagen fibers in human osteonal bone

During bone formation osteocytes get connected with each other via a dense network of canaliculi within the mineralized bone matrix. Important functions attributed to the osteocyte network include the control of bone remodeling and a contribution to mineral homeostasis. To detect structural clues of the formation and functionality of the network, this study analyzes the structure and orientation of the osteocyte lacuno-canalicular network (OLCN), specifically in relation to the concentric bone lamellae within human osteons. The network structure within 49 osteons from four samples of cortical bone from the femoral midshaft of middle-aged healthy women was determined by a combination of rhodamine staining and confocal laser scanning microscopy followed by computational image analysis. A quantitative evaluation showed that 64±1% of the canalicular length has an angle smaller than 30° to the direction towards the osteon center, while the lateral network - defined by an orientation angle larger than 60° - comprises 16±1%. With the same spatial periodicity as the bone lamellae, both radial and lateral network show variations in the network density and order. However, only the preferred orientation of the lateral network twists when crossing a lamella. This twist agrees with the preferred orientation of the fibrous collagen matrix. The chirality of the twist was found to be individual-specific. The coalignment between network and matrix extends to the orientation of the elongated osteocyte lacunae. The intimate link between OLCN and collagen matrix implies an interplay between osteocyte processes and the arrangement of the surrounding collagen fibers during osteoid formation.

Abuse and Neglect of the Elderly

Violence may affect everyone, even elderly people, whether man or woman. As a result of reduced physical strength, older people are rather helpless when it comes to defending themselves, and frailty, illness, and care dependency entail an increased risk for serious health damage as a result of physical violence or trauma. Elderly people mostly have more grave and long-standing effects to bear than younger victims of violence. In case of acute injuries of chronic and psychological consequences of violence, most victims do turn to medical help. However, the truth about the triggers of their suffering is often withheld, because of shame, fear, or dependence upon their tormentors, or it may not be voiced because of an underlying disease. It is therefore crucial to detect violence as the cause behind visible symptoms and complaints, not only to ensure the correct medical treatment and adequate support of victims but also to prevent further abuse.