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A bone biopsy of the anterior iliac crest is the gold standard for the evaluation of bone disease in chronic kidney disease (CKD). The bone histomorphometric analysis consists of a detailed description of bone histology, including skeletal remodeling, bone mineralization, and bone microarchitecture. Like many histopathological diagnostic classifications (e.g. Banff), bone histomorphometry is based on a semi-quantitative assessment, utilizing both quantitative cutoffs of key parameters and a qualitative interpretation by an experienced physician.

Renal osteodystrophy (ROD) is a heterogeneous group of metabolic bone diseases complicating progressive chronic kidney disease (CKD). Bone biomarkers and bone imaging techniques may help to assess bone health and predict fractures in CKD but do have important inherent limitations. By informing on bone turnover and mineralization, a bone biopsy may help to guide prevention and treatment of ROD and its consequences. According to a recent survey conducted among European nephrologists, bone biopsies are performed rather exceptionally, both for clinical and research purposes. Obviously, clinical research in the field of ROD is threatened by vanishing clinical and pathological expertise, small patient cohorts and scientific isolation. In March 2016, the European Renal Osteodystrophy (EU-ROD) initiative was created under the umbrella of the ERA-EDTA CKD-mineral and bone disorder (MBD) Working Group to revitalize bone biopsy as a clinically useful tool in the diagnostic workup of CKD-MBD and to foster research on the epidemiology, implications and reversibility of ROD. As such, the EU-ROD initiative aims to increase the understanding of ROD and ultimately to improve outcomes in CKD patients. info:eu-repo/semantics/publishedVersion

International audience; The objective of this study was to develop and validate a technique for both 3D imaging and quantification of the vascular network of bone tissue in the rat. Five month-old male Wistar rats were divided into tail-suspension (21 days) and control groups. Sixty percent barium sulfate solution was infused into the vena cava. The tibiae were evaluated in 2D and 3D before and after decalcification, using conventional microcompu-terized tomography (lCT) at 10 and 5 lm resolution and synchrotron radiation (SR) lCT. The perfusion technique and tomography exhibited excellent bone vasculature imaging. Significant positive correlations were observed between 2D histomorphometric and 3D lCT vascular parameters (P < 0.05). 3DlCT discriminated significant changes of vessel structures in unloading condition: vessel number decreased by 25%, (P < 0.005), vessel separation increased by 27%, P < 0.01. SRlCT could image sinusoid clusters in bone. lCT is an accurate and reproducible technique for 3D quantitative evaluation of long bone vascularisation in the rat. Anat Rec, 293:215-224

Sclerostin is an osteocyte-specific glycoprotein secreted by the osteocyte and involved in the regulation of bone mass. High sclerostin levels are associated with osteoporosis, whereas low sclerostin levels are correlated with higher bone mineral density. It seems interesting to investigate a potential association between sclerostin levels and vascular calcifications since sclerostin is considered as a potent inhibitor of bone formation. In chronic kidney disease, serum sclerostin levels rise as renal function declines. Preliminary studies show a positive association between serum sclerostin and vascular calcification, but the link between sclerostin and survival of patients remains unclear in the absence of large-scale studies.

The fibrobalst growth factor 23 (FGF23) is a protein secreted in the plasma by bone cells. FGF23 controls phosphate and calcitriol plasma concentration. Its main physiological targets are the kidney and the parathyroid gland. Its production is stimulated by phosphate intake and plasma calcitriol levels. FGF23 binds a receptor at the cell surface that is composed by the association of a FGF receptor with the protein Klotho. Klotho expression is restricted to few organs including the kidney and the parathyroid gland. Klotho, which is expressed at the cell surface, can also be released in the plasma after an enzymatic cleavage. The role of the circulating form of Klotho is unknown. In addition to its role as a co-receptor Klotho may control the stability of the renal sodium phosphate transporter NPT2a and the calcium channel TRPV5 via enzymatic properties. FGF23 or Klotho gene disruption in mice leads to similar phenotypes except for the levels of FGF23, undetectable or increased respectively. FGF23 plasma concentration rises from the early stage of renal insufficiency to prevent plasma phosphate concentration from increasing. In parallel Klotho production seems to diminish. These modifications trigger the secondary hyperparathyroidism observed in renal insufficiency. The increase in FGF23 concentration and the decrease in Klotho levels are associated in animals and in human as well to an augmentation of mortality especially from cardiovascular causes and to heart modification: hypertrophy or dysfunction.

Bone is a composite organ that fulfils several interconnected functions, which may conflict with each other in pathological conditions. Bone vascularization is at the interface between these functions. The roles of bone vascularization are better documented in bone development, growth and modeling than in bone remodeling. However, every bone remodeling unit is associated with a capillary in both cortical and trabecular envelopes. Here we summarize the most recent data on vessel involvement in bone remodeling, and we present the characteristics of bone vascularization. Finally, we describe the various techniques used for bone vessel imaging and quantitative assessment, including histology, immunohistochemistry, microtomography and intravital microscopy. Studying the role of vascularization in adult bone should provide benefits for the understanding and treatment of metabolic bone diseases.

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against α1β1 integrin. RA up-regulates steady-state levels of α1 mRNA without affecting β1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins. J. Cell. Physiol. 179:267–275, 1999. © 1999 Wiley-Liss, Inc.

International audience; The osteocyte cell network in bone tissue is thought to orchestrate tissue adaptation and remodeling, thus holding responsibility for tissue quality. Previously, this structure has been studied mainly in 2D and its architecture and functions are not fully elucidated. The assessment of the osteocyte system is prerequisite for deeper understanding of bone remodeling and for advances in management of bone diseases. Our goal is to enable 3D isotropic imaging of bone at cellular level and to develop algorithms for quantitative image analysis of the cell network. We recently demonstrated accurate 3D imaging of this cell structure with synchrotron radiation tomography at submicrometric scale. Due to the limited spatial resolution of the imaging system and the constraints in terms of radiation dose, the images suffer from low signal to noise ratio and the detection of the cell dendrites is challenging. Here we detail a method for enhancement of the osteocyte network in human bone from 3D microtomography images. The approach combines Hessian-based 3D line enhancement and bilateral filtering. Our method enables extraction of the interconnected cells from noisy images, preserving the integrity of the cells and of their slender dendrites. Qualitative and quantitative results are presented

International audience; We present a method for phase microtomography based on X-ray propagation based phase contrast imaging for multi-material objects. Previously, homogeneous composition assumptions have been in the Radon domain to overcome low sensitivity in the low frequency range. Here, we introduce a prior in the object domain based on multiple, but known, materials in the sample. This is achieved by first reconstructing a tomographic attenuation scan, introducing the prior by segmentation, and finally forward projecting this initial object estimate to yield a priori phase maps. The method is applied to the imaging of a mouse knee where analysis of both soft and hard tissue is of interest, and is shown to perform better than previously proposed methods.

International audience; A new method for simultaneous 3D imaging and analysis of microvascularization and bone microstructure in rat bone is developed. The method is based on the use of quantitative synchrotron micro-computed tomography (SR-microCT) coupled to an automatic image analysis procedure. Analysis of bone microvascularization is generally performed from 2D histology. The proposed method enables for the first time the simultaneous 3D analysis of microvascularization and bone microstructure in a rat model. It was applied to investigate the effect of intermittent parathyroid hormone (PTH) administration on angiogenesis and osteogenesis in rats. Rats were posthumously injected with a contrast agent and subsequently imaged. The algorithm allowed the reconstruction and the extraction of 3D quantitative parameters both on bone microstructure and microvascularization. Due to the short acquisition times of SR-microCT and the efficiency of the image analysis algorithm, a large data set was analyzed, which permitted statistical analysis of the measured parameters. Statistical analysis confirmed that treatment with PTH significantly increased bone volume and thickness, but decreased bone mineralization. It was further revealed that treatment with PTH significantly increased average vessel thickness.

International audience; A method for simultaneous 3D imaging and analysis of microvascularization and bone microstructure in rat bone is presented. The method is based on the use of quantitative synchrotron micro-computed tomography (SR-¿CT) coupled to an automatic image analysis procedure. Previously, analysis of bone microvascularization has generally been performed from 2D histology. The proposed method enables for the first time to simultaneously analyze in 3D the microvascularization and bone microstructure in a rat model. We also propose a new parameter, utilizing the availablilty of both microstructures to relate the two, which we dub the vascular-trabecular interdistance (VTI). The proposed method was applied to investigate the effect of intermittent parathyroid hormone (PTH) administration on angiogenesis and osteogenesis in rats. It was possible to extract 3D quantitative parameters both on bone microstructure and micro-vascularization. Due to the short acquisition times of SR-¿CT and the efficiency of the image analysis algorithm, a large data set was analyzed, which permitted statistical analysis of the measured parameters. Statistical analysis showed that treatment with PTH significantly modulated several bone and vessel parameters, including the VTI.

We report a case of arthritis, hypercalcemia, and lytic bone lesions that occurred shortly after repeated administration of recombinant hepatitis B virus (HBV) vaccine in a 44-year-old man who had had myasthenia gravis 20 years earlier. He presented with ankle and knee arthritis and hypercalcemia. Radiographs revealed small lytic lesions and densitometry showed severe osteopenia. Quantimetric bone biopsy confirmed major bone loss and showed dramatic increase in bone turnover, as well as an unusual periosteal apposition of woven bone. Short term treatment with prednisone and furosemide and longterm treatment with clodronate allowed rapid improvement. After one year, the patient remains clinically asymptomatic. Despite negative immunologic investigations to sustain the hypothesis of HBV vaccination as a causal factor, we believe the bone lesions could be attributed to unusual bone "hyperremodeling" triggered by an immune process in a predisposed individual.

The effects of antiresorptive drugs on bone loss remain unclear. Using three-dimensional microtomography, dual X-ray/densitometry, and histomorphometry, we evaluated tiludronate effects in the bone loss model of immobilization in tail-suspended rats after 7, 13, and 23 days. Seventy-eight 12-week-old Wistar male rats were assigned to 13 groups: 1 baseline group, and for each time point, 1 control group treated with vehicle and three tail-suspended groups treated with either tiludronate (0.5 or 5 mg/kg) or vehicle, administered s. c. every other day, during the last week before sacrifice. In primary spongiosa (ISP), immobilization-induced bone loss plateaued after day 7 and was prevented by tiludronate. In secondary spongiosa (IISP), bone loss appeared at day 13 with a decrease in trabecular thickness and trabecular number (Tb.N) as assessed by three-dimensional microtomography. Osteoclastic parameters did not differ in tail-suspended rats versus control rats, whereas bone formation showed a biphasic pattern: after a marked decrease at day 7, osteoblastic activity and recruitment normalized at days 13 and 23, respectively. At day 23, the 80% decrease in bone mass was fully prevented by high-dose tiludronate with an increase in Tb.N without preventing trabecular thinning. In summary, at day 7, tiludronate prevented bone loss in ISP. After day 13, tiludronate prevented bone loss in ISP and IISP despite a further decrease in bone formation. Thus, the preventive effects of tiludronate in this model may be related to the alteration in bone modeling with an increase in Tb.N in ISP and subsequently in IISP.

Osteoblast morphology and attachment were studied during parabolic flight and during centrifugation. Cultures of osteosarcoma cells were exposed to gravitational changes and then analyzed for morphological changes and stained using immunofluorescence staining for vinculin. Changes in cell adhesion parameters and focal contact topography are presented and discussed.