Synthesis and use of pHEMA microbeads with human EA.hy 926 endothelial cells

Cancer has become a major problem in public health and the resulting bone metastases a worsening factor. Facing it, different strategies have been proposed and mechanisms involved in tumor angiogenesis are being studied. Enhanced permeability retention (EPR) effect is a key step in designing new anticancer drugs. We have prepared poly 2-hydroxyethyl methacrylate (pHEMA) microbeads to target human endothelial EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells. Microbeads were synthesized by emulsion precipitation method and carried positive or negative charges. EA.hy 926 cells were cultured in 24-well plates and microbeads were deposited on cells at various times. Scanning and transmission electron microscopy, flow cytometry, confocal microscopy, and three-dimensional (3D) reconstruction were used to characterize microbeads and their location outside and inside cells. Microbeads were uptaken by endothelial cells with a better internalization for negatively charged microbeads. 3D reconstruction of confocal optical sections clearly evidenced the uptake and internalization of microbeads by endothelial cells. pHEMA microbeads could represent potential drug carrier in tumor model of metastases

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities. Methodology/Principal Findings We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter. Conclusion This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time

New laboratory tools in the assessment of bone quality

International audienceBone quality is a complex set of different factors that are interdependent. The bone matrix organization can be described at five different levels of anatomical organization: nature (organic and mineral), texture (woven or lamellar), structure (osteons in the cortices and arch-like packets in trabecular bone), microarchitecture and macroarchitecture. Any change in one of these levels can alter bone quality. An altered bone remodeling can affect bone quality by influencing one or more of these factors. Here, we have reviewed the main methods that can be used in the laboratory to explore bone quality on bone samples. Bone remodeling can be evaluated by histomorphometry, microarchitecture is explored in 2D on histological sections and in 3D by microCT or synchrotron. Microradiography and scanning electron microscopy in the backscattered electron mode can measure the mineral distribution; Raman and Fourier transformed infra-red spectroscopy and imaging can simultaneously explore the organic and mineral phase of the matrix on multispectral images; scanning acoustic microscopy and nanoidentation provide biomechanical informations on individual trabeculae. Finally, some histological methods (polarization, surface staining, fluorescence, osteocyte staining) may also be of interest in the undestanding of quality as a component of bone fragility. Conclusion. A growing number of laboratory techniques are now available. Some of them have been described many years ago and can find a new youth, others having benefit from improvements in physical and computer techniques are now available

Iron inhibits hydroxyapatite crystal growth in vitro

International audienceHemochromatosis is a known cause of osteoporosis in which the pathophysiology of bone loss is largely unknown and the role of iron remains questionable. We have investigated the effects of iron on the growth of hydroxyapatite crystals in vitro on carboxymethylated poly(2-hydroxyethyl methacrylate) pellets. This noncellular and enzyme-independent model mimics the calcification of woven bone (composed of calcospherites made of hydroxyapatite crystals). Polymer pellets were incubated with body fluid containing iron at increasing concentrations (20, 40, 60 μmol/L). Hydroxyapatite growth was studied by chemical analysis, scanning electron microscopy, and Raman microscopy. When incubated in body fluid containing iron, significant differences were observed with control pellets. Iron was detected at a concentration of 5.41- to 7.16-fold that of controls. In pellets incubated with iron, there was a ∼3- to 4-fold decrease of Ca and P and a ∼1.3- to 1.4-fold increase in the Ca/P ratio. There was no significant difference among the iron groups of pellets, but a trend to a decrease of Ca with the increase of iron concentration was noted. Calcospherite diameters were significantly lower on pellets incubated with iron. Raman microspectroscopy showed a decrease in crystallinity (measured by the full width of the half height of the 960 Δcm−1 band) with a significant increase in carbonate substitution (measured by the intensity ratio of 1071 to 960 Δcm−1 band). Energy dispersive x-ray analysis identified iron in the calcospherites. In vitro, iron is capable to inhibit bone crystal growth with significant changes in crystallinity and carbonate substitution.</p

Fetuin and osteocalcin interact with calcospherite formation during the calcification process of poly(2-hydroxyethylmethacrylate)in vitro: a Raman microspectroscopic monitoring

International audienceCalcification is a complex process implying numerous proteins acting as nucleators, ion transporters and crystal growth regulators. Several proteins impair mineralization, such as fetuin and osteocalcin [(bone Gla protein), BGP]. We have evaluated their effets on the biomimetic calcification of carboxymethylated poly(2-hydroxyethyl methacrylate). Polymer pellets were incubated in synthetic body fluid for 4 days at 37 °C to induce nucleation. They were transferred for 11 days in a fresh medium containing fetuin (5 mg/ml) or BGP (1 mg/ml) or a combination of boths. Pellets were examined by scanning and transmission electron microscopy. Detection of proteins was done by immunogold and Raman microspectroscopy. Calcospherites were dissolved, Ca and P were dosed. BGP did not modify the amount of Ca[BOND]P or the Ca/P ratio, but the mean size of calcospherites was two times larger than controls. Fetuin reduced the number of calcospherites and the amount of Ca[BOND]P but increased the Ca/P ratio. Ca[BOND]P deposition was reduced on pellets incubated with both proteins, and calcospherites appeared considerably smaller. Immunogold and Raman spectroscopy identified both proteins adsorbed on hydroxyapatite (HA) tablets. Noncollagenous proteins control HA crystal growth in a different manner. The interaction between fetuin and BGP reduced the amount of calcified material deposited but also affected the morphology of the calcospherites

Effects of risedronate in a rat model of osteopenia due to orchidectomy and disuse: Densitometric, histomorphometric and microtomographic studies.: Bone loss due to orchidectomy and localized disuse.

International audienceDual energy X-ray absorptiometry (DXA), histomorphometry and X-ray microtomography (microCT) were used to assess effects of risedronate and testosterone in a combined rat model of orchidectomy (ORX) and local paralysis induced by botulinum neurotoxin (BTX). Four groups of mature rats were studied for 1 month: SHAM operated; ORX and right hindlimb immobilization (BTX); ORX+BTX+risedronate or testosterone. Changes in bone and body composition were measured by DXA (BMC, lean and fat mass), histomorphometry (BV/TV(2D), Tb.Th and microarchitectural parameters) and microCT (BV/TV(3D), SMI and cortical parameters). ORX and BTX had additive effects on bone loss since differences were maximized on the immobilized bone. The decrease in BMC on the tibial metaphysis reached -33.6% vs. -11.3% in the non-immobilized limb. BV/TV and Tb.N decreased and Tb.Sp increased in both hindlimbs whereas Tb.Th was significantly lower only in the immobilized limb. Decrease of tibial cortical area and thickness was greater in the immobilized limb. Risedronate prevented BMC, BV/TV and architecture loss but not reduction in Tb.Th. Cortical bone was preserved only in the non-immobilized limb. Testosterone was unable to prevent trabecular and cortical bone loss, but it prevents loss of whole body lean mass. In conclusion, ORX and BTX resulted in additive effects on bone loss. Risedronate had protective effects on trabecular bone loss but was less effective on cortical bone

Trabecular bone microarchitecture is related to the number of risk factors and etiology in osteoporotic men.

International audienceMicroarchitecture of trabecular bone is a very important component of bone quality in osteoporosis and a determinant of vertebral fracture in men with low bone mineral density (BMD). In contrast to women, male osteoporosis is, in most cases, secondary. The relationships between microarchitecture and different risk factors have never been evaluated in men. About 152 men with low BMD at the lumbar spine or hip (BMD, T-score < -2.5) were included in this study. Risk factors were: age, BMI, alcohol intake, corticosteroid therapy, hypogonadism, and chronic diseases. Transiliac bone biopsies were obtained and histomorphometry was done on an image analyzer; the following parameters were measured: cortical thickness (Ct.Th), trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), separation (Tb.Sp) and number (Tb.N), interconnectivity Index (ICI), star volume of the bone marrow, and strut analysis with node and free-end count. The 50 men with two risk factors had a lower BMD, lower Ct.Th and a significant higher star volume than those with one factor or idiopathic osteoporosis. The 26 men with at least three risk factors, had a lower BMD, a reduction of BV/TV and Ct.Th and a marked disorganization of the trabecular network (increased Tb.Sp, ICI, star volume, and free-end to free-end struts). The prevalence of vertebral fractures was higher in these patients. When the main risk factor was considered, a marked decrease in trabecular bone connectivity was observed in hypogonadic men. In osteoporotic men, higher the number of risk factors, lower the connectivity of trabecular network and higher the vertebral fracture risk