Pharmacologic inhibitors of IκB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo

The NF-κB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IκB kinase (IKK), a key component of NF-κB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1β and tumor necrosis factor α–induced IκB phosphorylation and prevented nuclear translocation of NF-κB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKα and IKKβ, and celastrol inhibited IKKα/β activation by preventing the phosphorylation of TAK1, a key receptor–associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer

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

Modèles animaux d'hyper-résorption osseuse (méthodes d'étude et physiopathologie)

Plusieurs modèles animaux d'hyper-résorption osseuse ont été étudiés : modèle du rat orchidectomisé (ORX), modèle murin de myélome (5T2MM) combiné ou non à une ovariectomie (OVX). Nous avons évalué la performance et la sensibilité de différentes méthodes d'étude de la perte osseuse, et caractérisé la physiopathologie de l'hyper-résorption. Des mesures densitométriques (DXA) du contenu minéral osseux (CMO) d'os de rats contrôles, ont été obtenues avec une bonne reproductibilité et une bonne exactitude sur 3 générations de densitomètres, avec cependant une influence du poids de l'os sur le CMO. Des mesures de CMO, effectuées sur des os de rats ORX et ORX traités par bisphosphonate, ont montré qu' une large distribution du CMO n'altère pas l'exactitude des mesures de DXA. Cette méthode est cependant moins sensible que l'histomorphométrie pour apprécier le degré de perte osseuse dans le modèle du rat ORX. Chez le rat ORX, l'histomorphométrie a mis en évidence une altération précoce de la microarchitecture osseuse précédent la perte osseuse. Dans le modèle murin de myélome 5T2MM, l'hyper-résorption se traduit par une disparition de l'os trabéculaire et la présence de nombreuses perforations corticales. Nous avons élaboré un modèle associant OVX au modèle 5T2MM. L'OVX induit un hyper-remodelage associé à une augmentation de la progression tumorale et une apparition plus précoce des lésions ostéolytiques. Ce résultat pourrait expliquer le passage brutal d'un myélome indolent à un myélome agressif chez l'homme lorsqu'une modification du niveau de remodelage survient.Several animal models, with a high bone resorption level, were studied : the orchidectomized (ORX) rat model, the 5T2MM murine myeloma model with or without ovariectomy (OVX). We have first examined reproducibility, accuracy and sensibility of several methods used to evaluate bone loss. Then, we have studied the physiopathology of high remodeling rate in these animal models. Densitometric measurements (DXA) of bone mineral content (BMC) were done in control rats. Precise and accurate BMC measurements were obtained on 3 different generations of densitometer. However, discrepancy of BMC was dependent on the net weight of the bone. BMC measurements were performed on bone of ORX rat and ORX treated with a bisphosphonate. Accuracy was not affected by a large distribution of BMC values. DXA appeared to be less sensitive than bone histomorphometry to appreciate bone loss in the ORX rat model. In the ORX rat, histomorphometry evidenced alteration of trabecular bone architecture before bone loss occured. In the 5T2MM murine myeloma model, the increase of bone resorption induced disaparition of trabecular bone and numerous cortical perforations. We have proposed a combined animal model in which OVX was performed in mice prior inoculation of 5T2MM cells. OXV induced an increase bone remodeling which was associated with an increase of tumor growth and earlier development of osteolytic lesions. This result could explain some sudden burden of indolent MM into aggressive MM in man when a modification of mode remodeling happens.ANGERS-BU Médecine-Pharmacie (490072105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Osteomorphs as a tool for personalized medicine

International audienceMcDonald and colleagues reported osteoclast-related dynamic mechanisms that lead, by fission, to osteomorphs; motile, fusion-competent cells capable of forming bone-resorbing osteoclasts. scRNA-seq analyses revealed that osteomorphs are transcriptionally distinct from osteoclasts and macrophages and might be implicated in rare and common bone diseases in humans

Analysis of β-tricalcium phosphate granules prepared with different formulations by nano-computed tomography and scanning electron microscopy

International audienceAmong biomaterials used for filling bone defects, beta-tricalcium phosphate (β-TCP) is suitable in non-bearing bones, particularly in dental implantology, oral and maxillofacial surgery. When β-TCP granules are placed in a bone defect, they occupy the void 3D volume. Little is known about the 3D arrangement of the granules, which depends on the nature and size of the granules. The aim of this study was to examine the 3D architecture of porous β-TCP granules. Granules were prepared with different concentrations of β-TCP powder in slurry (10, 11, 15, 18, 21, and 25&nbsp;g of β-TCP powder in distilled water). Granules were prepared by the polyurethane foam method. They were analyzed by nano-computed tomography (nanoCT) and compared with scanning electron microscopy (SEM). Commercial granules of hydroxyapatite-β-TCP prepared by the same methodology were also used. The outer and inner architectures of the granules were shown by nanoCT which evidenced macroporosity, internal porosity and microporosity between the sintered grains. Macroporosity was reduced at high concentration and conversely, numerous concave surfaces were observed. Internal porosity, related to the sublimation of the polyurethane foam, was present in all the granules. Microporosity at the grain joints was evidenced by SEM and on 2D nanoCT sections. Granules presented a heterogeneous aspect due to the different mineralization degree of the sintered powder grains in the β-TCP granules; the difference between hydroxyapatite and β-TCP was also evidenced. NanoCT is an interesting method to analyze the fine morphology of biomaterials with a resolution close to synchrotron and better than microcomputed tomography.</p

Adsorption and release of strontium from hydroxyapatite crystals developed in Simulated Body Fluid (SBF) on poly (2-hydroxyethyl) methacrylate substrates.

International audiencePoly (2-hydroxy ethyl) methacrylate (PHEMA) is a polymer that can be carboxymethylated to induce calcification at on its surface. This mimics the calcification of bone matrix since the polymer surface induces the deposit of large hydroxyapatite calcospherites. We investigated the effect of Sr2+ on hydroxyapatite crystals developed on PHEMA pellets. Pellets were incubated for 1 week in a synthetic body fluid (SBF) to induce mineralization, then 2 weeks in SBF containing 0, 130, 260 or 390 µM of Sr2+ allowing growth and maturation of calcospherites. Calcospherites were dissolved in HCl and Ca2+, PO43- and Sr2+ content was measured. Sr2+ release was assessed by transferring other pellets in saline which was collected at regular intervals to measure Sr2+ release. Hydroxyapatite was characterized by SEM, X-ray diffraction, FTIR and Raman microspectroscopy. After the maturation period, Sr2+ was incorporated into hydroxyapatite crystals as a function of its concentration in SBF. However, size of the calcospherites decreased as a function of the strontium concentration. During the release phase, the slope of Sr2+ elution was progressive and similar independently of the initial concentration; ~30% Sr2+ was released after 61 days. XRD showed that incorporation of Sr2+ produced no significant change in crystal lattice parameters or cristallinity. A progressive release of Sr2+occurred from the crystals. Strontium can adsorb rapidly on hydroxyapatite crystals and can be released easily. Carboxymethylated PHEMA can be used to study the effect of chemical compounds on the growth of hydroxyapatite nodules and their release in a second time

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

In vivo osseointegration and erosion of nacre screws in an animal model

International audienceThe use of resorbable devices for osteosynthesis has become a subject of interest. Nacre has been proposed as a resorbable and osteoconductive material favoring bone apposition without triggering an inflammatory reaction. We compared the in vivo osseointegration and erosion of nacre screws in an animal model with titanium screws. Implantation of similar nacre and titanium screws was performed in the femoral condyles of adult rats. Animals (n = 41) were randomized in four groups sacrificed at day one, 1, 6, and 12 months. Microcomputed tomography (microCT) allowed 3D morphometry of erosion of nacre. Osseointegration was measured as the volume of trabecular bone bone volume/tissue volume (BV/TV) in a standardized volume of interest around each screw. Undecalcified bone histol-ogy was also done. Gross examination revealed a similar clinical osseointegration for titanium and nacre screws. A progressive erosion of nacre screws, but no erosion of titanium screws, was observed in microCT. The volume of nacre screws progressively decreased over time whereas no modification occurred for titanium. For titanium screws, BV/TV remained stable throughout the study. For nacre screws, the BV/TV decrease was not statistically different. A significant difference was found between nacre and titanium screws at 6 months but not at 12 months. The screw heads, outside the bone shaft, were not eroded even after 12 months. Erosion of nacre occurred during the entire study period, only within the bone shaft in direct contact with bone marrow. Bone apposition was observed on nacre surfaces without signs of erosion. Nacre is a promising biomaterial in maxillofacial surgery. K E Y W O R D S erosion, maxillofacial surgery, mother of pearl, nacre, osseointegration, resorbable device, titanium scre

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