Double disruption of alpha(2A)- and alpha(2C)-adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via beta(2)-adrenoceptor (beta(2)-AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, alpha(2A)-AR and alpha(2C)-AR(alpha(2A)/alpha(2C)-ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In alpha(2A)/alpha(2C)-ARKO versus wild-type (WT) mice, micro-computed tomographic (mu CT) analysis showed increased, better connected, and more plate-shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate-resistant acid phosphatase (TRACP) and receptor activator of NF-kappa B (RANK), which are osteoclast-related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine-regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial beta(2)-AR mRNA expression also was similar in KO and WT littermates, whereas alpha(2A)-, alpha(2B)- and alpha(2C)-AR mRNAs were detected in the tibia of WT mice and in osteoblast-like MC3T3-E1 cells. By immunohistochemistry, we detected alpha(2A)-, alpha(2B)-, alpha(2C)- and beta(2)-ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5-day-old mouse fetuses and 35-day-old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective alpha(2)-AR agonist clonidine and to the nonspecific alpha-AR antagonist phentolamine. These findings suggest that beta(2)-AR is not the single adrenoceptor involved in bone turnover regulation and show that alpha(2)-AR signaling also may mediate the SNS actions in the skeleton. (c) 2011 American Society for Bone and Mineral Research.

LOW-INTENSITY PULSED ULTRASOUND PRODUCED AN INCREASE OF OSTEOGENIC GENES EXPRESSION DURING THE PROCESS OF BONE HEALING IN RATS

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. (E-mail: a.renno@unifesp.br) (C) 2010 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology.

Is Nitric Oxide a Mediator of the Effects of Low-Intensity Electrical Stimulation on Bone in Ovariectomized Rats?

Low-intensity electrical stimulation (LIES) may counteract the effects of ovariectomy (OVX) on nitric oxide synthase (NOS) expression, osteocyte viability, bone structure, and microarchitecture in rats (Lirani-Galvo et al., Calcif Tissue Int 84:502-509, 2009). The aim of the present study was to investigate if these effects of LIES could be mediated by NO. We analyzed the effects of NO blockage (by l-NAME) in the response to LIES on osteocyte viability, bone structure, and microarchitecture in OVX rats. Sixty rats (200-220 g) were divided into six groups: sham, sham-l-NAME (6 mg/kg/day), OVX, OVX-l-NAME, OVX-LIES, and OVX-LIES-l-NAME. After 12 weeks, rats were killed and tibiae collected for histomorphometric analysis and immunohistochemical detection of endothelial NOS (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). In the presence of l-NAME, LIES did not counteract the OVX-induced effects on bone volume and trabecular number (as on OVX-LIES). l-NAME blocked the stimulatory effects of LIES on iNOS and eNOS expression of OVX rats. Both l-NAME and LIES decreased osteocyte apoptosis. Our results showed that in OVX rats l-NAME partially blocks the effects of LIES on bone structure, turnover, and expression of iNOS and eNOS, suggesting that NO may be a mediator of some positive effects of LIES on bone.

Low-Intensity Electrical Stimulation Counteracts the Effects of Ovariectomy on Bone Tissue of Rats: Effects on Bone Microarchitecture, Viability of Osteocytes, and Nitric Oxide Expression

Low Intensity Electrical Stimulation (LIES) has been used for bone repair, but little is known about its effects on bone after menopause. Osteocytes probably play a role in mediating this physical stimulus and they could act as transducers through the release of biochemical signals, such as nitric oxide (NO). The aim of the present study was to investigate the effects of LIES on bone structure and remodeling, NOS expression and osteocyte viability in ovariectomized (OVX) rats. Thirty rats (200-220 g) were divided into 3 groups: SHAM, OVX, and OVX subjected to LIES (OVX + LIES) for 12 weeks. Following the protocol, rats were sacrificed and tibias were collected for histomorphometric analysis and immunohistochemical detection of endothelial NO synthase (eNOS), inducible NOS (iNOS), and osteocyte apoptosis (caspase-3 and TUNEL). OVX rats showed significant (p < 0.05 vs. SHAM) decreased bone volume (10% vs. 25%) and trabecular number (1.7 vs. 3.9), and increased eroded surfaces (4.7% vs. 3.2%) and mineralization surfaces (15.9% vs. 7.7%). In contrast, after LIES, all these parameters were significantly different from OVX but not different from SHAM. eNOS and iNOS were similarly expressed in subperiosteal regions of tibiae cortices of SHAM, not expressed in OVX, and similarly expressed in OVX + LIES when compared to SHAM. In OVX, the percentage of apoptotic osteocytes (24%) was significantly increased when compared to SHAM (11%) and OVX + LIES (8%). Our results suggest that LIES counteracts some effects of OVX on bone tissue preserving bone structure and microarchitecture, iNOS and eNOS expression, and osteocyte viability.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

Performance of a solar energy powered falling film evaporator with film promoter

A solar energy powered failing film evaporator with film promoter was developed for concentrating diluted solutions (industrial effluents). The procedure proposed here does not emit CO(2), making it a viable alternative to the method of concentrating solutions that uses vapor as a heat source and releases CO(2) from burning fuel oil in a furnace, in direct opposition to the carbon reduction agreement established by the Kyoto protocol. This novel device consists of the following components: a flat plate solar collector with adjustable inclination, a film promoter (adhering to the collector), a liquid distributor, a concentrate collector. and accessories. The evaporation rate of the device was found to be affected both by the inclination of the collector and by the feed flow. The meteorological variables cannot be controlled, but were monitored constantly to ascertain the behavior of the equipment in response to the variations occurring throughout the day. Higher efficiencies were attained when the inclination of the collector was adjusted monthly, showing up to 36.4% higher values than when the collector remained in a fixed position. (c) 2008 Elsevier Ltd. All rights reserved.

Surface modification of bovine bone ash prepared by milling and acid washing process

Bovine bone ash is the main raw material for fabrication of bone china, a special kind of porcelain that has visual and mechanical advantages when compared to usual porcelains. The properties of bone china are highly dependent on the characteristics of the bone ash. However, despite a relatively common product, the science behind formulations and accepted fabrication procedures for bone china is not completely understood and deserves attention for future processing optimizations. In this paper, the influence of the preparation steps (firing, milling, and washing of the bones) on the physicochemical properties of bone ash particles was investigated. Bone powders heat-treated at temperatures varying from 700 to 1000 degrees C were washed and milled. The obtained materials were analyzed in terms of particle size distribution, chemical composition, density, specific surface area, FTIR spectroscopy, dynamic electrophoretic mobility, crystalline phases and scanning electron microscopy. The results indicated that bone ash does not significantly change in terms of chemistry and physical features at calcination temperatures above 700 degrees C. After washing in special conditions, one could only observe hydroxyapatite in the diffraction pattern. By FTIR it was observed that carbonate seems to be mainly concentrated on the surface of the powders. Since this compound can influence in the dispersion stability, and consequently in the quality of the final bone china product, and considering optimal washing parameters based on the dynamic electrophoretic mobility results, we describe a procedure for surface cleaning. (c) 2009 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Hemoglobin Regeneration Efficiency in Anemic Rats: Effects on Bone Mineral Composition and Biomechanical Properties

This study reports the effects of dietary iron (Fe) deficiency and recovery on bone mineral composition and strength in anemic rats submitted to a hemoglobin (Hb) repletion assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulfate (n = 8) or ferric pyrophosphate (FP; n = 12). At final day of each period (depletion and repletion), Fe-adequate animals were also euthanized. Iron status (blood Hb, Hb Fe pool, Hb regeneration efficiency), tibia mineral concentrations (Ca, Mg, Fe, Cu, and Zn) and biomechanical properties were evaluated. Iron-deficient rats had lower tibia Fe and Mg levels and bone strength when compared to controls. Yield load and resilience were positively related to tibia Mg levels (r = 0.47, P = 0.02 and r = 0.56, P = 0.004, respectively). Iron repletion did not recover tibia Mg concentrations impaired by Fe deficiency. Moreover, bone elastic properties were negatively affected by FP consumption. In conclusion, bone mineral composition and strength were affected by Fe deficiency, whereas dietary Fe source influenced tibia Mg and resistance in the period during which rats were recovering from anemia.

Changes in bone mass, biomechanical properties, and microarchitecture of calcium- and iron-deficient rats fed diets supplemented with inulin-type fructans

Feeding mineral-deficient diets enhances absorptive efficiency as an attempt of the body to compensate for the lack of an essential nutrient. Under certain circumstances, it does not succeed, and nutritional deficiency is produced Our hypothesis was that mulin-type fructans (ITF), which arc known to affect mineral absorption, could increase Ca and Fe bioavailability in Ca- and Fe-deficient rats. Male Wistar rats (n = 48, 4 weeks old) were assigned to I of 8 groups derived from 2 x 2 x 2 factorial design with 2 levels of added Fe (0 and 35 mg/kg), Ca (0 and 5 g/kg), and ITF (0 and 100 g/kg) for 33 days. The Fe status (hemoglobin, serum Fe, total Fe-binding capacity, transferrin saturation, liver minerals) was evaluated. Tibia minerals (Ca, Mg, and Zn), bone strength, and histomorphometry were determined In nondeficient rats, ITF supplementation did not affect Fe status or organ minerals, with the exception of tibia Mg Moreover, ITF improved bone resilience and led to a reduction in eroded surface per body surface and number of osteoclasts per area In Ca-deficient rats, ITF increased liver (Fe and Zn) and tibia (Zn) mineral levels but impaired tibia Mg, yield load, and resilience. In conclusion, ITF worsened the tibia Mg levels and elastic properties when supplemented in Ca-deficient diets In contrast, although bone Ca was not affected in nondeficient rats under the present experimental conditions, bone quality improved, as demonstrated by a moderate reduction in femur osteoclast resorption and significant increases in tibia Mg content and elasticity. (C) 2009 Elsevier Inc. All rights reserved.

Study of lymphocyte subpopulations in bone marrow in a model of protein-energy malnutrition

Objective: Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. Hematopoietic tissue requires a high nutrient supply, and a reduction in leukocytes, especially lymphocytes, suggests that some nutritional deficiencies might be altering bone marrow function and decreasing its ability to produce lymphocytes. In this study, we evaluated the effect that PEM has on lymphocyte subtypes and the cell cycle of CD5(+) cells. Methods: Swiss mice were subjected to PEM using a low-protein diet containing 4% protein. When the experimental group had lost about 20% of their original body weight, we collected blood and bone marrow cells and evaluated the hemogram, the myelogram, bone marrow lymphoid markers using flow cytometry, and the cell cycle in CD5(+) bone marrow. Results: Malnourished animals presented anemia, reticulocytopenia, and leukopenia with lymphopenia. The bone marrow was hypocellular, and flow cytometric analyses of bone marrow cells showed cells that were CD45(+) (91.2%), CD2(+) (84.9%), CD5(+) (37.3%), CD3(+) (23.5%), CD19(+) (43.3%), CD22(+) (34.7%), CD19(+)/CD2(+) (51.2%), CD19(+)/CD3(+)(24.0%), CD19(+)/CD5(+) (13.2%), CD22(+)/CD2(+) (40.1%), CD22(+)/CD3(+) (30.3%), and CD22(+)/CD5(+) (1.1%) in malnourished animals and CD45(+) (97.5%), CD2(+) (42.9%), CD5(+) (91.5%), CD3(+) (92.0%), CD19(+) (52.0%), CD22(+) (75.6%), CD19(+)/CD2(+) (62.0%), CD19(+)/CD3(+) (55.4%), CD19(+)/CO5(+) (6.7%), CD22(+)/CD2(+) (70.3%), CD22(+)/CD3(+) (55.9%), and CD22(+)/ CD5(+) (8.4%) in control animals. Malnourished animals also presented more CD5(+) cells in the G0 phase of cell cycle development. Conclusion: Malnourished animals presented bone marrow hypoplasia, maturation interruption, prominent lymphopenia with depletion in the lymphoid lineage, and changes in cellular development. We suggest that these changes are some of the primary causes of lymphopenia in cases of PEM and partly explain the increase in susceptibility to infections found in malnourished individuals. Published by Elsevier Inc.

Methylenedioxymethamphetamine (Ecstasy) Decreases Neutrophil Activity and Alters Leukocyte Distribution in Bone Marrow, Spleen and Blood

Objective: Looking for possible neuroimmune relationships, we analyzed the effects of methylenedioxymethamphetamine (MDMA) administration on neuroendocrine, neutrophil activity and leukocyte distribution in mice. Methods: Five experiments were performed. In the first, mice were treated with MDMA (10 mg/kg) 30, 60 min and 24 h prior to blood sample collection for neutrophil activity analysis. In the second experiment, the blood of nave mice was collected and incubated with MDMA for neutrophil activity in vitro analysis. In the third and fourth experiments, mice were injected with MDMA (10 mg/kg) and 60 min later, blood and brain were collected to analyze corticosterone serum levels and hypothalamic noradrenaline (NA) levels and turnover. In the last experiment, mice were injected with MDMA 10 mg/kg and 60 min later, blood, bone marrow and spleen were collected for leukocyte distribution analysis. Results: Results showed an increase in hypothalamic NA turnover and corticosterone serum levels 60 min after MDMA (10 mg/kg) administration, a decrease in peripheral blood neutrophil oxidative burst and a decrease in the percentage and intensity of neutrophil phagocytosis. It was further found that MDMA (10 mg/kg) treatment also altered leukocyte distribution in blood, bone marrow and spleen. In addition, no effects were observed for MDMA after in vitro exposure both in neutrophil oxidative burst and phagocytosis. Conclusion: The effects of MDMA administration (10 mg/kg) on neutrophil activity and leukocyte distribution might have been induced indirectly through noradrenergic neurons and/or hypothalamic-pituitary-adrenal axis activations. Copyright (C) 2009 S. Karger AG, Basel

Capacity of the extracellular matrix of the bone marrow to induce proliferation of myeloid cells in vitro in model of protein malnutrition in mice

The aim of this study was to verify the capacity of the extracellular matrix (ECM) obtained from bone marrow of malnourished mice to sustain survival and to induce the proliferation of myeloid cells. We also verified the capacity of the tests to interact with in vitro hematopoietic cytokines. Male ""Swiss"" mice were submitted to protein malnutrition with a diet contents of 4% casein until they lost 20% of the original weight, while the group-control was kept with a diet content of 14% of casein. The bone marrow was extracted with 1.0 mg of aprotinin/mL in PBS. The proliferation tests were carried out with myeloid cell line FDCP-1, by the colorimetric method of reduction of the MTT. The obtained ECM from nourished and undernourished mice induced cellular proliferation in vitro. Tests performed with Il-3 and GM-CSF cytokines in a concentration of 10 and 500 rho g/mL displayed synergic and regulatory effects respectively. The ECM obtained from the malnourished group submitted to the binding to GM-CSF demonstrated higher cellular proliferation than the ECM obtained from the control group (p<0.05). The results suggest that the alterations in the composition of ECM of bone marrow caused by malnutrition might lead to modification of the GM-CSF activity modulation.

Sustained release carriers used to delivery bone morphogenetic proteins in the bone healing process

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor beta superfamily, especially BMP-2, induce bone formation in vivo, and clinical application in repair of bone fractures and defects is expected. However, appropriate systems to delivery BMPs for practical use need to be developed with the objective to heal cartilage and bone-related diseases in medical, dental and veterinary practice. Thus, the aim of this article was to present an overview of the principals carriers used to delivery BMPs and alternative delivery systems for these proteins.

Modulation of doxorubicin-induced clastogenesis in Wistar rat bone marrow cells by vitamin B(6)

Vitamin B(6) has shown to be a potentially effective antioxidant agent, and dietary antioxidants are also frequently valuable inhibitors of clastogenesis and carcinogenesis. The purpose of the present work was to study the clastogenicity of different doses of vitamin B6 and to examine the possible modulating effect of this vitamin on chromosomal damage induced by the antitumor agent doxorubicin in Wistar rats. Experimental groups were set up for pre-and simultaneous treatment with vitamin B6 alone or in combination with DXR. The data obtained from administering diVerent doses of vitamin B(6) (12.5-100 mg/kg b. w.) showed no signigicant increase in total chromosomal aberrations when compared with the negative control. The administration of two doses of 25 mg/kg b. w. or one dose of 50 mg/kg b. w. of vitamin B6 before doxorubicin injection seemed equally effective in protecting cells against doxorubicin clastogenicity. The anticlastogenic effect of vitamin B(6) on DXR-induced chromosomal damage could be ascribed to its antioxidant properties. Vitamin B6 was not clastogenic or cytotoxic in rat bone marrow cells and it plays a role in inhibiting the clastogenicity induced by DXR.

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet`s effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet`s effects on genomic stability and DNA methylation. (C) 2011 Elsevier ay. All rights reserved.