Elevated levels of active Transforming Growth Factor ß1 in the subchondral bone relate spatially to cartilage loss and impaired bone quality in human knee osteoarthritis.

OBJECTIVE: The association between the spatially distributed level of active TGFß1 in human subchondral bone, and the characteristic structural and cellular parameters of human knee OA, was assessed. DESIGN: Paired subchondral bone samples from 35 OA arthroplasty patients, (15 men and 20 women, aged 69 ± 9 years) were obtained from beneath macroscopically present (CA+) or denuded cartilage (CA-) to determine the concentration of active TGFß1 (ELISA) and its relationship to bone quality (synchrotron micro-CT), cellularity, and vascularization (histology). RESULTS: Bone samples beneath (CA-) regions had significantly increased concentrations of active TGFß1 protein (mean difference: 26.4; 95% CI: [3.2, 49.7]), when compared to bone in CA + regions. Trabecular Bone below (CA-) regions had increased bone volume (median difference: 4.3; 96.49% CI: [-1.7, 17.8]), increased trabecular number (1.5 [0.006, 2.6], decreased trabecular separation (-0.05 [-0.1,-0.005]), and increased bone mineral density (394.5 [65.7, 723.3]) comparing to (CA+) regions. Further, (CA-) bone regions showed increased osteocyte density (0.012 [0.006, 0.018]), with larger osteocyte lacunae (39.8 [7.8, 71.7]) that were less spherical (-0.02 [-0.04, -0.003]), and increased bone matrix vascularity (12.4 [0.3, 24.5]) compared to (CA+). In addition, increased levels of active TGFß1 related to increased bone volume (0.04 [-0.11, 0.9]), while increased OARSI grade associated with lacunar volume (-44.1 [-71.1, -17.2]), and orientation (2.7 [0.8, 4.6]). CONCLUSION: Increased concentration of active TGFß1 in the subchondral bone of human knee OA associates spatially with impaired bone quality and disease severity, suggesting that TGFß1 is a potential therapeutic target to prevent or reduce human OA disease progression.

Measurement of single- and double-escape HPGe efficiency ratios for 60Co.

As a by-product of another measurement, ratios of the single-escape (SE) and double-escape (DE) efficiencies relative to the full-energy-peak efficiency (FE) have been measured for two HPGe detectors for 60Co. For a 2.5-cm-thick 95 cm3 crystal the results were SE/FE = 0.000 48 ± 0.000 20 and 0.003 25 ± 0.000 24 for 1173 and 1332 keV gamma-rays, respectively, and DE/FE = 0.000 90 ± 0.000 17 and 0.003 41 ± 0.000 11 for 1173 and 1332 keV, respectively. For a 3.0-cm-thick 84 cm3 crystal the results were SE/FE = 0.000 67 ± 0.000 32 and 0.003 79 ± 0.000 27 for 1173 and 1332 keV respectively, and DE/FE = 0.001 05 ± 0.000 28 and 0.004 29 ± 0.000 16 for 1173 and 1332 keV, respectively. These measurements may be of relevance in connection with Monte Carlo calculations of HPGe detector efficiencies, and may also suggest a path towards improved atomic cross-section measurements.

Bone marrow lesions in knee osteoarthritis: regional differences in tibial subchondral bone microstructure and their association with cartilage degeneration.

OBJECTIVE: The aim of this study was to investigate how bone microstructure within bone marrow lesions (BMLs) relates to the bone and cartilage across the whole human tibial plateau. DESIGN: Thirty-two tibial plateaus from patients with osteoarthritis (OA) at total knee arthroplasty and eleven age-matched non-OA controls, were scanned ex vivo by MRI to identify BMLs and by micro CT to quantitate the subchondral (plate and trabecular) bone microstructure. For cartilage evaluation, specimens were processed histologically. RESULTS: BMLs were detected in 75% of the OA samples (OA-BML), located predominantly in the anterior-medial (AM) region. In contrast to non-OA control and OA-no BML, in OA-BML differences in microstructure were significantly more evident between subregions. In OA-BML, the AM region contained the most prominent structural alterations. Between-group comparisons showed that the AM region of the OA-BML group had significantly higher histological degeneration (OARSI grade) (P < .0001, P < .05), thicker subchondral plate (P < .05, P < .05), trabeculae that are more anisotropic (P < .0001, P < .05), well connected (P < .05, P = n.s), and more plate-like (P < 0.05, P < 0.05), compared to controls and OA-no BML at this site. Compared to controls, OA-no BML had significantly higher OARSI grade (P < .0001), and lower trabecular number (P < .05). CONCLUSION: In established knee OA, both the extent of cartilage damage and microstructural degeneration of the subchondral bone were dependent on the presence of a BML. In OA-no BML, bone microstructural alterations are consistent with a bone attrition phase of the disease. Thus, the use of BMLs as MRI image-based biomarkers appear to inform on the degenerative state within the osteochondral unit.

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of ß-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released ß-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.

Gamma-ray spectroscopy for probing highly radioactive items behind thick shields?

In favourable circumstances, including the availability of prior knowledge, a potential use of gamma-ray spectroscopy with an HPGe detector for probing highly radioactive items behind thick shields is described.

Osteocyte regulation of bone mineral: a little give and take.

Osteocytes actively participate in almost every phase of mineral handling by bone. They regulate the mineralisation of osteoid during bone formation, and they are also a major RANKL-producing cell. Osteocytes are thus able to liberate bone mineral by regulating osteoclast differentiation and activity in response to a range of stimuli, including bone matrix damage, bone disuse and mechanical unloading, oestrogen deficiency, high-dose glucocorticoid and chemotherapeutic agents. At least some of these activities may be regulated by the osteocyte-secreted product, sclerostin. There is also mounting evidence that in addition to regulating phosphate homeostasis systemically, osteocytes contribute directly to calcium homeostasis in the mature skeleton. Osteocyte cell death and the local loss of control of bone mineralisation may be the cause of focal hypermineralisation of bone and osteopetrosis, as seen in aging and pathology. The sheer number of osteocytes in bone means that "a little give and take" in terms of regulation of bone mineral content translates into a powerful whole organism effect.

Unexpected 13N concentrations in ISIS synchrotron room air.

The specific activity of air in the large open room housing the 800-MeV proton synchrotron of the ISIS Spallation Neutron and Muon Source has been measured. Air from several positions within the ISIS synchrotron room was sucked through a long flexible tube, and run past a shielded HPGe gamma-ray detector outside the synchrotron room. In spite of an expectation that 13N should be the largest component of the overall activity in the air, the results of the measurements are consistent with the presence in the air of 11C and 41Ar only, and suggest that the activity in the air is mostly created not in the synchrotron room itself but in the massive shielding monoliths around the neutron-producing targets, monoliths through which ventilation air is drawn into the synchrotron room. Typical specific activities of 11C and 41Ar in the air in the synchrotron room are ∼0.10 and ∼0.03 Bq cm-3 respectively, the upper limit for 13N being at most ∼0.01 Bq cm-3.

Relationship between serum RANKL and RANKL in bone.

It is now well accepted that the molecule receptor activator of NFκB ligand (RANKL) and osteoprotegerin play key roles in regulating physiological and pathological bone turnover. There are a large number of published reports of circulating RANKL levels in both health and pathology. However, interpretation of these data has been elusive, and the relationship between circulating RANKL and RANKL levels in bone is still not clear. This review explores this subject, documenting the possible origins of circulating RANKL and suggesting additional information that is required before serum RANKL levels can provide useful diagnostic or research information.

Eutrophication of lakes cannot be controlled by reducing nitrogen input: results of a 37-year whole-ecosystem experiment.

Lake 227, a small lake in the Precambrian Shield at the Experimental Lakes Area (ELA), has been fertilized for 37 years with constant annual inputs of phosphorus and decreasing inputs of nitrogen to test the theory that controlling nitrogen inputs can control eutrophication. For the final 16 years (1990-2005), the lake was fertilized with phosphorus alone. Reducing nitrogen inputs increasingly favored nitrogen-fixing cyanobacteria as a response by the phytoplankton community to extreme seasonal nitrogen limitation. Nitrogen fixation was sufficient to allow biomass to continue to be produced in proportion to phosphorus, and the lake remained highly eutrophic, despite showing indications of extreme nitrogen limitation seasonally. To reduce eutrophication, the focus of management must be on decreasing inputs of phosphorus.

Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors.

We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.

Strontium ranelate treatment of human primary osteoblasts promotes an osteocyte-like phenotype while eliciting an osteoprotegerin response.

SUMMARY: The effect of strontium ranelate (SR) on human osteoblast differentiation was tested. SR induced osteoblastic proliferation, in vitro mineralization, and increased the expression of osteocyte markers. SR also elicited an osteoprotegerin (OPG) secretory response. We conclude that SR promotes the osteoblast maturation and osteocyte differentiation while promoting an additional antiresorptive effect. INTRODUCTION: SR is a new treatment for osteoporosis that reduces the risk of hip and vertebral fractures in postmenopausal women. This study sought to investigate the extent, to which SR modulates human osteoblast differentiation. METHODS: Adult human primary osteoblasts (NHBC) were exposed to SR under mineralizing conditions in long-term cultures. Osteoblast differentiation status was investigated by cell-surface phenotypic analysis. Expression of genes associated with osteoblast/osteocyte differentiation was examined using real-time RT-PCR. Secreted OPG was assayed by enzyme-linked immunosorbent assay. RESULTS: SR significantly increased osteoblast replication. SR time- and dose-dependently induced an osteocyte-like phenotype, as determined by cell surface alkaline phosphatase and STRO-1 expression. SR at 5 mM or greater dramatically increased in vitro mineralization. In parallel, mRNA levels of dentin matrix protein (DMP)-1 and sclerostin were higher under SR treatment, strongly suggestive of the presence of osteocytes. SR also increased the OPG/RANKL ratio throughout the culture period, consistent with an effect to inhibit osteoblast-induced osteoclastogenesis. CONCLUSIONS: This study suggests that SR can promote osteoblast maturation and an osteocyte-like phenotype. Coupled with its effect on the OPG/RANKL system, these findings are consistent with in vivo effects in patients receiving SR for the treatment of osteoporosis.

Effects of climatic warming on lakes of the central boreal forest.

Twenty years of climatic, hydrologic, and ecological records for the Experimental Lakes Area of northwestern Ontario show that air and lake temperatures have increased by 2 degrees C and the length of the ice-free season has increased by 3 weeks. Higher than normal evaporation and lower than average precipitation have decreased rates of water renewal in lakes. Concentrations of most chemicals have increased in both lakes and streams because of decreased water renewal and forest fires in the catchments. In Lake 239, populations and diversity of phytoplankton also increased, but primary production showed no consistent trend. Increased wind velocities, increased transparency, and increased exposure to wind of lakes in burned catchments caused thermoclines to deepen. As a result, summer habitats for cold stenothermic organisms like lake trout and opposum shrimp decreased. Our observations may provide a preview of the effects of increased greenhouse warming on boreal lakes.

Long-term ecosystem stress: the effects of years of experimental acidification on a small lake.

Experimental acidification of a small lake from an original pH value of 6.8 to 5.0 over an 8-year period caused a number of dramatic changes in the lake's food web. Changes in phytoplankton species, cessation of fish reproduction, disappearance of the benthic crustaceans, and appearance of filamentous algae in the littoral zone were consistent with deductions from synoptic surveys of lakes in regions of high acid deposition. Contrary to what had been expected from synoptic surveys, acidification of Lake 223 did not cause decreases in primary production, rates of decomposition, or nutrient concentrations. Key organisms in the food web leading to lake trout, including Mysis relicta and Pimephales promelas, were eliminated from the lake at pH values as high as 5.8, an indication that irreversible stresses on aquatic ecosystems occur earlier in the acidification process than was heretofore believed. These changes are caused by hydrogen ion alone, and not by the secondary effect of aluminum toxicity. Since no species of fish reproduced at pH values below 5.4, the lake would become fishless within about a decade on the basis of the natural mortalities of the most long-lived species.

Contemporary limnology of the rapidly changing glacierized watershed of the world's largest High Arctic lake.

Glacial runoff is predicted to increase in many parts of the Arctic with climate change, yet little is known about the biogeochemical impacts of meltwaters on downstream freshwater ecosystems. Here we document the contemporary limnology of the rapidly changing glacierized watershed of the world's largest High Arctic lake (Lake Hazen), where warming since 2007 has increased delivery of glacial meltwaters to the lake by up to 10-times. Annually, glacial meltwaters accounted for 62-98% of dissolved nutrient inputs to the lake, depending on the chemical species and year. Lake Hazen was a strong sink for NO3--NO2-, NH4+ and DOC, but a source of DIC to its outflow the Ruggles River. Most nutrients entering Lake Hazen were, however, particle-bound and directly transported well below the photic zone via dense turbidity currents, thus reinforcing ultraoligotrophy in the lake rather than overcoming it. For the first time, we apply the land-to-ocean aquatic continuum framework in a large glacierized Arctic watershed, and provide a detailed and holistic description of the physical, chemical and biological limnology of the rapidly changing Lake Hazen watershed. Our findings highlight the sensitivity of freshwater ecosystems to the changing cryosphere, with implications for future water quality and productivity at high latitudes.

Vascular pathology and osteoarthritis.

There is mounting evidence that vascular pathology plays a role in the initiation and/or progression of the major disease of joints: osteoarthritis (OA). Potential mechanisms are: episodically reduced blood flow through the small vessels in the subchondral bone at the ends of long bones, and related to this, reduced interstitial fluid flow in subchondral bone. Blood flow may be reduced by venous occlusion and stasis or by the development of microemboli in the subchondral vessels. There are several likely effects of subchondral ischaemia: the first of these is compromised nutrient and gas exchange into the articular cartilage, a potential initiator of degradative changes in the cartilage. The second is apoptosis of osteocytes in regions of the subchondral bone, which would initiate osteoclastic resorption of that bone and at least temporarily reduce the bony support for the overlying cartilage. It may be important to recognize these potential aetiological factors in order to develop more effective treatments to inhibit the progression of OA.

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells.

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.

A simple representation of spallation neutron energy spectra for 'back of the envelope' estimates.

A simple approximate easy-to-use analytic formula is suggested for the absolute integrated-over-angle energy spectrum of neutrons from a high-Z spallation neutron target driven by 500-1500MeV proton beams. Overall, the formula is probably reliable to within a factor ~2-3, and could be useful for quickly making rough 'back of the envelope' estimates.

Experimental verification of spallation inventory calculations.

Monte Carlo calculations and gamma-ray spectrometry measurements have been made for a highly irradiated tungsten target from a proton-driven spallation neutron source. A comparison of the calculated and measured activities of 60Co and 172Lu in the radionuclide inventory shows surprisingly good agreement.

Estimation of anisotropic permeability in trabecular bone based on microCT imaging and pore-scale fluid dynamics simulations.

In this paper, a comprehensive framework is proposed to estimate the anisotropic permeability matrix in trabecular bone specimens based on micro-computed tomography (microCT) imaging combined with pore-scale fluid dynamics simulations. Two essential steps in the proposed methodology are the selection of (i) a representative volume element (RVE) for calculation of trabecular bone permeability and (ii) a converged mesh for accurate calculation of pore fluid flow properties. Accurate estimates of trabecular bone porosities are obtained using a microCT image resolution of approximately 10 µm. We show that a trabecular bone RVE in the order of 2 × 2 × 2 mm3 is most suitable. Mesh convergence studies show that accurate fluid flow properties are obtained for a mesh size above 125,000 elements. Volume averaging of the pore-scale fluid flow properties allows calculation of the apparent permeability matrix of trabecular bone specimens. For the four specimens chosen, our numerical results show that the so obtained permeability coefficients are in excellent agreement with previously reported experimental data for both human and bovine trabecular bone samples. We also identified that bone samples taken from long bones generally exhibit a larger permeability in the longitudinal direction. The fact that all coefficients of the permeability matrix were different from zero indicates that bone samples are generally not harvested in the principal flow directions. The full permeability matrix was diagonalized by calculating the eigenvalues, while the eigenvectors showed how strongly the bone sample's orientations deviated from the principal flow directions. Porosity values of the four bone specimens range from 0.83 to 0.86, with a low standard deviation of ± 0.016, principal permeability values range from 0.22 to 1.45 ⋅ 10 -8 m2, with a high standard deviation of ± 0.33. Also, the anisotropic ratio ranged from 0.27 to 0.83, with high standard deviation. These results indicate that while the four specimens are quite similar in terms of average porosity, large variability exists with respect to permeability and specimen anisotropy. The utilized computational approach compares well with semi-analytical models based on homogenization theory. This methodology can be applied in bone tissue engineering applications for generating accurate pore morphologies of bone replacement materials and to consistently select similar bone specimens in bone bioreactor studies.

Regulation of cell growth mediated by the calcitonin receptor.

Calcitonin (CT) is a 32 amino acid peptide hormone of thyroidal origin, whose main recognised physiological role is the inhibition of osteoclast--mediated bone resorption. There is also evidence that CT might modulate bone formation. However, both CT and its receptors (CTR) have also been identified in a large number of other cell types and tissue sites, suggesting roles for the CT/CTR system distinct from those involving calcium homeostasis. Evidence has accumulated consistent with the involvement of CT in cell growth and differentiation and in tissue development and remodelling. The close proximity of cells expressing CT, or CT receptors (CTR), during development, and during pregnancy and lactation, is consistent with important roles for CT in morphogenesis. It thus appears that, in tissues such as the uterus, breast and pituitary, CT acts in a paracrine manner to Influence cell proliferation and function, as distinct from its endocrine actions to regulate calcium stores in the skeleton. In vitro studies have shown that CT can be either mitogenic or can inhibit cell proliferation, depending on the cell type and the conditions of the experiment. More recently, evidence has also been obtained for a role for CT in cell survival, in cells as diverse as osteoblast--like and osteocyte--like cells, osteoclasts and neurons.