Alternative radiopacifiers for polymethyl methacrylate bone cements: Silane-treated anatase titanium dioxide and yttria-stabilised zirconium dioxide.

Poly (methyl methacrylate) (PMMA) bone cement is widely used for anchoring joint arthroplasties. In cement brands approved for these procedures, micron-sized particles (usually barium sulphate, BaSO4) act as the radiopacifier. It has been postulated that these particles act as sites for crack initiation and subsequently cement fatigue. This study investigated whether alternative radiopacifiers, anatase titanium dioxide (TiO2) and yttria-stabilised zirconium dioxide (ZrO2), could improve the in vitro mechanical, fatigue crack propagation and biological properties of polymethyl methacrylate (PMMA) bone cement and whether their coating with a silane could further enhance cement performance. Cement samples containing 0, 5, 10, 15, 20 and 25%w/w TiO2 or ZrO2 and 10%w/w silane-treated TiO2 or ZrO2 were prepared and characterised in vitro in terms of radiopacity, compressive and bending strength, bending modulus, fatigue crack propagation, hydroxyapatite forming ability and MC3T3-E1 cell attachment and viability. Cement samples with greater than 10%w/w TiO2 and ZrO2 had a similar radiopacity to the control 10%w/w BaSO4 cement and commercial products. The addition of TiO2 and ZrO2 to bone cement reduced the bending strength and fracture toughness and increased fatigue crack propagation due to the formation of agglomerations and voids. Silane treating TiO2 reversed this effect, enhancing the dispersion and adhesion of particles to the PMMA matrix and resulted in improved mechanical properties and fatigue crack propagation resistance. Silane-treated TiO2 cements had increased nucleation of hydroxyapatite and MC3T3-E1 cell attachment in vitro, without significantly compromising cell viability. This research has demonstrated that 10%w/w silane-treated anatase TiO2 is a promising alternative radiopacifier for PMMA bone cement offering additional benefits over conventional BaSO4 radiopacifiers.

A large-conductance (BK) potassium channel subtype affects both growth and mineralization of human osteoblasts.

The pharmacology of the large-conductance K(+) (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM K(+)/140 mM K(+)) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM K(+)/140 mM K(+). The selectivity ratio (ratio of K(+) to Na(+) permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 microM), and paxilline (10 microM) and activated by isopimaric acid (20 microM). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 microM, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 microM, respectively). Neither iberiotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimaric acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function.

Osteopenia in Sparc (osteonectin)-deficient mice: characterization of phenotypic determinants of femoral strength and changes in gene expression.

Sparc null mutants have been generated independently via targeted mutations in exons 4 and 6. Previous studies have identified low-turnover osteopenia in the 129Sv/C57BL/6 exon 4 knockout. Since both Sparc null mutations result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background (strain) and/or linkage disequilibrium effects can influence phenotype. Different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the 129Sv/EvSparc(tm1cam) exon 6 knockout at 4 and 9 mo, using physical measurement, mechanical strength tests, and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue metabolism. 129Sv/EvSparc(tm1cam) null mice show decreased bone mineral density and bone mineral content and increased mechanical fragility of bone, in line with previous studies. Differences were also noted. Increased body weight and levels of bone marrow adiposity but decreased circulating leptin concentrations were identified at 4, but not 9 mo, and 129Sv/EvSparc(tm1cam) null mice also had shorter femurs. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semiquantitative RT-PCR validation. We identified 429 genes highly expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4, two ESTs) are differentially regulated in 129Sv/EvSparc(tm1cam) cortical femur vs. 129Sv/Ev controls. We confirm low-turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis.

Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin.

UNLABELLED: We showed that human osteoprogenitor cells produced adenosine and expressed ecto-5'-nucleotidase and all four adenosine receptor subtypes. Adenosine stimulated IL-6 but inhibited osteoprotegerin secretion, suggesting that adenosine is a newly described regulator of progenitor cell function. INTRODUCTION: Maintaining skeletal homeostasis relies on there being a balance between bone formation and resorption; an imbalance between these processes can lead to diseases such as osteoporosis and rheumatoid arthritis. Recent reports showed that locally produced ATP, acting through P2 receptors, has pronounced effects on bone formation. However, ATP can be enzymatically cleaved to adenosine that has little or no activity at P2 receptors but mediates its action through the P1 family of receptors. We studied whether adenosine may also have an important role in controlling bone cell differentiation and function. MATERIALS AND METHODS: Extracellular adenosine levels were analyzed by high-performance liquid chromatography in HCC1 and bone marrow stromal (BMS) cells. Ecto-5'-nucleotidase (CD73) expression and activity was determined by RT-PCR, immunocytochemistry, and the cleavage of etheno-AMP to ethenoadenosine. Adenosine receptor expression and activity were determined by RT-PCR and cAMP measurements. The effects of adenosine receptor agonists on IL-6, osteoprotegerin (OPG), and RANKL expression were determined by ELISA and QRT-PCR. RESULTS: HCC1 and BMS cells produce adenosine and express CD73 and all four adenosine receptor subtypes. The A2b receptor was shown to be functionally dominant in HCC1 cells, as determined by cAMP production and in its stimulation of IL-6 secretion. Adenosine receptor agonism also inhibited OPG secretion and OPG but not RANKL mRNA expression. CONCLUSIONS: Our findings show that HCC1 and primary BMS cells produce adenosine, express CD73 and all four adenosine receptor subtypes. In HCC1 cells, adenosine has a potent stimulatory action on IL-6 secretion but an inhibitory action on OPG expression. These data show for the first time that adenosine may be an important regulator of progenitor cell differentiation and hence an important local contributor to the regulation of bone formation and resorption.

Novel tetralone-derived retinoic acid metabolism blocking agents: synthesis and in vitro evaluation with liver microsomal and MCF-7 CYP26A1 cell assays.

The potent inhibitory activity of novel 2-benzyltetralone and 2-benzylidenetetralone derivatives vs liver microsomal retinoic acid metabolizing enzymes and a MCF-7 CYP26A1 cell assay is described. In the liver microsomal assay, the 2-biphenylmethyl-6-hydroxytetralone derivatives 16a and 16b were found to be potent inhibitors (IC50 = 0.5 and 0.8 microM) compared with the broad spectrum P450 inhibitor ketoconazole and the retinoid mimetic R115866 (IC50 = 18.0 and 9.0 microM, respectively). In the MCF-7 CYP26A1 cell assay, the 2-(4-hydroxybenzyl)-6-methoxytetralone 5 and unsaturated benzylidene precursor 6 were found to be the most potent (IC50 = 7 and 5 microM, respectively), which was comparable with liarozole (7 microM) but considerably less active than R115866 (IC50 = 5 nM). With a CYP26A1 homology model, the tetralones were shown to be positioned in a hydrophobic tunnel with additional interactions, e.g., transition metal coordination and hydrogen-bonding interactions with GLY300, observed for the potent 4-hydroxyphenyl substituted inhibitors.

The Death Receptor 3-TNF-like protein 1A pathway drives adverse bone pathology in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints that is associated with cartilage and bone destruction. Death Receptor 3 (DR3), a tumor necrosis factor (TNF) receptor superfamily member, has recently been associated with the pathogenesis of RA. We demonstrate that absence of DR3 confers resistance to the development of adverse bone pathology in experimental antigen-induced arthritis (AIA). DR3(ko) mice exhibited a reduction in all histopathological hallmarks of AIA but, in particular, failed to develop subchondral bone erosions and were completely protected from this characteristic of AIA. In contrast, TNF-like protein 1A (TL1A), the ligand for DR3, exacerbated disease in a dose- and DR3-dependent fashion. Analysis of osteoclast number within AIA joint revealed a reduction in areas susceptible to bone erosion in DR3(ko) mice, whereas in vitro osteoclastogenesis assays showed that TL1A could directly promote osteoclastogenesis in mouse and man. Treatment with antagonistic anti-TL1A mAb protected animals in a systemic model of RA disease collagen-induced arthritis. We therefore conclude that the DR3-TL1A pathway regulates joint destruction in two murine models of arthritis and represents a potential novel target for therapeutic intervention in inflammatory joint disease.

Adiposity profile in the dwarf rat: an unusually lean model of profound growth hormone deficiency.

This study describes the previously uncharacterized ontogeny and regulation of truncal adipose reserves in the profoundly GH-deficient dwarf (dw/dw) rat. We show that, despite normal proportionate food intake, dw/dw rats develop abdominal leanness and hypoleptinemia (circulating leptin halved in dw/dw males, P < 0.05) during puberty. This contrasts with the hyperleptinemia seen in moderately GH-deficient Tgr rats (circulating leptin doubled at 6 wk of age, P < 0.05) and in GH receptor-binding protein (GHR/BP)-null mice (circulating leptin doubled; P < 0.05). This lean/hypoleptinemic phenotype was not completely normalized by GH treatment, but dw/dw rats developed abdominal obesity in response to neonatal MSG treatment or maintenance on a high-fat diet. Unlike Tgr rats, dw/dw rats did not become obese with age; plasma leptin levels and fat pad weights became similar to those in wild-type rats. In contrast with truncal leanness, tibial marrow adiposity was normal in male and doubled in female dwarves (P < 0.01), this increase being attributable to increased adipocyte number (P < 0.01). Neonatal MSG treatment and high-fat feeding elevated marrow adiposity in dw/dw rats by inducing adipocyte enlargement (P < 0.05). These results demonstrate that, despite lipolytic influence of GH, severe GH deficiency in dw/dw rats is accompanied by a paradoxical leanness. This lean/hypoleptinemic phenotype is not solely attributable to reduced GH signaling and does not appear to result from a reduction in nutrient intake or the ability of dw/dw adipocytes to accumulate lipid. Disruption of preadipocyte differentiation or adipocyte proliferation in the dw/dw rat may lead to the development of this unusually lean/hypoleptinemic phenotype.