The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca2+ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.

A chondrocyte and its surrounding pericellular matrix (PCM) are defined as a chondron. Single chondrocytes and chondrons isolated from bovine articular cartilage were compressed by micromanipulation between two parallel surfaces in order to investigate their biomechanical properties and to discover the mechanical significance of the PCM. The force imposed on the cells was measured directly during compression to various deformations and then holding. When the nominal strain at the end of compression was 50 per cent, force relaxation showed that the cells were viscoelastic, but this viscoelasticity was generally insignificant when the nominal strain was 30 per cent or lower. The viscoelastic behaviour might be due to the mechanical response of the cell cytoskeleton and/or nucleus at higher deformations. A finite-element analysis was applied to simulate the experimental force-displacement/time data and to obtain mechanical property parameters of the chondrocytes and chondrons. Because of the large strains in the cells, a nonlinear elastic model was used for simulations of compression to 30 per cent nominal strain and a nonlinear viscoelastic model for 50 per cent. The elastic model yielded a Young's modulus of 14 + 1 kPa (mean + s.e.) for chondrocytes and 19 + 2 kPa for chondrons, respectively. The viscoelastic model generated an instantaneous elastic modulus of 21 + 3 and 27+ 4 kPa, a long-term modulus of 9.3 + 0.8 and 12 + 1 kPa and an apparent viscosity of 2.8 + 0.5 and 3.4 + 0.6 kPa s for chondrocytes and chondrons, respectively. It was concluded that chondrons were generally stiffer and showed less viscoelastic behaviour than chondrocytes, and that the PCM significantly influenced the mechanical properties of the cells.

BackgroundTranscatheter closure of coronary artery fistulae (CAF) has emerged as an alternative to surgery, but there are no long‐term outcome results.ObjectiveWe report immediate and long‐term results in 24 patients after transcatheter closure of congen‐ ital CAF.MethodsA total of 24 patients aged 5 to 56 years old with congenital CAF underwent attempted percutaneous transcatheter closure using various devices between November 1998 and August 2008. The immediate closure results and clinical follow‐up were reviewed.ResultsOf the 24 patients, 20 (83.3%) underwent successful transcatheter closure using various devices. An angiogram right after device deployment revealed complete occlusion in 15 patients (75%) and trivial‐ to mild‐ residual flow in 5 patients (25%). Four patients (20%) had transient ST‐T wave changes after the procedure. The left ventricular end‐diastolic volume decreased from 165±31.4 mm3 to 128.6±24.4 mm3 (P = 0.012) 24 hours after procedure, and the cardiothoracic ratio from 0.57±0.02 to 0.53±0.01 (P = 0.003). Follow‐up was 100% complete and ranged from 3 months to 10 years. There were no early or late deaths. All patients were asymptomatic with complete closure of CAF except 1 patient (5%) who had a recurrence of shunt at 6‐month follow‐up, which was re‐closed by percutaneous technique.ConclusionTranscatheter closure of CAF is feasible and safe in anatomically suitable vessels and is a promising alternative to surgery in most patients. Copyright © 2009 Wiley Periodicals, Inc.

Poly­(vinylphosphonic acid-co-acrylic acid) (PVPA-co-AA) has recently been identified as a possible candidate for use in bone tissue engineering. It is hypothesized that the strong binding of PVPA-co-AA to calcium in natural bone inhibits osteoclast activity. The free radical polymerization of acrylic acid (AA) with vinylphosphonic acid (VPA) has been investigated with varying experimental conditions. A range of copolymers were successfully produced and their compositions were determined quantitatively using 31P NMR spectroscopy. Monomer conversions were calculated using 1H NMR spectroscopy and a general decrease was found with increasing VPA content. Titration studies demonstrated an increase in the degree of dissociation as a function of VPA in the copolymer. However, a VPA content ca. 30 mol % was found to be the optimum for calcium chelation, suggesting that this composition is the most promising for biomaterials applications. Assessment of cell metabolic activity showed that PVPA-co-AA has no detrimental effect on cells, regardless of copolymer composition.

This is the first study to quantitate and profile the glycosaminoglycan (GAG) composition of the pericellular matrix (PCM) of chondrons and chondrocytes using the highly sensitive technique; fluorophore-assisted carbohydrate electrophoresis (FACE). Bovine articular chondrocytes and chondrons were isolated enzymatically. High cell yield and viability were obtained for both preparations. Chondrons had strong immunofluorescent labeling for keratan sulphate and chondroitin-6 sulphate but no labeling for hyaluronan. We compared the immunofluorescent data with FACE. The quantities of total keratan sulphate were determined to be 0.013 ± 0.002 pg cell -1 and 0.032 ± 0.003 pg cell -1 in the chondrocyte and chondron preparations, respectively. Four internal keratan sulphate sugars were detected (gal β 1 , 4 glcNAc6S, gal6S β 1 , 4 glcNAc6S, glcNAc β 1 , 3 gal and glcNAc6S β 1 , 3 gal) for both preparations but they were present at significantly higher concentrations in chondron preparations ( P < 0.01). Total chondroitin sulphate (CS) was determined to be 0.054 ± 0.004 pg cell -1 and 0.077 ± 0.005 pg cell -1 for chondrocyte and chondron preparations, respectively. Unsulphated CS disaccharide levels were similar but chondrons had significantly more chondroitin-4 sulphated disaccharides and chondroitin-6 sulphated disaccharides ( P < 0.05). Hyaluronan acid was present at low concentrations (0.010 ± 0.001 pg cell -1 ) in both chondrocytes and chondrons. In this study, enzyme digestion coupled with FACE separation revealed new information about the differences in GAGs from isolated chondrocyte and chondron preparations. Further investigation of the differences in GAGs from chondrocytes and chondrons from different zones of articular cartilage may be useful for tissue engineering approaches.