Expression and localization of TASK-1, -2 and -3 channels in MG63 human osteoblast-like cells.

It is well known that a number of ion channels are involved in the proliferation, migration and invasion of tumor cells. TASK channels, an acid-sensitive subgroup of the two-pore-domain K(+) channel (K2P) family, are expressed in numerous types of tissue and exhibit various physiological functions depending on the cell type. In the present study, we employed RT-PCR and western blot analysis to determine the expression of TASK-1, -2 and -3 at the mRNA and protein levels in MG63 human osteoblast-like cells. Immunofluorescence with specific antibodies against the TASK channels revealed the localization patterns at the plasma membrane and the juxtanuclear compartment. The induced fluctuations in the extracellular pH from 7.4 to 6.9 and to 6.4 significantly reduced the proliferation rate of MG63 cells by 44.3 and 90.1%, respectively. These data revealed the expression of TASK-1, -2 and -3, and the correlation between TASK channels and cell proliferation in MG63 cells, suggesting that these channels may be involved in the tumorigenesis of osteosarcoma.

17ß-Estradiol inhibits outward voltage-gated K⁺ currents in human osteoblast-like MG63 cells.

Previous studies have shown that 17ß-estradiol has a pivotal function by blocking voltage-gated K⁺ (Kv) channels in several different types of cells such as cardiac myocytes and neurons. Outward Kv currents can also be measured in osteoblasts, although little is known about the effects of 17ß-estradiol on these currents. In human osteoblast-like MG63 cells, we found that 17ß-estradiol inhibits peak and end Kv currents, with IC50 values of 480 and 325 nM, respectively. To elucidate the mechanism of inhibition, the kinetics of Kv currents were investigated. The half-maximum activation potential (V(½)) was 1.3 mV and was shifted left to -4.4 mV after application of 500 nM 17ß-estradiol. For steady-state inactivation, the V(½) was -55.0 mV and weakly shifted left to -58.2 mV. To identify the molecular basis of outward Kv currents in MG63 cells, we performed RT-PCR analyses. The expression of Kv2.1 channels appeared to dominate over that of other Kv channels in MG63 cells. In COS-7 cells with heterologously expressed Kv2.1 channels, 17ß-estradiol also inhibits macroscopic currents of Kv2.1. Our data indicate that 17ß-estradiol inhibits Kv currents in human osteoblast-like MG63 cells and that Kv2.1 is a potential molecular correlate of outward Kv currents in these cells.

Synthesis and characterization of UPPE-PLGA-rhBMP2 scaffolds for bone regeneration.

A novel unsaturated polyphosphoester (UPPE) was devised in our previous research, which is a kind of promising scaffold for improving bone regeneration. However, the polymerization process of UPPE scaffolds was unfavorable, which may adversely affect the bioactivity of osteoinductive molecules added if necessary, such as recombinant human bone morphogenetic protein-2 (rhBMP2). The purpose of this study was to build a kind of optimal scaffold named UPPE-PLGA-rhBMP2 (UPB) and to investigate the bioactivity of rhBMP2 in this scaffold. Furthermore, the cytotoxicity and biocompatibility of UPB scaffold was assessed in vitro. A W1/O/W2 method was used to fabricate PLGA-rhBMP2 microspheres, and then the microspheres were added to UPPE for synthesizing UPB scaffold. The morphological characters of PLGA-rhBMP2 microspheres and UPB scaffolds were observed under the scanning electron microscopy and laser scanning confocal microscopy. The cumulative release of UPB scaffolds was detected by using ELISA. The cytotoxicity and biocompatibility of UPB scaffolds were evaluated through examining the adsorption and apoptosis of bone marrow stromal cells (bMSCs) seeded on the surface of UPB scaffolds. The bioactivity of rhBMP2 in UPB scaffolds was assessed through measuring the alkaline phosphates (ALP) activity in bMSCs seeded. The results showed that UPB scaffolds sequentially exhibited burst and sustained release of rhBMP2. The cytotoxicity was greatly reduced when the scaffolds were immersed in buffer solution for 2 h. bMSCs attached and grew on the surface of soaked UPB scaffolds, exerting well biocompatibility. The ALP activity of bMSCs seeded was significantly enhanced, indicating that the bioactivity of rhBMP2 remained and still took effect after the unfavorable polymerization process of scaffolds. It was concluded that UPB scaffolds have low cytotoxicity, good biocompatibility and preserve bioactivity of rhBMP2. UPB scaffolds are promising in improving bone regeneration.