Chondro-protective effects of low intensity pulsed ultrasound.

OBJECTIVES: Cartilage is a highly mechano-responsive tissue. Chondrocytes undergo a series of complex changes, including proliferation and metabolic alteration as the target of external biomechanical and biochemical stimuli. IL-1ß is known to regulate chondrocyte metabolism and plays an important role in the pathogenesis of osteoarthritis (OA). The objective of this study was to employ low-intensity pulsed ultrasound (LIPUS) as a localized mechanical stimulus and assess its effects on chondrocyte migration, proliferation, metabolism, and differentiation, as well as its ability to suppress IL-1ß mediated catabolism in cartilage. METHODS: Human cartilage explants and chondrocytes were stimulated by LIPUS in the presence and absence of IL-1ß to asses cartilage degradation, chondrocytes metabolism, migration, and proliferation. Western blot analyses were conducted to study IL-1ß the associated NFκB pathway in chondrocytes. RESULTS: LIPUS stimulation increased the proteoglycan content in human cartilage explants and inhibited IL-1ß induced loss of proteoglycans. LIPUS stimulation increased rates of chondrocyte migration and proliferation, and promoted chondrogenesis in mesenchymal stem cells (MSC). Further, LIPUS suppressed IL-1ß induced activation of phosphorylation of NFκB-p65 and IĸBα leading to reduced expression of MMP13 and ADAMT5 in chondrocytes. CONCLUSIONS: Collectively, these data demonstrate the potential therapeutic effects of LIPUS in preventing cartilage degradation and treating OA via a mechanical stimulation that inhibits the catabolic action of IL-1ß and stimulates chondrocyte migration, proliferation, and differentiation.

Simultaneous determination of delta(15)N and delta(18)O of N2O and delta(13)C of CH4 in nanomolar quantities from a single water sample.

We have developed a rapid, sensitive, and automated analytical system to simultaneously determine the concentrations and stable isotopic compositions (delta(15)N, delta(18)O, and delta(13)C) of nanomolar quantities of nitrous oxide (N(2)O) and methane (CH(4)) in water, by combining continuous-flow isotope-ratio mass spectrometry and a helium-sparging system to extract and purify the dissolved gases. Our system, which is composed of cold traps and a capillary gas chromatograph that use ultra-pure helium as the carrier gas, achieves complete extraction of N(2)O and CH(4) in a water sample and separation among N(2)O, CH(4), and the other component gases. The flow path following exit from the gas chromatograph was periodically changed to pass the gases through the combustion furnace to convert CH(4) and the other hydrocarbons into CO(2), or to bypass the combustion furnace for the direct introduction of eluted N(2)O into the mass spectrometer, for determining the stable isotopic compositions through monitoring the ions of m/z 44, 45, and 46 of CO(2) (+) and N(2)O(+). The analytical system can be operated automatically with sequential software programmed on a personal computer. Analytical precisions better than 0.2 per thousand and 0.3 per thousand and better than 1.4 per thousand and 2.6 per thousand were obtained for the delta(15)N and delta(18)O of N(2)O, respectively, when more than 6.7 nmol and 0.2 nmol of N(2)O, respectively, were injected. Simultaneously, analytical precisions better than 0.07 per thousand and 2.1 per thousand were obtained for the delta(13)C of CH(4) when more than 5.5 nmol and 0.02 nmol of CH(4), respectively, were injected. In this manner, we can simultaneously determine stable isotopic compositions of a 120 mL water sample with concentrations as low as 1.7 nmol/kg for N(2)O and 0.2 nmol/kg for CH(4).

NADPH oxidase 1 plays a critical mediating role in oncogenic Ras-induced vascular endothelial growth factor expression.

Reactive oxygen species (ROS)-generating enzyme Nox1 is important in the induction of oncogenic Ras transformation phenotypes, but it is not defined whether Nox1 is involved in Ras-induced upregulation of vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. Here we describe that ablation of the Nox1 activity by Nox1 small-interference RNAs (siRNAs) or diphenylene iodonium (DPI) inhibited synthesis of both VEGF proteins and VEGF mRNAs in K-Ras transformed normal rat kidney (KNRK) cells. Nox1siRNAs and DPI suppressed extracellular signal-regulated kinase (ERK)-dependent phosphorylation of a transcription factor Sp1 and Sp1 binding to a VEGF promoter. Furthermore, tumors derived from Nox1siRNA-transfected KNRK cells markedly decreased neovascularization. The Nox1 activity was required for VEGF production in human colon cancer CaCO-2 cells, as in the case of KNRK cells. However, since overexpression of Nox1 in normal rat kidney cells failed to induce VEGF, the Nox1 activity alone was not sufficient to upregulate VEGF expression, which suggests that unlike the previously proposed model, Nox1 may act in concert with other effectors integrated into the Ras network. We propose that Nox1 mediates oncogenic Ras-induced upregulation of VEGF and angiogenesis by activating Sp1 through Ras-ERK-dependent phosphorylation of Sp1.

Dependence of the photoelectrochemical performance of sensitised ZnO on the crystalline orientation in electrodeposited ZnO thin films.

The influence of the crystal orientation in porous crystalline films of ZnO electrodeposited on the photoelectrochemical characteristics of the films is studied. For differently oriented ZnO thin films following removal of the respective structure-directing agent (SDA) and adsorption of a sensitiser, time-resolved photocurrent measurements, intensity modulated photocurrent spectroscopy (IMPS), intensity modulated photovoltage spectroscopy (IMVS) and current-voltage curves were measured in acetonitrile-based electrolytes containing I(3)(-)/I(-) as the redox electrolyte. The crystal orientation has a significant influence on the charge transport across such films and hence is reflected in the observed electrode kinetics. Films originally grown in the presence of, e.g., Coumarin 343 as a SDA, showed a significantly faster response to illumination. Increased electron diffusion coefficients and diffusion lengths were calculated from the results of IMPS and IMVS, caused by a faster electron movement in the films. Implications of these findings on further improvements of sensitised ZnO films prepared by electrochemical deposition are discussed.

Activation of the transcription factor HIF-1 and its target genes, VEGF, HO-1, iNOS, during fracture repair.

One of the immediate sequelae of bone fracture is regional hypoxia resulting from vasculature disruption. Hypoxia stabilizes and activates the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), which ultimately leads to HIF-1-regulated gene expression. Because nothing is known about HIF-1 involvement in bone regeneration, we performed a series of experiments to elucidate the expression pattern of HIF-1alpha and selected HIF-1 target genes using a rat femoral fracture model. Callus samples were obtained on postfracture days (PFD) 3, 5, 7, 10, 14, and 21. Quantitative RT-PCR (qRT-PCR) was employed to quantify the temporal mRNA expression patterns of HIF-1alpha, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1). Elevated HIF-1alpha and VEGF expression was seen at all time points, with peak increases of approximately 6- and 2-fold relative to the intact bone present on PFD 10 for HIF-1alpha and VEGF, respectively. Robust activation of iNOS was detected solely on PFD 10 (6.8-fold) with all other time points showing slight downregulation. HO-1 expression peaked on PFD 3 (4.5-fold) with no significant changes on any other PFD. Western blot analysis verified the temporal expression patterns with HIF-1alpha protein expression showing a steady rise to a PFD 10 peak of approximately 18-fold. Similarly, the expression patterns for VEGF and HO-1 showed increases of approximately 4-fold at their PFD 10 and PFD 3 peaks, respectively. Immunohistochemical analysis of PFD 10 callus sections revealed coexpression of HIF-1alpha and VEGF in proliferating chondrocytes and active osteoblasts. Immunostaining for HO-1 on PFD 3 callus sections demonstrated strong expression in hematoma macrophages and vascular endothelial cells. Taken together, these experiments demonstrate for the first time that HIF-1alpha is upregulated at both transcriptional and translational levels in the fracture callus and indicate that PFD 10 may be a key angiogenic time point in the developing rat fracture callus.