Researchers obtained permits from the diagnostic centers to use the tumor samples

cts were diluted 1:5 in DNAse-free water prior to storage at 220uC until quantitative PCR analysis. Real-time PCR analysis using SsoAdvanced SYBR Green Supermix, and pre-designed, and pre-optimized qSTAR qPCR primer pairs was performed on a CFX96 Connect RT-PCR detection system with CFX Manager software version 3.0 and Precision melt analysis version 1.2. Primer sequences are listed in irregular but dense decoration of gold at the surface of these particles, consistent with a fetuin-A-containing proteinacious `shell’. Electron diffraction patterns of particles yielded a series of concentric rings, confirming the presence of material in a polycrystalline form. Energy-dispersive X-ray spectral analysis showed strong peaks for Ca, P and O, with a calcium-tophosphate molar ratio of 1.6760.03, consistent with calcium apatite-like material. Given the limited availability of serum-derived CPP material, and the potential for co-purification of bioactive contaminants, subsequent experiments were performed using synthetic secondary CPP, unless otherwise indicated. This also allowed us to specifically evaluate the effect of fetuin-A on crystal handling. Synthetic CPP, isolated from precipitation medium 24 h after mixing, showed near-identical morphology, size and a JW-55 chemical information similar diffraction pattern to serum-derived particles. As expected, EDX analysis showed strong peaks for Ca, P 15120495 and O, with a Ca/P of 1.6760.02. Synthetic CPP were found to remain intact and stable in culture medium for at least 5 days at 37uC, with ionic calcium and phosphate concentrations remaining unchanged over this period. Pro-inflammatory Cytokine Expression is Attenuated by Fetuin-A-containing CPP Compared to Naked Hydroxyapatite Crystals Several studies have reported that BCP nanocrystals strongly stimulate the secretion of TNF-a and IL-1b by monocyte-macrophage. First, we tested whether CPP induce a similar response in RAW 264.7 macrophages. Treatment of macrophages with CPP for 24 h resulted in a dose-dependent increase in the expression of TNF-a and IL-1b, however this effect only became significant at CPP levels above 80 mg/mL. Up-regulated cytokine expression was found to persist at 48 and 72 h post-treatment with 80 mg/mL CPP. Consistent 22286128 with the increase in gene expression, protein levels of TNF-a and IL-1b in the culture medium also increased significantly with higher CPP levels, but likewise only became significantly elevated at CPP levels above 60 mg/mL. Comparison of cells treated with either synthetic or serumderived CPP showed a parallel trend towards increased TNF-a expression with increasing CPP levels. Serum-derived CPP, however, elicited significantly lower levels of TNF-a expression over synthetic CPP at 100 mg/mL. We next sought to compare the effect of CPP and HAP of equivalent size and calcium content on cytokine production after 24 h treatment in respective test media. Critically, compared to CPP-treated cells, HAPtreated cells consistently yielded higher cytokine concentrations in the culture medium across the concentration range tested, particularly above 60 mg/mL. Statistical Analysis All experiments were performed in triplicate for each treatment and repeated in a minimum of 3 independent experiments. Data are expressed as mean 6 SD unless otherwise stated. Unpaired Student’s t tests were used to compare two groups and one-way ANOVA with Tukey post-test was performed for the comparison of three or more groups. P values,0.05 were considered significant.