Journal: Journal of Cellular Physiology

Article Title: Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

doi: 10.1002/jcp.25041

Figure Lengend Snippet: The effect of pH on mineralisation by primary rat osteoblasts and ecto‐nucleotidase mRNA expression. Osteoblasts were cultured at pH 6.9 or 7.4 for up to 14 days. A: Low power scans demonstrate a lack of alizarin red staining at pH 6.9 compared to pH 7.4. Phase contrast micrographs show the presence of collagenous matrix but the absence of alizarin red staining at pH 6.9 indicates that mineralisation has failed to occur. Scale bars: tissue culture wells = 5 mm, phase contrast micrographs = 500 μm. B: Culture at pH 6.9 results in the complete abolition of mineralised nodule formation. Values are means ± SEM (n = 6 replicate wells), *** P < 0.001. C, D: Ecto‐nucleotidase mRNA expression was investigated in differentiating (day 7) and mature, bone forming (day 14) osteoblasts at physiological pH7.4 using qRT‐PCR. Expression levels are given as relative to TNAP ( Alpl ) expression. The rank order of mRNA expression in differentiating osteoblasts was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > Alpl > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2 . In mature, bone forming osteoblasts the rank order of expression was NTPdase 6 > Enpp1 > NTPdase 4 > Alpl > NTPdase 5 > Enpp3 > NTPdase 3 > Enpp2 > NTPdase 1 > NTPdase 2 . E: RT‐PCR analysis of mRNA at day 7, 10 and 14 showed that compared to pH 7.4 culture at pH 6.9 upregulated the expression of Enpp1 mRNA in osteoblasts, at all stages of differentiation. In contrast, Alpl , Enpp2 , Enpp3 and NTPdase 2 mRNA expression was decreased in acid. mRNA expression of NTPdase1, 3‐6 and Ank was unchanged. F: qRT‐PCR analysis of osteoblasts at day 10 (the stage when the largest effects were seen with RT‐PCR) showed that culture under acid conditions resulted in a 2.8‐fold increase in Enpp1 mRNA expression; expression of Alpl and Enpp3 was decreased 3.2‐ and 2.5‐fold, respectively. The mRNA expression of the other ecto‐nucleotidases was not significantly altered. All qPCR reactions were carried out in triplicate using RNAs derived from four different osteoblast cultures. Values are means ± SEM, * P < 0.05.

Article Snippet: Primary rat osteoblast cells were obtained from 2‐day‐old neonatal Sprague–Dawley rats as described previously (Orriss et al., ; Taylor et al., ).

Techniques: Expressing, Cell Culture, Staining, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Journal of Cellular Physiology

Article Title: Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

doi: 10.1002/jcp.25041

Figure Lengend Snippet: Acidosis increases NPP1 protein levels and total NPP activity in osteoblasts. A: Western blot analysis demonstrated increased NPP1 protein expression by osteoblasts grown in acid conditions. B: Immunocytochemistry demonstrated increased NPP1 expression (green) by osteoblasts cultured at pH 6.9 for 7 days compared to those cultured at pH 7.4. DAPI staining of cell nuclei is blue. Scale bar = 50 μm. C: The NPP activity was examined in osteoblast whole cell lysates after 7, 10 or 14 days of culture at pH 7.4 or 6.9. Total cellular NPP activity was doubled after 7 days at pH 6.9 and increased by ∼45% after 10 days. No differences in NPP activity were observed after 14 days in culture. Values are means ± SEM (n = 6 replicate wells), *** P < 0.001, * P < 0.05.

Article Snippet: Primary rat osteoblast cells were obtained from 2‐day‐old neonatal Sprague–Dawley rats as described previously (Orriss et al., ; Taylor et al., ).

Techniques: Activity Assay, Western Blot, Expressing, Immunocytochemistry, Cell Culture, Staining

Journal: Journal of Cellular Physiology

Article Title: Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

doi: 10.1002/jcp.25041

Figure Lengend Snippet: Osteoblasts from Enpp1 −/− mice display resistance to the inhibitory effects of acidosis on bone mineralisation. Osteoblasts were isolated from Enpp1 +/+ or Enpp1 −/− mice and cultured for 28 days at pH 7.4 or 6.9. A: Representative phase contrast micrographs showing alizarin red stained osteoblast cell layers from Enpp1 +/+ and Enpp1 −/− mice at pH 7.4 and pH 6.9. Enpp1 −/− osteoblasts display increased mineralisation at pH 7.3 and resistance to the inhibitory effects of acidosis on bone mineralisation. Scale bar = 500 μm. B: Bone mineralisation is increased 2.5‐fold in Enpp1 −/− osteoblasts at pH7.4, compared to Enpp1 +/+ . Culture at pH 6.9 abolishes bone mineralisation in Enpp1 +/+ osteoblasts, whilst in Enpp1 −/− osteoblasts mineralisation was only reduced by 75%. Values are means ± SEM (n = 6 replicate wells), *** P < 0.001.

Article Snippet: Primary rat osteoblast cells were obtained from 2‐day‐old neonatal Sprague–Dawley rats as described previously (Orriss et al., ; Taylor et al., ).

Techniques: Isolation, Cell Culture, Staining

Journal: Journal of Cellular Physiology

Article Title: Acidosis Is a key regulator of osteoblast ecto‐nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity

doi: 10.1002/jcp.25041

Figure Lengend Snippet: Acidosis does not affect ATP release from osteoblasts. Osteoblasts were cultured for up to 14 days at pH 7.4 or 6.9 and ATP release measured at 4, 7, 10 and 14 days. A: Basal ATP release increased with differentiation being eightfold higher in mature, bone forming osteoblasts relative to pre‐osteoblasts. Acidosis did not influence ATP release at any stage of culture. Values are means ± SEM (n = 8–10 replicate wells), *** P < 0.001. B: Osteoblasts cultured for 10 days at pH 7.4 or 6.9 showed similar levels of quinacrine staining. Scale bar = 50 μm.

Article Snippet: Primary rat osteoblast cells were obtained from 2‐day‐old neonatal Sprague–Dawley rats as described previously (Orriss et al., ; Taylor et al., ).

Techniques: Cell Culture, Staining

Journal: PLoS ONE

Article Title: Effect of Change in Spindle Structure on Proliferation Inhibition of Osteosarcoma Cells and Osteoblast under Simulated Microgravity during Incubation in Rotating Bioreactor

doi: 10.1371/journal.pone.0076710

Figure Lengend Snippet: Cell-cycle histograms of MG-63(A), U-2 OS (B) cells and rat calvaria primary osteoblast(C) under simulated microgravity for 72 hours. The cycle changes of MG-63(D), U-2 OS(E), hFOB1.19(F) cells and rat calvaria primary osteoblast(G) under simulated microgravity. For MG-63 and U-2 OS the cell number in G1 phase decreases and increases in S and G2/M phase under simulated microgravity. For hFOB1.19 cells, increase in S phase under simulated microgravity. Rat calvaria primary osteoblasts increases only in G2 phases under simulated microgravity for 72 hours. The data shown represent the mean± S.E. (n = 3). *0.01< P <0.05, and ** P <0.01 ( t -test as compared to untreated control group).

Article Snippet: And primary osteoblasts derived from fetal Sprague-Dawley rat calvaria are cultured in DMEM (Gibco-BRL) with 10% (v/v) FBS (Gibco-BRL) .

Techniques: Control

Journal: PLoS ONE

Article Title: Effect of Change in Spindle Structure on Proliferation Inhibition of Osteosarcoma Cells and Osteoblast under Simulated Microgravity during Incubation in Rotating Bioreactor

doi: 10.1371/journal.pone.0076710

Figure Lengend Snippet: In hFOB1.19 cells (A and B), early apoptosis and late death mean extremely significant differences between untreated control and simulated microgravity for 24–72 hours. In rat calvaria primary osteoblasts (C and D), early apoptosis and late death mean significant differences between untreated control and simulated microgravity for 72 hours.The data shown represent the mean± S.E. (n = 3). *0.01< P <0.05, and ** P <0.01 ( t -test as compared to untreated control group).

Article Snippet: And primary osteoblasts derived from fetal Sprague-Dawley rat calvaria are cultured in DMEM (Gibco-BRL) with 10% (v/v) FBS (Gibco-BRL) .

Techniques: Control

Journal: PLoS ONE

Article Title: Effect of Change in Spindle Structure on Proliferation Inhibition of Osteosarcoma Cells and Osteoblast under Simulated Microgravity during Incubation in Rotating Bioreactor

doi: 10.1371/journal.pone.0076710

Figure Lengend Snippet: The spindles are stained green by anti-α-tubulin first antibody and FITC-conjugated goat anti-mouse IgG. A and B. Bipolar spindle of MG-63(A) cell and rat calvaria primary osteoblast (B) under 1 g condition. C–E. Multipolar spindle under simulated microgravity in MG-63 cell (C. three polar spindles, D. four polar spindles) and hFOB1.19 cell (E. five polar spindles). F–H. Microtubule arrangement change under simulated microgravity (F. MG-63, G. U-2 OS, H. rat calvaria primary osteoblast).

Article Snippet: And primary osteoblasts derived from fetal Sprague-Dawley rat calvaria are cultured in DMEM (Gibco-BRL) with 10% (v/v) FBS (Gibco-BRL) .

Techniques: Staining

Journal: PLoS ONE

Article Title: Effect of Change in Spindle Structure on Proliferation Inhibition of Osteosarcoma Cells and Osteoblast under Simulated Microgravity during Incubation in Rotating Bioreactor

doi: 10.1371/journal.pone.0076710

Figure Lengend Snippet: The results demonstrate significant increase in multipolar spindle incidence along with simulated microgravity duration, and exhibit significant difference resulting from being cultured for 72 hours(MG-63, A), 48 hours(U-2 OS(B) and hFOB1.19(C)), or even 24 hours(rat calvaria primary osteoblast, D). The data shown represent the mean±S.E. (n = 3). *0.01< P <0.05, ** P <0.01, and *** P <0.001 ( -test as compared to rotation control group).

Article Snippet: And primary osteoblasts derived from fetal Sprague-Dawley rat calvaria are cultured in DMEM (Gibco-BRL) with 10% (v/v) FBS (Gibco-BRL) .

Techniques: Cell Culture, Control