Journal: Archives of Toxicology

Article Title: Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models

doi: 10.1007/s00204-020-02906-z

Figure Lengend Snippet: Limitations of using mitochondrial activity for normalization. Mitochondrial activity was assessed by Resazurin conversion assay. a Mitochondrial activity varies between different osteogenic cell lines. Exemplary, 5 * 10 4 cells of the SCP-1, MG-63, Cal-72, and SaOS-2 cell line were seeded, and after 24 h, mitochondrial activity was determined. b Mitochondrial activity was measured in THP-1 and SaOS-2 cell cultures both in mono-culture and direct co-culture. c Surface stiffness affects mitochondrial activity. 5 * 10 4 cells of the same cell line were seeded on surfaces with different stiffness, and after 24 h, mitochondrial activities were measured. Experiments were repeated three times (N = 3) in triplicates (n = 3). Comparison of groups was performed by Kruskal–Wallis test followed Dunn’s multiple comparison test

Article Snippet: SaOS-2 cell line was transfected to over-express a red fluorescent protein (RFP: Addgene plasmid #54642 tdTomato-N1) or a green-fluorescent protein (GFP: Addgene plasmid #54737 sfGFP_N1). a RFP-overexpressing SaOS-2 cells (red) were directly co-cultured with THP-1 cells in a ratio of 1:2.

Techniques: Activity Assay, Co-Culture Assay

Journal: Archives of Toxicology

Article Title: Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models

doi: 10.1007/s00204-020-02906-z

Figure Lengend Snippet: Using fluorescent labels for normalization. SaOS-2 cell line was transfected to over-express a red fluorescent protein (RFP: Addgene plasmid #54642 tdTomato-N1) or a green-fluorescent protein (GFP: Addgene plasmid #54737 sfGFP_N1). a RFP-overexpressing SaOS-2 cells (red) were directly co-cultured with THP-1 cells in a ratio of 1:2. After 4 days of conventional 2D culture viable cells were visualized with Calcein-AM (green). Nuclei were counterstained with Hoechst 33342 (blue). b Schematic overview on the respective 3D culture. c Mitochondrial activity of the un-/labelled SaOS-2 cells on day 4 of culture. d ALP activity of the un-/labelled SaOS-2 cells on day 4 of culture. e Mineralized matrix formed by the un-/labelled SaOS-2 cells after 10 days of culture. Experiments were repeated three times (N = 3) in triplicates (n = 3). Comparison of groups was performed by Kruskal–Wallis test followed by Dunn’s multiple comparison test

Article Snippet: SaOS-2 cell line was transfected to over-express a red fluorescent protein (RFP: Addgene plasmid #54642 tdTomato-N1) or a green-fluorescent protein (GFP: Addgene plasmid #54737 sfGFP_N1). a RFP-overexpressing SaOS-2 cells (red) were directly co-cultured with THP-1 cells in a ratio of 1:2.

Techniques: Transfection, Plasmid Preparation, Cell Culture, Activity Assay

Journal: Journal of cellular biochemistry

Article Title: Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2

doi: 10.1002/jcb.26832

Figure Lengend Snippet: To identify genes that immediately respond to osteogenic stimuli, we retrieved microarray data were retrieved for experiments with mouse C2C12 mesenchymal cells that were treated with 300 ng/ml of BMP-2 and analysed at distinct time points (0, 4, 8, 12, 16, 20, and 24 h). Data on ADAM genes were filtered for genes that show more than a 2 fold change in gene expresseion. This analysis revealed that the Adam17, Adam10 and Adam9 genes are upregulated for more than 2-fold (A), while three others are downregulated (Adam8, Adam15, Adam19) (B). (C) The bar graph shows average expression values (in RPKM; STD as error bar for n=3 human donors) that were rank ordered for relative expression based on RNA-seq analysis of human osteoblastic bone-derived cells from three different donors. The pie chart presented in the inset shows that the six most highly expressed ADAM genes (including the BMP-responsive ADAMs ADAM17, ADAM10 and ADAM9, which are presented in color) account for almost all (~97%) ADAM-related transcripts. (D) Results of RNA-seq analysis for each individual donor and select osteosarcoma cell lines (SaOS-2, MG63 and U2OS) as indicated. (E) Visual presentation and validation of gene expression data using semi-quantitative RT-PCR and ethidium bromide staining. ADAM17, ADAM10 and ADAM9 gene expression was assessed as indicated in human SAOS-2, MG63, U2OS, HOS, G292 and 143B immature osteoblast cells. The data shown are representative of three experiments with similar outcomes. The graphs show quantification of the RT-PCR data relative to Gapdh mRNA (D). All data are presented as mean ± SEM from three independent experiments.

Article Snippet: Mouse MC3T3–E1 osteoblasts, mouse pre-myogenic mesenchymal C2C12 precursor cells, rat osteosarcoma ROS17/2.8 cells and human osteosarcoma cell lines (SAOS-2, MG63, U2OS, G292, HOS and 143B cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Microarray, Expressing, RNA Sequencing, Derivative Assay, Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of cellular biochemistry

Article Title: Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2

doi: 10.1002/jcb.26832

Figure Lengend Snippet: Adam17 and Runx2 expression was assessed in mouse Runx2-null osteoprogenitor cells (Runx2−/−) and mouse MC3T3–E1 pre-osteoblasts cells, as well as rat ROS17/2.8 and human SAOS-2 osteosarcoma cells. Protein and mRNA levels were evaluated by western blot analysis (A, and down graph B) and RT-PCR (C, and down graph D), respectively. Runx2-null cells were infected with an adenovirus vector expressing RUNX2 or GFP (control) as indicated (E and G). Alternativelly, cells were transiently transfected with different concentrations (0.5, 1, 2.5, 5, and 10 μg of DNA) of pcDNA-Runx2 or pcDNA-empty vector (control) (I). Runx2-null cells expressing Adam17 and Runx2 mRNA (E, I, and down graph F and J) and protein levels (G, and down graph H) were evaluated by RT-PCR and western blot analysis, respectively. The data shown are representative of three experiments with similar outcomes. Adam17 and Runx2 mRNA and protein values were normalized to Gapdh and Actin, respectively. All data are presented as mean ± SEM from three independent experiments. *P<0.05 and **P<0.01.

Article Snippet: Mouse MC3T3–E1 osteoblasts, mouse pre-myogenic mesenchymal C2C12 precursor cells, rat osteosarcoma ROS17/2.8 cells and human osteosarcoma cell lines (SAOS-2, MG63, U2OS, G292, HOS and 143B cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Control, Transfection

Journal: Journal of Functional Biomaterials

Article Title: Cytocompatibility Assessment of L-PBF-Manufactured Zinc–Silver–Copper Alloys for Customized Biodegradable Medical Implants

doi: 10.3390/jfb17030146

Figure Lengend Snippet: ( A ) Representative images of the direct cytocompatibility contact test with SAOS-2 cells are presented. The top row ( a ) shows the tested sample plates (black) at the top of the image, with cells growing in direct contact on the bottom of the six-well plate (five times magnification). ( b ) The cell density at a 12 mm distance from the test plates (ten times magnification). ( B , D ) Three different extract concentrations (undiluted, diluted 1:3 and diluted 1:10) from the three L-PBF-manufactured Zn alloys (ZnAgCu, ZnAgCuMn, and ZnAgCuTi), rolled Zn, Cu, and Ti controls were tested, and the proliferation of L929 ( B ) and SAOS-2 ( D ) cells was determined after 24 h. ( C , E ) Live/dead staining of L929 ( C ) and SAOS-2 ( E ) cells growing directly on top of the samples is shown. Red and yellow staining indicate dead or dying cells, respectively, while living cells appear green. Each test was carried out four times with four replicates per sample. Shown are the mean values with their corresponding standard deviations. All results were normalized to control cells grown in cell culture medium without extract (proliferation equals 100%). To calculate statistical significance, a one-way ANOVA followed by a Tukey test was carried out (** p < 0.01 and **** p < 0.0001).

Article Snippet: In addition, the human primary osteogenic sarcoma cell line SAOS-2 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), cultured in McCoy’s 5A medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) supplemented with 15% FBS (Bio&SELL GmbH) and 1% L-glutamine (Thermo Fisher Scientific Inc.), was used.

Techniques: Staining, Control, Cell Culture

Journal: Journal of Functional Biomaterials

Article Title: Cytocompatibility Assessment of L-PBF-Manufactured Zinc–Silver–Copper Alloys for Customized Biodegradable Medical Implants

doi: 10.3390/jfb17030146

Figure Lengend Snippet: Time dependency of ion release and cytocompatibility of undiluted extracts tested for 24 h with SAOS-2 cells from untreated ( A ) and polished ( B ) L-PBF-manufactured plates. Extracts collected daily over a period of 10 days. ( C ) OM of untreated and polished plates before and after 10 days of immersion. The mean proliferation values with the corresponding standard deviation of independent experiments ( n = 4) are plotted graphically. All results were normalized to the control cells grown in cell culture medium without extract (=100%). Significance was determined using one-way ANOVA followed by a Tukey test (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001). ( D , E ) ICP-OES ion concentration measurement. ( D ) Zn 2+ release of the different alloys into daily renewed McCoy’s medium from day 1 to day 10. The extracts were taken from untreated and freshly polished samples. The mean values with the corresponding standard deviations of five independent experiments are plotted. ( E ) Ion concentrations of Zn 2+ , Ag + , Cu 2+ , and Mn 2+ release of ZnAgCuMn into the daily changed McCoy’s medium ( n = 5).

Article Snippet: In addition, the human primary osteogenic sarcoma cell line SAOS-2 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), cultured in McCoy’s 5A medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) supplemented with 15% FBS (Bio&SELL GmbH) and 1% L-glutamine (Thermo Fisher Scientific Inc.), was used.

Techniques: Standard Deviation, Control, Cell Culture, Concentration Assay

Journal: Journal of Functional Biomaterials

Article Title: Cytocompatibility Assessment of L-PBF-Manufactured Zinc–Silver–Copper Alloys for Customized Biodegradable Medical Implants

doi: 10.3390/jfb17030146

Figure Lengend Snippet: Cytocompatibility extract test with SAOS-2 cells cultivated for 24 h in extracts from untreated, new polished and aged polished samples. The mean proliferation values with the corresponding standard deviation of independent experiments ( n = 8) are plotted graphically. All results were normalized to the control cells grown in cell culture medium without extract (=100%). Significance was determined using one-way ANOVA followed by a Tukey test (* p < 0.05; ** p < 0.01).

Article Snippet: In addition, the human primary osteogenic sarcoma cell line SAOS-2 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH), cultured in McCoy’s 5A medium (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) supplemented with 15% FBS (Bio&SELL GmbH) and 1% L-glutamine (Thermo Fisher Scientific Inc.), was used.

Techniques: Standard Deviation, Control, Cell Culture

Journal: Disease Models & Mechanisms

Article Title: Loss of MMP-2 in murine osteoblasts upregulates osteopontin and bone sialoprotein expression in a circuit regulating bone homeostasis

doi: 10.1242/dmm.007914

Figure Lengend Snippet: SaOS2 stable siMMP2 clones overexpress OPN and BSP. (A) Approximately 70% knockdown of mRNA encoding MMP-2 (above) and MMP-2 protein activity levels (below) were achieved in isolated clones A and B. E, siLuc-transfected control cell line. (B,C) SiMMP-2 stable clones showed high upregulation of OPN and BSP at mRNA (B) and protein levels (C). Quantification of immunoblots are shown in italics below the blots. Values represent the average of three separate experiments; error bars represent standard deviations; * P <0.05.

Article Snippet: Serum samples were collected from age- and sex-matched mice and SaOS2 cell extracts were harvested in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology) using a standard protocol.

Techniques: Clone Assay, Knockdown, Activity Assay, Isolation, Transfection, Control, Western Blot