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| In this study we used the human osteosarcoma cell line Saos-2. This line was first established from an osteosarcoma isolated from an 11 year old female patient in 1973 [1][2]. The line has been commonly used to study extracellular matrix and mechanical mechanisms in both expression and mineralisation studies [3] although the utility of Saos-2 as a human model system for normal osteoblast formation and function has been debated. Varying cellular morphology and proliferation has been observed [4], but Saos-2 demonstrates physiological levels of multiple osteoblastic markers including, osteocalcin (OC), bone sialoprotein (BSP) and decorin (DCN) [3]. In addition, active levels of alkaline phosphatase (ALPL) and the capacity to mineralize render Saos-2 a useful model system in the study of osteoblast function and ECM formation [5].
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| This experiment was designed to determine promoter specificity and gene expression patterns of key regulators early and late in the mineralization process using this human model system. To control for effects of serum cell proliferation and hyperconfluence we also submitted two control samples: mock-treated Saos-2 cells that had medium changes at the same time points but were not treated with BGP/ascorbic acid and MG63 osteosarcoma cells that were treated but failed ot mineralise. MG63 cells lack high levels of alkaline phosphatase and are not able to mineralise under these conditions, but they may respond to the signal by upregulating some required genes. [3][6].
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| The experiment was designed across 18 time points: 0, 15, 30, 45, 60, 80,100,120,150 and 180 minutes, 4, 8, and 24 hour as well as 4, 7, 14, 21, and 28 days following calcification induction. Cells were plated into 6 well plates (NUNC) or flasks (T25 or T75) at approximately 100,000 cells / well and mineralization was induced two days later with 50µg/ml ascorbic acid (Sigma) and 2.5mM ß-glycerophosphate (BGP) (Sigma) in medium with 10% serum. Medium was replaced with fresh medium including 10% serum and BGP/ascorbic acid every two to three days. All time point samples were processed for RNA at the same time relative to medium-change. RNA from the 18 time point samples were isolated through the following methods. The cells were lifted using 1x Trypsin-EDTA solution (Sigma) and RNA was isolated using the RNA Bee protocol (Ambisco). The sample RNA was quantified using a Nanodrop spectrophotometer (Nanodrop, USA). Three biological replicates were performed.<br><br>
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| Reference:<br>
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| [1] Rodan S.B. et al., Characterizatin of a human osteosarcoma cell line (Saos-2) with osteoblastic properties. Cancer Res, 1987. 46(18): p. 4961-6.<br>
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| [2] Fogh, J., J.M. Fogh, and T. Orfeo, One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst, 1977. 59(1): p. 221-6.<br>
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| [3] Pautke, C., et al., Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer Res, 2004. 24(6): p. 3743-8.<br>
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| [4] Boskey, A.L. and R. Roy, Cell culture systems for studies of bone and tooth mineralization. Chem Rev, 2008. 108(11): p. 4716-33.<br>
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| [5] McQuillan, D.J., et al., Matrix Deposition by a Calcifying Human Osteogenic Sarcoma Cell Line (SAOS-2).Bone, April 1995. 16(4):p.415-26<br>
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| [6] Billiau, A., et al., Human interferon: mass production in a newly established cell line, MG-63. Antimicrob Agents Chemother, 1977. 12(1): p. 11-5. <br>
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| Mineralization was verified using alizarin red staining of calcium at time point 0, 7, 14, 21 and 28 days. The cells were fixed with 4% paraformaldehyde (Sigma) for 10 minutes at room temperature, and rinsed with 1% PBS solution. The cells were stained using 1ml of 2% Alizarin Red (pH 4.2) for 5 minutes at room temperature. The wells were washed 3 times with dH20. The wells were extracted with 10% cetypyridium chloride and the extract used for quantification. The optical density was recorded using Multiskan ascent (Thermo) plate reader at a wavelength of 570nm.
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