The condyles of the proximal and distal femurs were removed and the bone marrow was flushed out with phosphate buffered saline. The cell suspension was filtered through a 70μm filter and plated into a dish. The first media change was done less than 72 hours after plating the cells in the flask. These bone marrow stromal cells (BMSCs) were maintained in stem cell medium for 10 days and then were stimulated to differentiate into osteoblasts in osteogenic media containing 50 μg/ml ascorbic acid and 10 mM β-glycerol phosphate for 3 days (Life Technologies, Carlsbad, CA, USA) before being used for RNA isolation and sequencing (n=3–4/genotype/sex).
β glycerol phosphate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, Switzerland
β-glycerol phosphate is a chemical compound that serves as a buffer in various laboratory applications. It maintains a stable pH in aqueous solutions, supporting the optimal conditions for various biochemical reactions and cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Thermo Fisher Scientific (3 mentions)
Ascorbic acid, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ascorbic acid 2 phosphate, by Thermo Fisher Scientific (2 mentions)
Alizarin red s solution, by Merck Group (2 mentions)
Ix51 microscope, by Olympus (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase alp activity kit, by Merck Group (1 mentions)
Dmem low glucose lg, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Thermo Fisher Scientific (1 mentions)
α mem, by Merck Group (1 mentions)
Mc3t3 e1, by American Type Culture Collection (1 mentions)
Penicillin streptomycin fungizone, by Thermo Fisher Scientific (1 mentions)
P s f, by Thermo Fisher Scientific (1 mentions)
Lithium bromide libr, by Thermo Fisher Scientific (1 mentions)
Methanol meoh, by Merck Group (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Ascorbate, by Thermo Fisher Scientific (1 mentions)
10 protocols using β glycerol phosphate
1
Osteoblast Differentiation from BMSCs
2
Metformin's Impact on DPSC Proliferation and Differentiation
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Considering the dose-dependent effect of metformin, 100 µmol/L metformin hydrochloride (Alborz Pharmaceutical Co., Tehran, Iran) was chosen based on a previous study. 19 (link) The positive effect of this concentration of metformin on the proliferation and differentiation of DPSCs was evaluated. In brief, the cells at a density of 10 4 were seeded in 48-well plates with 100 μmol/L metformin hydrochloride, and either the standard medium was added for the proliferation assay or the osteogenic medium for the differentiation assay. Proliferation was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Sigma Aldrich, St. Louis, USA) assay after 1, 3 and 7 days of treatment with or without metformin. Osteogenic differentiation was evaluated using an alkaline phosphatase (ALP) activity kit (Sigma Aldrich) after 3, 7 and 14 days. Similar to the proliferation assay, DPSCs cultured in the osteogenic medium only (DMEM-low glucose (LG), 0.2 mol/L ascorbic acid 2-phosphate, 10 -8 mol/L dexamethasone, and 10 mmol/L β-glycerol phosphate; Life Technologies), without metformin, were used as a control for pairwise comparisons at each time point.
3
DPSC Osteogenic Differentiation with FDBA and Metformin
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Particulate cortical/cancellous mineralized 1-2-millimeter FDBA granules (Tissue Regeneration Corporation, Tehran, Iran) were placed in 48-well plates and a cell suspension including 2.5 × 10 4 DPSCs was loaded into the wells. The plates were incubated at 37°C for 1 h. Then, according to the type of evaluation, 2 kinds of media (standard or osteogenic) were added to the wells. The osteogenic medium included DMEM-LG, 0.2 mol/L ascorbic acid 2-phosphate, 10 -8 mol/L dexamethasone, and 10 mmol/L β glycerol phosphate (Life Technologies). Next, 100 μmol/L metformin hydrochloride was added to the samples. The study groups and the content of the wells are summarized in Table 1.
4
Preosteoblastic Cell Culture Protocol
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The MBG disks were sterilized under ultraviolet light for 7 min each side [31] . After that, disks were stabilized in complete medium that consist on: α-Modified Eagle's Medium (α-MEM, Sigma Chemical Company, St. Louis, MO, USA) with 10% fetal bovine serum (FBS, Gibco, BRL), 1 mM L-glutamine, penicillin/streptomycin (400 mg/mL BioWhittaker Europe, Belgium), under a CO 2 (5%) atmosphere at 37°C for 24 h in 24-well tissue culture plates (Cultek SLU, Spain). Complete medium was supplemented with β-glycerolphosphate (50 mg, Life Technologies SA, Spain) and Lascorbic acid (Life Technologies SA, Spain) for ALP test. Cell culture experiments were performed using the well-characterized mouse preosteoblastic cell line MC3T3-E1 (subclone 4, CRL-2593; ATCC, Mannassas, VA). Controls in tissue culture plastic in the absence of samples were always carried out.
5
Osteogenic Differentiation of Rat BMSCs
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BMSCs were obtained from the femur and tibia of rats at the age of 3 (young), 12 (middle-aged) and 18- (old-aged) months. The procedure was carried out as follows: i) firstly, the medullary cavity was thoroughly flushed the using an α-modified Eagle's medium (αMEM; Invitrogen; Thermo Fisher Scientific, Inc.) without ascorbic acid to collect the cells. The cells were then allowed attach in complete αMEM with 10% FBS, 1% penicillin and streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.) at 37°C, 5% CO2 for 72 h. Following this step, BMSCs were finally obtained by replacing the fresh culture medium. For the induction of osteogenic differentiation, BMSCs were cultured for 16 days in osteogenesis induction αMEM medium with 10% FBS, 300 ng/ml bone morphogenetic protein 2 (Gibco; Thermo Fisher Scientific, Inc.), 50 µg/ml ascorbic acid (Gibco; Thermo Fisher Scientific, Inc.) and 5 mM β-glycerolphosphate (Gibco; Thermo Fisher Scientific, Inc.).
6
Osteogenic Differentiation Assay
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Expanded cells around 95% confluence were incubated for 21 days in osteogenic medium containing DMEM, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL L-ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerol phosphate (Thermo Fisher Scientific). For evaluation of matrix mineralization, induced cells (n = 4) were fixed with 70% ice-cold ethanol for 1 h and then incubated in 1% Alizarin Red S (ARS) solution (pH = 4.3; MilliporeSigma) for 20 min at room temperature with agitation. After rinsing three times with PBS, images of calcium deposition were taken using an Olympus IX51 microscope (Olympus America Inc., Center Valley, PA, USA). Osteogenic marker genes were quantified using RT-qPCR (see Section 2.2.3 for details).
7
Osteogenic Differentiation Assay
8
Silk-based Scaffold Preparation and Characterization
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Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS; order number 10270–06), penicillin-streptomycin-fungizone (P/S/F), nonessential amino acids (NEAA), basic fibroblast growth factor (bFGF), β-glycerolphosphate (βGP), ascorbic acid (AA), dexamethasone (Dex), alamarBlue® solution and Quant-iTTM PicoGreen® double stranded DNA (dsDNA) reagent kit were from Gibco (Zug, Switzerland). 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was from abcr GmbH & Co. (Karlsruhe, Germany). Methanol (MeOH) was from Merck (Zug, Switzerland) and Lithium Bromide (LiBr) from Thermo Fisher Scientific (Reinach, Switzerland). All other substances were of analytical grade and were purchased from Sigma (Buchs, Switzerland). Silkworm cocoons were kindly supplied by Trudel Silk Inc (Zurich, Switzerland).
9
Osteogenic and Adipogenic Differentiation of HAFSCs
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HAFSCs were maintained in osteogenic and adipogenic induction media 3 weeks. Osteogenic induction media consisted of α-MEM supplemented with 10% FBS, 0.1 μmol/l dexamethasone, 10mmol/l β-glycerol phosphate, 50μmol/l ascorbate (Gibco). Adipogenic induction media contained α-MEM supplemented with 10% FBS, 1μmol/l dexamethasone, 5μg/ml insulin, 0.5 mmol/l isobutylmethylxanthine and 60μmol/l indomethacin (Gibco). Alizarin red and Oil Red was utilized to identify osteogenic and adipogenic differentiation, respectively.
10
Osteogenic Activity Assessment of Cell Scaffolds
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For measurement of the osteogenic activity of cells on the scaffolds, alkaline phosphatase (ALP) activity and mineralization assays were performed. Cells were seeded at a density of 1 x 104 and cultured in osteogenic media (α‐MEM with 15% FBS supplemented with 0.2 mM ascorbic acid [Gibco] and 10 mM β‐glycerol phosphate [Gibco]). On days 7 and 14 of culture, ALP activity was measured using an ALP Activity Assay Kit (AnaSpec Inc.,Fremont., CA) and a Protein Assay Kit (iNtRON Biotechnology, Seoul, Korea). ALP activity was normalized as the concentration of p‐nitrophenol (pNP) to the protein amounts. On the day 21, mineralization analysis was performed. Fixation with 95% cold ethanol was done. Cells were stained with a 1% alizarin red S solution (Wako Chemicals, Osaka, Japan) for 5 min.
Cells were fixed in 95% cold ethanol and stained with the images of stained plates were observed. For quantitative measurement, an elution procedure with cetylpyridinium chloride in 10 mM sodium phosphate was performed and measured using an automatic microplate reader at 540 nm wavelength.
Cells were fixed in 95% cold ethanol and stained with the images of stained plates were observed. For quantitative measurement, an elution procedure with cetylpyridinium chloride in 10 mM sodium phosphate was performed and measured using an automatic microplate reader at 540 nm wavelength.
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