Primary calvarial osteoblasts were isolated from calvaria of 5-day-old neonates using collagenase type II (50 mg/ml, Worthington, LS004176)/dispase II (100 mg/ml, Roche, 10165859001). Osteoblasts were maintained in alpha-minimal essential medium (aMEM) (Gibco) containing 10% FBS (Gibco), 2mM L-glutamine (Corning), 1% penicillin/streptomycin (Corning), and 1% nonessential amino acids (Corning) and differentiated with ascorbic acid (200 uM, Sigma, A8960). and β-glycerophosphate (10 mM, Sigma, G9422). To induce osteogenic differentiation, the growth media was supplemented with ascorbic acid (200 mM, Sigma, A8960) and beta-glycerophosphate (10 mM, Sigma, G9422).
β glycerophosphate
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, Italy, China, Sao Tome and Principe, France, Canada, Australia, Macao, India, Senegal, Ireland, Spain, Denmark, Belgium
β-glycerophosphate is a chemical compound that serves as a buffering agent and source of phosphate for cell culture media. It helps maintain a stable pH environment for cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (1044 mentions)
Ascorbic acid, by Merck Group (711 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (243 mentions)
L ascorbic acid, by Merck Group (175 mentions)
Indomethacin, by Merck Group (142 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (129 mentions)
Insulin, by Merck Group (126 mentions)
Alizarin red s, by Merck Group (119 mentions)
L ascorbic acid 2 phosphate, by Merck Group (114 mentions)
Ascorbic acid 2 phosphate, by Merck Group (99 mentions)
Streptomycin, by Thermo Fisher Scientific (89 mentions)
α mem, by Thermo Fisher Scientific (88 mentions)
Alizarin red, by Merck Group (88 mentions)
Penicillin, by Thermo Fisher Scientific (85 mentions)
Oil red o, by Merck Group (82 mentions)
Ascorbate 2 phosphate, by Merck Group (73 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (59 mentions)
Fetal bovine serum (fbs), by Merck Group (49 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (46 mentions)
Isobutylmethylxanthine, by Merck Group (39 mentions)
1 734 protocols using β glycerophosphate
1
Isolation and Osteogenic Differentiation of Primary Calvarial Osteoblasts
2
Osteogenic Differentiation of MSCs
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MSCs were plated in flat-bottom plates in triplicates at 6,000 or 1,000 cells/cm2 for early and late analyses, respectively. Cells were grown until confluency and then exposed to different experimental conditions. For osteogenic differentiation, growth medium (see above) was supplemented with 10 mM of β-glycerophosphate (Sigma-Aldrich) and rhBMP-2 (750 ng/mL, InductOS, Wyeth/Pfizer, New York, NY, USA). In addition, osteogenic differentiation medium (ODM) was used, consisting of 10 mM of β-glycerophosphate and 10 nM dexamethasone/0.2 mM L-ascorbic acid 2-phosphate (Sigma-Aldrich). Medium was changed every 3–4 days.
3
Aortic Smooth Muscle Cell Mineralization
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Primary mouse aortic smooth muscle cells (MAoSMCs) were isolated from Asm−/− and Asm+/+ mice and routinely cultured (passages from 3 to 7) as described previously [50 (link)]. MAoSMCs were treated with 2 mM β-glycerophosphate (Sigma–Aldrich) and/or 0.01 U/ml sphingomyelinase (Staphylococcus aureus) (Enzo Life Sciences). For analysis of mineralization, treatment with calcification medium containing 10 mM β-glycerophosphate and 1.5 mM CaCl2 (Sigma–Aldrich) was used. For long-term experiments, fresh medium with agents was added every 2–3 days.
4
Picoberin Effect on Osteogenic Mineralization
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To investigate the effect
of Picoberin on matrix mineralization, 6 × 104 C3H10T1/2
cells per well were seeded into 12-well plates (Sarstedt, #83.3921)
and incubated at 37 °C and 5% CO2 for 48 h to a confluence
of 80%. Cells were then treated with mineralization medium (Shh-conditioned
medium containing 50 μg/mLl -ascorbic-3-phosphate (Sigma-Aldrich,
#8960), 10 mM β-glycerophosphate (Sigma-Aldrich, #G9422), and
10% heat-inactivated FCS) or control medium (empty vector-conditioned
medium containing 50 μg/mLl -ascorbic-3-phosphate,
10 mM β-glycerophosphate, and 10% heat-inactivated FCS) and
different concentrations of the compounds or DMSO as a control. The
medium was refreshed every 3–4 days for 21 days. Cells were
then fixed using 3.7% formaldehyde for 10 min and washed twice with
ddH2O. Afterward, mineralized nodules were stained with
40 mM Alizarin Red S solution (ChemCruz, #sc-205998A, pH = 4.1, adjusted
using 10% ammonium hydroxide) for 40 min. To remove unspecific staining,
cells were washed five times with ddH2O prior to microscopy
analysis using the Zeiss Observer Z1 (Carl Zeiss, Germany).
of Picoberin on matrix mineralization, 6 × 104 C3H10T1/2
cells per well were seeded into 12-well plates (Sarstedt, #83.3921)
and incubated at 37 °C and 5% CO2 for 48 h to a confluence
of 80%. Cells were then treated with mineralization medium (Shh-conditioned
medium containing 50 μg/mL
#8960), 10 mM β-glycerophosphate (Sigma-Aldrich, #G9422), and
10% heat-inactivated FCS) or control medium (empty vector-conditioned
medium containing 50 μg/mL
10 mM β-glycerophosphate, and 10% heat-inactivated FCS) and
different concentrations of the compounds or DMSO as a control. The
medium was refreshed every 3–4 days for 21 days. Cells were
then fixed using 3.7% formaldehyde for 10 min and washed twice with
ddH2O. Afterward, mineralized nodules were stained with
40 mM Alizarin Red S solution (ChemCruz, #sc-205998A, pH = 4.1, adjusted
using 10% ammonium hydroxide) for 40 min. To remove unspecific staining,
cells were washed five times with ddH2O prior to microscopy
analysis using the Zeiss Observer Z1 (Carl Zeiss, Germany).
5
ALP Activity of BMSCs in Osteogenic Media
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Three groups were used in the experiment: the control group, the APS group, and the OHS group. In 24-well plates, BMSCs were cultured at a density of 2 × 104 mL-1. Dexamethasone, L-ascorbic acid, and β-glycerophosphate (Sigma-Aldrich, USA) were added to the total medium, APS and OHS extract to reach concentrations of 8–10 M Dexamethasone, 5 mg/mL L-ascorbic acid, and 5 mM β-glycerophosphate soon after incubation for a day, and the medium was exchanged. After that, the frequency of changing the medium was the same as that of the cytotoxicity test. On the 14th day, cells were lysed with radioimmunoprecipitation assay buffer (Lablead, China), and the lysate was added to a 96-well plate, followed by p-nitrophenyl phosphate (pNpp). ALP can catalyze the reaction of pNpp to generate p-nitrophenol, with the strongest absorbance at a wavelength of 405 nm. To assess the ALP activity, a microplate reader was used to obtain the absorbance readings, and a standard curve was created using a standard. To determine the amount of total cellular protein, a BCA kit (Yeasen, China) was used.
6
Vascular Smooth Muscle Cell Culture and Calcification
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HAoSMCs (Fisher Scientific and Sigma-Aldrich) were routinely cultured as described previously [1 (link), 28 (link), 75 (link)] and used in experiments up to passage 12. N indicates the number of independent experiments performed at different passages of the cells. HAoSMCs were treated with the indicated concentrations or 1 μM isoproterenol (stock in PBS; Sigma-Aldrich) [70 (link)], 2 mM β-glycerophosphate (Sigma-Aldrich) [69 (link), 74 (link)] or 1 μM ICI 118,551 (stock in DMSO; MedChemExpress) [78 (link)]. Equal amounts of vehicle were used as control. For calcification analysis, HAoSMCs were treated with calcification medium supplemented with 10 mM β-glycerophosphate and 1.5 mM CaCl2 (Sigma-Aldrich) [28 (link), 75 (link)]. For long-term treatments, fresh medium with agents was added every 2–3 days. Where indicated, HAoSMCs were transfected with 10 nM PRKACA siRNA (ID:s11066), 10 nM CREB1 siRNA (ID:s3490), 10 nM ADRB2 siRNA (ID:s1122), or 10 nM negative control siRNA (ID:4390843) by using siPORT amine transfection agent (all from Fisher Scientific) [28 (link)].
7
Osteoblast Mineralization Assay
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Osteoblasts at P3 were cultured in 6-well plates with a seeding density of 1×105 cells/well if Bio-Oss (1 mg) was added or 2×104 cells/well if α-GGRP-Bio-Oss (1 mg) was used. When the cells reached 80% con-fluency, the cell culture media was changed to mineralized induction solution, which contained ascorbic acid (10 mg/ml), β-glycerophosphate (10 mM) and dexamethasone (0.1 µmol/l; all from Sigma-Aldrich; Merck KGaA) for culturing osteoblasts for up to 21 days. Subsequent to three washes in PBS, cells were fixed with 4% paraformaldehyde for 10 min. Alizarin Red-S solution (BestBio, Inc.) was added and cells were cultured for 30 min at 37°C.
8
Osteoblast Co-Culture with Stem Cells
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OB were seeded at a density of 3 × 104 cells/well in 12-well plates (12-well companion plate, Corning, Kaiserslautern, Germany). NPC, AFC and CEPC were encapsulated separately in 1.2% alginate at a density of 4 Mio/mL by using a syringe (22 G needle). Cell-alginate suspension was then dropped in 102 mM CaCl2 solution [40 (link),41 (link)]. The alginate beads from all three cell types were distributed in different quantities (6, 9 or 12 beads) in culture inserts (0.4 µm pore size, high pore density, polyethylene terephthalate track-etched, Becton, Dickinson and Company, Allschwil, Switzerland) and co-cultured with the OB monolayer. The experiment included a positive control with OB and culture insert containing empty beads only. The experimental groups and the positive control group were cultured in α-MEM (Gibco) supplemented with 10% FBS, P/S, 50 µg/mL l -ascorbic acid-2-phosphate, 10 nM dexamethasone and 5 mM β-glycerophosphate (all purchased from Sigma-Aldrich). The negative control group included OB cultured without culture inserts and beads with α-MEM without any osteogenic supplements (Figure 5 ). The experiment was run for 21 days under hypoxic conditions. The medium was changed every second to third day.
9
Osteogenic Potential of Dentin Matrix Molecules
10
Modulating Osteoblast Differentiation via miR-19a/b Inhibition
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Example 3
To test whether the differentiation of calvarial osteoblasts can be influenced by interfering with miR-19a/b expression, the cells were transfected with oligonucleotides that are designed to bind to and inhibit endogenous miR-19a (anti-miR19a: 6-FAM/TGCATAGATTTGCAC) and miR-19b (anti-miR-19b: 6-FAM/TGCATGGATTTGCAC). These synthetic oligonucleotides contain phosphorothioate backbone bonds for optimal use in functional studies as well as optimized pharmacokinetic and pharmacodynamic properties and minimal toxicity. Furthermore, the 5′ fluorescein FAM label allows monitoring of transfection efficiency in vitro and tissue delivery in vivo. The cells were transfected with anti-miR-19a/b (50 nM) using the Neon electroporation system (Invitrogen). Osteoblast differentiation was induced by supplementing α-MEM with 0.2 mM L-ascorbic acid and 10 mM β-glycerophosphate (both Millipore). Osteoblast differentiation was determined by alkaline phosphatase (ALP) staining after fixing the cells in 4% neutrally buffered formaldehyde solution. For ALP staining, cells were incubated with naphthol AS-MX/Fast Blue (both Sigma-Aldrich) in Tris-HCl solution for 15 min at room temperature.
Results:
It was found that differentiation of calvarial osteoblasts was enhanced by transfection with inhibitors against miR-19a and miR-19b in a synergistic way (
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