Two parallel samples (composite bone cement columns) for each hydrogel ratio of the PLGA-PEG-PLGA/C3S/C2S/POP were ground into powders. The mixed culture liquid was sterilized at a concentration of 10 mg/mL. The stem cells were seeded in 24-well plates. After 24 h, the old culture medium was removed, and 0.5 mL of osteogenic induction medium (Sigma, Hiroshima, Japan) was added. Induction was performed for 14 d. The cells were then fixed for 15 min with 4% paraformaldehyde (Sigma, Japan) and stained with Alizarin Red solution (Beyotime, Petaluma, CA, USA) for 3 min, after which they were observed under a microscope and photographed. Subsequently, hexadecyl chloride was added and left at room temperature for 30 min, the supernatant was drawn, and the absorbance of the sample was measured at 560 nm using a microplate reader (Biotech, Shanghai, China), while simultaneously measuring the absorbance of the supernatant of a group of simple cells. The difference between the absorbances of the sample and the simple cell groups was recorded.
Osteogenic induction medium
Manufactured by Merck Group
Sourced in Japan, United States
Osteogenic induction medium is a laboratory reagent used to promote the differentiation of cells, such as mesenchymal stem cells, into osteoblasts, which are responsible for bone formation. The medium contains a specific combination of growth factors, nutrients, and other components that support the osteogenic differentiation process.
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Lab products found in correlation
Alizarin red solution, by Beyotime (1 mentions)
Glycerophosphate, by Merck Group (1 mentions)
Alkaline phosphatase kit, by Merck Group (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Adipogenic medium, by Merck Group (1 mentions)
Oil red staining, by Merck Group (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Dexamethasone (dex), by BD (1 mentions)
Sodium pyruvate, by BD (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Adipogenic induction medium, by Merck Group (1 mentions)
7 protocols using osteogenic induction medium
1
Osteogenic Differentiation of Stem Cells
2
Alizarin Red Staining of Osteogenic OP-ASCs
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After transfection with various lentiviral vectors (OE‐Wnt10b, KD‐Wnt10b and Mock), these modified OP‐ASCs were inoculated in six‐well plates, then cultured in the osteogenic induction medium (Sigma‐Aldrich). On the 14th day of osteogenesis induction, the alizarin red staining was utilized to examine these cells' osteogenic capacities. Briefly, 4% paraformaldehyde was used to fix these modified OP‐ASCs for 30 min, followed by washing the cells three times with PBS, and 30 min at room temperature incubation with alizarin red dye. The mineralized nodules formed in each group were identified and videotaped under the bright‐field view of the inverted microscope, with at least three random images taken for each well.
3
Mesenchymal Stem Cell Differentiation
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For cell proliferation, cells were seeded in a 24-well plate at the density of 1×104 cells per well in cultured medium. Cells were counted every two days for 8 days. The mean cell number was calculated for each triplicate. For differentiation, passage 3 (P3) cells were seeded in a 24-well plate at the density of 1×104 cells per well overnight. They were then induced with two media: one osteogenic induction medium, which contains 0.1 mM dexamethasome, 50 mM ascorbate-2 phosphate, 10 mM glycerophosphate (Sigma-Aldrich); and the other adipogenic induction medium, which contains 1 mM dexamethasone, 200 mM indomethacin, 0.5 mM 3-isobutyl-1-methyl-xanthine, and 10 μg/ml insulin (Sigma-Aldrich). In vitro osteogenesis, cells were cultured for 0, 3, 7, 10 days, and then stained for alkaline phosphatase (ALP) activity, using the alkaline phosphatase kit (Sigma-Aldrich). In vitro adipogenesis, cells were cultured for 0, 3, 7, 10 days, and then stained for fat droplets, using the Oil-Red-O (Sigma-Aldrich). In addition, P1 cells were seeded in a 24-well plate at the density of 1×104 cells per well on carbon nanotube (CNT) for 3 days, and were analyzed for the expression of cardiac troponin T (cTnT), GATA4, and Nkx2.5 by qRT-PCR.
4
Adipogenic and Osteogenic Induction of hADSCs
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Adipogenic induction of hADSCs followed a previous reported procedure 33 . Human adipose‐derived stem cells at P2 were induced in an adipogenic induction medium (Sigma‐Aldrich) for 12 days. Then, the cells were fixed with 10% paraformaldehyde for 30 min. and washed three times with PBS. The cells were stained with 2% fresh Oil red‐O solutions (Sigma‐Aldrich) for 10 min. The cells were washed with 70% alcohol and were imaged using an inverted microscope (OLYPUS, Tokyo, Japan).
Osteogenic induction of hADSCs was performed according to a previously reported method34 . hADSCs at P2 were induced in an osteogenic induction medium (Sigma‐Aldrich) for 12 days. Then, the cells were fixed with 4% paraformaldehyde for 10 min. and washed three times with PBS. The cells were stained with 10 mg/ml alizarin red (Sigma‐Aldrich) for 10 min. The cells were gently washed three times with deionized water and imaged with an inverted microscope.
Osteogenic induction of hADSCs was performed according to a previously reported method
5
Multilineage Differentiation Potential of WJ-MSCs
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P4 cells were used to investigate the WJ-MSCs’ differentiation potential. The cells were seeded at 10,000 cells/cm2 in 12-well plates. After reaching approximately 70% confluence, a specific induction medium was added to each well. For the osteogenesis assay, the cells were grown in an osteogenic induction medium (Sigma-Aldrich, USA) that contained DMEM, 10% FBS, 10 nM Dexamethasone, 0.2 mM ascorbic acid 2-phosphate, and 10 mM glycerol phosphate. Two times a week for 21 days, the medium was changed. The induction media was withdrawn after the cells had been exposed to it for 21 days. Then, the cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at 4°C, and stained with 2% alizarin red at pH=7.2. For adipogenesis induction, the cells were cultured in an adipogenic medium (Sigma-Aldrich, USA) that contained DMEM enriched with 10% FBS, indomethacin (50 μg/ml) dexamethasone (1 μM), insulin (5 μg/ml), and isobutylmethylxanthine (0.5 mM).
The culture medium was changed every 3~4 days for up to 21 days. In the following, the cells were fixed in 4% paraformaldehyde and visualized with Oil Red staining (Sigma-Aldrich, USA). An inverted microscope (Olympus, Japan) was used to observe calcium deposits and lipid droplets.
The culture medium was changed every 3~4 days for up to 21 days. In the following, the cells were fixed in 4% paraformaldehyde and visualized with Oil Red staining (Sigma-Aldrich, USA). An inverted microscope (Olympus, Japan) was used to observe calcium deposits and lipid droplets.
6
Alizarin Red S Staining for Osteogenic Differentiation
7
Multilineage Potential of Mesenchymal Stem Cells
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Multi-differentiation potential of MMSCs and BMSCs in vitro were tested for adipogenesis, chondrogenesis, and osteogenesis. Both types of cells at passage 2 were seeded either on plastic surfaces in 6-well plates at a density of 2.4 × 105 cells/well or in 24-well plates at a density of 6 × 104 cells/well in basic growth medium consisting of low glucose DMEM, 10% heat inactivated FBS, 100U/ml penicillin, and 100 μg/ml streptomycin. To test adipogenic potential, cells were cultured in adipogenic induction medium (Millipore, Billerica, MA) consisting of basic growth medium added with dexamethasone (1 μM), insulin (10 μg/ml), indomethacin (100 μM), and isobutylmethylxanthine (0.5 mM). As a test of chondrogenic potential, two kinds of MSCs were cultured in basic growth medium supplemented with prolin (40 μg/ml), dexamethasone (39 ng/ml), TGF-β3 (10 ng/ml), ascorbate 2-phosphate (50 μg/ml), sodium pyruvate (100 μg/ml), and insulin transferrin-selenious acid mix (50 mg/ml) (BD Bioscience, Bedford, MA). Finally, the osteogenic potential of MMSCs and BMSCs was tested by culturing them in osteogenic induction medium (Millipore, Billerica, MA) consisting of basic growth medium supplemented with dexamethasone (0.1 μM), ascorbic 2-phosphate (0.2 mM), and glycerol 2-phosphate (10 mM).
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