The biomaterials used are made of type III and I collagen sponges (95% of type I collagen; diameter 5 mm, thickness 2 mm) manufactured by “Symatèse Biomatériaux” company (Chaponost, France). The cells were seeded into sponges at P4 (passage 4) at the density of 0.5 million cells per sponges and plated in a 48-well plate at 37 °C in humidified atmosphere containing 5% CO2 (v/v). The sponges were divided into two groups and cultured in normoxia (20% O2, v/v) or in hypoxia (5% O2, v/v). The chondrogenic differentiation medium was DMEM-HG (4.5 g/L) supplemented with 1% glutamine (Gibco), 1% streptomycin and penicillin (Gibco), 40 μg/ml proline (Sigma), 50 μg/ml l -ascorbic acid-2-phosphate (Sigma), 10−7 M dexamethasone (Sigma), and 1% sodium pyruvate (Gibco). For both groups (normoxia and hypoxia), the sponges were treated from day 3 either (i) with ITS 1% (Insulin-Transferrin-Selenium; ITS+premix, BD) as control, versus (ii) ITS 1% + 100 ng/mL BMP-2 (Miltenyi Biotec) or (iii) ITS 1% + 10 ng/mL TGF-β1 (Miltenyi Biotec) or (iv) ITS + TGF-ß1 + BMP-2 in combination. The sponges were harvested on D28 for analysis.
Bmp 2
Manufactured by Miltenyi Biotec
Sourced in Germany
BMP-2 is a recombinant human bone morphogenetic protein-2 product produced by Miltenyi Biotec. It is a growth factor that plays a role in bone and cartilage formation.
Automatically generated - may contain errors
Lab products found in correlation
Tgf β1, by Miltenyi Biotec (2 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
Proline, by Merck Group (1 mentions)
Its premix, by BD (1 mentions)
Tgf 1, by Miltenyi Biotec (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Gw1929, by Cayman Chemical (1 mentions)
Naphthol as mx phosphate, by Nacalai Tesque (1 mentions)
9 cis retinoic acid, by Fujifilm (1 mentions)
Gw4064, by Cayman Chemical (1 mentions)
Gw7647, by Cayman Chemical (1 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
Lithocholic acid, by Merck Group (1 mentions)
Insulin transferrin selenium solution, by Thermo Fisher Scientific (1 mentions)
Dmem f12 basal medium, by Thermo Fisher Scientific (1 mentions)
3 protocols using bmp 2
1
Chondrogenic Differentiation of Cells in Collagen Sponges
2
Assay for Nuclear Receptor Activation
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GW4064, (Z)-guggulsterone (GS), T0901317, 1α,25-vitamin D3, GW7647, and GW1929 were obtained from Cayman Chemical (Ann Arbor, MI, USA). CDCA, lithocholic acid, and Fast-blue BB salt were purchased from Sigma (St. Louis, MO, USA). GW501516 was from ChemScene LLC (Newark, NJ, USA). All-trans retinoic acid and 9-cis-retinoic acid were from FUJIFILM Wako Pure Chemical (Osaka, Japan). BMP-2 was from Miltenyi Biotec. (Bergisch Gladbach, Germany). Naphthol AS-MX phosphate was from Nacalai Tesque (Kyoto, Japan). Amino acid derivative and the coupling reagents were from Watanabe Chemical (Hiroshima, Japan) or Peptide Institute (Osaka, Japan). All other chemicals were from Tokyo Chemical Industry (Tokyo, Japan), FUJIFILM Wako Pure Chemical, and Aurora Fine Chemicals (San Diego, CA, USA) unless otherwise stated.
3
Chondrocyte and Fibroblast Cell Culture
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The cells were cultured in Cell Culture Inserts for 6-well plates (Corning, Cat.No. 353090) or tissue culture dishes with growth or differentiation media. Growth medium was prepared by adding fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.01% ascorbic acid phosphate magnesium salt n-hydrate (Wako Chemicals, Cat.No. 013-12061), FGF-2 (Fiblast spray, Kaken Pharmaceutical), and TGF-β1 (Miltenyi Biotec, Cat.No. 130-095-066) to DMEM-F12 basal medium (Thermo Fisher Scientific, Cat.No. 11320-082). The concentrations of FBS, TGF-β1, and FGF-2 were determined in the first experiment (10% FBS, 10 ng/mL FGF-2, and 1 ng/mL TGF-β1). Differentiation medium was prepared by adding 1% FBS, 1% penicillin-streptomycin, 1% insulin-transferrin-selenium solution (Thermo Fisher Scientific, Cat.No. 41400-045), 0.01% ascorbic acid phosphate magnesium salt n-hydrate, 10 ng/mL TGF-β1, 10 ng/mL bone morphogenetic protein 2 (BMP-2) (Miltenyi Biotec, Cat.No. 130-094-616), and 10 ng/mL growth and differentiation factor 5 (GDF-5) (BioVision, Cat.No. 4667-50). Thawed NHACs and NHDFs were seeded at 1 × 104 cells/cm2 in growth medium. For re-differentiation, the medium was changed to differentiation medium at Day 7. In all experiments, cells were cultured in a humidified incubator at 37 °C under 5% CO2 and the medium was changed 2 or 3 times in a week.
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