A 250 μL volume of cell suspension (containing 2.5 × 105 cells, including both living and dead cells) was added to six 15 mL tubes (Falcon) containing a chondrogenic induction medium consisting of Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific), 10 ng/mL transforming growth factor-β3 (TGF-β3, Miltenyi Biotec, Bergisch Gladbach, Germany), 500 ng/mL bone morphogenetic protein 2 (BMP-2, Medtronic, Minneapolis, MN, USA), 40 μg/mL proline (Merck), 100 nM dexamethasone, 100 μg/mL pyruvate (Merck), 1% antibiotic–antimycotic, 50 μg/mL ascorbate-2-phosphate, and 1% ITS + Premix (Becton Dickinson, San Jose, CA, USA). The cells were centrifuged at 450×g for 10 min to form cell pellets, which were cultured for 21 days. The cultured cell pellets were photographed and weighed with a semi micro balance (CPA225D, Sartorius, Gottingen, Germany). The pellets were cut into 5 µm sections and stained with safranin O and toluidine blue.
Bmp 2
Manufactured by Medtronic
Sourced in United States, Ireland
BMP-2 is a recombinant human bone morphogenetic protein-2 produced in E. coli cells. It is a growth factor that stimulates bone and cartilage formation.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Tgf β3, by Miltenyi Biotec (2 mentions)
Its premix, by Corning (2 mentions)
Pdgf bb, by R&D Systems (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Proline, by Merck Group (1 mentions)
Pyruvate, by Merck Group (1 mentions)
15 ml tube, by BD (1 mentions)
Its premix, by BD (1 mentions)
Cpa225d, by Sartorius (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
L proline, by Merck Group (1 mentions)
Tgf β1, by Merck Group (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Bmp buffer, by Medtronic (1 mentions)
Human bmp 2 quantikine elisa kit, by R&D Systems (1 mentions)
14 protocols using bmp 2
1
Chondrogenic Differentiation of Cells
2
Chondrogenic Differentiation of Cells
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To obtain cell pellets, 2 × 105 cells were centrifuged in polypropylene tube and cultured in growth medium. The next day, the medium was exchanged to chondrogenic medium (DMEM, 1% ITS+Premix (Corning, NY, USA), 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich), 40 μg/mL L-proline (Sigma-Aldrich)) supplemented with 100 ng/mL BMP2 (Medtronic, Dublin, Ireland), and 10 ng/mL TGFβ1 (ORIENTAL YEAST, Tokyo, Japan). Dexamethasone (Sigma-Aldrich) was used with TGFβ1 at concentration of 10 nM [19 ]. The cell pellets were maintained with 0.5 mL medium at 37 °C in a humidified atmosphere with 5% CO2. The medium was replaced twice per week.
3
BMSC Osteogenesis Induction Protocol
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BMSCs were seeded at 6 × 104 cells/cm2 in 12-well plates for RNA collection (Nunc) and in 24-well plates (Nunc) for mineralization and adipogenic assays. Osteogenic induction medium contained HG-DMEM supplemented with 10% FBS, 1% PenStrep, 10 mM β-glycerol phosphate (BGP, Sigma-Aldrich), 100 nM dexamethasone, and 50 μM L-ascorbic acid-2-phosphate. Where indicated, 100 ng/mL BMP-2 (Medtronic) was added to the medium. Where indicated, LDN 193189 was added to medium at a concentration of 0.1, 1, 10, 100, or 1,000 nM. Control medium contained osteogenic induction medium components and 0.1% DMSO without BMP-2 or LDN 193189. Cultures were maintained for 9 days to prevent complete delamination, and the medium was exchanged every 3 days. The concentration of BMP-2 added to the BMSC osteogenic cultures was determined by doing a titration experiment wherein increasing concentrations of BMP-2 were added to BMSC cultures for 14 days.
4
BMP-2 Release Kinetics from Bone Grafts
5
MSC Spheroid Formation with BMP-2 Loaded HA Nanoparticles
6
Alginate Hydrogels with MSCs for Bone Repair
7
Stem Cell Differentiation and Bone Formation
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Collagenase P (Roche, IN, USA), hyaluronidase (Sigma Aldrich, St Louis, MO, USA), Ad-GFP (Vector Biolabs, Cat No. 1060), Ad-Cre (Vector Biolabs, Cat No. 1045), PDGF-BB (R&D Systems, Minneapolis, MN, USA), recombinant human BMP2 (PeproTech, Rocky Hill, NJ, USA), TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), ImProm II kit (Promega, USA), TaqMan Universal Mastermix (Thermo Fisher Scientific, UK), SYBR green-based assay (Thermo Fsher, Waltham, MA, USA; Life Technologies, Grand Island, NY, USA), Protease Inhibitor cocktail (Roche), tamoxifen (Sigma Aldrich, St. Louis, MO, USA), corn oil (Acros Organics, Belgium), Cryomatrix (Thermo Scientific, Waltham, MA, USA), Leukocyte acid phosphatase kit (Sigma Aldrich, St. Louis, MO, USA), BMP2 (Medtronic, MN, USA) and PDGF BB (R&D, 520-BB-050/CF), methyl methacrylate bone cement (Orthodontic Resin, Dentisply Caulk Inc. Milford, DE, USA). Antibodies used are listed in Supplementary Table 1 .
8
Osteogenic Differentiation Assay Protocol
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Osteogenic differentiation assays were performed as previously reported79 (link) in non-collagen coated flasks. Briefly, 100,000 cells were plated in 6-well plates and cultured under specific osteogenic conditions in DMEM supplemented with 10% FBS (Sigma-Aldrich), 100 U/mL penicillin/streptomycin solution (Sigma-Aldrich), 0.1 mM dexamethasone (Sigma-Aldrich), 50 μM ascorbate-2-phosphate (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich), and 100 ng/mL bone morphogenetic protein-2 (BMP2; Medtronic), with or without 10 nM rapamycin, and incubated at 37°C in 5% CO2. Mineralization was assessed by Alizarin Red staining on day 21. The cultures were rinsed twice with PBS, fixed in 100% ethanol for 30 min, and stained with 1% Alizarin Red (Hartman Leddon, Philadelphia, PA, USA) in 0.28% ammonia water for 10 min at room temperature. The stained cell layers were washed twice with distilled water and air dried for micro-/macroscopic analyses. For quantification, Alizarin Red dye was extracted from the stained cell layer with 5% formic acid solution, and the optical density was measured with a photometer (microplate reader Model 680; Bio-Rad, Tokyo, Japan) at a 415 nm wavelength. Total RNA was extracted for qRT-PCR analysis. The expression patterns of osteogenic genes expressing collagen type 1 alpha 1 (Col1A1) and osteocalcin were analyzed.
9
Osteogenic Differentiation of Cntnap4-Knockdown MC3T3-E1 Cells
10
Dicalcium Phosphate Scaffolds for Bone Regeneration
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For all the implants a dicalciumphosphate (DCP) scaffold CopiOs® (Zimmer, Warsaw, Indiana, USA) was used. From the original scaffold blocks, rings with a diameter of 2 cm and a central hole of 6 mm and a thickness of 4 mm were shaped. For the 3 cm FD group 8 rings were used, for the 4.5 cm BED group 12 rings.
For the combination of scaffold plus BMP, the rings were coated with either BMP-2 (Medtronic, Minneapolis, USA) or BMP-6 (Genera Inc., Kalinovica, Rakov Potok, Croatia) according to the subgroups.
For the FD group, the amount of BMP-6 coated onto each scaffold ring was 43 μg (total of 344 μg). For the BED group the amount of BMP-2 coated onto each scaffold ring was 125 μg (total of 1500 μg). For BMP-6125 μg and respectively 317 μg per ring was used for the two subgroups (total of 1500 μg and 3800 μg respectively). Subsequently, the scaffolds rings were incubated for 1 h at 37 °C and seeded with 51. 10 6 cells (total amount of cells 408 million for FD and 612 million for BED group) in 240 μl of DMEM with 10% autologous sheep serum and once again incubated for 1 h at 37 °C. At last 25 ml of DMEM with 10% autologous sheep serum was added for transport and implantation was performed within 3 h after assembly of the ATMP. The residual transport medium was centrifuged for calculating of cell seeding efficiency by counting the remaining cells.
For the combination of scaffold plus BMP, the rings were coated with either BMP-2 (Medtronic, Minneapolis, USA) or BMP-6 (Genera Inc., Kalinovica, Rakov Potok, Croatia) according to the subgroups.
For the FD group, the amount of BMP-6 coated onto each scaffold ring was 43 μg (total of 344 μg). For the BED group the amount of BMP-2 coated onto each scaffold ring was 125 μg (total of 1500 μg). For BMP-6125 μg and respectively 317 μg per ring was used for the two subgroups (total of 1500 μg and 3800 μg respectively). Subsequently, the scaffolds rings were incubated for 1 h at 37 °C and seeded with 51. 10 6 cells (total amount of cells 408 million for FD and 612 million for BED group) in 240 μl of DMEM with 10% autologous sheep serum and once again incubated for 1 h at 37 °C. At last 25 ml of DMEM with 10% autologous sheep serum was added for transport and implantation was performed within 3 h after assembly of the ATMP. The residual transport medium was centrifuged for calculating of cell seeding efficiency by counting the remaining cells.
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