SD-MBG was sterilized by heating at 160 °C for 2 h before BMP-2 immobilization. Then, 500 μg BMP-2 (pure rhBMP-2 from E. coli, Peprotech, Hamburg, Germany) was dissolved in 1 mL of ultrapure water to form BMP-2 solution (500 μg/mL). Next, 20 μL of BMP-2 solution was added to 0.75 mg of SD-MBG. After adsorption for 30 min, another 20 μL of BMP-2 solution was added. This process was repeated until 50 μg of BMP-2 was added to 0.75 mg of MBG particles. Repetitions of loading small volumes of BMP-2 solution were performed in order to homogenously and precisely load the particles with BMP-2. For in vitro analysis of bioactivity and morphology, 15 µg BMP-2/0.75 mg SD-MBG were loaded. The BMP-2 loaded SD-MBG (SD-MBG + BMP-2) was then dried overnight at 37 °C under sterile conditions. BMP-2 loaded SD-MBG was stored at −20 °C until usage.
Bmp 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The BMP-2 is a lab equipment product manufactured by Thermo Fisher Scientific. It is a recombinant human bone morphogenetic protein-2 used for in vitro research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Tgf β1, by Thermo Fisher Scientific (11 mentions)
Tgf β3, by Thermo Fisher Scientific (7 mentions)
Ascorbic acid, by Merck Group (7 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (7 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (7 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Activin a, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (4 mentions)
Na pyruvate, by Thermo Fisher Scientific (4 mentions)
Insulin, by Merck Group (4 mentions)
Pdgf bb, by Thermo Fisher Scientific (4 mentions)
Sodium pyruvate, by Merck Group (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Pdgf aa, by Thermo Fisher Scientific (3 mentions)
97 protocols using bmp 2
1
BMP-2 Immobilization on Silica Microparticles
2
Fabrication of Bone-Mimetic Scaffold
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Acetic acid solution was diluted in deionized water to a concentration of 0.005 mol/l, and COL (10 mg; Shengyou Biotechnology Co., Ltd., Hangzhou, China) was subsequently added into 10 ml acetic acid solution and stirred (JB-2A; Bante Instruments Ltd., Shanghai, China) for 50 min. nHAP (10 mg; Emperor Nano Material Co., Ltd., Nanjing, China) was added to this solution and stirred overnight. The quality ratio was determined to be 1:1.5 (nHAP:COL). BMP-2 (PeproTech, Inc., Rocky Hill, NJ, USA) was dissolved using PeproTech protein solution to 10 ng/µl (21 (link)), and added to the scaffold solution prior to stirring for 50 min at 4°C, resulting in a final BMP-2 concentration of 100 ng/ml. The solution was added into a 24-well Teflon™ culture plate and frozen at −20°C for 24 h, and lyophilized at −80°C for 48 h (VFD-2000; Boyikang Laboratory Instruments, Co., Ltd., Beijing, China) to form BMP-2-nHAP-COL scaffolds. Scaffold morphology and microstructure was observed by scanning electron microscopy (SEM).
3
Hepatic Differentiation of Human ES Cells
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Undifferentiated human H1 ES cells (WiCell) were maintained in monolayer culture on Matrigel (BD Biosciences) in mTeSR1 medium (Stemcell Technologies, 05850) at 37 °C with 5% CO2. Cells were manually passaged at 1:4 to 1:6 split ratios every 3–5 days. For hepatic differentiation, cells were cultured for 3 days in RPMI/B27 medium (Insulin minus, Gibco, A18956-01) supplemented with 100 ng/ml Activin A (Peprotech, 120-14E), followed by 4 days with 20 ng/mL BMP2 (Peprotech, 120-02) and 30 ng/mL FGF-4 (Peprotech, 100-31) in RPMI/B27 (complete with Insulin, Gibco, 17504-044) medium, then 6 days with 20 ng/mL HGF (Peprotech, 100-39) and KGF (Peprotech, 100-19) in RPMI/B27 (complete with Insulin), then 8 days with 20 ng/mL Oncostatin-M (R&D Systems, 295-OM/CF) in hepatocyte culture media (Lonza, cc-3198) supplemented with SingleQuots (without EGF). All cell lines used were negative for mycoplasma contamination. For 3D culture, cells were dissociated as small patch at day 16 using accutase and manual pipetting and add to a solution of 40% Matrigel containing hepatocyte culture medium supplemented with SingleQuots (without EGF). Then cells were placed as drop in eight-well Lab-Tek II chamber slides and culture for 6 days before fixation.
4
Multi-lineage Differentiation Potential of hDIAS Cells
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To determine the multi-lineage differentiation potential of cells from each tissue type, hDIAS cells from each donor were grown in established adipogenic, osteogenic, and chondrogenic culture conditions [10 (link)]. For adipogenic and osteogenic differentiation, 1.5x104 cells from each donor were plated in each well of a 24-well plate. Adipogenic differentiation medium consisted of DMEM with high glucose/GlutaMAX™-I, 5% FBS, 1% P/S/F, 1% NEAA, 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine (Sigma-Aldrich), and 0.2 mM indomethacin (Sigma-Aldrich). Osteogenic differentiation medium consisted of DMEM with high glucose/GlutaMAX™-I, 10% FBS, 1% P/S/F, 1% NEAA, 100 nM dexamethasone, 10 mM β-glycerophosphate (Sigma-Aldrich), 250 mM ascorbate-2-phosphate, and 50 ng/mL bone morphogenetic protein-2 (BMP-2) (Peprotech). For chondrogenic differentiation, 2.5x105 cells from each donor were added to a round bottom polystyrene 96-well plate (Costar 3799, Corning, NY) 0.2 mL of CHG, supplemented with 50 ng/mL BMP-2, and 10 ng/mL transforming growth factor beta-1 (TGF-β1) (Peprotech), and centrifuged at 500 xg for 5 minutes to form cell pellets [30 (link)]. All groups were then maintained at 37°C and 5% CO2 for 4 weeks, with media exchanged every other day.
5
Differentiation of PGCLCs from 4i-state Cells
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To induce PGCLC differentiation, 4i-state cells were dissociated with TrypLE Express (GIBCO), then the cells were seeded into ultra-low cell attachment U-bottom 96-well plates (Corning) at a density of 3000 cells/well in 200 μL PGCLC differentiation medium containing Glasgow’s MEM (GMEM, GIBCO), 15% KOSR, 0.1 mM nonessential amino acids, 0.1 mM β-mercaptoethanol, 1% penicillin–streptomycin-L-glutamate, 1 mM sodium pyruvate, 250 ng/mL BMP4 (R&D Systems), 250 ng/mL BMP2 (PeproTech), 1 μg/mL human LIF (PeproTech), 100 ng/mL SCF (PeproTech), 50 ng/mL EGF (PeproTech), and 10 μM ROCK inhibitor, Y-27632 (Selleck). The medium was changed daily.
6
Chondrogenic Differentiation of cyiPSCs
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The cyiPSCs were chondrogenically differentiated to produce cartilage using a previously described method for human iPSCs9 with one minor modification. In brief, after chondrogenic differentiation, the cells were transferred into suspension culture to induce ECM secretion and form cartilaginous particles 1–3 mm in diameter. Cells and particles were cultured in chondrogenic medium [Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis) with 1% ITS-X (Thermo Fisher Scientific, Waltham), 1% fetal bovine serum (FBS) (Thermo Fisher Scientific), 1 × 10−4 M nonessential amino acids (Thermo Fisher Scientific), 1 mM Na pyruvate (Thermo Fisher Scientific), 50 U penicillin, 50 mg/mL streptomycin (1% PC/SM, Thermo Fisher Scientific), 0.25 μg/mL amphotericin B (Thermo Fisher Scientific), 50 μg/mL ascorbic acid (Nacalai), 1 μM rosuvastatin (BioVision, Milpitas), 10 ng/mL BMP2 (Peprotech), 10 ng/mL transforming growth factor beta 1 (TGFβ1) (Peprotech), and 10 ng/mL GDF5 (BioVision)]. For human iPSCs, the chondrogenic medium is typically replaced with the conventional medium (DMEM supplemented with 10% FBS and 1% PC/SM) at 6 weeks after the chondrogenic differentiation.9 In contrast, cyiPSCs were kept in chondrogenic medium after the chondrogenic differentiation until the analysis.
7
Tuning BMP-2 Release in Laponite Bioink
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To investigate whether the addition of laponite can tailor the growth factor release profile over a long culture period, a base bioink (Fast BMP-2 Release) and a laponite bioink (Slow BMP-2 Release) were compared. For both growth factor release profiles, a dual-syringe approach was used to deliver BMP-2 (200 ng/ml; PeproTech, UK) to the solutions before precross-linking with 60 mM CaSO4 to make the bioinks. These were printed into a 100 mM CaCl2 soak agarose mold to generate final constructs of Ø 6 mm by 6 mm high. In addition to comparing the growth factor release profile of the two bioinks, the degradation rate of the bioinks was also investigated. These scaffolds were cultured in normoxic conditions for up to 35 days and media from each sample were changed weekly. For BMP-2 release study, medium samples were taken (days 0, 5, 7, 14, 21, and 35) and snap-frozen at −80°C. Printed hydrogels were also snap-frozen at −80°C on day 0 to quantify the concentration of growth factor present in the constructs directly after printing. For the degradation study, samples were washed and snap-frozen at −80°C and each time point (days 0, 5, 7, 14, and 21). Samples were lyophilized by placing the samples in a freeze dryer (FreeZone Triad, Labconco, Kansas City, USA). Each sample was then weighed using an analytical balance (Mettler Toledo, XS205).
8
Mammary Epithelial Cell Culture and Hormone Treatment
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HC11 mouse mammary epithelial cells were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). HC11 cells were cultured at 37°C with 5% CO2 in RPMI-1640 (Gibco, Grand Island, NY, United States) medium supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), 5 μg/mL insulin (Sigma) and 10 ng/mL epithelial growth factor (EGF, Gibco). BMP4 (Abcam, Cambridge, MA, United States) is obtained from Escherichia coli, reconstituted in 10 mM citric acid and suitable for functional studies. BMP2 (PeproTech, Rocky Hill, NJ, United States) is obtained from Escherichia coli, reconstituted in deionized water containing 0.1% BSA and biological activity verification. For BMPs treatment, HC11 cells were serum starved overnight and then treated with BMP4 (50 ng/mL) or BMP2 (50 ng/mL) at the indicated time points. 10 mM citric acid was used as vehicle control for BMP4, deionized water containing 0.1% BSA was used as vehicle control for BMP2. For hormone stimulation, HC11 cells were grown to 70% confluence and serum starved overnight, then stimulated with 100% ethanol-dissolved 10 nM β-estradiol (E2, Sigma) and/or 100 nM progesterone (Pg, Sigma), 100% ethanol was used as vehicle control, the cells were harvested at 24 h after this treatment.
9
Cartilage Tissue Engineering Reagents
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Gelatin was purchased from Nitta Gelatin Inc. (Osaka, Japan). Low molecular weight heparin (LMWH), bovine serum albumin (BSA), and dexamethasone were purchased from Sigma-Aldrich (USA). Recombinant GDF-5, transforming growth factor beta 1 (TGF-β1), and bone morphogenetic protein 2 (BMP2) were from PeproTech (Rocky Hill, USA). The antibodies against aggrecan and COL2 were purchased from Abcam (UK). All reagents used were of at least ACS grade.
10
Chondrogenic Differentiation of Cells
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