Recombinant human BMP-2 (R&D Systems, Minneapolis, MN) was dissolved at 100 μg/mL in sterile water and single use aliquots were frozen until use. For loading and release studies, 0.1 mg of each MP formulation and 100 ng BMP-2 were incubated in 0.5 mL 0.5% BSA PBS solution for 16 h at 4°C. The amount of BMP-2 was chosen to ensure that the concentration of BMP-2 released from the MPs falls within the linear range of the BMP-2 bioactivity assay. After 16 hours, MPs were centrifuged at 15,000 RCF for 3 min, the supernatant was removed, and MPs were re-suspended in 0.5 mL fresh 0.5% BSA solution. The supernatant removed at 16 and 19 h was used to quantify loading onto MPs via BMP-2 ELISA (R&D Systems). Assuming that BMP-2 released over the initial 3 h was not specifically bound to the MPs, the BMP-2 in the supernatant at 16 and 19 h was subtracted from the amount of BMP-2 in BMP-2 samples that had been incubated for the same time without MPs to determine the amount of BMP-2 bound/loaded onto MPs. Following this loading procedure, MPs were incubated at 37°C and supernatant was collected on days 1, 2, 4, 7, and 10 to quantify BMP-2 release (n = 3–5).
Recombinant human bmp 2
Manufactured by R&D Systems
Sourced in United States
Recombinant human BMP-2 is a growth factor produced in a cell expression system. It is a member of the bone morphogenetic protein family and plays a role in bone and cartilage development.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (3 mentions)
Bmp 2, by R&D Systems (3 mentions)
Sb431542, by Merck Group (2 mentions)
Dmh 1, by R&D Systems (2 mentions)
Dorsomorphin dihydrochloride dorsomorphin, by R&D Systems (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
α mem, by Fujifilm (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions)
Vivotag 750, by PerkinElmer (2 mentions)
Bmp 2 elisa, by R&D Systems (1 mentions)
5α dihydrotestosterone dht, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Igf 1, by Merck Group (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Normal goat igg, by R&D Systems (1 mentions)
Heparin, by Merck Group (1 mentions)
Goat anti rabbit igg h l hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l hrp, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
37 protocols using recombinant human bmp 2
1
Quantifying BMP-2 Loading and Release from Microparticles
2
Cell Culture Reagent Analysis
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Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM)-α, penicillin-streptomycin solution, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). 5α-dihydrotestosterone (DHT), recombinant human somatotropin (GH), and IGF-I were also obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Recombinant human BMP-2, anti-mouse IGF-I antibody, and normal goat IgG were purchased from R & D Systems Inc. (Minneapolis, MN, USA).
3
Regulation of BMP2-Induced Osteogenesis
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Recombinant human BMP2, DMH-1 (4-[6-[4-(1-methylethoxy) phenyl] pyrazolo [1,5-a]pyrimidin-3-yl]-quinoline), and dorsomorphin dihydrochloride (dorsomorphin) were obtained from R&D Systems (Minneapolis, MN, United States). SB431542 (catalog no. S4317) was purchased from the Sigma-Aldrich Corp. (St. Louis, MO, United States). A polyclonal goat anti-COX-1 (catalog no. sc-1752), polyclonal rabbit anti-SMAD1/5/8 (N-18; sc-6031-R), monoclonal mouse anti-α-tubulin (B-5-1-2; catalog no. sc-23948) and monoclonal mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G-9; sc-365062) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). A polyclonal rabbit anti-phospho-SMAD1 (Ser463/465)/SMAD5 (Ser463/465)/SMAD8 (Ser465/467) (D5B10) antibody was obtained from Cell Signaling Technology (Beverly, MA, United States). Horseradish peroxidase-conjugated rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Bio-Rad (Richmond, CA, United States).
4
Recombinant BMP-2 Biomaterial Evaluation
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Recombinant human BMP-2 was obtained from R&D Systems (Minneapolis, MN, USA). PAA (catalog molecular weight: 5000) was obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Heparin, PMAA (catalog molecular weight: 7750), PAsp (poly(α,β-dl -aspartic acid); catalog molecular weight: 2000 to 11,000), and PGlu (poly(l -glutamic acid); catalog molecular weight: 1500 to 5500) were obtained from Sigma-Aldrich (Milwaukee, WI, USA).
5
Antibody Validation Protocols for Cellular Signaling
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The following antibodies (Abs) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA): p-SMAD1 (Ser206), p-SMAD2 (Ser465/467), p-SMAD3 (Ser423/425), SMAD2/3, HA-Tag, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-MAPK14 (Thr180/Tyr182), MAPK14, and p-JNK1/2 (Thr183/Tyr185). Abs against GAPDH, RUNX2, COL10A1, MMP13, SMAD1, SMAD4, ITGB1, JNK1/2, THOC1, p-SMAD1 (Ser463/Ser465), and anti-phospho-serine/threonine antibody were from Abcam (Cambridge, UK). Abs against COL2A1, BMPR1A, and BMPR1B were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). p-ITGB1 (Thr788) and Flag-Tag Abs were obtained from Sigma-Aldrich (St. Louis, MO, USA). Goat anti-rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP) secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA). Goat anti-rabbit IgG (H+L), F(ab′)2 fragment (Alexa Fluor® 555 Conjugate) secondary antibodies were from CST. Anti-human IgG antibody was purchased from Abcam.
Blocking antibodies against human integrin beta1, recombinant human interleukin-1 beta, and recombinant human BMP2 were obtained from R&D systems. Purified COL2A1 was purchased from Chondrex, Inc. (Redmond, WA, USA). U0126 and SP600125 were from CST. SB505124 was obtained from MedChemExpress (Monmouth Junction, NJ, USA).
Blocking antibodies against human integrin beta1, recombinant human interleukin-1 beta, and recombinant human BMP2 were obtained from R&D systems. Purified COL2A1 was purchased from Chondrex, Inc. (Redmond, WA, USA). U0126 and SP600125 were from CST. SB505124 was obtained from MedChemExpress (Monmouth Junction, NJ, USA).
6
Osteoblastic Differentiation of Hair Follicle Cells
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Proliferative hair follicle cells (5×105 cells/dish) were cultured in α Minimum Essential Medium (αMEM; Wako Pure Chemical Industries) containing 10% fetal calf serum (FCS) and 200 ng/mL recombinant human BMP-2 (R&D Systems, Inc., MN, USA) in 6-cm collagen Type I-coated dishes (IWAKI). Fresh medium was added on day 3 of the culturing process. Osteoblastic differentiation was determined by measuring ALP activity, ALP staining, and expression of osteoblast-related genes (Runx2, ALP, osteocalcin, osterix). To confirm calcification, proliferative hair follicle cells (2×104 cells/well) were cultured in αMEM containing 10% FCS, 200 ng/mL recombinant human BMP-2, 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 mg/mL ascorbic acid (Sigma-Aldrich), and 10−8 M dexamethasone (Sigma-Aldrich) in 96-well collagen Type I-coated plates (IWAKI) for 20 days. Calcification was detected by von Kossa and alizarin red staining.
7
Probing BMP2 Signaling Pathways with Kinase Inhibitors
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The polyclonal anti-SMAD4 (9515) antibody was purchased from Cell Signaling Technology (Danvers, MA). Monoclonal anti-α-tubulin (sc-23,948) and polyclonal anti-CTGF (sc-14,939) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin G were obtained from Bio-Rad Laboratories (Hercules, CA). HRP-conjugated donkey anti-goat immunoglobulin G was obtained from Santa Cruz Biotechnology. Recombinant human BMP2, dorsomorphin dihydrochloride (dorsomorphin), and dorsomorphin homolog 1 (DMH-1; 4-[6-[4-(1-methylethoxy) phenyl] pyrazole [1, 5-a] pyrimidin-3-yl]-quinoline) were obtained from R&D Systems (Minneapolis, MN). CTGF overexpression and CONTROL vectors were obtained from Gene Copoeia (Rockville, MD). In this study, two specific protein kinase inhibitors DMH-1 (an inhibitor of ALK2/3) and dorsomorphin (an inhibitor of ALK2/3/6) were used to conduct the loss of function experiment by blocking the downstream signaling pathways of ALK2/36. Specifically, the cells were pretreated with DMH-1 (0.25 μM) or dorsomorphin (10 μM) for 1 h, and cells were then treated with a vehicle control or 50 ng/ml BMP2 for 6 h (for examining mRNA levels) or 12 h (for examining protein levels).
8
Osteoblast Differentiation Protocol
9
Bone Marrow Stromal Cell Differentiation
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Bone marrow stromal cells and OBs were treated with OB differentiation medium containing 50 μg·mL−1 ascorbic acid and 10 mm β‐glycerophosphate for 0, 7, or 14 days 23 . BMSCs, OBs, OCYs, or 8‐week‐old male mice femoral bones were treated with 100 ng·mL−1 recombinant human BMP2 (R&D Systems, Minneapolis, MN, USA), several concentrations (0, 10, 20, 50, 100, or 200 ng·mL−1) of rhWnt3a (R&D Systems), 100 ng·mL−1 rhDkk1 (R&D Systems), 100 ng·mL−1 rhWnt1 (R&D Systems), 100 ng·mL−1 rhWnt5a (R&D Systems), 10 μm KCl (Sigma Aldrich Chemicals, St. Louis, MO, USA), or 10 μm LiCl (Sigma Aldrich Chemicals) for 1 day. HEK293T cells were maintained and cultured as reported previously 24 .
10
Osteogenic Differentiation of hMSCs
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Recombinant human BMP2 was purchased from R&D Systems (Minneapolis, MN). E2 was purchased from Steraloids, Inc. (Newport, RI). Estradiol 17β cypionate was purchased from Pfizer Company (Kalamazoo, MI). Phenol red-free DMEM, linolenic acid, bovine serum albumin (BSA), and fine chemicals were purchased from Sigma Chemical Company (St. Louis, MO). LDN193,189 was purchased from Sigma Chemical Co. (St. Louis, MO), ICI182,780 was purchased from Tocris Bioscience (Bristol, UK), and human holo-transferrin was obtained from BD pharmaceuticals (San Jose, CA). Plastic embedding medium and supplies were obtained from Electron Microscopy Sciences (Hatfield, Pa). Quantitative RT-PCR primers and probes were synthesized by Sigma Chemical Company. RNeasy mini kit and Taq DNA polymerase were from Qiagen, Inc. (Valencia, CA). All other molecular biology grade chemicals were obtained from Sigma Chemical Company or Fisher Scientific (Pittsburgh, PA).
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